Performance evaluation of a non-invasive one-step multiplex RT-qPCR assay for detection of SARS-CoV-2 direct from saliva.

Polymerase chain reaction (PCR) has proven to be the gold-standard for SARS-CoV-2 detection in clinical settings. The most common approaches rely on nasopharyngeal specimens obtained from swabs, followed by RNA extraction, reverse transcription and quantitative PCR. Although swab-based PCR is sensitive, swabbing is invasive and unpleasant to administer, reducing patient compliance for regular testing and resulting in an increased risk of improper sampling. To overcome these obstacles, we developed a non-invasive one-step RT-qPCR assay performed directly on saliva specimens. The University of Nottingham Asymptomatic Testing Service protocol simplifies sample collection and bypasses the need for RNA extraction, or additives, thus helping to encourage more regular testing and reducing processing time and costs. We have evaluated the assay against the performance criteria specified by the UK regulatory bodies and attained accreditation (BS EN ISO/IEC 17,025:2017) for SARS-CoV-2 diagnostic testing by the United Kingdom Accreditation Service. We observed a sensitivity of 1 viral copy per microlitre of saliva, and demonstrated a concordance of > 99.4% between our results and those of other accredited testing facilities. We concluded that saliva is a stable medium that allows for a highly precise, repeatable, and robust testing method.

The active form of Helicobacter pylori vacuolating cytotoxin induces decay-accelerating factor CD55 in association with intestinal metaplasia in the human gastric mucosa.

High-level expression of decay-accelerating factor, CD55, has previously been found in human gastric cancer (GC) and intestinal metaplasia (IM) tissues. Therapeutic effects of CD55 inhibition in cancer have been reported. However, the role of Helicobacter pylori infection and virulence factors in the induction of CD55 and its association with histological changes of the human gastric mucosa remain incompletely understood. We hypothesised that CD55 would be increased during infection with more virulent strains of H. pylori, and with more marked gastric mucosal pathology. RT-qPCR and immunohistochemical analyses of gastric biopsy samples from 42 H. pylori-infected and 42 uninfected patients revealed that CD55 mRNA and protein were significantly higher in the gastric antrum of H. pylori-infected patients, and this was associated with the presence of IM, but not atrophy, or inflammation. Increased gastric CD55 and IM were both linked with colonisation by vacA i1-type strains independently of cagA status, and in vitro studies using isogenic mutants of vacA confirmed the ability of VacA to induce CD55 and sCD55 in gastric epithelial cell lines. siRNA experiments to investigate the function of H. pylori-induced CD55 showed that CD55 knockdown in gastric epithelial cells partially reduced IL-8 secretion in response to H. pylori, but this was not due to modulation of bacterial adhesion or cytotoxicity. Finally, plasma samples taken from the same patients were analysed for the soluble form of CD55 (sCD55) by ELISA. sCD55 levels were not influenced by IM and did not correlate with gastric CD55 mRNA levels. These results suggest a new link between active vacA i1-type H. pylori, IM, and CD55, and identify CD55 as a molecule of potential interest in the management of IM as well as GC treatment. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Direct RT-qPCR Assay for the Detection of SARS-CoV-2 in Saliva Samples.

Since mid-2020 there have been complexities and difficulties in the standardisation and administration of nasopharyngeal swabs. Coupled with the variable and/or poor accuracy of lateral flow devices, this has led to increased societal 'testing fatigue' and reduced confidence in test results. Consequently, asymptomatic individuals have developed reluctance towards repeat testing, which remains the best way to monitor COVID-19 cases in the wider population. On the other hand, saliva-based PCR, a non-invasive, highly sensitive, and accurate test suitable for everyone, is gaining momentum as a straightforward and reliable means of detecting SARS-CoV-2 in symptomatic and asymptomatic individuals. Here, we provide an itemised list of the equipment and reagents involved in the process of sample submission, inactivation and analysis, as well as a detailed description of how each of these steps is performed.

Analysis of T Cell Responses to Helicobacter pylori Infection.

Analysis of human and mouse T-helper (Th) cell subset responses is key to understanding how the inflammatory response in the stomach is regulated, and links with pathology and disease outcomes. Here, we present methods for extracting cells from blood and tissue, and analyzing the balance of Th1, Th2, Th17, and regulatory T cell (Treg) subsets using flow cytometry and cytokine ELISA .

Helicobacter: Inflammation, immunology and vaccines.

Helicobacter pylori is usually acquired in early childhood and the infection persists lifelong without causing symptoms. In a small of cases, the infection leads to gastric or duodenal ulcer disease, or gastric cancer. Why disease occurs in these individuals remains unclear, however the host response is known to play a very important part. Understanding the mechanisms involved in maintaining control over the immune and inflammatory response is therefore extremely important. Vaccines against H. pylori have remained elusive but are desperately needed for the prevention of gastric carcinogenesis. This review focuses on research findings which may prove useful in the development of prognostic tests for gastric cancer development, therapeutic agents to control immunopathology, and effective vaccines.

The Human Stomach in Health and Disease: Infection Strategies by Helicobacter pylori.

Helicobacter pylori is a bacterial pathogen which commonly colonizes the human gastric mucosa from early childhood and persists throughout life. In the vast majority of cases, the infection is asymptomatic. H. pylori is the leading cause of peptic ulcer disease and gastric cancer, however, and these outcomes occur in 10-15% of those infected. Gastric adenocarcinoma is the third most common cause of cancer-associated death, and peptic ulcer disease is a significant cause of morbidity. Disease risk is related to the interplay of numerous bacterial host and environmental factors, many of which influence chronic inflammation and damage to the gastric mucosa. This chapter summarizes what is known about health and disease in H. pylori infection, and highlights the need for additional research in this area.

Helicobacter pylori-Mediated Protection from Allergy Is Associated with IL-10-Secreting Peripheral Blood Regulatory T Cells.

Helicobacter pylori infections are usually established in early childhood and continuously stimulate immunity, including T-helper 1 (Th1), Th17, and regulatory T-cell (Treg) responses, throughout life. Although known to be the major cause of peptic ulcer disease and gastric cancer, disease occurs in a minority of those who are infected. Recently, there has been much interest in beneficial effects arising from infection with this pathogen. Published data robustly show that the infection is protective against asthma in mouse models. Epidemiological studies show that H. pylori is inversely associated with human allergy and asthma, but there is a paucity of mechanistic data to explain this. Since Th1 and Treg responses are reported to protect against allergic responses, we investigated if there were links between the human systemic Th1 and Treg response to H. pylori and allergen-specific IgE levels. The human cytokine and T-cell responses were examined using peripheral blood mononuclear cells (PBMCs) from 49 infected and 58 uninfected adult patients. Concentrations of total and allergen-specific plasma IgE were determined by ELISA and ImmunoCAP assays. These responses were analyzed according to major virulence factor genotypes of the patients' colonizing H. pylori strains. An in vitro assay was employed, using PBMCs from infected and uninfected donors, to determine the role of Treg cytokines in the suppression of IgE. Significantly higher frequencies of IL-10-secreting CD4(+)CD25(hi) Tregs, but not H. pylori-specific Th1 cells, were present in the peripheral blood of infected patients. Total and allergen-specific IgE concentrations were lower when there was a strong Treg response, and blocking IL-10 in vitro dramatically restored IgE responses. IgE concentrations were also significantly lower when patients were infected with CagA(+) strains or those expressing the more active i1 form of VacA. The systemic IL-10(+) Treg response is therefore likely to play a role in H. pylori-mediated protection against allergy in humans.

Selective Inhibition of Membrane Type 1 Matrix Metalloproteinase Abrogates Progression of Experimental Inflammatory Arthritis: Synergy With Tumor Necrosis Factor Blockade.

OBJECTIVE: In rheumatoid arthritis (RA), destruction of articular cartilage by the inflamed synovium is considered to be driven by increased activities of proteolytic enzymes, including matrix metalloproteinases (MMPs). The purpose of this study was to investigate the therapeutic potential of selective inhibition of membrane type 1 MMP (MT1-MMP) and its combination with tumor necrosis factor (TNF) blockage in mice with collagen-induced arthritis (CIA). METHODS: CIA was induced in DBA/1 mice by immunization with bovine type II collagen. From the onset of clinical arthritis, mice were treated with MT1-MMP selective inhibitory antibody DX-2400 and/or TNFR-Fc fusion protein. Disease progression was monitored daily, and serum, lymph nodes, and affected paws were collected at the end of the study for cytokine and histologic analyses. For in vitro analysis, bone marrow-derived macrophages were stimulated with lipopolysaccharide for 24 hours in the presence of DX-2400 and/or TNFR-Fc to analyze cytokine production and phenotype. RESULTS: DX-2400 treatment significantly reduced cartilage degradation and disease progression in mice with CIA. Importantly, when combined with TNF blockade, DX-2400 acted synergistically, inducing long-term benefit. DX-2400 also inhibited the up-regulation of interleukin-12 (IL-12)/IL-23 p40 via polarization toward an M2 phenotype in bone marrow-derived macrophages. Increased production of IL-17 induced by anti-TNF, which correlated with an incomplete response to anti-TNF, was abrogated by combined treatment with DX-2400 in CIA. CONCLUSION: Targeting MT1-MMP provides a potential strategy for joint protection, and its combination with TNF blockade may be particularly beneficial in RA patients with an inadequate response to anti-TNF therapy.

Basal localization of MT1-MMP is essential for epithelial cell morphogenesis in 3D collagen matrix.

The membrane-anchored collagenase membrane type 1 matrix metalloprotease (MT1-MMP) has been shown to play an essential role during epithelial tubulogenesis in 3D collagen matrices; however, its regulation during tubulogenesis is not understood. Here, we report that degradation of collagen in polarized epithelial cells is post-translationally regulated by changing the localization of MT1-MMP from the apical to the basal surface. MT1-MMP predominantly localizes at the apical surface in inert polarized epithelial cells, whereas treatment with HGF induced basal localization of MT1-MMP followed by collagen degradation. The basal localization of MT1-MMP requires the ectodomains of the enzyme because deletion of the MT-loop region or the hemopexin domain inhibited basal localization of the enzyme. TGFß is a well-known inhibitor of tubulogenesis and our data indicate that its mechanism of inhibition is, at least in part, due to inhibition of MT1-MMP localization to the basal surface. Interestingly, however, the effect of TGFß was found to be bi-phasic: at high doses it effectively inhibited basal localization of MT1-MMP, whereas at lower doses tubulogenesis and basal localization of MT1-MMP was promoted. Taken together, these data indicate that basal localization of MT1-MMP is a key factor promoting the degradation of extracellular matrix by polarized epithelial cells, and that this is an essential part of epithelial morphogenesis in 3D collagen.

cis-Urocanic acid enhances prostaglandin E2 release and apoptotic cell death via reactive oxygen species in human keratinocytes.

Urocanic acid (UCA) is a major UVR-absorbing skin molecule that undergoes trans to cis photoisomerization in the epidermis following UVR exposure. Murine studies have established that cis-UCA is an important mediator of UVR-induced immune suppression, but little is known about its signaling pathway. We have previously demonstrated that treatment of normal human epidermal keratinocytes with cis-UCA resulted in increased synthesis of prostaglandin E(2) (PGE(2)) and cell death. Here, using immortalized human keratinocytes, we report that cis-UCA but not trans-UCA generates reactive oxygen species (ROS) in a dose-dependent manner and that the natural antioxidant α-tocopherol can reduce this ROS generation, subsequent PGE(2) release, and apoptotic cell death. Western blot analysis revealed that cis-UCA leads to a transient phosphorylation of EGFR as well as downstream mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK) and p38. Pharmacological inhibition of their activity attenuated PGE(2) release induced by cis-UCA. After transient activation, cis-UCA downregulated EGFR protein expression that corresponded to activation of caspase-3. In addition, pretreatment with α-tocopherol inhibited EGFR downregulation and caspase-3 activation induced by cis-UCA. These results suggest that cis-UCA exerts its effects on human keratinocytes via intracellular ROS generation that modulates EGFR signaling and subsequently induces PGE(2) synthesis and apoptotic cell death.

Differences in the clinical characteristics of Pneumocystis jirovecii pneumonia in immunocompromized patients with and without HIV infection.

BACKGROUND AND OBJECTIVE: The incidence of Pneumocystis jirovecii pneumonia (PCP) in patients with predisposing immunodeficiencies other than AIDS is growing. Knowing the different characteristics and outcomes of PCP according to HIV status would help physicians manage and treat patients with PCP. METHODS: The medical charts of all patients with a proven first episode of PCP, diagnosed between 1997 and 2007 were retrospectively reviewed, and clinical and laboratory data abstracted. RESULTS: Of the 35 patients with PCP, 18 were HIV-positive and 17 were HIV-negative with other immunosuppressive conditions. HIV-negative patients were significantly older than HIV-positive patients. The WCC (10 952 +/- 5669 vs 9750 +/- 3133/microL; P = 0.015), neutrophil counts (9631 +/- 5421 vs 5680 +/- 2628/microL; P = 0.01) and CD4+ lymphocyte counts (329 +/- 502 vs 47 +/- 50/microL; P < 0.001) were significantly higher in HIV-negative patients. Six of the 17 HIV-negative patients had a CD4+ lymphocyte count >300/microL. Serum IgG levels were lower in HIV-negative patients (943 +/- 379 vs 1635 +/- 657 mg/dL; P = 0.017). Mortality was higher in HIV-negative (52.9%) than in HIV-positive patients (0%). On univariate analysis, risk factors for mortality from PCP were the presence of underlying pulmonary disease (odds ratio 4.000, 95% CI: 1.501-10.658) and HIV-negative status (odds ratio 2.125, 95% CI: 1.283-3.518). CONCLUSIONS: The characteristics and outcomes of PCP differ significantly depending on HIV status. The existence of underlying pulmonary diseases may be associated with the prognosis of HIV-negative patients with PCP.

cis-Urocanic acid stimulates primary human keratinocytes independently of serotonin or platelet-activating factor receptors.

Urocanic acid (UCA) is a major epidermal chromophore that undergoes trans to cis isomerization after ultraviolet radiation (UVR). cis-UCA suppresses cell-mediated immunity. Recent studies suggest that cis-UCA binds to serotonin (5-hydroxytryptamine) 2A (5-HT(2A)) receptor and that antagonists of 5-HT(2A) and the platelet-activating factor (PAF) receptor can block cis-UCA-induced immune suppression in mice. Here, we examined the involvement of 5-HT(2A) and PAF receptors in the ability of cis-UCA to stimulate immunomodulatory mediator production in primary human keratinocytes. Using real-time reverse transcription-PCR (RT-PCR), PAF but not 5-HT(2A) receptor mRNA was constitutively expressed in primary human keratinocytes. Treatment with cis-UCA increased prostaglandin E(2) (PGE(2)), tumor necrosis factor-alpha (TNF-alpha), and IL-6 secretion, whereas 5-HT only stimulated IL-6 production. Pretreatment with a 5-HT receptor antagonist partially inhibited IL-6 increase by 5-HT, but did not inhibit mediator production by cis-UCA. Similarly, a PAF receptor antagonist did not inhibit cis-UCA-induced increase in PGE(2). Intracellular calcium mobilization studies using a human epithelial cell line stably transfected with PAF receptor also showed little evidence that cis-UCA stimulated PAF receptor and it did not bind to this receptor. Thus, cis-UCA stimulates mediator production by a pathway that is independent of these receptors in human keratinocytes, and these cells may not be the major target for cis-UCA-induced immune suppression.

[Clinical statistics of outpatients investigated for asbestos-related lung diseases at a general hospital in Tokyo].

We evaluated 122 outpatients who visited our hospital for examination of asbestos-related diseases between November, 2005 and October, 2006. Patients were divided into three groups; occupational exposure, non-occupational exposure and non-exposure groups. The occupational exposure group showed a significantly higher rate of asbestos-related abnormal findings than the non-occupational exposure plus the non-exposure group (33% vs. 5%, respectively; P = 0.001). Pleural plaque was the most common abnormal finding related to asbestos. Only four of 24 patients with pleural plaques could obtain personal health records for workers enjoyed in dangerous work, whereas the rest of them were not able to mainly because they were self-employed. A health support system is necessary to also cover non-employees.

[A case of bird related hypersensitivity pneumonitis had a progressive course].

A 50-year-old woman was admitted due to productive cough continuing for 3 years. A chest computed tomography showed appearance of nonspecific interstitial pneumonia (NSIP). Thoracoscopic lung biopsy specimens showed mainly a pattern of NSIP with multinucleated cells and cholesterol clefts. She was not a bird fancier, but had indirect exposure to birds in her living environment, and had been using feather-filled duvets for a long time. We established a diagnosis of bird-related hypersensitivity pneumonitis based on antibodies in serum positive to pigeon dropping extracts. She was treated by coadministration of corticosteroids and immunosuppressants, and avoidance of bird-related antigens, but had a progressive course and died of respiratory failure.

cis-Urocanic acid initiates gene transcription in primary human keratinocytes.

It is well established that solar UV radiation (UVR) suppresses cutaneous cell-mediated immunity in humans. trans-Urocanic acid (trans-UCA) is a major UVR-absorbing skin molecule that undergoes a photoisomerization to its cis-isomer following UVR exposure. Animal studies have demonstrated that cis-UCA plays a role in UVR-induced immune suppression, but the molecular mechanisms of action of cis-UCA are not fully understood. In this study, we examined changes in gene expression and synthesis of cytokines and PGE2 following UCA treatment of primary human keratinocytes. A limited microarray analysis of keratinocytes from two donors indicated that approximately 400 genes were induced by solar-simulated radiation (SSR), 16 of which were also up-regulated by cis-UCA. In contrast, trans-UCA had little or no effect on gene expression. The genes up-regulated by both cis-UCA and SSR were associated with apoptosis, cell growth arrest, cytokines, and oxidative stress. Further studies using primary keratinocytes from four new donors showed that PG-endoperoxide synthase-2 was dramatically induced by cis-UCA, resulting in an enhanced secretion of PGE2 into the cell culture supernatant. cis-UCA also increased cytokine protein production such as that of TNF-alpha, IL-6, and IL-8 in a dose-dependent manner. SSR had the same effect as cis-UCA, but trans-UCA had no effect. In addition, activation of NF-kappaB and lipid peroxidation were induced by cis-UCA and SSR, but not trans-UCA, suggesting possible upstream events of the gene expression changes. The data suggest that the induction of immune suppression by cis-UCA may involve the initiation of gene transcription of immunomodulatory mediators in primary human keratinocytes.

T cell receptor-interacting molecule acts as a chaperone to modulate surface expression of the CTLA-4 coreceptor.

The costimulatory molecule CTLA-4 is a potent downregulator of T cell responses. Although localized mostly in intracellular compartments, little is understood regarding the mechanism that regulates its transport to the cell surface. In this study, we demonstrated that the adaptor TRIM (T cell receptor-interacting molecule) bound to CTLA-4 in the trans Golgi network (TGN) and promoted transport of CTLA-4 to the surface of T cells. Increased TRIM expression augmented surface CTLA-4 expression, and pulse-chase analysis showed a more rapid transport of CTLA-4 to the cell surface. A reduction of TRIM expression by small hairpin RNAs reduced the expression of surface CTLA-4. This resulted in a more localized pattern of CTLA-4 in the TGN. Altered CTLA-4 expression by TRIM was accompanied by corresponding changes in coreceptor-mediated effects on cytokine production and proliferation. Our findings identify a role for TRIM as a chaperone in regulating CTLA-4 expression and function by enhancing CTLA-4 transport to the surface of T cells.