Antibodies

Evolutionary pressure has selected antibodies as key immune molecules acting against foreign pathogens. The development of monoclonal antibody technology has allowed their widespread use in research, real-time diagnosis and treatment of multiple diseases, including cancer. However, compared with hematologic malignancies, solid tumors have often proven to be relatively resistant to antibody-based therapies. In an attempt to improve the tumor-targeting efficacy of antibodies, new formats with modified, multivalent properties have been generated. Initially, these formats imitated the structure of native IgG, creating mostly monospecific, bivalent antibodies. Recently, novel trivalent antibodies have been developed to maximize tumor targeting capabilities through enhanced biodistribution and functional affinity. We review recent advances in the engineering of multivalent antibodies and further discuss their promise as agents for in vivo diagnostics and therapy.

Neutrophil infiltration into inflammatory sites is one of the hallmarks of acute inflammation. Locally produced chemotactic factors are presumed to mediate the sequence of events leading to the infiltration at inflammatory sites. Interleukin-8 (IL-8), a novel leukocyte chemotactic activating cytokine (chemokine), is produced by various types of cells upon stimulation with inflammatory stimuli and exerts a variety of functions on leukocytes, particularly, neutrophils in vitro. However, no definitive evidence has been presented on its role in recruiting and activating neutrophils in the lesions of various types of inflammatory reactions. We administered a highly specific neutralizing antibody against IL-8 in several types of acute inflammatory reactions, including lipopolysaccharide (LPS)-induced dermatitis, LPS/IL-1-induced arthritis, lung reperfusion injury, and acute immune complex-type glomerulonephritis. Anti-IL-8 treatment prevented neutrophil-dependent tissue damage as well as ...

Filamin, also called actin binding protein-280, is a dimeric protein that cross-links actin filaments in the cortical cytoplasm. In addition to this ubiquitously expressed isoform (FLN1), a second isoform (ABP-L/␥-filamin) was recently identified that is highly expressed in mammalian striated muscles. A monoclonal antibody was developed, that enabled us to identify filamin as a Z-disc protein in mammalian striated muscles by immunocytochemistry and immunoelectron microscopy. In addition, filamin was identified as a component of intercalated discs in mammalian cardiac muscle and of myotendinous junctions in skeletal muscle. Northern and Western blots showed that both, ABP-L/␥-filamin mRNA and protein, are absent from proliferating cultured human skeletal muscle cells. This muscle specific filamin isoform is, however, up-regulated immediately after the induction of differentiation. In cultured myotubes, ABP-L/␥-filamin localises in Z-discs already at the first stages of Z-disc formation, suggesting that ABP-L/␥-filamin might play a role in Z-disc assembly.

Surfaces that resist protein adsorption are important for many bioanalytical applications. Bovine serum albumin (BSA) coatings and multi-arm poly(ethylene glycol) (PEG) coatings display low levels of non-specific protein adsorption and have enabled highly quantitative single-molecule (SM) protein studies. Recently, a method was developed for coating a glass with PEG-BSA nanogels, a promising hybrid of these two low-background coatings. We characterized the nanogel coating to determine its suitability for SM protein experiments. SM adsorption counting revealed that nanogel-coated surfaces exhibit lower protein adsorption than covalently coupled BSA surfaces and monolayers of multi-arm PEG, so this surface displays one of the lowest degrees of protein adsorption yet observed. Additionally, the nanogel coating was resistant to DNA adsorption, underscoring the utility of the coating across a variety of SM experiments. The nanogel coating was found to be compatible with surfactants, whereas the BSA coating was not. Finally, applying the coating to a real-world study, we found that single ligand molecules could be tethered to this surface and detected with high sensitivity and specificity by a digital immunoassay. These results suggest that PEG -BSA nanogel coatings will be highly useful for the SM analysis of proteins.

Five New World (NW) arenaviruses cause human hemorrhagic fevers. Four of these arenaviruses are known to enter cells by binding human transferrin receptor 1 (hTfR1). Here we show that the fifth arenavirus, Chapare virus, similarly uses hTfR1. We also identify an anti-hTfR1 antibody, ch128.1, which efficiently inhibits entry mediated by the glycoproteins of all five viruses, as well as replication of infectious Junín virus. Our data indicate that all NW hemorrhagic fever arenaviruses utilize a common hTfR1 apical-domain epitope and suggest that therapeutic agents targeting this epitope, including ch128.1 itself, can be broadly effective in treating South American hemorrhagic fevers.

Formation of antibodies against botulinum toxin type A has been observed following treatment of Cervical Dystonia (CD). We present the immunological findings from two 12-week Phase III prospective, randomized, doubleblind, single-dose, placebo-controlled studies (Study 1, n = 116; Study 2, n = 136). Patients in both studies were administered abobotulinumtoxinA 500U or placebo intramuscularly at baseline. Patients could receive up to three or four additional treatments (250-1000U) in an open-label follow-up period. Blood samples were collected at baseline and during treatment to test for antibodies to abobotulinumtoxinA using a radioimmunoprecipitation assay (Study 2 only) and a mouse protection assay. Loss of response was predefined using the Toronto Western Spasmodic Torticollis Rating Scale (TWSTRS) total score at 4 weeks following injection. No subjects in Study 1 and one individual in Study 2 developed neutralizing antibodies (nABs) during the double-blind treatment phase; the individual who developed immunoresistance had received botulinum toxin type A treatment prior to the study and did not respond to treatment. Two subjects demonstrated a change in nAB status during open-label treatment and overall responsiveness was maintained in these patients. In conclusion, the development of immunoresistance was rare and, in the presence of circulating nABs, patients may still gain benefit from intramuscular abobotulinumtoxinA treatment.

Silent coeliac disease is reported in 1% of Caucasian populations, but there is a lack of knowledge of its natural course and the risk of complications. The need for population screening is debated. We sought for complications of untreated coeliac disease in a well-defined cohort of Finnish adults. Subjects (n=2427, ages 24-39 years) attending the 21-year follow-up visit of the study "Cardiovascular Risk in Young Finns" completed an extensive questionnaire on their health, diet, social situation and family life, and were given a medical examination. Measurement of serum IgA-transglutaminase and IgA-endomysium antibodies identified 21 subjects with silent coeliac disease. The subjects with silent coeliac disease did not differ from the rest of the cohort in age, gender, stature, weight, medical diagnoses (autoimmune, malignant), health concerns, use of alternative medications, physical activity, or in the cause of death their parents. They had lower serum HDL-cholesterol (1...

The present investigation was executed to study the sero-surveillance of hemorrhagic septicemia (HS) in buffaloes and cattle in district Dera-Ghazi-Khan, Punjab, Pakistan. The average geometric mean titers (GMT) recorded against HS in diseased buffaloes and cattle were 5.7 and 6.1, respectively. The morbidity, mortality and case fatality rates were 57.58, 52.30 and 90.83% in young buffalo calves; and 3.17, 1.92 and 60.65%, in adult buffaloes, respectively. Whereas, in case of young cattle calves, morbidity, mortality and case fatality rates were 8.63, 5.27 and 61.11%, respectively. While in adult cattle, morbidity, mortality and case fatality rates were 4.83, 2.18 and 45.23%, respectively. The present study revealed that the mortality, morbidity and case fatality rates due to HS were greater in young calves than the adults both in buffaloes and cattle. Furthermore, buffaloes were found to be more susceptible to the disease than the cattle.

This work examines the cellular localization of holocarboxylase synthetase (HCS) and its association to chromatin during different stages of development of Drosophila melanogaster. While HCS is well known for its role in the attachment of biotin to biotin-dependent carboxylase, it also regulates the transcription of HCS and carboxylases genes by triggering a cGMP-dependent signal transduction cascade. Further, its presence in the nucleus of cells suggests additional regulatory roles, but the mechanism involved has remained elusive. In this study, we show in D. melanogaster that HCS migrates to the nucleus at the gastrulation stage. In polytene chromosomes, it is associated to heterochromatin bands where it co-localizes with histone 3 trimethylated at lysine 9 (H3K9met3) but not with the euchromatin mark histone 3 acetylated at lysine 9 (H3K9ac). Further, we demonstrate the association of HCS with the hsp70 promoter by immunofluorescence and chromatin immuno-precipitation (ChIP) of associated DNA sequences. We demonstrate the occupancy of HCS to the core promoter region of the transcriptionally inactive hsp70 gene. On heat-shock activation of the hsp70 promoter, HCS is displaced and the promoter region becomes enriched with the TFIIH subunits XPD and XPB and elongating RNA pol II, the latter also demonstrated using ChIP assays. We suggest that HCS may have a role in the repression of gene expression through a mechanism involving its trafficking to the nucleus and interaction with heterochromatic sites coincident with H3K9met3.

The 8th Banff Conference on Allograft Pathology was held in Edmonton, Canada, 15-21 July 2005. Major outcomes included the elimination of the non-specific term 'chronic allograft nephropathy' (CAN) from the Banff classification for kidney allograft pathology, and the recognition of the entity of chronic antibody-mediated rejection. Participation of B cells in allograft rejection and genomics markers of rejection were also major subjects addressed by the conference.

Steroid-responsive encephalopathy associated with autoimmune thyroiditis (SREAT) presents with focal or diffuse nonenhancing MR imaging abnormalities in 50% of patients with SREAT during subacute exacerbation. Vasculitic changes in biopsy studies as well as the elevation of antithyroid antibodies and CSF protein suggests an inflammatory cause. We report the case of a patient with SREAT with changes on diffusion-weighted MR imaging, which improved with corticosteroid therapy and plasmapheresis, supporting the theory of inflammatory changes in exacerbation of presumptive SREAT.

Neuromyelitis optica (NMO) is characterized by severe optic neuritis and longitudinally extensive transverse myelitis. The discovery of an NMO-specific autoantibody to the aquaporin-4 (AQP4) water channel has improved knowledge of NMO pathogenesis. Many studies have focused on inflammatory and pathological biomarkers of NMO, including cytokines and chemokines. Increased concentrations of T helper (Th)17-and Th2-related cytokines and chemokines may be essential factors for developing NMO inflammatory lesions. For example, interleukin-6 could play important roles in NMO pathogenesis, as it is involved in the survival of plasmablasts that produce anti-AQP4 antibody in peripheral circulation and in the enhancement of inflammation in the central nervous system. Therefore, assessment of these useful biomarkers may become a supportive criterion for diagnosing NMO. Significant advances in the understanding of NMO pathogenesis will lead to the development of novel treatment strategies. This review focuses on the current advances in NMO immunological research, particularly that of cytokines and chemokines.

Protein A chromatography is widely employed for the capture and purification of antibodies and Fc-fusion proteins. Due to the high cost of protein A resins, there is a significant economic driving force for using these chromatographic materials for a large number of cycles. The maintenance of column performance over the resin lifetime is also a significant concern in large-scale manufacturing. In this work, several statistical methods are employed to develop a novel principal component analysis (PCA)-based tool for predicting protein A chromatographic column performance over time. A method is developed to carry out detection of column integrity failures before their occurrence without the need for a separate integrity test. In addition, analysis of various transitions in the chromatograms was also employed to develop PCA-based models to predict both subtle and general trends in real-time protein A column yield decay. The developed approach has significant potential for facilitating timely and improved decisions in large-scale chromatographic operations in line with the process analytical technology (PAT) guidance from the Food and Drug Administration (FDA).

The co-occurrence of autoimmune diseases such as rheumatoid arthritis (RA) and type 1 diabetes mellitus (DM) has been reported in individuals and families. In this study, the strength and nature of this association were investigated at the population level in a Swedish case-control cohort. Methods. For this case-control study, 1,419 patients with incident RA diagnosed between 1996 and 2003 were recruited from university, public, and private rheumatology units throughout Sweden; 1,674 matched control subjects were recruited from the Swedish national population registry. Sera from the subjects were tested for the presence of antibodies to cyclic citrullinated peptide (anti-CCP), rheumatoid factor (RF), and the 620W PTPN22 allele. Information on a history of diabetes was obtained by questionnaire, telephone interview, and/or medical record review. The prevalence of type 1 DM and type 2 DM was compared between patients with incident RA and control subjects and further stratified for the presence of anti-CCP, RF, and the PTPN22 risk allele. Results. Type 1 DM was associated with an increased risk of RA (odds ratio [OR] 4.9, 95% confidence interval [95% CI] 1.8-13.1), and this association was specific for anti-CCP-positive RA (OR 7.3, 95% CI 2.7-20.0), but not anti-CCP-negative RA. Further adjustment for the presence of PTPN22 attenuated the risk of anti-CCP-positive RA in patients with type 1 DM to an OR of 5.3 (95% CI 1.5-18.7). No association between RA and type 2 DM was observed. Conclusion. The association between type 1 DM and RA is specific for a particular RA subset, anti-CCPpositive RA. The risk of developing RA later in life in patients with type 1 DM may be attributed, in part, to the presence of the 620W PTPN22 allele, suggesting that this risk factor may represent a common pathway for the pathogenesis of these 2 diseases. Autoimmune diseases such as rheumatoid arthritis (RA) and type 1 diabetes mellitus (DM) have been observed to co-occur within individuals and families (1-4). Although the exact etiologies of RA and type 1 DM are unknown, they are likely attributable to a combination of genetic susceptibility and interactions between environmental risk factors and genes. Thus far, one established genetic risk factor is shared by RA and type 1 DM: the 620W allele of the protein tyrosine phosphatase N22 gene (PTPN22) (5-9). Other genes involved in the pathogenesis of RA and type 1 DM have

Modification of proteins with polyethylene glycol (PEG) has been shown to result in a decrease in immunogenicity. Superoxide dismutase (SOD) and catalase were modified with PEG and used to immunize mice. Antibody titers against the antigens were determined by ELBA. Mice immunized with PEG-SOD had antibody titers 0.03%-0.07% of that seen in mice with SOD, while mice immunized with PEG-catalase developed titers 0.02%-0.09% of that seen in mice with catalase. The modified enzymes retained the ability to react with preformed antibodies to the unmodified antigens. Antibodies to SOD reacted equally well with the PEG-SOD or SOD antigen. Antibodies to catalase reacted to PEG-catalase but at only 0.02% of the reaction with catalase antigen. In reciprocal studies, antisera against the PEG-proteins failed to react to an appreciable level with the corresponding unmodified protein. Modification with PEG resulted in a decrease in immunogenicity of both SOD and catalase.

Nanoemulsions are adjuvants that enhance antigen penetration in the nasal mucosa, increase cellular uptake of antigens by both epithelial dendritic cells, and promote migration of antigen-loaded dendritic cells to regional lymph nodes within a day of vaccine administration. The objective of this study was to determine whether the W 80 5EC nanoemulsion adjuvant enhances immune response not only by direct uptake of antigen by dendritic cells, but also indirectly, by phagocytosis of antigen-primed, apoptotic, epithelial cells. Consistent with this, we show that exposure of both epithelial cells (TC-1s) and dendritic cells (JAWS II or bone marrow derived dendritic cells (BMDCs)) to nanoemulsion exhibited augmented antigen uptake in cell culture. TC-1 cells subsequently underwent G 2 /M cell cycle arrest and apoptosis, and when co-cultured with JAWS II or BMDCs were rapidly engulfed by the dendritic cells, which responded by up-regulating dendritic cell maturation marker CD86. Altogether these results suggest that the effectiveness of nanoemulsions as adjuvants stems, at least in part, from the engulfment of antigen-loaded epithelial cells, leading to enhanced antigen processing and a strong and balanced mucosal and systemic immune response.

To accurately phenotype red cells from patients with a positive direct antiglobulin test (DAT), nonlytic elution procedures were assessed for their ability to dissociate IgG from antibody-coated red cells without altering red cell antigen expression. STUDY DESIGN AND METHODS: Antibodies coating red cells that were sensitized in vivo (warm-reactive autoantibodies: 8 patients) or in vitro were eluted by using glycine-HCI and EDTA (acid/ EDTA), heat (56"C, 10 min), or chloroquine method. RESULTS: Acid/EDTA elution gave the best results, reducing DAT positivity to microscopic levels or rendering the DAT negative in 48 of 50 instances, whereas 4 samples remained resistant to heat elution and 24 to chloroquine. Standard DAT agglutination scores demonstrated that both acid/EDTA and heat elution were superior to the chloroquine method (p<O.OOOl). With the gel low-ionic-strength saline indirect antiglobulin test, acid/ EDTA was superior to heat (p<O.OOl). Overall, acid/ EDTA elution dissociated more antibodies than heat (p<O.OOOl), especially for Kell system (K, k, Kpa, Kpb) alloantibodies. Common red cell antigens, other than Kell system antigens, were unaffected by acid/EDTA elution. In contrast, the expression of most blood group antigens was diminished after heat elution. However, it was possible to type red cell antigens by using gel low-ionicstrength saline indirect antiglobulin tests or tube agglutination methods.

High titers of anti-GD,, antibodies have been found in patients with Guillain-Bar& syndrome or motor neuropathy. To determine the possible diagnostic relevance of these antibodies, we measured serum anti-GD,, IgG and IgM antibodies by enzyme-linked immunosorbent assay in 195 patients with different motor syndromes and in 335 control subjects. Moderately high antibody titers (I/ 1,280-1/5,120) were occasionally found in patients with chronic inflammatory demyelinating polyneuropathy (5%), multifocal motor neuropathy (18%), lower motor neuron disease (3.8%), or amyotrophic lateral sclerosis (1.8%) and in immunological control subjects (1.2%), while titers of 1/20,480 or higher were only found in 2 patients with Guillain-Barri! syndrome (IgG in both) and 2 with motor neuropathy and IgMX monoclonal gammopathy improving with immunotherapy. In both motor neuropathy patients and the Guillain-Bar& syndrome patient who were retested during recovery, anti-GD,, titers decreased concomitantly with clinical improvement. High anti-GD,, antibody titers may be found in several motor syndromes but only markedly increased anti-GD,, titers are strictly associated with potentially treatable dysimmune neuropathies. Carpo M, Nobile-Orazio E, Meucci N, Camba M, Barbieri S, Allaria S, Scarlato G. Anti-GD,, ganglioside antibodies in peripheral motor syndromes. Ann Neurol 1996;33:539-543 High titers of serum antibodies to the ganglioside GD,, were demonstrated recently in some patients with Guillain-Bark syndrome (GBS) [l-51 and in a patient with motor neuropathy (MN) associated with IgM monoclonal gammopathy [6], whereas lower antibody levels were detected in patients with motor neuron disease (MND) [7, 81. The anti-GD,, antibodies were mostly IgG in patients with GBS and IgM in those with MN or M N D and the titers, at least in GBS patients [l, 21, decreased concomitantly with clinical improve-From the

A general problem in immunocytochemistry is the development of a reliable multiple immunolabeling method with primary antibodies originating from the same host species. Here, we briefly outline different approaches intended to close this technological gap and focus on multiple immunolabeling with monoclonal primary antibodies. To this end, we generated a basic universal protocol for the use of secondary antibodies selectively recognizing different isotypes/subclasses of monoclonal primary antibodies. This approach is widely applicable and offers a simple procedure for simultaneously detecting two or more antigens.

The epidermal growth factor receptor 1 (EGFR) is overexpressed in various malignancies and is associated with a poor patient prognosis. A small, receptor-specific, high-affinity imaging agent would be a useful tool in diagnosing malignant tumors and in deciding upon treatment and assessing the response to treatment. We describe here the affinity maturation procedure for the generation of Affibody molecules binding

Interferon (IFN)-β is among the first-line therapies for multiple sclerosis (MS), however the clinical response varies among individuals, and early identification of responders and non-responders would be of critical importance. Here we show that before treatment circulating lymphocytes of MS patients who will respond to IFN-β have higher mRNA levels for the D 1 -like dopaminergic receptor (DR) D 5 in comparison to cells from those who will not respond. Lymphocyte DR D 5 is reduced in MS and IFN-β restores their expression and responsiveness. DR D 5 mRNA levels in circulating lymphocytes might represent an early marker of response to IFN-β in MS patients.

Effective small interfering RNA (siRNA)-mediated therapeutics require the siRNA to be delivered into the cellular RNA-induced silencing complex (RISC). Quantitative information of this essential delivery step is currently inferred from the efficacy of gene silencing and siRNA uptake in the tissue. Here we report an approach to directly quantify siRNA in the RISC in rodents and monkey. This is achieved by specific immunoprecipitation of the RISC from tissue lysates and quantification of small RNAs in the immunoprecipitates by stem-loop PCR. The method, expected to be independent of delivery vehicle and target, is label-free, and the throughput is acceptable for preclinical animal studies. We characterized a lipid-formulated siRNA by integrating these approaches and obtained a quantitative perspective on siRNA tissue accumulation, RISC loading, and gene silencing. The described methodologies have utility for the study of silencing mechanism, the development of siRNA therapeutics, and ...

hCAP-18 is a newly described protein of human neutrophilic granulocytes which belongs to the cathelicidin family of antimicrobial proteins. Members of this protein family share a common N-terminal sequence followed by a highly diverse antimicrobial, cationic C-terminus. The present work describes the production of recombinant hCAP-18, the generation of antibodies to the protein and the development of an accurate, sensitive and specific ELISA for the detection of hCAP-18 in cells, plasma and urine with a detection limit of 0.084 ngrml. The amount of hCAP-18 in neutrophils is 0.627 mg protein per 10 6 cells. The plasma level is 1.18 mgrml which is several fold higher than for other neutrophil specific granule proteins. hCAP-18 is present in plasma as high molecular weight complexes. In accordance with this, hCAP-18 is barely excreted in the urine. The bone marrow appears to be the major source of plasma hCAP-18. The high level of hCAP-18 in plasma may provide an important defense against microorganisms and endotoxins. q 1997 Elsevier Science B.V.

Aleutian mink disease parvovirus (ADV) infects macrophages but infection is restricted. Using the human macrophage cell line U937, we reported infection to be antibody-dependent [22, 23]. Recently, we learned that the U937 cells were contaminated with the human cell line, K562[12]. To clarify which cell line actually supported ADV infection, we studied the antibody-dependent enhancement (ADE) of ADV infection in pure lines of U937 and K562. After infection with ADV complexed with mink anti-ADV antibody, up to 10% of the K562 cells, but none of the U937 cells showed evidence of infection. These results indicated that K562 cells, but not U937 cells, were susceptible to ADV. ADVinfected cultures of K562 were sorted by flow cytometry after staining with a murine monoclonal antibody (IV.3) directed against Fc(gamma)RII. Only cells that were IV.3 positive supported ADV gene expression. Also, preincubation of K562 cells with IV.3 blocked ADV infection by more than 90%. These results established that the ADE of ADV in K562 is Fc(gamma)RIIA-mediated. * Aleutian mink disease parvovirus (ADV), family Parvoviridae, genus Parvovirus, causes persistent infection and progressive immune dysfunction in adult mink. The Aleutian disease syndrome is characterized by plasmacytosis, polyclonal hypergammaglobulinemia, lymphadenopathy, splenomegaly, and mesangioproliferative glomerulonephritis [6, 20]. In vivo, ADV infects macrophages in a semipermissive fashion [24, 35]. Since macrophages are important as antigenpresenting cells and modulate the immune response through cytokine production, ADV-infected macrophages may be involved in the disturbances of the immune system that are characteristic of Aleutian disease [23].

The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the original docum and are linked to publications on ResearchGate, letting you access and read them immediately. Abstract-l. Exposure of the rotifer Brachionusplicutilis to elevated temperature resulted in the synthesis of a number of proteins, including a prominent one of 58,000 Da (SP58).

One of the key stimulators of intestinal development in the chick is physical exposure to feed, while feed withholding delays the onset of gut development. A delay of 24–72 h in onset of feeding is quite common in the poultry industry due to variation in hatching time and hatchery treatments. As intestinal development occurs in concert with the development of the gut associated lymphoid tissue (GALT), we investigated the effects of short term feed withholding on development of GALT in broiler hatchlings. GALT activity was determined by antibody production (systemic and locally in the gut), distribution of B and T lymphocytes in the gut, expression of lymphocyte specific genes, and distribution of B and T lymphocytes in the cloacal bursa. Our findings show that while development of GALT in the foregut (duodenum, jejunum, ileum) was only slightly and temporarily impeded by feed withholding, GALT activity in the hindgut and the gut-related cloacal bursa was significantly delayed during the first 2 weeks of life: Systemic and intestinal antibody responses following rectal immunization to antigen were lower, colonization of the hindgut (cecum and colon) by T and B lymphocytes was delayed, as well as the expression of chIL-2 mRNA in hindgut T lymphocytes. We also found that the increase of B and T population size in the cloacal bursa was delayed with time. Full recovery occurred from 2 weeks of age. The 2-week vulnerable period should be seriously considered in circumstances where hatchlings are in transit for extended periods from hatcheries to farms.

A method is described which produces a high, permanent antibody response following a single injection of venom. Animals (mice, rabbits, sheep) were given intravenous, subcutaneous or orally administered Nigerian Echis carinatus (carpet viper) venom which had been incorporated into sphingomyelin-cholesterol liposomes whose membranes had been stabilized by cross-linking adjacent molecules of sphingomyelin using osmium tetroxide. Before use the preparations were thoroughly dialysed to remove any unbound osmium tetroxide. Antibody levels were estimated using enzyme immunoassay. Venom treated in this way and administered i.v. or s.c. produced a powerful, sustained and protective antibody response lasting for the lifetime of a mouse. We also report the development of significant antibody responses after oral administration of liposome-entrapped but not free venom.

It has long been suggested that the overall shape of the antigen combining site (ACS) of antibodies is correlated with the nature of the antigen. For example, deep pockets are characteristic of antibodies that bind haptens, grooves indicate peptide binders, while antibodies that bind to proteins have relatively flat combining sites. In 1996, MacCallum, Martin and Thornton used a fractal shape descriptor and showed a strong correlation of the shape of the binding region with the general nature of the antigen.

Intact CD3-specific antibody transiently depletes large numbers of T cells and subsequently induces long-term immune tolerance. The underlying mechanisms for the systemic tolerance, however, remain unclear. We show here that treatment of normal mice with intact antibody to CD3 increases systemic transforming growth factor-b (TGF-b) produced by phagocytes exposed to apoptotic T cells. Among the phagocytes, macrophages and immature dendritic cells (iDCs) secrete TGF-b upon ingestion of apoptotic T cells, which induces CD4 + Foxp3 + regulatory T cells in culture and contributes to immune tolerance mediated by CD3-specific antibody in vivo. In accordance with these results, depletion of macrophages and iDCs not only abrogates CD3-specific antibody-mediated prevention of myelin oligodendrocyte glycoprotein-induced acute experimental autoimmune encephalomyelitis (EAE), but also reverses the therapeutic effects of antibody to CD3 on established disease in a model of relapsing-remitting EAE. Thus, CD3-specific antibody-induced immune tolerance is associated with TGF-b production in phagocytes involved in clearing apoptotic T cells, which suggests that apoptosis is linked to active suppression in immune tolerance.

Nonsegmental vitiligo is considered to be an autoimmune disease and is known to be associated with other autoimmune diseases, particularly affecting the thyroid. Screening patients with nonsegmental vitiligo for thyroid function and for the presence of thyroid autoantibodies has been recommended. To investigate the prevalence of thyroid dysfunction and thyroid peroxidase-specific (TPO) antibodies in a large cohort of patients with nonsegmental vitiligo in order to help decide whether routine screening is justified. A total of 434 adults with nonsegmental vitiligo who were referred to our institute were enrolled. Thyroid function and anti-TPO antibody titres were assessed in those patients who had no history of thyroid disease or recent thyroid screening. Forty-three patients had already been diagnosed with thyroid dysfunction, and in 27 patients the general practitioner had performed a thyroid function test with negative results &lt;3months previously. In these patients, thyroid fun...

Extracellular matrix (ECM) conformation is regulated by a variety of stimuli in vivo, including mechanical forces and allosteric binding partners, and these conformational changes contribute to the regulation of cell behavior. Heparin and heparan sulfate, for example, have been shown to regulate the sequestration and presentation of numerous growth factors, including vascular endothelial growth factor, on the heparin 2 binding domain in fibronectin (Fn). However, mechanical force also alters Fn conformation, indicating that the growth factor binding region may be co-regulated by both heparin and mechanical force. Herein, we describe a simple antibody-based method for evaluating the conformation of the heparin 2 binding domain in Fn, and use it to determine the relative contributions of heparin and mechanical strain to the regulation of Fn conformation. We achieved specificity in quantifying conformational changes in this region of Fn by measuring the ratio of two fluorescent monoclonal antibodies, one that is insensitive to Fn conformational changes and a second whose binding is reduced or enhanced by non-equilibrium conformational changes. Importantly, this technique is shown to work on Fn adsorbed on surfaces, single Fn fibers, and Fn matrix fibers in cell culture. Using our dual antibody approach, we show that heparin and mechanical strain co-regulate Fn conformation in matrix fibrils, which is the first demonstration of heparin-dependent regulation of Fn in its physiologically-relevant fibrillar state. Furthermore, the dual antibody approach utilizes commercially available antibodies and simple immunohistochemistry, thus making it accessible to a wide range of scientists interested in Fn mechanobiology.

Systemic lupus erythematosus (SLE) is a multifactorial chronic autoimmune disease, marked by the presence of autoantibodies to nuclear antigens belonging to different isotype classes. For several years, IgE antibodies have been incriminated in the development of allergic diseases and parasitic infections and different anti-IgE therapies have been developed to encounter the pathogenic role of IgE in these pathologies. Recently, multiple studies showed the presence of elevated total IgE levels and demonstrated a pathogenic role of autoreactive IgE in SLE. This review aims to summarize the findings incriminating IgE and autoreactive IgE in the pathophysiology of SLE, to describe their functional outcomes on their targeted cells as well as to discuss different IgE-related therapeutic modalities that emerged and that may be beneficial for SLE patient care.