Residual IgM Research Articles

Oropharyngeal microbial ecosystem perturbations influence the risk for acute respiratory infections in common variable immunodeficiency.

The respiratory tract microbiome is essential for human health and well-being and is determined by genetic, lifestyle, and environmental factors. Patients with Common Variable Immunodeficiency (CVID) suffer from respiratory and intestinal tract infections, leading to chronic diseases and increased mortality rates. While CVID patients' gut microbiota have been analyzed, data on the respiratory microbiome ecosystem are limited. This study aims to analyze the bacterial composition of the oropharynx of adults with CVID and its link with clinical and immunological features and risk for respiratory acute infections. Oropharyngeal samples from 72 CVID adults and 26 controls were collected in a 12-month prospective study. The samples were analyzed by metagenomic bacterial 16S ribosomal RNA sequencing and processed using the Quantitative Insights Into Microbial Ecology (QIME) pipeline. Differentially abundant species were identified and used to build a dysbiosis index. A machine learning model trained on microbial abundance data was used to test the power of microbiome alterations to distinguish between healthy individuals and CVID patients. Compared to controls, the oropharyngeal microbiome of CVID patients showed lower alpha- and beta-diversity, with a relatively increased abundance of the order Lactobacillales, including the family Streptococcaceae. Intra-CVID analysis identified age >45 years, COPD, lack of IgA, and low residual IgM as associated with a reduced alpha diversity. Expansion of Haemophilus and Streptococcus genera was observed in patients with undetectable IgA and COPD, independent from recent antibiotic use. Patients receiving azithromycin as antibiotic prophylaxis had a higher dysbiosis score. Expansion of Haemophilus and Anoxybacillus was associated with acute respiratory infections within six months. CVID patients showed a perturbed oropharynx microbiota enriched with potentially pathogenic bacteria and decreased protective species. Low residual levels of IgA/IgM, chronic lung damage, anti antibiotic prophylaxis contributed to respiratory dysbiosis.

Comparison of Contaminant Profiles and Biochemical Properties for Commercial Preparations of Immune Globulin Intravenous (IGIV)

Immune globulin intravenous (IGIV) has grown steadily in the volume of use and numbers of clinical indications since the first modern commercial preparation was approved by FDA in 1981. All US-marketed IGIV products are approved by FDA to treat primary immune deficiency; seven are also approved for immune thrombocytopenia purpura. Currently, most IGIV infusions are for treating autoimmune diseases.IGIV has historically been isolated from pools of human plasma (at least 1,000 donors, but typically ranging from 10,000 donations in the US up to 60,000 donations in some EU nations) using Cohn-Oncley cold ethanol fractionation. Most firms now supplement this process with chromatographic steps to remove Factor XIa, other promoters of complement activation, and other Ig isotypes.Despite high purity, a range of adverse symptoms, varying widely by product, is associated with IGIV infusions. To date, a possible correlation between adverse event profiles and contaminant profiles for IGIV products has not been studied and remains poorly understood. FDA requires donor blood tests for absence of hepatitis, HIV and other viral diseases. Final container immunoglobulin products must show potency against measles, diphtheria and polio virus and contain no prekallikrein activator. However, no further characterization of contaminant proteins is currently required.We therefore undertook comparative component analyses of 5% and 10% solution IGIV products approved or in development for the US market. Component analysis was performed using high resolution mass spectrometry (HRMS) and very sensitive multiplexed ELISA (mELISA) surveys to identify and quantitate trace contaminants in individual products. Factor XIa antigen and activity tests as well as functional assays were performed to assess the thrombogenic potential of the products. Subgroup and Ig isotype distribution were assessed by ELISA, and size exclusion chromatography was used to determine the extent of molecular aggregation.HRMS and mELISA are complementary but fundamentally different survey methods. HRMS is less “biased” in that it will detect any molecular entity more abundant than the limit of detection, whereas mELISA detected primarily biomarkers included in lists of assays preset by the vendor. Among these were several tumor markers, a wide range of inflammatory modulators, and a number of metabolic hormones.For a complex biologic such as polyclonal IGIV, HRMS typically detects entities present at levels of 10 - 20 molar ppm, while the lower limit of quantitation (LLOQ) of the mELISA is typically 3 - 5 orders of magnitude lower. Therefore, the mELISA survey of each IGIV product typically detects a substantially greater number of contaminant entities than the HRMS assay. If mELISA found a protein at ≥ 10 ppm for a product, it was generally also among the contaminants detected by HRMS. Thus, HRMS and mELISA results correlated satisfactorily. With both methods, detected levels of any given contaminant varied up to several orders of magnitude among the IGIV products tested.The most abundant contaminants in most preparations were IgA, IgD, β 2-glycoprotein-1, α 2-macroglobulin and albumin. Residual albumin and IgM were found in some but not all products. Inflammatory modulators were largely absent from the IGIV products surveyed. The interleukins IL6, IL-8 and IL-10 were detected in a few products, but in no case more than 4-fold above the mELISA LLOQ. The tumor markers carcinoembryonic antigen (CEA), α-fetoprotein (αFP), CA-125, CA 19-9 and tumor necrosis factor-α were not found in any product.The results of these assays are discussed with respect to their potential relationship to the observed adverse event and safety profiles of the individual products. DisclosuresAbramson: GC Mogam, Incorporated: Consultancy, Research Funding. Van Cott: GC Mogam, Incorporated: Research Funding.

PB2017 SEQUENTIAL IMPROVEMENTS IN THE OUTCOME OF INTENSIVE INDUCTION CHEMOIMMUNOTHERAPY FOLLOWED BY AUTOLOGOUS STEM CELL TRANSPLANTATION IN PATIENTS WITH UNTREATED WALDENSTROM MACROGLOBULINEMIA.

Background:Waldenstrom Macroglobulinemia (WM) is still considered an incurable IgM‐secreting lymphoplasmacytic lymphoma. The role of intensive induction chemoimmunotherapy followed by autologous stem cell transplantation (ASCT) in the management of patients with WM has not been established.Aims:To study the effectiveness of intensive induction chemoimmunotherapy and its combination with ibrutinib followed by ASCT in patients with untreated WM.Methods:A total of 8 treatment naive patients with WM were enrolled in our study between March 2017 and February 2019 years. Median age was 55.5 years (range 34‐62 years) and 6 (75%) were men. Three patients were treated with 4 cycles of alternating of R‐EPOCH (rituximab, doxorubicin, etoposide, vincristine, cyclophosphamide, prednisolone) in conventional doses and R‐BAC (rituximab, bendamustin, cytarabine) in conventional doses – group №1. Five patients were treated with 4 cycles of alternating of R‐EPOCH + ibrutinib 420 mg daily and R‐BAC + ibrutinib 420 mg daily – group №2. Response was assessed using modified IWWM‐6 criteria.Results:According to IPSSWM – 1,4 and 3 patients, were considered as being at low, intermediate, and high risk respectively. Indications for treatment included hyperviscosity ‐ related symptoms in 8 (100%) patients, anemia in 8 (100%), lymphadenopathy and splenomegaly in 7 (87.5%), constitutional symptoms in 6 (75%) and thrombocytopenia 2 (25%). At baseline, median serum total protein was 115 g/l (range 101‐130 g/l), median serum IgM monoclonal protein was 53 g/l (range 39‐74 g/l), median hemoglobin was 7.8 g/dl (range 5.6‐10.5 g/dl), median β‐2‐microglobulin was 4.32 mg/l (2.8‐9.3 mg/l). High serum LDH activity was in 3 (37.5%) patients, albumin less than 30 g/l was in 3 (37.5%) patients. In 6 (75%) patients the MYD88L265P mutation was detected and CXCR4 mutation was not detected in any patients. Deletion of 17p was identified in one patient but mutation of the TP53 gene determined using by Sanger sequencing of the coding region (exons 1(2)‐11) was not identified in any patients. All evaluable patients have completed 4 cycles of induction therapy. Partial response (PR) was attained in 8 (100%). Median hemoglobin increased to 12.7 g/dl (range 12.1‐ 13.7 g/dl) and regression of all baseline symptoms occurred in all cases. Two patients in group №1 and 3 in group №2 subsequently underwent ASCT, the preparative regimen was CEAM (lomustine, etoposide, cytarabine, melphalan). Very good partial response was achieved in one patient after ASCT and in 4 patients serum IgM monoclonal protein continued to decline (tab.). Median observation without progression from the end of therapy is 8 (range 1‐17) months and we continue our research.Summary/Conclusion:The combination of intensive immunochemotherapy with ibrutinib leads to rapid and deep decline of serum IgM monoclonal protein and regression of baseline symptoms of WM. Applying of ibrutinib as part of chemoimmunotherapy for a limited period of time results in a lower financial burden compared to its long‐term use. ASCT as part of first line therapy leads to improve in the outcomes and may be a safe, feasible, and reasonable therapeutic option for young WM patients. Perhaps, residual serum IgM monoclonal protein is due to the presence of clonal plasma cells in the bone marrow after treatment and is considered as residual disease. Applying targeted therapy might possibly to eliminate this pathological clone.image

Interpretation of very low avidity indices acquired with the Liaison XL Toxo IgG avidity assay in dating toxoplasmosis infection.

Congenital toxoplasmosis is an important cause of complications in pregnancy. Toxoplasmosis is often asymptomatic and thus serological tests are usually performed to screen for it. A first serum which exhibit both IgG and IgM may be due to nascent toxoplasmosis seroconversion, non-specific IgM reaction, or residual IgM. The IgG avidity test has been proposed to identify latent infections. A high index excludes recent toxoplasmosis whereas an intermediate or low index only suggests a recent infection, the caveats being that some people with latent Toxoplasma gondii infection show IgG with low or intermediate avidity. In this study, we investigated the ability of the Liaison XL Toxo IgG avidity (DiaSorin, Saluggia, Italy) assay to confirm recent infection when IgG avidity index is very low (≤ 0.1). Four thousand two hundred ninety-seven sera exhibiting both IgG and IgM were included and avidity was performed on the Liaison device according to the manufacturer's recommendations. One hundred twenty-six sera on the 297 sera which exhibited very low IgG avidity indices (≤ 0.1) could be exploited: 97% of sera with IgG avidity indices < 0.05 actually corresponded to recent infection (less than 3months). A similar but less pronounced trend was observed for the sera exhibiting indices between 0.05 and 0.1 (69% corresponded to recent infections). The IgG avidity index data we obtained with the Liaison XL Toxo device are similar to those obtained with other devices. This body of consistent results underlines the interest of very low IgG avidity indices as a sign of probable recent toxoplasmosis.

Performance of the BioPlex 2200 Flow Immunoassay in Critical Cases of Serodiagnosis of Toxoplasmosis

The BioPlex 2200 automated analyzer (Bio-Rad Laboratories, Hercules, CA) is a recently developed multiplex analyzer that enables the detection of anti-Toxoplasma, -rubella, and -cytomegalovirus antibodies in the same assay. The aim of this study was to compare this new technology (using the BioPlex 2200 ToRC IgG/IgM kit) in critical cases of serodiagnosis of toxoplasmosis (acute, chronic, or congenital infections and cases with discrepant results) to the technologies used in our routine practice, i.e., the Platelia IgG/IgM enzyme-linked immunosorbent assays (ELISAs) (Bio-Rad Laboratories) and the Toxo-Screen direct agglutination assay (bioMérieux, Lyon, France). Overall, most cases of false-positive/negative results obtained with the Platelia IgG or Toxo-Screen assay were corrected by the BioPlex 2200 ToRC IgG (87.5%). Furthermore, the analysis of 35 sequences of sera showed a trend toward a more rapid decrease of IgM titers by BioPlex 2200 than by Platelia. These results for IgM detection can be explained by a weaker detection of residual IgM. Indeed, among 23 serum samples from patients with probable past infection with long-lasting IgM (Platelia M positive and IgG avidity index, ≥0.5), the BioPlex 2200 Toxoplasma IgM assay was positive for only 11 serum samples. In our panel of critical cases comprising 156 serum and 6 cord blood samples from 103 patients with acute, chronic, or congenital infection, the BioPlex 2200 IgG assay was a sensitive (97.8%) and specific (91.3%) method for IgG detection. The high specificity (97.4%) of IgM detection combined with the shorter kinetics of IgM titers may considerably reduce the number of residual IgM detections, thus yielding more precise diagnoses of acute infections.

Comparison of Four Commercially Available Avidity Tests for Toxoplasma gondii-Specific IgG Antibodies

Toxoplasma infection in pregnant women may cause congenital toxoplasmosis. Diagnosis of infection is based on serological tests aimed at detecting IgM and IgG antibodies against Toxoplasma gondii. However, IgM antibodies are not an accurate marker for discriminating between acute and latent infection. Detection of residual or persistent IgM may occur months or even years after primary infection, while the IgG avidity test is a rapid means of identifying latent infections in pregnant women who exhibit both IgG and IgM anti-Toxoplasma antibodies on initial testing during pregnancy. In this study, we assessed and compared the performances of four commercially available Toxoplasma IgG avidity tests in immunocompetent and immunocompromised patients with acute and latent toxoplasmosis. The positive predictive value of high avidity to confirm latent toxoplasmosis was 100% for all the assays, indicating that high avidity is a hallmark of latent infection. However, the negative predictive value of high avidity ranged from 99.2% (bioMérieux) to 95.3% (Abbott), indicating that acute toxoplasmosis could not be reliably diagnosed based on low IgG avidity alone. Thus, the avidity test provides a rapid means for identifying latent Toxoplasma infection in immunocompetent pregnant women presenting both IgG and IgM anti-Toxoplasma antibodies on initial testing. In terms of cost-effectiveness, avidity testing is a powerful tool that optimizes screening and follow-up of pregnant women while minimizing the costs of screening by avoiding subsequent costly maternal and fetal investigation and unnecessary treatment. The cheapest assay, Vidas Toxo IgG Avidity, also had the best performance for the diagnosis of latent toxoplasmosis.

Flow cytometry-based algorithm to analyze the anti-fixed Toxoplasma gondii tachyzoites IgM and IgG reactivity and diagnose human acute toxoplasmosis

In the present study we evaluated the performance of a flow cytometry-based algorithm as a new serological approach to detect antibodies to T. gondii and specific IgG avidity to diagnose acute toxoplasmosis. The results showed that using FC-AFTA-IgM assay, all serum samples from patients with acute toxoplasmosis demonstrated seropositivity, whereas 90% of patients with chronic infection and 100% of non-infected individuals presented negative results. Thus, only 10% of patients with chronic toxoplasmosis showed residual IgM, in contrast with other methodologies used to diagnosis acute toxoplasmosis. On the order hand, FC-AFTA-IgG assay as well as FC-AFTA-IgG subclasses is unlikely to discriminate acute from chronic toxoplasmosis. We have also evaluated the performance of FC-AFTA-IgG avidity as a tool to exclude chronic toxoplasmosis in patients with positive FC-AFTA-IgM. Our data showed an excellent performance of FC-AFTA-IgG avidity employing the cut-off of 60% for Avidity Index (AI) with sensitivity and specificity of 100%. All serum samples from patients presenting acute toxoplasmosis showed low avidity index (AI≤60%), whereas all chronic patients showed high avidity index (AI>60%). The outstanding performance indexes of this novel flow cytometry-based algorithm support its use as a non-conventional alternative serological approach to diagnose human acute toxoplasmosis.

Sérologie de la toxoplasmose chez la femme enceinte : caractéristiques et pièges

Screening program of congenital toxoplasmosis depends principally on serological testing and follow-up of pregnant women with negative serology from the onset of pregnancy. This program is particularly efficient because the majority of cases of obstetrical seroconversion are diagnosed and followed up. Serological techniques have evolved with time and are many on the market. The "IgG" tests are more and more specific at the detriment of their sensitivity. The latter is variable from one technique to the other. In 2008, despite the presence of an international standard, serological titrations are particularly variable making interpretation of the kinetics impossible between two different series, two techniques or two laboratories. In cases of interpretation discordance or borderline values, it is useful to do a confirmatory technique. The dye test or the western blot are useful in these cases and prevent up to 2 to 8% of unnecessary obstetric follow up. IgM tests are becoming more and more sensitive and precocious. These tests frequently detect "non specific IgM" as well as "residual IgM" (associated with a stable positive IgG titre). In such cases and in the absence of a preceding positive result, a complementary test done by specialized laboratories (such as ISAgA IgM or IgA, IgG avidity test, differential agglutination, etc.) is indispensable in order to prevent any error of interpretation. These complementary tests as well as the serological evolution (control done in 15 days) permit, in a large number of cases, to reassure the patient with full confidence.

Evaluation of DPC immulite 2000®Toxoplasma quantitative IgG/IgM kits for automated toxoplasmosis serology with immulite 2000®

In this study, we evaluated a new immunoassay for the automated detection of anti-toxoplasma IgG and IgM with Immulite 2000 (DPC-Siemens, La Garenne-Colombes, France). We tested 280 sera from 112 patients with past infection (PI), 40 PI with residual IgM, 75 seronegatives, 16 infants (31 sera) monitored for neonatal screening of congenital toxoplasmosis, and 13 patients with recent seroconversion (SC) (22 sequential sera). Detection sensitivity and specificity for IgG were 99 and 100%, respectively. IgG titers obtained with Immulite 2000 were higher than with Vidas (BioMérieux, Marcy I'Etoile, France) and Access (Beckman-Coulter, Villepinte, France) (paired Wilcoxon test z=4.44 and z=3.67, respectively, P<0.001). IgM specificity was 100%. Detection sensitivity for IgM was 100% in the SC group, 86% in congenitally infected infants, and 75% in PI with persistent IgM. IgM detection seemed less prolonged in time than with the IgM Access and ISAGA IgM techniques.

Association of the presence of residual anti-Toxoplasma gondii IgM in pregnant women and their respective family groups in Miracema, Northwest Rio de Janeiro, Brazil

The seroprevalence of toxoplasmosis in 832 pregnant women in Miracema, Rio de Janeiro, was determined and 75.1% (625) and 2.0% (17) were anti-Toxoplasma gondii IgG and IgM positive, respectively. Out of the 17 IgM positive pregnant women, only one had low avidity IgG corresponding to the acute phase of the infection. All the other women presented with high avidity IgG and also presented with residual IgM anti-T. gondii. Of this sample, 106 received home visits (this includes 11 family nuclei of pregnant women with residual IgM anti-T. gondii, 68 nuclei of only IgG positive pregnant women and 27 nuclei of pregnant women with no antibodies to anti-T. gondii), resulting in 267 individuals visited. Out of these 267 individuals, 21 were positive for IgG and IgM anti-T. gondii and were candidates for the IgG avidity test. All of them presented with high avidity IgG and residual IgM. Five of these IgM+ individuals were (5/238; 2.1%) relatives of IgM negative pregnant women. The other 16 (16/29; 55.2%) were relatives of IgM+ pregnant women who were positive for residual IgM anti-T. gondii. This association was statistically significant (p = 0.0000). The analysis presented herein raises questions regarding the presence of residual IgM anti-T. gondii such as genetic determinants or even constant antigenic stimuli for the same family cluster.

Avidity of IgG antibodies against excreted/secreted antigens of Toxoplasma gondii: immunological marker for acute recent toxoplasmosis

Detection of anti-toxoplasma IgM antibodies has frequently been used as a serological marker for diagnosing recently acquired toxoplasmosis. However, the persistence of these antibodies in some patients has complicated the interpretation of serological results when toxoplasmosis is suspected. The purpose of the present study was to evaluate the avidity of IgG antibodies against excreted/secreted antigens of Toxoplasma gondii by means of immunoblot, to establish a profile for acute recent infection in a single serum sample and confirm the presence of residual IgM antibodies obtained in automated assays. When we evaluated the avidity of IgG antibodies against excreted/secreted antigens of Toxoplasma gondii by means of immunoblot, we observed phase-specific reactivity, i.e. cases of acute recent toxoplasmosis presented low avidity and cases of non-acute recent toxoplasmosis presented high avidity towards the 30 kDa protein fraction, which probably corresponds to the SAG-1 surface antigen. Our results suggest that the avidity of IgG antibodies against excreted/secreted antigens of Toxoplasma gondii is an important immunological marker for distinguishing between recent infections and for determining the presence of residual IgM antibodies obtained from automated assays.

Diagnóstico molecular da toxoplasmose: revisão

A toxoplasmose infecta milhões de pessoas no mundo inteiro, sendo que a prevalência da infecção humana na maioria dos países está entre 40% e 50%. No Brasil essa taxa aumenta até 80%, dependendo da área estudada. Na maioria dos hospedeiros a infecção é assintomática. A mulher grávida com sorologia negativa pode contribuir para o incremento da morbidade, transmitindo o Toxoplasma gondii para o feto, através da placenta, se adquirir toxoplasmose aguda durante a gravidez. O diagnóstico da toxoplasmose aguda é baseado na detecção de IgM anti-Toxoplasma gondii circulante. A alta sensibilidade das técnicas sorológicas atuais trouxe a realidade da presença de IgM residuais confundindo muitas vezes o diagnóstico final. Nesse sentido, as técnicas moleculares, tais como a reação em cadeia da polimerase (PCR), podem ajudar a uma melhor interpretação do estado real da interação parasito/homem, embora sejam ainda pouco validadas para uso na rotina de diagnóstico laboratorial da toxoplasmose.

EVALUATION OF AN ELISA ASSAY USING SYNTHETIC A AND B CARBOHYDRATES TO ASSESS ANTIBODY LEVELS IN ABO INCOMPATIBLE KIDNEY TRANSPLANT PATIENTS

A282 While the outcomes of ABO incompatible kidney transplantation generally correlate with the baseline antibody level as determined by hemagglutination, we have observed several occurrences when patients with low hemagglutination titers have developed humoral rejection. We hypothesized that the hemagglutination assay (HA) may underestimate the level of anti-AB antibodies and may provide targets for antibodies other than A and B. The Aim of this study was to develop a specific ELISA assay for anti-AB antibodies using synthetic A and B carbohydrates and to compare the results with HA. Methods: ELISA plates with low (LB) or high (HB) protein binding capacity were coated with polyacril-amid (PAA)-A or B antigens. Uncoated wells, wells coated with PAA, and with PAA-irrelevant trisacharide (PAA-Ir) served as controls for nonspecific binding (NSB). Human serum samples were applied in 2 fold dilutions followed by incubations with monoclonal antibodies against IgM, IgG, IgA classes/subclasses, anti-mouse-peroxidase antibody and ABTS substrate. The positive result was determined as serum dilution (titer) after the NSB of the controls was deducted. HA was performed as immediate spin (IS) agglutination and anti-human globulin (AHG) test. Results: The LB plates showed low NSB but also low specific binding, which accounted for false negative results. The HB plates showed higher background, which was similar in the uncoated wells, PAA wells and PAA-Ir wells which demonstrates that the NSB is caused by absorption of proteins on the plastic. The NSB was corrected by using the intra assay coefficient of variation (IACV) based on the formula ODAB –2IACV>ODContr+2IACV. Interassay CV for anti-AB IgM/G/A was from 4.6 to 9.7%. The specificity of the assay was confirmed by negative results with PAA-Ir. The sensitivity of the ELISA assay was 2.77 two-fold dilutions higher than the HA (p<0.0001). IgM ELISA titers correlated with the IS results (p < 0.001). No correlation was found between IgG ELISA titers and AHG. Titers for IgG2 were generally higher than IgG1. Anti-AB IgA was found mostly in blood type O. We examined the ELISA levels before and after plasmapheresis in 5 patients. ELISA showed measurable anti-AB antibody levels after plasmapheresis when the HA titers were zero. The levels of IgM anti-AB antibodies after plasmapheresis in 2 patients were ≥ 128; one of these patients later developed antibody mediated rejection and the other developed chronic allograft nephropathy. Conclusions: 1) The sensitivity of ELISA was significantly higher than HA. 2) IgM anti-AB antibody levels showed correlation with IS while IgG did not show correlation with AHG, which might be due to residual IgM antibodies participating in AHG agglutination. 3) Preliminary data suggest that ELISA IgM anti-AB levels after plasmapheresis when HA levels are not measurable may predict negative outcome. Corollary: These data supported that the ELISA assay is sensitive and specific and would allow for better evaluation of the role of anti-AB antibodies in ABO incompatible grafts.

Xenotransplant issues

To determine the role of the terminal α-galactosyl residue in the endothelial damage mediated by human xenoreactive natural antibodies (IgM and IgG), we treated porcine endothelial cells in culture with green coffee bean α-galactosidase. A practically complete removal of terminal α-Gal residues (as evaluated by flow cytometry with Bandeiraea simplicifolia isolectin B4) and concomitant exposure of N-acetyllactosamine were obtained without altering cell viability. A dramatic decrease in IgM and IgG binding (from a pool of human sera) was observed, confirming the key role of the α-galactosyl residues. The enzyme treatment did not induce any nonspecific immunoglobulin binding sites but led to the exposure of new epitopes for a minor fraction of IgM. The main residual IgM and IgG binding could be due to xenoantigens other than the α-galactosyl residues. When α-galactosidase-treated endothelial cells were used as targets in cytotoxicity experiments, they were less susceptible than untreated cells to complement-mediated cytotoxicity induced by fresh human serum. In contrast, they did not acquire resistance to human IgG-dependent cellular cytotoxicity, despite the decrease in IgG binding. Because it is known that antibody-dependent cytotoxicity mediated by CD 16 NK cells is dependent on IgG1 and IgG3, and not IgG2 or IgG4, which was confirmed by blocking experiments, we studied the binding of all four subclasses to intact and α-galactosidasetreated endothelial cells. Two major subclasses, IgG1 and IgG2, bound to untreated endothelial cells, whereas IgG3 binding was low and IgG4 binding was negligible. A decrease in IgG1, IgG2, and IgG3 binding was observed upon α-galactosidase treatment, indicating that antibodies belonging to these three subclasses recognize α-galactosyl residues. The decrease in IgG2 binding was more pronounced than the decrease in IgG1 binding. Collectively, these data indicate that IgG1 xenoreactive natural antibodies, including those which are not directed at the α-galactosyl residues, could play a major role in the early delayed vascular rejection of pig xenografts. Fetal porcine islet-like cell clusters (ICC) were transplanted under the renal capsule of normoglycemic normal or athymic (nu/nu) C57BL/6 mice. Control animals were implanted with allogeneic minced kidney tissue from C57BL/Ks mice. The animals were killed 6 or 14 days after transplantation and the grafts were processed for flow cytometric analyses or immunohistochemistry. Xenograft destruction was evident in normal mice on day 6 after transplantation. The majority of infiltrating cells were macrophage-like cells expressing the F4/80 antigen. Lymphocytes expressing the CD3 antigen. were in the minority and mainly located in the peripheral parts of the ICC xenograf. The frequency and distribution of CD4+ cells were found to resemble those of the CD3+ cells. A large number of infiltrating cells, including several macrophage-like cells, expressed the Thy 1.2 antigen. Flow cytometry of infiltrating cells in the ICC xenograft revealed that approximately half of the cells expressing the F4/80 antigen also expressed Thy 1.2 and/or CD4. No cells were found expressing both the F4/80 and CD8 antigens. Both the F4/80 single-positive and the F4/80, CD4 doublepositive cells were found to be large and more granular than the CD4 single-positive cells No co-expression of CD4 or Thy 1.2 with the F4/80 antigen was detected on cells infiltrating allogeneic tissue grafts. Moreover, a relative large number of cells (=15%) in the xenograft expressed the NK 1.1 antigen as determined by flow cytometry. The role of natural killer (NK) cells in islet xenograft rejection was further evaluated in mice depleted of NK cells, using intraperitoneal injections of the monoclonal antibody NK 1.1. The simultaneous inoculation and subsequent growth of the NK cell-sensitive β2- microglobulin-deficient mutant, C4.4–25, lumphoma cell line EL-4, served as an in vivo control of NK cell depletion. However, all NK cell-depleted mice rejected the ICC xenograft. In contrast, athymic mice permanently accepted the porcine ICC xenograft but readily rejected the NK cell-sensitive lymphoma cell line. taken together, ICC xenograft rejection in mice seems to be T Cell-dependent, as evidenced in the nude mice model, while the main effector cel appears to be a macrophage with a unique phenotype.

Removal of terminal alpha-galactosyl residues from xenogeneic porcine endothelial cells. Decrease in complement-mediated cytotoxicity but persistence of IgG1-mediated antibody-dependent cell-mediated cytotoxicity.

To determine the role of the terminal alpha-galactosyl residue in the endothelial damage mediated by human xenoreactive natural antibodies (IgM and IgG), we treated porcine endothelial cells in culture with green coffee bean alpha-galactosidase. A practically complete removal of terminal alpha-Gal residues (as evaluated by flow cytometry with Bandeiraea simplicifolia isolectin B4) and concomitant exposure of N-acetyllactosamine were obtained without altering cell viability. A dramatic decrease in IgM and IgG binding (from a pool of human sera) was observed, confirming the key role of the alpha-galactosyl residues. The enzyme treatment did not induce any nonspecific immunoglobulin binding sites, but led to the exposure of new epitopes for a minor fraction of IgM. The main residual IgM and IgG binding could be due to xenoantigens other than the alpha-galactosyl residues. When alpha-galactosidase-treated endothelial cells were used as targets in cytotoxicity experiments, they were less susceptible than untreated cells to complement-mediated cytotoxicity induced by fresh human serum. In contrast, they did not acquire resistance to human IgG-dependent cellular cytotoxicity, despite the decrease in IgG binding. Because it is known that antibody-dependent cytotoxicity mediated by CD16+ NK cells is dependent on IgG1 and IgG3, and not on IgG2 or IgG4, which was confirmed by blocking experiments, we studied the binding of all four subclasses to intact and alpha-galactosidase-treated endothelial cells. Two major subclasses, IgG1 and IgG2, bound to untreated endothelial cells, whereas IgG3 binding was low and IgG4 binding was negligible. A decrease in IgG1, IgG2, and IgG3 binding was observed upon alpha-galactosidase treatment, indicating that antibodies belonging to these three subclasses recognize alpha-galactosyl residues. The decrease in IgG2 binding was more pronounced than the decrease in IgG1 binding. Collectively, these data indicate that IgG1 xenoreactive natural antibodies, including those which are not directed at the alpha-galactosyl residues, could play a major role in the early delayed vascular rejection of pig xenografts.

Isotype commitment of human B cells that are transformed by Epstein‐Barr virus

Epstein-Barr virus (EBV) can transform a subpopulation of preactivated B cells thus promoting their growth and differentiation into plasma cells. In EBV-transformed clones of IgM-producing cells, the heavy chain constant region (CH) genes on the productive allele are fixed in germ-line configuration, whereas in isotype-switched clones the CH genes proximal to the expressed CH gene are deleted. In order to define more precisely the EBV-susceptible B cells, we sorted subpopulations of B cells on the basis of their cell surface Ig (sIg) isotypes, infected them with EBV, and determined which isotypes they could produce following transformation. Most precursors of IgM-producing plasma cells expressed both IgM and IgD on their surface, while a minority expressed IgM alone. Some B cell precursors of IgG- and IgA-producing cells also expressed sIgM, but surprisingly none expressed IgD. Those precursors of IgG and IgA producers, which bore sIgM, expressed it in relatively low levels, whereas B cells expressing high levels of sIgM were incapable of generating IgG and IgA producers. All of the precursors of IgG and IgA plasma cells expressed these isotypes on their cell surface. Interestingly, precursor B cells capable of producing the IgG3 and IgA2 subclasses could be respectively enriched on the basis of the presence or absence of cell sIgM. These results demonstrate the isotype precommitment of EBV-transformable B cells. They further suggest that residual IgM is transiently expressed on the surface of the IgG- and IgA-committed B cell precursors, whereas sIgD expression is extinguished earlier in the process of isotype switching via CH gene deletion.

Antibody capture radioimmunoassay (MACRIA) for coxsackievirus B4 and B5-specific IgM

An antibody capture radioimmunoassay was established for the detection of coxsackievirus B4 and B5-specific IgM. A significant feature of the assay was the use of an unrefined coxsackievirus B (CBV) antigen. The antigen was prepared by freeze thawing, ultrasonication and low speed centrifugation of infected Vero cells with no purification or concentration of the antigen being performed. Results of sera tested were expressed as a serum ratio (SR) by comparison with a low positive control serum. To establish an SR indicating positivity in the assays, 100 antenatal sera collected in late February were tested. The mean SR was calculated and the mean plus three standard deviations was taken as the minimum SR indicating positivity. Although resulting in a relatively insensitive assay, such a value was required to exclude sera giving a low level of reactivity which may be due to residual enterovirus-specific IgM resulting from a remote infection.The homologous CBV-IgM assay was positive in four cases of CBV4 infection and six cases of CBV5 infection. For the CBV4 IgM assay, ten of 20 (50%) sera from infections with CBV other than CBV4 were positive and nine of the 13 (69%) sera from infections with echoviruses or coxsackieviruses A were positive. Five of 18 (27%) sera with an elevated CBV neutralization titre were positive in the CBV4-IgM assay. For the CB5-IgM assay seven of 18 (39%) sera from infections with CBV other than CBV5 were positive and nine of 13 (69%) sera from infections with echoviruses or coxsackieviruses A were positive. The nine sera that were positive from this group in the CBV5-IgM assay were the same nine as were positive in the CBV4-IgM assay. Two of the 18 (11%) sera with an elevated CBV neutralization titre were positive in the CBV5-IgM assay. These two sera were also positive in the CBV4-IgM assay and had an elevated monotypic CBV4 neutralization titre. None of the sera giving positive results gave significant reactivity when tested with control antigen. Twelve rheumatoid factor containing sera and 46 sera from other infections were negative in both assays. Of 24 sera from confirmed CBV infection, seven gave a positive monotypic CBV4 or 5-IgM response, ten were positive in both assays and seven were negative in both assays. The positive results seen with sera from cases of heterologous enterovirus infection may result from an anamnestic IgM response or, more likely, IgM directed against enterovirus cross-reacting antigens. The absence of homologous neutralizing antibody at a dilution of 1:20 in nine of 20 sera that gave a positive CBV-IgM result and the presence of positive results for CBV4 and 5-IgM in a 14 month old infant who had echovirus 7 infection indicates that the IgM need not be directed against neutralizing antigens.Thus the CBV4 and 5-IgM assays developed appeared to be specific for an enterovirus infection but because of the cross-reactivity were not type-specific or group-specific.

Détermination du type IgM ou IgG des immunoglobulines spécifiques anti-HBc: Relations avec les autres marqueurs du virus HB

A procedure for determining anti-HBc of IgM class is described herein. After IgG anti-HBc antibodies have been preferentially absorbed with Staphylococcus aureus cells positive for protein A, we have tested for residual IgM anti-HBc in the supernatant (absorbed serum) by radioimmunoassay. The occurrence and time course of anti-HBc, studied in 3 patients with ongoing infection, show that IgM anti-HBc persist for about 2 months in cases of acute hepatitis. IgM anti-HBc --marker for a recent HBV infection-- was found to be a useful tool in diagnosis of an unapparent hepatitis with transient or undetectable HBs antigenemia (case no 5). The presence of IgM or IgG anti-HBc, HBeAg and anti-HBe was determined by radioimmunoassay in 68 patients HBsAg positive. The immunoglobulin classes (IgM or IgG) of anti-HBc are dependent on the phase of hepatitis B. Of the 29 IgM anti-HBc positive specimens, 28 were found to be HBeAg positive, 18 of these patients were hemodialysed. 62 among 63 HBsAg positive blood donors had IgG anti-HBc, 6 associated with HBeAg and 56 with anti-HBe. All anti-HBc of anti-HBs positive sera were of IgG class (patients or blood donors). In order to estimate the anti-HBc titer, we have determined the per cent inhibition of 134 HBsAg and 46 anti-HBs positive sera diluted to 1 : 100. We correlate the presence of HBsAg --regardless of the level of titer of it --with high titers of anti-HBc and the presence of anti-HBs with low titers (P less than less than 0,0001). These results are very instructive with regard to the problem of anti-HBc titer and possibility of persisting HBV and we support the hypothesis that HBsAg negative but strongly anti-HBc positive blood might be infectious. IgM anti-HBc are on the average of lower titer than IgG anti-HBc, but we did not observe difference in IgG anti-HBc titer between HBeAg positive sera and anti-HBe, HBsAg positive sera.

A propos du diagnostic de l'hépatite virale A: Mise en évidence des anticorps anti-hépatite A de type IgM

A Bradley's modified procedure is described herein. It allows the diagnosis of acute viral hepatitis A by evidence of IgM anti HAV: after IgG anti-HAV have been preferentially absorbed with Staphylococcus aureus cells positive for protein A, we test for residual IgM anti-HAV in the supernatant (absorbed serum) by radioimmunoassay. We have documented the ability of this procedure to differentially detect IgM or IgG anti-HAV by analyzing 308 sera. This simple method clearly distinguishes between recent (28 cases) and past HAV infection which is very frequent in France: 80% in adults.

Staphylococcal protein A adsorption of neonatal serum to facilitate early diagnosis of congenital infection

The early diagnosis of congenital infection frequently depends on the ability to distinguish between infant IgM and maternal IgG antibodies. Staphylococcal protein A, which specifically binds IgG, removed maternal IgG from the serum of newborn infants. Residual IgM antibodies to CMV, rubella, toxoplasmosis, and syphilis were then identified by routine serologic techniques. Persistence of greater than or equal to 25% of the original antibody titer following SPA adsorption distinguished the sera of infants with congenital infection from those of healthy infants. No false negative results were encountered. Specificity of the serologic results of SPA-treated infant sera correlated with IgM-specific identification of the causative agent. Potentially false positive titers were identified by concurrent elevation of IgA or rheumatoid factor. Adsorption of cord or neonatal serum with SPA facilitates accurate serologic diagnosis of congenital infection.