Infectivity titration of guinea-pig inclusion conjunctivitis agent in irradiated McCoy cells

Estimation of the infectivity of the agent of guinea-pig inclusion conjunctivitis for irradiated McCoy cells, assayed as inclusion-forming units, was influenced by the age of cells after irradiation, the maturation time of the inclusions, the centrifugal force and the centrifugation temperature. Agent passaged through irradiated McCoy cells or guinea-pig conjunctivae showed a greater capacity to infect irradiated McCoy cells without centrifuging than agent grown in a chick embryo. The nature of the change and the mechanism of infectivity enhancement by centrifuging are discussed.     

Use of a live chlamydial vaccine to prevent ovine enzootic abortion

A lyophilised chlamydial vaccine was prepared from the 1B temperature-sensitive strain of ovine Chlamydia psittaci. Ewes inoculated with a low titre of the live vaccine four weeks before artificial insemination were challenged on day 70 of gestation with five UK field isolates of C psittaci, including strains A22 and S26/3 previously incorporated into a commercial inactivated vaccine. There was a significantly lower chlamydial abortion rate after challenge in the vaccinated group (7.1 per cent) than in the unvaccinated group (80 per cent). All the lambs born to the vaccinated ewes were viable and of good quality. The vaccine also reduced the number of infected ewes in the group and the severity of the infection. The compatibility of the chlamydial vaccine and a toxoplasma vaccine was also tested. The abortion rate of ewes vaccinated with the two vaccines at separate injection sites (16 per cent) was less than that of ewes vaccinated with both vaccines at one site (32 per cent).     

Studies on the early phase of the pathogenesis of ovine enzootic abortion in the non-pregnant ewe

The initial phase of infection of non-pregnant sheep by Chlamydia psittaci (ovis) was studied by inoculating na?ve and previously exposed sheep by the oro-nasal and subcutaneous routes with the BS isolate of C. psittaci (ovis). Naive animals exhibited a marginal rise in temperature and seroconversion was detected by enzyme-linked immunosorbent assay (ELISA) as early as 9 days after inoculation. Chlamydaemia was detected using culture and an antigen detection ELISA. No faecal shedding of chlamydiae was detected in ewes kept up to 3 weeks after infection. Although chlamydial antigen was demonstrated in epithelial cells or lymphocytes in lungs, liver, spleen, kidney, abomasum, jejunum, tonsils and suprapharyngeal, mandibular, parotid and mesenteric lymph nodes of some of the na?ve sheep using an ELISA and streptavidin-biotin and immunofluorescent staining techniques, the organism could not be cultured from these tissues. No chlamydial antigen was demonstrated in any of the tissues of the previously exposed sheep nor in uninoculated controls. It is concluded that previously infected sheep are capable of completely eliminating subsequent infection and that chlamydiae localize in a variety of tissues of infected na?ve sheep, especially within epithelial cells and lymphocytes.     

Effects of growth factors on wound healing in serum-deprived kitten corneal endothelial cell cultures

The influence of epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and insulin-like growth factor (IGF) I and II on wound healing was investigated in a corneal endothelial system with minimal mitotic activity, using serum-deprived kitten corneal endothelial-cell cultures. After wounding, growth factors were added and wound diameter was evaluated. The DNA synthesis was determined by 3H-thymidine labeling. Wounds did not close in the control cultures grown in serum-free medium without growth factors. The IGF I or II, alone (10 and 100 ng/ml) or added (10 ng/ml) to EGF or bFGF, had no significant effect on wound closure or thymidine uptake. With EGF or bFGF (10 ng/ml), wounds closed after 15 days. Wounds closed after 10 days with EGF or bFGF (100 ng/ml) alone or with the combination of EGF and bFGF (each at 10 ng/ml). Combined EGF and bFGF (each at 100 ng/ml) did not enhance wound closure further. Thymidine uptake was significantly higher in cultures treated with EGF or bFGF (10 ng/ml) than in controls. The uptake could be increased, if both growth factors were combined, but only to the same level achieved with a single factor at 100 ng/ml. This study showed that EGF and bFGF, but not IGF I or II, enhanced wound closure and DNA synthesis in a corneal endothelial cell system that had minimal mitotic activity.     

Experience with the McCoy laryngoscope in difficult laryngoscopy

We report our experience with the McCoy levering laryngoscope in 48 patients who were a Cormack and Lehane grade 3 or grade 4 view at direct laryngoscopy. The view with the blade in neutral position was grade 3 in 39 patients and grade 4 in nine patients. Elevation of the levered tip of the blade in the grade 3 group improved the view to grade 2 in 17 patients (44%), in 17 patients (44%) the view remained unchanged and in five patients (12%) the view deteriorated to grade 4. In the patients initially grade 4, the view improved to grade 3 in one patient and remained unchanged in eight patients. The McCoy laryngoscope is a useful tool to aid intubation in about half of patients who are a grade 3 view at laryngoscopy. Our experience indicates it is unlikely to improve a grade 4 view.     

The role of the gas phase in the greening and growth of illuminated cell suspension cultures of spinach (Spinacia oleracea, L.)

The gas phase developed above spinach suspension cultures critically affected their growth and greening. Ethylene accumulation inhibited greening; this effect of ethylene was antagonised when the culture gas phase was enriched with carbon dioxide. Greening was enhanced by reducing the partial pressure of oxygen below the air level; this effect was observed when oxygen supply did not restrict growth.     

Pregnancy-specific alterations in the expression of the insulin-like growth factor system during early placental development in the ewe

The placenta is recognized as an important determinant of fetal growth rate, yet the factors regulating its proliferation remain poorly understood. Components of the insulin-like growth factor (IGF) system were localized in the ovine uterus using in situ hybridization between days 13-55 of gestation, the period of implantation and placentome formation. IGF-II messenger RNA (mRNA) expression was intense in the fetal mesoderm, particularly at the tips of the invading placentome villi. Moderate levels of IGF-II mRNA were also observed in the maternal caruncular stroma. In contrast, expression of IGF-1 mRNA was low (compared to estrous levels) and ubiquitous decreasing as gestation advanced. IGF-binding protein-2 (IGFBP-2 mRNA was not detected until day 29 of gestation, when it appeared restricted to the dense caruncular-like stroma lining the luminal epithelium, colocalized with IGFBP-4. High concentrations of IGFBP-4 mRNA expression were also found in the placentome capsule. IGFBP-3 mRNA expression was intense in the luminal epithelium between days 13-15 of gestation. Subsequently, levels in this region dropped significantly (P < 0.001). IGFBP-3 mRNA expression was also high in the maternal placentome villi, where photographic emulsions localized expression to blood vessel walls. Peak expression of IGF type 1 receptor (IGF-1R) mRNA was found in the deep uterine glands, with intermediate expression in the superficial uterine glands. Moderate expression of IGF-1R mRNA was initially recorded in caruncular stroma, but levels in this region decreased significantly (P < 0.001) to below the detection limit of the technique after interdigitation by the fetal allantochorion. Furthermore, IGF-1R mRNA could not be detected in any fetal placentome tissue. This study, therefore, has established the pattern of expression of the IGFs, IGF-1R, and three of the IGFBPs during establishment of the ovine placenta. It will form the basis for future work to investigate how this system is regulated and to determine the role of the IGFs in placental development.     

The evaluation of the cytotoxicity of preparations with anticarcinogenic action in human cell cultures

An approach for determination of cytotoxicity of chemical and biological drugs which combine the estimation of quality and quantity of cells after their treatment are proposed by the authors. The visual control of cellular minicultures permits one: 1) to register specific and pathologic alterations of cell morphology, 2) to trace their development in dynamics under the action of individual or complex drugs and 3) to choose the optimum time for the experiment fixation. The special staining of the treated cells and measurement of the optical density of absorbed histological dye permits one to make a conclusion about the changing of the cells quantity. A combined method of double estimation gives the opportunity to detect the artefacts taking place after staining the cells treated by some drugs and extracts of natural origin in high concentrations.     

Reproductive wastage in the androstenedione-immune ewe

An experiment was carried out using 320 adult Merino ewes to examine the effects of immunization against an androstenedione human serum albumin conjugate (Fecundin) on ovulation rate, fertilization rate and embryo viability at days 2, 9 and 13-14 after fertilization. The ovulation rate of immunized ewes (2.19 +/- 0.06) was greater (P < 0.001) than that of control ewes (1.43 +/- 0.04). The recovery rate of embryos or of unfertilized oocytes on day 2 was reduced in immunized ewes, but fertilization rate of recovered oocytes was unaffected by immunization. The mean number of normal embryos per ewe pregnant (prolificacy) was higher and the proportion of ewes pregnant (fertility) was lower in immunized than in the control ewes. The distribution of the number of cells per embryo showed no differences in developmental age over the period of sampling, the majority of embryos at this time being at the two- to four-cell stage of development. At day 9 of pregnancy, blastocyst recovery rates were lower in immunized than in control ewes. About 90% of blastocysts recovered were developing normally in control ewes compared with 64% in immunized ewes. The majority of blastocysts recovered on day 9 had hatched from the zona pellucida prior to recovery (mean values were 94.2% and 87.8% for control and immunized groups, respectively). In control ewes single blastocysts were larger than twin blastocysts, but for the immunized ewes this difference was not significant. Both single blastocysts (P < 0.01) and twin blastocysts (P < 0.05) from immunized ewes were smaller than those from control ewes.(ABSTRACT TRUNCATED AT 250 WORDS)     

The mechanism of beta-glycerophosphate action in mineralizing chick limb-bud mesenchymal cell cultures

Differentiating chick limb-bud mesenchymal cells plated in micromass culture form a cartilage matrix that can be mineralized in the presence of 4 mM inorganic phosphate (Pi), and 1 mM calcium. Previous studies showed that when beta-glycerophosphate (beta GP) is used in place of Pi, the mineral crystals formed are larger and differ in distribution. The present study shows that the difference in distribution is not associated with alterations in cell proliferation, protein synthesis, or with collagen, proteoglycan core protein, or alkaline phosphatase gene expression. Cultures with 2.5, 5, and 10 mM beta GP did show different levels of alkaline phosphatase activity, and in the presence of low (0.3 mM) Ca had different Pi contents (4, 6 and 9 mM, respectively), indicating that the increase in CaxP product may in part be responsible for the altered pattern of mineralization. However, cultures with beta GP in which alkaline phosphatase activity was inhibited with levamisole still had an altered mineral distribution as revealed by Fourier transform-infrared (FT-IR) microspectroscopy. The presence of a casein kinase II-like activity in the mineralizing cultures, the ability of specific inhibitors of this enzyme to block mineralization, and the known ability of beta GP to block phosphoprotein phosphatase activity suggests that altered patterns of matrix protein phosphorylation may influence mineral deposition in these cultures.     

A new antitumor agent amrubicin induces cell growth inhibition by stabilizing topoisomerase II-DNA complex

Amrubicin is a novel, completely synthetic 9-aminoanthracycline derivative. Amrubicin and its C-13 alcohol metabolite, amrubicinol, inhibited purified human DNA topoisomerase II (topo II). Compared with doxorubicin (DXR), amrubicin and amrubicinol induced extensive DNA-protein complex formation and double-strand DNA breaks in CCRF-CEM cells and KU-2 cells. In this study, we found that ICRF-193, a topo II catalytic inhibitor, antagonized both DNA-protein complex formation and double-strand DNA breaks induced by amrubicin and amrubicinol. Coordinately, cell growth inhibition induced by amrubicin and amrubicinol, but not that induced by DXR, was antagonized by ICRF-193. Taken together, these findings indicate that the cell growth-inhibitory effects of amrubicin and amrubicinol are due to DNA-protein complex formation followed by double-strand DNA breaks, which are mediated by topo II.     

Inhibition of growth of primary human tumour cell cultures by a 4-anilinoquinazoline inhibitor of the epidermal growth factor receptor family of tyrosine kinases

The epidermal growth factor receptor (EGFR) is thought to mediate the action of the mitogens EGF and tumour growth factor-alpha (TGF-alpha) in a variety of cancers, including those of the lung, breast and ovary. A number of new selective inhibitors of EGFR tyrosine kinase have now been developed as potential new antitumour agents. We used a potent inhibitor of this tyrosine kinase, 6-amino-4-[(3-bromophenyl)amino]-7-(methylamino)quinazoline (SN 25531; PD 156273), to determine the responses of primary cultures derived from patients with cancer of the lung, ovary, breast, cervix and endometrium. Cells were cultured in 96-well plates and proliferation assessed by incorporation of 3H-thymidine. Measured growth inhibitory concentrations IC50 values) varied from 1 nM to 14 microM with a 1000-fold differential between sensitive and resistant cultures. Results were compared with rates of proliferation, estimated using a paclitaxel-based method. We also measured the IC50 values for the tyrosine kinase inhibitor using a number of established human cell lines, and compared them with EGFR content using fluorescent antibody staining and flow cytometry. The presence of EGFR was found to be necessary, but not sufficient, for in vitro response. Only a small number of cell lines (3 of 7 for lung, 1 of 7 for ovarian, 2 of 3 squamous cell and 0 of 12 for melanoma) were sensitive to the tyrosine kinase inhibitor. In contrast, 40 of the 50 primary cultures (including 14 of 15 lung cancer samples and 14 of 19 ovarian cancer samples) were sensitive.     

The production of cricket paralysis virus in suspension cultures of insect cell lines

We have observed seven initially obese individuals who, during the course of a strenuous weight-reduction program, developed diabetes mellitus: non-insulin-dependent diabetes mellitus in five cases and insulin-dependent diabetes mellitus in two cases. None had any sign of prior diabetic symptoms. Although weight reduction is encouraged in obesity, crash diets without proper medical surveillance may have deleterious effects. This sequence of induction of diabetes has not previously been reported in the medical literature. The metabolic situation in extremely low-calorie diets may be comparable to that in starvation. An attempt is made to explain our observation concerning the induction of a diabetic state during such diets, on the basis of increased insulin resistance in states of starvation and anorexia nervosa, with a concomitant role in stress hormones.     

The importance of the density of spleen cell suspensions in cultures for development of immune reactions

A relationship was revealed between the number of cells in the suspension of the splenic tissue taken for induction of primary immunization of the reaction outside the organism and the number of the plaque-forming cells (PFC) formed by the end of incubation. It was shown that with increase of the cell density in the culture in the bottom of the incubation flask there was a 10-100 fold decrease of the PFC response; as to the cell viability--it was not affected or decreased but slightly. This effect was observed in using sheep red blood cells and water-soluble antigen extracted from them as an antigen. The effect was independent of shortage of the antigen or of the nutrient substances; it was accompanied by a general reduction of the 3H-thymidine incorporation into cultured cells.     

Cross contamination of continuous cell cultures

The possibility of air-droplet cell transmission during cell transfer was experimentally confirmed, this appearing to be the most probable route of cross contamination of cell cultures. The consequences of such contamination are assessed and measures reducing the risk of intercellular contamination proposed.