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Biomedical subjects

Steven H Sacks

Publications and source records attributed to Steven H Sacks.

At least 19 recordsLinked to original sources
PubMed2006

Controlling the generation and function of human CD8+ memory T cells in vitro with immunosuppressants.

BACKGROUND: Memory T cells play a pivotal role in acute and chronic rejection of transplanted organs. Novel therapies such as alemtuzumab and the identification of heterologous memory have highlighted their importance following transplantation. Unlike naive T cells, the influence of immunosuppressive agents on the de novo generation of memory T cells and on the function of pre-existing memory T cells is poorly understood. METHOD: CD8 effector memory T cells (Tem, CD45RA, CCR7) were generated by stimulating peripheral blood mononuclear cells for five days with anti-CD3 and anti-CD28 monoclonal antibodies, followed by nine days of rest. The influence of immunosuppression on this was assessed by flow cytometry. Tem that had been naturally formed in vivo were used to study the influence of the same agents on the function (intracellular interferon-gamma production) of Tem cells. RESULTS: Whereas all the immunosuppressive agents tested inhibited the expansion of CD8 Tem population by influencing their proliferation and apoptosis, the calcineurin inhibitors were better at controlling their function. Furthermore, Tem generated despite the presence of tacrolimus were functional and did not exhibit drug resistance. CONCLUSIONS: Immunosuppressive agents differ in their ability to control the generation and function of CD8 Tem; the calcineurin inhibitors being the most efficacious. This may be of importance when tailoring immunosuppressants for transplant recipients, particularly in those where there is pre-existing immunological memory or in those receiving T-cell depleting therapies, which skews the immune repertoire towards memory.

Antigens, CD↗
PubMed2006

CD46 (membrane cofactor protein) acts as a human epithelial cell receptor for internalization of opsonized uropathogenic Escherichia coli.

Escherichia coli is a common urinary pathogen whose uptake into epithelial cells is mediated by attachment through type 1 fimbriae. In this study, we show by using using human urinary tract epithelial cells that maximal internalization of E. coli is achieved only when bacteria are opsonized with complement. The concentrations of complement proteins in the urine rise sufficiently during infection to allow bacterial opsonization. The complement regulatory protein, CD46 (membrane cofactor protein), acts in cohort with fimbrial adhesion to promote the uptake of pathogenic E. coli. This uptake is inhibited by RNA interference to lower the expression of CD46 and by soluble CD46 that will competitively inhibit opsonized bacteria binding to cell surface CD46. We propose that efficient internalization of uropathogenic E. coli by the human urinary tract depends on cooperation between fimbrial-mediated adhesion and C3 receptor (CD46)-ligand interaction. Complement receptor-ligand interaction could pose a new target for interrupting the cycle of reinfection due to intracellular bacteria.

Antibodies, Bacterial↗
PubMed2006

Rapamycin at subimmunosuppressive levels inhibits mesangial cell proliferation and extracellular matrix production.

In view of its proven antiproliferative effects, rapamycin offers potential in the treatment of mesangioproliferative disease. Previous data have shown an effect of rapamycin on mesangial cell proliferation at high doses and have not explored the mechanism of action. Therefore, we explored the effects and mechanism of action of low levels of rapamycin on mesangial cell proliferation. Primary cultures of mouse mesangial cells were grown in medium containing serum with differing concentrations of rapamycin. A rapamycin concentration of 0.1 ng/ml caused a decrease in cell number and DNA synthesis with no effect on apoptosis. Type IV collagen protein production was inhibited at 0.01 ng/ml rapamycin, although gene expression was unaffected. P70S6K phosphorylation was inhibited in parallel with the effects on cell number and DNA synthesis in a dose-dependent manner, but no effect was seen at 0.01 ng/ml rapamycin. These data show an effect on mesangial cell proliferation and p70S6 kinase phosphorylation of 0.1 ng/ml rapamycin and an effect on collagen IV production of 0.01 ng/ml rapamycin. We suggest that further in vivo studies should explore the potential for low-dose rapamycin in the treatment of mesangioproliferative glomerulonephritis.

Animals↗
PubMed2006

TLR2 stimulation of intrinsic renal cells in the induction of immune-mediated glomerulonephritis.

Infection may exacerbate organ-specific autoimmune disease such as glomerulonephritis. This may occur in the absence of a measurable effect on the adaptive immune response, and the mechanisms responsible are not fully understood. To investigate this, we have studied the effect of TLR2 ligation by the synthetic ligand Pam(3)CysSK(4) on the development of glomerulonephritis in mice. We demonstrated that glomerular inflammation induced by passive administration of nephrotoxic Ab does not occur in the absence of TLR2 stimulation, with a strong synergy when Ab deposition and TLR2 stimulation occur together. Parameters of glomerular inflammation were neutrophil influx, thrombosis, and albuminuria. To investigate the relative contribution of TLR2 on bone marrow-derived cells and intrinsic renal cells, we constructed bone marrow chimeras. Nephrotoxic Ab and TLR2 ligation caused a neutrophil influx in both types of chimera above [corrected] that seen in sham chimeras totally TLR2 deficient [corrected] Albuminuria was seen in both types of chimera above that seen in sham chimeras that were totally TLR2 deficient. This was greater in chimeras with TLR2 present on bone marrow-derived cells. To find a potential mechanism by which intrinsic renal cells may contribute toward disease exacerbation, mesangial cells were studied and shown to express TLR2 and MyD88. Wild-type but not TLR2-deficient mesangial cells produced CXC chemokines in response to stimulation with Pam(3)CysSK(4). These results demonstrate that TLR2 stimulation on both bone marrow-derived and resident tissue cells plays a role in amplifying the inflammatory effects of Ab deposition in the glomerulus.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Role of dendritic cell synthesis of complement in the allospecific T cell response.

Although extrahepatic synthesis of complement and particularly C3 has been widely studied in most cells and tissues, new information is emerging on dendritic cells (DCs). This research has shown that mouse bone marrow (BM) derived DCs are able to synthesise C3 and this synthesis has a substantial impact on DC activation, affecting the diverse range of DC functions relevant to the allospecific T cell response. Thus, local production of C3 appears to regulate the capacity of DCs to trigger the primary T cell response against donor alloantigen. Understanding of the key mechanisms by which complement activation modulates DC maturation could lead to the development of therapeutic strategies to down regulate DC activation thus reduce allograft rejection.

Animals↗
PubMed2006

Toll-like receptor 2 agonists exacerbate accelerated nephrotoxic nephritis.

Glomerulonephritis can be exacerbated by infection. This study investigated the effect of N-palmitoyl-S-(2,3-bis(palmitoyloxy)-(2R,S)-propyl)-(R)-cysteinyl-seryl-(lysyl)3-lysine (Pam3CysSK4), a synthetic Toll-like receptor 2 (TLR2) ligand, that was given at immunization on disease severity in accelerated nephrotoxic nephritis. Stimulation of TLR by microbial constituents is known to influence the development of the adaptive immunity. It was hypothesized that the TLR2 ligand Pam3CysSK4 can modulate the development of disease in accelerated nephrotoxic nephritis by influencing the development of adaptive immunity. It is shown that Pam3CysSK4, when given at immunization, can increase profoundly the severity of disease, and with the use of TLR2-deficient mice, it is shown that this disease exacerbation depends on the presence of TLR2. Wild-type mice that were given Pam3CysSK4 at immunization and had more severe disease also had a greater amount of antigen-specific IgG1, IgG2b, and IgG3 in the serum and more IgG2b and IgG3 deposited within the glomerulus. They also had increased numbers of glomerular CD4-positive T cells. Therefore, the more severe disease that was seen in the group that was immunized with lipopeptide can be attributed to an influence on the adaptive immune response.

Animals↗
PubMed2006

Influence of donor C3 allotype on late renal-transplantation outcome.

BACKGROUND: The complement system has a critical role in both the innate and the adaptive immune responses. In humans, C3 exists as two main allotypes, F (fast) and S (slow), which are known to affect the incidence of inflammatory disease. We conducted a study to address the influence of these alleles on late renal-graft outcome. METHODS: We determined the C3 allotypes of 662 pairs of adult kidney donors and recipients from 1993 through 2002 and then related C3F/S polymorphism status to demographic and clinical outcome data. The median length of follow-up was 3.3 years. RESULTS: Analysis of 513 pairs of white donors and recipients identified 113 C3S/S recipients of a C3S/F or a C3F/F kidney and 179 C3S/S recipients of a C3S/S kidney. Graft survival was significantly better with a C3F/F or C3F/S donor allotype than a C3S/S allotype (P=0.05). The hazard ratio for graft loss of C3S/S kidneys, as compared with C3F/F or C3F/S kidneys, was 2.21 (95 percent confidence interval, 1.04 to 4.72; P=0.04). The graft function of C3F/F or C3F/S donor kidneys was significantly better than that of C3S/S donor kidneys (P<0.001). The effect of the C3F allele was specific to recipients who did not themselves possess this allele. Multivariate analysis excluded effects of other factors known to influence graft outcome. CONCLUSIONS: Expression of C3 alleles by donor renal cells appears to have a differential effect on late graft outcome. Among white C3S/S recipients, receipt of a C3F/F or C3F/S donor kidney, rather than a C3S/S donor kidney, is associated with a significantly better long-term outcome. These findings suggest that the two alleles have functional differences.

Adult↗
PubMed2006

Dendritic cell synthesis of C3 is required for full T cell activation and development of a Th1 phenotype.

Previous studies have found that deficiency of complement component C3 is associated with reduced T cell responses in several disease models including viral infection, autoimmune disease, and transplantation. However, the underlying mechanism is unclear. In this study, we demonstrate that dendritic cells (DCs) are able to synthesize C3 and this synthesis is required for the capacity of DCs to stimulate alloreactive T cell responses in vitro and in vivo. Compared with C3-producing DCs, C3-nonproducing DCs exhibit reduced potency to stimulate an alloreactive T cell response, favor the polarization of CD4(+) T cells toward Th2 phenotype, and have regulatory T cell-driving capacity. In addition, priming mice with C3-deficient DCs compared with wild-type DCs led to delayed skin allograft rejection. Our findings that nonproduction of C3 by DCs significantly reduced T cell stimulation and impaired allograft rejection provide a potentially important explanation of how C3-deficient mice develop reduced T cell responses and of how C3-deficient donor kidney is protected from T cell-mediated graft rejection.

Animals↗
PubMed2006

Therapeutic strategy with a membrane-localizing complement regulator to increase the number of usable donor organs after prolonged cold storage.

A shortage of donor organs and increasing dependence on marginal grafts with prolonged ischemic times have meant that new methods are needed to prevent postischemic damage. Herein is reported a new strategy aimed to protect donor kidney from complement-mediated postischemic damage and therefore increase the number of successful transplants. Rat donor kidneys were perfused with a membrane-localizing complement regulator derived from human complement receptor type 1 (APT070) and then subjected to prolonged periods of cold storage (at 4 degrees C). A relationship was found between the duration of cold ischemia and the extent of complement-mediated tubule damage and loss of graft function. After 16 h of cold storage, APT070-treated kidneys that were transplanted into syngeneic recipients showed a significant increase in the number of surviving grafts, compared with control-treated grafts (63.6 versus 26.3%). Surviving grafts also displayed less acute tubular injury and better preservation of renal function. These results not only enhance the understanding of the mechanism by which prolonged cold ischemia reduces immediate graft survival but also provide essential information about the effectiveness of membrane-localizing complement regulator with prolonged cold storage. This could lead to more effective strategies for improving the use of severely ischemic donor organs.

Animals↗
PubMed2006

Accumulation of immune complexes in glomerular disease is independent of locally synthesized c3.

Although complement activation can make immune complex glomerulonephritis worse, the third complement component also can solubilize immune complexes and thus reduce the severity of disease. How C3 that is produced within the kidney contributes to this balance is unknown. This study therefore investigated the relative roles of systemic and local C3 production in a model of glomerular immune complex disease. Injection of sheep anti-glomerular basement membrane antibody into preimmunized mice resulted in accumulation of immune complexes and progressive loss of function over 14 d that was much more marked in C3-deficient (C3-/-) mice. In C3-sufficient mice that received a transplant of a C3-/- mouse kidney and in C3-/- mice with C3-sufficient mouse kidney transplants, the severity and the pattern of injury went with the complement status of the recipient. That is, mice with deficient circulating C3 developed severe glomerular immune complex disease, whereas those with a high level of circulating C3 had well-preserved glomerular structure and function. It is concluded that circulating C3 is a critical factor in reducing the glomerular accumulation of immune complexes. Local synthesis of C3 did not have a major influence on this aspect of glomerular disease.

Analysis of Variance↗
PubMed2006

Local extravascular pool of C3 is a determinant of postischemic acute renal failure.

The third complement component (C3) is an acute phase protein that plays a central role in reperfusion injury in several organ models. To investigate the contribution of local synthesis of C3 and distinguish it from that of circulating complement mainly produced by hepatic synthesis, we employed a mouse renal isograft model. Our model demonstrated a close relationship between the extent of intrarenal expression of C3 and cold-ischemia induced injury. Ischemic C3-positive donor kidneys transplanted into C3-positive or C3-negative recipients developed widespread tissue damage and severe acute renal failure. In contrast, ischemic C3-negative isografts exhibited only mild degrees of functional and structural disturbance, even when transplanted into normal C3-positive recipients. Thus local synthesis of C3, mostly identified in the tubular epithelium, was essential for complement-mediated reperfusion damage, whereas circulating C3 had a negligible effect. Our results suggest a two-compartment model for the pathogenic function of C3, in which the extravascular compartment is the domain of local synthesis of C3, and where the role of circulating C3 is redundant. Our data cast new light on the mechanism of complement-mediated tissue injury in nonimmunological disorders, and challenges the longstanding dogma that circulating components are the main complement effectors of extravascular tissue damage.

Animals↗
PubMed2006

Deficiency of C4 from donor or recipient mouse fails to prevent renal allograft rejection.

Complement effector products generated in the transplanted kidney are known to mediate transplant rejection, but which of the three main activation pathways of complement trigger this response is unclear. Here we assessed the role of the classical and lectin pathways by studying the common component C4 in mouse kidney transplant rejection. We transplanted wild-type or C4-null H-2(b) donor kidneys into H-2(k) or H-2(d) recipients, or vice-versa, to assess the roles of donor kidney and recipient expression of complement. Intragraft C4 gene expression rose substantially during rejection. However, we found no significant association between graft acceptance and the presence of C4 in either the donor kidney or recipient mouse. At the time of rejection, we found no significant differences in alloantibody response in the different groups. Tubular deposition of C3 to C9 occurred regardless of the absence or presence of C4 in either the donor or recipient mouse, indicating that C4 was dispensable for complement activation at this site. These data suggest that complement activation and renal allograft rejection are independent of the classical and lectin pathways in these models, implying that in the absence of these pathways the alternative pathway is the main trigger for complement-mediated rejection.

Animals↗
PubMed2005

Macrophages from C3-deficient mice have impaired potency to stimulate alloreactive T cells.

Impaired T-cell reactivity is a feature of C3-deficient mice in several disease models. The mechanism behind the reduced T-cell response is, however, poorly understood. We explored the hypothesis that antigen-presenting cells (APCs) from C3-/- mice have impaired potency to stimulate antigen-specific T cells, in an alloantigen-dependent model. Our results show that C3-/- macrophages have reduced ability to elicit alloreactive T-cell responses in vitro and in vivo, affecting both the primary and secondary responses. The C3 status of donor macrophages had a major impact on the CD4 T-cell response. The impaired CD4 T-cell response was associated with reduced expression of MHC class II on the surface of C3-/- macrophages, without loss of class II gene expression. Furthermore, inhibition of C3 gene expression in C3+/+ macrophages reduced their ability to stimulate alloreactive T cells, suggesting that endogenous production of C3 could in part contribute to the potency of APCs. Our data provide compelling evidence that C3 deficiency modulates the potency of APCs to stimulate the T-cell response, suggesting a critical role for complement in the maintenance of APC function. This could offer a partial explanation as to why the T-cell response is impaired in C3-/- mice.

Animals↗
PubMed2005

Allograft rejection: effect of local synthesis of complement.

The complement system is known for its ability to participate in non-specific inflammation and membrane injury as well as contributing to antigen-specific immune stimulation. In renal transplantation, the complement cascade behaves true to form in that both non-immune- and immune-mediated destruction of the renal tubules are complement dependent. What is remarkable, however, is the extent of involvement of local synthesis of complement in both of these injuries, suggesting that the extravascular tissue compartment is the domain of local synthesis, whereas the effect of circulating complement is much less. This creates a new paradigm for studying the influence of local synthesis of complement in other organ-based diseases and underlines the need for tissue-targeting strategies in successful therapeutic development.

Animals↗
PubMed2004

Independent pathways of P-selectin and complement-mediated renal ischemia/reperfusion injury.

Evidence from in vitro studies indicates that complement activation regulates the expression of P-selectin on endothelial cells. This suggests that in disorders such as ischemia/reperfusion injury, in which both complement and P-selectin have been shown to play a role, complement activation is a primary event and the effects of P-selectin are secondary. To test this hypothesis in vivo, we examined a mouse kidney model of ischemia/reperfusion injury. Surprisingly, the time course and extent of expression of P-selectin was unaltered in C3-deficient mice compared with wild-type mice, in which there was rapid but transient up-regulation of P-selectin on capillary walls and slower accumulation of complement split product on the tubular epithelium. In addition, treatment with anti-P-selectin antibody to reduce the neutrophil-mediated reperfusion damage was equally effective in the absence of C3. These data imply that complement and P-selectin-mediated pathways of renal reperfusion injury are mutually independent, a conclusion that is possibly explained by the differences in the location and time kinetics of complement activation and P-selectin expression. We conclude that in vivo interaction between complement and P-selectin is limited because of time and spatial considerations. Consequently, complement and P-selectin pose distinct targets for therapy.

Animals↗
PubMed2004

CD59a deficiency exacerbates ischemia-reperfusion injury in mice.

The terminal complement components C5a and the membrane attack complex are involved in the pathogenesis of ischemia-reperfusion injury in many organs. CD59 is the major regulator of membrane attack complex formation. Mice deficient in the Cd59a gene (mCd59a-/-) were used to investigate the role of CD59 in renal ischemia-reperfusion injury. Unilateral ischemia-reperfusion injury was induced by clamping the left renal pedicle for 30 minutes under general anesthetic. Mice were studied at 72 hours and 2 weeks after ischemia-reperfusion injury. mCd59a-/- mice developed significantly greater tubular injury (P = 0.01), tubulointerstitial apoptosis (P = 0.02), and neutrophil influx (P = 0.04) than controls at 72 hours after ischemia-reperfusion. Two weeks after ischemia-reperfusion, mCd59a-/- mice exhibited more severe tubular damage predominantly in a corticomedullary distribution than controls (P = 0.02). Quantification of interstitial leukocytes revealed significantly greater numbers of infiltrating lymphocytes (but not macrophages) in mCd59a-/- mice than controls (P = 0.04) at 2 weeks. At both time points, significantly more C9 (as a marker of membrane attack complex) deposition occurred in a peritubular distribution in mCd59a-/- mice than controls. In conclusion, these results demonstrate that the lack of CD59a, by allowing unregulated membrane attack complex deposition, exacerbates both the tubular injury and the interstitial leukocyte infiltrate after ischemia-reperfusion injury in mice.

Animals↗
PubMed2004

Complement activation regulates the capacity of proximal tubular epithelial cell to stimulate alloreactive T cell response.

Tissue expression of C3 is an unexpected regulator of the alloimmune response in mouse kidney transplantation. It is unclear, however, whether a direct or an indirect action of complement on the host immune response is involved. Also unknown is which of the complement effector products, cleaved C3, cleaved C5, or C5b-9, is responsible. Proximal tubular epithelial cells (PTEC) not only constitute a major target of the alloimmune response but also produce substantial amounts of C3. This study investigated the property of mouse PTEC to stimulate alloreactive T cells in a complement-dependent manner. The proliferative and cytokine responses of primed alloreactive T cells were measured after exposure to donor-specific PTEC that had been pretreated with normal mouse serum, heat-inactivated mouse serum, or complement- deficient (C3, C5, or C6) mouse sera to differentially deposit complement components. PTEC were able to stimulate alloreactive T cells in an antigen-specific manner. Complement activation leading to the deposition of cleaved C3 on PTEC enhanced the alloreactive T cell response. This complement-mediated stimulation of the T cell response was dependent on C3 but not on C5 or C6. The primary influence of tissue-bound complement was on CD4(+) T cells. Moreover, the effect of complement on alloreactive T cells was B7 dependent, shown by inhibition studies with CTLA4-Ig. These results suggest that donor epithelium-bound C3 can upregulate the alloimmune response. It is postulated that surface-bound C3 interacts with complement receptors on alloreactive T cells or on antigen presenting cells to increase allo-immune stimulation.

Animals↗