Histological studies on cultured canine heart valves recovered from −196 °C

Adult canine heart valves have been frozen to ?196 °C, (0.5 to 0.7 °C/min from 0 to ?100 °C) with 10% DMSO ($\text{v}\text{v}$), stored, thawed at ~150 °C/min, and then cultured for 9 to 12 days. A histological analysis of sections derived from several valves indicates viability, but with a not inconsiderable loss of stromal fibrocytes and some damage to the endothelial lining. The practicality of freezing valve tissue for banking will have to be looked at critically, before valve transplants can be considered as a possible alternative to the well established use of mechanical valve prosthesis. However, demonstrating viability of heart valve tissue extends the range of tissues that are amenable to cryopreservation.     

Permeability of the 17-day fetal rat pancreas to glycerol and dimethylsulfoxide

Experimentally induced diabetes in rats can be reversed by the transplantation of several fresh or frozen-thawed fetal pancreases. An important question to both the mechanistic and practical aspects of cryobiology is the role played by the permeation of protective additives during freezing, thawing, and subsequent dilution. Answers require knowledge of the kinetics of permeation of the specific additive into the cell or tissue. In this paper, we report isotopic measurements of the rate of permeation of 2 M glycerol and 1 and 2 M dimethylsulfoxide (Me₂SO) into 17-day fetal pancreases at 0 and 22 °C. In Me₂SO, equilibrium was achieved in about 10–15 min at 0 °C and in less than 10 min at 22 °C. In glycerol, equilibrium was attained in about 60 min at 22 °C; but at 0 °C permeation was only 65% complete after 180 min. In general, Me₂SO permeated 10–30 times more rapidly than glycerol at 0 °C, and glycerol permeated about 10 times more rapidly at 22 than at 0 °C.The kinetics of permeation were more characteristic of a two-compartment than a single-compartment system. In all probability, the two compartments are the intercellular space and the intracellular space. The permeability data suggest that each compartment occupies about half the total volume.     

Progesterone in milk: a simple experiment illustrating the estrous cycle and enzyme immunoassay

Experiments designed for students in reproductive physiology are rare. Here, we describe a simple experiment concerning a physiological aspect of the reproductive system. Milk samples are obtained from cows in estrus, in midcycle, 21 days after insemination, and in gestation. With these samples, the gestation or estrous stage is determined according to the progesterone level in milk that is measured using enzyme immunoassay. This experiment can therefore be used to demonstrate assay techniques and to illustrate the variations in progesterone concentrations during an estrous cycle and gestation. This exercise should be given after the reproduction section of the animal physiology course so that students can apply their knowledge concerning hormonal profiles during an estrous cycle.     

The Gla domain of factor IXa binds to factor VIIIa in the tenase complex

During blood coagulation factor IXa binds to factor VIIIa on phospholipid membranes to form an enzymatic complex, the tenase complex. To test whether there is a protein-protein contact site between the gamma-carboxyglutamic acid (Gla) domain of factor IXa and factor VIIIa, we demonstrated that an antibody to the Gla domain of factor IXa inhibited factor VIIIa-dependent factor IXa activity, suggesting an interaction of the factor IXa Gla domain with factor VIIIa. To study this interaction, we synthesized three analogs of the factor IXa Gla domain (FIX1-47) with Phe-9, Phe-25, or Val-46 replaced, respectively, with benzoylphenylalanine (BPA), a photoactivatable cross-linking reagent. These factor IX Gla domain analogs maintain native tertiary structure, as demonstrated by calcium-induced fluorescence quenching and phospholipid binding studies. In the absence of phospholipid membranes, FIX1-47 was able to inhibit factor IXa activity. This inhibition is dependent on the presence of factor VIIIa, suggesting a contact site between the factor IXa Gla domain and factor VIIIa. To demonstrate a direct interaction we did cross-linking experiments with FIX1-479BPA, FIX1-4725BPA, and FIX1-4746BPA. Covalent cross-linking to factor VIIIa was observed primarily with FIX1-4725BPA and to a much lesser degree with FIX1-4746BPA. Immunoprecipitation experiments with an antibody to the C2 domain of factor VIIIa indicate that the factor IX Gla domain cross-links to the A3-C1-C2 domain of factor VIIIa. These results suggest that the factor IXa Gla domain contacts factor VIIIa in the tenase complex through a contact site that includes phenylalanine 25 and perhaps valine 46.     

A sequential sampling scheme for detecting infestation levels of tracheal mites (Heterostigmata: Tarsonemidae) in honey bee (Hymenoptera: Apidae) colonies

The introduction of parasitic honey bee mites, the tracheal mite, Acarapis woodi (Rennie) in 1984 and the Varroa mite, Varroa jacobsoni, in 1987, has dramatically increased the winter mortality of honey bee, Apis mellifera L., colonies in many areas of the United States. Some beekeepers have minimized their losses by routinely treating their colonies with menthol, currently the only Environmental Protection Agency-approved and available chemical for tracheal mite control. Menthol is also expensive and can interfere with honey harvesting. Because of inadequate sampling techniques and a lack of information concerning treatment, this routine treatment strategy has increased the possibility that tracheal mites will develop resistance to menthol. It is important to establish economic thresholds and treat colonies with menthol only when treatment is warranted rather than treating all colonies regardless of infestation level. The use of sequential sampling may reduce the amount of time and effort expended in examining individual colonies and determining if treatment is necessary. Sequential sampling also allows statistically based estimates of the percentage of bees in standard Langstroth hives infested with mites while controlling for the possibility of incorrectly assessing the amount of infestation. On the average, sequential sampling plans require fewer observations (bees) to reach a decision for specified probabilities of type I and type II errors than are required for fixed sampling plans, especially when the proportion of infested bees is either very low or very high. We developed a sequential sampling decision plan to allow the user to choose specific economic injury levels and the probability of making type I and type II errors which can result inconsiderable savings in time, labor and expense.     

Suppression of islet allogeneic immune response by indoleamine 2,3 dioxygenase-expressing fibroblasts

Success of transplantation of pancreatic islets which is a promising way for restoring efficient insulin regulation in type 1 diabetes depends on lifelong use of immunosuppressive drugs. To eliminate the use of systemic immunosuppressive drugs for islet transplantation, we examined the potential use of a local immunosuppressive factor, indoleamine 2,3-dioxygenase (IDO). Thus, the aim of this study was to determine whether local expression of IDO in bystander syngeneic fibroblasts could prevent islet allogeneic immune response in vitro. C57BL/6 (B6) mouse fibroblasts were induced to express IDO by either IFN-gamma treatment or transduction with an adenoviral vector and were co-cultured with B6 mouse lymphocytes and BALB/c mouse pancreatic islets in the presence or absence of an IDO inhibitor. Proliferation of lymphocytes were then assessed using [(3)H]-thymidine incorporation assay. IDO-expression by co-cultured syngeneic fibroblasts resulted in a five-fold decrease in lymphocyte proliferation rate upon stimulation of lymphocytes by allogeneic mouse pancreatic islets (21.9% +/- 5.3 and 22.1% +/- 4.9 in the preparations with IFN-gamma treated and genetically modified IDO-expressing fibroblasts, respectively vs. 100% in control groups, P < 0.01). Allogeneic response was restored when IDO inhibitor was added to the culture indicating that suppression was due to IDO. In conclusion, this study shows that local expression of IDO by syngeneic bystander fibroblasts can suppress in vitro proliferation of lymphocytes in response to stimulation with allogeneic pancreatic islets. This local immunosuppressive function of IDO may be employed for development of a novel alternative strategy for preventing allogeneic islet graft rejection.     

Influence of biotic and abiotic factors on the persistence of a Beauveria bassiana biopesticide in laboratory and high-rise poultry house settings

Recent studies suggest the potential for use of oil formulations of the entomopathogenic fungus, Beauveria bassiana, as residual sprays for control of house flies in poultry production facilities. The current study investigated the influence of biotic and abiotic factors on biopesticide persistence. We found that flies physically removed and deactivated conidia, with higher fly densities and greater cumulative exposure hastening the decline. Nonetheless, very low densities of viable conidia were still able to cause rapid mortality, suggesting the potential for relatively long re-treatment intervals as fly populations are controlled. Considering abiotic factors, we found that fungal spray treatments remained viable for up to 13 weeks under laboratory conditions. Periodic exposure of flies to the spray residue showed high levels of mortality, with very little decline in mortality rate over time. Equivalent treatments placed in a commercial poultry house showed much more rapid decline. One trial at the end of summer showed conidia to remain viable up to seven weeks. However, repeats during the winter months revealed decay in 1–2 weeks, with fly mortality rates influenced accordingly. The exact reasons for the more rapid decay remain unclear but could be linked to high concentrations of ammonia in the basement areas, especially during winter when ventilation is minimal. The combined data suggest the potential for adaptive treatment regimes with weekly spray intervals in conditions with very high fly populations and/or high ammonia levels, and potentially monthly spray intervals when fly populations and ammonia levels are reduced.     

Early behavioral changes and quantitative analysis of neuropathological features in murine prion disease: Stereological analysis in the albino Swiss mice model

Behavioral and neuropathological changes have been widely investigated in murine prion disease but stereological based unbiased estimates of key neuropathological features have not been carried out. After injections of ME7 infected (ME7) or normal brain homogenates (NBH) into dorsal CA1 of albino Swiss mice and C57BL6, we assessed behavioral changes on hippocampal-dependent tasks. We also estimated by optical fractionator at 15 and 18 weeks post-injections (w.p.i.) the total number of neurons, reactive astrocytes, activated microglia and perineuronal nets (PN) in the polymorphic layer of dentate gyrus (PolDG), CA1 and septum in albino Swiss mice. On average, early behavioral changes in albino Swiss mice start four weeks later than in C57BL6. Cluster and discriminant analysis of behavioral data in albino Swiss mice revealed that four of nine subjects start to change their behavior at 12 w.p.i. and reach terminal stage at 22 w.p.i and the remaining subjects start at 22 w.p.i. and reach terminal stage at 26 w.p.i. Biotinylated dextran-amine BDA-tracer experiments in mossy fiber pathway confirmed axonal degeneration and stereological data showed that early astrocytosis, microgliosis and reduction in the perineuronal nets are independent of a change in the number of neuronal cell bodies. Statistical analysis revealed that the septal region had greater levels of neuroinflammation and extracellular matrix damage than CA1. This stereological and multivariate analysis at early stages of disease in an outbred model of prion disease provided new insights connecting behavioral changes and neuroinflammation and seems to be important to understand the mechanisms of prion disease progression.Key words: prion disease, optical fractionator, neuropathology, behavioral changes, albino Swiss mice     

Sphingosine kinase 1 is a negative regulator of CD4+ Th1 cells

CD4+ Th1 cells produce IFN-gamma, TNF-alpha, and IL-2. These Th1 cytokines play critical roles in both protective immunity and inflammatory responses. In this study we report that sphingosine kinase 1 (SPHK1), but not SPHK2, is highly expressed in DO11.10 Th1 cells. The expression of SPHK1 in Th1 cells requires TCR signaling and new protein synthesis. SPHK1 phosphorylates sphingosine to form sphingosine-1-phosphate. Sphingosine-1-phosphate plays important roles in inhibition of apoptosis, promotion of cell proliferation, cell migration, calcium mobilization, and activation of ERK1/2. When SPHK1 expression was knocked down by SPHK1 short interfering RNA, the production of IL-2, TNF-alpha, and IFN-gamma by Th1 cells in response to TCR stimulation was enhanced. Consistently, overexpression of dominant-negative SPHK1 increased the production of IL-2, TNF-alpha, and IFN-gamma in Th1 cells. Furthermore, overexpression of SPHK1 in Th1 and Th0 cells decreased the expression of IL-2, TNF-alpha, and IFN-gamma. Several chemokines, including Th2 chemokines CCL17 and CCL22, were up-regulated by SPHK1 short interfering RNA and down-regulated by overexpression of SPHK1. We also showed that Th2 cells themselves express CCL17 and CCL22. Finally, we conclude that SPHK1 negatively regulates the inflammatory responses of Th1 cells by inhibiting the production of proinflammatory cytokines and chemokines.     

Identification of a novel human granzyme B inhibitor secreted by cultured sertoli cells

Sertoli cells have long since been recognized for their ability to suppress the immune system and protect themselves as well as other cell types from harmful immune reaction. However, the exact mechanism or product produced by Sertoli cells that affords this immunoprotection has never been fully elucidated. We examined the effect of mouse Sertoli cell-conditioned medium on human granzyme B-mediated killing and found that there was an inhibitory effect. We subsequently found that a factor secreted by Sertoli cells inhibited killing through the inhibition of granzyme B enzymatic activity. SDS-PAGE analysis revealed that this factor formed an SDS-insoluble complex with granzyme B. Immunoprecipitation and mass spectroscopic analysis of the complex identified a proteinase inhibitor, serpina3n, as a novel inhibitor of human granzyme B. We cloned serpina3n cDNA, expressed it in Jurkat cells, and confirmed its inhibitory action on granzyme B activity. Our studies have led to the discovery of a new inhibitor of granzyme B and have uncovered a new mechanism used by Sertoli cells for immunoprotection.