Georgios Kartalis

A Novel C5a Receptor-Tissue Factor Cross-Talk in Neutrophils Links Innate Immunity to Coagulation Pathways

Neutrophils and complement are key sentinels of innate immunity and mediators of acute inflammation. Recent studies have suggested that inflammatory processes modulate thrombogenic pathways. To date, the potential cross-talk between innate immunity and thrombosis and the precise molecular pathway by which complement and neutrophils trigger the coagulation process have remained elusive. In this study, we demonstrate that antiphospholipid Ab-induced complement activation and downstream signaling via C5a receptors in neutrophils leads to the induction of tissue factor (TF), a key initiating component of the blood coagulation cascade. TF expression by neutrophils was associated with an enhanced procoagulant activity, as verified by a modified prothrombin time assay inhibited by anti-TF mAb. Inhibition studies using the complement inhibitor compstatin revealed that complement activation is triggered by antiphospholipid syndrome (APS) IgG and leads to the induction of a TF-dependent coagulant activity. Blockade studies using a selective C5a receptor antagonist and stimulation of neutrophils with recombinant human C5a demonstrated that C5a, and its receptor C5aR, mediate the expression of TF in neutrophils and thereby significantly enhance the procoagulant activity of neutrophils exposed to APS serum. These results identify a novel cross-talk between the complement and coagulation cascades that can potentially be exploited therapeutically in the treatment of APS and other complement-associated thrombotic diseases.

MEFV alterations and population genetics analysis in a large cohort of Greek patients with familial Mediterranean fever

Familial Mediterranean fever (FMF) is a disease characterized by recurrent, self‐limiting bouts of fever and serositis and caused by altered pyrin due to mutated MEFV gene. FMF is common in the Mediterranean Basin populations, although with varying genetic patterns. The spectrum and clinical significance of MEFV alterations in Greece has yet not been elucidated. The aim of this study was to analyze the spectrum of MEFV alterations in FMF patients and healthy individuals in Greece. A cohort of 152 Greek FMF patients along with 140 Greek healthy controls was enrolled. Non‐isotopic RNase cleavage assay (NIRCA) and sequencing allowed mutational and haplotypic analysis of the entire coding sequence of MEFV. The arlequin 2.0, dnasp 4.0 and phylip software were used for population genetics analysis. Among patients, 127 (83.6%) carried at least one known mutation. The most common mutations identified were M694V (38.1%), M680I (19.7%), V726A (12.2%), E148Q (10.9%) and E230K (6.1%). The total carrier rate among healthy individuals was 0.7%. The presence of R202Q homozygosity in 12 of the remaining 25 MEFV negative FMF patients might be considered as disease related in Greeks. Population genetics analysis revealed that Greeks rely closer to the eastern rather than western populations of the Mediterranean Basin.

Increased frequency of mutations in the gene responsible for familial Mediterranean fever (MEFV) in a cohort of patients with ulcerative colitis: evidence for a potential disease-modifying effect?

The MEFV gene, responsible for familial Mediterranean fever (FMF), is involved in inflammatory reactions through altered leukocyte apoptosis, secretion of interleukin (IL)-1β, and activation of the NF-κ B pathway. Ulcerative Colitis (UC) and FMF are both characterized by a recurrent pattern of presentation with periods of remission and flares associated with neutrophilic infiltration at the site of injury. The aim of this study was to investigate the possible correlation between UC and MEFV gene alterations. Twenty-five consecutive, first-diagnosed and untreated UC patients, 28 control patients with rheumatoid arthritis, and 65 normal individuals were analyzed. Nonisotopic RNase Cleavage Assay (NIRCA) was applied as a first-step mutational screening method of exons 10 and 2 of MEFV gene; direct sequencing was subsequently performed to confirm the results. MEFVmutations were identified in 7 (3 M694V/0, 2 M680I/0, 1 E148Q/E148Q, and 1 A744S/0) out of 25 UC patients versus 1 (M694V/0) out of 28 rheumatoid arthritis patients (P = .0199) and 1 (M694V/0) out of 65 healthy controls (P = .0004). Four out of 7 patients with MEFVmutations had inflammatory arthritis, a clinical finding that was not observed in the 18 UC patients with unmutated MEFV (P = .0028). Patients with UC almost universally carried the T A C G MEFV exon 2 haplotype in contrast with normal individuals (P < .0001) and FMF patients (P = .0310). In conclusion the increased frequency of mutations of MEFV in UC patients, especially in those with episodic arthritis, suggests a possible modifying effect of MEFV in the disease process and its localization within the joint. The difference in distribution of MEFV exon 2 haplotypes between UC patients and both FMF patients and normal individuals, suggests that UC patients constitute a genetically distinct population. Larger, longitudinal studies are needed to confirm these initial findings.

Leptin induces the expression of functional tissue factor in human neutrophils and peripheral blood mononuclear cells through JAK2-dependent mechanisms and TNFα involvement

INTRODUCTION Leptin is an adipocyte-derived cytokine primarily involved in the regulation of body weight and energy balance. In vivo studies suggest that leptin promotes platelet aggregation and thrombosis. Neutrophils are involved in the crosstalk between inflammation and thrombosis in clinical disorders. Leptin is also involved in the regulation of inflammation. AIM We examined the in vitro effects of leptin on the expression of tissue factor (TF), the primary initiator of coagulation, in healthy neutrophils. MATERIALS AND METHODS/RESULTS The effects on TF expression were assayed functionally using a modified prothrombin time (mPT), as well as at mRNA and protein levels. The same experiments were performed in parallel with PBMC. Leptin induced functional TF and increased TF mRNA and protein expression in both cell types, as determined by mPT, real-time RT-PCR, western blot, flow cytometry, immunocytochemistry. Inhibition studies revealed that the effect of leptin on TF expression is mediated, at least in part, by JAK2 and PI3K. Our findings, after neutralising TNFalpha in supernatants of leptin-treated cells, also suggest the involvement of TNFalpha in the leptin-induced TF expression in leukocytes. CONCLUSIONS This study indicates a novel link between inflammation, obesity and thrombosis by showing that leptin is able to trigger the extrinsic coagulation cascade. This work suggests a possible mechanism of the thrombotic effects of hyperleptinemic-associated clinical disorders.

Low expression of interferon regulatory factor-1 and identification of novel exons skipping in patients with chronic myeloid leukaemia

Summary. Chronic myeloid leukaemia (CML) is a malignant clonal disorder of the haematopoietic stem cell. Treatment of CML patients with interferon alpha (IFN‐α) has induced haematological and cytogenetic remission. Interferons transcriptionally activate target genes through the JAK–STAT and interferon regulated factors (IRFs) family pathways. Interferon regulated factor‐1 (IRF‐1) is a transcriptional activator of genes critical for cell growth, differentiation and apoptosis. The skipping of exons 2 or 2 and 3 of IRF‐1 in patients with myelodysplastic syndromes and acute myelogenous leukaemia suggests that this factor may have a critical role in leukaemogenesis. The role of IRF‐1 in CML is currently unknown. Therefore, mutational analysis of IRF‐1 was performed and its expression pattern was also studied in CML patients. We studied IRF‐1 in peripheral blood mononuclear cells of 21 patients in chronic phase CML. No point mutations were identified at the cDNA level. Surprisingly, fourfold reduction of full‐length IRF‐1 mRNA expression was established in 17/21 patients compared with normal individuals. Low expression of full‐length IRF‐1 was observed in conjunction with high levels of aberrantly spliced mRNAs, reported for the first time. In three patients who were also analysed during blastic transformation, further reduction of full‐length IRF‐1 mRNA was observed. These findings demonstrate that, in CML patients, IRF‐1 can produce high levels of aberrant spliced mRNAs with subsequent reduction in the levels of full‐length IRF‐1 mRNA. This observation is consistent with the notion that exon skipping may constitute another mechanism of tumour suppressor gene inactivation in this disease.

Predisposing factors and clinical symptoms in HIV-negative patients with Candida oesophagitis: are they always present?

Candida oesophagitis (CO) is scarce among immunocompetent patients. This study aimed at evaluating predisposing factors, clinical symptoms and endoscopic findings in this group. We retrospectively reviewed 55 patients diagnosed as CO endoscopically (whitish plaques) and cytologically (fungal mycelia on brush cytology). Carcinoma, diabetes, acid suppression, steroids, gastric surgery and oesophageal motility disorders were considered as predisposing factors. Twenty of 55 patients lacked any predisposing factor for CO. These patients were more frequently asymptomatic (8/20) when compared with those with known predisposing factors (5/35) (p = 0.031). Moreover, dysphagia was more prevalent in the latter group (24/35 vs. 8/20; p = 0.039). Endoscopic findings correlated with the presence of neither predisposing factors nor symptoms (Wilcoxon p > 0.05). Thus, CO can be discovered in patients without apparent predisposing risk factors and clinical symptoms. Further studies are needed to elucidate the mechanisms of transition from colonisation to infection.

The usefulness of PCR amplification of the IS6110 insertion element of M. tuberculosis complex in ascitic fluid of patients with peritoneal tuberculosis

Background: The diagnosis of tuberculous peritonitis (TP) may be difficult and elusive. The present study was designed to demonstrate the diagnostic usefulness of a nested polymerase chain reaction (PCR) assay, specific for the IS6110 insertion element of M. tuberculosis complex, in patients with ascites who were suspected of having TP in order to achieve a more timely diagnosis and treatment. Methods: Three HIV-negative patients suffering from fever and ascites were evaluated for suspected TP. Specimens were obtained from ascitic fluid, bone marrow, and peripheral blood and analyzed by both conventional methods and nested PCR for the presence of bacilli. Response to antituberculous treatment was considered as the final criterion for diagnosis of peritoneal tuberculosis. Results: All three patients had an excellent response to antituberculous therapy. Our PCR-based protocol detected M. tuberculosis complex DNA in the ascitic fluid of all patients, whereas conventional methods failed to establish the disease. Furthermore, in one patient, M. tuberculosis was also detected in both bone marrow and peripheral blood. Conclusions: PCR amplification of the IS6110 sequence of M. tuberculosis complex in ascitic fluid is a useful tool when peritoneal tuberculosis is suspected. However, its validity still needs to be established.

Absence of Bruton's tyrosine kinase (Btk) mutations in patients with acute myeloid leukaemia

Brutons tyrosine kinase (Btk) is a non‐receptor protein tyrosine kinase (PTK) that is expressed in all haemopoietic lineages except mature T cells and plasma cells. Despite the broad range of expression, mutations that inactivate this molecule affect primarily the development of the B‐cell lineage. As a PTK, Btk could potentially be involved directly or indirectly in the processes that relate to the malignant transformation of all the cell lineages where this molecule is expressed. Previous studies have failed to demonstrate mutations in patients with B‐cell origin acute lymphoblastic leukaemia (ALL).

Eplerenone relieves spironolactone-induced painful gynaecomastia in patients with decompensated hepatitis B-related cirrhosis

TO THE EDITOR: One of the main therapeutic goals in cirrhotic ascites is to compromise secondary aldosteronism. Spironolactone has been extensively used for this purpose, despite its potential side effects, including hyperkalaemia, gynaecomastia and impotence. Recently, a second aldosterone antagonist, eplerenone, has been introduced mainly for the treatment of essential hypertension and congestive heart failure. Nevertheless, the efficacy and safety of eplerenone in the treatment of cirrhotic ascites has not been evaluated yet. We report on the case of four patients with chronic hepatitis B-induced cirrhosis who were admitted to our Department from January to March 2007 with decompensated ascites and painful bilateral gynaecomastia. The patients were receiving treatment with spironolactone (100 mg b.i.d.) plus furosemide (40 mg b.i.d.) for at least 6 months. A switch of spironolactone to eplerenone at 50 mg b.i.d. was introduced. After 3 months of follow-up, breast size was clearly decreased and all the patients reported complete relief of pain. Child-Pugh score and bodyweight remained unchanged. Although spironolactone is the current drug of choice for the treatment of cirrhotic ascites, the potentially serious side effects might limit its use. More specifically, gynaecomastia is doseand timedependent and may be clinically pronounced. A number of mechanisms have been proposed to explain this phenomenon, including a dose-dependent reduction of microsomal cytochrome P450, alterations in the testosterone/oestrogen ratio, a peripheral conversion of testosterone to oestradiol, a decrease in testosterone or increase in oestrone and oestradiol serum levels [1]. In contrast to spironolactone, eplerenone is 40 times less potent in blocking aldosterone activation of the mineralocorticoid receptor and 370 times less potent in blocking dihydrotestosterone activation of androgen receptors. The limited sexual side effects of eplerenone have been documented in the EPHESUS (Eplerenone Post Acute Myocardial Infarction Heart Failure Efficacy and Survival Study) study [2]. In this study, the incidence of gynaecomastia in men was 0.5% (comparable with placebo). In contrast, in the RALES (Randomized Aldactone Evaluation Study) study [3], evaluation of spironolactone, gynaecomastia as well as breast pain was significantly more frequent (9% in treated patients versus 1% in the placebo group). In conclusion, we report for the first time that a highly selective aldosterone antagonism, by means of eplerenone, could be a more appropriate therapeutic tool in comparison with spironolactone in cirrhotic patients with ascites. Further studies are needed to establish the efficacy and safety in this group of patients.

Primary hepatic gastrinoma: Report of a case and review of literature

Primary hepatic gastrinoma is a very rare ectopic gastrinoma with less than 20 cases reported worldwide. We report the case of a patient with hypergastrinemia who was subjected to exhaustive preoperative and intraoperative imaging and also careful surgical exploration of the duodenum and pancreas which failed initially to identify the primary tumour. Eventually the patient was subjected to left liver lobectomy, as a small palpable lesion was noted intraoperatively. The diagnosis of gastrinoma requires a high index of clinical suspicion and the flawless cooperation of many specialties.