Ten-year Experiences on Initial Genetic Examination in Childhood Acute Lymphoblastic Leukaemia in Hungary (1993–2002). Technical Approaches and Clinical Implementation

A nationwide study was started in 1993 to provide genetic diagnosis for all newly diagnosed childhood ALL cases in Hungary using cytogenetic examination, DNA-index determination, FISH (aneuploidy, ABL/BCR, TEL/AML1) and molecular genetic tests (ABL/BCR, MLL/AF4, TEL/AML1). Aim of the study was to assess the usefullness of different genetic methods, to study the frequency of various aberrations and their prognostic significance. Results were synthesized for genetic subgrouping of patients. To assess the prognostic value of genetic aberrations overall and event-free survival of genetic subgroups were compared using Kaplan-Meier method. Prognostic role of aberrations was investigated by multivariate analysis (Cox’s regression) as well in comparison with other factors (age, sex, major congenital abnormalities, initial WBC, therapy, immunophenotype). Five hundred eighty-eight ALL cases were diagnosed between 1993–2002. Cytogenetic examination was performed in 537 (91%) (success rate 73%), DNA-index in 265 (45%), FISH in 74 (13%), TEL/AML1 RT-PCR in 219 (37%) cases producing genetic diagnosis in 457 patients (78%). Proportion of subgroups with good prognosis in prae-B-cell ALL was lower than expected: hyperdiploidB 18% (73/400), TEL/AML1+ 9% (36/400). Univariate analysis showed significantly better 5-year EFS in TEL/AML1+ (82%) and hyperdiploidB cases (78%) than in tetraploid (44%) or pseudodiploid (52%) subgroups. By multivariate analysis main negative prognostic factors were: congenital abnormalities, high WBC, delay in therapy, specific translocations. Conclusion: Complementary use of each of genetic methods used is necessary for reliable genetic diagnosis according to the algorythm presented. Specific genetic alterations proved to be of prognostic significance.     

Human dirofilariosis in Hungary: an emerging zoonosis in central Europe

INTRODUCTION: In the temperate regions of Europe, mainly Italy, France, Greece, and in recent years Hungary, a specific filarioid worm, Dirofilaria (Nochtiella) repens, a parasite of dogs, cats and some other carnivores, transmitted by mosquitoes, has occasionally been contracted, causing dirofilariosis in humans. We report a case of dirofilariosis and review all previously observed cases in Hungary. MATERIALS, METHODS AND RESULTS: The authors present a case of human subconjunctival dirofilariosis in a 56-year-old man with mild itching and a burning sensation in his right eye. A slow-moving, whitish, thread-like worm was detected by slit-lamp examination. The worm was surgically removed and parasitological examination identified it as an immature male Dirofilaria repens. A review of Hungarian cases from 1880 to 2000 is presented, with updates of earlier reports. In addition, 16 new cases (8 men; 8 women) diagnosed between 2001 and 2006 are reviewed. The mean age of these 16 patients was 60 years; eight cases had ocular localization, seven were subcutaneous and one was diagnosed in a histopathological section of removed axillary lymph node in a patient with lymphoid leukemia. Twelve of the 16 identified Dirofilariae were females, four were males. According to their recorded histories the patients had not traveled internationally. CONCLUSION: Most of the above cases of dirofilariosis are certainly autochthonous infections. The increasing number of diagnosed cases suggests that this zoonosis is becoming more important in central Europe and that the incidence may increase with the improvement of clinical diagnosis.     

Predictive role of evoked potential examinations in patients with clinically isolated optic neuritis in light of the revised McDonald criteria

To analyse the sensitivity and role of somatosensory and motor evoked potentials (EP) in patients with a first episode of clinically isolated optic neuritis (ON) in predicting the development and course of multiple sclerosis (MS), 27 patients with ON underwent EP and magnetic resonance imaging (MRI) examinations at presentation. Follow-up MRI scans were also performed (mean: 20, range: 4-48 months). It was found that 2/27 patients did not fulfill the MRI (McDonald) and clinical criteria of MS upon follow-up and also had normal EP results. Abnormal EP results were found in 6/27 patients and all of them had follow-up MRI results fulfilling the revised McDonald criteria of MS; 4/6 patients in this group were also diagnosed as clinically definitive MS. The majority, 19/27 patients had normal EP results, but went on to develop MS based on follow-up MRI results and McDonald criteria. Of these patients, however, only 3/19 converted to clinically definitive MS as well. The baseline MRI was abnormal in similar proportions (4/6 and 12/19) in these last two groups of patients. Thus, abnormal EP examinations at the first episode of ON can be considered as a predictive factor only for the earlier clinical conversion to MS - in this respect, however, being more sensitive than the initial MRI - and as such they may contribute to the delineation of the patient group who may benefit from early immunomodulatory treatment. They do not however have a predictive value for the development of MS itself as diagnosed by the McDonald criteria.     

Hyaluronan-induced masking of ErbB2 and CD44-enhanced trastuzumab internalisation in trastuzumab resistant breast cancer

Although trastuzumab, a recombinant humanised anti-ErbB2 antibody, is widely used in the treatment of breast cancer, neither its mechanism of action, nor the factors leading to resistance are fully understood. We have previously shown that antibody-dependent cellular cytotoxicity is pivotal in the in vivo effect of trastuzumab against JIMT-1, a cell line showing in vitro resistance to the antibody, and suggested that masking of the trastuzumab-binding epitope by MUC-4, a cell surface mucin, took place. Here, we further explored the role of masking of ErbB2 in connection with CD44 expression and synthesis of its ligand, hyaluronan. We show that high expression of CD44 observed in JIMT-1 cells correlates with ErbB2 downregulation in vivo, while siRNA-mediated inhibition of CD44 expression leads to decreased rate of trastuzumab internalisation and low cell proliferation in vitro. An inhibitor of hyaluronan synthesis, 4-methylumbelliferon (4-MU) significantly reduced the hyaluronan level of JIMT-1 cells both in vivo and in vitro leading to enhanced binding of trastuzumab to ErbB2 and increased ErbB2 down-regulation. Furthermore, the inhibitory effect of trastuzumab on the growth of JIMT-1 xenografts was significantly increased by 4-MU treatment. Our results point to the importance of the CD44-hyaluronan pathway in the escape of tumour cells from receptor-oriented therapy.     

Avemar, a nontoxic fermented wheat germ extract, induces apoptosis and inhibits ribonucleotide reductase in human HL-60 promyelocytic leukemia cells

Avemar (MSC) is a nontoxic fermented wheat germ extract demonstrated to significantly improve the survival rate in patients suffering from various malignancies. We investigated its effects in human HL-60 promyelocytic leukemia cells. After 24, 48, and 72 h of incubation, Avemar inhibited the growth of HL-60 cells with IC₅₀ values of 400, 190, and 160 μg/ml, respectively. Incubation with MSC caused dose-dependent induction of apoptosis in up to 85% of tumor cells. In addition, Avemar attenuated the progression from G2–M to G0–G1 phase of the cell cycle and was also found to significantly reduce the in situ activity of ribonucleotide reductase, the key enzyme of de novo DNA synthesis. We conclude that Avemar exerts a number of beneficial effects which could support conventional chemotherapy of human malignancies.     

Semaphorin 6D regulates the late phase of CD4+ T cell primary immune responses

The semaphorin and plexin family of ligand and receptor proteins provides important axon guidance cues required for development. Recent studies have expanded the role of semaphorins and plexins in the regulation of cardiac, circulatory and immune system function. Within the immune system, semaphorins and plexins regulate cell–cell interactions through a complex network of receptor and ligand pairs. Immune cells at different stages of development often express multiple semaphorins and plexins, leading to multivariate interactions, involving more than one ligand and receptor within each functional group. Because of this complexity, the significance of semaphorin and plexin regulation on individual immune cell types has yet to be fully appreciated. In this work, we examined the regulation of T cells by semaphorin 6D. Both in vitro and in vivo T cell stimulation enhanced semaphorin 6D expression. However, semaphorin 6D was only expressed by a majority of T cells during the late phases of activation. Consequently, the targeted disruption of semaphorin 6D receptor–ligand interactions inhibited T cell proliferation at late but not early phases of activation. This proliferation defect was associated with reduced linker of activated T cells protein phosphorylation, which may reflect semaphorin 6D regulation of c-Abl kinase activity. Semaphorin 6D disruption also inhibited expression of CD127, which is required during the multiphase antigen-presenting cell and T cell interactions leading to selection of long-lived lymphocytes. This work reveals a role for semaphorin 6D as a regulator of the late phase of primary immune responses.     

Brain endothelial cell production of a neuroprotective cytokine, interleukin-6, in response to noxious stimuli

Brain endothelial cells (BECs), specialized cells of the blood–brain barrier (BBB), are ideally positioned to monitor and respond to events in the periphery. The present study examined their potential role in transducing immune signals to the brain and in responding to noxious stimuli. BECs were isolated from rhesus monkeys at 3 age points (fetal/neonatal, adult, and very old animals). Cells were then challenged in vitro with either an immune stimulus (interleukin-1β (IL-1β), or lipopolysaccharide (LPS)) or an oxidative challenge (hypoxia). BECs released interleukin-6 (IL-6), which is known to have neurotrophic and neuroprotective functions. Furthermore, higher amounts of IL-6 were released in both baseline and stimulated conditions by BECs derived from aged animals. This research indicates a pathway whereby immune signals may be communicated to the CNS and has revealed one way that the BBB may protect neuronal survival under challenge conditions.     

Synergistic action of resveratrol, an ingredient of wine, with Ara-C and tiazofurin in HL-60 human promyelocytic leukemia cells

OBJECTIVE: Resveratrol, a naturally occurring stilbene derivative, is a potent free-radical scavenger causing a number of biochemical and antineoplastic effects. It was shown to induce differentiation and apoptosis in leukemia cells. Resveratrol was also identified as an inhibitor of ribonucleotide reductase (RR), a key enzyme of DNA synthesis. We report about the biochemical effects of resveratrol on the concentration of deoxyribonucleoside triphosphates (dNTPs), the products of RR, and on the incorporation of 14C-labeled cytidine into the DNA of HL-60 human promyelocytic leukemia cells. MATERIALS AND METHODS: Incorporation of 14C-labeled cytidine into the DNA of resveratrol-treated HL-60 cells was measured. Concentration of dNTPs was determined by a HPLC method. Cytotoxic effects of resveratrol, Ara-C, and tiazofurin were analyzed using growth inhibition and clonogenic assays. Induction of apoptosis was studied using a Hoechst/propidium iodide staining method. RESULTS: We found that resveratrol effectively inhibited incorporation of 14C-labeled cytidine into DNA. Furthermore, incubation of HL-60 cells with resveratrol significantly decreased intracellular dCTP, dTTP, dATP, and dGTP concentrations. Based on these results, we investigated the combination effects of resveratrol with Ara-C or tiazofurin, both antimetabolites, which are known to exhibit synergistic effects in combination with other inhibitors of RR. In growth inhibition, apoptosis, and clonogenic assays, resveratrol acted synergistically with both Ara-C and tiazofurin in HL-60 cells. CONCLUSIONS: We conclude that resveratrol could become a viable candidate as one compound in the combination chemotherapy of leukemia and therefore deserves further testing.     

Comparison of Ropivacaine and Lidocaine for Intravenous Regional Anesthesia in Volunteers: A Preliminary Study on Anesthetic Efficacy and Blood Level

Background: Ropivacaine may be useful for intravenous regional anesthesia, but its anesthetic effectiveness and toxicity have not been evaluated.

Methods: Two doses of ropivacaine (1.2 and 1.8 mg/kg) and one dose of lidocaine (3 mg/kg) were compared for intravenous regional anesthesia in 15 volunteers. An arm tourniquet was inflated for 30 min after injection and then deflated in two cycles. Sensory block was measured by response to touch, cold, pinprick, and transcutaneous electric stimulation, and motor function was measured by hand grip strength and muscle power. Median, ulnar, radial, and musculocutaneous nerve functions were tested before local anesthetic injection and then at 5-min intervals until blocks resolved. The plasma ropivacaine and lidocaine concentrations were determined from arterial and venous blood samples drawn from the unanesthetized arm.

Results: Sensory and motor blocks were complete within 25 min and 30 min, respectively, in all three treatment groups. However, recovery of sensory and motor block after tourniquet release was slowest in the high-dose ropivacaine group. Anesthesia to pinprick and transcutaneous electric stimulation was sustained in all the volunteers in the high-dose ropivacaine group for 55 min and 85 min, respectively, whereas complete recovery was observed in the lidocaine group (P = 0.008) and partial recovery in the low-dose ropivacaine group (P < 0.05) during the same period. Motor block also was sustained in the high-dose ropivacaine group for 70 min, which was significantly longer than in the lidocaine group (P < 0.05). All volunteers (five of five) given lidocaine and one volunteer given high-dose ropivacaine reported light-headedness and hearing disturbance during tourniquet release when the arterial plasma lidocaine and ropivacaine concentrations were 4.7 +/- 2.1 [micro sign]g/ml (mean) and 2.7 [micro sign]/ml, respectively.