A mini review of Liparis species: an ornamental genus with considerable medicinal value.

The genus Liparis, a group of perennial ornamental herbs in the family Orchidaceae, is widely distributed in tropical and subtropical regions. Many species of the genus Liparis have been commonly used as traditional herbal medicines for the treatment of menorrhagia, haemoptysis, traumatic bleeding, snake bites, and pneumonia. This review describes the ornamental value of plants of the genus Liparis and summarises the chemical constituents and pharmacological activities reported during the last decade. The main chemical constituents of this genus are phenolic acids, alkaloids, flavonoids, etc. Most phenolic acids and alkaloids have a nervogenic acid skeleton, and most alkaloids also have a pyrrolizidine skeleton. Extracts from the genus Liparis plants showed significant haemostatic, antitumor, anti-inflammatory, hypolipidemic, antioxidant, and antibacterial activities. This paper proposed ideas and research directions for the future study of plants in the genus Liparis, providing valuable information for the development of new drugs and promoting their utilisation.

HIPK3 maintains sensitivity to platinum drugs and prevents disease progression in gastric cancer.

In the realm of cancer therapeutics and resistance, kinases play a crucial role, particularly in gastric cancer (GC). Our study focused on platinum-based chemotherapy resistance in GC, revealing a significant reduction in homeodomain-interacting protein kinase 3 (HIPK3) expression in platinum-resistant tumors through meticulous analysis of transcriptome datasets. In vitro and in vivo experiments demonstrated that HIPK3 knockdown enhanced tumor proliferation and metastasis, while upregulation had the opposite effect. We identified the myocyte enhancer factor 2C (MEF2C) as a transcriptional regulator of HIPK3 and uncovered HIPK3's role in downregulating the morphogenesis regulator microtubule-associated protein (MAP7) through ubiquitination. Phosphoproteome profiling revealed HIPK3's inhibitory effects on mTOR and Wnt pathways crucial in cell proliferation and movement. A combined treatment strategy involving oxaliplatin, rapamycin, and IWR1-1-endo effectively overcame platinum resistance induced by reduced HIPK3 expression. Monitoring HIPK3 levels could serve as a GC malignancy and platinum resistance indicator, with our proposed treatment strategy offering novel avenues for reversing resistance in gastric cancer.

Epstein-Barr virus-based prognostic model in nodular sclerosis classic Hodgkin lymphoma.

The role of Epstein-Barr virus (EBV) in lymphoma cells of nodular sclerosis classic Hodgkin lymphoma (NScHL) is controversial. Our aim was to explore this and establish a clinically feasible model for risk stratification. We interrogated data from 542 consecutive subjects with NScHL receiving ABVD therapy and demonstrated EBV-infection in their lymphoma cells with EBV-encoded small RNAs (EBERs) in situ hybridization. Subjects were divided into training and validation datasets. As data from the training dataset suggested EBERs-positivity was the only independent prognostic factor for both progression-free survival (PFS) and overall survival (OS), we developed corresponding prognostic models based on it. Our models showed excellent performance in both training and validation cohort. These data indicate the close association of EBV infection and the outcomes of persons with NScHL receiving ABVD. Additionally, our newly developed models should help physicians estimate prognosis and select individualized therapy.

Factors influencing loyalty to online health consultation platform: a cross-strait cultural perspective.

BACKGROUND: The geographical, cultural, and linguistic proximity between Taiwan and Mainland China has facilitated rapid growth of cross-strait interactions. Both countries have developed online health consultation platforms on the Internet for the public to access healthcare related information. This study examines factors that influence loyalty to a specific online health consultation platform (OHCP) from a cross-strait perspective. METHODS: Based on the Expectation Confirmation Theory and the combined Trust, Perceived Health Risks and Culture, we examine factors that influence loyalty to OHCPs among cross-strait users by investigating the roles of trust, perceived health risks, and culture. Data was collected through a questionnaire survey. RESULTS: The research models used provide a high-power explanation of loyalty to OHCPs. Results generally align with those of previous studies, with the exception of the relationships between Perceived Health Risks and Perceived Usefulness, Perceived Usefulness and Loyalty, Confirmation and Satisfaction, and Trust and Loyalty. In other words, culture may have moderated these relationships. CONCLUSIONS: Findings can help promote OHCPs among cross-strait users to make things easier for patients, and further reduce the load on the emergency department, especially in view of the still ongoing issues related to global outbreak of Coronavirus disease by facilitating early detection of potential cases.

miR-29a-3p promotes the regulatory role of eicosapentaenoic acid in the NLRP3 inflammasome and autophagy in microglial cells.

Eicosapentaenoic acid (EPA) has been reported to play an anti-inflammatory and antioxidative stress role in a series of human diseases, including major depressive disorder. However, its exact mechanism is still largely unknown. Mouse BV-2 cells were treated with lipopolysaccharide (LPS) to induce an in vitro inflammatory cell model of depression. Cytotoxic effects were assessed with MTT and lactate dehydrigebase release assays. Cytokine mediators were elevated by western blot and enzyme-linked immunosorbent assays. Autophagy-relators were determined by immunofluorescence and western blot analyses. Interaction relationships among molecules were evaluated utilizing chromatin immunoprecipitation and dual luciferase assays. Methylated miR-29a-3p was detected via methylation-specific polymerase chain reaction. EPA treatment at 60 µM had no cytotoxic effects on BV2 cells and significantly inhibited the LPS-induced inflammatory response and NLRP3 inflammasome but activated autophagy, while all these effects were reversed by the autophagy inhibitor 3-MA. Importantly, miR-29a-3p exhibited a role similar to that of EPA in LPS-treated BV2 cells. Mechanistically, EPA treatment elevated miR-29a-3p by repressing its promoter methylation. MAPK8 was a direct target of miR-29a-3p. Inhibition of miR-29a-3p greatly diminished the regulatory roles mediated by EPA in LPS-treated BV2 cells, while these roles were further impeded after MAPK8 silencing. To conclude, our data demonstrated that EPA treatment alleviated LPS-induced NLRP3 inflammasomes by activating autophagy via regulation of miR-29a-3p/MAPK8 signaling, which further elucidates the potential antidepressant mechanism of EPA.

Leukemic cells resist lysosomal inhibition through the mitochondria-dependent reduction of intracellular pH and oxidants.

Acidic lysosomes are indispensable for cancer development and linked to chemotherapy resistance. Chloroquine (CQ) and functional analogues have been considered as a potential solution to overcome the cancer progression and chemoresistance by inhibiting the lysosome-mediated autophagy and multidrug exocytosis. However, their anti-cancer efficacy in most clinical trials demonstrated modest improvement. In this study, we investigated the detailed mechanisms underlying the acquired resistance of K562 leukemic cells to CQ treatment. In response to 5-80 µM CQ, the lumen pH of endosomal-lysosomal system immediately increased and gradually reached dynamic equilibrium within 24 h. Leukemic cells produced more acidic organelles to tolerate 5-10 µM CQ. CQ (20-80 µM) concentration-dependently triggered cytosolic pH (pHi) rise, G0/G1 arrest, mitochondrial depolarization/fragmentation, and necrotic/apoptotic cell death. Oxidant induction by CQ was responsible for the mitochondria-dependent cytotoxicity and partial pHi elevation. Cells that survived the CQ cytotoxicity were accompanied with increased mitochondria. Under the 80 µM CQ challenge, co-treatment with the inhibitor of F0 part of mitochondrial H+-ATP synthase, oligomycin (40 nM), prevented the elevation of oxidants as well as pHi, and attenuated stresses on mitochondria, cell survival, and cell proliferation. Besides, oligomycin-treated cells obviously displayed the lysosomal peripheralization and plasma membrane blebbing, suggesting that these cells were in process of lysosomal exocytosis and microvesicle release. Enhanced motion of these secretory processes allowed the cells to exclude CQ and repair necrotic injury. Together, the oxidant production and the proton dynamic interconnection among lysosomes, mitochondria, and cytosol are crucial for leukemic susceptibility to lysosomotropic chemotherapeutics.

The genomic architecture of EBV and infected gastric tissue from precursor lesions to carcinoma.

BACKGROUND: Epstein-Barr virus (EBV)-associated gastric carcinomas (EBVaGCs) present unique molecular signatures, but the tumorigenesis of EBVaGCs and the role EBV plays during this process remain poorly understood. METHODS: We applied whole-exome sequencing, EBV genome sequencing, and whole-genome bisulfite sequencing to multiple samples (n = 123) derived from the same patients (n = 25), which covered saliva samples and different histological stages from morphologically normal epithelial tissues to dysplasia and EBVaGCs. We compared the genomic landscape between EBVaGCs and their precursor lesions and traced the clonal evolution for each patient. We also analyzed genome sequences of EBV from samples of different histological types. Finally, the key molecular events promoting the tumor evolution were demonstrated by MTT, IC50, and colony formation assay in vitro experiments and in vivo xenograft experiments. RESULTS: Our analysis revealed increasing mutational burden and EBV load from normal tissues and low-grade dysplasia (LD) to high-grade dysplasia (HD) and EBVaGCs, and oncogenic amplifications occurred late in EBVaGCs. Interestingly, within each patient, EBVaGCs and HDs were monoclonal and harbored single-strain-originated EBV, but saliva or normal tissues/LDs had different EBV strains from that in EBVaGCs. Compared with precursor lesions, tumor cells showed incremental methylation in promotor regions, whereas EBV presented consistent hypermethylation. Dominant alterations targeting the PI3K-Akt and Wnt pathways were found in EBV-infected cells. The combinational inhibition of these two pathways in EBV-positive tumor cells confirmed their synergistic function. CONCLUSIONS: We portrayed the (epi) genomic evolution process of EBVaGCs, revealed the extensive genomic diversity of EBV between tumors and normal tissue sites, and demonstrated the synergistic activation of the PI3K and Wnt pathways in EBVaGCs, offering a new potential treatment strategy for this disease.

CEP55 Positively Affects Tumorigenesis of Esophageal Squamous Cell Carcinoma and Is Correlated with Poor Prognosis.

Centrosomal protein 55 (CEP55) is a centrosome- and midbody-associated protein that is overexpressed in several cancers. However, the underlying molecular mechanism of CEP55-mediated progression and metastasis of esophageal squamous cell carcinoma (ESCC) is not clear. In the current study, we detected CEP55 mRNA by qRT-PCR while protein expression was detected by western blot analysis and immunohistochemistry (IHC). In addition, we knocked down CEP55 and investigated the ability of CEP55 to affect colony formation and migration. Here, we report that CEP55 mRNA and protein expression was significantly increased in ESCC. IHC staining showed that CEP55 expression correlated with TNM stage (p=0.046) and lymph node metastases (p=0.024). According to overall survival (OS) and disease-free survival (DFS), patients whose tumors expressed a higher level of CEP55 had a poorer prognosis than those with low expression level of CEP55. A multivariate analysis revealed that CEP55 expression was an independent prognostic indicator for patients with ESCC. Knockdown of CEP55 decreased the colony formation ability and migration of ESCC cells and also reduced the phosphorylation of Src, FAK, and ERK. Therefore, our study implied that CEP55 may be a valuable biomarker and a potential target in the treatment of patients with ESCC.

[Relationship between PMP, FN, vWF and Bleeding Degree in Patients with Acute Leukemia].

OBJECTIVE: To detect the serum levels of platelet microparticle (PMP), fibronectin (FN), and von Willebrand Factor (vWF) in acute leukemia (AL) patients with thrombocytopenic and to analyze the relationship of the serum levels of PMP, FN and vWF with bleeding degree. METHODS: One hundred and one newly diagnosed AL patients from May 2014 to May 2017 were enrolled the AL group. According to the WHO standard of bleeding stratification, 101 AL patients were divided into 5 sub groups: 0, 1, 2, 3 and 4 score groups; 52 normal persons subjected to physical examination were enrolled in control group. The PMP level was detected by flow cytometry; the FN and vWF levels were detected by ELISA. The levels of PMP, FN and vWF were compared between the AL group and the control group. The serum levels of PMP, FN and vWF were compared according to bleeding degree group. The relationship of bleeding degree with the serum levels of PMP, FN and vWF was analyzed. RESULTS: The patients with newly diagnosed acute leukemia aged 18 to 60, and accounted for 61.39%. The degree of bleeding was mainly 1 score, which accounted for 38.61%. The serum levels of PMP, vWF and FN AL groups were significantly higher than those in control group (6.06%±4.38% vs 0.89%±0.50%, 205.82±24.89 vs 58.04±13.35 µg/L, 398.29±46.93 vs 311.37±26.02 µg/L)(P<0.001). The serum levels of PMP, FN and vWF were different among 5 subgroup (P<0.01); the level of PMP and FN were the highest in 0 score group and the lowest in 4 score group; the vWF level was the highest in 4 score groups and the lowest in 0 score group. The bleeding degree in the patients with acute leukemia negatively correlated with PMP level, and positively with NF and vWF levels (r=-0.753, r=0.648, r=0.805). CONCLUSION: According to the relationship of the bleeding degree with serum levels of PMP, FN, vWF in patients, the detection of PMP, vWF and FN levels can help to evaluale the bleeding degree in the patients.

Entinostat reverses cisplatin resistance in esophageal squamous cell carcinoma via down-regulation of multidrug resistance gene 1.

Cisplatin resistance frequently occurs in esophageal squamous cell carcinoma (ESCC). The underlying mechanism for cisplatin resistance in ESCC remains largely obscure. Here we report that entinostat reversed cisplatin resistance in ESCC both in vitro and in vivo by induction of apoptosis and inhibition of cell proliferation, accompanied by a decrease of multidrug resistance gene 1 (MDR1), P-Src, Mcl-1, Cyclin D1 and an increase of cleaved PARP. MDR1 expression was associated with worsen survival of ESCC patients with cisplatin-based chemotherapy. Dasatinib potentiated entinostat to overcome cisplatin resistance. By inhibiting Src, dasatinib reduced the expression of MDR1 and Mcl-1. Furthermore, Obatoclax, an inhibitor of Mcl-1, obviously decreased the expression of MDR1, suggesting that entinostat might surmount cisplatin resistance in ESCC via a Src-Mcl-1-MDR1 pathway. Interestingly, cisplatin also enhanced the effect of entinostat both in vitro and in vivo. Our data disclose a molecular basis that entinostat reverses cisplatin resistance, and provide a promising strategy with combinatorial drugs to treat cisplatin resistant ESCC patients.

Genomic comparison of esophageal squamous cell carcinoma and its precursor lesions by multi-region whole-exome sequencing.

Esophageal squamous dysplasia is believed to be the precursor lesion of esophageal squamous cell carcinoma (ESCC); however, the genetic evolution from dysplasia to ESCC remains poorly understood. Here, we applied multi-region whole-exome sequencing to samples from two cohorts, 45 ESCC patients with matched dysplasia and carcinoma samples, and 13 tumor-free patients with only dysplasia samples. Our analysis reveals that dysplasia is heavily mutated and harbors most of the driver events reported in ESCC. Moreover, dysplasia is polyclonal, and remarkable heterogeneity is often observed between tumors and their neighboring dysplasia samples. Notably, copy number alterations are prevalent in dysplasia and persist during the ESCC progression, which is distinct from the development of esophageal adenocarcinoma. The sharp contrast in the prevalence of the 'two-hit' event on TP53 between the two cohorts suggests that the complete inactivation of TP53 is essential in promoting the development of ESCC.The pathogenesis of oesophageal squamous cell carcinoma is a multi-step process but the genetic determinants behind this progression are unknown. Here the authors use multi-region exome sequencing to comprehensively investigate the genetic evolution of precursor dysplastic lesions and untransformed oesophagus.

Effects of hydrotalcite combined with esomeprazole on gastric ulcer healing quality: A clinical observation study.

AIM: To evaluate the effects of hydrotalcite combined with esomeprazole on gastric ulcer healing quality. METHODS: Forty-eight patients diagnosed with gastric ulcer between June 2014 and February 2016 were randomly allocated to the combination therapy group or monotherapy group. The former received hydrotalcite combined with esomeprazole, and the latter received esomeprazole alone, for 8 wk. Twenty-four healthy volunteers were recruited and acted as the healthy control group. Endoscopic ulcer healing was observed using white light endoscopy and narrow band imaging magnifying endoscopy. The composition of collagen fibers, amount of collagen deposition, expression of factor VIII and TGF-ß1, and hydroxyproline content were analyzed by Masson staining, immunohistochemistry, immunofluorescent imaging and ELISA. RESULTS: Following treatment, changes in the gastric microvascular network were statistically different between the combination therapy group and the monotherapy group (P < 0.05). There were significant differences (P < 0.05) in collagen deposition, expression level of Factor VIII and TGF-ß1, and hydroxyproline content in the two treatment groups compared with the healthy control group. These parameters in the combination therapy group were significantly higher than in the monotherapy group (P < 0.05). The ratio of collagen I to collagen III was statistically different among the three groups, and was significantly higher in the combination therapy group than in the monotherapy group (P < 0.05). CONCLUSION: Hydrotalcite combined with esomeprazole is superior to esomeprazole alone in improving gastric ulcer healing quality in terms of improving microvascular morphology, degree of structure maturity and function of regenerated mucosa.

Distinct patterns and prognostic values of tumor-infiltrating macrophages in hepatocellular carcinoma and gastric cancer.

BACKGROUND: Macrophages (Mφs) constitute a major component of the leukocyte infiltrate and perform distinct roles in different tumor microenvironments. This study aimed to characterize the distribution, composition and prognostic value of Mφs in hepatocellular carcinoma (HCC) and gastric cancer (GC). METHODS: Immunohistochemistry and immunofluorescence were used to identify Mφ subsets in HCC and GC tissues. Kaplan-Meier analysis and Cox regression models were applied to estimate the overall survival (OS) for HCC and GC patients. RESULTS: The results showed that the density of Mφs decreased in the intra-tumor region (IT) of HCC, but remarkably increased in the IT of GC, as compared with their non-tumor regions (NT). In HCC, most CD68+ Mφs were CD204+ and CD169+ cells in the NT region; however, there was a significant decrease in the percentage of CD169+ Mφ in the IT region. In contrast, CD68+ Mφs comprised a smaller percentage of CD204+ than the CD169+ subpopulation in the NT region, while more CD204+ but fewer CD169+ cells were present in the IT region of GC. The density of CD204+ Mφs correlated with poor prognosis in HCC, and CD169+ Mφs were associated with good survival in both HCC and GC. Moreover, the combination of low numbers of CD204+ and high numbers of CD169+ Mφs was associated with improved OS in both GC and HCC. CONCLUSIONS: Mφs display tissue-specific distributions and distinct composition patterns in HCC and GC tissues. Our results suggested that different types of tumors might use diverse strategies to reconstitute Mφ patterns to promote tumor progression.

Poly(I:C) Induces Human Lung Endothelial Barrier Dysfunction by Disrupting Tight Junction Expression of Claudin-5.

Viral infections are often accompanied by pulmonary microvascular leakage and vascular endothelial dysfunction via mechanisms that are not completely defined. Here, we investigated the effect of the Toll-like receptor 3 (TLR3) ligand polyinosinic-polycytidylic acid [Poly(I:C)], a synthetic analog of viral double-stranded RNA (dsRNA) commonly used to simulate viral infections, on the barrier function and tight junction integrity of primary human lung microvascular endothelial cells. Poly(I:C) stimulated IL-6, IL-8, TNFα, and IFNß production in conjunction with the activation of NF-κB and IRF3 confirming the Poly(I:C)-responsiveness of these cells. Poly(I:C) increased endothelial monolayer permeability with a corresponding dose- and time-dependent decrease in the expression of claudin-5, a transmembrane tight junction protein and reduction of CLDN5 mRNA levels. Immunofluorescence experiments revealed disappearance of membrane-associated claudin-5 and co-localization of cytoplasmic claudin-5 with lysosomal-associated membrane protein 1. Chloroquine and Bay11-7082, inhibitors of TLR3 and NF-κB signaling, respectively, protected against the loss of claudin-5. Together, these findings provide new insight on how dsRNA-activated signaling pathways may disrupt vascular endothelial function and contribute to vascular leakage pathologies.

Decreased Expression of FADS1 Predicts a Poor Prognosis in Patients with Esophageal Squamous Cell Carcinoma.

FADS1 (fatty acid desaturase 1) plays a crucial role in fatty acid metabolism, and it was recently reported to be involved in tumorigenesis. However, the role of FADS1 expression in esophageal squamous cell carcinoma (ESCC) remains unknown. In the current study, we investigated the expression and clinical pathologic and prognostic significance of FADS1 in ESCC. Immunohistochemical analyses revealed that 58.2% (146/251) of the ESCC tissues had low levels of FADS1 expression, whereas 41.8% (105/251) exhibited high levels of FADS1 expression. In positive cases, FADS1 expression was detected in the cytoplasm of cells. Correlation analyses demonstrated that FADS1 expression was significantly correlated with tumor location (p=0.025) but not with age, gender, histological grade, tumor status, nodal status or TNM staging. Furthermore, patients with tumors expressing high levels of FADS1had a longer disease-free survival time (p<0.001) and overall survival time (p<0.001). Univariate and multivariate analyses revealed that, along with nodal status, FADS1 expression was an independent and significant predictive factor (p<0.001). In conclusion, our study suggested that FADS1 might be a valuable biomarker and potential therapeutic target for ESCC.

SIRT3 is a novel prognostic biomarker for esophageal squamous cell carcinoma.

Sirtuin-3 (SIRT3) protein is a member of Sirtuins family. It functions as a critical mitochondrial deacetylase, which is involved in a variety of diseases, such as cancer, cardiovascular disease and diabetes. However, its role in esophageal squamous cell carcinoma (ESCC) has never been investigated before. With the help of immunohistochemistry, we studied the clinical significance of SIRT3 expression in ESCC. The receiver operating characteristic method was used to define the SIRT3 IRS cutoff value. The correlations between SIRT3 expression and clinicopathological variables were assessed using Pearson's χ (2) test. To evaluate the clinical significance of SIRT3 expression, Kaplan-Meier analysis was used to compare postoperative survival between different SIRT3 expression groups of ESCC patients. High expression of SIRT3 in ESCC tissues was more frequently observed than corresponding adjacent non-malignant esophageal mucosa tissues. No correlations were found between SIRT3 expression and clinical parameters. Multivariate analysis revealed that SIRT3 expression was an independent prognostic factor in ESCC (HR 1.454, P = 0.034). Increased SIRT3 expression suggests unfavorable prognosis for ESCC patients. Further studies are warranted.

Use of toll-like receptor assays to detect and identify microbial contaminants in biological products.

Toll-like receptor (TLR)-expressing cells, for the first time, detected and identified a microbial contaminant in a product made in Escherichia coli using an old manufacturing process. It was suspected of having a microbial contaminant(s) because, although it tested negative by standard pyrogen assays, it was associated with adverse events in early clinical trials. The assay readout is the induction of NF-kappaB and/or cytokines in response to TLR activation. Four coded samples, labeled A to D, including a sample prepared by the older manufacturing process, were submitted. The cell lines were activated only by samples B and D. Sample D stimulated only Mono-Mac 6 and HEK-human TLR4 (hTLR4) cells and was later identified as lipopolysaccharide. Except for TLR3 cells, sample B stimulated cells bearing the different TLRs (TLRs 2, 4, 5, 7, 8, and 9) and nontransfected HEK293 cells. These data suggested that flagellin was the microbial contaminant, since TLR5, the receptor for flagellin, is known to be expressed constitutively on HEK293 cells. Moreover, purified flagellin from Salmonella enterica serovar Typhimurium behaved like sample B, stimulating HEK293 and HEK-hTLR5 cells but not HEK-hTLR3 cells, and this stimulation by flagellin and sample B was blocked by an anti-hTLR5 neutralizing antibody. Western blots showed bands positive for flagellin and sample B with the molecular sizes expected for the flagellins from S. Typhimurium and E. coli, respectively. Mass spectrometry data were consistent with the presence of flagellin in the manufacturer's sample B. Taken together, these data indicate that the microbial contaminant in sample B was flagellin and may have been associated with adverse events when the recombinant product was administered.

MyD88-dependent signaling contributes to protection following Bacillus anthracis spore challenge of mice: implications for Toll-like receptor signaling.

Bacillus anthracis is a spore-forming, gram-positive organism that is the causative agent of the disease anthrax. Recognition of Bacillus anthracis by the host innate immune system likely plays a key protective role following infection. In the present study, we examined the role of TLR2, TLR4, and MyD88 in the response to B. anthracis. Heat-killed Bacillus anthracis stimulated TLR2, but not TLR4, signaling in HEK293 cells and stimulated tumor necrosis factor alpha (TNF-alpha) production in C3H/HeN, C3H/HeJ, and C57BL/6J bone marrow-derived macrophages. The ability of heat-killed B. anthracis to induce a TNF-alpha response was preserved in TLR2-/- but not in MyD88-/- macrophages. In vivo studies revealed that TLR2-/- mice and TLR4-deficient mice were resistant to challenge with aerosolized Sterne strain spores but MyD88-/- mice were as susceptible as A/J mice. We conclude that, although recognition of B. anthracis occurs via TLR2, additional MyD88-dependent pathways contribute to the host innate immune response to anthrax infection.

Th1-like cytokine induction by heat-killed Brucella abortus is dependent on triggering of TLR9.

In this report we provide evidence, for the first time, that bacterial DNA in the context of heat-killed Brucella abortus (HKBA) engages TLR9 in dendritic cells (DC), resulting in a Th1-like cytokine response. This is based on the findings that HKBA induction of IL-12p40 is: 1) abolished in DC from TLR9(-/-) mice; 2) blocked by suppressive oligodeoxynucleotides; 3) simulated by bacterial DNA derived from HKBA; and 4) abrogated by DNase or methylation of the DNA from HKBA. Furthermore, the effect of HKBA can be inhibited by chloroquine, indicating that endosomal acidification is required and supporting the notion that DNA from HKBA is interacting with TLR9 at the level of the endosome, as is the case with CpG oligodeoxynucleotides. In addition to DC, HKBA can elicit IL-12p40 secretion from macrophages, in which case the effect is wholly MyD88 dependent but only partially TLR9 dependent. This probably explains why HKBA effects in vivo are only partially reduced in TLR9(-/-), but absent in MyD88(-/-) mice. Because of their intimate interactions with T cells, the DC response is most likely to be critical for linking innate and adaptive immune responses, whereas the macrophage reaction may play a role in enhancing NK cell and bystander immune responses. In addition to IL-12p40, HKBA induces other Th1-like cytokines, namely, IFN-alpha and IFN-gamma, in a TLR9-dependent manner. These cytokines are important in protection against viruses and bacteria, and their induction enhances HKBA as a potential carrier for vaccines.