Culture Lysates Research Articles

Conjunctivitis caused by a swine Chlamydia trachomatis-like organism in gnotobiotic pigs.

The objective of this study was to determine whether a chlamydial strain recovered from growing and finishing swine with conjunctivitis or keratoconjunctivitis could cause the same infections in gnotobiotic pigs. The strain shares biological characteristics with Chlamydia trachomatis. After propagation in Vero cells and preparation of the inoculum (10(7) inclusion-forming units/ml), chlamydial strain H7 was instilled into the ventral conjunctival sac (0.15 ml/sac) of 12 anesthetized 3-day-old gnotobiotic piglets. Four age-matched gnotobiotic piglets were anesthetized and sham infected with uninfected cell culture lysates. None of the principal piglets developed clinical symptoms of conjunctivitis or keratoconjunctivitis. Principal piglets necropsied 7 days postinfection (DPI) had histologic lesions of mild or moderate conjunctivitis; immunohistochemical evaluation revealed chlamydial antigen in conjunctival epithelium. A majority of principal piglets necropsied at 14-28 DPI had histologic lesions of mild conjunctivitis, but chlamydial antigen was not detected by immunohistochemistry. The results indicated that chlamydial strain H7 can cause mild or occasionally moderate conjunctivitis in gnotobiotic pigs, but the conjunctival infection is asymptomatic.

Evaluation of the purity of a purified, inactivated hepatitis A vaccine (VAQTA TM)

Manufacture of VAQTA TM, an inactivated hepatitis A virus vaccine, includes extensive purification of the intact virus particle to remove endogenous components from the host cell culture lysate as well as compounds introduced in the upstream purification process. Analysis of the final purified hepatitis A virus product by SDS-PAGE prior to inactivation shows that greater than 95% of the protein in the preparation is found in four protein bands, which have been confirmed to be hepatitis A virus capsid proteins VP0, VP1, VP2 and VP3 based on Western blot and mass spectrometry analyses. Validation of the manufacturing process and direct analysis of the final product were used to demonstrate that no other specific host cell-derived components are detected and that process residuals are all below the limits of detection of the assays used. Establishment of a rigorous standard of high purity for this product was pursued to minimize the impact of impurities during clinical development of this product and will facilitate the incorporation of this product into combination vaccines.

Intracellular Measurement of Prostaglandin E2: Effect of Anti-inflammatory Drugs on Cyclooxygenase Activity and Prostanoid Expression

Cyclooxygenase (COX) converts arachidonic acid to prostaglandin (PG) H2, which is further metabolized to various prostaglandins, prostacyclin and thromboxane A2. COX exists in at least two different isoforms. COX-1 is constitutively expressed, whereas COX-2 is induced by proinflammatory stimuli. Prostaglandin E2 is a major metabolite of COX activation. In order to compare the activity of target ligands and COX inhibitors on PGE2 synthesis and release, the responsiveness of several cell lines to the calcium ionophore A23187, bacterial lipopolysaccharide (LPS), nonsteroidal anti-inflammatory drugs (NSAIDs), and the glucocorticoid, dexamethasone, were investigated. For intracellular measurements, the culture supernatant was aspirated, and the cells were thoroughly washed and lysed with dodecyltrimethylammonium bromide. Intracellular and secreted PGE2 were measured with an enzyme immunoassay. A23187 and LPS increased intracellular PGE2 in a dose-dependent manner. Kinetic experiments with A23187-stimulated mouse 3T3 fibroblast cells revealed a distinct biphasic response in COX activity. In the presence of NSAIDs or dexamethasone, there was a dose-dependent inhibition in intracellular PGE2 with A23187-stimulated 3T3 cells. Inhibitory studies demonstrated an apparent increased sensitivity of COX activity to the action of inhibitors when measuring intracellular PGE2 compared with using cell culture supernatants. Indeed, intracellular PGE2 levels were comprehensively reduced in the presence of low concentrations of inhibitor. The utilization of cell culture lysates and, in particular, measurement of intracellular PGE2 should prove useful for identifying new COX inhibitors.

Regulation of iron metabolism by oxalomalate

It has been investigatedin vitro the effect of oxalomalate (OMA) on the ferritin mRNA binding capacity of the bifunctional protein aconitase/IRP-1 contained in lysates of confluent cultures of 3T3-L1 fibroblasts. After short time of incubation with OMA at 37 °C, the gel mobility-shift analysis showed that the binding capacity of IRP-1 diminished and after 1 hour practically disappeared, indicating that the interaction of OMA with the aconitase counterpart reduced the aliquot of IRP-1 available to mRNA binding.

Divergent differentiation paths in airway smooth muscle culture: induction of functionally contractile myocytes.

We tested the hypothesis that prolonged serum deprivation would allow a subset of cultured airway myocytes to reacquire the abundant contractile protein content, marked shortening capacity, and elongated morphology characteristic of contractile cells within intact tissue. Passage 1 or 2 canine tracheal smooth muscle (SM) cells were grown to confluence, then serum deprived for up to 19 days. During serum deprivation, two differentiation pathways emerged. One-sixth of the cells developed an elongated morphology and aligned into bundles. Elongated myocytes contained cables of contractile myofilaments, dense bodies, gap junctions, and membrane caveoli, ultrastructural features of contractile SM in tissue. These cells immunostained intensely for SM alpha-actin, SM myosin heavy chain (MHC), and SM22 (an SM-specific actin-binding protein), and Western analysis of culture lysates disclosed 1.8 (SM alpha-actin)-, 7.7 (SM MHC)-, and 5.8 (SM22)-fold protein increases during serum deprivation. Immunoreactive M3 muscarinic receptors were present in dense foci distributed throughout elongated, SM MHC-positive myocytes. ACh (10(-3) M) induced a marked shortening (59.7 +/- 14.4% of original length) in 62% of elongated myocytes made semiadherent by gentle proteolytic digestion, and membrane bleb formation (a consequence of contraction) occurred in all stimulated cells that remained adherent and so did not shorten. Cultured airway myocytes that did not elongate during serum deprivation instead became short and flattened, lost immunoreactivity for contractile proteins, lacked the M3 muscarinic-receptor expression pattern seen in elongated cells, and exhibited no contractile response to ACh. Thus we demonstrate that prolonged serum deprivation induces distinct differentiation pathways in confluent cultured tracheal myocytes and that one subpopulation acquires an unequivocally functional contractile phenotype in which structure and function resemble contractile myocytes from intact tissue.

Expression of G-protein subtypes in cultured cerebral endothelial cells

This paper describes Western-blotting evidence for the presence of various guanine nucleotide binding proteins, G-proteins in cultured rat cerebral endothelial cells (CECs) and two immortalized cerebral endothelial cell lines, RBE4 and GP8. By using specific antibodies raised against known sequences of appropriate G-protein types that were previously characterized, we demonstrated the presence of Gsalpha, Gi2alpha, Gi3alpha, Gq/11alpha, Goalpha and Gbeta in cell lysates of primary cultures of CECs, and plasma membranes of RBE4 and GP8 cells. The appearance of Goalpha proteins in CECs might be of special importance, since they were not detected in peripheral endothelial cells in previous studies. Isoproterenol and bradykinin displayed significant, dose-dependent stimulation of [35S]GTPgammaS binding above basal values. This assay, reflecting the GDP-GTP exchange reaction on Galpha-subunits by receptor agonists, suggested that there were functional, G-protein coupled beta-adrenergic and bradykinin receptors in these systems. No significant stimulation of [35S]GTP7gammaS binding was noted with serotonin under our experimental conditions. Since stimulation of [35S]GTPgammaS binding by isoproterenol and bradykinin was additive, it was concluded that different Galpha proteins were activated by these two ligands. In analogy to other systems, activation of Gs is most likely by isoproterenol, while Gi and/or Gq/11 proteins might be activated by bradykinin receptors. The possible significance of the receptors and G-proteins detected is being discussed in the functioning of cerebral endothelium, and thus the blood-brain barrier.

Viruses and cells with mutations affecting viral entry are selected during persistent rotavirus infections of MA104 cells.

To better understand mechanisms of persistent rotavirus infections of cultured cells, we established independent, persistently infected cultures of MA104 cells, using rotavirus strain SA11. The cultures were either passaged when the cells reached confluence or supplemented with fresh medium every 7 days. Viral titers in culture lysates varied from 10(4) to 10(7) PFU per ml during 350 days of culture maintenance. Trypan blue staining indicated that 72 to 100% of cells in the cultures were viable, and immunocytochemical staining using a monoclonal antibody directed against viral protein VP6 demonstrated that 38 to 63% of the cells contained rotavirus antigen. We tested the capacity of rotaviruses isolated from the persistently infected cultures (PI viruses) to infect cells cured of persistent infection. Although wild-type (wt) and PI viruses produced equivalent yields in parental MA104 cells, PI viruses produced greater yields than wt virus in cured cells, which indicates that viruses and cells coevolve during persistent rotavirus infections of MA104 cells. To determine whether mutations in viruses and cells selected during these persistent infections affect viral entry, we tested the effect of trypsin treatment of the viral inoculum on growth of wt and PI viruses. Trypsin pretreatment is required for postattachment penetration of rotavirus virions into cells. In contrast to the case with wt virus, PI viruses produced equivalent yields with and without trypsin pretreatment in parental MA104 cells. However, PI viruses required trypsin pretreatment for efficient growth in cured cells. These results indicate that mutant viruses and cells are selected during maintenance of persistent rotavirus infections of MA104 cells and suggest that mutations in each affect trypsin-dependent steps in rotavirus entry.

Three-dimensional Type I Collagen Lattices Induce Coordinate Expression of Matrix Metalloproteinases MT1-MMP and MMP-2 in Microvascular Endothelial Cells

Matrix metalloproteinases (MMPs) are hypothesized to play a key role in the processes of endothelial cell migration and matrix remodeling during angiogenesis. We utilized an in vitro model of microvascular endothelial cell angiogenesis, cells cultured within a collagen matrix, to investigate the MMP profile of endothelial cells undergoing angiogenesis. We demonstrated by gelatin zymography that monolayer cultures (two-dimensional) of endothelial cells constitutively expressed low levels of latent MMP-2, but that culture in a three-dimensional collagen matrix increased the total amount of MMP-2 mRNA and protein. Furthermore, 51% of total MMP-2 protein was activated in the three-dimensional culture lysates, compared with 3.5% in two-dimensional culture. The mRNA and protein of MT1-MMP, the putative activator of MMP-2, were up-regulated in endothelial cells cultured in three-dimensional as compared with two-dimensional culture. Treatment of cultures with MMP inhibitors blocked activation of MMP-2 and inhibited formation of endothelial cell networks within the collagen gel. Induction of MT1-MMP and MMP-2 appeared to be specific to collagen, inasmuch as culture of the endothelial cells on top of, or within, a Matrigel(R) matrix neither increased total MMP-2 nor increased activation of MMP-2. These results suggest that MT1-MMP activation of MMP-2 occurs in endothelial cells undergoing angiogenesis, that this activation has a functional role in endothelial cell organization, and that specific matrix interactions may be critical for the increased expression of MT1-MMP and MMP-2.

Infectious bursal disease virus polyprotein processing does not involve cellular proteases.

The larger genome segment, segment A, of infectious bursal disease virus (IBDV) encodes VP2, VP3 and VP4 as a precursor polyprotein. The viral protease, VP4, is responsible for self-processing of the polyprotein, however, there are additional secondary precursor products such as VPX whose further processing has not been defined. Expression of IBDV cDNAs in vitro with rabbit reticulocyte lysates in a coupled transcription-translation system and in the Sindbis virus expression system (with BHK-21 and Vero cell cultures) were used to study processing of the polyprotein. In both expression systems, we identified three main gene products with molecular masses of 48, 34, and 30.5 kDa corresponding to VPX, VP3, and VP4, respectively, as found in IBDV-infected Vero cell cultures, although the amount of each product was variable. A translational time course of the polyprotein gene and analyses of products specified by various sub-clones of the full-length cDNA were used to distinguish primary processing products of translation from secondary products generated by proteolytic processing during in vitro coupled transcription-translation expression. The VPX, VP3 and VP4, which are the primary processing products, first appeared after 20 min of incubation and their production was maximum by 75 min of the coupled transcription-translation reaction. Cycloheximide chases demonstrated that there is no secondary processing of VPX (or VP3 and VP4). Thus VP2, the major capsid protein in virions, was not detected either in translation products of rabbit reticulocyte lysates or in lysates of Sindbis virus recombinant-infected cell cultures indicating the absence of secondary processing of VPX to VP2 during foreign expression of the segment A cDNA. We conclude that VPX maturation to VP2 does not involve cellular proteases.

Rapid PCR-based monitoring of infectious enteroviruses in drinking water

Currently, the standard method for the detection of enteroviruses and hepatitis A virus in water involves cell culture assay which is expensive and time consuming. Direct RT-PCR offers a rapid and sensitive alternative to virus detection but sensitivity is often reduced by PCR inhibitory substances and the requirement for small reaction volumes. Rapid methods for detection of infectious enteroviruses in PCR inhibitory environmental samples are being developed utilising an integrated cell culture/PCR approach (ICC/PCR). With this approach, 300–4001 of water were concentrated using charged filters followed by a modified 11, 1.5% BEV/glycine elution and organic flocculation reconcentration. Water concentrates were analysed by direct RT-PCR, conventional cell culture and ICC/PCR. For ICC/PCR, sample concentrates were incubated with BGM or FRhK cells for 24–48h. The cell culture lysates were collected following freeze-thaw cycles, centrifuged, resin column purified and PCR amplified. In this study viruses known to be present by cell culture analysis could not be detected by direct PCR. Using the integrated method, virus concentrations as low as 0.001MPN/l of original water were detected in samples which were previously inhibitory to direct PCR. In addition, confirmed enterovirus results were achieved as soon as 48h against 5–16d with cell culture alone. Therefore, the integrated approach overcame some of the traditional problems associated with conventional cell culture and direct RT-PCR by allowing rapid, confirmed detection of low levels of enteroviruses in PCR inhibitory samples.

Isolation of a virus infectious to the harmful bloom causing microalga Heterosigma akashiwo (Raphidophyceae)

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 13:135-140 (1997) - doi:10.3354/ame013135 Isolation of a virus infectious to the harmful bloom causing microalga Heterosigma akashiwo (Raphidophyceae) Nagasaki K, Yamaguchi M A virus infecting the harmful bloom causing microalga Heterosigma akashiwo (Raphidophyceae) was isolated from the coastal water of Nomi Bay, Japan, in July 1996. The isolate caused lysis in 2 strains of H. akashiwo tested and numerous virus-like particles (VLPs) appeared in the lysed algal culture, whereas virus multiplication was not detected in the healthy culture of H. akashiwo without its inoculation. Thus, fulfilling Koch's postulate, it was considered to be a virus and designated HaV (Heterosigma akashiwo virus) clone GSNOU-30. The virus particle is icosahedral, lacking a tail, and 202 ± 6 nm (average ± standard deviation) in diameter with an electron-dense roundish core that is distinct from the capsid. The virus stained positive with DAPI, indicating that it possesses a double stranded DNA genome. The virus proliferated in the protoplasm of the host cell as had previously been observed in H. akashiwo cells from a natural red tide population. The virus did not cause lysis of Chattonella antiqua, C. verruculosa or Fibrocapsa japonica (Raphidophyceae) as well as 15 strains of phytoplankton belonging to other classes. It is most noteworthy that 3 strains of H. akashiwo isolated from Hiroshima Bay, Japan, were resistant to GSNOU-30, suggesting that the viral infectivity is not species-specific but strain-specific. These results suggest that the virus is involved in the population dynamics of H. akashiwo, playing a role as a selector to increase genetic diversity of a host species. Red tide · Harmful algal bloom · Lytic virus · Heterosigma akashiwo · Raphidophyceae · Host specificity · HaV Full text in pdf format NextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 13, No. 2. Publication date: August 21, 1997 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 1997 Inter-Research.

Efficiency of viral entry determines the capacity of murine erythroleukemia cells to support persistent infections by mammalian reoviruses.

To determine mechanisms by which persistent viral infections are established and maintained, we initiated persistent infections of murine erythroleukemia (MEL) cells by using reovirus strains type 3 Abney and type 3 Dearing. Establishment of persistent reovirus infections of MEL cells was not associated with a significant cytopathic effect despite the presence of high titers of infectious virus in the cultures (>10(5) PFU/ml of culture lysate). Maintenance of persistently infected MEL-cell cultures was associated with coevolution of mutant viruses and cells. Mutant viruses produced greater yields than the parental wild-type (wt) strains in MEL cells cured of persistent infection and in cells treated with ammonium chloride, a weak base that blocks viral disassembly. Mutant cells supported growth of wt infectious subvirion particles, which are disassembly intermediates generated in vitro by treatment of virions with chymotrypsin, substantially better than growth of wt virions. These findings indicate that viral and cellular mutations selected during maintenance of persistently infected MEL-cell cultures affect acid-dependent proteolysis of virions during entry into cells. We also found that wt infectious subvirion particles produce greater yields than wt virions in wt MEL cells, which suggests that inefficient viral disassembly in MEL cells favors establishment of persistent infection. Therefore, steps in reovirus replication leading to viral disassembly appear to be critical determinants of the capacity of MEL cells to support both establishment and maintenance of persistent reovirus infections.

Rapid PCR-based monitoring of infectious enteroviruses in drinking water

Currently, the standard method for the detection of enteroviruses and hepatitis A virus in water involves cell culture assay which is expensive and time consuming. Direct RT-PCR offers a rapid and sensitive alternative to virus detection but sensivity is often reduced by PCR inhibitory substances and the requirement for small reaction volumes. Rapid methods for detection of infectious enteroviruses in PCR inhibitory environmental samples are being developed utilising an integrated cell culture/PCR approach (ICC/PCR). With this approach, 300–4001 of water were concentrated using charged filters followed by a modified II, 1.5% BEV/glycine elution and organic flocculation reconcentration. Water concentrates were analysed by direct RT-PCR, Conventional cell culture and ICC/PCR. For ICC/PCR, sample concentrates were incubated with BGM or FRhK cells for 24–48h. The cell culture lysates were collected following freeze-thaw cycles, centrifuged, resin column purified and PCR amplified. In this study viruses known to be present by cell culture analysis could not be detected by direct PCR. Using the integrated method, virus Concentrations as low as 0.001MPN/l of original water were detected in samples which were previously inhibitory to direct PCR. In addition, confirmed enterovirus results were achieved as soon as 48h against 5–l6d with cell culture alone. Therefore, the integrated approach overcame some of the traditional problems associated with conventional cell culture and direct RT-PCR by allowing rapid, confirmed detection of low levels of enteroviruses in PCR inhibitory samples.

Vascular reactivity during combined ultrafiltration-hemodialysis: influence of dialysate-derived contaminants.

It has been suggested that hemodynamic instability and impaired vascular reactivity during combined ultrafiltration-hemodialysis are related to bioincompatibility factors such as dialysate-derived contaminants or the dialyzer. The study presented here investigated whether vascular reactivity could be improved by the use of sterile dialysate. Forearm vascular resistance and venous tone (measured by strain-gauge plethysmography) as well as arterial blood pressure (by Dinamap) and heart rate (by electrocardiogram) were measured in ten stable dialysis patients (age range, 28 to 71 yr) during 2 h of combined ultrafiltration-hemodialysis (bicarbonate; ultrafiltration rate 1.0 L/h). In addition, a dialysate sample was obtained for culture and limulus amebocyte lysate testing while blood was withdrawn for the estimation of plasma bactericidal/permeability increasing factor (measured by ELISA) and the soluble tumor necrosis factor receptor p75 (measured by ELISA). Patients served as their own control, comparing dialysis with nonsterile and sterile dialysate. No bacterial growth was observed in sterile dialysate, whereas all samples were positive for Pseudomonas in culture in nonsterile dialysis. All limulus amebocyte lysate tests were negative. Bactericidal/permeability increasing factor tended to increase during nonsterile dialysis (P = 0.063) and remained unchanged during sterile dialysis. In both treatments, tumor necrosis factor receptor p75 increased significantly (P < 0.01). There were no significant differences in hemodynamic parameters between the treatment modalities. Despite use of sterile dialysate, forearm vascular resistance remained unchanged whereas venous tone decreased significantly. These results indicate that vascular reactivity during combined ultrafiltration-hemodialysis is not improved by the use of sterile dialysate.

Intestinal lesions caused by two swine chlamydial isolates in gnotobiotic pigs.

The objective of this study was to determine whether 2 distinct chlamydial isolates recovered from the intestines and feces of diarrheic nursery pigs could cause intestinal lesions in gnotobiotic pigs. Both isolates share biological characteristics with Chlamydia trachomatis. Chlamydial isolates R27 and R19 were propagated in Vero cells or embryonated eggs, respectively, and suspended in sucrose-phosphate-glutamine buffer with 10% fetal bovine serum for inoculation. Sham inocula were prepared from uninfected cell culture lysates and from uninfected eggs. Each piglet was fed 1 ml of inoculum or sham inoculum at 3-4 days of age. Ten piglets were each fed 10(9) inclusion-forming-units (IFU) and 14 piglets were each fed 10(6) IFU of isolate R27; 5 control piglets were fed sham inoculum. Twenty piglets were each fed 10(5) IFU R19; 5 control piglets were fed sham inoculum. All infected piglets developed diarrhea 4-5 days postinfection (DPI). Most piglets fed 10(9) IFU R27 became anorexic, dehydrated, and weak and were necropsied 4-7 DPI. Piglets fed 10(6) IFU R27 or 10(5) IFU R19 were necropsied 4, 7, 10, 14, and 18 DPI. Diarrhea, although never profuse, persisted in the piglets fed 10(6) IFU R27 or 10(5) IFU R19 through 12 DPI. At necropsy, all diarrheic piglets had watery colonic contents with flecks of undigested curd. In small intestine, histologic lesions were seen most consistently in distal jejunum and ileum. Distal jejunum and ileum from piglets fed 10(9) IFU R27 and necropsied 4-5 DPI were characterized by villus atrophy and multifocal necrosis of villi; necrosis was limited to the tips or apical one half of villi. Mild to severe villus atrophy, lymphangitis, and perilymphangitis were seen in the distal jejunum and ileum from all infected piglets 7 and 10 DPI. Colon from 1 infected piglet necropsied 10 DPI had mild focal serositis; significant colonic lesions were not seen in the other infected piglets. Immunostaining done on sections of distal jejunum and ileum revealed chlamydial antigen in villus enterocytes, occasional goblet cells, and occasional cryptal enterocytes and in foci of lymphangitis and perilymphangitis; the amount of detectable chlamydial antigen decreased after 4 DPI. In colon, sparse positive staining was seen in surface enterocytes and cryptal enterocytes. Ultrastructural examination of ileal villus enterocytes revealed chlamydiae, often together with glycogen particles, in vacuoles or occasionally free in the cytoplasm. The results indicated that the swine chlamydial isolates used in this study are intestinal pathogens in gnotobiotic pigs.

Product Purification by Reversible Phase Transition FollowingEscherichia coliExpression of Genes Encoding up to 251 Repeats of the Elastomeric Pentapeptide GVGVP

By constructing a basic gene unit encoding (GVGVP)10, it was possible to build concatemer genes with as many as 25 repeats of the monomer unit encoding a protein-based polymer with a molecular weight of greater than 100,000 Da. This employed the use of terminal cloning adaptor oligonucleotides as chain terminators to enhance the desired polymer gene size distribution. These genes have been expressed inEscherichia coliand the products have been purified from the culture lysates using a simple centrifugation method which relies upon the inverse temperature transitional properties of these elastomeric protein-based polymers. At 4°C, the polymers are soluble; on raising the temperature above 26°C, the onset temperature (Tt) for the (GVGVP)251inverse temperature transition, the polymer separates out as the more dense phase. Upon shifting the temperature between 4 and 37°C, the recombinant elastomeric protein-based polymers undergo reversible phase transitions from soluble (4°C) to insoluble (37°C) allowing their separation from other cellular components by several cycles of centrifugations at alternate transitional states. Additional centrifugation, at a temperature just belowTt, allows for dramatic lowering of endotoxin levels. Furthermore, many ways of varying the value ofTt, such as adding salt to lowerTtor changing the degree of ionization in polymers with functional side chains, can be used to achieve purification of more complex polymers.

Lung and nasal lesions caused by a swine chlamydial isolate in gnotobiotic pigs.

The objective of this study was to determine whether a chlamydial isolate recovered from nasal swabs from swine with pneumonia could cause pneumonia and rhinitis in gnotobiotic pigs. The identity of the isolate currently is unknown, but it shares characteristics with Chlamydia trachomatis. After propagation in Vero cells and preparation of the inoculum (2.5 x 10(10) inclusion-forming units/ml), chlamydiae were instilled into nostrils (1.0 ml/nostril) and lungs (2.0 ml intralaryngeally) of 15 anesthetized 3-day-old gnotobiotic piglets. Five age-matched gnotobiotic piglets were anesthetized and sham infected with uninfected cell culture lysates. Two treated piglets were moribund and 2 were severely dyspneic prior to necropsy 7 days postinfection (DPI), whereas remaining treated piglets showed mild dyspnea upon exertion throughout the study. All treated piglets developed diarrhea. All treated piglets necropsied 7-21 DPI had extensive consolidation in cranial, middle, and accessory lung lobes; a majority of these piglets also had extensive consolidation in the caudal lobes. Treated piglets necropsied 28 and 35 DPI had a lobular pattern of consolidation in all lung lobes. Histologically, lesions in lungs from treated piglets necropsied 7, 14, and 21 DPI were characterized by bronchointerstitial pneumonia with foci of type II pneumocyte hypertrophy and hyperplasia; pneumocytes and bronchial and bronchiolar epithelial cells were markedly vacuolated. Alveolar macrophages, peribronchitis, peribronchiolitis, and perivasculitis were seen in lungs from treated piglets necropsied 28 and 35 DPI; those necropsied 28 DPI also had foci of lymphohistiocytic and plasmacytic infiltrates. Turbinate lesions in all treated piglets were characterized by mild multifocal lymphoplasmacytic and occasionally neutrophilic rhinitis. Immunohistochemistry detected chlamydial antigen in bronchial and bronchiolar epithelial cells, pneumocytes, and inflammatory cells in treated piglets necropsied 7, 14, and 21 DPI. Positive staining was limited to alveolar macrophages in treated piglets necropsied 28 and 35 DPI. Chlamydial antigen was detected in turbinate epithelial cells at all necropsy intervals. Ultrastructurally, chlamydiae were seen with glycogen particles in vacuoles or free in the cytoplasm of bronchial and bronchiolar epithelial cells and pneumocytes. The results indicated that the chlamydial isolate used in this study is a pathogen in gnotobiotic pigs.

Eradication of Persistent Reovirus Infection from a B-Cell Hybridoma

The 2G10 B-cell hybridoma was found to be persistently infected with reovirus serotype 3 (RV3). The persistently infected 2G10 culture produced approximately 1 × 10 8 plaque-forming units of virus per milliliter of culture lysate, and a majority of cells in the culture were infected, as determined by infectious center assay and immunocytochemistry. Cure of the persistent infection was achieved by passaging 2G10 cells for 33 days (12 passages) in medium containing polyclonal anti-RV3 antiserum and a monoclonal antibody specific for the RV3 attachment protein. After several passages in antibody-free medium, cured 2G10 cells had (1) nondetectable levels of RV3 in cell-culture lysates, (2) no infectious centers per 3 × 10 5 cells, (3) no immunocytochemically detectable RV3 antigen, and (4) no detectable reovirus-specific RNA by reverse transcription-polymerase chain reaction amplification. Additionally, mice inoculated with cured 2G10 cell lysates did not generate antibodies directed against RV3. These observations demonstrate that persistent reovirus infection of a B-cell hybridoma can be cured by passage in medium containing anti-reovirus antibodies and suggest that the maintenance of this persistent infection is dependent on horizontal cell-to-cell transmission of virus in the culture.

Neuroprotective sigma ligands interfere with the glutamate‐activated NOS pathway in hippocampal cell culture

We studied neuroprotective properties of 12 structurally different sigma site ligands in primary rat hippocampal cell cultures and analyzed whether they interfere with glutamate-induced activation of the nitric oxide synthase (NOS) pathway. Neurotoxicity was triggered with 1 mM glutamate on day 8 of culture. Cells were treated with various concentrations of the compounds for 7 days before glutamate exposure (prolonged pretreatment), or during glutamate exposure (acute treatment). Protection was seen after prolonged pretreatment (long-term protection) with sabeluzole, opipramole, haloperidol, ifenprodil, fenpropimorph, carbetapentane, and tiospirone, with pIC50s of 7.30, 7.15, 6.87, 6.68, 6.66, 6.39, and 6.34, respectively. There was no protection with PD 128298, 1,3-ortho-di-tolylguanidine, BMY-14802, (+)3-(3-hydroxyphenyl)-N-1(propyl) piperidine, or dextromethorphan. Upon acute treatment, only ifenprodil was protective. Interference of the drugs with glutamate activation of the NOS pathway was determined by measuring glutamate-activated cGMP formation and citrulline levels. Glutamate-activated cGMP formation was reduced by all neuroprotective sigma ligands after prolonged pretreatment but not after acute treatment. Sigma ligands added to cell culture lysate did not reduce citrulline formation, evidence that there was no direct effect on the NOS enzyme. We conclude that some but not all sigma ligands exert long-term protective properties against glutamate-induced neurotoxicity in primary hippocampal cultures, and that this protection is accompanied by attenuation of cGMP formation in the NOS pathway. However, inhibition of cGMP formation by itself appeared not sufficient for obtaining neuroprotective effects, as inhibition of glutamate-activated cGMP formation by N omega-nitro-L-arginine, haemoglobin, or PD128298 did not provide neuroprotection.

Selective lysis of cultures and cell walls of penicillin-resistant but not penicillin-susceptible Streptococcus pneumoniae strains by a murein hydrolase complex

A murein hydrolase complex selectively lysed cultures of penicillin-resistant pneumococci and their cell walls in which the majority of muropeptide subunits were indirectly cross-linked through oligopeptide substituents (alanyl-alanine or alanyl-serine) on the epsilon-amino group of the stem peptide lysine residues. Walls of penicillin-susceptible strains were not hydrolyzed.