Basic fibroblast growth factor prolongs the proliferation of rat cortical progenitor cells in vitro without altering their cell cycle parameters

Basic fibroblast growth factor (bFGF) has been shown to influence the survival, proliferation and differentiation of a variety of cell types in the nervous system. In this investigation we have examined the action of bFGF on: (i) the rate of proliferation; (ii) cell cycle parameters; (iii) the maintenance of cell division; (iv) the recruitment of quiescent cells; and (v) the degree of differentiation of cortical progenitor cells in cultures prepared from E16 rat embryos. The proliferation rate (labelling index) of cortical progenitor cells doubled in the presence of bFGF over 48 h. However, the lengths of the cell cycle phases were unchanged. Clones marked with a recombinant retrovirus on the first day in vitro (DIV) grew significantly larger in the presence of bFGF. Furthermore, many of the clones examined in control cultures had ceased to divide after a maximum of four cell cycles, whereas almost all clonally related cells were still dividing in the presence of bFGF 4 days later, i.e. for at least six cell cycles. Basic FGF also stimulated the division of quiescent progenitor cells, which otherwise would have differentiated or undergone cell death. The degree of neuronal and glial differentiation was studied after 5 DIV using MAP-2 and GFAP immunocytochemistry. In the presence of bFGF, the percentage of MAP-2-labelled cells was less than half that of control cultures, whereas the number of cells immunoreactive for nestin (a marker of progenitor cells) remained very high. Cells immunoreactive for GFAP were present in bFGF-treated cultures, yet were extremely rare in control conditions. These experiments show that bFGF, a potent mitogen for cortical progenitor cells, has no effects on the parameters of their cell cycle but extends their proliferative capability, promotes their survival and delays their differentiation into neurons.      

Clinical relevance of P-glycoprotein expression in de novo acute myeloid leukemia

Cytofluorimetric detection of the multidrug resistance (MDR)-associated membrane protein (P-170) was performed at the time of diagnosis in 158 patients with acute myeloid leukemia using the C219 monoclonal antibody (MoAb). In 108 of these cases the JSB1 MoAb was also tested. An improved histogram subtraction analysis, based on curve fitting and statistical test was applied to distinguish antigen-positive from antigen-negative cells. A marker was considered positive when more than 20% of the cells were stained. At onset, P-170 was detected in 43% of cases with C219 and in 73% of cases with JSB1. There was a strict correlation between C219 and JSB1 positivity, as all C219+ cases were also positive for JSB1 MoAb (P < .001). No relationship was found between sex, age, organomegaly, and MDR phenotype. Significant correlation was found between CD7 and both C219 and JSB1 expression (P < .001 and .001, respectively). C219-negative phenotype was more often associated with a normal karyotype (24 of 55 with P = .030). Rhodamine 123 (Rh123) staining and flow cytometry analysis showed a significantly decreased mean fluorescence in 51 C219+ and 38 JSB1+ patients compared to 42 MDR negative ones (P < .001). The rate of first complete remission (CR) differed both between C219+ and C219- cases and between JSB+ and JSB- ones (30.9% v 71.1% and 35.4% v 93.1%, respectively, P < .001). Of the 21 C219+ patients who had yielded a first CR, 19 (90.4%) relapsed, compared with 28 of 64 (43.7%) C219- patients (P < .001). Of the 28 JSB1+ patients in first CR, 17 (60.7%) relapsed relative to 8 (29.6%) of 27 JSBI- ones (P = .021). A higher rate of relapses among MDR+ compared with MDR- patients was observed both for C219 and JSB1 MoAbs taken separately (C219 80% v 44%; JSB1 52% v 27%), with no relationship to age. The survival rates (Kaplan-Meyer method) were significantly shorter both in C219+ patients and in JSB1+ cases (P < .001). Disease-free survival curves followed this same trend. The combination (C219- JSB1+) identified a subset of patients with an intermediate outcome compared to C219 positive cases. The prognostic value of both markers (C219 and JSB1) was confirmed in multivariate analysis. These results suggest that the assessment of MDR phenotype by flow cytometry may be an important predictor of treatment outcome.     

Contribution of hydrazines-derived alkyl radicals to cytotoxicity and transformation induced in normal c-myc-overexpressing mouse fibroblasts

Several hydrazine derivatives (HD) tested so far have pharmacological activities, but many also have toxic side effects, including carcinogenesis. Their toxicity has been ascribed to carbocations (via formation of azoxy intermediates), alkyl radicals or reactive oxygen species. Cytotoxicity and transformation by carbocations is widely accepted, but the role of alkyl radicals is still questioned. We have investigated the cytotoxicity of HD to mouse fibroblasts in three activation systems in which enhanced alkyl radical formation is demonstrated by electron spin resonance/spin-trapping. Cytotoxicity was assayed by inhibition of [3H-methyl]thymidine uptake into DNA of Balb/c 3T3 and/or Myc 9E fibroblasts (normal Balb/c 3T3 cells over-expressing the c-myc proto-oncogene). Based on the results obtained in the cytotoxicity assays we also investigated the transforming potential of procarbazine (PCZ) and methylhydrazine (MeH) activated by horseradish peroxidase (HRP) using the Myc 9E cell line, which aims at the activation of a second cooperating oncogene. Our results show that: (i) cytotoxicity of HD to mouse fibroblasts is increased by HRP activation of MeH, phenelzine and PCZ, which displayed enhanced alkyl radical formation, but not of 1,2-dimethylhydrazine (DMH), which did not produce increased alkyl radical formation under these conditions; (ii) cytotoxicity of neutrophil-activated MeH (producing a 10-fold higher concentration of methyl radicals), is more pronounced than DMH; (iii) MeH and DMH activated by prolonged auto-oxidation in 24-h incubations have comparable cytotoxicity and alkyl radical formation; and (iv) PCZ and MeH activation by HRP to alkyl radicals increased the transformation induced in Myc 9E cells. Taken together, our results strongly support a role for hydrazine-derived alkyl radicals in HD- induced cytotoxicity and cell transformation.      

Projections to layer VI of the posteromedial barrel field in the rat: a reappraisal of the role of corticothalamic pathways

The present study bears on afferents that terminate in layer VI of the posteromedial barrel field in the rat. Their origin was determined by the retrograde transport of cholera toxin, and their axonal arborizations were revealed by targeting injections of biotinylated dextran amine in regions that contained retrogradely labeled neurons. Afferents to lamina VI arise from the thalamus (the ventral posteromedial, the posterior group and the intralaminar nuclei), the claustrum and the infragranular layers of other somatomotor regions of the neocortex (the motor, second somatosensory and perirhinal cortices). Among these afferent systems, corticocortical axons, particularly those issuing from the motor cortex, give rise to the most profuse projections in layer VI, whereas thalamic and claustral afferents form sparse terminal fields. Because corticothalamic cells represent approximately 50% of the neuronal population in lamina VI and 73% of their dendritic processes are deployed locally, it seems likely that afferents arising from the infragranular layers of the motor cortex may directly influence the firing of these neurons. These anatomical data suggest that the role of corticothalamic pathways should be studied from the viewpoint that sensory perception is an active process which operates under the guidance of motor activities.      

Dental erosions and gastro-oesophageal reflux disease in institutionalized intellectually disabled individuals

OBJECTIVE: Both exogenous acids, from the diet, and endogenous acids, from stomach juice, can dissolve the enamel mineral, resulting in dental erosionS. Gastric acid may reach the mouth by gastro-oesophageal reflux disease (GERD), recurrent vomiting, rumination and regurgitation. These conditions are frequently found in the intellectually disabled population. Therefore, we investigated the presence of dental erosions in combination with GERD among intellectually disabled inhabitants, with an IQ<50, taken from three Dutch institutes.
MATERIALS AND METHODS: At random 63 individuals underwent an oesophageal pH test and dental screening and possible predisposing and attributable factors were determined. An abnormal pH level was defined as a pH <4, >4.5% of the measured time. Subjects with dental erosions were compared to those without dental erosions.
RESULTS: In 29 out of 63 (46.0%) cases evidence of dental erosions was found. In 19 of these 29 subjects with erosions (65.5%) GERD was diagnosed, compared to nine (26.5%) out of 34 subjects without erosions ( P = 0.04). In the subjects with erosions mean duration of pH <4 was 15.6% (range: 0.5–90.5) compared to 6.3% (range 0–40.4) in subjects without erosions ( P = 0.02).An IQ <35 was found to be predisposing ( P <0.001).
CONCLUSION: In this population of 63 institutionalised intellectually disabled persons dental erosions were diagnosed in 46%.Sixty-five per cent of them had GERD. Individuals with longer duration of pH <4 than 6.3% of the measured time and with an IQ < 35 were at higher risk to develop dental erosions. This study shows that dental erosions in the intellectually disabled population might be an oral manifestation of GERD.     

A systematic review of the use of simulated patients and pharmacy practice research

Objective The use of simulated patients to assess current practice, or to derive outcome measures for pharmacy practice research, has received much attention in recent years. A simulated patient is an individual who is trained to visit a pharmacy to enact a scenario testing specific behaviour of the pharmacist or pharmacy staff. The aim of this study was to provide a definitive review of the use of simulated patients as a methodological tool for pharmacy practice research. Method A systematic review was undertaken to identify all pharmacy practice studies that had used simulated patient methodology. The electronic databases searched to identify relevant studies were MEDLINE, EMBASE and CINAHL. Articles fulfilling all the following criteria were considered for inclusion in the review: primary reports of trials conducted in community pharmacy and drug store settings which used simulated patients to derive outcome measures. The review was not restricted by language or by country. The review was restricted to publications from 1976 to May 2005. Key findings In total, 56 full publications were retrieved for further examination, of which 46 studies were included in the review, including: nine randomised controlled trials, three controlled trials, 30 cross‐sectional, two time‐series and two ‘other’ studies. Ten publications were excluded: seven reviews, one laboratory‐based study, one telephone survey and one study presented only as an abstract. Conclusions There has been steady growth in the use of simulated patient methodology over the past 30 years. Although simulated patients have received negative attention in the pharmaceutical media, they can be a rigorous and robust method of measuring practice if used appropriately. This review demonstrates the range of activities for which this method can be used, including the assessment of counselling and advice provision, the treatment of minor and major illness, and the assessment of the public health activities of pharmacy and drug store staff. Simulated patient methodology has been used in developing countries to a similar, if not greater extent, than the developed world, demonstrating its versatility and applicability to pharmacy practice research globally.     

Increased frequency of wild-type arylamine-N-acetyltransferase allele NAT2*4 homozygotes in Portuguese patients with colorectal cancer

Here we report that colorectal cancer patients show a markedly higher frequency (3-fold) of wild-type NAT2*4 allele homozygotes than the control population. However, a marked difference in NAT2*4/NAT2*4 genotype frequency associated with the patients gender was observed pointing to a male-specific effect of this genotype as a risk factor in colon cancer. The arylamine-N-acetyltransferase (E.C. 2.3.1.5.) NAT2, a phase II detoxification enzyme, has been implicated in procarcinogen activation, namely from food contained arylamines, cigarette smoking, as well as environmental amines of various types. NAT2 is encoded by a polymorphic gene presenting several allelic variants encoding partially inactive enzymes expressed in human liver and colon. Epidemiological studies based on phenotype determination have long indicated the importance of the NAT2 active phenotype as a susceptibility factor in colorectal cancer. In the present study we investigated the NAT2 allelic frequencies and genotype distribution in a group of 114 unrelated colorectal cancer patients, in parallel with 201 healthy Portuguese subjects. We first demonstrate that the frequency of the wild-type NAT2*4 allele in the Portuguese sample population (23.4%) does not significantly differ from the values described for other Europeans. Besides the 3-fold higher frequency of NAT2*4 homozygotes found in colorectal cancer subjects, the NAT2*4/NAT2*5A compound genotype, known to determine a faster acetylator phenotype than other heterozygotic combinations, also increased by the same order of magnitude. These two genotypes represent 32% of the patients population versus 11% of the healthy controls. Taken together, our results strongly indicate that NAT2 genotype, particularly NAT2*4 allele zygosity, constitutes an individual susceptibility trait associated with sporadic colorectal cancer development, probably due to the local dietary habits in Portugal.