PubMed Health⌕ Search

Biomedical subjects

B Bonavida

Publications and source records attributed to B Bonavida.

At least 145 records · Page 8Linked to original sources
PubMed1986

Inhibition of IA positive reticulum cell sarcoma tumor cell growth in syngeneic SJL/J mice by passive administration of monoclonal anti-IA antibody.

SJL/J (H-2s) mice develop spontaneous reticulum cell sarcoma (RCS) tumors at the age of 8-11 months. The RCS tumor expresses IA antigens on the cell surface, stimulates syngeneic T-cell proliferation, and appears to depend on host cells' participation for its own growth. The present study investigates the role of passively administered monoclonal anti-IA antibody on RCS tumor growth. The administration of monoclonal anti-IAs antibody into SJL/J mice prior to tumor inoculation or at the same time as tumor transplantation resulted in a significant inhibition of tumor growth. Furthermore, pretreatment of RCS tumor with antibody prior to inoculation also resulted in tumor growth inhibition. The inhibition seen in all cases studied was tumor specific, since the use of normal ascites on antibody directed against unrelated antigens resulted in no inhibition of tumor growth. Examination of tumor cells derived from spleen and lymph nodes of antibody treated mice demonstrated that the observed inhibition of tumor growth was the result of a significant depletion of IA positive tumor cells. In contrast to other tumor systems studied to date whereby anti-IA antibody promotes tumor growth, the present findings demonstrate that passive administration of anti-IA antibodies inhibit RCS tumor growth in syngeneic mice. The possible mechanisms involved are discussed.

Animals↗
PubMed1985

Use of a self-generating percoll gradient and single cell cytotoxicity assay to identify tumor-lytic properties of inflammatory neutrophils.

Within a murine model of regional immunotherapy, the cytolytic potential of peritoneal neutrophils could not be confirmed or quantified using routine techniques of cell separation and chromium release assays. We, therefore, developed procedures for the enrichment of neutrophils and estimation of the frequency of killer cells. Peritoneal exudate cells from mice injected with Corynebacterium parvum were fractionated on a self-generating Percoll gradient to enrich for neutrophils and deplete macrophages. A significant enrichment of neutrophils (greater than 90%) was obtained in a band corresponding to a density of 1.088 with a recovery of 35-50% of input. Neutrophil-enriched cell populations were then mixed with tumor cells to examine neutrophil-target interactions at the single cell level. Conjugates of neutrophils and tumor targets were obtained and the majority were lytic. With the aid of trypan blue staining and safranin counterstaining, it was possible to distinguish effector cells from targets and neutrophils from other host cells. The frequency of conjugates was dependent upon the effector to target cell ratio and was not affected by changes in temperature (range 4-30 degrees C). The post-binding lytic events were initiated rapidly after conjugation and tumor lysis was completed within 30 min. The lytic events occurred optimally between 25 degrees and 37 degrees C. The present studies support the role of neutrophils in tumor lysis following administration of an immunoadjuvant. The techniques described are important to further study the role of neutrophils in disease states as well as the underlying mechanisms of neutrophil-mediated tumor cytotoxicity.

Animals↗
PubMed1985

Dose-response effects of 4-hydroperoxycyclophosphamide on human T and B cell function in vitro.

4-Hydroperoxycyclophosphamide (4-OOH-CYP) is spontaneously converted in aqueous solution to 4-hydroxycyclophosphamide (4-OH-CYP), the major active metabolic of cyclophosphamide. We studied the dose related effects of in vitro treatment with 4-OOH-CYP on human T- and B cell-mediated immune responses. T-cell proliferation to mitogens and alloantigens was only partially inhibited even relatively high-doses of 4-OOH-CYP (greater than 6-12 micrograms/ml). In contrast cytotoxic functions of activated T-cells and natural killer (NK) cells were inhibited at lower doses (3-6 micrograms/ml). PWM induced in vitro synthesis of IgG by B-cells was inhibited at less than 3 micrograms/ml of 4-OOH-CYP. These data indicate that 4-HOO-CYP has selective, dose-dependent effects on human T and B cells in vitro.

B-Lymphocytes↗
PubMed1985

Studies on the mechanism of natural killer cell-mediated cytotoxicity. VI. Characterization of human, rat, and murine natural killer cytotoxic factors.

Recent evidence has implicated natural killer cytotoxic factors (NKCF) as the lytic mediators of NK cell-mediated cytotoxicity reactions. The objective of this study was to examine and compare some of the biochemical and functional characteristics of human, rat, and murine NKCF. Supernatants containing NKCF were generated by stimulating effector cells with Con A or U937 (for human PBL) or YAC-1 (for rodent spleen cells) and tested for cytotoxic activity in a 20-hour (rodent) or 24-hour (human) 51Cr release assay. NKCF activity was inactivated by heating to 63 degrees C, 8 M urea, pH 2, and reduction and alkylation. These factors were highly sensitive to trypsin, moderately sensitive to papain and resistant to neuraminidase. Adsorption of human NKCF to U937 cells is inhibited by mannose-6-phosphate and adsorption of rodent NKCF to YAC-1 cells is inhibited by alpha-methyl-D-mannoside and fructose-6-phosphate. Oxidation of NKCF with sodium periodate abolished lytic activity. Pretreatment of NKCF with Con A but not pretreatment of target cells inhibited lytic activity. NKCF activity eluted in a single broad band of apparent MW of 15,000-40,000 after fractionation by HPLC gel permeating chromatography. Pooled fractions containing NKCF activity were subjected to some of the same tests performed on whole supernatants. Test result with semipurified NKCF confirmed that these factors are inactivated by trypsin or sodium periodate and that mannose-6-phosphate inhibits their binding to target cells. There were no major differences observed in NKCF produced by the three different species whether stimulated by Con A or NK-sensitive tumor cells. The evidence indicates that NKCF are glycoproteins in which disulfide bonding is essential for lytic activity. Furthermore, it appears that carbohydrate residues expressed on NKCF molecules are involved in the binding of these factors to the target cell membrane.

Animals↗
PubMed1985

Effect of altered membrane fluidity on NK cell-mediated cytotoxicity. I. Selective inhibition of the recognition or post recognition events in the cytolytic pathway of NK cells.

NK cell-mediated cytotoxicity results from membrane interactions between NK effector and target cells. The role of membrane fluidity in these events is not known. The present study was undertaken to investigate the effect of changes in membrane lipid fluidity of NK effector and NK-sensitive target cells on the lytic pathway of NK cell-mediated cytotoxicity. Fluidity was modulated by various lipids and measured by fluorescence polarization. NK effector cells treated with phosphatidylcholine complexed with polyvinylpyrrolidone (PVP) and bovine serum albumin (BSA) showed increased membrane fluidity. This fluidization of the effector cell membrane resulted in a significant inhibition of cytotoxic activity in the 51Cr-release assay. Single cell analysis revealed that the inhibition was due to a decrease in the frequency of NK target conjugates and reduced killing of conjugated targets. Rigidification of the NK effector cell membranes by treatment with cholesteryl hemisuccinate complexed with PVP and BSA also resulted in inhibition of cytotoxicity. This inhibition was post binding, because binding was increased and lysis was abrogated. Fluidization of K562 target cell membranes caused a slight but insignificant increase in their lysis by NK cells without affecting the binding step. On the other hand, rigidification of K562 membranes decreased the sensitivity of these target cells to lysis. Single cell analysis revealed that this inhibition of NK lysis is post binding, because the frequency of killers was significantly decreased. It was also shown that membrane rigidification of target cells that were programmed for lysis during the lethal hit stage and subsequently separated from effector cells, rendered the programmed cells resistant to killing during the killer cell-independent lysis step. These results demonstrate that fluidization or rigidification of the plasma membrane of either effector or target cells affect different stages of the NK cell-mediated cytolytic events.

Adult↗
PubMed1985

Studies on the induction and expression of T cell-mediated immunity. XV. Role of non-MHC papain-sensitive target structures and Lyt-2 antigens in allogeneic and xenogeneic lectin-dependent cellular cytotoxicity (LDCC).

The present study examines the role of nonclass I MHC target membrane structures involved in lysis by cytotoxic lymphocytes in the lectin-dependent cellular cytotoxicity (LDCC) system. Cytotoxic cells of rodents and humans have been shown to nonspecifically lyse target cells of different origins and species. The mechanism by which such cytotoxicity takes place is not known, although several hypotheses have been put forth. This study examines the role of class I MHC expression on target cells and Lyt-2 antigens on the cytotoxic cells in both allogeneic and xenogeneic LDCC systems. Human peripheral blood lymphocytes, and murine-allosensitized cytotoxic T lymphocytes (CTL) are used as effector cells, and class I MHC-negative Daudi and class I MHC-positive Raji human target cells are used. The studies reveal that in LDCC, human lymphocytes lyse HLA-negative Daudi targets to the same extent as HLA-positive Raji targets. This was shown by the 51Cr-release assay as well as the single cell assay. Furthermore, allosensitized murine CTL lyse Daudi and Raji in LDCC, demonstrating that lysis of class I negative targets is achieved by xenogeneic CTL. Target cells treated with papain were found to be resistant to lysis in LDCC. However, incubation of papain-treated targets for 1 to 2 hr resulted in recovery of cytotoxicity. In the single cell assay, the papain treatment of targets slightly reduces the frequency of binders but significantly reduces the frequency of killers, revealing that the papain-sensitive structures are essential during the lethal hit stage of lysis. Monoclonal anti-Lyt-2 antibody inhibits lysis of both Daudi and Raji targets by murine CTL. The lack of discrimination between mouse and human targets by murine CTL suggests that the Lyt-2 molecule may have a different role than merely interacting with the target cell. In the LDCC system, we propose that class I MHC molecules are not essential for lysis. Several papain-sensitive and -insensitive target cell structures may participate in the cytolytic mechanism, and we propose that both the lectin and Lyt-2 molecules are involved in the perturbation of cell membrane conformation essential for lysis.

Animals↗
PubMed1985

Regulation of reticulum cell sarcoma tumor growth in SJL/J mice by a serum inhibitor affecting T-cell proliferation.

The reticulum cell sarcoma (RCS) tumor in SJL/J mice has been shown to stimulate a strong syngeneic proliferative response. A unique characteristic of the RCS tumor is that it requires host T-cells for growth. Consequently, factors that inhibit host T-cell proliferation may have a profound effect on tumor cell growth in vivo. This report investigates the relationship between a serum inhibitor of T-cell proliferation and tumor growth. Inhibition was measured in an interleukin 2-dependent proliferation of the CTLL-2 line. Sera from mice with RCS transplantable lines or from mice with spontaneous tumors were much less inhibitory than were sera from normal syngeneic SJL/J mice or from allogeneic mice. The inhibitory activity appears to follow a circadian rhythm, because serum derived in the morning was more inhibitory than was serum derived in the evening. Serum from female mice was less inhibitory than serum from male mice. In contrast to male mice, serum from 35-week-old female mice was as inhibitory as was that from young (8- to 12-week-old) mice. The mechanism of serum inhibition in tumor-bearing mice was examined. The serum was tested for the presence of interleukin 2 which could decrease inhibitory activity, and no interleukin was found. Furthermore, absorption of the serum with CTLL-2 cells did not enhance the inhibitory effect to the level of normal mouse serum. These results suggest that tumor growth in vivo coincides with less serum inhibitor, providing an adequate T-cell response requisite for tumor growth. This corroborates the notion of RCS-host T-cell interaction necessary for tumor growth which is in part regulated by a serum inhibitor.

Animals↗
PubMed1985

Non-H-2-linked control of in vivo growth of SJL/J-derived reticulum cell sarcoma in recombinant inbred strains between BALB/cKe and SJL/J mice.

This report investigated the growth of reticulum cell sarcoma (RCS) in several congenic and recombinant inbred strains between BALB/cKe female (H-2d) and SJL/J male (H-2s) mice. SJA20 mice congenic with SJL/J except at the Igh locus supported RCS growth. Recombinant mice of the H-2d haplotype did not support RCS growth. However, recombinant inbred mice of the H-2s haplotype varied in their susceptibility to permit RCS growth in vivo. These results supported the role of non-H-2 gene(s) controlling the growth of RCS. Since the recombinant strains of mice exhibited different immunologic characteristics and since RCS tumor growth depended on the ability of the mice to develop a strong antitumor proliferative response, the findings reported here suggested that non-H-2 genes control the magnitude of the syngeneic proliferative response and consequently regulate RCS growth in vivo.

Animals↗
PubMed1984

Mechanism of cell-mediated cytotoxicity at the single-cell level. VII. Trigger of the lethal hit event is distinct for NK/K and LDCC effector cells as measured in the two-target conjugate assay.

Normal human peripheral blood lymphocytes (PBL) express several in vitro cytotoxic functions, among which are natural killer (NK), antibody-dependent cellular cytotoxicity (ADCC), and lectin-dependent cellular cytotoxicity (LDCC). The relationship of these various cytotoxic functions and the identity of cells involved has been a subject of controversy. Recently it was reported that NK and K for ADCC can be mediated by the same cell, suggesting that they constitute in large part a single subpopulation with multiple cytotoxic functions. The ability of this NK/K effector cell to mediate LDCC was examined here using the two target conjugate assay. The effector cells were Ficoll-Hypaque PBL or LGL-enriched fractions. The targets used were K562 or MOLT for NK, RAJI coated with antibody for ADCC, and RAJI coated with PHA or Con A or modified by NaIO4 for LDCC. In the two-target conjugate assay, one of the targets is fluorescein labeled for identification. The results show that (a) LDCC copurifies with NK/K and is enriched in the LGL fraction, as measured in both the 51Cr-release assay and the single-cell assay for cytotoxicity; (b) single effector cells simultaneously bind to NK or ADCC and LDCC targets, revealing that single cells bear binding receptors for all targets; and (c) single lymphocytes were not able to kill both bound NK/K and LDCC targets. However, significant two-target killing was obtained when both targets were NK targets, ADCC targets, LDCC targets, or one NK and one ADCC target. These results demonstrate that the NK and LDCC effector cells are distinct subpopulations copurified in the LGL fraction. In addition, the results show that lectin is unable to trigger globally an NK effector cell to mediate cytotoxicity against a bound NK insensitive target. Thus, although both NK and LDCC effector cells are present in the LGL fraction and can bind to both types of targets, the trigger of the lethal hit event is the function of specialized effector cells.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1984

Enzyme-linked immunosorbent assay for determination of HLA: gene dose effect.

In a previous publication we demonstrated that polymorphic HLA antigens could be detected on fresh and dried peripheral blood lymphocytes using the enzyme-linked immunosorbent assay (ELISA) with HLA alloantisera (Bishara et al. 1983). In the present study we investigated whether the ELISA technique can be used in determination of gene-dose effect for antigens of the HLA-A and B loci. Lymphocytes from HLA-A1 and HLA-B14 homozygous individuals are shown to bind significantly more anti-HLA alloantibodies than their heterozygous siblings for the same HLA antigens. These results indicate that ELISA is a sensitive and reliable technique for the qualitative and quantitative assay of polymorphic HLA determinants.

Adult↗
PubMed1984

Mechanism of inhibition of human natural killer activity by ultraviolet radiation.

Ultraviolet radiation (UVR) has been shown to inhibit various immune functions in vivo and in vitro. We have confirmed that UVR inhibits human natural killer (NK) activity in vitro and have shown that UVR inhibits human ADCC. In this report, the mechanism by which UVR inhibits NK function was investigated by analyzing the stage at which the inhibiting activity occurs and the ability of the NK cells to release cytotoxic factors previously shown to be involved in CMC. Single cell assays in Agarose revealed that inhibition of NK activity was localized at the postbinding lethal hit stage rather than the initial recognition or binding stage of lysis. We then examined whether UV-treated cells were able to release cytotoxic factors after stimulation with target cells. As expected, stimulated cells released cytotoxic factors, yet, surprisingly, these factors were also released in the absence of stimulator cells. The spontaneous release was detectable in the supernatants as early as 30 min after UV irradiation. The lytic material examined in 48- to 72-hr viability assays was not NK specific, because lysis was obtained with a wide range of NK sensitive and resistant target cells. These results demonstrate that UVR does not alter the capacity of the cells to secrete cytotoxic material, but in fact enhances its release. Several possible mechanisms are proposed to explain the UVR-induced NK inhibition.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1984

Studies on the mechanism of natural killer cell-mediated cytotoxicity. V. Lack of NK specificity at the level of induction of natural killer cytotoxic factors in cultures of human, murine, or rat effector cells stimulated with mycoplasma-free cell lines.

We have proposed that lysis of target cells by NK cells is mediated by NK cytotoxic factors (NKCF). According to our model, for a target cell to be NK-sensitive, it must be recognized by the NK cell, it must stimulate the release of NKCF, and it must be sensitive to lysis by these factors. This report examines whether the ability to stimulate release of NKCF is a characteristic restricted to NK-sensitive tumor cells or whether it is also a property of NK-resistant target cells. Many different types of cell lines were tested for their ability to stimulate release of NKCF in the human, rat, and murine systems. It was found that mycoplasma-free NK-sensitive cell lines, resistant cell lines, and Con A could stimulate the release of NKCF. Many different types of cell lines grown in suspension or in monolayers were found to be effective stimulators, including T or B lymphoid, myeloid, and those of histiocytic origin. Cells cultured in the absence of serum stimulated NKCF release, thus ruling out the possible involvement of serum components in stimulation. NKCF was also produced by xenogeneic combinations of effector and stimulator cells, demonstrating lack of species specificity in NKCF production. Factors stimulated by NK-resistant cell lines or by Con A exhibited the same NK target specificity as supernatants stimulated by NK-sensitive tumor cells. The finding that many different NK-resistant cell lines can stimulate the release of NKCF indicates that there is no apparent NK specificity at the level of induction of NKCF release from human, rat, or murine effector cells. Therefore, the NK specificity of a target cell is determined ultimately by its sensitivity to lysis by NKCF.

Animals↗
PubMed1984

Mechanism of cell-mediated cytotoxicity at the single cell level. VI. Direct assessment of the cytotoxic potential of human peripheral blood non-lytic effector-target cell conjugates.

Single cell cytotoxicity assays reveal that a large percentage of lymphocytes are unable to kill attached targets in a 4- to 18-hr assay. Additional signals (in the form of lectin or anti-target antibody) delivered to target-bound lymphocytes enable these previously non-lytic lymphocytes to kill attached target cells. This finding was obtained by using a modification of the single cell assay, in which lectin or target cell antibody is incorporated into agarose with preformed lymphocyte-target conjugates. Human peripheral blood lymphocytes (PBL) or Percoll density gradient-enriched large granular lymphocytes (LGL) were used as effector cells in natural killer (NK), antibody-dependent cellular cytotoxicity (LDCC) assay systems. The targets used were NK-sensitive K562 and Molt-4 and NK-insensitive Raji. Several findings were made in the modified single cell assay, namely a) the frequency of cytotoxic NK or ADCC effector cells was not augmented, suggesting that the initial trigger was sufficient for lytic expression in these instances. Furthermore, these results showed that the NK-sensitive targets used do not bind nonspecifically to the LDCC effector cells. K562 coated with Con A, however, serve as LDCC targets. b) The frequency of two target conjugate lysis by NK/K effectors was not augmented by Con A. These results suggest that Con A does not potentiate the killing of multiple targets bound to a single cytotoxic lymphocyte. c) Although conjugates formed between LGL or PBL and NK-insensitive Raji are non-lethal, significant lysis was observed when these conjugates were suspended in Con A or antibody agarose. These results demonstrate that Raji bind to cytotoxic NK, K, and LDCC effector cells, but are lysed only when the appropriate trigger is provided. d) The cytotoxic potential of non-lytic conjugates appears to lie within the low density Percoll fraction, although the high density lymphocytes are able to nonlethally bind to targets. Altogether the results demonstrate that target recognition and/or binding by the effector cells is a distinct event from the trigger or lytic process. The implications of these findings are discussed.

Animals↗