Household Transmission of Rotavirus in a Community with Rotavirus Vaccination in Quininde,Ecuador

Background

We studied the transmission of rotavirus infection in households in peri-urban Ecuador in the vaccination era.

Methods

Stool samples were collected from household contacts of child rotavirus cases, diarrhea controls and healthy controls following presentation of the index child to health facilities. Rotavirus infection status of contacts was determined by RT-qPCR. We examined factors associated with transmissibility (index-case characteristics) and susceptibility (household-contact characteristics).

Results

Amongst cases, diarrhea controls and healthy control household contacts, infection attack rates (iAR) were 55%, 8% and 2%, (n = 137, 130, 137) respectively. iARs were higher from index cases with vomiting, and amongst siblings. Disease ARs were higher when the index child was <18 months and had vomiting, with household contact <10 years and those sharing a room with the index case being more susceptible. We found no evidence of asymptomatic infections leading to disease transmission.

Conclusion

Transmission rates of rotavirus are high in households with an infected child, while background infections are rare. We have identified factors associated with transmission (vomiting/young age of index case) and susceptibility (young age/sharing a room/being a sibling of the index case). Vaccination may lead to indirect benefits by averting episodes or reducing symptoms in vaccinees.     

Infection in Health Personnel with High and Low Levels of Exposure in a Hospital Setting during the H1N1 2009 Influenza A Pandemic

A novel H1N1 influenza A virus caused the first pandemic of the 21^st century in 2009. Hospitals had an increased demand of health consultations, that made it difficult to estimate the incidence of infection in hospital personnel due to asymptomatic presentations and the under notification of cases. To estimate and compare the rate of exposure of high versus low risk health personnel to 2009 pandemic H1N1 (H1N1pdm2009) influenza A virus in a University Hospital in Chile, we performed a comparative and prospective study. Serum samples were obtained from 117 individuals that worked in the emergency room (ER) and the operating room (OR) during the peak of the pandemic. Antibody titers were determined by the hemagglutination inhibition (HI) assay. Of the samples analyzed, 65% were workers at the ER and 35% at the OR. Of the total number of the subjects tested, 29.1% were seropositive. One out of 3 (36.8%) workers at the ER had positive HI titers, meanwhile only 1 out of 7 (14.6%) workers from the OR was seropositive to the virus. The possibility of being infected in the ER as compared to the OR was 3.4 times greater (OR 3.4; CI 95%, 1.27–9.1), and the individuals of the ER had almost twice as much antibody titers against H1N1pdm2009 than the personnel in the OR, suggesting the potential of more than one exposure to the virus. Of the 34 seropositive subjects, 12 (35.3%) did not develop influenza like illness, including 2 non-clinical personnel involved in direct contact with patients at the ER. Considering the estimated population attack rate in Chile of 13%, both groups presented a higher exposure and seropositive rate than the general population, with ER personnel showing greater risk of infection and a significantly higher level of antibodies. This data provide a strong rationale to design improved control measures aimed at all the hospital personnel, including those coming into contact with the patients prior to triage, to prevent the propagation and transmission of respiratory viruses, particularly during a pandemic outbreak.     

Yol082p, a novel CVT protein involved in the selective targeting of aminopeptidase I to the yeast vacuole

The yeast vacuolar enzyme aminopeptidase I (API) is synthesized in the cytoplasm as a precursor (pAPI). Upon its assembly into dodecamers, pAPI is wrapped by double-membrane saccular structures for its further transport within vesicles that fuse with the vacuolar membrane and release their content in the vacuolar lumen. Targeting of API to the vacuole occurs by two alternative transport routes, the cvt and the autophagy pathways, which although mechanistically similar specifically operate under vegetative growth or nitrogen starvation conditions, respectively. We have studied the role of Yol082p, a protein identified by its ability to interact with API, in the transport of its precursor to the vacuole. We show that Yol082p interacts with mature API, an interaction that is strengthened by the amino extension of the API protein. Yol082p is required for targeting of pAPI to the vacuole, both under growing and short term nitrogen starvation conditions. Absence of Yol082p does not impede the assembly of pAPI into dodecamers, but precludes the enclosure of pAPI within transport vesicles. Microscopy studies show that during vegetative growth Yol082p is distributed between a cytoplasmic pool and a variable number of 0.13--0.27-microm round, mobile structures, which are no longer observed under conditions of nitrogen starvation, and become larger in cells expressing the inactive Yol082 Delta C32p, or lacking Apg12p. In contrast to the autophagy mutants involved in API transport, a Delta yol082 strain does not lose viability under nitrogen starvation conditions, indicating normal function of the autophagy pathway. The data are consistent with a role of Yol082p in an early step of the API transport, after its assembly into dodecamers. Because Yol082p fulfills the functional requisites that define the CVT proteins, we propose to name it Cvt19.     

Thrombin induces proteinase-activated receptor-1 gene expression in endothelial cells via activation of Gi-linked Ras/mitogen-activated protein kinase pathway

We addressed the mechanisms of restoration of cell surface proteinase-activated receptor-1 (PAR-1) by investigating thrombin-activated signaling pathways involved in PAR-1 re-expression in endothelial cells. Exposure of endothelial cells transfected with PAR-1 promoter-luciferase reporter construct to either thrombin or PAR-1 activating peptide increased the steady-state PAR-1 mRNA and reporter activity, respectively. Pretreatment of reporter-transfected endothelial cells with pertussis toxin or co-expression of a minigene encoding 11-amino acid sequence of COOH-terminal Galphai prevented the thrombin-induced increase in reporter activity. Pertussis toxin treatment also prevented thrombin-induced MAPK phosphorylation, indicating a role of Galphai in activating the downstream MAPK pathway. Expression of constitutively active Galphai2 mutant or Gbeta1gamma2 subunits increased reporter activity 3-4-fold in the absence of thrombin stimulation. Co-expression of dominant negative mutants of either Ras or MEK1 with the reporter construct inhibited the thrombin-induced PAR-1 expression, whereas constitutively active forms of either Ras or MEK1 activated PAR-1 expression in the absence of thrombin stimulation. Expression of dominant negative Src kinase or inhibitors of phosphoinositide 3-kinase also prevented the MAPK activation and PAR-1 expression. We conclude that thrombin-induced activation of PAR-1 mediates PAR-1 expression by signaling through Gi1/2 coupled to Src and phosphoinositide 3-kinase, and thereby activating the downstream Ras/MAPK cascade.     

Intramembrane Charge Movement Associated with Endogenous K+ Channel Activity in HEK-293 Cells

1. The use of molecular biology in combination with electrophysiology in the HEK-293 cell line has given fascinating insights into neuronal ion channel function. Nevertheless, to fully understand the properties of channels exogenously expressed in these cells, a detailed evaluation of endogenous channels is indispensable. 2. Previous studies have shown the expression of endogenous voltage-gated K+, Ca2+, and Cl- channels and this predicts that changes in membrane potential will cause intramembrane charge movement, though this gating charge translocation remain undefined. Here, we confirm this prediction by performing patch-clamp experiments to record ionic and gating currents. Our data show that HEK-293 cells express at least two types of K+-selective endogenous channels which sustain the majority of the ionic current, and exclude a significant contribution from Ca2+ and Cl- channels to the whole-cell current. 3. Gating currents were unambiguously resolved after ionic current blockade enabling this first report of intramembrane charge movement in HEK-293 cells arising entirely from endogenous K+ channel activity, and providing valuable information concerning the activation mechanism of voltage-gated K+ channels in these cells.     

Amino-terminal and midmolecule parathyroid hormone-related protein,phosphatidylcholine, and type II cell proliferation in silica-injured lung

Acute silica lung injury is marked by alveolar phospholipidosis and type II cell proliferation. Parathyroid hormone-related protein (PTHrP) 1-34 could have a regulatory role in this process because it stimulates phosphatidylcholine secretion and inhibits type II cell growth. Other regions of the PTHrP molecule may have biological activity and can also exert pulmonary effects. This study examined the temporal pattern for expression of several regions of PTHrP after silica lung injury and evaluated the effects of changes in expression on cell proliferation and lung phospholipids. Expression of all PTHrP regions fell at 4 days after injury. Reversing the decline in PTHrP 1-34 or PTHrP 67-86 with one intratracheal dose and four daily subcutaneous doses of PTHrP 1-34 or PTHrP 67-86 stimulated bronchoalveolar lavage disaturated phosphatidylcholine (DSPC) levels. Cell culture studies indicate that the peptides exerted direct effects on DSPC secretion by type II cells. Neither peptide affected type II cell proliferation with this dosing regimen, but addition of an additional intratracheal dose resulted in significant inhibition of growth, consistent with previous effects of PTHrP 1-34 in hyperoxic lung injury. These studies establish a regulatory role for PTHrP 1-34 and PTHrP 67-86 in DSPC metabolism and type II cell proliferation in silica injury. Growth inhibitory effects of PTHrP could interact with phospholipid stimulation by affecting type II cell numbers. Further studies are needed to explore the complex interactions of PTHrP-derived peptides and the type II cell response at various stages of silica lung injury.     

Differentiation between acetylcholinesterase and the organophosphate-inhibited form using antibodies and the correlation of antibody recognition with reactivation mechanism and rate

Two types of polyclonal antibodies were generated from (a) a decapeptide sequence that includes the active site serine of acetylcholinesterase (anti-AChE10S) and (b) the identical decapeptide sequence phosphorylated at the active site serine of acetylcholinesterase (anti-AChE10SP). The anti-AChE10S antiserum was found to specifically recognize native, control, and vehicle-treated recombinant mouse AChE (rMoAChE) but did not recognize rMoAChE that was phosphorylated by the four organophosphate (OP) compounds tested. Conversely the anti-AChE10SP antiserum recognized phosphoserine rMoAChE that resulted from reaction with phosphorous oxychloride (POCl3) but did not recognize native or vehicle-treated rMoAChE. Anti-AChE10SP also did not recognize OP-AChE conjugates that resulted from the reaction of rMoAChE with other OP compounds that afford neutral or monoanionic phosphoserine groups thereby indicating a high specificity for a precise OP conjugate. Antisera recognition correlated well with the rates of enzyme inhibition, aging, and oxime-induced reactivation indicating these antisera can both quantify the extent and type of inhibition and also differentiate between select mechanisms of inhibition. The ability to discern mechanistic differences between native AChE and OP-AChE conjugates suggests that these antisera can be used to identify biomarkers of OP exposure in a mechanism-based approach.     

Functional studies of the kidney of living animals using multicolor two-photon microscopy

Optical microscopy, when applied to livinganimals, provides a powerful means of studying cell biology in the mostphysiologically relevant setting. The ability of two-photon microscopyto collect optical sections deep into biological tissues has opened upthe field of intravital microscopy to high-resolution studies of the brain, lens, skin, and tumors. Here we present examples of the way inwhich two-photon microscopy can be applied to intravital studies ofkidney physiology. Because the kidney is easily externalized withoutcompromising its function, microscopy can be used to evaluate variousaspects of renal function in vivo. These include cell vitality andapoptosis, fluid transport, receptor-mediated endocytosis, blood flow, and leukocyte trafficking. Efficient two-photon excitation of multiple fluorophores permits comparison of multiple probes andsimultaneous characterization of multiple parameters and yields spectral information that is crucial to the interpretation of imagescontaining uncharacterized autofluorescence. The studies described heredemonstrate the way in which two-photon microscopy can provide a levelof resolution previously unattainable in intravital microscopy,enabling kinetic analyses and physiological studies of the organs ofliving animals with subcellular resolution.

     

Distal recognition site for classical pathway convertase located in the C345C/netrin module of complement component C5

Previous studies focused on indels in the complement C345 protein family identified a number of potential protein-protein interaction sites in components C3 and C5. Here, one of these sites in C5, near the alpha-chain C terminus, was examined by alanine-scanning mutagenesis at 16 of the 18 non-alanine residues in the sequence KEALQIKYNFSF RYIYPLD. Alanine substitutions affected activities in the highly variable manner characteristic of binding sites. Substitutions at the lysine or either phenylalanine residue in the central KYNFSF sequence had the greatest effects, yielding mutants with <20% of the normal activity. These three mutants were also resistant to the classical pathway (CP) C5 convertase, with sensitivities roughly proportional to their hemolytic activities, but had normal susceptibilities to the cobra venom factor (CVF)-dependent convertase. Synthetic peptide MGKEALQIKYNFS-NH2 was found similarly to inhibit CP but not CVF convertase activation, and the effects of alanine substitutions in this peptide largely reflected those of the equivalent mutations in C5. These results indicate that residues KYNFSF form a novel, distal binding site for the CP, but not CVF convertase. This site lies approximately 880 residues downstream of the convertase cleavage site within a module that has been independently named C345C and NTR; this module is found in diverse proteins including netrins and tissue inhibitors of metalloproteinases.