In vitro inhibition of promyelocytic leukemia/retinoic acid receptor-alpha (PML/RARalpha) expression and leukemogenic activity by DNA/LNA chimeric antisense oligos

Acute promyelocytic leukemia (APL) is a subtype of myeloid leukemia characterized by the chromosomal translocation t(15:17) that leads to the expression of promyelocytic leukemia/retinoic acid receptor-alpha (PML/ RARalpha) oncofusion protein. The block of differentiation at the promyelocytic stage of the blasts and their increased survival induced by PML/RARalpha are the principal biological features of the disease. Therapies based on pharmacological doses of retinoic acid (RA, 10(-6) M) are able to restore APL cell differentiation in most cases, but not to achieve complete hematological remission because retinoic acid resistance occurs in many patients. In order to elaborate alternative therapeutic approaches, we focused our attention on the use of antisense oligonucleotides as gene-specific drug directed to PML/RARalpha mRNA target. We used antisense molecules containing multiple locked nucleic acid (LNA) modifications. The LNAs are nucleotide analogues that are able to form duplexes with complementary DNA or RNA sequences with highly increased thermal stability and are resistant to 3'-exonuclease degradation in vitro. The DNA/LNA chimeric molecules were designed on the fusion sequence of PML and RARalpha genes to specifically target the oncofusion protein. Cell-free and in vitro experiments using U937-PR9-inducible cell line showed that DNA/LNA oligonucleotides were able to interfere with PML/RARalpha expression more efficiently than the corresponding unmodified DNA oligo. Moreover, the treatment of U937-PR9 cells with these chimeric antisense molecules was able to abrogate the block of differentiation induced by PML/RARalpha oncoprotein. These data suggest a possible application of oligonucleotides containing LNA in an antisense therapeutic strategy for APL.     

Macrophage depletion induced by clodronate-loaded erythrocytes

Given the important role of macrophages in various disorders, the transient and organ specific suppression of their functions may benefit some patients. Until now, liposome-encapsulated bisphosphonate clodronate has been extensively proposed to this end. In this paper, we demonstrate that erythrocytes loaded with clodronate can also be effective in macrophage depletion. Here, clodronate was encapsulated in erythrocytes through hypotonic dialysis, isotonic resealing and reannealing to final concentrations of 4.1 +/- 0.4 and 10.1 +/- 0.8 micromol/ml of human and murine erythrocytes, respectively. The ability of clodronate-loaded erythrocytes to deplete macrophages was evaluated both in vitro and in vivo. In vitro studies on human macrophages showed that a single administration of engineered erythrocytes was able to reduce cell adherence capacity in a time-dependent manner, reaching 50 +/- 4% reduction, 13 days post treatment. The administration of loaded erythrocytes to cultures of murine peritoneal macrophages was able to reduce macrophage adhesion 67 +/- 3%, 48 h post treatment. In vivo, the ability of clodronate-loaded erythrocytes to deplete macrophages was evaluated both in Swiss and C57BL/6 mice. Swiss mice received 125 microg of clodronate through erythrocytes and 6 days post treatment 69 +/- 7% reduction in the number of adherent peritoneal macrophages and 75 +/- 5% reduction in number of spleen macrophages were observed. C57BL/6 mice received 220 microg clodronate by RBC and 3 and 8 days post treatment 65 +/- 7% reduction in the number of spleen macrophages and the complete depletion of liver macrophages were obtained. In summary, our results indicate that clodronate selectively targeted to the phagocytic cells by a single administration of engineered erythrocytes is able to deplete macrophages, even if not completely. The transient suppression of macrophage functions through clodronate-loaded erythrocytes can be used in many biomedical phenomena and research applications.     

Craniofacial analysis of the Tweed Foundation in Angle Class II, division 1 malocclusion

This study has defined the cephalometric values of the Craniofacial Analysis of the Tweed Foundation for a sample of Brazilian subjects. The sample consisted of 211 cephalometric radiographs from subjects aged 12-15, which were divided into two groups: Class II group, with 168 lateral teleradiographs (cephalograms) of white Brazilian subjects, with Angle Class II, division 1 malocclusion, of both genders (82 males and 86 females); and the Control Group, with 43 lateral teleradiographs (cephalograms) of subjects whose occlusion was clinically excellent, and also of both genders (21 males and 22 females). The teleradiographs were selected from the files of the Department of Orthodontics, School of Dentistry of Piracicaba, State University of Campinas, previously to the orthodontic treatment. The results demonstrated no sexual dimorphism for each group, as attested by the Student's t-test. The exploratory analysis (+/- 0.5 standard deviation) enabled the tolerance limits to be determined and a Craniofacial Analysis Table to be constructed using the respective cephalometric intervals. In addition, the difference between the two groups was not statistically significant according to the maxilla position. The maxilla was in a good position in relation to the cranial base. On the other hand, the mandible was retruded in relation to the cranial base in the Class II cases. The skeletal pattern was not defined because only the Facial Height Index (FHI) showed a vertical pattern in Class II subjects, while the Y Axis, SN.PlO, SN.GoMe and FMA values did not show any statistically significant difference between the groups. The Class II division 1 subjects showed lower incisors more labially tipped and a convex facial profile.     

Noradrenergic modulation of 4-aminopyridine-induced acetylcholine release from rat cerebral cortex

The noradrenergic influence on cortical acetylcholine (ACh) release was investigated by the cortical cup technique in urethane anaestetized rats treated with 4-aminopyridine (4-AP). The following results were obtained: 1) The increase in ACh release induced by 4-AP (3 mg/kg i.p.) was strongly potentiated by pretreatment with -methyl-p-tyrosine (alpha-MPT) which inhibits catecholamine biosynthesis or by N-(2-chloroethyl)-N-ethyl-bromobenzylamine (DSP4) bringing about a selective degeneration of noradrenergic fibres. Neither pretreatment enhanced the spontaneous ACh output. 2) Pretreatment with p-chlorophenylalanine (PCPA), an inhibitor of serotonin synthesis, did not modify 4-AP effect on ACh output. 3) The alpha blockers, yohimbine (1 mg/kg i.p.) and prazosin (4 mg/kg i.p.), did not enhance the 4-AP effect on ACh release but only delayed its onset. 4) Yohimbine (7 mg/kg i.p.) completely reversed 4-AP effect on ACh release which was significantly decreased. It is concluded therefore that pretreatments with alpha-MPT and DSP4 remove an inhibitory noradrenergic control on cortical ACh release. On the other hand, the alpha blockers might interfere with the ionic mechanisms underlaying the 4-AP effect thus, masking the removal of the noradrenergic control, due to an alpha blockade.     

Assessment of the nasolabial angle in young Brazilian black subjects with normal occlusion

Black individuals present craniofacial characteristics which differ from those of other races, especially the white race, whose cephalometric analyses are usually considered as the standard in routine orthodontic diagnosis and treatment planning. Further studies are therefore needed to enable more accurate and specific diagnoses for this ethnic group. The present study was conducted in order to assess average values for the nasolabial angle in young Brazilian black individuals with normal occlusion, and to assess the occurence of sexual dimorphism. Thirty-six lateral skull, extraoral radiographs from Brazilian black individuals were selected from the archives of the Scientific Recordings Department, Orthodontics Graduate Program, School of Dentistry of Piracicaba, State University of Campinas (UNICAMP). The patients' ages varied from 10 to 14 years, they presented normal occlusion upon clinical examination, and had not been submitted to orthodontic treatment. The cephalometric landmarks from which the nasolabial angle was obtained and measured were traced by a single researcher. Statistical analysis and evaluation of the results led to the conclusion that the nasolabial angle of young Brazilian black individuals is sharper, i.e., the soft tissue profile is more protruded. The average value for the whole sample was 88.14 degrees +/- 12.52 degrees. The nasolabial angle was statistically smaller among females (p < 0.05), demonstrating the occurrence of sexual dimorphism.     

The echo-guided treatment of calcific tendinitis of the shoulder

The authors report the results of percutaneous mini-invasive treatment of chronic calcific tendinitis of the rotator cuffs. A total of 39 patients have been treated by echo-guided injection under local anaesthesia since June 2000 with a follow-up of about 2 years. Considerable reduction in symptoms was obtained in 34 patients within a few days of treatment; improvement was moderate in 5 cases, there were no complications in any of the cases. Complete regression of calcification was observed in 21 patients, there was a more than 60% reduction in calcific deposits in 11 patients, there was little reduction in 5, and the calcification remained unchanged in 2. The method, based on our experience, proved to be simple to execute, low-cost and easily repeatable, offering good results from a symptomatological point of view as well. It is the purpose of this study to determine and maximize mini-invasive treatment that will allow for the elimination or reduction of calcifications by means of percutaneous and echo-guided access.     

Reassessing the level of unintended pregnancy and its correlates in Vietnam

Despite rapidly increasing contraceptive use and rapidly declining fertility, unintended pregnancy and induced abortion remain common in Vietnam. This study reassesses the level of unintended pregnancy in Vietnam and its correlates, drawing on retrospective calendar data gathered for the Vietnam Demographic and Health Survey II. Data from 13,540 "segments" of outcomes and contraceptive practice were analyzed. Based on the calendar data, 40 percent of pregnancies during the 1994-97 period are estimated to have been unintended, a proportion 48 percent higher than the prevailing estimate calculated from the reported intendedness of live births. When concealment of pregnancies ending in induced abortions is taken into account, the unintended pregnancy rate in Vietnam is likely to approach levels found only in developing countries. Unintended pregnancy was found to be associated with age, early marriage, spousal age difference, number of living sons, past unintended pregnancy, geographic region, contraceptive use prior to pregnancy, and the family planning supply environment. The findings suggest that broadening the method mix at the community level, targeting high-risk and underserved groups, and expanding postabortion counseling and services are likely to have a dramatic impact on the unintended pregnancy rate in Vietnam.