Biotechnology therapeutics最新文献

It has long been suggested that malignant melanomas, cutaneous as well as uveal, are responsive to human immune-mediated host defenses. We report here 5 consecutive cases of posterior choroidal melanomas which were treated with hyperthermia generated by high-intensity focused ultrasound. Patient immune function was monitored by determination of T-cell helper/suppressor (CD4/CD8) ratios immediately before and approximately 1 week following hyperthermia treatment. All 5 patients had normal total T-cell counts as measured by the pan-T-cell marker CD3. Two patients were noted to have inverted CD4/CD8 ratios (< 1:1) before hyperthermia. In both these cases, the ratio reverted to normal (> 1:1) 1 week following treatment. One patient whose CD4/CD8 ratio was not inverted was noted to have a further increase in his CD4 T cells relative to his CD8 (37% increase). Two patients with initially normal CD4/CD8 demonstrated no significant change following hyperthermia. It appears that ultrasonic hyperthermia may induce a systemic immunomodulatory effect in patients with posterior choroidal melanoma and inverted T-cell helper/suppressor resulting in a normalization of T-cell subset ratios.

The capacity of alpha-interferon (alpha-IFN) to induce lymphokine activated killer (LAK) cytotoxicity in the absence of interleukin-2 (IL2) has prompted us to test whether its ability to reduce dramatically the number of Ph1+ clones in chronic myelogenous leukemia (CML) patients is in part mediated through the generation of natural killer (NK) or LAK activity. The latter were tested using NK-sensitive (K562) and NK-resistant (Raji) cell lines in a target-cell colony-growth inhibition assay. Effector cells (E) were patient blood mononuclear cells (MC) without in vitro activation prior to their coculture with targets (T). Here we report that cytogenetic remission in alpha-IFN-treated patients is associated with significantly enhanced NK and LAK activities. Nevertheless, some patients under alpha-IFN therapy were found to develop lymphoid blast crisis despite high levels of NK and LAK activities, and partial or total cytogenetic remission. In contrast, most of the patients who developed nonlymphoid blast crisis presented 100% Ph1+ cells and displayed defective NK and/or LAK activity. These observations could favor the hypothesis that there is an indirect but complex effect of alpha-IFN on leukemic cells, mediated by cells involved in immune surveillance; and also that lymphoid blast cells may actually escape LAK cytotoxicity.

Lipophilic prodrugs of 3'-azido-3'-deoxythymidine (AZT) and of 2',3'-didehydro-3'-deoxythymidine (D4T) have been synthesized. 3 beta-(2'-carboxymethoxy)-cholest-5-ene acid, palmitic acid, linolenic acid, linoleic acid, and cholanic acid have been covalently bound to AZT and D4T. In some experiments the fluorescent molecule NBD was simultaneously linked. These prodrugs were incorporated into LDL or acetylated LDL. The best incorporation was obtained with drugs presenting a steroid moiety (cholesterol derivative or cholanic acid) in their structure. The incorporation of prodrugs into LDL was estimated as approximately 200 molecules of prodrug per LDL particle. Cytofluorimetric studies clearly show that the NBD-steroid LDL or NBD-steroid acetylated LDL are bound and then internalized by the B-E receptor (U937) or the scavenger receptor (mouse peritoneal macrophage), respectively. The antiretroviral activity of palmitate-D4T, cholanic-AZT, and cholanic-AZT-LDL complex was similar to the activity of free D4T and free AZT, respectively. Development of lipid nucleoside-LDL complexes to attach specifically to cells involved in HIV infection might have a direct clinical relevance.

The glutathione redox cycle plays an important role in antioxidant and detoxification mechanisms. We recently reported that a calf thymic peptide (TP) protected vascular endothelial cells from oxidant injury induced by hydrogen peroxide. Using electrophoresis and amino acid sequencing analysis, we have now shown that TP consists of two peptides. The fast-moving peptide has 9 amino acid residues at the NH2 terminal and accounts for 92% of total quantity, while the other peptide has 18 amino acid residues at the NH2 terminal and amounts to 8%. The present study investigated the effect of TP on glutathione redox cycle. Confluent monolayers of bovine pulmonary artery endothelial cells (PAEC) were incubated with TP (12.5-100 micrograms/mL) for 24-48 h. TP caused a dose-dependent increase in glutathione (GSH) level and glutathione disulfide reductase activity but no significant change in GSH peroxidase activity. Exposure of PAEC to an organic oxidant t-butyl hydroperoxide (tBHP) resulted in decreased GSH level, increased lipid peroxidation, and elevated leakage of intracellular lactate dehydrogenase. Preincubation of PAEC with TP prevented these changes induced by tBHP. The data suggest that the antioxidant effect of TP may be due, at least in part, to its modulation of the GSH redox cycle in vascular endothelial cells. TP may thus be considered a new antioxidant with novel activities in addition to being an immune regulator.

The active oxygen induced and free radical mediated oxidation of biological molecules, membranes, and tissues has been suggested as a major cause of cancer, atherosclerosis, and aging. Damage of endothelial cells may lead to cardiovascular and cerebrovascular diseases. In the present study, the antioxidant effect of pycnogenol (procyanidins extracted from Pinus maritima) was investigated in vitro using vascular endothelial cells. Confluent monolayers of bovine pulmonary artery endothelial cells (PAEC) were preincubated with different concentrations of pycnogenol for 16 h, washed, and then exposed to an organic oxidant t-butyl hydroperoxide (tBHP) for 3 or 4 h. Cellular injury was assessed by measuring cell viability with methylthiazol tetrazolium (MTT) assay and by determining the release of intracellular lactate dehydrogenase (LDH). Lipid peroxidation products of PAEC were monitored as malondialdehyde (MDA) with a thiobarbituric acid fluorometric assay. Incubation of tBHP (75, 100, or 125 microM) with PAEC decreased cell viability, increased LDH release, and elevated MDH production. Preincubation of PAEC with pycnogenol (10-80 micrograms/mL) before tBHP exposure significantly increased cell viability, decreased LDH release, and reduced MDA production. These results demonstrate that pycnogenol can protect vascular endothelial cells from oxidant injury. The data thus suggest that pycnogenol may be useful for the prevention of disorders associated with oxidative damage.

This study examines the interaction of TNF with its receptor(s) in the presence of antibodies which have been previously found to enhance the in vivo antiviral and antitumor activities of this cytokine. The presence of Mab 32 has been previously shown to enhance the binding of 125I-TNF to the surface L929 cells (13), and this property was also found in the present study for HeLa cells. In addition to this, Mab 32 was found to enhance the internalization of the TNF ligand into both L929 and HeLa cells compared to control treated cultures. The consequences of such enhanced TNF-binding on TNF antiviral activity were examined in both L929 cells and HeLa cells. It was discovered that the similarities in antibody-enhanced TNF binding and internalization contrasted dramatically with the sensitivities of these two cell lines to the antiviral actions of TNF, both with and without Mab 32 (viz., L929 cells were sensitive; HeLa cells were resistant). It has been proposed that the modulation of TNF-R expression, particularly by IFNs, is an important factor in TNF's biological effects. It has been shown that the presence of IFN-gamma, with TNF plus specific enhancing antibodies, further augmented antiviral activity in vivo (13). This finding stimulated interest in examining IFN-gamma modulation of TNF-R as a factor in the antiviral activity of TNF. The expression of TNF receptor(s) in TNF- and/or IFN-gamma-exposed cells, both with and without HSV-1 infection, was therefore examined. TNF alone could induce a dose-dependent increase in receptor expression which was not significantly increased by Mab 32. Exposure of L929 cells to IFN-gamma alone also induced TNF receptor expression in mock-infected cells. HSV-1 infection of L929 cells resulted in a significant upregulation of TNF-R expression which was reversed if the cells had been preexposed to IFN-gamma. The inclusion of TNF with IFN-gamma before infection restored TNF-R expression but did not show any further synergistic or additive effects on TNF-R expression. Some minor increases in TNF-R expression were seen if Mab 32 was included with these two cytokines.

The role of cells of lymphoid and myeloid origin as effectors in the immunosurveillance network is based upon their recognition of surface structures on target cells. The work described here, however, has focused on an alternative class of target molecules, that is, soluble intracellular factors, as potential immunogens. Results from this study, which was aimed at a biochemical definition of the tumor immunoenvironment, demonstrate that the soluble intracellular contents of a tumor cell line are significantly more effective than the extracellular medium conditioned by the same cells in both stimulating the growth of a human T-lymphocyte line and inducing differentiation markers in a human myeloid leukemic cell line. A proposal is made for a restructuring of the way in which the immunosurveillance network is considered. Specifically, it is suggested that the soluble intracellular components of tumor cells may serve as immunogens in the immunosurveillance network. It is further proffered that an understanding of the physical and chemical states of molecules which under pathologic conditions become exposed to effector components of the immunosurveillance network will give rise to new immunotherapeutic venues.

This present study examines Il-4 regulation of perforin gene expression and cytolytic granule production in alpha CD3-induced activated killer cells CD3-AK. After stimulation of resting T cells with alpha CD3, proliferative response could be detected at 1 day after activation. The expression of perforin mRNA and production of cytolytic granules (using BLT-E as indicator) was detected on days 2-4, and this time course correlated with the generation of lytic CD3-AK cells. These findings indicate that killer cells generation is a late event during the course of alpha CD3 activation. Generation of CD3-AK cells is primarily PKC dependent and is blocked by the depletion or inhibition of PKC by PMA or SSP. These changes are accompanied by the suppression of perforin gene expression (mRNA) and BLT-E production. However, adding IL-4 into the cultures restored the perforin mRNA expression and BLT-E production, and also the cytolytic activity of the CD3-AK cells. Furthermore, for preactivated CD3-AK cells cultured in IL-2, SSP also suppressed the perforin mRNA and BLT-E with the concomitant reduction of cytolytic activity. Similar to the resting T cells, in the SSP-maintained preactivated CD3-AK cells, switching the cytokine from IL-2 to IL-4/IL-2 restored perforin mRNA expression and BLT-E production, with concomitant restoration of the cytolytic activity. In contrast, switching from IL-4/IL-2 gave the opposite effect. These results could be reproduced by using amiloride which also inhibited PKC activity but did not affect the growth of preactivated CD3-AK cells. These findings indicate that IL-4 may play a role in the late stage of alpha CD3 activation to regulate the expression of perforin gene and probably the translation process during the generation of activated killer cells.

Recombinant yeast-derived granulocyte-macrophage colony-stimulating factor (GM-CSF) was administered to 10 patients after autologous bone marrow transplantation for Hodgkin's disease given as a 24-h continuous intravenous infusion from the day of marrow infusion until the patient had obtained an absolute neutrophil count of 1.5 x 10(9)/L for 2 consecutive days or until day 30, whichever occurred first. Results were compared with results from 18 historical control patients who did not receive GM-CSF but were otherwise treated in a similar fashion. The infusion of GM-CSF led to a significantly faster neutrophil and monocyte recovery compared to the patients in the historical control group. The median days to achieve an absolute neutrophil count for the GM-CSF group and the control group were 0.5 x 10(9)/L; 9.5 and 14 days; 1.0 x 10(9)/L: 10 and 18 days; 1.5 x 10(9)/L: 11 and 29 days. No significant difference was found with respect to platelet engraftment and red cell transfusion requirements. GM-CSF therapy was discontinued at a median of 12 days. Hospitalization was also shorter for the GM-CSF group (22.5 vs. 26.5 days) and no patient in the GM-CSF group had to be readmitted after initial discharge. The incidence of documented infections was similar among both patient groups and no difference was noted in terms of antimicrobial usage. Some side effects occurred with the continuous infusion of GM-CSF, particularly fluid retention, dyspnea, fever, diarrhea, and bone pain leading to early discontinuation of GM-CSF in 2 patients. The data suggest that a continuous 24-h infusion of GM-CSF significantly accelerates myeloid engraftment, leading to earlier discharge from the hospital.

Recently many biologic response modifiers have been discovered or produced, but their effects are incompletely understood. The effects of these agents are complicated by their dependence not merely upon the administered dose, nor even the interval of time between administration and observation of effect, but also because of the cascade of interactions that can be invoked. Conventional experimental designs that test a specific combination looking for statistical significance, and that involve doing a series of studies varying one parameter at a time, are likely to require large numbers of animals or patients to answer the question of possible effect, and have a high likelihood of missing the effect altogether because of not selecting the correct combination of parameter levels. Response surface designs are intended for simultaneous multiparameter evaluation and so are less likely to miss an effect. However, if fixed sample designs are employed, the number of subjects required may still be quite large. Sequential designs based on response surface models have been used in other fields but are only now being applied in the fields of biology and medicine. We have explored the use of these designs to evaluate the potential usefulness of interleukin-2 (IL-2) to increase tumor uptake of subsequently administered antibody in nude mice. We found that these methods enabled us to efficiently determine optimal combinations of dose of IL-2 and interval of time between IL-2 and antibody administration for enhancement. Enhancement of tumor uptake of the antibody by IL-2 was substantial in amount and sufficient to stimulate studies in patients where we will use similar sequential methodology for trial design.