Current State of Precision Medicine in Primary Systemic Vasculitides

Precision medicine (PM) is an emerging data-driven health care approach that integrates phenotypic, genomic, epigenetic, and environmental factors unique to an individual. The goal of PM is to facilitate diagnosis, predict effective therapy, and avoid adverse reactions specific for each patient. The forefront of PM is in oncology; nonetheless, it is developing in other fields of medicine, including rheumatology. Recent studies on elucidating the genetic architecture of polygenic and monogenic rheumatological diseases have made PM possible by enabling physicians to customize medical treatment through the incorporation of clinical features and genetic data. For complex inflammatory disorders, the prevailing paradigm is that disease susceptibility is due to additive effects of common reduced-penetrance gene variants and environmental factors. Efforts have been made to calculate cumulative genetic risk score (GRS) and to relate specific susceptibility alleles for use of target therapies. The discovery of rare patients with single-gene high-penetrance mutations informed our understanding of pathways driving systemic inflammation. Here, we review the advances in practicing PM in patients with primary systemic vasculitides (PSVs). We summarize recent genetic studies and discuss current knowledge on the contribution of epigenetic factors and extracellular vesicles (EVs) in disease progression and treatment response. Implementation of PM in PSVs is a developing field that will require analysis of a large cohort of patients to validate data from genomics, transcriptomics, metabolomics, proteomics, and epigenomics studies for accurate disease profiling. This multi-omics approach to study disease pathogeneses should ultimately provide a powerful tool for stratification of patients to receive tailored optimal therapies and for monitoring their disease activity

Whole genome sequencing of experimental hybrids supports meiosis-like sexual recombination in Leishmania

Hybrid genotypes have been repeatedly described among natural isolates of Leishmania, and the recovery of experimental hybrids from sand flies co-infected with different strains or species of Leishmania has formally demonstrated that members of the genus possess the machinery for genetic exchange. As neither gamete stages nor cell fusion events have been directly observed during parasite development in the vector, we have relied on a classical genetic analysis to determine if Leishmania has a true sexual cycle. Here, we used whole genome sequencing to follow the chromosomal inheritance patterns of experimental hybrids generated within and between different strains of L. major and L. infantum. We also generated and sequenced the first experimental hybrids in L. tropica. We found that in each case the parental somy and allele contributions matched the inheritance patterns expected under meiosis 97–99% of the time. The hybrids were equivalent to F1 progeny, heterozygous throughout most of the genome for the markers that were homozygous and different between the parents. Rare, non-Mendelian patterns of chromosomal inheritance were observed, including a gain or loss of somy, and loss of heterozygosity, that likely arose during meiosis or during mitotic divisions of the progeny clones in the fly or culture. While the interspecies hybrids appeared to be sterile, the intraspecies hybrids were able to produce backcross and outcross progeny. Analysis of 5 backcross and outcross progeny clones generated from an L. major F1 hybrid, as well as 17 progeny clones generated from backcrosses involving a natural hybrid of L. tropica, revealed genome wide patterns of recombination, demonstrating that classical crossing over occurs at meiosis, and allowed us to construct the first physical and genetic maps in Leishmania. Altogether, the findings provide strong evidence for meiosis-like sexual recombination in Leishmania, presenting clear opportunities for forward genetic analysis and positional cloning of important genes.</div

Human glycated albumin affects glucose metabolism in L6 skeletal muscle cells by impairing insulin-induced insulin receptor substrate (IRS) signaling through a protein kinase C alpha-mediated mechanism.

Nonenzymatic glycation is increased in diabetes and leads to increased levels of glycated proteins. Most studies have focused on the role of glycation products in vascular complications. Here, we have investigated the action of human glycated albumin (HGA) on insulin signaling in L6 skeletal muscle cells. Exposure of these cells to HGA inhibited insulin-stimulated glucose uptake and glycogen synthase activity by 95 and 80%, respectively. These effects were time- and dose-dependent, reaching a maximum after 12 h incubation with 0.1 mg/ml HGA. In contrast, exposure of the cells to HGA had no effect on thymidine incorporation. Further, HGA reduced insulin-stimulated serine phosphorylation of PKB and GSK3, but did not alter ERK1/2 activation. HGA did not affect either insulin receptor kinase activity or insulin-induced Shc phosphorylation on tyrosine. In contrast, insulin-dependent IRS-1 and IRS-2 tyrosine phosphorylation was severely reduced in cells preincubated with HGA for 24 h. Insulin-stimulated association of PI3K with IRS-1 and IRS-2, and PI3K activity were reduced by HGA in parallel with the changes in IRS tyrosine phosphorylation, while Grb2-IRS association was unchanged. In L6 myotubes, exposure to HGA increased PKC activity by 2-fold resulting in a similar increase in Ser/Thr phosphorylation of IRS-1 and IRS-2. These phosphorylations were blocked by the PKC inhibitor bisindolylmaleimide (BDM). BDM also blocked the action of HGA on insulin-stimulated PKB and GSK3 alpha. Simultaneously, BDM rescued insulin-stimulation of glucose uptake and glycogen synthase activity in cells exposed to HGA. The use of antibodies specific to PKC isoforms shows that this effect appears to be mediated by activated PKC alpha, independent of reactive oxygen species production. In summary, in L6 skeletal muscle cells, exposure to HGA leads to insulin resistance selectively in glucose metabolism with no effect on growth-related pathways regulated by the hormone

Lessons from the first ecancer symposium on angiogenesis in gastric cancer

In March 2015, ecancer hosted a symposium at the European Institute of Oncology in Milan, Italy on the topic of angiogenesis in gastric cancer. During this meeting, leaders in the field focused on the latest research on the topic of angiogenesis in gastric cancer, delivering lectures combined with interactive question and answer (Q & A) sessions and a roundtable discussion with the meeting's chairs. Topics covered included biomarkers, imaging, and the current state of antiangiogenic drugs in gastric cancer. This report will provide an understanding of the relevance of angiogenesis in gastric cancer research, and clinical experiences from diverse perspectives

Nursing care postpartum women seropositive for hiv before the inability to natural breastfeeding

Objectives: to know the expertise of nurses in caring for postpartum women seropositive for HIV on breastfeeding; identify the interaction of nurses with women with HIV about the impossibility of breastfeeding. Method: this was a descriptive, exploratory and qualitative in nature, with twenty-three women in the rooming Antônio Pedro University Hospital (HUAP) through structured interviews and analyzed with the precepts of content analysis in thematic, after approval by the Ethics Committee of the HUAP, under nº 254.060/13. Results: the following categories emerged: disparities in the guidelines at the rooming set: natural breastfeeding; interaction of nurses rooming with HIV-positive mothers for HIV on the impossibility of breastfeeding. Conclusion: the need for guidance and awareness of women about their reasons and issues related to the inability to breastfeed

In Situ

Chagas' disease is caused by the protozoan parasite Trypanosoma cruzi. The immune system plays an important role in the reduction of parasite load, but may also contribute to the development of lesions observed during the chronic phase of the disease. We analyzed cytokines produced by inflammatory heart cells in 21 autopsy samples obtained from patients with Chagas' disease divided according to the presence or absence of heart failure (HF). Left ventricular sections were analyzed by immunohistochemistry using antibodies against human IL-4, IFN-γ, TGF-β, TNF-α, and NOS2. In situ mRNA expression was quantified by a Low Density Array. The number of IFN-γ-positive cells was significantly higher than IL-4 positive cells. TNF-α, TGF-β and NOS2 were detected in 65%, 62% and 94% of samples respectively. There was an association between TNF-α-producing cells and the presence of HF. Subjects with HF presented higher levels of STAT4 mRNA, whereas FoxP3 and STAT6 levels were similar in the two groups. A Th1 cytokine pattern predominated in the cardiac inflammatory cell infiltrate of Chagas’ disease patients associated with HF. High degree of fibrosis was associated with low NOS2 expression. These results support the idea that Th1 immune responses are involved in heart lesions of Chagas' disease patients

Malat1 Suppresses Immunity to Infection through Promoting Expression of Maf and IL-10 in Th Cells.

Despite extensive mapping of long noncoding RNAs in immune cells, their function in vivo remains poorly understood. In this study, we identify over 100 long noncoding RNAs that are differentially expressed within 24 h of Th1 cell activation. Among those, we show that suppression of Malat1 is a hallmark of CD4+ T cell activation, but its complete deletion results in more potent immune responses to infection. This is because Malat1-/- Th1 and Th2 cells express lower levels of the immunosuppressive cytokine IL-10. In vivo, the reduced CD4+ T cell IL-10 expression in Malat1-/- mice underpins enhanced immunity and pathogen clearance in experimental visceral leishmaniasis (Leishmania donovani) but more severe disease in a model of malaria (Plasmodium chabaudi chabaudi AS). Mechanistically, Malat1 regulates IL-10 through enhancing expression of Maf, a key transcriptional regulator of IL-10 Maf expression correlates with Malat1 in single Ag-specific Th cells from P. chabaudi chabaudi AS-infected mice and is downregulated in Malat1-/- Th1 and Th2 cells. The Malat1 RNA is responsible for these effects, as antisense oligonucleotide-mediated inhibition of Malat1 also suppresses Maf and IL-10 levels. Our results reveal that through promoting expression of the Maf/IL-10 axis in effector Th cells, Malat1 is a nonredundant regulator of mammalian immunity

Inhibitor of serine peptidase 2 enhances Leishmania major survival in the skin through control of monocytes and monocyte-derived cells

Leishmania major is the causative agent of the neglected tropical disease, cutaneous leishmaniasis. In the mouse, protective immunity to Leishmania is associated with inflammatory responses. Here, we assess the dynamics of the inflammatory responses at the lesion site during experimental long-term, low-dose intradermal infection of the ear, employing noninvasive imaging and genetically modified L. major Significant infiltrates of neutrophils and monocytes occurred at 1-4 d and 2-4 wk, whereas dermal macrophage and dendritic cell (DC) numbers were only slightly elevated in the first days. Quantitative whole-body bioluminescence imaging of myeloperoxidase activity and the quantification of parasite loads indicated that the Leishmania virulence factor, inhibitor of serine peptidase 2 (ISP2), is required to modulate phagocyte activation and is important for parasite survival at the infection site. ISP2 played a role in the control of monocyte, monocyte-derived macrophage, and monocyte-derived DC (moDC) influx, and was required to reduce iNOS expression in monocytes, monocyte-derived cells, and dermal DCs; the expression of CD80 in moDCs; and levels of IFN-γ in situ. Our findings indicate that the increased survival of L. major in the dermis during acute infection is associated with the down-regulation of inflammatory monocytes and monocyte-derived cells via ISP2.-Goundry, A., Romano, A., Lima, A. P. C. A., Mottram, J. C., Myburgh, E. Inhibitor of serine peptidase 2 enhances Leishmania major survival in the skin through control of monocytes and monocyte-derived cells

Fatal progression of experimental visceral leishmaniasis is associated with intestinal parasitism and secondary infection by commensal bacteria, and is delayed by antibiotic prophylaxis.

Leishmania donovani causes visceral leishmaniasis (VL), which is typically fatal without treatment. There is substantial variation between individuals in rates of disease progression, response to treatment and incidence of post-treatment sequelae, specifically post-kala-azar dermal leishmaniasis (PKDL). Nevertheless, the majority of infected people are asymptomatic carriers. Hamsters and mice are commonly used as models of fatal and non-fatal VL, respectively. Host and parasite genetics are likely to be important factors, but in general the reasons for heterogeneous disease presentation in humans and animal models are poorly understood. Host microbiota has become established as a factor in cutaneous forms of leishmaniasis but this has not been studied in VL. We induced intestinal dysbiosis in mice and hamsters by long-term treatment with broad-spectrum antibiotics in their drinking water. There were no significant differences in disease presentation in dysbiotic mice. In contrast, dysbiotic hamsters infected with L. donovani had delayed onset and progression of weight loss. Half of control hamsters had a rapid progression phenotype compared with none of the ABX-treated animals and the nine-month survival rate was significantly improved compared to untreated controls (40% vs. 10%). Antibiotic-treated hamsters also had significantly less severe hepatosplenomegaly, which was accompanied by a distinct cytokine gene expression profile. The protective effect was not explained by differences in parasite loads or haematological profiles. We further found evidence that the gut-liver axis is a key aspect of fatal VL progression in hamsters, including intestinal parasitism, bacterial translocation to the liver, malakoplakia and iron sequestration, none of which occurred in non-progressing murine VL. Diverse bacterial genera were cultured from VL affected livers, of which Rodentibacter was specifically absent from ABX-treated hamsters, indicating this pathobiont may play a role in promoting disease progression. The results provide experimental support for antibiotic prophylaxis against secondary bacterial infections as an adjunct therapy in human VL patients

The mating competence of geographically diverse Leishmania major strains in their natural and unnatural sand fly vectors

Invertebrate stages of Leishmania are capable of genetic exchange during their extracellular growth and development in the sand fly vector. Here we explore two variables: the ability of diverse L. major strains from across its natural range to undergo mating in pairwise tests; and the timing of the appearance of hybrids and their developmental stage associations within both natural (Phlebotomus duboscqi) and unnatural (Lutzomyia longipalpis) sand fly vectors. Following co-infection of flies with parental lines bearing independent drug markers, doubly-drug resistant hybrid progeny were selected, from which 96 clonal lines were analyzed for DNA content and genotyped for parent alleles at 4-6 unlinked nuclear loci as well as the maxicircle DNA. As seen previously, the majority of hybrids showed '2n' DNA contents, but with a significant number of '3n' and one '4n' offspring. In the natural vector, 97% of the nuclear loci showed both parental alleles; however, 3% (4/150) showed only one parental allele. In the unnatural vector, the frequency of uniparental inheritance rose to 10% (27/275). We attribute this to loss of heterozygosity after mating, most likely arising from aneuploidy which is both common and temporally variable in Leishmania. As seen previously, only uniparental inheritance of maxicircle kDNA was observed. Hybrids were recovered at similar efficiencies in all pairwise crosses tested, suggesting that L. major lacks detectable 'mating types' that limit free genetic exchange. In the natural vector, comparisons of the timing of hybrid formation with the presence of developmental stages suggest nectomonads as the most likely sexually competent stage, with hybrids emerging well before the first appearance of metacyclic promastigotes. These studies provide an important perspective on the prevalence of genetic exchange in natural populations of L. major and a guide for experimental studies to understand the biology of mating