Intra-articularly localized bacterial DNA containing CpG motifs induces arthritis.

Unmethylated CpG motifs are often found in bacterial DNA, and exert immunostimulatory effects on hematopoietic cells. Bacteria produce severe joint inflammation in septic and reactive arthritides; bacterial DNA may be involved in this process. We injected bacterial DNA originating from Escherichia coli and Staphylococcus aureus and oligonucleotides containing CpG directly into the knee joints of mice of different strains. Arthritis was seen by histopathology within 2 hours and lasted for at least 14 days. Unmethylated CpG motifs were responsible for this induction of arthritis, as oligonucleotides containing these motifs produced the arthritis. The arthritis was characterized by an influx of monocytic, Mac-1+ cells and by a lack of T lymphocytes. Depletion of monocytes resulted in abrogation of the synovial inflammation. Tumor necrosis factor (TNF)-alpha, a cytokine produced by cells of the monocyte/macrophage lineage, is an important mediator of this disease, as expression of mRNA for TNF-alpha was evident in the inflamed joints, and the CpG-mediated inflammation was abrogated in mice genetically unable to produce this cytokine. These findings demonstrate that bacterial DNA containing unmethylated CpG motifs induces arthritis, and indicate an important pathogenic role for bacterial DNA in septic arthritis.

Staphylococcus aureus resistance to human defensins and evasion of neutrophil killing via the novel virulence factor MprF is based on modification of membrane lipids with l-lysine.

Defensins, antimicrobial peptides of the innate immune system, protect human mucosal epithelia and skin against microbial infections and are produced in large amounts by neutrophils. The bacterial pathogen Staphylococcus aureus is insensitive to defensins by virtue of an unknown resistance mechanism. We describe a novel staphylococcal gene, mprF, which determines resistance to several host defense peptides such as defensins and protegrins. An mprF mutant strain was killed considerably faster by human neutrophils and exhibited attenuated virulence in mice, indicating a key role for defensin resistance in the pathogenicity of S. aureus. Analysis of membrane lipids demonstrated that the mprF mutant no longer modifies phosphatidylglycerol with l-lysine. As this unusual modification leads to a reduced negative charge of the membrane surface, MprF-mediated peptide resistance is most likely based on repulsion of the cationic peptides. Accordingly, inactivation of mprF led to increased binding of antimicrobial peptides by the bacteria. MprF has no similarity with genes of known function, but related genes were identified in the genomes of several pathogens including Mycobacterium tuberculosis, Pseudomonas aeruginosa, and Enterococcus faecalis. MprF thus constitutes a novel virulence factor, which may be of general relevance for bacterial pathogens and represents a new target for attacking multidrug resistant bacteria.

The rat antigen-presenting lectin-like receptor complex influences innate immunity and development of infectious diseases.

Genetic variation in the antigen-presenting lectin-like receptor gene complex (APLEC) associates with autoimmunity and arthritis in rats and humans. We hypothesized that the encoded C-type lectin-like receptors might influence innate immunity and responses to infectious agents. To test this hypothesis, we compared in vivo and in vitro phenotypes in DA rats and APLEC-congenic rats. Survival rates following infection with Staphylococcus aureus and Herpes simplex virus differed significantly between the two strains. Likewise, differential delayed type hypersensitivity (DTH), an immunological reaction involving T lymphocytes and macrophages, was observed in response to provocation with the chemical oxazolone. Unstimulated bone marrow-derived macrophages from the two strains appeared to already have polarized activation states with different mRNA levels of CD163 and Dectin-1 receptors. Following stimulation with a panel of microbial agents, differences in induced mRNA and protein levels were shown for interleukin (IL)-6 and IL-10 following stimulation with lipopolysaccharide, mannan and beta-glucan. Expression levels of APLEC gene mRNAs also differed, and both strains had a notably dichotomous expression of the genes, with general downregulation of all four Dcir genes and upregulation of Mincle and Mcl. We suggest that human APLEC genes may similarly regulate infectious diseases, DTH and general macrophage activation status.

Impact of short-term therapies with biologics on prothrombotic biomarkers in rheumatoid arthritis.

BACKGROUND: Imbalance of haemostasis in patients with rheumatoid arthritis (RA) contributes to future risk of cardiovascular diseases (CVD). Prothrombotic molecules, e.g. fibrinogen, D-dimer, and tPA are elevated in plasma of RA patients, being associated to CVD. There is no imformation about the influence of biological drugs, e.g. anti-CD20 and tumor necrosis factor (TNF) antibodies on these prothrombotic molecules. OBJECTIVE: To assess whether anti-TNF and anti-CD20 therapies modify the profiles of cardiovascular risk factors in patients with RA. METHODS: The expression of prothrombotic molecules in plasma was investigated in 10 RA patients before and after treatment with TNF-alpha antibodies and in another 12 RA patients before and after anti-CD20 treatment. RESULTS: Both anti-TNF and anti-CD20 infusions gave rise to clear clinical improvement. However, only anti-CD20 infusion significantly (p=0.05) reduced concentration of fibrinogen (p=0.05), D-dimer (p<0.001), as well as tPA levels (p<0.01). In contrast, in TNF antibody treated patients only tPA levels were significantly decreased following the treatment (p<0.05). CONCLUSIONS: Infusion of CD20 antibodies to the patients with active RA led to a clearly reduced plasma levels of predictors of CVD indicating that this treatment, apart from its anti-inflammatory properties, may reduce the risk for future CVD in RA.

The impact of substance P signalling on the development of experimental staphylococcal sepsis and arthritis.

Substance P (SP), acting on the neurokinin-1 receptor (NK-1R), is a neuropeptide, involved in the inflammatory processes. It promotes vasodilatation and increases vasopermeability, thus ensuing extravasation and accumulation of leucocytes at sites of injury. The aim of this study was to assess the impact of SP signalling on the responses during staphylococcal infection and the accompanying arthritis. Three experiments were performed where NK-1R-/- mice and controls were intravenously infected with different doses of Staphylococcus aureus. Clinical assessment of arthritis was performed as well as histological analysis of bone and cartilage destruction in the joints. In addition, the impact of NK-1R mutation on bacterial load in the kidneys as well as the phagocytic capacity of blood leucocytes were studied. Mice lacking the NK-1R displayed significantly higher bacterial load in the kidneys and significantly more severe synovitis and cartilage/bone destruction than the controls when inoculated with 1.4 x 10(7) staphylococci. Infection with 3.5 x 10(8) CFU/mouse induced sepsis. Thus, 11 days after bacterial inoculation 15 of 19 mice in the NK-1R-/- group had died versus 8 of 15 in the control group. Phagocytosis test revealed that significantly fewer macrophages from NK-1R-/- mice were able to phagocytose S. aureus when compared with macrophages from congenic control mice. Blocking the biological responses to substance P via its receptor NK-1R results in a less efficient clearance of bacteria leading to more severe arthritic lesions in mice.

Circulating survivin indicates severe course of juvenile idiopathic arthritis.

BACKGROUND: Survivin is an anti-apoptotic protein that has been recently suggested as a predictive marker of joint destruction in adult rheumatoid arthritis. We assessed the presence of extracellular survivin in patients with juvenile idiopathic arthritis (JIA). METHODS: Survivin levels were assessed in the circulation of 46 patients with JIA and in the age- and gender-matched controls (n=46) having no inflammatory disease, by ELISA. Survivin levels were analyzed with respect to the onset type and the activity of the joint disease. The intensity of inflammation and cartilage turnover was measured as levels of IL-6, serum amyloid A protein (SAA), and cartilage oligomeric matrix protein (COMP), respectively. RESULTS: The levels of extracellular survivin were significantly higher in JIA compared to the controls (p=0.0002). High levels of survivin (above mean + 2SD of the controls) were detected in 8/46 (17% JIA patients. High survivin expression was associated with polyarticular onset, active phase of arthritis. In contrast, survivin was neither related to the levels of IL-6, SAA, nor to COMP. CONCLUSION: Circulating survivin is expressed in a significant group of patients with JIA being associated to a severe course of the disease. It may be potentially used to select children with unfavorable prognosis of JIA who are in need of active pharmacologic treatment.

Vaccination with a recombinant fragment of collagen adhesin provides protection against Staphylococcus aureus-mediated septic death.

Staphylococcus aureus is a major cause of nosocomial and community-acquired infections. Morbidity and mortality due to infections such as sepsis, osteomyelitis, septic arthritis, and invasive endocarditis remain high despite the use of antibiotics. The emergence of antibiotic resistant super bugs mandates that alternative strategies for the prevention and treatment of S. aureus infections are developed. We investigated the ability of vaccination with a recombinant fragment of the S. aureus collagen adhesin to protect mice against sepsis-induced death. Actively immunized NMRI mice were intravenously inoculated with the S. aureus clinical isolate strain Phillips. 14 d after inoculation, mortality in the collagen adhesin-vaccinated group was only 13%, compared with 87% in the control antigen immunized group (P < 0.001). To determine if the protective effect was antibody mediated, we passively immunized naive mice with collagen adhesin-specific antibodies. Similar to the active immunization strategy, passive transfer of collagen adhesin-specific antibodies protected mice against sepsis-induced death. In vitro experiments indicated that S. aureus opsonized with sera from collagen adhesin immunized mice promoted phagocytic uptake and enhanced intracellular killing compared with bacteria opsonized with sera from control animals. These results indicate that the collagen adhesin is a viable target in the development of immunotherapeutics against S. aureus.

TNF-alpha mediated suppression of tissue type plasminogen activator expression in vascular endothelial cells is NF-kappaB- and p38 MAPK-dependent.

BACKGROUND: Several proatherothrombotic conditions are associated with enhanced levels of circulating proinflammatory cytokines, which are believed to impair endothelial fibrinolytic capacity. OBJECTIVE: This study aims at investigating how tumor necrosis factor (TNF)-alpha regulates endothelial gene expression of the key fibrinolytic enzyme tissue-type plasminogen activator (t-PA). METHODS: Cultured human umbilical vein endothelial cells were pretreated with selective inhibitors of the three major inflammatory signaling pathways activated by TNF-alpha; the nuclear factor kappa-B (NF-kappaB), the p38 mitogen-activated protein kinase (p38 MAPK), and the c-jun N-terminal kinase (JNK) pathways. Following TNF-alpha stimulation, effects on t-PA gene expression were evaluated with real-time reverse transcriptase polymerase chain reaction and interactions of nuclear proteins with potential gene regulatory elements were studied with electrophoretic mobility shift assays. RESULTS: Approximately 50% suppression of t-PA gene expression was observed after prolonged stimulation with TNF-alpha (> or =24 h). The repression was shown to be preferentially dependent on NF-kappaB activation, but also on p38 MAPK signaling. Further, we provide evidence for a TNF-alpha induced binding of NF-kappaB to the recently described kappaB site in the t-PA gene and of cyclic adenosine monophosphate response element binding protein (CREB) to the t-PA CRE-like site. CONCLUSIONS: We conclude that TNF-alpha impairs fibrinolytic capacity in vascular endothelial cells by a NF-kappaB and p38 MAPK-dependent suppression of t-PA. This mechanism sheds a light on how inflammation contributes to the pathogenesis of cardiovascular diseases.

Model systems: modeling human staphylococcal arthritis and sepsis in the mouse.

The staphylococci have been recognized as serious pathogens for over a century and are the etiological agent of a variety of diseases ranging from mild cutaneous infections to often fatal forms of septic arthritis, endocarditis, toxic shock syndrome and sepsis. Despite intensive efforts to halt their spread, they remain the most common cause of community- and nosocomially acquired bacteremia. Murine models of Staphylocococus aureus-mediated arthritis and sepsis exist and are being used to gain a better understanding of the host-bacterium relationship as well to develop better methods of prevention and treatment.

Mouse chimaeras revisited: recollections and reflections.

This very personal and subjective article briefly describes the evolution of techniques used for obtaining mouse chimaeras from various sources (embryos, EC, ES and EG cells), summarizes studies on inter-specific chimaeras, mentions some of other applications ('rescuing' chimaeras), presents the contribution of Ralph Brinster to this area and tries to estimate whether the expectations I expressed in 1961 as to the usefulness of making and studying chimaeras turned out to be correct. Tribute is paid mainly to those, who as the first, contributed to various aspects of these studies.

How many blastomeres of the 4-cell embryo contribute cells to the mouse body?

The aim of this study was to estimate how many blastomeres of the 4-cell mouse embryo contribute cells to the embryo proper and finally to the animal. To this end, 4-cell embryos of pigmented and albino genotypes were disaggregated and single blastomeres (henceforth called '1/4' or 'quarter' blastomeres) were reaggregated in the following combinations: one 'pigmented' blastomere + three 'albino' blastomeres or vice versa (henceforth called '1+3') and two pigmented blastomeres + two albino blastomeres (henceforth called '2+2'). The aggregations were cultured in vitro and transferred as blastocysts either to the oviduct or uterus of pseudopregnant females. Recipients were allowed to litter naturally, or the foetuses were removed by Caesarian section and raised by lactating foster mothers. Chimaerism was assessed on the basis of coat (adults) or eye pigmentation (dead neonates). Among 28 '1+3' animals, there were 13 chimaeric and 15 non-chimaeric individuals. The pigmentation of non-chimaeras was always concordant with the genotype of the three 1/4 blastomeres and not with the genotype of the single blastomere in the given aggregation. These results make rather unlikely the possibility that the mouse is built of cells derived either from one or all four 1/4 blastomeres. Both two remaining options (2 or 3 1/4 blastomeres) are conceivable but the observed ratio of chimaeras to non-chimaeras among '1+3' animals (13:15) fits better the assumption of two 1/4 blastomeres contributing cells to the animal body. This assumption finds additional support in the observation that among '2+2' animals there were non-chimaeras (5 out of 7) and these would not have been expected should three 1/4 blastomeres contribute cells to the mouse body.

Mouse singletons and twins developed from isolated diploid blastomeres supported with tetraploid blastomeres.

The aim of this study was to obtain mice, hopefully identical multiplets, from single diploid blastomeres isolated at the 4-cell stage, or from pairs of sister blastomeres isolated at the 8-cell stage. To this end isolated blastomeres were aggregated with one or two tetraploid carrier embryos produced by electrofusion of 2-cell embryos. Diploid embryos were albino and homozygous for the "a" allele of glucose-phosphate isomerase (GPI-1a1a) and tetraploid embryos were pigmented and GPI-1b1b. The aggregates were cultured in vitro up to the blastocyst stage. Each quartet (occasionally triplet or doublet) of chimaeric blastocysts was transplanted to the oviduct of a separate pseudopregnant recipient. Altogether 62 blastocysts were transplanted to 17 recipients. Eight full-term foetuses (two singletons and three pairs of twins) were rescued by Caesarian section on day 19, 20 or 21 of pregnancy. Three young (one singleton and twins) were successfully reared by foster mothers and proved to be normal and fertile females. All foetuses and animals were albino. In five individuals only the 1-A form of GPI (characteristic for 2n blastomere) was found. In one adult female traces of the 1-B form of GPI (characteristic for 4n carrier blastomeres) were detected in the heart and the lungs while 4 other organs contained only the 1-A form. These observations strongly suggest that the majority of foetuses/animals produced according to our experimental system are 'pure' diploids rather than 2n/4n chimaeras, and that the described method can be used in future to produce twins, triplets and quadruplets in the mouse. Our study confirms earlier work by Kelly (1975, 1977) that 'quarter' blastomeres of the mouse are still totipotent.

Remodelling of thymocyte nuclei in activated mouse oocytes: an ultrastructural study.

Oocyte-thymocyte mouse cell hybrids were produced using polyethylene glycol (PEG) and examined at the ultrastructural level. Fusion was accomplished either before or after activation of metaphase II oocytes. In both experimental variants thymocyte nuclei undergo remodelling which comprises the following sequence of events: nuclear envelope breakdown, initial chromatin condensation, and subsequent decondensation, nuclear envelope reformation and formation of nucleoli. In hybrids produced before oocyte activation but activated within a short time and cultured for several hours the thymocyte nuclei become identical to the female pronucleus. In the second variant (fusion with activated oocytes) the degree of remodeling of thymocyte nuclei is variable. Our observations demonstrate that between metaphase II, telophase of meiosis and early female pronuclear stages the mouse oocyte contains all "factors" necessary for remodelling of differentiated somatic nuclei and their development as if they were pronuclei.

An experimental model of cutaneous infection induced by superantigen-producing Staphylococcus aureus.

Skin infections caused by Staphylococcus aureus, such as erysipelas, are commonly occurring, painful, and costly for society. Despite the high prevalence of this condition, little is known about the host immune responsiveness and bacterial virulence factors during S. aureus dermatitis. We present here a mouse model of infectious dermatitis in which S. aureus is inoculated by an intracutaneous injection to the shaved back of NMRI mice. Visible skin inflammation, characterized by redness and swelling, was noted 48 h after inoculation of staphylococci in mice that received 2 x 108 colony-forming units of S. aureus. Microscopic evaluation revealed a dermal and subcutaneous infiltrate rich in macrophages and neutrophilic granulocytes already within 6 h after inoculation. A sparse influx of T lymphocytes was noted somewhat later. Bacterial cultures from skin revealed high numbers of staphylococci early after inoculation, with a successive decline during 2 wk follow-up. Total white blood cell count as well as the number of polymorphonuclear leukocytes peaked 2 d after bacterial inoculation. Also, serum interleukin-6 levels peaked within 2 d, with a 10-fold increase compared to non-infected control mice, indicating a systemic reaction to skin infection. The role of toxic shock syndrome toxin 1 in the pathogenesis of the dermatitis was assessed using isogenic S. aureus strains. Even though the gross inflammatory skin reaction was similar for mice infected with either of the strains, it was apparent that bacteria secreting toxic shock syndrome toxin 1 preferentially triggered influx of T lymphocytes to the skin. In addition, mice inoculated with staphylococci producing toxic shock syndrome toxin 1 showed a weight decrease during the experiment whereas mice inoculated with the isogenic strain showed a weight increase. This model of staphylococcal dermatitis will enable future in-depth studies regarding the host-bacterium relationship.

Integrin-associated protein (IAP)-deficient mice are less susceptible to developing Staphylococcus aureus-induced arthritis.

The integrin-associated protein (IAP) has been shown to function in a signaling complex with beta3 integrins, influencing the migration of phagocytic cells into inflamed tissues. We have previously shown that gene-targeted mice deficient for IAP succumbed to peritonitis when inoculated with gram-negative bacteria. The aim of this study was to assess the role of IAP in our recently established model of haematogenously induced Staphylococcus aureus septicaemia and arthritis. In this model, neutrophils play a crucial role in the early phase of the infection. Mice lacking IAP and congenic controls were intravenously inoculated with S. aureus LS-1. The IAP-/- mice were resistant to developing clinical signs of arthritis compared with their IAP-expressing littermates. The clinical findings were corroborated by histopathological evaluation indicating that the IAP-/- mice had less cartilage and bone destruction in the joints. We believe that a delayed migration of leukocytes into the joints of mice lacking IAP expression leads to decreased susceptibility to develop S. aureus-induced arthritis.

Impact of transcription factors AP-1 and NF-kappaB on the outcome of experimental Staphylococcus aureus arthritis and sepsis.

Staphylococcus aureus infection is, despite adequate antibiotic treatment, a disease characterized by high mortality. The bacterium triggers an exaggerated immune response in the host, which on the one hand acts as an efficient defense, but on the other hand gives rise to tissue damage. In this study we have modulated the hosts response to S. aureus by inhibition of nuclear factor kappaB (NF-kappaB) and activator protein-1 (AP-1)-triggered release of pro-inflammatory cytokines and tissue-destructive proteins, respectively. Mice were administered with antisense oligonucleotides (ODN) to the p65 subunit of NF-kappaB and/or a double-stranded oligonucleotide (mCoAP-1) with homology to the murine AP-1 binding site of collagenase IV gene (metalloproteinase-9; MMP-9), solely or in combination with antibiotics. In mice systemically treated with antisense ODN to NF-kappaB p65 alone, the bacterial burden in the kidneys was significantly increased (P = 0.04) The same tendency was seen when mCoAP-1 was administered either alone or in combination with antibiotics. We also found significantly (P = 0.04) elevated levels of IL-6 in p65 antisense treated mice. Surprisingly, this p65 antisense therapy approach, which has turned out to be highly efficient in amelioration of aseptic arthritis and colitis, failed to change the clinical course of either septic arthritis or sepsis. We suggest that interaction with transcription factors leads to increased bacterial burden in vivo, abrogating the potential benefits of the anti-inflammatory properties exerted by these compounds.

A demineralization procedure for immunohistopathological use. EDTA treatment preserves lymphoid cell surface antigens.

An evaluation of the usefulness of EDTA treatment for decalcification of murine bone tissue in order to preserve both morphological details and immunologically intact cell surface antigens has been performed. The ABC immunohistochemical staining technique employing monoclonal antibodies to subsets of T-lymphocytes, B-lymphocytes and to Ia antigens was used on frozen sections. Treatment of mouse hindlegs with EDTA for 14 days resulted in an efficient decalcification and good preservation of morphological details. When lymphoid tissues were handled in the same manner monoclonal antibodies, defining Ly 1, Ly 2, L3T4, MAS 034 and Ia molecules, were shown to retain their reactivity comparable to that of directly frozen tissues. In contrast, formic acid, the commonly used decalcification agent, destroyed most of the antigenic reactivity. We conclude that EDTA treatment of non-fixed, bone-containing tissues provides a suitable demineralization procedure in the immunohistochemical study of, e.g., arthritis and periodontitis.

False positive results in class-specific rheumatoid factor (RF) assays due to interaction between RF and Fc fragments of anti-immunoglobulin indicator reagents.

A recently developed, simple immunological method, diffusion-in-gel ELISA, has been adapted for class-specific determination of rheumatoid factor (RF). Evidence was obtained of an analytical error due to interaction between RF and antigenic determinants on the Fc part of the indicator immunoglobulin (Ig) molecules used. This was eliminated by replacing the usual indicator reagent, complete Ig molecules, by conjugated Fab or F(ab')2 fragments. The findings imply that unless the RF-Fc interaction mentioned is avoided in the assaying technique, false positive results may be obtained for, e.g. IgG RF and IgA RF in sera containing IgM RF.

Solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of IgG rheumatoid factor-secreting cells.

Although IgG rheumatoid factor may play an essential role in the pathogenesis of rheumatoid arthritis, there is no precise method for its specific detection at the cellular level. A modification of the recently developed enzyme-linked immunospot assay has been devised for enumeration of cells secreting IgG rheumatoid factor (IgG RF) and simultaneous quantitation of the IgG RF secreted. Specific, sensitive and simple, this new assay should provide a valuable tool for study of isotype-specific RF secretion by single cells.

An MTT-based method for the in vivo quantification of myotoxic activity of snake venoms and its neutralization by antibodies.

The reduction of the tetrazolium compound MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) was used as the basis for the development of a simple method for the quantitative estimation of metabolically active skeletal muscle tissue remaining after in vivo venom-induced myonecrosis. Using the venom of the snake Micrurus nigrocinctus as a potent myotoxic agent, this MTT-based technique was evaluated in comparison with available methods based on the measurement of creatine kinase (CK) activity, and a quantitative histological technique considered as a reference. Homogenates of the gastrocnemius muscle prepared in the presence of 1% Triton X-100 reduced MTT and this activity correlated closely with the number of viable cells in the tissue, as determined by histological evaluation. After venom injection, residual MTT-reducing activity of muscle homogenates showed higher correlation to the myonecrosis index obtained by histological analysis, than residual muscle CK activity. Using the new MTT-based assay, the ability of an anti-M. nigrocinctus equine antivenom to neutralize venom myotoxins was studied. The myotoxic activity of the venom was completely neutralized using 4 ml antivenom/mg venom, with a 50% effective dose (ED50) value of about 2.5 ml/mg. The MTT-based method described should be useful in the estimation and standardization of anti-myotoxic potency of antivenoms, and in the screening of other neutralizing agents, as a convenient and reliable alternative to the time-consuming quantitative histological methods.