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S C Wright

Publications and source records attributed to S C Wright.

70 records · Page 4Linked to original sources
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↗
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↗
PubMed1983

Partial purification of estradiol receptor from Xenopus laevis liver and levels of receptor in relation to estradiol concentration.

We have used ammonium sulphate precipitation followed by affinity chromatography to partially purify the estrogen receptor from Xenopus laevis liver which may control the genes for vitellogenin, the precursor of the egg yolk proteins. The rate at which receptor binds estradiol explains the kinetics of the induction of vitellogenin synthesis by estradiol, and the dissociation constant (0.5 X 10(-9) M) explains the concentration dependence of the response, which has a threshold of 10(-9) M estradiol, when 67% of the receptor is bound to estradiol. The estradiol concentration in male liver, which does not make vitellogenin, is 0.18 X 10(-9) M, sufficient to saturate 26% of the receptor, while in female liver, which makes vitellogenin continuously, the estradiol concentration is 3.5 X 10(-9) M, giving 88% saturation of receptor, suggesting that the proportion of occupied receptor decides whether or not the vitellogenin genes are active. In the physiological concentration range, estradiol modulates the level of receptor, which varies between 100 binding sites per nucleus in males and 440 in females, but artificially high concentrations of estradiol raise the level to approximately 1000 sites per nucleus. This suggests that the small increase in vitellogenin mRNA induced by physiological concentrations of estradiol is due to pre-existing receptor and that the much larger increases induced by very high concentrations depends on newly-synthesized receptor.

Animals↗
PubMed1983

YAC-1 variant clones selected for resistance to natural killer cytotoxic factors are also resistant to natural killer cell-mediated cytotoxicity.

The possible involvement of natural killer cytotoxic factors (NKCF) in the lytic mechanism of natural killer (NK) cell-mediated cytotoxicity (CMC) was investigated by studying the mechanism of NK resistance of variant clones of the YAC-1 cell line. The NK-resistant YAC-1 (YAC-R) clones were generated by prolonged culture of the initially NK-sensitive YAC-1 cell line in the presence of NKCF. The YAC-R clones were resistant to lysis by NKCF as well as lysis by NK cells in a CMC assay. The defect was specific for NK CMC because the YAC-R clones could still be lysed by alloimmune cytotoxic T lymphocytes. Experiments to determine the mechanism of NK resistance of the YAC-R clones indicated that they still possessed the NK recognition structures because they formed a normal number of conjugates with murine spleen cells. In addition, the YAC-R clones, like the parental YAC-1 cell line, were able to stimulate the release of NKCF during co-culture with spleen cells. However, the YAC-R clones, in contrast to YAC-1 cells, were unable to adsorb NKCF from cell-free supernatants of such cultures. Thus, it appears that the YAC-R clones are NK resistant due to a deficiency of NKCF binding sites. The present studies demonstrate that a NK-sensitive target cell must not only be recognized by the NK cell and stimulate release of NKCF but it must also bind NKCF for cell lysis to ultimately result. These findings support our model for the mechanism of NK CMC in which it is proposed that target cell lysis is mediated by NKCF released from the effector cell.

Animals↗
PubMed1983

Studies on the mechanism of natural killer cytotoxicity. III. Activation of NK cells by interferon augments the lytic activity of released natural killer cytotoxic factors (NKCF).

The mechanism by which interferon (IFN) pretreatment of effector cells augments natural killer (NK) cell-mediated cytotoxicity (CMC) was examined by determining whether IFN has any effect on the production of natural killer cytotoxic factors (NKCF). NKCF are released into the supernatant of co-cultures of murine spleen cells and YAC-1 stimulator cells, and their lytic activity is measured against YAC-1 target cells. It was demonstrated that pretreatment of effector cells with murine fibroblast IFN or polyinosinic-polycytidylic acid (pIC) resulted in the release of NKCF with augmented lytic activity. Evidence indicated that the IFN-induced augmentation of NKCF activity required protein synthesis during the IFN pretreatment period, because concurrent pretreatment with both IFN and cycloheximide abrogated the IFN effect. Protein synthesis, however, is not required for the production of base levels of NKCF because emetine pretreatment of normal spleen cells did not result in a decrease in NKCF production. Furthermore, substantial levels of NKCF activity could be detected in freeze-thaw lysates of freshly isolated spleen cells. Cell populations enriched for NK effector cells, such as nylon wool-nonadherent nude mouse spleen cells, produced lysates with high levels of NKCF activity, whereas lysates of CBA thymocytes were devoid of NKCF activity. Pretreatment of spleen cells with either IFN or pIC resulted in an augmentation of the NKCF activity present in their cell lysates. Taken altogether, these findings suggest that freshly isolated NK cells contain preformed pools of NKCF. Pretreatment of these cells with IFN causes de novo synthesis of additional NKCF and/or activation of preexisting NKCF. According to our model for the mechanism of NK CMC, target cell lysis is ultimately the result of transfer of NKCF from the effector cell to the target cell. The evidence presented here suggests that the IFN-induced augmentation of NK activity could be accounted for by an increase in the synthesis, activation, and/or release of NKCF.

Animals↗
PubMed1983

Studies on the mechanism of natural killer cell-mediated cytotoxicity. IV. Interferon-induced inhibition of NK target cell susceptibility to lysis is due to a defect in their ability to stimulate release of natural killer cytotoxic factors (NKCF).

The effects of interferon (IFN) at the level of the target cell in the natural killer cytotoxic factors (NKCF) system have been examined to determine whether they correlate with the effects of IFN in natural killer (NK) cell-mediated cytotoxicity (CMC). NKCF are released into the supernatant of co-cultures of murine spleen cells and YAC-1 stimulator cells, and their lytic activity is measured against YAC-1 target cells. It was found that IFN-pretreated YAC-1 target cells were still sensitive to lysis by NKCF, although their ability to stimulate release of NKCF from murine spleen cells was impaired. This defect was not due to a lack of NK target structures, because IFN-pretreated YAC-1 cells form a normal number of conjugates with spleen cells. The defect also could not be attributed to an IFN-induced inhibition of protein synthesis, because emetine-pretreated YAC-1 stimulator cells can still induce the release of high levels of NKCF. Cellfree supernatants containing NKCF also contain low levels of endogenous IFN (10 to 50 U/ml). Although this concentration of IFN itself is not toxic to YAC-1 cells, it may still interact with NKCF and/or the target cell to enhance cytotoxicity mediated by NKCF. Evidence in support of this possibility is derived from experiments that demonstrated that addition of exogenous IFN to NKCF resulted in a synergistic enhancement of cytotoxicity. Taken together, these findings can account for the IFN-induced inhibition of target cell susceptibility to lysis in the NK CMC system. In addition, the evidence supports our model for the role of NKCF in the mechanism of NK CMC. According to this model, the effector cell must first bind to the target cell; then the target cell delivers a signal to the effector cell to activate the NKCF release mechanism. The effector cell releases NKCF that then bind to the target cell and mediate cell lysis. The mechanism by which IFN-pretreated target cells become relatively NK-resistant appears to be a defect in their ability to stimulate the effector cell to release NKCF after the initial effector-target cell binding.

Animals↗
PubMed1983

Studies on the mechanism of natural killer cytotoxicity. II. coculture of human PBL with NK-sensitive or resistant cell lines stimulates release of natural killer cytotoxic factors (NKCF) selectively cytotoxic to NK-sensitive target cells.

This investigation has employed the "innocent bystander" type of experimental design to determine whether soluble cytotoxic factor(s) are released during interactions between human peripheral blood lymphocytes (PBL) and NK-sensitive target cells. PBL cocultured with NK-sensitive Molt-4 or K562 target cells in the lower well of a miniaturized Marbrook culture released natural killer cytotoxic factors (NKCF), which diffused across a 0.2-mu Nucleopore membrane and lysed Molt-4 or K562 target cells cultured in the upper chamber. Coculture of PBL with the NK-resistant Raji or WI-L2 cell lines also induced release of NKCF. These factors were selectively cytotoxic to NK-sensitive targets and lysed Molt-4 and, to a lesser extent, K562 cells. However, Raji, WI-L2, and RPMI 1788 cells were all resistant to lysis. In addition, low density fractions from Percoll density gradients that were enriched for NK effector cells also released increased levels of NKCF during coculture with Molt-4 cells. Lysis of Molt-4 and K562 targets was observed after exposure to NKCF for 48 hr and 60 to 70 hr, respectively. Cellfree supernatants containing NKCF were obtained after a short time of incubation (i.e., within 5 hr of coculture of PBL with NK target cells). The factors were nondialyzable, stable at 56 degrees C for 3 hr, and showed partial loss of activity on storage at 4 degrees C or -20 degrees C for 7 days. These data suggest that NKCF may be involved in the lytic mechanism of human NK cell-mediated cytotoxicity.

Cell Line↗
PubMed1982

Soluble cytotoxic factors and the mechanism of NK cell mediated cytotoxicity.

Soluble cytotoxic factors from mouse spleen cells have been shown to selectively lyse NK sensitive target cells. Lysis of target cells is assessed by trypan blue uptake or 51Cr--release assay in a 16-48 hour assay. The possible role of such natural killer cytotoxic factors (NKCF) in the mechanism of natural killer cell-mediated cytotoxicity (NKCMC) has been examined. Several lines of evidence are presented which indicate that there exists a strong correlation between lysis by NKCF and lysis in NKCMC. For instance, (1) NKCF are generated following stimulation of mouse spleen cells with NK sensitive targets; (2) Lysis of NKCF is selective for NK sensitive targets and is species specific; (3) Mice with poor NK activity, such as Bg/Bg mice, produce poor NKCF: (4) There is concomittant inhibition of NKCMC and NKCF activities by blocking RAT serum; and (5) Several known characteristics of the mechanism of NKCMC are shown to be shared in the NKCF system. Based on these findings, we propose a model for NKCMC in which lysis by NK effector cells is the result of multiple steps, namely target binding to an NK effector cell, activation of the lytic mechanism, and involvement of NKCF to mediate lysis. Accordingly, for targets to be NK sensitive, they ought to be able to interact and bind with NK effectors, activate the NK cells, bind NKCF, and be sensitive to the NKCF lytic activity.

Animals↗
PubMedDate not supplied

Distal splenorenal shunt vs. portal-systemic shunt: current status of a controlled trial.

A prospective controlled comparison of portal-systemic (PSS) and distal splenorenal shunts (DSRS) in cirrhotic patients who had survived hemorrhage from esophagogastric varices was undertaken 5 yr ago at five hospitals by the Boston-New Haven Collaborative Liver Group. The clinical and endoscopic criteria for massive hemorrhage were satisfied in 155 patients. Thirty-four patients were excluded, primarily because of uncontrolled hemorrhage. Thirty-four were rejected because the were poor operative risks and 21 because they did not satisfy criteria. Thirteen patients refused to participate; the remaining 53 were randomized; 29 to receive PSS and 24, DSRS. The two groups were similar in clinical, laboratory, and manometric characteristics. The DSRS group was older and tended to have had more previous hemorrhages. Followup ranged from 1 to 56 months (mean 21). After PSS, which was performed by 10 different surgeons, 6 patients died during the hospital admission (21%) compared to 2 after DSRS (12%). There were 6 late deaths in the PSS group and 4 in the DSRS group. Portal-systemic encephalopathy occurred in 5 of the 23 survivors of PSS (23%), and in 6 of the 19 who survived DSRS (32%. Two patients in the PSS group bled (9%), 1 after thrombosis and 1 after stenosis of the shunt. Three patients in the DSRS group bled (16%) and all had thrombosis of the shunt. PSS was associated with an unexplained but inordinately high operative mortality. Although the DSRS was accomplished with an acceptably low operative mortality, it was associated with frequent portal-systemic encephalopathy, shunt occlusion, and recurrent hemorrhage. Similar incidences of portal-systemic encephalopathy, shunt occlusion, and recurrent hemorrhage were observed in the PSS group. More patients and longer followup are necessary to determine which of these portal decompressive procedures is superior.

Clinical Trials as Topic↗
PubMedDate not supplied

Distal splenorenal vs. portal-systemic shunts after hemorrhage from varices: a randomized controlled trial.

Between 1975 and 1983, 303 cirrhotic patients with endoscopically proven major variceal hemorrhage were admitted to the participating hospitals of the Boston-New Haven Collaborative Liver Group. Of these, 274 were evaluated for admission to a prospective, randomized controlled trial comparing portal-systemic shunts with distal splenorenal shunts. The criteria for inclusion were as follows: (i) a portohepatic pressure gradient greater than or equal to 12 mmHg; (ii) angiographic evidence of antegrade portal venous flow; (iii) angiographic demonstration that the inferior vena cava and portal, splenic and left renal veins were anatomically suitable for either a portal-systemic or distal splenorenal shunt, and (iv) the patient was a reasonable operative risk. Eighty-one patients from the six participating hospitals fulfilled the criteria and consented to participate. Thirty-eight patients were randomly assigned to have portal-systemic shunt and 43 to have distal splenorenal shunt. After a follow-up period of 11 years (mean = 3.5 years for all patients), survival was found to be similar in the two groups of patients. The 30-day operative mortality was 13% for the portal-systemic shunt group and 9% for the distal splenorenal shunt patients. Late mortality was 55% for the portal-systemic shunt and 37% for the distal splenorenal shunt group. Total mortality was 68% for the portal-systemic shunt and 46% for the distal splenorenal shunt group. None of these differences is statistically significant. In those patients who survived greater than 30 days after surgery, recurrent variceal hemorrhage occurred in four (12%) in the portal-systemic shunt group compared to seven in the distal splenorenal shunt group (18%) (NS).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗