Differences in the Cellular Immune Response during and after Treatment of Sudanese Patients with Post-kala-azar Dermal Leishmaniasis, and Possible Implications for Outcome.

BACKGROUND: The host cellular immune response associated with two treatments for post-kala-azar dermal leishmaniasis (PKDL) - paromomycin plus miltefosine (Arm 1), and liposomal amphotericin B plus miltefosine (Arm 2) - was examined in Sudanese patients before treatment (D0), at the end of treatment (D42), and during the post-treatment period (D180). METHODS: Whole blood samples were stimulated with soluble Leishmania antigen for 24 h (whole blood assay [WBA]) and the concentrations of Th1/Th2/Th17-associated cytokines, IP-10, PDL-1 and granzyme B were determined. RESULTS: The Arm 1 treatment (98.2% cure rate) induced a Th1/Th2/Th17 response, while the Arm 2 treatment (80% cure rate) induced a Th1/Th2 response. Five Arm 2 patients relapsed and showed lower IFN-γ, TNF and IL-1ß concentrations at D0 than non-relapsers in this Arm. In patients with low-IFN-γ-production at D0, Arm 1 treatment led to a better host immune response and clinical outcome than Arm 2 treatment. CONCLUSIONS: A Th1/Th2/Th17 response was associated with a higher cure rate. Patients with low IFN-γ, TNF and IL-1ß before treatment are more likely to relapse if they undergo Arm 2-type treatment. Determining IFN-γ, TNF and IL-10 levels prior to treatment could help predict patients at higher risk of relapse/recovery from PKDL. TRIAL REGISTRATION: ClinicalTrials.gov NCT03399955, Registered 17 January 2018, https://clinicaltrials.gov/study/ NCT03399955.

Near-field detection and peak frequency metric for substrate and activation mapping of ventricular tachycardias in two- and three-dimensional circuits.

AIMS: Successful ventricular arrhythmia (VA) ablation requires identification of functionally critical sites during contact mapping. Estimation of the peak frequency (PF) component of the electrogram (EGM) may improve correct near-field (NF) annotation to identify circuit segments on the mapped surface. In turn, assessment of NF and far-field (FF) EGMs may delineate the three-dimensional path of a ventricular tachycardia (VT) circuit. METHODS AND RESULTS: A proprietary NF detection algorithm was applied retrospectively to scar-related re-entry VT maps and compared with manually reviewed maps employing first deflection (FDcorr) for VT activation maps and last deflection (LD) for substrate maps. Ventricular tachycardia isthmus location and characteristics mapped with FDcorr vs. NF were compared. Omnipolar low-voltage areas, late activating areas, and deceleration zones (DZ) in LD vs. NF substrate maps were compared. On substrate maps, PF estimation was compared between isthmus and bystander sites. Activation mapping with entrainment and/or VT termination with radiofrequency (RF) ablation confirmed critical sites. Eighteen patients with high-density VT activation and substrate maps (55.6% ischaemic) were included. Near-field detection correctly located critical parts of the circuit in 77.7% of the cases compared with manually reviewed VT maps as reference. In substrate maps, NF detection identified deceleration zones in 88.8% of cases, which overlapped with FDcorr VT isthmus in 72.2% compared with 83.3% overlap of DZ assessed by LD. Applied to substrate maps, PF as a stand-alone feature did not differentiate VT isthmus sites from low-voltage bystander sites. Omnipolar voltage was significantly higher at isthmus sites with longer EGM durations compared with low-voltage bystander sites. CONCLUSION: The NF algorithm may enable rapid high-density activation mapping of VT circuits in the NF of the mapped surface. Integrated assessment and combined analysis of NF and FF EGM-components could support characterization of three-dimensional VT circuits with intramural segments. For scar-related substrate mapping, PF as a stand-alone EGM feature did not enable the differentiation of functionally critical sites of the dominant VT from low-voltage bystander sites in this cohort.

Monitoring the Phenolic and Terpenic Profile of Olives, Olive Oils and By-Products throughout the Production Process.

Olive oil is a food of great importance in the Mediterranean diet and culture. However, during its production, the olive oil industry generates a large amount of waste by-products that can be an important source of bioactive compounds, such as phenolic compounds and terpenes, revalorizing them in the context of the circular economy. Therefore, it is of great interest to study the distribution and abundance of these bioactive compounds in the different by-products. This research is a screening focused on phytochemical analysis, with particular emphasis on the identification and quantification of the phenolic and terpenic fractions. Both the main products of the olive industry (olives, olive paste and produced oil) and the by-products generated throughout the oil production process (leaf, "alpeorujo", liquid and solid residues generated during decanting commonly named "borras" and washing water) were analyzed. For this purpose, different optimized extraction procedures were performed for each matrix, followed by high-performance liquid chromatography coupled with electrospray time-of-flight mass spectrometry (HPLC-ESI-TOF/MS) analysis. Although no phenolic alcohols were quantified in the leaf and the presence of secoiridoids was low, this by-product was notable for its flavonoid (720 ± 20 µg/g) and terpene (5000 ± 300 µg/g) contents. "Alpeorujo" presented a complete profile of compounds of interest, being abundant in phenolic alcohols (900 ± 100 µg/g), secoiridoids (4500 ± 500 µg/g) and terpenes (1200 ± 100 µg/g), among others. On the other hand, while the solid residue of the borras was the most abundant in phenolic alcohols (3700 ± 200 µg/g) and secoiridoids (680 ± 20 µg/g), the liquid fraction of this waste was notable for its content of elenolic acid derivatives (1700 ± 100 µg/mL) and phenolic alcohols (3000 ± 300 µg/mL). Furthermore, to our knowledge, this is the first time that the terpene content of this by-product has been monitored, demonstrating that it is an important source of these compounds, especially maslinic acid (120 ± 20 µg/g). Finally, the phytochemical content in wash water was lower than expected, and only elenolic acid derivatives were detected (6 ± 1 µg/mL). The results highlighted the potential of the olive by-products as possible alternative sources of a wide variety of olive bioactive compounds for their revalorization into value-added products.

A panel of recombinant Leishmania donovani cell surface and secreted proteins identifies LdBPK_323600.1 as a serological marker of symptomatic infection.

Visceral leishmaniasis is a deadly infectious disease and is one of the world's major neglected health problems. Because the symptoms of infection are similar to other endemic diseases, accurate diagnosis is crucial for appropriate treatment. Definitive diagnosis using splenic or bone marrow aspirates is highly invasive, and so, serological assays are preferred, including the direct agglutination test (DAT) or rK39 strip test. These tests, however, are either difficult to perform in the field (DAT) or lack specificity in some endemic regions (rK39), making the development of new tests a research priority. The availability of Leishmania spp. genomes presents an opportunity to identify new diagnostic targets. Here, we use genome data and a mammalian protein expression system to create a panel of 93 proteins consisting of the extracellular ectodomains of the Leishmania donovani cell surface and secreted proteins. We use these panel and sera from murine experimental infection models and natural human and canine infections to identify new candidates for serological diagnosis. We observed a concordance between the most immunoreactive antigens in different host species and transmission settings. The antigen encoded by the LdBPK_323600.1 gene can diagnose Leishmania infections with high sensitivity and specificity in patient cohorts from different endemic regions including Bangladesh and Ethiopia. In longitudinal sampling of treated patients, we observed reductions in immunoreactivity to LdBPK_323600.1 suggesting it could be used to diagnose treatment success. In summary, we have identified new antigens that could contribute to improved serological diagnostic tests to help control the impact of this deadly tropical infectious disease. IMPORTANCE: Visceral leishmaniasis is fatal if left untreated with patients often displaying mild and non-specific symptoms during the early stages of infection making accurate diagnosis important. Current methods for diagnosis require highly trained medical staff to perform highly invasive biopsies of the liver or bone marrow which pose risks to the patient. Less invasive molecular tests are available but can suffer from regional variations in their ability to accurately diagnose an infection. To identify new diagnostic markers of visceral leishmaniasis, we produced and tested a panel of 93 proteins identified from the genome of the parasite responsible for this disease. We found that the pattern of host antibody reactivity to these proteins was broadly consistent across naturally acquired infections in both human patients and dogs, as well as experimental rodent infections. We identified a new protein called LdBPK_323600.1 that could accurately diagnose visceral leishmaniasis infections in humans.

Transcriptomic Analysis of Extracellular Vesicles in the Search for Novel Plasma and Thrombus Biomarkers of Ischemic Stroke Etiologies.

Accurate etiologic diagnosis provides an appropriate secondary prevention and better prognosis in ischemic stroke (IS) patients; still, 45% of IS are cryptogenic, urging us to enhance diagnostic precision. We have studied the transcriptomic content of plasma extracellular vesicles (EVs) (n = 21) to identify potential biomarkers of IS etiologies. The proteins encoded by the selected genes were measured in the sera of IS patients (n = 114) and in hypertensive patients with (n = 78) and without atrial fibrillation (AF) (n = 20). IGFBP-2, the most promising candidate, was studied using immunohistochemistry in the IS thrombi (n = 23) and atrium of AF patients (n = 13). In vitro, the IGFBP-2 blockade was analyzed using thromboelastometry and endothelial cell cultures. We identified 745 differentially expressed genes among EVs of cardioembolic, atherothrombotic, and ESUS groups. From these, IGFBP-2 (cutoff > 247.6 ng/mL) emerged as a potential circulating biomarker of embolic IS [OR = 8.70 (1.84-41.13) p = 0.003], which was increased in patients with AF vs. controls (p < 0.001) and was augmented in cardioembolic vs. atherothrombotic thrombi (p < 0.01). Ex vivo, the blockage of IGFBP-2 reduced clot firmness (p < 0.01) and lysis time (p < 0.001) and in vitro, diminished endothelial permeability (p < 0.05) and transmigration (p = 0.06). IGFBP-2 could be a biomarker of embolic IS and a new therapeutic target involved in clot formation and endothelial dysfunction.

Staphylococcus aureus Panton-Valentine Leukocidin worsens acute implant-associated osteomyelitis in humanized BRGSF mice.

Staphylococcus aureus is the most common pathogen that causes implant-associated osteomyelitis, a clinically incurable disease. Immune evasion of S. aureus relies on various mechanisms to survive within the bone niche, including the secretion of leukotoxins such as Panton-Valentine leukocidin (PVL). PVL is a pore-forming toxin exhibiting selective human tropism for C5a receptors (C5aR1 and C5aR2) and CD45 on neutrophils, monocytes, and macrophages. PVL is an important virulence determinant in lung, skin and soft tissue infections. The involvement of PVL in S. aureus pathogenesis during bone infections has not been studied extensively yet. To investigate this, humanized BALB/c Rag2-/-Il2rg-/-SirpaNODFlk2-/- (huBRGSF) mice were subjected to transtibial implant-associated osteomyelitis with community-acquired methicillin-resistant S. aureus (CA-MRSA) USA300 wild type strain (WT), an isogenic mutant lacking lukF/S-PV (Δpvl), or complemented mutant (Δpvl+pvl). Three days post-surgery, Δpvl-infected huBRGSF mice had a less severe infection compared to WT-infected animals as characterized by 1) improved clinical outcomes, 2) lower ex vivo bacterial bone burden, 3) absence of staphylococcal abscess communities (SACs) in their bone marrow, and 4) compromised MRSA dissemination to internal organs (liver, kidney, spleen, heart). Interestingly, Δpvl-infected huBRGSF mice had fewer human myeloid cells, neutrophils, and HLA-DR+ monocytes in the bone niche compared to WT-infected animals. Expectedly, a smaller fraction of human myeloid cells were apoptotic in the Δpvl-infected huBRGSF animals. Taken together, our study highlights the pivotal role of PVL during acute implant-associated osteomyelitis in humanized mice.

Phospholipase C epsilon-1 (PLCƐ1) mediates macrophage activation and protection against tuberculosis.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) infects up to a quarter of the world's population. Although immune responses can control Mtb infection, 5%-10% of infected individuals can progress to active TB disease (progressors). A myriad of host factors regulate disease progression in TB and a better understanding of immune correlates of protection and disease is pivotal for the development of new therapeutics. Comparison of human whole blood transcriptomic metadata with that of macaque TB progressors and Mtb-infected diversity outbred mice (DO) led to the identification of differentially regulated gene (DEG) signatures, associated with TB progression or control. The current study assessed the function of Phospholipase C epsilon (PLCƐ1), the top downregulated gene across species in TB progressors, using a gene-specific knockout mouse model of Mtb infection and in vitro Mtb-infected bone marrow-derived macrophages. PLCƐ1 gene expression was downregulated in TB progressors across species. PLCε1 deficiency in the mouse model resulted in increased susceptibility to Mtb infection, coincident accumulation of lung myeloid cells, and reduced ability to mount antibacterial responses. However, PLCε1 was not required for the activation and accumulation of T cells in mice. Our results suggest an important early role for PLCƐ1 in shaping innate immune response to TB and may represent a putative target for host-directed therapy.

Recurrent bacteremia and endocarditis due to Staphylococcus capitis in a patient with Bowen's disease: a case report.

This case report details the management of a 79-year-old male with recurrent methicillin-resistant Staphylococcus capitis bacteremia and endocarditis. The patient's clinical journey encompassed multiple hospital admissions, with challenges in managing endocarditis, pacemaker replacements, and potential cutaneous sources of infection. The treatment regimen included intravenous antibiotic therapy during hospitalization and suppressive antibiotic treatment upon discharge, alongside a decolonization strategy for his scalp lesions.

Subjective identification and ablation of drivers improves rhythm control in patients with persistent atrial fibrillation. The CHAOS-AF study.

INTRODUCTION AND OBJECTIVES: The optimal approach for persistent atrial fibrillation (AF) ablation remains unknown. In patients with persistent AF, we compared an ablation strategy based on pulmonary vein isolation (PVI) plus ablation of drivers (PVI+D), with a conventional PVI-only approach performed in a 1:1 propensity score-matched cohort. METHODS: Drivers were subjectively identified using conventional high-density mapping catheters (IntellaMap ORION, PentaRay NAV or Advisor HD Grid), without dedicated software, as fractionated continuous or quasicontinuous electrograms on 1 to 2 adjacent bipoles, which were ablated first; and as sites with spatiotemporal dispersion (the entire cycle length comprised within the mapping catheter) plus noncontinuous fractionation, which were only targeted in patients without fractionated continuous electrograms, or without AF conversion after ablation of fractionated continuous electrograms. Ablation included PVI plus focal or linear ablation targeting drivers. RESULTS: A total of 50 patients were included in each group (61±10 years, 25% women). Fractionated continuous electrograms were found and ablated in 21 patients from the PVI+D group (42%), leading to AF conversion in 7 patients. In the remaining 43 patients, 143 sites with spatiotemporal dispersion plus noncontinuous fractionation were targeted. Globally, AF conversion was achieved in 21 patients (42%). The PVI+D group showed lower atrial arrhythmia recurrences at 1 year of follow-up (30.6% vs 48%; P=.048) and at the last follow-up (46% vs 72%; P=.013), and less progression to permanent AF (10% vs 40%; P=.001). CONCLUSIONS: Subjective identification and ablation of drivers, added to PVI, increased 1-year freedom from atrial arrhythmia and decreased long-term recurrences and progression to permanent AF.

Portable smartphone-based molecular test for rapid detection of Leishmania spp.

PURPOSE: Leishmaniasis, caused by the parasite of the genus Leishmania, is a neglected tropical disease which is endemic in more than 60 countries. In South-East Asia, Brazil, and East Africa, it mainly occurs as kala-azar (visceral leishmaniasis, VL), and subsequently as post kala-azar dermal leishmaniasis (PKDL) in a smaller portion of cases. As stated per WHO roadmap, accessibility to accurate diagnostic methods is an essential step to achieve elimination. This study aimed to test the accuracy of a portable minoo device, a small battery-driven, multi-use fluorimeter operating with isothermal technology for molecular diagnosis of VL and PKDL. METHODS: Fluorescence data measured by the device within 20 min are reported back to the mobile application (or app) via Bluetooth and onward via the internet to a backend. This allows anonymous analysis and storage of the test data. The test result is immediately returned to the app displaying it to the user. RESULTS: The limit of detection was 11.2 genome copies (95% CI) as determined by screening a tenfold dilution range of whole Leishmania donovani genomes using isothermal recombinase polymerase amplification (RPA). Pathogens considered for differential diagnosis were tested and no cross-reactivity was observed. For its diagnostic performance, DNA extracted from 170 VL and PKDL cases, comprising peripheral blood samples (VL, n = 96) and skin biopsies (PKDL, n = 74) from India (n = 108) and Bangladesh (n = 62), was screened. Clinical sensitivity and specificity were 88% and 91%, respectively. CONCLUSION: Minoo devices can offer a convenient, cheaper alternative to other molecular diagnostics. Its easy handling makes it ideal for use in low-resource settings to identify parasite burden.

Potential selection of antimony and methotrexate cross-resistance in Leishmania infantum circulating strains.

BACKGROUND: Visceral leishmaniasis (VL) resolution depends on a wide range of factors, including the instauration of an effective treatment coupled to a functional host immune system. Patients with a depressed immune system, like the ones receiving methotrexate (MTX), are at higher risk of developing VL and refusing antileishmanial drugs. Moreover, the alarmingly growing levels of antimicrobial resistance, especially in endemic areas, contribute to the increasing the burden of this complex zoonotic disease. PRINCIPAL FINDINGS: To understand the potential links between immunosuppressants and antileishmanial drugs, we have studied the interaction of antimony (Sb) and MTX in a Leishmania infantum reference strain (LiWT) and in two L. infantum clinical strains (LiFS-A and LiFS-B) naturally circulating in non-treated VL dogs in Spain. The LiFS-A strain was isolated before Sb treatment in a case that responded positively to the treatment, while the LiFS-B strain was recovered from a dog before Sb treatment, with the dog later relapsing after the treatment. Our results show that, exposure to Sb or MTX leads to an increase in the production of reactive oxygen species (ROS) in LiWT which correlates with a sensitive phenotype against both drugs in promastigotes and intracellular amastigotes. LiFS-A was sensitive against Sb but resistant against MTX, displaying high levels of protection against ROS when exposed to MTX. LiFS-B was resistant to both drugs. Evaluation of the melting proteomes of the two LiFS, in the presence and absence of Sb and MTX, showed a differential enrichment of direct and indirect targets for both drugs, including common and unique pathways. CONCLUSION: Our results show the potential selection of Sb-MTX cross-resistant parasites in the field, pointing to the possibility to undermine antileishmanial treatment of those patients being treated with immunosuppressant drugs in Leishmania endemic areas.

Sex-specific differences in intestinal microbiota associated with cardiovascular diseases.

BACKGROUND: Cardiovascular diseases (CVD), including coronary heart disease (CHD), display a higher prevalence in men than women. This study aims to evaluate the variations in the intestinal microbiota between men and women afflicted with CHD and delineate these against a non-CVD control group for each sex. METHODS: Our research was conducted in the framework of the CORDIOPREV study, a clinical trial which involved 837 men and 165 women with CHD. We contrasted our findings with a reference group of 375 individuals (270 men, 105 women) without CVD. The intestinal microbiota was examined through 16S metagenomics on the Illumina MiSeq platform and the data processed with Quiime2 software. RESULTS: Our results showed a sex-specific variation (beta diversity) in the intestinal microbiota, while alpha-biodiversity remained consistent across both sexes. Linear discriminant analysis effect size (LEfSe) analysis revealed sex-centric alterations in the intestinal microbiota linked to CVD. Moreover, using random forest (RF) methodology, we identified seven bacterial taxa-g_UBA1819 (Ruminococcaceae), g_Bilophila, g_Subdoligranulum, g_Phascolarctobacterium, f_Barnesiellaceae, g_Ruminococcus, and an unknown genus from the Ruminococcaceae family (Ruminococcaceae incertae sedis)-as key discriminators between men and women diagnosed with CHD. The same taxa also emerged as critical discriminators between CHD-afflicted and non-CVD individuals, when analyzed separately by sex. CONCLUSION: Our findings suggest a sex-specific dysbiosis in the intestinal microbiota linked to CHD, potentially contributing to the sex disparity observed in CVD incidence. Trial registration Clinical Trials.gov.Identifier NCT00924937.

The frequency with which cardiovascular diseases occur differs in men and women as it appears with greater frequency in men. Moreover, it has been known for years that the community of bacteria living in our intestine, also known as the gut microbiota, influences the development of these diseases. Indeed, nowadays it known the influence of the intestinal microbiota in the development of atherosclerosis, the pathological process which is responsible for the three main causes of cardiovascular diseases: coronary heart disease, cerebrovascular disease and peripheral arterial disease. This study shows the differences in the community of bacteria living in the gut of men and those living in the gut of women, so that these differences could explain, at least in part, the differences in the frequency with which cardiovascular diseases appear between men and women. Our results suggest that the dysbiosis of the intestinal microbiota associated with CHD seems to be partially sex-specific, which may influence the sexual dimorphism in its incidence. Moreover, the identification of the mechanisms responsible for sexual dimorphism in the incidence of metabolic and cardiovascular disease is of particular importance when developing effective strategies and therapies aimed at reducing their incidence and recurrence. Indeed, the strategies and therapies used to treat the dysbiosis of the gut microbiota should be sex-specific.

Delivery of a sebum modulator by an engineered skin microbe in mice.

Microorganisms can be equipped with synthetic genetic programs for the production of targeted therapeutic molecules. Cutibacterium acnes is the most abundant commensal of the human skin, making it an attractive chassis to create skin-delivered therapeutics. Here, we report the engineering of this bacterium to produce and secrete the therapeutic molecule neutrophil gelatinase-associated lipocalin, in vivo, for the modulation of cutaneous sebum production.

Experimental Tumor Induction and Evaluation of Its Treatment in the Chicken Embryo Chorioallantoic Membrane Model: A Systematic Review.

The chorioallantoic membrane (CAM) model, generated during avian development, can be used in cancer research as an alternative in vivo model to perform tumorigenesis in ovo due to advantages such as simplicity, low cost, rapid growth, and being naturally immunodeficient. The aim of this systematic review has been to compile and analyze all studies that use the CAM assay as a tumor induction model. For that, a systematic search was carried out in four different databases: PubMed, Scopus, Cochrane, and WOS. After eliminating duplicates and following the established inclusion and exclusion criteria, a total of 74 articles were included. Of these, 62% use the in ovo technique, 13% use the ex ovo technique, 9% study the formation of metastasis, and 16% induce tumors from patient biopsies. Regarding the methodology followed, the main species used is chicken (95%), although some studies use quail eggs (4%), and one article uses ostrich eggs. Therefore, the CAM assay is a revolutionary technique that allows a simple and effective way to induce tumors, test the effectiveness of treatments, carry out metastasis studies, perform biopsy grafts of patients, and carry out personalized medicine. However, unification of the methodology used is necessary.

Role of factor XIII in ischemic stroke: a key molecule promoting thrombus stabilization and resistance to lysis.

BACKGROUND: Active coagulation factor XIII (FXIII) catalyzing crosslinking of fibrin and other hemostatic factors plays a key role in clot stability and lysis. OBJECTIVES: To evaluate the effect of FXIII inhibition in a mouse model of ischemic stroke (IS) and the role of activated FXIII (FXIIIa) in clot formation and lysis in patients with IS. METHODS: A ferric chloride IS murine model was performed before and after administration of a FXIIIa inhibitor (FXIIIinh). Thromboelastometry in human and mice blood was used to evaluate thrombus stiffness and lysis with FXIIIinh. FXIIIa-dependent fibrin crosslinking and lysis with fibrinolytic drugs (tissue plasminogen activator and tenecteplase) were studied on fibrin plates and on thrombi and clotted plasma of patients with IS. Finally, circulating and thrombus FXIIIa were measured in 85 patients with IS. RESULTS: FXIIIinh administration before stroke induction reduced infarct size, α2-antiplasmin (α2AP) crosslinking, and local microthrombosis, improving motor coordination and fibrinolysis without intracranial bleeds (24 hours). Interestingly, FXIII blockade after stroke also reduced brain damage and neurologic deficit. Thromboelastometry in human/mice blood with FXIIIinh showed delayed clot formation, reduced clot firmness, and shortened tissue plasminogen activator lysis time. FXIIIa fibrin crosslinking increased fibrin density and lysis resistance, which increased further after α2AP addition. FXIIIinh enhanced ex vivo lysis in stroke thrombi and fibrin plates. In patients with IS, thrombus FXIII and α2AP were associated with inflammatory and hemostatic components, and plasma FXIIIa correlated with thrombus α2AP and fibrin. CONCLUSION: Our results suggest a key role of FXIIIa in thrombus stabilization, α2AP crosslinking, and lysis resistance, with a protective effect of FXIIIinh in an IS experimental model.

Immunosuppressants alter the immune response associated with Glucantime® treatment for Leishmania infantum infection in a mouse model.

Background: Immunosuppression is a major risk factor for the development of visceral leishmaniasis (VL). The number of patients receiving immunosuppressant drugs such as TNF antagonist (anti-TNF) and methotrexate (MTX) is increasing. In these patients, VL is more severe, their response to treatment poorer, and they are at higher risk of relapse, a consequence (largely) of the poor and inappropriate immune response they develop. Objectives: To examine the effect of immunosuppressive treatment on the host immune response and thus gain insight into the reduced efficacy of pentavalent antimonials in these patients. Experiments were performed using BALB/c mice immunosuppressed with anti-TNF or MTX, infected with Leishmania infantum promastigotes, and then treated with Glucantime® at clinical doses. Results: Immunosuppression with both agents impeded parasite elimination from the spleen and bone marrow. Low pro-inflammatory cytokine production by CD4+ and CD8+ T cells was detected, along with an increase in PD-1 and IL-10 expression by B and T cells in the immunosuppressed groups after treatment. Conclusion: The immunosuppressed mice were unable to develop specific cellular immunity to the parasite, perhaps explaining the greater risk of VL relapse seen in pharmacologically immunosuppressed human patients.

Sodium-Glucose Cotransporter-2 Inhibitor, Empagliflozin, Suppresses the Inflammatory Immune Response to Influenza Infection.

Influenza is a highly contagious, acute respiratory disease that causes significant public health and economic threats. Influenza infection induces various inflammatory mediators, IFNs, and recruitment of inflammatory cells in the host. This inflammatory "cytokine storm" is thought to play a role in influenza-induced lung pathogenesis. Empagliflozin is a drug primarily used to lower blood glucose in type II diabetes patients by inhibiting the sodium-glucose cotransporter-2 (SGLT-2) found in the proximal tubules in the kidneys. In this study, we have investigated the effects of empagliflozin on the pulmonary immune response to influenza infection. C57BL/6 mice (wild type) were infected with influenza A/PR/8/34 and treated with empagliflozin, and the disease outcomes were analyzed. Empagliflozin treatment decreased the expression of the inflammatory cytokines IL-1ß, IL-6, and CCL2; the percentage of inflammatory monocytes and inducible NO synthase-positive macrophages; and IFN response genes Stat1 and CXCL9 during influenza infection. Further, empagliflozin treatment decreases the expression of IL-6, CCL2, and CCL5 in RAW264.7 macrophages and bone marrow-derived macrophages. However, empagliflozin treatment increased influenza viral titer during infection. Despite fostering an increased viral burden, treatment with empagliflozin decreases the mortality in wild type and high fat diet-induced atherosclerotic LDLR-/- mice. Based on our findings, empagliflozin may have therapeutic implications for use in patients to prevent lung damage and acute respiratory illness.

Synthetic genetic oscillators demonstrate the functional importance of phenotypic variation in pneumococcal-host interactions.

Phenotypic variation is the phenomenon in which clonal cells display different traits even under identical environmental conditions. This plasticity is thought to be important for processes including bacterial virulence, but direct evidence for its relevance is often lacking. For instance, variation in capsule production in the human pathogen Streptococcus pneumoniae has been linked to different clinical outcomes, but the exact relationship between variation and pathogenesis is not well understood due to complex natural regulation. In this study, we use synthetic oscillatory gene regulatory networks (GRNs) based on CRISPR interference (CRISPRi) together with live cell imaging and cell tracking within microfluidics devices to mimic and test the biological function of bacterial phenotypic variation. We provide a universally applicable approach for engineering intricate GRNs using only two components: dCas9 and extended sgRNAs (ext-sgRNAs). Our findings demonstrate that variation in capsule production is beneficial for pneumococcal fitness in traits associated with pathogenesis providing conclusive evidence for this longstanding question.