iNOS expression in dystrophinopathies can be reduced by somatic gene transfer of dystrophin or utrophin

BACKGROUND: Nitric oxide (NO) is an inorganic gas produced by a family of NO synthase (NOS) proteins. The presence and the distribution of inducible-NOS (NOS II or iNOS), and NADPH-diaphorase (NADPH-d), a marker for NOS catalytic activity, were determined in muscle sections from control, DMD, and BMD patients. MATERIALS AND METHODS: NADPH-d reactivity, iNOS- and nNOS (NOS I)-immunolocalization were studied in muscles from mdx mice before and after somatic gene transfer of dystrophin or utrophin. RESULTS: In control patients, few fibers (<2%) demonstrated focal accumulation of iNOS in sarcolemma. In DMD patients, a strong iNOS immunoreactivity was observed in some necrotic muscle fibers as well as in some mononuclear cells, and regenerating muscle fibers had diffusely positive iNOS immunoreactivity. In DMD patients, NADPH-d reactivity was increased and mainly localized in regenerating muscle fibers. In mdx mice quadriceps, iNOS expression was mainly observed in regenerating muscle fibers, but not prior to 4 weeks postnatal, and was still present 8 weeks after birth. The expression of dystrophin and the overexpression of utrophin using adenovirus-mediated constructs reduced the number of iNOS-positive fibers in mdx quadriceps muscles. The correction of some pathology in mdx by dystrophin expression or utrophin overexpression was independent of the presence of nNOS. CONCLUSIONS: These results suggest that iNOS could play a role in the physiopathology of DMD and that the abnormal expression of iNOS could be corrected by gene therapy.     

Cross-presentation by dendritic cells of tumor antigen expressed in apoptotic recombinant canarypox virus-infected dendritic cells

We have investigated the possible usefulness of recombinant canarypox virus (ALVAC) encoding the melanoma-associated Ag, Melan-A/MART-1 (MART-1), in cancer immunotherapy, using a dendritic cell (DC)-based approach. ALVAC MART-1-infected DC express, and are able to process and present, the Ag coded by the viral vector. One consistent feature of infection by ALVAC is that these viruses induce apoptosis, and we show cross-presentation of Ag when uninfected DC are cocultured with ALVAC MART-1-infected DC. Uptake of apoptotic virally infected DC by uninfected DC and subsequent expression of tumor Ag in the latter were verified by flow cytometry analysis, image cytometry, and confocal microscopy. Functional activity was monitored in vitro by the stimulation of a MART-1-specific cytotoxic T cell clone. Heightened efficiency in Ag presentation is evidenced in the 2- to 3-fold increase in IFN-gamma production by the T cell clone, as compared with the ALVAC-infected DC alone. Cocultures of ALVAC MART-1-infected and uninfected DC are able to induce MART-1-specific T cell immune responses, as assessed by HLA class I/peptide tetramer binding, IFN-gamma ELISPOT assays, and cytotoxicity tests. Overall, our data indicate that DC infected with recombinant canarypox viruses may represent an efficient presentation platform for tumor Ags, which can be exploited in clinical studies.     

Membrane potential can be determined in individual cells from the nernstian distribution of cationic dyes.

The distribution of a selection of cationic fluorescent dyes can be used to measure the membrane potential of individual cells with a microfluorometer. The essential attributes of these dyes include membrane permeability, low membrane binding, spectral properties which are insensitive to environment, and, of course, strong fluorescence. A series of dyes were screened on HeLa cells for their ability to meet these criteria and several commercially available dyes were found to be satisfactory. In addition, two new dyes were synthesized for this work by esterification of tetramethyl rhodamine. The analysis of the measured fluorescent intensities requires correction for fluorescence collected from outside the plane of focus of the cell and for nonpotentiometric binding of the dye. The measurements and analysis were performed on three different cell types for which there exists a body of literature on membrane potential; the potentials determined in this work were always within the range of literature values. The rhodamine esters are nontoxic, highly fluorescent dyes which do not form aggregates or display binding-dependent changes in fluorescence efficiency. Thus, their reversible accumulation is quantitatively related to the contrast between intracellular and extracellular fluorescence and allows membrane potentials in individual cells to be continuously monitored.     

Sublethal,whole-body ionizing irradiation can be tumor promotive or tumor destructive depending on the stage of development of underlying antitumor immunity

Summary It was shown that sublethal (500 rads), whole-body -irradiation of mice bearing an established i.d. immunogenic tumor can result, after several days delay, in complete tumor regression and long-term survival, but only if radiation is given after the tumor is established and growing progressively. Exposing mice to the same dose of radiation several hours after tumor cells were implanted resulted, in contrast, in enhanced growth of the primary tumor and in earlier death from systemic disease. Irradiation-induced tumor regression failed to occur in mice that were incapable of generating antitumor immunity, because of having been made T cell deficient by thymectomy and irradiation. Again, irradiation-induced tumor regression could be blocked by infusion of spleen cells from donor mice bearing a well-established tumor. These and previously published results support the view that sublethal, whole-body ionizing irradiation causes tumor regression by preferentially destroying radiosensitive suppressor T cells, thereby enabling the host to generate a therapeutic level of concomitant immunity. It is suggested that the preferential destruction of suppressor cells by irradiation depends on the acquisition, during immunologic induction, of radioresistance by antigen-activated effector T cells, and that this is the reason irradiation causes regression only of established tumors. Not all tumors tested were immunogenic enough to undergo regression in response to -irradiation.This study was supported by Grants CA-16642 and CA-27794 from the National Cancer Institute, Grant RR-05705 from the Division of Research Resources, NIH; and a Grant-in-aid from RJR Nabisco     

The receptor specificity of bacteriophages can be determined by a tail fiber modifying protein.

T-Even type bacteriophages recognize their cellular receptors with the distal ends of their long tail fibers. The distal part of these fibers consists of a dimer of gene product (gp) 37. The assembly of this gp to a functional dimer requires the action of two other proteins, gp57 and gp38. Genes (g) 38 have been cloned from five T-even type phages which use the Escherichia coli outer membrane protein OmpA as a receptor. The phages used differ in their ability to infect a series of ompA mutants producing altered OmpA proteins, i.e., each phage has a specific host range for these mutants. The cloned genes 38 complemented g38 amber mutants of phage T2, which uses the outer membrane protein OmpF as a receptor. The complemented phages had become phenotypically OmpA-dependent and, with one exception, OmpF-independent, but regained the host range of T2 upon growth in a host lacking the cloned g38. The host range of the complemented phages, as determined on the ompA mutants, was identical to, similar to, or different from that of the phage, from which the cloned g38 originated. The results presented show that gp38 from one phage can phenotypically 'imprint', in a finely-tuned manner, a host range onto gp37 of another phage with a different host specificity. In view of the extreme diversity of host ranges observed, it is suggested that gp38 of T2 and of the OmpA-specific phages may remain attached to gp37 in the phage particle and in cooperation with gp37 determine the host range.     

Split immunity: immune inhibition of rat gliomas by subcutaneous exposure to unmodified live tumor cells

Gliomas that grow uninhibited in the brain almost never metastasize outside the CNS. The rare occurrences of extracranial metastasis are usually associated with a suppressed immune system. This observation raises the possibility that some gliomas might not grow outside the CNS due to an inherent immune response, We report in this study that the highly malignant F98 Fischer rat undifferentiated glioma, which grows aggressively in the brain, spontaneously regresses when injected live s.c. We found that this regression is immune-mediated and that it markedly enhances the survival or cures rats challenged with the same tumor intracranially either before or after the s.c. live-cell treatment. Adoptive transfer experiments showed the effect was immune-mediated and that the CD8 T cell fraction, which exhibited direct tumor cytotoxicity, was more effective than the CD4 T cell fraction in mediating resistance to intracranial challenge of naive rats. Brain tumors from treated rats exhibited enhanced CD3(+)CD8(+)CD4(-) and CD3(+)CD4(+)CD8(-) T cell infiltration and IFN-γ secretion. The results in the F98 glioma were corroborated in the Lewis rat CNS-1 astrocytoma. In both tumor models, s.c. treatment with live cells was significantly better than immunization with irradiated cells. We propose in this study a location-based immunotherapeutic phenomenon we term "split immunity": a tumor that thrives in an immune-privileged site may be inhibited by injecting live, unmodified tumor cells into a site that is not privileged, generating protective immunity that spreads back to the privileged site. Split immunity could explain several long-standing paradoxes regarding the lack of overt extracranial metastasis in patients with primary brain tumors.     

Active cystathionine beta-synthase can be expressed in heme-free systems in the presence of metal-substituted porphyrins or a chemical chaperone

Cystathionine beta-synthase (CBS), a key enzyme in the metabolism of homocysteine, has previously been shown to require a heme co-factor for maximal activity. However, the biochemical function of the CBS heme is not well defined. Here, we show that expression of human CBS in heme-deficient strains of Saccharomyces cerevisiae and Escherichia coli results in production of an enzyme that is misfolded and degraded. Addition of exogenous heme, porphyrins with non-iron metal, or porphyrin lacking metal entirely produced stable and active CBS enzyme. Purification of recombinant CBS enzyme expressed in the presence of various metalloporphyrins confirmed that Mn(III) and Co(III) had 30-60% of the specific activity of Fe(III)-CBS, and still responded to allosteric activation by S-adenosyl-L-methionine. Treatment of S. cerevisiae with the chemical chaperone trimethylamine-N-oxide resulted in near complete restoration of function to human CBS produced in a heme-deficient strain. Taken together, these results suggest that porphyrin moiety of the heme plays a critical role in proper CBS folding and assembly, but that the metal ion is not essential for this function or for allosteric regulation by S-adenosyl-L-methionine.     

基因枪接种乙型肝炎表面抗原促进DNA疫苗诱生的免疫应答转换

目的 为了克服基因枪接种乙型肝炎表面抗原(HBsAg)DNA疫苗诱生的免疫应答以Th2为主的缺点,在基因枪接种质粒HBsAg DNA疫苗的同时共导入或共表达乙型肝炎病毒壳(HBV core)基因作为佐剂,以促进其所诱生的HBsAg特异性的Th2型免疫应答向Tn1型转换。方法 构建可单独或共同表达HBsAg或核心抗原(HBcAg)的DNA免疫用载体pIRKS／core、pIRES／C149、pIRES／S、pIRES／S／Core和pIRES／S／C149,并在真核细胞进行表达验证。对BALB／c雌鼠进行免疫并检测小鼠免疫后的特异性体液免疫和细胞免疫指标。结果 共导入或共表达HBV core基因能增强基因枪接种HBsAg DNA疫苗诱生的Th1型免疫应答水平,包括HBsAg特异的IgG2a应答、CTL活性、IFN-γ产生能力等。结论 以HBV core基因为佐剂能促进基因枪接种HBsAg DNA疫苗诱生的Th2型免疫应答向Th1型免疫应答转换。     

Reproductive refractoriness in the Welsh Mountain ewe induced by a short photoperiod can be overridden by exposure to a shorter photoperiod

The objectives were to determine if relative lengths of photoperiods that induce reproductive cycles in ewes affect the length of the subsequent breeding season, if duration of the refractoriness that terminates breeding is affected by photoperiod length, and if the resulting refractoriness to an inductive photoperiod is absolute. Groups of Welsh Mountain ewes were exposed to either 12L:12D (n = 12) or 8L:16D (n = 6) photoperiods beginning at the summer solstice when daylengths reach a maximum of 17.5 h at Bristol, England. A control group (n = 10) was exposed to natural daylengths. Ovarian cycles in the controls, as judged by monitored plasma progesterone levels, commenced in early October, about 1 mo later (p less than 0.001 in both cases) than in sheep exposed to 12L:12D or 8L:16D. The advancement in cycle onset was similar under 12L:12D and 8L:16D (69 +/- 2 and 77 +/- 4 days after the summer solstice compared with 102 +/- 2 days in the controls). Duration of the breeding season (100 +/- 4 days) in ewes exposed to 12L:12D was significantly shorter (p less than 0.001 in both cases) than in ewes exposed to natural daylengths or 8L:16D (153 +/- 3 and 133 +/- 5 days, respectively). Approximately 70 days after the ending of ovulatory cycles in the 12L:12D group, half of the animals (n = 6) were transferred to 8L:16D. This treatment greatly (p less than 0.001) reduced the duration of anestrus and cycles began again 62 +/- 4 days after transfer to 8L:16D, or about 90 days earlier than in ewes (n = 6) remaining in 12L:12D.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Myxococcus xanthus developmentally expressed asgB-dependent genes can be targets of the A signal-generating or A signal-responding pathway.

Functional Myxococcus xanthus A signal-generating and A signal-responding pathways are required for the progression through early multicellular development. To identify genes responsive to these pathways, the expression of eight early developmental genes was analyzed. This examination identified one gene as a target of the A signal-generating pathway and four genes as targets of the A signal-responding pathway.     

Development of systemic immunity to glioblastoma multiforme using tumor cells genetically engineered to express the membrane-associated isoform of macrophage colony-stimulating factor.

We investigated the ability of Fischer rat T9 glioblastoma cells transduced with cDNA genes for the secreted (s) or membrane-associated (m) isoform of M-CSF to elicit an antitumor response when implanted into syngeneic animals. Intracranial (i.c.) implantation of 1 x 10(5) T9 cells expressing mM-CSF (T9/mM-CSF) resulted in 80% tumor rejection. Electron microscopy of the T9/mM-CSF tumor site, 2-4 days postimplantation, showed marked infiltration by macrophages, many of which were in physical contact with the T9/mM-CSF cells. Animals that rejected T9/mM-CSF cells were resistant to i.c. rechallenge with T9 cells, but not syngeneic MadB106 breast adenocarcinoma cells, suggesting that T9-specific immunity can be generated within the brain via the endogenous APCs. Intracranial injection of parental T9, vector control (T9/LXSN), or T9 cells secreting M-CSF (T9/sM-CSF) was 100% fatal. Subcutaneous injection of 1 x 10(7) T9/sM-CSF, T9/LXSN, or parental T9 cells resulted in progressive tumors. In contrast, T9/mM-CSF cells injected s.c. were destroyed in 7-10 days and animals developed systemic immunity to parental T9 cells. Passive transfer of CD3+ T cells from the spleens of immune rats into naive recipients transferred T9 glioma-specific immunity. In vitro, splenocytes from T9/mM-CSF-immunized rats specifically proliferated in response to various syngeneic glioma stimulator cells. However, only marginal T cell-mediated cytotoxicity was observed by these splenocytes in a CTL assay against T9 target cells, regardless of restimulation with T9 cells. Subcutaneous immunization with viable T9/mM-CSF cells was effective in eradicating i.c. T9 tumors.     

Avian sarcoma-leukosis virus pol-endo proteins expressed independently in mammalian cells accumulate in the nucleus but can be directed to other cellular compartments.

Eucaryotic expression vectors have been used to study transient expression of the avian sarcoma-leukosis retrovirus pol-endo protein in COS cells. The constructs encode proteins with N termini identical to that of authentic viral pp32 endonuclease with the exception of a single met residue encoded by the initiator AUG. The C termini correspond to unprocessed viral pol protein, authentic processed pp32, or a derivative which includes eight amino acids from the unprocessed portion. All three proteins localize to the nucleus. However, when the pol-endo domain is fused to a secretory signal peptide, the protein is found in medium and appears also to localize in the Golgi bodies and the cell membrane. These and derivative vectors will make it possible to assess the consequence of retroviral pol gene expression in eucaryotic cells.     

Complement-dependent lysis of tumor cells by a baboon IgM antibody to a tumor-associated antigen

Summary We developed a high-titer polyclonal antiserum to a glycoprotein tumor-associated antigen (TAA) by immunization of a baboon with the purified glycoprotein antigen. The baboon serum was fractionated into IgG and IgM components by DEAE Affi-Gel blue chromatography. The ability of the baboon IgM anti-TAA antibody to effect tumor cell lysis in the presence of complement was tested using a chromium-release assay. The baboon antibody was able to lyse melanoma target cells (20.8%–71.4% cytolysis), breast carcinoma cells (36.5%–38.9% cytolysis), and a neuroblastoma cell line (35.5% cytolysis) in the presence of complement but did not effect significant lysis of autologous lymphoblastoid cell lines (4.9% cytolysis) or peripheral blood lymphocytes from healthy volunteers (12.6% cytolysis). Cytolysis of melanoma target cells was completely inhibited by preabsorption of the IgM anti-TAA antibody with UCLA-SO-M14 (M14) cells and partially inhibited by preabsorption with several other melanoma cell lines. There was no significant inhibition of tumor cell lysis after preabsorption of the antibody with lymphoblastoid cell lines. Complement-dependent lysis of M14 targets could be blocked by addition of the purified antigen to the antibody prior to incubation with the tumor cells. Our results suggest that the glycoprotein TAA resides on the tumor cell surface and that the baboon IgM anti-TAA antibody recognizes the antigen on the cell surface and is able to fix complement and effect the lysis of the tumor cells.     

Antigen-specific sIalo B cells do not secrete IgG2a in response to TH1 cells and antigen: responsiveness can be restored by IL-4

The ability of Th cells, type 1 (TH1), to activate and induce differentiation of B cells into antibody-secreting cells is controversial because 1) some clones of TH1 cells provide help while others do not, and 2) by using the same TH1 clone, different laboratories disagree on whether they provide help to B cells. One possible explanation for the latter is the variability in the activation status of the B cells used in different laboratories. In the present studies, we have used Ag-specific B cells from athymic (nu/nu) mice, or sterilely housed nu/+ mice to study the TH1-mediated activation of B cells that had received little or no prior help from T cells and/or antigen in vivo. These B cells express low levels of surface Ia (sIa) Ag, and fail to secrete IgG2a in response to TH1 cells plus Ag; in contrast, responses to TH2 cells plus Ag are normal. To explore this observation further, we prepared "surface(s) Ia1o" B cells from conventionally housed BALB/c mice by sorting spleen cells on the fluorescence-activated cell sorter. This sIalo population also failed to produce IgG2a in response to TH1 cells plus Ag. In contrast, the sIahi, (presumably more mature) B cells, responded to both the TH1 and TH2 cells. The addition of LPS, TH2 cells or the lymphokine, IL-4, to cultures of sIalo B cells from normal or nu/nu mice (plus Ag and TH1 cells), restored IgG2a responses to control levels. Low sIa levels were not the sole cause of nonresponsiveness of the nu/nu B cells because a 24-h pulse with IL-4 restored sIa to control levels without restoring IgG2a production after activation with TH1 cells plus Ag. These data support the conclusion that sIalo B cells are immature and require an activation/maturation signal from IL-4 in vivo in order to respond to TH1 cells and Ag in vitro.     

Thymus-repopulating capacity of cells that can be induced to differentiate to T cells in vitro.

It was established previously that committed precursors of T cells, which reside in bone marrow and spleen and lack T cell surface differentiation antigens, can be induced by thymopoietin and certain other agents to differentiate rapidly in vitro into T cells bearing typical surface antigens, including Thy-1 and TL (Komuro-Boyse assay). To relate this differentiative step observed in vitro to physiologic events in vivo, a system was devised to trace the migration of precursor cells to the thymus, and their maturation to T cells. Lethally irradiated mice of a TL- strain received spleen cells from TL+ hybrids i.v., and the TL+ population of the thymus was enumerated 13 to 20 days later. Donor TL+ cells first became detectable at 13 days and increased thereafter. Preliminary tests showed that cells capable of migrating to the thymus have a similar density to the cells that are inducible in the Komuro-Boyse assay, this being lower than that of mature of T cells. The thymus-repopulating properties of the donor spleen population were not affected by: 1) pre-treatment in vitro with thymus extract or thymopoietin, which initiates differentiation of T cells precursors, nor b) pre-treatment with anti Thy-1 serum plus complement, which eliminates differentiated T cells. But pre-treatment a) and b) applied in sequence markedly reduced the capacity of spleen cells to repopulate the thymus. These results can be interpreted as follows: induction of Thy-1-TL- precursor cells (pro-thymocyte) in vitro yields Thy-1+TL+ cells (early thymocytes) which have not yet lost their property of repopulating the thymus; therefore, thymus-repopulation was not depleted by treatment a) alone, which induced Thy-1 +TL+ cells, nor by treatment b) alone, which did not affect thymus-repopulation by Thy-1-TL- cells, although treatments a) plus b) did eliminate the newly induced Thy-1+TL+ cells and thus impaired repopulation of the thymus. We conclude that the cell which responds to thymopoietin in the Komuro-Boyse assay by expressing the T cell surface phenotype is the same cell (pro-thymocyte) that normally migrates in vivo from hemopoietic tissues to the thymus and is there induced by thymopoietin to express the phenotype of an early T cell.     

WISP-2 is a secreted protein and can be a marker of estrogen exposure in MCF-7 cells

As many structurally diverse chemicals have been reported to function as estrogens, evaluations for estrogenicity of compounds are of widespread concern. Recently, we identified WISP-2 (Wnt-1 inducible signaling pathway protein 2) as a novel estrogen-inducible gene in human breast cancer cells. In this study, we examined whether WISP-2 could be utilized as a marker for screening environmentally relevant compounds for estrogenicity. In MCF-7 cells, progesterone, dexamethasone, tri-iodothyronine, and 2,3,7,8-tetrachlorodibenzo-p-dioxin did not regulate the expression of WISP-2, indicating that its induction is highly specific for hormones that interact with the estrogen receptor. Western blot analysis detected WISP-2 protein induced by 17-beta-estradiol (E2), not only in the cell lysates but also in the culture supernatant of exposed cells, indicating that WISP-2 was a secreted protein. The induction of WISP-2 protein by E2 in the culture supernatant was dose-dependent with estimated EC(50) levels between 10 and 100 pM. Our results demonstrated the capacity to screen environmental compounds for estrogenicity via WISP-2 induction.     

A different intracellular distribution of a single reporter protein is determined at steady state by KKXX or KDEL retrieval signals

To establish the specific contribution to protein topology of KKXX and KDEL retrieval motifs, we have determined by immunogold electron microscopy and cell fractionation the intracellular distribution at steady state of the transmembrane and anchorless versions of human CD8 protein, tagged with KKXX (CD8-E19) and KDEL (CD8-K), respectively, and stably expressed in epithelial rat cells (Martire, G., Mottola, G., Pascale, M. C., Malagolini, N., Turrini, I., Serafini-Cessi, F., Jackson, M. R., and Bonatti, S. (1996) J. Biol. Chem. 271, 3541-3547). The CD8-E19 protein is represented by a single form, initially O-glycosylated: only about half of it is located in the endoplasmic reticulum, whereas more than 30% of the total is present in the intermediate compartment and cis-Golgi complex. In the latter compartments, CD8-E19 colocalizes with beta-coat protein (COP) (COPI component) and shows the higher density of labeling. Conversely, about 90% of the total CD8-KDEL protein is localized in clusters on the endoplasmic reticulum, where significant co-localization with Sec-23p (COPII component) is observed, and unglycosylated and initially O-glycosylated forms apparently constitute a single pool. Altogether, these results suggest that KKXX and KDEL retrieval motifs have different topological effects on theirs own at steady state: the first results in a specific enrichment in the intermediate compartment and cis-Golgi complex, and the latter dictates residency in the endoplasmic reticulum.     

Lymphocyte transformation induced by autologous cells. III. Lymphoblast-induced lymphocyte to stimulation does not correlate with EB viral antigen expression or immunity.

Human mitogen-induced and cell line B lymphoblasts stimulate the proliferation of allogeneic and autologous lymphocytes in culture. The role in thes reaction of EB viral determinants on the stimulating cells and immunity of the lymphocyte donor to the EB virus has been studied. The stimulatory capacity of cultured cell line lymphoblasts is not inhibited by incubating lymphoblasts with antisera to EB viral determinants. Cultured cell line B lymphoblasts stimulate as much thymidine incorporation by lymphocytes from donors with or without immunity to the EB virus. Further, a B lymphoblast cell line (U-698) which lacks the EB viral genome stimulated as much lymphocyte proliferation as did B lymphoblasts with the EB genome. Cultured T lymphoblast cell lines do not stimulate allogeneic lymphocyte proliferation. These cells appear to lack the determinants which stimulate lymphocyte transformation. No evidence was found that cultured cell line T lymphoblasts suppressed allogeneic lymphocyte proliferation. Mitogeninduced lymphoblasts from EB-immune and non-immune subjects stimulated the proliferation of autologous lymphocytes comparably. It is concluded that neither immunity to the EB virus nor expression of EB viral antigens on mitogen-induced on cell line lymphoblasts is necessary for the stimulation of lymphocyte proliferation.