Eri Hagiwara

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.

Structural and functional characterization of the USP11 deubiquitinating enzyme, which interacts with the RanGTP-associated protein RanBPM.

RanBPM is a RanGTP-binding protein required for correct nucleation of microtubules. To characterize the mechanism, we searched for RanBPM-binding proteins by using a yeast two-hybrid method and isolated a cDNA encoding the ubiquitin-specific protease USP11. The full-length cDNA of USP11 was cloned from a Jurkat cell library. Sequencing revealed that USP11 possesses Cys box, His box, Asp and KRF domains, which are highly conserved in many ubiquitin-specific proteases. By immunoblotting using HeLa cells, we concluded that 921-residue version of USP11 was the predominant form, and USP11 may be a ubiquitous protein in various human tissues. By immunofluorescence assay, USP11 primarily was localized in the nucleus of non-dividing cells, suggesting an association between USP11 and RanBPM in the nucleus. Furthermore, the association between USP11 and RanBPM in vivo was confirmed not only by yeast two-hybrid assay but also by co-immunoprecipitation assays using exogenously expressed USP11 and RanBPM. We next revealed proteasome-dependent degradation of RanBPM by pulse-chase analysis using proteasome inhibitors. In fact, ubiquitinated RanBPM was detected by both in vivo and in vitro ubiquitination assays. Finally, ubiquitin conjugation to RanBPM was inhibited in a dose-dependent manner by the addition of recombinant USP11. We conclude that RanBPM was the enzymic substrate for USP11 and was deubiquitinated specifically.

Safety and efficacy of pirfenidone in idiopathic pulmonary fibrosis in clinical practice

BACKGROUND Previous pirfenidone trials have only involved patients with mild-to-moderate idiopathic pulmonary fibrosis (IPF). The aim of this study was to investigate the safety and efficacy of pirfenidone in patients with mild-to-severe IPF in clinical practice. METHODS The clinical records of 76 patients who were diagnosed with IPF and received pirfenidone were reviewed. RESULTS The most frequent adverse event was anorexia, although the grade of anorexia in most patients was mild. Dose reduction of pirfenidone improved anorexia in 84% affected patients, which resulted in a high medication compliance rate. The mean forced vital capacity (FVC) at the initiation of pirfenidone therapy in this study was approximately 10% lower than that in previous clinical trials. The mean change in FVC during the 6-month period prior to the therapy initiation was -188 mL, which improved to -19 mL during the 6-month period after therapy. Significant attenuation in percentage predicted diffusion capacity of the lung for carbon monoxide decline was also achieved after pirfenidone therapy initiation. The efficacy of pirfenidone in attenuating the degree of FVC decline was higher in the group with FVC decline of ≥150 mL during the 6-month period prior to therapy initiation. The levels of serum markers, such as KL-6 and SP-D, were also lowered by the therapy. CONCLUSIONS These results showed that pirfenidone was well-tolerated and had beneficial effects in patients with mild-to-severe and/or progressive IPF. The degree of disease progression prior to the initiation of pirfenidone therapy had an impact on the response to the therapy.

Zymosan enhances the immune response to DNA vaccine for human immunodeficiency virus type-1 through the activation of complement system.

In the present study, the adjuvant effect of zymosan on human immunodeficiency virus type‐1 (HIV‐1)‐specific DNA vaccine and the mechanism of this enhancement were studied in a murine model. We coinoculated zymosan with our candidate HIV‐1 specific DNA vaccine (pCMV160IIIB) into skeletal muscles of BALB/c mice. Higher levels of both humoral immune response and HIV‐specific delayed‐type hypersensitivity (DTH) response were observed when zymosan was coinoculated with pCMV160IIIB compared with that obtained using pCMV160IIIB alone. HIV‐specific cytotoxic T lymphocyte (CTL) activity was also enhanced. This enhancing activity was suppressed when coinoculated to the fifth complement (C5)‐deficient DDD and AKR mice. The enhanced activity was also suppressed when anti‐C3 antibody was inoculated to mice intramuscularly. There was significant induction of immunoglobulin G2a (IgG2a) and interferon‐γ (IFN‐γ) in pCMV160IIIB vaccine with zymosan. These results suggest that zymosan‐mediated DNA vaccination enhances helper T cell (Th) 1‐mediated immunity. The effect is suggested to be based on the consequences of its recruitment and activation of macrophages, dendritic cells or antigen‐presenting cells (APC) through complement activation, especially through the alternative pathway. Taken together, these results suggest that zymosan can be an effective immunological adjuvant in DNA vaccination against HIV‐1.

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.

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 Peyers 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.

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 × 107 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.

Prognostic Factors in Interstitial Lung Disease Associated with Primary Sjögren's Syndrome: A Retrospective Analysis of 33 Pathologically-Proven Cases

Introduction Interstitial lung disease associated with primary Sjögren’s syndrome (pSS–ILD) shows several patterns such as nonspecific interstitial pneumonia (NSIP) and usual interstitial pneumonia (UIP). Although UIP is a well–recognized prognostic determinant in idiopathic interstitial pneumonias, whether this is also the case in pSS–ILD is unclear. The objectives of this study were to evaluate the prognostic effect of UIP, and to identify the prognostic factors in pSS–ILD. Methods A retrospective review of medical records identified 33 consecutive patients with pathologically–proven pSS–ILD. Each patient was classified into each ILD pattern by multidisciplinary analysis. Baseline clinical–radiologic–pathologic characteristics and survival rates were compared between the ILD patterns. Finally, the prognostic factors in pSS–ILD were assessed by univariate and subsequent multivariate analyses using Cox’s proportional hazards regression model. Results pSS–ILD patients were diagnosed with NSIP (n = 22) or UIP (n = 11). The median follow–up period was 110 months, and five-year survival rate was 87.3% in the total patient population. The prognosis of the UIP patients was not significantly different from that of the NSIP patients (NSIP to UIP, hazard ratio [HR]: 0.77, 95% confidence interval [CI]: 0.18–3.36, P = 0.73). Multivariate analysis identified PaCO2 (HR: 1.68 per 1 Torr increase, 95% CI: 1.24–2.28, P < 0.01), extent of reticular abnormality on high–resolution CT (HR: 4.17 per 1-grade increment, 95% CI: 1.18–14.73, P = 0.03), and severity of fibroblastic foci (HR: 9.26 per 1-grade increment, 95% CI: 1.74–49.35, P < 0.01) as prognostic factors in pSS–ILD. Conclusions UIP in pSS–ILD was not related to poorer prognosis than NSIP. Assessment of detailed clinical–radiologic–pathologic findings is more important than distinguishing UIP to evaluate prognosis in this disease.

Quantitation of autoantibody-secreting B cells in systemic lupus erythematosus

An ELISA spot assay was used to quantitate the number of autoantibody-secreting B cells in the peripheral blood of patients with systemic lupus erythematosus. Patients with active disease had 20 fold more anti-DNA, 4 fold more anti-actin and 3 fold more anti-myosin secreting lymphocytes than controls but normal numbers of anti-cardiolipin and anti-transferrin secreting B cells. 60% of SLE patients had increased numbers of B cells reactive with multiple autoantigens. These data suggest that B cell activation in SLE may be influenced by both antigen-specific and antigen-independent factors.

Distinct features of distant metastasis and lymph node stage in lung adenocarcinoma patients with epidermal growth factor receptor gene mutations.

BACKGROUND The influence of epidermal growth factor receptor (EGFR) mutation status on distant and lymph node metastasis is not fully understood. METHODS Ninety-five consecutive patients with stage IV lung adenocarcinoma, who had been examined for the EGFR mutation status, were retrospectively analyzed with regard to numbers of distant metastasis and clinical stage of lymph node metastasis at the time of diagnosis. RESULTS While EGFR mutation status did not influence the presence or absence of distant metastasis in the lung, brain, or liver, patients with EGFR mutations demonstrated a significantly greater number of metastatic lesions in the lung (median: 85 vs. 4, P=0.01) and the brain (11 vs. 3.5, P=0.04). On the other hand, patients with EGFR mutations showed a significantly lower lymph node staging (P<0.01). CONCLUSION The presence of EGFR mutations in patients with lung adenocarcinoma correlates with lower lymph node stage and a greater number of metastatic lesions in the lung and brain.