John U. Balis

Human umbilical cord blood mononuclear cells for the treatment of acute myocardial infarction.

Cell transplantation is a new treatment to improve cardiac function in hearts that have been damaged by myocardial infarction. We have investigated the use of human umbilical cord blood mononuclear progenitor cells (HUCBC) for the treatment of acute myocardial infarction. The control group consisted of 24 normal rats with no interventions. The infarct + vehicle group consisted of 33 rats that underwent left anterior descending coronary artery (LAD) ligation and after 1 h were given Isolyte in the border of the infarction. The infarct + HUCBC group consisted of 38 rats that underwent LAD ligation and after 1 h were given 106 HUCBC in Isolyte directly into the infarct border. Immunosuppression was not given to any rat. Measurements of left ventricular (LV) ejection fraction, LV pressure, dP/dt, and infarct size were determined at baseline and 1, 2, 3, and 4 months. The ejection fraction in the controls decreased from 88 ± 3% to 78 ± 4% at 4 months (p = 0.03) as a result of normal aging. Following infarction in the infarct + vehicle group, the ejection fraction decreased from 87 ± 4% to 51 ± 3% between 1 and 4 months (p < 0.01). In contrast, the ejection fraction of the infarcted + HUCBC-treated rat hearts decreased from 87 ± 4% to 63 ± 3% at 1 month, but progressively increased to 69 ± 6% at 3 and 4 months, which was different from infarct + vehicle group rats (p < 0.02) but similar to the controls. At 4 months, anteroseptal wall thickening in infarct + HUCBC group was 57.9 ± 11.6%, which was nearly identical to the control anteroseptal thickening of 59.2 ± 8.9%, but was significantly greater than the infarct + vehicle group, which was 27.8 ± 7% (p < 0.02). dP/dtmax increased by 130% in controls with 5.0 μg of phenylephrine (PE)/min (p < 0.001). In the infarct + vehicle group, dP/dtmax increased by 91% with PE (p = 0.01). In contrast, in the infarct + HUCBC group, dP/dtmax increased with PE by 182% (p < 0.001), which was significantly greater than the increase in dP/dtmax in the infarct + vehicle group (p = 0.03) and similar to the increase in the controls. Infarct sizes in the infarct + HUCBC group were smaller than the infarct + vehicle group and averaged 3.0 ± 2.8% for the infarct + HUCBC group versus 22.1 ± 5.6% for infarct + vehicle group at 3 months (p < 0.01); at 4 months they averaged 9.2 ± 2.0% for infarct + HUCBC group versus 40.0 ± 9.2% for the infarct + vehicle group (p < 0.001). The present experiments demonstrate that HUCBC substantially reduce infarction size in rats without requirements for immunosuppression. As a consequence, LV function measurements, determined by LV ejection fraction, wall thickening, and dP/dt, are significantly greater than the same measurements in rats with untreated infarctions.

Biochemical composition of adult human lung surfactant.

Surfactant was isolated from minced adult human lung tissues by repetitive centrifugation on NaBr density gradients. The surface active fraction contained 12 mg phospholipid per mg of protein. Eighty percent of the phospholipid was phosphatidylcholine and 9% was phosphatidylglycerol. The phosphatidylcholines, 55% of which were disaturated, contained more than 70% palmitic acid. In contrast, phosphatidylglycerol contained 22% palmitic acid and 52% oleic acid, suggesting that the importance of phosphatidylglycerol in surfactant function relates to its acidic head group. The most abundant proteins in human surfactant were high molecular weight (>400,000) glycoproteins which on reduction with dithiothreitol yielded peptides of 34,000 daltons. Antibodies to the high molecular weight proteins were prepared in rabbits and using immunoperoxidase methods were shown to stain alveolar type II cells and the alveolar and bronchial surfaces, but no other human tissues. A low molecular weight protein was also present in lung surfactant and was most apparent in surfactant subfractions which had higher phospholipid to protein ratios. The results of this study provide a basis to further investigate the role of normal and altered surfactant constituents in disease states of the human lung.

Human umbilical cord blood progenitor cells are attracted to infarcted myocardium and significantly reduce myocardial infarction size.

We are investigating the effects of human umbilical cord blood mononuclear progenitor cells (HUCBC) for the treatment of acute myocardial infarction because human cord blood is a readily available and an abundant source of primitive cells that may be beneficial in myocardial repair. However, there is currently no scientific consensus on precisely when to inject stem/progenitor cells for the optimal treatment of acute myocardial infarction. We used an in vitro assay to determine the attraction of infarcted rat myocardium at 1, 2, 2.5, 3, 6, 12, 24, 48, and 96 h after left anterior descending coronary artery (LAD) occlusion from 45 rats for HUCBC in order to determine the optimal time to transplant HUCBC after myocardial infarction. Our assay is based on the migration of fluorescent DAPI-labeled HUCBC from wells in an upper chamber of a modified Boyden apparatus through a semiporous polycarbonate membrane into wells in a lower chamber that contain either normal or infarcted myocardium. DAPI-labeled HUCBC (100,000) were placed in each of the separate wells above the membrane that corresponded to normal or infarct homogenate in the lower wells. The greatest HUCBC migration to infarcted myocardium occurred at 2 h and 24 h after LAD occlusion in comparison with normal controls. A total of 76,331 ± 3384 HUCBC migrated to infarcted myocardium at 2 h and 69,911 ±2732 at 24 h after LAD occlusion (both p < 0.001) and significantly exceeded HUCBC migration to normal heart homogenate. The HUCBC migration remained greatest at 2 and 24 h after LAD occlusion when the number of migrated cells was adjusted for the size of each myocardial infarction. Injection of 106 HUCBC in saline into infarcted myocardium of non immunosuppressed rats within 2 h (n = 10) or at 24 h (n = 5) after LAD occlusion resulted in infarction sizes 1 month later of 6.4 ± 0.01% and 8.4 ± 0.02% of the total left ventricular muscle area, respectively, in comparison with infarction sizes of 24.5 ± 0.02% (n = 10) in infarcted rat hearts treated with only saline (p < 0.005). Acute myocardial infarction in rats treated with only saline increased the myocardial concentration of tumor necrosis factor-α (TNF-α) from 6.9 ± 0.8% to 51.3 ± 4.6%, monocyte/macrophage chemoattractant protein (MCP-1) from 10.5 ± 1.1% to 39.2 ± 2.0%, monocyte inflammatory protein (MIP) from 10.6± 1.6% to 23.1 ± 1.5%, and interferon-? (INF-?) from 8.9 ± 0.3% to 25.0 ± 1.7% between 2 and 12 h after coronary occlusion in comparison with known controls (all p < 0.001). In contrast, the myocardial concentrations of these cytokines in rat hearts treated with HUCBC did not significantly change from the controls at 2, 6, 12, and 24 h after coronary occlusion. The present investigations suggest that infarcted myocardium significantly attracts HUCBC, that HUCBC can substantially reduce myocardial infarction size, and that HUCBC can limit the expression of TNF-α, MCP-1, MIP, and INF-? in acutely infarcted myocardium.

Lung surfactant phospholipids in different animal species

A comparative study of adult mammalian lung surfactants was undertaken to determine which animal species might serve as appropriate models for surfactant alterations in human lung diseases. Phosphatidylcholine (PC) comprised 80–87% of the phospholipid and contained more than 65% palmitic acid in all species studied. Phosphatidylglycerol (PG) was found to vary significantly in fatty acid composition among the species. Rabbit, dog and rat surfactant PG contained 50–60% palmitic acid, while human and cat surfactant contained much lower levels of saturated fatty acids. Both the PC and PG of all species contained 2 positional isomers of fatty acids with 16 carbons and one double bond, but the relative amounts of the unusual isomer, 16∶1Δ7, and palmitoleic, acid, 16∶1Δ9, varied among the different animal species. Only cat and dog surfactant phospholipids contained 18∶1Δ5. Cat surfactant phospholipids also differed by the absence of 20∶4 and the presence of small amounts of several 20- and 22-carbon fatty acids. These results explain some discrepancies found in the literature concerning surfactant composition and delineate limiting factors in extrapolating results from animal studies for the evaluation of maturation and pathological alterations in human surfactant.

Pregnancy-induced hypertension: Development of a model in the pregnant primate (Papio anubis)

Experiments were performed on two groups of pregnant baboons. In the experimental group, the subrenal aortic blood flow was reduced by 58% of its original value at 100 days of gestational age. In the control group, the blood flow was measured but not restricted. In the experimental group fetal death occurred in three of 12 animals following the use of a single left-flank incision to approach both the renal artery and the abdominal aorta. In the control group, pregnancies in eight of nine animals went successfully to 165 days. In the experimental group the development of hypertension, a decrease in plasma renin activity, an increase in renal resistance, an increase in serum uric acid, and the development of glomerular changes consistent with those seen in human pregnancy-induced hypertension were noted. These studies demonstrate that pregnancy-induced hypertension can be produced experimentally in a pregnant baboon, and this model should prove useful in expediting studies on the pathophysiologic features and treatment of this condition.

PULMONARY TOXICITY OF SIMULATED LUNAR AND MARTIAN DUSTS IN MICE: I. HISTOPATHOLOGY 7 AND 90 DAYS AFTER INTRATRACHEAL INSTILLATION

NASA is contemplating sending humans to Mars and to the moon for further exploration. Volcanic ashes from Arizona and Hawaii with mineral properties similar to those of lunar and Martian soils, respectively, are used to simulate lunar and Martian environments for instrument testing. Martian soil is highly oxidative; this property is not found in Earths volcanic ashes. NASA is concerned about the health risk from potential exposure of workers in the test facilities. Fine lunar soil simulant (LSS), Martian soil simulant (MSS), titanium dioxide, or quartz in saline was intratracheally instilled into groups of 4 mice (C57BL/6J) at 0.1 mg/mouse (low dose, LD) or 1 mg/mouse (high dose, HD). Separate groups of mice were exposed to ozone (0.5 ppm for 3 h) prior to MSS instillation. Lungs were harvested for histopathological examination 7 or 90 days after the single dust treatment. The lungs of the LSS-LD groups showed no evidence of inflammation, edema, or fibrosis; clumps of particles and an increased number of macrophages were visible after 7 days but not 90 days. In the LSS-HD-7d group, the lungs showed mild to moderate alveolitis, and perivascular and peribronchiolar inflammation. The LSS-HD-90d group showed signs of mild chronic pulmonary inflammation, septal thickening, and some fibrosis. Foci of particle-laden macrophages (PLMs) were still visible. Lung lesions in the MSS-LD-7d group were similar to those observed in the LSS-HD-7d group. The MSS-LD-90d group had PLMs and scattered foci of mild fibrosis in the lungs. The MSS-HD-7d group showed large foci of PLMs, intra-alveolar debris, mild-to-moderate focal alveolitis, and perivascular and peribronchiolar inflammation. The MSS-HD-90d group showed focal chronic mild-to-moderate alveolitis and fibrosis. The findings in the O 3 -MSS-HD-90d group included widespread intra-alveolar debris, focal moderate alveolitis, and fibrosis. Lung lesions in the MSS groups were more severe with the ozone pretreatment. The effects of O 3 and MSS coexposure appeared to be more than additive. Results for the TiO 2 and quartz controls were consistent with the known pulmonary toxicity of these compounds. The overall severity of lung injury was TiO 2 < LSS < MSS < O 3 + MSS < quartz. Except for TiO 2, the increased duration of dust presence in the lung from 7 to 90 days transformed the acute inflammatory response to a chronic inflammatory lesion. This study showed that LSS and MSS are more hazardous in the lungs than nuisance dusts.

Ultrastructural and immunohistochemical studies of bronchiolo‐alveolar carcinoma

A detailed ultrastructural study was made of seven cases of bronchiolo‐alveolar carcinoma, and the findings were correlated with histochemical and immunohistochemical data. By electron microscopic examination all seven tumors displayed glandular differentiation, manifested by the presence of microvilli and intercellular junctions, with or without mucin production. Variable proportions of tumor cells retained ultrastructural characteristics of alveolar type II cells and Clara cells. In addition, some tumor cells revealed desmosomes and tonofilaments consistent with squamous differentiation. Immunohistochemical evaluation was carried out using a peroxidase—antiperoxidase technique and specific antibodies against surfactant high molecular weight glycoproteins, keratin proteins, IgA + secretory piece, carcinoembryonic antigen (CEA), human chorionic gonadotropin (HCG), and alpha‐fetoprotein (AFP). Four tumors with type II cell‐like differentiation stained with anti‐surfactant glycoprotein sera. All seven tumors stained focally with anti‐keratin and IgA + anti‐surfactant piece antibodies, and diffusely with CEA. These tumors failed to stain with antisera against HCG and AFP. It is concluded that bronciolo‐alveolar carcinomas are primarily composed of cells with alveolar and bronchiolar cell differentiation. Adequate criteria were established for ultrastructural identification of tumor cells with differentiation to type II alveolar cell or Clara cell. Moreover, the findings of this study indicate that the surfactant glycoprotein marker, when present in a given tumor either diffusely or focally, is diagnostic of bronchiolo‐alveolar carcinoma.

Acute ozone-induced lung injury in rats: structural-functional relationships of developing alveolar edema.

As part of a study on the effects of acute ozone stress on the lung surfactant system, we correlated morphometric, biochemical, and functional indices of lung injury using male rats exposed to 3 ppm ozone for 1, 2, 4, and 8 hr. Evaluation of lung mechanics, using the Pulmonary Evaluation and Diagnostic Laboratory System, revealed a significant decrease in dynamic lung compliance (ml/cmH2O/kg) from a control value of 0.84 +/- 0.02 (SEM) to 0.72 +/- 0.04 and 0.57 +/- 0.06 at 4 and 8 hr, respectively. At 2 hr there was a transient increase in PaO2 to 116 torr (control = 92 torr) followed by a decrease at 4 hr (65 torr) and 8 hr (55 torr). Morphometry of lung tissue, fixed by perfusion of fixative via the pulmonary artery at 12 cm H2O airway distending pressure, demonstrated an increase in the area of the intravascular compartment at 8 hr, in association with a 65 and 39% replacement of the alveolar area by fluid in ventral and dorsal lung regions, respectively. There was a positive correlation (r = 0.966) between alveolar edema and transudated proteins in lavage fluid. A stepwise multiple regression model, with edema as the dependent variable, suggested that pulmonary vasodilatation, hypoxemia, and depletion of surfactant tubular myelin in lavage fluid were indices for predicting alveolar edema. In a second model, with lavage protein concentration as the dependent variable, decreasing dynamic compliance and hypoxemia were predictors of progressive, intraalveolar transudation of plasma proteins. The above structural-functional relationships support the concept that ozone-induced high-protein alveolar edema is pathogenetically linked to pulmonary hyperemia, deficiency of surfactant tubular myelin, and associated lung dysfunctions.

Surfactant-associated glycoproteins accumulate in alveolar cells and secretions during reparative stage of hyaline membrane disease

Surfactant-associated (SA) glycoproteins are lung-specific proteins produced in the human lung by alveolar type II cells and Clara cells. The distribution of these proteins was studied immunohistochemically in lung tissue obtained postmortem from 12 stillborn fetuses and 49 infants with hyaline membrane disease (HMD). By 21 weeks of gestation, SA glycoproteins were detected in the fetal alveolar epithelium and within Clara cells. The staining increased in intensity and extent with advancing gestational age. Infants with HMD who survived less than 48 hours did not generally exhibit stainable material either within type II cells or secretions, but staining was often noted in Clara cells as well as focally beneath hyaline membranes. In infants surviving more than 48 hours, intense staining of hyaline membranes, alveolar secretions, proliferating alveolar type II cells, and Clara cells was evident. Immunoreactivity was intense in hypertrophic type II cells that formed a continuous alveolar epithelial lining in lungs with bronchopulmonary dysplasia. Included in the population of infants with HMD were 15 infants with pulmonary hypoplasia. The lungs of these infants showed minimal staining for SA glycoproteins regardless of postnatal survival time. The results provide an immunomorphologic basis for defining normal and abnormal lung maturation. They also indicate that enhanced SA glycoprotein production is a sustained response of regenerating and hypertrophic type II cells in premature infants.

Ozone-induced alterations of lamellar body lipid and protein during alveolar injury and repair

Alveolar Type II cells in the rat respond to severe, acute ozone injury (3 ppm ozone for eight hours) by increasing their intracellular pool of surfactant; however, the newly stored surfactant is abnormal in composition. Lamellar bodies isolated between 24 and 96 hours after ozone exposure contained significantly more cholesterol in relation to phosphatidylcholine than did controls. By contrast, the cholesterol content of surfactant isolated from alveolar lavage remained unchanged throughout an 8-day post-ozone period. The total protein content of lamellar bodies in relation to phosphatidylcholine was significantly decreased at 24 and 48 hours post-ozone. Analysis of lamellar body proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the amount of a 14 kDa proteolipid was greatly reduced at the end of the eight-hour ozone exposure and remained low for at least 48 hours. This proteolipid appeared to be a specific lamellar body component since it was not detected in extracellular surfactant. The findings indicate that oxidative alveolar stress initiates characteristic alterations in both lipid and protein constituents of stored surfactant, without perturbation in the composition of extracellular surfactant.