Evidence that recipient CD8+ T cell depletion does not alter development of chronic vascular rejection in a rat heart allograft model

Progressive chronic vascular rejection is a central feature of indefinitely surviving WF.1L LEW/Gut (RT1(1)) heart grafts transplanted to LEW (RT1(1)) recipients in unmodified donor-recipient combinations. At 70 days posttransplantation, large vessels of the grafts are characterized by the presence of vasculitis, vasculitis with associated variable myointimal thickening, and occlusive myointimal thickening with minimal or absent concomitant vasculitis. To assess the potential role of CD8+ T cells as critical effectors of chronic vascular rejection in this model, LEW recipients of WF.1L heart grafts were effectively depleted of CD8+ T cells as a result of prior thymectomy and anti-CD8 (MRC OX8) monoclonal antibody administration prior to transplantation. WF.1L heart grafts transplanted to LEW recipients that had undergone prior sham thymectomy and MRC OX8 administration, or thymectomy and administration of antibody-free culture supernatant, provided appropriate controls. At 70 days posttransplantation, large vessels of WF.1L heart grafts in all 3 transplantation groups showed similar morphologic features, which were comparable to those observed in heart grafts of long-surviving unmodified donor-recipient pairs. This study has shown that profound selective depression of recipient CD8+ T cells does not alter the characteristic features of chronic vascular rejection in this rat cardiac model, and provides evidence that CD8+ T cells play no critical role in the initiation or development of progressive vascular damage in this setting.     

Natural killer cell depletion fails to influence initial CD4 T cell commitment in vivo in exogenous antigen-stimulated cytokine and antibody responses

The role played by NK- and NK1.1-expressing T cells in CD4 T cell activation and induction of immune responses in vivo is controversial. These effector cells of the innate immune response are hypothesized to play a pivotal role in shaping initial T cell activation, with some groups reporting that classical NK cells are required for optimal Th1-like T cell activation, and others supporting a role for NK1.1+ alphabeta T cells in Th2 generation. Here, we examine the impact of in vivo NK cell depletion on the development of exogenous Ag-specific cytokine and Ab responses using a murine model of human immediate hypersensitivity. OVA-specific immune responses were induced in 1) C57Bl/6 bg/bg and bg/+ mice, 2) BALB/c mice pretreated with anti-asialoGM1 or control Ab, and 3) C57Bl/6 mice depleted of NK1.1-expressing cells by in vivo administration of anti-NK1.1 mAb PK136. Depletion efficacy was assessed by functional assays and flow cytometric analysis. Each of these approaches indicated that depletion of NK cells and NK1.1+ CD4+ T cells fails to alter the Th1:Th2 balance of Ag-driven cytokine synthesis, as indicated by OVA-stimulated cytokine synthesis in primary bulk culture. Similarly, the kinetics and intensity of effector responses such as OVA-specific IgG2a and IgE synthesis were neither increased nor decreased in any of the three models examined. The results argue that NK cells and peripheral NK1.1+ T cells do not play an essential role in shaping the induction of Ag-specific immune responses to soluble exogenous Ags, the most common class of inhalant allergen.     

Effect of IL-4 deletion on cardiac allograft survival in the BALB/c to 129Sv x C57BL/6 strain combination

Calcified brain metastases are rare. They are unique or multiple. The primary site is lung, breast, gastro-intestinal tract, uterine cervix, bone or may be unidentified. It corresponds pathologically to an adenocarcinoma, a sarcoma or to a squamous cell carcinoma. We report a case of multiple calcified brain metastases discovered before primary tumour (bronchial adenocarcinoma), raising the problem of differential diagnosis resolved by stereotaxic brain biopsy.     

The intragraft CD8+ T cell response in renal allograft rejection in the mouse

To identify the role of donor class I alloantigens in regulating the CD8+ T cell response to a kidney allograft, we analyzed and compared the CD8+ infiltrate in kidney transplants from MHC class I-deficient (class I-) mouse donors and class I+ controls. One week after transplantation, there was a prominent CD8+ infiltrate in control allografts, whereas CD8+ T cells were virtually absent in grafts from class I- donors. In class I+ allografts, infiltrating CD8+ cells utilized a wide range of T cell receptor (TCR) Vbeta families and their Vbeta usage was similar to that of the systemic CD8+ population. However, there was a modest but significant overrepresentation of cells bearing Vbeta8 in the graft compared with the spleen due to an expansion of CD8+ Vbeta8.3+ cells. This could be detected as early as 1 week and became more pronounced by 3 weeks after transplantation. In 3-week allografts, only 52% of CD8+ cells expressed alphabetaTCR. Among T cells isolated from class I+ grafts, the CD8+ Vbeta8+ cells demonstrated allospecific responses that were numerically larger than responses of the CD8+ Vbeta8- population. In contrast to the early (1 week) time point, significant numbers of CD8+ cells could be isolated from class I- grafts by 3 weeks after transplantation and their Vbeta repertoire resembled that seen in controls. While increasing numbers of CD8+ Vbeta8+ were present in the class I- grafts at 3 weeks, this increase was not statistically significant. Thus, expression of class I alloantigens on a kidney graft plays an important role in regulating the rate of accumulation of CD8+ T cells in rejecting kidney grafts. However, the TCR Vbeta repertoire of the CD8+ T cell infiltrate is largely determined by factors that are independent of normal class I expression on the graft.     

T cell responses to myelin basic protein in experimental autoimmune encephalomyelitis-resistant BALB/c mice

In strains of mice that are susceptible to experimental autoimmune encephalomyelitis (EAE), cloned CD4+ T cells reactive with autologous myelin basic protein (MBP) have been shown to cause disease when transferred to naive syngeneic recipients. Recent reports indicate that under particular experimental conditions, 'resistant' strains of mice can also develop EAE, although cloned cells have not been isolated and characterized. An analysis of the characteristics of a panel of MBP-specific T cells and the antigen presenting capability of CNS-derived cells obtained from the resistant strain BALB/c is presented here. The data demonstrate that immunization of EAE-resistant BALB/c mice results in the activation of a heterogeneous group of T cells reactive with autologous MBP. Both peripheral antigen presenting cells, as well as microglia isolated from brains of BALB/c mice, are capable of stimulating these cloned MBP-specific T cells to proliferate. When optimally activated in vitro and then injected in vivo into syngeneic BALB/c recipients, three clones studied induced severe cachexia, resulting in loss of up to 35% of body weight before death. Two of the clones also induced clinical and histological EAE, while the third induced only occasional histological evidence of disease. Differences in epitope recognition, T cell receptor usage, cytokine profiles or regulatory mechanisms of self tolerance, may play important roles in preventing potentially destructive autoimmune reactions by these T cells capable of recognizing autologous myelin in the central nervous system.     

cis-dominant regulation of CD4 and CD8 gene expression in rat/mouse T cell heterohybridomas

To determine whether expression of CD4 and CD8 molecules on T cells is determined-solely by transacting regulators, we examined heterohybridomas derived from the fusion of a rat CD4+ T cell line and the CD4- CD8- mouse thymoma BW5147. The majority of hybrid offspring expressed rat CD4. However, a fraction of the cell lines obtained expressed not only rat CD4 but also various amounts of mouse CD4 and CD8 molecules from both species. Cloning of two of these heterogeneous lines revealed that expression of all four Ag varied not only between different clones but also within clonal populations. The expression of Ag not present on the parental cells suggested an alteration in the normally stable regulatory mechanisms present in those cells. Moreover, a lack of concordant expression between the rat and mouse loci was observed, indicating that active and silent homologous loci can exist together in single nuclei. Expression of CD4 and CD8 in these cells, therefore, cannot be solely mediated by trans-acting diffusible regulators but must also depend on cis-dominant effects on the loci themselves. The phenotypic heterogeneity of clonal populations was found to result from unpredictable shifts, both positive and negative, in the expression of CD4 and CD8 over time, indicating that the cis-dominant effects were only quasistable. Preliminary examinations of the density of 5-methylcytosine within the CD4 and CD8 loci in various phenotypic populations separated by FACS from within heterogeneous clones revealed a correlation between surface expression of the mouse CD8 protein and a lack of methylation around the mouse CD8 gene. In contrast, the CD4 gene remained extensively methylated regardless of its surface expression.     

CXCR4 utilization is sufficient to trigger CD4+ T cell depletion in HIV-1-infected human lymphoid tissue

The human chemokine receptors CCR5 and CXCR4 have emerged as the predominant cofactors, along with CD4, for cellular entry of HIV-1 in vivo whereas the contribution of other chemokine receptors to HIV disease has not been yet determined. CCR5-specific (R5) viruses predominate during primary HIV-1 infection whereas viruses with specificity for CXCR4 (R5/X4 or X4 viruses) often emerge in late stages of HIV disease. The evolution of X4 viruses is associated with a rapid decline in CD4+ T cells, although a causative relationship between viral tropism and CD4+ T cell depletion has not yet been proven. To rigorously test this relationship, we assessed CD4+ T cell depletion in suspensions of human peripheral blood mononuclear cells and in explants of human lymphoid tissue on exposure to paired viruses that are genetically identical (isogenic) except for select envelope determinants specifying reciprocal tropism for CXCR4 or CCR5. In both systems, X4 HIV-1 massively depleted CD4+ lymphocytes whereas matched R5 viruses depleted such cells only mildly despite comparable viral replication kinetics. These findings demonstrate that the coreceptor specificities of HIV-1 are a causal factor in CD4+ T cell depletion ex vivo and strongly support the hypothesis that the evolution of viral envelope leading to usage of CXCR4 in vivo accelerates loss of CD4+ T cells, causing immunodeficiency.     

Distribution of cholecystokinin immunoreactivity in the BALB/c mouse forebrain: an immunocytochemical study

The present study describes cholecystokinin (CCK) immunoreactivity (CCK-IR) distribution in the brains of control and colchicine-treated mice. In the brains of control mice, the CCK-IR strongly revealed numerous axons and terminals. Perikarya exhibiting a faint to moderate immunoreactivity were also observed in areas such as cortices, hippocampus, amygdala, septum, and thalamus. The colchicine treatment did not seem to notably affect the brain CCK-IR innervation, but resulted in profound changes of the perikaryal staining. Indeed, the regions, which contained numerous moderately stained perikarya in the control animals, exhibited after colchicine treatment immunoreactive perikarya intensely stained but only in moderate number. This feature obviously appeared in the cortex in which, in addition to strongly stained perikarya, colchicine induced the appearance of numerous CCK-IR hillocks. In the lateral amygdala and thalamus of colchicine-treated animals, the somatic immunoreactivity was considerably decreased. The regions, such as paraventricular hypothalamic nucleus and bed nucleus of the stria terminalis, which in the control animals did not exhibit any stained perikaryon, showed a high number of strongly stained cell bodies after colchicine treatment. This study, mapping the mouse forebrain CCK-IR, demonstrated a wide distribution of this peptide. Moreover, CCK-IR is spontaneously visible in neurons of untreated mouse in some brain areas previously shown in the rat to exhibit CCK mRNA, but no clear perikaryal CCK-IR even after colchicine treatment.     

Gastrointestinal T lymphocytes retain high potential for cytokine responses but have severe CD4(+) T-cell depletion at all stages of simian immunodeficiency virus infection compared to peripheral lymphocytes

Gastrointestinal complications in human immunodeficiency virus (HIV) infection are indicative of impaired intestinal mucosal immune system. We used simian immunodeficiency virus (SIV)-infected rhesus macaques as an animal model for HIV to determine pathogenic effects of SIV on intestinal T lymphocytes. Intestinal CD4(+) T-cell depletion and the potential for cytokine responses were examined during SIV infection and compared with results for lymphocytes from lymph nodes and blood. Flow cytometric analysis demonstrated severe depletion of CD4(+)CD8(-) single-positive T cells and CD4(+)CD8(+) double-positive T cells in intestinal lamina propria lymphocytes (LPL) and intraepithelial lymphocytes (IEL) during primary SIV infection which persisted through the entire course of SIV infection. In contrast, CD4(+) T-cell depletion was gradual in peripheral lymph nodes and blood. Flow cytometric analysis of intracellular gamma interferon (IFN-gamma) and interleukin-4 (IL-4) production following short-term mitogenic activation revealed that LPL retained same or higher capacity for IFN-gamma production in all stages of SIV infection compared to uninfected controls, whereas peripheral blood mononuclear cells displayed a gradual decline. The CD8(+) T cells were the major producers of IFN-gamma. There was no detectable change in the frequency of IL-4-producing cells in both LPL and peripheral blood mononuclear cells. Thus, severe depletion of CD4(+) LPL and IEL in primary SIV infection accompanied by altered cytokine responses may reflect altered T-cell homeostasis in intestinal mucosa. This could be a mechanism of SIV-associated enteropathy and viral pathogenesis. Dynamic changes in intestinal T lymphocytes were not adequately represented in peripheral lymph nodes or blood.     

Flow cytometric single-cell analysis of cytokine production by CD4+ T cells in synovial tissue and peripheral blood from patients with rheumatoid arthritis

BACKGROUND AND PURPOSE: To detect areas of cerebral perfusion from bypass arteries after vascular reconstruction, we administered selective intraarterial microsphere tracer into the external carotid arteries and determined (via single-photon emission computed tomography [IA-SPECT]) whether the distribution of radiotracer matched the arteriographic distribution of contrast material as shown on external carotid angiograms. METHODS: We compared the extent of regional distribution of tracer after external carotid artery injection of 20 to 40 MBq of 99mTc-HMPAO or 99mTc-ECD with that of contrast medium on the external carotid angiograms in 582 cortical regions in 12 patients with atherosclerotic occlusive disease and in 18 patients with moyamoya disease. RESULTS: Marked accumulation of tracer was found only in the expected, specific, newly developed areas of cerebral perfusion from bypass arteries. The regional distribution of tracer corresponded to that of contrast medium in 523 regions (90%) and did not correspond in 59 regions (10%). Significant overestimation of the distribution of contrast material relative to that of tracer was observed in the patients with moyamoya disease. CONCLUSION: SPECT showed slightly different distribution of tracer from that predicted by conventional angiography. IA-SPECT should enhance the analysis of newly developed areas of cerebral perfusion from the bypass arteries.     

The IL-4 rapidly produced in BALB/c mice after infection with Leishmania major down-regulates IL-12 receptor beta 2-chain expression on CD4+ T cells resulting in a state of unresponsiveness to IL-12

Within 1 day of infection with Leishmania major, susceptible BALB/c mice produce a burst of IL-4 in their draining lymph nodes, resulting in a state of unresponsiveness to IL-12 in parasite-specific CD4+ T cells within 48 h. In this report we examined the molecular mechanism underlying this IL-12 unresponsiveness. Extinction of IL-12 signaling in BALB/c mice is due to a rapid down-regulation of IL-12R beta2-chain mRNA expression in CD4+ T cells. In contrast, IL-12R beta2-chain mRNA expression was maintained on CD4+ T cells from resistant C57BL/6 mice. The down-regulation of the IL-12R beta2-chain mRNA expression in BALB/c CD4+ T cells is a consequence of the early IL-4 production. In this murine model of infection, a strict correlation is shown in vivo between expression of the IL-12R beta2-chain in CD4+ T cells and the development of a Th1 response and down-regulation of the mRNA beta2-chain expression and the maturation of a Th2 response. Treatment of BALB/c mice with IFN-gamma, even when IL-4 has been produced for 48 h, resulted in maintenance of IL-12R beta2-chain mRNA expression and IL-12 responsiveness. The data presented here support the hypothesis that the genetically determined susceptibility of BALB/c mice to infection with L. major is primarily based on an up-regulation of IL-4 production, which secondarily induces extinction of IL-12 signaling.     

CD4 T cell anergy in murine AIDS: costimulation via CD28 and the addition of IL-12 are not sufficient to rescue anergic CD4 T cells

Murine acquired immunodeficiency syndrome (MAIDS) is a fatal disease induced by a mixture of retroviruses known as BM5. It is characterized by splenomegaly, lymphadenopathy, hypergammaglobulinemia, loss of T and B cell function, and development of B cell lymphomas. As the disease progresses, by wk 8 of infection, CD4 T cell response to Ags and mitogens is severely curtailed and the CD4 T cell population becomes anergic. We examined responses of anergic CD4 T cells upon addition of a costimulatory signal (anti-CD28) and a cytokine (IL-12), which might help to restore the function of cells. We report that proliferation and cytokine production were restored in the early stages of infection by the strategies we tested, but not at later stages when anergy was well established. We also examined the effect of the same treatments on anergy of CD4 T cells from thymectomized, BM5-infected mice to determine whether the rescue seen was due to cells freshly derived from the thymus. We report that proliferation and cytokine production decreased in thymectomized mice even at wk 4 of infection, indicating that cells that are freshly derived from thymus are the ones responding to treatment. This study indicates that once anergy has been established in MAIDS, it cannot be reversed by providing costimulation via CD28 and IL-12. Anergy of CD4 T cells in MAIDS appears to be different from that seen in other systems, both in underlying cause and in the ability of the cells to revert to a normal state.     

Dermal microvascular injury in the human peripheral blood lymphocyte reconstituted-severe combined immunodeficient (HuPBL-SCID) mouse/skin allograft model is T cell mediated and inhibited by a combination of cyclosporine and rapamycin

We have analyzed the mechanism of human endothelial injury in a human peripheral blood lymphocyte-severe combined immunodeficient (huPBL-SCID) mouse/human skin graft model of allograft injury and examined the effect of immunosuppressive drugs on this process. In this model, split-thickness human skin containing the superficial dermal microvessels was grafted onto immunodeficient C.B-17 SCID or SCID/beige mice and allowed to heal. Human peripheral blood mononuclear cells (PBMCs) allogeneic to the skin, when subsequently introduced by intraperitoneal injection, caused destruction of the human dermal microvasculature by day 16, evident as endothelial cell sloughing and thrombosis. In the same specimens, mouse microvessels that invaded the human skin graft were uninjured. Human microvascular cell injury was accompanied by a mononuclear cell infiltrate consisting of approximately equal numbers of human CD4+ and CD8+ T cells, some of which contained perforin-positive granules. We found no evidence of human natural killer cells and noted occasional human, but not mouse, macrophages at a frequency indistinguishable from that resident in skin on animals not receiving human PBMCs. These human T cell infiltrates did not extend into adjacent mouse skin. Human immunoglobulin G antibody was detected in the blood and was diffusely present throughout mouse and human tissues in SCID mice receiving PBMCs. Mouse C3 was detected on human dermal vessels in both unreconstituted control animals and those that received PBMCs. Blood and tissues from mice injected with PBMCs depleted of B cells showed no human immunoglobulin, but circulating CD3+ cells were detected by flow cytometry at levels comparable with those of animals receiving whole PBMCs. Significantly, skin graft infiltration by human T cells and human dermal microvascular injury were equivalent in the B cell-depleted and whole-PBMC-reconstituted mice. Mice inoculated with PBMCs depleted of CD8+ T cells developed microvascular injury and infiltrates containing perforin-expressing CD4+ T cells. These data suggested a cytolytic T cell-dependent mechanism of microvessel injury. We then tested the ability of T cell immunosuppressants, cyclosporine and rapamycin, to attenuate vessel damage. Neither cyclosporine nor rapamycin alone effectively reduced either mononuclear cell infiltration or vascular injury. However, a combination of the two agents reduced both parameters. We conclude that the huPBL-SCID/skin allograft model may be used both to study cytolytic T cell-mediated rejection and to test the effect of immunosuppressive drug strategies in vivo in a small-animal model of human immune responses.     

Highly active antiretroviral therapy results in a decrease in CD8+ T cell activation and preferential reconstitution of the peripheral CD4+ T cell population with memory rather than naive cells

Criteria for detection of chromosome aberrations by Comparative Genomic Hybridization (CGH) are not standardized and improvement of this part of the analysis is of paramount importance to the applicability of the technique. The aim of this work was to suggest CGH detection criteria that increase the specificity and sensitivity and at the same time include chromosome regions previously excluded from CGH analysis. We analyzed 33 hybridizations with normal DNA and modified our CGH software in order to use a selection of these normal analyses as a model for interpretation of analyses of unknown samples. This approach was successfully tested on 14 samples with known aberrations.     

Defective Th1 and Th2 cytokine synthesis in the T-T cell presentation model for lack of CD40/CD40 ligand interaction

In this study, T or NK cell clones used as antigen-presenting cells (T- or NK-APC) were shown to be significantly less efficient than professional APC in inducing Th1 and Th2 cytokines by antigen-specific T cell clones. This phenomenon was not related to a limited engagement of TCR by T-APC, since comparable thresholds of TCR down-regulation were shown when antigen was presented by either T-APC or professional APC. Rather, the stimulatory T-APC weakness was due to their inability, because they are CD40-, to provide the appropriate co-stimuli to responder T cells both indirectly via IL-12, and partially via direct CD40L triggering on T cells. Indeed, the simultaneous addition of IL-12 and reagents directly engaging CD40L on responder T cells restored T cell cytokine synthesis when antigen was presented by T-APC. In addition, either IL-12 production or blocking of T cell cytokine synthesis by anti-IL-12 p75 antibodies was evident only when professional APC were used in our antigen-specific system. The down-regulation of cytokine synthesis in the system of T-T cell presentation could represent a novel mechanism of immune regulation, which may intervene to switch off detrimental Th1- or Th2-mediated responses induced by antigen presentation among activated T cells infiltrating inflamed tissues.     

Antigen specificity of CD4 T cell response in the central nervous system of mice infected with mouse hepatitis virus

Previously, we showed that the transmembrane (M) and surface (S) glycoproteins were recognized by splenic CD4 T lymphocytes harvested from mice infected intraperitoneally with mouse hepatitis virus, strain JHM (MHV-JHM), whereas only the S protein was recognized by splenocytes derived from mice with MHV-induced chronic demyelination. From these results, it could not be determined which proteins were recognized by T cells localized in the infected central nervous system (CNS). Herein, we show that CD4 T cells responding to both the M and S proteins can be detected in the CNS of mice with either acute encephalitis or the chronic demyelinating disease. As part of these analyses, two CD4 T cell epitope regions encompassing residues 328-347 and 358-377 within the S protein were identified. Both epitopes, as well as a previously identified M-specific epitope, were recognized by the CNS-derived lymphocytes. Finally, viral RNA harvested from mice with chronic demyelination was analyzed for mutations in the S specific CD4 T cell epitopes since changes resulting in escape from CD8 T cell surveillance were previously identified in these samples. A mutation in epitope region S(328-347) (ala to thr at position 337) was detected in a minority of samples but this change did not abrogate recognition of the epitope and therefore was unlikely to contribute to virus persistence. In conclusion, these studies identify epitopes recognized by MHV-specific CD4 T cells in the infected CNS and show that these cells are preferentially located at the site of infection in mice with clinical disease.     

Frequent development of murine T-cell lymphomas with TcR alpha/beta+, CD4-/8- phenotype after implantation of human inflammatory breast cancer cells in BALB/c nude mice

Tumors developed quite frequently in some of the visceral organs, including spleen and liver, in BALB/c nude mice upon subcutaneously xenografting surgical specimens from five different inflammatory breast cancer patients. All of these tumors developed within two and a half months to one year after the subcutaneous inoculation of surgical specimens. From these tumors, five independent transplantable tumors, including tMK-2, tHK-1, tYK-1, tYK-2 and tTY-1 have been established. Chromosome analysis, morphologic studies by light and electron microscopy and phenotype analysis indicated that these tumors are of mouse origin. The tMK-2 tumor was highly metastatic to the spleen and liver when it was subcutaneously transplanted into the right scapular region. In addition, the region where the tMK-2 tumor cells were subcutaneously inoculated showed an apparently inflammatory process represented by erythema. After subcutaneous inoculation into the right scapular region, tHK-1, tYK-1, 2, and tTY-1 tumors also metastasized to some of the visceral organs, including spleen and liver. From these tumors, in vitro cell lines were established. The cells grew in a stromal-cell dependent manner under in vitro culture conditions. The cells were again tumorigenic at the inoculated region and metastasized to various organs, including liver and spleen, of BABL/c nude mice. Histological examination revealed that the tumors showed features of malignant lymphoma. Phenotypically, these five tumors expressed early T lymphocyte markers as revealed by anti-mouse anti-TcR alpha/beta, anti-CD3, CD4 and CD8 monoclonal antibodies. To our knowledge, these cell lines are the first T-cell lines showing the phenotype of extrathymically differentiated T-cells in the liver.     

Monoclonal antibody to the HLA class I alpha 3 domain inhibits T cell activation and prolongs cardiac allograft survival in HLA-transgenic mice

Workload measurement is one of the most reliable tools in perspective payment systems, to evaluate clerk and technical personnel needs in a Surgical Pathology Department. In 1992 Italian Government began a full-comprehensive modification of financial support in National Health Care System, and during the last four years Public Hospitals and Local Health Units modified their financial organization with the introduction of workload measurement for the personnel needs. We propose a synthetic method for the workload measurement in Surgical Pathology Departments, similar to that proposed for perspective payment of surgical pathology services. The method is reliable and simple representing the full performance to obtain pathological report.     

Identification of two NF-kappa B sites in mouse CD95 ligand (Fas ligand) promoter: functional analysis in T cell hybridoma

Fas ligand (FasL) gene expression is critically involved in peripheral T cell tolerance and lymphocyte homeostasis. Previous studies have suggested that nuclear translocation of NF-kappaB during T cell activation is a critical event for FasL gene activation. In the present study we have identified two NF-kappaB sites (designated FasL-kappaB1 and FasL-kappaB2) on the promoter (approximately 700 bp) of FasL. The NF-kappaB sites were identified by electrophoretic mobility shift assay. Transient transfection reporter analyses showed that the FasL promoter activity was comparable between a construct that contains both sites and a shorter construct (433 bp) that contains only the FasL-kappaB1 site. Furthermore, elimination of FasL-kappaB1 by site-directed mutagenesis significantly inhibited FasL promoter activity. These observations provide strong evidence that NF-kappaB directly binds to the FasL-kappaB1 site and up-regulates FasL gene expression.