Flt3 ligand plus IL-7 supports the expansion of murine thymic B cell progenitors that can mature intrathymically

Flt3 ligand (flt3L) has potent effects on hemopoietic progenitors, dendritic cells, and B lymphopoiesis. We have investigated the effects of flt3L on intrathymic precursors. The addition of flt3L + IL-7 to lobe submersion cultures of murine fetal thymic lobes resulted in the expansion of an immature population of Thy-1(low), CD44(high), HSA(high) cells. This population contained cells with precursor activity, as determined by their capacity to repopulate deoxyguanosine-treated fetal thymic lobes. Upon reentry to the thymic lobe, flt3L + IL-7-cultured Thy-1(low), CD44(high), HSA(high) cells underwent expansion and differentiation into B cells. Two weeks after fetal thymic organ culture following thymic lobe reconstitution, intrathymic cells were Thy-1-, B220+, and a subset was sIgM+. The intrathymic B cells shared features of adult thymic B cells, including CD5 expression and proliferative responses to IL-4 + IL-5 + CD40 ligand, but not to LPS or soluble anti-IgM. Ig production was noted upon stimulation with IL-4 + IL-5 + LPS and IL-4 + IL-5 + CD40 ligand. In conclusion, we have demonstrated that flt3L + IL-7 supports the expansion of a subset of progenitors present in the fetal thymus. The cultured progenitors can repopulate a fetal thymic lobe and develop into mature functional B cells, demonstrating that the fetal thymus is able to support B cell as well as T cell development.     

Use of cultured thymic tissues for the regeneration of the thymus

An account of research conducted on the transplantation of thymic cells and tissues in order to restore the functional activity of the thymus is reviewed, and discussed in the context of current concepts. Although most rodent work has been conducted on the transplantation of cultured fragments under the kidney capsule, human transplantation studies and models have used other sites or other species such as the severe combined immunodeficient mouse as hosts. The factors affecting the growth of thymic cells in culture is considered in detail since the methodology can strongly influence the cells favoured under culture conditions. An extension of this work to characterize both thymic fragments implanted under the kidney capsule of rats and cultured thymic cells has recently led to the appreciation that the adult thymus must contain a small number of neural crest-like cells. These cells have a high level of proliferation in the implanted fragments, expand in culture, and belong to a family of cytokeratin-positive cells exhibiting immunoreactivity for a wide range of neuropeptides and transmitters. Thus primary cultures of thymus can contain a wide range of glia-like cells. This can be explained by the fact that the thymus, in addition to having a mesenchymal neural crest component, is probably derived from cardiac neural crest. Closely associated neural crest also has glia-like properties (the supporting cells of the enteric nervous system). These finding can account for the large number of reports of epithelial cells of the adult thymus being immunoreactive to antibodies raised to neuroendocrine and neurotransmitters. Neuroimmune interactions in the thymus are more fundamental than previous work had suggested.     

MR volumetric analysis of the human entorhinal, perirhinal, and temporopolar cortices

Cell-adhesion molecules (CAMs) are thought to play crucial roles in development and plasticity in the nervous system. This study tested for a role for cell adhesion and in particular, the recognition of two glycosyl epitopes (HNK-1 and oligomannoside) in the activity-driven sharpening of the retinotopic map formed by the regenerating retinal fibers of goldfish. HNK-1 is a prominent glycosyl epitope on many CAMs and extracellular matrix (ECM) molecules, including NCAM, L1, ependymin, and integrins, which have all been implicated in synaptic plasticity. To test for a role of HNK-1 in the sharpening process, we used osmotic minipumps to infuse HNK-1 antibodies for 7-21 days into the tectal ventricle starting at 18 days after optic nerve crush. Retinotopic maps recorded at 76-86 days postcrush showed a lack of sharpening similar to that seen previously with two antibodies to ependymin, an HNK-1-positive ECM component present in cerebrospinal fluid. The multiunit receptive fields at each point averaged 26 degrees versus 11-12 degrees in regenerates infused with control antibodies or Ringer's alone. The HNK-1 epitope also binds to the G2 domain of laminin to mediate neuron-ECM adhesion. To test for a role for laminin, a polyclonal antibody was similarly infused and also prevented sharpening to approximately the same degree. The results support a role for the HNK-1 epitope and laminin in retinotectal sharpening. The oligomannoside epitope (recognized by monoclonal antibody L3) on the CAM L1 interacts with NCAM on the same cell to promote stronger L1 homophilic interactions between cells. Both an L1-like molecule and NCAM are prominently reexpressed in the regenerating retinotectal system of fish. Infusion of oligomannosidic glycopeptides resulted in decreased sharpening, with multiunit receptive fields that averaged 22.7 degrees. Infusions of mannose-poor glycopeptides less prominently disrupted sharpening, with average multiunit receptive fields of 18 degrees. Thus, oligomannosidic glycans in particular may play a role in retinotopic sharpening. Blocking glycan-mediated interactions between CAMs and ECM molecules could decrease the extent of exploratory growth of retinal axon collaterals, preventing them from finding their retinotopic sites, or could interfere with L1 or NCAM and laminin binding at the synaptic densities preventing stabilization of retinotopically appropriate synapses. Together, these results support a prominent role for cell adhesion and glycan epitopes in visual synaptic plasticity.     

Recognition of human recombinant myelin associated glycoprotein by anti-carbohydrate antibodies of the L2/HNK-1 family

The L2 and HNK-1 monoclonal antibodies recognize carbohydrate determinants containing sulfate-3-glucuronate that are prominent on cells of neural crest lineages. In humans these epitopes are most abundant on the Myelin Associated Glycoprotein and it was assumed that they co-localize on the same molecules. Recently, in vitro synthesized carbohydrates have provided a basis for the different recognition requirements of these two antibodies. We now provide in vivo evidence that a human melanoma cell line can produce glycoproteins such as fibronectin, which is recognized by both the L2 and HNK-1 antibodies, and simultaneously a transfected Myelin Associated Glycoprotein carrying only L2-type carbohydrates. Conceivably, the differential expression of L2- and HNK-1 type glycans could have a role in development.     

Recent thymic emigrants in the rat express a unique antigenic phenotype and undergo post-thymic maturation in peripheral lymphoid tissues

Studies of the functional properties and developmental potentials of immediate post-thymic cells have been hampered by the lack of reliable markers with which to distinguish recent thymic emigrants (RTE) from the bulk of peripheral T cells. In the present study, the intrathymic FITC-labeling technique was used in concert with three-color flow-cytometric analysis to identify, phenotypically characterize, and study the short term fate of RTE in normal rats. The results indicated that between 3 and 4% of total T cells in lymph node and spleen of 5- to 8-wk-old rats had been released from the thymus within the preceding 24 h. Unlike the bulk of the peripheral T cells, which had a Thy1-, CD45RC+, and/or RT6+ phenotype, these RTE were Thy1+, CD45RC-, and RT6-. Furthermore, two discrete subsets of RTE were defined: a major subset (approximately 95%) of CD4+ or 8+ (single positive), TCR-alpha beta hi T cells that resembled medullary thymocytes; and a minor subset (approximately 5%) of CD4+ and 8+ (double positive), TCR-alpha beta low T cells that resembled cortical thymocytes. The following evidence suggested that most if not all Thy1+ T cells in young adult rats are RTE and their immediate descendants: 1) thymectomy (but not sham thymectomy) selectively depleted Thy1+ T cells from lymph node within 3 to 7 days, even in adrenalectomized rats; 2) most FITC-labeled RTE differentiated into Thy1-, CD45RC+, RT6+ T cells within 7 days of release from the thymus; 3) transitional phenotypes of Thy1+ T cells, including Thy1low, CD45RC+, and RT6+, were observed in normal, as well as in intrathymic, FITC-injected rats; 4) most T cells in neonatal rats were Thy1+ and RT6-, whereas their descendants were Thy1- and RT6+. These experiments demonstrate that most RTE in normal rats are phenotypically (and presumably developmentally) immature at the time of release from the thymus, and progressively acquire the phenotypic attributes of more mature T cells post-thymically. These unique phenotypic attributes should expedite the isolation of RTE and their immediate descendants for definitive studies of their developmental and functional properties.     

The role of zinc in pre- and postnatal mammalian thymic immunohistogenesis

Histogenetically, the thymus is the primary lymphopoietic organ and provides an optimal microenvironment for the differentiation of T lymphocytes, independently of the influence of foreign antigens. Lymphocytes with diverse potential are produced in a protective microenvironment optimal for their maturation, whose dual cellular network is provided by endodermally derived RE cells and numerous ectomesenchymal cells derived from the neural crest. The full development of intrathymic hematopoiesis depends upon the successful completion of a series of well coordinated cellular interactions between widely divergent (in terms of origin) cells [epithelium (primitive pharynx); ectomesenchyrne (neural crest); and PHSCs (yolk sac, fetal liver)]. The cells of the thymic epithelial primordium do not proliferate in the absence of "inductive" interactions with the ectomesenchyme. Moreover, the nature of the mesenchyme determines the behavior of the thymic epithelial anlagen. The ectomesenchymal origin of chemotactic stem cell factor secretion, responsible for hemopoietic stem cell immigration, is a distinct possibility. The human thymus is a generalized hematopoietic tissue with between 7 to 9 weeks of ontogenesis. In human and dog fetuses various elements of mammalian hematopoiesis were identified intrathymically: B lymphocytes, plasma cells, erythropoietic and granulocytopoietic (neutrophils and eosinophils) cells, antigen presenting dendritic cells, and mast cells. Our light and ultrastructural (transmission and scanning), as well as immunocytochemical observations have established that during the embryonal and fetal period, the thymus is seeded by pluripotent, yolk sac derived PHSCs characterized by the following immunophenotype CD34+CD43+CD38-Lin-HLA-DR+CD69+. Stem cell c-kit tyrosine kinase (also referred to as mast cell growth factor, stem cell factor, or steel factor) in combination with autocrine and paracrine growth factors and cytokines (IL-3, IL-4, IL-5, IL-6, IL-7, G-CSF, etc.) stimulates myelopoiesis, including erythropoiesis, as well as lymphopoiesis. These hematopoietic growth factors are produced by activated lymphoblastic cells and stromal RE cells under the influence of immunoneuroendocrine regulation, supported by the finding that experimental or spontaneous, in vivo neural crest ablation during early mammalian ontogenesis always results in an abnormal development of the thymus, as well as the heart and great vessels, thyroid, and parathyroid glands.     

The drug abuse problem in Peninsular Malaysia: parent and child differences in knowledge, attitudes and perceptions

Neural crest cell migration in the gut and the growth of the mid- and hindgut of avian embryos was investigated by a combination of whole-mount immunofluorescence of the HNK-1 neural crest marker epitope, chorioallantoic membrane grafting and morphometry. HNK-1-labelled cells advanced rostrocaudally in the gut of quail embryos (to the duodenum by stage HH 21, to the umbilicus by HH 25, to the ceca by HH 27, to the cloaca by HH 33). The timetable in chick embryos appeared to be slightly slower, but neural cells were obscured by background fluorescence in this species. More rostral regions of the gut commenced rapid growth earlier than more caudal regions (preumbilical small intestine after HH 26, postumbilical small intestine after HH 27 and colorectum after HH 28), and the small intestine and ceca grew most rapidly in length while the colorectum grew most rapidly in diameter. The rates of growth of the gut were low prior to the stage when HNK-1-labelled cells normally arrive in the small intestine, ceca and rostral colorectum, but increased dramatically after arrival. In the caudal colorectum rapid growth had commenced at the time of arrival of these cells. These data are consistent with the idea that a delay in arrival of vagal neural crest cells at any point in the intestine could jeopardize the ability of the cells to fully populate the remainder of the gut, due to the normal growth spurt causing the migration end-point to recede faster than the rate of neural crest cell migration. Thus, a mismatch in timing of neural crest cell migration and gut growth could play a role in the etiology of some forms of Hirschsprung's disease.     

Vasoactive intestinal polypeptide (VIP) innervation of rat spleen, thymus, and lymph nodes

In the thymus, VIP-positive (+) fibers were found in the capsular/septal system, cortex, and medulla. In the spleen, VIP+ nerves coursed along large arteries and central arterioles, and in the white pulp, venous/trabecular system, and red pulp. Splenic VIP innervation was more robust in Long-Evans hooded rats than in Fischer 344 rats. VIP+ nerves in mesenteric lymph nodes were found in the cortex, and along the cortical vasculature and medullary cords. No VIP innervation was observed in popliteal lymph nodes. Immunocytes also were VIP+, suggesting that both neural and cellular synthesis of VIP contributes to VIP concentration in lymphoid organs. Surgical sympathectomy did not alter splenic or thymic VIP content, respectively, and VIP innervation of these organs was not altered, suggesting an origin for VIP+ nerves other than the sympathetic nervous system.     

Rhesus thymic/liver xenografts in severe combined immunodeficient mice: immunologic reconstitution and intrathymic infection with simian immunodeficiency virus

By serving as host recipients of xenografts from both humans and animals, severe combined immunodeficient (SCID) mice have become valuable to many laboratories interested in examining the pathophysiology of different diseases. To gain insight into the usefulness of the SCID mutation in retrovirus research, rhesus monkey fetal hematolymphoid tissues (liver and thymus) were used to construct a SCID-rhesus chimeric mouse (SCID-rh) and were engrafted in the renal capsule. The size and maturation of the thymic engrafts were monitored grossly, histologically, and immunologically. SCID mice were tolerant to rhesus tissues, and thymic engrafts contained thymocytes at different stages of maturation and differentiation that had morphologic features similar to age-matched rhesus thymus. Mature single positive CD2+, CD4+, and CD8+ T lymphocytes that were phenotypically similar to rhesus T lymphocytes were present at low levels (2% to 5%) in the peripheral blood and at moderately higher levels (7% to 15%) in the spleens of SCID-rh mice obtained between 12 and 15 weeks after thymus/liver engraftment. Within 3 weeks after engraftment, > 85% of the thymocytes in the thymic engrafts were immature double positive CD4+CD8+ T cells. The highest number of positive cells were seen in thymic engrafts obtained at 12 to 18 weeks. During these weeks, > 90% of the cells were double positive (CD2+CD4+, CD2+CD8+, and CD4+CD8+). After infection of the engrafted thymus tissue with simian immonodeficiency virus (SIVmac239), PCR analysis revealed successful viral infection of engrafts at 2 and 4 weeks after infection. No significant histopathologic and flow cytometric changes were observed in the thymic engrafts at 2 and 4 weeks after infection. An unrelated lesion of thymic lymphomas involving the SCID host thymus was seen in 12% of the mice. The data presented herein suggest that the SCID-rh is a valuable model for specific studies related to thymus-retrovirus interaction and that it could be used for further studies. The results are discussed in relation to current knowledge of thymus involvement during simian and human immunodeficiency virus infection.     

Human natural killer cell committed thymocytes and their relation to the T cell lineage

Recent studies have demonstrated that mature natural killer (NK) cells can be grown from human triple negative (TN; CD3-, CD4-, CD8-) thymocytes, suggesting that a common NK/T cell precursor exists within the thymus that can give rise to both NK cells and T cells under appropriate conditions. In the present study, we have investigated human fetal and postnatal thymus to determine whether NK cells and their precursors exist within this tissue and whether NK cells can be distinguished from T cell progenitors. Based on the surface expression of CD56 (an NK cell-associated antigen) and CD5 (a T cell-associated antigen), three phenotypically distinctive populations of TN thymocytes were identified. CD56+, CD5-; CD56-, CD5-, and CD56-, CD5+. The CD56+, CD5- population of TN thymocytes, although displaying a low cytolytic function against NK sensitive tumor cell targets, were similar in antigenic phenotype to fetal liver NK cells, gave rise to NK cell clones, and were unable to generate T cells in mouse fetal thymic organ cultures (mFTOC). This population of thymocytes represents a relatively mature population of lineage-committed NK cells. The CD56-, CD5- population of TN thymocytes were similar to thymic NK cells in antigenic phenotype and NK cell clonogenic potential. Clones derived from this population of TN thymocytes acquired CD56 surface expression and NK cell cytolytic function. CD56-, CD5- TN thymocytes thus contain a novel population of NK cell-committed precursors. The CD56-, CD5- population of TN thymocytes also contains a small percentage of CD34+ cells, which demonstrate no in vitro clonogenic potential, but possess T cell reconstituting capabilities in mFTOC. The majority of TN thymocytes do not express CD56, but coexpress CD34 and CD5. These CD56-, CD5+, CD34+ cells demonstrate no NK or T cell clonogenic potential, but are extremely efficient in repopulating mFTOC and differentiating into CD3+, CD4+, CD8+ T cells. The results of this investigation have identified NK cells and NK cell precursors in the human thymus and have shown that these cell types are unable to differentiate along the T cell lineage pathway. Thus, while a common NK/T cell progenitor likely exists, once committed to the NK cell lineage these cells no longer have the capacity to develop along the T cell developmental pathway.     

Estrogen blocks early T cell development in the thymus

PROBLEM: Pregnancy and estrogen are known to suppress B lymphopoiesis as well as lead to thymic involution in the mouse. Additionally, estrogen deficiency by oophorectomy reportedly causes a selective increase in the B220+ B cells in the murine bone marrow. The purpose of this study was to determine if estrogens played a regulatory role in T cell development. METHODS: The first experimental group consisted of 5-6-week-old Balb/c mice that received subcutaneous pellets of placebo, estriol, estradiol, or progesterone. The thymus glands were examined 2-4 weeks after treatment. The second group consisted of 6-week-old Balb/c mice who underwent either bilateral oophorectomy or a sham procedure. Two weeks after the surgery, extensive phenotypic characterization of the thymus and spleen cells was performed by flow cytometry using monoclonal antibodies to surface markers of T cell subsets. RESULTS: Estrogen treatment causes a dramatic reduction of thymic size and cellularity. All defined T cell subsets of CD4 and CD8 were reduced, with a disproportionate loss of CD4+CD8+ double positive cells. Examination of the triple negative (CD3-CD4-CD8-) subset revealed a striking loss of TN developmental progression of the early precursor cells. Based on the expression of CD44 (pgp-1) and CD25 (IL-2R alpha) markers, the TN thymic compartment was composed almost entirely of the earliest population (CD44+, CD25-), with the remaining maturational stages (CD44+, CD25+; CD44-, CD25+; CD44-, CD25-) depleted. In contrast, all T cell developmental stages in the thymus were found to be in normal proportions in the oophorectomized mice, with no differences in the splenic T and B cell subsets. CONCLUSIONS: The study demonstrates that estrogen but not progesterone blocks T cell development in the thymus. However, contrary to our expectation, estrogen deprivation by oophorectomy does not enhance T cell development.     

Distribution of the perivascular nerve Ca2+ receptor in rat arteries

We recently showed that perivascular sensory nerves of mesenteric branch arteries express a receptor for extracellular Ca2+ (CaR), and reported data indicating that this CaR mediates relaxation induced by physiologic levels of Ca2+. We have now tested whether the perivascular sensory nerve CaR-linked dilator system is a local phenomenon restricted to the mesentery, or is present in other circulations. Vessels from the mesenteric, renal, coronary, and cerebral circulations were studied. Immunocytochemical analysis was performed using anti-CaR and anti-neural cell adhesion molecule (NCAM) antibodies. Wire myography was used to assess contraction and relaxation. Although perivascular nerves of all arteries stained for CaR protein, there were regional differences. A morphometric method used to estimate CaR positive nerve density revealed the following rank order: mesenteric branch artery > basilar artery = renal interlobar artery > main renal trunk artery > left anterior descending coronary artery. Vessels from the mesentery, renal, coronary, and cerebral circulations showed nerve-dependent relaxation in response to electrical field stimulation (EFS) when precontracted with serotonin in the presence of guanethidine. The degree of Ca2+-induced relaxation of mesenteric, renal, and cerebral arteries positively correlated with the magnitude of EFS-induced relaxation. In contrast, coronary arteries contracted at Ca2+ levels between 1.5 and 3 mmol L(-1), and relaxed to a small degree to 5 mmol L(-1) Ca2+. Thus, a functional perivascular sensory nerve CaR-linked dilator system is present to varying degrees in the mesenteric, renal, and cerebral circulations, but only to a very limited extent in the coronary circulation.     

A tumor-specific Th2 clone initiating tumor rejection via primed CD8+ cytotoxic T-lymphocyte activation in mice

We established a CD4+ T-cell clone specific for syngeneic methylcholanthrene-induced sarcoma, S1509a raised in an A/J mouse, involved in tumor regression. The phenotype of the T-cell clone was CD3+, TCR-beta+, CD4+, CD45RB+, LFA-1+, ICAM-1+, CD44+, and VLA-4+. The CD4+ T-cell clone specifically proliferated through antigen stimulation with attenuated S1509a in the presence of syngeneic accessory cells, and this antigen-induced proliferation was inhibited with anti-CD4 and anti-I-Ek monoclonal antibodies. The CD4+ T-cell clone designated YS1093 secreted interleukin (IL) 4, IL-5, and IL-6, but not IFN-gamma, tumor necrosis factor alpha, or IL-2, thus indicating that the clone belongs to the Th2 type. YS1093 cells and their culture supernatant after antigen stimulation augmented the primed cytotoxic T lymphocyte killing activity at the effector phase. YS1093 cells having Th2-type characteristics made the homologous growing tumor regress in the tumor-bearing syngeneic mice when YS1093 cells were transferred into the tumor-bearing mice i.v. The in vivo tumor regression initiated by YS1093 cell transfer essentially required the presence of CD8+ T cells in the tumor-bearing hosts, thus suggesting that some specific Th2 cells are positively involved in tumor regression by activating primed CD8+ cytotoxic T lymphocytes against the homologous tumor in situ.     

Infantile muscle phosphorylase-b-kinase deficiency. A case report

A 13-year-old male developed thymic non-Hodgkin's lymphoma. Microscopically, the tumor was composed of large cells, resembling centroblasts. Immunohistochemically, the tumor demonstrated leukocyte common antigen+, L26 (B-cell)+, UCHL1 (T-cell)-, suggesting the B-cell phenotype. In contrast to the terminally differentiated phenotype (CD10-, surface immunoglobulin-) observed in adult cases, flow cytometric analysis showed that they were relatively immature: CD10+, CD19+, HLA-DR-, IgM+/-, kappa+. He was successfully treated with intensive chemotherapy. Since childhood thymic lymphomas are exclusively small non-cleaved cell lymphoma with T-cell phenotype, this case represents a unique entity in children.     

Characterization of CD34+ thymic stromal cells located in the subcapsular cortex of the human thymus

In this paper we report that suspensions of human fetal thymocytes contain cells that express high levels of CD34 and Thy-1. These cells were characterized with regard to location within the thymus, phenotype, and function. Confocal laser scan analysis of frozen sections of fetal thymus with anti-CD34 and Thy-1 antibodies revealed that the double-labeled cells were located in the pericortical area. In addition, it was found that the CD34+Thy-1+ cells lacked CD45 and CD50, indicating that these cells are not of hematopoietic origin; this was confirmed by the finding that these cells could be cultured as adherent cells in a medium with cholera toxin and dexamethasone, but failed to grow in mixtures of hematopoietic growth factors. Further analysis indicated that most cultured CD34+Thy-1+ cells expressed cytokeratin (CK) 14 but lacked CK 13, suggesting that these cells are immature epithelial cells. Cultured CD34+Thy-1+ cells were able to induce differentiation of CD1-CD34+CD3-CD4-CD8- thymic precursors into CD4+CD8+ cells in a reaggregate culture in the absence of exogenous cytokines. The CD4+CD8+ cells that developed in these cultures did not express CD3, indicating that CD34+Thy-1+ thymic stromal cells are not capable of completing full T cell differentiation of thymic hematopoietic progenitor cells.     

Thymic alterations in feline GM1 gangliosidosis

GM1 gangliosidosis is an inherited metabolic disease characterized by progressive neurological deterioration with premature death seen in children and numerous animals, including cats. We have observed that thymuses from affected cats greater than seven months of age (GM1 mutant cats) show marked thymic reduction compared to age-matched normal cats. The studies reported here were done to describe alterations in the thymus prior to (less then 90 days of age) and during the development of mild (90 to 210 days of age) to severe (greater than 210 days of age) progressive neurologic disease and to explore the pathogenesis of the thymic abnormality. Although histologic examination of the thymus from GM1 affected cats less than 210 days of age showed no significant differences from age-matched control cats, thymuses from GM1 mutant cats greater than 210 days of age were significantly reduced in size (approximately 3-fold). Histologic sections of lymph nodes, adrenal glands, and spleens from GM1 gangliosidosis-affected cats showed no significant differences. Flow cytometric analyses showed a marked decrease in the percentage of immature CD4+CD8+ thymocytes (p < 0.001) and significantly increased CD4-CD8+ cells (p < 0.01) in GM1 mutant cats greater than 210 days of age when compared to normal age matched cats. Co-labelling with CD4, CD8, and CD5 indicated an increase in the percentage of GM1 mutant cat thymocytes at this age which were CD5high, suggesting the presence of more mature cells. Cytometric analyses of subpopulations of peripheral lymphocytes indicated an increase in CD4-CD8+ cells (p < 0.05) with concurrent decreases in CD4+CD8- and CD4-CD8- cells (which were not significant). Similar analyses of thymocyte and lymphocyte subpopulations from cats < 210 days of age showed no significant differences between GM1 mutant and normal cells. GM1 mutant cats at all ages had increased surface binding of Cholera toxin B on thymocytes, indicating increased surface GM1 ganglioside expression. Increases were highly significant in GM1 mutant cats greater than 210 days of age. In situ labelling for apoptosis was increased in GM1 mutant cats between 90 to 200 days of age when thymic masses were within normal limits. In GM1 mutant cats over 200 days of age, decreased labelling was observed when thymic mass was reduced and the CD4+CD8+ subpopulation, known to be very susceptible to apoptosis, was significantly decreased. These data describe premature thymic involution in feline GM1 gangliosidosis and suggest that increased surface GM1 gangliosides alters thymocyte development in these cats.     

Differential distribution of the HNK-1 carbohydrate epitope in the vertebrate retina

The expression of the cell adhesion-related HNK-1 carbohydrate epitope in the retina and ciliary body was studied in different vertebrates and in man. A series of eyes from 4 fish, 5 bird, and 9 mammalian species was analyzed by immunohistochemistry with monoclonal antibodies (MAb) HNK-1 and VC1.1 to the HNK-1 epitope, and with MAb SY38 to synaptophysin. Additionally, 7 morphologically normal human eyes were studied. In all fishes, as well as in baboons and man, the radial glia and all retinal layers except the photoreceptor cell layer were immunoreactive for the HNK-1 epitope. In all birds, the nerve fiber layer and both plexiform layers were labelled. In nonprimate mammals only the plexiform layers were immunoreactive. Fine differences in this general immunoreaction pattern were seen in different species. Mab SY38 labeled both plexiform layers of mammals only. In the ciliary body, immunoreaction for the HNK-1 epitope was seen in the inner connective tissue layer only in man, but the ciliary nerves were labelled in all species except the mouse and rat. The HNK-1 epitope seems to be phylogenetically conserved in the retina, where the HNK-1 immunoreactive plexiform layers possibly are overlapped with HNK-1 reactive radial glial cells in fishes and primates. Instead in the inner connective tissue layer of the ciliary body, the HNK-1 epitope is not phylogenetically conserved.     

NCAM140 interacts with the focal adhesion kinase p125(fak) and the SRC-related tyrosine kinase p59(fyn)

Axonal growth cones respond to adhesion molecules and extracellular matrix components by rapid morphological changes and growth rate modification. Neurite outgrowth mediated by the neural cell adhesion molecule (NCAM) requires the src family tyrosine kinase p59(fyn) in nerve growth cones, but the molecular basis for this interaction has not been defined. The NCAM140 isoform, which is found in migrating growth cones, selectively co-immunoprecipitated with p59(fyn) from nonionic detergent (Brij 96) extracts of early postnatal mouse cerebellum and transfected rat B35 neuroblastoma and COS-7 cells. p59(fyn) did not associate significantly with the NCAM180 isoform, which is found at sites of stable neural cell contacts, or with the glycophosphatidylinositol-linked NCAM120 isoform. pp60(c-)src, a tyrosine kinase that promotes neurite growth on the neuronal cell adhesion molecule L1, did not interact with any NCAM isoform. Whereas p59(fyn) was constitutively associated with NCAM140, the focal adhesion kinase p125(fak), a nonreceptor tyrosine kinase known to mediate integrin-dependent signaling, became recruited to the NCAM140-p59(fyn) complex when cells were reacted with antibodies against the extracellular region of NCAM. Treatment of cells with a soluble NCAM fusion protein or with NCAM antibodies caused a rapid and transient increase in tyrosine phosphorylation of p125(fak) and p59(fyn). These results suggest that NCAM140 binding interactions at the cell surface induce the assembly of a molecular complex of NCAM140, p125(fak), and p59(fyn) and activate the catalytic function of these tyrosine kinases, initiating a signaling cascade that may modulate growth cone migration.