Recurrence of Bcl-2/IgH polymerase chain reaction positivity following a prolonged molecular remission can be unrelated to the original follicular lymphoma clone

OBJECTIVE: The aim of this study was to evaluate whether reappearance of polymerase chain reaction (PCR) positivity for the Bcl-2/IgH translocation following a phase of molecular remission in autografted follicular lymphoma (FL) patients is always associated with reappearance of the original neoplastic clone. PATIENTS AND METHODS: The molecular follow-up of 119 autografted Bcl-2/IgH positive patients was evaluated by nested PCR. In case of molecular recurrence, direct sequencing of involved rearrangements has been performed both at diagnosis and at the time of recurrence. The two sequences then were compared in terms of breakpoints, N insertions, and JH usage. RESULTS: Seventy-five patients achieving molecular remission were identified in our patient sample (63%). Of these patients, eight (10.6%) experienced molecular recurrence. Direct sequencing of the Bcl-2/IgH translocation performed at diagnosis and recurrence showed identical rearrangements in six subjects and unrelated rearrangements in two. As opposed to most true molecular relapses, unrelated rearrangements always occurred several years after transplantation. To date, the two subjects carrying unrelated rearrangements show no signs of active lymphoproliferative disease. CONCLUSIONS: This report is the first evidence that Bcl-2/IgH rearrangements unrelated to the original tumor clone can lead to false-positive results during the molecular follow-up of autografted FL patients. Based on these results, we recommend confirmation by direct sequencing, at least for patients experiencing molecular relapse 2 or more years after the end of treatment. This will be particularly important for patients enrolled in clinical trials that schedule additional treatment in case of molecular evidence of persistent disease activity.     

Fludarabine ability to down-regulate Bcl-2 gene product in CD5+ leukaemic B cells: in vitro/in vivo correlations

CD5⁺ B-chronic lymphocytic leukaemia (B-CLL) and mantle cell lymphoma (MCL) in leukaemic phase are characterized by defects in cell death induction that primarily involves the Bcl-2 family of genes. Fludarabine (9-β- D -arabinofuranosyl-2-fluoradenine, F-ara-A) is a potent inducer of apoptosis in CLL cells. This study aimed to determine whether F-ara-A-induced apoptosis might be related to Bcl-2 modifications and to evaluate in vitro/in vivo correlations.   Peripheral blood lymphocytes from eight B-CLL and four leukaemic MCL were cultured in the presence of different concentrations of F-ara-A ±methylprednisolone (MP). F-ara-A down-regulated the expression of Bcl-2 in 5/12 cases. mRNA down-regulation was maximal at 48 h; protein down-regulation was prominent after 48 h. Both events were dose-dependent. The amount of apoptosis was significantly higher in the samples treated with F-ara-A than in those exposed to MP alone. In the seven remaining cases, no Bcl-2 down-regulation was observed after exposure to F-ara-A and the degree of F-ara-A-induced apoptosis overlapped that induced by MP.   The in vivo outcome after treatment with three to six courses of F-ara-A was evaluable in 10 patients: 4/5 cases, whose cells had shown in vitro Bcl-2 down-regulation and prominent apoptosis after exposure to F-ara-A, had a complete response (CR) and a partial response (PR) was observed in the remaining patient. Of the five patients whose cells had shown no in vitro Bcl-2 modulation after exposure to F-ara-A, two had a PR, but the other three did not show any in vivo clinical response.     

Wild-type and e antigen-minus hepatitis B viruses and course of chronic hepatitis.

Using an oligonucleotide hybridization assay, we studied the clinical implication of wild-type hepatitis B virus (HBV) and a HBV mutant that is unable to secrete hepatitis B e antigen (HBeAg) because of a translational defect due to a stop codon in the pre-C region in 106 hepatitis B surface antigen-positive patients with chronic hepatitis B. Wild-type HBV was detected in 31 of 42 (73.8%) HBeAg-positive patients, whereas a mixed viral population was present in 10 (23.8%). Significant differences in the severity and outcome of liver disease were not observed in the two groups of patients. However, the emergence of HBeAg-minus HBV in wild-type HBV carriers was associated with an exacerbation of liver disease and was followed by the presence of antibodies against HBeAg (anti-HBe) in serum in 50% of the cases. In 61 of 64 (95.3%) anti-HBe-positive patients, HBeAg-minus HBV was the predominant virus: HBeAg-minus HBV was detected in 42 patients (65.6%), whereas both wild-type and HBeAg-minus HBV were present in 19 (29.7%). HBeAg-minus HBV was associated with a course of hepatitis characterized by flare-ups of liver cell necrosis interspersed with periods of asymptomatic HBV carriage (P less than 0.01). These data support the hypothesis that genetic heterogeneity of HBV significantly influences the course and outcome of chronic hepatitis B. Wild-type HBV secreting HBeAg induces immunologic tolerance and causes chronic infection. HBeAg-minus HBV might be unable to induce chronic infection without the helper function of wild-type HBV, but it appears to be more pathogenic. Once chronic infection is established, HBeAg-minus HBV variants may prevail and displace wild-type virus.     

The effect of losartan treatment on the response of diabetic cardiomyocytes to ATP depletion

The present work aimed to investigate the effect of losartan treatment of healthy and diabetic rats on cardiomyocyte response to ATP depletion.Cells were isolated from normoglycemic (N) and streptozotocin-injected (55 mg/kg) rats (D) treated or not treated with losartan (20 mg/kg/day in the drinking water; NL and DL, respectively) for 3 weeks. In each group of cells, enzyme activities such as glucose-6-phosphate (G6PDH) and glycerol-3-phosphate dehydrogenases (G3PDH), lactate/pyruvate, glycogen levels and citrate synthase were measured as an index of glycolytic dysregulation and mitochondrial mass, respectively.Cells were then challenged with NaCN (2 mM) in glucose-free Tyrode solution (metabolic intoxication, MI), a protocol to study ischemia at cell level. Under these conditions, the time to contractile failure up to rigor-type hyper-contracture in field-stimulated cells and K_ATP current activation by patch-clamp recordings were measured.In comparison with N and NL, D cells presented higher G6PDH and cytoplasmic G3PDH activities, lactate/pyruvate, glycogen content but similar levels of citrate synthase, and decay time of contraction. When subjected to MI, D cells showed delayed activation of the K_ATP current (25.7 ± 7.1 min; p < 0.001 vs. N and NL), increased time to contractile failure and rigor-type hyper-contracture (p < 0.001 vs. N and NL). In cells from DL rats both functional (time to rigor and to K_ATP current activation) and metabolic parameters, approached values similar to those measured in N and NL cells.These results demonstrate that diabetic cardiomyocytes from rats treated with losartan, maintain the capacity to respond promptly to ATP depletion reaching contractile failure, rigor-type hypercontracture and K_ATP opening with a similar timing of N cells.     

Activity and expression of semicarbazide-sensitive benzylamine oxidase in a rodent model of diabetes: interactive effects with methylamine and alpha-aminoguanidine

Previous data indicate that methylamine injection in fasted healthy mice produced a hypophagic effect dependent of neuronal K(v)1.6 channels expression and increased by alpha-aminoguanidine, an inhibitor of semicarbazide-sensitive benzylamine oxidase enzymes mainly involved in amine degradation. In the present work we have investigated: 1) the level of expression and activity of the semicarbazide-sensitive benzylamine oxidase; 2) the effect of methylamine alone and in the presence of alpha-aminoguanidine on food intake of genetic obese and type II diabetes mice (the db/db mice). Db/db mice showed higher levels of semicarbazide-sensitive benzylamine oxidase activities in adipose tissue and in plasma than their lean counterpart (db/db(+) mice). Methylamine (30-75 microg, i.c.v.) showed similar hypophagic effects in obese and lean mice consistently with the levels of neuronal K(v)1.6 found in both animal strains. Alpha-aminoguandine (50 mg/kg, i.p.) increased methylamine (i.c.v.) hypophagia in both obese and lean mice and only in obese mice when methylamine was given i.p. These results suggest a crucial role of semicarbazide-sensitive benzylamine oxidase activity in controlling methylamine hypophagia in hyperphagic diabetic mice.     

The additionally glycosylated variant of human sex hormone‐binding globulin (SHBG) is linked to estrogen‐dependence of breast cancer

Sex HormoneBinding Globulin (SHBG), the plasma carrier for androgens and estradiol, inhibits the estradiolinduced proliferation of breast cancer cells through its membrane receptor, cAMP, and PKA. In addition, the SHBG membrane receptor is preferentially expressed in estrogendependent (ER+/PR+) breast cancers which are also characterized by a lower proliferative rate than tumors negative for the SHBG receptor. A variant SHBG with a point mutation in exon 8, causing an aminoacid substitution (Asp 327 Asn) and thus, the introduction of an additional Nglycosylation site, has been reported. In this work, the distribution of the SHBG variant was studied in 255 breast cancer patients, 32 benign mammary disease patients, and 120 healthy women. The presence of the SHBG mutation was evaluated with PCR amplification of SHBG exon 8 and HinfI restriction fragment length polymorphism (RFLP) procedure. This technique allowed us to identify 54 SHBG variants (53 W/v and 1 v/v) in breast cancer patients (21.2%), 5 variants (4 W/v and 1 v/v) in benign mammary disease patients (15.6%), and 14 variants (W/v) in the control group (11.6%). The results of PCR and RFLP were confirmed both by nucleotide sequence of SHBG exon 8 and western blot of the plasma SHBG. No differences in the mean plasma level of the protein were observed in the three populations. The frequency of the SHBG variant was significantly higher in ER+/PR+ tumors and in tumors diagnosed in patients over 50 years of age than in the control group. This observation suggests the existence of a close link between the estrogendependence of breast cancer and the additionally glycosylated SHBG, further supporting a critical role of the protein in the neoplasm.     

Compliance in antiamblyopia occlusion therapy.

Antiamblyopia occlusion therapy relies on compliance. We retrospectively reviewed the charts of 496 amblyopic subjects. Measures of non-compliance included patient reporting and patient records of broken appointments. Out of 496 subjects, 92 (18%) failed to follow the occlusion regimen. Compliance with treatment was analyzed by age group, refractive error and type of strabismus. The failure rate was 82.6% for the unilateral high myopia group and 37.5% for the monofixation syndrome group. The risk for non-compliance appeared to be higher in the 1 to 2-year-old group (37%). The chi 2-test showed the differences to be highly significant (P less than 0.0001). Children having lower initial visual acuity were also significantly less complaint (P less than 0.007). Several factors such as age, parental understanding, initial visual acuity and improvement rate seem to be involved in compliance.     

Vascularized nerve graft: a clinical contribution

The authors present 4 cases of vascularized nerve graft. The results were better than those obtained with traditional grafting. The indication is a rare one, and the experimental results are contradictory. Indications are limited to Volkmann ischemic syndromes, post-actinic lesions of the brachial plexus, infections and finally, post-burning scarring. Nevertheless, traditional nerve grafts remain the treatment of choice for peripheral nerve lesions which cannot undergo direct suturing.