Functional interaction of HTLV-1 tax protein with the POZ domain of the transcriptional repressor BCL6.

The Tax protein encoded by human T-cell leukaemia virus type 1 (HTLV-1) has a pivotal role in T-cell transformation by deregulating cellular signalling pathways. Using the yeast two-hybrid system to screen a human leukocyte cDNA library, we identified BCL6 (B-cell lymphoma 6) as a cellular protein, which interacts with Tax 1. The BCL6 gene encodes a sequence-specific transcriptional repressor that contains a conserved N-terminal poxvirus and zinc finger (POZ) repressor domain and a C-terminal Kruppel-like zinc finger DNA binding domain. Using both in vivo and in vitro methods, we demonstrate that the POZ domain of BCL6 is sufficient for its interaction with Tax 1. Using functional assays, we demonstrate that Tax 1 enhanced the repressive activity of BCL6 and increased the levels of apoptosis induced by BCL6 in osteosarcoma cells indicating that both proteins cooperate in vivo to cause a physiological affect. Furthermore, BCL6 recruited Tax 1 into punctate nuclear structures, which suggests that Tax 1 colocalizes with BCL6 in repressor complexes in vivo. BCL6 expression significantly downregulated both basal and Tax-induced nuclear factor-kappaB and long terminal repeat activation. This suggests that the expression of BCL6 in HTLV infected cells may contribute to the silencing of viral gene expression and to the long clinical latency associated with HTLV infection.

Molecular evidence for infection by HTLV-2 among individuals with negative serological screening tests for HTLV antibodies.

Previous serological studies on the Arara do Laranjal Indian group revealed extensive HTLV-2 infections. A collection of 97 new samples from the Arara were found repeatedly negative using three different commercial enzyme immunoassays. Eight samples that exhibited optical density readings close to the cut-off value were re-evaluated using Western blot (GeneLab 2.4, Singapore) assay. One sample was found to be non-reactive, five exhibited indeterminate patterns, one was classified as HTLV, and one was confirmed as HTLV-2. Peripheral blood mononuclear cell DNA of the eight samples were subjected to nested PCR and restriction fragment length polymorphism (RFLP) analysis of the pX and env regions, and nucleotide sequencing of the 5'-LTR region. All produced amplification products of pX, but env could be amplified in only one sample with the commonly used primers. RFLP analysis of the pX region using TaqI confirmed HTLV-2 infection. Nucleotide sequencing of the 5'-LTR region was performed in three samples (HTLV-2, HTLV and indeterminate based on Western blot pattern). Phylogenetic analysis of a 449-nt fragment using the Neighbour-Joining method clearly demonstrated that the three samples clustered within the HTLV-2c molecular subtype. The present study confirms the wide dissemination of the HTLV-2c subtype among linguistically and culturally distinct Amazonian Indian groups, and emphasizes the unique occurrence of infection by this subtype in Brazil. Moreover, it emphasizes the limitation of employing the present serological screening assays in blood banks, epidemiological studies, and the importance of molecular assays in the confirmatory procedures for the primary detection of HTLV-2 infections.

In vivo induction of human immunodeficiency virus type 1 entry into nucleus-free cells by CD4 gene transfer to hematopoietic stem cells: a hypothetical possible strategy for therapeutic intervention.

As a useful alternative to employing soluble CD4 to inhibit binding of human immunodeficiency virus type 1 (HIV-1) to target cells, the introduction of CD4-bearing erythrocyte has been proposed by two study groups (see Refs. (5,6)). Prominently, Nicolau and colleagues demonstrated that the electroinserted CD4 molecules in the membranes of erythrocytes are capable of mediating HIV-1 entry. The implications of the studies are that inactivation of the integration-dependent retrovirus by the facilitation of entry into the nucleus-free cells, referred to as 'fake host trap' or 'host cell decoy', may be a possible therapeutic approach. Here we expand this concept to include genetic modification of autologous hematopoietic stem cells and review the relevant theoretical basis. Effective application of molecular technologies to induce partial replacement of hematopoiesis may be critical for this strategy.

Antenatal hepatitis B screening - is there a need for a national policy?

Routine antenatal testing for hepatitis B carriage with maternal consent was introduced at the Rotunda in January 1998. The uptake of testing has been excellent; 99.98% of women presenting for antenatal care accepted hepatitis B (HBV) screening in the 30-months from January 1998 through June 2000. The prevalence of HBV carriage was 0.35% (58 pregnancies of 16,222 tested) increasing from 0.25% in 1998 (16 of 6227) to 0.45% in the first six months of 2000 (16 of 3484). Fifty-five women had 58 pregnancies (three women had two pregnancies). Two of these were e-antigen positive. HBV carrier status was previously unknown in 48 (87%). Two additional women had acute HBV infection in pregnancy. Forty-five infants have been born to mothers included in this screening programme. Audit of infant outcome reveals excellent compliance with immunisation and follow-up: 29 (64%) have completed the 3 dose HBV vaccination schedule to date. Thirteen infants (31%) are still attending; three are lost to follow-up including one whose family has emigrated. Routine antenatal screening for hepatitis B carriage is cost-effective and should be considered a standard of care in maternity practice.

The tripeptide glycyl-prolyl-glycine amide does not affect the early steps of the human immunodeficiency virus type 1 replication.

OBJECTIVE: To determine whether the peptide glycyl-prolyl-glycine amide (GPG-NH2) corresponding to a conserved motif in the tip of the third hypervariable region of gp120 affected the early events in the human immunodeficiency virus type 1 (HIV-1) replication. DESIGN/METHODS: Glycyl-prolyl-glycine amide was tested for its effect on HIV-1 adsorption, co-receptor usage, proviral DNA synthesis, messenger RNA (mRNA) synthesis and splicing, translation, tat/TAR transactivation, and virus protease activity. RESULTS: Glycyl-prolyl-glycine amide did not appear to affect the early events of the virus replication. HIV-1 having glycine-leucine-glycine instead of GPG in the V3 loop and the mutants deleted of the GPG motif were still inhibited by the peptide. Glycyl-prolyl-glycine-NH2 had no discernible effect on any of the other steps in the virus replication cycle tested. The only effect observed was an increased sodium dodecyl sulfate polyacrylamide amide gel electrophoresis mobility of gp160/120 at high concentrations of GPG-NH2. CONCLUSIONS: The tripeptide GPG-NH2 is a nontoxic compound that inhibits the replication of HIV-1 by an apparently new mode of action.

Molecular evidence of mother-to-child transmission of HTLV-IIc in the Kararao Village (Kayapo) in the Amazon region of Brazil.

Blood samples from native Indians in the Kararao village (Kayapo), were analysed using serological and molecular methods to characterize infection and analyse transmission of HTLV-II. Specific reactivity was observed in 3/26 individuals, of which two samples were from a mother and child. RFLP analysis of the pX and env regions confirmed HTLV-II infection. Nucleotide sequence of the 5' LTR segment and phylogenetic analysis showed a high similarity (98%) between the three samples and prototype HTLV-IIa (Mot), and confirmed the occurrence of the HTLV-IIc subtype. There was a high genetic similarity (99.9%) between the mother and child samples and the only difference was a deletion of two nucleotides (TC) in the mother sequence. Previous epidemiological studies among native Indians from Brazil have provided evidence of intrafamilial and vertical transmission of HTLV-IIc. The present study now provides molecular evidence of mother-to-child transmission of HTLV-IIc, a mechanism that is in large part responsible for the endemicity of HTLV in these relatively closed populations. Although the actual route of transmission is unknown, breast feeding would appear to be most likely.

Inactivation of p53 by HTLV type 1 and HTLV type 2 Tax trans-activators.

Human T cell lymphotropic virus type II (HTLV-2) was originally isolated from a patient with a hairy T cell leukemia. It has been associated with rare cases of CD8(+) T lymphoproliferative disorders, and has a controversial role as a pathogen. The loss of p53 function, as a consequence of mutation or inactivation, increases the chances of genetic damage. Indeed, the importance of p53 as a tumor suppressor is evident from the fact that over 60% of all human cancers have a mutant or inactive p53. p53 status has been extensively studied in HTLV-1-infected cell lines. Interestingly, despite the fact that p53 mutations have been found in only a minority of cells, the p53 functions were found to be impaired. We have analyzed the functional activity of the p53 tumor suppressor in cells transformed with HTLV-2 subtypes A and B. As with HTLV-1-infected cells, abundant levels of the p53 protein are detected in HTLV-2 virus-infected cell lines. Using p53 reporter plasmid or induction of p53-responsive genes in response to gamma-irradiation, the p53 was found to be transcriptionally inhibited in HTLV-2-infected cells. Interestingly, although Tax-2A and-2B inactivate p53, the Tax-2A protein appears to inhibit p53 function less efficiently than either Tax-1 or Tax-2B in T cells, but not in fibroblasts.

Spontaneous production of C-C chemokines by individuals infected with human T lymphotropic virus type II (HTLV-II) alone and HTLV-II/HIV-1 coinfected individuals.

To investigate the immunological features of human T lymphotropic virus type II (HTLV-II) infection and specific mechanisms whereby HTLV-II might influence the progression of HIV-1 disease in coinfected individuals, we have analyzed the production of the C-C chemokines RANTES and macrophage inflammatory proteins 1alpha and 1alpha (MIP-1alpha and MIP-1beta) by PBMCs from HTLV-II-infected and HTLV-II/HIV-1-coinfected individuals. We observed spontaneous production of significant levels of MIP-1alpha and -1beta and, to a lesser extent, RANTES, from individuals infected with HTLV-II alone or with concomitant HIV-1 infection. Spontaneous C-C chemokine production was not observed in PBMCs from uninfected or HIV-1-infected individuals. Although HTLV-II is known to preferentially infect CD8+ lymphocytes in vivo, we observed that whereas RANTES was produced exclusively by the CD8+-enriched fraction, MIP-1alpha and -1beta were produced by both the CD8+-enriched and CD8+-depleted fractions of HTLV-II-infected PBMCs. RT-PCR demonstrated active expression of the HTLV-II regulatory protein Tax in the infected CD8+ T lymphocyte population, and it was further shown that Tax transactivates the promoters of MIP-1beta and RANTES. Therefore, it appears that HTLV-II stimulates the production of C-C chemokines both directly at a transcriptional level via the viral transactivator Tax and also indirectly. Although the HTLV-II-infected individuals in this study are all virtually asymptomatic, they certainly display an abnormal immune phenotype. Moreover, our findings suggest that HTLV-II, via chemokine production, would be expected to alter the progression of HIV-1 infection in coinfected individuals.

Isolation, cloning, and complete nucleotide sequence of a phenotypically distinct Brazilian isolate of human T-lymphotropic virus type II (HTLV-II).

Analysis of human T-lymphotropic virus type II (HTLV-II) isolates from North America and Europe have demonstrated the existence of two molecular subtypes of the virus, HTLV-IIa and HTLV-IIb. Recently, studies on HTLV-II infections in Brazil have revealed isolates that are related phylogenetically to the HTLV-IIa subtype but have a HTLV-IIb phenotype with respect to the transactivating protein, tax. To more clearly define this relationship, HTLV-II was isolated from peripheral blood of an IVDA from Sao Paulo, Brazil (SP-WV), and the complete provirus was cloned and sequenced. Comparison of HTLV-II(SP-WV) nucleotide sequences to other available complete HTLV-II proviral sequences revealed that HTLV-II(SP-WV) is most closely related to HTLV-II(Mo), the prototypic HTLV-IIa subtype sequence. Phylogenetic analysis of LTR, env, and tax regions unequivocally demonstrated that HTLV-II(SP-WV) and all other Brazilian sequences examined are members of the IIa subtype. The predicted amino acid sequences of the major coding regions of HTLV-II(SP-WV) are also most closely related to HTLV-II(Mo), with the important exception of tax. The tax protein encoded by HTLV-II(SP-WV) is 96-99% identical to the tax of IIb isolates and is similar in that it has an additional 25 amino acids at the carboxy-terminus compared to the HTLV-II(Mo) tax with which it shares 91% identity. Analysis of tax stop codon usage of a number of HTLV-IIa isolates from North American, Europe, and Brazil demonstrated that isolates from the last region appear to be unique in their extended tax phenotype. It could be demonstrated that the extended tax proteins in the HTLV-IIb and Brazilian isolates had equivalent ability to transactivate the viral LTR, and studies with deletion mutants indicated that the extended C-terminus is not essential for transactivation. In contrast, the HTLV-IIa tax was found to have a greatly diminished ability to transactivate the viral LTR, which appeared to be a consequence of reduced expression of the protein. The studies show that although the Brazilian strains do not represent an entirely new subtype based on nucleotide sequence analysis they are a phenotypically unique molecular variant within the HTLV-IIa subtype.

Transcriptional activation of JC virus by human T-lymphotropic virus type I Tax protein in human neuronal cell lines.

Polyomavirus JC (JCV) causes the human demyelinating disease, progressive multifocal leukoencephalopathy (PML). The recent demonstration of cases of PML in association with human T-lymphotropic virus type I (HTLV-I) infection prompted us to examine whether the HTLV-I-encoded regulatory protein Tax activates JCV transcription. By employing a dual luciferase assay, we initially found that the expression of Tax activated the transcriptional potential of both early and late promoters of JCV in human neuronal but not in non-neuronal cells. We subsequently analyzed the mechanism of Tax-induced activation of the JCV promoter in neuronal cells with the following results: 1) the JCV promoter that lacks the NF-kappaB-binding motif could not be activated by Tax; 2) the overexpression of IkappaBalpha abolished Tax-induced transcriptional activation of the JCV promoter; 3) a Tax mutant (M22) lacking the potential for activation via the NF-kappaB pathway did not activate the JCV promoter. Furthermore, Tax enhances the gene expression of JCV T antigen and VP1. We examined mechanisms of the cell-specific activation of the JCV promoter by Tax. Electrophoretic mobility shift assay demonstrated the presence of Tax-bound protein(s) that were specifically present in non-neuronal cells. This study is the first demonstration of the activation of JCV promoter by HTLV-I Tax in an NF-kappaB-dependent manner.

Endoscopic harvest of the rectus abdominis muscle.

The rectus abdominis muscle is a versatile muscle with many applications. The use of this muscle is often limited by its considerable donor site morbidity. This study reports a minimally invasive technique to harvest the rectus abdominis muscle. The described technique has been used successfully in 5 patients who required a superiorly based flap for reconstruction of a sternal defect. All patients have had long-term flap survival and resolution of their sternal osteomyelitis. Although initially lengthy, harvest times have been less than 1 hour for the last 3 patients. Patients report minimal discomfort at their operative site. To date there have been no hernias or other complications. The rectus abdominis muscle can be harvested successfully endoscopically. With no other modification other than port site placement, this technique could be used to harvest free flaps or harvest inferiorly based rectus flaps. This technique is learned easily, is safe, and should reduce substantially the donor site morbidity associated with more traditional harvesting techniques.

Microvascular anastomosis using 2-octyl cyanoacrylate in the rat femoral artery.

Patency of the microvascular anastomosis is the most important requirement for tissue survival in free tissue transfer and in replantation. In efforts to improve on the standard suture method of microvascular anastomosis, new techniques such as limited-suture sleeve anastomoses and histoacryl glue anastomoses have been employed experimentally. However, as a result of factors such as tissue toxicity and suboptimal outcome, cyanoacrylates have not enjoyed clinical use. In addition, sleeve anastomoses continue to utilize suture, increasing the risks of intimal damage, platelet adhesion, and thrombosis. In an attempt to surmount these problems, the authors investigated the use of a new 2-octyl cyanoacrylate glue and a sutureless sleeve anastomosis. Anastomosis of 20 rat femoral arteries with a sutureless sleeve technique bonded with glue resulted in an 80% patency rate at 1 day to 3 weeks. Failures occurred in the first few attempts as the technique was evolving. These encouraging results suggest that 2-octyl cyanoacrylate may have applicability in quick, sutureless microvascular anastomoses.

Different population dynamics of human T cell lymphotropic virus type II in intravenous drug users compared with endemically infected tribes.

The phylogeny of human T cell lymphotropic virus type II (HTLV-II) was investigated by using strains isolated from Amerindian and Pygmy tribes, in which the virus is maintained primarily through mother-to-child transmission via breast-feeding, and strains from intravenous drug users (IDUs), in which spread is mainly blood-borne via needle sharing. Molecular clock analysis showed that HTLV-II has two different evolutionary rates with the molecular clock for the virus in IDUs ticking 150-350 times faster than the one in endemically infected tribes: 2.7 x 10(-4) compared with 1.71/7.31 x 10(-7) nucleotide substitutions per site per year in the long terminal repeat region. This dramatic acceleration of the evolutionary rate seems to be related with the mode of transmission. Mathematical models showed the correlation of these two molecular clocks with an endemic spread of HTLV-II in infected tribes compared with the epidemic spread in IDUs. We also noted a sharp increase in the population size of the virus among IDUs during the last decades probably caused by the worldwide increase in intravenous drug use.

High rate of human T lymphotropic virus type IIa infection in HIV type 1-infected intravenous drug abusers in Ireland.

Serological and molecular analyses of a cohort of HIV-1-infected intravenous drug abusers (IVDAs) (n = 103) in Dublin, Ireland have demonstrated that 15 of 103 (14.6%) were infected with HTLV-II, which is the highest infection rate yet recorded for any European country. Restriction fragment length polymorphism (RFLP) analysis of the env region of the provirus demonstrated that the infection involved only the HTLV-IIa subtype; the HTLV-IIb subtype was not detected. Phylogenetic analysis of the nucleotide sequences of the long terminal repeat (LTR) confirmed infection with the HTLV-IIa subtype, and demonstrated that the viruses clustered closely with HTLV-IIa isolates from North American IVDAs. Previous observations that IVDAs in southern Europe, specifically Spain and Italy, appear to be infected predominantly with the HTLV-IIb subtype, along with the present report and evidence that IVDAs in Sweden are infected with the HTLV-IIa subtype, suggest different origins of HTLV-II infection in Europe.

Correction of areolar depression in postsurgically treated gynecomastic patients.

Nipple-areolar depression after resection for gynecomastia can be avoided by preserving an ample amount of breast tissue and fat under the areola. If a marked depression of the areola occurs, correction can be difficult. Each patient requires an individual assessment, but the principle of rotation of soft tissue under the areolar complex is usually necessary. An effective way to accomplish this is by deepithelialization of a semilunar area inferior to the original subareolar scar, freeing it at its caudal boarder and advancing this deepithelialized flap under the areola and repairing the wound. The technique is simple in design and may be a useful tool in dealing with this difficult problem.

Clonal expansion within CD4+ and CD8+ T cell subsets in human T lymphotropic virus type I-infected individuals.

To investigate the diversity of the T cell repertoire involved in human T lymphotropic virus type I (HTLV-I) infections, peripheral blood T cell subsets were analyzed by using a PCR-based assay that permits determination of complementarity-determining region 3 (CDR3) length variation in TCR Vbeta transcripts. In two of four asymptomatic HTLV-I carriers and in four of five patients with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), mono- or oligoclonal expansions were detected in the CD4+ T cell subset. In one patient with adult T cell leukemia, a specific clone bearing Vbeta7 was detected in the CD4+ T cell subset. In contrast, clonal expansion was not observed in the CD4 T cell subsets of three individuals with asymptomatic HTLV-II infection or in our previous studies of a large number of uninfected individuals. Oligoclonal expansions in the CD8+ T cell subset were detected in all subjects, including the patient with adult T cell leukemia. No differences in the number of expanded clones were noted between asymptomatic carriers and in patients with HAM/TSP and there was no obvious restriction in the TCR V region usage. Direct sequencing revealed no significant bias in the CDR3 motifs utilized by the predominant clones. This report is the first direct demonstration of clonal expansions within fractionated T cell subsets (CD4+ and CD8+) in HTLV-I infections and suggests that 1) clonal expansion of CD4+ T lymphocytes likely occurs as a direct result of infection and 2) polyclonal CD8+ T cell expansion occurs frequently and independently of disease association.

Serological evidence of HTLV-I and HTLV-II coinfections in HIV-1 positive patients in Belém, State of Pará, Brazil

The occurence of HTLV-I/II and HIV-1 coinfections have been shown to be frequent, probably in consequence of their similar modes of transmission. This paper presents the prevalence of coinfection of HTLV among HIV-1 infected and AIDS patients in Belém, State of Pará, Brazil. A group of 149 patients attending the AIDS Reference Unit of the State Department of Health was tested for the presence of antibodies to HTLV-I/II using an enzyme immunoassay and the positive reactions were confirmed with a Western blot that discriminates between HTLV-I and HTLV-II infections. Four patients (2.7 per cent) were positive to HTLV-I, seven (4.7 per cent) to HTLV-II and one (0.7 per cent) showed an indeterminate pattern of reation. The present results show for the first time in Belém not only the occurrence of HTLV-II/HIV-1 coinfections, but also a higher prevalence of HTLV-II in relation to HTLV-I. Furthermore. it also enlarges the geographical limits of the endemic area for HTLV-II in the Amazon region of Brazil.

Negative-pressure dressings as a bolster for skin grafts.

Contoured wounds needing closure with skin grafts are often located in complex anatomic regions or are in unusual positions, which make conventional skin graft stabilization techniques cumbersome and ineffective. Often after 72 hours, a skin graft covered with a bolstered dressing has poor take secondary to shear stresses, as well as hematoma formation or serum collection, negating the effectiveness of the stabilizing dressing. The Food and Drug Administration has recently approved vacuum-assisted closure (V.A.C.), an innovative technique using negative pressure, for closure of chronic wounds. This reportedly leads to enhanced granulation tissue formation and consequently more rapid reepithelialization of wounds compared with conventional packing with saline-moistened gauze. Experimental studies have demonstrated increased oxygen tension, decreased bacterial counts, and increased granulation formation occurring under negative-pressure systems. Extending the use of this concept, we have coupled skin grafting with negative-pressure dressings for closure of large, complex open wounds. Our results indicate greater than 95% graft take in all patients in this study. This technique is extremely efficacious, with increased graft take due to total immobilization of the graft, thereby limiting shear forces, elimination of fluid collections, bridging of the graft, and decreased bacterial contamination. Moreover we have noted decreased edema in rotated muscle flaps, improved contour conformity, and shortened hospitalizations.