Synthesis of immunoglobulins by human endocervix in organ culture.

The synthesis of immunoglobulins by the uterine cervix was investigated in an endocervical organ-culture system. Using Ouchterlony immunodiffusion gels immunoglobulin G, immunoglobulin A and secretory piece were detected in washings of endocervical explants and in explant incubation medium. Synthesis of immunoglobulin in the organ-culture system was investigated by polyacrylamide-gel electrophoresis of radiolabelled polypeptides; 2 polypeptides co-migrated with the heavy and light chains of a reference polyclonal immunoglobulin G and were confirmed, by use of anti-human globulin and iodinated staphylococcal protein A, to be the heavy and light chains of immunoglobulin G. This experimental system will provide a useful model in future investigations of the efficacy of a local vaccine in human subjects.     

Hematopoiesis in human embryonic liver organ culture

It was shown by the method of multiple organ cultures on Millipore filters that hematopoiesis of predominantly erythroid type is maintained for a long time (over 1.5 months) in cultures of human embryonic liver. The general morphology of 7–50-day-old cultures was studied and described. The myeloid population of cells (the number of colony-forming units) was virtually exhausted by the 14th–16th day of culture.Laboratory of Bone Marrow Culture and Transplantation, Central Institute of Hematology and Blool Transfusion, Ministry of Health of the USSR. Laboratory of Pediatric Hematology, N. I. Pirogov Second Moscow Medical Institute. First Gynecological Department, No. 49 City Hospital, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR, N. A. Fedorov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 4, pp. 460–462, April, 1977.     

Mucin gene expression in the human endocervix

In the human uterine endocervix, five out of the six human mucingenes investigated (MUC 2, MUC 3, MUC 4, MUC 5AC, MUC 5B andMUC 6) are expressed, whereas in some other mucosae the hybridizationpattern demonstrates the expression of only two or three ofthese genes. The most intense labelling by in-situ hybridizationis obtained significantly with MUC 4, predominantly during theluteal phase. The expression of the MUC 4 gene appears to beinfluenced by the oestro-progesterone ratio. During the ovulatorycycle, there are only a few differences concerning the variationsof expression of all other MUC genes.     

Maturation of human fetal esophagus maintained in organ culture

The purpose of this work was to study the human fetal esophagus maintained in organ culture. Esophageal explants from 8 fetuses aged from 12 to 16 weeks of gestation were cultured up to 21 days at 37 degrees C in Leibovitz L-15 serum-free medium. Between 12 and 16 weeks of gestation, the esophagus has a stratified columnar ciliated epithelium, and glycogen aggregates are present in all cell layers. This morphology remains the same up to 5 days in culture. After 7 to 9 days, a vacuolization in the upper half layer occurs, leading to a lifting off of the ciliated layer and a flattening of the subjacent cells. After 15 days of culture, the esophageal epithelium is stratified squamous and the cells are exfoliated at the surface of the explants. Glycogen aggregates are still present in all layers. Islets of ciliated cells resting on the basal cell layers develop within the squamous epithelium. With the extension of the culture period up to 21 days, the general morphology of the epithelium does not change. The ultrastructural features of the newly formed squamous epithelium, with its basal lamina, are similar to that reported for human adult esophageal epithelium. During the course of the culture, the DNA synthesis continues as determined by autoradiography. It is concluded that it is possible to maintain viable human fetal esophagus in organ culture and that an accelerated maturation takes place leading to the formation of the adult esophageal epithelium.     

Nonadherent,stationary organ culture of human respiratory mucosa

Summary Fragments of human adenoid tissue were grown in a tissue culture system where all artificial tissue handling, except for the explanation procedures and tissue culture condition in itself, was excluded. The fragments formed spheroids that were covered by a pseudostratified, ciliated epithelium. The epithelium rested on a basement membrane. The central parts of the spheroids consisted of fibroblasts and collagen fibers. This structure remained unchanged for the 40 days the fragments were observed in culture.     

Freeze-fracture observations on human fetal kidney in serum-free organ culture

The current study was undertaken to examine and characterize junctional complexes, through freeze-fracture, in developing human fetal kidney and in cultured renal explants maturing in vitro. Tissue specimens were cultured for 7 days in Leibovitz's L-15 medium in the absence of serum or hormones. In uncultured explants, cells in the different nephron segments were joined by zonulae occludentes which consisted of ridges on the P-face and grooves on the E-face of lateral membranes. Tight junction composition was heterogeneous and complexity increased from proximal to collecting tubules. Proximal tubule cells were also characterized by the presence of gap junctions and a brush border. Podocytes were joined by macular junctions, while zipper-like junctions were observed between collecting duct cells. Intercalated cells were decorated with rod-shaped intramembrane particles on lateral and apical membranes, instead of the usual spherical particles present in other cells. All these structures could be observed at various intervals during tissue culture, indicating the preservation of ultrastructural integrity of the explants. These observations extend and support previous studies made at the light and electron microscopic levels. Thence, the present culture model constitutes a valuable tool to study the direct effect of growth factors on nephrogenesis.     

Synthesis of taurocholate by rat fetal liver in organ culture: effects of cortisol in vitro.

Taurocholate production by fetal hepatic organ cultures was measured by radioimmunoassay. Taurocholate production was maximal on day 1 of in vitro incubation, but was demonstrable in organ cultures maintained for periods up to 15 days. Explants obtained from fetuses of 18 gestational days of age produced only 82 pmol taurocholate per milligram dry weight of tissue during the first 24 h of incubation. Explants obtained from fetuses 21 gestational days of age produced 1,043 pmol taurocholate per milligram dry weight. The presence of cortisol (2.0 X 10(-6) M) in the incubation medium increased synthesis of taurocholate by rat fetal liver in which total taurocholate rose 50-fold above control after 120 h of incubation. In increasing concentrations from 2.0 X 10(-9) M to 2.0 X 10(-7) M, cortisol produced an incremental rise in taurocholate. However, additional increases in cortisol dose failed to provide further stimulation, and taurocholate production was inhibited by cortisol concentrations of 2.0 X 10(-5) M. The results provide further validation for the technique of fetal hepatic organ culture. They demonstrate that taurocholate synthesis is increasing rapidly during the final stages of gestation and show that cortisol augments taurocholate synthesis in a dose-response pattern.     

Intimal proliferation in an organ culture of human saphenous vein.

This study investigated whether intimal proliferation, the characteristic feature of the response of human saphenous vein to arterial implantation, also occurs in organ culture. Vein segments were maintained for 14 days in medium supplemented with 30% fetal bovine serum. Tissue viability (measured by adenosine triphosphate [ATP] concentration) decreased only 20% from 280 +/- 20 to 220 +/- 20 nmol/g wet weight. In veins prepared for culturing, endothelial loss (approximately 20%) was confined to near the cut edges. Cultured veins retained an endothelial layer in the initially undamaged areas, while the initially injured areas became covered by a mixture of endothelial and vascular smooth muscle cells. Autoradiography in conjunction with scanning electron microscopy showed the presence of proliferating cells on the intimal surface. Transverse sections of cultured veins showed the development of a new intima containing vascular smooth muscle cells identified by immunocytochemistry with anti-alpha-actin. There were also endothelial cells identified with Ulex europaeus lectin arranged in capillarylike structures. Pulse or continuous labeling of cultures with [3H]thymidine showed that proliferating cells were confined to the new intima and suggested that the smooth muscle cells in this layer arose from both immigration and proliferation. The results demonstrate that intimal proliferation occurs in organ culture of human saphenous veins.     

Characteristics of nonmalignant and malignant human prostate in organ culture.

Prostate tissue was obtained from 52 radical prostatectomies immediately upon surgery. From each specimen, a small piece of tissue was fixed in 10% buffered formalin and used for histology, cytokeratin staining, staining with the antibodies to the proliferation-associated antigen (Ki-67), and histochemical evaluation of the epithelial-stromal basement membrane. A second piece was used for the isolation of epithelial cells and stromal cells in monolayer culture. The remainder of each specimen was cut into cubes (approximately 1 mm on a side) and incubated in organ culture for up to 20 days. At the end of the incubation period, tissue was fixed in 10% buffered formalin and examined as described above with zero-time tissue. These studies showed that normal epithelial and stromal elements survived in organ culture in the presence of a serum-free medium containing a mixture of growth factors (epidermal growth factor, insulin, pituitary extract, and dihydrotestosterone). In many of the tissues examined at 4 days, individual glands resembled those seen immediately after surgery, with a single layer of basal epithelial cells and a layer of secretory cells above. By Day 8, the secretory epithelium was lost in many places and basal cells proliferated to fill in the lumens of the glands. All of the nonmalignant glands were reactive with the anti-cytokeratin antibody (K903), and there was a large increase in the number of cells staining for Ki-67 as compared with zero-time tissue. Staining with the Periodic Acid Schiff (PAS) and PAS-methenamine silver (PASME) reagents revealed an intact basement membrane around virtually all of the epithelial structures. The basement membrane appeared to be thickened in some areas. In places where a gland was cut during the processing of the tissue, epithelial cells migrated out of the gland and covered the cut surface of the tissue piece. There was no detectable basement membrane separating the epithelium from the stroma at these sites. Whereas nonmalignant epithelial cells were preserved in the growth factor- and dihydrotestosterone-supplemented culture medium, most of the malignant cells rapidly lysed under the same conditions. However, when phorbol myristate acetate was included in the culture medium, many of the tumor cells remained viable. This was seen with the more well-differentiated tumors as well as with tumors that were highly anaplastic. All of the tumor cells were nonreactive with anti-cytokeratin antibody, and only a few cells stained for Ki-67. The basement membrane surrounding malignant cells was thin and, in places, appeared to be discontinuous. Where malignant glands were cut in the processing of the tissue, cells did not migrate out over the cut surface. In summary, this study identifies culture conditions for the successful maintenance of human prostate tissue for several days in organ culture. Histological/histochemical features that distinguish nonmalignant and malignant tissue are present in this model.     

Ultrastructural study of viral replication in human fetal intestinal organ culture

Summary Human fetal intestinal organ cultures were employed to study the growth patterns of various viruses and resultant cytoarchitectural changes using electron microscopic techniques. Representative strains of adenovirus, adenovirusassociated virus, herpes simplex virus, poliovirus, and echovirus were chosen for the study. The intracellular localization of these viruses was demonstrated, and the occurrence and rate of cellular destruction were observed. The difficulty of visualizing smaller viruses represented was emphasized. The use of these techniques to detect certain fastidious viruses of the gastrointestinal tract was suggested, since human fetal intestinal organ culture provides differentiated cell types which may be important for the initiation and support of viral replication.     

Metabolic effects of nicotine on human adipose tissue in organ culture

Summary Fragments of human adipose tissue were maintained in culture for 1 week in a medium containing 1 mU/ml insulin and 100 ng/ml dexamethasone. Under these conditions lipoprotein lipase activity was present in human adipose tissue fragments which converted [¹⁴C]glucose to ¹⁴C0₂ and [¹⁴C]triglyceride. Both metabolic parameters studied were affected by human tumor necrosis factor and brefeldin A. When fragments of human adipose tissue after 1 week in culture were incubated with nicotine tartrate for 20 h, a slight but significant increase in lipoprotein lipase activity was observed, and an increased conversion of [¹⁴C]glucose to ¹⁴CO₂ and [¹⁴C] triglyceride occurred. Nicotin was taken up by human adipose tissue, but no conversion to cotinine was observed. Our data demonstrate a direct effect of nicotine on human adipose tissue metabolism. Furthermore, it is suggested that weight loss in smokers is a multifactorial phenomenon, and one of the important factors to be considered is the direct effect of nicotine within the tissue.Abbreviations LPL lipoprotein lipase - MEM minimal essential medium - rHuTNF recombinant human tumor necrosis factor - BFA brefeldin A - TG triglyceride     

Synthesis of human rotavirus polypeptides in cell culture

The replication strategy of a human serotype 1 rotavirus, adapted to rapid growth in CV-1 cells, was investigated. A single cycle growth curve revealed eclipse and latent periods of 3 and 4 hours, respectively. Although the extent of reduction of host cell protein synthesis was directly related to the multiplicity of infection of the virus, incorporation of actinomycin D and excess NaCl into the medium resulted in significant reduction in host cell background and enabled observation of viral polypeptides as early as 2 hours post infection. Five polypeptides were found to be structural components of the virion, and a further eight appeared to be nonstructural proteins or intermediates. Five polypeptides were glycosylated during virus replication, but only one of these, VP7, was a definite structural glycoprotein. Pulse-chase experiments revealed that four low molecular weight polypeptides underwent post-translational modifications.     

Cell kinetics of human gastric mucosa and gastric cancer in organ culture

Summary The cell kinetics of human gastric epithelium in organ culture have been measured using flash labelling with tritiated thymidine and the metaphase arrest technique to estimate cell birth rates. Normal gastric antral and body mucosa have been compared with mucosa showing gastritis and gastric carcinoma. Labelling indices with tritiated thymidine in normal gastric mucosa declined over a 48-h period suggesting that essential growth factors were lacking. Labelling indices and cell birth rates were higher in gastritis than in normal mucosa and highest in gastric carcinoma. Labelling indices were higher in intestinal-type gastric carcinoma than diffuse carcinoma. In metaphase arrest experiments carcinomas showed on average an eightfold increase in resistance to the metaphase-arresting properties of vincristine when compared with normal mucosa. The validity of using the metaphase arrest technique to measure cell birth rate in gastric cancers in view of this vincristine resistance is discussed.     

Tumour fragment spheroids from human non-small-cell lung cancer maintained in organ culture

Biopsy material from 17 human non-small-cell lung carcinomas (NSCLC) was maintained in agar overlay culture as tumour fragment spheroids for 40 days. A practical procedure for the formation of spheroids and organ culture is described. The mechanically dissociated tumour specimens showed a variation in their ability to generate spheroids that was not related to the ploidy or the histological differentiation of the biopsies. Light microscopic observations revealed a heterogeneous spheroid population with a mixture of tumour cells and stromal elements. Most of the histological elements normally found in human NSCLC could be seen in the spheroids. The cellular components in the spheroids varied between highly cellular to sparsely cellular, dominated by stromal elements. The squamous carcinomas were in general found to generate highly cellular spheroids more often than the adenocarcinomas. Spheroids with a different cellular content could be selected in vitro by using a morphometric technique. Diameter measurements showed a large variability in spheroid growth. Most of the spheroids decreased in size although bromodeoxyuridine labelling indicated active cell proliferation in the specimens. Frequent changes of medium did not affect spheroid growth. The culture system presented provides a model for studying the cellular heterogeneity as well as the biological characteristics of tumour tissue from individual patients in vitro.