Donor-derived Kaposi's sarcoma in a liver-kidney transplant recipient.

Human herpes virus 8 (HHV-8), also known as Kaposi's sarcoma associated herpesvirus (KSHV), is an oncogenic virus that can cause Kaposi's sarcoma (KS). KS can develop following organ transplantation through reactivation of the recipient's latent HHV-8 infection, or less commonly through donor-derived infection which has higher risk for severe illness and mortality. We describe a case of probable donor-derived KS in the recipient of a liver-kidney transplant. The donor had multiple risk factors for HHV-8 infection. The KS was successfully treated by switching immunosuppression from tacrolimus to sirolimus. With an increasing number of human immunodeficiency virus (HIV)-positive persons seeking organ transplantation and serving as organ donors for HIV-positive recipients, HHV-8 prevalence among donors and recipients will likely increase and with that the risk for post-transplant KS. Predetermination of HHV-8 status can be useful when considering organ donors and recipients with risk factors, although there are currently no validated commercial tests for HHV-8 antibody screening.

Tumor necrosis factor alpha-induced apoptosis in human neuronal cells: protection by the antioxidant N-acetylcysteine and the genes bcl-2 and crmA.

Tumor necrosis factor alpha (TNF-alpha) is a candidate human immunodeficiency virus type 1-induced neurotoxin that contributes to the pathogenesis of AIDS dementia complex. We report here on the effects of exogenous TNF-alpha on SK-N-MC human neuroblastoma cells differentiated to a neuronal phenotype with retinoic acid, TNF-alpha caused a dose-dependent loss of viability and a corresponding increase in apoptosis in differentiated SK-N-MC cells but not in undifferentiated cultures. Importantly, intracellular signalling via TNF receptors, as measured by activation of the transcription factor NF-kappa B, was unaltered by retinoic acid treatment. Finally, overexpression of bcl-2 or crmA conferred resistance to apoptosis mediated by TNF-alpha, as did the addition of the antioxidant N-acetylcysteine. These results suggest that TNF-alpha induces apoptosis in neuronal cells by a pathway that involves formation of reactive oxygen intermediates and which can be blocked by specific genetic interventions.

Comparison of a microtiter plate system to Southern blot for detection of human herpesvirus 8 DNA amplified from blood and saliva.

The recent discovery of human herpesvirus 8 (HHV-8) as the etiologic agent of Kaposi's sarcoma (KS) has led to the interest in the development of PCR for this virus that is accurate, rapid, and convenient. We developed a sensitive PCR assay for HHV-8 with microtiter plate detection of amplimers. DNA was purified from white blood cells and saliva from HIV-infected men with and without Kaposi's sarcoma and one-step PCR was undertaken with primer sets specific for the N-terminal region of the glycoprotein B gene and open reading frame (orf) 26 of HHV-8. PCR was performed on 40 clinical specimens, followed by Southern blot and microtiter plate detection of amplimers. Results from the two methods of detection were nearly identical. Sensitivity for both methods based on serial dilution of a known standard was five to ten copies of HHV-8 per 400 ng of cellular DNA. In conclusion, microtiter plate detection of HHV-8 PCR amplimers is as sensitive and specific as Southern blot with much faster turnaround time at comparable cost, and utilizes common laboratory equipment.

Regulation of the human papillomavirus type 11 E6 promoter by viral and host transcription factors in primary human keratinocytes.

Human papillomavirus (HPV) type 11 is strictly trophic for epithelial cells and induces benign condylomata of the external genitalia and also causes laryngeal papillomas. Primary keratinocytes are the appropriate hosts for studies of HPV gene regulation, but they are not frequently used, owing to difficulties in culturing and low transfection efficiencies. By modifying a Polybrene transfection procedure, we achieved consistently high transfection efficiencies in primary human foreskin keratinocytes and characterized the HPV type 11 enhancer in the context of the homologous E6 promoter. Contrary to previous studies with immortalized human cervical carcinoma C-33A cells, constitutive enhancer element II in the upstream regulatory region conferred no enhancer activity and did not abrogate repression by the homologous E2 protein. Rather, repression was strong, ranging from 5.6- to 20-fold for the various enhancer deletion mutations. By deletion analysis, a strong enhancer that included three nuclear factor 1 sites and one nuclear factor 1-associated factor-binding site was localized to a 45-bp region within constitutive enhancer element I, and it showed some degree of tissue specificity.

Identification of a lytic-phase origin of DNA replication in human herpesvirus 6B strain Z29.

DNA sequences which have structural features suggestive of their functioning as an origin of lytic-phase DNA replication were previously identified in both human herpesvirus 6B strain Z29 [HHV-6B (Z29)] and in HHV-6A (U1102). Plasmid constructs containing the putative HHV-6B (Z29) oriLyt element were replicated after transfection into permissive T cells, when trans-acting factors were provided by HHV-6B (R-1) infection. By using this assay, the HHV-6B (Z29) oriLyt was mapped to a minimal region of approximately 400 bp which lies upstream of the gene that is homologous to herpes simplex virus UL29, a region that carries an origin in other betaherpesviruses and in some alphaherpesviruses.

Epithelial-specific gene expression during differentiation of stratified primary human keratinocyte cultures.

Cultured epithelial cells are used to generate extensive patches of autologous skin equivalent for patients with burns or wounds and to investigate the growth and differentiation of epithelia in vitro. We have undertaken a comprehensive study of the morphological and molecular events that occur during culturing of human foreskin keratinocytes at the liquid-air interface on a dermal equivalent consisting of a collagen matrix containing fibroblasts. Using radioactively labeled RNA probes for mRNAs and monoclonal antibodies for proteins, we found that the expression of a comprehensive set of differentiation stage-specific genes was affected by the type of fibroblasts included in the matrix as well as by the age of the culture. The expression of these genes was not always coordinated and could not be predicted from the histological appearance of the stratified epithelium. Surprisingly, the mouse fibroblasts promoted epithelial differentiation much more closely resembling foreskin than did the homologous primary foreskin fibroblasts.

Enhanced responsiveness to nuclear factor kappa B contributes to the unique phenotype of simian immunodeficiency virus variant SIVsmmPBj14.

Infection with a variant of simian immunodeficiency virus, SIVsmmPBj14, leads to severe acute disease in macaques. This study was designed to investigate the functional significance of previously described mutations in the viral long terminal repeat (LTR) and to elucidate their contribution to the unique phenotype of SIVsmmPBj14. LTR-directed transcription was measured by using luciferase reporter constructs that were transiently transfected into cultured cells. In a wide range of cell types, the basal transcriptional activity of the LTR from SIVsmmPBj14 was found to be 2- to 4.5-fold higher than that of an LTR from a non-acutely pathogenic strain. These LTRs differ by five point mutations and a 22-bp duplication in SIVsmmPBj14, which includes a nuclear factor kappa B (NF kappa B) site. Transcriptional differences between these LTRs were further enhanced by two- to threefold upon treatment of cells with phorbol ester or tumor necrosis factor alpha or by cotransfection with plasmids expressing NF kappa B subunits. Mutagenesis studies, and the use of a reporter construct containing an enhancerless promoter, indicate that these transcriptional effects are due principally to the 22-bp sequence duplication and the NF kappa B site contained within it. Finally, infectious virus stocks that were isogenic except for the LTR were generated. The LTR from SIVsmmPBj14 was found to confer an increase in the kinetics of virus replication in cultured cells. Inclusion of this LTR in recombinant SIVs also resulted in a two- to threefold rise in the extent of cellular proliferation that was induced in quiescent simian peripheral blood mononuclear cells. These studies are consistent with the hypothesis that LTR mutations assist SIVsmmPBj14 in responding efficiently to cellular stimulation and allow it to replicate to high titers during the acute phase of viral infection.

Characterization of an HPV type 11 isolate propagated in human foreskin implants in nude mice.

Human papillomavirus type 11-Hershey (HPV-11-H) from an extract of genital warts has been serially passaged by infecting human foreskin chips that are then implanted in athymic mice (J. W. Kreider, M. K. Howett, A. E. Leure-Dupree, R. J. Zaino, and J. A. Weber (1987), J. Virol. 61, 590). Subsequent attempts to propagate HPVs present in other human lesions have not been successful. In an effort to identify the basis for the seemingly unique ability of HPV-11-H to propagate in the xenografts, we carried out extensive physical and functional characterizations of the cloned DNA, including restriction digestions, DNA sequencing of transcriptional control regions, and E2 trans-activation of the upstream regulatory region. We uncovered no significant differences compared to the prototype HPV-11 (K. Dartmann, E. Schwarz, L. Gissmann, and H. zur Hausen (1986) Virology 151, 124; H. Hirochika, T. R. Broker, and L. T. Chow (1987) J. Virol. 61, 2599). Moreover, viral enhancer assays indicated that the observed stimulatory effect of pregnancy on condylomata and of estradiol on experimental cysts is likely an indirect one and could not be attributed to up-regulation of the HPV-11 transcriptional enhancer.

Activation of nuclear factor kappa B in brains from children with HIV-1 encephalitis.

Human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system is associated with decreased neuronal density in discrete areas of the brain. Neuronal loss may occur via apoptosis, initiated by soluble neurotoxic factors secreted from HIV-1 infected macrophages and microglia. To examine further the molecular events involved in HIV-1 neuropathogenesis, we assessed the activity of NF kappa B, an inducible transcription factor involved in the activation of multiple proinflammatory, and potentially neurotoxic, genes. NF kappa B was analysed by immunocytochemistry using specific antisera to the NF kappa B p. 50 and p. 65 subunits. Brains from children with HIV-1 encephalitis and progressive encephalopathy were found to contain increased numbers of NF kappa B immunoreactive cells, relative to control brains (HIV-1 negative, or HIV-1 positive without encephalitis). Double-labelling studies using antibodies to CD68, or RCA-1 lectin, markers for cells of monocyte/macrophage lineage, revealed an increase in the number of microglia and macrophages with nuclear immunoreactivity for NF kappa B in association with HIV-1 encephalitis. NF kappa B positive multinucleated giant cells were also detected, as were cells which contained both NF kappa B and HIV-1 antigen. In contrast, the number of neurons and GFAP-positive astrocytes that were immunoreactive for NF kappa B was approximately the same in all groups of subjects. These data are consistent with the hypothesis that persistent, high-level activation of NF kappa B may promote the sustained production of neurotoxins by microglia and macrophages during HIV-1 encephalitis.

New immunofluorescence assays for detection of Human herpesvirus 8-specific antibodies.

Several assays have been developed for detection of immunoglobulin G antibodies to Human herpesvirus 8 (HHV-8), including immunofluorescence assays (IFAs) and enzyme-linked immunosorbent assays (ELISAs). However, the specificity and sensitivity of these assays are not completely defined due to the lack of a "gold standard." Although IFAs based on primary effusion lymphoma (PEL) cell lines are used widely, the assays can be confounded by nonspecific reactions against cellular components and potential cross-reaction with antibodies against other herpesviruses. To provide more reliable IFAs, we established recombinant Semliki Forest viruses (rSFVs) expressing the HHV-8-specific proteins ORF73 and K8.1 and used BHK-21 cells infected with these rSFVs for IFA (ORF73-IFA and K8.1-IFA). Expression of the HHV-8-specific proteins at very high levels by the rSFV system allowed easy scoring for IFA and thereby increased specificity. The rSFV system also allowed detection of antibodies against glycosylation-dependent epitopes of K8.1. Titers measured by rSFV-based IFAs and PEL-based IFAs correlated well (correlation coefficients of >0.9), and concordances of seroreactivities between rSFV-based and PEL-based IFAs were >97% (kappa > 0.93). K8.1-IFA was more sensitive than either ORF73-IFA or peptide ELISAs. Using PEL-based lytic IFA as a reference assay, the sensitivity and specificity of K8.1-IFA were estimated to be 94 and 100%, respectively. HHV-8 prevalences determined by K8.1-IFA among the human immunodeficiency virus (HIV)-positive (HIV(+)) Kaposi's sarcoma (KS) patients, HIV(+) KS(-) patients, and healthy controls were 100, 65, and 6.7%, respectively, which were consistent with prior reports. Therefore, our rSFV-based IFAs may provide a specific and sensitive method for use in epidemiology studies. In addition, they will provide a basis for further development of diagnostic tests for HHV-8 infection.

Production of human papillomavirus and modulation of the infectious program in epithelial raft cultures. OFF.

Human papillomaviruses trophic for anogenital epithelia cause benign warts, and certain genotypes are closely associated with cervical neoplasia. By using our modifications of the epithelial raft culture system, we were able to recapitulate and modulate the infectious program of a papillomavirus in vitro for the first time. Small pieces of a condyloma containing human papillomavirus type 11 were explanted onto a dermal equivalent consisting of a collagen matrix with fibroblasts and were cultured at the medium-air interface. The infected stem cells proliferated rapidly across the matrix, stratified, and differentiated, as judged by histology. The results correlated with the state of epithelial differentiation, which, in turn, was dependent on the type of fibroblast in the matrix. Under conditions where the epithelial outgrowth underwent terminal differentiation, the entire productive program took place, leading to virion assembly. In contrast, using an alternative condition where the outgrowth failed to achieve terminal differentiation, only the E-region RNAs from the E1 promoter accumulated to any appreciable extent. The proliferating cell nuclear antigen was induced in the differentiated suprabasal cells in the productive cyst growth, which also exhibited high copy viral DNA and abundant E6-E7 RNAs. Comparable cells in the nonproductive cyst outgrowth were negative for all three. These results suggest that the E6 and E7 proteins may play a role in establishing a cellular environment conducive to vegetative viral replication. The culture conditions described should be useful for genetic analysis of this family of important human pathogens and for testing potential pharmacological agents.

Blood-borne and sexual transmission of human herpesvirus 8 in women with or at risk for human immunodeficiency virus infection.

BACKGROUND: Human herpesvirus 8 (HHV-8), the causal agent of Kaposi's sarcoma, is transmitted sexually among homosexual men, but little is known of its transmission among women. Although HHV-8 has been detected in blood, there has been no clear evidence of blood-borne transmission. METHODS: We identified risk factors for HHV-8 infection in 1295 women in Baltimore, Detroit, New York, and Providence, Rhode Island, who reported high-risk sexual behavior or drug use. HHV-8 serologic studies were performed with two enzyme-linked immunosorbent assays. RESULTS: In univariate analyses, HHV-8 was associated with black race, Hispanic ethnic background, a lower level of education, and infection with syphilis, the human immunodeficiency virus (HIV), hepatitis B virus (HBV), or hepatitis C virus (HCV). The risk of seropositivity for HHV-8 increased with the frequency of injection-drug use (P<0.001); HHV-8 seroprevalence among the women who used drugs daily was three times that among women who never injected drugs. Among the women with a low risk of sexual transmission, HHV-8 seroprevalence was 0 percent in those who had never injected drugs and 36 percent in those who had injected drugs (P<0.001). However, injection-drug use was linked less strongly to HHV-8 infection than to infection with HBV or HCV. In a multivariate analysis, independent predictors of HHV-8 seropositivity included HIV infection (odds ratio, 1.6; 95 percent confidence interval, 1.1 to 2.2), syphilis infection (odds ratio, 1.8; 95 percent confidence interval, 1.1 to 2.8), and daily injection-drug use (odds ratio, 3.2; 95 percent confidence interval, 1.4 to 7.6). CONCLUSIONS: Both injection-drug use and correlates of sexual activity were risk factors for HHV-8 infection in the women studied. The independent association of HHV-8 infection with injection-drug use suggests that HHV-8 is transmitted through needle sharing, albeit less efficiently than HBV, HCV, or HIV.

Comparison of human herpesvirus 8 and Epstein-Barr virus seropositivity among children in areas endemic and non-endemic for Kaposi's sarcoma.

Human herpesvirus 8 (HHV-8) is the etiologic agent of Kaposi's sarcoma (KS). Several studies indicate horizontal HHV-8 transmission among children in areas where KS is endemic, but few studies have assessed acquisition of HHV-8 by children in low seroprevalence areas. Antibody screening was carried out for HHV-8 and Epstein-Barr virus (EBV) on 787 serum specimens from children living in two areas where HHV-8 is not endemic, the United States (US) and Germany, and on 184 specimens from children living in a KS-endemic area (Nigeria). For children in the US and Germany, the results showed low HHV-8 seroprevalence rates (3-4%). However, US children aged 6 months to 5 years had higher HHV-8 antibody titers than did 6-17-year-old children (P < 0.01), a finding consistent with more recent infections being detected in the younger children. Compared with seroprevalence rates and antibody titers in US and German children, those in Nigerian children were significantly higher, and seroprevalence increased with age. There was no evidence of cross-reactivity between assays for HHV-8 and EBV, despite the genetic similarity of these two herpesviruses. The data indicate that HHV-8 transmission among children where HHV-8 is not endemic occurs, but is uncommon. The findings also suggest that HHV-8 antibodies, as measured by current tests, may not persist for long periods in populations at low risk for KS and that vertical transmission is rare, although longitudinal studies are necessary to address directly these issues.

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.

Multicenter comparison of serologic assays and estimation of human herpesvirus 8 seroprevalence among US blood donors.

BACKGROUND: As part of assessing the possibility of transfusion transmission of human herpesvirus 8 (HHV-8 or Kaposi's sarcoma-associated herpesvirus), HHV-8 seroprevalence was estimated among US blood donors, the performance of HHV-8 serologic tests was compared, and the presence of HHV-8 DNA was tested for in donated blood. STUDY DESIGN AND METHODS: Replicate panels of 1040 plasma specimens prepared from 1000 US blood donors (collected in 1994 and 1995) and 21 Kaposi's sarcoma patients were tested for antibodies to HHV-8 in six laboratories. HHV-8 PCR was performed on blood samples from 138 donors, including all 33 who tested seropositive in at least two laboratories and 22 who tested positive in at least one. RESULTS: The estimated HHV-8 seroprevalence among US blood donors was 3.5 percent (95% CI, 1.2%-9.8%) by a conditional dependence latent-class model, 3.0 percent (95% CI, 2.0%-4.6%) by a conditional independence latent-class model, and 3.3 percent (95% CI, 2.3%-4.6%) by use of a consensus-derived gold standard (specimens positive in two or more laboratories); the conditional dependence model best fit the data. In this model, laboratory specificities ranged from 96.6 to 100 percent. Sensitivities ranged widely, but with overlapping 95 percent CIs. HHV-8 DNA was detected in blood from none of 138 donors evaluated. CONCLUSIONS: Medical and behavioral screening does not eliminate HHV-8-seropositive persons from the US blood donor pool, but no viral DNA was found in donor blood. Further studies of much larger numbers of seropositive individuals will be required to more completely assess the rate of viremia and possibility of HHV-8 transfusion transmission. Current data do not indicate a need to screen US blood donors for HHV-8.