Checkpoint-mediated control of replisome-fork association and signalling in response to replication pausing

The replication checkpoint controls the integrity of replicating chromosomes by stabilizing stalled forks, thus preventing the accumulation of abnormal replication and recombination intermediates that contribute to genome instability. Checkpoint-defective cells are susceptible to rearrangements at chromosome fragile sites when replication pauses, and certain human cancer prone diseases suffer checkpoint abnormalities. It is unclear as to how the checkpoint stabilizes stalled forks and how cells sense replication blocks. We have analysed the checkpoint contribution in controlling replisome-fork association when replication pauses. We show that in yeast wild-type cells, stalled forks exhibit stable replisome complexes and the checkpoint sensors Ddc1 and Ddc2, thus activating Rad53 checkpoint kinase. Ddc1/Ddc2 recruitment on stalled forks and Rad53 activation are influenced by the single-strand-binding protein replication factor A (RFA). rad53 forks exhibit a defective association with DNA polymerases alpha, epsilon and delta. Further, in rad53 mutants, stalled forks progressively generate abnormal structures that turn into checkpoint signals by accumulating RFA, Ddc1 and Ddc2. We suggest that, following replication blocks, checkpoint activation mediated by RFA-ssDNA filaments stabilizes stalled forks by controlling replisome-fork association, thus preventing unscheduled recruitment of recombination enzymes that could otherwise cause the pathological processing of the forks.     

Prevention of restenosis after stenting: the emerging role of inflammation

Restenosis after stent implantation is mainly caused by neointimal proliferation through the stent struts. Experimental studies indicate a marked activation of inflammatory cells at the site of stent struts, which are likely to play a key role in the process of neointimal proliferation and restenosis. Coronary stenting is a strong inflammatory stimulus and the acute systemic inflammatory response to local inflammation produced by coronary stenting is highly individual and predicts restenosis and event-free survival. The benefit of anti-inflammatory therapy during the periprocedural period and long-term follow-up is dependent on the inflammatory status. Measurement of cytokine and acute phase proteins, such as C-reactive protein, may be important to identify high-risk subjects and develop specific treatment tailored to the individual patient.     

Avian adenovirus CELO recombinants expressing VP2 of infectious bursal disease virus induce protection against bursal disease in chickens

To develop a CELO virus vector that can induce protection against infectious bursal disease, CELO viruses expressing the host-protective antigen VP2 of infectious bursal disease virus (IBDV) were constructed. In the engineered recombinants, the VP2 gene (the 441-first codons of the IBDA polyprotein) was placed under the control of the CMV promoter. Two positions in the CELO genome were chosen to insert the VP2 expression cassette. The recombinants were found apathogenic, when inoculated by different routes and even at high doses (up to 10(8) per animal). Chickens vaccinated oro-nasally with these different recombinants and challenged with very virulent IBDV were found to be poorly protected. In contrast, when inoculated with one or two (subcutaneous or intradermic) injections of CELOa-VP2, the chickens showed no clinical signs and no mortality after challenge. In the vaccinated chickens, the titers of neutralization antibody reached 7-9 values, showing that protection could be explained by the induction of a sufficient humoral response. After challenge, the weight ratio Bursa of Fabricius/body was about 2.5 per thousand, a value similar to that obtained with the commercial Bur706 vaccine. However, histological lesions in the Bursa of Fabricius were observed, showing that a complete protection was not totally achieved. Contact transmission was evidenced. Protection was also obtained when inoculation of CELOa-VP2 was carried out in ovo. Prime-boost strategies were also tested with the CELOa-VP2 vector used in association with the purified VP2 antigen, or DNA encoding VP2 or a CELO vector expressing chicken myeloid growth factor (cMGF). None of these regimens were shown to substantially increase the level of protection when compared to double CELOa-VP2 inoculations. These results indicate that CELO-based vectors are useful to safely induce a strong protective immunity against vvIBDV in chickens.     

Two regions of deletion in 9p22- p24 in neuroblastoma are frequently observed in favorable tumors

Neuroblastoma is a tumor of infancy that presents several chromosomal abnormalities. Nonrandom deletion of chromosome arm 9p has been identified in primary neuroblastoma suggesting the presence of a tumor suppressor gene located on this chromosome. In previous work, we showed that CDKN2A and CDKN2B genes, mapped at 9p21, were not deleted in neuroblastoma cells. In the present article, we refine the deleted region of 9p using polymerase chain reaction-based analysis of highly polymorphic simple sequence repeats and a two color fluorescence in situ hybridization technique on interphase nuclei. We analyzed 71 primary tumors of patients at the onset of the disease. We found loss of heterozygosity (LOH) in 16 of 71 (23%) cases; the frequency of LOH for 9p was higher (28%) in favorable stages 1, 2, and 4s than in unfavorable stages 3 and 4 (14%). Our results identify two regions of frequent allelic loss: the first at the locus D9S1849 and the second at the locus D9S157. These regions appear to be distant from CDKN2A and CDKN2B loci suggesting that other genes may be involved in 9p deletion. Finally, our data show that 9p deletion is more frequent in tumors of patients with a favorable prognosis, indicating that deleted genes may not be crucial for tumor progression.     

Prevalence estimates for chronic diseases in Italy: exploring the differences between self-report and primary care databases

BACKGROUND: The aim of this study was to describe population and primary care morbidity and to examine how the differences vary across the diseases and are influenced by patients' demographic characteristics. METHODS: A comparison of the prevalence of four chronic conditions for 432747 patients from the Health Search Database (HSD) and 119799 individuals from a Health Interview Survey was carried out. A linear regression was performed to study the associations between age and difference in morbidity. RESULTS: Similar prevalence was found for diabetes and hypertension, whereas for chronic obstructive pulmonary disease (COPD) and gastroduodenal ulcer lower HSD prevalence was reported. Among females, age was always associated with morbidity difference. Among males, significant associations were found only for COPD (R2 = 0.81; p = 0.001) and gastroduodenal ulcer (R2 = 0.93; p < 0.001). CONCLUSIONS: The difference between population and primary care morbidity is affected by disease under investigation and patients' demographic characteristics. Therefore, in choosing the more cost-effective approach to collect data such evidence should be taken into account, and results should be interpreted with great caution.     

The cyclopentenone prostaglandin 15-deoxy-delta(12,14)- PGJ2 attenuates the development of colon injury caused by dinitrobenzene sulphonic acid in the rat

1. Inflammatory bowel disease (IBD) is characterized by oxidative and nitrosative stress, leukocyte infiltration, and increased expression of the adhesion molecules intercellular adhesion molecule 1 (ICAM-1) in the colon. Recent evidence also suggests that the cyclopentenone prostaglandin (PG) 15-deoxy-delta(12,14)-PGJ(2) (15d- PGJ(2)) functions as an early anti-inflammatory signal. 2. The aim of the present paper is to investigate the effects of 15d-PGJ(2) in rats subjected to experimental colitis. 3. Colitis was induced in rats by intra-colonic instillation of dinitrobenzene sulphonic acid (DNBS). 15d-PGJ(2) was administered daily as intraperitoneal injection (20 or 40 microg kg(-1)). On day 4, animals were sacrificed and tissues were taken for histological and biochemical analysis. 4. 15d-PGJ(2) significantly reduced the degree of haemorrhagic diarrhoea and weight loss caused by administration of DNBS. 15d-PGJ(2) also caused a substantial reduction of (i) the degree of colonic injury, (ii) the rise in myeloperoxidase (MPO) activity (mucosa), (iii) the increase in the tissue levels of malondialdehyde (MDA) and (iv) of the pro-inflammatory cytokines tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta). 5. Furthermore, 15d-PGJ(2) reduced the increase in immunohistochemical staining for (i) inducible nitric oxide synthase (iNOS), (ii) nitrotyrosine and (iii) poly (ADP-ribose) polymerase (PARP), as well as (iv) the increased expression of ICAM-1 caused by DNBS in the colon. 6. Electrophoresis mobility shift assay (EMSA) of inflamed colon revealed that 15d- PGJ(2) also caused a substantial reduction of the activation of nuclear factor-kappaB (NF-kappaB). Furthermore, 15d-PGJ(2) stimulates the activation of heat shock protein 72 (hsp72) in the inflamed colon, as assessed by Western blot analysis. 7. In conclusion, 15d-PGJ(2) reduces the development of experimental colitis.     

Relationship between AgNORs,MIB-1 and oncogene expression in male breast carcinoma and papillary superficial bladder neoplasm

The expression of p53, c-erbB-2, bcl-2 and c-myc proteins was compared to the quantity of the nucleolar organiser regions (AgNORs) and MIB-1 antigen to elucidate the relationship between oncogene expression and rapidity of cell proliferation and tumor growth fraction. Sections from 50 male breast carcinomas (MBC) and 62 superficial papillary bladder neoplasias were stained with the standardised AgNOR method and monoclonal antibodies MIB-1, DO7, CB11, bcl-2 124 and 9E11. p53 immunopositivity was associated with high AgNOR quantity and MIB-1 scores both in MBC and bladder neoplasm. c-erbB-2 expression was associated with high AgNOR quantity in bladder neoplasm. bcl-2 expression was associated with low AgNOR quantity in MBC. c-myc expression was associated with high AgNOR quantity in MBC. MBC patients with low AgNOR quantity, and p53, c-erbB-2 and c-myc immunonegativity had the longest overall survival. Patients with bladder neoplasia with low AgNOR quantity, negative p53 and positive c-erbB-2 immunostaining had the longest disease-free survival time. Our results indicate that p53 overexpression reflects both the rapidity of cell proliferation, as assessed by AgNOR quantity, and tumor growth fraction, as assessed by MIB-1 scores, while c-erbB-2, c-myc and bcl-2 expression mainly reflects the rapidity of cell proliferation. The combination of AgNOR quantity and oncogene expression may stratify patients into different risk groups.     

Microalbuminuria, renal function and development of sustained hypertension: a longitudinal study in the early stage of hypertension

OBJECTIVE: Microalbuminuria (MA) is a marker of adverse outcome in hypertension. The aim of this study was to investigate the association of MA with cardiovascular risk factors and glomerular hyperfiltration in the early stage of hypertension and to assess its predictive value for the development of sustained hypertension requiring antihypertensive treatment. DESIGN AND PARTICIPANTS: We studied 1041 young stage 1 hypertensive subjects. Study variables were 24-h ambulatory blood pressure and heart rate, anthropometric measures, metabolic variables, creatinine clearance and lifestyle factors analyzed as a function of ascending urinary albumin measured from 24-h collections. Subjects were followed until they developed sustained hypertension and were eligible for antihypertensive medication according to current guidelines. SETTING: Seventeen outpatient clinics in Italy. RESULTS: Eighty-five percent of the subjects were normoalbuminuric, 9% had borderline MA, and 6% had overt MA. No between-group differences were found for age, body mass index, heart rate, lifestyle factors and biochemistry in both genders. Creatinine clearance was greater in the subjects with overt MA and borderline MA than in the normoalbuminuric subjects (P = 0.003 and 0.011, respectively). In a two-way ANCOVA, microalbuminuric subjects both with hyperfiltration (P < 0.001) and with normal filtration (P = 0.04) had higher 24-h systolic blood pressure than subjects with normoalbuminuria and normal filtration. In a Cox analysis, neither MA nor hyperfiltration were significant predictors of development of sustained hypertension. CONCLUSION: MA is not associated with an adverse metabolic risk profile in the early stage of hypertension. MA is associated with greater hemodynamic load and with glomerular hyperfiltration in this clinical setting, but does not help in predicting those subjects destined to develop sustained hypertension requiring antihypertensive therapy.     

Factors associated with growth patterns from birth to 18 months in a Beninese cohort of children

The aim of this study was to analyze factors influencing the growth pattern of children from birth to 18 months. A longitudinal prospective study was conducted in three maternity wards in Southern Benin. Inclusion took place between June 2007 and July 2008; children were followed-up until 18 months of age. Height-for-age and weight-for-height Z-scores were computed using the newborn's anthropometric measurements taken at delivery, every month up to 6 months and then quarterly. Infant and young child feeding (IYCF) practices and malarial morbidity were recorded. Gestational age was estimated using the Ballard method; William's sex-specific reference curve of birth weight-for-gestational-age was used to determine intrauterine growth retardation (IUGR). Analyses were performed on 520 children using a linear mixed model. Low birth weight (coef = −0.43; p = 0.002), IUGR (coef = −0.49; p < 0.001), maternal short stature (coef = −0.25; p = 0.001) and maternal low weight status (coef = −0.19; p = 0.006) were significantly associated with growth impairment. Only LBW (coef = −0.28; p = 0.05) and maternal low weight status (coef = −0.23; p = 0.004) were associated with wasting. A good IYCF score was positively associated with weight gain (coef = 0.14; p < 0.001) whereas we found a paradoxical association with length (coef = −0.18; p < 0.001). Malaria morbidity was not associated with growth. LBW, IUGR and maternal low weight status and height were important determinants of children's growth. These results reinforce and justify continuing public health initiatives to fight IUGR and LBW and break the intergenerational cycle of malnutrition.