Y-Chromosome Analysis in Individuals Bearing the Basarab Name of the First Dynasty of Wallachian Kings

Vlad III The Impaler, also known as Dracula, descended from the dynasty of Basarab, the first rulers of independent Wallachia, in present Romania. Whether this dynasty is of Cuman (an admixed Turkic people that reached Wallachia from the East in the 11^th century) or of local Romanian (Vlach) origin is debated among historians. Earlier studies have demonstrated the value of investigating the Y chromosome of men bearing a historical name, in order to identify their genetic origin. We sampled 29 Romanian men carrying the surname Basarab, in addition to four Romanian populations (from counties Dolj, N = 38; Mehedinti, N = 11; Cluj, N = 50; and Brasov, N = 50), and compared the data with the surrounding populations. We typed 131 SNPs and 19 STRs in the non-recombinant part of the Y-chromosome in all the individuals. We computed a PCA to situate the Basarab individuals in the context of Romania and its neighboring populations. Different Y-chromosome haplogroups were found within the individuals bearing the Basarab name. All haplogroups are common in Romania and other Central and Eastern European populations. In a PCA, the Basarab group clusters within other Romanian populations. We found several clusters of Basarab individuals having a common ancestor within the period of the last 600 years. The diversity of haplogroups found shows that not all individuals carrying the surname Basarab can be direct biological descendants of the Basarab dynasty. The absence of Eastern Asian lineages in the Basarab men can be interpreted as a lack of evidence for a Cuman origin of the Basarab dynasty, although it cannot be positively ruled out. It can be therefore concluded that the Basarab dynasty was successful in spreading its name beyond the spread of its genes.     

True 3q Chromosomal Amplification in Squamous Cell Lung Carcinoma by FISH and aCGH Molecular Analysis: Impact on Targeted Drugs

Squamous lung carcinoma lacks specific “ad hoc” therapies. Amplification of chromosome 3q is the most common genomic aberration and this region harbours genes having role as novel targets for therapeutics. There is no standard definition on how to score and report 3q amplification. False versus true 3q chromosomal amplification in squamous cell lung carcinoma may have tremendous impact on trials involving drugs which target DNA zones mapping on 3q. Forty squamous lung carcinomas were analyzed by FISH to assess chromosome 3q amplification. aCGH was performed as gold-standard to avoid false positive amplifications. Three clustered patterns of fluorescent signals were observed. Eight cases out of 40 (20%) showed ≥8 3q signals. Twenty out of 40 (50%) showed from 3 to 7 signals. The remaining showed two fluorescent signals (30%). When corrected by whole chromosome 3 signals, only cases with ≥8 signals maintained a LSI 3q/CEP3 ratio >2. Only the cases showing 3q amplification by aCGH (+3q25.3−3q27.3) showed ≥8 fluorescent signals at FISH evidencing a 3q/3 ratio >2. The remaining cases showed flat genomic portrait at aCGH on chromosome 3. We concluded that: 1) absolute copy number of 3q chromosomal region may harbour false positive interpretation of 3q amplification in squamous cell carcinoma; 2) a case results truly “amplified for chromosome 3q” when showing ≥8 fluorescent 3q signals; 3) trials involving drugs targeting loci on chromosome 3q in squamous lung carcinoma therapy have to consider false versus true 3q chromosomal amplification.     

Point-of-Care Urine Tests for Smoking Status and Isoniazid Treatment Monitoring in Adult Patients

Background

Poor adherence to isoniazid (INH) preventive therapy (IPT) is an impediment to effective control of latent tuberculosis (TB) infection. TB patients who smoke are at higher risk of latent TB infection, active disease, and TB mortality, and may have lower adherence to their TB medications. The objective of our study was to validate IsoScreen and SmokeScreen (GFC Diagnostics, UK), two point-of-care tests for monitoring INH intake and determining smoking status. The tests could be used together in the same individual to help identify patients with a high-risk profile and provide a tailored treatment plan that includes medication management, adherence interventions, and smoking cessation programs.

Methodology/Principal Findings

200 adult outpatients attending the TB and/or the smoking cessation clinic were recruited at the Montreal Chest Institute. Sensitivity and specificity were measured for each test against the corresponding composite reference standard. Test reliability was measured using kappa statistic for intra-rater and inter-rater agreement. Univariate and multivariate logistic regression models were used to explore possible covariates that might be related to false-positive and false-negative test results. IsoScreen had a sensitivity of 93.2% (95% confidence interval [CI] 80.3, 98.2) and specificity of 98.7% (94.8, 99.8). IsoScreen had intra-rater agreement (kappa) of 0.75 (0.48, 0.94) and inter-rater agreement of 0.61 (0.27, 0.90). SmokeScreen had a sensitivity of 69.2% (56.4, 79.8), specificity of 81.6% (73.0, 88.0), intra-rater agreement of 0.77 (0.56, 0.94), and inter-rater agreement of 0.66 (0.42, 0.88). False-positive SmokeScreen tests were strongly associated with INH treatment.

Conclusions

IsoScreen had high validity and reliability, whereas SmokeScreen had modest validity and reliability. SmokeScreen tests did not perform well in a population receiving INH due to the association between INH treatment and false-positive SmokeScreen test results. Development of the next generation SmokeScreen assay should account for this potential interference.     

Research questions and priorities for tuberculosis: a survey of published systematic reviews and meta-analyses

Background

Systematic reviews are increasingly informing policies in tuberculosis (TB) care and control. They may also be a source of questions for future research. As part of the process of developing the International Roadmap for TB Research, we did a systematic review of published systematic reviews on TB, to identify research priorities that are most frequently suggested in reviews.

Methodology/Principal Findings

We searched EMBASE, MEDLINE, Web of Science, and the Cochrane Library for systematic reviews and meta-analyses on any aspect of TB published between 2005 and 2010. One reviewer extracted data and a second reviewer independently extracted data from a random subset of included studies. In total, 137 systematic reviews, with 141 research questions, were included in this review. We used the UK Health Research Classification System (HRCS) to help us classify the research questions and priorities. The three most common research topics were in the area of detection, screening and diagnosis of TB (32.6%), development and evaluation of treatments and therapeutic interventions (23.4%), and TB aetiology and risk factors (19.9%). The research priorities determined were mainly focused on the discovery and evaluation of bacteriological TB tests and drug-resistant TB tests and immunological tests. Other important topics of future research were genetic susceptibility linked to TB and disease determinants attributed to HIV/TB. Evaluation of drug treatments for TB, drug-resistant TB and HIV/TB were also frequently proposed research topics.

Conclusions

Systematic reviews are a good source of key research priorities. Findings from our survey have informed the development of the International Roadmap for TB Research by the TB Research Movement.     

Intestinal Cytokine mRNA Expression in Canine Inflammatory Bowel Disease: A Meta-Analysis with Critical Appraisal

Data implicating mucosal cytokines in the pathogenesis of canine inflammatory bowel disease (IBD) are limited. The aims of the present study were to report new findings of intestinal cytokine expression in dogs with IBD and to compare these data with previous studies through meta-analysis. Cytokine mRNA abundance in intestinal biopsies collected prospectively was evaluated by using a semiquantitative RT-PCR technique. For meta-analysis, an electronic database search revealed 3 clinical trials, all of which were nonrandomized (type III) case series. Prospective analysis showed that the intestines of healthy dogs and those with IBD express numerous cytokines and that a proinflammatory expression profile is not a feature of small or large-intestinal IBD. The meta-analysis data included 158 dogs characterized as healthy (n = 45), diarrheic nonIBD dogs (n = 6), nonresponders (n = 2), small-intestinal IBD (n = 41), colonic IBD (n = 25), and chronic enteropathy (n = 39). German shepherd dogs were overrepresented in 3 of the 4 studies. Healthy dogs showed mRNA expression for most cytokines including IL2, IL4, IL5, IL10, IL12, IFNγ, TNFα, and TGFβ. Only IL12 mRNA expression was increased consistently in small-intestinal IBD, whereas IBD colitis lacked consistent patterns of expression. In summary, dogs with IBD fail to express a predominant Th1- or Th2 cytokine bias in inflamed mucosa. Heterogeneity of results among these studies might be explained by numerous factors including the method of mRNA quantification, stage of disease, and demographic differences in study populations.Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IBD, inflammatory bowel disease; LP, lymphocytic–plasmacyticIdiopathic inflammatory bowel disease (IBD) in dogs is a chronic immune-mediated disorder empirically defined by clinical, histologic, and therapeutic features.^{18,26,27,29,45} Evidence suggests that intestinal inflammation in IBD results from altered interaction between the resident microflora and mucosa in a susceptible host.^48,53 Aggressive host immune responses directed against commensal bacteria play a central role in the pathogenesis of chronic mucosal inflammation. The concept of impaired immunoregulation in canine IBD is supported by observations of increased numbers of immunoglobulin-containing cells and T cells in inflamed tissues,^16,28,30,49 upregulated mucosal and luminal expression of nitric oxide metabolites,^20,29 and altered serum concentrations of select acute phase proteins, such as C-reactive protein, in diseased dogs.³¹ C-reactive protein is a marker of inflammation and tissue injury and is produced by the liver in response to stimulation by IL6, IL1β, and TNFα.^14,51Cytokines play a key role in the modulation of the mucosal immune system of humans. To maintain gut homeostasis, the normal mucosal immune system balances a network of inflammatory mediators, including proinflammatory, antiinflammatory, and regulatory cytokines.⁴⁷ Cytokines are synthesized rapidly and secreted on stimulation and induce the production of adhesion molecules and other inflammatory mediators including reactive oxygen species, nitric oxide metabolites, and lipid products such as prostaglandins, leukotrienes, and platelet-activating factor. Cytokine-producing cells induce, amplify, prolong, and mediate intestinal mucosal injury.¹³ Disturbances in the balance of proinflammatory (Th1/Th17-derived) and immunoregulatory (Th2/Tr1-derived) cytokines occur in humans with IBD as well as numerous animal models of intestinal inflammation.^16,39,44Data evaluating the role of CD4⁺ T cells and mucosal cytokines in the pathogenesis of canine IBD are limited. Lymphocytes expressing CD4⁺ are either increased¹⁶ or decreased²⁸ in dogs having small-intestinal IBD, whereas mucosal CD4⁺ T cells are increased in dogs with IBD colitis.^30,49 A recent study¹⁵ described a balance between proinflammatory and antiinflammatory cytokine mRNA expression in dogs with small-intestinal enteropathies but included only 4 dogs diagnosed with IBD. A separate investigation⁴⁴ evaluating mucosal cytokine mRNA expression in dogs with lymphocytic–plasmacytic colitis reported upregulated expression of proinflammatory cytokines IL2 and TNFα. Yet another study⁴¹ reported no difference in cytokine expression in the duodenal mucosa of dogs with or without chronic diarrhea; however, the dogs of this report were not subdivided in terms of response to therapy as having idiopathic IBD, antibiotic-responsive diarrhea, or food-responsive enteropathy. Because of these varied observations, it is unclear which cytokines, if any, control or enhance the local immune response of canine IBD because 1) few dogs with IBD have been evaluated, 2) German shepherd dogs with enteropathies were over-represented in most studies, 3) various measures of cytokine mRNA expression were used, and (4) distinctly variable patterns of cytokine expression were present among these earlier studies.The objectives of this study were to 1) assess cytokine mRNA expression in the intestinal mucosa of dogs diagnosed with small- and large-intestinal IBD by using an RT-PCR technique and 2) compare these data with previously published data to determine the putative role of cytokine expression in the pathogenesis of canine IBD and other forms of chronic enteropathy through meta-analysis of combined findings.     

Glomerular abundance of complement proteins characterized by proteomic analysis of laser-captured microdissected glomeruli associates with progressive disease in IgA nephropathy

Background

The clinical course of IgA nephropathy (IgAN) is variable and complement activation may predict prognosis. The present study investigated whether glomerular abundance of complement proteins associates with progression to end-stage renal disease (ESRD) in patients for whom prognosis could not be predicted based on clinical variables.

Methods

Based on data from the Norwegian Kidney Biopsy Registry and the Norwegian Renal Registry, three groups were included: IgAN patients with (n = 9) or without (n = 16) progression to ESRD during 10 years, and controls (n = 15) with a normal kidney biopsy. IgAN patients had eGFR > 45 ml/min/1.73 m² and non-nephrotic proteinuria at time of biopsy. Using stored formalin-fixed paraffin embedded kidney biopsy tissue, about 100 glomerular cross sections were microdissected for each patient. Samples were analyzed by liquid chromatography–tandem mass spectrometry and relative abundances of complement proteins were compared between groups.

Results

Proteomic analyses quantified 2018 proteins, of which 28 proteins belong to the complement system. As compared to IgAN patients without progressive disease, glomeruli from patients with progressive IgAN had significantly higher abundance of components of the classical and the terminal complement pathways, and inhibitory factors such as Factor H and factor H related proteins. Abundance of complement proteins classified progressors from non-progressors with an area under ROC curve of 0.91 (p = 0.001). Clinical and morphological data were similar between the two patient groups and could not predict progressive IgAN.

Conclusions

In conclusion, higher glomerular abundance of complement proteins was associated with a progressive clinical course in IgAN and are candidate biomarkers to predict prognosis.

     

Kinetics and persistence of cellular and humoral immune responses to SARS‐CoV‐2 vaccine in healthcare workers with or without prior COVID‐19

SARS‐CoV‐2 vaccines are highly efficient against severe forms of the disease, hospitalization and death. Nevertheless, insufficient protection against several circulating viral variants might suggest waning immunity and the need for an additional vaccine dose. We conducted a longitudinal study on the kinetics and persistence of immune responses in healthcare workers vaccinated with two doses of BNT162b2 mRNA vaccine with or without prior SARS‐CoV‐2 infection. No new infections were diagnosed during follow‐up. At 6 months, post‐vaccination or post‐infection, despite a downward trend in the level of anti‐S IgG antibodies, the neutralizing activity does not decrease significantly, remaining higher than 75% (85.14% for subjects with natural infection, 88.82% for vaccinated after prior infection and 78.37% for vaccinated only). In a live‐virus neutralization assay, the highest neutralization titres were present at baseline and at 6 months follow‐up in persons vaccinated after prior infection. Anti‐S IgA levels showed a significant descending trend in vaccinated subjects (p < 0.05) after 14 weeks. Cellular immune responses are present even in vaccinated participants with declining antibody levels (index ratio 1.1–3) or low neutralizing activity (30%–40%) at 6 months, although with lower T‐cell stimulation index (p = 0.046) and IFN‐γ secretion (p = 0.0007) compared to those with preserved humoral responses.