Quantitative real-time polymerase chain reaction as an efficient molecular tool for detecting minimal residual disease in Moroccan chronic myeloid leukemia patients.

Chronic myeloid leukemia (CML) is characterized by BCR-ABL translocation and an increased number and migration of immature myeloid cells into the peripheral blood. The detection limit of the BCR-ABL transcript, particularly after treatment, is controversial. In the present study, we used quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR) to monitor BCR-ABL expression in Moroccan CML patients undergoing imatinib treatment, and compared the results with those of conventional PCR and fluorescence in situ hybridization (FISH). The aim of this study was to establish a new molecular tool for in vitro diagnosis of CML. In a retrospective comparative analysis, 20 CML Moroccan patients who had received imatinib treatment (N = 20) were analyzed by real-time PCR, conventional RT-PCR, and FISH. Half of the samples analyzed (N = 10) were positive for BCR-ABL gene expression, while the other half (N = 10) were negative according to conventional PCR. Interestingly, 5 of the 10 samples shown to be negative by conventional PCR showed positive expression of the BCR-ABL gene according to RT-qPCR. The RT-qPCR results were confirmed by FISH, which revealed a high concordance (100%) rate. We found that real-time RT-qPCR is more reliable and should be used in Moroccan biomedical analysis laboratories to monitor CML progression, particularly for minimal residual disease, following imatinib treatment.

[Paraneoplastic Cushing's syndrome, a real diagnostic and therapeutic challenge: A case report and literature review]. / Le syndrome de Cushing paranéoplasique, un vrai défi diagnostique et thérapeutique: à propos d'un cas et revue de la littérature.

INTRODUCTION: Paraneoplastic Cushing's syndrome is a rare cause of endogenous hypercortisolism attributable to ectopic ACTH secretion by non-pituitary tumors. Imaging and biochemical results are often inconclusive and differential diagnosis with Cushing's disease can then be challenging. Moreover, these tumors may be occult and difficult to find and thus the need of new imaging tools such as (18)FDG-PET scan and (18)DOPA-PET scan. CASE REPORT: We report a 50-year-old man who presented with very aggressive clinical features related to Cushing's syndrome. Biological work-up confirmed the hypercortisolism and was consistent with an ectopic ACTH secretion. Conventional localization techniques failed to show any tumor and bilateral adrenalectomy was performed because of life-threatening complications. Two years later, thoracic computed tomography reveals an 11 mm mass in the left lower pulmonary lobe, (18)FDG-PET scan found a non-specific mild hypermetabolism of the lung nodule, and the (18)DOPA-PET scan confirmed the high uptake of this nodule suggesting an endocrine carcinoma. Histology confirmed a typical carcinoid tumor. The tumor cells stained positive for ACTH, CD56, chromogranin and synaptophysin. CONCLUSION: This case illustrates the dilemma between the need for morphological diagnosis of the ectopic ACTH source and control of the life-threatening hypercortisolism. (18)FDG-PET scan and (18)DOPA-PET scan should be considered early as a secondary diagnostic tool when conventional imagery fails to show any tumor.

Specific BRCA1 gene variations amongst young Moroccan breast cancer patients.

Germline mutations in the BRCA1 gene are known predictive markers for the development of hereditary breast cancer. Nevertheless, no comprehensive study has been performed targeting the presence and relevance of BRCA1 mutations in Moroccan breast cancer patients. We here present an analysis of BRCA1 gene regions (exon 2 and exon 11a/b) of 50 female Moroccan breast cancer patients with early disease onset (≤ 40 years) or familial disease backgrounds. Results showed that no mutation was present in either exon 2 or exon 11a of the BRCA1 gene in any of the 50 patients analysed. However, in exon 11b, a mutation generated by a nucleotide exchange was detected in 8% of patients, most of whom were young women (≤ 40). This mutation leads to substitution of the amino acid glutamine by an arginine at position 356 of the polypeptide sequence (Q356R). Although this mutation was previously characterised at a lower frequency in western populations, our study is the first to describe it in a young Moroccan population. Furthermore, another mutation was detected with a high frequency (4%) on exon 11b of the BRCA1 gene in exclusively young patients (≤ 40). This mutation was silent, encoding the same threonine residue at position 327 (T327T) as the wild type. The present study is the first to describe this mutation as well, particularly in a young Moroccan population. Analysis of a larger population is required in order to highlight the relevance of the Q356R and T327T mutations in young Moroccan breast cancer patients.

Role of both actin-myosin cross bridges and NO-cGMP pathway modulators in the contraction and relaxation of human placental stem villi.

INTRODUCTION: Human placental stem villi (PSV) present contractile properties. We studied the role of actin-myosin cross bridges (CBs) and the effects of NO-cGMP pathway modulators in the PSV contraction and relaxation. METHODS: In vitro contractile properties were investigated in 71 PSV from term human placentas studied according to their long axis. Contraction was induced by both KCl and electrical tetanic stimulation. Relaxation was induced by inhibiting the CB cycle with either 2,3-butanedione monoxime (BDM) or blebbistatin (BLE) and by activating the NO-cGMP pathway with isosorbide dinitrate (ISDN), sildenafil (SIL) or ISDN + SIL. RESULTS: PSV tension slowly increased by 140% of the basal tone after KCl exposure and by 85% after tetanus. The addition of BDM, BLE, ISDN, SIL and ISDN + SIL induced a relaxation of PSV, the overall time course of relaxation (in s) was respectively (means ± SD) 3412 ± 1904, 14,250 ± 3095*, 3813 ± 1383, 2883 ± 1188 and 2440 ± 477; significantly longer in BLE compared with BDM, ISDN, SIL and ISDN + SIL:*p < 0.001). the overall time course of relaxation (in s) was respectively (means ± SD) 3412 ± 1904, 14,250 ± 3095*, 3813 ± 1383, 2883 ± 1188 and 2440 ± 477; significantly longer in BLE compared with BDM, ISDN, SIL and ISDN + SIL:*p < 0.001). These relaxation kinetics were particularly slow. Other relaxation parametres, i.e., maximum lengthening, -peak dT/dt, and resting tension, did not differ between these 5 subgroups. DISCUSSION AND CONCLUSION: Isolated human PSV were able to contract after both KCl exposure and tetanus. This increase in contractility was reversed by inhibiting the CB cycle with BDM or BLE and by stimulating the NO-cGMP pathway with ISDN or SIL. The association ISDN + SIL did not potentiate the relaxing processes.

Chemotherapy-mediated p53-dependent DNA damage response in clear cell renal cell carcinoma: role of the mTORC1/2 and hypoxia-inducible factor pathways.

The DNA-damaging agent camptothecin (CPT) and its analogs demonstrate clinical utility for the treatment of advanced solid tumors, and CPT-based nanopharmaceuticals are currently in clinical trials for advanced kidney cancer; however, little is known regarding the effects of CPT on hypoxia-inducible factor-2α (HIF-2α) accumulation and activity in clear cell renal cell carcinoma (ccRCC). Here we assessed the effects of CPT on the HIF/p53 pathway. CPT demonstrated striking inhibition of both HIF-1α and HIF-2α accumulation in von Hippel-Lindau (VHL)-defective ccRCC cells, but surprisingly failed to inhibit protein levels of HIF-2α-dependent target genes (VEGF, PAI-1, ET-1, cyclin D1). Instead, CPT induced DNA damage-dependent apoptosis that was augmented in the presence of pVHL. Further analysis revealed CPT regulated endothelin-1 (ET-1) in a p53-dependent manner: CPT increased ET-1 mRNA abundance in VHL-defective ccRCC cell lines that was significantly augmented in their VHL-expressing counterparts that displayed increased phosphorylation and accumulation of p53; p53 siRNA suppressed CPT-induced increase in ET-1 mRNA, as did an inhibitor of ataxia telangiectasia mutated (ATM) signaling, suggesting a role for ATM-dependent phosphorylation of p53 in the induction of ET-1. Finally, we demonstrate that p53 phosphorylation and accumulation is partially dependent on mTOR activity in ccRCC. Consistent with this result, pharmacological inhibition of mTORC1/2 kinase inhibited CPT-mediated ET-1 upregulation, and p53-dependent responses in ccRCC. Collectively, these data provide mechanistic insight into the action of CPT in ccRCC, identify ET-1 as a p53-regulated gene and demonstrate a requirement of mTOR for p53-mediated responses in this tumor type.

Elevated levels of IFNγ and LIGHT in the spinal cord of patients with sporadic amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a paralytic and fatal neurodegenerative disorder caused by the gradual loss of both upper and lower motoneurons. There is compelling evidence from ALS experimental models that neuroinflammation actively contributes to motoneuron damage. We recently proposed that interferon gamma (IFNγ), a potent proinflammatory cytokine, induces motoneuron death by eliciting the activation of the lymphotoxin beta receptor (LT-ßR) through its ligand LIGHT. Here, we explore the pertinence of this non-cell-autonomous mechanism in human ALS. METHODS: The levels and expression pattern of IFNγ, LIGHT, and LT-ßR were investigated by Western blot and immunohistochemical analysis in spinal cord of patients with sporadic ALS. RESULTS: We observed significant increased levels of IFNγ in human ALS spinal cords compared to control cases. We found that large ventral horn neurons as well as glial cells were immunoreactive for IFNγ in sporadic ALS spinal cord. We further observed that LIGHT and LT-ßR were expressed mainly by motoneurons in both ALS and control cases, and while LT-ßR levels remained constant between ALS and control cases, LIGHT levels were increased in human ALS spinal cords. CONCLUSION: These findings in sporadic ALS cases, which are consistent with the observation made in ALS experimental models, propose that the IFNγ-triggered LIGHT/LT-ßR-mediated death pathway may contribute to human ALS pathogenesis.

IFNγ triggers a LIGHT-dependent selective death of motoneurons contributing to the non-cell-autonomous effects of mutant SOD1.

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease that primarily affects motoneurons in the brain and spinal cord. Dominant mutations in superoxide dismutase-1 (SOD1) cause a familial form of ALS. Mutant SOD1-damaged glial cells contribute to ALS pathogenesis by releasing neurotoxic factors, but the mechanistic basis of the motoneuron-specific elimination is poorly understood. Here, we describe a motoneuron-selective death pathway triggered by activation of lymphotoxin-ß receptor (LT-ßR) by LIGHT, and operating by a novel signaling scheme. We show that astrocytes expressing mutant SOD1 mediate the selective death of motoneurons through the proinflammatory cytokine interferon-γ (IFNγ), which activates the LIGHT-LT-ßR death pathway. The expression of LIGHT and LT-ßR by motoneurons in vivo correlates with the preferential expression of IFNγ by motoneurons and astrocytes at disease onset and symptomatic stage in ALS mice. Importantly, the genetic ablation of Light in an ALS mouse model retards progression, but not onset, of the disease and increases lifespan. We propose that IFNγ contributes to a cross-talk between motoneurons and astrocytes causing the selective loss of some motoneurons following activation of the LIGHT-induced death pathway.

Ruminal fermentation, microbial growth and amino acid flow in single-flow continuous culture fermenters fed a diet containing olive leaves.

Six single-flow continuous culture fermenters were used to determine fermentation profile, microbial growth and amino acid (AA) flow promoted by olive leaves supplemented with barley grains and faba beans (OLSUP), and alfalfa hay (AH). Two incubation runs were carried out with three fermenters inoculated with ruminal fluid from wethers and three from goats. The inoculum source did not affect (p = 0.059 to 0.980) any of the parameters. Daily volatile fatty acid (VFA) production and carbohydrate digestibility were greater (p = 0.009 and 0.024, respectively) for AH, therefore the pH values were lower (p = 0.015) than for OLSUP. Acetate was greater (p < 0.001) and isobutyrate, isovalerate and caproate lower (p < 0.001 to 0.006) for AH with greater acetate/propionate (p = 0.014) and 'VFA/digested carbohydrate' (p = 0.026) ratios. Daily microbial N flow and efficiency were greater (p = 0.016 and p = 0.041) for diet AH. Individual AA flows were greater (p < 0.001 to 0.016) for AH, but microbial essential AA proportion was greater for OLSUP (p = 0.015). The results indicate that OLSUP promoted lower bacterial growth and AA flow than AH, which could have been partially due to a limitation of N availability to ruminal microbes.

Comparison of bacterial pellets and microbial markers for the estimation of the microbial nitrogen and amino acids flows from single flow continuous culture fermenters fed diets containing two-stage olive cake.

The effects of using effluent bacteria (EB) and solid- (SAB) and liquid- (LAB) associated bacteria and diaminopimelic acid (DAPA) or purine bases (PB) and partially substituting alfalfa hay (AH) by a concentrate including olive cake on the microbial N flow (MNF) and amino acids (AA) flow were investigated with continuous culture fermenters fed AH and a mixture of AH and a concentrate containing barley grains and two-stage olive cake (2:1 ratio) without (AHCO) or with polyethylene glycol (PEG) (AHCOP). The MNF was not different among diets with SAB or LAB (p = 0.302 and 0.203, respectively) and DAPA, but differed with PB (p = 0.021 and 0.014, respectively). With EB both markers detected similar differences, AHCOP showing a higher value (p < 0.05) than AH and AHCO. The MNF was higher (p < 0.001) with PB than DAPA. Daily flow of non-essential AA was not different (p = 0.356) among diets but essential AA flow was higher (p < 0.05) for AH and AHCOP than for AHCO. The SAB presented lower (p < 0.05) total AA than LAB and higher total AA (p < 0.05) for diet AH than AHCO. The AA profile of EB was similar to that of LAB, but alanine and leucine were higher (p < 0.05) in EB than in LAB. Microbial contribution to AA flow was 45.4%, 55.6% and 58.1% for diets AH, AHCO and AHCOP respectively. With both markers, microbial AA flow was higher (p < 0.05) for diet AHCOP compared with AH (451 and 355 mg/day, respectively), but not different (p > 0.05) for AHCOP and AHCO (389 mg/day). The results would indicate that olive cake could be used in the practical feeding of small ruminants without negatively affecting microbial AA N supply.

Fermentation characteristics and microbial growth promoted by diets including two-phase olive cake in continuous fermenters.

Two-phase olive cake (2POC) is the by-product obtained from the so called 'two-phase' procedure to extract olive oil by mechanical methods. After the mechanical extraction the 2POC is dried and most of the remaining oil extracted by chemical means. The production of the crude by-product may reach more than 4 millions t/year in Spain (MAPA, 2003), most of it in areas (Southeast) with shortage of pastures and conventional feeds for ruminants. Six continuous fermenters, inoculated with ruminal liquor from wethers or goats, were fed lucerne hay (LH); LH plus a concentrate including dried two-phase olive cake (LHCO) and; diet LHCO added with polyethylene glycol (LHCOP). The highest pH values and ammonia N (NH(3)-N) output were found in fermenters fed diet LH (6.19 and 6.35 for pH, and 53.7 and 68.9 mg NH(3)N/day, respectively, in fermentes inoculated with rumen liquor from sheep and goats) without differences due to the inoculum origin. The digestibility of carbohydrates (CHO) was affected (p < 0.001) by inoculum (67.0 and 58.8%, respectively, for goats and wethers) the lowest values being for diet LHCO (53.2 and 57.0% with inoculum from wethers and goats, respectively). The main volatile fatty acid (VFA) was acetic acid with higher (p < 0.01) values in fermenters with inoculum from goats than from wethers (80.2 and 63.0 mmol/day respectively). The efficiency of bacterial protein synthesis (EBS) was not different (p > 0.05) with inoculum from wethers and goats [26.4 and 28.1 and 35.2 and 33.5 g bacterial N/kg digested CHO, respectively, obtained by using diamino pimelic acid (DAPA) and purine bases (PB) as microbial markers]. The lowest (p < 0.05) values were found in fermenters fed diets LHCOP and LH, estimated, respectively, from DAPA and PB (21.9 and 29.0 g bacterial N/kg digested CHO). The substitution of a part of lucerne hay by a concentrate including dried 2POC does not seem to cause important differences in efficiency of VFA production. Results concerning bacterial protein synthesis are not so clear since values estimated from DAPA and PB did not show similar trends. Neither inoculum origin nor PEG had important effects on fermentation characteristics. The DAPA could be an adequate marker in continuous fermenters, with comparative purposes, as protozoa are not present and, estimated protein synthesis values are similar to those obtained in vivo with similar diets using urinary excretion of PD (Yáñez Ruiz et al., 2004b). Further research is needed to state the optimal proportion of 2POC in practical diets for ruminants at both maintenance and production stages.

Ruminal fermentation and degradation patterns, protozoa population and urinary purine derivatives excretion in goats and wethers fed diets based on olive leaves.

Olives leaves, accrued during the processing of olive harvests for oil extraction, are poor in N, rich in crude fat and ADF (1.19, 8.03 and 28.2 g/100 g of DM, respectively), and relatively low in condensed tannins (11.1 mg/g of DM). Three experiments were conducted in a 2 x 3 (two animal species: goats vs. wethers; and three experimental diets: olive leaves without or with polyethylene glycol supply and olive leaves supplemented with barley and faba beans) factorial design to evaluate ruminal degradation and passage kinetics (Exp. 1), fermentation pattern and protozoa population (Exp. 2), and urinary purine derivatives excretion (Exp. 3). Polyethylene glycol was supplied to evaluate the effects of condensed tannins contained in olive leaves. Ruminal degradability of CP was low in both goats and wethers, although goats showed higher (P < 0.05) values than wethers. Supplementation of olive leaves with barley and faba beans increased (P < 0.001) ruminal degradability of DM and CP. Both goats and wethers fed olive leaves showed similarly low particulate fractional passage rates (0.021 and 0.023/h, respectively). Ingestion of olive leaves promoted low NH3-N and VFA concentrations, which reflect poor microbial activity. These concentrations, especially that of VFA, increased when barley and faba beans were added. Ingestion of olive leaves affected ruminal protozoa: Entodiniomorphida showed low concentrations and Holotricha completely disappeared. When animals received a diet based on olive leaves, barley, and faba beans, Holotricha appeared in the ruminal liquor and Entodiniomorphida increased (P < 0.001). In goats and wethers fed olive leaves alone, urinary allantoin excretion was very low (163 and 164 micromol/kg BW0.75 in goats and wethers, respectively), and moderate values (352 and 389 micromol/kg BW0.75 in goats and wethers, respectively) were observed when a diet of olive leaves, barley, and faba beans was fed. The polyethylene glycol supply did not have an effect in goats or in wethers, indicating the lack of an effect of condensed tannins in olive leaves. Ingestion of olive leaves promotes a low microbial activity, although its supplementation with readily degraded carbohydrates and protein improves microbial activity and, as a consequence, increases its ruminal degradation. In general, for most of the measured variables, there were no animal species x diet interactions. Thus, goats and wethers had similar ruminal activities when fed diets based on olive leaves.

Ruminal fermentation and degradation patterns, protozoa population, and urinary purine derivatives excretion in goats and wethers fed diets based on two-stage olive cake: effect of PEG supply.

Three experiments were conducted in Granadina goats and Segureña wethers fed at maintenance level to evaluate the effect of including a mixture of barley and a new by-product derived from olive oil extraction (two-stage dried olive cake) on ruminal degradation and passage kinetics (Exp. 1), fermentation pattern and protozoa population (Exp. 2), and urinary purine derivatives excretion (Exp. 3). Polyethylene glycol was supplied to the animals to evaluate the effects of tannins contained in the by-product. The experimental diets were as follows: alfalfa hay and alfalfa hay plus a concentrate, formulated with two-stage dried olive cake, barley, and a mineral-vitamin mixture either with or without the addition of polyethylene glycol to the drinking water. The inclusion of two-stage dried olive cake in the diet resulted in an increase of condensed tannins. Ruminal VFA concentration in goats and wethers increased (P < 0.05) and ammonia N (NH3-N) concentration decreased (P < 0.05). The inclusion of two-stage dried olive cake decreased (P < 0.001) urinary allantoin excretion only in wethers. Ruminal degradation profiles and fractional passage rates were similar in goats and wethers. The polyethylene glycol supply increased (P < 0.001) DM and N degradation rates in both animal species but did not modify the fractional passage rate. Ruminal fermentation patterns were also similar in goats and wethers and were affected by polyethylene glycol supply. In general, Entodiniomorphida and Holotricha protozoa counts were higher (P < 0.05) in the rumen of goats than of wethers. Protozoa count in wethers responded more to polyethylene glycol supply than in goats. The present work presents the first data obtained from a comparative study with sheep and goats concerning urinary excretion of purine derivatives. The excretion was similar in both animal species when fed alfalfa hay; however, polyethylene glycol affected only urinary allantoin excretion in wethers. Results suggest a greater sensitivity of wethers than of goats to two-stage olive cake condensed tannins.

The HIV-1 repeated sequence R as a robust hot-spot for copy-choice recombination.

Template switching during reverse transcription is crucial for retroviral replication. While strand transfer on the terminal repeated sequence R is essential to achieve reverse transcription, template switching from internal regions of the genome (copy choice) leads to genetic recombination. We have developed an experimental system to study copy-choice recombination in vitro along the HIV-1 genome. We identify here several genomic regions, including the R sequence, where copy choice occurred at high rates. The frequency of copy choice occurring in a given region of template was strongly influenced by the surrounding sequences, an observation that suggests a pivotal role of the folding of template RNA in the process. The sequence R, instead, constituted an exception to this rule since it was a strong hot-spot for copy choice in the different sequence contexts tested. We suggest therefore that the structure of this region has been optimised during viral evolution to ensure efficient template switching independently from the sequences that might surround it.

Influence of phase duration of biphasic waveforms on defibrillation energy requirements with a 70-microF capacitance.

BACKGROUND: Phase duration of biphasic shocks may be an important determinant of defibrillation success. The purpose of this study was to investigate the effect of changing phase duration of biphasic pulses delivered by 70-microF capacitors on defibrillation energy requirements. This may be clinically relevant for the optimization of implantable cardioverter-defibrillator design and programming. METHODS AND RESULTS: Defibrillation thresholds (DFTs) were determined for 13 waveforms in 13 pigs by application of a 70-microF capacitance and a transvenous/submuscular lead system. In part I, phase-1 duration varied, preserving a phase-1/phase-2 duration ratio of 60%/40%. The phase-1 durations were 1, 2, 3, 4, 5, and 6 ms. The DFT was lowest (22.9+/-7 J) for phase 1=3 ms compared with phase 1=1 ms (36.4+/-7.5 J), 2 ms (25+/-6.5 J), 4 ms (25+/-7.6 J), 5 ms (30.7+/-7.3 J), or 6 ms (32.9+/-8.1 J) (P<.001). In part II, phase-1 duration was 3 ms but phase-2 duration varied: 0.7, 1.3, 2, 2.7, 3.3, 4, and 6 ms. Significant DFT minima were found at phase 2=2 ms (22.5+/-4.2 J) and phase 2=4 ms (22.5+/-4.2 J) compared with phase 2=0.7 ms (31.7+/-9.3 J), phase 2=3.3 ms (26.7+/-6.1 J), or phase 2=6 ms (28.3+/-6.8 J) (P<.05). CONCLUSIONS: The strength-duration curve of biphasic defibrillation shocks demonstrates a single optimum for phase-1 duration. In contrast, two optima with minimal energy requirements were found for phase-2 duration. Optimization of both phases of low-capacitance biphasic shocks may reduce energy requirements for defibrillation.

Epidemiology of hydatidosis/echinococcosis in Ouarzazate, the pre-Saharian region of Morocco.

In the Ouarzazate province of Southern Morocco, 1085 cattle and 358 sheep were examined for hydatid cysts. The prevalence was 44.6% (range 8.3-83.4%) in cattle and 5.3% (range 1.3-28.6%) in sheep. The prevalence increased with the age of the animals. The lung was the predominant site of infection, followed by the liver. In cattle, 14.2% of hydatid cysts were fertile and 16.4% had degenerative changes. The mean loss per head of cattle slaughtered was about 1 kg of liver and 900 g of lungs in the urban abattoirs. In monetary terms at the current price of 1980, this meant a loss of U.S. +4.5 at urban and +1.6 at rural abattoirs. The mean prevalence of infection in 61 stray dogs was 50.8%, with a mean worm burden of 413. The urban dogs had a lower infection rate (42.9%) than the rural dogs (61.5%). There were 130 dogs per 1000 inhabitants. The human population was ignorant of the life-cycle of Echinococcus granulosus and was not aware of risk of infection through dogs. Dogs become infected at abattoirs, at home slaughter and in the field by consuming dead carcasses. The maintenance and transmission of E. granulosus in animals and man is related to social, religious and cultural factors.