Plasma brain natriuretic peptide and the evaluation of volume overload in infants and children with congenital heart disease

This study was designed to explore whether it was possible to evaluate the severity of VSD, PDA, and ASD by measuring brain natriuretic peptide (BNP) levels. We also investigated normal BNP levels in children to provide a baseline for our study. We measured BNP levels in 253 normal children, including 11 normal neonates, and in 91 VSD patients, 29 PDA patients, and 34 ASD patients. BNP levels showed no age-related differences in normal children (the mean value: 5.3 +/- 3.8 pg/ml). In the healthy neonates, BNP levels rose from 10.4 +/- 11.9 pg/ml in cord blood to 118.8 +/- 83.2 pg/ml on day 0, then fell to 15.3 +/- 7.8 pg/ml by day 7. In VSD and PDA patients, BNP levels correlated significantly with Qp/Qs, LVEDV, and peak RVP/LVP. In ASD patients, BNP levels correlated with Qp/Qs and RVEDV. Especially, in VSD patients, as an index corresponding to 1.5-2.0 of the Qp/Qs ratio, BNP levels of 20-35 pg/ml were found to be best with regard to both sensitivity and specificity. In the healthy neonates, BNP levels changed rapidly after birth. In VSD, PDA, and ASD patients, BNP levels were well-correlated with the severity of the disease. Especially, in VSD patients, it that appears BNP levels may be useful in evaluating surgical indications, with 20-35 pg/ml levels being the appropriate cut-off value.     

Identification and characterization of temperature-sensitive mild mutations in three Japanese patients with nonsevere forms of very-long-chain acyl-CoA dehydrogenase deficiency

Very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is clinically classified into severe, intermediate, and myopathic forms. We identified mutations in three unrelated Japanese patients with VLCAD deficiency: two with the myopathic form and one with the intermediate form, all compound heterozygotes of K264E/M437V, A416T/1798delA, and P89S/IVS16-3delAA, respectively. We characterized four missense mutations, K264E, M437V, A416T, and P89S, by transisent expression analysis, using SV40-transformed fibroblasts derived from a VLCAD-null patient, as recipient cells. In transient expression of the wild-type VLCAD cDNA, VLCAD activity at 30 degrees C was higher than at 37 degrees C. Moreover, this temperature-sensitive character is more evident in all the mutant proteins tested than in wild type. Based on characterization of the five missense mutations identified in four Japanese patients, including data on one patient with the myopathic form previously reported, patients with the nonsevere forms (intermediate or myopathic forms) have missense mutations with residual activities in at least one allele. Expression analysis at 30 degrees C may be more useful for evaluating these missense mutations, compared with that at 37 degrees C.     

Induction of biologically active antineutrophil cytoplasmic antibodies by immunization with human apoptotic polymorphonuclear leukocytes

Translocation of intracellular components to the cell surface during the priming or apoptosis of polymorphonuclear leukocytes (PMN) is an important mechanism for interaction of antineutrophil cytoplasmic antibodies (ANCA) with these antigens. To test the capacity of apoptotic PMN to trigger production of ANCA, six groups of mice were immunized with either live or apoptotic lymphocytes, or with live, apoptotic, formalin-fixed, or lysed PMN. Mice immunized with both live and apoptotic neutrophils developed high titers of antibodies which gave a granular cytoplasmic immunofluorescent pattern. These antibodies were specific for lactoferrin and myeloperoxidase. Following a second intravenous infusion of apoptotic PMNs, mice developed anti-PR3 antibodies. Vasculitis lesions were not found in mice which developed ANCA. The ANCA-containing IgG fraction induced superoxide production by human PMNs. These results support the hypothesis that neutrophil-specific antigens presented on the cell membranes of apoptotic PMN may induce ANCA in the proper conditions.     

Differential expression of PD-L1 and PD-L2, ligands for an inhibitory receptor PD-1, in the cells of lymphohematopoietic tissues

PD-1 is a member of the immunoglobulin superfamily expressed on immune cells, including T and B cells, and is involved in the delivery of inhibitory signal upon engagement of its ligands, PD-L1 and PD-L2. While the expression profile of PD-1 has been well documented, the analysis of PD-L1 and PD-L2 distributions on a protein basis has not been carried out because of the lack of available monoclonal antibodies specific for the molecules. In this study, we established two monoclonal antibodies, 1-111A and 122, specific for murine PD-L1 and PD-L2, respectively, and examined their expression profiles. Based on flow cytometric analyses, the expression of PD-L1 was detected in a variety of lymphohematopoietic cell types, including a minor proportion of T and B cells in the spleen, majority of pre-B cells and myeloid cells in bone marrow and subsets of thymocytes, while the expression of PD-L2 was not observed in the lymphohematopoietic cells at all. Notably, a significant proportion of the most immature lineage-marker-negative and c-Kit-positive bone marrow cells containing stem cells did express PD-L1. Following mitogenic stimulation, essentially all lymphocytes expressed PD-L1. Furthermore, a variety of leukemic lines also expressed PD-L1, while none of them did PD-L2. Thus, present results demonstrate the distinct expression patterns of PD-L1 and PD-L2 with the cells of lymphohematopoietic tissues exclusively expressing the former.     

Distinct progression pathways involving the dysfunction of DUSP6/MKP-3 in pancreatic intraepithelial neoplasia and intraductal papillary-mucinous neoplasms of the pancreas.

DUSP6/MKP-3 is identified as a candidate tumor suppressor gene for pancreatic cancer. The aim of this study was to elucidate the roles of DUSP6 in the pancreatic carcinogenesis through the pancreatic intraepithelial neoplasia and/or intraductal papillary-mucinous neoplasms, both of which are considered to be precursor lesions of invasive carcinoma of the pancreas, by comparing with involvements of other major tumor suppressive pathways. Expressions of DUSP6, CDKN2A, TP53, and SMAD4 were investigated by immunohistochemistry in a total of 206 lesions of dysplastic ductal precursors and carcinomas retrieved from 52 pancreata with invasive ductal carcinomas and 51 of those with intraductal papillary-mucinous neoplasms. The intensity of staining was evaluated in lesions at different atypical grades and statistically compared among them. Mutations of KRAS2 were analyzed by methods of the allele-specific oligonucleotide hybridization and nucleotide sequencing. In pancreata with invasive ductal carcinomas, expressions of DUSP6 were abrogated exclusively in the invasive carcinoma cells in contrast to its fairly preserved expressions in pancreatic intraepithelial neoplasia. In pancreata with intraductal papillary-mucinous neoplasms, abrogated expressions of DUSP6 were observed in a relatively small fraction of intraductal adenoma/borderlines and intraductal carcinomas. Most of the intraductal adenoma/borderline lesions with abrogation of DUSP6 harbored mutations of KRAS2. None of the molecules was associated with each other in any grade of lesions. Morphological variations of papillae of the intraductal papillary-mucinous neoplasms were evaluated and analyzed for their associations with abrogations of the molecules, which resulted in finding of no significant associations. Our results suggest that the abrogation of DUSP6 is associated exclusively with progression from pancreatic intraepithelial neoplasia to the invasive ductal carcinoma while it is potentially associated with initiation of intraductal papillary-mucinous neoplasms with mutated KRAS2, which is independent of other major tumor suppressive pathways in both types of neoplasms.     

Dietary nucleic acid and intestinal microbiota synergistically promote a shift in the Th1/Th2 balance toward Th1-skewed immunity

BACKGROUND: Intestinal microbiota are known to play an important role in the establishment of oral tolerance, thereby protecting the organism from food allergies. Dietary intake of nucleic acid (NA) is also reported to have such an anti-allergic effect; however, one unsolved question is whether or not dietary NA would act through a process of toll-like receptor 9 signaling activated by DNA containing a CpG motif, a well-known sequence leading to immunostimulatory activity. In this study, we focused on the question of whether the addition of dietary NA lacking CpG motifs would allow continued modulation of the Th1/Th2 balance. METHODS: Germ free (GF) and Bifidobacterium-infantis-monoassociated BALB/c mice were maintained on either an NA-free casein diet or on an NA-supplemented casein diet for 4 weeks. Thereafter, both the in vivo anti-casein antibody levels and in vitro splenocyte cytokine secretion pattern were evaluated. RESULTS: Feeding with a casein diet elicited a substantial increase in the serum anti-casein-specific IgG1, IgG2a, and IgE levels of GF mice fed the NA free-diet. The in vitro cytokine production profile showed that enhanced IL-4 production in the GF mice fed the NA free-diet was markedly reduced by the supplementation with dietary NA in both the GF and B.-infantis-monoassociated mice. In addition, IFN-gamma secretion increased in the B.-infantis-reconstituted mice fed the diet containing NA. CONCLUSIONS: These results suggest that dietary intake of NA devoid of CpG motifs may prevent the development of allergies via acceleration of Th1-dominant immunity.     

Use of glycopeptidolipid core antigen for serodiagnosis of mycobacterium avium complex pulmonary disease in immunocompetent patients

We report the development of a serodiagnostic method for Mycobacterium avium complex (MAC) disease with an enzyme immunoassay (EIA) with the MAC-specific glycopeptidolipid (GPL) core as the antigen. In this study, we confirmed by EIA that the GPL core antibody was in the sera of immunocompetent patients with MAC disease. The EIA for quantifying the GPL core antibody was evaluated as a clinical tool for serodiagnosis of pulmonary MAC disease. A significant increase in GPL core antibodies (immunoglobulins G, A, and M) was detected in sera of patients with MAC pulmonary diseases when they were compared to patients who were colonized with MAC, patients with Mycobacterium kansasii disease or tuberculosis, and healthy subjects. The sensitivities and specificities of the GPL core-based EIA for diagnosis of MAC pulmonary disease were 72.6% and 92.2%, respectively, for IgG, 92.5% and 95.1%, respectively, for IgA, and 78.3% and 91.0%, respectively, for IgM. The best sensitivity and specificity were obtained by measuring immunoglobulin A antibodies against GPL core antigen. The level of GPL core antibodies reflected disease activity, since it decreased in cured MAC patients who had responded to chemotherapy. Measurement of serum antibodies against GPL core is useful for both diagnosis and assessment of disease activity in MAC disease of the lung.