Lymphatic endothelium-specific hyaluronan receptor LYVE-1 is expressed by stabilin-1+, F4/80+, CD11b+ macrophages in malignant tumours and wound healing tissue in vivo and in bone marrow cultures in vitro: implications for the assessment of lymphangiogenesis.

Lymphangiogenesis is a novel prognostic parameter for several cancers that is preferentially quantified by immunohistochemistry of the lymphatic endothelium-specific hyaluronan receptor LYVE-1. Recently, the specificity of LYVE-1 was challenged by serendipitous observations of LYVE-1 expression in rare tissue macrophages. As expression of the hyaluronan receptor-like molecule stabilin-1 is shared by sinusoidal endothelium and macrophages, a thorough analysis of LYVE-1 expression was performed using macrophage-specific markers in vivo and in vitro. In murine tumour models and excisional wound healing, LYVE-1 expression occurred in a subset of CD11b(+), F4/80(+) tissue macrophages that preferentially co-expressed stabilin-1. Upon comparison of single- and double-labelling immunofluorescence, it became apparent that LYVE-1(+) macrophages mimic sprouting and collapsed lymphatic vessels. In vitro, LYVE-1 expression was induced in 25-40% of murine bone marrow-derived macrophages upon exposure to B16F1 melanoma-conditioned medium and IL-4/dexamethasone. By FACS analysis, 11.5% of bone marrow-derived macrophages were LYVE-1(+), stabilin-1(+) double-positive, while 9.9% were LYVE-1(+), stabilin-1(-) and 33.5% were LYVE-1(-), stabilin-1(+). Northern and western analyses confirmed expression of LYVE-1 mRNA and protein in bone marrow-derived macrophages. In the light of the current debate about true endothelial trans-differentiation versus endothelial mimicry of monocytes/macrophages, LYVE-1(+), stabilin-1(+) non-continuous endothelial-like macrophages will require further developmental and functional analyses. In conclusion, the findings imply that LYVE-1 staining must be supplemented by double labelling with macrophage markers in order to differentiate clearly between LYVE-1(+) lymphatics and LYVE-1(+) tumour-infiltrating macrophages. This improved approach will help to clarify the prognostic significance of lymphangiogenesis in malignant tumours.

Heterogeneity of non-insulin-dependent diabetes expressed as variability in insulin sensitivity, beta-cell function and cardiovascular risk profile.

AIMS: The present study investigated the variability in insulin sensitivity and beta-cell function and their relationship to anti-glutamic acid decarboxylase (GAD) positivity and the metabolic syndrome in a group of patients with non-insulin-dependent diabetes mellitus (NIDDM). METHODS: Fifty-four subjects aged 59.5 +/- 6.1 (mean +/- SD) years with NIDDM for 7.9 +/- 3.9 years referred to hospital due to poor glycaemic control, were investigated. Insulin sensitivity was determined by the euglycaemic hyperinsulinaemic glucose clamp technique as the glucose disposal rate relative to the insulin level obtained (GDRI), and also estimated with the homeostasis model assessment (HOMA-S). beta-cell function was measured by assaying the fasting and glucagon-stimulated C-peptide levels and with the HOMA-B. RESULTS: The insulin sensitivity varied 18-fold between subjects when estimated with the clamp and six-fold when estimated with HOMA-S, and was lower the more criteria for the metabolic syndrome present (P = 0.0001 by anova). The beta-cell function varied four-fold when measured as stimulated C-peptide, and eight-fold when estimated with the HOMA-B. The levels of fasting C-peptide and HOMA-B values tended to be lower in anti-GAD+ (n = 11) than in anti-GAD-subjects (P = 0.06 and P = 0.08, respectively). From previously published coronary risk charts, we estimated the 10-year risk of a coronary heart disease (CHD) event to be > 20% in 17 of 39 patients free from cardiovascular disease at the time of study, 16 of whom qualified for a diagnosis of the metabolic syndrome. CONCLUSIONS: The wide variations in insulin sensitivity and beta-cell function found among subjects with NIDDM support the notion that the disorder is highly heterogeneous. Reduced insulin sensitivity was clearly related to the metabolic syndrome and an increased risk for CHD.

NK- and CD8(+) T cell-mediated eradication of established tumors by peritumoral injection of CpG-containing oligodeoxynucleotides.

Unmethylated cytosine-phosphorothioate-guanine (CpG) containing oligodeoxynucleotides (CpG-ODN) are known to act as adjuvants and powerful activators of the innate immune system. We investigated the therapeutic effect of CpG-ODN on a variety of established mouse tumors including AG104A, IE7 fibrosarcoma, B16 melanoma, and 3LL lung carcinoma. These tumors are only weakly immunogenic and notoriously difficult to treat. Repeated peritumoral injection of CpG-ODN resulted in complete rejection or strong inhibition of tumor growth, whereas systemic application had only partial effects. The CpG-ODN-induced tumor rejection was found to be mediated by both NK and tumor-specific CD8(+) T cells. Comparison of parental tumors and variants rendered more antigenic by transfection with tumor Ags suggested that the efficiency of the CpG-ODN therapy correlated with the antigenicity of the tumors. Peritumoral CpG-ODN treatment was even effective in a situation where the immune system was tolerant for the tumor Ag, as shown by breakage of tolerance and tumor elimination. These results suggest that peritumoral application of CpG-ODN acts locally by inducing NK cells, and also leads to efficient presentation of tumor Ags and stimulation of CD8(+) effector and memory T cells, thus providing a powerful antitumor therapy that can be also applied without knowledge of the tumor Ag.

Autoaggression and tumor rejection: it takes more than self-specific T-cell activation.

Establishment of self-tolerance prevents autoaggression against organ-specific self-antigens. This beneficial effect, however, may in turn be responsible for tumor immune evasion. Thus, dissecting the mechanisms leading to the breakdown of self-tolerance in autoimmune diseases might provide insights for successful antitumor immune therapies. In a variety of animal models, organ- or tumor-specific immunity has been described, focusing on antigen-specific T-cell activation. Here, we discuss two transgenic mouse models which demonstrate that both autoaggression and tumor rejection require more than activated, self-reactive T cells. TCR transgenic mice, which are tolerant to a liver-specific MHC class I antigen, Kb, can be activated to reject Kb-positive grafts, but fail to attack Kb-expressing liver. However, autoaggression occurs when activated T cells are combined with "conditioning" of the target organ by irradiation or infection with a liver-specific pathogen. Similarly, in a mouse model of islet cell carcinoma, neither co-stimulatory tumor cells nor highly activated antitumor lymphocytes provoke an effective immune response against the tumor. Instead, a combination of activated lymphocytes and irradiation is required for lymphocyte infiltration into solid tumors. Both model systems provide evidence that although activated antigen-specific lymphocytes are a prerequisite for autoaggression, effector cell extravasation and appropriate interaction with the target organ/tumor are equally important. Thus, we propose that the organ/tumor microenvironment is a critical parameter in determining the effectiveness of an anti-self immune response.

Cimetidine reduces weight and improves metabolic control in overweight patients with type 2 diabetes.

OBJECTIVE: To investigate the weight-reducing effect of cimetidine in overweight patients with Type 2 diabetes. DESIGN: A 12-week clinical intervention study of 400 mg cimetidine prescribed three times daily in a randomised, double-blind, placebo-controlled design. SUBJECTS: Forty-three overweight patients with Type 2 diabetes (age 18-65 y, body mass index (BMI) 27.2-48.2 kg/m2). MEASUREMENTS: Body weight, BMI, body fat, waist and hip circumference, waist/hip ratio, blood pressure, fasting blood glucose, HbA1c, plasma concentrations of insulin, insulin/glucose ratio and lipids at the start and after 12 weeks, and daily recordings of appetite. RESULTS: Subjects given cimetidine (n = 19) and placebo (n = 24) lost 5.0 +/- 2.2 kg (mean +/- s.d.) and 1.3 +/- 1.1 kg, respectively. Significant reductions were observed in appetite, body fat (29.9 +/- 6.6% to 25.3 +/- 7.4%), waist circumference (111.5 +/- 10.3 cm to 107.4 +/- 10.6 cm), waist/hip ratio (0.96 +/- 0.08 to 0.94 +/- 0.08), and systolic and diastolic blood pressure (reductions of 6.9 +/- 11.4 mm Hg and 6.0 +/- 6.6 mm Hg, respectively) in cimetidine group only. Significant decreases in fasting concentrations of blood glucose, HbA1c, plasma insulin, insulin/glucose ratio, plasma triglycerides and a significant increase in plasma high-density lipoprotein cholesterol were observed in the cimetidine group only. CONCLUSIONS: Cimetidine reduces appetite and body weight, and improves metabolic control in overweight subjects with Type 2 diabetes.

Tumor microenvironment can restrict the effectiveness of activated antitumor lymphocytes.

Transgenic mice expressing the oncogene SV40 T antigen (Tag) in the insulin-producing beta cells of the pancreas develop islet cell carcinomas. Expression of the oncogene beginning in adult life leads to autoimmunity and lymphocytic infiltration of premalignant lesions. Nevertheless, Tag-expressing solid tumors escape the immune surveillance and are devoid of infiltrating lymphocytes. Attempting to elicit a tumor inflammatory response, we have both expressed a potent costimulator in oncogene-expressing beta cells and increased the abundance of reactive T cells. Coexpression of the costimulator B7.1 and the Tag oncoprotein leads to destruction of normal and premalignant islets and severe diabetes. Nevertheless, Tag+ tumors eventually develop, evidencing significantly reduced B7.1 expression and no infiltration. Another approach, whereby the abundance of reactive T cells was increased in double transgenic mice expressing Tag and a Tag-specific, CD4+-restricted T-cell receptor, was similarly unable to elicit tumor infiltration and destruction. Thus, neither costimulatory tumor cells nor hyperactivated antitumor lymphocytes were sufficient to produce an effective tumor immune response. In contrast, adoptive transfer of lymphocytes activated ex vivo did result in modest tumor infiltration with a limited induction of high endothelial venules on tumor vasculature, provided that T cells were transferred into irradiated recipients. However, adoptive transfer of ex vivo activated lymphocytes did not produce the dramatic inflammation seen in premalignant lesions. Thus, in addition to the parameters of activation and abundance of antitumor lymphocytes, the tumor microenvironment is evidently a critical parameter that can suppress lymphocyte extravasation and/or function inside tumors, likely in part via distinctive properties of the tumor vasculature.

The mouse tyrosinase gene. Promoter modulation by positive and negative regulatory elements.

Tyrosinase, the key enzyme in melanin synthesis, is expressed specifically in pigment-producing cells. Studies with transgenic mice and gene transfer experiments have demonstrated that the 270-base pair 5'-flanking sequence of the mouse tyrosinase gene leads to weak but cell type-specific and developmentally regulated expression. To elucidate the underlying transcriptional control, we focused on the identification of cis-acting elements within this 270-base pair minimal promoter. We also addressed the potential role of promoter elements in the control of cAMP regulation of the tyrosinase gene. Deletion and linker scanning mutagenesis revealed that promoter activity is modulated by two positive elements and one negative element. One of the positive elements includes the M-box, a sequence motif shared with the promoter of two other melanocyte-specific genes, trp-1 and trp-2. Cotransfection experiments provide evidence that a basic helix-loop-helix-zipper protein, encoded at the microphthalmia gene locus, transactivates the tyrosinase promoter, probably by binding to the M-box. Activating cis elements are bound by nuclear factors in vitro and confer increased expression to a reporter gene both in melanoma cells and in fibroblasts. We therefore suggest that the positive promoter elements modulate tyrosinase expression rather than determine cell specificity in vivo, whereas the negative element acts cell type specifically.

Analysis of the mouse tyrosinase promoter in vitro and in vivo.

The restricted expression of the tyrosinase gene in cells producing pigment suggests the presence of cis-regulatory elements and trans-acting tissue-specific factors. Since 270 bp upstream of the transcriptional start site contain sufficient information for tissue-specific and developmentally regulated expression, we confined our analyses to this region. In this article, we discuss the recent results we have obtained on the regulation of the mouse tyrosinase gene expression demonstrating the existence of one negative and two positive-acting elements in vitro. We have evidence that the positive elements do not determine pigment production in vivo but rather modulate transcription of the mouse tyrosinase gene.

The cyclic adenosine 3',5'-monophosphate- and the glucocorticoid-dependent enhancers are targets for insulin repression of tyrosine aminotransferase gene transcription.

The pathway of gluconeogenesis is activated in liver shortly after birth and is controlled by glucagon and glucocorticoids, which stimulate, and insulin, which inhibits, the expression of genes coding for gluconeogenic enzymes. To understand the molecular basis of this cell type-specific and coordinate control, we analyzed the cis-regulatory elements of the tyrosine aminotransferase gene, which confer liver cell-specific expression in dependence of these hormones. The cAMP-responsive element (CRE) of the TAT gene is an essential element within a liver-specific enhancer and is recognized by the CRE-binding protein (CREB) in a phosphorylation-dependent manner. The glucocorticoid response is mediated by a complex regulatory unit comprised of the glucocorticoid receptor and other transcription factor-binding sites. Here, we show that both the cAMP- and glucocorticoid-inducible enhancers are targets for the antagonistic effects of insulin. The insulin-responsive sequences coincide with the CREB-binding site of the cAMP-responsive enhancer and a hepatocyte nuclear factor-3-binding site within the glucocorticoid-responsive unit. This design of the hormone-dependent enhancers reflects the molecular mechanism underlying the onset of tyrosine aminotransferase expression at birth when insulin levels decrease and concentrations of glucagon and glucocorticoids increase.

A cell-specific enhancer far upstream of the mouse tyrosinase gene confers high level and copy number-related expression in transgenic mice.

The tyrosinase gene encodes the key enzyme of melanin production and is tightly regulated during development. A yeast artificial chromosome covering the mouse tyrosinase gene has been shown to rescue completely the albino phenotype of recipient mouse strains, conferring copy number-dependent, position-independent expression. To investigate the presence of cis-acting regulatory elements responsible for the appropriate expression of the tyrosinase gene, DNase I hypersensitive site mapping was performed. A melanoma cell-specific DNase I hypersensitive site was identified at -12 kb upstream of the tyrosinase gene. Functional analysis of the corresponding cis-acting element in transgenic mice and transient transfection assays revealed properties of a strong cell-specific enhancer. RNA expression levels of the transgene correlate with copy number, which is reflected in coat colour and eye pigmentation of transgenic mice. Full enhancer activity in transient transfections is obtained with a minimal sequence of 200 bp. Protein binding analysis reveals the presence of a melanoma cell-specific complex which might contribute to the faithful expression of the tyrosinase gene.

Targeted mutation of the CREB gene: compensation within the CREB/ATF family of transcription factors.

The cAMP response element binding protein (CREB) has been implicated as a key regulator in the transcriptional control of many genes. To assess the functional importance of CREB in vivo and its role in development, we used gene targeting to generate mice with a disruption of the CREB gene. Homozygous mutant mice appeared healthy and exhibited no impairment of growth or development. In this report we demonstrate that CREB and two other members of the CREB/ATF family, cAMP response element modulation protein (CREM) and activating transcription factor 1 (ATF1), appear to form a unique subgroup within this extensive class of transcription factors. Examination of CREM mRNA and protein levels in CREB mutant mice demonstrated overexpression of CREM in all tissues examined, but no change in ATF1 levels. These data demonstrate that CREB is not the sole mediator of cAMP-dependent transcriptional regulation and probably acts in concert with a specific subset of cAMP response element-binding proteins to transduce the cAMP signal and, in its absence, these same proteins can compensate for CREB function in vivo.

24,25-dihydroxy vitamin D3 treatment inhibits parathyroid-stimulated adenylate cyclase in iliac crest biopsies from uremic patients.

Renal osteodystrophy with increased bone resorption is a major clinical problem in patients with chronic renal failure. Previous reports have shown that treatment with 24,25-dihydroxy vitamin D3 (24,25(OH)2D3) may result in decreased bone resorption. The present study addresses basic mechanisms for the action of 24,25(OH)2D3 in bone of patients with elevated serum parathyroid hormone (PTH) levels due to chronic renal disease. Twenty-four patients 56 +/- 17 years old (mean +/- SE) with chronic kidney disease in the predialytic state (serum creatinine > 150 mumol/l) and elevated serum midregion PTH > 1.2 micrograms/l were randomly assigned to oral treatment with either 1,25-dihydroxy vitamin D3 (1,25(OH)2D3) (0.25-0.50 microgram/day), 24,25(OH)2D3 (daily dose of 15 micrograms), or a combination of the two vitamin D3 analogs. The control group received calcium carbonate (maximal dosage of 1 g x 3). Selected variables in serum and urine as well as hormone sensitive adenylate cyclase (AC) in iliac crest biopsies were assessed before treatment and during follow-up after two and six months. Serum levels of 1,25(OH)2D3 and 24,25(OH)2D3 were significantly (P < 0.05) increased after two and six months in the respective treatment groups. Net bone PTH-enhanced AC (PTH-AC) fell abruptly (P < 0.01) after two months of treatment and was nearly abolished (P < 0.01) after six months with 24,25(OH)2D3 given alone or in combination with 1,25(OH)2D3. An inverse relationship (r = -0.57, P < 0.05, n = 48) between net PTH-AC in bone and serum levels of 24,25(OH)2D3 was demonstrated. In all groups, serum total calcium (s-Ca) was maintained within normal range.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular characterization of the mouse tyrosinase gene: pigment cell-specific expression in transgenic mice.

Tyrosinase is the key enzyme in melanin synthesis, and is expressed in the pigment epithelium of the retina, a cell layer derived from the optic cup; and in neural crest-derived melanocytes of skin, hair follicle, choroid, and iris. The tyrosinase gene has been cloned and shown to map to the well-characterized c-locus (albino locus) of the mouse. Subsequent studies demonstrated that a functional tyrosinase minigene was able to rescue the albino phenotype in transgenic mice. The transgene was expressed in a cell type-specific manner in skin and eye. During development of the mouse, the tyrosinase gene is expressed in the pigment epithelium of the retina as early as day 10.5 of gestation. In the hair follicle, tyrosinase gene expression is detected from day 16.5 onwards. This cell-type-specific expression is largely reproduced in transgenic mice. Our results suggest that sequences in the immediate vicinity of the mouse tyrosinase gene are sufficient to provide cell type-specificity and developmental regulation in melanocytes and the pigment epithelium.

Prevention of hemodialysis associated hypoxemia by use of low-concentration bicarbonate dialysate.

Hypoxemia during acetate dialysis is caused by hypoventilation due to bicarbonate loss across the dialyzer and its regeneration from acetate by a CO2 consuming process. Loss of bicarbonate is prevented by using a bicarbonate containing dialysate, but hypoxemia is still found by many authors. In the current study, ten patients were dialyzed twice against acetate dialysate, high concentration bicarbonate (36 mmol/L), and low concentration bicarbonate (29 mmol/L) dialysates. A significant decrease in PO2 was found during both acetate and high concentration bicarbonate dialysis. Hypoxemia was prevented by low concentration bicarbonate dialysate. A possible explanation for the hypoxemia in high concentration bicarbonate dialysis may be hypoventilation induced by alkalosis. It was concluded that low concentration bicarbonate dialysate prevents hypoxemia during hemodialysis.

Removal of chelated aluminium during haemodialysis using polysulphone high-flux dialysers.

Polysulphone high-flux dialysers were used for removal of chelated aluminium in desferrioxamine-treated patients on maintenance haemodialysis. When compared with charcoal haemoperfusion in series with a cuprophane dialyser, the same aluminium clearance was obtained (34% of blood flow). During 4 h of haemodialysis serum aluminium was reduced to the concentration seen before desferrioxamine infusion. We conclude that high-flux polysulphone dialysers remove chelated aluminium as efficiently as does charcoal haemoperfusion, and at a lower cost.