Histologic classification and pathologic findings for endometrial biopsy specimens obtained from 2964 perimenopausal and postmenopausal women undergoing screening for continuous hormones as replacement therapy (CHART 2 Study).

OBJECTIVE: The aim of this study was to screen for disorders and to histologically classify endometrial biopsy specimens from 2964 perimenopausal and postmenopausal women who were candidates for hormonal replacement therapy. STUDY DESIGN: Endometrial biopsy specimens were obtained with a Vabra aspiration curette, processed by standard methods, and stained by hematoxylin and eosin and special methods to reveal subtle features of the endometrium. RESULTS: Of the endometrial biopsy specimens, 68.7% were atrophic, 23.5% were proliferative, 0.5% were secretory, 0.6% were hyperplastic, 0.07% were adenocarcinoma, and 6.6% were insufficient for classification. Three independent senior microscopists agreed on the classification of each biopsy specimen. CONCLUSION: The number of patients is the largest ever screened for a single hormone replacement therapy study. The low yield of endometrial cancer indicates that biopsies are unnecessary before hormone replacement therapy is initiated in asymptomatic women.

Response of leiomyosarcoma to cryosurgery: clinicopathological and ultrastructural study.

Two elderly patients with primary leiomyosarcoma (LMS) of the scalp were treated cryosurgically. Complete involution of both tumours with full epithelialization of the affected sites was achieved. Pretreatment biopsies and sequential biopsies obtained after treatment allowed observation of microscopical changes taking place during tumour involution. Gradual shrinkage of both LMS, closely monitored under the operating microscope, started immediately after the initial freezing. Light and electron microscopic observation of the shrinking LMS revealed a rapid disappearance of the tumoral architecture. Early accumulation of eosinophils and erythrocytes was followed by migration of lymphocytes and plasma cells. Capillary neoformation, fibroblasts and newly formed connective tissue fibres became apparent during the later stages of healing. Two years after treatment, both patients showed no signs of recurrence. These results suggest cryosurgery--performed in an extended protracted fashion--can be a valuable therapeutic choice in the management of LMS, particularly when surgical excision is not feasible.

Pretreatment with the adenosine A1 selective agonist, 2-chloro-N6-cyclopentyladenosine (CCPA), causes a sustained limitation of infarct size in rabbits.

OBJECTIVE: The highly selective adenosine A1 receptor agonist, 2-chloro-N6-cyclopentyl-adenosine (CCPA), has been shown to be as cardioprotective as ischaemic preconditioning when evaluated with an early staining method using tetrazolium. However, tetrazolium-positive tissue measured 3 h after reperfusion may still overestimate the long term salvage. To test for this possible artefact, a 72 h reperfusion rabbit model of myocardial infarction was used, and infarct size was assessed by histology. METHODS: Myocardial infarction was induced by a 30 min coronary occlusion. Rabbits were assigned to a control group receiving no treatment, pretreatment with 0.125 mg.kg-1 CCPA, or 0.25 mg.kg-1 pretreatment with CCPA (0.25 mg.kg-1) followed by an A1 selective antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) 30 min after reperfusion to reverse the haemodynamic side effects. RESULTS: In the 0.125 mg.kg-1 CCPA group, 30.8(SEM 4.2)% of the ischaemic zone was infarcted, which was significantly less than that seen in the control group [46.5(3.0)%; p < 0.01]. Reversing the side effects of CCPA by giving DPCPX soon after reperfusion did not block the protective effects [26.2(1.9)% infarction; p < 0.01 v control]. CONCLUSIONS: This finding confirms a genuine anti-infarct effect of adenosine A1 receptor stimulation when given prior to the onset of ischaemia. Furthermore blocking the A1 receptors soon after reperfusion reverses the side effects but does not block protection.

Tissue response to bioerodible, subcutaneous drug implants: a possible determinant of drug absorption kinetics.

The fibrous tissue compartments that develop in response to the subcutaneous implantation of bioerodible heat-fused rods of norethindrone and cholesterol (85 and 15%, respectively) were studied by light and electron microscopy at various intervals after implantation to determine whether the biological inflammatory response may play a role in drug absorption. Thirty-five regularly menstruating, sterilized (tubal ligation), healthy females each received four Annuelle rods. The microanatomy of seven of the largest implants (135 mg norethindrone) was studied. A dense fibrous biological compartment was found to surround each rod. By light microscopy no abnormal tissue response was revealed. Scanning and transmission electron microscopy showed that the surfaces of the rods were covered by a cellular matrix of mononuclear cells. The fibrous compartment was composed of a loose cellular bed immediately surrounding the norethindrone rod, a dense fibrous connective tissue envelope containing blood and lymphatic vessels, and an outer fatty connective tissue layer. Transmission electron microscopy confirmed that the cellular tissue immediately surrounding the rods was composed mainly of lipid laden macrophages. Norethindrone levels in tissue capsules at 3 and 10.5 months were 0.05 and 8.4% by weight, respectively. These observations suggest that the local inflammatory response plays a role in the active processing of this delivery system. This picture is qualitatively different from the general view of the fibrous capsule as a simple rate limiting membrane. The effects observed in this study suggest that a more complex, functional biological system develops in response to the subcutaneous introduction of a drug delivery device.

Reversal of increased microvascular permeability associated with ischemia-reperfusion: role of cAMP.

Ischemia-reperfusion (IR) is a form of oxidant injury known to increase microvascular permeability in the lung. Agents that increase adenosine 3',5'-cyclic monophosphate (cAMP) levels have been shown to have beneficial effects in several models of oxidant lung injury associated with increased microvascular permeability. We investigated the role of adenylate cyclase activation with isoproterenol (ISO) or forskolin (FSK) in reversing the increased microvascular permeability associated with IR. ISO or FSK administered after 45 min of ischemia and 46 min of reperfusion caused a reduction in the capillary filtration coefficient (Kfc) from 1.25 +/- 0.13 to 0.53 +/- 0.08 and 0.55 +/- 0.10 ml.min-1.cmH2O-1.100 g tissue-1, respectively, at 90 min of reperfusion. This reduction in Kfc was accompanied by a rise in perfusate cAMP levels from 16.5 +/- 4.9 and 31.2 +/- 11.9 pmol/ml at 45 min of reperfusion to 444.2 +/- 147.8 and 276.1 +/- 91.0 pmol/ml at 105 min of reperfusion in lungs treated with ISO or FSK, respectively, at 46 min of reperfusion. Dibutyryl cAMP (DBcAMP), a membrane-permeable cAMP analogue, mimicked the permeability effect by reducing Kfc to 0.67 +/- 0.15 at 90 min of reperfusion. Significant hemodynamic changes occurred but were small and cannot explain the observed effect on Kfc. Photomicrographs from lungs treated with ISO or FSK revealed a reversal of the morphological manifestations of increased microvascular permeability. We conclude that the increased microvascular permeability associated with IR can be reversed by ISO, FSK, and DBcAMP and that cAMP produced by the lung contributes to the observed reversal.

Tetrazolium artifactually indicates superoxide dismutase-induced salvage in reperfused rabbit heart.

We tested the ability of a single dose of superoxide dismutase to induce salvage of reperfused rabbit myocardium. Infarct size was measured by tetrazolium method following 3, 24, or 72 h of reperfusion. In addition, the 24 h reperfused hearts were examined to determine if the drug induced salvage in those hearts was reflected in the histology. A coronary arterial branch was occluded for 45 min and then allowed to reperfuse for 3, 24 or 72 h. At the end of the reperfusion period the hearts were removed, perfusion stained with triphenyl tetrazolium, and fixed in buffered formalin. The hearts were sectioned and infarct size was determined in all groups. In addition, the 24 h heart slices were prepared for histology with H&E staining. The results revealed that 5 mg/kg hSOD treatment was associated with smaller infarcts in the 3 and 24 h groups but that differences were no longer apparent in the 72 h group. The 24 h control hearts showed good correlation between infarct size by TTC and that by conventional histology. In the 24 h treatment hearts, however, infarcts by TTC averaged only about 1/2 the size of those by conventional histology. We conclude that a single dose of hSOD fails to offer a sustained reduction of infarct size. Furthermore, histology from the 24 h reperfused group revealed that hSOD did not delay the onset of necrosis but rather simply caused dead tissue to retain its ability to reduce the tetrazolium salts.

Chronic portal hypertension: effects on gastrointestinal blood flow distribution.

The intramural distribution of blood flow in the gastrointestinal tract was measured in shamoperated control and portal vein-stenosed rats. Total organ blood flow, measured via the radioactive microsphere technique, was elevated in the esophagus (66%), stomach (102%), duodenum (42%), jejunum (52%), ileum (54%), and colon (79%) of portal-hypertensive rats. Histological evaluation of carbonized nonradioactive 15-microns microspheres allowed for fractionation of blood flow within the wall (mucosa, submucosa, and muscularis externa) of each organ. The microsphere distribution pattern indicates that intramural blood flow distribution in all organs was not dramatically affected by chronic portal hypertension. These findings further define the characteristics of the factors responsible for the gastrointestinal hyperemia produced by chronic portal hypertension.

Fungus-host relationship in candidiasis. A brief review.

Candidiasis and its causative agent, Candida albicans, have been under continuous study in our clinics and laboratories for the past 20 years. Cultured cells of C albicans and tissues from natural and experimental infections were used for observations by light microscopy, transmission electron microscopy, scanning electron microscopy and freeze fracture techniques. In cultures, the cells of C albicans revealed a more complex cell wall, plasma membrane, intracellular organelles, and biochemical organization than those described in classic text-books on mycology. In infected tissues, noteworthy characteristics of C albicans were prominent vacuoles and invasion of host cells with subsequent intracellular localization and lysis of tissues surrounding the fungus. These findings are discussed in relation to their importance in the pathogenesis and management of candidiasis and to the mechanism of action of anticandida agents.

Clinical evaluation of an improved injectable microcapsule contraceptive system.

Pharmacokinetics and pharmacodynamics of a long-acting injectable microcapsule, poly(DL-lactide-co-glycolide), delivery system were tested in 10 women. Two doses (75 or 100 mg of norethindrone) were administered by intramuscular injection. Treatment suppressed ovarian function and inhibited ovulation for 3 months in all subjects. Levels of norethindrone in subjects who received the 100 mg dose were proportionately higher than those in subjects who received the 75 mg dose. Subsequent to the injection, there was a rapid rise in the serum levels of norethindrone followed by a gradual decline until 8 to 10 weeks. Between 10 and 20 weeks after treatment, there was a secondary rise and fall in the serum levels of norethindrone. Treatment caused suppression of the endometrium for 3 months, and, except for spotting and irregular menstrual cycles, there were no adverse side effects. Treatment had no significant effect on serum lipids.

Scanning electron microscopy of human spermatozoa after incubation with the spermicide nonoxynol-9.

The most frequently utilized spermicide in vaginal contraceptives is No-9. Schill and Wolff used TEM to demonstrate the focal effects of No-9 on human sperm and reported that No-9 damaged cell membranes and acrosomal membrane complexes. The present study by SEM was made to assess the extent of membrane damage due to the direct action of No-9 during an incubation period of only 5 minutes. SEM revealed that No-9 caused loosening and detachment of acrosomal, neck, and midpiece membranes of all sperm even at the lowest concentration tested (0.05%). The severity of membrane alterations observed in any of these regions would render sperm immotile and unable to penetrate the ovum. The fact that these alterations are produced within 5 minutes after exposure to No-9 attests to the effectiveness of No-9 as a vaginal contraceptive.

PIP: Scanning electron microscopy was used to assess the extent of membrane damage due to the direct action of nonoxynol-9 during an incubation period of 5 minutes. Nonoxynol-9 is the most frequently utilized spermicide in vaginal contraceptives. Nonoxynol-9 disrupted sperm membranes in all regions except the postacrosomal region and tail. It caused loosening and detachment of acrosomal, neck, and midpiece membranes of all sperm even at the lowest concentration tested (0.05%). Damage to all membranes was 1st evident as vesiculations, then membranes became loose and detached. The severity of membrane alterations observed in any of these regions would render sperm immotile and unable to penetrate the ovum. A dose-response relationship was not apparent, and there was no variation in response among sperm donors. The fact that these alterations are produced within 5 minutes confirms the effectiveness of nonoxynol-9 as a vaginal contraceptive. Scanning electron microscopy proved to be well suited for evaluating the extent of damage caused by nonoxynol-9 and provided more information than transmission electron microscopy.

Mechanisms of uterine bleeding in postmenopausal patients receiving estrogen alone or with a progestin.

The response of postmenopausal endometrium to cyclic estrogen and progestin and cyclic estrogen alone was studied in 75 biopsies and over 2000 preparations using standard histologic, histochemical, and scanning and transmission electron microscopic techniques. Estrogen and a progestin caused the atrophic endometrium to assume normal proliferative and secretory phases and to develop nucleolar channel systems. Cyclic unopposed estrogen produced unphysiologic responses in the glands, stromal cells, and vessels. The concept of a progestin or progesterone producing a "medical curettage" should be reappraised. Cyclic estrogen and progestin therapy do not cause all the endometrium to desquamate to the basalis layer. The combination therapy is associated with increased glycoprotein production in the gland and stromal cells, and an orderly regression and remodeling of the endometrium upon hormonal withdrawal. Cyclic estrogen alone causes irregular and unpredictable breakdown, which may or may not extend to the basalis. The stimulation of the endometrium by estrogen alone may allow the endometrium to use the majority of its energy for growth, which may lead to hyperplasia and neoplasia.

Ultrastructure of endometrium of postmenopausal patients on two modes of hormonal therapy: estrone sulfate with or without MPA.

The response of human postmenopausal endometrium to standard, oral doses of an estrogen alone (estrone sulfate) and to an estrogen plus a progestin (medroxyprogesterone acetate; MPA) was evaluated by scanning electron microscopy and transmission electron microscopy. Endometrial biopsies were obtained over a 4-year period from 12 patients with well-established ovarian failure. Biopsies were taken before the initiation of therapy and during each mode of hormonal treatment. The ability of estrone sulfate alone to stimulate growth of the endometrium was shown during the 1st treatment cycle when the atrophic epithelial cells transformed into tall columnar cells which synthesized and released some secretory products. Unopposed estrogen therapy led to excessive ciliation and breakthrough bleeding. Addition of MPA to the estrone sulfate regimen produced a progestational endometrium. The epithelial cells had ultrastructural features of those in normal postovulatory endometrium, including nucleolar channel systems. MPA elicited deciliation and transformation of ciliated cells into secretory cells. Stromal cells hypertrophied, the vascular endothelium thickened, and bleeding subsided. Estrogen-progestin therapy as tremendously more physiological than unopposed estrogen therapy.

Differences in the ultrastructure of the submandibular glands of baboon and Rhesus monkey revealed by the use of different fixatives.

Submandibular glands of adult baboon and Rhesus monkeys were compared after different methods of fixation. In both species, serous acinar cells outnumber mucous acinar cells. In the baboon, serous cells contain secretory granules showing dense cores, moderately dense crescents, and flocculent material. In mucous cells, secretory granules vary in appearance from amorphous to highly ordered depending on fixation. In the Rhesus monkey, serous cells contain the same 3 components of secretory granules as in the baboon. Additionally, a fourth component is represented by a layer of electron-dense material between the crescent and flocculent material. Mucous cells contain electron-dense granules when fixed in Millonig's buffered fixatives, but when Clarke's or Sorensens' buffer is used the granules resemble more typical mucous granules. Duct systems of the two species are similar, but differ mainly in that large foci of glycogen are present in the striated duct cells of the Rhesus monkey.

Pathology of an experimental extradural spinal T cell tumor.

Syngeneic mice injected intravenously with a T cell tumor line (line 13) induced by Gross' murine leukemia virus developed paraparesis and sensory loss below the midthoracic level 2 to 3 weeks after inoculation. Although signs of systemic disease coexisted, the animals survived through the development of the neurologic symptoms, and treatment with cytotoxic agents was not required. Pathologic study of the spinal cord and brain revealed tumoral infiltration of the meninges, confined to the extradural spaces, more markedly at spinal than cerebral levels. Equally severe infiltrates occurred in the paravertebral musculature. No leptomeningeal or parenchymal involvement was present, irrespective of the severity of the extradural infiltration. Marked bone marrow and visceral infiltration coexisted with central nervous system involvement. The topography of the extradural and muscular tumor cells collections related to the proximity of the involved bone marrow and areas of direct communication between these spaces were repeatedly identified. On the other hand, line 13 cells injected directly into the brain substance produced diffuse leptomeningeal tumoral infiltration without extradural involvement. These findings suggest that the pathogenesis of this model of spinal T cell tumor proliferation involves a first stage of bone marrow infiltration, followed by extradural involvement. This occurs by direct migration of bone marrow tumor cells through gaps in the vertebral bone. This model offers the opportunity for the study of malignancies that produce bone destruction as a mechanism for tumoral spread.

Filiform papillae of cat tongue.

A pronounced species variation in the organization of filiform papillae has been observed. Preliminary histologic studies of cat tongue revealed marked regional variations in size, shape and organization of filiform papillae. Therefore, a correlated light microscopic, transmission and scanning electron microscopic study of tissue samples of representative areas of the cat tongue was undertaken for further elucidation. Results showed that filiform papillae on the tip of the tongue were short and exhibited several conical processes from the base of each papilla. In contrast, filiform papillae in the midportion of the dorsum of the tongue consisted of a large mound with a single sharp spinous process projecting posteriorly. In the region of the vallate papillae, the filiform papillae were shorter and more conical than those on the midportion of the tongue. In addition, keratohyalin granules in filiform papillae were comprised of an eosinophil spheroid with small basophil attachments.

Effects of net transmucosal volume flux on lymph flow in the canine colon. Structural-functional relationship.

The effects of net transmucosal fluid flux on lymph flow, lymphatic protein flux, and lymph oncotic pressure were analyzed in an isolated autoperfused canine colon preparation. Active fluid absorption and secretion were induced by intraluminal instillation of Tyrode's solution alone and Tyrode's solution containing 40 mM theophylline, respectively. In contrast to previous observations in the small bowel, colonic lymph flow, lymph protein flux, or lymph oncotic pressure was not affected by net transmucosal volume flux (absorptive or secretory). Ultrastructural analyses of the lymphatic and capillary microcirculations of the mucosal regions of the colon and ileum indicate that, relative to the small intestine, the colonic mucosa is characterized by (a) lymphatic vessels that are smaller in caliber and do not extend beyond the bottom third of the mucosa and (b) blood capillaries that are situated half as far from the base of the epithelial cells. These results indicate that (a) the inability of net transmucosal fluid movement to alter colonic lymph flow can be attributed to the paucity of lymphatic drainage in the colonic mucosa and (b) blood capillaries are the sole conduits by which absorbed volume is removed from the colonic interstitium, a task facilitated by the close proximity of the fenestrated capillaries to the absorptive epithelium.

Adherence and colonization of Mycoplasma pulmonis to genital epithelium and spermatozoa in rats.

Mycoplasma pulmonis is the etiological agent of a naturally occurring genital disease in rats. Transmission and scanning electron microscopic evaluations of the genital tracts of naturally and experimentally infected female rats show M. pulmonis in close association with both squamous and nonsquamous epithelial cells, although more frequently with the latter. In contrast to other species of mycoplasmas, M. pulmonis adhesion to epithelial cells appears to be mediated by a generalized interaction of the mycoplasma membrane with the host cell membrane, rather than by a specialized attachment tip. Extensive studies of all levels of the male genital tract have not yet been performed, but M. pulmonis can be demonstrated in the urethra and epididymis in animals showing evidence of chronic inflammation. Adherence of M. pulmonis to rat spermatozoa in vitro is associated with a decrease in motility. Addition of anti-M. pulmonis antibody following organism attachment results in marked agglutination of the spermatozoa. Further study of mechanisms involved in M. pulmonis adherence and subsequent mycoplasma host cell interactions is expected to contribute to an understanding of mechanisms involved in reproductive failure.

Effect of atropine on bile-oleic acid-induced alterations in dog jejunal hemodynamics, oxygenation, and net transmucosal water movement.

The effects of intraluminal bile-oleic acid on jejunal blood flow, oxygen uptake, net transmucosal fluid flux, and mucosal ultrastructure were studied in isolated autoperfused jejunal segments before and after cholinergic blockage with atropine. Bile-oleic acid increased jejunal blood flow (40%), decreased oxygen extraction (24%), and increased oxygen uptake (15%). Cholinergic blockade did not abolish the bile-oleic acid-induced changes in these parameters. Bileoleic acid induced net fluid secretion or significantly depressed absorption. Cholinergic blockade reversed net volume secretion to absorption or attenuated the depression of absorption produced by bile-oleic acid. Ultrastructural analyses of tissue samples taken during bile-oleic acid-induced secretion indicate major structural damage to the mucosal membrane which was not affected by cholinergic blockade. The physiologic and ultrastructural data acquired in this study suggest that: (a) the effects of bile-oleic acid on net transmucosal water movement are reversed by cholinergic blockade and (b) the hyperemia, increased oxygen consumption, and morphologic alterations induced by bile-oleic acid are not affected by cholinergic blockade. The results of this study may have important implications in steatorrheal diseases in that, atropine may alleviate the diarrhea induced by the presence of excess lipid in chyme without compromising the intestinal hyperemia and enhanced oxygen delivery required to meet the increased metabolic demands during nutrient absorption.