Detection, isolation, and analysis of a released Bordetella pertussis product toxic to cultured tracheal cells.

Cultured hamster trachea epithelial cells were selected as an in vitro model system to study Bordetella pertussis in the respiratory tract. DNA synthesis by serum-stimulated tracheal cells, in contrast to other cell types tested, was inhibited by the supernatant from log-phase B. pertussis broth cultures. A sensitive microassay with these tracheal cells permitted the development of a chromatographic purification scheme based on aggregation of the biological activity under salt-free conditions. The active fraction from this first stage of purification caused a dose-dependent inhibition of DNA synthesis without a similar effect on RNA or protein synthesis. Organ cultures of hamster tracheal rings, when exposed to this partially purified fraction, developed epithelial cytopathology comparable to that seen during B. pertussis infection. Ciliary activity showed and eventually ceased as ciliated cells were extruded from the ring, leaving an intact but mostly nonciliated epithelium. Further purification of this biological activity was achieved with preparative-scale high-voltage paper electrophoresis. Based on ninhydrin staining and the radioactive profile of material purified from radiolabeled B. pertussis cultures, four fractions were eluted from the paper by descending chromatography. Only component B caused a dose-dependent inhibition of cultured tracheal cell DNA synthesis and epithelial cytopathology in tracheal rings. Combination experiments also demonstrated enhanced inhibition by component B in the presence of component G (oxidized glutathione), a copurifying molecule from the growth medium. Amino acid analysis (five residues), glycine (two residues), cysteine (two residues), and diaminopimelic acid (one residue), as well as muramic acid and glucosamine

Scaling and the Metal-Insulator Transition in Si/SiGe Quantum Wells

The existence of a metal-insulator transition at zero magnetic field in two- dimensional electron systems has recently been confirmed in high mobility Si-MOSFETs. In this work, the temperature dependence of the resistivity of gated Si/SiGe/Si quantum well structures has revealed a similar metal- insulator transition as a function of carrier density at zero magnetic field. We also report evidence for a Coulomb gap in the temperature dependence of the resistivity of the dilute 2D hole gas confined in a SiGe quantum well. In addition, the resistivity in the insulating phase scales with a single parameter, and is sample independent. These results are consistent with the occurrence of a metal-insulator transition at zero magnetic field in SiGe square quantum wells driven by strong hole-hole interactions.Comment: 3 pages, 3 figures, LaTe

Mucus and surfactant synthesis and secretion by cultured hamster respiratory cells

Procedures for the selective isolation and cultivation in monolayer of respiratory cells have been developed. This technique requires repeated protease treatment and gradient centrifugation of hamster tracheal or lung tissues and permits the establishment of proliferating cultures of epithelial cells with biologic specialization. Mucus synthesis was monitored in cultured tracheal cells by incorporation of 3H-labeled N-acetyl-D-galactosamine and 14C-serine into glycoprotein as determined by trichloroacetic acid precipitation of growth medium followed by acrylamide gel electrophoresis. For comparative purposes tracheal explants and several established cell lines were also examined. Synthesis and secretion of the glycoprotein macromolecule by tracheal cell monolayers appeared to be regulated by vitamin A since its addition to the culture medium significantly increased both the number of cell-associated granules and glycoprotein secretion. Lung-originated cell cultures were grown to confluence and radio-labeled with 3H-choline in serum-free medium for 24 hr to examine surfactant synthesis. Cell monolayers and growth medium were then extracted by the Folch method, and total radioactive phosphatidylcholine as well as disaturated phosphatidylcholine were determined by thin-layer chromatography and alumina gel fractionation of osmium tetroxide-reactive phospholipid, respectively. Data indicate that these cultures have a marked ability to synthesize and secrete surfactant when compared to other established cell lines. In addition, naturally transformed cells that arose during passage and senescence of the primary cultures were analyzed for their biosynthetic capabilities.ImagesFIGURE 1.FIGURE 3.FIGURE 4

Public Health Emergency Preparedness and Response Communications with Health Care Providers: A Literature Review

<p>Abstract</p> <p>Background</p> <p>Health care providers (HCPs) play an important role in public health emergency preparedness and response (PHEPR) so need to be aware of public health threats and emergencies. To inform HCPs, public health issues PHEPR messages that provide guidelines and updates, and facilitate surveillance so HCPs will recognize and control communicable diseases, prevent excess deaths and mitigate suffering. Public health agencies need to know that the PHEPR messages sent to HCPs reach their target audience and are effective and informative. Public health agencies need to know that the PHEPR messages sent to HCPs reach their target audience and are effective and informative. We conducted a literature review to investigate the systems and tools used by public health to generate PHEPR communications to HCPs, and to identify specific characteristics of message delivery mechanisms and formats that may be associated with effective PHEPR communications.</p> <p>Methods</p> <p>A systematic review of peer- and non-peer-reviewed literature focused on the following questions: 1) What public health systems exist for communicating PHEPR messages from public health agencies to HCPs? 2) Have these systems been evaluated and, if yes, what criteria were used to evaluate these systems? 3) What have these evaluations discovered about characterizations of the most effective ways for public health agencies to communicate PHEPR messages to HCPs?</p> <p>Results</p> <p>We identified 25 systems or tools for communicating PHEPR messages from public health agencies to HCPs. Few articles assessed PHEPR communication systems or messaging methods or outcomes. Only one study compared the effectiveness of the delivery format, device or message itself. We also discovered that the potential is high for HCPs to experience "message overload" given redundancy of PHEPR messaging in multiple formats and/or through different delivery systems.</p> <p>Conclusions</p> <p>We found that detailed descriptions of PHEPR messaging from public health to HCPs are scarce in the literature and, even when available are rarely evaluated in any systematic fashion. To meet present-day and future information needs for emergency preparedness, more attention needs to be given to evaluating the effectiveness of these systems in a scientifically rigorous manner.</p

Transient Acquired Hemophilia Associated with Mycoplasma Pneumoniae Pneumonia

Acquired hemophilia is a rare disorder caused by autoantibodies to factor VIII (FVIII) (also referred to as factor VIII inhibitors or anti-FVIII) and may be associated with pregnancy, underlying malignancy, or autoimmune disorders. A 33-month-old girl who presented with hematochezia and ecchymotic skin lesions was diagnosed with Mycoplasma pneumoniae pneumonia by serology and polymerase chain reaction. Hematologic studies showed a prolonged activated partial thromboplastin time (aPTT), partially corrected mixing test for aPTT, reduced levels of FVIII, and the presence of antibodies against FVIII. She was treated conservatively with prednisone and intravenous immunoglobulin (IVIG) without FVIII transfusion and recovered without sequelae. This report provides the first description of acquired hemophilia due to anti-FVIII in association with M. pneumoniae in Korea. We discuss this case in the context of the current literature on acquired hemophilia in children

Identification of an N-terminal 27 kDa fragment of Mycoplasma pneumoniae P116 protein as specific immunogen in M. pneumoniae infections

<p>Abstract</p> <p>Background</p> <p><it>Mycoplasma pneumoniae </it>is an important cause of respiratory tract infection and is increasingly being associated with other diseases such as asthma and extra-pulmonary complications. Considerable cross-reactivity is known to exist between the whole cell antigens used in the commercial serological testing assays. Identification of specific antigens is important to eliminate the risk of cross-reactions among different related organisms. Adherence of <it>M. pneumoniae </it>to human epithelial cells is mediated through a well defined apical organelle to which a number of proteins such as P1, P30, P116 and HMW1-3 have been localized, and are being investigated for adhesion, gliding and immunodiagnostic purposes.</p> <p>Methods</p> <p>A 609 bp fragment P116_(N-27),corresponding to the N-terminal region of <it>M. pneumoniae </it>P116 gene was cloned and expressed. A C-terminal fragment P1_(C-40),of P1 protein of <it>M. pneumoniae </it>was also expressed. Three IgM ELISA assays based on P116_(N-27),P1_(C-40)and (P116 _(N-27)+ P1_(C-40)) proteins were optimized and a detailed analysis comparing the reactivity of these proteins with a commercial kit was carried out. Comparative statistical analysis of these assays was performed with the SPSS version 15.0.</p> <p>Results</p> <p>The expressed P116_(N-27)protein was well recognized by the patient sera and was immunogenic in rabbit. P1_(C-40)of <it>M. pneumoniae </it>was also immunogenic in rabbit. In comparison to the reference kit, which is reported to be 100% sensitive and 75% specific, ELISA assay based on purified P116_(N-27),P1_(C-40)and (P116_(N-27)+ P1_(C-40)) proteins showed 90.3%, 87.1% and 96.8% sensitivity and 87.0%, 87.1% and 90.3% specificity respectively. The p value for all the three assays was found to be < 0.001, and there was a good correlation and association between them.</p> <p>Conclusion</p> <p>This study shows that an N-terminal fragment of P116 protein holds a promise for serodiagnosis of <it>M. pneumoniae </it>infection. The IgM ELISA assays based on the recombinant proteins seem to be suitable for the use in serodiagnosis of acute <it>M. pneumoniae </it>infections. The use of short recombinant fragments of P116 and P1 proteins as specific antigens may eliminate the risk of cross-reactions and help to develop a specific and sensitive immunodiagnostic assay for <it>M. pneumoniae </it>detection.</p

Methionine Sulfoxide Reductase A (MsrA) Deficient Mycoplasma genitalium Shows Decreased Interactions with Host Cells

Mycoplasma genitalium is an important sexually transmitted pathogen that affects both men and women. In genital-mucosal tissues, it initiates colonization of epithelial cells by attaching itself to host cells via several identified bacterial ligands and host cell surface receptors. We have previously shown that a mutant form of M. genitalium lacking methionine sulfoxide reductase A (MsrA), an antioxidant enzyme which converts oxidized methionine (Met(O)) into methionine (Met), shows decreased viability in infected animals. To gain more insights into the mechanisms by which MsrA controls M. genitalium virulence, we compared the wild-type M. genitalium strain (G37) with an msrA mutant (MS5) strain for their ability to interact with target cervical epithelial cell lines (HeLa and C33A) and THP-1 monocytic cells. Infection of epithelial cell lines with both strains revealed that MS5 was less cytotoxic to HeLa and C33A cell lines than the G37 strain. Also, the MS5 strain was more susceptible to phagocytosis by THP-1 cells than wild type strain (G37). Further, MS5 was less able to induce aggregation and differentiation in THP-1 cells than the wild type strain, as determined by carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling of the cells, followed by counting of cells attached to the culture dish using image analysis. Finally, MS5 was observed to induce less proinflammatory cytokine TNF-α by THP-1 cells than wild type G37 strain. These results indicate that MsrA affects the virulence properties of M. genitalium by modulating its interaction with host cells

Critical Role of Macrophages and Their Activation via MyD88-NFκB Signaling in Lung Innate Immunity to Mycoplasma pneumoniae

Mycoplasma pneumoniae (Mp), a common cause of pneumonia, is associated with asthma; however, the mechanisms underlying this association remain unclear. We investigated the cellular immune response to Mp in mice. Intranasal inoculation with Mp elicited infiltration of the lungs with neutrophils, monocytes and macrophages. Systemic depletion of macrophages, but not neutrophils, resulted in impaired clearance of Mp from the lungs. Accumulation and activation of macrophages were decreased in the lungs of MyD88−/− mice and clearance of Mp was impaired, indicating that MyD88 is a key signaling protein in the anti-Mp response. MyD88-dependent signaling was also required for the Mp-induced activation of NFκB, which was essential for macrophages to eliminate the microbe in vitro. Thus, MyD88-NFκB signaling in macrophages is essential for clearance of Mp from the lungs

Complete Genome Sequence of Treponema paraluiscuniculi, Strain Cuniculi A: The Loss of Infectivity to Humans Is Associated with Genome Decay

Treponema paraluiscuniculi is the causative agent of rabbit venereal spirochetosis. It is not infectious to humans, although its genome structure is very closely related to other pathogenic Treponema species including Treponema pallidum subspecies pallidum, the etiological agent of syphilis. In this study, the genome sequence of Treponema paraluiscuniculi, strain Cuniculi A, was determined by a combination of several high-throughput sequencing strategies. Whereas the overall size (1,133,390 bp), arrangement, and gene content of the Cuniculi A genome closely resembled those of the T. pallidum genome, the T. paraluiscuniculi genome contained a markedly higher number of pseudogenes and gene fragments (51). In addition to pseudogenes, 33 divergent genes were also found in the T. paraluiscuniculi genome. A set of 32 (out of 84) affected genes encoded proteins of known or predicted function in the Nichols genome. These proteins included virulence factors, gene regulators and components of DNA repair and recombination. The majority (52 or 61.9%) of the Cuniculi A pseudogenes and divergent genes were of unknown function. Our results indicate that T. paraluiscuniculi has evolved from a T. pallidum-like ancestor and adapted to a specialized host-associated niche (rabbits) during loss of infectivity to humans. The genes that are inactivated or altered in T. paraluiscuniculi are candidates for virulence factors important in the infectivity and pathogenesis of T. pallidum subspecies