Clonal evolution through genetic bottlenecks and telomere attrition: Potential threats to in vitro data reproducibility

Tissue cultures of immortalized human cells, also known as established cell lines, are broadly accessible and cost‐efficient tools for biomedical research. We here review potential genetic sources of systematic error in cell line experiments due to clonal evolution in vitro. In particular, the authors highlight alterations in telomere function over prolonged culture and population bottlenecks, respectively, as two commonly overlooked phenomena that can result in significant alterations in cell line genotypes over just one or a few passages in vitro. These alterations may include changes in mutation status of oncogenes and large scale chromosomal imbalances. We introduce a simple list of factors to be avoided in order to reduce the risk of data misinterpretation due to clonal evolution, including unacknowledged in vitro selection pressures, prolonged culture per se, harsh population size reductions, experiments at early phases after establishment, and the employment of cell lines not sufficiently analyzed by high resolution genetic techniques.     

Colorectal cancer morbidity in Poland and Japan in the years 1999–2006 – a comparison

Introduction

The frequency of colorectal cancer (CRC) is dependent on sex and age. The illness more often affects men than women, and a considerable increase in morbidity is noted after 65 years of age. The aim of the study was to assess the influence of various risk factors on CRC morbidity, statistical data from countries varied in cultural, socioeconomic and biological terms need to be studied. Poland and Japan can serve as examples of such contrasts.

Material and methods

The basis for the analysis of CRC morbidity was statistical data from Poland and Japan gathered in the period 1999–2006. They were expressed in absolute numbers and were related to the total morbidity, as well as morbidity according to sex and age.

Results

In the years 1999–2006, the total morbidity in Japan was on average 2.3 times higher than in Poland. The value was 2.5 times higher among men and 2.1 times among women when compared to Poland. In both countries morbidity is closely related to age and it reaches a peak between 65 and 74 years of age. Although the change in morbidity over the period was the same for Poland and Japan, there are considerable differences concerning the sex of the patients.

Conclusions

There is a substantial increase in CRC morbidity in both countries, especially among elderly people, which may point to the predominant influence of environmental factors and population aging in both countries. The much higher increase in morbidity among Polish men when compared to Polish women, not observed in the Japanese population, requires further study.     

Recombinant protein-based assays for detection of antibodies to severe acute respiratory syndrome coronavirus spike and nucleocapsid proteins

Recombinant severe acute respiratory syndrome (SARS) nucleocapsid and spike protein-based immunoglobulin G immunoassays were developed and evaluated. Our assays demonstrated high sensitivity and specificity to the SARS coronavirus in sera collected from patients as late as 2 years postonset of symptoms. These assays will be useful not only for routine SARS coronavirus diagnostics but also for epidemiological and antibody kinetic studies.     

Lactobacillus rhamnosus CNCM I-3690 and the commensal bacterium Faecalibacterium prausnitzii A2-165 exhibit similar protective effects to induced barrier hyper-permeability in mice

Impaired gut barrier function has been reported in a wide range of diseases and syndromes and in some functional gastrointestinal disorders. In addition, there is increasing evidence that suggests the gut microbiota tightly regulates gut barrier function and recent studies demonstrate that probiotic bacteria can enhance barrier integrity. Here, we aimed to investigate the effects of Lactobacillus rhamnosus CNCM I-3690 on intestinal barrier function. In vitro results using a Caco-2 monolayer cells stimulated with TNF-α confirmed the anti-inflammatory nature of the strain CNCM I-3690 and pointed out a putative role for the protection of the epithelial function. Next, we tested the protective effects of L. rhamnosus CNCM I-3690 in a mouse model of increased colonic permeability. Most importantly, we compared its performance to that of the well-known beneficial human commensal bacterium Faecalibacterium prauznitzii A2-165. Increased colonic permeability was normalized by both strains to a similar degree. Modulation of apical tight junction proteins expression was then analyzed to decipher the mechanism underlying this effect. We showed that CNCM I-3690 partially restored the function of the intestinal barrier and increased the levels of tight junction proteins Occludin and E-cadherin. The results indicate L. rhamnosus CNCM I-3690 is as effective as the commensal anti-inflammatory bacterium F. prausnitzii to treat functional barrier abnormalities.     

Determinants of Plasma Renin Activity: Role of a Human Renin Gene Variant as a Genetic Factor

The plasma renin activity (PRA) is affected by a number of environmental factors. However, significant heritability has been shown for the activity. A hypothesis that a candidate regulatory single-nucleotide polymorphism, C-5312T, of human renin gene should have a significant effect on PRA was elucidated and updating of independent determinants of PRA was attempted.Cross sectional study.Outpatient study.We enrolled consecutive 810 subjects who had consulted our hospitals for lifestyle-related diseases.Genotypes were assayed with genomic DNA for C-5312T. Among the genetic variants, the difference of PRA was evaluated. Monovariate linear regression analysis was performed to test the correlation between PRA and clinical variables. Finally, stepwise multiple regression analysis was performed to evaluate the independent determinants.On comparing 2 genotype groups, CC/CT and T allele homozygote, the geometric means of PRA were 0.778 and 0.941 ng/ml/h, respectively (F = 5.992, P = 0.015). Monovariate linear regression analysis revealed that a number of variables have a significant correlation with the activity, including urinary salt excretion. A stepwise multivariate regression analysis revealed that renin C-5312T variant (TT) is one of the independent determinants of PRA.Thus, for the first time, a human renin gene variant was associated with a significant increase in PRA as a genetic factor and the independent determinants for the activity were updated including genetic factor.     

Monoclonal antibodies against carcinoembryonic antigen (CEA) used in a solid-phase enzyme immunoassay. First clinical results

A solid-phase enzyme immunoassay using both mouse monoclonal and goat polyclonal antibodies against carcinoembryonic antigen (CEA) was developed. The assay detects 0.6 to 1.2 ng of CEA per ml of serum and has 3 incubation steps which can be performed in 1 day. Polystyrene balls coated with polyclonal goat anti-CEA antibodies are first incubated with heat-extracted serum samples. Bound CEA is then detected by addition of mouse monoclonal antibodies, followed by goat IgG anti-mouse IgG1 coupled to alkaline phosphatase. Results with this enzyme immunoassay using monoclonal antibodiies (M-EIA) have been compared with those obtained by the conventional inhibition radioimmunoassay (RIA) using goat antiserum. Three hundred and eighty serum samples from 167 patients with malignant or non-malignant diseases and from 134 normal individuals with or without heavy smoking habits were analyzed by the 2 assays. Excellent correlation between the results of the 2 assays was obtained, but the M-EIA, using monoclonal antibodies from a single hybridoma, did not discriminate better than the conventional RIA between CEA produced by different types of carcinoma and between CEA associated with malignant or non-malignant diseases. Follow-up studies of several patients by sequential CEA determinations with the 2 assays showed that the M-EIA was as accurate as the RIA for the detection of tumor recurrences.     

IgG4-related renal disease: clinical and pathological characteristics

IgG4-related disease is a recently established systemic condition. Tubulointerstitial nephritis is the most common renal manifestation. Glomerular lesions, particularly membranous glomerulonephritis, can develop simultaneously. Some patients present with serological renal dysfunction associated with elevated IgG or IgE levels and hypocomplementemia, while others are incidentally found to have abnormalities in kidneys on imaging. A majority of patients with IgG4-related kidney disease have similar lesions at other anatomical sites, which help us to suspect this condition. Serum IgG4 elevation (>135 mg/dL) is the most, although not entirely, specific marker for the diagnosis. Imaging findings varies from small nodules to bilateral diffuse abnormalities. In addition to the renal parenchyma, the renal pelvis and perirenal adipose tissue can be affected. Histological features include dense lymphoplasmacytic infiltration, storiform or “bird’s eye” fibrosis (highlighted by PAM stain), and IgG4-positive plasma cell infiltration (>10 cells/high-power field and IgG4/IgG-positive cell ratio >40%). Immune complex deposition is detectable in the tubular basement membrane by immunofluorescence and/or electron microscopy. Patients usually respond well to corticosteroids, but highly active diseases may require other immunosuppressive therapies. Further investigations will be required to fully understand pathophysiology underlying this emerging condition.     

海藻糖对生物人工肝用人永生化肝细胞系C3A细胞低温的保存

背景：随着生物人工肝在临床上的应用,需要有一种方便、有效、实用的保护生物反应器装载细胞活力及功能的方法,设计有自主知识产权的生物人工肝用肝细胞的低温保护液迫在眉梢。 目的：验证海藻糖作为低温保护剂在4 ℃低温保存肝细胞的作用。 方法：采用C3A人永生化肝细胞系在不同低温保存液(DMEM培养液,乳酸钠林格氏液,加有不同浓度海藻糖的乳酸钠林格氏液,UW液)4 ℃保存24,48,72 h。复苏后行细胞活力、细胞损伤指标、氧自由基代谢相关指标等检测,并通过荧光双染法观察细胞凋亡情况。 结果与结论：不同低温保存液保存不同时间C3A细胞的活力、细胞损伤及细胞凋亡表现均有所不同,其中同时间点不同浓度海藻糖组均优于单用乳酸钠林格氏液组,但不如UW液组(P < 0.01)。而保存24 h的不同浓度海藻糖组及UW液组与乳酸钠林格氏液组相比差异无显著性意义(P > 0.05),提示海藻糖能有效减轻低温对肝细胞凋亡及缺血再灌注损伤的程度,可成为低温保存液中有效及重要的保护剂组分。     

A TLR4 polymorphism is associated with asthma and reduced lipopolysaccharide-induced interleukin-12(p70) responses in Swedish children

BACKGROUND: Bacterial signals play an important role in the maturation of the immune system. Polymorphisms in genes coding for receptors to bacterial components can alter the immune responsiveness of the host to microbial agents and may indicate the development of aberrant immune responses that are associated with immune-mediated diseases such as atopic diseases. OBJECTIVE: The study's objective was to investigate the relationship between TLR4 and CD14 gene polymorphisms, the LPS responsiveness of PBMCs, and the presence of asthma and allergic rhinoconjunctivitis in children. METHODS: The TLR4 (Asp299Gly) and CD14/-159 polymorphisms were determined in 115 Swedish children aged 8 and 14 years. LPS-induced IL-12(p70), IL-10, and IFN-gamma responses of PBMCs from 69 of the children were analyzed by means of ELISA. The levels of soluble CD14 in serum samples were analyzed by means of ELISA, and the total IgE levels were analyzed by means of UniCAP Total IgE (Pharmacia Diagnostics, Uppsala, Sweden). RESULTS: Decreased LPS-induced IL-12(p70) and IL-10 responses were associated with the TLR4 (Asp299Gly) polymorphism and independently with asthma, especially atopic asthma. The TLR4 (Asp299Gly) polymorphism was associated with a 4-fold higher prevalence of asthma in school-aged children (adjusted odds ratio 4.5, 95% CI 1.1-17.4) but not to allergic rhinoconjunctivitis. CONCLUSION: A TLR4 polymorphism modifies innate immune responses in children and may be an important determinant for the development of asthma. This may influence the outcome of intervention studies that use microbial stimuli as immune modulators.