Critical role of the major histocompatibility complex and IL-10 in matrilin-1-induced relapsing polychondritis in mice

Relapsing polychondritis (RP) is an autoimmune disease that affects extra-articular cartilage. Matrilin-1-induced relapsing polychondritis (MIRP) is a model for RP and is useful for studies of the pathogenic mechanisms in this disease. There are indications that the major histocompatibility complex (MHC) class II plays a major role in RP, since DR4⁺ patients are more commonly affected than controls. We have now addressed the role of the MHC region, as well as the non-MHC contribution, using congenic mouse strains. Of the MHC congenic strains, B10.Q (H2 ^q ) was the most susceptible, the B10.P (H2 ^p ) and B10.R (H2 ^r ) strains developed mild disease, while B10 strains carrying the v, b, f, or u H2 haplotypes were resistant. A slight variation of susceptibility of H2 ^q strains (B10.Q> C3H.Q> DBA/1) was observed and the (B10.Q × DBA/1)F₁ was the most susceptible of all strains. Furthermore, macrophages and CD4⁺ T cells were the most prominent cell types in inflammatory infiltrates of the tracheal cartilage. Macrophages are the major source of many cytokines, such as interleukin-10 (IL-10), which is currently being tested as a therapeutic agent in several autoimmune diseases. We therefore investigated B10.Q mice devoid of IL-10 through gene deletion and found that they developed a significantly more severe disease, with an earlier onset, than their heterozygous littermates. In conclusion, MHC genes, as well as non-MHC genes, are important for MIRP induction, and IL-10 plays a major suppressive role in cartilage inflammation of the respiratory tract.     

IRAK1 deletion disrupts cardiac Toll/IL-1 signaling and protects against contractile dysfunction

Myocardial contractile dysfunction accompanies both systemic and cardiac insults. Septic shock and burn trauma can lead to reversible contractile deficits, whereas ischemia and direct inflammation of the heart can precipitate transient or permanent impairments in contractility. Many of the insults that trigger contractile dysfunction also activate the innate immune system. Activation of the innate immune response to infection is coordinated by the conserved Toll/interleukin-1 (IL-1) signal transduction pathway. Interestingly, components of this pathway are also expressed in normal and failing hearts, although their function is unknown. The hypotheses that Toll/IL-1 signaling occurs in the heart and that intact pathway function is required for contractile dysfunction after different insults were tested. Results from these experiments demonstrate that lipopolysaccharides (LPS) activate Toll/IL-1 signaling and IL-1 receptor-associated kinase-1 (IRAK1), a critical pathway intermediate in the heart, indicating that the function of this pathway is not limited to immune system tissues. Moreover, hearts lacking IRAK1 exhibit impaired LPS-triggered downstream signal transduction. Hearts from IRAK1-deficient mice also resist acute LPS-induced contractile dysfunction. Finally, IRAK1 inactivation enhances survival of transgenic mice that develop severe myocarditis and lethal heart failure. Thus the Toll/IL-1 pathway is active in myocardial tissue and interference with pathway function, through IRAK1 inactivation, may represent a novel strategy to protect against cardiac contractile dysfunction.     

Pulsed-field gel electrophoresis, plasmid profiles and phage types for the human isolates of Salmonella enterica serovar Enteritidis obtained over 13 years in Taiwan

AIMS: Plasmid profile, phage typing, and pulsed-field gel electrophoresis (PFGE) patterns of 124 Salmonella Enteritidis strains isolated in 1998-2002 in Taiwan were analysed and the results were compared with those of the 63 strains obtained in 1991-1997, so that molecular subtypes and epidemic strains for Salmonella Enteritidis over a 13-year period (1991-2002) could be elucidated. METHODS AND RESULTS: A total of 124 strains of Salmonella Enteritidis isolated from human in Taiwan between 1998 and 2002 were analysed by PFGE, plasmid analysis and phage typing. The results obtained were compared with those of the 63 strains obtained in 1991-1997, so that the clonal relationships for a total of 187 strains obtained over 13 years could be elucidated. For PFGE, restriction enzymes XbaI, SpeI and NotI were used for chromosomal DNA digestion. Results showed 28 PFGE pattern combinations for the 187 Salmonella strains. Of them, pattern X3S3N3 was the major subtype as 130 strains isolated from different locations during 1991-2002 showed this PFGE pattern. For all these 187 strains, the genetic similarity was higher than 80%. Plasmid analysis showed 17 distinct types, which consist of one to four plasmids and the predominant phage type of those strains was PT4 (71.6%) and PT6a (13.4%). The three methods identified different degrees of polymorphism in the following order: plasmid profile (18 types, D = 0.659) > PFGE (28 types, D = 0.512) > phage typing (13 types, D = 0.438). As PFGE patterns, phage type and plasmid profile were combined for subtyping, the 187 strains could be grouped into 46 subtypes and the discriminatory index was raised to 0.795. For these 46 subtypes, the predominant one was X3S3N3/P1/PT4, which contained 77 (41%) isolates. CONCLUSIONS: Most of the Salmonella Enteritidis strains from sporadic cases were with pattern X3S3N3. They were the prevalent and may be the epidemic strains found in Taiwan during 1991-2002. The present study suggested that the several variants were derived from a single clonal line and the genome for strains of Salmonella Enteritidis are highly conserved over a 13-year period (1991-2002). SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained here are useful for epidemiolgical study of salmonellosis caused by Salmonella Enteritidis in Taiwan. Comparing the data of the present study with those obtained for strains from other countries, the major subtypes for Salmonella Enteritidis infection in the world can be elucidated.     

The use of nucleotide analogs to evaluate the mechanism of the heterotropic response of Escherichia coli aspartate transcarbamoylase

As an alternative method to study the heterotropic mechanism of Escherichia coli aspartate transcarbamoylase, a series of nucleotide analogs were used. These nucleotide analogs have the advantage over site-specific mutagenesis experiments in that interactions between the backbone of the protein and the nucleotide could be evaluated in terms of their importance for function. The ATP analogs purine 5'-triphosphate (PTP), 6-chloropurine 5'-triphosphate (Cl-PTP), 6-mercaptopurine 5'-triphosphate (SH-PTP), 6-methylpurine 5'-triphosphate (Me-PTP), and 1-methyladenosine 5'-triphosphate (Me-ATP) were partially synthesized from their corresponding nucleosides. Kinetic analysis was performed on the wild-type enzyme in the presence of these ATP analogs along with GTP, ITP, and XTP. PTP, Cl-PTP, and SH-PTP each activate the enzyme at subsaturating concentrations of L-aspartate and saturating concentrations of carbamoyl phosphate, but not to the same extent as does ATP. These experiments suggest that the interaction between N6-amino group of ATP and the backbone of the regulatory chain is important for orienting the nucleotide and inducing the displacements of the regulatory chain backbone necessary for initiation of the regulatory response. Me-PTP and Me-ATP also activate the enzyme, but in a more complex fashion, which suggests differential binding at the two sites within each regulatory dimer. The purine nucleotides GTP, ITP, and XTP each inhibit the enzyme but to a lesser extent than CTP. The influence of deoxy and dideoxynucleotides on the activity of the enzyme was also investigated. These experiments suggest that the 2' and 3' ribose hydroxyl groups are not of significant importance for binding and orientation of the nucleotide in the regulatory binding site. 2'-dCTP inhibits the enzyme to the same extent as CTP, indicating that the interactions of the enzyme to the O2-carbonyl of CTP are critical for CTP binding, inhibition, and the ability of the enzyme to discriminate between ATP and CTP. Examination of the electrostatic surface potential of the nucleotides and the regulatory chain suggest that the complimentary electrostatic interactions between the nucleotides and the regulatory chain are important for binding and orientation of the nucleotide necessary to induce the local conformational changes that propagate the heterotropic effect.     

Bone marrow-derived cells contribute to contractile dysfunction in endotoxic shock

How infection precipitates depressed contractility is incompletely understood but may involve the immune, nervous, and endocrine systems as well as the heart itself. In this study, we examined the role of Toll-like receptor 4 (TLR4) in LPS-induced myocardial contractile depression. Eighteen hours following endotoxin challenge, we compared contractile responses in hearts from wild-type (WT) and TLR4-deficient mice using modified Langendorff preparations. Unlike hearts from WT mice, TLR4-deficient hearts did not reveal significant contractile dysfunction following LPS administration, as measured by decreased responses in maximal left ventricular pressure, +dP/dtmax, and -dP/dtmax in ex vivo Langendorff preparations. These findings indicate a requirement for TLR4 in LPS-induced contractile depression. To determine the contribution of bone marrow-derived TLR4 function to LPS-induced myocardial dysfunction, we generated TLR4 chimeras using adoptive transfer between histocompatible mouse strains: either TLR4-deficient mice with TLR4+/+ bone marrow-derived cells or TLR4+/+ animals lacking TLR4 in their hematopoietic cells. We then compared the contractile responses of engrafted animals after LPS challenges. Engraftment of TLR4-deficient mice with WT marrow restored sensitivity to the myocardial depressant effects of LPS in TLR4-deficient hearts (P < 0.05). Inactivation of bone marrow-derived TLR4 function, via transplantation of WT mice with TLR4-/- marrow, however, did not protect against the depressant effect of endotoxin. These findings indicate that bone marrow-derived TLR4 activity is sufficient to confer sensitivity to mice lacking TLR4 in all other tissues. However, because inactivation of marrow-derived TLR4 function alone does not protect against endotoxin-triggered contractile dysfunction, TLR4 function in other tissues may also contribute to this response.     

Discrimination of Bacillus cereus and Bacillus thuringiensis with 16S rRNA and gyrB gene based PCR primers and sequencing of their annealing sites

AIMS: To evaluate the possibility for discrimination of Bacillus cereus and B. thuringiensis using 16S rRNA and gyrB gene based PCR methods, and to obtain the sequences of the primer annealing sites so that the PCR results may be explained. METHODS AND RESULTS: Based on the sequence difference in the variable region (V1) of 16S rRNA and in the gyrB gene between B. cereus and B. thuringiensis, PCR primers specific to these Bacillus spp. were designed. When these primers were used to discriminate B. cereus and B. thuringiensis, six of 82 B. cereus strains were identified as B. thuringiensis while 67 of 73 B. thuringiensis strains were identified as B. cereus. Sequence analysis of the primer annealing sites showed that there is no clear-cut difference in the V1 region of 16S rRNA, and in the gyrB gene, between B. cereus and B. thuringiensis strains. CONCLUSIONS: Although 16S rDNA based probes and gyrB gene based PCR primers have been suggested for the discrimination of B. cereus and B. thuringiensis strains, when a large number of Bacillus strains was tested, results showed that discrimination between B. cereus and B. thuringiensis is difficult. Therefore, to distinguish B. thuringiensis from B. cereus, a single feature, such as the presence of a parasporal crystal protein or cry gene, may sometimes be reliable. SIGNIFICANCE AND IMPACT OF THE STUDY: Discrimination between B. cereus and B. thuringiensis is a challenging debate to which this paper makes a contribution.     

Development and use of 16S rRNA gene targeted PCR primers for the identification of Escherichia coli cells in water

The primary sequences of the V₃ and V₆ regions of the 16S rRNA gene of pathogenic and non-pathogenic strains of Escherichia coli were determined and compared with those obtained for a number of reference strains which belong to the family Enterobacteriaceae. Three oligonucleotide primers 16E1, 16E2 and 16E3 were designed and used in the polymerase chain reaction to identify specifically all E. coli isolates. When 16E1, 16E2 and 16E3 were used as primers for the identification of E. coli cells present in tap, underground and pond waters, as low as 1 cfu 100 ml⁻¹ of water could be detected if an 8 h pre-culture step was performed prior to the PCR reaction.