Vascular endothelial growth factor polymorphisms in mantle cell lymphoma.

In this study, we determined the allele and genotype frequencies of vascular endothelial growth factor (VEGF) G+405C, C-460T, C+936T and C-2578A single nucleotide polymorphisms (SNPs) in 32 patients affected by mantle cell lymphoma (MCL) and 58 healthy controls. Real-time PCR combined with melting curve analysis was used for the determination of SNP alleles. A significant difference in the allele frequency of VEGFC-460T and C+936T SNPs in MCL and healthy cases was not observed. On the contrary, VEGF G+405C and C-2578A SNP allele distribution was significantly lower in the patient group than among normal controls (p = 0.014, p = 0.001). This observation suggests that further investigation is warranted, both in vitro and in a larger series of patients, to further examine the role of VEGF polymorphisms in the pathogenesis of MCL. In addition, the use of quantitative real-time PCR combined with a melting curve analysis method in the detection of the 4 VEGF SNPs may have the potential to replace older and more time-consuming PCR-RFLP methods and bears further investigation.

Association of B-chronic lymphocytic leukemia and acute myeloid leukemia.

A 62-year-old man presented with fatigue, pallor and mild weight loss. Laboratory studies showed Hb 7.6 g/dl, Hct 21.8%, WBC 108x10(9)/1, PLT 143x10(9)/1. At morphological examination, circulating cells appeared as 60% blasts and 40% lymphocytes, with smudge cells. A bone marrow aspirate showed infiltration by blasts (50%) and lymphocytes (40%); alpha-naphtyl-acetate esterase was positive in 90% of blasts, while myeloperoxidase was positive in 10%. The immunologic phenotype of blasts was characterized by the co-expression of CD13, CD33, CD14, CD4, CD15, CD64, CD117, HLA-DR, CD11b. Lymphocytes were characterized by a B-CLL immunophenotype: CD19+, CD5+, CD23+, CD20+(dim), FMC7+(dim), K light chain+(dim). Karyotype was normal and PCR assays for AML-ETO, CBFbeta-MYH11, PML-RARalpha, BCR-ABL and bcl-1/JH translocation were negative. Coexistence of CLL and AML with monoblastic features was diagnosed. Simultaneous appearance of CLL and AML has rarely been described and represents a peculiar biological phenomenon.

Clinical and microbiological efficacy of colistin therapy alone or in combination as treatment for multidrug resistant Pseudomonas aeruginosa diabetic foot infections with or without osteomyelitis.

We retrospectively evaluated the safety and effectiveness of colistin alone or in combination with other antimicrobials in eight diabetic patients with severe diabetic foot infections due to multidrug resistant (MDR) Pseudomonas aeruginosa, complicated in 4 cases by osteomyelitis. All patients received colistin after other ineffective antimicrobial treatment, when MDR P. aeruginosa strains were isolated by cultural examination and together with a multidisciplinary care approach including revascularization, surgical debridement and adequate offloading. The mean duration of therapy was 72 +/- 52.9 days. Six out of 8 patients (75%) successfully benefited from colistin therapy, while 2 patients failed and/or experienced side effects that led to discontinuation of therapy. Serious adverse events (i.e. acute renal failure and pulmonary edema) were observed in 1 patient. Our data allow us to conclude that colistin, alone or in combination with other antimicrobials, is safe and effective when administered as part of a multidisciplinary approach, to promote healing of diabetic foot infection due to MDR P. aeruginosa.

Clinical efficacy of intravenous colistin therapy in combination with ceftazidime in severe MDR P. aeruginosa systemic infections in two haematological patients.

Nosocomial infections due to MDR P. aeruginosa are an increasing problem. Therapeutical options are few. We describe two haematological patients with severe neutropenia and systemic infection due to MDR P. aeruginosa treated successfully with colistin plus ceftazidime. Severe adverse events were not described.

Autografting followed by nonmyeloablative immunosuppressive chemotherapy and allogeneic peripheral-blood hematopoietic stem-cell transplantation as treatment of resistant Hodgkin's disease and non-Hodgkin's lymphoma.

PURPOSE: To investigate the use of a nonmyeloablative fludarabine-based immunosuppressive regimen to allow engraftment of HLA-sibling donors' mobilized stem cells and induction of a graft-versus-lymphoma effect for patients with advanced resistant Hodgkin's disease and non-Hodgkin's lymphoma. PATIENTS AND METHODS: Fifteen patients with Hodgkin's disease (n = 10) and non-Hodgkin's lymphoma (n = 5) were studied. All patients received cyclophosphamide and granulocyte colony-stimulating factor to mobilize autologous hematopoietic stem cells (HSCs). Subsequently, they received high-dose therapy with carmustine, etoposide, cytarabine, and melphalan and reinfusion of HSCs. At a median of 61 days after engraftment, patients were given fludarabine 30 mg/m(2) with cyclophosphamide 300 mg/m(2) daily for 3 days. Donor-mobilized HSC collections were prepared for fresh infusion and were not T-cell depleted. Methotrexate and cyclosporine were used to prevent graft rejection and as graft-versus-host disease (GVHD) prophylaxis. RESULTS: Combined treatment was well tolerated. After mini-allografting, hematologic recovery was prompt. Thirteen patients had 100% donor cell engraftment. Eleven patients achieved complete remission (CR) after the combined procedure. Nine patients, who were in partial remission after autografting, achieved CR after mini-allografting. Seven patients developed >/= grade 2 acute GVHD (aGVHD) and two developed extensive chronic GVHD (cGVHD). Three patients who received the highest number of donor lymphocyte infusions (DLIs) developed grade 3 GVHD (two patients) and extensive cGVHD (one patient). Ten patients are currently alive, and five are in continuous CR. Seven patients received DLI, with five CRs. Five patients died: one of progressive disease, two of progressive disease combined with aGVHD or cGVHD, one of extensive cGVHD, and one of infection. CONCLUSION: Fludarabine/cyclophosphamide was well tolerated and allowed consistent engraftment in lymphoma allografted patients. Response rates were high in this group of refractory and heavily pretreated patients. This dual procedure seems to be most promising in patients with end-stage malignant lymphomas.

The relevance of secondary radicals in the mode of action of anthralin.

The formation of free radicals during the reaction of anthralin analogues with peroxidizing polyunsaturated lipids was monitored by ESR spectroscopy. The biological effect of the different compounds was assessed by their ability to inhibit respiration of cultured human keratinocytes. C(10)-monosubstituted analogues of anthralin exhibited a strong antirespiratory effect and produced a cascade of radicals. Abstraction of the hydrogen atom at C(10) led to the generation of primary radicals which further decomposed into secondary radicals similar to those observed with anthralin itself. 10, 10'-disubstituted analogues of anthralin did not form any paramagnetic species during reaction with peroxidizing lipids while decomposition of a 2,7-disubstituted anthralin derivative under the same conditions resulted in primary, but not secondary radical species. Since both types of disubstituted analogues are devoid of antirespiratory activity we postulate that the antimitochondrial and thus antiproliferative activity of anthralin and its analogues is associated with their capacity to form secondary radicals during their decomposition.

Radical intermediates and antioxidants: an ESR study of radicals formed on carnosic acid in the presence of oxidized lipids.

Carnosic acid, an antioxidant extracted from rosemary, is shown to produce radicals when in contact with oxidized methyl oleate in the absence of air above 50 degrees C. Two radical species are formed: the first one, stable up to approximately 110 degrees C, is an hydroxy-phenoxy radical whose ESR spectrum was analyzed by studying its temperature dependence and its sensitivity to deuterium/proton exchange. The second species was observed above 110 degrees C, its ESR spectrum was identical to the spectrum obtained when carnosol, another antioxidant extracted from rosemary, was heated at the same temperature in the presence of oxidized lipid. This observation is probably due to the transformation of carnosic acid into carnosol; the analysis of the corresponding ESR spectrum suggests the formation of a keto phenoxy radical exhibiting a great delocalization of the unpaired electron.

Radical exchange reactions between vitamin E, vitamin C and phospholipids in autoxidizing polyunsaturated lipids.

Antioxidant reactions of mixtures of vitamin E, vitamin C and phospholipids in autoxidizing lipids at 90 degrees C have been studied by ESR spectroscopy. When the phospholipid contained a tertiary amine (e.g. phosphatidylcholine), the vitamin C and the vitamin E radicals were successively observed as these two vitamins were sequentially oxidised during lipid oxidation. In the presence of the primary amine contained in phosphatidylserine, the vitamin E oxidation was delayed for a few hours. In this case neither the vitamin C, nor the vitamin E radicals but a nitroxide radical derived from the phospholipid was observed. Similar results to those obtained with PS were obtained in the presence of either phosphatidylethanolamine or soybean lecithin. The participation in the radical reactions of phospholipids possessing a primary amine can therefore explain the synergistic effect of these phospholipids in a mixture of vitamins E and C.

The fate of antioxidant radicals during lipid autooxidation. I. The tocopheroxyl radicals.

Peroxidizing lipids were used to induce the formation of antioxidant radicals. It has been shown by electron spin resonance (ESR) spectroscopy and simulation of the first derivative ESR spectra that the radicals formed by this method are the already known tocopheroxyl radicals. dl-alpha-Tocopheroxyl radicals were formed in relatively high concentration but were rather rapidly destroyed as compared to the dl-delta-tocopheroxyl radicals, which were formed in rather low concentration and were destroyed rather slowly, dl-beta- and dl-gamma-tocopheroxyl radicals reacted in an intermediate way. Autooxidation induction times of the same lipids stabilized with the tocopherols show the well accepted series of antioxidant activities alpha less than beta congruent to gamma less than delta. Their relative antioxidant activity is nicely explained by the ESR experiment: the fast reacting dl-alpha-tocopherol is reacting more rapidly and traps the radicals more thoroughly and is therefore only available as an antioxidant for a short period of time as compared with the slowly reacting dl-delta-tocopherol. dl-beta- and dl-gamma-Tocopherols behave in an intermediate way.

Role of hydroxyl radicals and singlet oxygen in the formation of primary radicals in unsaturated lipids: a solid state electron paramagnetic resonance study.

Primary radicals were generated by UV photolysis of samples of trilinolein, at 77 K and under a controlled atmosphere. The resulting EPR spectra clearly show that the amount of radicals is dependent on the purity of the lipid, the exposure to visible light in the presence of a photosensitizer and oxygen, and, finally, the presence of an antioxidant. These solid state EPR experiments indicate that if all of the elements for the production of singlet oxygen (Rose Bengal, molecular oxygen, and visible light) are not present, primary radicals are practically not generated. They also point out the various steps of the oxidation mechanism: formation of singlet oxygen, which reacts with the lipid to form a hydroperoxide; and photolytic formation of the hydroxyl radical, which reacts with the frozen lipid to generate primary lipidic radicals. This constitutes a new method for investigating lipid oxidation and studying the influence of photosensitizers and molecules that are likely to react with singlet oxygen.

Plasmapheresis treatment in Guillain-Barré syndrome: potential benefit over intravenous immunoglobulin.

Guillain-Barré syndrome includes acute inflammatory demyelinating polyradiculoneuropathy, acute motor axonal neuropathy, acute motor and sensory axonal neuropathy, Miller Fisher syndrome and acute pandysautonomia. Plasma exchange was the first treatment in Guillain-Barrd syndrome proven to be superior to supportive treatment alone and intravenous immunoglobulin was subsequently shown to be equally effective and is now commonly used as first-line treatment. We describe a 78-year-old woman who presented with a two-day history of progressive generalised weakness and left facial nerve palsy, preceded by a flu-like illness lasting for one week. A five-day course of daily immunoglobulin (0.4 g/kg/day) was commenced without benefit and progressive clinical deterioration. Seven days after completion of immunoglobulin treatment, plasma exchange was started with an exchange of about three litres of plasma every day for three days and every second day on two further occasions. A gradual improvement of respiratory function and peripheral muscle strength was observed after the first plasma exchange and on the eighth day the patient was weaned off mechanical ventilation. This case suggests that patients with severe Guillain-Barrd syndrome may benefit from plasma exchange after immunoglobulin treatment in refractory cases. Plasma exchange should be considered early in Guillain-Barrc syndrome cases with axonal involvement, and in the recurrent or familial Guillain-Barré syndrome forms.

Shortened and intensified MJMA: an effective salvage therapy for relapsed and refractory lymphomas and a strong mobilizer of PBSCs.

There is great interest in chemotherapies for relapsed or refractory lymphomas that are both directly effective against the lymphoma and able to mobilize PBSCs for rescue after high-dose chemotherapy (HDC). Twenty-eight patients with relapsed or refractory lymphomas were treated with a shortened, intensified MJMA regimen (mitoxantrone 10 mg/m(2) i.v. day 1, carboplatin 200 mg/m(2) i.v. days 1-2, methylprednisolone 500 mg/m(2) i.v. days 1-3, cytarabine 2000 mg/m(2) i.v. day 3) for six cycles every 21 days. A median of five cycles/patient was administered. Nineteen patients had complete responses, seven partial responses and two no responses. The only remarkable toxicity was hematological. In 18 patients who were candidates for HDC, a mean of 10.45 x 10(6) CD34/kg of patients' body weight was collected (range: 3.70-24.88 x 10(6)/kg). Eleven patients underwent transplantation, which converted two of four partial responses into complete responses. The median follow-up was 49 months. Survival parameters were not related to relapsed/refractory status or to the time from the last chemotherapy, but were related only weakly to the number of prior chemotherapies. Outpatient MJMA is a feasible and very effective salvage chemotherapy per se. The complete response rate is high and it is a powerful PBSC mobilizer.

Three-times weekly teicoplanin as outpatient treatment of chronic osteoarticular infections.

Treatment of difficult-to-treat infections such as osteomyelitis or infections related to indwelling medical devices requires lengthy antibiotic therapy and adequate surgical debridement. Teicoplanin, a glycopeptide antibiotic with a long half-life, was used three-times weekly in the treatment of these infections. After a period of daily dosing with teicoplanin, patients were treated with an intravenous dose of 12 mg on mondays, wednesdays and fridays. A control group of patients were treated with teicoplanin daily. Teicoplanin levels were measured during the study. Thirty-six patients were enrolled in the study: 14 with vertebral osteomyelitis, 12 with infected orthopedic implants, 7 with osteomyelitis and 3 with arterial prosthetic infections. The duration of treatment ranged from 60 to 360 days. Cure was obtained in 21 (58%) patients and improvement in 15 (42%) patients. Trough and peak serum concentrations in three-time weekly patients were 16.2+/-7.2 mg/l and 58.7+/-14.4 mg/l. In the control group trough and peak serum concentrations were 18.9+/-13.6 mg/l and 52.2+/-27 mg/l. Adverse events occurred in 6 patients: mainly mild liver toxicity. Three times weekly teicoplanin seems to be a valuable option in the treatment of chronic infections.

The role of dose size in a chemotherapy regimen (ProMECE-CytaBOM) for the first-line treatment of large B-cell lymphomas: a randomized trial by the Gruppo Italiano Studio Linfomi (GISL).

BACKGROUND: It is still unclear the actual contribute of dose intensity (DI), dose size (DS) and dose density (DD) in the conventional chemotherapy of large, B-cell non-Hodgkin lymphomas. METHODS: A prospective, randomized trial compared the cyclic schedule of ProMECE-CytaBOM chemotherapy (cyc-PC, 6 cycles) with a modified version of it, which administered the same drugs sequentially (seq-PC), with the same planned cumulative DI and an 83% DD, within the same time frame (113 days), but with three times higher DS of all the drugs except vincristine. RESULTS: Fifty-six patients received cyc-PC and 52 seq-PC. The actual mean cumulative DI was 0.79 +/- 0.15 with cyc-PC, 0.78 +/- 0.17 with seq-PC. Response was complete in 59% and 52%, partial in 20% and 21%, null in 5% and 6%, respectively. There were four toxic deaths (two per arm). Relapses occurred in 36% and 37%, respectively. Toxicity was similar in both arms. Overall, failure-free, progression-free and disease-free survival (median follow-up: 54 months) were statistically indifferent. CONCLUSIONS: The very similar DI actually delivered in both arm seems to be the main common determinant of the indifferent results recorded. Increasing DS--at least within the limits clinically attainable without stem cell rescue--does not improve results.

Phase of second-harmonic light self-generated in a glass fiber.

Focusing an intense laser beam and its second harmonic into a glass fiber transforms the fiber into a frequency doubler. We measure the temporal evolution of both the amplitude and the phase of the second-harmonic light produced by a germanium-doped fiber and so determine the initial phase of the second-harmonic light to be Deltatheta= -71 degrees +/- 3 degrees . We demonstrate that the fiber-produced green light can exceed the seeding green light even if these two beams are 90 degrees out of phase. We also show that cross-phase modulation in the fiber can limit the maximum useful interaction length and consequently the ultimate efficiency of second-harmonic generation in fibers.

Measurement of the phase of second-harmonic generation in SK5 glass.

Focusing an intense laser beam and its second harmonic into a SK5 glass slab transforms the glass into a frequency doubler. We present a new method to measure the optical phase between the second-harmonic beam that transformed the glass and the second-harmonic beam subsequently generated by the glass. We find this phase shift to be Deltatheta= -90 degrees +/- 7 degrees . A spatial map of this phase confirms that the internal dc electric field locked inside the glass resembles a dipole electric field.

Effects of oxygen and antioxidants on the mitochondrial Ca-retention capacity.

2-Oxoglutarate-supported rat liver mitochondria were loaded with moderate amounts of calcium and submitted to O2 deprivation and reoxygenation. In the presence of acetoacetate, anaerobic energy production maintained Ca2+ retention by mitochondria during the anoxia period unless the Pi concentration of the incubation solution was raised to 4-6 mM. Acetoacetate prompted Ca2+ release from O2-deprived mitochondria at elevated Pi levels, presumably due to occurrence of a permeability transition of the inner membrane. Providing 3-hydroxybutyrate and malate, together with acetoacetate, was found to delay the permeability transition until O2 was reintroduced, i.e., O2 triggered a paradoxical release of Ca2+ from mitochondria under these conditions. Whether initiated by O2 in the presence of Pi or by Pi under aerobic conditions, Ca2+ release was initially activated and subsequently inhibited or reversed in the presence of alpha-tocopherol (10-90 mumol.g protein-1). Similar effects were exerted by alpha-tocopherol during Pi-induced Ca2+ release from oligomycin-treated mitochondria supported by succinate (+ rotenone). In addition, the permeability transition was delayed by retinol (3-30 mumol.g protein-1) while beta-carotene, ubiquinone, and water-soluble antioxidants, including Trolox C, were ineffective. Other observations suggest that the Ca(2+)-releasing and/or -retaining effects of alpha-tocopherol and retinol may be independent from pro- and/or antioxidant activities. Effects resembling those of alpha-tocopherol were exerted by alpha-tocopherol succinate, which is devoid of antioxidant activity. Our data indicate that the permeability transition of Ca(2+)-loaded liver mitochondria may be triggered by O2, in the presence of ketone bodies, and affected by lipid-soluble antioxidants through mechanisms seemingly unrelated to free-radical generation or scavenging.

Role of free radicals in the mode of action of anthralin.

Radical reactions of anthralin and its metabolites with skin have been studied by ESR spectroscopy. The influence of compounds which are known to suppress inflammation are described. The ESR spectra recorded during the reaction of anthralin with skin were essentially composed of one broad line centered at g = 2.0030. Similar but much weaker spectra were recorded with the dimer and no signal at all was obtained with 9,10-dihydroxyanthraquinone. The ESR response obtained with anthralin was neither affected by the radical scavengers 2-tert-butyl-4-methoxy-phenol (BHA), 2,6-di-tert-butyl-4-methyl-phenol (BHT) dl-alpha-tocopherol, nor by the antiinflammatory agents clobetasol-17-propionate or indomethacin, nor by potassium hydroxide. We infer that anthralin inflammation is not associated with the presence of anthralin-derived radicals in the skin.

Chemical evidence for interactions between vitamins E and C.

Experimental proof is provided for interactions between radicals of vitamin E/vitamin C as generated by air-oxidized lipids (liquid fraction of subcutaneous chicken fat). Using ESR spectroscopy, hydrogen atom exchange is shown to take place between vitamin C and the radical of vitamin E. Sequential consumption of these two vitamins in oxidized lipid, first vitamin C then vitamin E, is demonstrated by means of differential pulse polarography. These results elucidate the in vitro radical scavenging functions attributed to vitamin E and vitamin C as well as their synergism in lipid antioxidation.