IgM Fractions Research Articles

Clinical Use and Therapeutic Potential of IVIG/SCIG, Plasma-Derived IgA or IgM, and Other Alternative Immunoglobulin Preparations.

Intravenous and subcutaneous immunoglobulin preparations, consisting of IgG class antibodies, are increasingly used to treat a broad range of pathological conditions, including humoral immune deficiencies, as well as acute and chronic inflammatory or autoimmune disorders. A plethora of Fab- or Fc-mediated immune regulatory mechanisms has been described that might act separately or in concert, depending on pathogenesis or stage of clinical condition. Attempts have been undertaken to improve the efficacy of polyclonal IgG preparations, including the identification of relevant subfractions, mild chemical modification of molecules, or modification of carbohydrate side chains. Furthermore, plasma-derived IgA or IgM preparations may exhibit characteristics that might be exploited therapeutically. The need for improved treatment strategies without increase in plasma demand is a goal and might be achieved by more optimal use of plasma-derived proteins, including the IgA and the IgM fractions. This article provides an overview on the current knowledge and future strategies to improve the efficacy of regular IgG preparations and discusses the potential of human plasma-derived IgA, IgM, and preparations composed of mixtures of IgG, IgA, and IgM.

Generation of Anti-Boa Immunoglobulin Antibodies for Serodiagnostic Applications, and Their Use to Detect Anti-Reptarenavirus Antibodies in Boa Constrictor.

Immunoglobulins (Igs), the key effectors of the adaptive immune system, mediate the specific recognition of foreign structures, i.e. antigens. In mammals, IgM production commonly precedes the production of IgG in the response to an infection. The reptilian counterpart of IgG is IgY, but the exact kinetics of the reptilian immune response are less well known. Boid inclusion body disease (BIBD), an often fatal disease of captive boas and pythons has been linked to reptarenavirus infection, and BIBD is believed to be immunosuppressive. However, so far, the study of the serological response towards reptarenaviruses in BIBD has been hampered by the lack of reagents. Thus we set up a purification protocol for boa constrictor IgY and IgM, which should also be applicable for other snake species. We used centrifugal filter units, poly ethylene glycol precipitation and gel permeation chromatography to purify and separate the IgM and IgY fractions from boa constrictor serum, which we further used to immunise rabbits. We affinity purified IgM and IgY specific reagents from the produced antiserum, and labelled the reagents with horseradish peroxidase. Finally, using the sera of snakes with known exposure to reptarenaviruses we demonstrated that the newly generated reagents can be utilised for serodiagnostic purposes, such as immunoblotting and immunofluorescent staining. To our knowledge, this is the first report to show reptarenavirus-specific antibodies in boa constrictors.

Antibody repertoire of atlantic salmon: Ig-sec or high-throughput sequencing of the variable region of IgM heavy chain

Immunoglobulins (Ig) play a key role in protection of fish against bacterial and viral pathogens. Atlantic salmon has three types of Ig (M, D and T), of which IgM is the main actor of the adaptive humoral immunity. Ig heavy chains are encoded with two loci and their variable regions (VR) are produced by somatic recombination of J, D and V genes. While immunological methods detect specific antibodies, parallel sequencing of VR (Ig-seq) provides a comprehensive survey of the repertoire. We established a protocol for construction of IgM-VR libraries for Illumina sequencing that captures all transcripts and a bioinformatics pipeline Iguana (Ig Universe Analyses). The clonotypes (CLT) of transcripts are identified by combinations of J, D and V genes and sequences of J(D)V junctions or CDR3 – the highly variable region, which is responsible for antigen binding. We also developed metrics for characterization of the repertoire. The size of salmon IgM-V repertoire is relatively large – to this end, more than 1.4 million different CLT have been detected in 18.5 million transcripts. An intriguing finding was a large size of the public compartment, i.e. CLT shared by at least several individuals. While a vast majority of CLT was unique, all of the most frequent transcripts were public and comprised a substantial fraction of IgM; i.e. 5.7% CLT, which were found in at least two fish encoded 46.2% of all transcripts. The origin, properties and functional roles of public CLT in Atlantic salmon are unknown. It is possible that they are similar to natural polyreactive antibodies of warm blood animals, which are regarded as a part of the first line of defence against pathogens. Ig-seq was applied to monitor responses to viral infections (PMCV, PRV and SAV) and changes in course of Atlantic salmon development. Enhanced antibody production or clone expansion assessed by increased cumulative frequencies of most abundant CLT and Gini index was noticed after viral infection in the head kidney and to lesser extent in peripheral blood, but not in the spleen. Until present sequencing of IgM-V has not produced evidence for hyper mutations and affinity maturation. Communality or fraction of public CLT among the leaders showed markedly different changes after infection with SAV and PRV being respectively low in the former and high in the latter case. Intriguingly, salmon infected with PRV showed high resistance to secondary infection with SAV and serum of PRV infected fish was able to neutralize SAV in vitro. Results suggest that public IgM transcripts may encode polyreactive antibodies that provide an unspecific protection.

The expanding spectrum of HCV-related cryoglobulinemic vasculitis: a narrative review.

Cryoglobulinemic vasculitis (CV) is a small-to-medium-vessel vasculitis that appears in 10-15% of patients chronically infected with hepatitis C virus (HCV). The classic symptom triad of CV, purpura/asthenia/arthralgia, is accompanied by clinical features that include glomerulonephritis, neuropathy, interstitial pneumonitis, and cardiomyopathy, ranging in their severity from mild to life threatening. The risk of developing non-Hodgkin lymphoma is also higher. The cumulative 10-year survival rate of CV patients is significantly lower than in the age- and sex-matched general population, with death typically caused by nephropathy, malignancies, liver involvement, and severe infections. Unfailing serological stigmata include both a cryoglobulin IgM fraction with rheumatoid factor activity and decreased complement C4 levels. On peripheral B cells, the expression of the CD81 B cell receptor is reduced while that of the CD19 receptor is increased. A monoclonal B cell lymphocytosis develops in almost one-third of patients. HCV-related proteins (but not HCV-RNA genomic sequences) can be detected on biopsy samples by immunofluorescence and immunohistochemistry and involve the vessel lumen, vessel walls, and the perivascular spaces of the skin, kidney, and peripheral nerves, supporting the pathogenetic role of HCV in the onset of a widespread microvasculitis. Based on the demonstration of HCV infection in the large majority of CV patients, a therapeutic regimen consisting of once-weekly pegylated interferon-α and the daily administration of ribavirin results in a sustained virologic response in ~50% of patients. In those with refractory and relapsing disease, addition of the anti-CD20 monoclonal antibody rituximab has significantly increased the overall response rates. The extension to CV of latest-generation direct-acting antivirals, strikingly successful in non-CV HCV-positive patients, has yielded high complete response rates according to the few studies published thus far.

Recombinant carp IL-4/13B stimulates invitro proliferation of carp IgM(+) B cells.

Teleost IL-4/13B is a cytokine related to mammalian IL-4 and IL-13, of which hitherto the function had not been studied at the protein level. We identified an IL-4/13B gene in common carp (Cyprinus carpio) and expressed the recombinant protein (rcIL-4/13B). RcIL-4/13B was shown to stimulate proliferation of IgM(+) B cells, because after four days of stimulation the IgM(+) fraction of carp kidney and spleen leukocytes had formed many cell colonies, whereas such colonies were not found in the absence of rcIL-4/13B stimulation. After nine days of incubation with rcIL-4/13B these cells had proliferated to more than 3-to-7-fold higher numbers when compared to untreated cells. The proliferating cells contained a majority of IgM(+) cells but also other cells, as indicated by FACS and RT-PCR analyses. The important conclusion is that in fish not only IL-4/13A has B cell stimulating properties, as a previous publication has shown, but also IL-4/13B.

Functional characterization of IgM+ B cells and adaptive immunity in lumpfish (Cyclopterus lumpus L.)

The innate immune responses in lumpfish (Cyclopterus lumpus L.) have been shown to be functional, but little is currently known about the B cells, immunoglobulins or adaptive immune responses in this species. We have used anti-IgM antiserum to isolate B cells and compared them morphologically and functionally with other cell types. The fraction of IgM+ cells among isolated peripheral blood leukocytes (PBL), head kidney leukocytes (HKL) and spleen leukocytes (SL) was in the range of 40%, 12% and 34%, respectively. The IgM+ B cells had high phagocytic ability and were the predominant phagocytes in blood with higher capacity than IgM+ B cells in HKL. Interestingly, among PBL, the most potent phagocytes were, in addition to monocytes, some small agranular uncharacterized IgM− cells. The IgM+ B cells were positive for acid phosphatases (AcP), but negative for myeloperoxidase (MPO). Neutrophils were positive for MPO, while monocytes/macrophages and dendritic-like cells stained negatively. Monocytes/macrophages and the small, agranular IgM− cells stained most strongly positive for AcP corresponding to their high phagocytic capacity. Further, the ability to produce specific antibodies upon immunization verified adaptive immunity in the species.The high proportion of phagocytic IgM+ B cells and their phagocytic ability indicate a significant role of phagocytic B cells in lumpfish innate immunity. The present analyses also give strong indications that vaccination and immunostimulation of farmed lumpfish can be used to prevent disease and mortality caused by pathogenic organisms.

A temporal switch in the germinal center determines differential output of memory B and plasma cells (IRC3P.468)

Abstract Though memory B cells (MBCs) and long-lived plasma cells (LLPCs) are both thought to derive from the germinal center (GC) reaction, there is little insight into or agreement about the signals that control differentiation to one cell type or another. By performing BrdU pulse-labeling studies, GC disruption experiments and V gene sequencing, we found that the generation of these cell types is actually temporally controlled and separated during the immune response. We report that MBCs mainly derive from early GCs (much before GC peak size), while more affinity matured LLPCs are predominantly formed during late GCs - long after its peak in size. Based on these findings, we propose a new model that the GC response undergoes a temporal switch, functioning quite differently at early and late stages. Therefore the generation of MBCs and LLPCs is the consequence of a general shift in GC output over time rather than the result of specific instructive signals that are selectively delivered to GC B cells at any given time during the response. We also present direct evidence that a large fraction of long-lived IgM+ MBC, and even some IgG+ MBC, are formed at very early time points, prior to the existence of detectable GCs. The knowledge of when specific long-lived immune-effector cells are generated during an immune response has strong implications for vaccine design and understanding long-term pathogen immunity.

Autoantibodies to erythropoietin receptor in patients with immune‐mediated diseases: relationship to anaemia with erythroid hypoplasia

The prevalence, clinical associations and pathogenic role of newly identified autoantibodies to the erythropoietin receptor (EPOR) in patients with anaemia were investigated. Sera from 203 patients with immune-related or chronic kidney diseases were screened for anti-EPOR antibodies by enzyme-linked immunosorbent assay, and antibody specificity was evaluated by immunoprecipitating EPOR from AS-E2 cells using purified immunoglobulin (Ig) fractions. In addition, the pathogenic role of anti-EPOR antibodies was determined by examining their inhibitory effects on AS-E2 cell proliferation. Clinical findings were compared between patients with and without anti-EPOR antibodies, in all patients and those with systemic lupus erythematosus (SLE). Serum anti-EPOR antibodies were detected in 52 patients. Purified IgG or IgM fractions from anti-EPOR antibody-positive sera immunoprecipitated EPOR and inhibited the EPO-dependent proliferation of AS-E2 cells in a dose-dependent manner. Anti-EPOR antibodies were associated with low haemoglobin concentrations and reticulocytopenia in all patients enrolled and those with SLE. Further, there was a negative correlation between the levels of anti-EPOR antibodies and the number of bone marrow erythroblasts in patients who underwent bone marrow examinations. These findings suggest that EPOR autoantibodies are present in a subset of patients with anaemia and that impaired erythropoiesis can be mediated by anti-EPOR antibodies, which functionally neutralize EPO activity.

Patients with Chronic Lymphocyte Leukemia (CLL) Have Naturally Occurring Antibodies Against the Receptor Tyrosine Kinase (ROR1)

Abstract 1771 Background:The receptor tyrosine kinase ROR1 is a tumor-associated antigen which is highly expressed by leukemic cells from patients with chronic lymphocytic leukemia (CLL) and in a variety of other hematologic malignancies. ROR1 is not expressed in normal adult tissues with the exception for adipose cells and a subset of normal B cells during early ontogeny. ROR1 has important functions during embryogenesis. Thus ROR1 is considered an oncofetal antigen (Fakuda T et al. PNAS, 105, 3047, 2008). Aims:As ROR1 in adults represents a neo-antigen, we analysed spontaneously induced humoral immune response against ROR1 in CLL patients with non-progressive and progressive disease. Methods:Antibodies against ROR1 was analysed in 23 CLL patients with non-progressive (n=15) or progressive (n=8) diseases, as well as in 13 healthy donors by ELISA and Western blot, using a recombinant ROR1 protein. The recombinant protein contained 101 amino acids of the KNG domain of the extracellular ROR1 part equipped with a GST tag. Immunoprecipitated ROR1 from CLL cells was also used as an antigen to further define the presence of anti ROR1 antibodies. Highly purified IgG and IgM from sera of CLL patients as well as healthy donors were used in flowcytometry for staining of CLL cells, expressing the ROR1 molecule. Regulatory T cells frequency (CD4+CD25+FoxP3+ and CD8+CD25+FoxP3+) was determined by flowcytometry. The presence of soluble ROR1 molecule was also investigated in 9 CLL patients. To detect soluble ROR1 from 9 CLL patients, serum was passed through HiTrap protein G, A and IgM columns to delete the pre existing IgM and IgG antibodies. The effluent sera were concentrated by Amicon Ultra 50000 Dalton tubes and concentrated serum from each patient was analysed by Western blotting to detect ROR1 molecules, using goat and rat anti ROR1 antibodies. Results:In 3 out of the 23 CLL patients anti-ROR1 antibodies were detected by ELISA and Western blot using the recombinant ROR1 protein (containing only the KNG domain), all these 3 patients had non-progressive disease. Using immunoprecipitated ROR1 protein from CLL cells as antigen, revealed the presence of anti ROR1 antibodies in all non-progressive CLL patients (15/15) by Western blot. Antibodies were also detected in patients with progressive disease (5/8) but at a lower concentration, compared to non-progressive patients. Flowcytometry staining of ROR1 expressing CLL cells using whole sera of patients and healthy donors as well as the purified IgG and IgM fractions, revealed the presence of both IgG and IgM antibodies from CLL patients reacting with ROR1 expressing CLL cells. Both IgM and IgG antibodies were detected in patients with non-progressive disease, but in progressive CLL patients only IgM antibodies were found. None of the sera of healthy donors and the purified IgG and IgM antibodies showed reactivity with CLL cells.Analysis of the frequency of regulatory T cells revealed a significantly higher frequency of CD4+CD25+FoxP3+ cells in CLL patients compared to healthy donors. A higher frequency of these cells was observed in progressive compared to non-progressive CLL patients, but the differences were not statistically significant. Soluble ROR1 molecules of 105 and 130 KDa molecular weights were detected in sera of CLL patients (n=9) by goat (polyclonal) and rat (monoclonal) anti ROR1 antibodies using Western blot. Conclusion:Our results showed that anti ROR1 antibodies are present in the sera of CLL patients, particularly in those with non-progressive disease as compared to progressive disease. These antibodies were both of IgM and IgG isotypes. The main source of soluble ROR1 molecules is probably apoptotic or necrotic cells and may contribute to the induction of a humoral anti-ROR1 response in addition to lysed tumor cells taken up by dendritic cells. Together with our previous results, exploring the presence of spontaneous T cell immunity in CLL patients, the current results confirm that ROR1 may be an immunodominant antigen in CLL patients. Active immunotherapy using ROR1 as an antigen might be an interesting approach to test in the clinic. Disclosures:Hojjat-Farsangi:Kancera AB: Equity Ownership. Daneshmanesh:Kancera AB: Equity Ownership. Mellstedt:Kancera AB: Equity Ownership.

Autoimmune reactivity of IgM acquired after oxidation

ObjectivesRedox-reactive antibodies, mainly of the IgG class, gained a wide area of interest after their autoimmune reactivity was revealed following the application of chemical and physiological oxidants. In this study, we examined the susceptibility of IgMs to oxidation and evaluated their binding to the autoantigens important in some autoimmune diseases.MethodsIgM and IgG fractions, isolated from healthy individuals’ sera, were oxidized using direct electric current or physiological oxidant hemin. Specificities towards beta-2-glycoprotein I (β2-GPI), cardiolipin (CL), and rheumatoid factor were evaluated with the enzyme-linked immunosorbent assays (ELISAs). Post-translational modification was investigated by 2,4-dinitrophenylhydrazine reaction.ResultsElectrochemically oxidized IgM fractions exhibited altered immunoreactivity – low to medium titers in anti-CL and low positive titers in anti-β2-GPI ELISA but exhibited no rheumatoid factor reactivity. Oxidized IgG and IgM fractions exhibited 2.5- and 5-fold increase in the carbonyl content, respectively.DiscussionAn increase in the carbonyl content along with increased immunoreactivity after oxidation suggests modifications of the IgM paratopes. These results point towards possible modifications of native IgMs to their autoimmune state despite the fact that IgMs were less susceptible to oxidation than IgGs. The importance of an individual's redox status in maintenance of autoimmune reactions was emphasized by in vitro diagnostic tests.

A 40-year-old man with recurrent fainting, hypotension, lower limb edema and oliguria with body weight gain and secondary erythrocytosis

Dr. Berti de Marinis, Dr. Allemand: In November 2009, a 40-year-old man was urgently hospitalized after orthostatic syncope secondary to severe hypotension. Three days before admission, he had experienced profuse sweating and contracted diuresis after intense effort (a long run to reach a stadium to see a rugby match) followed by the onset of fever, sore throat and a flu-like syndrome associated with palpitations. The past medical history was relevant to hypothyroidism treated with hormone replacement therapy, and previous episodes of abdominal pain due to bilateral kidney stones, treated with lithotripsy. Physical examination on arrival at the emergency department showed pallor, body mass index 32.93, body weight 102 kg, redness of the pharynx, hardening of the calves with no signs of edema. The vital signs were: temperature 37.6 C, blood pressure (BP) 92/54 mmHg, heart rate (HR) 108 beats per min, respiratory rate 16 breaths per min, oxygen saturation by pulse oximetry 100% without oxygen supplementation. Laboratory analyses revealed: hemoglobin (Hb) 227 g/l, hematocrit (Ht) 64.3%, white blood cells (WBC) 18.84 9 10/l, neutrophils (N) 14.71 9 10/l, platelets 289 G/L, serum creatinine 151 lmol/l, blood urea nitrogen (BUN) 10.5 mmol/l, urine specific gravity 1084, and C-reactive protein (CRP) 13.82 mg/l, troponin 0.01 lg/l. Findings on electrocardiography, chest radiography and echocardiography were normal, there were no signs of deep vein thrombosis on compression ultrasound (CUS), and only a modest splenomegaly was found on abdominal ultrasound (diameter 12.5 cm). Given the increase in d-dimer levels (508 ng/l), pulmonary embolism was excluded by multislice computed tomography. The patient was administered 2.5 l of saline, and was admitted to our internal medicine ward with a diagnosis of ‘‘hemoconcentration secondary to dehydration’’. He arrived with sustained hypotension, tachycardia and fainting, prompting the intravenous infusion of more fluids (1,000 ml of saline and 500 ml of colloids), followed by a gradual return to normal blood pressure values and adequate diuresis. Urea, creatinine and urine specific gravity also returned to normal. Further laboratory and instrumental analyses performed on admission to the ward showed normal levels of IgG, IgA, IgM and complement fractions, and a low level of serum protein (49.2 g/l) with a normal albumin concentration, but no measurable IgG k monoclonal gammopathy with elevated b2 microglobulin (2.65 mg/l) and the absence of Bence Jones protein. Any presence of infection, renal insufficiency, nephrotic/nephritic syndrome or hepatic or cardiac insufficiency was ruled out. The hematocrit remained high (about 60%) despite the return to a normal plasma volume. Therapeutic phlebotomy was performed to prevent any complications of hyperviscosity with a return to a normal hematocrit. Erythropoietin assay was normal, and the search for the V617F Jak2 mutation was negative, excluding polycythemia vera. G. Berti de Marinis I. Bertozzi E. Allemand F. Tezza M. L. Randi F. Fabris (&) Department of Medical and Surgical Sciences, Padua University Medical School, via Giustiniani 2, 35128 Padova, Italy e-mail: fabrizio.fabris@unipd.it

Characterization of Anti- Salmonella enterica Serotype Typhi Antibody Responses in Bacteremic Bangladeshi Patients by an Immunoaffinity Proteomics-Based Technology

Salmonella enterica serotype Typhi is the cause of typhoid fever and a human-restricted pathogen. Currently available typhoid vaccines provide 50 to 90% protection for 2 to 5 years, and available practical diagnostic assays to identify individuals with typhoid fever lack sensitivity and/or specificity. Identifying immunogenic S. Typhi antigens expressed during human infection could lead to improved diagnostic assays and vaccines. Here we describe a platform immunoaffinity proteomics-based technology (IPT) that involves the use of columns charged with IgG, IgM, or IgA antibody fractions recovered from humans bacteremic with S. Typhi to capture S. Typhi proteins that were subsequently identified by mass spectrometry. This screening tool identifies immunogenic proteins recognized by antibodies from infected hosts. Using this technology and the plasma of patients with S. Typhi bacteremia in Bangladesh, we identified 57 proteins of S. Typhi, including proteins known to be immunogenic (PagC, HlyE, OmpA, and GroEL) and a number of proteins present in the human-restricted serotypes S. Typhi and S. Paratyphi A but rarely found in broader-host-range Salmonella spp. (HlyE, CdtB, PltA, and STY1364). We categorized identified proteins into a number of major groupings, including those involved in energy metabolism, protein synthesis, iron homeostasis, and biosynthetic and metabolic functions and those predicted to localize to the outer membrane. We assessed systemic and mucosal anti-HlyE responses in S. Typhi-infected patients and detected anti-HlyE responses at the time of clinical presentation in patients but not in controls. These findings could assist in the development of improved diagnostic assays.

Imatinib Mesylate Treatment Increases Lymphoplasmocytoid Cells through SDF-1 and BMP4/7 Production in the Bone Marrow of Patients with Chronic Myelogenous Leukaemia: Relationship with Clinical/Haematological Response.

Abstract 3263Poster Board III-1▪▪This icon denotes an abstract that is clinically relevant.In the last years, tyrosine kinase inhibitors, as imatinib mesylate (Gleevec, Novartis, formerly known as STI571) are confirmed the first line treatment of Chronic Myelogenous Leukemia (CML) and of a rare form of gastroenteric stromal cancer. In the latter case, the response to the drug in vivo is mainly due to immunocompetent cells, able to produce cytokines with antineoplastic activity. In this study, 30 CML patients, prior and during treatment with imatinib mesylate, underwent bone marrow (BM) aspirates every 6 months to assess morphologic phenotypic, cytogenetic and biomolecular patterns, compared to peripheral blood. Plasma from BM and peripheral blood (PB) was also tested for cytokines able to induce B lymphocytes differentiation, such as interleukin IL-4, IL-6 (whose receptor is CD126), IL-3, IL-10 or IL-21 (by ELISPOT and real time polymerase chain reaction) togheter with the expression of MCP-1, SDF-1, BMP4, BMP7, IP-10 and IL-8 . We report that in 24 out of 30 CML patients a significant increase in the percentage of BM lymphoplasmocytoid cells was observed upon treatment with imatinib mesylate, with >10% (range 8–12%) of CD20+CD126+cells. Among this population, two third of cells coexpressed IgM and one third was IgD+, whereas a lower fraction of IgM+CD126+CD20- (3–4%) or IgD+CD126+CD20- (2–3%) cells was detected , too. In all these patients SDF1 increased in the BM plasma after imatinib (from 10–80pg/ml to 150–450pg/ml) and its receptor CXCR4 was up-regulated on CD20+CD126+cells. In some cases also IP-10 and its receptor CXCR3 were up-.regulated. No significant increase in transcription and secretion of IL-3, IL-4, IL-6, IL-10, IL-21, IL-8 or MCP1 were observed. The lasting 6 patients had<5% of CD20 +CD126+ lymphocytes (range2-4%), 2/3 coexpressing IgM and 1/3 coexpressing IgD. Every patients with increased number of CD126+ B lymphocytes achieved hematologic remission, most of them complete cytogenetic and molecular remission. On the other hand, among the patients with low or undetectable CD20+CD126+cells, only 4 obtained hemathological remission and none of them stable cytogenetyc and molecular remission. In 4 patients relapsed after six months of treatment, the fraction of BM CD20+CD126+ lymphocytes decreased from 11% and 8% to 7 and 5%, respectively, with undetectable IgM+ CD126+CD20- or IgD+ CD126+CD20- cells. The increased production of SDF-1,(partially mediated by production of BMP4 and BMP7), following imatinib administration might increase BM lymphoplasmocitoid cells, thanks to the double proliferative/chemotactic effect of the cytokine on B cells, with redistribution and in situ differentiation of CD20+ CD126+ lymphocytes. These findings shed some light on the possibility that, even in CML,immunological events may play a role in disease control ; moreover they could be useful in monitoring disease outcome . Disclosures:No relevant conflicts of interest to declare.

Functional, Non-Clonal IgMa-Restricted B Cell Receptor Interactions with the HIV-1 Envelope gp41 Membrane Proximal External Region

The membrane proximal external region (MPER) of HIV-1 gp41 has several features that make it an attractive antibody-based vaccine target, but eliciting an effective gp41 MPER-specific protective antibody response remains elusive. One fundamental issue is whether the failure to make gp41 MPER-specific broadly neutralizing antibodies like 2F5 and 4E10 is due to structural constraints with the gp41 MPER, or alternatively, if gp41 MPER epitope-specific B cells are lost to immunological tolerance. An equally important question is how B cells interact with, and respond to, the gp41 MPER epitope, including whether they engage this epitope in a non-canonical manner i.e., by non-paratopic recognition via B cell receptors (BCR). To begin understanding how B cells engage the gp41 MPER, we characterized B cell-gp41 MPER interactions in BALB/c and C57BL/6 mice. Surprisingly, we found that a significant (∼7%) fraction of splenic B cells from BALB/c, but not C57BL/6 mice, bound the gp41 MPER via their BCRs. This strain-specific binding was concentrated in IgMhi subsets, including marginal zone and peritoneal B1 B cells, and correlated with enriched fractions (∼15%) of gp41 MPER-specific IgM secreted by in vitro-activated splenic B cells. Analysis of Igha (BALB/c) and Ighb (C57BL/6) congenic mice demonstrated that gp41 MPER binding was controlled by determinants of the Igha locus. Mapping of MPER gp41 interactions with IgMa identified MPER residues distinct from those to which mAb 2F5 binds and demonstrated the requirement of Fc CH regions. Importantly, gp41 MPER ligation produced detectable BCR-proximal signaling events, suggesting that interactions between gp41 MPER and IgMa determinants may elicit partial B cell activation. These data suggest that low avidity, non-paratopic interactions between the gp41 MPER and membrane Ig on naïve B cells may interfere with or divert bnAb responses.

Evidence for Increased Bone Marrow Lymphoplasmocytoid Cells and SDF1 Secretion in imatinib Treated CML. Relationship with Clinical/hemathological Response

Tyrosine kinase inhibitors, such as imatinib mesylate (Gleevec, Novartis, formerly known as STI571) are confirmed to be the first line treatment of Chronic Myelogenous Leukemia (CML) and of a rare form of gastroenteric stromal cancer. It has been reported that in the latter case, the response to the drug in vivo is mainly due to immunocompetent cells, able to produce cytokines with antineoplastic activity. In this study, peripheral blood and bone marrow of 20 CML patients were studied, prior and during treatment with imatinib, to assess morphologic, phenotypic, cytogenetic and biomolecular patterns. Plasma from BM and PB was also tested to evaluate (by ELISPOT and real time polymerase chain reaction) cytokines able to induce B lymphocytes differentiation, and/or proliferation, such as interleukin (IL)-4, IL-6 (ligand for CD126), IL-3, IL-10 or IL-21 together with chemokines MCP-1, SDF-1, IP-10 and IL-8. In 14 out of 20 CML patients a significant increase in the percentage of BM lymphoplasmocytoid cells was observed on imatinib treatment with >10% (range 8–12%) of CD20+CD126+cells. Among this population, two third of cells coexpressed IgM and one third was IgD+, moreover a lower fraction of IgM+CD126+CD20− (3–4%) or IgD+CD126+CD20− (2–3%) cells was found too. In all these patients SDF1 increased in the BM plasma after imatinib (from 10–80pg/ml to 150–450pg/ml) and its receptor CXCR4 was up-regulated on CD20+CD126+cells. In 4 patients the amount of IP-10 in BM plasma and the expression of its receptor CXCR3 were also increased. No significant modification in transcription and secretion of IL-3, IL-4, IL-6, IL-10, IL-21, IL-8 or MCP1 were observed. The lasting 6 patients had<5% of CD20 +CD126+ lymphocytes (range2–4%), 2/3 coexpressing IgM and 1/3 coexpressing IgD. Every patients with increased number of CD126+ B lymphocytes achieved hemathologic remission, 7 of them complete cytogenetic and biomolecular remission. On the other hand, among the patients with low or undetectable CD20+CD126+cells, 2 obtained only hemathological remission. In the 2 relapsed patients, BM CD20+CD126+ lymphocytes decreased from 11% and 8% to 7 and 5%, respectively, with undetectable IgM+ CD126+CD20− or IgD+ CD126+CD20− cells. The increased production of SDF-1, following imatinib administration, might increase BM lymphoplasmocitoid cells, thanks to the double proliferative/chemotactic effect of the cytokine on B cells, with redistribution and in situ differentiation of CD20+ CD126+ lymphocytes. These findings shed some light on the possibility that, even in CML, immunological events may play a role in disease control;moreover they could be useful in monitoring disease outcome.

Label-free cancer markers detection by capacitance biochip

This paper investigates technological advancements in developing reliable technologies for label free detection of cancer markers. Capacitance detection was used successfully to develop an immunosensor. Rigorous probe immobilization was implemented to obtain reliable capacitance measurements. The commonly considered alkanethiols for proteins immobilization do not allow sufficiently stable capacitance measurements. Therefore, new ethylene-glycol functionalized alkanethiols were used as more suitable materials to develop lab-on-a-chip detection of cancer markers. Results from AFM, Chemi-luminescence, Fluorescence, Cyclic Voltammetry, QCM, and capacitance measurements showed that three (ethylene-glycol) alkanethiols highly stabilized the sensing surfaces in time. The measured capacitance on the antibody layers was in the range of 60–70 nF/cm 2 and 80–115 nF/cm 2 for the antigen detection. The developed system was utilized to detect the Hepatocarcinoma marker SCCA, and the whole IgM fraction from Hepatocarcinoma patient’s serum. Electrode by electrode reproducibility within the same chip increased by one order of magnitude (variations were reduced from 30–40% down to less than 2%) using these new alkanethiols, while the reached limit in sensitivity was close to 2.43 μg/ml.

Cross-reactive idiotypes in sera from patients with leprosy, lupus and Lyme disease and from healthy individuals

Monoclonal IgM autoantibodies have previously been generated from a patient with lepromatous leprosy. Polyclonal anti-idiotypes raised against two of these monoclonal antibodies (8E7 and TH9) were used in an immunoassay to detect the presence of idiotype in human serum. The anti-idiotypes recognize different but overlapping sets of idiotypic determinants, some of which are present on antibodies which bind to Mycobacterium leprae. Sera were tested from 16 individuals with leprosy, 45 with systemic lupus erythematosus, 20 with Lyme disease, and 80 healthy subjects. Positive sera were detected in all groups (seven, two, three, and four, respectively). In most cases the serum bound to both anti-idiotypes, the idiotype being present in the IgM and/or IgG fraction. Levels of the two idiotypes varied independently of total serum IgG concentration and, in serial samples from one patient, independently of each other. The results indicate that 8E7 and TH9 may be representative of serum antibodies which are commonly expressed in leprosy, but may also be expressed in other diseases and in health; and they suggest that such serum antibodies are encoded by a widely shared set of variable region genes.

Clinical relevance of immunoglobulin M antibody fraction against hepatitis B core antigen (anti-HBc-IgM) (author's transl)

Forty-four anti-HBc positive patients with various liver diseases were followed up over 2-3 years with enzyme-immunological determinations of the IgM fraction of anti HBc (anti-HBc-IgM). Patients with acute virus hepatitis B (n = 10) had anti-HBc-IgM titres greater than or equal to 10(-3) in the acute phase. In the reconvalescent phase the titre decreased significantly but was still detectable at a level of 10(-1) two years after the onset of the disease. In patients with HBs-antigen positive chronic active hepatitis (n = 25) the anti-HBc-IgM was always detectable when HBe-antigen was positive. Patients with HBe-antigen negative chronic active hepatitis B showed various anti-HBc-IgM findings. Patients with liver diseases not due to hepatitis B virus infections (n = 9) were always anti-HBc-IgM negative. Thus with the help of anti-HBc-IgM determinations acute hepatitis B virus infection can be diagnosed in HBs-antigen negative people. On the other hand it is not suitable for differentiation between acute and chronic hepatitis B infections in HBs-antigen positive persons.

Relationship between Clinical/Hematological Response and Increase of Plamacells in the Bone Marrow of Patients with Chronic Myelogenous Leukemia Imatinib Mesylate Treatment (631).

Tyrosine kinase inhibitors, such as imatinib mesylate (Gleevec, Novartis, formerly known as STI571) are the first line treatment of Chronic Myelogenous Leukemia (CML) and of a rare form of gastroenteric stromal cancer. It has been recently reported that in the latter case, tumor cells are refractory to imatinib antiproliferative effect in vitro and the response to the drug in vivo is due to immunocompetent cells, able to produce cytokines with antineoplastic activity. In this study, 20 CML patients, prior and during treatment with imatinib mesylate, underwent bone marrow (BM) aspirates every 6 months, including: morphologic and phenotypic analysis, cytogenetic and biomolecular evaluation, compared to peripheral blood. Plasma from BM and peripheral blood was also recovered for cytokyne-chemokine dosage. We report that in 12 out of 20 CML patients a significant increase in the percentage of BM lymphoplasmocytoid cells was observed upon treatment with imatinib mesylate, with >10% (range 10–16%) of CD20+CD126+cells. Among this population, two third of cells coexpressed IgM and one third was IgD+, while a smaller fraction of IgM+CD126+CD20– (3–4%) or IgD+CD126+CD20- (2–3%) cells was also found. The lasting 8 patients had<5% of CD20 +CD126+ lymphocytes (range2–4%), 2/3 coexpressing IgM and 1/3 coexpressing IgD. All patients with increased number of CD126+ B lymphocytes underwent hematologic remission, 7 of them with complete molecular and cytogenetic remission. On the other hand, among the patients with low or undetectable CD20+CD126+cells, only 4 underwent hemathological remission and none of them displayed stable cytogenetyc and molecular remission. In two patients relapsed after six months of treatment, the fraction of BM CD20+CD126+ lymphocytes decreased from 16% and 11% to 7 and 5%, respectively, with undetectable IgM+ CD126+CD20- or IgD+ CD126+CD20- cells. These data suggest that this population of lymphoplasmocytoid B cells depends on or contribute to the pharmacological response; by the way, this phenomenon might help in monitoring the outcome of disease and the response to treatment. To check this item and understand the biochemical mechanisms substaining the observed increase in BM lymphoplasmocitoid cells on imatinib treatment, we wonder if the production of cytokines able to induce B lymphocytes differentiation, such as interleukin (IL)-4, IL-6 (whose receptor is CD126), IL-3, IL10 or IL-21 was affected by imatinib administration. To this aim, both soluble cytokines (by ELISPOT) and their mRNA (by real time polymerase chain reaction) were evaluated in the BM of these patients: moreover, the expression of MCP-1, SDF-1, IP-10 and IL-8 were also measured, to verify whether the increse in BM CD20+ CD126+ lymphocytes was due to a redistribution rather than to “in situ” differentiation. Preliminary results seem to indicate that the latter hypothesis is unlikely; in addition, when CD20+ CD126+ were increased in the BM, they also raised in the peripheral blood. These immunological events might have a role in the response to tyrosine kinase inhibitor and need further investigations.

Critical evaluation of the biological role of IgM in cerebrospinal fluid in inflammatory and other diseases of the nervous system

In a group of 10,156 patients with neurological diseases, the IgM level was assessed (using laser nephelometry) both in cerebrospinal fluid (CSF) and serum; concentration of other 17 protein fractions was also simultaneously determined: albumin, immunoglobulins, acute phase reactants, complement components, apolipoproteins, proteinase inhibitors and alpha1-microglobulin. Total protein, element counts and glucose level were also evaluated. In patients with normal CSF findings, only limited statistically significant correlations were demonstrated between IgM and other CSF protein fractions while, in the group of patients with pathological CSF findings, significant correlations were found between CSF(IgM) and other immunoglobulins, complement fractions and the rate of intrathecal synthesis of immunoglobulins. Correlations were also found between CSF(IgM) and CSF antithrombin-III and alpha1-microglobulin. Correlations between CSF(IgM) and CSF apolipoproteins support the theory of CNS tissue destruction whenever the concentration of CSF apolipoproteins is elevated. Our data substantially contribute to establishing the diagnosis in patients with neurological diseases; simultaneous measurement of a high number of CSF proteins is becoming inevitable for a reasonable assessment of the CSF Protein Status.