Inhibitory CD161 receptor identified in glioma-infiltrating T cells by single-cell analysis.

T cells are critical effectors of cancer immunotherapies, but little is known about their gene expression programs in diffuse gliomas. Here, we leverage single-cell RNA sequencing (RNA-seq) to chart the gene expression and clonal landscape of tumor-infiltrating T cells across 31 patients with isocitrate dehydrogenase (IDH) wild-type glioblastoma and IDH mutant glioma. We identify potential effectors of anti-tumor immunity in subsets of T cells that co-express cytotoxic programs and several natural killer (NK) cell genes. Analysis of clonally expanded tumor-infiltrating T cells further identifies the NK gene KLRB1 (encoding CD161) as a candidate inhibitory receptor. Accordingly, genetic inactivation of KLRB1 or antibody-mediated CD161 blockade enhances T cell-mediated killing of glioma cells in vitro and their anti-tumor function in vivo. KLRB1 and its associated transcriptional program are also expressed by substantial T cell populations in other human cancers. Our work provides an atlas of T cells in gliomas and highlights CD161 and other NK cell receptors as immunotherapy targets.

An Integrative Model of Cellular States, Plasticity, and Genetics for Glioblastoma.

Diverse genetic, epigenetic, and developmental programs drive glioblastoma, an incurable and poorly understood tumor, but their precise characterization remains challenging. Here, we use an integrative approach spanning single-cell RNA-sequencing of 28 tumors, bulk genetic and expression analysis of 401 specimens from the The Cancer Genome Atlas (TCGA), functional approaches, and single-cell lineage tracing to derive a unified model of cellular states and genetic diversity in glioblastoma. We find that malignant cells in glioblastoma exist in four main cellular states that recapitulate distinct neural cell types, are influenced by the tumor microenvironment, and exhibit plasticity. The relative frequency of cells in each state varies between glioblastoma samples and is influenced by copy number amplifications of the CDK4, EGFR, and PDGFRA loci and by mutations in the NF1 locus, which each favor a defined state. Our work provides a blueprint for glioblastoma, integrating the malignant cell programs, their plasticity, and their modulation by genetic drivers.

Cohesin Loss Eliminates All Loop Domains.

The human genome folds to create thousands of intervals, called "contact domains," that exhibit enhanced contact frequency within themselves. "Loop domains" form because of tethering between two loci-almost always bound by CTCF and cohesin-lying on the same chromosome. "Compartment domains" form when genomic intervals with similar histone marks co-segregate. Here, we explore the effects of degrading cohesin. All loop domains are eliminated, but neither compartment domains nor histone marks are affected. Loss of loop domains does not lead to widespread ectopic gene activation but does affect a significant minority of active genes. In particular, cohesin loss causes superenhancers to co-localize, forming hundreds of links within and across chromosomes and affecting the regulation of nearby genes. We then restore cohesin and monitor the re-formation of each loop. Although re-formation rates vary greatly, many megabase-sized loops recovered in under an hour, consistent with a model where loop extrusion is rapid.

Losartan controls immune checkpoint blocker-induced edema and improves survival in glioblastoma mouse models.

Immune checkpoint blockers (ICBs) have failed in all phase III glioblastoma trials. Here, we found that ICBs induce cerebral edema in some patients and mice with glioblastoma. Through single-cell RNA sequencing, intravital imaging, and CD8+ T cell blocking studies in mice, we demonstrated that this edema results from an inflammatory response following antiprogrammed death 1 (PD1) antibody treatment that disrupts the blood-tumor barrier. Used in lieu of immunosuppressive corticosteroids, the angiotensin receptor blocker losartan prevented this ICB-induced edema and reprogrammed the tumor microenvironment, curing 20% of mice which increased to 40% in combination with standard of care treatment. Using a bihemispheric tumor model, we identified a "hot" tumor immune signature prior to losartan+anti-PD1 therapy that predicted long-term survival. Our findings provide the rationale and associated biomarkers to test losartan with ICBs in glioblastoma patients.

Aspirin 15cH has Different Effects on Morphology and Function of Lipopolysaccharide-Challenged RAW 264.7 Macrophages In Vitro Compared to a Pharmacological Dose of Aspirin.

INTRODUCTION: Aspirin is one of the most commonly used drugs worldwide. It is known to present antipyretic, anti-inflammatory and anti-thrombotic actions, making it extremely useful in a wide range of clinical contexts. Interestingly, homeopathically prepared Aspirin 15cH has been found to have a pro-thrombotic effect in rats, raising the hypothesis that Aspirin 15cH could also modulate the activity of inflammatory cells in different pathological processes. OBJECTIVE: Our objective was to assess what effect Aspirin 15cH has on RAW 264.7 macrophages in vitro. METHODS: The effects of Aspirin 15cH on biochemical and morphological activities of lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages were evaluated. These effects were compared with unchallenged macrophages (negative control), untreated LPS-stimulated macrophages, macrophages treated with succussed water (vehicle control), or aspirin 200 µg/mL (pharmacological inhibitor of LPS activity). Cell morphology (adhered cell area and cytoskeleton arrangements), cell viability, toll-like receptor-4 (TLR-4) expression, and the production of nitric oxide, cytokines and intracellular reactive oxygen species were assessed. RESULTS: Aspirin 15cH reduced the number of cells expressing TLR-4 on the surface (p = 0.03) and induced a "columnar" morphology of macrophage pseudopods, indicating changes in cytoskeleton arrangement. When cells were treated with both Aspirin 15cH and LPS, cell morphology became heterogeneous, suggesting that sub-populations of cells had differing sensitivities to LPS or Aspirin 15cH. Exposure of the cells to LPS alone, succussed water or aspirin 200 µg/mL produced effects consistent with the literature. CONCLUSION: Aspirin 15cH, aspirin 200 µg/mL, LPS and succussed water appear to act as independent stimuli able to induce different patterns of macrophage response. Aspirin 15cH induced changes suggestive of M2 polarization of the macrophages (i.e., toward a wound healing or tissue repair, rather than inflammatory, phenotype). These preliminary findings need to be confirmed in further specific studies.

LincRNA-Cox2 Regulates Smoke-induced Inflammation in Murine Macrophages.

Cigarette smoke (CS) exposure is a risk factor for many chronic diseases, including chronic obstructive pulmonary disease, but the mechanism by which smoke exposure can alter homeostasis and bring about chronic inflammation is poorly understood. Here, we showcase a novel role for smoke in regulating long noncoding RNAs, showing that it activates lincRNA-Cox2, which we previously characterized as functional in inflammatory regulation. Exposing lincRNA-Cox2 murine models to smoke in vivo confirmed lincRNA-Cox2 as a regulator of inflammatory gene expression in response to smoke both systemically and within the lung. We also report that lincRNA-Cox2 negatively regulates genes in smoked bone marrow-derived macrophages exposed to LPS stimulation. In addition to the effects on long noncoding RNAs, we also report dysregulated transcription and splicing of inflammatory protein-coding genes in the bone marrow niche after CS exposure in vivo. Collectively, this work provides insights into how innate immune signaling from gene expression to splicing is altered after in vivo exposure to CS and highlights an important new role for lincRNA-Cox2 in regulating immune genes after smoke exposure.

BID expression determines the apoptotic fate of cancer cells after abrogation of the spindle assembly checkpoint by AURKB or TTK inhibitors.

BACKGROUND: Drugs targeting the spindle assembly checkpoint (SAC), such as inhibitors of Aurora kinase B (AURKB) and dual specific protein kinase TTK, are in different stages of clinical development. However, cell response to SAC abrogation is poorly understood and there are no markers for patient selection. METHODS: A panel of 53 tumor cell lines of different origins was used. The effects of drugs were analyzed by MTT and flow cytometry. Copy number status was determined by FISH and Q-PCR; mRNA expression by nCounter and RT-Q-PCR and protein expression by Western blotting. CRISPR-Cas9 technology was used for gene knock-out (KO) and a doxycycline-inducible pTRIPZ vector for ectopic expression. Finally, in vivo experiments were performed by implanting cultured cells or fragments of tumors into immunodeficient mice. RESULTS: Tumor cells and patient-derived xenografts (PDXs) sensitive to AURKB and TTK inhibitors consistently showed high expression levels of BH3-interacting domain death agonist (BID), while cell lines and PDXs with low BID were uniformly resistant. Gene silencing rendered BID-overexpressing cells insensitive to SAC abrogation while ectopic BID expression in BID-low cells significantly increased sensitivity. SAC abrogation induced activation of CASP-2, leading to cleavage of CASP-3 and extensive cell death only in presence of high levels of BID. Finally, a prevalence study revealed high BID mRNA in 6% of human solid tumors. CONCLUSIONS: The fate of tumor cells after SAC abrogation is driven by an AURKB/ CASP-2 signaling mechanism, regulated by BID levels. Our results pave the way to clinically explore SAC-targeting drugs in tumors with high BID expression.

MYO1F Regulates IgE and MRGPRX2-Dependent Mast Cell Exocytosis.

The activation and degranulation of mast cells is critical in the pathogenesis of allergic inflammation and modulation of inflammation. Recently, we demonstrated that the unconventional long-tailed myosin, MYO1F, localizes with cortical F-actin and mediates adhesion and migration of mast cells. In this study, we show that knockdown of MYO1F by short hairpin RNA reduces human mast cell degranulation induced by both IgE crosslinking and by stimulation of the Mas-related G protein-coupled receptor X2 (MRGPRX2), which has been associated with allergic and pseudoallergic drug reactions, respectively. Defective degranulation was accompanied by a reduced reassembly of the cortical actin ring after activation but reversed by inhibition of actin polymerization. Our data show that MYO1F is required for full Cdc42 GTPase activation, a critical step in exocytosis. Furthermore, MYO1F knockdown resulted in less granule localization in the cell membrane and fewer fissioned mitochondria along with deficient mitochondria translocation to exocytic sites. Consistent with that, AKT and DRP1 phosphorylation are diminished in MYO1F knockdown cells. Altogether, our data point to MYO1F as an important regulator of mast cell degranulation by contributing to the dynamics of the cortical actin ring and the distribution of both the secretory granules and mitochondria.

Increased susceptibility to encephalitozoonosis associated with mixed Th1/Th2 profile and M1/M2 profile in mice immunosuppressed with cyclophosphamide.

Encephalitozoon cuniculi is a unicellular, spore-forming, obligate intracellular eukaryote belonging to the phylum Microsporidia. It is known to infect mainly immunocompromised and immunocompetent mammals, including humans. The parasite-host relationship has been evaluated using both in vitro cell culturing and animal models. For example, Balb/c and C57BL/6 mouse strains have been used interchangeably, although the latter has been considered more susceptible due to the higher fungal load observed after infection. In the present study, we identified the characteristics of the immune response of C57BL/6 mice treated or not with the immunosuppressant cyclophosphamide (Cy) and challenged with E. cuniculi by intraperitoneal route. After 14 days of infection, serum was collected to analyze Th1, Th2, and Th17 cytokine levels. In addition, peritoneal washes were performed, and the spleen sample was collected for immune cell phenotyping, whereas liver, spleen, kidney, lung, intestine, and central nervous system (CNS) samples were collected for histopathological analysis. Although infected mice displayed a reduced absolute number of macrophages, they showed an M1 profile, an elevated number of CD4+T, CD8+T, B-1, and B-2 lymphocytes, with a predominance of Th1 inflammatory cytokines (interferon [IFN]-γ, tumor necrosis factor [TNF]-α, and interleukin [IL]-2) and Th17. Furthermore, Cy-Infected mice showed a reduced absolute number of macrophages with an M1 profile but a reduced number of CD4+T, CD8+T, B-1, and B-2 lymphocytes, with a predominance of Th1 inflammatory cytokines (IFN-γ, TNF-α, and IL-2) and Th2 (IL-4). This group displayed a higher fungal burden as well and developed more severe encephalitozoonosis, which was associated with a reduced number of T and B lymphocytes and a mixed profile of Th1 and Th2 cytokines.

PED in 2021: a major update of the protein ensemble database for intrinsically disordered proteins.

The Protein Ensemble Database (PED) (https://proteinensemble.org), which holds structural ensembles of intrinsically disordered proteins (IDPs), has been significantly updated and upgraded since its last release in 2016. The new version, PED 4.0, has been completely redesigned and reimplemented with cutting-edge technology and now holds about six times more data (162 versus 24 entries and 242 versus 60 structural ensembles) and a broader representation of state of the art ensemble generation methods than the previous version. The database has a completely renewed graphical interface with an interactive feature viewer for region-based annotations, and provides a series of descriptors of the qualitative and quantitative properties of the ensembles. High quality of the data is guaranteed by a new submission process, which combines both automatic and manual evaluation steps. A team of biocurators integrate structured metadata describing the ensemble generation methodology, experimental constraints and conditions. A new search engine allows the user to build advanced queries and search all entry fields including cross-references to IDP-related resources such as DisProt, MobiDB, BMRB and SASBDB. We expect that the renewed PED will be useful for researchers interested in the atomic-level understanding of IDP function, and promote the rational, structure-based design of IDP-targeting drugs.

MITF Downregulation Induces Death in Human Mast Cell Leukemia Cells and Impairs IgE-Dependent Degranulation.

Activating mutations in KIT (CD117) have been associated with several diseases, including gastrointestinal stromal tumors and mastocytosis. Rapidly progressing pathologies or drug resistance highlight the need for alternative treatment strategies. Previously, we reported that the adaptor molecule SH3 binding protein 2 (SH3BP2 or 3BP2) regulates KIT expression at the transcriptional level and microphthalmia-associated transcription factor (MITF) expression at the post-transcriptional level in human mast cells and gastrointestinal stromal tumor (GIST) cell lines. Lately, we have found that the SH3BP2 pathway regulates MITF through miR-1246 and miR-5100 in GIST. In this study, miR-1246 and miR-5100 were validated by qPCR in the SH3BP2-silenced human mast cell leukemia cell line (HMC-1). MiRNA overexpression reduces MITF and MITF-dependent target expression in HMC-1. The same pattern was observed after MITF silencing. In addition, MITF inhibitor ML329 treatment reduces MITF expression and affects the viability and cell cycle progression in HMC-1. We also examine whether MITF downregulation affected IgE-dependent mast cell degranulation. MiRNA overexpression, MITF silencing, and ML329 treatment reduced IgE-dependent degranulation in LAD2- and CD34+-derived mast cells. These findings suggest MITF may be a potential therapeutic target for allergic reactions and deregulated KIT mast-cell-mediated disorders.

Surface-enhanced Raman spectroscopy and ultrastructural analysis of penicillin-producing Penicillium rubens strains.

Raman spectroscopy, transmission electron microscopy (TEM) and atomic force microscopy (AFM) techniques can perform chemical analyses and acquire high-resolution images of cell samples. For this reason, in this study, semi-thin sections of a single Penicillium rubens cell were analysed by Raman enhanced surface spectroscopy. The spectra showed peaks corresponding to the macromolecules that make up the cellular components. In addition, the various organelles were analysed by TEM and AFM to observe the cellular nanostructures. With the use of these techniques, it is possible to identify molecules in semi-thin sections, which provides a wide potential for biomedical applications and for the analysis of cell dynamics. The observation of the most detailed possible structure of cells is used as a starting point in numerous studies to identify and localise some biochemical processes. Given that the function of eukaryotic cells depends on the location, shape, structure and function of the subcellular organelles (and on the interaction between them), the sum of the data obtained allows a complete analysis of what happens in the cell. This article addresses, from a multidisciplinary point of view, what happens in a single cell of a filamentous fungus (Penicillium rubens) while it is in a physiological moment (secondary metabolism) that allows the biosynthesis of an antibiotic (penicillin). For this purpose, different types of microscopies were used (TEM: transmission electron microscopy, and AFM: atomic force microscopy, which allow visualising small details in the cell) and a spectroscopy method (Raman, which allows detecting certain characteristics of the macromolecules and some stretching bonds). Regarding the results, during the synthesis of penicillin, the antibiotic-producing Penicillium rubens cells showed significant changes compared to the non-producing cells: the cell wall is observed to be significantly thickened in the production phase, organelles such as peroxisomes grow in number and size since it is known that the final route of metabolite synthesis takes place in them. When penicillin is released from peroxisomes, they must be degraded to release the load from the cell; this is done by vacuoles, which are active and engulf peroxisomes. The newly synthesised penicillin is found within secretory vesicles that travel towards the cell membrane and both membranes fuse creating ripples. On the other hand, and given that a single cell is being studied, it is essential to increase the signal to detect biomolecules employing the Raman-SERS technique, using a silver substrate to obtain the increased signal.

Hepcidin Is Essential for Alveolar Macrophage Function and Is Disrupted by Smoke in a Murine Chronic Obstructive Pulmonary Disease Model.

Chronic obstructive pulmonary disease (COPD) is a debilitating lung disease associated with cigarette smoking. Alterations in local lung and systemic iron regulation are associated with disease progression and pathogenesis. Hepcidin, an iron regulatory peptide hormone, is altered in subjects with COPD; however, the molecular role of hepcidin in COPD pathogenesis remains to be determined. In this study, using a murine model of smoke-induced COPD, we demonstrate that lung and circulating hepcidin levels are inhibited by cigarette smoke. We show that cigarette smoke exposure increases erythropoietin and bone marrow-derived erythroferrone and leads to expanded but inefficient erythropoiesis in murine bone marrow and an increase in ferroportin on alveolar macrophages (AMs). AMs from smokers and subjects with COPD display increased expression of ferroportin as well as hepcidin. Notably, murine AMs exposed to smoke fail to increase hepcidin in response to Gram-negative or Gram-positive infection. Loss of hepcidin in vivo results in blunted functional responses of AMs and exaggerated responses to Streptococcus pneumoniae infection.

Local regulation of gene expression by lncRNA promoters, transcription and splicing.

Mammalian genomes are pervasively transcribed to produce thousands of long non-coding RNAs (lncRNAs). A few of these lncRNAs have been shown to recruit regulatory complexes through RNA-protein interactions to influence the expression of nearby genes, and it has been suggested that many other lncRNAs can also act as local regulators. Such local functions could explain the observation that lncRNA expression is often correlated with the expression of nearby genes. However, these correlations have been challenging to dissect and could alternatively result from processes that are not mediated by the lncRNA transcripts themselves. For example, some gene promoters have been proposed to have dual functions as enhancers, and the process of transcription itself may contribute to gene regulation by recruiting activating factors or remodelling nucleosomes. Here we use genetic manipulation in mouse cell lines to dissect 12 genomic loci that produce lncRNAs and find that 5 of these loci influence the expression of a neighbouring gene in cis. Notably, none of these effects requires the specific lncRNA transcripts themselves and instead involves general processes associated with their production, including enhancer-like activity of gene promoters, the process of transcription, and the splicing of the transcript. Furthermore, such effects are not limited to lncRNA loci: we find that four out of six protein-coding loci also influence the expression of a neighbour. These results demonstrate that cross-talk among neighbouring genes is a prevalent phenomenon that can involve multiple mechanisms and cis-regulatory signals, including a role for RNA splice sites. These mechanisms may explain the function and evolution of some genomic loci that produce lncRNAs and broadly contribute to the regulation of both coding and non-coding genes.

"Oxidative stress induced by phthalates in mammals: State of the art and potential biomarkers".

BACKGROUND: Phthalates, plasticizers that are widely used in consumer products including toys, cosmetics, and food containers, have negative effects in liver, kidney, brain, lung and reproductive system of humans and other mammals. OBJECTIVES: To summarize, describe and discuss the available information on the effects of phthalate exposure in mammals, with emphasis on oxidative stress, and to suggest potential biomarkers of the health risks associated with phthalate exposure. METHODS: An assessment of scientific journals was performed using the PRISMA model for systematic reviews. Manuscripts reporting effects of phthalate exposure on mammalian health published in the last decade were selected according to originality, content, and association to health hazards. RESULTS AND DISCUSSION: We identified 25 peer-reviewed articles published between January 1st, 2010 and June 1st, 2021 that fit the aims and selection criteria. Phthalates induce oxidative stress and cell degenerative processes by increasing intracellular reactive species. Antioxidant cytoprotective systems decrease with time of exposure; conversely, oxidative damage markers, including thiobarbituric acid-reactive substances (TBARS), 8-hydroxy-2'-desoxyguanosine (8-OHdG) and malondialdehyde (MDA), increase. Phthalates were associated with endocrine system disfunction, metabolic disorders, infertility, nonviable pregnancy, cell degeneration, growth impairment, tumor development, and cognitive disorders. Phthalates can also aggravate health conditions such as asthma, hepatitis, diabetes, allergies, chronic liver and kidney diseases. Among humans, the more vulnerable subjects to phthalate exposure effects were children and individuals with a prior health condition. CONCLUSION: Chronic exposure to phthalates induces oxidative stress in mammals with concomitant adverse effects in reproductive, respiratory, endocrine, circulatory, and central nervous systems in both in vitro and in vivo trials. Oxidative damage markers and phthalate metabolites levels were the most common biomarkers of phthalate exposure effects. Studies in free-ranging and wild mammals are nil. Further studies on the pathways that lead to metabolic disruption are needed to identify potential treatments against phthalate-induced detrimental effects.

DisProt: intrinsic protein disorder annotation in 2020.

The Database of Protein Disorder (DisProt, URL: https://disprot.org) provides manually curated annotations of intrinsically disordered proteins from the literature. Here we report recent developments with DisProt (version 8), including the doubling of protein entries, a new disorder ontology, improvements of the annotation format and a completely new website. The website includes a redesigned graphical interface, a better search engine, a clearer API for programmatic access and a new annotation interface that integrates text mining technologies. The new entry format provides a greater flexibility, simplifies maintenance and allows the capture of more information from the literature. The new disorder ontology has been formalized and made interoperable by adopting the OWL format, as well as its structure and term definitions have been improved. The new annotation interface has made the curation process faster and more effective. We recently showed that new DisProt annotations can be effectively used to train and validate disorder predictors. We believe the growth of DisProt will accelerate, contributing to the improvement of function and disorder predictors and therefore to illuminate the 'dark' proteome.

Frontiers in human factors: integrating human factors and ergonomics to improve safety and quality in Latin American healthcare systems.

BACKGROUND: The importance of human factors/ergonomics (HFE) is well established in all high-reliability systems but only applied in the healthcare sector relatively recently. Across many sectors, low-/middle-income countries (LMICs) lag behind more economically developed countries in their application of this safety science, due to resource and, in some cases, awareness and expertise. Most previous applications of HFE related to occupational ergonomics rather than healthcare safety. METHODS: The paper details how the reputation of HFE is being developed within healthcare communities of Latin America (LatAm), through increasing awareness and understanding of its role as safety science in the healthcare sector. It starts by articulating the need for HFE and then provides examples from Mexico, Colombia and Peru. RESULTS: The practical examples for research and education illustrate a developing awareness of the relevance of HFE to the healthcare sectors in LatAm and an appreciation of its worth to improve health service quality and patient safety through healthcare community engagement. A new LatAm Network of HFE in Healthcare Systems (RELAESA) was formed in 2019, which has provided a platform for HFE advice during the COVID-19 pandemic. CONCLUSION: There is a real opportunity in LatAm and other LMIC health services to make more rapid and sustainable progress in healthcare-embedded HFE than has been experienced within healthcare services of more developed nations.

Anaphylaxis: Focus on Transcription Factor Activity.

Anaphylaxis is a severe allergic reaction, rapid in onset, and can lead to fatal consequences if not promptly treated. The incidence of anaphylaxis has risen at an alarming rate in past decades and continues to rise. Therefore, there is a general interest in understanding the molecular mechanism that leads to an exacerbated response. The main effector cells are mast cells, commonly triggered by stimuli that involve the IgE-dependent or IgE-independent pathway. These signaling pathways converge in the release of proinflammatory mediators, such as histamine, tryptases, prostaglandins, etc., in minutes. The action and cell targets of these proinflammatory mediators are linked to the pathophysiologic consequences observed in this severe allergic reaction. While many molecules are involved in cellular regulation, the expression and regulation of transcription factors involved in the synthesis of proinflammatory mediators and secretory granule homeostasis are of special interest, due to their ability to control gene expression and change phenotype, and they may be key in the severity of the entire reaction. In this review, we will describe our current understanding of the pathophysiology of human anaphylaxis, focusing on the transcription factors' contributions to this systemic hypersensitivity reaction. Host mutation in transcription factor expression, or deregulation of their activity in an anaphylaxis context, will be updated. So far, the risk of anaphylaxis is unpredictable thus, increasing our knowledge of the molecular mechanism that leads and regulates mast cell activity will enable us to improve our understanding of how anaphylaxis can be prevented or treated.

Silicea terra and Zincum metallicum Modulate the Activity of Macrophages Challenged with BCG In Vitro.

BACKGROUND: The homeopathic medicines Silicea terra (Sil) and Zincum metallicum (Zinc) modulate macrophage activity and were assessed in an experimental study in-vitro for their effects on macrophage-BCG (Bacillus Calmette-Guérin) interaction. METHODS: RAW 264.7 macrophages were infected with BCG, treated with different potencies of Sil and Zinc (6cH, 30cH and 200cH) or vehicle, and assessed 24 and 48 h later for bacilli internalization, hydrogen peroxide (H2O2) and cytokine production, and lysosomal activity. RESULTS: Treatment with vehicle was associated with non-specific inhibition of H2O2 production to the levels exhibited by uninfected macrophages. Sil 200cH induced significant reduction of H2O2 production (p < 0.001) compared with the vehicle and all other treatments, as well as higher lysosomal activity (p ≤ 0.001) and increased IL-10 production (p ≤ 0.05). Such effects were considered specific for this remedy and potency. The number of internalized bacilli was inversely proportional to Zinc potencies, with statistically significant interaction between dilution and treatment (p = 0.003). Such linear-like behavior was not observed for Sil dilutions: peak internalization occurred with the 30cH dilution, accompanied by cellular degeneration, and IL-6 and IL-10 increased (p ≤ 0.05) only in the cells treated with Sil 6cH. CONCLUSION: Sil and Zinc presented different patterns of potency-dependent effect on macrophage activity. Bacterial digestion and a balanced IL-6/IL-10 production were related to Sil 6cH, though reduced oxidative stress with increased lysosomal activity was related to Sil 200cH. Degenerative effects were exclusively related to Sil 30cH, and potency-dependent phagocytosis was related only to Zinc.

Risk factors associated with testing positive for tuberculosis in high-yielding Holstein cows.

Tuberculosis remains a significant infectious disease of farm animals and humans worldwide. The objective of this study was to assess various risk factors associated with testing positive for bovine tuberculosis (bTB) in high-yielding Holstein cows in an intensive dry-lot dairy operation. In a retrospective observational study, 9312 records from Holstein cows from a large dairy herd in northern Mexico were used. The incidence rate of lactating cows reactor to bTB was 7.3 cases/100 cow years (95% CI = 6.7-7.9%). Multiple logistic regression models indicated that cows with total milk yield during the first lactation >10,200 kg were 1.3 times (95% confidence intervals (CI) for odds ratio (OR) = 1.2-1.6) more likely to be detected as bTB reactors than cows with total milk yield <10,200 kg (8.2 vs. 6.4%; P < 0.01). The risk of being reactor to bTB increased with x̅ total milk yield/lactation >11,500 kg (OR=2.7, 95% CI = 2.3-3.3; 10.4 vs. 4.2%) compared with cows with x̅ total milk yield/lactation <11,500 kg. Cows with >10,200 kg 305-day milk yield in the last lactation was associated (P < 0.01) with 1.3 times higher odds (95% CI for OR = 1.1-1.5) of being classed as reactors than cows with <10,200 kg. Cows with peak milk yield >48 kg were 1.9 times (95% CI for OR = 1.6-2.2) more likely to be reactor to bTB than cows with peak milk yield <48 kg (9.2 vs. 5.1%; P < 0.01). Cows with either puerperal metritis (OR = 0.07, 95% CI = 0.5-0.9) or carrying twins (OR = 0.05, 95% CI = 0.01-0.19) had a protective role for being reactor to bTB. This study showed that increased milk production was associated with a higher risk of becoming positive to tuberculin skin test in high-yielding Holstein cows.