Matricellular protein SPARCL1 regulates tumor microenvironment-dependent endothelial cell heterogeneity in colorectal carcinoma.

Different tumor microenvironments (TMEs) induce stromal cell plasticity that affects tumorigenesis. The impact of TME-dependent heterogeneity of tumor endothelial cells (TECs) on tumorigenesis is unclear. Here, we isolated pure TECs from human colorectal carcinomas (CRCs) that exhibited TMEs with either improved (Th1-TME CRCs) or worse clinical prognosis (control-TME CRCs). Transcriptome analyses identified markedly different gene clusters that reflected the tumorigenic and angiogenic activities of the respective TMEs. The gene encoding the matricellular protein SPARCL1 was most strongly upregulated in Th1-TME TECs. It was also highly expressed in ECs in healthy colon tissues and Th1-TME CRCs but low in control-TME CRCs. In vitro, SPARCL1 expression was induced in confluent, quiescent ECs and functionally contributed to EC quiescence by inhibiting proliferation, migration, and sprouting, whereas siRNA-mediated knockdown increased sprouting. In human CRC tissues and mouse models, vessels with SPARCL1 expression were larger and more densely covered by mural cells. SPARCL1 secretion from quiescent ECs inhibited mural cell migration, which likely led to stabilized mural cell coverage of mature vessels. Together, these findings demonstrate TME-dependent intertumoral TEC heterogeneity in CRC. They further indicate that TEC heterogeneity is regulated by SPARCL1, which promotes the cell quiescence and vessel homeostasis contributing to the favorable prognoses associated with Th1-TME CRCs.

Comparison between bortezomib and rituximab in the treatment of antibody-mediated renal allograft rejection.

BACKGROUND: Antibody-mediated rejection (ABMR) following kidney transplantation is associated with poor allograft survival. Conventional treatment based on plasmapheresis (PPH) and the administration of intravenous immunoglobulins (IVIG) is not satisfactory. Two compounds, more specifically targeting B cells and plasma cells, may help to improve the prognosis: rituximab, a B-cell-depleting monoclonal antibody, and bortezomib, a proteasome inhibitor causing apoptosis of plasma cells. METHODS: Starting in February 2009, we treated 10 consecutive patients with ABMR according to current Banff criteria with one cycle of bortezomib [1.3 mg/m(2) intravenously (i.v.), Day 1, 4, 8, 11]. This group was compared to a historical control group of patients (n = 9) treated with a fixed single dose of rituximab (500 mg i.v.). All patients received PPH (6×) and IVIG (30 g). Patients with acute ABMR additionally received methylprednisolone (3 × 500 mg i.v.). RESULTS: Patient survival in both groups was 100%. At 18 months after treatment, graft survival was 6/10 in the bortezomib group as compared to 1/9 functioning grafts in the rituximab group (P = 0.071). Renal function was superior in patients treated with bortezomib as compared to rituximab-treated patients (serum creatinine at 9 months: 2.5 ± 0.6 versus 5.1 ± 2.1 mg/dL, P = 0.008). Proteinuria was not different between both groups (9 months: 1.3 ± 1.0 versus 1.6 ± 1.6 g/day, P = n.s.). CONCLUSIONS: Treatment of ABMR with bortezomib in addition to standard therapy was partially effective, whereas treatment with a fixed dose of rituximab in addition to standard therapy with PPH and IVIG did not result in sufficient long-term graft survival. In the future, new strategies including the combination of both substances and the application of higher doses must be discussed.

Antihumoral rejection therapy by proteasome inhibitor bortezomib: a case series.

These three case reports strongly support previously published data on the usefulness of bortezomib as an innovative and potent antihumoral rejection therapeutical. Contrary to the current, conventional therapeutic regimens including rituximab, PPH and IVIg the administration of bortezomib led to significantly decreased DSA levels facilitating prolonged allograft survival in two out of three cases. The present data might also suggest that the time point of first administration of bortezomib as therapeutic agent is of high importance for the outcome since progressed graft deterioration due to long persisting DSA cannot be reversed.

Differential effects of single dose FTY720 on CD62L+ B-cells in stable renal allograft recipients.

FTY720, a sphingosine-1-phosphate receptor agonist, is the archeotype of a new class of immune modulators, which redirects lymphocytes from the peripheral blood into secondary lymphatic tissue. Previously, it was shown that FTY720 differentially decreases peripheral T-cells, expressing specific chemokine and adhesion receptors. Here, we investigated the effect of single doses FTY720 on peripheral B-cells expressing CD62L, CD11a, CD49d and CXCR4 in stable human renal allograft recipients. Peripheral blood lymphocytes were isolated by Ficoll density centrifugation and stained with monoclonal antibodies against CD3 or CD19 and CD62L, CD11a, CD49d, CXCR4 to determine the percentage of these T- and B-cell subpopulations. Total lymphocyte counts were measured by routine laboratory diagnostics to calculate absolute lymphocyte subset counts. In FTY720 treated patients, total lymphocyte counts decreased by 31.8% (0.25-2 mg) and 60.4% (3.5 mg), and total T-cell counts by 38.8% (0.25-2 mg) and 70.9% (3.5 mg). In comparison, total B-cell counts decreased by 32.2% (0.25-2 mg) and 61.1% (3.5 mg). The reduction of CD62L+ B-cells was less pronounced as compared to CD62L+ T-cells (0.25-2 mg: 15.7% vs. 57.3%; 3.5 mg: 57.2% vs. 86.9%). CD11a+ B-cells decreased by 15.4% (0.25-2 mg) and 57.1% (3.5 mg), and CD49d+ B-cells by 15.0% (0.25-2 mg) and 56.7% (3.5 mg). CXCR4+ B-cells decreased by 19.9% (0.25-2 mg) and 57.2% (3.5 mg). In vitro experiments showed that FTY720 did not change the mean expression of CD62L, CD11a, CD49d and CXCR4 on CD19+ B-cells. In conclusion FTY720 treatment reduces B-cells expressing CD62L to a significant lesser degree than T-cells expressing CD62L.

FTY720 (fingolimod) in renal transplantation.

FTY720 (Fingolimod) is a novel immunomodulator with a mode of action that is completely different from classical immunosuppressants. FTY is a structural and functional analogue of the natural serum lipid, sphingosine, and is the first in a new class of drugs called sphingosine 1-phosphate receptor (S1P-R) modulators. This review discusses the recent findings on the mechanism of action, preclinical models and outlines the results of the ongoing clinical development program. FTY is highly effective in prolonging allograft survival in preclinical models of transplantation and in experimental models of autoimmune diseases. In clinical trials, this novel compound was investigated in de novo renal transplantation and in multiple sclerosis. Pharmacokinetics are characterized by a prolonged absorption phase, a large volume of distribution, and a long elimination half-life. FTY induces a rapid and transient decrease in lymphocyte counts, which supports the modulatory effects of the drug on lymphocyte sequestration. The most common adverse event was asymptomatic transient bradycardia, a pharmacodynamic effect modulated by atrial S1 P-R. FTY failed to show an improvement in efficacy for the prevention of renal allograft rejection in two large phase III studies. FTY treatment regimens were associated with impaired renal function and the development of macula edema. Consequently, the further development in renal transplantation was stopped. Because initial clinical studies strongly suggest that FTY is highly effective in multiple sclerosis FTY is now being explored in phase III studies for the treatment of demyelinating diseases, Ongoing studies in multiple sclerosis are eagerly awaited because they may provide novel therapeutic options for patients with autoimmune diseases.

Limitations of C2 monitoring in renal transplant recipients.

BACKGROUND: Recent developments have proposed the cyclosporin (CsA) concentration at 2 h post-dose (C(2)) as the best single time-point predictor of the extent of CsA exposure and as the optimal basis for monitoring immunosuppressive therapy in renal transplant patients. The present study sought to validate the cornerstones of the current concept of C(2) monitoring. METHODS: We assessed the predictive value, dose proportionality and intrapatient variability of C(2) levels in 41 de novo renal transplant recipients treated with CsA microemulsion, steroids, mycophenolate sodium and basiliximab. RESULTS: Patients with rejection and patients with CsA nephrotoxicity had lower C(2) (P = NS) and absorption (P<0.05 for toxicity), while C(0) did not show any significant difference. Receiver operating characteristic analysis did not detect discriminative C(2) values as a predictor of rejection or toxicity. In a substantial number of patients (29%) we observed poor and/or slow absorption, with C(0) >300 ng/ml and C(2) levels <800 ng/ml during the first month and a high rate of complications in these patients (18% rejection, 64% toxicity). Absorption increased over the first month post-transplant. Analysis of dose changes indicated that C(2) levels are not dose-proportional. Intrapatient variability of C(2) was as high as that of C(0). CONCLUSIONS: C(2) levels do not predict rejection or toxicity. C(2) monitoring alone does not detect toxicity in poor and/or slow absorbers, who constitute a significant proportion of patients. Changes in absorption over time, high intrapatient variability and lack of dose proportionality constitute further limitations of the C(2) monitoring concept in the early post-transplant phase.

FTY720 mediates apoptosis-independent lymphopenia in human renal allograft recipients: different effects on CD62L+ and CCR5+ T lymphocytes.

BACKGROUND: The sphingolipid FTY720 (FTY), a novel immune modulator, induces lymphopenia and prevents allograft rejection. This study was designed to study the effect of FTY on lymphocyte subpopulations and apoptosis in stable renal allograft recipients. METHODS: Stable renal allograft recipients received a single oral dose of 0.25 to 3.5 mg of FTY (n= 13) or placebo (n= 3). Whole blood was drawn immediately before and at 4, 8, 12, 24, 72, and 96 hr after administration. The number of lymphocyte subpopulations, with an emphasis on surface markers involved in lymphocyte migration, was analyzed by flow cytometry. Apoptotic lymphocytes were detected following Annexin V-FITC/PI staining. Lymphocyte mobility was investigated in a modified Boyden chamber. RESULTS: FTY induced a transient lymphopenia by an apoptosis-independent mechanism. In vitro experiments with peripheral blood mononuclear cells (PBMC) confirmed that clinically relevant concentrations of FTY (0.1 microM) increased lymphocyte mobility, whereas only suprapharmacologic concentrations of FTY (10 microM) could induce apoptosis. FTY-treated patients had reversible changes in the composition of peripheral lymphocyte subpopulations. CD62L+ T cells decreased to the greatest extent (-57%). In contrast, CCR5+ T-cell counts declined only marginally (-10%). In vitro, treatment of PBMC with FTY (1 mM-10 microM) did not induce changes in the expression of these surface markers. CONCLUSIONS: The data indicate that FTY mediates apoptosis-independent lymphopenia in human renal allograft recipients. FTY-induced lymphopenia preferentially affects CD62L+ and CCR5- T-lymphocyte subpopulations.