Partial RAG deficiency in humans induces dysregulated peripheral lymphocyte development and humoral tolerance defect with accumulation of T-bet+ B cells.

The recombination-activating genes (RAG) 1 and 2 are indispensable for diversifying the primary B cell receptor repertoire and pruning self-reactive clones via receptor editing in the bone marrow; however, the impact of RAG1/RAG2 on peripheral tolerance is unknown. Partial RAG deficiency (pRD) manifesting with late-onset immune dysregulation represents an 'experiment of nature' to explore this conundrum. By studying B cell development and subset-specific repertoires in pRD, we demonstrate that reduced RAG activity impinges on peripheral tolerance through the generation of a restricted primary B cell repertoire, persistent antigenic stimulation and an inflammatory milieu with elevated B cell-activating factor. This unique environment gradually provokes profound B cell dysregulation with widespread activation, remarkable extrafollicular maturation and persistence, expansion and somatic diversification of self-reactive clones. Through the model of pRD, we reveal a RAG-dependent 'domino effect' that impacts stringency of tolerance and B cell fate in the periphery.

Mutations in yeast are deleterious on average regardless of the degree of adaptation to the testing environment.

The role of spontaneous mutations in evolution depends on the distribution of their effects on fitness. Despite a general consensus that new mutations are deleterious on average, a handful of mutation accumulation experiments in diverse organisms instead suggest that beneficial and deleterious mutations can have comparable fitness impacts, i.e. the product of their respective rates and effects can be roughly equal. We currently lack a general framework for predicting when such a pattern will occur. One idea is that beneficial mutations will be more evident in genotypes that are not well adapted to the testing environment. We tested this prediction experimentally in the laboratory yeast Saccharomyces cerevisiae by allowing nine replicate populations to adapt to novel environments with complex sets of stressors. After >1000 asexual generations interspersed with 41 rounds of sexual reproduction, we assessed the mean effect of induced mutations on yeast growth in both the environment to which they had been adapting and the alternative novel environment. The mutations were deleterious on average, with the severity depending on the testing environment. However, we found no evidence that the adaptive match between genotype and environment is predictive of mutational fitness effects.

Preemptive analgesia in dental implant surgery: A systematic review and meta-analysis of randomized controlled trials.

BACKGROUND: To assess the effectiveness of preemptive analgesia in dental implant surgery in randomized controlled trials (RCTs). MATERIAL AND METHODS: The present study was conducted in accordance with the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement and registered in PROSPERO database CRD42020168757. A search without restrictions regarding language or date of publication was conducted in six databases and gray literature. A random effect meta-analysis compared the efficacy of preemptive analgesia compared to placebo through pooled OR and 95%CI. The interpretation of results followed the certainty of evidence using the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) approach together with the magnitude of the effect according to GRADE guidelines. RESULTS: Four studies were included in the review and three were incorporated into the meta-analysis. All studies demonstrated that preemptive analgesia contributed to a significant improvement in the postoperative pain control. However, the overall pooled standard mean difference (SMD) showed that preemptive analgesia had small effects compared to placebo in reducing pain (SMD: -0.45; IC: -0.83; -0.08) with low certainty of the evidence. Our meta-analysis showed that the magnitude of the effect was bigger six to eight hours after the surgery (large effect), compared to the time of one to two hours after the surgery (small effect). CONCLUSIONS: Preemptive analgesia may have a positive effect in reducing pain compared to not using preemptive medication, but the evidence is very uncertain.

Human mast cells present antigen to autologous CD4+ T cells.

BACKGROUND: Mast cells (MCs), the primary effector cell of the atopic response, participate in immune defense at host/environment interfaces, yet the mechanisms by which they interact with CD4+ T cells has been controversial. OBJECTIVE: We used in situ-matured primary human MCs and matched CD4+ T cells to diligently assess the ability of MCs to act as antigen-presenting cells. METHODS: We examined mature human skin-derived MCs using flow cytometry for expression of antigen-presenting molecules, for their ability to stimulate CD4+ T cells to express CD25 and proliferate when exposed to superantigen or to cytomegalovirus (CMV) antigen using matched T cells and MCs from CMV-seropositive or CMV-seronegative donors, and for antigen uptake. Subcellular localization of antigen, HLA molecules, and tryptase was analyzed by using structured illumination microscopy. RESULTS: Our data show that IFN-γ induces HLA class II, HLA-DM, CD80, and CD40 expression on MCs, whereas MCs take up soluble and particulate antigens in an IFN-γ-independent manner. IFN-γ-primed MCs guide activation of T cells by Staphylococcus aureus superantigen and, when preincubated with CMV antigens, induce a recall CD4+ TH1 proliferation response only in CMV-seropositive donors. MCs co-opt their secretory granules for antigen processing and presentation. Consequently, MC degranulation increases surface delivery of HLA class II/peptide, further enhancing stimulation of T-cell proliferation. CONCLUSIONS: IFN-γ primes human MCs to activate T cells through superantigen and to present CMV antigen to TH1 cells, co-opting MC secretory granules for antigen processing and presentation and creating a feed-forward loop of T-cell-MC cross-activation.

Correction to: Respiratory infections are temporally associated with initiation of type 1 diabetes autoimmunity: the TEDDY study.

The authors regret that the SNP in SH2B3 was incorrectly referred to as rs3184505 instead of rs3184504 on both mentions in this paper (Methods section and Table 1).

Perinatal and maternal morbidity and mortality among term singletons following midcavity operative vaginal delivery versus caesarean delivery.

OBJECTIVE: To quantify severe perinatal and maternal morbidity/mortality associated with midcavity operative vaginal delivery compared with caesarean delivery. DESIGN: Population-based, retrospective cohort study. SETTING: British Columbia, Canada. POPULATION: Term, singleton deliveries (2004-2014) by attempted midcavity operative vaginal delivery or caesarean delivery in the second stage of labour, stratified by indication for operative delivery (n = 10 901 deliveries; 5057 indicated for dystocia, 5844 for fetal distress). METHODS: Multinomial propensity scores and mulitvariable log-binomial regression models were used to estimate adjusted rate ratios (ARR) and 95% confidence intervals (95% CI). MAIN OUTCOME MEASURES: Composite severe perinatal morbidity/mortality (e.g. convulsions, severe birth trauma and perinatal death) and severe maternal morbidity (e.g. severe postpartum haemorrhage, shock, sepsis and cardiac complications). RESULTS: Among deliveries with dystocia, attempted midcavity operative vaginal delivery was associated with higher rates of severe perinatal morbidity/mortality compared with caesarean delivery (forceps ARR 2.11, 95% CI 1.46-3.07; vacuum ARR 2.71, 95% CI 1.49-3.15; sequential ARR 4.68, 95% CI 3.33-6.58). Rates of severe maternal morbidity/mortality were also higher following midcavity operative vaginal delivery (forceps ARR 1.57, 95% CI 1.05-2.36; vacuum ARR 2.29, 95% CI 1.57-3.36). Among deliveries with fetal distress, there were significant increases in severe perinatal morbidity/mortality following attempted midcavity vacuum (ARR 1.28, 95% CI 1.04-1.61) and in severe maternal morbidity following attempted midcavity forceps delivery (ARR 2.34, 95% CI 1.54-3.56). CONCLUSION: Attempted midcavity operative vaginal delivery is associated with higher rates of severe perinatal morbidity/mortality and severe maternal morbidity, though these effects differ by indication and instrument. TWEETABLE ABSTRACT: Perinatal and maternal morbidity is increased following midcavity operative vaginal delivery.

Respiratory infections are temporally associated with initiation of type 1 diabetes autoimmunity: the TEDDY study.

AIMS/HYPOTHESIS: Respiratory infections and onset of islet autoimmunity are reported to correlate positively in two small prospective studies. The Environmental Determinants of Diabetes in the Young (TEDDY) study is the largest prospective international cohort study on the environmental determinants of type 1 diabetes that regularly monitors both clinical infections and islet autoantibodies. The aim was to confirm the influence of reported respiratory infections and to further characterise the temporal relationship with autoantibody seroconversion. METHODS: During the years 2004-2009, 8676 newborn babies with HLA genotypes conferring an increased risk of type 1 diabetes were enrolled at 3 months of age to participate in a 15 year follow-up. In the present study, the association between parent-reported respiratory infections and islet autoantibodies at 3 month intervals up to 4 years of age was evaluated in 7869 children. Time-dependent proportional hazard models were used to assess how the timing of respiratory infections related to persistent confirmed islet autoimmunity, defined as autoantibody positivity against insulin, GAD and/or insulinoma antigen-2, concordant at two reference laboratories on two or more consecutive visits. RESULTS: In total, 87,327 parent-reported respiratory infectious episodes were recorded while the children were under study surveillance for islet autoimmunity, and 454 children seroconverted. The number of respiratory infections occurring in a 9 month period was associated with the subsequent risk of autoimmunity (p < 0.001). For each 1/year rate increase in infections, the hazard of islet autoimmunity increased by 5.6% (95% CI 2.5%, 8.8%). The risk association was linked primarily to infections occurring in the winter (HR 1.42 [95% CI 1.16, 1.74]; p < 0.001). The types of respiratory infection independently associated with autoimmunity were common cold, influenza-like illness, sinusitis, and laryngitis/tracheitis, with HRs (95% CI) of 1.38 (1.11, 1.71), 2.37 (1.35, 4.15), 2.63 (1.22, 5.67) and 1.76 (1.04, 2.98), respectively. CONCLUSIONS/INTERPRETATION: Recent respiratory infections in young children correlate with an increased risk of islet autoimmunity in the TEDDY study. Further studies to identify the potential causative viruses with pathogen-specific assays should focus especially on the 9 month time window leading to autoantibody seroconversion.

Novel Molecular Insights into Classical and Alternative Activation States of Microglia as Revealed by Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)-based Proteomics.

Microglia, the resident immune cells of the brain, have been shown to display a complex spectrum of roles that span from neurotrophic to neurotoxic depending on their activation status. Microglia can be classified into four stages of activation, M1, which most closely matches the classical (pro-inflammatory) activation stage, and the alternative activation stages M2a, M2b, and M2c. The alternative activation stages have not yet been comprehensively analyzed through unbiased, global-scale protein expression profiling. In this study, BV2 mouse immortalized microglial cells were stimulated with agonists specific for each of the four stages and total protein expression for 4644 protein groups was quantified using SILAC-based proteomic analysis. After validating induction of the various stages through a targeted cytokine assay and Western blotting of activation states, the data revealed novel insights into the similarities and differences between the various states. The data identify several protein groups whose expression in the anti-inflammatory, pro-healing activation states are altered presumably to curtail inflammatory activation through differential protein expression, in the M2a state including CD74, LYN, SQST1, TLR2, and CD14. The differential expression of these proteins promotes healing, limits phagocytosis, and limits activation of reactive nitrogen species through toll-like receptor cascades. The M2c state appears to center around the down-regulation of a key member in the formation of actin-rich phagosomes, SLP-76. In addition, the proteomic data identified a novel activation marker, DAB2, which is involved in clathrin-mediated endocytosis and is significantly different between M2a and either M1 or M2b states. Western blot analysis of mouse primary microglia stimulated with the various agonists of the classical and alternative activation states revealed a similar trend of DAB2 expression compared with BV2 cells.

AcmB Is an S-Layer-Associated ß-N-Acetylglucosaminidase and Functional Autolysin in Lactobacillus acidophilus NCFM.

UNLABELLED: Autolysins, also known as peptidoglycan hydrolases, are enzymes that hydrolyze specific bonds within bacterial cell wall peptidoglycan during cell division and daughter cell separation. Within the genome of Lactobacillus acidophilus NCFM, there are 11 genes encoding proteins with peptidoglycan hydrolase catalytic domains, 9 of which are predicted to be functional. Notably, 5 of the 9 putative autolysins in L. acidophilus NCFM are S-layer-associated proteins (SLAPs) noncovalently colocalized along with the surface (S)-layer at the cell surface. One of these SLAPs, AcmB, a ß-N-acetylglucosaminidase encoded by the gene lba0176 (acmB), was selected for functional analysis. In silico analysis revealed that acmB orthologs are found exclusively in S-layer- forming species of Lactobacillus Chromosomal deletion of acmB resulted in aberrant cell division, autolysis, and autoaggregation. Complementation of acmB in the ΔacmB mutant restored the wild-type phenotype, confirming the role of this SLAP in cell division. The absence of AcmB within the exoproteome had a pleiotropic effect on the extracellular proteins covalently and noncovalently bound to the peptidoglycan, which likely led to the observed decrease in the binding capacity of the ΔacmB strain for mucin and extracellular matrices fibronectin, laminin, and collagen in vitro These data suggest a functional association between the S-layer and the multiple autolysins noncovalently colocalized at the cell surface of L. acidophilus NCFM and other S-layer-producing Lactobacillus species. IMPORTANCE: Lactobacillus acidophilus is one of the most widely used probiotic microbes incorporated in many dairy foods and dietary supplements. This organism produces a surface (S)-layer, which is a self-assembling crystalline array found as the outermost layer of the cell wall. The S-layer, along with colocalized associated proteins, is an important mediator of probiotic activity through intestinal adhesion and modulation of the mucosal immune system. However, there is still a dearth of information regarding the basic cellular and evolutionary function of S-layers. Here, we demonstrate that multiple autolysins, responsible for breaking down the cell wall during cell division, are associated with the S-layer. Deletion of the gene encoding one of these S-layer-associated autolysins confirmed its autolytic role and resulted in reduced binding capacity to mucin and intestinal extracellular matrices. These data suggest a functional association between the S-layer and autolytic activity through the extracellular presentation of autolysins.

Functional Analysis of an S-Layer-Associated Fibronectin-Binding Protein in Lactobacillus acidophilus NCFM.

Bacterial surface layers (S-layers) are crystalline arrays of self-assembling proteinaceous subunits called S-layer proteins (Slps) that comprise the outermost layer of the cell envelope. Many additional proteins that are associated with or embedded within the S-layer have been identified in Lactobacillus acidophilus NCFM, an S-layer-forming bacterium that is widely used in fermented dairy products and probiotic supplements. One putative S-layer-associated protein (SLAP), LBA0191, was predicted to mediate adhesion to fibronectin based on the in silico detection of a fibronectin-binding domain. Fibronectin is a major component of the extracellular matrix (ECM) of intestinal epithelial cells. Adhesion to intestinal epithelial cells is considered an important trait for probiotic microorganisms during transit and potential association with the intestinal mucosa. To investigate the functional role of LBA0191 (designated FbpB) in L. acidophilus NCFM, an fbpB-deficient strain was constructed. The L. acidophilus mutant with a deletion off bpB lost the ability to adhere to mucin and fibronectin in vitro Homologues off bpB were identified in five additional putative S-layer-forming species, but no homologues were detected in species outside theL. acidophilus homology group.

Conserved S-Layer-Associated Proteins Revealed by Exoproteomic Survey of S-Layer-Forming Lactobacilli.

The Lactobacillus acidophilus homology group comprises Gram-positive species that include L. acidophilus, L. helveticus, L. crispatus, L. amylovorus, L. gallinarum, L. delbrueckii subsp. bulgaricus, L. gasseri, and L. johnsonii. While these bacteria are closely related, they have varied ecological lifestyles as dairy and food fermenters, allochthonous probiotics, or autochthonous commensals of the host gastrointestinal tract. Bacterial cell surface components play a critical role in the molecular dialogue between bacteria and interaction signaling with the intestinal mucosa. Notably, the L. acidophilus complex is distinguished in two clades by the presence or absence of S-layers, which are semiporous crystalline arrays of self-assembling proteinaceous subunits found as the outermost layer of the bacterial cell wall. In this study, S-layer-associated proteins (SLAPs) in the exoproteomes of various S-layer-forming Lactobacillus species were proteomically identified, genomically compared, and transcriptionally analyzed. Four gene regions encoding six putative SLAPs were conserved in the S-layer-forming Lactobacillus species but not identified in the extracts of the closely related progenitor, L. delbrueckii subsp. bulgaricus, which does not produce an S-layer. Therefore, the presence or absence of an S-layer has a clear impact on the exoproteomic composition of Lactobacillus species. This proteomic complexity and differences in the cell surface properties between S-layer- and non-S-layer-forming lactobacilli reveal the potential for SLAPs to mediate intimate probiotic interactions and signaling with the host intestinal mucosa.

Buprenorphine: a new player in neonatal withdrawal syndrome.

OBJECTIVE: To study the incidence and patient characteristics of Neonatal Abstinence Syndrome (NAS) in infants who were born to opioid addicted mothers and required NICU admission over the three year period. STUDY DESIGN: From 2009 to 2011, data of infants admitted in WVUH NICU with history of maternal drug exposure were extracted and reviewed. Infants born to mothers treated with buprenorphine (BPN) and those with methadone (MTD) were compared. RESULTS: Incidence of drug exposure infants significantly increased in 2011. BPN exposure infants increased substantially while the number of MTD exposure infants did not significantly change. Eighty-one percent of those MTD exposure infants required drug treatment for NAS compared to 26% of BPN exposure infants. CONCLUSION: Significant increase in opioid exposure infants with NAS was observed in our unit in 2011. Although, the incidence of BPN exposure with NAS increased significantly, only 25% of them required drug treatment.

Biomarker discovery study design for type 1 diabetes in The Environmental Determinants of Diabetes in the Young (TEDDY) study.

AIMS: The Environmental Determinants of Diabetes in the Young planned biomarker discovery studies on longitudinal samples for persistent confirmed islet cell autoantibodies and type 1 diabetes using dietary biomarkers, metabolomics, microbiome/viral metagenomics and gene expression. METHODS: This article describes the details of planning The Environmental Determinants of Diabetes in the Young biomarker discovery studies using a nested case-control design that was chosen as an alternative to the full cohort analysis. In the frame of a nested case-control design, it guides the choice of matching factors, selection of controls, preparation of external quality control samples and reduction of batch effects along with proper sample allocation. RESULTS AND CONCLUSION: Our design is to reduce potential bias and retain study power while reducing the costs by limiting the numbers of samples requiring laboratory analyses. It also covers two primary end points (the occurrence of diabetes-related autoantibodies and the diagnosis of type 1 diabetes). The resulting list of case-control matched samples for each laboratory was augmented with external quality control samples.

Impact of genomics on the field of probiotic research: historical perspectives to modern paradigms.

For thousands of years, humans have safely consumed microorganisms through fermented foods. Many of these bacteria are considered probiotics, which act through diverse mechanisms to confer a health benefit to the host. However, it was not until the availability of whole-genome sequencing and the era of genomics that mechanisms of probiotic efficacy could be discovered. In this review, we explore the history of the probiotic concept and the current standard of integrated genomic techniques to discern the complex, beneficial relationships between probiotic microbes and their hosts.

Oral gavage delivery of Cornus officinalis extract delays type 1 diabetes onset and hyperglycemia in non-obese diabetic (NOD) mice.

Type 1 diabetes (T1D) is an autoimmune disease initiated by genetic predisposition and environmental influences, which result in the specific destruction of insulin-producing pancreatic ß-cells. Currently, there are over 1.6 million cases of T1D in the United States with a worldwide incidence rate that has been increasing since 1990. Here, we examined the effect of Cornus officinalis (CO), a well-known ethnopharmacological agent, on a T1D model of the non-obese diabetic (NOD) mouse. A measured dose of CO extract was delivered into 10-week-old NOD mice by oral gavage for 15 weeks. T1D incidence and hyperglycemia were significantly lower in the CO-treated group as compared to the water gavage (WT) and a no handling or treatment control group (NHT) following treatment. T1D onset per group was 30%, 60% and 86% for the CO, WT and NHT groups, respectively. Circulating C-peptide was higher, and pancreatic insulitis was decreased in non-T1D CO-treated mice. Our findings suggest that CO may have therapeutic potential as both a safe and effective interventional agent to slow early stage T1D progression.

Loss of feedback regulation between FAM3B and androgen receptor driving prostate cancer progression.

BACKGROUND: Although the fusion of the transmembrane serine protease 2 gene (TMPRSS2) with the erythroblast transformation-specific-related gene (ERG), or TMPRSS2-ERG, occurs frequently in prostate cancer, its impact on clinical outcomes remains controversial. Roughly half of TMPRSS2-ERG fusions occur through intrachromosomal deletion of interstitial genes and the remainder via insertional chromosomal rearrangements. Because prostate cancers with deletion-derived TMPRSS2-ERG fusions are more aggressive than those with insertional fusions, we investigated the impact of interstitial gene loss on prostate cancer progression. METHODS: We conducted an unbiased analysis of transcriptome data from large collections of prostate cancer samples and employed diverse in vitro and in vivo models combined with genetic approaches to characterize the interstitial gene loss that imposes the most important impact on clinical outcome. RESULTS: This analysis identified FAM3B as the top-ranked interstitial gene whose loss is associated with a poor prognosis. The association between FAM3B loss and poor clinical outcome extended to fusion-negative prostate cancers where FAM3B downregulation occurred through epigenetic imprinting. Importantly, FAM3B loss drives disease progression in prostate cancer. FAM3B acts as an intermediator of a self-governing androgen receptor feedback loop. Specifically, androgen receptor upregulates FAM3B expression by binding to an intronic enhancer to induce an enhancer RNA and facilitate enhancer-promoter looping. FAM3B, in turn, attenuates androgen receptor signaling. CONCLUSION: Loss of FAM3B in prostate cancer, whether through the TMPRSS2-ERG translocation or epigenetic imprinting, causes an exit from this autoregulatory loop to unleash androgen receptor activity and prostate cancer progression. These findings establish FAM3B loss as a new driver of prostate cancer progression and support the utility of FAM3B loss as a biomarker to better define aggressive prostate cancer.

Methods, quality control and specimen management in an international multicentre investigation of type 1 diabetes: TEDDY.

BACKGROUND: The vast array and quantity of longitudinal samples collected in The Environmental Determinants of Diabetes in the Young study present a series of challenges in terms of quality control procedures and data validity. To address this, pilot studies have been conducted to standardize and enhance both biospecimen collection and sample obtainment in terms of autoantibody collection, stool sample preservation, RNA, biomarker stability, metabolic biomarkers and T-cell viability. RESEARCH DESIGN AND METHODS: The Environmental Determinants of Diabetes in the Young is a multicentre, international prospective study (n = 8677) designed to identify environmental triggers of type 1 diabetes (T1D) in genetically at-risk children from ages 3 months until 15 years. The study is conducted through six primary clinical centres located in four countries. RESULTS: As of May 2012, over three million biological samples and 250 million total data points have been collected, which will be analysed to assess autoimmunity status, presence of inflammatory biomarkers, genetic factors, exposure to infectious agents, dietary biomarkers and other potentially important environmental exposures in relation to autoimmunity and progression to T1D. CONCLUSIONS: Detailed procedures were utilized to standardize both data harmonization and management when handling a large quantity of longitudinal samples obtained from multiple locations. In addition, a description of the available specimens is provided that serve as an invaluable repository for the elucidation of determinants in T1D focusing on autoantibody concordance and harmonization, transglutaminase autoantibody, inflammatory biomarkers (T-cells), genetic proficiency testing, RNA lab internal quality control testing, infectious agents (monitoring cross-contamination, virus preservation and nasal swab collection validity) and HbA1c testing.

The changing landscape of type 1 diabetes: recent developments and future frontiers.

Type 1 diabetes (T1D) research has made great strides over the past decade with advances in understanding the pathogenesis, natural history, candidate environmental exposures, exposure triggering time, disease prediction, and diagnosis. Major monitoring efforts have provided baseline historical measures, leading to better epidemiological studies incorporating longitudinal biosamples (ie, biobanks), which have allowed for new technologies ('omics') to further expose the etiological agents responsible for the initiation, progression, and eventual clinical onset of T1D. These new frontiers have brought forth high-dimensionality data, which have furthered the evidence of the heterogeneous nature of T1D pathogenesis and allowed for a more mechanistic approach in understanding the etiology of T1D. This review will expand on the most recent advances in the quest for T1D determinants, drawing upon novel research tools that epidemiology, genetics, microbiology, and immunology have provided, linking them to the major hypotheses associated with T1D etiology, and discussing the future frontiers.

The Personal Suicide Stigma Questionnaire (PSSQ): Relation to Self-Esteem, Well-Being, and Help-Seeking.

Two studies are reported that extend the evidence base for use of the Personal Stigma of Suicide Questionnaire (PSSQ). In the first study (N = 117), the Rosenberg Self-Esteem Scale, the WHO-5 measure of well-being, as well as measures of suicidality were examined in relation to the PSSQ. A self-selected sub-sample (N = 30) completed the PSSQ after an interval of two months. In line with the stigma internalization model, when demographic variables and suicidality were accounted for, the PSSQ self-blame subscale was the most significant predictor of self-esteem. As for well-being, the rejection subscale was involved as well as self-blame. The retest stability of the PSSQ for the sub-sample was 0.85 and coefficient alpha for the total sample was 0.95, indicating both good stability and internal consistency for the scale. In the second study (N = 140), PSSQ was studied in relation to intention to seek help from four sources in the case of suicidal ideation. The strongest relationship with PSSQ was with intention not to seek help from anyone (r = 0.35). When other variables were included in the prediction of help-seeking from a general medical practitioner, family or friends, or from nobody, the only significant PSSQ correlate was minimization. For help-seeking from a psychologist or psychiatrist, the most significant predictor was judged helpfulness of prior contact with them. The results from these studies strengthen previous findings of the construct validity of the PSSQ and point to its utility in understanding barriers to help-seeking among those experiencing suicidality.