Sodium Sulfite Exacerbates Allograft Vasculopathy and Affects Tryptophan Breakdown in Murine Heterotopic Aortic Transplantation.

Graft vasculopathy is the main feature of chronic rejection in organ transplantation, with oxidative stress being a major trigger. Inflammation-associated prooxidant processes may be controlled by antioxidants; however, interference with redox-regulated mechanisms is a complex endeavor. An essential feature of the cellular immune response is the acceleration of tryptophan (Trp) breakdown, leading to the formation of several bioactive catabolites. Long-term activation of this immunobiochemical pathway contributes to the establishment of a tolerogenic environment, thereby supporting allograft survival. Herein, the impact of the antioxidant sodium sulfite on the development of graft vasculopathy was assessed in murine aortic transplantation. Allogeneic (BALB/c to C57BL/6) heterotopic murine aortic transplantations were performed. Animals were left untreated or were treated with 10 µl of 0.1 M, of 0.01 M sodium sulfite, or of 0.1 M sodium sulfate, intraperitoneally once/day, until postoperative day (POD) 100. Grafts were assessed by histology, immunohistochemistry, and adhesion molecule gene expression. Serum concentrations of tryptophan and its catabolite kynurenine (Kyn) were measured. On day 100, graft vasculopathy was significantly increased upon treatment with 0.1 M sodium sulfite, compared to allogeneic untreated controls (p = 0.004), which correlated with a significant increase of α-smooth-muscle-actin, Vcam-1, and P-selectin. Serum Kyn concentrations increased in the allogeneic control group over time (p < 0.05, POD ≥ 50), while low-dose sodium sulfite treatment (0.01 M) treatment resulted in a decrease in Kyn levels over time (p < 0.05, POD ≥ 10), compared to the respective baselines (p < 0.05). Longitudinal analysis of serum metabolite concentrations in the different treatment groups further identified an overall effect of sodium sulfite on Kyn concentrations. Antioxidative treatment may result in ambivalent consequences. Our data reveal that an excess of antioxidants like sodium sulfite can aggravate allograft vasculopathy, which further highlights the challenges associated with interventions that interfere with the complex interplay of redox-regulated inflammatory processes.

Autoantigen-specific immunosuppression with tolerogenic peripheral blood cells prevents relapses in a mouse model of relapsing-remitting multiple sclerosis.

BACKGROUND: Dendritic cells (DCs) rendered suppressive by treatment with mitomycin C and loaded with the autoantigen myelin basic protein demonstrated earlier their ability to prevent experimental autoimmune encephalomyelitis (EAE), the animal model for multiple sclerosis (MS). This provides an approach for prophylactic vaccination against autoimmune diseases. For clinical application such DCs are difficult to generate and autoantigens hold the risk of exacerbating the disease. METHODS: We replaced DCs by peripheral mononuclear cells and myelin autoantigens by glatiramer acetate (Copaxone(®)), a drug approved for the treatment of MS. Spleen cells were loaded with Copaxone(®), incubated with mitomycin C (MICCop) and injected into mice after the first bout of relapsing-remitting EAE. Immunosuppression mediated by MICCop was investigated in vivo by daily assessment of clinical signs of paralysis and in in vitro restimulation assays of peripheral immune cells. Cytokine profiling was performed by enzyme-linked immunosorbent assay (ELISA). Migration of MICCop cells after injection was examined by biodistribution analysis of (111)Indium-labelled MICCop. The number and inhibitory activity of CD4(+)CD25(+)FoxP3(+) regulatory T cells were analysed by histology, flow cytometry and in vitro mixed lymphocyte cultures. In order to assess the specificity of MICCop-induced suppression, treated EAE mice were challenged with the control protein ovalbumin. Humoral and cellular immune responses were then determined by ELISA and in vitro antigen restimulation assay. RESULTS: MICCop cells were able to inhibit the harmful autoreactive T-cell response and prevented mice from further relapses without affecting general immune responses. Administered MICCop migrated to various organs leading to an increased infiltration of the spleen and the central nervous system with CD4(+)CD25(+)FoxP3(+) cells displaying a suppressive cytokine profile and inhibiting T-cell responses. CONCLUSION: We describe a clinically applicable cell therapeutic approach for controlling relapses in autoimmune encephalomyelitis by specifically silencing the deleterious autoimmune response.

Immunosuppressive properties of mitomycin C-incubated human myeloid blood cells (MIC) in vitro.

Previous animal studies showed that donor-derived blood cells treated with mitomycin C (MMC) prolong allograft survival when injected into recipients. This model was effective with whole blood, peripheral blood mononuclear cells (PBMC) (monocytes being the active cell subpopulation) or dendritic cells. In view of a potential clinical application, we study now the immunosuppressive properties of human myeloid cells in vitro. Mature dendritic cells (generated from naïve monocytes) or monocytes treated with mitomycin C do not or only weakly inhibit allogeneic T cells in vitro, whereas cells in an early differentiation state between monocytes and DC exert suppressive activity when treated with MMC. In contrast, DC generated from MMC-treated monocytes show the morphology and phenotype of early immature DC (iDC) and suppress T-cell responses. It is known that untreated monocytes injected into a recipient encounter a cytokine milieu which differentiates them to stimulatory DC. In our in vitro experiment MMC-treated monocytes cultured in a DC-maturing milieu transform themselves into suppressive early iDC. This reproduces a process which takes place when administering MMC-monocytes to a recipient. In conclusion, human MMC-DC or MMC-monocytes are not or only weakly suppressive in vitro. When MMC-monocytes are differentiated to DC the resulting cells become suppressive.

Generation of suppressive blood cells for control of allograft rejection.

Our previous studies in rats showed that incubation of monocytic dendritic cells (DCs) with the chemotherapeutic drug mitomycin C (MMC) renders the cells immunosuppressive. Donor-derived MMC-DCs injected into the recipient prior to transplantation prolonged heart allograft survival. Although the generation of DCs is labour-intensive and time-consuming, peripheral blood mononuclear cells (PBMCs) can be easily harvested. In the present study, we analyse under which conditions DCs can be replaced by PBMCs and examine their mode of action. When injected into rats, MMC-incubated donor PBMCs (MICs) strongly prolonged heart allograft survival. Removal of monocytes from PBMCs completely abrogated their suppressive effect, indicating that monocytes are the active cell population. Suppression of rejection was donor-specific. The injected MICs migrated into peripheral lymphoid organs and led to an increased number of regulatory T-cells (Tregs) expressing cluster of differentiation (CD) markers CD4 and CD25 and forkhead box protein 3 (FoxP3). Tolerance could be transferred to syngeneic recipients with blood or spleen cells. Depletion of Tregs from tolerogenic cells abrogated their suppressive effect, arguing for mediation of immunosuppression by CD4⁺CD25⁺FoxP3⁺ Tregs. Donor-derived MICs also prolonged kidney allograft survival in pigs. MICs generated from donor monocytes were applied for the first time in humans in a patient suffering from therapy-resistant rejection of a haploidentical stem cell transplant. We describe, in the present paper, a simple method for in vitro generation of suppressor blood cells for potential use in clinical organ transplantation. Although the case report does not allow us to draw any conclusion about their therapeutic effectiveness, it shows that MICs can be easily generated and applied in humans.

Single-incision laparoscopic cholecystectomy: will it succeed as the future leading technique for gallbladder removal?

BACKGROUND: The quest for less traumatic abdominal approaches is changing paradigms in times of minimally invasive surgery. While natural orifice translumenal endoscopic surgery remains experimental, the single-incision approach could be the future of gallbladder surgery. METHODS: Prospectively collected data from 875 patients subjected to conventional single-incision laparoscopic cholecystectomy (SILC) or 4-port [laparoscopic cholecystectomy (LC)] were retrospectively analyzed and discussed with the current literature. RESULTS: Between 2008 and 2011, 201 (23%) SILCs and 674 (77%) LCs were performed. Mean age was 51.7±17.5 years (SILC: 45.1 vs. LC: 53.7 y). Patients were predominantly female (SILC: 75.1% vs. LC: 56.5%). Preoperative body mass index was 27.4±9.1 (SILC: 26.4 vs. LC: 27.8; P<0.05) and American Society of Anesthesiologists' score counted 1.67±0.57 in SILC and 1.86±0.7 in LC patients. Acute inflammation of the gallbladder (AIG) was not considered as a contraindication for SILC (AIG in SILC: 17.4% vs. LC: 35.5%). The mean operative time was significantly lower in the SILC group (SILC: 71±31 vs. LC: 79±27 min) and duration of postoperative hospital stay was shorter (SILC: 3.2±1.7 vs. LC: 4.5±2.6 d). No significant difference was observed between SILC and LC in any of the registered complications, including postoperative bleeding, trocar hernias, wound infection, abdominal abscess formation, bile duct injury, or cystic duct leakage. CONCLUSIONS: In the near future SILC could overrule conventional LC as the leading technique for gallbladder surgery. Our data reconfirm an excellent risk profile for SILC that is equal to that of LC. Large multicenter randomized controlled trials will be required to finally legitimize SILC as the succeeding principal method.

Single-incision laparoscopic sleeve gastrectomy versus multiport laparoscopic sleeve gastrectomy: analysis of 80 cases in a single center.

BACKGROUND: Through efficacy and improved safety, multiport laparoscopic sleeve gastrectomy (LAPS-G) has emerged as an important and broadly available treatment option for people with severe and complex obesity. Because a single-incision laparoscopic sleeve gastrectomy (SILS-G) would be less invasive, we applied this novel surgical technique for a selected number of patients enrolled into our minimally invasive bariatric program. SUBJECTS AND METHODS: A retrospective review of prospectively collected data from 80 morbidly obese patients who qualified for SILS-G or LAPS-G was performed from January 2011 to May 2012. RESULTS: SILS-G and LAPS-G were performed in 40 patients, respectively. All patients were female. Mean age was 41 (range, 19-73) years (SILS-G, 37 [19-62] years; LAPS-G, 43 [24-73] years; P=not significant). Preoperative body mass index was 40.8 (35.1-45.0) kg/m(2) in the SILS-G group and 43.8 (35.0-47.8) kg/m(2) in the LAPS-G group (P=not significant). Total operative time was significantly lower in the SILS-G group (85±21 minutes) compared with the LAPS-G group (97±26 minutes) (P<.05). Median percentage excess weight loss was comparable in both groups (SILS-G, 57.2%; LAPS-G, 53.7%) at 6.6 months after surgery. Mean hospital stay was 5 days (SILS-G, 5 [4-24] days; LAPS-G, 6 [4-14] days; P=not significant). Complication rates were low in both groups: leakage, 2.5% in SILS-G and 0% in LAPS-G; bleeding, 2.5% in SILS-G and 2.5% in LAPS-G; and trocar-site hernia, 0% in both groups. Patients operated on with single-incision laparoscopy had a significantly better cosmetic outcome as assessed by a scar satisfaction assessment questionnaire (P<.01). CONCLUSIONS: SILS-G is a feasible and safe operative procedure that leads to a significant reduction of total operative time compared with a multiport access procedure. Further potential benefits associated with single-incision laparoscopic surgery remain to be investigated objectively.

On the importance of tail ratios for psychological science.

Even small group-mean differences (whether combined with variance differences or not) or variance differences alone (absent mean differences) can generate marked and sometimes surprising imbalances in the representation of the respective groups compared in the distributional tail regions. Such imbalances in group representation, quantified as tail ratios, have general importance in the context of any threshold, susceptibility, diathesis-stress, selection, or similar models (including the study of sex differences), as widely conceptualized and applied in the psychological, social, medical, and biological sciences. However, commonly used effect-size measures, such as Cohen's d, largely exploit data information around the center of distributions, rather than from the tails, thereby missing potentially important patterns found in the tail regions. This account reviews the background and history of tail ratios, emphasizes their importance for psychological research, proposes a consensus approach for defining and interpreting them, introduces a tail-ratio calculator, and outlines future research agenda.

Evaluation of porcine mesenchymal stem cells for therapeutic use in human liver cancer.

Mesenchymal stem cell (MSC) transplantation is suggested for therapy of end-stage liver disease, due to e.g. liver cancer and metastasis. Liver transplantation is the only therapeutic option so far but donor organs are short. Also, the availability of allogeneic human MSCs for liver regeneration is limited. Therefore, we evaluated the suitability of porcine bone marrow MSCs from semi-adult pigs and found that morphology, surface expression pattern and multilineage differentiation are similar to those of human MSCs. Porcine MSCs differentiated to a hepatocyte-like phenotype and expressed porcine mRNA of typical liver proteins. However, hepatocyte-like MSCs failed to express the corresponding proteins and did not produce glycogen and urea as primary porcine hepatocytes do. Porcine MSCs were immunotolerated, since they did not activate resting human PBMCs, and were not attacked by human activated PBMCs. However, porcine MSCs led to enhanced proliferation of human pre-activated PBMCs suggesting that immunotoleration of porcine MSCs in the human system has limitations. Together, the potential of porcine MSCs for xenogenous use in human liver therapy is promising but needs further evaluation prior to clinical use.

Mitomycin C-treated antigen-presenting cells as a tool for control of allograft rejection and autoimmunity: from bench to bedside.

Cells have been previously used in experimental models for tolerance induction in organ transplantation and autoimmune diseases. One problem with the therapeutic use of cells is standardization of their preparation. We discuss an immunosuppressive strategy relying on cells irreversibly transformed by a chemotherapeutic drug. Dendritic cells (DCs) of transplant donors pretreated with mitomycin C (MMC) strongly prolonged rat heart allograft survival when injected into recipients before transplantation. Likewise, MMC-DCs loaded with myelin basic protein suppressed autoreactive T cells of MS patients in vitro and prevented experimental autoimmune encephalitis in mice. Comprehensive gene microarray analysis identified genes that possibly make up the suppressive phenotype, comprising glucocorticoid leucine zipper, immunoglobulin-like transcript 3, CD80, CD83, CD86, and apoptotic genes. Based on these findings, a hypothetical model of tolerance induction by MMC-treated DCs is delineated. Finally, we describe the first clinical application of MMC-treated monocyte-enriched donor cells in an attempt to control the rejection of a haploidentical stem cell transplant in a sensitized recipient and discuss the pros and cons of using MMC-treated antigen-presenting cells for tolerance induction. Although many questions remain, MMC-treated cells are a promising clinical tool for controlling allograft rejection and deleterious immune responses in autoimmune diseases.

Interleukin-1 regulates keratinocyte expression of T cell targeting chemokines through interleukin-1 receptor associated kinase-1 (IRAK1) dependent and independent pathways.

IL-1 is a potent pro-inflammatory cytokine that activates intracellular signaling cascades some of which may involve IL-1 receptor associated kinase-1 (IRAK1). Psoriasis is a T cell dependent chronic inflammatory condition of the skin of unknown cause. IL-1 has been implicated in psoriasis pathology, but the mechanism has not been elucidated. Interestingly, expression of IRAK1 is elevated in psoriatic skin. To identify a potential link between IL-1, keratinocytes and T cells in skin inflammation we employed pathway-focused microarrays to evaluate IL-1 dependent gene expression in keratinocytes. Several candidate mRNAs encoding known T cell chemoattractants were identified in primary keratinocytes and the stable keratinocyte cell line HaCaT. CCL5 and CCL20 mRNA and protein levels were confirmed up-regulated by IL-1 in concentration and time-dependent manners. Furthermore IL-1 synergized with IFN-gamma and TNF-alpha. Expression of CXCL9, CXCL10 and CXCL11 mRNAs was also increased in response to IL-1, but protein could only be detected in medium from cells treated with IFN-gamma alone or in combination with IL-1. Over-expression of IRAK1 led to increased constitutive and cytokine induced production of CCL5 and CCL20. Inhibition of IRAK1 activity through RNAi or expression of a dominant negative mutant blocked production of CCL5 and CCL20 but had no effect upon the IL-1 enhancement of IFN-gamma induced CXCL9, CXCL10 and CXCL11 production. In conclusion IL-1 regulates T cell targeting chemokine production in keratinocytes through IRAK1 dependent and independent pathways. These pathways may contribute to acute and chronic skin inflammation.

Functional polymorphisms of folate-metabolizing enzymes in relation to homocysteine concentrations in systemic lupus erythematosus.

OBJECTIVE: To determine if functional polymorphisms of folate/homocysteine pathway enzymes are associated with homocysteine concentrations and/or coronary artery calcification (CAC) scores in patients with systemic lupus erythematosus (SLE) and controls. METHODS: We investigated 163 SLE patients and 160 controls. Functional polymorphisms in 6 genes in the folate/homocysteine pathway were genotyped: 5,10-methylenetetrahydrofolate reductase (MTHFR) 677C>T, MTHFR 1298A>C, cystathionine ss-synthase (CBS) 844ins68, methionine synthase (MTR) 2756A>G, methionine synthase reductase (MTRR) 66A>G, thymidylate synthase (TYMS) 1494del6, and dihydrofolate reductase (DHFR) c.86+60_78. RESULTS: Homocysteine levels were higher in African American SLE patients than Caucasian patients and African American controls. Genotype distributions were significantly different in African American and Caucasian controls for 6 of the 7 polymorphisms. Genotype distributions for each polymorphism did not differ significantly between SLE patients and controls even after stratification by race. Glomerular filtration rate was strongly negatively correlated to homocysteine levels, and was therefore adjusted for as a covariate in the models of the effects of the polymorphisms on homocysteine levels. In SLE patients none of the 7 polymorphisms was associated with homocysteine concentrations. In Caucasian controls only MTHFR 677C>T and 1298A>C showed effects on homocysteine similar to what would be expected from the literature. There were no genotypic associations with median CAC scores in SLE patients or controls with and without stratification by race. CONCLUSION: Polymorphisms in folate/homocysteine metabolizing enzymes do not predict higher homocysteine levels or CAC scores in patients with SLE.