c-Ki-ras gene amplification and malignant behavior in murine embryonal carcinoma cell lines.

Embryonal carcinoma (EC) cells are malignant components of murine teratoma tumors. To extend our earlier findings concerning c-Ki-ras amplification in the embryonal carcinoma PCC4 cell line, we examined the c-Ki-ras protooncogene and its expression in other EC cell lines. We report here that c-Ki-ras amplification is not a general feature of EC cell lines since neither PCC3, PCC6, nor F9 cell lines have an amplified copy number of this protooncogene. Furthermore, molecular analysis of three independently passaged PCC4 cell lines showed marked heterogeneity for c-Ki-ras amplification. Two PCC4 cell lines showed amplified copy number and elevated expression of c-Ki-ras whereas the original one does not, suggesting that this gene amplification occurred through laboratory passage. Malignancy in syngeneic mice of PCC4 with or without an amplified c-Ki-ras gene was also examined. Our results indicate that c-Ki-ras amplification alone is not a determining factor in the malignant behavior of EC cells.

Serotonin and histamine production by human carcinoid cells in culture.

We are reporting on the first human carcinoid cells ever cultured in vitro. These cells, termed CGP, originated from a jejunal carcinoid tumor. Before tumor resection, the 29-year old male patient presented high levels of blood serotonin and histamine and also of urinary serotonin and 5-hydroxyindole-acetic acid; values returned to normal 9 days after resection. CGP cells exhibit a very slow multiplication rate; generation time is about 10 days. From the first subculture, the main cytological, ultrastructural, and biochemical features of CGP cultures remain unchanged. The cells show most of the enterochromaffin cell histomorphological characteristics; for example, cytoplasmic granulations, specific of argyrophilic cells, can be seen both by electron microscopy and by light microscopy (preceded by silver impregnation). The high amounts of serotonin and histamine found by highly specific radioenzymatic assay in the supernatant of CGP cultures indicate that, after 6 months (25 subcultures), CGP cells have retained the main metabolic characteristics of the original tumor, i.e., the ability to synthesize, store, and release both serotonin and histamine.

Epidemiology and immunovirology of human T-cell leukemia/lymphoma virus type I-associated adult T-cell leukemia and chronic myelopathies as seen in France.

Seventeen patients with adult T-cell leukemia (ATL) and 21 with tropical spastic paraparesis/human T-cell leukemia/lymphoma virus type I (HTLV-I)-associated myelopathy (TSP/HAM) were observed during a 3-yr survey (1986-1988) in some hospitals in Paris, France. Most of them were black, originating from high-HTLV-I-endemic areas (West Indies or Africa), but two cases of TSP/HAM occurred in French Caucasians. In one case, the patient acquired the virus from a transfusion during a cardiac transplantation. Most of the ATL cases were diagnosed as acute leukemia or lymphoma, with a proliferation of CD2+, CD3+, CD4+, CD8-, DR+, and CD25+ lymphoid cells. Only three cases were diagnosed as a smoldering ATL. All of the TSP/HAM cases exhibited a spastic paraparesis with a chronic and slow evolution and high HTLV-I antibody titers in serum and cerebrospinal fluid, with a high HTLV-I antibody index and specific HTLV-I immunoglobulin = oligoclonal bands. In TSP/HAM, a high percentage of DR-expressing cells (15 to 40%) was found, with a slightly elevated CD4/CD8 ratio. This was associated with the presence of 1 to 10% abnormally shaped nuclei in lymphoid cells and a polyclonal integration of HTLV-I proviruses in these peripheral blood mononuclear cells. On the contrary, a clonal integration was always found in the ATL malignant cells (leukemic, lymph node, and cutaneous infiltrate). Long-term interleukin 2-dependent T-cell lines (CD2+, CD3+, CD4+, and WT31+) with activated T-cell markers (CD25+ and DR+) producing HTLV-I were established from ATL and TSP/HAM peripheral blood mononuclear cells.

Recent insights into the biology of the human foamy virus.

Human foamy virus (HFV) is a complex retrovirus with structural similarities to HIV and human T cell leukemia virus. There have been considerable advances in understanding the biology of HFV in cell cultures. However, viral behavior in vivo, and even viral detection, are still poorly understood. While HFV-transgenic mice develop neuromuscular disorders, attempts to associate HFV with a specific human disease have given inconclusive results.

High level of HTLV-I specific protein expression in a patient with adult T-cell leukemia, chronic progressive myelopathy and Kaposi's sarcoma.

Analysis was made of serum anti-HTLV-I antibodies, virus-specific proteins in peripheral blood lymphocytes (PBL) and proviruses in lymphocyte DNA of a patient with adult T-cell leukemia (ATL), Kaposi's sarcoma, and chronic myelopathy. Using Western blot and PCR (with HIV-1 specific primers), it was shown that Kaposi's sarcoma was not linked to HIV infection. Western blot analysis of serum revealed antibodies against p19, p24 and Pr 53 of HTLV-I. Examination of proteins in fresh PBL by Western blot revealed a high level of HTLV-I specific protein expression. Southern blot analysis of the patient's DNA revealed two different sites for HTLV-I provirus integration.

HTLV-1-like particles and HTLV-1-related DNA sequences in an unambiguous case of Sèzary syndrome.

An unambiguous case of Sèzary syndrome associated with the presence of unusual retroviral infection markers is described. The blood smear showed 15% typical Sèzary cells but also rare atypical lymphocytes with convoluted nuclei, evocative of characteristic adult T-cell leukemia (ATL) flower cells. However, the patient did not present any clinical or biological manifestations of ATL, and human T-cell leukemia virus type 1 (HTLV-1) serology was consistently negative. After being cultured for 4 months, peripheral blood mononuclear cells (PBMC) produced typical type C retrovirus-like particles with budding forms strongly resembling HTLV-1 virions. The producer cells did not express HTLV-1-specific antigens detectable by indirect immunofluorescence (IIF). Southern blotting of uncultured PBMC DNA, submitted to digestion with the restriction enzymes PstI and SacI, and hybridized with a full genomic HTLV-1 probe, showed the presence of specific homologous sequences, absent in all of the healthy donor control PBMC DNAs. These HTLV-1-like sequences presented a restriction enzyme pattern distinct from that of the HTLV-1 prototype genome and of other HTLV-1 proviruses studied up to now. Polymerase chain reaction (PCR) with highly conserved HTLV-1 derived pol and env primers was consistently negative with the patient's DNA. All these results taken together suggest that our patient carries a retroviral agent partially homologous to, but probably different from HTLV-1. The possibility is discussed that this type of retroviral agent might be associated with a subtype of cutaneous T-cell lymphoma (CTCL) represented by a typical Sèzary syndrome with a very low percentage of ATL-like flower cells in the blood smear.

No evidence for HTLV-I infection in 24 cases of French and Portuguese mycosis fungoïdes and Sezary syndrome (as seen in France).

A survey in search of evidence for HTLV-I infection was conducted on French and Portuguese patients residing in France with a diagnosis of mycosis fungoïdes or Sezary syndrome. Methods used in this investigation included serological assays (ELISA, Western blot, particle agglutination, indirect immunofluorescence) and DNA molecular studies (Southern blot and polymerase chain reaction). Cultures of peripheral blood mononuclear cells were performed and checked by electron microscopy and reverse transcriptase assay. The results indicate that neither HTLV-I nor a closely related retrovirus are associated with mycosis fungoïde or Sezary syndrome in the cases studied.

Sequence analysis of the simian foamy virus type 1 genome.

We have cloned the simian foamy virus type 1 genome (SFV1) and determined its nucleotide sequence. Analysis of this genome reveals, in addition to the usual genes encoding retroviral capsid, reverse transcriptase, and envelope protein (respectively, gag, pol, and env), two open reading frames (ORFs) between env and the long terminal repeat with partial homology to the human foamy virus (HFV) bel1 and bel2 genes. The first ORF could code for a polypeptide of 312 amino acids (aa) showing 40% homology with the HFV bel1 putative gene product. A more detailed analysis showed that the protein encoded by this ORF would have features characteristic of known trans-activating proteins. The second ORF could code for a polypeptide of 403 aa showing 38% homology with the putative HFV bel2 gene product. Moreover, the 5' extremity of the RNA genome can be folded into a secondary structure identical to the Tat-response element of human immunodeficiency viruses. A phylogenetic tree of retroviruses, including SFV1 and HFV, was constructed. It showed at the molecular level that Spumavirinae, previously classified on the basis of their morphology and their biological properties, constitute a separate group. The homology between SFV1 and HFV reaches 89% in the reverse transcriptase domain of the pol gene. but is much smaller in other parts of the genome.

Functional comparison between HTLV-I envelopes originating from TSP/HAM or ATL cell lines.

The human T-cell leukemia type I (HTLV-I) virus is associated with two different diseases, adult T-cell leukemia (ATL) and tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM). We have compared the viral envelopes originating from TSP/HAM and ATL patients, using the capacity of infected cells to form syncytia with receptor-expressing cells. We show that like the ATL cell lines, the TSP/HAM ones can form syncytia with a large panel of human target cells, including a variety of hematopoietic cell lines, as well as cell lines of neuroectodermal origin. None of the target cell lines tested was able to discriminate between TSP/HAM- and ATL-infected cell lines. When infected cells of TSP/HAM origin are cocultivated with cells of ATL origins, syncytia are never observed. This interference phenomenon suggests that the viruses expressed by the different cell lines utilize the same receptor.

Decreased whole blood 5-hydroxytryptamine (serotonin) in AIDS patients.

Significantly (p less than 0.001) decreased whole blood unconjugated serotonin levels were detected in AIDS patients as compared to patients with advanced cancers and to healthy individuals.

Amplification and rearrangement of Ki-ras oncogene in human teratocarcinoma-derived cell lines.

The structure of the ras gene family was analyzed in two human teratocarcinoma-derived cell lines, Tera I and Tera II, by DNA restriction enzyme digestion and Southern blot. We report here a ten-fold amplification of the c-Ki-ras-2 gene in these cell lines, whereas no structural alterations seem to occur either in c-Ha-ras-1 or N-ras. We also provide evidence indicating that no point mutation at codon 12, specifically recognized by Sac I, was detected. Moreover, DNA rearrangement, due to the loss of a Pvu II site located in the intervening sequences between the third and the fourth exon, has been found in both Tera I and Tera II.

Murine retrovirus genome directs the synthesis of gag protein precursor early after infection.

Incoming type C retroviral genomic 35S RNA is present in polysomes of undifferentiated and differentiated murine teratocarcinoma cell lines at 4 hours after infection. At the same time a 65,000 daltons viral specific protein is produced by the infected cells. These data present evidence that incoming viral RNA serves as messenger for the synthesis of gag protein precursor Pr65 early in the infectious cycle of ecotropic murine retrovirus.

Inhibition of the in vitro infectivity and cytopathic effect of human foamy virus by dideoxynucleosides.

Human foamy virus (HFV) is a human retrovirus that has not been clearly associated with human disease. In this study, we tested the capacity of nucleoside derivatives to inhibit the infectivity and cytopathic effect of HFV in T-lymphoblastoid cells in vitro. H9 cells showed a dramatic cytopathic effect 3 weeks after exposure to HFV. At this time, viral infection was demonstrated by detection of viral antigens by immunofluorescence staining, release of reverse transcriptase activity (RT) in the supernatant, detection of typical viral particles by electron microscopy, and presence of proviral DNA by Southern blot analysis. H9 cells were pretreated with dideoxycytidine (ddC), dideoxyinosine (ddI), or azidothymidine (AZT) at various concentrations before HFV infection. ddC could not completely suppress viral replication at low concentrations, and inhibited cell proliferation at higher concentrations. ddI partially inhibited the formation of giant cells at 10 microM, with 95% inhibition of RT in the supernatant. AZT induced a complete inhibition of cytopathic effect at concentrations > or = 1 microM, with more than 95% inhibition of RT in the supernatant. Moreover, the synthesis of proviral DNA was completely suppressed by 10 microM AZT. These results show that AZT and ddI can inhibit HFV replication in vitro.

Anti HIV-2 serological screening in Portuguese populations native from or having had close contact with Africa.

PIP: To gather epidemiologic information on the spread of human immunodeficiency virus (HIV)-2 in Portugal, sera were collected in 1985 from 156 healthy adults currently living in Portugal but natives of Guinea Bissau, Cape Verde Islands, Saint Tome/Prince, Angola, and Mozambique and from 321 native Portuguese men and women who had close contact with local African populations. As a control, sera were collected from 102 health Portuguese with no previous contact with Africa or African natives. The enzyme-linked immunosorbent assay (ELISA) developed by Diagnostic Pasteur was used to screen for antibodies to HIV. No positive reactions were recorded in the control population. In contracts, 9 (6%) of the African natives and 7 (2%) of the contacts of Africans were HIV-positive, 6 of the positive sera were from women and 10 were from men. Significantly, 1 of the HIV-2-positive serum samples was from a native of Mozambique and 3 were from natives of Angola. This suggests that HIV-2 infection may have spread to other former Portuguese colonies, and foreign army soldiers who were at 1 time residents of Mozambique or Angola should be considered a risk group capable of spreading HIV-2 infection to other countries.^ieng

Molecular differences between two immunologically related spumaretroviruses: the human prototype HSRV and the chimpanzee isolate SFV6.

A comparative study at the genomic and protein level was performed between two immunologically related spumaretroviruses, the human HSRV and the simian SFV6. Cross immunoprecipitation analysis with specific polyclonal and monoclonal antisera indicates shared antigenic determinants. However, restriction analysis of the viral DNAs and thermal stability of the hybrids demonstrate that HSRV and SFV6 are two different isolates.

Human foamy virus DNA forms and expression in persistently infected Dami megakaryocytic cells.

We have characterized human foamy virus (HFV) proviral DNA and determined HFV expression in a persistent infection model, the Dami megakaryocytic cell line. Molecular studies were performed on parental persistently infected cells (Dami-P), as well as on derived clones (Dami-Cl). We report that in these nonlytic and non-HFV producer cells, viral DNA was found to be integrated into the cellular genome and that the few free proviral forms detected in Dami-P cells were deleted in their 5' LTR. Our molecular analysis indicates the presence of undeleted 5' LTR forms in the integrated provirus within a proviral population mainly composed of deleted forms. In addition, the deletion in the bel1 trans-activator gene, previously described by Saïb et al., was found to be highly predominant. However, in 5-iodo-2'-deoxyuridine treated Dami-Cl cultures, virus production occurred, providing evidence for the presence of complete viral genome. Analysis of HFV expression in Dami-Cl cells, by Northern blot and immunoprecipitation, shows that the most striking difference between cytolytic and persistent HFV infection was the lack of expression of structural viral proteins, in contrast with Bet protein expression, which is maintained. Our data suggest that the Bet protein could be involved in the maintenance of viral persistency and that the persistently infected Dami system provides a suitable model for clarifying its function.

The diffuse neuroendocrine (APUD) system.

The bulk of experimental evidence indicates that the APUD series of cells is a distinct system based upon common pathophysiological features. The diffuse nature of this system with elements in the central and peripheral nervous system suggests a more complex interaction of the body's homeostasis than has been established. It is probable that as radioimmunological and radioenzymatic assays become more widely available and standardized, other apudomas will be described. Finally, an understanding of the APUD concept, with its peculiar pluripotential for the production of biogenic amines and peptides, the multicentric nature of the disease and the possibility of multiple cell involvement, is of key importance in managing patients. Studies of the apudomas will also advance the understanding of the normal physiologic interrelationships of the APUD cells.

Paradoxical effects of recombinant mouse beta-interferon on different expression levels of intracisternal A particle genes.

We have studied the effect of mouse recombinant beta-Interferon on the expression of the intracisternal A particle (IAP) genes in a transformed mouse fibroblast cell line. Northern and immunoprecipitation analysis showed an increase in specific transcripts and polypeptides. Run-on experiments on isolated nuclei and transfection assays with an IAP long terminal repeat construct, coupled with the chloramphenicol acetyl transferase gene, showed no enhancement of the transcription rate following beta-IFN treatment. This suggests that the increase in IAP expression depends on a post-transcriptional event. Electron microscopic analysis revealed that in spite of the higher levels of IAP related polypeptides, the number of particles was significantly reduced by interferon.

Comparative studies on IL-6 production status in HTLV-1 chronically infected cell lines derived from different HTLV-1 associated pathologies.

We have comparatively studied the qualitative and quantitative status of Interleukin 6 (IL-6) production in several HTLV-1 chronically infected T-cell lines derived from patients suffering either of adult-T-cell leukemia/lymphoma or of tropical spastic paraparesis (TSP). Two other HTLV-1 chronically infected cell lines, coming from healthy seropositive carriers, were also analyzed. Our results demonstrate that, independently of the origin, all these T cell lines, as compared to uninfected HTLV-1 T cell ones, which were always IL-6 non producers, synthesize and yield comparable amounts of IL-6 synthesis, he nosological origin of infecting viral strains does not seem to play a differential role on IL-6 qualitative or quantitative production parameters.