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Healthy liver, intestine, lung, and skin harbor resident lymphocytes with conventional and unconventional phenotypes. Lymphocytes also have been detected in healthy mice kidneys; however, these cells have not been well studied and have been largely overlooked. To better characterize the intra-renal lymphocytes, we extensively perfused C57BL/6J mice with PBS and then isolated mononuclear cells for flow cytometry analysis. We observed T cells, B cells, and NK cells in normal mice kidneys after extensive perfusion. Approximately 50% of kidney T lymphocytes expressed intermediate levels of CD3 (CD3int T cells). Similar to liver and lung, a high percentage of unconventional CD3+CD4−CD8− double-negative T cells was observed in normal mice kidneys, from which 11% expressed B220 antigen. Unlike the spleen and blood, the classic CD4+ and CD8+ T lymphocytes in the kidney had a high proportion of activated CD69+ and effector/memory CD44CD62L ligand phenotypes. Also, a small percentage of CD4+CD25+forkhead box p3+ and NKT cells was observed in perfused and exanguinated kidneys. In addition, a distinct TCR repertoire was found on intra-renal conventional and unconventional T cells compared with those from the spleen. Finally, after 24 h of renal ischemia reperfusion injury (IRI), increased production of cytokines IFN-γ and TNF-α by CD4+ and CD8+ T cells, isolated from perfused kidneys, was observed. These data suggest that some of these cells harbored in the kidney could be implicated in the immune response of the IRI pathogenic process.

Treatment with an anti-inflammatory Salmonella vaccine expressing enterotoxigenic Escherichia coli colonization factor Ag 1 (CFA/I) proved effective in stimulating protective, potent CD25+CD4+ regulatory T (Treg) cells in susceptible mice challenged with experimental autoimmune encephalomyelitis (EAE). Because the Salmonella vector was considerably less protective, we questioned whether altering fimbrial subunit expression to resemble conventional Salmonella expression may impact Treg cell potency. The Salmonella-CFA/I vaccine was modified to limit fimbrial subunit expression to the intracellular compartment (Salmonella-CFA/IIC). SJL mice were challenged with proteolipid protein peptide 139–151 to induce EAE and orally treated with one of three Salmonella vaccines 6 days postchallenge. Treatment with Salmonella-CFA/IIC greatly reduced clinical disease, similarly as Salmonella-CFA/I, by subduing IL-17 and IL-21; however, mechanisms of protection differed as evident by increased IL-13 and IFN-γ but diminished TGF-β production by Treg cells from Salmonella-CFA/IIC-treated mice. Adoptive transfer of Treg cells from both CFA/I-expressing constructs was equivalent in protecting against EAE, showing minimal disease. Although not as potent in its protection, CD25−CD4+ T cells from Salmonella-CFA/IIC showed minimal Th2 cells, but vaccination did prime these Th2 cells rendering partial protection against EAE challenge. In vivo IL-13 but not IFN-γ neutralization compromised protection conferred by adoptive transfer with Salmonella-CFA/IIC-induced Treg cells. Thus, the Salmonella-CFA/IIC vaccine elicits Treg cells with attributes from both the Salmonella vector and Salmonella-CFA/I vaccines. Importantly, these Treg cells can be induced to high potency by simply vaccinating against irrelevant Ags, offering a novel approach to treat autoimmune diseases independently of the autoantigen.

T and B lymphocytes have been implicated in the pathogenesis of renal ischemia reperfusion injury (IRI). The trafficking of lymphocytes into kidneys during IRI has been postulated to underlie this effect, but has not been rigorously studied. We therefore characterized the lymphocyte populations infiltrating into mouse kidneys 3 and 24 h after renal IRI. Immunohistochemistry and flow cytometry staining of kidney lymphocytes showed increased trafficking of CD3+ T cells and CD19+ B cells in both sham-operated and IRI mice 3 h after renal IRI. In the IRI mice, increased infiltration of NK1.1+ and CD4+NK1.1+ cells compared with normal and sham-operated mice was observed 3 and 24 h after renal IRI, respectively. After 24 h of renal IRI, the decreased percentages of CD3+, CD19+, and NK1.1+ populations in the IRI mice compared with control groups were observed. Increased TNF-α and IFN-γ production of kidney infiltration CD3+ T cells in IRI mice but not sham-operated mice was found. Unexpectedly, isolation and transfer of kidney-infiltrating lymphocytes 24 h after renal IRI into T cell-deficient mice reduced their functional and histological injury after renal IRI, suggesting that kidney-infiltrating lymphocytes could have a protective function. These quantitative, qualitative, and functional changes in kidney lymphocytes provide mechanistic insight into how lymphocytes modulate IRI, as well as demonstrating that abdominal surgery alone leads to lymphocyte changes in kidney.

Antibodies to K99 fimbriae afford protection to F5+bovine enterotoxigenicEscherichia coli(ETEC). Previous studies show that murine dams immunized withSalmonellavaccine vectors stably expressing K99 fimbriae confer protection to ETEC-challenged neonatal pups. To begin to address adaptation of the K99 scaffold to display heterologous B- and T-cell epitopes, studies were conducted to determine how much of the assembled K99 fimbria is required to maintain protective immunity. Sequential deletions in the K99 gene clusters were made, resulting in diminished localization of the K99 fimbrial subunit in the outer membrane. As placement of the K99 fimbrial subunit became progressively contained within the vaccine vector, diminished immunoglobulin A (IgA) and IgG1 antibody titers, as well as diminished Th2-type cytokine responses, were observed in orally immunized mice. Deletion offanGH, which greatly reduced the export of the fimbrial subunit to the outer membrane, showed only partial reduction in protective immunity. By contrast, deletion offanDEFGH, which also reduced the export of the fimbrial subunit to the outer membrane but retained more subunit in the cytoplasm, resulted in protective immunity being dramatically reduced. Thus, these studies showed that retention of K99 fimbrial subunit as native fimbriae or with the deletion offanGHis sufficient to confer protection.

To evaluate the role of the zinc-binding metalloprotease inBacteroides fragilistoxin (BFT) processing and activity, the zinc-binding consensus sequences (H348, E349, H352, G355, H358, and M366) were mutated by site-directed-mutagenesis. Our results indicated that single point mutations in the zinc-binding metalloprotease motif do not affect BFT processing but do reduce or eliminate BFT biologic activity in vitro.

Chloroaromatic compounds are xenobiotics that cause great concern. The degradation of a model molecule, 3,4-dichlorobenzoate (3,4-DCB), was studied using three aerobic (AE)-anaerobic (AN) biofilm reactor systems: a coupled aerobic-anaerobic recycle biofilm reactor (CAR) system, an in-series anaerobic-aerobic biofilm reactor (SAR) system; and an independent aerobic and anaerobic biofilm reactor (IAR) system. In all three systems the inlet substrate concentration was 2.0 g/l and the dilution rates ranged from 0.045 to 0.142 per hour. The results show that the degradation efficiency of the CAR system (expressed as dechlorination and xenobiotic disappearance efficiencies, and biomass yield), was higher at all dilution rates tested than in both SAR and IAR systems. Moreover, dechlorination and xenobiotic disappearance efficiencies for resting suspended aerobic and anaerobic cells or mixed aerobic-anaerobic growing cells under anaerobic conditions were higher than under aerobic conditions. These results suggest that a "cooperative metabolism" between aerobic and anaerobic bacteria (caused by an exchange of cells and metabolites between AE and AN reactors) in the CAR system overcame the metabolic and kinetic limitations of aerobic and anaerobic bacteria in the AE and AN reactors of IAR and SAR systems. Therefore, the degradation efficiency of persistent and recalcitrant chloroaromatic xenobiotic compounds could be enhanced by using a CAR system.

Acute kidney injury (AKI) is a frequent event associated with decreased allograft survival in patients with transplanted kidneys and high mortality in patients with native kidneys (1,2). AKI is a common complication in hospitalized patients, and its incidence has risen substantially over the past 15 years (1-3). As a conservative estimate, roughly 17 million admissions annually in the United States are complicated by AKI, resulting in over $10 billion in costs to the health care system (4). Kidney transplants from living unrelated donors (not well HLA matched) with minimal ischemic injury have improved allograft survival, compared with grafts from well matched cadaveric donors with significant ischemia (5, 6). This implies that renal ischemia reperfusion injury (IRI) can have important consequences on long-term graft survival and native kidneys. IRI is a highly complex cascade of events that includes interactions between vascular endothelium, interstitial compartments, circulating cells, and numerous mediator molecules (7). Renal ischemic injury has been found to permanently damage peritubular capillaries causing hypoxia, which may be involved in the progression of chronic renal disease after AKI (7, 8). Tubulointerstitial influx of inflammatory cells is found in many forms of chronic renal diseases, including ‘nonimmune’ diseases such as diabetes and hypertension (9). Tlymphocyte infiltration has also been observed early after moderate ischemia injury (10,11) however, the dynamics of infiltrating lymphocyte populations long term after moderate or severe ischemic injury is not very clear. It has been demonstrated that T and B lymphocytes are important mediators in the pathogenesis of renal IRI (10, 12) however, the mechanisms by which these cells induce kidney injury is largely unknown. The trafficking of pathogenic lymphocytes into kidneys after moderate and severe ischemic injury has been postulated to contribute to kidney damage (11, 13, 14) however the physiologic state and the dynamics of trafficking of these populations long term after ischemia have not been rigorously studied. Furthermore, the activation and expression of the effector-memory phenotype by infiltrated lymphocytes suggests the possibility that these lymphocytes are responding to an injury-associated antigen (15, 16). In addition, these lymphocytes are responsible to produce inflammatory mediators not only causing local kidney structure damage, but also the severe effects

It is well-established that significant ischemia-reperfusion injury during kidney transplantation results in increased incidence of long-term fibrosis and rejection. To test for a role of T cell infiltration and activation following ischemic injury, we induced both bilateral and unilateral renal ischemia in mice, followed by reperfusion, and then isolated mononuclear cells. Analysis of these cells by flow cytometry showed that 2 weeks after bilateral ischemia there was a significant increase of CD8(+) T cells. Furthermore, both CD4(+) and CD8(+) T cells infiltrated the injured kidney 6 weeks after unilateral ischemia. These T cells had increased expression of CD69(+) and CD44(hi)CD62L(-), markers of activation and effector-memory, respectively. CD4(+)NK1.1(+) and CD19(+) B cells were decreased in percentage both 6 and 11 weeks after bilateral or unilateral injury. There was a significant upregulation of IL-1beta, IL-6, TNF-alpha, IFN-gamma, MIP-2, and RANTES expression, measured by real-time PCR, 6 weeks after unilateral renal ischemia, further indicating T cell activation. Depletion of CD4(+) and CD8(+) T cells before ischemia caused less medullary damage and reduced kidney IFN-gamma expression, whereas their depletion following ischemia increased kidney IL-1beta; however, depletion of these cells had no effect on histological damage to the kidney. Our study demonstrates that moderate or severe kidney ischemia induces long-term T lymphocyte infiltration and cytokine/chemokine upregulation, leading to kidney structural changes.