Lead induced increase of blood pressure in female lead workers.

AIMS: Although lead exposure has, in the absence of mathematical modelling, been believed to elevate blood pressure in females, it is necessary to clarify the relation between lead and blood pressure by eliminating confounding factors in the analysis. METHODS: Blood lead was measured in 193 female workers, including 123 lead exposed workers. Possible confounding factors were controlled by multiple regression analyses. RESULTS AND CONCLUSIONS: Blood lead above 40 micro g/dl was found to be the most potent factor for elevating systolic/diastolic blood pressure. Aging, urine protein, and plasma triglyceride also contributed to systolic/diastolic/pulse pressure increase, but hypertensive heredity did not. Data suggested that lead induced changes in lipoprotein metabolism may play an important role in the lead induced blood pressure increase in female workers.

Increased secretion of IL-18 in vitro by peripheral blood mononuclear cells of patients with bronchial asthma and atopic dermatitis.

This study was performed to determine whether or not IL-18, formerly called IFN-gamma-inducing factor, is involved in the pathogeneses of allergic disorders. Peripheral blood mononuclear cells (PBMC) were obtained from patients with allergic bronchial asthma (BA), patients with atopic dermatitis (AD) and controls who did not have any allergic disease, and then cultured with lipopolysaccharide (LPS) or phytohaemagglutinin (PHA). The concentrations of IL-18, IFN-gamma and IL-13 in supernatant fluids were determined by enzymatic immunoassaying, and the expression of IFN-gamma messenger (m) RNA in the cells was measured by colorimetric microplate assaying. IL-18 secretion in the BA patients (geometric mean (gm) = 189 pg/ml) and AD patients (gm = 172 pg/ml) was significantly higher than that in non-allergic controls (gm = 118 pg/ml). In contrast, IFN-gamma secretion in the BA patients (gm = 7.3 IU/ml) and AD patients (gm = 6.8 IU/ml) was significantly lower than that in non-allergic controls (gm = 20.7 IU/ml). The amounts of IL-13 in supernatant fluids and IFN-gamma mRNA in cells were not statistically different among the BA patients, AD patients and non-allergic controls. The possible involvement of IL-18 in allergic disorders is discussed.

PU.1 is involved in the regulation of B lineage-associated and developmental stage-dependent expression of the germinal center-associated DNA primase GANP.

Germinal center-associated DNA primase (GANP) associated with MCM3 of the DNA replication complex is up-regulated selectively in germinal center B cells. We studied promoter activity of the 5' region involved in the developmental stage-dependent expression in B lineage cells by luciferase reporter assay. Selective regulation of ganp expression was observed in the -737-bp promoter region in B and plasma cell lines but was significantly low in pre-B and T cell lines. The deletion constructs displayed a gap decrease after shortening the region from -134 to -108 bp. Further narrowing suggested the involvement of the PU.1 consensus sequence at -126 bp by electrophoretic mobility shift assay. The protein component PU.1 complex is not inhibited with mutated probes at the consensus site but is inhibited with the known PU.1 probe of CD72 and with anti-PU.1 antibody. Moreover, introduction of PU.1 cDNA enhanced the reporter gene activity in a dose-dependent manner in B cells, whereas the reporter construct with the mutated PU.1 site did not respond. Anti-CD40 stimulation induced the reporter activity with a 100% increase, which is not observed with the PU.1-mutated reporter construct. These results demonstrate that the germinal center-associated DNA primase expression is partly regulated by the transcription factor PU.1 expressed in B lineage cells.

Treatment of developmental dysplasia of the hip with the Pavlik harness: factors for predicting unsuccessful reduction.

To evaluate the risk factors for unsuccessful reduction, we reviewed 100 hips with developmental dysplasia of the hip treated with the Pavlik harness. We divided them into a successful reduction group (reduced without avascular necrosis) and an unsuccessful reduction group (reduced followed by avascular necrosis or unreduced). We compared Yamamuro's distance A, the acetabular angle and age at initial application of the Pavlik harness between the two groups. Our data suggest that a Yamamuro's distance A of 7 mm or smaller, an acetabular angle of 36 degrees or greater, and an age at initial application of 4 months or older are risk factors.

Human CC chemokine liver-expressed chemokine/CCL16 is a functional ligand for CCR1, CCR2 and CCR5, and constitutively expressed by hepatocytes.

Liver-expressed chemokine (LEC)/CCL16 is a human CC chemokine selectively expressed in the liver. Here, we investigated its receptor usage by calcium mobilization and chemotactic assays using mouse L1.2 pre-B cell lines stably expressing a panel of 12 human chemokine receptors. At relatively high concentrations, LEC induced calcium mobilization and chemotaxis via CCR1 and CCR2. LEC also induced calcium mobilization, but marginal chemotaxis via CCR5. Consistently, LEC was found to bind to CCR1, CCR2 and CCR5 with relatively low affinities. The binding of LEC to CCR8 was much less significant. In spite of its binding to CCR5, LEC was unable to inhibit infection of an R5-type HIV-1 to activated human peripheral blood mononuclear cells even at high concentrations. In human liver sections, hepatocytes were strongly stained by anti-LEC antibody. HepG2, a human hepatocarcinoma cell line, was found to constitutively express LEC. LEC was also present in the plasma samples from healthy adult donors at relatively high concentrations (0.3--4 nM). Taken together, LEC is a new low-affinity functional ligand for CCR1, CCR2 and CCR5, and is constitutively expressed by liver parenchymal cells. The presence of LEC in normal plasma at relatively high concentrations may modulate inflammatory responses.

Chemokines in immunity.

Chemokines are a superfamily of small, heparin-binding cytokines that induce directed migration of various types of leukocytes through interactions with a group of seven-transmembrane G protein-coupled receptors. At present, over 40 members have been identified in humans. Until a few years ago, chemokines were mainly known as potent attractants for leukocytes such as neutrophils and monocytes, and were thus mostly regarded as the mediators of acute and chronic inflammatory responses. They had highly complex ligand-receptor relationships and their genes were regularly mapped on chromosomes 4 and 17 in humans. Recently, novel chemokines have been identified in rapid succession, mostly through application of bioinformatics on expressed sequence tag databases. A number of surprises have followed the identification of novel chemokines. They are constitutively expressed in lymphoid and other tissues with individually characteristic patterns. Most of them turned out to be highly specific for lymphocytes and dendritic cells. They have much simpler ligand-receptor relationships, and their genes are mapped to chromosomal loci different from the traditional chemokine gene clusters. Thus, the emerging chemokines are functionally and genetically quite different from the classical "inflammatory chemokines" and may be classified as "immune (system) chemokines" because of their profound importance in the genesis, homeostasis and function of the immune system. The emergence of immune chemokines has brought about a great deal of impact on the current immunological research, leading us to a better understanding on the fine traffic regulation of lymphocytes and dendritic cells. The immune chemokines and their receptors are also likely to be important future targets for therapeutic intervention of our immune responses.

A novel recombinant adeno-associated virus vaccine induces a long-term humoral immune response to human immunodeficiency virus.

Recombinant adeno-associated virus (AAV) has attracted tremendous interest as a promising vector for gene delivery. In this study we have developed an HIV-1 vaccine, using an AAV vector expressing HIV-1 env, tat, and rev genes (AAV-HIV vector). A single injection of the AAV-HIV vector induced strong production of HIV-1-specific serum IgG and fecal secretory IgA antibodies as well as MHC class I-restricted CTL activity in BALB/c mice. The titer of HIV-1-specific serum IgG remained stable for 10 months. When AAV-HIV vector was coadministered with AAV-IL2 vector, the HIV-specific cell-mediated immunity (CMI) was significantly enhanced. Boosting with AAV-HIV vector strongly enhanced the humoral response. Furthermore, the mouse antisera neutralized an HIV-1 homologous strain, and BALB/c mice immunized via the intranasal route with an AAV vector expressing the influenza virus hemagglutinin (HA) gene showed protective immunity against homologous influenza virus challenge. These results demonstrate that AAV-HIV vector immunization may provide a novel and promising HIV vaccination strategy.

Organization of the chemokine genes in the human and mouse major clusters of CC and CXC chemokines: diversification between the two species.

Chemokines are a family of small cytokines that play essential roles in the directed migration of various types of leukocytes. Based on the arrangement of the conserved cysteine residues, they are classified into two major subfamilies, CXC and CC, and two minor subfamilies, C and CX3C. So far, more than 40 members of this family have been identified in humans. Strikingly, the majority of CXC chemokine genes and that of CC chemokine genes are closely clustered at chromosomes 4q12-21 and 17q11.2, respectively. Similarly, the mouse major CXC and CC chemokine gene clusters are located on chromosomes 5 and 11, respectively. In order to understand the evolutionary processes that generated large numbers of CXC and CC chemokine genes in the respective chromosomal sites, we have constructed BAC and YAC contigs covering the human and mouse major clusters of CXC and CC chemokine genes. The results reveal that the organizations of CXC and CC chemokine genes in the major clusters are quite diverged between the two species most probably due to very recent gene duplications and rearrangements. Our results provide an important insight into the evolutionary processes that generated the major chemokine gene clusters and also valuable information in assigning the orthologues between human and mouse major cluster chemokines.

Induction of acyl-coenzyme A:cholesterol acyltransferase-1 by 1,25-dihydroxyvitamin D(3) or 9-cis-retinoic acid in undifferentiated THP-1 cells.

We have previously shown that acyl-coenzyme A: cholesterol acyltransferase-1 (ACAT-1) protein content increases significantly during the human monocyte-macrophage differentiation process. To gain further insight, we used undifferentiated human monocytic THP-1 cells as a model system with which to examine whether ACAT-1 mRNA and protein content can be increased by treating cells with 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] or with 9-cis-retinoic acid (9-cis-RA), two agents known to upregulate the expression of various genes during the monocyte-macrophage differentiation process. Immunoblot analysis with anti-human ACAT-1 antibodies revealed that ACAT-1 protein was increased by 2.6-fold, using 1,25-(OH)(2)D(3) at a physiological concentration (100 pM). ACAT-1 protein was also increased when using 9-cis-RA, but only at relatively high concentrations (0.1;-1 microM). Northern blot analysis revealed that among the four ACAT-1 mRNA transcripts (2.8, 3.6, 4.2, and 7.0 kb) examined, only the 2.8- and 3.6-kb transcripts were selectively increased. On the basis of enzyme assays in vitro, ACAT activity was increased 3.0-fold by using 100 nM 1,25-(OH)(2)D(3), and 1.8-fold by using 1 microM 9-cis-RA. Together, our results suggest that 1,25-(OH)(3) participates in ACAT-1 gene expression during the monocyte-macrophage differentiation process.

WECHE: a novel hematopoietic regulatory factor.

Previously, we described AGM-derived endothelial cell lines that either inhibited or permitted the development of erythroid or B cells. We utilized a differential gene expression method to isolate a chemokine, termed WECHE, from one of these cell lines. WECHE inhibited the formation of erythroid cells but had no effect on either myeloid or B cell formation. WECHE repressed BFU-E development from either mouse fetal liver or bone marrow progenitor cells but had no effect on colony formation induced by IL-3 or IL-7. WECHE reduced HPP-CFC production from fetal liver-derived stem cells. WECHE hindered the growth of yolk sac-derived endothelial cells. WECHE was also chemotactic for bone marrow cells. Thus, WECHE is a novel chemokine that regulates hematopoietic differentiation.

Molecular cloning of a novel CC chemokine, interleukin-11 receptor alpha-locus chemokine (ILC), which is located on chromosome 9p13 and a potential homologue of a CC chemokine encoded by molluscum contagiosum virus.

Molluscum contagiosum virus (MCV) encodes a CC chemokine MC148R which is likely to have been acquired from the host. By a homology search employing MC148R as a probe, we have identified a novel CC chemokine whose gene exists next to the IL-11 receptor alpha (IL-11Ralpha) gene in both humans and mice. Thus, this chemokine maps to chromosome 9p13 in humans where IL-11Ralpha has been assigned. We term this novel chemokine IL-11Ralpha-locus chemokine (ILC). ILC has the highest homology to MC148R among the known human CC chemokines. Furthermore, ILC is strongly and selectively expressed in the skin where infection of MCV also takes place. Thus, ILC is likely to be the original chemokine of MC148R.

Molecular cloning of a novel human CC chemokine (Eotaxin-3) that is a functional ligand of CC chemokine receptor 3.

Previously, we mapped the novel CC chemokine myeloid progenitor inhibitory factor 2 (MPIF-2)/eotaxin-2 to chromosome 7q11.23 (Nomiyama, H., Osborne, L. R., Imai, T., Kusuda, J., Miura, R., Tsui, L.-C., and Yoshie, O. (1998) Genomics 49, 339-340). Since chemokine genes tend to be clustered, unknown chemokines may be present in the vicinity of those mapped to new chromosomal loci. Prompted by this hypothesis, we analyzed the genomic region containing the gene for MPIF-2/eotaxin-2 (SCYA24) and have identified a novel CC chemokine termed eotaxin-3. The genes for MPIF-2/eotaxin-2 (SCYA24) and eotaxin-3 (SCYA26) are localized within a region of approximately 40 kilobases. By Northern blot analysis, eotaxin-3 mRNA was constitutively expressed in the heart and ovary. We have generated recombinant eotaxin-3 in a baculovirus expression system. Eotaxin-3 induced transient calcium mobilization specifically in CC chemokine receptor 3 (CCR3)-expressing L1.2 cells with an EC(50) of 3 nM. Eotaxin-3 competed the binding of (125)I-eotaxin to CCR3-expressing L1.2 cells with an IC(50) of 13 nM. Eotaxin-3 was chemotactic for normal peripheral blood eosinophils and basophils at high concentrations. Collectively, eotaxin-3 is yet another functional ligand for CCR3. The potency of eotaxin-3 as a CCR3 ligand seems, however, to be approximately 10-fold less than that of eotaxin. Identification of eotaxin-3 will further promote our understanding of the control of eosinophil trafficking and other CCR3-mediated biological phenomena. The strategy used in this study may also be applicable to identification of other unknown chemokine genes.

Erythrocyte delta-aminolevulinic acid dehydratase genotype and other mechanisms affecting workers' susceptibility to lead.

In this study, the role of delta-aminolevulinic acid dehydratase (ALAD) variants in lead susceptibility was examined. The study subjects comprised 223 male workers, and the relationship between their blood lead level and erythrocyte ALAD activity or plasma/urine delta-aminolevulinic acid level was studied. Leukocyte specimens from 11 workers, whose erythrocyte ALAD activities were as low as one-fifth that of the other normal workers, were subjected to analyses of their ALAD and ALAD alleles. Further, the entire exon fragment of the ALAD gene was analyzed by polymerase chain reaction, and the reaction product was used as a target for direct DNA sequencing. Genomic DNA analysis revealed that all 11 workers had the ALAD allele, whereas the entire ALAD gene analysis failed to indicate other variants, except for the Rsa I site. The depletion in erythrocyte ALAD activity was not found to be caused by the ALAD allele.

Mechanism of hepatorenal syndrome in rats of Long-Evans Cinnamon strain, an animal model of fulminant Wilson's disease.

Rats of Long-Evans Cinnamon (LEC) strain were used as a hepatorenal syndrome model of fulminant Wilson's disease. Copper levels in the kidneys increased markedly from 16 to 126 microg Cu/g from 12 to 16 weeks, and remained at the same level at 16 and 19 weeks when the rats suffered from severe renal dysfunction and also at 20 weeks in some other normal rats. The above findings imply that the renal dysfunction may have been induced independently of the copper level in the kidneys. The present study suggested the following mechanism: immediately after copper-induced hepatic dysfunction, plasma copper-metallothionein (CuMT), which was released from the liver, became elevated. The elevation was closely related to the increases in alkaline phosphatase, glucose and amino acids, all in the urine. The above findings suggest that plasma CuMT, which was released from the liver into the blood upon copper-induced hepatic dysfunction, was subsequently filtered at the glomeruli due to its smaller molecular weight, and then caused dysfunction of the brush border membrane of the renal proximal tubules probably after splitting into radical copper and amino acids in acidic vesicles close to the membrane. The critical concentration of plasma CuMT required to induce renal dysfunction was estimated as 1 microg Cu/l.

Genomic organization of the genes for human and mouse CC chemokine LEC.

Liver-expressed chemokine (LEC) is a CC chemokine that is selectively expressed in the liver. We report here the structures of the human and mouse genes for LEC. The human LEC gene (SCYA16) was isolated from a bacterial artificial chromosome (BAC) clone that also contained CC chemokine genes for MPIF-1/Ckbeta8, HCC-2/Lkn-1/MIP-5/MIP-1delta, and HCC-1. The LEC gene is approximately 5.0 kb in length and has a three-exon and two-intron structure common to most CC chemokine genes. However, the promoter region is devoid of a typical TATA box, and transcription initiates at multiple sites. The gene for CC chemokine HCC-1, which is most similar to LEC, is located approximately 2.2 kb upstream from the 5' end of the LEC gene in a head-to-tail fashion. The mouse DNA fragment that hybridized with the human LEC cDNA was isolated from a BAC clone that also contained the CC chemokine genes for C10, MRP-2/CCF18/MIP-1gamma, and RANTES. Sequence analysis revealed that the isolated gene does not encode a functional chemokine because of deletions, insertions, and base changes. Southern blot analysis revealed that the sequence isolated from the BAC clone was the only one hybridizing with human LEC cDNA in the mouse genome. Therefore, mice may have only an LEC pseudogene.

Organization of the chemokine gene cluster on human chromosome 17q11.2 containing the genes for CC chemokine MPIF-1, HCC-2, HCC-1, LEC, and RANTES.

To understand the organization of the human CC chemokine gene cluster on chromosome 17q11.2, we determined the nucleotide sequence of a region 181 kb long containing five CC chemokine genes, MPIF-1 (SCYA23), HCC-2 (SCYA15), HCC-1 (SCYA14), LEC (SCYA16), and RANTES (SCYA5), by the random shot-gun method. The four CC chemokine genes, MPIF-1, HCC-2, HCC-1, and LEC, are clustered within a region 40 kb long, whereas the RANTES gene is located approximately 10 kb apart from the four chemokine gene minicluster. These chemokine genes are arranged in the same orientation, and their sizes are relatively long, 3.1 (HCC-1)-8.8 kb (RANTES) when compared with other CC chemokine genes, such as MIP-1alpha/LD78alpha (SCYA3) (1.9 kb) and MCP-1 (SCYA2) (1.5 kb). In contrast to most other human CC chemokine genes that consist of three exons, the MPIF-1 and HCC-2 genes, separated by 12 kb, have four exons. When the nucleotide sequences of the MPIF-1 and HCC-2 genes are compared, they are well conserved, including introns and flanking sequences, except for the middle region of the long first intron, indicating that they have been generated recently in evolutionary terms by duplication. In addition to the CC chemokine genes, more than 30 exons are identified in the sequenced region by similarity search against expressed sequence tags (ESTs) and also by the gene prediction program GenScan. This indicates that the chemokine cluster sequenced in this study is a gene-rich region in the human genome.

Selective expression of liver and activation-regulated chemokine (LARC) in intestinal epithelium in mice and humans.

The liver and activation-regulated chemokine (LARC), also termed MIP-3alpha and Exodus, is a novel human CC chemokine with a selective chemotactic activity for lymphocytes and dendritic cells. Here we describe genomic and cDNA clones encoding the murine orthologue of LARC (mLARC). The gene consists of four exons and three introns. The 5'-noncoding region of about 400 bp contains typical TATA and CAAT boxes but no other potential regulatory elements so far described. The cDNA encodes a CC chemokine of 97 amino acid residues with the highest homology to human LARC (64% amino acid identity). The 3'-noncoding region contains as many as five potential mRNA destabilization signals. mLARC was strongly and transiently induced in the murine monocytoid cell line J774 by lipopolysaccharide (LPS) but not by cytokines such as TNF-alpha, IFN-gamma, IL-1beta or IL-4. In normal mice, mLARC mRNA was expressed selectively in intestinal tissues such as small intestine and colon. Upon treatment with LPS, mLARC expression was enhanced in intestinal tissues and induced in some lymphoid tissues such as lymph nodes. Because of alternative splicing, there are two types of transcripts encoding mLARC and its variant mLARCvar with and without an N-terminal alanine in the mature protein, respectively. Both types of transcripts appeared to be expressed in various mouse tissues. In situ hybridization revealed that epithelial cells of intestinal tissues, especially those lining lymphoid follicles, expressed mLARC. Localization of LARC mRNA in epithelial cells was also demonstrated in a human appendix. Furthermore, mLARC was efficiently chemotactic for cells such as gammadelta type T cells in intestinal epithelium and naive B cells in Peyer's patches. Thus, in both humans and mice, LARC may be physiologically involved in formation and function of the mucosal lymphoid tissues by attracting lymphocytes and dendritic cells toward epithelial cells.

Chemokine PARC gene (SCYA18) generated by fusion of two MIP-1alpha/LD78alpha-like genes.

Two loci in the human genome, chromosomes 4q12-q21 and 17q11.2, contain clusters of CXC and CC chemokine subfamily genes, respectively. Since mice appear to contain fewer chemokine genes than humans, numerous gene duplications might have occurred in each locus of the human genome. Here we describe the genomic organization of the human pulmonary and activation-regulated CC chemokine (PARC), also known as DC-CK1 and AMAC-1. Despite high sequence similarity to a CC chemokine macrophage inflammatory protein-1alpha (MIP-1alpha)/LD78alpha, PARC is chemotactic for lymphocytes and not for monocytes and does not share its receptor with MIP-1alpha. Analyses of the BAC clones containing the human PARC gene indicated that the gene is located most closely to MIP-1alpha (HGMW-approved symbol SCYA3) and MIP-1beta (HGMW-approved symbol SCYA4) on chromosome 17q11.2. Dot-plot comparison suggested that the PARC gene had been generated by fusion of two MIP-1alpha-like genes with deletion and selective usage of exons. Base changes accumulated before and after the fusion might have adapted the gene to a new function. Since there are variably duplicated copies of the MIP-1alpha gene called LD78beta (HGMW-approved symbol SCYA3L) in the vicinity of the MIP-1alpha gene, the locus surrounding the MIP-1alpha gene seems to be a "hot spring" that continuously produces new family genes. This evidence provides a new model, duplication and fusion, of the molecular basis for diversity within a gene family.