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Biomedical subjects

J Alexander

Publications and source records attributed to J Alexander.

At least 397 records · Page 22Linked to original sources
PubMed1990

An analysis of insulin receptor expression and binding on MHC class I positive and negative human lymphoblastoid cells.

Recent studies have demonstrated a specific membrane association between the polymorphic class I antigens of the MHC and the insulin receptor (IR). In some reports, variation in IR binding is attributed to the class I phenotype. To extend these observations, we have examined whether MHC class I products influence IR function, in particular, high affinity ligand-specific binding. Using two independent methods, we have compared IR affinity and density on a human B-lymphoblastoid cell line expressing HLA-A, -B products vs a null mutant and a set of transfectants where HLA-A3, -B7, or -Bw58 expression has been restored through gene transfer. The results from radioreceptor assay and RIA measuring IR binding and expression indicate that although there is a decrease in high affinity insulin binding sites in the HLA-A, -B null mutant, ligand binding cannot be restored in the transfectants expressing HLA-A or -B alleles. We conclude that the MHC class I products do not determine the insulin-binding phenotype of the cells examined in this study. Alternatively, we propose that insulin receptor heterogeneity in affinity and density may be influenced by other undetermined factors inherent to clonally expanded cells, thereby complicating dissection of IR/MHC interactions.

Cell Line↗
PubMed1990

Immunoprotective Leishmania major synthetic T cell epitopes.

Using the predictive algorithm of Rothbard and Taylor (1988. EMBO J. 7:93) and the primary structure of gp63 (Button, L., and M.R. McMaster. 1988. J. Exp. Med. 167:724; Miller, R.A., S.G. Reed, and M. Parsons. 1990. Mol. Biochem. Parasitol. 39:267) we have been able to delineate the structures of a number of gp63 T-cell epitopes which stimulate the proliferation of CD4+ cells. One of these synthetic antigens, inoculated subcutaneously with adjuvant, was shown to specifically induce proliferation of the Th1 subset and provided immunoprotection against two species of Leishmania parasites.

Algorithms↗
PubMed1990

The electrocardiogram at rest and exercise during a simulated ascent of Mt. Everest (Operation Everest II).

To evaluate the effect of extreme altitude on cardiac function in normal young men, electrocardiograms were recorded at rest and during maximal exercise at several simulated altitudes up to the equivalent of the summit of Mt. Everest (240 torr or 8,848 m). The subjects spent 40 days in a hypobaric chamber as the pressure was gradually reduced to simulate an ascent. Changes in the resting electrocardiogram were evident at 483 torr (3,660 m) and were more marked at 282 torr (7,620 m) and 240 torr (8,848 m). They consisted of an increase in resting heart rate from 63 +/- 5 to a maximum of 89 +/- 8 beats/min; increase in P-wave amplitude in inferior leads; right-axis shift in the frontal plane; increased S/R ratio in the left precordial leads; and increased T negativity in V1 and V2. No significant arrhythmias or conduction defects were observed. Most changes reverted to normal within 12 hours of return to sea level, with the exception of the frontal-plane axis and T-wave alterations. Maximal cycle ergometer exercise at 282 torr (7,620 m) and 240 torr (8,848 m) resulted in a heart rate of 138 +/- 7 and 119 +/- 6 beats/min at the 2 altitudes, respectively. No ST depression or T-wave changes suggestive of ischemia occurred despite a mean arterial oxygen saturation of 49% and a mean pH of 8 during peak exercise. Occasional ventricular premature beats were observed during exercise in 2 subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1990

Presentation of viral antigen controlled by a gene in the major histocompatibility complex.

We describe a mutant human cell line (LBL 721.174) that has lost a function required for presentation of intracellular viral antigens with class I molecules of the major histocompatibility complex (MHC), but retains the capacity to present defined epitopes as extracellular peptides. The cell also has a defect in the assembly and expression of class I MHC molecules, which we show can be restored by exposure of the cells to a peptide epitope. This phenotype suggests a defect in the association of intracellular antigen with class I molecules similar to that described for the murine mutant RMA-S (ref. 5), but in the present case the genetic defect can be mapped within the MHC locus on human chromosome 6.

Antigen-Presenting Cells↗
PubMed1990

Characterization of the cytolytic activity of CD4+ and CD8+ tumor-infiltrating lymphocytes in human renal cell carcinoma.

Previously we showed that IL2 expanded tumor-infiltrating lymphocytes (TILs) from renal cell carcinoma mediated non-major histocompatibility complex-restricted cytotoxicity. Phenotypic analysis showed that cultured TILs were composed mostly of T-lymphocytes with varying numbers of CD4+, CD8+, and CD56+ (Leu19+) populations. Here we compared the cytolytic activity of the two predominant TIL subsets, CD3+CD4+ and CD3+CD8+, to that of the CD56+ populations. Using magnetic beads coated with antibodies to either CD4 or CD8, CD3+CD4+, and CD3+CD8+ TILs were isolated in a highly enriched form (greater than 92%) and could be expanded for over 40 days in vitro with 1000 units/ml IL2. In a 4-h 51Cr release assay the CD4+ and CD8+ TILs showed minimal lytic activity, whereas unseparated cells exhibited significant levels of non-major histocompatibility complex-restricted cytotoxicity. The lytic activity seen in the 4-h assay with unseparated TILs appeared to be related to the presence of CD56+ populations. With one exception none of the purified CD4+ or CD8+ TILs expressed any significant levels of CD56, while the unseparated TILs contained varying numbers of CD3+CD56+ and CD3-CD56+ populations. Cell-sorting experiments verified that the CD56+ populations were responsible for most of the lytic activity in 4 h even though CD3+CD56- cells represented the predominant cell type. Although CD3+CD56- TILs were minimally lytic in 4 h, we show here that both CD3+CD4+ and CD3+CD8+ subsets displayed substantial cytotoxicity in long-term assays. In the 18-h 51Cr release assay 5 of 6 CD4+ and 2 of 3 CD8+ TILs were lytic for the autologous tumor. In two cases, restimulation with the autologous tumor induced augmented cytolytic activity of TIL subsets and in one case induced lytic activity in 4 h. The cytotoxic activity of TIL subsets was further examined using a 72-h assay in which TILs were cocultured with a confluent layer of tumor cells. The degree of cytotoxicity was quantitated by measuring the amount of crystal violet dye that was incorporated by tumor cells which remained after the incubation period. CD4+ and CD8+ TILs typically caused greater than a 50% reduction of tumor cells in 3 days and the level of reduction was increased when IL2 was added to the cultures. All the CD4+ and CD8+ subset preparations were cytotoxic in the 3-day assay even though some were not lytic for certain targets in the 18-h 51Cr release assay.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1990

Characterization of a transferrin-independent uptake system for iron in HeLa cells.

HeLa cells incubated in serum-free medium accumulated 59Fe ("non-transferrin iron") when incubated with either 59Fe-citrate, 59Fe-nitrilotriacetate, or 59Fe dissolved in Tricine ascorbate. Accumulation of iron was time-, concentration-, and Ca2+-dependent and was saturable. Uptake of non-transferrin (non-Tf) iron was transferrin-independent because of the fact that uptake occurred at pH 5.5, a pH at which transferrin binds iron poorly and at which transferrin is not internalized by cells. Uptake of non-Tf iron was less affected than uptake of transferrin iron by 1) exposure of cells to trypsin, a maneuver that cleaves Tf receptors, or 2) incubation of cells with phenylarsine oxide, an agent that inhibits both fluid- and receptor-mediated internalization. After exposure of cells to non-Tf iron at 37 degrees C, most of the cell-associated radioactivity was recovered in heme and ferritin, demonstrating that iron gained access to intracellular compartments and was not simply adsorbed to the cell surface. Uptake of non-Tf iron could be partially blocked by Cu2+ in a dose-dependent manner, while the accumulation of transferrin-bound iron was unaffected by Cu2+. Other transition metals, such as Zn2+, Cd2+, and Mn2+ were able to inhibit the uptake of non-Tf iron to different degrees. The accumulation of 109Cd was inhibited by incubation of cells with non-Tf iron, Cu2+, or Mn2+. The extent of inhibition was concentration- and metal-dependent. A number of cultured cell lines including HeLa, human skin fibroblasts, and Chinese hamster ovary cells demonstrated uptake of non-Tf iron and 109Cd. Additionally, an endosome acidification mutant of Chinese hamster ovary cells, which exhibited an increase in non-Tf iron uptake, also exhibited an increase in the uptake of Cd2+. These observations suggest that the characteristics of the non-Tf iron transport system in HeLa cells are similar if not identical to those reported for perfused rat liver (Wright, T. L., Brissot, P., Ma, W.-L., and Weisiger, P. A. (1986) J. Biol. Chem. 261, 10909-10914) and suggest the existence of a family of transition metal transport systems, each with a different metal specificity.

Arsenicals↗
PubMed1990

TIL from renal-cell carcinoma: restimulation with tumor influences proliferation and cytolytic activity.

TIL were cultured from human renal-cell carcinoma (RCC) in 1,000 U/ml rIL2 and restimulated with autologous tumor in efforts to establish which conditions would best expand the number of lymphocytes cytotoxic for autologous tumor. Greater cell yields resulted from multiple restimulations of TIL with autologous tumor. In most instances, these TIL lysed autologous tumor better than TIL grown in rIL2 alone. Enhanced proliferation was seen also after restimulation of TIL with allogeneic RCC as well as with tumor cells of non-renal origin. Although in some instances lysis of autologous tumor appeared to be specific, restimulation with autologous tumor did not consistently result in the generation of specific cytolytic T cells. Attempts to culture more specific cytolytic T cells by using 50 U/ml rIL2 were successful in expanding TIL with enhanced lytic activity; however, this activity was not specific for autologous tumor. The phenotype of the tumor-restimulated TIL generally did not change. In most of the TIL cultures, CD3+CD4+ cells were predominant with low numbers of CD3+Leu19+ cells and minimal numbers of CD3-Leu19+ cells. Thus, the cytotoxic response to tumor was mediated by T cells and not NK cells. Overall, our data indicate that restimulation of TIL with autologous tumor may be beneficial for growing larger numbers of cells which have increased lytic activity and for prolonging the presence of lytic activity among the expanded TIL.

Adult↗
PubMed1990

Unna's boot vs polyurethane foam dressings for the treatment of venous ulceration. A randomized prospective study.

Recent reports have suggested that polyurethane foam dressings provide a more rapid and comfortable healing of venous stasis cutaneous ulcerations than standard semirigid impregnated gauze dressings. This multi-institutional study consists of a randomized, prospective comparison of 36 consecutive patients who were treated with either polyurethane foam dressings (group 1, n = 17) or Unna's boot (group 2, n = 19) for venous ulceration of the lower extremities. Ulcer size ranged from 6.0 to 270 cm2 (mean, 32.2 cm2) for group 1 and 0.2 to 600 cm2 (mean, 76.0 cm2) for group 2. Nine (52.9%) of 17 group 1 patients withdrew from the study due to wound odor, while there was 100% compliance in group 2. Overall wound healing was superior in group 2 (18 [94.7%] of 19) as compared with group 1 (7 [41.2%] of 17) (chi 2 = 8.2). The rate of healing was also better in group 2 (0.5 cm2/d) than in group 1 (0.07 cm2/d). Contrary to published European trials, impregnated gauze dressings exhibited superior treatment results when compared with polyurethane foam dressings in the current study.

Bandages↗
PubMed1990

The transport of class I major histocompatibility complex antigens is determined by sequences in the alpha 1 and alpha 2 protein domains.

The transport of human-mouse hybrid class I histocompatibility antigens has been studied in a mutant human cell line, 174 X CEM.T2 (T2). T2, a somatic cell hybrid of human B- and T-lymphoblastoid cell lines (B-LCL and T-LCL, respectively), synthesized HLA-A2 and HLA-B5 glycoproteins, but expresses only low levels of A2 and undetectable levels of B5 at the cell surface. We have previously shown that the products of human class I genes introduced into T2 by transfection behave like the endogenous HLA-B5 glycoproteins, while the products of mouse class I alleles similarly introduced are transported normally to the cell surface. We have now determined that the surface expression of class I glycoproteins in T2 depends on the origin of the alpha 1 and alpha 2 domains. Human (HLA-B7) and mouse (H-2Dp) hybrid class I genes, encoding the leader, alpha 1, and alpha 2 sequences of one species fused to the alpha 3, transmembrane, and cytoplasmic domains of the other, were transfected into T2. Normal surface expression of the hybrid class I molecule was observed in T2 only when the leader, alpha 1, and alpha 2-encoding exons were derived from the mouse gene. The reciprocal construct, encoding human leader, alpha 1, and alpha 2 domains fused to the mouse alpha 3, transmembrane, and cytoplasmic regions, resulted in biosynthesis of a hybrid glycoprotein which was not transported to the cell surface. The products of both constructs were expressed normally in control cells. The effects of glycosylation on class I antigen transport were also studied using mutant class I constructs with altered glycosylation sites. Two mutant B7 genes encoding either an extra glycosylation site at position 176 or no glycosylation sites were transfected into T2. These mutant products were expressed at the cell surface in control cells, but were synthesized and not surface-expressed in T2. These data demonstrate that the HLA/H-2 transport dichotomy in T2 is a function of the origin of the alpha 1 and/or alpha 2 domains of the class I glycoprotein, and is not a reflection of glycosylation differences between the human and mouse molecules.

Amino Acid Sequence↗
PubMed1990

Mutations in the alpha 1 domain of a class I gene define residues important for specific allorecognition.

Our strategy to use saturation mutagenesis to produce an unbiased collection of major histocompatibility class I mutants has resulted in unpredicted mutant phenotypes. First, we have shown data supporting our earlier work of the Dp20(Y27N) mutant. Allorecognition is altered at the clonal level while no variation in lymphocytic choriomeningitis virus (LCMV)-restricted recognition is observed. The defect does not destroy the integrity of this class I protein on the basis of three observations: (i) LCMV self-restricted recognition is not impaired, (ii) beta 2 microglobulin still associates with Dp20(Y27N) at the cell surface, and (iii) this mutant can stimulate a primary MLR. Thus, we believe Dp20(Y27N) specifically affects allorecognition, perhaps by altering self peptide associations. The Dp14(A11V;E32Q) mutant appears to interact with T cell receptors (TCR) from a cloned cytotoxic T lymphocyte, but is altered in inducing a wild type signal into the responding cell. This is presumably due to decreased interaction at the cell surface between Dp14(A11V;E32Q) and wild type-specific TCR such that variations are detected in how a cell perceives extracellular signals. Analysis of additional mutants suggests that mutant Dp163(N66S) alters the binding site for monoclonal antibodies 7-16.10 and 135, while leaving unaltered the binding site for monoclonal antibodies 34-1.2 and 11-20.3. This maps the residue responsible for 7-16.10 and 135 binding to the region of Dp163(N66S).

Animals↗