Bortezomib downregulates MGMT expression in T98G glioblastoma cells.

The efficacy of treatment for glioblastoma multiforme is currently limited by the development of resistance, particularly, but not exclusively, due to the expression of the DNA repair enzyme O6-methylguanine methyltransferase (MGMT) in a significant proportion of astrocytic tumors. MGMT is post-translationally regulated by the 26S proteasome, a multi-subunit organelle responsible for degradation of misfolded cellular proteins. The boronic acid dipeptide bortezomib is the first and only proteasome inhibitor in clinical use so far, and has been reported as a strategy to restrict growth and promote apoptosis of glioblastoma cells. In this study we investigated the effect of bortezomib on MGMT expression in T98G cells, looking for an effect on the nuclear factor kappa B (NFκB) pathway, which is a major player in MGMT regulation and is also under tight control by the ubiquitin-proteasome system. Administration of bortezomib led to a significant reduction of T98G cell viability and induction of DNA fragmentation. These effects coincided with reduced expression of MGMT transcript levels, and a decrease in cellular amount and IκBα-mediated, proteasomal activity-dependent nuclear translocation of NFκB. In addition, bortezomib-induced phosphorylation of the translation initiation factor 2alpha (eIF2α) was in parallel with translational repression of MGMT. Taken together, these results suggest a novel role for bortezomib as a potent MGMT inhibitor and support its ongoing testing as a chemosensitizer in glioblastoma.

High rates of obesity prevalence in adults living in central Greece: data from the ARGOS study.

OBJECTIVE: To investigate the prevalence of obesity in adults of a large region of Central Greece. DESIGN: The target group was adults aged 18 to 79 years who were residents of the region of Thessaly for at least one year. A sample of 852 individuals stratified for sex and age were included. Each subject underwent a thorough physical examination and body mass index (BMI) was calculated from body weight and height. Waist and hip circumferences as well as body fat content were additionally measured. RESULTS: Mean (SD) BMI for the total population was 27.5+/-5.5 and was significantly higher in males than in females (28.2+/-4.4 vs. 26.9+/-6.2, p<0.001). The overall prevalence of obesity was 26.6% distributed equally between men (27.8%) and women (25.6%), whereas prevalence of overweight was 39.4% with male predominance (50.8% vs. 29.3%, p<0.001). Morbid obesity (MO) was found in 3.5% with female predominance. The prevalence of central obesity, using waist circumference cut-off points (>102cm for men, >88cm for women), was comparable in males (40.4%) and females (35.3%). There was a positive association between obesity, central obesity, and age. The prevalence of overweight (19.5%) and obesity (9.4%) in the age-range of 18-29 years almost doubled in the next decade of age and attained the highest value, respectively, in the age-range of 50 to 59 (48.2%), and of 60 to 70 years group (38.9%). CONCLUSIONS: The rates of overweight and obesity in the population of Thessaly are relatively high with overweight being more prominent in males than in females, whereas MO was higher in females compared to males.

Additive inhibition of colorectal cancer cell lines by aspirin and bortezomib.

PURPOSE: To investigate the effect of cyclooxygenase-2 (Cox-2) inhibitor aspirin (acetylsalicylic acid, ASA) and proteasome inhibitor bortezomib in the proliferation and apoptosis of colorectal cancer cell lines. METHODS: MTT assay, trypan blue exclusion and DNA fragmentation have been used to investigate cell proliferation and apoptosis in the presence of drugs. For the determination of Cox activity a colorimetric method was used. Western blotting was used for the measurement of the effect of the drugs in different proteins expression. RESULTS: Bortezomib together with aspirin inhibit the growth of colorectal cancer cell lines HCT116, HT-29, and CaCo2 more than each drug alone. In the first two cell lines ASA inhibitory effects are Cox-2 independent because HCT116 cells do not express the enzyme while in HT-29 cells, Cox-2 has no activity as shown by a Cox activity assay. In CaCo2 cells that express enzymatically active Cox-2 this activity is inhibited by ASA. ASA is also able to suppress the increase in Cox-2 activity induced by bortezomib in these cells. Cell cycle inhibitors p21 and p27 are induced in the three cell lines by bortezomib and the combination treatment. Akt1 kinase is down-regulated in all three lines by the same treatments. Transcription factor NF-kappaB is retained in the cytoplasm by drug treatment in cell lines HCT116 and HT-29, a fact that may play a role in their pro-apoptotic activity. Pro-apoptotic bcl-2 family member, bad is down-regulated in cell lines HCT116 and CaCo2 by bortezomib treatment, a neoplasia-promoting event that is reversed by combination treatment. CONCLUSION: The combination of bortezomib and ASA cooperates to decrease proliferation and induce apoptosis in three human colorectal cell lines with different genetic lesions. These effects are at least in some cases Cox-2 independent and involve common and diverse mechanisms in the three lines.

Hematogenous spinal infection in central Greece.

STUDY DESIGN: We retrospectively analyzed spinal infection (SpI), in a teaching Hospital, in Central Greece. OBJECTIVE: To study presentation, etiology, and outcome of SpI in Central Greece. SUMMARY OF BACKGROUND DATA: SpI most frequently involves the intervertebral disc and adjacent vertebral bodies and can cause neurologic impairment. METHODS: Thirty three patients (23 men; age [mean +/- standard deviation], 60.6 +/- 11.3 years; disease duration, 44.5 [+/-54.7] days) hospitalized with SpI between January 2000 and December 2007 were included in the study. All patients had magnetic resonance imaging of the spine. RESULTS: Nineteen patients had pyogenic SpI (57.6%) and 14 patients had granulomatous SpI, 11 due to Brucella spp (34.4%), 3 due to Mycobacterium tuberculosis (9.4%). Staphylococcus aureus was the most frequent cause of pyogenic SpI, and spondylodiscitis (SpD) was the most frequent localization. Epidural entension was found in 8 of 17 pyogenic SpD and in 2 of 11 brucellar SpD patients. Subdural extension was detected in 3 patients with pyogenic SpD. Blood cultures were positive in 17 of 19 patients with pyogenic SpI. Two patients had concomitant endocarditis (staphylococcal 1, enterococcal 1). The most common associated disease was diabetes mellitus. All but 2 patients received medical treatment alone. Two patients died of uncontrollable sepsis. CONCLUSION: Back pain in presence of fever, constitutional symptoms, and/or high inflammation markers should alert physicians for spinal infection. In endemic areas, Brucella is a frequent cause of SpI.

Legionella species colonization of water distribution systems, pools and air conditioning systems in cruise ships and ferries.

BACKGROUND: Legionnaires' disease continues to be a public health concern in passenger ships. This study was scheduled in order to investigate Legionella spp. colonization of water distribution systems (WDS), recreational pools, and air-conditioning systems on board ferries and cruise ships in an attempt to identify risk factors for Legionella spp. colonization associated with ship water systems and water characteristics. METHODS: Water systems of 21 ferries and 10 cruise ships including WDS, air conditioning systems and pools were investigated for the presence of Legionella spp. RESULTS: The 133 samples collected from the 10 cruise ships WDS, air conditioning systems and pools were negative for Legionella spp. Of the 21 ferries WDS examined, 14 (66.7%) were legionellae-positive. A total of 276 samples were collected from WDS and air conditioning systems. Legionella spp. was isolated from 37.8% of the hot water samples and 17.5% of the cold water samples. Of the total 96 positive isolates, 87 (90.6%) were L. pneumophila. Legionella spp. colonization was positively associated with ship age. The temperature of the hot water samples was negatively associated with colonization of L. pneumophila serogroup (sg) 1 and that of L. pneumophila sg 2 to 14. Increases in pH >/=7.8 and total plate count > or =400 CFU/L, correlated positively with the counts of L. pneumophila sg 2 to 14 and Legionella spp. respectively. Free chlorine of > or =0.2 mg/L inhibited colonization of Legionella spp. CONCLUSION: WDS of ferries can be heavily colonized by Legionella spp. and may present a risk of Legionnaires' disease for passengers and crew members. Guidelines and advising of Legionnaires' disease prevention regarding ferries are needed, in particular for operators and crew members.

Immunological features of visceral leishmaniasis may mimic systemic lupus erythematosus.

OBJECTIVE: Visceral leishmaniasis (VL), caused by the intracellular parasite Leishmania, can present with fever, splenomegaly, pancytopenias, hypergammaglobulinemia, and autoantibody production. These features may mimic systemic lupus erythematosus (SLE). The objective was to study features of VL that shared with and differed from SLE. DESIGN AND METHODS: A small retrospective study of six patients with VL diagnosed in a University Hospital between 2001 and 2007. RESULTS: All patients had cytopenias, firm splenomegaly, high acute phase reactants, and activation of the coagulation cascade. Hypergammaglobulinemia was detected in five patients. Direct Coombs test was positive in all patients, anti-nuclear antibodies were detected in five patients, anti-smooth muscle antibodies (ASMA) in four patients, and IgM rheumatoid factor (RF) in four patients. Anti-dsDNA antibodies were detected in one patient and IgM anti-cardiolipin antibodies were detected in one patient. CONCLUSION: Autoantibodies are frequently detected in VL and may mimic SLE, but massive firm splenomegaly, very high acute phase reactants, and activation of coagulation system with high D-dimers point toward infection.

Transferrin receptor-1 and 2 expression in chronic lymphocytic leukemia.

Transferrin receptor (TfR)-1 and 2 mRNA and CD71 (TfR1) expression was analyzed in 118 CLL patients. Ninety-five out of 109 analyzed cases expressed CD71, mostly at a high level; 60% of CD71 (+) cases were IGH-mutated. All samples were TfR1 mRNA (+); TfR2-alpha/beta mRNA was detected in, respectively, 52/102 and 100/109 cases. Competitive RT-PCR showed widely divergent levels of TfR1 mRNA in cases with high CD71 expression, alluding to post-transcriptional control of TfR1 expression in CLL. The almost uniformly high CD71 expression in CLL is in keeping with the activated status of neoplastic cells, regardless of IGH mutational load.

Primary biliary cirrhosis presented as peripheral eosinophilia in asymptomatic women with or without elevated alkaline phosphatase.

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease characterized by the destruction of biliary epithelial cells, presumably by autoimmune mechanism(s). Although lymphocytes play a pivotal role in the pathogenesis of PBC, the possible involvement of eosinophils has also been suggested. Recent studies have shown that eosinophilia possibly occurs in the peripheral blood of PBC patients. We present four cases of asymptomatic middle-aged women with moderate-to-high eosinophilia observed during random investigation. Alkaline phosphatase (ALP) was increased in two of them. As a result of clinical and laboratory evaluations the early stages of PBC were diagnosed in all the patients, as attested by the detection of high titres of anti-mitochondrial antibodies and the characteristic lesions on liver biopsies. Liver function tests and eosinophils returned within normal limits after 2 months of treatment with ursodeoxycholic acid, suggesting that its potential immunomodulatory actions may extend to eosinophils. Our report further supports the possibility that eosinophilia may occur in PBC, especially in its early stages. From the clinical point of view, we believe that PBC should be considered in the differential diagnosis of eosinophilia with an otherwise unknown origin. In particular, PBC should be suspected in a patient when other causes of eosinophilia have been excluded, irrespective of the presence or absence of symptoms, or the presence or absence of elevated ALP. In such cases further evaluation for anti-mitochondrial antibodies should be done. These observations might assist the development of future therapeutic concepts in the management of PBC, at least for patients in early stages of the disease.

Hemopoietic progenitor cells and bone marrow stromal cells in patients with autoimmune hepatitis type 1 and primary biliary cirrhosis.

BACKGROUND/AIMS: Autologous hematopoietic stem cell transplantation has been used in severe cases of autoimmunity. We investigated whether hemopoietic progenitor cells and/or bone marrow (BM) microenvironment are affected in autoimmune hepatitis type-1 (AIH-1) and primary biliary cirrhosis (PBC). METHODS: We studied 13 AIH-1 patients, 13 PBC patients, 12 cirrhotic controls (CC) and ten healthy controls (HC). Flow cytometry, expansion cultures, long-term BM cultures and clonogenic progenitor cell assays were used. Stromal cell function was assessed in long-term BM cultures recharged with normal CD34+ cells. RESULTS: AIH-1 had increased CD34+, CD34+/CD38+ and CD34+/CD38- cells compared to all groups (P<0.001). PBC had lower progenitor cells compared to controls (P<0.005). No differences were found between CC and HC. Committed progenitor cells in non-adherent cell fraction were increased in AIH-1 (P<0.05) but decreased in PBC compared to controls (P<0.05). Granulocyte-macrophage colony forming units (CFU) and erythroid-burst CFU were increased in AIH-1 compared to all groups (P<0.001). PBC had these CFUs decreased compared to controls (P<0.005). Stromal cells failed to support normal hemopoiesis in PBC. CONCLUSIONS: We demonstrated for the first time that AIH-1 had increased hemopoietic progenitor cells and normal stromal function. In PBC, progenitor cells and BM microenvironment were defective. Further studies will determine the significance of these novel findings.

Diabetes mellitus and lung function.

OBJECTIVES: To assess the nature of pulmonary dysfunction in type 1 diabetes and the relationship of pulmonary function tests to diabetic factors and complication. SUBJECTS AND METHODS: Sixteen type 1 diabetic patients and 26 control subjects matched for age and sex were studied. We performed spirometry measurements and measured pulmonary diffusing capacity (DL(CO)) in sitting and supine position by the single-breath method corrected by alveolar volume (VA). Glycosylated hemoglobin (HbA(Ic)), retinopathy and microalbuminuria were included as parameters of metabolic control and diabetic complications. RESULTS: Diabetic patients showed a significant reduction of the following pulmonary function tests (% predicted value) as compared with control subjects: total lung capacity (TLC, 92.6 +/- 14.5 vs. 113.9 +/- 17.5, p < 0.001), lung diffusing capacity in sitting position (DL(CO), 90.4 +/- 21.1 vs. 107.7 +/- 15.6, p = 0.004), lung diffusing capacity in supine position (DL(CO), 88.3 +/- 19.3 vs. 111.9 +/- 19.9, p = 0.001). The differences in diffusing capacity corrected by alveolar volume in sitting and supine position (DL(CO)/VA) were not significant. By changing the posture from sitting to supine position both diabetic patients and control subjects significantly increased DL(CO)/VA (103.4 +/- 17.7 vs. 112.7 +/- 22.3, p = 0.046 and 99.5 +/- 13.4 vs. 114.4 +/- 13, p < 0.001, respectively). There was no correlation between pulmonary function tests and diabetic complications. CONCLUSION: These data indicate that type 1 diabetic patients showed reduced TLC and DL(CO), features of pulmonary restrictive dysfunction. There was no correlation between abnormal pulmonary function and the presence of other diabetic complications.