Innate biosignature of treatment failure in human cutaneous leishmaniasis.

The quality and magnitude of the immune and inflammatory responses determine the clinical outcome of Leishmania infection, and contribute to the efficacy of antileishmanial treatments. However, the precise immune mechanisms involved in healing or in chronic immunopathology of human cutaneous leishmaniasis (CL) are not completely understood. Through sequential transcriptomic profiling of blood monocytes (Mo), neutrophils (Nφ), and eosinophils (Eφ) over the course of systemic treatment with meglumine antimoniate, we discovered that a heightened and sustained Type I interferon (IFN) response signature is a hallmark of treatment failure (TF) in CL patients. The transcriptomes of pre-treatment, mid-treatment and end-of-treatment samples were interrogated to identify predictive and prognostic biomarkers of TF. A composite score derived from the expression of 9 differentially expressed genes (common between Mo, Nφ and Eφ) was predictive of TF in this patient cohort for biomarker discovery. Similarly, machine learning models constructed using data from pre-treatment as well as post-treatment samples, accurately classified treatment outcome between cure and TF. Results from this study instigate the evaluation of Type-I IFN responses as new immunological targets for host-directed therapies for treatment of CL, and highlight the feasibility of using transcriptional signatures as predictive biomarkers of outcome for therapeutic decision making.

Microanalytical Mass Spectrometry with Super-Resolution Microscopy Reveals a Proteome Transition During Development of the Brain's Circadian Pacemaker.

During brain development, neuronal proteomes are regulated in part by changes in spontaneous and sensory-driven activity in immature neural circuits. A longstanding model for studying activity-dependent circuit refinement is the developing mouse visual system where the formation of axonal projections from the eyes to the brain is influenced by spontaneous retinal activity prior to the onset of vision and by visual experience after eye-opening. The precise proteomic changes in retinorecipient targets that occur during this developmental transition are unknown. Here, we developed a microanalytical proteomics pipeline using capillary electrophoresis (CE) electrospray ionization (ESI) mass spectrometry (MS) in the discovery setting to quantify developmental changes in the chief circadian pacemaker, the suprachiasmatic nucleus (SCN), before and after the onset of photoreceptor-dependent visual function. Nesting CE-ESI with trapped ion mobility spectrometry time-of-flight (TOF) mass spectrometry (TimsTOF PRO) doubled the number of identified and quantified proteins compared to the TOF-only control on the same analytical platform. From 10 ng of peptide input, corresponding to <∼0.5% of the total local tissue proteome, technical triplicate analyses identified 1894 proteins and quantified 1066 proteins, including many with important canonical functions in axon guidance, synapse function, glial cell maturation, and extracellular matrix refinement. Label-free quantification revealed differential regulation for 166 proteins over development, with enrichment of axon guidance-associated proteins prior to eye-opening and synapse-associated protein enrichment after eye-opening. Super-resolution imaging of select proteins using STochastic Optical Reconstruction Microscopy (STORM) corroborated the MS results and showed that increased presynaptic protein abundance pre/post eye-opening in the SCN reflects a developmental increase in synapse number, but not presynaptic size or extrasynaptic protein expression. This work marks the first development and systematic application of TimsTOF PRO for CE-ESI-based microproteomics and the first integration of microanalytical CE-ESI TimsTOF PRO with volumetric super-resolution STORM imaging to expand the repertoire of technologies supporting analytical neuroscience.

Capturing discrete latent structures: choose LDs over PCs.

High-dimensional biological data collection across heterogeneous groups of samples has become increasingly common, creating high demand for dimensionality reduction techniques that capture underlying structure of the data. Discovering low-dimensional embeddings that describe the separation of any underlying discrete latent structure in data is an important motivation for applying these techniques since these latent classes can represent important sources of unwanted variability, such as batch effects, or interesting sources of signal such as unknown cell types. The features that define this discrete latent structure are often hard to identify in high-dimensional data. Principal component analysis (PCA) is one of the most widely used methods as an unsupervised step for dimensionality reduction. This reduction technique finds linear transformations of the data which explain total variance. When the goal is detecting discrete structure, PCA is applied with the assumption that classes will be separated in directions of maximum variance. However, PCA will fail to accurately find discrete latent structure if this assumption does not hold. Visualization techniques, such as t-Distributed Stochastic Neighbor Embedding (t-SNE) and Uniform Manifold Approximation and Projection (UMAP), attempt to mitigate these problems with PCA by creating a low-dimensional space where similar objects are modeled by nearby points in the low-dimensional embedding and dissimilar objects are modeled by distant points with high probability. However, since t-SNE and UMAP are computationally expensive, often a PCA reduction is done before applying them which makes it sensitive to PCAs downfalls. Also, tSNE is limited to only two or three dimensions as a visualization tool, which may not be adequate for retaining discriminatory information. The linear transformations of PCA are preferable to non-linear transformations provided by methods like t-SNE and UMAP for interpretable feature weights. Here, we propose iterative discriminant analysis (iDA), a dimensionality reduction technique designed to mitigate these limitations. iDA produces an embedding that carries discriminatory information which optimally separates latent clusters using linear transformations that permit post hoc analysis to determine features that define these latent structures.

Early Leukocyte Responses in Ex-Vivo Models of Healing and Non-Healing Human Leishmania (Viannia) panamensis Infections.

Early host-pathogen interactions drive the host response and shape the outcome of natural infections caused by intracellular microorganisms. These interactions involve a number of immune and non-immune cells and tissues, along with an assortment of host and pathogen-derived molecules. Our current knowledge has been predominantly derived from research on the relationships between the pathogens and the invaded host cell(s), limiting our understanding of how microbes elicit and modulate immunological responses at the organismal level. In this study, we explored the early host determinants of healing and non-healing responses in human cutaneous leishmaniasis (CL) caused by Leishmania (Viannia) panamensis. We performed a comparative transcriptomic profiling of peripheral blood mononuclear cells from healthy donors (PBMCs, n=3) exposed to promastigotes isolated from patients with chronic (CHR, n=3) or self-healing (SH, n=3) CL, and compared these to human macrophage responses. Transcriptomes of L. V. panamensis-infected PBMCs showed enrichment of functional gene categories derived from innate as well as adaptive immune cells signatures, demonstrating that Leishmania modulates adaptive immune cell functions as early as after 24h post interaction with PBMCs from previously unexposed healthy individuals. Among differentially expressed PBMC genes, four broad categories were commonly modulated by SH and CHR strains: cell cycle/proliferation/differentiation, metabolism of macromolecules, immune signaling and vesicle trafficking/transport; the first two were predominantly downregulated, and the latter upregulated in SH and CHR as compared to uninfected samples. Type I IFN signaling genes were uniquely up-regulated in PBMCs infected with CHR strains, while genes involved in the immunological synapse were uniquely downregulated in SH infections. Similarly, pro-inflammatory response genes were upregulated in isolated macrophages infected with CHR strains. Our data demonstrate that early responses during Leishmania infection extend beyond innate cell and/or phagocytic host cell functions, opening new frontiers in our understanding of the triggers and drivers of human CL.

HLA and autoantibodies define scleroderma subtypes and risk in African and European Americans and suggest a role for molecular mimicry.

Systemic sclerosis (SSc) is a clinically heterogeneous autoimmune disease characterized by mutually exclusive autoantibodies directed against distinct nuclear antigens. We examined HLA associations in SSc and its autoantibody subsets in a large, newly recruited African American (AA) cohort and among European Americans (EA). In the AA population, the African ancestry-predominant HLA-DRB1*08:04 and HLA-DRB1*11:02 alleles were associated with overall SSc risk, and the HLA-DRB1*08:04 allele was strongly associated with the severe antifibrillarin (AFA) antibody subset of SSc (odds ratio = 7.4). These African ancestry-predominant alleles may help explain the increased frequency and severity of SSc among the AA population. In the EA population, the HLA-DPB1*13:01 and HLA-DRB1*07:01 alleles were more strongly associated with antitopoisomerase (ATA) and anticentromere antibody-positive subsets of SSc, respectively, than with overall SSc risk, emphasizing the importance of HLA in defining autoantibody subtypes. The association of the HLA-DPB1*13:01 allele with the ATA+ subset of SSc in both AA and EA patients demonstrated a transancestry effect. A direct correlation between SSc prevalence and HLA-DPB1*13:01 allele frequency in multiple populations was observed (r = 0.98, P = 3 × 10-6). Conditional analysis in the autoantibody subsets of SSc revealed several associated amino acid residues, mostly in the peptide-binding groove of the class II HLA molecules. Using HLA α/ß allelic heterodimers, we bioinformatically predicted immunodominant peptides of topoisomerase 1, fibrillarin, and centromere protein A and discovered that they are homologous to viral protein sequences from the Mimiviridae and Phycodnaviridae families. Taken together, these data suggest a possible link between HLA alleles, autoantibodies, and environmental triggers in the pathogenesis of SSc.

Genome-wide scans of myopia in Pennsylvania Amish families reveal significant linkage to 12q15, 8q21.3 and 5p15.33.

Myopia is one of the most common ocular disorders in the world, yet the genetic etiology of the disease remains poorly understood. Specialized founder populations, such as the Pennsylvania Amish, provide the opportunity to utilize exclusive genomic architecture, like unique haplotypes, to better understand the genetic causes of myopia. We perform genetic linkage analysis on Pennsylvania Amish families that have a strong familial history of myopia to map any potential causal variants and genes for the disease. 293 individuals from 25 extended families were genotyped on the Illumina ExomePlus array and merged with previous microsatellite data. We coded myopia affection as a binary phenotype; myopia was defined as having a mean spherical equivalent (MSE) of less than or equal to - 1 D (diopters). Two-point and multipoint parametric linkage analyses were performed under an autosomal dominant model. When allowing for locus heterogeneity, we identified two novel genome-wide significantly linked variants at 12q15 (heterogeneity LOD, HLOD = 3.77) in PTPRB and at 8q21.3 (HLOD = 3.35) in CNGB3. We identified further three genome-wide significant variants within a single family. These three variants were located in exons of SLC6A18 at 5p15.33 (LODs ranged from 3.51 to 3.37). Multipoint analysis confirmed the significant signal at 5p15.33 with six genome-wide significant variants (LODs ranged from 3.6 to 3.3). Further suggestive evidence of linkage was observed in several other regions of the genome. All three novel linked regions contain strong candidate genes, especially CNGB3 on 8q21.3, which has been shown to affect photoreceptors and cause complete color blindness. Whole genome sequencing on these regions is planned to conclusively elucidate the causal variants.

Brief Report: Whole-Exome Sequencing to Identify Rare Variants and Gene Networks That Increase Susceptibility to Scleroderma in African Americans.

OBJECTIVE: Whole-exome sequencing (WES) studies in systemic sclerosis (SSc) patients of European American (EA) ancestry have identified variants in the ATP8B4 gene and enrichment of variants in genes in the extracellular matrix (ECM)-related pathway that increase SSc susceptibility. This study was undertaken to evaluate the association of the ATP8B4 gene and the ECM-related pathway with SSc in a cohort of African American (AA) patients. METHODS: SSc patients of AA ancestry were enrolled from 23 academic centers across the US under the Genome Research in African American Scleroderma Patients consortium. Unrelated AA individuals without serologic evidence of autoimmunity who were enrolled in the Howard University Family Study were used as unaffected controls. Functional variants in genes reported in the 2 WES studies in EA patients with SSc were selected for gene association testing using the optimized sequence kernel association test (SKAT-O) and pathway analysis by Ingenuity Pathway Analysis in 379 patients and 411 controls. RESULTS: Principal components analysis demonstrated that the patients and controls had similar ancestral backgrounds, with roughly equal proportions of mean European admixture. Using SKAT-O, we examined the association of individual genes that were previously reported in EA patients and none remained significant, including ATP8B4 (P = 0.98). However, we confirmed the previously reported association of the ECM-related pathway with enrichment of variants within the COL13A1, COL18A1, COL22A1, COL4A3, COL4A4, COL5A2, PROK1, and SERPINE1 genes (corrected P = 1.95 × 10-4 ). CONCLUSION: In the largest genetic study in AA patients with SSc to date, our findings corroborate the role of functional variants that aggregate in a fibrotic pathway and increase SSc susceptibility.

Case Management Reduces Drinking During Pregnancy among High Risk Women.

AIM: Estimate the efficacy of Case Management (CM) for women at high risk for bearing a child with Fetal Alcohol Spectrum Disorders (FASD). DESIGN: Women were recruited from antenatal clinics and engaged in 18 months of CM. SETTING: A South African community with a subculture of heavy, regular, weekend, recreational drinking and high documented rates of FASD. PARTICIPANTS: Forty-one women who were high risk for bearing a child with FASD. MEASURES: Statistical analysis of trends in drinking and other risk factors. FINDINGS: At intake 87.8% were pregnant, most had previous alcohol-exposed pregnancies, most/all of their friends drink alcohol (67.5%), and 50.0% had stressful lives. CM was particularly valuable for pregnant women, as statistically significant reductions in alcohol risk were obtained for them in multiple variables: total drinks on weekends after six months of CM (p = .026) and estimated peak blood alcohol concentration (BAC) at six (p < .001) and 18 months (p < .001). For participants completing 18 months of CM, AUDIT scores improved significantly by 6-month follow-up (from 19.8 to 9.7, p = .000), and even though rising at 12 and 18 months, AUDIT scores indicate that problematic drinking remained statistically significantly lower than baseline throughout CM. Happiness scale scores correlated significantly with reduced drinking in most time periods. CONCLUSIONS: An enduring change in drinking behavior is difficult in this social setting. Yet, CM provided by skilled and empathic case managers reduced maternal drinking at critical times, and therefore, alcohol exposure levels to the fetus.

Neutrophils are associated with capillary closure in spontaneously diabetic monkey retinas.

Type 2 diabetes develops spontaneously in obese aging rhesus monkeys (Macaca mulatta). This study investigates the association between polymorphonuclear leukocytes and development of retinopathy. Blood pressure and plasma glucose levels were determined in 15 diabetic and 6 nondiabetic monkeys. The plasma levels of total cholesterol, LDL cholesterol, HDL cholesterol, and triglycerides were determined just before the start of the animal's final decline and elective necropsy. Retinas were incubated for ADPase (labels viable retinal blood vessels) and nonspecific esterase (labels neutrophils) activities. Polymorphonuclear leukocytes were counted per millimeter squared of retina. After the retina was flat-embedded in glycol methacrylate, tissue sections were taken through areas of interest and observed microscopically. Elevated numbers of intravascular polymorphonuclear leukocytes were present adjacent to areas with retinal capillary nonperfusion. There were significantly more polymorphonuclear leukocytes per millimeter squared in diabetic retinas (6.91 +/- 5.01) compared with normal retinas (1.45 +/- 1.62, P = 0.018). Severity of hypertension in diabetes was also significantly associated with greater numbers of polymorphonuclear leukocytes (P = 0.02). There was a significant positive exponential correlation between the number of polymorphonuclear leukocytes per millimeter squared and the level of total cholesterol (R = 0.907), LDL cholesterol (R = 0.875), the total cholesterol-to-HDL cholesterol ratio (R = 0.86), and total triglycerides (R = 0.888). This study demonstrates that severity of diabetes and the development of retinopathy are associated with increased numbers of polymorphonuclear leukocytes in the retina of diabetic monkeys. Hypertension, high plasma levels of LDL cholesterol and triglycerides, and low plasma levels of HDL cholesterol also are associated with increased polymorphonuclear leukocytes in retina.

Retinopathy in monkeys with spontaneous type 2 diabetes.

PURPOSE: Type 2 diabetes occurs spontaneously in rhesus monkeys and shows an extraordinary similarity to human diabetes in clinical features and relative time course. The purpose of this study was to investigate clinically and histopathologically the ocular changes in these monkeys. METHODS: Ophthalmoscopic examinations were performed on aged normal and diabetic monkeys. Retinas from 16 diabetic monkeys and 6 nondiabetic monkeys were incubated postmortem for adenosine diphosphatase (ADPase) activity (labels viable retinal blood vessels) and flat-embedded in JB-4. Tissue sections were cut through areas of interest. RESULTS: Cotton-wool spots, intraretinal hemorrhages, and hard exudates in the macula were observed by ophthalmoscopy in some diabetic monkeys. Dot/blot hemorrhages, cotton-wool spots, and small nonperfused areas were the earliest histologically documented changes in the retinas. Large nonperfused areas extending from optic disc to midfovea were observed in four diabetic monkeys. Formation of small intraretinal microvascular abnormalities (IRMAs) and microaneurysms were associated with the areas of nonperfusion. There were apparent fluid-filled spaces in the outer plexiform layer in three of these maculas, suggesting macular edema. There was a significant correlation between the occurrence of retinopathy and hypertension (P = 0.037 for systolic pressure; P = 0.019 for diastolic pressure). In elastase-digested retinas, the ratio of pericytes to endothelial cells was 0.66:1 in diabetic and 0.64:1 in nondiabetic (P = 0.75) retinas. CONCLUSIONS: This is the first detailed analysis of retinopathy in a colony of spontaneous type 2 diabetic monkeys. Monkeys with type 2 diabetes have many of the angiopathic changes associated with human diabetic retinopathy. Hypertension correlates with the severity of the diabetic retinopathy.

Minimally invasive surgery via laparoscopy for intra-abdominal biopsy in obese rhesus macaques (Macaca mulatta).

PURPOSE: To determine the safety and effectiveness of laparoscopy for repeated intra-abdominal biopsy of liver and omental adipose tissue (AT) in obese rhesus monkeys (Macaca mulatta). METHODS: Nine obese rhesus monkeys were studied by use of 18 laparoscopic procedures (two procedures each, approx. six weeks apart). Time-sensitive liver and omental AT specimens were obtained from monkeys under general anesthesia, using a three-port approach with a roticulating endoscopic stapler/divider and a monopolar electrosurgery for hemostasis. RESULTS: All subjects tolerated the initial and repeat laparoscopic procedures well. Liver specimens weighed a mean +/- SEM of 3.8 +/- 0.5 g, and omental AT specimens weighed 16.6 +/- 0.8 g. Compared with previous studies of conventional laparotomy with liver wedge resection, the monkeys experienced faster postoperative recovery via laparoscopy, with rapid return to normal food intake and activity. Minimal to no adhesions were observed by use of the repeat procedure in all monkeys, with no major complications. CONCLUSIONS: Laparoscopy in obese rhesus monkey (ranging from young to older-aged), with repeated intra-abdominal liver and omental AT biopsy, was an excellent minimally invasive surgical method. In contrast to laparotomy with wedge resection, this approach greatly decreases operative time and stress, provides generous tissue specimens in a time-efficient manner, and facilitates rapid and full recovery of the nonhuman primate.

Mortality and morbidity in laboratory-maintained Rhesus monkeys and effects of long-term dietary restriction.

Mortality and morbidity were examined in 117 laboratory-maintained rhesus monkeys studied over approximately 25 years (8 dietary-restricted [DR] and 109 ad libitum-fed [AL] monkeys). During the study, 49 AL monkeys and 3 DR monkeys died. Compared with the DR monkeys, the AL monkeys had a 2.6-fold increased risk of death. Hyperinsulinemia led to a 3.7-fold increased risk of death (p <.05); concordantly, the risk of death decreased by 7%, per unit increase in insulin sensitivity (M). There was significant organ pathology in the AL at death. The age at median survival in the AL was approximately 25 years compared with 32 years in the DR. The oldest monkey was a diabetic female (AL) that lived to be 40 years of age. These results suggest that dietary restriction leads to an increased average age of death in primates, associated with the prevention of hyperinsulinemia and the mitigation of age-related disease.