Inhibition of group A streptococcal infection by Melaleuca alternifolia (tea tree) oil concentrate in the murine model.

AIMS: To investigate the effect of a water-soluble Melaleuca alternifolia concentrate (MAC) on group A streptococcus (GAS; Streptococcus pyogenes)-induced necrotizing fasciitis. METHODS AND RESULTS: MAC pretreatment (1% and 2% v/v) was able to protect mice from GAS infection in an air pouch model. GAS-induced mouse death and skin injury were inhibited dose dependently by MAC. Administration of MAC at 6 h post-GAS infection partially delayed mouse death. Surveys of the exudates of the air pouch of MAC-treated mice revealed that the survival of infiltrating cells was prolonged, the bacteria were eliminated, and the production of inflammatory cytokines was inhibited. MAC could directly inhibit the growth of GAS in vitro, and the minimal inhibitory concentration (MIC) of MAC for GAS was determined as 0.05% v/v using the time-kill assay. Furthermore, a sub-MIC dose of MAC not only enhanced the bactericidal activity of RAW264.7 macrophage cells against GAS but also increased susceptibility of GAS for blood clearance. CONCLUSIONS: These results suggest that MAC may inhibit GAS-induced skin damage and mouse death by directly inhibiting GAS growth and enhancing the bactericidal activity of macrophages. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results provide scientific data on the use of MAC for the treatment of GAS-induced necrotizing fasciitis in the murine model.

Phagocytosis and immune response studies of Macrophage-Nanodiamond Interactions in vitro and in vivo.

The applications of nanodiamond as drug delivery and bio-imaging can require the relinquishing ND-drug conjugate via blood flow, where interaction with immune cells may occur. In this work, we investigated the ND penetration in macrophage and the immune response using the tissue-resident murine macrophages (RAW 264.7). Confocal fluorescence imaging, immunofluorescence analysis of nuclear translocation of interferon regulatory factor IRF-3 and transcriptional factor NF-κΒ, analysis of pro-inflammatory cytokines production IL-1ß, IL-6 IL-10 with a reverse transcription-polymerase chain reaction technique were applied. The TNF-α factor production has been studied both in vitro at ND interaction with the macrophage and in vivo after ND injection in the mice blood system using immunoassay. The macrophage antibacterial function was estimated through E. coli bacterial colony formation. ND didn't stimulate the immune response and functionality of the macrophage was not altered. Using MTT test, ND was found negligibly cytotoxic to macrophages. Thus, ND can serve as a biocompatible platform for bio-medical applications. Left: Graphic representation of Nanodiamond internalization in macrophage. Right: (a) Fluorescence images of lysosomes, (b) nanodiamond and (c) merged image of nanodiamond internalization in macrophage.

Identification of the bases in the ompF regulatory region, which interact with the transcription factor OmpR.

Expression of the outer membrane protein OmpF of Escherichia coli K-12 is influenced by a variety of environmental signals. Most of the signals are thought to regulate OmpF expression at the level of transcription initiation. A key element of this regulation is the interaction between the transcriptional factor OmpR and the cis-acting regulatory region of ompF. In this study, we used a combination of DNase I, dimethyl sulfate and hydroxyl radical footprinting analysis and DNA migration retardation assays to identify the bases within the ompF regulatory region that are in contact with OmpR. Our results indicate that the -107 to -39 region of ompF contains three individual binding sites and that a single OmpR-binding site is capable of interacting with two OmpR molecules. We also establish that a single OmpR-binding site is composed of two half-sites and that both half-sites are required for the formation of stable OmpR/DNA complexes. Comparisons of the sequences protected by OmpR indicate that an OmpR-binding site spans approximately 18 bp and has two highly conserved G/C base-pairs that are separated by three nucleotides. Although the three OmpR-binding sites we identified exhibit limited sequence similarity, this may reflect the fact that two of the sites are incapable of binding OmpR independently and can bind OmpR only if adjacent to another OmpR-binding site. Finally, our DNA migration retardation assays suggest that phosphorylation stimulates the cooperative interactions between OmpR molecules bound at neighboring sites. Therefore, this study provides a detailed understanding of how OmpR interacts with its binding sites immediately upstream of ompF and serves as a foundation for studying how phosphorylation of OmpR results in the regulation of ompF expression in response to environmental signals.

A new cell-based assay for measuring the forward mutation rate of HIV-1.

Over 20 years into the ever-worsening AIDS pandemic, genetic variation remains the greatest obstacle for treating and preventing HIV-1 infection. Mutation rate assays for HIV-1 have been reported; however, none measure directly the forward mutation rate during replication of the virus in cell culture while still retaining the ability to propagate and further study mutant proviruses. Therefore, the objective of the current study was to develop such a phenotypic cell-based assay for measuring the forward mutation rate of HIV-1. Conventional recombinant DNA techniques and polymerase chain reaction were used to create a replication defective HIV-1 vector, pNL4-3Delta+cass, which is based on the NL4-3 strain and contains the thymidine kinase gene from human herpes virus type 1 as the mutational target. A series of transfection and infection steps were used to introduce the vector into 143B cells, which are negative for thymidine kinase function, and produce vector virus for a single cycle of replication. Viral titers were measured by counting the number of drug resistant colonies on the assay plates, and forward mutation rates were calculated from the viral titers. Mutant proviruses were sequenced to determine the types of genetic alterations that occurred. The average forward mutation rate for HIV-1 was 2.2 x 10(-5)mutations/base/cycle. The majority of mutations were base substitutions, including high frequencies of C-->U and G-->A transitions. Single adenosine insertions were also observed frequently. The new assay is economical and provides a direct measurement of the mutation rate during a single cycle of viral replication. Target cells containing mutant proviruses survive the drug selection process and may be propagated for further analysis. The new assay is novel and has many advantages over previous mutation rate assays and thus will be very useful in future studies on genetic variation of HIV-1.

Induction of thymocyte apoptosis in mice by Yersinia enterocolitica products.

In-vivo administration of the culture supernates from Yersinia enterocolitica resulted in thymus atrophy in C3H/HeJ mice, known to be lipopolysaccharide (LPS)-nonresponders. The thymocytes underwent apoptosis as characterised by fragmented DNA ladders on agarose gel electrophoresis, a cell death detection ELISA and a morphological study by the TUNEL reaction. As a control, LPS treatment did not induce thymocyte apoptosis in C3H/HeJ mice. Flow cytometric analysis indicated that thymus atrophy was due predominantly to the deletion of CD4+ CD8+ T cells. When cells were undergoing apoptosis, an elevation in the percentage of T-cell receptor (TCR)-alphabeta(high) cells was observed at 24 h, which was correlated with the increase in the percentages of cells expressing high levels of the Vbeta6 and Vbeta8 TCR. Gel electrophoretic analysis demonstrated the presence of protein bands with mol.wts ranging from 17 to 65 kDa in Y. enterocolitica culture supernates.

Preparation of desmopressin-containing liposomes for intranasal delivery.

The loading and leakage characteristics of the desmopressin-containing liposomes and the effect of liposomes on the nasal mucosa permeation and antidiuresis of desmopressin were investigated. Higher loading efficiency of desmopressin for positively charged liposomes than negatively charged liposomes was obtained, and neutral liposomes resulted in a similar loading efficiency as that of positively charged liposomes. Greater leakage of desmopressin from negatively charged liposomes than from positively charged and neutral liposomes was shown. The increase of permeability of desmopressin through the nasal mucosa indicated positively charged liposomes>negatively charged liposomes>solution. It was suggested that the enhanced contact time of positively charged liposomes with negatively charged nasal mucosa led to a high local desmopressin concentration on the penetration site to promote an effective penetration of desmopressin through the nasal mucosa. The desmopressin antidiuresis result after intranasal administration was in good agreement with the permeability result in the order of positively charged liposomes>negatively charged liposomes>solution. One of the mechanisms for the explanation of the best result on the enhancement of antidiuresis for positively charged liposomes may be the bioadhesive effect of the liposomes on the negatively charged nasal mucosa.

Acyclovir-containing liposomes for potential ocular delivery. Corneal penetration and absorption.

The in vitro corneal penetration and in vivo corneal absorption of acyclovir from an acyclovir-containing liposome system were investigated. Results of in vitro corneal penetration demonstrated that positively charged liposomes resulted in a penetration rate lower than those of negatively charged liposomes and free acyclovir in solution. An in vivo study indicated that the extent of acyclovir absorption from positively charged liposomes was higher that those from negatively charged liposomes and free acyclovir. The acyclovir concentration in the cornea after administration of positively charged liposomes showed that an acyclovir deposition in the cornea was greater than those of negatively charged liposomes and free acyclovir. From morphological observation of the cornea surface treated with liposomes, it was suggested that positively charged liposomes formed a completely coated layer on the cornea surface. These liposomes would bind intimately on the cornea surface, leading to an increase of residence time. Therefore, positively charged liposomes resulted in an increase of acyclovir (ACV) absorption.

Bovine ephemeral fever in Taiwan.

Bovine ephemeral fever (BEF) is a vector-borne disease of cattle, spanning tropical and subtropical zones of Asia, Australia, and Africa, caused by Ephemerovirus of the Rhabdoviridae. Taiwan has had 3 BEF epizootics, occurring in 1989, 1996, and 1999, since the vaccination regimen was initiated in 1984, given once a year in the spring with a single-dose formaldehyde-inactivated vaccine using the 1983 isolate as the seed virus. This study evaluated the 1999 population immunity against BEF virus in Taiwanese dairy cows with a neutralization test and whether the recent BEF virus isolates have mutated significantly from the vaccine virus. In March 1999, before vaccination, 94% of the animals studied were already seropositive, suggestive of an endemic or persistent infection from the previous year. By June 1999, when 51% of herds had been vaccinated, the antibody level rose, and by September 1999, the serum-neutralizing antibody (SNA) level fell to a minimum, preceding the outbreak of BEF in October 1999, during which the antibody levels of vaccinated cows continued to decline while those of unvaccinated cows rose sharply. The results suggest that, in 1999, vaccine-induced immunity was partially protective against BEE Because the current single-dose vaccination regimen resulted in minimal population immunity by September, a booster vaccination given in late summer may be advisable for future disease control. Analysis of the glycoprotein gene of Taiwanese isolates between 1983 and 1999 showed a 97.4-99.6% homology, with an alteration of 4 amino acids in antigenic sites G1, G3b, and G3c. Phylogenetic analysis of Taiwanese isolates revealed at least 2 distinct clusters: the 1983-1989 isolates and the 1996-1999 isolates. Both were distinct from 2 Japanese strains and the Australian BB7721 strain. Thus, at least 2 distinct BEF viruses, which had diverged before 1983, existed in Taiwanese dairy cows.

In vitro comparison of the activity of various antifungal drugs against new yeast isolates causing thrush in poultry.

Of 44 representative isolates of yeast isolated from the poultry upper digestive tract from 40 clinical thrush cases in Taiwan during 1985-87, 39 (89%) isolates were classified as Candida albicans, and 5 (11%) were classified as Torulopsis pintolopesii. Fifteen commercial antifungal drugs, incorporated individually in Sabouraud's dextrose agar by serial twofold dilutions, were tested for their inhibitory effect against the 44 isolates. The MIC50 of these drugs in increasing order was less than or equal to 2 ppm GV-11, 6 ppm gentian violet, less than 16 ppm amphotericin B, 16 ppm hyamine 1622, 25 ppm econazole, 35 ppm chlorohydroxyquinoline, 40 ppm nystatin, 64 ppm miconazole, 747 ppm malachite green, 1550 ppm benzoic acid, 1536 ppm copper sulfate, 3144 ppm Monoprop, 4951 ppm Mold Zap, greater than 16,384 ppm propionic acid, and greater than 16,384 ppm sodium propionate.

Classification, pathogenicity, and drug susceptibility of hemolytic gram-negative bacteria isolated from sick or dead chickens.

Fifteen hemolytic gram-negative bacteria were isolated from the respiratory tracts of sick birds suffering from a long-lasting respiratory syndrome or from the bone marrow of dead birds distributed in the southern part of Taiwan. These were classified as Pseudomonas aeruginosa (10 isolates), Pseudomonas fluorescens (2 isolates), Pseudomonas stutzeri (1 isolate), Pasteurella haemolytica (1 isolate), and Proteus morganii (1 isolate). Each isolate was inoculated intraperitoneally into one group of ten 4-week-old male white leghorn chickens. Mortality and lesions were scored daily for 1 week. Three of the 10 isolates of Pseudomonas aeruginosa caused 100% mortality. Six other isolates of Pseudomonas aeruginosa and the one isolate of Proteus morganii caused 50% mortality. The remaining isolates induced less than 30% mortality. The sole nonpathogenic sample was one isolate of Pseudomonas fluorescens. When therapeutic levels of 22 antibiotics or sulfa drugs were evaluated for their inhibitory activity against the 15 isolates, the most effective were apramycin (15/15), gentamicin (15/15), spectinomycin (13/15), oxytetracycline (8/15), and sulfachloropyrazine (7/15). The least effective were ampicillin, cloxacillin, and tiamulin, which were not effective against any of the isolates. The 14 other drugs were of very low (> 4/15) effectiveness. Most of the isolates studied were virulent for chickens and very resistant to currently used drugs.

[Teratogenicity study on bromperidol in rats].

A teratogenicity study on bromperidol, a neuroleptic, was carried out in Wistar-Imamichi rats. Bromperidol was administered orally at the dose levels of 0.2, 1.5 and 10.0 mg/kg/day for 11 days from day 7 to day 17 of gestation. About two-thirds of pregnant females in each group were sacrificed on day 21 of gestation, and their fetuses were examined. The remaining dams were allowed to litter naturally, and the post-natal development of the offspring was observed. Treatment of bromperidol caused sedation of pregnant females in 1.5 and 10.0 mg/kg groups with concomitant decrease in body weight and in food and water intake in dose-dependent manner. Effect of bromperidol treatment on F1 generation was observed mainly in animals of 10.0 mg/kg group, suggesting overall growth retardation such as decrease in live fetal weight, retarded ossification of metacarpals and metatarsals, a slight decrease in day 1 viability index, a marginal delay in eyelid opening, reduction in growth rate and decrease in organ weight. No treatment-related external and internal abnormalities were observed in fetuses (F1), and no apparent effect on behavior, learning ability and reproductive function of offspring (F1) was found.

Enhancement of nasal absorption of calcitonin loaded in liposomes.

Intranasal administration of calcitonin-containing liposomes in rabbits was investigated to evaluate the in vivo calcitonin absorption performance. Plasma calcitonin concentrations and calcium levels were measured and pharmacokinetic parameters were calculated. The bioavailability of calcitonin resulted from the intranasal delivery formulations demonstrated an order of calcitonin-containing positively charged liposomes > calcitonin-containing negatively charged liposomes > calcitonin solution. The significant enhancement of bioavailability of calcitonin for positively charged liposomes may be due to the charge interaction of positively charged liposomes with the negatively charged mucosa surface. Marked accumulation of positively charged liposomes was found on the negatively charged nasal mucosa surface. The retention of positively charged liposomes on the nasal mucosa resulted in an increase of residence time with high local concentration of calcitonin for increase of absorption.

Cancer of the colon during pregnancy: report of a case and review of the literature.

A case of adenocarcinoma of the sigmoid colon during pregnancy is reported. The patient presented with anemia and a painless mass over the left abdomen without gastrointestinal discomfort, making this case different from 25 previously reported cases of colon carcinoma above the peritoneal reflection associated with pregnancy.

A study on the ecological habit of Armigeres subalbatus in Dawa area of the Mengshan Mountain in Shandong Province.

Armigeres subalbatus (A.s.) was reported in Shandong Province for the first time in 1965 and found in five counties of south Shandong including Pingyi, Linyi etc. in 1986. In Dawa area of the Mengshan mountain A.s. alults could be found in the first ten days of May, which increased in number in July, and become the dominant species in mosquito colonies in Aug. and Sept., then decreased gradually in number in Oct. and disappeared in Nov. There were two peaks of activity and blood-sucking behavior during the 24 hours of a day, one at dusk and the other at dawn. When the temperature dropped to 16 degrees C and below in the last ten days of Oct., the wigglers began their diapause period. The survival ratio reached 90.5% after 12 h freezing at -5 degrees C and none survived after 60 hours freezing. When the temperature rose to 17 degrees C and above the over-winterting larvae developed into adults, which could suck blood only at the temperature above 17.5 degrees C.

Streptococcal pyrogenic exotoxin B antibodies in a mouse model of glomerulonephritis.

Streptococcal pyrogenic exotoxin B is an extracellular cysteine protease. Only nephritis-associated strains of group A streptococci secrete this protease and this may be involved in the pathogenesis of post-streptococcal glomerulonephritis. Mice were actively immunized with a recombinant protease inactive exotoxin B mutant or passively immunized with exotoxin B antibody. Characteristics of glomerulonephritis were measured using histology, immunoglobulin deposition, complement activation, cell infiltration, and proteinuria. None of the mice given bovine serum albumin or exotoxin A as controls showed any marked changes. Immunoglobulin deposition, complement activation, and leukocyte infiltration occurred only in the glomeruli of exotoxin B-hyperimmunized mice. One particular anti-exotoxin B monoclonal antibody, 10G, was cross-reactive with kidney endothelial cells and it caused kidney injury and proteinuria when infused into mice. This cross-reactivity may be involved in the pathogenesis of glomerulonephritis following group A streptococcal infection.

Effects of lysine-sensitive aspartokinase III on lysine biosynthesis in Escherichia coli K-12.

A promoterless lysC gene, coding for Escherichia coli aspartokinase III (AKase III), has been cloned by phenotypic complementation using plasmid pUC19 as the vector. The hybrid plasmid obtained, pUC19AK3, preserved the ribosome binding site and transcriptional termination signal of the gene but with a lac promoter. E. coli strains containing the recombinant plasmid had high levels of AKase III activity. AKase III activity from expressing strains was inhibited by lysine, leucine, and S-(2-aminoethyl)-L-cysteine (AEC) but not by threonine and methionine. The overexpressed AKase III enzyme had a molecular weight of about 50 kD from SDS-polyacrylamide gel electrophoresis. N-terminal amino acid sequence analysis confirmed that the product from the hybrid plasmid was identical to native AKase III rather than a fusion protein. Moreover, overexpression of AKase III significantly increased lysine excretion in the plasmid-harboring E. coli strain DH1. This increase in the level of AKase III activity also affected other metabolites than lysine. Addition of aspartate to the medium brought about significant increases in lysine excretion. A maximum increase (about 8-fold) in lysine accumulation was observed 45 minutes after incubation in minimal medium containing 0.2% aspartate as compared to aspartate-free medium.

A distant upstream site involved in the negative regulation of the Escherichia coli ompF gene.

The two-component regulatory system, OmpR-EnvZ, of Escherichia coli K-12 regulates the expression of the major outer membrane porin protein, OmpF. OmpR is a DNA-binding protein which acts as both an activator and a repressor to control ompF transcription. In this article, we describe a new OmpR-binding site that is located between 384 to 351 bp upstream from the ompF start point of transcription. Inactivation of this site by insertion of a 22-bp fragment prevents the repression of ompF expression conferred by the dominant negative mutation, envZ473. On the basis of the location of this binding site, the presence of bent DNA in the ompF regulatory region (T. Mizuno, Gene 54:57-64, 1987), and the fact that mutations altering integration host factor result in constitutive ompF expression (P. Tsui, V. Helu, and M. Freundlich, J. Bacteriol. 170:4950-4953, 1988), we propose that the negative regulation of ompF involves a DNA loop structure.

Phosphorylation stimulates the cooperative DNA-binding properties of the transcription factor OmpR.

The two-component regulatory proteins OmpR and EnvZ of Escherichia coli K-12 regulate expression of the major outer membrane porin protein, OmpF. OmpR is a DNA-binding protein that is involved in both the positive and negative control of ompF transcription. EnvZ is a histidine kinase that phosphorylates OmpR in response to environmental signals. We used DNA migration retardation analysis to examine the interactions of OmpR and the phosphorylated form of OmpR (OmpR-P) with the regulatory region immediately upstream of the ompF promoter. Our results indicate that the binding of OmpR to this regulatory region is cooperative and that phosphorylation significantly stimulates these cooperative interactions. Moreover, although phosphorylation increases the intrinsic binding of OmpR to a single OmpR-binding site, the primary role of phosphorylation in ompF regulation is to facilitate cooperative interactions between OmpR molecules bound at adjacent sites. Based on these results, we propose a model to explain how the phosphorylation of OmpR could stimulate the occupancy of specific sites in the ompF regulatory region, thereby resulting in the activation or repression of ompF transcription under the appropriate environmental conditions.

Stability of desmopressin loaded in liposomes.

Desmopressin-containing liposome formulations have been developed for intranasal administration previously. Positively charged liposomes were found to be an efficient delivery system for desmopressin. In this study, stability of the loaded desmopressin in positively charged liposomes was further investigated. Comparison of the stability of desmopressin in solution and liposomes was made. Degradation of desmopressin was shown to follow a pseudo-first-order reaction. Degradation of desmopressin in both solution and liposomes demonstrated the same kinetic behavior and exhibited no significant difference in half-lives. Similar v-shape pH-rate profile was found for desmopressin degradation in solution and liposomes. At pH 4.0, the inflection point of the v-shape pH-rate curve, the reaction rate of desmopressin was lowest and the stability was greatest. The stability of lipid ingredients of dioleoylphosphatidylcholine (DOPC), cholesterol (C), and stearylamine (S) in the liposome dispersion at pH 4.0 was studied. Results demonstrated that DOPC, C, and S were relatively stable in the liposome structure when formulated with desmopressin. The degradation of desmopressin in solution and liposomes in the presence of alpha-chymotrypsin was investigated. A longer half-life for desmopressin in liposomes than in solution was observed. It was suggested that desmopressin was protected by the liposomes against alpha-chymotrypsin digestion.

An antigen-specific hypersensitivity which does not fit into traditional classification of hypersensitivity.

A unique type of Ag-specific hypersensitivity was induced by challenging the Ag-sensitized mice at the ear. It was elicited within 1 h after the Ag challenge, and thus was distinct from either the delayed-type hypersensitivity (DTH) which developed in 24 h or the immune complex-mediated hypersensitivity which evolved in 4 to 6 h. This hypersensitivity was referred to as early-type hypersensitivity (ETH). The time required for these types of hypersensitivity to develop after immunization was also different; DTH required 4 to 6 days, ETH 9 to 11 days, whereas plasma protein-induced immune complex-mediated hypersensitivity needed 18 to 21 days. The ETH could be induced by a smaller amount of Ag than DTH, and unlike DTH could be transferred by either immune sera or T cell-derived culture factor which was small m.w. Although the ETH developed later than DTH after sensitization, it lasted longer once developed and the pattern of response was inversely related to DTH. Furthermore, the denatured hepatitis B surface Ag induced DTH but not ETH, in contrast to native hepatitis B surface Ag that induced both, suggesting that the epitopes recognized by TETH cells were distinct from those recognized by TDTH cells. The ETH could be induced by most Ag tested including poly(Glu60Ala10Tyr10, L-lactic dehydrogenase, insulin, chicken egg white lysozyme, polymerized human serum albumin, horse gamma-globulin, transferrin, fibrinogen, and plasminogen, but not by purified protein derivative. Because poly(Glu60Ala10Tyr10, L-lactic dehydrogenase, egg white lysozyme and insulin were under the Ir gene control and the inducibility of ETH was Ag dependent and was closely correlated with that of DTH, the expression of ETH also must be regulated by Ir gene. The histopathologic changes in ETH consisted of capillary congestion and edema. The vasopermeability was increased and there was the leakage of plasma proteins into the tissue. Based on these data, we concluded that the ETH reported in this study was a novel type of Ag-specific hypersensitivity.