Chlamydial Lipopolysaccharide Research Articles

AAUS guidelines 2021 revision sexually transmitted infection (STIs) diagnostic strategy for STI

AAUS guidelines 2021 revision sexually transmitted infection (STIs) diagnostic strategy for STI

Metabolic adaptation of Chlamydia trachomatis to mammalian host cells.

Metabolic adaptation is a key feature for the virulence of pathogenic intracellular bacteria. Nevertheless, little is known about the pathways in adapting the bacterial metabolism to multiple carbon sources available from the host cell. To analyze the metabolic adaptation of the obligate intracellular human pathogen Chlamydia trachomatis, we labeled infected HeLa or Caco-2 cells with 13 C-marked glucose, glutamine, malate or a mix of amino acids as tracers. Comparative GC-MS-based isotopologue analysis of protein-derived amino acids from the host cell and the bacterial fraction showed that C. trachomatis efficiently imported amino acids from the host cell for protein biosynthesis. FT-ICR-MS analyses also demonstrated that label from exogenous 13 C-glucose was efficiently shuffled into chlamydial lipopolysaccharide probably via glucose 6-phosphate of the host cell. Minor fractions of bacterial Ala, Asp, and Glu were made de novo probably using dicarboxylates from the citrate cycle of the host cell. Indeed, exogenous 13 C-malate was efficiently taken up by C. trachomatis and metabolized into fumarate and succinate when the bacteria were kept in axenic medium containing the malate tracer. Together, the data indicate co-substrate usage of intracellular C. trachomatis in a stream-lined bipartite metabolism with host cell-supplied amino acids for protein biosynthesis, host cell-provided glucose 6-phosphate for cell wall biosynthesis, and, to some extent, one or more host cell-derived dicarboxylates, e.g. malate, feeding the partial TCA cycle of the bacterium. The latter flux could also support the biosynthesis of meso-2,6-diaminopimelate required for the formation of chlamydial peptidoglycan.

Resveratrol Inhibits Propagation ofChlamydia trachomatisin McCoy Cells

Resveratrol (RESV), an antifungal compound from grapes and other plants, has a distinct ability to inhibit the Chlamydia (C.) trachomatis developmental cycle in McCoy cells, a classic cell line used for chlamydial research. Inoculation of C. trachomatis with increasing amounts of RESV (from 12.5 to 100 μM) gave a dose-dependent reduction in the number of infected McCoy cells visualized by using monoclonal antibodies against chlamydial lipopolysaccharide. A similar trend has been observed with immunoassay for major outer membrane protein (MOMP). Furthermore, there was a step-wise reduction in the number of C. trachomatis infective progenies caused by the increasing concentrations of RESV. The ability of RESV to arrest C. trachomatis growth in McCoy cells was confirmed by a nucleic acid amplification protocol which revealed dose-dependent changes in mRNAs for different genes of chlamydial developmental cycle (euo, incA, and omcB). Although the precise nature of the antichlamydial activity of RESV is yet to be determined and evaluated in future studies, the observed effect of RESV on C. trachomatis infection was not related to its potential effect on attachment/entry of the pathogen into eukaryotic cells or RESV toxicity to McCoy cells. Similar inhibitory effect was shown for C. pneumoniae and C. muridarum.

ON THE ASSESSMENT AND TREATMENT OF PATIENTS WITH CHRONIC PROSTATITIS IN PERSISTENT UROGENITAL CHLAMYDIOSIS

Актуальность. Урогенитальный хламидиоз – наиболее распространенная бактериальная инфекция, передаваемая половым путем, сопровождающаяся высокой частотой осложнений (хронический простатит, сальпингоофорит и др.) и неблагоприятных последствий (нарушение половой и репродуктивной функций). В последние годы отмечается нарастание персистирующей хламидийной инфекции, которая не поддается лечению антибиотиками и диагностируется на основании обнаружения методом иммуноферментного анализа антител иммуноглобулина (Ig) класса G к белку теплового шока хламидий с молекулярной массой 60 кД (HSP60) и определения секреторного IgA к Chlamydia trachomatis в семенной плазме. Цель – выявление частоты пер- систирующей хламидийной инфекции у больных хроническим простатитом путем определения сывороточных антител IgG к HSP60 C. trachomatis и наличия противохламидийных IgA в семенной плазме. Материал и методы. В рамках проспективного исследования мы провели обследование 18 больных хроническим простатитом в возрасте от 21 до 58 лет, в контрольную группу вошли 30 человек без хламидийной урогенитальной патологии. Клиническое обследование включало сбор анамнеза, инструментальное обследование, ультразвуковое исследование предстательной железы и семенных пузырьков. Серологические исследования проводились с помощью иммуноферментного анализа в четырех тест-системах фирмы “Medac” (Германия) к двум антигенам: родоспецифическому липополисахариду хламидий r-ELISA (антитела классов IgM, IgG и IgA к Chlamydia) и основному белку наружной мембраны (МОМР) C. trachomatis (анти- тела классов IgG и IgA). Антитела к HSP60 хлами- дий определяли в тест-системе c HSP60-IgG-ELISA фирмы “Medac”. Определение секреторного IgA в семенной жидкости проводили с помощью тест-системы Chlamydia trachomatis-IgA-pELISA. Результаты. В основной группе в 13 (72,2%) из 18 случаев выявлен хронический хламидийный простатит, который в 7 (38,9%) случаях протекал на фоне персистирующей хламидийной инфекции, сопровождаясь одновременным повышением титра сывороточных антител IgG к HSP60 C. trachomatis и наличием противохламидийных антител IgA в семенной плазме. У 30 че- ловек контрольной группы сывороточные антитела IgG к C. trachomatis обнаружены не были. Заключение. Полученные данные подтверждают диагностическую ценность определения присутствия IgA к С. trachomatis с целью установления этиологии воспалительного процесса в мужском генитальном тракте, а также свидетельствуют о высокой частоте персистирующей хламидийной инфекции у больных хроническим простатитом и целесообразности использования серологического обследования на наличие сывороточных антител IgG к HSP60 C. trachomatis и противохламидийных IgA в семенной плазме.

M2 Polarization of Human Macrophages Favors Survival of the Intracellular Pathogen Chlamydia pneumoniae.

Intracellular pathogens have developed various strategies to escape immunity to enable their survival in host cells, and many bacterial pathogens preferentially reside inside macrophages, using diverse mechanisms to penetrate their defenses and to exploit their high degree of metabolic diversity and plasticity. Here, we characterized the interactions of the intracellular pathogen Chlamydia pneumoniae with polarized human macrophages. Primary human monocytes were pre-differentiated with granulocyte macrophage colony-stimulating factor or macrophage colony-stimulating factor for 7 days to yield M1-like and M2-like macrophages, which were further treated with interferon-γ and lipopolysaccharide or with interleukin-4 for 48 h to obtain fully polarized M1 and M2 macrophages. M1 and M2 cells exhibited distinct morphology with round or spindle-shaped appearance for M1 and M2, respectively, distinct surface marker profiles, as well as different cytokine and chemokine secretion. Macrophage polarization did not influence uptake of C. pneumoniae, since comparable copy numbers of chlamydial DNA were detected in M1 and M2 at 6 h post infection, but an increase in chlamydial DNA over time indicating proliferation was only observed in M2. Accordingly, 72±5% of M2 vs. 48±7% of M1 stained positive for chlamydial lipopolysaccharide, with large perinuclear inclusions in M2 and less clearly bordered inclusions for M1. Viable C. pneumoniae was present in lysates from M2, but not from M1 macrophages. The ability of M1 to restrict chlamydial replication was not observed in M1-like macrophages, since chlamydial load showed an equal increase over time for M1-like and M2-like macrophages. Our findings support the importance of macrophage polarization for the control of intracellular infection, and show that M2 are the preferred survival niche for C. pneumoniae. M1 did not allow for chlamydial proliferation, but failed to completely eliminate chlamydial infection, giving further evidence for the ability of C. pneumoniae to evade cellular defense and to persist in human macrophages.

Human blood monocytes support persistence, but not replication of the intracellular pathogen C. pneumoniae.

BackgroundIntracellular pathogens have devised various mechanisms to subvert the host immune response in order to survive and replicate in host cells. Here, we studied the infection of human blood monocytes with the intracellular pathogen C. pneumoniae and the effect on cytokine and chemokine profiles in comparison to stimulation with LPS.ResultsMonocytes purified from peripheral blood mononuclear cells by negative depletion were infected with C. pneumoniae. While immunofluorescence confirmed the presence of chlamydial lipopolysaccharide (LPS) in the cytoplasm of infected monocytes, real-time PCR did not provide evidence for replication of the intracellular pathogen. Complementary to PCR, C. pneumoniae infection was confirmed by an oligonucleotide DNA microarray for the detection of intracellular pathogens. Raman microspectroscopy revealed different molecular fingerprints for infected and non-infected monocytes, which were mainly due to changes in lipid and fatty acid content. Stimulation of monocytes with C. pneumoniae or with LPS induced similar profiles of tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6, but higher levels of IL-1β, IL-12p40 and IL-12p70 for C. pneumoniae which were statistically significant. C. pneumoniae also induced release of the chemokines MCP-1, MIP-1α and MIP-1β, and CXCL-8, which correlated with TNF-α secretion.ConclusionInfection of human blood monocytes with intracellular pathogens triggers altered cytokine and chemokine pattern as compared to stimulation with extracellular ligands such as LPS. Complementing conventional methods, an oligonucleotide DNA microarray for the detection of intracellular pathogens as well as Raman microspectroscopy provide useful tools to trace monocyte infection.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-014-0060-1) contains supplementary material, which is available to authorized users.

Synthesis of chlamydia lipopolysaccharide haptens through the use of α-specific 3-iodo-Kdo fluoride glycosyl donors.

A scalable approach towards high-yielding and (stereo)selective glycosyl donors of the 2-ulosonic acid Kdo (3-deoxy-D-manno-oct-2-ulosonic acid) is a fundamental requirement for the development of vaccines against Gram-negative bacteria. Herein, we disclose a short synthetic route to 3-iodo Kdo fluoride donors from Kdo glycal esters that enable efficient α-specific glycosylations and significantly suppress the elimination side reaction. The potency of these donors is demonstrated in a straightforward, six-step synthesis of a branched Chlamydia-related Kdo-trisaccharide ligand without the need for protecting groups at the Kdo glycosyl acceptor. The approach was further extended to include sequential iteration of the basic concept to produce the linear Chlamydia-specific α-Kdo-(2→8)-α-Kdo-(2→4)-α-Kdo trisaccharide in a good overall yield.

Interferon-γ expression in trophoblast cells in pregnant ewes challenged with Chlamydophila abortus

Pregnant ewes were challenged with Chlamydia abortus at 91-98 days of gestation and euthanised at 14, 21 and 28 days post-challenge. IFNγ mRNA labelling appeared to be co-localised with Chlamydial lipopolysaccharide within trophoblast cells in discrete areas lining the primary villi in the limbus and hilar zone of the placentomes from challenged sheep on days 21 and 28 post-infection. The presence of IFNγ was also demonstrated by immunohistochemistry. No labelling was seen in tissues from the non-infected ewes. The presence of IFNγ in trophoblast cells from infected ewes may indicate an attempt to restrict the replication of the organism and be an important trigger for the inflammatory responses that develop on the fetal side of the placenta in enzootic abortion.

Structural insights into parallel strategies for germline antibody recognition of lipopolysaccharide from Chlamydia

The structure of the antigen-binding fragment from the monoclonal antibody S64-4 in complex with a pentasaccharide bisphosphate fragment from chlamydial lipopolysaccharide has been determined by x-ray diffraction to 2.6 Å resolution. Like the well-characterized antibody S25-2, S64-4 displays a pocket formed by the residues of germline sequence corresponding to the heavy and light chain V gene segments that binds the terminal Kdo residue of the antigen; however, although S64-4 shares the same heavy chain V gene segment as S25-2, it has a different light chain V gene segment. The new light chain V gene segment codes for a combining site that displays greater affinity, different specificity, and allows a novel antigen conformation that brings a greater number of antigen residues into the combining site than possible in S25-2. Further, while antibodies in the S25-2 family use complementarity determining region (CDR) H3 to discriminate among antigens, S64-4 achieves its specificity via the new light chain V gene segment and resulting change in antigen conformation. These structures reveal an intriguing parallel strategy where two different combinations of germline-coded V gene segments can act as starting points for the generation of germline antibodies against chlamydial antigens and show how anti-carbohydrate antibodies can exploit the conformational flexibility of this class of antigens to achieve high affinity and specificity independently of CDR H3.

Monoclonal Antibodies Against Lipopolysaccharide ofChlamydia trachomatiswith Cross Reactivity to Human ApoB

Splenocytes obtained from mice immunized with whole purified elementary bodies of Chlamydia trachomatis were used for hybridoma construction. The resulting clones were screened with ELISA using chlamydial lipopolysaccharide (LPS) and full-length human apolipoprotein B (ApoB). One analyzed clone producing IgG1 (MAb 7B5) showed simultaneous recognition of chlamydial LPS and human ApoB, suggesting the presence of common antigenic epitopes in their structures. MAb 7B5 exhibited agreeable activity in immunoblot analysis conducted using chlamydial extracts or full-length human ApoB as well as in immunofluorescence (IF) detecting typical inclusion bodies of C. trachomatis and C. pneumoniae in the infected eukaryotic host cells. The removal of LPS from chlamydial suspensions with lauroyl sarcosyl led to a complete disappearance of IF associated with the elementary bodies of C. trachomatis. Therefore, immunologic response to chlamydial antigen may be associated with the generation of ApoB-specific antibody. Molecular mimicry and subsequent formation of cross-reactive antibodies might be an essential mechanism explaining the appearance of circulating auto-antibodies against low density lipoproteins (LDL) in patients with atherosclerosis. Moreover, newly generated MAb 7B5 can be a useful tool in the laboratory diagnosis of chlamydial infections.

A Common NH53K Mutation in the Combining Site of Antibodies Raised against Chlamydial LPS Glycoconjugates Significantly Increases Avidity

The crystal structures of the antigen-binding fragment of the murine monoclonal antibody (mAb) S25-39 in the presence of several antigens representing chlamydial lipopolysaccharide (LPS) epitopes based on the bacterial sugar 3-deoxy-α-D-manno-oct-2-ulosonic acid (Kdo) have been determined at resolutions from 2.4 to 1.8 Å. The antigen-binding site of this antibody differs from the well-characterized antibody S25-2 by a single mutation away from the germline of asparagine H53 to lysine, yet this one mutation results in a significant increase in avidity across a range of antigens. A comparison of the two antibody structures reveals that the mutated Lys H53 forms additional hydrogen bonds and/or charged-residue interactions with the second Kdo residue of every antigen having two or more carbohydrate residues. Significantly, the NH53K mutation results from a single nucleotide substitution in the germline sequence common among a panel of antibodies raised against glycoconjugates containing carbohydrate epitopes of chlamydial LPS. Like S25-2, S25-39 displays significant induced fit of complementarity determining region (CDR) H3 upon antigen binding, with the unliganded structure possessing a conformation distinct from those reported earlier for S25-2. The four different observed conformations for CDR H3 suggest that this CDR has evolved to exploit the recognition potential of a flexible loop while minimizing the associated entropic penalties of binding by adopting a limited number of ordered conformations in the unliganded state. These observations reveal strategies evolved to balance adaptability and specificity in the germline antibody response to carbohydrate antigens.

Chlamydia trachomatis antigens in enteroendocrine cells and macrophages of the small bowel in patients with severe irritable bowel syndrome

BackgroundInflammation and immune activation have repeatedly been suggested as pathogentic factors in irritable bowel syndrome (IBS). The driving force for immune activation in IBS remains unknown. The aim of our study was to find out if the obligate intracellular pathogen Chlamydia could be involved in the pathogenesis of IBS.MethodsWe studied 65 patients (61 females) with IBS and 42 (29 females) healthy controls in which IBS had been excluded. Full thickness biopsies from the jejunum and mucosa biopsies from the duodenum and the jejunum were stained with a monoclonal antibody to Chlamydia lipopolysaccharide (LPS) and species-specific monoclonal antibodies to C. trachomatis and C. pneumoniae. We used polyclonal antibodies to chromogranin A, CD68, CD11c, and CD117 to identify enteroendocrine cells, macrophages, dendritic, and mast cells, respectively.ResultsChlamydia LPS was present in 89% of patients with IBS, but in only 14% of healthy controls (p < 0.001) and 79% of LPS-positive biopsies were also positive for C. trachomatis major outer membrane protein (MOMP). Staining for C. pneumoniae was negative in both patients and controls. Chlamydia LPS was detected in enteroendocrine cells of the mucosa in 90% of positive biopsies and in subepithelial macrophages in 69% of biopsies. Biopsies taken at different time points in 19 patients revealed persistence of Chlamydia LPS up to 11 years. The odds ratio for the association of Chlamydia LPS with presence of IBS (43.1; 95% CI: 13.2-140.7) is much higher than any previously described pathogenetic marker in IBS.ConclusionsWe found C. trachomatis antigens in enteroendocrine cells and macrophages in the small bowel mucosa of patients with IBS. Further studies are required to clarify if the presence of such antigens has a role in the pathogenesis of IBS.

Chlamydia trachomatisgrowth inhibition and restoration of LDL-receptor level in HepG2 cells treated with mevastatin

BackgroundPerihepatitis is rare but consistently occurring extragenital manifestation of untreated Chlamydia trachomatis infection. Despite of possible liver involvement in generalized C. trachomatis infection, the ability of the pathogen to propagate in the hepatic cells and its impact on liver functions is not thoroughly investigated. The effect of mevastatin, an inhibitor of 3-hydroxy-3-methylglutaryl CoA reductase, on C. trachomatis growth in human hepatoma cell line HepG2 has been studied. Bacterial growth was assessed by immunostaining with FITC-labeled monoclonal antibody against chlamydial lipopolysaccharide and by RT-PCR for two chlamydial genetic markers (16S rRNA and euo).ResultsChlamydial inclusion bodies were seen in approximately 50% of hepatocytes at 48 hours in the post infection period. Lysates obtained from infected hepatocytes were positive in the infective progeny test at 48 and especially in 72 hours after infection initiation. It has been shown that chlamydial infection in hepatocytes also leads to the decline of LDL-receptor mRNA which reflects infection multiplicity rate. Additions of mevastatin (1, 20 and 40 μM) 1 hour before inoculation restored and upregulated LDL-receptor mRNA level in a dose-dependent manner. Mevastatin treatment had no effect on internalization of chlamydial particles. However it reduced drastically the number of chlamydial 16S rRNA and euo transcripts as well as overall infection rate in HepG-2 cells. Complete eradication of infection has been seen by immunofluorescent staining at 40 μM mevastatin concentration, when expression level of chlamydial 16S rRNA and euo was undetectable. Lower concentration of mevastatin (20 μM) promoted euo expression level and the appearance of atypically small chlamydial inclusions, while there was a noticeable reduction in the number of infected cells and 16S rRNA transcripts.ConclusionsC. trachomatis can efficiently propagate in hepatocytes affecting transcription rate of some liver-specific genes. Ongoing cholesterol synthesis is essential for chlamydial growth in hepatocytes. Inhibitors of cholesterol biosynthesis can supplement conventional strategy in the management of C. trachomatis infection.

Antibodies Raised Against Chlamydial Lipopolysaccharide Antigens Reveal Convergence in Germline Gene Usage and Differential Epitope Recognition

The structures of antigen-binding fragments from two related monoclonal antibodies have been determined to high resolution in the presence of several carbohydrate antigens raised against chlamydial lipopolysaccharide. With the exception of CDR H3, antibodies S54-10 and S73-2 are both derived from the same set of germline gene segments as the previously reported structures S25-2 and S45-18. Despite this similarity, the antibodies differ in specificity and the mechanism by which they recognize their cognate antigen. S54-10 uses an unrelated CDR H3 to recognize its antigen in a fashion analogous to S45-18; however, S73-2 recognizes the same antigen as S45-18 and S54-10 in a wholly unrelated manner. Together, these antibody-antigen structures provide snapshots into how the immune system uses the same set of inherited germline gene segments to generate multiple possible specificities that allow for differential recognition of epitopes and how unrelated CDR H3 sequences can result in convergent binding of clinically relevant bacterial antigens.

Chlamydial lipopolysaccharide (cLPS) is present in atherosclerotic and aneurysmal arterial wall—cLPS levels depend on disease manifestation

The role of Chlamydia pneumoniae in peripheral atherosclerosis disease and abdominal aortic aneurysm (AAA) remains unclear. Chlamydial lipopolysaccharide (cLPS) detection is a method used conventionally in routine chlamydial diagnosis of gynecological or ophthalmic samples. We compared cLPS concentrations, as well as other markers of bacterial load, in plaques and sera of patients operated on for carotid artery stenosis (n=110), aorto-occlusive disease (n=22), or AAAs (n=50) at the Helsinki University Central Hospital. The median levels of cLPS in plaques were 2.28, 0.80, and 0.29 ng/ml in AAA, aorto-occlusive disease, and carotid artery stenosis patients, respectively (P<.001, Kruskal-Wallis). cLPS in serum correlated with LPS binding protein levels (Spearman's rho=0.52, P<.001), suggesting that the presence of chlamydiae is sufficient to produce an innate immune response reaction in these patients. Serum inflammatory markers interleukin 6 and highly sensitive C-reactive protein also correlate with cLPS (Spearman's rho=0.42 and 0.51, respectively, P<.001). cLPS is present in arterial disease, and the potential role of C. pneumoniae in the pathogenesis of both peripheral atherosclerosis disease and AAA should not be forgotten. cLPS has a positive correlation with serum inflammatory markers, but this is no proof of a causal association.

Chlamydia pneumoniae DNA is present in peripheral blood mononuclear cells during acute coronary syndrome and correlates with chlamydial lipopolysaccharide levels in serum

Chlamydia pneumoniae can possibly trigger and maintain inflammation in coronary arteries. Chlamydia pneumoniae DNA and chlamydial lipopolysaccharide (cLPS) were measured 3 times during a 1-y period in 97 patients with acute coronary syndrome. Chlamydia pneumoniae DNA in peripheral blood mononuclear cells was detected in 8 (8.2%) patients at the initial hospitalization and in 9 (10.6%) patients at 3 months. One y after the acute coronary syndrome, Chlamydia pneumoniae DNA was not found in any patients. Serum cLPS levels were elevated at inclusion, and declined significantly during follow-up (1.40 microg/ml; (0.20-2.91), median; (range of 25th to 75th percentiles) at inclusion, 0.44 microg/ml; (0.00-1.39) at 1 y; ANOVA p<0.0001). cLPS levels correlated significantly to Chlamydia pneumoniae DNA positivity at 3 months (p=0.003). In conclusion, Chlamydia pneumoniae DNA is present during acute coronary syndrome and in the recovery period, but declines in stable state, suggesting a role of the bacterium in the acute phase of coronary syndrome.

Induction of Interleukin-18 in Atherosclerotic Patients: A Role for Chlamydia pneumoniae

Objectives: The present work explored gene expression and spontaneous induction of the inflammatory cytokine interleukin-18 (IL-18) in atherosclerotic patients. In addition, the effect of the chlamydial antigen heat shock protein 60 (HSP60) and lipopolysaccharide (LPS) on the induction of this mediator was examined. Subjects and Methods: Detection of IL-18 mRNA and protein level were assessed by in situ hybridization and immunohistochemistry, respectively, in 15 patients with coronary artery disease undergoing angiograms and 15 matching controls. Results: These experiments showed significantly high levels of spontaneously expressed IL-18 mRNA and high protein levels in patients compared to healthy controls (p < 0.0005). Cells stimulated with chlamydial HSP60 (CHSP60) and LPS showed a significantly high expression of IL-18 at the mRNA level (p < 0.0005 for CHSP60 and p < 0.005 for LPS) and an increased production of IL-18 at protein level (p < 0.0005 for CHSP60 and p < 0.005 for LPS). Conclusion: This study demonstrated de novo synthesis of the inflammatory cytokine IL-18 in atherosclerosis and, furthermore, that chlamydial antigens might play a role in the immunopathological events in this disease by generating more inflammatory mediators such as IL-18.

Parachlamydia acanthamoebaeInfection and Abortion in Small Ruminants

<i>Parachlamydia acanthamoebae</i>Infection and Abortion in Small Ruminants

Chlamydial LPS and high-sensitivity CRP levels in serum are associated with an elevated body mass index in patients with cardiovascular disease

Seropositivity for Chlamydia pneumoniae has been associated with an elevated body mass index (BMI). Our aim was to study if serum chlamydial lipopolysaccharide (cLPS), C. pneumoniae antibodies and high-sensitivity C-reactive protein (hsCRP) levels are associated with BMI. The study population consisted of 174 patients with symptomatic carotid stenosis, abdominal aortic aneurysm or occlusive aortic disease. Information on BMI, diabetes, smoking, hypercholesterolemia, and statin medication was available. Serum C. pneumoniae IgG and IgA antibodies, cLPS, hsCRP and total endotoxin activity (totLPS) were measured. BMI correlated with cLPS (r = 0.197; P < 0.01) and with hsCRP (rho = 0.195; P < 0.01); in addition, there was a positive correlation between cLPS and hsCRP (rho = 0.499; P < 0.01). A trend of an increasing proportion of C. pneumoniae IgG positivity (titre > or = 64; P = 0.018) and higher serum cLPS (P = 0.01) and hsCRP (P = 0.01) concentrations was observed across the BMI groups (BMI < or = 24.9 kg/m(2), BMI = 25.0-29.9 kg/m(2), and BMI > or = 30.0 kg/m(2)). Among the three BMI groups, 24.6%, 38.8%, and 48.3% were C. pneumoniae IgG-positive and the median (IQR) cLPS concentrations (ng/ml) of the groups were: 92.6 (50.8-167.0), 128.9 (76.4-163.9), and 146.4 (105.8-175.8), respectively. The median (IQR) hsCRP (mg/l) concentrations of the groups were: 1.70 (0.70-3.05) 1.70 (0.80-5.20), and 3.40 (1.45-8.55), respectively. These associations remained statistically significant in a multivariate analysis. Elevated serum cLPS levels were associated with an elevated BMI. This is a novel finding and it strengthens the link between chlamydial infection and obesity. A lack of association between totLPS and BMI suggests that the association between infection and an elevated BMI may be specific to certain pathogens.

Pseudo-symmetry and twinning in crystals of homologous antibody Fv fragments

A difference of seven conservative amino-acid substitutions between two single-chain antibodies (scFvs) specific for chlamydial lipopolysaccharide does not significantly affect their molecular structures or packing contacts, but dramatically affects their crystallization. The structure of the variable domain (Fv) of SAG173-04 was solved to 1.86 A resolution and an R(cryst) of 18.9% in space group P2(1)2(1)2(1). Crystals of the homologous SAG506-01 diffracted to 1.95 A resolution and appeared at first to have Patterson symmetry I4/m or P4/mmm; however, no solution could be found in space groups belonging to the former and refinement in the only solution corresponding to the latter (in space group P4(3)2(1)2) stalled at R(free) = 30.0%. Detailed examination of the diffraction data revealed that the crystal was likely to be twinned and that the correct space group was P2(1)2(1)2(1). Both translational pseudo-symmetry and pseudo-merohedral twinning were observed in one crystal of SAG506-01 and pseudo-merohedral twinning was observed for a second crystal. The final R factor for SAG506-01 after refinement in P2(1)2(1)2(1) was 20.5%.