Philip Ruiz

Complement component C3 is not required for full expression of immune complex glomerulonephritis in MRL/lpr mice.

Complement activation and tissue deposition of complement fragments occur during disease progression in lupus nephritis. Genetic deficiency of some complement components (e.g., Factor B) and infusion of complement inhibitors (e.g., Crry, anti-C5 Ab) protect against inflammatory renal disease. Paradoxically, genetic deficiencies of early components of the classical complement pathway (e.g., C1q, C4, and C2) are associated with an increased incidence of lupus in humans and lupus-like disease in murine knockout strains. Complement protein C3 is the converging point for activation of all three complement pathways and thus plays a critical role in biologic processes mediated by complement activation. To define the role of C3 in lupus nephritis, mice rendered C3 deficient by targeted deletion were backcrossed for eight generations to MRL/lpr mice, a mouse strain that spontaneously develops lupus-like disease. We derived homozygous knockout (C3−/−), heterozygous (C3+/−), and C3 wild-type (C3+/+) MRL/lpr mice. Serum levels of autoantibodies and circulating immune complexes were similar among the three groups. However, there was earlier and significantly greater albuminuria in the C3−/− mice compared with the other two groups. Glomerular IgG deposition was also significantly greater in the C3−/− mice than in the other two groups, although overall pathologic renal scores were similar. These results indicate that C3 and/or activation of C3 is not required for full expression of immune complex renal disease in MRL/lpr mice and may in fact play a beneficial role via clearance of immune complexes.

The Role of Donor Bone Marrow Infusions in Withdrawal of Immunosuppression in Adult Liver Allotransplantation

We investigated the role of donor bone marrow cell (DBMC) infusions in immunosuppression withdrawal in adult liver transplantation. Patients enrolled were at least 3 years post‐transplantation, with stable graft function. Forty‐five (study group: G1) received DBMC, and 59 (control group: G2) did not. Immunosuppression was reduced by one third upon enrollment, by another third the second year of the study and was completely withdrawn the third year.

The first case report of the use of a zoom videoendoscope for the evaluation of small bowel graft mucosa in a human after intestinal transplantation

BACKGROUND Control of allograft rejection remains the most difficult dilemma in intestinal transplantation. Standard endoscopic surveillance to date has not been always accurate in the diagnosis of rejection. We describe the first application of a zoom video endoscope in monitoring graft mucosa in humans after intestinal transplantation. METHOD A zoom video endoscope, which can magnify the image up to 100-fold, was used in this study. The patient was a 31-year-old man who received an isolated intestinal transplant. Surveillance endoscopy with the zoom video endoscope was performed through the ileostomy. Endoscopic biopsies were done at the same time. RESULTS The zoom video endoscope showed the microscopic architecture of the graft mucosa such as villi and crypts with outstanding quality. We found that an enlargement of the crypt areas appeared to correlate with morphologic changes of early rejection. This finding was reversed with the treatment of rejection. CONCLUSIONS The zoom video endoscope successfully showed the detailed information of intestinal mucosa. The ability to visualize a more representative view of the graft mucosa could lead to better detection of early rejection. A greater experience with this unique method will provide more accurate assessment of the intestinal allograft.

A role for the thromboxane receptor in L-NAME hypertension.

Actions of the lipid mediator thromboxane (Tx) A2 acting through the TP receptor contribute to the pathogenesis of cardiovascular disease. To further explore the role of TxA2 in hypertension, we examined the consequences of deficiency of the TP receptor on the course of hypertension associated with endothelial dysfunction and salt sensitivity. To this end, the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) was administered to TP-deficient (Tp-/-) and wild-type (Tp+/+) control mice in drinking water for 21 wk along with a high-salt (HS; 6% NaCl) diet. Administration of L-NAME increased urinary excretion of TxB2 to a similar extent in both Tp+/+ and Tp-/- animals. L-NAME also caused significant and sustained elevations in blood pressure that reached a maximum between weeks 3 and 6. However, the severity of hypertension was attenuated in the Tp-/- mice throughout the study period (P<0.001). At the end of the study, the wild-type mice developed significant cardiac hypertrophy (23.6+/-2% increase in heart-to-body weight ratio). The severity of cardiac hypertrophy was attenuated in the TP-deficient group (11.1+/-2.6%; P<0.05). In contrast, kidney hypertrophy was exaggerated in the Tp-/- mice compared with controls (37.1+/-5.4 vs. 12.3+/-2.3%; P<0.01). Moreover, the severity of glomerulosclerosis, tubule vacuolization, and interstitial chronic inflammation was also enhanced in the Tp-/- group (P<0.01). Thus, in L-NAME hypertension, TP receptors contribute to elevated blood pressure and cardiac hypertrophy. In this model, TP receptors also provided unexpected protection against kidney injury.

Differential effect of allogeneic versus syngeneic mesenchymal stem cell transplantation in MRL/lpr and (NZB/NZW)F1 mice.

MSC are being explored as a promising novel treatment for SLE. In this study, we: 1) assessed the differential effects of allogeneic versus syngeneic MSC transplantation on lupus-like disease, 2) explored the mechanisms by which MSC modulate disease, and 3) investigated whether lupus-derived-MSC have intrinsic immunomodulatory defects. We showed that in MRL/lpr mice and (NZB/NZW)F1 mice, both B6-MSC and lupus-MSC from young mice ameliorated SLE-like disease and reduced splenic CD3+CD4+ T lymphocytes and CD19+CD21+ B lymphocytes. However, lupus-MSC from older (NZB/NZW)F1 mice did not reduce spleen weights, glomerular IgG deposits, renal pathology, interstitial inflammation, CD3+CD4+ T lymphocytes or CD19+CD21+ B lymphocytes significantly. Thus MSC transplantation ameliorates SLE-like disease partly through decreasing CD4+ T cell and naïve mature B cell numbers. Allogeneic MSC may be preferred over syngeneic lupus-derived-MSC given the decreased overall effectiveness of post-lupus-derived-MSC, which appears partially due to disease and not exclusively intrinsic defects in the MSC themselves.

Fecal calprotectin level measurements in small bowel allograft monitoring: a pilot study.

Background. Protocol endoscopy with biopsy is currently the gold standard of small bowel transplantion (SBTx) monitoring, however it is invasive, costly, needs skilled operator, may require anesthesia and may cause complications. We investigated fecal calprotectin level (FCL) as a candidate noninvasive marker for monitoring patients after SBTx. Methods. A pilot study was performed to test the use of FCL measurement in following up SBTx patients. Ileostomy effluents were collected at various postoperative days before endoscopy and biopsy. FCLs were measured by enzyme-linked immunosorbent assay and a cut-off level of 100 ng/mg was considered positive. The results were retrospectively evaluated in combination with clinical, endoscopic, and histopathological findings. FCLs are presented as median nanogram per milligram. Results. FCLs were measured in 122 samples that were obtained from 29 patients after SBTx. Only 1 of 69 positive FCL did not accompany abnormal findings. Retrospective evaluation showed that 11 samples from six patients (FCL: 217) coincided with rejection episodes, six samples from three patients (FCL: 125) coincided with viral enteritis, 51 samples from 21 patients (FCL: 207) coincided with nonspecific inflammation, 11 samples from two patients (FCL: 998) coincided with chronic intestinal ulceration, and finally 50 samples from 19 patients (FCL: 43) coincided with normal findings. No significant FCL difference was found between rejection, infection, and inflammation. FCL evolution in individuals showed that FCL can predict rejection days before histopathological diagnosis. Conclusion. FCL is a sensitive test for ongoing organic intestinal allograft pathologies. It might be useful as prescreening marker to avoid unnecessary endoscopies.

Immunohistochemical localization, purification, and characterization of human urinary bladder glutathione S-transferases

This study describes immunohistochemical localization, purification and characterization of glutathione S-transferase (GST) of human urinary bladder. Even though all the three major classes of isoenzymes (alpha, mu, and pi) were expressed in human bladder, more than 90% of total GST activity was accounted for by a pi class anionic form. Human bladder alpha, mu, and pi class GSTs were immunologically related to respective isoenzymes of other human tissues. GST pi was present in all 13 samples analyzed, whereas GST alpha and mu were detected in nine and eleven samples, respectively. GST alpha of human bladder appeared to be unique, because unlike this class of GSTs of other human tissues, bladder enzyme had lower affinity for GSH linked to epoxy-activated Sepharose 6B affinity resin. Immunohistochemical staining indicated localization of GST alpha in epithelial surface cells, underlying submucosa and smooth muscle, whereas mu and pi class isoenzymes were predominantly distributed in epithelial surface cells. These results suggest that human bladder GSTs may play an important role in providing protection against xenobiotics because epithelium is considered a target for several carcinogens and all the three classes of isoenzymes are expressed in these cells.

Inducible Nitric Oxide Synthase Inhibitors Reduce Urinary Markers of Systemic Oxidant Stress in Murine Proliferative Lupus Nephritis

Background Proliferative lupus nephritis (PLN) is characterized by increased expression of inducible nitric oxide (NO) synthase (iNOS). Inhibition of iNOS with NG-monomethyl L-arginine (L-NMMA) abrogates renal disease in two models of murine PLN, but the mechanism of this effect is unknown. Reactive oxygen species have both direct and indirect pathogenic effects in inflammatory lesions and are therefore potentially an important therapeutic target in PLN. We hypothesized that inhibition of iNOS activity would reduce ROS production in murine PLN. Methods A dose escalation of L-NMMA (0, 20, 100, and 500 mg/kg/day) was performed in New Zealand Black × New Zealand White F1 (NZB/W) mice with active renal disease. Twenty-four-hour urine nitrate + nitrite (NOX) was measured with a chemiluminescence NO analyzer. Twenty-four-hour urine 8-isoprostane F2α (8-iso-PGF2α) was measured by gas chromatography-negative ion chemical ionization mass spectrometry. MRL-MpJFASlpr (MRL/lpr) and NZB/W mice were divided into three groups and given either L-NMMA, L-N6-iminoethyl-lysine (L-NIL), or distilled water for 2 weeks. Urine NOX and 8-iso-PGF2α were determined after 2 weeks. Results L-NMMA reduced both urine NOX and 8-iso-PGF2α levels in a dose-dependent fashion in NZB/W and MRL/lpr mice. Urine NOX and 8-iso-PGF2α levels were highly correlated. Both specific (L-NIL) and nonspecific (L-NMMA) iNOS inhibition reduced urine NOX and 8-iso-PGF2α levels in both models of murine PLN. Conclusion These findings suggest that iNOS activity is a major source of reactive oxidant stress in these models of murine PLN. Future studies will address the pathogenic role of reactive oxygen stress in PLN.

Role of MHC-linked genes in autoantigen selection and renal disease in a murine model of systemic lupus erythematosus

We previously described a renal protective effect of factor B deficiency in MRL/lpr mice. Factor B is in the MHC cluster; thus, the deficient mice were H2b, the haplotype on which the knockout was derived, whereas the wild-type littermates were H2k, the H2 of MRL/lpr mice. To determine which protective effects were due to H2 vs factor B deficiency, we derived H2b congenic MRL/lpr mice from the 129/Sv (H2b) strain. Autoantibody profiling using autoantigen microarrays revealed that serum anti-Smith and anti-small nuclear ribonucleoprotein complex autoantibodies, while present in the majority of H2k/k MRL/lpr mice, were absent in the H2b/b MRL/lpr mice. Surprisingly, 70% of MRL/lpr H2b/b mice were found to be serum IgG3 deficient (with few to no IgG3-producing B cells). In addition, H2b/b IgG3-deficient MRL/lpr mice had significantly less proteinuria, decreased glomerular immune complex deposition, and absence of glomerular subepithelial deposits compared with MRL/lpr mice of any H2 type with detectable serum IgG3. Despite these differences, total histopathologic renal scores and survival were similar among the groups. These results indicate that genes encoded within or closely linked to the MHC region regulate autoantigen selection and isotype switching to IgG3 but have minimal effect on end-organ damage or survival in MRL/lpr mice.

Allogeneic uterus transplantation in baboons: surgical technique and challenges to long-term graft survival.

The authos declare no funding or conflicts of interest. Address correspondence to: James Fernandez, M.D., Ph.D., Department of Allergy and Clinical Immunology, Cleveland Clinic Foundation, Cleveland OH, 9500 Euclid Ave, A90, Cleveland, OH, 44195. E-mail: fernandezj2@ccf.org Received 21 May 2014. Accepted 22 May 2014. Copyright * 2014 by Lippincott Williams & Wilkins ISSN: 0041-1337/14/9805-e50 DOI: 10.1097/TP.0000000000000320