A perspective on In vitro developmental neurotoxicity test assay results: An expert panel review.

For decades, there has been increasing concern about the potential developmental neurotoxicity (DNT) associated with chemicals. Regulatory agencies have historically utilized standardized in vivo testing to evaluate DNT. Owing to considerations including higher-throughput screening for DNT, reduction in animal use, and potential cost efficiencies, the development of alternative new approach methods (NAMs) occurred; specifically, the advent of the DNT in vitro test battery (DNT IVB). SciPinion convened an expert panel to address specific questions related to the interpretation of in vitro DNT test data. The consensus of the expert panel was that the DNT IVB might be used during initial screening, but it is not presently a complete or surrogate approach to determine whether a chemical is a DNT in humans. By itself, the DNT IVB does not have the ability to capture nuances and complexity of the developing nervous system and associated outcomes including behavioral ontogeny, motor activity, sensory function, and learning/memory. Presently, such developmental landmarks cannot be adequately assessed in the DNT IVB or by other NAMs. The expert panel (all who serve as co-authors of this review) recommended that additional data generation and validation is required before the DNT IVB can be considered for application within global regulatory frameworks for decision-making.

Interleukin (IL)-17A, F and AF in inflammation: a study in collagen-induced arthritis and rheumatoid arthritis.

Interleukin (IL)-17 plays a critical role in inflammation. Most studies to date have elucidated the inflammatory role of IL-17A, often referred to as IL-17. IL-17F is a member of the IL-17 family bearing 50% homology to IL-17A and can also be present as heterodimer IL-17AF. This study elucidates the distribution and contribution of IL-17A, F and AF in inflammatory arthritis. Neutralizing antibody to IL-17A alone or IL-17F alone or in combination was utilized in the mouse collagen-induced arthritis (CIA) model to elucidate the contribution of each subtype in mediating inflammation. IL-17A, F and AF were all increased during inflammatory arthritis. Neutralization of IL-17A reduced the severity of arthritis, neutralization of IL-17A+IL-17F had the same effect as neutralizing IL-17A, while neutralization of IL-17F had no effect. Moreover, significantly higher levels of IL-17A and IL-17F were detected in peripheral blood mononuclear cells (PBMC) from patients with rheumatoid arthritis (RA) in comparison to patients with osteoarthritis (OA). IL-17A and AF were detected in synovial fluid mononuclear cells (SFMC) in RA and OA, with IL-17A being significantly higher in RA patients. Enriched CD3(+) T cells from RA PBMCs produced singnificantly high levels of IL-17A and IL-17AF in comparison to OA peripheral blood CD3(+) T cells. IL-17A, F and AF were undetectable in T cells from SFMCs from RA and OA. While IL-17A, F, and AF were all induced during CIA, IL-17A played a dominant role. Furthermore, production of IL-17A, and not IL-17F or IL-17AF, was elevated in PBMCs, SFMCs and enriched peripheral blood CD3(+) T in RA.

The role of T helper type 17 cells in inflammatory arthritis.

While T cells have been implicated in the pathogenesis of inflammatory arthritis for more than three decades, the focus on the T helper type 17 (Th17) subset of CD4 T cells and their secreted cytokines, such as interleukin (IL)-17, is much more recent. Proinflammatory actions of IL-17 were first identified in the 1990s, but the delineation of a distinct Th17 subset in late 2005 has sparked great interest in the role of these cells in a broad range of immune-mediated diseases. This review summarizes current understanding of the role of Th17 cells and their products in both animal models of inflammatory arthritis and human immune-driven arthritides.

Heavy metals affect rod, but not cone, photoreceptors.

Low concentrations of lead, mercury, or cadmium depress the amplitude of the rod receptor potential in the perfused bullfrog retina. Responses from the cones were not affected. The data implicate the rods as a lesion site in animals exhibiting scotopic vision deficits as a result of heavy metal poisoning.

Monocyte-endothelial adhesion in chronic rheumatoid arthritis. In situ detection of selectin and integrin-dependent interactions.

Blood monocytes are the principal reservoir for tissue macrophages in rheumatoid synovitis. Receptor-mediated adhesive interactions between circulating cells and the synovial venules initiate recruitment. These interactions have been studied primarily in cultured endothelial cells. Thus the functional activities of specific adhesion receptors, such as the endothelial selectins and the leukocytic integrins, have not been evaluated directly in diseased tissues. We therefore examined monocyte-microvascular interactions in rheumatoid synovitis by modifying the Stamper-Woodruff frozen section binding assay initially developed to study lymphocyte homing. Specific binding of monocytes to venules lined by low or high endothelium occurred at concentrations as low as 5 x 10(5) cells/ml. mAbs specific for P-selectin (CD62, GMP-140/PADGEM) blocked adhesion by > 90% in all synovitis specimens examined. In contrast, P-selectin-mediated adhesion to the microvasculature was either lower or absent in frozen sections of normal foreskin and placenta. mAbs specific for E-selectin (ELAM-1) blocked 20-50% of monocyte attachment in several RA synovial specimens but had no effect in others. mAbs specific for LFA-1, Mo1/Mac 1, the integrin beta 2-chain, and L-selectin individually inhibited 30-40% of adhesion. An mAb specific for the integrin beta 1-chain inhibited the attachment of elutriated monocytes up to 20%. We conclude that P-selectin associated with the synovial microvasculature initiates shear-resistant adhesion of monocytes in the Stamper-Woodruff assay and stabilizes bonds formed by other selectins and the integrins. Thus the frozen section binding assay permits direct evaluation of leukocyte-microvascular adhesive interactions in inflamed tissues and suggests a prominent role for P-selectin in monocyte recruitment in vivo.

Dendritic cells genetically engineered to express IL-4 inhibit murine collagen-induced arthritis.

Dendritic cells (DCs) are specialized antigen-presenting cells that migrate from the periphery to lymphoid tissues, where they activate and regulate T cells. Genetic modification of DCs to express immunoregulatory molecules would provide a new immunotherapeutic strategy for autoimmune and other diseases. We have engineered bone marrow-derived DCs that express IL-4 and tested the ability of these cells to control murine collagen-induced arthritis (CIA), a model for rheumatoid arthritis in which Th1 cells play a critical role. IL-4-transduced DCs inhibited Th1 responses to collagen type II in vitro. A single injection of IL-4-transduced DCs reduced the incidence and severity of CIA and suppressed established Th1 responses and associated humoral responses, despite only transient persistence of injected DCs in the spleen. In contrast, control DCs and IL-4-transduced T cells or fibroblastic cells failed to alter the course of the disease. The functional effects correlated well with the differential efficiency of DC migration from various sites of injection to lymphoid organs, especially the spleen. The ability of splenic T cells to produce IL-4 in response to anti-CD3 was enhanced after the administration of IL-4-transduced DCS: These results support the feasibility of using genetically modified DCs for the treatment of autoimmune disease.

Activation pathways of synovial T lymphocytes. Expression and function of the UM4D4/CDw60 antigen.

Accumulating evidence implicates a central role for synovial T cells in the pathogenesis of rheumatoid arthritis, but the activation pathways that drive proliferation and effector function of these cells are not known. We have recently generated a novel monoclonal antibody against a rheumatoid synovial T cell line that recognizes an antigen termed UM4D4 (CDw60). This antigen is expressed on a minority of peripheral blood T cells, and represents the surface component of a distinct pathway of human T cell activation. The current studies were performed to examine the expression and function of UM4D4 on T cells obtained from synovial fluid and synovial membranes of patients with rheumatoid arthritis and other forms of inflammatory joint disease. The UM4D4 antigen is expressed at high surface density on about three-fourths of synovial fluid T cells and on a small subset of synovial fluid natural killer cells; in synovial tissue it is present on more than 90% of T cells in lymphoid aggregates, and on approximately 50% of T cells in stromal infiltrates In addition, UM4D4 is expressed in synovial tissue on a previously undescribed population of HLA-DR/DP-negative non-T cells with a dendritic morphology. Anti-UM4D4 was co-mitogenic for both RA and non-RA synovial fluid mononuclear cells, and induced IL-2 receptor expression. The UM4D4/CDw60 antigen may represent a functional activation pathway for synovial compartment T cells, which could play an important role in the pathogenesis of inflammatory arthritis.

Identification of two related markers for common acute lymphoblastic leukemia as heat shock proteins.

By direct analysis of the polypeptide constituents of leukemic cells, we have previously detected several polypeptides that are restricted in their expression to acute lymphoblastic leukemia (ALL). In this study, we provide evidence that two polypeptides designated L2 and L4 are structurally related and represent novel markers for common ALL. Partial amino acid sequence analysis did not uncover differences between L2 and L4. The sequences obtained correspond to a previously cloned human gene designated hsp 27 that is expressed, following heat shock treatment, in a variety of cells. 32Pi incorporation studies indicate that L4 is an unphosphorylated form and L2 is a phosphorylated form of hsp27. The two forms were inducible by heat shock in leukemic and nonleukemic lymphoid cells. Thus, in acute leukemia, the common ALL subtype is uniquely characterized by the constitutive expression of a polypeptide that represents a major cellular phosphoprotein.

Cell density dependence of focus formation in the C3H/10T1/2 transformation assay.

Some of the dynamics of neoplastic transformation in vitro have been studied with the use of benzo(a)pyrene as the carcinogen in the C3H/10T1/2 morphological transformation assay. Experiments that involved the dsipersion of cells into new culture dishes at various times after carcinogen treatment have shown that no change in the fraction of potentially transformed cells occurs while cultures grow to form a confluent monolayer, that little or no change in the fraction of potentially transformed cells occurs for approximately 3 weeks after confluence is attained, and that this fraction increases rapidly some 7 weeks after BP treatment. When confluent benzo(a)pyrene-treated cultures are dispersed in new culture dishes prior to the onset of growth toward focus formation, the formation of transformed foci is suppresssed at high cell densities of seeding. This phenomenon is independent of the total number of divisions undergone by cells after treatment. We suggest that phenotypic expression of morphological transformation is dependent on colony interactions in the C3H/10T1/2 system, which we do not yet understand, but which are independent of time posttreatment either in cell generations or absolute time.

Cytokine blockade as a new strategy to treat rheumatoid arthritis: inhibition of tumor necrosis factor.

Rheumatoid arthritis (RA) is a common, frequently severe, chronic inflammatory disease. Although the cause of RA remains unknown, recent advances in understanding its pathogenesis have been substantial. Despite the use of a variety of medications, particularly methotrexate, treatment of RA is not fully effective in most patients. Until recently, insights into inflammatory mechanisms in RA had not been successfully translated into novel classes of therapeutic agents. This gap now will likely be bridged in the form of a new strategy for treating RA-cytokine blockade. Although a variety of cytokines are important in the pathogenesis of RA, tumor necrosis factor (TNF) seems to play a pivotal role. Neutralizing TNF in patients with RA, by means of soluble TNF receptors or anti-TNF monoclonal antibodies, has proven to be a powerful means of controlling disease activity. Studies are in progress to obtain additional information regarding long-term safety of TNF blockade and its effects on disease progression.

UM4D4+ (CDw60) T cells are compartmentalized into psoriatic skin and release lymphokines that induce a keratinocyte phenotype expressed in psoriatic lesions.

UM4D4 (CDw60), the surface molecule of a novel antigen-independent T-cell activation pathway, was found to be highly expressed on lesional psoriatic T cells. To examine whether UM4D4 represents a T-cell activation pathway for psoriatic T cells, a T-cell line was initiated from an acute skin lesion and cloned by limiting dilution. Clonality was verified by analysis of T-cell receptor gene rearrangement. All T-cell clones tested, whether CD4+2H4+CD8-, CD4+2H4-CD8-, or CD4-CD8+CD11b-, expressed UM4D4 and were activated by the monoclonal antibody anti-UM4D4. Lesional psoriatic T-cell clones were heterogeneous in the degree of anti-UM4D4-induced proliferation and in their production of IL-2 and gamma-interferon. Lymphokines released by anti-UM4D4 activation were capable of inducing ICAM-1 and HLA-DR expression on cultured normal keratinocytes. Thus, the high expression of UM4D4 on T-cells in psoriatic skin provides an alternative mechanism for T-cell activation that may be operative in the psoriatic lesional milieu. Indeed, activation of lesional T-cells through the UM4D4 molecule resulted in release of lymphokines that directly induced keratinocytes to express a phenotype displayed in psoriatic skin lesions.

Leukemic T cells from patients with cutaneous T-cell lymphoma demonstrate enhanced activation through CDw60, CD2, and CD28 relative to activation through the T-cell antigen receptor complex.

Antigen-dependent activation of T cells occurs through the T-cell antigen-receptor complex (TCR/CD3). Antigen-independent T-cell activation may occur through the surface molecules CDw60, CD2, and CD28. We wished to determine whether these antigen-independent T-cell-activation pathways could be involved in proliferation of leukemic T cells from patients with cutaneous T-cell lymphoma (CTCL). Whereas CDw60 was only expressed on 28% +/- 7% (mean +/- SEM) of blood T cells obtained from healthy control subjects (n = 4), CDw60 was expressed on 94% +/- 3% of blood T cells obtained from patients with CTCL (n = 4). Dual color immunofluorescence microscopy of the T-cell infiltrate in involved skin of these patients demonstrated that almost 100% of the T cells expressed CDw60. Not only did T cells in the patients with CTCL express CDw60, but triggering of the T cells with anti-CDw60 resulted in enhanced proliferation relative to anti-TCR/CD3 and mitogenic lectins. Other antigen-independent pathways also appeared highly active in the T cells from patients with CTCL because enhanced proliferation relative to anti-TCR/CD3 or mitogenic lectins was found when anti-CD2 or anti-CD28 plus phorbol ester was used as stimulant. Despite the brisk proliferation induced by anti-CDw60, anti-CD2, or anti-CD28, T cells from the patients did not produce detectable amounts of gamma-interferon. The inability to produce gamma-interferon correlates with our finding of absent (n = 3) or weak (n = 1) intercellular adhesion molecule-1 expression in the lesional keratinocytes in these patients. In conclusion, T cells of patients with CTCL demonstrate elevated expression of a T-cell-independent signaling molecule CDw60 and respond to antigen-independent activating signals.

Biological therapies: a novel approach to the treatment of autoimmune disease.

Biological therapies for rheumatoid arthritis (RA) make use of molecules (including derivative and recombinant forms) produced by cells of the immune system or by cells that participate in inflammatory reactions. Development of monoclonal antibodies against cell-surface structures that are lineage or subset specific has led to trials of anti-T-cell reagents in RA, but results thus far must be regarded as a significant therapeutic disappointment. A monoclonal antibody designed to interfere with the action of a cytokine, tumor necrosis factor alpha (TNF-alpha), has been studied in both open and controlled trials. Treatment with this antibody resulted in marked changes in indices of inflammation, but duration of responses may be limited by the eventual development of antibodies to the anti-TNF-alpha antibody. Immunomodulatory strategies that use the immune system to regulate autoimmune activity have been developed based on animal studies, and evaluation of oral collagen as a treatment in RA is currently underway. If successful, this approach would represent a new direction in the treatment of human autoimmune disease. In the future, use of gene therapy directed to the joint could be a powerful approach to treatment of RA. Rational use of biological therapies in RA will depend, in part, on improved understanding of the pathogenesis of this condition.

Abnormalities in CD4+ T-lymphocyte subsets in inflammatory rheumatic diseases.

The monoclonal antibodies anti-2H4 and anti-4B4 identify the suppressor-inducer (CD4+2H4+) and helper-inducer (CD4+4B4+) subpopulations of CD4 (T4+) lymphocytes, respectively. The cell surface phenotype of peripheral blood lymphocytes and synovial fluid lymphocytes in patients with rheumatoid arthritis and other inflammatory joint diseases was analyzed by use of these and other well-characterized anti-T-cell monoclonal antibodies. In the synovial fluid of patients with rheumatoid arthritis, there was a markedly decreased percentage of T4+2H4+ suppressor-inducer cells (3.1 +/- 1 percent) and an increased percentage of T4+4B4+ helper-inducer cells (29.1 +/- 9 percent) as compared with the proportions found in the peripheral blood of normal individuals (T4+2H4+: 19.0 +/- 6 percent, T4+4B4+: 23.0 +/- 7 percent). Moreover, patients with other chronic and acute inflammatory joint diseases exhibited highly similar synovial T-cell findings to those of the patients with rheumatoid arthritis (T4+2H4+: 4.2 +/- 3 percent, T4+4B4+: 33.1 +/- 9 percent). In contrast, there were no significant differences between the normal control subjects and patients with rheumatoid arthritis in the percentage of T4+2H4+ cells in peripheral blood lymphocytes, nor were there significant differences between normal control subjects, patients with rheumatoid arthritis, and patients with other joint diseases (osteoarthritis, gout, B27+ spondyloarthropathy, and psoriatic arthritis) in the number of T4+4B4+ cells or in the T4/T8 ratio of peripheral blood lymphocytes. However, very low numbers of T4+2H4+ (suppressor-inducer) peripheral blood lymphocytes were seen in a subgroup of patients, including five of seven with Reiter's syndrome and several patients with systemic rheumatic disease syndromes. In addition, although the percentage of T4+2H4+ cells in peripheral blood lymphocytes of patients with osteoarthritis (13.7 +/- 7 percent) and gout (14.3 +/- 7 percent) was decreased compared with that of normal controls (19.0 +/- 6 percent) (osteoarthritis versus normal controls p less than 0.025), this difference appeared to reflect alterations due to age rather than disease. Consistent with the phenotypic changes observed, synovial T cells were also functionally defective, since autologous mixed lymphocyte reaction-activated T4 cells from the synovial fluid of patients with rheumatoid arthritis failed to exhibit suppressor-inducer activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Prenatal ethanol exposure alters scotopic and photopic components of adult rat electroretinograms.

Ocular malformations and visual deficits are pathognomic of fetal alcohol syndrome (FAS). However, there are no reports on retinal visual function. To determine if prenatal (human second-trimester equivalent) or prenatal plus 10 days of postnatal (human third-trimester equivalent) ethanol exposure produced scotopic and/or photopic deficits in adult hooded rats, electroretinography (ERG) was used to examine rod and cone, increment threshold, dark adaptation, and paired-flash amplitude recovery functions. The rhodopsin content per eye also was determined. Five main results were found. First, voltage-log intensity and latency-log intensity functions, generated from single-flash ERGs in fully dark-adapted rats, showed increases in absolute threshold and latency and decreases in response amplitude. Second, cone ERGs had latency increases. Third, there were decreases in the scotopic and photopic critical flicker-fusion frequencies, increment threshold functions, and absolute and relative refractory periods. Fourth, rod sensitivity, range, and rate of dark adaptation were decreased. Fifth, rhodopsin content per eye was decreased. These data showed that prenatal ethanol exposure produces long-term deficits in retinal sensitivity, amplitude, light and dark adaptation, temporal processing, and excitability. Larger deficits occurred in the scotopic than photopic system and were produced with an additional 10 days of postnatal ethanol exposure. Alterations in photoreceptors and other cells of the distal retina probably contributed to these deficits. The relevance and applicability of these data to FAS and subclinical alcohol embryopathy have yet to be demonstrated; however, they suggest that similar retinal alterations may occur in human FAS.

Retinoic acid produces rod photoreceptor selective apoptosis in developing mammalian retina.

PURPOSE: All-trans retinoic acid (ATRA) or 9-cis retinoic acid (9CRA), added to dissociated developing neural retinal cells, induces progenitor cells to adopt the rod cell's fate. Retinoic acid (RA) also produces apoptotic cell death in developing tissues. The effects of retinoids on mouse retinal development were examined. METHODS: Retinas were explanted on postnatal day (PN)1 and cultured with or without the retinal pigment epithelium (RPE) attached. Retinas were cultured for 3 weeks in the absence or presence of 100 or 500 nM ATRA or 9CRA. Morphologic development and apoptotic cell death were examined using cell-specific immunocytochemical markers, the TdT-dUTP terminal nick-end labeling (TUNEL) method, and a caspase assay. RESULTS: Retinal explants, with and without RPE, had similar age-dependent increases in opsin expression. In contrast, explants with RPE had less apoptosis during the first week than retinas without RPE. In explants with RPE, ATRA or 9CRA produced rod-selective apoptotic cell death in which 20% to 25% were lost by PN7 with no further loss by PN21. 9CRA-treated explants without RPE had a decreased number of apoptotic cells and a higher number of (rhod)opsin-positive cells at PN3. CONCLUSIONS: Factors in RPE appear to regulate rod apoptosis in developing retina. Retinoids produce rod-selective apoptotic cell death during normal rod differentiation. In contrast, retinoids accelerate the expression of opsin in retinas without RPE. These differential effects of RA on rod photoreceptors-apoptosis and differentiation-are similar to those observed in other developing tissues and play an important role in both normal and pathologic development.

The retina of c-fos-/- mice: electrophysiologic, morphologic and biochemical aspects.

PURPOSE: Mice without a functional c-Fos protein (c-fos-/- mice) do not exhibit light-induced apoptotic cell death of rods in contrast to their wild-type littermates (c-fos+/+ mice). To analyze the consequences of the absence of c-fos in the retina, we investigated whether the retinas of c-fos-/- mice have a reduced capacity to absorb and transduce light compared with c-fos+/+ mice. METHODS: Retinal function was evaluated in dark-adapted mice by full-field electroretinograms (ERGs) over more than 6 log units of intensity. Retinal morphology was studied by light- and electron microscopy. Arrestin and the heat shock protein 70 (Hsp70) were detected by Western blot analysis. The rhodopsin content and the kinetics of rhodopsin regeneration were determined in retinal extracts. RESULTS: Although the configuration of the ERGs was comparable in both groups of mice, c-fos-/- mice showed a marked variability in all quantitative ERG-measures with lower mean amplitudes, longer latencies, and a 0.9-log-unit lower b-wave sensitivity on average. Morphometry showed that c-fos-/- mice have 23% fewer rods on average, whereas the number of cones was comparable among c-fos+/+ and c-fos-/- mice. Arrestin levels appeared slightly reduced in c-fos-/- mice when compared with c-fos+/+ mice, whereas Hsp70 levels were comparable in both genotypes. The kinetics of rhodopsin regeneration were similar, but c-fos-/- mice had a 25% lower rhodopsin content on average. CONCLUSIONS: Compared with c-fos+/+ mice, retinal function in c-fos-/- mice is attenuated to a variable but marked degree, which may be, at least in part, related to the reduced number of rods and the reduced rhodopsin content. However, c-fos does not appear to be essential for the ability to absorb photons, nor for phototransduction or the function of second-order neurons. The resistance to light-induced apoptosis of photoreceptor cells in c-fos-/- mice may result from the acute deficit of c-fos in the apoptotic cascade rather than from developmental deficits affecting rod photoreceptor function.

Fine mapping of monoclonal antibody epitopes on human von Willebrand factor using a recombinant peptide library.

A recombinant human von Willebrand factor (vWF) cDNA fragment library was constructed in lambda gt11 for the localization of anti-vWF monoclonal antibody epitopes. Twelve of 21 monoclonal antibodies screened identified epitopes expressed in lambda gt11 as beta-galactosidase fusion proteins. By sequence analysis, these antigenic determinants were localized to segments ranging from 17 to 105 amino acids in length. Four epitopes apparently shared by more than one antibody were identified, suggesting the presence of immuno-dominant epitopes within vWF. Monoclonal antibody C3, which blocks factor VIII (FVIII) binding to vWF, bound to the same epitope previously identified by a second monoclonal antibody which also blocks this function, suggesting that this region may be at or near the vWF/FVIII binding domain. Three antibodies recognize the same region within the vWF A2 repeat. Mutations near this region appear to be responsible for Type IIA von Willebrand's disease. The co-localization of these antibodies suggests that this domain might be exposed on the surface of vWF, consistent with its apparent increased sensitivity to plasma proteases.

CD6 dependent interactions of T cells and keratinocytes: functional evidence for a second CD6 ligand on gamma-interferon activated keratinocytes.

The CD6 glycoprotein is expressed by T lymphocytes and is hypothesized to interact with one or more ligands expressed on antigen presenting cells (APCs). We show that CD6 mediates binding of the transformed CD4+ T cell line Hut 78 to gamma-interferon activated keratinocytes (KCs). A recombinant CD6-Ig fusion protein has been reported to bind to a CD6 ligand ALCAM, but this is the first demonstration that cell-cell adhesion of human T lymphocytes can be CD6 dependent. The known CD6 ligand ALCAM (CD166) is expressed on cultured KCs but does not appear to mediate KC-Hut 78 binding, suggesting the existence of additional CD6 ligands expressed on KCs. In functional studies using autologous KCs as APCs for tetanus toxoid specific T cell clones, KCs +/- gamma-interferon are unable to stimulate autologous T cells with recall antigen. Therefore interaction of T cell CD6 with CD6 ligands on KCs does not provide sufficient co-stimulation of primed T cells to support responses to nominal antigen.

Spatial and temporal expression of AP-1 responsive rod photoreceptor genes and bZIP transcription factors during development of the rat retina.

PURPOSE: The promoter region of the rod-specific beta subunit of cGMP PDE (beta-PDE) and opsin genes contains highly conserved cis-acting elements, which include an AP-1 and/or Nrl response element (NRE: An extended AP-1 like sequence). Transactivation of AP-1 or NRE appears necessary to drive expression of these rod-specific genes during adulthood, however, their role during development is relatively unknown. Therefore, we determined the spatial and temporal relationships between rod morphological and functional development, rod-specific gene expression, and expression of the bZIP transcription factors c-fos, junD and Nrl. METHODS: Retinas from 0-45 day old (PN0-45) dark- and light-adapted Long-Evans rats were used. Morphological development was monitored by light and electron microscopy. Whole retinal trypsin-activated cGMP-PDE activity and rhodopsin content were measured biochemically. The expression of opsin, beta-PDE, c-fos, junD and Nrl mRNAs were determined by Northern blot analysis. The cellular localization of Nrl was examined with in situ hybridization. RESULTS: The mRNAs for opsin, beta-PDE and c-fos were observed at PN0-2, while cGMP-PDE activity and rhodopsin were detected first at PN5: coincident with rod outer segment development. The developmental pattern of cGMP-PDE activity and rhodopsin accumulation paralleled the expression of beta-PDE and opsin mRNA and all reached their maximal levels by PN45. Nrl expression, for all three transcripts found in the rat retina, was low on PN2 and reached its maximal level at PN14. The c-fos and Nrl expression preceded beta-PDE and opsin mRNA expression by 1-2 days. Nrl expression was detected first in the distal post-mitotic retina at PN5 and then in all nuclear layers during retinal development. Maximal expression shifted from the ganglion cells to the outer nuclear layer as the neural retina matured. In contrast, junD expression was highest at PN0 and declined to a stable level by PN10. CONCLUSIONS: Colocalization of Nrl and c-Fos suggests that expression of rod-specific genes, which utilize AP-1 or NRE sites in their promoter, could be regulated through the formation of Nrl-Fos dimers. We hypothesize that Nrl and c-Fos play a fundamental role in the initiation and regulation of the rod-specific gene expression in developing and adult rod photoreceptors.