The novel role of SERPINB9 in cytotoxic protection of human mesenchymal stem cells

Clinical trials using allogeneic mesenchymal stem cells (MSCs) are ongoing for the purpose of providing therapeutic benefit for a variety of human disorders. Pertinent to their clinical use are the accessibility to sufficient quantities of these cells allowing for repetitive administration, as well as a better understanding of the specific mechanisms by which allogeneic MSCs evade host immune responses that in turn influence their life span following administration. In this report, we sought to characterize and compare human peripheral blood MSCs (hPB-MSCs) with bone marrow-derived MSCs. hPB-MSCs met the established criteria to characterize this cellular lineage, including capacity for self-renewal, differentiation into tissues of mesodermal origin, and expression of phenotypic surface markers. In addition, hPB-MSCs suppressed alloreactive proliferation as well as the production of proinflammatory cytokines. Examination of the mechanisms by which allogeneic MSCs evade the host immune response, which is crucial for their therapeutic use, demonstrated that constitutive expression of serine protease inhibitor 9 (PI-9) on hPB-MSCs and bone marrow-derived MSCs is a major defense mechanism against granzyme B-mediated destruction by NK cells. Similarly, MSCs treated with small interfering RNA for PI-9 increased MSC cellular death, whereas expression of transgenic PI-9 following retroviral transduction protected MSCs. These data significantly advance our understanding of the immunomodulatory role for hPB-MSCs as well as the mechanisms by which they evade host immune responses. These findings contribute to the development of MSC-based therapies for diseases.     

Myosin 3A Kinase Activity Is Regulated by Phosphorylation of the Kinase Domain Activation Loop

Class III myosins are unique members of the myosin superfamily in that they contain both a motor and kinase domain. We have found that motor activity is decreased by autophosphorylation, although little is known about the regulation of the kinase domain. We demonstrate by mass spectrometry that Thr-178 and Thr-184 in the kinase domain activation loop and two threonines in the loop 2 region of the motor domain are autophosphorylated (Thr-908 and Thr-919). The kinase activity of MYO3A 2IQ with the phosphomimic (T184E) or phosphoblock (T184A) mutations demonstrates that kinase activity is reduced 30-fold as a result of the T184A mutation, although the Thr-178 site only had a minor impact on kinase activity. Interestingly, the actin-activated ATPase activity of MYO3A 2IQ is slightly reduced as a result of the T178A and T184A mutations suggesting coupling between motor and kinase domains. Full-length GFP-tagged T184A and T184E MYO3A constructs transfected into COS7 cells do not disrupt the ability of MYO3A to localize to filopodia structures. In addition, we demonstrate that T184E MYO3A reduces filopodia elongation in the presence of espin-1, whereas T184A enhances filopodia elongation in a similar fashion to kinase-dead MYO3A. Our results suggest that as MYO3A accumulates at the tips of actin protrusions, autophosphorylation of Thr-184 enhances kinase activity resulting in phosphorylation of the MYO3A motor and reducing motor activity. The differential regulation of the kinase and motor activities allows for MYO3A to precisely self-regulate its concentration in the actin bundle-based structures of cells.     

FasL‐triggered death of Jurkat cells requires caspase 8‐induced,ATP‐dependent cross‐talk between fas and the purinergic receptor P2X7

Fas ligation via the ligand FasL activates the caspase‐8/caspase‐3‐dependent extrinsic death pathway. In so‐called type II cells, an additional mechanism involving tBid‐mediated caspase‐9 activation is required to efficiently trigger cell death. Other pathways linking FasL–Fas interaction to activation of the intrinsic cell death pathway remain unknown. However, ATP release and subsequent activation of purinergic P2X₇ receptors (P2X₇Rs) favors cell death in some cells. Here, we evaluated the possibility that ATP release downstream of caspase‐8 via pannexin1 hemichannels (Panx1 HCs) and subsequent activation of P2X₇Rs participate in FasL‐stimulated cell death. Indeed, upon FasL stimulation, ATP was released from Jurkat cells in a time‐ and caspase‐8‐dependent manner. Fas and Panx1 HCs colocalized and inhibition of the latter, but not connexin hemichannels, reduced FasL‐induced ATP release. Extracellular apyrase, which hydrolyzes ATP, reduced FasL‐induced death. Also, oxidized‐ATP or Brilliant Blue G, two P2X₇R blockers, reduced FasL‐induced caspase‐9 activation and cell death. These results represent the first evidence indicating that the two death receptors, Fas and P2X₇R connect functionally via caspase‐8 and Panx1 HC‐mediated ATP release to promote caspase‐9/caspase‐3‐dependent cell death in lymphoid cells. Thus, a hitherto unsuspected route was uncovered connecting the extrinsic to the intrinsic pathway to amplify death signals emanating from the Fas receptor in type II cells. J. Cell. Physiol. 228: 485–493, 2013. © 2012 Wiley Periodicals, Inc.     

Aminoacetone Induces Loss of Ferritin Ferroxidase and Iron Uptake Activities

The effects of inorganic phosphate (P_i), the main intracellular membrane permeable anion capable of altering mitochondrial pH gradients (ΔpH), were measured on mitochondrial H₂O₂ release. As expected, P_i decreased ΔpH and increased the electric membrane potential (ΔΨ). Mitochondrial H₂O₂ release was stimulated by P_i and also by its structural analogue arsenate. However, acetate, another membrane-permeable anion, did not stimulate mitochondrial H₂O₂ release. The stimulatory effect promoted by P_i was prevented by CCCP, which decreases transport of P_i across the inner mitochondrial membrane, indicating that P_i must be in the mitochondrial matrix to stimulate H₂O₂ release. In conclusion, we found that P_i and arsenate stimulate mitochondrial reactive oxygen release, an effect that may contribute towards oxidative stress under conditions such as ischemia/reperfusion, in which high-energy phosphate bonds are hydrolyzed.     

Amblyomma triste (Koch, 1844) (Acari: Ixodidae): morphological description of the ovary and of vitellogenesis

The present study presents the morphology, histology, and the dynamics of vitellogenesis in females of the tick Amblyomma triste. The ovary in this species is of the panoistic type, therefore it lacks nurse cells. It is composed of a layer of epithelial cells that outwardly form the wall of the ovary, but also originate the pedicel, the structure that attaches the oocytes to its external margin, as well the oocytes themselves. In Amblyomma triste, the oocytes develop in four synchronic stages, which differs from the process in other tick species. The classification of the stages of the oocytes was carried out based on the presence of four morphologic characteristics: cytoplasm appearance; site of the germ vesicle; presence, quantity, and constitution of the yolk granules and presence of chorium.     

Copulation is necessary for the completion of a gonotrophic cycle in the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae)

Ovarian development and egg maturation are essential stages in animal reproduction. For bisexual ixodid ticks, copulation is an important prerequisite for the completion of the gonotrophic cycle. In this study, we aimed to characterize the morpho-histological changes in the ovary and oocytes of the tick Rhipicephalus sanguineus, together with the identification of feeding and reproductive parameters associated with mating. Virgin and cross-mated females (with R. turanicus males) weighed 60% less at full engorgement than females mated conspecifically. In addition, the oocytes of these females did not develop to the same advanced stages as those of the conspecifically mated females. Sequencing of a 250-bp ITS-2 fragment in eggs that originated from a cross between an R. sanguineus female and an R. turanicus male showed a genotype similar (except by a deletion of 1 thymine) to that observed in the mother, arguing against fertilization by a trans-specific male. These findings suggest that male sex peptides are species-specific molecules that influence both full engorgement and oocyte maturation. Mechanical stimulation of the gonopore alone was insufficient for the completion of the entire process of vitellogenesis.     

Serum calcitonin negative Medullary thyroid carcinoma

Background

Epithelial ovarian neoplasms are an important cause of morbidity and mortality in women. The surgical management of ovarian neoplasms depends on their correct categorization as benign, borderline or malignant. This study was undertaken to evaluate the accuracy of intra-operative frozen section in the diagnosis of various categories of ovarian neoplasms.

Methods

Intraoperative frozen section diagnosis was retrospectively evaluated in 217 patients with suspected ovarian neoplasms who underwent surgery as primary line of therapy at our institution. This was compared with the final histopathologic diagnosis on paraffin sections.

Results

In 7 patients (3.2%) no opinion on frozen section was possible. In the remaining 210 patients frozen section report had a sensitivity of 100%, 93.5% and 45.5% for benign, malignant and borderline tumors. The corresponding specificities were 93.2%, 98.3% and 98.5% respectively. The overall accuracy of frozen section diagnosis was 91.2%. The majority of cases of disagreement were in the mucinous and borderline tumors.

Conclusion

Intraoperative frozen section has high accuracy in the diagnosis of suspected ovarian neoplasms. It is a valuable tool to guide the surgical management of these patients and should be routinely used in all major oncology centers.     

Phylogenetic relationships in genus <Emphasis Type="Italic">Arachis</Emphasis> based on ITS and 5.8S rDNA sequences

Background  

The genus Arachis comprises 80 species and it is subdivided into nine taxonomic sections (Arachis, Caulorrhizae, Erectoides, Extranervosae, Heteranthae, Procumbentes, Rhizomatosae, Trierectoides, and Triseminatae). This genus is naturally confined to South America and most of its species are native to Brazil. In order to provide a better understanding of the evolution of the genus, we reconstructed the phylogeny of 45 species using the variation observed on nucleotide sequences in internal transcribed spacer regions (ITS1 and ITS2) and 5.8 S of nuclear ribosomal DNA.     

Markers of cell death in salivary glands of males of the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari, Ixodidae)

The salivary glands of Rhipicephalus sanguineus males at stages: unfed (control), at day seven post-attachment, and at days three and seven post-detachment from the host were examined using methods of enzymatic analysis and cell viability. At these stages of feeding, different staining patterns were observed in the cells of type IV, III, II and I acini, which were affected by degeneration in this sequence. Acid phosphatase reaction was inversely proportional to that of ATPase, while ATPase reaction was proportional to membrane integrity. Salivary gland cells of unfed males exhibited intact nucleus and plasma membrane, suggesting that the acid phosphatase detected may participate in the normal physiology of acini. In males at day seven post-attachment, intact membranes were observed in almost all types of acini, as well as stronger reaction for acid phosphatase, nuclear changes, and decrease in ATPase reaction, changes associated with the degenerative process. At days three and seven post-detachment degeneration progress, being observed loss of membrane integrity, nuclear changes, prominent decrease in ATPase reaction, and an increase in acid phosphatase reaction in the first case and a decreased of it at day seven post-detachment from the host. During cell death, alterations occurred in the following sequence: a) nuclear changes, b) loss of ATPase reaction, c) loss of integrity of the plasma membrane, and d) increase of acid phosphatase. The latter might be associated with the late degradation of cytoplasmic remnants, characterizing the process of cell death in glands of R. sanguineus males as atypical or non-classic apoptosis.     

Morphological changes in the salivary glands of Amblyomma cajennense females (Acari: Ixodidae) in different feeding stages on rabbits at first infestation

This study examined salivary glands of unfed, partially engorged, and engorged females of the tick Amblyomma cajennense on rabbits at first infestation using histological and histochemical techniques. In type I acini, no significant changes were observed among the three feeding conditions. In type II acini of unfed females, c1, c2, and c4 cells were described for the first time in this species. In a comparison among the three feeding conditions, an increase in this acinus was observed, due to the increase in secretion in c1, c2, and c4 cells and the appearance of c3 cells. In engorged females, some cells were still active. Type III acini presented cells d, e, and f containing secretion in unfed females. In partially engorged females, these cells were devoid of secretion. In engorged females, type III acini exhibited a reduced lumen. After engorgement, all acini underwent a degenerative process, as observed in females after two to five days post-engorgement.