Differences in Colistin-resistant Acinetobacter baumannii Clinical Isolates Between Patients With and Without Prior Colistin Treatment

BACKGROUND: The increasing morbidity and mortality rates associated with Acinetobacter baumannii are due to the emergence of drug resistance and the limited treatment options. We compared characteristics of colistin-resistant Acinetobacter baumannii (CR-AB) clinical isolates recovered from patients with and without prior colistin treatment. We assessed whether prior colistin treatment affects the resistance mechanism of CR-AB isolates, mortality rates, and clinical characteristics. Additionally, a proper method for identifying CR-AB was determined. METHODS: We collected 36 non-duplicate CR-AB clinical isolates resistant to colistin. Antimicrobial susceptibility testing, Sanger sequencing analysis, molecular typing, lipid A structure analysis, and in vitro synergy testing were performed. Eleven colistin-susceptible AB isolates were used as controls. RESULTS: Despite no differences in clinical characteristics between patients with and without prior colistin treatment, resistance-causing genetic mutations were more frequent in isolates from colistin-treated patients. Distinct mutations were overlooked via the Sanger sequencing method, perhaps because of a masking effect by the colistin-susceptible AB subpopulation of CR-AB isolates lacking genetic mutations. However, modified lipid A analysis revealed colistin resistance peaks, despite the population heterogeneity, and peak levels were significantly different between the groups. CONCLUSIONS: Although prior colistin use did not induce clinical or susceptibility differences, we demonstrated that identification of CR-AB by sequencing is insufficient. We propose that population heterogeneity has a masking effect, especially in colistin non-treated patients; therefore, accurate testing methods reflecting physiological alterations of the bacteria, such as phosphoethanolamine-modified lipid A identification by matrix-assisted laser desorption ionization-time of flight, should be employed.

Distinct Effects of Monophosphoryl Lipid A, Oligodeoxynucleotide CpG, and Combination Adjuvants on Modulating Innate and Adaptive Immune Responses to Influenza Vaccination

Monophosphoryl lipid A (MPL) and oligodeoxynucleotide CpG are toll-like receptor (TLR) 4 and 9 agonist, respectively. Here, we investigated the effects of MPL, CpG, and combination adjuvants on stimulating in vitro dendritic cells (DCs), in vivo innate and adaptive immune responses, and protective efficacy of influenza vaccination. Combination of MPL and CpG was found to exhibit distinct effects on stimulating DCs in vitro to secrete IL-12p70 and tumor necrosis factor (TNF)-α and proliferate allogeneic CD8 T cells. Prime immunization of mice with inactivated split influenza vaccine in the presence of low dose MPL+CpG adjuvants increased the induction of virus-specific IgG and IgG2a isotype antibodies. MPL and CpG adjuvants contribute to improving the efficacy of prime influenza vaccination against lethal influenza challenge as determined by body weight monitoring, lung function, viral titers, and histology. A combination of MPL and CpG adjuvants was effective in improving vaccine efficacy as well as in reducing inflammatory immune responses locally and in inducing cellular immune responses upon lethal influenza virus challenge. This study demonstrates unique adjuvant effects of MPL, CpG, and combination adjuvants on modulating innate and adaptive immune responses to influenza prime vaccination.

Induction of Bronchial Tolerance After 1 Cycle of Monophosphoryl-A-Adjuvanted Specific Immunotherapy in Children With Grass Pollen Allergies

PURPOSE: Subcutaneous allergen-specific immunotherapy (SCIT) is a well-established and clinically effective method to treat allergic diseases, such as rhinitis and asthma. It remains unclear how soon after initiation of an ultra-short course of grass pollen immunotherapy adjuvanted with monophosphoryl lipid A (MPL)-specific bronchial tolerance can be induced. METHODS: In a prospective study of 69 children double-sensitized to birch and grass pollens (51 males, average age 11.1 years), development of bronchial tolerance after 1 cycle of SCIT for grass was evaluated. In all the patients, the bronchial allergen provocation test (BAP) was performed before and after treatment. According to the results of the first BAP, the patients were divided into 2 groups: those showing a negative BAP with a decrease in FEV1 of or =20% (SAR with allergic asthma [SAR and Asthma] group, n=22). All the patients received MPL-adjuvanted, ultra-short course immunotherapy for birch, but only those with a positive BAP to grass received MPL-SCIT for grass. RESULTS: After the pollen season, the BAP in the SAR group remained unchanged, while it was improved in the SAR and Asthma group (decrease in FEV1 of 28.8% vs 12.5%, P<0.01). The IgG4 levels increased after SCIT (median before SCIT 0.34 to 11.4 after SCIT), whereas the total and specific IgE levels remained unchanged. CONCLUSIONS: After 1 cycle of MPL-SCIT, specific bronchial tolerance may be significantly induced, whereas in patients without SCIT, bronchial hyperactivity may remain unchanged.

Effect of Intravenous Lipid Emulsion in the Patient with Acute Poisoning : A Systematic Review

PURPOSE: The purpose of this study was to evaluate the usefulness of intravenous lipid emulsion as well as adverse events in acute poisoning patients. METHODS: Literature was accessed through PubMed, EMBASE, Cochrane library, Web of science, and KoreaMed. All forms of literatures relevant to human use of intravenous lipid emulsion for acute poisoning were included. Cases reports or letters without description of clinical outcomes for each case were excluded. The literature search was conducted by two investigators in March, 2015, with publication language restricted to English and Korean. The effect, onset time, and adverse event of lipid emulsion and final outcome of each case were analyzed. RESULTS: Eighty-one published articles were included, excluding articles whose title and abstract were not relevant to this study. No articles were classified as high level of evidence. Sixty-eight case reports were identified, consisting of 25 local anesthetics and 43 other drugs, such as tricyclic antidepressants and calcium channel blockers. Although most cases described significant clinical improvements, some of them showed no beneficial effect or worsening of clinical course. Several adverse events including hyperamylasemia and laboratory interference were reported. CONCLUSION: Although there were many case reports illustrating successful use of lipid for various drug poisonings, the effect cannot be estimated due to significant possibility of publication bias. Therefore, lipids might be considered in severe hemodynamic instability resulting from lipophilic drug poisoning, however further studies should follow to establish the use of lipid as the standard of care.

Lipid A as a Drug Target and Therapeutic Molecule

In this review, lipid A, from its discovery to recent findings, is presented as a drug target and therapeutic molecule. First, the biosynthetic pathway for lipid A, the Raetz pathway, serves as a good drug target for antibiotic development. Several assay methods used to screen for inhibitors of lipid A synthesis will be presented, and some of the promising lead compounds will be described. Second, utilization of lipid A biosynthetic pathways by various bacterial species can generate modified lipid A molecules with therapeutic value.

Infección por Brucella canis en humanos: propuesta de un modelo teórico de infección a través de la ruta oral / Brucella canis infection in humans: Proposal of a theoretical model of infection through the oral route

La infección por Brucella canis en los humanos se ha reconocido recientemente como una zoonosis, pero frecuentemente es sub reportada debido a que los síntomas pueden confundirse con los de un resfriado común u otras infecciones causadas por otros patógenos. Los caninos son los hospederos primarios de Brucella canis ; el incremento en la tendencia de tener perros como mascotas podría también aumentar la posibilidad de transmisión de la infección a los humanos por el estrecho contacto entre la mascota infectada y su propietario. En Colombia, hay reportes de aislamientos de B. canis de caninos de criaderos y de un humano en contacto con perros infectados, al igual que reportes de caninos seropositivos a la infección. Sin embargo, no hay mucha información disponible sobre los mecanismos de interacción hospedero-patógeno que conduzcan al establecimiento de la infección por Brucella canis en perros y en humanos esta información es todavía menor. En esta revisión se propone un modelo para la infección humana con Brucella canis a través de la ruta oral utilizando la información disponible para otras especies de Brucella que infectan al humano, incluyendo B. abortu s y B. melitensis , que difieren de B. canis en la composición estructural de su lipopolisacárido. También se hipotetiza el mecanismo de infección celular que es usado por B. canis para invadir y establecer la infección en células no fagocíticas y fagocíticas.

Brucella canis infection in humans has recently been recognized as a zoonosis, but it is frequently under reported because the flu-like symptoms are often confused with the presence of other disease-causing pathogens. Dogs are the primary hosts for Brucella canis ; the increasing trend to adopt dogs as pets also enhances the likelihood of transmission of Brucella canis infection through contact between infected dogs and owners. In Colombia, there are reports of isolates of B. canis from kennel dogs and also from one human being along with seropositive results from dogs and humans. However, the mechanism of hostpathogen interactions leading to the infection of Brucella canis in dogs is still unknown and even less is known about human infections. This review proposes a model for human infection with Brucella canis through the oral route. We use the information available for other human-infecting Brucella species, including B. abortu s and B. melitensis, which differ from B. canis in the structural composition of the lipopolysaccharide molecule. The mechanism of cellular infection used by B. canis to invade and establish infection in nonphagocytic and phagocytic cells is also hypothesized.

Safety and skin delayed-type hypersensitivity response in vervet monkeys immunized with Leishmania donovani sonicate antigen delivered with adjuvants / Segurança e reação de hipersensibilidade tardia na pele de macacos vervet imunizados com antígeno sonicado de Leishmania donovani junto com adjuvantes

In this study, we report on the safety and skin delayed-type hypersensitivity (DTH), responses of the Leishmania donovani whole cell sonicate antigen delivered in conjunction with alum-BCG (AlBCG), Montanide ISA 720 (MISA) or Monophosphoryl lipid A (MPLA) in groups of vervet monkeys. Following three intradermal injections of the inoculums on days 0, 28 and 42, safety and DTH responses were assessed. Preliminary tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) levels were also measured and these were compared with DTH. Only those animals immunized with alum-BCG reacted adversely to the inoculum by producing ulcerative erythematous skin indurations. Non-parametric analysis of variance followed by a post-test showed significantly higher DTH responses in the MISA+Ag group compared with other immunized groups (p < 0.001). The MPLA+Ag group indicated significantly lower DTH responses to the sonicate antigen compared with the AlBCG+Ag group. There was a significant correlation between the DTH and cytokine responses (p < 0.0001). Based on this study we conclude that Leishmania donovani sonicate antigen containing MISA 720 is safe and is associated with a strong DTH reaction following immunization.

Neste estudo reportamos segurança e resposta de hipersensibilidade tardia (DTH) do antígeno sonicado de células totais de Leishmania donovani introduzidos juntamente com alume-BCG (AIBCG) Montanide ISA 720 (MISA) ou lípide A monofosforilado (MPLA) em grupos de macacos vervet. Depois de três injeções intradérmicas do inóculo nos dias 0, 28 e 42 segurança e resposta DTH foram avaliados. Preliminarmente níveis de fator de necrose tumoral alfa (TNF-α) e interferon gama (IFN-γ) foram também medidos e comparados com o DTH. Somente os animais imunizados com alume-BCG reagiram de maneira diversa ao inóculo produzindo indurações ulceradas e eritematosas na pele. Análise não paramétrica de variação seguida por um teste posterior mostraram resposta significantemente mais alta do DTH no grupo MISA + Ag quando comparado com outros grupos imunizados (p < 0.001). O grupo MPLA + Ag demonstrou resposta DTH significantemente menor do antígeno sonicado comparado com o grupo AIBCG + Ag. Houve correlação significante entre o DTH e a resposta às citocinas (p < 0.0001). Baseados neste estudo concluímos que o antígeno sonicado de Leishmania donovani contendo MISA 720 é seguro e está associado com forte reação DTH após imunização.

The role of toll like receptor-4 signal pathways activation in ischemia-reperfusion injury of island skin flap / 中华整形外科杂志

<p><b>OBJECTIVE</b>To determine the role of toll like receptor-4 signal pathways activation in ischemia-reperfusion injury of island skin flap.</p><p><b>METHODS</b>A totol of 50 adult male SD rats were randomized into 3 groups: sham-operated group (n=10), ischemia/reperfusion group (n=20) and TLR4 inhibitor-eritoran tetrasodium (E5564)-treated group (n=20). The inguinal island skin flaps models were set up. A bolus of E5564 (5 mg/kg) was infused intravenously 60 min before reper fusionm. TLR4 binding activity in flap tissue was analyzed at 1, 2, 4 and 6 h of reperfusion by immunohistochemical technique and flaps were assessed histologically at 6 h of reperfusion. The viability of flaps was assessed 7 days postoperatively.</p><p><b>RESULTS</b>Exprerssion TLR4 in skin flap tissue was significantly increased in I/R group, compared with E5564-treated group. Immunohistochemical exam showed TLR4 mainly expressed in skin flap vessel wall and PMN membrane. Marked neutrophil infiltration and edema was observed in I/R group, while less neutrophil infiltration was observed in E5564-treated group. In the E5564-treated group, the survival of flaps was (80.31 +/- 11.63)%, which was significantly greater than that in the I/R group (51.70 +/- 7.62)% (P < 0.01).</p><p><b>CONCLUSION</b>After ischemia-reperfusion injury in rats, the expression of TLR4 increased in the skin flap tissue with excessive neutrophil infiltration. Administration of E5564 can significantly improve flap survival by regulating the early activation of TLR4 and suppressing neutrophil infiltration within the flap.</p>

Interactions of complement proteins C1q and factor H with lipid A and Escherichia coli: further evidence that factor H regulates the classical complement pathway

Proteins of the complement system are known to interact with many charged substances. We recently characterized binding of C1q and factor H to immobilized and liposomal anionic phospholipids. Factor H inhibited C1q binding to anionic phospholipids, suggesting a role for factor H in regulating activation of the complement classical pathway by anionic phospholipids. To extend this finding, we examined interactions of C1q and factor H with lipid A, a well-characterized activator of the classical pathway. We report that C1q and factor H both bind to immobilized lipid A, lipid A liposomes and intact Escherichia coli TG1. Factor H competes with C1q for binding to these targets. Furthermore, increasing the factor H: C1q molar ratio in serum diminished C4b fixation, indicating that factor H diminishes classical pathway activation. The recombinant forms of the Cterminal, globular heads of C1q A, B and C chains bound to lipid A and E. coli in a manner qualitatively similar to native C1q, confirming that C1q interacts with these targets via its globular head region. These observations reinforce our proposal that factor H has an additional complement regulatory role of down-regulating classical pathway activation in response to certain targets. This is distinct from its role as an alternative pathway down-regulator. We suggest that under physiological conditions, factor H may serve as a downregulator of bacterially-driven inflammatory responses, thereby fine-tuning and balancing the inflammatory response in infections with Gram-negative bacteria.

Histological analysis antimetastatic effect of intera-venus injection of staphylococcal enterotoxin b and monophosphoryl lipid a against fibrosarchoma in lung tissue

Staphylococcal enterotoxin B [SEB] is a potent inducer of cytotoxic T-cell activity, cytokine production and necrosis induction in vivo. Monophosphoryl lipid A [MPL] is an adjuvant derived from the lipopolysaccharide of E.coli, Salmonella Minnesota Re595 and other gram negative bacteria. In this research, The antitumor and antimetastatic effect of intra-venus injection of Monophosphoryl Lipid A [MPL], Staphylococcal enterotoxin B [SEB] and SEB+ MPL was evaluated using Balb/C male mice bearing inoculable mice Fibrosarcoma. The anti tumor effect of SEB+ MPL, SEB and MPL in mice with inoculated fibrosarchoma tumor [Wehi-164] was examined by IV injection and the sizes of the inoculated tumors were determined. The inoculated tumors were also examined histologically. Moreover, histophatologic study in lung tissue didn't showed any metastasis. In the mice IV injected group with SEB4- MPL, reduction of tumor size show a significant difference compared with mice in the SEB and MPL injected and negative control group. A significantly higher frequency of necrosis in tumor tissues was also observed in mice in the IV [SEB+ MPL]-injected group in comparison with other group. Moreover, histophatologic study in lung tissue didn't show any metastasis Our findings suggest that tumor cell death and the prevention of metastasis be caused by increased Cytotoxic T-cell activity in response to IV injection of SEB+ MPL that need to more investigation

Genetic Variants Associated with Calorie and Macronutrient Intake in a Genome-Wide Association Study

There has been no genome-wide association study (GWAS) for macronutrient intake as a quantitative trait. To explore genetic loci associated with total calorie and macronutrient intake, genome-wide association data of autosomal single nucleotide polymorphisms (SNPs) from Korean adults were analyzed. We conducted a GWAS in 3,690 men and women aged 40 to 60 years from an urban population-based cohort. At the baseline examination (June 18, 2001 through January 29, 2003), DNA samples of the study subjects were collected and analyzed for genotyping. The information of average daily consumption of total calorie, carbohydrate, protein, and fat was obtained from a semi-quantitative food frequency questionnaire and transformed by natural logarithm for analyses after adjustment of calorie intake. Using multivariate linear regression analysis adjusted for age, sex, and height, we tested for 352,021 SNPs and found weak associations, which do not reach genome-wide association significance, with calorie and macronutrient intake. However, a number of SNPs were found to have potential associations with macronutrient intake; in particular, signals in SORBS1 and those in PRKCB1 were likely associated with carbohydrate and fat intake, respectively. We observed an inverse association between the minor allele of the SNPs in these genes and the amount of consumption of carbohydrate or fat. Our GWAS identified loci and minor alleles weakly associated with macronutrient intake. Because SORBS1 and PRKCB1 are reportedly associated with the metabolism of glucose and lipid as well as with obesity-related diseases, further investigations on biological and functional roles of polymorphism of these genes in the relation to macronutrient intake are warranted.

Vaccination with a Human Papillomavirus (HPV)-16/18 AS04-Adjuvanted Cervical Cancer Vaccine in Korean Girls Aged 10-14 Years

The human papillomavirus (HPV)-16/18 AS04-adjuvanted cervical cancer vaccine has been demonstrated to be highly efficacious and immunogenic with a favorable safety profile. This study assessed the immunogenicity and safety of the HPV-16/18 AS04-adjuvanted vaccine in healthy Korean girls aged 10-14 yr. This multi-center, observer-blind trial randomly assigned 321 healthy girls to receive three doses (0, 1, 6-month schedule) of HPV-16/18 AS04-adjuvanted vaccine or hepatitis A vaccine. Immunogenicity against vaccine antigens was assessed one month post-Dose 3. Solicited and unsolicited adverse events (AEs) and serious AEs (SAEs) were recorded. In the according-to-protocol analysis, all initially seronegative subjects vaccinated with the HPV-16/18 AS04-adjuvanted vaccine had seroconverted at Month 7, with a peak geometric mean titer (GMT) that was 600-fold higher than the natural infection titer of 29.8 EU/mL for HPV-16 and a peak GMT that was 400-fold higher than the natural infection titer of 22.6 EU/mL for HPV-18. The vaccine was well tolerated with no increase in reactogenicity with subsequent doses and no reports of vaccine-related SAEs. In conclusion, the HPV-16/18 AS04-adjuvanted vaccine is shown to be highly immunogenic and generally well-tolerated in Korean girls aged 10-14 yr.

Discovery of Diagnostic Biomarkers for Legionnaires' Disease: Virulence Gene Expression Profiling of Legionella pneumophila serogroup 1 in A/J Mouse Model / 감염과화학요법

BACKGROUND: Legionella pneumophila is the causative agent of Legionnaires' disease, a severe form of pneumonia. After L. pneumophila is inhaled through contaminated aerosols, it is phagocytized by alveolar macrophages, multiplies in a specialized phagosome approximately 10 h postinfection, and eventually leads to the death of host cells. Currently available diagnostic tests for Legionella pneumonia have some limitations. This study was conducted to find diagnostic biomarkers for Legionella pneumonia using virulence gene expression profiling in a murine experimental model. MATERIALS AND METHODS: A/J mice were intranasally inoculated with L. pneumophila serogroup 1, and lungs were harvested 4, 8, 24, and 48 h postinfection. The strain grown in buffered yeast extract broth was used as reference samples. Cy-dye labeled cDNA samples were prepared with total RNA from lungs or broth culture, and hybridized on the oligo-microarray slide containing 2,895 genes of L. pneumophila serogroup 1. Virulence gene expression patterns were analyzed using a MIDAS software from TIGR (www.tigr.org). RESULTS: Among a total of 332 virulence genes examined, 17 genes including sidA, lepB, the genes related to flagella assembly (fliR and fliP), LPS lipid A biosynthesis, and the enhanced entry protein EnhA were up-regulated at all four time points. We further confirmed by quantitative real-time reverse transcription PCR that the expression of fliP gene was highly expressed in lung tissue as well as in bronchoalveolar lavage fluids from the mouse infected with L. pneumophila serogroup 1. CONCLUSIONS: Through gene expression analysis of L. pneumophila in a mouse model, several candidate biomarkers for diagnosing Legionnaires' disease could be identified.

Porphyromonas gingivalis resistance to polymyxin B is determined by the lipid A 4'-phosphatase, PGN_0524 / 国际口腔科学杂志·英文版

<p><b>AIM</b>To elucidate the genetic basis for the pronounced resistance that the oral pathogen, Porphyromonas gingivalis (P. gingivalis), exhibits towards the cationic antimicrobial peptide, polymyxin B.</p><p><b>METHODOLOGY</b>A genetic screen of P. gingivalis clones generated by a Tn4400'-based random insertion mutagenesis strategy was performed to identify bacteria harboring novel genetic mutations that render P. gingivalis susceptible to killing by the cationic antimicrobial peptide, polymyxin B (PMB, 50 microg x mL(-1)).</p><p><b>RESULTS</b>P. gingivalis (ATCC 33277) is unusually resistant to the cationic antimicrobial peptide, PMB at relatively high concentrations (200 microg x mL(-1)). Approximately 2,700 independent Tn4400'-derived mutants of P. gingivalis were examined for increased sensitivity to PMB killing at a relatively low dose (50 microg x mL(-1)). A single PMB-sensitive mutant was obtained in this phenotypic screen. We determined that the Tn4400' transposon was integrated into the gene encoding the lipid A 4'-phosphatase, PGN_0524, demonstrating that this insertion event was responsible for its increased susceptibility of this clone to PMB-dependent killing. The resulting mutant strain, designated 0524-Tn4400', was highly sensitive to PMB killing relative to wild-type P. gingivalis, and exhibited the same sensitivity as the previously characterized strain, 0524KO, which bears a genetically engineered deletion in the PGN_0524 locus. Positive ion mass spectrometric structural (MALDI-TOF MS) analyses revealed that lipid A isolates from 0524-Tn4400' and 0524KO strains displayed strikingly similar MALDI-TOF MS spectra that were substantially different from the wildtype P. gingivalis lipid A spectrum. Finally, intact 0524-Tn4400' and 0524KO mutant bacteria, as well as their corresponding LPS isolates, were significantly more potent in stimulating Toll-like receptor 4 (TLR4)-dependent E-selectin expression in human endothelial cells relative to intact wild-type P. gingivalis or its corresponding LPS isolate.</p><p><b>CONCLUSION</b>The combined molecular evidence provided in this report suggests that PGN_0524, a lipid A 4'-phosphatase, is the sole genetic element conferring the ability of the periodontopathogen, P. gingivalis, to evade the killing activity of cationic antimicrobial peptides, such as PMB. These data strongly implicate PGN_0524 as a critical virulence factor for the ability of P. gingivalis to evade front-line host innate defenses that are dependent upon cationic antimicrobial peptide activity and TLR 4 sensing.</p>

Polymyxin B antagonizing biological activity of lipopolysaccharide / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the mechanism of polymyxin B (PMB) antagonizing the biological activity of lipopolysaccharide (LPS).</p><p><b>METHODS</b>The affinity of PMB for LPS and lipid A was assayed by biosensor, and the neutralization of PMB for LPS (2 ng/ml) was detected by kinetic turbidimetric limulus test. The releases of TNF-alpha and IL-6 in murine peritoneal macrophages a (PMphi) after exposure to LPS (100 ng/ml) were detected, and the expression levels of TLR4, TNF-alpha and IL-6 mRNA in PMphi induced by LPS (100 ng/ml) were measured by RT-PCR.</p><p><b>RESULTS</b>PMB had high-affinity to LPS and lipid A with dissociation equilibrium constants of 18.9 nmol/L and 11.1 nmol/L, respectively, and neutralized LPS in a dose-dependent manner. Furthermore, PMB could markedly inhibit the expressions of TLR4, TNF-alpha and IL-6 mRNA and the release of cycokines in LPS-stimulated murine peritoneal macrophages.</p><p><b>CONCLUSIONS</b>PMB neutralizes LPS and inhibites the expression and release of cycokines in macrophages, in which the affinity of PMB for lipid A plays an important role.</p>

Study of the anti-endotoxin effect of beta-1, 2, 3, 4, 6-penta-O-galloyl-D-glucopyranose in vitro / 中华烧伤杂志

<p><b>OBJECTIVE</b>To study the anti-endotoxin effect of beta-1, 2, 3, 4, 6-penta-O-galloyl-D-glucopyranose (PGG) in vitro.</p><p><b>METHODS</b>The affinity of PGG with lipid A was determined with biosensor technology, and the endotoxin-neutralizing effect was assayed with LAL. Human peripheral blood mononuclear cells (hPBMC) were separated from healthy donors and cultured in vitro. The effect of different concentrations of PGG on the release of TNF-alpha and hIL-6 from LPS-stimulated hPBMC were measured by ELISA method.</p><p><b>RESULTS</b>Lipid A was combined with different concentrations of PGG. The combination speed was shortened with the increase in PGG concentration. The KD value between PGG and Lipid A was 5.2 x 10(-7) mol/L. The release of TNF-alpha and IL-6 of hPBMC under LPS stimulation (958 +/- 234 ng/L vs 1 351 +/- 99 ng/L) was obviously inhibited by PGG in the concentration of higher than 20 mg/L compared with that without PGG treatment (1 788 +/- 171 ng/L vs 1 965 +/- 232 ng/L, P < 0.05).</p><p><b>CONCLUSION</b>PGG show an anti-endotoxin effect in vitro, which may be associated with its ability to combine and neutralize endotoxin.</p>

Study of biosensor technology on the detection of endotoxin-neutralizing materials / 中华烧伤杂志

<p><b>OBJECTIVE</b>To explore the application of biosensor technology in the determination of endotoxin-neutralizing materials.</p><p><b>METHODS</b>After mixing polymyxin B (PMB) with endotoxin in certain concentration, the neutralizing ratio of PMB to endotoxin was assessed by biosensor technique and limulus amebocyte lysate test respectively. The results from the two methods were compared.</p><p><b>RESULTS</b>The neutralizing ratio of PMB to endotoxin as assessed by biosensor technology was 0.35 microg to 1 ng, while that by dynamic turbidimetric and chromogenic limulus amebocyte lysate (LAL) technique was 0.5 mg to 1 ng and 1 mg to 1 ng, respectively. The results obtained by biotechnology were similar to that by biosensor technique.</p><p><b>CONCLUSION</b>Biosensor technology was an accurate, convenient and rapid method for the determination of potency of endotoxin-neutralizing materials.</p>

A Current Research Insight into Function and Development of Adjuvants

In recent years, adjuvants have received much attention because of the development of purified subunit and synthetic vaccines which are poor immunogens and require adjuvants to evoke the immune response. Therefore, immunologic adjuvants have been developed and testing for most of this century. During the last years much progress has been made on development, isolation and chemical synthesis of alternative adjuvants such as derivatives of muramyl dipeptide, monophosphoryl lipid A, liposomes, QS-21, MF-59 and immunostimulating complexes (ISCOMS). Biodegradable polymer microspheres are being evaluated for targeting antigens on mucosal surfaces and for controlled release of vaccines with an aim to reduce the number of doses required for primary immunization. The most common adjuvants for human use today are aluminum hydroxide and aluminum phosphate. Calcium phosphate and oil emulsions have been also used in human vaccination. The biggest issue with the use of adjuvants for human vaccines is the toxicity and adverse side effects of most of the adjuvant formulations. Other problems with the development of adjuvants include restricted adjuvanticity of certain formulations to a few antigens, use of aluminum adjuvants as reference adjuvant preparations under suboptimal conditions, non-availability of reliable animal models, use of non-standard assays and biological differences between animal models and humans leading to the failure of promising formulations to show adjuvanticity in clinical trials. The availability of hundreds of different adjuvants has prompted a need for identifying rational standards for selection of adjuvant formulations based on safety and sound immunological principles for human vaccines. The aim of the present review is to put the recent findings into a broader perspective to facilitate the application of these adjuvants in general and experimental vaccinology.

In-vivo 1H MR Spectroscopy in Abscessess Induced in Rabbit Thighs: Evaluation of Treatment Effect

PURPOSE: To analyze the serial changes of proton magnetic resonance (MR) spectra in the abscess and to determine the effect of the antibiotic treatment on the metabolite patterns. MATERIALS AND METHODS: MR imaging and MR spectroscopy of an experimentally induced abscess were performed sequentially for four weeks at interval of one week in both the control group (n=5) and the antibiotic treatment group (n=5). On MR imaging, the shape and the size of the abscess were analyzed. On MR spectroscopy, the resonance peaks of metabolites were assigned on the basis of reported peaks in the literature. The metabolite ratios measured by using N-acetyl alanine as an external reference and by using lipid as an internal reference were compared in both the control and treatment groups. RESULTS: The abscesses were seen as cystic masses on MR imaging. On MR spectroscopy, the variable peaks of acetate, succinate and various amino acids, which are the metabolites of infection, were identified in the control and antibiotic treatment groups. The most frequent peak was that of acetate at 1.92 ppm (70%). Both the peak ratios of acetate to lipid and acetate to external reference tended to decrease in the treatment group while the ratios did not change significantly in the control group. CONCLUSION: MR spectroscopy is useful not only for the diagnosis of abscess but also for monitoring the evolution of the abscess by using the acetate peak.

Monophosphoryl lipid A (MPL) upregulates major histocompatibility complex (MHC) class I expression by increasing interferon-gamma (IFN-gamma)

Tumor immunity is primarily mediated by cells as CD8+ cytotoxic T lymphocytes (CTL) recognize tumor antigen by MHC class I molecules. But most tumors are associated with a decreased expression of MHC class I to escape the antitumor immunity of the host. Our previous data have demonstrated that MPL has an antitumor effect on metastatic lung cancer of B16 melanoma with enhancing cytotoxicity due to increase of IFN-gamma and IL-2, and decrease of IL-4, which indicates the stimulation of type 1 helper T cells (Th1). To determine the effects of MPL, IFN-gamma, TNF-alpha, and IL-1 alpha on MHC class I expression of B16 melanoma cells, we evaluated the expression of MHC class I molecules with treatments of MPL, IFN-gamma, TNF-alpha, and IL-1 alpha by flow cytometry. The supernatant of MPL-treated spleen cells in vitro upregulated the expression of MHC class I molecules of B16 melanoma cells compared to the control supernatant of spleen cells. The MHC class I expression of B16 melanoma cells treated with IFN-gamma, but not TNF-alpha or IL-1 alpha, increased in a time-dependent manner. In conclusion, MPL upregulated MHC class I expression of B16 melanoma cells by activating spleen cells via IFN-gamma. These data suggest that increased IFN-gamma by MPL is responsible for the upregulation of MHC class I expression to augment cytotoxicity. Therefore, we suggest that MPL could play an important role in immunotherapy.