Methotrexate restores the function of peripheral blood regulatory T cells in psoriasis vulgaris via the CD73/AMPK/mTOR pathway.

BACKGROUND: Methotrexate (MTX) is used to treat psoriasis, a chronic inflammatory skin disease. OBJECTIVES: To investigate the molecular mechanism of MTX in the treatment of psoriasis. METHODS: Regulatory T cells (Tregs) and effector T (Teff) cells were isolated from the blood of patients with psoriasis and healthy controls. The proliferation of Teff cells was detected by carboxyfluorescein succinimidyl ester assay. The interferon (IFN)-γ and interleukin (IL)-17 levels were analysed by enzyme-linked immunosorbent assay. The expression of CD73 and FoxP3 were determined by flow cytometry. The expression of proteins in the AMPK/mTOR pathway were detected by Western blot analysis. RESULTS: The data suggested that patients with psoriasis have Tregs with decreased immune suppression function and reduced expression of CD73 compared with healthy controls. Moreover, MTX could significantly restore the immunosuppressive function of IL-17-secreting Tregs. This, in turn, inhibits aberrant proliferation of Teff cells in patients with psoriasis, reverses downregulation of CD73, upregulates phosphorylated AMPK and inhibits phosphorylated mTOR, and downregulates IL-17 and IFN-γ levels. CONCLUSIONS: We speculate that MTX can restore the immunosuppressive function of Tregs through upregulating CD73, activating AMPK and inactivating the mTOR pathway. These findings may partly explain the mechanism by which MTX treats psoriasis.

Dexamethasone and sodium carboxymethyl cellulose prevent postoperative intraperitoneal adhesions in rats

We aimed to evaluate the effects of the barrier agent sodium carboxymethyl cellulose (SCMC) with and without dexamethasone for the prevention of postoperative adhesion formation in a rat model of postoperative peritoneal adhesion. A total of 160 three-month old male and female Wistar rats underwent a laparotomy, and adhesions were induced by ileocecal abrasion. Rats were randomly assigned to 4 groups (n=40 each): group A, untreated; group B, treated with SCMC only; group C1, treated with SCMC + 3 mg dexamethasone, and group C2, treated with SCMC + 8 mg dexamethasone. After 12 days, adhesion formation and histopathological changes were compared. In groups A, B, C1, and C2, the mortality rates were 10, 5, 5, and 5%, respectively. In groups C1 and C2, the adhesions were filmy and easy to dissect and were milder compared with those in groups A and B. The total adhesion score in group C1 (3.38±0.49) was significantly lower than that of group B (6.01±0.57; P<0.01) or group A (8.01±0.67; P<0.05). There was no significant difference in adhesion formation between groups C1 and C2. Compared with groups A and B, groups C1 and C2 exhibited milder histopathological changes. SCMC in combination with dexamethasone can prevent adhesion formation and is a better barrier agent than SCMC alone. The safety and feasibility of SCMC in combination with dexamethasone to prevent adhesion formation after abdominal surgery warrants further clinical study.

Dexamethasone and sodium carboxymethyl cellulose prevent postoperative intraperitoneal adhesions in rats.

We aimed to evaluate the effects of the barrier agent sodium carboxymethyl cellulose (SCMC) with and without dexamethasone for the prevention of postoperative adhesion formation in a rat model of postoperative peritoneal adhesion. A total of 160 three-month old male and female Wistar rats underwent a laparotomy, and adhesions were induced by ileocecal abrasion. Rats were randomly assigned to 4 groups (n=40 each): group A, untreated; group B, treated with SCMC only; group C1, treated with SCMC + 3 mg dexamethasone, and group C2, treated with SCMC + 8 mg dexamethasone. After 12 days, adhesion formation and histopathological changes were compared. In groups A, B, C1, and C2, the mortality rates were 10, 5, 5, and 5%, respectively. In groups C1 and C2, the adhesions were filmy and easy to dissect and were milder compared with those in groups A and B. The total adhesion score in group C1 (3.38±0.49) was significantly lower than that of group B (6.01±0.57; P<0.01) or group A (8.01±0.67; P<0.05). There was no significant difference in adhesion formation between groups C1 and C2. Compared with groups A and B, groups C1 and C2 exhibited milder histopathological changes. SCMC in combination with dexamethasone can prevent adhesion formation and is a better barrier agent than SCMC alone. The safety and feasibility of SCMC in combination with dexamethasone to prevent adhesion formation after abdominal surgery warrants further clinical study.

Serial analysis of gene expression in progressing and regressing mouse tumors implicates the involvement of RANTES and TARC in antitumor immune responses.

Previously we demonstrated that gene transduction of the granulocyte-macrophage colony stimulating-factor (GM-CSF) gene into mouse tumor cells eliminated tumorigenicity in vivo. The rejection process of the subcutaneous tumor was as follows: transient tumor growth peaked around 10 days after tumor injection, then the tumors were rejected within a week. In this paper, we analyzed the gene expression of the transiently established tumor masses by the serial analysis of gene expression method to identify molecules associated with the antitumor effect. We then screened those genes that were differentially expressed between the parental and the GM-CSF-transduced tumors and identified a group of genes that are suggested to have a relationship with tumor rejection, including a cytokine receptor, adhesion molecules, chemokines, cytotoxicity-related molecules, and others. Focusing on the chemokine genes TARC and RANTES, which were preferentially expressed in the GM-CSF-transduced tumors, their forced expression on mouse tumor cells showed moderate suppression of tumor formation. Transduction of GM-CSF in combination with either the TARC or the RANTES gene into tumor cells profoundly inhibited tumor establishment. Histological findings suggested the significant contribution of CD4+ T cells to tumor regression in both TARC/GM-CSF- and RANTES/GM-CSF-transduced tumor cells, in excess of that seen with GM-CSF transduction alone.

Prime-boost vaccination with plasmid DNA and a chimeric adenovirus type 5 vector with type 35 fiber induces protective immunity against HIV.

Immunization involving a DNA vaccine prime followed by an adenovirus type 5 (Ad5) boost elicited a protective immune response against SHIV challenge in monkeys. However, the hepatocellular tropism of Ad5 limits the safety of this viral vector. This study examines the safety and immunogenicity of a replication-defective chimeric Ad5 vector with the Ad35 fiber (Ad5/35) in BALB/c mice and rhesus monkeys. This novel Ad5/35 vector showed minimal hepatotoxicity after intramuscular administration with the novel Ad5/35 vector. In addition, an Ad5/35 vector expressing HIV Env gp160 protein (Ad5/35-HIV) generated strong HIV-specific immune responses in both animal models. Priming with a DNA vaccine followed by Ad5/35-HIV boosting yielded protection against a gp160-expressing vaccinia virus challenge in BALB/c mice. The Ad5/35-HIV vector was significantly less susceptible to the pre-existing Ad5 immunity than a comparable Ad5 vector. These findings indicate that an Ad5/35 vector-based HIV vaccine may be of considerable value for clinical use.

Research on wastewater reuse planning in Beijing central region.

The need to implement wastewater reuse in Beijing is discussed. Based on the investigation of the built wastewater reuse projects in Beijing, the differences between small wastewater reuse system and large systems were analyzed according to the technical, economical and social issues. The advantages and disadvantages of the small system and the large system were then given. In wastewater reuse planning in Beijing urban region, the large system was adopted. The rations of reclaimed water for difference land use type, including industrial reuse, municipal reuse, grass irrigation, and scenes water reuse were determined. Then according to the land use information in every block in central Beijing, using GIS techniques, the amounts of the reclaimed water needed in every block were calculated, and the main pipe system of reclaimed water was planned.

Immunomodulation by apoptosis-inducing caspases for an influenza DNA vaccine delivered by gene gun.

Apoptosis-inducing caspases have been tested for immunomodulatory effect on a gene gun-delivered DNA vaccine which expresses influenza hemagglutinin. Attenuated murine caspase 2 and a chimera of murine caspase 2 prodomain and human caspase 3 strongly enhanced humoral and cell-mediated immune response to hemagglutinin when they were co-administered with an immunogen DNA. In contrast, wild-type caspases did not enhance the DNA-raised immune response. Caspase dose-dependent antibody response curve revealed that the antibody level was in inverse relation to the amount of administered caspase. These findings indicate that bland apoptosis of antigen-harboring cells can elicit enhanced immune responses. Extensive apoptosis interferes with the generation of immune response. Gene gun delivery involving caspases elicited type-2 immune responses that characterized with dominant IL-4 and IgG1 production. ELISPOT assays showed that CD4 T cells were preferentially activated, while CD8 T cell response remained at marginal level. Using attenuated caspases for gene gun DNA vaccination is a useful approach to amplify type-2 immune responses.

Transplacental genetic immunization after intravenous delivery of plasmid DNA to pregnant mice.

A number of factors influence the development of tolerance, including the nature, concentration, and mode of Ag presentation to the immune system, as well as the age of the host. The studies were conducted to determine whether immunizing pregnant mice with liposome-encapsulated DNA vaccines had an effect on the immune status of their offspring. Two different plasmids (encoding Ags from HIV-1 and influenza virus) were administered i.v. to pregnant mice. We examined the uptake of plasmid DNA by the fetuses until the 21st postcoital day, but little such transfer occurred in early pregnancy. At 9.5 days postconception with cationic liposomes, injected plasmid was present in the tissues of the fetus, consistent with transplacental transfer. When the offspring of vaccinated dams were immunized with DNA vaccine, they mounted stronger Ag-specific immune responses than controls, and were protected against challenge by homologous influenza virus after vaccination. Moreover, such immune responses were strong in the offspring of mothers injected with DNA plasmid 9.5 days after coitus. These results suggest that DNA-vaccinated mothers confer the Ag-specific immunity to their progeny.

Topical application of HIV DNA vaccine with cytokine-expression plasmids induces strong antigen-specific immune responses.

The topical application of DNA vaccine to the skin is a useful method of immunization because of its simplicity, painlessness and economy. But the immune responses that it elicits are relatively low. In this study, we administered human immunodeficiency virus type-1 (HIV-1) DNA vaccine with cytokine-expressing plasmids to the skin of mice by a new topical application technique involving prior elimination of keratinocytes using fast-acting adhesive. Our results revealed that the topical application of HIV-1 DNA vaccine induced high levels of both humoral and cell-mediated immune activity against HIV-1 envelope antigen. Co-administration of the DNA vaccine with cytokine expression plasmids of IL-12 and granulocyte-macrophage colony-stimulating factor (GM-CSF) by this new method raised the levels of both the HIV-specific cytotoxic T lymphocyte (CTL) response and delayed-type hypersensitivity (DTH) and facilitated the induction of substantial immune responses by DNA vaccine. Skin biopsy sections, thus, immunized showed significant increases of S-100 protein-positive dendritic cells (DCs). These results suggest that the topical application method described here is an efficient route of DNA vaccine administration and that the immune response may be induced by DNA plasmids taken in by DCs, Langerhans cells (LCs), or others such as antigen-presenting cells. This new topical application is likely to be of benefit in clinical use.

Protection against influenza virus challenge by topical application of influenza DNA vaccine.

We studied the use of a DNA vaccine expressing the matrix (M) gene of the influenza virus A/PR/8/34. Mice were immunized by painting the DNA vaccine three times on the skin after removal of its keratinocytic layers. Immunization by this method produced M-specific antibodies and cytotoxic T lymphocyte (CTL) response, and acquired resistance against influenza virus challenge. This protection was abrogated by the in vivo injection of anti-CD8 or anti-CD4 monoclonal antibody. We further found that simultaneous topical application (t.a.) of GM-CSF expression plasmid (pGM-CSF) or liposomes plus mannan produced stronger immune response competence and enhanced the protective effect against influenza virus challenge. The present study revealed that administering DNA vaccine by topical application can elicit both humoral and cell-mediated immunity (CMI).

Activation of HIV-1-specific immune responses to an HIV-1 vaccine constructed from a replication-defective adenovirus vector using various combinations of immunization protocols.

We constructed a recombinant replication defective adenovirus vector containing the env gene (Ad-Bal) derived from macrophage-trophic HIV-1 (HIV-1 Bal). We then immunized mice with this vector using several administration routes and protocols, and examined the immune response. When the Ad-Bal viral vector (over 1 x 10(7) pfu) was injected subcutaneously, both humoral and cell-mediated immunities were induced. However, immune response induced by the Ad-Bal vector alone was weaker than that induced by the recombinant vaccinia viral vector. We then employed the following three immunization protocols: (l) DNA vaccination followed by immunization with the Ad-Bal; (2) vaccination using the Ad-Bal vector followed by DNA vaccination; and (3) DNA vaccination followed by Ad-Bal infection and passive transfer of dendritic cells (DCs) infected with the Ad-Bal. Among the three protocols, the last gave the strongest humoral and cell-mediated immunity. These results suggest that the combination of DNA vaccination, Ad-Bal vector infection and passive transfer of Ad-Bal-infected DCs can induce strong immunity against HIV-1 Bal.

Protective immunity against influenza A virus induced by immunization with DNA plasmid containing influenza M gene.

DNA vaccination is characterized by its preferential induction of the cytotoxic T cell lymphocyte (CTL) response and is expected to be a useful means of protection against viral infection. We examined the protective effect of an expression plasmid (pME18S-M) containing M1 and M2 genes of influenza A/PR/8/34. We detected the CTL activity by introducing these plasmids into BALB/c mice by either the intramuscular or the intranasal route. The influenza-specific antibody response was also induced, although its neutralizing effect against influenza virus was not observed. From 70 to 80% protection was observed in the mice immunized with the pME18S-M plasmid followed by lethal infection with influenza viruses of the A/WSN/33 and A/PR/8/34 strains, whereas all mice without the plasmid vaccination failed to survive. This protective activity was significantly weakened when the CD8(+) cells of these immunized mice were eliminated by several injections of anti-CD8 antibody. The protective activity was also weakened when anti-CD4 antibody was injected in the early phase of DNA vaccination. These data suggest that the pME18S-M plasmid is useful as a DNA vaccine for overcoming highly mutational influenza viruses.

A novel recombinant adeno-associated virus vaccine induces a long-term humoral immune response to human immunodeficiency virus.

Recombinant adeno-associated virus (AAV) has attracted tremendous interest as a promising vector for gene delivery. In this study we have developed an HIV-1 vaccine, using an AAV vector expressing HIV-1 env, tat, and rev genes (AAV-HIV vector). A single injection of the AAV-HIV vector induced strong production of HIV-1-specific serum IgG and fecal secretory IgA antibodies as well as MHC class I-restricted CTL activity in BALB/c mice. The titer of HIV-1-specific serum IgG remained stable for 10 months. When AAV-HIV vector was coadministered with AAV-IL2 vector, the HIV-specific cell-mediated immunity (CMI) was significantly enhanced. Boosting with AAV-HIV vector strongly enhanced the humoral response. Furthermore, the mouse antisera neutralized an HIV-1 homologous strain, and BALB/c mice immunized via the intranasal route with an AAV vector expressing the influenza virus hemagglutinin (HA) gene showed protective immunity against homologous influenza virus challenge. These results demonstrate that AAV-HIV vector immunization may provide a novel and promising HIV vaccination strategy.

Immunologic characterization of HIV-specific DNA vaccine.

We developed a method for applying HIV-1 DNA vaccine topically in mice. Topical application of DNA vaccine to the skin is useful against infections. To find a less expensive and less cumbersome vaccination method, we administered HIV-1 DNA vaccine to the skin of mice after elimination of keratinocytes using a fast-acting adhesive. HIV-1 DNA vaccine induced high levels of both humoral and cell-mediated immune activity against HIV-1 envelope antigen. A high level of HIV-1-specific cytotoxic T lymphocyte response was also observed, and a high level of IFN-gamma and IL-4 production was induced by the improved skin application of DNA vaccine. High levels of both HIV-specific cytotoxic T lymphocyte and delayed type hypersensitivity in topical application were induced by coadministration of the DNA vaccine with IL-12 expression plasmids and granulocyte-macrophage colony-stimulating factor expression plasmids. These immune responses were inhibited by intradermal injection of anti-CD11c or anti-I-A/I-E antibody. Therefore, topical administration of DNA vaccine is an effective route, and may be very useful for the prevention of infectious diseases.

8 Br-cAMP enhances both humoral and cell-mediated immune responses induced by an HIV-1 DNA vaccine.

From a series of preclinical studies and animal experiments, we have been able to demonstrate that DNA vaccines are a promising tool in strategies for protecting hosts from a variety of infectious diseases. Since the promoter activity of the human cytomegalovirus immediate-early promoter/ enhancer (CMV promoter) is known to be responsive to an elevation in the level of intracellular cAMP, we hypothesized that use of cAMP analogue (8-Bromo adenosine 3'5'-cyclic monophosphate, 8 Br-cAMP) would increase the level of transgene expression supported by the CMV, and enhance the ability of DNA vaccines to evoke an immune response against the transgene product in vivo. To evaluate this hypothesis, immune responses against HIV-1 envelope protein, gp160, an immunogenic HIV-1 component expressed under the control of the CMV promoter, were evaluated in BALB/c mice with or without stimulation by 8 Br-cAMP. DNA vaccine with 8 Br-cAMP was intramuscularly (i.m.) or intranasally (i.n.) administered to BALB/c mice twice on days 0 and 14. Regardless of which route was used, the combination increased the serum IgG antibody (Ab) titer, HIV-1-specific cytotoxic T lymphocyte (CTL) activity and the delayed-type hypersensitivity (DTH) response, compared with the effect of using the vaccine alone. When administered via the i.n. route, the combination also remarkably increased the titer of secretory IgA (sIgA). Moreover, it induced increased production of interferon-gamma with reduction in IL-4 synthesis, and decreased the ratio of serum IgG1/IgG2a. However, these enhancements were not observed when 8 Br-cAMP was coadministered with peptide vaccine or protein antigen. These data suggest that 8 Br-cAMP is able to enhance both humoral and cellular immune responses induced by the DNA vaccine. The induction of T helper type 1 (Th1) immunity against HIV-1 was also enhanced by coadministration of 8 Br-cAMP. A CAT assay study demonstrated that the adjuvant effect of 8 Br-cAMP may be due to the activation of the CMV promoter in the DNA vaccine. The virus challenge experiment in a mouse influenza model also proved our hypothesis.

The timing of GM-CSF expression plasmid administration influences the Th1/Th2 response induced by an HIV-1-specific DNA vaccine.

The mechanism of immune activation induced by a plasmid-encoding GM-CSF (pGM-CSF), administered in combination with a DNA vaccine encoding the envelope of HIV, was studied. Injecting pGM-CSF i.m. into mice 3 days before DNA vaccination primarily induced a Th2 response. Simultaneous administration of the DNA vaccine plus pGM-CSF activated both a Th1 and a Th2 response. When the plasmid was injected 3 days after DNA vaccination, enhancement of Th1 immunity predominated. These results suggest that the timing of cytokine expression determines the phenotype of the resultant Th response. After 3 days of pGM-CSF injection, the increased percentages of CD11c+, CD8+ cells were observed in the regional lymph nodes. In addition, many infiltrated cells, including S-100 protein-positive cells, were found in the pGM-CSF-injected tissue. The importance of these S-100+ cells or both CD8+ and CD11c+ cells, especially that of dendritic cells (DCs), was also studied. DCs derived from bone marrow and cultured in RPMI 1640 medium containing IL-4 and GM-CSF were incubated with DNA vaccine and then transferred into naive mice. Mice receiving DCs showed strong HIV-1-specific Th2 immune responses. Our results suggest that DCs play important roles in the activation or modification of the Th2-type immune response induced by DNA vaccination.

Rectal and vaginal immunization with a macromolecular multicomponent peptide vaccine candidate for HIV-1 infection induces HIV-specific protective immune responses.

An effective vaccine for human immunodeficiency virus (HIV) is needed to stimulate the immune response of the genital mucus to prevent mucosal transmission of the virus. We have developed a macromolecular multicomponent peptide vaccine candidate, VC1. Both rectal and vaginal immunization of VC1 mixed with cholera toxin (CT) induced HIV-1-specific IgA antibody in mouse fecal extract solution and vaginal wash. These antibody productions were enhanced by the combination with IL-4 or GM-CSF expressing plasmids. Either fecal extract or vaginal wash solution from immunized mice inhibited production of HIV-1IIIB p24 protein. The mononuclear cells from spleen, intestinal lymph nodes, or Peyer's patches from VC1- and CT-immunized mice released IFN-gamma or IL-4, when these cells were co-cultured with VC1 antigen. In addition, the regional lymphoid cells from rectal and vaginal region of mice immunized with VC1 and CT also elicited a substantial level of HIV-1-specific cytotoxic T cell (CTL) response. This CTL response was enhanced by the addition of IL-12 expressing plasmid. Our results clearly demonstrated that both rectal and vaginal immunization could induce systemic and mucosal immunities specific for HIV-1.

Evidence of HIV type 1 glycoprotein 120 binding to recombinant N-methyl-D-aspartate receptor subunits expressed in a baculovirus system.

Activation of the N-methyl-D-aspartate (NMDA) receptor by HIV-1 envelope glycoprotein 120 (gp120) is thought to represent at least one of the pathways causing neuronal damage in AIDS patients. In the present study, recombinant gp120 binding to NMDA receptor subunits expressed in a baculovirus system was examined by immunocytochemistry and a binding assay, using horseradish peroxidase (HRP)-conjugated and 125I-labeled recombinant gp120, respectively. We found that recombinant gp120 binds to Sf21 cells expressing epsilon1/zeta1 or epsilon2/zeta1 combined NMDA receptor subunits, but not to Sf21 cells infected with mock virus or Sf21 cells expressing a single epsilon1, epsilon2, or zeta1 NMDA receptor subunit. The binding was strongly blocked by unlabeled recombinant gp120, monoclonal anti-HIV-1 gp160 antibody, and a mixture of anti-epsilon1/epsilon2 and anti-zeta1 antibodies. The same results were obtained by flow cytometric analysis. These data suggest that HIV-1 gp120 may directly bind to the NMDA receptor. This evidence enhances our understanding of the mechanism of HIV-1-induced neuronal damage in AIDS patients.

Erythrocyte delta-aminolevulinic acid dehydratase genotype and other mechanisms affecting workers' susceptibility to lead.

In this study, the role of delta-aminolevulinic acid dehydratase (ALAD) variants in lead susceptibility was examined. The study subjects comprised 223 male workers, and the relationship between their blood lead level and erythrocyte ALAD activity or plasma/urine delta-aminolevulinic acid level was studied. Leukocyte specimens from 11 workers, whose erythrocyte ALAD activities were as low as one-fifth that of the other normal workers, were subjected to analyses of their ALAD and ALAD alleles. Further, the entire exon fragment of the ALAD gene was analyzed by polymerase chain reaction, and the reaction product was used as a target for direct DNA sequencing. Genomic DNA analysis revealed that all 11 workers had the ALAD allele, whereas the entire ALAD gene analysis failed to indicate other variants, except for the Rsa I site. The depletion in erythrocyte ALAD activity was not found to be caused by the ALAD allele.