Transcription of human herpesvirus-like agent (HHV-8) in Kaposi's sarcoma.

Recently, DNA sequences of what appear to be a unique human herpesvirus-like agent (HHV-8) have been detected in different types of Kaposi's sarcoma (KS) tumors (Chang, Y., E.C. Cesarman, M.S. Pessin, F. Lee, J.C. Culpepper, D.M. Knowles, and P.S. Moore. 1994. Science (Wash. DC). 266:1865-1869). To further elucidate the possibility that HHV-8 plays a role in the pathogenesis of KS, the expression of HHV-8 RNA was examined in fresh KS tissue specimens which were found to harbor HHV-8 DNA by polymerase chain reaction (PCR). The transcription of HHV-8 RNA was detected by RT-PCR in 26 of 29 specimens (89.7%) of the KS tumors including 2 of 3 CKS and 24 of 26 AIDS-KS. No positive signal was detected in eight biopsy specimens of normal skin from healthy donors. By Northern blot analysis, the expression of HHV-8 was detected in 2 of 10 KS tumors examined. Furthermore, the RNA transcripts were observed in endothelial cells lining the irregular vascular spaces and perivascular spindle-shaped cells histologically characteristic of KS in 2 out of 8 different KS specimens examined by in situ hybridization using an antisense probe specific of HHV-8. The detection of RNA expression of HHV-8 in KS tumors further supports the possible etiopathogenic role of this virus in the development of KS.

Expression of int-2 oncogene in Kaposi's sarcoma lesions.

Fibroblast growth factors (FGFs), such as basic FGF, have been implicated in the growth of Kaposi's sarcoma (KS) cells in vitro. In the evaluation of the expression of the various genes of the different members of the FGF family and their receptors in fresh KS tissue specimens, int-2 was found to be expressed in more than half of the KS tumors examined. Using reverse transcription PCR, the expression of int-2 was detected in 21 of 38 (55.2%) fresh KS biopsy specimens. In contrast, int-2 mRNA transcripts were not found in normal appearing skin from the same patients except in one sample which was obtained from an AIDS patient with disseminated KS lesions. Sequence data confirmed that the amplified sequences were derived from int-2 mRNA with proper splicing. In addition, 12 nucleic acid alterations were identified in eight out of nine KS tumor samples sequenced. Using immunohistochemical methods, int-2 protein was detected in some of the spindle-shaped tumor cells surrounding the abnormal endothelial-lined vascular slits histologically characteristic of KS. Int-2 specific immunostaining was shown to be present in both the nuclei and cytoplasm of these spindle cells but was more pronounced in the nuclei. Neither amplification nor gross rearrangement of the int-2 gene was detected in KS lesions by Southern blot analysis. These results suggest that the expression of int-2 may play a role in the pathogenesis KS by stimulating local angiogenesis and cell proliferation.

The absence of Tat sequences in tissues of HIV-negative patients with epidemic Kaposi's sarcoma.

OBJECTIVE: Tat, an essential regulatory protein of HIV, acts as a growth factor for Kaposi's sarcoma (KS)-derived cells in culture. We tested the hypothesis that HIV-negative epidemic KS patients who are also at high risk for HIV disease might have been infected with a defective HIV-1 virus that retained the ability to express Tat. METHODS: We evaluated the presence of Tat sequences in KS tissue and peripheral blood mononuclear cells (PBMC) of HIV-1-negative individuals with epidemic KS who had risk factors for HIV infection by polymerase chain reaction using specific primers for the Tat region of HIV-1. RESULTS: No evidence for the presence of Tat-1 sequences or for Tat-expressing defective HIV-1 virus was found. CONCLUSION: These results suggest that HIV-1 Tat does not play a role in the initiation of KS in HIV-1-negative individuals. Tat might play an indirect role in epidemic KS in HIV-infected patients.

HIV-1 infection and modulation of cytokine and growth factor expression in Kaposi's sarcoma-derived cells in vitro.

OBJECTIVES: HIV-1 transcripts have been detected in AIDS-related Kaposi's sarcoma (KS) tissues within the factor XIIIa + dermal dendrocytes present in the tumor. Various cytokines and growth factors have been shown to influence the growth of KS-derived cells in vitro. HIV-1 preferentially infects CD4+ cells and has also been found to infect some CD4- cells in vitro. The susceptibility of cultured KS cells in vitro to infection with HIV-1 and the expression of interleukin (IL)-1 beta, IL-6 and basic fibroblast growth factor (bFGF) after exposure to HIV-1 was examined. METHODS: The susceptibility of two different KS-derived cell cultures to HIV-1 infection was examined by the expression of p24 antigen, detection of proviral sequence and electron microscopy. The expression of IL-1 beta, IL-6 and bFGF was detected by enzyme-linked immunosorbent assay and reverse transcriptase polymerase chain reaction. RESULTS: KS-derived cells can be infected by HIV-1 in vitro. Both KS-derived cells were found to express CD4 mRNA. The expression of IL-1 beta and IL-6 was increased, whereas the expression of bFGF was not stimulated after exposure of KS cells to HIV-1. CONCLUSION: These experiments describe the in vitro infection of KS-derived cells by HIV-1 and the expression of various cytokines and growth factor following infection. The increased production of cytokines observed following such infection may be involved in the pathogenesis of AIDS-related KS.

A randomized, controlled, safety study using imiquimod for the topical treatment of anogenital warts in HIV-infected patients. Imiquimod Study Group.

OBJECTIVE: To assess the safety of imiquimod, an immune response modifier, in the topical treatment of external anogenital warts in HIV-infected patients. SETTING: Clinical sites in the United Kingdom (eight) and the United States (five). DESIGN: A prospective, randomized, double-blind, vehicle-controlled study of imiquimod 5% cream or vehicle applied for 8+/-2 h three times per week for a maximum of 16 weeks in HIV-seropositive males (n = 97) and females (n = 3) aged 18 years or more with clinically diagnosed external anogenital warts, CD4 T lymphocyte count of > or = 100 x 10(6) cells/l and Karnofsky score > or = 70. MAIN OUTCOME MEASURES: Safety was assessed through the incidence and severity of local skin reactions and other adverse events, and through clinical laboratory tests. Wart clearance was documented by two-dimensional measurements of warts and by photography. RESULTS: Among the patients treated with imiquimod (n = 65) and vehicle (n = 35), the most common local skin reaction was erythema, (41.9 and 26.7%, respectively) and the incidence of patients reporting at least one adverse event was 69.2 and 65.7%, respectively. No clinically meaningful differences or changes in laboratory values were observed between treatment groups, nor were drug-related adverse effects observed in regard to HIV disease. While there was no significant difference between treatment groups in the number of patients who totally cleared their baseline warts (imiquimod 11% versus vehicle 6%, P = 0.488), more imiquimod-treated patients experienced a > or = 50% reduction in baseline wart area (38% versus 14%, P = 0.013). CONCLUSION: Most local skin reactions were mild and no adverse effects on HIV disease were observed. Topically applied imiquimod 5% cream reduced wart area and may have clinical utility in treating external anogenital warts in some HIV-infected patients.

Urine-based diagnostic technologies.

The worldwide dissemination of infectious agents has created a demand for simple diagnostic tests. Urine-based testing makes use of non-invasive collection of specimens, and there is no need for expensive facilities and equipment, or for highly trained personnel. As urine antibodies retain activity under normal conditions of transport and storage, such tests appear to have widespread application. Urine-based antibody tests have also indicated a compartmentalized antibody response to HIV-1 infection. Urine studies suggest that antibodies to the products of endogenous viral genes may be involved in the pathogenesis of chronic diseases of suspected viral etiology.

Effect of eight antiviral drugs on the reactivation of herpes simplex virus in explant cultures of latently infected mouse trigeminal ganglia.

The effect of several antiviral drugs on the reactivation of herpes simplex virus type 1 in explant cultures of latently infected mouse trigeminal ganglia was investigated. Phosphonoacetate and phosphonoformate, which act directly on the virus-induced DNA polymerase, require a drug concentration of 400 micrograms/ml for the inhibition of virus reactivation in latently infected ganglia. Arabinosyladenine and arabinosyladenine monophosphate, which are phosphorylated to triphosphates by cellular enzymes and inhibit virus synthesis either by blocking the DNA polymerase or by incorporation into viral DNA, require a concentration of only 100 micrograms/ml for the inhibition of the reactivation process. Drugs that are phosphorylated by the virus-induced thymidine kinase, such as acyclovir, arabinosylthymine, bromovinyldeoxyuridine, and three fluorinated pyrimidine nucleosides require the lowest drug concentrations for complete inhibition of virus reactivation in latently infected ganglia explant cultures. Our data suggest that the inhibition of virus reactivation is dependent not only on drug concentration, but also on the number of latently infected neurons in the ganglia.

Phosphonoacetic acid treatment of shope fibroma and vaccinia virus skin infections in rabbits.

The antiviral efficacy of phosphonoacetic acid (PAA) was studied in localized skin lesions of rabbits produced by the intradermal inoculation of vaccinia virus (VV) and of Shope fibroma virus (SFV). Systemic administration of PAA by intraperitoneal injections had no significant effect on the pustular lesions induced by VV or on the benign skin tumors caused by SFV. A complete suppression of the appearance of VV-induced pustular lesions was achieved by 2% PAA ointment applied twice daily for 4 days, starting 24 hr after virus inoculation. A significant effect against SFV-induced tumors was obtained by PAA ointment applied beginning either 24 or 72 hr after virus inoculation. A complete suppression of SFV-induced tumors was observed when a dose of 10 mg PAA was injected intralesionally once daily for 5 days, beginning treatment 24 hr after virus inoculation. A significant reduction of the intensity of the tumors was seen following the same treatment schedule but with a delay of 72 hr after virus inoculation or by reducing the length of treatment to 3 days or with a dose of 1 mg injected intradermally daily for 5 days. After the healing of the lesions, PAA-treated rabbits were resistant to reinfections to the same extent as those in which spontaneous healing had occurred.

Clinical, histologic, microbiologic, and biochemical characterization of the causative agent of bacillary (epithelioid) angiomatosis: a rickettsial illness with features of bartonellosis.

It has been suggested that bacillary (epithelioid) angiomatosis (BEA) is a manifestation of cat scratch disease (CSD). Because of clinical similarity between this condition and the verruga peruana phase of bartonellosis, we sought to further characterize this disease as well as its causative agent and to compare it to bartonellosis. We isolated a small flagellated pleomorphic bacillus from skin lesions of two patients with BEA. Organisms were stained successfully with Warthin-Starry silver stains, but immunohistochemistry failed to demonstrate binding with a polyclonal antibody directed against the cat scratch bacillus. Whole cell fatty-acid gas chromatography performed on both BEA organisms and Bartonella bacilliformis demonstrated marked similarity between the two. Electron microscopy of BEA organisms in tissue and in suspension revealed features characteristic of a gram negative bacillus. Based on these findings, we propose that this unusual rickettsial infectious disease with vascular proliferation may represent an unusual variant of infection with a bartonella-like organism rather than a manifestation of cat scratch disease.

Tumor-associated antigen is expressed on lymphocytes from patients with acquired immunodeficiency syndrome.

Monoclonal antibody BE2 recognizes an antigen found on malignant T4+ lymphocytes from cutaneous T-cell lymphoma patients (CTCL). Normal peripheral blood lymphocytes do not express detectable levels of BE2 antigen. Forty-eight percent of patients with the acquired immunodeficiency syndrome (AIDS) had lymphocyte populations that were reactive with monoclonal antibody BE2. Peripheral blood lymphocytes from healthy homosexuals, patients with classical Kaposi's sarcoma or viral syndromes, and healthy normal controls were BE2-. Double-labeling studies demonstrated that BE2+ cells were T lymphocytes. This observation demonstrates that some AIDS patients as well as CTCL patients have circulating cells that express a common lymphocyte abnormality.

HIV-1 neutralizing antibodies in urine from seropositive individuals.

HIV-1 neutralizing activity was demonstrated in serum and 200-fold concentrated urine from individuals who were HIV-1 antibody positive in both their serum and urine, including AIDS-KS, AIDS-OI, ARC, and asymptomatic patients. Virus neutralization activity was detected in 23 of 56 (41.1%) of the serum samples and in 19 of 56 (33.9%) of the urine samples tested, with titers ranging from 1:8 to 1:256 and 1:1 to 1:4, respectively. The highest frequency of HIV-1 neutralizing activity (87.5%) and the highest mean neutralization titers (1:65) were found in the ARC patients. A high prevalence of p24 antigen in serum and low numbers of T4-lymphocytes correlated with a low frequency of neutralizing activity in either serum or urine in the infected individuals. HIV-1 neutralizing activity in the urine was shown to be due to immunoglobulins using a Sephadex G-100 filtration gel. All 19 urine samples with neutralizing activity contained antibodies reactive with envelope glycoproteins gp160, gp120, and gp41 by Western blot, similar to that seen with serum. The frequency of HIV-1 neutralizing activity in the urine concentrates was generally associated with high titers of neutralizing antibody in the corresponding serum. These findings suggest that HIV-1 neutralizing antibodies are lost in the urine by an as yet unknown mechanism.

Epidemic Kaposi's sarcoma.

No significant impact of available treatments on survival among patients with epidemic KS has been demonstrated. Therefore, antitumor therapy now should be considered palliative. In the early stages of the disease, systemic treatment may not be needed, whereas advanced disease requires systemic treatment with one or more agents known to have antitumor activity. A complete therapeutic response is difficult to achieve and if such response is obtained, maintenance therapy may be necessary. The overall prognosis for survival in patients with epidemic KS appears to depend on the severity of immune suppression and HIV infection rather than on the neoplastic proliferation and tumor load. This is reflected in the new staging proposals for KS. Ultimately, the ideal treatment for the AIDS patient with KS will be a combination of antiretroviral therapy to suppress further effects of HIV, biological therapy to reverse the immunologic defects, chemotherapy to control tumor development, and hematopoietic growth factors to ameliorate treatment toxicities.

Intravenous acyclovir therapy of first episodes of genital herpes: a multicenter double-blind, placebo-controlled trial.

PURPOSE: A collaborative multicenter double-blind, placebo-controlled trial of intravenous acyclovir treatment of first-episode genital herpes was performed in order to substantiate previous findings on the efficacy and safety of this drug, to evaluate the influence of parenteral therapy on recurrence frequency, and to obtain further data on the natural history of genital herpes. PATIENTS AND METHODS: Eighty-two patients with first episodes of genital herpes simplex virus (HSV) infection were randomly assigned in a double-blind fashion to treatment with intravenous acyclovir (5 mg/kg every eight hours) or placebo for five days. Before therapy, all lesions in the genital/perineal area and in extragenital sites were cultured. New lesions appearing in both areas after the onset of therapy were cultured separately. Lesions in all groups were cultured until completely healed. Sera were collected from all patients on entry to the study and on Day 21 to determine presence or absence of antibodies to HSV-1 and HSV-2. Time to healing, time to crusting, time to cessation of viral shedding, and appearance of new lesions during therapy were compared for each treatment group. RESULTS: Patients receiving acyclovir experienced a significant reduction in the median duration of pain (4.3 versus 4.8 days, p = 0.019), viral shedding (1.9 versus 8.4 days, p less than 0.001), and time to healing (8.4 versus 11.5 days, p = 0.02) compared with placebo recipients. These differences were largely attributable to the effect of therapy in the subset of patients with primary disease in whom acyclovir reduced the median duration of pain from 10.6 days to 4.2 days, the median duration of viral shedding from 17.1 days to 1.9 days, and the median time to healing from 14.2 days to 8.3 days. The rate of subsequent recurrence of genital herpes was not altered by acyclovir treatment: 24 of 32 acyclovir recipients (75 percent) experienced one or more recurrences during a mean follow-up of 14 months compared with 19 of 27 placebo recipients (70 percent). Among patients experiencing recurrences, the mean number of recurrences per month among acyclovir recipients was 0.25 compared with 0.19 for patients given placebo. CONCLUSION: This multicenter trial confirms the efficacy of intravenous acyclovir in the management of first-episode genital herpes, especially in patients with primary infection. However, therapy did not alter the frequency of recurrences.

Cutaneous disease resembling mycosis fungoides in HIV-infected patients whose skin and blood cells also harbor proviral HTLV type I.

Two homosexual HIV-infected patients with lymphocyte counts of < 50 presented with intense pruritus, hyperpigmentation, and skin lesions clinically suggestive of the cutaneous T cell lymphoma, mycosis fungoides. On light microscopy, the skin biopsies were difficult to interpret because of the sparseness of the lymphocytic infiltrates. However, electron microscopy revealed typical Sézary cells in the peripheral blood and skin. Cultures of blood mononuclear cells of one of the patients generated HTLV-I-like particles. Although both patients lacked antibodies to HTLV, their blood and skin specimens proved to harbor tax and pol HTLV-I proviral sequences as shown by the polymerase chain reaction and Southern blot analysis. Dual infection with HIV and HTLV should be considered in the diagnostic work-up of patients at risk, even in the absence of demonstrable antibodies. Dual infections could result in clinical manifestations and evolution of disease not anticipated in patients who harbor only one of these retroviruses.

Antibodies to human immunodeficiency virus type 1 in the urine specimens of HIV-1-seropositive individuals.

Specific antibodies to human immunodeficiency virus type 1 (HIV-1) were detected in 200-fold concentrated urine samples, but none were detected in unconcentrated urine specimens, from 100 randomly selected HIV-1--seropositive individuals by enzyme-linked immunosorbent assay (ELISA) and Western blot techniques using the manufacturer's recommended procedures. Using modified methods for both the ELISA and Western blot tests, antibodies to HIV-1 have also been detected in the unconcentrated urine specimens from the same HIV-1--seropositive individuals. No difference in the frequency of antibodies to HIV-1 were found between unconcentrated and 200-fold concentrated urine samples when tested by the modified methods. HIV-1 core antigen (p24) was not detected in either the concentrated or the unconcentrated HIV-1--seropositive adult urine samples; none of these individuals showed overt clinical or laboratory evidence of renal dysfunction. The titer of the antibodies to HIV-1 found in the urine specimens was found to be parallel with the titer of antibodies to HIV-1 in the corresponding individual's serum. Further elucidation of the pathophysiology and the nature of the specific antibodies to HIV-1 observed in the urine of HIV-1--seropositive individuals is under investigation in our laboratories.

Group specific component (Gc) and HIV diseases.

A previous report suggested a relationship between particular phenotypes of group specific component (Gc), and susceptibility or resistance to HIV infection, whereas other recent investigations failed to corroborate this finding. The present study demonstrated that Gc allele frequencies in white homosexual men corresponded to those expected, regardless of HIV serology and related disease. Assignment of phenotype was not influenced by Gc complexing secondary to tissue damage, or the process of infection per se. However, Gc allele frequencies in black patients with AIDS were significantly different from those in black control subjects, suggesting that the previously observed results might be in part explicable on the basis of gene admixture in ethnically mixed populations.

Arabinosyladenine monophosphate in genital herpes: a double-blind, placebo-controlled study.

A double-blind, placebo-controlled study was performed in 55 male patients with recurrent herpes simplex genitalis. The 29 patients who received topical arabinosyladenine monophosphate (ara-AMP) showed no significant difference in viral shedding, duration of pain, healing time or development of new lesions as compared to 26 placebo-treated patients. Ara-AMP was well-tolerated when topically applied. Serum neutralizing antibody titers did not change significantly during the acute and convalescent periods of the patient's recurrent HSG attacks. We conclude that ara-AMP, when applied topically as a 10% gel five times a day within 24 h of onset of recurrent HSG, does not influence the virologic and clinical evolution of the recurrent episode.

Evaluation of the tumorigenic and angiogenic potential of human fibroblast growth factor FGF3 in nude mice.

Recently, the expression of fibroblast growth factor 3 (FGF3) was found in 55% of human Kaposi's sarcoma (KS) tumor tissues examined, while almost no expression of FGF3 was found in normal skin. To further these studies, human FGF3 cDNA were constructed by the overlap-extension method. The proteins translated from two FGF3 cDNA, which differ only in the sequences preceding the AUG presumed to be the initiation codon, were shown to have the same molecular mass. This result suggests that translation of human FGF3, which is different from mouse FGF3, begins only at the AUG site. The human FGF cDNA was transfected into NIH3T3 cells. The NIH 3T3 cells transformed by FGF3 were then injected subcutaneously into athymic nude mice. Nodular lesions developed at the injection sites in all seven mice injected with the F3-1 cell clone, which showed high expression of FGF3, and in two out of six mice injected with the F3-2 cell clone, which expressed a low level of FGF3. Histopathological features of these tumors contained fascicles of spindle-shaped cells surrounding irregular endothelial lined vascular clefts, similar to those observed in human KS lesions. Immunohistochemical staining for factor V111 antigen revealed reactivity in multiple areas, especially in abundant vascular structures of the tumor sections examined. The expression of FGF3 together with the FGF receptors FGFR1, FGFR2, and FGFR3, was detected in the mouse tumors by Northern blot analysis. Our results indicate that tumors induced by FGF3-transformed NIH3T3 cells show some similarities to human KS tumors. In conclusion, our results demonstrate the potential tumorigenic and angiogenic role of human FGF3.

Rapid detection of Epstein-Barr virus DNA in clinical samples of oral hairy leukoplakia with HRP-labeled DNA probes and in situ hybridization.

An in situ hybridization technique, using horseradish peroxidase (HRP)-labeled DNA probes containing a portion of the Epstein-Barr virus (EBV) genome, was used to detect EBV DNA in tongue sections and smears from patients with oral lesions resembling the clinical features of oral hairy leukoplakia (HL). Eleven biopsy specimens (six consistent with HL, four normal tongue controls, and one leukoplakia) and 11 tongue smears were evaluated for the presence of EBV, cytomegalovirus (CMV), herpes simplex virus (HSV), and human papillomavirus (HPV) type 16. Following hybridization, six biopsy specimens and 10 tongue smears were found positive for EBV. All biopsy cases were negative for CMV, HSV, HPV-16 and the negative control probe. The specificity of the in situ hybridization assay was 100%. These results suggest that in situ hybridization using HRP-labeled DNA probes may be useful as a rapid diagnostic method for the detection of EBV in tongue sections or smears from patients diagnosed with HL.

Factor VIII-related antigen in Kaposi's sarcoma in young homosexual men.

This study investigated the histogenesis of the Kaposi's sarcomas occurring in young homosexual men. Paraffin sections of seven tumors were stained for factor VIII-related antigen by the unlabeled peroxidase antiperoxidase method. Both the spindle cell component and the cells lining vascular channels contained factor VIII-related antigen, a marker for endothelial cells. Our study supports the hypothesis that both components of Kaposi's sarcoma are of endothelial cell origin.