A cDNA library from phorbol ester-induced human herpesvirus-8 (HHV-8) carrying BCBL-1 cells was screened with an HIV+KS+ serum, and several cDNA clones encoding HHV-8 proteins were identified. Sequence analysis of two full-length cDNA clones show open reading frames (ORFs) encoded by spliced messages originating from the HHV-8 K8.1 gene. One cDNA encodes an ORF of 228 amino acids, designated K8. 1.A, with a cleavable signal sequence, a transmembrane domain, and four N-glycosylation sites. The splicing event generated the transmembrane domain in the ORF not seen in the genomic K8.1 ORF. Another cDNA encodes an ORF of 167 amino acids, designated K8.1.B, that shares similar amino and carboxyl termini with ORF K8.1.A but with an in-frame deletion. The primary translation products of ORF K8.1A (34 kDa) and K8.1B (20 kDa) in the in vitro-transcription-translation experiments shifted into glycosylated species of 43 and 32 kDa, respectively, in the presence of microsomal membranes. This suggested that the ORF K8.1A and K8.1B encode for glycoproteins. Riboprobes from the K8.1A cDNA insert hybridized with an HHV-8-specific 0.9-kb abundant transcript from BCBL-1 cells. Synthesis of this RNA was eliminated in the presence of a DNA synthesis inhibitor, suggesting that this RNA was a late gene transcript. Because ORFs K8.1A and K8.1B are unique for HHV-8, human sera were tested in Western blot reactions for antibodies against glutathione-S-transferase-ORF K8.1A fusion protein. All sera that were positive for HHV-8 antibodies in immunofluorescence assays with phorbol ester-induced BCBL-1 cells were also positive for anti-ORF K8.1A antibodies. This suggests that measurement of anti-ORF K8.1A antibodies would provide an HHV-8-specific serological assay. Further work is needed to define the biological role of the HHV-8 ORF K8.1A and K8.1B glycoproteins.

These data indicate that infusion of IA-PBMC cell suspension in experimental AMI circumvented inflammation, caused preferential homing of regenerative EPC and replaced infarcted myocardium.

Although Francisella tularensis subsp. tularensis is known to cause extensive tissue necrosis, the pathogenesis of tissue injury has not been elucidated. To characterize cell death in tularemia, C57BL/6 mice were challenged by the intranasal route with type A F. tularensis, and the pathological changes in infected tissues were characterized over the next 4 days. At 3 days postinfection, well-organized inflammatory infiltrates developed in the spleen and liver following the spread of infection from the lungs. By the next day, extensive cell death, characterized by the presence of pyknotic cells containing double-strand DNA breaks, was apparent throughout these inflammatory foci. Cell death was not mediated by activated caspase-1, as has been reported for cells infected with other Francisella subspecies. Mouse macrophages and dendritic cells that had been stimulated with type A F. tularensis did not release interleukin-18 in vitro, a response that requires the activation of procaspase-1. Dying cells within type A F. tularensis-infected tissues expressed activated caspase-3 but very little activated caspase-1. When caspase-1-deficient mice were challenged with type A F. tularensis, pathological changes, including extensive cell death, were similar to those seen in infected wild-type mice. In contrast, type A F. tularensis-infected caspase-3-deficient mice showed much less death among their F4/80 ؉ spleen cells than did infected wild-type mice, and they retained the ability to express tumor necrosis factor alpha and inducible NO synthase. These findings suggest that type A F. tularensis induces caspase-3-dependent macrophage apoptosis, resulting in the loss of potentially important innate immune responses to the pathogen.

Polymerase chain reaction (PCR) was used to examine human herpesvirus 8 (HHV-8) DNA from Kaposi's sarcoma (KS) lesions, normal skin, and peripheral blood mononuclear cells (PBMC) from human immunodeficiency virus (HIV)-infected patients who did or did not have KS. Of 9 KS biopsies, 8 were positive for five HHV-8 open-reading frames and ranged from 1 viral genome per 2.5-12.7 cells. Two putative replicative gene RNAs were detected by reverse transcription-PCR at low levels in 1 KS lesion. HHV-8 DNA was detected in 4 of 8 PBMC samples from patients with KS and in 2 of 18 PBMC samples from patients without KS. Sera were tested for reactivity with BCBL-1 cells (HHV-8 positive): High immunofluorescence antibody titers against HHV-8 lytic and latent antigens were detected in samples from KS-positive patients, and >20 polypeptides from induced BCBL-1 cells were recognized. Sera from 6 of 18 patients without KS showed low levels of antibodies against HHV-8 lytic and latent antigens.

The development of reliable, sensitive, and specific serological methods for the detection of human herpesvirus-8 (HHV-8) antibodies is critical for a thorough understanding of HHV-8 prevalence and pathogenesis. To evaluate the potential usefulness of HHV-8 proteins in measuring the responses against both latent and lytic antigens, we selected 1 latent [open reading frame (ORF) 73] antigen and 3 HHV-8 lytic antigens (ORFs 65, K8.1A, and K8.1B) previously identified as immunogenic [Virology (1998) 243, 208-217]. Full-length genomic ORF 73 and full-length ORFs 65, K8.1A, and K8.1B from the cDNA clones were cloned, expressed in bacterial and baculovirus-insect cell expression systems, and purified as GST fusion proteins. These recombinant proteins were used in Western blot reactions to test sera from 104 human immunodeficiency virus (HIV)+/Kaposi's sarcoma (KS)+ homosexual men, 77 HIV+/KS- homosexual men, and 84 age-matched HIV-/KS- men. These sera were also tested in immunofluorescence assays (IFAs) with uninduced and 12-O-tetradecanoylphorbol-13-acetate-induced B cell lymphoma-1 cells to detect antibodies against latency-associated nuclear antigens (LANA) and antibodies against lytic antigens (cytoplasmic fluorescence). These sera exhibited differential reactivities reflecting different titers of antibodies against HHV-8 proteins, and variable reactivities were seen more commonly with the sera from HIV-/KS- adult men. In the Western blot assay, 89% (93 of 104) of HIV+/KS + sera, 60% (46 of 77) of HIV+/KS- sera, and 7% (6 of 84) HIV+/KS- sera were reactive with both latent and lytic recombinant antigens. Western blot reactions with ORF 73 protein were more sensitive than LANA-IFA results. The lytic IFA and lytic Western blot (ORFs 65 and K8.1A) assays were more sensitive than the ORF 73 Western blots and LANA-IFA. With an exception of 2 sera from the HIV-/KS- group, all sera positive for lytic IFA antibodies and ORF 65 and K8.1A antibodies were also positive for latent antibodies. With few exceptions, sera positive for ORF 65 antibodies were also positive for K8.1A antibodies, and sera recognized the K8.1A protein more often than the K8.1B protein. There is a high degree of concordance between IFA and Western blot reactions, suggesting that this panel of HHV-8 recombinant proteins could detect a majority of the HHV-8-seropositive individuals. These results suggest that IFA followed by confirmation with the Western blot reactions with a panel of latent and lytic immunogenic antigens would provide a reliable, sensitive, and specific method for the detection of HHV-8 antibodies.

The reactivates of human sera with uninduced and phorbol ester (TPA)-induced human herpesvirus-8 (HHV-8)-infected BCBL-1 cells were examined by immunofluorescence assay (IFA) and by radioimmunoprecipitation reactions (RIP). The seroprevalence of HHV-8 infections is low in the United States general population and only low levels of HHV-8 antibodies were detected in the seropositive sera. In contrast, high levels of antibodies against HHV-8 lytic and latent antigens were detected by IFA in the sera from HIV+ Kaposi's sarcoma (KS)-positive individuals. These sera recognized several proteins and glycoproteins from BCBL-1 cells in RIP reactions. Two types of antibody responses were detected in the sera from HIV+ KS- homosexual men. In majority of the sera with and without detectable HHV-8 DNA in the peripheral blood mononuclear cells (PBMC), significantly low levels of HHV-8 antibodies were detected by IFA. These sera recognized only a subset of HHV-8 proteins and glycoproteins in RIP reactions. In contrast, in a subgroup of sera from HIV+ KS- homosexual men, higher levels of IFA antibodies against HHV-8 lytic and latent antigens were detected. These sera also recognized several viral proteins and glycoproteins in RIP reactions. These results suggest that antibody response profiles to HHV-8 infection vary significantly and serologic assays to detect antibody responses to a panel of both lytic and latent antibodies may be required for maximum sensitivity. Screening of a cDNA library from TPA-induced BCBL-1 cells with an HIV+ KS+ serum identified cDNAs encoding 12 HHV-8 proteins. Further characterization of these HHV-8 proteins would define the HHV-8 antigens useful for seroepidemiological studies and in discriminating lytic, latent, past, and/or reactivation infections.

Lack of standardization in antibiogram (ABGM) preparation (the overall profile of antimicrobial susceptibility results of a microbial species to a battery of antimicrobial agents) has not been addressed until recently. The objective of this study was to analyze current antibiograms using the recently published NCCLS M39-A guidelines for preparation of antibiograms to identify areas for improvement in the reporting of antibiogram susceptibility data. Antibiograms from across the United States were obtained by various methods, including direct mailings, Internet searches, and professional contacts. Each ABGM collected was analyzed using prospectively defined elements from the M39-A guidelines. Additionally, seven quality indicators were also evaluated to look for the reporting of any atypical or inappropriate susceptibility data. The 209 antibiograms collected from 149 institutions showed at least 85% compliance to 5 of the 10 M39-A elements analyzed. Clinically relevant elements not met included annual analysis, duplicate isolate notation, and the exclusion of organisms with fewer than 10 isolates. As for the quality indicators evaluated, unexpected results included the 7% of antibiograms that reported <100% vancomycin susceptibility for Staphylococcus aureus, 24% that had inconsistent betalactam susceptibility for Staphylococcus aureus, 20% that reported <100% imipenem susceptibility for Escherichia coli, and 37% that reported >0% ampicillin susceptibility for Klebsiella pneumoniae. These findings suggest that antibiograms should be reviewed thoroughly by infectious disease specialists (physicians and pharmacists), clinical microbiologists, and infection control personnel for identification of abnormal findings prior to distribution.

Pseudomonas aeruginosa alkaline protease and elastase are thought to contribute to bacterial invasiveness, tissue damage, and immune suppression in animals and patients infected with the bacterium. This study examined the ability of the two proteases to inactivate a number of cytokines that mediate immune and inflammatory responses. Human recombinant gamma interferon (rIFN-y) and human recombinant tumor necrosis factor alpha were inactivated by both proteases. Murine rIFN-,y was relatively resistant to alkaline protease but was inactivated by elastase, and human recombinant interleukin-la and recombinant interleukin-lo were resistant to the effects of both proteases. Western immunoblots suggested that cytokine inactivation by these proteases, where it occurred, required only limited proteolysis of the polypeptides. The ability of different P. aeruginosa strains to inactivate IFN--y appeared to require the production of both proteases for optimum activity. These results indicate that in vitro cytokine inactivation by Pseudomonas proteases is selective, requires only limited proteolysis, and in certain instances reflects the cooperative effects of both proteases.Pseudomonas aeruginosa is an important pulmonary pathogen in conditions like cystic fibrosis (15,42). It has been suggested that the ability of P. aeruginosa to establish itself in the respiratory tract may be promoted by its suppressive effects on pulmonary immune responses (2,16,40). The mechanisms of this immunosuppression are not entirely understood, but proteolytic enzymes secreted by the bacterium have been shown to degrade surface receptors on hematogenous cells (36, 43) and inactivate interleukin-2 (IL-2) and gamma interferon (IFN--y) (16,17,41).In the case of human IFN--y, cytokine inactivation was caused by either Pseudomonas alkaline protease (AP) (17) or elastase (E) (16). Significant reductions in antiviral and immunomodulatory activities were associated with limited proteolysis of IFN--y. Of particular interest were the synergistic effects on IFN--y seen when both purified proteases were added to reaction mixtures (16). These results would predict that Pseudomonas strains that produce both enzymes should be particularly immunosuppressive, a property that may aid the bacterium in establishing initial colonization by significantly altering immune and inflammatory responses in infections like those seen in cystic fibrosis.This study was undertaken to address three questions relating to the effects of Pseudomonas protease on cytokines. First, what are the specificities of these proteases relative to the inactivation of cytokines that might be involved in Pseudomonas infections? Second, is limited proteolysis of the type seen with human IFN--y sufficient for the inactivation of other cytokines? Third, is the ability to inactivate cytokines a common property of Pseudomonas strains, and how does this property relate to their production of the two proteases? MATERIALS AND METHODSHuman subjects. The studies reported here were approved by the Human Subj...