Monoclonal IgA Research Articles

Similarity of Fine Specificity of IgA Anti-gliadin Antibodies between Patients with Celiac Disease and Humanized α1KI Mice

Gliadins, and primarily α-gliadins containing several sequences such as aa 31-49, aa 56-88 (33-mer), aa 57-68, and aa 69-82, are critical in the induction of immune response or toxic reaction leading to the development of celiac disease (CLD). The role of IgA anti-gliadin antibodies (IgA AGA) is unknown. To this end, we prepared several humanized monoclonal IgA AGA using transgenic α1KI mice. Employing Pepscan with overlapping decapeptides of α-gliadin we observed a robust similarity between the specificity of humanized mouse monoclonal IgA AGA and IgA AGA from patients with florid CLD. The common immunodominant region included several sequential epitopes localized in the N-terminal part of α-gliadin (QFQGQQQPFPPQQPYPQPQPFP, aa 29-50, and QPFPSQQPYLQL, aa 47-58). Notably, IgA AGA produced by clones 8D12, 15B9, 9D12, and 18E2 had significant reactivity against sequences localized in the 33-mer, LQLQPFPQPQ (aa 56-65) and PQLPYPQPQPFL (aa 69-80). Humanized mouse monoclonal IgA AGA that have a known specificity are suitable as standard in ELISAs to detect serum IgA AGA of CLD patients and for studying the AGA pathogenic role in CLD, especially for analyzing the translocation of complex of specific IgA antibodies and individual gliadin peptides through enterocyte barrier.

Heavy Chain Ratio (HLCR) IgGκ/IgGλ or IgAκ/IgAλ: Experience and Clinical Implications In Multiple Myeloma at Diagnosis and During Disease Course

AbstractAbstract 5019A new method for the quantification of IgGκ, IgGλ, IgAκ and IgAλ individually and of deriving IgGκ/IgGλ and IgAκ/IgAλ ratios (HLCR) has been recently made available, allowing measurements of the monoclonal component alone as well as of the polyclonal, non-tumor immunoglobulins (Ig) in plasma cell dyscrasias. The purpose of the present study was to retrospectively determine HLCR and to investigate its contribution in a series of 98 multiple myeloma patients at presentation and in 38 of them, during their disease's course. There were 45 women and 53 men (median age 67 years). Disease stages were evenly distributed (36%, 30%, and 34% in ISS stage 1, 2, and 3 respectively). Paraprotein was IgG in 74 patients (50 IgGκ, 24 IgGλ) and IgA in 24 (12 IgAκ, 12 IgAλ) of them. Seventy-one symptomatic patients received conventional treatment while the other 27 were asymptomatic and were regularly followed only. Of the 38 patients also studied during disease course, 28 had IgG- and 10 IgA-MM; the median number of disease fluctuation events studied (remision-relapse) was 3 (range 2–9). Patients' median follow-up was 31 months. HLC analyses were performed on a Dade Behring BN™II nephelometer using polyclonal sheep antibodies (Hevylite™, Binding Site, UK) and HLCR was calculated with the involved Ig (either G or A) as numerator. HLC and HLCR values were then compared with disease parameters and survival. Statistical analysis was performed by standard methods. At patients' presentation, median monoclonal HLC IgG and IgA were 25 g/L and 32 g/L respectively while in patients with IgG-MM median IgG HLCR was 24.5 and IgA HLCR 60 in IgA-MM. HLCR was not correlated to b2-microglobulin, hypercalcemia, LDH or bone disease, FLCs or FLCR. It strongly correlated with time to treatment (p=0.0000093) and with a shorter overall survival (p=0.03) using as cut-0ff HLCR values in the upper tertile of symptomatic patients (>56 for IgG and >228 for IgA). Of patients also studied during their disease course, 25 out of 38 patients responded at least partially (PR) to first line treatment and 9 to a subsequent one; 20 relapsed and then responded again. In most patients HLCR followed disease fluctuations. It normalized in 9 patients and was below normal in 1 patient that achieved a complete (CR) or stringent response (sCR). In some patients HLCR allowed a better evaluation of disease status. In 5 thought to be responding, ηLCR remained stable or increased and disease course confirmed the fragility of the believed response. Interestingly, more than 50% of patients that received VAD showed greater reduction of the polyclonal Ig than the tumor Ig during treatment and sometimes thereafter. In conclusion, we provide evidence that HLCR correlates with survival in MM and allows, in some patients, better disease monitoring. Disclosures:Mirbahai:Binding Site Group Ltd: Employment. Bradwell:Binding Site: Equity Ownership, Patents & Royalties. Harding:Binding Site Group Ltd: Employment.

Impact of Human IgA Antibodies on Complement-Dependent Cytotoxicity Mediated by Combinations of EGF-R-Directed Antibodies

Dual combinations of non-crossblocking epidermal growth factor receptor (EGF-R)-directed monoclonal antibodies were demonstrated to effectively induce complement-dependent cytotoxicity (CDC) of tumor cells, whereas individual antibodies were ineffective. Here the modulating effects of different antibody isotypes on CDC were studied by adding them as a third antibody. Two different combinations of non-crossblocking EGF-R antibodies of human IgG1 isotype, 018/003 and 425/005, were investigated against the A431 and A1207 cell lines. As a third antibody, human IgG1, IgA1, and IgA2 isotype variants of the therapeutic EGF-R antibody 225 were employed that bind to an EGF-R epitope distinct from the other EGF-R antibodies. In this model, the human IgG1 antibody proved to further enhance CDC, whereas both IgA antibodies significantly blocked CDC. The IgG1 and IgA variants increased target opsonization at similar levels, but the isotypes differed in their effects on C1q fixation. Addition of IgG1 significantly enhanced complement factor binding on the target surface, whereas both IgA antibodies reduced complement binding. Control experiments revealed this blocking effect to be not specific to IgA antibodies, but to antibody constructs incapable of activating the complement system. Interestingly, the effects caused by the IgA2 isotype were consistently stronger than those by IgA1, which may be caused by stronger steric hindrance due to its reduced hinge flexibility. These results demonstrate that monoclonal IgA antibodies inhibit IgG-mediated complement activation in vitro and suggest that the appearance of IgA antibodies within a polyclonal immune response might inhibit complement activation in vivo.

Renal involvement of monoclonal immunoglobulin deposition disease associated with an unusual monoclonal immunoglobulin A glycan profile

A 38-year-old man was admitted to the hospital for the evaluation of proteinuria, microscopic hematuria, and monoclonal IgA-kappa gammopathy. The initial renal pathological findings showed mesangial proliferative glomerulonephritis with endocapillary proliferation, a necrotizing lesion, and cellular crescent formation accompanied by IgA1-kappa deposition in the mesangium. Neither typical immune-complex deposits nor organized-structure deposits were detected. We diagnosed the patient with monoclonal immunoglobulin deposition disease (MIDD) associated with monoclonal IgA (mIgA). After the initiation of a monthly treatment with melphalan and predonisolone (MP therapy), the patient's serum IgA levels declined, and clinical remission was ultimately achieved. The follow-up renal biopsy showed reduced IgA-kappa staining, and both the endocapillary proliferation and the necrotizing lesion had disappeared. To elucidate the mechanism of IgA deposition, we investigated the glycan profile of the patient's serum mIgA using a mass spectrometry technique. The results revealed an unusual N-glycan profile compared to that of another patient with circulating mIgA lacking renal involvement and that of a healthy control. mIgA deposition in the mesangial area is a rare disease, and the glycan profiling of MIDD with renal involvement has not been reported previously. Thus, the present case suggests that any variation in Ig glycosylation may be a step in the pathogenesis of MIDD with renal involvement and/or contribute to some cases of IgA nephropathy.

IgA pemphigus associated with monoclonal gammopathy completely resolved after achievement of complete remission of multiple myeloma with bortezomib, cyclophosphamide and dexamethasone regimen

Monoclonal gammopathy-associated IgA pemphigus is a debilitating skin disorder with inconsistent response to treatment. A 61-year-old woman with IgA pemphigus and monoclonal gammopathy of unknown significance had been treated unsuccessfully with cyclophosphamide/dexamethasone and then with rituximab. When the monoclonal gammopathy progressed to multiple myeloma, the patient received treatment with cyclophosphamide/doxorubicin/dexamethasone but there was no clinical response. Second-line therapy with a thalidomide/cyclophosphamide/dexamethasone combination led to severe exacerbation of the skin disorder. However, therapy with a combination regimen that included bortezomib, cyclophosphamide and dexamethasone resulted in complete and durable remission of multiple myeloma and IgA pemphigus. This suggests that bortezomib-based therapy is useful for the treatment of the rare dermatologic disorder associated with IgA gammopathy.

Acquired hemophilia associated with smoldering myeloma: demonstration that the monoclonal gammopathy acts as the factor VIII inhibitor

Abstract Abstract 1307 Poster Board I-329 A 44 years old woman presented with metrorrhagia. Coagulation tests revealed a prolongation of activated partial thromboplastin time up to 77 seconds (control 33 seconds) which could not be corrected by mixing with normal plasma. The factor VIII activity level was reduced to 0.06 IU/mL. The Bethesda assay demonstrated the presence of a factor VIII inhibitor at 29 Bethesda units per ml (BU/mL). Other coagulations studies were within the normal ranges. Serum protein electrophoresis revealed a monoclonal band in the gamma region. IgA kappa monoclonal gammopathy was confirmed by immunofixation. The concentration of IgA kappa was 2.1 g/L. The haemoglobin level was 13.1 g/dL, creatininemia 80 μmol/L and calcemia 2.38 mmol/L. Standard bone X Rays and MRI of spine showed no lytic bone lesions. Bone marrow aspiration showed 11% plasma cell infiltration. A diagnosis of IgA kappa smoldering multiple myeloma associated with acquired hemophilia was made. The patient initially received four Rituximab infusions without significant effect. Several episodes of moderate uterine bleedings were treated with recombinant activated Factor VII infusions. It is noteworthy that she never had any other bleeding. Finally, electrocoagulation of endometrium definitively stopped metrorrhagia despite persistence of factor VIII inhibitor activity. Given that the acquired haemophilia was no longer symptomatic and that there were no other complications associated with multiple myeloma no specific treatment of multiple myeloma was proposed. To investigate the link between the IgA kappa smoldering myeloma and the factor VIII inhibitor activity, polyclonal antibodies that are specific for IgA kappa or IgA lambda were used ex vivo. These antibodies target unique conformational epitopes located at the junctions of heavy chains and light chains of immunoglobulins (Binding Site Group Ltd). Bethesda assay was performed on patient's plasma diluted at 1/30 (patient's monoclonal IgA kappa final concentration: 0.07 g/L) mixed with either anti IgA kappa or anti IgA lambda antibodies (final concentration: 2.5 g/L). The anti factor VIII activity was decreased with addition of anti IgA kappa antibody (7.2 UB/ml) but not with anti IgA lambda antibodies (24 UB/ml). We confirmed these results by using a Polyethyleneglycol (PEG) precipitation step. Patient's plasma was mixed with anti IgA Kappa antibodies (final concentration 9.2 g/L) and PEG (3%). After centrifugation, the anti factor VIII inhibitor activity in supernatant was totally suppressed ( Acquired hemophilia is a rare autoimmune disorder in which the patients develop an autoantibody directed against coagulation factor VIII leading to a clinically significant bleeding diathesis. In 50% of the cases there is no detectable underlying disease; however in 10% of cases there is an association with an underlying malignancy, either solid or haematological. Concurrent acquired hemophilia and monoclonal gammopathy is rare and few reports have associated monoclonal proteins to factor VIII inhibition but without any in vitro confirmation. In this report, we described a case of an IgA kappa smoldering myeloma patient who presented with an acquired haemophilia and demonstrated that the IgA monoclonal protein was acting as the factor VIII inhibitor. A broad range of autoimmune phenomena frequently complicate hematologic malignancies and acquired hemophilia is considered to be the consequence of immune deregulation with usually oligoclonal anti-Factor VIII profile. Understanding of the underlying pathogenic mechanisms responsible for the acquired haemophilia could help to optimize treatment management. To the best of our knowledge, this is the first report of multiple myeloma associated acquired hemophilia with demonstration that monoclonal gammopathy could be responsible of factor VIII inhibitor activity. Disclosures Harding: The Binding Site Group Ltd: Employment. Bradwell: The Binding Site Group Ltd: Chairman.

Elevated fructosamine concentrations caused by IgA paraproteinemia in two dogs

An 8-year-old male Austrian Pinscher and a 14-year-old male Golden Retriever were presented for evaluation due to unexplainable high fructosamine values despite euglycemia and epistaxis in combination with polydipsia/polyuria, respectively. Blood analysis revealed severe hyperglobulinemia, hypoalbuminemia and markedly elevated fructosamine concentrations in both dogs. Multiple myeloma with IgA-monoclonal gammopathy was diagnosed by serum and urine electrophoresis including immunodetection with an anti-dog IgA antibody and bone marrow aspirations. Diabetes mellitus was excluded by repeated plasma and urine glucose measurements. Fructosamine values were positively correlated with globulin, but negatively correlated with albumin concentrations. These cases suggest that, as in human patients, monoclonal IgA gammopathy should be considered as a possible differential diagnosis for dogs with high fructosamine concentrations.

Secretory IgA: Arresting Microbial Pathogens at Epithelial Borders

Secretory IgA (SIgA) is the predominant class of antibody found in intestinal secretions. Although SIgA's role in protecting the intestinal epithelium from the enteric pathogens and toxins has long been recognized, surprisingly little is known about the molecular mechanisms by which this is achieved. The present review summarizes the current understanding of how SIgA functions to prevent microbial pathogens and toxins from gaining access to the intestinal epithelium. We also discuss recent work from our laboratory examining the interaction of a particular protective monoclonal IgA with Salmonella and propose, based on this work, that SIgA has a previously unrecognized capacity to directly interfere with microbial virulence at mucosal surfaces.

Abnormal Serum IgA Kappa / IgA Lambda Ratios at Maximum Response Predict Poor Progression Free Survival in Myeloma Patients.

Abstract 4879For some myeloma patients, co-migration of IgA monoclonal proteins with other proteins can make serum protein electrophoresis (SPE) densitometry inaccurate. Immunofixation (IFE) will identify the monoclonal immunoglobulins, however, it is not quantitative and requires skilful interpretation. Measurement of total IgA can also be used but has limited value when the monoclonal protein level is low. Antibodies which target the heavy and light chains of the immunoglobulin have now been developed, making it possible to quantify IgAk and IgA» in serum and to calculate the IgAk/IgA» ratio. Here we describe 2 patient groups where archived sera have been analysed to evaluate the utility of IgAk/IgA» ratios for monitoring response to treatment in myeloma patients. We also compared progression free survival (PFS) or overall survival (OS) for patients who did / did not achieve a normal IgAk/IgA» ratio at maximum response.Sequential, archived sera were analysed from 31 patients treated in the UK Myeloma VII trial (21 IgAk /10 IgA») and 27 patients treated at The Centre for Oncology and Haematology, Vienna (14 IgAκ /13 IgA»). Treatments utilised varied from combinations of cyclophosphamide, vincristine, doxorubicin and methylprednisolone with/without high-dose melphalan/stem cell rescue (UK) to VAD, Thalidomide-Dexamethasone, VMCP, DCEP with/without high dose melphalan/stem cell rescue (Vienna). Between 3 and 51 (median=6) sera were available for each patient. Serum IgAk and IgA» measurements were performed on a Siemens Dade-Behring BN™II nephelometer in UK and on a Roche Cobas Integra 800™ immunturbimeter in Vienna. IgAk/IgA» ratios were compared with a normal range derived from measurement of >100 blood donor sera. SPE and IFE were performed on a SEBIA Hydrasys™ electrophoresis system (UK) and on a SEBIA Kapillarys™ capillary electropheresis system and a Helena SPE 2000™(Vienna). IgAk/IgA» results were compared retrospectively to clinical assessment and outcome. Kaplan Meier curves and Cox regression analysis were performed using SPSS v14.0.For all 31 UK patients, changes in the IgAk/IgA» ratio were in accordance with the clinical assessments. With regard to the detection of residual disease, the IgAk/IgA» ratios remained abnormal beyond the time when SPE results had normalised and appeared approximately equal to IFE in sensitivity. The IgAk/IgA» ratio remained abnormal in all sera from the 13/31 patients who achieved a partial response (PR) or less. In 10/31 patients, SPE densitometry could not accurately quantify the monoclonal protein, either because there was no obvious monoclonal protein (5/10) or the monoclonal protein was obscured by other serum proteins (5/10). 18/31 patients achieved a complete response (CR) but 9/18 still had abnormal IgAk/IgA» ratios at this time and had a shorter progression free survival (PFS; 277 days v 912 days: p=0.01). Similarly, for the 27 Viennese patients, changes in the IgAκ/IgA» ratios were in accordance with overall clinical assessments. In 18/27 patients, where clinically determined maximum response was reached, an abnormal IgAk /IgA» was associated with a shorter overall survival (344 days v 2241 days: p=0.028).Monoclonal IgA proteins can be difficult to identify and measure using electrophoresis techniques because of their co-migration with other serum proteins. The results from this preliminary study indicate that the use of IgAk/IgA» ratios may offer an alternative technique for monitoring these myeloma patients. Furthermore, the IgAk/IgA» ratio may provide prognostic information at the point of maximum response. DisclosuresLudwig:Celgene: Honoraria; Mundipharma: Honoraria; AMGEN: Honoraria; Ortho-Biotech : Honoraria; Janssen-Cilag: Research Funding; Roche: Honoraria. Harding:The Binding Site Group Ltd: Employment. Bradwell:The Binding Site Group Ltd: Shareholder.

Acquired Hemophilia Associated with Smoldering Myeloma: Demonstration That the Monoclonal Gammopathy Acts as the Factor VIII Inhibitor.

Abstract 1307Poster Board I-329A 44 years old woman presented with metrorrhagia. Coagulation tests revealed a prolongation of activated partial thromboplastin time up to 77 seconds (control 33 seconds) which could not be corrected by mixing with normal plasma. The factor VIII activity level was reduced to 0.06 IU/mL. The Bethesda assay demonstrated the presence of a factor VIII inhibitor at 29 Bethesda units per ml (BU/mL). Other coagulations studies were within the normal ranges. Serum protein electrophoresis revealed a monoclonal band in the gamma region. IgA kappa monoclonal gammopathy was confirmed by immunofixation. The concentration of IgA kappa was 2.1 g/L. The haemoglobin level was 13.1 g/dL, creatininemia 80 μmol/L and calcemia 2.38 mmol/L. Standard bone X Rays and MRI of spine showed no lytic bone lesions. Bone marrow aspiration showed 11% plasma cell infiltration. A diagnosis of IgA kappa smoldering multiple myeloma associated with acquired hemophilia was made. The patient initially received four Rituximab infusions without significant effect. Several episodes of moderate uterine bleedings were treated with recombinant activated Factor VII infusions. It is noteworthy that she never had any other bleeding. Finally, electrocoagulation of endometrium definitively stopped metrorrhagia despite persistence of factor VIII inhibitor activity. Given that the acquired haemophilia was no longer symptomatic and that there were no other complications associated with multiple myeloma no specific treatment of multiple myeloma was proposed. To investigate the link between the IgA kappa smoldering myeloma and the factor VIII inhibitor activity, polyclonal antibodies that are specific for IgA kappa or IgA lambda were used ex vivo. These antibodies target unique conformational epitopes located at the junctions of heavy chains and light chains of immunoglobulins (Binding Site Group Ltd). Bethesda assay was performed on patient's plasma diluted at 1/30 (patient's monoclonal IgA kappa final concentration: 0.07 g/L) mixed with either anti IgA kappa or anti IgA lambda antibodies (final concentration: 2.5 g/L). The anti factor VIII activity was decreased with addition of anti IgA kappa antibody (7.2 UB/ml) but not with anti IgA lambda antibodies (24 UB/ml). We confirmed these results by using a Polyethyleneglycol (PEG) precipitation step. Patient's plasma was mixed with anti IgA Kappa antibodies (final concentration 9.2 g/L) and PEG (3%). After centrifugation, the anti factor VIII inhibitor activity in supernatant was totally suppressed (< 0.4 UB/mL). As a control, the titre was unchanged (24 BU/mL) when anti IgA Lambda were used following the same protocol. These results suggest that the IgA monoclonal protein is responsible of factor VIII inhibitor activity.Acquired hemophilia is a rare autoimmune disorder in which the patients develop an autoantibody directed against coagulation factor VIII leading to a clinically significant bleeding diathesis. In 50% of the cases there is no detectable underlying disease; however in 10% of cases there is an association with an underlying malignancy, either solid or haematological. Concurrent acquired hemophilia and monoclonal gammopathy is rare and few reports have associated monoclonal proteins to factor VIII inhibition but without any in vitro confirmation. In this report, we described a case of an IgA kappa smoldering myeloma patient who presented with an acquired haemophilia and demonstrated that the IgA monoclonal protein was acting as the factor VIII inhibitor. A broad range of autoimmune phenomena frequently complicate hematologic malignancies and acquired hemophilia is considered to be the consequence of immune deregulation with usually oligoclonal anti-Factor VIII profile. Understanding of the underlying pathogenic mechanisms responsible for the acquired haemophilia could help to optimize treatment management. To the best of our knowledge, this is the first report of multiple myeloma associated acquired hemophilia with demonstration that monoclonal gammopathy could be responsible of factor VIII inhibitor activity. DisclosuresHarding:The Binding Site Group Ltd: Employment. Bradwell:The Binding Site Group Ltd: Chairman.

Development of Recombinant Human IgA for Anticardiolipin Antibodies Assay Standardization

Controls and calibrators in autoimmune assays are typically developed from patient sera. However, the use of sera is accompanied by a number of disadvantages, such as lack of monospecificity, lack of assay comparability, and supply limitations. Ideally, the control reagent would be an antigen-specific human monoclonal antibody preparation that is defined and pure, easy to produce without any supply limitations, and of defined isotype (IgG, IgM, or IgA). The generation of antigen-specific human monoclonal antibodies has been complicated, but recent advances in development of fully human antibodies by means of in vitro antibody gene library selection has opened a way for the isolation of human antibodies to virtually any antigen, including self-antigens. Such antibodies can be converted to any isotype by gene cloning. Here we developed a set of human monoclonal IgA antibodies specific for the cardiolipin-beta2-glycoprotein 1 complex, using the HuCAL technology. We evaluated the IgA variants of those antibodies for their use as standards in IgA anticardiolipin antibody assays and compared these reagents with serum controls. Such recombinant antibodies may ultimately replace patient sera as assay control and calibration reagents.

Highs and lows of coeliac screening

Coeliac screening is one of the most frequently requested investigations in immunology laboratories. This study evaluates seven commercial anti-tissue transglutaminase (tTG) enzyme-linked immunosorbent assay (ELISA) kits across a varied population of 80 routine samples received for coeliac screening. This investigation assesses whether or not absorbance levels can be used to detect low serum IgA and whether or not raised IgA concentrations can interfere with IgA-tTG results. Sensitivity and specificity varied hugely and four out of the seven methods required cut-off value modification to attain 100% sensitivity with endomysial testing. Four of the seven kits identified low IgA samples with greater than 90% accuracy, but sensitivity dropped to 75% in others. All the kits were affected by raised serum IgA concentrations, leading to false-positive results. There was notable variation between the seven kits in respect of high IgA concentrations and positive IgA-tTG results, with concordance analysis indicating a weak linear relationship between IgA concentration and tTG value. This study concludes that there is significant variability between the commercial tTG assays in the diagnostic market. Laboratories should be aware of their kit’s limitations and may need to adjust cut-off values to maximise sensitivity. It is possible to identify IgA deficiency from the tTG values, but the ability to do this varies between manufacturers. Raised IgA levels continue to affect the specificity of IgA-tTG assays and interference by polyclonal and monoclonal IgA should be considered in samples with positive tTG and negative endomysial results.

ANTIBODI MONOKLONAL STREPTOKOKUS MUTANS 1(c) 67 kDa DALAM PASTA GIGI BAHAN DASAR UNTUK MENGHAMBAT PERTUMBUHAN STREPTOKOKUS MUTANS

Prevention of dental caries is still continuing, because the prevalency caries is high. There was many methods to prevent dental caries etc. dental education, oral hygiene, special method on tooth brushing, water fluoridation, fissure sealant and later on the passive immunization with monoclonal antibodies. The purpose of this study was to investigate about monoclonal antibodies IgA, IgG 1 and IgG 3 against Streptococcus mutans 1(c) in basic paste for inhibiting the growth Streptococcus mutans . The monoclonal antibodies were IgA Ab, IgG 1 Ab and IgG 3 Ab. Formula basic paste from PT “X” contained Aqua, Sorbitol, Nipagin, Dicalcium Phosphat, Titanium Dioxid, Sodium Carboxyl Methyl Sel. Sodium Lauryl Sulfate and Sacarin. Basic paste was mixed with monoclonal antibodies IgA, IgG 1 and IgG 3 in room temperature (27 o C) then to investigate zone of inhibition from these tooth paste with Wistreich and Lechman methods. The data obtained in this study was analyzed with one way Anova and LSD. The result showed that there was a significant differences between basic paste with or without monoclonal antibodies. From the data analyzed in this study it can be concluded that monoclonal antibodies against S. mutans 1( c ) could be formulation with basic paste.

Prevention of the post-chemotherapy relapse of tuberculous infection by combined immunotherapy

We report that a recently developed combined immunotherapy (CIT) has the capacity to prevent a spontaneous relapse of replicating Mycobacterium tuberculosis bacilli in the lungs of BALB/c, C57Bl/6 or C3H/HeJ strains of mice, following 4weeks of non-sterilising treatment with isoniazid and rifampicin. The CIT regimen, represented by recombinant IFNgamma, anti-alpha crystalline monoclonal IgA antibody and IL-4 neutralizing polyclonal antibody, reduced the 8-week relapse of viable bacterial counts in the lungs most significantly, when CIT was inoculated during the 5th week post infection, i.e. during the 3rd week of chemotherapy. Although CIT enhanced lung granuloma area, nitric oxide, cytokine and chemokine levels in lung washings significantly, these could not be directly associated with the beneficial effect of CIT on the degree of relapse in the lungs. These results represent a proof-of-principle, that the described CIT, when combined with chemotherapy, could have potential for future development of a shorter regimen of tuberculosis treatment.

Inhibition of Salmonella enterica Serovar Typhimurium Motility and Entry into Epithelial Cells by a Protective Antilipopolysaccharide Monoclonal Immunoglobulin A Antibody

Secretory immunoglobulin A (SIgA) antibodies directed against the O antigen of lipopolysaccharide (LPS) are the primary determinants of mucosal immunity to gram-negative enteric pathogens. However, the underlying mechanisms by which these antibodies interfere with bacterial colonization and invasion of intestinal epithelial cells are not well understood. In this study, we report that Sal4, a protective, anti-O5-specific monoclonal IgA, is a potent inhibitor of Salmonella enterica serovar Typhimurium flagellum-based motility. Using video light microscopy, we observed that Sal4 completely and virtually instantaneously "paralyzed" laboratory and clinical strains of serovar Typhimurium. Sal4-mediated motility arrest preceded and occurred independently of agglutination. Polyclonal anti-LPS IgG antibodies and F(ab)(2) fragments were as potent as was Sal4 at impeding bacterial motility, whereas monovalent Fab fragments were 5- to 10-fold less effective. To determine whether motility arrest can fully account for Sal4's protective capacity in vitro, we performed epithelial cell infection assays in which the requirement for flagellar motility in adherence and invasion was bypassed by centrifugation. Under these conditions, Sal4-treated serovar Typhimurium cells remained noninvasive, revealing that the monoclonal IgA, in addition to interfering with motility, has an effect on bacterial uptake into epithelial cells. Sal4 did not, however, inhibit bacterial uptake into mouse macrophages, indicating that the antibody interferes specifically with Salmonella pathogenicity island 1 (SPI-1)-dependent, but not SPI-1-independent, entry into host cells. These results reveal a previously unrecognized capacity of SIgA to "disarm" microbial pathogens on mucosal surfaces and prevent colonization and invasion of the intestinal epithelium.

An acute model for IgA-mediated glomerular inflammation in rats induced by monoclonal polymeric rat IgA antibodies.

An acute model for IgA-mediated glomerular inflammation in rats was induced by the in situ deposition of IgA directly into the glomerular mesangium. F(ab')2 anti-Thy1 MoAb was used to anchor an antigen, DNP (2,4-dinitrophenol), in the glomeruli of rats. Subsequent infusion of rat polymeric (p-) or monomeric (m-) IgA MoAb with specificity for DNP resulted in mesangial deposition of IgA in both groups of rats. However, acute proteinuria was observed only in p-IgA-treated rats and not in PBS- or m-IgA-treated rats. Immunofluorescence analysis revealed deposition of C3 in an identical pattern to that of IgA in the glomeruli of p-IgA-treated rats. No mesangial deposits of C4 or C1q were seen in these animals. Rats receiving m-IgA or PBS displayed no detectable C3, C4 or C1q deposition. The amount of proteinuria in p-IgA-treated rats was related to the amount of deposited C3. The presence of intraglomerular monocytes was only observed 2 days after p-IgA injection. By light microscopy, aneurysm formation, mesangial hypercellularity and matrix expansion were seen only in p-IgA-treated rats. However, by 37 days post-injection complete resolution of the lesions was observed. No histological renal changes were observed in PBS- or m-IgA-treated rats. In conclusion, an acute form of IgA-mediated nephritis in rats was induced by p-IgA but not by m-IgA. This reproducible model provides a basis for further study into the mechanisms of IgA-mediated glomerular inflammation.

Immunoglobulin A pemphigus associated with immunoglobulin A gammopathy and lung cancer

Immunoglobulin (Ig)A pemphigus is a rare disease marked by a vesiculopustular eruption characterized by intercellular IgA deposition in the epidermis. It has clinical and histopathological heterogeneity and encompasses two subgroups: subcorneal pustular dermatosis type and intraepidermal neutrophilic IgA dermatosis type. IgA pemphigus has been rarely associated with monoclonal IgA paraprotein, myeloma and B-cell lymphoma in the past. We report the first case, to our knowledge, of a 47-year-old male patient with a subcorneal pustular dermatosis type of IgA pemphigus associated with IgA gammopathy and lung cancer.

Recombinant Human Monoclonal IgA Antibody Against CEA to Recruit Neutrophils to CEA-Expressing Cells

IgA is able to trigger antibody-dependent cellular cytotoxicity (ADCC) by recruiting neutrophils expressing the Fc receptor for the CHalpha chain. We herein describe the preparation of a human recombinant anti-CEA IgA antibody to kill carcinoembryonic antigen (CEA)-expressing tumor cells via ADCC by neutrophils. A single chain Fv (scFv) gene was constructed using a cDNA library of a hybridoma clone that produces a human anti-CEA monoclonal IgG4 (C2-45). The scFv gene, linked with a CHalpha gene, was inserted into the pBK283 vector, which was cotransfected into BmN4 insect cells with the wild-virus BmNPV. After cloning and amplification, the recombinated virus was injected into silkworm larvae. The resulting human recombinant IgA, designated as 45scFvLCHalpha, was purified from hemolymph by Ni-affinity chromatography and characterized by ELISA, Western blotting, and the ADCC assay. 45scFvLCHalpha with an IgA antigenicity was bound to CEA and showed effective killing of the CEA-expressing cells in the presence of IFN-gamma-activated neutrophils. These data suggest the recombinant anti-CEA IgA antibody recruiting neutrophils maybe a useful means for the antibody-based immunotherapy of human CEA-expressing tumors.

Subcorneal pustular dermatosis: 50 years on

We review the key developments in our understanding of subcorneal pustular dermatosis (SCPD, also known as Sneddon-Wilkinson disease) over the past 50 years. SCPD is a rare, chronic, sterile pustular eruption that was first described by Sneddon and Wilkinson in 1956. The primary lesions are pea-sized pustules classically described as half-pustular, half-clear flaccid fluid blisters. Histologically the salient feature is a subcorneal accumulation of neutrophils, suggesting the presence of chemoattractants such as tumour necrosis factor (TNF)alpha in the uppermost epidermis. However, to date its exact pathophysiology is unknown. Cases in association with pyoderma gangrenosum, benign monoclonal IgA gammopathy and multiple myeloma are well documented. There are anecdotal reports of SCPD associated with other internal malignancies such as chronic lymphocytic leukaemia, thymoma, apudoma and epidermoid carcinoma of the lung. The treatment of choice is dapsone. Therapeutic alternatives include retinoids, phototreatment with psoralen ultraviolet (UV) A, broadband or narrow band UVB and corticosteroids. Anecdotal uses of tacalcitol, ketoconazole, azithromycin, tetracycline, minocycline, vitamin E, ciclosporin, colchicine, mizoribine, mebhydrolin, infliximab and adalimumab with mycophenolate mofetil have all been reported.

Protective Anti‐Lipopolysaccharide Monoclonal IgA Antibodies Interferes with Salmonella Motility and Invasion of Epithelial cells, Independent of Bacterial Agglutination

Mucosal immunity to enteric bacterial pathogens, including Salmonella enterica serovar Typhimurium (S.Typhimurium) and Shigella flexneri is mediated by secretory IgA (SIgA) antibodies directed against bacterial lipopolysaccharide (LPS). It has long been assumed that these antibodies protect intestinal epithelial cells from microbial colonization and invasion by simply promoting bacterial agglutination and entrapment in mucus. In this study, we challenge this paradigm by demonstrating that protective, serotype‐specific, anti‐LPS monoclonal IgA antibodies (IgA mAbs) at sub‐agglutinating concentrations interfere with bacterial motility and type three‐mediated secretion of proteins necessary for invasion of epithelial cells. These effects occurred within minutes of antibody exposure and at IgA concentrations sufficient to coat less than 5% of the bacterial LPS. By scanning electron microscopy we observed anti‐LPS IgA mAbs induced dramatic morphological changes in the surface topology of the bacterial outer membranes, suggesting motility arrest and inhibition of type‐three secretion may be the result of physical changes in the LPS layer. These data reveal a novel and previously unrecognized capacity of SIgA to effectively “disarm” microbial pathogens on mucosal surfaces and render them unable to colonize or invade host intestinal epithelial cells. This work was supported by New Investigator funds from the Wadsworth Center, New York State Department of Health.