The aging process in the kidneys has been well studied. It is known that the glomerular filtration rate (GFR) declines with age in subjects older than 50-60 years. However, there is still insufficient knowledge regarding the response of the aged kidney to environmental toxicants such as mercury, cadmium, and lead. Here, we present a review on the functional decline and proposed mechanisms in the aging kidney as influenced by metal pollutants. Due to the prevalence of these toxicants in the environment, human exposure is nearly unavoidable. Further, it is well known that acute and chronic exposures to toxic metals may be detrimental to kidneys of normal adults, thus it may be hypothesized that exposure of individuals with reduced GFR will result in additional reductions in renal function. Individuals with compromised renal function, either from aging or from a combination of aging and disease, may be particularly susceptible to environmental toxicants. The available data appear to show an association between exposure to mercury, cadmium and/or lead and an increase in incidence and severity of renal disease in elderly individuals. Furthermore, some physiological thiols, as well as adequate selenium status, appear to exert a protective action. Further studies providing improved insight into the mechanisms by which nephrotoxic metals are handled by aging kidneys, as well as possibilities of therapeutic protection, are of utmost importance.

Background The impact of intestinal helminth infection on the clinical presentation and immune response during active tuberculosis (TB) infection is not well characterized. Our aim was to investigate whether asymptomatic intestinal helminth infection alters the clinical signs and symptoms as well as the cell mediated immune responses in patients with active TB.

Methodology Consecutive, newly diagnosed TB patients and healthy community controls (CCs) were recruited in North-west Ethiopia. TB-score, body mass index and stool samples were analyzed. Cells from HIV-negative TB patients (HIV-/TB) and from CCs were analyzed for regulatory T-cells (Tregs) and cytokine responses using flow cytometry and ELISPOT, respectively.

Results A significantly higher ratio of helminth co-infection was observed in TB patients without HIV (Helm+/HIV-/TB) compared to HIV negative CCs, (40% (121/306) versus 28% (85/306), p = 0.003). Helm+/HIV-/TB patients showed significantly increased IL-5 secreting cells compared to Helm-/HIV-/TB (37 SFU (IQR:13-103) versus 2 SFU (1-50); p = 0.02, n = 30). Likewise, levels of absolute Tregs (9.4 (3.2-16.7) cells/mu l versus 2.4 (1.1-4.0) cells/mu l; p = 0.041) and IL-10 secreting cells (65 SFU (7-196) versus 1 SFU (0-31); p = 0.014) were significantly higher in Helm+/HIV-/TB patients compared to Helm-/HIV-/TB patients. In a multivariate analysis, a lower rate of sputum smear positivity for acid fast bacilli, lower body temperature, and eosinophilia were independently associated with helminth infection in TB patients.

Conclusions Asymptomatic helminth infection is associated with increased regulatory T-cell and Th2-type responses and a lower rate of sputum smear positivity. Further studies are warranted to investigate the clinical and immunological impact of helminth infection in TB patients.

Procalcitonin (PCT) protein has recently been identified as a clinical marker for bacterial infections based on its better sepsis sensitivity. Thus, an increased level of PCT could be linked with disease diagnosis and therapeutics. In this study, we describe the construction of the photoelectrochemical (PEC) PCT immunosensing platform based on it situ grown photo-active CuWO4 nanospheres over reduced graphene oxide layers (CuWO4@rGO). The in situ growth strategy enabled the formation of small nanospheres (diameter of 200 nm), primarily composed of tiny self-assembled CuWO4 nanoparticles (2-5 nm). The synergic coupling of CuWO4 with rGO layers constructed an excellent photo-active heterojunction for photoelectrochemical (PEC) sensing. The platform was then considered for electrocatalytic (EC) mechanism-based detection of PCT, where inhibition of the photocatalytic oxidation signal of ascorbic acid (AA), subsequent to the antibody-antigen interaction, was recorded as the primary signal response. This inhibition detection approach enabled sensitive detection of PCT in a concentration range of 10 pgmL(-1) to 50 ng.mL(-1) with signal sensitivity achievable up to 0.15 pgmL(-1). The proposed PEC hybrid (CuWO4@rGO) could further be engineered to detect other clinically important species.

The inflammatory microenvironment affects breast cancer progression. Proteins that govern the inflammatory response are secreted into the extracellular space, but this compartment still needs to be characterized in human breast tissues in vivo. Dense breast tissue is a major risk factor for breast cancer by yet unknown mechanisms and no non-toxic prevention for these patients exists. Here, we used the minimal invasive technique of microdialysis for sampling of extracellular proteins in live tissues in situ in breast cancers of women before surgery and in healthy women having dense or non-dense breast tissue on mammography. Proteins were profiled using a proximity extension assay. Out of the 32 proteins assessed, 26 exhibited similar profiles in breast cancers and dense breast tissues; CCL-4, -7, -8, -11, -15, -16, -22, -23, and -25, CXCL-5, -8, -9, -16 as well as sIL-6R, IL-18, vascular endothelial growth factor, TGF-a, fibroblast growth factor 19, matrix metalloproteinase (MMP)-1, -2, -3, and urokinase-type plasminogen activator were all increased, whereas CCL-3, CX3CL1, hepatocyte growth factor, and MMP-9 were unaltered in the two tissues. CCL-19 and -24, CXCL-1 and -10, and IL-6 were increased in dense breast tissue only, whereas IL-18BP was increased in breast cancer only. Our results provide novel insights in the inflammatory microenvironment in human breast cancer in situ and define potential novel therapeutic targets. Additionally, we show previously unrecognized similarities of the pro-inflammatory microenvironment in dense breast tissue and breast cancer in vivo suggesting that anti-inflammatory breast cancer prevention trials for women with dense breast tissue may be feasible.

BACKGROUND:

Low total diversity of the gut microbiota during the first year of life is associated with allergic diseases in infancy, but little is known how early microbial diversity is related to allergic disease later in school age.

OBJECTIVE:

To assess microbial diversity and characterize the dominant bacteria in stool during the first year of life in relation to the prevalence of different allergic diseases in school age, such as asthma, allergic rhinoconjunctivitis (ARC) and eczema.

METHODS:

The microbial diversity and composition was analysed with barcoded 16S rDNA 454 pyrosequencing in stool samples at 1 week, 1 month and 12 months of age in 47 infants which were subsequently assessed for allergic disease and skin prick test reactivity at 7 years of age (ClinicalTrials.gov ID NCT01285830).

RESULTS:

Children developing asthma (n = 8) had a lower diversity of the total microbiota than non-asthmatic children at 1 week (P = 0.04) and 1 month (P = 0.003) of age, whereas allergic rhinoconjunctivitis (n = 13), eczema (n = 12) and positive skin prick reactivity (n = 14) at 7 years of age did not associate with the gut microbiota diversity. Neither was asthma associated with the microbiota composition later in infancy (at 12 months). Children having IgE-associated eczema in infancy and subsequently developing asthma had lower microbial diversity than those that did not. There were no significant differences, however, in relative abundance of bacterial phyla and genera between children with or without allergic disease.

CONCLUSION AND CLINICAL RELEVANCE:

Low total diversity of the gut microbiota during the first month of life was associated with asthma but not ARC in children at 7 years of age. Measures affecting microbial colonization of the infant during the first month of life may impact asthma development in childhood.

Limited microbial exposure is suggested to underlie the increase of allergic diseases in affluent countries, and bacterial diversity seems to be more important than specific bacteria taxa. Prospective studies indicate that the gut microbiota composition during the first months of life influences allergy development, and support the theory that factors influencing the early maturation of the immune system might be important for subsequent allergic disease. However, recent research indicates that microbial exposure during pregnancy may be even more important for the preventative effects against allergic disease. This review gives a background of the epidemiology, immunology, and microbiology literature in this field. It focuses on possible underlying mechanisms such as immune-regulated epigenetic imprinting and bacterial translocation during pregnancy, potentially providing the offspring with a pioneer microbiome. We suggest that a possible reason for the initial exposure of bacterial molecular patterns to the fetus in utero is to prime the immune system and/or the epithelium to respond appropriately to pathogens and commensals after birth.

Background: During the initial phase of an infection, there is an upregulation of inducible nitric oxide synthase in the macrophages for the production of nitric oxide. This is followed by the recruitment of polymorphonuclear leukocytes (neutrophils) which release arginase. Arginase competes with inducible nitric oxide synthase for a common substrate L-arginine. Objective: To investigate whether the entry of neutrophils and release of arginase can interfere with nitric oxide production from stimulated mouse macrophages. Methods: Neutrophils were isolated from human blood and stimulated with cytodex-3 beads. Cultured macrophages were stimulated with lipopolysaccharide and interferon gamma with or without N (G)-nitro-L-arginine methyl ester or N (omega)-hydroxy-nor-L-arginine. Measurement of NO2-/NO3- and urea were done using the spectrophotometer. Results: A significantly higher level of nitric oxide production from stimulated macrophages was observed compared to control. There was a decrease in nitric oxide production when stimulated macrophages were treated with the supernatant from activated neutrophils (pless than0.05). Conclusion: Arginase from neutrophils can modulate nitric oxide production from activated macrophages which may affect the course of infection by intracellular bacteria.

MicroRNA can be found in various body fluids, including breast milk. MicroRNA may be transferred from mother to infant via breast milk and potentially regulate the development of the infants immune system on a post-transcriptional level. This study aimed to determine the microRNA extraction efficiency of five RNA extraction kits from human skim milk samples. Their efficiency was determined by comparing microRNA concentrations, total RNA yield and purity. Furthermore, hsa-miR-148a-3p expression and the recovery of an exogenous control, cel-miR-39-3p, were quantified using qPCR. Each kit extracted different amounts of microRNA and total RNA, with one kit tending to isolate the highest amount of both RNA species. Based on these results, the extraction kit ReliaPrep (TM) miRNA Cell and Tissue Miniprep System from Promega was found to be the most appropriate kit for microRNA extraction from human skim milk. Moreover, further research is needed to establish a standardized protocol for microRNA extraction from breast milk.

Antimicrobial resistance (AMR) in bacterial pathogens threatens global health, though the spread of AMR bacteria and AMR genes between humans, animals, and the environment is still largely unknown. Here, we investigated the role of wild birds in the epidemiology of AMR Escherichia coli. Using next-generation sequencing, we characterized cephalosporin-resistant E. coli cultured from sympatric gulls and bald eagles inhabiting a landfill habitat in Alaska to identify genetic determinants conferring AMR, explore potential transmission pathways of AMR bacteria and genes at this site, and investigate how their genetic diversity compares to isolates reported in other taxa. We found genetically diverse E. coli isolates with sequence types previously associated with human infections and resistance genes of clinical importance, including blaCTX-M and blaCMY. Identical resistance profiles were observed in genetically unrelated E. coli isolates from both gulls and bald eagles. Conversely, isolates with indistinguishable core-genomes were found to have different resistance profiles. Our findings support complex epidemiological interactions including bacterial strain sharing between gulls and bald eagles and horizontal gene transfer among E. coli harboured by birds. Results suggest that landfills may serve as a source for AMR acquisition and/or maintenance, including bacterial sequence types and AMR genes relevant to human health.

Although beta-hydroxybutyrate (BHB) analysis has proved its importance in forensic pathology, its effects on cause-of-death diagnostics are unaddressed. Therefore, this study aims at evaluating the effects of BHB analysis on the number of deaths by DKA (diabetes ketoacidosis), AKA (alcoholic ketoacidosis), HHS (hyperosmolar hyperglycaemic state), hypothermia, diabetes, alcoholism, and acidosis NOS (not otherwise specified). All 2900 deaths from 2013 through 2019 in which BHB was analysed at the National Board of Forensic Medicine, and 1069 DKA, AKA, HHS, hypothermia, diabetes, alcoholism, and acidosis cases without BHB analysis were included. The prevalence of BHB-positive cases for each cause of death, and trends and proportions of different BHB concentrations, were investigated. The number of BHB analyses/year increased from 13 to 1417. AKA increased from three to 66 and acidosis from one to 20. The deaths from alcoholism, DKA, and hypothermia remained stable. It is unclear why death from alcoholism remained stable while AKA increased. The increase in unspecific acidosis deaths raises the question why a more specific diagnosis had not been used. In conclusion, BHB analysis is instrumental in detecting AKA and acidosis. The scientific basis for the diagnosis of DKA and hypothermia improved, but the number of cases did not change.

Background In countries with a high prevalence of tuberculosis there is high coincident of helminth infections that might worsen disease outcome. While Mycobacterium tuberculosis (Mtb) gives rise to a pro-inflammatory Th1 response, a Th2 response is typical of helminth infections. A strong Th2 response has been associated with decreased protection against tuberculosis. Principal findings We investigated the direct effect of helminth-derived antigens on human macrophages, hypothesizing that helminths would render macrophages less capable of controlling Mtb. Measuring cytokine output, macrophage surface markers with flow cytometry, and assessing bacterial replication and phagosomal maturation revealed that antigens from different species of helminth directly affect macrophage responses to Mtb. Antigens from the tapeworm Hymenolepis diminuta and the nematode Trichuris muris caused an anti-inflammatory response with M2-type polarization, reduced macrophage phagosome maturation and ability to activate T cells, along with increased Mtb burden, especially in T. muris exposed cells which also induced the highest IL-10 production upon co-infection. However, antigens from the trematode Schistosoma mansoni had the opposite effect causing a decrease in IL-10 production, M1-type polarization and increased control of Mtb. Conclusion We conclude that, independent of any adaptive immune response, infection with helminth parasites, in a species-specific manner can influence the outcome of tuberculosis by either enhancing or diminishing the bactericidal function of macrophages.

In this study, using the Hain GenoType MTBDRsl assays (versions 1 and 2), we found that some nonsynonymous and synonymous mutations in gyrA in Mycobacterium tuberculosis result in systematic false-resistance results to fluoroquinolones by preventing the binding of wild-type probes. Moreover, such mutations can prevent the binding of mutant probes designed for the identification of specific resistance mutations. Although these mutations are likely rare globally, they occur in approximately 7% of multidrug-resistant tuberculosis strains in some settings.

Selenium (Se) is an essential dietary trace element that plays an important role in the prevention of inflammation, cardiovascular diseases, infections, and cancer. Selenoproteins contain selenocysteine in the active center and include, i.a., the enzymes thioredoxin reductases (TXNRD1-3), glutathione peroxidases (GPX1-4 and GPX6) and methionine sulfoxide reductase, involved in immune functions, metabolic homeostasis, and antioxidant defense. Ageing is an inevitable process, which, i.a., involves an imbalance between antioxidative defense and reactive oxygen species (ROS), changes in protein and mitochondrial renewal, telomere attrition, cellular senescence, epigenetic alterations, and stem cell exhaustion. These conditions are associated with mild to moderate inflammation, which always accompanies the process of ageing and age-related diseases. In older individuals, Se, by being a component in protective enzymes, operates by decreasing ROS-mediated inflammation, removing misfolded proteins, decreasing DNA damage, and promoting telomere length. Se-dependent GPX1-4 and TXNRD1-3 directly suppress oxidative stress. Selenoprotein H in the cell nucleus protects DNA, and selenoproteins residing in the endoplasmic reticulum (ER) assist in the removal of misfolded proteins and protection against ER stress. In this review, we highlight the role of adequate Se status for human ageing and prevention of age-related diseases, and further its proposed role in preservation of telomere length in middle-aged and elderly individuals.

The development of alternative ecological and effective antifouling technologies is still challenging. Synthesis of nature-inspired compounds has been exploited, given the potential to assure commercial supplies of potential ecofriendly antifouling agents. In this direction, the antifouling activity of a series of nineteen synthetic small molecules, with chemical similarities with natural products, were exploited in this work. Six (4,5,7,10,15and17) of the tested xanthones showed in vivo activity toward the settlement ofMytilus galloprovincialislarvae (EC50: 3.53-28.60 mu M) and low toxicity to this macrofouling species (LC50> 500 mu M and LC50/EC50: 17.42-141.64), and two of them (7and10) showed no general marine ecotoxicity (Artemia salinamortality) after 48 h of exposure. Regarding the mechanism of action in mussel larvae, the best performance compounds4and5might be acting by the inhibition of acetylcholinesterase activity (in vitro and in silico studies), while7and10showed specific targets (proteomic studies) directly related with the mussel adhesive structure (byssal threads), given by the alterations in the expression ofMytiluscollagen proteins (PreCols) and proximal thread proteins (TMPs). A quantitative structure-activity relationship (QSAR) model was built with predictive capacity to enable speeding the design of new potential active compounds.

Our aim was to develop a Mycobacterium tuberculosis (Mtb) growth inhibition assay (MGIA) as a summary estimate of host immune control of virulent Mtb. Mycobacterial growth inhibition (MGI) using previously frozen human PBMCs infected with H37Rv was assessed by live-cell imaging (Incucyte (c)) complemented by imaging flow cytometry analysis of phagocytosis. MGI measured as relative fluorescence units (RFU) was calibrated to time to positive culture (TTP) in BACTEC 960 MGIT. At a MOI (multiplicity of infection) of 5, there was a wide range of MGI of blood donors (1.1*10(6)-2.7*10(6) RFU, n = 14). Intra- and inter-assay variability were at most 17.5 and 20.7 CV%. Cell viability at day 5 was 57 and 62% monitored by the LDH and Draq7 assays respectively. There was a strong correlation between a readout for Mtb growth using CFU counts or TTP compared to RFU (r2 >= 0.96). Our MGIA enabling live-cell imaging and monitoring of cell viability was able to detect a wide range of Mtb growth inhibition by PBMCs and was calibrated to several readout options for bacterial growth. This MGIA may be valuable as a surrogate marker of host immunity in a personalized medicine approach.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a chronic disease presenting with severe fatigue, post-exertional malaise, and cognitive disturbances-among a spectrum of symptoms-that collectively render the patient housebound or bedbound. Epigenetic studies in ME/CFS collectively confirm alterations and/or malfunctions in cellular and organismal physiology associated with immune responses, cellular metabolism, cell death and proliferation, and neuronal and endothelial cell function. The sudden onset of ME/CFS follows a major stress factor that, in approximately 70% of cases, involves viral infection, and ME/CFS symptoms overlap with those of long COVID. Viruses primarily linked to ME/CFS pathology are the symbiotic herpesviruses, which follow a bivalent latent-lytic lifecycle. The complex interaction between viruses and hosts involves strategies from both sides: immune evasion and persistence by the viruses, and immune activation and viral clearance by the host. This dynamic interaction is imperative for herpesviruses that facilitate their persistence through epigenetic regulation of their own and the host genome. In the current article, we provide an overview of the epigenetic signatures demonstrated in ME/CFS and focus on the potential strategies that latent viruses-particularly Epstein-Barr virus-may employ in long-term epigenetic reprograming in ME/CFS. Epigenetic studies could aid in elucidating relevant biological pathways impacted in ME/CFS and reflect the physiological variations among the patients that stem from environmental triggers, including exogenous viruses and/or altered viral activity. image

Prions are proteinaceous infectious particles that replicate by structural conversion of the host-encoded cellular prion protein (PrPC), causing fatal neurodegenerative diseases in mammals. Species-specific amino acid substitutions (AAS) arising from single nucleotide polymorphisms within the prion protein gene (Prnp) modulate prion disease patho-genesis, and, in several instances, reduce susceptibility of homo-or heterozygous AAS carriers to prion infection. However, a mechanistic understanding of their protective effects against clinical disease is missing. We generated gene-targeted mouse infection models of chronic wasting disease (CWD), a highly contagious prion disease of cervids. These mice express wild-type deer or PrPC harboring the S138N substitution homo-or heterozygously, a polymorphism found exclusively in reindeer (Rangifer tarandus spp.) and fallow deer (Dama dama). The wild-type deer PrP-expressing model recapitulated CWD pathogenesis including fecal shedding. Encoding at least one 138N allele pre-vented clinical CWD, accumulation of protease-resistant PrP (PrPres) and abnormal PrP deposits in the brain tissue. However, prion seeding activity was detected in spleens, brains, and feces of these mice, suggesting subclinical infection accompanied by prion shedding. 138N-PrPC was less efficiently converted to PrPres in vitro than wild-type deer (138SS) PrPC. Heterozygous coexpression of wild-type deer and 138N-PrPC resulted in dominant-negative inhibition and progressively diminished prion conversion over serial rounds of protein misfolding cyclic amplification. Our study indicates that heterozy-gosity at a polymorphic Prnp codon can confer the highest protection against clinical CWD and highlights the potential role of subclinical carriers in CWD transmission.

An important aspect of microbiological surveillance is the ability to access to live viruses for microneutralization assays, which enables the study of viral characteristics and mechanisms in vitro and production of positive controls for diagnostic methods. During the COVID-19 pandemic, the Public Health Agency of Sweden established a protocol for the rapid collection of clinical samples and subsequent isolation of novel virus variants.

Shiga toxin-producing Escherichia coli (STEC) are important foodborne pathogens that can cause human infections ranging from asymptomatic carriage to bloody diarrhea (BD) and fatal hemolytic uremic syndrome (HUS). However, the molecular mechanism of STEC pathogenesis is not entirely known. Here, we demonstrated a large scale of molecular epidemiology and in-depth genomic study of clinical STEC isolates utilizing clinical and epidemiological data collected in Region Jonkoping County, Sweden, over a 15-year period. Out of 184 STEC isolates recovered from distinct patients, 55 were from patients with BD, and 129 were from individuals with non-bloody stools (NBS). Five individuals developed HUS. Adults were more associated with BD. Serotypes O157:H7, O26:H11, O103:H2, O121:H19, and O104:H4 were more often associated with BD. The presence of Shiga toxin-encoding gene subtypes stx(2a), stx(2a) + stx(2c), and stx(1a) + stx(2c) was associated with BD, while stx(1)(a) was associated with milder disease. Multiplex virulence and accessory genes were correlated with BD; these genes encode toxins, adhesion, autotransporters, invasion, and secretion system. A number of antimicrobial resistance (AMR) genes, such as aminoglycoside, aminocoumarin, macrolide, and fluoroquinolone resistance genes, were prevalent among clinical STEC isolates. Whole-genome phylogeny revealed that O157 and non-O157 STEC isolates evolved from distinct lineages with a few exceptions. Isolates from BD showed more tendency to cluster closely. In conclusion, this study unravels molecular trait of clinical STEC strains and identifies genetic factors associated with severe clinical outcomes, which could contribute to management of STEC infections and disease progression if confirmed by further functional validation.

Background: Detection of active tuberculosis (TB) before antiretroviral therapy (ART) initiation is important, but optimal diagnostic methods for use in resource-limited settings are lacking. We assessed the prevalence of TB, evaluated the diagnostic yield of Xpert MTB/RIF in comparison with smear microscopy and culture, and the impact of Xpert results on clinical management in HIV-positive adults eligible for ART at health centers in a region of Ethiopia. Methods: Participants were prospectively recruited and followed up at 5 health centers. Trained nurses collected data on socio-demographic characteristics, medical history and symptoms, and performed physical examination. Two paired morning sputum samples were obtained, and lymph node aspirates in case of lymphadenopathy. Diagnostic yield of Xpert MTB/RIF in sputum was compared with smear microscopy and liquid culture. Results: TB was diagnosed in 145/812 participants (17.9%), with bacteriological confirmation in 137 (16.9%). Among bacteriologically confirmed cases, 31 were smear-positive (22.6%), 96 were Xpert-positive (70.1%), and 123 were culture-positive (89.8%). Xpert MTB/RIF increased the TB detection rate by 64 cases (47.4%) compared with smear microscopy. The overall sensitivity of Xpert MTB/RIF was 66.4%, and was not significantly lower when testing one compared with two samples. While Xpert MTB/RIF was 46.7% sensitive among patients with CD4 cell counts greater than200 cells/mm(3), this increased to 82.9% in those with CD4 cell counts less than= 100 cells/mm(3). Compared with Xpert-positive TB patients, Xpert-negative cases had less advanced HIV and TB disease characteristics. Conclusions: Previously undiagnosed TB is common among HIV-positive individuals managed in Ethiopian health centers. Xpert MTB/RIF increased TB case detection, especially in patients with advanced immunosuppression. An algorithm based on the use of a single morning sputum sample for individuals with negative sputum smear microscopy could be considered for intensified case finding in patients eligible for ART. However, technical and cost-effectiveness issues relevant for low-income countries warrant further study.

ObjectiveTo assess the diagnostic performance of urine lipoarabinomannan (LAM) detection for TB screening in HIV-positive adults in Ethiopia. MethodsTesting for LAM was performed using the Determine TB-LAM lateral flow assay on urine samples from participants in a prospective cohort with baseline bacteriological categorisation for active TB in sputum. Characteristics of TB patients with regard to LAM status were determined. Participants were followed for 6months to evaluate survival, retention in care and incident TB. ResultsPositive LAM results were found in 78/757 participants. Among 128 subjects with definite (confirmed by culture and/or Xpert MTB/RIF) TB, 33 were LAM-positive (25.8%); the respective figure for clinically diagnosed cases was 2/20 (10%). Five of the remaining 43 LAM-positive individuals had died during the 6-month follow-up period, whereas 38 remained in care without clinical signs of TB. The overall sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were 25.8%, 92.9%, 42.3% and 86.0%, respectively. Among TB patients, LAM positivity was associated with higher WHO clinical stage, lower body mass index (BMI), CD4 cell and haemoglobin levels, and with increased mortality. A combination algorithm of urine LAM testing and sputum smear microscopy detected 49 (38.2%) of definite TB cases; among those with CD4 count 100cells/mm(3), this proportion was 66.7%. ConclusionsThe performance of urine LAM testing for TB detection was poor in this population. However, this was improved among subjects with CD4 count 100cells/mm(3). In combination with sputum microscopy urine, LAM could be considered for targeted TB screening in this subgroup.

Human strains of rotavirus A (RVAs) recognize fucosylated glycans belonging to histo-blood group antigens (HBGAs) through their spike protein VP8*. Lack of these ligands due to genetic polymorphisms is associated with resistance to gastroenteritis caused by P[8] genotype RVAs. With the aim to delineate the contribution of HBGAs in the process, we analyzed the glycan specificity of VP8* proteins from various P genotypes. Binding to saliva of VP8* from P[8] and P[4] genotypes required expression of both FUT2 and FUT3 enzymes, whilst binding of VP8* from the P[14] genotype required FUT2 and A enzymes. We further defined a glycan motif, GlcNAc beta 3Gal beta 4GlcNAc, recognized by P[6] clinical strains. Conversion into Lewis antigens by the FUT3 enzyme impaired recognition, explaining their lower binding to saliva of Lewis positive phenotype. In addition, the presence of neutralizing antibodies was associated with the presence of the FUT2 wild type allele in sera from young healthy adults. Nonetheless, in vitro infection of transformed cell lines was independent of HBGAs expression, indicating that HBGAs are not human RV receptors. The match between results from saliva-based binding assays and the epidemiological data indicates that the polymorphism of human HBGAs controls susceptibility to RVAs, although the exact mechanism remains unclear.

Periodontitis is a chronic inflammatory disease that is characterised by accumulation of pathogenic bacteria, including Porphyromonas gingivalis, in periodontal pockets. The lack of effective treatments has emphasised in an intense search for alternative methods to prevent bacterial colonisation and disease progression. Bacteriocins are bacterially produced antimicrobial peptides gaining increased consideration as alternatives to traditional antibiotics. We show rapid permeabilisation and aggregation of P. gingivalis by the two-peptide bacteriocin PLNC8 alpha beta. In a cell culture model, P. gingivalis was cytotoxic against gingival fibroblasts. The proteome profile of fibroblasts is severely affected by P. gingivalis, including induction of the ubiquitin-proteasome pathway. PLNC8 alpha beta enhanced the expression of growth factors and promoted cell proliferation, and suppressed proteins associated with apoptosis. PLNC8 alpha beta efficiently counteracted P. gingivalis-mediated cytotoxicity, increased expression of a large number of proteins and restored the levels of inflammatory mediators. In conclusion, we show that bacteriocin PLNC8 alpha beta displays dual effects by acting as a potent antimicrobial agent killing P. gingivalis and as a stimulatory factor promoting cell proliferation. We suggest preventive and therapeutical applications of PLNC8 alpha beta in periodontitis to supplement the host immune defence against P. gingivalis infection and support wound healing processes.

Objectives Obstructive sleep apnea (OSA) has an impact on an individuals quality of life and general health, and can also affect their oral health. The patients experiences, together with intraoral signs and symptoms could indicate the presence of OSA. Knowledge that the patient has, or is at high risk for having OSA can help the dental healthcare provider maintain the oral health and general health for these patients. The purpose was to explore dentists and dental hygienists experiences when encountering adult patients with potential, untreated and treated OSA. Methods A qualitative inductive approach was used. Experienced dentists and dental hygienists working within Swedish Public Dental Service were strategically selected. Semi-structured face-to-face interviews were performed followed by qualitative content analysis. Results Interviews from 13 participants, seven dental hygienist and six dentists, led to three areas describing varied experience: Importance of the patient encounter and identifying intraoral signs both of which describe experiences related to the importance of the initial unstructured conversation and focused clinical assessments, and strategies for nurturing care which point to interest about care, treatment, and collaborations with medical health care providers. Conclusions Dental professionals are not able to consistently recognize patients who have, or are at high risk for OSA. During the patient encounter, is it important to determine if a patient is at risk for, or has oral signs of OSA.

Aim: To characterize extended-spectrum -lactamase-producing Escherichia coli harboring the colistin resistance gene mcr-1 from human fecal samples collected in 2012 in a rural area of Shandong province, PR China. Materials amp; methods: Whole-genome sequencing and antimicrobial susceptibility testing was performed on 25 mcr-1-positive isolates to determine carriage of antibiotic resistance and virulence genes, diversity and antibiotic resistance profiles. Results: The isolates were highly genetically diverse and carried a large variety of different antibiotic resistance genes. The multidrug-resistance rate was high (96%). Virulence genes associated with intestinal pathogenic E. coli were carried by 32% of the isolates. Conclusion: Further monitoring of the epidemiological situation is necessary to ensure a preparedness for potential emergence of novel, difficult-to-treat strains and awareness of available treatment options.

Since its initial discovery in China in 2015, the plasmid-mediated colistin resistance gene mcr-1 has been reported in Escherichia coli isolated from clinical samples, animals and meat worldwide. In this study, 706 extended-spectrum beta-lactamase (ESBL)-producing E. coli from 411 persons were detected in a collection of faecal samples from 1000 rural residents in three counties in Shandong Province, China. These isolates were screened for mcr-1 and phenotypic colistin resistance. The gene was found in 3.5% of the isolates (from 4.9% of persons) from all three counties. All isolates with phenotypic colistin resistance carried mcr-1. These data indicate that commensal carriage of ESBL-producing E. coli with mcr-1 among persons in rural China was already present in 2012 and that mcr-1 was the most important colistin resistance mechanism. Interventions are necessary to minimise further dissemination of mcr-1, which would limit the future usefulness of colistin as a last-resort antibiotic. (C) 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

Vascular endothelial growth factor (VEGF) is an angiogenic factor involved in tumor growth and metastasis. Gremlin has been proposed as a novel therapeutic pathway for the treatment of renal inflammatory diseases, acting via VEGFR 2 receptor. To date, most FDA-approved tyrosine kinase (TK) inhibitors have been reported as dual inhibitors of EGFR and VEGFR 2. The aim of the present study was to find the potent and selective inhibitor of VEGFR 2 specifically for the treatment of renal cancer. Fourteen previously identified anti-inflammatory compounds i.e., 1, 3, 4 oxadiazoles derivatives by our own group were selected for their anti-cancer potential, targeting the tyrosine kinase (TK) domain of VEGFR2 and EGFR. A detailed virtual screening-based study was designed viz density functional theory (DFT) study to find the compounds stability and reactivity, molecular docking for estimating binding affinity, SeeSAR analysis and molecular dynamic simulations to confirm protein ligand complex stability and ADMET properties to find the pharmacokinetic profile of all compounds. The DFT results suggested that among all the derivatives, the 7g, 7j, and 7l were chemically reactive and stable derivatives. The optimized structures obtained from the DFTs were further selected for molecular docking, and the results suggested that 7g, 7j and 7l derivatives as the best inhibitors of VEGFR 2 with binding energy values -46.32, -48.89 and -45.01 kJ/mol. The Estimated inhibition constant (IC50) of hit compound 7j (0.009 mu M) and simulation studies of its complexes confirms its high potency and best inhibitor of VEGFR2. All the derivatives were also docked with EGFR, where they showed weak binding energies and poor interactions, important compound 7g, 7j and 7i exhibited binding energy of -31.01, -33.23 and -34.19 kJ/mol respectively. Furthermore, the anticancer potential of the derivatives was confirmed by cell viability (MTT) assay using breast cancer and cervical cancer cell lines. At the end, the results of ADMET studies confirmed these derivatives as drug like candidates. Conclusively, the current study suggested substituted oxadiazoles as the potential anticancer compounds which exhibited more selectivity towards VEGFR2 in comparison to EGFR. Therefore, the identified lead molecules can be used for the synthesis of more potent derivatives of VEGFR2, along with extensive in vitro and in vivo experiments, that can be used to treat various cancers, especially renal cancers, and to prevent angiogenesis due to aberrant expression of VEGFR2.

Introduction: Several novel vaccine platforms aim at mucosal immunity in the respiratory tract to block SARS-CoV-2 transmission. Standardized methods for mucosal sample collection and quantification of mucosal antibodies are therefore urgently needed for harmonized comparisons and interpretations across mucosal vaccine trials and real-world data. Methods: Using commercial electrochemiluminescence antibody panels, we compared SARS-CoV-2 spike-specific IgA and IgG in paired saliva, nasal secretions, and serum from 1048 healthcare workers with and without prior infection. Results: Spike-specific IgA correlated well in nasal secretions and saliva (r>0.65, p<0.0001), but the levels were more than three-fold higher in nasal secretions as compared to in saliva (p<0.01). Correlations between the total population of spike-specific IgA and spike-specific secretory IgA (SIgA) were significantly stronger (p<0.0001) in nasal secretions (r=0.96, p<0.0001) as opposed to in saliva (r=0.77, p<0.0001), and spike-specific IgA correlated stronger (p<0.0001) between serum and saliva (r=0.73, p<0.001) as opposed to between serum and nasal secretions (r=0.54, p<0.001), suggesting transudation of monomeric spike specific IgA from the circulation to saliva. Notably, spike-specific SIgA had a markedly higher SARS-CoV-2 variant cross-binding capacity as compared to the total population of spike specific IgA and IgG in both nasal secretions, saliva and serum, (all p<0.0001), which emphasizes the importance of taking potential serum derived monomeric IgA into consideration when investigating mucosal immune responses. Discussion: Taken together, although spike-specific IgA can be reliably measured in both nasal secretions and saliva, our findings imply an advantage of higher levels and likely also a larger proportion of SIgA in nasal secretions as compared to in saliva. We further corroborate the superior variant cross-binding capacity of SIgA in mucosal secretions, highlighting the potential protective benefits of a vaccine targeting the upper respiratory tract.

Response to BA.2.86 Infection after Vaccination Infection with the BA.2.86 subvariant in a vaccinated and previously infected person yielded a strong induction of mucosal IgA binding to the wild-type and BA.5 spike (factor change, 3.6 and 3.4, respectively).

Myalgic encephalomyelitis, also referred to as chronic fatigue syndrome (ME/CFS) is a debilitating disease characterized by myalgia and a sometimes severe limitation of physical activity and cognition. It is exacerbated by physical and mental activity. Its cause is unknown, but frequently starts with an infection. The eliciting infection (commonly infectious mononucleosis or an upper respiratory infection) can be more or less well diagnosed. Among the human herpesviruses (HHV-1 -8), HHV-4 (Epstein-Barr virus; EBV), HHV-6 (including HHV-6A and HHV-6B), and HHV-7, have been implicated in the pathogenesis of ME/CFS. It was therefore logical to search for serological evidence of past herpesvirus infection/reactivation in several cohorts of ME/CFS patients (all diagnosed using the Canada criteria). Control samples were from Swedish blood donors. We used whole purified virus, recombinant proteins, and synthetic peptides as antigens in a suspension multiplex immunoassay (SMIA) for immunoglobulin G (IgG). The study on herpesviral peptides based on antigenicity with human sera yielded novel epitope information. Overall, IgG anti-herpes-viral reactivities of ME/CFS patients and controls did not show significant differences. However, the high precision and internally controlled format allowed us to observe minor relative differences between antibody reactivities of some herpesviral antigens in ME/CFS versus controls. ME/CFS samples reacted somewhat differently from controls with whole virus HHV-1 antigens and recombinant EBV EBNA6 and EA antigens. We conclude that ME/CFS samples had similar levels of IgG reactivity as blood donor samples with HHV-1-7 antigens. The subtle serological differences should not be over-interpreted, but they may indicate that the immune system of some ME/CFS patients interact with the ubiquitous herpesviruses in a way different from that of healthy controls.

Apoptosis, or programmed cell death, is a controlled process by which aged or damages cells are eliminated in multicellular organisms. Neutrophils, short-lived phagocytes of the innate immune system, are highly equipped effectors that can sense, locate, ingest and kill bacterial pathogens. Inflammatory mediators and the presence of bacterial products at the foci of infection regulate the function and life span of these cells. Modulation of neutrophil apoptosis and the subsequent clearance by scavenger cells, such as macrophages, is part of a balanced inflammatory process leading to resolution of inflammation. Many pathogens are capable of modulating host cell apoptosis, and thereby influence the progression of disease. Hence, this thesis was aiming at elucidating mechanisms involved in pathogen- and host-modulated apoptosis and its contribution to the inflammatory process.

We found that different routes of bacterial entry, i.e. through invasion or by receptor-mediated phagocytosis, triggered different signaling pathways within phagocytes. Invasion of virulent Salmonella caused apoptosis, a process requiring activation of the Rho GTPases Rac1 and Cdc42. On the other hand, phagocytosis of the non-invasive Salmonella inhibited apoptosis despite similar intracellular survival as the invasive bacteria. Protection against phagocytosis-induced apoptosis was regulated by tyrosine- and PI3-kinase-dependent activation of AKT (also called PKB for protein kinase B). Furthermore, inhibiting the intraphagosomal production of reactive oxygen species (ROS) in neutrophils during phagocytosis of E. coli decreased apoptosis below spontaneous apoptosis, further indicating that both pro- and anti-apoptotic pathways are triggered by receptor-mediated phagocytosis.

Type 1 fimbria-expressing E. coli adhering to neutrophils resisted ingestion, and induced a ROS-dependent apoptosis by a cooperative effect of the FimH adhesin and LPS. To explore how compartmentalization of ROS during neutrophil activation was involved in modulating apoptosis, we evaluated the stability of lysosomes. In contrast to phagocytosis of E. coli, the adhesive strain induced intracellular non-phagosomal ROS production which triggered early permeabilization and release of lysosomal enzymes to the cytosol. Cathepsin B and/or L were responsible for targeting of the pro-apoptotic Bcl-2 protein Bid, thereby inducing mitochondrial damage, and apoptosis. These data propose a novel pathway for ROS-induced apoptosis in human neutrophils, where the location of the ROS rather than production per se is important.

Moreover, we found that pathogen-induced apoptotic neutrophils, in contrast to uninfected apoptotic neutrophils, activated blood-monocyte derived macrophages to increase their FcγRI surface expression and to produce large quantities of the pro-inflammatory cytokine TNF-α. This demonstrates that during the early phase of infection, pathogen-induced neutrophil apoptosis will help local macrophages to gain control over the microbes. Furthermore, we suggest that heat shock protein 60 and 70 represent a stress signal that enables macrophages to distinguish between, and react differently to, uninfected and inflammatory apoptotic neutrophils.

Faecal bacteria from 49 Gentoo penguins on the Antarctic Peninsula were identified by biochemical methods and sequencing, and tested for antibiotic susceptibility using agar dilution. Of the 42 Enterobacteriaceae isolates found, 39 belonged to the genus Edwardsiella. All isolates were susceptible to the 17 antibiotics tested. This implies that antibiotic selection pressure is a prerequisite to a high prevalence of antibiotic resistance, and in the absence of contact with human activities, antibiotic resistance in Enterobacteriaceae remains undetectable.

Faecal bacteria from 49 Gentoo penguins on the Antarctic Peninsula were identified by biochemical methods and sequencing, and tested for antibiotic susceptibility using agar dilution. Of the 42 Enterobacteriaceae isolates found, 39 belonged to the genus Edwardsiella. All isolates were susceptible to the 17 antibiotics tested. This implies that antibiotic selection pressure is a prerequisite to a high prevalence of antibiotic resistance, and in the absence of contact with human activities, antibiotic resistance in Enterobacteriaceae remains undetectable.

Hantaviridae is a family for negative - sense RNA viruses with genomes of about 10.5-14.6 kb. These viruses are maintained in and/or transmitted by fish, reptiles, and mammals. Several orthohantaviruses can infect humans, causing mild, severe, and sometimes - fatal diseases. Hantavirids produce enveloped virions containing three single- stranded RNA segments with open reading frames that encode a nucleoprotein (N), a glycoprotein precursor (GPC), and a large (L) protein containing an RNAdirected RNA polymerase (RdRP) domain. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Hantaviridae, which is available at ictv.global/report/hantaviridae.

Tuberculosis (TB) is an infectious disease caused by the bacterium Mycobacterium tuberculosis, and the cause of 1.5 million deaths in 2018. During a pulmonary TB infection, the bacterium reaches the lungs and is phagocytosed by cells of the innate immune system, primarily macrophages. The macrophages are either able to eradicate the bacteria or the bacteria start to replicate, and the following immune response leads to the formation of a large cluster of different cell types called a granuloma. In the granuloma the mycobacteria are contained in a latent infection, or they can start to replicate causing rupture of the granuloma and spread of the disease. Neutrophils are also innate immune cells that are rapidly recruited to the site of infection. They are phagocytes, but they also exert extracellular effector mechanisms by their release of microbicidal granule proteins, reactive oxygen species and neutrophil extracellular traps. M. tuberculosis has co-evolved and adapted to the human host making it ingenious at exploiting the human immune response, promoting its survival and replication in human host cells. The human immune system has also evolved mechanisms to limit M. tuberculosisreplication and spread. This thesis covers work on the innate immune response to TB and how neutrophils and macrophages respond to a mycobacterial infection and can control M. tuberculosis-replication.

Neutrophils and macrophages can respond to M. tuberculosis by releasing extracellular traps. We demonstrated that neutrophil extracellular traps contain the danger signal heat-shock protein 72 when induced by mycobacteria, which subsequently mediate a proinflammatory activation of adjacent macrophages. Macrophages can also release extracellular traps, and we observed the release of macrophage extracellular traps in response to M. tuberculosis that grow in cord-structures. We further demonstrated that the induction of extracellular traps also required the mycobacterial virulence factor ESAT-6.

Trained immunity is an epigenetically regulated memory of the innate immune system that results in a heightened response to a later encounter of the same or different pathogen. β-glucans are structural components of microbial cell walls and known inducers of trained immunity. We studied the effects of β-glucan from a bacterial source (curdlan from Alcaligenes faecalis), from yeast (WGP dispersible from Saccharomyces cerevisiae) and from the supernatant of a multicellular fungi (Alternaria) in search of functional changes in human macrophages which enhanced their anti-mycobacterial capacity. M. tuberculosis growth reduction was observed in WGP dispersible-trained macrophages when co-cultured with neutrophils. We also discovered that the interferon-gamma (IFNγ) signaling pathway, which is important for mycobacterial control, is hypomethylated in the WGP dispersible-trained macrophages. Since hypomethylation of genes typically is associated with gene activation, this suggests a more active IFNγ signaling in response to β-glucan innate immune training.

Most of our studies were performed using in vitro culturing of primary human macrophages and neutrophils. However, an in vitro 3D tissue model is a valuable tool when studying complex events that occur during a TB infection that involves both multiple cell types and requires knowledge of the spatial movement of cells. In this thesis we also describe an in vitro lung tissue model, which we could use to observe the clustering of monocytes around mycobacteria and quantify the size and number of macrophage clusters.

In conclusion, this thesis comprises work on innate immune functions during tuberculosis infection. We describe extracellular trap formation in macrophages and neutrophils in response to M. tuberculosis. We also explore trained immunity and how β-glucan training can enhance mycobacterial growth restriction.

Rotaviruses (RVs) are a major cause of severe diarrhoea in young children. Nicaragua introduced routine immunization with the pentavalent RV vaccine (RV5) in 2006, which greatly reduced the incidence of diarrhoea. A remaining concern has been the possible emergence of new RV strains to which the vaccination has less effect. In this study, 837 children with diarrhoea in hospital settings were investigated for RV between May 2011 and July 2013. RVs were subsequently typed by multiplex PCR and/or sequencing. Fecal anti-RV IgA titres for a subset of RV-infected (n = 137) and noninfected children (n = 52) were determined with an in-house enzyme-linked immunosorbent assay. The RV detection rate was 8% in 2011, followed by a sharp increase to 29% in 2012 and 19% in 2013. This was associated with emergence and predominance of genotype G12 RV, from 0% in 2011 to 66% in 2012 and 82% in 2013, infecting children from 1 month to 10 years of age. Two sequenced G12 strains showed a Wa-like genome with genotype G12-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1, similar to the globally emerging G12 strains. Fecal anti-RV IgA analysis showed that most G12-infected and noninfected children had been in contact with either vaccine or wild RV strains, but such antibodies did not prevent symptomatic G12 infection. A marked increase of RV was evident in the hospital setting associated with a nationwide emergence and predominance of RV G12 genotype in a population with high RV5 vaccine coverage.

Host genetic resistance to Norovirus (NoV) has been observed in challenge and outbreak studies in populations from Europe, Asia, and USA. In this study, we have investigated if histo-blood group antigens can predict susceptibility to diarrhea caused by NoV in Nicaragua, Central America, and if this can be reflected in antibody-prevalence and titer to NoV among individuals with different histo-blood group antigen phenotypes. Investigation of 28 individuals infected with NoV and 131 population controls revealed 6% of non-secretors in the population and nil non-secretors among patients infected with NoV, suggesting that non-secretors may be protected against NoV disease in Nicaragua. Surprisingly, 25% of the population was Lewis negative (Le(a-b-)). NoV infections with genogroup I (GI) and GII occurred irrespective of Lewis genotype, but none of the Lewis a positive (Le(a + b-)) were infected. The globally dominating GII.4 virus infected individuals of all blood groups except AB (n = 5), while the GI viruses (n = 4) infected only blood type O individuals. Furthermore, O blood types were susceptible to infections with GI.4, GII.4, GII.7, GII.17, and GII.18-Nica viruses, suggesting that secretors with blood type O are susceptible (OR = 1.52) and non-secretors resistant. The overall antibody-prevalence to NoV GII.3 VLP was 62% with the highest prevalence among blood type B carriers (70%) followed by A (68%) and O (62%). All four investigated individuals carrying blood type AB were antibody-negative. Among secretors, 63% were antibody-positive compared to 33% among non-secretors (P = 0.151). This study extends previous knowledge about the histo-blood group antigens role in NoV disease in a population with different genetic background than North American and European.

Recent studies have investigated whether the human histo-blood group antigen (HBGAs) could affect the effectiveness of the oral rotavirus vaccines, suggesting secretor positive individuals develop a more robust response. We investigated the Rotavirus A (RVA) shedding in association with the host susceptibility profile in children from a birth community-cohort in Rio de Janeiro, Brazil, from 2014 to 2018. A total of 132 children were followed-up between 0 to 11-month-old, stool samples were collected before/after the 1(st)/2(nd) RV1 vaccination doses and saliva samples were collected during the study. RVA shedding was screened by RT-qPCR and G/P genotypes determined by multiplex RT-PCR and/or Sanger nucleotide sequencing. The sequencing indicated an F167L amino acid change in the RV1 VP8* P[8] in 20.5% of shedding follow-ups and these mutant subpopulations were quantified by pyrosequencing. The HBGA/secretor status was determined and 80.3% of the children were secretors. Twenty-one FUT2 gene SNPs were identified and two new mutations were observed. The mutant F167L RV1 VP8* P[8] was detected significantly more in Le (a+b+) secretors (90.5%) compared to non-secretors and even to secretors Le (a-b+) (9.5%). The study highlights the probable association between RV1 shedding and HBGAs as a marker for evaluating vaccine strain host susceptibility.

Eosinophils have been linked to functional dyspepsia; however, less is known about their role in irritable bowel syndrome (IBS). This study tested the hypothesis of alterations in levels of fecal eosinophil-derived neurotoxin (F-EDN) and eosinophil density and degranulation within the colonic mucosa of IBS patients compared with healthy controls (HC). Colonic biopsies were collected from 37 IBS patients and 20 HC and analyzed for eosinophil numbers and local degranulation of eosinophil cationic protein (ECP) by histologic procedures. Fecal samples were collected for F-EDN and microbiota analysis. Differentiated 15HL-60 cells were used in vitro to investigate the direct effect of live bacteria on eosinophil activation measured by a colorimetric assay with o-phenylenediamine (OPD) substrate. We observed a higher number of eosinophils and increased extracellular ECP in the mucosa of IBS patients compared with HC. Moreover, F-EDN levels in IBS samples were elevated compared with HC and positively correlated to extracellular ECP. Metagenomic analysis showed significant correlations between bacterial composition and eosinophil measurements in both HC and IBS patients. In vitro experiments revealed an increased degranulation of 15HL-60 after stimulation with Salmonella typhimurium, Salmonella enterica, and Yersinia enterocolitica. To conclude, we could demonstrate alterations related to eosinophils in IBS, and, for the first time, a positive correlation between F-EDN levels and degranulated eosinophils in the colonic mucosa of IBS patients. Together our results suggest that eosinophils play a role in the pathophysiology of IBS and the mechanisms might be linked to an altered microbiota.

A method was developed and validated for the detection of colistin-resistant Escherichia coli containing mcr-1 in the feces of feral swine. Following optimization of an enrichment method using EC broth supplemented with colistin (1 mu g/mL) and vancomycin (8 mu g/mL), aliquots derived from 100 feral swine fecal samples were spiked with of one of five different mcr-1 positive E. coli strains (between 10(0) and 10(4) CFU/g), for a total of 1110 samples tested. Enrichments were then screened using a simple boil-prep and a previously developed real-time PCR assay for mcr-1 detection. The sensitivity of the method was determined in swine feces, with mcr-1 E. coli inocula of 0.1-9.99 CFU/g (n = 340), 10-49.99 CFU/g (n = 170), 50-99 CFU/g (n = 255), 100-149 CFU/g (n = 60), and 200-2200 CFU/g (n = 175), which were detected with 32%, 72%, 88%, 95%, and 98% accuracy, respectively. Uninoculated controls (n = 100) were negative for mcr-1 following enrichment.

Background While a few case-control studies indicated a possible correlation of IgG N-glycosylation patterns with pancreatitis, their restricted sample sizes and methodologies prevented conclusive insights into causality or distinguishing traits across pancreatitis types.Method We conducted a two-sample Mendelian Randomization (MR) analysis to investigate the causal relationship between 77 IgG N-glycosylation traits and various types of pancreatitis, including acute pancreatitis (AP), chronic pancreatitis (CP), alcohol acute pancreatitis (AAP), and alcohol chronic pancreatitis (ACP). This analysis utilized summary-level data from genome-wide association studies (GWAS), employing methods such as IVW, MR-Egger, and weighted median. To ensure the robustness of our findings, several sensitivity analyses, including Cochran's Q statistic, leave-one-out, MR-Egger intercept, and MR-PRESSO global test were conducted.Result Our study uncovered the causal relationship between specific IgG N-glycosylation traits and various types of pancreatitis. Notably, an increase in genetically predicted IGP7 levels was associated with a decreased risk of developing AP. For CP, our data suggested a protective effect associated with higher levels of both IGP7 and IGP31, contrasting with increased levels of IGP27 and IGP65, which were linked to a heightened risk. Moreover, in the case of AAP, elevated IGP31 levels were causatively associated with a lower incidence, while higher IGP26 levels correlated with an increased risk for ACP.Conclusion This study establishes causal relationship between specific IgG N-glycosylation patterns and varying risks of different pancreatitis forms, underscoring their potential as predictive biomarkers. These findings necessitate further exploration into the underlying mechanisms, promising to inform more personalized diagnostic and therapeutic strategies in pancreatitis management.

In this thesis, we have studied the immunological processes of joint inflammation that may be targets for future treatment of patients with arthritis. We focus on the immune-modulating properties of interferon-α (IFN-α) and uridine in experimental arthritis. The nucleoside uridine, which is regarded a safe treatment has anti-inflammatory properties notably by inhibiting tumor necrosis factor (TNF) release. Because the inflamed synovium in rheumatoid arthritis (RA) is characterised by pathogenic TNF-production, uridine could potentially be away to ameliorate arthritis. Systemic administration of uridine had no effect on antigeninduced arthritis (AIA), which is a T-cell dependent model where animals are immunized twice (sensitization) with bovine serum albumin (mBSA), before local triggering of arthritis by intra-articular antigen (mBSA) re-challenge. In contrast, intra-articular administration of uridine clearly down modulated development of AIA in a dose dependent manner and inhibited the expression of synovial adhesion molecules, influx of inflammatory leukocytes and synovial expression of TNF and interleukin 6, but did not affect systemic levels of proinflammatory cytokines or antigen-specific T-cell responses. Local administration of uridine may thus be a viable therapeutic option for treatment of arthritis in the future.

Viral double-stranded deoxyribonucleic acid (dsRNA), a common nucleic acid found in most viruses, can be found in the joints of RA patients and local deposition of such viral dsRNA induces arthritis by activating IFN-α. Here we show that arthritis induced by dsRNA can be mediated by IFN-producing dendritic cells in the joint and this may thus explain why viral infections are sometimes associated with arthritis.

Earlier, to study the effect of dsRNA and IFN-α in an arthritis model, that like RA, is dependent on adaptive immunity, dsRNA and IFN-α were administered individually during the development of AIA. Both molecules clearly protected against AIA in a type I IFN receptor-dependent manner but were only effective if administered in the sensitization phase of AIA. Here we show that the anti-inflammatory effect of IFN-α is critically dependent on signalling via transforming growth factor β (TGF-β) and the enzymatic activity of indoleamine 2,3 dioxygenase 1 (IDO). The IDO enzyme is produced by plasmacytoid DC and this cell type was critically required both during antigen sensitization and in the arthritis phase of AIA for the protective effect of IFN-α against AIA. In contrast, TGF-β and the enzymatic activity of IDO were only required during sensitization, which indicate that they are involved in initial steps of tolerogenic antigen sensitization. In this scenario, IFN- α first activates the enzymatic activity of IDO in pDC, which converts Tryptophan to Kynurenine, which thereafter activates TGF-β. Common for IDO-expressing pDC, Kyn and TGF-β is their ability to induce development of regulatory T cells (Tregs). We found that Tregs were crucial for IFN-α-mediated protection against AIA, but only in the arthritis phase. In line with this, adoptive transfer of Tregs isolated from IFN-α treated mice to recipient animals in the arthritis phase clearly protected against AIA. The numbers of Tregs were not significantly altered by IFN-α but IFN-α increased the suppressive capacity of Tregs against antigen-induced proliferation. This enhanced suppressive activity of Tregs in the arthritis phase was dependent on the earlier activated enzyme IDO1 during the sensitization phase of AIA. Thus, presence of IFN-α at the time of antigen sensitization activates the enzymatic activity of IDO, which generates Tregs with enhanced suppressive capacity that upon antigen re-challenge prevents inflammation. We have thus identified one example of how immune tolerance can be developed, that may be a future way to combat autoimmunity.

Klebsiella pneumoniae is a gram-negative, opportunistic pathogen, and a common cause of healthcare-associated infections such as pneumonia, septicemia, and urinary tract infection. The purpose of this study was to survey the occurrence of and characterize K. pneumoniae in different environmental sources in a rural area of Shandong province, China. Two hundred and thirty-one samples from different environmental sources in 12 villages were screened for extended-spectrum beta-lactamase-(ESBL)-producing K. pneumoniae, and 14 (6%) samples were positive. All isolates were multidrug-resistant and a few of them belonged to clinically relevant strains which are known to cause hospital outbreaks worldwide. Serotypes, virulence genes, serum survival, and phagocytosis survival were analyzed and the results showed the presence of virulence factors associated with highly virulent clones and a high degree of phagocytosis survivability, indicating the potential virulence of these isolates. These results emphasize the need for further studies designed to elucidate the role of the environment in transmission and dissemination of ESBL-producing K. pneumoniae and the potential risk posed to human and environmental health.

During an expedition to the Southern Argentinean town of Ushuaia, the Antarctic Peninsula, Antarctic Islands and the Falkland Islands, we collected 94 faecal specimens from wild birds to screen for yeast within the different bird species. The yeast species were identified by morphological features and commercial characterisation kits. From 54% of the specimens, we isolated 122 strains representing 29 yeast species. Debaryomyces hansenii, Candida lambica and Candida krusei were the most frequently isolated species. We found a plethora of yeasts in birds living in proximity to humans, whereas birds living in more remote areas were colonised with a lower number of fungal species.

Following the outbreak of novel severe acute respiratory syndrome (SARS)-coronavirus (CoV)2, the majority of nations are struggling with countermeasures to fight infection, prevent spread and improve patient survival. Considering that the pandemic is a recent event, no large clinical trials have been possible and since coronavirus specific drug are not yet available, there is no strong consensus on how to treat the coronavirus disease 2019 (COVID-19) associated viral pneumonia. Coronaviruses code for an important multifunctional enzyme named papain-like protease (PLP), that has many roles in pathogenesis. First, PLP is one of the two viral cysteine proteases, along with 3-chymotripsin-like protease, that is responsible for the production of the replicase proteins required for viral replication. Second, its intrinsic deubiquitinating and deISGylating activities serve to antagonize the hosts immune response that would otherwise hinder infection. Both deubiquitinating and deISGylating functions involve the removal of the small regulatory polypeptides, ubiquitin and ISG15, respectively, from target proteins. Ubiquitin modifications can regulate the innate immune response by affecting regulatory proteins, either by altering their stability via the ubiquitin proteasome pathway or by directly regulating their activity. ISG15 is a ubiquitin-like modifier with pleiotropic effects, typically expressed during the host cell immune response. PLP inhibitors have been evaluated during past coronavirus epidemics, and have showed promising results as an antiviral therapy in vitro. In this review, we recapitulate the roles of PLPs in coronavirus infections, report a list of PLP inhibitors and suggest possible therapeutic strategies for COVID-19 treatment, using both clinical and preclinical drugs.

Herpes virus type 2 (HSV2) is one of the most common sexually transmitted infections globally with a very high prevalence in many countries. During HSV2 infection viral particles become coated with complement proteins and antibodies, both existent in the genital fluids, which could influence the activation of the immune responses. In genital mucosa, the primary target cells for HSV2 infection are epithelial cells, but resident immune cells such as dendritic cells (DCs) are also infected. The DCs are the activators of the ensuing immune responses directed against HSV2, and the aim of this study was to examine the effects opsonization of HSV2, either with complement alone or with complement and antibodies, had on the infection of immature DCs and their ability to mount inflammatory and antiviral responses. Complement opsonization of HSV2 enhanced both the direct infection of immature DCs and their production of new infectious viral particles. The enhanced infection required activation of the complement cascade and functional complement receptor 3. Furthermore, HSV2 infection of DCs required endocytosis of viral particles and their delivery into an acid endosomal compartment. The presence of complement in combination with HSV1 or HSV2 specific antibodies more or less abolished the HSV2 infection of DCs.Our results clearly demonstrate the importance of studying HSV2 infection under conditions that ensue in vivo, i.e. when the virions are covered in complement fragments and complement fragments and antibodies, as this will shape the infection and the subsequent immune response and needs to be further elucidated.

IMPORTANCE: During HSV2 infection viral particles should become coated with complement proteins and antibodies, both existent in the genital fluids, which could influence the activation of the immune responses. The dendritic cells are the activators of the immune responses directed against HSV2, and the aim of this study was to examine the effects of complement alone or complement and antibodies, on the HSV2 infection of dendritic cells and their ability to mount inflammatory and antiviral responses.Our results demonstrate that the presence of antibodies and complement in the genital environment can influence HSV2 infection under in vitro conditions that reflect the in vivo situation. We believe that our findings are highly relevant for the understanding of HSV2 pathogenesis.

Genital herpes is a common sexually transmitted infection caused by herpes simplex virus type 2 (HSV-2). Genital herpes significantly enhances the acquisition and transmission of HIV-1 by creating a microenvironment that supports HIV infection in the host. Dendritic cells (DCs) represent one of the first innate cell types that encounter HIV-1 and HSV-2 in the genital mucosa. HSV-2 infection has been shown to modulate DCs, rendering them more receptive to HIV infection. Here, we investigated the potential mechanisms underlying HSV-2-mediated augmentation of HIV-1 infection. We demonstrated that the presence of HSV-2 enhanced productive HIV-1 infection of DCs and boosted inflammatory and antiviral responses. The HSV-2 augmented HIV-1 infection required intact HSV-2 DNA, but not active HSV-2 DNA replication. Furthermore, the augmented HIV infection of DCs involved the cGAS-STING pathway. Interestingly, we could not see any involvement of TLR2 or TLR3 nor suppression of infection by IFN-beta production. The conditioning by HSV-2 in dual exposed DCs decreased protein expression of IFI16, cGAS, STING, and TBK1, which is associated with signaling through the STING pathway. Dual exposure to HSV-2 and HIV-1 gave decreased levels of several HIV-1 restriction factors, especially SAMHD1, TREX1, and APOBEC3G. Activation of the STING pathway in DCs by exposure to both HSV-2 and HIV-1 most likely led to the proteolytic degradation of the HIV-1 restriction factors SAMHD1, TREX1, and APOBEC3G, which should release their normal restriction of HIV infection in DCs. This released their normal restriction of HIV infection in DCs. We showed that HSV-2 reprogramming of cellular signaling pathways and protein expression levels in the DCs provided a setting where HIV-1 can establish a higher productive infection in the DCs. In conclusion, HSV-2 reprogramming opens up DCs for HIV-1 infection and creates a microenvironment favoring HIV-1 transmission.

Mycobacterium tuberculosis (M.tb), the causative agent of Tuberculosis, is an intracellular bacterium well known for its ability to subvert host energy and metabolic pathways to maintain its intracellular survival. For this purpose, the bacteria utilize various mechanisms of which extracellular vehicles (EVs) related mechanisms attracted more attention. EVs are nanosized particles that are released by almost all cell types containing active biomolecules from the cell of origin and can target bioactive pathways in the recipient cells upon uptake. It is hypothesized that M.tb dictates the processes of host EV biogenesis pathways, selectively incorporating its molecules into the host EV to direct immune responses in its favor. During infection with Mtb, both mycobacteria and host cells release EVs. The composition of these EVs varies over time, influenced by the physiological and nutritional state of the host environment. Additionally, different EV populations contribute differently to the pathogenesis of disease at various stages of illness participating in a complex interplay between host cells and pathogens. These interactions ultimately influence immune responses and disease outcomes. However, the precise mechanisms and roles of EVs in pathogenicity and disease outcomes remain to be fully elucidated. In this review, we explored the properties and function of EVs in the context of M.tb infection within the host microenvironment and discussed their capacity as a novel therapeutic strategy to combat tuberculosis.

Sapovirus is an important etiological agent of acute gastroenteritis (AGE), mainly in children under 5 years old living in lower-income communities. Eighteen identified sapovirus genotypes have been observed to infect humans. The aim of this study was to identify sapovirus genotypes circulating in the Amazon region. Twenty-eight samples were successfully genotyped using partial sequencing of the capsid gene. The genotypes identified were GI.1 (n = 3), GI.2 (n = 7), GII.1 (n = 1), GII.2 (n = 1), GII.3 (n = 5), GII.5 (n = 1), and GIV.1 (n = 10). The GIV genotype was the most detected genotype (35.7%, 10/28). The phylogenetic analysis identified sapovirus genotypes that had no similarity with other strains reported from Brazil, indicating that these genotypes may have entered the Amazon region via intense tourism in the Amazon rainforest. No association between histo-blood group antigen expression and sapovirus infection was observed.

Recent studies show that rectal colonization with low-level ciprofloxacin-resistant Escherichia coli (ciprofloxacin minimal inhibitory concentration (MIC) above the epidemiological cutoff point, but below the clinical breakpoint for resistance), i.e., in the range amp;gt; 0.06-0.5 mg/L is an independent risk factor for febrile urinary tract infection after transrectal ultrasound-guided biopsy (TRUS-B) of the prostate, adding to the other risk posed by established ciprofloxacin resistance in E. coli (MIC amp;gt; 0.5 mg/L) as currently defined. We aimed to identify the quinolone that by disk diffusion best discriminates phenotypic wild-type isolates (ciprofloxacin MIC amp;lt;= 0.06 mg/L) of E. coli from isolates with acquired resistance, and to determine the resistance genotype of each isolate. The susceptibility of 108 E. coli isolates was evaluated by ciprofloxacin, levofloxacin, moxifloxacin, nalidixic acid, and pefloxacin disk diffusion and correlated to ciprofloxacin MIC (broth microdilution) using EUCAST methodology. Genotypic resistance was identified by PCR and DNA sequencing. The specificity was 100% for all quinolone disks. Sensitivity varied substantially, as follows: ciprofloxacin 59%, levofloxacin 46%, moxifloxacin 59%, nalidixic acid 97%, and pefloxacin 97%. We suggest that in situations where low-level quinolone resistance might be of importance, such as when screening for quinolone resistance in fecal samples pre-TRUS-B, a pefloxacin (S amp;gt;= 24 mm) or nalidixic acid (S amp;gt;= 19 mm) disk, or a combination of the two, should be used. In a setting where plasmid-mediated resistance is prevalent, pefloxacin might perform better than nalidixic acid.