Identification and characterization of novel multiple bacteriocins produced by Leuconostoc pseudomesenteroides QU 15.

AIM: To characterize novel multiple bacteriocins produced by Leuconostoc pseudomesenteroides QU 15. METHODS AND RESULTS: Leuconostoc pseudomesenteroides QU 15 isolated from Nukadoko (rice bran bed) produced novel bacteriocins. By using three purification steps, four antimicrobial peptides termed leucocin A (ΔC7), leucocin A-QU 15, leucocin Q and leucocin N were purified from the culture supernatant. The amino acid sequences of leucocin A (ΔC7) and leucocin A-QU 15 were identical to that of leucocin A-UAL 187 belonging to class IIa bacteriocins, but leucocin A (ΔC7) was deficient in seven C-terminal residues. Leucocin Q and leucocin N are novel class IId bacteriocins. Moreover, the DNA sequences encoding three bacteriocins, leucocin A-QU 15, leucocin Q and leucocin N were obtained. CONCLUSIONS: These bacteriocins including two novel bacteriocins were identified from Leuc. pseudomesenteroides QU 15. They showed similar antimicrobial spectra, but their intensities differed. The C-terminal region of leucocin A-QU 15 was important for its antimicrobial activity. Leucocins Q and N were encoded by adjacent open reading frames (ORFs) in the same operon, but leucocin A-QU 15 was not. SIGNIFICANCE AND IMPACT OF STUDY: These leucocins were produced concomitantly by the same strain. Although the two novel bacteriocins were encoded by adjacent ORFs, a characteristic of class IIb bacteriocins, they did not show synergistic activity.

Malaria parasite induces tryptophan-related immune suppression in mice.

Plasmodium spp. cause the worst parasitic diseases in humans and evade host immunity in complicated ways. Activated catabolism of tryptophan in dendritic cells is thought to suppress immunity, which is mediated by an inducible rate-limiting enzyme of tryptophan catabolism, indoleamine 2,3 dioxygenase (IDO), via both tryptophan depletion and production of toxic metabolites. In various infections, including malaria, IDO is known to be activated but its biological significance is unclear; therefore, we investigated whether malaria parasites induce IDO to suppress host immune responses. We found that enzymatic activity of IDO was elevated systematically in our mouse malaria model, and was abolished by in vivo IDO inhibition with 1-methyl tryptophan. Experimental infection with Plasmodium yoelii showed that IDO inhibition slightly suppressed parasite density in association with enhanced proliferation and IFN-gamma production by CD4+ T cells in response to malaria parasites. Our observations suggest that induction of IDO is one of the immune mechanisms of malaria parasites.

A novel DNA vaccine based on ubiquitin-proteasome pathway targeting 'self'-antigens expressed in melanoma/melanocyte.

Cancer vaccine that targets 'self'-antigens expressed at high levels in tumor cells is a potentially useful immunotherapy, but immunological tolerance often defeats this strategy. Here, we describe the use of a naked DNA vaccine encoding a self tumor antigen, tyrosinase-related protein 2, to whose N-terminus ubiquitin is fused in a 'nonremovable' fashion. Unlike conventional DNA vaccines, this vaccine broke the tolerance and induced protective immunity to melanoma in C57BL/6 mice, as evaluated by tumor growth, survival rate and lung metastasis. The protective immunity was cancelled in the proteasome activator PA28alpha/beta knockout mice. Moreover, this vaccination exhibited therapeutic effects on melanoma implanted before vaccination. Our findings provide evidence for the first time that naked DNA vaccines encoding a ubiquitin-fused self-antigen preferentially induce the main effector CD8+ T cells through efficient proteolysis mediated by the ubiquitin-proteasome pathway, and lead the way to strategies aimed at targeting tissue differentiation antigens expressed by tumors.

IL-18 gene therapy develops Th1-type immune responses in Leishmania major-infected BALB/c mice: is the effect mediated by the CpG signaling TLR9?

IL-18 regulates either Th1 or Th2 responses depending on the cytokine microenvironment. Administration of recombinant IL-18 (rIL-18) alone does not promote Th1 response, but rather induces Th2 response and exacerbates Leishmania major infection in susceptible BALB/c mice. Here, we treated BALB/c mice with an IL-18-expressing plasmid by using a gene gun weekly after L. major infection. This gene therapy resulted in improved pathogenic process and preferential induction of Th1 responses by inducing the expression of IL-12 p40, but treatment with rIL-18 did not. Notably, simultaneous administration of rIL-18 with an empty plasmid vector rendered BALB/c mice resistant to the infection, despite the fact that treatment with either rIL-18 alone or the plasmid vector alone did not influence the susceptibility. The synergistic role of the vector with rIL-18 was found to depend on CpG motifs, which enhanced expression of proinflammatory cytokines, especially IL-12, from APCs through Toll-like receptor (TLR) 9 ligation. Treatment with methylated plasmid vector in which CpG was disrupted could no longer prevent the disease development in coadministration with rIL-18. Taken together, IL-18 gene therapy was shown to develop Th1-type protective immunity in L. major-infected BALB/c mice without the requirement of exogenous IL-12, probably via CpG-TLR9 signaling pathway.

Experimental study of combined treatment with tacrolimus and donor splenocytes via the portal vein in small bowel transplantation.

We previously reported that the combined treatment of perioperative administration of donor splenocytes via the recipient's portal vein (DSPV) and a short-course Tacrolimus significantly prolonged the survival of fully allogenic grafts in rat small bowel transplantation (SBTX). In the present study we examined whether this effect depended on the quantity of the administered alloantigens in DSPV. In addition, we examined the expression of the surface antigen on T cells of the splenocytes and the induced toleragenic factor, according to the tolerant recipients which in our previous report had shown the prolongation of allogenic transplant small bowel graft survival by the combined treatment of DSPV (1 x 10(8) donor splenocytes) and a short-course Tacrolimus. Donor splenocytes were prepared from Brown-Norway (BN (RT1n)) rat spleens for Lewis (LEW (RT1l)) recipients. The recipients (n = 10), treated with a short course of Tacrolimus (0.5 mg/kg, 0 to 3 days postoperatively) only showed graft rejection with an average of 6.3 +/- 1.0 days postoperatively. However, the combined treatment, consisting of DSPV of 1 x 10(8) donor splenocytes and a short course Tacrolimus significantly prolonged graft survival to 12.7 +/- 2.1 days (n = 12, P < 0.01). DSPV of less than 1 x 10(8) donor splenocytes (5 x 10(7) cells and 2.5 x 10(7)) could not prolong the graft or animal survival under a short-course Tacrolimus treatment. In the tolerant recipients, the CD4 and CD8 percentages of splenocytes were not significantly different from those of control rats or recipients that were treated with short-course Tacrolimus alone. Nevertheless, the percentage of Tcr-alpha beta+ cells expressing IL-2 receptor (R) was significantly lower than in either control rats or the recipients with short-course Tacrolimus. In the suppression assay to one-way mixed lymphocyte response, a toleragenic factor was suggested to the present in the serum of the tolerant recipients. In the present study, it was suggested that the effects of the combined treatment of DSPV and short-course Tacrolimus for the prolongation of graft survival in the rat allogenic SBTX should depended on the quantity of the antigens administered into the portal vein. The beneficial effects of this treatment were reflected in the suppression of IL-2R on the recipient's splenocytes, and tolerogenic factor(s) might subsequently be induced in the tolerant recipient's serum.

Nippocystatin, a cysteine protease inhibitor from Nippostrongylus brasiliensis, inhibits antigen processing and modulates antigen-specific immune response.

During infection, parasites evade the host immune system by modulating or exploiting the immune system; e.g., they suppress expression of major histocompatibility complex class II molecules or secrete cytokine-like molecules. However, it is not clear whether helminths disturb the immune responses of their hosts by controlling the antigen-processing pathways of the hosts. In this study, we identified a new cysteine protease inhibitor, nippocystatin, derived from excretory-secretory (ES) products of an intestinal nematode, Nippostrongylus brasiliensis. Nippocystatin, which belongs to cystatin family 2, consists of 144 amino acids and is secreted as a 14-kDa mature form. In vivo treatment of ovalbumin (OVA)-immunized mice with recombinant nippocystatin (rNbCys) profoundly suppressed OVA-specific proliferation of splenocytes but not non-antigen-specific proliferation of splenocytes. OVA-specific cytokine production was also greatly suppressed in rNbCys-treated mice. Although the serum levels of both OVA-specific immunoglobulin G1 (IgG1) and IgG2a were not affected by rNbCys treatment, OVA-specific IgE was preferentially downregulated in rNbCys-treated mice. In vitro rNbCys inhibited processing of OVA by lysosomal cysteine proteases from the spleens of mice. Mice with anti-nippocystatin antibodies became partially resistant to infection with N. brasiliensis. Based on these findings, N. brasiliensis appears to skillfully evade host immune systems by secreting nippocystatin, which modulates antigen processing in antigen-presenting cells of hosts.

Granule-dependent killing of Toxoplasma gondii by CD8+ T cells.

Immunization of mice with live bradyzoites of a low-virulent Beverley strain of Toxoplasma gondii has been shown to increase CD8+ T-cell mediated immunity against a highly virulent RH strain. We found that preimmunization with an RH homogenate further enhanced this immunity. Using this model, we investigated the mechanism of CD8+ T-cell mediated protection against T. gondii infection. Splenic cells from mice immunized with RH homogenate and live bradyzoites stimulated apoptosis of RH-infected J774A.1 macrophages in vitro, and at the same time, the immunization significantly suppressed the proliferation of parasites within macrophages, as assessed by measuring 3H-uracil uptake by the parasites. Splenic cells from the immunized mice produced larger amounts of interferon-gamma (IFN-gamma) than did naive splenic cells; however, the production of nitric oxide (NO) by RH-infected macrophages was not enhanced. The elimination of CD8+ T cells from splenic cells significantly reduced their inhibitory action on parasite proliferation as well as their cytotoxic activity against RH-infected macrophages, but it did not affect the production of IFN-gamma. Treatment of CD8+ T-enriched splenic cells from the immunized mice with concanamycin A, but not an anti-Fas ligand monoclonal antibody, significantly reduced their anti-proliferative and killing capabilities, suggesting that the CD8+ T cells induced by immunization with RH antigen and live bradyzoites of the Beverley strain may exert protection against T. gondii infection at least in part through granule-dependent cytotoxic activities.

CD4+ T cells are required for HSP65 expression in host macrophages and for protection of mice infected with Plasmodium yoelii.

We have reported that macrophages expressing heat-shock protein 65 play an essential role in protection of mice infected with Plasmodium yoelii. In this study, we investigated the function and expression mechanism of HSP65 in macrophages of mice infected with P. yoelii. C57BL/6 (B6) mice are susceptible to infection with the lethal (L) strain but resistant to infection with the non-lethal (NL) strain of P. yoelii. The percentage of apoptotic macrophages in mice infected with the L strain was higher than that in mice infected with the NL strain. However, the percentage was low in L strain infected mice if they acquired resistance to the infection by primary infection with the NL strain. That apoptosis was reversely correlated with HSP65 expression in splenic macrophages from mice infected with P. yoelii suggests HSP65 may contribute to protective immunity by preventing apoptosis of macrophages in malarial infection. Cell depletion/transfer experiments showed that CD4+ T cells, but not CD8+ T cells, gammadelta T cells, NK cells or NK T cells, were required for HSP65 expression in macrophages as well as for protection of mice infected with P. yoelii. In conclusion, HSP65 may play a role in preventing apoptosis of macrophages in mice infected with P. yoelii. CD4+ T cells are required for HSP65 expression and for protective immunity against P. yoelii infection.

Th1-cytokine induction and anti-tumor effect of 55 kDa protein isolated from Aeginetia indica L., a parasitic plant.

We have isolated a 55 kDa protein from the seed extract of Aeginetia indica L. (AIL), a parasitic plant, by affinity chromatography on an N-hydroxysuccinimide-activated Sepharose High Performance column bound with F3, a monoclonal antibody that neutralizes the cytokine-inducing and anti-tumor effect of AIL. In the present study, we examined this protein (AILb-A) for cytokine induction and anti-tumor effects by animal study, using syngeneic Meth-A tumor-bearing BALB/c mice, in which the Th2 response is genetically dominant. AILb-A administration resulted in markedly increased levels of Th1 cytokines [interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, interleukin (IL)-2, IL-12 and IL-18] in the sera derived from Meth-A-bearing mice. The in vitro re-stimulation with AILb-A of splenocytes derived from AILb-A-primed mice also selectively induced Th1-type cytokines and antigen-specific killer cell activity. The neutralizing test using cytokine-specific antibodies revealed that AILb-A-induced IL-18 plays a most significant role for and killer cell-inducing activities. Furthermore, IL-12 and IL-18 induced by AILb-A inhibited specifically IL-10 and IL-4 production, respectively. Finally, we examined the anti-tumor effect of AILb-A in both Meth-A-bearing BALB/c mice and Meth-A-bearing nude mice with BALB/c background. AILb-A exhibited a striking anti-tumor effect in normal BALB/c mice inoculated with Meth-A cells. In athymic nude mice, the anti-tumor effect of AILb-A was relatively weak. These findings strongly suggested that AILb-A is a potent Th1 inducer and may be a useful immunotherapeutic agent for patients with malignant diseases.

Treatment with cathepsin L inhibitor potentiates Th2-type immune response in Leishmania major-infected BALB/c mice.

Prior to the activation of CD4 (+) T cells, exogenous proteins must be digested by endo/lysosomal enzymes in antigen-presenting cells (APC) to produce antigenic peptides that are able to be presented on class II molecules of the MHC. Studies described here inspect the functional significance of cathepsin L inhibition for antigen processing and T (h) 1/T (h) 2 differentiation in experimental leishmaniasis. We first demonstrated using in vitro systems that cathepsin L is one of the candidate endo/lysosomal enzymes in processing of soluble Leishmania antigen (SLA) and that its specific inhibitor, CLIK148, modulated the processing of SLA. BALB/c mice are known to be susceptible to infection with Leishmania major. Interestingly, treatment of BALB/c mice with CLIK148 exacerbated the infection by enhancing the development of SLA-specific T (h) 2-type response such as production of IL-4 and generation of T (h) 2-dependent specific IgE/IgG1 antibodies. Moreover, addition of CLIK148 in incubation of a SLA-specific CD4 (+) T cell line with APC up-regulated the production of IL-4. However, CLIK148 did not exert any direct influence on the function of T cells themselves. Taken together, these findings suggest that treatment of host mice with CLIK148 affects the processing of SLA in APC, resulting in the potentiation of T (h) 2-type immune responses and thus leading to exacerbation of the infection. Furthermore, endo/lysosomal cathepsin L was found to be functionally distinct from previously described cathepsins B and D.

Molecular characterization of a 2-Cys peroxiredoxin from the human malaria parasite Plasmodium falciparum.

We have identified the 2-Cys peroxiredoxin (PfPrx-1) from the human malaria parasite Plasmodium falciparum. The PfPrx-1 showed the highest identity at amino acid level to the type II Prx among the currently known six subfamilies of mammalian Prx. The sequence identity between the PfPrx-1 and the previously reported 1-Cys Prx of P. falciparum (PfPrx-2), which corresponded to mammalian type VI Prx, was 25%. This suggests that the parasite possesses two Prx subfamilies. The PfPrx-1 showed significant sequence similarities with those of 2-Cys peroxiredoxins of plants in the BLASTX search. This may reflect the consequences of a genetic transfer from an algal endosymbiont to the parasite nucleus during evolution. The recombinant PfPrx-1 protein (rPfPrx-1) was expressed as a histidine fusion protein in Escherichia coli and purified with Ni chromatography. The rPfPrx-1 existed as dimers under non-reducing conditions and dissociated into monomers in the presence of dithiothreitol. The PfPrx-1 protein also exists as a dimer in the parasites themselves. The reduction of the oxidized enzyme by the donation of electrons from E. coli thioredoxin (Trx)/Trx reductase system was demonstrated in its reaction with H(2)O(2), using the rPfPrx-1 protein. These results suggested that the PfPrx-1 can act as a terminal peroxidase of the parasite Trx system. An elevated expression of the PfPrx-1 protein seen in the trophozoite, the stage with active metabolism, suggests an association of the parasite Trx system with its intracellular redox control.

Hypoleptinemia, but not hypoinsulinemia, induces hyperphagia in streptozotocin-induced diabetic rats.

To assess the dominance between hypoinsulinemia and hypoleptinemia as factors in the development of hyperphagia in streptozotocin (STZ)-induced diabetes mellitus (STZ-DM) rodents with respect to hormone-neuropeptide interactions, changes in gene expression of agouti gene-related protein (AGRP) in the arcuate nucleus of the hypothalamus were investigated using STZ-DM rats, fasting Zucker fa/fa rats and STZ-DM agouti (STZ-DM A(y)/a) mice. AGRP mRNA and neuropeptide Y mRNA were both significantly up-regulated in STZ-DM rats, which are associated with body weight loss, hyperglycemia, hypoinsulinemia and hypoleptinemia. We proceeded to analyze whether insulin or leptin played the greater role in the regulation of AGRP using Zucker fa/fa rats. The AGRP mRNA did not differ significantly between fasted fa/fa rats, which have both leptin-insensitivity and hypoinsulinemia, and fed Zuckers, which have leptin-insensitivity and hyperinsulinemia. We further found that up-regulation of AGRP expression was normalized by infusion of leptin into the third cerebroventricle (i3vt), but not by i3vt infusion of insulin, although up-regulation of AGRP was partially corrected by systemic insulin infusion. The latter finding supports hypoleptinemia as a key-modulator of STZ-DM-induced hyperphagia because systemic insulin infusion, at least partially, restored hypoleptinemia through its acceleration of fat deposition, as demonstrated by the partial recovery of lost body weight. After STZ-DM induction, A(y)/a mice whose melanocortin-4 receptor (MC4-R) was blocked by ectopic expression of agouti protein additionally accelerated hyperphagia and up-regulated AGRP mRNA, implying that the mechanism is triggered by a leptin deficit rather than by the main action of the message through MC4-R. Hypoleptinemia, but not hypoinsulinemia per se, thus develops hyperphagia in STZ-DM rodents. These results are very much in line with evidence that hypothalamic neuropeptides are potently regulated by leptin as downstream targets of its actions.

Splenic cathepsin L is maturated from the proform by interferon-gamma after immunization with exogenous antigens.

The processing of foreign protein antigens into peptides requires the participation of various endo/lysosomal proteases in antigen-presenting cells (APCs). In this study, a proenzyme of cathepsin L, procathepsin L, was found to be present in the spleens of naive mice, as demonstrated by immunoblotting. Interestingly, the maturation of cathepsin L from procathepsin L was strongly induced when the host BALB/c mice were immunized with ovalbumin or soluble leishmanial antigen, despite the fact that mouse albumin, a kind of self-antigen, did not have such a potential. Furthermore, foreign antigens, but not self-antigens, could increase the activity of cathepsin L, probably being mediated by interferon-gamma, as demonstrated by in vivo and in vitro experiments. As cathepsin L matured, the efficiency of antigen processing was increased in APCs. These results suggest that endo/lysosomal cathepsin L plays an important role in the immune regulation via antigen processing even in peripheral lymphoid tissues as well as in the thymus.

Antibodies specific for heat shock proteins in human and murine malaria.

Heat shock proteins (HSPs) are immunodominant antigens recognized by the host immune system in various infectious diseases. We analyzed HSP-specific antibodies, including immunoglobulin G (IgG), IgM and IgA, in sera from malaria patients in Thailand by using an enzyme-linked immunosorbent assay. All of the antibodies to HSP90 were remarkably increased in the patients compared with those in controls, while only IgM to HSP70 or IgA to HSP65 was significantly elevated. Further experiments showed that anti-HSP IgG was significantly increased in C57BL/6 mice infected with a non-lethal strain of Plasmodium yoelii, with anti-HSP90 IgG being the most elevated. These results suggest that the antigenic potential of HSP90 is higher than those of HSP70 and HSP65 in malaria infection.

Role of innate immune cells in protection against Toxoplasma gondii at inflamed site.

The intraperitoneal infection with Toxoplasma gondii (T. gondii) caused accumulation of gamma delta T, NK, NK1.1+T-like (NKT) cells at inflamed sites. To clarify the roles of these cells in protection against T. gondii at the inflamed sites, BALB/c mice were depleted of gamma delta T, NK, NK and NKT cells by treatment with antibody against TCR-gamma delta, asialoGM1 or Interleukin-2 receptor beta-chain (IL-2 R beta), respectively, prior to infection. Mice treated with anti-TCR-gamma delta monoclonal antibody (mAb) became more susceptible to infection, whereas mice treated with anti-IL-2R beta mAb acquired resistance. Treatment with anti-asialoGM1 Ab showed no effect. We previously reported that heat shock protein 65 (HSP65) in macrophages induced by gamma delta T cells plays an essential role in protective immunity against T. gondii infection, by preventing apoptotic death of infected macrophages. In the present study, we showed that treatment with anti-IL-2R beta mAb, but not with anti-asialoGM1 Ab, enhanced the HSP65 induction in macrophages, and inhibited Interleukin-4 (IL-4) expression in nonadherent peritoneal exudate cells. Furthermore, neutralization of endogenous IL-4 by anti-IL-4 mAb enhanced the HSP65 induction in macrophages. These findings suggest that NKT cells, but not NK cells, negatively regulate the protective immunity against T. gondii infection possibly by producing IL-4 and suppressing HSP65 induction.

Molecular cloning of a cystatin from parasitic intestinal nematode, Nippostrongylus brasiliensis.

A novel member of the cystatin family, nippocystatin (NbCys), was identified from excretory-secretory (ES)-products of a nematode Nippostrongylus brasiliensis, and the cDNA was cloned and sequenced. The mRNA of NbCys was confirmed to be expressed in both larvae and adults of the parasite. NbCys was translated as a proform with a single domain for secretion and was detected as a 14-kDa mature form in ES-products of the adult worm. Recombinant protein of NbCys profoundly inhibited the activity of cysteine proteases such as cathepsin L and B, but not that of cathepsin D, an aspartic protease. Furthermore, the ES-products had also been confirmed to inhibit cysteine proteases. Taken together, NbCys may play a role in evasion of N. brasiliensis from host defense systems, since cysteine proteases are known to participate in immune systems of infected hosts.

Different cytokines are required for induction and maintenance of the Th2 type response in DBA/2 mice resistant to infection with Leishmania major.

Experimental cutaneous leishmaniasis is a useful model in studying the mechanism regulating immune responses between T helper type 1 (Th1) and Th2. Mice susceptible to Leishmania major infection such as BALB/c (H-2(d)) are associated with the induction of the disease-promoting Th2 response, while the resistant mice such as DBA/2 (H-2(d)) develop the protective Th1 response. To understand the induction mechanism of Th1 and Th2 responses, it is necessary to establish an immunization scheme by which the induction of each Th response can be easily and experimentally controlled. Adjuvants are known to enhance the immune responses through the combined effect of several factors: prolonged release of antigen, migration of cells, mitogenic effect and so forth. When the genetically resistant DBA/2 mice were immunized twice with soluble leishmanial antigen (SLA), emulsified in incomplete Freund's adjuvant (IFA) before L. major inoculation, these mice mounted a Th2 cell response and suffered from progressive infection. While IL-4 and IL-13 were upregulated early after the infection in both healer and non-healer groups of mice, IL-5 and IL-10 were upregulated only in non-healer mice. From these results, IL-5 and IL-10 appear to have an important role, at least in the early phases of the infection, rather than IL-4 and IL-13 in establishing the disease-promoting Th2 response in leishmaniasis. Further, IL-9 was found to be expressed in both BALB/c and DBA/2 mice immunized with IFA/SLA. This cytokine may support the establishment of a Th2 response in these mice. Therefore it is suggested that Th2 cytokines play different roles between priming and maintaining the Th2 immune response after the infection.

Pepstatin A-sensitive aspartic proteases in lysosome are involved in degradation of the invariant chain and antigen-processing in antigen presenting cells of mice infected with Leishmania major.

We previously reported that CA074, a specific inhibitor of cathepsin B, significantly deviated immune responses from the disease-promoting Th2 type to the protective Th1 type in BALB/c mice infected with Leishmania major. Herein, we found that pepstatin A-sensitive aspartic proteases (PSAP) in lysosomes seem to play a different role from that of cathepsin B in antigen-processing and Ii-degradation. That is, cathepsin B appears to digest 16-, 28-, and 31-kDa peptides of soluble leishmania antigen (SLA), whereas PSAP seems to process mainly 28-kDa peptides. Furthermore, the latter protease contributed to the degradation of Ii but cathepsin B did not. Following treatment with pepstatin A, both Th1 and Th2 responses were profoundly suppressed in resistant DBA/2 mice (H-2(d)) and in susceptible BALB/c mice (H-2(d)), and both strains of mice became markedly susceptible compared with the untreated groups, probably owing to failure in degradation of Ii and partly to failure in digestion of 28-kDa peptide.

Purification and characterization of cytokine-inducing protein of seed extract from Aeginetia indica L., a parasitic plant.

We have isolated 55 kDa protein from the seed extract of Aeginetia indica L. (AIL), a parasitic plant, by an affinity chromatography on N-hydroxysuccinimide (NHS)-activated Sepharose High Performance column bound F3 monoclonal antibody which neutralizes cytokine-inducing and antitumor effect of AIL. In in vitro model using human peripheral blood mononuclear cells (PBMC), the 55 kDa protein (AILb-A) induced multiple cytokines, such as IFN-gamma, tumor necrosis factor (TNF)-alpha, granulocyte macrophage-colony stimulating factor (GM-CSF), IL-2, IL-6, IL-10, IL-12 and IL-18, and also accelerated killer cell activities of PBMC. When compared with a commonly used immunotherapeutic agent OK-432, AILb-A induced Th1 cytokines are greater than OK-432. Of the Th2 cytokines, the amounts of IL-6 and IL-10 induced by AILb-A were lower than those by OK-432. No significant induction of IL-4 and IL-13 was observed in AILb-A-stimulated PBMC. TNF family including TNF-alpha, TNF-beta, Fas ligand (FasL) and TNF-related apoptosis-inducing ligand (TRAIL) were suggested to be important for AILb-A-induced killing activity of PBMC by reverse transcription-polymerase chain reaction (RT-PCR) analysis. Furthermore, the neutralizing test using cytokine-specific antibodies demonstrated that IL-18 plays a most significant role for IFN-gamma- and killer cell-inducing ability of AILb-A among the cytokines tested. These findings clearly indicated that AILb-A, a 55 kDa protein of AIL, is a potent Th1 cytokine inducer and may be a useful immunotherapeutic agent for the patients with malignancies.

CD4(+) v(alpha)14 NKT cells play a crucial role in an early stage of protective immunity against infection with Leishmania major.

The roles of gamma delta T, NK and NKT cells in an early stage of protective immunity against infection with Leishmania major were investigated. Further, the contribution of these innate cells to the expression of 65 kDa heat shock protein (HSP65) in host macrophages was examined, since we found previously that this expression prevents apoptotic death of infected macrophages and is a crucial step in the acquisition of protective immunity against infection with various obligate intracellular protozoa including L. major. C57BL/6 and DBA/2 mice were found to be resistant against the infection on the basis of the parasite burden in their regional lymph nodes, and to strongly express HSP65 in their macrophages, whereas BALB/c mice were susceptible and barely expressed the HSP65. In those resistant mice, CD4(+) NKT cells prominently increased in their regional lymph node and were the main effector cells at least for an early stage of the protective immunity and for the HSP65 expression, whereas this subset did not increase in susceptible BALB/c mice. Further, neither gamma delta T nor NK cells in resistant mice contributed to those protective immune responses. The NKT cell subset bore CD3, CD4, TCR alpha beta, IL-2R beta and NK1.1 but scarcely asialo-GM(1). Moreover, this effector subset was confirmed to be V(alpha)14 NKT cells by using J(alpha)281(-/-) mice.