Enzyme histochemistry of the mesenteric and dorsal root ganglion cells of cat and squirrel monkey

Summary A detailed histochemical study has been made on the mesenteric ganglia of the cat, and dorsal root ganglia of the squirrel monkey by the use of appropriate histochemical techniques accompanied by appropriate controls for phosphatases, esterases, and oxidative enzymes. The different neurons of a particular ganglion show varied amounts of enzyme activity at a particular time depending upon the functional state of the neurons. SDH, CYO and LDH reaction is prominent in the cytoplasm of the neurons, gliocytes and satellite cells, whereas the MAO preparations generally show a weak reaction. The AK is prominent in the neuropil, cell membranes and peripheral part of cytoplasm, whereas ATPase activity has been observed in blood vessels as well. In AC preparations the area of lipofuscin concentration shows more intense reaction than the rest of the cytoplasm. The activity of AChE and BChE varies from mild, to moderate to strong. The TPPase preparations show morphologically different types and amounts of TPPase positive Golgi material even in the adjoining cells. The relationship between the TPPase Golgi material and various oxidative and dephosphorylating enzymes has been briefly discussed.Abbreviations used AC Acid phosphatase - AChE Acetyl-cholinesterase (specific) - AK Alkaline phosphatase - AMPase Adenosine monophosphatase (5-nucleotidase) - ATPase Adenosine triphosphatase - BChE Butyryl-cholinesterase (nonspecific) - CYO Cytochrome oxidase - DPN-D DPN-diaphorase - G6P Glucose-6-phosphatase - LDH Lactic dehydrogenase - MAO Monoamine oxidase - MDH Malic dehydrogenase - NAD-D NAD-diaphorase - SDH Succinic dehydrogenase - SE Simple esterase - TPPase thiamine pyrophosphatase T. R. Shanthaveerappa in previous publications.     

Pachytene chromosomes in male and female mice heterozygous for the Is(7;1)40H insertion

Pairing of pachytene chromosomes was studied in oocytes and spermatocytes of mice heterozygous for the male-sterile Is(7;1)40H insertion using light and electron microscopy for synaptonemal complex analysis in surface-spread, silver-stained preparations. The data comprised four males and four female embryos. The insertion/deletion configurations appeared as either two bivalents or one quadrivalent in both sexes, but the proportion of bivalents was higher in oocytes. Some insertion and deletion bivalents showed synaptic adjustment. The insertion/deletion configurations were associated with, or adjacent to, the XY bivalent in the majority of spermatocytes. End-to-end association of different bivalents was more frequent in oocytes than in spermatocytes. It is suggested that physiological differences between male and female gametocytes may lead to the difference in their reproductive potential.The authors warmly dedicate this paper to the Founder and Senior Editor of Chromosoma, Professor Hans Bauer, on the occasion of his 80th birthday.     

Histology and histochemistry of the oviductal sperm storage pockets of the agamid lizard Calotes versicolor

Some reptiles are known to possess sperm storage in the female genital tract. Examination of the oviduct of the lizard Calotes versicolor shows the presence of sperm in storage pockets of the uterovaginal region throughout the reproductive cycle. Histochemical studies indicate that the epithelium of these pockets resembles the local oviductal epithelium and that a considerable amount of activity of steroidogenic and metabolic enzymes exists.     

Application of Bioluminescence Imaging to the Prediction of Lethality in Vaccinia Virus-Infected Mice

To find an alternative endpoint for the efficacy of antismallpox treatments, bioluminescence was measured in live BALB/c mice following lethal challenge with a recombinant WR vaccinia virus expressing luciferase. Intravenous vaccinia immunoglobulin treatments were used to confer protection on a proportion of animals. Using known lethality outcomes in 200 animals and total fluxes recorded daily in live animals, we performed univariate receiver operating characteristic (ROC) curve analysis to assess whether lethality can be predicted based on bioluminescence. Total fluxes in the spleens on day 3 and in the livers on day 5 generated accurate predictive models; the area under the ROC curve (AUC) was 0.91. Multiple logistic regression analysis utilizing a linear combination of six measurements: total flux in the liver on days 2, 3, and 5; in the spleen on days 1 and 3; and in the nasal cavity on day 4 generated the most accurate predictions (AUC = 0.96). This model predicted lethality in 90% of animals with only 10% of nonsurviving animals incorrectly predicted to survive. Compared with bioluminescence, ROC analysis with 25% and 30% weight loss as thresholds accurately predicted survival on day 5, but lethality predictions were low until day 9. Collectively, our data support the use of bioimaging for lethality prediction following vaccinia virus challenge and for gaining insight into protective mechanisms conferred by vaccines and therapeutics.In 1980, the World Health Organization declared that the world was finally free of smallpox as an extant human disease, and routine smallpox vaccination was discontinued. However, concerns that variola virus (the causative agent of smallpox) might be reintroduced into the human population as a bioterrorist agent has intensified research targeted toward the development of novel antiviral therapies and safe and effective vaccines (3, 14). The global eradication program was accomplished using several vaccines based on attenuated replicating vaccinia virus strains, including Dryvax—a vaccine based on live vaccinia virus derived from the New York City Board of Health strain (NYCBOH) and prepared from calf lymph (Wyeth Laboratories) that was used in the Unites States and West Africa (22). For a long time, Dryvax was the only U.S. licensed vaccine for human use against the smallpox virus and was considered the “gold standard” due to the long-lasting virus-neutralizing antibodies it generated (1, 13). However, due to substantial risks of developing adverse reactions, such as progressive vaccinia, eczema vaccinatum, and severe generalized vaccinia, vaccination is contraindicated in people with compromised immune systems and individuals with eczema. In addition, the Dryvax vaccine was also shown to induce transient myopericarditis in a small proportion of healthy recipients, and therefore, it is no longer recommended for general use (2). The smallpox ACAM2000 vaccine utilizing the same NYCBOH strain of vaccinia virus and propagated in African green monkey cells (Vero cells) is manufactured by Acambis/Baxter Pharmaceuticals and was recently licensed by the Food and Drug Administration for clinical use. A total of six clinical trials (phases I to III) with ACAM2000 vaccine were performed, and the results showed that vaccine-emergent reactions were slightly reduced in the ACAM2000 recipients (11). However, concerns related to stability, inability to be diluted, and decreased numbers of takes in ACAM2000 vaccine-experienced subjects suggested that further developments are needed to ensure the maintenance of a potent and safe vaccine stockpile (11). Several alternative vaccine modalities that are expected to have better safety profiles are under development or in clinical trials, such as modified vaccinia Ankara (5), DNA plasmids, and recombinant proteins. It is conceivable that new immunotherapies are needed to ensure safe and efficient treatment of complications associated with live vaccinia virus-based vaccines and for treatment of subjects exposed to variola virus.Intravenous vaccinia virus immunoglobulin (VIGIV; Cangene Corporation) is a new product that replaced intramuscular vaccinia virus immunoglobulin (VIGIM), which had been used for treatment of complications related to smallpox vaccinations since the 1950s. VIGIV is prepared from the purified gamma immunoglobulin (immunoglobulin G) fraction of plasma taken from healthy donors previously vaccinated with Dryvax who demonstrate high titers of vaccinia virus neutralizing antibody. This product was licensed for the treatment of patients who develop complications following Dryvax vaccination. It has been suggested that VIGIV might also be life saving for unvaccinated persons who have come into contact with people exposed to variola virus itself and may help to limit the spread of the disease (27). The efficacy of VIGIV has not been tested in many clinical situations. The clinical pharmacology and pharmacodynamics of VIGIV were tested in two phase I clinical trials, which showed that the product was well tolerated, with all adverse effects related to VIGIV being typical of those expected following intravenous administration of the protein (27). The recommended dosage of VIGIV for the management of several complications of smallpox (vaccinia) vaccinations is 6,000 U/kg body weight (4).The development of novel vaccines against smallpox and of new pre- or postexposure therapies critically depends on the use of animal models for the initial preclinical testing. Several animal models are employed as surrogate models of variola virus infection in humans, including infection of macaques with monkeypox virus, which has been used as a tool to dissect the immune responses to poxviruses (21). Importantly, experiments with rhesus macaques demonstrated a critical role for antibodies in the protection of vaccinated animals and helped select an optimal vaccine regimen utilizing DNA-coded monkeypox virus proteins in proof-of-concept studies (6, 15). However, due to the expense and the requirement for biosafety level 3 facilities, nonhuman primate models cannot be widely used for smallpox vaccine development. The majority of preclinical testing and initial characterization of smallpox vaccines and therapies is performed with the Western Reserve (WR) strain of vaccinia virus or with ectomelia virus, which is highly lethal in mice. Various endpoints are used to follow infections in mice, including weight loss, pox lesion scoring, and viral-load measurements by plaque formation on sensitive cell lines. These endpoints are not optimal, as they cannot avoid morbidity or accurately predict lethality in individual animals. They require very large number of animals in order to determine survival rates and to quantify viral loads in internal organs. Lethality was frequently used as an endpoint in the past but is no longer acceptable.Whole-animal bioimaging has been widely used for studies of microbial and viral pathogens in small-animal models (16, 19). This technology allows monitoring of pathogen dissemination in real time, locating pathogens residing in unexpected anatomical sites, and greatly reducing the numbers of animals required per study by providing spatial and temporal information for individual animals (16, 19). For this purpose, genes coding for luciferase enzymes are expressed in bacterial or viral pathogens, and dissemination of the recombinant pathogen is recorded by detecting light emitted from the tissues of infected live animals (19). Using bioimaging of live animals, bioluminescence of poxviruses expressing luciferase was employed to evaluate mucosal vaccinia virus vaccine (7), to dissect the roles of type I interferons and innate immunity in controlling viral replication and spread (18), and to characterize the tissue distribution of and immune responses induced by viral strains used for the development of vectored vaccines against other pathogens (9, 23). Measurements of photon fluxes have been shown to correlate in linear fashion with viral loads in internal organs in vaccinia virus-infected mice, suggesting that bioluminescence provides a direct measure of viral dissemination (19). However, whether bioluminescence signals derived from luciferase-expressing vaccinia virus can be used to predict lethality in infected mice has not yet been investigated.To that end, we used a recombinant WR vaccinia virus expressing luciferase to record bioluminescence in healthy mice after intranasal (i.n.) infection with 1 to 5 50% lethal doses (LD₅₀). Groups of animals were immunized with Dryvax or pretreated with VIGIV. We used acquired images from surviving and nonsurviving mice to calculate total fluxes in internal organs and applied receiver operating characteristic (ROC) curve analysis to generate predictive models. Our data showed that measurements of total fluxes in the spleen and liver 3 and 5 days postinfection provided strong predictive models of lethality. The predictive power of bioimaging was further investigated when bioluminescences from several organs and multiple time points were combined in multiple logistic regression analysis.     

Development of mammalian production cell lines expressing CNTO736, a glucagon like peptide‐1‐MIMETIBODYTM: Factors that influence productivity and product quality

In an attempt to develop a high producing mammalian cell line expressing CNTO736, a Glucagon like peptide‐1‐antibody fusion protein (also known as a Glucagon like peptide‐1 MIMETIBODY^TM), we have noted that the N‐terminal GLP‐1 portion of the MIMETIBODY^TM was susceptible to proteolytic degradation during cell culture, which resulted in an inactive product. Therefore, a number of parameters that had an effect on productivity as well as product quality were examined. Results suggest that the choice of the host cell line had a significant effect on the overall product quality. Product expressed in mouse myeloma host cell lines had a lesser degree of proteolytic degradation and variability in O‐linked glycosylation as compared to that expressed in CHO host cell lines. The choice of a specific CHOK1SV derived clone also had an effect on the product quality. In general, molecules that exhibited minimal N‐terminal clipping had increased level of O‐linked glycosylation in the linker region, giving credence to the hypothesis that O‐linked glycosylation acts to protect against proteolytic degradation. Moreover, products with reduced potential for N‐terminal clipping had longer in vivo serum half‐life. These findings suggest that early monitoring of product quality should be an essential part of production cell line development and therefore, has been incorporated in our process of cell line development for this class of molecules. Biotechnol. Bioeng. 2009;103: 162–176. © 2008 Wiley Periodicals, Inc.