加藤 大智

Feline coronavirus (FCoV) is divided into two types I and II, based on their growth in vitro and antigenicity. In this study, virus neutralization (VN) test was applied for type differentiation of FCoV infections. Sera of cats which were clinically and serologically diagnosed as feline infectious peritonitis (FIP) possessed significantly higher VN titers to type I FCoV, and sera from cats experimentally infected with FIPV type II had high VN titers to type II but not type I viruses. A total of 79 cat sera collected in the years between 2004 and 2005 were examined to evaluate seroprevalence by the VN test, showing the following results: (1) 50 cats (63.3%) were sero-positive to FCoV; (2) of the 50 FCoV positive cat serum samples, 49 (98%) showed significantly higher titers to type I virus and only one (2%) for type II virus. These results indicate that the VN test described here can be used for serological differentiation of FCoV infections of cats, and that FCoV type I is a dominant type in recent years of Japan.

OBJECTIVES: To determine the efficacy and the immunomodulatory function of Z-100 alone or combined with meglumine antimoniate on Leishmania amazonensis infection. METHODS: The effect of the compounds was evaluated by microscopic counting of intracellular amastigotes in macrophages stained with Giemsa, or axenic promastigotes, and IC(50) was determined by linear regression. The antileishmanial effect of the compounds was assessed in infected BALB/c mice by a limiting dilution analysis and the production of gamma interferon (IFN-gamma), interleukin 10 (IL-10), IL-4, IgG1 and IgG2a was measured by ELISA. RESULTS: In vitro, Z-100 showed antileishmanial activity against intracellular amastigotes of L. amazonensis with an IC(50) of 13 mg/L. Moreover, infected macrophages treated with Z-100 (12 mg/L) showed smaller parasitophorous vacuoles with fewer parasites than the control. In addition, the efficacy of Z-100 plus meglumine antimoniate [14 mg/L pentavalent antimony (Sb(v))] was higher (46% inhibition) than either Z-100 or meglumine antimoniate alone. Nevertheless, no effect of Z-100 on axenic promastigotes was observed. Infected BALB/c mice treated with Z-100 (100 microg/kg) alone did not show any antileishmanial effects in comparison with the control group, and IFN-gamma, as well as IL-10 and IL-4, was up-regulated by the treatment. In addition, both IgG1 and IgG2a were also increased by the Z-100 treatment. Although Z-100 plus meglumine antimoniate (14 or 28 mg/kg Sb(v)) controlled both the parasite load and the footpad swelling in comparison with control mice, no significant differences were found with meglumine antimoniate alone. CONCLUSIONS: In vitro, Z-100 alone or combined with meglumine antimoniate showed an antileishmanial effect on L. amazonensis. However, no effect was observed in infected BALB/c mice treated with Z-100, suggesting that the up-regulation of IL-10 and IL-4 production by the treatment could be interfering with the development of a protective Th1-type response. For further understanding of the effects of Z-100 in vivo, another strain of mice such as C57BL/6 should be tested in future.

The area around Rio Blanco, in the OrAn department in the north of the Argentinian province of Salta, is endemic for American tegumentary leishmaniasis. In an attempt to facilitate the identification of the Lutzomyia species in this area, sequences of the gene coding for the 18S ribosomal RNA (rRNA) of sandflies caught in a Shannon trap were explored, by a combination of PCR and analysis of restriction-fragment-length polymorphism (RFLP). The products from the PCR, which employed two primers developed specifically for this study (Lu.18S 1S and Lu.18S AR), were cloned into a commercial vector (pGEM-T Easy) so that their nucleotide sequences could be investigated. In the RFLP analysis, the products of single and double digestion with the AfaI and HapII restriction enzymes were separated by electrophoresis in 3% or 4% agarose. Taken together with the results of a morphological investigation of the flies, the resultant DNA fragment patterns were sufficient to identify most of the sandflies caught as Lu. neivai. Although two other species, Lu. cortelezzii and Lu. sallesi, were collected, they were relatively rare and only identified morphologically. A single digestion of the 18S-rRNA gene sequences with AfaI or HapII appeared sufficient and useful for the identification of Lu. neivai from the north of Salta province, and for several other Lutzomyia species.

Two transcripts coding for an adenosine deaminase (ADA) were identified by sequencing a Phlebotomus duboscqi salivary gland cDNA library. Adenosine deaminase was previously reported in the saliva of the sand fly Lutzomyia longipalpis but it was not present in the saliva of the sand flies Phlebotomus papatasi, P. argentipes, P. perniciosus and P. ariasi, suggesting that this enzyme is only present in the saliva of sand flies from the genus Lutzomyia. In the present work, we tested the hypothesis that the salivary gland transcript coding for ADA in Phlebotomus duboscqi, a sister species of Phlebotomus papatasi, produces an active salivary ADA. Salivary gland homogenates of P. duboscqi converted adenosine to inosine, suggesting the presence of ADA activity in the saliva of this species of sand fly; furthermore, this enzymatic activity was significantly reduced when using either salivary glands of recently blood-fed sand flies or punctured salivary glands, suggesting that this enzyme is secreted in the saliva of this insect. This enzymatic activity was absent from the saliva of P. papatasi. In contrast to other Phlebotomus sand flies, we did not find AMP or adenosine in P. duboscqi salivary glands as measured by HPLC-photodiode array. To confirm that the transcript coding for ADA was responsible for the activity observed in the saliva of this sand fly, we cloned this transcript into a prokaryotic expression vector and produced a soluble and active recombinant protein of approximately 60 kDa that was able to convert adenosine to inosine. Extracts of bacteria transformed with control plasmids did not show this activity. These results suggest that P. duboscqi transcripts coding for ADA are responsible for the activity detected in the salivary glands of this sand fly and that P. duboscqi acquired this activity independently from other Phlebotomus sand flies. This is another example of a gene recruitment event in salivary genes of blood-feeding arthropods that may be relevant for blood feeding and, because of the role of ADA in immunity, it may also play a role in parasite transmission.

Norovirus (NV) is a causative agent of acute gastroenteritis in humans, and shellfishes including oysters act as major vehicles of the virus. To investigate the genetic characteristics of NVs, we collected 1,512 oysters for raw consumption between October 2002 and March 2005 from two distinct areas (area A: the Sanriku Sea area; area B: the Setouchi Sea area). We detected the capsid gene and subjected it to phylogenetic analysis. By further quantification of the copy number of the genome by using real-time PCR, the NV capcid gene was detected in approximately 5% of the oysters, and they showed wide diversity. Two percent of the oysters from area B showed relatively large number of NVs, i.e., over 100 copies of capsid gene/oyster, whereas this was not observed in area A. Most of the detected NVs from oysters and humans were genetically related when the capsid region was compared. These results suggested that NVs obtained from humans and those obtained from oysters showed a potential relationship to each other and that some populations of Japanese oysters accumulated a relatively large number of NVs.

インターロイキン2(IL-2)はTh0およびTh1型ヘルパーT細胞によって放出されるT細胞の増殖因子であり、免疫反応に必須のサイトカインである。本研究において、組み換えグルタチオンS転移酵素(GST)-モルモットIL-2(GPIL-2)融合タンパク質を大腸菌(E.coli)により調製し、同タンパク質を免疫原として用いてGPIL-2に対するモノクローナル抗体(mAb)を産生し、モルモットにおける免疫反応についての研究の基礎確立を試みた。安定したハイブリドーマ細胞株3株を樹立し、E.coliにより産生した組み換えGPIL-2および組み換えバキュロウイルスに感染した昆虫細胞への各mAbの特異結合が酵素免疫測定法(ELISA)および/または免疫ブロット法により示された。これらのmAbのイソタイプ解析より、3種のmAbすべてがIgG1であり、κ鎖を有することが判明した。さらに、競合結合法によるエピトープの評価より、本研究で得られたmAbは異なる3エピトープに結合することが明らかとなった。以上、異なるGPIL-2エピトープに特異的なmAb2種に基づく、約0.3ng/mlのGPIL-2の感度閾値を有するサンドイッチELISAをGPIL-2検出用に開発した。

BACKGROUND: Determinants of the clinical presentation of the leishmaniases are poorly understood but Leishmania species and strain differences are important. To examine the relationship between clinical presentation, species and isoenzyme polymorphisms, 56 Leishmania isolates from distinct presentations of American tegumentary leishmaniasis (ATL) from Ecuador were analyzed. METHODS: Isolates were characterized by multilocus enzyme electrophoresis for polymorphisms of 11 isoenzymes. Patients were infected in four different ecologic regions: highland and lowland jungle of the Pacific coast, Amazonian lowlands and Andean highlands. RESULTS: Six Leishmania species constituting 21 zymodemes were identified: L. (Viannia) panamensis (21 isolates, 7 zymodemes), L. (V.) guyanensis (7 isolates, 4 zymodemes), L. (V.) braziliensis (5 isolates, 3 zymodemes), L. (Leishmania) mexicana (11 isolates, 4 zymodemes), L. (L.) amazonensis (10 isolates, 2 zymodemes) and L. (L.) major (2 isolates, 1 zymodeme). L. panamensis was the species most frequently identified in the Pacific region and was associated with several clinical variants of cutaneous disease (CL); eight cases of leishmaniasis recidiva cutis (LRC) found in the Pacific highlands were associated with 3 zymodemes of this species. Mucocutaneous leishmaniasis found only in the Amazonian focus was associated with 3 zymodemes of L. braziliensis. The papular variant of CL, Uta, found in the Andean highlands was related predominantly with a single zymodeme of L. mexicana. CONCLUSION: Our data show a high degree of phenotypic variation within species, and some evidence for associations between specific variants of ATL (i.e. Uta and LRC) and specific Leishmania zymodemes. This study further defines the geographic distribution of Leishmania species and clinical variants of ATL in Ecuador.

BACKGROUND: Salivary proteins from sandflies are potential targets for exploitation as vaccines to control Leishmania infection; in this work we tested the hypothesis that salivary proteins from geographically distant Phlebotomus duboscqi sandfly populations are highly divergent due to the pressure exerted by the host immune response. Salivary gland cDNA libraries were prepared from wild-caught P. duboscqi from Mali and recently colonised flies of the same species from Kenya. RESULTS: Transcriptome and proteome analysis resulted in the identification of the most abundant salivary gland-secreted proteins. Orthologues of these salivary proteins were identified by phylogenetic tree analysis. Moreover, comparative analysis between the orthologues of these two different populations resulted in a high level of protein identity, including the predicted MHC class II T-cell epitopes from all these salivary proteins. CONCLUSION: These data refute the hypothesis that salivary proteins from geographically distinct populations of the same Phlebotomus sandfly species are highly divergent. They also suggest the potential for using the same species-specific components in a potential vector saliva-based vaccine.

Seventeen Leishmania stocks isolated from cutaneous lesions of Pakistani patients were studied by multilocus enzyme electrophoresis and by polymerase chain reaction amplification and sequencing of the cytochrome b (Cyt b) gene. Eleven stocks that expressed nine zymodemes were assigned to L. (Leishmania) major. All of them were isolated from patients in the lowlands of Larkana district and Sibi city in Sindh and Balochistan provinces, respectively. The remaining six, distributed in two zymodemes (five and one), isolated from the highland of Quetta city, Balochistan, were identified as L. (L.) tropica. The same result at species level was obtained by the Cyt b sequencing for all the stocks examined. No clear-cut association between the clinical features (wet or dry type lesions) and the Leishmania species involved was found. Leishmania (L.) major was highly polymorphic compared with L. (L.) tropica. This difference may be explained by the fact that humans may act as a sole reservoir of L. (L.) tropica in anthroponotic cycles; however, many wild mammals can be reservoirs of L. (L.) major in zoonotic cycles.

In recent times, there has been an increase in the number of reports for new and rare variants of cutaneous leishmaniasis (CL). Here, we describe three unusual clinical forms of CL identified in Ecuadorian children. A total of 131 patients with CL were diagnosed over a 2-year period of active search. In 3 (2.29%), the lesions were very unusual; these included erysipeloid, recidiva cutis (LRC), and disseminated leishmaniasis (DL). The erysipeloid case is characterized by erythematous and indurated plaque seen on the face of a 5-year-old boy; the LRC one is differentiated by slowly progressing red-brown papules around large scars of healed sores in a 6-year-old girl, and the DL case is characterized by dozens of cutaneous ulcers distributed in the whole body of a 1-year-old girl. Leishmania parasites were isolated by lesion aspirate and analyzed by the technique multilocus enzyme electrophoresis (MLEE). All three isolates were identified as Leishmania (Viannia) panamensis. These distinct clinical variants rarely have been reported previously in the American cutaneous leishmaniasis, and for the first time L. (V.) panamensis was identified as the etiologic agent. Our cases extend the spectrum of clinical presentations in New World leishmaniasis.

Leishmaniasis, a zoonotic protozoan disease, starts with the inoculation of the Leishmania promastigotes into the skin at the time of blood ingestion by a female sandfly. The infection of leishmaniasis is established when the Leishmania organisms start their own intracellular multiplication after having been phagocytized by the host's macrophages. In the earliest stage of the infection, therefore, the attachment of the promastigates to the macrophages is essential. We incubated a mixed culture of macrophages (JM774-1A) and Leishmania (Leishmania) major for 6 hours in vitro and observed the process of the attachment between the parasite and host cell by scanning electron microscope. We found for the first time that the attachment between the two occurred at the site of the parasite body, in addition to the previously reported sites of the flagellar tip, flagellar base, and aflagellar tip (posterior pole).

Interleukin 4 (IL-4) is an important regulatory cytokine produced by activated T lymphocytes and mast cells, and regulates the growth and differentiation of cells such as B and T lymphocytes. In the present study, recombinant thioredoxin (Trx)-porcine IL-4 (pIL-4) fusion protein was prepared by Escherichia coli (E. coli), and by using this protein as an immunogen, monoclonal antibodies (mAbs) against pIL-4 were produced to establish a basis for a research on immune responses in pigs. Six stable hybridoma cell lines were successfully established and specific binding of each mAb to recombinant pIL-4 produced by E. coli and insect cells infected with recombinant baculovirus was shown by enzyme-linked immunosorbent assay (ELISA) and/or immunoblot analysis. Isotype analyses of these mAbs revealed that the subclass of 5 out of 6 mAbs was IgG1 and the rest was IgG2b. Further, assessment of their epitopes by competition binding assay indicated that the mAbs obtained in this study bound to 4 different epitopes. The recombinant proteins and mAbs produced in this study will be useful tools for the assessment of porcine immune system.

The surveillance of prevalent Leishmania and sand fly species in endemic areas is important for prediction of the risk and expansion of leishmaniasis. In this study, we developed a polymerase chain reaction (PCR)-based method for detection of Leishmania minicircle DNA within individual sand flies. Using this method, we detected minicircle DNA in 6 (3.3%) of 183 sand flies, while 5 (3.5%) of 143 were positive for Leishmania promastigotes in the same areas by microscopic examination. The species were identified as Leishmania (Leishmania) mexicana by nucleotide sequencing of the cytochrome b gene. Additionally, all the Leishmania-positive sand flies were identified as Lutzomyia ayacuchensis by the restriction enzyme digestion of the PCR-amplified 18S ribosomal RNA gene fragments. Since this combined method is relatively easy and can process a large number of samples, it will be a powerful tool for the rapid identification of prevalent sand fly and Leishmania species as well as monitoring the infection rate in sand fly populations in endemic areas.

Porcine interleukin-6 (PIL-6) protein without signal peptide was expressed as a glutathione S-transferase (GST) fusion protein in Escherichia coli. The fusion protein was expressed in an insoluble fraction, however, it was solubilized by refolding procedure using urea. From the solubilized protein, the recombinant PIL-6 (rPIL-6) was purified by a batch method using glutathione sepharose 4B and PreScission protease cleavage. By the B3B1 hybridoma cell proliferation assay, biological activity of the purified rPIL-6 was confirmed. Three monoclonal antibodies (MAbs) named 2B-1, 5A-8 and 4C-3 were generated by using the rPIL-6 as an immunogen. Immunoglobulin isotypes of the MAbs were IgG2a (4C-3) and IgG2b (2B-1 and 5A-8). For the epitope analysis, additive enzyme-linked immunosorbent assay and immunoblot analysis using deletion mutants of PIL-6 were performed. These experiments revealed that the two MAbs (2B-1 and 5A-8) recognize an overlapped epitope and the other (4C-3) recognizes a distinct epitope, and all epitopes reside in the region of aa26-64 of PIL-6.

Adult T-cell leukemia (ATL) occurs in a small population of human T-cell leukemia virus type 1 (HTLV-1)-infected individuals. Although the critical risk factor for ATL development is not clear, it has been noted that ATL is incidentally associated with mother-to-child infection, elevated proviral loads, and weakness in HTLV-1-specific T-cell immune responses. In the present study, using a rat system, we investigated the relationships among the following conditions: primary HTLV-1 infection, a persistent HTLV-1 load, and host HTLV-1-specific immunity. We found that the persistent HTLV-1 load in orally infected rats was significantly greater than that in intraperitoneally infected rats. Even after inoculation with only 50 infected cells, a persistent viral load built up to considerable levels in some orally infected rats but not in intraperitoneally infected rats. In contrast, HTLV-1-specific cellular immune responses were markedly impaired in orally infected rats. As a result, a persistent viral load was inversely correlated with levels of virus-specific T-cell responses in these rats. Otherwise very weak HTLV-1-specific cellular immune responses in orally infected rats were markedly augmented after subcutaneous reimmunization with infected syngeneic rat cells. These findings suggest that HTLV-1-specific immune unresponsiveness associated with oral HTLV-1 infection may be a potential risk factor for development of ATL, allowing expansion of the infected cell reservoir in vivo, but could be overcome with immunological strategies.

Our past studies showed that Peyer's patches were required for the induction of oral tolerance to the protein antigen ovalbumin (OVA), but not to the hapten 2,4,6-trinitrobenzene sulfonic acid (TNBS). In the present study, the effects of immunosenescence on oral tolerance induction were assessed with these two toleragens. Significant reductions in OVA-specific serum IgG antibody and CD4(+) T cell responses to subsequent challenge were observed in OVA-fed, young adult mice. Importantly, these reduced anti-OVA antibody responses were associated with delayed-type hypersensitivity, and antigen-induced CD4(+) T(h)1- and T(h)2-type cytokine responses. On the other hand, aged mice fed OVA failed to develop oral tolerance. Thus, CD4(+) T cells from Peyer's patches produced selected T(h)2- but no T(h)1-type cytokines. The TNP-specific serum IgG antibody and T cell responses were significantly diminished by prior TNBS feeding in young adult, 6- to 8-month-old and 12- to 14-month-old, but not in senescent, 2-year-old mice. Finally, we have directly assessed dendritic cell subsets and T cell responses in Peyer's patches, and their function in tolerance induction was impaired at an earlier stage of life. These results suggest that lack of oral tolerance to the protein OVA during aging is the result of dysfunctional Peyer's patches.

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL) in infected individuals after a long incubation period. Despite the apparent transforming ability of HTLV-1 under experimental conditions, most HTLV-1 carriers are asymptomatic. These facts suggest that HTLV-1 is controlled by host immunity in most carriers. To understand the interplay between host immunity and HTLV-1-infected cells, in this study, we isolated several HTLV-1 Tax-specific cytotoxic T-lymphocyte (CTL) lines from rats inoculated with Tax-coding DNA and investigated the long-term effects of the CTL on syngeneic HTLV-1-infected T cells. Our results demonstrated that long-term mixed culture of these CTL and the virus-infected T cells led to the emergence of CTL-resistant HTLV-1-infected cells. Although the Tax expression level in these resistant cells was equivalent to that in the parental cells, expression of surface major histocompatibility complex class I (MHC-I) was significantly downregulated in the resistant cells. Downregulation of MHC-I was more apparent in RT1.A(l), which presents a Tax epitope recognized by the CTL established in this study. Moreover, peptide pulsing resulted in killing of the resistant cells by CTL, indicating that resistance was caused by a decreased epitope density on the infected cell surface. This may be one of the mechanisms for persistence of HTLV-1-infected cells that evade CTL lysis and potentially develop ATL.

Background: Human T-cell leukemia virus type I (HTLV-1) is etiologically linked to adult T-cell leukemia (ATL). The disease has a high mortality rate and is resistant to chemotherapy; therefore, immunologic approaches to treatment could be of interest. We have previously shown that athymic rats inoculated with a syngeneic (i.e., with the same genetic background) HTLV-I-infected T-cell line (FPM1-V1AX) develop ATL-like disease and that the transfer of T cells from normal syngeneic rats immunized with FPM1-V1AX cells prevents disease development. In this study, we further characterized the host antitumor immunity to explore the possibility of peptide-based vaccination against the ATL-like disease. Methods: Immune T cells from rats immunized with FPM1-V1AX cells were analyzed for their phenotypes and cytotoxic properties. The epitope recognized by the T cells was analyzed by fine mapping. To evaluate the antitumor effects of a peptide-based vaccine, normal rats were immunized with synthetic oligopeptides corresponding to the epitope, the T cells were transferred to athymic rats inoculated with HTLV-1 infected cells, and tumor size was monitored. Results: Both CD4(+) and CD8(+) T-cell populations from rats immunized with FPM1-V1AX cells inhibited the growth of FPM1-V1AX cell-induced lymphomas in vivo. Long-term culture of splenic T cells from the immunized rats repeatedly resulted in establishment of CD8(+) HTLV-I-specific cytotoxic T lymphocyte (CTL) lines restricted to the rat major histo compatibility complex class I molecule, RT1.A(1). The cytotoxicity of these lines was directed against the HTLV-1 regulatory protein Tax and, specifically, against the epitope, amino acids 180-188 (GAFLT-NVPY). Adoptive transfer of the Tax 180-188-specific CTL line or freshly prepared T cells from rats vaccinated with the Tax 180-188 oligopeptide prevented the development of FPM1-V1AX-cell induced lymphomas in athymic rats in comparison with control groups (two rats in each group). Conclusions: This study indicated a potential therapeutic effect of peptide-based vaccination against HTLV-1-induced lymphoproliferative disease.

Induction of mucosal immunity by oral immunization with protein antigen alone is difficult: potent mucosal adjuvants vectors, or other special delivery systems are required. Cholera toxin (CT) has been shown to be an effective adjuvant for the development of mucosal vaccines and, when given with vaccine, induces both mucosal and systemic immune responses via a Th2 cell-dependent pathway. However, and in addition to potential type-I hypersensitivity, a major concern for use of mucosal adjuvants such as CT is that this molecule is not suitable for use in humans because of its inherent toxicity. When we examined the potential toxicity of CT for the central nervous system, both CT and CT-B accumulated in the olfactory nerves/epithelium and olfactory bulbs of mice when given by the nasal route. The development of effective mucosal vaccines for the elderly is also an important issue; however, only limited information is available. When mucosal adjuvanticity of CT was evaluated in aged mice, an early immune dysregulation was evident in the mucosal immune system. The present review discusses these potential problems for effective mucosal vaccine development. Tolerance represents the most common and important response of the host to environmental antigens, including food and commensal bacterial components, for the maintenance of an appropriate immunological homeostasis. We have examined whether Peyer patches could play a more important role for the maintenance of oral tolerance. Using Peyer patch-null mice, we found that mice lacking this gut-associated lymphoid tissue retained their capability to produce secretory IgA antibodies but did not develop normal oral tolerance to protein antigens.

Oral delivery of a large dose or prolonged feeding of protein Ags induce systemic unresponsiveness most often characterized as reduced Ige and IgE Ab- and Ag specific CD4+ T cell responses. It remains controversial whether oral tolerance extends to diminished mucosal IgA responses in the gastrointestinal tract. To address this issue, mice were given a high oral dose of OVA or PBS and then orally immunized with OVA and cholera toxin as mucosal adjuvant, and both systemic and mucosal immune responses were assessed. OVA-specific serum IgG and IgA and mucosa) IgA Ab levels were markedly reduced in mice given OVA orally compared with mice fed PBS, Furthermore, when OVA-specific Ah-forming cells (AFCs) in both systemic and mucosa-associated tissues mere examined, IgG AFCs in the spleen and IgA AFCs in the gastrointestinal tract lamina propria of mice given OVA orally were dramatically decreased. Furthermore, marked reductions in OVA-specific CD4+ T cell proliferative and cytokine responses in spleen and Peyer's patches were seen in mice given oral OVA but were unaffected in PBS-fed mice. We conclude that high oral doses of protein induce both mucosal and systemic unresponsiveness and that use of mucosal adjuvants that induce both parenteral and mucosal immunity may be a better way to assess oral tolerance,

Despite recent advances in the cellular and molecular analysis of induction and regulation of mucosal immune responses, little is yet known about differences which occur in aging. To address this important issue, we have compared the mucosal and systemic immune responses of aged (12- to 14-mo- or 2-year-old) and young adult (6- to 8-wk-old) C57BL/6 mice. Both aged and young mice were immunized weekly with three oral doses of 1 mg of OVA and 10 mug of cholera toxin (CT) as mucosal adjuvant. Both groups of mice over 1 or 2 years of age showed reduced levels of Ag-specific mucosal or systemic immune responses at day 21, An Ag-specific B cell enzyme-linked immunospot assay confirmed these results at the cellular level, When the Ag-induced cytokine responses were examined at both protein and mRNA levels, CD4(+) T cells from spleen and Peyer's patches of young adult mice revealed elevated levels of IL-4 production; however, these cytokine responses were significantly diminished in aged mice. In contrast to mucosal immunization, mice s.c. immunized with OVA plus CT resulted in impaired OVA-specific but intact CT B subunit-specific immune responses in 12- to 14-mo-old mice although the responses to both Ags were depressed in 2-year-old mice, These results provide the first evidence that the development of age-associated alterations possibly occurs earlier in the mucosal immune system than in the systemic immune compartment.

The level of host immune responses against human T cell leukemia virus type 1 (HTLV-1) varies among HTLV-1-infected individuals, In the present study, we investigate the role of host immunity on HTLV-1 leukemogenesis in vivo by using animal models. At first, we examined the effect of the routes of HTLV-1 transmission on the host anti-HTLV-1 immune responses. When immune competent adult rats were inoculated with HTLV-1-infected cells, the orally infected rats were persistently infected with HTLV-1 without humoral and cellular immune responses against HTLV-1, whereas all intravenously or intraperitoneally inoculated rats showed significant levels of immune responses. Next, we examined in vivo tumorigenicity of HTLV-1-immortalized cells in the absence of T cell immunity, by using athymic F344/N Jcl-rnu/rnu (nu/nu) rats. When inoculated into nu/nu rats, not all but some HTLV-1-immortalized rat cell lines including syngeneic FPM1-V1AX could grow and form T cell lymphoma in vivo, This syngeneic lymphoma formation was inhibited by adoptively transferred immune T cells. Furthermore, immunocompetent rats allowed in vivo growth of HTLV-1-infected lymphoma, when treated with antibodies that block costimulatory signals for T cell activation. These observations Indicated that (1) host anti-HTLV-1 immunity can be affected by the conditions of the primary infection, (2) under the low pressure of anti-HTLV-1 immunity, some HTLV-1-infected cell clones grow in vivo, and (3) T cell immunity is required for in vivo surveillance against these HTLV-1-infefted cell clones.

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL) in infected individuals after a long incubation period. To dissect the mechanisms of the development of the disease, we have previously established a rat model of ATL-like disease which allows examination of the growth and spread of HTLV-1 infected tumor cells, as well assessment of the effects of immune T cells on the development of the disease. In the present study, we induced HTLV-1 Tax-specific cytotoxic T lymphocyte (CTL) immunity by vaccination with Tax-coding DNA and examined the effects of the DNA vaccine in our rat ATL-like disease model. Our results demonstrated that DNA vaccine with Tax effectively induced Tax-specific CTL activity in F344/N Jcl-rnu/+ (nu/+) rats and that these CTLs were able to lyse HTLV-1 infected syngeneic T cells in vitro. Adoptive transfer of these immune T cells effectively inhibited the in vivo growth of HTLV-1-transformed tumor in F344/N Jcl-rnu/rnu (nu/nu) rats inoculated with a rat HTLV-1 infected T cell line. Vaccination with mutant Tax DNA lacking transforming ability also induced efficient anti-tumor immunity in this model. Our results indicated a promising effect for DNA vaccine with HTLV-1 Tax against HTLV-1 tumor development in vivo.

Objective-To evaluate the mechanism of multidrug resistance in feline lymphoma cell lines. Sample Population-A feline lymphoma cell line (FT-1) and its adriamycin (ADM)-resistant subline (FT-1/ADM). Procedures-The FT-1 cell line was cultivated in the presence of a gradually increasing concentration of ADM to generate its ADM-resistant subline (FT-1/ADM). Susceptibility of cells from the parental FT-1 cell line and the FT-1/ADM subline to antineoplastic drugs was determined. From the complementary DNA (cDNA) template of FT-1/ADM cells, feline MDR1 cDNA was amplified by use of polymerase chain reaction (PCR) and sequenced. Reverse transcription (RT)-PCR and Western blot analyses were performed to assess expression of the MDR1 gene and P-glycoprotein (P-gp) in FT-1/ADM cells, compared with that in FT-I cells. Results-A drug sensitivity assay revealed that FT-1/ADM cells were much more resistant to ADM and vincristine than the parental FT-1 cells. The feline MDR1 cDNA amplified by use of PCR was 3,489 irs long, corresponding to approximately 90% of the whole open reading frame of human MDR1 cDNA; its amino acid sequence was 91.5, 87.0, and 79.4% identical to that of human MDR1, mouse mdr1a, and mdr1b cDNA, respectively. By RT-PCR analysis, expression of MDR1 messenger RNA was clearly detected in FT-1/ADM cells but not in the parental FT-1 cells. Western blot analysis also revealed the expression of P-gp encoded by the MDR1 gene in FT-1/ADM cells but not in FT-1 cells. Conclusions-The basic structure of the feline MDR1 gene was essentially the same as that of multidrug-resistance genes of other species. Expression of P-gp appeared to be one of the mechanisms responsible for the development of multidrug resistance in feline lymphoma cell lines in vitro.

Host immunity influences clinical manifestations of human T-cell leukemia virus type 1 (HTLV-1) infection. In this study, we demonstrated that HTLV-1-transformed tumors could develop in immunocompetent rats by blocking a costimulatory signal for T-cell immune responses, Four-week-old WKA/HKm rats were treated with monoclonal antibodies (MAbs) to CD80 and CD86 and subcutaneously inoculated with syngeneic HTLV-1-infected TARS-1 cells, During MAb treatment for 14 days, TARS-1 inoculation resulted in the development of solid tumors at the site of inoculation, which metastasized to the lungs. In contrast, rats not treated with MAbs promptly rejected tumor cells. Splenic T cells from MAb-treated rats indicated impairment of proliferative and cytotoxic T-lymphocyte responses against TARS-1 in vitro compared to untreated rats. However, tumors grown in MAb-treated rats regressed following withdrawal of MAb therapy. Recovery of TARS-1-specific T-cell immune responses was associated with tumor repression in these rats. Our results suggest that HTLV-1-specific cell-mediated immunity plays a critical role in immunosurveillance against HTLV-1-transformed tumor development in vivo.

Human T-cell leukemia virus type 1 (HTLV-1) causes T-cell malignancies in a small percentage of the population infected with the virus after a long carrier state. In the present study, we established a seronegative HTLV-1 carrier state in rats inoculated with a newly established HTLV-1-infected rat T cell line, FPM1. FPM1 originated from rat thymocytes cocultured with a human HTLV-1 producer, MT-2 cells, and expressed rat CD4, CD5, CD25, and HTLV-1 Tax. However, FPM1 scarcely expressed other major HTLV-1 structural proteins and failed to induce typical antibody responses against HTLV-1 in inoculated rats. In contrast, control rats inoculated with MT-2 cells generated significant levels of anti-HTLV-1 antibodies. HTLV-1 proviruses were detected in peripheral blood cells of syngeneic rats inoculated with FPM1 for more than 1 year. Analysis of the flanking region of HTLV-1 provirus integrated inter host cells suggested that FPM1 cells remained in these animals over a relatively long period of time. However, a similar seronegative HTLV-1 carrier state was induced in the rats inoculated with mitomycin C-treated FPM1 cells and also in FPM1-inoculated allogeneic rats, suggesting that FPM1 could also transmit HTLV-1 into host cells in vivo, Our findings indicated that (i) HTLV-1-immortalized T cells which preferentially express HTLV-1 Tax persisted in vivo hut failed to induce any diseases in immunocompetent syngeneic rats and that (ii) suboptimal levels of HTLV-1 for antibody responses allowed the establishment of persistent HTLV-1 infection.

Human T-cell leukemia virus type 1 (HTLV-1) has been shown to be the etiologic agent of adult T-cell leukemia (ATL), but the in vivo mechanism by which the virus causes the malignant transformation is largely unknown. In order to investigate the mechanisms of HTLV-1 leukemogenesis, we developed a rat model system in which ATL-like disease was reproducibly observed, following inoculation of various rat HTLV-1-immortalized cell lines. When previously established cell lines, F344-S1 and TARS-1, but not TART-1 or W7TM-1, were inoculated, systemic multiple tumor development was observed in adult nude (nu/nu) rats. FPM1 cells, newly established from a heterozygous (nu/+) rat syngeneic to nu/nu rats, caused transient tumors only at the injection site in adult nu/nu rats, but could progressively grow in newborn nu/nu rats and metastasize in lymph nodes. The derivative cell line (FPM1-V1AX) serially passed through newborn nu/nu rats acquired the potency to grow in adult nu/nu rats. These results indicated that only some with additional changes but not all of the in vitro HTLV-1-immortalized cell lines possessed in vivo tumorigenicity. Using the syngeneic system, we further showed the inhibition of tumor development by transferring splenic T cells from immunized rats, suggesting the involvement of T cells in the regression of tumors. This novel and reproducible nude rat model of human ATL would be useful for investigation of leukemogenesis and antitumor immune responses in HTLV-1 infection.

CD8(+) T lymphocytes of asymptomatic human immunodeficiency virus type 1 (HIV-1) carriers (ACs) are capable of suppressing HIV-1 replication in CD4(+) peripheral blood mononuclear cells (PBMC) by a variety of known and unknown mechanisms. In the present study, cell contact-dependent, major histocompatibility complex type I (MHC I)-unrestricted, CD8(+) cell-mediated suppression of HIV-1 LAI replication was detected. CD8+ PBMC of ACs suppressed HIV-1 replication more efficiently in MHC I-matched CD4(+) PBMC than in mismatched cells. However, even when MHC I was totally mismatched, CD8(+) cells still suppressed replication to a considerable extent in CD4(+) PBMC, This MHC I-unrestricted, CD8(+) cell-mediated HIV-1 suppression required cell contact and was not effective against cells of the established T cell line ILT-KK, In contrast, MHC I-restricted HIV-1 suppression by CD8(+) T cells was detected when ILT-KK cells were used as a target. By using these systems, we examined MHC I-restricted and -unrestricted suppressive activities of CD8(+) cells in various donors in more detail. Although both types of CD8(+) cell-mediated HIV-1 suppression diminished at the advanced stage of the infection, MHC I-unrestricted suppression diminished earlier than MHC I-restricted suppression, in parallel with the decline in CD4(+) T cells. These results suggest that suppression by the MHC I-restricted mechanism alone may fail to protect against CD4(+) T-cell loss at the late stage of infection.

Feline thrombopoietin (TPO) was molecularly cloned to establish a basis for cytokine therapy of thrombocytopenia in cats. cDNA clones covering the whole coding sequence of feline TPO were isolated from feline liver. The feline TPO cDNA obtained in this study contained an open reading frame encoding 349 amino acid residues. The predicted amino acid sequence of feline TPO shared 78.7, 69.9, 72.9 and 83.0% similarity with sequences of human, murine, rat and canine TPO, respectively. Four cysteine residues and two of four N-glycosylation sites that are conserved among species were also found at the corresponding positions in feline TPO. The feline TPO cDNA fragment encoding the whole amino acid coding region was recloned into an expression vector, and the resulting vector was transfected into 293T cells using the calcium phosphate method. The supernatant of the transfected 293T cells stimulated the proliferation of a human megakaryoblastic leukemia cell line (UT-7/TPO) cells in a dose dependent manner, indicating that the feline TPO cDNA obtained in this study encodes biologically active feline TPO. (C) 1998 Elsevier Science B.V. All rights reserved.

The major route of human T-cell leukemia virus type 1 (HTLV-1) infection is mother-to child transmission caused by breast-feeding. We investigated the host immune responses to orally established persistent HTLV-1 infection in adult rats. HTLV-l-producing MT-2 cells,were inoculated into immunocompetent adult rats either orally, intravenously, or intraperitoneally. HTLV-1 proviruses were detected in the peripheral blood and several organs for at least 12 weeks. Transmission of HTLV-1 to these animals was confirmed by analysis of HTLV-1 flanking regions. Despite persistent HTLV-1 presence, none of the orally inoculated rats produced detectable levels of anti-HTLV-l antibodies, whereas all intravenously or intraperitoneally inoculated rats showed significant anti-HTLV-l antibody responses. T-cell proliferative responses against HTLV-1 were also absent in orally inoculated rats. Our findings suggest that gastrointestinal exposure of adult rats to HTLVI-infected cells induces persistent HTLV-1 infection in the absence of both humoral and cellular immune responses against HTLV-1. This immune unresponsiveness at primary infection may subsequently affect the host defense ability against HTLV-1.

Stromal cell-derived factor 1 (SDF-1) inhibits T-cell tropic (T-tropic) HIV-1 infection in vitro. In this study, we examined the regulatory role of SDF-1 on HIV-1 replication in peripheral blood mononuclear cells (PBMC) of HIV-infected individuals. We found that the amount of SDF-1 mRNA in freshly isolated PBMC of HIV-1 carriers was higher than in healthy donors. Moreover, PBMC from some asymptomatic carriers (ACs) exhibited high levels of SDF-1 mRNA expression. The level of SDF-1 expression in PBMC did not correlate with the magnitude of CD8(+) T cell-mediated suppression of HIV-1 among ACs. SDF-1 inhibited HIV-1 replication at the viral entry step, whereas a single-cycle HIV-I infection system showed that the major part of the CD8(+) T-cell-mediated suppression occurs after intracellular penetration of the virus. Our results suggest that SDF-1 acts as a suppressor of virus replication in a CD8(+) T-cell-independent mechanism in HIV-infected individuals. (C) 1998 Academic Press.

Equine interleukin-1 receptor antagonist (IL-1ra) was molecularly cloned to establish a basis for cytokine therapy of acute and chronic inflammatory diseases in the horse. cDNA clones encoding the whole coding sequence of equine IL-1ra were isolated from equine peripheral blood mononuclear cells (PBMC) that had been stimulated with lipopolysaccharide (LPS). The equine IL-1ra cDNA obtained in this study contained an open reading frame encoding 177 amino acid residues. The predicted amino acid sequence of equine IL-1ra shared 75.7, 75.3 and 76.3% similarity with sequences of human, murine and rabbit IL-1ras, respectively, An N-glycosylation site and five cysteine residues conserved in human, murine and rabbit IL-1ras were also found at the corresponding positions in equine IL-1ra. Recombinant glutathione S-transferase (GST)-equine IL-1ra fusion protein produced by Esherichia coli was purified. This protein was shown to inhibit the cytostatic or cytotoxic activity of IL-1 on A375S2 cells, indicating that the equine IL-1ra cDNA obtained in this study encodes biologically active equine IL-1ra. (C) 1997 Elsevier Science B.V.

Ig-alpha of the B-cell antigen receptor complex forms a heterodimeric structure with Ig-beta on the plasma membrane of B-lymphocytes and is apparently involved in signal transduction during the activation of B-cells. Bovine leukemia virus (BLV) is predominantly a B-cell tropic retrovirus, which induces persistent lymphocytosis and leukemia/lymphoma of B-cell lineage in cattle. To understand the mechanisms of proliferation and tumorigenesis of bovine B-cells that are associated with BLV infection, we investigated the B-cell antigen receptor complex, especially bovine mb-1 encoding the bovine Ig-alpha protein. We isolated a full-length bovine mb-1 cDNA clone encoding 223 amino acid residues. The deduced amino acid sequence of the bovine mb-1 showed extensive homology with those of human and murine mb-1. The cytoplasmic tail of the bovine mb-1 also contained a consensus motif (D/E-X7-D/E-X2-L/I-X7-Y-X2-L/I) that may interact with the SH2 domain of src-type kinase. Interestingly, a similar consensus sequence motif was found in the BLV gp30(enu), although the overall sequence similarity between bovine mb-1 and BLVgp30 was not significant Furthermore, elevated levels of mb-1 transcript were detected in various bovine leukemia/lymphoma cell lines. These results indicated that the proliferation of B-cells associated with BLV-infection may be related to abnormal signal transduction through the B-cell antigen receptor complex.

Levels of granulocyte-colony stimulating factor (G-CSF) in the blood of horses were measured before and after a long-distance transportation to clarify the pathogenesis of transportation-induced fever. The serum G-CSF level was measured by its ability to stimulate growth in a mouse myeloblastic cell line, NFS-60. Of 26 horses transported for a long distance, 9 had fever more than 39.0 degrees C during or after transportation. After transportation, the serum G-CSF level significantly increased in horses with transportation-induced fever but not in those without fever, and the serum G-CSF level correlated positively with the peak body temperature and with an increase in peripheral white blood cell count. These data indicate that microbial infection, which is closely related to the elevation of the serum G-CSF level, is the causative factor of transportation-induced fever.

Feline immunodeficiency virus (FIV) was first isolated from cats with immunodeficiency syndrome. Recently, neurological abnormalities and brain lesions were shown in cats infected with FIV. To investigate the FIV genome associated with central nervous system (CNS) lesions, proviral DNA sequences from the V3-V6 region of the FIV env gene were directly amplified from uncultured necropsy tissues of a 2-year-old naturally FIV-infected cat with marked neurological symptoms and. encephalitis. By in situ hybridization, FIV RNA was detected mainly in the astrocytes. Fifteen clones isolated from cerebrum, bone marrow and lymph node samples showed only a small number of mutations or deletions in this reg:ion. A representative clone, JN-BR1, was distantly related to the previous Japanese strain (TM2) belonging to the subtype B. However, it was relatively close to the Petaluma strain which is known to infect feline brain-derived culture cells and induce brain lesions in inoculated cats. By phylogenetic analysis, the JN-BR1 strain was placed in subtype A that included Petaluma strain and several other American and European strains. The JN-BR1 strain derived from brain with encephalitis in this study and the Petaluma strain may share a common genetic structure that is related to their neuropathogenicity.

A 12-year-old neutered male mixed-breed dog was referred to hospital for evaluation of chronic diarrhea. Cellulose acetate electrophoresis of its serum revealed two monoclonal peaks in the gamma-globulin fraction. On immunoelectrophoretic analysis, the two monoclonal peaks in the gamma-globulin region were strongly precipitated with anti-dog IgA serum. On sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis, the fractions corresponding to these two peaks were shown to be dimer and trimer or tetramer of immunoglobulin consisting of heavy and light chains. These results indicated that the studied dog had gammopathy with two M-components with dimer and trimer or tetramer of IgA. Accumulations of large amounts of these immunoglobulins with very high molecular weight in the serum were concluded to induce the hyperviscosity syndrome in this dog in the terminal stage.

Molecular cloning of canine interleukin-8 (IL-8) was performed to establish a basis for its investigation in the canine immune system. From a cDNA pool constructed from LPS-stimulated popliteal lymph node cells, canine IL-8 cDNA covering the whole coding region was amplified by polymerase chain reaction. The nucleotide sequence of a canine IL-8 clone, designated pcIL-8#38, was highly similar to those of human, rabbit and porcine IL-8, and comprised 353 bp with an open reading frame that encoded 101 amino acids. Analysis of the deduced amino acid sequence of insert DNA in pcIL-8#38 showed 76.5, 80.2, and 87.0% similarities with human, rabbit and porcine IL-8 proteins, respectively. Insert DNA of pcIL-8#38 was transferred to a mammalian expression vector, pcDL-SR alpha 296, and transfected into Cos7 cells. The supernatant of the transfectant had neutrophil chemotactic activity when it was examined by the neutrophil migration assay, suggesting that our cloned cDNA was biologically active. The cloned canine IL-8 cDNA will be useful for canine inflammatory disease and comparative immunology research.