Human lymphoblastoid cell lines secreting antibodies with restricted HLA specificity

Peripheral B lymphocytes obtained from three healthy individuals who had been immunized against peripheral blood lymphocytes from appropriate HLA-incompatible donors were transformed by the use of Epstein-Barr virus. The transformed blastoid B cells were repeatedly subcultured by means of cluster picking, and the HLA antibody-producing cultures were identified by testing the culture supernatants by means of the cytotoxicity assay, using the corresponding donor cells. Thus far, four cell lines that secrete cytotoxic HLA antibodies (MP1, 3, 4, and 5) have been established. Specific immunoabsorption experiments revealed that the antibody activity is carried by lambda-type IgM for MP1, by kappa-type IgM for MP3 and MP5, and by both for MP4. Specificity analysis of a panel of HLA-pretyped cells indicated that MP1 detects DQw2, whereas MP5 recognizes B7. The specificity of MP3 was similar to a DQ specificity termed DC5 (probably equivalent to TA10) but not the same. In the case of MP4, both of the lambda-type and kappa-type antibodies appeared to be directed toward new HLA class 11 determinants.Abbreviations used in this paper HLA human major histocompatibility - EBV Epstein-Barr virus - B-LCL Blymphoblastoid cell line - NA not absorbed - PBS phosphate-buffered saline - SPA Sepharose protein A - NRS normal rabbit serum     

Immunohistochemical heterogeneity of alpha-tubulin in human epithelia revealed with monoclonal antibodies

Summary Four monoclonal antibodies raised to alpha subunit of pig brain tubulin (TU-01, TU-02, TU-03, TU-04) were used to study immunohistochemical heterogeneity of alpha tubulin in human epithelia. Selective reactivity was detected in the skin and trachea/bronchi, whereas all other epithelia investigated reacted uniformly with all four monoclonal antibodies. In the skin TU-01 reacted very strongly with all layers except the basal layer; TU-02 reacted strongly with granular layer and was unreactive or only weakly reactive with others; TU-03 reacted very strongly with basal layer and weakly to moderately with superficial layers; TU-04 reacted strongly with the granular layer of epidermis and was unreactive with other layers. In the trachea and major bronchi TU-01 reacted with the entire epithelial layer; TU-02 reacted only with superficial layer; TU-03 reacted with superficial and basal layer; TU-04 reacted only with superficial layer. Different staining patterns obtained with these four monoclonal antibodies indicate that there is immunohistochemical heterogeneity of alpha tubulin in some but not all normal human epithelia.     

Stimulation of pancreatic secretory process in the rat by low-molecular weight proteinase inhibitor

Oral application of a single dose of a new synthetic proteinase inhibitor Camostate (Foy-305) in male Wistar rats was carried out together with studies of in vitro amino acid incorporation followed by separation of proteins by two-dimensional gel electrophoresis. The aim of this experiment was to analyze changes produced by the inhibitor in total protein and individual enzyme biosynthesis. Administration of 100 mg/kg Foy-305 resulted in significant inhibition of total pancreatic protein synthesis, without changes in fractional rates for individual enzymes. 50 mg/kg Foy-305 induced a 10-fold elevation of cholecystokinin (CCK) levels in serum; this persisted for 3 h and led to a significant increase in the total rate of protein synthesis with peak values at 6 and 9 h (78% and 84% above control levels, respectively), returning to control by 15 h. Changes in fractional rates of synthesis occurred with a latency of 6 h and were restricted to amylase and the anionic form of trypsinogen and chymotrypsinogen. Amylase biosynthesis decreased by about 40% from control levels at 9 h to return to control levels by 15 h. Increased synthesis of trypsinogen and chymotrypsinogen was observed; this was also phasic. The results show similar enzyme-specific regulation as previously described for exogenous CCK stimulation and for the adaptation of the pancreas to diets enriched in protein. They demonstrate the effectiveness of pulsatory endogenous hormone release in the regulation of protein synthesis.     

The highly modified membrane of cornified cells in stratified squamous epithelia: A comparison of heterogenous deposits in keratinized and nonkeratinized epithelia

Summary The chemical nature of the thickened plasma membrane of cornified cells in stratified squamous epithelium was investigated in comparison with that in noncornified epithelium. Localizations of transglutaminase, molecular weight 92000 daltons, and detection of epidermal cysteine proteinase inhibitor were effected with a monoclonal antibody and a monospecific rabbit anti-inhibitor immunoglobulin, respectively, directed to the antigens. N-(7-dimethylamino-4-methylcoumarinyl) maleimide was used to demonstrate S-S cross-linking. In all keratinizing epithelia, the enzyme and inhibitor were deposited on membranes of granular cells. S-S bonds were formed in cornification with the appearance of electron-dense material by the inner leaflet. Both enzyme and inhibitors occurred on the corneal epithelium, but S-S linkage and the thickened plasma membrane did not form even at the last stage of maturation. On the other hand, the internal vaginal epithelium in the proestrous stage without keratinization contained the enzyme, but neither inhibitor nor S-S linkage. Both antigens and S-S bonds were detected when keratinization proceeded during estrus. The staining patterns in the epithelium near the vaginal introitus were identical to those in the skin. Cuboidal and simple epithelia exhibited none of those constituents. The findings indicated that heterogenous components contribute to modification of the plasma membrane of cornified cells, but S-S cross-linkages are associated exclusively with formation of the ultrastructurally unique membrane structure. In addition, findings suggested hormonal regulation in the chemical modification of the membrane in estrogen-sensitive internal vaginal epithelium.     

Arterial supply of the choriocapillaris of anuran amphibians (Rana temporaria,Rana esculenta)

Summary The pattern of the vascular supply to the choroid of the frog eye was studied in toto with the use of the injection-replication-SEM technique. The choroid of anuran amphibians is composed mainly of the choriocapillaris. In both species studied (Rana temporaria, Rana esculenta), an independent arterial supply to the choriocapillaris supplemented that from the ciliary arteries. This additional vascular route arises from the optic artery, a separate branch of the arteria infundibularis superficialis. The optic artery, accompanied by its vein within the vascular sheath of the optic nerve, joins the rich arterial capillary network of the choriocapillaris and supplies the posterior pole of the ocular bulb. The superficial capillary network displays a dense collar around the entrance of the optic nerve into the eye and is composed of a circular meshwork of small capillaries, several layers deep. More peripherally, however, it becomes single layered. This capillary network, as a whole, establishes numerous connections with the adjacent choriocapillaris at the posterior pole of the ocular bulb. In anuran amphibians the complex arrangement of both arterial systems supporting the choriocapillaris may be regarded as a more complete equivalent of the short posterior ciliary arteries of mammals.     

Immunohistochemical localization of oxytocin and vasopressin in the adrenal glands of rat,cow, hamster and guinea pig

Summary The distribution of oxytocin and vasopressin in the adrenals of rat, cow, hamster and guinea pig has been studied by use of immunohistochemical techniques. In all the species studied the adrenal cortex contained both peptides; the staining in the zona glomerulosa being more intense than that in zona fasciculata or zona reticularis. The medulla, however, showed considerable species variation. In the cow, both peptides appear to be present in the adrenergic and noradrenergic cells, though staining was particularly prominent in cortical islands interspersed within the medullary tissue. In the rat, groups of medullary cells positive for both peptides were found, though it was not possible to associate these groups with particular chromaffin cell types. In the hamster oxytocin was present only in adrenaline-containing cells, whereas vasopressin was present in all medullary cells. The guinea pig medulla, which contains only adrenaline-secreting cells, was positive for both peptides. The possibilities that vasopressin and oxytocin have an autocrine or paracrine role in functioning of the adrenal gland is discussed.     

Structure of isolated sensilla and sensory neurons in vitro: Observations on developing silkmoth antennae

Summary By combined enzymatic and mechanical treatment, it was possible to dissociate the sensory epithelium of developing antennae of male Antheraea polyphemus and A. pernyi silkmoths from the stage of separation of the antennal branches up to the early stages of cuticle deposition. Large numbers of entire developing trichoid sensilla were isolated. These are characterized by a large trichogen cell with a long apical, hair-forming process and a large nucleus. A cluster of 2–3 sensory neurons, enclosed by the thecogen cell, is situated in the basal region. The dendrites run past the nucleus of the trichogen cell into the apical process from which they protrude laterally. The nuclei of the tormogen and a 4^th enveloping cell can be distinguished near the base of the prospective hair. After further dissociation, only the neuron clusters remain, still enclosed by their thecogen cell and often attached to the antennal branch nerve via their axons. It is finally possible to disrupt the thecogen cells and the axons, leaving the sensory neurons with inner dendritic segments and axon stumps. The majority of these neurons can be expected to be olfactory.