Lymphocyte subsets show marked differences in their distribution between blood and the afferent and efferent lymph of peripheral lymph nodes.

The surface phenotypes (CD1, CD4, CD5, CD8, SBU-T19, MHC class I, MHC class II, and sIg) of cells in blood, lymph nodes, and lymph were determined to examine simultaneously the distribution of lymphocyte subsets circulating in blood, afferent lymph, and efferent lymph of a peripheral lymph node. Marked differences in the percentage of certain lymphocyte subsets were apparent within the compartments examined, suggesting that lymphocyte subsets leave the blood with differing efficiencies. Lymphocyte subsets also appeared to be extracted from the blood at different rates by lymph node as opposed to subcutaneous vascular endothelium. Endothelial cells in different vascular beds may express different numbers of molecules complementary to a set of migration-related cell surface molecules specific for each lymphocyte subset. Accordingly, the vascular endothelium would be the key factor in regulating nonrandom cell migration.

MRC OX-22, a monoclonal antibody that labels a new subset of T lymphocytes and reacts with the high molecular weight form of the leukocyte-common antigen.

A mouse monoclonal antibody (MRC OX-22) is described that labels rat T cells which mediate graft-versus-host reactions and those responsible for the suppression of antibody synthesis in hosts undergoing these reactions. In contrast, most of the T cells that provide help for B cells are MRC OX-22 negative. These results, taken together with those published previously, demonstrate that the rat contains at least three phenotypically and functionally distinct subsets of T cells. The MRC OX-22 antibody also labels all B cells, 50% of bone marrow cells, but only 2% of thymocytes. Of these latter cells about half are found at the edge of the medulla and the remainder are randomly distributed throughout the cortex and medulla. These findings lend support to the view that mature thymocytes leave the thymus at the cortico-medullary junction, and also suggest that both cortex and medulla may be sites where thymocytes mature. Biochemical studies showed that the MRC OX-22 antibody reacts with the high molecular weight form of the leukocyte-common antigen (L-CA). Comparison with data on human L-CA suggests that the molecular and antigenic heterogeneity of this set of glycoproteins has been conserved between rat and man.

New murine model for hepatocellular carcinoma: transgenic mice expressing metallothionein-ovine growth hormone fusion gene.

Hepatocellular carcinomas developed at a high frequency in the livers of transgenic (C57BL/6 X SJL/J)F1 mice under the influence of growth hormone. Three lines of giant transgenic mice expressing a mouse metallothionein-ovine growth hormone fusion gene were generated. The giant mice weighed twice as much as control littermates. The three lines of giant mice expressing very high levels of growth hormone were bred over several generations. Mice from all three lines developed hepatocellular tumors, including adenoma and carcinoma. The occurrence of tumors was age-dependent, and their incidence increased to 70% of the mice studied after 43 weeks of age. Pathologic changes in the livers resembled those observed in rats in which hepatocellular carcinomas are induced chemically. Transgenic mice carrying the metallothionein-ovine growth hormone fusion gene represent a new model for hepatocellular carcinogenesis. This model exemplifies the oncogenic potential for a sustained proliferative growth stimulus within an organ.

A spectroscopic and equilibrium binding analysis of cationic detergent-protein interactions using soluble and insoluble recombinant porcine growth hormone.

Overexpression of cloned eukaryote genes in bacteria often leads to the formation of insoluble refractile bodies which require solubilization by harsh denaturants or detergents. We describe the conformational changes associated with the binding of a surfactant, cetyltrimethylammonium chloride (CTAC) to recombinant porcine growth hormone (PGH). The stoichiometry of binding by CTAC to the soluble and insoluble forms of recombinant PGH was also assessed. Optimum CTAC binding and protein solubilisation were obtained at 50 degrees C and at extreme pH. Increased ionic strength and changes in pH towards the isoelectric point of PGH (pH 6) decreased both the binding of CTAC and the efficiency of solubilising PGH from inclusion bodies. The positive charge on the quaternary ammonium head group of CTAC was found to be critical in the binding of CTAC to PGH and for the subsequent solubilisation of inclusion bodies. The binding of CTAC to the soluble form of PGH caused appreciable changes to the tertiary structure of the protein but did not significantly alter secondary structure, or cause complete unfolding. These observations help to explain earlier results which demonstrate that urea, guanidine hydrochloride and CTAC solubilized recombinant PGH molecules behave differently during in vitro refolding (Puri, N.K., Crivelli, E.C., Cardamone, M., Fiddes, R., Bertolini, J., Ninham, B. and Brandon, M.R. (1992) Biochem. J. 285, 871-879.).

Antigens of activated rat T lymphocytes including a molecule of 50,000 Mr detected only on CD4 positive T blasts.

Mouse monoclonal antibodies (MAbs) have been prepared against rat T cell blasts. One MAb called MRC OX-40 recognized an antigen that differed from any previously described in that its expression was detected only on T blasts that also expressed the CD4 antigen. The OX-40 MAb did not detect an activation determinant of CD2 or CD4 molecules but recognized a distinct chain of mol. wt 50,000. The OX-40 MAb augmented T cell proliferation at late stages on in vitro responses. Other MAbs without obvious counterparts in other species were MRC OX-48 and MRC OX-49,50 which recognized cell surface molecules of mol. wts of about 95,000 and 90,000, respectively. The OX-48 antigen was not expressed on resting lymphocytes but was found on a subset of T and B blasts and also on other leucocytes. The OX-49,50 antigen was found on most haemopoietic cells but was expressed at greatly increased levels after lymphocyte activation and this was also the case for MRC OX-47 antigen which is of unknown Mr. The MRC OX-39 MAb was found to bind the rat IL-2 receptor; expression of this antigen was detected on thymic dendritic cells as well as on T blasts. The phenotype of rat T blasts compared to resting cells was also examined and changes in expression of L-CA, Thy-1, OX-2 and CD8 antigens were seen in addition to the changes found with the above MAbs.

The thyroxine-binding properties of rat and rabbit serum proteins.

The thyroxine (T4) binding properties of rat and rabbit serum proteins were studied using a gel equilibration technique, gel filtration and polyacrylamide gel electrophoresis. In both species two different T4 binding molecules were identified in whole serum and in Cohn fraction V preparations. Only one of these binding species demonstrated the characteristics of specific binding, i.e., high affinity for the hormone and binding site saturability. The mean (+/- SD) apparent association constants (ka) at 37 C for the specific binding proteins in whole rat and rabbit serum were 3.5 +/- 0.5 x 10(8)M-1 and 2.8 +/- 0.5 x 10(8)M-1, respectively. The mean T4 binding capacities of these proteins were 4.3 +/- 1.2 x 10(-6)M and 7.4 +/- 1.0 x 10(-6)M for rat and rabbit serum, respectively. Non-specific binding was due to serum albumin. Rat albumin (ka = 6.1 +/- 1.6 x 10(5)M-1, at 37 C) bound T4 significantly more strongly than did rabbit albumin (ka = 2.3 +/- 0.4 x 10(5)M-1, at 37 C) when it was assumed that there was only one T4 binding site/molecule of albumin. The ability of rat albumin to bind more T4 could also be explained by a greater number of T4 binding sites/molecule. Partial separation of the two T4 binding species was attained by gel filtration on Sephadex G200 columns. The specific binding protein of both rat and rabbit serum was eluted slightly later than the corresponding serum albumin. Polyacrylamide gel electrophoresis resulted in the separation of two distinct T4 binding peaks in the rat, but with rabbit serum clear separation of the two T4 binding molecules was not attained. In both species the specific binding protein migrated anodal to serum albumin.

The expression of a metallothionein-ovine growth hormone fusion gene in transgenic mice does not impair fertility but results in pathological lesions in the liver.

The physiological effects of high serum levels of ovine GH (oGH) were studied in three generations of transgenic mice carrying a metallothionein 1-(MT)oGH fusion gene. Livers of mice expressing oGH were enlarged, irrespective of the level of serum oGH detected. In mice expressing high levels of oGH, direct measurements of hepatocytes in liver sections revealed that cell and nuclear size were abnormally large. Hepatocytes of different transgenic mice varied from 1.4-2.2 times normal size and hepatocyte nuclei varied from 1.7-2.4 times normal size. In addition, intranuclear inclusions were observed in hepatocytes of transgenic mice and their presence was always associated with high serum levels of oGH. In contrast to female transgenic mice containing mouse MT-human, rat, or bovine GH fusion genes female mice containing the MT oGH fusion gene were fertile and their pituitary glands showed synthesis of GH.

Elevation of growth hormone (GH) and prolactin receptors in transgenic mice expressing ovine GH.

The effect of elevated serum ovine GH (oGH) concentration on liver somatotrophic and lactogenic receptors was studied in transgenic mice expressing a metallothionein 1(MT)-oGH fusion gene. The mice belonged to three different pedigrees and were killed between 14 and 63 weeks of age. The levels of GH receptor (GH-R) and PRL receptor (PRL-R) determined by competitive binding assays were similar to those observed in late pregnant, nontransgenic mice. This observation was made for all transgenic mice expressing elevated serum oGH levels, irrespective of sex, final size, or age. Cross-linking studies revealed that binding occurred predominantly to a Mr 48,000 polypeptide with a small amount of binding to polypeptides of Mr 60,000, 70,000, and 100,000 in transgenic mice as well as in a late pregnant, nontransgenic mouse. Total cellular RNA was isolated from livers of transgenic and nontransgenic mice and analyzed on Northern blots using probes specific for GH-R and PRL-R. Results showed that the levels of messenger RNA for both GH-R and PRL-R were elevated in transgenic mice expressing high levels of serum oGH. Since levels of PRL in these mice were within the normal range, these results demonstrate that oGH is capable of inducing hepatic GH-R and PRL-R in vivo and that PRL is not required for the induction of its own receptor. These data also demonstrate, for the first time, the suitability of transgenic mice expressing a foreign GH for the study of the regulation of hepatic GH and PRL receptors.

Production of methionyl-minus ovine growth hormone in Escherichia coli and one-step purification.

The overproduction of ovine growth hormone (oGH) in Escherichia coli is described, achieved in part by alteration of the codon usage for nine of the first 15 amino acids (aa) of the mature hormone. Recombinant oGH (re-oGH), representing 12% of the total cellular protein, was isolated from inclusion bodies by solubilisation using the cationic surfactant cetyltrimethylammonium chloride (CTAC). The hormone was refolded and subsequently purified to greater than 95% homogeneity in a single step using preparative reverse phase high performance liquid chromatography. The aa sequence analysis revealed that the N-terminus of the E. coli-derived polypeptide was identical to that of pituitary-derived oGH, and re-oGH displayed potent somatotropic activity in vivo.

Expression and characterization of biologically active ovine FSH from mammalian cell lines.

Stably transfected cell lines expressing the alpha subunit, beta subunit and alpha/beta heterodimer of ovine (o)FSH have been established following the transfection of Chinese hamster ovary cells with alpha and beta subunit cDNA expression vectors. In the absence of the alpha subunit, FSH beta subunit polypeptides were inefficiently secreted and displayed a short intracellular half-life, while free alpha subunits were readily secreted in the absence of the beta subunit. Cotransfection of oFSH alpha and beta subunit cDNAs led to heterodimer assembly and secretion. While alteration of the nucleotide sequence flanking the beta subunit AUG initiation codon did not appreciably enhance heterodimer biosynthesis and secretion, the replacement of the 5' untranslated and signal peptide-coding regions of the beta subunit cDNA with the corresponding sequences from an oGH cDNA clone was associated with a twofold increase in oFSH heterodimer secretion. The recombinant oFSH had a higher molecular weight than pituitary-derived oFSH, and was more acidic than the native hormone when analysed using isoelectric focusing, suggesting a greater degree of sialylation of the recombinant hormone. A comparison of the activities of the recombinant and native hormones in the porcine testis radioreceptor assay and in the in vitro Sertoli cell bioassay revealed that the recombinant oFSH displayed enhanced biological activity in the Sertoli cell assay when compared with the native hormone.

Antibody secreting cells as specific probes for antigen identification.

Parasite specific antibody probes were prepared by harvesting lymphocytes from infected tissue and incubating them in vitro to allow the spontaneous secretion of antibodies in the culture medium by antibody secreting B cells present in the lymphocyte cultures. The culture supernatant was then used to screen Western blots of parasite antigens and resulted in the detection of antigens specific for the parasite stages present in the tissue at the time of sampling. Similar antigen recognition patterns were also observed when the cells were taken from the draining lymph nodes but the same pattern was not observed with serum taken from the same animal. The use of antibody probes obtained from in vivo induced antibody secreting B cells (ASC probes) offers a unique and universal approach to study local antibody recognition during infection.

Monoclonal antibody (SBU-1 and SBU-3) identification of cells dissociated from the sheep placentomal trophoblast.

We studied the localization of alpha-keratin in the sheep placenta using an alpha-keratin-specific monoclonal antibody (MAb) SBU-1, and examined the feasibility of using this MAb as a marker for determining the purity of isolated uninucleate cells from the placentomal trophoblast. At about 30-50 days of gestation the placentomal and interplacentomal uninucleate cells and some binucleate cells were stained by SBU-1, whereas only the apical region of the syncytial cytoplasm was stained with this MAb. Other cells stained included the uterine and endometrial glandular epithelial cells and fibroblast-like cells in the endometrium and chorionic villi. At about 100-130 days of gestation only the trophoblast uninucleate cells were stained by SBU-1. Approximately 60% of cells isolated from placentomes at 100-130 days of gestation were stained by SBU-1, and they had similar morphological features to the trophoblast uninucleate cells. The number of binucleate cells present was confirmed by their affinity for MAb SBU-3. These results show that MAb SBU-1 is an excellent marker for trophoblast uninucleate cells from placenta of sheep at the later stages of pregnancy.

Characterization of a monoclonal antibody (SBU-1) made to the thymic rudiment of sheep.

A monoclonal antibody (SBU-1) was raised to sheep thymic rudiment by fusion of NSI myeloma cells with spleen cells from BALB/c mice immunized with thymic rudiment isolated from fetal sheep between 25-30 days of gestation. By employing the indirect immunoperoxidase technique the antigen recognized by SBU-1 was found to be present in the epithelial reticular cells of the fetal sheep thymus. The intensity of staining decreased as gestation progressed. In the adult thymus the antigen was mainly restricted to Hassall's corpuscles and occasional epithelial cells in the medulla. In addition, the antigen was also shown to be present in epithelial cells of the small intestine, the bronchiole, the keratinized epithelium of the rumen, and the epithelial cells of the kidney tubules. By use of immunofluorescence the antigen was shown to be present in most of the cells of wool follicles and the cortex of developing wool fibers. Western blotting of SBU-1 against the low-sulfur alpha-keratin proteins of wool confirmed that the antigen recognized by SBU-1 belongs to a family of keratins. It was concluded that SBU-1 was raised against alpha-keratin expressed by the epithelial cells of the thymic rudiment and that the expression of this antigen on the reticular network of the thymus declined with advancement of pregnancy.

Monoclonal antibodies to sheep MHC class II molecules recognize all HLA-D or subsets of HLA-D region products.

Six murine monoclonal antibodies raised against sheep MHC class II molecules were analyzed for reactivity with HLA-D subregion products. All the antibodies reacted with human peripheral blood lymphocytes, monocytes, and B-lymphoblastoid cell lines homozygous for various HLA-DR specificities, suggesting that the antibodies recognized nonpolymorphic determinants on HLA class II molecules. SDS-PAGE and two-dimensional NEPHGE/SDS-PAGE analyses of molecules immunoprecipitated from 35S-methionine-labeled, DR-homozygous B-lymphoblastoid cell lines showed that the monoclonal antibodies precipitated typical class II molecules (Mr 32-34K and 25-29K). From comparison with antibodies of known HLA-D subregion specificity, two of the sheep antibodies appeared to react with the products of single HLA-D subregions, while another showed balanced reactivity with all HLA-D molecules. Antibody SBU.II 38-27 reacted exclusively with HLA-DQ molecules, antibody SBU.II 28-1 with HLA-DP molecules, and antibody SBU.II 49-1 with HLA-DR, -DQ, and -DP molecules. However, analysis of immunoprecipitates from surface-iodinated WT-49 cells (DR3 homozygous) using SBU.II 28-1 and the DP-specific monoclonal antibody B7/21, suggested that the two antibodies immunoprecipitated different alpha polypeptides. The two antibodies SBU.II 38-27 and 28-1 appear to be at least as specific as existing reagents, if not more so. As such, they are of value in their potential contribution to our understanding of the molecular characteristics and ultimately the functions of the HLA-DQ and -DP subregion products, as well as the identification/characterization of HLA-D equivalents in other species.

Thymus and the endocrine system: ovarian dysgenesis in neonatally thymectomized rats.

By 10 days after neonatal thymectomy, the areas of lymphocytic depletion were being repopulated by plasma or reticular cells. Subsequently, ovarian dysgenesis was observed in 50-day-old thymectomized female rats as hyperplasia of ovarian interstitial cells an an enhanced degeneration of follicles. Thymectomized rats at 130 and 170 days of age showed complete ovarian dysgenesis, testicular atrophy, hypertrophy of pituitary beta-cells with pronounced cytoplasmic haloes, and lymphocytic infiltration in the pituitary, thyroid and prostate glands. These results suggest that neonatal thymectomy may initially influence the lymphoid organs (i.e. the immune system) and that changes in the immune system may result in the autoimmune-like damage in the endocrine organs and the prostate gland. Associated with these changes, the concentrations of plasma progesterone and 17 alpha-hydroxyprogesterone were significantly reduced in 130-day-old thymectomized female rats with respect to the sham-thymectomized controls. The concentration of plasma oestradiol-17 beta in thymectomized female rats was the same as in sham-thymectomized female rats. These results suggest that gestagens may normally suppress immune responses but that, in the case of thymectomized female rats, the reduced levels of gestagens may lead to enhanced immune responses.

Effect of ionic strength and ionic composition of assay buffers on the interaction of thyroxine with plasma proteins.

When plasma proteins are diluted with buffer the ionic strength and ionic composition of that buffer affects the interactions between thyroxine (T4) and its plasma protein-binding sites. Increases in phosphate, chloride or barbiturate ion concentration from 50 to 200 mmol/l caused a significant decrease in the affinity of plasma proteins for T4, and a concurrent increase in the concentration of unbound T4. These results cannot be completely accounted for by changes in ionic strength since at the same ionic strength different anions caused quantitatively different effects on unbound T4 concentration. The degree of depression of T4 binding by the three anions studied was in the order barbiturate greater than chloride greater than phosphate. The results of a systematic study on the composition of diluent buffer systems indicated that when a 50 mM-sodium phosphate-100 mM-NaCl buffer (pH 7-4) was used as a plasma diluent, there were unlikely to be gross changes in the T4-binding properties of plasma proteins with dilution.

Systemic immunization of sheep with surface antigens from Haemonchus contortus larvae.

Sheep immunized systemically with a surface extract from third-stage H. contortus larvae showed high serum antibody reactivity against surface antigens and whole, viable larvae. After a first infection, no significant difference was found between the mean egg counts of the vaccinates and controls although most vaccinated sheep seemed to show an increased susceptibility to infection. The local abomasal response was stimulated by giving both vaccinated and control sheep a large, abbreviated infection cured after 11 days by drenching. Thereafter, a second challenge infection was given. This immunization regime resulted in seven of the nine vaccinated sheep showing clear protection against the second challenge infection.

Ultrastructural immunogold investigation of the function and diversity of binucleate cells in the ovine placenta using a monoclonal antibody.

Ultrastructural immunogold labelling of ovine placentomes demonstrated that the molecule recognized by the monoclonal antibody SBU-3 is restricted to the fetal binucleate cell granules and Golgi body, and granules of similar size in the syncytium. Quantitative examination shows that the percentage of placentomal mature binucleate cells that are SBU-3-positive increases rapidly from a low level at 29 days of pregnancy to a plateau at virtually 100 per cent from 41 days to term, whereas interplacentomal binucleate cells rarely show label at any stage. There were no detectable differences in ultrastructure between SBU-3-positive or -negative binucleate cells. These results corroborate the hypothesis of syncytium formation by migration of binucleate cells and indicate local control of SBU-3 production and its possible role in villus formation.

Characterization of a sheep trophoblast-derived antigen first appearing at implantation.

A monoclonal antibody designated SBU-3 was produced by the fusion of mouse NS-1 myeloma cells with spleen cells from a BALB/c mouse immunized with sheep trophoblast microvilli. Lee et al (1985) have reported the immunohistological staining of sheep trophoblast with SBU-3 showing that, as early as 21 days of gestation, the monoclonal antibody recognizes an antigen restricted to the binucleate cells of the trophoblast which are located only at sites of invasion of the underlying uterine tissue. Subsequently the antigen appears in the maternal syncytial layer. Immunoprecipitation of 125I-labelled microvilli by SBU-3, characterization of the antigen on immunoblots, and biochemical analysis all suggest that this monoclonal antibody specifically recognizes a carbohydrate epitope on a series of glycoproteins of molecular weights between 30 000 and 200 000. SBU-3 antigen is present in allantoic fluid but is not detectable in any fetal or adult tissue studied, including maternal and fetal sera. It is suggested that this antigen may have a role in the placentation process.

Localization and purification of SBU-4--a pregnancy specific protein of the ovine uterus.

In order to elucidate the function of molecules of the ovine maternal-fetal interface a monoclonal antibody was produced to intact interplacentomal trophoblast membranes. Extensive immunohistological studies revealed that the monoclonal antibody recognizes a protein designated SBU-4 which originates in the intercaruncular regions of the gravid sheep uterus at about the time of implantation and increases in concentration throughout gestation. The data suggest that SBU-4 is produced by endometrial epithelial cells and that adjacent uninucleate cells of the trophoblast acquire the antigen by endocytosis. Initial biochemical analysis of the purified SBU-4 molecule prepared by monoclonal antibody immunoaffinity chromatography indicates that SBU-4 is high molecular weight glycoprotein complex comprising several sub-units.