Cytokine-related and sodium channel polymorphism as candidate predisposing factors for childhood encephalopathy FIRES/AERRPS.

Febrile infection-related epilepsy syndrome (FIRES), or acute encephalitis with refractory, repetitive partial seizures (AERRPS), is an epileptic encephalopathy beginning with fever-mediated seizures. The etiology remains unclear. To elucidate the genetic background of FIRES/AERRPS (hereafter FIRES), we recruited 19 Japanese patients, genotyped polymorphisms of the IL1B, IL6, IL10, TNFA, IL1RN, SCN1A and SCN2A genes, and compared their frequency between the patients and controls. For IL1RN, the frequency of a variable number of tandem repeat (VNTR) allele, RN2, was significantly higher in the patients than in controls (p=0.0067), and A allele at rs4251981 in 5' upstream of IL1RN with borderline significance (p=0.015). Haplotype containing RN2 was associated with an increased risk of FIRES (OR 3.88, 95%CI 1.40-10.8, p=0.0057). For SCN1A, no polymorphisms showed a significant association, whereas a missense mutation, R1575C, was found in two patients. For SCN2A, the minor allele frequency of G allele at rs1864885 was higher in patients with borderline significance (p=0.011). We demonstrated the association of IL1RN haplotype containing RN2 with FIRES, and showed a possible association of IL1RN rs4251981 G>A and SCN2A rs1864885 A>G, in Japanese patients. These preliminary findings suggest the involvement of multiple genetic factors in FIRES, which needs to be confirmed by future studies in a larger number of FIRES cases.

Differential stimulation requirements for IL-12 production among clones of macrophage hybridomas.

We have previously established cloned macrophage hybridomas by somatic cell fusion of the macrophage tumor P388D1 of DBA/2 (H-2d) origin with splenic adherent cells of CKB mice (H-2k). Several cloned lines displayed the serologic and functional characteristics of macrophages. In this study, we evaluated the ability of these hybridomas to produce IL-12 after combined stimulation with IFN-gamma and lipopolysaccharide (LPS). The patterns of IL-12 production by these cloned macrophages fell into three groups. The first group produced IL-12 on stimulation with LPS in combination with IFN-gamma pretreatment, the second group produced IL-12 on stimulation with LPS regardless of the pretreatment with IFN-gamma, and the third group did not produce IL-12 at all on stimulation with IFN-gamma and LPS. None of the macrophage clones tested produced IL-12 constitutively. The results correlated well with IL-12 p40 mRNA expression in those macrophages as detected by RT-PCR. These results suggest the differential stimulation requirements for IL-12 production among macrophages at a clonal level.

A case of severe progressive early-onset epileptic encephalopathy: unique GABAergic interneuron distribution and imaging.

Early-onset epileptic encephalopathies include various diseases such as early-infantile epileptic encephalopathy with suppression burst. We experimentally investigated the unique clinicopathological features of a 28-month-old girl with early-onset epileptic encephalopathy. Her initial symptom was intractable epilepsy with a suppression-burst pattern of electroencephalography (EEG) from 7 days of age. The suppression-burst pattern was novel, appearing during sleep, but disappearing upon waking and after becoming 2 months old. The EEG showed multifocal spikes and altered with age. Her seizures demonstrated various clinical features and continued until death. She did not show any developmental features, including no social smiling or head control. Head MRI revealed progressive atrophy of the cerebral cortex and white matter after 1 month of age. (123)IMZ-SPECT demonstrated hypo-perfusion of the cerebral cortex, but normo-perfusion of the diencephalon and cerebellum. Such imaging information indicated GABA-A receptor dysfunction of the cerebral cortex. The genetic analyses of major neonatal epilepsies showed no mutation. The neuropathology revealed atrophy and severe edema of the cerebral cortex and white matter. GAD-immunohistochemistry exhibited imbalanced distribution of GABAergic interneurons between the striatum and cerebral cortex. The results were similar to those of focal cortical dysplasia with transmantle sign and X-linked lissencephaly with ARX mutation. We performed various metabolic examinations, detailed pathological investigations and genetic analyses, but could not identify the cause. To our knowledge, her clinical and pathological courses have never been described in the literature.

Delayed progression of murine AIDS in C57BL/6 mice pre-immunized with a highly antigenic 10-mer peptide encoded by the murine AIDS defective virus gag p12 gene.

C57BL/6 (B6) mice were immunized with a highly antigenic 10-mer peptide (P12-10), which is encoded by the murine AIDS (MAIDS) defective virus gag p12 gene, emulsified in incomplete Freund's adjuvant (ICFA). One week later, the mice were inoculated with the MAIDS virus to see if the immunization affects progression of MAIDS. It was demonstrated that the immunization significantly delayed progression of MAIDS, although it failed to induce appreciable cytotoxic T lymphocyte (CTL) responses against the P12-10 antigen. In contrast, immunization of B6 mice with the P12-10 coupled with liposome induced substantial CTL responses but failed to protect the mice against MAIDS development. This segregation between CTL activity and in vivo protection efficacy might be worth considering when we exploit vaccines for augmenting cellular immunity mediated by CD8+ T cells.

IgE-specific unresponsiveness in mice induced by ovalbumin coupled with murine red blood cells.

Ovalbumin (OVA) was coupled with murine red blood cells (MRBC) using glutaraldehyde. OVA-MRBC conjugate induced anti-OVA IgG antibody production in mice at almost the same level as OVA in alum. However, no IgE antibody production specific for OVA was observed in OVA-MRBC-injected mice. IgE-specific unresponsiveness was also observed in mice injected with OVA coupled with sheep red blood cells (OVA-SRBC), suggesting that our present observation was not restricted to the property of MRBC. These results show the potential ability of antigen-RBC conjugate for the development of vaccine that induces sufficient IgG antibody production without IgE synthesis.

Biotin deficiency in an infant fed with amino acid formula and hypoallergenic rice.

An amino acid formula produced in Japan is not supplemented with biotin since biotin is not permitted as a food additive. Biotin deficiency developed in an 11-month-old Japanese infant who had been diagnosed as a neonate with cow milk and soy bean allergy and fed with an amino acid formula and hypoallergenic rice processed by protease. Serum levels of zinc, essential fatty acids and biotinidase were within the normal range while that of biotin was below the normal range. Urinary 3-hydroxy-isovalerate and slightly elevated levels of plasma branched-chain amino acids disappeared 1 week after oral supplementation with 1 mg day-1 of biotin as did the symptoms of orificial skin lesions, lethargy, hypotonia and alopecia later. In summary, to prevent biotin deficiency, biotin should be added to the Japanese amino acid formula.

Inhibition of tumor cell growth by murine splenic adherent cells stimulated with IFN-gamma.

We have previously reported that the growth of lymphocytes and tumor cells with lymphocyte lineage was strongly inhibited by a part of cloned macrophage hybridomas. This growth inhibition was accomplished by cell-to-cell contact and found to be attributed to lipid-like molecule(s) in a macrophage hybridoma cell membrane fraction. Instead of macrophage hybridomas, in the present study we utilized splenic adherent cells (SACs) that had been stimulated with IFN-gamma to see whether they inhibited tumor cell growth or not. The results demonstrated that IFN-gamma-stimulated but not unstimulated SACs showed a significant growth inhibition of BW-5147 tumor cells. This growth inhibition was not mainly mediated by prostaglandin E2 secreted from macrophages, since the inhibition was not reduced in the presence of indomethacin. Furthermore, as was reported previously in the case of macrophage hybridomas, the inhibitory activity resides in a lipid fraction of IFN-gamma-stimulated SAC membrane.

Macrophage inhibition of lymphocyte and tumor cell growth is mediated by 25-hydroxycholesterol in the cell membrane.

We have previously reported that a lipid molecule in the membrane fraction of cloned macrophage hybridomas inhibited the growth of lymphocytes and several tumor cell lines. In this study, the inhibitory lipid molecule in the membrane fraction of macrophages was analyzed by thin-layer chromatography and identified as 25-hydroxycholesterol, a family of oxysterols. This conclusion was confirmed by analysis using gas chromatography-mass spectrometry. In addition, both 25-hydroxycholesterol and the lipid molecule recovered from macrophage cell membrane induced apoptosis of the murine T cell lymphoma, BW-5147. These results suggest that an oxysterol expressed in the macrophage cell membrane may participate in the regulation of cell growth through cell contact.

Ovalbumin-liposome conjugate induces IgG but not IgE antibody production.

Antibody response after immunization with surface-coupled ovalbumin (OVA) of liposomes was investigated in mice. OVA was coupled to the surface of liposome via amino groups using glutaraldehyde. OVA-liposome conjugate induced a significant anti-OVA IgG antibody production in mice. However, no IgE antibody production specific for OVA was observed. Immunization with OVA-liposome induced IgE-specific unresponsiveness even after the subsequent challenge with OVA adsorbed with with aluminium hydroxide (OVA-alum), which induces a high level of IgE antibody production. Furthermore, following the primary immunization with OVA-alum, a secondary challenge with OVA-liposome boosted anti-OVA IgG but not anti-OVA IgE antibody production. These results show the potential of the antigen-liposome conjugate for the development of a vaccine with the least allergic reaction and also for the application of immunotherapy.

Evaluation of current sterility tests for human live viral vaccines.

Current sterility tests for human viral vaccines were evaluated. A total of 43 lots of bulk suspension of live viral vaccines (measles, mumps, rubella and oral poliomyelitis) produced by six manufacturers in Japan were evaluated for bacteriostatic and mycoplasmastatic activities. Some of them showed fairly high bacteriostatic and mycoplasmastatic activities, due to antibiotics added during vaccine production. It was concluded that the current sterility test for mycoplasmas is not reliable to detect viable mycoplasmas in live viral vaccines.

Application of PCR for detection of mycoplasma DNA and pestivirus RNA in human live viral vaccines.

PCR techniques were applied for the detection of mycoplasma DNA and pestivirus RNA to 43 lots of live viral vaccines (measles, mumps, rubella, and oral poliomyelitis) produced by six manufacturers in Japan. Although mycoplasma DNA was not detected in any of the vaccines tested, pestivirus RNA was detected in 12 lots (28%). The incidence of contamination among the four viral vaccines was in the range of 20 to 37%, and the incidence among the six manufacturers varied from 0 to 56%.

Ovalbumin coupled either with murine red blood cells or liposome induces IgG but not IgE antibody production.

Ovalbumin (OVA) was coupled with murine red blood cells (MRBC) using glutaraldehyde. The OVA-MRBC conjugate induced anti-OVA IgG antibody in mice at almost the same level as OVA in alum. However, no IgE antibody production specific for OVA was observed in OVA-MRBC-injected mice. A significant increase in IGG2a production was obtained with OVA-MRBC immunization, whereas the production of IgG1 predominated in OVA in alum immunization. Am OVA-liposome conjugate induced IgE-specific unresponsiveness in mice in the same manner as OVA-MRBC. Similar results were obtained when antigens other than OVA, such as tetanus toxoid or diphtheria toxoid, were coupled to liposome. These results show the potential of antigen-liposome conjugates for the development of vaccine that induces sufficient IgG antibody production without IgE synthesis.

Antigen-specific, IgE-selective unresponsiveness induced by antigen-liposome conjugates. Comparison of four different conjugation methods for the coupling of antigen to liposome.

BACKGROUND: We have previously reported that ovalbumin (OVA) coupled with liposome via glutaraldehyde (GA) induced OVA-specific- and IgE-selective unresponsiveness in mice. METHODS: In this study, OVA-liposome conjugates were made using four different coupling protocols: via GA, N-(6-maleimidocaproyloxy) succinimide (EMCS), disuccinimidyl suberate (DSS) and N-succimidyl-3(2-pyridyldithio)propionate (SPDP) and the induction of antigen-specific IgG and IgE antibody production was investigated for each. In addition, antigen-specific cytokine production by spleen cells of mice immunized either with OVA-liposome or with OVA adsorbed with aluminum hydroxide was investigated. RESULTS: OVA-liposome conjugates coupled via GA or DSS did not induce anti-OVA IgE antibody production but induced substantial anti-OVA IgG antibody production. On the other hand, the induction of anti-OVA IgE unresponsiveness by OVA-liposome conjugates coupled via EMCS or SPDP was incomplete. The amount of interleukin 4 (IL-4) produced by spleen cells stimulated in vitro with OVA correlated well with anti-OVA IgE antibody production in donor mice. However, the production of no other cytokine, i.e., IL-2, IL-5, IL-10 or interferon-gamma, was correlated with in vivo IgE antibody production. CONCLUSION: OVA-liposome coupled via GA or DSS induced complete suppression of anti-OVA IgE production. The results in this study further suggest that the regulation of IgE antibody production does not necessarily correlate with so-called Th1 cytokine production.

Protection against verocytotoxin in mice induced by liposome-coupled verocytotoxin.

Purified verocytotoxins (VTs), VT1 and VT2, were coupled to liposomes via glutaraldehyde. During the coupling procedure, both VT1 and VT2 were detoxified. Intraperitoneal injection in BALB/c mice with either VT1-liposome or VT2-liposome induced a substantial amount of anti-VT1 or anti-VT2 IgG antibody production, respectively. Mice immunized with VT2-liposome were protected against intravenous challenge with a lethal dose of VT2 and the degree of protection correlated well with the amount of IgG induced against VT2. Although VT1-liposome failed to induce protection against VT1, the decrease of the body weight observed after the toxin challenge correlated inversely with the amount of anti-VT1 IgG induced, suggesting that VT1 neutralizing antibody was present in VT1-liposome-immune mice. In addition, VT-liposome conjugate induced no detectable anti-VT IgE antibody production. These results demonstrate the potential ability of VT-liposome conjugates for the production of VT vaccine which induces protection against VTs.

Induction of protection against tetanus toxin in mice by tetanus toxoid-liposome conjugate.

Tetanus toxoid (Ttd) was coupled to liposomes via glutaraldehyde. Intraperitoneal injection in BALB/c mice with Ttd-liposomes induced a substantial amount of anti-Ttd IgG antibody production and an extremely low level of anti-Ttd IgE antibody production. Mice immunized with Ttd-liposomes were successfully protected against a subsequent challenge with a lethal dose of tetanus toxin (Ttx). On the other hand, aluminum hydroxide-adsorbed Ttd (Ttd-alum) and plain Ttd solution induced the production of both IgG and IgE antibodies against Ttd. Moreover, secondary immunization with Ttd-liposomes in mice, in which anti-Ttd IgE antibody production was induced by Ttd-alum led to enhanced anti-Ttd IgG and a limited anti-Ttd IgE antibody production. When Ttd-liposome preparation was lyophilized, the efficacy of Ttd-liposomes was maintained for 6 months at 37 C, suggesting that this vaccine preparation would be stable without refrigeration. These results demonstrate the potential ability of Ttd-liposome conjugates to produce a tetanus vaccine which provides protection against (Ttx) while inducing the least amount of anti-Ttd IgE antibodies.

Induction of protection against oral infection with cytotoxin-producing Escherichia coli O157:H7 in mice by shiga-like toxin-liposome conjugate.

We have previously reported that purified Shiga-like toxins (SLT), SLT-I and SLT-II coupled with liposomes induced a substantial amount of anti-SLT-I and anti-SLT-II IgG antibody production, respectively, in mice. The levels of anti-SLT antibody in the sera of SLT-liposome-immune mice correlated well with the protection against subsequent challenge with SLT. In this study, mice were immunized intraperitoneally with the mixture of SLT-I-liposome and SLT-II-liposome and protection against oral infection with cytotoxin-producing Escherichia coli O157:H7 was evaluated. All of the mice that received immunization with the mixture of SLT-I-liposome and SLT-II-liposome were protected against subsequent intravenous challenge with 10 LD50 of either SLT-I or SLT-II. Eight weeks after primary immunization, mice were inoculated intragastrically with 10(9) CFU of E. coli O157:H7 strain 96-60. All SLT-liposome-immune mice tested survived without any apparent symptom while control mice died within 5 days. In addition, as shown by other antigen-liposome conjugates, SLT-liposome induced undetectable anti-SLT IgE antibody production while they induced substantial amounts of anti-SLT IgG antibodies. These results suggest that SLT-liposome conjugate may serve as a candidate vaccine that induces protection against cytotoxin-producing E. coli infection.

Suppression of specific IgE antibody responses by liposome-conjugated ovalbumin in mice sensitized with ovalbumin via the respiratory tract.

BACKGROUND: Previously we have shown that intranasal administration of ovalbumin (OVA) together with cholera toxin (CT) abrogates nasal tolerance to OVA, resulting in the induction of specific IgE antibody (Ab) responses, and that intraperitoneal injection of OVA coupled with liposomes (OVA-liposomes) induces a selective suppression of IgE Ab responses to OVA. Whether OVA-liposomes suppress anti-OVA IgE Ab responses in mice sensitized with CT-combined OVA via the respiratory tract remains to be clarified. METHODS: In some experiments, mice were given OVA, liposomes or OVA-liposomes with or without CT intranasally three times, at 2-week intervals (weeks 0, 2 and 4). In other experiments, mice were given OVA-liposomes intranasally 2 days before or 1 and 3 weeks after CT-combined OVA (week 0), which was administered intranasally three times, at 2-week intervals (weeks 0, 2 and 4). Two weeks after the third administration of CT-combined OVA (week 0), nasal wash and serum IgA, IgG and IgE Ab responses were assayed. RESULTS: Pretreatment with OVA-liposomes suppressed IgE Ab responses to CT-combined OVA, with a significantly high production of both nasal IgA and serum IgG Abs. Moreover, treatment with OVA-liposomes 1 and 3 weeks after CT-combined OVA administration also suppressed IgE Ab responses. The suppression of anti-OVA IgE Ab production by OVA-liposomes was accompanied by a simultaneous enhancement of specific IgA and IgG (IgG1, and especially IgG2a) Ab production. CONCLUSIONS: Postimmunization treatment with OVA-liposomes, as well as preimmunization treatment, suppressed specific IgE Ab responses in mice sensitized intranasally with CT-combined OVA. Allergens conjugated to liposomes may be appropriate for preventing the development of allergies to inhaled or dietary antigens in humans.