Circulating exosomes deliver free fatty acids from the bloodstream to cardiac cells: Possible role of CD36.

Regulation of circulating free fatty acid (FFA) levels and delivery is crucial to maintain tissue homeostasis. Exosomes are nanomembranous vesicles that are released from diverse cell types and mediate intercellular communication by delivering bioactive molecules. Here, we sought to investigate the uptake of FFAs by circulating exosomes, the delivery of FFA-loaded exosomes to cardiac cells and the possible role of the FFA transporter CD36 in these processes. Circulating exosomes were purified from the serum of healthy donors after an overnight fast (F) or 20 minutes after a high caloric breakfast (postprandial, PP). Western blotting, Immunogold Electron Microscopy and FACS analysis of circulating exosomes showed that CD36 was expressed under both states, but was higher in postprandial-derived exosomes. Flow cytometry analysis showed that circulating exosomes were able to take-up FFA directly from serum. Importantly, preincubation of exosomes with a blocking CD36 antibody significantly impeded uptake of the FFA analogue BODIPY, pointing to the role of CD36 in FFA exosomal uptake. Finally, we found that circulating exosomes could delivery FFA analogue BODIPY into cardiac cells ex vivo and in vivo in a mice model. Overall, our results suggest a novel mechanism in which circulating exosomes can delivery FFAs from the bloodstream to cardiac tissue. Further studies will be necessary to understand this mechanism and, in particular, its potential involvement in metabolic pathologies such as obesity, diabetes and atherosclerosis.

Central neurocytoma presenting with intraventricular hemorrhage: case report and review of literature.

We report a case of a 25 year old man presenting with acute headache, vomiting and nuchal rigidity. Computed Tomography (CT) scan and MRI without contrast showed a right ventricular hemorrhage surrounding a mass lesion. The tumor and hematoma were completely removed by neurosurgical transcortical-transventricular approach. Anatomopathological analysis revealed a central neurocytoma. Central neurocytoma seldom present with hemorrhage. We review 16 cases of neurocytoma with hemorrhage. It is important to recognize central neurocytoma as a cause of intraventricular hemorrhage, especially in adolescents and young adults. Outcome is often favorable when the tumor is completely removed. In some patients the clinical course is more aggressive and additional treatment such as radiotherapy, radiosurgery or chemotherapy is needed.

Homotypic cluster formation of dendritic cells, a close correlate of their state of maturation. Defects in the biobreeding diabetes-prone rat.

Aggregation of dendritic cells (DCs) in homotypic clusters has been described in vivo in lymph and skin, and here we report studies on homotypic clustering of rat splenic (s) DCs in vitro. Wistar rat sDCs readily formed homotypic clusters in culture, which increased in number and size over time (with a peak at t = 3 h). Keeping the cells at higher densities or treatment with anti-CD43 induced more and larger homotypic clusters. After such enhanced clustering the DCs had increased their T cell stimulating capabilities in syngeneic mixed lymphocyte reaction, and had a higher expression of CD80 and CD86 (signs of maturation). Ag transfer from bovine serum albumin-fluorescein isothiocyanate-pulsed to unpulsed DCs was observed during clustering. Here we also show that sDCs of the biobreeding diabetes-prone (BB-DP) rat, a model of autoimmune diabetes/thyroiditis, formed fewer and smaller clusters than Wistar sDCs, and that DC-DC clustering resulted in only a modest maturation of the cells (as determined in syn MLR and by phenotyping). Anti-CD43 completely restored the clustering defect BB-DP DCs in vitro, yet T cell-stimulating capability was only restored to a limited extent. Ag transfer in BB-DP DC clusters was similar.

Glucocorticoids hamper the ex vivo maturation of lung dendritic cells from their low autofluorescent precursors in the human bronchoalveolar lavage: decreases in allostimulatory capacity and expression of CD80 and CD86.

Dendritic cells (DCs) were prepared from human bronchoalveolar lavage (BAL) cells. We previously reported that, in particular, the CD1a fraction of the low autofluorescent (LAF) cells contains the precursors for DCs: after overnight culture, 40% of the LAF cells change into functionally and phenotypically prototypic dendritic/veiled cells. There are, as yet, no data on the modulatory effects of glucocorticoids (GC) on the maturation and function of such DCs isolated from the human lung. Functional tests (allogeneic mixed lymphocyte reaction: allo-MLR) were therefore performed with CD1a+ LAF cells at different stimulator-to-T-cell ratios and after preincubation with different dexamethasone (DEX) concentrations. DEX caused suppression of the T-cell stimulatory capacity of CD1a+ LAF cells, which was dose-dependent, and more evident at the higher stimulator-to-T-cell ratios. Here, we also show that CD80 and CD86 are normally expressed at low levels on CD1a+ LAF cell-derived DCs compared to other DC populations. This low-level expression of costimulatory molecules is discussed here in relation to the previously reported low-level expression of CD80 (and CD86) on lung DCs in experimental animals. This appears to play a role in a predominant Th2 cell stimulating potential of DC from the lung environment. DEX exposure of CD1a+ LAF cells prevented the upregulation of even this low-level expression of CD80 and CD86. The veiled/dendritic morphology and the expression of other relevant cell surface markers and adhesion molecules was not affected by DEX exposure. It is concluded that DEX hampers the maturation of CD1a+ LAF cells into active lung DCs.

A functional and phenotypic study on immune accessory cells isolated from the thyroids of Wistar and autoimmune-prone BB-DP rats.

Dendritic cells (DCs) comprise a small population of cells in the normal thyroid. These excellent antigen-presenting cells (APCs) are thought to be involved in the initiation of thyroid autoimmune reactions. However it is not known whether the APCs involved in this process are indeed DCs, or thyrocytes. Our aims were as follows: (1) to isolate DCs from the thyroid of normal Wistar rats and BB-DP rats prior to the development of lymphocytic thyroiditis; (2) to determine the T-cell stimulatory capability of such isolated thyroid DCs and to compare this capability to that of BB-DP thyrocytes and splenic DCs; and (3) to investigate the phenotype of isolated thyroid DCs and to compare it to that of splenic DCs; and (4) to investigate the capability of such thyroid DCs to regulate thyrocyte growth and function, and to compare it to our earlier reports demonstrating such capability with splenic DCs. Leukokcytic cell fractions were isolated from the thyroids of BB-DP and control Wistar rats of 7-20 weeks of age. The isolation steps included gentle enzymatic tissue disruption, the collection of non-plastic adherent cells and density gradient centrifugation of these cells to yield a low and a high density non-adherent fraction. The low density cell (LDC) fraction was composed of 50-75% leukocytes in both strains. These leukocytes were almost exclusively ED1+ monocytes or MHC-class II+ DC. The high density cell (HDC) fractions of both strains were composed of about 70% MHC-class II-negative thyrocytes and 30% ED1+ monocytes. The thyroid LDCs of both strains had an APC capability in syngeneic(syn)-MLR comparable to that of splenic DCs. However, the HDCs were extremely poor in syngeneic T cell stimulation. There was a difference in composition between the Wistar and the BB-DP LDC fractions: The Wistar LDCs were composed of 30-35% ED1+ monocytes and 15-20% typical MHC-II+ DCs, while BB-DP LDC fractions contained more ED1+ monocytes (about 70%), but fewer DCs (5-10%). In comparison to splenic DCs, thyroid DCs had a low CD80 and CD86 expression in both strains (i.e., an 'immature' phenotype). The LDCs of both animal strains were shown to decrease both basal and TSH-stimulated thyrocyte proliferation and T(3)release by about half. This report shows that a cell fraction enriched for monocytes and DCs can be isolated from the thyroids of both Wistar and BB-DP rats. The cells in this fraction were as capable as splenic DCs to act as T cell stimulators in syn-MLR. Since the thyroid HDCs (predominantly thyrocytes) were very poor at such T cell stimulation, thyroid monocytes and DCs (and not thyrocytes) are the prime candidates to act as immune accessory cells in the initiation of thyroid autoimmunity in the rat. Wistar thyroid LDCs differed in phenotype from BB-DP LDCs. The latter contained a lower percentage of DCs and a higher percentage of their precursors, the monocytes. Interestingly, a defect in the transition of monocytes to DCs has been described in another animal model of autoimmune thyroiditis/insulitis (the NOD mouse), as well as in thyroiditis and diabetic patients.

Signs of immaturity of splenic dendritic cells from the autoimmune prone biobreeding rat: consequences for the in vitro expansion of regulator and effector T cells.

From the biobreeding-diabetic prone (BB-DP) rat, an animal model for endocrine autoimmunity, phenotype and function of splenic dendritic cells (DC) were studied. Furthermore, the suppressive effect of peritoneal macrophages (pMphi) from the BB-DP rat in the MLR was investigated. Lower numbers of splenic DC were isolated from BB-DP rats than from control Wistar rats. In the preautoimmune phase, DC of the BB-DP rat had a lower surface MHC class II expression (and in preliminary data, a lower CD80 expression), ingested more bacteria, and had a lower stimulatory potency in the syngeneic (syn)MLR as compared with control DC. During disease development, the MHC class II expression further decreased, and a low stimulatory activity became evident in the allogeneic (allo)MLR. With regard to the expansion of suppressor/regulatory T cells, a lower percentage of RT6+ T cells but higher percentages of CD45RClow T cells were induced by BB-DP DC in synMLR, but not in alloMLR. An increase in the CD4/CD8 T cell ratio was observed in both the syn- and alloMLR due to a relative weak expansion of CD8+ T cells with DC of the BB-DP rat. Resident pMphi isolated from BB-DP or Wistar rats were equally effective in suppressing the DC-driven synMLR. In conclusion, splenic DC from the BB-DP rat have a lower accessory cell function already at young age, before the development of disease, and expanded different subsets of effector/suppressor T cells in vitro as compared with those from Wistar rats. The dysfunction of DC from BB-DP rats is likely to be caused by their relative immaturity as indicated by their low class II and costimulatory molecule expression and relatively high phagocytic activity.

Antigen-presenting dendritic cells as regulators of the growth of thyrocytes: a role of interleukin-1beta and interleukin-6.

An accumulation of antigen-presenting dendritic cells (DC) in the thyroid gland, followed by thyroid autoimmune reactivity, occurs in normal Wistar rats during iodine deficiency, and spontaneously in diabetic-prone Biobreeding rats. This intrathyroidal DC accumulation coincides with an enhanced growth rate and metabolism of the thyrocytes, suggesting that both phenomena are related. Because DC are known to regulate the hormone synthesis and growth in other endocrine systems (i.e. the pituitary, the ovary, and the testis), we tested the hypothesis that DC, known for their superb accessory cell function in T cell stimulation, act as regulators of thyrocyte proliferation (and hormone secretion). We investigated the effect of (Nycodenz density gradient) purified splenic DC from Wistar rats on the growth rate of and thyroid hormone secretion by Wistar thyroid follicles (collagenase dispersion) in culture. Various numbers of DC and follicles were cocultured during 24 h. The proliferative capacity of thyrocytes was measured by adding tritiated thymidine (3H-TdR) and bromodeoxyuridine, the hormone secretion into the culture fluid was measured by using a conventional T3 RIA. Furthermore, antibodies directed against interleukin-1beta (IL-1beta), IL-6, and tumor necrosis factor-alpha (TNF-alpha) were added to these cocultures to determine the role of these cytokines in a possible DC regulation of thyrocyte growth. Cocultures were also carried out in the presence of antimajor histocompatibility complex-class I (MHC I), anti-MHC II, antiintercellular adhesion molecule-1 (ICAM-1), and antilymphocyte function-associated antigen-1alpha (LFA-1alpha) antibodies to possibly interfere with DC-thyrocyte interactions. The addition of DC to thyroid follicles clearly inhibited their 3H-TdR uptake, particularly at a 10:1 ratio, in comparison to follicle cultures alone, both under basal conditions and after TSH stimulation (75 +/- 7% and 49 +/- 11% reduction, respectively, n = 4). The follicle T3 secretion (after TSH stimulation) was also suppressed by DC in this system, but to a lesser extent (at best at an 1:1 ratio, 25 +/- 7% reduction, n = 4). The DC-induced inhibition of thyroid follicle growth was totally abrogated after addition of anti-IL-1beta antibodies; anti-IL-6 only had effect on the DC inhibition of non-TSH-stimulated thyrocytes, whereas anti-TNF-alpha demonstrated no effect at all. The antibodies to MHC and to adhesion molecules had also no effect on this DC-induced growth inhibition. The effect of the different anti-cytokine and anti-adhesion antibodies on the T3 secretion from thyroid follicles was not investigated. The clear inhibition of thyrocyte growth by splenic DC (classical antigen-presenting cells) again demonstrates the regulatory role of DC in endocrine systems. Proinflammatory cytokines such as IL-1beta and IL-6 are important mediators in this regulation. The here shown dual role of DC represents a link between the immune and endocrine system, which may form the gateway to the understanding of the initiation of thyroid autoimmune reactions and the thyroid autoimmune phenomena seen in iodine deficiency.

Pre-autoimmune thyroid abnormalities in the biobreeding diabetes-prone (BB-DP) rat: a possible relation with the intrathyroid accumulation of dendritic cells and the initiation of the thyroid autoimmune response.

Thyroid autoimmune reactions start with an accumulation of mainly dendritic cells in the thyroid. There is increasing evidence that, apart from being antigen-presenting cells, they are also able to control the growth and hormone synthesis of neighbouring endocrine cells. The questions thus arise: are dendritic cells accumulating in the pre-autoimmune thyroid in response to an altered proliferative or metabolic activity of thyrocytes, and do cytokines, monocyte chemoattractants, or both, have a role in their accumulation? We have investigated these questions in thyrocytes of the biobreeding diabetes-prone (BB-DP) rat in relation to the start of the intrathyroid accumulation of dendritic cells--that is, at about 9 weeks of age. BB-DP rats and Wistar rats (controls) were studied from 3 to 20 weeks of age. Hyperplastic goitre development was studied by assessing the thyroid weight and by measuring the number of thyrocyte nuclei per 0.01 mm2 thyroid section. In addition, the in situ expression of interleukin-6 (IL-6), tumour necrosis factor-alpha (TNF-alpha), monocyte-chemotactic protein-1 (MCP-1), and intercellular adhesion molecule-1 (ICAM-1) were studied by immunohistochemistry. The in vitro proliferative capacity of BB-DP and Wistar thyrocytes was measured by tritiated-thymidine ([3H]TdR) and bromodeoxyuridine (BrdU) incorporation into reconstituted, TSH- and non-TSH-stimulated, cultured thyroid follicles. Further in vitro studies consisted of measurement of the production of thyroxine (T4), triiodothyronine (T3), thyroglobulin, IL-6, TNF-alpha and MCP-1 by the thyroid follicles. BB-DP rats developed a small hyperplastic goitre between the ages of 9 and 12 weeks. The in vitro proliferative rate of thyrocytes isolated from hyperplastic BB-DP thyroids was significantly lower than that of Wistar thyrocytes. This phenomenon also occurred in follicles isolated from BB-DP rats before hyperplastic goitre development, which produced significantly less T4, but more T3, than did Wistar follicles of the same age. At the time of and after hyperplastic goitre development, BB-DP follicles exhibited altered metabolic behaviour and produced significantly more T4, but equal amounts of T3 compared with both Wistar follicles of the same age and follicles of younger BB-DP rats (both under basal conditions and TSH-stimulated). In vitro IL-6 production by these BB-DP thyroid follicles was also increased. There was no noteworthy difference in production of thyroglobulin and MCP-1 between BB-DP and Wistar follicles at any age. TNF-alpha was not produced by BB-DP or Wistar thyroid follicles. Immunohistochemistry revealed the expression of IL-6 by both BB-DP and Wistar thyroid follicle cells at all times of sampling. MCP-1 and TNF-alpha were expressed only when infiltrates were present in BB-DP thyroids (restricted to leucocytes, ages > 18 weeks). Modest ICAM-1 expression was restricted to large blood vessels in both BB-DP and Wistar thyroids; in the case of infiltrates (BB-DP rat) alone, high ICAM-1 expression was found on blood vessels and leucocytes in these infiltrations. At the time of intrathyroidal dendritic cells accumulation, BB-DP rats develop a small hyperplastic goitre. At that time there is also in vitro evidence for a shift to a higher production of thyroxine and IL-6 from thyrocyte follicles. The in vitro proliferation rate of BB-DP thyrocytes is, however, abnormally low (both in the pre- and hyperplastic period). Similar pre-autoimmune thyroid growth abnormalities have been described in another animal model of thyroid autoimmune disease, the obese strain chicken.

Enzyme-inducing effects of bicalutamide in mouse, rat and dog.

1. Bicalutamide, a non-steroidal antiandrogen, produced dose-related increases in total cytochrome P450 (P450) and aldrin epoxidase, but had no effect on ethoxyresorufin O-deethylase, when administered for 10 weeks at 0, 25, 75 and 150 mg/kg/day to the male dog. 2. In the male and female mouse, bicalutamide, administered orally at 75 mg/kg/day for 3 months, produced marked induction of total P450, ethoxycoumarin O-deethylase, pentoxyresorufin O-dealkylase and aldrin epoxidase. Immunoblotting showed that bicalutamide produced substantial induction of CYP2B isoforms, with lower increases in CYP3A. Immunohistochemistry of mouse liver sections also showed marked increases in the level of CYP2B isoforms, with an increase in the extent of distribution from centrilobular to panlobular; CYP3A isoforms were also increased, but to a lesser degree. 3. Bicalutamide, administered as 14 daily oral doses (250 mg/kg) to groups of male rats, produced increases primarily in ethoxycoumarin O-deethylase and erythromycin N-demethylase, together with smaller increases in ethoxyresorufin O-deethylase and pentoxyresorufin O-dealkylase; these changes were reversible within 7 days. Immunoblotting of microsomes and immunocytochemistry of liver sections showed that bicalutamide markedly induced CYP3A1, but had little effect on CYP2B1 in rat. Compared with dexamethasone, bicalutamide is a more selective inducer of CYP3A1 in rat. 4. Bicalutamide, administered to rats as 14 daily oral doses of 10 mg/kg, induced its own metabolism by stimulating both aromatic hydroxylation and direct glucuronidation. This effect was apparently offset by a concomitant decrease in hydrolysis of bicalutamide, resulting in no marked change in total amounts of dose eliminated over 2 days. 5. Although the secondary effects of enzyme induction result in thyroid hypertrophy and adenoma in rat and hepatocellular carcinoma in mouse following chronic administration of bicalutamide, these changes are considered to have little clinical relevance. In any case, bicalutamide does not produce enzyme induction in man at clinically relevant dose levels.

The effects of thymostimulin on immunological function in patients with head and neck cancer.

Patients with head and neck carcinoma show deficits of cellular immunity, probably due to low molecular mass factors (LMMFs) released by the tumour. Thymostimulin (TP1) restores the defective monocyte chemotaxis and dendritic cell clustering capability in the presence of the tumour when administered pre-operatively. In the present study we investigated these immune parameters in 39 patients treated with TP1 for 10 days pre- and 6 months post-operatively and in 22 patients who were not treated with TP1 for 6 months after operation. Removal of the tumour in non-TP1-treated patients also resulted in a restoration of monocyte and dendritic cell functions, while TP1 treatment gave no additional effect. LMMF levels in the blood of both TP1-treated and non-TP1-treated patients remained elevated even after removal of the tumour, and it is therefore concluded that it is unlikely that depression of cellular immunity is a direct effect of these LMMFS.

Histomorphological aspects of the development of thyroid autoimmune diseases: consequences for our understanding of endocrine ophthalmopathy.

In this short review we will first evaluate the histomorphological aspects of the human and spontaneous animal thyroid autoimmune diseases. These diseases include Hashimoto goiter, primary myxedema, Graves' disease, and the spontaneous forms of thyroiditis in the Bio Breeding (BB) rat, the nonobese diabetic (NOD) mouse, and the obese strain (OS) of chicken. Based on sequential histomorphological events in the animal models of thyroid autoimmune disease, a mechanism for the pathogenesis of thyroid autoimmune disease is proposed. Since one of the human thyroid autoimmune diseases, specifically Graves' disease, is often associated with ophthalmopathy, the histomorphological aspects of the ophthalmopathic process are also evaluated to consider its possible autoimmune character.

Thymostimulin enhancement of T-cell infiltration into head and neck squamous cell carcinoma.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) produces immunosuppressive low-molecular-mass factors (LMMFs) responsible for defects in the cell-mediated immune system. These defects include impaired monocyte chemotaxis and an impaired capability of dendritic cells (DC) to form cellular clusters. It has been shown previously that the immunomodulating drug thymostimulin (TP1) restores these defects in vitro. METHODS: An immunohistochemical study was performed on tumors of 18 patients with HNSCC who had preoperatively been treated with TP1 in one of three dosages (0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg body weight). Additionally, tumors of 4 patients who had been treated with a placebo and 12 patients who had not received any preoperative treatment were studied. A relative surface area of infiltration, meaning the percentage of stromal or epithelial tissue covered by infiltrating cells in histological sections, was calculated using an image analysis system (VIDAS RT) for CD3+ T-cells, CD14+/CD68+ monocytes/macrophages and L25+/CD1a+ dendritic cells for each tumor. RESULTS: A highly significant, denser T-cell infiltration into the stromal tissue area of tumors of patients who had been treated with TP1 when compared with tumors of non-TP1-treated patients was observed for all three dosages. None of the other tumor-infiltrating cell types was affected by TP1. In addition, a correlation was found between the tumor T-cell infiltration and capability of DCs in the peripheral blood to form clusters with T-cells. No correlation existed between CD3+ T-cell numbers in peripheral blood and T-cell infiltration into the tumor; nor were monocyte chemotactic functions in peripheral blood correlated with tumor infiltration by monocytes or monocyte-derived macrophages and DCs. CONCLUSIONS: Preoperative treatment of HNSCC patients with TP1 appears to strongly enhance tumor--T-cell infiltration. The number of tumor-infiltrating DCs was not affected by TP1, but a positive correlation between tumor--T-cell infiltration and DC clustering capability suggests that the functional status of DCs is important in improved cell-mediated immunity.

Enantioselective metabolism and pharmacokinetics of Casodex in the male rat.

1. Casodex, a non-steroidal antiandrogen, is a racemic mixture of R-Casodex, the pharmacologically active (-)-enantiomer, and S-Casodex, the inactive (+)-enantiomer. Single oral doses of pseudo-racemic 14C-Casodex (10 mg/kg), prepared from mixtures of either 14C-labelled R-Casodex and unlabelled S-Casodex, or 14C-S-Casodex and unlabelled R-Casodex, were administered to the intact and bile duct-cannulated male rat. 2. Neither enantiomer underwent stereochemical inversion, but the pharmacokinetics of Casodex showed marked enantioselectivity. 3. After dosing R-labelled Casodex, plasma concentrations of R-Casodex increased slowly to reach a peak of 3.50 +/- 0.05 micrograms/ml (mean +/- SEM) at 12 h and, thereafter, declined monoexponentially with an elimination half-life of 24 h. Plasma concentrations of S-Casodex rose rapidly to reach a much lower peak of 0.97 +/- 0.06 microgram/ml at 3 h and, thereafter, declined rapidly, although there were insufficient data to determine the half-life. R-Casodex had a much higher AUC0-24 (66 micrograms.h/ml) than S-Casodex (12 micrograms.h/ml). Plasma drug concentrations measured using an achiral assay were in very good agreement with the sum of the enantiomer concentrations throughout the profile. R-Casodex comprised 94% of the total plasma radioactivity at 12 h, decreasing to 75% at 120 h. 4. Plasma concentration data generated after administration of S-Casodex were similar to those observed after dosing R-labelled Casodex. S-Casodex comprised about 74% of the total plasma radioactivity at 6 h and only 41% at 24 h. 5. The urine of intact animals contained 36 +/- 2 and 48 +/- 3% of the dose respectively up to 48 and 120 h after dosing with R-labelled Casodex, and 33 +/- 4 and 34 +/- 4% respectively after dosing with S-labelled Casodex. The urine and bile of the cannulated rat contained 43 +/- 2 and 21 +/- 2% of the dose respectively up to 48 h after dosing with R-labelled Casodex and 37 (n = 2) and 50% respectively after dosing with S-labelled Casodex. 6. After dosing with R- or S-labelled Casodex, the urinary radioactivity consisted of the carboxylic acid metabolite formed by hydrolytic cleavage at the amide, whereas biliary radioactivity consisted of hydroxy-Casodex and Casodex, mainly conjugated with glucuronic acid. The clearance of R-Casodex by each of these pathways of metabolism was less than that of S-Casodex, with direct glucuronidation and hydroxylation showing greater enantioselectivity than hydrolysis.

In vivo effects of thymostimulin treatment on monocyte polarization, dendritic cell clustering and serum p15E-like trans-membrane factors in operable head and neck squamous cell carcinoma patients.

Head and neck squamous cell carcinoma patients have been characterized by impairments in their cell-mediated immune system, particularly by decreased chemotactic function of monocytes and impairments in the function of the monocyte-derived dendritic cells (viz, a decreased capability to form cell "clusters"). These impairments are thought to be due to immunosuppressive factors of low molecular mass released by tumor, the so-called p15E-like factors. These suppressive effects of p15-like factors can be neutralized in vitro by thymic peptides, such as thymostimulin (TP1). In a randomized double-blind, placebo-controlled multicenter trial in the Netherlands, 41 patients with operable head and neck squamous cell carcinomas (HNSCC) were treated for 10 days prior to surgery with intramuscular TP1 in one of three dosages (0.5 mg/kg; 1.0 mg/kg or 2.0 mg/kg body weight) or treated with placebo. Assessment of monocyte chemotaxis, the capability of dendritic cells to form clusters and the presence of p15E-like low-molecular-mass factors (LMMFs) in serum was performed before TP1 treatment and on the day of surgery. Findings demonstrated that TP1 in a dose of 1.0 mg/kg and 2.0 mg/kg resulted in normalization of impaired monocyte chemotactic capability. Although the cluster capability of dendritic cells after TP1 treatment improved, values only reached statistical significance for the 0.5 mg/kg group. Serum p15E-like LMMF levels were not affected by TP1 treatment in any of the patient groups. Contrary to expectations we found no correlation between elevated immunosuppressive LMMFs and defective monocyte chemotaxis or cluster capability of dendritic cells. We conclude that treatment with TP1 can improve monocyte chemotaxis in HNSCC patients but an effect on the production of p15E-like factors by carcinoma cells could not be demonstrated.

Use of proton nmr spectroscopy to measure propofol metabolites in the urine of the female Caucasian patient.

1. Six female Caucasian patients received i.v. doses of propofol (2.7 +/- 0.3 (SE) mg/kg) for induction of anaesthesia. Anaesthesia was maintained with nitrous oxide, oxygen and enflurane for periods up to 3 h. A total urine collection was made from each subject for 24 h after administration of propofol; samples were concentrated and analysed for propofol metabolites by nmr spectroscopy. 2. By 24 h, 50.9 +/- 4.0% of the dose of propofol was recovered as metabolites. The proportions of propofol metabolites, 1-quinol glucuronide (1-QG), 4-quinol glucuronide (4-QG), propofol glucuronide (PG) and 4-quinol sulphate (4-QS) recovered in urine (0-24 h) of the patients were 12 +/- 1, 8 +/- 2, 76 +/- 4 and 4 +/- 1% respectively. However, one patient showed a metabolite profile in which PG comprised 93% and 1-QG 7% of the total metabolites. 3. Nmr spectroscopy has been shown to be a satisfactory method for the quantification of propofol metabolites in urine without the necessity for reference samples.

New monoclonal antibodies against the putative immunosuppressive site of retroviral p15E.

Both retroviral infections as well as human tumors may cause immunosuppression. One of the factors involved in immunosuppression in patients with squamous cell carcinoma of the head and neck (SCC-HN) is a protein related to the retroviral protein p15E. A conserved, 17-amino acid sequence represents the immunosuppressive epitope of retroviral p15E. In order to study the relationship between SCC-HN associated immunosuppression and retroviral p15E, we produced three new monoclonal antibodies (MAbs; ER-IS1, ER-IS2, and ER-IS5) directed against the immunosuppressive synthetic CKS-17 peptide. These MAbs react with the immunosuppressive peptide (in enzyme-linked immunosorbent assay), with human tumor cell lines (in FACScan analysis), with retroviral p15E (on Western blot), and with cryostat sections of SCC-HN tumor tissue. In addition, the MAbs neutralize the immunosuppressive low molecular weight factors present in sera of patients with SCC-HN. These results show that retroviral p15E and the immunosuppressive factors associated with SCC-HN share a conserved immunosuppressive epitope and that MAbs against this epitope can be used for detection and neutralization of the tumor-associated immunosuppressive protein(s).