Altered apoptosis and proliferation in milk cells and PBMc from BLV-infected bovines with different proviral loads: Possible role of the BCL-2 family proteins, TNF-alpha, and receptors.

Bovines infected by bovine leukemia virus (BLV) are characterized by presenting low proviral load (LPL) or high proviral load (HPL). It is reported that animals with HPL in peripheral blood mononuclear cells (PBMCs) present a decrease in apoptosis, an increase in viability and the proliferation rate, while animals that maintain an LPL have an intrinsic ability to control the infection, presenting an increased apoptosis rate of their PBMCs. However, there is little information on the effect of BLV on these mechanisms when the virus infects somatic milk cells (SC). This study investigates the mechanisms underlying apoptosis in milk and blood from BLV-infected animals with HPL and LPL. Relative levels of mRNA of tumor necrosis factor-α (TNF-α), TNF receptor 1 (TNF-RI), TNF receptor 2 (TNF-RII), anti-apoptotic B-cell lymphoma 2 protein (Bcl-2), and pro-apoptotic Bcl-2-like protein 4 (Bax) were measured in SC and PBMCs using quantitative reverse transcription-polymerase chain reaction (RT-qPCR) assay. A significant decrease in the expression of TNF-α in SC from HPL animals vs non-infected bovines was observed, but the infection in SC with BLV did not show a modulation on the expression of TNF receptors. A significant increase in TNF-RI expression in PBMCs from HPL bovines compared to LPL bovines was observed. No significant differences in PBMCs between HPL and LPL compared to non-infected animals concerning TNF-α, TNF-RI, and TNF-RII expression were found. There was a significant increase of both Bcl-2 and Bax in SC from LPL compared to non-infected bovines, but the Bcl-2/Bax ratio showed an anti-apoptotic profile in LPL and HPL bovines compared to non-infected ones. Reduced mRNA expression levels of Bax were determined in the PBMCs from HPL compared to LPL subjects. In contrast, BLV-infected bovines did not differ significantly in the mRNA expression of Bax compared to non-infected bovines. Our data suggest that the increased mRNA expression of Bax corresponds to the late lactation state of bovine evaluated and the exacerbated increase of mRNA expression of Bcl-2 may be one of the mechanisms for the negative apoptosis regulation in the mammary gland induced by BLV infection. These results provide new insights into the mechanism of mammary cell death in HPL and LPL BLV-infected bovine mammary gland cells during lactation.

Modulation of cathelicidins, IFNß and TNFα by bovine alpha-herpesviruses is dependent on the stage of the infectious cycle.

Production of antimicrobial peptides cathelicidins, interferons and cytokines is an important feature in airway epithelial host defense. The innate immune response to alpha-herpesvirus infection at the sites of primary replication has not been fully studied. Thus, the aim of this study was to determine the expression of innate immune components, cathelicidins, IFNß, TNFα and TNF receptors (TNFRI and TNFRII) during acute infection and reactivation of bovine herpesvirus type 1 (BoHV-1) and 5 (BoHV-5) in the respiratory tract and lymphoid tissue of their natural host. We found that BoHV infection modulates mainly the expression of BMAP28, a key cathelicidin in cattle. It was downregulated by both viruses in retropharyngeal lymph nodes of acutely infected-calves, and it was accompanied by a lower expression of IFNß, TNFα and TNFRI. BoHV-5 showed a pronounced role in the downregulation of BMAP28, even in nasal mucosa and lung. However, during reactivation, BoHV-5 upregulated both BMAP28 and IFNß in retropharyngeal lymph nodes. Acute replication induced also TNFα mRNA and protein synthesis, and expression of TNFRI and II was positively regulated during both acute infection and reactivation, particularly in the trachea. Moreover, BMAP27 was detected during BoHV-1 reactivation suggesting a potential role at this stage. Thus, cathelicidins are implicated in alpha-herpesvirus infections of the bovine respiratory system and the response is distinct during BoHV-1 and BoHV-5 acute infection and reactivation. This demonstrates that these viruses modulate differentially the components of innate immune response, possibly influencing their pathogenesis. This study provides an initial pilot analysis of factors that might be implicated in alpha-herpesvirus infection of the bovine respiratory system.

Antibody response against three widespread bovine viruses is not impaired in Holstein cattle carrying bovine leukocyte antigen DRB3.2 alleles associated with bovine leukemia virus resistance.

Due to the wide dissemination of bovine leukemia virus (BLV) infection among dairy cattle, control and eradication programs based on serological detection of infected cattle and subsequent culling face a major economic task. In Argentina, genetic selection of cattle carrying alleles of the bovine leukocyte antigen (BoLA) DRB3.2 gene associated with BLV-infection resistance, like *0902, emerges as the best additional tool toward controlling virus spread. A potential risk in expanding or segregating BoLA selected populations of cattle is that it might increase susceptibility to other common viruses. Special concern raises the strong association found between low proviral load and low antibody titer against major BLV structural proteins. This phenomenon might depend on host genetic factors influencing other viruses requiring, unlike BLV, strong and long-lasting humoral immune response to prevent infection. In this study, we demonstrate that there is no association among neutralizing antibody titers against foot and mouth disease virus, bovine viral diarrhea virus, or bovine herpesvirus type 1 and polymorphism of the BoLA DRB3.2 gene. Conversely, there is strong association between BoLA DRB3.2*0902 and low antibody titers against 2 BLV structural proteins--env gp51 and gag p24--to date, the best BLV resistance marker. There is also significant association between low antibody titers against gp51 and p24 and BoLA DRB3.2*1701 and low antibody titers against p24 and BoLA DRB3.2*1101 or 02. Our data suggest that increasing BoLA-selected BLV-resistant cattle or segregating BoLA-associated alleles to BLV susceptibility would not affect the resistance or the predisposition to bovine viral diarrhea virus, bovine herpesvirus type 1, or foot and mouth disease virus infection.

[Prevalence of human immunodeficiency virus (HIV)-Trypanosoma cruzi co-infection and injectable-drugs abuse in a Buenos Aires health center]. / Prevalencia de la coinfección virus de la inmunodeficiencia humana (VIH)-Trypanosoma cruzi e impacto del abuso de drogas inyectables en un centro de salud de la ciudad de buenos aires.

The aim of this study was to evaluate the prevalence of human immunodeficiency virus (HIV)-Trypanosoma cruzi co-infection in a Buenos Aires health center. A retrospective analysis of the clinical charts of 602 HIV-infected patients was performed. Only 51.3% of the patients were evaluated against T. cruzi. The global co-infection prevalence was 4.2%, being more frequent among injectable drug users (IDU) (8.9% vs. 2.6%, < 0.05). The indication of T. cruzi testing should be stressed for HIV-infected patients, especially in those centers where IDU are assisted.

Prevalencia de la coinfección virus de la inmunodeficiencia humana (VIH)- Trypanosoma cruzi e impacto del abuso de drogas inyectables en un centro de salud de la ciudad de Buenos Aires / Prevalence of human immunodeficiency virus (HIV)- Trypanosoma cruzi co-infection and injectable-drugs abuse in a Buenos Aires health center

Se evaluó la prevalencia de coinfección virus de la inmunodeficiencia humana (VIH)- Trypanosoma cruzi ( T. cruzi) en pacientes atendidos en un centro asistencial de Buenos Aires, Argentina. Se realizó un análisis retrospectivo de las historias clínicas de 602 individuos VIH positivos. Sólo en el 51,3% de estos pacientes se había investigado la presencia de T. cruzi. La prevalencia global de coinfección fue del 4,2%, siendo más elevada en usuarios de drogas inyectables (UDI) (8,9% vs. 2,6%, p<0,05). Sobre la base de estos resultados, concluimos que debería enfatizarse el cumplimiento de la indicación de diagnóstico para la enfermedad de Chagas en pacientes VIH positivos, especialmente en UDI.

The aim of this study was to evaluate the prevalence of human immunodeficiency virus (HIV)- Trypanosoma cruzi co-infection in a Buenos Aires health center. A retrospective analysis of the clinical charts of 602 HIV-infected patients was performed. Only 51.3% of the patients were evaluated against T. cruzi. The global co-infection prevalence was 4.2%, being more frequent among injectable drug users (IDU) (8.9% vs. 2.6%, p<0.05). The indication of T. cruzi testing should be stressed for HIV-infected patients, especially in those centers where IDU are assisted.

Association of BLV infection profiles with alleles of the BoLA-DRB3.2 gene.

Bovine leukaemia virus (BLV) causes lymphosarcoma and persistent lymphocytosis (PL). Some MHC class II gene polymorphisms have been associated with resistance and susceptibility to the development of lymphosarcoma and PL, as well as with a reduced number of circulating BLV-infected lymphocytes. Previously, 230 BLV-infected Holstein cattle were classified into two infection profiles characterized by low and high proviral loads (LPL and HPL respectively). Here, the influence of the polymorphism at the BoLA-DRB3.2* gene of these animals was examined. After genotyping, the association between the BoLA-DRB3.2* alleles and the BLV infection profile was determined as the odds ratio (OR). Two subtypes of allele *11 were identified (ISAG*0901 and *0902). Allele ISAG*0902 showed a stronger association with the LPL profile (OR = 8.24; P < 0.0001) than allele *11 itself (OR = 5.82; P < 0.0001). Allele ISAG*1701 (*12) also showed significant association with the LPL profile (OR = 3.46; P < 0.0055). Only one allele, ISAG*1501 or 03 (*16), showed significant association with HPL (OR = 0.36; P < 0.0005). The DRB3.2* alleles were assigned to three categories: resistant (R), susceptible (S) and neutral (N). Based on their DRB3 genotypes, cattle were classified as homozygous or heterozygous. The RR and RN genotypes were associated with the LPL profile, while the SS and NS genotypes were associated with the HPL profile. The RS genotype could not be associated with any particular profile. Our results show that allele ISAG*0902 appears to be the best BLV resistance marker in Holstein cattle.

Two immunomodulators, curcumin and sulfasalazine, enhance IDV antiretroviral activity in HIV-1 persistently infected cells.

Since the appearance of resistance to antiretroviral treatment is unavoidable, the host cell's transcription factor NF-kappaB is a novel HIV target. The goal of this study was to characterize the effect of two immunomodulators, curcumin (Cur) and sulfasalazine (Sul), with a protease inhibitor, indinavir (IDV), on HIV-1 persistently infected CD4+ T-cells. Viral p24 antigen production, viral infectivity (tested on MAGI cells) and viral relative infectivity (viral infectivity/p24) were analysed. When used alone, both immunomodulators were able to reduce viral infectivity. When in combination, both 10 microM IDV plus 10 microM Cur and 10 microM IDV plus 250 microM Sul showed a significant reduction in viral infectivity and viral relative infectivity when compared to the reduction produced by IDV alone. Thus, the use of immunomodulators with IDV could help to reduce HIV-1 production in persistently infected cells.

Characterization of the main placental cytokine profiles from HIV-1-infected pregnant women treated with anti-retroviral drugs in France.

Cytokines are involved in regulating HIV-1 infection. They are also placental environment major components. We assessed the potential impact of HIV-1 infection and/or anti-retroviral drugs on the placental cytokine profiles that may be involved in controlling HIV-1 placental dissemination. Placental explants were obtained after elective caesarean section from anti-retroviral-treated HIV-1-infected pregnant women and from HIV-1 non-infected pregnant women. The main placental cytokines were assessed for protein secretion in the supernatants of 24-h placental culture explants and/or in uncultured placental explants for mRNA expression levels. The cytokine profiles were different between the HIV-1-infected and the non-infected groups. Higher medians of leukaemia inhibiting factor (LIF), tumour necrosis factor (TNF)-alpha and interleukin (IL)-8 secretion were found in the 24-h culture supernatant of term placenta from HIV-1-infected women. High median levels of IL-16 and regulated upon activation normal T cell expressed and secreted (RANTES) levels were found in both groups. The mRNA expression medians were lower for TNF-alpha and IL-8 and higher for stromal cell-derived factor-1 (SDF-1) in uncultured placental explants from HIV-1-infected women. In the HIV-1-infected group, but not in the non-infected group, the secretion levels of TNF-alpha and IL-8, as well as their mRNA expression levels, were highly positively correlated; furthermore, their secretion levels were correlated positively with LIF and IL-10 secretion levels. We found no correlation between the cytokine levels and the immunovirological status of the HIV-1-infected mothers or the type or duration of treatment. These results highlight the potential impact of HIV-1 and of the anti-retroviral treatments on the placental cytokines pattern, independently of their anti-viral activity.

Down modulation of TNF-alpha mRNA placental expression by AZT used for the prevention of HIV-1 mother-to-child transmission.

Mechanisms of HIV-1 in utero mother-to-child transmission (MTCT) protection provided by AZT are not completely understood. The placental cytokine network is involved in the control of HIV-1 in utero transmission but the effect of AZT on this network is unknown. To evaluate the effects of AZT on placental cytokine expression, the chorionic villi from HIV-1 uninfected women term placentae were cultured with 0, 100, and 2,000 ng/ml AZT. Tissue fragments were harvested at days 1, 4, and 7 to determine the level of cytokine mRNA by real-time RT-PCR. The viability and morphology of the placental histocultures were monitored by the expression of beta-human chorionic gonadotropin (beta-hCG) gene, lipopolysaccharide (LPS) activation, and microscopic examination. AZT at 2,000 ng/ml significantly down-regulated TNF-alpha mRNA expression at day 1 and day 4, but had no effect on beta-hCG, stromal cell-derived factor 1 (SDF-1), and IL-10 gene expression. AZT did not induce any deleterious impact on placental tissue structure. Furthermore, activation of chorionic villi by LPS for 24 h up-regulated IL-10 and TNF-alpha mRNA expression. Down-regulation of TNF-alpha mRNA could represent a mechanism through which AZT can decrease the risk of HIV-1 MTCT, in addition to its direct effect on HIV-1 replication.

Evaluation of the placental environment with a new in vitro model of histocultures of early and term placentae: determination of cytokine and chemokine expression profiles.

We aimed to set up and validate a new in vitro model of placental histocultures, for the evaluation of cytokine and chemokine profiles of the placental environment, over a long culture period. Micro-explant cultures from 6 early and 6 term placentae were set up on collagen sponge gel supports at a liquid/air interface. At various times during culture, we analyzed tissue morphology and cell death by microscopy and quantified beta-hCG production and mRNA levels for beta-hCG and insulin-like 4 (INSL4). Levels of IL-6, LIF, TNF alpha, IL-10, IFN-gamma, IL-16 and RANTES in the medium were measured by ELISA on days 1, 4 and 7 of culture. SDF-1 mRNA expression was determined by real-time PCR at the same time points. Histocultures from early and term placentae remained viable until day 10. High levels of IL-6 and LIF production, low levels of TNF alpha, IL-10 and IFN-gamma production and significant SDF-1 expression were observed. These data indicate that placental histoculture is a suitable and reliable in vitro model for studying the placental environment.

Cell-free HIV type 1 infection is restricted in the human trophoblast choriocarcinoma BeWo cell line, even with expression of CD4, CXCR4 and CCR5.

The restriction of cell-free HIV-1 infection has been demonstrated in placental trophoblast choriocarcinoma BeWo cells. We tried to determine the level of the viral replication cycle at which this restriction occurs. BeWo cells produce infectious viruses after transfection with HIV-1 plasmids, independently of viral tropism. CCR5 and CXCR4, but not the CD4 molecule, were detected at the cell surface. We therefore derived CD4-expressing clones from transfected BeWo cells. Cell-free virus infection of these clones resulted in neither virus production nor viral sequence integration, indicating that the restriction occurs before integration of the virus. If we used luciferase reporter viruses pseudotyped with HIV-1 Env R5 and X4 for infection, no luciferase activity was detected, even in the BeWo-CD4+ clone, in contrast to what was observed in VSV-G pseudotyped virus infection. Our results show that infection of trophoblast-derived cells with cell-free virus is at least restricted at the level of entry. Thus, BeWo is an interesting human placental cell line that is resistant to HIV-1, even if CD4, CXCR4, and CCR5 are expressed.

Development and evaluation of a highly sensitive and specific blocking enzyme-linked immunosorbent assay and polymerase chain reaction assay for diagnosis of bovine leukemia virus infection in cattle.

OBJECTIVE: To develop a blocking ELISA for detection of bovine leukemia virus (BLV) antibodies that is comparable to a radioimmunoprecipitation (RIP) assay, to evaluate use of this ELISA for identification of BLV-infected herds, and to develop a polymerase chain reaction (PCR) assay for direct diagnosis of infection with BLV. SAMPLE POPULATION: Serum samples and pooled bulk-tank milk samples from cattle. PROCEDURE: The blocking ELISA was developed, using BLV gp51 as antigen, captured by a selected bovine polyclonal serum. A nested PCR was conducted with primers specific for a segment of the pol region of the BLV genome. RESULTS: Sensitivity and specificity of the ELISA were comparable to those of the RIP assay. Use of the ELISA on pooled milk samples allowed identification of herds in which prevalence of BLV infection among lactating cows was as low as 2.5%. Pooled milk samples from BLV-free herds did not react in the ELISA. All cattle that had positive results for the nested PCR had BLV antibodies, but cattle with consistantly low antibody titers required examination of sequential DNA samples to detect viral sequences. None of the 63 antibody-negative cattle had positive results for the PCR. CONCLUSIONS AND CLINICAL RELEVANCE: This ELISA is a highly specific and sensitive assay for the detection of BLV antibodies in serum and milk samples of cattle. Examination of pooled milk samples with the ELISA provides a reliable, practical, and economic procedure for identification of BLV-infected herds. The nested PCR also constitutes a specific procedure for direct diagnosis of infection with BLV.

The complete genomic sequence of a BLV strain from a Holstein cow from Argentina.

DNA was extracted from the peripheral blood of a seropositive, PCR-positive, BLV-infected Holstein cow (No. 38) from Argentina. The DNA was amplified via PCR with a series of overlapping primers encompassing the entire BLV proviral DNA. The amplified BLV ARG 38 DNA was cloned, sequenced, and compared phylogenetically to three other full-length BLV sequences. Characterization of its deduced proteins and its relationship to other members of the PTLV/BLV genus of retroviruses are discussed.

Evidence of the existence of a new circulating recombinant form of HIV type 1 subtype A/J in Cameroon. The European Network on the Study of In Utero Transmission of HIV-1.

Several genetic subtypes and circulating recombinant forms (CRFs) of HIV-1 have been identified. The greatest degree of genetic diversity is displayed by variants from Central and West Africa. HIV-1 env C2-V5 and protease sequences were obtained from 15 HIV-1-infected pregnant women, who were selected from a larger cohort study in Yaoundé, Cameroon. Fourteen of 15 virus variants were shown to be recombinant, whereas a single variant appeared to be nonrecombinant subtype A. Five viruses were subtype A/J recombinants, with env genes derived from subtype A and protease genes derived from subtype J. Seven viruses clustered with reference sequences for CRF02 AG(IbNG) in both the env and protease gene fragments, and were thus subtype A/G recombinants. Two variants displayed even more complex recombination patterns. Phylogenetic analyses indicated that the five subtype A/J recombinants might be the first representatives of a previously unrecognized CRF.

Coreceptor usage of HIV-1 isolates representing different genetic subtypes obtained from pregnant Cameroonian women. European Network for In Utero Transmission of HIV-1.

In this study, coreceptor usage of HIV-1 other than subtype B in relation to HIV-1 transmission from mother to child was investigated. Repeated sampling of 42 HIV-1-seropositive, asymptomatic women in Cameroon during the second and third trimesters of pregnancy, at delivery, and 6 months postpartum were performed. Env subtyping was carried out from uncultured peripheral blood mononuclear cells (PBMCs) by heteroduplex mobility assay and, whenever necessary, by DNA sequencing. Virus isolates were tested for coreceptor usage on human cell lines-U87.CD4 and GHOST(3)-engineered to express stably CD4 and the chemokine receptors CCR1, CCR2b, CCR3, CCR5, or CXCR4, or the orphan receptors BOB/gpr15 or Bonzo/STRL33/TYMSTR. Transmission rate was 11.9%. Viruses were predominantly envelope subtype A and used CCR5 as coreceptor and, surprisingly, 4 of 28 (14.2%) isolates from mothers and 1 of 3 isolates from children used the orphan receptor Bonzo as well. In 2 transmitting mothers from whom sequential HIV-1 isolates were available, viral coreceptor usage evolved from CCR5 monotropic to CCR5/Bonzo dual tropic during pregnancy, and in 1 case transmission of this virus could be documented. Our data suggest that evolution of HIV-1 coreceptor usage to dual (or multi-) tropism may occur during pregnancy.

[Closure of patent ductus arteriosus in thoracoscopy: analysis of the experience in the Gaslini Institute of Genoa]. / Chiusura del dotto arterioso persistente in toracoscopia: analisi dell'esperienza dell'istituto Gaslini di Genova.

In 1993, Laborde et al described a new surgical approach for closure of the patent ductus arteriosus (PDA), using a videoassisted technique. For the first time, videosurgery was utilized in pediatric cardiovascular surgery. The advantages of this new method are that it is minimally invasive (thoracotomy is replaced by three small holes), it reduces postoperative pain, recovery is more rapid with a shorter hospital stay and convalescence, and last but not least, it offers better aesthetic results. Between July 1994 and June 1996, we utilized the videoassisted thoracoscopic technique described by Laborde in 23 patients with PDA (age: 8 m-12 yrs, mean 45.2 m +/- 37.8; weight: 5.3-32 kg, mean 23 kg +/- 10.3). In two patients, we converted the procedure to standard thoracotomy due to anatomic problems. Echo-doppler evaluation, performed in the operating room shortly after the procedure, confirmed the complete closure of the PDA in all patients. There was neither morbidity nor mortality. The average hospital stay was four days. At a mean follow-up interval of 7.2 months (range 1-13 months), all patients showed excellent cosmetic results. We conclude that thoracoscopic closure of PDA is a valuable therapeutic option that can be used as an alternative to open-chest surgery and interventional cardiology.

[Bovine leukemia virus (BLV): prevalence in the Cuenca Lechera Mar y Sierras from 1994 to 1995]. / Virus de la leucosis bovina (BLV): prevalencia en la Cuenca Lechera Mar y Sierras entre 1994 y 1995.

The Cuenca Lechera Mar y Sierras (CLMS) includes about 300 dairy farms located in the counties of Tandil, Balcarce, Juarez, Ayacucho, General Pueyrredón, Gonzalez Chavez and Necochea, in the province of Buenos Aires. The purpose of this investigation was to determine the prevalence of infection caused by Bovine Leukemia Virus (BLV) in the CLMS. We investigated the presence of anti-BLV antibodies in 4,203 milk samples taken from 73 dairy farms belonging to the CLMS. An indirect ELISA, which is described and evaluated in this paper, was used to test the antibodies in milk. We classified the dairy farms according to their rate of infection. The percentage of dairy farms free of infection resulted in 31.50. On the other hand, 49.40% of the dairy farms showed a figure between 1% and 15% of infected cattle; 17.80% between 16% and 30%, and the remaining 1.30% turned out more than 30% of infected cattle. If compared with data obtained in the 1979-1981 period, which showed that 95.65% of the dairy farms was BLV-free, it is clear that a dramatic progress of the BLV infection has occurred for the last 15 years. Nevertheless, the CLMS is in a privileged position so as to incorporate an inexpensive control plan to eradicate the BLV infection, as almost 1/3 of its dairy farms is still BLV-free and 49.40% still has a low rate of BLV infection. Only about 20% of the dairy farms would require costly strategies of control.