Overproduction of cyclo-oxygenase-2 (COX-2) is involved in the resistance to apoptosis in vascular smooth muscle cells from diabetic patients: a link between inflammation and apoptosis.

AIMS/HYPOTHESIS: Inflammation is a common feature in cardiovascular diseases, including diabetes mellitus. In addition to the well-known inflammatory role of cyclo-oxygenase-2 (COX-2), this protein has also been implicated in apoptosis resistance in tumour cells. Vascular smooth muscle cells (VSMC) from diabetic patients are also resistant to apoptosis because of an increased abundance of B cell lymphoma 2 protein (BCL2). In this work, we investigated whether overproduction of COX-2 was involved in the resistance to apoptosis in VSMC from diabetic patients. METHODS: VSMC were obtained from internal mammary arteries from patients who had undergone coronary artery bypass graft surgery. Apoptosis was measured by DNA fragmentation, BCL2 degradation and cytochrome c release. RESULTS: Apoptosis induced by C-reactive protein in cells from non-diabetic patients was mediated by COX-2. VSMC from diabetic patients showed higher basal levels of COX-2 compared with those from non-diabetic patients. Transfection of VSMC from non-diabetic patients with a plasmid containing COX-2 (also known as PTGS2) increased basal production of COX-2 and BCL2 and mimicked the resistance to apoptosis that occurs in diabetic patients. We also found a significant correlation (R = 0.846, p = 0.016) between COX-2 and BCL2 production in arterial rings from diabetic patients measured by confocal microscopy. However, inhibition of COX-2 production by small interfering RNA proved unable to reverse BCL2 production in diabetic VSMC. CONCLUSIONS/INTERPRETATION: These results suggest a link between inflammation (COX-2) and apoptosis resistance (BCL2) in the arteries of diabetic patients. This relationship is not causative and the common production of these two proteins may be co-regulated by shared regulatory elements in diabetes.

Antimicrobial efficacy in vivo of a new formulation of 2-butanone peroxide in n-propanol: comparison with commercial products in a cross-over trial.

The use of hand rub to obtain maximum decrease in bacterial load is important because the reduction needed to avoid transmission is unknown. The monomer of 2-butanone peroxide is a peroxygen derivative with potential biocidal use in hospitals. The aim of this study was to compare the efficacy of hand rub with an alcoholic solution of peroxide 2-butanone versus five antiseptic products, against E. coli K12 (CECT 433) transient flora acquired by hand immersion in a broth culture following the UNE-EN-1500 standard. Isopropanol 60% (control) obtained 99.99% reductions, driving down the bacterial load from 10(6) cfu/mL in the initial inocula to <100 cfu/mL. Products A, B and C (different alcoholic solutions ranging from 65% to 75% with low amounts of biguanidines and/or quaternary ammonium compounds) resulted in significantly lower amounts, reducing initial inocula to approximately 500 cfu/mL. Products D and E (70-75% alcohol solutions containing higher amounts of different quaternary ammonium compounds and triclosan in the case of product E) produced reductions similar to that of isopropanol, with significantly larger reductions than products A, B and C. The product with the solution of 2-butanone peroxide produced the same effect as products D and E with mean reductions of approximately 4log(10) (99.99%), driving the initial inocula down to < or = 100 cfu/mL, despite the low concentration (35%) of propanol in the solution. This novel peroxygen biocide offers high in-vivo cidal activity against acquired E. coli transient flora, offering an alternative to products with higher alcohol concentrations.

Assessing the residual antibacterial activity of clinical materials disinfected with glutaraldehyde, o-phthalaldehyde, hydrogen peroxide or 2-bromo-2-nitro-1,3-propanediol by means of a bacterial toxicity assay.

This study investigated the use of a rapid bacterial toxicity test for detecting disinfectant residues released by disinfected materials. The test substances included an environmental disinfectant used in hospitals in high-risk areas, such as critical care units or emergency services, and three disinfectants used on clinical devices when a high level of disinfection is required. The test materials were polyurethane, polypropylene, glass, latex and cotton from different instruments and utensils used in hospitals. Of the four test disinfectants, o-phthalaldehyde (OPA) and 2-bromo-2-nitro-1,3-propanediol (BNP) showed the greatest inhibitory activity (as much as 300-fold greater than hydrogen peroxide in the case of OPA) according to the toxicity text. However, with the exception of hydrogen peroxide on latex, it was the most porous test materials, namely latex and cotton, that accumulated the least residue. BNP was the disinfectant that left the least residue on the five test materials, while the greatest residual concentration was left by hydrogen peroxide on latex (as much as 5 microg/cm2). The biotest used in this study permitted the detection of disinfectant residues released by different types of previously disinfected clinical materials, and can be adapted to simulate elution conditions similar to those existing in routine hospital practice.

Early and delayed consolidation chemotherapy significantly improves the outcome of children with intermediate risk acute lymphoblastic leukemia. Final results of the prospective randomized PETHEMA ALL-89 TRIAL.

BACKGROUND AND OBJECTIVES: To evaluate the impact of early and delayed consolidation chemotherapy on the outcome of children with acute lymphoblastic leukemia (ALL) stratified according to risk groups. DESIGN AND METHODS: From 1989 to 1994, 195 children (< or = 15 years old) diagnosed as having ALL (ALL-L3 excluded) in 15 Spanish hospitals entered the prospective, randomized PETHEMA ALL-89 trial. Patients were stratified into low-risk (LR), intermediate-risk (IR) and high-risk (HR) groups according to their initial features and the rate of response to induction therapy. LR-ALL patients were randomized to receive or not early consolidation chemotherapy (C-1). After receiving C-1, IR patients were randomized to receive or not delayed consolidation chemotherapy (C-2). HR patients received C-1 and C-2 chemotherapy. Standard maintenance chemotherapy was administered to all patients for 2 years. High doses of intravenous methotrexate and 12 triple intrathecal doses were given as prophylaxis against central nervous system (CNS) disease. RESULTS: The mean (and standard deviation) age was 6 (4) years and 120 patients were males. Fourteen patients had early pre-B-ALL, 149 common or pre-B-ALL, and 32 T-ALL. Complete remission (CR) was attained in 189 patients (97%), 11 of whom (6%) had a slow response. Risk group stratification after CR was: LR 89, IR 50 and HR 56 patients (including a subset of 26 patients at very high risk). Ten-year event-free survival (EFS) and overall survival (OS) probabilities for the whole series were 58% (95% CI: 52-64%) and 69% (61-77), respectively, with a median follow-up of 8.7 years. Dividing the patients according to risk group, the 10-year EFS and OS probabilities in the LR group were 71% (63-79) and 86% (80-92), respectively; in the IR group 69% (57-81) and 76% (64-88), respectively, and in the HR group 30% (18-42) and 44% (32-57), respectively. For LR patients receiving C-1, EFS and OS were 79% (57-92) and 90% (82-98), respectively, versus 62% (48-76) and 66% (51-81) in patients not receiving C-1 (p= 0.06). For IR patients, EFS and OS were significantly improved in those receiving early and delayed consolidation (EFS 87% (74-88) vs. 52% (41-70), and OS 92% (87-97) vs. 61% (51-71)(p=0.036). Prognostic factors for EFS identified in multivariable analyses were: age >10 years in the LR group (OR 3.5, 95% CI 1.3-9.5, p=0.01), and treatment with C-2 in IR patients (OR 5.0, 95% CI 1.4-17.8, p=0.01). The CNS relapse rate was 4% for all the series (including the HR subset). Tolerance to treatment was good. INTERPRETATION AND CONCLUSIONS: In this study, early consolidation seemed to improve the prognosis of children with LR-ALL, but differences in EFS were not significant. Delayed consolidation had a favorable influence on the outcome of IR-ALL. CNS preventive treatment without cranial irradiation was effective in all the groups of ALL patients.

Increased conventional chemotherapy does not improve survival in multiple myeloma: long-term results of two PETHEMA trials including 914 patients.

BACKGROUND: Melphalan and prednisone (MP) has been the standard treatment for multiple myeloma (MM) for the last 30 years. Combination chemotherapy at conventional doses has not shown a significant prolongation of survival when compared to MP. There are few data comparing conventional chemotherapy at standard doses with conventional treatment at higher doses. We present the long-term outcome of 914 patients from two randomized trials comparing three different dose intensity regimens. METHODS: From 1 January, 1985 to 31 December, 1989, 487 patients were randomized between MP (melphalan 9 mg/m(2) p.o. and prednisone 60 mg/m(2) days 1-4) and alternating VCMP (vincristine 1 mg i.v. on day 1, cyclophosphamide 500 mg/m(2) i.v. on day 1, melphalan 6 mg/m(2) p.o. on days 1-4, and prednisone 60 mg/m(2) on days 1-4) and VBAP (vincristine 1 mg i.v. on day 1, BCNU and doxorubicin 30 mg/m(2) i.v. each on day 1, and prednisone 60 mg/m(2) on days 1-4). From 1 January, 1990 to 31 May, 1994, 427 patients were randomized between VCMP/VBAP at the above detailed doses (VCMP/VBAP 'SD') and the same regimen increasing the doses of cyclophosphamide and doxorubicin from 500 to 1200 mg/m(2) and from 30 to 50 mg/m(2), respectively (VCMP/VBAP 'HD'). RESULTS: Increasing dose intensity produced a significantly higher partial response rate (31% vs 45% vs 51% for MP, VCMP/VBAP 'SD', and VCMP/VBAP 'HD', respectively; P < 0.01). However, a significantly early death rate was observed in the HD arm (7.7, 7.5 and 12.1% for MP, VCMP/VBAP 'SD', and VCMP/VBAP 'HD', respectively; P = 0.05). Median duration of response (20 vs 18 vs 19 months for MP, VCMP/VBAP 'SD', and VCMP/VBAP 'HD', respectively; P = NS) and median survival (25 vs 31 vs 29 months for MP, VCMP/VBAP 'SD', and VCMP/VBAP 'HD', respectively; P = NS) were similar in the three groups. MP produced a higher degree of thrombocytopenia than combination chemotherapy at standard (P = 0.002) or high dose (P = 0.01), this leading to a significantly higher dose reduction in the MP arm (P < 0.001 and P = 0.003 for VCMP/VBAP 'SD' and VCMP/VBAP 'HD', respectively). CONCLUSION: In these trials the response rate significantly correlated with the regimen intensity. However, no significant differences in response duration and survival were found. This highlights the limited role of conventional chemotherapy in MM and the need for further trials, aimed at determining the impact of new treatment approaches such as high-dose therapy/autotransplantation.

Maintenance treatment with interferon alpha-2b in multiple myeloma: a prospective randomized study from PETHEMA (Program for the Study and Treatment of Hematological Malignancies, Spanish Society of Hematology).

The objectives of the present study were to investigate whether interferon alpha (IFN) maintenance could prolong response duration and survival in patients with multiple myeloma (MM) in objective response and to analyze the characteristics of relapse and subsequent survival. From January 1991 to November 1994, 92 patients from the Spanish Cooperative Group PETHEMA with MM in objective response after 12 courses of VCMP/VBAP chemotherapy were randomized to receive IFN maintenance vs no treatment until relapse. Prognostic factors at diagnosis were similar in both groups. IFN was administered at a starting dose of 3 mU/m2 three times per week. The IFN toxicity was moderate with granulocytopenia and fatigue being the most common adverse effects. Median duration of response from randomization until relapse was 13 months in the IFN group vs 7.7 months in the no treatment arm (P = 0.042). Median survival from randomization was 38.8 months for patients given IFN vs 32.7 months for those allocated to the no treatment arm (P = 0.12). Features at relapse were similar in patients who received IFN maintenance and in those assigned to no treatment. Finally, survival from relapse was identical in both groups. In summary, our results show a significant prolongation of response in patients maintained with IFN with no significant influence on survival. In addition, in our series features at relapse and subsequent outcome were similar in both groups.

Evaluation of the prevalence of sarcoptic mange in slaughtered fattening pigs in southeastern Spain.

In this study the prevalence of sarcoptic mange in fattening pigs in Murcia, southeastern Spain was investigated. Results showed that 37% of the 1318 slaughtered pigs examined were positive for Sarcoptes scabiei var. suis. Skin lesions potentially attributable to this mite were present in 92.80% of animals, but the parasite could be detected in only 38.60% of them. The condition of the ear (clean/dirty) was not a defining characteristic for the diagnosis of this swine disease. A sucrose flotation-concentration technique was more effective than direct microscopy in finding the mite in ear scrapings.

A trial to compare the effects of pH, buffer concentration, and NaCl, on one fluorescent and two bioluminescent bacterial tests for acute toxicity.

Of all the bioassays to determine acute toxicity described in the literature, those that employ bacteria as indicator organisms are usually the most rapid and the most economic, although alone they cannot predict the possible toxic effect of any type of substance. When bioassays are employed to test the toxicity of known substances and of compounds in samples from waste discharges they have to work in very different conditions from those for which they are designed. The effects of three factors, pH, buffer concentration, and NaCl, on the performance of a fluorogenic bioassay based on the beta-glucuronidase activity of Escherichia coli were investigated. The results of this test were compared with those of two known biluminescent bacteria tests. The fluorogenic bioassay has a more restricted optimum pH range, while the influence of buffer concentration was similar for the three tests. E. coli glucuronidase activity was affected at a concentration as low as 128 mg/l of NaCl. Changes in the pH or buffer concentrations or chloride ions, greatly influenced the respectives toxicities of four substances, acridine orange, TEMED, 2-mercaptoethanol, and mercuric chloride.

Analysis of natural killer-associated antigens in peripheral blood and bone marrow of multiple myeloma patients and prognostic implications.

The aim of this study was to analyse the expression of NK-associated antigens in both peripheral blood and bone marrow lymphocytes from a large series of newly diagnosed multiple myeloma patients. 112 patients with untreated multiple myeloma (MM) were included in the study. 36 sex- and age-matched healthy volunteers were used as controls for peripheral blood (PB) studies and 14 for the bone marrow (BM) studies. Simultaneous stainings with the CD3/CD56, CD2/CD16 and CD8/CD57 monoclonal antibodies were systematically performed in PB and CD3/CD56 and CD2/CD16 in BM in order to analyse their relationship with the clinical and biological characteristics of the disease and survival. The expression of NK-associated antigens (CD56, CD16 and CD57) assessed within the lymphoid gate, was significantly increased (P < 0.001) in the PB of MM patients both in relative and absolute numbers. In the BM a significant increase in the percentage of CD56+ lymphocytes (P < 0.001) was also observed; in contrast, the proportion of CD16+ cells did not differ significantly from that of normal BM samples. The number of CD56+CD3- lymphocytes increased significantly within high-risk patients (869 +/- 671) as compared to intermediate (388 +/- 212) and low-risk patients (274 +/- 199) (P = 0.04). Moreover, patients with high values of CD56+CD3- lymphocytes showed a statistically significant association with several adverse prognostic factors including anaemia, hypoalbuminaemia, renal failure, high beta 2M, DNA diploidy and high S-phase plasma cells. In addition, patients with higher absolute numbers of PB CD56+CD3-lymphocytes displayed a poorer prognosis, whereas patients with higher values of CD57+CD8- cells had a better outcome.

Prognostic implications of DNA aneuploidy in 156 untreated multiple myeloma patients. Castelano-Leonés (Spain) Cooperative Group for the Study of Monoclonal Gammopathies.

In this study the incidence of DNA aneuploidy in a large series of untreated multiple myeloma (MM) patients was assessed in order to determine its clinical and prognostic significance. A total of 156 MM patients were included in the study. DNA measurements were performed in all cases at diagnosis using two different flow cytometry methods: (1) propidium iodide (PI) staining on isolated nuclei, and (2) CD38/PI double staining on whole cells. The DNA ploidy status was correlated with the most relevant clinical and haematological disease characteristics. From the 156 cases analysed, 91 (58%) were aneuploid (56% hyperdiploid and 2% hypodiploid). The correlation between the two techniques on the detection of DNA aneuploidy was excellent, although CD38/PI double staining would be preferable in cases with < 5% of DNA aneuploid plasma cells (PC). Upon comparing the clinical and haematological disease characteristics of hyperdiploid versus diploid cases, the former group was characterized by a lower age, reduced incidence of anaemia, lower beta 2M levels, higher proliferative activity within the residual normal haemopoietic cells, increased expression of CD56 antigen in PC, and higher proportion of PB CD4+ T cells. In contrast, diploid cases had a higher expression of the CD10, CD20 and CD15 antigens and greater numbers of PB CD56+CD3- NK cells (P < 0.05). Circulating PC were identified in six cases, all being diploid. Overall survival was significantly longer in hyperdiploid compared to diploid MM (P = 0.02). These results show that over 50% of MM patients are aneuploid, almost all of them being hyperdiploid. This characteristic is associated with better prognosis.

Evaluation of the toxicity of several heavy metals by a fluorescent bacterial bioassay.

This new bioassay determined the toxicity of chemical compounds dissolved in water by measuring the degree of inhibition of the ultraviolet light-stimulated fluorescence of Escherichia coli in a culture medium in which 4-methylumbelliferyl beta-D-glucuronide was the only carbon source. Inhibition produced by one of five heavy-metal salts (Cd2+, Cr6+, Hg2+, Pb2+ or Zn2+) was the end-point and comparison standard to determine the EC50 and minimum effective concentration (MEC) that produced a decrease of E. coli growth rate, increased doubling time and percentage inhibition and reduced numbers of generations; all these values were derived from the fluorescence signals. Only Cr6+ and Hg2+ at two concentrations (0.25 and 0.5 mg l-1) almost completely inhibited this E. coli strain. All toxicant concentrations tested produced at least partial inhibitions of growth; Cr6+, Hg2+ and Cd2+, in that order, were most toxic, and Pb2+ the least. Zn2+ gave higher EC50 values at 3 h of incubation than at 4 h. The method was simple, rapid and inexpensive and would permit a large number of samples to be tested quickly.

A new staging system for multiple myeloma based on the number of S-phase plasma cells.

In the present study, we analyzed the cell cycle distribution of bone marrow (BM) cells in 120 untreated multiple myeloma patients using a DNA/CD38 double-staining technique at flow cytometry in which plasma cells (PCs) can be clearly discriminated from residual BM cells based on their CD38 expression. This approach allows us to determine the proliferative activity of both PCs and residual normal BM cells. The percentage of S-phase cells in the myelomatous population was found to be significantly lower than that of the residual normal BM cells (P < .001). Regarding the proliferative activity of myelomatous cells, patients with a high number of S-phase PCs (> 3%) showed a significantly (P < .05) increased incidence of anemia and hypercalcemia; higher values of beta 2-microglobulin (beta 2M), urea, and creatinine; and higher numbers of peripheral blood natural killer cells, as well as a poor prognosis as assessed both by response duration and overall survival. With respect to the residual BM normal fraction, a low proliferative activity was significantly (P < .05) associated with the presence of anemia and neutropenia together with increased numbers of BM PCs, a higher incidence of Bence Jones myelomas, and DNA diploidy. Multivariate analysis showed that the number of S-phase PCs was the most important independent prognostic factor, allowing us to discriminate two subgroups of patients with different prognoses, even within the same clinical stage. Moreover, the S-phase PCs, together with beta 2M, age, and performance status, represent the best combination of disease characteristics for stratifying patients according to prognosis and allow the establishment of a simple and powerful staging system for multiple myeloma patients. In addition, this classification can be used for planning treatment in patients who are candidates for transplantation.

New toxicity determination method that uses fluorescent assay of Escherichia coli.

We describe a new method that uses a fluorogenic bioassay of the beta-glucuronidase conversion of 4-methylumbelliferyl beta-D-glucuronide (MUG) to 4-methylumbelliferone to evaluate the individual toxic effects on Escherichia coli of Al3+, Cr6+, Hg2+ and Li+. This work was designed to examine the effectiveness of this method to measure the effects of five ionic concentrations of either Al3+, Cr6+, Hg2+ or Li+, on the growth of E. coli in a minimal medium that had MUG as the only source of carbon. This method was simple and fast, and its toxicity detection sensitivity was equal to, or greater than, existing bacterial bioassays. The use of the MUG substrate minimized the danger of interference by bacteria other than E. coli. Evaluations of toxicity in samples of public drinking water proved equally sensitive.

Genetic stability of African swine fever virus grown in monkey kidney cells. Brief report.

Viral DNA subpopulations were produced when the ASFV was grown in monkey kidney MS cells. They were detected after 44 passages but not during the first 14 passages or in the unadapted ASFV E 70 strain grown in pig leukocytes. Different viral variants were isolated and their genomes were characterized. Restriction enzyme site variations were detected in both terminal fragments, Cla I-M and Sal I-F, and in the internal fragments Clal-O and Sma I-H. These variations result in changes in the size of the viral genome which ranges from 156 Kbp to 170 Kbp.

African swine fever virus DNA: deletions and additions during adaptation to growth in monkey kidney cells.

Restriction enzyme cleavage maps for the fragments produced by Cla I, Sal I and Sma I have been constructed for African swine fever virus (ASFV) DNA grown in pig leukocytes (strain E70 L6) and after adaptation to growth in MS monkey kidney cells (strain E70MS14). The mapping data revealed that before adaptation to growth in MS cells, the size of the DNA from ASFV strain E70 L6 was l73 Kbp and after adaptation it was only l56 Kbp. The decrease in size was produced by deletions and additions mainly in the terminal regions of the genome. These genetic variations were located between 0.0 to 0.01 m.u. (Cla I-M1 fragment), 0.04 to 0.14 m.u. (Sma I-B1, Sal I-A1 fragments), 0.51 to 0.52 m.u. (Cla I-O fragment), 0.84 to 0.86 m.u. (Sma I-H1), 0.95 to 0.97 m.u. (Cla I-A1, Cla I-G1 fragments) and 0.99 to 1.0 m.u. (Cla I-G1) on viral genome of ASFV grown in pig leukocytes.

Neutralization of African swine fever virus by sera from African swine fever-resistant pigs.

Sera from African swine fever-resistant pigs with infection-inhibitory activity decreased virus replication in infected porcine buffy coat cultures. This same effect was observed even after virus was adsorbed. The infection-inhibition was not reversed by removing the immune serum from the assay cultures. Reduction of African swine fever virus replication by immune sera was demonstrated by fluorescent focus assay on MS cell line cultures. Virus-neutralization tests showed a persistent fraction of non-neutralized virus, which was not demonstrable by infection-inhibition tests. One hypothesis for explaining this difference is proposed.