Tissue distribution of MHC class II--positive cells, their down-manipulation by monoclonal antibodies and potential role in organ allograft immunogenicity.

This review article presents current state of knowledge of tissue distribution of MHC class II antigens in rat kidney. Our attention focuses particularly on down manipulation of these antigens by monoclonal antibodies. The results of organ perfusion procedure on prolongation of graft survival and perspectives of further studies are also discussed.

Influence of recipient pretreatment with donor spleen cells and 15-deoxyspergualin on rat skin graft survival.

Experimental studies performed in rat kidney transplantation showed that treatment with 15-deoxyspergualin (15-DOS) for 14 days after grafting may induce both permanent graft function and specific immune tolerance. The aim of this study was to check if donor spleen cell transfer and 15-DOS pretreatment before transplantation prolong skin graft survival. Pretreatment of the recipient with donor cells induced slight prolongation of subsequent skin graft survival. Addition of 15-DOS pretreatment for 14 days in a dose of 2.0 mg/kg b.w. to cell transfer did not influence survival of the graft. On the other hand, in the recipients receiving allogeneic spleen cells before transplantation and subtherapeutic doses of 15-DOS directly after grafting the prolongation of skin graft survival was observed. These results indicate that pretreatment of the recipients with donor cells before allogeneic skin transplantation allows for reduction of immunosuppression after grafting.

Hematological effects of the new immunosuppressive drug 15-deoxyspergualin.

Since systematic hematological studies on blood and bone marrow changes after treatment with 15-Deoxyspergualin (DOS) are lacking, a quantitative assessment was performed fourteen or twenty eight days after intraperitoneal application of DOS to rats. Further observations done 7 and 14 days after discontinuation of DOS administration allowed analysis of bone marrow regeneration. DOS induced lymphocytopenia, granulocytopenia and anemia with a decrease of bone marrow cellularity due to suppression of cell maturation. The effect was dose-dependent and bone marrow as well as blood changes were observed in animals treated with doses from 0.5 to 10.0 mg/kg DOS. Within 14 days after termination of the treatment, rapid recovery with normalization of all hematological parameters was observed. In the light of our data, these hematological side effects may not be a major disadvantage, if DOS is used in doses below 2.5 mg/kg, and for a course of therapy which is limited to 7 to 14 days.

Cell migration between graft and host--an analysis with monoclonal antibodies after allogeneic rat kidney transplantation.

In order to analyse migration patterns of donor MHC class II cells out of transplanted kidney and accumulation of host cells within the graft, immunomorphological studies were performed using monoclonal antibodies in rat allogeneic kidney transplantation model. To answer the question of how many donor cells migrate out of the renal cortex MRC 0 x 3 monoclonal antibody (MoAb) against LEW MHC class II antigens was used. In the grafts explanted after 4,24 48 and 73 h, a slow reduction of donor class II cells was observed and some areas in cortex showed only very few, if any, donor cells. At the same time, starting from day 2 after transplantation accumulation of donor cells was found in perivascular spaces. Spleen sections stained at 24, 48, 72 and 96 h after transplantation revealed donor cells present in recipient's spleen. They were detected up to day 3 after surgery. Their numbers, however, decreased after day 2. After 2 and 3 days, accumulations of recipient's cells between tubules were detected. It was found that many cells in infiltrations were stained with anti-T lymphocyte MoAb. Expression of class II antigen on rat kidney cells increases significantly from the day 4 after transplantation.

Analysis of tolerance inducing mechanism after application of oncogen-transformed macrophages.

The purpose of this study was to examine the expression of T cell receptors (TCR) and their V beta subclasses under the influence of the parental cell line P388D1 and its clones mos2 and mos3, using a mouse model. It was shown, that v-mos oncogene-transformed cells of this line (mos2) induced selective immunological unresponsiveness in vitro. Because the induction of tolerance is of a central importance for the organ transplantation, this phenomenon, found in vitro, was also studied in vivo. We found that the in vivo injection of mos2 cells into mice induced a state of selective noncreativity. To further analyse these effects, we studied whether specific tolerance is the consequence of a decreased number of essential receptors or receptor families. For this purpose C57BL/6 mice were immunized with cells of the parental line P388D1 or mos2 and mos3 clones. Their spleen and thymus cells were examined phenotypically. The most impressive result of this study was a clearly changed amount of T cells receptors in mos2 immunized mice, in which a state of tolerance was induced. In these mice only the expression of CD3 T receptors as well as that of the V beta 11 chains was reduced. In spleen of these mice the CD3 expression was decreased, compared to D1 or nonimmunized control animals by 54-58% and compared to mos3 mice by 38-40%. Even though the differences in the thymus were not very pronounced, we still saw a decrease in CD3 stained cells selective in mos2 immunized C57B1/6. The expression of V beta 11 chains on the surface of spleen cells of mos2 animals was reduced by 33.3%, on the thymocytes even by 50% comparing to that in nonimmunized mice. Whether the reduced expression of T receptor V beta families is due to changes in the genetic material (cDNA), has to be studied.

Induction of specific tolerance by 15-deoxyspergualin (DOS) treatment after rat skin and kidney transplantation in rats. Analysis of effectivity of various protocols of DOS application.

The purpose of this study was to analyse the effect of various protocols of 15-deoxyspergualin (DOS) application on skin or kidney graft survival. Following rat strain combinations were used: AS-->LEW (MHC identical/non-MHC-different) and DA-->LEW (MHC-different/non-MHC-different). Reference DOS dose was 2.5 mg/kg, i.p. It was shown that the effect of DOS depended on multiple factors, such as: type of tissue or organ, onset of treatment, drug dose and length of drug application. In skin transplantation graft survival was 32-34 days in AS-->LEW and 24-26 days in DA-->LEW. Kidney graft survived more than 150 days. DOS prolonged skin survival when the application was started earlier than day 8, whereas kidney graft survived only when DOS treatment was started not later than 3-4 days after transplantation. In skin transplantation a dose of 0.3 mg/kg had a small effect-prolongation graft survival up to 4 days. Higher doses induced longer graft survival, however, maximal survival of allogeneic skin was 22 days. In kidney transplantation a dose of 0.3 mg/kg led to prolonged graft survival-up to 150 days. Doses of 2.0-2.5 mg/kg were able to induce specific tolerance. The optimal skin or kidney graft survival was obtained when DOS was applied for 14 days. Shorter than 12-day treatment with DOS led to a shorter graft survival. When donor was pretreated with DOS prolongation of non-allogeneic graft survival was observed. Our results showed that short-term application of DOS is safe and effective. To obtain optimal DOS effect the drug application must be started directly after transplantation.

In vitro analysis of the mode of action of the immuno-suppressive drug 15-deoxyspergualin.

The purpose of the study was to analyse the action of the immunosuppressive drug 15-deoxyspergualin (DOS) in vitro. We studied: a) the influence of DOS alone and DOS in combination with various monoclonal antibodies on alloantigen stimulation in the mixed lymphocyte culture (MLC), b) the influence of DOS treatment on the MHC class I and II expression of splenocytes, lymph node cells and peritoneal macrophages, c) the influence of DOS treatment on a suppressor cell population. Our study showed that: a) DOS inhibits interleukin 1 (IL-1) secretion by macrophages, leading to reduction of immune response to alloantigens. This effect was neutralized by addition of IL-1; b) DOS treatment has no influence on MHC class II antigen expression, but induces changes of MHC class I expression. After DOS application in a population of spleen macrophages a subpopulation of cells with reduced MHC class I antigen expression appeared. Down-regulation of these molecules was also observed in immunomorphological studies of kidney graft sections of rats treated with DOS after transplantation; c) after DOS treatment suppressor cells were detected in "suppressor" MLC, 16-33 days after kidney transplantation. Their activity was confirmed 137 days after treatment with DOS, but were inactive in the case of third party cells. These results suggest that DOS action is based on a blockade of antigen presentation by reducing IL-1 production, down-regulation of MHC class I antigen and by inducing suppressor cell population.

Influence of a rat kidney perfusion using the anti-MHC class II monoclonal antibody and reduced dose of 15-deoxyspergualine on the allogeneic graft survival.

One of the main factors inducing rejection of the allogenic graft are the donor MHC-class II antigens. In this study, the allogenic rat renal graft survival after the blockage of MHC-class II positive cells was analyzed and compared with the effectiveness of the recipient treatment with 15-deoxyspergualine (15-DOS). It was found that the DA (RT1 a) rat kidney perfusion with the anti-MHC class II monoclonal antibody (MoAb 29A1--Kiel) allowed to prolong survival of the graft in the LEW (RT1 1) recipient (9.6 +/- 0.8 vs. 7.7 +/- 0.5 days). Our another study demonstrated that the 14 day treatment of the LEW recipient with 15-DOS at the dose of 0.5 mg/kg body weight can induce tolerance to the grafted kidney from the DA strain. The dose of 0.2 mg/kg body weight of 15-DOS prolonged the graft survival only to a small extend (16.5 +/- 0.5 days). In contrast, the combination of the graft pretreatment with MoAb 29A1 with the application of the reduced dose of 15-DOS to the LEW recipient allowed to further prolong the graft survival (97.4 +/- 59.0 days, n = 5). In 3 cases, the long-time (close to 150 days) graft survival was obtained. The above presented results suggest that the blockade of the MHC-class II antigens can reduce the immunogenicity of the graft. Although this procedure was not sufficient to induce tolerance, it allowed to minimize the immunosuppressive treatment.

Morphometric analysis in the diagnosis and differentiation of certain glomerulopathies (MCD, GNMes, FSG)

Morphometric analysis was used to evaluate mesangial components in MCD (minimal changes disease), GNMes (mesangial glomerulonephritis), FSG (focal segmental glomerulosclerosis). In GNMes the increase of matrix was found to be generally proportional to the amount of mesangial cells. There are clear statistically significant differences in the ratio of matrix volume to cell component volume and of matrix volume to the whole mesangial area in MCD and GNMes as compared with FSG. In the case of GNMes where morphometric results resemble those in FSG cautious prognosis is recommended as there is a possibility of FSG. This has been confirmed both by the course of the disease and the results of repeated biopsy.

Use of radioimmunoassay for the detection of circulating antistreptococcal antibody in patients with glomerulonephritis.

A method of radioimmunologic quantitation of antibodies to streptococcal antigen separated from the cell wall extract of group A type T12 strain has been developed. The highest values of radioactive antigen binding were observed in acute glomerulonephritis (75%), as compared to chronic glomerulonephritis in which values of 25% to 56% were found depending on the morphology of renal changes. It was shown that none of the patients with pyelonephritis, Alport's syndrome, lupoid nephritis and polycystic renal disease had elevated antistreptococcal antibody levels. In contrast to this, all patients with tonsillitis and proteinuria exhibited increased titre of this antibody. It was shown that the antigen is related neither to M-protein nor to group A polysaccharide and that it is not type-specific because the binding of antigen T12 may be inhibited by the antigen produced from strain T5. Although the antigen is not type-specific, some differences in the response to antigens prepared from various types of streptococci in patients with different forms of chronic glomerulonephritis are observed.

Humoral immunity to streptococcal antigen in acute and chronic glomerulonephritis.

A study was carried out to verify the clinical usefulness of the elaborated method for the measurement of antistreptococcal antibody in revealing the streptococcal etiology of glomerulonephritis. In 158 patients with glomerulonephritis antistreptococcal antibody (ASA), circulating immune complexes (CIC) and haemolytic activity of the complement were measured. On the basis of immune complex formation it has been concluded that streptococcal infection may cause glomerulonephritis. Serial determinations of ASA and CIC are helpful in establishing the streptococcal etiology of glomerulonephritis and in monitoring the course of the disease.

[Anti-HCV antibodies among hemodialysed and kidney-transplanted patients]. / Markery hepatitis C (anty-HCV) u chorych dializowanych i po przeszczepie nerek.

We have studied from two hemodialysed centers to determine the prevalence of hepatitis C virus infection in hemodialysis 142 patients and 147 renal-transplant patients. Serum samples were tested for antibody by "Ortho HCV 2.0 ELISA test System" second generation assay. Antibody to hepatitis C virus was detected in 81 (57.04%) and 85 (57.82%) patients respectively. Age-related differences were not clear, but significantly were noted in hemodialysis patients. Patients with a history blood transfusion in both groups tended to have significantly high anti-HCV positivity rate. There was a tendency for a longer duration of hemodialysis to coincide with higher positivity anti-HCV.

[The role of tubulointerstitial changes in progression of kidney function failure in patients with chronic glomerulonephritis (GN)]. / Rola zmian cewkowo-sródmiazszowych w postepujacym uposledzeniu funkcji nerek u chorych na przewlekle klebuszkowe zapalenie nerek (KZN).

In most cases of glomerulonephritis (GN) long-term course lead to chronic renal failure. The cause of inevitably gradually progress of GN to end-stage renal disease (ESRD) is unclear. The histological abnormalities seen in patients with progressive renal failure consist of focal and segmental glomerulosclerosis and tubulointerstitial nephritis. At present it is considered that tubulointerstitial changes attends almost all forms of progressive glomerular and vascular injury. It was known that chronic tubulointerstitial nephritis is characterized morphologically by tubular atrophy, interstitial fibrosis and interstitial inflammation of variable severity. The pathomechanism of this changes is complicated. Tubular ischaemia results from obliteration of peritubular capillaries, adaptation of tubular function with increased oxygen consumption and increased glomerular capillary permeability to macromolecules are reasons of chronic tubular damage. Injured tubules release growth factors and cytokines, which induce interstitial fibroblast proliferation, chemo-attraction and proliferation of infiltrating cells, and disruption of the balance between synthesis and degradation of cellular constituents. The consequences of these processes are tubular atrophy and interstitial fibrosis. Because of many studies concurred that tubulointerstitial changes determinant the progression of GN, tubular injury markers were searched for. Although over 50 enzymes were detected in human urine, only a few have been used for diagnosis in renal disease. The most widely used are lysosomal enzyme N acetyl-beta-D-glucosaminidase (NAG) and brush border enzymes alanine-aminopeptidase (AAP) and gamma-glutamyltransferase (GGT). tubular damage in hypertension, diabetes and in diagnostics of renal disease. AAP and GGT, brush border enzymes seem to be sensitive markers of renal injury too. Pathological value of GGT was observed even in the early stage of disease. Measurement of urinary excretion of low molecular weight proteins was valuable supplement in estimation of tubulointerstitial system malfunction. These proteins are readily filtered by normal glomeruli and virtually completely reabsorbed by normal proximal tubules. Favour are alpha-1-microglobulin (alpha-1-m) and retinol-binding protein (RBP) because they are less affected than beta-2-microglobulin (beta-2-m) by low urine pH. Above presented review confirm that further research in correlation between activity of disease, histological picture, deterioration in renal function and changes in urinary excretion of markers proteins (for example alpha-1-m, AAP, NAG, GGT) is advisable, and can contribute to use in clinic diagnostics of GN.

[Urinary excretion of alpha-1-microglobulin and complement components in patients with chronic glomerulonephritis]. / Wydalanie alpha-1-mikroglobuliny i skladowych dopelniacza z moczem chorych na prezewlekle klebkowe zapalenie nerek.

The urinary excretion of alpha-1-micro-albumin (alpha 1m) and complement components (CCs) was evaluated in the urine of 49 patients suffering from chronic glomerulonephritis (GN). Nephrotic syndrome (NS) was shown in 18 cases and increased serum creatinine level (Pcr: 115.0-159.1 mumol/l) in 9 patients. The most frequent CCs presence and the highest values of alpha 1m excretion were found in patients with membranous GN. In the early phase of the disease the alpha 1m urinary excretion was higher in subjects with NS than in those not showing the feature of it, independently of the morphological basis of the disease. Also CCs were detected mainly in the nephrotic patients. As the glomerular filtration improved a significant decrease in the urinary alpha 1m excretion was observed. The application of steroid immunosuppressive therapy resulted in the decrease of alpha 1m as well as CCs excretion. The results seem to point out that the increased alpha 1m and CCs excretion may be secondary to the activity of glomerular alternations as well as to the disturbances in glomerular blood flow.

[Prolongation of allogenic rat renal graft survival after perfusion the organ with anti-MHC class II antibody]. / Przedluzenie przezycia allogenicznego przeszczepu nerki szczura pod wplywem perfuzji narzadu przeciwcialem monoklonalnym skierowanym przeciwko antygenom zgodnosci tkankowej klasy II.

One of the main factor inducing rejection of the allogenic graft are the donor MHC-class II antigens. The cytotoxic damage or the elimination of these cells from the organ may influence his function after grafting. In this study, the influence of the continuous perfusion of the rat kidney with monoclonal antibody against rat MHC-class II antigens (MoAb 25D5) on graft survivals was investigated. For the perfusion, the HTK solution containing MoAb 25D5, rat complement (C) and Pentoxifilline (Ptx) was used. After one-hour perfusion at 21 degrees C kidney were transplanted to the syngenic (LEW-LEW) or allogenic (Da-LEW) recipients, kidney perfused with the solution containing MoAb 25D5 and C survived statistically longer (14.0 +/- 2.4 days) than in the control group without perfusion (7.7 +/- 0.5 days) or after perfusion with the solution containing only Ptx (7.8 +/- 0.8 days). It seems likely that the continuous perfusion of the rat kidney with HTK protective solution containing MoAb against MHC-class II antigens and C can reduce the immunogenicity of the graft.