Frank M. Unger

Artificial antigens. Synthesis of polyacrylamide copolymers containing 3-deoxy-d-manno-2-octulopyranosylonic acid (KDO) residues

Starting from an anomeric mixture of methyl (allyl 4,5,7,8-tetra-O-acetyl-3-deoxy-alpha- and -beta-D-manno-2-octulopyranosid)onates, the glycosides sodium (allyl 3-deoxy-alpha- and -beta-D-manno-2-octulopyranosid)onate, sodium O-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-(2----4)-[allyl 3-deoxy-alpha-D-manno-2-octulopyranosid]onate and sodium (allyl 3-deoxy-7-O-beta-D-ribofuranosyl-beta-D-manno-2-octulopyranosid)++ +onate were prepared in several steps. Radical copolymerization of the allyl glycosides with acrylamide afforded linear macromolecular antigens containing mono- and di-saccharide residues corresponding to the KDO-region of Salmonella minnesota rough-form lipopolysaccharide and to partial structures of the capsular polysaccharide from Escherichia coli K 23, respectively. The copolymers were substituted by KDO-residues in a ratio of 1:18 +/- 2 (based on acrylamide) and had molecular masses of 60-100 kdaltons.

Randomized, double-blind, placebo-controlled study of oral lactobacilli to improve the vaginal flora of postmenopausal women.

OBJECTIVE The purpose of this randomized, double-blind, placebo-controlled study was to evaluate the influence of the orally administered probiotic strains Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 on the quality of the vaginal flora in postmenopausal women. STUDY DESIGN Postmenopausal women with Nugent scores between 4 and 6 in initial vaginal swab, were randomized into two groups. Women in the intervention group received probiotic capsules containing 2.5x10(9)CFU (colony forming units) each of lyophilized L. rhamnosus GR-1 and L. reuteri RC-14 and women in the control group received an oral placebo once daily, in both groups for 14 days. Final vaginal swabs were taken 1 day after the last administration of the medication. The primary efficacy variable was a change in the Nugent score between baseline and the end of the study of at least two grades in each individual patient. RESULTS Seventy two women were recruited in the study, 35 assigned to the intervention group and 37 to the control group. Twenty-one of the 35 subjects (60%) in the intervention group and 6 of the 37 subjects (16%) in the control group showed a reduction in the Nugent score by at least two grades. The difference in the number of patients with improvement was highly significant (p=0.0001). The median difference in Nugent scores between baseline and the end of the study was 3 in the intervention group and 0 in the control group (p=0.0001). CONCLUSION Our results provide evidence for an alternative modality to restore the normal vaginal flora using specific probiotic strains administered orally.

Selection of reliable reference genes for qPCR studies on chondroprotective action

BackgroundChondroprotective agents (CPA) such as glucosamine, curcumin and diacerein represent potential remedies for the management of osteoarthritis and several studies have been performed on their effects in-vitro and in-vivo. For the investigation of chondroprotective action on chondrocyte gene expression, quantitative real-time RT-PCR is the method of choice. However, validation of applied normalization strategies represents a crucial and sometimes neglected step in the analysis process. Therefore, the present study aimed to determine the expression stability of common reference genes (ACTB, Beta actin; GAPDH, Glyceraldehyde-3-phosphate; B2M, Beta-2-microglobulin; HPRT1, Hypoxanthine phosphoribosyl-transferase I; SDHA, Succinate dehydrogenase complex, subunit A; YWHAZ, Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide) under the influence of glucosamine, curcumin and diacerein in the IL-1β-stimulated C-28/I2 chondrocyte model, using the geNorm software tool.ResultsCPA treatment of C-28/I2 chondrocytes significantly affected the expression level of many reference genes (p < 0.05). According to their expression stability, geNorm analysis revealed rankings of the 3 most stable genes (from most stable to least stable) as follows: GAPDH, B2M and SDHA in glucosamine treated samples and HPRT1, GAPDH and B2M in curcumin or diacerein treated samples. Interestingly, ACTB was one of the most variably expressed genes throughout all experiments.ConclusionOur study points out the problem of relying on commonly used reference genes without an accurate validation process. For normalization purposes in gene profiling studies on glucosamine action, the genes GAPDH, B2M and SDHA are recommended as single reference genes depending on the expression level of the target gene or more favourably in combination. For experiments with curcumin and diacerein the use of HPRT1, GAPDH and B2M should be considered.

Structure of de-o-acylated lipopolysaccharide from the escherichia coli re mutant strain F 515

Abstract The structure of the oligosaccharide portion of an E.coli Re lipopolysaccharide was determined as α-KDO-(2→4)-α-KDO-(2→6)-β-GlcN-α(1→6)-α-GlcN, bisphosphorylated at positions 1 and 4′. Taking into account the previous determination of the acylation pattern of the GlcN disaccharide, the total structure of E.coli Re LPS was thus established.

Comparison between chondroprotective effects of glucosamine, curcumin, and diacerein in IL-1β-stimulated C-28/I2 chondrocytes

OBJECTIVE To compare the effects of glucosamine (GlcN), curcumin, and diacerein in immortalized human C-28/I2 chondrocytes at the cellular and the gene expression level. This study aimed to provide insights into the proposed beneficial effects of these agents and to assess the applicability of the C-28/I2 cell line as a model for the evaluation of chondroprotective action. METHODS Interleukin-1beta (IL-1beta)-stimulated C-28/I2 cells were cultured in the presence of GlcN, curcumin, and diacerein prior to the evaluation of parameters such as viability, morphology and proliferation. The impact of GlcN, curcumin, and diacerein on gene expression was determined using quantitative real-time RT-PCR (qPCR). RESULTS At the transcriptional level, 5 mM GlcN and 50 microM diacerein increased the expression of cartilage-specific genes such as aggrecan (AGC) and collagen type II (COL2), while reducing collagen type I (COL1) mRNA levels. Moreover, the IL-1beta-mediated shift in gene expression pattern was antagonized by GlcN and diacerein. These effects were associated with a significant reduction in cellular proliferation and the development of chondrocyte-specific cell morphology. In contrast, curcumin was not effective at lower concentrations but even damaged the cells at higher amounts. CONCLUSIONS Both GlcN and diacerein promoted a differentiated chondrocytic phenotype of immortalized human C-28/I2 chondrocytes by altering proliferation, morphology, and COL2/COL1 mRNA ratios. Moreover, both agents antagonized inhibitory effects of IL-1beta by enhancing AGC and COL2 as well as by reducing COL1 mRNA levels.

Apoptosis of human breast cancer cells induced by microencapsulated betulinic acid from sour jujube fruits through the mitochondria transduction pathway

Betulinic acid (BA), a natural pentacyclic triterpenoid, was isolated from sour jujube fruits for the first time. An inclusion complex comprising BA and β-cyclodextrin (β-CD) was formed to improve the dissolution of BA, but little is known about its anticancer effect. In this study, the anti-proliferative and apoptosis mechanisms of BA-β-CD on human breast cancer MCF-7 cells were further investigated. Experimental results confirmed that the complexation model inhibited the growth of MCF-7 cells in a dose-dependent manner, arrested cell cycle in the G2/M phase and induced apoptosis via the mitochondria transduction pathway. Gene and protein analyses showed that the complexation model significantly inhibited Bcl-2 expression and promoted Bax expression, causing caspase-3 and caspase-9 cascade activation. These findings corroborated evidence on microencapsulated BA as an apoptosis inducer in MCF-7 cells. Thus, sour jujube fruits may have potential use as a breast cancer chemotherapeutic agent.

Immunoreactivity of allergen (Bet v 1) conjugated to crystalline bacterial cell surface layers (S-layers)

BACKGROUND Crystalline cell surface layers (S-layers) from Gram-positive eubacteria had been demonstrated as carrier/adjuvants for chemically synthesized tumor-associated oligosaccharide haptens and capsular polysaccharide antigens of Streptococcus pneumoniae strains. OBJECTIVES The applicability of S-layers as vaccine carrier for treatment of Type I allergy was investigated. STUDY DESIGN Native or cross-linked S-layer self-assembly products and cell wall preparations from Bacillus sphaericus CCM 2177 and Thermoanaerobacter thermohydrosulfuricus L111-69 and L110-69 were used for immobilization of recombinant major birch pollen allergen Bet v 1. RESULTS AND CONCLUSIONS Depending on the carrier used, amounts of approximately 20-40 micrograms allergen per mg conjugate could be immobilized. By application of L-glutamic acid dimethyl ester as a spacer, this value could be increased approximately 10-fold. The functionality of the rBet v 1-conjugates was assessed in immunological systems. (i) The presence of intact B-cell epitopes was demonstrated in inhibition experiments using human Bet v 1-specific IgE. (ii) The rBet v 1-S-layer conjugates were immunogenic in mice. (iii) The proliferation of rBet v 1-specific T-cell clones suggested that the peptides created by processing of immobilized Bet v 1 were similar to those derived from natural allergen. (iv) Stimulation of human allergen-specific TH2 lymphocytes with S-layer-conjugated Bet v 1 led to a modulation of the cytokine production pattern from TH2 to TH0/TH1. This study indicates that S-layers may be suitable carriers for few immunotherapeutical vaccines for Type 1 hypersensitivity.

Enzymic synthesis of 5-acetamido-9-azido-3,5,9-trideoxy--glycero--galacto-2-nonulosonic acid, a 9-azido-9-deoxy derivative of N-acetylneuraminic acid

Abstract N-Acetylneuraminate synthase from Neisseria meningitidis 6OE catalyzes the conversion of phosphoenolpyruvate and 2-acetamido-6-azido-2,6-dideoxy- -mannose into 5-acetamido-9-azido-3,5,9-trideoxy- - glycero - - galacto -2-nonulosonic acid. The product, a 9-azido-9-deoxy derivative of N-acetylneuraminic acid, is indistinguishable from a chemically synthesized sample by i. the thiobarbituric acid assay, ii. paper electrophoresis, and iii. paper electrophoresis following sodium borohydride reduction. Both the chemically and the enzymically synthesized samples are substrates of the reaction catalyzed by CTP:CMP-N-acetylneuraminate cytidylyl-transferase from Neisseria meningitidis 6OE

Anti-inflammatory activity of an ethanolic Caesalpinia sappan extract in human chondrocytes and macrophages

ETHNOPHARMACOLOGICAL RELEVANCE Caesalpinia sappan is a common remedy in Traditional Chinese Medicine and possesses diverse biological activities including anti-inflammatory properties. Osteoarthritis (OA) is a degenerative joint disease with an inflammatory component that drives the degradation of cartilage extracellular matrix. In order to provide a scientific basis for the applicability of Caesalpinia sappan in arthritic diseases, the present study aimed to assess the effects of an ethanolic Caesalpinia sappan extract (CSE) on human chondrocytes and macrophages. MATERIALS AND METHODS Primary human chondrocytes were isolated from cartilage specimens of OA patients. Primary cells, SW1353 chondrocytes and THP-1 macrophages were serum-starved and pretreated with different concentrations of CSE prior to stimulation with 10 ng/ml of interleukin-1beta (IL-1β) or lipopolysaccharide (LPS). Following viability tests, nitric oxide (NO) and tumor necrosis factor-alpha (TNF-α) were evaluated by Griess assay and ELISA, respectively. Using validated real-time PCR assays, mRNA levels of IL-1β, TNF-α, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) were quantified. SW1353 cells were cotransfected with a COX-2 luciferase reporter plasmid and nuclear factor-kappa-B (NF-κB) p50 and p65 expression vectors in the presence or absence of CSE. RESULTS CSE dose-dependently inhibited the expression of pro-inflammatory cytokines IL-1β and TNF-α in IL-1β-stimulated chondrocytes and LPS-stimulated THP-1 macrophages. CSE further suppressed the synthesis of NO in primary OA chondrocytes by blocking iNOS mRNA expression. The inhibition of COX-2 transcription was found to be related with the CSE inhibition of the p65/p50-driven transactivation of the COX-2 promoter. CONCLUSIONS The present report is first to demonstrate the anti-inflammatory activity of CSE in an in vitro cell model of joint inflammation. CSE can effectively abrogate the IL-1β-induced over-expression of inflammatory mediators at the transcriptional level in human chondrocytes and macrophages, most likely by inhibiting NF-κB (p65/p50) signaling. Blockade of IL-1β-induced NF-κB signaling and its downstream pro-inflammatory targets by CSE may be beneficial for reducing cartilage breakdown in arthritis.

Induction of T-cell immunity to oligosaccharide antigens immobilized on crystalline bacterial surface layers (S-layers)

Immunization of Balb/c mice with conjugates of oligosaccharide haptens and crystalline bacterial surface-layer proteins (S-layers) primed the mice for a strong, hapten-specific, delayed-type hypersensitivity (DTH) response. Conjugates of haptens with bovine serum albumin produced only weak DTH responses but, when mixed with aluminium hydroxide, elicited DTH responses comparable to those against S-layer conjugates. Surface-layer conjugates also elicited strong anti-hapten DTH responses when administered by an oral/nasal route. Apparently, the natural assembly of S-layer proteins into large, two-dimensional arrays endows them with intrinsic adjuvant properties.