High-contrast 10 fs OPCPA-based front end for multi-PW laser chains.

Applications using multi-PW lasers necessitate high temporal pulse quality with a tremendous contrast ratio (CR). The first crucial prerequisite to achieve multi-PW peak power is the generation of ultrashort pulses with good spectral phase quality. Second, to avoid any deleterious pre-ionization effect on targets, nanosecond contrast better than 1012 is also targeted. In the framework of the Apollon 10 PW French laser program, we present a high-contrast 10 fs front-end design study to inject highly energetic Ti:sapphire PW lasers. The CR has been measured and analyzed in different time ranges highlighting the different major contributions for each scale.

Repetition rate increase and diffraction-limited focal spots for a nonthermal-equilibrium 100-TW Nd:glass laser chain by use of adaptive optics.

Dynamic wave-front correction is applied before each shot on a 100-TW, 30-J/300-fs high-power laser facility by use of an adaptive-optics system. This system allows us to increase the repetition rate of high-energy lasers while maintaining excellent and constant beam focusability with a Strehl ratio of >0.75 despite the amplifiers' not being in thermal equilibrium. The best results in terms of the highest Strehl ratio and intensities are obtained when locking the system on wave-front sensing after pulse recompression.

Discordant cellular and humoral immune responses to cytomegalovirus infection in healthy blood donors: existence of a Th1-type dominant response.

Previous studies have documented discordant cellular and humoral immune responses to subjects exposed to HIV-1, and that the nature of such responses may determine susceptibility and resistance to disease. We determined whether there is a spectrum of cellular versus humoral immunodominant responses to cytomegalovirus (CMV) infection. Blood samples from 50 healthy blood donors were tested for anti-CMV IgG antibodies and for proliferative responses of peripheral blood mononuclear cells (PBMC) to CMV antigens. Four patterns of immune responses to CMV were found: no detectable response (30%, Ab(-)/Tc(-)), anti-CMV IgG only (28%, Ab(+)/Tc(-)), both anti-CMV IgG and T lymphocyte proliferation to CMV antigens (18%, Ab(+)/Tc(+)), and, interestingly, T lymphocyte proliferation to CMV only (24%, Ab(-)/Tc(+)). To determine whether these immunodominant phenotypes correlate with the ability of PBMC to secrete IL-2 and IFN-gamma in response to CMV antigens, we found that a greater percentage of individuals with a T cell proliferative response to CMV antigens (Ab(-)/Tc(+) and Ab(+)/Tc(+)) responded with increased IL-2 (P = 0.001) and IFN-gamma levels (P = 0.002), compared to those without a proliferative response (Ab(-)/Tc(-) and Ab(+)/Tc(-)). Our data therefore demonstrate that different individuals exhibit different immunodominant patterns of response to CMV. In particular, some individuals who are exposed to CMV fail to develop an antibody response but do develop cellular immunity. Whether these different patterns predict susceptibility or resistance to CMV-induced disease remains to be determined.

IL-12 as well as IL-2 upregulates CCR5 expression on T cell receptor-triggered human CD4+ and CD8+ T cells.

The expression of chemokine receptors on leukocytes is related to their activation state. However, the exact mechanism underlying the induction of each chemokine receptor is poorly understood. Here, we investigated how CCR5, a chemokine receptor implicated in T cell trafficking and HIV infection, is induced in human T cells. CCR5 was marginally detected on a freshly prepared human peripheral blood mononuclear cell (PBMC) population. Long-term (8-day) stimulation of PBMC with IL-2 resulted in high levels of CCR5 expression on T cells. IL-12 failed to induce CCR5 on T cells in such a directly stimulated PBMC population. Stimulation of PBMC T cells with anti-CD3 plus anti-CD28 induced detectable albeit very low levels of CCR5 along with the induction of IL-12 receptor. However, these TCR-triggered T cells expressed much higher levels of CCR5 when stimulated with IL-12. Although IL-2 also induced CCR5 expression, CCR5 expression was more potent in IL-12 than IL-2 stimulation. These results indicate that, in addition to IL-2, IL-12 plays an important role in the induction of CCR5 expression on T cells, particularly TCR-triggered T cells.

IFN-alpha acts on T-cell receptor-triggered human peripheral leukocytes to up-regulate CCR5 expression on CD4+ and CD8+ T cells.

Interleukin-12 (IL-12) as well as IL-2 was recently shown to up-regulate CCR5 expression on T-cell receptor (TCR)-triggered human T cells. Because of the functional similarity between interferon-alpha (IFN-alpha) and IL-12, the present study investigated whether IFN-alpha also up-regulates T cell CCR5 expression. CCR5 was marginally detected on T cells from unstimulated human peripheral blood leukocytes (PBLs) and only slightly induced on PBL T cells following stimulation with anti-CD3 plus anti-CD28 monoclonal antibodies (mAbs). When anti-CD3/anti-CD28-triggered PBLs were exposed to IFN-alpha, T cells expressed high levels of CCR5. The levels of CCR5 expression were comparable to those induced by IL-12. However, when purified T cells instead of unfractionated PBL were stimulated with anti-CD3/CD28 and then exposed to IL-12 or IFN-alpha, CCR5 expression was induced by IL-12 but not by IFN-alpha. IFN-alpha was found to act on anti-CD3/anti-CD28-stimulated PBL to promote their IL-12 production. Moreover, addition of anti-IL-12 mAb to IFN-alpha-stimulated cultures of anti-CD3/CD28-pretreated PBL resulted in considerable inhibition of CCR5 expression. Together, these results indicate that IFN-alpha as well as IL-12 up-regulates CCR5 expression on TCR-triggered T cells and that IFN-alpha functions not by acting directly on T cells but via enhancing IL-12 production by PBL.

Effects of human cytomegalovirus immediate-early proteins on p53-mediated apoptosis in coronary artery smooth muscle cells.

BACKGROUND: Restenotic and atherosclerotic lesions often contain smooth muscle cells (SMCs), which display high rates of proliferation and apoptosis. Human cytomegalovirus (HCMV) may increase the incidence of restenosis and predispose to atherosclerosis. Although the mechanisms contributing to these processes are unclear, studies demonstrate that one of the immediate-early (IE) gene products of HCMV, IE2-84, binds to and inhibits p53 transcriptional activity. Given the role of p53 in mediating apoptosis, we studied the ability of IE2-84 to inhibit p53-dependent apoptosis in human coronary artery SMCs. METHODS AND RESULTS: Apoptosis of SMCs was induced either by use of an adenovirus vector encoding human wild-type p53 protein or by treatment with doxorubicin. HCMV IE1-72 and IE2-84, the major IE proteins of HCMV, were overexpressed separately with adenovirus vectors encoding each protein, and the effects on p53-induced apoptosis were examined by both nick end-labeling (TUNEL) assay and flow cytometry. Expression of IE2-84, but not IE1-72, protected SMCs from p53-mediated apoptosis. CONCLUSIONS: These data indicate that an HCMV IE protein antagonizes p53-mediated apoptosis, suggesting a pathway by which HCMV infection predisposes to SMC accumulation and thereby contributes to restenosis and atherosclerosis.

Human glioma-induced immunosuppression involves soluble factor(s) that alters monocyte cytokine profile and surface markers.

Patients with gliomas exhibit deficient in vitro and in vivo T cell immune activity, and human glioblastoma culture supernatants (GCS) inhibit in vitro T lymphocyte responses. Because APC are essential for initiating and regulating T cell responses, we investigated whether GCS would affect cytokines produced by monocytes and T cells from healthy donors of PBMC. Incubation of PBMC with GCS decreased production of IL-12, IFN-gamma, and TNF-alpha, and increased production of IL-6 and IL-10. The GCS-induced changes in IL-12 and IL-10 occurred in monocytes, and involved changes in IL-12 p40 and IL-10 mRNA expression. Incubation with GCS also resulted in reduced expression of MHC class II and of CD80/86 costimulatory molecules on monocytes. The immunosuppressive effects were not the result of IL-6 or TGF-beta1 that was detected in GCS. However, it was due to a factor(s) that is resistant to pH extremes, differentially susceptible to temperature, susceptible to trypsin, and has a minimum molecular mass of 40 kDa. Our findings show that glioblastoma-generated factors that are known to suppress T cell responses alter the cytokine profiles of monocytic APC that, in turn, inhibit T cell function. This model indicates that monocytes can serve as an intermediate between tumor-generated immune-suppressive factors and the T cell responses that are suppressed in gliomas.

Structure and expression of variant BRCA2a lacking the transactivation domain.

BRCA1 and BRCA2 are tumor suppressor genes shown to be involved in 90% of familial breast cancers and also known to be involved in ovarian and prostate cancers. Both BRCA1 and BRCA2 gene products are regulated in a cell cycle-dependent manner and have potential transactivation function. Here, we show that BRCA2 undergoes differential splicing giving rise to a novel variant protein BRCA2a, lacking putative transcriptional activation domain. Both BRCA2a and BRCA2 are expressed at high levels in thymus and testis but moderate levels in mammary gland and prostate suggesting that BRCA2a and BRCA2 may have a role in the development and differentiation of these tissues.

B cell-mediated down-regulation of IFN-gamma and IL-12 production induced during anti-tumor immune responses in the tumor-bearing state.

Unfractionated spleen cells taken from tumor-bearing mice contained tumor-primed T cells which produced lymphokines such as IFN-gamma and IL-2 through collaboration with antigen-presenting cells (APC) binding tumor antigens when cultured in vitro. Here, we investigated the regulatory mechanisms underlying IFN-gamma production by T-APC interactions. Elimination of B cells from a splenic population of tumor-bearing mice resulted in enhanced IFN-gamma production. Adding B cells back into cultures down-regulated IFN-gamma production to almost the same levels as those induced by unfractionated spleen cells. IL-2 production was not enhanced by B cell depletion, but rather was significantly suppressed. IFN-gamma-selective up-regulation was due to an enhancement of IL-12 production because IL-12 was detected in B cell-depleted cultures and enhanced IFN-gamma production was prevented by addition of anti-IL-12 mAb or anti-CD40 ligand (CD40L) mAb capable of inhibiting CD40L-induced IL-12 production. These results indicate that B cells interfere with IFN-gamma production induced through interactions between anti-tumor T cells and APC, and this suppressive effect is based on the capacity of CD40+ B cells to down-regulate the CD40L-induced IL-12 production by APC.

Retroviral transduction of TLS-ERG initiates a leukemogenic program in normal human hematopoietic cells.

Many chimeric oncogenes have been identified by virtue of the association between chromosomal translocation and specific human leukemias. However, the biological mechanism by which these oncogenes disrupt the developmental program of normal human hematopoietic cells during the initiation of the leukemogenic process is poorly understood due to the absence of an appropriate experimental system to study their function. Here, we report that retroviral transduction of TLS-ERG, a myeloid leukemia-associated fusion gene, to human cord blood cells results in altered myeloid and arrested erythroid differentiation and a dramatic increase in the proliferative and self-renewal capacity of transduced myeloid progenitors. Thus, TLS-ERG expression alone induced a leukemogenic program that exhibited similarities to the human disease associated with this translocation. These results provide an experimental examination of the early stages of the human leukemogenic process induced by a single oncogene and establish a paradigm to functionally assay putative leukemogenic genes in normal human hematopoietic cells.

The BRCA2 is a histone acetyltransferase.

Patients carrying mutations in BRCA1 or BRCA2 tumor suppressor genes have shown to have high risk in developing breast and ovarian cancers. Two potential functions of BRCA2 were proposed which includes role in the regulation of transcription and also in DNA repair. Forty-five-amino acid region encoded by exon 3 of BRCA2 was shown to have transcriptional activation function. Recent studies of the several enzymes involved in acetylation and deacetylation of histone residues have revealed a possible relationship between gene transcriptional activation and histone acetylation. Since BRCA2 appear to function as a transcriptional factor, we have tested for Histone acetyl transferase (HAT) activity of BRCA2. Here, we present evidence that BRCA2 has intrinsic HAT activity, which maps to the amino-terminal region of BRCA2. Our results demonstrate that BRCA2 proteins acetylate primarily H3 and H4 of free histones. These observations suggest that HAT activity of BRCA2 may play an important role in the regulation of transcription and tumor suppressor function.

Enhanced induction of antitumor T-cell responses by cytotoxic T lymphocyte-associated molecule-4 blockade: the effect is manifested only at the restricted tumor-bearing stages.

Cytotoxic T lymphocyte-associated molecule-4 (CTLA-4), a second counterreceptor for the B7 family of costimulatory molecules, functions as a negative regulator of T-cell activation. Here, we investigated whether the blockade of the CTLA-4 function leads to enhancement of antitumor T-cell responses at various stages of tumor growth. Unfractionated spleen cells taken from CSAIM fibrosarcoma-bearing mice 1-2 weeks after CSA1M cell implantation (early tumor-bearing mice) contained tumor-primed T cells that produced interleukin 2 and IFN-gamma through collaboration with antigen-presenting cell-binding tumor antigens when cultured in vitro. However, this initial lymphokine-producing capacity decreased at later stages of tumor growth (7-10 weeks after tumor cell implantation). Anti-CTLA-4 monoclonal antibody (mAb) was added to whole-spleen cell cultures from early or late tumor-bearing mice. Spleen cells from early tumor-bearing mice exhibited enhanced production of interleukin 2 and IFN-gamma upon in vitro culture in the presence of anti-CTLA-4 mAb. However, addition of anti-CTLA-4 mAb to whole-spleen cell cultures from late tumor-bearing mice failed to display such an enhancement. Consistent with these in vitro results, the in vivo antitumor effect of anti-CTLA-4 administration was observed in a tumor-bearing stage-restricted manner; in vivo administration of anti-CTLA-4 (1 mg/mouse, three times at 1-week intervals) into early tumor-bearing mice resulted in regression of growing tumors, whereas the same treatment did not affect tumor growth when performed for late tumor-bearing mice. Similar anti-CTLA-4 effect was observed in another tumor (OV-HM ovarian carcinoma) model. These in vitro and in vivo results indicate that CTLA-4 blockade in tumor-bearing individuals enhances the capacity to generate antitumor T-cell responses, but the expression of such an enhancing effect is restricted to early stages of tumor growth.

Distamycin-A derivatives potentiate tumor-necrosis-factor activity via the modulation of tyrosine phosphorylation.

The cytotoxic activities of 2 novel distamycin-A derivatives, FCE 24517 and FCE 25450A, alone and in combination with tumor-necrosis factor-alpha (TNF), were studied. Both drugs, especially FCE 25450A, analyzed extensively here, inhibited the growth of HL60 promyelocytic cells, and human SV80 and murine L929 transformed fibroblasts in a dose-dependent manner. The growth-inhibitory potential of sequential exposure to the distamycin-A analogs and TNF was determined. A 4-hr treatment of L929 fibroblasts with 100-1,000 ng/ml FCE 25450A, followed by 2 ng/ml TNF, resulted in a synergistic anti-proliferative effect. The synergism of FCE 24517 with TNF was less profound. Experiments to elucidate the mechanism underlying the cooperation revealed that FCE 25450A pre-treatment almost completely abolished the elevated tyrosine phosphorylation of a 137-kDa and other membranal proteins and prevented the de-phosphorylation of another protein band observed in L929 cells in the presence of TNF. FCE 25450A alone induced no changes in the phosphotyrosine profile of the cells. The effect of FCE 25450A was counteracted by the tyrosine-phosphatase inhibitor orthovanadate. In parallel, the inhibitor also diminished the antiproliferative action of the FCE 25450A/TNF combination. These findings suggest that, beyond their cytotoxic effects as single agents, the distamycin derivatives increase the sensitivity of cells to TNF. This effect is governed via the inhibition of TNF-induced tyrosine phosphorylation of specific proteins which are probably involved in the development of TNF resistance. Thus, protein de-phosphorylation might provide an additional mechanism of action of these novel distamycin-A-derived drugs.

Enhanced induction of very late antigen 4/lymphocyte function-associated antigen 1-dependent T-cell migration to tumor sites following administration of interleukin 12.

Administration of interleukin 12 (IL-12) into mice bearing CSA1M, OV-HM, Meth A, or MCH-1-A1 tumor induced complete regression of CSA1M and OV-HM tumors but induced only a slight growth inhibition of Meth A and MCH-1-A1 tumors. These effects of IL-12 were associated with high and only marginal levels of T-cell infiltration into CSA1M/OV-HM and Meth A/MCH-1-A1 tumor masses, respectively. Here, we investigated the role of IL-12 in the induction of T-cell migration. Spleen cells from untreated or IL-12-treated CSA1M-bearing mice were stained in vitro with a fluorescein chemical and transferred i.v. into IL-12-untreated CSA1M-bearing mice. Migration of donor cells was quantitated by counting the number of fluorescent cells on cryostat sections of tumor masses. Although only a slight migration was detected for spleen cells from IL-12-untreated CSA1M-bearing as well as IL-12-treated or untreated normal mice, enhanced migration was observed for cells from IL-12-treated CSA1M-bearing mice. A similar enhanced migration was observed for the OV-HM model. In contrast, such an enhancement was only marginal in the Meth A and MCH-1-A1 models. Immunohistochemical studies of tumors from IL-12-treated mice revealed that the predominant T-cell subset was CD4+ in CSA1M and CD8+ in OV-HM tumor masses. Consistent with this observation, the dominant subset of migrating T cells was found to be CD4+ in the CSA1M and CD8+ in the OV-HM models. T-cell migration was inhibited by pretreatment of recipients with either combination of anti-very late antigen 4 + anti-vascular cell adhesion molecule 1 or anti-lymphocyte function-associated antigen 1 + anti-intercellular adhesion molecule 1 monoclonal antibody. These results indicate that IL-12 can confer T cells with a capacity to migrate to tumor sites through very late antigen 4/lymphocyte function-associated antigen 1 adhesion pathways and that the in vivo acquisition of such a capacity following IL-12 treatment correlates with the induction of tumor regression.

[Clinical study on treating asthma of cold type with wenyang tongluo mixture].

Sixty-eight asthma patients of Cold type were randomly divided into two groups, 34 for each group. The treated group was treated with Chinese herbal medicine Wenyang Tongluo Mixture (WYTLM), the control group was treated with Salbutamol orally and beclomethasone dipropionate aerosol. After 8 weeks of treatment, the results showed that there was no significant difference between the short-term total effective rate of the two groups (P > 0.05). Results of followup 1 year after withdrawal of treatment, showed that 9 patients (26.47%) in the treated group and 2 (5.88%) in the control group were cured clinically, it indicated that the long-term curative rate of the former group was higher than that of the latter group significantly (P < 0.05). And the effect of treated group on eliminating Asthenia-Cold symptoms, improving pulmonary ventilation function, regulating adrenergic beta-receptors of peripheral blood lymphocyte and decreasing the serum level of 5-hydroxytryptamine was more superior to that of control group (P < 0.05-0.01). This study provided some objective basis for using WYTLM in preventing and treating asthma of Cold type.

Interferon-independent activation of (2'-5') oligoadenylate synthetase in Friend erythroleukemia cell variants exposed to HMBA.

To provide evidence for the implication of interferon (IFN)-induced proteins in the regulation of cell growth during differentiation, the activation of (2'-5') oligoadenylate synthetase (2-5A synthetase) as well as of PKR, two IFN-induced proteins, during differentiation of Friend erythroleukemia cells, was studied. Two cell variants were used. The first (FL) was completely susceptible to hexamethylene bis-acetamide (HMBA)-treatment and responded in both growth-retardation and hemoglobin synthesis. The second (R1) failed to synthesize hemoglobin in response to HMBA although cell growth was still inhibited. In both cell variants, 2-5A synthetase enzyme activity was induced in a similar fashion, reaching a peak at 26 hours after treatment with HMBA. However, the down regulation of activity thereafter was not identical in both cases. In R1 cells, the reduction was much slower compared to FL cells. A similar pattern was observed with the appearance of the 43 kDa isoform of 2-5A synthetase in immunoblots. An analysis of 2-5A synthetase gene expression revealed the presence of 1.7 kb transcripts which peaked at 16 hours after HMBA-treatment in both cell variants. Again, the down-regulation in expression was slower in R1 than in FL cells. Addition of anti-murin alpha/beta-IFN antibodies did not reduce the level of either 2-5A synthetase expression or enzyme activity in either cell variant. Interestingly, the presence of antibodies also did not affect the pattern of pRb phosphorylation in the cell variants exposed to HMBA. In both cell variants, an increase in the amount of the phosphorylated form (ppRb) was observed in immunoblots after 4 hours. This form was gradually transformed to the underphosphorylated molecule (pRb) with time in culture, even in the presence of antibodies. This further substantiates the notion that IFN-induced regulation of pRb phosphorylation is mediated by IFN-induced proteins. The basal level of either expression or ezymatic activity of PKR detected in untreated FL or R1 cells, was relatively high. Treatment with HMBA did not result in further induction of PKR in either cell variant.

Molecular mechanisms underlying IFN-gamma-mediated tumor growth inhibition induced during tumor immunotherapy with rIL-12.

The present study investigates the molecular mechanisms by which IFN-gamma produced as a result of in vivo IL-12 administration exerts its anti-tumor effects. rIL-12 was administered three or five times into mice bearing CSA1M fibrosarcoma, OV-HM ovarian carcinoma or MCH-1-A1 fibrosarcoma. This regimen induced complete regression of CSA1M and OV-HM tumors but only transient growth inhibition of MCH-1-A1 tumors. The anti-tumor effects of IL-12 were associated with enhanced induction of IFN-gamma because these effects were abrogated by pretreatment of hosts with anti-IFN-gamma antibody. Exposure in vitro of the three types of tumor cells to rRFN-gamma resulted in moderate to potent inhibition of tumor cell growth. IFN-gamma stimulated the expression of mRNAs for an inducible type of NO synthase (iNOS) in CSA1M cells and indoleamine 2,3-dioxygenase (IDO), an enzyme capable of degrading tryptophan, in OH-HM cells, but induced only marginal levels of these mRNAs in MCH-1-A1 cells. In association with iNOS gene expression, IFN-gamma-stimulated CSA1M cells produced a large amount of NO which functioned to inhibit their own growth in vitro. Although OV-HM and MCH-1A1 cells did not produce NO, they also exhibited NO susceptibility. Whereas the tumor masses from IL-12-treated CSA1M-bearing or OV-HM-bearing mice induced higher levels of iNOS (for CSA1M) or IDO and iNOS (for OV-HM) mRNAs, the MCH-1-A1 tumor mass expressed lower levels of iNOS mRNA alone. Moreover, massive infiltration of CD4(+) and CD8(+) T cells and Mac-1(+) cells was seen only in the CSA1M and OV-HM tumors. Thus, these results indicate that IFN-gamma produced after IL-12 treatment induces the expression of various genes with potential to modulate tumor cell growth by acting directly on tumor cells or stimulating tumor-infiltrating lymphoid cells and that the effectiveness of IL-12 therapy is associated with the operation of these mechanisms.

Administration of recombinant interleukin 12 prevents outgrowth of tumor cells metastasizing spontaneously to lung and lymph nodes.

The present study investigates the ability of recombinant interleukin 12 (rIL-12) to modulate the growth of a primary tumor as well as the outgrowth of metastatic tumor cells in an ovarian carcinoma (OV-HM) model. This aggressive tumor displayed rapid growth of the primary tumor mass, high incidence of metastases to lung and lymph nodes, and invasion from the primary s.c. site to the peritoneal cavity. Starting 12 days after s.c. tumor cell implantation, several i.p. injections of rIL-12 at 2-3 day intervals resulted in regression of growing tumors. These treated mice did not show signs of metastases or tumor recurrence at the original site. One month after tumor implantation, untreated mice did not have visible lung metastasis, but some did have palpable lymph nodes. At this stage, the primary tumors of animals without palpable lymph nodes were surgically resected. When examined 2 months later, most animals had developed lymph node and lung metastases. In contrast, rIL-12 injections after tumor resection inhibited the development of metastases in both lung and lymph nodes. This contrasted with the failure of IL-2 to prevent metastases. Even for mice already showing signs of lymph node metastases or invasion of the abdominal wall, rIL-12 administration after tumor resection prevented further invasion to the peritoneal cavity and growth of metastatic tumor cells in lung. It was somewhat surprising that the IL-12 treatment of animals after 1 month of tumor growth without resection also resulted in complete tumor regression, as well as eradication of micrometastasis that would have occurred before the treatment. Moreover, they exhibited resistance to a rechallenge with the same tumor but not with a second tumor. Thus, this tumor system provides a relevant model to clinical situations in terms of treatment of advanced tumors and metastases. These results also indicate that IL-12 can induce a curative immune response, even in the face of an aggressive micrometastasizing tumor.