Hypoxia induced apoptosis of rat gastric mucosal cells by activating autophagy through HIF-1α/TERT/mTORC1 pathway

The pathogenesis of high altitude-related gastric mucosal injury remains poorly understood, this study aimed to investigate the role of autophagy in hypoxia-induced apoptosis of rat gastric mucosal cells. Rats were randomized into four groups which were maintained at an altitude of 400 m (P) or received no treatment (H), autophagy inducer rapamycin (H+AI) or autophagy inhibitor 3-MA (H+AB) at an altitude of 4,300 m for 1, 7, 14 and 21 days, respectively, and the morphology, ultrastructure, autophagy, and apoptosis of gastric mucosal tissues were examined. Gastric mucosal epithelial cells CC-R039 were cultured under conditions of normoxia, 2% O2 (hypoxia), or 2% O2+anti-mTORC1 for 0, 24, 48, and 72 h, respectively, and the autophagy and apoptosis were analyzed. CC-R039 cells were transfected with siHIF-1α, siTERT, or siRNA and the autophagy was examined. The results showed that the exposure to hypoxia increased the autophagy and apoptosis of gastric mucosal cells in rats, and apoptosis was aggravated by rapamycin treatment but alleviated by 3-MA treatment. Increased duration of hypoxia from 0 to 72 h could increase the autophagy and apoptosis but decrease the proliferation of gastric mucosal cells. Inhibition of mTORC1 with rapamycin led to further increase in apoptosis and even substantial cell death, and inhibition of HIF- 1α and TERT increased mTORC1 expression and reduced autophagy. Moreover, the inhibition of HIF-1α reduced TERT expression. In conclusion, hypoxia could induce apoptosis of rat gastric mucosal cells by activating autophagy through HIF-1α/TERT/mTORC1 pathway     

Regulation of gene expression for neurotransmitters during adaptation to hypoxia in oxygen-sensitive neuroendocrine cells

Reduced oxygen tension (hypoxia) in the environment stimulates oxygen-sensitive cells in the carotid body (CB). Upon exposure to hypoxia, the CB immediately triggers a reflexive physiological response, thereby increasing respiration. Adaptation to hypoxia involves changes in the expression of various CB genes, whose products are involved in the transduction and modulation of the hypoxic signal to the central nervous system (CNS). Genes encoding neurotransmitter-synthesizing enzymes and receptors are particularly important in this regard. The cellular response to hypoxia correlates closely with the release and biosynthesis of catecholamines. The gene expression of tyrosine hydroxylase (TH), the rate-limiting enzyme for catecholamine biosynthesis, is regulated by hypoxia in the CB and in the oxygen-sensitive cultured PC12 cell line. Recently, genomic microarray studies have identified additional genes regulated by hypoxia. Patterns of gene expression vary, depending on the type of applied hypoxia, e.g., intermittent vs. chronic. Construction of a hypoxia-regulated, CB-specific, subtractive cDNA library will enable us to further characterize regulation of gene expression in the CB.     

Nitric oxide averts hypoxia-induced damage during reoxygenation in rat heart

Nitric oxide (NO), synthesized by the hemoproteins NO synthases (NOS), is known to play important roles in physiological and pathological conditions in the heart, including hypoxia/reoxygenation (H/R). This work investigates the role that endogenous NO plays in the cardiac H/R-induced injury. A follow-up study was conducted in Wistar rats subjected to 30 min of hypoxia, with or without prior treatment using the nonselective NOS inhibitor L-NAME (1.5 mM). The rats were studied at 0 h, 12 h, and 5 days of reoxygenation, analysing parameters of cell, and tissue damage (lipid peroxidation, apoptosis, and protein nitration), as well as in situ NOS activity and NO production (NOx). The results showed that after L-NAME administration, in situ NOS activity was almost completely eliminated in all the experimental groups, and consequently, NOx levels fell. Contrarily, the lipid peroxidation level and the percentage of apoptotic cells rose throughout the reoxygenation period. These results reveal that NOS inhibition exacerbates the peroxidative and apoptotic damage observed before the treatment with L-NAME in the hypoxic heart, pointing to a cardioprotective role of NOS-derived NO against H/R-induced injury. These findings could open the possibility of future studies to design new therapies for H/R-dysfunctions based on NO-pharmacology.     

IR780 loaded hollow MnO₂ nanoparticles for dual-mode imaging and enhanced photodynamic therapy of oral squamous cell carcinoma

Photodynamic therapy (PDT) has emerged as a novel therapeutic modality for cancer treatment, but its therapeutic efficacy is severely limited by the hypoxic tumor microenvironment (TME). Here we designed an innovative multifunctional nano-platform which consists of a hollow MnO₂ shell and internal photosensitizer IR780. It is not only used for multimodal imaging of oral squamous cell carcinoma (OSCC), but also for adjustment hypoxic TME to enhance cancer treatment. Hollow MnO₂ can promote decomposition of tumor endogenous H₂O₂ to relieve tumor hypoxia, thereby enhancing the effect of photodynamic therapy. Photosensitizer IR780 generates singlet oxygen under laser irradiation to kill tumor cells, playing photodynamic effect, can also act as the contrast agents for photoacoustic and fluorescence multiple imaging, providing potential imaging capability for cancer therapeutic guidance and monitoring. Our research results in this article show that HMnO₂-IR780 nanocomposite exhibits good biocompatibility and nontoxicity, strong PA/FL imaging contrast, excellent oxygen production capacity and outstanding photodynamic therapy effect. This finding provides a new idea for multimodal imaging-guided nanotherapy for OSCC.     

Agmatine pretreatment protects retinal ganglion cells (RGC-5 cell line) from oxidative stress <i>in vitro</i>

Agmatine, 2-(4-aminobutyl)guanidine, has been reported to have neuroprotective effects against various neuronal damages. In this study it was investigated whether agmatine pretreatment rescues the retinal ganglion cells from oxidative injury in vitro. After differentiation of transformed rat retinal ganglion cells (RGC-5 cell line) with staurosporine, agmatine (0.0 to 100.0 μM) pretreatment was performed for 2 hours. Subsequently, they were exposed to hydrogen peroxide (0.0 to 2.5 mM) as an oxidative stress. Cell viability was monitored for up to 48 hours with the lactate dehydrogenase (LDH) assay and apoptosis was examined by the terminal deoxynucleotide transferase-mediated terminal uridine deoxynucleotidyl transferase nick end-labeling (TUNEL) method. As a result, differentiated RGC-5 cells were found to have decreased viability after addition of hydrogen peroxide in a dose-dependent manner. This hydrogen peroxide induced cytotoxicity caused apoptosis characterized by DNA fragmentation. Agmatine pretreatment not only increased cell viability but also attenuated DNA fragmentation. In conclusion, agmatine pretreatment demonstrated neuroprotective effects against oxidative stress induced by hydrogen peroxide in differentiated RGC-5 cells in vitro. This suggests a novel therapeutic strategy rescuing retinal ganglion cells from death caused by oxidative injury     

Hypoxia-induced differential apoptosis in the central nervous system of the sturgeon (Acipenser shrenckii)

Hypoxia is a frequent challenge to aquatic vertebrates as compared with that for their terrestrial counterparts. All vertebrates respond to hypoxia in a similar, but not identical manner, indicating that these responses appeared early in the evolution of vertebrates. The aim of this study is to find out the effects of hypoxia on apoptosis in the central nervous system (CNS) of sturgeon, an archaic fish. With the regional specialization of the CNS, we hypothesize that if cell death does occur, the response will vary between regions, i.e., some CNS areas will be more susceptible to hypoxia than the others would. Sturgeons (Acipenser shrenckii) were subjected to hypoxia by exposure to either air or hypoxic water. After 6- or 30-h recovery they were sacrificed and the following regions of the CNS: retina, olfactory lobe, optic tectum, pituitary, cerebellum, pons/medulla, and spinal cord were examined by the terminal transferase mediated dUTP nick end labeling technique and for the cleaved fragment of activated caspase-3 by Western blotting. In hypoxia-treated sturgeons, the retina, optic tectum, pituitary, and spinal cord were found to have significantly more apoptotic cells than did untreated sturgeons at both 6 and 30 h after the hypoxic insults, indicating prolonged damage. Apoptosis was confirmed by Western blotting of the cleaved fragment of activated caspase-3. Olfactory lobe, cerebellum, and pons/medulla had relatively few apoptotic cells. The CNS of sturgeon showed a differential pattern of apoptosis in response to hypoxia.     

Gene expression in peripheral arterial chemoreceptors

The peripheral arterial chemoreceptors of the carotid body participate in the ventilatory responses to hypoxia and hypercapnia, the arousal responses to asphyxial apnea, and the acclimatization to high altitude. In response to an excitatory stimuli, glomus cells in the carotid body depolarize, their intracellular calcium levels rise, and neurotransmitters are released from them. Neurotransmitters then bind to autoreceptors on glomus cells and postsynaptic receptors on chemoafferents of the carotid sinus nerve. Binding to inhibitory or excitatory receptors on chemoafferents control the electrical activity of the carotid sinus nerve, which provides the input to respiratory-related brainstem nuclei. We and others have used gene expression in the carotid body as a tool to determine what neurotransmitters mediate the response of peripheral arterial chemoreceptors to excitatory stimuli, specifically hypoxia. Data from physiological studies support the involvement of numerous putative neurotransmitters in hypoxic chemosensitivity. This article reviews how in situ hybridization histochemistry and other cellular localization techniques confirm, refute, or expand what is known about the role of dopamine, norepinephrine, substance P, acetylcholine, adenosine, and ATP in chemotransmission. In spite of some species differences, review of the available data support that 1). dopamine and norepinephrine are synthesized and released from glomus cells in all species and play an inhibitory role in hypoxic chemosensitivity; 2). substance P and acetylcholine are not synthesized in glomus cells of most species but may be made and released from nerve fibers innervating the carotid body in essentially all species; 3). adenosine and ATP are ubiquitous molecules that most likely play an excitatory role in hypoxic chemosensitivity.     

MiR-194-5p suppresses the warburg effect in ovarian cancer cells through the IGF1R/PI3K/AKT axis

Background: The Warburg effect is considered as a hallmark of various types of cancers, while the regulatory mechanism is poorly understood. Our previous study demonstrated that miR-194-5p directly targets and regulates insulin-like growth factor1 receptor (IGF1R). In this study, we aimed to investigate the role of miR-194-5p in the regulation of the Warburg effect in ovarian cancer cells. Methods: The stable ovarian cell lines with miR-194-5p overexpression or silencing IGF1R expression were established by lentivirus infection. ATP generation, glucose uptake, lactate production and extracellular acidification rate (ECAR) assay were used to analyze the effects of aerobic glycolysis in ovarian cancer cells. Gene expression was analyzed by quantitative polymerase chain reaction (qPCR) and western blot. Immunohistochemistry assays were performed to assess the expression of the IGF1R protein in ovarian cancer tissues. Results: Overexpression of miR-194-5p or silencing IGF1R expression in ovarian cancer cells decreases ATP generation, glucose uptake, lactate production, and ECAR and inhibits both the mRNA and protein expression of PKM2, LDHA, GLUT1, and GLUT3. While the knockdown of miR-194-5p expression led to opposite results. Overexpression of miR-194-5p or silencing IGF1R expression suppressed the phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) pathway, whose activation can sustain aerobic glycolysis in cancer cells, and the knockdown of miR-194-5p expression promoted the activation of the PI3K/AKT pathway. Conclusion: Our results suggest that miR- 194-5p can inhibit the Warburg effect by negative regulation of IGF1R and further repression of the PI3K/AKT pathway, which provides a theoretical basis for further test of miR-194-5p as a target in the treatment of ovarian cancer.     

Tumor necrosis factor modulates CD 20 expression on cells from chronic lymphocytic leukemia: a new role for TNF alpha?

Tumor necrosis factor alpha (TNF alpha) is a pleiotropic cytokine that is constitutively produced by leukemic cells in B Chronic Lymphocytic Leukemia (B-CLL). It has been shown to have autocrine and paracrine functions in normal B cells and in B lymphoproliferative diseases. This study was conducted to determine the effect of TNF alpha (in vitro) on CD20 expression on cells from patients with B-CLL. Currently, anti-CD20 monoclonal antibody therapy is becoming a second line treatment in the management of B cell disorders like low-grade non-Hodgkin's lymphoma (NHL) and B-CLL. Our results demonstrate amply that very low doses of TNF alpha (0. 0125 ng/ml) can be used to significantly increase CD20 expression on cells from patients of B-CLL as evidenced by increases in both percentage positivity and mean fluorescence intensity. The upregulation is evident as early as 24 hours and is maintained for up to 72 hours. We propose that the upregulation is a direct result of in vitro differentiation stimulated by TNF alpha. The results presented can be exploited in the designing of priming protocols prior to antibody therapy and this is discussed.     

Cardioprotective effect of ivabradine via the AMPK/SIRT1/PGC-1α signaling pathway in myocardial ischemia/reperfusion injury induced in H9c2 cell

Post-resuscitation myocardial dysfunction (PRMD) is the most severe myocardial ischemia-reperfusion injury (MIRI) and is characterized by difficult treatment and poor prognosis. Research has shown the protective effects of the rational use of ivabradine (IVA) against PRMD; however, the molecular mechanisms of IVA remain unknown. In this study, an ischemia-reperfusion injury (IRI) model was established using hypoxic chambers. The results demonstrated that pretreatment with IVA reduced IRI-induced cytotoxicity and apoptosis. IVA attenuated mitochondrial damage, eliminated excess reactive oxygen species (ROS), suppressed IRI-induced ATP and NAD⁺ , and increased the AMP/ATP ratio. We further found that IVA increased the mRNA levels of sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) and upregulated the expression levels of phosphorylated AMP-activated protein kinase (p-AMPK)/AMPK, SIRT1, and PGC-1α proteins. Interestingly, no change in AMPK mRNA levels was observed. Cardiomyocyte energy metabolism significantly changed after IRI. The aim of this study was to demonstrate the cardioprotective effect of Ivabradine via the AMPK/SIRT1/PGC-1α signaling pathway in myocardial ischemia/reperfusion injury-induced in H9c2 cell.     

Comparison of three transmittance oximeters

Estimates of oxygenation from three transmittance oximeters, Hewlett-Packard 47201A, Nellcor, and Biox III, were obtained simultaneously in ten healthy human subjects during isocapnic progressive hypoxia and under conditions of episodic hypoxemia. In the oxygen saturation range studied (approximately 95-75%), results showed that values from the Nellcor finger oximeter were 2-4% lower than values from the Hewlett-Packard ear oximeter. Values from the Biox III oximeter, using either the ear or finger probe, were 3-7% higher than those from the Hewlett-Packard, and the biggest discrepancies were seen as oxygen saturation fell below 80%. This often resulted in an estimation of the ventilatory response to hypoxia as measured using the Biox III oximeter that was higher than estimates using the Nellcor or the Hewlett-Packard (P less than 0.005). Hypoxic ventilatory responses using the Nellcor and the Hewlett-Packard oximeters gave similar values. During rapidly changing oxygenation, the distribution of values for oxygen saturation were significantly different for the three oximeters. For example, the median of the distribution for Nellcor values was 89 +/- 1.5% (mean +/- SEM); for the Hewlett-Packard, it was 92 +/- 1.3%; and for the Biox III, it was 93 +/- 0.8% (finger) and 95 +/- 1.6% (ear). The authors conclude that differences between the three non-invasive transmittance oximeters will affect clinical measurement of the ventilatory responsiveness to hypoxia and the estimation of the stimulus for hypoxic complications in respiratory diseases.     

Inhibition of H2O2-induced apoptosis of lymphocytes by calorie restriction during aging

Calorie restriction (CR) is known to delay the aging process in rodents and is postulated to act by decreasing free radical generation and increasing antioxidant enzyme activity. The present study was designed to investigate the effect of CR and age on oxidative stress-induced apoptosis and associated changes in the levels of TNF-alpha, and Bcl-2 in splenic T lymphocytes. Ad libitum (AL)- or CR-fed C57BL/6J mice were sacrificed either at 6 (young) or 18 (old) months and splenic lymphocytes were incubated with or without 25 micro M H2O2 to induce apoptosis. Apoptosis increased with age in cells of AL-fed mice incubated with H2O2. CR prevented this rise in apoptosis in total splenic lymphocytes and in CD4(+) and CD8(+) T lymphocyte subsets either with or without H2O2. Free radicals increased and mitochondrial membrane potential decreased in aged mice. CR prevented these changes and also prevented the age-associated increase in TNF-alpha and loss of Bcl-2 in total splenic lymphocytes and in CD4(+) and CD8(+) lymphocyte subsets. In summary, lymphocytes in aged AL-fed mice were much more susceptible to oxidative stress-induced apoptosis whereas CR normalized apoptosis by preventing the increase in TNF-alpha and the decrease in Bcl-2 associated with aging.     

Ang-(1-7) exerts anti-inflammatory and antioxidant activities on high glucose-induced injury by prohibiting NF-κB-IL-1β and activating HO-1 pathways in HUVECs

Previous reports have suggested that Ang-(1-7) may have a protective effect in endothelial cells against high glucose (HG)-induced cell injury thanks to a modulatory mechanism in the NF-κB signaling pathway. In this study, we have examined whether NF-κB-IL-1β and Heme oxygenase-1 (HO-1) pathways contribute to the protection of Ang-(1-7) against hyperglycemia-induced inflammation and oxidative stress in human umbilical vein endothelial cells (HUVECs). Our results indicate that time-varying exposures of HUVECs, from 1 h to 24 h, to high glucose concentrations result in an increased expression of phosphorylated (p)-p65 and HO-1 in a time-dependent manner. As an inhibitor of NF-κB, pyrrolidinedithiocarbamic acid (PDTC) suppressed IL-1β production induced by HG. Of note, HUVECs previously treated with Ang-(1-7) (2 μM) for 30 min before being exposed to HG concentrations significantly ameliorated the HG-increased in p-p65 and IL-1β expression; whereas obviously up-regulated the level of HO-1, along with inhibition of oxidative stress, inflammation, and the HG-induced cytotoxicity. Importantly, when HUVECs were previously treated either with PDTC or IL-1Ra for 30 min before being exposed to HG, it significantly prevented damages caused by high glucose concentrations mentioned above, while the treatment of HO-1 inhibitor Sn-protoporphyrin (SnPP) before exposure to both HG and Ang-(1-7) significantly blocked the protective effect exerted by Ang-(1-7) on endothelial cells against injuries induced by HG mentioned above. To conclude, the data of this study showed that activation and inhibition of the NF-κB-IL-1β pathway and HO-1 pathway may constitute an important defense mechanism against endothelial cell damage caused by HG concentrations. We additionally gave new evidence showing that exogenous Ang-(1-7) exerts a protective effect on HUVECs against the HG-induced cell injury via the inhibition and the activation of the NF-κB-IL-1β pathway and the HO-1 pathway, respectively.     

Deconstructing the molecular mechanisms of cell cycle control in a mouse adrenocortical cell line: roles of ACTH

This is a progress report of an attempt to deconstruct the signaling network underlying cell cycle control in the mouse Y1 adrenocortical cell line, aiming to uncover ACTH growth regulatory pathways. Y1 adrenocortical tumor cells possess amplified and overexpressed c-Ki-ras proto-oncogene. Despite this oncogenic lesion, Y1 cells retain tight regulatory mechanisms of cell cycle control typified by the sequential events comprising the mitogenic response triggered by FGF2 in G0/G1-arrested Y1 cells: 1) activation of ERK1/2 and PI3K, by 5 minutes; 2) induction of c-Fos and c-Myc proteins by 2 hours; 3) induction of cyclin D1 protein by 5 hours; 4) phosphorylation of Rb protein between 6 and 8 hours; 5) onset of DNA synthesis by 8-9 hours. In this cell line, ACTH-receptor (ACTH-R) activates contradictory pathways of growth regulation. First, ACTH coordinately induces fos and jun gene families via activation of both ERK1/2 and cAMP/PKA pathways, resembling a mitogen. Second, ACTH-R triggers cAMP/PKA-mediated antimitogenic mechanisms comprised of Akt/PKB dephosphorylation/deactivation, c-Myc protein degradation, and p27(Kip1) protein induction. Induction of cyclin D1 depends on activation of both ERK1/2 and PI3K, but is not affected by ACTH action. As a consequence, ACTH antagonizes FGF2 mitogenic activity but ectopic expression of the c-Myc protein (via MycER fusion protein) is sufficient to abrogate this ACTH antagonistic effect over FGF2 mitogenic activity. Ectopic expression of both c-Myc and cyclin D1 is not sufficient to drive G0/G1-arrested Y1 cells into S phase, but when the sustained expression of these two proteins is complemented by ACTH treatment it promotes G1 phase progression and DNA synthesis initiation. In conclusion, ACTH-receptor lacks signaling potential sufficient to initiate a mitogenic response in Y1 adrenocortical cells and, therefore, cannot substitute for bona fide mitogens like FGF2.     

Decreased serum HMGB1 associated with M2 macrophage polarization and patients with calcific aortic valve disease

Except for the standard aortic valve replacement, no effective medical treatment is available to prevent or delay calcific aortic valve disease (CAVD) progression. Recently, macrophages and high-mobility group box 1 (HMGB1) are the most intriguing candidates in various inflammatory disorders. However, the association between serum HMGB1, CAVD, and macrophage polarization remains unclear. Therefore, we examined whether the level of serum HMGB1 is clinically associated with aortic valve calcification and whether HMGB1 treatment can promote macrophage differentiation toward M1 or M2 phenotype. This experimental study included 19 CAVD patients and 20 healthy controls whose serum HMGB1 levels were examined by ELISA assay. THP-1 macrophage polarization system was established to test the polarization capability of HMGB1 treatment. The results showed that serum levels of HMGB1 were significantly reduced in patients with CAVD. HMGB1 treatment promoted M2 macrophage polarization but not M1 phenotype with increased IL-10 expression and reduced inducible nitric oxide synthase (iNOS) expression. Our findings suggest that serum HMGB1 is negatively associated with the development of aortic valve calcification, and HMGB1 treatment may facilitate M2 macrophage polarization for reducing aortic valve calcification.     

Advanced glycation end-products change placental barrier function and tight junction in rats with gestational diabetes mellitus via the receptor for advanced glycation end products/nuclear factor-κB pathway

The placenta plays an important role in nutrient transport to maintain the growth and development of the embryo. Gestational diabetes mellitus (GDM), the most common complication during pregnancy, highly affects placental function in late gestation. Advanced glycation end-products (AGEs), a complex and heterogeneous group of compounds engaged by the receptor for AGEs (RAGE), are closely associated with diabetes-related complications. In this study, AGEs induced a decrease in the expression of tight junction (TJ) proteins in BeWo cells and increased the paracellular permeability of trophoblast cells by regulating RAGE/NF-κB. Sprague-Dawley (SD) rats injected with 100 mg/kg AGEs-rat serum albumin (RSA) via the tail vein from embryo day 2 were set as the placental barrier dysfunction model group (n = 10). The effect of AGEs on placental permeability was determined using the EvansBlue dye extravasation method. The ultrastructure of the placenta samples was observed by transmission electron microscopy. The effects of AGEs on the placenta were confirmed by treating rats with RAGE antagonist FPS-ZM1 and soluble forms of RAGE (sRAGE). AGEs treatment increased placental permeability and disrupted the tight junctions in pregnant rat placenta, but has no effect on blood glucose. The expression of TJ-related proteins, including ZO-1, Occludin, and Claudin 5, were downregulated after AGEs treatment. Further, AGEs treatment increased the expression of RAGE and nuclear factor-κB in the placenta of rats and upregulated the levels of vascular endothelial growth factor. The effects of AGEs on the placenta were blocked by RAGE antagonist FPS-ZM1 and sRAGE. This study demonstrates the mechanism underlying AGEs-induced disturbance in placental function in pregnant rats and highlights the potential of AGEs in the treatment of GDM.     

TNF-alpha gene polymorphism: clinical and biological implications

TNF-alpha is a proinflammatory cytokine that has been implicated in the severity of different immune-regulated diseases including autoimmune diseases and transplantation. The gene for TNF-alpha is located within the MHC region on chromosome 6p21.3. This is a highly polymorphic region, and the TNF-alpha itself contains a large number of polymorphisms. Some of these polymorphisms form extended haplotypes with the HLA class I and II alleles. TNF polymorphisms have been investigated in different diseases and most often whenever there is an HLA association with the disease (for example IDDM and RA) association(s) with TNF polymorphisms has been described. There are many studies on the function of the TNF polymorphisms showing the influence of the different alleles on the in vitro and in vivo levels of TNF production. However, recent studies in animal models suggest that not only polymorphisms within the TNF cluster are important in the regulation of TNF production but also the receptors as well (TNF R). This suggests that investigating polymorphisms within the TNF cluster and TNF receptors will be important in understanding the role of TNF regulation in a given disease.