HTLV-V: a new human retrovirus isolated in a Tac-negative T cell lymphoma/leukemia.

A new human retrovirus was isolated from a continuous cell line derived from a patient with CD4+ Tac- cutaneous T cell lymphoma/leukemia. This virus is related to but distinct from human T cell leukemia/lymphoma virus types I and II (HTLV-I and HTLV-II) and human immunodeficiency virus (HIV-1). With the use of a fragment of provirus cloned from one patient with T cell leukemia, closely related sequences were found in DNA of the cell line and of tumor cells from seven other patients with the same disease; these sequences were only distantly related to HTLV-I. The phenotype of the cells and the clinical course of the disease were clearly distinguishable from leukemia associated with HTLV-I. All patients and the wife of one patient showed a weak serological cross-reactivity with both HTLV-I and HIV-1 antigens. None of the patients proved to be at any apparent risk for HIV-1 infection. The name proposed for this virus is HTLV-V, and the date indicate that it may be a primary etiological factor in the major group of cutaneous T cell lymphomas/leukemias, including the sporadic lymphomas known as mycoses fungoides.

Placenta growth factor is a survival factor for human malignant mesothelioma cells.

Placenta growth factor (PlGF) is a key regulator of pathological angiogenesis and its overexpression has been linked to neoplastic progression. To assess whether PlGF could have a role in malignant mesothelioma (MM), we analyzed the expression of PlGF, VEGF, and their cognate receptors (VEGF-R1 and VEGF-R2) and co-receptors (neuropilin-1 and neuropilin-2) in MM cell lines as well as in resected MM tissues, hyperplastic/reactive mesothelium and normal mesothelium. MM cell cultures expressed both ligands and the associated receptors to a variable extent and released different amounts of PlGF. As assessed by immunohistochemistry, PlGF expression was switched on in hyperplastic/reactive compared to normal mesothelium. Moreover, 74 and 94 percent of MM tissues overexpressed PlGF and VEGF-R1, respectively (p<0.05 MM vs normal mesothelium). Administration of recombinant PlGF-2 did not elicit a significant stimulation of MM cell growth, while it was associated with a transient phosphorylation of Akt, suggesting that PlGF-2 could activate downstream effectors of proliferative and cytoprotective signals via VEGF-R1 in MM cells. Indeed, the administration of an anti-PlGF antibody was found to cause a significant reduction of MM cell survival. In conclusion, our data demonstrate that, by acting as a survival factor, PlGF can play a role which goes beyond the stimulation of angiogenesis in MM. This evidence could help the rational design of new therapeutic interventions for this aggressive tumor.

4-Hydroxynonenal, a product of cellular lipid peroxidation, which modulates c-myc and globin gene expression in K562 erythroleukemic cells.

Several studies point to the existence of an inverse correlation between cellular lipid peroxidation and both cell proliferation and neoplastic transformation. Here, we show that 4-hydroxynonenal (HNE) concentrations close to the level found in normal cells (in the range of 1 and 3 microM) can specifically induce changes in the expression of c-myc and gamma-globin mRNA in K562 cells, without inducing any toxic effects or affecting cell viability. Since we have determined that K562 cells have undetectable levels of endogenous lipid peroxidation, all these effects can be assigned to the exogenous HNE treatment. After a 1-h treatment with 1 microM HNE, c-myc mRNA levels decrease transiently during the first 4 h, rebounding later to higher levels, and normalizing to basal expression after 4 days. Run-on experiments show a transient transcriptional block 20 min after HNE treatment and subsequent posttranscriptional regulation. According to S1 mapping, mRNA changes are exerted on c-myc transcripts initiated from both the principal constitutive start sites (P1 and P2). gamma-Globin mRNA levels concomitantly increase 3- to 4-fold, but no significant changes of housekeeping gene expression are observed. On the basis of these results it appears that the restoration in human erythroleukemic K562 cells of HNE concentrations closer to the level in normal cells can modulate the expression of specific genes.

Inflammatory cytokines stimulate vascular smooth muscle cells locomotion and growth by enhancing alpha5beta1 integrin expression and function.

The formation of atherosclerotic lesions requires the migration of vascular smooth muscle cells from the media into the intima of the artery and their proliferation. These events, which are preceded and accompanied by inflammation, are modulated by integrin receptors linking vascular smooth muscle cells to extracellular matrix molecules. Among them, fibronectin induces vascular smooth muscle cells to acquire the phenotype they show in the atherosclerotic plaque. Here we show that amounts of interleukin-1 beta, tumor necrosis factor alpha and interferon-gamma as possibly released by activated immune cells infiltrating atherosclerotic lesions, upregulate vascular smooth muscle cell expression of the alpha5beta1 integrin, a fibronectin receptor. This improves vascular smooth muscle cell capability of migrating toward soluble or anchored fibronectin and of adhering to immobilized fibronectin. The latter effect, in turn, augments vascular smooth muscle cell proliferative response to mitogens, as suggested by the increase of intracellular pH. Finally, the effects that inflammatory cytokines have on vascular smooth muscle cell locomotion and growth, are specifically blocked by anti-alpha5beta1 antibodies. As fibronectin and alpha5beta1 levels are augmented in vivo in the atherosclerotic plaques, these findings support the use of integrin antagonists as potential adjuvants in atherosclerosis treatment.

HTLV-I and HIV-1 infection in patients with lymphadenopathy syndrome detected during routine breast screening at a tumor prevention center.

Lymphadenopathy with no apparent cause had been reported in a group of women participating in a mammary tumor prevention program. A screening for retrovirus infection was organized to detect the virus as possible etiological agents. Data show a high percentage of positivity for HIV-1 among these lymphadenopathy patients, and surprisingly for HTLV-I, while no such positivity for either virus was found in matched controls or in patients where a different causal agent for lymphadenopathy was found. Of 26 seropositives, 23 deny any risk factor for HIV-1 and do not come from a HTLV-I known endemic area, but while it is impossible to exclude their knowledge of risk factors, it is worth noting that none of them presented a HTLV-I/HIV-1 double infection, which is very frequent in intravenous drug abusers, the major risk group in Italy. On the basis of these data spread of HTLV-I and HIV-1 appears to be more important in Italy than previously thought, and not confined to well-defined groups or, at least, among those who believe they do not belong to a risk group and therefore can represent a major vehicle for virus diffusion. Institution of screening for HTLV-I in blood donors should be taken immediately, and retrovirus infection risk criteria must be revised.

A rapid and sensitive assay for proviral sequences of a human retrovirus (HTLV) in leukemic cells.

Whole cells of cellular DNA from leukemia patients were used in blot hybridization to cloned probes of a human retrovirus HTLV. The results demonstrated the facility of screening large numbers of samples of limited material for the presence of low copy number of HTLV sequences.

Involvement of bcl-2 and bax gene expression in apoptosis and differentiation of the non-tumorigenic murine hematopoietic cell line, 32DC13(G).

32DCl3(G) is an interleukin-3 (IL-3) dependent, non-tumorigenic murine hematopoietic cell line which undergoes terminal differentiation into granulocytes when exposed to granulocyte-colony stimulating factor (G-CSF). This line therefore offers a convenient system to study the expression of genes involved in apoptosis and differentiation. In our experiments we have acquired evidence that during the differentiation pathway, likewise in apoptosis induced by IL-3 deprivation, detectable levels of bax mRNA appear, while bcl-2 expression decreases. These events are under the control of the p53 tumor-suppressor gene. In these cells, an overexpression of exogenous wild-type p53 leads to a decrease in bcl-2 mRNA and to the appearance of box mRNA, which instead is absent in the parental cells growing in IL-3 conditioned medium. Furthermore, results from experiments on p53 transfected cells demonstrate that excess wild-type p53 activity, on its own, fails to elicit apoptosis as long as IL-3 is present and does not induce differentiation if G-CSF is not added to the culture medium. We conclude that in apoptosis and differentiation of 32DCl3(G) the alterate ratio of bcl-2 and box gene expression, modulated by p53, is an early event dependent on IL-3 withdrawal and that the appearance of bax and the decrease of bcl-2 expression are necessary, but not sufficient for the acquisition of a completely mature granulocytic phenotype.

Epstein-Barr virus serology in nasopharyngeal carcinomas and other head and neck neoplasms in Italy.

This paper reports the results of the EBV-specific antibody response in 17 Italian nasopharyngeal carcinoma (NPC) patients, 15 other head and neck tumor patients and 15 normal controls. Nucleic acid hybridization has been performed on the biopsy tissue from 4 of the NPC patients, and EBV-DNA was present in two undifferentiated (WHO 3) tumors, and absent in two samples of the keratinizing (WHO 1) type. EBV serology appears to be specifically related to NPC, more evidently for VCA-IgA and EA-IgG antibodies, and useful as an aid in diagnosis of NPC. However, in order to assess a prognostic value of the above markers, a greater number of patients followed for a longer period of time (at least 5 years) is needed, and is currently being pursued.

Human immunodeficiency virus (HIV) and Epstein-Barr virus (EBV) antigens and genome in lymph nodes of HIV-positive patients affected by persistent generalized lymphadenopathy (PGL).

The presence of human immunodeficiency virus (HIV) and Epstein-Barr virus (EBV) antigens and genome has been investigated in 50 lymph nodes involved by persistent generalized lymphadenopathy (PGL). All the patients were HIV infected and most of them (42 of 50) also had anti-EBV serum antibodies. At lymph node level, HIV and EBV antigens were studied by immunohistochemistry using monoclonal antibodies directed against viral core proteins. The HIV p24 protein was detected in 43 of 50 lymph nodes within the B-cell germinal centers with a reticular pattern. Few cells with positive results for EBV antigens were found in only 2 of 50 lymph nodes. These rare EBV-positive centrocyte-like cells were mainly located in the germinal centers. The presence of HIV and EBV genome was also studied in lymph nodes involved by PGL, with the use of in situ and Southern blot hybridization. A positive reaction for HIV genome was detected in only 1 of 14 lymph nodes with the Southern blot hybridization, and the presence of EBV genome was never demonstrated in these lymph nodes with the use of both in situ and Southern blot hybridization. The expression of EBV antigens and genome was also investigated in the peripheral blood of 15 patients with PGL in which cells with positive results for EBV antigens were detected in a single case with a frequency of 1 X 10(-4). No evidence of EBV genome was found with the use of the in situ hybridization. These results suggest that EBV is not present in lymph nodes during the PGL phase and that its possible implication in the pathogenesis of acquired immune deficiency syndrome (AIDS)-associated lymphoma might be a late event.

Amplifications of multiple regions of the HTLV-I genome from DNA of an Italian spastic paraparesis patient but not from DNA of multiple sclerosis patients.

We searched for evidence of infection by the human T-cell lymphoma/leukemia virus type I (HTLV-I) in patients with multiple sclerosis (40 cases); brainstem encephalitis (1 case); Friedreich's ataxia (1 case); spastic paraparesis of unknown etiology (1 case). All patients were from the region of Abruzzo, Italy. Sera were all negative for anti-HTLV-I reactivity by the Western blotting (WB) analysis. DNAs from peripheral blood mononuclear cells were amplified using the polymerase chain reaction (PCR) technique with primers specific for the HTLV-I gag, pol, and env proviral regions. HTLV-I sequences were amplified only in the patient with spastic paraparesis of unknown etiology. In this case, HTLV-I infection might have been related to blood transfusions received 2 years prior to the onset of the neurologic symptoms. Members of the patient's family were negative for HTLV-I by PCR and WB. These data indicate that HTLV-I associated myelopathy is present also in Italy, but fail to substantiate an association of HTLV-I with multiple sclerosis.

Italian population data on the polymarker system and on the five short tandem repeat loci CSF1PO, TPOX, TH01, F13B, and vWA.

A population study on five short tandem repeat (STR) loci and five sequence specific polymorphism loci was performed on unrelated Italian Caucasians. Separation and detection of the amplified STR fragments were carried out by high resolution vertical denaturing polyacrylamide gel electrophoresis (PAGE) and silver staining, respectively. The sequence specific loci were analyzed using the AmpliType PM Typing Kit (Perkin Elmer, Foster City, CA). All loci, except Gc (p = 0.031), meet Hardy-Wienberg expectations. In addition, there is no evidence for association of alleles between pairs of loci. The combined power of discrimination for the five STR loci is 0.9999862 and for the PM loci is 0.99503. The results suggest that these loci may be useful for human identification cases in Italy.

Globin mRNA sequences in polyadenylated and nonpolyadenylated nuclear precursor-messenger RNA from avian erythroblasts.

Nuclear RNA from immature duck erythrocytes was fractionated into polyadenylated and nonpolyadenylated fractions, and globin mRNA sequences were determined by hybridization to DNA complementary to globin mRNA. 80--90% of labeled nuclear RNA is found to be nonpolyadenylated, and 70--80% of the globin mRNA sequences present in the nucleus are found in nonpolyadenylated molecules. These data suggest that polyadenylation does not specifically select for globin mRNA sequences. The nonpolyadenylated globin mRNA sequences present in the nucleus are found mostly in molecules of small size, close to the size of polyribosomal globin mRNA, suggesting that polyadenylation is a later event in globin mRNA formation.

Plasma amino acid values and pancreatic beta-cell function in phenylketonuria.

In 16 phenylketonuric (PKU) patients aged 5-12 years, plasma glucose, immunoreactive insulin (IRI), C-peptide (CP) and plasma amino acids were measured in basal conditions and under a standard oral glucose tolerance test (OGTT). The beta-cell response to OGTT was higher in PKU patients than in normal subjects as demonstrated by peak levels and areas under the curves of plasma concentrations of IRI and of CP. A significant correlation was observed between plasma phenylalanine values and both IRI and CP 'output' in PKU patients. Mean concentrations of branched chain amino acids and tyrosine in plasma decreased significantly during OGTT, while phenylalanine values increased in PKU subjects.

Molecular cloning of a unique human T-cell leukemia virus (HTLV-IIMo).

Human T-cell leukemia virus IIMo (HTLV-IIMo) is a human retrovirus isolated from a patient with a T-cell hairy cell leukemia. This virus has been shown to have core protein (gag) antigens similar to, but distinct from, those of all known isolates of the prototype human T-cell leukemia virus (HTLV-I). We have used a subgenomic clone of the HTLV-I env-pX region to detect and characterize HTLV-IIMo proviral sequences by performing Southern blot hybridization under conditions of low stringency. Using the HTLV-I probe, we cloned a partial integrated HTLV-IIMo provirus from a genomic library of the producer Mo cell line. These sequences could be characterized by low stringency hybridization with different subgenomic clones of HTLV-I. An HTLV-IIMo-specific subclone was made by isolating a 3.6-kilobase BamHI fragment of the partial provirus. This was used to clone two full-length integrated viral genomes. Using the HTLV-IIMo viral probe, we also showed by hybridization under stringent conditions to DNA and RNA of various infected and uninfected cell lines that these HTLV-IIMo sequences are unique.

Common site of integration of HTLV in cells of three patients with mature T-cell leukaemia-lymphoma.

Human T-cell leukaemia-lymphoma virus (HTLV) was first isolated in the United States from a patient with an aggressive form of cutaneous T-cell lymphoma (CTCL) and was later found associated with clusters of adult T-cell leukaemia-lymphomas (ATL) in various parts of the world, including Japan and the Caribbean. Leukaemic cells of the HTLV-positive patients seem to be clonal expansions of single infected cells since the provirus(es) are found at the same sites in a given patient. In avian leukosis virus-induced B-cell lymphomas, the provirus very frequently integrates at several discrete sites in a common domain near the cellular gene, c-myc, that it activates and it has been speculated that the same would hold true for other chronic leukaemia viruses. We report here that cultured cells from two US patients with CTCL and fresh leukaemia cells of a Japanese patient with ATL contained an HTLV provirus integrated at the same site. In addition, a cord blood T-lymphocyte cell line established by co-cultivation with one of the two HTLV-positive CTCL cell lines also contained HTLV provirus contiguous with the same flanking cellular sequences. Ten other HTLV-positive cell samples did not show integration of HTLV at this site, suggesting that there is more than one discrete site of HTLV integration in tumour cells.

Simultaneous detection of reverse transcriptase and high molecular weight RNA in tissue of patients with Hodgkin's disease and patients with leukemia.

Complexes of high-molecular-weight RNA and reverse transcriptase (RNA-dependent DNA nucleotidyltransferase) have been detected in 14(77.8%) of 18 spleen from patients with Hodgkin's disease and in all samples tested of peripheral leukocytes and spleens from leukemic patients. The enzyme and its template are localized in a particle having a density between 1.16 and 1.19 g/ml. These observations describe characteristic features of RNA tumor viruses.

Effect of 4-hydroxynonenal on c-myc expression.

The 4-hydroxynonenal aldehyde (HNE), a product of lipid peroxidation with high biological activity, inhibits cancerous growths in vivo and in vitro. The mechanism by which this aldehyde acts is not yet understood. The c-myc oncogene seems to be involved in the regulation of cellular multiplication and transformation. We evaluated the c-myc expression and the RNA, DNA and protein synthesis in K562 cells. These cells were incubated for 1 hour in presence of several aldehyde concentrations (range 5.10(-7) to 10(-4)), then washed and kept for 20 hours in a growth medium until used. HNE inhibited both the nucleic acids and protein synthesis in a dose dependent manner, and c-myc expression was evaluated in the K562 cells after incubation with 10(-4) M or 10(-6) M HNE. HNE inhibited c-myc expression only at the highest dose. These preliminary results may suggest that the inhibition of c-myc expression is related to nucleic acid synthesis inhibition following HNE exposure.

T-helper phenotype chronic lymphocytic leukemia (Thp-CLL): characterization of an Italian case with particular biological findings.

A patient with Chronic Lymphocytic Leukemia (CLL) characterized by an expansion of helper phenotype mature T lymphocytes is here described. The phenotype of these cells was OKT3+, OKT4+, Leu 9+, 5/9+, OKT8-, Tac- and functional studies showed a strong helper activity on B cell differentiation; an "in vivo" presence of an IgG-lambda paraproteinaemia has been demonstrated. Cytogenetic studies showed multiple clonal, numerical and structural rearrangements which included a tandem t(14;14) (q11;32) translocation. Hybridization showed HTLV I related specific bands indicating the presence of exogenous sequences related to prototype virus but derived from a different Retrovirus (HTLV 1c). The clinical course was aggressive and unsuccessful treatments with various polichemotherapeutic protocols, associated with multiple leukaphereses, were performed. The authors underline that despite the morphological, immunological, biological and virological heterogeneity, the common feature of T-helper CLL is the inexorable clinical course which needs a new therapeutic approach.