Treatment of multiple sclerosis (MS) with interferon beta (IFNbeta) reduces relapse rate, magnetic resonance imaging (MRI) activity and progression of disability. It has been suggested that this beneficial effect is paralleled by an inhibition of proinflammatory cytokines such as interferon gamma (IFNgamma) and tumor necrosis factor alpha (TNFalpha) and an induction of anti-inflammatory cytokines such as interleukin-4 (IL-4) and interleukin-10 (IL-10). In this study, we record a reduced number of spontaneously IFNgamma mRNA-expressing cerebrospinal fluid mononuclear cells (CSF-MC) and IFNgamma, TNFalpha and IL-10 mRNA-expressing peripheral blood mononuclear cells (PBMC) after 6 months of IFNbeta-1a treatment, paralleled by a decreased purified protein derivate (PPD)-stimulated and unstimulated IFNgamma secretion by PBMC. These effects were not apparent after 2 weeks of treatment, and IFNbeta-1a induced IFNgamma production by naive PBMC in vitro. We did not record increased numbers of IL-4 mRNA-expressing CSF-MC or PBMC, increased plasma IL-10 levels, increased numbers of IgG, A or M secreting plasma cells or in vitro induction of IL-10 production by IFNbeta-1a. We conclude that long-term cytokine modulation by IFNbeta-1a differs from acute effects and that downregulation of both pro- and anti-inflammatory cytokines, rather than a shift in the cytokine profile, is apparent after 6 months of IFNbeta-1a treatment of MS patients.

This paper shows that melatonin regulates both thymosin alpha1 and thymulin production as well as the expression of the prothymosin alpha gene. The results revealed the following facts: (a) The concentrations of thymosin alpha1 in both serum and thymus of rat showed a nyctohemeral profile with peak values late at night and basal values during the day. The concentrations of thymulin in rat serum also showed a 24-h rhythm with an increase in their values at night. This rhythmical character for thymosin alpha1, and thymulin was also found in the human serum. (b) Rats injected with melatonin during the day exhibited a significant increase in the concentrations of both peptides. Moreover, continuous light exposure on the animals at daytime and pinealectomy cause a decrease in thymosin a1 and thymulin concentrations with regards to those found in control rats. (c) Melatonin regulates the expression of the prothymosin alpha gene, analyzed by Northern blot. These results suggest that melatonin may be involved in the regulation of immune functions by increasing the thymic peptides production.

Angiogenesis is crucial for tumor growth and metastasis. In several tumors, microvascular density has been shown to correlate with metastasis and aggressiveness. Basic fibroblast growth factor (bFGF) has potent angiogenic activity and has been identified in a wide variety of malignancies including head and neck squamous cell carcinomas (HNSCC). Material and Methods Frozen sections of 50 HNSCC were immunostained for von Willebrand factor and bFGF. Microvessels were counted by light microscopy; bFGF expression was studied at the light and electron microscopic level. Laryngeal cancer cell line HlaC79 was incubated with interferon (IFN) alpha and beta. bFGF quantification was performed by ELISA, and antiproliferative effects were determined by BrdU assay.

In this study, the roles of oestrogen and progesterone in the regulation of oxytocin gene expression in equine endometrium were examined. Anoestrous mares (n=19) were assigned randomly to one of the following treatment groups: control (vehicle control for 1 day; n=3); progesterone (250 mg progesterone per day for 6 days; n=4); oestradiol (5 mg beta-oestradiol 17-valerate per day for 6 days; n=4); oestradiol plus short duration progesterone (5 mg beta-oestradiol 17-valerate per day for 6 days followed by 250 mg progesterone per day for 6 days; n=4); and oestradiol plus long duration progesterone (5 mg beta-oestradiol 17-valerate per day for 6 days followed by 250 mg progesterone per day for 12 days; n=4). Jugular venous blood samples were obtained for oestrogen and progesterone radioimmunoassays. Endometrial biopsies were obtained and total RNA was extracted. Expression of mRNA for oxytocin and glyceraldehyde 3'-phosphate dehydrogenase was assessed by RT-PCR and Southern blotting. Oxytocin mRNA abundance was significantly higher (P < 0.05) in the oestrogen-treated group than in all other groups. These data demonstrate that oestradiol priming for 6 days upregulated expression of the endometrial oxytocin gene. Progesterone treatment for either 6 or 12 days after oestradiol priming returned oxytocin mRNA abundance to levels similar to those of controls.

Eletriptan (Relpax) is a novel 5-hydroxytryptamine (serotonin)(1D/1B) agonist currently in development for the acute treatment of migraine. The aim of this work was to evaluate the relative induction potency of eletriptan in vitro compared with well characterized cytochrome P-450 (CYP) inducers with primary cultures of human hepatocytes and to relate this to the situation in vivo. Eletriptan was a weak inducer of CYP3A4 protein and cyclosporin A oxidation in four of the six cultures used, whereas rifampicin was a potent inducer in all cultures. Induction was concentration dependent and not detectable at eletriptan concentrations of 5 microM and lower. The amplitude of the increase in CYP3A4 protein and activity by 25 microM eletriptan was significantly lower, with a mean of 19 (P =.0015) and 26% (P =.0002), respectively, of that observed in response to 25 microM rifampicin. CYP2A6, a protein with minor pharmacological implication, also was induced by eletriptan and rifampicin in two cultures but was not detected in the others. The levels of other CYP proteins, including CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP2E1, were not affected by eletriptan. Because the maximum blood concentration of eletriptan in humans after a therapeutic dose (maximum 80 mg) is 0.5 microM, the in vitro model would predict no clinically significant induction of CYP3A4 protein in vivo. This has been confirmed subsequently in a clinical study, with 6beta-hydroxycortisol/cortisol ratios as marker of CYP3A4 activity. Eletriptan is therefore not an inducer of CYP3A4 at clinical doses.

In a double-blind, placebo-controlled, randomized study, 10 healthy men received either a single dose of 480 microg granulocyte colony-stimulating factor (G-CSF) or saline. Blood taken from the volunteers was stimulated with 10 microg/mL endotoxin and released cytokines were measured by enzyme-linked immunosorbent assay. Expression of G-CSF receptors on leukocytes was examined by flow cytometry and reverse transcriptase-polymerase chain reaction. Functional activity of these receptors was tested by challenging isolated leukocyte populations to release cytokines with endotoxin in the presence of G-CSF. The G-CSF treatment attenuated the release of the proinflammatory cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-12, IL-1beta, and interferon (IFN)-gamma in ex vivo lipopolysaccharide (LPS)-stimulated whole blood. In blood from untreated volunteers the presence of G-CSF in vitro also attenuated the LPS-stimulated release of these cytokines. G-CSF in vitro also attenuated TNF-alpha release from elutriation-purified monocytes. In the presence of 10 ng/mL recombinant TNF-alpha, the attenuation of LPS-inducible IFN-gamma release by G-CSF was blunted in whole blood. However, G-CSF had no such effect on IFN-gamma release from isolated lymphocytes stimulated with anti-CD3 or a combination of TNF-alpha and IL-12. G-CSF receptor expression was detected in human neutrophils and monocytes but not in lymphocytes by means of RT-PCR as well as flow cytometry. These results indicate that G-CSF receptors expressed on monocytes are functional in modulating monokine release. We conclude that the attenuation of IFN-gamma release from lymphocytes is not a direct effect of G-CSF on these cells but is rather due to the inhibition of monocytic IL-12 and TNF-alpha release by G-CSF. (Blood. 2000;95:270-276)

The allergen-induced late nasal response is associated with a high local expression of interleukin (IL) -4, a TH2-type cytokine implicated in immunoglobulin (Ig) E production, tissue eosinophilia and other events considered to be relevant to allergic inflammation. Interaction of IL-4 with its receptor activates at least two distinct signalling pathways that culminate in the transcription of specific target genes. One pathway involves the activation of a transcription factor termed signal transducer and activator of transcription factor 6 (STAT6).

Psoriasis is a chronic inflammatory skin disease in which epidermal hyperplasia results from skin infiltration by type I T lymphocytes and release of associated cytokines. A multifunctional cytokine, rhIL-11, modulates macrophage and type I T-lymphocyte function in cell culture and shows anti-inflammatory activity in animal models. We are testing subcutaneous delivery of rhIL-11 to patients with psoriasis in a phase 1 open-label dose-escalation clinical trial. Tissue was obtained from lesional and uninvolved skin before and during treatment with rhIL-11 and was examined by histology/immunohistochemistry and quantitative RT-PCR. Expression of over 35 genes was examined in all patients, and multiple genetic markers of psoriasis were identified. Expression of numerous proinflammatory genes was elevated in psoriatic tissue compared with nonlesional skin. Seven of 12 patients responded well to rhIL-11 treatment. Amelioration of disease by rhIL-11, as shown by reduced keratinocyte proliferation and cutaneous inflammation, was associated with decreased expression of products of disease-related genes, including K16, iNOS, IFN-gamma, IL-8, IL-12, TNF-alpha, IL-1beta, and CD8, and with increased expression of endogenous IL-11. We believe that this is the first study in humans to indicate that type I cytokines can be selectively suppressed by an exogenous immune-modifying therapy. The study highlights the utility of pharmacogenomic monitoring to track patient responsiveness and to elucidate anti-inflammatory mechanisms.

Leader binding protein-1 (LBP-1)/late SV40 factor (LSF) and ying yang-1 (YY1) transcription factors are involved in the regulation of HIV expression. In particular, YY1 and LBP-1 have been shown to cooperate in repressing HIV-1-long terminal repeat reporter gene expression by in vitro cotransfection experiments. However, no information is available on the levels of expression and activation of these transcription factors in PBMC of HIV-infected individuals. Therefore, we have evaluated the expression and DNA binding activity of YY1 and LBP-1 (LSF) in PBMC of HIV-infected individuals before, during, and after administration of IL-2 in association with antiretroviral therapy (ART), a regimen under consideration for broad clinical use in this disease based on its ability to stably raise the absolute number of circulating CD4+ T lymphocytes. Both YY1- and LBP-1 (LSF)-DNA binding were profoundly down-modulated during administration of IL-2/ART, and a proteolytic activity probably responsible for the reduced expression of the two cellular transcription factors was found activated in PBMC of individuals receiving the immunotherapeutic regimen. This study is the first evidence of modulation of cellular transcription factors following IL-2/ART administration and provides a potential correlate of the transient raises in plasma viremia early reported in patients receiving IL-2 in the absence of ART, thus underscoring the importance of always administering this cytokine to HIV-infected individuals together with potent antiretrovirals.

The newly described uncoupling proteins, UCP2 and UCP3, may play a role in regulating energy expenditure (EE) in humans. GH deficiency (GHD) is associated with decreased lean body mass, increased adiposity, and reduced EE, which are reversed by GH treatment. In the present study we investigated whether GH treatment for 4 months influenced the expression of UCPs in skeletal muscle and adipose tissue in 22 GHD patients who were investigated before and after GH (n = 11) or placebo (n = 11) treatment. GH treatment increased the amount of lean body mass by 4.5% (P < 0.05) and decreased body fat mass by 12% (P < 0.05), whereas no changes in these parameters were observed after placebo treatment. The level of UCP3 messenger ribonucleic acid (mRNA) increased 3-fold (P < 0.005) in skeletal muscle and almost 2-fold (P < 0.05) in adipose tissue after GH treatment, with no changes observed after placebo treatment. Skeletal muscle UCP2 mRNA was slightly (25%), but significantly (P < 0.05), decreased, whereas the level of UCP2 mRNA in adipose tissue was unaffected after GH treatment. The T4 level was positively correlated with skeletal muscle UCP2 and UCP3 expression (r = 0.518; P < 0.05 and r = 0.463; P < 0.05, respectively). Furthermore, plasma free fatty acids were positively correlated with the expression of UCP2 (r = 0.573; P < 0.01) and UCP3 (r = 0.518; P < 0.05) in skeletal muscle. The marked increase in UCP3 expression after GH treatment indicates that the UCPs might play a role in the effects of GH on EE in GHD patients. Finally, the strong association between thyroid hormone and skeletal muscle UCP and the correlation between plasma free fatty acids and UCP expression in skeletal muscle indicate that these hormones/metabolites might influence UCP expression in humans as previously demonstrated in rodents.

This trial was designed to test the safety and efficacy of a tumor-specific genetic prodrug activation therapy targeted by use of the human erbB-2 gene promoter. The erbB-2 oncogene is overexpressed in approximately 20% of cases of breast cancer and is associated with poor prognosis.

Transcription factors adjust the up- and downregulation of inflammatory genes. Within the bounds of the SIC-study (Selenium in Intensive Care) the authors will investigate the role of transcription factors NF-kappa B and AP-1 in S.I.R.S/septic patients. The goal of these investigations is the corroboration of therapy of septic patients with sodium selenite at the molecular biological level.

This study presents the kinetics of CD4/CD25 cell numbers, serum sCD25 levels, and intracellular production and release of interleukin-2 (IL-2) and interleukin-16 (IL-16) in 11 HIV+ patients treated with six cycles of highly active antiretroviral therapy (HAART) plus six MUI of subcutaneous IL-2 compared to 10 HIV+ patients treated with HAART alone. IL-2 therapy induced moderate effects on CD4 T cell recovery and increased CD4/CD25+ cells and sCD25 levels after 2 weeks, while intracellular and secreted IL-2 was reduced and IL-16 was increased at the same time point. After 24 weeks, while HAART-treated patients had increased IL-2 production, in IL-2 treated patients, cytokine production was unaltered compared to pretreatment values. Decreased in vitro IL-2 production may depend on a feedback inhibition by IL-2 infusion. Because of its known antiviral effects, the increased IL-16 production seen after 2 weeks in IL-2-treated individuals may produce beneficial effects on HIV disease. The kinetics of cytokine production may serve to define better the use IL-2 in clinical trials.

The BCR/ABL hybrid gene plays a central role in the pathogenesis of the chronic phase of chronic myeloid leukemia (CML). We used a very sensitive quantitative reverse transcriptase-polymerase chain reaction to investigate the levels of hybrid BCR/ABL mRNA in bone marrow cells of 20 patients with Philadelphia positive (Ph(+)) CML treated with interferon-alpha (IFN-alpha) as a single agent. Bone marrow samples were collected at diagnosis and at hematologic remission induced by IFN-alpha, or by hydroxyurea in case of resistance to IFN-alpha. The mean levels of BCR/ABL transcripts in bone marrow mononuclear cells of patients who showed a complete hematologic response to IFN-alpha were significantly reduced with respect to those at diagnosis (48 x 10(3) v 168 x 10(3); P <.001), whereas no difference was detected between the values at diagnosis and at hematologic remission in patients resistant to IFN-alpha. In cell culture experiments, IFN-alpha priming significantly reduced the levels of BCR/ABL hybrid transcripts in a dose-dependent manner in Ph+ bone marrow precursors obtained at diagnosis from patients who subsequently responded to IFN-alpha treatment (P < .005). No downmodulation was observed in bone marrow precursors from patients who subsequently proved to be IFN-resistant. These results indicate that downmodulation of BCR/ABL gene expression could be one of the mechanisms involved in the response of CML patients to IFN-alpha treatment.

This publication is the fourth in a series of safety evaluations performed by the Expert Panel of the Flavour and Extract Manufacturers' Association (FEMA). In 1993, the Panel initiated a comprehensive program to re-evaluate the safety of more than 1700 GRAS flavouring substances under conditions of intended use. In this review, scientific data relevant to the safety evaluation of trans-anethole (i.e. 4-methoxypropenylbenzene) as a flavouring substance is critically evaluated by the FEMA Expert Panel. The evaluation uses a mechanism-based approach in which production of the hepatotoxic metabolite anethole epoxide (AE) is used to interpret the pathological changes observed in different species and sexes of laboratory rodents in chronic and subchronic dietary studies. Female Sprague Dawley rats metabolize more trans-anethole to AE than mice or humans and, therefore, are the most conservative model for evaluating the potential for AE-induced hepatotoxicity in humans exposed to trans-anethole from use as a flavouring substance. At low levels of exposure, trans-anethole is efficiently detoxicated in rodents and humans primarily by O-demethylation and omega-oxidation, respectively, while epoxidation is only a minor pathway. At high dose levels in rats, particularly females, a metabolic shift occurs resulting in increased epoxidation and formation of AE. Lower activity of the "fast" acting detoxication enzyme epoxide hydrolase in the female is associated with more pronounced hepatotoxicity compared to that in the male. The continuous intake of high dose levels of trans-anethole (i.e. cumulative exposure) has been shown in dietary studies to induce a continuum of cytotoxicity, cell necrosis and cell proliferation. In chronic dietary studies in rats, hepatotoxicity was observed when the estimated daily hepatic production of AE exceeded 30 mg AE/kg body weight. In female rats, chronic hepatotoxicity and a low incidence of liver tumours were reported at a dietary intake of 550 mg trans-anethole/kg body weight/day. Under these conditions, daily hepatic production of AE exceeded 120 mg/kg body weight. Additionally, neither trans-anethole nor AE show any evidence of genotoxicity. Therefore, the weight of evidence supports the conclusion that hepatocarcinogenic effects in the female rat occur via a non-genotoxic mechanism and are secondary to hepatotoxicity caused by continuous exposure to high hepatocellular concentrations of AE. trans-Anethole was reaffirmed as GRAS (GRASr) based on (1) its low level of flavour intake (54 microg/kg body weight/day); (2) its metabolic detoxication pathway in humans at levels of exposure from use as a flavouring substance; (3) the lack of mutagenic or genotoxic potential; (4) the NOAEL of 120 mg trans-anethole/kg body weight/day in the female rat reported in a 2 + -year study which produces a level of AE (i.e. 22 mg AE/kg body weight/day) at least 10,000 times the level (0.002 mg AE/kg body weight day) produced from the intake of trans-anethole from use as a flavouring substance; and (5) the conclusion that a slight increase in the incidence of hepatocellular tumours in the high dose group (550 mg trans-anethole/kg body weight/day) of female rats was the only significant neoplastic finding in a 2+ -year dietary study. This finding is concluded to be secondary to hepatotoxicity induced by high hepatocellular concentrations of AE generated under conditions of the study. Because trans-anethole undergoes efficient metabolic detoxication in humans at low levels of exposure, the neoplastic effects in rats associated with dose-dependent hepatotoxicity are not indicative of any significant risk to human health from the use of trans-anethole as a flavouring substance.

The influence of clopidogrel 75 mg, given once daily for 10 days on hepatic P-450 mixed function oxidases, was examined by assessing its effect on the disposition of antipyrine, on urinary 6-betahydroxycortisol (6beta-OHC) and on the plasma activity of gamma-glutamyl transpeptidase. This double-blind, randomized, placebo-controlled study was conducted in two parallel groups of 10 healthy young volunteers. Subjects were required to fast for 12 hours before and for 4 hours after dosing. Antipyrine 10 mg/kg was administered in the morning, two days before treatment (day -2) and 24 hours after the last dose of clopidogrel or placebo. Plasma levels of antipyrine, and urinary excretion of antipyrine, 3-hydroxymethyl-antipyrine and nor-antipyrine were measured over 36 hours post-drug for pharmacokinetic determinations. Bleeding time and platelet aggregation induced by 5 microM of ADP were measured before treatment (baseline) and at regular intervals after dosing during treatment. Clopidogrel treatment had a marked effect on platelet aggregation and bleeding time. No significant change in the disposition of antipyrine was observed after the ingestion of clopidogrel over 10 days: mean AUC ratio (+/-SEM) for plasma antipyrine was 1.021+/-0.023 for the clopidogrel group versus 1.001+/-0.019 for the placebo group; mean day 10/day -2 t 1/2 ratios were 1.019+/-0.018 and 1.027+/-0.023, respectively. Urinary excretions of antipyrine and metabolites were unchanged by clopidogrel compared to placebo. The changes in plasma cortisol concentrations, 6beta-OHC excretion and serum gamma-glutamyl transpeptidase activities observed at the end of treatment were fully comparable between the two treatment groups. Thus, the different tests showed no evidence of hepatic enzyme induction by clopidogrel in a pharmacologically effective dose regimen.

Retinoids can reverse neoplastic lesions and prevent second primary tumors in the aerodigestive tract. These effects are thought to be mediated by nuclear retinoic acid receptors (RARs) and retinoid X receptors (RXRs), each receptor group including three subtypes (alpha, beta, and gamma). Previously, we found that RARbeta expression was suppressed in lung cancer. In this study, we investigated whether expression of RARbeta is modulated by chemopreventive intervention.

Retinoic acid receptor-beta (RAR-beta) mRNA is not expressed by retinoid-resistant renal cancer cell lines but is present in retinoid-sensitive SK-RC-06 renal cancer cells and increases following incubation with retinoic acid (RA), suggesting that the antitumor action of RA is mediated through RAR-beta (A. D. Hoffman et al., Clin. Cancer Res., 2: 1077-1082, 2996). To determine whether RAR-beta expression correlates in vivo with major clinical response to patients with renal cell carcinoma (RCC) who were treated with retinoid-based therapy, we used in situ hybridization to analyze RAR-beta expression in tumor specimens obtained from patients who were treated on a clinical trial with 13-cis-RA and IFN-alpha. Thirty-three tissue specimens were analyzed (23 pretreatment and 10 on-treatment). mRNA expression was based on staining intensity, with scores within tumor cells ranging from 0 to 2, where a score of 0 indicated absence of staining, a score of 1 indicated weak staining, and a score of 2 indicated strong staining. RAR-beta expression was present in 22 of 23 (96%) pretreatment and 9 of 10 (90%) on-treatment specimens. Pretreatment levels of expression did not associate with the site of biopsy and did not predict for major clinical response to RA plus IFN-alpha therapy (two-sided Fisher's exact test, P = 0.826). However, an increase in the intensity of RAR-beta mRNA expression was detected in four of five (80%) patients who achieved a major response but in none of the five patients with progressive disease in whom sequential biopsies were available (two-sided Fisher's exact test, P = 0.048). These data show that RAR-beta transcripts increase in tumor cells of RCC patients who clinically respond to retinoid-based therapy. Retinoids that potently induce RAR-beta expression should be evaluated in the treatment of advanced RCC.

A substantial number of studies have demonstrated increased imidazoline receptors (I1 binding sites) on platelets of depressed patients and downregulation following antidepressant treatments. Herein, imidazoline receptor binding protein (IRBP) antiserum was used to quantify imidazoline receptors on platelets of depressed patients before and after treatment with the atypical aminoketone antidepressant, bupropion. Western blots revealed an increase in IRBP-immunodensity (p = 0.01, two-tailed) in a 33 kDa protein band in untreated depressed patients (n = 21) as compared with controls (n = 17). This band has been positively correlated with I1 binding sites on platelets. Following 6 weeks' treatment with bupropion, IRBP-immunodensity was downregulated in depressed patients (p = 0.03, paired t-test); predominantly in responders (p = 0.005). Patients non-responsive to bupropion (n = 5) were significantly different from responders (p = 0.05) by exhibiting no elevation in IRBP-immunodensity at pre-treatment and no downregulation of the 33 kDa band after treatment. IRBP-immunodensity was negatively correlated (r = -0.79, p = 0.01) with plasma concentrations of bupropion and its metabolites at week-4 of BUP treatment. Thus, a 33-kDa IRBP on platelet plasma membranes is elevated in depression and normalized in responders to bupropion.