Oxidants, antioxidants, and respiratory tract lining fluids.

Respiratory tract lining fluids (RTLFs) are a heterogeneous group of substances covering the respiratory tract epithelial cells (RTECs) from nasal mucosa to alveoli. Antioxidant contained in the RTLFs can be expected to provide an initial defense against inhaled environmental toxins. The major antioxidants in RTLF include mucin, uric acid, protein (largely albumin), ascorbic acid, and reduced glutathione (GSH). RTLF antioxidants can be augmented by such processes as transudation/exudation of plasma constituents; RTEC secretory processes, including glandular mucus secretion; and cellular antioxidants derived from lysis of RTECs and of inflammatory cells. The antioxidant composition of RTLFs and their role in modulating normal and pathophysiologic RTEC functions under conditions of oxidative stress are yet to be fully characterized

Impaired Chromatin Remodelling at STAT1-Regulated Promoters Leads to Global Unresponsiveness of Toxoplasma gondii-Infected Macrophages to IFN-γ

Intracellular pathogens including the apicomplexan and opportunistic parasite Toxoplasma gondii profoundly modify their host cells in order to establish infection. We have shown previously that intracellular T. gondii inhibit up-regulation of regulatory and effector functions in murine macrophages (MΦ) stimulated with interferon (IFN)-γ, which is the cytokine crucial for controlling the parasites' replication. Using genome-wide transcriptome analysis we show herein that infection with T. gondii leads to global unresponsiveness of murine macrophages to IFN-γ. More than 61% and 89% of the transcripts, which were induced or repressed by IFN-γ in non-infected MΦ, respectively, were not altered after stimulation of T. gondii-infected cells with IFN-γ. These genes are involved in a variety of biological processes, which are mostly but not exclusively related to immune responses. Analyses of the underlying mechanisms revealed that IFN-γ-triggered nuclear translocation of STAT1 still occurred in Toxoplasma-infected MΦ. However, STAT1 bound aberrantly to oligonucleotides containing the IFN-γ-responsive gamma-activated site (GAS) consensus sequence. Conversely, IFN-γ did not induce formation of active GAS-STAT1 complexes in nuclear extracts from infected MΦ. Mass spectrometry of protein complexes bound to GAS oligonucleotides showed that T. gondii-infected MΦ are unable to recruit non-muscle actin to IFN-γ-responsive DNA sequences, which appeared to be independent of stimulation with IFN-γ and of STAT1 binding. IFN-γ-induced recruitment of BRG-1 and acetylation of core histones at the IFN-γ-regulated CIITA promoter IV, but not β-actin was diminished by >90% in Toxoplasma-infected MΦ as compared to non-infected control cells. Remarkably, treatment with histone deacetylase inhibitors restored the ability of infected macrophages to express the IFN-γ regulated genes H2-A/E and CIITA. Taken together, these results indicate that Toxoplasma-infected MΦ are unable to respond to IFN-γ due to disturbed chromatin remodelling, but can be rescued using histone deacetylase inhibitors

Genome-wide screens identify Toxoplasma gondii determinants of parasite fitness in IFNγ-activated murine macrophages

Macrophages play an essential role in the early immune response against Toxoplasma and are the cell type preferentially infected by the parasite in vivo. Interferon gamma (IFNγ) elicits a variety of anti-Toxoplasma activities in macrophages. Using a genome-wide CRISPR screen we identify 353 Toxoplasma genes that determine parasite fitness in naїve or IFNγ-activated murine macrophages, seven of which are further confirmed. We show that one of these genes encodes dense granule protein GRA45, which has a chaperone-like domain, is critical for correct localization of GRAs into the PVM and secretion of GRA effectors into the host cytoplasm. Parasites lacking GRA45 are more susceptible to IFNγ-mediated growth inhibition and have reduced virulence in mice. Together, we identify and characterize an important chaperone-like GRA in Toxoplasma and provide a resource for the community to further explore the function of Toxoplasma genes that determine fitness in IFNγ-activated macrophages

Diagnóstico broncoscópico das pneumonias em doentes queimados com lesão por inalação de fumo

RESUMO: A broncofibroscopia (BFB) com culturas bacteriológicas quantitativas das secreções brônquicas (SB) e do lavado broncoalveolar (LBA) são úteis no diagnóstico das pneumonias em doentes internados em unidades de cuidados intensivos.Este estudo teve como objectivo determinar a utilidade das culturas quantitativas obtidas por BFB em doentes queimados que foram sujeitos a inalação de fumo. Os autores relacionaram os resultados com as alterações radiológicas, com as atitudes terapêuticas e com o outcome.O estudo foi retrospectivo, tendo sido revistos 34 processos clínicos de doentes queimados com lesões por inalação de fumo, internados numa unidade de queimados de um hospital universitário. Foram analisadas as radiografias do tórax, os resultados das BFB com as respectivas culturas quantitativas das SB e LBA, a clínica e o outcome.Todos os doentes com suspeita de PNM foram sujeitos a BFB, não havendo complicações com a técnica. A pneumonia foi confirmada por culturas quantitativas das SB e LBA em 58% dos doentes, em que houve suspeição clínica de PNM, e em 22,8% dos doentes queimados com lesões por inalação de fumo.A bacteriologia foi relacionada com as alterações radiológicas, sendo as culturas quantitativas das SB e do LBA mais positivas (p=0,0045) do que as alterações radiológicas. A flora nosocomial (os Gram â e o staphilococus) aumenta com a duração da hospitalização (p<0,05) e está mais vezes relacionada com o aparecimento de opacidades pulmonares (p=0,026).Os resultados bacteriológicos das culturas quantitativas das SB e LBA levaram ao ajustamento da antibioterapia em 72% dos doentes.A mortalidade nas unidades de queimados ronda os 12%, aumentando para 23% quando há lesões por inalação de fumo. Nos doentes queimados com PNM diagnosticada, a mortalidade foi de 23% neste estudo.A BFB nos doentes queimados com lesões por inalação de fumo tem o objectivo de diagnóstico das lesões provocadas pela inalação de fumo e de diagnóstico das sobreinfecções responsáveis pelas PNM.Não se conseguiram tirar conclusões sobre o custo/efectividade desta estratégia e sobre o outcome destes doentes. COMENTÃRIO: A PNM é uma complicação tardia nos doentes queimados, particularmente nos que foram sujeitos a inalação de fumo. A mortalidade nestes casos pode mesmo duplicar, conforme algumas séries publicadas.Os doentes queimados com lesões provocadas por inalação de fumo podem apresentar infiltrados pulmonares, secreções brônquicas abundantes, febre, leucocitose e alterações inflamatórias de fase aguda, alterações que podem dificultar o diagnóstico das pneumonias.Analisados estes aspectos e os resultados deste trabalho, parecenos que a BFB tem um papel importante não só no diagnóstico das lesões endobrônquicas provocadas pela inalação de fumo, mas também no diagnóstico das sobreinfecções responsáveis pelas pneumonias, permitindo ainda fazer a toilette da árvore brônquica, eventualmente queimada.Os doentes sujeitos a ventilação mecânica podem desenvolver quadros clínicos de Síndrome de Dificuldade Respiratória no Adulto (SDRA), de edema cardiogénico, de atelectasia, o que dificulta o eventual diagnóstico das pneumonias, pelo que a BFB vai permitir objectivar, ou não, a existência das sobreinfecções e subsequente PNM.Apesar da controvérsia que existe sobre a valorização destes dados, não podemos deixar de salientar que este grupo de trabalho alterou a estratégia terapêutica antibiótica em 72% dos casos, permitindo uma abordagem mais rigorosa e o uso mais selectivo dos antibióticos, com as vantagens conhecidas â diminuir a probalidade de resistências microbianas e a diminuição de custos.Pensamos que o desenvolvimento deste estudo permitirá avaliar e estabelecer o valor preditivo desta estratégia, assim como tirar conclusões sobre o outcome destes doentes, o que neste trabalho foi impossí-vel.As limitações de todos estes aspectos não nos impede de considerar que a BFB é não só um método útil de diagnóstico das lesões endobrônquicas provocadas pela inalação de fumo, mas também na detecção das sobreinfecções responsáveis pelo desenvolvimento das PNM nestes doentes. Palavras-chave: Pneumonia (PNM), lesão por inalação de fumo, culturas bacteriológicas quantitativas, broncofibroscopia (BFB), queimado

AMPA GluA1-flip targeted oligonucleotide therapy reduces neonatal seizures and hyperexcitability

<div><p>Glutamate-activated α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPA-Rs) mediate the majority of excitatory neurotransmission in brain and thus are major drug targets for diseases associated with hyperexcitability or neurotoxicity. Due to the critical nature of AMPA-Rs in normal brain function, typical AMPA-R antagonists have deleterious effects on cognition and motor function, highlighting the need for more precise modulators. A dramatic increase in the flip isoform of alternatively spliced AMPA-R GluA1 subunits occurs post-seizure in humans and animal models. GluA1-flip produces higher gain AMPA channels than GluA1-flop, increasing network excitability and seizure susceptibility. Splice modulating oligonucleotides (SMOs) bind to pre-mRNA to influence alternative splicing, a strategy that can be exploited to develop more selective drugs across therapeutic areas. We developed a novel SMO, GR1, which potently and specifically decreased GluA1-flip expression throughout the brain of neonatal mice lasting at least 60 days after single intracerebroventricular injection. GR1 treatment reduced AMPA-R mediated excitatory postsynaptic currents at hippocampal CA1 synapses, without affecting long-term potentiation or long-term depression, cellular models of memory, or impairing GluA1-dependent cognition or motor function in mice. Importantly, GR1 demonstrated anti-seizure properties and reduced post-seizure hyperexcitability in neonatal mice, highlighting its drug candidate potential for treating epilepsies and other neurological diseases involving network hyperexcitability.</p></div

Using cost and health impacts to prioritize the targeted testing of tuberculosis in the United States

Purpose: Evaluation improves efficiency and effectiveness. Current U.S. tuberculosis (TB) control policies emphasize the treatment of latent TB infection (LTBI). However, this policy, if not targeted, may be inefficient. We determined the efficiency of a state-law mandated TB screening program and a non state-law mandated one in terms of cost, morbidity, treatment, and disease averted. Methods: We evaluated two publicly funded metropolitan TB prevention and control programs through retrospective analyses and modeling. Main outcomes measured were TB incidence and prevalence, TB cases averted, and cost. Results: A non state-law mandated TB program for homeless persons in Tarrant County screened 4.5 persons to identify one with LTBI and 82 persons to identify one with TB. A state-law mandated TB program for jail inmates screened 109 persons to identify one with LTBI and 3274 persons to identify one with TB. The number of patients with LTBI treated to prevent one TB case was 12.1 and 15.3 for the homeless and jail inmate TB programs, respectively. Treatment of LTBI by the homeless and jail inmate TB screening programs will avert 11.9 and 7.9 TB cases at a cost of $14,350 and$34,761 per TB case, respectively. Conclusions: Mandated TB screening programs should be risk-based, not population-based. Non mandated targeted testing for TB in congregate settings for the homeless was more efficient than state-law mandated targeted testing for TB among jailed inmates

A single ICV injection of GR1 provided a long-lasting reduction in GluA1-flip expression without adverse effects on cognition.

<p>Mice were a given ICV injection of GR1 (4 μg) at P10, <b>(a)</b> Real-time PCR showed GR1 reduced GluA1-flip in the cortex and hippocampus at all time-points from 2–60 d after administration (n = 4–5; p < 0.001). <b>(b)</b> GluA1-flop expression levels, measured in same samples as in panel <b>a</b>, were only marginally different between the GR1-treated and saline-treated controls. <b>(c)</b> GR1-treated mice did not show impairment in Y maze performance when tested at P30, compared to saline-treated controls (p > 0.05; n = 8–9). <b>(d)</b> In the object recognition paradigm, GR1-injected mice showed similar preferences for exploring novel objects as saline-injected controls (p > 0.05 between groups; same cohort of mice as in panel <b>c</b>). GluA1-flip expression was analyzed by one-sample Student’s t-test with bonferoni correction, novel object preference by unpaired two-sample two-tailed t-tests, and Y-maze performance by Fischer’s exact test.</p

CA1 synaptic function measured extracellularly in P10 hippocampal slices from mice injected at P1, P3, and P5 with saline or GR1 (2 μg).

<p><b>(a).</b> Stimulus-response curves for fEPSPs at CA1 synapses showed a reduction in fEPSP amplitude in slices from GR1-treated mice (p < 0.01). <b>(b)</b> Paired-pulse facilitation was normal after GR1 treatment (p > 0.05). <b>(c)</b> LTP was normal in GR1-treated mice. Data shows % baseline slope before and after two high frequency trains. No difference was observed between slices from saline and GR1-treated mice (p > 0.05). <b>(d)</b> LTD was normal in GR1-treated mice. Data shows % baseline slope before and after a 15 min 1 Hz train. No difference was observed between slices from saline and GR1-treated mice (p > 0.05). In all panels <b>a-c</b>, n = 7 for saline and GR1-treated groups. In panel <b>d</b>, saline n = 8, GR1-treated n = 7. Asterisks indicate significant difference between GR1 and saline-treated groups. Two-factor ANOVA was performed on extracellular recording data.</p

Anti-seizure effects of GR1.

<p>GR1 protected neonatal mice from KA-induced seizures and prevented SE-induced increase in aEPSC amplitude. <b>(a)</b> Three 2 μg ICV doses of GR1 given at P1, P3, and P5 significantly reduced the percentage of mice progressing to severe seizure stages after a single dose of KA (3 mg/kg- IP) at P10, compared to saline-treated controls (n = 11 per group; p < 0.001 by Fisher’s exact test). <b>(b-c)</b> Total KA dose required to elicit SE and latency to reach individual seizures stages at P10 was significantly greater in mice treated with 2 μg (x3 doses) of GR1, compared to saline-treated controls (n = 6 per group; p < 0.05 and p < 0.01 respectively) as analyzed by two-factor ANOVA. <b>(d)</b> A single 4 μg dose of GR1, given 2 hours post-P10 SE, prevented the increased susceptibility to “double hit” KA seizure observed for saline-treated SE-experienced mice at P12, compared to naïve controls (*p < 0.05, n = 7 per group). <b>(e)</b> Whole-cell patch-clamp recordings of aEPSCs from CA1 pyramidal neurons in P12 mice treated as in panel <b>d</b>. Recordings from double hit SE-experience mice demonstrated a large increase in aEPSC amplitude compared to naïve (no SE) mice (p < 0.001) This SE-induced potentiation of aEPSCs was completely prevented by injection of 4 μg GR1 at 2 hr post-SE onset at P10 (n = 5–7 mice per group). Representative traces of maximal aEPSCs are shown for saline and GR1 treated mice post-SE (scale bars: 50 ms, 200 pA). Panels d and e were assessed by unpaired two-tailed t-test.</p