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PubMed1995

Clinical, immunologic, and virologic observations related to human immunodeficiency virus (HIV) type 1 infection in a volunteer in an HIV-1 vaccine clinical trial.

A vaccine breakthrough occurred in a phase 1 clinical trial of a human immunodeficiency virus (HIV) type 1 candidate subunit vaccine. The vaccine antigen, gp120SF2, is a fully glycosylated protein produced in mammalian cells from the HIVSF2 isolate. After 4 immunizations, the subject developed neutralizing antibodies and lymphoproliferative responses to the gp120 protein. About 18 weeks after the last immunization, the subject became HIV infected. During the acute phase of infection, there was high virus burden, a decline in CD4+ T lymphocytes, increases in rgp120SF2-binding antibodies and HIVSF2- and HIVMN-neutralizing antibodies, and transient lymphoproliferative responses to HIV-1 envelope and core proteins. The nucleotide sequence of the V3 loop from 2 virus isolations displayed close similarity to the V3 sequence of the vaccine antigen. Thus, the immunologic responses induced by the vaccine in this subject did not protect him from HIV-1 infection.

AIDS Vaccines↗

PubMed1996

Neutralization of macrophage inflammatory protein-2 attenuates neutrophil recruitment and bacterial clearance in murine Klebsiella pneumonia.

The role of macrophage inflammatory protein-2 (MIP-2) in bacterial pneumonia was characterized. Mice were challenged with Klebsiella pneumoniae intratracheally, and organs were harvested at 8, 24, and 48 h. Inoculation with K. pneumoniae resulted in the time-dependent expression of MIP-2 mRNA and protein within the lung, which was maximal 48 h after inoculation. Mice were then passively immunized with rabbit anti-murine MIP-2 serum intraperitoneally 2 h before administration of K. pneumoniae. Treatment with anti-MIP-2 serum resulted in a 60% decrease in lung neutrophil (PMNL) influx and a significant increase in K. pneumoniae colony-forming units in both lung and liver homogenates. Finally, treatment with anti-MIP-2 serum decreased early (48-72 h) but not late (after 72 h) survival in animals with Klebsiella pneumonia. This study indicates that MIP-2 is produced during Klebsiella pneumonia and inhibition of MIP-2 bioactivity in vivo results in decreased PMNL influx and lung bacterial clearance in murine Klebsiella pneumonia. MIP-2 is produced during Klebsiella pneumonia and inhibition of MIP-2 bioactivity in vivo results in decreased PMNL influx and lung bacterial clearance in murine Klebsiella pneumonia.

Animals↗

PubMed1996

Antiretroviral monotherapy in early stage human immunodeficiency virus disease has no detectable effect on virus load in peripheral blood and lymph nodes.

Initiation of antiretroviral monotherapy early in the course of infection with human immunodeficiency virus may result in a temporary slowing in the rate of disease progression; however, little is known about the virologic effects of early therapy. Virus load was measured in peripheral blood and lymph nodes from 16 antiretroviral-naive patients with a mean CD4 T lymphocyte count of 659 cells/microliter at baseline and after 8 weeks of either no treatment or zidovudine therapy. CD4 T lymphocyte counts and all virologic parameters examined remained unchanged regardless of zidovudine treatment status. Histopathology and virus distribution within lymph nodes remained constant between baseline and week 8 in each patient, indicating that the virologic and histologic parameters examined in a single lymph node are representative of a systemic process. Early antiretroviral monotherapy with zidovudine had no effect on virologic parameters in this group of patients with relatively high CD4 T lymphocyte counts and low measures of virus load at baseline.

Adult↗

PubMed2001

In vivo gene repair of point and frameshift mutations directed by chimeric RNA/DNA oligonucleotides and modified single-stranded oligonucleotides.

Synthetic oligonucleotides have been used to direct base exchange and gene repair in a variety of organisms. Among the most promising vectors is chimeric oligonucleotide (CO), a double-stranded, RNA-DNA hybrid molecule folded into a double hairpin conformation: by using the cell's DNA repair machinery, the CO directs nucleotide exchange as episomal and chromosomal DNA. Systematic dissection of the CO revealed that the region of contiguous DNA bases was the active component in the repair process, especially when the single-stranded ends were protected against nuclease attack. Here, the utility of this vector is expanded into Saccharomyces cerevisiae. An episome containing a mutated fusion gene encoding hygromycin resistance and eGFP expression was used as the target for repair. Substitution, deletion and insertion mutations were corrected with different frequencies by the same modified single-stranded vector as judged by growth in the presence of hygromycin and eGFP expression. A substitution mutation was repaired the most efficiently followed by insertion and finally deletion mutants. A strand bias for gene repair was also observed; vectors designed to direct the repair of nucleotide on the non-transcribed (non-template) strand displayed a 5-10-fold higher level of activity. Expanding the length of the oligo-vector from 25 to 100 nucleotides increases targeting frequency up to a maximal level and then it decreases. These results, obtained in a genetically tractable organism, contribute to the elucidation of the mechanism of targeted gene repair.

Base Sequence↗

PubMed1995

Entry of Listeria monocytogenes into hepatocytes requires expression of inIB, a surface protein of the internalin multigene family.

The intracellular bacterium Listeria monocytogenes can invade several types of normally non-phagocytic cells. Entry into cultured epithelial cells requires the expression of inIA, the first gene of an operon, comprising two genes: inIA, which encodes internalin, an 800-amino-acid protein, and inIB, which encodes a 630-amino-acid protein. Several genes homologous to inIA are detected in the genome of L. monocytogenes; InIB is one of them. We have assessed the role of inIB in invasiveness of L. monocytogenes by constructing isogenic chromosomal deletion mutants in the inIAB locus. Our findings indicate that: i) inIB is required for entry of L. monocytogenes into hepatocytes, but not into intestinal epithelial cells; ii) inIB encodes a surface protein; iii) internalin plays a role for entry into some hepatocyte cell lines. These results provide the first insight into the cell tropism displayed by L. monocytogenes.

Amino Acid Sequence↗

PubMed2006

The dctA gene of Pseudomonas chlororaphis O6 is under RpoN control and is required for effective root colonization and induction of systemic resistance.

Transcription from the dctA gene, which encodes an organic acid transporter in the root-colonizing bacterium Pseudomonas chlororaphis O6, is under complex regulatory control. Promoter sequence analysis revealed an RpoN binding site. The regulation of transcript accumulation by the level of ammonium ions in the growth medium confirmed RpoN regulation, even in the presence of glucose. A dctA mutant colonized tobacco roots to a lesser extent than the wild-type mutant during early seedling development. Colonization by the dctA mutant, as compared to the wild type, also reduced the level of systemically induced resistance against the soft rot pathogen Erwinia carotovora SCC1. We ascribe this reduced colonization to the inability of the mutant to utilize certain organic acid components in the root exudates. The dctA mutant failed to grow on succinate and fumarate, and showed reduced growth on malate. All altered properties of the mutant were complemented by the full-length dctA gene. We propose that organic acids in root exudates may provide important nutrient sources for the beneficial root-colonizing pseudomonad.

Ammonium Chloride↗

PubMed1998

Combined molecular and conventional analyses of nitrifying bacterium diversity in activated sludge: Nitrosococcus mobilis and Nitrospira-like bacteria as dominant populations.

The ammonia-oxidizing and nitrite-oxidizing bacterial populations occurring in the nitrifying activated sludge of an industrial wastewater treatment plant receiving sewage with high ammonia concentrations were studied by use of a polyphasic approach. In situ hybridization with a set of hierarchical 16S rRNA-targeted probes for ammonia-oxidizing bacteria revealed the dominance of Nitrosococcus mobilis-like bacteria. The phylogenetic affiliation suggested by fluorescent in situ hybridization (FISH) was confirmed by isolation of N. mobilis as the numerically dominant ammonia oxidizer and subsequent comparative 16S rRNA gene (rDNA) sequence and DNA-DNA hybridization analyses. For molecular fine-scale analysis of the ammonia-oxidizing population, a partial stretch of the gene encoding the active-site polypeptide of ammonia monooxygenase (amoA) was amplified from total DNA extracted from ammonia oxidizer isolates and from activated sludge. However, comparative sequence analysis of 13 amoA clone sequences from activated sludge demonstrated that these sequences were highly similar to each other and to the corresponding amoA gene fragments of Nitrosomonas europaea Nm50 and the N. mobilis isolate. The unexpected high sequence similarity between the amoA gene fragments of the N. mobilis isolate and N. europaea indicates a possible lateral gene transfer event. Although a Nitrobacter strain was isolated, members of the nitrite-oxidizing genus Nitrobacter were not detectable in the activated sludge by in situ hybridization. Therefore, we used the rRNA approach to investigate the abundance of other well-known nitrite-oxidizing bacterial genera. Three different methods were used for DNA extraction from the activated sludge. For each DNA preparation, almost full-length genes encoding small-subunit rRNA were separately amplified and used to generate three 16S rDNA libraries. By comparative sequence analysis, 2 of 60 randomly selected clones could be assigned to the nitrite-oxidizing bacteria of the genus Nitrospira. Based on these clone sequences, a specific 16S rRNA-targeted probe was developed. FISH of the activated sludge with this probe demonstrated that Nitrospira-like bacteria were present in significant numbers (9% of the total bacterial counts) and frequently occurred in coaggregated microcolonies with N. mobilis.

Ammonia↗

PubMed1999

Comparative diversity of ammonia oxidizer 16S rRNA gene sequences in native, tilled, and successional soils.

Autotrophic ammonia oxidizer (AAO) populations in soils from native, tilled, and successional treatments at the Kellogg Biological Station Long-Term Ecological Research site in southwestern Michigan were compared to assess effects of disturbance on these bacteria. N fertilization effects on AAO populations were also evaluated with soils from fertilized microplots within the successional treatments. Population structures were characterized by PCR amplification of microbial community DNA with group-specific 16S rRNA gene (rDNA) primers, cloning of PCR products and clone hybridizations with group-specific probes, phylogenetic analysis of partial 16S rDNA sequences, and denaturing gradient gel electrophoresis (DGGE) analysis. Population sizes were estimated by using most-probable-number (MPN) media containing varied concentrations of ammonium sulfate. Tilled soils contained higher numbers than did native soils of culturable AAOs that were less sensitive to different ammonium concentrations in MPN media. Compared to sequences from native soils, partial 16S rDNA sequences from tilled soils were less diverse and grouped exclusively within Nitrosospira cluster 3. Native soils yielded sequences representing three different AAO clusters. Probes for Nitrosospira cluster 3 hybridized with DGGE blots from tilled and fertilized successional soils but not with blots from native or unfertilized successional soils. Hybridization results thus suggested a positive association between the Nitrosospira cluster 3 subgroup and soils amended with inorganic N. DGGE patterns for soils sampled from replicated plots of each treatment were nearly identical for tilled and native soils in both sampling years, indicating spatial and temporal reproducibility based on treatment.

Ammonia↗

PubMed2005

Responses of Baltic Sea ice and open-water natural bacterial communities to salinity change.

To investigate the responses of Baltic Sea wintertime bacterial communities to changing salinity (5 to 26 practical salinity units), an experimental study was conducted. Bacterial communities of Baltic seawater and sea ice from a coastal site in southwest Finland were used in two batch culture experiments run for 17 or 18 days at 0 degrees C. Bacterial abundance, cell volume, and leucine and thymidine incorporation were measured during the experiments. The bacterial community structure was assessed using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified partial 16S rRNA genes with sequencing of DGGE bands from initial communities and communities of day 10 or 13 of the experiment. The sea ice-derived bacterial community was metabolically more active than the open-water community at the start of the experiment. Ice-derived bacterial communities were able to adapt to salinity change with smaller effects on physiology and community structure, whereas in the open-water bacterial communities, the bacterial cell volume evolution, bacterial abundance, and community structure responses indicated the presence of salinity stress. The closest relatives for all eight partial 16S rRNA gene sequences obtained were either organisms found in polar sea ice and other cold habitats or those found in summertime Baltic seawater. All sequences except one were associated with the alpha- and gamma-proteobacteria or the Cytophaga-Flavobacterium-Bacteroides group. The overall physiological and community structure responses were parallel in ice-derived and open-water bacterial assemblages, which points to a linkage between community structure and physiology. These results support previous assumptions of the role of salinity fluctuation as a major selective factor shaping the sea ice bacterial community structure.

Colony Count, Microbial↗

PubMed1995

Active immunity to Pneumocystis carinii reinfection in T-cell-depleted mice.

Immunocompetent C.B-17 mice were immunized against Pneumocystis carinii by several intratracheal inoculations with infective P. carinii. These mice and another group of C.B-17 mice naive to P. carinii were then depleted of CD4+ cells by treatment with both anti-CD4 and anti-Thy1 monoclonal antibodies. Both groups of mice were then challenged with an infective inoculum containing 10(7) P. carinii organisms by intratracheal instillation. The mean log10 counts of P. carinii nuclei in the lungs of the nonimmune mice were 4.98, 5.89, and 6.77 when they were killed at 4, 10, and 19 days, respectively, after challenge. The P. carinii counts in the lungs of the immune mice were significantly lower at each time point and below detectable levels at 10 and 19 days. Analysis of P. carinii DNA by PCR revealed no detectable P. carinii in the lungs of the immunized mice at either 10 or 19 days, whereas all of the nonimmunized mice contained P. carinii DNA at all time points. The sera of immune but not nonimmune mice contained P. carinii-specific immunoglobulin G. These results indicate that immunization of an immunocompetent host against P. carinii can protect against P. carinii pneumonia even after the host is depleted of CD4+ cells. In addition, the results are consistent with the possibility that antibodies were responsible for the observed protection against P. carinii.

Animals↗

PubMed1997

Identification of four new members of the internalin multigene family of Listeria monocytogenes EGD.

Listeria monocytogenes is a bacterial pathogen that is able to invade nonphagocytic cells. Two surface proteins, internalin, the inlA gene product, and InlB, play important roles in the entry into cultured mammalian cells. These proteins also have extensive sequence similarities. Previously, Southern hybridization predicted the existence of an internalin multigene family. Recently, InlC, a secreted protein of 30 kDa homologous to InlA and InlB, was identified. In this work, we identified and characterized four new members of the internalin multigene family, inlC2, inlD, inlE, and inlF which encode proteins of 548, 567, 499, and 821 amino acids respectively. inlC2, inlD, and inlE are contiguous on the chromosome of L. monocytogenes EGD, whereas inlF is located in a different chromosomal region. These four inl gene products display the principal features of internalin, namely, a signal sequence, two regions of repeats (or LRR and B repeats), and a putative cell wall anchor sequence containing the sorting motif LPXTG. The four inl genes were maximally expressed albeit at a low level during early exponential growth in bacterial medium at 37 degrees C. The role of these inl genes in L. monocytogenes invasion was assessed by constructing isogenic chromosomal deletion mutants and testing them for entry into various nonphagocytic cells. Unexpectedly, the inlC2, inlD, inlE, and inlF null mutants were not affected for entry into any of the cell lines tested, raising the possibility that these genes are needed for an aspect of pathogenicity other than invasion. The identity of such an aspect remains to be determined.

Amino Acid Sequence↗

PubMed1999

A novel Campylobacter jejuni two-component regulatory system important for temperature-dependent growth and colonization.

Campylobacter jejuni colonizes the intestines of domestic and wild animals and is a common cause of human diarrheal disease. We identified a two-component regulatory system, designated the RacR-RacS (reduced ability to colonize) system, that is involved in a temperature-dependent signalling pathway. A mutation of the response regulator gene racR reduced the organism's ability to colonize the chicken intestinal tract and resulted in temperature-dependent changes in its protein profile and growth characteristics.

Animals↗

PubMed1998

Genetic variation in a human immunodeficiency virus type 2 live-virus Macaca nemestrina vaccine model.

Four pigtailed macaques were inoculated with an infectious, apathogenic human immunodeficiency virus type 2 (HIV-2) molecular clone (HIV-2KR) and subsequently challenged with a highly pathogenic strain, HIV-2287, together with two naive control animals. After challenge, two animals inoculated with a high dose of the immunizing strain were protected from CD4 decline and immunodeficiency. To examine the role of genetic heterogeneity in protection, fragments of the env gene were amplified from peripheral blood mononuclear cell DNA and plasma RNA of challenged animals by PCR, examined by using a heteroduplex tracking assay (HTA), and sequenced. By HTA, variation was detected principally within the V1 and V2 regions of envelope. Extent of variation in viral DNA clones as assessed by HTA correlated with inoculum size, as did the degree of variation in sequences of clones derived from viral DNA. Conversely, a rapid reduction in the number of plasma viral RNA variants was noted by HTA at 8 weeks postinfection in protected animals; this reduction was not present in naive or unprotected macaques. Sequences derived from plasma viral RNA were found to be more closely related than corresponding viral DNA sequences, and protection correlated with a significant reduction in variation in plasma RNA sequences in animals given the identical inocula of HIV-2287. Nonsynonymous mutations were significantly less prevalent in the protected animals. An additional potential glycosylation site was predicted to be present in the V2 region in all but one clone, and amino acid signatures related to protection were identified in viral DNA and RNA clones within both the V1 and V2 regions. Examination of the role of viral variation in this HIV-2 live-virus vaccine model may provide valuable insights into immunopathogenesis.

Amino Acid Sequence↗

PubMed1998

Delayed infection after immunization with a peptide from the transmembrane glycoprotein of the feline immunodeficiency virus.

Recent advances in the quantitative assessment of viral burden, by permitting the extension of criteria applied to assess the efficacy of vaccines from all-or-none protection to diminution of the viral burden, may allow the identification of original immunogens of value in combined vaccines. Peptides corresponding to three domains of the envelope glycoproteins of feline immunodeficiency virus that are recognized during natural infection were used to immunize cats. After challenge with a primary isolate of feline immunodeficiency virus, the development of acute infection was monitored by quantitative assessment of the viral burden in plasma and tissues by competitive reverse transcription-PCR, by measurement of the humoral response developed to viral components, and by lymphocyte subset analysis. Whereas immunization with two peptides derived from the surface glycoprotein had no effect on the early course of infection, immunization with a peptide derived from the transmembrane glycoprotein delayed infection, as reflected by a diminished viral burden in the early phase of primary infection and delayed seroconversion. This peptide, located in the membrane-proximal region of the extracellular domain, has homology to an epitope of human immunodeficiency virus type 1 recognized by a broadly neutralizing monoclonal antibody. These results suggest that lentivirus transmembrane glycoproteins share a determinant in the juxtamembrane ectodomain which could be of importance in the design of vaccines against AIDS.

Acute Disease↗

PubMed2005

Idiopathic bilateral optic atrophy in the rhesus macaque.

PURPOSE: To document the existence of idiopathic bilateral optic atrophy (BOA) in rhesus macaque monkeys and to characterize the structural and functional consequences of this condition. METHODS: In vivo assessment of retinal and optic nerve structure included fundus biomicroscopy and stereophotography. Functional analyses included transient pattern-reversal electroretinography (PERG) and full-field flash ERG, with both white flashes while dark adapted and red flashes on a blue background used to assess the photopic negative response (PhNR). Also measured were visual evoked cortical potentials (VEPs) and multifocal (mf)ERGs, with both a standard fast and slowed (7F) stimulation sequence. Post mortem histologic evaluation was performed on a subset of five animals with BOA and compared with data from 22 healthy normal animals. Blood tests, including vitamin E, B(12), folate, lead, and complete blood cell count with differential were obtained on the four animals that remained alive. RESULTS: Animals with BOA showed temporal pallor of the optic nerve head and thinning of the retinal nerve fiber layer (RNFL) between the temporal vascular arcades (i.e., of the papillomacular bundle). Severity of optic atrophy and RNFL loss varied between animals from mild to severe, but was similar in the two eyes of each animal. Functional changes included greater loss of the PERG N95, compared with the P50 component and substantial reduction of mfERG high-frequency components. The mfERG low-frequency components were slightly larger than normal. None of the full-field flash ERG amplitudes (a-wave, b-wave, oscillatory potentials, or PhNR) was significantly different from normal. There were no consistent abnormalities found in the results of any blood test. Histologic findings included axonal loss and gliosis limited to the temporal optic nerve, reduction of nuclei within the retinal ganglion cell layer, and thinning of the temporal retinal RNFL. CONCLUSIONS: The existence of BOA in nonhuman primates warrants caution on the part of investigators who use these animals in experimental models of ophthalmic disease.

Animals↗

PubMedDate not supplied

The effectiveness of anti-retroviral drug therapy for HIV-1 is associated with HIV-1 proviral DNA levels and viral selection.

The effect of combination anti-retroviral therapy regimens on HIV-1 proviral DNA levels in peripheral blood mononuclear cells was examined in 12 HIV-1-positive patients, using endpoint dilution polymerase chain reaction and serial cloning, and sequencing of the gag region of HIV-1. The major clone was defined as the most numerous of 10 analysed clones, and observation periods ranged from 8 months to 32 months (mean 19.7 +/- 10.2 months). In five patients (one with primary-stage HIV-1 infection) receiving three anti-retroviral drugs, HIV-1 RNA reduced to undetectable levels (i.e. < or = 100 copies/ml). HIV-1 proviral DNA and the number of major clones reduced in four of these patients. HIV-1 RNA levels reduced, but remained detectable, in five other patients. In the two remaining patients (both receiving two rather than three anti-retroviral drugs), HIV-1 RNA levels increased. These results suggest that the population of major clones may be affected when HIV-1 RNA levels reduce following combination regimens of anti-retroviral therapy.

Adult↗

PubMed2002

Vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating peptide receptor 2 deficiency in mice results in growth retardation and increased basal metabolic rate.

Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) are two closely related peptides that bind two homologous G protein-coupled receptors, VIP/PACAP receptor 1 (VPAC1R) and VIP/PACAP receptor II (VPAC2R), with equally high affinity. Recent reports suggest that VPAC2R plays a role in circadian rhythm and T cell functions. To further elucidate the functional activities of VPAC2R, we generated VPAC2R-deficient mice by deleting exons VIII-X of the VPAC2R gene. The VPAC2R-deficient mice showed retarded growth and had reduced serum IGF-I levels compared with gender-matched, wild-type siblings. The mutant mice appeared healthy and fertile at a young adult age. However, older male mutant mice exhibited diffuse seminiferous tubular degeneration with hypospermia and reduced fertility rate. The mutant mice appeared to have an increase in insulin sensitivity. VPAC2R-deficient mice had increased lean mass and decreased fat mass with reduced serum leptin levels. Indirect calorimetry experiments showed that the respiratory quotient values immediately following the transition into the dark cycle were significantly higher in male knockout mice for about 4 h. Additionally, male and female VPAC2R-deficient mice presented an increased basal metabolic rate (23% and 10%, respectively) compared with their wild-type siblings. Our results suggest that VPAC2R plays an important role in growth, basal energy expenditure, and male reproductive functions.

Amino Acid Sequence↗

PubMed2006

santa and valentine pattern concentric growth of cardiac myocardium in the zebrafish.

During embryogenesis, the myocardial layer of the primitive heart tube grows outward from the endocardial-lined lumen, with new cells added to generate concentric thickness to the wall. This is a key evolutionary step, demarcating vertebrates from more primitive chordates, and is essential for normal cardiac function. Zebrafish embryos with the recessive lethal mutations santa (san) and valentine (vtn) do not thicken, but do add the proper number of cells to the myocardium. Consequently, the heart chambers are huge, constituted of a monolayered myocardium lined by endocardium. This phenotype is similar to that of the heart of glass (heg) mutation, which we described previously as a novel endocardial expressed gene. By positional cloning, we here identify san as the zebrafish homolog of human CCM1, and vtn as the homolog of human CCM2. Dominant mutations of either in humans cause vascular anomalies in the brain, known as cerebral cavernous malformations. The synergistic effects of morpholino pairs indicate that san, vtn and heg are in a genetic pathway, and san and vtn contain protein motifs, NPxY and PTB domain, respectively, known to interact. This suggests that concentric growth of the myocardium, crucial for blood pressure generation, is dictated by a heg-san-vtn signaling pathway.

Amino Acid Sequence↗