Significance of antibodies of IgG, IgA and IgM isotypes against peptidylarginine deiminase 4 in early rheumatoid arthritis / 中华风湿病学杂志

Objective To evaluate the prevalence and clinical significance of IgG,IgA and IgM isotypes of anti-peptidylarginine deiminase 4 (anti-PAD4) antibodies in early rheumatoid arthritis (RA).Methods IgG,IgA and IgM isotypes of anti-PAD4 antibodies were measured in the sera of 88 RA patients with disease duration less than 2 years,62 patients with other rheumatic diseases and 57 healthy subjects.The diagnostic performance of IgG,IgA and IgM isotypes of anti-PAD4 antibodies and their relationship with disease duration,DAS28,ESR,CRP,anti-CCP antibodies and rheumatoid factor (RF) were evaluated.Data analysis were performed using t test,U test and Spearman's association analysis.Results ① The sensitivities of IgG,IgA,and IgM isotypes for early RA were 28.41％,36.36％ and 9.09％ respectively.The specificities of IgG,IgA and IgM isotypes were 94.12％,93.28％ and 95.80％ respectively.② IgA isotype was positively correlated with age (r=0.234,P=0.028),DAS28 (r=0.309,P=0.007),ESR (r=0.382,P=0.000) and CRP (r=0.291,P=0.008),while negatively correlated with disease duration (r=-0.295,P=0.006).③ IgA isotype was positively correlated with IgG isotype (r=0.451,P＜0.01).In the IgG negative patients,the positivity of IgA isotype was 29％(18/63),which indicated that the IgA isotype might be helpful in diagnosing RA in IgG isotype negative patients.Conclusion Anti-PAD4 antibodies can be detected in early RA,primarily with IgA and IgG isotypes.IgA isotype has negative correlation with disease duration,indicating that IgA isotype of anti-PAD4 antibody may play a role in the very early stage RA.

Alum Directly Modulates Murine B Lymphocytes to Produce IgG1 Isotype

Aluminum hydroxide (alum) is the most widely used adjuvant in human vaccines. Nevertheless, it is virtually unknown whether alum acts on B cells. In the present study, we explored the direct effect of alum on Ig expression by murine B cells in vitro. LPS-activated mouse spleen B cells were cultured with alum, and the level of isotype-specific Ig secretion, IgG1 secreting cell numbers, and Ig germ-line transcripts (GLT) were measured using ELISA, ELISPOT, and RT-PCR, respectively. Alum consistently enhanced total IgG1 production, numbers of IgG1 secreting cells, and GLTgamma1 expression. These results demonstrate that alum can directly cause IgG1 isotype switching leading to IgG1 production.

Immunoglobulin Isotype Switching in a Plasma Cell Myeloma Patient Treated with High-dose Chemotherapy and Hematopoietic Stem Cell Transplantation

A malignant plasma cell clone usually produces a single abnormal monoclonal protein with a constant isotype. However, switching of paraprotein isotype has been reported to be a transient phenomenon associated with the recovery of B-cell function, and, in some cases, the switching might be misinterpreted as relapse. In August 2008, we encountered a case of a 59-year-old man with proteinuria and high IgG level (5.6 g/dL), kappa free light chain level of 2,660 mg/L, reversed A/G ratio (0.51), and multiple osteolytic lesions. Plasma cells, which accounted for 57% of all the nucleated cells, in bone marrow aspirates were positive for kappa immunostaining. Serum protein electrophoresis showed one M-spike, concentration of 4.87 g/dL in the beta region. Immunofixation electrophoresis revealed the peak as an IgG-kappa monoclonal protein; therefore, a diagnosis of plasma cell myeloma was made. Complete remission was achieved after chemotherapy, and autologous peripheral stem cell collection was performed. In March 2009, the patient underwent high-dose chemotherapy and autologous peripheral stem cell transplantation support. After 2 months, serum protein electrophoresis showed 2 M-spikes in the gamma region with positive IgM-lambda, IgG-lambda, and IgG-kappa, and these bands persisted. The electrophoretic mobility of the IgG-kappa protein was different from that of the original disease protein, and bone marrow results were the same as the previous ones. Although immunoglobulin isotype switch is known to have a benign course, it always requires careful monitoring because, in rare cases, true clonal switching may occur.

Vitamin C acts indirectly to modulate isotype switching in mouse B cells / 대한해부학회지

Vitamin C, one of essential micronutrients, has been reported to modulate the humoral immune responses in some mammals. We investigated whether vitamin C might modulate this response in mice by directly affecting B cells. Splenic B cells were isolated and activated by CD40- and B cell receptor-ligation in vitro. The cells were cultured with a pretreatment of vitamin C from 0 to 1 mM of concentrations. Vitamin C slightly increased apoptosis of B cells dose-dependently and behaved as an antioxidant. We found that in vivo administration of vitamin C by intraperitoneal injection affected isotype switching as previously reported: the titer of antigen-specific IgG1 antibody was decreased, while that of IgG2a was unaffected. Somewhat different from those observed in vivo, in vitro exposure to vitamin C slightly decreased isotype switching to IgG1 and increased isotype switching to IgG2a. Pretreatment with vitamin C in the safe range did not affect either proliferation of cultured B cells or the expression of CD80 and CD86 in those cells. Taken together, in vivo results suggest that vitamin C acts to modulate isotype switching in the mouse. However, because of our in vitro results, we suggest that the modulation exerted by vitamin C in vivo is by indirectly affecting B cells, perhaps by directly influencing other immune cells such as dendritic cells.

Immunological imbalance between IFN-³ and IL-10 levels in the sera of patients with the cardiac form of Chagas disease

The immune response is crucial for protection against disease; however, immunological imbalances can lead to heart and digestive tract lesions in chagasic patients. Several studies have evaluated the cellular and humoral immune responses in chagasic patients in an attempt to correlate immunological findings with clinical forms of Chagas disease. Moreover, immunoglobulins and cytokines are important for parasitic control and are involved in lesion genesis. Here, cytokine and IgG isotype production were studied, using total epimastigote antigen on sera of chagasic patients with indeterminate (IND, n = 27) and cardiac (CARD, n = 16) forms of the disease. Samples from normal, uninfected individuals (NI, n = 30) were use as controls. The results showed that sera from both IND and CARD patients contained higher levels of Trypanosoma cruzi-specific IgG1 (IgG1) antibodies than sera from NI. No difference in IgG2 production levels was observed between NI, IND and CARD patients, nor was a difference in IL-10 and IFN-³ production detected in the sera of IND, CARD and NI patients. However, IND patients displayed a positive correlation between IL-10 and IFN-³ levels in serum, while CARD patients showed no such correlation, indicating an uncontrolled inflammatory response in CARD patients. These findings support the hypothesis that a lack of efficient regulation between IFN-³ and IL-10 productions in CARD patients may lead to cardiac immunopathology.

Conjugação e validação de controle isotípico IgG1-FITC para uso em citometria de fluxo / Conjugation and validation of IgG1-FITC isotype control to be used in flow cytometry

Em meados da década de 50 iniciou-se o desenvolvimento da citometria de fluxo, tecnologia que permite verificar características físico-químicas de células ou partículas suspensas em meio fluido. Esta tecnologia utiliza anticorpos monoclonais marcados com fluorocromos como ferramenta de investigação em diversas análises e necessita de controles isotípicos para definição da região negativa (background). Estes controles são constituídos por imunoglobulinas de mesmo isotipo e fluorocromo dos anticorpos testes, sendo o isotiocianato de fluoresceína (FITC) o marcador fluorescente mais utilizado na conjugação de anticorpos. Os controles isotípicos têm como função definir a fluorescência inespecífica (células negativas) e as regiões fluorescentes (células positivas). No presente estudo foi selecionado anticorpo monoclonal murino (AcMm) dirigido contra antígeno eritrocitário canino, produzido no Laboratório de Anticorpos Monoclonais do Hemocentro de Botucatu, o qual reage positivamente com hemácias de cães, mas nunca com leucócitos humanos, tendo, portanto, potencial utilidade como controle negativo em citometria de fluxo. A purificação do AcMm da subclasse IgG1 foi feita por cromatografia de afinidade em Proteína-A Sepharose, e o controle da purificação realizado por eletroforese em géis de ágarose e poliacrilamida (SDS-PAGE). A imunoglobulina purificada foi conjugada ao FITC e filtrado em coluna de Sephadex G-25 para separação das proteínas marcadas e não-marcadas. O AcMm conjugado foi testado contra hemácias de cães, e o êxito da conjugação comprovado por testes de fluorescência, sendo a mediana de positividade de 94,70. Frente a leucócitos humanos a mediana de positividade foi 0,03 contra 0,50 dos reagentes comerciais. Os testes estatísticos não-paramétricos de Wilcoxon e correlação de Spearman comprovaram a eficiência e validam o controle isotípico produzido em comparação aos reagentes comerciais testados.

It was during the 1950's that the development of flow cytometry started, technology that allow to measure physiochemical characteristics of cells or suspended particles in fluid. This technology uses monoclonal antibodies labeled by fluorochromes as investigation tool in several analysis and needs isotype controls to define the negative region (background). These controls are constituted by immunoglobulins of the same isotype and fluorochrome from test antibodies, being fluorescein isothiocyanate (FITC) the most fluorescent marker used in antibody conjugations. The isotype controls have the function to define the unspecific fluorescence (negative cells) and the fluorescent regions (positive cells). In this study was selected monoclonal antibody (mAb) against canine erythrocyte antigen, produced in the Monoclonal Antibodies Laboratory - Blood Center of Botucatu, which reacts positively with dog red blood cells, but never with human leukocytes, having therefore, utility potential as negative control in flow cytometry. The purification mAb of IgG1 subclass was made by affinity chromatography in Sepharose Protein-A and the purification control was performed by electrophoresis in ágarose and polyacrylamide gels (SDS-PAGE). The purified immunoglobulin was conjugated to FITC and after was filtered in Sephadex G25 column to separation of labeled and unlabeled proteins. The conjugated mAb was tested against dog red blood cells and the conjugation success was verified by fluorescence tests, being the median positivity of 94.70. To the human leucocytes the positivity median was 0.03 against 0.50 of the commercial reagents. The nonparametric statistical tests of Wilcoxon and the correlation Spearman showed the efficiency and validate the isotype control produced in relation to the tested commercial reagents.

Neoglycoprotein preparation of YCP, a polysaccharide from Phoma herbarum YS4108 and immunogenicity evaluation in mice / 中国药科大学学报

Aim:To characterize the immune response to YCP, a polysaccharide (PS) isolated from marine filamentous fungus Phoma herbarum YS4108. Methods:YCP was coupled to bovine serum albumin (BSA) to construct three neoglycoproteins which are different in their degrees of substitution (DSs; ≈ 3, ≈ 6, or ≈ 10 mol of BSA carrier/mol of YCP hapten, respectively), and their immunogenicities were evaluated in mice following subcutaneous immunization,respectively. IgG subclasses against the PS and the carrier protein were measured in addition to the total IgG and IgM antibodies for YCP induced by the neoglycoproteins. Results: While unconjugated PS was weakly immunogenic, theneoglycoprotein induced vigorous primary IgM and booster IgG responses to PS and the carrier protein. Interestingly,the IgG subclass distribution was different between PS and the carrier protein; for PS, the IgG response was predominant of IgG2a subclass with approximately low levels of IgG2b and IgG1, while the response to the carrier protein was mainly of the IgG1 subclass with relatively low levels of IgG2a and IgG2b, and the lowest of IgG3. Conclusion:YCP has the potential to elicit preferentially IgG2a as the dominant isotype antibody in mice.

Characterization of Mouse B Lymphoma Cells (CH12F3-2A) for the Study of IgA Isotype Switching

BACKGROUND: It is well known that IgA isotype switching is induced by TGF-beta1. LPS-activated mouse normal B cells well differentiate into IgA secreting plasma cells under the influence of TGF-beta1. Nevertheless, there are lots of difficulties in studying normal B cells in detail because it is not simple to obtain highly purified B cells, showing low reproducibility and transfection efficacy, moreover impossible to keep continuous culture. To overcome these obstacles, it is desperately needed to develop B cell line which acts like normal B cells. In the present study, we investigated whether CH12F3-2A lymphoma cells are appropriate for studying IgA isotype switching event. METHODS: CH12F3-2A B cell line was treated with LPS and TGF-beta1, then levels of germ-line (GL) transcripts were measured by RT-PCR, and GLalpha promoter activity was measured by luciferase assay. In addition, membrane IgA (mIgA) expression and IgA secretion were determined by FACS and ELISA, respectively. RESULTS: TGF-beta1, regardless of the presence of LPS, increased level of GLalpha transcripts but not GLgamma2b transcripts. However, IgA secretion was increased dramatically by co-stimulation of LPS and TGF-beta1. Both mIgA and IgA secretion in the presence of TGF-beta1 were further increased by over-expression of Smad3/4. Finally, GLalpha promoter activity was increased by TGF-beta1. CONCLUSION: CH12F3-2A cell line acts quite similarly to the normal B cells which have been previously reported regarding IgA expression. Thus, CH12F3-2A lymphoma cell line appears to be adequate for the investigation of the mechanism(s) of IgA isotype switching at the cellular and molecular levels.

The Isotype of Antiperinuclear Factor in Rheumatoid Arthritis / 대한류마티스학회지

OBJECTIVE: Antiperinuclear factor (APF)is regarded as a marker antibody with high sensitivity and specificity for rheumatoid arthritis (RA).However, the precise role of APF in the pathogenesis of RA has not yet been elucidated. Studying the isotype of APF may be a step in revealing the nature of this anti-body,but such studies have been rare,and none have been done in Korea.The present study is set out to identify the isotype of APF from sera of Korean patients with RA,and to determine whether certain isotypes are related to specific clinical aspects. METHODS: A total of 114 APF positive RA sera were tested against IgG,IgA and IgM separately by indirect immunofluorescence method,and the medical records were retrospectively reviewed. RESULTS: All 114 serum samples were positive for IgG-APF,100 positive for only IgG-APF (G group,87.7%),9 positive for IgG and IgA-APF (GA group, 7.9%),4 positive for IgG and IgM-APF (GM group,3.5%),and one sample was positive for IgG,IgA and IgM-APF (0.9%).The levels of rheumatoid factor (RF)and C-reactive protein (CRP)were different between the 3 groups (ANOVA test),and the RF and CRP of the GM group was higher than the other 2 groups (Bonferroni test). CONCLUSION: The APF identified in Korean patients with RA was always IgG-APF.IgM-APF may be used as a serological marker to assess disease activity in conjunction with CRP.The differences in the laboratory parameters between each isotype groups indicate the possibility of utilizing the isotype of APF to determine the disease activity or prognosis of RA.

Apolipoprotein E isotype distribution amongst Ramgharias of Punjab.

Apolipoprotein E is a major constituent of chylomicrons and HDL fractions of the normal blood plasma involved in lipid transfer systems. The three common alleles apoE2, apoE3 and apoE4 with six possible phenotypes have been identified at this locus in all the populations of the world studied so far, with apoE3 being the most common. The present study employed PCR restriction isotyping techniques to estimate allele frequency distribution amongst Ramgharias an artisan caste group of Punjab in North India. The methodology was developed to visualise the isotypes on PAGE using silver staining. The major genotypes observed were E2/3, E3/3 and E3/4 with allele frequencies APO E2=0.107, APOE3=0.714 AND APO E4=0.179.

Chromosome Analysis of Abortuses in Recurrent Spontaneous Aborters with Serum Anticardiolipin Antibodies / 대한산부인과학회잡지

OBJECTIVE: It has been suggestes that various mechanism of fetal loss are associated with anticardiolipin(ACA) and humoral immunity in the patients with recurrent spontaneous abortion. Thus we have investigated the relationship between ACA and chromosomal anomaly to know the clinical impact of ACA to early fetal loss as comparing to the chromosomal anomaly in the patients of recurrent spontaneous abortions. MATERIALS AND METHODS: Patients(n=88) with a history of recurrent spontaneous abortion (2 or more) between January 1, 1994 and June 30, 1999 were included in this study. Quantitative measurement of serum ACA was performed by ELISA and chromosomal analysis of chorionic villi obtained from aborted conceptuses was done by using standard G-banding technique. RESULTS: The incidence rate of ACA positive was 27%(24/88) and that of chromosomal abnormality was 57%(50/88). The incidence rate of abnormal karyotype was 54% (13/24) in ACA positive. Among 24 ACA positive, 10 had IgG-ACA positive, 10 had IgM-ACA positive and 4 had both types of ACA. The incidence rate of chromosomal anomalies was 30% (3/10) in IgG-ACA positive, 90%(9/10) in IgM-ACA positive and there was significant difference between these two groups (p=0.02). The incidence rate of chromosomal trisomy was 59% (23/37) in ACA negative, 62% (8/13) in ACA positive and there was no significance between two groups. CONCLUSIONS: The significantly low incidence of chromosomal abnormalities in conceptal products of patients with IgG-ACA comparing that of patients with IgM-ACA suggests that this isotype of antibody have influence on the genesis of spontaneous abortions in genetically normal pregnancy. In further studies, additional trials are mandatory for obtaining a definitive conclusions about relationship between pathologic changes of conceptal products and pathophysiologic effects of IgG-ACA.

T Cell Dependent Antigen-Induced Immunoglobulin Isotype Swiching and Diifferentiation of Lymph Node / 대한면역학회지

Lymph nodes, one of peripheral lymphoid organs, are the sites, where the lymphocytes receive their initial instructions for producing effector functioning resulting in humoral or cell-mediated immunity. Each lymph node consists of an outer cortex in which there are aggregates of cells constituting the follicles, B-cell areas. Some follicles have central areas called germinal centers, which stain lightly. Germinal centers are B lymphoblast cell areas arising eccentrically in primary lymphoid follicles in response to T-cell dependent antigenic stimulation and are the generally accepted sites of generation of memory B cells and undergoing isotype switching and somatic mutation. We observed the morphologic, cellular, protein and molecular events arising in mouse popliteal lymph nodes in response to T-cell dependent antigenic stimulation. In this study mice were immunized into footpads with TNP-chicken ovalbumin. The germinal center formation in primary follicles of popliteal lymph nodes was first observed 6 days after immunization and germinal centers persisted until 24 days of immunization. Lymph node cells were stained with PE-labeled anti-B220 antibody and/or FITC labeled PNA and analyzed by using FACScan. B cells (B220(+) cell) in lymph node increased after 3 days and peaked between 6 and 18 days after immunization. The proportion of germinal center B cells (B220, PNA(high) cells) among lymph node B cells was low (2%) before immunization but increased at day 6 (9%) and reached the peak (30%) at day 18. The expression of IgG1 productive mRNAs and germline transcripts were observed by using RT-PCR. The expression of IgG1 productive mRNA was detected at day 10 and continued until 24 days after immunization. The expression of IgG1 germline transcripts was observed 10 days after immunization and rapidly declined over the next one week. IgG1 anti-TNP antibody, main isotype of anti-TNP antibodies, was first detected at day 14 and reached the peak level 24 days after immunization. Taken these data together, we can conclude that the first immunological event observed from mouse popliteal lymph node in response to T-cell dependent antigenic stimulation is the increase in the number of B cells, and this event is followed by appearance of germinal center B cells and at the same time by the formation of germinal center in primary lymphoid follicles. Once the germinal center is formed, the process of isotype switching to IgG1 occurs in lymph node and antigen-specific IgG1 antibody is produced.

The Re-evaluation of the Serologic ELISA tests for the Diagnosis of Helicobacter pylori Infection / 대한임상병리학회지

BACKGROUND: Helicobacter pylori (H. pylori), a Gram negative spiral bacilli, is known to be the cause of chronic gastritis and peptic ulcer disease and is strongly associated with gastric cancer. Therefore, the rapid detection of H. pylori infection is necessary for prevention and treatment. Of the diagnostic tests currently used, the serologic tests, which makes use of the immune response, do not need a biopsy specimen. This method is relatively accurate, rapid, simple and inexpensive. We re-evaluated the clinical usefulness of the isotypes of H. pylori (IgG, IgA, IgM) antibodies for the detection of H. pylori infection. MATERIALS AND METHODS: Serum samples were obtained from 1,851 patients confirmed to have gastric or duodenal disease by gastric endoscopy from June, 1993 to December, 1994 at Hanyang University Hospital. The phenol-red urease test was done during endoscopy and the H-E stain on the gastric biopsies. Serologic tests (GAP Test IgG, IgA, IgM kits) were performed with patient sera. RESULTS: The sensitivities of the GAP EIA were 80% for IgG, 27% for IgA, and 85% for IgM. The specificities were 33%, 79%, and 14%, respectively. The detection rates of H. pylori were highest for the phenol-red urease test (88%), followed by IgM by ELISA (86%), IgG (72%), H-E stain (43%), and IgA (21%). The serum levels of IgG and IgA antibodies were higher in those with H. pylori infection than in those without, but there was no difference in IgM levels. And, no difference of serologic antibody levels according to disease state. Where follow-up was possible, the majority of IgG levels decreased, but IgA or IgM levels are not changed. CONCLUSION: A positive serologic test is incapable of discriminating between actual infection and normal bacterial colonization, or between recent and past infections. Therefore a serologic test seems unsatisfactory for confirming a diagnosis of H. pylori infection, but because the serial IgG levels of treated patients decreased significantly, we believe that this test may be used as an indirect means of assessing the response to therapy.

Effect of Anti - idiotype Antibody on Anti - DNA Antibody Production by Hybridoma Cells / 대한면역학회지

Anti-idiotype antibody (anti-id Ab) which recognizes idiotope in the variable region of immunoglobulin (Ig) can regulate Ab production by B cells in vivo and in vitro. Although it has been reported that anti-id Ab can suppress IgM production by lymphocytes or hybridoma cells without suppression of cell proliferation, the regulatory mechanism of anti-id Ab is not completely understood. We studied the effects of anti-id Ab on the production of IgG class anti-DNA Ab by hybridoma cells, on the proliferation of cells, and on the transcription levels of Ig genes. In contrast to suppressive effect of anti-id Ab on the production of IgM previously reported by others, stimulatory effects of anti-id Ab on the production of IgG by hybridoma cells as well as the proliferation of these .cells were observed. However, little effect of anti-id Ab on the transcription levels of Ig genes was observed. These results suggest that anti-id Ab can increase Ab production by stimulation of cell proliferation. Furthermore, these results suggest that the effect of anti-id Ab on the production of Ab may be determined by the difference in class of Ab produced by hybridoma cells following the treatment with anti-id Ab.