Spatially correlated phenotyping reveals K5-positive luminal progenitor cells and p63-K5/14-positive stem cell-like cells in human breast epithelium.

Understanding the mechanisms regulating human mammary epithelium requires knowledge of the cellular constituents of this tissue. Different and partially contradictory definitions and concepts describing the cellular hierarchy of mammary epithelium have been proposed, including our studies of keratins K5 and/or K14 as markers of progenitor cells. Furthermore, we and others have suggested that the p53 homolog p63 is a marker of human breast epithelial stem cells. In this investigation, we expand our previous studies by testing whether immunohistochemical staining with monospecific anti-keratin antibodies in combination with an antibody against the stem cell marker p63 might help refine the different morphologic phenotypes in normal breast epithelium. We used in situ multilabel staining for p63, different keratins, the myoepithelial marker smooth muscle actin (SMA), the estrogen receptor (ER), and Ki67 to dissect and quantify the cellular components of 16 normal pre- and postmenopausal human breast epithelial tissue samples at the single-cell level. Importantly, we confirm the existence of K5+ only cells and suggest that they, in contrast to the current view, are key luminal precursor cells from which K8/18+ progeny cells evolve. These cells are further modified by the expression of ER and Ki67. We have also identified a population of p63+K5+ cells that are only found in nipple ducts. Based on our findings, we propose a new concept of the cellular hierarchy of human breast epithelium, including K5 luminal lineage progenitors throughout the ductal-lobular axis and p63+K5+ progenitors confined to the nipple ducts.

Interlaboratory variability of Ki67 staining in breast cancer.

BACKGROUND: Postanalytic issues of Ki67 assessment in breast cancers like counting method standardisation and interrater bias have been subject of various studies, but little is known about analytic variability of Ki67 staining between pathology labs. Our aim was to study interlaboratory variability of Ki67 staining in breast cancer using tissue microarrays (TMAs) and central assessment to minimise preanalytic and postanalytic influences. METHODS: Thirty European pathology labs stained serial slides of a TMA set of breast cancer tissues with Ki67 according to their routine in-house protocol. The Ki67-labelling index (Ki67-LI) of 70 matched samples was centrally assessed by one observer who counted all cancer cells per sample. We then tested for differences between the labs in Ki67-LI medians by analysing variance on ranks and in proportions of tumours classified as luminal A after dichotomising oestrogen receptor-positive cancers into cancers showing low (<14%, luminal A) and high (≥14%, luminal B HER2 negative) Ki67-LI using Cochran's Q. RESULTS: Substantial differences between the 30 labs were indicated for median Ki67-LI (0.65%-33.0%, p < 0.0001) and proportion of cancers classified as luminal A (17%-57%, p < 0.0001). The differences remained significant when labs using the same antibody (MIB-1, SP6, or 30-9) were analysed separately or labs without prior participation in external quality assurance programs were excluded (p < 0.0001, respectively). CONCLUSION: Substantial variability in Ki67 staining of breast cancer tissue was found between 30 routine pathology labs. Clinical use of the Ki67-LI for therapeutic decisions should be considered only fully aware of lab-specific reference values.

Exercise intensity: platelet function and platelet-leukocyte conjugate formation in untrained subjects.

INTRODUCTION: Strenuous and exhaustive exercise intensifies platelet activity as shown in the literature but effects of moderate exercise are still in discussion. The present study investigated effects of two different standardised exercise intensities controlled by individual anaerobic threshold (IAT) on platelet function and conjugate formation. METHODS: 20 healthy male non-smokers underwent two exercises at 80% (moderate) of IAT which corresponded to about 57% of peak oxygen consumption (peak VO(2)) in our subjects and 100% (strenuous) of IAT, corresponding to about 69% peak VO(2). Blood samples were taken after 30 min rest and immediately after exercise. CD62P expression and differentiated platelet-leukocyte conjugates (CD45, CD14, CD41) as well as microparticles and platelet-platelet aggregates were detected flow cytometrically with and without TRAP-6-stimulation. RESULTS: CD62P expression and the number of aggregates were increased (P< or =0.05) after exercise in the TRAP-stimulation experiment independent of exercise intensity. The number of platelet-granulocyte (rest 5.7+/-1.8 to post 8.1+/-1.7 (80%) vs. 6.2+/-1.9 to 10.3+/-2.0 (100%)), platelet-monocyte (5.3+/-3.6 to 8.5+/-3.7 (80%) vs. 7.4+/-3.5 to 11.7+/-4.8 (100%)), and platelet-lymphocyte conjugates (4.4+/-1.2 to 6.4+/-1.3 (80%) vs. 4.6+/-1.7 to 7.8+/-1.8% positive cells (100%)) were also higher after both exercises but increased significantly weaker (P< or =0.05) after moderate exercise. These results were confirmed by the TRAP-stimulation experiment. CONCLUSION: Although moderate exercise led to an increase in platelet reactivity and platelet-leukocyte conjugate formation the changes in conjugate formation were significantly weaker compared to strenuous exercise. Therefore it is recommended that submaximal endurance performance should be individually developed in order for everyone to be able to carry out normal daily activities and also to exercise well below the IAT.

The influence of bromelain on platelet count and platelet activity in vitro.

Bromelain is a general name for a family of sulfhydryl-containing, proteolytic enzymes from the pineapple plant. The aim of the present study was to investigate the influence of bromelain on platelet count, platelet aggregation and platelet activity in vitro. Blood samples were taken from the antecubital vein of 10 healthy male non-smokers. Platelet count decreased after incubation with 2.5 and 5 mg bromelain/ml from 277 +/- 17 platelets/nl before to 256 +/- 21 and 247 +/- 19 platelets/nl after the treatment. The ADP and TRAP-6 induced platelet aggregation led to a significant decrease after the incubation with 2.5 mg (ADP: 48.6 +/- 25.7%; TRAP-6: 49.6 +/- 28.9%) or 5 mg (ADP: 5.0 +/- 4.6%; TRAP-6: 9.0 +/- 4.9%) bromelain/ml in comparison to control (ADP: 81.4 +/- 5.0%; TRAP-6: 77.4 +/- 10.4%). The percentage of unstimulated CD62P positive platelets which were investigated by flow cytometry was minimally higher after incubation with 5 mg bromelain/ml (0.57 +/- 0.48% PC) in comparison to control (0.22 +/- 0.11% PC), but after TRAP-6 stimulation the incubation with 5 mg bromelain/ml led to a remarkable decrease in comparison to the untreated control (50.4 +/- 20.2 to 0.9 +/- 0.8% PC). The changes of CD62P (TRAP-stimulated) and the results of platelet aggregation after incubation with bromelain in vitro may demonstrate the potential of bromelain as a substance for platelet inhibition.

Transcription in response to physical stress--clues to the molecular mechanisms of exercise-induced asthma.

To clarify stress-induced immunological reactions and molecular events during exercise and the potential relevance to exercise-induced bronchoconstriction, transcriptional responses to standardized physical stress were determined. Six healthy, young volunteers underwent an endurance exercise of 90% of their individual anaerobic threshold for 90 min. Time-dependent alterations in the expression pattern of leukocytes from healthy, trained subjects were analyzed by DNA microarrays before and 2 h and 6 h after exercise. Starting out from a large collection of cDNA library clones comprising more than 70,000 human expressed sequence tags, we selected, designed, and immobilized oligonucleotide probes (60-70mers) for transcripts of 5000 stress- and inflammation-relevant genes. Exercise-induced stress provoked changes in the expression of 433 gene activities 2 h and/or 6 h after exercise, which could be grouped into six clusters. The most prominent feature was an enhanced transcription of two genes, coding for 5-lipoxygenase (ALOX5) and ALOX5-activating protein. Moreover, enhanced levels of leukotriene B4 (LTB4) and LTC4 (P<0.05) were detected in plasma after exercise. Our data demonstrate that exercise alters the activities of a distinct number of genes. In particular, they possibly provide novel insights into the molecular mechanisms of exercise-induced bronchoconstriction and suggest that enhanced transcription of ALOX5 and its activating protein together with a present predisposition of the subject critically contribute to exercise-induced asthma.

Pure eccentric exercise does not activate blood coagulation.

Eccentric exercise can cause skeletal muscle damage with ultrastructural disruption, inflammation and increased proteolytic enzyme activity. It may be possible that these changes are able to trigger blood coagulation in vivo. The aim of the study was to investigate changes in blood coagulation via the measurement of aPTT, the thrombin potential (total [TTP] and endogenous [ETP], both intrinsic [in] and extrinsic [ex]) and the thrombin generation (prothrombinfragment 1 + 2 [F1 + 2] and thrombin-antithrombin complex [TAT]) after pure eccentric exercise. Seventeen healthy non-smokers (28 +/- 6 years, VO2-peak 59 +/- 7 ml/min/kg) underwent pure eccentric down jumps (9 x 28 isolated down jumps in 90 min, drop from a height of 55 cm), a cycle exercise (90% of the individual anaerobic threshold for 60-90 min) and a control experiment on different days. Blood samples were drawn after a 30-min rest, immediately, and 2 h after exercise. After the cycle exercise, a clear shortening by 12% (P<0.001) in aPTT and an increase in TTPin (13%; P<0.05) and TAT (33%; P<0.05) in comparison to the control experiment were seen, while after eccentric exercise only minimal changes in aPTT and thrombin potential (TTPin, ETPin) and no thrombin generation (F1 + 2 and TAT) were found. In contrast to concentric dynamic exercise, e.g. cycle ergometry, only insignificant changes in thrombin potential and no thrombin generation could be observed after skeletal muscle damage induced by pure eccentric exercise. It can be concluded that the mechanical impact associated with eccentric exercise does not activate blood coagulation.

Differentiation of platelet-leukocyte conjugate formation by short term exercise.

Platelet-leukocyte conjugates are increased in cardiovascular disease, but exercise is also able to trigger platelet-leukocyte formation in healthy subjects. The aim was to investigate the heterogeneity of platelet-leukocyte conjugate formations triggered by short term exercise. 18 healthy non-smokers underwent a 90 second maximal test on a SRM cycle ergometry system and a control experiment. Blood samples were taken after 30 min rest, immediately before and after, 15 min and 1 h after exercise. The different platelet-leukocyte conjugates were detected by flow cytometry via CD45, CD14, CD16, CD41, together with CD62P antibodies for the investigation of platelet activation in the conjugates. In addition, a stimulation of conjugate formation in vitro with 8 microM TRAP-6 was initiated. Immediately after exercise platelet-granulocyte (+24%), and -lymphocyte (+17%) conjugates were increased (p<0.01), while the platelet-monocyte conjugates (+40%) were enhanced (p<0.05) 15 min after exercise. The differentiation after stimulation showed that the regular (CD14(+)16(-); +32%) and mature (CD14(+)16(+); +35%) monocytes were both increased after exercise (p<0.01) but the regular monocytes were preferred (p<0.001) in platelet-monocyte conjugate formation. In addition, these conjugates revealed the highest CD62P expression. Maximal short term exercise is useful for the investigation of platelet-leukocyte formation; e.g., it could be shown, that regular monocytes may be preferred in conjugate formation and that these conjugates revealed the highest CD62P expression.

Platelet activity, reactivity and platelet-leukocyte conjugate formation before and after exhaustive or moderate exercise in patients with IDDM.

Diabetes mellitus alters blood coagulation and platelet function which supports the suggestion that diabetes mellitus is a hypercoagulable state. Firstly the aim of the study was to investigate if differences in platelet activity, reactivity and platelet-leukocyte conjugate (PLC) formation can be observed in subjects with IDDM; secondly, if differences can be seen between the diabetic and control group concerning exercise-induced changes in platelet activation and conjugate formation; and thirdly, if different types of exercise lead to different patterns in platelet activation. Sixteen subjects with IDDM and 16 controls underwent a maximal step test and an endurance test (90% IAT, 45 min). Blood samples were taken after 30 min rest, and immediately and 1 h after completion of exercise. CD62P expression and differentiated platelet-leukocyte conjugates (CD45, CD14, CD41) were detected flow-cytometrically with and without stimulation with TRAP-6. The rest values of the platelet-granulocyte (PGC) and platelet-lymphocyte conjugates (PLyC) were higher (P < 0.05) in the diabetics. After exercise, platelet reactivity (CD62P-TRAP; P < 0.05) but not the activity (CD62P-unstimulated), as well as all different conjugates with or without stimulation were increased (P < 0.05) independently from the group. Differences according to the type of exercise were barely observable. IDDM without vascular complications leads to higher PCG and PLyC at rest and to identical increases in differentiated platelet-leukocyte formation after exercise in comparison with matched controls.

Blood coagulation and fibrinolysis before and after exhaustive exercise in patients with IDDM.

Diabetes mellitus involves changes in haemostasis which leads to the opinion that diabetes mellitus is a hypercoagulable state. However, little is known about the relationship of exercise and haemostasis in diabetics. Therefore, first of all the aim was to investigate if differences in blood coagulation and fibrinolysis can be demonstrated in subjects with insulin-dependent diabetes mellitus (IDDM) compared to controls and secondly, if differences concerning exercise induced changes can be seen in diabetics. 16 moderately fit subjects with IDDM and 16 matched controls underwent a maximal step test. Blood samples were taken after a 30 min rest, immediately and 1h after exercise and in addition after 30 min rest 7 days later at the same time of day. The rest values (mean of the two rest samples) in extrinsic total thrombin potential (TTPex, P=0.049), tPA-activity (P=0.007) were significantly higher and in PAI-1-antigen (P=0.002) -activity (P=0.049) lower in the diabetic group. APTT, PT, TAT (only control), TTPin, tPA-activity and -antigen and PAP were increased immediately and D-dimer (only control) 1 h after exercise, whereas PAI-1-activity and -antigen (only control) decreased immediately or 1 h after exercise (all minimal P<0.05). The increase of tPA-antigen and decrease in PAI-1-antigen after exercise were both lower in the diabetics (P<0.05). IDDM led to higher extrinsic total thrombin and fibrinolytic potential at rest, and reducing the exercise provoked distribution of tPA-antigen and decrease of PAI-1-antigen. Nevertheless a higher thrombotic risk after maximal exercise has not been investigated in young IDDM patients without complications and in good metabolic control.

Paraformaldehyde fixation induces a systematic activation of platelets.

In whole blood flow cytometric platelet assays sample fixation using paraformaldehyde (PFA) is considered very advantageous to prevent spontaneous activation of platelets in vitro. However, fixation is an important variable in activation assays and its influence on platelets is poorly understood. Using a direct immunofluorescence labelling technique and whole blood flow cytometry, the effect of PFA fixation was investigated for 4 different epitopes on platelet surface each of which mirrors a different aspect of platelet activation, namely P-selectin (CD62P), GP IIbIIa complex (CD41), the fibrinogen binding site of the activated GP IIaIIIb complex (PAC-1) and GP Ib-V-IX complex (CD42b). Platelets fixed with PFA (0.5%) before antibody labelling showed significant (P<0.01) increases in mean fluorescence intensity (MFI) of CD62P (1.10 +/- 0.14 vs. 0.94 +/- 0.12 arbitrary units of fluorescence), CD41 (27.3 +/- 6.3 vs. 15.6 +/- 2.1) and PAC-1 (6.21 +/- 1.25 vs. 0.55 +/- 0.12) when compared to unfixed samples. At the same time, MFI of CD42b was reduced from 28.2 +/- 1.6 to 22.6 +/- 2.3 (P<0.01). When fixation was initiated after antibody labelling, we observed less prominent increases in MFI of CD41 (P<0.05) and PAC-1 (P<0.05) while there was no significant difference for CD62P and rather a moderate rise in CD42b than a decrease (P<0.05). Because these alterations cannot be explained by unspecific effects only, it must be concluded that PFA induces a systematic stimulation of platelets. The lowest in vitro platelet activation was found when antibody labelling was started immediately after blood sampling and when samples were analysed within 10 minutes after being stored without fixation of 4 degrees C in the dark.

Blood coagulation and fibrinolysis after long-duration treadmill exercise controlled by individual anaerobic threshold.

For rehabilitation training it is recommended that the intensity of exercise should be clearly below the individual anaerobic threshold (IAT). We investigated blood coagulation, particularly endogenous thrombin potential (ETP) and fibrinolysis following a standardized treadmill (TR) ergometer test at 90% IAT for 60-120 min. Sixteen healthy male non-smokers underwent the TR test. Blood samples were taken after a 30-min rest, immediately after exercise, and 2 h after exercise completion. Extrinsic and intrinsic total (TTP(ex+in)) and endogenous (ETP(ex+in)) thrombin potential, prothrombin fragment 1+2 (F1+2), thrombin-antithrombin complex (TAT), plasmin-alpha2-antiplasmin complex (PAP), D-dimer, tissue plasminogen activator antigen and activity (tPA-AG and tPA-ACT) and plasminogen activator inhibitor type 1 antigen and activity (PAI-1-AG and PAI-1-ACT) were measured. Immediately after TR, F1+2, TAT and TTP(ex+in) were increased ( P<0.05) while ETP(ex+in) remained unchanged. In contrast, PAP, D-dimer, tPA-AG, tPA-ACT ( P<0.05) were distinctly enhanced while PAI-1-ACT was decreased ( P<0.05) immediately after exercise. The changes in tPA-AG, tPA-ACT, and PAI-1-ACT were reversed to nearly baseline while the enhancement in PAP and D-dimer was prolonged by more than 2 h after exercise. Long-duration exercise between 60 and 120 min controlled by IAT (90%) on a TR ergometer only implicates a small increase in thrombin generation markers and total (free and alpha(2)-macroglubulin-bound thrombin), but not in endogenous (free) thrombin potential alone. In contrast, fibrinolysis is distinctly increased after this type of exercise. Endurance exercise with an intensity below 90% IAT and a duration below 2 h generates a more favourable condition for fibrinolysis than for blood coagulation in healthy young subjects. Data are given as mean (SD).

Blood coagulation and fibrinolysis after extreme short-term exercise.

INTRODUCTION: Maximal exercise may be a trigger for cardiovascular events. The aim of the study was to investigate changes in blood coagulation and fibrinolysis following maximal short-term exercises with different durations up to 90 s. METHODS: A total of 15 healthy nonsmokers underwent three isokinetic maximal tests on an SRM cycle ergometry system with durations of 15, 45, and 90 s. Blood samples were taken after a 30-min rest, immediately before and after exercise, 15 min, and 1 h after completion of exercise. For the investigation of blood coagulation, prothrombin fragment 1+2 (F1+2), thrombin-antithrombin III complex (TAT), intrinsic and extrinsic total (TTPin+ex), and endogenous thrombin potential (ETPin+ex) were measured. For testing fibrinolysis, determinations of plasmin-alpha(2)-antiplasmin complex (PAP), tissue-type plasminogen activator (tPA)-antigen, plasminogen activator inhibitor (PAI)-1-antigen and D-dimer were used. RESULTS: Immediately after the exercise tests, only F1+2 (15- and 90-s test) and TTPin (45 and 90 s) showed a moderate increase (p<0.05), while TAT and ETP was unchanged. In contrast, a clear increase in PAP and tPA-antigen already after 15 s maximal exercise in relation to the exercise duration time could be investigated. These effects were not totally reversed to baseline 15 min after exercise; D-dimer and PAI-1-antigen still remained unchanged after these types of exercise. CONCLUSIONS: Maximal short-term exercise does not lead to a relevant activation of blood coagulation in healthy young subjects, it is only slightly altered within the normal range. In contrast, fibrinolysis is clearly activated, and the increase is directly dependent on exercise duration. Additionally, it could be shown for the first time that fibrinolysis is already activated after 15 s maximal exercise duration.

Platelet activity, sensitivity to agonist, and platelet--leukocyte conjugate formation after long-term exercise.

For rehabilitation training it is recommended that the intensity of exercise should be distinctly below the individual anaerobic threshold (IAT). We investigated platelet activity, reactivity and platelet-leukocyte conjugate formation following a stardardized treadmill (TR) ergometer test at 90% IAT for 60-120 min. Seventeen healthy male non-smokers underwent TR. Blood samples were taken after a 30-min rest, immediately after exercise, and 2 h after exercise completion. Platelets were detected flow cytometrically by CD41 in whole blood, activated platelets by CD62P. In addition, stimulation of platelets in vitro with 7.5 microM TRAP-6 was performed. For testing platelet-leukocyte conjugates, antibodies against CD45 and CD41 were used. After TR the percent of non-stimulated CD62P-positive platelets (%PC) remained unchanged (1.65 +/- 0.56 to 1.73 +/- 0.79%PC) (mean +/- SD). In contrast, an increase (P<0.05) from 31.9 +/- 13.5 to 37.4 +/- 15.0 %PC in CD62P, TRAP-6 stimulated and enhanced (P<0.01) platelet-leukocyte conjugates (11.7 +/- 3.7 to 16.1 +/- 6.9, CD41-%PC) after TR were observed. Both changes were independent of thrombin generation measured by F1+2 and TAT, and reversible after 2 h. Long-term exercise (90% IAT) on a treadmill ergometer only leads to a moderate increase of platelet reactivity and platelet-leukocyte conjugates. The determination of platelet-leukocyte conjugates may offer the possibility to detect an early activation stage of platelets in vivo.