Role of fibrinogen in acute ischemic kidney injury.

Renal ischemia-reperfusion (I/R) is associated with activation of the coagulation system and accumulation of blood clotting factors in the kidney. The aim of the present study was to examine the functional impact of fibrinogen on renal inflammation, damage, and repair in the context of I/R injury. In this study, we found that I/R was associated with a significant increase in the renal deposition of circulating fibrinogen. In parallel, I/R stress induced the de novo expression of fibrinogen in tubular epithelial cells, as reflected by RT-PCR, immunofluorescence, and in situ hybridization. In vitro, fibrinogen expression was induced by oncostatin M and hyper-IL-6 in primary tubular epithelial cells, and fibrinogen-containing medium had an inhibitory effect on tubular epithelial cell adhesion and migration. Fibrinogen(+/-) mice showed similar survival as wild-type mice but better preservation in early postischemic renal function. In fibrinogen(-/-) mice, renal function and survival were significantly worse than in fibrinogen(+/-) mice. Renal transplant experiments revealed reduced expression of tubular damage markers and attenuated proinflammatory cytokine expression but increased inflammatory cell infiltrates and transforming growth factor-ß expression in fibrinogen(-/-) isografts. These data point to heterogeneous effects of fibrinogen in renal I/R injury. While a complete lack of fibrinogen may be detrimental, partial reduction of fibrinogen in heterozygous mice can improve renal function and overall outcome.

New insights on molecular mechanisms of renal aging.

Long-term transplant outcome is importantly influenced by the age of the organ donor. The mechanisms how age carries out its pathophysiological impact on graft survival are still not understood. One major contributing factor for the observed poor performance of old donor kidneys seems in particular the age-related loss in renal regenerative capacity. In this review, we will summarize recent findings about the molecular basis of renal aging with specific focus on the potential role of somatic cellular senescence and mitochondrial aging in renal transplant outcome.

Protocol biopsy-driven interventions after pediatric renal transplantation.

The therapeutic value of protocol biopsies (PBs) in renal transplant recipients remains unclear. We performed protocol biopsies in 57 children six months after transplantation. We increased the CNI dose in patients with borderline findings. In cases of Banff grade Ia, six prednisolone IV-pulses were given and the CNI dose was increased. CNI toxicity and polyomavirus nephropathy led to a reduction in the CNI dose. GFR was compared with a control group of 51 children with no PBs transplanted in the same period. Forty-two percent of PBs had no pathological changes, 24% IF/TA. Borderline findings were detected in 11%, Banff grade Ia in 15% (CNI), toxicity in 8%, and one case showed polyomavirus nephropathy. GFR after 1.5 and 2.5 yr was similar in both groups. GFR 3.5 yr after transplantation was significantly higher in the intervention group (57 ± 17 vs. 46 ± 20). Patients treated with low-dose CNI and everolimus had a significantly lower number of pathological findings in PBs. The performance of protocol biopsies followed by a standardized treatment algorithm led to better graft function 3.5 yr after transplantation. Prospective randomized studies to confirm our findings are needed.

Effects of donor age and cell senescence on kidney allograft survival.

The biological processes responsible for somatic cell senescence contribute to organ aging and progression of chronic diseases, and this may contribute to kidney transplant outcomes. We examined the effect of pre-existing donor aging on the performance of kidney transplants, comparing mouse kidney isografts and allografts from old versus young donors. Before transplantation, old kidneys were histologically normal, but displayed an increased expression of senescence marker p16(INK4a). Old allografts at day 7 showed a more rapid emergence of epithelial changes and a further increase in the expression of p16(INK4a). Similar but much milder changes occurred in old isografts. These changes were absent in young allografts at day 7, but emerged by day 21. The expression of p16(INK4a) remained low in young kidney allografts at day 7, but increased with severe rejection at day 21. Isografts from young donors showed no epithelial changes and no increase in p16(INK4a). The measurements of the alloimmune response-infiltrate, cytology, expression of perforin, granzyme B, IFN-gamma and MHC-were not increased in old allografts. Thus, old donor kidneys display abnormal parenchymal susceptibility to transplant stresses and enhanced induction of senescence marker p16(INK4a), but were not more immunogenic. These data are compatible with a key role of somatic cell senescence mechanisms in kidney transplant outcomes by contributing to donor aging, being accelerated by transplant stresses, and imposing limits on the capacity of the tissue to proliferate.

Association of elevated blood levels of pentachlorophenol (PCP) with cellular and humoral immunodeficiencies.

It has long been suspected that pentachlorophenol (PCP) exerts a damaging influence on the immune system. In this study, the possible relationship between blood levels of PCP and immune function was studied in 190 patients who had been exposed for more than 6 mo to PCP-containing pesticides. The patients suffered from frequent respiratory infections and general fatigue. Lymphocyte subpopulations, in-vitro responses to mitogens, allogeneic stimulator cells, plasma neopterin, cytokines, soluble cytokine receptors, soluble adhesion molecules, and immunoglobulin autoantibodies were determined. A dose-response relationship between blood levels of PCP and cellular and humoral immune parameters was established. Blood levels of PCP were associated negatively with (a) total lymphocyte counts (p = .0002), CD4/CD8 ratios (p = .0015), and absolute counts of CD3+ (p < .0001), CD4+ (p < .0001), CD16+ (p < .0001), CD25+ (p = .0003), DR+ (p < .0001), CD8+/56+ (p = .020), and CD19+ cells (p = .092); (b) plasma levels of interleukin-2 (IL-2) (p < .0001), soluble IL-2R (p < .0001), IL-6 (p < .0001), IL-10 (p = .0039), interferon-gamma (IFN-gamma) (p < .0001), tumor necrosis factor-alpha (TNF-alpha) (p < .0001), transforming-growth factor-beta2 (p = .023), soluble IL-1 receptor antagonist (sIL-1 RA) (p < .0001), soluble intercellular adhesion molecule-1 (p = .0003); and (c) immunoglobulin (Ig) M-anti-Fab type autoantibodies (p = .0353). PCP levels were associated positively with (a) number of impaired stimulation assays per patient (p = .041); (b) number of circulating CD11b+ monocytes (p = .0015); and (c) plasma levels of neopterin (p < .0001), IL-4 (p = .020), and sIL-6R (p = .020). Compared with patients who had PCP plasma levels that were less than or equal to 10 microg/l, patients with blood levels of PCP that exceeded 10 microg/l experienced the following more often: low numbers of total blood lymphocytes (p = .054), CD3+ (p = .0014), CD4+ (p = .0001), DR+ (p = .0003), CD16+ (p = .0033), and CD25+ cells (p = .0033). In addition, the same aforementioned patients experienced the following more frequently: undetectable plasma levels of IL-2 (p = .0057), IL-6 (p = .042), IL-8 (p = .038), IL-10 (p = .0001), TNF-alpha (p = .0062), and IFN-gamma (p = .016); and impaired in-vitro responses of lymphocytes (p = .071). The authors concluded that increased blood levels of PCP were associated significantly with cellular and humoral immunodeficiencies. Recurrent respiratory infections and general fatigue could originate from PCP-associated immunosuppression.

Switch from cyclosporine A to tacrolimus in renal transplant recipients: impact on Th1, Th2, and monokine responses.

We showed previously that pretransplant CD4 helper defects and low in-vitro IL-10 responses predict a low risk of acute kidney graft rejection. To compare the effect of tacrolimus (Tacr) and cyclosporine A (CsA) on the humoral immune response we assessed T helper function, B cell/monocyte responses and in-vitro cytokine responses (TNF-alpha, GM-CSF, IL-1 beta, IL-2, IL-4, IL-6, IL-10) in 20 renal transplant recipients before and 3 months after they were switched from CsA to Tacr because of hyperlipoproteinemia, hirsutism, or gum hyperplasia. T helper function was assessed using a PWM-driven allogeneic coculture system of patient T cells together with control B cells. B cell/monocyte responses were determined using a PWM-stimulated allogeneic coculture system, SAC I-stimulated B-cell cultures and LPS-stimulated monocyte cultures. Immunoglobulin-secreting cell (ISC) responses were assessed in a reverse hemolytic plaque assay, and ELISA were used to determine cytokine secretion. Treatment with Tacr resulted in a decreased expression of costimulatory ligands and adhesion molecules (T cells: CD40L, p < 0.05; CD28 and CD54, p < or = 0.01; B cells: CD25, p = 0.05; CD40, p < 0.001; monocytes: CD40, p < 0.05), which coincided with decreased PHA-stimulated T cell IL-2 responses (398 +/- 153 versus 43 +/- 15 pg/ml, p < 0.05), impaired CD4 helper activity (117% +/- 22% versus 73% +/- 19%, p < 0.05) and increased CD4 suppressor activity (-120% +/- 28% versus -18% +/- 27%, p = 0.02). We observed enhanced CD4 IL-10 responses (p < 0.01) and LPS-stimulated monocyte responses (TNF-alpha, IL-1 beta, and IL-6, p < 0.005; IL-10, p < 0.05), indicating an increased humoral immune responsiveness under treatment with tacrolimus. Our data show that switching of immunosuppressive therapy from CsA to tacrolimus results in suppression of costimulatory ligands, adhesion molecules, Th1 responses and CD4 helper activity. However, enhanced humoral immune responses, Th2 and monokine responses, might have a negative impact on long-term graft function.

Reduction of viral load and immune complex load on CD4+ lymphocytes as a consequence of highly active antiretroviral treatment (HAART) in HIV-infected hemophilia patients.

BACKGROUND AND OBJECTIVES: Human immunodeficiency virus (HIV)-induced immune complex load on circulating CD4+ blood lymphocytes is associated with dysfunction and depletion of CD4+ lymphocytes and with increased monocyte/macrophage function. It was investigated whether HAART reduces both the viral load in plasma and the number of immune complex-coated CD4+ lymphocytes in the blood, and whether CD4+ counts are associated with viral load and/or immune complex load. MATERIALS AND METHODS: Twelve HIV+ hemophilia patients before and after conversion to HAART (group 1); eight HIV+ hemophilia patients without antiretroviral therapy (group 2). HIV-1 RNA copies in plasma using NASBA/Nuclisens kits; CD4+ lymphocytes coated in-vivo with immune complexes using flowcytometry on whole blood samples; in-vitro responses of immune complex-coated T lymphocytes in cell culture assays. RESULTS: After conversion to HAART there was a significant reduction of viral load, CD4+ gp120+, CD4+ IgM+, and CD4+ IgG+ circulating blood lymphocytes and plasma neopterin, paralleled by a significant increase of CD4+ and CD8+ counts. The percentage of immune complex-coated CD4+ lymphocytes of converted patients was significantly associated with CD4+ counts, in-vitro responses to concanavalin A (Con A), pokeweed mitogen (PWM), phytohaemagglutinin (PHA), anti-CD3 and pooled allogeneic stimulator cells, and with plasma neopterin levels. CONCLUSION: HAART reduces viral load and HIV-induced immune complex load on circulating CD4+ blood lymphocytes. The results of this study can be interpreted to suggest that HAART increases CD4+ lymphocyte counts in part by counteracting HIV-induced autoimmune phenomena.

Microchimerism in sensitized renal patients.

BACKGROUND: Patients exposed to allogeneic human tissue sometimes produce anti-HLA antibody for many years in the absence of further obvious antigen exposure. To investigate the mechanism of sustained sensitization, we identified females awaiting renal transplantation with high panel-reactive antibody but no exposure to allogeneic tissue for at least 1 month. METHODS: We analyzed peripheral blood microchimerism using nested polymerase chain reaction amplification specific for the SRY region of the Y chromosome. RESULTS: Microchimerism was detected in 3 of 10 patients but in none of 8 normal female subjects. In two cases, the amplified DNA polymerase chain reaction product was sequenced and was confirmed to be identical to the SRY gene. The estimated level of chimerism as compared with serial dilutions of DNA from male peripheral blood leukocytes was about 1/50000. CONCLUSION: These results do not establish causality but support the possibility that antigens from microchimeric donor cells may sustain the HLA antibody response in certain patients.

CD4 depletion in HIV-infected haemophilia patients is associated with rapid clearance of immune complex-coated CD4+ lymphocytes.

The predominant immunological finding in HIV+ haemophilia patients is a decrease of CD4+ lymphocytes during progression of the disease. Depletion of CD4+ lymphocytes is paralleled by an increase in the proportion of immune complex-coated CD4+ cells. We examined the hypothesis that the formation of immune complexes on CD4+ lymphocytes is followed by rapid clearance of immune complex-coated CD4+ lymphocytes from the circulation. In this study, the relationship of relative to absolute numbers of immune complex-loaded CD4+ blood lymphocytes and their association with viral load were studied. Two measurements of relative and absolute numbers of gp120-, IgG- and/or IgM-loaded CD4+ lymphocytes were analysed in HIV+ and HIV- haemophilia patients, with a median interval of approx. 3 years. Immune complexes on CD4+ lymphocytes were determined using double-fluorescence flow cytometry and whole blood samples. Viral load was assessed using NASBA and Nuclisens kits. Whereas the proportion of immune complex-coated CD4+ lymphocytes increased with progression of the disease, absolute numbers of immune complex-coated CD4+ lymphocytes in the blood were consistently low. Relative increases of immune complex-coated CD4+ blood lymphocytes were significantly associated with decreases of absolute numbers of circulating CD4+ lymphocytes. The gp120 load on CD4+ blood lymphocytes increased in parallel with the viral load in the blood. These results indicate that immune complex-coated CD4+ lymphocytes are rapidly cleared from the circulation, suggesting that CD4+ reactive autoantibodies and immune complexes are relevant factors in the pathogenesis of AIDS. Relative increases of immune complex-positive cells seem to be a consequence of both an increasing retroviral activity as well as a stronger loading with immune complexes of the reduced number of CD4+ cells remaining during the process of CD4 depletion. The two mechanisms seem to enhance each other and contribute to the progressive CD4 decrease during the course of the disease.

P-glycoprotein expression is not a useful predictor of acute or chronic kidney graft rejection.

Because of the role of P-glycoprotein (P-gp) in multidrug resistance (MDR), it has been suggested that P-gp might play a role in acute and chronic rejection after organ transplantation. The purpose of the present work was to investigate a possible relationship between graft outcome and P-gp expression on peripheral mononuclear cells of renal transplant recipients. We determined P-gp expression in 27 patients with long-term, stable graft function (ST) and in 15 patients with chronic deterioration of graft function (CR). In addition, 40 patients were studied prior to, and at intervals after, transplantation with 21 healthy individuals serving as controls. P-gp values were highest in healthy controls and in ST patients. We found no correlation between P-gp values and acute rejection. CR patients tended to have lower levels of P-gp expression. Our results contradict the opinion that an overexpression of P-gp induces acute or chronic rejection by inhibiting the efficacy of immunosuppressive treatment.

Association of viral load in plasma samples of HIV-infected hemophilia patients with autoantibodies and gp120-containing immune complexes on CD4+ lymphocytes.

OBJECTIVE: We investigated whether the induction of antilymphocyte autoantibodies and immune complexes is associated with the activity of HIV replication. METHODS: Viral HIV-1 RNA was measured in the plasma samples of 84 HIV+ hemophilia patients and correlated with the IgM, IgG, IgM/IgG and IgM/IgG/gp120 load of circulating CD4+ lymphocytes, CD4+ and CD8+ cell counts, plasma neopterin levels and in vitro T-cell responses to mitogens and pooled allogeneic stimulator cells. RESULTS: Compared to patients with no immune complexes, on circulating CD4+ lymphocytes, viral load was increased in patients with IgM, IgM/IgG or IgM/IgG/gp120 complexes. Sequential analysis of HIV+ patients showed that peaks of retroviral activity were associated with the subsequent formation of CD4+ lymphocyte-reactive IgM and IgG autoantibodies and gp120-containing immune complexes. CONCLUSION: The induction of autoantibodies and immune complexes attached to CD4+ lymphocytes is associated with periods of increased viral activity in HIV-infected patients.

Diagnostic and prognostic significance of anticardiolipin antibodies in patients with recurrent spontaneous abortions.

PROBLEM: The role of ACA in unexplained RSA is controversial. In the present study, diagnostic and prognostic aspects were investigated. METHOD: One hundred five nonpregnant patients with primary, 29 with secondary RSA, and 209 controls were investigated for IgG-ACA. Follow-up studies were done during pregnancy in 76 individuals. IgM-ACA were tested in a subset of patients. RESULTS: Elevated ACA levels were significantly more frequent in both patient groups (26 and 24%) than in controls (16%). However, there was no correlation of ACA with various parameters including pregnancy outcome. In ACA-positive patients with successful pregnancy a significant decrease of ACA values during pregnancy was observed, while ACA remained high in aborting patients. IgG- and IgM-ACA correlated well. CONCLUSIONS: Although the data from nonpregnant RSA patients does not allow diagnostic or prognostic conclusions to be drawn, sequential testing of ACA-positive individuals provides the possibility to foresee pregnancy outcome.

Immunogenetic and serological investigations in nonpregnant and in pregnant women with a history of recurrent spontaneous abortions. German RSA/IVIG Study Group.

In the context of a controlled multicenter study on intravenous immunoglobulin (IVIG) treatment of patients with a history of unexplained recurrent spontaneous abortions (RSA), a number of controversial immunological parameters were evaluated prior to and during pregnancy with respect to their diagnostic and/or prognostic significance. A total of 390 serum samples from 52 patients were investigated. Sharing of 2 or more HLA (A, B, DR, DQ) antigens was significantly more frequent in RSA couples than in controls. The rate of cytotoxic or Fc-receptor (FcR)-blocking antibodies was not significantly lower in RSA patients than in individuals with normal pregnancies. Both tumor necrosis factor-alpha (TNF-alpha) levels and IgG anticardiolipin antibodies (IgG-ACA) were significantly increased in the patient group. While the occurrence of HLA sharing, cytotoxic/FcR-blocking antibodies and IgG-ACA did not correlate with the outcome of pregnancy, TNF-alpha levels were found to be significantly higher in patients with subsequent miscarriage than in those with successful pregnancy. IgG-ACA, if present, significantly decreased during the course of successful pregnancy but remained high in patients with subsequent abortion. It is concluded that the diagnostic and/or prognostic value of HLA sharing and cytotoxic/FcR-blocking antibodies has been overestimated while TNF-alpha and ACA levels are potential diagnostic markers and/or exhibit prognostic significance in subgroups of RSA patients.

Effects of ketamine, naloxone, and physostigmine on flash evoked potentials in rat superior colliculus.

Flash evoked potentials were recorded from the superior colliculus of chronically implanted hooded rats at 5 and 20 min following IP injections of saline, ketamine (75 mg/kg), naloxone (10 mg/kg), or physostigmine (0.4 mg/kg) on separate days. Components in an early positive complex were unaffected by ketamine and naloxone, but were reduced in amplitude by physostigmine. A positive spike emerged from the middle of a later negative wave following ketamine administration, but the amplitude of the negative wave was unaltered by naloxone or physostigmine. A succeeding positive component was enhanced by both ketamine and physostigmine. Physostigmine produced the most consistent alterations in latency, with most components increasing in latency. Naloxone pretreatment did not alter ketamine's influence on evoked potential amplitudes. Pretreatment with physostigmine briefly decreased the amplitude of the ketamine-induced positive spike, augmented the amplitude of the succeeding positive component, and also increased most peak latencies. Ketamine, naloxone and physostigmine all produced approximately equivalent hypothermia. Physostigmine, but not naloxone, pretreatment augmented the ketamine-induced hypothermia. The body temperature data suggest that some of the observed latency alterations are secondary to hypothermia. The amplitude data indicate that ketamine and physostigmine produce a combination of similar, distinct, and antagonistic effects on evoked potentials.