The time required for radiographic union following femoral fracture increases with age in both humans and rats for unknown reasons. Since abnormalities in fracture innervation will slow skeletal healing, we explored whether abnormal mRNA expression of genes related to nerve cell activity in the older rats was associated with the slowing of skeletal repair.Simple, transverse, mid-shaft, femoral fractures with intramedullary rod fixation were induced in anaesthetized female Sprague-Dawley rats at 6, 26, and 52 weeks of age. At 0, 0.4, 1, 2, 4, and 6 weeks after fracture, a bony segment, one-third the length of the femur, centered on the fracture site, including the external callus, cortical bone, and marrow elements, was harvested. cRNA was prepared and hybridized to 54 Affymetrix U34A microarrays (3/age/time point).The mRNA levels of 62 genes related to neural function were affected by fracture. Of the total, 38 genes were altered by fracture to a similar extent at the three ages. In contrast, eight neural genes showed prolonged down-regulation in the older rats compared to the more rapid return to pre-fracture levels in younger rats. Seven genes were up-regulated by fracture more in the younger rats than in the older rats, while nine genes were up-regulated more in the older rats than in the younger.mRNA of 24 nerve-related genes responded differently to fracture in older rats compared to young rats. This differential expression may reflect altered cell function at the fracture site that may be causally related to the slowing of fracture healing with age or may be an effect of the delayed healing. Bone formation to bridge the fracture gap following skeletal fracture slows with age in both humans -6 and raThese earlier studies done with RT-PCR revealed a paucity of data for genes differentially expressed by age. We had hypothesized that bone formation to bridge the fracture gap would be under a negative-feedback control system. Thus, the genes which stimulate bone formation should be up-regulated in adult or older rats to attempt to accelerate their slower progression of bony healing. This was not observed in adult  or olderIn this project, mRNA gene expression was measured by DNA microarray technology at various time points after fracture for young, adult, and older rats. The goal was to identify genes whose expression following fracture was altered by age. Such genes may either show reduced expression, if the age-related slowing of healing is caused by inadequate expression levels, or they may show enhanced expression, in an attempt to stimulate some poorly responding pathway. Among the genes which were differentially expressed at the fracture site with age were genes related to nerve cell activity.In this study, we explored whether abnormal mRNA expression of genes related to nerve cell activity was associated with the slowing of skeletal repair in older rats. Abnormalities in the innervation of the fracture site will slow skeletal healing clinically -15 and ead libitum. The work was done in an AAALAC-accredited vivarium under protocols approved by our Institutional Animal Care and Use Committee.Intact female Sprague-Dawley rats (Harlan Sprague-Dawley, Inc., Indianapolis, IN) were purchased at one or six months of age and housed in our vivarium in pairs until they were the proper age for experimentation. The rats were fed Teklad Rodent Diet [W] (#8604, Harlan Teklad, Madison, WI) and tap water Intact female Sprague-Dawley rats at 6 (young), 26 (adult) or 52 (older) weeks of age, weighing 154 ± 11 g (mean ± SD), 281 ± 25 g, and 330 ± 30 g respectively, were anaesthetized with an intraperitoneal injection of ketamine and xylazine (30 mg and 5 mg/kg body weight respectively) as described earlier ,11. The ). The sample preparation is described here in brief. Total RNA was extracted from the tissue by TRIzol (Invitrogen Life Technologies, Carlsbad, CA) with disruption of the tissue in a Brinkman Polytron homogenizer. RNA from two rats of the same age and time point was pooled for each microarray sample. Samples with 30 μg RNA were purified on RNeasy columns by Qiagen  and then converted to double-stranded cDNA with a Superscript Double Stranded cDNA Synthesis Kit (Invitrogen Life Technologies, P/N 11917-010). The cDNA was then expressed as biotin-labeled cRNA by in vitro transcription (IVT) with the Enzo RNA Transcript Labeling Kit (Affymetrix, P/N 900182). Each sample was spiked with bioB, bioC, bioD, and cre (Affymetrix P/N 900299). The biotin-labeled cRNA was fragmented non-enzymatically. The fragmented cRNA was hybridized to 54 Rat U34A microarrays (Affymetrix P/N 900249) in the Affymetrix hybridization buffer for 16 hours at 45 C. The hybridized arrays were washed and stained in the Affymetrix Fluidics Station 400 to attach fluorescent labels to the biotin, followed by biotin-labeled antibody, and then a second staining with fluorescent labeling of the biotin. Each array was scanned twice by the Agilent GeneArray Scanner G2500A . Three arrays from three independent samples (six rats) were done for each age at each time point.Samples were prepared as described in the Affymetrix GeneChip Expression Analysis Technical Manual  of all genes to an average of 500 for each array. For each gene, the software reported a signal value and a Present/Marginal/Absent call. This latter algorithm was a statistical comparison of the variation among the several probe sets for each gene compared to the noise level and gave a call for each gene as Present, Marginal, or Absent. The program then compared the signal value of each gene in the fractured samples against the signal value of the same gene in the unfractured control sample. The difference between the two signal levels, relative to the variability between the multiple probes for each gene, yielded a probability of change due to chance alone. Genes with p less than 0.005 were judged significantly different from the same gene in the unfractured sample. This more conservative p value was employed to minimize false positive responses.The Data Mining Tool was used for cluster analysis with the Self Organizing Map (SOM) algorithm. The data were clustered on the signal values between 20 and 20,000 with the maximum/minimum ratio of at least 3.0 and the maximum – minimum difference of at least 100. One hundred clusters were specified. and in the PubMed database . GenBank accession numbers and names are shown for each gene. Each graph shows the average ± SEM of the three microarrays that were done for each time point for each age. Significant changes in gene expression were demonstrated by t test and linear regression [Nerve-related genes were identified by searches for nerve-related names in the gene descriptions of each gene on the microarray. This association was confirmed by a review of the information for that gene in the NetAffx web site gression .. A copy of the full microarray data set has been deposited in the NCBI Gene Expression Omnibus  as series GSE594.This report conforms to the MIAME standards of MGED In all young rats, bone bridged the fracture gap by four weeks after surgery. By six weeks after fracture, remodeling was beginning to obscure the fracture site Fig. . In contOn each array, on average, 5,200 genes were scored as absent, and 3,300 as present. Of these, 1,159 were significantly up-regulated and 928 were significantly down-regulated at two weeks after fracture in the adult rats of the first series see . Up-reguSOM clusters identified genes up- or down-regulated by fracture. Most genes affected by fracture followed the same time course at all three ages. These genes showed approximately the same peak expression level and regressed to baseline at about the same time point at all three ages. Among the genes affected by fracture were a number of genes associated with nerve cells. These were selected for more intense analysis.vs. 0.4 week , followed by a significant increase at 6 weeks after fracture compared to 0.4 week after fracture (P < 0.001 by t test for 9 samples).Up-regulated nerve-related genes are shown in Table vs. 4 and 6 week old).SOM cluster analysis identified three types of defects in the older rats. In the first type, a number of genes were down-regulated by fracture at all three ages. However, while genes in the younger rats were returning to pre-fracture expression levels by six weeks after fracture, there was less recovery in the older rats. These genes are shown in Table In the second type of defect, other genes were up-regulated by fracture, but the response was weaker in the older rats. These genes are shown in Table In the third type of defect, genes were also up-regulated by fracture. However, the response was stronger in the older rats than in the younger rats. These genes are shown in Table For each gene for each array, the Microarray Suite software reported a statistical decision as to whether the mRNA was Present, Marginal, or Absent. We have reviewed these calls for the genes shown in Figures In this study, as in our earlier work ,10-12, tMore than two-thirds of the detectable genes on the rat U34A microarray have a change in mRNA expression level following fracture . Most ofTo evaluate this hypothesis, nerve-related genes were studied from among the genes present on the Affymetrix Rat U34A microarray. Genes were identified for which the mRNA response to femoral fracture was changed in the older rats compared to the young rats. Three types of change with age were found:1. The mRNA expression levels of the genes shown in Table 2. Other nerve-related genes had strong up-regulation after fracture in young rats but only mild up-regulation in older rats. These are shown in Table 3. A third set of genes was increased in mRNA expression by fracture, but the increase was greater in the older rats. These are shown in Table All three classes of genes showed altered expression in the older rats compared to young rats. We hypothesize that bone fracture may physically disrupt nerve fibers in bone. A sub-population of these skeletal nerve fibers may regrow into the fracture site or regain function at a slower rate in older rats. This may account for the failure to recover from low mRNA values for the first group (prolonged down-regulation) or the failure to up-regulate mRNA expression adequately after fracture in the older rats in the second group (diminished up-regulation). Other genes in the third group with increased levels of mRNA after fracture in the older rats may represent attempts to stimulate nerve regrowth or other processes that are not responding. This may represent negative-feedback-induced up-regulation caused by effector cell resistance. Taken together, these changes in nerve cell function with age may contribute to the slowing of fracture repair in older rats. It must be pointed out that the associations noted here do not necessarily reflect cause and effect. It is also possible that the delayed re-innervation of the fracture site is an effect of the delayed healing in the older rats and not a cause of the delayed healing.et al. [Experimental studies have been done to detect the role of innervation on fracture healing. Studies of sectioning the sciatic nerve in concert with tibial fracture have been reported to speed fracture healing -33. Howeet al.  have repet al. . Direct et al. .et al. [et al. [et al. [In humans, abnormal bone healing is also associated with lack of nerve activity at the fracture site. Nagano et al.  have not [et al.  have fou [et al.  have fouHumans -6 show aThese changes with age are not limited to genes related to neuronal activity. We have also noted similar changes in genes related to mitochondrial activity . It is lThe specificity of these changes is seen in the majority of the nerve-related genes for which the expression pattern following fracture was unaffected by age. These transcripts had similar increases or decreases following fracture in the young, adult, and older rats. These uniform responses suggest that most metabolic patterns were unaffected by age. Nerve-related genes similarly up-regulated by femoral fracture at all three ages were broadly related to differentiation and growth of nerve cells, to known up-regulation following nerve injury, or to association with apoptosis . Good coIn summary, most genes respond to bone fracture with altered mRNA gene expression, including genes related to neuronal functioning. However, a number of these genes responded to fracture differently in older rats than in young rats. Such differential expression with age may reflect altered cell functioning at the fracture site that may be related to the slowing of fracture healing in older rats.None declared.MM, WE, and RM participated in the surgery and the sample collection. MM and WE prepared and hybridized the samples and analyzed the data. RM prepared the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: as samples GSM9028 (no-fracture sample) and GSM9031 (2-week sample).Additional File 1. Response of adult rats at two weeks after fracture. This spreadsheet is a listing of all genes altered by fracture in the first adult rat sample at two weeks after fracture compared to the first adult no-fracture sample. The data are sorted on the probability value for the comparison of each gene between the two arrays. For each array, the signal (mRNA transcript level), detection  and detection p-value are given. In addition, for the two-week sample, the change  and change p-value are given for the comparison of the two-week sample to the no-fracture sample. These two arrays are archived in the GEO repository Click here for file


Colorectal cancer is a common cancer all over the world. Aberrations in the cell cycle checkpoints have been shown to be of prognostic significance in colorectal cancer.cyclin D1, cyclin A, histone H3 and Ki-67 was examined in 60 colorectal cancer cases for co-regulation and impact on overall survival using immunohistochemistry, southern blot and in situ hybridization techniques. Immunoreactivity was evaluated semi quantitatively by determining the staining index of the studied proteins.The expression of cyclin D1 gene amplification and protein overexpression (concordance = 63.6%) and between Ki-67 and the other studied proteins. The staining index for Ki-67, cyclin A and D1 was higher in large, poorly differentiated tumors. The staining index of cyclin D1 was significantly higher in cases with deeply invasive tumors and nodal metastasis. Overexpression of cyclin A and D1 and amplification of cyclin D1 were associated with reduced overall survival. Multivariate analysis shows that cyclin D1 and A are two independent prognostic factors in colorectal cancer patients.There was a significant correlation between Cyclin A and D1 are superior independent indicators of poor prognosis in colorectal cancer patients. Therefore, they may help in predicting the clinical outcome of those patients on an individual basis and could be considered important therapeutic targets.Loss of cell cycle checkpoints control is common in colorectal cancer.  In Egyp kinases ,5. The cll cycle . Abnormacyclin D ,7.Cyclin D1 is a G1 cyclin that regulates the transition from G1 to S phase since its peak level and maximum activity are reached during the G1 phase of the cell cycle. Whereas cyclin A is regarded a regulator of the transition to mitosis since it reaches its maximum level during the S and G2 phases [cyclins include chromosomal translocations, gene amplification and aberrant protein overexpression [2 phases . The mecpression ,9.histone H3 mRNA expression can be used to identify the S phase fraction (SPF) through the in situ hybridization (ISH) technique [histone H3 mRNA reaches its peak during the S phase and then drops rapidly at the G2 phase [Several studies have shown that, echnique ,11. The G2 phase .Ki-67 (pan-cell cycle marker), cyclin D1 (G1 phase marker), histone H3 mRNA (S phase marker), cyclin A (S to G2 phase marker) in CRC. The expression level of these markers was correlated to the clinicopathologic features and the overall survival of patients.In face of the increasing incidence of CRC and its peculiar pattern in the Egyptian population, the present study was conducted to assess the role of Paraffin-embedded tumor tissues were obtained from 60 CRC patients  that were diagnosed and treated at the National Cancer Institute, Cairo, Egypt during the period from January, 1997 to June, 2002. Clinicopathological data of the studied cases are illustrated in table Ki-67 (MIB-1, Dako), cyclin A  and cyclin D1 (DCS-6, Dako). A case of invasive breast cancer was used as a positive control for Ki-67 and cyclin A whereas a case of mantle cell lymphoma was used as a control for cyclin D1. Negative controls were obtained by replacing the primary antibody by non-immunized rabbit or mouse serum.Four micron sections of each normal and tumor specimen were cut onto positive-charged slides; air dried overnight, de-paraffinized in xylene, hydrated through a series of graded alcohol and washed in distilled water and 0.01 PBS (pH 7.4). Slides were then processed for IHC as described by Handa et al. . using tBrown nuclear staining was regarded as a positive result for all studied markers. The proportion of positively-stained cells and the intensity of staining were scored in tumor and normal colorectal mucosal sections at medium power (×200). The degree of positive tumor staining (percentage of positive tumor cells in the examined section) was scored from 1–6 and the staining intensity was scored from 0–6 according to the pattern of staining in the examined section. Staining index (SI) was calculated by multiplying the cellularity and staining scores as described by King et al. .histone H3 mRNA by ISH using the commercially available 550 base fluorescein-labeled DNA probe (Dako, Carpinteria, CA) as described by Nagao et al., 1996. This probe hybridizes to the whole mRNA transcript of the human histoneH3 gene including the5' and 3' un-translated regions. Scoring of histone H3 mRNA was performed as for immunohistochemistry, however, hybridization signals were detected in the cytoplasm.All tumor samples and 5 normal controls were assessed for cyclin D1 if more than 75% of the examined sections were formed of neoplastic cells. Accordingly, 50 cases were eligible for the analysis. Ten micrograms of the extracted DNA was digested with EcoR1. DNA from selected cases was also digested with BglII and HindIII. Samples were separated on 0.8% agarose gels and transferred to Hybond-N membranes (Amersham Int., Amersham, UK). The membranes were hybridized with 50% formamide, 5 × SSC, 5 × Denhardt's, 500 μg/ml denatured salmon sperm DNA, 10% dextran sulphate and 106 cpm/ml of 32P-labeled PRAD-1 probe for 24 h. Membranes were washed with 2 × SSC, 0.1% SDS at room temperature for 30 min followed by 2 × SSC, 0.1% SDS at 60°C for 30 min and 0.1 × SSC, 0.1% SDS at 60°C for 1 h. Filters were autoradiographed using an intensifying screen at -70°C for 24–72 h. After being stripped free of the PRAD-1 probe, the same blots were hybridized with 32P-labeled B-actin probe to normalize against possible variations in the loading or transfer of DNA. The autoradiograms were analyzed using a densitometer. Intensities of PRAD-1/cyclin D1 were normalized to the β-actin control bands. The degree of amplification was calculated from these normalized values. Amplification was considered when the signal of the tumor band was ≥2-fold the value of the matched normal mucosa [High molecular weight DNA was extracted from paraffin-embedded tissues of the tumor and normal colorectal mucosal samples as previously described . The prol mucosa .p. values less than 0.05 were considered significant.The Mann-Whitney non-parametric test was used to compare the SIs of pairs of subjects whereas the Kruskal-wallis was used for categorial data. Correlation between indices was performed using a simple linear regression test. The Kaplan-Meier method was used to create survival curves which were analyzed by the log-rank test. The impact of different variables on survival was determined using the Cox proportional hazards model. p = 0.0001). Normal colorectal mucosa revealed positive imunostaining for Ki-67 in the lower half of the crypts only. A heterogeneous staining pattern was detected in the neoplastic cells of well and moderately-differentiated adenocarcinomas whereas a diffuse homogeneous staining pattern was detected in poorly-differentiated carcinomas. The SI ranged from 10–40.2 (mean: 24.6 ± 6.5).The results of IHC are illustrated in figures cyclin D1 was predominantly nuclear but cytoplasmic staining was detected in some cases. However, unless a nuclear staining was also detected, cases with cytoplasmic staining were considered negative. Normal colorectal mucosal samples were almost negative for cyclin D1 whereas 41 out of the 60 (68.3%) CRC cases were positive. Marked heterogeneity was observed in well- and moderately-differentiated adenocarcinomas even within the same tumor. Poorly-differentiated carcinomas revealed a diffuse staining pattern with more darkly-stained nuclei. The SI ranged from 0.5–28.6 (mean: 9.3 ± 4.2).Immunostaining for cyclin A was detected in 80% (48/60) of CRC cases and in all non-neoplastic control samples. Positively-stained nuclei were confined to the lower half of the crypts in normal colonic mucosa and diffusely-dispersed in carcinomas. The SI ranged from 3.3–30.2 (mean: 15.1 ± 6.6).Positive nuclear staining for Histone H3 mRNA was intensely expressed in the cytoplasm of all examined samples either neoplastic or non-neoplastic. The distribution of histone H3 mRNA was similar to that of cyclin A and Ki-67 however, the proportion of histone H3 mRNA positive cells was less than that of Ki-67. The SI ranged from 1.8–24.2 (mean: 12.4 ± 5.3).PRAD-1 probe detected 3 EcoRI fragments of 4.0, 2.2 and 2.0 and 1 BglII fragment of 15 Kb. PRAD-1/cyclin D1 gene amplification was detected in 22/50 (44%) cases analyzed. The degree of amplification was heterogeneous with 2–10 fold increase when compared to normal mucosal samples .There was a significant correlation between Ki-67 and cyclin D1, cyclin A, histone H3 as well as between the SIs of cyclin A and histone H3 (p = 0.008, 0.0001, and 0.0001 respectively) . In addition, SI of Ki-67 and histone H3 were higher in patients <50 years than in those ≥50 years (p < 0.05)  Figure . There wcyclin D1 SI and large, poorly-differentiated tumors, carcinomas with positive lymph node metastasis and deeply-invasive carcinomas (p < 0.05, p < 0.001, p < 0.05 and p < 0.05 respectively). Whereas cyclin D1 gene amplification was significantly associated with an advanced disease stage since amplification was detected in 10/15 (66.7%) of stage IV tumors compared to 12/45 (26.7%) of stage I-III tumors (p = 0.002). Similarly, DNA amplification was detected in 60.5% (26/43) of the carcinomas with extensive local invasion (beyond sm) but only in 23.5% (4/17) of the carcinomas with limited invasion (m, sm) (p = 0.001). A significant correlation was also present between cyclin D1 gene amplification and the presence of lymph node metastasis (p = 0.008) as well as between the SI of histone H3, the size of the tumor and the patient's age (p < 0.05, p < 0.001 respectively). The SI was higher in tumors >5 cm in diameter and in patients <50 years.In addition table cyclin A overexpression (our definition: SI ≥ 10.5), cyclin D1 overexpression (our definition: SI ≥ 6.1), poorly differentiated histology, lymph node metastasis, TNM stage, tumor size and depth of invasion were all significant prognostic variables for survival (Table Ki-67 overexpression (our definition: SI ≥ 11.5) and histone H3 overexpression (our definition: SI ≥ 8.2) tended to have poor prognosis but this did not reach a statistically significant level, however the overall survival was significantly lower in patient with cyclin A and cyclin D1 overexpression. Cox multivariate regression analysis revealed that lymph node metastasis, cyclin A and cyclin D1 overexpression were independent negative prognostic factors after adjustment for the depth of tumor invasion, age and sex of the patient . Eighteen of 60 patients had already died by the time the study was completed. We defined the cutoff level for overexpression of each cell cycle marker at the point that showed the maximum difference of survival rate between the 2 groups separated by that point. Cox regression analysis revealed that nt Table .cyclins or by flowcytometric determination of the SPF of the cell cycle [cyclin D1 gene amplification in a panel of 47 CRC cell lines; its protein was overexpressed in about 30% of CRC cases that were included in the studies of Bartakova et al. [cyclin D1 was aberrantly accumulated in a significant subset of human CRC cases and the cell lines derived from these cases were dependent on cyclin in their cell cycle progression. In the second study [cyclin D1 was detected in 30% of adenomatous polyps indicating that overexpression is a relatively early event in colon carcinogenesis which is possibly responsible for the pathological changes in the mucosa preceding neoplastic transformation. More recently, Holland et al. [cyclin D1 in 58.7%, 100% and 43% of their studied cases respectively.The proliferative activity of CRC cells has been investigated in several studies either by immunohistochemical determination of cell proliferation index using antibodies to some types of ll cycle . Althougll cycle  did not a et al.  and Arbea et al. . In the a et al. cyclin D1nd study , overexpd et al. , Pasz-Wad et al.  and Utsud et al.  reportedcyclin D1 was detected in 68.3% of the cases. The SI was significantly higher in carcinomas than in normal colorectal mucosa and in poorly-differentiated adenocarcinomas it was approximately twice that of other histological types. Amplification and/or overexpression of cyclin D1 significantly correlated with deeply invasive tumors and positive lymph node metastasis. Our results in this regards are consistent with previous studies [cyclin D1 and p21waf proteins are important in colorectal tumorigenesis and have implications for patient prognosis. Similarly McKay et al. [cyclin D1 was the only protein in their panel (cyclin D1, p53, p16, Rb-1, PCNA and p27) that correlated with improved outcome in CRC patients. However, few studies failed to detect any correlation between cyclin D1 overexpression and the clinicopathological factors in CRC [cyclin D1 overexpression and the high histological grade was also reported in other tumor types including non-small cell lung carcinomas [In the present study, up-regulation of  studies . In 2001 studies . demonsty et al.  found ths in CRC . This corcinomas  and squacyclin D1 was more common than gene amplification of the PRAD-1/cyclin D1 gene with a 63.6% concordance. This was similarly reported by Bartakova et al. [cyclin D1 is overexpressed without PRAD-1/cyclin D1 gene amplification. Consistent with this hypothesis are reports of elevated cyclin D1 mRNA levels and immunohistochemically detectable accumulation of the protein in over one third of breast cancer cases at a frequency significantly higher than that deduced from DNA amplification studies [cyclin D1 in solid tumors.In the present work, overexpression of  studies ,25. Thescyclin D1 overexpression in various carcinomas, including CRC [cyclin D1 overexpression tended to have poor prognosis.So far, several studies were done to reveal the prognostic significance of ding CRC . Howevercyclin A positive carcinomas had significantly shorter median survival times. Handa et al. [cyclin A overexpression in 77% of their CRC cases. They also demonstrated that, cylcin A could be used as a prognostic factor of CRC. More recently, Habermann et al. [cyclin A overexpression. They found that, cyclin A overexpression was higher in cases of ulcerative colitis with adenocarcinomas than in those without adenocarcinomas. Consequently, they concluded that, cyclin A could be used for monitoring ulcerative colitis patients and for the early detection of an emerging carcinoma in this high risk group of patients.It was reported that, patients with a et al.  were abln et al.  studied cyclin A was detected in 80% of the patients and Cox regression analysis showed that it could be used as a prognostic marker in CRC in addition to cyclin D1.In our study, cyclin A by another technique (DNA amplification). This would have added more information regarding the gene status on one hand and confirmed the results of IHC on the other hand. Unfortunately, this was not possible because in most of the cases included in the present work, the extracted DNA was not sufficient to study cyclin amplification after the assessment of cyclin D1.It would have been useful if we assessed the expression level of histone H3 labeling index significantly correlated with ki-67 immunostaining and was high in poorly differentiated human hepatocellular carcinoma. This was similarly reported in the present work since we found a significant correlation between the SI of histone H3 and Ki-67. However, no statistically significant correlation was found between histone H3 SI and any of the studied clinicopathological factors.In 1996, Nagao et al.  reportedKi-67 immunostaining reflects the proliferative activity of CRC, it has not been recognized as a significant prognostic factor in this type of tumors [Ki-67 labeling index and local invasion of CRC. In the present study there was a significant relationship between the SI of Ki-67, tumor size and grade. However, Kaplan-Meier survival curves showed no significant difference in survival rates between patients with- and without overexpression of Ki-67.Although f tumors ,28. Howef tumors  found a cyclin D1, cyclin A, histone H3 and Ki-67 are overexpressed in a subset of CRC, however only cyclin D1 and cyclin A overexpression correlates with poor differentiation and tumor progression. This indicates the superiority of cyclin A and cyclin D1 as indicators of poor prognosis compared to Ki-67 and histone H3 mRNA in CRC. Cyclin A and D1 could therefore be considered significant, independent prognostic factors in CRC patients. These findings are especially important in stage II patients since 25–30% of those patients have poor prognosis in spite of being node-negative. However, the standard clinicopathologic prognostic factors can not identify this subset accurately and therefore; there is a great demand for more accurate, individually-based, biological prognostic parameters that help in detecting this high risk group of patients who can benefit from an adjuvant therapy. If the findings of the present study are confirmed in a larger study, evaluation of cyclin A and D1 may be applicable to clinical management of CRC, allowing the identification of patients with poor prognosis.Our results demonstrate that The author(s) declare that they have no competing interests.CRC – Colorectal cancerOS – overall survivalSI – staining indexSPF – S phase fractionISH – in situ hybridizationm – muscularis mucosasm – invasion of the sub mucosaBA and ZA-R carried out the molecular genetic studies, designed, coordinated the study and drafted the manuscript. BA and El-HS carried out all the histopathological and immunohistochemical studies. El-SA participated in molecular genetic studies and drafted the manuscript. MM coordinated the study. El-SM carried out all the patient clinical data. All authors read and approved the final manuscriptThe pre-publication history for this paper can be accessed here:


Islet transplantation offers hope to many patients with diabetes, who envision a life free of glucose checks and insulin injections. What are the barriers to its widespread implementation? Treatment for, and the prognosis of, type-1 diabetes mellitus (T1DM) has progressed dramatically during the last century, but the disease remains a major cause of morbidity and mortality. Although precise figures are not available, over 1 million United States citizens currently live with the disease, with approximately 30,000 new cases diagnosed in the US each year. The total number of people with diabetes worldwide is expected to rise to 366 million in 2030, up from 171 million in 2000 The exact etiology of the disease remains uncertain, but extensive research suggests an interaction between genetic predisposition and environment. In fact, for unknown reasons, the incidence of T1DM is increasing Banting and Best's discovery of insulin in the early 1920s revolutionized diabetes treatment and greatly improved the prognosis for what had previously been a rapidly fatal disease. As shown by the Diabetes Control and Complications Trial and the more recent Epidemiology of Diabetes Interventions and Complications trial, insulin therapy has made such considerable advances (with better insulin formulations and delivery systems) that many patients can maintain their blood sugar levels within a tight range and thereby reduce their risk for the disease's long-term complications ,4,5. In Just as financial investors balance a portfolio, with some risky investments and others that are more secure, researchers will undoubtedly continue to further refine “secure” insulin-based regimens to help patients achieve even better glycemia control. At the same time, scientists are pursuing more high-risk, high-payoff approaches to revolutionize diabetes care. One such approach is the closed-loop insulin pump (i.e., a pump that continuously monitors blood glucose and concurrently converts that data into appropriate insulin dosing), which offers the potential to serve as a mechanical pancreas. However, such a mechanical system would need be fail-safe in order to avoid devastating effects . In other, similar scenarios with no tolerance for error, NASA (for instance) sets up systems in which two independent monitoring systems must come up with similar measurements before an action is taken. Perhaps the engineering obstacles that currently limit the closed-loop insulin pump can be overcome.Other research groups are investigating whether the insulin-producing cells within the pancreas , might be promoted to regenerate (in vitro or in vivo) to replace the pool of insulin-producing cells reduced by autoimmune destruction. Another promising approach for creating cells capable of physiologically regulated insulin secretion is to “coax” stem cells—undifferentiated cells with self-regenerative capacity—to differentiate into ß-like cells. Gene therapy approaches may overcome present obstacles and result in cells capable of physiologically regulated insulin secretion While such potential therapies remain experimental, pancreas transplantation is currently performed in patients with complicated diabetes. However, a recent report that shows benefit for patients with both diabetes and kidney failure who receive a combined pancreas and kidney transplant also found that an isolated pancreas transplant (for patients with preserved kidney function) actually worsened survival Islet transplantation has recently received considerable interest as a potentially definitive treatment for diabetes. The concept of islet transplantation is not new—investigators as early as the English surgeon Charles Pybus (1882–1975) attempted to graft pancreatic tissue to cure diabetes. Most, however, credit the recent era of islet transplantation research to Paul Lacy's studies dating back more than three decades. In 1967, Lacy's group described a novel collagenase-based method (later modified by Dr. Camillo Ricordi, then working with Dr. Lacy) to isolate islets, paving the way for future in vitro and in vivo islet experiments While significant progress has been made in the islet transplantation field Looked at another way, patients with heart, liver, lung, or kidney failure have a dismal prognosis for survival, so the toxicity associated with immunosuppression is warranted . But for the subset of patients with diabetes and preserved kidney function, even those with long-standing and difficult-to-control disease, the prognosis for survival is comparatively much better. In addition to the immunosuppressive toxicities, other risks are associated with the islet transplant procedure itself, including intra-abdominal hemorrhage following the transplant, and portal vein thromboses. The fact that there is already a good alternative to islet transplantation (i.e., the modern intensive insulin regimen) forces us to regard any newer, riskier interventions with a critical eye.Like all transplantation therapies, islet transplantation is also handicapped by the limited donor pool. The numbers are striking; at least 1 million Americans have T1DM, and only a few thousand donor pancreata are available each year. To circumvent this organ shortage problem, researchers continue to look for ways to grow islets—or at least cells capable of physiologically regulated insulin secretion—in vitro, but currently only islets from cadaveric donors can be used to restore euglycemia. Further exacerbating the problem (and unlike kidney, liver, and heart transplants, where only one donor is needed for each recipient) most islet transplant patients require islets from two or more donors to achieve euglycemia. Lastly, the current methods for islet isolation need improvement, since only about half of attempted isolations produce transplant-ready islets.While islet transplantation research has made important progress and the success stories are encouraging, the long-term safety and efficacy of the procedure remain unclear. Other concerns relating to the field include questions about the impact of having insulin-producing foreign cells within the hepatic parenchyma, the long-term consequences of elevated portal pressures resulting from the islet infusion, and the fact that islet recipients can be sensitized against donor tissue types, making it more difficult to find a suitable donor should another life-saving transplant be required in the future. Also, very few islet transplant recipients have remained euglycemic without the use of any exogenous insulin beyond four years post-transplant. Thus, while most islet recipients achieve better glycemia control and suffer less serious hypoglycemia, islet transplantation continues to fall short of the definitive diabetes cure.While no one suggests that the therapy is ready for widespread clinical application, another way of highlighting current problems is to focus on cost. Assuming present hurdles were cleared, islet transplantation costs approximately $150,000 per patient per transplant. With over 1 million Americans dealing with T1DM, it would cost over $100 billion to give each patient a single islet transplant, with little assurance as yet of any long-term benefit. In contrast, the annual direct cost of a proven therapy like intensive insulin treatment is about $3,500 per patient The limitations of islet transplantation force us to recognize that the therapy remains experimental, and that many questions must be answered before it is incorporated into general clinical practice. At the present time, we urge a focus on the selection of only those patients for whom this procedure offers the greatest likelihood of benefit. Most people with diabetes can, with diligence and perseverance, implement an insulin regimen that maintains tight glucose control while avoiding dangerous hypoglycemia. However, there are some patients who continue to have tremendous difficulty managing their disease despite optimal care and effort. Even the statement “despite optimal care and effort” is difficult to define, and we advocate that all patients being considered for an islet transplant first be referred for several months to specialty teams that are committed to diabetes care. Since such patients whose diabetes is the most difficult to control have a poor quality of life, islet transplantation offers potential benefit. Even a low baseline level of insulin production by the transplanted islets may lower the amount of insulin required, while reducing the number and severity of hypoglycemic events. We also believe the islet transplant risk-benefit ratio is favorable for those with both T1DM and kidney failure who are listed for a life-preserving kidney transplant; such patients will have to take immunosuppressive agents after transplant to preserve the kidney allograft function, so the islets can be added without too much additional risk.Where do we go from here? Just as early studies showed islet transplantation's promise, research must now overcome the hurdles revealed by the recent islet transplant experience. New immunomodulatory agents offer the greatest hope of revolutionizing the field. New drug regimens capable of inducing tolerance to the transplanted islets would allow recipients to maintain their grafts without general immunosuppression and its associated toxicities. While many targets are currently under investigation, none are ready for clinical use. We advocate that such immunomodulatory approaches be tested first in controlled models where the results can be appropriately attributed to the agent itself.Less than a century ago, T1DM was invariably a fatal disease. With the advent of insulin, the prognosis changed overnight, and we have continued to witness improvements in diabetes care and outcomes. Pancreatic islet transplant has offered renewed hope to many patients with diabetes, who envision a life free of glucose checks and insulin injections. Some transplanted patients have enjoyed “success” and are pleased with their decisions; unfortunately these results are not universal. Researchers must continue to look for ways to improve the procedure while protecting the welfare of each individual patient. The field has come a long way, but we must remain cautious, as we are treating a non-fatal disease for which there is a very effective standard therapy.


Advances in optical imaging modalities and the continued evolution of genetically-encoded fluorescent proteins are coming together to facilitate the study of cell behavior at high resolution in living organisms. As a result, imaging using autofluorescent protein reporters is gaining popularity in mouse transgenic and targeted mutagenesis applications.in vivo, and to evaluate fusion of a human histone protein to green fluorescent protein for ubiquitous fluorescent labeling of nucleosomes in mice. To this end we have generated embryonic stem cells and a corresponding strain of mice that is viable and fertile and exhibits widespread chromatin-localized reporter expression. High levels of transgene expression are maintained in a constitutive manner. Viability and fertility of homozygous transgenic animals demonstrates that this reporter is developmentally neutral and does not interfere with mitosis or meiosis.We have used embryonic stem cell-mediated transgenesis to label cells at sub-cellular resolution in situ. Since this reporter segments three-dimensional space, it permits the visualization of individual cells within a population, and so facilitates tracking cell position over time. It is therefore attractive for use in multidimensional studies of in vivo cell behavior and cell fate.Using various optical imaging modalities including wide-field, spinning disc confocal, and laser scanning confocal and multiphoton excitation microscopy, we can identify cells in various stages of the cell cycle. We can identify cells in interphase, cells undergoing mitosis or cell death. We demonstrate that this histone fusion reporter allows the direct visualization of active chromatin  Macro- and microscopic imaging are pivotal readouts in the field of biology both for determining the normal (baseline) course of events and for observing the effects of experimental perturbations and natural aberrations . Recent To construct high-resolution anatomical models of normal, mutant and pathological situations, we must establish technologies to identify and follow individual cells in three-dimensional (3D) space and in 3D over time, in four-dimensions (4D). Unfortunately, native fluorescent proteins permit tracking the position of any given cell over time only if the population of tagged cells is distributed among non-expressing cells by virtue of lineage or in a mosaic experimental situation -9. In siin toto imaging  of indiing mice -12. For As the principal structural proteins of eukaryotic chromosomes, histones are attractive targets for fluorescent nuclear labeling. Histone tagged fluorescent protein fusions have previously been shown to incorporate into chromatin without any adverse effects on the viability of cells in culture . When coTo date GFP fusions to several histones have been generated and used for labeling nuclei in live transgenic animals, including nematode worms, fruit flies and zebrafish ,14,16,17To evaluate histone-tagged fluorescent protein fusions in embryonic stem (ES) cells and mice, we generated constructs comprising an N-terminally positioned human H2B sequence followed at the C-terminus by sequences for various fluorescent proteins both GFP and DsRed-based. We previously reported that DsRed1 was not amenable to use in ES cells or mice , howeverThe H2B-fluorescent protein fusions we generated were introduced into vectors utilizing the CAG promoter  designedn = 30) we calculated the progression from early prophase to cytokinesis to take less than one hour .H2B-EGFP expressing ES cells are shown in Fig. our Fig. c. No losWe next tested the effects of widespread expression of an H2B fusion protein in mice. We generated germ line chimeras and established transgenic lines of mice constitutively expressing H2B-EGFP. We were able to breed this transgene to homozygosity, resulting in viable and fertile animals exhibiting widespread expression with no overt morphological abnormalities. The transgene has been maintained for over three years in a breeding colony of homozygous mice with no apparent effect on viability, breeding performance or lifespan. We therefore infer that this fusion protein is developmentally neutral and does not interfere with either mitosis or meiosis. Wide-field microscopic analysis of both mouse embryos and adult organs demonstrates widespread expression of the H2B-EGFP fusion in all types of nucleated cells.z-stacks) that can be used to generate high-resolution anatomical volumetric  images with details of interphase chromatin in addition to mitotic chromosomes and fragmenting nuclei. To do this, stacks of sequential optical sections are reconstructed into 3-dimensional projections. This methodology can be used to generate 3-dimensional (3D) image sets not only of cells propagated in culture but also of cells in situ in living animals and is illustrated here by imaging whole mouse embryos at the 4-cell stage, the blastocyst stage, and the pre-gastrula stage  7.5 embryo, cells of the definitive endoderm, mesoderm and embryonic ectoderm can be distinguished solely on the basis of nuclear morphology and orientation in addition to their expected position Fig. . Low magryo Fig. i reveal ryo Fig. j, and elryo Fig.  and 5, iryo Fig. l.Wide-field microscopic examination of organs from adult animals revealed widespread fluorescence as has been reported for animals expressing native fluorescent proteins under the regulation of the CAG promoter ,22,27. LFinally we investigated whether we could follow cell movement, division, and death in time-lapse experiments using various imaging modalities. We cultured ES cells and embryos on the stages various each of which had been modified to permit culture under physiological conditions. The different types of data routinely generated using different optical imaging modalities that are widely used and commercially available are illustrated in Figure in vivo through the generation of transgenic embryonic stem (ES) cells and mice having widespread expression of this reporter.Here we report the evaluation of a chromatin localized histone fusion fluorescent reporter in vivo data and allows it to be integrated onto a high-resolution anatomical framework. This type of multidimensional data is complex and thus difficult to digitize and compile into a standardized and integratable format. In toto imaging of fluorescent protein expressing specimens on a large-scale could be used for generating high-resolution digitally recorded anatomical databases where the baseline (wild-type) cell behaviors and cell fates can be contrasted to those observed in mouse mutants. However, developing in toto imaging technologies for acquiring large amounts of data will necessitate improving the speed and throughput of microscopic image acquisition and analysis. This would also be coincident with the ongoing development of improved computational approaches to mine and integrate this type of data [discussed in ref. [The transgenic mice that we have generated provide a new tool for high-resolution live imaging of a genetically tractable mammalian model organism ,11. TheyMuch of the information generated using a fluorescent fusion reporter such as the H2B-EGFP fusion is analogous to conventional histology ,31 excepin vitro culture and optical imaging [The future development and availability of mouse strains constitutively expressing spectral variant histone H2B fusions should prove useful for visualizing anatomy and tracking different populations of cells in multiple dimensions at high-resolution in mice as has previously been demonstrated in other organisms which are classically perceived as being more amenable to  imaging ,16. They imaging , in addi imaging . Also, s imaging . Additioin vivo imaging using genetically-encoded reporters in mice to sub-cellular resolution. The development of additional strains permitting spectrally-distinct high-resolution live cell in vivo imaging, coupled to advances in optical imaging modalities and the development of improved computational methods to mine imaging data should pave the way for a multidimensional understanding of biological processes.The CAG::H2B-EGFP strain that we have generated takes in vivo imaging approaches using transgenic animals expressing genetically-encoded fluorescent proteins will not only provide high-resolution information on cell behavior in specific biological processes [in vivo biological information which could mirror the recent explosion of available genomic data. Therefore recent advances in live imaging will need to be paired with developments in computational biology, as appropriate informatics methods will need to be developed and implemented in order to mine, present and integrate this type of in vivo biological data.It is anticipated that in the future, rocesses ,33, but Pfx Polymerase (Invitrogen). The resulting product was cloned into pCR4 TOPO (Invitrogen) to generate pH2B. The H2B fragment was then cloned into plasmids pEGFP-N1, pDsRed2-N1 pDsRedExpress-N1 (BD Biosciences, Inc) in order to generate plasmids pH2B-EGFP, pH2B-DsRed2 and pH2B-DsRedExpress (oligonucleotide sequences are available upon request). The resulting fusions were then re-amplified by PCR and cloned into the XhoI site of pCAGGS [The coding sequence for the human histone H2B gene (X57127) was amplified from genomic DNA by PCR using f pCAGGS  to generin vitro under both stem cell conditions and conditions employed to promote their differentiation were maintained further. For stem cell conditions ES cells were grown on gelatin in the presence of LIF. For differentiation, ES cells were grown on bacteriological Petri dishes in the absence of LIF for 2–5 days to promote embryoid body formation. Thereafter embryoid bodies were re-plated onto tissue culture dishes in the presence of factors promoting directed differentiation.Transgenic ES cell lines constitutively expressing H2B-EGFP were generated by co-electroporation of the linearized reporter construct and a circular PGK-Puro-pA plasmid  conferriTo assess whether an H2B fusion can continue to be widely expressed and transmitted through the germline of mice we used H2B-EGFP expressing ES cells for chimera generation by injection into C57BL/6 blastocysts using standard procedures . Chimera2) chamber (Bioptechs Inc. or Solent Sci Ltd. or home-made). For cytoplasmic staining, samples were incubated in Cell Tracker Orange  for 10–20 minutes. Embryos were kept in a standard tissue culture incubator at 37°C during staining. Samples were then washed twice with warm dissecting or culture media prior to imaging.Embryos and organs were dissected in HEPES buffered DMEM media containing 10% fetal calf serum, then cultured either in a standard tissue culture incubator or on a microscope stage under standard conditions promoting the culture of mouse embryos ,36 in 50All images shown  fitted on a Zeiss Axiovert 200M microscope with illumination from a 488 nm Argon laser (Melles Griot). Laser scanning confocal and multiphoton excitation data were taken on a Zeiss LSM510 NLO on a Zeiss Axioscop 2 FS MOT microscope. Objective lenses used on the Axiovert 200M and Axioscop 2 were plan-apochromat 63x/NA1.4, C-apochromat 40x/NA1.2, a plan-apochromat 20x/NA0.75 and a fluar 5x/NA0.25. For laser scanning microscopy GFP was excited using either a 488 nm Argon laser (Lassos, Inc) at 488 nm (for single-photon excitation) or a Titanium:Sapphire laser (Coherent Mira 900F with Verdi 5W pump laser) tuned between 860 and 890 nm (for two-photon excitation). Cell Tracker Orange was excited using a 543 nm HeNe laser (for single-photon use).z-stacks comprising sequential x-y sections taken at 0.1–2 μm z-intervals. Raw data was processed using a variety of packages including Zeiss AIM software (Carl Zeiss Microsystems at ), Image J (NIH at ) and Volocity (Improvision at ). Each image series was re-animated using software to make the time-lapse movies that are available as additional files. Both rendered volume and time-lapse movies were assembled in QuickTime Player (Apple Computer, Inc at ).Images were acquired as ).The CAG::H2B-EGFP strain of mice generated in this study will be made available through the Jackson Laboratories Induced Mutant Resource  half blastocyst. Generated from the same raw data set used to compile Supplementary File 1. Supplementary to stills presented in Fig. Click here for fileRotating 3D projection the node region of a live CAG::H2B-EGFP Tg/+ E7.5 embryo. Supplementary to stills presented in Fig. Click here for fileRotating 3D projection of the notochord of a live CAG::H2B-EGFP Tg/+ E10.5 embryo. Supplementary to Fig. Click here for fileRotating 3D projection of the branchial region of a live CAG::H2B-EGFP Tg/+ E10.5 embryo. Supplementary to stills presented in Fig. Click here for fileTime-lapse sequence of CAG::H2B-EGFP Tg/+ ES cells. Supplementary to stills presented in Fig. Click here for fileTime-lapse sequence of CAG::H2B-EGFP Tg/+ preimplantation embryos. Supplementary to stills presented in Fig. Click here for fileTime-lapse sequence of a CAG::H2B-EGFP Tg/+ postimplantation embryo. Supplementary to stills presented in Fig. Click here for fileRotating 3D projection of a fixed CAG::H2B-EGFP Tg/+ E8.5 embryo. Supplementary to stills presented in Fig. Click here for fileHigh-magnification rotating 3D projection of the headfold region of the same fixed CAG::H2B-EGFP Tg/+ E8.5 embryo as shown in Movie 7. Supplementary to stills presented in Fig. Click here for fileHigh-magnification rotating 3D projection of the allantois and posterior tail region of the same fixed CAG::H2B-EGFP Tg/+ E8.5 embryo as shown in Movie 7. Supplementary to stills presented in Fig. Click here for file


Hypertriglyceridemia in combination with low HDL cholesterol levels is a risk factor for cardiovascular disease. Our objective was to evaluate the efficacy of ciprofibrate for the treatment of this form of dyslipidemia and to identify factors associated with better treatment response.Multicenter, international, open-label study. Four hundred and thirty seven patients were included. The plasma lipid levels at inclusion were fasting triglyceride concentrations between 1.6–3.9 mM/l and HDL cholesterol ≤ 1.05 mM/l for women and ≤ 0.9 mM/l for men. The LDL cholesterol was below 4.2 mM/l. All patients received ciprofibrate 100 mg/d. Efficacy and safety parameters were assessed at baseline and at the end of the treatment. The primary efficacy parameter of the study was percentage change in triglycerides from baseline.2) compared to the rest of the population (8.2 vs 19.7%, p < 0.001). In contrast, cases with excess body weight had a larger decrease in non-HDL cholesterol levels (-20.8 vs -10.8%, p < 0.001). There were no significant complications resulting from treatment with ciprofibrate.After 4 months, plasma triglyceride concentrations were decreased by 44% (p < 0.001). HDL cholesterol concentrations were increased by 10% (p < 0.001). Non-HDL cholesterol was decreased by 19%. A greater HDL cholesterol response was observed in lean patients (body mass index < 25 kg/mCiprofibrate is efficacious for the correction of hypertriglyceridemia / low HDL cholesterol. A greater decrease in non-HDL cholesterol was found among cases with excess body weight. The mechanism of action of ciprofibrate may be influenced by the pathophysiology of the disorder being treated. Hypertriglyceridemia in combination with abnormally low concentrations of HDL cholesterol (High Density Lipoprotein Cholesterol) is one of the most common and atherogenic profiles of lipid metabolism . In the In the Veteran Affairs HDL Intervention Trial (VAHIT), the use of a fibrate (gemfibrozil) resulted in a 22% reduction in the incidence of cardiovascular events in subjects with low HDL cholesterol and a broad range of triglyceride values . The benThe trial included men and post-menopausal or non-pregnant women aged between 30 and 70 years who had hypertriglyceridemia (fasting concentrations between 1.68–3.9 mM/l (150 – 350 mg/dl) and hypoalphalipoproteinemia (HDL cholesterol ≤ 1.05 mM/l (40 mg/dl) for women and ≤ 0.92 mM/l (35 mg/dl) for men). The LDL cholesterol had to be lower than 4.2 mM/l (160 mg/dl). Patients were excluded if they had an acute coronary event during the three months preceding the study, type 1 diabetes, uncontrolled hypertension, severe renal dysfunction, nephrotic syndrome or aspartate aminotransferase (AST) or alanine aminotransferase (ALT) levels > 1.5 × the upper limit of normal (ULN), or if their creatine phosphokinase (CPK) levels were > 3 × ULN. Consumption of any lipid-altering drug within the previous 4 weeks (6 months for probucol) prevented entry into the study. Patients could be receiving other concomitant medication as long as the dosage was not modified during the study.The Ethics Committee in each institution approved the protocol and every patient provided witnessed, written informed consent prior to entering the study.This was a multicenter, international, open-label study. Patients were recruited from 25 lipid clinics from México (n = 152), Brazil n = 129), Chile (n = 78) and Colombia (n = 78). Cases fulfilling the inclusion criteria were invited to participate. The initial visit included a medical evaluation, a physical examination and the prescription of an isocaloric diet consisting of 50% carbohydrate, 30% fat, 20% protein with a cholesterol content of 200 mg [, Chile . In Brazil, the local laboratory of every center was used instead. Blood samples were taken after an overnight fast (≥ 9 hours). Measurements were performed during the first 24 hours after the blood was drawn; blood samples were kept at 4°C until the analysis. All laboratory analyses were performed with commercially available standardized methods. Glucose was measured using the glucose oxidase method. Total serum cholesterol and triglycerides levels were measured using an enzymatic method. HDL cholesterol levels were assessed using phosphotungstic acid and Mg2+.Statistical analysis was performed using SPSS for Windows version 10. An intention to treat analysis was used. Two sided ANOVA tests were used for assessing differences between groups for continuous variables. All categorical variables were analyzed using the chi squared test. Multiple logistic regression models were constructed for the identification of variables associated with the achievement of treatment goals.2 (n = 221); an additional 32.5% were obese (n = 142) Diabetes was present in 125 subjects (28.7%).Four hundred thirty seven patients were included. The clinical characteristics of the study subjects are shown in table Both evaluations were completed in every case. The medication was stopped before the trial was completed by 117 subjects (26.7%). In the majority of cases this was not related to side effects (see below). In addition, 46 cases (10.5%) had poor compliance to the medication. The body weight remained constant in all patients. The alcohol and tobacco consumption was not modified during the study.Ciprofibrate treatment and diet had a significant beneficial effect on the lipid profile, as shown in table The achievement of treatment goals is the ultimate aim of lipid-lowering therapy. Almost half of the cases had reduced their triglyceride concentrations below 1.68 mM/l (150 mg/dl) (n= 191(43.7%). HDL cholesterol levels above 1.05 mM/l (40 mg/dl) were found in 51% of the cases (n = 223). Also, a significant proportion of the subjects (63.15%, n = 276) achieved the non-HDL cholesterol goal 4.2 mM/l (< 160 mg/dl). The LDL cholesterol goal < 3.4 mM/l (< 130 mg/dl) was attained by 56.2 % (n = 246). A full correction of the hypertrigliceridemia / low HDL cholesterol occurred in 129 subjects (29.5%). Many of them also had a non-HDL cholesterol level below 4.2 mM/l (160 mg/dl) (n = 101, 23.1%)2. As is shown in figure The lipid response during treatment differed between cases with a body mass index above or below 25 kg/mPatients with diabetes had moderate hyperglycemia during the study. Their fasting glycemia was 7.7 ± 2.6 mM/l (139 ± 47 mg/dl) at baseline. A small but statistically significant decrease in glucose concentration was observed at the end of the trial (5.7 ± 3 mM/l, 104 ± 54 mg/dl p < 0.001). At baseline, their lipid profile differed, from the non-diabetic subjects, only with regards to higher triglyceride concentrations (3.13 ± 0.97 vs 2.95 ± 0.66 mM/l, p < 0.001). The lipid response to ciprofibrate and dietary treatment did not differ from that observed in the whole group. Individuals with a family history of dyslipidemia (n = 171) had higher cholesterol, triglycerides and LDL cholesterol at baseline compared to the rest of the population. The lipid response to treatment was similar to that reported in the whole group.2, the triglyceride response and the coexistence of cholesterol above 5.2 mM/l (200 mg/dl) (mixed hyperlipidemia) were predictors for a greater non-HDL cholesterol response  in comparison to that found in lean individuals. Since excess body weight is strongly associated with insulin resistance [A large range of lipid responses was observed between individuals. There are few reports designed to analyse the determinants of the lipid response to fibrates. Robins reported a lower HDL cholesterol elevation with a fibrate when insulin resistance is present . In thissistance ,17, our sistance ,19. The sistance -22 or bysistance . Genes esistance . AdditioSeveral limitations of the study must be recognized. The uncontrolled design resulted in a relatively high rate of drug discontinuation. However, this phenomenon is a common finding in studies done in open populations assessing the adherence to different lipid-lowering medications . To overIn conclusion, ciprofibrate is effective in the treatment of patients with hypertriglyceridemia / low HDL cholesterol. Significant reductions in triglycerides and non-HDL cholesterol resulted from ciprofibrate therapy. In addition, higher HDL cholesterol levels were found at the end of the treatment. Excess body weight alters the lipid response to ciprofibrate. A greater non-HDL cholesterol lowering is achieved in subjects with excess body weight compared to that found in lean individuals. Controlled trials are needed to compare the lipid-lowering effects of ciprofibrate in groups of subjects defined by their adiposity or other markers of insulin resistance.This study was supported by an educational grant provided by Sanofi-Synthelabo. It included the study expenses and it will cover the article processing charge. No other competing interest need to be declaredCAAS participated in the design of the study, performed the statistical analysis and drafted the manuscript. AALV participated in the design of the study and in the preparation of the manuscript. FJGP participated in the design of the study and in the preparation of the manuscript. All other authors were responsible of the inclusion and follow-up of the study subjects. All authors read and approved the final manuscript.


The consistent finding of higher prevalence of hypertension in US blacks compared to whites has led to speculation that African-origin populations are particularly susceptible to this condition. Large surveys now provide new information on this issue.Using a standardized analysis strategy we examined prevalence estimates for 8 white and 3 black populations (N = 85,000 participants).The range in hypertension prevalence was from 27 to 55% for whites and 14 to 44% for blacks.These data demonstrate that not only is there a wide variation in hypertension prevalence among both racial groups, the rates among blacks are not unusually high when viewed internationally. These data suggest that the impact of environmental factors among both populations may have been under-appreciated. Population surveys in the US from early in the last century have consistently documented higher blood pressures and related cardiovascular sequelae in blacks compared to whites ,2. The eInternational comparative studies on hypertension have been seriously limited by the absence of a valid method of standardization. In the last decade, however, high quality population surveys have been conducted in a wide range of populations that used either careful internal standardization or sufficiently comparable methods -15. We rBlack populations were drawn from the International Collaborative Study on Hypertension (ICSHIB) and the National Health and Nutrition Survey III ,16. A prThe surveys that formed the basis of ICSHIB were conducted in localized communities by door-to-door screening ,7. In sund BP from the clinic visit was used to create the mean for the age-gender groups, except for England where the 2nd home BP was used. Hypertension was defined as BP ≥ 140/90 mmHg or current use of antihypertensive medication.The examination methods have been reported in detail previously ,7,16. InBP, body mass index (BMI), and hypertension prevalence were calculated for 5-year age-gender groups and aggregated as the primary data file. To achieve maximum overlap we restricted the analysis to 35–74 years for age-specific estimates of BP and hypertension prevalence, and 35–64 years for age-adjusted results. In the US NHANES whites and blacks were analyzed separately with the appropriate weighting for population size. As previously reported, the prevalence estimates obtained for US blacks from ICSHIB were virtually identical to those from NHANES [The age-averaged BPs and BMIs are presented for each survey, by gender, in Table Hypertension prevalence, which accounts for the effect of treatment, follows a similar pattern although the east-west gradient among the African-origin groups is more consistent Table . Among tThe only etiological factor on which standardized information was available was obesity, measured by its proxy BMI. The correlation between average BMI and hypertension prevalence was 0.6 (p < 0.01), all populations combined. Within the black populations the same correlation was observed between mean BMIs and hypertension prevalence (r = 0.6). Among whites, however, the relationship was weaker (r = 0.3). Of course, since obesity will be correlated with many other aspects of lifestyle, it is difficult to infer whether weight gain itself is playing a less important role in determining the variation among white populations. The contrasts noted above in hypertension prevalence by gender are consistent with the relative excess of obesity in women compared to men among Jamaicans and US blacks ,6.Comparisons of BP distributions across populations are made difficult by the requirement of comparability of the survey methods. In the last two decades, however, adoption of standardized protocols along with rigorous training have greatly improved the quality of epidemiological studies of hypertension ,17-19. AThe data presented here demonstrate a two-fold variation in prevalence of hypertension in both European- and African-origin populations. The prevalences are similar in blacks in the US and whites in Europe, although important gender differences are apparent. Although not a systematic sample, the populations that are included generally reflect the characteristic social setting in which these groups are found around the world. Summed across all groups, the white populations on average have a substantially higher burden of hypertension. This result can be attributed in large part to the inclusion of several black samples from developing countries where risk factors for hypertension are presently at a lower level. In the only head-to-head comparison within the same survey, US blacks have a prevalence that is 50% higher than among whites. Data from the UK, including the national survey, also demonstrate higher BPs and more hypertension among blacks of Caribbean and African descent -27. On tAre these findings merely artifactual, reflecting either methodological error or the sampling process? The most unexpected features of the data presented here are the high rates of hypertension in Europe, when contrasted to whites in Canada and the US. These results have been reported in greater detail in an earlier publication . It is bThe primary purpose of this analysis was to provide descriptive results and very limited information was available on factors that might explain the findings we observed. The gradient among the black populations is consistent with the transition to an industrialized lifestyle and is thereby collinear with most known risk factors . BMI is Treatment guidelines and practice patterns vary widely among these countries -19. WideIf the North American-European contrasts are occurring in genetically homogeneous populations, large environmental influences must be at work that are not apparent on the surface. A similar process could be taking place across the social environments into which persons of African origin are assorted within societies such as the US and the UK. The debate over inherent susceptibility cannot be resolved with these data since neither the genetic nor the environmental influences can be held constant, allowing a test of the relative influence of the other factor. In fact, the question of inherent susceptibility is probably non-testable under any circumstances -37. WhilThe author(s) declare that they have no competing interests.RC, KWM, AL and JB were responsible for study concept, design and supervision. RC, KWM, JB, SG, MJ, AA, TF MK, PP, BS and MT were involved in data acquisition. RC, KWM, JB, MJ, PP and AL were responsible for analysis and interpretation of data. RC and KWM drafted the manuscript. RC, JB, SG, AL, AA, TF, MJ, MK, PP, BS and MT were involved in critical revision of the manuscript for important intellectual content. Statistical expertise was provided by MJ. Administrative, technical and material support was provided by KWM, MK, and BS.The pre-publication history for this paper can be accessed here:


Analyses of genetic data at the level of haplotypes provide increased accuracy and power to infer genotype-phenotype correlations and evolutionary history of a locus. However, empirical determination of haplotypes is expensive and laborious. Therefore, several methods of inferring haplotypes from unphased genotypic data have been proposed, but it is unclear how accurate each of the methods is or which methods are superior. The accuracy of some of the leading methods of computational haplotype inference (PL-EM, Phase, SNPHAP, Haplotyper) are compared using a large set of 308 empirically determined haplotypes based on 15 SNPs, among which 36 haplotypes were observed to occur. This study presents several advantages over many previous comparisons of haplotype inference methods: a large number of subjects are included, the number of known haplotypes is much smaller than the number of chromosomes surveyed, a range in values of linkage disequilibrium, presence of rare SNP alleles, and considerable dispersion in the frequencies of haplotypes.In contrast to some previous comparisons of haplotype inference methods, there was very little difference in the accuracy of the various methods in terms of either assignment of haplotypes to individuals or estimation of haplotype frequencies. Although none of the methods inferred all of the known haplotypes, the assignment of haplotypes to subjects was about 90% correct for individuals heterozygous for up to three SNPs and was about 80% correct for up to five heterozygous sites. All of the methods identified every haplotype with a frequency above 1%, and none assigned a frequency above 1% to an incorrect haplotype.All of the methods of haplotype inference have high accuracy and one can have confidence in inferences made by any one of the methods. The ability to identify even rare (≥ 1%) haplotypes is reassuring for efforts to identify haplotypes that contribute to disease in a significant proportion of a population. Assignment of haplotypes is relatively accurate among subjects heterozygous for up to 5 sites, and this might be the largest number of SNPs for which one should define haplotype blocks or have confidence in haplotype assignments. Very rapid and inexpensive methods exist for determining the genotype of diploid organisms at single nucleotide polymorphisms (SNPs). Unfortunately, these high-throughput methods do not provide direct information on which SNP alleles at multiple sites coexist on the same chromosome. Instead, computational methods must be employed to infer the set of SNP alleles that are cosegregating on a single chromosome, referred to as haplotypes. However, the inference of haplotypes from phase-unknown data is computationally difficult, partly due to the fact that the number of possible haplotypes roughly increases as a power of 2 with each additional SNP.Interest in the accurate inference of haplotype structure from unphased genotypic data has increased tremendously in recent years for several reasons. Relative to analysis of single polymorphisms, haplotypes can greatly improve one's ability to infer the evolutionary history of a DNA region ,2. AdditThere are three principal computational approaches to inferring haplotypes from unphased SNP data. The most commonly used approach is implementation of the expectation-maximization (EM) algorithm . This meThe accuracy of computational haplotype inference improves as the magnitude of linkage disequilibrium (LD) among sites increases . Gene coThe 15 sites studied by Horan et al.  span 535Increasing sample size improves the accuracy of inferred haplotypes. In Horan's study  308 chroThe distribution of haplotype frequencies also influences the accuracy of haplotype inference in two ways. First, the presence of some haplotypes at comparatively high frequency increases the chances that those haplotypes can be unambiguously inferred from homozygotes, allowing the alternative haplotype to be inferred with high confidence in compound heterozygotes. Second, the presence of some haplotypes at near-zero frequency allows truly nonexistent haplotypes to be accurately estimated as having zero frequency. The empirical haplotype frequencies in this study exhibit considerable dispersion. Two haplotypes are relatively common . The latter analyses were performed to better approximate the characteristics of data sets that are typically collected in genetic epidemiological studies. Although the presence of rare alleles and haplotypes improves the accuracy of haplotype inference, sites with a low frequency minor allele are often ignored due to their reduced usefulness in mapping disease loci and the assumption that such loci will contribute little to population-wide predisposition to disease. Very little difference was observed in the accuracy of haplotype inference between the two data sets.Assignment of haplotypes to individuals was very accurate by all methods Table . ApproxiF. As measured by the mean squared error (MSE) the implementation of the program Phase that ignored linkage disequilibrium among sites gave marginally lower accuracy for the full data set and for the data set composed of higher frequency alleles, but the magnitude of the MSE was small for all methods and spanned only about a two-fold difference between the best and worst value. PL-EM successfully identified the largest number of correct haplotypes, but this success rate was accompanied by the burden of the highest number of incorrect haplotypes inferred. Indeed, the aggregate frequency of incorrect haplotypes inferred by PL-EM was about 1% higher than for the other methods. This observation may have practical value for the analysis of unphased genotypic data. PL-EM may be slightly advantageous if the analytical goal of identify the largest number of correct haplotypes is much more important than minimization of the number of incorrect haplotypes inferred, which may be the case in studies of functional genetics. However, if minimization of the number of incorrect low-frequency haplotypes is more important, as will usually be the case in genetic epidemiological studies, PL-EM may not be the optimal method. Unfortunately, none of the methods is clearly superior in minimizing the number of incorrect haplotypes inferred.Estimation of haplotype frequencies was also highly accurate, and there was no meaningful difference in accuracy among the methods as measured by the similarity index, IImportantly, none of the methods failed to identify haplotypes with frequencies above 1%. Conversely, no incorrect haplotype was assigned a frequency greater than 1%. Indeed, the aggregate frequency of incorrect haplotypes was ≤ 3.7% by all methods. These results are reassuring in two respects. First, it appears unlikely that any of the methods will fail to identify a haplotype that is a major contributor to disease risk within a study population. Second, it also is unlikely that an incorrect haplotype will be implicated as a significant disease risk.It has been noted previously ,18 that k, where k is the number of heterozygous sites. For this reason, the difficulty of correctly assigning haplotypes to subjects increases dramatically as those subjects become heterozygous at more sites. Therefore, the error rate for assigning haplotypes was evaluated based on the number of sites at which subjects were heterozygous. Up to 3 heterozygous sites, the error rate is below 10%. For 4 heterozygous sites the error rate is about 15% and exceeds 20% only when 6 sites are heterozygous. Phase was an odd exception to this pattern due to an unusually high error rate for four heterozygous sites, despite the lowest error rates for five and six heterozygous sites. On the assumption that an error rate not much larger than 10% is desirable for a genetic study, it appears that computationally assigned haplotypes for subjects heterozygous at more than four SNPs should be viewed with extreme caution. Similarly, there is a current effort to define haplotype blocks in the human genome to facilitate genome-wide scans for disease loci with a minimum number of sites that must be genotyped. If the results for this gene can be generalized, it would appear unwise to define haplotype blocks based on more than 4–5 SNPs.The number of possible haplotypes compatible with an individual's unphased genotype is 2All of the implementations of the EM and Bayesian methods of haplotype inference had high accuracy. Therefore, if this data set is representative of other SNP genotyping studies one can have high confidence in the assignment of haplotypes and estimation of haplotype frequencies produced by any one of the programs. Each method identified every haplotype with a frequency greater than 1%. Therefore, it is unlikely that any of the methods would fail to identify a haplotype contributing to disease risk in a significant proportion of a population. Conversely, no incorrect haplotype was assigned a frequency greater than 1%, indicating a low probability of an incorrect haplotype being identified as a significant disease risk factor. Assignment of haplotypes was very accurate for subjects heterozygous for up to three SNPs, and was at least 80% accurate for up to five heterozygous sites. This suggests that haplotype blocks should perhaps be defined based on no more than five sites and that this might be the practical limit at which one can have confidence in the assignment of haplotypes to subjects.The empirically determined set of haplotypes from Horan et al.  were kinThe full data set of Horan et al.  includesPairwise D' , the linThe accuracy of haplotype inference was examined by several metrics. The mean squared error (MSE)  is definpek and ptk are the inferred and empirically determined frequencies for the kth haplotype, and h is the number of haplotypes. IF and IH were proposed by Excoffier and Slatkin [IF is another measure of how closely the inferred and empirical haplotype frequencies correspond and is given bywhere  Slatkin . IF is aIF ranges from 0 to a maximum value of 1 when the frequencies match perfectly. IH compares the number of haplotypes inferred to the number actually known to occur and ranges from 0 to 1 (complete correspondence between inferred and true). IH is defined aswhere the variables are defined as above. mtrue is the number of haplotypes known to occur, mest is the number of inferred haplotypes with frequency ≥ 1/(2n), and mmissed is the number of known haplotypes that were not inferred. The error rate [where ror rate  is the p


Microglia make up the innate immune system of the central nervous system and are key cellular mediators of neuroinflammatory processes. Their role in central nervous system diseases, including infections, is discussed in terms of a participation in both acute and chronic neuroinflammatory responses. Specific reference is made also to their involvement in Alzheimer's disease where microglial cell activation is thought to be critically important in the neurodegenerative process. A role for immune responses, involving antigen presentation and immune-response-generating cytokines, in neurodegenerative diseases such as Alzheimer's disease was recognized for a decade before the term neuroinflammation came into widespread use ,2. A PubMany researchers now consider this innate immune response in the brain to be a potentially pathogenic factor in a number of CNS diseases that lack the prominent leukocytic infiltrates of adaptive immune responses, but that do have activated microglia and astrocytes, i.e., neuroinflammation.The idea that neuroinflammation is detrimental implies that glial cell activation precedes and causes neuronal degeneration , a sequeIn the following, we provide some basic definitions and discussion to more precisely define the idea of neuroinflammation as a CNS tissue response to injury, and the notion of neuroinflammation as a pathogenic factor in neurodegenerative diseases.Inflammation is a reaction of living tissues to injury . The disBefore "neuroinflammation" became a commonly used term, neuroscientists spoke of "reactive gliosis" in describing endogenous CNS tissue responses to injury. Reactive gliosis specifically referred to the accumulation of enlarged glial cells, notably microglia and astrocytes, appearing immediately after CNS injury has occurred. In contrast to glial reactivity, which suggests a largely passive response to injury; glial activation implies a more aggressive role in responding to activating stimuli: activated glial cells release factors that act on and engender responses in target cells analogous to the responses of activated immune cells in the periphery. Activation of immune cells in the periphery leads to leukocyte infiltration of tissues, but this is notably absent in the brain unless there has been destruction or compromise of the blood brain barrier ,8. In thIn limited, acute reactions to injury, in the absence of blood-brain barrier breakdown, there is the subtler response of the brain's own immune system, composed largely of rapid activation of glial cells. These responses represent the other end of the spectrum of CNS injury, where limited neuronal insults trigger glial cell activation without breakdown of the blood brain barrier and without concomitant leukocytic infiltration. This form of "pure" glial response occurs in neuronal injury caused by either loss of afferents  or loss Although such specific responses might, in a strict sense, be included in the term "neuroinflammation," neuroinflammation as generally used and understood applies to more chronic, sustained cycles of injury and response, in which the cumulative ill effects of immunological microglial and astrocytic activation contribute to and expand the initial neurodestructive effects, thus maintaining and worsening the disease process through their actions.The concept of chronic inflammation (as opposed to acute inflammation) is more relevant in the context of understanding CNS disease (as opposed to CNS injury), as the very term "disease" implies chronicity. Chronic multiple sclerosis is, of course, an unequivocal and long-recognized example of an inflammatory brain disease. Although the underlying cause(s) of multiple sclerosis have not been elucidated, it is probably safe to say that the persistent injurious stimulus that accounts for neuroinflammation in multiple sclerosis is a myelin-related protein that has escaped self-tolerance and become an autoimmunogen. Consistent with the chronic persistence of this autoimmunogen is a persistent accumulation of blood-derived mononuclear leukocytes in the CNS parenchyma, a phenomenon that is similar to what is found in other autoimmune diseases such as rheumatoid arthritis or polymyositis.Infections are another group of diseases that are classically recognized as inflammatory in nature, with meningeal, perivascular, or even parenchymal infiltrates of peripheral leukocytes. There are, however, exceptions. Rabies is a disease in which the peripheral immune response is slow and inadequate, and in which classic inflammatory changes are less striking than those found in other viral encephalidites. Babes, in 1897 , describvia myelomonocytic cells: monocytes, perivascular cells, and microglia [Today, the most important example of a chronic brain infection is human immunodeficiency virus (HIV). Chronic HIV encephalitis is characterized by the same nodules of activated microglia that Babes described in rabies. HIV enters and persists in the CNS icroglia . HIV infPrion diseases represent another chronic infectious CNS disease that is not accompanied by leukocytic infiltrates. Microglial activation, again, appears to be the most prominent inflammatory component of prion diseases ,14, althNeurodegenerative diseases – particularly Alzheimer's disease, but also amyotrophic lateral sclerosis, Parkinson's disease, and Huntington's disease – lack the prominent infiltrates of blood-derived mononuclear cells that characterize autoimmune diseases. On the other hand, there is abundant evidence that many substances involved in the promotion of inflammatory processes are present in the CNS of patients with such neurodegenerative diseases. By far the bulk of this body of evidence is related to studies in Alzheimer's disease . What diβ-Amyloid precursor protein βAPP functions as a neuronal acute-phase, injury-response protein. For instance, there is excessive expression of βAPP, accompanied by microglial activation and cytokine expression, after traumatic head injury . With heChronic microglial activation is an important component of neurodegenerative diseases, and this chronic neuroinflammatory component likely contributes to neuronal dysfunction, injury, and loss (and hence to disease progression) in these diseases. The recognition of microglia as the brain's intrinsic immune system, and the understanding that chronic activation of this system leads to pathologic sequelae, has led to the modern concept of neuroinflammation. This vision of microglia-driven neuroinflammatory responses, with neuropathological consequences, has extended the older vision of passive glial responses that are inherent in the concept of "reactive gliosis."Aβ: β-amyloid peptideβAPP: Aβ precursor proteinCNS: central nervous systemHIV: human immunodeficiency virusMS: multiple sclerosisNone declaredWJS conceived this review, wrote the initial draft, modified this with the comments of REM and WSTG, and wrote the final draft. REM and WSTG contributed particularly to the sections on infections and on Alzheimer's disease. All authors read and approved the final version.