Lassa fever is a neglected tropical disease with significant impact on the health care system, society, and economy of Western and Central African nations where it is endemic. Treatment of acute ...Lassa fever infections has successfully utilized intravenous administration of ribavirin, a nucleotide analogue drug, but this is not an approved use; efficacy of oral administration has not been demonstrated. To date, several potential new vaccine platforms have been explored, but none have progressed toward clinical trials and commercialization. Therefore, the development of a robust vaccine platform that could be generated in sufficient quantities and at a low cost per dose could herald a subcontinent-wide vaccination program. This would move Lassa endemic areas toward the control and reduction of major outbreaks and endemic infections. To this end, we have employed efficient mammalian expression systems to generate a Lassa virus (LASV)-like particle (VLP)-based modular vaccine platform.
A mammalian expression system that generated large quantities of LASV VLP in human cells at small scale settings was developed. These VLP contained the major immunological determinants of the virus: glycoprotein complex, nucleoprotein, and Z matrix protein, with known post-translational modifications. The viral proteins packaged into LASV VLP were characterized, including glycosylation profiles of glycoprotein subunits GP1 and GP2, and structural compartmentalization of each polypeptide. The host cell protein component of LASV VLP was also partially analyzed, namely glycoprotein incorporation, though the identity of these proteins remain unknown. All combinations of LASV Z, GPC, and NP proteins that generated VLP did not incorporate host cell ribosomes, a known component of native arenaviral particles, despite detection of small RNA species packaged into pseudoparticles. Although VLP did not contain the same host cell components as the native virion, electron microscopy analysis demonstrated that LASV VLP appeared structurally similar to native virions, with pleiomorphic distribution in size and shape. LASV VLP that displayed GPC or GPC+NP were immunogenic in mice, and generated a significant IgG response to individual viral proteins over the course of three immunizations, in the absence of adjuvants. Furthermore, sera from convalescent Lassa fever patients recognized VLP in ELISA format, thus affirming the presence of native epitopes displayed by the recombinant pseudoparticles.
These results established that modular LASV VLP can be generated displaying high levels of immunogenic viral proteins, and that small laboratory scale mammalian expression systems are capable of producing multi-milligram quantities of pseudoparticles. These VLP are structurally and morphologically similar to native LASV virions, but lack replicative functions, and thus can be safely generated in low biosafety level settings. LASV VLP were immunogenic in mice in the absence of adjuvants, with mature IgG responses developing within a few weeks after the first immunization. These studies highlight the relevance of a VLP platform for designing an optimal vaccine candidate against Lassa hemorrhagic fever, and warrant further investigation in lethal challenge animal models to establish their protective potential.
Hedgehog (Hh) signaling plays an important role in embryonic development and in the regulation of a variety of cellular functions. Aberrant activation of Hh signaling has been implicated in several ...human cancers including hepatocellular carcinoma (HCC). In this study we examined the pathobiological functions and molecular mechanisms of the Hh signaling pathway in HCC cells. Treatment of cultured human HCC cells (Huh7, Hep3B, and HepG2) with the Hh signaling ligand (recombinant Shh) or agonist, SAG and purmorphamine, prevented the induction of autophagy. In contrast, GANT61 (a small molecule inhibitor of Gli1 and Gli2) induced autophagy, as determined by immunoblotting for microtubule‐associated protein light chain 3 (LC3) and p62, GFP‐LC3 puncta, monodansylcadaverine (MDC) staining, and transmission electron microscopy. Hh inhibition‐induced autophagy was associated with up‐regulation of Bnip3, as determined by immunoblotting and real‐time polymerase chain reaction (PCR) assay. Knockdown of Bnip3 by RNAi impaired GANT61‐induced autophagy. Additionally, Hh inhibition‐induced autophagy was associated with Bnip3‐mediated displacement of Bcl‐2 from Beclin‐1, as determined by immunoblotting and immunoprecipitation assays. Furthermore, inhibition of Hh signaling increased HCC cell apoptosis and decreased cell viability, as determined by caspase and WST‐1 assays. Pharmacological or genetic inhibition of autophagy by 3‐methyladenine (3‐MA) or Beclin‐1 small interfering RNA (siRNA) partially suppressed GANT61‐induced cell apoptosis and cytotoxicity. In a tumor xenograft model using SCID mice inoculated with Huh7 cells, administration of GANT61 inhibited tumor formation and decreased tumor volume; this effect was partially blocked by the autophagy inhibitor, 3‐MA. Conclusion: These findings provide novel evidence that Hh inhibition induces autophagy through up‐regulation of Bnip3 and that this mechanism contributes to apoptosis. Therefore, the status of autophagy is a key factor that determines the therapeutic response to Hh‐targeted therapies. (Hepatology 2013;53:995–1010)
A murine model that demonstrated a placental barrier to fetal enterovirus infection in late pregnancy was extended to middle and early gestation. Inoculation with Theiler's murine encephalomyelitis ...virus (TMEV)in middle and early gestation infected 73% and 90% of placentas and 7% and 78% of fetuses, respectively. In situ hybridization (ISH) of tissues obtained after middle-gestation inoculation revealed TMEVin the placental decidua and spongiotrophoblast layers but generally not in the labyrinth (the layer adjacent to the fetus) or fetus (similar to late gestation). In contrast, ISH of placentas harvested after early-gestation inoculation identified TMEV predominantly in the labyrinth, in which vasculature was often replaced by hyalinized hemorrhagic tissu eand small cell infiltrates; fetuses contained virus in heart, pericardium, great vessels, lung, pleura, brain, and liver. The placental barrier to enterovirus transmission appears to develop between early and middle gestation.Enterovirus infection before this time may induce placental damage, fetal infection, or both.
The value of the ultrastructural study of the renal biopsy was investigated in a series of pediatric patients with nephrotic syndrome. Forty-eight cases of renal biopsies with clinical data were ...reviewed and divided into diagnostic groups. The contribution of electron microscopy to the final diagnosis was graded as essential diagnosis could not be reached without it; supportive it increased the level of confidence in the final diagnosis; and noncontributory. In this series of renal biopsies from 48 children with nephrotic syndrome resistant or nonresponsive to therapy, the mostfrequent diagnosis was minimal change disease, present in 42%of the patients. The contribution of the electron microscopic study to the final diagnosis was essential in 73%of the series, and was supportive in a further 27%.Therefore, it is concluded that the ultrastructural study was an essential component in the study of the renal biopsy in children with nephrotic syndrome, suggesting that electron microscopy needs to continue to be performed for all these patients.
Rabbit lung type II cell differentiation was evaluated by use of ultrastructural, morphometric techniques. Fetal lung epithelial cells decreased in size dramatically from day 19 to day 21 of ...gestation. Thereafter, the cell and cytoplasmic cross‐sectional area declined gradually until the neonatal time point. The tall columnar cell shape characteristic of fetal lung epithelial cells at early stages of development became cuboidal by day 24 of gestation. The number of mitochondria per μm2 cytoplasmic area in presumptive alveolar epithelial cells and the mitochondrial volume density increased toward the end of gestation. The volume density of glycogen pools within fetal lung epithelial cells reached a plateau on day 21 of gestation and then declined sharply on day 26 of gestation in lamellar body‐containing, type II epithelial cells. Lamellar bodies increased in number and volume density in epithelial cells starting on day 26 of gestation and peaked with respect to these parameters in the neonatal lung tissue. Multivesicular bodies, which are thought to be a precursor to the lamellar body, became more prominent in differentiated type II cells on day 26 of gestation and increased in volume density from day 28 of gestation to the adult time point. The distance between mesenchymal and epithelial cells in fetal lung tissue declined sharply between days 24 and 26 of gestation but remained relatively constant thereafter. Foot processes extending from connective tissue cells contiguous to the epithelium were generally more numerous than those extending from the basal plasma membrane of epithelial cells at every stage of development examined. These data quantitate for the first time key ultrastructural events that occur during the differentiation of fetal lung epithelial cells in vivo.
Maternal administration of glucocorticoids is known to stimulate fetal lung maturation. In the present study, we used microscopy and stereology to evaluate the morphological effects of maternal ...glucocorticoid treatment on rabbit fetal lung tissue. Betamethasone was administered to pregnant rabbits on days 25 and 26 of gestation at a dose of 0.2 mg/kg body weight. The animal were sacrificed on day 27 of gestation. Glucocorticoid treatment significantly increased the presumptive airspace in the fetal lung tissue but did not alter the relative proportion of epithelium, connective tissue, or vasculature in the tissue. In addition, glucocorticoid treatment significantly increased the proportion of type II cells in the prealveolar epithelium, increased the rate of phosphatidylcholine synthesis, and increased the content of the major surfactant‐associated protein, SP‐A, in the fetal lung tissue. We could detect no effect of betamethasone on lamellar body crosssectional area, numerical density, or volume density within fetal lung type II cells. Glucocorticoid treatment of the pregnant doe caused a decrease in the volume density of intracellular glycogen and an increase in the volume density of mitochondria in fetal lung type II cells. Betamethasone treatment did not alter the distance between fetal lung epithelial cells and subadjacent connective tissué cells. However, glucocorticoid treatment increased the number of connective tissue foot processes that pierced the epithelial basal lamina. Thus, glucocorticoid treatment of the pregnant doe results in structural changes in the fetal lung tissue, an acceleration of some aspects of type II cell defferentiation, and a concomitant increase in epithelial‐mesenchymal interactions.
X-linked alport syndrome in females Meleg-Smith, Suzanne; Magliato, Susan; Cheles, Mary ...
Human pathology,
04/1998, Letnik:
29, Številka:
4
Journal Article
Recenzirano
Alport syndrome (AS) is in the differential diagnosis of hematuria. Variability in clinical presentation and in the ultrastructural changes of the glomerulus can make the diagnosis of AS a challenge ...in female patients. The purpose of this report is to present immunostaining for glomerular basement membrane (GBM) expression of α5(IV) as an adjunctive diagnostic method. Renal biopsy specimens from eight female patients with clinical presentation suggestive of AS were studied. The patients were between 7 and 36 years of age; six were between 12 and 15 years. Light microscopy and immunohistochemistry using a monoclonal antibody to α5(IV) were performed. Controls showed a continuous linear pattern along the GBM in normal kidneys and absence in renal biopsy specimens from male X-linked AS patients. To express the variability of the ultrastructural GBM changes among the patients in the series, we developed a semi-quantitative Alport Index, obtained by quantification of severity and extent of ultrastructural GBM changes. With immunohistochemistry, we showed an interrupted, discontinuous linear pattern for α5(IV) in glomeruli from the eight patients in the series, confirming the diagnosis of X-linked AS. The ultrastructutal Alport Index varied between 6 and 47, showing the heterogeneity in the severity of the GBM changes, even among the six patients aged between 12 and 15 years. In three of the eight biopsy specimens, the predominant change was thin GBM, and the Alport Index was below 20. Immunohistochemistry for α5(IV) in renal biopsy specimens can identify female patients heterozygous for X-linked AS. In this series, the method led to the diagnosis of AS in female patients in whom the predominant ultrastructural change was thin basement membrane.
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