Efficient expansion of clinical-grade human fibroblasts on microcarriers: cells suitable for ex vivo expansion of clinical-grade hESCs

Human embryonic stem cells hold considerable potential for cell-based treatments of a variety of degenerative diseases, including diabetes, ischemic heart failure, and Parkinson's disease. However, advancing research to provide clinical-grade product requires scale-up to therapeutic quantities of stem cells and their differentiated progeny. Most human embryonic stem cell culture platforms require direct support by a fibroblast feeder layer or indirect support using fibroblast conditioned medium. Accordingly, large numbers of clinically compliant fibroblasts will be requisite for stem cell production. Published platforms for feeder production are insufficient for stem cell scale-up, being costly to operate and requiring considerable effort to prepare, maintain and harvest. Here we describe the expansion of cGMP-grade, FDA-approved human foreskin fibroblasts using cGMP-grade reagents and polystyrene-based cationic trimethyl ammonium-coated microcarriers in spinner flasks. Fibroblasts attach rapidly to the microcarriers (T(1/2)=75 min), and expand with a maximum doubling time of 22.5h. Importantly, microcarrier-expanded fibroblasts and their conditioned medium support pluripotent stem cell growth through >5 passages, enabling extended self-renewal and expansion while retaining full differentiation potential. In summary, the method described is an economical and cGMP-compliant means of producing human fibroblast cells in support of cGMP human embryonic stem cell culture.     

Tioman virus, a paramyxovirus of bat origin, causes mild disease in pigs and has a predilection for lymphoid tissues

Disease manifestation, pathology, and tissue tropism following infection with Tioman virus (TioPV), a newly isolated, bat-derived paramyxovirus, was investigated in subcutaneously (n = 12) and oronasally (n = 4) inoculated pigs. Pigs were either asymptomatic or developed pyrexia, but all of the animals produced neutralizing antibodies. The virus (viral antigen and/or genome) was detected in lymphocytes of the thymus, tonsils, spleen, lymph nodes and Peyer's patches (ileum), tonsillar epithelium, and thymic epithelioreticular cells. Virus was isolated from oral swabs but not from urine. Our findings suggest that the pig could act as an intermediate or amplifying host for TioPV and that oral secretion is a possible means of viral transmission.     

A sensitive and selective assay for chloramine production by myeloperoxidase

We describe a new assay for the chlorination activity of myeloperoxidase and detection of chloramines. Chloramines were detected by using iodide to catalyze the oxidation of either 3,3',5,5'-tetramethylbenzidine (TMB) or dihydrorhodamine to form strongly absorbing or fluorescent products, respectively. With TMB as little as 1 muM taurine chloramine could be detected. The sensitivity of the dihydrorhodamine assay was about 10-fold greater. The chlorination activity of myeloperoxidase was measured by trapping hypochlorous acid with taurine and subsequently using iodide to promote the oxidation reactions of the accumulated taurine chloramine. A similar approach was used to detect hypochlorous acid production by stimulated human neutrophils. Iodide-dependent catalysis distinguished N-chloramines from N-bromamines. This allows for discrimination between heme peroxidases that generate either hypochlorous acid or hypobromous acid. The assay has distinct advantages over existing assays for myeloperoxidase with regard to sensitivity, specificity, and its ease and versatility of use.     

Dopaminergic cell death precedes iron elevation in MPTP-injected monkeys

Though increasing lines of evidence suggest that iron accumulation and iron-induced oxidative stress might be important pathological factors responsible for substantia nigra (SN) cell death in Parkinson's disease (PD), it is still unknown whether iron accumulation is a primary cause or consequence of nigral cell death. Using nuclear microscopy, iron histochemistry, TUNEL method for apoptosis detection, and tyrosine hydroxylase (TH) immunohistochemistry, the present study investigated possible changes in iron contents in the SN and correlations of dopaminergic cell death progression with the process of iron accumulation in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-induced parkinsonian monkey from 1 d to 18 months after MPTP administration. Our study demonstrated that apoptosis occurred in the ipsilateral SN at 1 d after MPTP injection and the number of TH-positive cells decreased significantly from 1 week onward. However, iron content was significantly increased in the ipsilateral SN from 4.5 months to 18 months after MPTP injection, and the iron increase was significantly correlated to the extent of dopaminergic cell death. These results suggest that dopaminergic cell death induced by MPTP administration might lead to iron accumulation in the monkey SN, and increased iron might contribute to the progression of nigral degeneration.     

Plasma leptin in moderately obese men: independent effects of weight loss and aerobic exercise

The independent effects of weight loss and exercise on plasma leptin and total (AT), subcutaneous (SAT), and visceral (VAT) adipose tissue were investigated in 52 obese men. Subjects were randomly assigned to four 12-wk protocols: 1) control (C, n = 8), 2) diet-induced weight loss (DWL, n = 14), 3) exercise-induced weight loss (EWL, n = 14), and 4) exercise with weight maintenance (EWS, n = 16). Plasma leptin was unchanged in C (from 7.8 +/- 1.3 to 7.7 +/- 1.0 ng/ml). Equivalent weight loss (7.5 kg) decreased leptin significantly but similarly (DWL, from 8.5 +/- 1.0 to 4.8 +/- 0.6 ng/ml; EWL, from 10.1 +/- 1.0 to 5.0 +/- 0.6 ng/ml). Exercise in the absence of weight loss did not alter leptin levels (from 10.1 +/- 1. 3 to 9.2 +/- 1.2 ng/ml). Changes in leptin correlated with changes in AT and SAT (both P < 0.05) but not in VAT. We conclude that reduction in adipose tissue after weight loss results in a collateral decrease in circulating leptin, and exercise, independent of its effects on weight loss, has no profound influence on leptin secretion.     

Chromosomal 16p microdeletion in Rubinstein-Taybi syndrome detected by oligonucleotide-based array comparative genomic hybridization: a case report

Introduction

Chromosomal aberrations of chromosome 16 are uncommon and submicroscopic deletions have rarely been reported. At present, a cytogenetic or molecular abnormality can only be detected in 55% of Rubinstein-Taybi syndrome patients, leaving the diagnosis in 45% of patients to rest on clinical features only. Interestingly, this microdeletion of 16 p13.3 was found in a young child with an unexplained syndromic condition due to an indistinct etiological diagnosis. To the best of our knowledge, no evidence of a microdeletion of 16 p13.3 with contiguous gene deletion, comprising cyclic adenosine monophosphate-response element-binding protein and tumor necrosis factor receptor-associated protein 1 genes, has been described in typical Rubinstein-Taybi syndrome.

Case presentation

We present the case of a three-year-old Malaysian Chinese girl with a de novo microdeletion on the short arm of chromosome 16, identified by oligonucleotide array-based comparative genomic hybridization. Our patient showed mild to moderate global developmental delay, facial dysmorphism, bilateral broad thumbs and great toes, a moderate size atrial septal defect, hypotonia and feeding difficulties. A routine chromosome analysis on 20 metaphase cells showed a normal 46, XX karyotype. Further investigation by high resolution array-based comparative genomic hybridization revealed a 120 kb microdeletion on chromosomal band 16 p13.3.

Conclusion

A mutation or abnormality in the cyclic adenosine monophosphate-response element-binding protein has previously been determined as a cause of Rubinstein-Taybi syndrome. However, microdeletion of 16 p13.3 comprising cyclic adenosine monophosphate-response element-binding protein and tumor necrosis factor receptor-associated protein 1 genes is a rare scenario in the pathogenesis of Rubinstein-Taybi syndrome. Additionally, due to insufficient coverage of the human genome by conventional techniques, clinically significant genomic imbalances may be undetected in unexplained syndromic conditions of young children. This case report demonstrates the ability of array-based comparative genomic hybridization to offer a genome-wide analysis at high resolution and provide information directly linked to the physical and genetic maps of the human genome. This will contribute to more accurate genetic counseling and provide further insight into the syndrome.     

Morphometric variation in the pusillus group of the genus Rhinolophus (Mammalia: Chiroptera: Rhinolophidae) in East Asia

Based on 203 specimens belonging to the Rhinolophus "pusillus group" (Mammalia: Chiroptera: Rhinolophidae), univariate and multivariate morphometric analyses using 19 characters were performed to assess the confused species taxonomy. The results indicated that R. pusillus (including calidus, parcus, and szechuanus) in the continental region and Hainan Island of China and "R. cornutus" in Japan are morphologically divergent species. Rhinolophus cornutus should be further split into R. cornutus (including orii, pumilus, and miyakonis) in the main islands of Japan, the Amami and Okinawa Group of the central Ryukyu Archipelago, and Miyako Group of the southern Ryukyus; and R. perditus and R. imaizumii from the Yaeyama Group in the southern Ryukyus. Rhinolophus monoceros from Taiwan is morphologically more similar to species in Japan than to R. pusillus. In addition to R. pusillus, another form that is morphologically similar to species in Japan was recognized from Langzhong in Sichuan Province; this may represent an undescribed species, and further examination is necessary to determine its taxonomic status. Specimens from Guang'an in Sichuan Province, China, are also different from the others, and are characterized by the smallest skull size. Although further studies are required, these specimens were tentatively identified as R. subbadius.     

Butanol-extracted lipoteichoic acid induces in vivo leukocyte adhesion

Lipoteichoic acid (LTA), an immunostimulatory component of the cell walls of Gram positive bacteria, has pro-inflammatory effects in vitro and in vivo. However, one in vivo study concluded that LTA had no noticeable effects on leukocyte recruitment. In this study we investigated the effects of highly purified LTA, prepared by butanol extraction (Bu-LTA) at room temperature, on in vivo leukocyte adhesion. Using intravital microscopy we measured adhesion of leukocytes in mesenteric post-capillary venules of rats and mice. Topical superfusion of Bu-LTA (1 μg/ml) in rats significantly (p < 0.05) increased adhesion within 30 min. By contrast, hot phenol-extracted LTA did not increase adhesion. Alkaline hydrolysis of Bu-LTA removed alanine residues and prevented adhesion. Also, pre-administration of anti-rat β₂-integrin antibody abolished Bu-LTA-induced adhesion. Finally, intraperitoneal injection of Bu-LTA (100 μg/ml) into mice also significantly (p < 0.01) increased leukocyte adhesion measured at 60 min. In conclusion, Bu-LTA with intact alanine residues promotes β₂-integrin-dependent leukocyte adhesion in vivo.     

Peptide mimotopes of complex carbohydrates in Salmonella enterica serovar typhi which react with both carbohydrate-specific monoclonal antibody and polyclonal sera from typhoid patients

Polyclonal sera from typhoid patients and a monoclonal antibody, mAb ATVi, which recognizes the capsular polysaccharide Vi antigen (ViCPS), were used to select for peptides that mimic the ViCPS by using a phage-displayed random 12-mer peptide library. Two major common mimotopes selected from the library carried the amino acid sequences TSHHDSHGLHRV and ENHSPVNIAHKL. Enzyme-linked immunosorbent assays (ELISAs) showed that these peptides carry mimotopes to ViCPS. Phage clones that contained the 12-mer peptides were also tested against pooled/individual typhoid patients' sera and found to have 3 to 5 times higher binding compared to normal sera. By using Phage-ELISA assays, the derived synthetic peptides, TSHHDSHGLHRV and ENHSPVNIAHKL, were tested against a monoclonal antibody mAb ATVi and over 2-fold difference in binding was found between these peptides and a control unrelated peptide, CTLTTKLYC. Inhibition of the mAb's binding to ViCPS indicated that the synthetic peptides successfully competed with the capsular polysaccharide for antibody binding.