Immune evasion by Helicobacter pylori is mediated by induction of macrophage arginase II

Helicobacter pylori infection persists for the life of the host due to the failure of the immune response to eradicate the bacterium. Determining how H. pylori escapes the immune response in its gastric niche is clinically important. We have demonstrated in vitro that macrophage NO production can kill H. pylori, but induction of macrophage arginase II (Arg2) inhibits inducible NO synthase (iNOS) translation, causes apoptosis, and restricts bacterial killing. Using a chronic H. pylori infection model, we determined whether Arg2 impairs host defense in vivo. In C57BL/6 mice, expression of Arg2, but not arginase I, was abundant and localized to gastric macrophages. Arg2(-/-) mice had increased histologic gastritis and decreased bacterial colonization compared with wild-type (WT) mice. Increased gastritis scores correlated with decreased colonization in individual Arg2(-/-) mice but not in WT mice. When mice infected with H. pylori were compared, Arg2(-/-) mice had more gastric macrophages, more of these cells were iNOS(+), and these cells expressed higher levels of iNOS protein, as determined by flow cytometry and immunofluorescence microscopy. There was enhanced nitrotyrosine staining in infected Arg2(-/-) versus WT mice, indicating increased NO generation. Infected Arg2(-/-) mice exhibited decreased macrophage apoptosis, as well as enhanced IFN-γ, IL-17a, and IL-12p40 expression, and reduced IL-10 levels consistent with a more vigorous Th1/Th17 response. These studies demonstrate that Arg2 contributes to the immune evasion of H. pylori by limiting macrophage iNOS protein expression and NO production, mediating macrophage apoptosis, and restraining proinflammatory cytokine responses.     

Prediction of pharmacokinetic properties using experimental approaches during early drug discovery

There has been a significant increase in the number of compounds synthesized in early drug-discovery programs with the advances in combinatorial chemistry and high-throughput biological screening efforts. Various in silico, in vitro and in situ approaches have been described in literature that achieve higher throughput pharmacokinetic screening. In silico methodologies have mainly attempted to quantify the prospects of oral absorption of compounds based upon their physico-chemical properties. There is a greater availability of in vitro and in situ approaches to screen compounds for intestinal permeability (as a surrogate for absorption) and metabolic stability (as a surrogate for clearance). More recent modifications of the in vitro and in situ approaches to assess the potential of absorption and metabolism have enabled a higher throughput and an ability to correlate better with in vivo pharmacokinetics of compounds.     

Intermittent Right Ventricular Outflow Tract Capture due to Chronic Right Atrial Lead Dislodgement

A 58 year old male, known case of type 2 diabetes and hypertension, had undergone implantation of a dual chamber pacemaker(DDDR) in 2007 for complaints of recurrent syncope and trifascicular block with a normal ejection fraction andnormal coronaries. His post implantation parameters were normal at that time.He now presented to our pacemaker clinic where his ECG done showed two types o fpaced complexes. The first few complexes were consistent with atrial sensed right ventricular apical pacing with left superior axis. Later complexes showed loss of atrial sensing with pacing from right ventricular outflow tract(inferior axis) with subtle oscillation in it''s axis. On application of magnet, two pacemaker spikes were visible withinterspike interval of 120 ms and paced complexes with inferior axis starting from the first spike suggesting that the atrial lead was responsible for RVOT depolarization. On interrogation of the pacemaker, atrial EGM showed sensed activity from atrium followed by large sensed ventricular complex. Fluoroscopy confirmed that the atrial lead was dislodged and was intermittently prolapsing into the RVOT. Since the patient was asymptomatic, he refused any intervention and subsequentlyhis atrial lead was switched off by telemetry. The above case signifies that asymptomatic lead dislodgement is no talways manifested as loss of capture and even subtle variation of the axis o fthe paced complexes can provide us with a clue that can be confirmed by telemetry of the pacemaker and fluoroscopy.     

Carbon Dioxide is a Powerful Inducer of Monokaryotic Hyphae and Spore Development in Cryptococcus gattii and Carbonic Anhydrase Activity is Dispensable in This Dimorphic Transition

Cryptococcus gattii is unique among human pathogenic fungi with specialized ecological niche on trees. Since leaves concentrate CO₂, we investigated the role of this gaseous molecule in C. gattii biology and virulence. We focused on the genetic analyses of β-carbonic anhydrase (β-CA) encoded by C. gattii CAN1 and CAN2 as later is critical for CO₂ sensing in a closely related pathogen C. neoformans. High CO₂ conditions induced robust development of monokaryotic hyphae and spores in C. gattii. Conversely, high CO₂ completely repressed hyphae development in sexual mating. Both CAN1 and CAN2 were dispensable for CO₂ induced morphogenetic transitions. However, C. gattii CAN2 was essential for growth in ambient air similar to its reported role in C. neoformans. Both can1 and can2 mutants retained full pathogenic potential in vitro and in vivo. These results provide insight into C. gattii adaptation for arboreal growth and production of infectious propagules by β-CA independent mechanism(s).     

Caffeine abolishes the mammalian G(2)/M DNA damage checkpoint by inhibiting ataxia-telangiectasia-mutated kinase activity

Recent evidence indicates that arrest of mammalian cells at the G(2)/M checkpoint involves inactivation and translocation of Cdc25C, which is mediated by phosphorylation of Cdc25C on serine 216. Data obtained with a phospho-specific antibody against serine 216 suggest that activation of the DNA damage checkpoint is accompanied by an increase in serine 216 phosphorylated Cdc25C in the nucleus after exposure of cells to gamma-radiation. Prior treatment of cells with 2 mM caffeine inhibits such a change and markedly reduces radiation-induced ataxia-telangiectasia-mutated (ATM)-dependent Chk2/Cds1 activation and phosphorylation. Chk2/Cds1 is known to localize in the nucleus and to phosphorylate Cdc25C at serine 216 in vitro. Caffeine does not inhibit Chk2/Cds1 activity directly, but rather, blocks the activation of Chk2/Cds1 by inhibiting ATM kinase activity. In vitro, ATM phosphorylates Chk2/Cds1 at threonine 68 close to the N terminus, and caffeine inhibits this phosphorylation with an IC(50) of approximately 200 microM. Using a phospho-specific antibody against threonine 68, we demonstrate that radiation-induced, ATM-dependent phosphorylation of Chk2/Cds1 at this site is caffeine-sensitive. From these results, we propose a model wherein caffeine abrogates the G(2)/M checkpoint by targeting the ATM-Chk2/Cds1 pathway; by inhibiting ATM, it prevents the serine 216 phosphorylation of Cdc25C in the nucleus. Inhibition of ATM provides a molecular explanation for the increased radiosensitivity of caffeine-treated cells.     

Development and in vitro characterization of a bivalent DNA containing HN and F genes of velogenic Newcastle disease virus

Newcastle disease (ND) is highly contagious, economically important viral disease affecting most of avian species worldwide. Newcastle disease virus (NDV) has single stranded negative sense RNA genome which encodes for six structural and two non-structural proteins. Envelope glycoproteins i.e. hemagglutinin-neuraminidase (HN) and the fusion (F), elicit protective immune response. In this study, HN and F genes of velogenic (virulent) strain were amplified and cloned at multiple cloning sites A and B, respectively into pIRES bicistronic vector for use as bivalent DNA vaccine against ND. The recombinant plasmid was characterized for its orientation by restriction enzyme digestion and PCR. Expression of HN and F genes was assessed in transfected Vero cells at RNA level using RT-PCR in total RNA as well as protein level using IFAT, IPT and western blot using NDV specific antiserum. All these experiments confirmed that HN and F genes cloned in recombinant pIRES.nd.hn.f are functionally active. The recombinant construct is being evaluated as DNA vaccine against ND.     

Caulogenic Efficacy of Cytokinin in Internodal Explants of Rosmarinus officinalis L. as Affected by Auxin and Inorganic Salts

Caulogenesis took place in internodal stem segments of Rosmarinus officinalis in the presence of both 6-benzylaminopurine (BAP) and 3-indoleacetic acid (IAA). There existed a synergism between BAP and IAA, where lower concentration of 0.1 mg/l IAA increased the shoot bud inducing efficacy of BAP to about ten times, while at 0.25 mg/l IAA it was counteracted. Presence of both NH₄NO₃ and KNO₃ was essential for shoot bud differentiation. While caulogenesis greatly declined if any of the inorganic salts was lacking in the medium, it increased in the absence of CaCl₂. The regeneration potentiality of the first internode was more than the second internode and within the internode it was more in the upper end than the lower. Shoot meristems differentiated from the subepidermal tissue from the meristemoids, which were formed in close proximity with nests of tracheary tissue. There was no direct regeneration from the epidermal cells.