The aortas of 8 week old apoE-/-/LDLreceptor-/- mice displayed immunoreactivity for TNF-α as well as TNF p55 and p75 receptors , but did not have any detectable lesions. A marginal increase in TNF-α and TNF receptor immunoreactivity was observed at 12 weeks and atherosclerotic plaques were detected in 1 out of 5 animals. At 16 weeks TNF-α expression in the media was increased more than four-fold as compared with 8 week old mice, and atherosclerosis was widespread. TNF-α immunoreactivity was also observed in all plaques. In addition, at the same age a tendency towards increased TNF-α mRNA levels was detected in the double knockout mice compared to age-matched controls. A further increase in TNF-α and TNF receptor immunoreactivity as well as plaque size was observed at 20 weeks. With only a few exceptions, no TNF-α or TNF receptor immunoreactivity was detected in C57BL/6 control mice. These findings demonstrate that medial TNF-α and TNF receptor expression precedes lesion formation in apoE-/-/LDL receptor-/- mice.TNF-α is present in atherosclerotic lesions, activates endothelial adhesion molecule expression, stimulates the release of proinflammatory cytokines and matrix metalloproteinases and promotes smooth muscle cell proliferation and migration. Taken together these observations suggest that TNF-α may be functionally involved in early atherosclerosis development. To further evaluate this hypothesis we compared vascular TNF-α and TNF receptor expression in atherosclerosis-susceptible apoE TNF-α is a multifunctional cytokine that plays an important role in immune defense , 2. It hSeveral lines of evidence indicate that TNF-α, as well as other inflammatory cytokines, may also be involved in the development of atherosclerosis. TNF-α expressed in skeletal muscle and adipose tissue inhibits phosphorylation of the insulin receptor -9. By inTNF-α may also have a direct influence on development and progression of atherosclerotic lesions. It is expressed during intimal thickening in humans and in atherosclerotic plaques, and to a lesser extent also in the underlying media , 13. Exp-/-/LDL receptor-/-). This model has been found to have total cholesterol levels similar to the ones of apo E deficient mice, whereas LDL receptor deficient mice have much lower levels  on a C57BL/6 background and C57BL/6 (n=32), were obtained from Bomice . Animals were fed standard R3 chow .Male apolipoprotein E and LDL receptor gene double knockout mice  and exsanguinated by cardiac puncture on weeks 8, 12, 16 and 20. The blood was immediately placed on ice and allowed to coagulate overnight at 4°C. Hearts and aortas were surgically removed. Hearts, including the aortic arch and the upper part of descending aorta were washed in 0.9% saline containing 0.02 mM butylated hydroxytoluene (BHT) and fixed in 4% formaldehyde for 12h. For immunohistochemistry, the aortic arch, the thoracic and abdominal regions of the aorta above the renal artery were cut into equal pieces and mounted together in one paraffin block. The abdominal aorta below the renal artery and the common iliac arteries were immediately placed in liquid nitrogen and used for mRNA extraction.2O2 in 80% methanol for 30 min at room temperature. After washing, sections were blocked with 2-5% rabbit serum in PBS for 30 min. Primary antibodies against TNF-α or TNF receptor p55 and 75 (Santa Cruz Biotechnology) were diluted in PBS (1:100 dilution) and incubated for 16 h at 4°C in a humidified chamber. Sections were washed and incubated with biotinylated secondary rabbit anti-goat antibody  diluted in PBS (1:200 dilution) for 30 min and then washed. The sections were incubated for 30 min with a peroxidase labeled avidin-biotin complex (Vector) and washed again. Sections were developed using DAB detection kit (Vector) and counterstained in hematoxylin. Negative controls included substitution of the primary antibody with either PBS or irrelevant antibody.Tissue sections were deparaffinized with xylene and dehydrated with graded ethanol. The membranes were permeabilized in 0.2% Triton X-100. Endogenous peroxidase activity was quenched by incubating the sections in 0.3% HThe TNF immunoreactivity was evaluated by image analysis and expressed as percent positive immunostaining of total media or plaque area. A mean medial staining for each animal was calculated based on analyses of 2 sections from the aortic arch, 2 from the descending thoracic aorta and 2 from the abdominal aorta. A mean plaque area for each animal was calculated based on analyses of 6 sections from the aortic arch, 6 from the descending thoracic aorta and 6 from the abdominal aorta.Cholesterol and triglyceride concentrations in serum were assayed by colorimetric enzymatic techniques . After an overnight incubation at 4°C the blood samples were spun at 2000 rpm for 10 minutes after which the plasma was removed and stored at -70°C. For analysis, each sample was thawed and diluted (1:10) with PBS and assayed according to instruction using Sigma diagnostics kit.Total RNA was isolated from the aorta from each mouse using the protocol for FastPrep™ system  with small modifications. A speed rating of 6 for 45 seconds to lyse the aortas was used. The synthesis of cDNA was performed by mixing 1 µg of total RNA with random primers , DTT (GibcoBRL), dNTP , RNaseINH  and Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV-RT) (GibcoBRL). Tubes were incubated for 10 min at 30°C, 50 min at 42°C and 2 min at 94°C.-/- mice (B&M).TaqMan Universal PCR Master Mix  was used in an ABI PRISM 7700 Sequence Detection System  for amplification of cDNA according to the manufacturer’s protocol. Samples without MMLV-RT in the cDNA synthesis and wells with no template were used as controls. All samples were run in triplicates. Amplification of TNF-α and 18S rRNA was carried out by using cDNA and TaqMan Pre-developed Assay Reagents  and 2x TaqMan Universal PCR Master Mix. Amplification of TNF p55 and p75 receptor was carried out by using appropriate concentrations of specific primer pairs and probes and 2x TaqMan Universal PCR Master Mix. The endogenous control 18S rRNA was used for normalization of data. Calculations were performed using standard curves established by serial dilutions of cDNA synthesized from RNA extracted from aortas of seven months old apoEP≤0.05 was considered statistical significant.Values are given as mean ± SD. Differences between groups were calculated using ANOVA. -/-/LDL receptor-/- mice) with control mice that does not develop atherosclerosis unless challenged with a high-fat diet (C57BL/6). Plasma triacylglycerol and cholesterol levels in apoE-/-/LDL receptor-/- and C57BL/6 mice are presented in Table -/-/LDLR-/- mice than in the control mice. Both triacylglycerol and cholesterol levels increased with age in the double knockout mice.One of the aims of the present study was to compare vascular TNF-α and TNF receptor expression in a mouse strain that spontaneously develops atherosclerosis  and also the expression of TNF p55 and p75 receptors was significantly increased as compared with 8 week old animals . No significant differences in expression of TNF p55 and TNF p75 receptor mRNA levels were detected in the double knock-out mice compared to controls (Table -/-/LDL receptor-/- mice than in controls (Table The amounts of TNF-α mRNA transcripts assessed by Real Time PCR decreased with age in both apoEce Table . In 16-wls Table . There wls Table , whereasls Table .-/-/LDL receptor-/- mice. The expression is mainly located in vascular media. The medial TNF-α and TNF receptor expression in apoE-/-/LDL receptor-/- mice increased with age and in parallel with progression of lesions with the exception of the TNF p75 receptor that showed decreased staining at 20 weeks. Virtually no TNF-α or TNF receptor immunoreactivity was detected in C57BL/6 control mice. This may be an explanation for why depletion of TNF receptors in this non-atherosclerosis prone mouse model doesn’t fit other observations that TNF-α would be a pro-atherogenic cytokine (The present study demonstrates that vascular expression of TNF-α and TNF p55 and p75 receptors precedes development of atherosclerotic plaques in apoEcytokine .-/-/LDL receptor-/- mouse is well in line with previous studies showing expression of TNF-α mRNA and protein in medial SMC of 6 month old WHHL rabbits (A medial expression of TNF-α in non-atherosclerotic arteries of hypercholesterolemic animals such as the apoE rabbits . Using a rabbits . At this rabbits . However rabbits .Only a sub-population of medial SMC appears to express TNF-α and TNF receptors in response to hypercholesterolemia. Studies in balloon-injured rabbits have provided evidence suggesting that medial TNF-α is primarily produced by proliferating SMC . It has Previous studies of vascular inflammatory response to hypercholesterolemia have mainly focused on early up-regulation of adhesion molecules in the endothelium. Our present findings demonstrate presence of an early inflammatory reaction also in the media. The functional role of this inflammatory response remains to be elucidated, but it is likely to participate in activation of endothelial adhesion molecule expression. One possible scenario is that medial SMC respond to extensive accumulation of native, aggregated or oxidized LDL particles with stimulated TNF secretion resulting in signals to the endothelium to recruit monocytes for modified lipoprotein removal.In established lesions accumulation of B cells occurs primarily at sites where the surrounding cells express TNF-α and VCAM-1, suggesting that TNF-α-induced expression of adhesion molecules may be of importance for recruitment of mononuclear cells also within plaques .An important question is whether TNF-α in itself plays a critical role in the development of atherosclerosis. This issue is made even more relevant by the recent development of effective TNF-α-inhibitors for use in other chronic inflammatory diseases. TNF-α-inhibition could influence concomitant cardiovascular disease or even be used directly for treatment of atherosclerosis . A subst-/-/LDL receptor-/- mouse, whereas the aortas of control C57BL/6 mice were essentially negative for TNF-α and TNF receptor immunoreactivity. This suggests that lipid-induced activation of TNF-α expression may play a role in endothelial activation and other inflammatory processes during early stages of atherosclerosis.In summary, this study shows that medial TNF-α and TNF receptor expression precedes lesion formation in the atherosclerosis-susceptible apoE


The G-308A polymorphism of the tumour necrosis factor-α (TNF-α) gene, a variant that influences TNF-α transcription, may contribute to non-ischaemic dilated cardiomyopathy. To evaluate whether TNF-α genotyping may assist in identifying a subset of patients who could potentially benefit from immunomodulatory therapy, we assessed the relationship between the G-308A polymorphism of the TNF-α gene and changes in left ventricular (LV) chamber dimensions and systolic function in patients with idiopathic dilated cardiomyopathy (IDC) before and six months after diuretic, digoxin and angiotensin-converting enzyme inhibitor (ACEI) therapy.p < 0.01) and the TNF-12/22 (AG/GG) genotype  were associated with IDC. However, in 122 patients with IDC, the TNF-α genotype was not associated with plasma TNF-α concentrations. In 133 patients with IDC, the TNF-α genotype failed to predict either the severity of pump dysfunction and cardiac dilatation at baseline, or changes in pump function and cardiac dimensions after six months of medical treatment.In 331 patients with IDC and 349 controls, the TNF-2 (A) allele (odds ratio = 1.509, 95% CI = 1.130–2.015, We conclude therefore that although the TNF-α gene G-308A polymorphism may contribute to the development of IDC, it does not influence pump function or adverse cardiac remodelling in patients with IDC. Genotyping for this variant is therefore unlikely to assist in identifying patients with heart failure who may be particularly susceptible to novel immunomodulatory therapeutic strategies. Patients with heart failure have elevated plasma concentrations of the pro-inflammatory cytokine TNF-α.6Prior to further studies being performed with immunomodulatory therapy, it is therefore important to identify patients who are most likely to benefit from a decrease in TNF-α concentrations. In this regard, the genetic background may promote the deleterious effects of TNF-α in heart failure. Indeed, the TNF-2 -308A) allele of a G-308A polymorphism of the TNF-α gene has been shown to be associated with the presence of non-ischaemic dilated cardiomyopathy in some studies.A allele The TNF-2 allele is a more powerful transcriptional activator than the TNF-1 (G-308) allele and results in a seven-fold increase in induced TNF-α gene transcription.There are no prospective data to determine the impact of this polymorphism on outcomes or disease progression in heart failure. In the present study, we therefore explored the possibility that the TNF-2 allele of the G-308A polymorphism of the TNF-α gene is associated with changes in left ventricular (LV) dimensions and pump function after six months of medical therapy in patients with idiopathic dilated cardiomyopathy (IDC).The Committee for Research on Human Subjects of the University of the Witwatersrand approved the study . Subjects provided informed consent.The clinical component of the study was conducted between 1995 and 2001 when the use of β-AR blockers was not considered standard therapy for heart failure in South Africa. Three hundred and thirty-one patients with IDC and 349 age- and gender-matched control subjects of the same ethnic origin (African ancestry) were recruited. Patients were recruited if they were between 18 and 70 years of age, in NYHA functional class (FC) II or III heart failure of unknown aetiology, had a left ventricular ejection fraction (LVEF) < 40% as determined by radionuclide ventriculography, and had high-quality echocardiographic images with an LV end-diastolic diameter (LVEDD) > 5.5 cm. Exclusion criteria included evidence of another cause of heart failure and the presence of arrhythmias that could alter LVEF.After initial presentation, and following a diagnosis by clinical examination and echocardiography (screening visit), 176 of the 331 patients agreed to participate in a prospective study assessing the impact of the TNF-α gene polymorphism on LV dimensions and function. During the six-month period of follow up, 24 patients died and 20 were lost to follow up. Of the remaining 132 patients who were followed prospectively, 71 were newly diagnosed.All patients received treatment with digoxin and diuretics (furosemide) for seven days and then angiotensin-converting enzyme inhibitors (ACEI) were added to their therapy. The patients were followed for six months. Monthly visits were scheduled for clinical assessment and evaluation of the patients’ adherence to therapeutic agents. Clinical examinations, echocardiographic assessments and radionuclide studies were performed at baseline, and then repeated at six months.The primary endpoints were LVEF, determined using radionuclide ventriculography, and LV end-diastolic diameter (LVEDD), determined using echocardiography. Radionuclide ventriculography as opposed to echocardiography was used as the method of preference to assess the impact of TNF-α genotype on LV systolic function, as this measurement is not subject to observer bias. To show a 10-point difference in radionuclide LVEF between groups with 80% power after six months of therapy required a sample size of 21 patients in each group.A physician assessed the NYHA functional class of the patients during the baseline and follow-up visits. The same physician evaluated all patients. A multiple-gated equilibrium cardiac blood pool scintigraphic technique was used to measure LVEF (Elscint Apex 409).Two-dimensional targeted M-mode echocardiography with Doppler colour flow mapping was performed using a Hewlett Packard Sonos 5500 echocardiograph attached to a 2.5- or 3.5-MHz transducer. All studies were performed and interpreted by the same operator and recorded on videotape. Left ventricular dimensions were measured according to the American Society of Echocardiography guidelines.et al.2, 200 nM of each primer, 200 μM of each dNTP and 50 mM KCl. The G-308 substitution gives rise to a Nco I restriction enzyme recognition site and therefore, following PCR amplification, the 107 bp PCR product was digested with 5U of Nco I (Roche) for four hours at 37ºC and the resulting DNA fragments were visualised on a 3.5% agarose gel. The G allele gives rise to two digestion products of 87 and 20 bp, therefore TNF-11 homozygotes show two bands of 87 and 20 bp, TNF-12 heterozygotes show three bands of 107, 87 and 20 bp and TNF-22 homozygotes show a single band of 107 bp.Genomic DNA was extracted from white blood cells as described by Higuchi Plasma TNF-α concentration was determined in the first consecutive 122 patients using a high-sensitivity, human TNF-α-specific ELISA kit (Amersham) according to the manufacturer’s instructions.t-test or a Mann-Whitney test, depending on whether variables were nominal or ordinal (Bartlett’s test). To test for Hardy-Weinberg equilibrium, the expected genotype numbers were calculated from the allele frequencies and deviation from the observed genotype numbers was determined using a χ2 test. The relative risk of the presence of IDC with the TNF-α G-308A polymorphism was estimated from an odds ratio calculation using a Fisher’s exact test.Data are presented as mean ± SEM. Case and control group mean values were compared with the use of a two-sample Student’s t-test was used to detect changes from baseline. Analysis of covariance adjusting for baseline data, age, gender, disease duration and type of ACEI was employed to determine differences in changes in LV cavity size and function, and final LV cavity size and function between genotype groups. Genotype effects on final LV cavity size and function were assessed using MANCOVA with age, gender, BMI, disease duration and baseline data included as covariates. Data are expressed as mean ± standard error of the mean (SEM).To assess the effect of genotype on either baseline LVEF or cardiac dimensions, a MANCOVA was performed with age, gender, disease duration and BMI included in the regression model. A paired Student’s The characteristics of the patient and control groups are summarised in 2 value of 5.11, whereas the patient group, with a χ2 value of 23.72, was not in Hardy-Weinberg equilibrium. A dominant model of inheritance of the risk allele was assumed, and the TNF-12 and TNF-22 genotypes groups were pooled. A significant excess of both the TNF-12 and 22 genotypes  and the TNF-2 allele  was noted in the IDC group compared to the control group, suggesting that the TNF-2 allele is associated with the development of IDC in the population studied.The genotype and allele frequencies are shown in -1 vs TNF-12/22 = 7.2 ± 0.9 pg.ml-1).At baseline, the G-308A TNF-α polymorphism was not associated with either LV dimensions or systolic function p < 0.0001), LV end-diastolic diameter (LVEDD) decreased by 2.7 ± 0.6 mm (p < 0.02), and LV end-systolic diameter (LVESD) decreased by 4.3 ± 0.7 mm (p < 0.001) in all patients considered together. The increase in LVEF and decreases in LVEDD and LVESD were the same in TNF-α genotype-specific groups A similar percentage of patients in each TNF-α genotype-specific group died or was lost to follow-up (data not shown). There was also a similar percentage of newly diagnosed patients in each group. Both genotype groups received similar doses and type of drug therapy .The finding in the present study that both the TNF-12/22 genotype and the TNF-2 allele were associated with the presence of IDC in the population studied is consistent with previous studies,Assuming that the TNF-2 allele is a risk factor for IDC, a potential explanation for the association could be that in IDC, a trigger event such as a viral infection could lead to excess cardiac TNF-α production in individuals carrying the TNF-2 allele, which in turn could augment cardiac remodelling and pump dysfunction. Viral infection is currently considered to be a likely trigger for the development of many cases of IDC.20Although the present study was a prospective study where therapy was largely standardised between patients, there are still potential limitations to the study. In particular, prospective follow-up was for only six months, rather than a longer period. However, this period was selected because beyond six months, mortality and morbidity related to heart failure would have limited our ability to appropriately assess LV structural and functional changes.21The present study shows that although the TNF-2 allele may contribute to the development of IDC, this allele has no impact on in-treatment adverse chamber remodelling and pump dysfunction. Genotyping for this variant is therefore unlikely to assist in identifying patients with heart failure who may be particularly susceptible to novel immunomodulatory therapeutic strategies.


Paroxysmal depolarization shifts are the cellular representations of interictal spikes. Interictal spikes (IIS) have a long history in the diagnosis of epilepsy. Previously, it was thought that IIS are largely asymptomatic, but there is growing evidence that beside being involved in epileptogenesis, they are also involved in the pathogenesis of various other neurological diseases. However, the pathomechanisms leading to the formation of IIS and how IIS may lead to functional neuronal impairment are poorly understood. In a previous study  we showe1 mM caffeine was applied as an epileptogenic agent, and LTCC activity was modulated by Bay K8644 (BayK) and isradipine, respectively.2O2, DS appeared in a subpopulation of neurons. Testing both BayK and H2O2 on the same neurons we found that H2O2 evoked DS only in those neurons where BayK showed a clear DS-inducing effect. In those cells, where BayK failed to cause DS, H2O2 also had no effect.In contrast to earlier studies on hippocampal slices , caffeinOur data suggest that potentiation of LTCCs promotes the formation of depolarization shifts. Oxidative stress appears to be a pathogenic initiator of DS, and this activity may require LTCCs. Since LTCC augmentation is being considered as a pathological mechanism in neurodegenerative diseases, our data point to the possibility that the initiation of IIS may be a precipitating factor and that LTCC inhibition may provide a means to counteract neuropathogenic mechanisms.


TNF-α and/or LT-α gene polymorphisms since their products play a key role in inflammation. The aim of the study was to investigate the possible roles of TNF gene polymorphisms in CF disease phenotype and progression.It is well known that the disease progression in cystic fibrosis (CF) patients may be diverse in subjects with identical mutation in CFTR gene. It is quite possible that such heterogeneity is associated with TNF-α–308GA and LT-α + 252AG polymorphisms.198 CF patients and 130 control subjects were genotyped for both TNF-α–308A allele more frequently had asthma as compared to patients homozygous for the TNF-α–308 G allele. In 9 of 108 (8.3%) of LTα + 252AA carriers, tuberculosis infection has been documented, whereas there was no case of tuberculosis among patients, either homozygous or heterozygous for LTα +252 G alleles (p = 0.01). We never observed virus hepatitis among LTα + 252GA carriers. The genotypes TNF-α–308GG – LT-α + 252AA and TNF-α–308GA – LT-α + 252AG were unfavorable with regard to liver disease development (both p < 0.05). It was also shown that neutrophil elastase activity was higher in sputum specimens from high TNF producers with genotypes TNF-α–308GA or LT-α + 252GG. In addition the carriers of such genotypes demonstrated a higher risk of osteoporosis development .The carriers of the The carriers of genotypes, which are associated with higher TNF-α production, demonstrated increased frequency of asthma, higher levels of neutrophil elastase, and decrease of bone density. On the contrary, the carriers of genotypes associated with low TNF-α production showed a higher frequency of tuberculosis infection. TNFα and TNF-β (LTα) genes are located close to each other [The Major Histocompatibility Complex (MHC) contains genes essential to both the adaptive and innate immune systems. In humans, these genes are referred to as HLA genes. Genes within the MHC traditionally divided into three different subregions. Class I and II regions contain genes encoded molecules that are responsible for antigen presentation to T cells. The human Class III region is the most gene-dense and highly conserved region of the human genome . Within ch other . The gencystic fibrosis transmembrane conductance regulator (CFTR) gene, it is very difficult to connect the disease clinical course with the type of mutation. Indeed, it is well known that CF progression may be diverse among either siblings or unrelated patients with identical mutation in CFTR gene. Apparently there are other factors including genetic ones, which may determine the course of disease [TNFα and/or LTα gene polymorphisms. A polymorphism in the promoter region of theTNF-α, but LT-α is also important for the development of lymphoid organs -5. It is disease ,7. It isTNFα gene at nucleotide −308, relative to the transcription start site, may be important in determining the host TNF-α response [TNFα308 G and TNFα308A. In normal population TNFα308 G homozygosity is the predominant genotype. The TNFα308GA polymorphism has a small, but significant, functional effect, with the A allele being associated with higher constitutive and inducible levels of transcription for TNF-α than the G allele [LTα gene at position +252, there is NcoI restriction polymorphism, consisting of a Guanine (LTα + 252 G) on one allele and an Adenine (LTα + 252Α) on the alternate allele. The presence of G at this position defines the allele which is less frequent one in white subjects and is associated with higher TNF-α and TNF-β production [TNFα308A polymorphism [TNFα polymorphisms, other than −308 G/A polymorphic loci, are also associated with severity of CF lung disease in Czech and Belgian patients [G allele . Within oduction ,11. Recemorphism . It has patients .TNF gene polymorphisms in CF disease phenotype and progression. To address this issue, we genotyped 198 CF patients and 130 control subjects for both TNF-α–308GA and LT-α + 252AG polymorphisms.The aim of the study was to investigate the possible roles of CFTR alleles. Clinical, biological and functional data were obtained from hospital records from the previous 2 to 15 years. The data included date of birth, sex, CFTR genotype, pulmonary function tests, nutritional status, airways microbiology, CF and non-CF complications . Lung function was assessed by spirometry in children >4 years during periods of clinical stability. Respiratory microbial flora was determined by microscopy and culture of lower respiratory tract secretions or throat swabs realized every routine visit to the CF Department. Chronic airway colonization with Pseudomonas aeruginosa was defined by the persistence of the pathogen in at least three airway samples for at least 6 months. 127 individuals were chronically colonized with the mucoid form of P. aeruginosa. The CFTR genotype in 138 CF patients was homozygous or heterozygous for F508del (ΔF508). Forced expiratory volume in 1 sec (FEV1) and forced vital capacity (FVC) values averaged 78.1 ± 4.4 and 70.1 ± 4.0% predicted, respectively. Characteristics of patient groups with different TNF gene polymorphisms are presented in Additional file P. aeruginosa infection were treated by cephalosporins of third generation in combination with aminoglycosids or ciprofloxacin.A total of 198 patients  from the Cystic Fibrosis Department of the Research Centre for Medical Genetics (Moscow) were enrolled into the study. CF was diagnosed by increased chloride concentrations (>60 mmol/l) in a sweat test, typical clinical symptoms of the disease, and/or detection of mutations in both Blood was collected in tubes with heparin (25 IU/ml) by venipuncture. The sputum samples were placed into the container with ice and delivered to the laboratory within 1 h. The weight of each sputum sample was calculated. The same weight of phosphate-buffered saline without Ca2+ and Mg2+ was added to the sputum sample. The mixture placed on vortex for 10 sec and then on the rocker for 30 min. The sample was filtered through a 100 μm filter to remove the mucus. The filtrate has been centrifuged at 400 × g for 10 min at 4°C to pellet the cells. The supernatant has been harvested, aliquoted and stored at −60°C. Protein concentrations in the samples were measured by Bradford’s method.TNFα promoter gene polymorphism  was performed by polymerase chain reaction (PCR) amplification (using a 5′primer 5′-AGGCAATAGGTTTTGAGGGCCAT3′ and 5′-TCCTCCCTGCTCCGATTCCG3′ as the 3′primer) and NcoI digestion as described by Zhang DL et al. [′-deoxiribonucleoside 5′-triphosphate, 67 mM Tris–HCl, pH 8.4, 2 mM MgCl2, 16.6 mM (NH4)SO4, and 20 μg/ml BSA. The cycling condition consisted of an initial activation of Taq polymerase at 94°C for 5 min followed by 35 cycles of denaturation at 94°C for 45 sec, annealing at 60°C for 45 sec, and extension at 72°C for 45 sec. The PCR products were digested with 8 U of Nco I at 37°C for 6 h. Digested DNA was analyzed on 8% polyacrylamide gels. Ethidium bromide staining of the gel demonstrated the original 107 basepairs fragment , three fragments of 102, 87 and 20 basepairs (heterozygous patients), or two fragments of 87 and 20 basepairs of size . The Nco I polymorphism in intron 1 of the LTα  was determined by PCR-restriction fragment length polymorphism method. A 782 basepairs fragment of the LT-α genomic sequence, including the polymorphic NcoI site, was amplified with a sense primer (5′- CCGTGCTTCGTGCTTTGGACTA 3′) and an antisense primer (5′- AGAGGGGTGGATGCTTGGGTTC3′) [′-deoxiribonucleoside 5′-triphosphate, 67 mM Tris–HCl, pH 8.4, 2 mM MgCl2, 16.6 mM (NH4)SO4, and 20 μg/ml BSA. The cycling condition consisted of an initial activation of Taq polymerase at 94°C for 5 min followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 50°C for 1 min, and extension at 72°C for 1 min. The PCR products were digested with 8 U of Nco I at 37°C for 6 h. Digested DNA was analyzed on 8% polyacrylamide gels. Ethidium bromide staining of the gel demonstrated the original 782 basepairs fragment , three fragments of 782, 586 and 196 basepairs (heterozygous patients), or two fragments of 586 and 196 basepairs of size .Genomic DNA was extracted from anti-coagulated blood by a conventional proteinase K digestion/phenol-chloroform extraction method. Typing of L et al. . PCR wasGGTTC3′) ,15. PCR N-methoxysuccinyl-ala-ala-pro-val p-nitro anilide , forming a colored complex with maximum of absorbance at 410 nm [The method is based on the ability of neutrophil elastase to interact with specific chromogenic substrate t 410 nm . The stat 410 nm . FinallyFifty four patients were undergone to bone mineral density  assessment. BMD was assessed at the lumbar spine (L1-L4) by dual energy x-ray absorptiometry using a Lunar Prodigy Bone Densitometer . The results were expressed as Z score for age, sex and ethnicity according to the reference data given by the GE Lunar Corporation software. Z scores were calculated by subtracting the sex- and age-specific population mean BMD from the CF subject’s BMD; this value was then divided by the standard deviation (SD) of the sex and age-specific mean. Z scores were classified according to the standards recently proposed for pediatric subjects to define bone density reduction . We const-test. P values less than 0.05 were considered significant.The differences in allele/genotype frequencies between patients and controls were analyzed by the Fisher’s exact test. This test was also used for assessment of the differences in clinical course between patients with different genotypes. The levels of neutrophil elastase activity in sputa, the data of pulmonary function tests as well as z-score values were compared by unpaired Student’s The study was approved by the Ethics Committee of the Research Centre for Medical Genetics.TNF genotype frequencies .Followed the classical single polymorphism approach, we have found no association between individual n Figure . In the TNFα308A allele more frequently had asthma as compared to patients homozygous for the TNFα308 G allele   was associated with susceptibility to virus hepatitis in our CF patients. Indeed, among LTα + 252 G carriers the patients with virus hepatitis have not been found, and only patient with LTα + 252 G/G genotype were infected with HCV. Our data are in line with previous study by Goyal et. al. suggesting that LTα + 252AA allele is significantly more common in non-CF subjects infected with HCV as compared to healthy controls [Liver disease is the second leading cause of death in patients with CF . It is econtrols .TNF-α–308 G allele was significantly higher than that in CF carriers of the TNF-α–308A allele . Bone status of the two groups is shown in Figure TNF-α–308 G allele had a normal BMD, with a Z score higher than −1. A mild BMD reduction  was documented in 12 subjects . A condition of severe BMD reduction, with a lumbar spine Z score less than −2, was evident in 13 patients . At the same time 10 of 13 TNF-α–308A carriers  demonstrated a severe BMD reduction. It was also shown the statistically significant differences in bone density among patients homozygous for LT-α + 252A allele and LT-α + 252 G carriers. The last were found to demonstrate decreased values of lumbar spine BMD Z score  and increased osteoporosis frequency. Thus, 15 of 24 CF patients  with LT-α + 252 G had severe BMD reduction. In this patient group a mild BMD reduction and normal BMD were found in 4 (16.7%) and 5 (20.8%) subjects, respectively. In contrast, most of 30 patients homozygous for LT-α + 252A allele had normal BMD or mild BMD reduction . No statistical difference was found among the groups in terms of patient age.Mean lumbar spine BMD Z score in CF patients homozygous for the TNF-α and LT-α gene polymorphisms in the development of various diseases [TNF genes may be implicated in the pathogenic mechanisms of inflammatory diseases. In case of CF we could say only about CF progression and development of the disease complications, but not about predisposition to suffer from CF, since the etiology of it is absolutely clear and associated with mutation in both CFTR alleles. It might be supposed that the influence of TNF gene polymorphisms upon the disease progression in CF patients is associated with high or low TNF production in the carriers of different TNF genotypes. Indeed, it was shown that allele TNFαMany studies have investigated the potential role of diseases ,21,26-30308A has been associated with higher inducible levels of gene transcription and TNF-α protein production [LT-α gene LTα + 252 G allele is associated with higher TNF-α and TNF-β production [in vitro experiments on human cell lines. Thus, the results of meta-analysis carried out to explore the association between the TNFα –308GA polymorphism and asthma development suggested that TNFα –308A allele may be a risk factor in the etiology of the disease. In the subgroup analysis by atopic status, significant elevated risks of asthma were associated with A allele carriers in atopic population [TNFα308GG genotype may have a protective role in asthma pathogenesis [TNFα308A and LTα + 252 G alleles, is an important genetic modifier of lung disease in CF. Although 8.1 AH is associated with delayed onset of respiratory colonization with S. aureus and P. aeruginosa in young CF patients. An elevated inflammatory response being beneficial in the early stages of childhood CF becomes destructive as chronic infection ensure in older patients [TNFα308A allele [oduction ,31. Amonoduction ,11. Clinpulation . It was pulation . On the ogenesis . At the patients . On the TNF gene polymorphisms are the same that TNF gene associated diseases in general population. Simply, there is a strong possibility that such genetically predisposed diseases will be diagnosed in CF subjects in time since most of them undergo a medical examination at least twice a year.Our data received in the cohort of CF patients confirm these observations. Thus, the carriers of genotypes, which are associated with higher TNF production, demonstrated more frequency of asthma, higher levels of neutrophil elastase, and decrease of bone density. At the same time, low TNF producers showed a higher frequency of tuberculosis infection. We believe that the CF complications associated with TNF genotyping may be useful for prognosis and for choice of therapeutic strategy in CF patients.The work shows that the carriers of genotypes, which are associated with higher TNF production, demonstrate more frequency of asthma, higher levels of neutrophil elastase, and decrease of bone density. On the contrary, the carriers of genotypes associated with low TNF production show a higher frequency of tuberculosis and virus hepatitis infection. Thereby, The authors declare that they have no competing interests.GS and AP made an equal contribution in data analysis and manuscript planning and writing. VA participated manuscript writing and approved the final version. NP and EZ were responsible for genotyping. LA conducted the clinical examination and performed data collection. NK revised the manuscript critically. All authors read and approved the final manuscript.Table S1. Characteristics of the cystic fibrosis patients with different TNF gene polymorphisms.Click here for fileTable S2. Forced vital capacity (FVC and FEV1) in CF patients with different TNF genotypes.Click here for fileTable S3. Frequencies of various TNF genotypes among CF patients and Healthy subjects.Click here for fileTable S4. Contribution of individual TNF-α and LT-α gene polymorphisms in CF associated cirrhosis with portal hypertension.Click here for file


Aim. To evaluate maternal TNF-alpha and IL-6 plasma levels in normotensive pregnant women, women with preeclampsia, and to examine the temporal changes in their levels from theantepartum to the postpartum period correlated with the regression of preeclampsia. Method. A prospective study was performed in the 2nd Department of Obstetrics and Gynecology, University of Athens. Blood samples were obtained: (1) antepartum at the time of clinical diagnosis of the syndrome, 2. 12-14 weeks postpartum. Results. No statistically significant differences were found in IL-6 levels, whereas a difference was found in TNF-alpha levels between preeclamptic and controls in antepartum period . Long after delivery, TNF-alpha levels were significantly higher in preeclamptic compared to normotensive controls . No difference was observed in TNF-alpha before and after delivery in both groups. No difference was noticed in IL-6 levels in women of normotensive group long after delivery compared to that before delivery. Long after delivery IL-6 levels were statistically significant higher in preeclamptic women compared to normal controls . Conclusion. Preeclamptic women remain under a status of increased inflammatory stress up to 12-14 weeks postpartum despite the fact that all the other signs of preeclampsia are resolved. Although there is no systemic inflammation during pregnancy, circulating cytokines are found to be elevated in maternal plasma . More spα soluble receptors in systemic circulation may indicate either increased expression of the receptors on the cells' surface, and therefore the increased sensitivity and responsiveness of these cells to elevated levels of TNF-a, as it has been shown in the preeclamptic women of our study or increased presence of systemic circulation proteases of the extracellular part of transmembrane TNF-α receptor and (b) the presence of TNF-α soluble receptor in the systemic circulation which does not document their ability to bind TNF-α in systemic circulation, as the decay of the proteases is likely to affect the binding capacity. Furthermore, the poorly perfused and hypoxic placenta is thought to synthesize increased vasoactive factors, for example, soluble fms-like tyrosine kinase-1, cytokines , and angiotensin II type 1 receptor antibodies) [Maternal serum levels of IL-6 and TNF-alpha play a significant role in pathogenesis of preeclampsia . TNF-alpibodies) . Such elibodies) , 12. Iscibodies) –17.Conflicting results, however, are found regarding the role of circulating IL-6 and TNF-alpha in a review of the current literature. Many authors believe that circulating levels of TNF-alpha and IL-6 are enhanced in preeclamptic patients compared with normotensive and nonpregnant women . On the In the present study, we have evaluated maternal TNF-alpha and IL-6 plasma levels in normotensive pregnant women as well as in women with preeclampsia. We have also examined whether the temporal changes in plasma TNF-alpha and IL-6 levels from the antepartum to the postpartum period correlate with the regression of preeclampsia.Two groups of patients were studied: normal controls and women with preeclampsia. The preeclamptic group consisted of 17 normotensive women who later in their pregnancy developed preeclampsia. The diagnosis of preeclampsia was established according to the criteria developed by the National High Blood Pressure Education Programme Working Group : systoliAll women included in preeclamptic group had severe preeclampsia because they met one or more of the following criteria: systolic blood pressure >160 mm Hg, diastolic blood pressure >100 mg Hg, proteinuria >2 grams in a 24-hour urine collection, blurred vision, epigastric or upper quadrant abdominal pain, vomiting, or subnormal fetal growth. The control group consisted initially of 19 gestation-age-matched normotensive pregnant women. Three women were excluded from the study because one developed gestational hypertension and two gestational diabetes later in pregnancy. The rest of the women (16 cases) remained normotensive and without any complication throughout their pregnancy. All women had similar socioeconomic status and were nonsmokers. None was a permanent drug user or had any known metabolic or renal disease. All women had been receiving ferrous sulfate supplementation during pregnancy and had hemoglobin levels of more than 10.5 gr/dl.An informed consent was obtained from all women participating in the study. The study was approved by the ethics committee of our institution.Blood samples were obtained in two periods: (1) antepartum at the time of clinical diagnosis of the syndrome and (2) 12–14 weeks postpartum in normal women as well as in women with preeclampsia at a time when the harmful effects of preeclampsia were expected to be absent. Blood samples (15 ml) were collected into citrated vacutainers. The tubes remained for 30 minutes at 4°C, and the blood samples were centrifuged at 1600 g for 15 minutes at a temperature of 4°C. The supernatant plasma was separated and stored at −70°C. Enzyme-linked immunosorbent assay was used for quantitative detection of human TNF-alpha, Bender Medsystems GmbH (BMS223HS), Austria. The limit of detection of TNF-alpha was determined to be 0.13 pg/ml. The overall intraassay coefficient of variation has been calculated to be 8.5% while the overall interassay coefficient of variation has been calculated to be 9.8%. Enzyme-linked immunosorbent assay was used for quantitative detection of soluble human IL-6, Bender Medsystems GmbH (BMS213HS), Austria. The limit of detection of IL-6 was determined to be 0.02 pg/ml. The overall intraassay coefficient of variation has been calculated to be 6.9% while the overall interassay coefficient of variation has been calculated to be 8.0%.t-test or paired t-test was used as appropriate for comparison of normally distributed variables, while nonparametric Wilcoxon rank-sum test was used for comparison of nonnormally distributed variables, and values are expressed as mean ± SE for normally distributed and as median  ±95 confidence intervals for nonnormally distributed variables. A P level of <.05 was considered significant.Statistical analysis was performed with a commercially available statistical package (SPSS v 17.0 Chicago IL). Normality of the pregnancy distribution of the studied variables was checked by Kolmogorov-Smirnov test. Student's Demographic characteristics of the two groups are presented in Alterations in plasma IL-6 and TNF-alpha levels in antepartum (A) and postpartum (P) periods are shown in P : .9) in antepartum period. On the contrary, a statistically significant difference was found in TNF-alpha levels in women with preeclampsia compared to normal pregnant women .No statistically significant differences were found in plasma IL-6 levels between preeclamptic and controls . No difference was observed in maternal plasma TNF-alpha before and after delivery in both groups. A drop, but not a statistically significant one, was noticed in plasma IL-6 levels in women of normotensive group (mean ± SE: 1.69 ± 0.48 pg/ml) long after delivery compared to that (mean ± SE: 2.44 ± 0.85 pg/ml P : .4) before delivery.Long after delivery, TNF-alpha levels were significantly higher in preeclamptic women  compared to normotensive controls .An increase, but not a statistically significant one, was observed in plasma IL-6 levels of the preeclamptic women (mean ± SD: 3.53 ± 0.52 pg/ml) in postpartum period compared to that found before delivery .As shown in TNF-alpha is a polypeptide cytokine produced by monocytes and macrophages and has a potentially cytotoxic effect to trophoblasts as demonstrated by in vitro experiments . TNF-alpConflicting results are found regarding the role of IL-6 and TNF-alpha in a review of the current literature. Many believe that circulating levels of TNF-alpha and IL-6 are enhanced in preeclamptic patients compared with normotensive and nonpregnant women . In two α levels compared to those of controls, both directly and in the long period of time after completion of preeclampsia, could lead to increased stimulation of monocytes and macrophages, causing overproduction of free radicals: O2 and HOCl (based the oxidative stress theory). Thus, maintaining high TNF-α levels in systemic circulation may be responsible for the stimulation or disruption of vascular endothelium in the distant time after the removal of the causing factor of preeclampsia which could be expressed by increased production in the systemic circulation of IL -6. In this way, the longer-term clinical effects of preeclampsia in coronary vessels could be justified, as recent literature argues that preeclampsia is often followed in the future by coronary heart disease and cardiovascular events. Histological observations in near term placental bed biopsies, although still conflicting, indicate higher local expression of these cytokines in preeclamptic patients. Decidual monocytes and macrophages are also a rich source of TNF-alpha. The elevated secretion of TNF-alpha by placental villous tissue in response to hypoxia causes a reduction of endothelial cell viability and up-regulates the expression of the adhesion molecule E-selectin by the endothelial cell [According to our results, only the circulating levels of TNF-alpha and not those of IL-6 were enhanced in women with preeclampsia when compared to normotensive women. The source of excess TNF-alpha in preeclampsia remains unclear. The maintenance of elevated TNF-ial cell . It is pial cell , but an ial cell . Thus, tial cell . In paraial cell . Hypoxiaial cell . Additioial cell . On the ial cell , 32.Our results showing no changes of maternal circulating TNF-alpha and a drop, but not a significant one, of IL-6 levels long after delivery suggest that the source of the excessive production of TNF-alpha in preeclampsia may be monocytes, neutrophils, and unlikely placenta itself. This suggestion is in accordance to that of other authors .The similar concentrations of IL-6 in maternal circulations in both groups in antepartum period may indicate that the role of circulating IL-6 is limited in the pathogenesis of preeclampsia and it is opposite to the suggestions of Jirik et al.  that theAn interesting finding of this study is that elevated circulating TNF-alpha and IL-6 levels were detected long after delivery in women with preeclampsia. It is well known that the primary biological activities of proinflammatory cytokines include inflammation and endothelial cell activation . Our finIt is being increasingly recognized that women with a history of preeclampsia are at an increased risk for developing cardiovascular diseases , 42. SevOur study shows that circulating TNF-alpha, but not IL-6, levels are higher in preeclamptic women—at the time symptoms are presented—than in healthy pregnant women in the same period. So we may hypothesize that increased inflammatory factors seen in our material after delivery are likely to play a role in the pathogenesis of cardiovascular diseases seen late in life among women with preeclampsia.


Damage to the intestinal lining is thought to be mediated by increased shedding of epithelial cells and enhanced gut permeability. These processes remain poorly understood at the molecular level, in part because of a lack of suitable animal models. Now, Mark Pritchard and colleagues have established a robust model for examining intestinal epithelial cell shedding, using lipopolysaccharide (LPS) treatment to induce intestinal injury in mice. They show that LPS-treated mice develop diarrhoea, which correlates with intestinal epithelial cell shedding and caspase-3-driven apoptosis. By examining the effects of LPS in knockout mice, they demonstrate a role for TNFR1 and NFκB2 signalling in these molecular events. The model provides a powerful tool for investigating the mechanisms underlying intestinal epithelial damage, which could guide the development of new therapies for gastrointestinal disorders.


The RV144 clinical trial of a prime/boost immunizing regimen using recombinant canary pox (ALVAC-HIV) and two gp120 proteins (AIDSVAX B and E) was previously shown to have a 31.2% efficacy rate. Plasma specimens from vaccine and placebo recipients were used in an extensive set of assays to identify correlates of HIV-1 infection risk. Of six primary variables that were studied, only one displayed a significant inverse correlation with risk of infection: the antibody (Ab) response to a fusion protein containing the V1 and V2 regions of gp120 (gp70-V1V2). This finding prompted a thorough examination of the results generated with the complete panel of 13 assays measuring various V2 Abs in the stored plasma used in the initial pilot studies and those used in the subsequent case-control study. The studies revealed that the ALVAC-HIV/AIDSVAX vaccine induced V2-specific Abs that cross-react with multiple HIV-1 subgroups and recognize both conformational and linear epitopes. The conformational epitope was present on gp70-V1V2, while the predominant linear V2 epitope mapped to residues 165–178, immediately N-terminal to the putative α4β7 binding motif in the mid-loop region of V2. Odds ratios (ORs) were calculated to compare the risk of infection with data from 12 V2 assays, and in 11 of these, the ORs were ≤1, reaching statistical significance for two of the variables: Ab responses to gp70-V1V2 and to overlapping V2 linear peptides. It remains to be determined whether anti-V2 Ab responses were directly responsible for the reduced infection rate in RV144 and whether anti-V2 Abs will prove to be important with other candidate HIV vaccines that show efficacy, however, the results support continued dissection of Ab responses to the V2 region which may illuminate mechanisms of protection from HIV-1 infection and may facilitate the development of an effective HIV-1 vaccine. The RV144 HIV-1 vaccine trial was the first to demonstrate evidence of protection against HIV-1 infection, with an estimated vaccine efficacy of 31.2% In order to identify correlates of risk of HIV-1 infection in RV144, two sequential sets of analyses of plasma specimens from study participants were conducted inversely with the risk of infection. 2) The level of plasma IgA Abs reactive with a panel of 14 envelope glycoproteins correlated directly with risk of infection.Case-control specimens consisted of specimens drawn two weeks after the last immunization from 41 infected vaccinees (cases) and from 205 frequency-matched uninfected vaccinees (controls). Two of the six primary variables significantly correlated with HIV-1 infection risk in vaccine recipients: 1) The level of plasma IgG antibodies (Abs) reactive with gp70-V1V2, a scaffolded protein carrying the first and second variable regions of an HIV-1 gp120 envelope glycoprotein fused to murine leukemia virus gp70. Levels of Abs specific for gp70-V1V2 were correlated The participation of the V2 region of gp120 in the infectious process and the role of V2-specific Abs in protection from infection have been the subject of investigation and controversy for nearly two decades. Although, by definition, “variable” regions vary in amino acid (AA) sequence, many residues in these regions do not vary, or tolerate only conservative changes Abs specific for V2 occur in only ∼25–40% of HIV-infected individuals The RV144 clinical vaccine trial was registered with ClinicalTrials.gov; it was assigned NCT00223080 as the registration number. Written informed consent and counseling was conducted as described previously The initial pilot studies were performed using various sets of plasma from the RV144 participants which were selected randomly, evenly balanced for men and women, and derived from participants at visit 1 (pre-bleed), visit 8 (week 26 after the first immunization [two weeks after the last immunization]), and visit 9 (52 weeks). The pilot studies were performed with plasma sets C , A and L , and Z . Subsequently, case-control plasma specimens, described above, were tested for the primary and secondary variables selected as described above and in This assay was previously described This assay was previously described This method was previously described Linear Peptide ELISA (PB). These assays were performed as previously described V2 Direct binding ELISAs were conducted as previously described The arrays measured reactivity with 15-mer linear peptides with 12 residue overlaps. Raw peptide microarray data were processed and analyzed as described in Measurements were conducted with a Biacore T100 as previously described These assays were performed as previously described Immune parameters measured two weeks after the last immunizing dose were assessed as correlates of subsequent infection risk using the previously described statistical analysis plan http://www.r-project.org) was used to assess the ability of 12 V2 variables from The lasso model selection procedure In RV144, the V2 sequence in the recombinant ALVAC priming immunogen derived from subtype E strain 92TH023 was:157CSFNMTTELRDKKQKVHALFYKLDIVPIEDNTSS.SEYRLINC198.The V2 sequence in the protein boosting gp120 immunogen AIDSVAX E (strain A244) was:157CSFNMTTELRDKKQKVHALFYKLDIVPIEDNNDS.SEYRLINC198.The V2 sequence of the protein boosting gp120 immunogen AIDVAX B (strain MN) was:157CSFNITTSIGDKMQKEYALLYKLDIEPI.DN.DSTS.YRLISC198.Insertion of periods in the sequences allows for alignment. Numbering shown and used throughout this report is that assigned to strain HxB2 The antigenic reactivity of the V2 region in AIDSVAX B and E was assessed using human anti-V2 mAbs 697D and 2158 A gp70-V1V2 scaffolded protein carrying the V1 and V2 variable regions from a subtype B strain, case A2, was previously described The frequency of V2 responses detected with pilot study specimens derived from vaccinees varied with the assay used, ranging from 6% for IgA Abs reactive with a linear V2 peptide (K178) when measured by Luminex see  to 97% fFor fine mapping of linear V2 epitopes recognized by Abs in the plasma of RV144 vaccinees, four 21-mer peptides , which matches the results from the independent ELISA data described above. It is also noteworthy that the weakest reactivity was detected with the subtype B subset of V2 peptides, confirming the poor reactivity with Peptides 18 and 19  carried by glycosylated gp70-V1V2 containing a subtype B sequence, but not with linear or cyclic (non-glycosylated) peptides 169 and V172, were critical for the binding of vaccinees' plasma Abs to V2 peptides, and (f) the V2 epitope includes K169 which was identified by sieve analysis to be mismatched in vaccine breakthrough infections The reactivity of vaccinees' Abs with overlapping V2 peptides also correlated with reduced risk of infection Table 1,It is noteworthy that the single primary variable showing an inverse correlate of infection risk in the RV144 case-control study was Ab reactivity with gp70-V1V2 which contains the V1V2 sequence of case A2, a subtype B strain env gene of CRF01_AE, whose V2 region is nearly identical to that of the V2 of the A244 gp120 protein boost, and is extremely divergent from the V2 carried by the MN gp120 boost. Thus, it would appear that the Ab response to the CRF01_AE V2 was primed more effectively than the response to the V2 of subtype B. To address these issues, further studies of V2 responses with specimens from other vaccine trials, e.g., VAX003 and VAX004, are underway, along with assays using additional peptides, proteoglycans, and epitope-scaffolded proteins.The explanation for the strong induction of V2 Abs by the A244 subtype E gp120 immunogen compared to the weak response induced by the MN subtype B gp120 despite the similar antigenicity of the two proteins may be due to the fact that the V2 domain of A244 rgp120 is more immunogenic than that of MN rgp120, and that it usually takes three immunizations with protein, given at appropriate intervals, to elicit good seroconversion to the V2 domain Finally, the mechanisms by which anti-V2 Abs may reduce HIV infection have yet to be understood. As noted, anti-V2 mAbs can neutralize many Tier 1 pseudoviruses in the TZM.bl assay


Babesia. Tumour necrosis factor alpha (TNF-α) is a cytokine that plays a role in the pathogenesis of canine babesiosis. In this study, the authors determined the concentration of serum TNF-α in 11 dogs infected with Babesia canis and calculated Spearman’s rank correlations between the concentration of TNF-α and blood pressure, and between TNF-α and indices of renal damage such as: fractional excretion of sodium (FE(Na+)), urinary creatinine to serum creatinine ratio (UCr/SCr), renal failure index (RFI), urine specific gravity (USG) and urinary protein to urinary creatinine ratio (UPC). The results demonstrated statistically significant strong negative correlations between TNF-α and systolic arterial pressure (r = −0.7246), diastolic arterial pressure (r = −0.6642) and mean arterial pressure (r = −0.7151). Serum TNF-α concentration was also statistically significantly correlated with FE(Na+) (r = 0.7056), UCr/SCr (r = −0.8199), USG (r = −0.8075) and duration of the disease (r = 0.6767). The results of this study show there is an increase of serum TNF-α concentration during canine babesiosis, and the increased TNF-α concentration has an influence on the development of hypotension and renal failure in canine babesiosis. This probably results from the fact that TNF-α is involved in the production of nitric oxide and induction of vasodilation and hypotension, which may cause renal ischaemia and hypoxia, and finally acute tubular necrosis and renal failure.Canine babesiosis is a tick-borne disease caused by parasites of the genus  Babesia , were detected in Germany in dogs imported from Romania, and in Spain, respectively  is a proinflammatory cytokine that plays a significant role in the pathogenesis of babesiosis and malaria, and influences the severity of these diseases ), urinary creatinine to serum creatinine ratio (UCr/SCr), renal failure index (RFI), urine specific gravity (USG) and urinary protein to urinary creatinine ratio (UPC) are useful in the differentiation between prerenal and renal azotaemia in azotaemic dogs , renal failure indices and blood pressures in dogs infected with B. canis prior to treatment. Diagnosis of infection with B. canis was based on the results of a blood smear examination , UCr/SCr, RFI and UPC. The FE(Na+) and RFI were calculated using the following formulas: FE(Na+) = UNa × SCr × 100 % ÷ SNa × UCr and RFI = UNa × SCr ÷ UCr, respectively; where UNa is the urinary sodium concentration, SCr is the serum creatinine concentration, SNa is the serum sodium concentration and UCr is the urinary creatinine concentration  and 11 dogs infected with B. canis (group B). Spearman’s rank correlation coefficient was used to calculate correlations between serum TNF-α concentrations in dogs infected with B. canis and their blood pressures , serum urea and creatinine concentrations, FE(Na+), UCr/SCr, RFI USG, UPC and the duration of the disease before admission to the clinic. The value of p < 0.05 was considered significant.Blood, serum and urine samples were collected from 11 dogs of various breeds   Table . Statist+) Table .Fig. 2CoB. canis. A similar result has been observed in dogs experimentally infected with Rangelia vitalii, which is a protozoan parasite that causes canine piroplasmosis in Brazil  and UCr/SCr ratio result from the influences of decreased blood pressure and/or TNF-α on renal failure in canine babesiosis. In previous research, Tracey et al. (+) and RFI and decrease of the UCr/SCr ratio are indicative for acute tubular necrosis  into renal azotaemia (lower USG) with a concurrent increase of the serum TNF-α concentration. In this work, all azotaemic dogs had relatively low USG (1.013–1.016), but not isosthenuria. One dog had hyposthenuria , and serum levels of urea and creatinine were at the upper reference intervals in this dog; thus, it cannot be excluded that azotaemia was developing in this dog. It should also be mentioned that the USG value (decreasing) can be influenced by other factors such as cortisol or aldosterone Watson ; increasB. rossi. In theory, UPC is a useful tool in clinical recognition of damage of renal glomeruli when increasing above values of 0.5–1.0 in azotaemic dogs  with the results of previous work in which a lack of a correlation between UPC and decrease of blood pressure was observed (Buranakarl et al. There was no statistically significant correlation between the TNF-α concentration and UPC in this study. Lobetti and Jacobson  observedIn this work, there were strong positive correlations between serum TNF-α concentration and concentrations of serum urea and creatinine, which suggested that cytokine has an influence on the development of azotaemia in canine babesiosis. Additionally, the correlation between duration of the disease and TNF-α concentration suggested that this cytokine is involved in the development of pre-renal azotaemia, caused by hypotension, at the beginning of the disease and further, is involved in renal azotaemia caused by renal hypoxia when the disease is progressing.B. microti and Babesia duncani depend on the host’s immune response and that increased production of TNF-α plays an important role in the pathogenesis of the disease (Hemmer et al. The results of this study show there is an increase in the serum TNF-α concentration in canine babesiosis and that the increased TNF-α concentration influences the development of hypotension and renal failure in canine babesiosis. A previous study has shown that the severity and outcomes of babesiosis in mice experimentally infected with human babesial strains of


Tumour necrosis factor-α (TNF-α) plays a key role in the regulation of cardiac contractility. Although cardiomyocytes are known to express the TNF-α receptors (TNFRs), the mechanism of TNF-α signal transmission is incompletely understood. The aim of this study was to investigate whether the tumour suppressor Ras-association domain family protein 1 isoform A (RASSF1A) modulates TNF-α signalling in cardiomyocytes.−/−) mice and wild-type (WT) littermates in this study. Acute stimulation with a low dose of TNF-α (10 µg/kg iv) increased cardiac contractility and intracellular calcium transients' amplitude in WT mice. In contrast, RASSF1A−/− mice showed a blunted contractile response. Mechanistically, RASSF1A was essential in the formation of the TNFR complex (TNFRC), where it functions as an adaptor molecule to facilitate the recruitment of TNFR type 1-associated death domain protein and TNFR-associated factor 2 to form the TNF-α receptor complex. In the absence of RASSF1A, signal transmission from the TNF-α receptor complex to the downstream effectors, such as cytoplasmic phospholipase A2 and protein kinase A, was attenuated leading to the reduction in the activation of calcium handling molecules, such as L-type Ca2+ channel and ryanodine receptors.We used RASSF1A knockout (RASSF1AOur data indicate an essential role of RASSF1A in regulating TNF-α signalling in cardiomyocytes, with RASSF1A being key in the formation of the TNFRC and in signal transmission to the downstream targets. For example, treatment of adult cardiomyocytes with TNF-α for 12 h protects against hypoxic injury.5 Also, low dose of TNF-α treatment induces hypertrophic growth in isolated myocytes,6 whereas acute treatment with a low dose of TNF-α produces a positive inotropic effect in conscious dogs.7 Moreover, TNF-α appears to produce biphasic effects on cardiac contractility: at low concentrations, TNF-α increases the amplitude of [Ca2+]i transients and contraction, whereas at high concentrations TNF-α impairs electrically stimulated [Ca2+]i transients and contraction.9Tumour necrosis factor-α (TNF-α) is a pro-inflammatory cytokine that plays an important role in the heart, in particular, in the regulation of contractility and left ventricular (LV) remodelling. Mice with cardiomyocyte-specific overexpression of TNF-α developed dilated cardiomyopathy, altered cardiac contractility, and abnormal intracellular calcium dynamics.10 Serum TNF-α level is dramatically elevated in endotoxemia and may be important in determining cardiac contractile function in this condition.TNF-α is involved in a number of pathological conditions. For example, TNF-α is one of the key mediators of systemic endotoxemia.11 Upon binding to TNF-α, the TNFRs recruit a number of molecules to form the TNFR complex (TNFRC), which in turn initiates the activation of the downstream signalling cascade.12 Here, we discover for the first time that a Ras effector/interacting protein, the Ras-association domain family protein 1 isoform A (RASSF1A), is essential for the formation of the TNFRC in cardiomyocytes. RASSF1A is a tumour suppressor molecule that in non-cardiomyocytes regulates a number of important cellular processes such as apoptosis, cell growth and viability, and also the cell cycle. RASSF1A lacks enzymatic activity and it exerts its functions mainly via interaction with and modulation of other molecules.14 RASSF1A is expressed in the heart, but its involvement in the regulation of TNF-α signalling in cardiomyocytes is unknown.TNF-α mainly exerts its biological effects through binding to two different types of TNF-α receptors, TNFR1 and TNFR2, both of which are expressed in cardiomyocytes.In the present study, we used a RASSF1A knockout mouse model, as well as isolated cardiomyocytes with genetic ablation or overexpression of RASSF1A, to demonstrate the pivotal role of this molecule in mediating the TNF-α-induced contractile response in cardiomyocytes and in the whole heart.2.2.115 Plasmid containing human TNFR-associated factor 2 (TRAF2) cDNA was a gift of Dr John Kyriakis .16 Plasmids containing human TNFR1 and human TNFR type 1-associated death domain protein (TRADD) were obtained from Origene. For the NFκB activity assay, we used a luciferase construct containing four tandem repeats of NFκB-binding sites (Clontech). For RASSF1A gene silencing, we used shRNA targeting rat RASSF1A driven by the U6 promoter and shRNA containing scrambled sequence driven by the same promoter was used as the control.The human RASSF1A cDNA was a gift of Dr Geoffrey Clark . The generation of RASSF1A deletion mutants has been described previously.2.2Adenoviruses were generated by cloning cDNAs to the pAd/CMV/V5-DEST vector (Invitrogen) using the Gateway system following the manufacturer's recommended methods. pENTR11 vector (Invitrogen) was used as the shuttle system.2.3Rassf1a gene as described previously.17 All animal experiments were performed on 16- to 20-week-old mice in accordance with the UK Animals (Scientific Procedures) Act 1986 and were approved by the University of Manchester Ethics Committee.We used mice with systemic genetic ablation of the 2.4In vivo haemodynamic analyses were performed as described previously.18 Briefly, mice were anaesthetized by intraperitoneal injection of tribromoethanol [240 mg/kg body weight (BW)] and placed on a heat pad at 37°C. A 1.4-Fr pressure–volume catheter (Millar Instruments) was inserted into the left ventricle via the right carotid artery. Pressure–volume signals were recorded first under basal conditions and then recorded 30 min after intravenous injection of TNF-α (10 µg/kg BW).2.5−/− mice, using methods described previously.18 Neonatal rat cardiomyocytes were isolated from 1- to 3-day-old Sprague-Dawley rats. Details of the isolation methods are provided in Supplementary material online, Methods.Adult cardiomyocytes were isolated from 3- to 4-month-old wild-type (WT) or RASSF1A2.618 To assess the involvement of cytoplasmic phospholipase A2 (cPLA2), we treated cardiomyocytes with cPLA2 inhibitor AACOCF3  at a dose of 20 μM or cPLA2 activator peptide PLAP (Santa Cruz Biotechnology) at 1 μM. Details of calcium transient measurement are provided in Supplementary material online, Methods.Isolated adult cardiomyocytes were loaded with calcium ratiometric fluorescent dye (Indo-l). In order to measure the cytosolic calcium, the myocytes were perfused with Tyrode solution and then field stimulated at a frequency of 1 Hz. Calcium changes during myocyte contraction were recorded before and after stimulation with either TNF-α (10 ng/mL) or isoproterenol (100 nM) as previously described.2.7t-test, one- or two-way analysis of variance (ANOVA), where appropriate. Values were considered significant if P < 0.05 .Data are presented as mean ± SEM. Statistical analyses were carried out using the Student's 3.3.1−/− mice. We analysed pressure–volume loops to assess indices of contractility . There was no significant difference in basal haemodynamic indices between WT and RASSF1A−/− mice. However, paired analysis between basal and 30-min post-TNF-α infusion showed that acute TNF-α treatment significantly increased end-systolic LV pressure and decreased end-diastolic LV pressure without creating any changes in heart rate and other diastolic parameters in both WT and knockout mice (Table 1). The changes in indices of contractility (ΔdP/dtmax and ΔEes [end-systolic elastance]) in response to TNF-α were dramatically different between WT and RASSF1A−/− mice. Both Ees and dP/dtmax were significantly enhanced in WT animals , indicating that acute treatment with a low dose of TNF-α increased cardiac contractility, which was consistent with previously published data.9 Interestingly, RASSF1A−/− mice showed a blunted contractile response following treatment with the same dose of TNF-α as indicated by ΔdP/dtmax and ΔEes  . These data suggested that RASSF1A is involved in modulating the cardiac contractile response following TNF-α stimulation.To assess the involvement of RASSF1A in TNF-α signalling, we injected a low dose of TNF-α (10 µg/kg BW) intravenously in WT and RASSF1A3.2−/− cardiomyocytes. Consistent with the in vivo data, isolated adult cardiomyocytes from WT animals showed significantly higher calcium transient amplitude in response to TNF-α stimulation . However, RASSF1A−/− cardiomyocytes showed no significant change in calcium transient amplitude following similar stimulation . No changes in the calcium decay rate were observed in both RASSF1A−/− and WT mice . These data confirm the regulatory role of RASSF1A in cardiomyocytes and imply that the in vivo effect described in Figure 1 was likely due to the direct TNF-α effect on cardiomyocytes.TNF-α injection in mice could affect various cell types such as macrophages and endothelial cells, which could trigger an immune response. Therefore, to investigate whether RASSF1A mediates TNF-α-induced contractile response as a direct effect on cardiomyocytes, we assessed the effect of low-dose TNF-α treatment (10 ng/mL for 30 min) on isolated adult WT and RASSF1AFigure  showed that there was no difference between WT and RASSF1A−/− cardiomyocytes regarding the change in calcium amplitude and calcium decay rate following β-adrenergic agonist (isoproterenol) stimulation.Next, we analysed whether RASSF1A ablation alters the β-adrenergic pathway. Data presented in 3.319 a process which is essential in signal transmission to the intracellular effectors. To investigate the importance of RASSF1A in the formation of TNFRC, we conducted co-immunoprecipitation experiments in adult cardiomyocytes isolated from RASSF1A−/− mice and their WT littermates with or without TNF-α treatment. In WT cardiomyocytes, TNFR1 co-precipitated with both TRAF2 and TRADD and the amount of co-precipitated proteins increased following TNF-α treatment . In contrast, there was a marked reduction in co-immunoprecipitation between TNFR1 and TRAF2/TRADD in RASSF1A−/− myocytes before and after TNF-α treatment . The levels of TNFR1, TRADD, and TRAF2 expressions were not different between WT and RASSF1A−/− . These data suggest that RASSF1A might play an essential role in the recruitment of TRADD and TRAF2 to the TNFR and the formation of TNFRC following TNF-α stimulation.It has been described that, following activation by TNF-α, the TNFR recruits several molecules such as TRAF2 and TRADD to form a TNFRC,3.420 However, it is not known whether RASSF1A binds to TRAF2 and TRADD and whether it links these molecules to TNFR1 following TNF-α stimulation. We conducted co-immunoprecipitation experiments in isolated neonatal rat cardiomyocytes to examine the possible protein–protein interactions between different components of the TNFRC  and RASSF1A in the presence of 10 ng/mL of TNF-α. Results shown in Figure  revealed that endogenous RASSF1A co-precipitated with TNFR1, TRAF2, and TRADD in cardiomyocytes in the presence of TNF-α. These data strongly suggested that upon TNF-α stimulation RASSF1A was recruited to the TNFRC, where it might play an important role as an adapter molecule which links TRAF2 and TRADD to TNFR1.To further investigate the mechanism by which RASSF1A modulates the formation of TNFRC, we performed experiments to determine whether RASSF1A interacts with components of TNFRC. A previous report has shown the association of RASSF1A with TNFR1 in the U2OS osteosarcoma cell line.21 Therefore, it is important to examine whether TNFR2 is also involved in the RASSF1A-dependent TNF-α regulation. However, immunoprecipitation analysis showed that RASSF1A did not interact with TNFR2 . This suggests that the RASSF1A regulatory mechanism was unlikely via TNFR2.TNF-α receptor 2 (TNFR2) has been described to have a protective role in the heart.3.515 In brief, we have generated: (i) RASSF1A-ΔN, which has the N-terminal region (amino acids 1–132) deleted; (ii) RASSF1A-ΔC mutant, which lacks 53 amino acids at the carboxy terminal region, thereby removing the Salvador-RASSF-Hippo (SARAH) domain; and (iii) RASSF1A-ΔC + RA, which lacks both the RA (Ras binding) and SARAH domains . We generated adenoviruses to enable expression of these mutant proteins in neonatal rat cardiomyocytes, which was confirmed by western blot . Immunoprecipitation experiments showed that only the RASSF1A-ΔN co-precipitated with TRAF2 and TRADD. Both of the C-terminal deletion RASSF1A mutants did not bind to either TRAF2 or TRADD, indicating that the C-terminal region of RASSF1A is likely to be important in mediating interaction with both TRAF2 and TRADD .To further define the mechanism by which RASSF1A modulates TRAF2 and TRADD recruitment to TNFR1, we performed co-immunoprecipitation analysis using deletion mutants of the RASSF1A protein as described previously.3.6Figure  and B was the result of systemic RASSF1A knockout in mice. To assess the effects of acute inactivation of RASSF1A, we performed a RNAi gene silencing approach. Adenovirus expressing RASSF1A shRNA was used to effectively knockdown RASSF1A expression in cardiomyocytes . We then performed analyses to assess whether RASSF1A acute gene silencing altered the interaction between TNFR1 and TRAF2/TRADD. Co-immunoprecipitation experiments presented in Figure  showed that RASSF1A gene silencing markedly reduced TRAF2 and TRADD recruitment to TNFR1, demonstrating a consistent phenotype between systemic gene ablation in mice and acute gene silencing using the RNAi approach in isolated myocytes.The phenotype described in 3.722 To investigate whether alteration of RASSF1A expression modifies NF-κB signalling, we performed experiments using adenoviral-mediated NF-κB luciferase reporter. Using this system, we were able to detect activation of the NF-κB pathway in response to TNF-α in control cells. We found a significantly lower NF-κB luciferase activity in cardiomyocytes lacking RASSF1A following TNF-α treatment . Importantly, overexpressing human RASSF1A in cells lacking this molecule rescued the NF-κB luciferase activation to the same level as control cells . These data strongly support the notion of the important regulatory role of RASSF1A in TNF-α signalling.TNF-α regulates several important signalling pathways in cardiomyocytes, such as the NFκB pathway, through binding to the TNFRs.3.8−/− and WT mice and treated them with 10 ng/mL of TNF-α for 30 min. Measurement of cPLA2 and CaMKII activities showed that the activities of both enzymes were significantly increased in response to TNF-α treatment; however, only cPLA2 activity was markedly reduced in RASSF1A-deficient cardiomyocytes , suggesting that cPLA2 was likely the downstream effector of RASSF1A signalling. In cardiomyocytes, cPLA2 activation will enhance the formation of arachidonic acid (AA),7 which may eventually modulate intracellular calcium via a variety of systems, including ion channel activation and induction of protein kinase A (PKA) activity.24 Consistently, PKA activity was significantly reduced in RASSF1A−/− cardiomyocytes following TNF-α induction .Two possible downstream effectors of the TNF-α signalling pathway were investigated to gain mechanistic insights into the regulation of cardiomyocyte calcium dynamics, i.e. the cPLA2 and the CaMKII. We isolated adult cardiomyocytes from RASSF1A−/− cardiomyocytes .Next, we analysed activation of calcium handling proteins that are essential in regulating calcium transient amplitude. We focused on measuring the phosphorylation levels of L-type calcium channel (LTCC) and ryanodine receptor (RYR). Western blot analysis showed that TNF-α stimulation significantly increased the phosphorylation of LTCC and RYR in WT cardiomyocytes; however, this effect was completely ablated in RASSF1A−/− cardiomyocytes revealed that treatment with AACOCF3 reduced the TNF-α-induced elevation of Ca2+ transient amplitude in WT myocytes to a level comparable with that produced by RASSF1A−/− myocytes . No further effect of cPLA2 inhibition was observed in RASSF1A−/− myocytes. On the other hand, cPLA2 activator was able to rescue the reduction of TNF-α-induced elevation of Ca2+ transient amplitude in RASSF1A−/− myocytes . No difference was observed in calcium decay rate between WT and RASSF1A−/− after cPLA2 inhibition or activation . Moreover, inhibition of cPLA2 reduced phosphorylation of LTCC and RYR following TNF-α treatment in WT cardiomyocytes, whereas treatment with PLAP rescued the reduction in LTCC and RYR phosphorylation as well as the reduction in PKA activity in the RASSF1A−/− cardiomyocytes .To further ascertain if RASSF1A regulates TNF-α-dependent calcium transients via cPLA2, we performed analysis using cPLA2 inhibitor  as well as cPLA2 activator peptide . Analysis of calcium transients in WT and RASSF1AFigure ).Taken together, our results suggest that the downstream mechanism by which RASSF1A affects TNF-α contractile response is likely through cPLA2 and subsequently the phosphorylation of calcium handling proteins such as RYR and LTCC; via activation of PKA  was injected intraperitoneally. Cardiac function and serum TNF-α level were assessed 16 h after injection.Endotoxemia is an important pathophysiological condition affecting cardiac contractile function. TNF-α is one of the key mediators of systemic endotoxemia.−/− mice . Haemodynamic analysis showed that LPS-treated RASSF1A−/− mice exhibited a significantly lower contractile function compared with LPS-treated WT mice as indicated by Ees, dP/dtmax, and dP/dtmin values . We then treated isolated adult cardiomyocytes from WT and knockout mice with LPS (10 µg/mL) for 60 min and analysed the calcium transients . We found that there was no significant difference regarding the change in calcium amplitude as well as the rate of calcium decay following direct LPS treatment between WT and RASSF1A−/− cardiomyocytes. Furthermore, there was no difference in cPLA2 and PKA activities between WT and RASSF1A−/− cardiomyocytes . Consistently, western blot analysis suggested that the levels of LTCC and RYR phosphorylation were not altered in RASSF1A−/− cardiomyocytes following direct LPS treatment . We also performed immunoprecipitation analysis to assess whether RASSF1A interacts with LPS receptor, Toll-like receptor 4 (TLR4) in cardiomyocytes. We found that RASSF1A did not co-precipitate with TLR4 in the cardiomyocytes . Taken together, our data suggest that the difference in the in vivo contractility was unlikely due to the direct LPS effect on cardiomyocytes.The levels of serum TNF-α were massively increased following LPS injection; however, we did not observe any difference between WT and RASSF1A4.−/− cardiomyocytes, suggesting that the effect of RASSF1A ablation seems to be specific to TNF-α signalling.Our study shows that RASSF1A is an essential component for the formation of the TNFRC in cardiomyocytes and its subsequent downstream positive inotropic effects. We found that RASSF1A is a key adaptor molecule that facilitates the recruitment of TRAF2 and TRADD to the TNFR1 upon TNF-α stimulation, and this process is important in the regulation of downstream signal transmission. In the absence of RASSF1A, little if any TRAF2 and TRADD are recruited to the TNFRC. Functionally, the absence of RASSF1A virtually ablates the contractile response to TNF-α. Importantly, the β-adrenergic inotropic response was not altered in RASSF1A9 In RASSF1A−/− mice and adult cardiomyocytes isolated from RASSF1A−/−, these effects were abolished. We proposed that RASSF1A facilitates the association of TRAF2 and TRADD, two molecules essential for TNF signalling, with the TNFRC. In the absence of RASSF1A, less TRAF2 and TRADD were recruited to the complex upon stimulation with TNF-α, leading to a reduction in downstream phospholipase-mediated signal transmission and hence a reduction in the calcium amplitude and contractility. Previously, it has been described that the N-terminus of RASSF1A is responsible for the interaction with TNFR120 in the U2OS osteosarcoma cell line. In this study, we show the interaction of RASSF1A with TRAF2 and TRADD, which is likely mediated by the C-terminal region of RASSF1A. Therefore, we speculate that, in the process of TNFRC formation, RASSF1A may act as an adaptor molecule linking TRAF2/TRADD  to TNFR1 .Previous reports have shown that acute application of low doses of TNF-α produces an increase in calcium transient amplitude and cardiac contractility.25 It has been shown previously that cPLA2 activation requires phosphorylation by p38 and caspase-mediated cleavage for its activation.27 TRAF2 is important in the TNFα-induced p38 phosphorylation via receptor-interacting protein and germinal centre kinase,28 whereas TRADD is crucial in the TNFα-induced caspase activation.29 Further downstream, active cPLA2 induces the release of AA from the cell membrane and hence elevation of AA level.25 AA has the capability to induce calcium entry from the intracellular calcium store.23 This process is dependent on PKA and anchoring protein for PKA (AKAP) activities, since inhibition of PKA30 and disruption of AKAP function23 abolish AA-induced calcium release. Taken together, these reports suggest that cPLA2 may activate PKA via AA and AKAP may play an important role in the spatial regulation of this signal.In various cell types, including cardiomyocytes, TNF-α stimulation induces cPLA2 activation.−/− cardiomyocytes. This suggests that the TNF-α-induced CaMKII activation is unlikely to be mediated by the RASSF1A-dependent pathway. Indeed, it has been shown that the TNF-α-induced CaMKII activation is mostly mediated by oxidation and autophosphorylation processes due to the elevation of free radicals.32 This might explain our finding that the CaMKII activation was not different between WT and RASSF1A−/− cardiomyocytes.Another possible downstream effector of TNF-α stimulation is CaMKII. Although as expected we observed significant elevation of CaMKII activity following TNF-α stimulation, we did not find any difference between WT and RASSF1A2+ amplitude) as well as the phosphorylation of LTCC and RYR in WT cardiomyocytes, whereas treatment with cPLA2 activator (PLAP) rescued the phenotype of RASSF1A−/− myocytes. Taken together, our data present one possible mechanism explaining the reduction in contractile response and calcium transients in RASSF1A−/− cardiomyocytes following TNF-α stimulation: ablation of RASSF1A alters the formation of the TNFRC, in particular the recruitment and activation of TRAF2 and TRADD, which subsequently causes a reduction in cPLA2 activation. This will result in the decreased activity of the downstream effectors, possibly PKA, which will eventually lead to the reduction in LTCC and RYR phosphorylation.In this study, we found that ablation of RASSF1A led to the reduction in cPLA2 activity as well as decreased phosphorylation of major calcium handling proteins, such as LTCC and RYR, following TNF-α stimulation. In fact, cPLA2 inhibition using AACOCF3 abolished the TNF-α-induced inotropic response .−/− mice displayed a significant decrease in contractility compared with WTs. Interestingly, no differences in Ca2+ amplitude, cPLA2, and PKA activities or phosphorylation of LTCC and RYR were observed when we directly challenged isolated cardiomyocytes with LPS. Also, we found that RASSF1A did not interact with TLR4 in cardiomyocytes. Therefore, we speculated that this phenotype might be caused by the different response to TNF-α signalling. Both WT and RASSF1A−/− mice produced comparable levels of TNF-α in the serum following LPS injection; however, since RASSF1A−/− mice exhibited an attenuated inotropic response to TNF-α, this could account for the reduced contractility exhibited by RASSF1A−/− mice following LPS injection . It should be noted that further experiments need to be done to clarify the exact mechanism by which RASSF1A modulates LPS-induced cardiomyopathy. For example, a rescue experiment by activating the cPLA2–PKA pathway in RASSF1A−/− mice during LPS stimulation is needed to elucidate whether this pathway is responsible in mediating the observed phenotype.The present study also provides initial evidence of the possible involvement of RASSF1A in LPS-induced cardiomyopathy. In response to LPS injection, RASSF1AIn summary, our present work demonstrates a novel TNF-α effector pathway mediated by RASSF1A which transmits the positive inotropic effect of TNF-α in cardiomyocytes. The complete understanding of the effector pathways of TNF-α is pivotal that may enable us to develop molecules that preserve the beneficial effect of TNF-α in the heart. This could be very useful for the treatment of conditions involving acute TNF-α elevation such as endotoxemia.Cardiovascular Research online.Supplementary material is available at Conflict of interest: none declared.British Heart Foundation (BHF) Intermediate Fellowship Grant (FS/09/046/28043) and BHF Project Grant (PG/11/23/28801) to D.O, and a Medical Research Council (MRC) Research Grant (G0802004) to L.N.This work was supported by


Objective. The aim of this study was to quantitatively evaluate the salivary tumor necrosis factor-alpha (TNF-α) level in oral lichen planus patients and to compare the levels of TNF-α between saliva and serum of OLP and controls. Methods. Serum and whole saliva from 30 patients with active lesions of oral lichen planus (OLP) and 30 healthy persons were investigated for the presence of TNF-α by enzyme immunoassay. Student's independent t-test and two-sample binomial proportion test were used to calculate significance of the mean values of TNF-alpha in serum and saliva and to determine the proportions of the detected and nondetected samples in both groups. Results. Proportion of detection and the mean of detectability between saliva and serum of Group B show an almost equal value, which suggests that saliva can be a good alternate to serum to analyze TNF-α in oral lichen planus patients. Lichen planus (LP) is a chronic inflammatory mucocutaneous disease. It was first described by the British physician Erasmus Wilson in 1869. The clinical appearances of these lesions resembled lichens a primitive organism of symbiotic algae and fungi growing on rocks, hence designated as lichen planus [This disease primarily affects the skin and mucosal surfaces including the oral cavity. It can also involve other sites like the scalp and the nails . It is eOut of the six clinical forms of OLP the reticular, papular, and plaque type lesions are usually asymptomatic  whereas γ) and tumor necrosis factor-alpha (TNF-α) serve to stimulate T-cells, initiate their adhesion to blood vessels, and direct their migration from the blood vessels to the tissues [The exact cause of LP remains unclear; however several predisposing factors like stress, chronic liver disease, and genetics have been implicated in the pathogenesis . Current tissues .α) has been implicated in the pathogenesis of many precancerous, cancerous, autoimmune, and inflammatory diseases including oral submucous fibrosis, oral leukoplakia, oral squamous cell carcinoma, systemic lupus erythematosus, rheumatoid arthritis, psoriasis, and OLP. In addition to being cytotoxic for certain tumor cells, it also acts as an essential mediator in inflammatory and immunologic reactions during host defense by increasing the major histocompatibility complex (MHC) class I antigen and intercellular adhesion molecule (ICAM) expression on many cells [Tumor necrosis factor-alpha  and the results were interpreted by using spectrophotometry at 450 nm. On a linear graph, the optical density values were plotted for each of the calibrators (y-axis) against the corresponding standard concentration of TNF-α (x-axis), and a calibration curve was drawn. The TNF-α concentration of salivary and serum samples were calculated using the optical density and the concentration of the standard. The results were expressed as pg/mL.All reagents and samples were brought to room temperature (18–25°C) before use. The samples were subjected to analysis and TNF-t-test. The proportions of the detected and nondetected samples in both groups were determined using two-sample binomial proportion test.Statistical Package for Social Science (SPSS) software was used to analyze the data. Mean and standard deviation were estimated in each study group. The significance of the mean values of TNF-alpha in serum and saliva between the control and the patients were done using Student's independent t-test was used to compare the mean values of Group A (controls) and Group B (OLP) in Tables α in OLP patients was higher than the control group and it was statistically significant with a P value of 0.039.Students' independent α levels in OLP patients was higher than the control group and it was statistically very highly significant with a P value of 0.000697.As shown in α in salivary and serum samples.Two-sample binomial proportion tests were done to compare the proportion of detectability and nondetectability of TNF-α in saliva of OLP patients as shown in P value of 0.02.The proportion of detectability of TNF-α in serum of OLP patients as shown in P value of 0.001.The proportion of detectability of TNF-α in serum of OLP patients was high when compared with saliva from the same group but it was statistically not significant.As shown in t-test was used to compare the mean of detectability of TNF-α in saliva and serum of OLP patients. As shown in α in saliva and serum of OLP patients is same.Students' independent α in oral lichen planus patients.Since there is no significant difference between the proportion of detection  and the α in OLP patients were evaluated in this study.Oral lichen planus (OLP) is a chronic inflammatory disorder. Although the exact etiology of OLP remains unclear, immunological aberration plays a demanding role among the various etiological factors . Though α is a potentially important and regulatory cytokine in the initiation and progression of OLP, Sugermann et al. [α were detected in all patients with OLP in comparison with healthy controls. Simultaneously with the expression of other proinflammatory cytokines, OLP lesions have also been shown to contain cells with mRNA for TNF-α [α in OLP patients [α in saliva and serum in Indian population, the present study was carried out to evaluate and compare the levels of TNF-α in saliva and serum of oral lichen planus patients. The levels of TNF-α can be quantified using ELISA which is more sensitive, reliable, simple, and widely used and very little sample is required for estimation [α ELISA kit was used to quantify the levels of TNF-α.TNF-n et al. . Accordin et al.  and Sklan et al.  high seror TNF-α  and onlypatients –15. As ttimation . In the α levels are serum, saliva, and oral tissue transudates. The use of saliva as an adjunctive diagnostic tool has various advantages and supplements the current methodologies. Compared to serum, saliva has significant diagnostic and logistical advantage as a diagnostic fluid as it is less infective, noninvasive, relatively simple collected safely, collected repeatedly without discomfort to the patient, cost effective, easy and safe in disposal; does not require specialized training or special equipment; and lends itself readily for mass screening [Body fluids that can be used to estimate the TNF-creening . These aα from a variety of “resident” and “migratory” cells contributes to the total aggregation of bioactive TNF-α in OLP. Within the lesion T lymphocytes, mast cells and keratinocytes are considered to be the plausible cellular source of TNF-α [α enters saliva is by diffusion, active transport, and leakage through tissue transudates [TNF-of TNF-α . The exansudates .α in saliva.Unstimulated saliva better represents the physiological state when compared to a stimulated state . Hence iα value in Group B (OLP patients) ranged from 0 to 266.67 pg/mL in saliva and 0 to 246 pg/mL in serum. In Group A (controls) it ranged from 0 to 253.33 pg/mL in saliva and 0 to 236 pg/mL in serum.TNF-α was higher in Group B (OLP patients) when compared to Group A (controls) and it was statistically significant with a P value of 0.039. Similarly in serum, the mean value of TNF-α was higher in Group B (OLP patients) when compared to Group A (controls) and it was statistically very highly significant with a P value of 0.000697. These results were consistent with the previous studies done in saliva [α plays an important role in the pathogenesis of OLP. The proportion of detectability of TNF-α in saliva of Group B (OLP patients) was significantly high when compared to Group A (controls) with a P value of 0.02. Similarly the proportion of detectability of TNF-α in serum of Group B (OLP patients) was very highly significant when compared to Group A (controls) with a P value of 0.001. The proportion of nondetectable salivary and serum samples for TNF-α was less in Group B (OLP patients) when compared to Group A (controls). Comparing the proportion of detectability between serum and saliva of Group B, (OLP patients) the detectability was marginally high in serum and the mean detectability level of TNF-α in saliva and serum of Group B (OLP patients) was same. This signifies that saliva can be a good alternate for serum to analyze TNF-α in oral lichen planus patients.Results suggested that the mean salivary levels of TNF-n saliva –15 and sn saliva , 11, 19 α in Group B (OLP patients). Diets rich in curcumin and yogurt has been shown to inhibit the activity of TNF-α levels in saliva and serum [α in saliva and serum of Group B. Gavala et al. have reported that alcohol can inhibit the levels of TNF-α in blood [α can occur during storage as they were estimated after a specific period of time. This can also be considered as a reason for nondetectability of TNF-α in saliva and serum of OLP patients.23.3% in saliva and 16.7% in serum were not detected for TNF-nd serum , 21 whicin blood . Althougα. Though the exclusion criteria were strictly followed, it can be suggested that these patients would have had a local trauma, aphthous ulcer, upper respiratory tract infection, or any other subclinical infection in the recent past for having high levels of TNF-α in saliva and serum.Few salivary and serum samples in Group A (controls) showed elevated levels of TNF-α concentration between them.Although patients with 5 different clinical manifestations of OLP were enrolled in this study, no subgroups were formed to categorize the clinical types to evaluate and compare the significant difference of TNF-α in both the test specimens were almost equal. However larger sample size may be required to prove this in a conclusive manner. The study also points to a fact that saliva can be used as a test specimen in the evaluation of TNF-α.This study proves the fact that the expressions of TNF-