Effects of tampons and menses on the composition and diversity of vaginal microbial communities over time.

OBJECTIVE: To investigate the influence of menses on the vaginal microbiota and determine whether tampons that differ in material composition influence these bacterial communities in different ways. DESIGN: A single-centre trial with randomised, complete block design. SETTING: Procter & Gamble facility. SAMPLE: Seven self-declared healthy, female volunteers of reproductive age. METHODS: Volunteers used a pad and two types of tampons during the study, one product exclusively each month for three sequential menstrual cycles. During menses and once each mid-cycle, vaginal bacterial community composition was characterised by cultivation-independent methods based on pyrosequencing of V1-V2 variable regions of 16S ribosomal RNA genes. MAIN OUTCOME MEASURES: Changes in the species composition, abundance and diversity in vaginal bacterial communities over time and between treatments. RESULTS: The vaginal microbiotas of all seven women were dominated by Lactobacillus spp. at mid-cycle, and the compositions of those communities were largely consistent between cycles. Community dynamic patterns during menses varied considerably and were more or less individualised. In three of the seven women the community diversity during pad use was significantly different from at least one tampon cycle. CONCLUSIONS: Changes in the composition of the vaginal microbiota during menses were common, but the magnitude of change varied between women. Despite these changes, most communities were capable of resuming a composition similar to previous mid-cycle sampling times following menstruation. Overall we conclude that the two tampons tested do not significantly impact the vaginal microbiota in different ways; however, larger studies should be performed to confirm these findings.

Use of gene probes based on the insertion sequence IS986 to differentiate between BCG vaccine strains.

Gene probes derived from the insertion sequence IS986, which have previously been shown to differentiate isolates of Mycobacterium tuberculosis for epidemiological analysis, are also capable of distinguishing two groups of BCG vaccine strains. Most BCG strains have a single copy of IS986, at the same chromosomal site, while the Brazilian, Japanese and USSR strains have an additional copy at a different, common location. These results correlate with the results of previous antigenic analysis and may reflect a different clonal origin of the two groups of BCG strains.

Effect of the method of preparation of bacille Calmette-Guérin (BCG) vaccine on the properties of four daughter strains.

Samples of Bacille Calmette-Guérin (BCG) vaccines from four collaborating production laboratories, each of which had prepared vaccine from four different daughter strains of BCG, had previously been monitored for changes in colony morphology and the present study was undertaken to determine whether the changes observed were reflected in the patterns of protein secretion and lipid content. In the samples examined there was evidence for a correlation between colony morphology and the presence or absence of mycoside B. As the components of BCG that determine virulence and protective immunity are unknown, care must be taken to ensure constancy of the strains during the manufacture of vaccines.

Some effects of nutritional components on the morphology of BCG colonies.

Under appropriate conditions of growth colonies showing fine wrinkling (rugosity) of their surface and characteristic of certain BCG strains can be distinguished from colonies with a smoother non-rugose morphology that are characteristic of some other BCG strains. This study has been concerned with the influence of nutritional and other media constituents on the evolution of these colonies. In a preliminary investigation nutritional components such as enzymic digest of casein, asparagine and salts were systematically eliminated from an agar medium, separately and together, and in the presence or absence of glycerol. From this and supplementary experiments, it appeared that the nutritional element with most effect on the size and morphology of colonies is the carbohydrate. Small inocula of BCG will normally grow on simple agar media only in the presence of enrichments such as blood or albumin, or if charcoal has been added. However, in a second investigation it was found that individual colonies would develop from such inocula placed 2 cm or more away from a concentrated inoculum that had been seeded onto the medium approximately 7-14 days earlier. It appears that a diffusible agent is produced from multiplying bacilli within a colony which counteracts toxic factors in the medium and may also assist nutrition within the colony. To examine the effects of carbohydrate on the evolution of colonies, in a third investigation glycerol was added in increasing quantities to an agar medium enriched with increasing quantities of bovine albumin and/or 5% of blood, and suspensions of French (Pasteur) and British (Glaxo) BCG vaccines were seeded onto it. Individual colonies cultured from these two strains have a rugose and non-rugose morphology respectively, and to highlight the effects in question, the evolution over a prolonged period of concentrated 'drop-colonies' seeded onto the medium from each strain was studied. There was a very marked difference between the two strains in the evolution of such 'drop-colonies', and it appeared that the lateral spread of fine rugosity from those of the Pasteur strain represented an enhanced ability of small numbers of bacilli to take up the nutrient.

Changes in BCG strains.

BCG originated from a virulent bovine strain of the tubercle bacillus after prolonged serial subculture on a potato medium. Since attenuation was achieved, the BCG strain has been distributed to a large number of centres where BCG vaccine is produced. Many of these production laboratories have maintained their BCG lines by continuing serial transfers, but have employed a variety of media for this purpose, and have produced BCG vaccine by a variety of techniques. Distinct differences have developed between some of the daughter strains of BCG, but the mechanism through which these changes have occurred has not been clear. In recent years methods have been developed which have enabled changes taking place within some BCG strains during experimental serial subculture to be monitored. In this survey the relationship of the changes observed to the different techniques employed for the maintenance of BCG lines and for the preparation of vaccine is considered. It is suggested that selection of minority populations within BCG strains noted during experimental studies may provide an analogy with the mechanism through which the original attenuation of the virulent bovine strain was brought about. The relevance of small-scale laboratory investigations to full-scale production procedures is also discussed, and finally some additional measures that might be taken to minimise changes in BCG strains are proposed.

BCG vaccine: an investigation of colony morphology from four different strains after their introduction as seed for vaccine preparation in four production laboratories.

Four collaborating BCG production laboratories had each prepared vaccine from four different BCG strains. In this laboratory the morphology of colonies cultured from samples of the 16 vaccine lots thus available was examined, and it was found that changes monitored in earlier small-scale experimental reconstructions had also occurred during some of the full-scale production procedures, in accordance with prediction. In particular, a minority population carried by the Danish BCG strain had replaced the original majority when this strain had been employed as seed for the production of vaccine by the British procedure. Similarly, a minority carried by the Japanese strain had replaced the original majority when this strain was introduced as seed into the production procedures in the United Kingdom and France. Serial subcultures made in this laboratory showed, in accordance with previous experience, that the changes that had occurred in the Japanese strain could be completely reversed by serial subculture as a surface pellicle on Sauton medium.

Some factors influencing the tuberculin tine test.

Some physical factors influencing the performance of the tuberculin Tine Test have been investigated. Conditions under which tuberculin is removed from the tines were examined, by direct observation of elution with water, and by insertion into a number of materials, including human and animal skin. Uptake of tuberculin on to tines was also studied and quantitative estimates were obtained by dipping the tines in tuberculin mixed with 32P; it was found that the amount of tuberculin adhering to the tines was approximately doubled by applying a second coat after the first had dried. Tines coated with this mixture were applied to human skin from a cadaver, and after removal it was noted that approximately half the tuberculin orginally coating the tines had been deposited in the skin; the quantity inserted by doubly-coated tines was approximately double that deposited by singly-coated ones.

High-performance liquid chromatographic analysis of egg yolk phospholipids.

A rapid and efficient method for the separation of egg yolk phospholipids by normal-phase high-performance liquid chromatography has been developed. The separation is accomplished on an Ultrasil-NH2 column using hexane, isopropanol, methanol and water mixtures with direct ultraviolet detection at 206 nm. Phospholipids from a methanol extract of fresh lyophilized egg yolks were analyzed providing complete separation of (in order of elution) neutral lipids, phosphatidylcholine, sphingomyelin, lysophosphatidylcholine and phosphatidylethanolamine. Identification of eluting species was accomplished by comparative retention times of standard samples by thin-layer chromatographic analyses of collected fractions. Furthermore, a mixture of naturally occurring phospholipid standards from bovine and egg sources has been separated by this method. In addition to separating the individual classes of phospholipids, in some instances, separation of molecular species within a class was achieved as in the case of cerebrosides, sphingomyelin and partially with phosphatidylethanolamine.

Serial subcultivation of Czechoslovakian and Japanese BCG strains.

Changes in the Danish BCG strain under certain regimens of subculture have been shown in preceding studies to be associated with selection of a minority population. Three Czechoslovakian BCG strains, all originally derived from the Danish strain but thereafter and in distinction from it maintained on potato media, have now been investigated. Changes in the immunizing potency of two of these strains have been attributed by other workers to employment of the richer potato media in place of Sauton medium as used for maintenance of the parent Danish strain. However, results from the present study suggest rather that selection of a pre-existing minority genotype or of a new mutant occurred. This proposal is supported by the finding that the third strain has maintained characteristics similar to those of the Danish parent despite many previous transfers on potato media.Another BCG strain investigated was the Japanese which, like the three Czechoslovakian strains, had been previously maintained on potato media. This strain has been shown in the present study to resemble the Danish strain in supporting a minority population yielding non-spreading colonies. Czechoslovakian vaccine prepared with seed culture supplied from Tokyo has retained characteristics similar to those of the Japanese parent. Although a majority population yielding spreading colonies appears so far to have been retained in both centres, it is considered that selection of the minority could still occur in the course of routine transfer.

Serial subculture of BCG on solid and liquid media.

To follow an earlier study of the effects on BCG of deep subculture in liquid media, transfers on the surface of Löwenstein-Jensen and Sauton's media have been made. When a vaccine preparation that yields on culture 99+% of spreading-type colonies, with a small minority of non-spreading forms, is transferred on Löwenstein-Jensen medium, the minority population can be readily selected out. Growth of BCG on this medium is not homogeneous, and the selection of either the minority or majority populations appears fortuitous, whereas it was previously demonstrated that deep subculture in Dubos medium tends to favour selection of a minority yielding non-spreading colonies. When such a minority population has been favoured during transfer on Löwenstein-Jensen medium, subsequent subculture in Dubos medium usually accentuates the effect. Subculture as a surface pellicle on Sauton's medium has a powerful effect in reducing the relative size of a minority population yielding non-spreading colonies, and thereafter maintaining 99+% of spreading forms. The relationship between transfers on Löwenstein-Jensen and Sauton's media and those on potato-Sauton medium is briefly discussed.

Effect of 25-hydroxy-vitamin D3 on intestinal absorption of calcium in normal man and patients with renal failure.

The effects of short-term treatment with 25-hydroxy-vitamin D3 (25(OH)D3) on intestinal absorption of 47Ca were examined in 18 studies of normal subjects and 16 studies of patients with advanced renal failure. Doses of 25(OH)D3 were 20, 100, 500, or 1000 microgram/day given orally for 7--10 days. There was an increase in 47Ca absorption and urinary calcium in normal subjects receiving 20 microgram/day, while doses of 500 or 1000 microgram/day were required to augment 47Ca absorption in renal failure patients. During treatment, plasma levels of 25(OH)D increased to similar levels in both normal and uremic subjects. A comparison of the dose-response curves found 25(OH)D3 to be 1/125 as potent as 1,25-(OH)2D3 in the normal subjects and 1/400 as potent as 1,25(OH)2D3 in patients with chronic renal failure. Thus, pharmacologic doses of 25(OH)D3 are active in both normal and uremic patients, although relatively greater quantities are necessary in uremia. This difference in relative potency of 1,25(OH)2D3 and 25(OH)D3 may be explained by some conversion of 25(OH)D3 to 1,25(OH)2D3 in normal compared to uremic subjects, while 25(OH)D3 may act in large part via mass action in uremic patients.

Effects of BCG and C. parvum treatment on human melanoma xenografts.

CBA mice rendered immunodeficient by thymectomy, potentially lethal gamma-irradiation and reconstitution with bone marrow cells were used to grow a wide variety of human tumours as subcutaneous implants. Samples of human melanoma obtained at surgery were successfully passaged by transplantation and produced rapidly growing tumours, some of which metastasized to lung, lymph nodes and the para-aortic node; this system was used as a model for the study of immunotherapy of melanoma. Preliminary results show that intratumour injections of C. parvum retard or inhibit the growth of melanoma transplants and, therefore, do not require the involvement of T lymphocytes, whilst BCG has no effect on growth rate.

A study of some effects of subculture on two BCG strains.

A marked improvement in the ability to consistently distinguish between colonies cultured from BCG strain 1331 (Copenhagen), employed for the routine production of Danish freeze-dried BCG vaccine, and those cultured from BCG strain 1077 (Glaxo), employed for the routine production of British freeze-dried BCG vaccine, was obtained in our laboratory by modifying the constituents of a Dubos-type solid-medium. Several batches of British BCG vaccine had been specially prepared by the manufacturer, using strain 1331 as seed culture, and samples from these batches were cultured on this medium in our laboratory. Two of the batches produced, in place of the spreading colonies normally cultured from strain 1331, a large majority of nonspreading colonies, normally characteristic of strain 1077. To study this very rapid change, modifications of the system of serial transfers employed routinely by the manufacturer were set up in our laboratory, and the relative proportions of the two colony forms cultivated at each stage of transfer were noted. The rapid changes that had occurred during manufacture were reproduced under our experimental conditions, and it was observed that transfer through deep culture in liquid Dubos medium tended to shift the balance between the numbers of spreading and non-spreading colonies in favour of the latter, whilst the reverse was true of the transfers through deep culture in production medium. The ability to monitor changes occurring in BCG strains during serial transfer should facilitate the provision of measures to prevent such changes taking place.

The chemistry and conformational and biological analysis of vitamin D3, its metabolites and analogues.

The chemical properties, stereochemical relationships and solution conformation, as assessed in part by proton NMR spectroscopy, for vitamin D3, its major metabolites [including 1alpha,25-(OH)2D3, its hormonally active form] and a number of A-ring and side chain analogues are evaluated and discussed in relation to their biological activity. In particular the relative ability of many of these seco-steroids to compete both with 25-OHD3 for its chick serum binding protein and 1alpha,25-(OH)2-D3 for its chick intestinal cytosol-chromatin receptor system was quantitated, in vitro. Further, the relative effectiveness of all these metabolites and analogues to mediate in vivo intestinal calcium absorption and bone calcium mobilization was determined. Collectively these chemical and biological studies constitute a "systems analysis" of the various steroid structural parameters both required and tolerated by the multi-stepped endocrine system associated with the biological actions of vitamin D.