Identification of a specific Sertoli cell marker, Sox9, for use in transplantation.

The immunoprivileged environment of the testes was first described in the 1930s, and the Sertoli cell was later identified as the main cell type responsible for this phenomenon. Recent work has examined the possibility of recreating this immunoprivileged environment at heterotopic sites using isolated Sertoli cells. These studies have focused on protection of pancreatic islets and neuronal cells from immune destruction in the hopes of reversing type I diabetes and Parkinson's disease. The absence of a definitive marker for identifying Sertoli cells at the transplant site has been an obstacle to this research. The current study examines the presence of a nuclear transcription factor, Sox9, which is preferentially expressed in Sertoli cells. Syngeneic Lewis rat Sertoli cells were transplanted into the renal subcapsular space and a subcutaneous site in Lewis female rats and examined histologically 21 days later. In addition, porcine Sertoli cells were transplanted into the renal subcapsular space in female SCID mice. Control testes and the transplant sites were examined immunohistochemically using an antibody to Sox9. The results from the study demonstrate that Sox9 expression is restricted to the Sertoli cells of the neonatal rat and porcine testis, indicating high homology between species. In addition, Sox9 expression was also observed in the testicular-like tubules that formed in both syngeneic and xenogeneic heterotopic transplants in rats and SCID mice. The Sox9 expression was restricted to the regions where Sertoli cells would be found in the native testis. These results suggest that the Sox9 protein is a useful marker in identifying Sertoli cells in heterotopic transplants in a manner similar to insulin as a marker for pancreatic islets.

Selective mitomycin C and cyclophosphamide induction of apoptosis in differentiating B lymphocytes compared to T lymphocytes in vivo.

Differentiating B and T lymphocytes differ in sensitivity to a number of environmental toxins and anticancer agents. B lymphocytes are susceptible and T lymphocytes resistant to killing by cyclophosphamide (Cy) metabolites capable of forming DNA interstrand cross-links. However, the mechanisms responsible for the rapid killing and loss of bursal-resident B lymphocytes are unknown. Therefore, we investigated the cellular mechanisms of selective toxicity of two cross-linking drugs, mitomycin C (MMC) and Cy, towards differentiating B and T lymphocyte populations using the chicken embryo model system. Viability of bursal-resident B lymphocytes (bursacytes) decreased starting at 5 h post exposure (PE) to MMC, and was maximally reduced by 71.6% by 10 h PE at the highest dose examined (9.0 micrograms MMC/g). Dose-dependent increases in the percentage of apoptotic bursacytes were observed as early as 5 h PE, and increased to 72% by 10 h PE. This was accompanied by reductions in bursacyte numbers. Cy also induced apoptosis in bursacytes. In contrast, thymus-resident lymphocytes (thymocytes) were much more resistant to the toxic effects of MMC and Cy. Viability of thymocytes was reduced by only 10% in the 9.0 micrograms/g MMC treatment group. In addition, the percentage of thymocytes engaged in apoptosis was much lower than that for bursacytes. MMC induced modest cell cycle inhibition in bursacytes and thymocytes. These data strongly suggest that MMC and Cy-induced diferential toxicity involves primarily early and extensive triggering of apoptosis in differentiating B lymphocytes, leading to rapid reduction of lymphocyte numbers in the embryonic bursa.

Selective expression of major histocompatibility complex (MHC) antigens and modulation of T-cell differentiation in chickens with increased MHC-chromosome dosages.

Increased dosage of genes belonging to the immunoglobulin superfamily may be responsible for some of the less noticeable but targeted phenotypic disturbances seen in trisomy conditions of humans and animals. We used an avian aneuploidy model to study the specific effects of extra major histocompatibility complex (MHC)-microchromosome dosage on the progression of thymocyte differentiation through a broad period of embryonic and neonatal development. The particular goal in the present investigation was to determine whether a reduction in the number of thymocytes, previously observed in the developing thymus of MHC aneuploids, is accompanied by particular alterations in thymocyte differentiation. We hypothesized that the subpopulation structure and/or developmental pattern for thymocyte differentiation are characteristically perturbed (delayed or modified) by increased MHC-chromosome dosage in cells. The regulation of MHC surface antigen expression in aneuploid thymocytes was also studied to detect dosage-dependent expression for one and possibly more sub-regions (class I, II, IV) of the avian MHC. Surface densities of MHC class I antigens on thymocytes were increased significantly at all ages studied, for example by 15% and 45% in trisomics and tetrasomics, respectively at 22 days post-hatching. The surface density of CT1 antigen, a thymocyte-specific marker, was also increased in a dosage-dependent manner, but only in juveniles. Increases in the proportion of alpha beta 1, TCR+ and CD3+ thymocytes were observed in juveniles, with no alterations in other TCR-expressing thymocytes. No major alterations in CD4 and CD8 thymocyte populations were observed. These results demonstrate a targeted effect of extra MHC-chromosome dosage towards enhanced class I and CT1, and not class II or IV, expression. The increased MHC-microchromosome dosage appears to influence primarily immature thymocytes expressing alpha beta 1 TCR and CD3.

MHC dosage effects on primary immune organ development in the chicken.

Immune development in vertebrates is thought to be influenced by many factors including genotype. We used the Trisomic avian model to probe for influences of the major histocompatibility complex (MHC) on the development of the primary immune organs. Chickens were produced having two, three, and four copies of the MHC-encoding microchromosome. Studies of growth, immune organ development and structure, and lymphocyte populations were performed at three post-hatching time points. Chickens with three and four MHC copies (trisomic and tetrasomic) exhibited reduced overall growth and had smaller bursae and thymuses compared to disomic controls. However, the fold reductions in immune organ weights were much greater than for body weights. Histological analysis of the immune organs revealed dramatic alterations in follicular composition in tetrasomic bursae compared to disomic controls. No obvious alterations were observed in thymic and splenic histology of tetrasomics. Studies of organ lymphocyte numbers revealed a sharp reduction in bursal and thymic lymphocyte counts of trisomic and tetrasomic chickens. Based on these results, it appears that MHC dosage modulates, in a targeted fashion, the cellularity of the primary immune organs through an extended period in differentiation of B and T lymphocytes.

Distribution and inducibility of a P450I activity in cellular components of the avian immune system.

The level of expression of the cytochrome P450 system in an immune tissue could influence the sensitivity of that immune tissue to damage by xenobiotics. The capacity of immune organs and their cellular components for P450I-catalyzed metabolism was assayed in the 4-week-old chicken using the P450I-specific ethoxyresorufin-O-deethylase (EROD) assay and the P450I-inducer, 3,4,3',4'-tetrachlorobiphenyl (TCB). After induction by TCB, EROD was detectable in microsomes from whole thymus, bursa and in peritoneal exudate cells (containing primarily macrophages) at levels of 28.3, 7.2 and 1.3 pmol/mg microsomal protein/min, respectively; the level in control liver was 89.9 pmol/mg microsomal protein/min. No activity was detected in these immune tissues without induction. The P450I specific in vitro inhibitor, alpha-naphthoflavone (NF) inhibited the TCB-induced liver and immune tissue EROD by 50% at concentrations in the range of 0.07-0.1 microM. The cellular distribution of EROD in the bursa and thymus was studied in lymphocytes and supporting tissue cells after their separation by density gradient centrifugation. Much higher TCB-induced EROD was detected in immune tissue supporting cells than in lymphocytes, particularly in the thymus. The P450I in the supporting tissue of the bursa and thymus at 1 week post-hatch was also measured after eradication of the lymphocytes in both immune tissues by in ovo administration of CP. TCB-induced EROD was 12-fold higher in the lymphocyte-depleted thymus than in normal thymus, with a less marked but similar pattern in the bursa.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of eumelanic phenotypes on the expression of amelanosis in the Smyth chicken.

The Smyth line is characterized by an autoimmune loss of melanin in the feather and eye in association with a hypermelanizing melanocyte, which presumably triggers immune system intervention. Inheritance appears to be multigenic. The present study was designed to determine if eumelanin-enhancing modifiers influence the incidence and severity of the line-associated amelanosis. Smyth chicks with dark brown (eb) down had a higher incidence of amelanosis (P less than .01) than did their hatchmates with light brown down. Furthermore, parents with dark down at hatch produced a higher incidence of amelanotic progeny than parents with light down. Reciprocal crosses of the Smyth line to a highly eumelanized (eb/eb) Recessive Black (RB) line produced F1 amelanosis. However, sires from the Smyth line produced significantly more amelanotics than did RB males (P less than .01). The influence of dark down on amelanotic development was also apparent in the Smyth-RBF1. The use of amelanotic F1 parents produced a significantly higher incidence of affected F2 offspring than did the use of unaffected parents. A backcross to the Smyth line produced an incidence of 67.6% amelanosis, whereas only one chick (2.04%) developed amelanosis from an F1 x RB mating. The finding that dark-downed Smyth chicks exhibit, and subsequently produce, a significantly higher incidence of amelanosis supports ultrastructural observations that associate the Smyth line amelanosis with a hyperactive melanocyte. The unusually high expression of amelanosis (22.7%) in the Smyth-RB F1 suggests that the two lines share one or more common eumelanogenic genetic factors.