The use of cell cultures from the chorioallantoic membrane of chick embryos for studies of chlamydiae

Cells of the chorioallantoic membrane of chick embryos were processed into monolayer cell cultures by a recently described technique of Cursiefen and Brecht. The cultures provided a sensitive substrate for the quantification of infectivity of C. psittaci strain 6BC and meningo-pneumonitis.     

The use of PIXE in experimental studies of the physiology of human skin epidermis

In order to understand the normal and pathological physiologies of the epidermal cells, the simultaneous determination of several elements in the different cellular strata is of crucial importance. In recent years the electron microprobe (EMP) has become an established technique in this field. Its high spatial resolution, in principle, allows measurements of various cell organelles. However, the limited (intrinsic) sensitivity of the EMP represents a serious drawback to the technique. The introduction of the proton microprobe (PMP) has significantly improved the sensitivity, although the ultimate spatial resolution of the PMP is much less than that of the EMP. When studying the elemental profiles in skin epidermis, it is possible to use skin sections with a thickness of the order of 10 μm, then the spatial resolution of the PMP is equal to or better than that of the EMP since the electrons are scattered to a significant degree in the sample. The characteristics of the two methods have been compared by analysis of parallel duplicate freeze-dried sections of normal human skin. The distributions of the elements P, S, Cl, and K, obtained with the two techniques, were in good agreement. In addition, the PMP provided distributions of the important elements Ca, Fe, and Zn. In a recently started study, the useful features of the PMP will be used for studying how efficient a barrier the skin is to nickel and chromate ions. A preliminary experiment has been performed by exposing cadaverous skin, not older than 24-h postmortem, to solutions of the two ions. After an 18-h exposure, samples were prepared by shock-freezing and sectioning. The first results from PMP analysis of these samples demonstrate the presence of a nickel and chromium gradient in the outer strata in the epidermis (mainly stratum corneum). A third experiment deals with the physiology of psoriatic skin. Calcium is an important element in the differentiation. Hence, the higher sensitivity of the PMP has been used in analysis of sections from psoriatic skin epidermis. Preliminary results are presented.     

The epidemiologic significance of tuberculosis mycobacteria of the avian type]

Reaction to intradermal injection of tuberculin from Mycobacteria tuberculosis of the avian type proved to be negative in all the 505 students examined who never came in contact with fowl farms. Of 738 local residents it was found to be positive in 23.8% and of 320 workers of fowl farms--in 54.5% of cases. In response to the injection of Koch tuberculin and PPD-L the number of positive reactions in all the mentioned groups constituted 69-80%. A conclusion was drawn on the epidemiological danger of Mycobacteria of the avian type for the workers of fowl farms and, to a certain degree, for the surrounding population.     

The deoxyglucose method adapted for studies of glucose metabolism in the early chick embryo

14C-2-deoxyglucose (DG), currently employed in in vivo studies of brain glucose metabolism, has been used for determination of glucose consumption in the in vitro developing chick embryo. DG, presented in traces, accumulates in the embryo in proportion with incubation time. Analysis of tissue homogenates shows that the accumulated radioactivity is due to both phosphorylated (DGP) and nonphosphorylated DG. As it is only the radioactivity originating from the DGP that is proportional to glucose utilization, the nonphosphorylated DG must be washed out. The washout shows two distinct kinetics: a fast one corresponding to DG that has entered the cells but has not yet been phosphorylated and a slow one that is probably due to a dephosphorylated DGP coming from a different cellular compartment. On the basis of these results the optimal experimental conditions have been defined, allowing quantitative studies of glucose metabolism during the first day of development of the chicken embryo. From 18 to 24 hr of incubation (end of gastrulation), total glucose consumption increases from 50 nmol X h-1 at stage 3-4 to 90 nmol X h-1 at stage 6-7. This increase mainly reflects the growth of the blastodisc. Comparison with the values of O2 uptake measured at the same period of development suggests that only a fraction of the glucose consumed is oxidized, the major part being converted aerobically to lactate.     

The methylation state of the proviruses in avian sarcoma virus transformed chick and rat cells

The endogenous viruses in the avian cells are not completely methylated, nor are the Schmidt-Ruppin RSV-D (SRD) proviruses in the infected cells completely unmethylated. Avian sarcoma proviruses integrated in rat transformed cloned cells are heavily methylated. In these cells, a region in the 3' end of the env gene is unmethylated in all the src-containing proviruses but not in the transformed defective (td) proviruses. A possible role for the hypomethylation of the 3' end of the env region is proposed.     

Immunohistochemical studies on the development of avian embryo pituitary corticotrophs under normal and experimental conditions

Summary Immunofluorescent ACTH cells are present in the developing chick pituitary gland from the 9th day of incubation.Rathke pouch grafts from 4–5 day or 5.5 and 6.5 day-old chicks, grafted into chick chorioallantoic membrane and grown for 12 days, gave rise to tinctorially normal pituitary glands in both cases.The early grafts were of pouch epithelium alone, separated from mesenchyme by trypsinization. The later grafts were surrounded by their attached mesenchyme, from which they are virtually inseparable.In 17 out of 18 of the 4.5 day grafts no immunofluorescent ACTH cells developed. (In the 18th case a few feebly stained single cells). In 16 out of 30 of the 5.5 and 6.5 day grafts ACTH cells were present in normal numbers.Of the 3 hypotheses put forward to explain these findings only one appears valid. This is that the ACTH cells are contributed directly by the mesectoderm (neural crest) surrounding the 5.5 and 6.5 day pituitary primordia.     

The use of macroarrays for the identification of MDR Mycobacterium tuberculosis

The emergence of Mycobacterium tuberculosis (Mtb), resistant to both isoniazid (INH) and rifampicin (RIF) (MDR-TB), is an increasing threat to tuberculosis control programs. Susceptibility testing of Mtb complex isolates by phenotypic methods requires a minimum of 14 days from a primary specimen. This can be reduced significantly if molecular analysis is used. Low density oligonucleotide arrays (macroarrays) have been used successfully for the detection of RIF resistance in Mtb. We describe the use of macroarray technology to identify Mtb complex isolates resistant to INH and/or RIF. The macroarray MDR-Mtb screen has been designed to detect mutations in the RIF resistance determining region (RRDR) of Mtb rpoB and loci in katG and mabA-inhA associated with INH resistance. A panel of Mtb isolates containing 38 different RRDR genotypes, 4 different genotypes within codon 315 of katG and 2 genotypes at mabA-inhA was used to validate the macroarray. The wild type (WT) genotype was correctly identified at all three loci. Of the 37 mutant rpoB genotypes, 36 were correctly detected; the single mutant not detected contained a 9 base insertion. All mutations within katG and mabA-inhA were correctly identified. We conclude that this low cost, rapid system can usefully detect the mutations associated with the vast majority of MDR-Mtb.