René P. Michel

Expression of Endothelin-1 in the Lungs of Patients with Pulmonary Hypertension

BACKGROUND Pulmonary hypertension is characterized by an increase in vascular tone or an abnormal proliferation of muscle cells in the walls of small pulmonary arteries. Endothelin-1 is a potent endothelium-derived vasoconstrictor peptide with important mitogenic properties. It has therefore been suggested that endothelin-1 may contribute to increases in pulmonary arterial tone or smooth-muscle proliferation in patients with pulmonary hypertension. We studied the sites and magnitude of endothelin-1 production in the lungs of patients with various causes of pulmonary hypertension. METHODS We studied the distribution of endothelin-1-like immunoreactivity (by immunocytochemical analysis) and endothelin-1 messenger RNA (by in situ hybridization) in lung specimens from 15 control subjects, 11 patients with plexogenic pulmonary arteriopathy (grades 4 through 6), and 17 patients with secondary pulmonary hypertension and pulmonary arteriopathy of grades 1 through 3. RESULTS In the controls, endothelin-1-like immunoreactivity was rarely seen in vascular endothelial cells. In the patients with pulmonary hypertension, endothelin-1-like immunoreactivity was abundant, predominantly in endothelial cells of pulmonary arteries with medial thickening and intimal fibrosis. Likewise, endothelin-1 messenger RNA was increased in the patients with pulmonary hypertension and was expressed primarily at sites of endothelin-1-like immunoreactivity. There was a strong correlation between the intensity of endothelin-1-like immunoreactivity and pulmonary vascular resistance in the patients with plexogenic pulmonary arteriopathy, but not in those with secondary pulmonary hypertension. CONCLUSIONS Pulmonary hypertension is associated with the increased expression of endothelin-1 in vascular endothelial cells, suggesting that the local production of endothelin-1 may contribute to the vascular abnormalities associated with this disorder.

Application of the Cavalieri principle and vertical sections method to lung: estimation of volume and pleural surface area

A practical methodology is proposed for the stereological analysis of lung and other organs using recently developed unbiased procedures. This study concentrates on the unbiased estimation of lung volume using Cavalieris principle compared with the fluid displacement method and measurement of pleural surface area using vertical sections. Furthermore, the proposed design, in addition to the sampling of extensive slices for the initial steps, also allows sampling of vertical sections for light and electron‐microscopical stereology.

Hypertonic saline resuscitation attenuates neutrophil lung sequestration and transmigration by diminishing leukocyte-endothelial interactions in a two-hit model of hemorrhagic shock and infection.

BACKGROUND Hypertonic saline (HTS) attenuates polymorphonuclear neutrophil (PMN)-mediated tissue injury after hemorrhagic shock. We hypothesized that HTS resuscitation reduces early in vivo endothelial cell (EC)-PMN interactions and late lung PMN sequestration in a two-hit model of hemorrhagic shock followed by mimicked infection. METHODS Thirty-two mice were hemorrhaged (40 mm Hg) for 60 minutes and then given intratracheal lipopolysaccharide (10 microg) 1 hour after resuscitation with shed blood and either HTS (4 mL/kg 7.5% NaCl) or Ringers lactate (RL) (twice shed blood volume). Eleven controls were not manipulated. Cremaster intravital microscopy quantified 5-hour EC-PMN adherence, myeloperoxidase assay assessed lung PMN content (2 1/2 and 24 hours), and lung histology determined 24-hour PMN transmigration. RESULTS Compared with RL, HTS animals displayed 55% less 5-hour EC-PMN adherence (p = 0.01), 61% lower 24-hour lung myeloperoxidase ( p= 0.007), and 57% lower mean 24-hour lung histologic score ( p= 0.027). CONCLUSION Compared with RL, HTS resuscitation attenuates early EC-PMN adhesion and late lung PMN accumulation in hemorrhagic shock followed by inflammation. HTS resuscitation may attenuate PMN-mediated organ damage.

Scoring of p53, VEGF, Bcl-2 and APAF-1 immunohistochemistry and interobserver reliability in colorectal cancer.

Molecular tumor markers are often studied in colorectal cancer using immunohistochemistry to determine their prognostic or predictive value. Protein expression is typically assigned a ‘positive’ score based on a predetermined cutoff. A semiquantitative scoring method that evaluates the percentage of positive tumor cells (0–100%) may provide a better understanding of the prognostic or predictive significance of these markers. The aim of this study was to assess and compare the interobserver agreement of immunohistochemistry scores using a percentage scoring method and three categorical scoring systems. Immunohistochemistry for p53, Bcl-2, vascular endothelial growth factor (VEGF) and apoptotic protease activating factor-1 (APAF-1) was performed on 87 tumor biopsies from patients with rectal carcinoma and scored independently by four pathologists as the percentage of positive tumor cells. Interobserver agreement was assessed by the intraclass correlation coefficient. The intraclass correlation coefficients for p53 and VEGF (>0.6) indicate substantial agreement between observers. The distribution of Bcl-2 and APAF-1 scores in addition to weaker interobserver agreement by percentage scoring suggest that this approach may not be appropriate for these proteins. In conclusion, p53 and VEGF protein expression assessed by immunohistochemistry in colorectal cancer and scored as a percentage of positive tumor cells may be a viable alternative scoring method.

Combined analysis of VEGF and EGFR predicts complete tumour response in rectal cancer treated with preoperative radiotherapy

The ability to predict complete pathologic response or sensitivity to radiation before treatment would have a significant impact on the selection of patients for preoperative radiotherapy or chemo-radiation therapy schedules. The aim of this study was to determine the value of epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), p53, Bcl-2 and apoptosis protease-activating factor-1 (APAF-1) as predictors of complete pathologic tumour regression in patients undergoing preoperative radiotherapy for advanced rectal cancer. Pretreatment tumour biopsies from predominantly cT3 patients undergoing a preoperative high-dose-rate brachytherapy protocol were immunostained for EGFR, VEGF, p53, Bcl-2 and APAF-1. Immunoreactivity was evaluated by three pathologists. Cut-off scores for tumour marker positivity were obtained by receiver-operating characteristic (ROC) curve analysis. The association of marker expression with complete pathologic response was analysed in univariate and multivariable analysis. Multi-marker phenotypes of the independent protein markers were evaluated. In multivariable analysis, loss of VEGF (P-value=0.009; odds ratio (OR) (95% CI)=0.24 (0.08–0.69)) and positive EGFR (P-value=0.01; OR (95% CI)=3.82 (1.37–10.6)) both demonstrated independent predictive value for complete pathologic response. The odds of complete response were 12.8 for the multi-marker combination of VEGF-negative and EGFR-positive tumours. Of the 34 EGFR-negative- and VEGF-positive cases, 32 (94.1%) had no complete pathologic response. The combined analysis of VEGF and EGFR is predictive of complete pathologic response in patients undergoing preoperative radiotherapy. In addition, the findings of this study have identified a subgroup of simultaneous EGFR-negative and VEGF-positive patients who are highly resistant to radiotherapy and should perhaps be considered candidates for innovative neoadjuvant combined modalities.

Mechanisms for the diminished neutrophil exudation to secondary inflammatory sites in infected patients with a systemic inflammatory response (sepsis).

OBJECTIVE To determine the mechanism for the reduced polymorphonuclear neutrophil exudation to secondary inflammatory sites in critically ill patients with infection and systemic inflammatory response (sepsis). DESIGN Prospective cohort study. SETTING Research laboratory and integrated intensive care unit of a tertiary care university-affiliated teaching hospital. PATIENTS Healthy subjects or critically ill patients with confirmed infection and a systemic inflammatory response (septic patients). MEASUREMENTS AND MAIN RESULTS We found that polymorphonuclear neutrophil delivery to a secondary inflammatory site (skin window blisters) is reduced by >70% in humans with sepsis, defined as serious infection and a systemic inflammatory response compared with healthy controls. The expression of the endothelial adhesion molecules intercellular adhesion molecule-1, E-selectin and P-selectin in microvessels from skin biopsies was comparable in the two study groups. Also, CD11a and CD11b levels were equal in circulating polymorphonuclear neutrophils (PMNs) from both study groups. Both adhesion molecules were markedly and equally up-regulated during exudation. Circulating PMNs from septic patients showed marked shedding of L-selectin compared to those of healthy controls, with a corresponding increase in their plasma L-selectin levels. An increased concentration gradient between plasma and exudate fluid was found for tumor necrosis factor-alpha and interleukin-8 in septic patients, but not for C5a. The phagocytic and bactericidal capacity of septic patient circulating PMNs was higher then in healthy control patients, but these differences were lost after exudation. There were no major differences in oxidative burst or intracellular calcium flux of circulating PMNs from the two study groups. Polymorphonuclear neutrophil exudation primed both responses to different extents. CONCLUSIONS Septic patients deliver fewer PMNs to secondary inflammatory sites. In addition, neutrophil exudation results in loss of the small priming effect for phagocytosis and bactericidal function induced by sepsis. Failure to produce a gradient to C5a and intravascular shedding of L-selectin may be responsible for this sepsis-induced reduction in neutrophil exudation to secondary inflammatory sites.

Zonulae occludentes in alveolar epithelium and capillary endothelium of dog lungs studied with the freeze—fracture technique

Intercellular junctions both in the alveolar epithelium and in the pulmonary capillary endothelium of the alveolar—capillary wall of dog lungs were observed in the electron microscope using the freeze—fracture technique. Only zonulae occludentes (tight junctions) were observed between cells in both alveolar epithelium and capillary endothelium and no other type of junctions were found. The zonula occludens between epithelial cells was a well-developed, bubble-like assembly of compartments made up of interconnecting junctional strands. The endothelial junction, on the other hand, was rather poorly developed with an approximately parallel arrangement of fewer strands with less frequent interconnections. The number of strands and the depth of the junction were measured on 20 alveolar epithelial and 45 capillary endothelial junctions. In the epithelium the mean depth of the junction was 0.261 ± 0.023 (SE) μ m and the number of junctional strands varied from 2–3 to 4–10. Mean depth of the endothelial junction was 0.166 ± 0.011 (SE) μ m, the number of strands varied from 1–2 to 3–4 with the most frequent occurrence of 2–3 strands. These observations suggest that the alveolar epithelium is “tight” while capillary endothelium is “leaky.” The fine structure of the zonulae occludentes in the pulmonary capillary endothelium is in favor of the hypothesis that they are the site of the small pore system which has been proposed by physiologists.

Endothelin-1-Like Immunoreactivity in Postobstructive Pulmonary Vasculopathy

Postobstructive pulmonary vasculopathy (POPV) is produced by chronic ligation of one pulmonary artery and results in bronchial collateral vessel proliferation and pulmonary arterial abnormalities. The role of endothelin-1 (ET-1), a potent vasoconstrictor peptide, was investigated in a canine model of POPV using radioimmunoassay and immunohistochemistry. The left main pulmonary artery of 9 dogs was ligated, and 3 months (n = 3) and 15 months (n = 6) later, ET-1 levels were measured by radioimmunoassay of plasma samples from left and right pulmonary arteries and veins. In addition, tissues from control and ligated lungs were fixed in Bouins solution embedded in paraffin and stained with antiserum to ET-1. Plasma ET-1 levels distal to the ligation were not different from those of the control pulmonary artery (1.7 pg/ml in the ligated lungs vs. 1.4 pg/ml in the controls; NS). ET-1-like immunoreactivity was localized mainly to the epithelium in both control and ligated lungs, but the pulmonary arteries and the new bronchial vessels stained more intensely in the ligated lungs. The sections immunostained with ET-1 antiserum preabsorbed with synthetic ET-1 did not stain. These results suggest that ET-1 may play a role in the bronchial neovascularization and the pulmonary arterial thickening characteristic of POPV.

Mineral dusts in lungs with scar or scar cancer

Five lungs with small scars and five lungs with small scar associated cancers, were studied by light and scanning electron microscopy and x‐ray energy dispersive spectrometry. Six hundred particles were photographed and their physical and chemical properties analyzed from scar, cancer, or normal alveolar tissue on carbon planchet‐mounted, deparaffinized and low temperature‐ashed sections. Amosite/crocidolite fibers were accumulated only in one cancerous lung. All other lungs shared similar types of mineral particles. The lungs with noncancer scars, however, showed an increase in the ratio of aluminum and calcium salts (non‐silicates), while the lungs with scar cances had a higher ratio of silicates. These patterns of particle distribution were similar in different areas of the same lung, despite the fact that particles accumulated heavily in the scar and scarcely in the normal alveolus. Although the mechanism is unclear, these results are consistent with the possibility that the pattern of mineral particle distribution in a lung may influence the formation of cancer in a scar.

Immunoblastic lymphosarcoma: a light, immunofluorescence, and electron microscopic study.

Immunoblastic lymphosarcoma (ILS) is a newly recognized malignant lymphoreticular neoplasm and is included in the recent W.H.O. classification of lymphomas. This report concerns six cases of ILS studied by light, immunofluorescence (IF), and electron microscopy (EM). Four patients were female and all except one were over 50 years of age. Four patients had some immunological abnormality. Light microscopy showed a monomorphic population of immunoblasts with pyroninophilic cytoplasm and variable plasmacytoid differentiation. Intracytoplasmic IgG was demonstrated by IF in four cases, and IgA in one. Large lymphoid cells with varying proportions of polysomes, rough endoplasmic reticulum, and Golgi apparatus were seen by EM in four cases. Mean survival was 4.8 months in five cases; death in four was due to disseminated ILS. We concluded that our cases of ILS are of B cell origin, are often associated with immunological abnormalities, and carry a poor prognosis. Immunofluorescence and EM are helpful in its diagnosis.