Primary human herpesvirus 8 infection generates a broadly specific CD8(+) T-cell response to viral lytic cycle proteins.

Human herpesvirus 8 (HHV-8) is a recently discovered gammaherpesvirus that is the etiologic agent of Kaposi sarcoma (KS). The natural history of primary HHV-8 infection, including clinical outcome and host immune responses that may be important in preventing disease related to HHV-8, has not been elucidated. The present study characterized the clinical, immunologic, and virologic parameters of primary HHV-8 infection in 5 cases detected during a 15-year longitudinal study of 108 human immunodeficiency virus type 1 seronegative men in the Multicenter AIDS Cohort Study. Primary HHV-8 infection was associated with mild, nonspecific signs and symptoms of diarrhea, fatigue, localized rash, and lymphadenopathy. There were no alterations in numbers of CD4(+) or CD8(+) T cells or CD8(+) T-cell interferon gamma (IFN-gamma) production to mitogen or nominal antigen. CD8(+) cytotoxic T-lymphocyte precursor (CTLp) and IFN-gamma reactivity were detected during primary HHV-8 infection, with broad specificity to 5 lytic cycle proteins of HHV-8 encoded by open reading frame 8 (ORF 8; glycoprotein B homolog of Epstein-Barr virus), ORF 22 (gH homolog), ORF 25 (major capsid protein homolog), ORF 26 (a minor capsid protein homolog), or ORF 57 (an early protein homolog), in association with increases in serum antibody titers and appearance of HHV-8 DNA in blood mononuclear cells. CD8(+) T-cell responses to HHV-8 decreased by 2 to 3 years after primary infection. This antiviral T-cell response may control initial HHV-8 infection and prevent development of disease.

CD8+ cytotoxic T lymphocyte responses to lytic proteins of human herpes virus 8 in human immunodeficiency virus type 1-infected and -uninfected individuals.

T cell immunity to lytic proteins of herpesviruses is important in host control of infection. We have characterized the cytotoxic T lymphocyte (CTL) response to 5 human herpesvirus 8 (HHV-8) homologues of lytic proteins in HHV-8-seropositive individuals. HLA class I-restricted, CD8(+) CTL responses to >/=1 HHV-8 lytic protein were detected in all 14 HHV-8-seropositive study subjects tested, with or without human immunodeficiency virus type 1 (HIV-1) infection, but not in any of 5 HHV-8-seronegative individuals. Seven of these study subjects with both HHV-8 and HIV-1 infection had greater anti-CTL reactivity to glycoprotein H (open-reading frame 22) than did the 7 study subjects infected only with HHV-8. Moreover, there was a strong, inverse correlation between HIV-1 load and glycoprotein H-specific CTL lysis in the study subjects infected with both viruses. CTL reactivity to HHV-8 lytic proteins may be involved in host control of HHV-8-related diseases, such as Kaposi's sarcoma.

Comparison of serologic assays and PCR for diagnosis of human herpesvirus 8 infection.

A variety of assays for the diagnosis human herpesvirus 8 (HHV-8) infection have been reported. We compared several such assays with a panel of 88 specimens from human immunodeficiency virus (HIV)-infected patients with Kaposi's sarcoma (KS) (current-KS patients; n = 30), HIV-infected patients who later developed KS (later-KS patients; n = 13), HIV-infected patients without KS (no-KS patients; n = 25), and healthy blood donors (n = 20). PCR assays were also performed with purified peripheral blood mononuclear cells (PBMCs) to confirm positive serologic test results. The order of sensitivity of the serologic assays (most to least) in detecting HHV-8 infection in current-KS patients was the mouse monoclonal antibody-enhanced immunofluorescence assay (MIFA) for lytic antigen (97%), the orfK8.1 peptide enzyme immunoassay (EIA) (87%), the orf65 peptide EIA (87%), MIFA for latent antigen (83%), the Advanced Biotechnologies, Inc., EIA (80%), and the orf65 immunoblot assay (80%). Combination of the results of the two peptide EIAs (combined peptide EIAs) increased the sensitivity to 93%. For detection of infection in later-KS patients, the MIFA for lytic antigen (100%), the orfK8.1 peptide EIA (85%), and combined peptide EIAs (92%) were the most sensitive. Smaller percentages of no-KS patients were found to be positive (16 to 56%). Most positive specimens from the current-KS and later-KS groups were positive by multiple assays, while positive specimens from the no-KS group tended to be positive only by a single assay. PCR with PBMCs for portions of the HHV-8 orf65 and gB genes were positive for less than half of current-KS and later-KS patients and even fewer of the no-KS patients. The concordance between serologic assays was high. We propose screening by the combined peptide EIAs. For specimens that test weakly positive, we recommend that MIFA for lytic antigen be done. A positive result with a titer of >/=1:40 would be called HHV-8 positive. A negative or low titer would be called HHV-8 negative. If a population has a high percentage of persons who test positive by the combined peptide EIAs, then a MIFA could be performed with the negative specimens to determine if any positive specimens are being missed. Alternatively, if a population has a low percentage that test positive, then a MIFA could be performed with a subset of the negative specimens for the same reason. As described above, only a titer of >/=1:40 would be considered HHV-8 positive.

Glycoprotein B of human herpesvirus 8 is a component of the virion in a cleaved form composed of amino- and carboxyl-terminal fragments.

Human herpesvirus 8 (HHV-8) or Kaposi's sarcoma-associated herpesvirus (KSHV) is the only known human member of the Rhadinovirus genus of the gammaherpesvirus subfamily. Antibodies against peptides representing portions of the amino- and carboxyl-termini of HHV-8 gB were produced and used to detect gB expression in Vero cells transfected with the gB gene, in the HHV-8-harboring cell line, BCBL-1, and in purified virions. Expression of gB was detected in approximately 3% of uninduced BCBL-1 cells, while up to 30% of the cells expressed gB after 12-O-tetradecanoylphorbol-13-acetate (TPA) induction of virus replication. Indirect immunofluorescence assays and confocal microscopy showed that gB was distributed throughout the cytoplasm of BCBL-1 cells and transfected Vero cells. Immunoblot analyses of virion preparations revealed the presence of full-length as well as two smaller than full-length gB-derived species corresponding to the amino- and carboxy-terminal portions of gB, respectively. Biochemical analysis of the gB carbohydrate moieties using glycosylation inhibitors revealed that gB contained N-linked oligosaccharides of the high-mannose type, characteristic of precursor carbohydrate chains added in the endoplasmic reticulum.

Individuals from North America, Australasia, and Africa are infected with four different genotypes of human herpesvirus 8.

To study human herpesvirus 8 (HHV-8) transmission between individuals and in populations, we developed a system for genetic fingerprinting of HHV-8 strains based on variation in the HHV-8 K1, glycoprotein B (gB), and glycoprotein H (gH) genes. Using this system, we sequenced nearly the entire K1 gene (840 bp); two segments of the gB gene (open reading frame 8), totaling 813 bp; and a 702-bp segment of the gH gene (open reading frame 22) from blood and tissue samples obtained from 40 human immunodeficiency virus-infected and noninfected individuals, including those with Kaposi's sarcoma, primary effusion lymphoma, or Castleman's disease. The specimen collection was assembled from individuals living in diverse geographical locations, including the United States, Australia, New Zealand, Uganda, and Zambia. As reported by others, K1 was the most variable gene, with up to 16% variation at the nucleotide sequence level and up to 32% variation at the amino acid sequence level. Despite this extensive sequence variation, the K1 amino acid sequence contained 14 conserved cysteine sites, suggesting a conserved tertiary structure. gB and gH sequences were highly conserved, in most cases differing by <0.6% in pairwise comparisons. K1 was the most useful gene for strain discrimination, but the other genes enabled the discrimination of strains with identical K1 sequences. Individuals from diverse geographic locations were infected with four different HHV-8 genotypes; strains did not strictly segregate by continent of origin. The majority of HHV-8 strains from the United States and Europe were relatively closely related, whereas some strains identified from Uganda and Australia were phylogenetically distant. Genotype I strains were the most common and were found on three continents. Identical sequences were found in specimens obtained from different body sites and at different times from the same individual.

Suppression of the expression of a pancreatic beta-cell form of the kinesin heavy chain by antisense oligonucleotides inhibits insulin secretion from primary cultures of mouse beta-cells.

Granular/vesicular transport is thought to be supported by microtubule-based force-generating adenosine triphosphatases such as kinesin. Kinesin is a motor molecule that has been well studied in brain and other neuronal tissues. Although vesicular transport is important for pancreatic beta-cell secretory activities, the role of kinesin in beta-cell function has not been investigated. It is hypothesized that kinesin functions as a translocator that associates with both microtubules and insulin-containing granules in beta-cells and transports the secretory granules from deep within the cytoplasm, where insulin is synthesized and processed, to the surface of beta-cells upon secretory stimulation. To test this hypothesis, a mouse beta-cell kinesin heavy chain complementary DNA was cloned and sequenced. Kinesin expression in primary cultures of mouse beta-cells then was selectively suppressed by antimouse beta-cell kinesin heavy chain antisense oligonucleotide treatment. Analysis of insulin secretion determined that the basal level of insulin secretion from the treated cells was decreased by 50%. Furthermore, glucose-stimulated insulin release from treated beta-cells was reduced by almost 70% after suppression of kinesin expression by antisense treatment. The findings from this study provide the first direct evidence that kinesin, a microtubule-based motor protein, plays an important role in insulin secretion.

Effect of norethisterone enanthate on endometrial bleeding and changes in coagulation function, ovarian hormones and endometrial histology in Thai women.

Endometrial bleeding and alteration in blood coagulation, fibrinolysis, ovarian function and endometrial morphology were studied in twelve normally menstruating women who received the injectable contraceptive norethisterone enanthate (NET-EN) 200mg at 60-day intervals. Levels of clotting factor VII declined significantly during episodes of irregular bleeding compared to those during both normal pretreatment menstruation and the bleeding-free period during treatment. Antithrombin III and fibrinolytic activity, expressed by euglobulin lysis time, showed no marked change. The average level of progesterone during the bleeding-free period was slightly but significantly lower than that during the bleeding period. There were no significant alterations in the mean levels of estradiol and the ratio of estradiol to progesterone. The endometrial biopsies showed considerable individual variation and seem to be independent of the effects of NET-EN on ovarian function.

The effects of long-term using once-a-month injectable contraceptive norethisterone enanthate/estradiol valerate on coagulation in the Chinese.

Sixty-four Chinese women were studied for the effects of injecting once-a-month contraceptive norethisterone enanthate/estradiol valerate between 2 to 4 1/2 years on certain coagulation and fibrinolytic parameters, and 59 women not receiving any steroidal contraception were controls. In both groups there was no significant change in PT, fibrinogen, factor X activity, AT III antigen, AT III activity and fibrinolytic activity expressed by euglobulin lysis time. There was no significant difference in mean levels of factor VIIIR:Ag between the two groups, although a sub-group women in treatment group had higher than accepted levels of factor VIIIR:Ag.

Hemostatic changes in women using a monthly injectable contraceptive for one year.

The effect of consecutively injecting a one-a-month contraceptive (norethisterone enantate 50 mg with estradiol valerate 5 mg) for one year on haematological parameters was evaluated in 42 Chinese women. The healthy volunteers were randomly allocated to either the treatment group (22) or a control group (20). Blood samples were collected in the follicular and luteal phases of a pretreatment cycle, on days 28 +/- 3 after the 1st, 3rd, 6th, 12th injections and in the luteal phase of the post-treatment cycle. The results showed that in both groups, prothrombin time and fibrinogen fluctuated significantly, and leucocyte count was not significantly changed during the whole course. Factor VIII-related antigen and antithrombin III (AT-III) antigen showed minor changes, although in the 3rd treatment cycle, the differences between the two groups in both parameters reached statistical significance. A progressive and significant decrease in Factor X and AT-III functional activity occurred with the monthly injectable treatment, decreasing by about 14% and 20%, respectively, after 12 months of treatment. Haemoglobin levels were increased in the treatment group after the 3rd injection and remained at the higher level during the study period. It is doubtful whether these changes are likely to be of clinical relevance.