Predictability of the neurodevelopmental assessment based on the Kyoto Scale of Psychological Development 2001 at 18-24 months of corrected age and 3 years of age in identifying cognitive impairment at 6 years of age in very preterm infants: A retrospective study at a Japanese tertiary center.

BACKGROUND: It is unknown how accurately the current Japanese classification system for neurodevelopmental delay based on the assessment with the Kyoto Scale of Psychological Development (KSPD) at toddlerhood and pre-school periods predicts cognitive impairment at school age. METHODS: This single-center retrospective cohort study enrolled infants born at 22-29 weeks of gestational age. At 18-24 months of corrected age and 3 years of age, the patients were categorized according to the current Japanese criteria for neurodevelopmental delay based on their overall developmental quotient calculated using the KSPD-2001. Cognitive impairment at 6 years of age was classified according to the calculated or estimated full-scale intelligence quotient. The predictability of the current Japanese classification of neurodevelopmental delay for cognitive impairment at 6 years of age was investigated. RESULTS: Of 566 eligible patients, 364 (64 %) completed the protocol. The current classification for the neurodevelopmental delay showed significant agreement with the severity of cognitive impairment at 6 years of age. The sensitivity and specificity of the KSPD-2001-based assessment for any cognitive impairment at 6 years of age were 0.64 and 0.74 at 18-24 months of corrected age and 0.83 and 0.70 at 3 years of age. The corresponding sensitivity and specificity for moderate/severe cognitive impairment were 0.51 and 0.96 at 18-24 months of corrected age and 0.68 and 0.95 at 3 years of age. CONCLUSION: The KSPD-2001 is a useful tool to predict the severity of cognitive impairment at school age.

Diagnostic and prognostic factors for acute encephalopathy.

BACKGROUND: Acute encephalopathy has the possibility of sequelae. While early treatment is required to prevent the development of sequelae, differential diagnosis is of the utmost priority. The aim of this study was therefore to identify parameters that can facilitate early diagnosis and prediction of outcome of acute encephalopathy. METHODS: We reviewed the medical charts of inpatients from 2005 to 2011 and identified 33 patients with febrile status epilepticus. Subjects were classified into an acute encephalopathy group (n = 20) and a febrile convulsion group (n = 13), and the parameters serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), ammonia (NH3 ), cerebrospinal fluid (CSF) tau protein, and CSF interleukin-6 compared between them. Furthermore, the relationship between each parameter and prognosis was investigated in the encephalopathy group. RESULTS: Significant differences in serum AST, ALT, and LDH were observed between the febrile convulsion and acute encephalopathy group. Moreover, a significant difference in serum LDH was noted between the patients with and without developmental regression at the time of hospital discharge in the encephalopathy group. In particular, CSF tau protein was found to be highly likely to indicate progress, with CSF tau protein >1000 pg/dL associated with poor prognosis leading to developmental regression. CONCLUSION: Serum AST, ALT and LDH may be related to early diagnosis and prognosis, and should be carefully investigated in patients with encephalopathy. CSF tau protein could also be used as an indicator of poor prognosis in acute encephalopathy.

Environment-induced epigenetic reprogramming in genomic regulatory elements in smoking mothers and their children.

Epigenetic mechanisms have emerged as links between prenatal environmental exposure and disease risk later in life. Here, we studied epigenetic changes associated with maternal smoking at base pair resolution by mapping DNA methylation, histone modifications, and transcription in expectant mothers and their newborn children. We found extensive global differential methylation and carefully evaluated these changes to separate environment associated from genotype-related DNA methylation changes. Differential methylation is enriched in enhancer elements and targets in particular "commuting" enhancers having multiple, regulatory interactions with distal genes. Longitudinal whole-genome bisulfite sequencing revealed that DNA methylation changes associated with maternal smoking persist over years of life. Particularly in children prenatal environmental exposure leads to chromatin transitions into a hyperactive state. Combined DNA methylation, histone modification, and gene expression analyses indicate that differential methylation in enhancer regions is more often functionally translated than methylation changes in promoters or non-regulatory elements. Finally, we show that epigenetic deregulation of a commuting enhancer targeting c-Jun N-terminal kinase 2 (JNK2) is linked to impaired lung function in early childhood.

Persistent verbal and behavioral deficits after resection of the left supplementary motor area in epilepsy surgery.

An 8-year-old boy underwent a resection for focal cortical dysplasia at the left supplementary motor area (SMA) for the treatment of intractable epilepsy. The manifestations of SMA syndrome, such as transient mutism and right hemiparesis, resolved within a few weeks. Verbal disfluency and impaired executive function, accompanied by impulsivity and distractibility, persisted for more than 12months. The verbal and behavioral problems caused serious difficulties in the school life of the patient, until they became less evident at 18months after surgery. Tractography performed 18months after surgery revealed a defect in the subportion of fronto-parietal association fibers within the left superior longitudinal fascicles. Verbal influency can persist with unusually long duration after resection of SMA during childhood. Although not discernible on the routine neuroimaging, white matter damage beneath the SMA region could result in serious disabilities in executive function. These complications should be recognized for the prediction and assessment of deficits in children after surgical intervention involving this region.

Requirement of apoptosis-inducing kinase 1 for the induction of bronchial asthma following stimulation with ovalbumin.

BACKGROUND: Bronchial asthma is a chronic inflammatory disease of the airway. Apoptosis signal-regulating kinase 1 (ASK1), a member of the mitogen-activated protein kinase kinase kinase family, is activated by environmental stress and plays a crucial role in the induction of apoptosis and inflammation. To examine whether ASK1 is involved in the induction of bronchial asthma, we investigated the role of ASK1 using a genetic approach in the production of cytokines, as well as the development of airway hyperreactivity (AHR) and antibody responses using a murine airway inflammation model. METHODS: ASK1-deficient (ASK1(-/-)) and control wild-type (WT) mice were immunized with ovalbumin (OVA) without alum intraperitoneally, followed by intranasal administration of OVA. Airway infiltration of inflammatory cells, cytokine production, AHR and antibody production were assayed. The asthmatic phenotype was assessed following intranasal administration of IL-13 or TNF-α. RESULTS: ASK1(-/-) mice sensitized with OVA displayed an impaired inflammatory cell infiltration into airways and a decreased AHR relative to WT mice. Moreover, the production of OVA-specific IgE antibodies and proasthmatic cytokines (IL-5, IL-13 and TNF-α) was substantially reduced in OVA-stimulated ASK1(-/-) mice. Intranasal administration of IL-13 and OVA enhanced the accumulation of inflammatory cells in OVA-primed ASK1(-/-) mice. The OVA-induced AHR in response to methacholine was enhanced by IL-13 in WT mice but not ASK1(-/-) mice. CONCLUSIONS: The ASK1 signaling pathway regulates the OVA-induced asthmatic phenotype, specifically AHR sensitivity and cytokine production. Therefore, the ASK1 signaling pathway is a promising target for therapeutic intervention in some asthmatic patients.

Enhanced T cell-independent antibody responses in c-Jun N-terminal kinase 2 (JNK2)-deficient B cells following stimulation with CpG-1826 and anti-IgM.

Although c-Jun NH2-terminal kinase (JNK) 1 and JNK2 have been demonstrated to modulate T cell activation, role of JNKs in B cell activation remains largely unclear. Phosphorylation of JNK2 was increased in murine B cells following stimulation with either anti-IgM or CpG-1826 oligonucleotide (ODN) alone, with a further increase by a combined stimulation with anti-IgM and CpG-1826 ODN. In this study, we examined whether antibody production induced by CpG ODN and/or anti-IgM is affected in B cells from JNK2-deficient (JNK2-/-) mice. After stimulation with CpG ODN or both CpG ODN and anti-IgM, JNK2-/- B cells displayed an enhanced antibody production of IgG1 and IgG2a, with less pronounced in IgG2b production, as assessed by enzyme-linked immunoassay (ELISA). However, IgM production in JNK2-/- B cells by CpG ODN was comparable to that in WT B cells. TLR9 expression was increased in JNK2-/- B cells after stimulation with anti-IgM or both CpG ODN and anti-IgM, suggesting that the anti-IgM/CpG ODN-induced enhancement of antibody production is partly due to the increased expression of TLR9. The enhanced antibody production in JNK2-/- B cells by the combined stimulation does not appear to involve either increased class switch recombination or cell proliferation. Our results provide useful information on the role of JNK2 in antibody responses mediated by T cell-independent antigens.

Tumor necrosis factor-related apoptosis-inducing ligand induces apoptotic cell death through c-Jun NH2-terminal kinase activation in squamous cell carcinoma cells.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces apoptosis in MIT6 cells derived from primary oral squamous cell carcinoma (OSCC), whereas it does not induce apoptosis in MIL6 cells derived from metastases. The present studies were performed to examine whether activation of c-Jun NH2-terminal kinase (JNK) is implicated in the differential sensitivity to TRAIL-induced apoptosis. The TRAIL-induced JNK activation in MIT6 cells was stronger than in MIL6 cells, as assessed by Western blotting using antibodies specific for phospho-JNK. To evaluate the role of JNK1 in TRAIL-induced cell death, one clone expressing the dominant-negative form of JNK1 (dnJNK1) was established. The dnJNK1-expressing cells and MIL6 cells expressed TRAIL protein at levels similar to or even greater than MIT6 cells did. When cell death was assessed by annexin V staining and mitochondrial membrane potential, kinetic studies demonstrated that the dnJNK1-expressing cells were substantially more resistant to 100 ng/ml TRAIL, comparable to MIL6 cells, at 36 and 48 h after stimulation. Collectively, the primary OSCC cell line, MIT6, is sensitive to TRAIL but its metastatic line MIL6 is resistant to TRAIL exposure. Thus, the underlying molecular mechanism of TRAIL-induced cell death involves JNK activation. These results suggest that the acquisition of TRAIL resistance provides some metastatic capacity to primary tumors.

Apoptosis signal-regulating kinase (ASK)-1 mediates apoptosis through activation of JNK1 following engagement of membrane immunoglobulin.

Engagement of membrane immunoglobulin (mIg) on WEHI-231 mouse B lymphoma cells results in growth arrest at the G1 phase of the cell cycle, followed by a reduction of mitochondrial membrane potential (DeltaPsim) and apoptosis. WEHI-231 cells resemble immature B cells in terms of the cell surface phenotype and sensitivity to mIg engagement. However, the molecular mechanisms underlying mIg-induced loss of DeltaPsim and apoptosis have not yet been established. In this study, we show that apoptosis signal-regulating kinase 1 (ASK1)-c-Jun N-terminal kinase 1 (JNK1) signaling pathway participates in mIg-induced apoptosis through the generation of reactive oxygen species (ROS). Stimulation of WEHI-231 cells with anti-IgM induces phosphorylation and subsequent activation of ASK1, leading to JNK activation. Anti-IgM stimulation immediately (5 min) induces hydrogen peroxide (H2O2) production with a substantial increase during later time points (36-48 h), accompanied by loss of DeltaPsim and an increase in cells with sub-G1 DNA content. The anti-IgM-induced late-phase H2O2 production, loss of DeltaPsim, and increase in the sub-G1 fraction were all reduced substantially in WEHI-231 cells overexpressing a dominant-negative form of ASK1, compared with control vector alone, but enhanced substantially in cells overexpressing a constitutively active form of ASK1. These mIg-mediated events were also partially abrogated by ROS scavenger N-acetyl-L-cysteine (NAC). Taken together, these results suggest that mIg engagement induces H2O2 production leading to activation of ASK1-JNK1 pathway, creating a feedback amplification loop of ROS-ASK/JNK that leads to loss of DeltaPsim and finally apoptosis.

c-Jun NH2-terminal kinase (JNK)-dependent nuclear translocation of apoptosis-inducing factor (AIF) following engagement of membrane immunoglobulin on WEHI-231 B lymphoma cells.

WEHI-231 B lymphoma cells have been employed for analysis of antigen-induced B cell unresponsiveness because these cells undergo cell cycle arrest in G1, accompanied by induction of apoptosis. In the present study, we examined the requirement for toxic small molecules apoptosis-inducing factor (AIF) and cytochrome c, and subsequent caspase activation in apoptotic cell death in WEHI-231 and CH31 B lymphoma cells following engagement of membrane immunoglobulin (mIg). Pan-caspase inhibitor BD-fmk blocked mIg-mediated increase in cells with sub-G1 DNA content, whereas it did not affect mIg-mediated loss of mitochondrial membrane potential and phosphatidylserine exposure on B cell membrane. Dominant-negative form of c-Jun NH2-terminal kinase1 (JNK1) blocked the translocation of AIF into the nuclei and cytosol from the mitochondria in the WEHI-231 and CH31 cells following mIg engagement, whereas constitutively active form of JNK1 enhanced it. This AIF translocation was also blocked by Bcl-xL, but not by BD-fmk. Moreover, AIF-deficient clones via small interfering RNA (siRNA)-mediated method showed small increase in loss of mitochondrial membrane potential. After mIg engagement, the AIF-deficient clones displayed an enhanced sensitivity to mIg-mediated apoptosis, concomitant with translocation of a residual AIF into the nuclei, compared with control clone. Our findings are compatible with the notion that AIF has dual role, with a proapoptotic function in the nuclei and a distinct anti-apoptotic function in the mitochondria. These observations would be valuable for analysis of B cell unresponsiveness and hopefully for treatment of diseases involving B cell dysfunction.

c-Jun-NH2-terminal kinase potentiates apoptotic cell death in response to carboplatin in B lymphoma cells.

PURPOSE: Exposure to carboplatin (CBDCA) has been demonstrated to result in apoptotic and/or necrotic cell death, but molecular mechanisms underlying CBDCA-induced apoptosis or necrosis remain largely unclear. Here, we examined whether activation of c-Jun NH(2)-terminal kinase (JNK) modulates the mode of cell death induced by CBDCA in CD31 B lymphoma cells. METHODS: The mode of cell death (apoptosis versus necrosis) was investigated by flow cytometry using 7-amino-actinomycin D (7-AAD) and annexin-FITC probes. To evaluate the role of JNK1 in CBDCA-induced cell death, CH31 B lymphoma cells overexpressing dominant-negative form of JNK1 (dnJNK1) or constitutively active form of JNK1 (MKK7-JNK1) were established. Intracellular accumulation of superoxide anion (O(2) (-)) was determined by flow cytometry using the fluorescent probe dihydroethidium (DHE). RESULTS: The CBDCA-induced primary apoptosis and secondary necrosis were abrogated in the dnJNK1-overexpressing CH31 cells, while it was somewhat enhanced in the MKK7-JNK1-overexpressing cells. In contrast, the CBDCA-induced primary necrosis was reduced by MKK7-JNK1, with a concurrent decrease in production of O(2) (-). The superoxide anion scavenger for butylated hydroxyanisol (BHA) partially reduced the CBDCA-induced O(2) (-) production and necrotic, but not apoptotic, death in both wild type and dnJNK1-overexpressing CH31 cells. CONCLUSIONS: Prolonged activation of JNK1 appears to be involved in CBDCA-induced apoptosis with prevention of necrosis induction, and the induction of necrosis appears to correlate with CBDCA-induced O(2) (-) production, which is partially blocked by co-culture with BHA. These observations provide valuable information for understanding molecular mechanisms underlying CBDCA-induced cell death, and hopefully for the design of novel treatment modalities for patients with tumors.

Blockade of the OX40 ligand prolongs corneal allograft survival.

Although corneal transplantation is one of the most common tissue transplantations and is known to have a high graft acceptance rate, occasional corneal graft rejection remains a cause of blindness. OX40, a member of the TNF receptor superfamily, is expressed on activated T cells, and transmits a costimulatory signal by binding to OX40 ligand (OX40L) expressed on several cells with antigen-presenting functions. Using a blocking monoclonal antibody (mAb) against murine OX40L, we investigated the role of OX40 in a murine model of corneal transplantation. C3H/He mouse corneas were transplanted to BALB/c mice orthotopically. Administration of anti-OX40L mAb significantly reduced allograft rejection, and increased graft survival rate to 40% at 8 weeks after transplantation, while all corneas were rejected within 5 weeks in control IgG-treated mice. Similar reduced rejection was observed when wild-type donor corneas were transplanted to OX40L-deficient recipients. In vitro study revealed that the anti-OX40L mAb treatment reduced proliferative response and IFN-gamma production of draining lymph node cells in response to stimulation with donor alloantigen. These results demonstrate that OX40L blockade is effective for prolongation of corneal allograft survival by inhibiting recipient T cell activation.

C-terminal LRRs of human Toll-like receptor 3 control receptor dimerization and signal transmission.

Toll-like receptor (TLR)3 recognizes dsRNA and activates the signaling cascade leading to production of IFN-beta via an adaptor protein, TICAM-1 (also called TRIF). The interface between ligand recognition and signal transduction by TLR3 remains largely unknown. The crystalized ectodomain of human TLR3 revealed a horseshoe-shaped solenoid assembled from 23 leucine-rich repeats (LRRs). Here, we constructed LRR deletion mutants and tested the participation of each LRR in the IFN-inducing ability of TLR3. Only 3 of the 23 LRRs (LRR4, LRR11 and LRR17) were dispensable for the TLR3 function. Among the 20 dysfunctional mutants, LRR20- and LRR22-deleted mutants acted as dominant-negative inhibitors of wild-type TLR3. The LRR20-deleted mutant lost the poly(I:C)-binding ability, while LRR22-deleted mutant possessed it. Strikingly, the LRR21-deleted mutant functioned as a constitutively active form. These three mutants formed homodimers regardless of their different functional features and reacted with TLR3.7, a function-blocking anti-human TLR3 mAb whose epitope resided in LRR10-LRR16, suggesting that the intact conformation around the central solenoid was retained in the C-terminal mutants. These results suggest that the extracellular domains are a crucial trigger of cytoplasmic IFN signaling in TLR3. The altered molecular topology resulting from the deletion of LRR20, LRR21 or LRR22 critically affects the functional assembly of cytoplasmic TLR3, resulting in dysregulation of receptor-receptor association and signal transmission from the outside ectodomain to the inside TIR domain.

Requirement for JNK-dependent upregulation of BimL in anti-IgM-induced apoptosis in murine B lymphoma cell lines WEHI-231 and CH31.

The cross-linking of B cell receptor (BCR) undergoes growth arrest, accompanied by apoptosis, in the CH31 and WEHI-231 B lymphoma cells, a model representing primary immature B cells. We have previously demonstrated that sustained activation of c-Jun N-terminal kinase (JNK) is required for BCR-mediated apoptosis. In the present study, we examined how the anti-IgM-induced prolonged activation of JNK results in apoptosis. Anti-IgM upregulated the expression levels of three isoforms of Bim protein, especially BimL, which appeared to be dependent on JNK activation. In contrast to protein expression, BimL mRNA levels were down-regulated upon anti-IgM stimulation, suggesting that anti-IgM-induced upregulation of BimL is regulated through post-transcriptional control. Upon JNK activation, phosphorylated form of JNK, together with Bax migrated from cytosol to mitochondria. In unstimulated cells, BimL protein was complexed with Bcl-x(L) and changed the partner to associate with Bax on the mitochondrial membrane after ligation of BCR, leading to initiation of apoptotic processes. Retroviral transduction of BimL into WEHI-231 cells overexpressing dominant-negative form of JNK1 (dnJNK1) resulted in a comparable level of apoptotic cells to control cells, whereas the BimL-mediated apoptosis was partially prevented by Bcl-x(L). Taken together, engagement of BCR with anti-IgM results in association of Bax-alpha with BimL in the mitochondria, at least in part, through a sustained activation of JNK.

Interferon-beta-induced activation of c-Jun NH2-terminal kinase mediates apoptosis through up-regulation of CD95 in CH31 B lymphoma cells.

Type I interferon (IFN)-induced antitumor action is due in part to apoptosis, but the molecular mechanisms underlying IFN-induced apoptosis remain largely unresolved. In the present study, we demonstrate that IFN-beta induced apoptosis and the loss of mitochondrial membrane potential (delta psi m) in the murine CH31 B lymphoma cell line, and this was accompanied by the up-regulation of CD95, but not CD95-ligand (CD95-L), tumor necrosis factor (TNF), or TNF-related apoptosis-inducing ligand (TRAIL). Pretreatment with anti-CD95-L mAb partially prevented the IFN-beta-induced loss of delta psi m, suggesting that the interaction of IFN-beta-up-regulated CD95 with CD95-L plays a crucial role in the induction of fratricide. IFN-beta induced a sustained activation of c-Jun NH2-terminal kinase 1 (JNK1), but not extracellular signal-regulated kinases (ERKs). The IFN-beta-induced apoptosis and loss of delta psi m were substantially compromised in cells overexpressing a dominant-negative form of JNK1 (dnJNK1), and it was slightly enhanced in cells carrying a constitutively active JNK construct, MKK7-JNK1 fusion protein. The IFN-beta-induced up-regulation of CD95 together with caspase-8 activation was also abrogated in the dnJNK1 cells while it was further enhanced in the MKK7-JNK1 cells. The levels of cellular FLIP (c-FLIP), competitively interacting with caspase-8, were down-regulated by stimulation with IFN-beta but were reversed by the proteasome inhibitor lactacystin. Collectively, the IFN-beta-induced sustained activation of JNK mediates apoptosis, at least in part, through up-regulation of CD95 protein in combination with down-regulation of c-FLIP protein.

Modulation of mThy28 nuclear protein expression during thymocyte development.

We have recently shown that down-regulation of mouse Thy28 (mThy28) protein expression appears to be accompanied by apoptotic processes. Thymocytes from mice contain moderate amounts of mThy28 protein and undergo proliferation, differentiation, or apoptosis during murine thymic maturation. As a first step to examine the potential role of the mThy28 protein in the thymocyte development, such as positive-negative selection, the expression of mThy28 protein in the thymocyte subsets was examined. Thymocytes are separated into four subpopulations by the expression levels of CD4 and CD8: CD4-CD8- (DN), CD4+CD8+ (DP), and CD4+CD8- or CD4-CD8+ (SP). Flow cytometry analysis using three-color staining demonstrated that the mThy28 expression in immature DP cells is lower than that in DN and SP cells. The down-regulation of the mThy28 expression in the DP stage was also detected by Western blotting and reverse transcription-polymerase chain reaction (RT-PCR). The immunostaining method also showed that mThy28 protein was expressed in the medulla containing mature thymocytes, but not the cortex having immature thymocytes. The mThy28 protein in the thymocytes was mainly localized in the nucleus, as recently demonstrated in lymphoma cells, indicating that the mThy28 protein resides in the nucleus, irrespective of the cyclic or resting stage of the cell cycle. Together, the observation that mThy28 expression is down-modulated during the DP stage suggests that mThy28 protein might play some role in the positive-negative selection step in thymic maturation.

Chemotherapeutic agents sensitize sarcoma cell lines to tumor necrosis factor-related apoptosis-inducing ligand-induced caspase-8 activation, apoptosis and loss of mitochondrial membrane potential.

Chemotherapeutic agents have been used for the treatment of patients with osteosarcoma (OS). However, inherent or acquired resistance to these agents is a serious problem in the management of OS patients. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered to induce apoptosis in a variety of cancer cells but not normal cells. In the present study, we examined whether chemotherapeutic agents enhance TRAIL-induced apoptosis in the sarcoma cell lines MG-63 and SaOS-2. Pretreatment with sub-toxic or slightly toxic concentrations of chemotherapeutic agents (cis-diammine dichloroplatinum, CDDP and doxorubicin, DXR) sensitized both cell lines to TRAIL-induced apoptosis, as assessed by the propidium iodide or Annexin V-Cy5 staining method. These cell lines expressed death receptors TRAIL-receptor 1 (TRAIL-R1) and TRAIL-R2, which were unaltered by treatment with CDDP, as assessed by flow cytometry. The decoy receptors TRAIL-R3 and -R4 were barely detected in both cell lines. CDDP down-regulated c-FLIP, tending to lower the activation threshold required for TRAIL-induced caspase-8 activation. The CDDP-pretreated cells indeed demonstrated more increased TRAIL-mediated caspase-8 activation, loss of mitochondrial membrane potential (DeltaPsi(m)), and apoptosis than untreated cells. Consequently, the activated caspase-8 might lead to either activation of effector caspases such as caspase-3 or loss in DeltaPsi(m). Both the increased caspase activation and mitochondrial dysfunction induced by combination of CDDP and TRAIL would contribute to enhanced apoptotic cell death. The results of the present study would be valuable for the design of novel treatment modalities for patients with OS.

Thiamine-responsive pyruvate dehydrogenase deficiency in two patients caused by a point mutation (F205L and L216F) within the thiamine pyrophosphate binding region.

The human pyruvate dehydrogenase complex (PDHC) catalyzes the thiamine-dependent decarboxylation of pyruvate. Thiamine treatment is very effective for some patients with PDHC deficiency. Among these patients, five mutations of the pyruvate dehydrogenase (E1)alpha subunit have been reported previously: H44R, R88S, G89S, R263G, and V389fs. All five mutations are in a region outside the thiamine pyrophosphate (TPP)-binding region of the E1alpha subunit. We report the biochemical and molecular analysis of two patients with clinically thiamine-responsive lactic acidemia. The PDHC activity was assayed using two different concentrations of TPP. These two patients displayed very low PDHC activity in the presence of a low (1 x 10(-4) mM) TPP concentration, but their PDHC activity significantly increased at a high (0.4 mM) TPP concentration. Therefore, the PDHC deficiency in these two patients was due to a decreased affinity of PDHC for TPP. Treatment of both patients with thiamine resulted in a reduction in the serum lactate concentration and clinical improvement, suggesting that these two patients have a thiamine-responsive PDHC deficiency. The DNA sequence of these two male patients' X-linked E1alpha subunit revealed a point mutation (F205L and L216F) within the TPP-binding region in exon 7.

Oral administration of interferon-beta suppresses experimental autoimmune uveoretinitis.

BACKGROUND: The oral administration of type I interferons (IFNs) have been reported to reduce severity of inflammation in several animal models of autoimmune disease. This study examined whether oral administration of IFN-beta is capable of modulating inflammation in experimental autoimmune uveoretinitis (EAU). METHODS: EAU was induced in rats by immunization with interphotoreceptor retinoid-binding protein (IRBP) emulsified in complete Freund's adjuvant. Rats were treated with either varying doses (10(2), 10(3), 10(4) or 10(5)IU) of mouse recombinant IFN-beta or phosphate-buffered saline for control, via direct oropharyngeal application once a day for 28 days starting 7 days before IRBP immunization. Intraocular inflammation was assessed by slit-lamp biomicroscopy and histopathological examination. Spleen cell proliferation response and cytokine production under IRBP stimulation were assessed. Spleen cell subpopulations were also measured. RESULTS: IFN-beta at doses of either 10(4) or 10(5) IU significantly reduced both the clinical and histopathological severity of EAU. Spleen cell proliferation and IFN-gamma production from rats treated with 10(4) IU IFN-beta were significantly decreased compared with controls. Furthermore, the proportion of both NK cells and NKT cells in the spleen of rats treated with IFN-beta was increased compared with controls. CONCLUSION: These results suggest that the oral administration of IFN-beta reduces inflammation in IRBP-mediated EAU and that the mechanism of this action may involve NK cells and NKT cells.

Requirement of c-Jun N terminal kinase and P38 mitogen-activated protein kinase for anti-IgM-induced reduction in mitochondrial membrane potential in CH31 B lymphoma cells.

CH31 cells have been used for analysis of B cell tolerance, since engagement of membrane immunoglobulin (mIg) results in loss in mitochondrial membrane potential (DeltaPsim), followed by cell death. We have reported that the dominant-negative (dn) form of c-Jun N-terminal kinase (JNK) substantially prevented a loss of DeltaPsim at 24 h, with partial protection around 48 h after anti-IgM stimulation. In this study, we demonstrate that anti-IgM induced a sustained activation of p38 mitogen-activated protein (MAP) kinase. The p38MAP kinase inhibitor SB203580 substantially prevented loss of DeltaPsim at 14 h, with partial prevention (18-24 h) after anti-IgM stimulation. The dnJNK1-mediated prevention of anti-IgM-induced mitochondrial dissipation was enhanced by SB203580 at 42 h, but not 24 h, after stimulation, suggesting that activation of either p38 MAP kinase or JNK may be sufficient for the initiation of early phase of anti-IgM-induced loss of DeltaPsim while both may be necessary in the late phase.