Germinal centers and the B-cell system

Summary Germinal centers of the rabbit appendix were studied for the presence of complement receptors, immunoglobulin and alkaline phosphatase. In popliteal lymph nodes, de novo-developing germinal centers were studied with respect to these markers up to 21 days after sheep red blood cell (SRBC)-stimulation. In addition, the possible presence of antigen (SRBC) receptor-bearing cells in these germinal centers was investigated.The results may be summarized as follows: 1) Germinal centers in the appendix as well as those in popliteal lymph nodes were rich in complement receptor-bearing cells. Complement-receptor density did not significantly change during a germinal-center reaction. 2) Immunoglobulins were present only at very low densities on the surface of lymphoid cells in the densely populated area of germinal centers. In germinal centers of popliteal lymph nodes lymphoid cells in the thinly populated area again showed higher densities. Immunoglobulins possibly complexed with antigen on the surface of follicular dendritic cells were not observed in the initial phase of a germinal center reaction. In contrast, in germinal centers of the appendix, immunoglobulin was present in excessive amounts throughout the thinly populated area, possibly complexed with antigen, which is also abundantly present. 3) Reticular staining patterns of alkaline phosphatase were present in the densely populated area, but absent in the thinly populated area of germinal centers in both appendix and popliteal lymph nodes. Primary follicles and young germinal centers were alkaline phosphatase-negative. 4) Antigen receptor-bearing cells were detected in germinal centers of popliteal lymph nodes as early as 5 days after SRBC-stimulation, reaching a maximum at day 10.In conclusion, with the present experimental approach, microenvironmental differences were shown between the densely populated area and the thinly populated area of germinal centers. However, no indication was obtained for a postulated maturation event of the lymphocytes within germinal centers, or for functional differences that may exist between germinal centers in the appendix and popliteal lymph nodes.     

Mammalian Brain Alkaline Phosphatase: Expression of Liver/Bone/Kidney Locus Comparison of Fetal and Adult Activities

The alkaline phosphatases (EC 3.1.3.1) are determined by at least three gene loci, which can be sharply distinguished one from another by their sensitivity to inhibition with various amino acids and peptides and by ther-mostability. Alkaline phosphatase is present in the brains of guinea pig, rat, mouse, hamster, squirrel, rabbit, cat, sheep, cow, tamarin, baboon, and man. The gene locus coding for alkaline phosphatase in all these brains is the liver/ bone/kidney locus, as indicated by thermostability studies and by inhibition studies with L-phenylalanine, L-homoarginine, and L-phenylalanylglycylglycine. The average brain alkaline phosphatase activity is about 35% of the average for the livers and only 7.2% and 4.4% of the average kidney and placental activities, respectively. During growth and development, brain alkaline phosphatase activity decreases in the mammals studied. The amount of change is tissue- and species-dependent.     

Histopathological study on six cases of calf leukosis.

A pathological study was carried out on six calves 4 to 10 months of age affected with lymphoid tumors. Most of the lymph nodes enlarged in consequence of leukotic changes in all the calves. These changes occurred also in other various organs. When the leukotic lesions were investigated to clarify the distribution and histological manifestation, three pathological patterns were discriminated among them. One of the patterns was seen in four cases, in which leukotic lesions were present constantly in bone marrow, thymus, liver and kidney. In the lymph nodes, tonsils, and intestinal lymphatic apparatus, neoplastic cellular proliferation took place in paracortical or interfollicular areas and medullary cords obliterating the lymph follicles. Leukotic involvement was observed in the interstitial and the vascular connective tissue in the thymus, as well as in liver and kidney. A second pattern of lesion was revealed in one of the other two cases. Besides lymph nodes in which neoplastic proliferation was seen in the paracortical area, only the thymus manifested intralobular neoplastic involvement in this case. In the remaining case, the leukotic lesion was characterized by the presence of neoplastic cellular masses resembling large lymph follicles in appearance in the lymphatic tissues. It was manifested distinctly in the spleen. Severe thymic atrophy and granulocytic hyperplasia in bone marrow were present in this case.     

Contribution to the problem of clonation of lymphatic cellsin vivo

In transfers of lymph node or thymus cells from normal mice to irradiated recipients the application of phytohemagglutinin to the donors had no significant effect on proliferation of lymphatic elements. Phytohemagglutinin only partially stimulated hemopoiesis after transfer of spleen cells. On transfers of allogeneic lymphatic cells there is marked proliferation in the lymphatic organs having the character of the graft-versus-host reaction. For clonation of lymphatic cells a very suitable procedure was used in which the mouse spleen cells were transferred to syngeneic supralethally irradiated recipients which at the same time received as antigen sheep, or also rabbit erythrocytes. The clones of antibody producing cells were detected by the hemolytic activity of spleen fragments placed between two agarose plates with sheep and rabbit erythrocytes.     

Appendix and γM-antibody formation: VII. Rosette-forming cells in rabbits immunized with sheep erythrocytes

After intravenous immunization with sheep red blood cells the rabbit spleen shows a sharp rise in the number of plaque-forming cells but there is no detectable rise in PFC in the appendix or mesenteric lymph node of the same animals. Repeated immunization via an appendicostomy blind loop results in virtually no local PFC and only a small rise in splenic PFC.In lethally irradiated animals neither thymocytes nor appendix cells alone restore the splenic PFC response. Simultaneous injection of the two cell types restores both direct and indirect plaque formation. The injected cells were labeled with tritiated adenosine and a standard rosette assay for specific antigen-binding cells applied to recipients' spleen cells following immunization. Rosettes appeared by 3 days after immunization whether thymocytes or appendix cells were labeled. Labeled rosettes were observed only in animals receiving labeled appendix cells.This result demonstrates the presence of rosette forming cell precursors in rabbit appendix cell populations and suggests that the cells of gut-associated lymphoid tissues include antibody-forming cell precursors which are normally seeded to the spleen and draining lymph nodes.     

Identification of thymus-dependent areas in frozen sections of guinea pig lymphatic tissue by adherence of rabbit erythrocytes

Untreated rabbit erythrocytes adhere to thymus-dependent areas of guinea pig lymphatic tissues as shown with frozen sections. The adherence reaction is temperature dependent. Optimal results were obtained by incubation of the tissue section with the erythrocytes at temperatures between 0 ° and 4 °C. At 37 °C no adherence of erythrocytes was observed. Out of other erythrocytes tested (human, sheep, chicken, rat, mouse) only rat and mouse cells showed weak adherence to guinea pig thymus sections.     

Germinal centers and the B-cell system

Summary The migration pattern of germinal center cells of the rabbit appendix was studied and compared with that of appendix dome cells, spleen cells, thymus cells and thoracic duct lymphocytes. To discriminate T-and B-cell migration pathways, normal or T-cell-depleted rabbits were used as donors. Cell suspensions were labeled in vitro with ³H-leucine followed by intravenous transfer. The migration of labeled cells in lymphoid organs was studied using autoradiography, particular attention being paid to the spleen of the recipient. B-cells from the appendix dome, spleen and thoracic-duct lymph migrate to primary follicles or the corona of secondary follicles via thymus-dependent areas of peripheral lymphoid organs. In contrast, a B-cell subpopulation from the germinal centers of the appendix migrates to the center of splenic primary follicles and into germinal centers. The migration of germinal center cells to splenic follicle centers is not enhanced by specific antigens. The migration properties of B-cells, possibly changing during differentiation, may be instrumental in the two types of immune reactions, i.e., plasma-cell reaction and germinal-center reaction.     

An interspecies comparison of gangliosides and neutral glycolipids in adrenal glands

Gangliosides and neutral glycolipids of adrenal glands of mouse, rat, guinea pig, rabbit, cat, pig, cow, monkey, and chicken were analyzed by thin layer chromatography (TLC). The major gangliosides common to all species had lactosylceramide in their core structure. GM3 containing N-acetylneuraminic acid (NeuAc) was the major ganglioside in rat, guinea pig, rabbit, and cat, whereas GM3 containing N-glycolylneuraminic acid (NeuGc) was the major one in mouse, cow, and monkey. GD3 was also detected in all species except mouse and GD3(NeuAc)2 was the major in pig adrenal gland. GM4(NeuAc) was detected in the adrenal glands of guinea pig and chicken but not in those of the other species. In the neutral glycolipid fractions, galactosylceramide, glucosylceramide, lactosylceramide, globotriaosylceramide and globotetraosylceramide were detected and the proportions of these glycolipids varied among the species. Guinea pig and chicken adrenal glands contained large amounts of galactosylceramide, this being consistent with the presence of GM4 in these two species. Globopentaosylceramide was detected in mouse, guinea pig, cat, and chicken by the TLC-immunostaining procedure.     

Aldehyde dehydrogenase activity in blood from various vertebrates

1. Aldehyde dehydrogenase activity was determined in whole blood samples from 17 selected vertebrates of 5 classes, using 3,4-dihydroxyphenylacetaldehyde (the aldehyde derived from dopamine) as substrate. 2. Aldehyde dehydrogenase activity in blood was widely but unevenly distributed among the species studied. 3. Mean aldehyde dehydrogenase activities in the range of 40-140 nmol/min.ml blood (measured at 37 degrees C, pH 8.8) were found in blood from man, monkey, rabbit, guinea pig and mouse (C57BL and NMRI strains), with the highest activity in rabbit blood. 4. Much lower aldehyde dehydrogenase activities (0.5-7.5 nmol/min.ml blood) were found in blood from Sprague-Dawley and Wistar rat, dog, cat, horse, pig, chicken, caiman, frog and rainbow trout, whereas the activities in blood from DBA mouse, cow, sheep and crucian carp were close to the detection limit.     

Histochemistry of normal and diseased human lymph nodes.

Gomori's metal precipitate technique was used to demonstrate the phosphatase activity of the human cervical lymph node in health and disease, using four different phosphate esters (sodium beta-glycerophosphate and adenosine triphosphate at pH 9, riboflavin 5'-phosphate at pH 9.2 and 5'-monophosphoric acid at pH 8.3). In fetal lymph nodes, using 5'-monophosphoric acid, an outstanding positive activity was noticed in the lymphatic follicles. With the other three substrates there was either no nodular reaction or just a narrow rim of positive activity around the follicles, the internodular tissue being negative with all four substrates used. With chronic non-specific lymphadenitis the enzyme hydrolysing the three substrates (beta-glycerophosphate, riboflavin 5'-phosphate and adenosine triphosphate) began to make their appearance. It seems that with lymphadenitis, a qualitative change of the phosphatase activity takes place. A special characteristic pattern of phosphatase activity has been described in both 'early' and 'caseating' tuberculous lymphadenitis. In malignant lymphomas it was noticed that no activity was encountered with any of the four substrates in reticulum cell sarcoma. However, in lymphosarcoma a positive activity was obtained when either beta-glycerophosphate or adenosine triphosphate substrates was used, to the extent that one can depend upon this characteristic phosphatase activity in differentiating between reticulum cell sarcoma and lymphosarcoma. However, no enzymatic activity was obtained when the other two phosphate esters were used.     

Comparative studies on the gastric glycopeptide in eleven animal species.

1. Glycopeptides in the stomachs of eleven mammalian species, including human, rabbit, horse, cow, pig, goat, sheep, dog, cat, guinea pig and rat were assayed by determining the carbohydrate content of materials which remained after proteolysis. 2. The glycopeptide content was higher in the mucosa than in the muscular layer including serosa, especially in the porcine stomach and the fourth stomachs of the ruminants than in the stomachs of any other animals. 3. The glycopeptide, which was stained with both alcian blue and PAS, was absent or sparingly present in the mucosae of the human, rabbit, horse stomachs and in the mucosae of the first to third stomachs of the cow, goat and sheep, whereas in the mucosae of the pig, dog, cat, guinea pig and rat stomachs and in the mucosae of the fourth stomachs of the cow, goat and sheep, it was found in noticeable extents.     

Soluble and membrane-bound polyphosphoinositide phosphohydrolases in mammalian erythrocytes

Phosphatases and phosphodiesterases that hydrolyse polyphosphoinositides are described in both membrane and cytosol fractions of human, pig, rat, rabbit, and sheep erythrocytes using exogenous substrates. With suitably optimized assay conditions, Ca2+-dependent phosphatidylinositol bisphosphate (PIP2) phosphodiesterase activity was found in the hemoglobin-free cytosol fraction, as well as the membrane. Membrane activity is completely dependent upon Triton X-100 and salt and inhibited by cetyltrimethylammonium bromide (CTAB), while the soluble activity requires CTAB and is inhibited by Triton. A low Ca2+-dependent PIP2 phosphatase activity, not present in other tissues, was also detected. The cation-independent phosphatidylinositol phosphate (PIP) phosphatase is localized in the membrane in most species, while the diesterase and the PIP2 phosphatases (both Mg2+ and Ca2+ dependent) are localized in the cytosol. Rat and rabbit erythrocytes are atypical in having a substantial proportion of their Mg2+-dependent PIP2 phosphatase activities in the membrane. All activities are lowest in sheep erythrocytes, except the PIP phosphatase, most of which is soluble in this species. Ca2+-dependent PIP2 phosphatase activity is not correlated with the activity or subcellular distribution of any of the other hydrolases and seems to be a separate enzyme. All the phosphoinositide hydrolase activities, particularly the diesterase, are orders of magnitude lower in erythrocytes than in other tissues. Both soluble and membrane diesterase activities are lost as erythrocytes age. Soluble polyphosphoinositide diesterase does not seem to be active with membrane-bound substrate, since pig and sheep erythrocytes that have negligible membrane activity do not respond to Ca2+ loading, yet have substantial diesterase activity in the cytosol. This supports the view that the diesterase is not physiologically functional in normal erythrocytes.     

Quantitation and immunohistochemistry of catecholamines in the posterior segment of the eye

Dopamine, noradrenaline, and adrenaline were assayed with HPLC in the light adapted retinae of carp, frog, chicken, pigeon, rat, guinea-pig, rabbit, cat, pig and cow. Dopamine varied from 0.6 to 2.6 nmol/g wet weight and was not influenced by sympathectomy. The dopamine figures agree with previously published results. Noradrenaline concentrations varied from not detectable to 0.06 nmol/g wet weight in different species. Homolateral sympathectomy significantly decreased the noradrenaline figure in rabbits. There are no previous figures for noradrenaline for most of the species. Adrenaline was not detected in any species. Immunohistochemical analysis showed noradrenaline to be present in choroidal nerves, but noradrenaline immuno-reactivity was not seen in the retina (chicken, rat, guinea-pig, rabbit, cat, cow). It is concluded that dopamine is the major catecholamine in the retina. Noradrenaline was found present only in minute amounts in the assays, and much of its was likely to stem from sympathetic nerve fibres. The study did not demonstrate any noradrenergic neurons in the retina.     

Phosphatase activity in the lymph during a fever reaction]

Activity of acid and alkaline phosphatases, as well as isoenzymes of alkaline phosphatase in the lymph of the thoracic lymphatic duct, hepatic lymph and the peripheral blood have been studied on rabbits in the dynamics of the fever reaction of different duration. The fever reaction was followed by enzyme activity increase in all the body biologic fluids. However the degree of increase of their activity in the lymph was greater that that in the blood. Our data indicate that in the transport of phosphatases released from the tissues in the common circulation the essential role is played by the lymphatic system, the resorption and transport functions of which significantly characterise the dynamics and the level of their changes in the blood in fever reaction.     

Quantitation and immunohistochemistry of catecholamines in the posterior segment of the eye

Summary Dopamine, noradrenaline, and adrenaline were assayed with HPLC in the light adapted retinae of carp, frog, chicken, pigeon, rat, guinea-pig, rabbit, cat, pig and cow. Dopamine varied from 0.6 to 2.6 nmol/g wet weight and was not influenced by sympathectomy. The dopamine figures agree with previously published results. Noradrenaline concentrations varied from not detectable to 0.06 nmol/g wet weight in different species. Homolateral sympathectomy significantly decreased the noradrenaline figure in rabbits. There are no previous figures for noradrenaline for most of the species. Adrenaline was not detected in any species. Immunohistochemical analysis showed noradrenaline to be present in choroidal nerves, but noradrenaline immuno-reactivity was not seen in the retina (chicken, rat, guinea-pig, rabbit, cat, cow). It is concluded that dopamine is the major catecholamine in the retina. Noradrenaline was found present only in minute amounts in the assays, and much of its was likely to stem from sympathetic nerve fibres. The study did not demonstrate any noradrenergic neurons in the retina.     

Variability of oxygen radical absorbance capacity (ORAC) in different animal species

The oxygen radical absorbance capacity (ORAC) was measured both in whole (ORAC-T) and deproteinized (ORAC-AS) plasma samples of human, pig, cow, rabbit, dog, cat, sheep, horse, dolphin, turkey, guineahen and chicken. In the 12 species, ORAC-T data, expressed as micromoles of peroxyl radicals trapped by 11 of sample, were found scattered between 8,600 and 23,000 μmol/1. The species with the highest ORAC-T values were cat among mammals and chicken among avies. ORAC-AS values ranged between 600 and 2000 μmol/1, with the highest values found in dolphin and sheep among mammals, while chicken was first among avies. In the 12 species, the relative contribution of ORAC-AS in relation to ORAC-T ranged from 5% to 20%. Protein SH-groups and uric acid were measured in plasma of all species, but no significant correlation was found between thiols and ORAC-T values or between uric acid and ORAC-AS values. Our results show that: (1) the ORAC method is reproducible and sensitive enough to be used in the comparison of the peroxyl-radical absorbance capacity of protein and non-protein plasma components in different animal species; (2) both in mammals and in avies, there is a deep intra-class heterogeneity of ORAC-T and ORAC-AS values; (3) by considering most species, plasma proteins and lipoproteins account for about 85-90% of the overall peroxyl-radical trapping capacity. In the dolphin only, the protein contribution decreases to 80%; (4) uric acid accounts for about one-half of the ORAC-AS value in human, guinea-hen and for about one-third in chicken, while it provides a very limited contribution in other species. We conclude that species with the highest ORAC-T, like cat and chicken, or with the highest ORAC-AS, like dolphin, are interesting models to study the reasons of such a marked antioxidant defense in the plasma.     

Uptake and degradation of hyaluronan in lymphatic tissue.

Afferent lymph vessels entering popliteal lymph nodes of sheep were infused with [3H]acetyl-labelled hyaluronan of high Mr (4.3 x 10(6)-5.5 x 10(6)) and low Mr (1.5 x 10(5)). Analysis of efferent lymph and of residues in the nodes showed that hyaluronan presented by this route is taken up and degraded by lymphatic tissue. Labelled residues isolated in node extracts by gel chromatography and h.p.l.c. included N-acetylglucosamine, acetate, water and a fraction provisionally identified as N-acetylglucosamine 6-phosphate. Between 48 and 75% of the infused material was unrecovered, and had been presumably eliminated through the bloodstream as diffusible residues. Rates of degradation reached as high as 43 micrograms/h in a node of 2 g wt. infused with 56 micrograms/h. Some HA passed into efferent lymph and some was detected in the nodes, but fractions of Mr greater than 1 x 10(6) were not found in either. It is concluded that the amounts and Mr values of hyaluronan released from the tissues into peripheral lymph can be significantly underestimated by analysis of efferent lymph, i.e. lymph that has passed through lymph nodes. A substantial role in the normal metabolic turnover of at least one major constituent of intercellular matrix and connective tissue may now be added to the established functions of the lymphatic system.     

Characteristics of the immunosuppressive activity of lymphoid organ cells in the process of sensitization with ram erythrocytes and exposure to antilymphocyte serum

In the adoptive transfer of cells obtained from the thymus, lymph nodes and the spleen to intact syngeneic animals the suppression of immune response was induced by lymph node cells. If the donors were previously sensitized, the cells of the thymus and lymph nodes showed suppressive activity in the adoptive transfer test. A single injection of antilymphocytic serum to the donors of lymphoid cells, previously sensitized with sheep red blood cells, enhanced the immunosuppressing action of thymocytes and lymph node cells.     

Demonstration of 9-O-acetyl-N-acetylneuraminic acid in brain gangliosides from various vertebrates including man

Ganglioside fractions were isolated from brains of man, cow, horse, pig, sheep, cat, rabbit, rat, chicken and codfish. The acylneuraminic acid residues, liberated from these gangliosides by treatment with dilute aqueous acid or neuraminidase, were analysed by the thin-layer chromatography and combined gas-liquid chromatography/mass spectrometry. Small amounts (up to 20%) of 9-O-acetyl-N-acetylneuraminic acid, and in bovine and porcine brain gangliosides also traces of N-glycoloylneuraminic acid, were found in addition to N-acetylneuraminic acid.     

Comparative study of the autonomic innervation of the mammalian ovary,with particular regard to the follicular system

Summary The autonomic innervation of the ovary was studied in 12 mammalian species utilizing the cholinesterase method in combination with pseudocholinesterase inhibition for the cholinergic component, and glyoxylic acid histochemistry together with fluorometric determination of noradrenaline for the adrenergic component. Ovaries from cow, sheep, cat, and guinea pig were very richly supplied with adrenergic nerves in the cortical stroma, particularly enclosing follicles in various stages of development. In the follicular wall the nerve terminals were located in the theca externa, where they ran parallel to the follicular surface. Numerous adrenergic terminals also surrounded ovarian blood vessels. The adrenergic innervation was of intermediary density in the human ovary and in the pig, dog, cat, and opossum. Ovaries from rabbit, mouse and hamster had a sparse adrenergic nerve supply. The amount of intraovarian adrenergic nerves agreed well with the tissue concentration of noradrenaline in the various species. The cholinergic innervation was generally less well developed, but had the same distribution as the adrenergic system around blood vessels and in the ovarian stroma, including follicular walls.