(NZW x BXSB)F1 mouse. A new animal model of idiopathic thrombocytopenic purpura.

A decrease in thrombocyte count was observed in (NZW x BXSB)F1 (W/B F1) mice at the age of greater than 5 mo, whereas megakaryocyte counts were found to increase in such mice. FACS analyses revealed the presence of both platelet-associated antibodies (PAA) and circulating antiplatelet antibodies. There is a correlation between the presence of these antibodies and the degree of thrombocytopenia. The transplantation of normal bone marrow cells from BALB/c nu/nu mice to W/B F1 mice was found to have preventative and curative effects on thrombocytopenia; the mice showed normal platelet counts and no evidence of circulating antiplatelet antibodies. These results indicate that thrombocytopenia in W/B F1 mice is due to the presence of antibodies to platelets. We therefore think that W/B F1 mice serve as a useful animal model of idiopathic thrombocytopenic purpura (ITP) not only for elucidating the mechanism of the development of antiplatelet antibodies, but also for characterizing autoantibodies to platelets.

Role of CD4 molecule in the induction of interleukin 2 and interleukin 2 receptor in class II major histocompatibility complex-restricted antigen-specific T helper clones. T cell receptor/CD3 complex transmits CD4-dependent and CD4-independent signals.

The CD4 molecule plays an essential role in antigen-induced activation of T helper (Th) cells, but its contribution to signal transduction events resulting in physiologic T cell function is ill defined. By utilizing anti-CD4 monoclonal antibodies (MAbs) that recognize distinct epitopes of CD4, we have investigated the role of CD4 molecule in antigen-induced interleukin 2 (IL-2) and IL-2 receptor (IL-2R) alpha chain expression in class II major histocompatibility complex-restricted antigen-specific human Th clones. Pretreatment of the Th clones with Leu3a resulted in a dose-dependent suppression of antigen-induced proliferative responses, inositol phosphate accumulation, increase in free cytoplasmic calcium ions ([Ca2+]i), IL-2 mRNA accumulation, IL-2 secretion, and membrane IL-2R expression. IL-2R mRNA accumulation, however, was unaffected even at highest Leu3a concentrations. Leu3a treatment did not affect bypass activation of T cells with PMA plus ionomycin or activation via CD2 molecule. The MAb OKT4, which binds another domain of CD4, was not inhibitory. These results suggest that after T cell antigen receptor-CD3 activation, IL-2 gene induction, IL-2 secretion, and membrane IL-2R expression are absolutely dependent upon participation of CD4 molecules, phosphatidylinositol (PI) hydrolysis, and increase in [Ca2+]i. The requirement for IL-2R gene induction, however, occurs independently of CD4 molecule participation and PI hydrolysis.

Fas drives cell cycle progression in glioma cells via extracellular signal-regulated kinase activation.

Recent studies have revealed that a variety of malignant tumors express Fas and/or its ligand FasL. However, tumor cells expressing Fas are not always susceptible to Fas-mediated cell death, and the biological significance of simultaneous expression of Fas and FasL in the same tumor is not known. In the present study, we addressed this question in three glioma cells lines, A-172, T98G, and YKG-1, which express both Fas and FasL endogenously and their Fas transfectants. We report here that: (a) in gliomas, [3H]TdR incorporation was enhanced by anti-Fas IgM monoclonal antibody CH-11 and conversely inhibited by anti-FasL monoclonal antibody NOK-2; (b) cross-linking of Fas with CH-11 drove both cell cycle progression and apoptosis as demonstrated by the induction of the S-G2 phase of DNA and RNA and fragmented nuclei; (c) phosphorylation of extracellular signal-regulated kinase (ERK), but not of c-Jun NH2-terminal kinase or p38, was induced by cross-linking of Fas; (d) a mitogen-activated protein kinase/ERK kinase 1 (MEK1) inhibitor PD98059 completely blocked CH-11-induced ERK phosphorylation as well as cell cycle progression without affecting induction of apoptosis; and (e) a broad-spectrum caspase inhibitor Z-Asp-CH2-DCB inhibited CH-11-induced ERK phosphorylation, cell cycle progression, and apoptosis. These results indicate that Fas-mediated caspase activation elicits two independent cellular responses; one is to induce apoptosis and another is to promote cell cycle progression; the latter is closely linked to the MEK-ERK pathway. Together, our data strongly suggest that FasL may play a role as an autocrine growth factor in gliomas.

Intervention of T-cells in transportation of mouse mammary tumor virus (milk factor) to mammary gland cells in vivo.

Using BALB/c nu/nu, BALB/c nu/nufC3H (BALB/c nu/nu mice raised by C3H/HeN foster mother), BALB/c thymus-engrafted BALB/c nu/nufC3H, BALB/c nu/+, and BALB/c nu/+fC3H mice, we examined what kinds of cells are carriers of blood-borne mouse mammary tumor virus (B-MMTV). A radioimmunoassay and an immunoperoxidase assay revealed the presence of MMTV-gp52 antigen in the mammary glands of all BALB/c nu/+fC3H and BALB/c thymus-engrafted BALB/c nu/nufC3H mice (more than 60 days old) but only of some BALB/c nu/nufC3H mice (more than 120 days old): those that possessed a significant number of functional T-cells. BALB/c nu/+ mice did not show the antigen expression at any age. Transfer experiments of cells or plasma from young (less than 12 weeks) BALB/c nu/nufC3H to BALB/c +/+ virgins revealed that cells besides T-cells can also become carriers of B-MMTV. This was confirmed by Southern blotting analyses; exogenous provirus DNA sequences were found in B-cells as well as T-cells of BALB/c nu/+fC3H mice. However, when young BALB/c nu/nu mice were inoculated with BALB/c nu/nufC3H blood, they did not show the MMTV-gp52 antigen expression. Transfer experiments of purified T-cells, B-cells, natural killer cells, and macrophages from BALB/c fC3H mice to BALB/c nu/nu mice revealed that only T-cells have the ability to transfer viral activity to the mammary glands. These results suggest that B-MMTV is carried from the gastrointestinal tract to the mammary glands by lymphoid cells such as T-cells and B-cells, then transferred to the mammary gland cells by the T-cells.

Histogenesis of hemopoietic bone marrow in adult mice.

Based on the concept that the hierarchy of regulatory genes may specify mammalian development, we attempted to evaluate how morphological and functional phenotypes of hemopoietic bone marrow develop in murine models. From studies of local irradiation, s.c. implantation of bone with or without marrow, and renal subcapsular implantation of marrow plugs or bone without marrow, we identified four sequential (necrotic, clearing, stromal proliferating, and hemopoiesis-recovering) responses in the early phase of the reconstruction of hemopoietic marrow that spatially and temporally proceeded in this restricted order. In addition, characteristic hexagonal sinuses were found to participate in the development of hemopoietic marrow in the early phase. The development of hexagonally arranged sinuses seems to coincide with the formation of abundant osseous trabeculae and to precede the appearance of colony-forming units in culture (CFU-C) and spleen colony-forming units (CFU-S). These findings are discussed from the point of the hierarchy of regulation.

Nef protein of HIV-1 has B-cell stimulatory activity.

OBJECTIVE: To examine the B-cell stimulatory properties of the regulatory Nef protein of HIV-1. METHODS: The effect of the HIV-1 regulatory proteins Nef, Tat and Vif, were analyzed for their ability to induce differentiation of normal B lymphocytes into immunoglobulin secreting cells (ISC). RESULTS: A recombinant Nef protein, but neither Tat or Vif, was able to induce ISC in peripheral blood lymphocyte (PBL) cultures of HIV-1-seronegative donors. Another recombinant Nef protein, d-Nef, with a truncated amino terminal (deletion of 34 amino acids) failed to induce B-cell differentiation. Pretreatment of the Nef protein with a polyclonal anti-Nef-antibody abrogated its B-cell stimulatory activity. The Nef-induced B-cell differentiation was dependent on cell-to-cell contact. Cell surface molecules leukocyte function-associated molecule (LFA)-1, intracellular adhesion molecule (ICAM)-1, human lymphocyte antigen-DR and B7 were involved in the T-B-cell interaction because monoclonal antibodies to these molecules abrogated the Nef-induced B-cell differentiation response. The Nef protein was able to induce interleukin (IL)-6 messenger (m)RNA and IL-6 protein secretion in PBL, with monocytes as the primary source. CONCLUSIONS: These findings indicate that regulatory (Nef) proteins of HIV-1 contribute to the intense B-cell activation that occurs in association with HIV-1 infection. T-B-cell contact-dependent interaction and induction of IL-6 by these proteins appear to play major roles in this process.

Inhibitory influences of envelope glycoproteins of HIV-1 on normal immune responses.

Infection with the human immunodeficiency virus (HIV-1) leads to a wide range of immunological abnormalities. We have shown that native envelope glycoproteins (gp120) of HIV-1 inhibit antigen-specific and anti-CD3 induced lymphoproliferation of normal lymphocytes. We have demonstrated that gp120 binds to CD4 positive T lymphocytes and is internalized. These studies suggest that interaction of gp120 with the CD4 receptor may be sufficient to inhibit antigen-specific T cell responses that are mediated via the CD3-Ti receptor complex. Such an event could represent another mechanism by which the overall immune function of the CD4 positive T lymphocyte is depressed and may be relevant in planning investigations on the ongoing vaccine trial involving envelope glycoproteins.

Analyses of development of insulitogenic T lymphocytes in NOD mice by transplantation of bone marrow, thymus, and pancreas.

To estimate the functional participation of the insulitogenic genes in the development of insulitogenic lymphocytes, we attempted to induce insulitis in normal allogeneic BALB/c hosts by bone marrow transplantation from NOD mice. The development of the insulitogenic lymphocytes was histologically examined using the host's pancreas and pancreatic tissue from NOD mice which had been grafted under the renal capsules 2 weeks before sacrifice. Adult-thymectomized NOD mice that had been reconstituted with the thymus and bone marrow from NOD mice showed insulitis in both the host's pancreas and grafted pancreatic tissue from newborn NOD mice. When BALB/c mice were lethally irradiated and then reconstituted with NOD bone marrow cells, no insulitis developed in the pancreatic grafts from NOD and BALB/c mice or in the host's pancreas. Since the specificities and functions of T lymphocytes are controlled by the thymic microenvironment during the differentiation within the thymus, the thymus of BALB/c genotype may be responsible for the failure to induce the insulitogenic lymphocytes. Therefore, athymic BALB/c mice were reconstructed with bone marrow cells and thymus of NOD genotype. No insulitis developed, however, in either the host's pancreas or grafted pancreatic tissue. These results suggest that the bone marrow cells and thymus of NOD genotype are not sufficient to induce insulitogenic lymphocytes in the allogeneic BALB/c environment.

Ultraviolet-irradiated apoptotic lymphocytes produce interleukin-10 by themselves.

Since inflammatory responses are rarely associated with apoptotic cell death, it is plausible that cells undergoing apoptosis may signal the immune system to suppress inflammatory responses. By employing intracytoplasmic cytokine staining in conjunction with annexin V-binding, we examined the representative pro-inflammatory cytokine tumor necrosis factor-alpha (TNFalpha) and anti-inflammatory cytokine interleukin-10 (IL-10) expression in ultraviolet (UV)-irradiated lymphocytes and analyzed them with apoptosis induction at a single cell level. We show here that lymphocytes exposed to UV resulted in IL-10 expression with marginal TNFalpha expression, and these IL-10-expressing cells underwent apoptosis. Addition of inhibitors for caspases blocked UV-induced apoptosis but not IL-10. These results indicate that UV elicited at least two types of signals: one which was caspase dependent, leading to apoptosis; and another which was caspase independent, leading to IL-10 production. Lymphocyte apoptosis was thus found to link anti-inflammatory cytokine secretion, and thereby may contribute to preventing unwanted immune responses.

Human immunodeficiency virus type 1 RNA detection in peripheral blood mononuclear cells by polymerase chain reaction: enhanced sensitivity after mitogenic stimulation.

The aim of this study was to investigate whether stimulus-induced up-regulation of human immunodeficiency virus type 1 (HIV-1) expression in peripheral blood mononuclear cells (PBMC) could enhance the diagnostic sensitivity of the polymerase chain reaction (PCR). PBMC derived from 11 HIV-1-infected asymptomatic adults were cultured with a stimulus of phytohemagglutinin (PHA) plus phorbol 12-myristate 13-acetate (PMA) for 36 h prior to lysing the cells for PCR. In all 11 patients studied, the intensity of PCR-assisted HIV RNA amplification (RNA-PCR) performed on stimulated cells was significantly (p < 0.001) higher than that obtained on unstimulated cells. A comparison of conventional PCR-assisted DNA amplification (DNA-PCR) with that of RNA-PCR was made on seven patients. The sensitivity of DNA-PCR was also increased by prior stimulation of cells, although not to the same extent as was observed for RNA-PCR. The results of our study indicate that the sensitivity of PCR can be significantly enhanced by prior activation of cells with PHA and PMA.

Virus load as a marker of disease progression in HIV-infected children.

The relationship of virus load to clinical disease progression in HIV-infected children remains to be elucidated. In this study, HIV-1 proviral DNA load was determined in peripheral blood mononuclear cells (PBMCs) by the quantitative competitive DNA polymerase chain reaction assay (QC-PCR) in 47 HIV-infected children subdivided by age (group I, < or = 2 years; group II, > or = 5 years), who were further categorized to include 12 rapid progressors (RP, age < or = 2 years, Centers for Disease Control [CDC] defined clinical category C and/or immune category 3, or death before age 2 years) and slow progressors (SP, age > or = 5 years, excluding CDC categories C and/or immune category 3). Significantly higher mean proviral copies/10(3) PBMCs were detected in group I versus group II (75.4 +/- 104.3 and 13.0 +/- 17.8 respectively, p < 0.0001) and in RP (158.0 +/- 118.2) as compared to either SP (11.8 +/- 18.8, p < 0.0001) or other age-matched infected children (20.3 +/- 38.8, p < 0.0001). Thus HIV-infected children appear to have a higher cell-associated virus load early in life, especially in association with rapid disease progression.

Improved specificity of in vitro anti-HIV antibody production: implications for diagnosis and timing of transmission in infants born to HIV-seropositive mothers.

In vitro anti-HIV antibody production (IVAP), initially introduced as a method for diagnosis of human immunodeficiency virus type 1 (HIV-1) infection in infants, has been limited in its application because of poor specificity and sensitivity early in life. The aims of this study were to improve the specificity of the IVAP assay and to evaluate its sensitivity in conjunction with assays of HIV culture, polymerase chain reaction (PCR), and p24 antigen. To prevent false-positive reactions resulting from maternal serum-derived cytophilic anti-HIV IgG, additional preculture and washing steps for peripheral blood mononuclear cells (PBMCs) were introduced that resulted in dramatic improvement in specificity of IVAP. The sensitivity of the revised IVAP at age < 3 months in 20 infected infants was, however, only 25%; of 15 infected infants initially negative in IVAP, 13 became positive at a mean estimated age of 4.4 +/- 1.8 months. When correlated with virological assays, a failure to respond in IVAP at age < 1 month was often associated with negative virological identification, whereas a positive IVAP response at age < 3 months was always associated with positive results in all virological assays. Moreover, conversion from negative IVAP to positive responses occurred subsequent to, and not concurrently with, a positive virological identification of infected infants. The revised IVAP methodology renders this assay potentially useful as an additional tool not only for the diagnosis of HIV infection, but for estimating timing of maternal-infant HIV transmission as well.

Functional analyses of lpr gene in MRL/Mp-lpr/lpr mice. Role of lymph node stromal cells in lpr-lymphadenopathy.

To clarify the mechanism by which the lpr gene causes lymphadenopathy, we established an experimental system to induce lymph node (LN) swelling in unaffected mice. In MRL-(+)/+ mice that had been 5 Gy-irradiated and grafted with bone marrow cells (BMCs) plus LN from MRL-lpr/lpr mice, a remarkable enlargement of the LN grafts was seen. The enlarged grafts lacked normal LN structure and were indistinguishable from LNs of MRL-lpr/lpr mice. The induction of LN swelling by this method was achieved not only in [MRL-lpr/lpr-->MRL-(+)/+] but also in [MRL-lpr/lpr-->BALB/c], [MRL-lpr/lpr-->C3H], [B6-lpr/lpr-->B10.Thy1.1], and [B6-lpr/lpr-->BALB/c] combinations. Furthermore, the lpr/lpr LN grafts developed lymph node swelling even without the transplantation of BMCs. Most cells in the grafted LNs disappeared within a few days, and large clear fibroblast-like cells then became dominant for 1 to 4 weeks. Thereafter, lymphoid cells increased and had filled the graft by the 8th week. The LN grafts obtained from MRL-lpr/lpr (but not MRL-(+)/+) mice showed the ability to transfer LN node swelling into the secondary MRL-(+)/+ hosts two weeks after the primary transplantation. These results strongly suggest that the fibroblast-like LN stromal cells play a crucial role in lpr-associated lymphadenopathy.

Successful treatment of relapsed blastic natural killer cell lymphoma with unrelated cord blood transplantation.

The prognosis for blastic natural killer (NK) cell lymphoma is generally dismal. We report a patient who was successfully treated with unrelated cord blood transplantation (UCBT). A 15-year-old boy was diagnosed as having blastic NK cell lymphoma in the cervical lymph nodes. Autologous peripheral blood stem cell transplantation was performed on achieving a complete remission. However, the disease recurred in the bone marrow 6 months later. Chemotherapy induced a second remission and the patient received UCBT with a conditioning regimen consisting of total body irradiation, thiotepa and cyclophosphamide. Chronic GVHD of the lung occurred, but it was well controlled with steroids. At the time of writing, he remains in remission 18 months after UCBT with an excellent performance status. UCBT may be an option for patients with blastic NK cell lymphoma.

Proteasome inhibitor 1 enhances paclitaxel-induced apoptosis in human lung adenocarcinoma cell line.

Paclitaxel is a chemotherapeutic drug that induces apoptosis in tumor cells by stabilizing microtubules, prevents normal mitosis, and blocks the cell cycle at the G2/M phase. We have previously reported that the activation of caspase-3 and caspase-8 plays a crucial role in paclitaxel-induced apoptosis. Anti-tumor reagents including paclitaxel, irradiation, and other stimuli activate the transcription factor NF-kappaB, which has the ability to suppress the apoptotic potential of those stimuli. Using a human lung adenocarcinoma cell line (LC-2-AD), we therefore examined whether the inhibition of NF-kappaB activity by proteasome inhibitor 1 (PS1) could become a new adjuvant therapy for cancer. A synergistic effect on apoptosis induction was observed with the combination of more than 0.1 microg/ml paclitaxel and 0.5 microM PS1. An increase in the cell number of apoptotic cells is correlated with the loss of Deltaphim and the activation of caspase-3 and caspase-8. Furthermore, augmented apoptosis is related to NF-kappaB activation. Based on these findings, we propose that the combination of paclitaxel with PS1 could be a new strategy for cancer treatment.

Mechanism of apoptosis in peripheral blood mononuclear cells of HIV-infected patients.

Lymphocytes from patients with HIV-infection have been shown to undergo accelerated spontaneous apoptosis. Binding of CD4 molecules by HIV envelope protein gp120 and anti-gp120 antibodies can lead to crosslinking of CD4 molecules (CD4XL) in vitro and conceivably in vivo. We have recently shown that CD4XL in vitro, when performed in unfractioned peripheral blood mononuclear cells (PBMC) on normal HIV seronegative donors, is by itself sufficient to induce T cell apoptosis (Blood 82:3392, 1993). To further examine the mechanisms involved in apoptosis, we have examined the expression of Fas antigen (Fas) using 3 color flow cytometry. Fas is a cell surface molecule known to mediate apoptosis-triggering signals. We induced CD4XL in PBMC obtained from normal donors, either by anti-CD4 mAb Leu3a or by HIV-1 envelope protein gp160. PBMC subpopulations were examined for Fas Ag expression and for apoptosis induction by flow cytometry. CD4XL was found to result in increased Fas expression as well as Fas mRNA in lymphocytes and the up-regulated Fas Ag was closely correlated with apoptotic cell death. CD4XL in PBMC also resulted in induction of the cytokines INF-tau and TNF-alpha in the absence of IL-2 and IL-4 secretion. Both these cytokins contributed to Fas Ag up-regulation and antibodies to TNF-alpha and INF-tau abrogated CD4XL-induced Fas up-regulation and T-cell apoptosis. These findings suggest that CD4XL occurring in vivo might play an important role in inducing an abberant cytokine profile (which has been observed in HIV infected individuals) and also in triggering of T-cell apoptosis.

Role of apoptosis in HIV disease pathogenesis.

After approximately one and a half decades of intensive studies, the exact mechanisms to explain HIV-mediated cytopathicity are still enigmatic and need closer scrutiny. There has been a dichotomy between virological and immunological viewpoints in understanding HIV-mediated cytopathicity, the former emphasizing a killing of infected cells by HIV-1 and the latter emphasizing indirect mechanisms wherein HIV or its soluble component(s) alter CD4 T-cell function and induce susceptibility to apoptosis. Accumulating evidence points to the notion that apoptosis might be a major contributor to the depletion of CD4 T-cells in HIV infection. This review summarizes current information about the regulatory mechanisms of T-cell apoptosis and the role of apoptosis in HIV pathogenesis with the goal of providing an integrated view of HIV cytopathicity.