Homozygosity Haplotype and Whole-Exome Sequencing Analysis to Identify Potentially Functional Rare Variants Involved in Multiple Sclerosis among Sardinian Families

Multiple Sclerosis (MS) is a complex multifactorial autoimmune disease, whose sex- and age-adjusted prevalence in Sardinia (Italy) is among the highest worldwide. To date, 233 loci were associated with MS and almost 20% of risk heritability is attributable to common genetic variants, but many low-frequency and rare variants remain to be discovered. Here, we aimed to contribute to the understanding of the genetic basis of MS by investigating potentially functional rare variants. To this end, we analyzed thirteen multiplex Sardinian families with Immunochip genotyping data. For five families, Whole Exome Sequencing (WES) data were also available. Firstly, we performed a non-parametric Homozygosity Haplotype analysis for identifying the Region from Common Ancestor (RCA). Then, on these potential disease-linked RCA, we searched for the presence of rare variants shared by the affected individuals by analyzing WES data. We found: (i) a variant (43181034 T > G) in the splicing region on exon 27 of CUL9; (ii) a variant (50245517 A > C) in the splicing region on exon 16 of ATP9A; (iii) a non-synonymous variant (43223539 A > C), on exon 9 of TTBK1; (iv) a non-synonymous variant (42976917 A > C) on exon 9 of PPP2R5D; and v) a variant (109859349-109859354) in 3′UTR of MYO16.     

Biogeographic consequences of shifting climate for the western massasauga (Sistrurus tergeminus)

The western massasauga (Sistrurus tergeminus) is a small pit viper with an extensive geographic range, yet observations of this species are relatively rare. They persist in patchy and isolated populations, threatened by habitat destruction and fragmentation, mortality from vehicle collisions, and deliberate extermination. Changing climates may pose an additional stressor on the survival of isolated populations. Here, we evaluate historic, modern, and future geographic projections of suitable climate for S. tergeminus to outline shifts in their potential geographic distribution and inform current and future management. We used maximum entropy modeling to build multiple models of the potential geographic distribution of S. tergeminus. We evaluated the influence of five key decisions made during the modeling process on the resulting geographic projections of the potential distribution, allowing us to identify areas of model robustness and uncertainty. We evaluated models with the area under the receiver operating curve and true skill statistic. We retained 16 models to project both in the past and future multiple general circulation models. At the last glacial maximum, the potential geographic distribution associated with S. tergeminus occurrences had a stronghold in the southern part of its current range and extended further south into Mexico, but by the mid‐Holocene, its modeled potential distribution was similar to its present‐day potential distribution. Under future model projections, the potential distribution of S. tergeminus moves north, with the strongest northward trends predicted under a climate scenario increase of 8.5 W/m². Some southern populations of S. tergeminus have likely already been extirpated and will continue to be threatened by shifting availability of suitable climate, as they are already under threat from desertification of grasslands. Land use and habitat loss at the northern edge of the species range are likely to make it challenging for this species to track suitable climates northward over time.     

Galectin-9 recognizes and exhibits antimicrobial activity toward microbes expressing blood group–like antigens

While adaptive immunity recognizes a nearly infinite range of antigenic determinants, immune tolerance renders adaptive immunity vulnerable to microbes decorated in self-like antigens. Recent studies suggest that sugar-binding proteins galectin-4 and galectin-8 bind microbes expressing blood group antigens. However, the binding profile and potential antimicrobial activity of other galectins, particularly galectin-9 (Gal-9), has remained incompletely defined. Here, we demonstrate that while Gal-9 possesses strong binding preference for ABO(H) blood group antigens, each domain exhibits distinct binding patterns, with the C-terminal domain (Gal-9C) exhibiting higher binding to blood group B than the N-terminal domain (Gal-9N). Despite this binding preference, Gal-9 readily killed blood group B–positive Escherichia coli, whereas Gal-9N displayed higher killing activity against this microbe than Gal-9C. Utilization of microarrays populated with blood group O antigens from a diverse array of microbes revealed that Gal-9 can bind various microbial glycans, whereas Gal-9N and Gal-9C displayed distinct and overlapping binding preferences. Flow cytometric examination of intact microbes corroborated the microbial glycan microarray findings, demonstrating that Gal-9, Gal-9N, and Gal-9C also possess the capacity to recognize distinct strains of Providencia alcalifaciens and Klebsiella pneumoniae that express mammalian blood group–like antigens while failing to bind related strains that do not express mammalian-like glycans. In each case of microbial binding, Gal-9, Gal-9N, and Gal-9C induced microbial death. In contrast, while Gal-9, Gal-9N, and Gal-9C engaged red blood cells, each failed to induce hemolysis. These data suggest that Gal-9 recognition of distinct microbial strains may provide antimicrobial activity against molecular mimicry.     

NK cells and monocytes modulate primary HTLV-1 infection

We investigated the impact of monocytes, NK cells, and CD8⁺ T-cells in primary HTLV-1 infection by depleting cell subsets and exposing macaques to either HTLV-1 wild type (HTLV-1_WT) or to the HTLV-1_p12KO mutant unable to infect replete animals due to a single point mutation in orf-I that inhibits its expression. The orf-I encoded p8/p12 proteins counteract cytotoxic NK and CD8⁺ T-cells and favor viral DNA persistence in monocytes. Double NK and CD8⁺ T-cells or CD8 depletion alone accelerated seroconversion in all animals exposed to HTLV-1_WT. In contrast, HTLV-1_p12KO infectivity was fully restored only when NK cells were also depleted, demonstrating a critical role of NK cells in primary infection. Monocyte/macrophage depletion resulted in accelerated seroconversion in all animals exposed to HTLV-1_WT, but antibody titers to the virus were low and not sustained. Seroconversion did not occur in most animals exposed to HTLV-1_p12KO. In vitro experiments in human primary monocytes or THP-1 cells comparing HTLV-1_WT and HTLV-1_p12KO demonstrated that orf-I expression is associated with inhibition of inflammasome activation in primary cells, with increased CD47 “don’t-eat-me” signal surface expression in virus infected cells and decreased monocyte engulfment of infected cells. Collectively, our data demonstrate a critical role for innate NK cells in primary infection and suggest a dual role of monocytes in primary infection. On one hand, orf-I expression increases the chances of viral transmission by sparing infected cells from efferocytosis, and on the other may protect the engulfed infected cells by modulating inflammasome activation. These data also suggest that, once infection is established, the stoichiometry of orf-I expression may contribute to the chronic inflammation observed in HTLV-1 infection by modulating monocyte efferocytosis.     

Early endocytotic steps in elicited macrophages: omega-shaped plasma membrane vesicles at their cell surface.

Fluid-phase and receptor-mediated endocytosis were studied in Freund's adjuvant elicited macrophages. These cells were found to bind and internalize significantly larger amounts of peroxidase-antiperoxidase (PAP) immune complex than resident macrophages. Similarly the rate of the fluid-phase uptake was higher in elicited cells. When studying the early steps of endocytotic processes, omega-shaped plasma membrane pits (d～90 nm) were found at the macrophage cell surface. Although occurring occasionally in resident cells, their number was highly increased after elicitation in 30% of the macrophage cell population. The different morphology of these cells coincided with a lower endocytotic activity and a very strong ecto Ca²⁺-ATPase reaction. The present findings indicate that the elicited macrophage population is heterogenous and consists of different subclasses.