Jozef Stachowski

Relationship Between the Reactivity to Hepatitis B Virus Vaccination and the Frequency of MHC Class I, II and III Alleles in Haemodialysis Patients

To study the immunoreactivity genes in a heterogeneous human population needs a large number of individuals. Associations between HLA antigens and immunoresponse to viral or bacterial antigens have been studied with controversial results. As a homogeneous population, the MHC class I, II and III allele distribution was studied in 153 end‐stage renal disease patients (ESRD, average duration of renal replacement: 8.2+5.1 years) immunized with a recombinant hepatitis B vaccine in accordance to the standard vaccination schedule. Thirty‐four patients with an antibody titre of less than 10 U/l following the last booster injection were considered as non‐responders while 119 patients with antibody titre equal to or more than 10 U/l were considered as responders. The responder group was divided into two subgroups: low responders (antibody titre: 1000 U/l) and high responders (antibody titre: > 1000 U/ 1). Marked differences were observed between responders and non‐responders in the occurrence of carriers of different MHC class I, II and III alleles. Homozygotes for HLA—A1, HLA—B8, HLA—DR3 and HLA—DQ2 were found almost exclusively in the non‐responder group and significantly more heterozygotes for these alleles were found in the non‐responder group compared to the responders. Similar albeit less marked differences were found in the frequency of some MHC class III alleles (C4A*6, C4A*QO, Bf*F, BPS0.7). Within the responder group, carriers of HLA—A2, HLA—B7 and HLA—DR4 were found to be clustered in the low responder sub‐group whereas carriers of HLA—A1, HLA—B27, HLA—Cw2, C4A*6 and Bf*F were observed more frequently in the group of high responders. Similar differences were found with extended haplotypes as well. For example, the extended haplotypes HLA—Al, B8, BfS, C4AQO, C4B1, DR3, DQ2 and HLA—A1, B8, BfF, C4A6, C4B2, DR3, DQ2 were present in nine of 34 cases of non‐responders but only in one of 119 case of responders (P <0.000001). These observations indicate that the presence or absence of certain MHC alleles even in heterozygous form determine the responsiveness to hepatitis B vaccination in end‐stage renal disease patients, and among responders, the intensity of antibody response is also markedly influence by immunogenetic factors.

Th1/Th2 balance and CD45-positive T cell subsets in primary nephrotic syndrome

Abstract T cells are involved in the pathogenesis of nephrotic syndrome (NS). The aim of the study was to determine whether the activity of T-helper-1 (Th1) and T-helper-2 (Th2) cells and the distribution of the lymphocyte subsets, namely CD45RA+CD4+ (”naive” helper T cells, suppressor-inducer), CD45RA+CD8+ (”naive” suppressor T cells, suppressor-effector), CD45RO+CD4+ (”memory” helper T cells), are predictive for steroid sensitivity in children with primary NS. These parameters were assessed at the onset of disease, before initiation of steroid therapy. Two groups of NS children were retrospectively formed according to steroid sensitivity (SS) or resistance (SR). The activity of Th1 and Th2 cells was defined by the production of interleukin-2 (IL-2), interferon-γ, IL-4, and IL-10 in the supernatants of CD4+ T cell cultures activated with autologous monocytes presenting tetanus toxoid (TT). Peripheral lymphocyte subsets were determined using double- or triple-color flow cytometry. In SS children with NS we found a decreased proliferative response of CD4+ T cells to TT stimulation, cytokine synthesis indicating the predominance of Th2 activity, and an increased percentage of activated suppressor-inducer (CD45RA+ CD4+CD25+, 5.18±0.8, P<0.001) and suppressor-effector (CD45RA+CD8+CD25+, 2.05±0.6, P<0.01) cells, with the concomitant reduction of activated memory cells (CD45RO+CD4+CD25+, 0.2±0.1, P<0.001). In children with SRNS we found an increased proliferative response of CD4+ T cells to TT, a rise in activated memory (CD45RO+CD4+CD25+, 3.82±0.7, P<0.01) and suppressor-inducer peripheral T cells (CD45RA+ CD4+CD25+, 3.85±0.6, P<0.01), but a low percentage of activated suppressor-effector (CD45RA+CD8+ CD25+, 0.5±0.2, P<0.05) T cells. We conclude that prior to treatment the distribution of lymphocyte subpopulations in peripheral blood together with Th1 and Th2 cell activity provides a useful tool for evaluating the likelihood of steroid sensitivity in patients with primary NS.

Genetic regulation of the impaired immune response to hepatitis-B vaccine associated with low TCR density in end stage renal disease patients: contribution of complement C4 and factor B alleles.

We have studied the relationship between T-cell receptor (TCR) density, genetic factors and the specific immune response in 153 end stage renal disease (ESRD) patients on haemodialysis immunised with HBsAg vaccine. One-hundred and nineteen patients raised a protective (> 10 U/ml) antibody response to hepatitis-B vaccination (responder, R), while 34 patients were found to be non-responders (NR). The density of the T-cell receptors was determined by flow cytometry. Proliferation of the T-cells induced by autologous monocytes presenting HBsAg was also measured and expressed as a stimulation index (SI). MHC class I, II and III alleles of the patients were also determined. The densities of TCR/CD3 receptors in NR patients were found to be significantly decreased as compared to the R patients (189 +/- 22 vs. 282 +/- 58 arbitrary units, P = 1.3 x 10(-7). TCR/CD3 receptor densities were found to be strongly associated (Spearman correlation coefficient: 0.84, P < 0.000001) with the SI values. Both parameters were found to be under dual genetic control: (a) very low density of the TCR/CD3 receptors and very low SI were found mainly in NR patients carrying HLA-A1, HLA-B8 and HLA-DR3 alleles; and (b) TCR/CD3 densities and function in R group were found to be significantly lower in carriers than in non-carriers of two MHC class III complement protein alleles: C4A*6, and Bf*F. Non-responsiveness to hepatitis-B vaccination was found to be associated with extremely increased neopterin levels. These findings indicate that both genetic and acquired factors contribute to the hepatitis-B vaccination failure in ESRD patients.

Effect of Pseudomonas aeruginosa exotoxin A on IFN-γ synthesis: expression of costimulatory molecules on monocytes and activity of NK cells

The aim of the study was (1) to evaluate the effect of Pseudomonas aeruginosa Exotoxin A (P-ExA) on the production of IFN-gamma in anti-CD3 induced human peripheral blood mononuclear cells (PBMC) and (2) to establish the effect of P-ExA on the IFN-gamma dependent cellular activities such as the expression of costimulatory molecules on monocytes and cytotoxicity of NK cells. The toxin in a high dose (100 ng/ml) inhibited IFN-gamma synthesis. Inhibitory effect of P-ExA was abolished by IL-1alpha which in a combination with P-ExA exerted a strong synergistic effect on IFN-gamma synthesis. Other monokines such as IL-1beta, IL-6, TNF-alpha neither reversed the inhibitory effect of P-ExA nor induced production of IFN-gamma. P-ExA also inhibited IFN-gamma-induced cellular events: (1) expression of costimulatory molecules on monocytes (CD80, CD86, ICAM-1, HLA-DR); (2) cytotoxic activity of NK cells. Inhibition of NK cells activity by P-ExA was not reversed by cytokines such as IL-2, IFN-alpha and TNF-alpha, which are known to enhance effector functions of NK cells. From these results we conclude that: (1) inhibition of IFN-gamma synthesis, as well as IFN-gamma-induced expression of costimulatory molecules and NK-cell effector functions may lead to suppression of specific and non-specific defense mechanisms, respectively, which are necessary for elimination of PA bacteria; (2) enhancement of IFN-gamma synthesis induced by P-ExA in a combination with IL-1alpha may cause harmful, Th1 cells dependent, inflammatory reactions of the host (septic shock, tissue damage) during infection with Pseudomonas aeruginosa.

EFFECT OF PSEUDOMONAS AERUGINOSA EXOTOXIN A ON CD3-INDUCED HUMAN T-CELL ACTIVATION

The effect of Pseudomonas aeruginosa (PA) exotoxin A (P-ExA) on CD3-induced T-cell activation was studied on the level of T-cells (proliferation, synthesis of interleukin (IL)-2, expression of IL-2R complex, ICAM-1,2 and LFA-1 molecules), and on the level of monocytes (expression of ICAM-1,2, LFA-1 molecules, as well as FcRI and CD14 receptors). We found that: (1) P-ExA blocked T-cell proliferation and this effect was totally reversed by intact monocytes, and partially by IL-2 or TPA but not by costimulatory cytokines (IL-1alpha, IL-1beta, TNF-alpha or IL-6); (2) P-ExA transiently, in short-term cultures (48 h), inhibited synthesis of IL-2; (3) prolonged stimulation (96 h) of peripheral blood mononuclear cells (PBMC) or CD4 + T-cells with P-ExA in high or low doses (100 and 10 ng/ml, respectively), enhanced the level of IL-2 in the cultures; (4) P-ExA at low dose, combined with IL-1beta, TNF-alpha or IL-6, up-regulated synthesis of IL-2; and (5) stimulation of T-cells with anti-CD3 monoclonal antibody (mAb) and P-ExA at high dose diminished the expression of the p55 chain but not of the p75 chain of IL-2R complex and slightly affected the expression of CD3 complex, ICAM-1,2 and LFA-1 molecules. Hence, P-ExA can regulate the level of IL-2 in cultures of CD3-induced T-cells either by inhibition of IL-2 consumption (when P-ExA is applied in high dose), or by induction of IL-2 production (a costimulatory effect exerted by P-ExA in low dose in combination with monokines). Action of P-ExA on monocytes resulted in: (1) inhibition of the expression of ICAM-1,2 molecules and their ligand LFA-1 molecule; (2) low expression of FcRI receptor (a ligand for Fc part of CD3 mAb); and (3) inhibition (over 90%) of the expression of CD14 molecule. In conclusion, P-ExA-induced anergy of T-cells depends on: (a) decrease in the affinity of IL-2R complex on activated T-cells; and (b) inhibition of the accessory activities of monocytes.

Immunologische, alloantigenabhängige Faktoren in der chronischen Transplantatabstoßung

BACKGROUND The pathogenesis of chronic renal allograft rejection is still speculative. Amongst other factors immune-mediated graft injury is proposed. Since the allo-antigen is specifically recognized by the variable (V) alpha and beta chains of the T-cell receptor, a restricted T-cell repertoire might support the notion of allo-antigen involvement in chronic rejection. METHODS By the means of semiquantitative polymerase chain reaction the V beta families 1-20 were assessed in allograft biopsies with histologically confirmed chronic and acute rejection. At the same time the V beta repertoire was analyzed in PBMC. RESULT The intragraft V beta repertoire was limited to 1 to 3 dominant V beta families in chronic and acute rejection. The response was highly individual and did not correlate to the type or degree of HLA mismatches. The T-cell repertoire in PBMC was polyclonal and did not reflect the immune response in the graft. CONCLUSION The finding of a restricted V beta repertoire in both forms of rejection might indicate an immunological basis not only for acute, but also for ongoing chronic rejection. Tailor-made antibodies against the dominant V beta clones might provide a tool for selective immunosuppression in both entities of rejection targeting only those T cells which were activated by allo-antigens.Zusammenfassung□ HintergrundNeben nichtimmunologischen Faktoren werden in der Pathogenese der chronischen Transplantatabstoßung auch alloantigenabhängige, immunologische Faktoren diskutiert. Die T-Zelle ist der Promotor der akuten Abstoßung. Die variablen (V) Regionen der α- und β-Ketten des T-Zell-Rezeptors erkennen spezifisch das Antigen und bestimmen die Klonalität der T-Zelle. Eine auf wenige dominante Vβ-Klone limitierte T-Zell-Antwort könnte auf allo-HLA-Antigen vermittelte Immunprozesse in beiden Abstoßungsformen hinweisen.□ MethodikMittels der Polymerasekettenreaktion wurde in Biopsien von Transplantatnieren mit histologisch gesicherter chronischer oder akuter Abstoßung und in peripheren Lymphozyten das T-Zell-Repertoire Vβ 1–20 quantifiziert.□ ErgebnisPatienten sowohl mit akuter als auch mit chronischer Abstoßung zeigten ein auf eine bis drei dominante Vβ-Familien begrenztes Repertoire. Die Immunantwort war individuell und korrelierte nicht zu Art und Anzahl der HLA-Mismatches. Im peripheren Blut war die Vβ-Verteilung polyklonal und spiegelte somit nicht die Immunvorgänge im Transplantat wider.□ SchlußfolgerungDie limitierte T-Zell-Antwort nicht nur in der akuten, sondern auch in der chronischen Abstoßung weist auf immunvermittelte Prozesse in der Pathogenese der chronischen Transplantatabstoßung hin. Eine selektive Immunsuppression zum Beispiel in Form von anti-Vß-Antikörpern, die sich nur gegen die von allo-HLA-aktivierten dominanten T-Zell-Klone richten, könnte einen Therapieansatz für beide Abstoßungformen bilden.Summary□ BackgroundThe pathogenesis of chronic renal allograft rejection is still speculative. Amongst other factors immune-mediated graft injury is proposed. Since the allo-antigen is specifically recognized by the variable (V) α and βchains of the T-cell receptor, a restricted T-cell repertoire might support the notion of allo-antigen involvement in chronic rejection.□ MethodsBy the means of semiquantitative polymerase chain reaction the Vß families 1–20 were assessed in allograft biopsies with histologically confirmed chronic and acute rejection. At the same time the Vβ repertoire was analyzed in PBMC.□ ResultThe intragraft Vβ repertoire was limited to 1 to 3 dominant Vβ families in chronic and acute rejection. The response was highly individual and did not correlate to the type or degree of HLA mismatches. The T-cell repertoire in PBMC was polyclonal and did not reflect the immune response in the graft.□ ConclusionThe finding of a restricted Vß repertoire in both forms of rejection might indicate an immunological basis not only for acute, but also for ongoing chronic rejection. Tailor-made antibodies against the dominant Vβ clones might provide a tool for selective immunosuppression in both entities of rejection targeting only those T cells which were activated by allo-antigens.