Defensin Research Articles

Study on vaginal production of human defensins and the correlated pathogenetic factors of vulvovaginal candidiasis

To investigate the correlated pathogenetic factors and vaginal local immunity in vulvovaginal candidiasis (VVC). A case control study was conducted to compare VVC group (60 cases) with normal group (60 cases). All of the women filled up the specific questionnaires. Routine examination, pH test and bacterial culture were done on the vaginal discharge. Cytokines of the vaginal lavage were measured by enzyme linked immunosorbent assay. (1) Outcomes of the questionnaires: there was no significant difference between the two groups in educational background, knowledge of gynecologic infection, history of gynecologic infection, hygienic habit, sex life, or use of medicine (P > 0.05). The incidence of chronic cervicitis in normal group (43%, 26/60) was higher than in VVC group (22%, 13/60; P < 0.05). (2) There was no difference in vaginal pH between the two groups (P > 0.05). (3) Detection rate of candida albicans by vaginal discharge routine examination was 72% (43/60). (4) The concentrations of interleukin (IL) 2, and IL-4 in vaginal lavage did not show significant difference between the two groups (P > 0.05), but the concentrations of human defensin 5, human beta-defensin (HBD) 1, and HBD2 in VVC group [(0.94 +/- 0.44) mg/L, (3.1 +/- 0.4) microg/L, (10 +/- 6) microg/L] were higher than normal group (P < 0.05). VVC is a common vulvovaginitis. There is no significant correlation between the incidence of VVC and educational background, knowledge of gynecologic infection, history of gynecologic infection, hygienic habit, sex life, or use of medicine in the child-bearing period. Human defensin may be closely correlated with the pathogenesis of VVC.

Transfection of human beta defensin 2 gene into the lung by aerosol inhalation: experiment with rats

To investigate the feasibility to transfect human beta defensin 2 (hBD2) gene into the lung so as to enhance the endogenous hBD2 expression to defend infectious diseases. Recombinant plasmid containing hBD2 gene, pLXSN-hBD2, was mixed with a new cationic liposome prepared by film at different weight ratios. Sixteen Wistar rats were divided into 2 groups: experimental group (n = 11), undergoing aerosol inhalation of liposome/pLXSN-hBD2 complex, and control group (n = 5), undergoing aerosol inhalation of blank vector pLXSN. The tracheae were taken out from 5 rats of the experiment and control groups each 2 days later, and from 2 rats of the experimental group 6, 15, and 21 days later respectively so as to obtain the epithelial cells of trachea. DNA was extracted from the tracheal epithelial cells and PCR was used to examine the transfection and integration of hBD2. Western blotting was used to detect the protein expression of hBD2. Aerosolization impacted obviously the microcapsule structure of liposome/plasmid complexes, and there was the least structural destruction of complex at a ratio of 10:1 that suited for aerosolization best. After the inhalation the relevant plasmids were all successfully integrated into the epithelial cells in both groups. Protein expression of hBD2 was not detected in the control group and the hBD2 protein expression level 2 days after transfection of the experimental group was 4866.9 +/- 148.2, and then decreased gradually, and reached 3.2 +/- 1.5 twenty-one days after the transfection. The recombinant plasmid pLXSN-hBD2 can be transfect into the airway epithelial cells via aerosol inhalation and the expression of hBD2 sustains for a period of time.

Obtaining transgenic maize plants expressing the rabbit defensin (NP-1) gene and evaluation of their disease resistance

The rabbit defensin gene NP-1 was introduced into embryonic callus cultures of three maize (Zea mays L.) hybrid lines by particle bombardment, and transgenic plants were obtained. Genomic PCR and DNA dot blot analyses confirmed that the NP-1 gene was integrated into the genome of the regenerated T0 maize plants. Genomic PCR and Southern blotting results revealed that the NP-1 gene was transmitted stably from the T0 to the T1 generation. RNA dot blot analysis verified the transcription of the NP-1 gene in T1 plants. When challenged with northern corn leaf blight (Helminthosporium turcicum Pass), the T1 plants expressing the NP-1 gene showed greatly improved resistance to the fungal disease compared with the wild-type maize plants.

DNA sequence and analysis of human chromosome 8

The International Human Genome Sequencing Consortium (IHGSC) recently completed a sequence of the human genome. As part of this project, we have focused on chromosome 8. Although some chromosomes exhibit extreme characteristics in terms of length, gene content, repeat content and fraction segmentally duplicated, chromosome 8 is distinctly typical in character, being very close to the genome median in each of these aspects. This work describes a finished sequence and gene catalogue for the chromosome, which represents just over 5% of the euchromatic human genome. A unique feature of the chromosome is a vast region of approximately 15 megabases on distal 8p that appears to have a strikingly high mutation rate, which has accelerated in the hominids relative to other sequenced mammals. This fast-evolving region contains a number of genes related to innate immunity and the nervous system, including loci that appear to be under positive selection--these include the major defensin (DEF) gene cluster and MCPH1, a gene that may have contributed to the evolution of expanded brain size in the great apes. The data from chromosome 8 should allow a better understanding of both normal and disease biology and genome evolution.

103. PGK and CMV Promoters Exert the Strongest Activity in Lentiviral Gene Transduction of Myeloid Cells Including Mature Neutrophils

Gene therapy for the congenital deficiencies of neutrophil function such as chronic granulomatous disease (CGD) has been considered to be a curative approach. Lentiviral vectors are considered to be one of the most appropriate vectors for CGD gene therapy. In this study, we evaluated the activity of several promoters in myeloid cells including mature neutrophils. The VSV-pseudotyped lentiviral vectors (HIV vectors) used in this study consisted of the HIV-1 construct and GFP genes driven by promoters of cytomegalovirus (CMV), phosphoglycerokinase (PGK), elongation-factor 1|[alpha]| subunit (EF-1|[alpha]|), CAG compound (CAG) or human neutrophil specific |[alpha]|-defensin (DEF). Since the difference of promoter influences the transgene expression level and consequently affect the GFP titer, we adopted the DNA titer, which was not influenced by the internal promoter activity. At first, we transduced those HIV vectors into acute promyelocyte leukemia NB4 cells. The EF-1|[alpha]| and PGK were the strongest promoters in those cells, while the activities of these promoters became weak after the differentiation induced by all-trans retinoic acid. In contrast, the CMV promoter activity was the strongest in the differentiated cells. CAG and DEF showed similar activity patterns as CMV and EF-1|[alpha]|, respectively, but the expression level was low. Next, we transduced those HIV vectors into the CD34+ cord blood cells (CBCs) at an MOI of 1000, and these cells were cultured in the presence of cytokine cocktail (SCF, TPO, IL-3 and G-CSF) for 7 days followed by the 8 days culture with G-CSF. 7 days after transduction, the majority of cells ranged from promyelocytes to metamyelocytes, and the GFP+ rates of these cells transduced with the HIV vectors with PGK, EF-1|[alpha]|, CMV and DEF were 70%, 60%, 60% and 40%, respectively. Those GFP expressions were observed more strongly in CD34- cells than in CD34+ cells. As for the HIV vector with CAG, transduction was performed at an MOI of 100, because we could not produce high titer vectors, and the transduction efficiency was 50%. 15 days after transduction, 50 to 60% of cells were mature neutrophils and their GFP expression levels became weak regardless of vectors. However, the GFP+ rate with PGK and CMV promoter were relatively maintained with 50 and 40%, respectively. In addition, fluorescence intensity of GFP+ transduced with the HIV vector with CMV was the highest. Furthermore, the transduction of any kind of the HIV vectors did not affect significantly the growth, viability and functions of the differentiated neutrophils. In conclusion, as far as we could survey, PGK and CMV promoters were considered to be the most suitable for the lentiviral gene transduction to obtain appropriate gene expression of therapeutic level in both of immature and mature myeloid cells. We are now investigating the usefulness of those vectors for functional correction of neutrophils from CGD patients in vitro.

Polymorphic segmental duplications at 8p23.1 challenge the determination of individual defensin gene repertoires and the assembly of a contiguous human reference sequence

BackgroundDefensins are important components of innate immunity to combat bacterial and viral infections, and can even elicit antitumor responses. Clusters of defensin (DEF) genes are located in a 2 Mb range of the human chromosome 8p23.1. This DEF locus, however, represents one of the regions in the euchromatic part of the final human genome sequence which contains segmental duplications, and recalcitrant gaps indicating high structural dynamics.ResultsWe find that inter- and intraindividual genetic variations within this locus prevent a correct automatic assembly of the human reference genome (NCBI Build 34) which currently even contains misassemblies. Manual clone-by-clone alignment and gene annotation as well as repeat and SNP/haplotype analyses result in an alternative alignment significantly improving the DEF locus representation. Our assembly better reflects the experimentally verified variability of DEF gene and DEF cluster copy numbers. It contains an additional DEF cluster which we propose to reside between two already known clusters. Furthermore, manual annotation revealed a novel DEF gene and several pseudogenes expanding the hitherto known DEF repertoire. Analyses of BAC and working draft sequences of the chimpanzee indicates that its DEF region is also complex as in humans and DEF genes and a cluster are multiplied. Comparative analysis of human and chimpanzee DEF genes identified differences affecting the protein structure. Whether this might contribute to differences in disease susceptibility between man and ape remains to be solved. For the determination of individual DEF gene repertoires we provide a molecular approach based on DEF haplotypes.ConclusionsComplexity and variability seem to be essential genomic features of the human DEF locus at 8p23.1 and provides an ongoing challenge for the best possible representation in the human reference sequence. Dissection of paralogous sequence variations, duplicon SNPs ans multisite variations as well as haplotypes by sequencing based methods is the way for future studies of interindividual DEF locus variability and its disease association.

Structure of the Plant Defensin PhD1 from Petunia Hybrida

Structure of the Plant Defensin PhD1 from Petunia Hybrida

1H Chemical Shift Assignments and Interproton 3JNHA and 3JHAHB Coupling Constants of Aesculus hippocastanum Antimicrobial Protein 1 (Ah-AMP1), a Plant Defensin Isolated from Horse Chestnuts.

1H Chemical Shift Assignments and Interproton 3JNHA and 3JHAHB Coupling Constants of Aesculus hippocastanum Antimicrobial Protein 1 (Ah-AMP1), a Plant Defensin Isolated from Horse Chestnuts.

Malaria infection of the mosquitoAnopheles gambiaeactivates immune-responsive genes during critical transition stages of the parasite life cycle

Six gene markers have been used to map the progress of the innate immune response of the mosquito vector, Anopheles gambiae, upon infection by the malaria parasite, Plasmodium berghei. In addition to four previously reported genes, the set of markers included NOS (a nitric oxide synthase gene fragment) and ICHIT (a gene encoding two putative chitin-binding domains separated by a polythreonine-rich mucin region). In the midgut, a robust response occurs at 24 h post-infection, at a time when malaria ookinetes traverse the midgut epithelium, but subsides at later phases of malaria development. In contrast, the salivary glands show no significant response at 24 h, but are activated in a prolonged late phase when sporozoites are released from the midgut into the haemolymph and invade the glands, between 10 and 25 days after blood feeding. Furthermore, the abdomen of the mosquito minus the midgut shows significant activation of immune markers, with complex kinetics that are distinct from those of both midgut and salivary glands. The parasite evidently elicits immune responses in multiple tissues of the mosquito, two of which are epithelia that the parasite must traverse to complete its development. The mechanisms of these responses and their significance for malaria transmission are discussed.

Plasma defensin concentrations are elevated in patients with septicemia or bacterial meningitis.

We measured concentrations of defensins (human neutrophil peptides) in the plasma of healthy volunteers and patients with sepsis and meningitis. When a sensitive enzyme immunoassay was used, defensins were detected in plasma samples from 13 of 24 healthy blood donors, with a mean +/- SD of 42 +/- 53 ng/ml. Defensin levels in plasma samples from seven patients with sepsis at the onset of disease ranged from 900 ng/ml to 170,000 ng/ml. In 10 patients with meningitis in the initial phase of disease, plasma defensin concentrations ranged from 120 ng/ml to 910 ng/ml. Defensin concentrations in the plasma of both patient groups were significantly higher than those in healthy blood donors (p << 0.01), and patients with sepsis had higher defensin levels than patients with meningitis (p < 0.01). Defensin levels were significantly (p < 0.01) lower after the beginning of specific antibiotic therapy. Defensin concentrations in the plasma of patients with sepsis and meningitis correlated only weakly (r = 0.38) with blood neutrophil count. In vitro studies of defensin added to plasma indicated that all defensin was bound to plasma proteins. At high concentrations (1000 micrograms/ml), defensins caused precipitation of plasma proteins. Because plasma defensin levels may reflect neutrophil activation at sites of infection and inflammation, studies of the clinical utility of defensin ELISA are indicated.