β-N-acetylglucosaminidase grafted on mesoporous silica nanoparticles. A bionanoantibiotic system against Staphylococcus aureus bacteria

A bionanoantibiotic system based on beta-N-acetylglucosaminidase (Ami) and Lysozyme (Lyz) enzymes grafted on the external surface of amino functionalized mesoporous silica nanoparticles, having a radial arrangement of pores (MSNr-NH2), was prepared and fully characterized. Before the enzyme grafting the nanoparticles were also loaded with the antibiotic drug levofloxacin (Levo) to explore the possible synergic effect with the enzymes. MSNr-NH2-Lyz-Levo and MSNr-NH2-Ami-Levo did not show any activity against S. aureus. On the contrary, in the absence of the antibiotic, both Lyz and Ami immobilized on MSNr were able to destroy S. aureus cells, suggesting an inhibiting action of the antibiotic on the enzymes. Although the loading of immobilized Lyz was higher than that of Ami (76 vs. 20 mg/g, respectively), the highest antibacterial efficacy was found for MSNr-NH2-Ami nanoantibiotic. Moreover, MSNr-NH2-Ami was active against S. aureus even at very low concentration (12.5 mu g/ mL) with a bactericidal activity (79%), higher than that determined for MSNr-NH2 loaded with levofloxacin (54%). These results suggest the possibility of using enzyme grafted MSNr as a bionanoantibiotic drug with high efficiency even at low nanoparticles concentration

Spatio-temporal activation of caspase-8 in myeloid cells upon ischemic stroke

Ischemic stroke (caused by thrombosis, embolism or vasoconstriction) lead to the recruitment and activation of immune cells including resident microglia and infiltrating peripheral macrophages, which contribute to an inflammatory response involved in regulation of the neuronal damage. We showed earlier that upon pro-inflammatory stimuli, the orderly activation of caspase-8 and caspase-3/7 regulates microglia activation through a protein kinase C-δ dependent pathway. Here, we present in vivo evidence for the activation of caspase-8 and caspase-3 in microglia/macrophages in post-mortem tissue from human ischemic stroke subjects. Indeed, CD68-positive microglia/macrophages in the ischemic peri-infarct area exhibited significant expression of the cleaved and active form of caspase-8 and caspase-3. The temporal and spatial activation of caspase-8 was further investigated in a permanent middle cerebral artery occlusion mouse model of ischemic stroke. Increasing levels of active caspase-8 was found in Iba1-positive cells over time in the peri-infarct area, at 6, 24 and 48 h after artery occlusion. Analysis of post-mortem brain tissue from human subject who suffered two stroke events, referred as recent and old stroke, revealed that expression of cleaved caspase-8 and -3 in CD68-positive cells could only be found in the recent stroke area. Analysis of cleaved caspase-8 and -3 expressions in a panel of human stroke cases arranged upon days-after stroke and age-matched controls suggested that the expression of these caspases correlated with the time of onset of stroke. Collectively, these data illustrate the temporal and spatial activation of caspase-8 and -3 in microglia/macrophages occurring upon ischemic stroke and suggest that the expression of these caspases could be used in neuropathological diagnostic work.J.R. is supported by a doctoral fellowship from the Karolinska Institutet Foundations; M.A.B. is supported by a postdoctoral fellowship from Swedish Research Council. This work has been supported by grants from the Swedish Research Council, the Swedish Brain Foundation, the Parkinson foundation in Sweden, the Spanish MINECO/FEDER/UE and the Karolinska Institutet Foundations

Treatment outcomes of new tuberculosis patients hospitalized in Kampala, Uganda: a prospective cohort study.

BACKGROUND: In most resource limited settings, new tuberculosis (TB) patients are usually treated as outpatients. We sought to investigate the reasons for hospitalisation and the predictors of poor treatment outcomes and mortality in a cohort of hospitalized new TB patients in Kampala, Uganda. METHODS AND FINDINGS: Ninety-six new TB patients hospitalised between 2003 and 2006 were enrolled and followed for two years. Thirty two were HIV-uninfected and 64 were HIV-infected. Among the HIV-uninfected, the commonest reasons for hospitalization were low Karnofsky score (47%) and need for diagnostic evaluation (25%). HIV-infected patients were commonly hospitalized due to low Karnofsky score (72%), concurrent illness (16%) and diagnostic evaluation (14%). Eleven HIV uninfected patients died (mortality rate 19.7 per 100 person-years) while 41 deaths occurred among the HIV-infected patients (mortality rate 46.9 per 100 person years). In all patients an unsuccessful treatment outcome (treatment failure, death during the treatment period or an unknown outcome) was associated with duration of TB symptoms, with the odds of an unsuccessful outcome decreasing with increasing duration. Among HIV-infected patients, an unsuccessful treatment outcome was also associated with male sex (P = 0.004) and age (P = 0.034). Low Karnofsky score (aHR = 8.93, 95% CI 1.88 - 42.40, P = 0.001) was the only factor significantly associated with mortality among the HIV-uninfected. Mortality among the HIV-infected was associated with the composite variable of CD4 and ART use, with patients with baseline CD4 below 200 cells/µL who were not on ART at a greater risk of death than those who were on ART, and low Karnofsky score (aHR = 2.02, 95% CI 1.02 - 4.01, P = 0.045). CONCLUSION: Poor health status is a common cause of hospitalisation for new TB patients. Mortality in this study was very high and associated with advanced HIV Disease and no use of ART

Aeromonas spp.-mediated cell-contact cytotoxicity is associated with the presence of type III secretion system

In the study we examined the production of cytotonic and cytotoxic toxins and the presence of a type III secretion system (TTSS) in 64 Aeromonas spp. strains isolated from fecal specimens of patients with gastroenteritis. We observed that contact of the bacteria with host epithelial cells is a prerequisite for their cytotoxicity at 3 h incubation. Cell-contact cytotoxic activity of the strains was strongly associated with the presence of the TTSS. Culture supernatants of the strains induced low cytotoxicity effects at the same time of incubation. Cell-free supernatants of 61 (95%) isolates expressed cytotoxic activity which caused the destruction of HEp-2 cells at 24 h. Moreover, 44% strains were cytotonic towards CHO cells and 46% of strains invaded epithelial cells

Adjuvant interferon gamma in patients with drug – resistant pulmonary tuberculosis: a pilot study

BACKGROUND: Tuberculosis (TB) is increasing in the world and drug-resistant (DR) disease beckons new treatments. METHODS: To evaluate the action of interferon (IFN) gamma as immunoadjuvant to chemotherapy on pulmonary DR-TB patients, a pilot, open label clinical trial was carried out in the Cuban reference ward for the management of this disease. The eight subjects existing in the country at the moment received, as in-patients, 1 × 10(6 )IU of recombinant human IFN gamma intramuscularly, daily for one month and then three times per week up to 6 months as adjuvant to the indicated chemotherapy, according to their antibiograms and WHO guidelines. Sputum samples collection for direct smear observation and culture as well as routine clinical and thorax radiography assessments were done monthly. RESULTS: Sputum smears and cultures became negative for acid-fast-bacilli before three months of treatment in all patients. Lesion size was reduced at the end of 6 months treatment; the lesions disappeared in one case. Clinical improvement was also evident; body mass index increased in general. Interferon gamma was well tolerated. Few adverse events were registered, mostly mild; fever and arthralgias prevailed. CONCLUSIONS: These data suggest that IFN gamma is useful and well tolerated as adjunctive therapy in patients with DR-TB. Further controlled clinical trials are encouraged

An evaluation of factors associated with taking and responding positive to the tuberculin skin test in individuals with HIV/AIDS

<p>Abstract</p> <p>Background</p> <p>The tuberculin skin test (TST) is still the standard test for detecting latent infection by <it>M tuberculosis </it>(LTBI). Given that the Brazilian Health Ministry recommends that the treatment of latent tuberculosis (LTBI) should be guided by the TST results, the present study sets out to describe the coverage of administering the TST in people living with HIV at two referral health centers in the city of Recife, where TST is offered to all patients. In addition, factors associated with the non-application of the test and with positive TST results were also analyzed.</p> <p>Methods</p> <p>A cross-sectional study was carried out with HIV patients, aged 18 years or over, attending outpatient clinics at the Correia Picanço Hospital/SES/PE and the Oswaldo Cruz/UPE University Hospital, who had been recommended to take the TST, in the period between November 2007 and February 2010. Univariate and multivariate logistic regression analyses were carried out to establish associations between the dependent variable - taking the TST (yes/no), at a first stage analysis, and the independent variables, followed by a second stage analysis considering a positive TST as the dependent variable. The odds ratio was calculated as the measure of association and the confidence interval (CI) at 95% as the measure of accuracy of the estimate.</p> <p>Results</p> <p>Of the 2,290 patients recruited, 1087 (47.5%) took the TST. Of the 1,087 patients who took the tuberculin skin test, the prevalence of TST ≥ 5 mm was 21.6% among patients with CD4 ≥ 200 and 9.49% among those with CD4 < 200 (p = 0.002). The patients most likely not to take the test were: men, people aged under 39 years, people with low educational levels and crack users. The risk for not taking the TST was statiscally different for health service. Patients who presented better immunity (CD4 ≥ 200) were more than two and a half times more likely to test positive that those with higher levels of immunodeficiency (CD4 < 200).</p> <p>Conclusions</p> <p>Considering that the TST is recommended by the Brazilian health authorities, coverage for taking the test was very low. The most serious implication of this is that LTBI treatment was not carried out for the unidentified TST-positive patients, who may consequently go on to develop TB and eventually die.</p

The CXCL12γ Chemokine Displays Unprecedented Structural and Functional Properties that Make It a Paradigm of Chemoattractant Proteins

The CXCL12γ chemokine arises by alternative splicing from Cxcl12, an essential gene during development. This protein binds CXCR4 and displays an exceptional degree of conservation (99%) in mammals. CXCL12γ is formed by a protein core shared by all CXCL12 isoforms, extended by a highly cationic carboxy-terminal (C-ter) domain that encompass four overlapped BBXB heparan sulfate (HS)-binding motifs. We hypothesize that this unusual domain could critically determine the biological properties of CXCL12γ through its interaction to, and regulation by extracellular glycosaminoglycans (GAG) and HS in particular. By both RT-PCR and immunohistochemistry, we mapped the localization of CXCL12γ both in mouse and human tissues, where it showed discrete differential expression. As an unprecedented feature among chemokines, the secreted CXCL12γ strongly interacted with cell membrane GAG, thus remaining mostly adsorbed on the plasmatic membrane upon secretion. Affinity chromatography and surface plasmon resonance allowed us to determine for CXCL12γ one of the higher affinity for HS (Kd = 0.9 nM) ever reported for a protein. This property relies in the presence of four canonical HS-binding sites located at the C-ter domain but requires the collaboration of a HS-binding site located in the core of the protein. Interestingly, and despite reduced agonist potency on CXCR4, the sustained binding of CXCL12γ to HS enabled it to promote in vivo intraperitoneal leukocyte accumulation and angiogenesis in matrigel plugs with much higher efficiency than CXCL12α. In good agreement, mutant CXCL12γ chemokines selectively devoid of HS-binding capacity failed to promote in vivo significant cell recruitment. We conclude that CXCL12γ features unique structural and functional properties among chemokines which rely on the presence of a distinctive C-ter domain. The unsurpassed capacity to bind to HS on the extracellular matrix would make CXCL12γ the paradigm of haptotactic proteins, which regulate essential homeostatic functions by promoting directional migration and selective tissue homing of cells

Stratification of radiosensitive brain metastases based on an actionable S100A9/RAGE resistance mechanism

Whole-brain radiotherapy (WBRT) is the treatment backbone for many patients with brain metastasis; however, its efficacy in preventing disease progression and the associated toxicity have questioned the clinical impact of this approach and emphasized the need for alternative treatments. Given the limited therapeutic options available for these patients and the poor understanding of the molecular mechanisms underlying the resistance of metastatic lesions to WBRT, we sought to uncover actionable targets and biomarkers that could help to refine patient selection. Through an unbiased analysis of experimental in vivo models of brain metastasis resistant to WBRT, we identified activation of the S100A9–RAGE–NF-κB–JunB pathway in brain metastases as a potential mediator of resistance in this organ. Targeting this pathway genetically or pharmacologically was sufficient to revert the WBRT resistance and increase therapeutic benefits in vivo at lower doses of radiation. In patients with primary melanoma, lung or breast adenocarcinoma developing brain metastasis, endogenous S100A9 levels in brain lesions correlated with clinical response to WBRT and underscored the potential of S100A9 levels in the blood as a noninvasive biomarker. Collectively, we provide a molecular framework to personalize WBRT and improve its efficacy through combination with a radiosensitizer that balances therapeutic benefit and toxicity.We thank all members of the Brain Metastasis Group and A. Chalmers, E. Wagner, O. Fernández-Capetillo, R. Ciérvide and A. Hidalgo for critical discussion of the manuscript; the CNIO Core Facilities for their excellent assistance; and Fox Chase Cancer Center Transgenic Facility for generation of S100A9 mice. We thank EuCOMM repository for providing S100A9 targeted embryonic stem cells. We also thank J. Massagué (MSKCC) for some of the BrM cell lines and M. Bosenberg (Yale) for the YUMM1.1 cell line. Samples from patients included in this study that provided by the Girona Biomedical Research Institute (IDIBGI) (Biobanc IDIBGI, B.0000872) are integrated into the Spanish National Biobanks Network and in the Xarxa de Bancs de Tumors de Catalunya (XBTC) financed by the Pla Director d’Oncologia de Catalunya. All patients consented to the storage of these samples in the biobank and for their use in research projects. This study was funded by MINECO (SAF2017-89643-R) (M.V.), Fundació La Marató de TV3 (201906-30-31-32) (J.B.-B., M.V. and A.C.), Fundación Ramón Areces (CIVP19S8163) (M.V.) and CIVP20S10662 (E.O.P.), Worldwide Cancer Research (19-0177) (M.V. and E.C.-J.M.), Cancer Research Institute (Clinic and Laboratory Integration Program CRI Award 2018 (54545) (M.V.), AECC (Coordinated Translational Groups 2017 (GCTRA16015SEOA) (M.V.), LAB AECC 2019 (LABAE19002VALI) (M.V.), ERC CoG (864759) (M.V.), Portuguese Foundation for Science and Technology (SFRH/bd/100089/2014) (C.M.), Boehringer-Ingelheim Fonds MD Fellowship (L.M.), La Caixa International PhD Program Fellowship-Marie Skłodowska-Curie (LCF/BQ/DI17/11620028) (P.G.-G.), La Caixa INPhINIT Fellowship (LCF/BQ/DI19/11730044) (A.P.-A.), MINECO-Severo Ochoa PhD Fellowship (BES-2017-081995) (L.A.-E.) and an AECC postdoctoral fellowship (POSTD19016PRIE) (N.P.). M.V. is an EMBO YIP member (4053). Additional support was provided by Gertrud and Erich Roggenbuck Stiftung (M.M.), Science Foundation Ireland Frontiers for the Future Award (19/FFP/6443) (L.Y.), Science Foundation Ireland Strategic Partnership Programme, Precision Oncology Ireland (18/SPP/3522) (L.Y.), Breast Cancer Now Fellowship Award with the generous support of Walk the Walk (2019AugSF1310) (D.V.), Science Foundation Ireland (20/FFP-P/8597) (D.V.), Paradifference Foundation (C.F.-T.), “la Caixa” Foundation (ID 100010434) (A.I.), European Union’s Horizon 2020 research and innovation programme under Marie Skłodowska-Curie grant agreement 847648 (CF/BQ/PI20/11760029) (A.I.), Champalimaud Centre for the Unknown (N.S.), Lisboa Regional Operational Programme (Lisboa 2020) (LISBOA01-0145-FEDER-022170) (N.S.), NCI (R01 CA227629; R01 CA218133) (S.I.G.), Fundació Roses Contra el Càncer (J.B.-B.), Ministerio de Universidades FPU Fellowship (FPU 18/00069) (P.T.), MICIN-Agencia Estatal de Investigación Fellowships (PRE2020-093032 and BES-2017-080415) (P.M. and E. Cintado, respectively), Ministerio de Ciencia, Innovación y Universidades-E050251 (PID2019-110292RB-I00) (J.L.T.), FCT (PTDC/MED-ONC/32222/2017) (C.C.F.), Fundação Millennium bcp (C.C.F.), private donations (C.C.F.) and the Foundation for Applied Cancer Research in Zurich (E.L.R. and M.W.)

Treatment outcomes in multidrug resistant tuberculosis-human immunodeficiency virus Co-infected patients on anti-retroviral therapy at Sizwe Tropical Disease Hospital Johannesburg, South Africa

BACKGROUND: Multidrug resistant-tuberculosis (MDR-TB) is a threat to global tuberculosis control which is worsened by human immune-deficiency virus (HIV) co-infection. There is however paucity of data on the effects of antiretroviral treatment (ART) before or after starting MDR-TB treatment. This study determined predictors of mortality and treatment failure among HIV co-infected MDR-TB patients on ART. METHODS: A retrospective medical record review of 1200 HIV co-infected MDR-TB patients admitted at Sizwe Tropical Disease Hospital, Johannesburg from 2007 to 2010 was performed. Chi-square test was used to determine treatment outcomes in HIV co-infected MDR-TB patients on ART. Multivariable logistic regression and Poisson models were used to determine predictors of mortality and treatment failure respectively. RESULTS: Mortality was higher (21.8 % vs. 15.4 %) among patients who started ART before initiating MDR-TB treatment compared with patients initiated on ART after commencing MDR-TB treatment (p = 0.013). Factors significantly associated with mortality included: the use of ART before starting MDR-TB treatment (OR 1.65, 95 % CI 1.02–2.73), severely-underweight (OR 3.71, 95 % CI 1.89–7.29) and underweight (OR 2.35, 95 % CI 1.30–4.26), cavities on chest x-rays at baseline (OR 1.76, 95 % CI 1.08–2.94), presence of other opportunistic infections (OR 1.80, 95 % CI 1.10–2.94) and presence of other co-morbidities (OR 2.26, 95 % CI 1.20–4.21). Factors predicting failure were severe anaemia (IRR (OR 4.72, 95 % CI 1.47–15), other co-morbidities (OR 2.39, 95 % CI 1.05–5.43) and modified individualised regimen at baseline (OR 2.15, 95 % CI 0.98–4.71). CONCLUSIONS: High mortality among patients already on ART before initiating MDR-TB treatment is a worrisome development. Management of adverse-events, opportunistic infections and co-morbidities in these patients is important if the protective benefits of being on ART are to be maximized. There is the need to intensify intervention programmes targeted at early identification of MDR-TB, treatment initiation, drug monitoring and increasing adherence among HIV co-infected MDR-TB patients