C4d-negative antibody-mediated rejection with high anti-angiotensin II type I receptor antibodies in absence of donor-specific antibodies.

Science.gov (United States)

Fuss, Alexander; Hope, Christopher M; Deayton, Susan; Bennett, Greg Donald; Holdsworth, Rhonda; Carroll, Robert P; Coates, P Toby H

2015-07-01

Acute antibody-mediated rejection can occur in absence of circulating donor-specific antibodies. Agonistic antibodies targeting the anti-angiotensin II type 1 receptor (anti-AT1 R) are emerging as important non-human leucocyte antigen (HLA) antibodies. Elevated levels of anti-angiotensin II receptor antibodies were first observed in kidney transplant recipients with malignant hypertension and allograft rejection. They have now been studied in three separate kidney transplant populations and associate to frequency of rejection, severity of rejection and graft failure. We report 11 cases of biopsy-proven, Complement 4 fragment d (C4d)-negative, acute rejection occurring without circulating donor-specific anti-HLA antibodies. In eight cases, anti-angiotensin receptor antibodies were retrospectively examined. The remaining three subjects were identified from our centre's newly instituted routine anti-angiotensin receptor antibody screening. All subjects fulfilled Banff 2013 criteria for antibody-mediated rejection and all responded to anti-rejection therapy, which included plasma exchange and angiotensin receptor blocker therapy. These cases support the routine assessment of anti-AT1 R antibodies in kidney transplant recipients to identify subjects at risk. Further studies will need to determine optimal assessment protocol and the effectiveness of pre-emptive treatment with angiotensin receptor blockers. © 2015 Asian Pacific Society of Nephrology.

Development of antibodies against the rat brain somatostatin receptor.

Science.gov (United States)

Theveniau, M; Rens-Domiano, S; Law, S F; Rougon, G; Reisine, T

1992-05-15

Somatostatin (SRIF) is a neurotransmitter in the brain involved in the regulation of motor activity and cognition. It induces its physiological actions by interacting with receptors. We have developed antibodies against the receptor to investigate its structural properties. Rabbit polyclonal antibodies were generated against the rat brain SRIF receptor. These antibodies (F4) were able to immunoprecipitate solubilized SRIF receptors from rat brain and the cell line AtT-20. The specificity of the interaction of these antibodies with SRIF receptors was further demonstrated by immunoblotting. F4 detected SRIF receptors of 60 kDa from rat brain and adrenal cortex and the cell lines AtT-20, GH3, and NG-108, which express high densities of SRIF receptors. They did not detect immunoreactive material from rat liver or COS-1, HEPG, or CRL cells, which do not express functional SRIF receptors. In rat brain, 60-kDa immunoreactivity was detected by F4 in the hippocampus, cerebral cortex, and striatum, which have high densities of SRIF receptors. However, F4 did not interact with proteins from cerebellum and brain stem, which express few SRIF receptors. Immunoreactive material cannot be detected in rat pancreas or pituitary, which have been reported to express a 90-kDa SRIF receptor subtype. The selective detection of 60-kDa SRIF receptors by F4 indicates that the 60- and 90-kDa SRIF receptor subtypes are immunologically distinct. The availability of antibodies that selectively detect native and denatured brain SRIF receptors provides us with a feasible approach to clone the brain SRIF receptor gene(s).

Binding-site analysis of opioid receptors using monoclonal anti-idiotypic antibodies

International Nuclear Information System (INIS)

Conroy, W.G.

1988-01-01

Structural relatedness between the variable region of anti-ligand antibodies and opioid binding sites allowed the generation of anti-idiotypic antibodies which recognized opioid receptors. The IgG 3 k antibodies which bound to opioid receptors were obtained when an anti-morphine antiserum was the idiotype. Both antibodies bound to opioid receptors, but only one of these blocked the binding of [ 3 H]naloxone. The antibody which did not inhibit the binding of [ 3 H]naloxone was itself displaced from the receptor by opioid ligands. The unique binding properties displayed by this antibody indicated that anti-idiotypic antibodies are not always a perfect image of the original ligand, and therefore may be more useful than typical ligands as probes for the receptor. An auto-anti-idiotypic technique was successfully used to obtain anti-opioid receptor antibodies. Another IgG 3 k antibody that blocked the binding of [ 3 H]naloxone to rat brain opioid receptors was obtained when a mouse was immunized with naloxone conjugated to bovine serum albumin. These data confirmed that an idiotype-anti-idiotype network which can generate an anti-receptor antibody normally functions when an opioid ligand is introduced into an animal in an immunogenic form

Nogo-A expression dynamically varies after spinal cord injury

Directory of Open Access Journals (Sweden)

Jian-wei Wang

2015-01-01

Full Text Available The mechanism involved in neural regeneration after spinal cord injury is unclear. The myelin-derived protein Nogo-A, which is specific to the central nervous system, has been identified to negatively affect the cytoskeleton and growth program of axotomized neurons. Studies have shown that Nogo-A exerts immediate and chronic inhibitory effects on neurite outgrowth. In vivo, inhibitors of Nogo-A have been shown to lead to a marked enhancement of regenerative axon extension. We established a spinal cord injury model in rats using a free-falling weight drop device to subsequently investigate Nogo-A expression. Nogo-A mRNA and protein expression and immunoreactivity were detected in spinal cord tissue using real-time quantitative PCR, immunohistochemistry and western blot analysis. At 24 hours after spinal cord injury, Nogo-A protein and mRNA expression was low in the injured group compared with control and sham-operated groups. The levels then continued to drop further and were at their lowest at 3 days, rapidly rose to a peak after 7 days, and then gradually declined again after 14 days. These changes were observed at both the mRNA and protein level. The transient decrease observed early after injury followed by high levels for a few days indicates Nogo-A expression is time dependent. This may contribute to the lack of regeneration in the central nervous system after spinal cord injury. The dynamic variation of Nogo-A should be taken into account in the treatment of spinal cord injury.

Nogo-B (Reticulon-4B) functions as a negative regulator of the apoptotic pathway through the interaction with c-FLIP in colorectal cancer cells.

Science.gov (United States)

Kawaguchi, Nao; Tashiro, Keitaro; Taniguchi, Kohei; Kawai, Masaru; Tanaka, Keitaro; Okuda, Junji; Hayashi, Michihiro; Uchiyama, Kazuhisa

2018-08-01

Nogo-B is a member of the Nogo/Reticulon-4 family and has been reported to be an inducer of apoptosis in certain types of cancer cells. However, the role of Nogo-B in human cancer remains less understood. Here, we demonstrated the functions of Nogo-B in colorectal cancer cells. In clinical colorectal cancer specimens, Nogo-B was obviously overexpressed, as determined by immunohistochemistry; and Western blot analysis showed its expression level to be significantly up-regulated. Furthermore, knockdown of Nogo-B in two colorectal cancer cell lines, SW480 and DLD-1, by transfection with si-RNA (siR) resulted in significantly reduced cell viability and a dramatic increase in apoptosis with insistent overexpression of cleaved caspase-8 and cleaved PARP. The transfection with Nogo-B plasmid cancelled that apoptosis induced by siRNogoB in SW480 cells. Besides, combinatory treatment with siR-Nogo-B/staurosporine (STS) or siR-Nogo-B/Fas ligand (FasL) synergistically reduced cell viability and increased the expression of apoptotic signaling proteins in colorectal cancer cells. These results strongly support our contention that Nogo-B most likely played an oncogenic role in colorectal cancer cells, mainly by negatively regulating the extrinsic apoptotic pathway in them. Finally, we revealed that suppression of Nogo-B caused down-regulation of c-FLIP, known as a major anti-apoptotic protein, and activation of caspase-8 in the death receptor pathway. Interaction between Nogo-B and c-FLIP was shown by immunoprecipitation and immunofluorescence studies. In conclusion, Nogo-B was shown to play an important negative role in apoptotic signaling through its interaction with c-FLIP in colorectal cancer cells, and may thus become a novel therapeutic target for colorectal cancer. Copyright © 2018 Elsevier B.V. All rights reserved.

Chemokine Receptor-Specific Antibodies in Cancer Immunotherapy: Achievements and Challenges

Science.gov (United States)

Vela, Maria; Aris, Mariana; Llorente, Mercedes; Garcia-Sanz, Jose A.; Kremer, Leonor

2015-01-01

The 1990s brought a burst of information regarding the structure, expression pattern, and role in leukocyte migration and adhesion of chemokines and their receptors. At that time, the FDA approved the first therapeutic antibodies for cancer treatment. A few years later, it was reported that the chemokine receptors CXCR4 and CCR7 were involved on directing metastases to liver, lung, bone marrow, or lymph nodes, and the over-expression of CCR4, CCR6, and CCR9 by certain tumors. The possibility of inhibiting the interaction of chemokine receptors present on the surface of tumor cells with their ligands emerged as a new therapeutic approach. Therefore, many research groups and companies began to develop small molecule antagonists and specific antibodies, aiming to neutralize signaling from these receptors. Despite great expectations, so far, only one anti-chemokine receptor antibody has been approved for its clinical use, mogamulizumab, an anti-CCR4 antibody, granted in Japan to treat refractory adult T-cell leukemia and lymphoma. Here, we review the main achievements obtained with anti-chemokine receptor antibodies for cancer immunotherapy, including discovery and clinical studies, proposed mechanisms of action, and therapeutic applications. PMID:25688243

Optimal no-go theorem on hidden-variable predictions of effect expectations

Science.gov (United States)

Blass, Andreas; Gurevich, Yuri

2018-03-01

No-go theorems prove that, under reasonable assumptions, classical hidden-variable theories cannot reproduce the predictions of quantum mechanics. Traditional no-go theorems proved that hidden-variable theories cannot predict correctly the values of observables. Recent expectation no-go theorems prove that hidden-variable theories cannot predict the expectations of observables. We prove the strongest expectation-focused no-go theorem to date. It is optimal in the sense that the natural weakenings of the assumptions and the natural strengthenings of the conclusion make the theorem fail. The literature on expectation no-go theorems strongly suggests that the expectation-focused approach is more general than the value-focused one. We establish that the expectation approach is not more general.

Resolution of Disulfide Heterogeneity in Nogo Receptor 1 Fusion Proteins by Molecular Engineering

Energy Technology Data Exchange (ETDEWEB)

P Weinreb; D Wen; F Qian; C Wildes; E Garber; L Walus; M Jung; J Wang; J Relton; et al.

2011-12-31

NgRI (Nogo-66 receptor) is part of a signalling complex that inhibits axon regeneration in the central nervous system. Truncated soluble versions of NgRI have been used successfully to promote axon regeneration in animal models of spinal-cord injury, raising interest in this protein as a potential therapeutic target. The LRR (leucine-rich repeat) regions in NgRI are flanked by N- and C-terminal disulfide-containing 'cap' domains (LRRNT and LRRCT respectively). In the present work we show that, although functionally active, the NgRI(310)-Fc fusion protein contains mislinked and heterogeneous disulfide patterns in the LRRCT domain, and we report the generation of a series of variant molecules specifically designed to prevent this heterogeneity. Using these variants we explored the effects of modifying the NgRI truncation site or the spacing between the NgRI and Fc domains, or replacing cysteines within the NgRI or IgG hinge regions. One variant, which incorporates replacements of Cys{sup 266} and Cys{sup 309} with alanine residues, completely eliminated disulfide scrambling while maintaining functional in vitro and in vivo efficacy. This modified NgRI-Fc molecule represents a significantly improved candidate for further pharmaceutical development, and may serve as a useful model for the optimization of other IgG fusion proteins made from LRR proteins.

Serum Nogo-A levels are not elevated in amyotrophic lateral sclerosis patients.

Science.gov (United States)

Harel, Noam Y; Cudkowicz, Merit E; Brown, Robert H; Strittmatter, Stephen M

2009-09-01

Improved biomarkers would facilitate the diagnosis and treatment of amyotrophic lateral sclerosis (ALS). Muscle content of the neuritic outgrowth inhibitor Nogo-A is increased in patients with ALS and other denervating conditions. Seeking a less invasive diagnostic method, we sought to determine whether or not Nogo increases in the serum of ALS patients. We developed a dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) protocol to screen serum samples from 172 ALS patients and 172 healthy controls for Nogo-A immunoreactivity. Unexpectedly, there was a trend toward decreased levels of serum Nogo-A in ALS. Mean serum Nogo-A level in ALS patients was 0.71 nM (95% confidence interval (CI) 0.42-1.00), as opposed to 1.15 nM (95% CI 0.72-1.59) in healthy controls. A significantly larger percentage of healthy control sera (11.0% vs 4.7%) displayed markedly elevated levels of Nogo-A. Additional study is required to determine the factors that lead to elevated Nogo-A levels in a subset of both ALS patients and healthy controls.

Nogo receptor 1 limits tactile task performance independent of basal anatomical plasticity.

Directory of Open Access Journals (Sweden)

Jennifer I Park

Full Text Available The genes that govern how experience refines neural circuitry and alters synaptic structural plasticity are poorly understood. The nogo-66 receptor 1 gene (ngr1 is one candidate that may restrict the rate of learning as well as basal anatomical plasticity in adult cerebral cortex. To investigate if ngr1 limits the rate of learning we tested adult ngr1 null mice on a tactile learning task. Ngr1 mutants display greater overall performance despite a normal rate of improvement on the gap-cross assay, a whisker-dependent learning paradigm. To determine if ngr1 restricts basal anatomical plasticity in the associated sensory cortex, we repeatedly imaged dendritic spines and axonal varicosities of both constitutive and conditional adult ngr1 mutant mice in somatosensory barrel cortex for two weeks through cranial windows with two-photon chronic in vivo imaging. Neither constant nor acute deletion of ngr1 affected turnover or stability of dendritic spines or axonal boutons. The improved performance on the gap-cross task is not attributable to greater motor coordination, as ngr1 mutant mice possess a mild deficit in overall performance and a normal learning rate on the rotarod, a motor task. Mice lacking ngr1 also exhibit normal induction of tone-associated fear conditioning yet accelerated fear extinction and impaired consolidation. Thus, ngr1 alters tactile and motor task performance but does not appear to limit the rate of tactile or motor learning, nor determine the low set point for synaptic turnover in sensory cortex.

Reduced expression of Nogo-A leads to motivational deficits in rats

Directory of Open Access Journals (Sweden)

Thomas eEnkel

2014-01-01

Full Text Available Nogo-A is an important neurite growth-regulatory protein in the adult and developing nervous system. Mice lacking Nogo-A, or rats with neuronal Nogo-A deficiency, exhibit behavioral abnormalities such as impaired short-term memory, decreased prepulse inhibition and behavioral inflexibility. In the current study we extended the behavioral profile of the Nogo-A deficient rat line with respect to reward sensitivity and motivation and determined the concentrations of the monoamines dopamine and serotonin in the prefrontal cortex, dorsal striatum and nucleus accumbens. Using a limited access consumption task, we found similar intake of a sweet condensed milk solution following ad libitum or restricted feeding in wild-type and Nogo-A deficient rats, indicating normal reward sensitivity and translation of hunger into feeding behavior. When tested for motivation in a spontaneous progressive ratio task, Nogo-A rats exhibited lower break points and tended to have lower ‘highest completed ratios’. Further, under extinction conditions responding ceased substantially earlier in these rats. Finally, in the prefrontal cortex we found increased tissue levels of serotonin, while dopamine was unaltered. Dopamine and serotonin levels were also unaltered in the dorsal striatum and the nucleus accumbens. In summary, these results suggest a role for Nogo-A regulated processes in motivated behavior and related neurochemistry. The behavioral pattern observed resembles aspects of the negative symptomatology of schizophrenia.

Netrin-1 receptor antibodies in thymoma-associated neuromyotonia with myasthenia gravis.

Science.gov (United States)

Torres-Vega, Estefanía; Mancheño, Nuria; Cebrián-Silla, Arantxa; Herranz-Pérez, Vicente; Chumillas, María J; Moris, Germán; Joubert, Bastien; Honnorat, Jérôme; Sevilla, Teresa; Vílchez, Juan J; Dalmau, Josep; Graus, Francesc; García-Verdugo, José Manuel; Bataller, Luis

2017-03-28

To identify cell-surface antibodies in patients with neuromyotonia and to describe the main clinical implications. Sera of 3 patients with thymoma-associated neuromyotonia and myasthenia gravis were used to immunoprecipitate and characterize neuronal cell-surface antigens using reported techniques. The clinical significance of antibodies against precipitated proteins was assessed with sera of 98 patients (neuromyotonia 46, myasthenia gravis 52, thymoma 42; 33 of them with overlapping syndromes) and 219 controls (other neurologic diseases, cancer, and healthy volunteers). Immunoprecipitation studies identified 3 targets, including the Netrin-1 receptors DCC (deleted in colorectal carcinoma) and UNC5A (uncoordinated-5A) as well as Caspr2 (contactin-associated protein-like 2). Cell-based assays with these antigens showed that among the indicated patients, 9 had antibodies against Netrin-1 receptors (7 with additional Caspr2 antibodies) and 5 had isolated Caspr2 antibodies. Only one of the 219 controls had isolated Caspr2 antibodies with relapsing myelitis episodes. Among patients with neuromyotonia and/or myasthenia gravis, the presence of Netrin-1 receptor or Caspr2 antibodies predicted thymoma ( p myasthenia gravis, and neuromyotonia, often with Morvan syndrome ( p = 0.009). Expression of DCC, UNC5A, and Caspr2 proteins was demonstrated in paraffin-embedded thymoma samples (3) and normal thymus. Antibodies against Netrin-1 receptors (DCC and UNC5a) and Caspr2 often coexist and associate with thymoma in patients with neuromyotonia and myasthenia gravis. This study provides Class III evidence that antibodies against Netrin-1 receptors can identify patients with thymoma (sensitivity 21.4%, specificity 100%). © 2017 American Academy of Neurology.

Clinical implications of plasma Nogo-A levels in patients with coronary heart disease.

Science.gov (United States)

Ding, Yu; Gao, Bei-Bei; Zhou, Liang; Ye, Xian-Hua; Li, Hong; Lai, Lei; Huang, Jin-Yu

2017-06-01

Nogo-A is an important neurite growth-regulatory protein in the adult and developing nervous system. Recently, increasing evidence has shown that Nogo-A plays important roles in cardiac development and may act as a potential indicator for heart failure. In addition, increased oxidative stress has been found in individuals with cardiovascular diseases. However, not much is known regarding the expression levels of Nogo-A and reactive oxygen species (ROS) in patients with coronary heart disease (CHD). Therefore, we sought to investigate the relationship between Nogo-A, ROS levels and CHD. The plasma Nogo-A and ROS concentrations of 122 acute coronary syndrome (ACS), 101 unstable angina pectoris (UAP), and 21 acute myocardial infarction (AMI) patients and 56 healthy controls were measured by enzyme-linked immunosorbent assay (ELISA). We further generated a receiver operating characteristic (ROC) curve to assess the diagnostic accuracy of Nogo-A and ROS in CHD. The Nogo-A and ROS levels were significantly higher in patients with CHD than those in healthy controls. In addition, multivariate logistic regression analysis revealed that the level of Nogo-A (odds ratio (OR) = 1.624, 95% confidence interval: 1.125-2.293, p = 0.009) is a risk factor for prediction of CHD. Nogo-A has diagnostic value, with an optimal threshold of 5.466 ng/ml for maximized diagnostic performance (59% sensitivity and 78.6% specificity, area under curve, p < 0.05). However, ROS concentration is not a risk factor for prediction of CHD (OR = 0.999, 95% confidence interval: 0.997-1.001, p = 0.320). Increased plasma Nogo-A level may be associated with CHD.

Agonistic effects of a monoclonal antibody specific for the interleukin-2 receptor

International Nuclear Information System (INIS)

Eardley, D.D.; Makrides, V.

1986-01-01

Interleukin-2 (IL-2) mediated immune responses can be blocked by monoclonal antibodies to the IL-2 receptor. The monoclonal antibody, M720, is defined as specific for the IL-2 receptor because it blocks 35 S-IL-2 binding to Con A blasts, reacts with lymphoblasts but not resting splenocytes, and inhibits IL-2 induced proliferation to mitogen, antigen, or allogeneic stimuli. Under appropriate culture conditions, the IL-2 receptor-specific antibody can act like IL-2 in that it will induce proliferation in T cells in the absence of additional antigen or mitogen. This agonistic effect is dependent on time, dose of antibody, and requires fetal calf serum (FCS) in the media. Because the FCS is not mitogenic by itself, the authors propose that the FCS components act as incomplete mitogen to induce appearance of IL-2 receptors but lack a factor which would push the majority of the cells into the S phase of the cell cycle. This factor is usually IL-2, but in the authors experiments, the IL-2 receptor-specific antibody can provide the same stimulus. These data indicate that factors like FCS can induce IL-2 receptors, but without additional IL-2 or receptor triggering, the cells will not proceed through the synthetic and proliferative phases of cell growth

Anti-NMDA-receptor encephalitis: case series and analysis of the effects of antibodies.

Science.gov (United States)

Dalmau, Josep; Gleichman, Amy J; Hughes, Ethan G; Rossi, Jeffrey E; Peng, Xiaoyu; Lai, Meizan; Dessain, Scott K; Rosenfeld, Myrna R; Balice-Gordon, Rita; Lynch, David R

2008-12-01

A severe form of encephalitis associated with antibodies against NR1-NR2 heteromers of the NMDA receptor was recently identified. We aimed to analyse the clinical and immunological features of patients with the disorder and examine the effects of antibodies against NMDA receptors in neuronal cultures. We describe the clinical characteristics of 100 patients with encephalitis and NR1-NR2 antibodies. HEK293 cells ectopically expressing single or assembled NR1-NR2 subunits were used to determine the epitope targeted by the antibodies. Antibody titres were measured with ELISA. The effect of antibodies on neuronal cultures was determined by quantitative analysis of NMDA-receptor clusters. Median age of patients was 23 years (range 5-76 years); 91 were women. All patients presented with psychiatric symptoms or memory problems; 76 had seizures, 88 unresponsiveness (decreased consciousness), 86 dyskinesias, 69 autonomic instability, and 66 hypoventilation. 58 (59%) of 98 patients for whom results of oncological assessments were available had tumours, most commonly ovarian teratoma. Patients who received early tumour treatment (usually with immunotherapy) had better outcome (p=0.004) and fewer neurological relapses (p=0.009) than the rest of the patients. 75 patients recovered or had mild deficits and 25 had severe deficits or died. Improvement was associated with a decrease of serum antibody titres. The main epitope targeted by the antibodies is in the extracellular N-terminal domain of the NR1 subunit. Patients' antibodies decreased the numbers of cell-surface NMDA receptors and NMDA-receptor clusters in postsynaptic dendrites, an effect that could be reversed by antibody removal. A well-defined set of clinical characteristics are associated with anti-NMDA-receptor encephalitis. The pathogenesis of the disorder seems to be mediated by antibodies.

Response inhibition in borderline personality disorder: event-related potentials in a Go/Nogo task.

Science.gov (United States)

Ruchsow, M; Groen, G; Kiefer, M; Buchheim, A; Walter, H; Martius, P; Reiter, M; Hermle, L; Spitzer, M; Ebert, D; Falkenstein, M

2008-01-01

Borderline personality disorder (BPD) has been related to a dysfunction of anterior cingulate cortex, amygdala, and prefrontal cortex and has been associated clinically with impulsivity, affective instability, and significant interpersonal distress. We examined 17 patients with BPD and 17 age-, sex-, and education matched control participants with no history of Axis I or II psychopathology using event-related potentials (ERPs). Participants performed a hybrid flanker-Go/Nogo task while multichannel EEG was recorded. Our study focused on two ERP components: the Nogo-N2 and the Nogo-P3, which have been discussed in the context of response inhibition and response conflict. ERPs were computed on correct Go trials (button press) and correct Nogo trials (no button press), separately. Groups did not differ with regard to the Nogo-N2. However, BPD patients showed reduced Nogo-P3 amplitudes. For the entire group (n = 34) we found a negative correlation with the Barratt Impulsiveness Scale (BIS-10) and Becks's depression inventory (BDI). The present study is the first to examine Nogo-N2 and Nogo-P3 in BPD and provides further evidence for impaired response inhibition in BPD patients.

Measurement of anti- acetylcholine receptor auto-antibodies in ...

African Journals Online (AJOL)

auto-antibodies in myasthenia gravis. K. J. Steenkamp, W. Duim, M. s. Myer,. S. C. K. Malfeld, R. Anderson. Two different acetylcholine receptor (AChR) preparations derived from ... the detection of AChR auto-antibodies in serum specimens from 20 ... 4°C. Thereafter, 1 ml of washing solution (phosphate- buffered saline ...

The Value of Circulating Nogo-B for Evaluating Hepatic Functional Reserve in Patients with Cirrhosis

Directory of Open Access Journals (Sweden)

Maoyao Wen

2015-01-01

Full Text Available Objective. To examine Nogo-B in liver tissues and plasma of patients with liver cirrhosis and associate them with various clinical parameters. Materials and Methods. Nogo-B protein expression was examined by immunohistochemistry in 24 human fibrotic/cirrhotic liver specimens and 10 healthy controls. We determined plasma Nogo-B levels by enzyme-linked immunosorbent assay in 301 patients with liver cirrhosis and 153 healthy controls, and then analyzed various clinical parameters. Results. Nogo-B was mainly expressed in nonparenchymal cells in the liver and was marked increased in liver with significant fibrosis/cirrhosis compared to controls. Moreover, Metavir F4 showed a higher level of expression than F2. Plasma Nogo-B levels were significantly higher in cirrhotic patients than in healthy controls and were the highest in Child-Pugh class C patients. Plasma Nogo-B levels were positively correlated with Child-Pugh scores. However, there was no relationship between plasma Nogo-B levels and etiology of liver diseases, ALT, AST, platelet counts, and the severity of esophagogastric varices. Conclusions. Nogo-B is mainly expressed in hepatic nonparenchymal cells and is present in plasma. Abnormally high plasma levels of Nogo-B are associated with hepatic cirrhosis and Child-Pugh score, but not correlated with the grade of liver inflammation or portal hypertension. Plasma Nogo-B may be a novel surrogate marker to reflect liver function reserve.

Antibodies to the α-subunit of insulin receptor from eggs of immunized hens

International Nuclear Information System (INIS)

Song, C.; Yu, J.; Bai, D.H.; Hester, P.Y.; Kim, K.

1985-01-01

Simple methods for the generation, purification, and assay of antibodies to the α-subunit of insulin receptor from eggs of immunized hen have been described. Chicken antibodies against the α-subunit inhibit insulin binding to the receptor and stimulate glucose oxidation as well as autophosphorylation of the β-subunit. Thus the properties of chicken antibodies are very similar to those of antibodies found in human autoimmune diseases and different from rabbit antibodies obtained against the same antigen

NOGO-A induction and localization during chick brain development indicate a role disparate from neurite outgrowth inhibition

Directory of Open Access Journals (Sweden)

Liwnicz Boleslaw H

2007-04-01

Full Text Available Abstract Background Nogo-A, a myelin-associated protein, inhibits neurite outgrowth and abates regeneration in the adult vertebrate central nervous system (CNS and may play a role in maintaining neural pathways once established. However, the presence of Nogo-A during early CNS development is counterintuitive and hints at an additional role for Nogo-A beyond neurite inhibition. Results We isolated chicken NOGO-A and determined its sequence. A multiple alignment of the amino acid sequence across divergent species, identified five previously undescribed, Nogo-A specific conserved regions that may be relevant for development. NOGO gene transcripts (NOGO-A, NOGO-B and NOGO-C were differentially expressed in the CNS during development and a second NOGO-A splice variant was identified. We further localized NOGO-A expression during key phases of CNS development by in situ hybridization. CNS-associated NOGO-A was induced coincident with neural plate formation and up-regulated by FGF in the transformation of non-neural ectoderm into neural precursors. NOGO-A expression was diffuse in the neuroectoderm during the early proliferative phase of development, and migration, but localized to large projection neurons of the optic tectum and tectal-associated nuclei during architectural differentiation, lamination and network establishment. Conclusion These data suggest Nogo-A plays a functional role in the determination of neural identity and/or differentiation and also appears to play a later role in the networking of large projection neurons during neurite formation and synaptogenesis. These data indicate that Nogo-A is a multifunctional protein with additional roles during CNS development that are disparate from its later role of neurite outgrowth inhibition in the adult CNS.

Upregulated expression of Nogo-A and NgR in an experimental model of focal microgyria regulates the migration, proliferation and self-renewal of subventricular zone neural progenitors

Energy Technology Data Exchange (ETDEWEB)

Yu, Sixun; Shu, Haifeng; Yang, Tao; Huang, Haidong [Department of Neurosurgery, General Hospital of the People' s Liberation Army Chengdu Military Region, Chengdu, Sichuan, 610083 (China); Li, Song [Department of Neurosurgery, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037 (China); Zhao, Ziyi [Central Laboratory, Teaching Hospital of Chengdu University of Traditional Chinese Medicine, 610075 (China); Kuang, Yongqin, E-mail: kuangyongqin@163.com [Department of Neurosurgery, General Hospital of the People' s Liberation Army Chengdu Military Region, Chengdu, Sichuan, 610083 (China)

2016-04-29

Nogo-A and its receptor (NgR) were first described as myelin-associated inhibitors of neuronal regeneration in response to injury. In recent years, knowledge about the important role of the Nogo-A protein in several neuronal pathologies has grown considerably. Here, we employed a neonatal cortex freeze-lesion (NFL) model in neonatal rats and measured the expression of Nogo-A and NgR in the resulting cerebrocortical microdysgenesis 5–75 days after freezing injury. We observed marked upregulation of Nogo-A and NgR in protein levels. Furthermore, the migration of neural precursor cells (NPCs) derived from the subventricular zone (SVZ) toward the sits of injury was perturbed by treatment of NgR antagonist peptide NEP1-40. In vitro analysis showed that the knockdown of NgR by lentivirus-delivered siRNA promoted in axonal regeneration and SVZ-derived neural stem cell/progenitor cell (SVZ-NPCs) adhesion and migration, findings which were similar to the effects of NEP1-40. Taken together, our results indicate an important role for NgR in regulating the physiological processes of SVZ-NPCs. The observation of upregulated Nogo-A/NgR in lesion sites in the NFL model suggest that the effects of the perturbed Nogo-A are a key feature during the development and/or the progression of cortical malformation. - Highlights: • NFL model is an accurate experimental reproduction of focal microgyria of FCD. • The increase of the Nogo-A Levels occurs in response to freeze-induced focal lesioning. • Nogo-A/NgR may play a critical role for in the pathologic progression of FCD. • Nogo-A is associated with the migration, proliferation and self-renewal of SVZ-NPCs.

Ion channels in EEG: isolating channel dysfunction in NMDA receptor antibody encephalitis.

Science.gov (United States)

Symmonds, Mkael; Moran, Catherine H; Leite, M Isabel; Buckley, Camilla; Irani, Sarosh R; Stephan, Klaas Enno; Friston, Karl J; Moran, Rosalyn J

2018-04-30

Neurological and psychiatric practice frequently lack diagnostic probes that can assess mechanisms of neuronal communication non-invasively in humans. In N-methyl-d-aspartate (NMDA) receptor antibody encephalitis, functional molecular assays are particularly important given the presence of NMDA antibodies in healthy populations, the multifarious symptomology and the lack of radiological signs. Recent advances in biophysical modelling techniques suggest that inferring cellular-level properties of neural circuits from macroscopic measures of brain activity is possible. Here, we estimated receptor function from EEG in patients with NMDA receptor antibody encephalitis (n = 29) as well as from encephalopathic and neurological patient controls (n = 36). We show that the autoimmune patients exhibit distinct fronto-parietal network changes from which ion channel estimates can be obtained using a microcircuit model. Specifically, a dynamic causal model of EEG data applied to spontaneous brain responses identifies a selective deficit in signalling at NMDA receptors in patients with NMDA receptor antibody encephalitis but not at other ionotropic receptors. Moreover, though these changes are observed across brain regions, these effects predominate at the NMDA receptors of excitatory neurons rather than at inhibitory interneurons. Given that EEG is a ubiquitously available clinical method, our findings suggest a unique re-purposing of EEG data as an assay of brain network dysfunction at the molecular level.

The main immunogenic region of acetylcholine receptors does not provoke the formation of antibodies of a predominant idiotype.

Science.gov (United States)

Killen, J A; Hochschwender, S M; Lindstrom, J M

1985-08-01

Anti-idiotype antibodies were induced in rats by immunization with rat monoclonal antibodies to the main immunogenic region of acetylcholine receptors. These anti-idiotype antibodies showed very little crossreaction with other rat monoclonal antibodies which bind to the same region of the receptor. When the rats producing these anti-idiotype antibodies were immunized with receptor, they showed no net decrease in anti-receptor antibody production. These data indicate that, although more than half of the antibodies produced by rats immunized with receptor are directed at a small region, many anti-receptor idiotypes are involved in this response and anti-idiotype therapy is not beneficial.

Nogo-A couples with Apg-1 through interaction and co-ordinate expression under hypoxic and oxidative stress.

Science.gov (United States)

Kern, Florian; Stanika, Ruslan I; Sarg, Bettina; Offterdinger, Martin; Hess, Daniel; Obermair, Gerald J; Lindner, Herbert; Bandtlow, Christine E; Hengst, Ludger; Schweigreiter, Rüdiger

2013-10-15

Nogo-A is the largest isoform of the Nogo/RTN4 (reticulon 4) proteins and has been characterized as a major myelin-associated inhibitor of regenerative nerve growth in the adult CNS (central nervous system). Apart from the myelin sheath, Nogo-A is expressed at high levels in principal neurons of the CNS. The specificity of Nogo-A resides in its central domain, NiG. We identified Apg-1, a member of the stress-induced Hsp110 (heat-shock protein of 110 kDa) family, as a novel interactor of NiG/Nogo-A. The interaction is selective because Apg-1 interacts with Nogo-A/RTN4-A, but not with RTN1-A, the closest paralogue of Nogo-A. Conversely, Nogo-A binds to Apg-1, but not to Apg-2 or Hsp105, two other members of the Hsp110 family. We characterized the Nogo-A-Apg-1 interaction by affinity precipitation, co-immunoprecipitation and proximity ligation assay, using primary hippocampal neurons derived from Nogo-deficient mice. Under conditions of hypoxic and oxidative stress we found that Nogo-A and Apg-1 were tightly co-regulated in hippocampal neurons. Although both proteins were up-regulated under hypoxic conditions, their expression levels were reduced upon the addition of hydrogen peroxide. Taken together, we suggest that Nogo-A is closely involved in the neuronal response to hypoxic and oxidative stress, an observation that may be of relevance not only in stroke-induced ischaemia, but also in neuroblastoma formation.

Antibody-induced dimerization activates the epidermal growth factor receptor tyrosine kinase

NARCIS (Netherlands)

Spaargaren, M.; Defize, L. H.; Boonstra, J.; de Laat, S. W.

1991-01-01

The relationship between epidermal growth factor receptor (EGF-R) protein tyrosine kinase activation and ligand-induced receptor dimerization was investigated using several bivalent anti-EGF-R antibodies directed against various receptor epitopes. In A431 membrane preparations and permeabilized

Thymus cells in myasthenia gravis selectively enhance production of anti-acetylcholine-receptor antibody by autologous blood lymphocytes

International Nuclear Information System (INIS)

Newsom-Davis, J.; Willcox, N.; Calder, L.

1981-01-01

We investigated the role of the thymus in 16 patients with myasthenia gravis without thymoma by studying the production of anti-acetylcholine-receptor antibody by thymic and blood lymphocytes cultured alone or together. In 10 responders (with the highest receptor-antibody titers in their plasma), cultured thymic cells spontaneously produced measurable receptor antibody. Receptor-antibody production by autologous blood lymphocytes was enhanced by the addition of responder's thymic cells, irradiated to abrogate antibody production and suppression (P<0.01). This enhancement was greater and more consistent than that by pokeweed mitogen; it depended on viable thymic cells, appeared to be selective for receptor antibody, and correlated with the ratio of thymic helper (OKT4-positive or OKT4+) to suppressor (OKT8+) T cells (P<0.01). These results suggest that myasthenic thymus contains cell-bound acetylcholine-receptor-like material or specific T cells (or both) that can aid receptor-antibody production. This may be relevant to the benefits of thymectomy in myasthenia and to the breakdown in self-tolerance in this and other autoimmune diseases

Thymus cells in myasthenia gravis selectively enhance production of anti-acetylcholine-receptor antibody by autologous blood lymphocytes

Energy Technology Data Exchange (ETDEWEB)

Newsom-Davis, J.; Willcox, N.; Calder, L.

1981-11-26

We investigated the role of the thymus in 16 patients with myasthenia gravis without thymoma by studying the production of anti-acetylcholine-receptor antibody by thymic and blood lymphocytes cultured alone or together. In 10 responders (with the highest receptor-antibody titers in their plasma), cultured thymic cells spontaneously produced measurable receptor antibody. Receptor-antibody production by autologous blood lymphocytes was enhanced by the addition of responder's thymic cells, irradiated to abrogate antibody production and suppression (P<0.01). This enhancement was greater and more consistent than that by pokeweed mitogen; it depended on viable thymic cells, appeared to be selective for receptor antibody, and correlated with the ratio of thymic helper (OKT4-positive or OKT4+) to suppressor (OKT8+) T cells (P<0.01). These results suggest that myasthenic thymus contains cell-bound acetylcholine-receptor-like material or specific T cells (or both) that can aid receptor-antibody production. This may be relevant to the benefits of thymectomy in myasthenia and to the breakdown in self-tolerance in this and other autoimmune diseases.

Oligodendrocyte- and Neuron-Specific Nogo-A Restrict Dendritic Branching and Spine Density in the Adult Mouse Motor Cortex.

Science.gov (United States)

Zemmar, Ajmal; Chen, Chia-Chien; Weinmann, Oliver; Kast, Brigitt; Vajda, Flora; Bozeman, James; Isaad, Noel; Zuo, Yi; Schwab, Martin E

2018-06-01

Nogo-A has been well described as a myelin-associated inhibitor of neurite outgrowth and functional neuroregeneration after central nervous system (CNS) injury. Recently, a new role of Nogo-A has been identified as a negative regulator of synaptic plasticity in the uninjured adult CNS. Nogo-A is present in neurons and oligodendrocytes. However, it is yet unclear which of these two pools regulate synaptic plasticity. To address this question we used newly generated mouse lines in which Nogo-A is specifically knocked out in (1) oligodendrocytes (oligoNogo-A KO) or (2) neurons (neuroNogo-A KO). We show that both oligodendrocyte- and neuron-specific Nogo-A KO mice have enhanced dendritic branching and spine densities in layer 2/3 cortical pyramidal neurons. These effects are compartmentalized: neuronal Nogo-A affects proximal dendrites whereas oligodendrocytic Nogo-A affects distal regions. Finally, we used two-photon laser scanning microscopy to measure the spine turnover rate of adult mouse motor cortex layer 5 cells and find that both Nogo-A KO mouse lines show enhanced spine remodeling after 4 days. Our results suggest relevant control functions of glial as well as neuronal Nogo-A for synaptic plasticity and open new possibilities for more selective and targeted plasticity enhancing strategies.

No-go for tree-level R-symmetry breaking

Energy Technology Data Exchange (ETDEWEB)

Liu, Feihu [University of Electronic Science and Technology of China, School of Physical Electronics, Chengdu (China); Liu, Muyang [Sichuan University, Center for Theoretical Physics, College of Physical Science and Technology, Chengdu (China); Sun, Zheng [Sichuan University, Center for Theoretical Physics, College of Physical Science and Technology, Chengdu (China); Chinese Academy of Sciences, CAS Key Laboratory of Theoretical Physics, Institute of Theoretical Physics, Beijing (China)

2017-11-15

We show that in gauge mediation models with tree-level R-symmetry breaking where supersymmetry and R-symmetries are broken by different fields, the gaugino mass either vanishes at one loop or finds a contribution from loop-level R-symmetry breaking. Thus tree-level R-symmetry breaking for phenomenology is either no-go or redundant in the simplest type of models. Including explicit messenger mass terms in the superpotential with a particular R-charge arrangement is helpful to bypass the no-go theorem, and the resulting gaugino mass is suppressed by the messenger mass scale. (orig.)

Adult celiac disease with acetylcholine receptor antibody positive myasthenia gravis

Institute of Scientific and Technical Information of China (English)

Hugh J Freeman; Helen R Gillett; Peter M Gillett; Joel Oger

2009-01-01

Celiac disease has been associated with some autoimmune disorders. A 40-year-old competitive strongman with celiac disease responded to a glutenfree diet, but developed profound and generalized motor weakness with acetylcholine receptor antibody positive myasthenia gravis, a disorder reported to occur in about 1 in 5000. This possible relationship between myasthenia gravis and celiac disease was further explored in serological studies. Frozen stored serum samples from 23 acetylcholine receptor antibody positive myasthenia gravis patients with no intestinal symptoms were used to screen for celiac disease. Both endomysial and tissue transglutaminase antibodies were examined. One of 23 (or, about 4.3%) was positive for both IgA-endomysial and IgA tissue transglutaminase antibodies. Endoscopic studies subsequently showed duodenal mucosal scalloping and biopsies confirmed the histopathological changes of celiac disease. Celiac disease and myasthenia gravis may occur together more often than is currently appreciated. The presence of motor weakness in celiac disease may be a clue to occult myasthenia gravis, even in the absence of intestinal symptoms.

Nogo-receptor 1 antagonization in combination with neurotrophin-4/5 is not superior to single factor treatment in promoting survival and morphological complexity of cultured dopaminergic neurons.

Science.gov (United States)

Seiler, Stefanie; Di Santo, Stefano; Sahli, Sebastian; Andereggen, Lukas; Widmer, Hans Rudolf

2017-08-01

Cell transplantation using ventral mesencephalic tissue is an experimental approach to treat Parkinson's disease. This approach is limited by poor survival of the transplants and the high number of dopaminergic neurons needed for grafting. Increasing the yield of dopaminergic neurons in donor tissue is of great importance. We have previously shown that antagonization of the Nogo-receptor 1 by NEP1-40 promoted survival of cultured dopaminergic neurons and exposure to neurotrophin-4/5 increased dopaminergic cell densities in organotypic midbrain cultures. We investigated whether a combination of both treatments offers a novel tool to further improve dopaminergic neuron survival. Rat embryonic ventral mesencephalic neurons grown as organotypic free-floating roller tube or primary dissociated cultures were exposed to neurotrophin-4/5 and NEP1-40. The combined and single factor treatment resulted in significantly higher numbers of tyrosine hydroxylase positive neurons compared to controls. Significantly stronger tyrosine hydroxylase signal intensity was detected by Western blotting in the combination-treated cultures compared to controls but not compared to single factor treatments. Neurotrophin-4/5 and the combined treatment showed significantly higher signals for the neuronal marker microtubule-associated protein 2 in Western blots compared to control while no effects were observed for the astroglial marker glial fibrillary acidic protein between groups, suggesting that neurotrophin-4/5 targets mainly neuronal cells. Finally, NEP1-40 and the combined treatment significantly augmented tyrosine hydroxylase positive neurite length. Summarizing, our findings substantiate that antagonization of the Nogo-receptor 1 promotes dopaminergic neurons but does not further increase the yield of dopaminergic neurons and their morphological complexity when combined with neurotrophin-4/5 hinting to the idea that these treatments might exert their effects by activating common

Paired Immunoglobulin-like Receptor B Knockout Does Not Enhance Axonal Regeneration or Locomotor Recovery after Spinal Cord Injury*

OpenAIRE

Nakamura, Yuka; Fujita, Yuki; Ueno, Masaki; Takai, Toshiyuki; Yamashita, Toshihide

2010-01-01

Myelin components that inhibit axonal regeneration are believed to contribute significantly to the lack of axonal regeneration noted in the adult central nervous system. Three proteins found in myelin, Nogo, myelin-associated glycoprotein, and oligodendrocyte-myelin glycoprotein, inhibit neurite outgrowth in vitro. All of these proteins interact with the same receptors, namely, the Nogo receptor (NgR) and paired immunoglobulin-like receptor B (PIR-B). As per previous reports, corticospinal tr...

Measurement of antiacetylcholine receptor auto-antibodies in ...

African Journals Online (AJOL)

Two different acetylcholine receptor (AChR) preparations derived from amputated human muscle (AChRAMP) and from the human rhabdomyosarcoma cell line TE671 (AChRTE67,) were compared in radio-immunoprecipitation assays for the detection of AChR auto-antibodies in serum specimens from 20 patients with ...

Anti-idiotypic antibody: A new strategy for the development of a growth hormone receptor antagonist.

Science.gov (United States)

Lan, Hainan; Zheng, Xin; Khan, Muhammad Akram; Li, Steven

2015-11-01

In general, traditional growth hormone receptor antagonist can be divided into two major classes: growth hormone (GH) analogues and anti-growth hormone receptor (GHR) antibodies. Herein, we tried to explore a new class of growth hormone receptor (GHR) antagonist that may have potential advantages over the traditional antagonists. For this, we developed a monoclonal anti-idiotypic antibody growth hormone, termed CG-86. A series of experiments were conducted to characterize and evaluate this antibody, and the results from a competitive receptor-binding assay, Enzyme Linked Immunosorbent Assays (ELISA) and epitope mapping demonstrate that CG-86 behaved as a typical Ab2β. Next, we examined its antagonistic activity using in vitro cell models, and the results showed that CG-86 could effectively inhibit growth hormone receptor-mediated signalling and effectively inhibit growth hormone-induced Ba/F3-GHR638 proliferation. In summary, these studies show that an anti-idiotypic antibody (CG-86) has promise as a novel growth hormone receptor antagonist. Furthermore, the current findings also suggest that anti-idiotypic antibody may represent a novel strategy to produce a new class of growth hormone receptor antagonist, and this strategy may be applied with other cytokines or growth factors. Copyright © 2015 Elsevier Ltd. All rights reserved.

Adaptive strategies for the elderly in inhibiting irrelevant and conflict no-go trials while performing the go/no-go task

Directory of Open Access Journals (Sweden)

Shulan eHsieh

2016-01-01

Full Text Available This study aimed to differentiate whether or not older adults are more prone to distraction or conflict, as induced by irrelevant and conflict no-go stimuli (irNOGO and cfNOGO, respectively. This study also aimed to determine whether or not older adults would devote more effort to withholding a no-go trial in the higher-control demand condition (20% no-go trials’ probability as compared to the lower-control demand condition (50% and 80% no-go trials’ probability. A total of 96 individuals were recruited, and each of the three no-go trials’ probability conditions included 32 participants (16 younger adults and 16 older adults. Both behavioral and event-related potential (ERP data were measured. The behavioral results showed that the older adults performed more poorly than the younger adults for the go trials, as reflected by slower reaction times and higher numbers of omission errors in the go trials. In contrast, in the no-go trials, the older adults counter-intuitively exhibited similar behavioral performance (i.e., equivalent commission errors as compared to the younger adults. The ERP data further showed that the older adults (but not the younger adults exhibited larger P3 peak amplitudes for the irNOGO than cfNOGO trials. Yet, on the other hand, the older adults performed more poorly (i.e., had more commission errors in the cfNOGO than irNOGO trials. These results seem to suggest that the older adults recruited more control processes in order to conquer the commitment of responses in the no-go trials, especially in the irNOGO trials. This age-related compensatory response of recruiting more control processes was specifically seen in the 20% no-go trial probability condition. This study therefore provides a deeper understanding into how older adults adopt strategies for performing the go/no-go task such as devoting more control processes to inhibiting the irNOGO trials compared to the cfNOGO trials in order to cope with their deficient

Nogo-B Promotes Angiogenesis in Proliferative Diabetic Retinopathy via VEGF/PI3K/Akt Pathway in an Autocrine Manner

Directory of Open Access Journals (Sweden)

Yuelu Zhang

2017-10-01

Full Text Available Background/Aims: Nogo-B, a conservative protein of endoplasmic reticulum, is a member of the reticulon family of proteins. Proliferative diabetic retinopathy (PDR is the major concerning problem of diabetic retinopathy. This study explored the role of Nogo-B in the regulation of angiogenesis in PDR patients and primary human retinal endothelial cells (HRMECs. Methods: Nogo-B was down-regulated through the use of Lentivirus-NogoB-RNAi, the effects of Nogo-B on angiogenesis under high glucose stimulation were evaluated via CCK-8 assay, wound closure assay, transwell assay, and tube formation assay. Expression of Nogo-B, VEGF, PI3K and Akt were determined by western blotting, immunofluorescence, enzyme-linked immunosorbent assay (ELISA. Co-culture systerm was used to explore cell communication. Results: Nogo-B was highly enriched in ocular tissues of PDR patients and in HRMECs exposed to high glucose. Down-regulation of Nogo-B attenuated high glucose induced cell migration and tube formation in HRMECs. Mechanistically, in comparison with the negative control group, Lentivirus-NogoB-RNAi group had exhibited reduced VEGF secretion, weakened PI3K and Akt activation. Besides, high glucose treatment promoted the secretion of Nogo-B and presented as a “long-term memory”. Conclusions: These data collectively indicated that Nogo-B promoted angiogenesis in HRMECs via VEGF/PI3K/Akt pathway in an autocrine manner.

Anti-transferrin receptor antibody and antibody-drug conjugates cross the blood-brain barrier

International Nuclear Information System (INIS)

Friden, P.M.; Walus, L.R.; Musso, G.F.; Taylor, M.A.; Malfroy, B.; Starzyk, R.M.

1991-01-01

Delivery of nonlipophilic drugs to the brain is hindered by the tightly apposed capillary endothelial cells that make up the blood-brain barrier. The authors have examined the ability of a monoclonal antibody (OX-26), which recognizes the rat transferrin receptor, to function as a carrier for the delivery of drugs across the blood-brain barrier. This antibody, which was previously shown to bind preferentially to capillary endothelial cells in the brain after intravenous administration, labels the entire cerebrovascular bed in a dose-dependent manner. The initially uniform labeling of brain capillaries becomes extremely punctate ∼ 4 hr after injection, suggesting a time-dependent sequestering of the antibody. Capillary-depletion experiments, in which the brain is separated into capillary and parenchymal fractions, show a time-dependent migration of radiolabeled antibody from the capillaries into the brain parenchyma, which is consistent with the transcytosis of compounds across the blood-brain barrier. Antibody-methotrexate conjugates were tested in vivo to assess the carrier ability of this antibody. Immunohistochemical staining for either component of an OX-26-methotrexate conjugate revealed patterns of cerebrovascular labeling identical to those observed with the unaltered antibody. Accumulation of radiolabeled methotrexate in the brain parenchyma is greatly enhanced when the drug is conjugated to OX-26

BOLD data representing activation and connectivity for rare no-go versus frequent go cues

Directory of Open Access Journals (Sweden)

Harma Meffert

2016-06-01

Full Text Available The neural circuitry underlying response control is often studied using go/no-go tasks, in which participants are required to respond as fast as possible to go cues and withhold from responding to no-go stimuli. In the current task, response control was studied using a fully counterbalanced design in which blocks with a low frequency of no-go cues (75% go, 25% no-go were alternated with blocks with a low frequency of go cues (25% go, 75% no-go; see also “Segregating attention from response control when performing a motor inhibition task: Segregating attention from response control” [1]. We applied a whole brain corrected, paired t-test to the data assessing for regions differentially activated by low frequency no-go cues relative to high frequency go cues. In addition, we conducted a generalized psychophysiological interaction analysis on the data using a right inferior frontal gyrus seed region. This region was identified through the BOLD response t-test and was chosen because right inferior gyrus is highly implicated in response inhibition.

Anti-idiotypes against a monoclonal anti-haloperidol antibody bind to dopamine receptor

International Nuclear Information System (INIS)

Elazar, Z.; Kanety, H.; Schreiber, M.; Fuchs, S.

1988-01-01

Anti-idiotypic antibodies were raised in rabbits by immunization with a monoclonal anti-haloperidol antibody. Some of these anti-idiotypic antibodies bind in a concentration dependent manner to bovine striatal membranes. Following affinity purification, these antibodies inhibit haloperidol binding to striatal membranes and deplete [ 3 H]-spiperone binding sites from a solubilized preparation of striatal membranes. It is thus concluded that these anti-idiotypic antibodies are an internal image of haloperidol and as such can interact with D 2 -dopamine receptors

Growth inhibition of tumor cells in vitro by using monoclonal antibodies against gonadotropin-releasing hormone receptor.

Science.gov (United States)

Lee, Gregory; Ge, Bixia

2010-07-01

As the continuation of a previous study, synthetic peptides corresponding to the extracellular domains of human gonadotropin-releasing hormone (GnRH) receptor were used to generate additional monoclonal antibodies which were further characterized biochemically and immunologically. Among those identified to recognize GnRH receptor, monoclonal antibodies designated as GHR-103, GHR-106 and GHR-114 were found to exhibit high affinity (Kd L37), when cancer cells were incubated with GnRH or GHR-106. The widespread expressions of GnRH receptor in almost all of the studied human cancer cell lines were also demonstrated by RT-PCR and Western blot assay, as well as indirect immunofluorescence assay with either of these monoclonal antibodies as the primary antibody. In view of the longer half life of antibodies as compared to that of GnRH or its analogs, anti-GnRH receptor monoclonal antibodies in humanized forms could function as GnRH analogs and serve as an ideal candidate of anti-cancer drugs for therapeutic treatments of various cancers in humans as well as for fertility regulations.

Potentiated antibodies to mu-opiate receptors: effect on integrative activity of the brain.

Science.gov (United States)

Geiko, V V; Vorob'eva, T M; Berchenko, O G; Epstein, O I

2003-01-01

The effect of homeopathically potentiated antibodies to mu-receptors (10(-100) wt %) on integrative activity of rat brain was studied using the models of self-stimulation of the lateral hypothalamus and convulsions produced by electric current. Electric current was delivered through electrodes implanted into the ventromedial hypothalamus. Single treatment with potentiated antibodies to mu-receptors increased the rate of self-stimulation and decreased the threshold of convulsive seizures. Administration of these antibodies for 7 days led to further activation of the positive reinforcement system and decrease in seizure thresholds. Distilled water did not change the rate of self-stimulation and seizure threshold.

Zuo-Gui and You-Gui pills, two traditional Chinese herbal formulas, downregulated the expression of NogoA, NgR, and RhoA in rats with experimental autoimmune encephalomyelitis.

Science.gov (United States)

Kou, Shuang; Zheng, Qi; Wang, Yizhou; Zhao, Hui; Zhang, Qiuxia; Li, Ming; Qi, Fang; Fang, Ling; Liu, Lei; Ouyang, Junyao; Zhao, Haiyu; Wang, Lei

2014-12-02

Zuo-Gui pills (ZGPs) and You-Gui pills (YGPs) are 2 traditional Chinese herbal formulas used for treating multiple sclerosis (MS) in the clinical setting and have been shown to have neuroprotective effects in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. The aim of this study was to explore the mechanisms underlying the neuroprotective functions of ZGPs and YGPs. Female Lewis rats were randomly divided into normal control, EAE model, 2g/kg ZGP-treated EAE, 3g/kg YGP-treated EAE, and prednisone acetate-treated groups. EAE model was induced by subcutaneous injection of MBP68-86 antigen. The neurological function scores were estimated. Histological structures of the brains and spinal cords were observed, and myelinated and axons imaged. NogoA, Nogo receptor (NgR), and RhoA transcript and protein levels were measured by real-time quantitative RT-PCR and western blotting on postimmunization (PI) days 14 (acute stage) and 28 (remission stage). ZGPs and YGPs significantly reduced neurological functions scores and abrogated inflammatory infiltrates, demyelination, and axonal damage. Furthermore, treatment with ZGPs and YGPs inhibited NogoA, NgR, and RhoA mRNA and protein expression in rats at both the acute and remission stages. ZGPs exhibited stronger effects on NogoA and RhoA expressions, as well as neurological function, during the acute stage of EAE, while YGPs caused greater reductions in NogoA expression during the remission stage. Our findings suggested that ZGPs and YGPs exerted neuroprotective effects by downregulation of NogoA, NgR, and RhoA pathways, with differences in response times and targets observed between ZGPs and YGPs. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Development of cell-penetrating bispecific antibodies targeting the N-terminal domain of androgen receptor for prostate cancer therapy.

Science.gov (United States)

Goicochea, Nancy L; Garnovskaya, Maria; Blanton, Mary G; Chan, Grace; Weisbart, Richard; Lilly, Michael B

2017-12-01

Castration-resistant prostate cancer cells exhibit continued androgen receptor signaling in spite of low levels of ligand. Current therapies to block androgen receptor signaling act by inhibiting ligand production or binding. We developed bispecific antibodies capable of penetrating cells and binding androgen receptor outside of the ligand-binding domain. Half of the bispecific antibody molecule consists of a single-chain variable fragment of 3E10, an anti-DNA antibody that enters cells. The other half is a single-chain variable fragment version of AR441, an anti-AR antibody. The resulting 3E10-AR441 bispecific antibody enters human LNCaP prostate cells and accumulates in the nucleus. The antibody binds to wild-type, mutant and splice variant androgen receptor. Binding affinity of 3E10-AR441 to androgen receptor (284 nM) was lower than that of the parental AR441 mAb (4.6 nM), but could be improved (45 nM) through alternative placement of the affinity tags, and ordering of the VH and VK domains. The 3E10-AR441 bispecific antibody blocked genomic signaling by wild-type or splice variant androgen receptor in LNCaP cells. It also blocked non-genomic signaling by the wild-type receptor. Furthermore, bispecific antibody inhibited the growth of C4-2 prostate cancer cells under androgen-stimulated conditions. The 3E10-AR441 biAb can enter prostate cancer cells and inhibits androgen receptor function in a ligand-independent manner. It may be an attractive prototype agent for prostate cancer therapy. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Retrieval of estradiol receptor in paraffin sections of resting porcine uteri by microwave treatment. Immunostaining patterns obtained with different primary antibodies.

Science.gov (United States)

Sierralta, W D; Thole, H H

1996-05-01

The unmasking of estradiol receptor in paraffin sections of Bouin's-fixed uterine tissue from ovariectomized gilts was attained with microwave treatment. Immunocytochemistry of the receptor was performed using a polyclonal or five monoclonal antibodies, two of which are commercially available, reacting with different domains of the protein and an amplified-peroxidase system for detection. With five of the antibodies, a predominance of nuclear staining was observed in cells of endometrial glands, while one monoclonal antibody (13H2), reacting with the receptor's domain E, showed a preference for the cytoplasmic receptor. In stroma, all antibodies detected more receptor in nuclei than in cytoplasm. In epithelium, the commercially available antibody H222, our monoclonals 13H2 and HT65, and the polyclonal antibody 402 demonstrated more receptor in cytoplasmic than in nuclear areas. In myometrium, the nuclei from longitudinal and ring muscles were definitely stained with the antibodies. We conclude that the accessibilities of the antibody epitopes of the receptor differ according to the functional uterine cell type.

Glutamate receptor antibodies in neurological diseases: anti-AMPA-GluR3 antibodies, anti-NMDA-NR1 antibodies, anti-NMDA-NR2A/B antibodies, anti-mGluR1 antibodies or anti-mGluR5 antibodies are present in subpopulations of patients with either: epilepsy, encephalitis, cerebellar ataxia, systemic lupus erythematosus (SLE) and neuropsychiatric SLE, Sjogren's syndrome, schizophrenia, mania or stroke. These autoimmune anti-glutamate receptor antibodies can bind neurons in few brain regions, activate glutamate receptors, decrease glutamate receptor's expression, impair glutamate-induced signaling and function, activate blood brain barrier endothelial cells, kill neurons, damage the brain, induce behavioral/psychiatric/cognitive abnormalities and ataxia in animal models, and can be removed or silenced in some patients by immunotherapy.

Science.gov (United States)

Levite, Mia

2014-08-01

Glutamate is the major excitatory neurotransmitter of the Central Nervous System (CNS), and it is crucially needed for numerous key neuronal functions. Yet, excess glutamate causes massive neuronal death and brain damage by excitotoxicity--detrimental over activation of glutamate receptors. Glutamate-mediated excitotoxicity is the main pathological process taking place in many types of acute and chronic CNS diseases and injuries. In recent years, it became clear that not only excess glutamate can cause massive brain damage, but that several types of anti-glutamate receptor antibodies, that are present in the serum and CSF of subpopulations of patients with a kaleidoscope of human neurological diseases, can undoubtedly do so too, by inducing several very potent pathological effects in the CNS. Collectively, the family of anti-glutamate receptor autoimmune antibodies seem to be the most widespread, potent, dangerous and interesting anti-brain autoimmune antibodies discovered up to now. This impression stems from taking together the presence of various types of anti-glutamate receptor antibodies in a kaleidoscope of human neurological and autoimmune diseases, their high levels in the CNS due to intrathecal production, their multiple pathological effects in the brain, and the unique and diverse mechanisms of action by which they can affect glutamate receptors, signaling and effects, and subsequently impair neuronal signaling and induce brain damage. The two main families of autoimmune anti-glutamate receptor antibodies that were already found in patients with neurological and/or autoimmune diseases, and that were already shown to be detrimental to the CNS, include the antibodies directed against ionotorpic glutamate receptors: the anti-AMPA-GluR3 antibodies, anti-NMDA-NR1 antibodies and anti-NMDA-NR2 antibodies, and the antibodies directed against Metabotropic glutamate receptors: the anti-mGluR1 antibodies and the anti-mGluR5 antibodies. Each type of these anti

Clinical Value of Thyrotropin Receptor Antibodies for the Differential Diagnosis of Interferon Induced Thyroiditis.

Science.gov (United States)

Benaiges, D; Garcia-Retortillo, M; Mas, A; Cañete, N; Broquetas, T; Puigvehi, M; Chillarón, J J; Flores-Le Roux, J A; Sagarra, E; Cabrero, B; Zaffalon, D; Solà, R; Pedro-Botet, J; Carrión, J A

2016-01-01

The clinical value of thyrotropin receptor antibodies for the differential diagnosis of thyrotoxicosis induced by pegylated interferon-alpha remains unknown. We analyzed the diagnostic accuracy of thyrotropin receptor antibodies in the differential diagnosis of thyrotoxicosis in patients with chronic hepatitis C (CHC) receiving pegylated interferon-alpha plus ribavirin. Retrospective analysis of 274 patients with CHC receiving pegylated interferon-alpha plus ribavirin. Interferon-induced thyrotoxicosis was classified according to clinical guidelines as Graves disease, autoimmune and non- autoimmune destructive thyroiditis. 48 (17.5%) patients developed hypothyroidism, 17 (6.2%) thyrotoxicosis (6 non- autoimmune destructive thyroiditis, 8 autoimmune destructive thyroiditis and 3 Graves disease) and 22 "de novo" thyrotropin receptor antibodies (all Graves disease, 2 of the 8 autoimmune destructive thyroiditis and 17 with normal thyroid function). The sensitivity and specificity of thyrotropin receptor antibodies for Graves disease diagnosis in patients with thyrotoxicosis were 100 and 85%, respectively. Patients with destructive thyroiditis developed hypothyroidism in 87.5% of autoimmune cases and in none of those with a non- autoimmune etiology (pthyroid scintigraphy for the differential diagnosis of thyrotoxicosis in CHC patients treated with pegylated interferon. © Georg Thieme Verlag KG Stuttgart · New York.

Antibody Selection for Cancer Target Validation of FSH-Receptor in Immunohistochemical Settings

Directory of Open Access Journals (Sweden)

Nina Moeker

2017-10-01

Full Text Available Background: The follicle-stimulating hormone (FSH-receptor (FSHR has been reported to be an attractive target for antibody therapy in human cancer. However, divergent immunohistochemical (IHC findings have been reported for FSHR expression in tumor tissues, which could be due to the specificity of the antibodies used. Methods: Three frequently used antibodies (sc-7798, sc-13935, and FSHR323 were validated for their suitability in an immunohistochemical study for FSHR expression in different tissues. As quality control, two potential therapeutic anti-hFSHR Ylanthia® antibodies (Y010913, Y010916 were used. The specificity criteria for selection of antibodies were binding to native hFSHR of different sources, and no binding to non-related proteins. The ability of antibodies to stain the paraffin-embedded Flp-In Chinese hamster ovary (CHO/FSHR cells was tested after application of different epitope retrieval methods. Results: From the five tested anti-hFSHR antibodies, only Y010913, Y010916, and FSHR323 showed specific binding to native, cell-presented hFSHR. Since Ylanthia® antibodies were selected to specifically recognize native FSHR, as required for a potential therapeutic antibody candidate, FSHR323 was the only antibody to detect the receptor in IHC/histochemical settings on transfected cells, and at markedly lower, physiological concentrations (ex., in Sertoli cells of human testes. The pattern of FSH323 staining noticed for ovarian, prostatic, and renal adenocarcinomas indicated that FSHR was expressed mainly in the peripheral tumor blood vessels. Conclusion: Of all published IHC antibodies tested, only antibody FSHR323 proved suitable for target validation of hFSHR in an IHC setting for cancer. Our studies could not confirm the previously reported FSHR overexpression in ovarian and prostate cancer cells. Instead, specific overexpression in peripheral tumor blood vessels could be confirmed after thorough validation of the antibodies used.

Neural mechanisms underlying conflict monitoring over risky decision alternatives: evidence from ERP in a Go/Nogo task.

Science.gov (United States)

Wang, Shuzhen; Hui, Ning; Zhou, Xinsheng; He, Kaifeng; Yu, Yuanyuan; Shuai, Jing

2014-09-01

This study assessed conflict monitoring during presentation of risky decision alternatives, as indexed by the Nogo-N2, Nogo-P3, N2d and P3d event-related potentials (ERP). Decision-makers were tested on a Go/Nogo gambling task in which gain/loss outcomes as well as stimulus type (Go/Nogo) were equiprobable. Frontal-central Nogo-N2 and Nogo-P3 did not significantly differ across risky decision alternatives, whereas N2d and P3d amplitudes were more sensitive to the nature of risky decision alternatives. Frontal-central N2d was moderated by the magnitude of alternatives, with N2d amplitude greater for large than small alternatives, a result that suggests a greater degree of conflict monitoring for the former. Central P3d was associated with alternative valence, such that P3d amplitude was greater for loss than gain valences, again suggestive of more conflict monitoring for the former. The N2d and P3d potentials in risky decision alternatives are discussed in terms of the functional significance of the N2/P3 complex.

Immediate and Catastrophic Antibody-Mediated Rejection in a Lung Transplant Recipient With Anti-Angiotensin II Receptor Type 1 and Anti-Endothelin-1 Receptor Type A Antibodies.

Science.gov (United States)

Cozzi, E; Calabrese, F; Schiavon, M; Feltracco, P; Seveso, M; Carollo, C; Loy, M; Cardillo, M; Rea, F

2017-02-01

Preexisting donor-specific anti-HLA antibodies (DSAs) have been associated with reduced survival of lung allografts. However, antibodies with specificities other than HLA may have a detrimental role on the lung transplant outcome. A young man with cystic fibrosis underwent lung transplantation with organs from a suitable deceased donor. At the time of transplantation, there were no anti-HLA DSAs. During surgery, the patient developed a severe and intractable pulmonary hypertension associated with right ventriular dysfunction, which required arteriovenous extracorporeal membrane oxygenation. After a brief period of clinical improvement, a rapid deterioration in hemodynamics led to the patient's death on postoperative day 5. Postmortem studies showed that lung specimens taken at the end of surgery were compatible with antibody-mediated rejection (AMR), while terminal samples evidenced diffuse capillaritis, blood extravasation, edema, and microthrombi, with foci of acute cellular rejection (A3). Immunological investigations demonstrated the presence of preexisting antibodies against the endothelin-1 receptor type A (ET A R) and the angiotensin II receptor type 1 (AT 1 R), two of the most potent vasoconstrictors reported to date, whose levels slightly rose after transplantation. These data suggest that preexisting anti-ET A R and anti-AT 1 R antibodies may have contributed to the onset of AMR and to the catastrophic clinical course of this patient. © Copyright 2016 The American Society of Transplantation and the American Society of Transplant Surgeons.

Dual Mechanism of Interleukin-3 Receptor Blockade by an Anti-Cancer Antibody

Directory of Open Access Journals (Sweden)

Sophie E. Broughton

2014-07-01

Full Text Available Interleukin-3 (IL-3 is an activated T cell product that bridges innate and adaptive immunity and contributes to several immunopathologies. Here, we report the crystal structure of the IL-3 receptor α chain (IL3Rα in complex with the anti-leukemia antibody CSL362 that reveals the N-terminal domain (NTD, a domain also present in the granulocyte-macrophage colony-stimulating factor (GM-CSF, IL-5, and IL-13 receptors, adopting unique “open” and classical “closed” conformations. Although extensive mutational analyses of the NTD epitope of CSL362 show minor overlap with the IL-3 binding site, CSL362 only inhibits IL-3 binding to the closed conformation, indicating alternative mechanisms for blocking IL-3 signaling. Significantly, whereas “open-like” IL3Rα mutants can simultaneously bind IL-3 and CSL362, CSL362 still prevents the assembly of a higher-order IL-3 receptor-signaling complex. The discovery of open forms of cytokine receptors provides the framework for development of potent antibodies that can achieve a “double hit” cytokine receptor blockade.

Surveillance for Intracellular Antibody by Cytosolic Fc Receptor TRIM21

Directory of Open Access Journals (Sweden)

William A. McEwan

2016-11-01

Full Text Available TRIM21 has emerged as an atypical Fc receptor that is broadly conserved and widely expressed in the cytoplasm of mammalian cells. Viruses that traffic surface-bound antibodies into the cell during infection recruit TRIM21 via a high affinity interaction between Fc and TRIM21 PRYSPRY domain. Following binding of intracellular antibody, TRIM21 acts as both antiviral effector and sensor for innate immune signalling. These activities serve to reduce viral replication by orders of magnitude in vitro and contribute to host survival during in vivo infection. Neutralization occurs rapidly after detection and requires the activity of the ubiquitin-proteasome system. The microbial targets of this arm of intracellular immunity are still being identified: TRIM21 activity has been reported following infection by several non-enveloped viruses and intracellular bacteria. These findings extend the sphere of influence of antibodies to the intracellular domain and have broad implications for immunity. TRIM21 has been implicated in the chronic auto-immune condition systemic lupus erythematosus and is itself an auto-antigen in Sjögren’s syndrome. This review summarises our current understanding of TRIM21’s role as a cytosolic Fc receptor and briefly discusses pathological circumstances where intracellular antibodies have been described, or are hypothesized to occur, and may benefit from further investigations of the role of TRIM21.

Focal status epilepticus and progressive dyskinesia: A novel phenotype for glycine receptor antibody-mediated neurological disease in children.

Science.gov (United States)

Chan, D W S; Thomas, T; Lim, M; Ling, S; Woodhall, M; Vincent, A

2017-03-01

Antibody-associated disorders of the central nervous system are increasingly recognised in adults and children. Some are known to be paraneoplastic, whereas in others an infective trigger is postulated. They include disorders associated with antibodies to N-methyl-d-aspartate receptor (NMDAR), voltage-gated potassium channel-complexes (VGKC-complex), GABA B receptor or glycine receptor (GlyR). With antibodies to NMDAR or VGKC-complexes, distinct clinical patterns are well characterised, but as more antibodies are discovered, the spectra of associated disorders are evolving. GlyR antibodies have been detected in patients with progressive encephalopathy with rigidity and myoclonus (PERM), or stiff man syndrome, both rare but disabling conditions. We report a case of a young child with focal seizures and progressive dyskinesia in whom GlyR antibodies were detected. Anticonvulsants and immunotherapy were effective in treating both the seizures and movement disorder with good neurological outcome and with a decline in the patient's serum GlyR-Ab titres. Glycine receptor antibodies are associated with focal status epilepticus and seizures, encephalopathy and progressive dyskinesia and should be evaluated in autoimmune encephalitis. Copyright © 2016 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.

Monoclonal antibody to the rat glucocorticoid receptor. Relationship between the immunoreactive and DNA-binding domain

International Nuclear Information System (INIS)

Eisen, L.P.; Reichman, M.E.; Thompson, E.B.; Gametchu, B.; Harrison, R.W.; Eisen, H.J.

1985-01-01

The region of the glucocorticoid receptor that reacted with a monoclonal antibody (BUGR-1) was identified. In order to identify the immunoreactive region, the rat liver glucocorticoid receptor was subjected to limited proteolysis; immunoreactive fragments were identified by Western blotting. The monoclonal antibody reacted with both the undigested Mr approximately 97,000 receptor subunit and a Mr approximately 45,000 fragment containing the steroid-binding and DNA-binding domains. Digestion by trypsin also produced two steroid-binding fragments of Mr approximately 27,000 and 31,000 which did not react with the antibody and an immunoreactive Mr approximately 16,000 fragment. This Mr approximately 16,000 fragment was shown to bind to DNA-cellulose, indicating that it contained a DNA-binding domain of the receptor. The undigested receptor must have steroid associated with it to undergo activation to a DNA-binding form. However, the Mr approximately 16,000 immunoreactive fragment binds to DNA-cellulose even if it is obtained by digestion of the steroid-free holoreceptor which does not itself bind to DNA

Noncommutative gauge field theories: A no-go theorem

International Nuclear Information System (INIS)

Chaichian, M.; Tureanu, A.; Presnajder, P.; Sheikh-Jabbari, M.M.

2001-06-01

Studying the mathematical structure of the noncommutative groups in more detail, we prove a no-go theorem for the noncommutative gauge theories. According to this theorem, the closure condition of the gauge algebra implies that: 1) the local noncommutative u(n) algebra only admits the irreducible nxn matrix-representation. Hence the gauge fields, as elements of the algebra, are in nxn matrix form, while the matter fields can only be either in fundamental, adjoint or singlet states; 2) for any gauge group consisting of several simple group factors, the matter fields can transform nontrivially under at most two noncommutative group factors. In other words, the matter fields cannot carry more than two simple noncommutative gauge group charges. This no-go theorem imposes strong restrictions on the construction of the noncommutative version of the Standard Model and in resolving the standing problem of charge quantization in noncommutative QED. (author)

Fc receptors for mouse IgG1 on human monocytes: polymorphism and role in antibody-induced T cell proliferation.

Science.gov (United States)

Tax, W J; Hermes, F F; Willems, R W; Capel, P J; Koene, R A

1984-09-01

In previous studies, it was shown that there is polymorphism in the mitogenic effect of mouse IgG1 monoclonal antibodies against the T3 antigen of human T cells. This polymorphism implies that IgG1 anti-T3 antibodies are not mitogenic for T cells from 30% of healthy individuals. The present results demonstrate that this polymorphism is caused by polymorphism of an Fc receptor for mouse IgG1, present on human monocytes. The Fc receptor for murine IgG1 could be detected by a newly developed rosetting assay on monocytes from all individuals responsive to the mitogenic effect of IgG1 anti-T3 antibodies. This Fc receptor was not detectable on monocytes from those individuals exhibiting no mitogenic responses to IgG1 anti-T3 monoclonal antibodies. Cross-linking of T3 antigens appears to be essential for antibody-induced mitosis of T cells, because mononuclear cells that did not proliferate in response to WT 31 (an IgG1 antibody against T3 antigen) showed a proliferative response to Sepharose beads coated with WT 31. The Fc receptor--if functionally present--may be involved in the cross-linking of T3 antigens through anti-T3 antibodies. Further evidence for the involvement of this Fc receptor in antibody-induced T cell proliferation was provided by inhibition studies. Immune complexes containing IgG1 antibodies were able to inhibit the proliferative response to IgG1 anti-T3 antibodies. This inhibition by immune complexes appears to be mediated through the monocyte Fc receptor for mouse IgG1. These findings are important for the interpretation of previously described inhibitory effects of anti-T cell monoclonal antibodies on T cell proliferation, and show that such inhibitory effects may be monocyte-mediated (via immune complexes) rather than caused by a direct involvement of the respective T cell antigens in T cell mitosis. The Fc receptor for mouse IgG1 plays a role in antibody-induced T cell proliferation. Its polymorphism may have important implications for the

Blockade of human P2X7 receptor function with a monoclonal antibody.

Science.gov (United States)

Buell, G; Chessell, I P; Michel, A D; Collo, G; Salazzo, M; Herren, S; Gretener, D; Grahames, C; Kaur, R; Kosco-Vilbois, M H; Humphrey, P P

1998-11-15

A monoclonal antibody (MoAb) specific for the human P2X7 receptor was generated in mice. As assessed by flow cytometry, the MoAb labeled human blood-derived macrophage cells natively expressing P2X7 receptors and cells transfected with human P2X7 but not other P2X receptor types. The MoAb was used to immunoprecipitate the human P2X7 receptor protein, and in immunohistochemical studies on human lymphoid tissue, P2X7 receptor labeling was observed within discrete areas of the marginal zone of human tonsil sections. The antibody also acted as a selective antagonist of human P2X7 receptors in several functional studies. Thus, whole cell currents, elicited by the brief application of 2',3'-(4-benzoyl)-benzoyl-ATP in cells expressing human P2X7, were reduced in amplitude by the presence of the MoAb. Furthermore, preincubation of human monocytic THP-1 cells with the MoAb antagonized the ability of P2X7 agonists to induce the release of interleukin-1beta.

Anti-N-methyl-D-aspartate receptor encephalitis with serum anti-thyroid antibodies and IgM antibodies against Epstein-Barr virus viral capsid antigen: a case report and one year follow-up

Directory of Open Access Journals (Sweden)

Xu Chun-Ling

2011-11-01

Full Text Available Abstract Background Anti-N-methyl-D-aspartate receptor encephalitis is an increasingly common autoimmune disorder mediated by antibodies to certain subunit of the N-methyl-D-aspartate receptor. Recent literatures have described anti-thyroid and infectious serology in this encephalitis but without follow-up. Case presentation A 17-year-old Chinese female patient presented with psychiatric symptoms, memory deficits, behavioral problems and seizures. She then progressed through unresponsiveness, dyskinesias, autonomic instability and central hypoventilation during treatment. Her conventional blood work on admission showed high titers of IgG antibodies to thyroglobulin, thyroid peroxidase and IgM antibodies to Epstein-Barr virus viral capsid antigen. An immature ovarian teratoma was found and removal of the tumor resulted in a full recovery. The final diagnosis of anti-N-methyl-D-aspartate receptor encephalitis was made by the identification of anti-N-methyl-D-aspartate receptor antibodies in her cerebral spinal fluid. Pathology studies of the teratoma revealed N-methyl-D-aspartate receptor subunit 1 positive ectopic immature nervous tissue and Epstein-Barr virus latent infection. She was discharged with symptoms free, but titers of anti-thyroid peroxidase and anti-thyroglobulin antibodies remained elevated. One year after discharge, her serum remained positive for anti-thyroid peroxidase and anti-N-methyl-D-aspartate receptor antibodies, but negative for anti-thyroglobulin antibodies and IgM against Epstein-Barr virus viral capsid antigen. Conclusions Persistent high titers of anti-thyroid peroxidase antibodies from admission to discharge and until one year later in this patient may suggest a propensity to autoimmunity in anti- N-methyl-D-aspartate receptor encephalitis and support the idea that neuronal and thyroid autoimmunities represent a pathogenic spectrum. Enduring anti-N-methyl-D-aspartate receptor antibodies from admission to one year

Nogo-B regulates migration and contraction of airway smooth muscle cells by decreasing ARPC 2/3 and increasing MYL-9 expression

Directory of Open Access Journals (Sweden)

Cai Zailong

2011-01-01

Full Text Available Abstract Background Abnormal proliferation, apoptosis, migration and contraction of airway smooth muscle (ASM cells in airway remodeling in asthma are basically excessive repair responses to a network of inflammatory mediators such as PDGF, but the mechanisms of such responses remain unclear. Nogo-B, a member of the reticulum family 4(RTN4, is known to play a key role in arteriogenesis and tissue repair. Further studies are needed to elucidate the role of Nogo-B in airway smooth muscle abnormalities. Methods A mouse model of chronic asthma was established by repeated OVA inhalation and subjected to Nogo-B expression analysis using immunohistochemistry and Western Blotting. Then, primary human bronchial smooth muscle cells (HBSMCs were cultured in vitro and a siRNA interference was performed to knockdown the expression of Nogo-B in the cells. The effects of Nogo-B inhibition on PDGF-induced HBSMCs proliferation, migration and contraction were evaluated. Finally, a proteomic analysis was conducted to unveil the underlying mechanisms responsible for the function of Nogo-B. Results Total Nogo-B expression was approximately 3.08-fold lower in chronic asthmatic mice compared to naïve mice, which was obvious in the smooth muscle layer of the airways. Interference of Nogo-B expression by siRNA resulted nearly 96% reduction in mRNA in cultured HBSMCs. In addition, knockdown of Nogo-B using specific siRNA significantly decreased PDGF-induced migration of HBSMCs by 2.3-fold, and increased the cellular contraction by 16% compared to negative controls, but had limited effects on PDGF-induced proliferation. Furthermore, using proteomic analysis, we demonstrate that the expression of actin related protein 2/3 complex subunit 5 (ARPC 2/3 decreased and, myosin regulatory light chain 9 isoform a (MYL-9 increased after Nogo-B knockdown. Conclusions These data define a novel role for Nogo-B in airway remodeling in chronic asthma. Endogenous Nogo-B, which may exert

No-go theorems for the minimization of potentials

International Nuclear Information System (INIS)

Chang, D.; Kumar, A.

1985-01-01

Using a theorem in linear algebra, we prove some no-go theorems in the minimization of potentials related to the problem of symmetry breaking. Some applications in the grand unified model building are mentioned. Another application of the algebraic theorem is also included to demonstrate its usefulness

Single-domain antibodies that compete with the natural ligand fibroblast growth factor block the internalization of the fibroblast growth factor receptor 1

Energy Technology Data Exchange (ETDEWEB)

Veggiani, Gianluca; Ossolengo, Giuseppe; Aliprandi, Marisa; Cavallaro, Ugo [IFOM-IEO Campus, Via Adamello 16, 20139 Milano (Italy); Marco, Ario de, E-mail: ario.demarco@ung.si [IFOM-IEO Campus, Via Adamello 16, 20139 Milano (Italy); Dept. Environmental Sciences, University of Nova Gorica (UNG), Vipavska 13, P.O. Box 301-SI-5000, Rozna Dolina, Nova Gorica (Slovenia)

2011-05-20

Highlights: {yields} Recombinant antibodies for FGFR1 were isolated from a llama naive library in VHH format. {yields} These antibodies compete with the natural ligand FGF-2 for the same epitope on FGFR1. {yields} The antibody competition inhibits the FGF-2-dependent internalization of FGFR1. -- Abstract: Single-domain antibodies in VHH format specific for fibroblast growth factor receptor 1 (FGFR1) were isolated from a phage-display llama naive library. In particular, phage elution in the presence of the natural receptor ligand fibroblast growth factor (FGF) allowed for the identification of recombinant antibodies that compete with FGF for the same region on the receptor surface. These antibodies posses a relatively low affinity for FGFR1 and were never identified when unspecific elution conditions favoring highly affine binders were applied to panning procedures. Two populations of competitive antibodies were identified that labeled specifically the receptor-expressing cells in immunofluorescence and recognize distinct epitopes. Antibodies from both populations effectively prevented FGF-dependent internalization and nuclear accumulation of the receptor in cultured cells. This achievement indicates that these antibodies have a capacity to modulate the receptor physiology and, therefore, constitute powerful reagents for basic research and a potential lead for therapeutic applications.

Single-domain antibodies that compete with the natural ligand fibroblast growth factor block the internalization of the fibroblast growth factor receptor 1

International Nuclear Information System (INIS)

Veggiani, Gianluca; Ossolengo, Giuseppe; Aliprandi, Marisa; Cavallaro, Ugo; Marco, Ario de

2011-01-01

Highlights: → Recombinant antibodies for FGFR1 were isolated from a llama naive library in VHH format. → These antibodies compete with the natural ligand FGF-2 for the same epitope on FGFR1. → The antibody competition inhibits the FGF-2-dependent internalization of FGFR1. -- Abstract: Single-domain antibodies in VHH format specific for fibroblast growth factor receptor 1 (FGFR1) were isolated from a phage-display llama naive library. In particular, phage elution in the presence of the natural receptor ligand fibroblast growth factor (FGF) allowed for the identification of recombinant antibodies that compete with FGF for the same region on the receptor surface. These antibodies posses a relatively low affinity for FGFR1 and were never identified when unspecific elution conditions favoring highly affine binders were applied to panning procedures. Two populations of competitive antibodies were identified that labeled specifically the receptor-expressing cells in immunofluorescence and recognize distinct epitopes. Antibodies from both populations effectively prevented FGF-dependent internalization and nuclear accumulation of the receptor in cultured cells. This achievement indicates that these antibodies have a capacity to modulate the receptor physiology and, therefore, constitute powerful reagents for basic research and a potential lead for therapeutic applications.

HER2 monoclonal antibodies that do not interfere with receptor heterodimerization-mediated signaling induce effective internalization and represent valuable components for rational antibody-drug conjugate design.

Science.gov (United States)

de Goeij, Bart E C G; Peipp, Matthias; de Haij, Simone; van den Brink, Edward N; Kellner, Christian; Riedl, Thilo; de Jong, Rob; Vink, Tom; Strumane, Kristin; Bleeker, Wim K; Parren, Paul W H I

2014-01-01

The human epidermal growth factor receptor (HER)2 provides an excellent target for selective delivery of cytotoxic drugs to tumor cells by antibody-drug conjugates (ADC) as has been clinically validated by ado-trastuzumab emtansine (Kadcyla(TM)). While selecting a suitable antibody for an ADC approach often takes specificity and efficient antibody-target complex internalization into account, the characteristics of the optimal antibody candidate remain poorly understood. We studied a large panel of human HER2 antibodies to identify the characteristics that make them most suitable for an ADC approach. As a model toxin, amenable to in vitro high-throughput screening, we employed Pseudomonas exotoxin A (ETA') fused to an anti-kappa light chain domain antibody. Cytotoxicity induced by HER2 antibodies, which were thus non-covalently linked to ETA', was assessed for high and low HER2 expressing tumor cell lines and correlated with internalization and downmodulation of HER2 antibody-target complexes. Our results demonstrate that HER2 antibodies that do not inhibit heterodimerization of HER2 with related ErbB receptors internalize more efficiently and show greater ETA'-mediated cytotoxicity than antibodies that do inhibit such heterodimerization. Moreover, stimulation with ErbB ligand significantly enhanced ADC-mediated tumor kill by antibodies that do not inhibit HER2 heterodimerization. This suggests that the formation of HER2/ErbB-heterodimers enhances ADC internalization and subsequent killing of tumor cells. Our study indicates that selecting HER2 ADCs that allow piggybacking of HER2 onto other ErbB receptors provides an attractive strategy for increasing ADC delivery and tumor cell killing capacity to both high and low HER2 expressing tumor cells.

Diagnostic sensitivity of two radio receptor assays (TRAK Assay and TRAK Dyno human) for the detection of TSH receptor antibodies

International Nuclear Information System (INIS)

Paunkovic, N.; Paunkovic, J.

2003-01-01

Radio receptor assays for the detection of TSH receptor antibodies in serum are typically based on binding the competition of TSH-R antibodies and 125I -labelled-TSH for membrane preparation of thyrocytes (TBII tests). The sensitivity of the available tests utilizing porcine cell membranes was found to be around 80%. A new test (TRAK Dyno human, BRAHMS) utilizes human recombinant TSH receptor and human standard material that is supposed to improve the performance of the test. We have compared the results of these two assays. The sensitivity of the TRAK Assay tested in 356 patients with untreated Grave's disease was found to be 85%, and 97.5% for TRAK Dyno human in 111 newly diagnosed patients. Both tests were performed from the same serum specimen for 60 of the investigated patients. The TRAK Assay was positive in 50 patients (83.2%) and TRAK Dyno human in 59 patients (98.3%). The specificity of the new radio receptor assay was also improved. (author)

New-Onset Headache in Patients With Autoimmune Encephalitis Is Associated With anti-NMDA-Receptor Antibodies.

Science.gov (United States)

Schankin, Christoph J; Kästele, Fabian; Gerdes, Lisa Ann; Winkler, Tobias; Csanadi, Endy; Högen, Tobias; Pellkofer, Hannah; Paulus, Walter; Kümpfel, Tania; Straube, Andreas

2016-06-01

We tested the hypotheses (i) that autoimmune encephalitis is associated with new-onset headache, and (ii) that the occurrence of headache is associated with the presence of anti-N-methyl-D-aspartate (NMDA)-receptor antibodies. Autoimmune encephalitis presents with cognitive dysfunction as well as neuro-psychiatric symptoms. Its pathophysiology might involve antibody-mediated dysfunction of the glutamatergic system as indicated by the presence of anti-NMDA-receptor antibodies in some patients. In this cross-sectional study, patients with autoimmune encephalitis were assessed with a standardized interview for previous headache and headache associated with autoimmune encephalitis. Headache was classified according to the International Classification of Headache Disorders, second edition. Clinical and paraclinical findings were correlated with the occurrence of headache. Of 40 patients with autoimmune encephalitis, 19 did not have a history of headache. Of those, nine suffered from encephalitis-associated headache. Seven of these nine had anti-NMDA-receptor antibodies in contrast to only two among the remaining 10 patients without new-onset headache (P = .023, odds ratio: 14, 95% confidence interval: 1.5; 127). In most patients headache occurred in attacks on more than 15 days/month, was severe, and of short duration (less than 4 hours). International Headache Society criteria for migraine were met in three patients. New-onset headache is a relevant symptom in patients with autoimmune encephalitis who have no history of previous headache, especially in the subgroup with anti-NMDA-receptor antibodies. This indicates a thorough investigation for secondary headaches including anti-NMDA-R antibodies for patients with new-onset headache and neuropsychiatric findings. Glutamatergic dysfunction might be important for the generation of head pain but may only occasionally be sufficient to trigger migraine-like attacks in nonmigraineurs. © 2016 American Headache Society.

Subcellular localization of estradiol receptor in MCF7 cells studied with nanogold-labelled antibody fragments.

Science.gov (United States)

Kessels, M M; Qualmann, B; Thole, H H; Sierralta, W D

1998-01-01

Ultrastructural localization studies of estradiol receptor in hormone-deprived and hormone-stimulated MCF7 cells were done using F(ab') fragments of three different antibodies (#402, 13H2, HT277) covalently linked to nanogold. These ultra-small, non-charged immunoreagents, combined with a size-enlargement by silver enhancement, localized estradiol receptor in both nuclear and cytoplasmic areas of non-stimulated target cells; stimulation with the steroid induced a predominantly nuclear labelling. In the cytoplasm of resting cells, tagging was often observed at or in the proximity of stress fibers. In the nucleus a large proportion of receptor was found inside the nucleolus, specially with the reagent derived from antibody 13H2. We postulate that different accessibilities of receptor epitopes account for the different labelling densities observed at cytoskeletal elements and the nucleoli.

Nogo-A is a reliable oligodendroglial marker in adult human and mouse CNS and in demyelinated lesions

DEFF Research Database (Denmark)

Kuhlmann, Tanja; Remington, Leah; Maruschak, Brigitte

2007-01-01

to be strongly expressed in mature oligodendrocytes in vivo. In the present investigation we analyzed the expression patterns of Nogo-A in adult mouse and human CNS as well as in demyelinating animal models and multiple sclerosis lesions. Nogo-A expression was compared with that of other frequently used...... oligodendroglial markers such as CC1, CNP, and in situ hybridization for proteolipid protein mRNA. Nogo-A strongly and reliably labeled oligodendrocytes in the adult CNS as well as in demyelinating lesions and thus represents a valuable tool for the identification of oligodendrocytes in human and mouse CNS tissue...

Characterization of Notch1 antibodies that inhibit signaling of both normal and mutated Notch1 receptors.

Directory of Open Access Journals (Sweden)

Miguel Aste-Amézaga

2010-02-01

Full Text Available Notch receptors normally play a key role in guiding a variety of cell fate decisions during development and differentiation of metazoan organisms. On the other hand, dysregulation of Notch1 signaling is associated with many different types of cancer as well as tumor angiogenesis, making Notch1 a potential therapeutic target.Here we report the in vitro activities of inhibitory Notch1 monoclonal antibodies derived from cell-based and solid-phase screening of a phage display library. Two classes of antibodies were found, one directed against the EGF-repeat region that encompasses the ligand-binding domain (LBD, and the second directed against the activation switch of the receptor, the Notch negative regulatory region (NRR. The antibodies are selective for Notch1, inhibiting Jag2-dependent signaling by Notch1 but not by Notch 2 and 3 in reporter gene assays, with EC(50 values as low as 5+/-3 nM and 0.13+/-0.09 nM for the LBD and NRR antibodies, respectively, and fail to recognize Notch4. While more potent, NRR antibodies are incomplete antagonists of Notch1 signaling. The antagonistic activity of LBD, but not NRR, antibodies is strongly dependent on the activating ligand. Both LBD and NRR antibodies bind to Notch1 on human tumor cell lines and inhibit the expression of sentinel Notch target genes, including HES1, HES5, and DTX1. NRR antibodies also strongly inhibit ligand-independent signaling in heterologous cells transiently expressing Notch1 receptors with diverse NRR "class I" point mutations, the most common type of mutation found in human T-cell acute lymphoblastic leukemia (T-ALL. In contrast, NRR antibodies failed to antagonize Notch1 receptors bearing rare "class II" or "class III" mutations, in which amino acid insertions generate a duplicated or constitutively sensitive metalloprotease cleavage site. Signaling in T-ALL cell lines bearing class I mutations is partially refractory to inhibitory antibodies as compared to cell

Mouse Hepatitis Virus Strain A59 and Blocking Antireceptor Monoclonal Antibody Bind to the N-Terminal Domain of Cellular Receptor

Science.gov (United States)

Dveksler, Gabriela S.; Pensiero, Michael N.; Dieffenbach, Carl W.; Cardellichio, Christine B.; Basile, Alexis A.; Elia, Patrick E.; Holmes, Kathryn V.

1993-03-01

Mouse hepatitis virus (MHV) strain A59 uses as cellular receptors members of the carcinoembryonic antigen family in the immunoglobulin superfamily. Recombinant receptor proteins with deletions of whole or partial immunoglobulin domains were used to identify the regions of receptor glycoprotein recognized by virus and by antireceptor monoclonal antibody CC1, which blocks infection of murine cells. Monoclonal antibody CC1 and MHV-A59 virions bound only to recombinant proteins containing the entire first domain of MHV receptor. To determine which of the proteins could serve as functional virus receptors, receptor-negative hamster cells were transfected with recombinant deletion clones and then challenged with MHV-A59 virions. Receptor activity required the entire N-terminal domain with either the second or the fourth domain and the transmembrane and cytoplasmic domains. Recombinant proteins lacking the first domain or its C-terminal portion did not serve as viral receptors. Thus, like other virus receptors in the immunoglobulin superfamily, including CD4, poliovirus receptor, and intercellular adhesion molecule 1, the N-terminal domain of MHV receptor is recognized by the virus and the blocking monoclonal antibody.

Discovery of functional monoclonal antibodies targeting G-protein-coupled receptors and ion channels.

Science.gov (United States)

Wilkinson, Trevor C I

2016-06-15

The development of recombinant antibody therapeutics is a significant area of growth in the pharmaceutical industry with almost 50 approved monoclonal antibodies on the market in the US and Europe. Despite this growth, however, certain classes of important molecular targets have remained intractable to therapeutic antibodies due to complexity of the target molecules. These complex target molecules include G-protein-coupled receptors and ion channels which represent a large potential target class for therapeutic intervention with monoclonal antibodies. Although these targets have typically been addressed by small molecule approaches, the exquisite specificity of antibodies provides a significant opportunity to provide selective modulation of these target proteins. Given this opportunity, substantial effort has been applied to address the technical challenges of targeting these complex membrane proteins with monoclonal antibodies. In this review recent progress made in the strategies for discovery of functional monoclonal antibodies for these challenging membrane protein targets is addressed. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

N-methyl-d-aspartate receptor – nitric oxide synthase pathway in the cortex of Nogo-A-deficient rats in relation to brain laterality and schizophrenia

Czech Academy of Sciences Publication Activity Database

Krištofíková, Z.; Vrajová, M.; Šírová, J.; Valeš, Karel; Petrásek, Tomáš; Schönig, K.; Tews, B.; Schwab, M.; Bartsch, D.; Stuchlík, Aleš; Řípová, D.

2013-01-01

Roč. 7, Aug 12 (2013), s. 90 ISSN 1662-5153 R&D Projects: GA ČR(CZ) GBP304/12/G069 Institutional support: RVO:67985823 Keywords : Nogo-A * biochemic markers * rat Subject RIV: FH - Neurology Impact factor: 4.160, year: 2013

Drug of the year: programmed death-1 receptor/programmed death-1 ligand-1 receptor monoclonal antibodies.

Science.gov (United States)

Robert, Caroline; Soria, Jean-Charles; Eggermont, Alexander M M

2013-09-01

Programmed death-1 receptor (PD-1)/its ligand (PD-L1) antibodies have changed the landscape in oncology in 2013. The most mature results have been obtained in advanced melanoma patients. They indicate important response rates and high quality responses or prolonged duration. Also in renal cancer and in lung cancer remarkable activity has been demonstrated. Thus it is clear that these antibodies have a very broad potential and trials in many tumour types are being initiated. Breaking tolerance at the tumour site is a potent phenomenon and the potential for synergy with other checkpoint inhibitors such as ipilimumab have also been demonstrated in 2013. Long term tumour control now seems achievable and thus the concept of a clinical cure is emerging by modulation of the immune system. These antibodies bring immunotherapy to the forefront and indicate that immune-modulation will be a key component of therapeutic strategies from now on. Because of all these reasons PD-1/PD-L1 antibodies are considered 'drug of the year'. Copyright © 2013 Elsevier Ltd. All rights reserved.

Splitting spacetime and cloning qubits: linking no-go theorems across the ER=EPR duality

Energy Technology Data Exchange (ETDEWEB)

Bao, Ning [Institute for Quantum Information and Matter, California Institute of Technology, Pasadena, CA 91125 (United States); Walter Burke Institute for Theoretical Physics, California Institute of Technology, Pasadena, CA 91125 (United States); Pollack, Jason; Remmen, Grant N. [Walter Burke Institute for Theoretical Physics, California Institute of Technology, Pasadena, CA 91125 (United States)

2015-11-15

We analyze the no-cloning theorem in quantum mechanics through the lens of the proposed ER=EPR (Einstein-Rosen = Einstein-Podolsky-Rosen) duality between entanglement and wormholes. In particular, we find that the no-cloning theorem is dual on the gravity side to the no-go theorem for topology change, violating the axioms of which allows for wormhole stabilization and causality violation. Such a duality between important no-go theorems elucidates the proposed connection between spacetime geometry and quantum entanglement. (copyright 2015 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

A camelid single-domain antibody neutralizes botulinum neurotoxin A by blocking host receptor binding

Energy Technology Data Exchange (ETDEWEB)

Yao, Guorui; Lam, Kwok-ho; Weisemann, Jasmin; Peng, Lisheng; Krez, Nadja; Perry, Kay; Shoemaker, Charles B.; Dong, Min; Rummel, Andreas; Jin, Rongsheng (BCH); (Cornell); (Tufts CTSI); (UCI); (MHH)

2017-08-07

Antibody treatment is currently the only available countermeasure for botulism, a fatal illness caused by flaccid paralysis of muscles due to botulinum neurotoxin (BoNT) intoxication. Among the seven major serotypes of BoNT/A-G, BoNT/A poses the most serious threat to humans because of its high potency and long duration of action. Prior to entering neurons and blocking neurotransmitter release, BoNT/A recognizes motoneurons via a dual-receptor binding process in which it engages both the neuron surface polysialoganglioside (PSG) and synaptic vesicle glycoprotein 2 (SV2). Previously, we identified a potent neutralizing antitoxin against BoNT/A1 termed ciA-C2, derived from a camelid heavy-chain-only antibody (VHH). In this study, we demonstrate that ciA-C2 prevents BoNT/A1 intoxication by inhibiting its binding to neuronal receptor SV2. Furthermore, we determined the crystal structure of ciA-C2 in complex with the receptor-binding domain of BoNT/A1 (HCA1) at 1.68 Å resolution. The structure revealed that ciA-C2 partially occupies the SV2-binding site on H_CA1, causing direct interference of HCA1 interaction with both the N-glycan and peptide-moiety of SV2. Interestingly, this neutralization mechanism is similar to that of a monoclonal antibody in clinical trials, despite that ciA-C2 is more than 10-times smaller. Taken together, these results enlighten our understanding of BoNT/A1 interactions with its neuronal receptor, and further demonstrate that inhibiting toxin binding to the host receptor is an efficient countermeasure strategy.

Antibodies to a soluble form of a tumor necrosis factor (TNF) receptor have TNF-like activity

DEFF Research Database (Denmark)

Engelmann, H; Holtmann, H; Brakebusch, C

1990-01-01

Immunological cross-reactivity between tumor necrosis factor (TNF) binding proteins which are present in human urine (designated TBPI and TBPII) and two molecular species of the cell surface receptors for TNF is demonstrated. The two TNF receptors are shown to be immunologically distinct, to differ....... These antibodies are cytotoxic to cells which are sensitive to TNF toxicity, induce resistance to TNF toxicity, enhance the incorporation of thymidine into normal fibroblasts, inhibit the growth of chlamydiae, and induce the synthesis of prostaglandin E2. Monovalent F(ab) fragments of the polyclonal antibodies...

Synthetic α subunit peptide 125-147 of human nicotinic acetylcholine receptor induces antibodies to native receptor

International Nuclear Information System (INIS)

McCormick, D.J.; Griesmann, G.E.; Huang, Z.; Lennon, V.A.

1986-01-01

A synthetic peptide corresponding to residues 125-147 of the Torpedo acetylcholine receptor (AChR) α subunit proved to be a major antigenic region of the AChR. Rats inoculated with 50 μg of peptide (T α 125-147) developed T cell immunity and antibodies to native AChR and signs of experimental autoimmune myasthenia gravis. They report the synthesis and preliminary testing of a disulfide-looped peptide comprising residues 125-147 of the human AChR α subunit. Peptide H α 125-147 differs from T α 125-147 at residues 139 (Glu for Gln) and 143 (Ser for Thr). In immunoprecipitation assays, antibodies to Torpedo AChR bound 125 I-labelled Hα 125-147 antibody bound Hα 125-147, but monoclonal antibodies to an immunodominant region of native AChR bound neither Hα 125-147 nor T α 125-147. Rats immunized with H α 125-147 produced anti-mammalian muscle AChR antibodies that induced modulation of AChRs from cultured human myotubes. Thus, region 125-147 of the human AChR α subunit is extracellular in muscle, and is both antigenic and immunogenic. It remains to be determined whether or not autoantibodies to this region may in part cause the weakness or myasthenia gravis in man

Agonistic Human Monoclonal Antibodies against Death Receptor 4 (DR4) | NCI Technology Transfer Center | TTC

Science.gov (United States)

The National Cancer Institute is seeking parties interested in licensing human monoclonal antibodies (mAbs) that bind to death receptor 4 ("DR4"). The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and its functional receptors, DR4 and DR5, have been recognized as promising targets for cancer treatment.

Characterization of a purified nicotinic receptor from rat brain by using idiotypic and anti-idiotypic antibodies

International Nuclear Information System (INIS)

Abood, L.G.; Langone, J.J.; Bjercke, R.; Lu, X.; Banerjee, S.

1987-01-01

The availability of an anti-nicotine monoclonal antibody has made it possible to further establish the nature of the nicotine recognition proteins purified from rat brain by affinity chromatography and to provide a highly sensitive assay for determining [ 3 H]nicotine binding to the purified material. An enantiomeric analogue of nicotine. (-)-6-hydroxymethylnicotine, was used to prepare the affinity column. In addition, with the use of an anti-idiotypic monoclonal antibody, it was confirmed that the recognition site for nicotine resides on a protein complex composed of two components with molecular masses of 62 and 57 kDa. It was also demonstrated that the same two proteins could be purified by immunoaffinity chromatography with the use of an anti-idiotypic monoclonal antibody. With the use of the anti-nicotine antibody to measure [ 3 H]nicotine binding, the purified material was shown to bind 250 pmol/mg of protein. By utilizing a procedure in which the purified receptor protein was conjugated to membranes by disulfide bonds, a binding activity of 80 pmol/mg was obtained. With the availability of sterospecific monoclonal antibodies to (-)-nicotine as well as monoclonal anti-idiotypic antibodies derived when the anti-nicotine antibodies were used as immunogens, additional procedures became available for the further characterization of the purified nicotine receptor and examining its (-)-[ 3 H]nicotine-binding characteristics

Successful combination immunotherapy of anti-gamma aminobutyric acid (GABA)A receptor antibody-positive encephalitis with extensive multifocal brain lesions.

Science.gov (United States)

Fukami, Yuki; Okada, Hiroaki; Yoshida, Mari; Yamaguchi, Keiji

2017-08-31

A 78-year old woman who presented with akinetic mutism was admitted to our hospital. Brain MRI showed multifocal increased T 2 /FLAIR signal with extensive cortical-subcortical involvement. We suspected autoimmune encephalitis and the patient received methylprednisolone pulse. Her conscious level gradually recovered, but later relapsed again and presented with refractory status epilepticus. We treated her with intravenous immunoglobulin, plasma exchange and pulsed cyclophosphamide, with satisfactory response. A brain biopsy showed perivascular lymphocytic infiltrates and reactive gliosis. Anti-gamma aminobutyric acid (GABA) A receptor antibodies test came back to be positive after her recovery, and the diagnosis of anti-GABA A receptor antibody-positive encephalitis was made. This is a very rare case where brain biopsies were performed in a patient with anti-GABA A receptor antibody-positive encephalitis.

Prevalence of elevated serum anti-N-methyl-D-aspartate receptor antibody titers in patients presenting exclusively with psychiatric symptoms: a comparative follow-up study.

Science.gov (United States)

Ando, Yoshihito; Shimazaki, Haruo; Shiota, Katsutoshi; Tetsuka, Syuichi; Nakao, Koichi; Shimada, Tatsuhiro; Kurata, Kazumi; Kuroda, Jinichi; Yamashita, Akihiro; Sato, Hayato; Sato, Mamoru; Eto, Shinkichi; Onishi, Yasunori; Tanaka, Keiko; Kato, Satoshi

2016-07-08

Increasing numbers of patients with elevated anti-N-methyl-D-aspartate (NMDA) receptor antibody titers presenting exclusively with psychiatric symptoms have been reported. The aim of the present study was to clarify the prevalence of elevated serum anti-NMDA receptor antibody titers in patients with new-onset or acute exacerbations of psychiatric symptoms. In addition, the present study aimed to investigate the association between elevated anti-NMDA receptor titers and psychiatric symptoms. The present collaborative study included 59 inpatients (23 male, 36 female) presenting with new-onset or exacerbations of schizophrenia-like symptoms at involved institutions from June 2012 to March 2014. Patient information was collected using questionnaires. Anti-NMDA receptor antibody titers were measured using NMDAR NR1 and NR2B co-transfected human embryonic kidney (HEK) 293 cells as an antigen (cell-based assay). Statistical analyses were performed for each questionnaire item. The mean age of participants was 42.0 ± 13.7 years. Six cases had elevated serum anti-NMDA antibody titers (10.2 %), four cases were first onset, and two cases with disease duration >10 years presented with third and fifth recurrences. No statistically significant difference in vital signs or major symptoms was observed between antibody-positive and antibody-negative groups. However, a trend toward an increased frequency of schizophrenia-like symptoms was observed in the antibody-positive group. Serum anti-NMDA receptor antibody titers may be associated with psychiatric conditions. However, an association with specific psychiatric symptoms was not observed in the present study. Further studies are required to validate the utility of serum anti-NMDA receptor antibody titer measurements at the time of symptom onset.

Maintenance immunosuppression with intermittent intravenous IL-2 receptor antibody therapy in renal transplant recipients.

LENUS (Irish Health Repository)

Gabardi, Steven

2011-09-01

To report what we believe to be the first 2 cases of long-term (>24 months) intermittent intravenous interleukin-2 receptor antibody (IL-2RA) therapy for maintenance immunosuppression following renal transplantation.

The Roles of the TSH Receptor Antibodies in Autoimmune Thyroid Diseases

International Nuclear Information System (INIS)

Koh, Chang Soon

1986-01-01

To evaluate the clinical and pathogenetic roles of TSH receptor antibodies in autoimmune thyroid diseases, TBII were measured by TSH-radioreceptor assay methods in 352 patients with Graves disease, 108 patients with other thyroid diseases and 69 normal persons. The normal range of TBII activity was less than 15%. The frequencies of detectable TBIl in 169 patients with untreated Graves disease, 31 patients with hyperthyroidism under treatment and 70 patients with euthyroidism under treatment were 92.4%, 87.1% and 54.3% respectively. However 12 (21.8%) out of 55 patients who have been in remission more than one year after discontinuation of antithyroid drugs treatment had detectable TBII activities in their sera. In 196 patients with untreated Graves disease, the frequency of TBII increased by increasing size of goiter and the frequency of proptosis was significantly high in patients whose TBII activities were more than 60%. TBll activities were roughly correlated with total T 3 ,T 4 and free T 4 , index but low γ 2 value(less than 0.1). In 67 patients with Graves' disease who were positive TB1I before antithyroid drugs treatment, TBII activities began to decrease from the third months and it was converted to negative in 35.8% of patients at 12 months after treatment. There were no significant differences of the declining and disappearing rates of TBII activities between high dose and conventional dose groups. TBII activities were significantly increased initially (2-4 months) and then began to decrease from 5-9 months after 131 I treatment. There were two groups, one whose TBII activities decreased gradually and the other did not change until 12 months after subtotal thyroidectomy. Although preoperative clinical and laboratory findings of both groups were not different, TBII activities of non-decreasing group were significantly higher than those of decreasing group(74.6+18.6% vs 39.2+15.2%; P(0.01). Thirty three(55.9%) out of 59 patients with Graves disease

Antibody-induced activation of the epidermal growth factor receptor tyrosine kinase requires the presence of detergent

NARCIS (Netherlands)

Spaargaren, M.; Defize, L. H.; de Laat, S. W.; Boonstra, J.

1990-01-01

Activation of the epidermal growth factor receptor (EGF-R) tyrosine kinase was investigated in membrane preparations as well as intact A431 cells, using anti-EGF-R antibodies directed against extra- and intracellular receptor domains. In vitro assay conditions were mimicked on whole cells by a mild

Antibody guided diagnosis and therapy of brain gliomas using radiolabeled monoclonal antibodies against epidermal growth factor receptor and placental alkaline phosphatase

International Nuclear Information System (INIS)

Kalofonos, H.P.; Pawlikowska, T.R.; Hemingway, A.

1989-01-01

Twenty-seven patients with brain glioma were scanned using 123 I-labeled monoclonal antibodies against epidermal growth factor receptor (EGFR1) or placental alkaline phosphatase (H17E2). Successful localization was achieved in 18 out of 27 patients. Eleven out of 27 patients were also studied using a nonspecific control antibody (11.4.1) of the same immunoglobulin subclass and observable tumor localization was also achieved in five patients. The specificity of targeting was assessed by comparing images obtained with specific and nonspecific antibodies and by examining tumor and normal tissue biopsies after dual antibody administration. Ten patients with recurrent grade III or IV glioma who showed good localization of radiolabeled antibody were treated with 40-140 mCi of 131 I-labeled antibody delivered to the tumor area intravenously (n = 5) or by infusion into the internal carotid artery (n = 5). Six patients showed clinical improvement lasting from 6 mo to 3 yr. One patient continues in remission (3 yr after therapy), but the other five who responded initially relapsed 6-9 mo after therapy and died. No major toxicity was attributable to antibody-guided irradiation. Targeted irradiation by monoclonal antibody may be clinically useful and should be explored further in the treatment of brain gliomas resistant to conventional forms of treatment

Re-engineering therapeutic antibodies for Alzheimer's disease as blood-brain barrier penetrating bi-specific antibodies.

Science.gov (United States)

Pardridge, William M

2016-12-01

Therapeutic antibodies are large molecule drugs that do not cross the blood-brain barrier (BBB). Therefore, drug development of therapeutic antibodies for Alzheimer's disease (AD) requires that these molecules be re-engineered to enable BBB delivery. This is possible by joining the therapeutic antibody with a transporter antibody, resulting in the engineering of a BBB-penetrating bispecific antibody (BSA). Areas covered: The manuscript covers transporter antibodies that cross the BBB via receptor-mediated transport systems on the BBB, such as the insulin receptor or transferrin receptor. Furthermore, it highlights therapeutic antibodies for AD that target the Abeta amyloid peptide, beta secretase-1, or the metabotropic glutamate receptor-1. BSAs are comprised of both the transporter antibody and the therapeutic antibody, as well as IgG constant region, which can induce immune tolerance or trigger transport via Fc receptors. Expert opinion: Multiple types of BSA molecular designs have been used to engineer BBB-penetrating BSAs, which differ in valency and spatial orientation of the transporter and therapeutic domains of the BSA. The plasma pharmacokinetics and dosing regimens of BSAs differ from that of conventional therapeutic antibodies. BBB-penetrating BSAs may be engineered in the future as new treatments of AD, as well as other neural disorders.

The Anti-Acetylcholine Receptor Antibody Test in Suspected Ocular Myasthenia Gravis

Directory of Open Access Journals (Sweden)

Jung Jin Lee

2014-01-01

Full Text Available Aim. To estimate the clinical significance of anti-acetylcholine receptor antibody (anti-AChR-Ab levels in suspected ocular myasthenia gravis. Methods. In total, 144 patients complaining of fluctuating diplopia and ptosis were evaluated for serum levels of anti-acetylcholine receptor antibody and their medical charts were retrospectively reviewed. Subjects were classified into three groups: variable diplopia only, ptosis only, and both variable diplopia and ptosis. We investigated serum anti-AChR-Ab titer levels and performed thyroid autoantibody tests. Results. Patients’ chief complaints were diplopia (N=103, ptosis (N=12, and their concurrence (N=29. Abnormal anti-AChR-Ab was observed in 21 of 144 patients (14.1%. Between the three groups, mean age, number of seropositive patients, and mean anti-AChR-Ab level were not significantly different (P=0.224, 0.073, and 0.062, resp.. Overall, 27.5% of patients had abnormal thyroid autoantibodies. Conclusion. The sensitivity of anti-AChR-Ab was 14.1% in suspected ocular myasthenia gravis and seropositivity in myasthenia gravis patients showed a high correlation with the presence of thyroid autoantibodies.

Antibodies Against Hypocretin Receptor 2 Are Rare in Narcolepsy.

Science.gov (United States)

Giannoccaro, Maria Pia; Waters, Patrick; Pizza, Fabio; Liguori, Rocco; Plazzi, Giuseppe; Vincent, Angela

2017-02-01

Recently, antibodies to the hypocretin receptor 2 (HCRTR2-Abs) were reported in a high proportion of narcolepsy patients who developed the disease following Pandemrix® vaccination. We tested a group of narcolepsy patients for the HCRTR2-Abs using a newly established cell-based assay. Sera from 50 narcolepsy type 1 (NT1) and 11 narcolepsy type 2 (NT2) patients, 22 patients with other sleep disorders, 15 healthy controls, and 93 disease controls were studied. Cerebrospinal fluid (CSFs) from three narcoleptic patients were subsequently included. Human embryonic kidney cells were transiently transfected with human HCRTR2, incubated with patients' sera for 1 hr at 1:20 dilution and then fixed. Binding of antibodies was detected by fluorescently labeled secondary antibodies to human immunoglobulin G (IgG) and the different IgG subclasses. A nonlinear visual scoring system was used from 0 to 4; samples scoring ≥1 were considered positive. Only 3 (5%) of 61 patients showed a score ≥1, one with IgG1- and two with IgG3-antibodies, but titers were low (1:40-1:100). CSFs from these patients were negative. The three positive patients included one NT1 case with associated psychotic features, one NT2 patient, and an NT1 patient with normal hypocretin CSF levels. Low levels of IgG1 or IgG3 antibodies against HCRTR2 were found in 3 of 61 patients with narcolepsy, although only 1 presented with full-blown NT1. HCRTR2-Abs are not common in narcolepsy unrelated to vaccination. © Sleep Research Society 2016. Published by Oxford University Press on behalf of the Sleep Research Society. All rights reserved. For permissions, please e-mail journals.permissions@oup.com.

Differences in human skin between the epidermal growth factor receptor distribution detected by EGF binding and monoclonal antibody recognition

DEFF Research Database (Denmark)

Green, M R; Couchman, J R

1985-01-01

, the eccrine sweat glands, capillary system, and the hair follicle outer root sheath, generally similar in pattern to that previously reported for full-thickness rat skin and human epidermis. The same areas also bound EGF-R1 but in addition the monoclonal antibody recognized a cone of melanin containing......Two methods have been used to examine epidermal growth factor (EGF) receptor distribution in human scalp and foreskin. The first employed [125I]EGF viable explants and autoradiography to determine the EGF binding pattern while the second used a monoclonal antibody to the human EGF receptor to map...... whether EGF-R1 could recognize molecules unrelated to the EGF receptor, the EGF binding and EGF-R1 recognition profiles were compared on cultures of SVK14 cells, a SV40 transformed human keratinocyte cell line. EGF binding and EGF-R1 monoclonal antibody distribution on these cells was found to be similar...

A case of late-onset, thymoma-associated myasthenia gravis with ryanodine receptor and titin antibodies and concomitant granulomatous myositis.

Science.gov (United States)

Stefanou, M I; Komorowski, L; Kade, S; Bornemann, A; Ziemann, U; Synofzik, M

2016-09-13

Myasthenia gravis is an autoimmune neuromuscular disorder, which has only rarely been reported to co-manifest with myositis. The diagnosis of concomitant myositis in patients with myasthenia gravis is clinically challenging, and requires targeted investigations for the differential diagnosis, including EMG, autoantibody assays, muscle biopsy and, importantly, imaging of the mediastinum for thymoma screening. This report presents a case-vignette of a 72-year-old woman with progressive proximal muscle weakness and myalgias, diagnosed with thymoma-associated myasthenia and bioptically verified granulomatous myositis, with positive autoantibody status for ryanodine receptor and titin antibodies. The diagnosis of concurrent myositis and myasthenia gravis, especially in the presence of ryanodine receptor and titin antibodies, should lead neurologists to adopt different treatment strategies compared to those applied in myasthenia or myositis alone. Moreover, further evidence is warranted that titin and, particularly, ryanodine receptor antibodies may co-occur or be pathophysiologically involved in myasthenia-myositis cases.

Antibody guided irradiation of brain glioma by arterial infusion of radioactive monoclonal antibody against epidermal growth factor receptor and blood group A antigen

Energy Technology Data Exchange (ETDEWEB)

Epenetos, A.A.; Courtenay-Luck, N.; Pickering, D.; Hooker, G.; Lavender, J.P.; McKenzie, C.G. (Hammersmith Hospital, London (UK)); Durbin, H. (Imperial Cancer Research Fund, London (UK). Labs.)

1985-05-18

In a patient with recurrent grade IV glioma of the brain resistant to conventional treatment an antibody guided isotopic scan showed uptake by the tumour of a monoclonal antibody (9A) that was developed against epidermal growth factor receptor but cross reacted with blood group A antigen. As a therapeutic attempt antibody labelled with 1665 MBq (45.0 mCi) iodine-131 was delivered to the tumour area by infusion into the internal carotid artery. Computed tomography showed regression of the tumour after treatment, and an appreciable and sustained clinical improvement was noted without any toxicity. Delivery of irradiation guided by monoclonal antibody delivered by arterial infusion of the tumour area may be of clinical value in the treatment of brain gliomas resistant to conventional forms of treatment.

Synthetic. cap alpha. subunit peptide 125-147 of human nicotinic acetylcholine receptor induces antibodies to native receptor

Energy Technology Data Exchange (ETDEWEB)

McCormick, D.J.; Griesmann, G.E.; Huang, Z.; Lennon, V.A.

1986-03-05

A synthetic peptide corresponding to residues 125-147 of the Torpedo acetylcholine receptor (AChR) ..cap alpha.. subunit proved to be a major antigenic region of the AChR. Rats inoculated with 50 ..mu..g of peptide (T ..cap alpha.. 125-147) developed T cell immunity and antibodies to native AChR and signs of experimental autoimmune myasthenia gravis. They report the synthesis and preliminary testing of a disulfide-looped peptide comprising residues 125-147 of the human AChR ..cap alpha.. subunit. Peptide H ..cap alpha.. 125-147 differs from T ..cap alpha.. 125-147 at residues 139 (Glu for Gln) and 143 (Ser for Thr). In immunoprecipitation assays, antibodies to Torpedo AChR bound /sup 125/I-labelled H..cap alpha.. 125-147 antibody bound H..cap alpha.. 125-147, but monoclonal antibodies to an immunodominant region of native AChR bound neither H..cap alpha.. 125-147 nor T ..cap alpha.. 125-147. Rats immunized with H ..cap alpha.. 125-147 produced anti-mammalian muscle AChR antibodies that induced modulation of AChRs from cultured human myotubes. Thus, region 125-147 of the human AChR ..cap alpha.. subunit is extracellular in muscle, and is both antigenic and immunogenic. It remains to be determined whether or not autoantibodies to this region may in part cause the weakness or myasthenia gravis in man.

Shark Variable New Antigen Receptor (VNAR Single Domain Antibody Fragments: Stability and Diagnostic Applications

Directory of Open Access Journals (Sweden)

Stewart Nuttall

2013-01-01

Full Text Available The single variable new antigen receptor domain antibody fragments (VNARs derived from shark immunoglobulin new antigen receptor antibodies (IgNARs represent some of the smallest known immunoglobulin-based protein scaffolds. As single domains, they demonstrate favorable size and cryptic epitope recognition properties, making them attractive in diagnosis and therapy of numerous disease states. Here, we examine the stability of VNAR domains with a focus on a family of VNARs specific for apical membrane antigen 1 (AMA-1 from Plasmodium falciparum. The VNARs are compared to traditional monoclonal antibodies (mAbs in liquid, lyophilized and immobilized nitrocellulose formats. When maintained in various formats at 45 °C, VNARs have improved stability compared to mAbs for periods of up to four weeks. Using circular dichroism spectroscopy we demonstrate that VNAR domains are able to refold following heating to 80 °C. We also demonstrate that VNAR domains are stable during incubation under potential in vivo conditions such as stomach acid, but not to the protease rich environment of murine stomach scrapings. Taken together, our results demonstrate the suitability of shark VNAR domains for various diagnostic platforms and related applications.

Developmental wiring of specific neurons is regulated by RET-1/Nogo-A in Caenorhabditis elegans

DEFF Research Database (Denmark)

Torpe, Nanna; Nørgaard, Steffen; Høye, Anette M.

2017-01-01

Nogo-A is a membrane-bound protein that functions to inhibit neuronal migration, adhesion, and neurite outgrowth during development. In the mature nervous system, Nogo-A stabilizes neuronal wiring to inhibit neuronal plasticity and regeneration after injury. Here, we show that RET-1, the sole Nog...... present a previously unidentified function for RET-1 in the nervous system of C. elegans.......-A homolog in Caenorhabditis elegans, is required to control developmental wiring of a specific subset of neurons. In ret-1 deletion mutant animals, specific ventral nerve cord axons are misguided where they fail to respect the ventral midline boundary. We found that ret-1 is expressed in multiple neurons...

Graves' Disease Mechanisms: The Role of Stimulating, Blocking, and Cleavage Region TSH Receptor Antibodies

Science.gov (United States)

Morshed, S. A.; Davies, T. F.

2016-01-01

The immunologic processes involved in Graves' disease (GD) have one unique characteristic – the autoantibodies to the TSH receptor (TSHR) – which have both linear and conformational epitopes. Three types of TSHR antibodies (stimulating, blocking, and cleavage) with different functional capabilities have been described in GD patients, which induce different signaling effects varying from thyroid cell proliferation to thyroid cell death. The establishment of animal models of GD by TSHR antibody transfer or by immunization with TSHR antigen has confirmed its pathogenic role and, therefore, GD is the result of a breakdown in TSHR tolerance. Here we review some of the characteristics of TSHR antibodies with a special emphasis on new developments in our understanding of what were previously called “neutral” antibodies and which we now characterize as autoantibodies to the “cleavage” region of the TSHR ectodomain. PMID:26361259

Stereotypic Movements in Case of Sporadic Creutzfeldt-Jakob Disease: Possible Role of Anti-NMDA Receptor Antibodies

Directory of Open Access Journals (Sweden)

Michelle Molina

2012-12-01

Full Text Available Sporadic Creutzfeldt-Jakob disease (sCJD and anti-NMDA receptor antibody encephalitis (NMDAE can both produce a rapidly progressive dementia with resulting state of catatonia or akinetic mutism. Both are associated with movement disorders. In published case series, myoclonus appears to be the most frequent movement disorder in sCJD, while stereotypic, synchronized, one-cycle-per-second movements such as arm or leg elevation, jaw opening, grimacing, head turning, and eye deviation are seen in NMDAE. We report a case of a 59-year-old woman with rapidly worsening cognitive disturbance leading to a nearly catatonic state interrupted by stereotypic movements. sCJD was diagnosed via periodic sharp wave complexes on EEG as well as cerebrospinal fluid (CSF 14-3-3 and tau protein elevation. Characteristic movement disorder of NMDAE was present in absence of ovarian mass or CSF pleiocytosis. Given prior case reports of presence of anti-NMDA receptor antibodies in sCJD, we propose that the movement disorder in this case was caused by anti-NMDA receptor antibodies whose formation was secondary to neuronal damage from prion disease. It is important to consider sCJD even in cases that have some clinical features suggestive of NMDAE.

An assessment of radioimmunoassay procedures for determination of anti-acetylcholine receptor antibodies in the sera of patients with myasthenia gravis

International Nuclear Information System (INIS)

Carter, B.; Harrison, R.; Lunt, G.G.; Morris, H.; Savage-Marengo, T.; Stephenson, F.A.

1981-01-01

A reproducible radioimmunoassay procedure for the determination of anti-acetylcholine receptor antibodies in the sera of patients with myasthenia gravis is described and examined in detail. The assay combines features of a number of methods previously outlined and allows repeat determinations of antibody titre in a given myasthenic serum sample with coefficient of variation 6%. The mean +- standard deviation for normal human serum anti-acetylcholine receptor antibodies was found by this procedure to be 0.024 +- 0.033 nmol/l α-bungarotoxin binding sites whereas the range for myasthenic patients was 0-139.14 nmol/l with a mean value of 7.55 nmol/l α-bungarotoxin binding sites. (author)

Clinical Utility of Acetylcholine Receptor Antibody Testing in Ocular Myasthenia Gravis.

Science.gov (United States)

Peeler, Crandall E; De Lott, Lindsey B; Nagia, Lina; Lemos, Joao; Eggenberger, Eric R; Cornblath, Wayne T

2015-10-01

The sensitivity of acetylcholine receptor (AChR) antibody testing is thought to be lower in ocular myasthenia gravis (OMG) compared with generalized disease, although estimates in small-scale studies vary. There is little information in the literature about the implications of AChR antibody levels and progression from OMG to generalized myasthenia gravis. To test the hypothesis that serum AChR antibody testing is more sensitive in OMG than previously reported and to examine the association between AChR antibody levels and progression from OMG to generalized myasthenia gravis. A retrospective, observational cohort study was conducted of 223 patients (mean [SD] age, 59.2 [16.4] years; 139 [62.3%] male) diagnosed with OMG between July 1, 1986, and May 31, 2013, at 2 large, academic medical centers. Baseline characteristics, OMG symptoms, results of AChR antibody testing, and progression time to generalized myasthenia gravis (if this occurred) were recorded for each patient. Multiple logistic regression was used to measure the association between all clinical variables and antibody result. Kaplan-Meier survival analysis was performed to examine time to generalization. Among the 223 participants, AChR antibody testing results were positive in 158 participants (70.9%). In an adjusted model, increased age at diagnosis (odds ratio [OR], 1.03; 95% CI, 1.01-1.04; P = .007) and progression to generalized myasthenia gravis (OR, 2.92; 95% CI, 1.18-7.26; P = .02) were significantly associated with positive antibody test results. Women were less likely to have a positive antibody test result (OR, 0.36; 95% CI, 0.19-0.68; P = .002). Patients who developed symptoms of generalized myasthenia gravis had a significantly higher mean (SD) antibody level than those who did not develop symptoms of generalized myasthenia gravis (12.7 [16.5] nmol/L vs 4.2 [7.9] nmol/L; P = .002). We demonstrate a higher sensitivity of AChR antibody testing than previously reported in the

Conflict and inhibition differentially affect the N200/P300 complex in a combined go/nogo and stop-signal task.

Science.gov (United States)

Enriquez-Geppert, Stefanie; Konrad, Carsten; Pantev, Christo; Huster, René J

2010-06-01

Conflict and inhibition are considered to exert strong influences on the neurophysiological N200 and P300 brain responses as evoked in go/nogo and stop-signal tasks. In order to separate their underlying neural and functional mechanisms, the current experiment manipulated both conflict and inhibition. To do so, the go/nogo and stop-signal tasks were merged into one paradigm. Conflict was manipulated by varying go-trial frequencies across blocks (75% vs. 25%). Motor inhibition was manipulated by using go, nogo and stop trials each representing a different load of inhibition. Event-related potentials (ERPs) as well as current density reconstructions (CDRs) of fifteen healthy participants were analyzed. Overall, infrequent trials evoked significantly more pronounced N200s than frequent trials. The P300 predominantly revealed significant variations between trial types (go, nogo, stop). Estimated source activations of the MCC and the IFC supported the ERP results; N200-related effects were revealed in both regions, whereas the condition-specific variations of the P300 were only observed in the IFC. The results indicate that the N200 primarily reflects conflict-related effects whereas the P300 predominantly represents motor inhibition. Copyright 2010 Elsevier Inc. All rights reserved.

A Lettuce (Lactuca sativa) Homolog of Human Nogo-B Receptor Interacts with cis-Prenyltransferase and Is Necessary for Natural Rubber Biosynthesis*

Science.gov (United States)

Qu, Yang; Chakrabarty, Romit; Tran, Hue T.; Kwon, Eun-Joo G.; Kwon, Moonhyuk; Nguyen, Trinh-Don; Ro, Dae-Kyun

2015-01-01

Natural rubber (cis-1,4-polyisoprene) is an indispensable biopolymer used to manufacture diverse consumer products. Although a major source of natural rubber is the rubber tree (Hevea brasiliensis), lettuce (Lactuca sativa) is also known to synthesize natural rubber. Here, we report that an unusual cis-prenyltransferase-like 2 (CPTL2) that lacks the conserved motifs of conventional cis-prenyltransferase is required for natural rubber biosynthesis in lettuce. CPTL2, identified from the lettuce rubber particle proteome, displays homology to a human NogoB receptor and is predominantly expressed in latex. Multiple transgenic lettuces expressing CPTL2-RNAi constructs showed that a decrease of CPTL2 transcripts (3–15% CPTL2 expression relative to controls) coincided with the reduction of natural rubber as low as 5%. We also identified a conventional cis-prenyltransferase 3 (CPT3), exclusively expressed in latex. In subcellular localization studies using fluorescent proteins, cytosolic CPT3 was relocalized to endoplasmic reticulum by co-occurrence of CPTL2 in tobacco and yeast at the log phase. Furthermore, yeast two-hybrid data showed that CPTL2 and CPT3 interact. Yeast microsomes containing CPTL2/CPT3 showed enhanced synthesis of short cis-polyisoprenes, but natural rubber could not be synthesized in vitro. Intriguingly, a homologous pair CPTL1/CPT1, which displays ubiquitous expressions in lettuce, showed a potent dolichol biosynthetic activity in vitro. Taken together, our data suggest that CPTL2 is a scaffolding protein that tethers CPT3 on endoplasmic reticulum and is necessary for natural rubber biosynthesis in planta, but yeast-expressed CPTL2 and CPT3 alone could not synthesize high molecular weight natural rubber in vitro. PMID:25477521

Nogo stimuli do not receive more attentional suppression or response inhibition than neutral stimuli: evidence from the N2pc, PD and N2 components in a spatial cueing paradigm

Directory of Open Access Journals (Sweden)

Caroline eBarras

2016-05-01

Full Text Available It has been claimed that stimuli sharing the color of the nogo-target are suppressed because of the strong incentive to not process the nogo-target, but we failed to replicate this finding. Participants searched for a color singleton in the target display and indicated its shape when it was in the go color. If the color singleton in the target display was in the nogo color, they had to withhold the response. The target display was preceded by a cue display that also contained a color singleton (the cue. The cue was either in the color of the go or nogo target, or it was in an unrelated, neutral color. With cues in the go color, reaction times (RTs were shorter when the cue appeared at the same location as the target compared to when it appeared at a different location. Also, electrophysiological recordings showed that an index of attentional selection, the N2pc, was elicited by go cues. Surprisingly, we failed to replicate cueing costs for cues in the nogo color that were originally reported by Anderson and Folk (2012. Consistently, we also failed to find an electrophysiological index of attentional suppression (the PD for cues in the nogo color. Further, fronto-central ERPs to the cue display showed the same negativity for nogo and neutral stimuli relative to go stimuli, which is at odds with response inhibition and conflict monitoring accounts of the Nogo-N2. Thus, the modified cueing paradigm employed here provides little evidence that features associated with nogo-targets are suppressed at the level of attention or response selection. Rather, nogo-stimuli are efficiently ignored and attention is focused on features that require a response.

An Antibody Blocking Activin Type II Receptors Induces Strong Skeletal Muscle Hypertrophy and Protects from Atrophy

Science.gov (United States)

Minetti, Giulia C.; Sheppard, KellyAnn; Ibebunjo, Chikwendu; Feige, Jerome N.; Hartmann, Steffen; Brachat, Sophie; Rivet, Helene; Koelbing, Claudia; Morvan, Frederic; Hatakeyama, Shinji

2014-01-01

The myostatin/activin type II receptor (ActRII) pathway has been identified to be critical in regulating skeletal muscle size. Several other ligands, including GDF11 and the activins, signal through this pathway, suggesting that the ActRII receptors are major regulatory nodes in the regulation of muscle mass. We have developed a novel, human anti-ActRII antibody (bimagrumab, or BYM338) to prevent binding of ligands to the receptors and thus inhibit downstream signaling. BYM338 enhances differentiation of primary human skeletal myoblasts and counteracts the inhibition of differentiation induced by myostatin or activin A. BYM338 prevents myostatin- or activin A-induced atrophy through inhibition of Smad2/3 phosphorylation, thus sparing the myosin heavy chain from degradation. BYM338 dramatically increases skeletal muscle mass in mice, beyond sole inhibition of myostatin, detected by comparing the antibody with a myostatin inhibitor. A mouse version of the antibody induces enhanced muscle hypertrophy in myostatin mutant mice, further confirming a beneficial effect on muscle growth beyond myostatin inhibition alone through blockade of ActRII ligands. BYM338 protects muscles from glucocorticoid-induced atrophy and weakness via prevention of muscle and tetanic force losses. These data highlight the compelling therapeutic potential of BYM338 for the treatment of skeletal muscle atrophy and weakness in multiple settings. PMID:24298022

Characterization of ductal and lobular breast carcinomas using novel prolactin receptor isoform specific antibodies

Directory of Open Access Journals (Sweden)

Heger Christopher D

2010-12-01

Full Text Available Abstract Background Prolactin is a polypeptide hormone responsible for proliferation and differentiation of the mammary gland. More recently, prolactin's role in mammary carcinogenesis has been studied with greater interest. Studies from our laboratory and from others have demonstrated that three specific isoforms of the prolactin receptor (PRLR are expressed in both normal and cancerous breast cells and tissues. Until now, reliable isoform specific antibodies have been lacking. We have prepared and characterized polyclonal antibodies against each of the human PRLR isoforms that can effectively be used to characterize human breast cancers. Methods Rabbits were immunized with synthetic peptides of isoform unique regions and immune sera affinity purified prior to validation by Western blot and immunohistochemical analyses. Sections of ductal and lobular carcinomas were stained with each affinity purified isoform specific antibody to determine expression patterns in breast cancer subclasses. Results We show that the rabbit antibodies have high titer and could specifically recognize each isoform of PRLR. Differences in PRLR isoform expression levels were observed and quantified using histosections from xenografts of established human breast cancer cells lines, and ductal and lobular carcinoma human biopsy specimens. In addition, these results were verified by real-time PCR with isoform specific primers. While nearly all tumors contained LF and SF1b, the majority (76% of ductal carcinoma biopsies expressed SF1a while the majority of lobular carcinomas lacked SF1a staining (72% and 27% had only low levels of expression. Conclusions Differences in the receptor isoform expression profiles may be critical to understanding the role of PRL in mammary tumorigenesis. Since these antibodies are specifically directed against each PRLR isoform, they are valuable tools for the evaluation of breast cancer PRLR content and have potential clinical importance in

A control-theoretic outlook at the no-go zone in sailing vessels

DEFF Research Database (Denmark)

Yang, Bin; Xiao, Lin; Jouffroy, Jerome

2011-01-01

Sailing vessels, due to their particular propulsive mechanism, gradually lose power as they face the wind, i.e. when they are in the so-called “no-go zone”. Interestingly, dynamical models of sailing vessels, which are usually quite complex, all have in common this no-go zone effect. Using...

[Construction of a phage antibody library and screening of anti-epidermal growth factor receptor variant III single chain antibody].

Science.gov (United States)

Han, Dong-gang; Duan, Xiao-yi; Guo, You-min; Zhou, Qi; Wang, Quan-ying; Yang, Guang-xiao

2010-01-01

To obtain specific anti-epidermal growth factor receptor variant III (EGFRvIII) single chain antibody (ScFv) by phage antibody library display system. The total RNA was extracted from the spleen B cells of BALB/c mice immunized with pep-3-OVA protein, and the first-strand cDNA was synthesized by reverse transcription. Antibody VH and VL gene fragments were amplified and joined to a ScFv gene with the linker. The ScFv gene was ligated into the phagemid vector pCANTAB5E, which was transformed into competent E. coli TG1. The transformed cells were then infected with M13KO7 helper phage to yield the recombinant phage to construct the phage ScFv library. Pep-3-BSA protein was used to screen the phage antibody library and ELISA carried out to characterize the activity of the antibody. The VH and VL gene fragments of the antibody were about 350 bp and 320 bp in length as analyzed by agarose gel electrophoresis. The ScFv gene was 780 bp, consistent with the expected length. The recombinant phagemid with ScFv gene insert was rescued, and an immune phage ScFv library with the content of 5.0x10(6) was constructed. The recombinant ScFv phage had a titer of 3.0x10(4) cfu/ml, and the fourth phage harvest yielded 56 times as much as that of the first one. SDS-PAGE demonstrated a molecular mass of the soluble ScFv of about 28 kD. ELISA results indicated good specificity of the ScFv to bind EGFRvIII. An immune phage ScFv library is successfully constructed, and the ScFv antibody fragment is capable of specific binding to EGFRvIII.

The Roles of the TSH Receptor Antibodies in Autoimmune Thyroid Diseases

Energy Technology Data Exchange (ETDEWEB)

Koh, Chang Soon [Seoul National University College of Medicine, Seoul (Korea, Republic of)

1986-09-15

To evaluate the clinical and pathogenetic roles of TSH receptor antibodies in autoimmune thyroid diseases, TBII were measured by TSH-radioreceptor assay methods in 352 patients with Graves disease, 108 patients with other thyroid diseases and 69 normal persons. The normal range of TBII activity was less than 15%. The frequencies of detectable TBIl in 169 patients with untreated Graves disease, 31 patients with hyperthyroidism under treatment and 70 patients with euthyroidism under treatment were 92.4%, 87.1% and 54.3% respectively. However 12 (21.8%) out of 55 patients who have been in remission more than one year after discontinuation of antithyroid drugs treatment had detectable TBII activities in their sera. In 196 patients with untreated Graves disease, the frequency of TBII increased by increasing size of goiter and the frequency of proptosis was significantly high in patients whose TBII activities were more than 60%. TBll activities were roughly correlated with total T{sub 3},T{sub 4} and free T{sub 4}, index but low gamma{sup 2} value(less than 0.1). In 67 patients with Graves' disease who were positive TB1I before antithyroid drugs treatment, TBII activities began to decrease from the third months and it was converted to negative in 35.8% of patients at 12 months after treatment. There were no significant differences of the declining and disappearing rates of TBII activities between high dose and conventional dose groups. TBII activities were significantly increased initially (2-4 months) and then began to decrease from 5-9 months after {sup 131}I treatment. There were two groups, one whose TBII activities decreased gradually and the other did not change until 12 months after subtotal thyroidectomy. Although preoperative clinical and laboratory findings of both groups were not different, TBII activities of non-decreasing group were significantly higher than those of decreasing group(74.6+18.6% vs 39.2+15.2%; P(0.01). Thirty three(55.9%) out of 59

Human agonistic TRAIL receptor antibodies Mapatumumab and Lexatumumab induce apoptosis in malignant mesothelioma and act synergistically with cisplatin

Directory of Open Access Journals (Sweden)

Felley-Bosco Emanuela

2007-10-01

Full Text Available Abstract Background The incidence of malignant pleural mesothelioma (MPM is associated with exposure to asbestos, and projections suggest that the yearly number of deaths in Western Europe due to MPM will increase until 2020. Despite progress in chemo- and in multimodality therapy, MPM remains a disease with a poor prognosis. Inducing apoptosis by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL or agonistic monoclonal antibodies which target TRAIL-receptor 1 (TRAIL-R1 or TRAIL-R2 has been thought to be a promising cancer therapy. Results We have compared the sensitivity of 13 MPM cell lines or primary cultures to TRAIL and two fully human agonistic monoclonal antibodies directed to TRAIL-R1 (Mapatumumab and TRAIL-R2 (Lexatumumab and examined sensitization of the MPM cell lines to cisplatin-induced by the TRAIL-receptor antibodies. We found that sensitivity of MPM cells to TRAIL, Mapatumumab and Lexatumumab varies largely and is independent of TRAIL-receptor expression. TRAIL-R2 contributes more than TRAIL-R1 to death-receptor mediated apoptosis in MPM cells that express both receptors. The combination of cisplatin with Mapatumumab or Lexatumumab synergistically inhibited the cell growth and enhanced apoptotic death. Furthermore, pre-treatment with cisplatin followed by Mapatumumab or Lexatumumab resulted in significant higher cytotoxic effects as compared to the reverse sequence. Combination-induced cell growth inhibition was significantly abrogated by pre-treatment of the cells with the antioxidant N-acetylcysteine. Conclusion Our results suggest that the sequential administration of cisplatin followed by Mapatumumab or Lexatumumab deserves investigation in the treatment of patients with MPM.

Blood-brain barrier drug delivery of IgG fusion proteins with a transferrin receptor monoclonal antibody.

Science.gov (United States)

Pardridge, William M

2015-02-01

Biologic drugs are large molecules that do not cross the blood- brain barrier (BBB). Brain penetration is possible following the re-engineering of the biologic drug as an IgG fusion protein. The IgG domain is a MAb against an endogenous BBB receptor such as the transferrin receptor (TfR). The TfRMAb acts as a molecular Trojan horse to ferry the fused biologic drug into the brain via receptor-mediated transport on the endogenous BBB TfR. This review discusses TfR isoforms, models of BBB transport of transferrin and TfRMAbs, and the genetic engineering of TfRMAb fusion proteins, including BBB penetrating IgG-neurotrophins, IgG-decoy receptors, IgG-lysosomal enzyme therapeutics and IgG-avidin fusion proteins, as well as BBB transport of bispecific antibodies formed by fusion of a therapeutic antibody to a TfRMAb targeting antibody. Also discussed are quantitative aspects of the plasma pharmacokinetics and brain uptake of TfRMAb fusion proteins, as compared to the brain uptake of small molecules, and therapeutic applications of TfRMAb fusion proteins in mouse models of neural disease, including Parkinson's disease, stroke, Alzheimer's disease and lysosomal storage disorders. The review covers the engineering of TfRMAb-avidin fusion proteins for BBB targeted delivery of biotinylated peptide radiopharmaceuticals, low-affinity TfRMAb Trojan horses and the safety pharmacology of chronic administration of TfRMAb fusion proteins. The BBB delivery of biologic drugs is possible following re-engineering as a fusion protein with a molecular Trojan horse such as a TfRMAb. The efficacy of this technology will be determined by the outcome of future clinical trials.

Different mechanisms are involved in the antibody mediated inhibition of ligand binding to the urokinase receptor

DEFF Research Database (Denmark)

List, K; Høyer-Hansen, G; Rønne, E

1999-01-01

Certain monoclonal antibodies are capable of inhibiting the biological binding reactions of their target proteins. At the molecular level, this type of effect may be brought about by completely different mechanisms, such as competition for common binding determinants, steric hindrance or interfer......Certain monoclonal antibodies are capable of inhibiting the biological binding reactions of their target proteins. At the molecular level, this type of effect may be brought about by completely different mechanisms, such as competition for common binding determinants, steric hindrance......) can be employed as a highly useful tool to characterize the inhibitory mechanism of specific antagonist antibodies. Two inhibitory antibodies against uPAR, mAb R3 and mAb R5, were shown to exhibit competitive and non-competitive inhibition, respectively, of ligand binding to the receptor. The former...

A lettuce (Lactuca sativa) homolog of human Nogo-B receptor interacts with cis-prenyltransferase and is necessary for natural rubber biosynthesis.

Science.gov (United States)

Qu, Yang; Chakrabarty, Romit; Tran, Hue T; Kwon, Eun-Joo G; Kwon, Moonhyuk; Nguyen, Trinh-Don; Ro, Dae-Kyun

2015-01-23

Natural rubber (cis-1,4-polyisoprene) is an indispensable biopolymer used to manufacture diverse consumer products. Although a major source of natural rubber is the rubber tree (Hevea brasiliensis), lettuce (Lactuca sativa) is also known to synthesize natural rubber. Here, we report that an unusual cis-prenyltransferase-like 2 (CPTL2) that lacks the conserved motifs of conventional cis-prenyltransferase is required for natural rubber biosynthesis in lettuce. CPTL2, identified from the lettuce rubber particle proteome, displays homology to a human NogoB receptor and is predominantly expressed in latex. Multiple transgenic lettuces expressing CPTL2-RNAi constructs showed that a decrease of CPTL2 transcripts (3-15% CPTL2 expression relative to controls) coincided with the reduction of natural rubber as low as 5%. We also identified a conventional cis-prenyltransferase 3 (CPT3), exclusively expressed in latex. In subcellular localization studies using fluorescent proteins, cytosolic CPT3 was relocalized to endoplasmic reticulum by co-occurrence of CPTL2 in tobacco and yeast at the log phase. Furthermore, yeast two-hybrid data showed that CPTL2 and CPT3 interact. Yeast microsomes containing CPTL2/CPT3 showed enhanced synthesis of short cis-polyisoprenes, but natural rubber could not be synthesized in vitro. Intriguingly, a homologous pair CPTL1/CPT1, which displays ubiquitous expressions in lettuce, showed a potent dolichol biosynthetic activity in vitro. Taken together, our data suggest that CPTL2 is a scaffolding protein that tethers CPT3 on endoplasmic reticulum and is necessary for natural rubber biosynthesis in planta, but yeast-expressed CPTL2 and CPT3 alone could not synthesize high molecular weight natural rubber in vitro. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The Isolation of Novel Phage Display-Derived Human Recombinant Antibodies Against CCR5, the Major Co-Receptor of HIV

OpenAIRE

Shimoni, Moria; Herschhorn, Alon; Britan-Rosich, Yelena; Kotler, Moshe; Benhar, Itai; Hizi, Amnon

2013-01-01

Selecting for antibodies against specific cell-surface proteins is a difficult task due to many unrelated proteins that are expressed on the cell surface. Here, we describe a method to screen antibody-presenting phage libraries against native cell-surface proteins. We applied this method to isolate antibodies that selectively recognize CCR5, which is the major co-receptor for HIV entry (consequently, playing a pivotal role in HIV transmission and pathogenesis). We employed a phage screening s...

A panel of recombinant monoclonal antibodies against zebrafish neural receptors and secreted proteins suitable for wholemount immunostaining.

Science.gov (United States)

Staudt, Nicole; Müller-Sienerth, Nicole; Fane-Dremucheva, Alla; Yusaf, Shahnaz P; Millrine, David; Wright, Gavin J

2015-01-02

Cell surface receptors and secreted proteins play important roles in neural recognition processes, but because their site of action can be a long distance from neuron cell bodies, antibodies that label these proteins are valuable to understand their function. The zebrafish embryo is a popular vertebrate model for neurobiology, but suffers from a paucity of validated antibody reagents. Here, we use the entire ectodomain of neural zebrafish cell surface or secreted proteins expressed in mammalian cells to select monoclonal antibodies to ten different antigens. The antibodies were characterised by Western blotting and the sensitivity of their epitopes to formalin fixation was determined. The rearranged antigen binding regions of the antibodies were amplified and cloned which enabled expression in a recombinant form from a single plasmid. All ten antibodies gave specific staining patterns within formalin-treated embryonic zebrafish brains, demonstrating that this generalised approach is particularly efficient to elicit antibodies that stain native antigen in fixed wholemount tissue. Finally, we show that additional tags can be easily added to the recombinant antibodies for convenient multiplex staining. The antibodies and the approaches described here will help to address the lack of well-defined antibody reagents in zebrafish research. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Potent neutralization of VEGF biological activities with a fully human antibody Fab fragment directed against VEGF receptor 2

International Nuclear Information System (INIS)

Miao, H.-Q.; Hu, Kun; Jimenez, Xenia; Navarro, Elizabeth; Zhang, Haifan; Lu Dan; Ludwig, Dale L.; Balderes, Paul; Zhu Zhenping

2006-01-01

Compelling evidence suggest that vascular endothelial growth factor (VEGF) and its receptors, especially receptor 2 (VEGFR2, or kinase insert domain-containing receptor, KDR), play a critical role in angiogenesis under both physiological and pathological conditions, including cancer and angiogenic retinopathies such as age-related macular degeneration (AMD). To this end, inhibition of angiogenesis with antagonists to either VEGF or KDR has yielded significant therapeutic efficacy both in preclinical studies in animal models and in clinical trials in patients with cancer and AMD. We previously reported the identification of a high affinity, fully human anti-KDR antibody fragment, 1121B Fab, through a highly stringent affinity maturation process with a Fab originally isolated from a naive human antibody phage display library. In this study, we demonstrate that 1121B Fab is able to strongly block KDR/VEGF interaction, resulting in potent inhibition of an array of biological activities of VEGF, including activation of the receptor and its signaling pathway, intracellular calcium mobilization, and migration and proliferation of endothelial cells. Taken together, our data lend strong support to the further development of 1121B Fab fragment as an anti-angiogenesis agent in both cancer and angiogenic retinopathies

An MHC-restricted antibody-based chimeric antigen receptor requires TCR-like affinity to maintain antigen specificity

Directory of Open Access Journals (Sweden)

Marcela V Maus

2016-01-01

Full Text Available Chimeric antigen receptors (CARs are synthetic receptors that usually redirect T cells to surface antigens independent of human leukocyte antigen (HLA. Here, we investigated a T cell receptor-like CAR based on an antibody that recognizes HLA-A*0201 presenting a peptide epitope derived from the cancer-testis antigen NY-ESO-1. We hypothesized that this CAR would efficiently redirect transduced T cells in an HLA-restricted, antigen-specific manner. However, we found that despite the specificity of the soluble Fab, the same antibody in the form of a CAR caused moderate lysis of HLA-A2 expressing targets independent of antigen owing to T cell avidity. We hypothesized that lowering the affinity of the CAR for HLA-A2 would improve its specificity. We undertook a rational approach of mutating residues that, in the crystal structure, were predicted to stabilize binding to HLA-A2. We found that one mutation (DN lowered the affinity of the Fab to T cell receptor-range and restored the epitope specificity of the CAR. DN CAR T cells lysed native tumor targets in vitro, and, in a xenogeneic mouse model implanted with two human melanoma lines (A2+/NYESO+ and A2+/NYESO−, DN CAR T cells specifically migrated to, and delayed progression of, only the HLA-A2+/NY-ESO-1+ melanoma. Thus, although maintaining MHC-restricted antigen specificity required T cell receptor-like affinity that decreased potency, there is exciting potential for CARs to expand their repertoire to include a broad range of intracellular antigens.

Molecular and Therapeutic Characterization of Anti-ectodysplasin A Receptor (EDAR) Agonist Monoclonal Antibodies*

Science.gov (United States)

Kowalczyk, Christine; Dunkel, Nathalie; Willen, Laure; Casal, Margret L.; Mauldin, Elizabeth A.; Gaide, Olivier; Tardivel, Aubry; Badic, Giovanna; Etter, Anne-Lise; Favre, Manuel; Jefferson, Douglas M.; Headon, Denis J.; Demotz, Stéphane; Schneider, Pascal

2011-01-01

The TNF family ligand ectodysplasin A (EDA) and its receptor EDAR are required for proper development of skin appendages such as hair, teeth, and eccrine sweat glands. Loss of function mutations in the Eda gene cause X-linked hypohidrotic ectodermal dysplasia (XLHED), a condition that can be ameliorated in mice and dogs by timely administration of recombinant EDA. In this study, several agonist anti-EDAR monoclonal antibodies were generated that cross-react with the extracellular domains of human, dog, rat, mouse, and chicken EDAR. Their half-life in adult mice was about 11 days. They induced tail hair and sweat gland formation when administered to newborn EDA-deficient Tabby mice, with an EC50 of 0.1 to 0.7 mg/kg. Divalency was necessary and sufficient for this therapeutic activity. Only some antibodies were also agonists in an in vitro surrogate activity assay based on the activation of the apoptotic Fas pathway. Activity in this assay correlated with small dissociation constants. When administered in utero in mice or at birth in dogs, agonist antibodies reverted several ectodermal dysplasia features, including tooth morphology. These antibodies are therefore predicted to efficiently trigger EDAR signaling in many vertebrate species and will be particularly suited for long term treatments. PMID:21730053

Receptor-binding domain of SARS-CoV spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine

International Nuclear Information System (INIS)

He Yuxian; Zhou Yusen; Liu Shuwen; Kou Zhihua; Li Wenhui; Farzan, Michael; Jiang Shibo

2004-01-01

The spike (S) protein of severe acute respiratory syndrome (SARS) coronavirus (CoV), a type I transmembrane envelope glycoprotein, consists of S1 and S2 domains responsible for virus binding and fusion, respectively. The S1 contains a receptor-binding domain (RBD) that can specifically bind to angiotensin-converting enzyme 2 (ACE2), the receptor on target cells. Here we show that a recombinant fusion protein (designated RBD-Fc) containing 193-amino acid RBD (residues 318-510) and a human IgG1 Fc fragment can induce highly potent antibody responses in the immunized rabbits. The antibodies recognized RBD on S1 domain and completely inhibited SARS-CoV infection at a serum dilution of 1:10,240. Rabbit antisera effectively blocked binding of S1, which contains RBD, to ACE2. This suggests that RBD can induce highly potent neutralizing antibody responses and has potential to be developed as an effective and safe subunit vaccine for prevention of SARS

The clinical value of serum thyrotrophin receptor antibody level in patients with Graves ophthalmopathy

International Nuclear Information System (INIS)

Wang Chaodian; Shi Yuhong; Yan Bing

2013-01-01

Objective: To explore the value of serum thyrotrophin receptor antibody (TRAb) on the pathological mechanism of Graves ophthalmopathy. Methods: Two hundred and nineteen newly diagnosed Graves disease patients who were divided into Graves ophthalmopathy group (n=121) and without Graves ophthalmopathy group (n=98) were tested serum concentration with thyroid function, thyroperoxidase antibodies (TPOAb), thyroglobulin antibodies (TgAb) and TRAb. According to the consensus statement of the European Group on Graves ophthalmopathy, clinical activity score (CAS) and severity evaluation were carried out on Graves ophthalmopathy patients. Results: There was no significant difference in serum concentration of free thyroxine (FT 4 ), free triiodothyronine (FT 3 ), TPOAb and TRAb between the Graves ophthalmopathy group and the without Graves ophthalmopathy group. Serum concentration of TRAb was not correlated with the severity and CAS of Graves ophthalmopathy. Conclusions: The CAS and the severity of Graves ophthalmopathy were irrelevant to the serum concentration of TRAb. Therefore, the correlation between TRAb and Graves ophthalmopathy still needs further study. (authors)

Monoclonal antibody to the type I insulin-like growth factor (IGF-I) receptor blocks IGF-I receptor-mediated DNA synthesis: clarification of the mitogenic mechanisms of IGF-I and insulin in human skin fibroblasts

International Nuclear Information System (INIS)

Flier, J.S.; Usher, P.; Moses, A.C.

1986-01-01

Insulin and insulin-like growth factor type I (IGF-I) stimulate an overlapping spectrum of biological responses in human skin fibroblasts. Although insulin and IGF-I are known to stimulate the incorporation of [ 3 H]thymidine into DNA in these cells, the identify of the receptor(s) that mediates this effect has not been fully clarified. The mouse anti-human IGF-I receptor antibody αIR-3 binds with specificity to IGF-I but not to insulin receptors in human placental membranes; it also specifically inhibits the binding of 125 I-labeled IGF-I but not 125 I-labeled insulin to suspensions of human skin fibroblasts in a dose-dependent manner. αIR-3 competitively inhibits IGF-I-mediated stimulation of [ 3 H]thymidine incorporation into DNA. This inhibition is dependent on the concentration of αIR-3 and in the presence of a fixed antibody concentration can be partially overcome by high concentrations of IGF-I. In contrast, at concentrations of 3 H]thymidine incorporation is not inhibited by αIR-3. However, the incremental effects of higher concentrations (> 1 μg/ml) of insulin on [ 3 H]thymidine incorporation are inhibited by αIR-3. αIR-3 is a highly specific antagonist of IGF-I receptor-mediated mitogenesis in human skin fibroblasts. By using this antibody, it is shown directly that insulin can act through the IGF-I receptor to stimulate DNA synthesis but can also activate this effect through the insulin receptor itself

FcγRII-binding Centyrins mediate agonism and antibody-dependent cellular phagocytosis when fused to an anti-OX40 antibody.

Science.gov (United States)

Zhang, Di; Whitaker, Brian; Derebe, Mehabaw G; Chiu, Mark L

2018-04-01

Immunostimulatory antibodies against the tumor necrosis factor receptors (TNFR) are emerging as promising cancer immunotherapies. The agonism activity of such antibodies depends on crosslinking to Fc gamma RIIB receptor (FcγRIIB) to enable the antibody multimerization that drives TNFR activation. Previously, Fc engineering was used to enhance the binding of such antibodies to Fcγ receptors. Here, we report the identification of Centyrins as alternative scaffold proteins with binding affinities to homologous FcγRIIB and FcγRIIA, but not to other types of Fcγ receptors. One Centyrin, S29, was engineered at distinct positions of an anti-OX40 SF2 antibody to generate bispecific and tetravalent molecules named as mAbtyrins. Regardless of the position of S29 on the SF2 antibody, SF2-S29 mAbtyrins could bind FcγRIIB and FcγRIIA specifically while maintaining binding to OX40 receptors. In a NFκB reporter assay, attachment of S29 Centyrin molecules at the C-termini, but not the N-termini, resulted in SF2 antibodies with increased agonism owing to FcγRIIB crosslinking. The mAbtyrins also showed agonism in T-cell activation assays with immobilized FcγRIIB and FcγRIIA, but this activity was confined to mAbtyrins with S29 specifically at the C-termini of antibody heavy chains. Furthermore, regardless of the position of the molecule, S29 Centyrin could equip an otherwise Fc-silent antibody with antibody-dependent cellular phagocytosis activity without affecting the antibody's intrinsic antibody-dependent cell-meditated cytotoxicity and complement-dependent cytotoxicity. In summary, the appropriate adoption FcγRII-binding Centyrins as functional modules represents a novel strategy to engineer therapeutic antibodies with improved functionalities.

Evaluation of the specificity of antibodies raised against cannabinoid receptor type 2 in the mouse retina

DEFF Research Database (Denmark)

Cécyre, Bruno; Thomas, Sébastien; Ptito, Maurice

2014-01-01

Cannabinoid receptors (CB1R and CB2R) are among the most abundant G protein-coupled receptors in the central nervous system. The endocannabinoid system is an attractive therapeutic target for immune system modulation and peripheral pain management. While CB1R is distributed in the nervous system......, CB2R has traditionally been associated to the immune system. This dogma is currently a subject of debate since the discovery of CB2R expression in neurons using antibody-based methods. The localization of CB2R in the central nervous system (CNS) could have a significant impact on drug development...... because it would mean that in addition to its effects on the peripheral pain pathway, CB2R could also mediate some central effects of cannabinoids. In an attempt to clarify the debate over CB2R expression in the CNS, we tested several commercially or academically produced CB2R antibodies using Western...

Cetuximab in combination with anti-human IgG antibodies efficiently down-regulates the EGF receptor by macropinocytosis

International Nuclear Information System (INIS)

Berger, Christian; Madshus, Inger Helene; Stang, Espen

2012-01-01

The monoclonal antibody C225 (Cetuximab) blocks binding of ligand to the epidermal growth factor receptor (EGFR). In addition, it is known that incubation with C225 induces endocytosis of the EGFR. This endocytosis has previously been shown to be increased when C225 is combined with an additional monoclonal anti-EGFR antibody. However, the effects of antibody combinations on EGFR activation, endocytosis, trafficking and degradation have been unclear. By binding a secondary antibody to the C225-EGFR complex, we here demonstrate that a combination of antibodies can efficiently internalize and degrade the EGFR. Although the combination of antibodies activated the EGFR kinase and induced ubiquitination of the EGFR, the kinase activity was not required for internalization of the EGFR. In contrast to EGF-induced EGFR down-regulation, the antibody combination efficiently degraded the EGFR without initiating downstream proliferative signaling. The antibody-induced internalization of EGFR was found not to depend on clathrin and/or dynamin, but depended on actin polymerization, suggesting induction of macropinocytosis. Macropinocytosis may cause internalization of large membrane areas, and this could explain the highly efficient internalization of the EGFR induced by combination of antibodies. -- Highlight: ► Cetuximab induced endocytosis of EGFR increases upon combination with anti-human IgG. ► Antibody combination causes internalization of EGFR by macropinocytosis. ► Antibody-induced internalization of EGFR is independent of EGFR kinase activity. ► Antibody combination may have a zipper effect and cross-link EGFRs on neighboring cells.

A noncognate interaction with anti-receptor antibody-activated helper T cells induces small resting murine B cells to proliferate and to secrete antibody

DEFF Research Database (Denmark)

Owens, T

1988-01-01

on resting B cells (even in the presence of intact F23.1 antibody), but could induce antibody secretion by anti-Ig-preactivated B cells. Both F23.1+ clones (E9.D4 and 4.35F2) and one F23.1- clone (D2.2) could synergize with supernatants from activated E9.D4 T cells to induce B cell activation. F(ab')2......Culture of small resting allogeneic B cells (of an irrelevant haplotype) with two clones of T helper (Th) cells that were activated by the F23.1 anti-T cell receptor antibody led to the activation of B cells to proliferate and to secrete antibody. Th cell supernatants by themselves had no effect...... fragments of F23.1 induced E9.D4 to activate B cells as efficiently as intact F23.1 and B cell populations that had been incubated with F23.1 were not activated when cultured with E9.D4, although T cells recognized cell-presented F23.1 and were weakly activated. Reduction of the density of F23.1 adsorbed...

Ah receptor mediated suppression of the antibody response in mice is primarily dependent on the Ah phenotype of lymphoid tissue

International Nuclear Information System (INIS)

Silkworth, J.B.; Antrim, L.A.; Sack, G.

1986-01-01

Halogenated aromatic hydrocarbons act through the aromatic hydrocarbon (Ah) receptor in mice to produce a series of toxic effects of the immune system. The receptor protein is a product of the Ah gene locus. Ah responsive (Ahb/Ahb) mice express a high affinity receptor in both lymphoid and nonlymphoid tissues whereas nonresponsive Ahd/Ahd mice express a poor affinity receptor. To determine the role of the Ah receptor of lymphoid tissue relative to that of nonlymphoid tissue in the induction of immune impairment, bone marrow was used to reconstitute lethally irradiated mice of the same or opposite Ah phenotype. All mice were given 3,3',4,4'-tetrachlorobiphenyl (35 and 350 mumol/kg) ip 2 days before immunization with sheep erythrocytes (SRBC). The immune response to this T dependent antigen and organ weights were determined 5 or 7 days later in normal or chimeric mice, respectively. Monoclonal Lyt 1.1 and Lyt 1.2 antibodies were used to establish the origin of the cells which repopulated the chimeric thymuses. The immune responses of both BALB/cBy (Ahb/Ahb) and the BALB/cBy X DBA/2 hybrid, CByD2F1 (Ahb/Ahd), were significantly suppressed but DBA/2 mice were unaffected. The immune responses of chimeric BALB/cBy----BALB/cBy and BALB/cBy----DBA/2 (donor----recipient) mice were also significantly suppressed and thymic atrophy was observed in both cases. The serum anti-SRBC antibody titers of DBA/2----BALB/cBy chimeras were also significantly decreased although not to the same extent as in BALB/cBy----DBA/2 mice. Chimeric DBA/2----DBA/2 mice were not affected. These results indicate that the sensitivity to Ah receptor mediated suppression of the antibody response is primarily determined by the Ah phenotype of the lymphoid tissue

The PD-1/PD-L1 complex resembles the antigen-binding Fv domains of antibodies and T cell receptors

Energy Technology Data Exchange (ETDEWEB)

Lin, David Yin-wei; Tanaka, Yoshimasa; Iwasaki, Masashi; Gittis, Apostolos G.; Su, Hua-Poo; Mikami, Bunzo; Okazaki, Taku; Honjo, Tasuku; Minato, Nagahiro; Garboczi, David N. (NIH); (Kyoto)

2008-07-29

Signaling through the programmed death 1 (PD-1) inhibitory receptor upon binding its ligand, PD-L1, suppresses immune responses against autoantigens and tumors and plays an important role in the maintenance of peripheral immune tolerance. Release from PD-1 inhibitory signaling revives 'exhausted' virus-specific T cells in chronic viral infections. Here we present the crystal structure of murine PD-1 in complex with human PD-L1. PD-1 and PD-L1 interact through the conserved front and side of their Ig variable (IgV) domains, as do the IgV domains of antibodies and T cell receptors. This places the loops at the ends of the IgV domains on the same side of the PD-1/PD-L1 complex, forming a surface that is similar to the antigen-binding surface of antibodies and T cell receptors. Mapping conserved residues allowed the identification of residues that are important in forming the PD-1/PD-L1 interface. Based on the structure, we show that some reported loss-of-binding mutations involve the PD-1/PD-L1 interaction but that others compromise protein folding. The PD-1/PD-L1 interaction described here may be blocked by antibodies or by designed small-molecule drugs to lower inhibitory signaling that results in a stronger immune response. The immune receptor-like loops offer a new surface for further study and potentially the design of molecules that would affect PD-1/PD-L1 complex formation and thereby modulate the immune response.

Wormholes with fluid sources: A no-go theorem and new examples

Science.gov (United States)

Bronnikov, K. A.; Baleevskikh, K. A.; Skvortsova, M. V.

2017-12-01

For static, spherically symmetric space-times in general relativity (GR), a no-go theorem is proved: it excludes the existence of wormholes with flat and/or anti-de Sitter asymptotic regions on both sides of the throat if the source matter is isotropic, i.e., the radial and tangential pressures coincide. It explains why in all previous attempts to build such solutions it was necessary to introduce boundaries with thin shells that manifestly violate the isotropy of matter. Under a simple assumption on the behavior of the spherical radius r (x ), we obtain a number of examples of wormholes with isotropic matter and one or both de Sitter asymptotic regions, allowed by the no-go theorem. We also obtain twice asymptotically flat wormholes with anisotropic matter, both symmetric and asymmetric with respect to the throat, under the assumption that the scalar curvature is zero. These solutions may be on equal grounds interpreted as those of GR with a traceless stress-energy tensor and as vacuum solutions in a brane world. For such wormholes, the traversability conditions and gravitational lensing properties are briefly discussed. As a byproduct, we obtain twice asymptotically flat regular black hole solutions with up to four Killing horizons. As another byproduct, we point out intersection points in families of integral curves for the function A (x )=gt t, parametrized by its values on the throat.

Nodding syndrome in Tanzania may not be associated with circulating anti-NMDA-and anti-VGKC receptor antibodies or decreased pyridoxal phosphate serum levels-a pilot study.

Science.gov (United States)

Dietmann, Anelia; Wallner, Bernd; König, Rebekka; Friedrich, Katrin; Pfausler, Bettina; Deisenhammer, Florian; Griesmacher, Andrea; Seger, Christoph; Matuja, William; JilekAall, Louise; Winkler, Andrea S; Schmutzhard, Erich

2014-06-01

Nodding syndrome (NS) is a seemingly progressive epilepsy disorder of unknown underlying cause. We investigated association of pyridoxal-phosphate serum levels and occurrence of anti-neuronal antibodies against N-methyl-D-aspartate (NMDA) receptor and voltage gated potassium channel (VGKC) complex in NS patients. Sera of a Tanzanian cohort of epilepsy and NS patients and community controls were tested for the presence of anti-NMDA-receptor and anti-VGKC complex antibodies by indirect immunofluorescence assay. Furthermore pyridoxal-phosphate levels were measured. Auto-antibodies against NMDA receptor or VGKC (LG1 or Caspr2) complex were not detected in sera of patients suffering from NS (n=6), NS plus other seizure types (n=16), primary generalized epilepsy (n=1) and community controls without epilepsy (n=7). Median Pyridoxal-phosphate levels in patients with NS compared to patients with primary generalized seizures and community controls were not significantly different. However, these median pyridoxal-phosphate levels are significantly lower compared to the range considered normal in Europeans. In this pilot study NS was not associated with serum anti-NMDA receptor or anti-VGKC complex antibodies and no association to pyridoxal-phosphate serum levels was found.

Downregulation of transferrin receptor surface expression by intracellular antibody

International Nuclear Information System (INIS)

Peng Jilin; Wu Sha; Zhao Xiaoping; Wang Min; Li Wenhan; Shen Xin; Liu Jing; Lei Ping; Zhu Huifen; Shen Guanxin

2007-01-01

To deplete cellular iron uptake, and consequently inhibit the proliferation of tumor cells, we attempt to block surface expression of transferrin receptor (TfR) by intracellular antibody technology. We constructed two expression plasmids (scFv-HAK and scFv-HA) coding for intracellular single-chain antibody against TfR with or without endoplasmic reticulum (ER) retention signal, respectively. Then they were transfected tumor cells MCF-7 by liposome. Applying RT-PCR, Western blotting, immunofluorescence microscopy and immunoelectron microscope experiments, we insure that scFv-HAK intrabody was successfully expressed and retained in ER contrasted to the secreted expression of scFv-HA. Flow cytometric analysis confirmed that the TfR surface expression was markedly decreased approximately 83.4 ± 2.5% in scFv-HAK transfected cells, while there was not significantly decrease in scFv-HA transfected cells. Further cell growth and apoptosis characteristics were evaluated by cell cycle analysis, nuclei staining and MTT assay. Results indicated that expression of scFv-HAK can dramatically induce cell cycle G1 phase arrest and apoptosis of tumor cells, and consequently significantly suppress proliferation of tumor cells compared with other control groups. For First time this study demonstrates the potential usage of anti-TfR scFv-intrabody as a growth inhibitor of TfR overexpressing tumors

Development of therapeutic antibodies to G protein-coupled receptors and ion channels: Opportunities, challenges and their therapeutic potential in respiratory diseases.

Science.gov (United States)

Douthwaite, Julie A; Finch, Donna K; Mustelin, Tomas; Wilkinson, Trevor C I

2017-01-01

The development of recombinant antibody therapeutics continues to be a significant area of growth in the pharmaceutical industry with almost 50 approved monoclonal antibodies on the market in the US and Europe. Therapeutic drug targets such as soluble cytokines, growth factors and single transmembrane spanning receptors have been successfully targeted by recombinant monoclonal antibodies and the development of new product candidates continues. Despite this growth, however, certain classes of important disease targets have remained intractable to therapeutic antibodies due to the complexity of the target molecules. These complex target molecules include G protein-coupled receptors and ion channels which represent a large target class for therapeutic intervention with monoclonal antibodies. Although these targets have typically been addressed by small molecule approaches, the exquisite specificity of antibodies provides a significant opportunity to provide selective modulation of these important regulators of cell function. Given this opportunity, a significant effort has been applied to address the challenges of targeting these complex molecules and a number of targets are linked to the pathophysiology of respiratory diseases. In this review, we provide a summary of the importance of GPCRs and ion channels involved in respiratory disease and discuss advantages offered by antibodies as therapeutics at these targets. We highlight some recent GPCRs and ion channels linked to respiratory disease mechanisms and describe in detail recent progress made in the strategies for discovery of functional antibodies against challenging membrane protein targets such as GPCRs and ion channels. Copyright © 2016 Elsevier Inc. All rights reserved.

Cetuximab in combination with anti-human IgG antibodies efficiently down-regulates the EGF receptor by macropinocytosis

Energy Technology Data Exchange (ETDEWEB)

Berger, Christian [Department of Pathology, Oslo University Hospital, Rikshospitalet, Post box 4950 Nydalen, 0424 Oslo (Norway); Madshus, Inger Helene [Institute of Pathology, University of Oslo, Rikshospitalet, 0027 Oslo (Norway); Department of Pathology, Oslo University Hospital, Rikshospitalet, Post box 4950 Nydalen, 0424 Oslo (Norway); Stang, Espen, E-mail: espsta@rr-research.no [Department of Pathology, Oslo University Hospital, Rikshospitalet, Post box 4950 Nydalen, 0424 Oslo (Norway)

2012-12-10

The monoclonal antibody C225 (Cetuximab) blocks binding of ligand to the epidermal growth factor receptor (EGFR). In addition, it is known that incubation with C225 induces endocytosis of the EGFR. This endocytosis has previously been shown to be increased when C225 is combined with an additional monoclonal anti-EGFR antibody. However, the effects of antibody combinations on EGFR activation, endocytosis, trafficking and degradation have been unclear. By binding a secondary antibody to the C225-EGFR complex, we here demonstrate that a combination of antibodies can efficiently internalize and degrade the EGFR. Although the combination of antibodies activated the EGFR kinase and induced ubiquitination of the EGFR, the kinase activity was not required for internalization of the EGFR. In contrast to EGF-induced EGFR down-regulation, the antibody combination efficiently degraded the EGFR without initiating downstream proliferative signaling. The antibody-induced internalization of EGFR was found not to depend on clathrin and/or dynamin, but depended on actin polymerization, suggesting induction of macropinocytosis. Macropinocytosis may cause internalization of large membrane areas, and this could explain the highly efficient internalization of the EGFR induced by combination of antibodies. -- Highlight: Black-Right-Pointing-Pointer Cetuximab induced endocytosis of EGFR increases upon combination with anti-human IgG. Black-Right-Pointing-Pointer Antibody combination causes internalization of EGFR by macropinocytosis. Black-Right-Pointing-Pointer Antibody-induced internalization of EGFR is independent of EGFR kinase activity. Black-Right-Pointing-Pointer Antibody combination may have a zipper effect and cross-link EGFRs on neighboring cells.

Fcγ-receptor IIa-mediated Src Signaling Pathway Is Essential for the Antibody-Dependent Enhancement of Ebola Virus Infection.

Directory of Open Access Journals (Sweden)

Wakako Furuyama

2016-12-01

Full Text Available Antibody-dependent enhancement (ADE of Ebola virus (EBOV infection has been demonstrated in vitro, raising concerns about the detrimental potential of some anti-EBOV antibodies. ADE has been described for many viruses and mostly depends on the cross-linking of virus-antibody complexes to cell surface Fc receptors, leading to enhanced infection. However, little is known about the molecular mechanisms underlying this phenomenon. Here we show that Fcγ-receptor IIa (FcγRIIa-mediated intracellular signaling through Src family protein tyrosine kinases (PTKs is required for ADE of EBOV infection. We found that deletion of the FcγRIIa cytoplasmic tail abolished EBOV ADE due to decreased virus uptake into cellular endosomes. Furthermore, EBOV ADE, but not non-ADE infection, was significantly reduced by inhibition of the Src family protein PTK pathway, which was also found to be important to promote phagocytosis/macropinocytosis for viral uptake into endosomes. We further confirmed a significant increase of the Src phosphorylation mediated by ADE. These data suggest that antibody-EBOV complexes bound to the cell surface FcγRIIa activate the Src signaling pathway that leads to enhanced viral entry into cells, providing a novel perspective for the general understanding of ADE of virus infection.

T cell receptor-like recognition of tumor in vivo by synthetic antibody fragment.

Directory of Open Access Journals (Sweden)

Keith R Miller

Full Text Available A major difficulty in treating cancer is the inability to differentiate between normal and tumor cells. The immune system differentiates tumor from normal cells by T cell receptor (TCR binding of tumor-associated peptides bound to Major Histocompatibility Complex (pMHC molecules. The peptides, derived from the tumor-specific proteins, are presented by MHC proteins, which then serve as cancer markers. The TCR is a difficult protein to use as a recombinant protein because of production issues and has poor affinity for pMHC; therefore, it is not a good choice for use as a tumor identifier outside of the immune system. We constructed a synthetic antibody-fragment (Fab library in the phage-display format and isolated antibody-fragments that bind pMHC with high affinity and specificity. One Fab, fE75, recognizes our model cancer marker, the Human Epidermal growth factor Receptor 2 (HER2/neu peptide, E75, bound to the MHC called Human Leukocyte Antigen-A2 (HLA-A2, with nanomolar affinity. The fE75 bound selectively to E75/HLA-A2 positive cancer cell lines in vitro. The fE75 Fab conjugated with (64Cu selectively accumulated in E75/HLA-A2 positive tumors and not in E75/HLA-A2 negative tumors in an HLA-A2 transgenic mouse as probed using positron emission tomography/computed tomography (PET/CT imaging. Considering that hundreds to thousands of different peptides bound to HLA-A2 are present on the surface of each cell, the fact that fE75 arrives at the tumor at all shows extraordinary specificity. These antibody fragments have great potential for diagnosis and targeted drug delivery in cancer.

Consistent deformations of dual formulations of linearized gravity: A no-go result

International Nuclear Information System (INIS)

Bekaert, Xavier; Boulanger, Nicolas; Henneaux, Marc

2003-01-01

The consistent, local, smooth deformations of the dual formulation of linearized gravity involving a tensor field in the exotic representation of the Lorentz group with Young symmetry type (D-3,1) (one column of length D-3 and one column of length 1) are systematically investigated. The rigidity of the Abelian gauge algebra is first established. We next prove a no-go theorem for interactions involving at most two derivatives of the fields

Valence of acetylcholine-receptor-antibody-titers in myasthenia gravis

International Nuclear Information System (INIS)

Zeitlhofer, J.; Maida, E.M.; Mamoli, B.; Mayr, N.

1986-01-01

In a retrospective study in 47 patients with myasthenia gravis acetylcholine-receptor-antibody-titers (AChR-AB) were correlated with the severity of the disease. In 18 patients the course of titers was studied and two groups of patients could be differentiated: patients with relative constant and patients with fluctuating titers. Age, age of begin of myasthenia and sex did not influence the titers. Also the duration of the disease and the severity of symptoms did not influence the level of AChR-AB-titers. In this retrospective study the influence of immunsuppressive therapy on the intra-individual course of AB-titers and their correlation with the clinical symptoms could not be judged. Measurement of AChR-AB is of value for the diagnosis of myasthenia gravis and important for judging the clinical course and the effect of therapy. (Author)

Valuation of Go Stimuli or Devaluation of No-Go Stimuli? Evidence of an Increased Preference for Attended Go Stimuli Following a Go/No-Go Task.

Science.gov (United States)

Inoue, Kazuya; Sato, Nobuya

2017-01-01

Attentional inhibition that occurs during discrimination tasks leads to the negative evaluation of distractor stimuli. This phenomenon, known as the distractor devaluation effect also occurs when go/no-go tasks require response inhibition. However, it remains unclear whether there are interactions between attention and response controls when the distractor devaluation effect occurs. The aims of this study were to investigate whether attention to stimuli in the go/no-go task plays a facilitative role in distractor devaluation through response inhibition, and to clarify whether this effect reflects a decreased preference for no-go stimuli. Participants evaluated the preference for pictures before and after a go/no-go task. In Experiments 1 and 2, they made a go or no-go response depending on the category of pictures displayed (gummy candies or rice crackers), whereas in Experiment 3 they did on the basis digit category, even or odd numbers, superimposed on such pictures. Experiments 1 and 2 demonstrated that the pictures presented as no-go stimuli in the preceding go/no-go task were evaluated as less positive than the pictures presented as go stimuli. This devaluation effect reflected an increased preference for the go stimuli but not a decreased preference for the no-go stimuli. Experiment 3 indicated that response inhibition did not affect the preference for the pictures that had not received attention in a preceding go/no-go task. These results suggest that although attention plays an important role in differential ratings for go and no-go stimuli, such differences, in fact, reflect the valuation of go stimuli.

Fatigue and Use of Go/Nogo Pills in F-16 Pilots Subjected to Extraordinarily Long Combat Sorties

National Research Council Canada - National Science Library

Schultz, Darlene

2004-01-01

We lack knowledge of real world pilot fatigue and use of GO/NOGO pills. To analyze this, a forward-deployed flight surgeon studied fighter pilots subjected to combat sorties often longer than 8 hours in duration. Methods...

Engineering chimeric human and mouse major histocompatibility complex (MHC) class I tetramers for the production of T-cell receptor (TCR) mimic antibodies

Science.gov (United States)

Bentley, Carol; Yates, Jenna; Salimi, Maryam; Greig, Jenny; Wiblin, Sarah; Hassanali, Tasneem; Banham, Alison H.

2017-01-01

Therapeutic monoclonal antibodies targeting cell surface or secreted antigens are among the most effective classes of novel immunotherapies. However, the majority of human proteins and established cancer biomarkers are intracellular. Peptides derived from these intracellular proteins are presented on the cell surface by major histocompatibility complex class I (MHC-I) and can be targeted by a novel class of T-cell receptor mimic (TCRm) antibodies that recognise similar epitopes to T-cell receptors. Humoural immune responses to MHC-I tetramers rarely generate TCRm antibodies and many antibodies recognise the α3 domain of MHC-I and β2 microglobulin (β2m) that are not directly involved in presenting the target peptide. Here we describe the production of functional chimeric human-murine HLA-A2-H2Dd tetramers and modifications that increase their bacterial expression and refolding efficiency. These chimeric tetramers were successfully used to generate TCRm antibodies against two epitopes derived from wild type tumour suppressor p53 (RMPEAAPPV and GLAPPQHLIRV) that have been used in vaccination studies. Immunisation with chimeric tetramers yielded no antibodies recognising the human α3 domain and β2m and generated TCRm antibodies capable of specifically recognising the target peptide/MHC-I complex in fully human tetramers and on the cell surface of peptide pulsed T2 cells. Chimeric tetramers represent novel immunogens for TCRm antibody production and may also improve the yield of tetramers for groups using these reagents to monitor CD8 T-cell immune responses in HLA-A2 transgenic mouse models of immunotherapy. PMID:28448627

TITERS OF ANTIBODIES TO Β1-ADRENOCEPTOR AND M2 CHOLINERGIC RECEPTORS IN PATIENTS WITH VENTRICULAR ARRHYTHMIAS WITHOUT AN ORGANIC CARDIOVASCULAR DISEASE AND THEIR POSSIBLE CLINICAL SIGNIFICANCE

Directory of Open Access Journals (Sweden)

M. M. Rogova

2012-01-01

Full Text Available Aim. To identify the most promising epitopes that simulate various sites β1-adrenergic and M2-cholinergic receptors, and to evaluate their possible contribution to the development and maintenance of cardiac arrhythmias, particularly idiopathic ventricular arrhythmia. Material and methods. Patients with ventricular arrhythmias without organic cardiovascular disease (the study group; n=70 were included in the study. The control group consisted of 20 healthy volunteers. Evaluation of levels of antibodies to antigenic determinants, modeling various sites β1-adrenergic and M2-cholinergic performed in all patients. Causal treatment with clarithromycin and valacyclovir performed in part of patients. Results. Antibodies to different peptide sequences of β1-adrenergic and M2-cholinergic receptors have been identified in 25% of main group patients. A direct correlation between the frequency of episodes of ventricular tachycardia and IgG levels to MRI-MRIV (p=0.02 revealed. Increase in titre of antibodies to β1-adrenoceptors, to a peptide sequence β8 (p=0.02, and lower titers of antibodies to the M2 acetylcholine receptor — chimera MRI-MRIV IgM (p=0.06 and ARI-MRIV IgM (p=0.07 were observed when assessing the efficacy of the therapy in the causal dynamics in the group of "untreated" patients. IgG titer reduction of ARI-MRIV (p=0.02, which is 4 times out of 10 with reduction of ventricular ectopic activity , recorded after valacyclovir therapy. Clarithromycin therapy on the level of antibodies exerted no significant effect. Conclusion. Possible involvement of antibodies to β1-adrenoceptor and M2-cholinergic receptors in the development of idiopathic ventricular arrhythmias demonstrated. The relationship between the frequency of episodes of ventricular tachycardia and levels of antibody titers to M2-cholinergic receptors found. Attempt of causal treatment, depending on the possible mechanisms of the autoimmune process is executed. Further studies to

Modulation of the virus-receptor interaction by mutations in the V5 loop of feline immunodeficiency virus (FIV following in vivo escape from neutralising antibody

Directory of Open Access Journals (Sweden)

Samman Ayman

2010-04-01

Full Text Available Abstract Background In the acute phase of infection with feline immunodeficiency virus (FIV, the virus targets activated CD4+ T cells by utilising CD134 (OX40 as a primary attachment receptor and CXCR4 as a co-receptor. The nature of the virus-receptor interaction varies between isolates; strains such as GL8 and CPGammer recognise a "complex" determinant on CD134 formed by cysteine-rich domains (CRDs 1 and 2 of the molecule while strains such as PPR and B2542 require a more "simple" determinant comprising CRD1 only for infection. These differences in receptor recognition manifest as variations in sensitivity to receptor antagonists. In this study, we ask whether the nature of the virus-receptor interaction evolves in vivo. Results Following infection with a homogeneous viral population derived from a pathogenic molecular clone, a quasispecies emerged comprising variants with distinct sensitivities to neutralising antibody and displaying evidence of conversion from a "complex" to a "simple" interaction with CD134. Escape from neutralising antibody was mediated primarily by length and sequence polymorphisms in the V5 region of Env, and these alterations in V5 modulated the virus-receptor interaction as indicated by altered sensitivities to antagonism by both anti-CD134 antibody and soluble CD134. Conclusions The FIV-receptor interaction evolves under the selective pressure of the host humoral immune response, and the V5 loop contributes to the virus-receptor interaction. Our data are consistent with a model whereby viruses with distinct biological properties are present in early versus late infection and with a shift from a "complex" to a "simple" interaction with CD134 with time post-infection.

Modulation of the virus-receptor interaction by mutations in the V5 loop of feline immunodeficiency virus (FIV) following in vivo escape from neutralising antibody.

Science.gov (United States)

Willett, Brian J; Kraase, Martin; Logan, Nicola; McMonagle, Elizabeth L; Samman, Ayman; Hosie, Margaret J

2010-04-26

In the acute phase of infection with feline immunodeficiency virus (FIV), the virus targets activated CD4+ T cells by utilising CD134 (OX40) as a primary attachment receptor and CXCR4 as a co-receptor. The nature of the virus-receptor interaction varies between isolates; strains such as GL8 and CPGammer recognise a "complex" determinant on CD134 formed by cysteine-rich domains (CRDs) 1 and 2 of the molecule while strains such as PPR and B2542 require a more "simple" determinant comprising CRD1 only for infection. These differences in receptor recognition manifest as variations in sensitivity to receptor antagonists. In this study, we ask whether the nature of the virus-receptor interaction evolves in vivo. Following infection with a homogeneous viral population derived from a pathogenic molecular clone, a quasispecies emerged comprising variants with distinct sensitivities to neutralising antibody and displaying evidence of conversion from a "complex" to a "simple" interaction with CD134. Escape from neutralising antibody was mediated primarily by length and sequence polymorphisms in the V5 region of Env, and these alterations in V5 modulated the virus-receptor interaction as indicated by altered sensitivities to antagonism by both anti-CD134 antibody and soluble CD134. The FIV-receptor interaction evolves under the selective pressure of the host humoral immune response, and the V5 loop contributes to the virus-receptor interaction. Our data are consistent with a model whereby viruses with distinct biological properties are present in early versus late infection and with a shift from a "complex" to a "simple" interaction with CD134 with time post-infection.

Did hypocretin receptor 2 autoantibodies cause narcolepsy with hypocretin deficiency in Pandemrix-vaccinated children? Comment on “Antibodies to influenza nucleoprotein cross-react with human hypocretin receptor 2”

OpenAIRE

Vassalli Anne

2015-01-01

Abstract Did hypocretin receptor 2 auto antibodies cause narcolepsy with hypocretin deficiency in Pandemrix vaccinated children as suggested by Ahmed et al.? Using newly developed mouse models to report and inactivate hypocretin receptor expression Vassalli et al. now show that hypocretin neurons (whose loss causes narcolepsy) do not express hypocretin autoreceptors raising questions to the interpretation of Ahmed et al.’s findings. Mouse Genome Informatics: www.informatics.jax.org/reference/...

Investigating the Interaction between the Neonatal Fc Receptor and Monoclonal Antibody Variants by Hydrogen/Deuterium Exchange Mass Spectrometry

DEFF Research Database (Denmark)

Jensen, Pernille Foged; Larraillet, Vincent; Schlothauer, Tilman

2015-01-01

The recycling of immunoglobulins by the neonatal Fc receptor (FcRn) is of crucial importance in the maintenance of antibody levels in plasma and is responsible for the long half-lives of endogenous and recombinant monoclonal antibodies. From a therapeutic point of view there is great interest...... in understanding and modulating the IgG-FcRn interaction to optimize antibody pharmacokinetics and ultimately improve efficacy and safety. Here we studied the interaction between a full-length human IgG1 and human FcRn via hydrogen/deuterium exchange mass spectrometry and targeted electron transfer dissociation...... to map sites perturbed by binding on both partners of the IgG-FcRn complex. Several regions in the antibody Fc region and the FcRn were protected from exchange upon complex formation, in good agreement with previous crystallographic studies of FcRn in complex with the Fc fragment. Interestingly, we found...

CARbodies: Human Antibodies Against Cell Surface Tumor Antigens Selected From Repertoires Displayed on T Cell Chimeric Antigen Receptors

Directory of Open Access Journals (Sweden)

Vanesa Alonso-Camino

2013-01-01

Full Text Available A human single-chain variable fragment (scFv antibody library was expressed on the surface of human T cells after transduction with lentiviral vectors (LVs. The repertoire was fused to a first-generation T cell receptor ζ (TCRζ-based chimeric antigen receptor (CAR. We used this library to isolate antibodies termed CARbodies that recognize antigens expressed on the tumor cell surface in a proof-of-principle system. After three rounds of activation-selection there was a clear repertoire restriction, with the emergence dominant clones. The CARbodies were purified from bacterial cultures as soluble and active proteins. Furthermore, to validate its potential application for adoptive cell therapy, human T cells were transduced with a LV encoding a second-generation costimulatory CAR (CARv2 bearing the selected CARbodies. Transduced human primary T cells expressed significant levels of the CARbodies-based CARv2 fusion protein on the cell surface, and importantly could be specifically activated, after stimulation with tumor cells. This approach is a promising tool for the generation of antibodies fully adapted to the display format (CAR and the selection context (cell synapse, which could extend the scope of current adoptive cell therapy strategies with CAR-redirected T cells.

No-go theorems for R symmetries in four-dimensional GUTs

CERN Document Server

Fallbacher, Maximilian; Vaudrevange, Patrick K S

2011-01-01

We prove that it is impossible to construct a grand unified model, based on a simple gauge group, in four dimensions that leads to the exact MSSM, nor to a singlet extension, and possesses an unbroken R symmetry. This implies that no MSSM model with either a Z_{M>=3}^R or U(1)_R symmetry can be completed by a four-dimensional GUT in the ultraviolet. However, our no-go theorem does not apply to GUT models with extra dimensions. We also show that it is impossible to construct a 4D GUT that leads to the MSSM plus an additional anomaly-free symmetry that forbids the mu term.

Inhibition of fibroblast growth factor receptor 3-dependent lung adenocarcinoma with a human monoclonal antibody

Directory of Open Access Journals (Sweden)

Yongjun Yin

2016-05-01

Full Text Available Activating mutations in fibroblast growth factor receptor 3 (FGFR3 have been identified in multiple types of human cancer and in congenital birth defects. In human lung cancer, fibroblast growth factor 9 (FGF9, a high-affinity ligand for FGFR3, is overexpressed in 10% of primary resected non-small cell lung cancer (NSCLC specimens. Furthermore, in a mouse model where FGF9 can be induced in lung epithelial cells, epithelial proliferation and ensuing tumorigenesis is dependent on FGFR3. To develop new customized therapies for cancers that are dependent on FGFR3 activation, we have used this mouse model to evaluate a human monoclonal antibody (D11 with specificity for the extracellular ligand-binding domain of FGFR3, that recognizes both human and mouse forms of the receptor. Here, we show that D11 effectively inhibits signaling through FGFR3 in vitro, inhibits the growth of FGFR3-dependent FGF9-induced lung adenocarcinoma in mice, and reduces tumor-associated morbidity. Given the potency of FGF9 in this mouse model and the absolute requirement for signaling through FGFR3, this study validates the D11 antibody as a potentially useful and effective reagent for treating human cancers or other pathologies that are dependent on activation of FGFR3.

Paired immunoglobulin-like receptor B knockout does not enhance axonal regeneration or locomotor recovery after spinal cord injury.

Science.gov (United States)

Nakamura, Yuka; Fujita, Yuki; Ueno, Masaki; Takai, Toshiyuki; Yamashita, Toshihide

2011-01-21

Myelin components that inhibit axonal regeneration are believed to contribute significantly to the lack of axonal regeneration noted in the adult central nervous system. Three proteins found in myelin, Nogo, myelin-associated glycoprotein, and oligodendrocyte-myelin glycoprotein, inhibit neurite outgrowth in vitro. All of these proteins interact with the same receptors, namely, the Nogo receptor (NgR) and paired immunoglobulin-like receptor B (PIR-B). As per previous reports, corticospinal tract (CST) regeneration is not enhanced in NgR-knock-out mice after spinal cord injury. Therefore, we assessed CST regeneration in PIR-B-knock-out mice. We found that hindlimb motor function, as assessed using the Basso mouse scale, footprint test, inclined plane test, and beam walking test, did not differ between the PIR-B-knock-out and wild-type mice after dorsal hemisection of the spinal cord. Further, tracing of the CST fibers after injury did not reveal enhanced axonal regeneration or sprouting in the CST of the PIR-B-knock-out mice. Systemic administration of NEP1-40, a NgR antagonist, to PIR-B knock-out mice did not enhance the regenerative response. These results indicate that PIR-B knock-out is not sufficient to induce extensive axonal regeneration after spinal cord injury.

Antibodies to watch in 2014.

Science.gov (United States)

Reichert, Janice M

2014-01-01

Since 2010, mAbs has documented the biopharmaceutical industry's progress in transitioning antibody therapeutics to first Phase 3 clinical studies and regulatory review, and its success at gaining first marketing approvals for antibody-based products. This installment of the "Antibodies to watch" series outlines events anticipated to occur between December 2013 and the end of 2014, including first regulatory actions on marketing applications for vedolizumab, siltuximab, and ramucirumab, as well as the Fc fusion proteins Factor IX-Fc and Factor VIII-Fc; and the submission of first marketing applications for up to five therapeutics (secukinumab, ch14.18, onartuzumab, necitumumab, gevokizumab). Antibody therapeutics in Phase 3 studies are described, with an emphasis on those with study completion dates in 2014, including antibodies targeting interleukin-17a or the interleukin-17a receptor (secukinumab, ixekizumab, brodalumab), proprotein convertase subtilisin/kexin type 9 (alirocumab, evolocumab, bococizumab), and programmed death 1 receptor (lambrolizumab, nivolumab). Five antibodies with US Food and Drug Administration's Breakthrough Therapy designation (obinutuzumab, ofatumumab, lambrolizumab, bimagrumab, daratumumab) are also discussed.

Clinical significance of detection of antibodies to fetal and adult acetylcholine receptors in myasthenia gravis

Institute of Scientific and Technical Information of China (English)

Qi-Guang Shi; Zhi-Hong Wang; Xiao-Wei Ma; Da-Qi Zhang; Chun-Sheng Yang; Fu-Dong Shi; Li Yang

2012-01-01

Objective To evaluate the frequency,distribution and clinical significance of the antibodies to the fetal and/or adult acetylcholine receptor (AChR) in patients with myasthenia gravis (MG).Methods AChR antibodies were detected by cell-based assay in the serum of ocular MG (OMG) (n =90) and generalized MG (GMG) patients (n =110).The fetaltype (2α∶ β∶ γ∶ δ) and adult-type (2α∶ β∶ ε∶ δ) AChR were used as antigens,and their relevance to disease presentation was assessed.Results The overall frequencies of anti-adult and anti-fetal AChR antibodies were similar in all 200 patients examined,with 14 having serum specific to the AChR-γ subunit,and 22 to the AChR-ε subunit.The overall sensitivity when using the fetal and adult AChR antibodies was higher than that when using the fetal AChR antibody only (P =0.015).Compared with OMG patients,the mean age at disease onset and the positive ratio of antibodies to both isoforms of the AChR were significantly higher in patients who subsequently progressed to GMG.Older patients and patients with both anti-fetal and anti-adult AChR antibodies had a greater risk for developing generalized disease [odds ratio (OR),1.03;95％ confidence interval (CI),1.01-1.06 and OR,5.09;95％ CI,2.23-11.62].Conclusion Using both fetal-and adulttype AChRs as the antigens may be more sensitive than using either subtype.Patients with serum specific to both isoforms are at a greater risk of progressing to GMG.Patients with disease onset at an advanced age appear to have a higher frequency of GMG conversion.

Isolation of a human anti-epidermal growth factor receptor Fab antibody, EG-19-11, with subnanomolar affinity from naïve immunoglobulin repertoires using a hierarchical antibody library system.

Science.gov (United States)

Hur, Byung-ung; Yoon, Jae-bong; Liu, Li-Kun; Cha, Sang-hoon

2010-11-30

Specific antibodies that possess a subnanomolar affinity are very difficult to obtain from human naïve immunoglobulin repertoires without the use of lengthy affinity optimization procedures. Here, we designed a hierarchical phage-displayed antibody library system to generate an enormous diversity of combinatorial Fab fragments (6×10(17)) and attempted to isolate high-affinity Fabs against the human epidermal growth factor receptor (EGFR). A primary antibody library, designated HuDVFab-8L, comprising 4.5×10(9) human naïve heavy chains and eight unspecified human naïve light chains was selected against the EGFR-Fc protein by biopanning, and four anti-EGFR Fab clones were isolated. Because one of the Fab clones, denoted EG-L2-11, recognized a native EGFR expressed on A431 cells, the heavy chain of the Fab was shuffled with a human naïve light chain repertoire with a diversity of 1.4×10(8) and selected a second time against the EGFR-Fc protein again. One EG-L2-11 variant, denoted EG-19-11, recognized an EGFR epitope that was almost the same as that bound by cetuximab and had a K(D) of approximately 540 pM for soluble EGFR, which is about 7-fold higher than that of the FabC225 derived from cetuximab. This variant was also internalized by A431 cells, likely via receptor-mediated endocytosis, and it efficiently inhibited EGF-mediated tyrosine phosphorylation of the EGFR. These results demonstrate that the use of our hierarchical antibody library system is advantageous in generating fully human antibodies especially with a therapeutic purpose. Copyright © 2010 Elsevier B.V. All rights reserved.

A generalized quantitative antibody homeostasis model: maintenance of global antibody equilibrium by effector functions.

Science.gov (United States)

Prechl, József

2017-11-01

The homeostasis of antibodies can be characterized as a balanced production, target-binding and receptor-mediated elimination regulated by an interaction network, which controls B-cell development and selection. Recently, we proposed a quantitative model to describe how the concentration and affinity of interacting partners generates a network. Here we argue that this physical, quantitative approach can be extended for the interpretation of effector functions of antibodies. We define global antibody equilibrium as the zone of molar equivalence of free antibody, free antigen and immune complex concentrations and of dissociation constant of apparent affinity: [Ab]=[Ag]=[AbAg]= K D . This zone corresponds to the biologically relevant K D range of reversible interactions. We show that thermodynamic and kinetic properties of antibody-antigen interactions correlate with immunological functions. The formation of stable, long-lived immune complexes correspond to a decrease of entropy and is a prerequisite for the generation of higher-order complexes. As the energy of formation of complexes increases, we observe a gradual shift from silent clearance to inflammatory reactions. These rules can also be applied to complement activation-related immune effector processes, linking the physicochemical principles of innate and adaptive humoral responses. Affinity of the receptors mediating effector functions shows a wide range of affinities, allowing the continuous sampling of antibody-bound antigen over the complete range of concentrations. The generation of multivalent, multicomponent complexes triggers effector functions by crosslinking these receptors on effector cells with increasing enzymatic degradation potential. Thus, antibody homeostasis is a thermodynamic system with complex network properties, nested into the host organism by proper immunoregulatory and effector pathways. Maintenance of global antibody equilibrium is achieved by innate qualitative signals modulating a

Epitope mapping of the alpha-chain of the insulin-like growth factor I receptor using antipeptide antibodies.

Science.gov (United States)

Delafontaine, P; Ku, L; Ververis, J J; Cohen, C; Runge, M S; Alexander, R W

1994-12-01

Insulin-like growth factor I (IGF I) is an important mitogen for vascular smooth muscle cells (VSMC). The IGF I receptor (IGF IR) is a heterotetramer composed of two cross-linked extracellular alpha-chains and two membrane-spanning beta-chains that contain a tyrosine-kinase domain. It has a high degree of sequence similarity to the insulin receptor (IR), and the putative ligand-specific binding site has been localized to a cysteine-rich region (CRR) of the alpha-chain. To obtain insights into antigenic determinants of the IGF IR, we raised a panel of site-specific polyclonal antibodies against short peptide sequences N-terminal to and within the CRR. Several antibodies raised against linear epitopes within the CRR bound to solubilized and native rat and human IGF IR by ELISA, did not cross-react with IR, but unexpectedly failed to inhibit 125I-IGF I binding. A polyclonal antibody directed against a 48-amino acid synthetic peptide, corresponding to a region of the CRR postulated to be essential for ligand binding, failed to react with either solubilized, reduced or intact IGF IR. Three antibodies specific for the N-terminus of the alpha-chain reacted with solubilized and native IGF IR. One of these, RAB 6, directed against amino acids 38-44 of the IGF IR, inhibited 125I-IGF I binding to rat aortic smooth muscle cells (RASM) and to IGF IR/3T3 cells (overexpressing human IGF IR) by up to 45%. Immunohistochemical analysis revealed strong IGF IR staining in the medial smooth muscle cell layer of rat aorta. These findings are consistent with a model wherein conformational epitopes within the CRR and linear epitopes within the N-terminus of the alpha-chain contribute to the IGF I binding pocket. These antibodies should provide a valuable tool to study structure-function relationships and in vivo regulation of the IGF IR.

A monoclonal antibody against PDGF B-chain inhibits PDGF-induced DNA synthesis in C3H fibroblasts and prevents binding of PDGF to its receptor.

Science.gov (United States)

Vassbotn, F S; Langeland, N; Hagen, I; Holmsen, H

1990-09-01

A monoclonal antibody (MAb 6D11) against platelet-derived growth factor (PDGF) was studied. We found that the MAb 6D11 in concentrations equimolar to PDGF blocked the [3H]thymidine incorporation in C3H/10T1/2 C18 fibroblasts stimulated by PDGF B-B and PDGF A-B. This inhibition was overcome by high doses of PDGF. The [3H]thymidine incorporation stimulated by other growth factors (aFGF, bFGF and bombesin) was not inhibited by the antibody. The MAb 6D11 blocked receptor binding of PDGF B-B, but not PDGF A-A. These findings suggest that the MAb 6D11 abolishes PDGF-induced DNA synthesis by blocking PDGF receptor binding. In this communication we demonstrate an isoform-specific monoclonal antibody against PDGF.

Identification of endogenous opioid receptor components in rat brain using a monoclonal antibody

Energy Technology Data Exchange (ETDEWEB)

Bero, L.A.; Roy, S.; Lee, N.M.

1988-11-01

A monoclonal antibody generated against the tertiary structure of a partially purified opioid binding protein was used to probe the structure of the dynorphin and beta-endorphin receptors. The Fab fragment 3B4F11 inhibited completely the binding of 125I-beta-endorphin and (3H)dynorphin to rat brain P2 membranes with IC50 values of 26 ng/ml and 40 ng/ml, respectively. To explore further the interaction of 3B4F11 with the beta-endorphin receptor, the effect of the Fab fragment on 125I-beta-endorphin cross-linking to rat brain membranes was examined. 125I-beta-endorphin was covalently bound to three major species of approximate molecular weights 108,000, 73,000, and 49,000. The delta-selective ligand D-Pen2, D-pen5enkephalin was least effective at inhibiting the cross-linking of beta-endorphin, whereas the micro-selective ligand Tyr-D-Ala-Gly-NMe-Phe-Gly-ol and kappa-selective ligand U50488 inhibited beta-endorphin cross-linking to the 108,000 and 73,000 Da species. Both 3B4F11 and beta-endorphin prevented the covalent binding of 125I-beta-endorphin to all three labeled species. These findings suggest that micro and kappa receptor types might have some structural similarities, whereas the delta receptor type might differ in molecular size. In addition, the micro, kappa, and delta ligands might have different primary sequences, whereas their tertiary structures might share regions of molecular homology with all three receptor constituents labeled by 125I-beta-endorphin. 3B4F11 will be a valuable tool for the purification and isolation of the several components of the beta-endorphin receptor complex.

Clonal heterogeneity of thymic B cells from early-onset myasthenia gravis patients with antibodies against the acetylcholine receptor.

Science.gov (United States)

Vrolix, Kathleen; Fraussen, Judith; Losen, Mario; Stevens, Jo; Lazaridis, Konstantinos; Molenaar, Peter C; Somers, Veerle; Bracho, Maria Alma; Le Panse, Rozen; Stinissen, Piet; Berrih-Aknin, Sonia; Maessen, Jos G; Van Garsse, Leen; Buurman, Wim A; Tzartos, Socrates J; De Baets, Marc H; Martinez-Martinez, Pilar

2014-08-01

Myasthenia gravis (MG) with antibodies against the acetylcholine receptor (AChR-MG) is considered as a prototypic autoimmune disease. The thymus is important in the pathophysiology of the disease since thymus hyperplasia is a characteristic of early-onset AChR-MG and patients often improve after thymectomy. We hypothesized that thymic B cell and antibody repertoires of AChR-MG patients differ intrinsically from those of control individuals. Using immortalization with Epstein-Barr Virus and Toll-like receptor 9 activation, we isolated and characterized monoclonal B cell lines from 5 MG patients and 8 controls. Only 2 of 570 immortalized B cell clones from MG patients produced antibodies against the AChR (both clones were from the same patient), suggesting that AChR-specific B cells are not enriched in the thymus. Surprisingly, many B cell lines from both AChR-MG and control thymus samples displayed reactivity against striated muscle proteins. Striational antibodies were produced by 15% of B cell clones from AChR-MG versus 6% in control thymus. The IgVH gene sequence analysis showed remarkable similarities, concerning VH family gene distribution, mutation frequency and CDR3 composition, between B cells of AChR-MG patients and controls. MG patients showed clear evidence of clonal B cell expansion in contrast to controls. In this latter aspect, MG resembles multiple sclerosis and clinically isolated syndrome, but differs from systemic lupus erythematosus. Our results support an antigen driven immune response in the MG thymus, but the paucity of AChR-specific B cells, in combination with the observed polyclonal expansions suggest a more diverse immune response than expected. Copyright © 2013 Elsevier Ltd. All rights reserved.

The Impact of HLA Class I-Specific Killer Cell Immunoglobulin-Like Receptors on Antibody-Dependent Natural Killer Cell-Mediated Cytotoxicity and Organ Allograft Rejection.

Science.gov (United States)

Rajalingam, Raja

2016-01-01

Natural killer (NK) cells of the innate immune system are cytotoxic lymphocytes that play an important roles following transplantation of solid organs and hematopoietic stem cells. Recognition of self-human leukocyte antigen (HLA) class I molecules by inhibitory killer cell immunoglobulin-like receptors (KIRs) is involved in the calibration of NK cell effector capacities during the developmental stage, allowing the subsequent recognition and elimination of target cells with decreased expression of self-HLA class I (due to virus infection or tumor transformation) or HLA class I disparities (in the setting of allogeneic transplantation). NK cells expressing an inhibitory KIR-binding self-HLA can be activated when confronted with allografts lacking a ligand for the inhibitory receptor. Following the response of the adaptive immune system, NK cells can further destroy allograft endothelium by antibody-dependent cell-mediated cytotoxicity (ADCC), triggered through cross-linking of the CD16 Fc receptor by donor-specific antibodies bound to allograft. Upon recognizing allogeneic target cells, NK cells also secrete cytokines and chemokines that drive maturation of dendritic cells to promote cellular and humoral adaptive immune responses against the allograft. The cumulative activating and inhibitory signals generated by ligation of the receptors regulates mature NK cell killing of target cells and their production of cytokines and chemokines. This review summarizes the role of NK cells in allograft rejection and proposes mechanistic concepts that indicate a prominent role for KIR-HLA interactions in facilitating NK cells for Fc receptor-mediated ADCC effector function involved in antibody-mediated rejection of solid organ transplants.

The impact of HLA class I-specific killer cell immunoglobulin-like receptors on antibody-dependent natural killer cell-mediated cytotoxicity and organ allograft rejection

Directory of Open Access Journals (Sweden)

Raja Rajalingam

2016-12-01

Full Text Available Natural killer (NK cells of the innate immune system are cytotoxic lymphocytes that play important roles following transplantation of solid organs and hematopoietic stem cells. Recognition of self HLA class I molecules by inhibitory killer cell immunoglobulin-like receptors (KIR is involved in the calibration of NK cell effector capacities during a developmental stage, allowing the subsequent recognition and elimination of target cells with decreased expression of self HLA class I (due to virus infection or tumor transformation or HLA class I disparities (in the setting of allogeneic transplantation. NK cells expressing an inhibitory KIR binding self HLA can be activated when confronted with allografts lacking a ligand for the inhibitory receptor. Following the response of the adaptive immune system, NK cells can further destroy allograft endothelium by antibody-dependent cell-mediated cytotoxicity (ADCC, triggered through cross-linking of the CD16 Fc receptor by donor-specific antibodies bound to allograft. Upon recognizing allogeneic target cells, NK cells also secrete cytokines and chemokines that drive maturation of dendritic cells to promote cellular and humoral adaptive immune responses against the allograft. The cumulative activating and inhibitory signals generated by ligation of the receptors regulates mature NK cell killing of target cells and their production of cytokines and chemokines. This review summarizes the role of NK cells in allograft rejection and proposes mechanistic concepts that indicate a prominent role for KIR-HLA interactions in facilitating NK cells for Fc receptor-mediated ADCC effector function involved in antibody-mediated rejection of solid organ transplants.

Homogeneous plate based antibody internalization assay using pH sensor fluorescent dye.

Science.gov (United States)

Nath, Nidhi; Godat, Becky; Zimprich, Chad; Dwight, Stephen J; Corona, Cesear; McDougall, Mark; Urh, Marjeta

2016-04-01

Receptor-mediated antibody internalization is a key mechanism underlying several anti-cancer antibody therapeutics. Delivering highly toxic drugs to cancer cells, as in the case of antibody drug conjugates (ADCs), efficient removal of surface receptors from cancer cells and changing the pharmacokinetics profile of the antibody drugs are some of key ways that internalization impacts the therapeutic efficacy of the antibodies. Over the years, several techniques have been used to study antibody internalization including radiolabels, fluorescent microscopy, flow cytometry and cellular toxicity assays. While these methods allow analysis of internalization, they have limitations including a multistep process and limited throughput and are generally endpoint assays. Here, we present a new homogeneous method that enables time and concentration dependent measurements of antibody internalization. The method uses a new hydrophilic and bright pH sensor dye (pHAb dye), which is not fluorescent at neutral pH but becomes highly fluorescent at acidic pH. For receptor mediated antibody internalization studies, antibodies against receptors are conjugated with the pHAb dye and incubated with the cells expressing the receptors. Upon binding to the receptor, the dyes conjugated to the antibody are not fluorescent because of the neutral pH of the media, but upon internalization and trafficking into endosomal and lysosomal vesicles the pH drops and dyes become fluorescent. The enabling attributes of the pHAb dyes are the hydrophilic nature to minimize antibody aggregation and bright fluorescence at acidic pH which allows development of simple plate based assays using a fluorescent reader. Using two different therapeutic antibodies--Trastuzumab (anti-HER2) and Cetuximab (anti-EGFR)--we show labeling with pHAb dye using amine and thiol chemistries and impact of chemistry and dye to antibody ration on internalization. We finally present two new approaches using the pHAb dye, which will be

Myasthenic Crisis Complicated with Myxedema, Positive for Both Anti-acetylcholine Receptor and Anti-muscle-specific Tyrosine Kinase Antibodies.

Science.gov (United States)

Horiuchi, Kazuhiro; Nagai, Azusa; Wakita, Masahiro; Ito, Shotaro; Takamura, Kei; Houzen, Hideki

2018-01-15

We herein report the case of myasthenic crisis occurring in a 51-year-old man. He had experienced ptosis, increased body weight with edema, and fatigue with dyspnea. He presented at our emergency department with disturbed consciousness. He was originally diagnosed with myxedema coma, and he required artificial respiration. Because his weakness persisted and he was positive for anti-acetylcholine receptor antibodies and anti-muscle-specific tyrosine kinase antibodies, we diagnosed myasthenic crisis after various examinations. His clinical response to treatment was good and he was discharged in an ambulatory status 3 months after admission. This case demonstrates that myasthenic crisis may occur in association with myxedema.

Toll-like receptor activation enhances cell-mediated immunity induced by an antibody vaccine targeting human dendritic cells

Directory of Open Access Journals (Sweden)

Berger Marc A

2007-01-01

Full Text Available Abstract Previously, we have successfully targeted the mannose receptor (MR expressed on monocyte-derived dendritic cells (DCs using a fully human MR-specific antibody, B11, as a vehicle to deliver whole protein tumor antigens such as the human chorionic gonadotropin hormone (hCGβ. Since MRs play a role in bridging innate immunity with adaptive immunity we have explored several toll-like receptor (TLR-specific ligands that may synergize with MR targeting and be applicable as adjuvants in the clinic. We demonstrate that antigen-specific helper and cytolytic T cells from both healthy donors and cancer patients were effectively primed with B11-hCGβ-treated autologous DCs when a combination of one or several TLR ligands is used. Specifically, concomitant signaling of DCs via TLR3 with dsRNA (poly I:C and DC TLR 7/8 with Resiquimod (R-848, respectively, elicited efficient antigen presentation-mediated by MR-targeting. We demonstrate that MR and TLRs contribute towards maturation and activation of DCs by a mechanism that may be driven by a combination of adjuvant and antibody vaccines that specifically deliver antigenic targets to DCs.

Characterization and expression of the human T cell receptor-T3 complex by monoclonal antibody F101.01

DEFF Research Database (Denmark)

Geisler, C; Plesner, T; Pallesen, G

1988-01-01

A murine monoclonal antibody (MoAb) F101.01 reacting with the T cell receptor (TCR)-T3 complex is presented. Immunohistological studies showed that F101.01 specifically stains T-zone lymphocytes in lymph nodes, tonsils, and splenic tissue. Two-colour immunofluorescence and flow cytometry...... demonstrated co-expression of the antigen defined by F101.01 and the pan-T cell antigens defined by CD2, CD3, CD5, and CD7 antibodies. Cells stained with CD4 and CD8 antibodies were both included in the F101.01-positive population, whereas CD16-positive natural killer cells (NK), B cells (CD19 and CD20......), and myeloid cells (CD13 and CD33) were excluded. The target antigen of F101.01 co-modulated with the CD3-defined antigen (T3) and the TCR recognized by the MoAb WT-31. CD3 antibody and WT-31 both blocked binding of F101.01. F101.01 precipitated the TCR-T3 complex from lysates of 125I-labelled peripheral blood...

A monoclonal antibody TrkB receptor agonist as a potential therapeutic for Huntington's disease.

Directory of Open Access Journals (Sweden)

Daniel Todd

Full Text Available Huntington's disease (HD is a devastating, genetic neurodegenerative disease caused by a tri-nucleotide expansion in exon 1 of the huntingtin gene. HD is clinically characterized by chorea, emotional and psychiatric disturbances and cognitive deficits with later symptoms including rigidity and dementia. Pathologically, the cortico-striatal pathway is severely dysfunctional as reflected by striatal and cortical atrophy in late-stage disease. Brain-derived neurotrophic factor (BDNF is a neuroprotective, secreted protein that binds with high affinity to the extracellular domain of the tropomyosin-receptor kinase B (TrkB receptor promoting neuronal cell survival by activating the receptor and down-stream signaling proteins. Reduced cortical BDNF production and transport to the striatum have been implicated in HD pathogenesis; the ability to enhance TrkB signaling using a BDNF mimetic might be beneficial in disease progression, so we explored this as a therapeutic strategy for HD. Using recombinant and native assay formats, we report here the evaluation of TrkB antibodies and a panel of reported small molecule TrkB agonists, and identify the best candidate, from those tested, for in vivo proof of concept studies in transgenic HD models.

Targeting Malignant Brain Tumors with Antibodies

Directory of Open Access Journals (Sweden)

Rok Razpotnik

2017-09-01

Full Text Available Antibodies have been shown to be a potent therapeutic tool. However, their use for targeting brain diseases, including neurodegenerative diseases and brain cancers, has been limited, particularly because the blood–brain barrier (BBB makes brain tissue hard to access by conventional antibody-targeting strategies. In this review, we summarize new antibody therapeutic approaches to target brain tumors, especially malignant gliomas, as well as their potential drawbacks. Many different brain delivery platforms for antibodies have been studied such as liposomes, nanoparticle-based systems, cell-penetrating peptides (CPPs, and cell-based approaches. We have already shown the successful delivery of single-chain fragment variable (scFv with CPP as a linker between two variable domains in the brain. Antibodies normally face poor penetration through the BBB, with some variants sufficiently passing the barrier on their own. A “Trojan horse” method allows passage of biomolecules, such as antibodies, through the BBB by receptor-mediated transcytosis (RMT. Such examples of therapeutic antibodies are the bispecific antibodies where one binding specificity recognizes and binds a BBB receptor, enabling RMT and where a second binding specificity recognizes an antigen as a therapeutic target. On the other hand, cell-based systems such as stem cells (SCs are a promising delivery system because of their tumor tropism and ability to cross the BBB. Genetically engineered SCs can be used in gene therapy, where they express anti-tumor drugs, including antibodies. Different types and sources of SCs have been studied for the delivery of therapeutics to the brain; both mesenchymal stem cells (MSCs and neural stem cells (NSCs show great potential. Following the success in treatment of leukemias and lymphomas, the adoptive T-cell therapies, especially the chimeric antigen receptor-T cells (CAR-Ts, are making their way into glioma treatment as another type of cell

Expression of NgBR Is Highly Associated with Estrogen Receptor Alpha and Survivin in Breast Cancer

Science.gov (United States)

North, Paula; Kong, Amanda; Huang, Jian; Miao, Qing Robert

2013-01-01

NgBR is a type I receptor with a single transmembrane domain and was identified as a specific receptor for Nogo-B. Our recent findings demonstrated that NgBR binds farnesylated Ras and recruits Ras to the plasma membrane, which is a critical step required for the activation of Ras signaling in human breast cancer cells and tumorigenesis. Here, we first use immunohistochemistry and real-time PCR approaches to examine the expression patterns of Nogo-B and NgBR in both normal and breast tumor tissues. Then, we examine the relationship between NgBR expression and molecular subtypes of breast cancer, and the roles of NgBR in estrogen-dependent survivin signaling pathway. Results showed that NgBR and Nogo-B protein were detected in both normal and breast tumor tissues. However, the expression of Nogo-B and NgBR in breast tumor tissue was much stronger than in normal breast tissue. The statistical analysis demonstrated that NgBR is highly associated with ER-positive/HER2-negative breast cancer. We also found that the expression of NgBR has a strong correlation with the expression of survivin, which is a well-known apoptosis inhibitor. The correlation between NgBR and survivin gene expression was further confirmed by real-time PCR. In vitro results also demonstrated that estradiol induces the expression of survivin in ER-positive T47D breast tumor cells but not in ER-negative MDA-MB-468 breast tumor cells. NgBR knockdown with siRNA abolishes estradiol-induced survivin expression in ER-positive T47D cells but not in ER-negative MDA-MB-468 cells. In addition, estradiol increases the expression of survivin and cell growth in ER-positive MCF-7 and T47D cells whereas knockdown of NgBR with siRNA reduces estradiol-induced survivin expression and cell growth. In summary, these results indicate that NgBR is a new molecular marker for breast cancer. The data suggest that the expression of NgBR may be essential in promoting ER-positive tumor cell proliferation via survivin induction

The Isolation of Novel Phage Display-Derived Human Recombinant Antibodies Against CCR5, the Major Co-Receptor of HIV

Science.gov (United States)

Shimoni, Moria; Herschhorn, Alon; Britan-Rosich, Yelena; Kotler, Moshe; Benhar, Itai

2013-01-01

Abstract Selecting for antibodies against specific cell-surface proteins is a difficult task due to many unrelated proteins that are expressed on the cell surface. Here, we describe a method to screen antibody-presenting phage libraries against native cell-surface proteins. We applied this method to isolate antibodies that selectively recognize CCR5, which is the major co-receptor for HIV entry (consequently, playing a pivotal role in HIV transmission and pathogenesis). We employed a phage screening strategy by using cells that co-express GFP and CCR5, along with an excess of control cells that do not express these proteins (and are otherwise identical to the CCR5-expressing cells). These control cells are intended to remove most of the phages that bind the cells nonspecifically; thus leading to an enrichment of the phages presenting anti-CCR5-specific antibodies. Subsequently, the CCR5-presenting cells were quantitatively sorted by flow cytometry, and the bound phages were eluted, amplified, and used for further successive selection rounds. Several different clones of human single-chain Fv antibodies that interact with CCR5-expressing cells were identified. The most specific monoclonal antibody was converted to a full-length IgG and bound the second extracellular loop of CCR5. The experimental approach presented herein for screening for CCR5-specific antibodies can be applicable to screen antibody-presenting phage libraries against any cell-surface expressed protein of interest. PMID:23941674

Expression analysis and specific blockade of the receptor for human thymic stromal lymphopoietin (TSLP) by novel antibodies to the human TSLPRα receptor chain.

Science.gov (United States)

Borowski, Andreas; Vetter, Tina; Kuepper, Michael; Wohlmann, Andreas; Krause, Sebastian; Lorenzen, Thomas; Virchow, Johann Christian; Luttmann, Werner; Friedrich, Karlheinz

2013-02-01

Thymic stromal lymphopoietin (TSLP) is an interleukin-7 (IL-7)-like cytokine with a pivotal role in development and maintenance of atopic diseases such as allergic asthma and atopic dermatitis. Moreover, recent studies show an involvement of TSLP in the progression of various cancers. TSLP signaling is mediated by the TSLP receptor (TSLPR), a heterodimeric type I cytokine receptor. It consists of the IL-7 receptor alpha chain (IL-7Rα), which is shared with the IL-7 receptor, and the TSLPRα chain as a specific subunit. Blocking signal release by TSLP without affecting IL-7 function is a potentially interesting option for the treatment of atopic diseases or certain tumors. By employing the extracellular domain of human TSLPRα chain (hTSLPRα(ex)) as an antigen, we generated a set of monoclonal antibodies. Several binders to native and/or denatured receptor protein were identified and characterized by cytometry and Western blot analysis. A screen based on a STAT3-driven reporter gene assay in murine pro-B cells expressing a functional hTSLPR yielded two hybridoma clones with specific antagonistic properties towards hTSLP, but not IL-7. Kinetic studies measuring blockade of hTSLP-dependent STAT phosphorylation in a TSLP-responsive cell line revealed an inhibitory constant in the nanomolar range. Copyright © 2012 Elsevier Ltd. All rights reserved.

Visualization of Tumor Angiogenesis Using MR Imaging Contrast Agent Gd-DTPA-anti-VEGF Receptor 2 Antibody Conjugate in a Mouse Tumor Model

International Nuclear Information System (INIS)

Jun, Hong Young; Yin, Hong Hua; Kim, Sun Hee; Park, Seong Hoon; Kim, Hun Soo; Yoon Kwon Ha Yoon

2010-01-01

To visualize tumor angiogenesis using the MRI contrast agent, Gd- DTPA-anti-VEGF receptor 2 antibody conjugate, with a 4.7-Tesla MRI instrument in a mouse model. We designed a tumor angiogenesis-targeting T1 contrast agent that was prepared by the bioconjugation of gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) and an anti-vascular endothelial growth factor receptor-2 (VEGFR2) antibody. The specific binding of the agent complex to cells that express VEGFR2 was examined in cultured murine endothelial cells (MS-1 cells) with a 4.7-Tesla magnetic resonance imaging scanner. Angiogenesis-specific T1 enhancement was imaged with the Gd-DTPA-anti-VEGFR2 antibody conjugate using a CT-26 adenocarcinoma tumor model in eight mice. As a control, the use of the Gd-DTPA-anti-rat immunoglobulin G (Gd-DTPA-anti-rat IgG) was imaged with a tumor model in eight mice. Statistical significance was assessed using the Mann-Whitney test. Tumor tissue was examined by immunohistochemical analysis. The Gd-DTPA-anti-VEGFR2 antibody conjugate showed predominant binding to cultured endothelial cells that expressed a high level of VEGFR2. Signal enhancement was approximately three-fold for in vivo T1-weighted MR imaging with the use of the Gd-DTPA-anti-VEGFR2 antibody conjugate as compared with the Gd-DTPA-rat IgG in the mouse tumor model (p < 0.05). VEGFR2 expression in CT-26 tumor vessels was demonstrated using immunohistochemical staining. MR imaging using the Gd-DTPA-anti-VEGFR2 antibody conjugate as a contrast agent is useful in visualizing noninvasively tumor angiogenesis in a murine tumor model

Visualization of Tumor Angiogenesis Using MR Imaging Contrast Agent Gd-DTPA-anti-VEGF Receptor 2 Antibody Conjugate in a Mouse Tumor Model

Energy Technology Data Exchange (ETDEWEB)

Jun, Hong Young; Yin, Hong Hua; Kim, Sun Hee; Park, Seong Hoon; Kim, Hun Soo; Yoon Kwon Ha Yoon [Wonkwang University School of Medicine, Iksan (Korea, Republic of)

2010-08-15

To visualize tumor angiogenesis using the MRI contrast agent, Gd- DTPA-anti-VEGF receptor 2 antibody conjugate, with a 4.7-Tesla MRI instrument in a mouse model. We designed a tumor angiogenesis-targeting T1 contrast agent that was prepared by the bioconjugation of gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) and an anti-vascular endothelial growth factor receptor-2 (VEGFR2) antibody. The specific binding of the agent complex to cells that express VEGFR2 was examined in cultured murine endothelial cells (MS-1 cells) with a 4.7-Tesla magnetic resonance imaging scanner. Angiogenesis-specific T1 enhancement was imaged with the Gd-DTPA-anti-VEGFR2 antibody conjugate using a CT-26 adenocarcinoma tumor model in eight mice. As a control, the use of the Gd-DTPA-anti-rat immunoglobulin G (Gd-DTPA-anti-rat IgG) was imaged with a tumor model in eight mice. Statistical significance was assessed using the Mann-Whitney test. Tumor tissue was examined by immunohistochemical analysis. The Gd-DTPA-anti-VEGFR2 antibody conjugate showed predominant binding to cultured endothelial cells that expressed a high level of VEGFR2. Signal enhancement was approximately three-fold for in vivo T1-weighted MR imaging with the use of the Gd-DTPA-anti-VEGFR2 antibody conjugate as compared with the Gd-DTPA-rat IgG in the mouse tumor model (p < 0.05). VEGFR2 expression in CT-26 tumor vessels was demonstrated using immunohistochemical staining. MR imaging using the Gd-DTPA-anti-VEGFR2 antibody conjugate as a contrast agent is useful in visualizing noninvasively tumor angiogenesis in a murine tumor model

Antibody neutralization of retargeted measles viruses

Science.gov (United States)

Lech, Patrycja J.; Pappoe, Roland; Nakamura, Takafumi; Tobin, Gregory J.; Nara, Peter L.; Russell, Stephen J.

2014-01-01

The measles virus (MV) vaccine lineage is a promising oncolytic but prior exposure to the measles vaccine or wild-type MV strains limits treatment utility due to the presence of anti-measles antibodies. MV entry can be redirected by displaying a polypeptide ligand on the Hemagglutinin (H) C-terminus. We hypothesized that retargeted MV would escape neutralization by monoclonal antibodies (mAbs) recognizing the H receptor-binding surface and be less susceptible to neutralization by human antisera. Using chimeric H proteins, with and without mutations that ablate MV receptor binding, we show that retargeted MVs escape mAbs that target the H receptor-binding surface by virtue of mutations that ablate infection via SLAM and CD46. However, C-terminally displayed domains do not mediate virus entry in the presence of human antibodies that bind to the underlying H domain. In conclusion, utility of retargeted oncolytic measles viruses does not extend to evasion of human serum neutralization. PMID:24725950

Preparation and Characterization of an Antibody Antagonist That Targets the Porcine Growth Hormone Receptor

Directory of Open Access Journals (Sweden)

Huanzhong Cui

2016-10-01

Full Text Available A series of antagonists specifically targeting growth hormone receptors (GHR in different species, such as humans, rats, bovines, and mice, have been designed; however, there are currently no antagonists that target the porcine growth hormone (GH. Therefore, in this study, we developed and characterized a porcine GHR (pGHR antibody antagonist (denoted by AN98 via the hybridoma technique. The results from enzyme-linked immunosorbent assay, fluorescence activated cell sorter, indirect immunoinfluscent assay, and competitive receptor binding analysis showed that AN98 could specifically recognize pGHR, and further experiments indicated that AN98 could effectively inhibit pGH-induced signalling in CHO-pGHR cells and porcine hepatocytes. In addition, AN98 also inhibited GH-induced insulin-like growth factor-1 (IGF-1 secretion in porcine hepatocytes. In summary, these findings indicated that AN98, as a pGHR-specific antagonist, has potential applications in pGH-pGHR-related research on domestic pigs.

Antibodies against the melanocortin-4 receptor act as inverse agonists in vitro and in vivo.

Science.gov (United States)

Peter, Jean-Christophe; Nicholson, Janet R; Heydet, Déborah; Lecourt, Anne-Catherine; Hoebeke, Johan; Hofbauer, Karl G

2007-06-01

Functionally active antibodies (Abs) against central G-protein-coupled receptors have not yet been reported. We selected the hypothalamic melanocortin-4 receptor (MC4-R) as a target because of its crucial role in the regulation of energy homeostasis. A 15 amino acid sequence of the N-terminal (NT) domain was used as an antigen. This peptide showed functional activity in surface plasmon resonance experiments and in studies on HEK-293 cells overexpressing the human MC4-R (hMC4-R). Rats immunized against the NT peptide produced specific antibodies, which were purified and characterized in vitro. In HEK-293 cells, rat anti-NT Abs showed specific immunofluorescence labeling of hMC4-R. They reduced the production of cAMP under basal conditions and after stimulation with a synthetic MC4-R agonist. Rats immunized against the NT peptide developed a phenotype consistent with MC4-R blockade, that is, increased food intake and body weight, increased liver and fat pad weight, and elevated plasma triglycerides. In a separate experiment in rats, an increase in food intake could be produced after injection of purified Abs into the third ventricle. Similar results were obtained in rats injected with anti-NT Abs raised in rabbits. Our data show for the first time that active immunization of rats against the NT sequence of the MC4-R results in specific Abs, which appear to stimulate food intake by acting as inverse agonists in the hypothalamus.

A novel antibody engineering strategy for making monovalent bispecific heterodimeric IgG antibodies by electrostatic steering mechanism.

Science.gov (United States)

Liu, Zhi; Leng, Esther C; Gunasekaran, Kannan; Pentony, Martin; Shen, Min; Howard, Monique; Stoops, Janelle; Manchulenko, Kathy; Razinkov, Vladimir; Liu, Hua; Fanslow, William; Hu, Zhonghua; Sun, Nancy; Hasegawa, Haruki; Clark, Rutilio; Foltz, Ian N; Yan, Wei

2015-03-20

Producing pure and well behaved bispecific antibodies (bsAbs) on a large scale for preclinical and clinical testing is a challenging task. Here, we describe a new strategy for making monovalent bispecific heterodimeric IgG antibodies in mammalian cells. We applied an electrostatic steering mechanism to engineer antibody light chain-heavy chain (LC-HC) interface residues in such a way that each LC strongly favors its cognate HC when two different HCs and two different LCs are co-expressed in the same cell to assemble a functional bispecific antibody. We produced heterodimeric IgGs from transiently and stably transfected mammalian cells. The engineered heterodimeric IgG molecules maintain the overall IgG structure with correct LC-HC pairings, bind to two different antigens with comparable affinity when compared with their parental antibodies, and retain the functionality of parental antibodies in biological assays. In addition, the bispecific heterodimeric IgG derived from anti-HER2 and anti-EGF receptor (EGFR) antibody was shown to induce a higher level of receptor internalization than the combination of two parental antibodies. Mouse xenograft BxPC-3, Panc-1, and Calu-3 human tumor models showed that the heterodimeric IgGs strongly inhibited tumor growth. The described approach can be used to generate tools from two pre-existent antibodies and explore the potential of bispecific antibodies. The asymmetrically engineered Fc variants for antibody-dependent cellular cytotoxicity enhancement could be embedded in monovalent bispecific heterodimeric IgG to make best-in-class therapeutic antibodies. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

SINGLE CHAIN VARIABLE FRAGMENTS OF ANTIBODIES AGAINST DIPHTHERIA TOXIN B-SUBUNIT ISOLATED FROM PHAGE DISPLAY HUMAN ANTIBODY LIBRARY

Directory of Open Access Journals (Sweden)

Oliinyk O. S.

2014-02-01

Full Text Available Diphtheria toxin is an exoantigen of Corynebacterium diphtheriae that inhibits protein synthesis and kills sensitive cells. The aim of this study was to obtain human recombinant single-chain variable fragment (scFv antibodies against receptor-binding B subunit of diphtheria toxin. 12 specific clones were selected after three rounds of a phage display naїve (unimmunized human antibody library against recombinant B-subunit. scFv DNA inserts from these 12 clones were digested with MvaI, and 6 unique restriction patterns were found. Single-chain antibodies were expressed in Escherichia coli XL1-blue. The recombinant proteins were characterized by immunoblotting of bacterial extracts and detection with an anti-E-tag antibody. The toxin B-subunit-binding function of the single-chain antibody was shown by ELISA. The affinity constants for different clones were found to be from 106 to 108 М–1. Due to the fact, that these antibody fragments recognized epitopes in the receptor-binding Bsubunit of diphtheria toxin, further studies are interesting to evaluate their toxin neutralization properties and potential for therapeutic applications. Obtained scFv-antibodies can also be used for detection and investigation of biological properties of diphtheria toxin.

Comparison of monoclonal antibodies and tritiated ligands for estrogen receptor assays in 241 breast cancer cytosols

International Nuclear Information System (INIS)

Goussard, J.; Lechevrel, C.; Martin, P.M.; Roussel, G.

1986-01-01

Estrogen receptor determinations have been performed on 241 cytosols from 160 breast cancer tumors using both radioactive ligands ([ 3 H]-estradiol, [3H]R2858) and monoclonal antibodies (Abbott ER-EIA Kit) to compare the two methods and to evaluate the clinical usefulness of the new immunological, simplified assay. Intra- and interassay reproducibility of the enzyme immunoassay (EIA) method was studied during a 6-month period on 35 standard curves with 4 different batches of monoclonal antibodies. Intraassay coefficients of variation studied on duplicates were smaller than 5% in most cases and reproducibility of the curves showed coefficients of variation lower than 10% except for standard 0 and 5 fmol/ml. Pooled cytosols used as control for the dextran coated charcoal method had interassay variation coefficients between 3.8 and 11.4%. Reproducibility has been studied on clinical specimens assayed twice at two different periods with either EIA or dextran coated charcoal methods. Slopes obtained were 1.05 and 0.96, respectively. A good stability of EIA results was obtained with protein concentrations in the range 4-0.15 mg/ml cytosol. No significant effects of dithiothreitol or monothioglycerol (1 mM) on EIA and dextran coated charcoal assay were observed. Eighty breast cancer cytosols were assayed with both EIA and Scatchard analysis. The slope of the regression curve obtained was 1.04 (r = 0.963). Cytosols were assayed by EIA and by a saturating concentration of tritiated ligand (5 nM). With 153 cytosols the EIA/5 nM slope was 1.34 (r = 0.978). This slope can be compared with the slope Scatchard/5 nM obtained with 90 cytosols: 1.29 (r = 0.985). Absence of cross-reactivity of monoclonal ER antibodies with progesterone receptor was observed

Development and validation of receptor occupancy pharmacodynamic assays used in the clinical development of the monoclonal antibody vedolizumab.

Science.gov (United States)

Wyant, Tim; Estevam, Jose; Yang, Lili; Rosario, Maria

2016-03-01

Vedolizumab is a monoclonal antibody approved for use in ulcerative colitis and Crohn's disease. By specifically binding to α4 β7 integrin, vedolizumab prevents trafficking of lymphocytes to the gut, thereby interfering with disease pathology. During the clinical development program, the pharmacodynamic effect of vedolizumab was evaluated by 2 flow cytometry receptor occupancy assays: act-1 (ACT-1) and mucosal addressin cell adhesion molecule-1 (MAdCAM-1). Here we describe the development and validation of these assays. The ACT-1 assay is a receptor occupancy free-site assay that uses a monoclonal antibody with the same binding epitope as vedolizumab to detect free (unbound) sites on α4 β7 integrin. The MAdCAM-1 assay used a soluble version of the natural ligand for α4 β7 integrin to detect free sites. The assays were validated using a fit-for-purpose approach throughout the clinical development of vedolizumab. Both the ACT-1 assay and the MAdCAM-1 assay demonstrated acceptable reproducibility and repeatability. The assays were sufficiently stable to allow for clinical use. During clinical testing the assays demonstrated that vedolizumab was able to saturate peripheral cells at all doses tested. Two pharmacodynamic receptor occupancy assays were developed and validated to assess the effect of vedolizumab on peripheral blood cells. The results of these assays demonstrated the practical use of flow cytometry to examine pharmacodynamic response in clinical trials. © 2015 International Clinical Cytometry Society.

Negative correlation between bone mineral density and TSH receptor antibodies in long-term euthyroid postmenopausal women with treated Graves’ disease

DEFF Research Database (Denmark)

Ercolano, Monica A; Drnovsek, Monica L; Croome, Maria C

2013-01-01

Thyrotoxicosis is a cause of secondary osteoporosis. High concentrations of triiodotironine (T3) in Graves' disease stimulate bone turnover, but it is unclear if euthyroidism will always normalize bone metabolism. Thyrotropin (TSH) is known to affect directly the bone metabolism through the TSH...... receptor and TSH receptor antibodies (TRAb) may have an important role in bone turn-over.The aim of our study was to determine, in pre and postmenopausal euthyroidism patients with previous overt hyperthyroidism due to Graves' disease the bone mineral density (BMD) as well as factors that could affect BMD...

Antibody induction versus placebo, no induction, or another type of antibody induction for liver transplant recipients

DEFF Research Database (Denmark)

Penninga, Luit; Wettergren, André; Wilson, Colin H

2014-01-01

. All 19 trials were with high risk of bias. Of the 19 trials, 16 trials were two-arm trials, and three trials were three-arm trials. Hence, we found 25 trial comparisons with antibody induction agents: interleukin-2 receptor antagonist (IL-2 RA) versus no induction (10 trials with 1454 participants....... Furthermore, serum creatinine was statistically significantly higher when T-cell specific antibody induction was compared with no induction (MD 3.77 μmol/L, 95% CI 0.33 to 7.21; low-quality evidence), as well as when polyclonal T-cell specific antibody induction was compared with no induction, but this small...... T-cell specific antibody induction, drug-related adverse events were less common among participants treated with interleukin-2 receptor antagonists (RR 0.23, 95% CI 0.09 to 0.63; low-quality evidence), but this was caused by the results from one trial, and trial sequential analysis could not exclude...

Field flow fractionation for assessing neonatal Fc receptor and Fcγ receptor binding to monoclonal antibodies in solution.

Science.gov (United States)

Pollastrini, Joey; Dillon, Thomas M; Bondarenko, Pavel; Chou, Robert Y-T

2011-07-01

Analysis of the strength and stoichiometry of immunoglobulin G (IgG) binding to neonatal Fc receptor (FcRn) and Fcγ receptor (FcγR) is important for evaluating the pharmacokinetics and effector functions of therapeutic monoclonal antibody (mAb) products, respectively. The current standard for assessing FcγR and FcRn binding is composed of cell-based and surface plasmon resonance (SPR) assays. In this work, asymmetrical flow field flow fractionation (AF4) was evaluated to establish the true stoichiometry of IgG binding in solution. AF4 and liquid chromatography-mass spectrometry (LC-MS) were applied to directly observe IgG/FcγR and IgG/FcRn complexes, which were not observed using nonequilibrium size exclusion chromatography (SEC) analysis. Human serum albumin (HSA), an abundant component of human blood and capable of binding FcRn, was studied in combination with FcRn and IgG. AF4 demonstrated that the majority of large complexes of IgG/FcRn/HSA were at an approximate 1:2:1 molar ratio. In addition, affinity measurements of the complex were performed in the sub-micromolar affinity range. A significant decrease in binding was detected for IgG molecules with increased oxidation in the Fc region. AF4 was useful in detecting weak binding between full-length IgG/Fc fragments and Fc receptors and the effect of chemical modifications on binding. AF4 is a useful technique in the assessment of mAb product quality attributes. Copyright © 2011 Elsevier Inc. All rights reserved.

[N-methyl-D-aspartate receptor antibody encephalitis: value of immunomodulatory therapy].

Science.gov (United States)

Le Moigno, L; Ternant, D; Paintaud, G; Thibault, G; Cloarec, S; Tardieu, M; Lagrue, E; Castelnau, P

2014-06-01

Anti-N-methyl-D-aspartate receptor (NMDA-R) encephalitis is little known to pediatricians and likely underdiagnosed. The child's vital and cognitive prognosis is at stake. The use of immunomodulatory drugs, such as rituximab has led to spectacular results, but many questions remain about its mode of action in this type of pathology. We report the case of a 6-year-old girl with no medical history, admitted for status epilepticus preceded by behavior symptoms and sleep disorders. Gradually, the child became bedridden, mute, and animated by predominantly orofacial dyskinesia. Examinations were normal (cerebrospinal fluid [CSF] analysis, brain MRI). The diagnosis was established by the presence of NMDA-R antibodies in the CSF. After exclusion of a tumor-associated syndrome, treatment was started initially by intravenous immunoglobulins, then by plasma exchange, and finally rituximab. The patient was cured with rituximab despite an unusually early recovery of the B-cell pool. Anti-N-methyl-D-aspartate receptor (NMDA-R) encephalitis is a severe but potentially reversible neurologic disorder only recently described, even in childhood. It may be reversible without sequelae if diagnosed and treated early. The use of immunomodulatory therapy, such as rituximab seemingly improves the outcome. Immunological monitoring is needed to better understand its mechanism of action in autoimmune diseases of the nervous system in childhood. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Three Types of Striational Antibodies in Myasthenia Gravis

Directory of Open Access Journals (Sweden)

Shigeaki Suzuki

2011-01-01

Full Text Available Myasthenia gravis (MG is caused by antibodies that react mainly with the acetylcholine receptor on the postsynaptic site of the neuromuscular junction. A wide range of clinical presentations and associated features allow MG to be classified into subtypes based on autoantibody status. Striational antibodies, which react with epitopes on the muscle proteins titin, ryanodine receptor (RyR, and Kv1.4, are frequently found in MG patients with late-onset and thymoma. Antititin and anti-RyR antibodies are determined by enzyme-linked immunosorbent assay or immunoblot. More recently, a method for the detection of anti-Kv1.4 autoantibodies has become available, involving 12–15% of all MG patients. The presence of striational antibodies is associated with more severe disease in all MG subgroups. Anti-Kv1.4 antibody is a useful marker for the potential development of lethal autoimmune myocarditis and response to calcineurin inhibitors. Detection of striational antibodies provides more specific and useful clinical information in MG patients.

"Light switch" mental status changes and irritable insomnia are two particularly salient features of anti-NMDA receptor antibody encephalitis.

Science.gov (United States)

DeSena, Allen D; Greenberg, Benjamin M; Graves, Donna

2014-07-01

Anti-N-methyl-D-aspartate antibody encephalitis is becoming increasingly recognized as a cause of acute and subacute encephalopathy in both adults and children. The typical features of this disorder include some degree of encephalopathy, seizures, and often a movement disorder component. However, there is wide variability in its presentation, and diagnosis based on clinical features alone is often delayed. We report a series of four of 12 patients observed at our children's hospital between 2011 and 2013 that we chose as particularly representative examples of two distinct clinical features. In these individuals with anti-N-methyl-D-aspartate receptor antibody encephalitis, we note a very rapid on-off state between responsiveness and nonresponsiveness and/or insomnia accompanied by extreme irritability. We describe the abrupt mental status shift as "light switch" because the patients can awaken in seconds from a completely nonresponsive state. The insomnia noted in our patients was also impressive and often present early in the patients' courses. Light switch mental status changes and irritable insomnia are important early features of anti-N-methyl-D-aspartate receptor antibody encephalitis that can signal the presence of this disorder. The exact pathophysiology of these two symptoms has not been fully elucidated, and we feel that presence of one or both of these symptoms early in the disease course should prompt immediate concern for this disorder. Copyright © 2014 Elsevier Inc. All rights reserved.

Clinical study on antibody-associated limbic encephalitis

Directory of Open Access Journals (Sweden)

WANG Jia-wei

2013-01-01

Full Text Available In recent years, the antibody-associated limbic encephalitis (LE has attracted attentions of more and more clinicians. The associated antibodies mainly act on neuronal cell surface antigens, including the N-methyl-D-aspartate (NMDA receptor, the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA receptor, the γ-aminobutyric acid B (GABAB receptor, leucine-rich glioma-inactivated 1 (LGI1 and contactin-associated protein-like 2 (Caspr2 and so on. The clinical manifestation is primarily defined by the subacute onset of short-term memory loss, seizures, confusion and psychiatric symptoms suggesting the involvement of the limbic system. These severe and protracted disorders can affect children and young adults, occurring with or without tumor association. Routine detection of serum and cerebrospinal fluid (CSF and imaging tests show no specificity, but associated antibodies can be detected in serum and (or CSF. The patients respond well to tumor resection and immunotherapies, including corticosteroids, intravenous immunoglobulin (IVIg, plasma exchange or combination of them, but may relapse. This article aims to study the clinical features and treatment of antibody-associated limbic encephalitis and to improve the diagnosis and prognosis of these diseases.

Enzymatic Inactivation of Endogenous IgG by IdeS Enhances Therapeutic Antibody Efficacy.

Science.gov (United States)

Järnum, Sofia; Runström, Anna; Bockermann, Robert; Winstedt, Lena; Crispin, Max; Kjellman, Christian

2017-09-01

Endogenous plasma IgG sets an immunologic threshold that dictates the activity of tumor-directed therapeutic antibodies. Saturation of cellular antibody receptors by endogenous antibody limits antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Here, we show how enzymatic cleavage of IgG using the bacterial enzyme IdeS can be utilized to empty both high and low affinity Fcγ-receptors and clear the entire endogenous antibody pool. Using in vitro models, tumor animal models as well as ex vivo analysis of sera collected during a previous clinical trial with IdeS, we show how clearing of competing plasma antibody levels with IdeS unblocks cellular antibody receptors. We show that therapeutic antibodies against breast cancer (trastuzumab), colon cancer (cetuximab), and lymphomas (rituximab and alemtuzumab) can be potentiated when endogenous IgG is removed. Overall, IdeS is shown to be a potent tool to reboot the human antibody repertoire and to generate a window to preferentially load therapeutic antibodies onto effector cells and thereby create an armada of dedicated tumor-seeking immune cells. Mol Cancer Ther; 16(9); 1887-97. ©2017 AACR . ©2017 American Association for Cancer Research.

Targeting the MET oncogene by concomitant inhibition of receptor and ligand via an antibody-"decoy" strategy.

Science.gov (United States)

Basilico, Cristina; Modica, Chiara; Maione, Federica; Vigna, Elisa; Comoglio, Paolo M

2018-04-25

MET, a master gene sustaining "invasive growth," is a relevant target for cancer precision therapy. In the vast majority of tumors, wild-type MET behaves as a "stress-response" gene and relies on the ligand (HGF) to sustain cell "scattering," invasive growth and apoptosis protection (oncogene "expedience"). In this context, concomitant targeting of MET and HGF could be crucial to reach effective inhibition. To test this hypothesis, we combined an anti-MET antibody (MvDN30) inducing "shedding" (i.e., removal of MET from the cell surface), with a "decoy" (i.e., the soluble extracellular domain of the MET receptor) endowed with HGF-sequestering ability. To avoid antibody/decoy interaction-and subsequent neutralization-we identified a single aminoacid in the extracellular domain of MET-lysine 842-that is critical for MvDN30 binding and engineered the corresponding recombinant decoyMET (K842E). DecoyMET K842E retains the ability to bind HGF with high affinity and inhibits HGF-induced MET phosphorylation. In HGF-dependent cellular models, MvDN30 antibody and decoyMET K842E used in combination cooperate in restraining invasive growth, and synergize in blocking cancer cell "scattering." The antibody and the decoy unbridle apoptosis of colon cancer stem cells grown in vitro as spheroids. In a preclinical model, built by orthotopic transplantation of a human pancreatic carcinoma in SCID mice engineered to express human HGF, concomitant treatment with antibody and decoy significantly reduces metastatic spread. The data reported indicate that vertical targeting of the MET/HGF axis results in powerful inhibition of ligand-dependent MET activation, providing proof of concept in favor of combined target therapy of MET "expedience." © 2018 UICC.

Simultaneous Vascular Targeting and Tumor Targeting of Cerebral Breast Cancer Metastases Using a T-Cell Receptor Mimic Antibody

Science.gov (United States)

2014-05-01

in May 2013, the difference between nude mice (which lack T- cells , but still have a partially functional adaptive and innate immune system) and NSG...Mangada J, Greiner DL, Handgretinger R. Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human...Targeting of Cerebral Breast Cancer Metastases Using a T- Cell Receptor Mimic Antibody PRINCIPAL INVESTIGATOR: Ulrich Bickel

No-go theorem for bimetric gravity with positive and negative mass

International Nuclear Information System (INIS)

Hohmann, Manuel; Wohlfarth, Mattias N. R.

2009-01-01

We argue that the most conservative geometric extension of Einstein gravity describing both positive and negative mass sources and observers is bimetric gravity and contains two copies of standard model matter which interact only gravitationally. Matter fields related to one of the metrics then appear dark from the point of view of an observer defined by the other metric, and so may provide a potential explanation for the dark universe. In this framework we consider the most general form of linearized field equations compatible with physically and mathematically well-motivated assumptions. Using gauge-invariant linear perturbation theory, we prove a no-go theorem ruling out all bimetric gravity theories that, in the Newtonian limit, lead to precisely opposite forces on positive and negative test masses.

More on Weinberg's no-go theorem in quantum gravity

Science.gov (United States)

Nagahama, Munehiro; Oda, Ichiro

2018-05-01

We complement Weinberg's no-go theorem on the cosmological constant problem in quantum gravity by generalizing it to the case of a scale-invariant theory. Our analysis makes use of the effective action and the BRST symmetry in a manifestly covariant quantum gravity instead of the classical Lagrangian density and the G L (4 ) symmetry in classical gravity. In this sense, our proof is very general since it does not depend on details of quantum gravity and holds true for general gravitational theories which are invariant under diffeomorphisms. As an application of our theorem, we comment on an idea that in the asymptotic safety scenario the functional renormalization flow drives a cosmological constant to zero, solving the cosmological constant problem without reference to fine tuning of parameters. Finally, we also comment on the possibility of extending the Weinberg theorem in quantum gravity to the case where the translational invariance is spontaneously broken.

Experimental radioimmunotherapy of a xenografted human glioma using [sup 131]I-labeled monoclonal antibody to epidermal growth factor receptor

Energy Technology Data Exchange (ETDEWEB)

Takahashi, Hiroshi; Nakazawa, Shozo [Nippon Medical School, Tokyo (Japan); Herlyn, D

1993-09-01

[sup 131]I-labeled F (ab')[sub 2] fragments of murine monoclonal antibodies (MAb) 425 specific to the epidermal growth factor receptor expressed on human gliomas were used in experimental human malignant glioma immunotherapy. Two injections of 150 [mu]Ci [sup 131]I-labeled 425 F(ab')[sub 2] achieved growth inhibition of U-87MG human malignant glioma xenografts in nude mice. This radiolabeled specific MAb F(ab')[sub 2] was significantly superior to radiolabeled fragments of an anti-hepatitis virus control MAb A5C3 in influencing tumor growth. However, similar treatment of established human malignant glioma xenografts did not inhibit progressive tumor growth significantly. No clear tumor inhibition was produced by unlabeled MAb 425F(ab')[sub 2]. These studies suggest that [sup 131]I-labeled MAbs have a significant antitumor effect where unmodified antibody is ineffective. Multiple doses of antibody may achieve an increase in labeled MAb concentration in tumors. (author).

Muscle-Specific Tyrosine Kinase and Myasthenia Gravis Owing to Other Antibodies.

Science.gov (United States)

Rivner, Michael H; Pasnoor, Mamatha; Dimachkie, Mazen M; Barohn, Richard J; Mei, Lin

2018-05-01

Around 20% of patients with myasthenia gravis are acetylcholine receptor antibody negative; muscle-specific tyrosine kinase antibodies (MuSK) were identified as the cause of myasthenia gravis in 30% to 40% of these cases. Anti MuSK myasthenia gravis is associated with specific clinical phenotypes. One is a bulbar form with fewer ocular symptoms. Others show an isolated head drop or symptoms indistinguishable from acetylcholine receptor-positive myasthenia gravis. These patients usually respond well to immunosuppressive therapy, but not as well to cholinesterase inhibitors. Other antibodies associated with myasthenia gravis, including low-density lipoprotein receptor-related protein 4, are discussed. Copyright © 2018 Elsevier Inc. All rights reserved.

[Analysis of epitopes and function of anti-M3 muscarinic acetylcholine receptor antibodies in patients with Sjögren's syndrome].

Science.gov (United States)

Tsuboi, Hiroto; Matsuo, Naomi; Iizuka, Mana; Nakamura, Yumi; Matsumoto, Isao; Sumida, Takayuki

2010-01-01

Sjögren's syndrome (SS) is an autoimmune disease that affects exocrine glands including salivary and lacrimal glands. It is characterized by lymphocytic infiltration into exocrine glands, leading to dry mouth and eyes. A number of auto-antibodies, such as anti-SS-A and SS-B antibodies, are detected in patients with SS. However, no SS-specific pathologic auto-antibodies have yet been found in this condition. M3 muscarinic acetylcholine receptor (M3R) plays a crucial role in the secretion of saliva from salivary glands. It is reported that some patients with SS carried inhibitory auto-antibodies against M3R. To clarify the epitopes and function of anti-M3R antibodies in SS, we examined antibodies to the extracellular domains (N terminal region, the first, second, and third extracellular loop) of M3R by ELISA using synthesized peptide antigens encoding these domains in 42 SS and 42 healthy controls (HC). Titers and positivity of anti-M3R antibodies to every extracellular domain of M3R were significantly higher in SS than in HC. For functional analysis, human salivary gland (HSG) cells were pre-cultured with IgG from anti-M3R antibodies positive SS, negative SS, and HC. HSG cells were stimulated with cevimeline hydrochloride and intracellular calcium concentration ([Ca(2+)](i)) was measured. IgG from anti-M3R antibodies to the second loop positive SS inhibited the increase of [Ca(2+)](i), but IgG from antibodies to the N terminal or the first loop positive SS enhanced it, while IgG from antibodies to the third loop positive SS showed no effect on [Ca(2+)](i) as well as IgG from anti-M3R antibodies negative SS and HC. These findings indicated the presence of several B cell epitopes on M3R in SS and effect of anti-M3R antibodies on the salivary secretion might differ with these epitopes.

Sustained Brown Fat Stimulation and Insulin Sensitization by a Humanized Bispecific Antibody Agonist for Fibroblast Growth Factor Receptor 1/βKlotho Complex

Directory of Open Access Journals (Sweden)

Ganesh Kolumam

2015-07-01

Full Text Available Dissipating excess calories as heat through therapeutic stimulation of brown adipose tissues (BAT has been proposed as a potential treatment for obesity-linked disorders. Here, we describe the generation of a humanized effector-less bispecific antibody that activates fibroblast growth factor receptor (FGFR 1/βKlotho complex, a common receptor for FGF21 and FGF19. Using this molecule, we show that antibody-mediated activation of FGFR1/βKlotho complex in mice induces sustained energy expenditure in BAT, browning of white adipose tissue, weight loss, and improvements in obesity-associated metabolic derangements including insulin resistance, hyperglycemia, dyslipidemia and hepatosteatosis. In mice and cynomolgus monkeys, FGFR1/βKlotho activation increased serum high-molecular-weight adiponectin, which appears to contribute over time by enhancing the amplitude of the metabolic benefits. At the same time, insulin sensitization by FGFR1/βKlotho activation occurs even before the onset of weight loss in a manner that is independent of adiponectin. Together, selective activation of FGFR1/βKlotho complex with a long acting therapeutic antibody represents an attractive approach for the treatment of type 2 diabetes and other obesity-linked disorders through enhanced energy expenditure, insulin sensitization and induction of high-molecular-weight adiponectin.

Chimeric Monoclonal Antibody Cetuximab Targeting Epidermal Growth Factor-Receptor in Advanced Non-Melanoma Skin Cancer.

Science.gov (United States)

Wollina, Uwe; Tchernev, Georgi; Lotti, Torello

2018-01-25

Non-melanoma skin cancer (NMSC) is the most common malignancy in humans. Targeted therapy with monoclonal antibody cetuximab is an option in case of advanced tumor or metastasis. We present and update of the use of cetuximab in NMSC searching PUBMED 2011-2017. The monoclonal antibody cetuximab against epidermal growth factor receptor (EGFR) has been investigated for its use in NMSC during the years 2011 to 2017 by a PUBMED research using the following items: "Non-melanoma skin cancer AND cetuximab," "cutaneous squamous cell carcinoma AND cetuximab," and "basal cell carcinoma AND cetuximab", and "cetuximab AND skin toxicity". Available data were analyzed including case reports. Current evidence of cetuximab efficacy in NMSC was mainly obtained in cutaneous SCC and to a lesser extend in BCC. Response rates vary for neoadjuvant, adjuvant, mono- and combined therapy with cetuximab. Management of cutaneous toxicities is necessary. Guidelines are available. Cetuximab is an option for recurrent or advanced NMSC of the skin. It seems to be justified particularly in very high-risk tumors. There is a need for phase III trials.

Therapeutic antibodies as a treatment option for dengue fever.

Science.gov (United States)

Chan, Kuan Rong; Ong, Eugenia Z; Ooi, Eng Eong

2013-11-01

Dengue fever is the most prevalent mosquito-borne viral disease globally with about 100 million cases of acute dengue annually. Severe dengue infection can result in a life-threatening illness. In the absence of either a licensed vaccine or antiviral drug against dengue, therapeutic antibodies that neutralize dengue virus (DENV) may serve as an effective medical countermeasure against severe dengue. However, therapeutic antibodies would need to effectively neutralize all four DENV serotypes. It must not induce antibody-dependent enhancement of DENV infection in monocytes/macrophages through Fc gamma receptor (FcγR)-mediated phagocytosis, which is hypothesized to increase the risk of severe dengue. Here, we review the strategies and technologies that can be adopted to develop antibodies for therapeutic applications. We also discuss the mechanism of antibody neutralization in the cells targeted by DENV that express Fc gamma receptor. These studies have provided significant insight toward the use of therapeutic antibodies as a potentially promising bulwark against dengue.

Anti-PDGF receptor β antibody-conjugated squarticles loaded with minoxidil for alopecia treatment by targeting hair follicles and dermal papilla cells.

Science.gov (United States)

Aljuffali, Ibrahim A; Pan, Tai-Long; Sung, Calvin T; Chang, Shu-Hao; Fang, Jia-You

2015-08-01

This study developed lipid nanocarriers, called squarticles, conjugated with anti-platelet-derived growth factor (PDGF)-receptor β antibody to determine whether targeted Minoxidil (MXD) delivery to the follicles and dermal papilla cells (DPCs) could be achieved. Squalene and hexadecyl palmitate (HP) were used as the matrix of the squarticles. The PDGF-squarticles showed a mean diameter and zeta potential of 195 nm and -46 mV, respectively. Nanoparticle encapsulation enhanced MXD porcine skin deposition from 0.11 to 0.23 μg/mg. The antibody-conjugated nanoparticles ameliorated follicular uptake of MXD by 3-fold compared to that of the control solution in the in vivo mouse model. Both vertical and horizontal skin sections exhibited a wide distribution of nanoparticles in the follicles, epidermis, and deeper skin strata. The encapsulated MXD moderately elicited proliferation of DPCs and vascular endothelial growth factor (VEGF) expression. The active targeting of PDGF-squarticles may be advantageous to improving the limited success of alopecia therapy. Topical use of minoxidil is only one of the very few treatment options for alopecia. Nonetheless, the current delivery method is far from ideal. In this article, the authors developed lipid nanocarriers with anti-platelet-derived growth factor receptor ? antibody to target dermal papilla cells, and showed enhanced uptake of minoxidil. Copyright © 2015 Elsevier Inc. All rights reserved.

Ex-vivo expanded human NK cells express activating receptors that mediate cytotoxicity of allogeneic and autologous cancer cell lines by direct recognition and antibody directed cellular cytotoxicity

Directory of Open Access Journals (Sweden)

Campana Dario

2010-10-01

Full Text Available Abstract Background The possibility that autologous NK cells could serve as an effective treatment modality for solid tumors has long been considered. However, implementation is hampered by (i the small number of NK cells in peripheral blood, (ii the difficulties associated with large-scale production of GMP compliant cytolytic NK cells, (iii the need to activate the NK cells in order to induce NK cell mediated killing and (iv the constraints imposed by autologous inhibitory receptor-ligand interactions. To address these issues, we determined (i if large numbers of NK cells could be expanded from PBMC and GMP compliant cell fractions derived by elutriation, (ii their ability to kill allogeneic and autologous tumor targets by direct cytotoxitiy and by antibody-mediated cellular cytotoxicity and (iii defined NK cell specific receptor-ligand interactions that mediate tumor target cell killing. Methods Human NK cells were expanded during 14 days. Expansion efficiency, NK receptor repertoire before and after expansion, expression of NK specific ligands, cytolytic activity against allogeneic and autologous tumor targets, with and without the addition of chimeric EGFR monoclonal antibody, were investigated. Results Cell expansion shifted the NK cell receptor repertoire towards activation and resulted in cytotoxicity against various allogeneic tumor cell lines and autologous gastric cancer cells, while sparing normal PBMC. Blocking studies confirmed that autologous cytotoxicity is established through multiple activating receptor-ligand interactions. Importantly, expanded NK cells also mediated ADCC in an autologous and allogeneic setting by antibodies that are currently being used to treat patients with select solid tumors. Conclusion These data demonstrate that large numbers of cytolytic NK cells can be generated from PBMC and lymphocyte-enriched fractions obtained by GMP compliant counter current elutriation from PBMC, establishing the preclinical

Structure-based, targeted deglycosylation of HIV-1 gp120 and effects on neutralization sensitivity and antibody recognition

International Nuclear Information System (INIS)

Koch, Markus; Pancera, Marie; Kwong, Peter D.; Kolchinsky, Peter; Grundner, Christoph; Wang Liping; Hendrickson, Wayne A.; Sodroski, Joseph; Wyatt, Richard

2003-01-01

The human immunodeficiency virus (HIV-1) exterior envelope glycoprotein, gp120, mediates receptor binding and is the major target for neutralizing antibodies. Primary HIV-1 isolates are characteristically more resistant to broadly neutralizing antibodies, although the structural basis for this resistance remains obscure. Most broadly neutralizing antibodies are directed against functionally conserved gp120 regions involved in binding to either the primary virus receptor, CD4, or the viral coreceptor molecules that normally function as chemokine receptors. These antibodies are known as CD4 binding site (CD4BS) and CD4-induced (CD4i) antibodies, respectively. Inspection of the gp120 crystal structure reveals that although the receptor-binding regions lack glycosylation, sugar moieties lie proximal to both receptor-binding sites on gp120 and thus in proximity to both the CD4BS and the CD4i epitopes. In this study, guided by the X-ray crystal structure of gp120, we deleted four N-linked glycosylation sites that flank the receptor-binding regions. We examined the effects of selected changes on the sensitivity of two prototypic HIV-1 primary isolates to neutralization by antibodies. Surprisingly, removal of a single N-linked glycosylation site at the base of the gp120 third variable region (V3 loop) increased the sensitivity of the primary viruses to neutralization by CD4BS antibodies. Envelope glycoprotein oligomers on the cell surface derived from the V3 glycan-deficient virus were better recognized by a CD4BS antibody and a V3 loop antibody than were the wild-type glycoproteins. Absence of all four glycosylation sites rendered a primary isolate sensitive to CD4i antibody-mediated neutralization. Thus, carbohydrates that flank receptor-binding regions on gp120 protect primary HIV-1 isolates from antibody-mediated neutralization

Role of the Antigen Capture Pathway in the Induction of a Neutralizing Antibody Response to Anthrax Protective Antigen

Directory of Open Access Journals (Sweden)

Anita Verma

2018-02-01

Full Text Available Toxin neutralizing antibodies represent the major mode of protective immunity against a number of toxin-mediated bacterial diseases, including anthrax; however, the cellular mechanisms that lead to optimal neutralizing antibody responses remain ill defined. Here we show that the cellular binding pathway of anthrax protective antigen (PA, the binding component of anthrax toxin, determines the toxin neutralizing antibody response to this antigen. PA, which binds cellular receptors and efficiently enters antigen-presenting cells by receptor-mediated endocytosis, was found to elicit robust anti-PA IgG and toxin neutralizing antibody responses. In contrast, a receptor binding-deficient mutant of PA, which does not bind receptors and only inefficiently enters antigen-presenting cells by macropinocytosis, elicited very poor antibody responses. A chimeric protein consisting of the receptor binding-deficient PA mutant tethered to the binding subunit of cholera toxin, which efficiently enters cells using the cholera toxin receptor rather than the PA receptor, elicited an anti-PA IgG antibody response similar to that elicited by wild-type PA; however, the chimeric protein elicited a poor toxin neutralizing antibody response. Taken together, our results demonstrate that the antigen capture pathway can dictate the magnitudes of the total IgG and toxin neutralizing antibody responses to PA as well as the ratio of the two responses.

Biochemical and immunological studies of the Muscarinic acetylcholine receptor

International Nuclear Information System (INIS)

Gainer, M.W.

1985-01-01

Muscarinic acetylcholine receptors were solubilized from bovine brain membranes with 3[3-cholamidopropyl)dimethylammonio]propanesulfonate (CHAPS). A combination of 10 mM CHAPS and 1 M NaCl solubilized 15-40% of the specific receptor binding sites from these membranes. The solubilized receptors displayed high affinity binding of the muscarinic antagonist, [ 3 H]quinuclidinyl benzilate with a K/sub D/ = 300 pM. In addition, the solubilized and retained guanyl nucleotide regulation of agonist binding characteristic of membrane bound receptors. Gel filtration experiments showed that solubilized receptors from cortex and cerebellum had different elution profiles. Analysis by sucrose density gradient centrifugation showed that receptors in the lower molecular weight peak sedimented with a coefficient of 5S. Receptors in the larger molecular weight peak sedimented to the bottom of the gradient. Attempts to purify receptors by chromatography on propylbenzilycholine Sepharose were unsuccessful. The technique used to attach the ligand to the solid support, however, was used to synthesize a PrBCM-BSA conjugate and the conjugate used as an antigen in the production of anti-ligand antibodies. Two anti-PrBCM monoclonal antibodies were isolated that recognize muscarinic but not nicotinic cholinergic ligands. The abilities of the antibodies to recognize other muscarinic ligands indicated the antibodies recognized a portion of PrBCM involved in binding to the receptor. Construction of an antibody affinity resin resulted in the purification of this fragment a minimum of 170 fold

Stimulus-response correspondence in go-nogo and choice tasks: Are reactions altered by the presence of an irrelevant salient object?

Science.gov (United States)

Lien, Mei-Ching; Pedersen, Logan; Proctor, Robert W

2016-11-01

In 2-choice tasks, responses are faster when stimulus location corresponds to response location, even when stimulus location is irrelevant. Dolk et al. (J Exp Psychol Hum Percept Perform 39:1248-1260, 2013a) found this stimulus-response correspondence effect with a single response location in a go-nogo task when an irrelevant Japanese waving cat was present. They argued that salient objects trigger spatial coding of the response relative to that object. We examined this claim using both behavioral and lateralized readiness potential (LRP) measures. In Experiment 1 participants determined the pitch of a left- or right-positioned tone, whereas in Experiment 2 they determined the color of a dot within a centrally located hand pointing left, right, or straight ahead. In both experiments, participants performed a go-nogo task with the right-index finger and a 2-choice task with both index fingers, with a left-positioned Japanese waving cat present or absent. For the go-nogo task, the cat induced a correspondence effect on response times (RT) to the tones (Experiment 1) but not the visual stimuli (Experiment 2). For the 2-choice task, a correspondence effect was evident in all conditions in both experiments. Cat's presence/absence did not significantly modulate the effect for right and left responses, although there was a trend toward increased RT and LRP for right responses in Experiment 1. The results imply that a salient, irrelevant object could provide a reference frame for response coding when attention is available to process it, as is likely in an auditory task (Experiment 1) but not a visual task (Experiment 2).

Chronic cannabis use and ERP correlates of visual selective attention during the performance of a flanker go/nogo task.

Science.gov (United States)

Nicholls, Clare; Bruno, Raimondo; Matthews, Allison

2015-09-01

The aim of the study was to investigate the relationship between chronic cannabis use and visual selective attention by examining event-related potentials (ERPs) during the performance of a flanker go/nogo task. Male participants were 15 chronic cannabis users (minimum two years use, at least once per week) and 15 drug naive controls. Cannabis users showed longer reaction times compared to controls with equivalent accuracy. Cannabis users also showed a reduction in the N2 'nogo effect' at frontal sites, particularly for incongruent stimuli, and particularly in the right hemisphere. This suggests differences between chronic cannabis users and controls in terms of inhibitory processing within the executive control network, and may implicate the right inferior frontal cortex. There was also preliminary evidence for differences in early selective attention, with controls but not cannabis users showing modulation of N1 amplitude by flanker congruency. Further investigation is required to examine the potential reversibility of these residual effects after long-term abstinence and to examine the role of early selective attention mechanisms in more detail. Copyright © 2015 Elsevier B.V. All rights reserved.

Lack of antibodies to NMDAR or VGKC-complex in GAD and cardiolipin antibody-positive refractory epilepsy.

Science.gov (United States)

Liimatainen, Suvi; Peltola, Jukka; Hietaharju, Aki; Sabater, Lidia; Lang, Bethan

2014-03-01

Over the last few years autoantibodies against neuronal proteins have been identified in several forms of autoimmune encephalitis and epilepsy. NMDA receptor (NMDAR) and voltage gated potassium channel (VGKC) complex antibodies are mainly associated with limbic encephalitis (LE) whereas glutamic acid decarboxylase antibodies (GADA) and anticardiolipin (ACL) antibodies are more commonly detected in patients with chronic epilepsy. Clinical features vary between these antibodies suggesting the specificity of different neuronal antibodies in seizures. Serum samples of 14 GADA positive and 24 ACL positive patients with refractory epilepsy were analyzed for the presence of VGKC or NMDAR antibodies. No positive VGKC or NMDAR antibodies were found in these patients. The results confirm the different significance of these neuronal antibodies in seizure disorders. Different autoantibodies have different significance in seizures and probably have different pathophysiological mechanisms of actions. Copyright © 2014 Elsevier B.V. All rights reserved.

EFFICACY EVALUATION OF A MONOCLONAL ANTIBODY AGAINST THE EPIDERMAL GROWTH FACTORS RECEPTOR IN THE MODEL OF SUBCUTANEOUS XENOGRAFT IN IMMUNODEFICIENT MICE

Directory of Open Access Journals (Sweden)

Ya. Yu. Ustyugov

2015-01-01

Full Text Available This article presents the results of the comparative antitumor efficacy study of two test articles of therapeutic humanized monoclonal antibodies against epidermal growth factor receptor (EGFR manufactured by Russian biopharmaceutical company CJSC “Biocad” and the commercial drug “Erbitux®” (Merck, Germany in subcutaneous xenografts model using human epidermoid carcinoma A431NS cell line. EGFR overexpression in epithelial tumor cells is a commonly known fact that determines use of this receptor as a target for therapeutic monoclonal antibodies. The basic mechanism of action of such drugs is blocking of epithelial cells proliferation through competitive binding to EGFR. Evaluation of tumor growth dynamics in immunodeficient (Nu/Nu mice was performed during in vivo experiment using two parameters: tumor growth index and tumor growth inhibition (TGI, %. The results received with used study design show that antitumor effects of the test articles manufactured by CJSC “Biocad” and the commercial comparator drug “Erbitux®” estimated by values of TGI and tumor growth index are comparable.

Antibodies to voltage-gated potassium and calcium channels in epilepsy.

NARCIS (Netherlands)

Majoie, H.J.; Baets, M.H.V. de; Renier, W.O.; Lang, B.; Vincent, A.

2006-01-01

OBJECTIVE: To determine the prevalence of antibodies to ion channels in patients with long standing epilepsy. BACKGROUND: Although the CNS is thought to be protected from circulating antibodies by the blood brain barrier, glutamate receptor antibodies have been reported in Rasmussen's encephalitis,

Assessment of estrogen receptor-monoclonal antibody interaction by high performance liquid chromatography

International Nuclear Information System (INIS)

Brandt, D.W.; Wittliff, J.L.

1986-01-01

To define the interrelationships between the various isoforms of the estrogen receptors (ER), a monoclonal antibody-horse radish peroxidase conjugate H222 was used as a probe in conjunction with HPIEC (Synchrom AX-1000) and HPSEC (TSK-3000 SW Toyo Soda) columns. ER from breast tumors was assessed using [16α- 125 I]-iodoestradiol-17β (3nM) +/-200 fold excess estradiol-17β and excess H222. When ER was analyzed by HPSEC (size and shape), with 400 mM KCl which caused the dissociation of ER into 4S isoforms, a shift in retension time to higher molecular weight species was seen. The H222 appeared to interact with most isoforms of ER. However, when ER was analyzed by HPIEC (surface charge) with H222, a shift in virtually all of the high salt (150mM) isoform to the flow-through was observed with only 46% shift in elution of the low salt (60-70mM) isoforms. H222 did not alter total ER binding capacity. These data suggest that H222 recognized discrete forms of the ER. Therefore, modification in the receptor may have occurred which masks or removes the antigenic determinant limiting the specificity of H222. These results indicate that H222 may be employed as a tool to elucidate the interrelationships between these ER species

Radioimmunodetection of human leukemia with anti-interleukin-2 receptor antibody in severe combined immunodeficiency mice

International Nuclear Information System (INIS)

Hosono, Makoto; Takaori-Kondo, Akifumi; Zheng-Sheng, Yao; Kobayashi, Hisataka; Hosono, Masako N.; Sakahara, Harumi; Imada, Kazunori; Okuma, Minoru; Uchiyama, Takashi; Konishi, Junji

1995-01-01

Anti-Tac monoclonal antibody recognizes human interleukin-2 receptor, which is overexpressed in leukemic cells of most adult T-cell leukemia (ATL) patients. To examine the potency of anti-Tac for targeting of ATL, biodistributions of intravenously administered 125 I- and 111 In-labeled anti-Tac were examined in severe combined immunodeficiency (SCID) mice inoculated with ATL cells. Significant amounts of radiolabeled anti-Tac were found in the spleen and thymus. The trafficking of ATL cells in SCID mice was detected using 111 In-oxine-labeled ATL cells. These results were coincident with the histologically confirmed infiltration of ATL cells. The radiolabeled anti-Tac seemed potent for targeting of ATL

Designing two-in-one antibodies.

Science.gov (United States)

Valladares, Ignacio Garcia; Espinoza, Luis R

2009-09-01

Evaluation of: Bostrom J, Shang-Fan Y, Kan D et al.: Variants of the antibody Herceptin that interact with HER2 and VEGF at the antigen binding site. Science 323, 1610-1614 (2009). The longstanding held notion that one antibody equals one antigen and, hence, one function has been challenged in recent years. Improved technology in antibody production, especially the accumulation of sequence data of immunoglobulin genes and the advent of PCR have made it possible to clone antibody gene repertoires. The current paper provides further challenge to the notion of one antibody = one antigen by developing 'two-in-one' antibodies with an antigen-binding site that binds two distinct proteins with high affinity. A therapeutic variant antibody of Herceptin (Genentech, CA, USA) was isolated that binds the human EGF receptor (HER)2 and also to VEGF. This development may represent a breakthrough discovery and may have significant implications in the therapy of malignant, infectious, allergic and autoimmune disorders.

Shared fine specificity between T-cell receptors and an antibody recognizing a peptide/major histocompatibility class I complex

DEFF Research Database (Denmark)

Stryhn, A; Andersen, P S; Pedersen, L O

1996-01-01

Cytotoxic T cells recognize mosaic structures consisting of target peptides embedded within self-major histocompatibility complex (MHC) class I molecules. This structure has been described in great detail for several peptide-MHC complexes. In contrast, how T-cell receptors recognize peptide...... each other showing that peptide residues 1, 3, 4, 6, and 7 were exposed on the MHC surface and recognized by the T cells. Thus, the majority, and perhaps all, of the side chains of the non-primary anchor residues may be available for T-cell recognition, and contribute to the stringent specificity of T...... cells. A striking similarity between the specificity of the T cells and that of the pSAN antibody was found and most of the peptide residues, which could be recognized by the T cells, could also be recognized by the antibody....

Development of an affinity-matured humanized anti-epidermal growth factor receptor antibody for cancer immunotherapy.

Science.gov (United States)

Nakanishi, Takeshi; Maru, Takamitsu; Tahara, Kazuhiro; Sanada, Hideaki; Umetsu, Mitsuo; Asano, Ryutaro; Kumagai, Izumi

2013-02-01

We showed previously that humanization of 528, a murine anti-epidermal growth factor receptor (EGFR) antibody, causes reduced affinity for its target. Here, to improve the affinity of the humanized antibody for use in cancer immunotherapy, we constructed phage display libraries focused on the complementarity-determining regions (CDRs) of the antibody and carried out affinity selection. Two-step selections using libraries constructed in a stepwise manner enabled a 32-fold affinity enhancement of humanized 528 (h528). Thermodynamic analysis of the interactions between the variable domain fragment of h528 (h528Fv) mutants and the soluble extracellular domain of EGFR indicated that the h528Fv mutants obtained from the first selection showed a large increase in negative enthalpy change due to binding, resulting in affinity enhancement. Furthermore, mutants from the second selection showed a decrease in entropy loss, which led to further affinity maturation. These results suggest that a single mutation in the heavy chain variable domain (i.e. Tyr(52) to Trp) enthalpically contributed for overcoming the energetic barrier to the antigen-antibody interaction, which was a major hurdle for the in vitro affinity maturation of h528. We reported previously that the humanized bispecific diabody hEx3 Db, which targets EGFR and CD3, shows strong anti-tumor activity. hEx3 Db mutants, in which the variable domains of h528 were replaced with those of the affinity-enhanced mutants, were prepared and characterized. In a growth inhibition assay of tumor cells, the hEx3 Db mutants showed stronger anti-tumor activity than that of hEx3 Db, suggesting that affinity enhancement of h528Fv enhances the anti-tumor activity of the bispecific diabody.

A recombinant dromedary antibody fragment (VHH or nanobody) directed against human Duffy antigen receptor for chemokines.

Science.gov (United States)

Smolarek, Dorota; Hattab, Claude; Hassanzadeh-Ghassabeh, Gholamreza; Cochet, Sylvie; Gutiérrez, Carlos; de Brevern, Alexandre G; Udomsangpetch, Rachanee; Picot, Julien; Grodecka, Magdalena; Wasniowska, Kazimiera; Muyldermans, Serge; Colin, Yves; Le Van Kim, Caroline; Czerwinski, Marcin; Bertrand, Olivier

2010-10-01

Fy blood group antigens are carried by the Duffy antigen receptor for chemokines (DARC), a red cells receptor for Plasmodium vivax broadly implicated in human health and diseases. Recombinant VHHs, or nanobodies, the smallest intact antigen binding fragment derivative from the heavy chain-only antibodies present in camelids, were prepared from a dromedary immunized against DARC N-terminal extracellular domain and selected for DARC binding. A described VHH, CA52, does recognize native DARC on cells. It inhibits P. vivax invasion of erythrocytes and displaces interleukin-8 bound to DARC. The targeted epitope overlaps the well-defined DARC Fy6 epitope. K (D) of CA52-DARC equilibrium is sub-nanomolar, hence ideal to develop diagnostic or therapeutic compounds. Immunocapture by immobilized CA52 yielded highly purified DARC from engineered K562 cells. This first report on a VHH with specificity for a red blood cell protein exemplifies VHHs' potentialities to target, to purify, and to modulate the function of cellular markers.

Enavatuzumab, a Humanized Anti-TWEAK Receptor Monoclonal Antibody, Exerts Antitumor Activity through Attracting and Activating Innate Immune Effector Cells

Directory of Open Access Journals (Sweden)

Shiming Ye

2017-01-01

Full Text Available Enavatuzumab is a humanized IgG1 anti-TWEAK receptor monoclonal antibody that was evaluated in a phase I clinical study for the treatment of solid malignancies. The current study was to determine whether and how myeloid effector cells were involved in postulated mechanisms for its potent antitumor activity in xenograft models. The initial evidence for a role of effector cells was obtained in a subset of tumor xenograft mouse models whose response to enavatuzumab relied on the binding of Fc of the antibody to Fcγ receptor. The involvement of effector cells was further confirmed by immunohistochemistry, which revealed strong infiltration of CD45+ effector cells into tumor xenografts in responding models, but minimal infiltration in nonresponders. Consistent with the xenograft studies, human effector cells preferentially migrated toward in vivo-responsive tumor cells treated by enavatuzumab in vitro, with the majority of migratory cells being monocytes. Conditioned media from enavatuzumab-treated tumor cells contained elevated levels of chemokines, which might be responsible for enavatuzumab-triggered effector cell migration. These preclinical studies demonstrate that enavatuzumab can exert its potent antitumor activity by actively recruiting and activating myeloid effectors to kill tumor cells. Enavatuzumab-induced chemokines warrant further evaluation in clinical studies as potential biomarkers for such activity.

Nogo-A-deficient Transgenic Rats Show Deficits in Higher Cognitive Functions, Decreased Anxiety, and Altered Circadian Activity Patterns

Czech Academy of Sciences Publication Activity Database

Petrásek, Tomáš; Prokopová, Iva; Sládek, Martin; Weissová, Kamila; Vojtěchová, Iveta; Bahník, Štěpán; Zemanová, Anna; Schönig, K.; Berger, S.; Tews, B.; Bartsch, D.; Schwab, M. E.; Sumová, Alena; Stuchlík, Aleš

2014-01-01

Roč. 8, Mar 18 (2014), s. 90 ISSN 1662-5153 R&D Projects: GA MZd(CZ) NT13386 Grant - others:Rada Programu interní podpory projektů mezinárodní spolupráce AV ČR(CZ) M200111204; Univerzita Karlova(CZ) 365911 Institutional support: RVO:67985823 Keywords : behavior * Nogo-A * circadian system * learning * memory Subject RIV: FH - Neurology Impact factor: 3.270, year: 2014

Human Immunodeficiency Virus Proteins Mimic Human T Cell Receptors Inducing Cross-Reactive Antibodies

Directory of Open Access Journals (Sweden)

Robert Root-Bernstein

2017-10-01

Full Text Available Human immunodeficiency virus (HIV hides from the immune system in part by mimicking host antigens, including human leukocyte antigens. It is demonstrated here that HIV also mimics the V-β-D-J-β of approximately seventy percent of about 600 randomly selected human T cell receptors (TCR. This degree of mimicry is greater than any other human pathogen, commensal or symbiotic organism studied. These data suggest that HIV may be evolving into a commensal organism just as simian immunodeficiency virus has done in some types of monkeys. The gp120 envelope protein, Nef protein and Pol protein are particularly similar to host TCR, camouflaging HIV from the immune system and creating serious barriers to the development of safe HIV vaccines. One consequence of HIV mimicry of host TCR is that antibodies against HIV proteins have a significant probability of recognizing the corresponding TCR as antigenic targets, explaining the widespread observation of lymphocytotoxic autoantibodies in acquired immunodeficiency syndrome (AIDS. Quantitative enzyme-linked immunoadsorption assays (ELISA demonstrated that every HIV antibody tested recognized at least one of twelve TCR, and as many as seven, with a binding constant in the 10−8 to 10−9 m range. HIV immunity also affects microbiome tolerance in ways that correlate with susceptibility to specific opportunistic infections.

Structure of an isolated unglycosylated antibody CH2 domain

International Nuclear Information System (INIS)

Prabakaran, Ponraj; Vu, Bang K.; Gan, Jianhua; Feng, Yang; Dimitrov, Dimiter S.; Ji, Xinhua

2008-01-01

The crystal structure of an isolated unglycosylated antibody C H 2 domain has been determined at 1.7 Å resolution. The C H 2 (C H 3 for IgM and IgE) domain of an antibody plays an important role in mediating effector functions and preserving antibody stability. It is the only domain in human immunoglobulins (Igs) which is involved in weak interchain protein–protein interactions with another C H 2 domain solely through sugar moieties. The N-linked glycosylation at Asn297 is conserved in mammalian IgGs as well as in homologous regions of other antibody isotypes. To examine the structural details of the C H 2 domain in the absence of glycosylation and other antibody domains, the crystal structure of an isolated unglycosylated antibody γ1 C H 2 domain was determined at 1.7 Å resolution and compared with corresponding C H 2 structures from intact Fc, IgG and Fc receptor complexes. Furthermore, the oligomeric state of the protein in solution was studied using size-exclusion chromatography. The results suggested that the unglycosylated human antibody C H 2 domain is a monomer and that its structure is similar to that found in the intact Fc, IgG and Fc receptor complex structures. However, certain structural variations were observed in the Fc receptor-binding sites. Owing to its small size, stability and non-immunogenic Ig template, the C H 2-domain structure could be useful for the development by protein design of antibody domains exerting effector functions and/or antigen specificity and as a robust scaffold in protein-engineering applications

Mycoplasma infection of cell lines can simulate the expression of Fc receptors by binding of the carbohydrate moiety of antibodies.

Science.gov (United States)

Lemke, H; Krausse, R; Lorenzen, J; Havsteen, B

1985-05-01

During the production of Fc receptor (FcR)-bearing hybridomas it was observed with a particular monoclonal anti-sheep red blood cell antibody (anti-SRBC 1/5, IgG1) that the contamination with Mycoplasma arginini of in vitro cultured cell lines leads to an apparent FcR activity. This property did not correspond with the serological typing since other antibodies of the same isotype could not support FcR rosette formation. Another mycoplasma strain M. orale lacked this property. Analysis of the binding reaction revealed that M. arginini contains a lectin which binds the carbohydrate moiety of the anti-SRBC 1/5 antibody, i.e. anti-SRBC 1/5 synthesized under the influence of tunicamycin or deglycosylated by NaIO4 oxidation did not support rosette formation. These data suggest that binding of antibodies to certain mycoplasma strains may be a pathogenic factor during mycoplasma infections by masking the microorganisms with the host's own defense molecules. The experiments with M. arginini-infected cell lines gain immunological importance since we obtained identical results with staphylococcal protein A, as another bacteriological FcR, and cell lines expressing intrinsic membrane FcR. Although it is an open question whether the glycoconjugates are directly bound by the FcR or else by influencing the three-dimensional structure of the antibodies, it seems possible that FcR in general may be lectins.

Development of an EGFRvIII specific recombinant antibody

Directory of Open Access Journals (Sweden)

Li Gordon

2010-10-01

Full Text Available Abstract Background EGF receptor variant III (EGFRvIII is the most common variant of the EGF receptor observed in human tumors. It results from the in frame deletion of exons 2-7 and the generation of a novel glycine residue at the junction of exons 1 and 8. This novel juxtaposition of amino acids within the extra-cellular domain of the EGF receptor creates a tumor specific and immunogenic epitope. EGFRvIII expression has been seen in many tumor types including glioblastoma multiforme (GBM, breast adenocarcinoma, non-small cell lung carcinoma, ovarian adenocarcinoma and prostate cancer, but has been rarely observed in normal tissue. Because this variant is tumor specific and highly immunogenic, it can be used for both a diagnostic marker as well as a target for immunotherapy. Unfortunately many of the monoclonal and polyclonal antibodies directed against EGFRvIII have cross reactivity to wild type EGFR or other non-specific proteins. Furthermore, a monoclonal antibody to EGFRvIII is not readily available to the scientific community. Results In this study, we have developed a recombinant antibody that is specific for EGFRvIII, has little cross reactivity for the wild type receptor, and which can be easily produced. We initially designed a recombinant antibody with two anti-EGFRvIII single chain Fv's linked together and a human IgG1 Fc component. To enhance the specificity of this antibody for EGFRvIII, we mutated tyrosine H59 of the CDRH2 domain and tyrosine H105 of the CDRH3 domain to phenylalanine for both the anti-EGFRvIII sequence inserts. This mutated recombinant antibody, called RAbDMvIII, specifically detects EGFRvIII expression in EGFRvIII expressing cell lines as well as in EGFRvIII expressing GBM primary tissue by western blot, immunohistochemistry (IHC and immunofluorescence (IF and FACS analysis. It does not recognize wild type EGFR in any of these assays. The affinity of this antibody for EGFRvIII peptide is 1.7 × 107 M-1 as

Host-Primed Ebola Virus GP Exposes a Hydrophobic NPC1 Receptor-Binding Pocket, Revealing a Target for Broadly Neutralizing Antibodies.

Science.gov (United States)

Bornholdt, Zachary A; Ndungo, Esther; Fusco, Marnie L; Bale, Shridhar; Flyak, Andrew I; Crowe, James E; Chandran, Kartik; Saphire, Erica Ollmann

2016-02-23

The filovirus surface glycoprotein (GP) mediates viral entry into host cells. Following viral internalization into endosomes, GP is cleaved by host cysteine proteases to expose a receptor-binding site (RBS) that is otherwise hidden from immune surveillance. Here, we present the crystal structure of proteolytically cleaved Ebola virus GP to a resolution of 3.3 Å. We use this structure in conjunction with functional analysis of a large panel of pseudotyped viruses bearing mutant GP proteins to map the Ebola virus GP endosomal RBS at molecular resolution. Our studies indicate that binding of GP to its endosomal receptor Niemann-Pick C1 occurs in two distinct stages: the initial electrostatic interactions are followed by specific interactions with a hydrophobic trough that is exposed on the endosomally cleaved GP1 subunit. Finally, we demonstrate that monoclonal antibodies targeting the filovirus RBS neutralize all known filovirus GPs, making this conserved pocket a promising target for the development of panfilovirus therapeutics. Ebola virus uses its glycoprotein (GP) to enter new host cells. During entry, GP must be cleaved by human enzymes in order for receptor binding to occur. Here, we provide the crystal structure of the cleaved form of Ebola virus GP. We demonstrate that cleavage exposes a site at the top of GP and that this site binds the critical domain C of the receptor, termed Niemann-Pick C1 (NPC1). We perform mutagenesis to find parts of the site essential for binding NPC1 and map distinct roles for an upper, charged crest and lower, hydrophobic trough in cleaved GP. We find that this 3-dimensional site is conserved across the filovirus family and that antibody directed against this site is able to bind cleaved GP from every filovirus tested and neutralize viruses bearing those GPs. Copyright © 2016 Bornholdt et al.

Circulating angiotensin type II receptor: Possible marker for antibody mediated rejection after renal transplantation?

Science.gov (United States)

Kimball, Pamela M; Gupta, Gaurav; McDougan, Felecia

2017-10-01

Presence of antibody [Ab] against angiotensin receptor [AT1R] indicates heightened risk for antibody mediated rejection [AMR] after transplantation but is insufficient as a marker. We speculated AT1R might be released systemically because of AMR and might be a useful biomarker. AT1R was measured in blood from 73 Normals and 72 renal patients pre- and post-transplantation. Patients were stratified as AMR-free [Gp1], AMR1yr [Gp3]. AT1R was higher [13±26vs.367±537, p<0.01)] and more prevalent [20% vs. 92%, p<0.01] among renal patients than Normals. Pretransplant levels were similar [p=ns] between groups. One-year posttransplant levels approached [p<0.01] normalcy for Gps1+3 but spiked during AMR and remained elevated [155±58, p<0.01] for 50% Gp2 patients. One-year AT1R levels were higher among subsequent graft failures than surviving grafts [171±267vs. 38±50, p<0.01]. Pretransplant AT1R was abnormally elevated: possibly indicating ongoing tissue injury. Pretransplant AT1R didn't predict risk for AMR. However, AT1R spiked during early AMR and sustained elevations were associated with poorer outcomes. Copyright © 2017. Published by Elsevier Inc.

Humanized versus murine anti-human epidermal growth factor receptor monoclonal antibodies for immunoscintigraphic studies

Energy Technology Data Exchange (ETDEWEB)

Morales, Alejo A. Morales; Duconge, Jorge; Alvarez-Ruiz, Daniel; Becquer-Viart, Maria de Los Angeles; Nunez-Gandolff, Gilda; Fernandez, Eduardo; Caballero-Torres, Idania; Iznaga-Escobar, Normando

2000-02-01

The anti-human epidermal growth factor receptor (EGF-R) humanized antibody h-R3 (IgG{sub 1}), which binds to an extracellular domain of EGF-R, was used to evaluate the biodistribution on nude mice xenografted with A431 epidermoid carcinoma cell line. Results are compared with its murine version ior egf/r3 monoclonal antibody (mAb). Twenty-one athymic female 4NMRI nu/nu mice were injected intravenously with 10 {mu}g/100 {mu}Ci of {sup 99m}Tc-labeled mAbs. The mAb ior C5 that recognizes an antigen expressed preferentially on the surface of malignant and cytoplasm of normal colorectal cells was used as negative control. Immunoreactivity of {sup 99m}Tc-labeled mAbs was measured by enzyme linked immunosorbent assay on A431 cell line and the immunoreactive fractions determined by Lindmo method. Among all organs significant accumulation was found in tumor (6.14{+-}2.50 %ID/g, 5.06{+-}2.61 %ID/g for murine and humanized mAbs, respectively) 4 h after injection. The immunoreactive fractions were found to be 0.88 and 0.81 for murine and humanized mAb, respectively. Thus, we expect better results using the humanized mAb h-R3 for diagnostic immunoscintigraphy.

Humanized versus murine anti-human epidermal growth factor receptor monoclonal antibodies for immunoscintigraphic studies

International Nuclear Information System (INIS)

Morales, Alejo A. Morales; Duconge, Jorge; Alvarez-Ruiz, Daniel; Becquer-Viart, Maria de Los Angeles; Nunez-Gandolff, Gilda; Fernandez, Eduardo; Caballero-Torres, Idania; Iznaga-Escobar, Normando

2000-01-01

The anti-human epidermal growth factor receptor (EGF-R) humanized antibody h-R3 (IgG 1 ), which binds to an extracellular domain of EGF-R, was used to evaluate the biodistribution on nude mice xenografted with A431 epidermoid carcinoma cell line. Results are compared with its murine version ior egf/r3 monoclonal antibody (mAb). Twenty-one athymic female 4NMRI nu/nu mice were injected intravenously with 10 μg/100 μCi of 99m Tc-labeled mAbs. The mAb ior C5 that recognizes an antigen expressed preferentially on the surface of malignant and cytoplasm of normal colorectal cells was used as negative control. Immunoreactivity of 99m Tc-labeled mAbs was measured by enzyme linked immunosorbent assay on A431 cell line and the immunoreactive fractions determined by Lindmo method. Among all organs significant accumulation was found in tumor (6.14±2.50 %ID/g, 5.06±2.61 %ID/g for murine and humanized mAbs, respectively) 4 h after injection. The immunoreactive fractions were found to be 0.88 and 0.81 for murine and humanized mAb, respectively. Thus, we expect better results using the humanized mAb h-R3 for diagnostic immunoscintigraphy

ERBB oncogene proteins as targets for monoclonal antibodies.

Science.gov (United States)

Polanovski, O L; Lebedenko, E N; Deyev, S M

2012-03-01

General properties of the family of tyrosine kinase ERBB receptors are considered in connection with their role in the generation of cascades of signal transduction in normal and tumor cells. Causes of acquisition of oncogene features by genes encoding these receptors and their role in tumorigenesis are analyzed. Anti-ERBB monoclonal antibodies approved for therapy are described in detail, and mechanisms of their antitumor activity and development of resistance to them are reviewed. The existing and the most promising strategies for creating and using monoclonal antibodies and their derivatives for therapy of cancer are discussed.

The self-nonself discrimination and the nature and acquisition of the antibody repertoire.

Science.gov (United States)

Coutinho, A

1980-01-01

Network ideas are confronted with current hypotheses for the origin of antibody diversity and self-nonself discrimination. The difficulties of reconciling the promethean evolution of the antibody system with "germ line" theories are discussed, as well as the problems of "somatic" hypotheses to explain the completeness of the antibody repertoire. The formal incompatibility of the network theory with ideas basing self-nonself discrimination on the elimination of self-reactive cells is demonstrated, as well as the difficulties of these and other environment-dependent hypotheses for lymphocyte activation, to encompass the internal activity in the immune system. It is argued, on the other hand, that the limitations of the network theory in providing a functional basis for the idiotypic network and in accounting for self-nonself discrimination, can be solved by finding in a complete repertoire of antibody-combining sites the complementary structures to growth receptors on B lymphocytes, and by using these as internal mitogens in the expansion of the precursor cell pools and in the maintenance of the mature steady states. Letting self-nonself discrimination be accounted for by such growth receptors, both the integrity of the antibody repertoire and the internal activity in the system can also be ensured. Moreover, by postulating a germ line origin for the antireceptor antibodies and by accepting idiotypic cross-reactivity between growth receptors and other germ line antibodies, the possibilities are set for a phylogenetically and ontogenically autonomous immune system embodied with the capabilities for self-expansion, diversification and selection of available repertoires. Its promethean characteristics are explained by its completeness, and this is achieved by idiotypic interactions between growth receptors and a limited number of complementary or cross-reactive germ line antibodies, naturally selected on the basis of their structural relationships with growth receptors.

New epitopes and function of anti-M3 muscarinic acetylcholine receptor antibodies in patients with Sjögren's syndrome.

Science.gov (United States)

Tsuboi, H; Matsumoto, I; Wakamatsu, E; Nakamura, Y; Iizuka, M; Hayashi, T; Goto, D; Ito, S; Sumida, T

2010-10-01

M3 muscarinic acetylcholine receptor (M3R) plays a crucial role in the secretion of saliva from salivary glands. It is reported that some patients with Sjögren's syndrome (SS) carried inhibitory autoantibodies against M3R. The purpose of this study is to clarify the epitopes and function of anti-M3R antibodies in SS. We synthesized peptides encoding the extracellular domains of human-M3R including the N-terminal region and the first, second and third extracellular loops. Antibodies against these regions were examined by enzyme-linked immunosorbent assay in sera from 42 SS and 42 healthy controls. For functional analysis, human salivary gland (HSG) cells were preincubated with immunoglobulin G (IgG) separated from sera of anti-M3R antibody-positive SS, -negative SS and controls for 12 h. After loading with Fluo-3, HSG cells were stimulated with cevimeline hydrochloride, and intracellular Ca(2+) concentrations [(Ca(2+) )i] were measured. Antibodies to the N-terminal, first, second and third loops were detected in 42·9% (18 of 42), 47·6% (20 of 42), 54·8% (23 of 42) and 45·2% (19 of 42) of SS, while in 4·8% (two of 42), 7·1% (three of 42), 2·4% (one of 42) and 2·4% (one of 42) of controls, respectively. Antibodies to the second loop positive SS-IgG inhibited the increase of (Ca(2+) )i induced by cevimeline hydrochloride. Antibodies to the N-terminal positive SS-IgG and antibodies to the first loop positive SS-IgG enhanced it, while antibodies to the third loop positive SS-IgG showed no effect on (Ca(2+) )i as well as anti-M3R antibody-negative SS-IgG. Our results indicated the presence of several B cell epitopes on M3R in SS. The influence of anti-M3R antibodies on salivary secretion might differ based on these epitopes. © 2010 The Authors. Clinical and Experimental Immunology © 2010 British Society for Immunology.

Participation of L3T4 in T cell activation in the absence of class II major histocompatibility complex antigens. Inhibition by anti-L3T4 antibodies is a function both of epitope density and mode of presentation of anti-receptor antibody

DEFF Research Database (Denmark)

Owens, T; Fazekas de St Groth, B

1987-01-01

two monoclonal antibodies, KJ16-133.18 and F23.1, that recognize a determinant encoded by the T cell receptor V beta 8 gene family. These antibodies were used to select two clones of T cells with surface phenotype Thy-1.2+, L3T4+, Lyt-2-, KJ16-133.18+, F23.1+, IA-, IE-. One of these clones (E9.D4......The recognition of many class II major histocompatibility complex (MHC)-associated antigens by T cells requires the participation of the L3T4 molecule. It has been proposed that this molecule acts to stabilize low affinity binding to antigen in association with MHC and thereby increases the avidity...... of T cell/antigen interactions. By using antibodies against the T cell antigen receptor (TCR) to activate T cells, thereby circumventing the requirement for antigen presenting cells and MHC-associated antigen, we have been able to study the function of L3T4 in the absence of class II MHC. We have used...

Anti-Idiotypic Antibodies Specific to prM Monoantibody Prevent Antibody Dependent Enhancement of Dengue Virus Infection

Directory of Open Access Journals (Sweden)

Miao Wang

2017-05-01

Full Text Available Dengue virus (DENV co-circulates as four serotypes (DENV1-4. Primary infection only leads to self-limited dengue fever. But secondary infection with another serotype carries a higher risk of increased disease severity, causing life-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS. Serotype cross-reactive antibodies facilitate DENV infection in Fc-receptor-bearing cells by promoting virus entry via Fcγ receptors (FcγR, a process known as antibody dependent enhancement (ADE. Most studies suggested that enhancing antibodies were mainly specific to the structural premembrane protein (prM of DENV. However, there is still no effective drugs or vaccines to prevent ADE. In this study, we firstly confirmed that both DENV-2 infected human sera (anti-DENV-2 and DENV-2 prM monoclonal antibody (prM mAb could significantly enhance DENV-1 infection in K562 cells. Then we developed anti-idiotypic antibodies (prM-AIDs specific to prM mAb by immunizing of Balb/c mice. Results showed that these polyclonal antibodies can dramatically reduce ADE phenomenon of DENV-1 infection in K562 cells. To further confirm the anti-ADE effect of prM-AIDs in vivo, interferon-α and γ receptor-deficient mice (AG6 were used as the mouse model for DENV infection. We found that administration of DENV-2 prM mAb indeed caused a higher DENV-1 titer as well as interleukin-10 (IL-10 and alaninea minotransferase (ALT in mice infected with DENV-1, similar to clinical ADE symptoms. But when we supplemented prM-AIDs to DENV-1 challenged AG6 mice, the viral titer, IL-10 and ALT were obviously decreased to the negative control level. Of note, the number of platelets in peripheral blood of prM-AIDs group were significantly increased at day 3 post infection with DENV-1 compared that of prM-mAb group. These results confirmed that our prM-AIDs could prevent ADE not only in vitro but also in vivo, suggested that anti-idiotypic antibodies might be a new choice to be considered to

Anti-Idiotypic Antibodies Specific to prM Monoantibody Prevent Antibody Dependent Enhancement of Dengue Virus Infection.

Science.gov (United States)

Wang, Miao; Yang, Fan; Huang, Dana; Huang, Yalan; Zhang, Xiaomin; Wang, Chao; Zhang, Shaohua; Zhang, Renli

2017-01-01

Dengue virus (DENV) co-circulates as four serotypes (DENV1-4). Primary infection only leads to self-limited dengue fever. But secondary infection with another serotype carries a higher risk of increased disease severity, causing life-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Serotype cross-reactive antibodies facilitate DENV infection in Fc-receptor-bearing cells by promoting virus entry via Fcγ receptors (FcγR), a process known as antibody dependent enhancement (ADE). Most studies suggested that enhancing antibodies were mainly specific to the structural premembrane protein (prM) of DENV. However, there is still no effective drugs or vaccines to prevent ADE. In this study, we firstly confirmed that both DENV-2 infected human sera (anti-DENV-2) and DENV-2 prM monoclonal antibody (prM mAb) could significantly enhance DENV-1 infection in K562 cells. Then we developed anti-idiotypic antibodies (prM-AIDs) specific to prM mAb by immunizing of Balb/c mice. Results showed that these polyclonal antibodies can dramatically reduce ADE phenomenon of DENV-1 infection in K562 cells. To further confirm the anti-ADE effect of prM-AIDs in vivo , interferon-α and γ receptor-deficient mice (AG6) were used as the mouse model for DENV infection. We found that administration of DENV-2 prM mAb indeed caused a higher DENV-1 titer as well as interleukin-10 (IL-10) and alaninea minotransferase (ALT) in mice infected with DENV-1, similar to clinical ADE symptoms. But when we supplemented prM-AIDs to DENV-1 challenged AG6 mice, the viral titer, IL-10 and ALT were obviously decreased to the negative control level. Of note, the number of platelets in peripheral blood of prM-AIDs group were significantly increased at day 3 post infection with DENV-1 compared that of prM-mAb group. These results confirmed that our prM-AIDs could prevent ADE not only in vitro but also in vivo , suggested that anti-idiotypic antibodies might be a new choice to be considered to treat

The Role of Natural Antibodies to CC Chemokine Receptor 5 in HIV Infection

Directory of Open Access Journals (Sweden)

Assunta Venuti

2017-10-01

Full Text Available The CC chemokine receptor 5 (CCR5 is responsible for immune and inflammatory responses by mediation of chemotactic activity in leukocytes, although it is expressed on different cell types. It has been shown to act as co-receptor for the human and simian immunodeficiency viruses (HIV-1, HIV-2, and SIV. Natural reactive antibodies (Abs recognizing first loop (ECL1 of CCR5 have been detected in several pools of immunoglobulins from healthy donors and from several cohorts of either HIV-exposed but uninfected subjects (ESN or HIV-infected individuals who control disease progression (LTNP as well. The reason of development of anti-CCR5 Abs in the absence of autoimmune disease is still unknown; however, the presence of these Abs specific for CCR5 or for other immune receptors and mediators probably is related to homeostasis maintenance. The majority of anti-CCR5 Abs is directed to HIV binding site (N-terminus and ECL2 of the receptor. Conversely, it is well known that ECL1 of CCR5 does not bind HIV; thus, the anti-CCR5 Abs directed to ECL1 elicit a long-lasting internalization of CCR5 but not interfere with HIV binding directly; these Abs block HIV infection in either epithelial cells or CD4+ T lymphocytes and the mechanism differs from those ones described for all other CCR5-specific ligands. The Ab-mediated CCR5 internalization allows the formation of a stable signalosome by interaction of CCR5, β-arrestin2 and ERK1 proteins. The signalosome degradation and the subsequent de novo proteins synthesis determine the CCR5 reappearance on the cell membrane with a very long-lasting kinetics (8 days. The use of monoclonal Abs to CCR5 with particular characteristics and mode of action may represent a novel mode to fight viral infection in either vaccinal or therapeutic strategies.

Effect of producer cell line on functional activity of anti-D monoclonal antibodies destined for prevention of rhesus sensitization.

Science.gov (United States)

Olovnikova, N I; Ershler, M A; Belkina, E V; Nikolaeva, T L; Miterev, G Yu

2009-04-01

The ability of anti-D antibodies to cause antigen-specific immunosuppression depends on their interaction with low-affinity Fcgamma-receptors. Human monoclonal antibodies to D antigen of the rhesus system were investigated by antibody-dependent cytotoxicity assay in order to estimate their ability to induce hemolysis mediated by low-affinity Fcgamma receptors. We demonstrate that affinity of monoclonal antibodies to receptors of this type does not depend on primary structure of Fc-fragment, but depends on the producer cell line which expresses the antibodies. Monoclonal IgG1 antibodies interacting with FcgammaRIIa and FcgammaRIII lost this property, if they were secreted by human-mouse heterohybridoma, but not by human B-cell line. On the opposite, monoclonal antibodies that could not activate low-affinity Fcgamma receptors were highly active after human cells fusion with rat myeloma YB2/0. Hemolytic activity of IgG3 remained unchanged after fusion of human cells with rodent cells.

Receptor for advanced glycation end products (RAGE) functions as receptor for specific sulfated glycosaminoglycans, and anti-RAGE antibody or sulfated glycosaminoglycans delivered in vivo inhibit pulmonary metastasis of tumor cells.

Science.gov (United States)

Mizumoto, Shuji; Takahashi, Jun; Sugahara, Kazuyuki

2012-06-01

Altered expression of chondroitin sulfate (CS) and heparan sulfate (HS) at the surfaces of tumor cells plays a key role in malignant transformation and tumor metastasis. Previously we demonstrated that a Lewis lung carcinoma (LLC)-derived tumor cell line with high metastatic potential had a higher proportion of E-disaccharide units, GlcUA-GalNAc(4,6-O-disulfate), in CS chains than low metastatic LLC cells and that such CS chains are involved in the metastatic process. The metastasis was markedly inhibited by the pre-administration of CS-E from squid cartilage rich in E units or by preincubation with a phage display antibody specific for CS-E. However, the molecular mechanism of the inhibition remains to be investigated. In this study the receptor molecule for CS chains containing E-disaccharides expressed on LLC cells was revealed to be receptor for advanced glycation end products (RAGE), which is a member of the immunoglobulin superfamily predominantly expressed in the lung. Interestingly, RAGE bound strongly to not only E-disaccharide, but also HS-expressing LLC cells. Furthermore, the colonization of the lungs by LLC cells was effectively inhibited by the blocking of CS or HS chains at the tumor cell surface with an anti-RAGE antibody through intravenous injections in a dose-dependent manner. These results provide the clear evidence that RAGE is at least one of the critical receptors for CS and HS chains expressed at the tumor cell surface and involved in experimental lung metastasis and that CS/HS and RAGE are potential molecular targets in the treatment of pulmonary metastasis.

Anti-neuronal anti-bodies in patients with early psychosis.

Science.gov (United States)

Mantere, O; Saarela, M; Kieseppä, T; Raij, T; Mäntylä, T; Lindgren, M; Rikandi, E; Stoecker, W; Teegen, B; Suvisaari, J

2018-02-01

It may be challenging to distinguish autoimmune encephalitis associated with anti-neuronal autoantibodies from primary psychiatric disorders. Here, serum was drawn from patients with a first-episode psychosis (n=70) or a clinical high-risk for psychosis (n=6) and controls (n=34). We investigated the serum prevalence of 24 anti-neuronal autoantibodies: IgG antibodies for anti-N-methyl-d-aspartate-type glutamate receptor (anti-NMDAR), glutamate and γ-aminobutyric acid alpha and beta receptors (GABA-a, GABA-b), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA), glycine receptor (GlyR), metabotropic glutamate receptor 1 and 5 (mGluR1, mGluR5), anti-Tr/Delta/notch-like epidermal growth factor-related receptor (DNER), contactin-associated protein-like 2 (CASPR2), myelin oligodendrocyte glycoprotein (MOG), glutamic acid decarboxylase-65 (GAD65), collapsin response mediator protein 5/crossveinless-2 (CV2), aquaporin-4 (AQP4), anti-dipeptidyl-peptidase-like protein-6 (DPPX), type 1 anti-neuronal nuclear antibody (ANNA-1, Hu), Ri, Yo, IgLON5, Ma2, zinc finger protein 4 (ZIC4), Rho GTPase-activating protein 26, amphiphysin, and recoverin, as well as IgA and IgM for dopamine-2-receptor (DRD2). Anti-NMDA IgG antibodies were positive with serum titer 1:320 in one patient with a clinical high risk for psychosis. He did not receive a diagnosis of encephalitis after comprehensive neurological evaluation. All other antineuronal autoantibodies were negative and there were no additional findings with immunohistochemistry of brain issues. Copyright © 2017 Elsevier B.V. All rights reserved.

A first‐in‐human pharmacodynamic and pharmacokinetic study of a fully human anti‐glucagon receptor monoclonal antibody in normal healthy volunteers

Science.gov (United States)

Kostic, Ana; King, Thomas Alexander; Yang, Feng; Chan, Kuo‐Chen; Yancopoulos, George D.; Gromada, Jesper

2017-01-01

Aims Glucagon receptor (GCGR) blockers are being investigated as potential therapeutics for type 1 and type 2 diabetes. Here we report the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) of REGN1193, a fully human glucagon receptor blocking monoclonal antibody from a first‐in‐human healthy volunteer randomized double‐blinded trial. Methods Healthy men and women received single ascending doses of REGN1193 ranging from 0.05 to 0.6 mg/kg (n = 42) or placebo (n = 14) intravenously. Safety, tolerability and PK were assessed over 106 days. The glucose‐lowering effect of REGN1193 was assessed after induction of hyperglycaemia by serial glucagon challenges. Results REGN1193 was generally well tolerated. There were small (50 mg/dL, and did not require treatment or medical assistance. Concentration‐time profiles suggest a 2‐compartment disposition and marked nonlinearity, consistent with target‐mediated clearance. REGN1193 inhibited the glucagon‐stimulated glucose increase in a dose‐dependent manner. The 0.6 mg/kg dose inhibited the glucagon‐induced glucose area under the curve for 0 to 90 minutes (AUC0‐90 minutes) by 80% to 90% on days 3 and 15, while blunting the increase in C‐peptide. REGN1193 dose‐dependently increased total GLP‐1, GLP‐2 and glucagon, with plasma levels returning to baseline by day 29 in all dose groups. Conclusion REGN1193, a GCGR‐blocking monoclonal antibody, produced a safety, tolerability and PK/PD profile suitable for further clinical development. The occurrence of transient elevations in serum hepatic aminotransferases observed here and reported with several small molecule glucagon receptor antagonists suggests an on‐target effect of glucagon receptor blockade. The underlying mechanism is unknown. PMID:28755409

Somatostatin receptor scintigraphy in patients with cat-scratch disease

International Nuclear Information System (INIS)

Krause, R.; Schnedl, W.J.; Hoier, S.; Piswanger-Soelkner, C.; Lipp, R.W.; Daxboeck, F.; Reisinger, E.C.

2006-01-01

Aim: somatostatin receptor scintigraphy images various neoplastic, granulomatous, and auto-immun diseases. Cat-scratch disease in an infectious granulomatous disease usually affecting the lymphnodes. It is not known whether cat-scratch disease provides positive somatostatin receptor scintigrams. Patients, methods: twelve patients with lymphadenitis and suspected cat-scratch disease were investigated by immunofluorescence antibody testing and somatostatin receptor scintigraphy. Suppurated lymphnodes were extracted or drained and Bartonella henselae specific PCR was then performed. Results: eleven of 12 patients showed IgG antibodies against B. henselea. SRS showed positive scintigraphic results in 6 of 11 patients with CSD. B. henselae DNA was detected in tissue of lymphnodes from 4 of 5 patients with lymphnode extraction or lymphnode drainage. SRS demonstrated positive scintigrams in all patients with a positive PCR. In one patient with suspected CSD SRS was negative as well as antibody testing. Conclusion: somatostatin receptor scintigraphy correlated with positive Bartonella henselae specific PCR tests and positive Bartonella henselae specific antibody tests in patients with CSD. (orig.)

Somatostatin receptor scintigraphy in patients with cat-scratch disease

Energy Technology Data Exchange (ETDEWEB)

Krause, R.; Schnedl, W.J.; Hoier, S. [Div. of Infectious Diseases, Dept. of Internal Medicine, Univ. Graz (Austria); Piswanger-Soelkner, C.; Lipp, R.W. [Div. of Nuclear Medicine, Dept. of Internal Medicine, Univ. Graz (Austria); Daxboeck, F. [Clinical Inst. for Hygiene and Medical Microbiology, Div. of Hospital Hygiene, Univ. of Vienna (Austria); Reisinger, E.C. [Div. of Infectious Diseases and Tropical Medicine, Dept. of Internal Medicine, Univ. Rostock (Germany)

2006-07-01

Aim: somatostatin receptor scintigraphy images various neoplastic, granulomatous, and auto-immun diseases. Cat-scratch disease in an infectious granulomatous disease usually affecting the lymphnodes. It is not known whether cat-scratch disease provides positive somatostatin receptor scintigrams. Patients, methods: twelve patients with lymphadenitis and suspected cat-scratch disease were investigated by immunofluorescence antibody testing and somatostatin receptor scintigraphy. Suppurated lymphnodes were extracted or drained and Bartonella henselae specific PCR was then performed. Results: eleven of 12 patients showed IgG antibodies against B. henselea. SRS showed positive scintigraphic results in 6 of 11 patients with CSD. B. henselae DNA was detected in tissue of lymphnodes from 4 of 5 patients with lymphnode extraction or lymphnode drainage. SRS demonstrated positive scintigrams in all patients with a positive PCR. In one patient with suspected CSD SRS was negative as well as antibody testing. Conclusion: somatostatin receptor scintigraphy correlated with positive Bartonella henselae specific PCR tests and positive Bartonella henselae specific antibody tests in patients with CSD. (orig.)

Identification of antibody glycosylation structures that predict monoclonal antibody Fc-effector function.

Science.gov (United States)

Chung, Amy W; Crispin, Max; Pritchard, Laura; Robinson, Hannah; Gorny, Miroslaw K; Yu, Xiaojie; Bailey-Kellogg, Chris; Ackerman, Margaret E; Scanlan, Chris; Zolla-Pazner, Susan; Alter, Galit

2014-11-13

To determine monoclonal antibody (mAb) features that predict fragment crystalizable (Fc)-mediated effector functions against HIV. Monoclonal antibodies, derived from Chinese hamster ovary cells or Epstein-Barr virus-immortalized mouse heteromyelomas, with specificity to key regions of the HIV envelope including gp120-V2, gp120-V3 loop, gp120-CD4(+) binding site, and gp41-specific antibodies, were functionally profiled to determine the relative contribution of the variable and constant domain features of the antibodies in driving robust Fc-effector functions. Each mAb was assayed for antibody-binding affinity to gp140(SR162), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and for the ability to bind to FcγRIIa, FcγRIIb and FcγRIIIa receptors. Antibody glycan profiles were determined by HPLC. Neither the specificity nor the affinity of the mAbs determined the potency of Fc-effector function. FcγRIIIa binding strongly predicted ADCC and decreased galactose content inversely correlated with ADCP, whereas N-glycolylneuraminic acid-containing structures exhibited enhanced ADCP. Additionally, the bi-antenary glycan arm onto which galactose was added predicted enhanced binding to FcγRIIIa and ADCC activity, independent of the specificity of the mAb. Our studies point to the specific Fc-glycan structures that can selectively promote Fc-effector functions independently of the antibody specificity. Furthermore, we demonstrated antibody glycan structures associated with enhanced ADCP activity, an emerging Fc-effector function that may aid in the control and clearance of HIV infection.

Improved decision making for prioritizing tumor targeting antibodies in human xenografts: Utility of fluorescence imaging to verify tumor target expression, antibody binding and optimization of dosage and application schedule.

Science.gov (United States)

Dobosz, Michael; Haupt, Ute; Scheuer, Werner

2017-01-01

Preclinical efficacy studies of antibodies targeting a tumor-associated antigen are only justified when the expression of the relevant antigen has been demonstrated. Conventionally, antigen expression level is examined by immunohistochemistry of formalin-fixed paraffin-embedded tumor tissue section. This method represents the diagnostic "gold standard" for tumor target evaluation, but is affected by a number of factors, such as epitope masking and insufficient antigen retrieval. As a consequence, variances and discrepancies in histological staining results can occur, which may influence decision-making and therapeutic outcome. To overcome these problems, we have used different fluorescence-labeled therapeutic antibodies targeting human epidermal growth factor receptor (HER) family members and insulin-like growth factor-1 receptor (IGF1R) in combination with fluorescence imaging modalities to determine tumor antigen expression, drug-target interaction, and biodistribution and tumor saturation kinetics in non-small cell lung cancer xenografts. For this, whole-body fluorescence intensities of labeled antibodies, applied as a single compound or antibody mixture, were measured in Calu-1 and Calu-3 tumor-bearing mice, then ex vivo multispectral tumor tissue analysis at microscopic resolution was performed. With the aid of this simple and fast imaging method, we were able to analyze the tumor cell receptor status of HER1-3 and IGF1R, monitor the antibody-target interaction and evaluate the receptor binding sites of anti-HER2-targeting antibodies. Based on this, the most suitable tumor model, best therapeutic antibody, and optimal treatment dosage and application schedule was selected. Predictions drawn from obtained imaging data were in excellent concordance with outcome of conducted preclinical efficacy studies. Our results clearly demonstrate the great potential of combined in vivo and ex vivo fluorescence imaging for the preclinical development and characterization of

Growth promotion of genetically modified hematopoietic progenitors using an antibody/c-Mpl chimera.

Science.gov (United States)

Kawahara, Masahiro; Chen, Jianhong; Sogo, Takahiro; Teng, Jinying; Otsu, Makoto; Onodera, Masafumi; Nakauchi, Hiromitsu; Ueda, Hiroshi; Nagamune, Teruyuki

2011-09-01

Thrombopoietin is a potent cytokine that exerts proliferation of hematopoietic stem cells (HSCs) through its cognate receptor, c-Mpl. Therefore, mimicry of c-Mpl signaling by a receptor recognizing an artificial ligand would be attractive to attain specific expansion of genetically modified HSCs. Here we propose a system enabling selective expansion of genetically modified cells using an antibody/receptor chimera that can be activated by a specific antigen. We constructed an antibody/c-Mpl chimera, in which single-chain Fv (ScFv) of an anti-fluorescein antibody was tethered to the extracellular D2 domain of the erythropoietin receptor and transmembrane/cytoplasmic domains of c-Mpl. When the chimera was expressed in interleukin (IL)-3-dependent pro-B cell line Ba/F3, genetically modified cells were selectively expanded in the presence of fluorescein-conjugated BSA (BSA-FL) as a specific antigen. Furthermore, highly purified mouse HSCs transduced with the retrovirus carrying antibody/c-Mpl chimera gene proliferated in vitro in response to BSA-FL, and the cells retained in vivo long-term repopulating abilities. These results demonstrate that the antibody/c-Mpl chimera is capable of signal transduction that mimics wild-type c-Mpl signaling. Copyright © 2011 Elsevier Ltd. All rights reserved.

Impaired antibody response causes persistence of prototypic T cell-contained virus.

Directory of Open Access Journals (Sweden)

Andreas Bergthaler

2009-04-01

Full Text Available CD8 T cells are recognized key players in control of persistent virus infections, but increasing evidence suggests that assistance from other immune mediators is also needed. Here, we investigated whether specific antibody responses contribute to control of lymphocytic choriomeningitis virus (LCMV, a prototypic mouse model of systemic persistent infection. Mice expressing transgenic B cell receptors of LCMV-unrelated specificity, and mice unable to produce soluble immunoglobulin M (IgM exhibited protracted viremia or failed to resolve LCMV. Virus control depended on immunoglobulin class switch, but neither on complement cascades nor on Fc receptor gamma chain or Fc gamma receptor IIB. Cessation of viremia concurred with the emergence of viral envelope-specific antibodies, rather than with neutralizing serum activity, and even early nonneutralizing IgM impeded viral persistence. This important role for virus-specific antibodies may be similarly underappreciated in other primarily T cell-controlled infections such as HIV and hepatitis C virus, and we suggest this contribution of antibodies be given consideration in future strategies for vaccination and immunotherapy.

Fcγ receptor-mediated inflammation inhibits axon regeneration.

Directory of Open Access Journals (Sweden)

Gang Zhang

Full Text Available Anti-glycan/ganglioside antibodies are the most common immune effectors found in patients with Guillain-Barré Syndrome, which is a peripheral autoimmune neuropathy. We previously reported that disease-relevant anti-glycan autoantibodies inhibited axon regeneration, which echo the clinical association of these antibodies and poor recovery in Guillain-Barré Syndrome. However, the specific molecular and cellular elements involved in this antibody-mediated inhibition of axon regeneration are not previously defined. This study examined the role of Fcγ receptors and macrophages in the antibody-mediated inhibition of axon regeneration. A well characterized antibody passive transfer sciatic nerve crush and transplant models were used to study the anti-ganglioside antibody-mediated inhibition of axon regeneration in wild type and various mutant and transgenic mice with altered expression of specific Fcγ receptors and macrophage/microglia populations. Outcome measures included behavior, electrophysiology, morphometry, immunocytochemistry, quantitative real-time PCR, and western blotting. We demonstrate that the presence of autoantibodies, directed against neuronal/axonal cell surface gangliosides, in the injured mammalian peripheral nerves switch the proregenerative inflammatory environment to growth inhibitory milieu by engaging specific activating Fcγ receptors on recruited monocyte-derived macrophages to cause severe inhibition of axon regeneration. Our data demonstrate that the antibody orchestrated Fcγ receptor-mediated switch in inflammation is one mechanism underlying inhibition of axon regeneration. These findings have clinical implications for nerve repair and recovery in antibody-mediated immune neuropathies. Our results add to the complexity of axon regeneration in injured peripheral and central nervous systems as adverse effects of B cells and autoantibodies on neural injury and repair are increasingly recognized.

Anti-N-Methyl-D-Aspartate Receptor Encephalitis and Rasmussen-like Syndrome: An Association?

Science.gov (United States)

Gurcharran, Kevin; Karkare, Shefali

2017-01-01

N-methyl-D-aspartate (NMDA) receptor encephalitis is an immune-mediated condition that has a broad spectrum of manifestations, including seizures, coma, psychosis, and focal neurological deficits. Although usually a diffuse process, unihemispheric involvement mimicking early stages of Rasmussen encephalitis can occur. Rasmussen's encephalitis is a unique syndrome characterized by progressive hemiplegia, drug-resistant focal epilepsy, cognitive decline, and hemispheric brain atrophy contralateral to the hemiplegia. We describe a two-year-old girl with progressive right weakness and epilepsia partialis continua, concerning for early Rasmussen's encephalitis, who tested positive for anti-NMDA receptor antibodies. She experienced complete clinical recovery after immunotherapy. Anti-NMDA receptor antibodies were absent at three weeks and again at one year after the first treatment of intravenous immunoglobulin. There are few reports of Rasmussen-like encephalitis in individuals with anti-NMDA receptor antibody positivity. Thus the clinical significance of this association is yet to be determined. In addition, several other antibodies have been documented in individuals with Rasmussen encephalitis. The lack of a consistently reported antibody in Rasmussen encephalitis patients and the temporary nature of the anti-NMDA receptor antibody in our patient raise the following question: Is the presence of anti-NMDA receptor antibodies the cause of the symptoms or secondary to the pathogenic process? Copyright © 2016 Elsevier Inc. All rights reserved.

Acquired Antibody Responses against Plasmodium vivax Infection Vary with Host Genotype for Duffy Antigen Receptor for Chemokines (DARC)

Science.gov (United States)

Maestre, Amanda; Muskus, Carlos; Duque, Victoria; Agudelo, Olga; Liu, Pu; Takagi, Akihide; Ntumngia, Francis B.; Adams, John H.; Sim, Kim Lee; Hoffman, Stephen L.; Corradin, Giampietro; Velez, Ivan D.; Wang, Ruobing

2010-01-01

Background Polymorphism of the Duffy Antigen Receptor for Chemokines (DARC) is associated with susceptibility to and the severity of Plasmodium vivax malaria in humans. P. vivax uses DARC to invade erythrocytes. Individuals lacking DARC are ‘resistant’ to P. vivax erythrocytic infection. However, susceptibility to P. vivax in DARC+ individuals is reported to vary between specific DARC genotypes. We hypothesized that the natural acquisition of antibodies to P. vivax blood stages may vary with the host genotype and the level of DARC expression. Furthermore, high parasitemia has been reported to effect the acquisition of immunity against pre-erythrocytic parasites. We investigated the correlation between host DARC genotypes and the frequency and magnitude of antibodies against P. vivax erythrocytic stage antigens. Methodology/Findings We assessed the frequencies and magnitudes of antibody responses against P. vivax and P. falciparum sporozoite and erythrocytic antigens in Colombian donors from malaria-endemic regions. The frequency and level of naturally-acquired antibodies against the P. vivax erythrocytic antigens merozoite surface protein 1 (PvMSP1) and Duffy binding protein (PvDBP) varied with the host DARC genotypes. Donors with one negative allele (FY*B/FY*Bnull and FY*A/FY*Bnull) were more likely to have anti-PvMSP1 and anti-PvDBP antibodies than those with two positive alleles (FY*B/FY*B and FY*A/FY*B). The lower IgG3 and IgG1 components of the total IgG response may account for the decreased responses to P. vivax erythrocytic antigens with FY*A/FY*B and FY*B/FY*B genotypes. No such association was detected with P. falciparum erythrocytic antigens, which does not use DARC for erythrocyte invasion. Conclusion/Significance Individuals with higher DARC expression, which is associated with higher susceptibility to P. vivax infection, exhibited low frequencies and magnitudes of P. vivax blood-stage specific antibody responses. This may indicate that one of the

Acquired antibody responses against Plasmodium vivax infection vary with host genotype for duffy antigen receptor for chemokines (DARC.

Directory of Open Access Journals (Sweden)

Amanda Maestre

2010-07-01

Full Text Available Polymorphism of the Duffy Antigen Receptor for Chemokines (DARC is associated with susceptibility to and the severity of Plasmodium vivax malaria in humans. P. vivax uses DARC to invade erythrocytes. Individuals lacking DARC are 'resistant' to P. vivax erythrocytic infection. However, susceptibility to P. vivax in DARC+ individuals is reported to vary between specific DARC genotypes. We hypothesized that the natural acquisition of antibodies to P. vivax blood stages may vary with the host genotype and the level of DARC expression. Furthermore, high parasitemia has been reported to effect the acquisition of immunity against pre-erythrocytic parasites. We investigated the correlation between host DARC genotypes and the frequency and magnitude of antibodies against P. vivax erythrocytic stage antigens.We assessed the frequencies and magnitudes of antibody responses against P. vivax and P. falciparum sporozoite and erythrocytic antigens in Colombian donors from malaria-endemic regions. The frequency and level of naturally-acquired antibodies against the P. vivax erythrocytic antigens merozoite surface protein 1 (PvMSP1 and Duffy binding protein (PvDBP varied with the host DARC genotypes. Donors with one negative allele (FY*B/FY*Bnull and FY*A/FY*Bnull were more likely to have anti-PvMSP1 and anti-PvDBP antibodies than those with two positive alleles (FY*B/FY*B and FY*A/FY*B. The lower IgG3 and IgG1 components of the total IgG response may account for the decreased responses to P. vivax erythrocytic antigens with FY*A/FY*B and FY*B/FY*B genotypes. No such association was detected with P. falciparum erythrocytic antigens, which does not use DARC for erythrocyte invasion.Individuals with higher DARC expression, which is associated with higher susceptibility to P. vivax infection, exhibited low frequencies and magnitudes of P. vivax blood-stage specific antibody responses. This may indicate that one of the primary mechanisms by which P. vivax evades

Production and characterization of a murine monoclonal IgM antibody to human C1q receptor (C1qR)

International Nuclear Information System (INIS)

Ghebrehiwet, B.

1986-01-01

A hybridoma cell line that produces a monoclonal antibody (MAb) to cell surface C1q receptor (C1qr) has been produced by fusion of the P3 x 63-Ag8.653 mouse myeloma cell line with the spleen cells of a CD-1 mouse that had been hyperimmunized with viable Raji cell suspensions (5 x 10 7 cells/inoculum). This MAb, designated II1/D1, is an IgM antibody with lambda-light chain specificity. Radiolabeled or unlabeled, highly purified II1/D1 was used to determine that: a) this antibody competes for C1q binding sites on C1qR-bearing cells; b) the molecule recognized by this MAb is the C1qR; and c) cells that are known to bind C1q also bind II1/D1 in a specific manner. Western blot analysis of solubilized Raji, or U937 cell membranes, showed that the 125 I-MAb detected a major protein band of approximately 85000 m.w. in its unreduced state, indicating that the C1qR is similar, if not identical, in both types of cells. Analyses of 125 I-II/D1 binding experiments revealed that the antibody bound to Raji cells or u937 cells in a specific manner. Uptake of the antibody was saturable, with equilibrium virtually attained within 35 min. Scatchard analysis of the binding data using the intact MAb suggests that the affinity constant K/sub D/ is 2.9 x 10 -10 M, and at apparent saturation, 24.6 ng of the antibody were bound per 2 x 10 6 cells, giving an estimated 7.8 x 10 3 antibody molecules bound per cell. That the II1/D1 antibody is specifically directed to the C1q was further evidenced by an ELISA in which the ability of C1qR-bearing cells to bind the MAb was abrogated by c-C1q in a specific dose-dependent manner

Idiotypic network. Assay and use of anti-idiotype antibodies in medicine

International Nuclear Information System (INIS)

Revillard, J.P.; Oliva, Ph.

1988-01-01

After a brief history of idotypes, the structural basis of antibody and T cell receptor (Ti) diversity, the definition of various types of idiotopes, the idiotypic cascade and the network concept are presented. Some anti-idiotypic antibodies represent the internal image of the antigen and may be used to prepare anti-idiotypic vaccines. Other anti-idiotypic antibodies bind to cellular receptors and can mimick or antagonize the biological effects of the natural ligands (hormones, neurotransmitters etc...). The concept of regulatory idiotopes (Idx) and their use in the manipulation of the network offer new possibilities for the control for auto-antibody production. The main medical applications of idiotypy are briefly considered including cancer, transplantation, allergy and auto-immune diseases. Finally the methodology applicable to the detection and titration of anti-idiotypes is described [fr

Expression of inhibitory receptors on intratumoral T cells modulates the activity of a T cell-bispecific antibody targeting folate receptor

Science.gov (United States)

Schreiner, Jens; Thommen, Daniela S.; Herzig, Petra; Bacac, Marina; Klein, Christian; Roller, Andreas; Belousov, Anton; Levitsky, Victor; Savic, Spasenija; Moersig, Wolfgang; Uhlenbrock, Franziska; Heinzelmann-Schwarz, Viola A.; Umana, Pablo; Pisa, Pavel; von Bergwelt-Baildon, M.; Lardinois, Didier; Müller, Philipp; Karanikas, Vaios; Zippelius, Alfred

2016-01-01

ABSTRACT T-cell bispecific antibodies (TCBs) are a novel therapeutic tool designed to selectively recruit T-cells to tumor cells and simultaneously activate them. However, it is currently unknown whether the dysfunctional state of T-cells, embedded into the tumor microenvironment, imprints on the therapeutic activity of TCBs. We performed a comprehensive analysis of activation and effector functions of tumor-infiltrating T-cells (TILs) in different tumor types, upon stimulation by a TCB targeting folate receptor 1 and CD3 (FolR1-TCB). We observed a considerable heterogeneity in T-cell activation, cytokine production and tumor cell killing upon exposure to FolR1-TCB among different FolR1-expressing tumors. Of note, tumors presenting with a high frequency of PD-1hi TILs displayed significantly impaired tumor cell killing and T-cell function. Further characterization of additional T-cell inhibitory receptors revealed that PD-1hi TILs defined a T-cell subset with particularly high levels of multiple inhibitory receptors compared with PD-1int and PD-1neg T-cells. PD-1 blockade could restore cytokine secretion but not cytotoxicity of TILs in a subset of patients with scarce PD-1hi expressing cells; in contrast, patients with abundance of PD-1hi expressing T-cells did not benefit from PD-1 blockade. Our data highlight that FolR1-TCB is a promising novel immunotherapeutic treatment option which is capable of activating intratumoral T-cells in different carcinomas. However, its therapeutic efficacy may be substantially hampered by a pre-existing dysfunctional state of T-cells, reflected by abundance of intratumoral PD-1hi T-cells. These findings present a rationale for combinatorial approaches of TCBs with other therapeutic strategies targeting T-cell dysfunction. PMID:27057429

Influenza human monoclonal antibody 1F1 interacts with three major antigenic sites and residues mediating human receptor specificity in H1N1 viruses.

Directory of Open Access Journals (Sweden)

Tshidi Tsibane

Full Text Available Most monoclonal antibodies (mAbs to the influenza A virus hemagglutinin (HA head domain exhibit very limited breadth of inhibitory activity due to antigenic drift in field strains. However, mAb 1F1, isolated from a 1918 influenza pandemic survivor, inhibits select human H1 viruses (1918, 1943, 1947, and 1977 isolates. The crystal structure of 1F1 in complex with the 1918 HA shows that 1F1 contacts residues that are classically defined as belonging to three distinct antigenic sites, Sa, Sb and Ca(2. The 1F1 heavy chain also reaches into the receptor binding site (RBS and interacts with residues that contact sialoglycan receptors and determine HA receptor specificity. The 1F1 epitope is remarkably similar to the previously described murine HC63 H3 epitope, despite significant sequence differences between H1 and H3 HAs. Both antibodies potently inhibit receptor binding, but only HC63 can block the pH-induced conformational changes in HA that drive membrane fusion. Contacts within the RBS suggested that 1F1 may be sensitive to changes that alter HA receptor binding activity. Affinity assays confirmed that sequence changes that switch the HA to avian receptor specificity affect binding of 1F1 and a mAb possessing a closely related heavy chain, 1I20. To characterize 1F1 cross-reactivity, additional escape mutant selection and site-directed mutagenesis were performed. Residues 190 and 227 in the 1F1 epitope were found to be critical for 1F1 reactivity towards 1918, 1943 and 1977 HAs, as well as for 1I20 reactivity towards the 1918 HA. Therefore, 1F1 heavy-chain interactions with conserved RBS residues likely contribute to its ability to inhibit divergent HAs.

Monoclonal antibody 1.6.1 against human MPL receptor allows HSC enrichment of CB and BM CD34(+)CD38(-) populations.

Science.gov (United States)

Petit Cocault, Laurence; Fleury, Maud; Clay, Denis; Larghero, Jérôme; Vanneaux, Valérie; Souyri, Michèle

2016-04-01

Thrombopoietin (TPO) and its receptor Mpl (CD110) play a crucial role in the regulation of hematopoietic stem cells (HSCs). Functional study of Mpl-expressing HSCs has, however, been hampered by the lack of efficient monoclonal antibodies, explaining the very few data available on Mpl(+) HSCs during human embryonic development and after birth. Investigating the main monoclonal antibodies used so far to sort CD110(+) cells from cord blood (CB) and adult bone marrow (BM), we found that only the recent monoclonal antibody 1.6.1 engineered by Immunex Corporation was specific. Using in vitro functional assays, we found that this antibody can be used to sort a CD34(+)CD38(-)CD110(+) population enriched in hematopoietic progenitor stem cells, both in CB and in adult BM. In vivo injection into NSG mice further indicated that the CB CD34(+)CD38(-)CD110(+) population is highly enriched in HSCs compared with both CD34(+)CD38(-)CD110(-) and CD34(+)CD38(-) populations. Together our results validate MAb1.6.1 as an important tool, which has so far been lacking, in the HSC field. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

No-go theorem for passive single-rail linear optical quantum computing.

Science.gov (United States)

Wu, Lian-Ao; Walther, Philip; Lidar, Daniel A

2013-01-01

Photonic quantum systems are among the most promising architectures for quantum computers. It is well known that for dual-rail photons effective non-linearities and near-deterministic non-trivial two-qubit gates can be achieved via the measurement process and by introducing ancillary photons. While in principle this opens a legitimate path to scalable linear optical quantum computing, the technical requirements are still very challenging and thus other optical encodings are being actively investigated. One of the alternatives is to use single-rail encoded photons, where entangled states can be deterministically generated. Here we prove that even for such systems universal optical quantum computing using only passive optical elements such as beam splitters and phase shifters is not possible. This no-go theorem proves that photon bunching cannot be passively suppressed even when extra ancilla modes and arbitrary number of photons are used. Our result provides useful guidance for the design of optical quantum computers.

Therapeutically targeting glypican-2 via single-domain antibody-based chimeric antigen receptors and immunotoxins in neuroblastoma.

Science.gov (United States)

Li, Nan; Fu, Haiying; Hewitt, Stephen M; Dimitrov, Dimiter S; Ho, Mitchell

2017-08-08

Neuroblastoma is a childhood cancer that is fatal in almost half of patients despite intense multimodality treatment. This cancer is derived from neuroendocrine tissue located in the sympathetic nervous system. Glypican-2 (GPC2) is a cell surface heparan sulfate proteoglycan that is important for neuronal cell adhesion and neurite outgrowth. In this study, we find that GPC2 protein is highly expressed in about half of neuroblastoma cases and that high GPC2 expression correlates with poor overall survival compared with patients with low GPC2 expression. We demonstrate that silencing of GPC2 by CRISPR-Cas9 or siRNA results in the inhibition of neuroblastoma tumor cell growth. GPC2 silencing inactivates Wnt/β-catenin signaling and reduces the expression of the target gene N-Myc, an oncogenic driver of neuroblastoma tumorigenesis. We have isolated human single-domain antibodies specific for GPC2 by phage display technology and found that the single-domain antibodies can inhibit active β-catenin signaling by disrupting the interaction of GPC2 and Wnt3a. To explore GPC2 as a potential target in neuroblastoma, we have developed two forms of antibody therapeutics, immunotoxins and chimeric antigen receptor (CAR) T cells. Immunotoxin treatment was demonstrated to inhibit neuroblastoma growth in mice. CAR T cells targeting GPC2 eliminated tumors in a disseminated neuroblastoma mouse model where tumor metastasis had spread to multiple clinically relevant sites, including spine, skull, legs, and pelvis. This study suggests GPC2 as a promising therapeutic target in neuroblastoma.

Type-I Insulin-Like Growth Factor Receptor (IGF1R)-Estrogen Receptor (ER) Crosstalk Contributes to Antiestrogen Therapy Resistance in Breast Cancer Cells

Science.gov (United States)

2013-02-01

vitro have downregulated J GF1R making antibodies directed agai nst th is receptor ineffective. Inhlbition of IH may be necessary to manage ...monoclonal antibody to insulin-like growth factor receptor 1. J Clin Oncol 2009;27:580Q-7. 31. Drury s. Detre s. Leary A, Salter J, Reis-Filho J

Cell-induced potentiation of the plasminogen activation system is abolished by a monoclonal antibody that recognizes the NH2-terminal domain of the urokinase receptor

DEFF Research Database (Denmark)

Rønne, E; Behrendt, N; Ellis, V

1991-01-01

We have raised four monoclonal antibodies recognizing different epitopes within the human cell-surface receptor for urokinase-type plasminogen activator (u-PA). One of these antibodies completely abolishes the potentiation of plasmin generation observed upon incubation of the zymogens pro......-u-PA and plasminogen with U937 cells. This antibody, which is also the only one to completely inhibit the binding of DFP-inactivated [125I]-u-PA to U937 cells, is directed against the u-PA binding NH2-terminal domain of u-PAR, a well-defined fragment formed by limited chymotrypsin digestion of purified u......-PAR, demonstrating the functional independence of the u-PA binding domain as well as the critical role of u-PAR in the assembly of the cell-surface plasminogen activation system....

Modeling the interactions of a peptide-major histocompatibility class I ligand with its receptors. II. Cross-reaction between a monoclonal antibody and two alpha beta T cell receptors

DEFF Research Database (Denmark)

Rognan, D; Engberg, J; Stryhn, A

2000-01-01

-peptide pair into the Fab combining site. Interestingly, the most energetically favored binding mode shows numerous analogies to the recently determined recognition of class I MHC-peptide complexes by alpha beta T cell receptors (TCRs). The pSAN13.4.1 also binds diagonally across the MHC binding groove......The recombinant antibody, pSAN13.4.1, has a unique T cell like specificity; it binds an Influenza Hemagglutinin octapeptide (Ha255-262) in an MHC (H-2Kk)-restricted manner, and a detailed comparison of the fine specificity of pSAN13.4.1 with the fine specificity of two Ha255-262-specific, H-2Kk......-restricted T cell hybridomas has supported this contention. A three-dimensional model of pSAN13.4.1 has been derived by homology modeling techniques. Subsequently, the structure of the pSAN13.4.1 antibody in complex with the antigenic Ha-Kk ligand was derived after a flexible and automated docking of the MHC...

IBC's 23rd Annual Antibody Engineering, 10th Annual Antibody Therapeutics international conferences and the 2012 Annual Meeting of The Antibody Society: December 3-6, 2012, San Diego, CA.

Science.gov (United States)

Klöhn, Peter-Christian; Wuellner, Ulrich; Zizlsperger, Nora; Zhou, Yu; Tavares, Daniel; Berger, Sven; Zettlitz, Kirstin A; Proetzel, Gabriele; Yong, May; Begent, Richard H J; Reichert, Janice M

2013-01-01

The 23rd Annual Antibody Engineering, 10th Annual Antibody Therapeutics international conferences, and the 2012 Annual Meeting of The Antibody Society, organized by IBC Life Sciences with contributions from The Antibody Society and two Scientific Advisory Boards, were held December 3-6, 2012 in San Diego, CA. The meeting drew over 800 participants who attended sessions on a wide variety of topics relevant to antibody research and development. As a prelude to the main events, a pre-conference workshop held on December 2, 2012 focused on intellectual property issues that impact antibody engineering. The Antibody Engineering Conference was composed of six sessions held December 3-5, 2012: (1) From Receptor Biology to Therapy; (2) Antibodies in a Complex Environment; (3) Antibody Targeted CNS Therapy: Beyond the Blood Brain Barrier; (4) Deep Sequencing in B Cell Biology and Antibody Libraries; (5) Systems Medicine in the Development of Antibody Therapies/Systematic Validation of Novel Antibody Targets; and (6) Antibody Activity and Animal Models. The Antibody Therapeutics conference comprised four sessions held December 4-5, 2012: (1) Clinical and Preclinical Updates of Antibody-Drug Conjugates; (2) Multifunctional Antibodies and Antibody Combinations: Clinical Focus; (3) Development Status of Immunomodulatory Therapeutic Antibodies; and (4) Modulating the Half-Life of Antibody Therapeutics. The Antibody Society's special session on applications for recording and sharing data based on GIATE was held on December 5, 2012, and the conferences concluded with two combined sessions on December 5-6, 2012: (1) Development Status of Early Stage Therapeutic Antibodies; and (2) Immunomodulatory Antibodies for Cancer Therapy.

[International classification of various types of monoclonal antibodies].

Science.gov (United States)

Scheen, A J

2009-01-01

Significant advances in the development of monoclonal antibodies ("mabs") have been acknowledged during the last two decades. Successive developments led to the marketing of murine antibodies ("o-mab" first, followed by chimeric antibodies ("xi-mab"), humanised antibodies ("zu-mab") and, finally, human monoclonal antibodies ("u-mab"). In order to facilitate the distinction between the various monoclonal antibodies used in clinical practice, an international nomenclature has been proposed with the use of a specific suffix corresponding to the origine/source of "mabs" preceded by an infix referring to the medicine's target. The efforts in developing new types of monoclonal antibodies aimed at improving their pharmacokinetics (longer half-life), pharmacodynamics (better efficacy because of stronger affinity to human receptor), and safety profile (less antigenic and immunogenic reactions). These progresses could be obtained thanks to the remarkable development of molecular biotechnology.

Isolation and killing of candidate chronic myeloid leukemia stem cells by antibody targeting of IL-1 receptor accessory protein

DEFF Research Database (Denmark)

Järås, Marcus; Johnels, Petra; Hansen, Nils Gunder

2010-01-01

stem cells could be prospectively separated. In addition, by generating an anti-IL1RAP antibody, we provide proof of concept that IL1RAP can be used as a target on CML CD34(+)CD38(-) cells to induce antibody-dependent cell-mediated cytotoxicity. This study thus identifies IL1RAP as a unique cell...... will require full eradication of Ph chromosome-positive (Ph(+)) CML stem cells. Here we used gene-expression profiling to identify IL-1 receptor accessory protein (IL1RAP) as up-regulated in CML CD34(+) cells and also in cord blood CD34(+) cells as a consequence of retroviral BCR/ABL1 expression. To test...... whether IL1RAP expression distinguishes normal (Ph(-)) and leukemic (Ph(+)) cells within the CML CD34(+)CD38(-) cell compartment, we established a unique protocol for conducting FISH on small numbers of sorted cells. By using this method, we sorted cells directly into drops on slides to investigate...

Development of antibody-based c-Met inhibitors for targeted cancer therapy

Directory of Open Access Journals (Sweden)

Lee D

2015-02-01

Full Text Available Dongheon Lee, Eun-Sil Sung, Jin-Hyung Ahn, Sungwon An, Jiwon Huh, Weon-Kyoo You Hanwha Chemical R&D Center, Biologics Business Unit, Daejeon, Republic of Korea Abstract: Signaling pathways mediated by receptor tyrosine kinases (RTKs and their ligands play important roles in the development and progression of human cancers, which makes RTK-mediated signaling pathways promising therapeutic targets in the treatment of cancer. Compared with small-molecule compounds, antibody-based therapeutics can more specifically recognize and bind to ligands and RTKs. Several antibody inhibitors of RTK-mediated signaling pathways, such as human epidermal growth factor receptor 2, vascular endothelial growth factor, epidermal growth factor receptor or vascular endothelial growth factor receptor 2, have been developed and are widely used to treat cancer patients. However, since the therapeutic options are still limited in terms of therapeutic efficacy and types of cancers that can be treated, efforts are being made to identify and evaluate novel RTK-mediated signaling pathways as targets for more efficacious cancer treatment. The hepatocyte growth factor/c-Met signaling pathway has come into the spotlight as a promising target for development of potent cancer therapeutic agents. Multiple antibody-based therapeutics targeting hepatocyte growth factor or c-Met are currently in preclinical or clinical development. This review focuses on the development of inhibitors of the hepatocyte growth factor/c-Met signaling pathway for cancer treatment, including critical issues in clinical development and future perspectives for antibody-based therapeutics. Keywords: hepatocyte growth factor, ligands, receptor tyrosine kinase, signaling pathway, therapeutic agent

Monoclonal antibodies for treating cancer

International Nuclear Information System (INIS)

Dillman, R.O.

1989-01-01

The purpose of this study is to assess the current status of in-vivo use of monoclonal antibodies for treating cancer. Publications appearing between 1980 and 1988 were identified by computer searches using MEDLINE and CANCERLIT, by reviewing the table of contents of recently published journals, and by searching bibliographies of identified books and articles. More than 700 articles, including peer-reviewed articles and book chapters, were identified and selected for analysis. The literature was reviewed and 235 articles were selected as relevant and representative of the current issues and future applications for in-vivo monoclonal antibodies for cancer therapy and of the toxicity and efficacy which has been associated with clinical trials. Approaches include using antibody alone (interacting with complement or effector cells or binding directly with certain cell receptors) and immunoconjugates (antibody coupled to radioisotopes, drugs, toxins, or other biologicals). Most experience has been with murine antibodies. Trials of antibody alone and radiolabeled antibodies have confirmed the feasibility of this approach and the in-vivo trafficking of antibodies to tumor cells. However, tumor cell heterogeneity, lack of cytotoxicity, and the development of human antimouse antibodies have limited clinical efficacy. Although the immunoconjugates are very promising, heterogeneity and the antimouse immune response have hampered this approach as has the additional challenge of chemically or genetically coupling antibody to cytotoxic agents. As a therapeutic modality, monoclonal antibodies are still promising but their general use will be delayed for several years. New approaches using human antibodies and reducing the human antiglobulin response should facilitate treatment. 235 references

Role of Antineuronal Antibodies in Children with Encephalopathy and Febrile Status Epilepticus

Directory of Open Access Journals (Sweden)

Kuang-Lin Lin

2014-06-01

Full Text Available Status epilepticus in childhood is more common, with a different range of causes and a lower risk of death, than convulsive status epilepticus in adults. Acute central nervous system infections appear to be markers for morbidity and mortality. Nevertheless, central nervous infection is usually presumed in these conditions. Many aspects of the pathogenesis of acute encephalitis and acute febrile encephalopathy with status epilepticus have been clarified in the past decade. The pathogenesis is divided into direct pathogens invasion or immune-mediated mechanisms. Over the past few decades, the number of antineuronal antibodies to ion channels, receptors, and other synaptic proteins described in association with central nervous system disorders has increased dramatically, especially their role in pediatric encephalitis and status epilepticus. These antineuronal antibodies are divided according to the location of their respective antigens: (1 intracellular antigens, including glutamic acid decarboxylase and classical onconeural antigens such as Hu (antineuronal nuclear antibody 1, ANNA1, Ma2, Yo (Purkinje cell autoantibody, PCA1, Ri (antineuronal nuclear antibody 2, ANNA2, CV2/CRMP5, and amphiphysin; and (2 cell membrane ion channels or surface antigens including voltage-gated potassium channel receptor, N-methyl-d-aspartate receptor, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor, γ-aminobutyric acid(B receptor, leucine-rich glioma-inactivated protein 1, and contactin-associated protein-like 2. Identifying the mechanism of the disease may have important therapeutic implications.

TRAIL death receptors and cancer therapeutics

International Nuclear Information System (INIS)

Huang Ying; Sheikh, M. Saeed

2007-01-01

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) also known as Apo2L is an apoptotic molecule that belongs to the tumor necrosis factor superfamily of cytokines. It mediates its apoptotic effects via its cognate death receptors including DR4 and DR5. Agonistic monoclonal antibodies have also been developed that selectively activate TRAIL death receptors to mediate apoptosis. Multiple clinically relevant agents also upregulate the expression of TRAIL death receptors, and cooperate with TRAIL as well as DR4 and DR5-specific agonistic antibodies to exhibit tumor cell killing. TRAIL is currently in phase I clinical trials, whereas DR4 and DR5-specific agonistic antibodies have been tested in phase I and II studies. Thus, TRAIL has clearly distinguished itself from the other family members including TNF-alpha and FasL both of which could not make it to the clinic due to their toxic nature. It is therefore, evident that the future of TRAIL-based therapeutic approaches looks brighter

Antibody Engineering & Therapeutics 2016: The Antibody Society's annual meeting, December 11-15, 2016, San Diego, CA.

Science.gov (United States)

Larrick, James W; Alfenito, Mark R; Scott, Jamie K; Parren, Paul W H I; Burton, Dennis R; Bradbury, Andrew R M; Lemere, Cynthia A; Messer, Anne; Huston, James S; Carter, Paul J; Veldman, Trudi; Chester, Kerry A; Schuurman, Janine; Adams, Gregory P; Reichert, Janice M

Antibody Engineering & Therapeutics, the largest meeting devoted to antibody science and technology and the annual meeting of The Antibody Society, will be held in San Diego, CA on December 11-15, 2016. Each of 14 sessions will include six presentations by leading industry and academic experts. In this meeting preview, the session chairs discuss the relevance of their topics to current and future antibody therapeutics development. Session topics include bispecifics and designer polyclonal antibodies; antibodies for neurodegenerative diseases; the interface between passive and active immunotherapy; antibodies for non-cancer indications; novel antibody display, selection and screening technologies; novel checkpoint modulators / immuno-oncology; engineering antibodies for T-cell therapy; novel engineering strategies to enhance antibody functions; and the biological Impact of Fc receptor engagement. The meeting will open with keynote speakers Dennis R. Burton (The Scripps Research Institute), who will review progress toward a neutralizing antibody-based HIV vaccine; Olivera J. Finn, (University of Pittsburgh School of Medicine), who will discuss prophylactic cancer vaccines as a source of therapeutic antibodies; and Paul Richardson (Dana-Farber Cancer Institute), who will provide a clinical update on daratumumab for multiple myeloma. In a featured presentation, a representative of the World Health Organization's INN expert group will provide a perspective on antibody naming. "Antibodies to watch in 2017" and progress on The Antibody Society's 2016 initiatives will be presented during the Society's special session. In addition, two pre-conference workshops covering ways to accelerate antibody drugs to the clinic and the applications of next-generation sequencing in antibody discovery and engineering will be held on Sunday December 11, 2016.

Development and Characterization of Anti-Nitr9 Antibodies

Directory of Open Access Journals (Sweden)

Radhika N. Shah

2012-01-01

Full Text Available The novel immune-type receptors (NITRs, which have been described in numerous bony fish species, are encoded by multigene families of inhibitory and activating receptors and are predicted to be functional orthologs to the mammalian natural killer cell receptors (NKRs. Within the zebrafish NITR family, nitr9 is the only gene predicted to encode an activating receptor. However, alternative RNA splicing generates three distinct nitr9 transcripts, each of which encodes a different isoform. Although nitr9 transcripts have been detected in zebrafish lymphocytes, the specific hematopoietic lineage(s that expresses Nitr9 remains to be determined. In an effort to better understand the role of NITRs in zebrafish immunity, anti-Nitr9 monoclonal antibodies were generated and evaluated for the ability to recognize the three Nitr9 isoforms. The application of these antibodies to flow cytometry should prove to be useful for identifying the specific lymphocyte lineages that express Nitr9 and may permit the isolation of Nitr9-expressing cells that can be directly assessed for cytotoxic (e.g., NK function.

Characterization of the glucagon-like peptide-1 receptor in male mouse brain using a novel antibody and in situ hybridization

DEFF Research Database (Denmark)

Jensen, Casper Bo; Pyke, Charles; Rasch, Morten Grønbech

2017-01-01

was abundantly expressed in numerous regions including the septal nucleus, the hypothalamus and the brain stem. GLP-1R protein expression was also observed on neuronal projections in brain regions devoid of any mRNA which has not been observed in earlier reports. Taken together, these findings provide new......Glucagon-like peptide-1 (GLP-1) is a physiological regulator of appetite and long-acting GLP-1 receptor agonists (GLP-1RA) lower food intake and bodyweight in both human and animal studies. The effects are mediated through brain GLP-1Rs, and several brain nuclei expressing the GLP-1R may...... be involved. To date, mapping the complete location of GLP-1R protein in the brain has been challenged by lack of good antibodies and the discrepancy between mRNA and protein especially relevant in neuronal axonal processes. Here, we present a novel and specific monoclonal GLP-1R antibody...

Generation and Characterization of Novel Human IRAS Monoclonal Antibodies

Directory of Open Access Journals (Sweden)

Bo Wang

2009-01-01

Full Text Available Imidazoline receptors were first proposed by Bousquet et al., when they studied antihypertensive effect of clonidine. A strong candidate for I1R, known as imidazoline receptor antisera-selected protein (IRAS, has been cloned from human hippocampus. We reported that IRAS mediated agmatine-induced inhibition of opioid dependence in morphine-dependent cells. To elucidate the functional and structure properties of I1R, we developed the newly monoclonal antibody against the N-terminal hIRAS region including the PX domain (10–120aa through immunization of BALB/c mice with the NusA-IRAS fusion protein containing an IRAS N-terminal (10–120aa. Stable hybridoma cell lines were established and monoclonal antibodies specifically recognized full-length IRAS proteins in their native state by immunoblotting and immunoprecipitation. Monoclonal antibodies stained in a predominantly punctate cytoplasmic pattern when applied to IRAS-transfected HEK293 cells by indirect immunofluorescence assays and demonstrated excellent reactivity in flow immunocytometry. These monoclonal antibodies will provide powerful reagents for the further investigation of hIRAS protein functions.

Quantitative assessment of antibody internalization with novel monoclonal antibodies against Alexa fluorophores.

Science.gov (United States)

Liao-Chan, Sindy; Daine-Matsuoka, Barbara; Heald, Nathan; Wong, Tiffany; Lin, Tracey; Cai, Allen G; Lai, Michelle; D'Alessio, Joseph A; Theunissen, Jan-Willem

2015-01-01

Antibodies against cell surface antigens may be internalized through their specific interactions with these proteins and in some cases may induce or perturb antigen internalization. The anti-cancer efficacy of antibody-drug conjugates is thought to rely on their uptake by cancer cells expressing the surface antigen. Numerous techniques, including microscopy and flow cytometry, have been used to identify antibodies with desired cellular uptake rates. To enable quantitative measurements of internalization of labeled antibodies, an assay based on internalized and quenched fluorescence was developed. For this approach, we generated novel anti-Alexa Fluor monoclonal antibodies (mAbs) that effectively and specifically quench cell surface-bound Alexa Fluor 488 or Alexa Fluor 594 fluorescence. Utilizing Alexa Fluor-labeled mAbs against the EphA2 receptor tyrosine kinase, we showed that the anti-Alexa Fluor reagents could be used to monitor internalization quantitatively over time. The anti-Alexa Fluor mAbs were also validated in a proof of concept dual-label internalization assay with simultaneous exposure of cells to two different mAbs. Importantly, the unique anti-Alexa Fluor mAbs described here may also enable other single- and dual-label experiments, including label detection and signal enhancement in macromolecules, trafficking of proteins and microorganisms, and cell migration and morphology.

Quantitative assessment of antibody internalization with novel monoclonal antibodies against Alexa fluorophores.

Directory of Open Access Journals (Sweden)

Sindy Liao-Chan

Full Text Available Antibodies against cell surface antigens may be internalized through their specific interactions with these proteins and in some cases may induce or perturb antigen internalization. The anti-cancer efficacy of antibody-drug conjugates is thought to rely on their uptake by cancer cells expressing the surface antigen. Numerous techniques, including microscopy and flow cytometry, have been used to identify antibodies with desired cellular uptake rates. To enable quantitative measurements of internalization of labeled antibodies, an assay based on internalized and quenched fluorescence was developed. For this approach, we generated novel anti-Alexa Fluor monoclonal antibodies (mAbs that effectively and specifically quench cell surface-bound Alexa Fluor 488 or Alexa Fluor 594 fluorescence. Utilizing Alexa Fluor-labeled mAbs against the EphA2 receptor tyrosine kinase, we showed that the anti-Alexa Fluor reagents could be used to monitor internalization quantitatively over time. The anti-Alexa Fluor mAbs were also validated in a proof of concept dual-label internalization assay with simultaneous exposure of cells to two different mAbs. Importantly, the unique anti-Alexa Fluor mAbs described here may also enable other single- and dual-label experiments, including label detection and signal enhancement in macromolecules, trafficking of proteins and microorganisms, and cell migration and morphology.

Development of a complete human anti-human transferrin receptor C antibody as a novel marker of oral dysplasia and oral cancer

International Nuclear Information System (INIS)

Nagai, Kentaro; Nakahata, Shingo; Shimosaki, Shunsuke; Tamura, Tomohiro; Kondo, Yuudai; Baba, Takashi; Taki, Tomohiko; Taniwaki, Masafumi; Kurosawa, Gene; Sudo, Yukio; Okada, Seiji; Sakoda, Sumio; Morishita, Kazuhiro

2014-01-01

Oral squamous cell carcinoma (OSCC) is the sixth most common cancer worldwide. Up to 20% of oral dysplasia cases have been suggested to undergo malignant transformation to OSCC; however, there are no methods to predict OSCC development. In this study, to identify the genes associated with oral dysplasia progression, we performed genomic copy number analyses of genomic DNA samples isolated from primary oral dysplasia and OSCC via the microdissection method and found elevated expression of transferrin receptor C (TfR1/TFRC) with genomic amplification in oral dysplasia and OSCC. The expression rate of TFRC in OSCC was significantly higher than that in dysplasia, suggesting that OSCC disease progression might be related to TFRC expression. Additionally, we investigated the in vitro and in vivo impacts of a newly established anti-human TFRC monoclonal antibody, which was isolated from a human cDNA library using the phage-display method, on cell proliferation and survival. The anti-TFRC antibody blocked the interaction between transferrin and TFRC and consequently inhibited iron uptake, leading to the iron deprivation-mediated suppression of cell growth and induction of apoptosis. Moreover, we demonstrated that the anti-TFRC antibody efficiently inhibited tumor growth in a murine xenograft OSCC model. Therefore, we suggest our developed complete human anti-human TFRC antibody as a useful, novel treatment for oral dysplasia and OSCC

A "Trojan horse" bispecific-antibody strategy for broad protection against ebolaviruses.

Science.gov (United States)

Wec, Anna Z; Nyakatura, Elisabeth K; Herbert, Andrew S; Howell, Katie A; Holtsberg, Frederick W; Bakken, Russell R; Mittler, Eva; Christin, John R; Shulenin, Sergey; Jangra, Rohit K; Bharrhan, Sushma; Kuehne, Ana I; Bornholdt, Zachary A; Flyak, Andrew I; Saphire, Erica Ollmann; Crowe, James E; Aman, M Javad; Dye, John M; Lai, Jonathan R; Chandran, Kartik

2016-10-21

There is an urgent need for monoclonal antibody (mAb) therapies that broadly protect against Ebola virus and other filoviruses. The conserved, essential interaction between the filovirus glycoprotein, GP, and its entry receptor Niemann-Pick C1 (NPC1) provides an attractive target for such mAbs but is shielded by multiple mechanisms, including physical sequestration in late endosomes. Here, we describe a bispecific-antibody strategy to target this interaction, in which mAbs specific for NPC1 or the GP receptor-binding site are coupled to a mAb against a conserved, surface-exposed GP epitope. Bispecific antibodies, but not parent mAbs, neutralized all known ebolaviruses by coopting viral particles themselves for endosomal delivery and conferred postexposure protection against multiple ebolaviruses in mice. Such "Trojan horse" bispecific antibodies have potential as broad antifilovirus immunotherapeutics. Copyright © 2016, American Association for the Advancement of Science.

Murine B cell development and antibody responses to model antigens are not impaired in the absence of the TNF receptor GITR.

Directory of Open Access Journals (Sweden)

Lenka Sinik Teodorovic

Full Text Available The Glucocorticoid-Induced Tumor necrosis factor Receptor GITR, a member of the tumor necrosis factor receptor superfamily, has been shown to be important in modulating immune responses in the context of T cell immunity. B lymphocytes also express GITR, but a role of GITR in humoral immunity has not been fully explored. To address this question, we performed studies to determine the kinetics of GITR expression on naïve and stimulated B cells and the capacity of B cells to develop and mount antibody responses in GITR(-/- mice. Results of our studies indicate that all mature B cells express GITR on the cell surface, albeit at different levels. Expression of GITR on naïve mature B cells is upregulated by BCR signaling, but is counteracted by helper T cell-related factors and other inflammatory signals in vitro. In line with these findings, expression of GITR on germinal center and memory B cells is lower than that on naïve B cells. However, the expression of GITR is strongly upregulated in plasma cells. Despite these differences in GITR expression, the absence of GITR has no effect on T cell-dependent and T cell-independent antibody responses to model antigens in GITR(-/- mice, or on B cell activation and proliferation in vitro. GITR deficiency manifests only with a slight reduction of mature B cell numbers and increased turnover of naïve B cells, suggesting that GITR slightly contributes to mature B cell homeostasis. Overall, our data indicate that GITR does not play a significant role in B cell development and antibody responses to T-dependent and independent model antigens within the context of a GITR-deficient genetic background.

Production and radioiodination of monoclonal antibodies and its applications in nuclear medicine

International Nuclear Information System (INIS)

Toledo e Souza, I.T. de; Okada, H.

1988-12-01

The basis of the monoclonal antibody production methodology, some immunological concepts which are important for the understanding of what is a Monoclonal Antibody, its radioiodination and acceptance as receptor-specific radiopharmaceuticals in nuclear medicine are reviewed. (author) [pt

Chimeric opioid peptides: Tools for identifying opioid receptor types

International Nuclear Information System (INIS)

Xie, G.; Miyajima, A.; Yokota, T.; Arai, K.; Goldstein, A.

1990-01-01

The authors synthesized several chimeric [125J-labelled] peptides in which the N-terminal nine residues of dynorphin-32, a peptide selective for the κ opioid receptor, were replaced by opioid peptides selective for other opioid receptor types. Each chimeric peptide retained the high affinity and type selectivity characteristic of its N-terminal sequence. The common C-terminal two-thirds of the chimeric peptides served as an epitope recognized by the same monoclonal antibody. When bound to receptors on a cell surface or membrane preparation, these peptides could still bind specifically to the monoclonal antibody. These chimeric peptides should be useful for isolating μ, δ, and κ opioid receptors and for identifying opioid receptors on transfected cells in expression cloning procedures. The general approach using chimeric peptides should be applicable to other peptide receptors

Mapping of monoclonal antibody- and receptor-binding domains on human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) using a surface plasmon resonance-based biosensor.

Science.gov (United States)

Laricchia-Robbio, L; Liedberg, B; Platou-Vikinge, T; Rovero, P; Beffy, P; Revoltella, R P

1996-10-01

An automated surface plasmon resonance (SPR)-based biosensor system has been used for mapping antibody and receptor-binding regions on the recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) molecule. A rabbit antimouse IgG1-Fc antibody (RAM.Fc) was coupled to an extended carboxymethylated-hydrogel matrix attached to a gold surface in order to capture an anti-rhGM-CSF monoclonal antibody (MAb) injected over the sensing layer. rhGM-CSF was subsequently injected and allowed to bind to this antibody. Multisite binding assays were then performed, by flowing sequentially other antibodies and peptides over the surface, and the capacity of the latter to interact with the entrapped rhGM-CSF in a multimolecular complex was monitored in real time with SPR. Eleven MAb (all IgG1K), were analyzed: respectively, four antipeptide MAb raised against three distinct epitopes of the cytokine (two clones against residues 14-24, that includes part of the first alpha-helix toward the N-terminal region; one clone against peptide 30-41, an intrahelical loop; and one clone against residues 79-91, including part of the third alpha-helix) and seven antiprotein MAbs raised against the entire rhGM-CSF, whose target native epitopes are still undetermined. In addition, the binding capacity to rhGM-CSF of a synthetic peptide, corresponding to residues 238-254 of the extracellular human GM-CSF receptor alpha-chain, endowed with rhGM-CSF binding activity, was tested. The results from experiments performed with the biosensor were compared with those obtained by a sandwich enzyme-linked immunosorbent assay (ELISA), using the same reagents. The features of the biosensor technology (fully automated, measure in real time, sharpened yes/no response, less background disturbances, no need for washing step or labeling of the reagent) offered several advantages in these studies of MAb immunoreactivity and epitope mapping, giving a much better resolution and enabling more distinct

Infection of CD4+ T lymphocytes by the human T cell leukemia virus type 1 is mediated by the glucose transporter GLUT-1: Evidence using antibodies specific to the receptor's large extracellular domain

International Nuclear Information System (INIS)

Jin, Qingwen; Agrawal, Lokesh; VanHorn-Ali, Zainab; Alkhatib, Ghalib

2006-01-01

To analyze HTLV-1 cytotropism, we developed a highly sensitive vaccinia virus-based assay measuring activation of a reporter gene upon fusion of two distinct cell populations. We used this system in a functional cDNA screening to isolate and confirm that the glucose transporter protein 1 (GLUT-1) is a receptor for HTLV-1. GLUT-1 is a ubiquitously expressed plasma membrane glycoprotein with 12 transmembrane domains and 6 extracellular loops (ECL). We demonstrate for the first time that peptide antibodies (GLUT-IgY) raised in chicken to the large extracellular loop (ECL1) detect GLUT-1 at the cell surface and inhibit envelope (Env)-mediated fusion and infection. Efficient GLUT-IgY staining was detected with peripheral blood CD4 + lymphocytes purified by positive selection. Further, GLUT-IgY caused efficient inhibition of Env-mediated fusion and infection of CD4 + T and significantly lower inhibition of CD8 + T lymphocytes. The specificity of GLUT-IgY antibodies to GLUT-1 was demonstrated by ECL1 peptide competition studies. Grafting ECL1 of GLUT-1 onto the receptor-negative GLUT-3 conferred significant receptor activity. In contrast, grafting ECL1 of GLUT-3 onto GLUT-1 resulted in a significant loss of the receptor activity. The ECL1-mediated receptor activity was efficiently blocked with four different human monoclonal antibody (HMab) to HTLV-1 Env. The ECL1-derived peptide blocked HTLV-1 Env-mediated fusion with several nonhuman mammalian cell lines. The results demonstrate the utilization of cell surface GLUT-1 in HTLV-1 infection of CD4 + T lymphocytes and implicate a critical role for the ECL1 region in viral tropism

[Contributions of neuropsychology to anti-NMDA receptor antibody encephalitis: a literature review].

Science.gov (United States)

Luna-Lario, P; Hernaez-Goni, P; Tirapu-Ustarroz, J

2016-05-01

Limbic encephalitis generated by anti-N-methyl-D-aspartate (NMDA) receptor antibodies is an acute and severe neurological entity, which is more prevalent in young females and is associated to an underlying tumour. Since it leads to severe cognitive impairment, thought needs to be given to the contributions of neuropsychology to the diagnosis, development and treatment of the disease, which have received little attention from researchers to date. A review is conducted of the prior literature, evaluating the measurement of the cognitive symptoms (predominantly mnemonic and executive) associated to this disease. Valid, reliable neuropsychological instruments are proposed, and it is suggested that neuropsychological measures may be used as parameters to follow up these patients which help monitor their functionality in daily living once they have recovered from the acute phase. Similarly they can become a basis on which to assemble rehabilitation programmes that favour the accomplishment of personal autonomy and the patients' reintegration in the community. Nevertheless, we stress the need to include neuropsychologists and neuropsychiatrists in not only the detection but also the treatment of these patients so as to enable them to recover their personal independence and re-adapt to their natural settings.

A new monoclonal antibody detects downregulation of protein tyrosine phosphatase receptor type γ in chronic myeloid leukemia patients

Directory of Open Access Journals (Sweden)

Marzia Vezzalini

2017-06-01

Full Text Available Abstract Background Protein tyrosine phosphatase receptor gamma (PTPRG is a ubiquitously expressed member of the protein tyrosine phosphatase family known to act as a tumor suppressor gene in many different neoplasms with mechanisms of inactivation including mutations and methylation of CpG islands in the promoter region. Although a critical role in human hematopoiesis and an oncosuppressor role in chronic myeloid leukemia (CML have been reported, only one polyclonal antibody (named chPTPRG has been described as capable of recognizing the native antigen of this phosphatase by flow cytometry. Protein biomarkers of CML have not yet found applications in the clinic, and in this study, we have analyzed a group of newly diagnosed CML patients before and after treatment. The aim of this work was to characterize and exploit a newly developed murine monoclonal antibody specific for the PTPRG extracellular domain (named TPγ B9-2 to better define PTPRG protein downregulation in CML patients. Methods TPγ B9-2 specifically recognizes PTPRG (both human and murine by flow cytometry, western blotting, immunoprecipitation, and immunohistochemistry. Results Co-localization experiments performed with both anti-PTPRG antibodies identified the presence of isoforms and confirmed protein downregulation at diagnosis in the Philadelphia-positive myeloid lineage (including CD34+/CD38bright/dim cells. After effective tyrosine kinase inhibitor (TKI treatment, its expression recovered in tandem with the return of Philadelphia-negative hematopoiesis. Of note, PTPRG mRNA levels remain unchanged in tyrosine kinase inhibitors (TKI non-responder patients, confirming that downregulation selectively occurs in primary CML cells. Conclusions The availability of this unique antibody permits its evaluation for clinical application including the support for diagnosis and follow-up of these disorders. Evaluation of PTPRG as a potential therapeutic target is also facilitated by the

Neuronal antibodies in patients with suspected or confirmed sporadic Creutzfeldt-Jakob disease.

Science.gov (United States)

Rossi, Meghan; Mead, Simon; Collinge, John; Rudge, Peter; Vincent, Angela

2015-06-01

There have been reports of patients with antibodies to neuronal antigens misdiagnosed as sporadic Creutzfeldt-Jakob disease (sCJD). Conversely, low levels of antibodies to neuronal proteins have been reported in patients with sCJD. However, the frequency of misdiagnoses, or of antibodies in patients with subsequently confirmed sCJD, is not clear. We reviewed 256 consecutive cases of sCJD seen in the National Prion Clinic, of whom 150 had sera previously referred for selected antibody tests. Eighty-two available samples were retested for antibodies to N-methyl-d-aspartate receptor (NMDAR), the glycine receptor (GlyR), voltage-gated potassium channel (VGKC)-complex and the associated proteins, leucine-rich glioma inactivated 1 (LGI1) and contactin-associated protein 2 (CASPR2). Four of the initial 150 sera referred were positive; two had antibodies to NMDAR, and two to the VGKC-complex, one of which was also positive for GlyR antibodies. Of the 82 sCJD sera retested, one had VGKC-complex antibodies confirming the previous result, two had CASPR2 and GlyR antibodies and one had CASPR2 and NMDAR antibodies; all antibodies were at low levels. Over the same period three patients with autoimmune encephalitis and high VGKC-complex antibodies were initially referred as sCJD. This study indicates that VGKC-complex/LGI1 antibodies. Low titres of neuronal antibodies occur only rarely in suspected patients with sCJD and when present should be interpreted with caution. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Investigation of neuronal auto-antibodies in systemic lupus erythematosus patients with epilepsy.

Science.gov (United States)

Karaaslan, Zerrin; Ekizoğlu, Esme; Tektürk, Pınar; Erdağ, Ece; Tüzün, Erdem; Bebek, Nerses; Gürses, Candan; Baykan, Betül

2017-01-01

Epilepsy is an important feature for neuropsychiatric involvement in systemic lupus erythematosus (SLE) with unknown mechanism. Our aim was to investigate the presence of neuronal auto-antibodies (NAbs) in neuropsychiatric SLE (NPSLE). Eighteen SLE patients (17 females, 1 male) experiencing recurrent seizures were enrolled to this study. Their clinical characteristics, EEG and MRI findings and follow-up information were evaluated from their files. Antibodies against voltage-gated potassium channel (VGKC)-complex antigens, contactin-associated protein-like 2 (CASPR-2), leucine-rich, glioma inactivated 1 (LGI1), glutamic acid decarboxylase (GAD), N-methyl-d-aspartate receptor (NMDA-R), alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor (AMPA-R) and type B gamma aminobutyric acid receptors (GABA B -R) were screened in the sera of these patients. Moreover, indirect immunohistochemistry and immunocytochemistry tests were performed to reveal neuropil antibodies. Six out of 18 patients (33.3%) had various forms of NAbs. Among them, one patient had antibodies against GAD, one patient with hippocampal sclerosis on MRI was CASPR-2 antibody positive, whereas the remaining four patients showed hippocampal neuropil staining. We could not find a significant difference between seropositive and seronegative groups, regarding the clinical characteristics, EEG and MRI findings. This study is the first to show hippocampal neuronal staining (4/18) reflecting antibodies against unknown neuronal cell surface antigens in SLE patients with epilepsy, besides the rare occurrence of GAD and CASPR2 antibodies. Further prospective studies are needed to search for new NAbs and uncover their pathogenic role in SLE associated with epilepsy. Copyright © 2016 Elsevier B.V. All rights reserved.

Use of antibodies against the variable regions of the T-cell receptor alpha/beta heterodimer for the study of cutaneous T-cell lymphomas.

Science.gov (United States)

Ralfkiaer, E; Wollf-Sneedorff, A; Vejlsgaard, G L

1991-11-01

Recent studies have suggested that antibodies against the variable (V) regions of the T-cell antigen receptor (TCR) may be used as markers for clonality and malignancy in T-cell infiltrates. We have investigated this by examining biopsy samples from 45 patients with cutaneous T-cell lymphomas (CTCL) for reactivity with seven antibodies against different V-gene families on the TCR alpha/beta heterodimer, i.e. ICI (V beta 5a), W112 (V beta 5b), OT145 (V beta 6a), 16G8 (V beta 8a), S511 (V beta 12a), F1 (V alpha 2a) and LC4 (alpha beta Va). Serial biopsies were available in 13 patients and a total of 62 samples were studied. The neoplastic cells in five cases were positive for either V beta 5 (one case), V beta 6 (one case), V beta 8 (two cases) or V beta 12 (one case). In the remaining 40 cases, no staining was seen of the neoplastic cells. These findings indicate that while antibodies against the TCR V-regions may be used as clonotypic markers for certain T-cell neoplasms, there is as yet not a sufficient number of anti-TCR V-region antibodies available for the routine diagnosis of these conditions.

Identification of anti-CD98 antibody mimotopes for inducing antibodies with antitumor activity by mimotope immunization.

Science.gov (United States)

Saito, Misa; Kondo, Masahiro; Ohshima, Motohiro; Deguchi, Kazuki; Hayashi, Hideki; Inoue, Kazuyuki; Tsuji, Daiki; Masuko, Takashi; Itoh, Kunihiko

2014-04-01

A mimotope is an antibody-epitope-mimicking peptide retrieved from a phage display random peptide library. Immunization with antitumor antibody-derived mimotopes is promising for inducing antitumor immunity in hosts. In this study, we isolated linear and constrained mimotopes from HBJ127, a tumor-suppressing anti-CD98 heavy chain mAb, and determined their abilities for induction of antitumor activity equal to that of the parent antibody. We detected elevated levels of antipeptide responses, but failed to detect reactivity against native CD98-expressing HeLa cells in sera of immunized mice. Phage display panning and selection of mimotope-immunized mouse spleen-derived antibody Fab library showed that HeLa cell-reactive Fabs were successfully retrieved from the library. This finding indicates that native antigen-reactive Fab clones represented an undetectable minor population in mimotope-induced antibody repertoire. Functional and structural analysis of retrieved Fab clones revealed that they were almost identical to the parent antibody. From these results, we confirmed that mimotope immunization was promising for retrieving antitumor antibodies equivalent to the parent antibody, although the co-administration of adjuvant compounds such as T-cell epitope peptides and Toll-like receptor 4 agonist peptides is likely to be necessary for inducing stronger antitumor immunity than mimotope injection alone. © 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

Anti-Streptococcus IgM Antibodies Induce Repetitive Stereotyped Movements: Cell Activation and Co-Localization with Fcα/μ Receptors in the Striatum and Motor Cortex

Science.gov (United States)

Zhang, Danhui; Patel, Ankur; Zhu, Youhua; Siegel, Allan; Zalcman, Steven S.

2012-01-01

Group A beta-hemolytic streptococcus (GABHS) infections are implicated in neuropsychiatric disorders associated with an increased expression of repetitive stereotyped movements. Anti-streptococcus IgG presumably cross-reacts with elements on basal ganglia cells, modifies their function, and triggers symptoms. IgM may play a unique role in precipitating behavioral disturbances since variations in cortico-striatal activity occur in temporal congruity with peak IgM titers during an orchestrated immune response. We discovered in Balb/c mice that single subcutaneous injections of mouse monoclonal IgM antibodies to Streptococcus Group A bacteria induce marked dose-dependent increases in repetitive stereotyped movements, including head bobbing, sniffing, and intense grooming. Effects were antibody- and antigen-specific: anti-streptococcus IgG stimulated ambulatory activity and vertical activity but not these stereotypies, while anti-KLH IgM reduced activity. We suggest that anti-streptococcus IgM and IgG play unique roles in provoking GABHS-related behavioral disturbances. Paralleling its stereotypy-inducing effects, anti-streptococcus IgM stimulated Fos-like immunoreactivity in regions linked to cortico-striatal projections involved in motor control, including subregions of the caudate, nucleus accumbens, and motor cortex. This is the first evidence that anti-streptococcus IgM antibodies induce in vivo functional changes in these structures. Moreover, there was a striking similarity in the distributions of anti-streptococcus IgM deposits and Fos-like immunoreactivity in these regions. Of further importance, Fcα/μ receptors, which bind IgM, were present- and co-localized with anti-streptococcus IgM in these structures. We suggest that anti-streptococcus IgM-induced alterations of cell activity reflect local actions of IgM that involve Fcα/μ receptors. These findings support the use of anti-streptococcus monoclonal antibody administration in Balb/c mice to model GABHS

Safety and efficacy of eculizumab in anti-acetylcholine receptor antibody-positive refractory generalised myasthenia gravis (REGAIN)

DEFF Research Database (Denmark)

Howard, James F; Utsugisawa, Kimiaki; Benatar, Michael

2017-01-01

BACKGROUND: Complement is likely to have a role in refractory generalised myasthenia gravis, but no approved therapies specifically target this system. Results from a phase 2 study suggested that eculizumab, a terminal complement inhibitor, produced clinically meaningful improvements in patients...... with anti-acetylcholine receptor antibody-positive refractory generalised myasthenia gravis. We further assessed the efficacy and safety of eculizumab in this patient population in a phase 3 trial. METHODS: We did a phase 3, randomised, double-blind, placebo-controlled, multicentre study (REGAIN) in 76...... hospitals and specialised clinics in 17 countries across North America, Latin America, Europe, and Asia. Eligible patients were aged at least 18 years, with a Myasthenia Gravis-Activities of Daily Living (MG-ADL) score of 6 or more, Myasthenia Gravis Foundation of America (MGFA) class II-IV disease...

In vitro and in vivo imaging of prostate cancer angiogenesis using anti-vascular endothelial growth factor receptor 2 antibody-conjugated quantum dot

International Nuclear Information System (INIS)

Kwon, Haejin; Lee, Jiyeon; Song, Rita; Lee, Jung Han; Hwang, Sung Il; Lee, Hak Jong; Kim, Young Hwa

2013-01-01

Authors aimed to determine the targeting ability of vascular endothelial growth factor receptor 2 (VEGFR2)-conjugated quantum dots (QDs) in vitro, and apply it for a xenograft prostate cancer mouse model. Conjugation reaction of QDs was performed by using the N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and sulfo-(N-hydroxysulfosuccinimide) (Sulfo-NHS). The human umbilical vein cord endothelial cells (HUVECs) were incubated with QDs, conjugated with antiVGFR2, to see a specific binding in vitro. Fluorescent cell images were taken by a confocal microscope. The human prostate cancer cells (PC3) were injected to five nude mice on hind limbs to make the xenograft tumor model. QD-antiVEGFR2 antibody complex was injected into the tumor model and fluorescence measurements were performed at 1, 4, 9, 12, 15, and 24 hours after the injection. The specific interaction between HUVECs and QD-antiVEGFR2 antibody was clearly shown in vitro. The in vivo fluorescence image disclosed that there was an increased signal of tumor, 12 hours after the injection of QDs. By showing endothelial cells binding with QDs-antiVEGFR2 antibodyand an experimental application of the antibody for VEGFR2 imaging in the prostate cancer xenograft mouse model, we suggests that the antibody-conjugated QDs can be a potential imaging tool for angiogenesis of the cancer.

Panel of monoclonal antibodies to sperm surface proteins as a tool for monitoring localization and identification of sperm-zona pellucida receptors

Czech Academy of Sciences Publication Activity Database

Zigo, Michal; Dorosh, Andriy; Pohlová, Alžběta; Jonáková, Věra; Šulc, Miroslav; Maňásková-Postlerová, Pavla

March, č. 359 (2015), s. 895-908 ISSN 0302-766X R&D Projects: GA ČR(CZ) GA14-05547S; GA MŠk(CZ) ED1.1.00/02.0109; GA ČR(CZ) GAP503/12/1834 Institutional research plan: CEZ:AV0Z50520701 Institutional support: RVO:86652036 ; RVO:61388971 Keywords : zona pellucida-binding receptors * monoclonal antibodies against sperm surface proteins * sperm surface proteins * RAB-2A * lactahedrin P47 Subject RIV: CE - Biochemistry Impact factor: 2.948, year: 2015

Mining Naïve Rabbit Antibody Repertoires by Phage Display for Monoclonal Antibodies of Therapeutic Utility.

Science.gov (United States)

Peng, Haiyong; Nerreter, Thomas; Chang, Jing; Qi, Junpeng; Li, Xiuling; Karunadharma, Pabalu; Martinez, Gustavo J; Fallahi, Mohammad; Soden, Jo; Freeth, Jim; Beerli, Roger R; Grawunder, Ulf; Hudecek, Michael; Rader, Christoph

2017-09-15

Owing to their high affinities and specificities, rabbit monoclonal antibodies (mAbs) have demonstrated value and potential primarily as basic research and diagnostic reagents, but, in some cases, also as therapeutics. To accelerate access to rabbit mAbs bypassing immunization, we generated a large naïve rabbit antibody repertoire represented by a phage display library encompassing >10 billion independent antibodies in chimeric rabbit/human Fab format and validated it by next-generation sequencing. Panels of rabbit mAbs selected from this library against two emerging cancer targets, ROR1 and ROR2, revealed high diversity, affinity, and specificity. Moreover, ROR1- and ROR2-targeting rabbit mAbs demonstrated therapeutic utility as components of chimeric antigen receptor-engineered T cells, further corroborating the value of the naïve rabbit antibody library as a rich and virtually unlimited source of rabbit mAbs. Copyright © 2017 Elsevier Ltd. All rights reserved.

[Current Perspective on Voltage-gated Potassium Channel Complex Antibody Associated Diseases].

Science.gov (United States)

Watanabe, Osamu

2018-04-01

Voltage-gated potassium channel (VGKC) complex auto-antibodies were initially identified in Isaacs' syndrome (IS), which is characterized by muscle cramps and neuromyotonia. These antibodies were subsequently identified in patients with Morvan's syndrome (MoS), which includes IS in conjunction with psychosis, insomnia, and dysautonomia. The antibodies have also been detected in a patient with limbic encephalopathy (LE) presenting with prominent amnesia and frequent seizures. Typical cases of LE have adult-onset, with frequent, brief dystonic seizures that predominantly affect the arms and ipsilateral face, and has recently been termed faciobrachial dystonic seizures. Autoantibodies against the extracellular domains of VGKC complex proteins, leucine-rich glioma-inactivated 1 (LGI1), and contactin-associated protein-2 (Caspr2), occur in patients with IS, MoS, and LE. However, routine testing has detected VGKC complex antibodies without LGI1 or Caspr2 reactivities (double-negative) in patients with other diseases, such as Creutzfeldt-Jakob disease and amyotrophic lateral sclerosis. Furthermore, double-negative VGKC complex antibodies are often directed against cytosolic epitopes of Kv1 subunits. Therefore, these antibodies should no longer be classified as neuronal-surface antibodies and lacking pathogenic potential. Novel information has been generated regarding autoantibody disruption of the physiological functions of target proteins. LGI1 antibodies neutralize the interaction between LGI1 and ADAM22, thereby reducing the synaptic AMPA receptors. It may be that the main action is on inhibitory neurons, explaining why the loss of AMPA receptors causes amnesia, neuronal excitability and seizures.

Monoclonal antibodies in the treatment of metastatic colorectal cancer: a review

NARCIS (Netherlands)

Tol, Jolien; Punt, Cornelis J. A.

2010-01-01

Two groups of agents targeting either the vascular endothelial growth factor (VEGF) receptor or the epidermal growth factor receptor (EGFR) have been added to the therapeutic arsenal against metastatic colorectal cancer (mCRC). Currently available agents in these groups are the anti-VEGF antibody

Development and Characterization of a Camelid Single Domain Antibody-Urease Conjugate That Targets Vascular Endothelial Growth Factor Receptor 2.

Science.gov (United States)

Tian, Baomin; Wong, Wah Yau; Uger, Marni D; Wisniewski, Pawel; Chao, Heman

2017-01-01

Angiogenesis is the process of new blood vessel formation and is essential for a tumor to grow beyond a certain size. Tumors secrete the pro-angiogenic factor vascular endothelial growth factor, which acts upon local endothelial cells by binding to vascular endothelial growth factor receptors (VEGFRs). In this study, we describe the development and characterization of V21-DOS47, an immunoconjugate that targets VEGFR2. V21-DOS47 is composed of a camelid single domain anti-VEGFR2 antibody (V21) and the enzyme urease. The conjugate specifically binds to VEGFR2 and urease converts endogenous urea into ammonia, which is toxic to tumor cells. Previously, we developed a similar antibody-urease conjugate, L-DOS47, which is currently in clinical trials for non-small cell lung cancer. Although V21-DOS47 was designed from parameters learned from the generation of L-DOS47, additional optimization was required to produce V21-DOS47. In this study, we describe the expression and purification of two versions of the V21 antibody: V21H1 and V21H4. Each was conjugated to urease using a different chemical cross-linker. The conjugates were characterized by a panel of analytical techniques, including SDS-PAGE, size exclusion chromatography, Western blotting, and LC-MS E peptide mapping. Binding characteristics were determined by ELISA and flow cytometry assays. To improve the stability of the conjugates at physiologic pH, the pIs of the V21 antibodies were adjusted by adding several amino acid residues to the C-terminus. For V21H4, a terminal cysteine was also added for use in the conjugation chemistry. The modified V21 antibodies were expressed in the E. coli BL21 (DE3) pT7 system. V21H1 was conjugated to urease using the heterobifunctional cross-linker succinimidyl-[( N -maleimidopropionamido)-diethyleneglycol] ester (SM(PEG) 2 ), which targets lysine resides in the antibody. V21H4 was conjugated to urease using the homobifunctional cross-linker, 1,8-bis(maleimido)diethylene glycol

Acetylcholine receptor binding antibody-associated myasthenia gravis and rhabdomyolysis induced by nivolumab in a patient with melanoma.

Science.gov (United States)

Shirai, Takushi; Sano, Tasuku; Kamijo, Fuminao; Saito, Nana; Miyake, Tomomi; Kodaira, Minori; Katoh, Nagaaki; Nishie, Kenichi; Okuyama, Ryuhei; Uhara, Hisashi

2016-01-01

We reported an 81-year-old woman with metastatic melanoma, in whom myasthenia gravis and rhabdomyolysis developed after nivolumab monotherapy. The first symptom of myasthenia gravis was dyspnea. Ultrasonography detected hypokinesis of the bilateral diaphragm suggesting myasthenia gravis, although there was no abnormal finding of the lungs in computed tomography images. Acetylcholine receptor binding antibodies were low-titer positive in the preserved serum before administration of nivolumab, strongly suggesting that the myasthenia gravis was a nivolumab-related immune adverse event. Despite the remarkable clinical benefits of immune checkpoint inhibitors for patients with advanced melanoma, it is important to recognize unexpected immune-related adverse events. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

β-Adrenergic receptor-mediated suppression of interleukin 2 receptors in human lymphocytes

International Nuclear Information System (INIS)

Feldman, R.D.; Hunninghake, G.W.; McArdle, W.L.

1987-01-01

Adrenergic receptor agonists are know to attenuate the proliferative response of human lymphocytes after activation; however, their mechanism of action is unknown. Since expression of interleukin 2 (IL-2) receptors is a prerequisite for proliferation, the effect of β-adrenergic receptor agonists on lymphocyte IL-2 receptors was studied on both mitogen-stimulated lymphocytes and IL-2-dependent T lymphocyte cell lines. In both cell types the β-adrenergic receptor agonist isoproterenol blocked the expression of IL-2 receptors, as determined with the IL-2 receptor anti-TAC antibody. To determine the effect of β-adrenergic agonists on expression of the high affinity IL-2 receptors, [ 125 I]IL-2 binding studies were performed at concentrations selective for high affinity sites. No significant effect of β-adrenergic agonists on high affinity IL-2 receptor sites could be detected. The data demonstrate that β-adrenergic receptor agonists down-regulate IL-2 receptors primarily affecting low affinity sites

Fixation effect of SurePath preservative fluids using epidermal growth factor receptor mutation-specific antibodies for immunocytochemistry.

Science.gov (United States)

Kawahara, Akihiko; Taira, Tomoki; Abe, Hideyuki; Watari, Kosuke; Murakami, Yuichi; Fukumitsu, Chihiro; Takase, Yorihiko; Yamaguchi, Tomohiko; Azuma, Koichi; Akiba, Jun; Ono, Mayumi; Kage, Masayoshi

2014-02-01

Cytological diagnosis of respiratory disease has become important, not only for histological typing using immunocytochemistry (ICC) but also for molecular DNA analysis of cytological material. The aim of this study was to investigate the fixation effect of SurePath preservative fluids. Human lung cancer PC9 and 11-18 cell lines, and lung adenocarcinoma cells in pleural effusion, were fixed in CytoRich Blue, CytoRich Red, 15% neutral-buffered formalin, and 95% ethanol, respectively. PC9 and 11-18 cell lines were examined by ICC with epidermal growth factor receptor (EGFR) mutation-specific antibodies, the EGFR mutation DNA assay, and fluorescence in situ hybridization. The effect of antigenic storage time was investigated in lung adenocarcinoma cells in pleural effusion by ICC using the lung cancer detection markers. PC9 and 11-18 cell lines in formalin-based fixatives showed strong staining of EGFR mutation-specific antibodies and lung cancer detection markers by ICC as compared with ethanol-based fixatives. DNA preservation with CytoRich Blue and CytoRich Red was superior to that achieved with 95% ethanol and 15% neutral-buffered formalin fixatives, whereas EGFR mutations by DNA assay and EGFR gene amplification by fluorescence in situ hybridization were successfully identified in all fixative samples. Although cytoplasmic antigens maintained high expression levels, expression levels in nuclear antigens fell as storage time increased. These results indicate that CytoRich Red is not only suitable for ICC with EGFR mutation-specific antibodies, but also for DNA analysis of cytological material, and is useful in molecular testing of lung cancer, for which various types of analyses will be needed in future. © 2013 American Cancer Society.

Epitope mapping and characterization of a novel CD4-induced human monoclonal antibody capable of neutralizing primary HIV-1 strains

International Nuclear Information System (INIS)

Xiang Shihua; Wang Liping; Abreu, Mariam; Huang, C.-C.; Kwong, Peter D.; Rosenberg, Eric; Robinson, James E.; Sodroski, Joseph

2003-01-01

Human immunodeficiency virus (HIV-1) enters target cells by binding its gp120 exterior envelope glycoprotein to CD4 and one of the chemokine receptors, CCR5 or CXCR4. CD4-induced (CD4i) antibodies bind gp120 more efficiently after CD4 binding and block the interaction with the chemokine receptor. Examples of CD4i antibodies are limited, and the prototypes of the CD4i antibodies exhibit only weak neutralizing activity against primary, clinical HIV-1 isolates. Here we report the identification of a novel antibody, E51, that exhibits CD4-induced binding to gp120 and neutralizes primary HIV-1 more efficiently than the prototypic CD4i antibodies. The E51 antibody blocks the interaction of gp120-CD4 complexes with CCR5 and binds to a highly conserved, basic gp120 element composed of the β19-strand and surrounding structures. Thus, on primary HIV-1 isolates, this gp120 region, which has been previously implicated in chemokine receptor binding, is accessible to a subset of CD4i antibodies

Infantile Onset Myasthenia Gravis with MuSK Antibodies

Directory of Open Access Journals (Sweden)

J Gordon Millichap

2006-08-01

Full Text Available A late infantile case of myasthenia gravis (MG with anti-muscle-specific receptor tyrosine kinase (MuSK high antibody (Ab titer is reported from Kyushu University, Fukuoka; and Nagasaki University, Japan.

Limbic encephalitis associated with anti-NH2-terminal of α-enolase antibodies

Science.gov (United States)

Kishitani, Toru; Matsunaga, Akiko; Ikawa, Masamichi; Hayashi, Kouji; Yamamura, Osamu; Hamano, Tadanori; Watanabe, Osamu; Tanaka, Keiko; Nakamoto, Yasunari; Yoneda, Makoto

2017-01-01

Abstract Several types of autoantibodies have been reported in autoimmune limbic encephalitis (LE), such as antibodies against the voltage-gated potassium channel (VGKC) complex including leucine-rich glioma inactivated 1 (LGI1). We recently reported a patient with autoimmune LE and serum anti-NH2-terminal of α-enolase (NAE) antibodies, a specific diagnostic marker for Hashimoto encephalopathy (HE), who was diagnosed with HE based on the presence of antithyroid antibodies and responsiveness to immunotherapy. This case suggests that LE patients with antibodies to both the thyroid and NAE could be diagnosed with HE and respond to immunotherapy. The aim of this study was to clarify the clinicoimmunological features and efficacy of immunotherapy in LE associated with anti-NAE antibodies to determine whether the LE is a clinical subtype of HE. We examined serum anti-NAE antibodies in 78 LE patients with limbic abnormality on magnetic resonance imaging and suspected HE based on positivity for antithyroid antibodies. Nineteen of the 78 patients had anti-NAE antibodies; however, 5 were excluded because they were double positive for antibodies to the VGKC complex including LGI1. No antibodies against the N-methyl-D-aspartate receptor (NMDAR), contactin-associated protein 2 (Caspr2), γ-aminobutyric acid-B receptor (GABABR), or α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) were detected in the 19 patients. Among the remaining 14 who were positive only for anti-NAE antibodies, the median age was 62.5 (20–83) years, 9 (64%) were women, and 8 (57%) showed acute onset, with less than 2 weeks between onset and admission. Consciousness disturbance (71%) and memory disturbance (64%) were frequently observed, followed by psychiatric symptoms (50%) and seizures (43%). The frequency of these symptoms significantly differed between the acute- and subacute-onset groups. Abnormalities in cerebrospinal fluid and electroencephalogram were commonly observed (92

Host-Primed Ebola Virus GP Exposes a Hydrophobic NPC1 Receptor-Binding Pocket, Revealing a Target for Broadly Neutralizing Antibodies

Directory of Open Access Journals (Sweden)

Zachary A. Bornholdt

2016-02-01

Full Text Available The filovirus surface glycoprotein (GP mediates viral entry into host cells. Following viral internalization into endosomes, GP is cleaved by host cysteine proteases to expose a receptor-binding site (RBS that is otherwise hidden from immune surveillance. Here, we present the crystal structure of proteolytically cleaved Ebola virus GP to a resolution of 3.3 Å. We use this structure in conjunction with functional analysis of a large panel of pseudotyped viruses bearing mutant GP proteins to map the Ebola virus GP endosomal RBS at molecular resolution. Our studies indicate that binding of GP to its endosomal receptor Niemann-Pick C1 occurs in two distinct stages: the initial electrostatic interactions are followed by specific interactions with a hydrophobic trough that is exposed on the endosomally cleaved GP1 subunit. Finally, we demonstrate that monoclonal antibodies targeting the filovirus RBS neutralize all known filovirus GPs, making this conserved pocket a promising target for the development of panfilovirus therapeutics.

GABARAPL1 antibodies: target one protein, get one free!

Science.gov (United States)

Le Grand, Jaclyn Nicole; Chakrama, Fatima Zahra; Seguin-Py, Stéphanie; Fraichard, Annick; Delage-Mourroux, Régis; Jouvenot, Michèle; Risold, Pierre-Yves; Boyer-Guittaut, Michaël

2011-11-01

Atg8 is a yeast protein involved in the autophagic process and in particular in the elongation of autophagosomes. In mammals, several orthologs have been identified and are classed into two subfamilies: the LC3 subfamily and the GABARAP subfamily, referred to simply as the LC3 or GABARAP families. GABARAPL1 (GABARAP-like protein 1), one of the proteins belonging to the GABARAP (GABA(A) receptor-associated protein) family, is highly expressed in the central nervous system and implicated in processes such as receptor and vesicle transport as well as autophagy. The proteins that make up the GABARAP family demonstrate conservation of their amino acid sequences and protein structures. In humans, GABARAPL1 shares 86% identity with GABARAP and 61% with GABARAPL2 (GATE-16). The identification of the individual proteins is thus very limited when working in vivo due to a lack of unique peptide sequences from which specific antibodies can be developed. Actually, and to our knowledge, there are no available antibodies on the market that are entirely specific to GABARAPL1 and the same may be true of the anti-GABARAP antibodies. In this study, we sought to examine the specificity of three antibodies targeted against different peptide sequences within GABARAPL1: CHEM-CENT (an antibody raised against a short peptide sequence within the center of the protein), PTG-NTER (an antibody raised against the N-terminus of the protein) and PTG-FL (an antibody raised against the full-length protein). The results described in this article demonstrate the importance of testing antibody specificity under the conditions for which it will be used experimentally, a caution that should be taken when studying the expression of the GABARAP family proteins.

Total Thyroidectomy for Thyroid Cancer Followed by Thyroid Storm due to Thyrotropin Receptor Antibody Stimulation of Metastatic Thyroid Tissue

DEFF Research Database (Denmark)

Folkestad, Lars; Brandt, Frans; Brix, Thomas

2017-01-01

BACKGROUND: Graves disease (GD) is an autoimmune condition characterized by the presence of antibodies against the thyrotropin receptor (TRAB), which stimulate the thyroid gland to produce excess thyroid hormone. Theoretically, TRAB could stimulate highly differentiated thyroid cancer tissue and...... treatment continued until after the fourth RAI dose. Hypothyroidism did not occur until following the fifth RAI treatment. SUMMARY AND CONCLUSIONS: We present a patient initially diagnosed with thyrotoxicosis and subsequently with metastatic follicular variant of papillary thyroid cancer. It is suggested...... that TRAB stimulated the highly differentiated extrathyroidal metastatic thyroid tissue to produce excessive amounts of thyroid hormone, delayed diagnosis, and potential aggravation of the course of thyroid cancer....

Immunological studies on the structure and function of the nicotinic acetylcholine receptor in mammalian muscle

Energy Technology Data Exchange (ETDEWEB)

Gu, Y.

1989-01-01

The specificity of the antibodies in the serum of a patient with myasthenia gravis for a the {alpha}-bungarotoxin binding sites of the acetylcholine receptor (AChR) was examined using AChRs in the C2 mouse muscle cell line as a model. The antibodies were shown to be specific for one of the two toxin-binding sites. The effect of the antibodies in this myasthenic serum on the functional response of the receptor to cholinergic agonists was also examined using carbamylcholine-induced {sup 22}Na uptake into C2 myotubes as a measured of the receptor function. Antibodies specific for the {gamma}, {delta}, and {epsilon} subunit, respectively, of mammalian muscle AChRs were developed using subunit-specific synthetic peptides as antigens. Using these antibodies and monoclonal antibodies for other subunits as probes, I have identified four ({alpha}, {beta}, {gamma}, and {delta}) subunits of mammalian muscle AChRs on immunoblots. When AChRs from embryonic, neonatal, normal and denervated adult muscles were compared on immunoblots, the {alpha}, {beta}, and {delta} subunits were identical in all four receptor preparations, with or without endoglycosidase digestion. The spatial and temporal distribution of the {gamma}- and {epsilon}- AChRs in developing and in denervated muscles corresponds to the distribution of AChRs with slow and fast channels, respectively, and that the development changes in the channel properties of the receptor arise from a change in the subunit composition of the receptor, in which the {gamma} is replaced by {epsilon}.

Immunological studies on the structure and function of the nicotinic acetylcholine receptor in mammalian muscle

International Nuclear Information System (INIS)

Gu, Y.

1989-01-01

The specificity of the antibodies in the serum of a patient with myasthenia gravis for a the α-bungarotoxin binding sites of the acetylcholine receptor (AChR) was examined using AChRs in the C2 mouse muscle cell line as a model. The antibodies were shown to be specific for one of the two toxin-binding sites. The effect of the antibodies in this myasthenic serum on the functional response of the receptor to cholinergic agonists was also examined using carbamylcholine-induced 22 Na uptake into C2 myotubes as a measured of the receptor function. Antibodies specific for the γ, δ, and ε subunit, respectively, of mammalian muscle AChRs were developed using subunit-specific synthetic peptides as antigens. Using these antibodies and monoclonal antibodies for other subunits as probes, I have identified four (α, β, γ, and δ) subunits of mammalian muscle AChRs on immunoblots. When AChRs from embryonic, neonatal, normal and denervated adult muscles were compared on immunoblots, the α, β, and δ subunits were identical in all four receptor preparations, with or without endoglycosidase digestion. The spatial and temporal distribution of the γ- and ε- AChRs in developing and in denervated muscles corresponds to the distribution of AChRs with slow and fast channels, respectively, and that the development changes in the channel properties of the receptor arise from a change in the subunit composition of the receptor, in which the γ is replaced by ε

Neuronal surface antigen antibodies in limbic encephalitis

Science.gov (United States)

Graus, F; Saiz, A; Lai, M; Bruna, J; López, F; Sabater, L; Blanco, Y; Rey, M J.; Ribalta, T; Dalmau, J

2008-01-01

Objective: To report the frequency and type of antibodies against neuronal surface antigens (NSA-ab) in limbic encephalitis (LE). Methods: Analysis of clinical features, neuropathologic findings, and detection of NSA-ab using immunochemistry on rat tissue and neuronal cultures in a series of 45 patients with paraneoplastic (23) or idiopathic (22) LE. Results: NSA-ab were identified in 29 patients (64%; 12 paraneoplastic, 17 idiopathic). Thirteen patients had voltage-gated potassium channels (VGKC)-ab, 11 novel NSA (nNSA)-ab, and 5 NMDA receptor (NMDAR)-ab. nNSA-ab did not identify a common antigen and were more frequent in paraneoplastic than idiopathic LE (39% vs 9%; p = 0.03). When compared with VGKC-ab or NMDAR-ab, the nNSA associated more frequently with intraneuronal antibodies (11% vs 73%; p = 0.001). Of 12 patients (9 nNSA-ab, 2 VGKC-ab, 1 NMDAR-ab) with paraneoplastic LE and NSA-ab, concomitant intraneuronal antibodies occurred in 9 (75%). None of these 12 patients improved with immunotherapy. The autopsy of three of them showed neuronal loss, microgliosis, and cytotoxic T cell infiltrates in the hippocampus and amygdala. These findings were compatible with a T-cell mediated neuronal damage. In contrast, 13 of 17 (76%) patients with idiopathic LE and NSA-ab (8 VGKC-ab, 4 NMDAR-ab, 1 nNSA-ab) and 1 of 5 (20%) without antibodies had clinical improvement (p = 0.04). Conclusions: In paraneoplastic limbic encephalitis (LE), novel antibodies against neuronal surface antigens (nNSA-ab) occur frequently, coexist with antibodies against intracellular antigens, and these cases are refractory to immunotherapy. In idiopathic LE, the likelihood of improvement is significantly higher in patients with NSA-ab than in those without antibodies. GLOSSARY GAD = glutamic acid decarboxylase; LE = limbic encephalitis; NMDAR = N-methyl-D-aspartate receptor; NSA = neuronal surface antigens; nNSA = novel NSA; SCLC = small-cell lung cancer; VGKC = voltage-gated potassium channels

A radioligand immunoassay for 1,25-dihydroxyvitamin D3 receptors using monoclonal antibody: detection of a phenotypic receptor variant in vitamin D-dependency rickets (type II) which does not bind hormone

International Nuclear Information System (INIS)

Pike, J.W.; Dokoh, Shigeharu; Liberman, U.A.; Eil, C.; Haussler, M.R.; Marx, S.J.

1984-01-01

Vitamin D-dependency rickets, type II (VDDRII), is a well recognized heritable disorder characterized by peripheral target organ resistance to 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ), the hormonally active form of the vitamin. Recently, cultured skin fibroblasts obtained from a number of patients with VDDRII have been utilized to characterize the underlying molecular defects associated with this malady. Recently monoclonal antibodies to the vitamin D receptor have been generated, and a radioligand immunoassay (RLIA) for the detection of this molecule has been developed which is independent of its hormone-binding capacity. This report describes the application of the immunoassay in the detection of receptor-like molecules in fibroblasts derived from patients with VDDRII. The results indicate that the molecule is generally present in all patients, and provides a mechanism for individual responsiveness to pharmacologic treatment with vitamin D 3 metabolites. 8 refs.; 3 figs.; 1 table

Severe hypoglycaemia in a person with insulin autoimmune syndrome accompanied by insulin receptor anomaly type B.

Science.gov (United States)

Kato, T; Itoh, M; Hanashita, J; Itoi, T; Matsumoto, T; Ono, Y; Imamura, S; Hayakawa, N; Suzuki, A; Mizutani, Y; Uchigata, Y; Oda, N

2007-11-01

A rare case of the insulin autoimmune syndrome (IAS) accompanied by insulin receptor anomaly is reported. Antibodies to insulin and insulin receptor were determined in the patient with severe hypoglycaemia before and after the treatment with prednisolone. Titers of antibody to insulin and insulin receptors were 73.0% and 41.5%, respectively. Drug-induced lymphocyte stimulation tests were all negative for the suspicious drugs. Her HLA-DR was DRB1*0403/04051. Following steroid therapy, the formation of antibodies was suppressed and alleviated her symptoms. Scatchard analysis yielded findings specific to polyclonal antibodies. The changes in autoantibodies resulted in alleviation of the hypoglycemic symptoms as a result of steroid therapy.

α7 Nicotinic acetylcholine receptor-specific antibody induces inflammation and amyloid β42 accumulation in the mouse brain to impair memory.

Directory of Open Access Journals (Sweden)

Olena Lykhmus

Full Text Available Nicotinic acetylcholine receptors (nAChRs expressed in the brain are involved in regulating cognitive functions, as well as inflammatory reactions. Their density is decreased upon Alzheimer disease accompanied by accumulation of β-amyloid (Aβ42, memory deficit and neuroinflammation. Previously we found that α7 nAChR-specific antibody induced pro-inflammatory interleukin-6 production in U373 glioblastoma cells and that such antibodies were present in the blood of humans. We raised a hypothesis that α7 nAChR-specific antibody can cause neuroinflammation when penetrating the brain. To test this, C57Bl/6 mice were either immunized with extracellular domain of α7 nAChR subunit α7(1-208 or injected with bacterial lipopolysaccharide (LPS for 5 months. We studied their behavior and the presence of α3, α4, α7, β2 and β4 nAChR subunits, Aβ40 and Aβ42 and activated astrocytes in the brain by sandwich ELISA and confocal microscopy. It was found that either LPS injections or immunizations with α7(1-208 resulted in region-specific decrease of α7 and α4β2 and increase of α3β4 nAChRs, accumulation of Aβ42 and activated astrocytes in the brain of mice and worsening of their episodic memory. Intravenously transferred α7 nAChR-specific-antibodies penetrated the brain parenchyma of mice pre-injected with LPS. Our data demonstrate that (1 neuroinflammation is sufficient to provoke the decrease of α7 and α4β2 nAChRs, Aβ42 accumulation and memory impairment in mice and (2 α7(1-208 nAChR-specific antibodies can cause inflammation within the brain resulting in the symptoms typical for Alzheimer disease.

Thyroid-Stimulating Hormone Receptor Antibodies in Pregnancy: Clinical Relevance

Science.gov (United States)

Bucci, Ines; Giuliani, Cesidio; Napolitano, Giorgio

2017-01-01

Graves’ disease is the most common cause of thyrotoxicosis in women of childbearing age. Approximately 1% of pregnant women been treated before, or are being treated during pregnancy for Graves’ hyperthyroidism. In pregnancy, as in not pregnant state, thyroid-stimulating hormone (TSH) receptor (TSHR) antibodies (TRAbs) are the pathogenetic hallmark of Graves’ disease. TRAbs are heterogeneous for molecular and functional properties and are subdivided into activating (TSAbs), blocking (TBAbs), or neutral (N-TRAbs) depending on their effect on TSHR. The typical clinical features of Graves’ disease (goiter, hyperthyroidism, ophthalmopathy, dermopathy) occur when TSAbs predominate. Graves’ disease shows some peculiarities in pregnancy. The TRAbs disturb the maternal as well as the fetal thyroid function given their ability to cross the placental barrier. The pregnancy-related immunosuppression reduces the levels of TRAbs in most cases although they persist in women with active disease as well as in women who received definitive therapy (radioiodine or surgery) before pregnancy. Changes of functional properties from stimulating to blocking the TSHR could occur during gestation. Drug therapy is the treatment of choice for hyperthyroidism during gestation. Antithyroid drugs also cross the placenta and therefore decrease both the maternal and the fetal thyroid hormone production. The management of Graves’ disease in pregnancy should be aimed at maintaining euthyroidism in the mother as well as in the fetus. Maternal and fetal thyroid dysfunction (hyperthyroidism as well as hypothyroidism) are in fact associated with several morbidities. Monitoring of the maternal thyroid function, TRAbs measurement, and fetal surveillance are the mainstay for the management of Graves’ disease in pregnancy. This review summarizes the biochemical, immunological, and therapeutic aspects of Graves’ disease in pregnancy focusing on the role of the TRAbs in maternal and fetal

Thyroid-Stimulating Hormone Receptor Antibodies in Pregnancy: Clinical Relevance

Directory of Open Access Journals (Sweden)

Ines Bucci

2017-06-01

Full Text Available Graves’ disease is the most common cause of thyrotoxicosis in women of childbearing age. Approximately 1% of pregnant women been treated before, or are being treated during pregnancy for Graves’ hyperthyroidism. In pregnancy, as in not pregnant state, thyroid-stimulating hormone (TSH receptor (TSHR antibodies (TRAbs are the pathogenetic hallmark of Graves’ disease. TRAbs are heterogeneous for molecular and functional properties and are subdivided into activating (TSAbs, blocking (TBAbs, or neutral (N-TRAbs depending on their effect on TSHR. The typical clinical features of Graves’ disease (goiter, hyperthyroidism, ophthalmopathy, dermopathy occur when TSAbs predominate. Graves’ disease shows some peculiarities in pregnancy. The TRAbs disturb the maternal as well as the fetal thyroid function given their ability to cross the placental barrier. The pregnancy-related immunosuppression reduces the levels of TRAbs in most cases although they persist in women with active disease as well as in women who received definitive therapy (radioiodine or surgery before pregnancy. Changes of functional properties from stimulating to blocking the TSHR could occur during gestation. Drug therapy is the treatment of choice for hyperthyroidism during gestation. Antithyroid drugs also cross the placenta and therefore decrease both the maternal and the fetal thyroid hormone production. The management of Graves’ disease in pregnancy should be aimed at maintaining euthyroidism in the mother as well as in the fetus. Maternal and fetal thyroid dysfunction (hyperthyroidism as well as hypothyroidism are in fact associated with several morbidities. Monitoring of the maternal thyroid function, TRAbs measurement, and fetal surveillance are the mainstay for the management of Graves’ disease in pregnancy. This review summarizes the biochemical, immunological, and therapeutic aspects of Graves’ disease in pregnancy focusing on the role of the TRAbs in maternal and

Laminin binding protein, 34/67 laminin receptor, carries stage-specific embryonic antigen-4 epitope defined by monoclonal antibody Raft.2

International Nuclear Information System (INIS)

Katagiri, Yohko U.; Kiyokawa, Nobutaka; Nakamura, Kyoko; Takenouchi, Hisami; Taguchi, Tomoko; Okita, Hajime; Umezawa, Akihiro; Fujimoto, Junichiro

2005-01-01

We previously produced monoclonal antibodies against the detergent-insoluble microdomain, i.e., the raft microdomain, of the human renal cancer cell line ACHN. Raft.2, one of these monoclonal antibodies, recognizes sialosyl globopentaosylceramide, which has the stage-specific embryonic antigen (SSEA)-4 epitope. Although the mouse embryonal carcinoma (EC) cell line F9 does not express SSEA-4, some F9 cells stained with Raft.2. Western analysis and matrix-assisted laser desorption ionization-time of flight mass spectrometry identified the Raft.2 binding molecule as laminin binding protein (LBP), i.e., 34/67 laminin receptor. Weak acid treatment or digestion with Clostridium perfringens sialidase reduced Raft.2 binding to LBP on nitrocellulose sheets and [ 14 C]galactose was incorporated into LBP, indicating LBP to have a sialylated carbohydrate moiety. Subcellular localization analysis by sucrose density-gradient centrifugation and examination by confocal microscopy revealed LBP to be localized on the outer surface of the plasma membrane. An SSEA-4-positive human EC cell line, NCR-G3 cells, also expressed Raft.2-binding LBP

Clonal relationships between thyroid-stimulating hormone receptor-stimulating antibodies illustrate the effect of hypermutation on antibody function

DEFF Research Database (Denmark)

Padoa, Carolyn J; Larsen, Sanne L; Hampe, Christiane S

2009-01-01

relationship and derivation from a single precursor B-cell clone. The IGHV-region genes of the two mAbs underwent high degrees of somatic hypermutation by sharing numerous mutations before diverging, while the IGLV genes evolved separately. Interestingly, the mutations were present in both the complementarity......, in experimentally immunized mice, multiple pathogenic antibodies to TSHR can arise from a single clone by a series of somatic mutations in the V-region genes and may give an insight into how such antibodies develop spontaneously in autoimmune Graves' disease....

Monoclonal Antibodies, Derived from Humans Vaccinated with the RV144 HIV Vaccine Containing the HVEM Binding Domain of Herpes Simplex Virus (HSV) Glycoprotein D, Neutralize HSV Infection, Mediate Antibody-Dependent Cellular Cytotoxicity, and Protect Mice from Ocular Challenge with HSV-1.

Science.gov (United States)

Wang, Kening; Tomaras, Georgia D; Jegaskanda, Sinthujan; Moody, M Anthony; Liao, Hua-Xin; Goodman, Kyle N; Berman, Phillip W; Rerks-Ngarm, Supachai; Pitisuttithum, Punnee; Nitayapan, Sorachai; Kaewkungwal, Jaranit; Haynes, Barton F; Cohen, Jeffrey I

2017-10-01

The RV144 HIV vaccine trial included a recombinant HIV glycoprotein 120 (gp120) construct fused to a small portion of herpes simplex virus 1 (HSV-1) glycoprotein D (gD) so that the first 40 amino acids of gp120 were replaced by the signal sequence and the first 27 amino acids of the mature form of gD. This region of gD contains most of the binding site for HVEM, an HSV receptor important for virus infection of epithelial cells and lymphocytes. RV144 induced antibodies to HIV that were partially protective against infection, as well as antibodies to HSV. We derived monoclonal antibodies (MAbs) from peripheral blood B cells of recipients of the RV144 HIV vaccine and showed that these antibodies neutralized HSV-1 infection in cells expressing HVEM, but not the other major virus receptor, nectin-1. The MAbs mediated antibody-dependent cellular cytotoxicity (ADCC), and mice that received the MAbs and were then challenged by corneal inoculation with HSV-1 had reduced eye disease, shedding, and latent infection. To our knowledge, this is the first description of MAbs derived from human recipients of a vaccine that specifically target the HVEM binding site of gD. In summary, we found that monoclonal antibodies derived from humans vaccinated with the HVEM binding domain of HSV-1 gD (i) neutralized HSV-1 infection in a cell receptor-specific manner, (ii) mediated ADCC, and (iii) reduced ocular disease in virus-infected mice. IMPORTANCE Herpes simplex virus 1 (HSV-1) causes cold sores and neonatal herpes and is a leading cause of blindness. Despite many trials, no HSV vaccine has been approved. Nectin-1 and HVEM are the two major cellular receptors for HSV. These receptors are expressed at different levels in various tissues, and the role of each receptor in HSV pathogenesis is not well understood. We derived human monoclonal antibodies from persons who received the HIV RV144 vaccine that contained the HVEM binding domain of HSV-1 gD fused to HIV gp120. These antibodies were

Nuclear oncology with monoclonal antibodies and peptides

International Nuclear Information System (INIS)

Hosono, Makoto

1998-01-01

Imaging and therapy using radiolabeled monoclonal antibodies have proved useful in many clinical studies. However, immunogenicity of mouse antibodies to human and insufficient tumor-to-normal tissue ratios remained to be solved. Chimerization and humanization by genetic engineering, and multistep targeting techniques have enabled lower immunogenicity and higher tumor-to-normal tissue contrast. Peptides like somatostatin-analogs have been reportedly useful in imaging tumors, which are either somatostatin receptor positive or negative. Elevated normal tissue accumulation of radiolabeled peptides is a drawback in aiming internal radiation therapy. (author). 51 refs

Production of thyrotropin receptor antibodies in acute phase of infectious mononucleosis due to Epstein-Barr virus primary infection: a case report of a child.

Science.gov (United States)

Nagata, Keiko; Okuno, Keisuke; Ochi, Marika; Kumata, Keisuke; Sano, Hitoshi; Yoneda, Naohiro; Ueyama, Jun-Ichi; Matsushita, Michiko; Kuwamoto, Satoshi; Kato, Masako; Murakami, Ichiro; Kanzaki, Susumu; Hayashi, Kazuhiko

2015-01-01

Various autoantibodies have been reported to be detected during the progression of infectious mononucleosis. We observed a case of infectious mononucleosis due to Epstein-Barr virus primary infection for 2 months, and noticed the transiently increased titer of thyrotropin receptor autoantibodies detected at the acute phase on the 3rd day after admission. At that time, real-time quantitative PCR also revealed the mRNA expressions of an immediate early lytic gene, BZLF1, and a latent gene, EBNA2. The expression of BZLF1 mRNA means that Epstein-Barr virus infects lytically, and EBNA2 protein has an important role in antibody production as well as the establishment of Epstein-Barr virus latency. These results suggest that Epstein-Barr virus lytic infection is relevant to thyrotropin receptor autoantibody production. Thyrotropin receptor autoantibodies stimulate thyroid follicular cells to produce excessive thyroid hormones and cause Graves' disease. Recently, we reported the thyrotropin receptor autoantibody production from thyrotropin receptor autoantibody-predisposed Epstein-Barr virus-infected B cells by the induction of Epstein-Barr virus lytic infection in vitro. This case showed in vivo findings consistent with our previous reports, and is important to consider the pathophysiology of Graves' disease and one of the mechanisms of autoimmunity.

Binding affinities of anti-acetylcholine receptor autoantibodies in myasthenia gravis

Energy Technology Data Exchange (ETDEWEB)

Bray, J.J.; Drachman, D.B.

1982-01-01

Antibodies directed against acetylcholine (ACh) receptors are present in the sera of nearly 90% of patients with myasthenia gravis (MG), and are involved in the pathogenesis of this autoimmune disease. However, the antibody titers measured by the standard radioimmunoassay correspond poorly with the clinical severity of the disease. To determine whether this disparity could be accounted for by differences in the binding affinities of anti-ACh receptor antibodies in different patients, we have measured the binding affinities of these autoantibodies in 15 sera from MG patients. The affinity constants (K/sub o/), as determined by Scatchard analysis, were all in the range of 10/sup 10/ M/sup -1/, comparable to the highest values reported in immunized animals. The affinity constants were truly representative of the population of autoantibodies detected by the radioimmunoassay, as shown by the remarkable linearity of the Scatchard plots (r/sup 2/>0.90) and the close correlation between the antibody titers determined by extrapolation of the Scatchard plots and by saturation analysis (r = 0.99; p < 0.001). There was only a 6-fold variation in affinity constants measured in this series of patients despite widely differing antibody titers and severity of the disease. Factors other than the titer and affinity of anti-ACh receptor antibodies may correlate better with the clinical manifestations of MG.

Functional characterization of the high affinity IgG Receptor : making heads and tails of FcγRI

NARCIS (Netherlands)

van der Poel, C.E.

2011-01-01

This thesis focuses on human FcγRI, a high affinity receptor for antibodies of the IgG isotype. IgG is the most abundant antibody type in blood and all currently FDA approved therapeutic antibodies are of the IgG isotype. FcγRI, a member of the activating Fcγ receptors, exists as a complex of a

Epidemiology of myasthenia gravis with anti-muscle specific kinase antibodies in the Netherlands

NARCIS (Netherlands)

Niks, Erik H.; Kuks, Jan B. M.; Verschuuren, Jan J. G. M.

The epidemiology of myasthenia gravis subtypes and the frequency of antibodies to muscle-specific kinase (MuSK) was studied in patients with generalised myasthenia gravis without anti-acetylcholine receptor antibodies who had an onset of symptoms between 1990 and 2004 in a well-defined region in the

A novel llama antibody targeting Fn14 exhibits anti-metastatic activity in vivo.

Science.gov (United States)

Trebing, Johannes; Lang, Isabell; Chopra, Martin; Salzmann, Steffen; Moshir, Mahan; Silence, Karen; Riedel, Simone S; Siegmund, Daniela; Beilhack, Andreas; Otto, Christoph; Wajant, Harald

2014-01-01

Expression of fibroblast growth factor (FGF)-inducible 14 (Fn14), a member of the tumor necrosis factor receptor superfamily, is typically low in healthy adult organisms, but strong Fn14 expression is induced in tissue injury and tissue remodeling. High Fn14 expression is also observed in solid tumors, which is why this receptor is under consideration as a therapeutic target in oncology. Here, we describe various novel mouse-human cross-reactive llama-derived recombinant Fn14-specific antibodies (5B6, 18D1, 4G5) harboring the human IgG1 Fc domain. In contrast to recombinant variants of the established Fn14-specific antibodies PDL192 and P4A8, all three llama-derived antibodies efficiently bound to the W42A and R56P mutants of human Fn14. 18D1 and 4G5, but not 5B6, efficiently blocked TNF-like weak inducer of apoptosis(TWEA K) binding at low concentrations (0.2–2 μg/ml). Oligomerization and Fcγ receptor (FcγR) binding converted all antibodies into strong Fn14 agonists. Variants of 18D1 with enhanced and reduced antibody-dependent cell-mediated cytotoxicity (ADCC) activity were further analyzed in vivo with respect to their effect on metastasis. In a xenogeneic model using human colon carcinoma cancer cells, both antibody variants were effective in reducing metastasis to the liver. In contrast, only the 18D1 variant with enhanced ADCC activity, but not its ADCC-defective counterpart, suppressed lung metastasis in the RE NCA model. In sum, this suggests that Fn14 targeting might primarily act by triggering of antibody effector functions, but also by blockade of TWEA K-Fn14 interaction in some cases

Macrophages are critical effectors of antibody therapies for cancer.

Science.gov (United States)

Weiskopf, Kipp; Weissman, Irving L

2015-01-01

Macrophages are innate immune cells that derive from circulating monocytes, reside in all tissues, and participate in many states of pathology. Macrophages play a dichotomous role in cancer, where they promote tumor growth but also serve as critical immune effectors of therapeutic antibodies. Macrophages express all classes of Fcγ receptors, and they have immense potential to destroy tumors via the process of antibody-dependent phagocytosis. A number of studies have demonstrated that macrophage phagocytosis is a major mechanism of action of many antibodies approved to treat cancer. Consequently, a number of approaches to augment macrophage responses to therapeutic antibodies are under investigation, including the exploration of new targets and development of antibodies with enhanced functions. For example, the interaction of CD47 with signal-regulatory protein α (SIRPα) serves as a myeloid-specific immune checkpoint that limits the response of macrophages to antibody therapies, and CD47-blocking agents overcome this barrier to augment phagocytosis. The response of macrophages to antibody therapies can also be enhanced with engineered Fc variants, bispecific antibodies, or antibody-drug conjugates. Macrophages have demonstrated success as effectors of cancer immunotherapy, and further investigation will unlock their full potential for the benefit of patients.

Monoclonal Antibodies to the Thyrotropin Receptor

Directory of Open Access Journals (Sweden)

Takao Ando

2005-01-01

Full Text Available The thyrotropin receptor (TSHR is a seven transmembrane G-protein linked glycoprotein expressed on the thyroid cell surface and which, under the regulation of TSH, controls the production and secretion of thyroid hormone from the thyroid gland. This membrane protein is also a major target antigen in the autoimmune thyroid diseases. In Graves' disease, autoantibodies to the TSHR (TSHR-Abs stimulate the TSHR to produce thyroid hormone excessively. In autoimmune thyroid failure, some patients exhibit TSHR-Abs which block TSH action on the receptor. There have been many attempts to generate human stimulating TSHR-mAbs, but to date, only one pathologically relevant human stimulating TSHR-mAb has been isolated. Most mAbs to the TSHR have been derived from rodents immunized with TSHR antigen from bacteria or insect cells. These antigens lacked the native conformation of the TSHR and the resulting mAbs were exclusively blocking or neutral TSHR-mAbs. However, mAbs raised against intact native TSHR antigen have included stimulating mAbs. One such stimulating mAb has demonstrated a number of differences in its regulation of TSHR post-translational processing. These differences are likely to be reflective of TSHR-Abs seen in Graves' disease.

Nogo-A downregulation impairs place avoidance in the Carousel maze but not spatial memory in the Morris water maze

Czech Academy of Sciences Publication Activity Database

Petrásek, Tomáš; Prokopová, Iva; Bahník, Štěpán; Schönig, K.; Berger, S.; Valeš, Karel; Tews, B.; Schwab, M. E.; Bartsch, D.; Stuchlík, Aleš

2014-01-01

Roč. 107, Jan 2014 (2014), s. 42-49 ISSN 1074-7427 R&D Projects: GA ČR(CZ) GCP303/10/J032; GA ČR(CZ) GBP304/12/G069; GA MZd(CZ) NT13386; GA ČR(CZ) GA14-03627S Grant - others:Rada Programu interní podpory projektů mezinárodní spolupráce AV ČR(CZ) M200111204; Univerzita Karlova(CZ) 365911 Institutional support: RVO:67985823 Keywords : Nogo-A * spatial navigation * cognitive coordination * spatial mazes * transgenic rat model Subject RIV: FH - Neurology Impact factor: 3.652, year: 2014

Biodistribution mechanisms of therapeutic monoclonal antibodies in health and disease.

Science.gov (United States)

Tabrizi, Mohammad; Bornstein, Gadi Gazit; Suria, Hamza

2010-03-01

The monoclonal antibody market continues to witness an impressive rate of growth and has become the leading source of expansion in the biologic segment within the pharmaceutical industry. Currently marketed monoclonal antibodies target a diverse array of antigens. These antigens are distributed in a variety of tissues such as tumors, lungs, synovial fluid, psoriatic plaques, and lymph nodes. As the concentration of drug at the proximity of the biological receptor determines the magnitude of the observed pharmacological responses, a significant consideration in effective therapeutic application of monoclonal antibodies is a thorough understanding of the processes that regulate antibody biodistribution. Monoclonal antibody distribution is affected by factors such as molecular weight, blood flow, tissue and tumor heterogeneity, structure and porosity, target antigen density, turnover rate, and the target antigen expression profile.

Contribution of enhanced engagement of antigen presentation machinery to the clinical immunogenicity of a human interleukin (IL)-21 receptor-blocking therapeutic antibody.

Science.gov (United States)

Xue, L; Hickling, T; Song, R; Nowak, J; Rup, B

2016-01-01

Reliable risk assessment for biotherapeutics requires accurate evaluation of risk factors associated with immunogenicity. Immunogenicity risk assessment tools were developed and applied to investigate the immunogenicity of a fully human therapeutic monoclonal antibody, ATR-107 [anti-interleukin (IL)-21 receptor] that elicited anti-drug antibodies (ADA) in 76% of healthy subjects in a Phase 1 study. Because the ATR-107 target is expressed on dendritic cells (DCs), the immunogenicity risk related to engagement with DC and antigen presentation pathways was studied. Despite the presence of IL-21R on DCs, ATR-107 did not bind to the DCs more extensively than the control therapeutic antibody (PF-1) that had elicited low clinical ADA incidence. However, ATR-107, but not the control therapeutic antibody, was translocated to the DC late endosomes, co-localized with intracellular antigen-D related (HLA-DR) molecules and presented a dominant T cell epitope overlapping the complementarity determining region 2 (CDR2) of the light chain. ATR-107 induced increased DC activation exemplified by up-regulation of DC surface expression of CD86, CD274 (PD-L1) and CD40, increased expansion of activated DC populations expressing CD86(hi), CD40(hi), CD83(hi), programmed death ligand 1 (PD-L1)(hi), HLA-DR(hi) or CCR7(hi), as well as elevated secretion of tumour necrosis factor (TNF)-α by DCs. DCs exposed to ATR-107 stimulated an autologous T cell proliferative response in human donor cells, in concert with the detection of immunoglobulin (Ig)G-type anti-ATR-107 antibody response in clinical samples. Collectively, the enhanced engagement of antigen presentation machinery by ATR-107 was suggested. The approaches and findings described in this study may be relevant to identifying lower immunogenicity risk targets and therapeutic molecules. © 2015 British Society for Immunology.

B lymphocyte depletion with the monoclonal antibody rituximab in Graves' disease: a controlled pilot study

DEFF Research Database (Denmark)

El Fassi, Daniel; Nielsen, Claus H; Bonnema, Steen J

2007-01-01

Graves' disease (GD) is a common TSH receptor autoantibody (TRAb)-mediated disorder. Because B lymphocytes are important self-antigen presenting cells and precursors for antibody-secreting plasma cells, temporary B-lymphocyte depletion with the monoclonal antibody rituximab (RTX) might...

Antibody-dependent enhancement of HIV-1 infection in human term syncytiotrophoblast cells cultured in vitro.

Science.gov (United States)

Tóth, F D; Mosborg-Petersen, P; Kiss, J; Aboagye-Mathiesen, G; Zdravkovic, M; Hager, H; Aranyosi, J; Lampé, L; Ebbesen, P

1994-06-01

We examined if Fc receptor-mediated antibody-dependent enhancement (FcR-ADE) or complement-mediated antibody-dependent enhancement (C'-ADE) of virus infection can contribute to increasing replication of HIV-1 in human syncytiotrophoblast (ST) cells. Here we report that both FcR-ADE and C'-ADE may result in enhanced virus release from HIV-1-infected ST cells. We show that FcR-ADE of HIV-1 infection in ST cells is mediated by FcRIII and other FcR(s) belonging to undetermined Fc classes and does not require CD4 receptors, whereas C'-ADE uses both CD4 and CR2-like receptors. FcR-ADE seems to be more efficient in enhancing HIV-1 replication than C'-ADE. While FcR-ADE leads to increased internalization of HIV-1, C'-ADE does not result in enhanced endocytosis of the virus. In addition, antibodies mediating FcR-ADE are reactive with the gp120 viral envelope antigen, whereas antibodies involved in C'-ADE react with the viral transmembrane glycoprotein gp41. Data suggest that both FcR-ADE and C'-ADE may contribute to the spread of HIV-1 from mother to the fetus.

The collagen receptor uPARAP/Endo180 as a novel target for antibody-drug conjugate mediated treatment of mesenchymal and leukemic cancers

DEFF Research Database (Denmark)

Nielsen, Christoffer Fagernæs; van Putten, Sander Maarten; Lund, Ida Katrine

2017-01-01

A key task in developing the field of personalized cancer therapy is the identification of novel molecular targets that enable treatment of cancers not susceptible to other means of specific therapy. The collagen receptor uPARAP/Endo180 is overexpressed by malignant cells in several non-epithelia......A key task in developing the field of personalized cancer therapy is the identification of novel molecular targets that enable treatment of cancers not susceptible to other means of specific therapy. The collagen receptor uPARAP/Endo180 is overexpressed by malignant cells in several non...... into the endosomal-lysosomal system, thus opening a potential route of entry into receptor-positive cells. This combination of specific expression and endocytic function appears well suited for targeting of uPARAP/Endo180-positive cancers by antibody-drug conjugate (ADC) mediated drug delivery. Therefore, we...... model with human uPARAP/Endo180-positive leukemic cells, obtaining a complete cure of all tested mice following intravenous ADC treatment with no sign of adverse effects. Our study identifies uPARAP/Endo180 as a promising target for novel therapy against several highly malignant cancer types....

Development of EMab-51, a Sensitive and Specific Anti-Epidermal Growth Factor Receptor Monoclonal Antibody in Flow Cytometry, Western Blot, and Immunohistochemistry.

Science.gov (United States)

Itai, Shunsuke; Kaneko, Mika K; Fujii, Yuki; Yamada, Shinji; Nakamura, Takuro; Yanaka, Miyuki; Saidoh, Noriko; Handa, Saori; Chang, Yao-Wen; Suzuki, Hiroyoshi; Harada, Hiroyuki; Kato, Yukinari

2017-10-01

The epidermal growth factor receptor (EGFR) is a member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases and is involved in cell growth and differentiation. EGFR homodimers or heterodimers with other HER members, such as HER2 and HER3, activate downstream signaling cascades in many cancers. In this study, we developed novel anti-EGFR monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. First, we expressed the full-length or ectodomain of EGFR in LN229 glioblastoma cells and then immunized mice with LN229/EGFR or ectodomain of EGFR, and performed the first screening using enzyme-linked immunosorbent assays. Subsequently, we selected mAbs according to their efficacy in flow cytometry (second screening), Western blot (third screening), and immunohistochemical (fourth screening) analyses. Among 100 mAbs, only one clone EMab-51 (IgG 1 , kappa) reacted with EGFR in Western blot analysis. Finally, immunohistochemical analyses with EMab-51 showed sensitive and specific reactions against oral cancer cells, warranting the use of EMab-51 to detect EGFR in pathological analyses of EGFR-expressing cancers.

Imaging thrombus with radiolabelled monoclonal antibody to platelets

Energy Technology Data Exchange (ETDEWEB)

Peters, A.M.; Lavender, J.P.; Needham, S.G.; Loutfi, I.; Snook, D.; Epenetos, A.A.; Lumley, P.; Keery, R.J.; Hogg, N.

1986-12-13

A study was conducted evaluating a method of imaging thrombus with platelets radiolabelled with a /sup 111/In labelled monoclonal antibody, P256, directed to the platelet surface glycoprotein complex IIb/IIIa. when the number of receptors occupied by P256 was less than 3% of the total available on the platelet surface, platelet function was undisturbed. P256 was radiolabelled with /sup 111/In using diethylenetriaminepenta-acetic acid, which achieved a specific activity of 185 MBq (5 mCi)/mg. No impairment of immunoreactivity was detected at this specific activity. Platelets were labelled with radiolabelled monoclonal antibody in vitro in two patients at a receptor occupancy of 6% and in vivo in six patients at a receptor occupancy of 1%. In vivo recovery and biodistribution kinetics suggested that after in vitro labelling platelets were minimally activated. The /sup 111/In kinetics recorded after intravenous P256 suggested rapid and efficient radiolabelling of platelets and gave no indication of platelet activation. Of the six patients who received intravenous P256, three had documented thrombus, two of whom gave positive results on P256 platelet scintigraphy. The third had chronic deep venous thrombosis and was scintigraphically negative.

Inhibition of ligand exchange kinetics via active-site trapping with an antibody fragment.

Science.gov (United States)

Oyen, David; Steyaert, Jan; Barlow, John N

2014-04-01

We describe the first example of an inhibitory antibody fragment (nanobody ca1697) that binds simultaneously to an enzyme (the enzyme dihydrofolate reductase from Escherichia coli) and its bound substrate (folate). Binding of the antibody to the substrate causes a 20-fold reduction in the rate of folate exchange kinetics. This work opens up the prospect of designing new types of antibody-based inhibitors of enzymes and receptors through suitable design of immunogens.

Interplay between Natural Killer Cells and Anti-HER2 Antibodies: Perspectives for Breast Cancer Immunotherapy

Directory of Open Access Journals (Sweden)

Aura Muntasell

2017-11-01

Full Text Available Overexpression of the human epidermal growth factor receptor 2 (HER2 defines a subgroup of breast tumors with aggressive behavior. The addition of HER2-targeted antibodies (i.e., trastuzumab, pertuzumab to chemotherapy significantly improves relapse-free and overall survival in patients with early-stage and advanced disease. Nonetheless, considerable proportions of patients develop resistance to treatment, highlighting the need for additional and co-adjuvant therapeutic strategies. HER2-specific antibodies can trigger natural killer (NK cell-mediated antibody-dependent cellular cytotoxicity and indirectly enhance the development of tumor-specific T cell immunity; both mechanisms contributing to their antitumor efficacy in preclinical models. Antibody-dependent NK cell activation results in the release of cytotoxic granules as well as the secretion of pro-inflammatory cytokines (i.e., IFNγ and TNFα and chemokines. Hence, NK cell tumor suppressive functions include direct cytolytic killing of tumor cells as well as the regulation of subsequent antitumor adaptive immunity. Albeit tumors with gene expression signatures associated to the presence of cytotoxic lymphocyte infiltrates benefit from trastuzumab-based treatment, NK cell-related biomarkers of response/resistance to HER2-specific therapeutic antibodies in breast cancer patients remain elusive. Several variables, including (i the configuration of the patient NK cell repertoire; (ii tumor molecular features (i.e., estrogen receptor expression; (iii concomitant therapeutic regimens (i.e., chemotherapeutic agents, tyrosine kinase inhibitors; and (iv evasion mechanisms developed by progressive breast tumors, have been shown to quantitatively and qualitatively influence antibody-triggered NK cell responses. In this review, we discuss possible interventions for restoring/enhancing the therapeutic activity of HER2 therapeutic antibodies by harnessing NK cell antitumor potential through

Mechanisms of resistance to HER family targeting antibodies

Energy Technology Data Exchange (ETDEWEB)

Kruser, Tim J. [Department of Human Oncology, University of Wisconsin School of Medicine and Public Health, Madison, WI (United States); Wheeler, Deric L., E-mail: dlwheeler@wisc.edu [Department of Human Oncology, University of Wisconsin School of Medicine and Public Health, Madison, WI (United States)

2010-04-15

The epidermal growth factor (EGF) family of receptor tyrosine kinases consists of four members: EGFR (HER1/ErbB1), HER2/neu (ErbB2), HER3 (ErbB3) and HER4 (ErbB4). Receptor activation via ligand binding leads to downstream signaling that influence cell proliferation, angiogenesis, invasion and metastasis. Aberrant expression or activity of EGFR and HER2 have been strongly linked to the etiology of several human epithelial cancers including but not limited to head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC), colorectal cancer (CRC), and breast cancer. With this, intense efforts have been made to inhibit the activity of the EGFR and HER2 by designing antibodies against the ligand binding domains (cetuximab, panitumumab and trastuzumab) or small molecules against the tyrosine kinase domains (erlotinib, gefitinib, and lapatinib). Both approaches have shown considerable clinical promise. However, increasing evidence suggests that the majority of patients do not respond to these therapies, and those who show initial response ultimately become refractory to treatment. While mechanisms of resistance to tyrosine kinase inhibitors have been extensively studied, resistance to monoclonal antibodies is less well understood, both in the laboratory and in the clinical setting. In this review, we discuss resistance to antibody-based therapies against the EGFR and HER2, similarities between these resistance profiles, and strategies to overcome resistance to HER family targeting monoclonal antibody therapy.

G protein-coupled receptor 30 is an estrogen receptor in the plasma membrane

International Nuclear Information System (INIS)

Funakoshi, Takeshi; Yanai, Akie; Shinoda, Koh; Kawano, Michio M.; Mizukami, Yoichi

2006-01-01

Recently, GPR30 was reported to be a novel estrogen receptor; however, its intracellular localization has remained controversial. To investigate the intracellular localization of GPR30 in vivo, we produced four kinds of polyclonal antibodies for distinct epitopes on GPR30. Immunocytochemical observations using anti-GPR30 antibody and anti-FLAG antibody show that FLAG-GPR30 localizes to the plasma membrane 24 h after transfection. Treatment with estrogen (17β-estradiol or E2) causes an elevation in the intracellular Ca 2+ concentration ([Ca 2+ ] i ) within 10 s in HeLa cells expressing FLAG-GPR30. In addition, E2 induces the translocation of GPR30 from the plasma membrane to the cytoplasm by 1 h after stimulation. Immunohistochemical analysis shows that GPR30 exists on the cell surface of CA2 pyramidal neuronal cells. The images on transmission electron microscopy show that GPR30 is localized to a particular region associated with the plasma membranes of the pyramidal cells. These data indicate that GPR30, a transmembrane receptor for estrogen, is localized to the plasma membrane of CA2 pyramidal neuronal cells of the hippocampus in rat brain

Monoclonal antibodies targeting CD38 in hematological malignancies and beyond

DEFF Research Database (Denmark)

van de Donk, Niels W C J; Janmaat, Maarten L.; Mutis, Tuna

2016-01-01

CD38 is a multifunctional cell surface protein that has receptor as well as enzyme functions. The protein is generally expressed at low levels on various hematological and solid tissues, while plasma cells express particularly high levels of CD38. The protein is also expressed in a subset of hema...... strong anti-tumor activity in preclinical models. The antibody engages diverse mechanisms of action, including complement-dependent cytotoxicity, antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis, programmed cell death, modulation of enzymatic activity...... combination therapies with existing as well as emerging therapies, which are currently evaluated in the clinic. Finally, CD38 antibodies may have a role in the treatment of diseases beyond hematological malignancies, including solid tumors and antibody-mediated autoimmune diseases. © 2016 John Wiley & Sons A....../S. Published by John Wiley & Sons Ltd....

Role of polymorphic Fc receptor Fc gammaRIIa in cytokine release and adverse effects of murine IgG1 anti-CD3/T cell receptor antibody (WT31).

Science.gov (United States)

Tax, W J; Tamboer, W P; Jacobs, C W; Frenken, L A; Koene, R A

1997-01-15

Anti-CD3 monoclonal antibody (mAb) OKT3 is immunosuppressive, but causes severe adverse effects during the first administration ("first-dose reaction"). These adverse effects are presumably caused by cytokine release that results from T-cell activation. In vitro, T-cell activation by anti-CD3 mAb requires interaction with monocyte Fc receptors. The Fc receptor for murine IgG1, Fc gammaRIIa, is polymorphic. In some individuals, murine IgG1 anti-CD3 mAb causes T-cell proliferation and cytokine release in vitro (high responders [HR]), whereas in individuals with the low-responder (LR) phenotype it does not. We have now investigated the role of this Fc gammaRIIa polymorphism in the release of cytokines in vivo and the occurrence of adverse effects after the administration of WT31, a murine IgG1 anti-CD3/T cell receptor mAb. WT31 caused an increase of plasma tumor necrosis factor-alpha in all four HR patients and none of the five LR patients. In all HR patients except one, plasma gamma-interferon and interleukin 6 also increased, and a first-dose response was observed, whereas no cytokine release or adverse effects occurred in any of the LR patients. WT31 caused lymphopenia in all HR and none of the LR patients. FACS analysis demonstrated that in HR patients, after the initial disappearance of CD3+ cells from peripheral blood, modulation of CD3 occurred, whereas in LR patients a high degree of coating of the lymphocytes was observed. Surprisingly, WT31 also induced a marked granulocytopenia, as well as a decrease of thrombocytes, in three of the four HR patients (and in none of the LR patients). These data provide direct clinical evidence that Fc receptor interaction determines the release of cytokines and the occurrence of adverse effects after administration of anti-CD3/T cell receptor mAb. Furthermore, these data suggest that tumor necrosis factor-alpha by itself is not sufficient to induce the first-dose reaction.

Extra-epitopic hepatitis C virus polymorphisms confer resistance to broadly neutralizing antibodies by modulating binding to scavenger receptor B1.

Science.gov (United States)

El-Diwany, Ramy; Cohen, Valerie J; Mankowski, Madeleine C; Wasilewski, Lisa N; Brady, Jillian K; Snider, Anna E; Osburn, William O; Murrell, Ben; Ray, Stuart C; Bailey, Justin R

2017-02-01

Broadly-neutralizing monoclonal antibodies (bNAbs) may guide vaccine development for highly variable viruses including hepatitis C virus (HCV), since they target conserved viral epitopes that could serve as vaccine antigens. However, HCV resistance to bNAbs could reduce the efficacy of a vaccine. HC33.4 and AR4A are two of the most potent anti-HCV human bNAbs characterized to date, binding to highly conserved epitopes near the amino- and carboxy-terminus of HCV envelope (E2) protein, respectively. Given their distinct epitopes, it was surprising that these bNAbs showed similar neutralization profiles across a panel of natural HCV isolates, suggesting that some viral polymorphisms may confer resistance to both bNAbs. To investigate this resistance, we developed a large, diverse panel of natural HCV envelope variants and a novel computational method to identify bNAb resistance polymorphisms in envelope proteins (E1 and E2). By measuring neutralization of a panel of HCV pseudoparticles by 10 μg/mL of each bNAb, we identified E1E2 variants with resistance to one or both bNAbs, despite 100% conservation of the AR4A binding epitope across the panel. We discovered polymorphisms outside of either binding epitope that modulate resistance to both bNAbs by altering E2 binding to the HCV co-receptor, scavenger receptor B1 (SR-B1). This study is focused on a mode of neutralization escape not addressed by conventional analysis of epitope conservation, highlighting the contribution of extra-epitopic polymorphisms to bNAb resistance and presenting a novel mechanism by which HCV might persist even in the face of an antibody response targeting multiple conserved epitopes.

Structural basis for the antibody neutralization of Herpes simplex virus

Energy Technology Data Exchange (ETDEWEB)

Lee, Cheng-Chung; Lin, Li-Ling [Academia Sinica, Taipei 115, Taiwan (China); Academia Sinica, Taipei 115, Taiwan (China); Chan, Woan-Eng [Development Center for Biotechnology, New Taipei City 221, Taiwan (China); Ko, Tzu-Ping [Academia Sinica, Taipei 115, Taiwan (China); Academia Sinica, Taipei 115, Taiwan (China); Lai, Jiann-Shiun [Development Center for Biotechnology, New Taipei City 221, Taiwan (China); Ministry of Economic Affairs, Taipei 100, Taiwan (China); Wang, Andrew H.-J., E-mail: ahjwang@gate.sinica.edu.tw [Academia Sinica, Taipei 115, Taiwan (China); Academia Sinica, Taipei 115, Taiwan (China); Taipei Medical University, Taipei 110, Taiwan (China)

2013-10-01

The gD–E317-Fab complex crystal revealed the conformational epitope of human mAb E317 on HSV gD, providing a molecular basis for understanding the viral neutralization mechanism. Glycoprotein D (gD) of Herpes simplex virus (HSV) binds to a host cell surface receptor, which is required to trigger membrane fusion for virion entry into the host cell. gD has become a validated anti-HSV target for therapeutic antibody development. The highly inhibitory human monoclonal antibody E317 (mAb E317) was previously raised against HSV gD for viral neutralization. To understand the structural basis of antibody neutralization, crystals of the gD ectodomain bound to the E317 Fab domain were obtained. The structure of the complex reveals that E317 interacts with gD mainly through the heavy chain, which covers a large area for epitope recognition on gD, with a flexible N-terminal and C-terminal conformation. The epitope core structure maps to the external surface of gD, corresponding to the binding sites of two receptors, herpesvirus entry mediator (HVEM) and nectin-1, which mediate HSV infection. E317 directly recognizes the gD–nectin-1 interface and occludes the HVEM contact site of gD to block its binding to either receptor. The binding of E317 to gD also prohibits the formation of the N-terminal hairpin of gD for HVEM recognition. The major E317-binding site on gD overlaps with either the nectin-1-binding residues or the neutralizing antigenic sites identified thus far (Tyr38, Asp215, Arg222 and Phe223). The epitopes of gD for E317 binding are highly conserved between two types of human herpesvirus (HSV-1 and HSV-2). This study enables the virus-neutralizing epitopes to be correlated with the receptor-binding regions. The results further strengthen the previously demonstrated therapeutic and diagnostic potential of the E317 antibody.

Humanised IgG1 antibody variants targeting membrane-bound carcinoembryonic antigen by antibody-dependent cellular cytotoxicity and phagocytosis.

Science.gov (United States)

Ashraf, S Q; Umana, P; Mössner, E; Ntouroupi, T; Brünker, P; Schmidt, C; Wilding, J L; Mortensen, N J; Bodmer, W F

2009-11-17

The effect of glycoengineering a membrane specific anti-carcinoembryonic antigen (CEA) (this paper uses the original term CEA for the formally designated CEACAM5) antibody (PR1A3) on its ability to enhance killing of colorectal cancer (CRC) cell lines by human immune effector cells was assessed. In vivo efficacy of the antibody was also tested. The antibody was modified using EBNA cells cotransfected with beta-1,4-N-acetylglucosaminyltransferase III and the humanised hPR1A3 antibody genes. The resulting alteration of the Fc segment glycosylation pattern enhances the antibody's binding affinity to the FcgammaRIIIa receptor on human immune effector cells but does not alter the antibody's binding capacity. Antibody-dependent cellular cytotoxicity (ADCC) is inhibited in the presence of anti-FcgammaRIII blocking antibodies. This glycovariant of hPR1A3 enhances ADCC 10-fold relative to the parent unmodified antibody using either unfractionated peripheral blood mononuclear or natural killer (NK) cells and CEA-positive CRC cells as targets. NK cells are far more potent in eliciting ADCC than either freshly isolated monocytes or granulocytes. Flow cytometry and automated fluorescent microscopy have been used to show that both versions of hPR1A3 can induce antibody-dependent cellular phagocytosis (ADCP) by monocyte-derived macrophages. However, the glycovariant antibody did not mediate enhanced ADCP. This may be explained by the relatively low expression of FcgammaRIIIa on cultured macrophages. In vivo studies show the efficacy of glycoengineered humanised IgG1 PR1A3 in significantly improving survival in a CRC metastatic murine model. The greatly enhanced in vitro ADCC activity of the glycoengineered version of hPR1A3 is likely to be clinically beneficial.

Elucidation of Distinct Roles of Guinea Pig CXCR1 and CXCR2 in Neutrophil Migration toward IL-8 and GROα by Specific Antibodies.

Science.gov (United States)

Tanaka, Kento; Yoshitomi, Tomomi; Hirahara, Kazuki

2017-01-01

Chemokine receptors CXCR1 and CXCR2 are conserved between guinea pigs and humans, but the distinct role of each receptor in chemotactic responses of neutrophils against chemokine ligands has not been elucidated due in part to the lack of specific inhibitors against these receptors in guinea pigs. In this study, we investigated the roles of guinea pig CXCR1 and CXCR2 on neutrophils in chemotactic responses to guinea pig interleukin (IL)-8 and growth-regulated oncogene (GRO)α by using specific inhibitory antibodies against these receptors. Neutrophil migration induced by IL-8 was partially inhibited by either anti-CXCR1 antibody or anti-CXCR2 antibody. In addition, the migration was inhibited completely when both anti-CXCR1 and anti-CXCR2 antibodies were combined. On the other hand, neutrophil migration induced by GROα was not inhibited by anti-CXCR1 antibody while inhibited profoundly by anti-CXCR2 antibody. These results indicated that CXCR1 and CXCR2 mediated migration induced by the IL-8 synergistically and only CXCR2 mediated migration induced by GROα in guinea pig neutrophils. Our findings on ligand selectivity of CXCR1 and CXCR2 in guinea pigs are consistent with those in humans.

Pediatric Opsoclonus-Myoclonus-Ataxia Syndrome Associated With Anti-N-methyl-D-aspartate Receptor Encephalitis.

Science.gov (United States)

Player, Brittany; Harmelink, Matthew; Bordini, Brett; Weisgerber, Michael; Girolami, Michael; Croix, Michael

2015-11-01

The full clinical spectrum of anti-N-methyl-D-aspartate receptor encephalitis is unknown in the pediatric population. We describe a previously healthy 4-year-old girl presenting with opsoclonus-myoclonus together with ataxia who had NR1-specific, anti-N-methyl-D-aspartate receptor antibodies in the cerebral spinal fluid. The presence of NR1-specific, anti-N-methyl-D-aspartate receptor antibodies in the setting of opsoclonus-myoclonus and ataxia syndrome may represent an expansion of the clinical presentations of anti-N-methyl-D-aspartate receptor encephalitis. Copyright © 2015 Elsevier Inc. All rights reserved.

Establishment of H2Mab-119, an Anti-Human Epidermal Growth Factor Receptor 2 Monoclonal Antibody, Against Pancreatic Cancer.

Science.gov (United States)

Yamada, Shinji; Itai, Shunsuke; Nakamura, Takuro; Chang, Yao-Wen; Harada, Hiroyuki; Suzuki, Hiroyoshi; Kaneko, Mika K; Kato, Yukinari

2017-12-01

Human epidermal growth factor receptor 2 (HER2) is overexpressed in breast cancer and is associated with poor clinical outcomes. In addition, HER2 expression has been reported in other cancers, such as gastric, colorectal, lung, and pancreatic cancers. An anti-HER2 humanized antibody, trastuzumab, leads to significant survival benefits in patients with HER2-overexpressing breast cancers and gastric cancers. Herein, we established a novel anti-HER2 monoclonal antibody (mAb), H 2 Mab-119 (IgG 1 , kappa), and characterized its efficacy against pancreatic cancers using flow cytometry, Western blot, and immunohistochemical analyses. H 2 Mab-119 reacted with pancreatic cancer cell lines, such as KLM-1, Capan-2, and MIA PaCa-2, but did not react with PANC-1 in flow cytometry analysis. Western blot analysis also revealed a moderate signal for KLM-1 and a weak signal for MIA PaCa-2, although H 2 Mab-119 reacted strongly with LN229/HER2 cells. Finally, immunohistochemical analyses with H 2 Mab-119 revealed sensitive and specific reactions against breast and colon cancers but did not react with pancreatic cancers, indicating that H 2 Mab-119 is useful for detecting HER2 overexpression in pancreatic cancers using flow cytometry and Western blot analyses.

Clinical application of antibody monoclonal humanized anti-EGFrnimotuzumab labeled

International Nuclear Information System (INIS)

Perera Pintado, Alejandro; Peña Quián, Yamilé; Batista Cuéllar, Juan F.; Prats Capote, Anaís; Torres Aroche, Leonel A.; Casacó Santana, Caridad; Sánchez Mendosa, Elvia L.; Sánchez González, Yolaine; Romero Collado, Susana; Quesada Pozo, Rodobaldo; Valladares Oviedo, Lourdes; Masquida García, Elsa M.; Leyva Montaña, René; Casacó, Angel; Ramos Suzarte, Mayra; Crombet, Tania

2016-01-01

Most malignant tumors are of epithelial origin. These are characterized by overexpression of the receptor of epidermal growth factor (EGFR), which the neoplastic cells escape the regulatory mechanisms are allowed, so its high concentration of membrane is generally associated with a poor prognosis . By binding an antibody specifically to this receptor, preventing binding of EGF latter and activation mechanism tyrosine kinase inhibiting cell mitosis and apoptosis causing tumor cell. For this reason, the EGFr has been considered as an attractive target for anti-tumor therapy. The humanized monoclonal antibody anti-EGFr nimotuzumab was developed by the Center of Molecular Immunology (Havana, Cuba). Numerous clinical trials have been developed in the Department of Clinical Research Center Isotopes (Cuba), in which it has been applied this antibody, both labeled with 99mTc for immuno gammagraphic detection of tumors, as labeled with 188 Re for radioimmunotherapy of gliomas high degree of malignancy. The aim of this paper is to show the experience of the Department of Clinical Research of CENTIS in various clinical trials with marking for both immuno gammagraphics detection of tumors, such as for radioimmunotherapy nimotuzumab. (author)

Epidermal growth factor receptor antibody plus recombinant human endostatin in treatment of hepatic metastases after remnant gastric cancer resection

Institute of Scientific and Technical Information of China (English)

无

2007-01-01

We report a 55-year-old male who developed advanced hepatic metastasis and peritoneal carcinomatosis after resection of remnant gastric cancer resection 3 mo ago. The patient only received epidermal growth factor (EGF) receptor antibody (Cetuximab) plus recombinant human endostatin (Endostar).Anti-tumor activity was assessed by 18F-fluorodeoxyglucose (18F-FDG)positron emission tomography/computer tomography (PET/CT) at baseline and then every 4 wk. The case illustrates that 18FDG-PET/CT could make an early prediction of the response to Cetuximab plus Endostar in such clinical situations. 18FDG-PET/CT is a useful molecular imaging modality to evaluate the biological response advanced hepatic metastasis and peritoneal carcinomatosis to Cetuximab plus Endostar in patients after remnant gastric cancer resection.

B cell recognition of the conserved HIV-1 co-receptor binding site is altered by endogenous primate CD4.

Directory of Open Access Journals (Sweden)

Mattias N E Forsell

2008-10-01

Full Text Available The surface HIV-1 exterior envelope glycoprotein, gp120, binds to CD4 on the target cell surface to induce the co-receptor binding site on gp120 as the initial step in the entry process. The binding site is comprised of a highly conserved region on the gp120 core, as well as elements of the third variable region (V3. Antibodies against the co-receptor binding site are abundantly elicited during natural infection of humans, but the mechanism of elicitation has remained undefined. In this study, we investigate the requirements for elicitation of co-receptor binding site antibodies by inoculating rabbits, monkeys and human-CD4 transgenic (huCD4 rabbits with envelope glycoprotein (Env trimers possessing high affinity for primate CD4. A cross-species comparison of the antibody responses showed that similar HIV-1 neutralization breadth was elicited by Env trimers in monkeys relative to wild-type (WT rabbits. In contrast, antibodies against the co-receptor site on gp120 were elicited only in monkeys and huCD4 rabbits, but not in the WT rabbits. This was supported by the detection of high-titer co-receptor antibodies in all sera from a set derived from human volunteers inoculated with recombinant gp120. These findings strongly suggest that complexes between Env and (high-affinity primate CD4 formed in vivo are responsible for the elicitation of the co-receptor-site-directed antibodies. They also imply that the naïve B cell receptor repertoire does not recognize the gp120 co-receptor site in the absence of CD4 and illustrate that conformational stabilization, imparted by primary receptor interaction, can alter the immunogenicity of a type 1 viral membrane protein.

B cell recognition of the conserved HIV-1 co-receptor binding site is altered by endogenous primate CD4.

Science.gov (United States)

Forsell, Mattias N E; Dey, Barna; Mörner, Andreas; Svehla, Krisha; O'dell, Sijy; Högerkorp, Carl-Magnus; Voss, Gerald; Thorstensson, Rigmor; Shaw, George M; Mascola, John R; Karlsson Hedestam, Gunilla B; Wyatt, Richard T

2008-10-03

The surface HIV-1 exterior envelope glycoprotein, gp120, binds to CD4 on the target cell surface to induce the co-receptor binding site on gp120 as the initial step in the entry process. The binding site is comprised of a highly conserved region on the gp120 core, as well as elements of the third variable region (V3). Antibodies against the co-receptor binding site are abundantly elicited during natural infection of humans, but the mechanism of elicitation has remained undefined. In this study, we investigate the requirements for elicitation of co-receptor binding site antibodies by inoculating rabbits, monkeys and human-CD4 transgenic (huCD4) rabbits with envelope glycoprotein (Env) trimers possessing high affinity for primate CD4. A cross-species comparison of the antibody responses showed that similar HIV-1 neutralization breadth was elicited by Env trimers in monkeys relative to wild-type (WT) rabbits. In contrast, antibodies against the co-receptor site on gp120 were elicited only in monkeys and huCD4 rabbits, but not in the WT rabbits. This was supported by the detection of high-titer co-receptor antibodies in all sera from a set derived from human volunteers inoculated with recombinant gp120. These findings strongly suggest that complexes between Env and (high-affinity) primate CD4 formed in vivo are responsible for the elicitation of the co-receptor-site-directed antibodies. They also imply that the naïve B cell receptor repertoire does not recognize the gp120 co-receptor site in the absence of CD4 and illustrate that conformational stabilization, imparted by primary receptor interaction, can alter the immunogenicity of a type 1 viral membrane protein.

Chimeric antigen receptor (CAR-specific monoclonal antibody to detect CD19-specific T cells in clinical trials.

Directory of Open Access Journals (Sweden)

Bipulendu Jena

Full Text Available Clinical trials targeting CD19 on B-cell malignancies are underway with encouraging anti-tumor responses. Most infuse T cells genetically modified to express a chimeric antigen receptor (CAR with specificity derived from the scFv region of a CD19-specific mouse monoclonal antibody (mAb, clone FMC63. We describe a novel anti-idiotype monoclonal antibody (mAb to detect CD19-specific CAR(+ T cells before and after their adoptive transfer. This mouse mAb was generated by immunizing with a cellular vaccine expressing the antigen-recognition domain of FMC63. The specificity of the mAb (clone no. 136.20.1 was confined to the scFv region of the CAR as validated by inhibiting CAR-dependent lysis of CD19(+ tumor targets. This clone can be used to detect CD19-specific CAR(+ T cells in peripheral blood mononuclear cells at a sensitivity of 1∶1,000. In clinical settings the mAb is used to inform on the immunophenotype and persistence of administered CD19-specific T cells. Thus, our CD19-specific CAR mAb (clone no. 136.20.1 will be useful to investigators implementing CD19-specific CAR(+ T cells to treat B-lineage malignancies. The methodology described to develop a CAR-specific anti-idiotypic mAb could be extended to other gene therapy trials targeting different tumor associated antigens in the context of CAR-based adoptive T-cell therapy.

Structural basis for pH-insensitive inhibition of immunoglobulin G recycling by an anti-neonatal Fc receptor antibody

Energy Technology Data Exchange (ETDEWEB)

Kenniston, Jon A.; Taylor, Brandy M.; Conley, Gregory P.; Cosic, Janja; Kopacz, Kris J.; Lindberg, Allison P.; Comeau, Stephen R.; Atkins, Kateri; Bullen, Jameson; TenHoor, Christopher; Adelman, Burt A.; Sexton, Daniel J.; Edwards, Thomas E.; Nixon, Andrew E. (Beryllium); (Dyax)

2017-09-06

The neonatal Fc receptor FcRn plays a critical role in the trafficking of IgGs across tissue barriers and in retaining high circulating concentrations of both IgG and albumin. Although generally beneficial from an immunological perspective in maintaining IgG populations, FcRn can contribute to the pathogenesis of autoimmune disorders when an abnormal immune response targets normal biological components. We previously described a monoclonal antibody (DX-2507) that binds to FcRn with high affinity at both neutral and acidic pH, prevents the simultaneous binding of IgG, and reduces circulating IgG levels in preclinical animal models. Here, we report a 2.5 Å resolution X-ray crystal structure of an FcRn–DX-2507 Fab complex, revealing a nearly complete overlap of the IgG–Fc binding site in FcRn by complementarity-determining regions in DX-2507. This overlap explains how DX-2507 blocks IgG binding to FcRn and thereby shortens IgG half-life by preventing IgGs from recycling back into circulation. Moreover, the complex structure explains how the DX-2507 interaction is pH-insensitive unlike normal Fc interactions and how serum albumin levels are unaffected by DX-2507 binding. These structural studies could inform antibody-based therapeutic approaches for limiting the effects of IgG-mediated autoimmune disease.

Chimeric antigen receptor T cells secreting anti-PD-L1 antibodies more effectively regress renal cell carcinoma in a humanized mouse model.

Science.gov (United States)

Suarez, Eloah Rabello; Chang, De Kuan; Sun, Jiusong; Sui, Jianhua; Freeman, Gordon J; Signoretti, Sabina; Zhu, Quan; Marasco, Wayne A

2016-06-07

Advances in the treatment of metastatic clear cell renal cell carcinoma (ccRCC) have led to improved progression-free survival of many patients; however the therapies are toxic, rarely achieve durable long-term complete responses and are not curative. Herein we used a single bicistronic lentiviral vector to develop a new combination immunotherapy that consists of human anti-carbonic anhydrase IX (CAIX)-targeted chimeric antigen receptor (CAR) T cells engineered to secrete human anti-programmed death ligand 1 (PD-L1) antibodies at the tumor site. The local antibody delivery led to marked immune checkpoint blockade. Tumor growth diminished 5 times and tumor weight reduced 50-80% when compared with the anti-CAIX CAR T cells alone in a humanized mice model of ccRCC. The expression of PD-L1 and Ki67 in the tumors decreased and an increase in granzyme B levels was found in CAR T cells. The anti-PD-L1 IgG1 isotype, which is capable of mediating ADCC, was also able to recruit human NK cells to the tumor site in vivo. These armed second-generation CAR T cells empowered to secrete human anti-PD-L1 antibodies in the ccRCC milieu to combat T cell exhaustion is an innovation in this field that should provide renewed potential for CAR T cell immunotherapy of solid tumors where limited efficacy is currently seen.

[Construction and screening of phage antibody libraries against epidermal growth factor receptor and soluble expression of single chain Fv].

Science.gov (United States)

Sheng, Wei-Jin; Miao, Qing-Fang; Zhen, Yong-Su

2009-06-01

Recent studies have shown that epidermal growth factor receptor (EGFR) is an important target for cancer therapy. The present study prepared single chain Fv (scFv) directed against EGFR. Balb/c mice were immunized by human carcinoma A431 cells, and total RNA of the splenic cells was extracted. VH and VL gene fragments were amplified by RT-PCR and further joined into scFv gene with a linker, then scFv gene fragments were ligated into the phagemid vector pCANTAB 5E. The phagemid containing scFv were transformed into electro-competent E. coli TG1 cells. The recombinant phage antibody library was constructed through rescuing the transformed cells with help phage M13K07. The specified recombinant phages were enriched through 5 rounds of affinity panning and the anti-EGFR phage scFv clones were screened and identified with ELISA. A total of 48 clones from the library were selected randomly and 45 clones were identified positive. After infecting E. coli HB2151 cells with one positive clone, soluble recombinant antibodies about 27 kD were produced and located in the periplasm and the supernatant. The result of sequencing showed that the scFv gene was 768 bp, which encoded 256 amino acid residues. VH and VL including 3 CDRs and 4 FRs, respectively, were all homologous to mouse Ig. The soluble scFv showed the specific binding activity to purified EGFR and EGFR located in carcinoma cell membrane. The successful preparation of anti-EGFR scFv will provide an EGFR targeted molecule for the development of antibody-based drugs and biological therapy of cancer.

ChLpMab-23: Cancer-Specific Human-Mouse Chimeric Anti-Podoplanin Antibody Exhibits Antitumor Activity via Antibody-Dependent Cellular Cytotoxicity.

Science.gov (United States)

Kaneko, Mika K; Nakamura, Takuro; Kunita, Akiko; Fukayama, Masashi; Abe, Shinji; Nishioka, Yasuhiko; Yamada, Shinji; Yanaka, Miyuki; Saidoh, Noriko; Yoshida, Kanae; Fujii, Yuki; Ogasawara, Satoshi; Kato, Yukinari

2017-06-01

Podoplanin is expressed in many cancers, including oral cancers and brain tumors. The interaction between podoplanin and its receptor C-type lectin-like receptor 2 (CLEC-2) has been reported to be involved in cancer metastasis and tumor malignancy. We previously established many monoclonal antibodies (mAbs) against human podoplanin using the cancer-specific mAb (CasMab) technology. LpMab-23 (IgG 1 , kappa), one of the mouse anti-podoplanin mAbs, was shown to be a CasMab. However, we have not shown the usefulness of LpMab-23 for antibody therapy against podoplanin-expressing cancers. In this study, we first determined the minimum epitope of LpMab-23 and revealed that Gly54-Leu64 peptide, especially Gly54, Thr55, Ser56, Glu57, Asp58, Arg59, Tyr60, and Leu64 of podoplanin, is a critical epitope of LpMab-23. We further produced human-mouse chimeric LpMab-23 (chLpMab-23) and investigated whether chLpMab-23 exerts antibody-dependent cellular cytotoxicity (ADCC) and antitumor activity. In flow cytometry, chLpMab-23 showed high sensitivity against a podoplanin-expressing glioblastoma cell line, LN319, and an oral cancer cell line, HSC-2. chLpMab-23 also showed ADCC activity against podoplanin-expressing CHO cells (CHO/podoplanin). In xenograft models with HSC-2 and CHO/podoplanin, chLpMab-23 exerts antitumor activity using human natural killer cells, indicating that chLpMab-23 could be useful for antibody therapy against podoplanin-expressing cancers.

Coordinate phosphorylation of insulin-receptor kinase and its 175,000-Mr endogenous substrate in rat hepatocytes

International Nuclear Information System (INIS)

Okamoto, M.; Karasik, A.; White, M.F.; Kahn, C.R.

1991-01-01

To investigate the early events in insulin signal transmission in liver, isolated rat hepatocytes were labeled with 32 P, and proteins phosphorylated in response to insulin were detected by immunoprecipitation with anti-phosphotyrosine and anti-receptor antibodies and analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and autoradiography. In these cells, insulin rapidly stimulated tyrosine phosphorylation of the 95,000-Mr beta-subunit of the insulin receptor and a 175,000-Mr phosphoprotein (pp175). Both proteins were precipitated by anti-phosphotyrosine antibody, whereas only the insulin receptor was recognized with anti-insulin-receptor antibody. In the insulin-stimulated state, both pp175 and the receptor beta-subunit were found to be phosphorylated on tyrosine and serine residues. Based on precipitation by the two antibodies, receptor phosphorylation was biphasic with an initial increase in tyrosine phosphorylation followed by a more gradual increase in serine phosphorylation over the first 30 min of stimulation. The time course of phosphorylation of pp175 was rapid and paralleled that of the beta-subunit of the insulin receptor. The pp175 was clearly distinguished from the insulin receptor, because it was detected only when boiling SDS was used to extract cellular phosphoproteins, whereas the insulin receptor was extracted with either Triton X-100 or SDS. In addition, the tryptic peptide maps of the two proteins were distinct. The dose-response curve for insulin stimulation was shifted slightly to the left of the insulin receptor, suggesting some signal amplification at this step. These data suggest that pp175 is a major endogenous substrate of the insulin receptor in liver and may be a cytoskeletal-associated protein

Antiproliferative and apoptotic effects of a specific anti-insulin-like growth factor I receptor single chain antibody on breast cancer cells.

Science.gov (United States)

Motallebnezhad, Morteza; Younesi, Vahid; Aghebati-Maleki, Leili; Nickho, Hamid; Safarzadeh, Elham; Ahmadi, Majid; Movassaghpour, Ali Akbar; Hosseini, Ahmad; Yousefi, Mehdi

2016-11-01

Insulin-like growth factor I receptor (IGF-IR) is expressed on breast cancer cells and involves in metastasis, survival, and proliferation. Currently, application of IGF-IR-targeting monoclonal antibodies (mAbs), alone or in combination with other drugs, is a promising strategy for breast cancer therapy. Single-chain fragment variable (scFv) antibodies have been introduced as appropriate tools for tumor-targeting purposes because of their advantages over whole antibodies. In the present study, we employed a naïve phage library and isolated scFvs against a specific epitope from extracellular domain of IGF-IR by panning process. The selected scFvs were further characterized using polyclonal and monoclonal phage ELISA, soluble monoclonal ELISA, and colony PCR and sequencing. Antiproliferative and apoptotic effects of selected scFv antibodies on breast cancer cell lines were also evaluated by MTT and Annexin V/PI assays. The results of ELISA indicated specific reactions of the isolated scFvs against the IGF-IR peptide, and analyses of PCR product and sequencing confirmed the presence of full length V H and Vκ inserts. Treatment of MCF7 and SKBR3 cells with anti-IGF-IR scFv led to a significant growth inhibition. The results also showed that scFv treatment significantly augmented trastuzumab growth inhibitory effects on SKBR3 cells. The percentage of the apoptotic MCF7 and SKBR3 cells after 24-h treatment with scFv was 39 and 30.70 %, respectively. Twenty-four-hour treatment with scFv in combination with trastuzumab resulted in 44.75 % apoptosis of SKBR3 cells. Taken together, our results demonstrate that the targeting of IGF-IR by scFv can be an effective strategy in the treatment of breast cancer and provide further evidence for effectiveness of dual targeting of HER2 and IGF-IR in breast cancer therapy.

Intra-articular administration of an antibody against CSF-1 receptor reduces pain-related behaviors and inflammation in CFA-induced knee arthritis.

Science.gov (United States)

Alvarado-Vazquez, P A; Morado-Urbina, C E; Castañeda-Corral, G; Acosta-Gonzalez, R I; Kitaura, H; Kimura, K; Takano-Yamamoto, T; Jiménez-Andrade, J M

2015-01-01

Several studies have shown that blockade of colony stimulating factor-1 (CSF-1) or its receptor (CSF-1R) inhibits disease progression in rodent models of rheumatoid arthritis (RA); however, the role of the CSF-1/CSF-1R pathway in RA-induced pain and functional deficits has not been studied. Thus, we examined the effect of chronic intra-articular administration of a monoclonal anti-CSF-1R antibody (AFS98) on spontaneous pain, knee edema and functional disabilities in mice with arthritis. Unilateral arthritis was produced by multiple injections of complete Freund's adjuvant (CFA) into the right knee joint of adult male ICR mice. CFA-injected mice were then treated twice weekly from day 10 until day 25 with anti-CSF-1R antibody (3 and 10 μg/5 μL per joint), isotype control (rat IgG 10 μg/5 μL per joint) or PBS (5 μl/joint). Knee edema, spontaneous flinching, vertical rearing and horizontal exploratory activity were assessed at different days. Additionally, counts of peripheral leukocytes and body weight were measured to evaluate general health status. Intra-articular treatment with anti-CSF-1R antibody significantly increased horizontal exploratory activity and vertical rearing as well as reduced spontaneous flinching behavior and knee edema as compared to CFA-induced arthritis mice treated with PBS. Treatment with this antibody neither significantly affect mouse body weight nor the number of peripheral leukocytes. These results suggest that blockade of CSF-1R at the initial injury site (joint) could represent a therapeutic alternative for improving the functional disabilities and attenuating pain and inflammation in patients with RA. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Human oxidation-specific antibodies reduce foam cell formation and atherosclerosis progression

DEFF Research Database (Denmark)

Tsimikas, Sotirios; Miyanohara, Atsushi; Hartvigsen, Karsten

2011-01-01

We sought to assess the in vivo importance of scavenger receptor (SR)-mediated uptake of oxidized low-density lipoprotein (OxLDL) in atherogenesis and to test the efficacy of human antibody IK17-Fab or IK17 single-chain Fv fragment (IK17-scFv), which lacks immunologic properties of intact antibod...... antibodies other than the ability to inhibit uptake of OxLDL by macrophages, to inhibit atherosclerosis....

Two Unique Cases with Anti-GluR Antibody-Positive Encephalitis

Directory of Open Access Journals (Sweden)

Kosuke Matsuzono

2013-01-01

Full Text Available We report two cases of anti-glutamic acid receptor (anti-GluR antibody-positive encephalitis in males with symptoms such as Parkinsonism, urinary retention, and paralytic ileus. Although non-herpetic encephalitis typically shows magnetic resonance imaging (MRI lesions in the limbic system during early stages, the present cases showed MRI lesions during later stages in the bilateral claustrum and pons. In both cases, anti-GluRε2 and δ2 antibodies were later shown to be positive in the cerebrospinal fluid but negative in the serum. Although early detection of anti-GluR antibodies is essential, early treatment may be significantly more important.

Reactivity of eleven anti-human leucocyte monoclonal antibodies with lymphocytes from several domestic animals

DEFF Research Database (Denmark)

Aasted, Bent; Blixenkrone-Møller, Merete; Larsen, Else Bang

1988-01-01

Nine commercially available monoclonal antibodies and two monoclonal antibodies from The American Type Culture Collection, raised against various human leucocyte surface antigens, were tested on lymphocytes from cow, sheep, goat, swine, horse, cat, dog, mink, and rabbit as well as man. Four...... antibodies bound to lymphocytes from some of the animals. These were the antibodies against CD8 and CD4 antigen, the antibody to C3b-receptor, and the antibody to the HLA-DR antigen. The CD8 antigen-reactive antibody reacted with lymphocytes from mink, cat, dog, and sheep, while the CD4 antigen......-reactive antibody reacted with lymphocytes from mink. The anti-C3b-R antibody reacted with lymphocytes from horse, swine, dog, and cat, and the anti-HLA-DR reacted with lymphocytes from cow, goat, sheep, horse, dog, cat, and mink....

Generation and Characterization of Inhibitory Antibodies Specific to Guinea Pig CXCR1 and CXCR2.

Science.gov (United States)

Tanaka, Kento; Yoshimura, Chigusa; Shiina, Tetsuo; Terauchi, Tomoko; Yoshitomi, Tomomi; Hirahara, Kazuki

2017-04-01

CXCR1 and CXCR2 are chemokine receptors that have different selectivity of chemokine ligands, but the distinct role of each receptor is not clearly understood. This is due to the absence of specific inhibitors in guinea pigs, which are the appropriate species for investigation of CXCR1 and CXCR2 because of their functional similarity to humans. In this study, we generated and evaluated monoclonal antibodies that specifically bound to guinea pig CXCR1 (gpCXCR1) and guinea pig CXCR2 (gpCXCR2) for acquisition of specific inhibitors. To assess the activity of antibodies, we established CHO-K1 cells stably expressing either gpCXCR1 or gpCXCR2 (CHO/gpCXCR1 or CHO/gpCXCR2). CHO/gpCXCR1 showed migration in response to guinea pig interleukin (IL)-8, and CHO/gpCXCR2 showed migration in response to both guinea pig IL-8 and guinea pig growth-regulated oncogene α. The receptor selectivities of the chemokines of guinea pigs were the same as the human orthologs. The inhibitory activities of the anti-gpCXCR1 and anti-gpCXCR2 monoclonal antibodies on cell migration were observed in a concentration-dependent manner. In conclusion, we successfully obtained inhibitory antibodies specific to gpCXCR1 and gpCXCR2. These inhibitory antibodies will be useful to clarify the physiological roles of CXCR1 and CXCR2 in guinea pigs.

Imaging thrombus with radiolabelled monoclonal antibody to platelets

International Nuclear Information System (INIS)

Loutfi, I.; Peters, A.M.; Lavender, J.P.; Epenetos, A.A.

1988-01-01

Indium-111-hydroxyquinoline labelled platelets, though useful in the detection of thrombus, have not gained widespread use owing to the time and technical skill required for their preparation. A study was therefore conducted evaluating a new method of imaging thrombus with platelets radiolabelled with a 111 In labelled monoclonal antibody, P 256 , directed to the platelet surface glycoprotein complex IIb/IIIa. When the number of receptors occupied by P 256 was less than 3% of the total available on the platelet surface platelet function, as assessed by platelet aggregometry, was undisturbed. P 256 was radiolabelled with 111 In using diethylenetriaminepenta-acetic acid, which achieved a specific activity of 185 MBq (5 mCi)/mg. No impairment of immunoreactivity was detected at this specific activity. Platelets were labelled with radiolabelled monoclonal antibody in vitro in two patients at a receptor occupancy of 6% and in vivo - that is, by direct intravenous injection of P 256 - in six patients at a receptor occupancy of 1%. In vivo recovery and biodistribution kinetics suggested that after in vitro labelling platelets were minimally activated. The 111 In kinetics recorded after intravenous P 256 suggested rapid and efficient radiolabelling of platelets and gave no indication of platelet activation. Of the six patients who received intravenous P 256 , three had documented thrombus, tow of whom gave positive results on P 256 platelet scintigraphy. The third subject had chromic deep venous thrombosis and was scintigraphically negative. Imaging thrombus using a radiolabelled monoclonal antibody directed to platelets appears to offer great potential as a simple, non-invasive approach to the diagnosis of thrombosis. 3 refs. (Author)

Formation of infectious dengue virus-antibody immune complex in vivo in marmosets (Callithrix jacchus) after passive transfer of anti-dengue virus monoclonal antibodies and infection with dengue virus.

Science.gov (United States)

Moi, Meng Ling; Ami, Yasushi; Shirai, Kenji; Lim, Chang-Kweng; Suzaki, Yuriko; Saito, Yuka; Kitaura, Kazutaka; Saijo, Masayuki; Suzuki, Ryuji; Kurane, Ichiro; Takasaki, Tomohiko

2015-02-01

Infection with a dengue virus (DENV) serotype induces cross-reactive, weakly neutralizing antibodies to different dengue serotypes. It has been postulated that cross-reactive antibodies form a virus-antibody immune complex and enhance DENV infection of Fc gamma receptor (FcγR)-bearing cells. We determined whether infectious DENV-antibody immune complex is formed in vivo in marmosets after passive transfer of DENV-specific monoclonal antibody (mAb) and DENV inoculation and whether infectious DENV-antibody immune complex is detectable using FcγR-expressing cells. Marmosets showed that DENV-antibody immune complex was exclusively infectious to FcγR-expressing cells on days 2, 4, and 7 after passive transfer of each of the mAbs (mAb 4G2 and mAb 6B6C) and DENV inoculation. Although DENV-antibody immune complex was detected, contribution of the passively transferred antibody to overall viremia levels was limited in this study. The results indicate that DENV cross-reactive antibodies form DENV-antibody immune complex in vivo, which is infectious to FcγR-bearing cells but not FcγR-negative cells. © The American Society of Tropical Medicine and Hygiene.

Antibody-functionalized porous silicon nanoparticles for vectorization of hydrophobic drugs.

Science.gov (United States)

Secret, Emilie; Smith, Kevin; Dubljevic, Valentina; Moore, Eli; Macardle, Peter; Delalat, Bahman; Rogers, Mary-Louise; Johns, Terrance G; Durand, Jean-Olivier; Cunin, Frédérique; Voelcker, Nicolas H

2013-05-01

We describe the preparation of biodegradable porous silicon nanoparticles (pSiNP) functionalized with cancer cell targeting antibodies and loaded with the hydrophobic anti-cancer drug camptothecin. Orientated immobilization of the antibody on the pSiNP is achieved using novel semicarbazide based bioconjugate chemistry. To demonstrate the generality of this targeting approach, the three antibodies MLR2, mAb528 and Rituximab are used, which target neuroblastoma, glioblastoma and B lymphoma cells, respectively. Successful targeting is demonstrated by means of flow cytometry and immunocytochemistry both with cell lines and primary cells. Cell viability assays after incubation with pSiNPs show selective killing of cells expressing the receptor corresponding to the antibody attached on the pSiNP. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structural bases of coronavirus attachment to host aminopeptidase N and its inhibition by neutralizing antibodies.

Directory of Open Access Journals (Sweden)

Juan Reguera

Full Text Available The coronaviruses (CoVs are enveloped viruses of animals and humans associated mostly with enteric and respiratory diseases, such as the severe acute respiratory syndrome and 10-20% of all common colds. A subset of CoVs uses the cell surface aminopeptidase N (APN, a membrane-bound metalloprotease, as a cell entry receptor. In these viruses, the envelope spike glycoprotein (S mediates the attachment of the virus particles to APN and subsequent cell entry, which can be blocked by neutralizing antibodies. Here we describe the crystal structures of the receptor-binding domains (RBDs of two closely related CoV strains, transmissible gastroenteritis virus (TGEV and porcine respiratory CoV (PRCV, in complex with their receptor, porcine APN (pAPN, or with a neutralizing antibody. The data provide detailed information on the architecture of the dimeric pAPN ectodomain and its interaction with the CoV S. We show that a protruding receptor-binding edge in the S determines virus-binding specificity for recessed glycan-containing surfaces in the membrane-distal region of the pAPN ectodomain. Comparison of the RBDs of TGEV and PRCV to those of other related CoVs, suggests that the conformation of the S receptor-binding region determines cell entry receptor specificity. Moreover, the receptor-binding edge is a major antigenic determinant in the TGEV envelope S that is targeted by neutralizing antibodies. Our results provide a compelling view on CoV cell entry and immune neutralization, and may aid the design of antivirals or CoV vaccines. APN is also considered a target for cancer therapy and its structure, reported here, could facilitate the development of anti-cancer drugs.

Comparison of (/sup 125/I)beta-endorphin binding to rat brain and NG108-15 cells using a monoclonal antibody directed against the opioid receptor

Energy Technology Data Exchange (ETDEWEB)

Bidlack, J.M.; O' Malley, W.E.; Schulz, R.

1988-02-01

The properties of (/sup 125/I)beta h-endorphin-binding sites from rat brain membranes and membranes from the NG108-15 cell line were compared using a monoclonal antibody directed against the opioid receptor and opioid peptides as probes. The binding of (/sup 125/I)beta h-endorphin to both rat brain and NG108-15 membranes yielded linear Scatchard plots with Kd values of 1.2 nM and 1.5 nM, respectively, and Bmax values of 865 fmol/mg rat brain membrane protein and 1077 fmol/mg NG108-15 membrane protein. A monoclonal antibody, OR-689.2.4, capable of inhibiting mu and delta binding but not kappa binding to rat brain membranes, noncompetitively inhibited the binding of 1 nM (/sup 125/I)beta h-endorphin to rat brain and NG108-15 membranes with an IC50 value of 405 nM for rat brain membranes and 543 nM for NG108-15 membranes. The monoclonal antibody also inhibited the binding of 3 nM (/sup 3/H) (D-penicillamine2, D-penicillamine5) enkephalin to NG108-15 membranes with an IC50 value of 370 nM. In addition to blocking the binding of (/sup 125/I)beta h-endorphin to brain membranes, the antibody also displaced (/sup 125/I)beta h-endorphin from membranes. Site-specific opioid peptides had large variations in their IC50 values depending on whether they were inhibiting (/sup 125/I)beta h-endorphin binding to rat brain or the NG108-15 membranes. When the peptides were tested with the monoclonal antibody for their combined ability to inhibit (/sup 125/I)beta h-endorphin binding to both membrane preparations, the peptides and antibody blocked binding as though they were acting at allosterically coupled sites, not two totally independent sites. These studies suggest that mu-, delta-, and beta-endorphin-binding sites share some sequence homology with the 35,000-dalton protein that the antibody is directed against.

Meta-analysis of the association between N-methyl-d-aspartate receptor antibodies and schizophrenia, schizoaffective disorder, bipolar disorder, and major depressive disorder.

Science.gov (United States)

Pearlman, Daniel M; Najjar, Souhel

2014-08-01

N-methyl-d-aspartate receptor (NMDAR) antibodies have been documented in the serum of individuals with primary psychiatric disorders from several independent cohorts, but these findings have not been systematically assessed in aggregate or in relation to methodological covariates. We searched MEDLINE, EMBASE, and PsycINFO for studies in any language that provided data on NMDAR antibody seropositivity or absolute serum titers in schizophrenia or schizoaffective, bipolar, or major depressive disorders. We used a random effects model to pool estimates across studies. Nine studies met the eligibility criteria. Five studies (3387 participants) provided data on NMDAR antibody seropositivity in psychiatric versus control groups based on high-specificity seropositivity thresholds (cell-based assays [CBAs]: 1:320 dilution, 1:200 dilution, visual score>1; enzyme-linked immunosorbent assay [ELISA]: 90(th) percentile of control titers). Meta-analysis showed significantly higher odds of NMDAR antibody seropositivity among those with schizophrenia or schizoaffective, bipolar, or major depressive disorders compared with healthy controls (odds ratio [OR], 3.10; 95% confidence interval [CI], 1.04-9.27; P=.043; I(2)=68%). Four studies (3194 participants) provided outcome data for these groups based on low-specificity seropositivity thresholds (CBAs 1:10 dilution; ELISA: 75(th) percentile of control titers). Meta-analysis showed greater heterogeneity and no significant between-group difference (OR, 2.31; 95% CI, 0.55-9.73; P=.25; I(2)=90%). Seropositive participants in psychiatric groups had various combinations of IgG, IgM, and IgA class antibodies against NR1, NR1/NR2B, and NR2A/NR2B subunits. Subgroup analysis revealed significantly higher odds of seropositivity among all participants based on 1:10 versus 1:320 dilution seropositivity thresholds (OR, 4.56; 95% CI, 2.41-8.62; Pschizoaffective disorder (OR, 1.15; 95% CI, 0.19-7.24; P=.88, I(2)=43%, studies=2, n=1108). Average NR2A

CD14(hi)CD16+ monocytes phagocytose antibody-opsonised Plasmodium falciparum infected erythrocytes more efficiently than other monocyte subsets, and require CD16 and complement to do so.

Science.gov (United States)

Zhou, Jingling; Feng, Gaoqian; Beeson, James; Hogarth, P Mark; Rogerson, Stephen J; Yan, Yan; Jaworowski, Anthony

2015-07-07

With more than 600,000 deaths from malaria, mainly of children under five years old and caused by infection with Plasmodium falciparum, comes an urgent need for an effective anti-malaria vaccine. Limited details on the mechanisms of protective immunity are a barrier to vaccine development. Antibodies play an important role in immunity to malaria and monocytes are key effectors in antibody-mediated protection by phagocytosing antibody-opsonised infected erythrocytes (IE). Eliciting antibodies that enhance phagocytosis of IE is therefore an important potential component of an effective vaccine, requiring robust assays to determine the ability of elicited antibodies to stimulate this in vivo. The mechanisms by which monocytes ingest IE and the nature of the monocytes which do so are unknown. Purified trophozoite-stage P. falciparum IE were stained with ethidium bromide, opsonised with anti-erythrocyte antibodies and incubated with fresh whole blood. Phagocytosis of IE and TNF production by individual monocyte subsets was measured by flow cytometry. Ingestion of IE was confirmed by imaging flow cytometry. CD14(hi)CD16+ monocytes phagocytosed antibody-opsonised IE and produced TNF more efficiently than CD14(hi)CD16- and CD14(lo)CD16+ monocytes. Blocking experiments showed that Fcγ receptor IIIa (CD16) but not Fcγ receptor IIa (CD32a) or Fcγ receptor I (CD64) was necessary for phagocytosis. CD14(hi)CD16+ monocytes ingested antibody-opsonised IE when peripheral blood mononuclear cells were reconstituted with autologous serum but not heat-inactivated autologous serum. Antibody-opsonised IE were rapidly opsonised with complement component C3 in serum (t1/2 = 2-3 minutes) and phagocytosis of antibody-opsonised IE was inhibited in a dose-dependent manner by an inhibitor of C3 activation, compstatin. Compared to other monocyte subsets, CD14(hi)CD16+ monocytes expressed the highest levels of complement receptor 4 (CD11c) and activated complement receptor 3 (CD11b) subunits

Induction of IgG3 to LPS via Toll-like receptor 4 co-stimulation.

Directory of Open Access Journals (Sweden)

Francisco J Quintana

Full Text Available B-cells integrate antigen-specific signals transduced via the B-cell receptor (BCR and antigen non-specific co-stimulatory signals provided by cytokines and CD40 ligation in order to produce IgG antibodies. Toll-like receptors (TLRs also provide co-stimulation, but the requirement for TLRs to generate T-cell independent and T-cell dependent antigen specific antibody responses is debated. Little is known about the role of B-cell expressed TLRs in inducing antigen-specific antibodies to antigens that also activate TLR signaling. We found that mice lacking functional TLR4 or its adaptor molecule MyD88 harbored significantly less IgG3 natural antibodies to LPS, and required higher amounts of LPS to induce anti-LPS IgG3. In vitro, BCR and TLR4 signaling synergized, lowering the threshold for production of T-cell independent IgG3 and IL-10. Moreover, BCR and TLR4 directly associate through the transmembrane domain of TLR4. Thus, in vivo, BCR/TLR synergism could facilitate the induction of IgG3 antibodies against microbial antigens that engage both innate and adaptive B-cell receptors. Vaccines might exploit BCR/TLR synergism to rapidly induce antigen-specific antibodies before significant T-cell responses arise.

Photo-antagonism of the GABAA receptor.

Science.gov (United States)

Mortensen, Martin; Iqbal, Favaad; Pandurangan, Arun P; Hannan, Saad; Huckvale, Rosemary; Topf, Maya; Baker, James R; Smart, Trevor G

2014-07-29

Neurotransmitter receptor trafficking is fundamentally important for synaptic transmission and neural network activity. GABAA receptors and inhibitory synapses are vital components of brain function, yet much of our knowledge regarding receptor mobility and function at inhibitory synapses is derived indirectly from using recombinant receptors, antibody-tagged native receptors and pharmacological treatments. Here we describe the use of a set of research tools that can irreversibly bind to and affect the function of recombinant and neuronal GABAA receptors following ultraviolet photoactivation. These compounds are based on the competitive antagonist gabazine and incorporate a variety of photoactive groups. By using site-directed mutagenesis and ligand-docking studies, they reveal new areas of the GABA binding site at the interface between receptor β and α subunits. These compounds enable the selected inactivation of native GABAA receptor populations providing new insight into the function of inhibitory synapses and extrasynaptic receptors in controlling neuronal excitation.

Late manifestation of subclinical hyperthyroidism after goitrogenesis in an index patient with a N670S TSH receptor germline mutation masquerading as TSH receptor antibody negative Graves' disease.

Science.gov (United States)

Schaarschmidt, J; Paschke, S; Özerden, M; Jäschke, H; Huth, S; Eszlinger, M; Meller, J; Paschke, R

2012-12-01

In 27 families with familial non-autoimmune hyperthyroidism (FNAH) reported up to date, the onset of hyperthyroidism varies from 18 months to 60 years. Also the manifestation of goitres is variable in these families. A 74-year-old woman first presented at the age of 69 years with tachyarrhythmia and hypertension. After initial treatment of her hypertension and oral anticoagulation for her intermittent atrial fibrillation, a thyroid workup revealed a suppressed TSH and normal fT3 and fT4. TPO, TSH receptor (TSHR), and thyroglobulin antibodies were negative. Thyroid ultrasound revealed a thyroid volume of 102 ml with several nodules with diameters of up to 2.6 cm right and up to 1.8 cm left. Scintigraphy showed a homogeneous Technetium-99 m ((99 m)Tc) uptake of 1.27%. She was subsequently treated with 1 GBq radioiodine ((131)I). At the age of 74, her thyroid function was normal and her thyroid volume decreased to 90 ml. Because of the diffuse (99 m)Tc uptake and the negative TPO, TSHR, and thyroglobulin antibodies, genetic analysis of her TSHR gene was performed, in spite of her negative family history for hyperthyroidism. Sequencing revealed a N670S TSHR germline mutation. Previous in vitro characterisation of this TSHR mutation suggests a weak constitutive activity, yet the experimental data are ambiguous. This case illustrates the necessity to analyse patients with hyperthyroidism accompanied by diffuse (99 m)Tc uptake and negative TPO, TSHR, and thyroglobulin antibodies for TSHR germline mutations. Moreover, it demonstrates that TSHR germline mutations may first lead to longstanding nodular goitrogenesis before the late manifestation of subclinical hyperthyroidism. © Georg Thieme Verlag KG Stuttgart · New York.

Neutron capture therapy of epidermal growth factor receptor (EGFR)vIII positive gliomas using boronated monoclonal antibody L8A4

International Nuclear Information System (INIS)

Yang, Weilian; Barth, Rolf F.; Wu, Gong

2006-01-01

The purpose of the present study was to evaluate the EGFRvIII specific monoclonal antibody, L8A4 as a boron delivery agent for NCT of the receptor (+) rat glioma, F98 npEGFRvIII . A heavily boronated polyamidoamine (PAMAM) dendrimer (BD) was linked to L8A4 by means of heterobifunctional reagents. Wild type (F98 WT ) receptor(-) or EGFRvIII human gene transfected receptor(+) F98 npEGFRvIII glioma cells were implanted into the brains of Fischer rats. Biodistribution studies were initiated 14 d later. Animals received 125 I-labeled BD-L8A4 by either convection enhanced delivery (CED) or intratumoral(i.t.) injection and were euthanized 6, 12, 24 or 48 h later. At 6 h following CED, equivalent amounts of the bioconjugate were detected in receptor(+) and (-) tumors, but by 24 h the amounts retained by receptor(+) gliomas were 60.1% following CED and 43.7% following i.t. injection, compared to 14.6% ID/g by receptor(-) tumors. Tumor boron concentrations were 32.7 and 44.5 μg/g, respectively, for BD-L8A4 alone or in combination with i.v. BPA. BNCT was carried out at the MITR-II Reactor 24 h after CED of BD-L8A4 (∼40 μg 10 B/∼750 μg protein) and 2.5 h after i.v. injection of BPA (500 mg/kg). Rats that received BD-L8A4 alone or in combination with BPA had mean survival times of 70.4 and 85d, respectively, with 20% and 10% long term survivors, respectively, compared to 40.1 d for i.v. BPA and 30.3 and 26.3 d for irradiated and untreated controls, respectively. These data convincingly demonstrate the therapeutic efficacy of molecular targeting of EGFRvIII and should provide a platform for the future development of combinations of high and low molecular weight delivery agents for BNCT of brain tumors. (author)

SEARCH FOR TARGET TISSUE IN THE EYE ORBIT FOR AUTOIMMUNE AGGRESSION OF THYROID ANTIBODIES IN ENDOCRINE OPHTHALMOPATHY

Directory of Open Access Journals (Sweden)

V. G. Likhvantseva

2017-01-01

Full Text Available We searched for a possible target tissue in eye orbit for thyroid autoantibodies in endocrine ophthalmopathy (Graves’ disease, using correlation analysis method. We examined a group of 139 patients (278 eye orbits with thyroid-associated ophthalmopathy associated with diffuse toxic goiter. Serological parameters (antibodies to thyroid-stimulating hormone receptor; thyroglobulin, thyroid peroxidase were compared with instrumental diagnostic data (multi-layer CT, ultrasonography of eye orbit, and exophthalmometer, as well as clinical symptoms. Statistical correlation analysis enabled us to show different degrees of association between thyroid antibodies and clinical manifestations of Graves’ disease and eye orbit involvement. Especially, carriers of antibodies to TSH receptor and thyroglobulin (as compared to seronegative patients exhibited higher exophthalmos scores (19.16±0.26 mm, p < 0.001, and 19.41±0.40 mm, p < 0.05, respectively, and with total muscle index (2.42±0.05, p < 0.01, and 2.42±0.08, respectively. Meanwhile, eyelids in carriers of antibodies to TSH receptor and thyroid peroxidase proved to be more swollen (p < 0.001, p < 0.05, respectively. Carriage of antibodies to thyroglobulin was associated with synchronous involvement of two structures of the eye orbit: extraocular muscles and retrobulbar tissue, which is reflected by increase in the average ntegral exophthalmos index within the group.

Enhanced antibody-dependent cellular phagocytosis by chimeric monoclonal antibodies with tandemly repeated Fc domains.

Science.gov (United States)

Nagashima, Hiroaki; Ootsubo, Michiko; Fukazawa, Mizuki; Motoi, Sotaro; Konakahara, Shu; Masuho, Yasuhiko

2011-04-01

We previously reported that chimeric monoclonal antibodies (mAbs) with tandemly repeated Fc domains, which were developed by introducing tandem repeats of Fc domains downstream of 2 Fab domains, augmented binding avidities for all Fcγ receptors, resulting in enhanced antibody (Ab)-dependent cellular cytotoxicity. Here we investigated regarding Ab-dependent cellular phagocytosis (ADCP) mediated by these chimeric mAbs, which is considered one of the most important mechanisms that kills tumor cells, using two-color flow cytometric methods. ADCP mediated by T3-Ab, a chimeric mAb with 3 tandemly repeated Fc domains, was 5 times more potent than that by native anti-CD20 M-Ab (M-Ab hereafter). Furthermore, T3-Ab-mediated ADCP was resistant to competitive inhibition by intravenous Ig (IVIG), although M-Ab-mediated ADCP decreased in the presence of IVIG. An Fcγ receptor-blocking study demonstrated that T3-Ab mediated ADCP via both FcγRIA and FcγRIIA, whereas M-Ab mediated ADCP exclusively via FcγRIA. These results suggest that chimeric mAbs with tandemly repeated Fc domains enhance ADCP as well as ADCC, and that Fc multimerization may significantly enhance the efficacy of therapeutic Abs. Copyright © 2010 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Radiometric immunosorbent assay for the detection of anti-hormone-binding protein antibodies

International Nuclear Information System (INIS)

Pierce, E.A.; Dame, M.C.; DeLuca, H.F.

1986-01-01

A radiometric immunosorbent assay (RISA) for the detection of monoclonal antibodies to hormone-binding proteins has been developed. The assay involves incubating hybridoma supernatants in microtiter wells that have been coated with goat anti-mouse IgG antibodies. Any mouse IgG in the test supernatant is thus specifically retained in the wells. Radioactive ligand-binding protein complexes are then incubated in the wells. The presence of anti-binding protein antibodies in the supernatant is indicated by specific retention of radioactive ligand-binding protein complexes in the wells. Crude antigen preparations, such as tissue homogenates, can be used to detect antibodies. The assay is capable of detecting antibody at concentrations 20 ng/ml (approx. 100 pM IgG). The RISA has been used successfully to screen for monoclonal antibodies to the intracellular receptor for 1,25-dihydroxyvitamin D 3 and should be useful for the detection of antibodies to ligand-binding proteins in general

R1507, an Anti-Insulin-Like Growth Factor-1 Receptor (IGF-1R) Antibody, and EWS/FLI-1 siRNA in Ewing's Sarcoma: Convergence at the IGF/IGFR/Akt Axis

Science.gov (United States)

Rodon, Jordi; Sun, Michael; Kuenkele, Klaus-Peter; Parsons, Henrique A.; Trent, Jonathan C.; Kurzrock, Razelle

2011-01-01

A subset of patients with Ewing's sarcoma responds to anti-insulin-like growth factor-1 receptor (IGF-1R) antibodies. Mechanisms of sensitivity and resistance are unknown. We investigated whether an anti-IGF-1R antibody acts via a pathway that could also be suppressed by small interfering (si) RNA against the EWS/FLI-1 fusion protein, the hallmark of Ewing's sarcoma. The growth of two Ewing's sarcoma cell lines (TC-32 and TC-71) was inhibited by the fully human anti-IGF-1R antibody, R1507 (clonogenic and MTT assays). TC-32 and TC-71 cells express high levels of IGF-2, while RD-ES and A4573 Ewing's cell lines, which were less responsive to R1507 in our assays, express low or undetectable IGF-2, respectively. TC-71 cells also expressed high levels of IGF-1R, and R1507 decreased steady-state levels of this receptor by internalization/degradation, an effect which was associated with a decrease in p-IGF-1R, p-IRS-1, and p-Akt. EWS/FLI-1 siRNA also decreased p-Akt, due to its ability to increase IGF-BP3 levels and subsequently decrease IGF-1 and IGF-2 levels, thus inhibiting signaling through p-IGF-1R. This inhibition correlated with growth suppression and apoptosis. The attenuation of Akt activation was confirmed in TC-71 and HEK-293 (human embryonic kidney) cells by transfecting them with IGF-1R siRNA. We conclude that antibodies and siRNA to IGF-1R, as well as siRNA to EWS/FLI-1, act via intersecting IGF/IGF-1R signals that suppress a common point in this pathway, namely the phosphorylation of Akt. PMID:22022506

R1507, an anti-insulin-like growth factor-1 receptor (IGF-1R antibody, and EWS/FLI-1 siRNA in Ewing's sarcoma: convergence at the IGF/IGFR/Akt axis.

Directory of Open Access Journals (Sweden)

Helen J Huang

Full Text Available A subset of patients with Ewing's sarcoma responds to anti-insulin-like growth factor-1 receptor (IGF-1R antibodies. Mechanisms of sensitivity and resistance are unknown. We investigated whether an anti-IGF-1R antibody acts via a pathway that could also be suppressed by small interfering (si RNA against the EWS/FLI-1 fusion protein, the hallmark of Ewing's sarcoma. The growth of two Ewing's sarcoma cell lines (TC-32 and TC-71 was inhibited by the fully human anti-IGF-1R antibody, R1507 (clonogenic and MTT assays. TC-32 and TC-71 cells express high levels of IGF-2, while RD-ES and A4573 Ewing's cell lines, which were less responsive to R1507 in our assays, express low or undetectable IGF-2, respectively. TC-71 cells also expressed high levels of IGF-1R, and R1507 decreased steady-state levels of this receptor by internalization/degradation, an effect which was associated with a decrease in p-IGF-1R, p-IRS-1, and p-Akt. EWS/FLI-1 siRNA also decreased p-Akt, due to its ability to increase IGF-BP3 levels and subsequently decrease IGF-1 and IGF-2 levels, thus inhibiting signaling through p-IGF-1R. This inhibition correlated with growth suppression and apoptosis. The attenuation of Akt activation was confirmed in TC-71 and HEK-293 (human embryonic kidney cells by transfecting them with IGF-1R siRNA. We conclude that antibodies and siRNA to IGF-1R, as well as siRNA to EWS/FLI-1, act via intersecting IGF/IGF-1R signals that suppress a common point in this pathway, namely the phosphorylation of Akt.

A Prospective Investigation of Graves' Disease and Selenium: Thyroid Hormones, Auto-Antibodies and Self-Rated Symptoms

DEFF Research Database (Denmark)

Calissendorff, Jan; Mikulski, Emil; Larsen, Erik Huusfeldt

2015-01-01

-stimulating hormone (TSH), free thyroxine (FT4), free triiodothyronine (FT3), thyroid receptor antibodies and thyroid peroxidase auto-antibodies before medication and at 6, 18 and 36 weeks after commencing treatment with methimazole and levo-thyroxine, with a randomized blinded oral administration of 200 µg Se...

Receptors for insulin-like growth factors I and II: autoradiographic localization in rat brain and comparison to receptors for insulin

International Nuclear Information System (INIS)

Lesniak, M.A.; Hill, J.M.; Kiess, W.; Rojeski, M.; Pert, C.B.; Roth, J.

1988-01-01

Receptors for insulin-like growth factor I (IGF-I) in rat brain were visualized using autoradiography with [125I]IGF-I. The binding of the labeled peptide was competed for fully by high concentrations of unlabeled IGF-I. At intermediate concentrations of unlabeled peptide the binding of [125I]IGF-I was competed for by unlabeled IGF-I more effectively than by IGF-II or insulin, which is typical of receptors for IGF-I. Essentially every brain section shows specific binding of IGF-I, and the pattern of binding of IGF-I to its receptors correlated well with the cytoarchitectonic structures. In parallel studies we showed that [125I]IGF-II was bound to tissue sections of rat brain and that the binding was competed for by an excess of unlabeled IGF-II. However, intermediate concentrations of unlabeled peptides gave inconclusive results. To confirm that the binding of [125I]IGF-II was to IGF-II receptors, we showed that antibodies specific for the IGF-II receptor inhibited the binding of labeled IGF-II. Furthermore, the binding of the antibody to regions of the brain section, visualized by the application of [125I]protein-A, gave patterns indistinguishable from those obtained with [125I]IGF-II alone. Again, the binding was very widely distributed throughout the central nervous system, and the patterns of distribution corresponded well to the underlying neural structures. Densitometric analysis of the receptors enabled us to compare the distribution of IGF-I receptors with that of IGF-II receptors as well as retrospectively with that of insulin receptors

Growth factor receptors as molecular targets for cancer diagnosis and therapy

International Nuclear Information System (INIS)

Zalutsky, M. R.

1997-01-01

Growth factor receptors are of great interest as molecular targets for the diagnosis and treatment of cancer. Growth factor receptors are frequently over expressed on malignant cell populations since many cellular oncogenes encode either growth factors of their receptors. The wild-type epidermal growth factor receptor has a molecular weight of 170 kD and is over expressed on gliomas, bladder tumors, squamous cells carcinomas and breast carcinomas. Another growth factor oncogene, c-erb B-2, encodes a 185-kD glycoprotein found on the surface of gliomas, breast and ovarian cancers as well as other carcinomas of epithelial origin. In addition to causing over expression, oncogenic transformation also can result in genomic re-arrangements. An important example from the perspective of targeting is EGFRvIII, a deletion mutant which lacks amino acids 6-273 in the extracellular domain of the epiderma growth factor receptor. The EGFRvIII molecule (145 kD) may be of great value for targeting because it appears to be tumor-specific. Antibodies have been developed with specific reactivity with these growth factor receptors. Since these antibodies are internalized into the cell after receptor binding, it is necessary to use radiolabeling methods which residualize the radioactivity in the tumor cell after intracellular catabolism. To investigate this problem they have evaluated the effect of radioiodination method on the in vitro an in vivo properties of an anti-EGFRvIII antibody. Methods studied were Iodogen, tyramine-cellobiose, and N-succinimidyl 5-iodo-3-pyridine-carboxylate with the last offering optimal localization in a human xenograft model

Structure of a Human Astrovirus Capsid-Antibody Complex and Mechanistic Insights into Virus Neutralization

Energy Technology Data Exchange (ETDEWEB)

Bogdanoff, Walter A.; Campos, Jocelyn; Perez, Edmundo I.; Yin, Lu; Alexander, David L.; DuBois, Rebecca M. (UCSC)

2016-11-02

Human astroviruses (HAstVs) are a leading cause of viral diarrhea in young children, the immunocompromised, and the elderly. There are no vaccines or antiviral therapies against HAstV disease. Several lines of evidence point to the presence of protective antibodies in healthy adults as a mechanism governing protection against reinfection by HAstV. However, development of anti-HAstV therapies is hampered by the gap in knowledge of protective antibody epitopes on the HAstV capsid surface. Here, we report the structure of the HAstV capsid spike domain bound to the neutralizing monoclonal antibody PL-2. The antibody uses all six complementarity-determining regions to bind to a quaternary epitope on each side of the dimeric capsid spike. We provide evidence that the HAstV capsid spike is a receptor-binding domain and that the antibody neutralizes HAstV by blocking virus attachment to cells. We identify patches of conserved amino acids that overlap the antibody epitope and may comprise a receptor-binding site. Our studies provide a foundation for the development of therapies to prevent and treat HAstV diarrheal disease.

IMPORTANCEHuman astroviruses (HAstVs) infect nearly every person in the world during childhood and cause diarrhea, vomiting, and fever. Despite the prevalence of this virus, little is known about how antibodies in healthy adults protect them against reinfection. Here, we determined the crystal structure of a complex of the HAstV capsid protein and a virus-neutralizing antibody. We show that the antibody binds to the outermost spike domain of the capsid, and we provide evidence that the antibody blocks virus attachment to human cells. Importantly, our findings suggest that a subunit-based vaccine focusing the immune system on the HAstV capsid spike domain could be effective in protecting children against HAstV disease.

Mouse monoclonal antibodies against human c-Mpl and characterization for flow cytometry applications.

Science.gov (United States)

Abbott, Christina; Huang, Guo; Ellison, Aaron R; Chen, Ching; Arora, Taruna; Szilvassy, Stephen J; Wei, Ping

2010-04-01

Mouse monoclonal antibodies (MAbs) against human c-Mpl, the cognate receptor for thrombopoietin (TPO), were generated using hybridoma technology and characterized by various assays to demonstrate their specificity and affinity. Two such MAbs, 1.6 and 1.75, were determined to be superior for flow cytometry studies and exhibited double-digit picomolar (pM) affinities to soluble human c-Mpl protein. Both MAbs specifically bound to cells engineered to overexpress human c-Mpl protein, immortalized human hematopoietic cell lines that express endogenous c-Mpl, primary human bone marrow and peripheral blood-derived CD34(+) cells, and purified human platelets. No binding was detected on cell lines that did not express c-Mpl. Receptor competition and siRNA knock-down studies further confirmed the specificity of antibodies 1.6 and 1.75 for human c-Mpl. In contrast to these newly generated MAbs, none of eight commercially available anti-c-Mpl antibodies tested were found to bind specifically to human c-Mpl and were thus shown to be unsuitable for flow cytometry studies. Monoclonal antibodies 1.6 and 1.75 will therefore be useful flow cytometry reagents to detect cell surface c-Mpl expression.

ADCC responses and blocking of EGFR-mediated signaling and cell growth by combining the anti-EGFR antibodies imgatuzumab and cetuximab in NSCLC cells

NARCIS (Netherlands)

Kol, Arjan; Terwisscha van Scheltinga, Anton; Pool, Martin; Gerdes, Christian; de Vries, Elisabeth; de Jong, Steven

2017-01-01

Imgatuzumab is a novel glycoengineered anti-epidermal growth factor receptor (EGFR) monoclonal antibody optimized to induce both antibody-dependent cellular cytotoxicity (ADCC) and EGFR signal transduction inhibition. We investigated antiEGFR monoclonal antibodies imgatuzumab and cetuximab-induced

Potent neutralization of hepatitis A virus reveals a receptor mimic mechanism and the receptor recognition site.

Science.gov (United States)

Wang, Xiangxi; Zhu, Ling; Dang, Minghao; Hu, Zhongyu; Gao, Qiang; Yuan, Shuai; Sun, Yao; Zhang, Bo; Ren, Jingshan; Kotecha, Abhay; Walter, Thomas S; Wang, Junzhi; Fry, Elizabeth E; Stuart, David I; Rao, Zihe

2017-01-24

Hepatitis A virus (HAV) infects ∼1.4 million people annually and, although there is a vaccine, there are no licensed therapeutic drugs. HAV is unusually stable (making disinfection problematic) and little is known of how it enters cells and releases its RNA. Here we report a potent HAV-specific monoclonal antibody, R10, which neutralizes HAV infection by blocking attachment to the host cell. High-resolution cryo-EM structures of HAV full and empty particles and of the complex of HAV with R10 Fab reveal the atomic details of antibody binding and point to a receptor recognition site at the pentamer interface. These results, together with our observation that the R10 Fab destabilizes the capsid, suggest the use of a receptor mimic mechanism to neutralize virus infection, providing new opportunities for therapeutic intervention.

Treatment with the anti-IL-6 receptor antibody attenuates muscular dystrophy via promoting skeletal muscle regeneration in dystrophin-/utrophin-deficient mice.

Science.gov (United States)

Wada, Eiji; Tanihata, Jun; Iwamura, Akira; Takeda, Shin'ichi; Hayashi, Yukiko K; Matsuda, Ryoichi

2017-10-27

Chronic increases in the levels of the inflammatory cytokine interleukin-6 (IL-6) in serum and skeletal muscle are thought to contribute to the progression of muscular dystrophy. Dystrophin/utrophin double-knockout (dKO) mice develop a more severe and progressive muscular dystrophy than the mdx mice, the most common murine model of Duchenne muscular dystrophy (DMD). In particular, dKO mice have smaller body sizes and muscle diameters, and develop progressive kyphosis and fibrosis in skeletal and cardiac muscles. As mdx mice and DMD patients, we found that IL-6 levels in the skeletal muscle were significantly increased in dKO mice. Thus, in this study, we aimed to analyze the effects of IL-6 receptor (IL-6R) blockade on the muscle pathology of dKO mice. Male dKO mice were administered an initial injection (200 mg/kg intraperitoneally (i.p.)) of either the anti-IL-6R antibody MR16-1 or an isotype-matched control rat IgG at the age of 14 days, and were then given weekly injections (25 mg/kg i.p.) until 90 days of age. Treatment of dKO mice with the MR16-1 antibody successfully inhibited the IL-6 pathway in the skeletal muscle and resulted in a significant reduction in the expression levels of phosphorylated signal transducer and activator of transcription 3 in the skeletal muscle. Pathologically, a significant increase in the area of embryonic myosin heavy chain-positive myofibers and muscle diameter, and reduced fibrosis in the quadriceps muscle were observed. These results demonstrated the therapeutic effects of IL-6R blockade on promoting muscle regeneration. Consistently, serum creatine kinase levels were decreased. Despite these improvements observed in the limb muscles, degeneration of the diaphragm and cardiac muscles was not ameliorated by the treatment of mice with the MR16-1 antibody. As no adverse effects of treatment with the MR16-1 antibody were observed, our results indicate that the anti-IL-6R antibody is a potential therapy for muscular dystrophy

Liver-X-receptor activator prevents homocysteine-induced production of IgG antibodies from murine B lymphocytes via the ROS-NF-κB pathway

International Nuclear Information System (INIS)

Chang Lina; Zhang, Zhenmin; Li Wenjing; Dai Jing; Guan Youfei; Wang Xian

2007-01-01

Our previous study showed that homosysteine (Hcy) promotes proliferation of mouse splenic B lymphocytes. In this study, we investigated whether Hcy could stimulate the production of IgG antibodies. Hcy significantly increased the production of IgG antibodies from resting B lymphocytes. B lymphocytes from ApoE-knockout mice with hyperhomocysteinemia showed elevated IgG secretion at either the basal Hcy level or in response to lipopolysaccharide. Hcy promoted reactive oxygen species (ROS) formation, and free radical scavengers, MnTMPyP decreased Hcy-induced IgG secretion. The inhibitor of NF-κB (MG132) also significantly reduced Hcy-induced IgG secretion. Furthermore, Hcy-induced formation of ROS, activation of NF-κB, and secretion of IgG could be inhibited by the liver-X-receptor (LXR) agonist TO 901317. Thus, our data provide strong evidence that HHcy induces IgG production from murine splenic B lymphocytes both in vitro and in vivo. The mechanism might be through the ROS-NF-κB pathway and can be attenuated by the activation of LXR

Preparation and functional studies of hydroxyethyl chitosan nanoparticles loaded with anti-human death receptor 5 single-chain antibody

Directory of Open Access Journals (Sweden)

Yang J

2014-05-01

Full Text Available Jingjing Yang,1,3,* Xiaoping Huang,1,3,* Fanghong Luo,1 Xiaofeng Cheng,3 Lianna Cheng,3 Bin Liu,4 Lihong Chen,2 Ruyi Hu,1,3 Chunyan Shi,1,3 Guohong Zhuang,1,3 Ping Yin2 1Anti-Cancer Research Center, Medical College, Xiamen University, Fujian, People's Republic of China, 2The Department of Pathology, Zhongshan Hospital, Xiamen University, Xiamen, People's Republic of China, 3Organ transplantation institution, Xiamen University, Xiamen, People's Republic of China, 4Jilin Vocational College of Industry and Technology, Jilin, People's Republic of China  *These authors contributed equally to this work Objective: To prepare hydroxyethyl chitosan nanoparticles loaded with anti-human death receptor 5 single-chain antibody, and study their characteristics, functions, and mechanisms of action. Materials and methods: The anti-human death receptor 5 single-chain antibody was constructed and expressed. Protein-loaded hydroxyethyl chitosan nanoparticles were prepared, and their size, morphology, particle-size distribution and surface zeta potential were measured by scanning electron microscopy and laser particle-size analysis. Mouse H22 hepatocellular carcinoma cells were cultured, and growth inhibition was examined using the CellTiter-Blue cell-viability assay. Flow cytometry and Hoechst 33342 were employed to measure cell apoptosis. Kunming mice with H22 tumor models were treated with protein-loaded hydroxyethyl chitosan nanoparticles, and their body weight and tumor size were measured, while hematoxylin and eosin staining was used to detect antitumor effects in vivo and side effects from tumors. Results: The protein-loaded hydroxyethyl chitosan nanoparticles had good stability; the zeta potential was -24.2±0.205, and the dispersion index was 0.203. The inhibition of the protein-loaded hydroxyethyl chitosan nanoparticles on H22 growth was both time- and dose-dependent. Increased expressions of active caspase 8, active caspase 3, and BAX were detected

Clinical diagnosis of Graves’ or non-Graves’ hyperthyroidism compared to TSH receptor antibody test

Directory of Open Access Journals (Sweden)

Lauren Bell

2018-04-01

Full Text Available Background: TSH receptor antibody (TRAb is considered the gold standard diagnostic test for the autoimmunity of Graves’ disease (GD, which is commonly diagnosed clinically. Aim: To evaluate the true positive (sensitivity and true negative (specificity rates of clinical diagnosis of GD or non-GD hyperthyroidism compared to the TRAb test. Setting: University teaching hospital in North West England. Participants: Patients in the Endocrinology service who had a TRAb measurement between December 2009 and October 2015. Methods: Electronic patient records were studied retrospectively for a pre-TRAb clinical diagnosis of GD or non-GD hyperthyroidism. We examined descriptive statistics and binary classification tests; Fisher exact test was used to analyse contingency tables. Results: We identified 316 patients with a mean age of 45 (range, 17–89 years; 247 (78% were women. Compared to the TRAb result, clinical diagnosis had a sensitivity of 88%, specificity 66%, positive predictive value 72%, negative predictive value 84%, false negative rate 12%, false positive rate 34%, positive likelihood ratio 2.6 and negative likelihood ratio 0.2 (P < 0.0001. Conclusions: Clinicians were liable to both over- and under-diagnose GD. The TRAb test can help reduce the number of incorrect or unknown diagnoses in the initial clinical assessment of patients presenting with hyperthyroidism.

Studies of a murine monoclonal antibody directed against DARC: reappraisal of its specificity.

Directory of Open Access Journals (Sweden)

Dorota Smolarek

Full Text Available Duffy Antigen Receptor for Chemokines (DARC plays multiple roles in human health as a blood group antigen, a receptor for chemokines and the only known receptor for Plasmodium vivax merozoites. It is the target of the murine anti-Fy6 monoclonal antibody 2C3 which binds to the first extracellular domain (ECD1, but exact nature of the recognized epitope was a subject of contradictory reports. Here, using a set of complex experiments which include expression of DARC with amino acid substitutions within the Fy6 epitope in E. coli and K562 cells, ELISA, surface plasmon resonance (SPR and flow cytometry, we have resolved discrepancies between previously published reports and show that the basic epitope recognized by 2C3 antibody is 22FEDVW26, with 22F and 26W being the most important residues. In addition, we demonstrated that 30Y plays an auxiliary role in binding, particularly when the residue is sulfated. The STD-NMR studies performed using 2C3-derived Fab and synthetic peptide corroborated most of these results, and together with the molecular modelling suggested that 25V is not involved in direct interactions with the antibody, but determines folding of the epitope backbone.

TAM Receptors Are Not Required for Zika Virus Infection in Mice.

Science.gov (United States)

Hastings, Andrew K; Yockey, Laura J; Jagger, Brett W; Hwang, Jesse; Uraki, Ryuta; Gaitsch, Hallie F; Parnell, Lindsay A; Cao, Bin; Mysorekar, Indira U; Rothlin, Carla V; Fikrig, Erol; Diamond, Michael S; Iwasaki, Akiko

2017-04-18

Tyro3, Axl, and Mertk (TAM) receptors are candidate entry receptors for infection with the Zika virus (ZIKV), an emerging flavivirus of global public health concern. To investigate the requirement of TAM receptors for ZIKV infection, we used several routes of viral inoculation and compared viral replication in wild-type versus Axl -/- , Mertk -/- , Axl -/- Mertk -/- , and Axl -/- Tyro3 -/- mice in various organs. Pregnant and non-pregnant mice treated with interferon-α-receptor (IFNAR)-blocking (MAR1-5A3) antibody and infected subcutaneously with ZIKV showed no reliance on TAMs for infection. In the absence of IFNAR-blocking antibody, adult female mice challenged intravaginally with ZIKV showed no difference in mucosal viral titers. Similarly, in young mice that were infected with ZIKV intracranially or intraperitoneally, ZIKV replication occurred in the absence of TAM receptors, and no differences in cell tropism were observed. These findings indicate that, in mice, TAM receptors are not required for ZIKV entry and infection. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Human Neutralizing Monoclonal Antibody Inhibition of Middle East Respiratory Syndrome Coronavirus Replication in the Common Marmoset.

Science.gov (United States)

Chen, Zhe; Bao, Linlin; Chen, Cong; Zou, Tingting; Xue, Ying; Li, Fengdi; Lv, Qi; Gu, Songzhi; Gao, Xiaopan; Cui, Sheng; Wang, Jianmin; Qin, Chuan; Jin, Qi

2017-06-15

Middle East respiratory syndrome coronavirus (MERS-CoV) infection in humans is highly lethal, with a fatality rate of 35%. New prophylactic and therapeutic strategies to combat human infections are urgently needed. We isolated a fully human neutralizing antibody, MCA1, from a human survivor. The antibody recognizes the receptor-binding domain of MERS-CoV S glycoprotein and interferes with the interaction between viral S and the human cellular receptor human dipeptidyl peptidase 4 (DPP4). To our knowledge, this study is the first to report a human neutralizing monoclonal antibody that completely inhibits MERS-CoV replication in common marmosets. Monotherapy with MCA1 represents a potential alternative treatment for human infections with MERS-CoV worthy of evaluation in clinical settings. © Crown copyright 2017.

Thyrotropin receptor antibody activities significantly correlate with the outcome of radioiodine (131I) therapy for hyperthyroid Graves' disease

International Nuclear Information System (INIS)

Kaise, Kazuro; Kaise, Nobuko; Yoshida, Katsumi; Fukazawa, Hiroshi; Mori, Koki; Yamamoto, Makiko; Sakurada, Toshiro; Saito, Shintaro; Yoshinaga, Kaoru

1991-01-01

The outcome of 131 I therapy for 109 patients with Graves' disease was analysed according to pretreatment laboratory data including thyrotropin receptor antibody (TRAb) activities. Forty-five percent of patients became euthyroid, and 13% of patients became hypothyroid within one year after 131 I therapy. Forty-two percent of patients remained hyperthyroid one year after 131 I therapy. Pretreatment values for serum T 4 , T 3 , and the estimated weight of the thyroid were significantly higher in the hyperthyroid group. The mean for the TRAb index of the hyperthyroid group was significantly higher than that of the euthyroid group. Life table analysis revealed a significant effect of the TRAb index on the rate of hyperthyroidism after 3 months or later. These results appear to suggest that the TRAb index is one of the factors which influence the outcome of 131 I therapy for Graves' disease. (author)

Detection of thyroid stimulating hormone receptor antibodies (TRAb) by radioreceptor assay (RRA) and enzyme-linked immunosorbent assay (ELISA)

International Nuclear Information System (INIS)

Dumrongpisutikul, S.; Tuchinda, S.

1990-01-01

Thyroid stimulating hormone receptor antibodies (TRAb) were determined in 100 patients using radioreceptor assay (RRA) and enzyme-linked immunosorbent assay (ELISA). The sensitivity of RRA and ELISA were found to be 70.6% and 88.2% respectively (n=51). The specificity of both assays were 100% (n=16). With RRA as the standard test the sensitivity and specificity of ELISA were 75.8% and 86.8%. In the untreated hyperthyroid the RRA result which expressed as % specific 125 I-TSH inhibition was 33.6% (n=51), decline to 26.9% in the treated hyperthyroid (n=33) and 14.1% in the euthyroid (n=16). The mean 0.D 492nm of TRAb-ELISA were 0.861 in untreated hyperthyroid, 0.437 in treated hyperthyroid and 0.135 in euthyroid Phi coefficient analysis show that the RRA was 60.4% correlated to hyperthyroidism where as TRAb-ELISA was 80.1%

[Limbic encephalitis with antibodies against intracellular antigens].

Science.gov (United States)

Morita, Akihiko; Kamei, Satoshi

2010-04-01

Limbic encephalitis is a paraneoplastic syndrome that is often associated with small cell lung cancer (SCLC), breast cancer, testicular tumors, teratoma, Hodgkin's lymphoma and thymoma. The common clinical manifestations of limbic encephalitis are subacute onset, cognitive dysfunction, seizures and psychiatric symptoms. Paraneoplastic neurological disorders are considered to occur because of cytotoxic T cell responses and antibodies against target neuronal proteins that are usually expressed by an underlying tumor. The main intracellular antigens related to limbic encephalitis are Hu, Ma2, and less frequently CV2/CRMP5 and amphiphysin. The anti-Hu antibody, which is involved in cerebellar degeneration and extensive or multifocal encephalomyelitis such as limbic encephalitis is closely associated with a history of smoking and SCLC. The anti-Ma2 antibody is associated with encephalitis of the limbic system, hypothalamus and brain-stem. For this reason, some patients with limbic encephalitis have sleep disorders (including REM sleep abnormalities), severe hypokinesis and gaze palsy in addition to limbic dysfunction. In men aged less than 50 years, anti-Ma2 antibody encephalitis is almost always associated with testicular germ-cell tumors that are occasionally difficult to detect. In older men and women, the most common tumors are non-SCLC and breast cancer. Limbic encephalitis associated with cell-surface antigens (e.g., voltage-gated potassium channels, NMDA receptors) is mediated by antibodies and often improves after a reduction in the antibody titer and after tumor resection. Patients with antibodies against intracellular antigens, except for those with anti-Ma2 antibodies and testicular tumors, are less responsive. Early diagnosis and treatment with immunotherapy, tumor resection or both are important for improving or stabilizing the condition of limbic encephalitis.

Anterior cingulate serotonin 1B receptor binding is associated with emotional response inhibition

DEFF Research Database (Denmark)

da Cunha-Bang, Sofi; Hjordt, Liv Vadskjær; Dam, Vibeke Høyrup

2017-01-01

-offender controls, completed an emotional Go/NoGo task requiring inhibition of prepotent motor responses to emotional facial expressions. We also measured cerebral serotonin 1B receptor (5-HT1BR) binding with [11C]AZ10419369 positron emission tomography within regions of the frontal cortex. We hypothesized that 5......-HT1BR would be positively associated with false alarms (failures to inhibit nogo responses) in the context of aversive (angry and fearful) facial expressions. Across groups, we found that frontal cortex 5-HT1BR binding was positively correlated with false alarms when angry faces were go stimuli......Serotonin has a well-established role in emotional processing and is a key neurotransmitter in impulsive aggression, presumably by facilitating response inhibition and regulating subcortical reactivity to aversive stimuli. In this study 44 men, of whom 19 were violent offenders and 25 were non...

Stabilizing the CH2 Domain of an Antibody by Engineering in an Enhanced Aromatic Sequon.

Science.gov (United States)

Chen, Wentao; Kong, Leopold; Connelly, Stephen; Dendle, Julia M; Liu, Yu; Wilson, Ian A; Powers, Evan T; Kelly, Jeffery W

2016-07-15

Monoclonal antibodies (mAbs) exhibiting highly selective binding to a protein target constitute a large and growing proportion of the therapeutics market. Aggregation of mAbs results in the loss of their therapeutic efficacy and can result in deleterious immune responses. The CH2 domain comprising part of the Fc portion of Immunoglobulin G (IgG) is typically the least stable domain in IgG-type antibodies and therefore influences their aggregation propensity. We stabilized the CH2 domain by engineering an enhanced aromatic sequon (EAS) into the N-glycosylated C'E loop and observed a 4.8 °C increase in the melting temperature of the purified IgG1 Fc fragment. This EAS-stabilized CH2 domain also conferred enhanced stability against thermal and low pH induced aggregation in the context of a full-length monoclonal IgG1 antibody. The crystal structure of the EAS-stabilized (Q295F/Y296A) IgG1 Fc fragment confirms the design principle, i.e., the importance of the GlcNAc1•F295 interaction, and surprisingly reveals that the core fucose attached to GlcNAc1 also engages in an interaction with F295. Inhibition of core fucosylation confirms the contribution of the fucose-Phe interaction to the stabilization. The Q295F/Y296A mutations also modulate the binding affinity of the full-length antibody to Fc receptors by decreasing the binding to low affinity Fc gamma receptors (FcγRIIa, FcγRIIIa, and FcγRIIIb), while maintaining wild-type binding affinity to FcRn and FcγRI. Our results demonstrate that engineering an EAS into the N-glycosylated reverse turn on the C'E loop leads to stabilizing N-glycan-protein interactions in antibodies and that this modification modulates antibody-Fc receptor binding.

Ionotropic crustacean olfactory receptors.

Directory of Open Access Journals (Sweden)

Elizabeth A Corey

Full Text Available The nature of the olfactory receptor in crustaceans, a major group of arthropods, has remained elusive. We report that spiny lobsters, Panulirus argus, express ionotropic receptors (IRs, the insect chemosensory variants of ionotropic glutamate receptors. Unlike insects IRs, which are expressed in a specific subset of olfactory cells, two lobster IR subunits are expressed in most, if not all, lobster olfactory receptor neurons (ORNs, as confirmed by antibody labeling and in situ hybridization. Ligand-specific ORN responses visualized by calcium imaging are consistent with a restricted expression pattern found for other potential subunits, suggesting that cell-specific expression of uncommon IR subunits determines the ligand sensitivity of individual cells. IRs are the only type of olfactory receptor that we have detected in spiny lobster olfactory tissue, suggesting that they likely mediate olfactory signaling. Given long-standing evidence for G protein-mediated signaling in activation of lobster ORNs, this finding raises the interesting specter that IRs act in concert with second messenger-mediated signaling.

Antibody therapy of cancer : Fc receptor-mediated mechanisms of action

NARCIS (Netherlands)

Overdijk, M.B.

2013-01-01

Cancer, a class of malignant diseases characterized by unregulated cell growth, is still a leading cause of death worldwide. The high specificity of antibodies combined with the ability to engage multiple mechanisms of action (MoA) and minimal side-effects makes them attractive agents for targeted

A Single Domain–Based Anti-Her2 Antibody Has Potent Antitumor Activities

Directory of Open Access Journals (Sweden)

Xiaoqiong Wu

2018-04-01

Full Text Available Human epidermal growth factor receptor 2 (HER2 is overexpressed in approximately 20% to 30% of breast cancers and various other types of cancers, which plays a vital role in the cancer progression. Monoclonal antibodies targeting Her2 are now used in the clinic to treat Her2 overexpression cancer patients. However, relapse or resistance is frequent with the current therapies. To generate a new treatment avenue against Her2, we immunized and selected a specific anti-Her2 single domain antibody C3 for further studies. The C3-Fc antibody drove antibody-dependent cell-mediated cytotoxicity against Her2-positive tumor cells in vitro and resulted in potent antitumor growth in vivo. These data suggest that the C3-Fc antibody may provide an alternative avenue for Her2-positive cancer therapy.

Comparison between sensitivity of autologous skin serum test and autologous plasma skin test in patients with Chronic Idiopathic Urticaria for detection of antibody against IgE or IgE receptor (FcεRIα).

Science.gov (United States)

Sajedi, Vahid; Movahedi, Masoud; Aghamohammadi, Asghar; Aghamohamadi, Asghar; Gharagozlou, Mohammad; Ghareguzlou, Mohammad; Shafiei, Alireza; Soheili, Habib; Sanajian, Nahal

2011-06-01

Intradermal injection of autologous serum and plasma elicit a cutaneous reactivity in almost 45-60% of patients with Chronic Idiopathic Urticaria (CIU). This reactivity is associated with the presence of auto antibodies against IgE or IgE receptors. This study was carried out to compare the cutaneous reactivity of autologous serum and plasma skin tests in a series of patients with CIU for diagnosis of auto antibodies against IgE or IgE receptor. Fifty eight patients with CIU were injected intradermally with autologous serum and plasma (anticoagulated by citrate). Histamine was used as positive control and normal saline as negative control. The study group was checked by routine laboratory tests (CBC, U/A etc), allergens with skin prick tests, and serum IgE level, and auto antibodies against thyroid as well. Duration of urticaria was another factor which was assessed.There was no significant difference between positive ASST and positive APST patients for the above mentioned tests. 77.6% of the patients were Positive for APST and 65.5% were ASST positive. Duration of urticaria was longer in patients with positive ASST and APST than ASST and APST negative patients, although the difference was not statistically significant.Autologus serum skin test (ASST) and autologous plasma skin test (APST) could be used for estimation of duration and severity of urticaria and planning for the treatment.

PRODUCTION AND PURIFICATION OF IgY ANTIBODIES AS A NOVEL TOOL TO PURIFY THE NR1 SUBUNIT OF NMDA RECEPTO

Directory of Open Access Journals (Sweden)