The outcome of hematopoietic stem cell transplantation (HSCT) is controlled by genetic factors among which the leukocyte antigen human leukocyte antigen (HLA) matching is most important. In addition, ...minor histocompatibility antigens and non-HLA gene polymorphisms in genes controlling immune responses are known to contribute to the risks associated with HSCT. Besides single-nucleotide polymorphisms (SNPs) in protein coding genes, SNPs in regulatory elements such as microRNAs (miRNAs) contribute to these genetic risks. However, genetic risks require for their realization the expression of the respective gene or miRNA. Thus, gene and miRNA expression studies may help to identify genes and SNPs that indeed affect the outcome of HSCT. In this review, we summarize gene expression profiling studies that were performed in recent years in both patients and animal models to identify genes regulated during HSCT. We discuss SNP-mRNA-miRNA regulatory networks and their contribution to the risks associated with HSCT in specific examples, including forkheadbox protein 3 and regulatory T cells, the role of the miR-155 and miR-146a regulatory network for graft-versus-host disease, and the function of MICA and its receptor NKG2D for the outcome of HSCT. These examples demonstrate how SNPs affect expression or function of proteins that modulate the alloimmune response and influence the outcome of HSCT. Specific miRNAs targeting these genes and directly affecting expression of mRNAs are identified. It might be valuable in the future to determine SNPs and to analyze miRNA and mRNA expression in parallel in cohorts of HSCT patients to further elucidate genetic risks of HSCT.
Osteoarthritis (OA) is a degenerative joint disease, causing impaired mobility. There are currently no effective therapies other than palliative treatment. Mesenchymal stromal cells (MSCs) and their ...secreted extracellular vesicles (MSC-EVs) have shown promise in attenuating OA progression, promoting chondral regeneration, and modulating joint inflammation. However, the precise molecular mechanism of action driving their beneficial effects has not been fully elucidated. In this study, we analyzed MSC-EV-treated human OA chondrocytes (OACs) to assess viability, proliferation, migration, cytokine and catabolic protein expression, and microRNA and mRNA profiles. We observed that MSC-EV-treated OACs displayed increased metabolic activity, proliferation, and migration compared to the controls. They produced decreased proinflammatory (Il-8 and IFN-γ) and increased anti-inflammatory (IL-13) cytokines, and lower levels of MMP13 protein coupled with reduced expression of MMP13 mRNA, as well as negative microRNA regulators of chondrogenesis (miR-145-5p and miR-21-5p). In 3D models, MSC-EV-treated OACs exhibited enhanced chondrogenesis-promoting features (elevated sGAG, ACAN, and aggrecan). MSC-EV treatment also reversed the pathological impact of IL-1β on chondrogenic gene expression and extracellular matrix component (ECM) production. Finally, MSC-EV-treated OACs demonstrated the enhanced expression of genes associated with cartilage function, collagen biosynthesis, and ECM organization and exhibited a signature of 24 differentially expressed microRNAs, associated with chondrogenesis-associated pathways and ECM interactions. In conclusion, our data provide new insights on the potential mechanism of action of MSC-EVs as a treatment option for early-stage OA, including transcriptomic analysis of MSC-EV-treated OA, which may pave the way for more targeted novel therapeutics.
BACKGROUNDGraft-versus-host (GvH) disease (GvHD) remains a serious concern for patients undergoing antiviral cellular therapy. Despite the major improvements in cellular immunotherapy, the ...immunogenicity of virus-specific T cells has not yet been fully defined. This present study aims to examine how cytomegalovirus (CMV)-specific cytotoxic T lymphocytes (CTLs) respond to allogeneic antigen stimulation and whether they give rise to GvHD target tissue damage.
METHODSCytomegalovirus-specific CTLs were isolated by the IFN-γ secretion assay (gamma-catch) from healthy seropositive volunteers and expanded in vitro. The levels of intracellular IFN-γ, cytotoxic activity, IFN-γ and granzyme B secretion, and CD25 expression were measured using flow cytometry (fluorescence-activated cell sorting). The ability of CMV-CTLs to induce GvHD target tissue damage was evaluated using the human in vitro skin explant assay (skin explant assay).
RESULTSCytomegalovirus-specific CTLs responded specifically to CMV-phosphoprotein 65 stimulation by secreting IFN-γ and killing virus peptide loaded autologous phytohemagglutinin (PHA) blasts. Compared with unselected peripheral blood mononuclear cells, CMV-CTLs induced significantly less severe cutaneous GvH tissue damage. This observation coincided with low levels of CD25 expression, as well as IFN-γ and granzyme B secretion after allogeneic antigen stimulation in both the mixed lymphocyte reaction and in the skin explant assay.
CONCLUSIONSCytomegalovirus-specific CTLs isolated by the IFN-γ secretion assay from HLA-unmatched healthy donors exhibited a high level of anti-CMV potency without inducing significant cutaneous GvH tissue damage in vitro. This finding provides novel evidence supporting the safe use of in vitro expanded CMV-CTLs as an antiviral therapy in transplant patients with refractory CMV infections.
Rheumatoid arthritis (RA) and ankylosing spondylitis (AS) belong to the most common inflammatory rheumatic diseases. MicroRNAs (miRNAs) are small 18–22 RNA molecules that function as ...posttranscriptional regulators. They are abundantly present within extracellular vesicles (EVs), small intercellular communication vesicles that can be found in bodily fluids and that have key functions in pathological and physiological pathways. Recently, EVs have gained much interest because of their diagnostic and therapeutic potential. Using NanoString profiling technology, the miRNA repertoire of serum EVs was determined and compared in RA and AS patients before and after anti-TNF therapy to assess its potential use as a diagnostic and prognostic biomarker. Furthermore, possible functional effects of those miRNAs that were characterized by the most significant expression changes were evaluated using in silico prediction algorithms. The analysis revealed a unique profile of differentially expressed miRNAs in RA and AS patient serum EVs. We identified 12 miRNAs whose expression profiles enabled differentiation between RA and AS patients before induction of anti-TNF treatment, as well as 4 and 14 miRNAs whose repertoires were significantly changed during the treatment in RA and AS patients, respectively. In conclusion, our findings suggest that extracellular vesicle miRNAs could be used as potential biomarkers associated with RA and AS response to biological treatment.
Abstract Background aims Advanced therapy medicinal products (ATMP) are gene therapy, somatic cell therapy or tissue-engineered products regulated under (EC) No. 1394/2007 to ensure their free ...movement within the European Union while guaranteeing the highest level of health protection for patients. Academic good manufacturing practice (GMP) centers are major contributors in the development of ATMPs and this study assessed the impact of regulations on them. Methods European academic and non-industrial facilities (n = 747) were contacted, and a representative sample of 50 replied to a detailed questionnaire. Experienced centres were further selected in every Member State (MS) for semi-structured interviews. Indicators of ATMP production and development success were statistically assessed, and opinions about directive implementation were documented. Results Facilities experienced in manufacturing cell therapy transplant products are the most successful in developing ATMPs. New centres lacking this background struggle to enter the field, and there remains a shortage of facilities in academia participating in translational research. This is compounded by heterogeneous implementation of the regulations across MS. Conclusions GMP facilities successfully developing ATMPs are present in all MS. However, the implementation of regulations is heterogeneous between MS, with substantial differences in the definition of ATMPs and in the approved manufacturing environment. The cost of GMP compliance is underestimated by research funding bodies. This is detrimental to development of new ATMPs and commercialization of any that are successful in early clinical trials. Academic GMP practitioners should strengthen their political visibility and contribute to the development of functional and effective European Union legislation in this field.
Purpose: The HOX genes comprise a large family of homeodomain-containing transcription factors, present in four separate clusters, which are
key regulators of embryonic development, hematopoietic ...differentiation, and leukemogenesis. We aimed to study the role of
DNA methylation as an inducer of HOX gene silencing in leukemia.
Experimental Design: Three hundred and seventy-eight samples of myeloid and lymphoid leukemia were quantitatively analyzed (by COBRA analysis
and pyrosequencing of bisulfite-modified DNA) for methylation of eight HOXA and HOXB cluster genes. The biological significance of the methylation identified was studied by expression analysis and through re-expression
of HOXA5 in a chronic myeloid leukemia (CML) blast crisis cell line model.
Results: Here, we identify frequent hypermethylation and gene inactivation of HOXA and HOXB cluster genes in leukemia. In particular, hypermethylation of HOXA4 and HOXA5 was frequently observed (26-79%) in all types of leukemias studied. HOXA6 hypermethylation was predominantly restricted to lymphoid malignancies, whereas hypermethylation of other HOXA and HOXB genes was only observed in childhood leukemia. HOX gene methylation exhibited clear correlations with important clinical variables, most notably in CML, in which hypermethylation
of both HOXA5 ( P = 0.00002) and HOXA4 ( P = 0.006) was strongly correlated with progression to blast crisis. Furthermore, re-expression of HOXA5 in CML blast crisis
cells resulted in the induction of markers of granulocytic differentiation.
Conclusion: We propose that in addition to the oncogenic role of some HOX family members, other HOX genes are frequent targets for gene inactivation and normally play suppressor roles in leukemia development.
Heat shock protein 70 (HSP70) has previously been described as a potent antitumour vaccine. The mechanism relied on the ability of tumour derived HSP70 to associate with antigenic peptides, which, ...when cross presented, elicited a T cell mediated antitumour response. Subsequently, HSP70 was incorrectly described as a potent adjuvant of innate immunity, and although mistakes in the experimental approaches were exposed and associated with endotoxin contamination in the recombinant HSP70 specimen, questions still remain regarding this matter. Here we review only publications that have cautiously addressed the endotoxin contamination problem in HSP70 in order to reveal the real immunological function of the protein. Accordingly, “endotoxin free” HSP70 stimulates macrophages and delivers antigenic peptides to APCs, which effectively prime T cells mediating an antitumour reaction. Conversely, HSP70 has potent anti-inflammatory functions as follows: regulating T cell responses, reducing stimulatory capacity of DCs, and inducing development of immunosuppressive regulatory T cells. These activities were further associated with the immune evasive mechanism of tumours and implicated in the modulation of immune reactivity in autoimmune diseases and transplant-related clinical conditions. Consequently, the role of HSP70 in immune regulation is newly emerging and contrary to what was previously anticipated.
The SNAP (Smoking and Nicotine in Pregnancy) trial compared nicotine replacement therapy (NRT) patches with placebo in pregnant smokers; although NRT doubled cessation rates in the first 4 weeks, by ...delivery no differences in maternal smoking or birth outcomes were noted. As a result, NRT used in standard doses during pregnancy is considered ineffective for smoking cessation. Subsequent effects of NRT on the children of treated mothers are unknown because no trials have investigated the effect of gestational NRT use beyond birth. To assess whether NRT use in pregnancy might cause harm to infants, we aimed to compare effects of NRT and placebo on infant development 2 years after delivery.
1050 pregnant smokers aged 16–45 years, at 12–24 weeks' gestation, and smoking at least five cigarettes per day were recruited from seven hospitals in England between May 1, 2007, and Feb 26, 2010, and followed up until their infants were 2 years old. Participants were randomly assigned (1:1) via a computer-generated pseudorandom code with permuted blocks of randomly varying size to receive 8-week courses of NRT patches (15 mg/16 h) (n=521) or matched placebo (n=529); both groups received behavioural smoking cessation support. Randomisation was stratified by site with participants, health-care professionals, and research staff masked to treatment allocation. The primary results for participants and infants at delivery were published in 2012; we present results from the trial cohort 2 years after birth. After delivery, questionnaires were posted to participants and, if there was no response, to family physicians. The primary outcome at 2 years was infants' survival without developmental impairment (ie, no disability or problems with behaviour or development). Treatment groups were compared on an intention-to-treat basis. The trial is registered with Controlled-Trials.com, number ISRCTN07249128.
Questionnaires were returned at 2 years for 891 (88%) of 1010 live singleton births (445 of (88%) 503 given NRT and 446 (88%) of 507 given placebo). Because of missing data, developmental outcomes, including four infant deaths, were documented for 888 of (88%) 1010 singleton infants; 445 (88%) of 503 infants in NRT group and 443 (87%) of 507 infants in placebo. In the NRT group, 323 (73%) of 445 infants had no impairment compared with 290 (65%) of 443 infants in the placebo group (odds ratio OR 1·40, 95% CI 1·05–1·86, p=0·023). At 2 years, 15 (3%) of 521 mothers in the NRT group and nine (2%) of 529 mothers in the placebo groups self-reported prolonged smoking abstinence since a quit date set in pregnancy (OR 1·71, 95% CI 0·74–3·94, p=0·20). Adverse events were not collected after delivery, but previously reported adverse pregnancy and birth outcomes were similar in the two groups.
Infants born to women who used NRT for smoking cessation in pregnancy were more likely to have unimpaired development. NRT had no effect on prolonged abstinence from smoking but did cause a temporary doubling of smoking cessation shortly after randomisation during pregnancy, which could explain findings. If findings are confirmed by subsequent research, this has potential implications for the management of smoking in pregnancy.
National Institute for Health Research Health Technology Assessment Programme.
MicroRNAs (miRNA) have emerged as central regulators of diverse biological processes and contribute to driving pathology in several diseases. Acute graft-versus-host disease (aGvHD) represents a ...major complication after allogeneic hematopoietic stem cell transplantation, caused by alloreactive donor T cells attacking host tissues leading to inflammation and tissue destruction. Changes in miRNA expression patterns occur during aGvHD, and we hypothesized that we could identify miRNA signatures in target tissues of aGvHD that may potentially help understand the underlying molecular pathology of the disease. We utilized a rat model of aGvHD with transplantation of fully MHC-mismatched T cell depleted bone marrow, followed by infusion of donor T cells. The expression pattern of 423 rat miRNAs was investigated in skin, gut, and lung tissues and intestinal T cells with the NanoString hybridization platform, in combination with validation by quantitative PCR. MHC-matched transplanted rats were included as controls. In the skin, upregulation of miR-34b and downregulation of miR-326 was observed, while in the intestines, we detected downregulation of miR-743b and a trend toward downregulation of miR-345-5p. Thus, tissue-specific expression patterns of miRNAs were observed. Neither miR-326 nor miR-743b has previously been associated with aGvHD. Moreover, we identified upregulation of miR-146a and miR-155 in skin tissue of rats suffering from aGvHD. Analysis of intestinal T cells indicated 23 miRNAs differentially regulated between aGvHD and controls. Two of these miRNAs were differentially expressed either in skin (miR-326) or in intestinal (miR-345-5p) tissue. Comparison of intestinal and peripheral blood T cells indicated common dysregulated expression of miR-99a, miR-223, miR-326, and miR-345-5p. Analysis of predicted gene targets for these miRNAs indicated potential targeting of an inflammatory network both in skin and in the intestines that may further regulate inflammatory cytokine production. In conclusion, comprehensive miRNA profiling in rats suffering from aGvHD demonstrate tissue-specific differences in the expression patterns of miRNA that may not be detected by profiling of peripheral blood T cells alone. These tissue-specific miRNAs may contribute to distinct pathologic mechanisms and could represent potential targets for therapy.
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