Validation of the German Version of the P4 Suicidality Tool.

For general practitioners (GPs), it may be challenging to assess suicidal ideation (SI) in patients. Although promising instruments exist for the use in primary care, only a few have been validated in German. The objectives of this study were to examine the validity of the brief P4 screener for assessing SI in a cross-sectional study including outpatients. Inclusion criteria were a PHQ-9 score ≥ 10 or an affirmative answer to its SI item. Construct validity of the P4 was examined by comparison with the four-item Suicide Behaviors Questionnaire-Revised (SBQ-R), the PHQ-9 (convergent), and the positive mental health (PMH) scale (divergent). The study sample included 223 patients (mean age 47.61 ± 15 years; 61.9% women) from 20 primary care practices (104 patients) and 10 psychiatric/psychotherapeutic clinics (119 patients). The first three items of the P4 correlate positively with most of the four items of the reference standard SBQ-R (convergent validity); the fourth item of the P4 (preventive factors) correlates significantly with the PMH scale. The most common preventive factor (67%) is family or friends. The German P4 screener can be used to assess SI in outpatient care. It explores preventive or protective factors of suicide, which may support the GP's decision on treatment. We recommend a further clinical interview for patients flagged by P4 assessment in order to more formally assess suicidal risk.

CBT-Intervention for panic disorder in primary care: 5 years follow-up of a cRCT during the Covid-19 pandemic.

A practice team-based exercise programme with elements of cognitive behavioural therapy (CBT) and case management for patients with panic disorder with or without agoraphobia in primary care showed significant positive effects. Here, we analyse the long-term effects (>5 years) of this intervention in the stressful context of the Covid-19 pandemic. All participants of the original PARADIES cluster randomized controlled trial (cRCT; 2012-2016) were invited to participate in a follow-up during the Covid-19 pandemic. Clinical outcomes were anxiety symptoms, number and severity of panic attacks, agoraphobic avoidance behaviour, Covid-specific anxiety symptom severity, depression, and patient assessment of chronic illness care. Data were analysed cross-sectionally for group differences (intervention, control) and longitudinally (T0: baseline, T1: 6 months and TCorona: >60 months). Of the original 419 participants, 100 participated in the 60 months follow-up (October 2020-May 2021). In the cross-sectional analysis, the anxiety symptom severity in the intervention group was lower than in the control group (p = .011, Cohen's d = .517). In the longitudinal analysis, both groups showed an increase of anxiety and depression symptoms compared to pre-pandemic level. The intervention may have had a lasting impact regarding anxiety severity despite the challenging context of the Covid-19 pandemic. However, we cannot say to what extend the intervention still played a role in participants' lives; other factors may also have helped with coping. The increase of anxiety and depression symptoms in both groups over time could be attributed to external circumstances.

Primary Biliary Cirrhosis and Granulomatous Hepatitis After Immune Checkpoint Blockade in Patients With Metastatic Melanoma: Report of 2 Cases and Literature Discussion.

Immune-related adverse events (irAEs) of immune checkpoint inhibitors can potentially affect every organ system, are sometimes challenging, and require a multidisciplinary approach. Most common irAEs are very well characterized, but some other such rare autoimmune liver diseases are probably underdiagnosed and less explored. We present here the case of a 69-year-old man with metastatic melanoma developing a severe primary biliary cirrhosis under pembrolizumab, and of a 52-year-old woman with metastatic melanoma with granulomatous hepatitis in the context of an immune-related multiorgan inflammatory reaction due to ipilimumab and nivolumab. Both cases were in part steroid refractory and required a complex diagnostic assessment and long-term therapeutic management. The liver biopsy was crucial for ensuring a correct diagnosis. Clinicians should be aware of rare liver diseases in the context of increased liver enzymes under immune checkpoint inhibitors, especially if not responding to corticosteroids. The primary diagnostic workup should localize the liver damage (biliary or parenchymal) and distinguish irAEs from other pathologic conditions such as metastasis, second benign and malignant tumors, viral hepatitis, and cholelithiasis. If in doubt, a liver biopsy should be performed. Early diagnosis and accurate assessment of hepatic adverse events is necessary for prompt and effective treatment, with reduction of inappropriate discontinuation of immunotherapy, morbidity, and mortality.

Proton-pump inhibitors elevate infection rate in cardiothoracic surgery patients by influencing PMN function in vitro and in vivo.

Proton-pump inhibitors (PPI) as pantoprazole are highly effective acid suppressive agents that belong to the world's most sold medication. However, they are pronounced to have immunosuppressive aspects. In our study, a negative influence of PPI on functions of polymorphonuclear cells in vitro like phagocytosis, oxidative burst, chemotaxis, and killing activity was shown, whereas formation of neutrophil extracellular traps (NET)osis remained unaffected. Pantoprazole stimulation additionally reduced the production of the proinflammatory cytokine IL-1ß in whole blood assay as well as the production of IL-2 and IFN-γ after whole blood stimulation with phytohaemagglutinin. Moreover, IFN-γ feedback mechanisms and signaling by STAT-1 was impaired by PPI. Cardiac surgery is accompanied by developing systemic inflammatory response syndrome with immunosuppressive aspects. We exhibited reduced oxidative burst analyzing cardiac surgery patients' samples receiving or not receiving PPI. Furthermore, a higher rate of infections in patients receiving permanent PPI medication in retrospective analysis was uncovered. Patients undergoing cardiac surgery with cardiopulmonary bypass and regular PPI medication developed significant more infections retrospectively indicating a clinical impact of the immunosuppressive influence of PPI.

Reduction of T-Helper Cell Responses to Recall Antigen Mediated by Codelivery with Peptidoglycan via the Intestinal Nanomineral-Antigen Pathway.

Naturally occurring intestinal nanomineral particles constituently form in the mammalian gut and trap luminal protein and microbial components. These cargo loaded nanominerals are actively scavenged by M cells of intestinal immune follicles, such as Peyer's patches and are passed to antigen-presenting cells. Using peripheral blood mononuclear cell populations as an in vitro model of nanomineral uptake and antigen presentation, we show that monocytes avidly phagocytose nanomineral particles bearing antigen and peptidoglycan (PGN), and that the presence of PGN within particles downregulates their cell surface MHC class II and upregulates programmed death receptor ligand 1. Nanomineral delivery of antigen suppresses antigen-specific CD4+ T cell responses, an effect that is enhanced in the presence of PGN. Blocking the interleukin-10 receptor restores CD4+ T cell responses to antigen codelivered with PGN in nanomineral form. Using human intestinal specimens, we have shown that the in vivo nanomineral pathway operates in an interleukin-10 rich environment. Consequently, the delivery of a dual antigen-PGN cargo by endogenous nanomineral in vivo is likely to be important in the establishment of intestinal tolerance, while their synthetic mimetics present a potential delivery system for therapeutic applications targeting the modulation of Peyer's patch T cell responses.

Cell-type-specific modulation of innate immune signalling by vitamin D in human mononuclear phagocytes.

Vitamin D is widely reported to inhibit innate immune signalling and dendritic cell (DC) maturation as a potential immunoregulatory mechanism. It is not known whether vitamin D has global or gene-specific effects on transcriptional responses downstream of innate immune stimulation, or whether vitamin D inhibition of innate immune signalling is common to different cells. We confirmed vitamin D inhibition of nuclear factor-κB (NF-κB) and p38 mitogen-activated protein kinase (MAPK) signalling in monocyte-derived DC (MDDC) stimulated with lipopolysaccharide (LPS). This was associated with global but modest attenuation of LPS-induced transcriptional changes at genome-wide level. Surprisingly, vitamin D did not inhibit innate immune NF-κB activation in monocyte-derived macrophages. Consistent with our findings in MDDC, ex vivo vitamin D treatment of primary peripheral blood myeloid DC also led to significant inhibition of LPS-inducible NF-κB activation. Unexpectedly, in the same samples, vitamin D enhanced activation of both NF-κB and MAPK signalling in primary peripheral blood monocytes. In a cross-sectional clinical cohort, we found no relationship between peripheral blood vitamin D levels and LPS-inducible activation of NF-κB and MAPK pathways in monocytes of myeloid DC. Remarkably, however, in vivo supplementation of people with vitamin D deficiency in this clinical cohort also enhanced LPS-inducible MAPK signalling in peripheral blood monocytes. Therefore, we report that vitamin D differentially modulates the molecular response to innate immune stimulation in monocytes, macrophages and dendritic cells. These results are of importance in the design of studies on vitamin D supplementation in infectious and immunological diseases.

Synthetic mimetics of the endogenous gastrointestinal nanomineral: Silent constructs that trap macromolecules for intracellular delivery.

Amorphous magnesium-substituted calcium phosphate (AMCP) nanoparticles (75-150nm) form constitutively in large numbers in the mammalian gut. Collective evidence indicates that they trap and deliver luminal macromolecules to mucosal antigen presenting cells (APCs) and facilitate gut immune homeostasis. Here, we report on a synthetic mimetic of the endogenous AMCP and show that it has marked capacity to trap macromolecules during formation. Macromolecular capture into AMCP involved incorporation as shown by STEM tomography of the synthetic AMCP particle with 5nm ultra-fine iron (III) oxohydroxide. In vitro, organic cargo-loaded synthetic AMCP was taken up by APCs and tracked to lysosomal compartments. The AMCP itself did not regulate any gene, or modify any gene regulation by its cargo, based upon whole genome transcriptomic analyses. We conclude that synthetic AMCP can efficiently trap macromolecules and deliver them to APCs in a silent fashion, and may thus represent a new platform for antigen delivery.

Intestinal APCs of the endogenous nanomineral pathway fail to express PD-L1 in Crohn's disease.

Crohn's disease is a chronic inflammatory condition most commonly affecting the ileum and colon. The aetiology of Crohn's disease is complex and may include defects in peptidoglycan recognition, and/or failures in the establishment of intestinal tolerance. We have recently described a novel constitutive endogenous delivery system for the translocation of nanomineral-antigen-peptidoglycan (NAP) conjugates to antigen presenting cells (APCs) in intestinal lymphoid patches. In mice NAP conjugate delivery to APCs results in high surface expression of the immuno-modulatory molecule programmed death receptor ligand 1 (PD-L1). Here we report that NAP conjugate positive APCs in human ileal tissues from individuals with ulcerative colitis and intestinal carcinomas, also have high expression of PD-L1. However, NAP-conjugate positive APCs in intestinal tissue from patients with Crohn's disease show selective failure in PD-L1 expression. Therefore, in Crohn's disease intestinal antigen taken up by lymphoid patch APCs will be presented without PD-L1 induced tolerogenic signalling, perhaps initiating disease.

Diagnostic 'omics' for active tuberculosis.

The decision to treat active tuberculosis (TB) is dependent on microbiological tests for the organism or evidence of disease compatible with TB in people with a high demographic risk of exposure. The tuberculin skin test and peripheral blood interferon-γ release assays do not distinguish active TB from a cleared or latent infection. Microbiological culture of mycobacteria is slow. Moreover, the sensitivities of culture and microscopy for acid-fast bacilli and nucleic acid detection by PCR are often compromised by difficulty in obtaining samples from the site of disease. Consequently, we need sensitive and rapid tests for easily obtained clinical samples, which can be deployed to assess patients exposed to TB, discriminate TB from other infectious, inflammatory or autoimmune diseases, and to identify subclinical TB in HIV-1 infected patients prior to commencing antiretroviral therapy. We discuss the evaluation of peripheral blood transcriptomics, proteomics and metabolomics to develop the next generation of rapid diagnostics for active TB. We catalogue the studies published to date seeking to discriminate active TB from healthy volunteers, patients with latent infection and those with other diseases. We identify the limitations of these studies and the barriers to their adoption in clinical practice. In so doing, we aim to develop a framework to guide our approach to discovery and development of diagnostic biomarkers for active TB.

Identification of Therapeutic Targets of Inflammatory Monocyte Recruitment to Modulate the Allogeneic Injury to Donor Cornea.

PURPOSE: We sought to test the hypothesis that monocytes contribute to the immunopathogenesis of corneal allograft rejection and identify therapeutic targets to inhibit monocyte recruitment. METHODS: Monocytes and proinflammatory mediators within anterior chamber samples during corneal graft rejection were quantified by flow cytometry and multiplex protein assays. Lipopolysaccharide or IFN-γ stimulation of monocyte-derived macrophages (MDMs) was used to generate inflammatory conditioned media (CoM). Corneal endothelial viability was tested by nuclear counting, connexin 43, and propidium iodide staining. Chemokine and chemokine receptor expression in monocytes and MDMs was assessed in microarray transcriptomic data. The role of chemokine pathways in monocyte migration across microvascular endothelium was tested in vitro by chemokine depletion or chemokine receptor inhibitors. RESULTS: Inflammatory monocytes were significantly enriched in anterior chamber samples within 1 week of the onset of symptoms of corneal graft rejection. The MDM inflammatory CoM was cytopathic to transformed human corneal endothelia. This effect was also evident in endothelium of excised human cornea and increased in the presence of monocytes. Gene expression microarrays identified monocyte chemokine receptors and cognate chemokines in MDM inflammatory responses, which were also enriched in anterior chamber samples. Depletion of selected chemokines in MDM inflammatory CoM had no effect on monocyte transmigration across an endothelial blood-eye barrier, but selective chemokine receptor inhibition reduced monocyte recruitment significantly. CONCLUSIONS: We propose a role for inflammatory monocytes in endothelial cytotoxicity in corneal graft rejection. Therefore, targeting monocyte recruitment offers a putative novel strategy to reduce donor endothelial cell injury in survival of human corneal allografts.

An endogenous nanomineral chaperones luminal antigen and peptidoglycan to intestinal immune cells.

In humans and other mammals it is known that calcium and phosphate ions are secreted from the distal small intestine into the lumen. However, why this secretion occurs is unclear. Here, we show that the process leads to the formation of amorphous magnesium-substituted calcium phosphate nanoparticles that trap soluble macromolecules, such as bacterial peptidoglycan and orally fed protein antigens, in the lumen and transport them to immune cells of the intestinal tissue. The macromolecule-containing nanoparticles utilize epithelial M cells to enter Peyer's patches, small areas of the intestine concentrated with particle-scavenging immune cells. In wild-type mice, intestinal immune cells containing these naturally formed nanoparticles expressed the immune tolerance-associated molecule 'programmed death-ligand 1', whereas in NOD1/2 double knockout mice, which cannot recognize peptidoglycan, programmed death-ligand 1 was undetected. Our results explain a role for constitutively formed calcium phosphate nanoparticles in the gut lumen and show how this helps to shape intestinal immune homeostasis.

Artefactual nanoparticle activation of the inflammasome platform: in vitro evidence with a nano-formed calcium phosphate.

AIM: To determine whether in vitro experimental conditions dictate cellular activation of the inflammasome by apatitic calcium phosphate nanoparticles. MATERIAL & METHODS: The responses of blood-derived primary human cells to in situ-formed apatite were investigated under different experimental conditions to assess the effect of aseptic culture, cell rest and duration of particle exposure. Cell death and particle uptake were assessed, while IL-1ß and caspase 1 responses, with and without lipopolysaccharide prestimulation, were evaluated as markers of inflammasome activation. RESULTS: Under carefully addressed experimental conditions, apatitic nanoparticles did not induce cell death or engage the inflammasome platform, although both could be triggered through artefacts of experimentation. CONCLUSION: In vitro studies often predict that engineered nanoparticles, such as synthetic apatite, are candidates for inflammasome activation and, hence, are toxic. However, the experimental setting must be very carefully considered as it may promote false-positive outcomes.