TLR signaling is required for Salmonella typhimurium virulence.

Toll-like receptors (TLRs) contribute to host resistance to microbial pathogens and can drive the evolution of virulence mechanisms. We have examined the relationship between host resistance and pathogen virulence using mice with a functional allele of the nramp-1 gene and lacking combinations of TLRs. Mice deficient in both TLR2 and TLR4 were highly susceptible to the intracellular bacterial pathogen Salmonella typhimurium, consistent with reduced innate immune function. However, mice lacking additional TLRs involved in S. typhimurium recognition were less susceptible to infection. In these TLR-deficient cells, bacteria failed to upregulate Salmonella pathogenicity island 2 (SPI-2) genes and did not form a replicative compartment. We demonstrate that TLR signaling enhances the rate of acidification of the Salmonella-containing phagosome, and inhibition of this acidification prevents SPI-2 induction. Our results indicate that S. typhimurium requires cues from the innate immune system to regulate virulence genes necessary for intracellular survival, growth, and systemic infection.

Toll-like receptor 2 on inflammatory monocytes induces type I interferon in response to viral but not bacterial ligands.

Despite the paradigm that the innate immune system uses nucleic acid-specific receptors to detect viruses because of a lack of other conserved features, many viruses are recognized by Toll-like receptor 2 (TLR2) and TLR4. The relevance of this recognition for antiviral immunity remains largely unexplained. Here we report that TLR2 activation by viruses led to the production of type I interferon. TLR2-dependent induction of type I interferon occurred only in response to viral ligands, which indicates that TLR2 is able to discriminate between pathogen classes. We demonstrate that this specialized response was mediated by Ly6C(hi) inflammatory monocytes. Thus, the innate immune system can detect certain non-nucleic acid features of viruses and links this recognition to the induction of specific antiviral genes.

Transmembrane mutations in Toll-like receptor 9 bypass the requirement for ectodomain proteolysis and induce fatal inflammation.

Recognition of nucleic acids as a signature of infection by Toll-like receptors (TLRs) 7 and 9 exposes the host to potential self-recognition and autoimmunity. It has been proposed that intracellular compartmentalization is largely responsible for reliable self versus nonself discrimination by these receptors. We have previously shown that TLR9 and TLR7 require processing prior to activation, which may further reinforce receptor compartmentalization and tolerance to self, yet this possibility remains untested. Here we report that residues within the TLR9 transmembrane (TM) region conferred the requirement for ectodomain proteolysis. TLR9 TM mutants responded to extracellular DNA, and mice expressing such receptors died from systemic inflammation and anemia. This inflammatory disease did not require lymphocytes and appeared to require recognition of self-DNA by dendritic cells. To our knowledge, these results provide the first demonstration that TLR-intrinsic mutations can lead to a break in tolerance.

The ectodomain of Toll-like receptor 9 is cleaved to generate a functional receptor.

Mammalian Toll-like receptors (TLRs) 3, 7, 8 and 9 initiate immune responses to infection by recognizing microbial nucleic acids; however, these responses come at the cost of potential autoimmunity owing to inappropriate recognition of self nucleic acids. The localization of TLR9 and TLR7 to intracellular compartments seems to have a role in facilitating responses to viral nucleic acids while maintaining tolerance to self nucleic acids, yet the cell biology regulating the transport and localization of these receptors remains poorly understood. Here we define the route by which TLR9 and TLR7 exit the endoplasmic reticulum and travel to endolysosomes in mouse macrophages and dendritic cells. The ectodomains of TLR9 and TLR7 are cleaved in the endolysosome, such that no full-length protein is detectable in the compartment where ligand is recognized. Notably, although both the full-length and cleaved forms of TLR9 are capable of binding ligand, only the processed form recruits MyD88 on activation, indicating that this truncated receptor, rather than the full-length form, is functional. Furthermore, conditions that prevent receptor proteolysis, including forced TLR9 surface localization, render the receptor non-functional. We propose that ectodomain cleavage represents a strategy to restrict receptor activation to endolysosomal compartments and prevent TLRs from responding to self nucleic acids.

Borderline Personality Traits and Romantic Relationship Dissolution.

Many studies have found that borderline personality disorder (BPD) is associated with romantic relationship instability, with relationship dissolution being a recurring theme. Scant research, however, has examined the dissolution strategies and post-breakup outcomes for individuals with elevated levels of borderline traits. Findings from two studies revealed that there was an association between BPD criteria and tendency to employ less adaptive dissolution strategies when terminating a relationship. Furthermore, elevated levels of BPD traits were associated with less self-concept clarity and more unwanted pursuit of ex-partners. These findings both provide insight into how individuals with BPD traits experience relationship dissolution and suggest possible factors underlying the unstable relationship processes typically associated with borderline traits.

DNA tumor virus oncogenes antagonize the cGAS-STING DNA-sensing pathway.

Cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) detects intracellular DNA and signals through the adapter protein STING to initiate the antiviral response to DNA viruses. Whether DNA viruses can prevent activation of the cGAS-STING pathway remains largely unknown. Here, we identify the oncogenes of the DNA tumor viruses, including E7 from human papillomavirus (HPV) and E1A from adenovirus, as potent and specific inhibitors of the cGAS-STING pathway. We show that the LXCXE motif of these oncoproteins, which is essential for blockade of the retinoblastoma tumor suppressor, is also important for antagonizing DNA sensing. E1A and E7 bind to STING, and silencing of these oncogenes in human tumor cells restores the cGAS-STING pathway. Our findings reveal a host-virus conflict that may have shaped the evolution of viral oncogenes.

Methods of detection of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythensis in periodontal microbiology, with special emphasis on advanced molecular techniques: a review.

BACKGROUND: Certain specific bacterial species from the subgingival biofilm have demonstrated aetiological relevance in the initiation and progression of periodontitis. Among all the bacteria studied, three have shown the highest association with destructive periodontal diseases: Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg) and Tannerella forsythensis (Tf). Therefore, the relevance of having accurate microbiological diagnostic techniques for their identification and quantification is clearly justified. AIM: To evaluate critically all scientific information on the currently available microbial diagnostic techniques aimed for the identification and quantification of Aa, Pg and Tf. SUMMARY: Bacterial culturing has been the reference diagnostic technique for many years and, in fact, most of our current knowledge on periodontal microbiology derives from cultural data. However, the advent of new microbial diagnostics, mostly based on immune and molecular technologies, has not only highlighted some of the shortcomings of cultural techniques but has also allowed their introduction as easy and available adjunct diagnostic tools to be used in clinical research and practice. These technologies, mostly polymerase chain reaction (PCR), represent a field of continuous development; however, we still lack the ideal diagnostic to study the subgingival microflora. Qualitative PCR is still hampered by the limited information provided. Quantitative PCR is still in development; however, the promising early results reported are still hampered by the high cost and the equipment necessary for the processing. CONCLUSION: Quantitative PCR technology may have a major role in the near future as an adjunctive diagnostic tool in both epidemiological and clinical studies in periodontology. However, culture techniques still hold some inherent capabilities, which makes this diagnostic tool the current reference standard in periodontal microbiology.

Quantitative real-time polymerase chain reaction based on single copy gene sequence for detection of periodontal pathogens.

OBJECTIVE: To develop a method for quantification of Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg) and Tannerella forsythensis (Tf) from subgingival plaque samples based on TaqMan real-time polymerase chain reaction (PCR) technology. MATERIAL AND METHODS: Bacterial cells from these species were obtained after culturing reference strains and were counted microscopically. Cellular suspensions in Tris-EDTA buffer were used for DNA extraction after boiling for 20 min. Primers for PCR were selected from sequences of the LktC (Aa), Arg-gingipain (Pg) and BspA antigen (Tf) genes in order to yield amplicons below 100 bp. TaqMan-based real-time PCR was adjusted to quantify each species separately. Cycle threshold (C(T)) values were calculated for each species according to the initial number of copies. A reliability analysis was carried out using intra-class correlation coefficients (ICCs) with a two-way random effects model. RESULTS: A high sensitivity and specificity was obtained for the detection of the three bacterial species. The TaqMan real-time PCR technology yielded a good repeatability in the obtained cycle threshold (C(T)) values for each initial number of copies, demonstrating coefficients of variation below 5% for each bacteria. The reproducibility of the technique was also demonstrated by the high ICCs (>0.98; p<0.00001) obtained for each bacteria with and without the addition of subgingival plaque. CONCLUSION: A novel diagnostic method based on TaqMan real-time PCR was developed for the quantification of Aa, Pg and Tf. It has demonstrated good sensitivity and repeatability on pure cultures. Its diagnostic utility should be demonstrated in subgingival plaque samples.

Quantitative real-time polymerase chain reaction versus culture: a comparison between two methods for the detection and quantification of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythensis in subgingival plaque samples.

OBJECTIVE: The purpose of this investigation was to validate a real-time quantitative polymerase chain reaction (PCR) assay in identifying and quantifying Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythensis from subgingival plaque samples taken from subjects with different periodontal conditions, when compared with conventional cultural procedures. PATIENTS AND METHODS: Ninety-two adult subjects participated in this study, 32 with periodontitis, 30 with gingivitis and 30 healthy. A pooled subgingival sample was obtained from every patient. Culturing procedures were carried out using standard techniques. For real-time PCR analysis, primers were selected from sequences of the LktC (A. actinomycetemcomitans), Arg-gingipain (P. gingivalis) and BspA antigen (T. forsythensis) genes. Contingency tables were constructed to compare the qualitative results, while quantitative data were evaluated by paired t-test. RESULTS: A. actinomycetemcomitans was the least frequently recovered species with both techniques. Prevalence of P. gingivalis was low in healthy patients, increased in gingivitis and peaked in periodontitis patients. The frequency of detection of T. forsythensis showed marked differences between culture and PCR, although the same tendency of an increase in prevalence from health to gingivitis and to periodontitis was observed with both methods. Contingency tables demonstrated a good level of agreement between PCR and culture procedures for A. actinomycetemcomitans and P. gingivalis, especially in periodontitis patients. P. gingivalis culture counts were significantly higher than those obtained by PCR. The opposite was true for T. forsythensis, and statistically significant higher counts were obtained by PCR for gingivitis and periodontitis patients. CONCLUSION: This study demonstrated a good agreement between the quantitative PCR technology and the culture procedure. The high sensitivity and specificity of the quantitative PCR technology justify its use in epidemiological studies and as an adjunct in clinical diagnosis of periodontal patients.

Quantitative real-time PCR based on single copy gene sequence for detection of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis.

OBJECTIVE: To establish a method for quantification of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis from subgingival plaque by real-time polymerase chain reaction (PCR) technique. MATERIAL AND METHODS: Bacterial cells from both species were obtained from type culture and counted microscopically. Cellular suspension in sterile distilled water was used for DNA extraction by boiling for 20 min, with a mineral oil cover. Primers for PCR were selected from sequences of LktC gene (A. actinomycetemcomitans) and Arg-gingipain (P. gingivalis) to yield amplicons below 100 bp. SYBR Green I based real-time PCR was adjusted to quantify separately both species. RESULTS: A good sensitivity and specificity were obtained for both species, although the yield was better for A. actinomycetemcomitans. A good repeatability of cycle threshold (CT) was encountered, so coefficient of variation was below 6% at every initial copy number. CONCLUSION: A new method of quantification of A. actinomycetemcomitans and P. gingivalis based on SYBR Green real-time PCR is presented. Its good sensibility and repeatability will allow its application to analysis of subgingival plaque samples.