Chromosomal abnormalities and neurological outcomes in fetal cerebral ventriculomegaly: a retrospective cohort analysis.

INTRODUCTION: This study investigated the incidences of chromosomal abnormalities and the neurological outcomes according to the degree of fetal cerebral ventriculomegaly. METHODS: All women with antenatal ultrasound diagnosis of fetal cerebral ventriculomegaly were retrospectively identified from two maternal-fetal medicine units in Hong Kong from January 2014 to December 2018. Degrees of fetal ventriculomegaly were classified as mild (10-11.9 mm), moderate (12-14.9 mm), or severe (≥15 mm). Genetic investigation results were reviewed, including conventional karyotyping and chromosomal microarray analysis (CMA); correlations between chromosomal abnormalities and the degree of fetal ventriculomegaly were explored. The neurological outcomes of subsequent live births were analysed to identify factors associated with developmental delay. RESULTS: Of 84 cases (ie, pregnant women and their fetuses) included, 46 (54.8%) exhibited isolated fetal ventriculomegaly, 55 (65.5%) had mild cerebral ventriculomegaly, and 29 (34.5%) had moderate or severe cerebral ventriculomegaly. Overall, 20% (14/70) of cases had chromosomal abnormalities. Moreover, 12% (3/25) of mild isolated ventriculomegaly cases had abnormal karyotype or CMA results. The CMA provided an incremental diagnostic yield of 8.6% (6/70), compared with conventional karyotyping; 4.3% exhibited pathogenic variants and 4.3% exhibited variants of uncertain significance. Among the 53 live births in the cohort, fewer cases of mild isolated ventriculomegaly were associated with developmental delay than more severe isolated ventriculomegaly (9.7% vs 41.7%, P<0.03). CONCLUSIONS: Chromosomal microarray analysis testing should be offered to all women with fetal cerebral ventriculomegaly, including women with isolated mild ventriculomegaly. The incidence of developmental delay after birth increases with the degree of prenatal cerebral ventriculomegaly.

Expanded carrier screening using next-generation sequencing of 123 Hong Kong Chinese families: a pilot study.

INTRODUCTION: To determine the carrier frequency and common mutations of Mendelian variants in Chinese couples using next-generation sequencing (NGS). METHODS: Preconception expanded carrier testing using NGS was offered to women who attended the subfertility clinic. The test was then offered to the partners of women who had positive screening results. Carrier frequency was calculated, and the results of the NGS panel were compared with those of a target panel. RESULTS: One hundred twenty-three women and 20 of their partners were screened. Overall, 84 (58.7%) individuals were identified to be carriers of at least one disease, and 68 (47.6%) were carriers after excluding thalassaemias. The most common diseases found were GJB2-related DFNB1 nonsyndromic hearing loss and deafness (1 in 4), alpha-thalassaemia (1 in 7), beta-thalassaemia (1 in 14), 21-hydroxylase deficient congenital adrenal hyperplasia (1 in 13), Pendred's syndrome (1 in 36), Krabbe's disease (1 in 48), and spinal muscular atrophy (1 in 48). Of the 43 identified variants, 29 (67.4%) were not included in the American College of Medical Genetics and Genomics or American College of Obstetrics and Gynecology guidelines. Excluding three couples with alpha-thalassaemia, six at-risk couples were identified. CONCLUSION: The carrier frequency of the investigated members of the Chinese population was 58.7% overall and 47.6% after excluding thalassaemias. This frequency is higher than previously reported. Expanded carrier screening using NGS should be provided to Chinese people to improve the detection rate of carrier status and allow optimal pregnancy planning.

Effects of sarcopenia, hypoalbuminemia, and laparoscopic surgery on postoperative complications in elderly patients with colorectal cancer: A prospective study.

With the increasing number of elderly patients, the risk of diseases such as colorectal cancer (CRC) has increased. The objective of this prospective study was to explore the effects of sarcopenia, hypoalbuminemia, and laparoscopic surgery on postoperative complications among elderly patients who recently underwent colorectal surgery. Patients aged over 65 years who underwent surgery for CRC at the First Affiliated Hospital of Wenzhou Medical University were considered for this study. The demographical and clinical characteristics of these patients, as well as postoperative complications, were prospectively analyzed. The patients were divided into two groups depending on the diagnosis of sarcopenia, and the clinical variables corresponding to the two groups were compared. Further, the risk factors associated with postoperative complications were evaluated using univariate analysis and multivariate logistic regression analysis. A total of 360 patients fulfilled the inclusion criteria. Incidences of postoperative complications in the sarcopenia and non-sarcopenia groups were at 38.3% and 27.3%, respectively. In addition, sarcopenia (p=0.029) and hypoalbuminemia (p=0.010) were identified as independent risk factors, while laparoscopic surgery (p=0.023) was identified as a protective factor for postoperative complications. However, laparoscopic surgery was a protective factor for postoperative complications in the colon group only (p=0.001). Sarcopenia and hypoalbuminemia are independent risk factors that influence the probability of developing complications following CRC surgery. Laparoscopic surgery is a protective factor for postoperative complications of CRC patients, particularly colon cancer patients.

[Levels of dichlorodiphenyltrichloroethane internal exposure levels in pregnant women of Xiamen and influencing factors].

Objective: To investigate the level of and factors influencing internal exposure to dichlorodiphenyltrichloroethane (DDT) in pregnant women. Methods: In all, 1 064 pregnant women were recruited in a hospital of Xiamen. Participants were asked to complete a questionnaire to obtain data on sociodemographic characteristics and lifestyle. Peripheral venous blood and cord blood samples were collected. Of the 1 064 pregnant women, 600 were enrolled in this study after completing the questionnaire and providing peripheral venous blood and cord blood. Among those women, 150 were selected randomly using a systematic sampling method. A gas chromatography coupled electron capture detector was used to determine the concentration of six DDT homologues: p,p'-dichlorodiphenyltrichloroethane (p,p'-DDT), o,p'-dichlorodiphenyltrichloroethane (o,p'-DDT), p,p'-dichlorodiphenyldichloroethane (p,p'-DDD), o,p'-dichlorodiphenyldichloroethane (o,p'-DDD), p,p'-dichlorodiphenylethylene (p,p'-DDE), and o,p'-dichlorodiphenylethylene (o,p'-DDE) . Pregnant women were divided into two groups according to DDT concentration: a low concentration group (detection value≤P50) and a high concentration group (detection value>P50). multivariate logistic regression was used to analyze the association between the DDT levels and potential influencing factors which investigated in the questionnaire. Results: The detection rates of p,p'-DDT, o,p'-DDT, p,p'-DDD, o,p'-DDD, p,p'-DDE and o,p'-DDE in the peripheral venous blood samples from the 150 pregnant women were 83.3% (125), 29.3% (44), 58.0% (87), 24.0% (36), 82.0% (123), and 34.7% (52), respectively. The median concentrations were 1.56, 0.03, 0.07, 0.03, 0.93 and 0.03 µg/ml, respectively. The detection rates of p,p'-DDT, o,p'-DDT, p,p'-DDD, o,p'-DDD, p,p'-DDE and o,p'-DDE in the cord blood samples were 69.3% (104), 10.7% (16), 29.3% (44), 20.7% (31), 81.3% (122) and 45.3% (68), and the median concentrations were 0.41, 0.03, 0.03, 0.03, 0.42 and 0.03 µg/ml, respectively. The concentration ranges in the low and high DDT concentration groups which contained 75 respondents respectively were 0-3.69 and 3.74-82.09 µg/ml, respectively. In the single-factor analysis, the number (percentage) of those who consumed seafood " rarely" , "less than twice a week" , and " twice a week or more" was 15 (20.3%), 22 (29.7%), and 37 (50.0%), respectively, in the low concentration group, and 4(5.3%), 20(26.7% ), and 51(68.0% ) in the high concentration group (χ2=8.69, P=0.013). The results of the multivariate logistic regression analysis indicate that pregnant women who consume seafood less than twice a week, twice a week or more have higher peripheral blood DDT concentrations compared with those who rarely consume seafood. The OR (95% CI) values were 1.14 (1.08-1.21), 2.11 (1.55-2.85), respectively. Conclusion: The exposure level of pregnant women to DDTs in the Xiamen area is higher than that of women in other regions. High seafood intake is a risk factor for internal exposure to DDTs.

Intratumoral expression profiling of genes involved in angiogenesis in colorectal cancer patients treated with chemotherapy plus the VEGFR inhibitor PTK787/ZK 222584 (vatalanib).

The phase III CONFIRM clinical trials demonstrated that metastatic colorectal cancer patients with elevated serum lactate dehydrogenase (LDH) had improved outcome when the vascular endothelial growth factor receptor (VEGFR) inhibitor PTK/ZK (Vatalanib) was added to FOLFOX4 chemotherapy. We investigated the hypothesis that high intratumoral expression of genes regulated by hypoxia-inducible factor-1 alpha (HIF1α), namely LDHA, glucose transporter-1 (GLUT-1), VEGFA, VEGFR1, and VEGFR2, were predictive of outcome in CONFIRM-1. Tumor tissue was isolated by laser-capture microdissection from 85 CONFIRM-1 tumor specimens; FOLFOX4/placebo n=42, FOLFOX4/PTK/ZK n=43. Gene expression was analyzed using quantitative RT-PCR. In univariate analyses, elevated mRNA expression of LDHA, GLUT-1, and VEGFR1 were associated with response to FOLFOX4/PTK/ZK. In univariate and multivariate analyses, elevated LDHA and VEGFR1 mRNA levels were associated with improved progression-free survival in FOLFOX4/PTK/ZK patients. Furthermore, increased HIF1α and VEGFR2 mRNA levels were associated with decreased survival in FOLFOX/placebo patients but not in patients who received FOLFOX4/PTK/ZK. These are the first data suggesting intratumoral mRNA expression of genes involved in angiogenesis/HIF pathway may predict outcome to VEGFR-inhibitors. Biomarkers that assist in directing VEGFR-inhibitors toward patients with an increased likelihood of benefit will improve the cost-effectiveness of these promising agents.

Extracellular vesicles from hypoxia-pretreated adipose-derived stem cells regulate hypoxia/reoxygenation-induced human dermal microvascular endothelial apoptosis and autophagy in vitro.

Recent studies suggest hypoxia can promote adipose-derived stem cells (ADSCs) to attenuate hypoxia/reoxygenation (H/R)-induced damage to human dermal microvascular endothelial cells (HDMECs). Extracellular vesicles (EVs), isolated from ADSCs, play an-important role in the fields of regenerative medicine. Here, we aimed to investigate the effect of EVs isolated from hypoxia-pretreated ADSCs (ADSC-EVs[H]) on HDMECs to attenuate ischemia/reperfusion injury of free skin flaps. First, we characterized EVs isolated from normoxia-cultured ADSCs (ADSC-EVs[N]) and ADSC-EVs(H). Experimental data indicated that EVs isolated from ADSCs consisted of lipid-bilayer vesicles that exhibited positive expression of vascular endothelial growth factor (VEGF) and marker proteins CD9, CD63 and CD81, and the mean particle size of EVs in the hypoxia-pretreated ADSCs (ADSC[H]) group was smaller (74.17 nm) than in the normoxic-cultured ADSCs (ADSC[N]) group (93.87 nm). Hypoxic pretreatment increased the number of EVs. Later, we favorably constructed the co-culture model of EVs isolated from ADSCs (ADSC-EVs) and H/R-induced HDMECs. Cell counting kit-8, Ethynyldeoxyuridine assay, western blotting and immunofluorescence staining showed that ADSC-EVs(H) promoted the survival of HDMECs and increased LC3 level. Apoptosis, reactive oxygen species (ROS) and JC-1 mitochondrial membrane potential (MMP) assays revealed that ADSC-EVs(H) reduced the apoptosis rate and ROS accumulation and increased MMP level in HDMECs, indicating that ADSC-EVs(H) effectively attenuated H/R-induced damage in HDMECs through autophagy activation and the-inhibition of apoptosis and oxidative stress. This study confirmed that ADSC-EVs(H) could effectively regulate the proliferation, apoptosis, oxidative stress, and autophagy expression of H/R-induced HDMECs in vitro, and therefore the transplantation of ADSC-EVs(H) may provide novel insights for the transplantation of free skin flaps.

Vascular density analysis in colorectal cancer patients treated with vatalanib (PTK787/ZK222584) in the randomised CONFIRM trials.

BACKGROUND: Pharmacological inhibitors of vascular endothelial growth factor (VEGF) receptors, like vatalanib, have been tested in randomised trials (CONFIRM (Colorectal Oral Novel therapy For the Inhibition of Angiogenesis and Retarding of Metastases) 1 and 2) in colorectal cancer showing activity in a subgroup of patients with high serum LDH expression. In the current study, we assessed the predictive role of vascular density (VD) in patients treated in the above trials. METHODS: Paraffin-embedded materials from 141 patients were analysed with immunohistochemistry for the expression of the CD31 (pan-endothelial cell marker) and of phosphorylated pVEGFR2/KDR on endothelial cells. The VD was correlated with response to therapy and with progression-free (PFS) and overall survival (OS). RESULTS: A significant association of pVEGFR2/KDR+ VD with poor response in the placebo group was noted (response rates (RRs) 15% (3/20) when high VD vs 52% (26/50) when low VD; P=0.006). The RR increased from 15 (3/20) to 50% (11/22) in tumours with high VD when vatalanib was added to chemotherapy (P=0.02). A significantly improved PFS was noted in patients with high pVEGFR2/KDR+ VD when treated with vatalanib (P=0.002). A similar effect was also noted in patients with high CD31+ VD (P=0.07). Overall survival was marginally improved (P=0.07). CONCLUSION: Assessment of the activated vessel density may allow the stratification of patients recruited in randomised trials with VEGFR-targeting anti-angiogenic agents, unmasking their therapeutic potential and enabling their introduction in the clinical practice for the benefit of specific patient subgroups, at the same time reducing the cost of therapy.

Genotypic diversity enhances invasive ability of Spartina alterniflora.

Although genetic diversity is very important for alien species, which have to cope with new environments, little is known about the role that genetic diversity plays in their invasive success. In this study, we set up a manipulation experiment including three levels of genotypic diversity to test whether genotypic diversity can enhance the invasive ability of alien species, in our case the invasive Spartina alterniflora in China, and to infer the underlying mechanisms. There was no significant relationship between genotypic diversity and parameters of performance in the first year; however, from the summer of the second year onwards, genotypic diversity enhanced four of the six parameters of performance. After two growing seasons, there were significant positive relationships between genotypic diversity and maximum spread distance, patch size, shoot number per patch, and aboveground biomass. Moreover, abundance of the native dominant species Scirpus mariqueter was marginally significantly decreased with genotypic diversity of S. alterniflora, suggesting that enhanced invasive ability of S. alterniflora may have depressed the growth of the native species. There was no significant difference in most measures of performance among six genotypes, but we observed a transgressive over performance in four measures in multiple-genotype patches. At the end of the experiment, there were significant nonadditive effects of genotypic diversity according to Monte Carlo permutations, in six-genotype, but not three-genotype plots. Our results indicated that both additive and nonadditive effects played roles in the positive relationship between genetic diversity and invasion success, and nonadditive effects were stronger as duration increased.

Comparison of clinical efficacy and safety between denosumab and alendronate in postmenopausal women with osteoporosis: a meta-analysis.

The aim of this study was to perform a head-to-head comparison of efficacy and safety profile between 60 mg denosumab (Den) subcutaneously (SC) per 6 months (Q6M) and 70 mg alendronate (Aln) orally per week (QW) for postmenopausal women with low bone mineral density. We searched electronic databases comparing efficacy and safety of Den SC Q6M and Aln QW in postmenopausal women. The primary outcomes of efficacy evaluation in included trials were incidence of clinical fracture in both groups and bone mineral density (BMD) at different skeletal sites. And adverse events (AEs), including incidence of neoplasms and infections, were considered as secondary outcomes. Following the instructions of 'Cochrane Handbook for systematic Reviews of Interventions 5.0.2', we identified eligible studies, evaluated the methodological quality and abstracted relevant data. Four heterogeneous randomised controlled trials (RCTs) involving 1942 women were identified. The results of review showed low evidence quality that supported the hypothesis the denosumab vs. alendronate could reduce risk of fracture [OR (95% CI) 1.42 (0.84 to 2.40), 11 more women per 1000 (from 4 fewer to 36 more), p = 0.19] but the moderate to high quality evidence suggesting treatment with 60 mg Den SC Q6M was more effective for postmenopausal women in increasing BMD [at distal radius (DR), total hip (TH), lumbar spine (LS), and femoral neck (FN)]. Hazards of neoplasms [OR (95% CI) 1.10 (0.65 to 1.86), 3 more per 1000 (from 10 fewer to 24 more), p = 0.62] or infections [OR (95% CI) 0.95 (0.79 to 1.15), 12 fewer per 1000 (from 53 fewer to 33 more,), p = 0.62] were appeared to be similar.Our review suggested within 1 year 60 mg Den SC Q6M treatment was more effective in increasing bone mass but could not reduce the fracture risk to a greater extent than 70 mg Aln QW therapy. Also the Den SC Q6M therapy did not increase the risks of neoplasms and infections compared with Aln QW.

Protective effects of anti-C5a in sepsis-induced thymocyte apoptosis.

Multiorgan apoptosis occurs during sepsis. Following cecal ligation and puncture (CLP) in rats, thymocytes underwent apoptosis in a time-dependent manner. C5a blockade dramatically reduced thymocyte apoptosis as measured by thymic weight, binding of annexin V to thymocytes, and laddering of thymocyte DNA. When C5a was generated in vivo by infusion of purified cobra venom factor (CVF), thymocyte apoptosis was significantly increased. Similar results were found when CVF was injected in vivo during the early stages of CLP. In animals 12 hours after induction of CLP, there was an increase in the activities of caspase-3, -6, and -9, but not caspase-1 and -8. Cytosolic cytochrome c levels increased by twofold, whereas mitochondrial levels showed a 50% decrease. Western blot analysis revealed that the content of Bcl-X(L) (but not of Bcl-2, BAX, Bad, and Bim) significantly decreased in thymocytes after CLP. C5a blockade in the sepsis model almost completely inhibited caspase-3, -6, and -9 activation, significantly preserved cytochrome c in the mitochondrial fraction, and restored Bcl-X(L) expression. These data suggest that systemic activation of complement induces C5a-dependent apoptosis of thymocytes and that the blockade of C5a during sepsis rescues thymocytes from apoptosis.

Inhibition of radioligand binding to A1 adenosine receptors by Bay K8644 and nifedipine.

Two dihydropyridine compounds, Bay K8644 (a calcium entry activator) and nifedipine (a calcium entry blocker), were found to inhibit the binding of [3H]phenylisopropyladenosine ([3H]PIA) to A1 adenosine receptors in rat cerebral cortex membranes with comparable potencies (IC50 10-30 microM). Scatchard analyses indicated that both Bay K8644 and nifedipine inhibited the binding of [3H]PIA by increasing the KD but without significant effect on the Bmax. When tested at 100 microM, neither Bay K8644 nor nifedipine showed a significant effect on [3H]-p-aminoclonidine ([3H]PAC; alpha 2-adrenergic receptor), [3H]dihydroalprenolol ([3H]DHA; beta-adrenergic receptor), [3H]spiperone (dopamine receptor), and [3H]nitrobenzylthioinosine [( 3H]NBMPR; nucleoside transporter) binding. In the presence of 10 mM Mg2+, the ability of 2-chloroadenosine (2-Cl-Ad, an A1 adenosine receptor agonist) to displace [3H]PIA binding was increased. Conversely, the potencies of 1,3-diethyl-8-phenylxanthine (DPX; an A1 receptor antagonist), Bay K8644 and nifedipine in inhibiting [3H]PIA binding were unchanged. It is suggested that both Bay K8644 and nifedipine may act as antagonists of adenosine A1 receptors, in addition to their well-known effects on calcium channels.

Technologies for detecting genetic polymorphisms in pharmacogenomics.

BACKGROUND: Pharmacogenomics is an emerging scientific discipline examining the genetic basis for individual variations in response to therapeutics. METHODS AND RESULTS: Genetic polymorphisms are a major cause of individual differences in drug response. Metabolic phenotyping can be accomplished by administering a probe drug or substrate and measuring the metabolites and clinical outcomes. However, this approach tends to be labor intensive and requires repeated sample collection from the individual being tested. Alternatively, genotyping allows determination of individual DNA sequence differences for a particular trait. Commonly used genotyping methods include gel electrophoresis-based techniques, such as polymerase chain reaction (PCR) coupled with restriction fragment length polymorphism analysis, multiplex PCR, and allele-specific amplification. Fluorescent dye-based high-throughput genotyping procedures are increasing in popularity, including oligonucleotide ligation assay, direct heterozygote sequencing, and TaqMan (Perkin Elmer, Foster City, CA) allelic discrimination. High-density chip array and mass spectrometry technologies are the newest advances in the genotyping field, but their wide application is yet to be developed. Novel mutations/polymorphisms also can be identified by conformation-based mutation screening and direct high-throughput heterozygote sequencing. CONCLUSIONS: Rapid and accurate detection of genetic polymorphisms has great potential for application to drug development, animal toxicity studies, improvement of human clinical trials, and postmarket monitoring surveillance for drug efficacy and toxicity.

Genotyping for Functionally Important Human CYP2D6*4 (B) Mutation Using TaqMan Probes.

The microsomal enzyme cytochrome P450 2D6 (CYP2D6), also known as debrisoquine 4-hydroxylase, is involved in the oxidative metabolism of many widely used drugs, including neuroleptics, tricyclic antidepressants, antiarrhythmics, and ß-adrenergic blocking agents (1). Polymorphisms of CYP2D6 are the best characterized examples of genetically mediated effects on a drug-metabolizing enzyme of clinical importance (2). When a drug that is a CYP2D6 substrate is taken by different individuals, it is not uncommon to observe large differences in plasma concentrations at steady state. This is explained, in part, by the three clinically distinct phenotypes associated with the CYP2D6 gene, normal metabolizers, poor metabolizers, and rapid metabolizers. In normal metabolizers, steady-state plasma drug concentrations fall within the desired therapeutic range and toxic effects are nonexistent or minimal. In fast-metabolizer individuals, desired concentrations are below therapeutic, and these patients generally do not respond at the recommended dosing regimen. In poor-metabolizer individuals, drug concentrations are above therapeutic level and undesired toxicity can be evoked.

Regulation of chemokine mRNA expression in a rat model of vanadium-induced pulmonary inflammation.

Environmental and occupational exposure to vanadium dusts results in toxic effects mainly confined to the respiratory system. Using a rat model of acute lung inflammation induced by intratracheal instillation of sodium metavanadate (NaVO3) at the dose of 200 microg V/kg, we investigated the relationship between the cytologic characterization of pulmonary inflammation and the expression of chemokine mRNA. Significant polymorphonuclear leukocyte (PMN) influx (P < 0.01) into the lung was noted 4 h after NaVO3 instillation, whereas alveolar macrophages (AMs) in bronchoalveolar lavage (BAL) cells appeared to decrease significantly. In contrast, neither PMNs nor AMs changed substantially 1 h after NaVO3 instillation. By Northern analysis, macrophage inflammatory protein (MIP)-2 mRNA in BAL cells increased markedly 1 h after NaVO3 instillation and reduced a little bit at 4 h, whereas MIP-1alpha mRNA in BAL cells was expressed relatively high 1 h after NaVO3 instillation, although a basal expression was detected in control group, and returned rapidly nearly to control level at 4 h. Since MIP-2 is a potent PMN chemoattractant and MIP-1alpha is a potent macrophage/monocyte chemoattractant has been well known. The facts that PMN influx was preceded by increased MIP-2 mRNA expression, suggesting that MIP-2 is involved in the development of NaVO3-induced pulmonary inflammation, whereas increased MIP-1alpha mRNA expression was followed by decreased AMs in BAL cells, suggesting AMs might be activated by MIP-1alpha, adherent to the lining surface of the airways and then resistant to be washed out. To delineate the mechanisms of transcriptional activation, we recently cloned the 5'-flanking region of the MIP-2 gene. The promotor region contains consensus binding sites for transcription factor nuclear factor kappaB (NF-kappaB) and activator protein-1 (AP-1). Using electrophoretic mobility shift assay, increased nuclear NF-kappaB, not AP-1, binding activity was detected 1 h after NaVO3 instillation, which correlated with the induction of MIP-2 mRNA. p65 (Rel A) and p50 protein appears to be involved in MIP-2 NF-kappaB binding. Taken together, our studies suggest that MIP-2 is an important mediator of NaVO3-induced pulmonary inflammation in the rat model. In addition, elevated MIP-2 mRNA levels are accompanied by increased NF-kappaB binding activity in BAL cells, suggesting possible MIP-2 transcriptional regulation through NF-kappaB.

Functional characterization of recombinant rat macrophage inflammatory protein-1 alpha and mRNA expression in pulmonary inflammation.

Chemokines are important inflammatory mediators that function by activating and recruiting leukocytes to an inflamed tissue. We have recently cDNA cloned the rat chemokine macrophage inflammatory protein-1 alpha (MIP-1 alpha) (1). In the present study, we characterize the biological function of recombinant MIP-1 alpha protein and describe expression of its mRNA both in vitro and in a rat model of lung inflammation. In vitro rat rMIP-1 alpha protein was chemotactic for both polymorphonuclear leukocytes (PMNs) and macrophages with maximal activity at 50 nM for both cell types. In in vivo studies, we found that intratracheal instillation of 1 and 5 micrograms of rMIP-1 alpha resulted in a significant (P < 0.05) influx of cells, primarily monocytes/macrophages, into the airspace of the lungs after 6 h. Mean numbers of lavagable PMNs were not elevated significantly (P < 0.05) for either dose of MIP-1 alpha. As a model of inflammation, rats were intratracheally instilled with 0.1 mg/kg bacterial lipopolysaccharide (LPS). Bronchoalveolar lavage (BAL) was performed 3 h later. Instillation of LPS resulted in an acute neutrophilia, but no significant change in lavagable macrophages. BAL cells from control animals (saline instilled) displayed no basal mRNA expression of either MIP-1 alpha or MIP-2 (positive control). In contrast, both MIP-1 alpha and MIP-2 mRNA levels increased markedly in BAL cells from rats instilled with LPS. The rat alveolar macrophage cell line (NR8383) also showed increased MIP-1 alpha mRNA levels in response to LPS (10 micrograms/ml) with a maximal increase after 6-8 h. The induction of MIP-1 alpha mRNA expression by LPS in NR8383 cells was attenuated by cotreatment with the antioxidants N-acetylcysteine and dimethylsulfoxide, suggesting that the induction of MIP-1 alpha mRNA by LPS is mediated via the generation of reactive oxygen species. We conclude that MIP-1 alpha is a potent chemoattractant for macrophages in vivo, and its mRNA expression in macrophages and BAL cells in response to inflammatory stimuli suggests a fundamental role in acute pulmonary inflammation.

Association between TGM5, PPAP2B and PSMA4 polymorphisms and NSCLC in never-smoking Chinese population.

AIM: To explore the potential association between SNPs in transglutaminase 5 (TGM5), phosphatidic acid phosphatase type 2B (PPAP2B) and proteasome subunit, alpha type 4 (PSMA4) and non-small cell lung cancer (NSCLC) susceptibility in Chinese patients who were non-smokers. SETTING AND DESIGN: A case-controlled study was conducted among Chinese population. MATERIALS AND METHODS: Two hundred NSCLC patients and 200 healthy controls who were age and sex matched were genotyped for rs504417 of TGM5, rs1261411 of PPAP2B and rs7164594 of PSMA4. Genotyping was performed using the Sequenom MassARRAY system based on the chip-based matrix-assisted laser desorption ionization time-of-flight mass spectrometry platform. STATISTICAL ANALYSIS USED: The association between genotype and lung cancer risk was evaluated by computing the odds ratio (OR) and 95% confidence interval (CI) from multivariate unconditional logistic regression analyses. RESULTS: There was no significant difference for the TGM5 rs504417, PPAP2B rs1261411 and PSMA4 rs716459 in allele or genotype frequencies, whether between controls and NSCLC or between controls and subgroups. CONCLUSIONS: polymorphisms of TGM5, PPAP2B and PSMA4 are not major contributors to NSCLC susceptibility, this primarily be attributed to the significantly distinct genetic background of Asian populations from western populations.

Administration of probiotics reduces bacterial translocation after intestinal transplantation in rats.

Bacterial translocation (BT) has been suggested to be responsible for the high incidence of infections after intestinal transplantation (IT). The purpose of this study was to examine whether a probiotic supplement after orthotopic IT ameliorated ischemia-reperfusion injury and reduced BT at 4 or 6 days postoperative (sham or IT), as mesenteric lymph nodes (MLNs), liver, and splenic tissue samples from the six groups were assessed for BT by bacterial culture, measurement of tumor necrosis factor-α (TNF-α) in MLNs by competitive reverse transcription-polymerase chain reaction, and histological evaluation by Park's classification. Oral administration of probiotics after IT did not improve short-term survival rates compared with the transplant-only groups (P > .05). However, the BT rates and levels of TNF-α in MLNs in groups with IT only were higher than the probiotic cohorts (P < .05). Histological injuries were significantly ameliorated in the group with six days of probiotic treatment compared with that in the nontreated hosts (P < .05). These data indicated that administration of probiotics after IT improved graft histology and reduced BT in rats.

Enabling large-scale pharmacogenetic studies by high-throughput mutation detection and genotyping technologies.

BACKGROUND: Pharmacogenetics is a scientific discipline that examines the genetic basis for individual variations in response to therapeutics. Pharmacogenetics promises to develop individualized medicines tailored to patients' genotypes. However, identifying and genotyping a vast number of genetic polymorphisms in large populations also pose a great challenge. APPROACH: This article reviews the recent technology development in mutation detection and genotyping with a focus on genotyping of single nucleotide polymorphisms (SNPs). CONTENT: Novel mutations/polymorphisms are commonly identified by conformation-based mutation screening and direct high-throughput heterozygote sequencing. With a large amount of public sequence information available, in silico SNP mapping has also emerged as a cost-efficient way for new polymorphism identification. Gel electrophoresis-based genotyping methods for known polymorphisms include PCR coupled with restriction fragment length polymorphism analysis, multiplex PCR, oligonucleotide ligation assay, and minisequencing. Fluorescent dye-based genotyping technologies are emerging as high-throughput genotyping platforms, including oligonucleotide ligation assay, pyrosequencing, single-base extension with fluorescence detection, homogeneous solution hybridization such as TaqMan, and molecular beacon genotyping. Rolling circle amplification and Invader assays are able to genotype directly from genomic DNA without PCR amplification. DNA chip-based microarray and mass spectrometry genotyping technologies are the latest development in the genotyping arena. SUMMARY: Large-scale genotyping is crucial to the identification of the genetic make-ups that underlie the onset of diseases and individual variations in drug responses. Enabling technologies to identify genetic polymorphisms rapidly, accurately, and cost effectively will dramatically impact future drug and development processes.

[3H]dipyridamole binding to nucleoside transporters from guinea-pig and rat lung.

Membranes from guinea-pig lung exhibited high-affinity binding of [3H]dipyridamole, a potent inhibitor of nucleoside transport. Binding (apparent KD 2 nM) was inhibited by the nucleoside-transport inhibitors nitrobenzylthioinosine (NBMPR), dilazep and lidoflazine and by the transported nucleosides uridine and adenosine. In contrast, there was no detectable high-affinity binding of [3H]dipyridamole to lung membranes from the rat, a species whose nucleoside transporters exhibit a low sensitivity to dipyridamole inhibition. Bmax. values for high-affinity binding of [3H]dipyridamole and [3H]NBMPR to guinea-pig membranes were similar, suggesting that these structurally unrelated ligands bind to the NBMPR-sensitive nucleoside transporter with the same stoichiometry.