Localizing a putative mutation as the major contributor to the development of sporadic Hirschsprung disease to the RET genomic sequence between the promoter region and exon 2

Hirschsprung disease (HSCR), a congenital disorder characterized by intestinal obstruction due to absence of enteric ganglia along variable lengths of the intestinal tract, occurs both in familial and sporadic cases. RET mutations have been found in approximately 50% of the families, but explains only a minority of sporadic cases. This study aims at investigating a possible role of RET in sporadic HSCR patients. Haplotypes of 13 DNA markers, within and flanking RET, have been determined for 117 sporadic HSCR patients and their parents. Strong association was observed for six markers in the 5' region of RET. The largest distortions in allele transmission were found at the same markers. One single haplotype composed of these six markers was present in 55.6% of patients versus 16.2% of controls. Odds ratios (ORs) revealed a highly increased risk of homozygotes for this haplotype to develop HSCR (OR>20). These results allowed us to conclude that RET plays a crucial role in HSCR even when no RET mutations are found. An unknown functional disease variant(s) with a dosage-dependent effect in HSCR is likely located between the promoter region and exon 2 of RET.     

Walking modality affects respiratory muscle action and contribution to respiratory effort

We hypothesized that walking at increased speed or increasing gradient might have different effects on chest wall kinematics and respiratory muscle power components, and contribute differently to respiratory effort sensation. We measured the volumes of chest wall compartments by optoelectronic plethysmography, esophageal, gastric and transdiaphragmatic (P _di) pressures, and the sensation of the respiratory effort by a Borg scale in five normal subjects walking both at ascending gradient with constant speed (AG) and at ascending speed with constant gradient (AS). Chest wall kinematics, evaluated by displacement of chest wall compartments, did not show any significant difference between AS and AG. Muscle power, calculated as the product of mean flow and mean pressure, increased similarly, but its partitioning into pressure and velocity of shortening differed in the two modes. A greater increase in the pressure developed by the abdominal muscles (P _abm) (4.06-fold), and in the velocity of shortening of both rib cage inspiratory muscles (v _rcm,i) (2.01-fold) and the diaphragm (v _di) (1.90-fold) was associated with a lower increase in the pressure developed by the rib cage inspiratory muscles (P _rcm,i) (1.24-fold) and P _di (0.99-fold) with AG. Instead, with AS, a lower increase in P _abm (2.12-fold), v _rcm,i (1.66-fold) and v _di (1.54-fold) was associated with a greater increase in P _rcm,i (1.56-fold) and P _di (1.97-fold). A combination of P _abm and v _di during AG (Wald ²=23.19, P<0.0000), with the addition of P _rcm,i during AS (Wald ²=29.46, P<0.0000), was the best predictor of Borg score. In conclusion, the general strategy adopted by respiratory centers during different walking modes does not differ in terms of ventilation, chest wall kinematics, and respiratory muscle power production, whereas it does in terms of partitioning of power into pressure and velocity of shortening, and respiratory muscle contribution to respiratory effort sensation. Combinations of different patterns of flow and pressure generation made the respiratory effort sensation similar during AS and AG modes.     

Stem cell therapy for neurodegenerative diseases: the issue of transdifferentiation

In the past few years research on stem cells has exploded as a tool to develop potential therapies to treat incurable neurodegenerative diseases. Stem cell transplantation has been effective in several animal models, but the underlying restorative mechanisms are still unknown. Several events such as cell fusion, neurotrophic factor release, endogenous stem cell proliferation, and transdifferentiation (adult cell acquisition of new unexpected identities) may explain therapeutic success, in addition to replacement of lost cells. This issue needs to be clarified further to maximize the potential for effective therapies. Preliminary stem transplantation trials have already been performed for some neurodegenerative diseases. There is no effective pharmacological treatment for amyotrophic lateral sclerosis, but recent preliminary data both in experimental and clinical settings have targeted it as an ideal candidate disease for the development of stem cell therapy in humans. This review summarizes recent advances gained in stem cell research applied to neurodegenerative diseases with a special emphasis to the criticisms put forward.     

Endogenous levels of mRNA for IFNs and IFN-related genes in hepatic biopsies of chronic HCV-infected and non-alcoholic steatohepatitis patients

To investigate the intra-hepatic activation of the IFN system in patients affected by chronic HCV-infection in comparison with that observed in a non-infectious liver disease such as non-alcoholic steatohepatitis, we measured the liver steady state mRNA levels of interferon-alpha, interferon-beta and interferon-gamma as well as of IFN-related genes (IFNAR-1, STAT1alpha, PKR, 2-5 AS, IRF-1, ICE and IL-18). In HCV-infected subjects, possible correlations of these parameters with viral load and liver injury were also analyzed. Twenty-four chronic untreated HCV-infected subjects and seven patients with non-alcoholic steatohepatitis were enrolled in the study. Liver biopsies were graded according to Knodell scores. Intra-hepatic mRNA levels of IFNs and related genes were assessed by semi-quantitative RT-PCR. In comparison with non-alcoholic steatohepatitis, in HCV-infected subjects IFN-alpha and -beta mRNA levels were significantly lower, whereas IFN-gamma, IFNAR-1, STAT1alpha IRF-1, and IL-18 mRNA were upregulated. Moreover, IFN-gamma mRNA steady state levels were correlated positively with those of IFNAR-1, IRF-1, and IL-18, suggesting a coordinated induction of these genes. Although plasma viral load was correlated inversely with IL-18-specific mRNA, viral load was not related to liver injury. IFN-gamma and IRF-1 mRNA levels were correlated positively with ALT, but not with the grading or staging. Conversely, IFN-alpha and -beta mRNA levels were higher in livers with lower staging scores. These findings support the hypothesis that in chronic HCV infection there is an imbalance between an upregulated IFN-gamma system and a downregulated IFN-alpha and -beta system, probably due to a mixed effect exerted by HCV-specific and inflammatory non-specific factors.     

The nature of high frequency sister chromatid exchange cells (HFCs)

We employed the three-way differential staining technique (TWD),which allows SCEs to be distinguished on a per generation basisby scoring third metaphases (M3), in order to study the spontaneouslevels of SCEs in normal and high frequency cells (HFCs) thatoccurred in the first (S1), second (S2) and third (S3) S phases.Fifty one of 900 lymphocytes from 37 healthy donors were definedas HFCs by calculating the 95th percentile of the distributionof SCEs in S1 + S2. ‘Normal’ cells presented almostthe same number of SCEs after the first, second and third cellcycles (SCE averages of 2.43, 2.04 and 3.53 respectively). Incontrast, HFCs showed a higher SCE count in SI, which decreasedrapidly through the cycles and reached baseline level at S3(SCE averages of 7.18, 4.29 and 3.45 respectively). This wouldsuggest that the lesions responsible for the higher SCE frequencyin HFCs were effectively removed after two cell cycles and stronglysupport the hypothesis that HFCs are lymphocytes which accumulatehigher levels of DNA lesions through time. ¹To whom correspondence should be addressed     

Tumour necrosis factor β gene polymorphisms in myasthenia gravis

Genetic analyses indicate that genes within the major histocompatibility complex (MHC) can be involved in susceptibility to autoimmune disease. To investigate the role of the tumour necrosis factor beta (TNFB) gene in myasthenia gravis (MG) susceptibility, we analysed an NcoI polymorphism within the TNFB gene in 63 MG patients and 93 healthy individuals. When patients were subdivided according to thymic pathology, we found differences between MG patients with thymic hyperplasia and thymoma versus controls. In MG patients with thymic hyperplasia we found a positive association with the TNFB*1 allele [Relative risk (RR): 2.6; P<0.001] and phenotype (RR: 1.8; P<0.005) and a negative association with the TNFB*2/2 genotype (RR: 0.2; P<0.001) when compared to the controls. On the other hand, in MG patients with thymoma we found a positive association with the TNFB*2/2 genotype (RR: 5.6; P<0.01) and a negative association with the TNFB*1 allele (RR: 0.3; P<0.05) and *1/2 genotype (RR: 0.2; P<0.01). These data suggest that the two different forms of MG can have different pathogenesis and that the TNFB gene could influence susceptibility to MG.