Capillary Regeneration in Scleroderma: Stem Cell Therapy Reverses Phenotype?

Background

Scleroderma is an autoimmune disease with a characteristic vascular pathology. The vasculopathy associated with scleroderma is one of the major contributors to the clinical manifestations of the disease.

Methodology/Principal Findings

We used immunohistochemical and mRNA in situ hybridization techniques to characterize this vasculopathy and showed with morphometry that scleroderma has true capillary rarefaction. We compared skin biopsies from 23 scleroderma patients and 24 normal controls and 7 scleroderma patients who had undergone high dose immunosuppressive therapy followed by autologous hematopoietic cell transplant. Along with the loss of capillaries there was a dramatic change in endothelial phenotype in the residual vessels. The molecules defining this phenotype are: vascular endothelial cadherin, a supposedly universal endothelial marker required for tube formation (lost in the scleroderma tissue), antiangiogenic interferon α (overexpressed in the scleroderma dermis) and RGS5, a signaling molecule whose expression coincides with the end of branching morphogenesis during development and tumor angiogenesis (also overexpressed in scleroderma skin. Following high dose immunosuppressive therapy, patients experienced clinical improvement and 5 of the 7 patients with scleroderma had increased capillary counts. It was also observed in the same 5 patients, that the interferon α and vascular endothelial cadherin had returned to normal as other clinical signs in the skin regressed, and in all 7 patients, RGS5 had returned to normal.

Conclusion/Significance

These data provide the first objective evidence for loss of vessels in scleroderma and show that this phenomenon is reversible. Coordinate changes in expression of three molecules already implicated in angiogenesis or anti-angiogenesis suggest that control of expression of these three molecules may be the underlying mechanism for at least the vascular component of this disease. Since rarefaction has been little studied, these data may have implications for other diseases characterized by loss of capillaries including hypertension, congestive heart failure and scar formation.     

Telomerase-immortalized non-malignant human prostate epithelial cells retain the properties of multipotent stem cells

Understanding prostate stem cells may provide insight into the origin of prostate cancer. Primary cells have been cultured from human prostate tissue but they usually survive only 15-20 population doublings before undergoing senescence. We report here that RC-170N/h/clone 7 cells, a clonal cell line from hTERT-immortalized primary non-malignant tissue-derived human prostate epithelial cell line (RC170N/h), retain multipotent stem cell properties. The RC-170N/h/clone 7 cells expressed a human embryonic stem cell marker, Oct-4, and potential prostate epithelial stem cell markers, CD133, integrin alpha2beta1(hi) and CD44. The RC-170N/h/clone 7 cells proliferated in KGM and Dulbecco's Modified Eagle Medium with 10% fetal bovine serum and 5 microg/ml insulin (DMEM+10% FBS+Ins.) medium, and differentiated into epithelial stem cells that expressed epithelial cell markers, including CK5/14, CD44, p63 and cytokeratin 18 (CK18); as well as the mesenchymal cell markers, vimentin, desmin; the neuron and neuroendocrine cell marker, chromogranin A. Furthermore the RC170 N/h/clone 7 cells differentiated into multi tissues when transplanted into the sub-renal capsule and subcutaneously of NOD-SCID mice. The results indicate that RC170N/h/clone 7 cells retain the properties of multipotent stem cells and will be useful as a novel cell model for studying the mechanisms of human prostate stem cell differentiation and transformation.     

SIRT2 is a negative regulator of anoxia-reoxygenation tolerance via regulation of 14-3-3 zeta and BAD in H9c2 cells

Knockdown or inhibition of SIRT2 enhances biological stress-tolerance. We extend this phenotype showing that SIRT2 knockdown reduces anoxia-reoxygenation injury in H9c2 cells. Gene array analysis following SIRT2 siRNA knockdown identifies 14-3-3 zeta as the most robustly induced gene. SIRT2 knockdown evokes induction of this chaperone, facilitating cytosolic sequestration of BAD with a corresponding reduction in mitochondrial BAD localization. Concurrent siRNA against SIRT2 and 14-3-3 zeta abolishes the SIRT2-depleted cytoprotective phenotype. SIRT2 functions to moderate cellular stress-tolerance, in part, by modulating the levels of 14-3-3 zeta with the concordant control of BAD subcellular localization.     

RAB2A: a major subacrosomal protein of bovine spermatozoa implicated in acrosomal biogenesis

The perinuclear theca (PT) of mammalian sperm is a unique subcellular structure encapsulating the nucleus. Compositionally, the PT is made up of at least six prominent polypeptides (60, 36, 31, 28, 24, and 15 kDa), of which only two have been sequence identified, as well as many less prominent ones. As an ongoing process in unveiling the protein composition of the PT, we have uncovered the sequence identity of the prominent 24-kDa polypeptide (PT24). Initial N-terminal sequence analysis obtained by Edman degradation suggested that PT24 is a RAB2 protein. This was corroborated by mass spectrometric analyses of trypsin-digested fragments of PT24, identifying RAB2A of the RAB2 subfamily as the best sequence match. Quadrapole/time-of-flight analysis identified 72%% sequence coverage between PT24 and bull, human, mouse, or rabbit RAB2A. Since a genome search only identified two RAB2 subfamily members, RAB2A and RAB2B, the 72%% sequence coverage of PT24 provides assurance that this protein is RAB2A and not a new RAB2 subfamily member. Furthermore, commercial RAB2A antibodies, raised against oligopeptide fragments in the unique C-terminal region of RAB2A, specifically labeled PT24 on Western blot analysis of PT extracts. These anti-RAB2A antibodies, along with immune serum that we raised and affinity purified against isolated PT24, demonstrated at both light and electron microscope levels that RAB2 is associated with the periphery of the growing proacrosomic and acrosomic vesicles in the Golgi and cap phases of spermiogenesis and consequently assembled as part of the PT. This pattern of subacrosomal assembly is reminiscent of the pathway used by SubH2Bv (PT15), another prominent and exclusive subacrosomal protein, indicating a common route for subacrosomal-PT assembly. Traditionally somatic RAB2 proteins are involved in vesicular transport between the endoplasmic reticulum and the cis-side of the Golgi apparatus. Our study suggests an unprecedented direction of RAB2A-mediated vesicular transport in spermatids during acrosomal biogenesis, from the trans-side of the Golgi apparatus to the nuclear envelope.     

Stability ofAlternaria toxins in fruit juices and wine

Alternaria alternata is a common fungal parasite on fruits and other plants and produces a number of mycotoxins, including alternariol (3,7,9-trihydroxy-1-methyl-6H-dibenzo [b,d]pyran-6-one), alternariol monomethyl ether (3,7-dihydroxy-9-methoxy-1-methyl-6H-dibenzo[b,d]pyran-6-one), and the mutagen altertoxin I {[1S-(1α,12aβ,12bα)] 1,2,11,12,12a, 12b-hexahydro-1,4,9,12a-tetrahydroxy-3,10-perylenedione}. Alternariol and alternariol monomethyl ether have previously been detected in some samples of fruit beverages. Stability studies of these toxins as well as altertoxin I added to fruit juices and wine (10–100 ng/mL) were carried out. To include altertoxin I in the analysis, cleanup with a polymer-based Varian Abselut solid phase extraction column was used, as recoveries from C-18 columns were low. The stabilities of alternariol and alternariol monomethyl ether in a low acid apple juice containing no declared vitamin C were compared with those in the same juice containing added vitamin C (60 mg/175 ml); there were no apparent losses at room temperature over 20 days or at 80°C after 20 min. in either juice. Altertoxin I was moderately stable in pH 3 buffer (75% remaining after a two week period). Furthermore, altertoxin I was stable or moderately stable in three brands of apple juice tested over 1–27 day periods and in a sample of red grape juice over 7 days. It is concluded that altertoxin I is sufficiently stable to be found in fruit juices and should be included in methods for alternariol and alternariol monomethyl ether.     

The accuracy of the pepsin-cellulase technique for estimating the dry matter digestibility in vivo of grasses and legumes

A study was made of the accuracy of predicting dry matter digestibility (DMD) in vivo of 32 legume samples and 50 grass samples from the dry matter disappearance (DMS) of the samples in pepsin (48 h) followed by Onozuka cellulase (48 h). The effects of grinder screen size, incubation temperature and sample size on the accuracy of predicting dry matter digestibility were examined.The DMD in vivo of legumes and grasses was significantly correlated with DMS at 39° C (r = 0.91 and 0.94) and increasing the incubation temperature to 50° C had no effect on the correlations, although DMS was greater. There was no significant difference between the regression equations for grasses and legumes. The regression equation for the combined 82 samples had an RSD of ± 3.0 digestibility units. The corresponding RSD for regressions based on fermentation with rumen fluid in vitro was ± 2.6 digestibility units. Grinding the grass samples through a 0.4 mm screen prior to analysis resulted in regressions with a larger RSD (± 3.3) than when a 1.0 mm screen was used to prepare the samples (P < 0.001).It was concluded that the digestibility in vivo of both legumes and grasses could be predicted by the pepsin-cellulase method by using the same equation, but to eliminate biased results, samples of known digestibility in vivo similar to those being tested should be included as standards in each study. The analytical time could be reduced by 48 h if shorter incubation periods (24 h) were used, but only at the expense of slightly larger errors in predicting DMD in vivo (P < 0.01).     

Lake sturgeon population attributes and reproductive structure in the Namakan Reservoir,Minnesota and Ontario

Quantified were the age, growth, mortality and reproductive structure of lake sturgeon (Acipenser fulvescens) collected in the US and Canadian waters of the Namakan Reservoir. The hypotheses were tested that (i) age and growth of lake sturgeon in the Namakan Reservoir would differ by sex and reproductive stage of maturity, and (ii) that the relative strength of year‐classes of lake sturgeon in the reservoir would be affected by environmental variables. To quantify age, growth and mortality of the population, existing data was used from a multi‐agency database containing information on all lake sturgeon sampled in the reservoir from 2004 to 2009. Lake sturgeon were sampled in the Minnesota and Ontario waters of the Namakan Reservoir using multi‐filament gillnets 1.8 m high and 30–100 m long and varying in mesh size from 178 to 356 mm stretch. Reproductive structure of the lake sturgeon was assessed only during spring 2008 and 2009 using plasma testosterone and estradiol‐17β concentrations. Ages of lake sturgeon >75 cm ranged from 9 to 86 years (n = 533, mean = 36 years). A catch‐curve analysis using the 1981–1953 year classes estimated total annual mortality of adults to be 4.8% and annual survival as 95.2%. Using logistic regression analysis, it was found that total annual precipitation was positively associated with lake sturgeon year‐class strength in the Namakan Reservoir. A 10 cm increase in total annual precipitation was associated with at least a 39% increase in the odds of occurrence of a strong year class of lake sturgeon in the reservoir. Plasma steroid analysis revealed a sex ratio of 2.4 females: 1 male and, on average, 10% of female and 30% of male lake sturgeon were reproductively mature each year (i.e. potential spawners). Moreover, there was evidence based on re‐captured male fish of both periodic and annual spawning, as well as the ability of males to rapidly undergo gonadal maturation prior to spawning. Knowledge of lake sturgeon reproductive structure and factors influencing recruitment success contribute to the widespread conservation efforts for this threatened species.     

Dynamics of Newly Established Elk Populations

Abstract: The dynamics of newly established elk (Cervus elaphus) populations can provide insights about maximum sustainable rates of reproduction, survival, and increase. However, data used to estimate rates of increase typically have been limited to counts and rarely have included complementary estimates of vital rates. Complexities of population dynamics cannot be understood without considering population processes as well as population states. We estimated pregnancy rates, survival rates, age ratios, and sex ratios for reintroduced elk at Theodore Roosevelt National Park, North Dakota, USA; combined vital rates in a population projection model; and compared model projections with observed elk numbers and population ratios. Pregnancy rates in January (early in the second trimester of pregnancy) averaged 54.1% (SE = 5.4%) for subadults and 91.0% (SE = 1.7%) for adults, and 91.6% of pregnancies resulted in recruitment at 8 months. Annual survival rates of adult females averaged 0.96 (95% CI = 0.94-0.98) with hunting included and 0.99 (95% CI = 0.97-0.99) with hunting excluded from calculations. Our fitted model explained 99.8% of past variation in population estimates and represents a useful new tool for short-term management planning. Although we found no evidence of temporal variation in vital rates, variation in population composition caused substantial variation in projected rates of increase (Λ = 1.20-1.36). Restoring documented hunter harvests and removals of elk by the National Park Service led to a potential rate of Λ = 1.26. Greater rates of increase substantiated elsewhere were within the expected range of chance variation, given our model and estimates of vital rates. Rates of increase realized by small elk populations are too variable to support inferences about habitat quality or density dependence.