Immunoglobulin G antibodies against deamidated-gliadin-peptides outperform anti-endomysium and tissue transglutaminase antibodies in children <2 years age.

To investigate the usefulness of deamidated-gliadin-peptides-antibodies in the diagnosis of celiac disease, serology was tested in 212 children suspected with celiac disease who had undergone a small-intestinal-biopsy. For deamidated-gliadin-peptides-antibodies, two kits were tested. Positive and negative predictive values for IgA deamidated-gliadin-peptides-antibodies using the Bindazyme-kit were 89% and 74%, while the Quanta-Lite-kit had values of 89% and 85%, respectively. For the IgG subtype using the Bindazyme-kit, these values were 85% and 89%, while they were 85% and 91% for the Quanta-Lite-kit. The positive predictive values for endomysium and tissue-transglutaminase antibodies were disappointing (77% and 87%), although the negative predictive values were better (97% and 96%). When the analysis was restricted to the 41 children aged <2 years, no misclassifications occurred with IgG deamidated-gliadin-peptides-antibodies giving 100% accuracy in both kits. The positive predictive value reached 100% for tissue-transglutaminase antibodies and both kits for IgA deamidated-gliadin-peptides-antibodies, while the negative predictive value was 94% in these assays. Positive and negative predictive values for endomysium antibodies were 96% and 93%, respectively. In conclusion, although deamidated-gliadin-peptides-antibodies do not outperform anti-endomysium antibodies in the total study population, the IgG subtype seems to be the best test in children aged <2 years, reaching 100% accuracy.

T cells in cardiac allograft vasculopathy are skewed to memory Th-1 cells in the presence of a distinct Th-2 population.

Cardiac allograft vasculopathy (CAV) in heart transplantation (HTx) patients remains the major complication for long-term survival, due to concentric neointima hyperplasia induced by infiltrating mononuclear cells (MNC). Previously, we showed that activated memory T-helper-1 (Th-1) cells are the major component of infiltrating MNC in coronary arteries with CAV. In this study, a more detailed characterization of the MNC in human coronary arteries with CAV (n = 5) was performed and compared to coronary arteries without CAV (n = 5), by investigating MNC markers (CD1a, DRC-1, CD3, CD20, CD27, CD28, CD56, CD68, CD69, FOXP3 and HLA-DR), cytokines (IL-1A, 2, 4, 10, 12B, IFN-gamma, and TGF-beta1), and chemokine receptors (CCR3, CCR4, CCR5, CCR7, CCR8, CXCR3 and CX3CR1) by immunohistochemical double-labeling and quantitative PCR on mRNA isolated from laser microdissected layers of coronary arteries. T cells in the neointima and adventitia of CAV were skewed toward an activated memory Th-1 phenotype, but in the presence of a distinct Th-2 population. FOXP3 positive T cells were not detected and production of most cytokines was low or absent, except for IFN-gamma, and TGF-beta. This typical composition of T-helper cells and especially production of IFN-gamma and TGF-beta may play an important role in the proliferative CAV reaction.

TNFalpha in patients with end-stage heart failure on medical therapy or supported by a left ventricular assist device.

BACKGROUND: In the heart elevated levels of TNFalpha can cause lethal heart failure, like Dilated Cardiomyopathy (DCM). The level of TNFalpha production is in part determined by promoter gene polymorphisms. We investigated whether the TNFalpha promoter gene polymorphism is in this way involved in the outcome of end-stage heart failure and predicts whether patients require left ventricular assist device (LVAD) support or can be kept on medical therapy (MT)while awaiting heart transplantation (HTx). As most patients in this study received a heart transplant, the role of the TNFalpha polymorphisms in transplant rejection was studied as well. METHODS AND RESULTS: In twenty nine patients with DCM, 35 patients with Ischemic Heart Disease (IHD; both on MT), 26 patients on LVAD support and 61 cardiac transplant donors TNFalpha plasma level was detected by EASIA. In both patients groups high levels of TNFalpha plasma levels was observed however, in patients supported by LVAD this increase was much higher compared to patients on MT. Furthermore, this increase seems to be associated with the TNF 1 allele ('G' at position -308) instead of the TNF2 allele (A at position -308). The promoter polymorphisms at positions -238, -244 and -308 were observed by polymerase chain reaction and sequencing. Polymorphism at positions -238, -244 and -308 did not show any relevant differences between the groups. However, at position -308, a trend of a higher incidence of the TNF2 allele (an "A" at position -308) in DCM patients compared to donors was shown. The distribution of the TNF1 and TNF2 alleles was not different in patients on medical therapy compared to the patients supported by a LVAD. No association was found between patients' TNFalpha promoter gene polymorphism and rejection. However, patients that received a donor heart with the TNF2 allele developed more rejection episodes, compared to patients that received a donor heart with the TNF1 allele. CONCLUSION: TNFalpha levels are high in patients with end-stage heart failure on MT, but even higher in patients on LVAD support. These high TNFalpha plasma levels however, are not correlated with the TNF2 allele but seems to be associated with the TNF1 allele. Furthermore, in HTx the donor TNFalpha gene seem to play a more important role in severity of acute rejection than that of the patient.

Stem cell-derived cardiomyocytes after bone marrow and heart transplantation.

Cardiomyocytes are a stable cell population with only limited potential for renewal after injury. Tissue regeneration may be due to infiltration of stem cells, which differentiate into cardiomyocytes. We have analysed the influx of stem cells in the heart of patients who received either a gender-mismatched BMT (male donor to female recipient) or a gender-mismatched cardiac transplant (HTX; female donor to male recipient). The proportion of infiltrating cells was determined by Y-chromosome in situ hybridization combined with immunohistochemical cell characterization. In BM transplanted patients and in cardiac allotransplant recipients, cardiomyocytes of apparent BM origin were detected. The proportions were similar in both groups and amounted up to 1% of all cardiomyocytes. The number of stem cell-derived cardiomyocytes did not alter significantly in time, but were relatively high in cases where large numbers of BM-derived Y-chromosome-positive infiltrating inflammatory cells were present. The number of Y-chromosome-positive endothelial cells was small and present only in small blood vessels. The number of BM-derived cardiomyocytes in both BMT and HTX is not significantly different between the two types of transplantation and is at most 1%.

No association between IL-10 promoter gene polymorphism and heart failure or rejection following cardiac transplantation.

Expression of interleukin (IL)-10 influences the frequency of rejection events after organ transplantation. Therefore, 70 heart transplant patients were genotyped for three single nucleotide polymorphisms and a microsatellite polymorphism in the promotor region of the IL-10 gene. The promoter region was amplified by polymerase chain reaction and genotyped by a colorometric oligo ligation assay and gene scan analysis, respectively. Patient groups consisted of patients suffering from dilated cardiomyopathy or ischaemic heart disease. Cardiac donors served as control group. No correlation was found between genotypes and heart failure or rejection after heart transplantation. This may indicate that in heart transplantation, the total balance of cytokine production is more important for post-transplant rejection activities than the levels of IL-10 as such.

High frequency of IL-4 producing helper T lymphocytes associated with a reduced incidence of heart allograft rejection.

The reduction in the frequency of rejection episodes several months after heart transplantation (HTX) correlates with the development of donor-specific nonresponsiveness. This is reflected in a reduced frequency of donor-specific cytotoxic T cells (CTL) in the peripheral blood. We investigated whether the reduced CTL frequency and the incidence of rejection episodes coincided with a change in the frequency of either IL-2- or IL-4-producing helper T lymphocytes (HTL). We measured the frequency of HTL before and at several time points after HTX in the blood of ten recipients, using limiting dilution analysis for IL-2 and IL-4. In most patients, HTL frequencies dropped immediately after transplantation, but returned to pre-HTX values later after transplantation. No consistent decrease or increase in frequencies was observed long after HTX. In contrast to IL-2, the HTL frequencies for IL-4 before transplantation were significantly higher in patients without post-HTX rejection episodes requiring treatment than in patients with such episodes. This phenomenon was observed for the in vitro responses towards both donor and third-party cells. In conclusion, relatively high frequencies of IL-4-producing T cells may have a beneficial effect on the outcome of human heart transplantation, because they are associated with a reduced incidence of rejection episodes after transplantation.

The prognostic value of the antiperinuclear factor, anti-citrullinated peptide antibodies and rheumatoid factor in early rheumatoid arthritis.

OBJECTIVE: To study the prognostic value of the antiperinuclear factor (APF), determined by an indirect immunofluorescence test (IIF) and a recently developed anti-citrullinated cyclic peptide (CCP) ELISA, in combination with rheumatoid factor (RF) status, in early RA (< 1 year). METHODS: A total of 249 participants in a randomized trial of treatment strategies were divided into 4 groups according to their APF (or CCP) and RF status at baseline. Differences in disability, joint involvement and radiological damage over a 3-year period were analysed. RESULTS: APF-IIF results differed from CCP-ELISA in 42 cases (17%); 38 of the 42 had a positive IIF and negative ELISA value. Disability after 3 years did not differ significantly between the RF and APF groups. APF- patients had significantly lower Thompson joint scores compared to APF+ patients (6 vs 24 for CCP-ELISA; 2 vs 24 for IIF). RF+APF+ patients exhibited more radiological damage compared to RF-APF- patients. RF+APF- and RF-APF+ patients had intermediate scores. Within the RF+ and RF- groups, APF+ was associated with more radiological damage and thus yielded prognostic information in addition to RF. In this respect, the results of ELISA and IIF were comparable. Thirty percent of the RF+APF+ patients had a radiological score higher than 45, compared to 13% of the RF+APF-, none of the RF-APF+, and 2% of RF-APF- patients (p < 0.001). In addition, more large joints were affected in APF+ than in APF- patients, while no difference was observed between RF+ and RF- patients. CONCLUSION: APF has prognostic value in addition to RF for joint involvement and radiological damage in early RA. The CCP-ELISA technique for APF assessment may facilitate its use in clinical practice. However, the prognostic value of the two tests lies in their ability to predict mild disease. Reliable identification at baseline of individual patients with progressive disease is still not possible.

T cell apoptosis in human heart allografts: association with lack of co-stimulation?

It is unclear whether the intracardial immune reactivity after heart transplantation influences the peripheral immunological status (activation or nonresponsiveness) of the patient. Co-stimulation and activation-induced cell death (AICD) or apoptosis play an important role in determining the balance between lymphocyte reactivity and nonreactivity. Therefore, we studied the expression of co-stimulatory molecules and the process of apoptosis in biopsies of human heart allografts, using immunohistochemistry. Although a normal expression of co-stimulatory molecules on antigen-presenting cells was observed, the expression of their counter-structures on T cells was absent. This may be due to chronic T cell activation, which can lead to the induction of apoptosis via the Fas/Fas ligand pathway. In the infiltrates, a considerable percentage of the lymphocytes, but not the macrophages, were apoptotic. Apoptosis was confirmed by DNA fragmentation analysis. Increased numbers of Bax-expressing versus decreased numbers of Bcl2-expressing lymphocytes in comparison with normal lymphoid tissue confirmed a imbalance in favor of apoptosis. Apoptosis was biased towards CD4+ T cells (65.7% versus 26.6% in CD8+ T cells). Fas was expressed on most of the infiltrating cells. Fas ligand expression was also observed, not only on most of the T cells but also on all macrophages. Because macrophages were often detected in close contact with T cells, they may play a role in T cell regulation via the Fas/Fas ligand pathway. This study indicates that, during rejection, not only is tissue damage induced by infiltrating T cells, but also the infiltrating lymphocytes themselves are actively down-regulated (eg, AICD) by one another and by macrophages in the infiltrate. This regulatory process may affect the immunological status of the patient after heart transplantation.

Cytokine messenger RNA expression by donor-specific cytotoxic T-cell clones after allogeneic heart transplantation.

BACKGROUND: After heart transplantation, expression of various cytokines can be detected in endomyocardial biopsy specimens both in the presence and in the absence of rejection. In this study we have analyzed the contribution of donor-specific CD8+ cytotoxic T cells to intragraft cytokine expression. METHODS: T cells were propagated from endomyocardial biopsy specimens in medium containing interleukin-2 and interleukin-4. From the T-cell lines obtained, T-cell clones were generated by limiting dilution. A number of 15 CD8+ donor-specific cytotoxic T-cell clones were generated from a single T-cell line and were analyzed for their cytokine messenger RNA expression. The cytokine profile of the clones was studied on the mRNA level by reverse transcriptase polymerase chain reaction. RESULTS: Almost all clones expressed mRNA for interleukin-1 beta, interleukin-4, and interleukin-10, and 75% of the clones expressed mRNA for interleukin-1 alpha, interleukin-9, and tumor necrosis factor-beta. In about half of the clones expression of interleukin-2, interleukin-6, interleukin-8, and interferon-gamma was detected. Tumor necrosis factor-alpha was only detected in one of the clones. The cytokine profiles exhibited a considerable heterogeneity. The 15 clones had previously been analyzed for their T-cell receptor V beta-gene family expression and T-cell receptor V-D-J region sequence. Homology in the V-D-J regions of the clones indicated that the 15 clones were derived from a limited number of progenitor cells. Interestingly, clones derived from one progenitor expressed a different mRNA cytokine profile. CONCLUSIONS: This study shows that donor-specific cytotoxic T cells can contribute to the spectrum of locally produced cytokines. The cytokine expression of these cytotoxic T cells seems not to be limited to a distinct profile.

Decrease in peripheral type 1 over type 2 T cell cytokine production in patients with rheumatoid arthritis correlates with an increase in severity of disease.

OBJECTIVES: To compare peripheral type 1 (T1) and type 2 (T2) T cell activities in rheumatoid arthritis (RA) patients with that found for osteoarthritic (OA) patients and healthy controls and to correlate peripheral T1/T2 cell activity in RA with parameters of the disease. METHODS: Peripheral blood mononuclear cells were isolated from patients with RA (n = 66), OA (n = 19), and healthy controls (n = 15). Primary T cell activity in these mononuclear cells was enhanced by means of anti-CD3/anti-CD28, which mimicks stimulation of T cells by activation of the T cell receptor and a major co-stimulatory signal. Interferon gamma (IFN gamma) production and interleukin 4 (IL4) production in the three groups were quantified as measures of T1 and T2 cell activity, respectively, and compared. Serum tumour necrosis factor alpha (TNF alpha), erythrocyte sedimentation rate (ESR), C reactive protein (CRP), and joint destruction assessed radiographically of RA patients were determined as parameters of disease activity and correlated with T1/T2 cell activity. RESULTS: Peripheral T cells from RA patients produced significantly less IFN gamma and more IL4 than T cells from both age and sex matched OA patients and healthy controls. Moreover, in RA patients both a decrease in IFN gamma and an increase in IL4 production correlated with an increase in serum TNF alpha, ESR, CRP, and joint destruction. CONCLUSIONS: These results suggest a role for differential T cell activity in RA. In view of the intra-articular T1 cell predominance the results might be explained by selective T1 cell migration into the joint or peripheral suppression of T1 cell activity.

Search for the antigen-specificity of homogeneous IgG components (H-IgG) after allogeneic bone marrow transplantation.

After allogeneic BMT, transient homogeneous Ig components (H-Ig) can be detected in the sera of most graft recipients. So far, data on the antigen-specificity and therefore the function of these H-Ig are not available. Such information may be important for our understanding of the underlying mechanisms that are responsible for these excessive clonal B cell expansions, and it may help to delineate the functional antibody repertoire after BMT. In the present study, sera of 98 paediatric BM graft recipients were investigated for the potential presence of H-Ig of IgG isotype (H-IgG) with specificity towards a panel of antigens, including vaccine and herpes virus antigens, auto-antigens and allo-antigens. The vast majority of H-IgG in sera of BM graft recipients were unreactive when tested for this panel of antigens. However, in four cases, antigen-specificity of H-IgG to tetanus toxoid could be demonstrated after vaccination with that antigen. An explanation for the negative findings may be either that a restricted antibody production had been elicited by other non-tested antigens, eg substances of colonizing and translocating bacteria or of food antigens, or that the H-IgG components may have anti-idiotype or anti-'self' specificity.

Prevention and reversal of cartilage degradation in rheumatoid arthritis by interleukin-10 and interleukin-4.

OBJECTIVE: Inflammation-induced articular cartilage degradation is a major problem in rheumatoid arthritis (RA). Type 1 T cell activity (characterized by interferon-gamma/interleukin-2 [IL-2] production), and consequently, the production of the proinflammatory cytokines IL-1 and tumor necrosis factor alpha (TNF alpha), have been reported to play a major role in cartilage damage. IL-10 and IL-4, both produced by type 2 T cells, are cytokines with the capacity to down-regulate proinflammatory responses. The present study was undertaken to investigate the way in which these cytokines affect activated mononuclear cells (MNC) of RA patients in relation to human articular cartilage degradation in vitro. METHODS: MNC from synovial fluid and peripheral blood of RA patients were stimulated with bacterial antigen and treated with IL-10 and/or IL-4. Bacterial antigen is known to activate type 1 T cells and to induce proinflammatory IL-1/TNF alpha-dependent cartilage damage. Cytokine production and effects of conditioned media, as well as effects of IL-10 and IL-4 on proteoglycan (PG) turnover (as a measure for cartilage damage), were determined. RESULTS: IL-10 and IL-4 inhibited proinflammatory cytokine production of stimulated RA MNC and completely reversed inhibition of cartilage PG synthesis induced by these stimulated RA MNC. IL-10 was more potent than IL-4 in this respect, and the combination of IL-10 and IL-4 had an additive effect. In addition, IL-10 directly stimulated cartilage PG synthesis. CONCLUSION: IL-10 reverses the cartilage degradation induced by antigen-stimulated MNC, and IL-4 has an additive effect on this process. Furthermore, IL-10 has a direct stimulatory effect on PG synthesis, and IL-4, as a growth factor for type 2 T cells, can reduce the ratio of type 1 to type 2 T cell activity. These results provide evidence in favor of the use of a combination of the two cytokines in the treatment of RA.

Skewed distribution of IgG Fc receptor IIa (CD32) polymorphism is associated with renal disease in systemic lupus erythematosus patients.

OBJECTIVE: Fc gamma receptors of class IIa (Fc gamma RIIa) occur in 2 allelic forms, with either a low (IIa-R131) or a high (IIa-H131) affinity for complexed IgG2 and IgG3. This polymorphism might have implications for the handling of immune complexes. Therefore, we determined the distribution of the Fc gamma RIIa allotypes in patients with systemic lupus erythematosus (SLE), with or without a history of lupus nephritis. METHODS: We studied 95 unrelated white European patients with SLE, as defined by the American College of Rheumatology criteria, 50 of whom had a history of lupus nephritis, and 69 healthy white European control subjects. Fc gamma RIIa allotypes were determined by immunophenotyping of blood monocytes. RESULTS: It was found that lupus nephritis was significantly associated with the "low affinity" Fc gamma RIIa R/R131 allotype and with the R131 allele, compared with healthy controls. No significant association was found upon comparison of groups with and without nephritis. CONCLUSION: SLE patients with a history of lupus nephritis have an abnormal distribution of Fc gamma RIIa allotypes. Fc gamma RIIa may well play a role in the pathogenesis of lupus nephritis, since IIa-R/R131 SLE patients seem to have a higher incidence of developing this complication.

Prevention of donor-specific T-cell unresponsiveness after buffy-coat-depleted blood transfusion.

The immunosuppressive effect of blood transfusions has been demonstrated in several clinical studies. The effect is probably mediated by HLA-class-II-bearing donor leucocytes, because results from laboratory tests show specific down-regulation of the recipient's T-Cell response after administration of blood from donors sharing one HLA haplotype with the recipient. In the present study we evaluated the immunosuppressive potential of buffy-coat-depleted red cell transfusions in patients waiting for renal transplantation, by measuring the frequency of cytotoxic precursor T cells before and after transfusion. The buffy coat was removed from whole blood by the Optipress system and resulted in > 97% depletion of lymphocytes and monocytes. A single transfusion of HLA-haplotype-matched buffy-coat-depleted red cells induced donor-specific down-regulation of T-cell responses in only two of 14 patients. Since HLA-class-II-bearing cells are also involved in the induction of anti-HLA antibodies, we evaluated retrospectively the incident of HLA alloimmunization after a single transfusion of buffy-coat-depleted red cells. No anti-HLA antibodies were found in 140 patients at risk for primary immunization. We conclude that the poor immunological responses found after a single transfusion of HLA-haplotype-matched buffy-coat-depleted red cells is due to the small number of residual HLA-class-II-bearing donor cells. This blood component should not be used for induction of immunosuppression.

Reduction of donor-specific cytotoxic T lymphocyte precursors in peripheral blood of allografted heart recipients.

Tolerance to allografted hearts in human recipients has been observed both in clinical situations and in in vitro experiments. To elucidate whether a quantitative change of alloreactive CTL is one of the mechanisms accounting for this graft tolerance, CTL precursor (CTLp) frequencies in the peripheral blood of 10 heart recipients were measured against spleen cells from donors and HLA nonidentical third-party persons. In this longitudinal follow-up study, we showed that the rejection reaction(s) in the grafted heart correlated with CTLp frequencies in samples taken before transplantation against the donor spleen cells, but not with the CTLp frequencies against the spleen cells from the third-party persons. The CTLp frequencies against the spleen cells from donors decreased 4-6 months after transplantation, and remained at a low level afterward. However, the CTLp frequencies against spleen cells from third-party persons in blood samples obtained 1 year after transplantation were not significantly different from those before transplantation. Therefore, we conclude that donor-reactive CTLs are important in rejecting allografted heart. The decrease in donor-specific CTLp after transplantation could explain the donor-specific tolerance. The decrease may be due to homing of the specific CTLp to the graft, or by clonal deletion of the donor-reactive CTL caused by chronic alloantigen stimulation in the presence of immunosuppressive therapies.

Cytotoxic T lymphocytes infiltrating the human cardiac allograft show a restriction in T-cell receptor V beta gene usage: a study on serial biopsy and blood specimens.

T lymphocytes were propagated in vitro from endomyocardial biopsy specimens that were obtained weekly from four patients during the first 2 to 3 months after heart transplantation. The culture was performed in the presence of recombinant interleukin-2 and interleukin-4, with or without mitogen, in which especially CD8+ donor-specific cytotoxic T cells expanded. These cells, presumably reflecting an in vivo activated population, could even be cultured from biopsy specimens without histopathologic signs of rejection. A preferential expression of T cell receptor V beta gene families was found in these T-cell lines. This finding is in contrast with the heterogenous expression in peripheral blood T cells of the same patient. The restriction in V beta gene family expression was substantiated in the evaluation of clones obtained from two cell lines. Among 17 donor-specific cytotoxic T-cell clones derived from one cell line, only four V beta gene families were expressed. All five clones from the other cell line used the V beta 8 family. Some clones expressing a distinct V beta gene family used the same V-D-J junction sequence, indicative of their origin from the same precursor. With the use of oligonucleotide probes complementary to clone-specific V-D-J junction sequences, four of five clones were detected not only in the parent T-cell line but also in T-cell lines derived from biopsy specimens with rejection reactions taken 1 week earlier and 2 weeks later and in blood cells taken before and 0.5, 3, and 6 months after transplantation; these clones were not detected in blood cells harvested 12 months after transplantation. This study showed a restricted usage of the V beta gene families by activated donor-specific cytotoxic T lymphocytes in the heart transplant.

Cytoimmunologic monitoring as an adjunct in monitoring rejection after heart transplantation: results of a 6-year follow-up in heart transplant recipients.

The cytoimmunologic monitoring assay has been proposed as a useful noninvasive technique in the diagnosis of rejection and infection after heart transplantation. In this study, we have analyzed the diagnostic usefulness of cytoimmunologic monitoring in 73 patients after heart transplantation. For individual patients, the follow-up varied between 2 and 78 months. Data were related to histopathologic characteristics of the endomyocardial biopsy. Significantly different cytoimmunologic monitoring results were not observed between groups according to endomyocardial biopsy histopathologic evaluation. The diagnostic usefulness of cytoimmunologic monitoring depended on the cutoff value applied. With higher cutoff values, the sensitivity decreased and the specificity and predictive value increased. For the previously reported cutoff value of 5%, the sensitivity was 0.29, the specificity was 0.73, and the predictive value was 0.66. Values of sensitivity, specificity, and predictive value were similar when only the first acute rejection was taken into account, or when only data on the first 4 weeks and the first 6 months after transplantation were considered. In calculating the diagnostic usefulness of the sensitivity, specificity, and predictive values were observed. We concluded that cytoimmunologic monitoring has a limited value for diagnosing acute rejection after heart transplantation.