Association between mycobacterial genotypes and disease progression in Mycobacterium avium pulmonary infection.

BACKGROUND: Non-tuberculous mycobacterial lung disease, most commonly caused by Mycobacterium avium infection, tends to show variable disease progression, and significant disease predictors have not been adequately established. METHODS: Variable numbers of tandem repeats (VNTR) were evaluated in 16 mycobacterial interspersed repetitive unit (MIRU) loci from M avium isolates cultured from respiratory specimens obtained from 2005 to 2007. Specifically, the association between VNTR profiles and disease progression was assessed. RESULTS: Among the 37 subjects who provided positive respiratory cultures for M avium during the 2005-6 period, 15 subjects were treated within 10 months following a microbiological diagnosis of progressive M avium lung disease. Nine subjects underwent long-term follow-up (>24 months) without treatment for stable M avium lung disease. Based on a neighbour-joining cluster analysis used to classify M avium-positive subjects according to the VNTR profile, subjects with progressive versus stable lung disease were found to be grouped together in distinct clusters. Further analysis using logistic regression modelling showed that disease progression was significantly associated with the genetic distance of the M avium isolate from an appropriately selected reference (age-adjusted odds ratio 1.95; 95% confidence interval 1.16 to 3.30; p = 0.01 for the most significant model). A best-fit model could be used to predict the progression of M avium lung disease when subjects from the 2005-6 period were combined with those from 2007 (p = 0.003). CONCLUSION: Progressive lung disease due to M avium infection is associated with specific VNTR genotypes of M avium.

Recurrent pneumonia with mild hypogammaglobulinemia diagnosed as X-linked agammaglobulinemia in adults.

BACKGROUND: X-linked agammaglobulinemia (XLA) is a humoral immunodeficiency caused by disruption of the Bruton's tyrosine kinase (BTK) gene. Typical XLA patients suffer recurrent and severe bacterial infections in childhood. METHODS: Flow cytometric analysis of the peripheral monocytes using the anti-BTK antibody was used to characterize a 27 year old male patient with mild hypogammaglobulinemia (IgG, 635 mg/dl; IgM, 11 mg/dl; IgA, <5 mg/dl). He had suffered from frequent pneumonia since age 25 but had no history of frequent infections in his childhood or in adolescence. Sequencing of the BTK cDNA obtained from an Epstein-Barr virus-transformed B lymphoblastoid cell line derived from the bone marrow of the patient was performed to confirm a genetic defect. RESULTS: Flow cytometric analysis of cytoplasmic BTK protein in peripheral monocytes indicated that the patient presents a rare case of adult-onset XLA and that his mother is an XLA carrier. Sequencing of the BTK gene revealed a deletion of AG in the codon for Glu605 (AGT), resulting in an aberrant stop codon that truncates the BTK protein in its kinase domain. CONCLUSIONS: This case suggests that some XLA cases may remain undiagnosed because they only show mild hypogammaglobulinemia and they lack repeated infections in childhood. Flow cytometric analysis is a powerful method to screen these patients.

[Non-small-cell lung cancer (NSCLC) in an elderly patient--a case of squamous cell carcinoma successfully treated with chemotherapy using vinorelbine].

We report an elderly patient with squamous cell carcinoma who was successfully treated with chemotherapy using vinorelbine. A 76-year-old man was referred to our hospital for evaluation of a nodular shadow in the left lung. Chest CT scam showed a 3-cm tumor shadow in left S9 and a 1-cm small nodule in right S2. Transbronchial lung biopsy yielded a diagnosis of squamous cell carcinoma. The clinical stage was IV (cT2N2M1). The patient first underwent chemotherapy consisting of cisplatin (CDDP) 80 mg/m2 on day 1 and vinorelbine (VNB) 20 mg/m2 on days 1, 8, and 15, which generated tumor shrinkage of 48% as well as transient elevation of grade 1 in serum creatinine. The 2 cycles of chemotherapy using vinorelbine only (VNB 20 mg/m2 on days 1, 8, 15) produced a tumor reduction of 70% with grade-1 decrease of granulocytes. The low grade of toxicity enabled us to treat the patient in our outpatient office for the second cycle of the regimen. This case suggests that chemotherapy using low-dose vinorelbine might be suitable to treat elderly patients with NSCLC in outpatient settings.

Induction of antitumor immunity by combined immunogene therapy using IL-2 and IL-12 in low antigenic Lewis lung carcinoma.

Interleukin-2 (IL-2) and interleukin-12 (IL-12) are crucial cytokines that induce potent antitumor responses in a variety of animal cancer models. Although single gene transfer of either IL-2 or IL-12 exhibits limited antitumor effects, the combination of IL-2 and IL-12 has been shown to induce a stronger antitumor response and to cure tumor-bearing mice. To examine the conditions necessary for tumor rejection, we varied the local concentration of IL-2 and IL-12 by introducing these genes into Lewis lung carcinoma (LLC) cells via retroviral vectors and/or an adenoviral vector and evaluated the growth of inoculated LLC cells (5 x 10(5) cells). In contrast to the result when using a stepwise dose increase of IL-2 either without or with a fixed production of IL-12 (4-5 ng/5 x 10(5) cells/24 hours, insufficient for tumor rejection by itself), rejection of the tumor was achieved in 75% of the mice when the IL-12 secretion was combined with high and transient IL-2 production (42 ng/5 x 10(5) cells/24 hours) using additional adenoviral vector transduction (100 multiplicities of infection). An abundant infiltration of both CD4+ (47.4/mm2) and CD8+ (85.6/mm2) T cells was a characteristic finding in the dual gene-transfected LLC tumors. Importantly, consistent with the rejection of rechallenged parental cells, tumor-specific cytotoxic T lymphocytes were induced only from the splenocytes of mice inoculated with the dual gene-transduced LLC cells, suggesting the existence of protective antitumor memory. In addition, only vaccination of dual gene-transduced LLC cells inhibited the growth of pre-established LLC tumors. These results indicate that generation of a pivotal antitumor response likely depends on the synergistic combination and concentration of IL-2 and IL-12 in the local milieu by which tumor-specific immune memory is established.

Autologous high-killing cytotoxic T lymphocytes against human lung cancer are induced using interleukin (IL)-1beta, IL-2, IL-4, and IL-6: possible involvement of dendritic cells.

Although CTLs bear main immune responses in human tumors, stable CTL clones against human lung cancer have rarely been generated. Our previous study demonstrated efficient autologous CTL induction in human gastric cancer and glioblastoma by cytokine combination of interleukin (IL)-1beta (167 IU/ml), IL-2 (67 IU/ml), IL-4 (67 IU/ml), and IL-6 (134 IU/ml). In this study, we demonstrated successful induction of autologous stable CTLs in five of six patients with lung adenocarcinoma from mixed-lymphocyte tumor culture using this cytokine combination. All CTLs revealed potent and specific killing activity against autologous target cells (over 75% in CD8+ CTLs and over 50% in CD4+ CTLs at an E:T ratio of 10 for 24 h). Using a series of antibodies, CD8+ CTLs showed to recognize tumor-specific antigens of lung cancer cells through HLA class I. In the separate experiments, failure of CTL induction from monocyte-depleted peripheral blood mononuclear cells and appearance of cells with characteristics of dendritic cells from adherent peripheral blood mononuclear cells in the culture of the same concentration of IL-1beta, IL-4, and IL-6 indicated that CTLs can be efficiently generated by this cytokine combination via possible dendritic cell induction. This is the first study of an efficient and reproducible in vitro CTL induction against human lung cancer.

[Pulmonary thromboembolism associated with antiphospholipid syndrome in scleroderma].

A 51-year-old woman was referred to our hospital with dyspnea. Chest roentgenogram on admission showed dilation of the pulmonary arteries and hyperlucency in the lung fields. An ultrasonic cardiographic examination showed that the right atrium and ventricle were dilated. Pulmonary thromboembolism due to left popliteal vein thrombosis was diagnosed by perfusion scintigram of the lung, which showed multiple wedge-shaped defects, and by digital subtraction angiogram, which showed a filing defect in the left popliteal vein. Antiphospholipid syndrome was diagnosed after IgG anticardiolipin antibody was defected. Scleroderma was subsequently diagnosed because the patient exhibited Raynaud's phenomenon and proximal scleroderma. Although closely associated with lupus erythematosus and other lupus variants, antiphospholipid syndrome has not been recognized as a common complication of scleroderma. This is the first report of a patient with pulmonary thromboembolism associated with antiphospholipid syndrome and scleroderma.

Characterization of the complete genomic structure of human thromboxane synthase gene and functional analysis of its promoter.

Thromboxane A2 (TXA) is a potent vasoconstrictor and mediator of platelet aggregation. Thromboxane synthase (TXAS), which catalyzes the biosynthesis of TXA, is a member of the cytochrome P450 superfamily. We report here the complete genomic structure of the human TXAS gene. The gene contains 13 exons and is 193 kb long, the largest P450 gene ever isolated. The physical localization of the TXAS gene on the genetic map of human chromosome 7 was established. A major transcription start site is located at a motif similar to the initiator element where the transcription factor TFII-I binds. We have sequenced up to 1.6 kb of the 5'-flanking region of the TXAS gene and characterized the promoter activity in a human megakaryocyte cell line and endothelial cells. Our results demonstrated that the nucleotides -248/+137 relative to the major transcription start site conferred a full promoter activity of the TXAS gene. This region was regulated negatively by cis-elements located between -1562 and -248. Moreover, the regions outside -1562/+137 might control tissue-specific TXAS expression.

Alternate splicing of human thromboxane synthase mRNA.

Two species of human thromboxane synthase (TXS) cDNA, called TXS-I and -II, were previously isolated (K. Ohashi, K.-H. Ruan, R. J. Kulmacz, K. K. Wu, and L.-H. Wang, 1992, J. Biol. Chem. 267, 789-793). TXS-II differs from TXS-I by a 163-bp deletion near the 3'-end of the coding region. Both types of TXS mRNA have now been demonstrated to be present in various blood and lung cultured cells. Analysis of the exon-intron boundaries of TXS genomic DNA revealed that the two mRNAs are generated via alternate splicing: TXS-II is produced by skipping an entire 163-bp exon which encodes the polypeptide segment containing the heme-binding cysteine conserved among other P450s. The mechanism by which alternate splicing occurs is probably due to the presence of a more powerful potential as the 3' acceptor site in the intron following the 163-bp exon. When expressed in baculovirus system, recombinant TXS-I catalyzed the formation of thromboxane A2 and 12-hydroxyheptadecatrienoic acid (HHT), whereas recombinant TXS-II did not synthesize thromboxane A2 or HHT. Alternate splicing of TXS RNA transcript thus may provide a mechanism for limiting cellular biosynthesis of thromboxane A2.

Lunar base CELSS design and analysis.

This paper describes the conceptual development of a hybrid biological-physical/chemical (P/C) life support system model for a lunar outpost. It presents steps that lead to loop closure and determines mass flow characteristics for an inedible biomass enzyme reactor and an activated sludge bioreactor. Computer modeling techniques were used to determine that the cellulose reactor has the design capabilities to provide significant increases in the plant harvest index. Activated sludge was found to fit design demands for a small, continuous-flow, steady-state system. Systems analysis and component sizing for these two bioreactors and information regarding supporting bioregenerative and physical/chemical components are presented.

Characterization of the genomic structure, chromosomal location and promoter of human prostaglandin H synthase-2 gene.

Prostaglandin H synthase (PGHS) is the rate-limiting enzyme in the conversion of arachidonic acid to prostanoids. The human PGHS has two isoforms. PGHS-1 is a house keeping gene whereas PGHS-2 is an inducible gene. We reported here the isolation of the entire PGHS-2 gene and its 5'-flanking region from a human bacteriophage P1 genomic library. The gene containing 10 exons is 7.5 kb in length and located at chromosome 1. The transcriptional start site was mapped at 134 bases upstream from the ATG start codon. Nucleotide sequence of 1.8 kb promoter region contains a TATA box and a number of potential regulatory elements including CRE, NF-kappa B, Sp1 and AP2 sites. Studies of the promoter activity showed that the first 460 nucleotides of 5'-flanking region efficiently drove transcription of the luciferase reporter gene in human umbilical vein endothelial cells upon stimulation with phorbor ester.

Presence of functional cyclic AMP responsive element in the 3'-untranslated region of the human thrombomodulin gene.

We characterized the transcriptional regulatory function of the 3'-untranslated region of the thrombomodulin gene by transient transfection assays in human umbilical endothelial cells. Deletion analyses of the 3'-untranslated region indicated that the region containing the consensus sequence of the cyclic AMP (cAMP) responsive element (position 2092) showed an increased transcriptional activity in response to cAMP. Gel-shift analysis showed that a band representing the fragment containing position 2092 was retarded when incubated with nuclear extracts from the cells treated with cAMP. In addition, the region downstream of the cAMP responsive element was found to function negatively in the gene expression. These results indicate that the 3'-untranslated region has a functional cAMP responsive element and plays an important role in the regulation of the thrombomodulin gene expression.

Functional characterization of the 5'-regulatory region of the human thrombomodulin gene.

Thrombomodulin, a glycoprotein expressed in endothelial cells, has an important role in the blood coagulation system as a modulator. Functional characterization of the 5'-regulatory region of the human thrombomodulin gene was carried out to identify elements necessary for its expression. We used a series of dissected gene constructs containing the bacterial chloramphenicol acetyltransferase gene in transient transfection assays on human umbilical vein endothelial cells. The region extending from -290 to -33 of the 5' end flanking sequence is required for the full expression of this gene. Within this region, four potential Sp1 sites were found, and the sequences of Sp1 sites were mutated to identify their role in the promoter activity of the gene, showing that the two Sp1 sites at -207 and -141 are important for the full activity of the thrombomodulin promoter. Site-directed mutation analysis identified sequence elements GCAATC at -110 as a functioning CAAT box. Another three regions, -290 to -223, -99 to -68, and -67 to -33 have unidentified positively and negatively acting elements. A silencer element was located in the region spanning from -947 to -772 bases of the 5' end flanking region. These data indicate that the expression of the thrombomodulin gene is regulated by various elements which act positively or negatively.