Impact of bone marrow-derived mesenchymal stromal cells on experimental xenogeneic graft-versus-host disease

Background aimsGraft-versus-host disease (GVHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation caused by donor T cells reacting against host tissues. Previous studies have suggested that mesenchymal stromal cells (MSCs) could exert potent immunosuppressive effects.MethodsThe ability of human bone marrow derived MSCs to prevent xenogeneic GVHD in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice and in NOD/SCID/interleukin-2Rγ(null) (NSG) mice transplanted with human peripheral blood mononuclear cells (PBMCs) was assessed.ResultsInjection of 200 × 10⁶ human PBMCs intraperitoneally (IP) into sub-lethally (3.0 Gy) irradiated NOD/SCID mice also given anti-asialo GM₁ antibodies IP 1 day prior and 8 days after transplantation induced lethal xenogeneic GVHD in all tested mice. Co-injection of 2 × 10⁶ MSCs IP on day 0 did not prevent lethal xenogeneic GVHD induced by injection of human PBMCs. Similarly, injection of 30 × 10⁶ human PBMCs IP into sub-lethally (2.5 Gy) irradiated NSG mice induced a lethal xenogeneic GVHD in all tested mice. Injection of 3 × 10⁶ MSCs IP on days 0, 7, 14 and 21 did not prevent lethal xenogeneic GVHD induced by injection of human PBMCs.ConclusionsInjection of MSCs did not prevent xenogeneic GVHD in these two humanized mice models.     

Cell cycle regulators cyclin D1 and CDK4/6 have estrogen receptor-dependent divergent functions in breast cancer migration and stem cell-like activity

Cyclin D1 and its binding partners CDK4/6 are essential regulators of cell cycle progression and are implicated in cancer progression. Our aim was to investigate a potential regulatory role of these proteins in other essential tumor biological characteristics. Using a panel of breast cancer cell lines and primary human breast cancer samples, we have demonstrated the importance of these cell cycle regulators in both migration and stem-like cell activity. siRNA was used to target cyclin D1 and CDK4/6 expression, having opposing effects on both migration and stem-like cell activity dependent upon estrogen receptor (ER) expression. Inhibition of cyclin D1 or CDK4/6 increases or decreases migration and stem-like cell activity in ER−ve (ER-negative) and ER+ve (ER-positive) breast cancer, respectively. Furthermore, overexpressed cyclin D1 caused decreased migration and stem-like cell activity in ER−ve cells while increasing activity in ER+ve breast cancer cells. Treatment of breast cancer cells with inhibitors of cyclin D1 and CDK4/6 (Flavopiridol/PD0332991), currently in clinical trials, mimicked the effects observed with siRNA treatment. Re-expression of ER in two ER−ve cell lines was sufficient to overcome the effects of either siRNA or clinical inhibitors of cyclin D1 and CDK4/6.   In conclusion, cyclin D1 and CDK4/6 have alternate roles in regulation of migration and stem-like cell activity. Furthermore, these effects are highly dependent upon expression of ER. The significance of these results adds to our general understanding of cancer biology but, most importantly, could be used diagnostically to predict treatment response to cell cycle inhibition in breast cancer.     

ERCC1 function in nuclear excision and interstrand crosslink repair pathways is mediated exclusively by the ERCC1-202 isoform

ERCC1 (excision repair cross-complementation group 1) plays essential roles in the removal of DNA intrastrand crosslinks by nucleotide excision repair, and that of DNA interstrand crosslinks by the Fanconi anemia (FA) pathway and homology-directed repair processes (HDR). The function of ERCC1 thus impacts on the DNA damage response (DDR), particularly in anticancer therapy when DNA damaging agents are employed. ERCC1 expression has been proposed as a predictive biomarker of the response to platinum-based therapy. However, the assessment of ERCC1 expression in clinical samples is complicated by the existence of 4 functionally distinct protein isoforms, which differently impact on DDR. Here, we explored the functional competence of each ERCC1 protein isoform and obtained evidence that the 202 isoform is the sole one endowed with ERCC1 activity in DNA repair pathways. The ERCC1 isoform 202 interacts with RPA, XPA, and XPF, and XPF stability requires expression of the ERCC1 202 isoform (but none of the 3 others). ERCC1-deficient non-small cell lung cancer cells show abnormal mitosis, a phenotype reminiscent of the FA phenotype that can be rescued by isoform 202 only. Finally, we could not observe any dominant-negative interaction between ERCC1 isoforms. These data suggest that the selective assessment of the ERCC1 isoform 202 in clinical samples should accurately reflect the DDR-related activity of the gene and hence constitute a useful biomarker for customizing anticancer therapies.     

CpG Methylation Changes within the IL2RA Promoter in Type 1 Diabetes of Childhood Onset

None of the polymorphic variants of the IL2RA gene found associated with Type 1 Diabetes (T1D) was shown to have a functional effect. To test if the epigenetic variation could play a role at this locus, we studied the methylation of 6 CpGs located within the proximal promoter of IL2RA gene in 252 T1D patients compared with 286 age-matched controls. We found that DNA methylation at CpGs −373 and −456 was slightly but significantly higher in patients than in controls (40.4±4.6 vs 38.3±5.4, p = 1.4E4; 91.4±2.8 vs 89.5±5.3, p = 1.8E-6), while other CpG showed a strictly comparable methylation. Among 106 single nucleotide polymorphisms (SNPs) located in the neighboring 180kb region, we found that 28 SNPs were associated with DNA methylation at CpG −373. Sixteen of these SNPs were known to be associated with T1D. Our findings suggest that the effect of IL2RA risk alleles on T1D may be partially mediated through epigenetic changes.     

Campylobacteriosis in Urban versus Rural Areas: A Case-Case Study Integrated with Molecular Typing to Validate Risk Factors and to Attribute Sources of Infection

Campylobacter infection is a leading cause of bacterial gastroenteritis worldwide, and most clinical cases appear as isolated, sporadic infections for which the source is rarely apparent. From July 2005 to December 2007 we conducted a prospective case-case study of sporadic, domestically-acquired Campylobacter enteritis in rural versus urban areas and a prevalence study of Campylobacter in animal and environmental sources in the Eastern Townships, Quebec. Isolates were typed using Multilocus Sequence Typing (MLST) to reinforce the case-case findings and to assign a source probability estimate for each human isolate. The risk of human campylobacteriosis was 1.89-fold higher in rural than urban areas. Unconditional multivariate logistic regression analysis identified two independent risk factors associated with human Campylobacter infections acquired in rural area: occupational exposure to animals (OR = 10.6, 95% CI: 1.2–91, p = 0.032), and household water coming from a private well (OR = 8.3, 95% CI: 3.4–20.4, p<0.0001). A total of 851 C. jejuni isolates (178 human, 257 chicken, 87 bovine, 266 water, 63 wild bird) were typed using MLST. Among human isolates, the incidence rates of clonal complexes (CC) CC-21, CC-45, and CC-61 were higher in rural than urban areas. MLST-based source attribution analysis indicated that 64.5% of human C. jejuni isolates were attributable to chicken, followed by cattle (25.8%), water (7.4%), and wild birds (2.3%). Chicken was the attributable source for the majority of cases, independent of residential area, sex and age. The increased incidence in rural compared to urban areas was associated with occupational exposure to animals, particularly cattle among those aged 15–34 years, and with consumption of private well water. Both bovine and water exposure appeared to contribute to the seasonal variation in campylobacteriosis. These results provide a basis for developing public education and preventive programs targeting the risk factors identified.     

Predictive Factors of Herpes Zoster HIV-Infected Patients: Another Adverse Effect of Crack Cocaine

A retrospective cohort study was conducted on 1541 HIV-infected patients to determine variables associated with the incidence of herpes zoster. A single failure Cox model showed that herpes zoster incidence increased following the first 6 months of antiretroviral treatment adjusted hazard ratio (AHR)=5 (95%CI=2.6-9.2), P<0.001; in the >60 years age group AHR=2 (95%CI=1-4), P=0.04; in patients in the top CD8 quartile AHR=2.1 (95%CI=1.3-3.6), P<0.001; and in patients previously reported to use crack cocaine AHR=5.9, (95%CI=1.4-25), P=0.02. Herpes zoster incidence increased in patients with CD4 counts<500 per mm³ and gradually declined since 1992-1996, with AHR=0.3 (95%CI=0.2-0.5), P<0.001 for the 1997-2002 period and AHR=0.24 (95%CI=0.14-0.4), P<0.001 for the 2002-2008 period. Contrary to what has been described elsewhere, there was no specific effect of protease inhibitors on herpes zoster incidence. The present study is the first to suggest that crack cocaine is associated with an increased incidence of herpes zoster. The neurological or immunological effects of crack are discussed.     

The Cholinergic Anti-Inflammatory Pathway Delays TLR-Induced Skin Allograft Rejection in Mice: Cholinergic Pathway Modulates Alloreactivity

Activation of innate immunity through Toll-like receptors (TLR) can abrogate transplantation tolerance by revealing hidden T cell alloreactivity. Separately, the cholinergic anti-inflammatory pathway has the capacity to dampen macrophage activation and cytokine release during endotoxemia and ischemia reperfusion injury. However, the relevance of the α7 nicotinic acetylcholine receptor (α7nAChR)-dependent anti-inflammatory pathway in the process of allograft rejection or maintenance of tolerance remains unknown. The aim of our study is to investigate whether the cholinergic pathway could impact T cell alloreactivity and transplant outcome in mice. For this purpose, we performed minor-mismatched skin allografts using donor/recipient combinations genetically deficient for the α7nAChR. Minor-mismatched skin grafts were not rejected unless the mice were housed in an environment with endogenous pathogen exposure or the graft was treated with direct application of imiquimod (a TLR7 ligand). The α7nAChR-deficient recipient mice showed accelerated rejection compared to wild type recipient mice under these conditions of TLR activation. The accelerated rejection was associated with enhanced IL-17 and IFN-γ production by alloreactive T cells. An α7nAChR-deficiency in the donor tissue facilitated allograft rejection but not in recipient mice. In addition, adoptive T cell transfer experiments in skin-grafted lymphopenic animals revealed a direct regulatory role for the α7nAChR on T cells. Taken together, our data demonstrate that the cholinergic pathway regulates alloreactivity and transplantation tolerance at multiple levels. One implication suggested by our work is that, in an organ transplant setting, deliberate α7nAChR stimulation of brain dead donors might be a valuable approach for preventing donor tissue inflammation prior to transplant.