A morphologic and biochemical study of the fate of antibody-bearing liposomes.

The effect of the intravenous administration of gamma-globulins, entrapped or not within liposomes, was investigated in rodent liver. Desialation of gamma-globulins did not influence significantly their uptake by this organ. Fluorescein-labeled nonspecific gamma-globulins and antibodies raised against the lysosomal acid alpha-glucosidase were entrapped within two types of liposomes. The administration of negatively charged liposomes made of lecithin, cholesterol, and dicetylphosphate was badly tolerated. Positively charged liposomes, in which dicetylphosphate was replaced by stearylamine, allowed a considerably higher yield of entrapment and were well tolerated by the animals. Approximately half of the fluorescent gamma-globulins were recovered in the liver, and evidence is presented for the intralysosomal localization of positively charged liposomes, both in the reticuloendothelial cells and in the hepatocytes. A striking difference exists, nevertheless, in the appearance of the two types of cells, 1 hour and 7 1/2 hours after injection. The specific antibodies never inhibited more than 50% of the activity of liver acid alpha-glucosidase, and there was no significant modification in the glycogen content of this organ. This work points to the influence of the composition of liposomes, in determining the entrapment of proteins, the toxicity, the uptake by the liver, and the distribution of these lipidic spherules in the different types of cells. This factor will have to be taken into account if liposomes are to be used as vectors in the treatment of lysosomal storage diseases.     

Cation-anion gap and choleretic properties of rat bile.

To investigate whether bile contains choleretic anion(s) other than bile salts (BS) that could account for the observed cation-anion gap in bile, the choleretic properties of bile were investigated in the rat. Infusion of bile increased bile flow significantly more than did taurocholate (TC) (P less than 0.005). By contrast, TC increased BS excretion more substantially (P less than 0.01). This effect was dose dependent for both bile and TC. The choleretic principle had a molecular weight of less than 1,000 as estimated by ultrafiltration of bile. Infusion of bile that had been chromatographed on BioBeads SM-2 still elicited choleresis, whereas bile that had been chromatographed on Dowex 1 x 50W did not. Administration of bile in vivo did not affect Na+-K+-stimulated ATPase activity in liver plasma membrane fractions. These results suggest that bile contains anion(s) other than 3-hydroxy-BS, which increase bile flow in a dose-dependent fashion without affecting the permeability of the biliary tree. This putative choleretic appears to be anionic in nature, heat stable, and has an apparent molecular weight of less than 1,000. This finding suggested that bile salt-independent bile flow partly depends on the excretion of a currently undefined anion.     

Biochemical and morphologic studies on the effect of bile acids on the epithelium of the rat jejunum

The influence of bile salts on the mucosal surface of rat jejunum was tested with an in vivo technique of segmental perfusion. Sodium taurocholate and chenodesoxycholate were applied in a concentration of 3 mmol/l. The release of 5 brush border membrane enzymes, 5 cytosolic, 1 mitochondrial, and 2 lysosomal enzymes during a perfusion time of 150 min as well as morphological alterations after bile salt treatment were investigated. Among the membrane enzymes, due to their superficial localization, the solubilization of enteropeptidase and alpha-1,4-glucosidase was highest both in the control perfusion and in the presence of bile salts. At the same time, cytoplasmic enzyme activities were liberated extensively whereas lysosomal and mitochondrial enzymes were scarcely detectable. This disproves any serious injury of the enterocytes. Electronmicroscopic results supported this suggestion. After administration of taurocholate (in physiological concentration), only an occasional diminution of the glycocalyx was observed and even chenodeoxycholate (in an unphysiological concentration) caused only negligible destructions of intestinal brush borders. Investigations with ruthenium red to contrast the glycocalyx showed a partially unchanged structure. Microvesiculation from the microvilli was observed in many electron microscopic photographs. That is a possibility for the release of membrane-bound and cytosolic enzymes without destruction of enterocytes.     

Metaplastic lesions of the extrahepatic bile ducts: a morphologic and immunohistochemical study.

Although metaplastic changes can occur in the extrahepatic bile ducts, a detailed morphologic study of these lesions has not been done. We examined the bile duct mucosa in 42 pancreaticoduodenectomy specimens, 32 with neoplastic lesions and ten with inflammatory lesions of the extrahepatic bile ducts, to assess the prevalence and type of metaplastic lesions. For comparison, the common bile ducts from 10 autopsy cases were reviewed. Twenty of the 42 total cases (48%), 13 of the 32 neoplastic cases (40%), and 7 of the 10 inflammatory cases (70%) had metaplastic changes. Pyloric gland metaplasia was the most common type (16/20 cases; 80%), whereas intestinal metaplasia was seen in 1/20 cases (5%). A combination of pyloric gland and intestinal metaplasia occurred in 2/20 cases (10%), and squamous metaplasia plus the above-mentioned two types of metaplasia was seen in 1/20 cases (5%). None of the normal common bile ducts obtained from ten autopsies had metaplastic changes. Endocrine cells were identified in nine (56%) of 17 metaplastic lesions. In contrast, endocrine cells within the intramural glands were seen in only 2 of the 10 normal common bile ducts. Although a significant proportion of carcinomas (6/13 cases) was in close proximity to areas of metaplasia, we were unable to find dysplastic foci within the metaplastic glands or the metaplastic surface epithelium. Reactive atypical cells involved the surface biliary epithelium and intramural glands and were associated with inflammation and metaplastic changes. The presence of goblet, mucinous, squamous, and reactive atypical cells in association with hyperplasia of intramural glands in frozen sections or small biopsy specimens may be mistaken for malignancy; hence, recognition of these lesions is of diagnostic importance.     

Homozygous achondroplasia: morphologic and biochemical study of cartilage

We have performed histochemical, immunohistochemical, electron microscopic, and biochemical studies on the upper tibial cartilage from a case of homozygous achondroplasia. The growth zone was narrow and disorganized. Columnization was absent except for a few areas with short rows of cells. Hypertrophy was reduced to scattered clusters of cells. The provisional calcification was patchy and primary trabeculae were thick and irregularly arranged. Islands of fibrous or fibrocartilagineous tissue were found along the growth zone. The matrix did not stain with safranin O and lacked metachromasia, except for pericellular rims around the hypertrophic cell clusters. Staining with antibodies against the large proteoglycan monomers and chondroitin-4-sulfate was weakly positive. Electron microscopic examination showed that only a few cells had degenerative signs. In most areas of the matrix, proteoglycan granules were absent. Areas with dense collagen fibers were seen. In contrast to the growth zone, the cartilage of the remaining epiphyses had normal histochemical, immunohistochemical, and electron microscopic appearance. The large proteoglycan monomers had a normal composition and hydrodynamic size. Type II and XI collagen, pepsin fragments of type IX collagen, and several noncollagenous proteins extracted from cartilage had a normal electrophoretic migration. It is suggested that a mutation affecting a matrix component or a regulatory pathway present only or predominantly in the growth area of the chondroepiphysis might explain the findings.     

Microbial transformation of bile acids. A unified scheme for bile acid degradation,and hydroxylation of bile acids

Through the isolation and identification of a wide variety of degradation products formed from bile acids by microorganisms, a unified scheme for the complete degradation of bile acids to carbon dioxide and water has been proposed and discussed. The proposed degradative pathways mainly consist of the following steps: natural C₂₄ 3-hydroxy bile acids → 3-oxo bile acids → Δ⁴-3-oxo bile acids → → C₁₆ or C₁₈ perhydroindane derivative (at least in two ways) → → (4→)-4-methyl-5-oxo-octanedioic acid (at least in three ways) → → CO₂ and H₂O. A microbial hydroxylation method for the preparation of bile acid samples was investigated which could be used as reference standards in the analysis of bile acids in biological materials and also as materials for studying the function of bile acids. The particular fungi, Curcularia lunata NRRL-2380, Helicostylum piriforme ATTC-8992 and Pestalotia foedans ATCC-11817 effected the 1β-, 11β-, 12β-, 15α- or 15β-hydroxylation of certain bile acids and gave the following products: 1β,3α-, 3α,12β- and 3α,15β-dihydroxy-5β-cholan ?24-oic acids, 3α,12β,15α- and 3α,12β, 15β-trihydroxy-5β-cholan-24-oic acids and 12β,15β-dihydroxy-3-oxo-5β-cholan-24-oic acid from-lithocholic acid; 1β,3α,12α- and 3α,12α,15β-trihydroxy-5β-cholan-24-oic acids and 3α, 11β-dihydroxy-12-oxo-5β-cholan-24-oic acid from deoxycholic acid; 3α, 7α, 12β-trihydroxy-5β-cholan-24-oic acid 3α,7α,12β, 15α-tetrahydroxy-5β-cholan-24-oic acid from chenodeoxycholic acid; 3α-6α,12β- and 3α,6α, 15β-trihydroxy-5β-cholan-24-oic acids from hyodeoxycholic acid; 3α, 7β, 12β trihydroxy-5β-cholan-24-oic acid from ursodeoxycholic acid; 3α,12β-dihydroxy-7-oxo-5β-cholan-24-oic acid from 3α-hydroxy-7-oxo-5β-cholan-24-oic acid. Some of these products were new compounds and their structures were determine.     

Influence of the gallbladder on serum bile acids.

Serum bile acids (SBA) were studied after a standard fatty meal in patients with gallbladder disease. Early postprandial bile acid values were found to be greater in patients with non-functioning gallbladders. Higher postprandial SBA values were found after cholecystectomy. Serum bile acid measurements were of no value in the assessment of gallbladder function.     

Massive obesity and the kidney. A morphologic and statistical study.

The renal morphology of 5 grossly obese patients with normal renal function and many of the features of the Pickwickian syndrome was studied at autopsy. The most striking feature was that of increased glomerular size. Measurements of two parameters of glomerular areas indicated statistically significant glomerular enlargement for both as compared to controls. Glomerulomegaly was primarily the result of vascular dilatation and a variable mesangial component. This abnormality was related to several factors, including increased blood volume, hypoxia, and increased right ventricular pressure. Polycythemia, commonly noted in other similar conditions with glomerulomegaly, is believed to be of no importance in the pathogenesis of glomerular enlargement.     

Apocrine carcinoma of the breast. A morphologic and immunocytochemical study.

The mode of recognition and hence the frequency of apocrine differentiation in breast carcinomas, assessed on purely morphologic grounds, remains uncertain. One hundred consecutive cases of breast carcinoma were studied in order to establish the incidence of this type of tumor. With the use of an immunocytochemical method for the detection of GCDFP-15, a protein present in apocrine epithelium and in the fluid of tension cyst of the breast, the presence of apocrine differentiation was confirmed in 4 cases initially diagnosed as apocrine carcinomas on histologic grounds. Eight additional cases contained immunoreactive cells: 1 contained 10% of positive cells scattered throughout the tumor, and the other 7 cases were only focally positive. In 4 of these latter cases positive staining was confined to the in situ component. The ultrastructural findings in 2 cases of apocrine carcinoma are discussed in order to link the morphologic features for recognizing this tumor type and the presence of the antigenic apocrine marker.     

A combined biochemical and cytogenetic study of thioridazine-induced damage to nucleic acids

In this work the biochemical effects of thioridazine, a commonly used phenothiazine, have been studied upon native double- and single-stranded DNA and also upon a supercoiled plasmid. The results indicate that thioridazine causes damage and scissions to these nucleic acids but only at concentrations much higher than the one used in our cytogenetic experiments and that the damage seems to depend on the concentrations used. Furthermore, we studied the action of thioridazine alone or in combination with caffeine and/or melphalan upon human lymphocytes in vitro. Thioridazine and caffeine (a well-known inhibitor of cellular repair mechanisms) were shown to act synergistically to potentiate the cytogenetic effect of melphalan on human lymphocytes. It is suggested that thioridazine alone or in combination with caffeine may exert its synergistic effect on melphalan cytotoxicity to cultured human lymphocytes not only indirectly, i.e. as a strong calmodulin inhibitor by facilitating the intracellular retention of melphalan, but also directly by reaction with nucleic acids and by causing scissions in and damage to them. Therefore, thioridazine (as chlorpromazine) has some potential as an adjuvant chemotherapeutic agent for the treatment of human cancer.