Differential responses of estrogen target tissues in rats including bone to clomiphene, enclomiphene, and zuclomiphene

The substituted triphenylethylene antiestrogen clomiphene (CLO) prevents cancellous bone loss in ovariectomized (OVX'd) rats. However, CLO is a mixture of two stereoisomers, enclomiphene (ENC) and zuclomiphene (ZUC), which have distinctly different activities on reproductive tissues and tumor cells. The purpose of the present dose response study was to determine the effects of ENC and ZUC on nonreproductive estrogen target tissues. These studies were performed in 7-month-old female rats with moderate cancellous osteopenia that was established by ovariectomizing rats 1 month before initiating treatment. OVX resulted in increases in body weight, serum cholesterol, endocortical resorption, and indices of cancellous bone turnover, as well as decreases in uterine weight, uterine epithelial cell height, bone mineral density, bone strength, and cancellous bone area. Estrogen treatment for 3 months restored body weight, uterine histology, dynamic bone measurements, and osteoblast and osteoclast surfaces in OVX'd rats to the levels found in the age-matched sham-operated rats. In contrast, estrogen only partially restored cancellous bone volume and uterine weight, and it reduced serum cholesterol to subnormal values. CLO was a weak estrogen agonist on uterine measurements and a much more potent agonist on body weight, serum cholesterol, and dynamic bone measurements. CLO increased trabecular thickness in osteopenic rats and was the most effective treatment in improving cancellous bone volume and architecture. ZUC was a potent estrogen agonist on all tissues investigated and had dose-dependent effects. In contrast, ENC had dose-dependent effects on most measurements similar to CLO and decreased the uterotrophic effects of ZUC. It is concluded that ENC antagonizes the estrogenic effects of ZUC on the uterus but that the beneficial effects of CLO on nonreproductive tissues in OVX'd rats is conferred by both isomers. Furthermore, the combined actions of the two isomers on bone volume and architecture were more beneficial than either isomer given alone.     

Autologous, hapten-modified vaccine as a treatment for human cancers

PURPOSE: To investigate the cellular dynamics of vessel formation during corneal neovascularization in the living eye by confocal microscopy. METHODS: Corneal neovascularization was initiated by placing a 7-0 silk suture through the corneal stroma 3 mm from the limbus at the 12 o'clock position in both eyes of 10 New Zealand white rabbits. The corneas were examined for vessel ingrowth at intervals from 1 to 15 days after suture placement using a tandem scanning confocal microscope with a 20X water immersion objective, as well as a slit-lamp biomicroscope. Changes in the limbal vessels were recorded on videotape for later analysis. As early vessel growth appeared to be associated with corneal nerves, the total number of sprouts and the number of sprouts along nerves were counted in confocal images, and the results analyzed for statistical significance. Vessel growth and the structural relationship between vascular buds and the deep stromal nerves were examined by light and transmission electron microscopy. RESULTS: The early events of cell migration from the limbal microvessels were found to be associated with the deep stromal nerves; although this association was easily visualized by confocal microscopy, it could not be documented by slit-lamp biomicroscopy. By 18 h after suture placement, the limbal vessels were dilated and the first vascular buds appeared as short, pointed, or flat-topped protrusions from the deep limbal capillaries. By 96 h, the capillary buds had increased in density and had begun to form lumens. Movement of red blood cells was established between 72 and 80 h after the first signs of bud formation, at the same time that cells of immune origin were seen. Confocal microscopy revealed and transmission electron microscopy verified that new bud formation began with the formation of vascular tubes by endothelial migration along the deep stromal nerves. The total number of sprouts and the number of sprouts associated with stromal nerves were similar on days 1 and 2 but differed on days 3-7, suggesting an association between sprouts and nerves in the early stages of neovascularization. CONCLUSION: Using real-time white light confocal microscopy, we were able, for the first time, to observe the process of corneal neovascularization in the living eye, from the earliest stages within hours after initiation to 2 weeks. The deep stromal nerves appear to serve as a focus for the growth of new vessels, by attracting and supporting vessel growth and/or by providing a potential space for movement of the endothelial cells. Confocal microscopy may provide a new approach to achieving a better understanding of the mechanisms involved in corneal neovascularization.     

Contribution of dermal macrophage trafficking in the sensitization phase of contact hypersensitivity

We investigated cellular trafficking of dermal macrophages that express a macrophage calcium-type lectin (MMGL) during the sensitization of delayed-type hypersensitivity. In skin, dermal macrophages, but not epidermal Langerhans cells, have been shown to express MMGL. Epicutaneous sensitization by FITC produced a transient increase in MMGL-positive cells in regional lymph nodes. To directly investigate whether the increase was due to cell migration from dermis, MMGL-positive cells purified from skin were intradermally injected into syngeneic mice after labeling with a fluorescent cell tracer, followed by epicutaneous sensitization over the site of injection. MMGL-positive cells containing the tracer were found in the regional lymph nodes after sensitization. The majority of the MMGL-positive cell migrants were negative for FITC fluorescence despite the presence of FITC-labeled cells that included Langerhans cell migrants. Because the extent of MMGL-positive cell migration was greatly influenced by the selection of vehicles to dissolve FITC, the efficiency of sensitization was compared using the ear swelling test. Migration of both Langerhans cells (FITC-labeled cells) and MMGL-positive cells contributed positively to the efficiency of sensitization. Interestingly, MMGL-positive cell migration was induced by vehicle alone, even in the absence of FITC. These results suggest that migration of dermal MMGL-positive cells accounts for the adjuvant effects of vehicles at least in part.     

Clinically applicable bulk isolation of blood CD34+ cells for autografting in children

The quality of population-based cancer registries has been measured by the indices of the proportion of total incident cases (DCO%) registered by death certificate only (DCO), and the ratio of incidence to mortality (I/D ratio). Recently it has been recommended that DCO% should be used as an index for the reliability of diagnosing cancers and that the proportion of cases first notified via death certificate (DCN, DCN%) be used as an index for the completeness of registration. Parkin introduced a method to estimate the registration rate, the estimated proportion of the "true incidence" that are registered in population-based registries. We recommend a modified method for estimating the registration rate for cancer registries where DCN% is relatively high, as it is in Japan, as Parkin's method may overestimate the registration rate. The method is as follows: the registration rate = (1-DCN% x 1/D ratio)/(1-DCN%). The registration rates at the Osaka Cancer Registry between 1966 and 1992 were estimated using our method. During this period, the yearly registration rate was 74.6-78.4% for males and 69.1-73.3% for females. When the cancer cases were looked at according to site, the yearly registration rate was 74.2-81.6% for stomach cancer, 81.2-89.3% for lung cancer, and 71.3-76.9% for uterine cancer. These results show that the registration rate is high for cancers that have an unfavorable prognosis and low for cancers that have a favorable prognosis. We recommend that all cancer registries in Japan calculate the completeness of registration by utilizing DCN defined as the sum of DCO plus cases not reported as cancer but with supportive clinical information of such obtained through survey of the registry for DCN.     

Transformation of myelofibrosis into acute myelogenous leukemia (M0) with multiple tumor emboli

AIMS: To develop a DNA based plate hybridisation assay for the detection of polymerase chain reaction (PCR) products amplified from Aspergillus fumigatus DNA; and to determine the sensitivity of this technique and compare it with Southern blotting. METHODS: A half-log dilution series of DNA extracted from A fumigatus was amplified with specific primers, one of which was 5' end labelled with biotin. PCR products were subsequently detected by agarose gel electrophoresis, Southern blotting, and binding of the products to a streptavidin coated microtitre well, followed by non-radioactive colorimetric detection. Amplification was carried out 10 times for each DNA dilution and a plot of initial DNA concentration against signal intensity was made. RESULTS: A DNA concentration of 1.5 pg could be detected by agarose gel electrophoresis and Southern blotting with a non-radioactively labelled aspergillus specific probe; 1.5 pg was detectable by streptavidin binding of the PCR products to a microtitre plate. The signal from the microtitre plate detection was proportional to the amount of DNA in the PCR reaction on a log-log scale between 100 and 1 pg of DNA. CONCLUSIONS: A DNA based plate hybridisation assay for the detection of A fumigatus PCR products is as sensitive as Southern blotting. However, results are obtained in three hours rather than the three days required for agarose gel electrophoresis, blotting, hybridisation, and detection.     

3-FG as substrate for investigating flux through the polyol pathway in dog lens by 19F-NMR spectroscopy

PURPOSE: To investigate flux through the polyol pathway in the dog lens by 19F-nuclear magnetic resonance (19F-NMR) spectroscopy, using 3-fluoro-3-deoxy-D-glucose (3-FG) as a substrate. METHODS: 3-FG metabolism was monitored by 19F-NMR analysis. Dog lenses were incubated in Dulbecco's modified Eagle's medium containing 10 mM 3-FG. Enzymatic reductase and dehydrogenase activities were spectrophotometrically determined, whereas the analyses of 3-FG metabolites were conducted by 19F-NMR analysis. Aldose reductase (AR) was immunohistochemically localized in dog lens with antibodies raised against dog kidney AR. RESULTS: 19F-NMR spectra indicate that incubation of purified dog lenses AR with 3-FG results in the formation of 3-fluoro-3-deoxy-D-sorbitol (3-FS) and that incubation of dog liver sorbitol dehydrogenase (SDH) with 3-FS results in the formation of 3-fluoro-3-deoxy-D-fructose (3-FF). This confirms that 3-FG is metabolized to 3-FF by the polyol pathway enzymes. The affinity (Km) of AR for 3-FG is approximately 20-fold better than that for D-glucose, whereas the Km of SDH for 3-FS was fourfold less than for D-sorbitol. 3-FG in cultured dog lenses is metabolized primarily to 3-FS; however, small amounts of 3-FF and 3-fluoro-3-deoxy-D-gluconic acid (3-FGA) are also formed. 3-FS formation was reduced by the AR inhibitor AL 1576, and 3-FF formation was eliminated by the SDH inhibitor CP-166,572. In dog lens epithelial cells cultured with 3-FG, only 3-FS is formed. Similarly, only 3-FS is formed when lens capsule containing primarily epithelial lens contaminated with superficial epithelial cells was incubated in 3-FG. Similar incubation of the remaining cortex resulted primarily in the formation of 3-FS and 3-FGA. This enzymatic distribution was confirmed by spectrophotometric activity analysis and the immunohistochemical localization of AR. CONCLUSIONS: The data confirm that flux through the polyol pathway primarily results in sorbitol accumulation. The absence of fructose and gluconic acid from cultured lens epithelium suggests that the epithelial cells primarily contain AR, whereas differentiated fiber cells also contain SDH and glucose dehydrogenase.     

Differences in the extent of inflammation caused by intratracheal exposure to three ultrafine metals: role of free radicals

The Concorde trial compared immediate (Imm) with deferred (Def) AZT monotherapy in asymptomatic HIV-positive participants. Haematological and immunological markers and weight were measured throughout, and correlated with clinical endpoints. Markers associated with disease progression (CD4 lymphocyte count and percentage, platelets, p24 antigen and beta 2 microglobulin favoured Imm: those associated with toxicity (haemoglobin, neutrophils and white cell count) favoured Def. CD8 and total lymphocyte count did not differ significantly between groups. In multivariate analysis, the combination of baseline CD4, p24 antigen and beta 2m was the best baseline predictor of disease. Including change in CD4 and beta 2m at 12 weeks, or changes over follow-up in these markers significantly improved the fit. Markers were also incorporated into the definition of 'clinical' endpoints. Hazard ratio estimates from end-points that included CD4 < 50 and CD4 < 25 were closest to those for AIDS or death alone, but added very few extra events. Use of other landmark CD4 counts (100 or greater) or relative decreases in counts (25% or more) increased the number of events, but overestimated the effect of immediate AZT. Although AZT had a beneficial effect on the surrogate markers of efficacy evaluated, these changes did not predict clinical outcome, nor could the markers be usefully incorporated into an endpoint definition.     

Survival and disability in stroke by stroke subtype based on computed tomographic findings in three rural Japanese communities

PURPOSE: We conducted an epidemiological study of survival and disability in stroke in three Japanese communities to seek community strategies for improvement in survival and disability. METHODS: A total of 297 first-ever strokes were identified between 1988 and 1992 in three rural communities (total population = 47,000) located in Akita and Ibaraki. We analyzed survival rates and activity of daily living by sex, age-group and stroke subtypes. Successful review of computed tomography (CT) for 84 percent of the strokes (249 out of 297) was possible and the data were used for subtype analyses. RESULTS: For all strokes (n = 297) survival rates were 85% for 30 day, 70% for one year, 62% for three year. The rates tended to be lower in women than in men. The rates were lowest in ages less than 60 at thirty day, and in ages 80 and older at the end of the first and third year. Intracerebral hemorrhage with ventricular rupture, subarachnoid hemorrhage and cortical cerebral infarction had lower survival rates than intracerebral hemorrhage without ventricular rupture and lacunar infarction. Based on Cox's proportional hazard model, risk ratio for death was 2.07 in ages 70-79, and 3.80 in ages 80 and older compared with ages 60-69. The risk ratio was 3.46 for intracerebral hemorrhage with ventricular rupture, 3.38 for subarachnoid hemorrhage and 2.46 for cortical cerebral infarction compared with lacunar infarction. The proportion of stroke survivors who need assistance from others in the first and third years tended to be higher in women than in men. The proportion was higher in older patients than in the younger, and higher for intracerebral hemorrhage with ventricular rupture and cortical cerebral infarction than in other subtypes of stroke. From logistic regression analysis, the odds ratio for disability in the first year was 6.55 for ages 80 and older compared with ages 60-69. The odds ratio was 5.61 for intracerebral hemorrhage with ventricular rupture, 4.53 for cortical cerebral infarction compared with lacunar infarction. In the third year the odds ratio was significant for ages 70-79, and decreased for intracerebral hemorrhage with ventricular rupture (odds ratio = 2.98), and increased for cortical cerebral infarction (odds ratio = 6.06). CONCLUSIONS: Survival and disability in stroke depended on age and stroke subtypes. Even after age adjustment, stroke subtypes with large cerebral involvement had worse prognosis than stroke subtypes. Community-based hypertension control programs are important to prevent any subtypes of stroke. Stroke subtypes as well as age should be taken into account to develop effective care and medical treatments for strokes.