Lessons from KEEPS: the Kronos Early Estrogen Prevention Study.

The Kronos Early Estrogen Prevention Study (KEEPS) was a randomized, double-blind, placebo-controlled trial designed to determine the effects of hormone treatments (menopausal hormone treatments [MHTs]) on the progression of carotid intima-medial thickness (CIMT) in recently menopausal women. Participants less than 3 years from menopause and without a history of overt cardiovascular disease (CVD), defined as no clinical CVD events and coronary artery calcium < 50 Agatston units, received either oral conjugated equine estrogens (0.45 mg/day) or transdermal 17ß-estradiol (50 µg/day), both with progesterone (200 mg/day for 12 days/month), or placebo pills and patches for 4 years. Although MHT did not decrease the age-related increase in CIMT, KEEPS provided other important insights about MHT effects. Both MHTs versus placebo reduced the severity of menopausal symptoms and maintained bone density, but differed in efficacy regarding mood/anxiety, sleep, sexual function, and deposition of ß-amyloid in the brain. Additionally, genetic variants in enzymes for metabolism and uptake of estrogen affected the efficacy of MHT for some aspects of symptom relief. KEEPS provides important information for use of MHT in clinical practice, including type, dose, and mode of delivery of MHT recently after menopause, and how genetic variants in hormone metabolism may affect MHT efficacy on specific outcomes.

Endothelial function in women of the Kronos Early Estrogen Prevention Study.

OBJECTIVE: Endothelial dysfunction occurs early in the atherosclerotic disease process, often preceding clinical symptoms. Use of menopausal hormone treatment (MHT) to reduce cardiovascular risk is controversial. This study evaluated effects of 4 years of MHT on endothelial function in healthy, recently menopausal women. METHODS: Endothelial function was determined by pulse volume digital tonometry providing a reactive hyperemia index (RHI) in a subset of women enrolled in the Kronos Early Estrogen Prevention Study. RHI was measured before and annually after randomization to daily oral conjugated equine estrogen (oCEE, 0.45 mg), weekly transdermal 17ß-estradiol (tE2, 50 µg) each with intermittent progesterone (200 mg daily 12 days of the month) or placebo pills and patch. RESULTS: At baseline, RHI averaged 2.39 ± 0.69 (mean ± standard deviation; n = 83), and over follow-up did not differ significantly among groups: oCEE, 2.26 ± 0.48 (n = 26); tE2, 2.26 ± 0.45 (n = 24); and placebo, 2.37 ± 0.37 (n = 33). Changes in RHI did not correlate with changes in traditional cardiovascular risk factors, but may inversely correlate with carotid intima medial thickness (Spearman correlation coefficient ρ = -0.268, p = 0.012). CONCLUSION: In this 4-year prospective assessment of recently menopausal women, MHT did not significantly alter RHI when compared to placebo.

Passive protection of rabbits infected with toxic shock syndrome-associated strains of Staphylococcus aureus by monoclonal antibody to toxic shock syndrome toxin 1.

The effectiveness of passive immunization was assessed in an infection model of toxic shock syndrome (TSS) in which monoclonal antibody to TSS toxin 1 (TSST-1) was administered intravenously to rabbits. Previously implanted infection chambers were inoculated with Staphylococcus aureus strains RN4710 and D4508. The former strain carries the TSST-1 gene on plasmid pRN6201; the latter is a TSST-1-negative clinical isolate obtained from a patient with nonmenstrual TSS. Purified monoclonal antibody, MAb 8-5-7 (IgG), was administered in two doses of approximately 1.25 mg each 24 hours before and 24 hours after infection. MAb 8-5-7 provided complete protection against both the TSS-like syndrome and the mortality that occurred in unprotected rabbits infected with strain RN4710 but did not provide complete protection in rabbits infected with strain D4508; three of the five rabbits either displayed signs of illness or died despite treatment. Western-blot analyses of the extracellular proteins produced by strains RN4710 and D4508 that used MAb 8-5-7 as a probe revealed that only TSST-1 produced by RN4710 reacted with the antibody. Thus, if MAb 8-5-7 partially protected animals against infections with strain D4508, the protection appears to have been nonspecific.

Protection of rabbits in an infection model of toxic shock syndrome (TSS) by a TSS toxin-1-specific monoclonal antibody.

An anti-TSST-1-specific monoclonal antibody (MAb 8-5-7) was tested for its protective capacity in a rabbit infection model to toxic shock syndrome (TSS). The challenge strain of Staphylococcus aureus (RN4710), which contained a plasmid encoding TSS toxin-1, was introduced into previously implanted chambers. Purified monoclonal antibody (1.25 mg of immunoglobulin G) administered parenterally 1 day before and 1 day after initiation of infection provided complete protection against the TSS-like syndrome and the mortality which occurred in unprotected rabbits.

Immunological protection of rabbits infected with Staphylococcus aureus isolates from patients with toxic shock syndrome.

Toxic shock syndrome toxin-1 (TSST-1) isolated from the growth medium of Staphylococcus aureus 1169 and 555 was used to immunize male rabbits before infection with either a TSST-1+ or a TSST-1- strain of S. aureus isolated from cases of TSS. None of the immunized rabbits died as a result of the infections, whereas 50% of the nonimmunized rabbits infected with the TSST-1- strain, D4508, and 75% of those infected with the TSST-1+ strain, 555, died. Western blots of crude extracellular protein preparations probed with sera from immunized rabbits indicated that the TSST-1- strain produces a 30,000-molecular-weight protein that cross-reacts with antiserum to TSST-1. Because both organisms caused similar diseases in rabbits, we propose to designate the cross-reacting protein as TSST-2.

Hormonal influence on experimental infections by a toxic shock strain of Staphylococcus aureus.

Subcutaneous infection chambers in rabbits were infected with a strain of Staphylococcus aureus isolated from a patient with toxic shock syndrome. Estrogens (mestranol and 17-beta-estradiol) protected male rabbits and prolonged survival. Neither androgens (testosterone and dihydrotestosterone) nor progesterone affected the susceptibility of intact or ovarihysterectomized female rabbits.

Enhanced susceptibility of male rabbits to infection with a toxic shock strain of Staphylococcus aureus.

Artificial infection chambers in rabbits were infected with a toxic shock strain of Staphylococcus aureus in an attempt to determine the nature of the enhanced virulence of toxic shock strains relative to non-toxic shock strains of staphylococci. The results showed that rabbits immunized with either neutral or acidic proteins were protected from the lethal effects of these infections. Male rabbits were found to be significantly more susceptible to these infections than female rabbits. Castration rendered both sexes equally susceptible to lethal infections. Numerous tissues from all infected rabbits were examined histologically, and most of the pathological findings involved lymphoid tissue. Of special interest was the observation that unprotected male rabbits which died had evidence of lymphoid depletion and that surviving rabbits, both male and female, usually manifested lymphoid hyperplasia. No other pathological response was noted which would characterize these infections, but immunized rabbits had a diminished level of thymic cortex involution that was not different between the sexes.

Characterization of Staphylococcus aureus isolates from patients with toxic shock syndrome, using polyethylene infection chambers in rabbits.

Isolates of Staphylococcus aureus from patients with toxic shock syndrome (TSS) were compared with non-TSS strains of S. aureus with respect to their virulence in rabbits. When the organisms were injected into subcutaneous chambers (perforated polyethylene golf balls) to assess virulence, a rapid mortality was observed with TSS but not with non-TSS strains. Of 16 TSS strains, 11 caused lethal infections in 33 rabbits tested, and none of the 5 control strains caused mortality in 10 rabbits. This evidence of enhanced virulence associated with TSS strains did not appear to be associated with the size of the inoculum. In addition, strains which produced lethal infections appeared to do so despite a reduction in the size of the original inoculum during the first 24 h. All of the TSS strains and none of the non-TSS strains elaborated extracellular protein(s) with a neutral pI when grown in a dialyzed beef heart medium. No other physiological difference was noted between the TSS and non-TSS strains.

X-ray diagnosis of healing fractures in rabbits.

Do X-rays adequately reflect the strength of callus post fracture? Eleven rabbit tibiae were manually fractured and allowed to heal in plaster. At varying times post fracture, the rabbits were sacrificed and the tibiae were removed. AP and lateral X-rays of each tibia were obtained and shown to 27 orthopedists and radiologists who were asked to rank the bones in order of strength. The valid ranking was done using data obtained by loading the bones axially to failure in an Instron machine. Each physician's ranking was compared with that obtained from the Instron with a median correlation level p=0.0668. This implies that a physician, whether an orthopedist or radiologist, is not very reliable at determining early osseous union.

The mechanism of mitochondrial extrusion from phenylhydrazine-induced reticulocytes in the circulating blood.

The mechanism of mitochondrial extrusion from reticulocytes was studied in whole blood from dogs made anemic by treatment with phenylhydrazine hydrochloride. The initial stage of preparation for mitochondrial extrusion was attraction of vesicles to mitochondria. There was subsequent encirclement of the organelle and other bodies, such as ferritin, by coalesced vesicles forming double membrane-limited vacuoles. Large vacuoles were formed from the union of single vacuoles, and they were usually situated near the periphery of the cell. Fusion of the outer membrane of vacuoles with the plasmalemma of the reticulocyte provided a route for exposure and release of mitochondria and other material to an extracellular location. An extracellular mitochondrion, therefore, was confined by its original double membrane, and a third membrane was derived from the internal boundary of vacuoles.

The mechanism of denucleation in circulating erythroblasts.

Erythroblast denucleation in the peripheral blood was studied by electron microscopy. Blood was used from dogs anemic either by infection with Babesia canis or from injections of phenylhydrazine hydrochloride. One of the earliest stages of denucleation was the migration of nuclei to the plasmalemma. Mitochondria and coalesced vesicles, derived from the cell membrane of the erythroblast, congregated at the underside of the nuclear envelope unapposed by erythroblastic cell membrane. The coalesced vesicles apparently provided the limiting membrane which surrounded the deep circumference of the extruded nucleus and its associated hemoglobin rim, and also furnished a new plasma membrane for the cell in the area where the nucleus, in denucleation, had utilized the original erythroblastic plasmalemma.