Biomedical subjects
Publications and source records attributed to J A Roth.
Lymphocytes from 8 Holstein heifers were evaluated during the periparturient period for mitogen-induced lymphocyte blastogenic responses. Phytohemagglutinin P- and concanavalin A-induced lymphocyte blastogenesis increased 2 and 3 weeks before parturition, respectively. However, by the first week after parturition, lymphocyte blastogenesis was markedly impaired.
The immunological characterization of the different forms of phenol sulfotransferase (PST) in a variety of human and nonhuman tissues is described. Immunoblotting techniques revealed that polyclonal antibodies raised to human platelet MII-PST reacted with polypeptides of 32 and 34 kDa from human platelet 100,000 x g supernatant solution. Immunoblot analysis of platelet 100,000 x g supernatant solution that was fractionated over a DEAE-cellulose column indicated a close correspondence of P-PST activity, as measured by phenol sulfation, and M-PST activity, as assessed by dopamine sulfation, with the 32 and 34 kDa polypeptides, respectively. Examination of various human tissues revealed the presence of immunologically detectable levels of P-PST in liver and adrenal gland whereas both M- and P-PST were detected in placenta at a 1/10,000 dilution of the antisera. Under these conditions, PST was undetectable in human frontal cortex, pituitary gland, kidney, lung, and jejunum. Further evaluation of human liver samples from four individuals indicated a strong correlation (r = 0.94) between the amount of 32-kDa immunoreactive protein and P-PST activity. Analysis of liver samples from several animal species (monkey, rat, mouse, guinea pig, and frog) revealed the presence of immunoreactive proteins of various molecular masses, suggesting that considerable homology may exist between human and nonhuman forms of PST.
Twenty Holstein steers subclinically infected with coccidia were allotted to 2 groups of 10 steers each. One group received a diet containing 0.5 mg of decoquinate/kg of body weight. After 25 days on the diet, there was no difference between the groups in lymphocyte blastogenic responsiveness to mitogens; however, there were differences in neutrophil function. Lymphocytes from steers of the decoquinate-fed group had decreased random migration under agarose, enhanced cytochrome C reduction, and enhanced iodination activity. Other measures of neutrophil function evaluated (chemotactic index, Staphylococcus aureus ingestion, and antibody-dependent and -independent cell-mediated cytotoxicity) were not affected. After 30 days of decoquinate feeding, half of the cattle in each group received 5 daily IM injections of dexamethasone (0.04 mg/kg of body weight). The dexamethasone-treated steers from the group that did not have decoquinate in the diet developed clinical coccidiosis, whereas the decoquinate-treated steers remained clinically normal. Lymphocyte and neutrophil function were again evaluated for a 3-day period beginning 4 days after dexamethasone treatment was halted. Neutrophils from the steers that developed clinical coccidiosis after dexamethasone administration had significantly (P less than 0.05) inhibited random migration under agarose, cytochrome C reduction, and iodination activity, but significantly (P less than 0.01) enhanced S aureus ingestion. The feeding of decoquinate prevented the inhibition of neutrophil cytochrome C reduction and lessened the inhibition of neutrophil iodination in the dexamethasone-treated group. Dexamethasone treatment was associated with an inhibition of lymphocyte blastogenic responsiveness to phytohemagglutinin in principals as well as controls.
The sulfoconjugation of tyrosyl residues is a widespread post-translational modification of biologically active proteins and peptides. The rat liver Golgi enzyme tyrosylprotein sulfotransferase has previously been shown to catalyze the transfer of a sulfate moiety from 5'-phosphoadenosine-3'-phosphosulfate to the synthetic acidic polymer poly(Glu6, Ala3, Tyr1) (EAY). In this study, we further characterized the biochemical properties and the substrate specificity of rat liver tyrosylprotein sulfotransferase using a variety of synthetic peptides, including EAY, tert-butoxycarbonylcholecystokinin-8 (Boc-CCK), and two carboxy terminal peptide fragments of complement component C4. The data demonstrate that all substrates can be sulfated by an isolated Golgi membrane fraction. In addition, Boc-CCK was also found to be sulfated by a cytosolic sulfotransferase. Using an enriched Golgi fraction, rat liver tyrosylprotein sulfotransferase sulfation of Boc-CCK displayed a pH optimum between 6.0 and 6.5, was stimulated by 50-150 mM NaCl, and required either Mn2+ or Mg2+ for maximal activity. In contrast, EAY sulfation displayed a pH optimum of 6.7, was not stimulated by NaCl, and required Mn2+ for maximal activity. Both Boc-CCK and EAY sulfation were similarly decreased when the enriched Golgi preparation was preincubated at 45 degrees for varying lengths of time. The particulate tyrosylprotein sulfotransferase could be effectively solubilized by 1% Triton X-100 in the presence of 0.2 M NaCl and anion exchange chromatography of the solubilized enzyme yielded a single peak of Boc-CCK- and EAY-sulfating activities. Rat liver tyrosylprotein sulfotransferase was also shown to sulfate a variety of other acidic and basic synthetic tyrosine-containing polymers, as well as two synthetic peptides of 10 and 16 amino acid residues that corresponding to the C-terminal portion of C4.
The use of preoperative computed tomographic (CT) scans of the chest in carcinoma of the esophagus to associate preoperative staging with postoperative survival is controversial. Thirty-two patients who underwent esophagectomy and reconstruction were examined with respect to a variety of variables available on preoperative evaluation, including lesion width on CT scan, lesion length on CT scan, and barium esophagogram. Each variable was evaluated for its ability to predict the presence of extraesophageal spread of tumor, as determined pathologically by logistic regression analysis. Lesions greater than 3.0 cm in width by CT scan were associated with a significantly higher incidence of extraesophageal spread. Tumor location, gender, histologic grade, presence of in situ lesions, vascular invasion, eosinophilia, nodal metastases, and preoperative chemotherapy had no predictive value for survival. Duration of survival was affected by the presence of esophageal spread of disease and lesion width. These results indicate that preoperative CT scanning can predict extraesophageal spread of tumor, and this is associated with overall survival.
We developed a model to assess the therapeutic effects of the 45-2D9-ricin A-chain immunotoxin (RTA) on pulmonary metastases. The 45-2D9 mouse monoclonal antibody recognizes a Mr 74,000 glycoprotein highly expressed by rat fibroblasts transformed with the Kirsten sarcoma virus (transformed rat fibroblasts). These cells metastasize spontaneously and form lung colonies in nu/nu and irradiated BALB/c mice. Injection i.v. of 45-2D9-RTA specifically reduced formation of spontaneous pulmonary metastases and lung colonies originating from freshly disaggregated tumor cells or cultured cells. Antibody alone or mixed with unconjugated ricin A chain and an immunotoxin that recognizes a melanoma-associated antigen were ineffective. Unconjugated 45-2D9 antibody specifically blocked the 45-2D9-RTA activity in vivo. Administration of the lysosomotrophic agents ammonium chloride and chloroquine in vivo did not potentiate immunotoxin-mediated reduction in lung colonies although they were effective in vitro. Monensin potentiated 45-2D9-RTA activity in vitro and in vivo.
Seven cell specific marker enzymes in brain and optic nerve and morphological evaluation by light microscopy were used to characterize the neurotoxicity associated with exposure of rats to hexachlorophene (HCP; 40 mg/kg/day, po, for 9 days). In vitro exposure to HCP at concentrations up to 100 microM had no direct inhibitory effect on the marker enzymes, validating their use in evaluating brain function in vivo. Rats exhibited a reduction in body weight gain, weakness, and ataxia of the hind limbs by the ninth day of HCP exposure. At 24 hr following the last day of exposure to HCP, the activities of the three neuron specific enzymes, glutamic acid decarboxylase, tyrosine hydroxylase, and choline acetyltransferase, in rat brain were unchanged from those of the vehicle-treated control group. Of the two astroglial enzyme markers measured, a small but significant increase was observed in the activity of nonneuronal enolase in the cerebellum and glutamine synthetase in the hippocampus of HCP-treated rats. The optic nerve appeared to be the most sensitive tissue in that the activity of both the astroglial marker, nonneuronal enolase, and the myelin marker, 2',3'-cyclic nucleotide phosphohydrolase, was significantly decreased following HCP exposure. This decrease in enzyme activity is consistent with the histological observations demonstrating extensive vacuolization and edema in the optic nerve after exposure to HCP.
Between 1970 and 1986, 49 patients had resection of presumed pulmonary metastases from malignant melanoma. Sixteen patients were found to have benign disease only despite the appearance of a new nodule in 13. Patients with benign disease had a significantly longer mean survival (169 months) compared with the group with malignant disease (22 months). Median survival for all patients with malignant disease was 13 months. Survival after resection did not correlate with the Clark level of the original lesion, lymph node status, disease-free interval, or number of nodules on preoperative tomograms. Two of 10 patients with 1 nodule resected are long-term survivors (88 and 120 months). Exploration in patients with presumed pulmonary metastases from melanoma is justified to rule out benign disease even if a new solitary nodule is detected. There are no prognostic indicators predicting survival after resection of melanoma metastases, and a significant number of patients will have benign disease.
The chronic effects of oral leucine and leucine metabolites on sheep immune function were determined in two experiments. In replicate experiments, 30 mixed-breed ram lambs were individually fed diets supplemented with approximately 0.05% ruminally protected limestone (control), alpha-ketoisocaproate (KIC), isovalerate (IVA) or leucine (Leu). Serum titers of antibodies produced in response to Brucella abortus antigen and porcine red blood cells were determined. Mitogen-stimulated lymphocyte blastogenesis was determined in experiment 2 by adding phytohemagglutinin (PHA), concanavalin A (Con A) or pokeweed mitogen (PWM) to isolated lymphocytes and measuring [3H]thymidine incorporation. In both experiments, in lambs fed Leu, antibody production to porcine red blood cells was approximately 80% (P less than 0.05) of that in control animals. When KIC was fed, antibody titers to porcine red blood cells were approximately 120% (P less than 0.05) of that of controls. Compared to controls background lymphocyte blastogenesis was higher when KIC was fed, whereas background blastogenesis was lower when Leu was fed (KIC vs. Leu; P less than 0.05). IVA did not significantly affect either measurement. These data indicate that feeding Leu may adversely affect immune function by suppressing lymphocyte activity, whereas oral administration of KIC has a positive influence on immune function in sheep by increasing lymphocyte activity.
A 23-year-old man who presented clinically with unilateral gynecomastia was found to have secretory breast carcinoma. The patient is free of disease 4 years following a modified radical mastectomy. The indolent biological potential of this rare variant of primary mammary carcinoma is evidenced by the long clinical history of a breast mass in this patient and the absence of axillary lymph node metastases. Although secretory carcinoma occurs both in children and adults, most examples have been reported in female patients.
A pure "pattern-recognition" approach to lung diseases in immune compromised patients as seen on the chest film is of limited value since any infection can present with several different patterns, and processes such as embolism, aspiration, edema, and hemorrhage may give appearances similar to infection. However certain pulmonary infections do occur commonly in association with one type of immunosuppression but rarely with others. Knowledge of these associations assists in narrowing down the differential diagnosis and in deciding upon the most appropriate next confirmatory diagnostic step.
Subcellular preparations from rat liver, brain, and kidney and from human liver were tested for their ability to desulfate T3 sulfate (T3SO4). Activity was found associated with the microsomal fraction: rat liver was the most active, hydrolyzing 76 pmol/min.mg protein of T3SO4 while preparations from rat kidney and brain were about 1/5 and 1/20 as active. Microsomal preparations from human liver obtained at autopsy were as active as fresh rat preparations. Thyroxine sulfate was not an active substrate. Microsomes prepared with dithiothreitol and EDTA in order to detect deiodinating activity maintained T3SO4-desulfating activity. Cytosolic preparations containing arylsulfatase activities failed to desulfate T3SO4. Estrone sulfate, dehydroepiandrosterone sulfate, and nitrophenyl sulfate are known substrates for microsome-associated arylsulfatase activities, and these compounds were found to inhibit hydrolysis of T3SO4 to various extents. Of these competing sulfatase substrates, only dehydroepiandrosterone sulfate inhibits T3SO4 desulfation completely. In order to determine whether desulfation occurs in intact cells, isolated hepatocytes were incubated in the presence of 7 and 54 microM T3SO4. These cells were found to hydrolyze 1-1.5% of the sulfate ester/h for up to 3 h. The demonstration of this activity raises the possibility that these hepatic cells may be able to reactivate T3SO4, which has generally been regarded as an irreversibly inactivated metabolite.
This paper discusses the use of dogs as experimental models for neuroimmunomodulation and compares immunodeficient dwarfism in dogs to that in rodents. Immunodeficient dwarfism in dogs is reviewed including description of the clinical syndrome, immunologic characteristics, neuroendocrine abnormalities, thymus histopathology, and therapy.
Thirty-nine patients with potentially resectable cancer of the middle or lower esophagus who had not previously been treated were randomly assigned to receive either immediate operation (n = 20) or operation plus preoperative and postoperative therapy with cisplatin, vindesine, and bleomycin (n = 19). Patients were stratified by tumor size and location and by sex, with no significant differences appearing between the two treatment groups. Median follow-up for both groups was 30 months. The preoperative response rate to chemotherapy was 47%. The postoperative complication rate for patients in the operation-only group was 47%; it was 29% for patients receiving chemotherapy. The overall resectability rates were similar for the two groups. Patients responding to chemotherapy preoperatively had significantly prolonged survival (median greater than 20 months) when compared with either nonresponders (median 6.2 months) or patients receiving only operation (median 8.6 months). A highly significant correlation was noted between a weight loss of less than 10% and response to chemotherapy, which suggested that responses occurred in patients with less advanced disease. We conclude that preoperative and postoperative cisplatin, vindesine, and bleomycin chemotherapy has acceptable toxicity and does not increase the incidence of postoperative complications. The natural history of epidermoid carcinoma of the esophagus is altered and overall survival is prolonged for patients responding to preoperative chemotherapy. Potential responding patients can be identified by the degree of preoperative weight loss.
Nonopsonized Brucella abortus and bacteria treated with fresh antiserum, heat-inactivated antiserum, or normal bovine serum were evaluated for their ability to stimulate production of superoxide anion and myeloperoxidase-mediated iodination by neutrophils from cattle. Brucella abortus opsonized with fresh antiserum or heat-inactivated antiserum stimulated production of approximately 3 nmol of O2-/10(6) neutrophils/30 min. Similarly treated bacteria also stimulated the binding of approximately 4.3 nmol of NaI/10(7) neutrophils/h to protein. Significant (P less than 0.05) production of O2- and iodination activity by neutrophils were not stimulated by nonopsonized bacteria or organisms treated with normal bovine serum. Seemingly, B abortus stimulated oxidative metabolism in bovine neutrophils; however, the stimulation was dependent on the presence of bacterial associated opsonins.
The purification to homogeneity and physical characterization of a monoamine-sulfating form of phenol sulfotransferase (PST) from human platelets is described. DEAE-cellulose chromatography of a 100,000 x g supernatant solution of homogenized human platelets revealed the presence of two peaks of both dopamine- and phenol-sulfating activity, termed M- and P-PST, respectively. The latter dopamine-sulfating form eluting from the ion exchange column, MII-PST, was purified approximately 10,000-fold to electrophoretic homogeneity by Sephacryl S-200 HR and 3'-phosphoadenosine-5'-phosphate-agarose chromatography. The final specific activity of the enzyme was 930 nmol/min/mg of protein. As determined by the hydrodynamic properties of MII-PST, the native Mr was approximately 69,000. The frictional ratio (f/fo) was estimated to be 1.28, indicating that the enzyme possesses a relatively low degree of asymmetry. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the affinity-purified enzyme revealed the presence of single Mr species of approximately 34,000, suggesting that MII-PST exists as a homodimer in vivo. Isoelectric focusing of purified MII-PST yielded a single protein species with a pl of 4.7. The sulfhydryl-modifying reagent N-ethylmaleimide (50 microM) was found to inactivate MII-PST in a time-dependent manner. This inactivation was totally prevented by saturating concentrations of 3'-phosphoadenosine-5'-phosphosulfate, whereas dopamine bestowed only partial protection to the enzyme. These results suggest that at least one sulfhydryl moiety is present at the active site of MII-PST.
The cellular localization of the soluble and membrane-bound forms of the enzyme, arylsulfatase (ArS), in rat brain was investigated by measuring their activities in rat striatum after unilateral lesioning with the neurotoxin, kainic acid. Membrane-bound ArS (C form of ArS) activity was found to increase after lesioning and the increase paralleled that of the astroglial marker enzyme, glutamine synthetase. Total soluble ArS (A and B forms of ArS) was shown to decrease on day 2 after the kainic acid injection but rapidly increase thereafter. When the two soluble forms of arylsulfatase were measured separately, the activity associated with the A form was found to initially decrease followed by a rapid increase in activity, whereas the activity of the B form of the enzyme increased over the entire duration of the experiment. These data suggest that the ArS-C and B form of arylsulfatase predominate in proliferating astroglial cells, whereas the A form of arylsulfatase is present both in neuronal cell bodies and astroglia associated with the rat striatum.
Compared with neutrophils from healthy dogs, neutrophils from 2 dogs with primary ciliary dyskinesia had increased distance of random migration, but fewer of the neutrophils migrated. The affected dogs had an increase in the numbers of Staphylococcus aureus phagocytized. Lymphocyte blastogenesis in the affected dogs in response to standard mitogens was considered to be normal.