CT‐guided aspiration cytology of advanced silicosis and confirmation of the deposited zeolite nano particles through X ray diffraction: A novel approach

Silicosis is a common occupational lung disease, resulting in fibrotic nodular lesions in the upper lobes of the lung parenchyma. Most of the pneumoconioses are diagnosed on the basis of relevant history and clinico‐radiological correlation. Image‐guided aspiration cytology appears to be poorly yielding and is not usually considered as a diagnostic modality. However, silicosis may sometimes offer a diagnostic challenge because of its radiological resemblance and clinical overlap with pulmonary tuberculosis and neoplastic lesions. We present a unique situation where image‐guided fine needle aspiration cytology (FNAC) has been advised on the basis of nodular upper lobe opacities. The cytology smears revealed hypocellular granular material, while phase contrast and polarized light microscopy highlighted crystalline particles. History of silica dust exposure long back was available after the cytological evaluation, suggesting the diagnosis of pulmonary silicosis. X ray diffraction (XRD) crystallography was also possible on cytology smears, confirming zeolite nano particles of size as small as 40 ? 50 nm as the concerned agent for the first time. Cytological evaluation by phase contrast and polarized light microscopy may be useful for the confirmation of silicosis, supplemented by clinical history and radiological evaluation. XRD on smears may help in determination of chemical nature and particle size. Diagn. Cytopathol. 2016;44:246–249. © 2015 Wiley Periodicals, Inc.     

Leishmania donovani-induced ceramide as the key mediator of Akt dephosphorylation in murine macrophages: role of protein kinase Czeta and phosphatase

Leishmania donovani is an intracellular protozoan parasite that impairs the host macrophage immune response to render it suitable for its survival and establishment. L. donovani-induced immunosuppression and alteration of host cell signaling is mediated by ceramide, a pleiotropic second messenger playing an important role in regulation of several kinases, including mitogen-activated protein kinase and phosphatases. We observed that the endogenous ceramide generated during leishmanial infection led to the dephosphorylation of protein kinase B (PKB) (Akt) in infected cells. The study of ceramide-mediated Akt phosphorylation revealed that Akt was dephosphorylated at both Thr308 and Ser473 sites in infected cells. Further investigation demonstrated that ceramide was also responsible for the induction of PKCzeta, an atypical Ca-independent stress kinase, as well as the ceramide-activated protein phosphatases (e.g., protein phosphatase 2A [PP2A]). We found that Akt dephosphorylation was mediated by ceramide-induced PKCzeta-Akt association and PP2A activation. In addition, treatment of L. donovani-infected macrophages with PKCzeta-specific inhibitor peptide could restore the translocation of phosphorylated Akt to the cell membrane. This study also revealed that ceramide is involved in the inhibition of proinflammatory cytokine tumor necrosis factor alpha release by infected macrophages. These observations strongly suggest the importance of ceramide in the alteration of normal cellular functions, impairment of the kinase/phosphatase balance, and thereby establishment of leishmaniasis in the hostile macrophage environment.     

Formulation Development,Optimization, and In Vitro–In Vivo Characterization of Natamycin-Loaded PEGylated Nano-Lipid Carriers for Ocular Applications

The present study aimed at formulating and optimizing natamycin (NT)-loaded polyethylene glycosylated nano-lipid carriers (NT-PEG-NLCs) using Box-Behnken design and investigating their potential in ocular applications. Response surface methodology computations and plots for optimization were performed using Design-Expert^® software to obtain optimum values for response variables based on the criteria of desirability. Optimized NT-PEG-NLCs had predicted values for the dependent variables which are not significantly different from the experimental values. NT-PEG-NLCs were characterized for their physicochemical parameters; NT's rate of permeation and flux across rabbit cornea was evaluated, in vitro, and ocular tissue distribution was assessed in rabbits, in vivo. NT-PEG-NLCs were found to have optimum particle size (<300 nm), narrow polydispersity index, and high NT entrapment and NT content. In vitro transcorneal permeability and flux of NT from NT-PEG-NLCs was significantly higher than that of Natacyn^®. NT-PEG-NLC (0.3%) showed improved delivery of NT across the intact cornea and provided concentrations statistically similar to the marketed suspension (5%) in inner ocular tissues, in vivo, indicating that it could be a potential alternative to the conventional suspension during the course of fungal keratitis therapy.     

Using a microcantilever array for detecting phase transitions and stability of DNA

The authors report the extension of the microcantilever platform to study the thermal phase transition of biomolecules as they are heated. Microcantilever-based sensors directly translate changes in Gibbs free energy due to macromolecular interactions into mechanical responses. The authors observed surface stress changes in response to thermal dehybridization of double-stranded DNA oligonucleotides that are attached onto one side of a microcantilever. Once the cantilever is heated, the DNA undergoes a transition as the complementary strand melts, which results in changes in the cantilever deflection. This deflection is due to changes in the electrostatic, ionic, and hydration interaction forces between the remaining immobilized DNA strands. This new technique has allowed the authors to probe DNA melting dynamics and leads to a better understanding of the stability of DNA complexes on surfaces.     

The value of prognostic indices for pneumonia

One of the most important decisions in the management of community-acquired pneumonia is deciding the care site, which affects morbidity, mortality, and costs. Clinical judgment alone is difficult and imprecise. The Pneumonia Severity Index score and the CURB-65 (confusion, urea nitrogen, respiratory rate, blood pressure, 65 years of age and older) score are validated prognostic indices to predict mortality, and they can identify low-risk patients who may be eligible for outpatient management. However, limitations of the scoring systems preclude their isolated use, and they can only be recommended as an aid to guide hospital admission decisions. The Pneumonia Severity Index score is slightly better at identifying the lowest risk patients, whereas CURB-65 is much simpler to use. As an adjunct to clinical judgment, we consider CURB-65 to be the most useful prognostic index for identifying low-risk patients.     

Regulation of protein synthesis in rabbit reticulocyte lysates by the heme-regulated protein kinase: Inhibition of interaction of Met-tRNAfMet binding factor with another initiation factor in formation of Met-tRNAfMet·40S ribosomal subunit complexes

Protein synthesis in reticulocytes and their lysates is regulated by heme. In heme deficiency a heme-regulated translational inhibitor (HRI) that blocks initiation of polypeptide chains is activated. HRI is a protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) that specifically phosphorylates the 38,000-dalton subunit of the Met-tRNA(f) (Met) binding factor (IF), which forms a ternary complex with Met-tRNA(f) (Met) and GTP, a finding that suggests that the inhibition by HRI involves the phosphorylation of IF.We have investigated the effect of HRI in the partial reactions of protein chain initiation in which the IF-promoted binding of Met-tRNA(f) (Met) to 40S ribosomal subunits is enhanced by another initiation factor [ternary complex dissociation factor (TDF)] and AUG. The results show that HRI at very low concentrations markedly inhibits the binding of Met-tRNA(f) (Met) to 40S subunits. The inhibitory effect of HRI requires ATP. Under these conditions HRI phosphorylates only the 38,000-dalton subunit of IF.The TDF preparations not only promote the binding of the ternary complex to 40S subunits but also promote the dissociation of the ternary complex in the presence of 5 mM Mg(2+) at 0 degrees . The preincubation of purified IF alone with low concentrations of HRI and ATP does not significantly affect its capacity to form the ternary complex; however, the TDF-promoted dissociation of the ternary complex is inhibited. The nonhydrolyzable analog adenosine 5'-[beta,gamma-imido]triphosphate does not substitute for ATP. These findings suggest that phosphorylation causes a conformational modification in IF, which results in inhibition of the interaction between the ternary complex and TDF that is required for the binding of the ternary complex to 40S subunits.     

Leishmanial lipid suppresses tumor necrosis factor alpha, interleukin-1beta, and nitric oxide production by adherent synovial fluid mononuclear cells in rheumatoid arthritis patients and induces apoptosis through the mitochondrial-mediated pathway

OBJECTIVE: Leishmanial lipid is a strong immunosuppressor of host cells. Inhibition of the inflammatory responses of synovial cells through induction of apoptosis is one of the main targets of therapeutic intervention in rheumatoid arthritis (RA). This study was undertaken to examine the antiinflammatory and apoptosis-inducing effects of leishmanial lipid on adherent synovial fluid mononuclear cells (SFMCs) in patients with RA. METHODS: Lipid was extracted from a Leishmania donovani promastigote (MHO/IN/1978/UR6) by the Bligh and Dyer method. Nitric oxide (NO) was measured using the Griess reaction, and enzyme-linked immunosorbent assays for cytokines, NF-kappaB, and cytochrome c were performed. Levels of cytokines, inducible nitric oxide synthase, caspases, Bcl-2, Bax, t-Bid, and cytochrome c in the cell lysate and of NF-kappaB p65 in the nucleus were determined by Western blotting. Microscopic analysis, nuclear staining, DNA fragmentation assay, fluorescence-activated cell sorting, colorimetric assay for caspases, and fluorescent probe for measurement of mitochondrial membrane potential were used to study the leishmanial lipid-induced apoptotic pathway in SFMCs. RESULTS: Leishmanial lipid inhibited the release of tumor necrosis factor alpha, interleukin-1beta, and NO in the culture, decreased their cytosolic protein levels, and decreased NF-kappaB p65 levels in SFMCs, in a dose-dependent manner. It had the reverse effect on interleukin-10 levels. Leishmanial lipid-induced apoptosis involved the activation of caspase 3, caspase 9, and Bax, the release of cytochrome c, the alteration of mitochondrial membrane potential, and the down-regulation of Bcl-2. CONCLUSION: These results suggest that leishmanial lipid has strong antiinflammatory and apoptosis-inducing effects on SFMCs from patients with RA, and that apoptosis occurs via the mitochondrial pathway.     

Epidermal growth factor regulation of DNA synthesis in human colonic lamina propria lymphocytes

Epidermal growth factor (EGF) is a potent growth factor for many tissues including the gastrointestinal tract. EGF is present in the gut lumen and is absorbed through the mucosa in the developing animals. In addition, EGF has been found to alter the immune system. In this study, we investigated thein vitro effect of EGF on normal colonic lamina propria lymphocyte DNA synthesis and ornithine decarboxylase activity. Human colonic lamina propria lymphocytes were isolated by collagenase-EDTA digestion. The effect of EGF on Con A-stimulated lymphocyte thymidine incorporation was tested. We observed that EGF suppressed DNA synthesis and ornithine decarboxylase (ODC) activity in lamina propria lymphocytes. EGF did not alter the time course of thymidine incorporation into LPL stimulated by the combination of phorbol 12,13-dibutyrate (PDB) and ionomycin. Our data suggest that (1) EGF suppresses DNA synthesis in human colonic lamina propria lymphocytes as well as ODC activity and (2) this inhibition may be mediated through protein kinase C or calcium flux. We postulate that EGF may have a role in modulating the human gut immune system.This work was supported in part by grant CA43280 from the National Institutes of Health.