Antibodies targeting human endothelin-1 receptors reveal different conformational states in cancer cells.

The endothelin axis (endothelins and their receptors) is strongly involved in physiological and pathological processes. ET-1 plays a crucial role in particular in tumor diseases. Endothelin-1 receptors (ET(A) and ET(B)) are deregulated and overexpressed in several tumors such as melanoma and glioma. We studied the binding of 24 monoclonal antibodies directed against human ET(B) receptors (hET(B)) to different melanoma cell lines. Few of these mAbs bound to all the melanoma cell lines. One of them, rendomab B49, bound to ET(B) receptors expressed at the surface of human glioma stem cells. More recently, we produced new antibodies directed against human ET(A) receptor (hET(A)). Several antibodies have been isolated and have been screened on different tumoral cells lines. As for the mAbs directed against the hET(B) receptor only some of new antibodies directed against ET(A) receptor are capable to bind the human tumoral cell lines. Rendomab A63 directed against hET(A) is one of them. We report the specificity and binding properties of these mAbs and consider their potential use in diagnosis by an in vivo imaging approach.

Cognitive deficits and increases in creatine precursors in a brain-specific knockout of the creatine transporter gene Slc6a8.

Creatine transporter (CrT; SLC6A8) deficiency (CTD) is an X-linked disorder characterized by severe cognitive deficits, impairments in language and an absence of brain creatine (Cr). In a previous study, we generated floxed Slc6a8 (Slc6a8 flox ) mice to create ubiquitous Slc6a8 knockout (Slc6a8-/y ) mice. Slc6a8-/y mice lacked whole body Cr and exhibited cognitive deficits. While Slc6a8-/y mice have a similar biochemical phenotype to CTD patients, they also showed a reduction in size and reductions in swim speed that may have contributed to the observed deficits. To address this, we created brain-specific Slc6a8 knockout (bKO) mice by crossing Slc6a8flox mice with Nestin-cre mice. bKO mice had reduced cerebral Cr levels while maintaining normal Cr levels in peripheral tissue. Interestingly, brain concentrations of the Cr synthesis precursor guanidinoacetic acid were increased in bKO mice. bKO mice had longer latencies and path lengths in the Morris water maze, without reductions in swim speed. In accordance with data from Slc6a8 -/y mice, bKO mice showed deficits in novel object recognition as well as contextual and cued fear conditioning. bKO mice were also hyperactive, in contrast with data from the Slc6a8 -/y mice. The results show that the loss of cerebral Cr is responsible for the learning and memory deficits seen in ubiquitous Slc6a8-/y mice.

Increased expression of MDR1 mRNAs and P-glycoprotein in placentas from HIV-1 infected women.

P-glycoprotein transports several compounds including protease inhibitors, actually used in the clinical management of HIV-1 infection. Since P-glycoprotein is expressed in placental trophoblasts, its efflux activity could interfere with placental transfer of antiretrovirals. The purpose of this study was to investigate the expression of P-gp-encoding MDR1 gene and P-gp itself in full-term placentas from uninfected (n=35) and HIV-1 infected women (n=24). MDR1 transcripts were quantified by real-time PCR using relative (MDR1 normalized upon 28S levels) and absolute (copy number) determinations. P-glycoprotein localization and expression were evaluated by immunohistochemistry and western blot analysis, respectively. Relative or absolute PCR quantification showed a significant 3.3-fold (p<0.0009) or 3.7-fold (p<0.0002) mean increase in MDR1 placental transcription in HIV-infected compared to non-infected women, respectively. Ratios of individual HIV-positive values to HIV-negative mean ranged from 0.1 to 21.8. Moreover a significant 2.5-fold increased expression of immunoreactive P-glycoprotein was evidenced in placentas from HIV-infected women (p<0.0001). This MDR1 overexpression was observed in a similar extent in placentas from pregnant women treated with Zidovudine alone or in combination with Nelfinavir and/or Lamivudine. Our findings suggest that P-glycoprotein in placentas from HIV-infected women would contribute to modulate the materno-fetal transport of antiretrovirals across the placental barrier and consequently diminish fetal exposure to these compounds.

In vitro models for the blood-brain barrier.

The aim of the present study was to identify a model for the blood-brain barrier based on the use of a continuous cell line, and to investigate the specificity of this model. A set of test compounds, reflecting different transport mechanisms and different degrees of permeability, as well as different physiochemical properties was selected. In vivo data for transport across the blood-brain barrier of this set of test compounds was generated as part of the study using two different in vivo models. A computational prediction model was also developed, based on 74 proprietary Pharmacia compounds, previously tested in one of the in vivo models. Molsurf descriptors were calculated and 21 descriptors were correlated with log(Brain(conc.)/Plasma(conc.)) using partial least squares projection to latent structures (PLS). However, the correlation between predicted and measured values was found to be rather low and differed between one and two log units for several of the compounds. The test compounds were analyzed in vitro using primary bovine and human brain endothelial cells co-cultured with astrocytes, and also using two different immortalized brain endothelial cell lines, one originating from rat and one from mouse. Cell models using cells not derived from the blood-brain barrier, ECV/C6, MDCK and Caco-2 cell lines, were also used. No linear correlation between in vivo and in vitro permeability was found for any of the in vitro models when all compounds were included in the analysis. The highest r2 values were seen in the bovine brain endothelial cells (r2=0.43) and MDCKwt (r2=0.46) cell models. Higher correlations were seen when only passively transported compounds were included in the analysis, bovine brain endothelial cells (r2=0.74), MDCKwt (r2=0.65) and Caco-2 (r2=0.86). By plotting in vivo Papp values against logDpH7.4 it was possible to classify compounds into four different classes: (1) compounds crossing the blood-brain barrier by passive diffusion, (2) compounds crossing the blood-brain barrier by blood-flow limited passive diffusion, (3) compounds crossing the blood-brain barrier by carrier mediated influx, and (4) compounds being actively excreted from the brain by active efflux. Papp and Pe values obtained using the different in vitro models were also plotted against logDpH7.4 and compared to the plot obtained when in vivo Papp values were used. Several of the in vitro models could distinguish between passively distributed compounds and efflux substrates. Of the cell lines included in the present study, the MDCKmdr-1 cell line gave the best separation of passively and effluxed compounds. Ratios between AUC in brain and AUC in blood were also calculated for six of the compounds and compared to ratios between Pe or Papp for transport in the apical to basolateral and basolateral to apical direction. Again the MDCKmdr-1 cell line gave the best correlation with only one compound (AZT) giving large discrepancy between in vitro and in vivo data. None of the in vitro models could identify compounds known to be substrates for carrier mediated influxed as such, and the results indicate that a tighter in vitro blood-brain barrier model probably is needed in order to facilitate studies on carrier mediated influx. The findings presented also indicate that identification of "batteries" of in vitro tests are likely to be necessary in order to improve in vitro-in vivo correlations and to make it possible to perform acceptable predictions of in vivo brain distributions from in vitro data.

Expression of cell adhesion molecules at the surface of in vitro human immunodeficiency virus type 1-infected human monocytes: relationships with tumor necrosis factor alpha, interleukin 1beta, and interleukin 6 syntheses.

Further evidence suggests that cell adhesion molecules (CAMs) expressed on the surface of human immunodeficiency virus type 1 (HIV-1)-infected cells are regulated during lentiviral infection. To address this hypothesis we have investigated the kinetic pattern of CAM expression at the surface of HIV-1Ba.L-infected human monocytes during the first 72 hr of infection. A significantly lower expression of CD18 and CD54 as well as a decrease in CD44 expression level were observed at the surface of infected monocytes when compared with mock-infected cultures. No modification of CD11a, CD11b, CD11c, CD58, and CD62L expression was detected. Except for CD18, the expression of which at the cell surface is decreased, no modification of CD44 and CD54 expression was observed after heat-inactivated HIV-1 treatment of monocytes. Investigation of soluble forms of CAMs (sCAMs) and cytokine production in the culture supernatants of infected monocytes showed a peak of sCD44, TNF-alpha, IL-1beta, and IL-6 release between 2 and 24 hr after infection. Treatment of monocytes with monoclonal antibodies (MAbs) against CAMs showed that engagement of some CAMs may trigger TNF-alpha and IL-1beta production. In addition, pretreatment of infected monocytes with a TNF-alpha synthesis inhibitor, RP 55778, or with MAbs directed against IL-1beta, confirmed the role of TNF-alpha and IL-1beta in the regulation of CD18, CD44, and CD54 expression.

Anti-HIV potential of a new interferon, interferon-tau (trophoblastin).

Antiretroviral effects of a new class of interferon (IFN), IFN-tau, were compared with those of IFN-alpha in primary peripheral blood lymphocytes (PBLs) and monocyte-derived macrophages (MDMs), infected in vitro by human immunodeficiency viruses type 1, HIV-1/LAI, and HIV-1/DAS isolates, respectively. Cells were treated with recombinant IFN 24 h before or after HIV infection and then continuously exposed. Viral replication was monitored twice a week by quantifying the reverse transcriptase activity in cell culture supernatants. Integrated proviral DNA was monitored 24 h after infection in IFN-tau-pretreated MDMs, using specific gag gene amplification by the polymerase chain reaction. IFN-tau inhibited HIV-1 replication in both PBLs and MDMs as well as in peripheral blood mononuclear cells (PBMCs). IFN-tau was 35-fold more potent than IFN-alpha in PBLs and 100-fold more potent in MDMs. Differences were observed in the amount of integrated proviral DNA between untreated and 10 IU/ml IFN-tau-treated HIV-infected MDMs. IFN-tau exhibits significant anti-HIV activity in comparison to IFN-alpha, and like other IFNs, it seems to interact with several steps of HIV replication cycle.

Inhibition of human immunodeficiency virus infection by heparin derivatives.

Heparin (Hep) and sulfated polysaccharides (SPs) have been reported to inhibit HIV infection in vitro. In vivo, anticoagulant activity and reduced bioavailability were found to limit the antiviral effects of Hep. In this investigation, three nonanticoagulant N-acylated Hep conjugates [OI1:3Hep, Pal1:5Hep, and Pal1:5Hep(SO4)] were compared to Hep for their ability to interact with HIV replication in CD4-positive cell lines and PBMCs. Resulfated palmitoyl-Hep [Pal1:5Hep(SO4)] exhibited the strongest anti-HIV effects. For instance, no provirus HIV DNA was detected in the genome of HIV-1-LAI-infected PBMCs treated with this heparin derivative. Cell-to-cell fusion and RT activity were explored to explain these differences. Hep and Pal1:5Hep(SO4) derivative exerted identical effects on cell-to-cell fusion. On the other hand, Pal1:5Hep(SO4) displayed the strongest inhibitory effects in the acellular RT inhibition assay. This suggests that RT might be a second target for N-acylated Hep, even though SP uptake and the preferential effects of SPs on RT as opposed to DNA polymerase have not yet been demonstrated. Nevertheless, considering the anticoagulant, antiviral, and antiinflammatory effects of N-acylated Hep, the N-acylated Hep derivatives might be excellent candidates as new anti-HIV pharmacological tools.

Relationships between humoral factors in HIV-1-infected mothers and the occurrence of HIV infection in their infants.

Based on what is known about the biology of HIV-1 vertical transmission, the HIV burden of the mother, maternal immune factors and the integrity of the placental barrier are likely to play major roles. We therefore sought to determine whether the presence of antibodies in sera from 47 HIV-1-infected mothers, including 30 non-transmitting and 17 transmitting mothers, affected the risk of HIV-1 transmission to infants. Our findings showed no significant correlation between the capacity of antibodies to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) and their capacity to induce protection of the child from HIV-1 infection (P = 0.14). Furthermore, no correlation was found between the capacity of maternal antibodies to neutralize in vitro lymphocyte or macrophage heterologous viral infection and the occurrence of in vivo HIV-1 infection in the infant. Sera recovered from five of 12 transmitting mothers and from five of 11 non-transmitting mothers were compared in their capacity to neutralize the viruses drawn from the same individuals. Four out of five maternal isolates from transmitting mothers and all maternal isolates from non-transmitting mothers were sensitive to enhancement of infection mediated by the maternal serum.

Clearance of HIV infection in 12 perinatally infected children: clinical, virological and immunological data.

OBJECTIVE: A case of HIV infection clearance in a perinatally infected infant has been recently reported. We report here on the molecular, biological and clinical features of such virus clearance in 12 children. DESIGN AND METHODS: We performed a retrospective analysis of the diagnosis in our 6-year cohort of 188 children born to HIV-seropositive mothers. HIV-1 was detected by coculture of infant peripheral blood mononuclear cells (PBMC) with cord blood cells, direct culture of infant cells, and DNA polymerase chain reaction (PCR). The children were diagnosed three times during the first 3 months of life and then followed up over a postnatal period of 18-36 months. RESULTS: The 12 reverted children had at least two positive PCR in at least two amplified regions. Among them, six were tested positive in culture/coculture assay, and five were treated long-term with zidovudine. Thus, seven out of 12 reversions cannot be attributed to antiretroviral therapy. All the virological results became negative during the first year of life, and serology lowered to negative values between 9 and 23 months. We could not find any correlation between either neutralizing or antibody-dependent cellular cytotoxicity-mediating antibodies and HIV clearance. CONCLUSION: In our cohort, we showed that an unexpected number of children born to HIV-seropositive mothers (6.7%) cleared HIV infection during the first year of life, and subsequently became seronegative. Interestingly, most of these children exhibited unspecified clinical signs during the first months of life. Five of these children were tested positive only by PCR, which suggests a low virus load and could, at least partly, explain spontaneous clearance. However, 4 years later, among the seven remaining infants, two seronegative children presented recurrent hepatosplenomegaly, which may indicate the presence of hidden virus not detectable by peripheral blood testing.

Functional consequences of macrophage infection by human immunodeficiency virus: bispecific antibody targeting of HIV-1-infected cells to Fc gamma RI expressing effector cells.

Human monocytes/macrophages play a major role in pathogenesis of human immunodeficiency virus (HIV) infection. These cells have been suspected of acting as a reservoir for the virus and are important in viral dissemination and persistence in infected individual. Furthermore, several biologic and clinical features indicate that monocytes/macrophages from HIV-1-seropositive patients have characteristics of an activation status, including the ability to secrete high levels of cytokines. Dysregulation of the cytokine network may influence the level and the consequences of viral replication in infected monocytes/macrophages. Therefore, the development of virus-specific agents that may interfere with viral replication could help to slow down the fatal course of HIV infection. In this article, we try to further quantify the early and late kinetic patterns of the cytokine network during HIV-1 macrophage infection and report the biologic effects of virus-specific bispecific antibody (MDX-240) in HIV-1 macrophage infection.

Modulations of 92kDa gelatinase B and its inhibitors are associated with HIV-1 infection in human macrophage cultures.

The macrophage-secreted 92-kDa type IV collagenase and metalloproteinases play a critical role in cell microenvironment regulation and cell movement. HIV infection of macrophages might be capable of deregulating the expression of these gelatinases. Hence, human monocyte-derived-macrophages were infected by lymphotropic HIV-1/Lai and monocytropic HIV-1/DAS isolates. Gelatinase activity and gelatinase and inhibitor (TIMP, alpha 2M) biosyntheses were evaluated in supernatants and cellular extracts. Our data suggest that HIV infection facilitates gelatinase secretion and intracellular inhibitor retention. These argue for the increase of free proteinase that could degrade barriers, which would permit cell movement and viral dissemination into tissues.

Infection of human macrophages with an endogenous tumour necrosis factor-alpha (TNF-alpha)-independent human immunodeficiency virus type 1 isolate is unresponsive to the TNF-alpha synthesis inhibitor RP 55778.

Monocyte-derived macrophages (MDM) were demonstrated to be susceptible to productive infection by the monocytotropic human immunodeficiency virus type 1 (HIV-1) strain HIV-1/Ba-L and by three primary HIV-1 isolates, HIV-1/DAS, HIV-1/PAR and HIV-1/THI. Production of tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and IL-1 beta was monitored between days 3 and 26 after MDM infection. TNF-alpha and IL-6 were detected in cell culture supernatants from days 16 to 21 following HIV-1/DAS, HIV-1/PAR and HIV-1/Ba-L infection, at the time of high viral replication. IL-1 beta was not found at the same time points. TNF-alpha mRNA expression occurred around the peak of both TNF-alpha levels and supernatant RT activities. In HIV-1/THI-infected macrophage cultures no endogenously produced TNF-alpha was observed, despite high levels of HIV-1 in MDM. This result demonstrates that a primary isolate may replicate independently of TNF-alpha in MDM. To investigate the relationship between TNF-alpha and viral replication we used a TNF-alpha synthesis inhibitor, RP 55778. Treatment throughout the course of cell culture resulted in a significant decrease in both TNF-alpha levels and viral production in HIV-1/DAS-, HIV-1/PAR- and HIV-1/Ba-L-infected MDM cultures. This phenomenon is reversed by adding recombinant human TNF-alpha to the RP 55778-treated cell cultures from day 14 post-infection. No effect of RP 55778 was observed in MDM cultures infected with the primary isolate HIV-1/THI, whose replication is independent of TNF-alpha production and therefore remained unchanged after RP 55778 treatment. We conclude that the clinical value of such a drug is directly dependent on the ability of the HIV-1 strains involved to induce TNF-alpha production at the time of viral replication.

Antibody-dependent cellular cytotoxicity and neutralization of human immunodeficiency virus type 1 by high affinity cross-linking of gp41 to human macrophage Fc IgG receptor using bispecific antibody.

Human monocytes/macrophages, which express Fc receptors for IgG are involved in human immunodeficiency virus type 1 (HIV-1) infection and pathogenesis. These receptors are known to mediate numerous immunological functions including cell-mediated killing and possibly targeting of HIV to the lysophagosome monocyte-derived macrophage (MDM) entry route for virus neutralization. To study both activities in HIV-1 infection, MDM Fc gamma RI was specifically selected using bispecific antibody (Bs-Ab) containing whole human monoclonal antibody against gp41 and the Fab' fragment of murine anti-Fc gamma RI 22.2 antibody. Bs-Ab was found to mediate potent antibody-dependent cellular cytotoxicity and virus neutralization.

Effect of human immunodeficiency virus type 1 (HIV-1) monocyte-derived macrophages infection on the manganous superoxide dismutase gene expression.

Clinical and biological features indicate that a dysregulation of microbicidal activity occurs in the cells of mononuclear phagocytic lineage of HIV-1-infected patients. Thus, the regulation of MnSOD gene expression has been investigated during the 10 h following in vitro HIV macrophage infection. As previously reported, in HIV-1 LAI-infected macrophages a high expression of the MnSOD gene is observed 2 and 4 h after infection. These results are confirmed when cells are infected with three macrophage-tropic strains HIV-1, DAS, PAR and Bal. Moreover, the detection of the MnSOD gene expression in the macrophage cultures is associated with the cellular tropism of the viral strains used. The binding of recombinant GP160 by itself is not sufficient to induce MnSOD expression. In fact, the same MnSOD gene induction was obtained with the heat inactivated viral isolates, indicating that these phenomena are due to the viral entry. On the other hand, phagocytosis of latex beads triggers a high expression of the MnSOD gene in macrophages, showing that phagocytosis of HIV may be sufficient to induce the expression of that gene. Taken together, these results indicate that the MnSOD gene expression observed within 10 h following infection of macrophages is mainly related to membrane biophysical unspecific modifications.

Treatment of human monocyte-derived macrophages with a TNF alpha synthesis inhibitor prior to HIV1 infection: consequences on cytokine production and viral replication.

Human monocyte-derived macrophages (MDM) were infected with the viral strain HIV1/Ba-L and with the clinical isolates HIV1/DAS and HIV1/PAR. Kinetics of tumour necrosis factor alpha (TNF alpha) and interleukin-6 (IL6) production were investigated for 28 days after infection. At the early stages of infection we observed significant TNF alpha and IL6 secretion 2 to 10 h after infection, whatever the viral strain we used. During the late events of MDM infection, TNF alpha and IL6 were detected over 16 to 21 days following HIV1 infection, at the time of high viral replication. Pretreatment of MDM with a TNF alpha synthesis inhibitor, RP 55778, 4 h prior to HIV infection induced a modified cytokine pattern during the first ten hours of infection: TNF alpha production was totally inhibited despite comparable amounts of IL6. At the late phases of the cell culture, a decrease in magnitude of both viral and cytokine production as well as a delay in the appearance of reverse transcriptase activity and cytokine secretion peaks were observed in RP-55778-pretreated and HIV1-infected MDM cultures. Similar results were obtained after pretreatment of HIV1/DAS-infected MDM cultures with an anti-TNF alpha monoclonal antibody.

In vitro infection of human macrophages with human T-cell leukemia virus type 1.

The tropism of the human T-cell leukemia virus type 1 (HTLV-1) for the cells of monocyte-macrophage lineage was evaluated by the coculture of blood monocyte-derived macrophages, with irradiated cells of HTLV-1 producing cell lines MT2 or C91/PL. The susceptibility to HTLV-1 was assessed by the detection of viral DNA using the polymerase chain reaction method. HTLV-1 gene expression in the cells was detected using in situ hybridization and by immunofluorescent staining of viral antigen. The presence of type C virus-like particles detected by electron microscopy and the ability to infect normal cord blood lymphocytes demonstrated that the infected macrophages produced infectious virus. These results indicate that human macrophages are susceptible in vitro to productive HTLV-1 infection, and thus might be involved in the pathogenesis of HTLV-1-related diseases.

Bispecific antibody targeting of human immunodeficiency virus type 1 (HIV-1) glycoprotein 41 to human macrophages through the Fc IgG receptor I mediates neutralizing effects in HIV-1 infection.

Human monocyte-derived macrophages that express the CD4 molecule and the Fc receptor for IgG (Fc gamma R) play a major role in the pathogenesis of human immunodeficiency virus (HIV) infection. To explore this possibility further, human monoclonal antibody to glycoprotein 41 (gp41) was produced, and a heterobifunctional antibody composed of F(ab') x F(ab')2 fragments of monoclonal anti-gp41 and anti-Fc gamma RI 22.2 were constructed. Both antibodies were analyzed for neutralizing effects, and the role of the CD4 molecule in HIV infection was studied with human monocyte-derived macrophages. The bispecific antibody exhibited strong neutralizing properties, in contrast to the monoclonal anti-gp41 antibody. Moreover, in the presence of monoclonal anti-Leu-3a antibody, viral production was completely inhibited. These findings demonstrate the necessity of the CD4 molecule in HIV infection of human macrophages and emphasize the usefulness of such heterobifunctional antibody directed to virus and monocyte-derived macrophage Fc receptors in prevention of HIV infection.

Functional consequences of monocyte/macrophage infection by HIV1.

Monocyte/macrophage infection by human immunodeficiency virus type 1 (HIV1) was studied for its effects on the production of tumour necrosis factor alpha (TNF alpha) and the expression of the manganese superoxide dismutase (MnSOD) gene. For this purpose, human peripheral blood monocytes were obtained from healthy HIV1-seronegative donors by centrifugal elutriation and infected with either the HIV1/LAV1 strain or with the primary HIV1/DAS isolate. The results showed that (1) HIV1/LAV1-infected macrophages did not produce any biologically detectable TNF alpha during the few hours following lentiviral infection, despite rises in the TNF alpha mRNA level; (2) MnSOD gene transcription in the macrophages increased, as measured 2 and 4 h after infection; (3) the level of the MnSOD gene expression declined during the late phases of lentiviral infection, but TNF alpha synthesis and gene expression rose; and (4) bispecific antibody comprised of anti-Fc gamma RI (anti-CD64) and anti-gp41 monoclonal antibodies inhibited the in vitro infection of monocyte-derived macrophages by HIV1/DAS.

In vitro infection of macrophages by HIV: correlation with cellular activation, synthesis of tumour necrosis factor alpha and proteolytic activity.

Macrophages were obtained after differentiation of healthy donor monocytes. Seven to 9 days after isolation, cells were infected with HIV1. Tumour necrosis factor alpha (TNF alpha) biological activity, TNF alpha- and 1-6-fructose-diphosphatase-gene expression and gelatinase activity were sequentially determined and correlated with viral infection and replication. TNF alpha was only detectable when mature viral particles were isolated in cell culture supernatants; 1-6-fructose diphosphatase mRNA was hyperexpressed in infected cells and its proteolytic activity was tremendously decreased during the early days postinfection. These results would seem to indicate that in human macrophage activation, cytokine secretion and microbicidal proteolytic activity are strongly modified by HIV infection.