3-Dimensional configuration of perimysial collagen fibres in rat cardiac muscle at resting and extended sarcomere lengths.

1. We have used fluorescence confocal laser scanning microscopy to attain the three-dimensional (3-D) microstructure of perimysial collagen fibres over the range of sarcomere lengths (1.9-2.3 micrometers) in which passive force of cardiac muscle increases steeply. 2. A uniaxial muscle preparation (right ventricular trabecula of rat) was used so that the 3-D collagen configuration could be readily related to sarcomere length. Transmission electron microscopy showed that these preparations were structurally homologous to ventricular wall muscle. 3. Trabeculae were mounted on the stage of an inverted microscope and fixed at various sarcomere lengths. After a trabecula was stained with the fluorophore Sirius Red F3BA and embedded in resin, sequential optical sectioning enabled 3-D reconstruction of its perimysial collagen fibres. The area fraction of these fibres, determined from the cross-sections of seven trabeculae, was 10.5 +/- 3.9 % (means +/- s.d.). 4. The reconstructed 3-D images show that perimysial collagen fibres are wavy (as distinct from coiled) cords which straighten considerably as the sarcomere length is increased from 1.85 +/- 0.06 micrometer (near-resting length) to 2.3 +/- 0.04 micrometer (means +/- s.d., n = 4). These observations are consistent with the notion that the straightening of these fibres is responsible for limiting extension of the cardiac sarcomere to a length of approximately 2.3 micrometers.

Characterization of cytoplasmic secretory granules (PSG), in prostatic epithelium and their transformation-induced loss in dysplasia and adenocarcinoma.

Cytoplasmic clarity is a histological feature of normal prostatic secretory cells, but in this study, tissue fixation in strong (>2.5%) glutaraldehyde dramatically altered cytological staining. Secretory cytoplasm appeared red and granular on routine stains because of myriad intensely staining eosinophilic granules (PSG). Immunostaining for prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) showed their exclusive localization to the PSG. Electron microscopy confirmed these findings and also showed that after fixation in many agents, including formaldehyde, PSG appeared empty, accounting for the artefactual "clear cell" appearance on light microscopy. PSG were most densely concentrated apically in a bud-shaped luminal compartment in which cytokeratin was selectively absent. Normal exocrine secretion was visualized as detachment of apocrine buds or their in situ disintegration. Distinctively in dysplasia and almost all carcinomas, PSG were rare to absent, and proteases were free in the cytoplasm, often concentrated beneath the apical membrane. The apocrine compartment was absent, with no observed secretory mechanism. Tumor cells had dark amphiphilic cytoplasm after all fixatives. This provided a reliable method of distinguishing malignant from benign glands in tissues fixed in strong glutaraldehyde. Clear cell carcinomas, whose cytoplasm mimicked routinely fixed normal secretory cells, surprisingly had almost no PSG. Instead, their "granules" were lipid-filled vacuoles reflecting a secretory pathway not seen in normal cells, dysplasia, or the common "dark cell" carcinomas. These observations may define two distinctive biological pathways of prostate cancer evolution and may facilitate diagnostic decisions on needle biopsy samples.

Prediction of pathological stage and clinical outcome in prostate cancer: an improved pre-operative model incorporating biopsy-determined intraductal carcinoma.

OBJECTIVE: To relate the predictive value of serum prostate specific antigen (PSA) levels, histological grade and intraductal carcinoma (IDC-P, a possible marker of poor prognosis) to pathological stage and subsequent clinical outcome, and thus derive an improved predictive model to aid the decision to initiate potentially curative therapy in localized prostate cancer. MATERIALS AND METHODS: Fifty-nine radical prostatectomy specimens were histologically graded, allocated a pathological stage and the tumour volume determined by image analysis. Pre-operative (needle biopsy) tumour grade, the presence or absence of IDC-P, and serum PSA levels were correlated with the pathological stage. This was used to define the sequence and values that would be incorporated into a predictive model for pathological stage and clinical outcome. RESULTS: There were close correlations between cancer volume and tumour grade (P = 0.004) and between cancer volume and serum PSA level (P = 0.003). However, in tumours with IDC-P, serum PSA level did not correlate with tumour volume of IDC-P (P > 0.9). IDC-P was an independent variable that significantly improved the prediction of pathological stage and tumour volume, and furthermore, was closely related to (r = 0.53, P = 0.001) and accurately predicted treatment failure. CONCLUSION: The model which best predicted pathological stage and clinical outcome involved first identifying those cancers with IDC-P as having the poorest outcome. Cancers without IDC-P were then separated into low- and high-risk groups on the basis of serum PSA levels below and above 10 ng/mL, and those in the high-risk group further stratified using Gleason grading. Furthermore, the use of a sequential consideration of pre-operative variables including IDC-P allowed cases to be grouped which, after radical surgery, closely correlated with clinical outcome.

Microvascular involvement in cardiac pathology.

Abnormalities of the microvasculature are centrally involved in the pathogenesis of some forms of heart disease, but in others are consequences of it. Microvascular abnormalities may contribute to the progression of viral myocarditis and Chagas' disease. Focal abnormalities may occur early in some cardiomyopathies and do occur later in most types of myocarditis. The thickening of arteriolar walls in chronic hypertension is likely to contribute significantly to the impairment of coronary haemodynamics associated with adaptive ventricular hypertrophy and the consequent diminution of coronary reserve, increasing diffusion distances and failure of angiogenesis to compensate. However, the resulting myocyte necrosis stimulates inflammatory angiogenesis. When ischemic myocyte injury becomes irreversible there is a concomitant loss of capacity for reperfusion, the no-reflow phenomenon. Less severe temporary ischemia reduces the proportion of functional capillaries. Multiple mechanisms are involved in this microvascular stunning, including: reperfusion injury; leukocyte activation; adhesion and accumulation; and impaired endothelium-dependent vasodilation. Many of the microvascular changes are those of the inflammatory response to cell death and form part of a final common pathway in myocarditis, cardiomyopathy, cardiac hypertrophy and failure, and ischemic heart disease. Stimulation of angiogenesis prior to myocyte necrosis in hypertrophy and control of leukocyte activity in ischemic heart disease could minimize myocyte loss.

Leukemia inhibitory factor is mitogenic to osteoblasts.

Leukemia inhibitory factor (LIF) regulates cell growth and is produced by a variety of tissues, including bone. Previously we have shown that recombinant human LIF induced an increase in osteoclast number, bone formation, and DNA synthesis. In the present study, we have defined the cells in intact bone at which the proliferative effects of LIF occur, using simultaneous enzyme histochemistry and autoradiographic techniques. The area of alkaline phosphatase-positive staining was increased twofold (p = 0.0008) and the number of [3H]thymidine-positive cells was increased twofold (p = 0.0024) in LIF-treated bones. The radiolabeled cells either colocalized with alkaline phosphatase or were in the osteoprogenitor region. They were not found in the acid phosphatase-positive staining osteoclasts. These results indicate that cells which have a mitogenic response to LIF are bone-forming rather than bone-resorbing cells.

Laminar structure of the heart: ventricular myocyte arrangement and connective tissue architecture in the dog.

We have studied the three-dimensional arrangement of ventricular muscle cells and the associated extracellular connective tissue matrix in dog hearts. Four hearts were potassium-arrested, excised, and perfusion-fixed at zero transmural pressure. Full-thickness segments were cut from the right and left ventricular walls at a series of precisely located sites. Morphology was visualized macroscopically and with scanning electron microscopy in 1) transmural planes of section and 2) planes tangential to the epicardial surface. The appearance of all specimens was consistent with an ordered laminar arrangement of myocytes with extensive cleavage planes between muscle layers. These planes ran radially from endocardium toward epicardium in transmural section and coincided with the local muscle fiber orientation in tangential section. Stereological techniques were used to quantify aspects of this organization. There was no consistent variation in the cellular organization of muscle layers (48.4 +/- 20.4 microns thick and 4 +/- 2 myocytes across) transmurally or in different ventricular regions (23 sites in 6 segments), but there was significant transmural variation in the coupling between adjacent layers. The number of branches between layers decreased twofold from subepicardium to midwall, whereas the length distribution of perimysial collagen fibers connecting muscle layers was greatest in the midwall. We conclude that ventricular myocardium is not a uniformly branching continuum but a laminar hierarchy in which it is possible to identify three axes of material symmetry at any point.

Microvascular incompetence and the failure of hearts to recover contractile function after cardioplegia.

The relationship between the development of microvascular incompetence and the loss of potential for functional recovery following cardioplegia was investigated using St. Thomas' Hospital No. 2 solution (STH) in isolated working rat hearts. Cardiac function was measured prior to cardioplegia and again after 30 min of reperfusion at 37 degrees C following 1, 2 or 4 h arrest at 30 degrees C (n=5). The hearts were then fixed by perfusion with 2.5% glutaraldehyde and then nuclear track emulsion was perfused as an intravascular marker of competent capillaries. Following cardioplegia for 1 h hearts showed 95.4% recovery of aortic flow in the working mode, and a high proportion of the capillaries in the subendocardial (84.6 +/- 2.3%), middle (94.6 +/- 3.0%) and subepicardial (89.1 +/- 4.9%) thirds of the left ventricular myocardium transmitted perfusate. Two hours arrest resulted in significantly diminished recovery of left ventricular function (aortic flow: 56.6 +/- 7.6% and aortic pressure: 64.4 +/- 2.5% and heart rate 56.0 +/- 23.1%). This loss of the remaining two thirds of the potential for functional recovery was associated with significant (P<0.02) reductions in the proportions of competent capillaries (subendocardial, middle and subepicardial thirds to 10.9%, 19.2% and 14.2%, respectively). These non-functional capillaries had open lumina and showed no sign of structural alteration, obstruction or compression, although some focal collections of myocytes (<30%) showed evidence of reperfusion damage including contraction band necrosis. Despite reductions in microvascular competence overall, coronary flow rates (non-working) did not decline, suggesting shunting via large arterio-venous channels. It seems likely that the loss of the first third of the potential for rapid functional recovery following cardioplegia is due to loss of high energy phosphates, whereas the loss of the remaining two-thirds is associated with endothelial cell mediated constriction of small arterial vessels which produces the capillary incompetence demonstrated in this study.

Distribution of sialosyl Tn and Tn antigens within normal and malignant colorectal epithelium.

Expression of the core blood group structures sialosyl Tn (STn) and Tn is regarded as a colorectal cancer-specific change reflecting truncated synthesis of the oligosaccharide component of goblet cell mucin. The distribution of STn and Tn in normal and malignant epithelium has been studied in detail by a combination of mucin-, lectin-, and immunohistochemistry with and without pretreatment with potassium hydroxide (KOH), neuraminidase, and KOH-neuraminidase. When O-acetylated sialic acid (neuraminidase-resistant) is converted by saponification to non-O-acetylated sialic acid (neuraminidase-sensitive), normal colorectal goblet cells (mainly of the lower two-thirds of crypts) are immunoreactive with the monoclonal antibody TKH2 (specific for STn). This immunoreactivity is abolished by the interposition of neuraminidase, but goblet cells then become immunoreactive with Hb-Tn1 (specific for Tn). While colorectal cancer mucin expresses STn, expression of Tn is not seen in either goblet cell mucin or extracellular material showing the morphological and histochemical characteristics of secretory mucin. Tn expression in cancers is mainly limited to the Golgi zone and in a proportion of cases to cytoplasm and apical membrane (glycocalyx) of columnar cells and inspissated material within lumina. The material reacting with Hb-Tn1 may be upregulated, membrane-associated MUC1 glycoprotein rather than MUC2 or MUC4 goblet cell mucin. The presence of STn and cryptic Tn within normal colorectal goblet cells and the absence of Tn expression within colorectal cancer secretory mucin contradicts the generally accepted concept of cancer-specific incomplete glycoprotein synthesis within these neoplasms.

Primary plexogenic pulmonary hypertension shows imperfect formation of the internal elastic lamina of the pulmonary arteries.

Lung tissue from subjects dying from primary plexogenic pulmonary hypertension (PPH) has shown defects of elastin formation of the lung arteries. Lung vessels from 5 cases of PPH were compared with those of 9 age-matched normal subjects, and 24 individuals having secondary pulmonary hypertension (2 degrees PH). PPH cases and those with 2 degrees PH due to congenital heart disease with left-to-right shunts (2 degrees PH, LRS), showed active proliferation of medial smooth muscle cells (SMC) through defects of the internal elastic lamina (IEL) into the arterial lumen to form typical plexiform lesions. Larger arteries showed accelerated intimal thickening similar to normal aging. Plexiform lesions were not seen in normal subjects or in those developing high pulmonary pressures later in life. The observations showed that the development of discontinuities of the IEL of the pulmonary arteries and intimal thickening is accelerated in normal subjects by high pulmonary artery pressure, especially when this is established at a very young age. They suggest that such discontinuities occur in PPH due to inherent abnormality of the elastin of the arterial walls, with advanced early proliferation of medial SMC and obstruction of the pulmonary arterial circulation.

Schistosomiasis and prostate cancer.

In endemic geographical areas schistosomiasis has been implicated as an etiological agent in the pathogenesis of bladder, colorectal and renal carcinoma. In particular bladder cancer commonly occurs in such geographic locations almost 2 decades earlier than in non-endemic areas. A relationship between prostate cancer and bilharzial infestation is not established. This is a report of 3 cases of co-existent schistosomiasis and prostatic adenocarcinoma occurring in unusually young patients.

Diagnostic markers demonstrating successful culture of human meningioma cells.

The light microscopic and ultrastructural features of cultured meningioma cells were compared with those of surgically resected specimens. Meningioma cells normally show variable degrees of epithelial or mesenchymal differentiation. In cultured cells, well formed psammoma bodies and whorls were seen by light microscopy and specialised intercellular junctions were observed by electron microscopy. Cytogenetic analysis was also shown to be a useful diagnostic marker to differentiate cultured meningioma from contaminant proliferating fibroblastic and endothelial cells. These methods have demonstrated that the cells cultured from operative samples were predominantly meningioma cells. The ability to reproducibly cultivate populations of meningioma cells should facilitate in vitro assessment of potential adjunctive treatment modalities.

Protective effects of oxygenated St. Thomas' Hospital cardioplegic solution during ischaemic cardiac arrest: improved function, metabolism and ultrastructure.

The isolated working rat heart model was use to define the cardioprotective effects (function, metabolic and ultrastructure) of the oxygenated St. Thomas' Hospital No. 2 cardioplegic solution (STH) during lengthy, hypothermic ischaemia (20 degrees C, 4 hours and 5 hours). Hearts (n = 9 for each group) were arrested with and exposed to multidose reinfusion (2 min every 40 min interval) throughout the ischaemic period with the cold (4 degrees C) STH or oxygenated (95% O2:5% CO2) STH. Oxygenated STH significantly (p < 0.01) improved the postischaemic recovery of cardiac output from 49.5 +/- 11.1% to 96.8 +/- 1.5% (in 4 hours) and from 20.3 +/- 7.2% to 72.2 +/- 5% (in 5 hours). Other indices of functional recovery showed similar improved performance with the significant decrease in time from the onset of reperfusion to the return of regular sinus rhythm (57 +/- 8 v 495 +/- 150 s). The efflux of lactate during 5 hr ischaemic arrest was decreased (20.62 +/- 1.3 v 26.18 +/- 1.73 mumol/heart for oxygenated STH and STH, respectively, p < 0.05) and the progressive increase in the coronary vascular resistance was abolished in the oxygenated STH treated hearts. These improvements were associated with the reduction in the decline of the myocardial adenosine triphosphate (14.49 +/- 2 v 3.3 +/- 0.19 mumol/g dry wt), creatine phosphate (24.61 +/- 3.47 v 7.48 +/- 1.34 mumol/g dry wt) and guanosine triphosphate (1.69 +/- 0.2 v 0.84 +/- 0.08 mumol/g dry wt) during ischaemia, total resynthesis after reperfusion (ATP: 103% v 36%, CP: 105% v 69% and GTP: 203% v 61% of control) and the total absence of myocardial cells and microvasculature injuries in ischaemic (non-reperfused) hearts. These results confirm that the provision of additional oxygen to the St. Thomas' Hospital solution (with 95% O2:5% CO2) can meet the metabolic demand of the ischaemic myocardium and thus increase the safe duration of cardiac arrest.