Insulin-Producing Cells Differentiated from Human Bone Marrow Mesenchymal Stem Cells In Vitro Ameliorate Streptozotocin-Induced Diabetic Hyperglycemia

Background

The two major obstacles in the successful transplantation of islets for diabetes treatment are inadequate supply of insulin-producing tissue and immune rejection. Induction of the differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) into insulin-producing cells (IPCs) for autologous transplantation may alleviate those limitations.

Methods

hMSCs were isolated and induced to differentiate into IPCs through a three-stage differentiation protocol in a defined media with high glucose, nicotinamide, and exendin-4. The physiological characteristics and functions of IPCs were then evaluated. Next, about 3 × 10⁶ differentiated cells were transplanted into the renal sub-capsular space of streptozotocin (STZ)-induced diabetic nude mice. Graft survival and function were assessed by immunohistochemistry, TUNEL staining and measurements of blood glucose levels in the mice.

Results

The differentiated IPCs were characterized by Dithizone (DTZ) positive staining, expression of pancreatic β-cell markers, and human insulin secretion in response to glucose stimulation. Moreover, 43% of the IPCs showed L-type Ca²⁺ channel activity and similar changes in intracellular Ca²⁺ in response to glucose stimulation as that seen in pancreatic β-cells in the process of glucose-stimulated insulin secretion. Transplantation of functional IPCs into the renal subcapsular space of STZ-induced diabetic nude mice ameliorated the hyperglycemia. Immunofluorescence staining revealed that transplanted IPCs sustainably expressed insulin, c-peptide, and PDX-1 without apparent apoptosis in vivo.

Conclusions

IPCs derived from hMSCs in vitro can ameliorate STZ-induced diabetic hyperglycemia, which indicates that these hMSCs may be a promising approach to overcome the limitations of islet transplantation.     

<Emphasis Type="Italic">Bacillus subtilis</Emphasis> based-formulation for the control of postbloom fruit drop of citrus

Postbloom fruit drop (PFD) caused by Colletotrichum acutatum affects flowers and causes early fruit drop in all commercial varieties of citrus. Biological control with the isolate ACB-69 of Bacillus subtilis has been considered as a potential method for controlling this disease. This study aimed to develop and optimize a B. subtilis based-formulation with a potential for large-scale applications and evaluate its effect on C. acutatum in vitro and in vivo. Bacillus subtilis based-formulations were developed using different carrier materials, and their ability to control PFD was evaluated. The results of the assays led to the selection of the B. subtilis based-formulation with talc + urea (0.02 %) and talc + ammonium molybdate (1 mM), which inhibited mycelial growth and germination of C. acutatum. Studies with detached citrus flowers showed that the formulations were effective in controlling the pathogen. In field conditions, talc + urea (0.02 %) provided 73 % asymptomatic citrus flowers and 56 % of the average number of effective fruit (ANEF), equating with fungicide treatment. On the contrary, non-treated trees had 8.8 % of asymptomatic citrus flowers and 0.83 % ANEF. The results suggest that B. subtilis based-formulations with talc as the carrier supplemented with a nitrogen source had a high potential for PFD control.     

Effects of cytokines on human thymic epithelial cells in culture. II. Recombinant IL 1 stimulates thymic epithelial cells to produce IL6 and GM-CSF

Our earlier study reported the ability of interleukin 1 (IL1) to promote proliferation and to induce morphological changes of human thymic epithelial cells (TEC) in culture. The present study was undertaken to examine the effects of IL1 on the secretory function of TEC. Both human recombinant IL1 alpha and IL1 beta induced TEC to produce molecules in the culture supernatant fluids (TES) which displayed marked thymocyte proliferative capacities. This activity was specifically induced by IL1 since other TEC growth factors such as epidermal growth factor and a bovine pituitary extract had no effect on promoting secretion of T cell-activating molecules by TEC. Using specific radioimmunoassays for both forms of IL1, we found that unstimulated TEC produced negligible amounts of IL1 alpha and IL1 beta in TES, which were not increased by IL1 stimulation, and we concluded that the IL1-induced TES molecules were not IL1. IL1 induced TEC to produce IL6, as detected by the hybridoma growth factor biological activity. Neutralizing anti-IL6 antibodies completely blocked the thymocyte activating capacities of the IL1-induced TES thus implying a major role for IL6 in TEC-derived T cell activation. IL1 also induced TEC to produce GM-CSF as measured by bioassay and confirmed by an immunoenzymetric assay. Our results confirm that TEC are a source of cytokines and show that TEC respond to IL1 by producing cytokines with consequences on the thymic lymphoid population. This further emphasizes the importance and complexity of paracrine molecular interactions involved in intrathymic development.     

The impact of litter effects on dose-response modeling in teratology

The fitting of dose-response models to teratology data involving littermates in order to generate estimates of teratogenic risk is receiving increasing attention as a potential alternative to the "safety-factor" approach to risk estimation. In this paper, we utilize the beta-binomial distribution to introduce varying degrees of intralitter correlation, and, for purposes of illustration, consider a logistic dose-response model that describes the logit of risk as a straight-line function of ln(dose). The biases and (exact and asymptotic) variances of the maximum likelihood estimators of the intercept and slope are studied by simulation as a function of the intralitter correlation structure.     

Distinct cellular functions mediated by different VLA integrin alpha subunit cytoplasmic domains.

To characterize VLA alpha subunit cytoplasmic domain functions, unaltered alpha 2 cDNA (called X2C2) and two chimeric cDNAs (called X2C5 and X2C4) were constructed with extracellular alpha 2 domains and cytoplasmic alpha 2, alpha 5, and alpha 4 domains respectively. Upon transfection into rhabdomyosarcoma (RD) cells, each construct yielded comparable expression levels, immunoprecipitation profiles, and avidity for collagen and laminin. However, while RDX2C2 and RDX2C5 transfectants mediated collagen gel contraction, RDX2C4 and a mock transfectant (RDpF) did not. Conversely, only RDX2C4 cells (but not RDX2C2 or RDX2C5) showed enhanced cell migration on collagen and laminin compared with RDpF cells. This indicates markedly differing roles for integrin alpha subunit cytoplasmic domains in post-ligand binding events. Furthermore, stable exertion of physical force (collagen gel contraction) may involve fundamentally different cellular machinery than the transient adhesion occurring during cell migration. Finally, these findings provide insight into a functional flexibility perhaps resulting from multiple integrins binding to identical ligands.     

Analysis of dichotomous response data from certain toxicological experiments

In certain toxicological experiments with laboratory animals, littermate data are frequently encountered. It is generally recognized that one characteristic of this type of data is the "litter effect", i.e., the tendency for animals from the same litter to respond more alike than animals from different litters. In this paper attention is restricted to dichotomous response variables that frequently arise in toxicological studies, such as the occurrence of fetal death or a particular malformation. Various techniques for estimating the underlying probability of response are discussed. A number of generalized models that have recently been proposed to take the litter effect into account are breifly reviewed and compared to the simpler binomial and Poisson models. Various procedures for assessing the significance of treatment-control differences are presented and their relative merits discussed. Finally, future research needs in this area are outlined.     

Cloning and structure of the BepI modification methylase.

The gene coding for a CGCG specific DNA methylase has been cloned in E. coli from Brevibacterium epidermidis. The enzyme, named BepI methylase, is probably the cognate methylase of the FnuDII isoschizomer BepI endonuclease isolated from this strain. The expression of BepI methylase in E. coli is dependent on the orientation of the cloned fragment suggesting that the gene is transcribed from a promoter on the plasmid vector. No BepI endonuclease could be detected in the clones producing BepI methylase. The nucleotide sequence of the BepI methylase gene has been determined, it predicts a protein of 403 amino acids (MR: 45,447). Analysis of the amino acid sequence deduced from the nucleotide sequence revealed similarities between the BepI methylase and other cytosine methylases. M. BepI methylates the external cytosine in its recognition sequence.     

Comparison of non-linear and linear models for estimating haemoglobin adduct stability

According to the kinetic theory for the build-up and elimination of haemoglobin (Hb) adducts, unstable Hb adducts are simultaneously eliminated by zero-order Hb turnover and first-order chemical instability. Thus, the elimination of unstable Hb adducts is non-linear with respect to time. Nonetheless, many studies of Hb adduct stability have characterized the elimination of Hb adducts using linear zero-order or linear first-order models. This paper demonstrates the use of non-linear regression to estimate the first-order rate constant of Hb adduct instability (k) using data on the elimination of Hb adducts in rats dosed with benzene or ortho -toluidine. Results obtained using non-linear regression models are compared with results from the more commonly employed zero- and first-order linear models. It is shown that exposure estimates based on measured levels of unstable Hb adducts can be severely biased if zero-order turnover is assumed. Furthermore, based on published data, estimates of k are subject to estimated relative biases in the range of -4% to 96% when first-order linear models are used to characterize Hb adduct instability.     

Netherlands Twin Register: a focus on longitudinal research.

In 1986 we began The Netherlands Twin Register (NTR) by recruiting young twins and multiples a few weeks or months after birth. Currently we register around 50% of all newborn multiples in The Netherlands. Their parents receive a questionnaire at registration and afterwards when the children are 2, 3, 5, 7, 10 and 12 years of age. Teachers are asked to rate the behavior of the children at ages 7, 10 and 12 years. Adolescent and young-adult twins were recruited through City Councils in the early 1990s. These twins, their parents and siblings participate in longitudinal survey studies that include items about health, fertility, lifestyle, addiction, personality and psychopathology, religion, socioeconomic status, and educational attainment. The total number of twins and multiples registered with the NTR is currently over 60,000. Subgroups of twins and siblings take part in studies of cognitive development, brain function and neuropsychological indices of attention processes, and molecular genetic studies of classical and behavioral cardiovascular risk factors. DNA samples are currently collected in selected twin families for two large linkage studies, which aim to find QTLs for anxious depression and for nicotine addiction. Sisters who are mothers of DZ twins contribute DNA samples for a linkage study of DZ twinning. Large cohorts of phenotyped family members from the general population are very valuable for genetic epidemiological studies and permit selection of informative families for gene finding studies.