Chronic granulomatous disease: two decades of experience from a tertiary care centre in North West India.

Chronic granulomatous disease (CGD) results from an inherited defect in the phagocytic cells of the immune system. It is a genetically heterogenous disease caused by defects in one of the five major subunits of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. There is a paucity of data from India on CGD. We herein describe the clinical features in 17 children with CGD from a single tertiary referral center in India. A detailed analysis of the clinical features, laboratory investigations and outcome of 17 children 7 with X-linked (XL) and 10 with autosomal recessive (AR) form was performed. Diagnosis of CGD was based on an abnormal granulocyte oxidative burst evaluated by either Nitroblue Tetrazolium (NBT) test or flow cytometry based Dihyrorhodamine 123 assay or both. The molecular diagnosis was confirmed by genetic mutation analysis in 13 cases. The mean age at diagnosis and the age at onset of symptoms was significantly lower in children diagnosed with XL- CGD compared those with AR disease. Mutations were detected in CYBB gene in 6 patients with XL-CGD and NCF-1 gene mutations were observed in 7 cases of AR- CGD. The course and outcome of the disease was much worse in children diagnosed with X-linked form of disease compared to AR forms of the disease; 4/7 (57%) children with X-CGD were dead at the time of data analysis. This is one of the largest series on chronic granulomatous disease from any developing country.

Genetic effects of demographic bottleneck and recovery in Kinkazan Island and mainland populations of Japanese macaques (Macaca fuscata).

Populations of Japanese macaques were significantly reduced in most areas from the 1900s to the 1960s and then recovered mainly in the northeastern part of Honshu. A drastic reduction in population size reduces genetic variability through a bottleneck effect. Demographic expansion after the reduction that accumulates new mutations can reduce the bottleneck effects or drive the recovery of genetic variability. We examined the genetic status of a small island population (Kinkazan Island) and a larger mainland population (southern Tohoku) of Japanese macaques that experienced recent demographic bottlenecks and recovery using eight microsatellite loci. The two populations were significantly genetically different from each other. The Kinkazan population exhibited lower genetic variability, remarkable evidence of bottleneck (i.e., significant heterozygosity excess and lower frequency of rare alleles), and a considerably smaller effective population size based on genetic data than based on the current census size. These results indicate that the genetic status has not completely recovered from the demographic bottleneck despite a full recovery in census size on Kinkazan Island. New mutations might rarely have accumulated because of the small carrying capacity of the island. Therefore, the genetic variability of the population would have been restrained by the severe bottleneck size, small carrying capacity, and long-term isolation. On the other hand, the bottleneck effect seems to be limited in the southern Tohoku population considering higher genetic variability, non-significant heterozygosity excess in many mutation conditions, and the highest frequency of rare alleles.

Prolonged heart rate recovery time after 6-minute walk test is an independent risk factor for cardiac events in heart failure: A prospective cohort study.

OBJECTIVES: To determine whether the time for peak exercise heart rate to return to resting heart rate after the 6-minute walk test (6MWT) can predict cardiac events in patients with heart failure (HF) within 2 years. DESIGN: Prospective cohort study. SETTING: HF outpatient facility at a tertiary teaching hospital. PARTICIPANTS: Seventy-six patients with HF, New York Heart Association functional classification II and III, and left ventricular ejection fraction <50%. MAIN OUTCOME MEASURES: Patients used a heart rate monitor to measure the time for peak exercise heart rate to return to resting heart rate after the 6MWT. Data were analysed using Polar Pro-Trainer 5 software (Kempele, Finland). Patients were followed for >2 years for cardiac events (hospitalisations and death). RESULTS: Thirty-four patients had cardiac events during the 2-year follow-up period. However, there was a significant difference in the time to return to resting heart rate between the groups with and without cardiac events {with 3.6 (SD 1.1) vs without 2.8 (SD 1.1) minutes; mean difference of 0.79 (95% confidence interval (CI) of the difference 0.28 to 1.28; P=0.003}. No significant differences between patients with and without cardiac events were found for mean walking distance, mean heart rate recovery at 1minute and mean heart rate recovery at 2minutes. The receiver operating curve discriminated between patients with and without cardiac events (área under the curve 0.71, 95% CI 0.61 to 0.81; P<0.001). Using logistic regression analysis, prolonged time to return to resting heart rate (≥3minutes) independently increased the risk for cardiac events 6.9-fold (95% CI 2.34 to 20.12; P<0.001). The Kaplan-Meier curve showed more cardiac events in patients with prolonged time to return to resting heart rate (P=0.028). CONCLUSIONS: Prolonged time to return to resting heart rate (≥3minutes) after the 6MWT was an independent predictor of cardiac events in patients with HF.

Spatiotemporal classification of environmental monitoring data in the Yeongsan River basin, Korea, using self-organizing maps.

Environmental monitoring data for planning, implementing and evaluating the Total Maximum Daily Loads (TMDL) management system have been measured at about 8-day intervals in a number of rivers in Korea since 2004. In the present study, water quality parameters such as Suspended Solids (SS), Biochemical Oxygen Demand (BOD), Dissolved Oxygen (DO), Total Nitrogen (TN), and Total Phosphorus (TP) and the corresponding runoff were collected from six stations in the Yeongsan River basin for six years and transformed into monthly mean values. With the primary objective to understand spatiotemporal characteristics of the data, a methodologically systematic application of a Self-Organizing Map (SOM) was made. The SOM application classified the environmental monitoring data into nine clusters showing exclusively distinguishable patterns. Data frequency at each station on a monthly basis identified the spatiotemporal distribution for the first time in the study area. Consequently, the SOM application provided useful information that the sub-basin containing a metropolitan city is associated with deteriorating water quality and should be monitored and managed carefully during spring and summer for water quality improvement in the river basin.

Tumor necrosis factor alpha stimulates osteoclast differentiation by a mechanism independent of the ODF/RANKL-RANK interaction.

Osteoclast differentiation factor (ODF, also called RANKL/TRANCE/OPGL) stimulates the differentiation of osteoclast progenitors of the monocyte/macrophage lineage into osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF, also called CSF-1). When mouse bone marrow cells were cultured with M-CSF, M-CSF-dependent bone marrow macrophages (M-BMM phi) appeared within 3 d. Tartrate-resistant acid phosphatase-positive osteoclasts were also formed when M-BMM phi were further cultured for 3 d with mouse tumor necrosis factor alpha (TNF-alpha) in the presence of M-CSF. Osteoclast formation induced by TNF-alpha was inhibited by the addition of respective antibodies against TNF receptor 1 (TNFR1) or TNFR2, but not by osteoclastogenesis inhibitory factor (OCIF, also called OPG, a decoy receptor of ODF/RANKL), nor the Fab fragment of anti-RANK (ODF/RANKL receptor) antibody. Experiments using M-BMM phi prepared from TNFR1- or TNFR2-deficient mice showed that both TNFR1- and TNFR2-induced signals were important for osteoclast formation induced by TNF-alpha. Osteoclasts induced by TNF-alpha formed resorption pits on dentine slices only in the presence of IL-1alpha. These results demonstrate that TNF-alpha stimulates osteoclast differentiation in the presence of M-CSF through a mechanism independent of the ODF/RANKL-RANK system. TNF-alpha together with IL-1alpha may play an important role in bone resorption of inflammatory bone diseases.

Hyper IgM syndrome and complement Clq deficiency in an individual with systemic lupus erythematosus-like disease.

Many immunedeficiency syndromes are associated with autoimmune disorders. We here report on a girl with a systemic lupus erythematosus-like disease who suffered from both hyperimmunoglobulin M syndrome (HIGMS) and C1q deficiency. Despite severe central nervous system-lupus like disease, probably due to C1q deficiency, kidney function was relatively spared. IgM autoantibody might play a protective role against lupus-glomerulonephritis.

Ataxia-telangiectasia in a patient presenting with hyper-immunoglobulin M syndrome.

Ataxia-telangiectasia (AT) and hyper-immunoglobulin M (HIGM) syndrome are both primary immunodeficiency diseases caused by different genetic defects. While a small proportion of AT patients have increased serum immunoglobulin (Ig) M concentrations during the course of a disease, a high level of IgM at onset is rare. We report the case of an 8-year-old girl who had experienced recurrent respiratory infection, cutaneous abscesses, and hepatosplenomegaly since the age of 2 years. She was diagnosed with HIGM based on the results of immunological studies, including low IgG and IgA levels and raised serum IgM concentrations. However, at the age of 4 years, a neurological examination revealed gait disturbance and telangiectatic lesions on the conjunctiva; therefore, a diagnosis of AT was suggested. In spite of regular intravenous immunoglobulin infusions and antimicrobial prophylaxis, the patient experienced several episodes of respiratory infection and eventually died of respiratory failure at the age of 8 years. Further molecular analysis revealed a novel homozygous missense mutation in exon 53 (c.8250C>T, p.2622Ala>Val) of the ATM gene. Patients with AT and the HIGM phenotype may not develop clinical characteristics of AT for some time. While patients with AT and increased serum IgM levels could have a considerably more severe disease course and a shorter survival, IgM levels could be considered a prognostic factor.

Subchronic effects of nasally instilled diesel exhaust particulates on the nasal and airway epithelia in mice.

Diesel exhaust is the major source of ultrafine particles released during traffic-related pollution. Subjects with chronic respiratory diseases are at greater risk for exacerbations during exposure to air pollution. This study evaluated the effects of subchronic exposure to a low-dose of diesel exhaust particles (DEP). Sixty male BALB/c mice were divided into two groups: (a) Saline: nasal instillation of saline (n = 30); and (b) DEP: nasal instillation of 30 microg of DEP/10 microl of saline (n = 30). Nasal instillations were performed 5 days a week, over 30 and 60 days. Animals were anesthetized with pentobarbital sodium (50 mg/kg intraperitoneal [i.p.]) and sacrificed by exsanguination. Bronchoalveolar lavage (BAL) fluid was performed to evaluate the inflammatory cell count and the concentrations of the interleukin (IL)-4, IL-10, and IL-13 by enzyme-linked immunosorbent assay (ELISA). The gene expression of oligomeric mucus/gel-forming (Muc5ac) was evaluated by real-time polymerase chain reaction (PCR). Histological analysis in the nasal septum and bronchioles was used to evaluate the bronchial and nasal epithelium thickness as well as the acidic and neutral nasal mucus content. The saline group (30 and 60 days) did not show any changes in any of the parameters. However, the instillation of DEP over 60 days increased the expression of Muc5ac in the lungs and the acid mucus content in the nose compared with the 30-day treatment, and it increased the total leukocytes in the BAL and the nasal epithelium thickness compared with saline for 60 days. Cytokines concentrations in the BAL were detectable, with no differences among the groups. Our data suggest that a low-dose of DEP over 60 days induces respiratory tract inflammation.

Mammalian homologue of the Caenorhabditis elegans UNC-76 protein involved in axonal outgrowth is a protein kinase C zeta-interacting protein.

By the yeast two-hybrid screening of a rat brain cDNA library with the regulatory domain of protein kinase C zeta (PKCzeta) as a bait, we have cloned a gene coding for a novel PKCzeta-interacting protein homologous to the Caenorhabditis elegans UNC-76 protein involved in axonal outgrowth and fasciculation. The protein designated FEZ1 (fasciculation and elongation protein zeta-1) consisting of 393 amino acid residues shows a high Asp/Glu content and contains several regions predicted to form amphipathic helices. Northern blot analysis has revealed that FEZ1 mRNA is abundantly expressed in adult rat brain and throughout the developmental stages of mouse embryo. By the yeast two-hybrid assay with various deletion mutants of PKC, FEZ1 was shown to interact with the NH2-terminal variable region (V1) of PKCzeta and weakly with that of PKCepsilon. In the COS-7 cells coexpressing FEZ1 and PKCzeta, FEZ1 was present mainly in the plasma membrane, associating with PKCzeta and being phosphorylated. These results indicate that FEZ1 is a novel substrate of PKCzeta. When the constitutively active mutant of PKCzeta was used, FEZ1 was found in the cytoplasm of COS-7 cells. Upon treatment of the cells with a PKC inhibitor, staurosporin, FEZ1 was translocated from the cytoplasm to the plasma membrane, suggesting that the cytoplasmic translocation of FEZ1 is directly regulated by the PKCzeta activity. Although expression of FEZ1 alone had no effect on PC12 cells, coexpression of FEZ1 and constitutively active PKCzeta stimulated the neuronal differentiation of PC12 cells. Combined with the recent finding that a human FEZ1 protein is able to complement the function of UNC-76 necessary for normal axonal bundling and elongation within axon bundles in the nematode, these results suggest that FEZ1 plays a crucial role in the axon guidance machinery in mammals by interacting with PKCzeta.

IL-6-induced homodimerization of gp130 and associated activation of a tyrosine kinase.

The biological functions of interleukin-6 (IL-6) are mediated through a signal-transducing component of the IL-6 receptor, gp130, which is associated with the ligand-occupied IL-6 receptor (IL-6R) protein. Binding of IL-6 to IL-6R induced disulfide-linked homodimerization of gp130. Tyrosine kinase activity was associated with dimerized but not monomeric gp130 protein. Substitution of serine for proline residues 656 and 658 in the cytoplasmic motif abolished tyrosine kinase activation and cellular responses but not homodimerization of gp130. The IL-6-induced gp130 homodimer appears to be similar in function to the heterodimer formed between the leukemia inhibitory factor (LIF) receptor (LIFR) and gp130 in response to the LIF or ciliary neurotrophic factor (CNTF). Thus, a general first step in IL-6-related cytokine signaling may be the dimerization of signal-transducing molecules and activation of associated tyrosine kinases.

Physical function is weakly associated with angiotensin-converting enzyme gene I/D polymorphism in elderly Japanese subjects.

BACKGROUND: The turning point in the deterioration of physical function seems to occur between the ages of 70 and 80 years. In particular, muscle strength may decline even more in subjects older than 75. A recent study found that the angiotensin-converting enzyme (ACE) genotype also affects physiological left ventricular hypertrophy. A very limited number of papers have examined genetic differences in resistance and endurance forms of a single sporting discipline. OBJECTIVE: The purpose of this study was to evaluate the relationship between ACE genotype and physical function by controlling the known confounding factors including dental status. METHODS: We selected 431 subjects who were aged 76 years and did not require special care for their daily activities. We conducted a medical examination, followed by 5 physical function tests, as follows: (1) maximum hand grip strength, (2) maximal isometric knee extensor strength, (3) maximal stepping rate for 10 s, (4) one-leg standing time with eyes open and (5) 10-meter maximum walking speed. Subjects were genotyped for the ACE intron 16 Alu insertion. In addition, serum concentrations of total cholesterol, total protein, IgA and IgG were measured at a commercial laboratory. The Eichner index was used as an indicator of occlusal condition. Multiple linear regression analysis was performed to evaluate the relationship between the ACE gene insertion/deletion (I/D) polymorphism and physical function considering confounding factors. RESULTS: The ACE gene I/D polymorphism was positively associated with hand grip strength and 10-meter maximum walking speed. Betas of hand grip strength were 0.09 for I/D (p = 0.022) and 0.12 for insertion/insertion (I/I; p = 0.004). Betas of 10-meter walking speed were -0.11 for I/D (p = 0.093) and -0.14 for I/I (p = 0.039). Dental status such as Eichner index class C was significantly associated with one-leg standing time with eyes open (beta -0.11; p = 0.028). CONCLUSION: This study suggests that there is a significant relationship between ACE genotype and physical function. In particular, subjects with the ACE deletion/deletion genotype were associated with upper extremities.

Protein C inhibitor directly and potently inhibits activated hepatocyte growth factor activator.

BACKGROUND: Hepatocyte growth factor (HGF) plays an important role in tissue repair and regeneration. HGF activator (HGFA), a factor XIIa-like serine protease, activates HGF precursor to HGF. The precursor of HGFA, proHGFA, is activated by thrombin generated at sites of tissue injury. It is known that protein C inhibitor (PCI), an inhibitor of activated protein C (APC), also inhibits thrombin-thrombomodulin (TM) complex. OBJECTIVES: In the present study we evaluated the effect of PCI on thrombin-catalyzed proHGFA activation in the presence of TM, and on HGFA activity. RESULTS: PCI did not inhibit thrombin-TM-mediated proHGFA activation, but it directly inhibited activated HGFA by forming an enzyme inhibitor complex. The second-order rate constants (m(-1) min(-1)) of the reaction between HGFA and PCI in the presence or absence of heparin (10 U mL(-1)) were 4.3 x 10(6) and 4.0 x 10(6), respectively. The inhibition of HGFA by PCI resulted in a significant decrease of HGFA-catalyzed activation of HGF precursor. Exogenous HGFA added to normal human plasma formed a complex with plasma PCI, and this complex formation was competitively inhibited by APC in the presence of heparin, but very weakly in the absence of heparin. We also demonstrated using recombinant R362A-PCI that Arg362 residue of PCI is important for HGFA inhibition by PCI as judged from the three-dimensional structures constructed using docking models of PCI and HGFA or APC. CONCLUSION: These observations indicate that PCI is a potent inhibitor of activated HGFA, suggesting a novel function for PCI in the regulation of tissue repair and regeneration.

Molecular cloning of a novel human cDNA encoding a zinc finger protein that binds to the interleukin-3 promoter.

The CT/GC-rich region (-76 to -47) is one transcriptional regulatory region of the interleukin-3 (IL-3) gene which confers basic transcriptional activity and responds to trans-activation by human T-cell leukemia virus type I-encoded Tax. We isolated three types of cDNAs encoding Cys2/His2-type zinc finger proteins that bind to this region. Two were identical to known transcription factors, EGR1 and EGR2, and the other clone, named DB1, encoded a novel protein of 516 amino acids with six zinc finger motifs. DB1 mRNA was present in human tissues, ubiquitously. Two constitutive transcripts of 4.0 and 4.8 kb in length were present in Jurkat cells. Electrophoretic mobility shift assay, with specific antibodies, showed that DB1 constitutively binds to this region whereas EGR1 binds in a T-cell activation-dependent manner. Overexpression of DB1 in Jurkat cells had no detectable effect on the transcription activity of the IL-3 promoter, in a transient-transfection assay. EGR1 and EGR2 increased IL-3 promoter activity when the transfected cells were stimulated with phorbol-12-myristate-13-acetate and A23187. When DB1 was cotransfected with a Tax expression vector, transcription activity of the IL-3 promoter induced by Tax was significantly increased, while EGR1 and EGR2 were without effect. These results suggest that EGR1 has a role in inducible transcription of the IL-3 gene, while DB1 sustains basal transcriptional activity and also cooperates with Tax to activate the IL-3 promoter.

Calcineurin-dependent nuclear translocation of a murine transcription factor NFATx: molecular cloning and functional characterization.

Members of the nuclear factor of activated T cells (NFAT) are involved in the induction of a number of cytokine genes. We report here cDNA cloning and chromosomal localization of a murine homologue of human NFATx, designated as mNFATx1, and its splicing variants mNFATx2 and m delta NFATx. Northern blot analysis showed mNFATx1 to be predominantly expressed in the thymus. mNFATx1, but not m delta NFATx, produced in COS-7 cells, bound to all NFAT-binding sites of the interleukin (IL)-2 and IL-4 promoters tested. Immunofluorescence assay showed that both mNFATx1 and m delta NFATx introduced into COS-7 cells localized predominantly to the cytoplasm, but did translocate to the nucleus, either by cotransfection with an active form of calcineurin or wild-type calcineurin followed by stimulation with calcium ionophore. Translocation of mNFATx1 correlated well with activation of the murine IL-2 promoter; mNFATx1 translocated under conditions described above, in combination with phorbol 12-myristate 13-acetate, activated the transiently transfected murine IL-2 promoter. Thus, nuclear-translocated mNFATx1 is involved in activation of the IL-2 promoter. These results provide the first evidence for the requirement of calcineurin in the control of mNFATx imported from the cytoplasm to the nucleus and implies that mNFATx may possibly be a substrate of calcineurin in vivo.

Schizophrenia-like phenotypes in mice with NMDA receptor ablation in intralaminar thalamic nucleus cells and gene therapy-based reversal in adults.

In understanding the mechanism of schizophrenia pathogenesis, a significant finding is that drug abuse of phencyclidine or its analog ketamine causes symptoms similar to schizophrenia. Such drug effects are triggered even by administration at post-adolescent stages. Both drugs are N-methyl-d-aspartate receptor (NMDAR) antagonists, leading to a major hypothesis that glutamate hypofunction underlies schizophrenia pathogenesis. The precise region that depends on NMDAR function, however, is unclear. Here, we developed a mouse strain in which NMDARs in the intralaminar thalamic nuclei (ILN) were selectively disrupted. The mutant mice exhibited various schizophrenia-like phenotypes, including deficits in working memory, long-term spatial memory, and attention, as well as impulsivity, impaired prepulse inhibition, hyperlocomotion and hyperarousal. The electroencephalography analysis revealed that the mutant mice had a significantly reduced power in a wide range of frequencies including the alpha, beta and gamma bands, both during wake and rapid eye movement (REM) sleep, and a modest decrease of gamma power during non-REM sleep. Notably, restoring NMDARs in the adult ILN rescued some of the behavioral abnormalities. These findings suggest that NMDAR dysfunction in the ILN contributes to the pathophysiology of schizophrenia-related disorders. Furthermore, the reversal of inherent schizophrenia-like phenotypes in the adult mutant mice supports that ILN is a potential target site for a therapeutic strategy.

Late Graft Rejection in Association With T-Large Granular Lymphocyte Expansion of Recipient Origin After Human Leukocyte Antigen-Haploidentical Stem Cell Transplantation: A Case Report.

BACKGROUND: Large granular lymphocyte (LGL) expansion occasionally occurs after allogeneic stem cell transplantation (allo-SCT), and is thought to be a good prognostic sign that is associated with a lower relapse rate. However, there have been no reports of late graft failure (LGF) due to graft rejection in association with oligoclonal LGL expansion. We herein report a case of LGF associated with the transient expansion of recipient-derived T-LGL after allo-SCT. CASE REPORT: A 65-year-old man underwent peripheral blood stem cell transplantation (PBSCT) from his human leukocyte antigen (HLA)-haploidentical son for the treatment of acute myeloid leukemia, which had evolved from a myelodysplastic syndrome (MDS). Neutrophil engraftment occurred on day 20. A chimerism analysis on day 29 showed both granulocytes and mononuclear cells in the peripheral blood to be completely of donor origin. However, his neutrophil count gradually decreased and a chimerism analysis on day 61 showed that 84% of the patient's T cells were of recipient origin while the granulocytes were 100% donor-derived. His LGLs rapidly increased to 4.01 × 109/L on day 113 and decreased thereafter. The percentage of donor cells in his granulocytes gradually decreased, and the patient's leukocytes were completely replaced by recipient cells on day 177. CONCLUSIONS: The clinical course suggests that the expansion of recipient-derived T-LGLs after allo-SCT can be a sign of graft rejection. Early intervention may be needed if the LGL expansion is recipient-derived.