Michio Ishibashi

Human Herpesvirus 6 Infection In Renal Transplantation

The relationship between renal transplantation and human herpesvirus 6 (HHV-6) infection was studied. All 21 kidney donors examined had antibody to HHV-6 at the time of transplantation. The 21 kidney recipients also had detectable antibody to HHV-6 before transplantation--and, of these, 8 patients showed a significant increase of serum antibody titer against HHV-6 after transplantation. All these 8 recipients suffered severe kidney rejection. Furthermore, virus isolation from peripheral blood lymphocytes of 2 recipients who suffered rejection was attempted, and in both cases HHV-6 was isolated. Biopsy specimens of rejected kidneys of 9 other patients were examined for the presence of HHV-6 antigens, and in 5 of these specimens antigens were detected in the tubular epithelium, as well as in infiltrating histiocytes and lymphocytes. These results suggest that HHV-6 can infect renal tissues and that the infection may be correlated with rejection or with immunosuppressive therapy.

Prevention of functional, structural, and molecular changes of chronic rejection of rat renal allografts by a specific macrophage inhibitor

Chronic rejection is the primary cause of long-term allograft loss. Macrophages and their products have been shown to be critical in the development of this process in an established kidney allograft rat model. A new synthetic agent, Gamma lactone, is a specific inhibitor of macrophages and monocytes that inhibits the generation of these populations in vitro and their activities in the effector phase of host alloresponsiveness. We tested its effects on the development of chronic changes in the model. Untreated control allograft recipients developed increasing proteinuria after 12 weeks; progressive glomerulosclerosis, interstitial fibrosis, and arterial obliteration developed thereafter. Infiltrating ED1+ macrophages as noted by immunohistology increased dramatically between 12 and 16 weeks, localizing preferentially in glomeruli and perivascular areas. The presence of these cells was associated with dense expression of their products. Reverse transcription polymerase chain reaction confirmed and expanded the immunohistological findings, showing significant gene expression of macrophage-derived mediators. In contrast, recipients treated with G-Lac daily for 32 weeks never developed proteinuria; macrophage infiltration was dramatically reduced, and expression of their products was virtually absent. At 32 weeks, most glomeruli and arteries remained histologically normal. In another group in which treatment was stopped at 24 weeks, however, proteinuria began to develop by 32 weeks; macrophages infiltrated the organs and expression of their products became manifest. These results confirm the importance of macrophages and macrophage-derived factors in chronic rejection and suggest that a specific inhibitor of macrophage activation may be useful in the prevention of the process over the long term.

Analysis of the Arterial Blood Flow Patterns of Normal and Allografted Kidneys by the Directional Ultrasonic Doppler Technique

AbstractThe arterial blood flow of the allotransplanted kidney was examined 52 times in 40 recipients and that of the normal kidney 6 times in 6 donors by means of the directional ultrasonic Doppler technique.The blood flow patterns showed a rapid forward phase in systole and a slow forward phase in diastole but nothing indicative of a reverse flow was found. A significant correlation was observed between the acceleration time of flow component and graft function, while there were no correlations among diastole/systole ratio, appearance time and graft function. The arterial blood flow patterns of the grafts were classified into 3 groups based on acceleration time: I—excellent, II—intermediate and III—poor graft functions.Additionally, in pursuit of possible relationships between the ultrasonic Doppler flow patterns of the grafts and their morphological features, histopathology and angiography were done for 20 and 15 recipients, respectively. As a result the histologic vascular changes with interstitial da...

FK506-induced kidney tubular cell injury

Some renal changes associated with cyclosporine, such as tubular vacuolization and glomerular thrombosis, have also been reported with FK506. Furthermore, FK506 therapy is associated with a decrease in glomerular filtration rate and renal plasma flow and an increase in renal vascular resistance. We studied the in vitro tubular cell sensitivity to FK506 in comparison with CsA, the ultrastructural changes induced by FK506 and CsA, and the effect of both drugs on tubular cell growth in vitro. We also investigated whether FK506 and CsA induced endothelin-1 (ET-1) secretion of cultured tubular cells and whether this stimulatory effect coincided with a change in the endothelin systemic synthesis. Exposure of tubular cells to high concentrations of FK506 or CsA (10, 50, 100 μM) induced a time- and dose-dependent cell injury in vitro. The damage induced by FK506 and CsA was characterized by a direct cytotoxic effect on tubular cells, as expressed by release of 3H thymidine from prelabeled cells, N-acetyl-β-D-glu-cosaminidase release, and cell detachment. Ultrastructural changes (vacuolizations, swelling, and mitochondrial enlargement) and inhibition of the growth of cultured tubular cells were also observed at high concentrations of FK506 and CsA. Low concentrations of FK506 and CsA (1, 0.1, 0.01, 0.001 %mUM) were not cytotoxic and induced only a minimal inhibitory effect on the growth of tubular cells in vitro. We demonstrated that FK506 (1, 0.1, 0.01 μM) time-dependently stimulated the secretion of endothelin by cultured tubular cells. CsA 10, 1, 0.1, 0.01 also exerted an enhancing effect on ET-1 secretion in cultured tubular cells. We observed that the concentration of CsA that induced the most important enhancing effect was 10 or 100 times higher than that required for FK506 to observe the same effect. The concentrations of FK506 or CsA that induced ET-1 secretion were not cytolytic for tubular cells in vitro. FK506− or CsA-treated rats showed an increase in serum level of ET-1 in comparison with the control. Through the stimulatory effect on endothelin secretion by tubular cells, FK506 and CsA may induce a perturbation of renal hemodynamics. Concentrations of FK506 and CsA, higher than established serum levels but close to those reached in tissues, are cytotoxic for tubular cells and induced ultrastructural changes and a significant delayed regeneration.

Predictability of renal allograft prognosis during rejection crisis by ultrasonic Doppler flow technique

Using the ultrasonic Doppler technique, renal blood flow was measured in 67 patients who underwent living related renal transplantation from January, 1976 to December, 1979. In 58 of 67 cases, 81 acute and 9 chronic rejection episodes occurred. In the initial stage of acute rejection, there are no particular changes in the pattern of systolic blood flow and by contrast marked changes of diastolic flow. The disappearance of the diastolic phase is indicative of an advanced stage of rejection, the reappearance indicative of recovery from rejection, and persistent loss accompanied by changes of systolic flow indicative of an unfavorable prognosis of rejection. In chronic rejection, there are rapid changes of neither systolic nor diastolic flow though the acceleration time in the systolic phase lengthens gradually. The ultrasonic Doppler flow technique for blood flowmetry of a transplanted kidney is a useful means of knowing the prognosis of rejection and provides an index for corticosteroid bolus therapy.

Immunosuppressive dose of azathioprine inhibits replication of human cytomegalovirus in vitro

SummaryAzathioprine (Aza) was found to have anti-human cytomegalovirus (HCMV) activity in vitro at concentrations used for immunosuppression therapy. The dose of Aza for 50% plaque reduction was 0.592µg/ml for HCMV in human embryonic lung (HEL) cells, but those of Aza for 50% plaque reduction for herpes simplex virus (HSV) and varicella-zoster virus were more than 20µg/ml. The dose of Aza for 50% reduction of the HCMV yield in infected cells was 0.25µg/ml, while that for 50% reduction of the HSV yield in infected cells was more than 50µg/ml. The dose of Aza for 50% growth inhibition of HEL cells was 30µg/ml, and 50.7 and 120 times greater than the doses for 50% reduction of the plaque formation and the yield of HCMV, respectively. Thus Aza was found to have a strong anti-HCMV activity at concentrations used for immunosuppression. When HCMV infected cells were treated with cyclosporine (CsA: 0.2µg/ml) and prednisolone (Pred: 0.3µg/ml) simultaneously with Aza, the doses of Aza for 50% reduction of plaque formation and the yield of HCMV were 0.73 and 0.32µg/ml, respectively. Thus an inhibitory effect of Aza was also observed in HCMV-infected cells treated with CsA and Pred at their concentrations used for immunosuppression. Maintenance of an anti-HCMV dose of Aza in combination with CsA and Pred might establish not only satisfactory immunosuppression but also suppression of HCMV infection in transplant recipients.

THE IN VITRO IMMUNOSUPPRESSIVE EFFECT OF DEOXYMETHYLSPERGUALIN IN MAN AS COMPARED WITH FK506 AND CYCLOSPORINE

The effect of deoxymethylspergualin (MeDSG) on in vitro human lymphocyte response was assessed in comparison with FK506 and cyclosporine. Peripheral blood mononuclear cells from normal human volunteers were used for assay of mixed lymphocyte reaction, cell mediated lympholysis, and blastogenesis by PHA, IL-2, and OKT3. MeDSG suppressed only allogeneic stimulation (MLR and CML) and IL-2-induced blastogenesis, not PHA- or OKT3-induced blastogenesis, although the other immunosuppressive agents showed some suppressive effect for all assays. A kinetic study of MLR showed that the suppressive activity did not decrease even when MeDSG was added at day 3 or day 4. The other agents, however, showed a weak suppressive effect when added at a later phase of MLR.

The significant effect of HLA-DRB1 matching on long-term kidney graft outcome.

Serotyping and genotyping (polymerase chain reaction with sequence-specific oligonucleotide probes method) were conducted on 520 unrelated individuals to determine the linkage disequilibrium of HLA-B and HLA-DRB1. Analyses of 511 kidney transplants (300 related and 211 cadaver recipients) were carried out at 4 transplant centers using the linkage disequilibrium of HLA-B and HLA-DRB1 established previously. All transplant recipients received CsA immunosuppression and were transplanted from June 1983 to December 1991. There were 51 significant linkages formed between HLA-B and HLA-DRB1 alleles (P<0.05). DRB1-compatible transplants experienced a comparable 5-year graft success rate of 94% as did the HLA-identical recipients with a 100% 5-year success rate. However DRB1-incompatible recipients displayed a significantly reduced 5-year graft survival rate of 73% (73% vs. 94% P<0.01). The 5-year graft survival rate of HLA-DR-incompatible recipients of 71% was compatible to the 73% for HLA-DRBl-incompatible recipients. No variation of rejection rate for DRB1-compatible grafts was seen in any of the 4 transplant centers. The results also indicated that HLA-DRB1 compatibility was essential for optimal success rate, regardless of HLA class I mismatches. The overall conclusion was that matching for HLA-DR was important to achieve optimal kidney graft survival on the molecular level but not on the serotyping level.

The immunosuppressive mode of action of mizoribine.

Mizoribine (MIZ) suppressed the mitogen response and mixed lymphocyte reaction (MLR) significantly at doses of 100 micrograms/ml and 10 micrograms/ml in a dose-response analysis. The 50% inhibition dose (ID50) was between 10 micrograms/ml and 1.0 microgram/ml, both in the mitogen response and MLR. In a kinetic study of the MLR, the degree of suppression with MIZ at a given dosage was essentially the same as the degree of suppression observed in the dose-response analysis when MIZ was added to MLR cultures from day 0 to day 4. In addition, MLR was more susceptible to the suppressive activity of MIZ at 100 micrograms/ml when MIZ was added near the peak of lymphocyte proliferation. This experiment also showed that MLR suppression induced by MIZ at 10 micrograms/ml was reversible and MLR activity had completely recovered 6-8 hr after its removal. MIZ had no inhibitory action on MLR-derived cytotoxic cells or the effector phase of cell mediated lymphocytotoxicity. These results clearly demonstrate that MIZ suppresses lymphoproliferation, but has no effect on the recognition phase or effector phase of cytotoxic lymphocytes.

Interleukin-6 and interleukin-6 receptor are expressed by cultured glomerular epithelial cells.

Interleukin‐6 (IL‐6) has been extensively studied in mesangial cells but little is known about the expression of this cytokine and its receptor in glomerular epithelial cells (GEC). IL‐6 was detected in the culture supernatants of human GEC and its production was enhanced in time and dose dependent manner by lipopolysaccharide (LPS), interleukin‐1β (IL‐Iβ) and tumour necrosis alpha (TNF‐α). Quiescent, serum‐starved GEC did not express clearly IL‐6 mRNA. Stimulation of cells with LPS, TNF‐α or IL‐1β resulted in an increase of detectable IL‐6 mRNA. Interestingly, it was found that IL‐6 induced its own mRNA attesting that this cytokine was secreted in autocrine fashion by GEC. GEC expressed IL‐6 receptor (IL‐6R) as demonstrated directly by the existence of IL‐6R mRNA detected by northern blotting. Stimulation of GEC by pro‐inflammatory mediators such as LPS increased the expression of IL‐6R mRNA. The soluble form of IL‐6 receptor (sIL‐6R) was not detectable in the culture supernatants harvested from untreated or cytokine‐treated cells. We investigated further, whether IL‐6 may influence growth of cultured GEC. Incubation of GEC with recombinant (r) IL‐6 resulted in a dose dependent increase in 3H thymidine incorporation indicating that IL‐6 acts as an autocrine growth factor for GEC. We conclude that GEC are a potent source of IL‐6, the local excessive expression of IL‐6 and its receptor may play a substantive role in the regulation of processes which appear critical to the initiation of progressive glomerular disease such as cell proliferation.