The principal neutralizing domain (PND) of Human Immunodeficiency Virus type 1 (HIV-1) is mapped to a 24-amino acid sequence located in the hypervariable V3 region of the viral envelope protein. The ...PND of HIV-1 isolates from infected individuals corresponds mostly to that of the HIV-1 MN strain. We found that a peptide designed from the PND of HIV-1 MN virus greatly enhanced viral infection, while a peptide-derived PND of HTLV-IIIB virus showed at least 10-fold less efficient activity; no such effect was exhibited by the other peptides tested, including one designed from the PND of HIV-1 RF strain. The observed enhancing effect occurred in the early steps of viral infection and was not strain-restricted as both MN- and IIIB-derived peptides increased heterologous virus expression, including that of the RF strain. The MN- and, to a lesser extent, IIIB-derived peptides also increased CD4 expression on the cell membrane and differentially inhibited CD4 down-regulation induced by the phorbol ester TPA and/or by the monosialoganglioside GM1; the peptides showing no viral infection enhancement had no such effects. These findings demonstrate that the viral enhancement observed took place through a CD4-dependent mechanism and suggest that the PND is involved in HIV-1 infection and spread.
Neste trabalho buscamos fazer urna reflexão acerca da adoção do método funcional no trabalho da enfermagem, bem como uma crítica a esse mesmo modelo. Tentamos também, evidenciar como a equipe de ...enfermagem e, principalmente as enfermeiras, vem "subvertendo" esse modelo no seu quotidiano de trabalho.
The potentiation of monoclonal antibody/ligand toxin (immunotoxin) cytotoxicity by the ionophore monensin (Mo) or by human serum albumin-monensin (HSA-Mo) conjugates was investigated. Since disulfide ...cross-linked HSA-Mo (HSA-SPDP-Mo) is rapidly inactivated by human serum (M. Colombatti et al., Cancer Res., 50: 1385-1391, 1990), we synthesized thioether cross-linked HSA-Mo conjugates (HSA-SIA-Mo). HSA-SIA-Mo is resistant to treatment with reducing agents (e.g., glutathione, dithiothreitol) and shows potentiating activity identical to that of Mo or of HSA-SPDP-Mo, enhancing immunotoxin (IT) cytotoxicity 45-35,000-fold. Human leukemic and tumor cell lines are highly sensitive to treatment with IT in combination with Mo, HSA-SPDP-Mo, or HSA-SIA-Mo (concentration required to inhibit protein synthesis by 50%, 10(-10)-2.5 x 10(-13) M). IT potentiation by both types of HSA-Mo conjugates, however, is inhibited by whole human serum. In contrast, human cerebrospinal fluid has no effect on the potentiation of IT by Mo or HSA-Mo conjugates. The serum blocking factors reside mostly in a Mr 40,000-90,000 protein fraction. Serum components of low molecular weight (less than 10,000) show no detectable effect upon the stability of HSA-Mo conjugates. The toxicity of HSA-SIA-Mo in vivo was investigated by intrathecal injections in rats. Concentrations of up to 60 micrograms/kg can be injected into the brain with only transient neurological sequelae. We therefore conclude that if the systemic delivery of HSA-Mo conjugates for the potentiation of ricin A chain-IT presents some limitations due to the blocking effect of serum, the application of HSA-Mo conjugates in combination with ricin A chain-IT for regional tumor therapy in the brain appears more promising.
Human immunodeficiency virus type 1 (HIV-1) isolates from 25 perinatally HIV-1 infected children were classified according to their capacity to replicate in vitro as rapid (R), intermediate (S/R) and ...slow (S) variants. R-type viruses replicated on peripheral blood mononuclear cells (PBMCs) and grew better in T-lymphoid cells, even though 9 out of 12 isolates also maintained tropism for monocytoid cells. The S/R-type isolates replicated efficiently after several days of culture, while the S-type viruses displayed only a low and transient replication activity; however, both S/R- and S-type isolates exerted viral transactivation activity in an indicator monocytoid cell line. Replication patterns in vitro were significantly associated in vivo with the number of HIV-1 copies in PBMCs as determined by polymerase chain reaction: in children with R-type isolates, the number of HIV-1 proviral DNA molecules/10(5) PBMCs ranged from 62 to 571, and in children with S/R and S isolates the range was 5-43. Seven children had severe symptomatic HIV-1 infection, and in all an R-type virus was identified; 18 children had no or only mild symptoms, and among these, S-, S/R-, and R-type isolates were found in 5, 8, and 5 cases, respectively. Besides demonstrating HIV-1 variability in perinatal infection, these findings suggest that R-type virus might be a prerequisite for disease progression.
Four Epstein-Barr virus-positive lymphoblastoid cell lines (LCL) were successfully infected in vitro with immunodeficiency virus type 1 (HIV-1) as demonstrated by reverse transcriptase activity and ...p24 HIV antigen in culture supernatants, positive cell staining for gag-encoded HIV proteins, presence of viral HIV genome by Southern blot analysis and ulstrastructural observations. In addition, both HIV-1-infected B cells and their supernatants efficiently transactivated the chloramphenicol acetyl transferase reporter gene which is under the control of the HIV-1 long terminal repeat. The LCL cells displayed long-term HIV-1 infection and production, but no cytopathic effects were observed. Cytofluorimetric analysis did not detect membrane CD4 presence in the LCL cells before and after HIV-1 infection; moreover, a minute amount of CD4 mRNA was observed only in one of the LCL. A monoclonal antibody specific for the viral binding site of the CD4 molecule delayed, but did not block, HIV-1 infection of the LCL cells. Following HIV-1 infection, changes in LCL phenotype were observed, consisting of a decrease in CD23- and CD39-positive cells, and a concomitant increase of cells with surface CD10 and Bac-1. Furthermore, HIV-1-infected LCL cells did not grow in tight clumps, as usually observed in uninfected LCL, but as disperse suspensions, and formed more agar colonies than control LCL. However, despite this apparent acquisition of a malignant-like phenotype, c-myc proto-oncogene rearrangement was not detected. The appearance of cells with new characteristics did not seem due to clone selection by HIV-1 infection, since all the LCL conserved their clonotypic pattern of IgH chain rearrangement. The acquisition of malignant-like features by HIV-infected B cells might be clinically significant in terms of the pathogenesis of non-Hodgkin's B cell lymphomas, which occur frequently in AIDS patients.
We have evaluated the sensitivity to immunotoxins (IT) of monolayer and of 200-250 microns multicellular tumor spheroid (MTS) cultures obtained with human breast (MCF7) and glioblastoma (U118) tumor ...cells and with rat glioblastoma (9L) cells. Monolayer MCF7 and U118 cells were highly sensitive to antitransferrin receptor (anti-TfnR) ricin A chain (RTA)-IT (Tfn-RTA and MAb OKT9-RTA) treatment in the presence of the intracellular RTA-IT enhancing agent human serum albumin-monensin (HSA-Mo) conjugate. A 790- to 2000-fold higher concentration of anti-TfnR IT was instead required to reduce by 50% the volume of individually treated MCF7 spheroids, as evaluated by applying the Gompertz growth model. Monolayer 9L cells showed 230- to 5700-fold lower sensitivity to Tfn-RTA IT than MCF7 and U118 monolayers, yet 9L spheroid cells were almost as sensitive to anti-TfnR IT as monolayer 9L cultures. Binding studies performed with 125I-Tfn and FITC-labelled anti-TfnR MAb revealed that 9L monolayers and MTS expressed 4.1-fold and 8.8-fold lower amounts of TfnR than MCF7 monolayers and MTS, respectively. However, Tfn bound to TfnR sites of 9L and of MCF7 cells with comparable affinity. Experiments carried out with the diphtheria toxin mutant CRM107 linked to Tfn confirmed the pattern observed with RTA-IT. Monolayers and spheroids showed no considerable differences in sensitivity to ricin toxin. Collectively, these results indicated that the efficacy of IT against 3-D tumors is heavily influenced by the number of target Ag expressed by the tumor cells, as well as by the affinity of IT/toxin-cell interaction.
Control techniques that optimize the behaviour of pulse-width modulation (PWM) rectifiers for magnet supplies are examined. Different control strategies, both with preprogrammed and variable ...switching patterns, are examined and compared in terms of optimization of the system performance. Optimum digital PWM, multilevel delta modulation, and hybrid PWM/delta techniques are analyzed. The validity of the control methods was verified by simulation and experimental tests on a 60 kW prototype. It was observed that, in practice, digital PWM techniques are better suited for optimizing the AC converter performance, while delta modulation techniques give better results in terms of DC current and voltage distortion.< >
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