HLA Polymorphism in Regressive and Non-Regressive Autism: A Preliminary Study.

Autism spectrum disorders (ASD) comprises heterogeneous neurodevelopmental conditions with symptom onset usually during infancy. However, about 10%-30% of affected cases experience a loss of language and social skills around 18-30 months, so-called regressive autism. In this subset with regression, immune dysfunctions including inflammation and autoimmunity have been proposed to be at risk factors. Given the implication of the human histocompatibility antigens (HLA) system in various aspects of immune responses, including autoimmunity, and in ASD, we investigate here the distribution of the HLA Class I and Class II haplotypes in 131 children with ASD meeting DSM-IV TR criteria, with and without regression. We found that 62 of the 98 non-regressive ASD patients carry the HLA-DPA1*01-DPB1*04 sub-haplotype as compared to 14 of the 33 patients with regression (63% vs. 43% respectively, Pc = 0.02), suggesting that this HLA haplotype may exert a protective effect against regression. Similarly, the HLA-DPA1*01-DPB1*04 has also been found to be more represented in healthy controls as compared to patients affected with common nonpsychiatric autoimmune disorders. Overall our findings suggest a possible involvement of HLA polymorphism in the context of regressive ASD. Autism Res 2020, 13: 182-186. © 2019 The Authors. Autism Research published by International Society for Autism Research published by Wiley Periodicals, Inc. LAY SUMMARY: Immune dysfunctions including inflammatory and autoimmune processes have been reported in autism, particularly in regressive forms. In this study, we analyzed the distribution of HLA haplotypes among children with autism spectrum disorder (ASD), with and without regression from Sweden and observed that HLA-DPA1*01-DPB1*04 sub-haplotype was less represented in patients with regressive autism as compared with those without regression. Such possible protective effect, also observed in other common autoimmune disorders, may constitute a link between HLA-mediated immune processes and regressive ASD.

HLA-class II haplotypes and Autism Spectrum Disorders.

Infections and autoimmunity are associated with autism spectrum disorders (ASD), with both strongly influenced by the genetic regulation of the human leukocyte antigen (HLA) system. The relationship between ASD and the HLA genetic diversity requires further investigation. Using a case control design, the distribution of HLA class II-DRB1 and DQB1 alleles, genotypes and haplotypes were investigated in ASD patients, versus healthy controls (HC). ASD patients meeting DSM-IV TR criteria and HC (474 and 350 respectively) were genotyped at medium resolution using a Luminex-based SSO technology. Comparisons of genotypes, allele frequencies associated with a haplotype analysis were performed. Results indicate: (i) the HLA-DRB1 *11-DQB1*07 haplotype was more prevalent in ASD patients, versus HC (Pc = 0.001), partially replicating previous data and possibly linking to gastro-intestinal (GI)-related pro-inflammatory processes, given that this haplotype associates with pediatric celiac disorders; (ii) the HLA-DRB1 *17-DQB1*02 haplotype was higher in HC, versus ASD patients (Pc = 0.002), indicating that this is a protective haplotype. Using the Autism Diagnostic Interview to assess clinical dimensions, higher scores on social (Pc = 0.006) and non-verbal functioning (Pc = 0.004) associated with the DRB1 *11 DQB1*07 haplotype. Our results support HLA involvement in ASD, with possible relevance to GI and gut-brain axis dysregulation.

Association between CRP genetic diversity and bipolar disorder comorbid complications.

BACKGROUND: Chronic low-grade inflammation is believed to contribute, at least in a subset of patients, to the development of bipolar disorder (BD). In this context, the most investigated biological marker is the acute phase response molecule, C-reactive protein (CRP). While the genetic diversity of CRP was amply studied in various pathological settings, little is known in BD. METHODS: 568 BD patients along with 163 healthy controls (HC) were genotyped for the following single-nucleotide polymorphisms (SNPs) on the CRP gene: intron rs1417938 (+ 29) T/A, 3'-UTR rs1130864 (+ 1444) G/A, and downstream rs1205 (+ 1846) (C/T). The statistical analysis was performed using Chi-square testing and consisted of comparisons of allele/genotype frequencies between patients and controls and within patient sub-groups according to BD clinical phenotypes and the presence of thyroid disorders. RESULTS: We found that the frequencies of the studied SNPs were similar in BD and HC groups. However, the CRP rs1130864 A allele carrier state was significantly more frequent: (i) in BD patients with thyroid disorders than in those without (pc = 0.046), especially among females (pc = 0.01) and independently of lithium treatment, (ii) in BD patients with rapid cycling than in those without (pc = 0.004). CONCLUSIONS: Overall, our findings suggest the possibility that CRP genetic diversity may contribute to the development of auto-immune comorbid disorders and rapid cycling, both proxy of BD severity. Such findings, if replicated, may allow to predict complex clinical presentations of the disease, a possible step towards precision medicine in psychiatry.

Optimization and evaluation of Luminex performance with supernatants of antigen-stimulated peripheral blood mononuclear cells.

BACKGROUND: The Luminex bead-based multiplex assay is useful for quantifying immune mediators such as cytokines and chemokines. Cross-comparisons of reagents for this technique from different suppliers have already been performed using serum or plasma but rarely with supernatants collected from antigen-stimulated peripheral blood mononuclear cells (PBMC). Here, we first describe an optimization protocol for cell culture including quantity of cells and culture duration to obtain reproducible cytokine and chemokine quantifications. Then, we compared three different Luminex kit suppliers. RESULTS: Intraclass correlation coefficients (ICCs) for a 2-days stimulation protocol were >0.8 for IFNγ and Perforin. The specific concentration was maximal after two or five days of stimulation, depending on the analyte, using 0.5 million PBMC per well, a cell quantity that gave the same level of specific cytokine secretion as 1.0 million. In the second part of the study, Luminex kits from Millipore showed a better working range than Bio-Rad and Ozyme ones. For tuberculin purified protein derivative (PPD)-stimulated samples, the overall mean pooled coefficients of variation (CVs) for all donors and all cytokines was 17.2 % for Bio-Rad, 19.4 % for Millipore and 26.7 % for Ozyme. Although the different kits gave cytokine concentrations that were generally compatible, there were discrepancies for particular cytokines. Finally, evaluation of precision and reproducibility of a 15-plex Millipore kit using a "home-made" internal control showed a mean intra-assay CV <13 % and an inter-assay CV <18 % for each cytokine concentration. CONCLUSIONS: A protocol with a single round of stimulation but with two time points gave the best results for assaying different cytokines. Millipore kits appear to be slightly more sensitive than those from Bio-Rad and Ozyme. However, we conclude that the panel of analytes that need to be quantified should be the main determinant of kit selection. Using an internal control we demonstrated that a 15-plex magnetic Milliplex kit displayed good precision and reproducibility. Our findings should help optimize assays for evaluating immune responses during the course of disease or infection, or in response to vaccine or therapy.

Toxoplasma gondii exposure may modulate the influence of TLR2 genetic variation on bipolar disorder: a gene-environment interaction study.

BACKGROUND: Genetic vulnerability to environmental stressors is yet to be clarified in bipolar disorder (BD), a complex multisystem disorder in which immune dysfunction and infectious insults seem to play a major role in the pathophysiology. Association between pattern-recognition receptor coding genes and BD had been previously reported. However, potential interactions with history of pathogen exposure are yet to be explored. METHODS: 138 BD patients and 167 healthy controls were tested for serostatus of Toxoplasma gondii, CMV, HSV-1 and HSV-2 and genotyped for TLR2 (rs4696480 and rs3804099), TLR4 (rs1927914 and rs11536891) and NOD2 (rs2066842) polymorphisms (SNPs). Both the pathogen-specific seroprevalence and the TLR/NOD2 genetic profiles were compared between patients and controls followed by modelling of interactions between these genes and environmental infectious factors in a regression analysis. RESULTS: First, here again we observed an association between BD and Toxoplasma gondii (p = 0.045; OR = 1.77; 95 % CI 1.01-3.10) extending the previously published data on a cohort of a relatively small number of patients (also included in the present sample). Second, we found a trend for an interaction between the TLR2 rs3804099 SNP and Toxoplasma gondii seropositivity in conferring BD risk (p = 0.017, uncorrected). CONCLUSIONS: Pathogen exposure may modulate the influence of the immunogenetic background on BD. A much larger sample size and information on period of pathogen exposure are needed in future gene-environment interaction studies.

Nanotubular crosstalk with distressed cardiomyocytes stimulates the paracrine repair function of mesenchymal stem cells.

Mesenchymal stem cells (MSC) are known to repair broken heart tissues primarily through a paracrine fashion while emerging evidence indicate that MSC can communicate with cardiomyocytes (CM) through tunneling nanotubes (TNT). Nevertheless, no link has been so far established between these two processes. Here, we addressed whether cell-to-cell communication processes between MSC and suffering cardiomyocytes and more particularly those involving TNT control the MSC paracrine regenerative function. In the attempt to mimic in vitro an injured heart microenvironment, we developed a species mismatch coculture system consisting of terminally differentiated CM from mouse in a distressed state and human multipotent adipose derived stem cells (hMADS). In this setting, we found that crosstalk between hMADS and CM through TNT altered the secretion by hMADS of cardioprotective soluble factors such as VEGF, HGF, SDF-1α, and MCP-3 and thereby maximized the capacity of stem cells to promote angiogenesis and chemotaxis of bone marrow multipotent cells. Additionally, engraftment experiments into mouse infarcted hearts revealed that in vitro preconditioning of hMADS with cardiomyocytes increased the cell therapy efficacy of naïve stem cells. In particular, in comparison with hearts treated with stem cells alone, those treated with cocultured ones exhibited greater cardiac function recovery associated with higher angiogenesis and homing of bone marrow progenitor cells at the infarction site. In conclusion, our findings established the first relationship between the paracrine regenerative action of MSC and the nanotubular crosstalk with CM and emphasize that ex vivo manipulation of these communication processes might be of interest for optimizing current cardiac cell therapies.