Establishment of new murine embryonic stem cell lines for the generation of mouse models of human genetic diseases

Embryonic stem cells are totipotent cells derived from the inner cell mass of blastocysts. Recently, the development of appropriate culture conditions for the differentiation of these cells into specific cell types has permitted their use as potential therapeutic agents for several diseases. In addition, manipulation of their genome in vitro allows the creation of animal models of human genetic diseases and for the study of gene function in vivo. We report the establishment of new lines of murine embryonic stem cells from preimplantation stage embryos of 129/Sv mice. Most of these cells had a normal karyotype and an XY sex chromosome composition. The pluripotent properties of the cell lines obtained were analyzed on the basis of their alkaline phosphatase activity and their capacity to form complex embryoid bodies with rhythmically contracting cardiomyocytes. Two lines, USP-1 and USP-3, with the best in vitro characteristics of pluripotency were used in chimera-generating experiments. The capacity to contribute to the germ line was demonstrated by the USP-1 cell line. This cell line is currently being used to generate mouse models of human diseases.

Pharmacology of human experimental anxiety

This review covers the effect of drugs affecting anxiety using four psychological procedures for inducing experimental anxiety applied to healthy volunteers and patients with anxiety disorders. The first is aversive conditioning of the skin conductance responses to tones. The second is simulated public speaking, which consists of speaking in front of a video camera, with anxiety being measured with psychometric scales. The third is the Stroop Color-Word test, in which words naming colors are painted in the same or in a different shade, the incongruence generating a cognitive conflict. The last test is a human version of a thoroughly studied animal model of anxiety, fear-potentiated startle, in which the eye-blink reflex to a loud noise is recorded. The evidence reviewed led to the conclusion that the aversive conditioning and potentiated startle tests are based on classical conditioning of anticipatory anxiety. Their sensitivity to benzodiazepine anxiolytics suggests that these models generate an emotional state related to generalized anxiety disorder. On the other hand, the increase in anxiety determined by simulated public speaking is resistant to benzodiazepines and sensitive to drugs affecting serotonergic neurotransmission. This pharmacological profile, together with epidemiological evidence indicating its widespread prevalence, suggests that the emotional state generated by public speaking represents a species-specific response that may be related to social phobia and panic disorder. Because of scant pharmacological data, the status of the Stroop Color-Word test remains uncertain. In spite of ethical and economic constraints, human experimental anxiety constitutes a valuable tool for the study of the pathophysiology of anxiety disorders.

Epidemiological and functional implications of molecular variants of human papillomavirus

Human papillomavirus genomes are classified into molecular variants when they present more than 98% of similarity to the prototype sequence within the L1 gene. Comparative nucleotide sequence analyses of these viruses have elucidated some features of their phylogenetic relationship. In addition, human papillomavirus intratype variability has also been used as an important tool in epidemiological studies of viral transmission, persistence and progression to clinically relevant cervical lesions. Until the present, little has been published concerning the functional significance of molecular variants. It has been shown that nucleotide variability within the long control region leads to differences in the binding affinity of some cellular transcriptional factors and to the enhancement of the expression of E6 and E7 oncogenes. Furthermore, in vivo and in vitro studies revealed differences in E6 and E7 biochemical and biological properties among molecular variants. Nevertheless, further correlation with additional functional information is needed to evaluate the significance of genome intratypic variability. These results are also important for the development of vaccines and to determine the extent to which immunization with L1 virus-like particles of one variant could induce antibodies that cross-neutralize other variants.

Random amplification of polymorphic DNA reveals clonal relationships among enteropathogenic Escherichia coli isolated from non-human primates and humans

Enteropathogenic Escherichia coli (EPEC) strains are important agents of infantile diarrhea all over the world, gaining even greater importance in developing countries. EPEC have also been isolated from various animal species, but most isolates belong to serotypes that differ from those recovered from humans. However, it has been demonstrated that several isolates from non-human primates belong to the serogroups and/or serotypes related to those implicated in human disease. The objective of this study was to evaluate the genetic differences between thirteen strains isolated from non-human primates and the same number of strains isolated from human infections. Human isolates belonged to the same serogroup/serotype as the monkey strains and the evaluation was done by analysis of random amplified polymorphic DNA. Dendrogram analysis showed that there was no clustering between human and monkey strains. Human and non-human isolates of the EPEC serotypes O127:H40 and O128:H2 shared 90 and 87% of their bands, respectively, indicating strong genomic similarity between the strains, leading to the speculation that they may have arisen from the same pathogenic clone. To our knowledge, this study is the first one comparing genomic similarity between human and non-human primate strains and the results provide further evidence that monkey EPEC strains correlate with human EPEC, as suggested in a previous investigation.

Efficacy of HUMN criteria for scoring the micronucleus assay in human lymphocytes exposed to a low concentration of p,p'-DDT

The cytokinesis-block micronucleus (CBMN) assay is one of the standard cytogenetic tools employed to assess chromosomal damage subsequent to exposure to genotoxic/cytotoxic agents, and is widely applicable to plant, animal and human cells. In the present study, the CBMN assay was used to assess the baseline damage in binuclear human peripheral blood lymphocytes exposed to 25 µg/L p,p'-DDT for 1, 2, 24, and 48 h by measuring the frequency of micronuclei, nucleoplasmic bridges and nuclear buds. These new scoring criteria facilitated the detection of different types of clastogenic and aneugenic effects induced by this type of pollutant. With these criteria, CBMN can also be used to measure nucleoplasmic bridges which are considered to be consequences of chromosome rearrangements and nuclear buds which are biomarkers of altered gene amplification and gene dosage. The total number of micronuclei observed in binuclear human peripheral blood lymphocytes of the exposed samples (ranging from 32 to 47) was significantly greater (P < 0.05) than that detected in the unexposed (0 time) control sample, where the total number of micronuclei was 7. The number of nucleoplasmic bridges and nuclear buds obtained after 24 and 48 h was also significantly (P < 0.05) greater in the samples treated with p,p'-DDT than in the unexposed control samples. Thus, our results confirmed the usefulness of the new criteria applicable for the CBMN assay employed in measuring the DNA damage and its role of a sensitive cytogenetic biomarker.

In vivo fluctuation of Tax, Foxp3, CTLA-4, and GITR mRNA expression in CD4+CD25+ T cells of patients with human T-lymphotropic virus type 1-associated myelopathy

HTLV-1 Tax expression exerts an inhibitory effect on the Foxp3 transcription factor in CD4+CD25+ T-regulatory cells (Treg). For a better understanding of the role of Tax mRNA in the gene expression of cellular markers we measured Tax, Foxp3, CTLA-4, GITR, TGF-β, and IL-10 mRNA in Treg cells of 50 patients with human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP; 27 women and 23 men; mean age: 56.7 years). The control group consisted of 23 non-infected subjects (12 women and 11 men) with a mean age of 51.3 years. Real-time PCR was used to measure mRNA of Tax proteins and several cellular markers of Treg function. Determinations revealed a high level of Tax mRNA in HAM/TSP (124.35 copies/100 CD4+CD25+ T cells). Foxp3, GITR, and CTLA-4 mRNA levels were lower in HAM/TSP patients (mean ± SD, 22.07 ± 0.78, 9.63 ± 0.36, and 4.54 ± 0.39, respectively) than in non-infected controls (47.15 ± 12.94, 22.14 ± 1.91, and 21.07 ± 2.31). Both groups had similar levels of TGF-β and IL-10. An inverse relationship was found between Tax levels and Foxp3, CTLA-4, and GITR levels. Conversely, there was a direct correlation between levels of Foxp3, GITR, and CTLA-4. Disease severity and evolution time did not correlate with Tax or Foxp3 levels. The present results suggest that Tax and Foxp3 mRNA vary with the same degree of disease severity in HAM/TSP patients. Tax fluctuations may affect CTLA-4 and GITR expression via the Foxp3 pathway, causing virus-induced dysfunction of CD4+CD25+ T cells in HAM/TSP patients.

Clinicopathological significance of PTPN12 expression in human breast cancer

Protein tyrosine phosphatase non-receptor type 12 (PTPN12) is a recently identified tumor suppressor gene (TSG) that is frequently compromised in human triple-negative breast cancer. In the present study, we investigated the expression of PTPN12 protein by patients with breast cancer in a Chinese population and the relationship between PTPN12 expression levels and patient clinicopathological features and prognosis. Additionally, we explored the underlying down-regulation mechanism from the perspective of an epigenetic alteration. We examined PTPN12 mRNA expression in five breast cancer cell lines using semi-quantitative reverse-transcription PCR, and detected PTPN12 protein expression using immunohistochemistry in 150 primary invasive breast cancer cases and paired adjacent non-tumor tissues. Methylation-specific PCR was performed to analyze the promoter CpG island methylation status of PTPN12. PTPN12 was significantly down-regulated in breast cancer cases (48/150) compared to adjacent noncancerous tissues (17/150; P < 0.05). Furthermore, low expression of PTPN12 showed a significant positive correlation with tumor size (P = 0.047), lymph node metastasis (P = 0.001), distant metastasis (P = 0.009), histological grade (P = 0.012), and survival time (P = 0.019). Additionally, promoter CpG island hypermethylation occurs more frequently in breast cancer cases and breast cancer cell lines with low PTPN12 expression. Our findings suggest that PTPN12 is potentially a methylation-silenced TSG for breast cancer that may play an important role in breast carcinogenesis and could potentially serve as an independent prognostic factor for invasive breast cancer patients.

Laboratory animal: biological reagent or living being?

The duties of humans toward non-human animals and their rights in society have been debated for a long time. However, a discussion on the terminology used for the identification of laboratory animals is usually not considered, although the employment of inadequate terminology may generate disastrous consequences for the animals before, during, and after the experiment. This study intends to defend the use of appropriate terminology, call attention to an unethical attitude of certain professionals when dealing with experimental animals, and also propose operational mechanisms, which allow for those distortions to be corrected.

Expression, production, and renaturation of a functional single-chain variable antibody fragment (scFv) against human ICAM-1

Intercellular adhesion molecule-1 (ICAM-1) is an important factor in the progression of inflammatory responses in vivo. To develop a new anti-inflammatory drug to block the biological activity of ICAM-1, we produced a monoclonal antibody (Ka=4.19×10−8 M) against human ICAM-1. The anti-ICAM-1 single-chain variable antibody fragment (scFv) was expressed at a high level as inclusion bodies in Escherichia coli. We refolded the scFv (Ka=2.35×10−7 M) by ion-exchange chromatography, dialysis, and dilution. The results showed that column chromatography refolding by high-performance Q Sepharose had remarkable advantages over conventional dilution and dialysis methods. Furthermore, the anti-ICAM-1 scFv yield of about 60 mg/L was higher with this method. The purity of the final product was greater than 90%, as shown by denaturing gel electrophoresis. Enzyme-linked immunosorbent assay, cell culture, and animal experiments were used to assess the immunological properties and biological activities of the renatured scFv.

A forced running wheel system with a microcontroller that provides high-intensity exercise training in an animal ischemic stroke model

We developed a forced non-electric-shock running wheel (FNESRW) system that provides rats with high-intensity exercise training using automatic exercise training patterns that are controlled by a microcontroller. The proposed system successfully makes a breakthrough in the traditional motorized running wheel to allow rats to perform high-intensity training and to enable comparisons with the treadmill at the same exercise intensity without any electric shock. A polyvinyl chloride runway with a rough rubber surface was coated on the periphery of the wheel so as to permit automatic acceleration training, and which allowed the rats to run consistently at high speeds (30 m/min for 1 h). An animal ischemic stroke model was used to validate the proposed system. FNESRW, treadmill, control, and sham groups were studied. The FNESRW and treadmill groups underwent 3 weeks of endurance running training. After 3 weeks, the experiments of middle cerebral artery occlusion, the modified neurological severity score (mNSS), an inclined plane test, and triphenyltetrazolium chloride were performed to evaluate the effectiveness of the proposed platform. The proposed platform showed that enhancement of motor function, mNSS, and infarct volumes was significantly stronger in the FNESRW group than the control group (P<0.05) and similar to the treadmill group. The experimental data demonstrated that the proposed platform can be applied to test the benefit of exercise-preconditioning-induced neuroprotection using the animal stroke model. Additional advantages of the FNESRW system include stand-alone capability, independence of subjective human adjustment, and ease of use.

Investigation of cytotoxic and mutagenic effects of Malpighia glabra L. (barbados cherry) fruit pulp and vitamin C on plant and animal test systems

Fruits are important sources of nutrients in human diet, and Barbados Cherry (Malpighia glabra L.) is of particular interest due to its high content of antioxidants. Diets rich in fruits and vegetables protect individuals against diseases and cancer, but excessive intake of vitamins may act as pro-oxidant and generate changes in DNA. To evaluate the effect of different in natura (BAN) and frozen (BAF) Barbados Cherry pulp concentrations and synthetic vitamin C in liquid form (VC) on the chromosome level and the cell cycle division, root meristeme cells of Allium cepa L. and bone marrow cells of Wistar rats Rattus norvegicus, were used as test system. In Allium cepa L., BAN, at the highest concentration (0.4 mg.mL-1) and BAF, at the lowest concentration (0.2 mg.mL-1), inhibited cell division, and there was recovery of cell division after the recovery period in water only for BAN. In the Wistar rats, all treatments with Barbados Cherry, either acute or subchronic, were not cytotoxic or mutagenic; only the highest concentration of VC increased significantly the rate of chromosomal abnormalities. The data obtained are important to reinforce the use of Barbados Cherry fruit in the diet.