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Biomedical subjects

S L Freeman

Publications and source records attributed to S L Freeman.

33 records · Page 2Linked to original sources
PubMed1999

Thalidomide analogue CC1069 inhibits development of rat adjuvant arthritis.

The cytokine tumour necrosis factor-alpha (TNF-alpha) has been implicated in the aetiology of rheumatoid arthritis in humans as well as of experimental arthritis in rodents. Thalidomide, and to a greater extent the new thalidomide analogue CC1069, inhibit monocyte TNF-alpha production both in vitro and in vivo. The aim of the present study is to establish whether these drugs block production of TNF-alpha as well as IL-2 by rat leucocytes and whether this inhibition affects the development of rat adjuvant arthritis (AA). Cultured splenocytes were stimulated with either lipopolysaccharide (LPS) or concanavalin A (Con A) in the presence of thalidomide, CC1069, or solvent, and the production of TNF-alpha and IL-2 were compared. Next, adjuvant was injected into the base of the tail of rats without or with daily intraperitoneal injections with 100-200 mg/kg per day thalidomide or 50-200 mg/kg per day CC1069. Disease activity, including ankle swelling, hind limb radiographic and histological changes, weight gain, and ankle joint cytokine mRNA levels, were monitored. CC1069, but not the parent drug thalidomide, inhibited in vitro production of TNF-alpha and IL-2 by stimulated splenocytes in a dose-dependent manner. In vivo, a dose-dependent suppression of AA disease activity occurred in the CC1069-treated animals. In contrast, thalidomide-treated rats experienced comparable arthritis severity to placebo-treated animals. There was also a reduction in TNF-alpha and IL-2 mRNA levels in the ankle joints of CC1069-treated rats compared with thalidomide- and placebo-treated arthritic rats. Early initiation of CC1069 treatment suppressed AA inflammation more efficiently than delayed treatment. We conclude that thalidomide, which did not suppress TNF-alpha or IL-2 production in vitro by Lewis rat cells, did not suppress development of rat AA. However, the development of rat AA can be blocked by the thalidomide analogue CC1069, which is an efficient inhibitor of TNF-alpha production and IL-2 in vitro.

Animals↗
PubMed1999

Comparison of sedative effects of romifidine following intravenous, intramuscular, and sublingual administration to horses.

OBJECTIVE: To compare sedative effects of romifidine following IV, IM, or sublingual (SL) administration in horses. ANIMALS: 30 horses that required sedation for routine tooth rasping. PROCEDURE: Horses (n = 10/group) were given romifidine (120 microg/kg) IV, IM, or SL. Heart rate, respiratory rate, head height, distance between the ear tips, thickness of the upper lip, response to auditory stimulation, response to tactile stimulation, and degree of ataxia were recorded every 15 minutes for 180 minutes. Tooth rasping was performed 60 minutes after administration of romifidine, and overall adequacy of sedation was assessed. RESULTS: IV and IM administration of romifidine induced significant sedation, but SL administration did not induce significant sedative effects. Scores for overall adequacy of sedation after IV and IM sedation were not significantly different from each other but were significantly different from scores for horses given romifidine SL. Sedative and other effects varied among groups during the first 60 minutes after drug administration; thereafter, effects of IV and IM administration were similar. CONCLUSIONS AND CLINICAL RELEVANCE: Onset of action was fastest and degree of sedation was greater after IV, compared with IM, administration of romifidine, but duration of action was longer after IM administration. Sublingual administration did not result in clinically important sedative effects.

Acoustic Stimulation↗
PubMed1997

Interleukin-10 upregulates tumor necrosis factor receptor type-II (p75) gene expression in endotoxin-stimulated human monocytes.

Interferon-gamma (IFN-gamma) upregulates expression of certain genes in monocytes, including cell-surface molecules such as HLA class II, B7, and ICAM-1. IFN-gamma also potentiates production of cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and IL-12. Conversely, IL-10 downregulates expression of many of these same genes and often antagonizes the effects of IFN-gamma. IL-10 is known to inhibit TNF-alpha production in lipopolysaccharide (LPS)-stimulated monocytes; however, the effects of IL-10 on TNF receptor (TNF-R) expression are not well defined. We examined the effects of IL-10 on production of both membrane-associated (m) and soluble (s) TNF-R type II (sTNF-RII) by purified human CD14(+) monocytes. We also compared the effects of IFN-gamma and IL-10 on production of TNF-alpha and sTNF-RII by these cells. Monocytes constitutively expressed low levels of TNF-RII mRNA and mTNF-RII protein. LPS stimulation induced rapid, but transient loss (shedding) of mTNF-RII molecules and a delayed, but marked increase in TNF-RII mRNA levels. IL-10 increased expression of both mTNF-RII and sTNF-RII by LPS-stimulated monocytes, whereas IFN-gamma decreased their expression. The increased levels of sTNF-RII in cultures of IL-10-treated monocytes correlated directly with increased levels of TNF-RII mRNA and inversely with the levels of TNF-alpha mRNA. The ability of IL-10 to upregulate TNF-RII gene expression was transcriptionally mediated because actinomycin D blocked this effect. Furthermore, IL-10 treatment did not alter the half-life of TNF-RII mRNA transcripts in LPS-stimulated monocytes. To further examine the mechanism by which IL-10 potentiates TNF-RII gene expression, a 1.8-kb fragment of the human TNF-RII promoter cloned into a luciferase expression vector (pGL2-basic) was transfected into the IL-10-responsive macrophage cell line, RAW264.7. Although IL-10 alone induced only minimal promoter activity in these cells, it markedly increased the LPS-induced response, providing further evidence that the ability of IL-10 to amplify TNF-RII gene expression is transcriptionally controlled. Together, these findings demonstrate that IL-10 coordinately downregulates expression of TNF-alpha and upregulates expression of TNF-RII, particularly the soluble form of this receptor, in monocytes.

Cell Adhesion Molecules↗
PubMed1997

TCR vbeta usage of TSH receptor-specific CD4+ T cells in Graves' disease patients and healthy humans.

Healthy humans have CD4+ T cells specific for self-components. Since autoreactive T cells in autoimmune patients may use a limited number of TCR V-region genes, we investigated here whether this also occurs for the potentially autoreactive CD4+ cells present in healthy persons. We studied CD4+ cells specific for human TSH receptor (TSHr) sequences, that are present with high frequency in healthy subjects and, as expected, in Graves' disease (GD) patients. We used short-term CD4+ cell lines propagated from four GD patients and five healthy subjects by cycles of stimulation with a pool of overlapping synthetic peptides corresponding to the putative extracellular parts of the TSHr sequence. The lines recognized the pool of TSHr peptides specifically and vigorously. Their epitope repertoire had been characterized previously: each line recognized one or a few TSHr peptides, different for each subject. We determined their TCR Vbeta usage by a semi-quantitative reverse transcriptase PCR assay, using primers specific for each known human Vbeta region family, in conjunction with a constant region primer. Six lines preferentially used one Vbeta family (42-94%), different for each line. In all lines, three or less Vbeta families accounted for approximately 60% or more of the Vbeta usage. Different Vbeta regions were used by each subject. There was no obvious difference between the Vbeta usage of the lines from GD patients and healthy controls. These results suggest that a limited pool of potentially autoreactive T cells survives clonal deletion. The pathogenic CD4+ cells involved in autoimmune diseases are likely recruited from that pool, since they have similar characteristics of epitope and TCR repertoire as the CD4+ cells specific for the same autoantigen in healthy subjects.

Adult↗
PubMed1995

Inhibition of IL-10 expression by IFN-gamma up-regulates transcription of TNF-alpha in human monocytes.

Stimulation of human monocytes with LPS induces expression of multiple cytokines, including TNF-alpha, IL-1 beta, IL-6, and IL-10, IL-10 expression is delayed relative to that of TNF-alpha, IL-1 beta, and IL-6. Furthermore, IL-10 feedback inhibits expression of TNF-alpha, IL-1 beta, and IL-6, thus providing an efficient autocrine mechanism for controlling proinflammatory cytokine production in monocytes. The Th1-type lymphokine, IFN-gamma, markedly up-regulates TNF-alpha production in monocytes. However, the precise mechanism by which IFN-gamma mediates this effect is unknown. We examined the effects of IFN-gamma on IL-10 expression in LPS-stimulated monocytes, and the relationship between IL-10 and TNF-alpha production in these cells. LPS stimulation induced rapid, ordered expression of multiple cytokines. Steady-state mRNA levels for TNF-alpha increased rapidly, reached maximal levels by 2 to 3 h poststimulation, and then declined sharply. IL-1 beta and IL-6 mRNA levels also increased markedly following stimulation with LPS, but decreased more slowly than did TNF-alpha. Down-regulation of mRNA for TNF-alpha, IL-1 beta, and IL-6 coincided with a delayed and more gradual increase in IL-10 mRNA levels. Furthermore, neutralization of IL-10 with anti-IL-10 Abs prolonged TNF-alpha mRNA expression, and significantly increased net TNF-alpha production. IFN-gamma suppressed expression of IL-10 mRNA and protein in a dose-dependent manner. Moreover, inhibition of IL-10 production correlated with a marked increase in both the magnitude and duration of TNF-alpha expression. Thus, potentiation of TNF-alpha production by IFN-gamma in monocytes is coupled to inhibition of endogenous IL-10 expression.

Cells, Cultured↗
PubMed1995

IFN-gamma priming of monocytes enhances LPS-induced TNF production by augmenting both transcription and MRNA stability.

The induction of cytokine expression in monocytes/macrophages by bacterial endotoxin or lipopolysaccharide is a critical, highly regulated host defence response. The augmentation of LPS responses by interferon gamma (IFN-gamma), referred to as priming, is well established. However, the mechanism(s) by which priming occurs is poorly defined. Using tumour necrosis factor (TNF) induction as a model, experiments were designed to analyse in detail the priming effect on the LPS response in human monocytes. Priming by IFN-gamma was primarily manifested at the level of TNF mRNA accumulation. IFN-gamma pre-treatment affected the magnitude rather than the sensitivity of the LPS response. Priming occurred after several hours of treatment, and the primed state was induced by either IFN-gamma or GM-CSF, but not M-CSF. Primed monocytes transcribed TNF mRNA at a higher rate than freshly isolated monocytes upon activation with LPS. The increased transcriptional rate correlated with a marked increase in nuclear factor-kappa B activity in these cells as determined by electrophoretic mobility shift assay using a consensus NF-kappa B oligonucleotide. An additional significant finding was than TNF mRNA induced in primed cells was much more stable than in unprimed cells (T1/2 increased 6-8-fold). Consistent with the increased mRNA stability, the duration of mRNA accumulation was longer following LPS stimulation in primed monocytes, in addition to being of greater magnitude. Finally, primed and unprimed cells possessed a differential sensitivity to the kinase inhibitor H-89. H-89 substantially suppressed LPS-induced TNF mRNA accumulation in unprimed cells, but had no effect on primed monocytes following LPS stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗
PubMed1995

TSH receptor sequences recognized by CD4+ T cells in Graves' disease patients and healthy controls.

Twenty-nine overlapping synthetic peptides, twenty residues long, representing the entire extracellular sequence of the human thyroid stimulating hormone receptor (hTSHr), were used to test the epitope repertoire of CD4+ T lymphocytes from patients with Graves' disease and from healthy subjects. The peptides were used to propagate and test short term CD4+ T cell lines specific for hTSHr epitopes, and to directly test CD8+ depleted, CD4+ enriched peripheral blood lymphocytes. Analysis of the response of short-term CD4+ T cells lines and CD8+ depleted peripheral blood lymphocytes to the individual peptides revealed that 14 of the 15 patients and nine of the ten controls responded to at least one hTSHr peptide. There was no common response pattern, nor any region of the hTSHr sequence that was predominantly recognized. Several peptides were recognized by both patients and controls. These results support the notion that immunological tolerance to hTSHr is due to peripheral tolerance of potentially autoreactive CD4+ T cells, not their clonal deletion. The presence of self-reactive, hTSHr-specific CD4+ T cells in healthy individuals implies that these cells are not permanently anergized, since they can be activated in vitro.

Adult↗
PubMed1993

Tissue-specific regulation of IL-6 production by IL-4. Differential effects of IL-4 on nuclear factor-kappa B activity in monocytes and fibroblasts.

IL-4 inhibits production of certain proinflammatory cytokines, including IL-1 beta, TNF-alpha, and IL-6, by activated monocytes. Although monocytes are a major source of IL-6, other cell types such as fibroblasts and endothelial cells can also express this cytokine. To determine whether IL-4 inhibits IL-6 expression in non-hemopoietic cells, we investigated the effects of IL-4 on IL-6 production in both primary human fibroblasts and fibroblast lines. Rheumatoid synovial fibroblasts were evaluated in these studies because, like monocytes, they produce high levels of IL-6 when stimulated with IL-1. Although peripheral blood monocytes did not constitutively express IL-6 mRNA or protein, stimulation with IL-1 or LPS induced de novo IL-6 expression in these cells. In contrast, synovial fibroblasts displayed a significant basal level of IL-6 production, which was markedly increased after stimulation with IL-1. IL-4 suppressed IL-6 expression in monocytes, but did not inhibit IL-6 production in synovial fibroblasts. The inability of IL-4 to suppress IL-6 synthesis in rheumatoid synovial fibroblasts was not caused by a lack of IL-4R and was not unique to these cells because IL-4 also failed to inhibit IL-6 production in normal fibroblast lines derived from other tissues. Inhibition of IL-6 production by IL-4 in monocytes was associated with decreased nuclear NF-kappa B levels. However, IL-4 does not globally suppress the activity of all DNA-binding proteins because IL-4 treatment did not reduce the levels of NF-IL-6 or NF-IL-1 beta B in the same cells. Because NF-kappa B activation is required for transcription of many cytokine genes, including IL-6, the ability of IL-4 to suppress NF-kappa B activity in monocytes suggests a potential mechanism by which this molecule may inhibit the expression of multiple cytokines.

Base Sequence↗
PubMed1992

Hyperthyroidism due to inappropriate secretion of thyrotropin in 10 patients.

PURPOSE: The syndrome of inappropriate thyroid-stimulating hormone (TSH) secretion, characterized by elevated serum free thyroxine and triiodothyronine levels in association with measurable serum TSH concentrations, remains an uncommon cause of hyperthyroidism that is being recognized with increasing frequency. The hyperthyroidism may be due to either neoplastic pituitary TSH secretion or selective pituitary resistance to thyroid hormone. In an effort to better understand this rare cause of hyperthyroidism, we undertook a retrospective analysis of our institution's experience with this condition. PATIENTS: We reviewed our cumulative experience (10 patients) with hyperthyroidism due to the syndrome of inappropriate secretion of TSH. RESULTS: Six patients were diagnosed with TSH-secreting pituitary adenomas and four were found to have selective pituitary resistance to thyroid hormone. One patient with tumor had a TSH-secreting pituitary adenoma in the setting of multiple endocrine neoplasia syndrome. In all patients with tumor, hyperthyroidism was successfully treated with transsphenoidal adenomectomy with or without pituitary radiotherapy. All four patients with pituitary resistance had thyroid ablation or resection prior to their correct diagnosis. Therefore, therapy for this group of patients involved thyroid hormone replacement and efforts to suppress TSH hypersecretion. All 10 patients have done well clinically, with follow-up ranging from 2 weeks to 13 years. CONCLUSIONS: Adequate treatment exists for the two primary causes of TSH hypersecretion. TSH-secreting pituitary adenomas are treated with surgery and, if necessary, adjuvant pituitary radiotherapy. The results are generally good if the tumor is diagnosed and treated at an early stage. Primary therapy for hyperthyroidism due to selective pituitary resistance to thyroid hormone is aimed at suppression of pituitary TSH hypersecretion. The evaluation of any patient with hyperthyroidism must be thorough and, in some cases, should include measurement of TSH to determine the presence of inappropriate secretion. Eliminating this diagnosis will help avoid improper and potentially harmful treatment of hyperthyroid patients.

Adenoma↗
PubMed1992

Inhibition of TSH bio-activity by synthetic beta TSH peptides.

The in vitro bioactivity of the human beta TSH subunit was investigated utilizing eleven overlapping synthetic peptides representing the entire 112 residue sequence. The peptides were tested for both stimulatory and inhibitory activity in two sensitive bioassay systems: the first based on cAMP production in FRTL-5 rat thyroid cells, and the second based on stimulation of iodine trapping by the same continuous cell line. Peptides from three distinct regions of the beta-subunit showed concentration dependent inhibition of TSH bio-activity, including beta 1-15, beta 11-25, beta 31-45, beta 81-95, and beta 91-105 with IC50 values ranging from 150 to 304 microM. An additional peptide representing the entire sequence of the "intercysteine loop" region of beta TSH, beta 31-52, also inhibited TSH activity with somewhat higher potency than its fragment peptide beta 31-45 (IC50 of 87.5 +/- 14.7 microM for beta 31-52 versus 207 +/- 92.4 microM for beta 31-45). Three of these, beta 1-15, beta 31-45, and beta 31-52, also inhibited binding of TSH to the receptor in a radio-receptor assay, as previously reported (1), supporting their importance in receptor interaction. None of the synthetic peptides stimulated either cAMP production or iodine trapping. Two other overlapping peptides, beta 81-95 and beta 91-105, possessed bio-inhibitory activity but did not inhibit binding of labeled TSH. Computer analysis of this sequence predicted an extended turn structure for this region. This region has been referred to as the "determinant loop" as it is bounded by cysteine residues at positions 88 and 95 that many believe form a disulfide bond in the native subunit. The current data suggests the beta 88-95 region may play a role in receptor activation after initial binding of hormone to receptor.

Amino Acid Sequence↗
PubMed1991

Use of human ultralente as the basal insulin component in treatment of patients with IDDM.

To determine the suitability of a single subcutaneous evening injection of human ultralente (UL) as the basal component of an intensive insulin therapy program, insulin concentrations were measured in five insulin-dependent diabetic volunteers over a 40-h period. Each patient had been maintained on a human UL-based program for at least one month prior to the study. All short-acting insulin was withheld during the study. The onset of action of human UL was 2 to 4 h, and a broad, variable peak was observed between 6 and 12 h after each injection. We concluded that human UL does not provide constant basal insulin concentrations. When human UL is considered as part of an intensive insulin therapy program, this potential disadvantage must be weighed against the potential advantage of low antigenicity.

Adult↗
PubMed1981

Getting more mileage from patient care evaluation.

Patient care evaluation requires a considerable investment of staff time and energy. Trustees can get more mileage out of this investment by seeking additional uses for patient care evaluation studies, including quality control, employee education, evaluation of hospital management, and public relations.

Cost-Benefit Analysis↗