Release of tumour necrosis factor alpha (TNFalpha) by TNFalpha cleaving enzyme (TACE) in response to septic stimuli in vitro

Background. Tumour necrosis factor     

Chondrocyte-specific knockout of the G protein G(s)alpha leads to epiphyseal and growth plate abnormalities and ectopic chondrocyte formation.

G(s)alpha is a ubiquitously expressed G protein alpha-subunit that couples receptors to adenylyl cyclase. Mice with chondrocyte-specific ablation of the G(s)alpha gene had severe epiphyseal and growth plate abnormalities and ectopic cartilage formation within the metaphyseal region of the tibia. These results show that G(s)alpha negatively regulates chondrocyte differentiation and is the critical signaling mediator of the PTH/PTH-rP receptor in growth plate chondrocytes. INTRODUCTION: G(s)alpha is a ubiquitously expressed G protein alpha-subunit that mediates signaling through G protein-coupled receptors to activate the cAMP/protein kinase A signaling pathway. Although studies suggest an important role for G(s)alpha in regulating growth plate development, direct in vivo results examining this role are lacking. MATERIALS AND METHODS: The G(s)alpha gene was ablated in murine cartilage by mating mice with loxP sites surrounding the G(s)alpha promoter and first exon with collagen 2a1 promoter-Cre recombinase transgenic mice. Skeletal tissues were studied by gross and microscopic pathology, and gene expression was determined by in situ hybridization. RESULTS AND CONCLUSIONS: Mice with complete chondrocyte-specific G(s)alpha deficiency (homozygotes) died within minutes after birth and had severe epiphyseal and growth plate defects with shortening of the proliferative zone and accelerated hypertrophic differentiation of growth plate chondrocytes, a phenotype similar to that of PTH/PTH-related peptide (PTHrP) receptor knockout mice. Indian hedgehog and PTH/PTHrP receptor expression in prehypertrophic chondrocytes was unaffected in mutant mice. PTHrP expression in periarticular cartilage was increased in the mutant mice, probably because of the closer proximity of Ihh-secreting chondrocytes to the periarticular zone. In addition, these mice developed ectopic cartilage at the anterior side of the metaphyseal region in the tibia. Mice with partial G(s)alpha deficiency (heterozygotes) exhibited no phenotype. These results show that G(s)alpha negatively regulates chondrocyte differentiation and is the critical signaling mediator of the PTH/PTHrP receptor in epiphyseal and growth plate chondrocytes.     

Impairment of insulin signaling in human skeletal muscle cells by co-culture with human adipocytes

Adipocyte factors play a major role in the induction of insulin resistance in skeletal muscle. To analyze this cross-talk, we established a system of co-culture of human fat and skeletal muscle cells. Cells of three muscle donors were kept in co-culture with cells of various fat cell donors, and insulin signaling was subsequently analyzed in myocytes. Insulin-induced tyrosine phosphorylation of insulin receptor substrate (IRS)-1 was completely blocked, with unaltered expression of IRS-1. Troglitazone increased insulin action on IRS-1 phosphorylation, in both the absence and presence of co-culture. Insulin-regulated activation of Akt kinase in the myocytes was significantly reduced after co-culture, with troglitazone restoring insulin action. Addition of tumor necrosis factor (TNF)-alpha (2.5 nmol/l) to myocytes for 48 h reduced IRS-1 expression and inhibited IRS-1 and Akt phosphorylation comparable to the effect of co-culture. Lower doses of TNF-alpha were ineffective. After co-culture, TNF-alpha in the culture medium was below the detection limit of 0.3 pmol/l. A very low level of resistin was detected in the supernatant of myocytes, but not of adipocytes. In conclusion, the release of fat cell factors induces insulin resistance in human skeletal muscle cells; however, TNF-alpha and resistin appear not to be involved in this process.     

Poly(ADP-ribose) polymerase expression in kidney transplantation: from alfa (alpha) to Omega (Omega)

INTRODUCTION: Overactivation of the enzyme poly(ADP-ribose) polymerase (PARP-1) can be induced by ischemia-reperfusion and involved in the renal injury subsequent to kidney transplant. The poly(ADP-ribosy)lation mechanism alters free radical-induced DNA damage, which is repair by PARP-1 polymer. However, PARP-1 overexpression induces cellular necrosis. Our aim was to study the immunohistochemical PARP-1 expression in kidney transplant biopsies associated with various events. MATERIALS AND METHODS: We studied the nuclear expression of PARP-1 in kidney tubule cells by immunohistochemistry using the monoclonal antibody PAR01 in donor biopsies without acute tubular necrosis (ATN) (n = 60; controls), allografts that suffer ATN (n = 90) or an episode of acute humoral rejection (n = 12) or acute tubulointerstitial rejection (n = 25), or chronic allograft nephropathy (n = 25). Furthermore, we also studied protocol biopsies with subclinical rejection (n = 60). Renal lesions in transplant biopsies were graded blindly using 1997 Banff criteria without any clinical information. RESULTS: Biopsies without morphological features of ATN, namely acute tubulointerstitial rejection, borderline or subclinical rejection, showed lesser PARP-1 expression compared with biopsies with ATN or with ischemic mechanism of acute humoral rejection or chronic allograft nephropathys. We observed an inverse relation between PARP-1 expression and renal function (P < .001). Overall, renal biopsies showing ATN revealed greater expression of PARP-1 (r = 0.785, Pearson test). A significant relationship with PARP-1 expression was demonstrated with renal function (effective diuresis, serum creatinine levels) and pretransplant cold ischemia time (P < .001). CONCLUSION: Kidney transplant events including ischemia were associated with the highest PARP-1 expression and worse allograft renal function.     

Alpha(v)beta3 and alpha(v)beta5 integrin expression in meningiomas

OBJECTIVE: Integrins are emerging as alternative receptors capable of mediating several biological functions, such as cell-matrix and cell-cell adhesion, cell migration, signal transduction, and angiogenesis. Two alpha(v) integrins, i.e., alpha(v)beta3 and alpha(v)beta5, play critical roles in mediating these activities, particularly in tumors. No data are available on the expression of these integrins in meningiomas. METHODS: Using Western blot and immunohistochemical analyses with LM609 and PG32, two monoclonal antibodies capable of recognizing the functional integrin heterodimer, we evaluated the expression of alpha(v)beta3 and alpha(v)beta5 integrins in a series of 34 meningiomas of different histological subtypes and grades. We studied their expression in tumor cells and vasculature, as well as the expression of their related angiogenic factors (fibroblast growth factor 2 and vascular endothelial growth factor) and the alpha(v)beta3 ligand vitronectin. RESULTS: Alpha(v)beta3 and alpha(v)beta5 integrins were expressed by neoplastic vasculature and cells. Alpha(v)beta3 and alpha(v)beta5 expression was associated and correlated with that of their respective growth factors (fibroblast growth factor 2 and vascular endothelial growth factor) and microvessel counts and densities. Alpha(v)beta3 was more strongly expressed than alpha(v)beta5 in two cases of histologically benign meningiomas with aggressive clinical behavior. Alpha(v)beta3 expression was associated with that of its related ligand vitronectin and was also evident in small vessels of brain tissue closely surrounding meningiomas. CONCLUSION: Our data demonstrate the expression of alpha(v)beta3 and alpha(v)beta5 integrins in meningioma cells and vasculature. Our findings suggest a role for both of these integrins, and particularly alpha(v)beta3, in meningioma angiogenesis.     

Reliability of continuous cardiac output determination by pulse-contour analysis in porcine septic shock

BACKGROUND: Pulse-contour analysis represents a technique for cardiac output (CO)-measurement and allows continuously monitoring trends in CO. We evaluated reliability of pulse-contour CO (COpc) in septic shock. METHODS: Seventeen anaesthetized and mechanically ventilated pigs were investigated. After baseline measurements, 14 animals received 0.75 g/kg body weight faeces into the abdominal cavity to induce sepsis and were observed over 9 h, three animals served as controls. A central venous catheter was inserted into the jugular vein and an arterial catheter for thermodilution was inserted into the femoral artery. Two bedside computers were used for COpc. After induction of sepsis, COpc-computer No. 1 (COpcCAL) was recalibrated hourly. No further calibrations were performed in computer No. 2 (COpcNoCAL). We directly compared COpcCAL hourly before recalibration with COpcNoCAL. One hundred and seventy parallel triplicate determinations of CO were analysed using the method of Bland-Altman. RESULTS: Three hours after sepsis induction, correlation between recalibrated and non-recalibrated CO was r = 0.74, P < 0.01, at 5 h r = 0.59, P < 0.05 and 9 h r = 0.02, NS. Three hours after sepsis induction, bias +/- SD (limits of agreement) between both groups was 1.6 +/- 15.5 (-29.4-32.6) ml/kg/min, at 5 h -15.0 +/- 24.3 (-63.6-33.7) ml/kg/min and at 9 h -87.0 +/- 90.8 (-268.5-94.6) ml/kg/min. CONCLUSION: Continuous CO determination using pulse-contour analysis is a reliable method of assessing CO up to 5 h without recalibration in porcine septic shock. Thus, COpc may be a useful tool for assessment of unpredictable haemodynamic changes in sepsis.     

Modulation by interferon alpha of the decreased natural killer activity in patients with glioblastoma

Summary It is known that natural killer (NK) cells are involved in immunosurveillance against tumours. This study examines the NK activity of mononuclear cells (MNC) from the peripheral blood of patients with gliobalstoma. The cytotoxic inducer effect of interferonalpha (IFN-) upon these MNC has also been studied. A marked decrease in NK activity mediated by MNC from these patients was found. This functional defected in MNC is not due to a decrease in phenotypically defined NK cells. After long-term (5-day) incubation with IFN-, MNC from 5 out of 14 patients showed strong lytic activity against NK-sensitive target cells. In this system, IFN- failed to induce cytotoxic activity against NK-resistant target cells in MNC from all the patients studied. Thisin vitro induction of cytotoxic activity in MNC from some patients with glioblastoma by IFN- suggests a potential immunotherapeutic use of the lymphokine in these subjects.     

Alpha(v)beta3 and alpha(v)beta5 integrin expression in glioma periphery

OBJECTIVE: This study analyzed the expression of integrins alpha(v)beta3 and alpha(v)beta5 in glioma tissue and focused on the periphery of high-grade gliomas. METHODS: The analysis was performed with Western blot, immunohistochemistry, and immunofluorescence, by use of two monoclonal antibodies able to recognize the functional integrin heterodimer. The expression of integrin-related ligands and growth factors also was studied. Sections from the tumor periphery were classified as either tumor periphery (light tumor infiltrate or scant visible cells) or peritumor (heavy tumor infiltration). RESULTS: Our data on glioma tissues demonstrated that both integrins were expressed in glioma cells and vasculature and their expression correlated with the histological grade. Alpha(v)beta3 expression was prominent in astrocytic tumors. Both integrins were markers of tumor vasculature, particularly of endothelial proliferation. A high-grade glioma periphery demonstrated a prominent expression of integrin alpha(v)beta3. Cells demonstrating alpha(v)beta3 positivity were identified as tumor astrocytes and endothelial cells by double imaging. The same cells were surrounded by some alpha(v)beta3 ligands and co-localized fibroblast growth factor 2. Matrix metalloproteinase 2 also was found to be co-localized with alpha(v)beta3 in the same cells. Alpha(v)beta3 expression was more relevant in tumor astrocytes. Alpha(v)beta3 integrin and vascular endothelial growth factor expression increased from the periphery to the tumor center. CONCLUSION: Our data support the role of integrins alpha(v)beta3 and alpha(v)beta5 in glioma-associated angiogenesis. In addition, they suggest a role for integrin alpha(v)beta3 in neoangiogenesis and cell migration in high-grade glioma periphery.     

Effect of CO(2) pneumoperitoneum on early cellular markers of retinal ischemia in rabbits with alpha-chymotrypsin-induced glaucoma

BACKGROUND: Increased intraperitoneal pressure in the head-down position is associated with a significant increase in intraocular pressure (IOP) in rabbits with alpha-chymotrypsin-induced glaucoma. Also, the retinal cells are weakened by the induction of increased IOP, and/or glaucoma, even when IOP is controlled by adequate therapy; therefore, these cells need to be protected from any additional aggression. Actin and vimentin are proteins of the retinal cell cytoskeleton that react readily in response to retinal injuries, including ischemia and glaucoma. Early changes in these cytoskeleton proteins determine the morphological changes observed after retinal damage. Therefore, we set out to investigate intracytoplasmic changes in vimentin and actin after a 4-h CO(2) pneumoperitoneum in the head-down position in rabbits with alpha-chymotrypsin-induced glaucoma. METHODS: Twenty-one rabbits with alpha-chymotrypsin-induced glaucoma in one eye received general anesthesia for 4 h in the head-down position and were randomly allocated to have (a) no pneumoperitoneum, (b) a 10 mmHg CO(2) pneumoperitoneum, or (c) a 20 mmHg CO(2) pneumoperitoneum. At the end of the trial, both the right glaucomatous and the left control eyes were enucleated and investigated immunocytochemically for alterations in vimentin and actin, and morphologically for retinal layer disorganization. RESULTS: Except for the preexisting morphological changes induced by glaucoma, both the control and the glaucomatous eyes in all rabbits appeared normal in terms of retinal layer organization and the distribution of intracellular vimentin and actin whatever the intraperitoneal pressure level applied. CONCLUSION: In rabbits with alpha-chymotrypsin-induced glaucoma, a 4-h CO(2) pneumoperitoneum of 相似文献   

Changes in the expression of cellular alpha and beta tubulins in patients with sporadic type colorectal cancer

The aim of this preliminar report is to evaluate alfa and beta tubulins, components of cellular microtubules, alterated expression in sporadic colorectal cancer patients. The Authors considered 16 patients who underwent surgery for sporadic colorectal carcinoma with radical intent. Alfa and beta tubulins were evaluated in tumoral mucosa by immunohistochemistry. In 56.2% of the examined patients a low expression of alfa and beta tubulins was showed while the alteration of alfa tubulin was showed in 81.2% of the patients. This finding supports the hypothesis of Porter that alterations in microtubule structure might be part of the cellular response to DNA damage.     

Volatile anesthetics mimic cardiac preconditioning by priming the activation of mitochondrial K(ATP) channels via multiple signaling pathways

BACKGROUND: Volatile anesthetics induce pharmacological preconditioning in cardiac tissue. The purpose of this study was to test whether volatile anesthetics mediate this effect by activation of the mitochondrial adenosine triphosphate-sensitive potassium (mitoK(ATP)) or sarcolemmal K(ATP) (sarcK(ATP)) channel in rat ventricular myocytes and to evaluate the signaling pathways involved. METHODS: A cellular model of ischemia with subsequent hypoosmolar trypan blue staining served to determine the effects of 5-hydroxydecanoate, a selective mitoK(ATP) channel blocker, HMR-1098, a selective sarcK(ATP) channel blocker, diazoxide, a preconditioning mimicking agent, and various modulators of putative signaling pathways on cardioprotection elicited by sevoflurane and isoflurane. Microscopy was used to visualize and measure autofluorescence of flavoproteins, a direct index of mitoK(ATP) channel activity. RESULTS: Volatile anesthetics significantly enhanced diazoxide-mediated activation of mitoK(ATP) channels as assessed by autofluorescence of myocytes. Conversely, volatile anesthetics alone did not alter mitoK(ATP) channel activity, implying a priming effect of volatile anesthetics on mitoK(ATP) channels. Administration of the protein kinase C inhibitor chelerythrine completely blocked this effect. Also, pretreatment with volatile anesthetics potentiated diazoxide-mediated protection against ischemia, as indicated by a reduction in trypan blue-positive myocytes. Importantly, cardioprotection afforded by volatile anesthetics was unaffected by the sarcK(ATP) channel blocker HMR-1098 but sensitive to modulations of nitric oxide and adenosine-G(i) signaling pathways. CONCLUSIONS: Using autofluorescence in live cell imaging microscopy and a simulated model of ischemia, the authors present evidence that volatile anesthetics mediate their protection in cardiomyocytes by selectively priming mitoK(ATP) channels through multiple triggering protein kinase C-coupled signaling pathways. These observations provide important new insight into the mechanisms of anesthetic-induced preconditioning.     

Differential galactose α(1,3) galactose expression by porcine cardiac vascular endothelium

Galactose α(1,3) galactose (Gal) is the terminal carbohydrate moiety recognized by xenoreactive natural antibodies during hyperacute rejection (HAR). Binding of these antibodies in HAR triggers rapid microvascular thrombosis. We examined the distribution of Gal on the endothelium of porcine hearts before and after heterotopic xenotransplantation into baboons. We found that Gal is strongly expressed on the endothelium of porcine capillaries with less expression on the endothelium of larger vessels. The distribution of Gal staining remains unchanged after xenotransplantation and correlates with the intensity of IgM and membrane attack complex (MAC) deposition. Thus, the Gal epitope is differentially expressed in the pig vasculature, which affects the pattern of xenoreactive antibody and MAC deposition and directs the distribution of vascular thrombosis.     

Transforming growth factor alpha (TGF-alpha) expression in dysplastic oral leukoplakia: modulation by 13-cis retinoic acid.

BACKGROUND: Surrogate endpoint biomarkers (SEBs) are detectable molecular, cellular, and tissue changes that take place during tumorigenesis and can be modulated by a chemoprevention agent. METHOD: To identify candidate SEBs for invasive squamous cell carcinoma of the upper aerodigestive tract (SCC), we have studied the expression of transforming growth factor-alpha (TGF-alpha) and epidermal growth factor receptor (EGFr) in sequential biopsy specimens of dysplastic oral leukoplakia and adjacent normal-appearing mucosa. Biopsies were taken from patients before, during, and after treatment with 13-cis retinoic acid, a vitamin A derivative. Immunohistochemistry was performed using the Biogenex Super Sensitive Biotin-Streptavidin horseradish peroxidase detection system. RESULTS: The pretreatment expression of TGF-alpha and EGFr in dysplastic oral leukoplakia was increased when compared with their expression in adjacent normal-appearing mucosa (p = 0.001 and p = 0.01, respectively). Eleven of 14 patients enrolled in the study (78.6%) completed 3 months of treatment with 13-cis retinoic acid (1. 0 mg/kg/day). TGF-alpha expression in dysplastic oral leukoplakia, but not in adjacent normal-appearing mucosa, decreased during treatment (p < 0.05). CONCLUSIONS: TGF-alpha is a candidate SEB for future SCC chemoprevention trials.     

Ontogeny and cellular origin of a rat seminiferous tubule factor(s) that inhibits LH-dependent testosterone production by interstitial cells in vitro

Previous studies have shown the presence of a peptide in spent media from incubated seminiferous tubules (SMST), which inhibits LH stimulation of testosterone production by rat Leydig cells in vitro. The present study has investigated whether the secretion of this inhibitor changes during development in the rat. Seminiferous tubules obtained from rats aged 10, 20, 25, 30, 35, 40, 42, 50 or 60 days were incubated at 32 degrees C for 24 h. Spent media from these incubations were then added to interstitial cells isolated from the testes of rats aged 60 days. Spent media from rats aged 10-30 days had no effect on basal or oLH-stimulated testosterone production by interstitial cells during 3-h incubation. Significant inhibition of LH-stimulated testosterone production was, however, observed with SMST from rats aged 35-60 days. Spent media prepared using tubules from normal, prenatally irradiated (Sertoli cell-enriched) or seminiferous tubules, depleted of peritubular cells, had no effect on basal, but inhibited LH-stimulated, testosterone production. Spent media from peritubular cell cultures had no effect on basal or LH-stimulated testosterone production by interstitial cells. The inhibitory effect of SMST was also dependent on the age of the rats providing the target cells. Interstitial cells from rats aged 10, 20, 50 or 60 days were responsive to the inhibitor while cells from rats aged 30 and 40 days were not. The results of the present study demonstrate that the seminiferous tubule factor(s), which inhibits LH action on interstitial cells, is first secreted at 35 days, a time when the most mature germ cells present are in the early maturation phase. Moreover, interstitial cells are responsive to this factor in both immature (10-20 day-old) and mature (50-60 day-old) rats, but not at ages in between these times. It is suggested that the adult Sertoli cell is the major source of the interstitial cell inhibitor.