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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 Wileyamp; Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

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 sample collection. 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 (UoNATS) protocol simplifies sample collection and bypasses the need for RNA extraction, additives, or extraneous processing steps, 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 17025:2017) for SARS-CoV-2 diagnostic testing by the United Kingdom Accreditation Service (UKAS). 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 with surprising longevity, and allows for a highly precise, repeatable, and robust testing method.

Detection of SARS-COV-2 in Saliva Samples

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 .

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 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 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.

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. TGFβ 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 TGFβ 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.

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.

SummaryThe 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.

In this study, we investigated a change in the excretory content of 2,7,8-trimethyl-2(2'-carboxyethyl)-6-hydroxychroman (gamma-CEHC), a gamma-tocopherol (gamma-Toc) metabolite, in rat urine and bile by using a new high-performance liquid chromatography-electrochemical detection (HPLC-ECD) method. In this determination, CEHC [alpha- and gamma-CEHC, where alpha-CEHC = 2,5,7,8-tetramethyl-2(2'-carboxyethyl)-6-hydroxychroman] in the biological specimens were treated with 3 N methanolic HCl to hydrolyze conjugates and to promote esterification. The methylated samples were extracted by n-hexane/water (1:2). The analyses of the methyl esters of alpha-CEHC and gamma-CEHC were performed by an HPLC-ECD using an ODS-3 column at 35 degrees C. The mobile phase was acetonitrile/water (45:55, vol/vol) containing 50 mM sodium perchlorate. After rat urine and bile samples, respectively, were methylated as described above, methylated biliary metabolites were identified by liquid chromatography-mass spectrometry as methyl esters of gamma-CEHC. Furthermore, we examined the differences in the excretion of gamma-CEHC between rat urine and bile after an oral administration of gamma-Toc or alpha- + gamma-Toc by the above HPLC method. In the gamma-Toc group, each vitamin E-deficient rat was given 0.5 mL of a stripped corn oil preparation containing 10 mg of gamma-Toc. In the alpha- + gamma-Toc group, the rat was given 10 mg of alpha-Toc and 10 mg of gamma-Toc. The content of gamma-CEHC in rat urine from the alpha- + gamma-Toc group was increased more in comparison to the gamma-Toc group at 18-36 h after oral administration. Moreover, the content of gamma-CEHC in rat bile in the alpha- + gamma-Toc group was increased more in comparison to the gamma-Toc group at 6-18 h after oral administration. Therefore, we have suggested that gamma-CEHC was shifted mainly to urinary excretion after gamma-CEHC had been excreted into the bile. Furthermore, we assume that alpha-Toc may affect the metabolism of gamma-Toc to gamma-CEHC in the body.

We investigated the distribution and metabolism of SRR-α-tocopherol (SRR-α-Toc), synthetic α-Toc compared with RRR-α-Toc, in rats after a single oral administration of 2 mg (20 μCi) SRR- and RRR-α-[5-methyl-14C]Toc. In the liver, there was no difference in the recovery of radioactivity until 12 h after administration, and it reached a maximum of 4.4% of the dose after 12 h, but in other tissues, radioactivity derived from RRR-α-Toc was clearly higher than that derived from SRR-α-Toc after 12 h. For 96 h after administration, urinary excretions of SRR-α-Toc were 7.8% of the dose and significantly greater than that of RRR-α-Toc, which was 1.3% of the dose. On the other hand, total fecal excretions of SRR- and RRR-α-Toc were 87.6% and 83.0%, respectively. Therefore, radioactivity in the urine was assumed to have transferred out of the liver. Furthermore, the urine samples were hydrolyzed with 3 N methanolic HCl and analyzed by high performance liquid chromatography (HPLC) and liquid chromatography/mass spectrometry. The results showed that about 73% of the total radioactivity injected into HPLC was found to be 2,5,7,8-tetramethyl-2-(2′-carboxyethyl)-6-hydroxy chroman (α-CEHC), as well as RRR-α-Toc. Thus, there is no difference between SRR-α-Toc and RRR-α-Toc in metabolic pathways, and it is suggested that SRR-α-Toc discriminated in the liver is rapidly metabolized by the liver and excreted as the conjugate of α-CEHC in the urine. —Kaneko, K., C. Kiyose, T. Ueda, H. Ichikawa, and O. Igarishi. Studies of the metabolism of α-tocopherol stereoisomers in rats using [5-methy1-14C]SRR- and RRR-α-tocopherol. J. Lipid Res. 2000. 41: 357–367.

We established a method to simultaneously determine RRR- and SRR-alpha-tocopherol (alpha-Toc) and their quinones in biological samples by chiral-phase high-performance liquid chromatography (HPLC). Alpha-Toc had a shorter retention time than alpha-tocopherylquinones (alpha-TQ), and 2-ambo-alpha-Toc was completely separated into two peaks; the first peak was RRR-alpha-Toc and the second SRR-isomer by chiral HPLC connected Chiralcel OD-H column and Sumichiral OA4100 column. In contrast, of the two peaks of alpha-TQ, the first was the SRR-isomer. We also investigated differences in the distribution of RRR- and SRR-alpha-TQ in rat tissues after oral administration of 2-ambo-alpha-Toc by the above HPLC method. Rats deficient in vitamin E were divided into two groups, control and experimental, and tissues were collected at 3, 6, and 24 h after oral 2-ambo-alpha-Toc administration. The concentrations of RRR- and SRR-alpha-Toc and their quinones in plasma and each tissue were determined. The concentration of SRR-alpha-TQ in plasma and adrenal glands was not significantly different from RRR-alpha-TQ. However, the concentration of SRR-alpha-TQ in liver up to 6 h after oral administration was higher than that of RRR-alpha-TQ, and SRR- and RRR-alpha-TQ levels were similar at 24 h after oral administration. Therefore, we may assume that the formation of alpha-TQ in vivo was not different between RRR- and SRR-isomer and that it was not affected by the presence of alpha-Toc stereoisomers.

Raman and IR spectra of uncomplexed 1,7,10,16-tetraoxa-4,13-diazacyclooctadecane (4,13-diaza18-crown-6) have been measured in the solid state. Normal coordinate calculations have been carried out on the C2h conformation of this macrocyclic compound. The force field of the diazacrown ether has been determined by adjusting several important several force constants associated with the imino group, other force constants being constrained, as transferred, to those of 18-crown-6. The vibrational assignments of 4,13-diaza-18-crown-6 are made on the basis of the previous assignments of a KSCN complex of 18-crown-6 with a similar ring conformation and of the results of the normal coordinate calculations. The normal modes of low-wavenumber vibrations are depicted. The lowest two vibrations represent out-of-plane deformations of folding the macrocyclic ring double. These modes are favourable to complexation of the macrocycle with guest. It is likely that these low-wavenumber vibrations are possible paths of the inclusion of the guest by the macrocyclic ring.

Raman and IR spectra of uncomplexed 1,7,10,16-tetraoxa-4,13-diazacyclooctadecane (4,13-diaza18-crown-6) have been measured in the solid state. Normal coordinate calculations have been carried out on the C2h conformation of this macrocyclic compound. The force field of the diazacrown ether has been determined by adjusting several important several force constants associated with the imino group, other force constants being constrained, as transferred, to those of 18-crown-6. The vibrational assignments of 4,13-diaza-18-crown-6 are made on the basis of the previous assignments of a KSCN complex of 18-crown-6 with a similar ring conformation and of the results of the normal coordinate calculations. The normal modes of low-wavenumber vibrations are depicted. The lowest two vibrations represent out-of-plane deformations of folding the macrocyclic ring double. These modes are favourable to complexation of the macrocycle with guest. It is likely that these low-wavenumber vibrations are possible paths of the inclusion of the guest by the macrocyclic ring.

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.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.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; P0.001) were significantly higher in HIV-negative patients. Six of the 17 HIV-negative patients had a CD4+ lymphocyte count300/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).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.

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 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.

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 ∼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-α, 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-κB 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.

We present a statistical mechanical analysis of electron-ion recombination processes in weakly ionized nonpolar dense media, being based on the model which can explain the external electric field dependence of electron mobility in these media. We derive a diffusion equation by a projection technique from the phase space to the coordinate space where the stationary distribution of electron momentum in an external electric field has been realized. Then we estimate the recombination rate constant from this equation. Here we deal with the system classically as a three-body recombination process.

The degree of twitch depression induced by nondepolarizing neuromuscular blocking drugs is known to be dependent on the stimulation frequency employed. Train-of-four (TOF) stimulations with different frequencies (0.67, 1.33 and 2.0 Hz) were delivered to a sciatic nerve of a rat and series of four twitch heights of a tibialis anterior muscle were measured after d-tubocurarine (d-TC) administration. With a decrease of stimulus interval, twitch heights were intensely depressed. We hypothesized that the oservations are due to the changes of released amount of neuromuscular transmitter, acetylcholine, dependent on stimulus interval, and a pharmacokinetic/pharmacodynamic model based on the hypothesis was proposed. The model allowed simultaneous fitting of the twitch height depression after d-TC administration. It also could give a rationale to the fact that TOF stimulation at 2.0 Hz is a more sensitive monitoring method of neuromuscular function than single twitch stimulation (0.1-0.2 Hz).

Effects of acebutolol on carbohydrate and lipid metabolism in rats and on adenylate cyclase of heart and liver in dogs were investigated to determine the beta receptor blocking properties of the compound. Acebutolol exhibited the beta blocking activity and inhibited the increase of serum lactate concentration induced by adrenaline. This inhibition was about one-sixth as potent as that of propranolol. In hyperglycemic and free fatty acid effects of adrenaline, acebutolol inhibited the adrenaline-induced free fatty acid increase more effectively than hyperglycemia induced by adrenaline. In the inhibition of stimulated adenylate cyclase activity in the heart and liver, acebutolol was more active on the heart than on liver. Relative beta1 specificity of acebutolol was 93.2. Inhibition of propranolol on adenylate cyclase activity was more potent than that of acebutolol on both tissues, but showed no specificity. These results suggest that acebutolol is beta1 selective, although its beta blocking potency is less than that of propranolol.

The motion of electrons in nonpolar dense media is studied in the framework of gas kinetic transport theory. The observed external electric field dependence of electron mobility is explained by means of the solution of Boltzmann‐type transport equation. Numerical calculations of the mobility, mean energy, and energy distribution function of electrons in liquid Ar and in liquid CH4 are presented and a good agreement with experimental data is obtained. Also the cases of gaseous Ar are studied.

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.