Paracrine Way Research Articles

Non-Hodgkin lymphoma expressing high levels of the danger-signalling protein HMGB1

HMGB1 is a high mobility group protein that can act either as a DNA binding protein or extracellularly as a cytokine-like danger signal. Extracellular HMGB1, either actively secreted or passively released by necrotic cells, is linked to inflammation and cancer. Herein, the results of a study to quantify the expression of HMGB1 in lymphomas by quantitative real-time RT-PCR are presented. HMGB1 expression was analysed in 18 non-Hodgkin lymphomas and two lymphoma cell lines. 11/18 primary lymphomas expressed HMGB1 mRNA at a level exceeding the average of normal lymph nodes. Immunohistochemistry showed that HMGB1 positivity is confined to the lymphoma cells. No correlation between HMGB1 expression and grading was found. However, a high percentage of lymphomas is overexpressing a danger-signalling protein. This protein can support the growth and angiogenesis of lymphoma cells in a paracrine way when released e.g. due to necrosis. Thus it constitutes an interesting therapeutic target as well.

Caffeine reduces TNFα up-regulation in human adipose tissue primary culture

Adipose tissue secretions play an important role in the development of obesity-related pathologies such as diabetes. Through inflammatory cytokines production, adipose tissue stromavascular fraction cells (SVF), and essentially macrophages, promote adipocyte insulin resistance by a paracrine way. Since xanthine family compounds such as caffeine were shown to decrease inflammatory production by human blood cells, we investigated the possible effect of caffeine on Tumor Necrosis Factor alpha (TNFalpha) and Interleukin-6 (IL-6) expression by human adipose tissue primary culture. For that purpose, human subcutaneous adipose tissue obtained from healthy non-obese women (BMI: 26.7 +/- 2.2 kg/m2) after abdominal dermolipectomy, was split into explants and cultured for 6 hours with or without caffeine. Three different concentrations of caffeine were tested (0.5 microg/mL, 5 microg/mL and 50 microg/mL). After 6 hours of treatment, explants were subjected to collagenase digestion in order to isolate adipocytes and SVF cells. Then, TNFalpha and IL-6 mRNA were analysed by real-time PCR alternatively in adipocytes and SVF cells. In parallel, we checked gene expression of markers involved in adipocyte differenciation and in SVF cells inflammation and proliferation. Our findings show a strong and dose dependent down-regulation of TNF-alpha gene expression in both adipocyte and SVF cells whereas IL-6 was only down regulated in SVF cells. No effect of caffeine was noticed on the other genes studied. Thus, caffeine, by decreasing TNFalpha expression, could improve adipose tissue inflammation during obesity.

P2 receptors in the regulation of renal transport mechanisms

Extracellular nucleotides (e.g., ATP) regulate physiological and pathophysiological processes through activation of nucleotide P2 receptors in the plasma membrane. Examples include such diverse processes as communication from taste buds to gustatory nerves, platelet aggregation, nociception, or neutrophil chemotaxis. Over approximately the last 15 years, evidence has also accumulated that cells in renal epithelia release nucleotides in response to physiological stimuli and that these nucleotides act in a paracrine and autocrine way to activate P2 receptors and play a significant role in the regulation of transport mechanisms and cell volume regulation. This review discusses potential stimuli and mechanisms involved in nucleotide release in renal epithelia and summarizes the available data on the expression and function of nucleotide P2 receptors along the native mammalian tubular and collecting duct system. Using established agonist profiles for P2 receptor subtypes, significant insights have been gained particularly into a potential role for P2Y(2)-like receptors in the regulation of transport mechanisms in the collecting duct. Due to the lack of receptor subtype-specific antagonists, however, the in vivo relevance of P2 receptor subtypes is unclear. Studies in gene knockout mice provided first insights including an antihypertensive activity of P2Y(2) receptors that is linked to an inhibitory influence on renal Na(+) and water reabsorption. We are only beginning to unravel the important roles of extracellular nucleotides and P2 receptors in the regulation of the diverse transport mechanisms of the kidney.

DIFFERENTIAL LOCALIZATION OF VIP AND VIP RECEPTOR IN CHICKEN GONADS DURING DEVELOPMENT

In avian species, gonadal differentiation not only depends on genetic information, but on the trophic action of numerous sex-determining factors and neuronal survival. Vasoactive Intestinal Peptide (VIP) participates in several functions, including sexual behavior, and has a neuroendocrine effect on the gonads. In gonadal tissue, VIP function is regulated via G-protein-coupled receptors and is mainly exerted through the VIP receptor type I (VIPR-I). In domestic hens, the VIP intercellular signal regulates the expression of enzymes that promote steroid production, such as progesterone and androgen in preovulatory follicles. In order to determine the sites of VIP synthesis and to compare it with VIPR-I localization during gonadal morphogenesis, pre and post-hatching left and right ovaries, as well as testicles were used. The patterns of VIP and VIPR-I localization were analyzed with immunohistochemistry techniques. From the 12th day of incubation, VIP distribution was observed in celomic epithelium, in testis and right and left ovaries. Moreover, VIP immunostaining was detected in the ovarian cortex in pregranulosa cells and shortly after, a gradient of VIP was seen from the cortex to the medulla. In the right ovary, the cortex is not found because it degenerates early in development. In ovarian and testicular medulla VIP was found in interstitial steroidogenic cumuli and in abundant nerve fibers. VIPergic fibers were more evident in ovaries than in testicles. In post-hatching left ovaries, VIP was found in the granulosa sheet and in some oocytes of immature follicles. In postnatal stages of males, VIP immunostaining was found in Leydig cells and spermatogonia. VIPR-I was observed in testicular and ovarian cortex and some epithelial cells during embryogenesis. In the ovary, VIPR-I was localized in the cytoplasm of granulosa cells and oocytes. In post-hatching females, up to the 7th day of incubation, VIPR-I immunostaining was detected mainly in the cortex, in oocytes and the granulosa sheet of developing follicles and in neuronal somas and nerve fibers. In embryonic stages of testis, VIPR-I was detected in Leydig cells and nerve fibers. Throughout maturation, VIPR-I was found in spermatogonia, Leydig cells and nerve fibers localized between the spermatic chords. With the findings of our work, we propose a regulatory action of VIP and VIPR-I on gonadal development. In the granulosa sheet of developing follicles it could be acting in a paracrine way, promoting oocyte maturation. We propose that VIP acts as a modulator of gonadal differentiation and steroidogenic activity. We thank to Verónica Rodríguez Mata for her technical assistance and Pablo G Hofmann Salcedo for reviewed the manuscript. Supported by DGAPA IN 207905. Supported by DGAPA IN206 007 (poster)

Selective 5-Lipoxygenase Expression in Langerhans Cells and Impaired Dendritic Cell Migration in 5-LO-Deficient Mice Reveal Leukotriene Action in Skin

5-Lipoxygenase (5-LO) catalyzes the initial steps in the formation of leukotrienes (LTs), which are implicated in immune reactions. Recently, it was shown that FITC-triggered epidermal Langerhans cell (LC) emigration to draining lymph nodes (LNs) is impaired in LTC4 export pump (multidrug resistance-associated protein 1)-deficient mice. Here, we sought genetic evidence for a role of endogenous LTs in dendritic cell function through the study of 5-LO-deficient mice. Though DC numbers in skin, spleen, and peripheral LNs were similar in both 5-LO-deficient and wild-type (WT) mice, DC homing from skin to draining LNs induced by FITC was reduced by 75% in 5-LO-deficient mice. Moreover, in WT mice, all epidermal LCs, dermal langerin+ LCs, and subsets of dermal macrophages and langerin+ LCs in T-cell areas of skin-draining LNs markedly expressed 5-LO. However, the enzyme was noticeably absent in all DC subsets of the dermis, thymus, spleen, Peyer's patches, mesenteric LNs, and mucosal surfaces of lung and intestine. As all epidermal cells other than LCs lacked 5-LO and because differentiation and activation of DCs generated from 5-LO-deficient mice in vitro were normal, these data support a selective role of endogenous LTs in DC homing following skin sensitization.

SLURP-1 and -2 in normal, immortalized and malignant oral keratinocytes

The secreted mammalian Ly-6/urokinase plasminogen activator receptor-related proteins (SLURP)-1 and -2 are produced by keratinocytes comprising the mucocutaneous epithelium. They regulate in autocrine and paracrine ways cell growth and differentiation through the nicotinic acetylcholine receptors (nAChRs) expressed on the plasma membrane. Keratinocyte nAChRs are targeted by tobacco-derived carcinogenic nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N′-nitrosonornicotine (NNN) that can induce tumorigenic transformation of Het-1A keratinocytes. In this study we asked if SLURPs could abolish tumorigenic effects of nitrosamines. Preincubation with either recombinant SLURP-1 or -2 in both cases considerably reduced the number of colonies in soft agar, and the number of tumor nodules >0.5 cm in diameter in Nu/Nu mice produced by Het-1A cells treated with nitrosamines. The levels of SLURP-1 and -2 mRNA transcripts in nitrosamine-transformed Het-1A cells as well as in the tumor cell lines SCC-25 and FaDu were significantly (p<0.05) less compared to normal gingival keratinocytes, which are probably the major source of the secreted SLURPs found in a sample of human saliva. The expression of SLURPs was decreased due to gene silencing of different nAChR α subunits with small hairpin RNA, suggesting that a positive feedback regulation is altered in malignant cells. Thus, SLURP-1 and -2 are efficient autocrine and paracrine ligands of keratinocyte nAChRs capable of preventing tobacco nitrosamine-induced malignant transformation of oral cells. These “proof-of-concept” preliminary results have salient clinical implications.

Évolution chez l'homme de l'Insulin-Like Growth Factor-I dans le liquide interstitiel musculaire suite à un exercice en résistance

Introduction The impact of resistance exercise on the growth of the vastus lateralis was assessed through interstitial microdialysis measurement of free Insulin-like Growth Factor-I at +3 h30, +4 h50 and +6 h after exercise in 4 healthy subjects. Results We observed 3 h30 after exercise an interstitial increase in 3 over 4 subjects, and thereafter a progressive decrease. In the mean time, free or total plasma concentrations did not change. Conclusion Resistance exercise seemed to induce an early increase of muscle interstitial free Insulin-like Growth Factor-I in order to initiate muscle growth by an autocrine – paracrine way.

Serotonergic neuron system in the spinal cord of the gar Lepisosteus oculatus (Lepisosteiformes, Osteichthyes) with special regard to the juxtameningeal serotonergic plexus as a paracrine site

Immunohistochemical and electron microscopic studies were carried out to elucidate the structure of the serotonergic neuron system in the spinal cord of the spotted gar, Lepisosteus oculatus, a nonteleost actinopterygian. Serotonin-immunoreactive (5HT-IR) cell bodies and fibers were widely distributed in the spinal cord, constituting an intrinsic neuron system. This system comprised three anatomical cell groups in different portions of the spinal cord, i.e., the rostromedial cell group, the paired ventrolateral cell groups, and the ventral superficial cell group. The rostromedial cell group included cerebrospinal fluid-contacting neurons with intraventricular processes. The immunostained fibers projecting from all three of these cell groups ran in various directions, mainly ventrally and ventrolaterally, and partly gave rise to a dense plexus at the ventrolateral surface of the spinal cord. Immunoelectron microscopy of the relevant portion demonstrated many varicose fibers containing 5HT-immunopositive vesicles. Conventional electron microscopy of the plexus showed that the constituent varicose fibers were unmyelinated and frequently made a direct contact with the basement membrane contiguous to the leptomeniges (meninx primitiva). There, exocytotic figures of cytoplasmic vesicles were demonstrated, suggesting that 5HT may be secreted, in a paracrine way, into the extraspinal space. This specialized area in the gar spinal cord may be referred to as the juxtameningeal serotonergic plexus.

Sinusoidal remodeling and angiogenesis: A new function for the liver-specific pericyte?

Sinusoidal remodeling and angiogenesis: A new function for the liver-specific pericyte?

Neuron-derived D-Serine Release Provides a Novel Means to Activate N-Methyl-D-aspartate Receptors

D-serine is a coagonist of N-methyl-D-aspartate (NMDA) receptors that occurs at high levels in the brain. Biosynthesis of D-serine is carried out by serine racemase, which converts L- to D-serine. D-serine has been demonstrated to occur in glial cells, leading to the proposal that astrocytes are the only source of D-serine. We now report significant amounts of serine racemase and D-serine in primary neuronal cultures and neurons in vivo. Several neuronal culture types expressed serine racemase, and D-serine synthesis was comparable with that in glial cultures. Immunohistochemical staining of brain sections with new antibodies revealed the presence of serine racemase and D-serine in neurons. Cortical neurons expressing serine racemase also expressed the NR2a subunit in situ. Neuron-derived D-serine contributes to NMDA receptor activation in cortical neuronal cultures. Degradation of endogenous D-serine by addition of the recombinant enzyme D-serine deaminase diminished NMDA-elicited excitotoxicity. Release of neuronal D-serine was mediated by ionotropic glutamate receptor agonists such as NMDA, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, and kainate. Removal of either external Ca2+ or Na+ blocked D-serine release. Release of D-serine was mostly through a cytosolic route because it was insensitive to bafilomycin A1, a potent inhibitor of vesicular neurotransmitter uptake. D-serine was also not transported into purified synaptic vesicles under conditions optimal for the uptake of known transmitters. Our results suggest that neurons are a major source of D-serine. Glutamate-induced neuronal D-serine release provides a novel mechanism for activating NMDA receptors by an autocrine or paracrine way.

Chromogranin-A expression in the bovine testis

Chromogranin-A (CgA) is the most distributed member of the granin family. Chromogranins are soluble anionic glycoproteins, found in the majority of the neuroendocrine and neural cells, co-stored with other endocrine substances (like insulin, glucagon, FSH and LH or NPY) in secretory granules. Outside the cell, it has been suggested that this peptide or one of its fragments, obtained by proteolytic cleavage, could act in an autocrine or paracrine way, regulating either the cell function or the contractibility of vascular segments. The purpose of the present study is to determine the distribution of chromogranin-A in the structures of the bovine testis. Immunohistochemical analysis was performed employing the biotin–streptavidin-peroxidase immunostaining technique in tissue specimens obtained at a local abbatoir. A CgA expression was found in the germinal epithelium at several stages of differentiation. Generally the strongest positive reaction was consistently observed in the basal compartment of the seminiferous tubules, with spermatogonia presenting a dense granular immunostaining pattern; a less intense reaction was also consistently recorded in type II spermatocytes and in round spermatids, which showed a more scattered disposition of CgA-positive granules. Clusters of Leydig cells also displayed a faint and homogeneous cytoplasmatic immunoreactivity for chromogranin-A. These results demonstrate a widely distribution of CgA-positive cells in the organism, and its presence in the testis raises the possibility of its participation in the cohort of local factors involved in the regulation of spermatogenesis.

Essential Role of Angiogenic Factors and Bone Marrow-Derived Endothelial/Hematopoietic Cells in the Growth of Solid Tumors

The current thesis examines the role of vascular endothelial growth factor receptor-1 (VEGFR-1) autocrine and paracrine loops in different subsets of cells, namely, breast tumor, endothelial and bone marrow (BM)-derived cells, and their role on tumor growth regulation. These studies highlight the functional relevance of the biological pathways mediated by this receptor and the importance of therapies against VEGFRs as antitumor and antiangiogenesis targets. These findings open new perspectives for the study and development of novel and more effective strategies to fight tumor growth and invasion, and lay the foundation for further development of clinical trials. It is well established that angiogenesis is essential for tumor development. Although the process is controlled by several molecules, the vascular endothelial growth factor (VEGF), by signaling through its receptors, VEGFR-1 and VEGFR-2, seems to be the main mediator. Whereas VEGFR-2 biological functions on tumor endothelium are clearly defined, VEGFR-1 roles are mostly unknown. Moreover, besides being initially described as endothelial specific, VEGFR-1 has also been reported in other types of cells of nonendothelial origin, such as subsets of breast tumor cells and hematopoietic stem and progenitor cells. These observations led us to hypothesize the following: (1) If breast tumor cells express VEGFR-1 and produce VEGF, VEGF/VEGFR-1 autocrine loops could occur and promote breast tumor growth in an endothelial cell-independent fashion. (2) Tumorproduced VEGF could also activate, in a paracrine fashion, VEGFR-1 on tumorassociated endothelium promoting angiogenesis, a feature still unclarified. (3) Tumor cells, by releasing VEGF, may be able to induce in a paracrine way the mobilization of populations of VEGFR-1, expressing BM-derived hematopoietic cells that, in addition to VEGFR-2+ endothelial progenitor cells (EPCs) recruitment to tumor sites, might influence malignant cell growth and neovascularization. The in vitro and in vivo functional expression of VEGFR-1 in human breast cancer cells, and the relevance of the specific impairment of this receptor as a therapeutic target, using monoclonal antibodies (mAbs) as strategy, was first evaluated. VEGFR-1 was functionally expressed on subsets of primary human invasive ductal breast carcinoma tissues and breast cancer cell lines, directly promoting their in vitro proliferation by the

Differential expression of melanoma-associated growth factors in keratinocytes and fibroblasts by ultraviolet A and ultraviolet B radiation

Besides the direct DNA-damaging effects of ultraviolet (UV) radiation on cells, indirect effects on the microenvironment of the skin may facilitate melanoma development. A stimulation of growth factor production by cells in the immediate environment of melanocytes may lead to a paracrine activation and proliferation of melanocytes that in turn become more susceptible to transformation. We investigated whether the expression of growth factors for melanocytes can be modulated in keratinocytes and fibroblasts by UVA or UVB. After irradiation with different doses of UVA or UVB, protein expression of basic fibroblast growth factor (bFGF), endothelin (ET)-1, transforming growth factor (TGF)-beta1, platelet-derived growth factor (PDGF)-AA, stem cell factor (SCF) and hepatocyte growth factor (HGF) was analysed by quantitative enzyme-linked immunosorbent assay. The mRNA expression of bFGF and ET-1 was analysed by quantitative real-time reverse transcriptase-polymerase chain reaction. In keratinocytes, UVB and UVA increased bFGF protein levels up to 2.6-fold. This increase was paralleled by elevated mRNA levels. UVB also induced ET-1 protein up to 1.8-fold, while UVA led to an 80% decrease. Secreted TGF-beta1 and PDGF-AA were downregulated by UVA by less than 50%, while there was no significant alteration by UVB. Secreted SCF was not changed significantly by UVA or UVB. In fibroblasts, bFGF protein levels were increased 11-64-fold by UVA and 34-61-fold by UVB. This was paralleled by elevated mRNA levels for bFGF up to 2.7-fold. HGF protein was stimulated by UVA up to 2.8-fold and by UVB up to 6.7-fold, while TGF-beta1 protein was increased up to 2.7-fold by UVB and 1.7-fold by UVA. UVA and UVB can stimulate and inhibit the production of growth factors for melanocytes in keratinocytes and fibroblasts dependent on the cell type and wavelength. We show for the first time that UVA and UVB can activate bFGF, HGF and TGF-beta1 in fibroblasts, while bFGF was the most inducible factor both in fibroblasts and in keratinocytes. The induction of bFGF and HGF in fibroblasts by UVA suggests that stroma cells in the dermis may be involved in the UV activation of melanocytes via paracrine ways and thus promote melanoma development.

Síndromes hormonales paraneoplásicos

We can define paraneoplastic syndromes as a combination of effects occurring far from the original location of the tumour and independently from the local repercussion of its metastases. Paraneoplastic hormonal syndromes depend on the secretion of hormonal peptides or their precursors, cytokines and, more rarely, thyroidal hormones and Vitamin D, which act in an endocrine, paracrine or autocrine way. Sometimes, paraneoplastic syndromes can be more serious than the consequences of the primary tumour itself and can precede, develop in parallel, or follow the manifestations of this tumour. It is important to recognise a paraneoplastic hormonal syndrome for several reasons, amongst which we would draw attention to three: 1) It can lead to the diagnosis of a previously undetected, underlying malign or benign neoplasia; 2) It can dominate the clinical picture and thus lead to errors with respect to the origin and type of primary tumour; and 3) It can follow the clinical course of the underlying tumour and thus be useful for monitoring its evolution. The molecular mechanisms responsible for the development of these syndromes are not well-known, but it is believed that they might be inherent to the mutations responsible for the primary tumour or depend on epigenetic factors such as methylation. In this review, we consider the following paraneoplastic hormonal syndromes: malign hypercalcaemia, hyponatraemia (inappropiate secretion of the antidiuretic hormone), ectopic Cushing's syndrome, ectopic acromegaly, hypoglycaemia due to tumours different from those of the islet cells and paraneoplastic gynaecomastia; we make a brief final reference to other hormones (calcitonin, somatostatin, and VIP).

Effect of testosterone on insulin-like growth factor-I, androgen receptor, and myostatin gene expression in splenius and semitendinosus muscles in sheep

Testosterone is known to act differentially on skeletal muscle from different regions. Two genes likely to mediate the testosterone effect are IGF-I, an important growth regulator acting in an autocrine and paracrine way, and androgen receptor (AR), as receptor density could account for differential muscle growth. Another muscle-specific gene that may play a role in differential muscle growth is myostatin, a member of the transforming growth factor-beta superfamily, shown to be a negative regulator of skeletal muscle mass. The objective of this study was to quantify and compare the expression of these three genes in two different skeletal muscles in sheep. East Friesian x Dorset-sired ram lambs from Dorset ewes were used in a 2 x 4 factorial experiment. Eighteen sets of twins were assigned to four age groups corresponding to 77, 105, 133, and 161 d of age, and one individual from each set was castrated at birth. Total RNA was extracted from samples of splenius (SP) and semitendinosus muscles collected at the time of slaughter. Insulin-like growth factor-I mRNA was measured using competitive reverse-transcription PCR. Androgen receptor and myostatin mRNA were measured by ribonuclease protection assay with standard curves. Weight of SP was greater than semitendinosus in rams compared with wethers at 105, 133, and 161 d (P = 0.05, P = 0.04, and P = 0.02, respectively). The difference in IGF-I mRNA levels between the two muscles was greater in rams than in wethers at 133 (P = 0.001) and 161 d (P = 0.014), and the difference in AR mRNA levels was greater in rams than in wethers at 105, 133, and 161 d (P = 0.002, P < 0.001, and P < 0.001, respectively), with greater abundance in the SP. No difference was found in myostatin mRNA level between the two muscles in rams and wethers at any age. These results suggest that locally produced IGF-I and the regulation of AR expression are important for sexually dimorphic muscle growth patterns.

Angiostatin inhibits pancreatic cancer cell proliferation and growth in nude mice

To observe the biologic behavior of pancreatic cancer cells in vitro and in vivo, and to explore the potential value of angiostatin gene therapy for pancreatic cancer. The recombinant vector pcDNA3.1(+)-angiostatin was transfected into human pancreatic cancer cells PC-3 with Lipofectamine 2000, and paralleled with the vector and mock control. Angiostatin transcription and protein expression were determined by immunofluorescence and Western blot. The stable cell line was selected by G418. The supernatant was collected to treat endothelial cells. Cell proliferation and growth in vitro were observed under microscope. Cell growth curves were plotted. The troms-fected or untroms-fected cells overexpressing angiostatin vector were implanted subcutaneously into nude mice. The size of tumors was measured, and microvessel density count (MVD) in tumor tissues was assessed by immunohistochemistry with primary anti-CD34 antibody. After transfected into PC-3 with Lipofectamine 2000 and selected by G418, macroscopic resistant cell clones were formed in the experimental group transfected with pcDNA 3.1(+)-angiostatin and vector control. But untreated cells died in the mock control. Angiostatin protein expression was detected in the experimental group by immunofluorescence and Western-blot. Cell proliferation and growth in vitro in the three groups were observed respectively under microscope. After treatment with supernatant, significant differences were observed in endothelial cell (ECV-304) growth in vitro. The cell proliferation and growth were inhibited. In nude mice model, markedly inhibited tumorigenesis and slowed tumor expansion were observed in the experimental group as compared to controls, which was parallel to the decreased microvessel density in and around tumor tissue. Angiostatin does not directly inhibit human pancreatic cancer cell proliferation and growth in vitro, but it inhibits endothelial cell growth in vitro. It exerts the anti-tumor functions through antiangiogenesis in a paracrine way in vivo.

CD4+ CCR5+ and CD4+ CCR3+ lymphocyte subset and monocyte apoptosis in patients with acute visceral leishmaniasis.

The potential involvement of apoptosis in the pathogenesis of visceral leishmaniasis (VL) was examined by studying spontaneous and Leishmania antigen (LAg)-induced apoptosis using cryopreserved peripheral blood mononuclear cells (PBMC) of Sicilian patients with VL. Results indicate that monocytes and T lymphocytes from acute VL patients show a significantly higher level of apoptosis compared with that observed in healed subjects. The percentage of apoptotic cells was higher in monocytes than in T lymphocytes. T cells involved in programmed cell death (PCD) were mainly of the CD4(+) phenotype. In particular, the T helper 1-type (Th1) subset, as evaluated by chemokine receptor-5 (CCR5) expression, is involved in this process. Cell death in Th1-type uses a CD95-mediated mechanism. Furthermore, Th1-type CCR5(+) cells are prone to cell suicide in an autocrine or paracrine way, as attested by enhanced expression of CD95L in acute VL patients. The reduction in Th1-type cells by apoptosis was confirmed by the decrease in interferon-gamma secretion. In conclusion, apoptosis of monocytes, CD4(+) and CD4(+) CCR5(+) T cells could be involved in the failure of cell mediated immunity that is responsible for severe immune-depression in VL.

Lowered tumor necrosis factor receptors, but not increased insulin sensitivity, with infliximab.

To verify whether Infliximab could modify insulin sensitivity and TNF-alpha and GLUT4 mRNA expression in muscle and adipose tissue of morbidly obese subjects. Soluble TNF receptors I and II (TNFR-I and TNFR-II) were also assayed. Six obese subjects were investigated before and 2 weeks after a single intravenous administration of 5 mg/kg Infliximab; insulin sensitivity was evaluated by euglycemic hyperinsulinemic clamp, and TNF-alpha and GLUT4 mRNA expression were assessed by reverse-transcriptase polymerase chain reaction on muscle and adipose tissue. TNF-alpha, TNFR-I, and TNFR-II were determined using the ELISA technique. Infliximab infusion did not affect fasting plasma insulin or fasting plasma glucose levels; whole body glucose uptake did not change significantly. TNF-alpha and GLUT4 mRNA did not show any significant change in muscle or adipose tissue. Serum TNF-alpha was undetectable before and after treatment, whereas TNFR-I and TNFR-II concentrations significantly decreased (p < 0.01). An explanation for the absence of effect of Infliximab on insulin resistance in morbidly obese subjects may be the paracrine way of action of this cytokine. Because Infliximab is predominantly distributed within the vascular compartment, its effectiveness in penetrating muscle and adipose tissue is potentially low. The significant decrease of TNFR-I and TNFR-II might be ascribed to a targeted effect of Infliximab on the immune system.

Regulation of 25-hydroxyvitamin D3-1α-hydroxylase and production of 1α,25-dihydroxyvitamin D3 by human dendritic cells

Abstract25-Hydroxyvitamin D3-1α-hydroxylase (25(OH)D3-1α-hydroxylase), the key enzyme of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) production, is expressed in monocyte-derived macrophages (MACs). Here we show for the first time constitutive expression of 25(OH)D3-1α-hydroxylase in monocyte-derived dendritic cells (DCs), which was increased after stimulation with lipopolysaccharide (LPS). Accordingly, DCs showed low constitutive production of 1,25(OH)2D3, but activation by LPS increased 1,25(OH)2D3 synthesis. In addition, 25(OH)D3-1α-hydroxylase expression was found in blood DCs but not in CD34+-derived DCs. Next we analyzed the functional consequences of these results. Addition of 1,25(OH)2D3 at concentrations comparable with those produced by DCs inhibited the allostimulatory potential of DCs during the early phase of DC differentiation. However, terminal differentiation decreased the responsiveness of DCs to 1,25(OH)2D3. In conclusion, DCs are able to produce 1,25(OH)2D3 especially following stimulation with LPS. Terminal maturation renders DCs unresponsive to the effects of 1,25(OH)2D3, but those cells are able to suppress the differentiation of their own precursor cells in a paracrine way through the production of 1,25(OH)2D3.

Corticotropin-releasing hormone (CRH)-induced thyrotropin release is directly mediated through CRH receptor type 2 on thyrotropes.

CRH is known as the main stimulator of ACTH release. In representatives of all nonmammalian vertebrates, CRH has also been shown to induce TSH secretion, acting directly at the level of the pituitary. We have investigated which cell types and receptors are involved in CRH-induced TSH release in the chicken (Gallus gallus). Because a lack of CRH type 1 receptors (CRH-R1) on the chicken thyrotropes has been previously reported, two hypotheses were tested using in situ hybridization and perifusion studies: 1) TSH secretion might be induced in a paracrine way involving melanocortins from the corticotropes; and 2) thyrotropes might express another type of CRH-R. For the latter, we have cloned a partial cDNA encoding the chicken CRH-R2. Neither alpha-melanotropin (alpha-MSH) nor its powerful analog Nle4,d-Phe7-MSH could mimic the in vitro TSH-releasing effect of ovine CRH. The nonselective melanocortin receptor blocker SHU91199 did not influence CRH- or TRH-induced TSH secretion. On the other hand, we have found that thyrotropes express CRH-R2 mRNA. The involvement of this CRH receptor in the response of thyrotropes to CRH was further confirmed by the fact that TSH release was stimulated by human urocortin III, a CRH-R2-specific agonist, whereas the TSH response to CRH was completely blocked by the CRH-R blocker astressin and the CRH-R2-specific antagonist antisauvagine-30. We conclude that CRH-induced TSH secretion is mediated by CRH-R2 expressed on thyrotropes.