Correlation between AIDS-related Kaposi sarcoma histological grade and in vitro behavior: reduced exogenous growth factor requirements for isolates from high grade lesions.

Kaposi sarcoma, the most common AIDS-associated malignancy, affects 10-30% of all AIDS patients. To date, research into the biological characteristics of AIDS-related Kaposi sarcoma (AIDS-KS) derived cell lines has been based on cultures established from skin explants or pleural effusions/peritoneal fluids. We have established several AIDS-KS lines from biopsy confirmed oral mucosal and epidermal AIDS-KS lesions and have found a correlation between AIDS-KS lesional grade and in vitro cellular growth characteristics. In comparison to epidermal AIDS-KS lesions, mucosal AIDS-KS lesions frequently possessed both a more advanced histologic grade and demonstrated a greater capacity to proliferate in minimal medium. We report the ability of AIDS-KS isolates from high grade lesions to sustain proliferation (greater than 60 population doubling levels) in medium not supplemented with endothelial cell growth supplement and/or cytokine rich conditioned medium. These findings indicate that AIDS-KS cells isolated from high grade lesions have reduced requirements for exogenously provided growth supplements, and suggest that increased autologous cytokine production accompanies AIDS-KS lesional progression.

Comparison of constitutive cytokine release in high and low histologic grade AIDS-related Kaposi's sarcoma cell strains and in sera from HIV+/KS+ and HIV+/KS- patients.

Kaposi's sarcoma (KS) is both an AIDS-defining disease and the most common HIV-associated malignancy. A cytokine-mediated pathogenesis for AIDS-KS is implicated because AIDS-KS-derived cell strains both respond to and express a variety of cytokines. We have reported the establishment of several (n = 18) AIDS-KS cell strains and determined that reduced exogenous growth factors are necessary to sustain proliferation in isolates from high histologic grade KS lesions. This current investigation explored the possibility that there are histologic grade-associated differences in either the qualitative and/or quantitative constitutive release of AIDS-KS growth stimulatory cytokines. Our findings showed that the incorporation of HTLV-II cytokine-rich conditioned media induced both qualitative and significant quantitative cytokine release, suggesting that exogenous growth promoters stimulate constitutive cytokine release. ELISA of our AIDS-KS cell strains demonstrated constitutive release of IL-6 (seven of seven), FGF-2 (five of seven), GM-CSF (three of seven), and IL-1 beta (one of seven). None of our AIDS-KS cell strains constitutively released detectable levels of Onco-M, IL-4, PDGF, TNF-alpha, or TNF-beta. In addition, we report that the method of cytokine result quantitation significantly affects reported cytokine levels. We determined that there was no significant histologic grade-dependent difference in the constitutive release of soluble cytokines by in vitro grown cultures of AIDS-KS cells. The presence of HIV influenced the sera cytokine profiles by elevating IL-6 and decreasing PDGF concentrations of HIV+ individuals relative to HIV- healthy controls. However, the presence of KS was not associated with unique serum cytokine profiles, because no differences were noted in comparisons of HIV+/KS+ versus HIV+/KS- individuals. Our findings suggest that the local environment is key in modulating AIDS-KS cytokine expression and that KS growth-promoting factors function at the local or paracrine, not the systemic, level. In conclusion, our previous results demonstrated a histologic grade-associated difference in the in vitro growth capacity of AIDS-KS cells; with high histologic grade isolates displaying a marked growth advantage during culture in minimally supplemented media. Findings from this current study reveal that although the potential for a constitutive growth loop exists in the high-grade isolates, it is not reflected in the free levels of soluble cytokines secreted into the culture medium.

Plasmid profiles of Klebsiella pneumoniae isolated from horses.

Plasmid profiles of Klebsiella pneumoniae isolated from horses were examined. Thirty-nine strains of K. pneumoniae capsular type 1 (K1) isolated from cervical swabs of mares suffering from metritis, and from semen of stallions showed similar plasmid profile patterns, and all strains possessed a 125 megadaltons (Md) plasmid. There was no difference in plasmid profiles between the heavily-encapsulated and the less heavily-encapsulated strains of K. pneumoniae K1. Non-capsulated variants derived from the strains of K1 showed the same plasmid profile pattern as the parent strains. Plasmid profiles of K. pneumoniae other than K1 were various, and none of these strains possessed the 125 Md plasmid.

Immune recognition of genetically diverse simian T-cell lymphotropic virus type I isolates.

Nucleotide sequence analysis of selected regions of the gag, pol, env and pX genes of simian T-cell lymphotropic virus type I (STLV-I) strains indicated that African isolates were more closely related to human T-cell lymphotropic virus type I (HTLV-I) than Asian isolates. Despite these recent comparative studies on nucleotide sequence homology between HTLV-I and STLV-I isolates, only limited information is available regarding the influence of genetic differences on antigen-antibody recognition of distinct STLV-I strains. In this study, we demonstrated that sera from STLV-I-infected yellow baboons (Papio cynocephalus) reacted strongly with env gp62/68 from HTLV-I-infected cell lines MT-2 and C10/MJ. In contrast, sera from Japanese macaques (Macaca fuscata) naturally infected with Asian STLV-I had weak reactivity to env gp62/68 of these prototypic HTLV-I strains. Pst-1 restriction enzyme analysis of proviral DNA indicated that the baboon virus isolates were more closely related to HTLV-I than the Japanese isolates. These results indicate that nucleotide sequence diversity, correlates with variations in proviral restriction enzyme sites and antibody recognition of viral envelope proteins. These differences in immunoreactivity may have important implications for serologic diagnosis, as well as epidemiological and vaccine studies of STLV-I infection.

Lack of evidence for human T cell lymphotrophic virus type I or II infection in patients with systemic lupus erythematosus or rheumatoid arthritis.

OBJECTIVE: Human retroviruses including human immunodeficiency virus (HIV) and human T cell lymphotrophic virus Types I and II (HTLV-I/II) have been associated with forms of connective tissue autoimmune diseases including systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). We looked for evidence of HTLV-I/II infection in a large population of SLE, RA, and control patients. METHODS: One hundred fifteen patients with connective tissue autoimmune disease and other rheumatological disorders were screened for antibodies to HTLV-I/II by Western immunoblots (WIB). Due to the transforming characteristic of these retroviruses, the patients' peripheral blood mononuclear cells (PBMNC) were cultured in attempts to establish continuous cell lines. Furthermore, PBMNC culture supernatants were analyzed for reverse transcriptase activity and/or HTLV-I/II gag antigen production. The presence of HTLV-I/II proviral sequences in short term culture and fresh PBMNC was determined by Southern blot analysis and polymerase chain reaction (PCR), respectively. respectively. RESULTS: All 115 patients were HTLV-I/II and HIV seronegative. Seventy-four attempts to establish PBMNC cell lines from 65 patients were unsuccessful with a mean culture survival time of 3.6 (+/- 1.4) months. Reverse transcriptase activity and HTLV-I/II gag antigen production were not detected in 51 and 16 culture supernatants tested, respectively. Cells from 11 patients tested by Southern blot analysis and from 57 patients tested by PCR were negative for HTLV-I/II related sequences. CONCLUSION: Our results failed to establish an association between human retroviruses (HTLV-I/II and HIV) and SLE, RA, or other rheumatological disorders. However, these results do not rule out other exogenous or endogenous retroviruses that may play a role in the initiation and/or promotion of these diseases.

Sexual transmission of simian T-lymphotropic virus type I: a model of human T-lymphotropic virus type I infection.

Simian T-lymphotropic virus type-I (STLV-I) seronegative females placed together with seropositive males for breeding purposes were followed from 1984-1990 to determined seroconversion rates by enzyme immunoassay and western immunoblot analysis. Two of 26 females and 1 of 4 males previously negative for antibodies to STLV-I seroconverted during the study period. Statistical analysis of sexual encounters indicated that the probability of a seronegative female testing positive for STLV-I after a sexual encounter with a seropositive male is less than 4%. These data indicate that even though sexual contact is important in the transmission of STLV-I, it may not be an efficient mode of viral infection. These data also suggest that female-to-male transmission of STLV-I occurs, as recently reported for human T-lymphotropic virus type-I (HTLV-I) infection. These results are important because HTLV-I and STLV-I share many features in common including routes of viral transmission. In addition, the difficulty of clearly quantitating the risk of sexual transmission in humans makes the primate animal model a valuable alternative to study the human infection.

Genetic analysis and molecular phylogeny of simian T-cell lymphotropic virus type I: evidence for independent virus evolution in Asia and Africa.

Type C retroviruses, designated simian T-cell lymphotropic virus type I (STLV-I), have been isolated from several genera of Old World monkeys and apes, but not from New World monkeys and prosimians. To determine the genomic diversity and molecular evolution of STLV-I and to clarify their genetic relationship to human T-cell lymphotropic virus type I (HTLV-I), we enzymatically amplified, then directly sequenced selected regions of the gag, pol, env, and pX genes of STLV-I strains from Asia and Africa. STLV-I strains Si-2, Matsu, and JM86 from Japanese macaques, which exhibited sequence similarities ranging from 98.5 to 99.8% among themselves, diverged by 12.9 to 13.3% from STLV-I strain MM39-83 from a naturally infected rhesus macaque, by 9.7 to 11.2% from STLV-I strains from Africa, and by 8.8 to 11.2% from HTLV-I strains originating in Japan, India, Africa, the Caribbean, the Americas, Polynesia, and Melanesia. By contrast, the interspecies nucleotide sequence similarity among African STLV-I strains from green monkey, yellow baboon, sooty mangabey, and common chimpanzee was remarkably high, ranging from 96.9 to 97.4%, and these STLV-I strains diverged by only 2.2 to 2.8% from HTLV-I strain EL from equatorial Zaire. Phylogenetic trees constructed by using the neighbor-joining and maximum parsimony methods indicated that the Asian STLV-I strains diverged from the common ancestral virus prior to African STLV-I and cosmopolitan and Melanesian HTLV-I strains. Thus, our data are consistent with an archaic presence of STLV-I in Asia, probably predating macaque speciation, with subsequent independent virus evolution in Asia and Africa.

Fusion activity dissociated from replication ability in feline immunodeficiency virus (FIV) in human cells.

Multinucleated-giant-cell formation followed by cell killing was observed after cocultivation of the feline immunodeficiency virus (FIV)-producing feline T-cell line 3201/FIV with various human cells, including T-cell lines carrying human T-cell lymphotropic virus type I (HTLV-I). The susceptibility to giant cell formation varied with the cell lines tested. Cocultivation of irradiated 3201/FIV cells with MT-2 cells resulted in giant cell formation as early as 2 h in culture, with striking resemblance to that induced by human immunodeficiency virus (HIV). MT-4 cells (HTLV-I positive) and H9 cells (HTLV-I negative) were less susceptible than MT-2 to the induction of syncytia. MOLT-4 cells (HTLV-I negative) had intermediate sensitivity to syncytia formation. No syncytia were observed in the monocytic cell line U-937 (HTLV-I negative). Syncytia formation between 3201/FIV and MT-2 cells was inhibited by polyclonal cat anti-FIV antisera but not polyclonal cat anti-feline leukemia virus (FeLV) antisera, goat anti-FeLV, uninfected specific-pathogen-free cat serum, human anti-HTLV-I antisera, or normal human and goat serum. Concentrated cell-free FIV supernatant from 3201/FIV also induced giant cells of MT-2 cells that were indistinguishable from those induced by cocultivation. Giant cells and extensive cell killing associated with giant cell formation declined and disappeared within 10 days. Surviving cells appeared to be of normal size and grew continuously without expressing FIV antigen or releasing infective virus. Although Southern blot analysis using probes specific for FIV could not detect proviral DNA in any of the five human cell lines cocultured with irradiated 3201/FIV cells, the polymerase chain reaction (PCR) technique detected FIV-specific DNA in MOLT-4 cells. DNA from the FIV-PCR positive MOLT-4 cells was PCR negative for endogenous FeLV-specific sequences, indicating that the MOLT-4 cell DNA was not contaminated with DNA from feline cells (i.e., 3201 cells). The FIV-MOLT-4 cells remained PCR positive for FIV after 40 passages, suggesting stable integration in the human cell line. These findings indicate that FIV is capable of forming proviral DNA in human T-lymphoid cells by cocultivation, although this FIV-carrying human cell line failed to produce replication-competent viruses.

Anti-human immunodeficiency virus (HIV) agents are also potent and selective inhibitors of feline immunodeficiency virus (FIV)-induced cytopathic effect: development of a new method for screening of anti-FIV substances in vitro.

The ability of several known anti-HIV substances to inhibit feline immunodeficiency virus (FIV) was tested. The results showed that FIV infection of feline T-cells was almost completely blocked in the presence of all of the agents tested. However, FIV-induced syncytium formation between a human T-cell line (MT-2 cells) and a FIV-infected feline lymphocyte cell line (3201/FIV) was inhibited only by dextran sulfate and pradimicin A. The assay used to measure syncytium inhibition was rapid and did not use potentially hazardous human immunodeficiency virus (HIV)-infected cells. The efficacy results coincided with those of HIV studies.

Detection of simian T-lymphotropic virus type I using the polymerase chain reaction.

To develop the polymerase chain reaction (PCR) for the detection of simian T-lymphotropic virus type I (STLV-I) infection, cell lines or peripheral-blood mononuclear cells (PBMC) from 2 non-human primate species [African green monkeys (AGM), Cercopithecus aethiops; baboon, Papio cynocephalus] were evaluated for their STLV-I status using oligonucleotide primer pairs and probes specific for the tax and pol gene regions of the closely related human T-lymphotropic virus type I (HTLV-I). These PCR results were compared with serologic (Western blot assay) and viral culture (p24-antigen capture assay) data. PCR products for both gene regions were detected in established baboon, Japanese macaque and rhesus macaque STLV-I-producing cell lines. STLV-I tax and pol products were also detected in PBMC from 4 of 4 infected AGM and 4 of 4 infected baboons, each of which were also Western-blot-positive and p24-antigen-capture-positive. Of the remaining AGM (n = 7) and baboon (n = 1) which were PCR-negative, each was also Western-blot-negative and p24-antigen-capture-negative. Two seronegative and virus-culture-negative AGM were classified as PCR indeterminate with weak reactivity using tax primers. These primer pairs failed to amplify DNA from uninfected human PBMC, an uninfected human lymphoid cell line, a simian immunodeficiency virus macaque (SIVmac251)-infected cell line and a simian-retrovirus-type-D(SRV-D)-infected cell line. HTLV-II-pol-specific primer pairs failed to amplify DNA from STLV-I-infected cell lines and PBMC from STLV-I-infected monkeys. Further, HTLV-I pol and tax primer pairs successfully amplified RNA from HTLV-I- and STLV-I-infected cell lines by reverse transcriptase (RT)-PCR. We have demonstrated excellent specificity in the detection of STLV-I by PCR using these HTLV-I-derived primers and probes. Additionally, our data suggest that the tax and pol gene regions are conserved between HTLV-I and STLV-I strains found among these diverse species of non-human primates.

Zinc effects on nickel dermatitis in the guinea pig.

Prevention of NiSO4 induced allergic contact dermatitis (ACD) using ZnSO4 in drinking water was studied in a guinea pig model. Without ZnSO4 interventions, nickel (Ni)-exposure resulted in significantly higher (p less than 0.05) stimulation indices (SIs) as compared to non-exposed controls, using NiSO4 as an allergen in the lymphocyte transformation test (LTT). Oral intake of ZnSO4 at both 250 micrograms/ml double-distilled deionized water (DDD) and 500 micrograms/ml DDD resulted in lower SIs than those of control guinea pigs drinking only DDD; the 250 micrograms/ml group had significantly lower SIs (p = 0.025) than controls. There was no significant correlation between intradermal test responses and the SI values of individual guinea pigs exposed to NiSO4. Mean zinc (Zn) concentrations in skin and in whole blood were not statistically different between the NiSO4 exposed control and Zn supplemented groups, nor between Ni-sensitive and non-sensitive animals within groups. The rôle of Zn homeostasis, rôle of the Langerhans cell, effect of Zn supplementation on Ni ACD in other species, and possible blocking effects of other metals should be investigated in future studies.

The effects of tumor promoters on HTLV-1 expression in a low-virus producer and a non-virus producer cell line.

Chemical agents including 5-iodo-2'-deoxyuridine (IUdR), 4-O-methyl 12-O-tetradecanoyl phorbol-13-acetate (TPA), phorbol, and the tumor promoters teleocidin and TPA were tested for their ability to induce Human T-cell leukemia-lymphoma virus Type I (HTLV-I) expression in the low-virus producer MT-1 cell line and in the non-virus producer C63/CRII-2 cell line, which contains at least one integrated HTLV-I genome equivalent per cell [26]. Viral antigen expression increased from 0.3% to 8.7% in the MT-1 cell line after treatment with IUdR, while TPA and teleocidin were marginally or non-effective as inducers. Chemicals did not induce HTLV-I antigen expression in the C63/CI(II-2) cell line. No enhancement of reverse transcriptase activity or alteration of proviral organization was observed after chemical treatment of MT-1 cells.

Expression, release, and characterization of soluble human T-lymphotropic virus-I (HTLV-I) antigens from an infected cell line.

The Human T-lymphotropic virus type I (HTLV-I) infected cell line MT-2 was studied to obtain HTLV-I or related proteins for the purpose of producing an effective vaccine for HTLV-I infection. The cells were characterized as to HTLV-I antigen expression during the cell cycle and antigens released into the culture fluids. MT-2 cell grown in fetal calf supplemented media produced more HTLV-I related antigens during the G2/M phase of the cell cycle. To determine the conditions for maximal release and harvest of HTLV-I associated proteins, the MT-2 cells were grown in RPMI 1640 medium supplemented with serum-free medium. Cell- and virus-free supernatants were collected on day 4, lyophilized, and concentrated 50-fold. The proteins in these supernatants were characterized using SDS-PAGE and western blot using rabbit anti-HTLV-I sera and human adult T-cell leukemia sera. The western-blot analysis indicated that the supernatants obtained from the MT-2 cells grown in serum-free supplemented medium contained detectable amounts of proteins which reacted with human ATL and rabbit anti-HTLV-I sera. The molecular weights of these proteins observed are 68kd, 46kd, 28kd, 24kd, 19kd, and 15kd indicating that gag, env, and pX gene products are present.

Chronic fatal disease in gorillas seropositive for simian T-lymphotropic virus I antibodies.

Sera from 10 gorillas were tested by indirect immunofluorescent antibody assay, Western blotting and ELISA to Human T-lymphotropic viruses for cross-reacting with antibodies to Simian T-lymphotropic virus I (STLV-I). Four were antibody positive. Of the 4 seropositive gorillas, one has remained healthy, while 3 have died with similar disease problems as reconstructed from clinical records. It is not known whether a causal relationship exists between these diseases and STLV-I retrovirus infection.

Persistent infection of Vero cells by paramyxoviruses. A morphological and immunoelectron microscopic investigation.

Two Vero cell lines persistently infected with canine distemper virus (CDV) or with both CDV and canine parainfluenza (CPI) viruses were investigated. Cells in the CPI-CDV cell line were 90-100% positive for CPI antigen and exhibited 10-80% CPI hemadsorption. Cytoplasmic CDV antigen expressed in both singly and dually infected monolayers varied weekly from 1 to 100%. Numerous cytolytic crises were observed in both cell lines. Cell replication was severely depressed in both cell lines when compared with uninfected Vero cells. Infrequent interfering activity against lytic CPI virus was present in the CPI-CDV cell line but not between lytic CDV and progeny virus from the CDV or the CPI-CDV cell line. Ultrastructurally, Vero cells persistently infected with both paramyxoviruses contained two types of viral nucleocapsids (NC). By immunoelectron microscopy, smooth NC were identified as CPI virus and rough NC were of CDV origin. The viral NC never intermingled but rather were restricted to discrete cytoplasmic areas containing either one type of NC or the other.

Serological and virological evidence of human T-lymphotropic virus in systemic lupus erythematosus.

We present evidence that systemic lupus erythematosus (SLE) patients, but not other mixed connective tissue disease patients, have been exposed to a retrovirus similar or identical to known human T-lymphotropic viruses (HTLV). Serological studies demonstrated a high incidence of seropositivity in SLE patients to HTLV-I antigens by indirect immunofluorescence and Western blot analysis, and to HTLV-III by Western blot analysis. Furthermore, peripheral blood mononuclear cells from four patients with SLE cultivated in vitro expressed HTLV-I antigens after 3 days or more in culture, and reverse transcriptase activity was detected in supernatants from short-term cultures.

Oncogenic viruses of domestic animals.

The objective of this article is to review the relevant oncogenic viruses of small animals. Basic scientific principles that have been discovered by research into feline leukemia and other animal cancer viruses have enhanced our understanding of oncogenesis and have led to practical methods of cancer control and prophylaxis.

Human T-cell leukemia virus I induction by 5-iodo-2'-deoxyuridine and N-methyl-N'-nitro-N-nitrosoguanidine: inhibition by retinoids, L-ascorbic acid, and DL-alpha-tocopherol.

Human T-cell leukemia virus type I was induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and 5-iodo-2'-deoxyuridine (ldUrd) in the MT-1 cell line. Virus expression was monitored by immunofluorescence microscopy with GIN-14, mouse monoclonal antibodies directed toward Mr 19,000 and Mr 28,000 protein-specific virus polypeptides. MNNG (0.1 micrograms/ml) and ldUrd (50 micrograms/ml) both induced virus synthesis in MT-1 cells. MNNG-induced virus expression peaked between 24 and 48 h of incubation, whereas ldUrd induced maximum virus expression between 48 and 72 h of incubation. Superinduction resulted when MNNG was added to cells induced 48 h previously with ldUrd, but not with concomitant treatment. 13-cis-Retinoic acid, retinol, retinol aldehyde, and retinol acetate (10(-6) to 10(-9)M) were concomitantly added with ldUrd to MT-1 cells for 24, 48, and 72 h incubation. All inhibited virus induction to various degrees. The retinoids were ranked as to inhibitory activity: retinol greater than retinoic acid greater than retinol aldehyde greater than retinol acetate. The most sensitive period for inhibiting ldUrd induction by retinoic acid was 24 h postinduction or with concomitant treatment. Vitamin C and vitamin E inhibited ldUrd induction most effectively with 48 h incubation. Retinol and vitamin C also inhibited virus induction by MNNG. None of the retinoids, vitamin C, or vitamin E significantly inhibited virus expression in noninduced cells or were toxic to the cells at the concentrations used in these experiments.