Bernard P. Lane

Regeneration of Rat Tracheal Epithelium After Mechanical Injury. I. The Relationship Between Mitotic Activity and Cellular Differentiation

Summary The time of appearance of cytologic specialization in cells of regenerating rat tracheal epithelium was compared to the time of maximal mitotic response. Trauma sufficient to lethally injure most cells reaching the surface but sparing most basal cells resulted in a peak of mitotic activity from the 26th to the 30th hour following injury. The cells in the hyperplastic regenerating epithelium at the peak of mitosis had high nuclear-cytoplasmic ratios with little cytoplasmic or surface specialization. Ciliogenesis and mucinogen formation were not. seen in most cells until the 60th hr after the injury and there was little cell division at that time or later. Since cessation of mitosis does not follow or even coincide with the replenishment of the ciliated or goblet cell populations, the data does not support the hypothesis that control of cell division resides in the elaboration or release of a suppressor substance by the mature cells.

Characterization of a bovine synovial fluid lubricating factor III. The interaction with hyaluronic acid

Although hyaluronate is not a boundary lubricant in cartilaginous and latex:glass bearings, a distinct interaction with purified synovial lubricating factor (PSLF) was demonstrated by three means: 1) enhancement of lubricating ability in an artificial test system; 2) viscometry; 3) electron microscopy. The interaction was of a physical rather than a specific chemical type; it varied with the degree of purification of PSLF and of hyaluronate. The interaction accounts for retention of the relatively small PSLF molecule (approximately 280 kDa) with the synovial mucin on a 0.22 microns filter. The data provide evidence that hyaluronate and PSLF act synergistically in the boundary lubricating activity of animal joints.

Gallbladder dyskinesia in chronic acalculous cholecystitis

To test the hypothesis that there is an early stage of cholesterol gallstone formation in man characterized by symptoms of chronic cholecystitis, poor gallbladder emptying, and biliary cholesterol crystals, we studied cholecystokinin-stimulated gallbladder emptying by DISIDA scintigraphy and examined bile for cholesterol crystals in symptomatic patients with normal oral cholecystography and gallbladder sonography. Of 36 patients studied, 16 had biliary cholesterol crystals; their mean 30-min gallbladder ejection fraction was 25.9±14.8%. Among the 20 patients without crystals, the mean ejection fraction was 60.3±23.3%. Fifteen patients, 11 with crystals and four without, had cholecystectomy because of persistent symptoms. All with crystals preoperatively and three without had chronic cholecystitis histologically. One patient without crystals had normal histology. We conclude that poor gallbladder contractility, well-established as an etiologic factor in animal models of cholesterol cholelithiasis, is now linked to acalculous cholecystitis, an early stage of human cholesterol cholelithiasis.

Fcγ Receptor Mediated Phagocytosis by Human Neutrophil Cytoplasts

Neutrophils utilize Fcγ Receptors (FcγR) to bind and internalize antibody coated cells or immune complexes. We have compared ultrastructurally FcγR-mediated phagocytosis by neutrophil cytoplasts (enucleated and granule-free neutrophils) prepared either by treatment with cytochalasin B (CB-cytoplasts) followed by ultracentrifugation or by brief heating at 45°C in suspension followed by ultracentrifugation. The phagocytosis of antibody-coated erythrocytes (EIgG) or the internalization of crosslinked IgG complexes by cytoplasts prepared by brief heating was comparable to that of untreated neutrophils. In contrast, CB-cytoplasts bound but failed to internalize EIgG or crosslinked IgG complexes. Comparison of these two methods for the preparation of neutrophil cytoplasts may assist in clarifying the signal transduction requirements involved following ligand binding to FcγR which initiate phagocytosis.

Predictors of proximal neoplasia in patients without distal adenomatous pathology

BACKGROUND:Previous colorectal cancer screening studies have observed that some patients may have advanced proximal neoplasia without distal findings. Since these studies have included only gender, age, and family history as risk factors, they are limited in their ability to identify predictors of isolated proximal neoplasia.METHODS:Data were collected from the charts of 1,988 patients who presented for colonoscopy. Information gathered included endoscopic findings, histology, known risk factors for colorectal neoplasia, and smoking pattern. Our main outcome was the presence of proximal adenomatous neoplasia in patients who had no distal adenomas. We defined significant neoplasia as adenocarcinoma, high-grade dysplasia, villous polyps, adenomas 1 cm or greater or more than two adenomas of any size.RESULTS:Fifty-five patients had isolated significant proximal neoplasia that would have been missed on a flexible sigmoidoscopy. While patients older than 60 yr had a greater risk for this neoplasia (odds ratio = 3.01: 95% CI = 1.66–4.23; p < 0.001), those who took a daily aspirin had a reduced risk (OR = 0.60; 95% CI = 0.30–0.88; p < 0.05). A family history of colorectal cancer increased the patients risk of having any adenomas (OR = 2.01; 95% CI = 1.33–3.40; p < 0.01) or villous tissue (OR = 2.03; 95% CI = 1.27–3.51; p < 0.05) in the proximal colon without distal findings. Smoking was associated with an increased risk of large (> 1 cm) isolated proximal tubular polyps (OR = 2.71; 95% CI = 1.64–4.46; p < 0.01) as well as isolated significant proximal neoplasia (OR = 2.30; 95% CI = 1.59–3.31; p < 0.01).CONCLUSIONS:Age greater than 60 yr, a history of at least 10 pack-years of smoking, and a family history of colorectal cancer increased the risk of finding significant proximal polyps in patients without distal pathology.

Role of steroids in secretion—modulating effect of triamcinolone and estradiol on protein synthesis and secretion from the rat exocrine pancreas

Following adrenalectomy of male rats, or adrenalectomy and ovariectomy of females, there was marked depletion of zymogen granules in acinar cells of the pancreas. Within 9 h after treatment with either triamcinolone or 17 beta-estradiol, complete restoration of these secretory vesicles was observed. This repletion was not inhibited by actinomycin-D. Supernatant fractions (100,000 g, 60 min) of rat pancreas, from both normal and surgically altered animals, contained proteins that bound [3H]-triamcinolone and [3H]-estradiol, suggesting that the action of these hormones is exerted directly on the pancreas. Binding of both steroid hormones required the presence of an additional coligand referred to as accessory factor. In addition, the binding proteins for [3H]-triamcinolone and [3H]-estradiol eluted in similar positions after Sephadex G-200 and CM Affi-gel Blue chromatography. It is uncertain, however, whether a single protein binds both steroid hormones since they had different binding isotherms. Scatchard analysis of binding of [3H]-estradiol yielded a single straight line of negative slope from which it was calculated that there were about 4.4 pmol of binding sites per mg protein, having an average apparent Kd of about 5 X 10(-8) M. Similar analysis of the data for [3H]-triamcinolone yielded a straight line of zero slope indicating nonsaturable binding of hormone at concentrations as high as 10 microM. Since both [14C]-L-leucine incorporation into protein and amylase secretion were affected markedly by the steroid-hormonal status of the animal, it is presumed that steroid-bound complexes in acinar cells of the pancreas modulate synthesis and secretion of protein.

Identification of contractile proteins in basal bodies of ciliated tracheal epithelial cells.

To determine the molecular composition of the components of basal bodies and the interbasal body apparatus of ciliated cells in rat tracheal epithelium, we used rabbit anti-actin, anti-alpha-actinin, anti-tropomyosin, and anti-myosin as primary antisera applied to the tissue in an indirect immunoperoxidase technique. The antisera was proven to be monospecific by elution of antibody after affinity chromatography. Sheep anti-rabbit immunoglobulin Fab fragments coupled to peroxidase were used for ultrastructural localization of the bound rabbit antibody. Antibodies against alpha-actinin were demonstrated around peripheral microtubules of cilia and linking these microtubules to central doublet and plasma membrane. Alpha-actinin was also shown in the basal foot processes. Anti-actin antibodies were associated with microtubules of the cilium and basal bodies, except in the region of the ciliary necklace. The antibodies directed against actin also had affinity for rootlets, basal foot processes, and communications between basal bodies and foot processes. Both anti-myosin and anti-tropomyosin antibodies were localized to part of the region of the constriction of the cilium, to the central basal density and the outer surfaces of basal body microtubules, and to the basal foot processes together with their communications to the basal body. The data suggest active contractile function of basal bodies.

Regeneration of Rat Tracheal Epithelium: Changes in Gap Junctions During Specific Phases of the Cell Cycle

Uninjured mitotically inactive tracheal epithelium has virtually no gap junctions. When the epithelium is stimulated to proliferate synchronously through a single wave of DNA synthesis and cell division, study of thin sections and freeze-fracture replicas by conventional transmission electron microscopy reveals that gap junctions are formed by the end of the DNA synthetic (S) phase. During the period of mitosis (M), the gap junctions disappear and are not again observed as the superficial cells undergo mucous and ciliary differentiation and normal pseudostratified architecture is restored. If the regenerative cells are continuously stimulated so they go through at least one additional S phase, gap junctions begin to reappear in G1 and reach peak numbers at the end of the second S phase. The correlation of the appearance of gap junctions in regenerating tracheal epithelium with a specific phase of the cell cycle and the absence of these junctions in cells in other phases of the cycle and in the differentiated progeny cells suggests that this surface feature plays a role in control of mitotic activity.

Therapeutic effects of water and milk for acute alkali injury of the esophagus

STUDY BACKGROUND Alkali ingestions cause progressive and devastating injury to the esophagus by liquefaction necrosis. However, the therapeutic efficacy of water or milk dilution for alkali-induced esophageal injury has not been determined. This study used our previously reported model of alkali-induced esophageal injury to evaluate the effectiveness of water and milk dilution. HYPOTHESIS Early dilution with water or milk is efficacious in decreasing esophageal damage from alkali exposure. METHODS The esopgagi of 75 Sprague-Dawley rats were harvested, and each end was cannulated with a 20-gauge catheter. Specimens were maintained in an oxygenated saline solution (at 37 degrees C) during a 60-minute experimental period and then fixed immediately in 10% Formalin solution for histologic examination. Esophagi from six experimental groups (total of 60) were perfused with 50% NaOH solution at time 0. Water or milk dilution was performed immediately at 0 minutes, 5 minutes after injury, and 30 minutes after injury. Blinded pathologic examination was performed using a score of 0 (no injury), 1 (minimal), 2 (moderate), or 3 (severe) for the following six histologic categories: epithelial viability, cornified epithelial cell differentiation, granular cell differentiation, epithelial cell nuclei, muscle cells, and muscle cell nuclei. RESULTS Positive and negative controls showed expected outcomes. Significant progressions of injury over time were seen for every histologic category for both water and milk dilution. The injury scores for the milk-treated group at 0 minutes were less than or equal to the injury score for the water-treated group for all categories. However, these differences were significant only for the cornified epithelial cells. CONCLUSION Early dilution therapy with water or milk reduces acute alkali injury of the esophagus and supports use of these forms of emergency treatment.

Effective treatment of acute alkali injury of the rat esophagus with early saline dilution therapy

BACKGROUND Controversy persists regarding the appropriate treatment of acute alkali injury to the esophagus. The current study establishes a controlled model of alkali esophageal injury and examines the efficacy of saline dilution therapy. STUDY HYPOTHESIS Early saline dilution therapy effectively reduces esophageal injury resulting from acute alkali exposure. METHODS The esophagi were harvested from 60 Sprague-Dawley rats. Each end was cannulated with a 20-gauge catheter. Specimens were maintained in an oxygen-perfused saline bath (37 C) during a 60-minute experimental period and then fixed immediately in 10% formalin solution for histologic examination. Three experimental groups (A, B, and C) were perfused with 50% NaOH solution at time zero. Treatment with saline perfusion was performed immediately in group A, five minutes after injury in group B, and 30 minutes after injury in group C. The positive control group D was perfused with saline at time zero. A negative control, group E, was perfused with 50% NaOH at time zero. This group did not receive subsequent treatment with saline. Pathologic examination was performed in a blinded fashion using a score of 0 to 3 (0, no injury; 1, minimal; 2, moderate; 3, severe) for seven histologic criteria: epithelial viability, extent of injury, cornified epithelial cell differentiation, granular cell differentiation, epithelial cell nuclei, muscle cells, and muscle cell nuclei. RESULTS The positive control group demonstrated scores of zero. Nonparametric analysis showed a significant difference among treatment groups for each injury category. Trend analysis revealed a significant progression of injury for each category associated with time to treatment. Discriminant analysis indicated that the muscle cells category was the most useful category with which to distinguish injury among groups. CONCLUSION In our model, saline lavage decreased objective evidence of esophageal injury after a severe alkaline exposure, and early therapy enhanced this beneficial effect.