Use of an adaptable cell culture kit for performing lymphocyte and monocyte cell cultures in microgravity

The results of experiments performed in recent years on board facilities such as the Space Shuttle/Spacelab have demonstrated that many cell systems, ranging from simple bacteria to mammalian cells, are sensitive to the microgravity environment, suggesting gravity affects fundamental cellular processes. However, performing well-controlled experiments aboard spacecraft offers unique challenges to the cell biologist. Although systems such as the European 'Biorack' provide generic experiment facilities including an incubator, on-board 1-g reference centrifuge, and contained area for manipulations, the experimenter must still establish a system for performing cell culture experiments that is compatible with the constraints of spaceflight. Two different cell culture kits developed by the French Space Agency, CNES, were recently used to perform a series of experiments during four flights of the 'Biorack' facility aboard the Space Shuttle. The first unit, Generic Cell Activation Kit 1 (GCAK-1), contains six separate culture units per cassette, each consisting of a culture chamber, activator chamber, filtration system (permitting separation of cells from supernatant in-flight), injection port, and supernatant collection chamber. The second unit (GCAK-2) also contains six separate culture units, including a culture, activator, and fixation chambers. Both hardware units permit relatively complex cell culture manipulations without extensive use of spacecraft resources (crew time, volume, mass, power), or the need for excessive safety measures. Possible operations include stimulation of cultures with activators, separation of cells from supernatant, fixation/lysis, manipulation of radiolabelled reagents, and medium exchange. Investigations performed aboard the Space Shuttle in six different experiments used Jurkat, purified T-cells or U937 cells, the results of which are reported separately. We report here the behaviour of Jurkat and U937 cells in the GCAK hardware in ground-based investigations simulating the conditions expected in the flight experiment. Several parameters including cell concentration, time between cell loading and activation, and storage temperature on cell survival were examined to characterise cell response and optimise the experiments to be flown aboard the Space Shuttle. Results indicate that the objectives of the experiments could be met with delays up to 5 days between cell loading into the hardware and initial in flight experiment activation, without the need for medium exchange. Experiment hardware of this kind, which is adaptable to a wide range of cell types and can be easily interfaced to different spacecraft facilities, offers the possibility for a wide range of experimenters successfully and easily to utilise future flight opportunities.     

Assessment of the indoor environment in respiratory allergy

Many types of allergens may be present in the indoor environment and may lead to sensitization and respiratory allergy. Common indoor allergens include dust mites, animal dander, cockroach exposure and molds. Exposure to indoor pollutants, such as tobacco smoke, wood-burning stoves or fireplaces and chemical sprays, can precipitate and exacerbate symptoms. An allergic reaction in the airways caused by natural exposure to allergens has been shown to lead to an increase in inflammatory reaction, increased airway hyperresponsiveness and increased eosinophils in bronchoalveolar lavage. Other research has demonstrated that asthma symptoms correlate with levels of domestic dust mite and cockroach exposure. In the case of dust mites, ending exposure results in symptomatic relief.     

Expression of Hras-p21 and keratin K13 in UVR-induced skin tumors in Sencar mice

An ultraviolet radiation (UVR)-induced Sencar mouse skin carcinogenesis model was established to investigate the expression of Hras-p21 and keratin K13 in different stages of carcinogenesis, including UV-exposed nontumor skin, papillomas, squamous-cell carcinomas (SCCs), and malignant spindle-cell tumors (SCTs). Expression of Hras-p21 and K13 was examined in paraffin-embedded tumor sections by using immunohistochemical, immunofluorescent, and double staining techniques with specific antibodies. Positive Hras-p21 staining was detected in 1/3 (33%) papillomas, 24/36 (67%) of SCCs, but not in UVR-exposed nontumor skin or SCTs. Positive staining of the malignant progression marker K13 was found in 22/36 (61%) of SCCs only. Coexpression of Hras-p21 and K13 was found in 17/36(47%) SCCs. H-ras exons 1 and 2 were amplified from skin/tumor sections by using nested polymerase chain reaction (PCR). PCR-based single-strand conformation polymorphism (SSCP) analysis and gene sequencing revealed three point mutations, one in UVR-exposed nontumor skin (codon 56), and two in SCCs (codons 13 and 21). There were no clear relationships between point mutations of H-ras and the positive staining of Hras-p21 and K13. These results indicate that overexpression of ras-p21 in conjunction with aberrant expression of K13 is a frequent event in UVR-induced SCCs in Sencar mouse skin. Point mutation of the H-ras gene appeared to be a rare event in UVR skin carcinogenesis and not to be responsible for overexpression of Hras-p21.     

Pathogenicity of Mycoplasma synoviae in broiler chickens

Six isolates of Mycoplasma synoviae, identified as WVU 1853, K1968, K1858, 92D8034, F10-2AS, and FMT, were compared for pathogenicity in broiler chickens. Specific-pathogen-free chickens were inoculated, in two groups of 20, with each isolate by footpad or eyedrop inoculation at 1 day of age and were examined at necropsy 7, 14, 28, and 42 days postinoculation. Specimens were taken for histopathology, culture, polymerase chain reaction assay, and hemagglutination-inhibition serology. Isolates were grouped according to pathogenicity on the basis of differences in lesion development and tissue distribution in the respiratory system, other viscera, and the skeletal system. K1968 (pathogenic) induced lesions in all sites examined in both the footpad and eyedrop inoculation groups. It was detected in all sites following footpad inoculation and in all sites except viscera following eyedrop inoculation. WVU 1853, K1858, and 92D8034 (moderately pathogenic) induced lesions and were detected in all sites following footpad inoculation. With eyedrop inoculation, lesions were identified only in upper and lower respiratory sites, and organisms were detected only in upper respiratory sites. F10-2AS (moderately pathogenic) was similar; however, footpad inoculation failed to induce visceral lesions or permit organism detection in any site. F10-2AS was detected in upper and lower respiratory tissues following eyedrop inoculation. FMT (mildly pathogenic) induced only upper respiratory lesions when either footpad or eyedrop inoculation was used, and detection was restricted to upper respiratory sites following eyedrop inoculation. These results are useful in comparative evaluations of the virulence of other M. synoviae isolates and form a basis for characterization of virulence factors of M. synoviae.     

Estradiol-mediated suppression of apoptosis in the rabbit corpus luteum is associated with a shift in expression of bcl-2 family members favoring cellular survival

In the rabbit, estradiol is the primary luteotropic hormone. Estradiol withdrawal results in a rapid decline in serum progesterone and eventually in corpus luteum (CL) regression. The objective of this study was to determine whether estradiol modulates luteal cell apoptosis. In the first experiment, rabbits were randomly assigned to one of five experimental groups. An empty capsule (control) or estradiol-filled Silastic capsule was inserted s.c. on Day 0 of pseudopregnancy (day of hCG administration). On Day 11 of pseudopregnancy, some of the group I (control) and group II (estradiol capsule) rabbits were subjected to laparotomy, and one ovary from each rabbit was perfused in vitro to determine progesterone secretion rates. The CL from the contralateral ovary were dissected, snap-frozen, and stored at -70 degrees C until analyzed for internucleosomal DNA cleavage (apoptosis). Estradiol-containing capsules were removed from some of the remaining rabbits on Days 8, 9, and 10 to initiate estradiol deprivation. Rabbits were then subjected to laparotomy 24, 48, or 72 h after capsule removal (groups III, IV, and V, respectively), and ovaries or CL were processed as described above. Deprivation of estradiol for 24 (group III), 48 (group IV), or 72 (group V) h in vivo reduced in vitro progesterone secretion rates by more than 90% as compared to that in ovaries collected from estradiol capsule-intact animals. After in vivo endogenous estradiol suppression, withdrawal of exogenous estradiol resulted in luteal cell apoptosis, which increased in a time-dependent manner. Northern blot analysis revealed an increase in bax mRNA levels and a decrease in bcl-x mRNA levels coincident with luteal cell apoptosis induced by estradiol withdrawal. These data demonstrate that changes in progesterone production caused by estradiol exposure and deprivation are in part related to luteal cell apoptosis, and alterations in the expression of bcl-2 gene family members may be one of the mechanisms by which estradiol exerts its luteotropic effect in the rabbit CL.     

Hypermethylation can selectively silence individual p16ink4A alleles in neoplasia

OBJECTIVES: To determine the incidence of and risk factors associated with pneumothorax after donor nephroureterectomy and to determine the utility of postoperative chest roentgenography. METHODS: A retrospective review was made of 130 living donor nephroureterectomies performed at one institution (Yale-New Haven Hospital) using an extraperitoneal flank incision. RESULTS: Incidental pleurotomy occurred in 11 cases (8.5%). Rib resection was associated with pleurotomy. Patient age, sex, and side of operation were not associated with pleurotomy. Ten (91 %) of the 11 cases were identified intraoperatively. One unrecognized pneumothorax was identified postoperatively with chest roentgenography; no specific intervention was necessary. CONCLUSIONS: The extraperitoneal flank incision poses a significant risk for pneumothorax. Most pneumothoraces will be recognized intraoperatively. No adverse effects were noted secondary to pneumothorax.     

Decrease of capsular opacification with adjunctive mitomycin C in combined glaucoma and cataract surgery

OBJECTIVE: The authors investigated the incidence of capsular opacification requiring YAG capsulotomy after primary trabeculectomy combined with phacoemulsification and implantation of all polymethylmethacrylate intraocular lenses. DESIGN: A prospective randomized study. PARTICIPANTS: One hundred seventy-four eyes of 174 nonselected patients with primary open-angle glaucoma (POAG) were randomized to either no adjunctive mitomycin C (MMC) control group of 93 eyes of 93 patients) or adjunctive subconjunctival MMC (MMC group of 81 eyes of 81 patients) during the primary glaucoma triple procedure (PGTP). INTERVENTION: Primary glaucoma triple procedure with and without MMC and YAG laser capsulotomy for posterior capsular opacification (PCO) was performed. MAIN OUTCOME MEASURES: The incidences of YAG capsulotomy for PCO were compared between the control and MMC groups and also between the control group and the MMC subgroups (1 minute, 3 minutes, and 5 minutes of MMC application) using Kaplan-Meier analysis with Mantel-Cox log-rank test. Cox proportional hazard regression analysis also was performed to identify significant factors affecting capsular opacification. RESULTS: The control and MMC groups were similar in preoperative characteristics. However, the probability of PCO requiring YAG capsulotomy was significantly lower in the MMC group than in the control group (P = 0.004). Among the MMC subgroups, MMC application for 3 minutes was most effective and significant when compared with that of the control group (P = 0.002). Although not as significant as the intraoperative use of MMC (P = 0.002), old age (P = 0.026) and presence of diabetes mellitus (P = 0.035) were also identified as significant beneficial factors for decreasing the incidence of YAG capsulotomy for PCO in Cox proportional hazard regression analysis. CONCLUSION: Intraoperative subconjunctival MMC application during combined glaucoma and cataract surgery has a beneficial effect of inhibiting PCO after combined surgery in patients with POAG. Thus, after intraoperative subconjunctival application of MMC at the concentration of 0.5 mg/ml for 3 minutes, the aqueous MMC level must have been great enough to inhibit the lens epithelial cell proliferation to result in a long-term decrease in PCO.