Evaluation of pathotype marker genes in Streptococcus suis isolated from human and clinically healthy swine in Thailand.

BACKGROUND: Streptococcus suis is a zoonotic pathogen that causes substantial economic losses in the pig industry and contributes to human infections worldwide, especially in Southeast Asia. Recently, a multiplex polymerase chain reaction (PCR) process was developed to distinguish disease-associated and non-disease-associated pathotypes of S. suis European strains. Herein, we evaluated the ability of this multiplex PCR approach to distinguish pathotypes of S. suis in Thailand. RESULTS: This study was conducted on 278 human S. suis isolates and 173 clinically healthy pig S. suis isolates. PCR identified 99.3% of disease-associated strains in the human isolates and 11.6% of non-disease-associated strains in the clinically healthy pig isolates. Of the clinically healthy pig S. suis isolates, 71.1% were classified as disease-associated. We also detected undetermined pathotype forms in humans (0.7%) and pigs (17.3%). The PCR assay classified the disease-associated isolates into four types. Statistical analysis revealed that human S. suis clonal complex (CC) 1 isolates were significantly associated with the disease-associated type I, whereas CC104 and CC25 were significantly associated with the disease-associated type IV. CONCLUSION: Multiplex PCR cannot differentiate non-disease-associated from disease-associated isolates in Thai clinically healthy pig S. suis strains, although the method works well for human S. suis strains. This assay should be applied to pig S. suis strains with caution. It is highly important that multiplex PCR be validated using more diverse S. suis strains from different geographic areas and origins of isolation.

Proteomics Analysis of Andrographolide-Induced Apoptosis via the Regulation of Tumor Suppressor p53 Proteolysis in Cervical Cancer-Derived Human Papillomavirus 16-Positive Cell Lines.

Regardless of the prophylactic vaccine accessibility, persistent infections of high-risk human papillomaviruses (hr-HPVs), recognized as an etiology of cervical cancers, continues to represent a major health problem for the world population. An overexpression of viral early protein 6 (E6) is linked to carcinogenesis. E6 induces anti-apoptosis by degrading tumor suppressor proteins p53 (p53) via E6-E6-associated protein (E6AP)-mediated polyubiquitination. Thus, the restoration of apoptosis by interfering with the E6 function has been proposed as a selective medicinal strategy. This study aimed to determine the activities of andrographolide (Androg) on the disturbance of E6-mediated p53 degradation in cervical cancer cell lines using a proteomic approach. These results demonstrated that Androg could restore the intracellular p53 level, leading to apoptosis-induced cell death in HPV16-positive cervical cancer cell lines, SiHa and CaSki. Mechanistically, the anti-tumor activity of Androg essentially relied on the reduction in host cell proteins, which are associated with ubiquitin-mediated proteolysis pathways, particularly HERC4 and SMURF2. They are gradually suppressed in Androg-treated HPV16-positive cervical cancer cells. Collectively, the restoration of p53 in HPV16-positive cervical cancer cells might be achieved by disruption of E3 ubiquitin ligase activity by Androg, which could be an alternative treatment for HPV-associated epithelial lesions.

E6D25E, HPV16 Asian variant shows specific proteomic pattern correlating in cells transformation and suppressive innate immune response.

HPV16 Asian variant (HPV16As) containing E6D25E oncogene, is commonly associated with cervical cancers of Asian populations. To explore a mechanism of E6D25E oncoprotein in carcinogenesis, we compared protein profiles in human keratinocytes expressing E6D25E with E6 of HPV16 prototype (E6Pro). A human cervical keratinocyte cell line, HCK1T, was transduced with retroviruses containing E6D25E or E6Pro genes. Biological properties of E6D25E or E6Pro transduced HCK1T cells were characterized. Protein profiles of the transduced HCK1T cells were analyzed using 2D-PAGE and characterized by mass spectrometry and western blotting. Reactomes of modulated proteins were analyzed by using the Reactome Knowledgebase. The E6D25E and E6Pro oncoproteins were comparable for their abilities to degrade p53 and suppress the induction of p21, and induce cell proliferation. Interestingly, the protein profiles of the HCK1T cells transduced with E6D25E showed specific proteomic patterns different from those with E6Pro. Among altered proteins, more than 1.5-fold up- or down- regulation was observed in E6D25E-expressing cells for gp96 and keratin7 which involved in activation of TLR signaling and transformation of squamocolumnar junction cells, respectively. This report describes new cellular proteins specifically targeted by E6D25E oncoprotein that may contribute to impair immune response against viral infection and cell transformation associated with oncogenic property of HPV16As variant.

HPV16 E2 protein promotes innate immunity by modulating immunosuppressive status.

The balance between active immune responses against human papillomavirus (HPV) and HPV-induced immune escape regulates viral clearance and carcinogenesis. To understand the role of the early viral protein HPV16 E2 in host innate immune responses, the HPV16 E2-transfected murine squamous cell carcinoma cell line SCCVII (SCC/E2) was generated and anti-tumor responses in T-cell-depleted mice were evaluated. Tumor growth of SCC/E2 was markedly reduced. Cytotoxicity against the NK-sensitive targets YAC-1 and SCCVII was clearly enhanced in SCC/E2-inoculated mice. Despite the comparable ratio of NK cells, the proportion of CD11b(+)Gr-1(+) myeloid-derived suppressor cells (MDSCs) was significantly decreased in SCC/E2-inoculated mice. The transcription of MDSC-related mediators such as inducible nitric oxide synthase, indoleamine 2,3-dioxygenase, and heme oxygenase-1 was significantly impaired in the SCC/E2-inoculated tumor tissues on day 3. Our results suggest that HPV16 E2 promotes anti-tumor innate effector function by modulating immunoregulatory events mediated by MDSCs and their mediators. This report describes a new role for HPV16 E2 as a local immunomodulator at infected sites.

Influence of goat management systems on hematological, oxidative stress profiles, and parasitic gastrointestinal infection.

Background and Aim: Good management in goats is known for good quality health and increasing productivity. The physiological change studies in goats are limited despite some existing studies on the relationship of various patterns to growth rates. This study aimed to determine the hematological parameters, oxidative stress, and parasitic infection in three management systems in Thai native goats. Materials and Methods: A total of 18 male goats were randomly assigned to the three systems: The free-range model (FREE), the semi-intensive model (SEMI), and the kept-in-a-cage model (BARN) for 35 days. Blood, fecal sampling, and weight data were collected and monitored every 5 days for analysis. Results: No statistical differences were found in the FREE and SEMI groups, but significance was observed in the BARN group. The body weight of the goats gradually reduced from 13.0 ± 2.44 kg to 10.18 ± 2.61 kg (mean ± standard deviation). In contrast, the significantly increasing red blood cells, packed-cell volume, white blood cells, neutrophil-to-lymphocyte (N/L) ratio, cortisol hormone, and antioxidation profiles were observed to be higher in BARN than in FREE and SEMI groups. The intensity of the parasite eggs was remarkably significant. It was observed in the BARN group between the beginning and end of the experiment (supported by a high level of eosinophils). Conclusion: These data can be applied for the realistic evaluation and improvement of management practices, especially fully restrained management (BARN) for monitoring the health status of goats.

Genomic characterization of vancomycin-resistant Enterococcus faecium clonal complex 17 isolated from urine in tertiary hospitals in Northeastern Thailand.

Vancomycin-resistant Enterococci (VREs) have increasingly become a major nosocomial pathogen worldwide, earning high-priority category from the World Health Organization (WHO) due to their antibiotic resistance. Among VREs, vancomycin-resistant Enterococcus faecium (VREfm) is particularly concerning, frequently isolated and resistant to many antibiotics used in hospital-acquired infections. This study investigated VREfm isolates from rural tertiary hospitals in Northeastern Thailand based both antibiotic susceptibility testing and whole-genome sequencing. All isolates showed resistance to vancomycin, ampicillin, erythromycin, tetracycline, ciprofloxacin, norfloxacin, and rifampin. Nitrofurantoin and tigecycline resistance were also observed in nearly all isolates. Conversely, all isolates remained susceptible to chloramphenicol, daptomycin, and linezolid. Genomic characterization revealed that all VREfm isolates belonged to clonal complex 17 (CC17), primarily consisting of sequence type (ST) 80, followed by ST17, ST761, and ST117. Additionally, all isolates harbored numerous antimicrobial-resistant genes, including vanA, tet(L), tet(M), aac(6')-li, ant(6)-Ia, aph(3')-III, aac(6')-aph(2″), aph(2″)-la, ant(9)-la, erm(B), msr(C), erm(T), erm(A), fosB, dfrG, and cfr(B). Notably, all isolates contained virulence genes, for collagen adhesin (acm) and cell wall adhesin (efafm), while hylEfm (glycosyl hydrolase) was detected in VREfm ST80. This study provided important information for understanding the genomic features of VREfm isolated from urine.

Treatment efficacy of Thunbergia laurifolia, Curcuma longa, Garcinia mangostana, and Andrographis paniculata extracts in Staphylococcus aureus-induced rabbit dermatitis model.

Background and Aim: Dermatitis is a soft-tissue infection caused by Staphylococcus aureus. The recurrence of inflammatory skin is linked to clinical manifestations. Anti-inflammatory cytokines, which are essential for tissue damage, are released by bacteria through skin tissues. Oxidative stress causes inflammatory cells to necrotize and reduces their antioxidant profile, resulting in toxic damage to surrounding tissues. Although studies on the antibacterial effects of Thunbergia laurifolia Lindl., Curcuma longa L., Garcinia mangostana L., and Andrographis paniculata (Burm.). Bacterial infection of S. aureus have been conducted, most of these studies have been in vitro and were not related to the rabbit model. In addition, anti-inflammatory and antioxidant studies need to be evaluated. Thus, this study aims to compare the antibacterial, anti-inflammatory, and antioxidant properties of four local herbs with a standard antibiotic in S. aureus-induced rabbit dermatitis model. Materials and Methods: The skin of New Zealand white rabbits were artificially wounded using a sterile blade and then infected with S. aureus. The rabbits were divided into seven groups, each with three rabbits (Total 21 rabbits): The first group was the no infection group (no infection and no treatment with scarification), the second group was the no treatment group (S. aureus infection of the wound but no treatment), and the other five treated groups were T. laurifolia, C. longa, G. mangostana, A. paniculata, and bacitracin cream, all of which involved wound infection and treatments. The treatment lasted for 7 days. The antibacterial, anti-inflammatory, and antioxidant properties after treatment were measured. Results: The efficacy of T. laurifolia, C. longa, G. mangostana, and A. paniculata was similar to that of an antioxidant and free radical scavenging property. The bacterial infection process gradually reduced the activities of antioxidant systems (i.e., enzymatic levels and gene expressions) and total glutathione. However, the activities of the antioxidant system were steadily increased when treated with herbal extracts. During bacterial invasion of the skin, the concentration of thiobarbituric acid reactive molecules, the level of lipid peroxidation, and the expression of anti-inflammatory cytokine genes were increased. All these were decreased when herbal extracts were used to treat the lesion. Conclusion: It can be concluded that T. laurifolia, C. longa, G. mangostana, and A. paniculate extract have antibacterial, anti-inflammatory, and antioxidant properties and are effective antibacterial agents. G. mangostana is the most effective herbal extract for antidermatitis and has the potential to be used as an alternative topical treatment.

The effects of mulberry (Morus alba Linn.) leaf supplementation on growth performance, blood parameter, and antioxidant status of broiler chickens under high stocking density.

Background and Aim: A stocking density system in boilers is well known for increasing productivity. However, this system increases stress and affects the growth performance of broilers. Mulberry is a valuable plant with therapeutic applications in traditional medicine; moreover, it reduces free radicals and improves growth performance in broilers. This study was conducted to investigate the effects of mulberry on the blood biochemistry parameters and the antioxidant status of broilers exposed to various raising systems. Materials and Methods: Two hundred and seventy-six 3-week-old male broilers were randomly assigned to nine categories composed of three growing systems: Semi-intensive, low stocking density, and high stocking density. Each group was fed with a control diet mixed with and without 10% mulberry leaf extract; the positive control group was provided with vitamin C. During the study, phytochemical screening of mulberry leaf extract, growth performances, hematological parameters, and antioxidant profiles were measured over the 4 weeks of the treatment. Results: In the high stocking density group, lipid peroxidation gradually increased while antioxidant activities decreased; however, the level of lipid peroxidation was reduced, whereas catalase and superoxide dismutase activities were significantly increased. The growth performance and blood biochemistry were improved after being fed with 10% mulberry leaf extract. Conclusion: This finding indicates that mulberry leaf extract reduced oxidative stress, activated antioxidant enzyme activities, and enhanced broilers' growth performance when raised under stress conditions.

Combinatorial sympathetic and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) blockades inhibit the murine melanoma growth by targeting infiltrating T cells.

BACKGROUND: Failure of the proliferation and infiltration of tumor-specific T cells in tumor site has been considered as one of important reasons induce the inefficiencies of immune checkpoint therapies in advanced cancers. Therefore, we aimed to demonstrate how combinatorial sympathetic and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) blockade affects the tumor growth of melanoma-bearing mice and potential mechanisms. METHODS: Tumor growth was measured and the infiltrating immune cell populations were observed with flow cytometry in B16-F10 melanoma-bearing mice treated with combined sympathetic and immune checkpoint blockade, using anti-CTLA-4 antibodies. The expression of adrenergic receptors was investigated in human peripheral blood mononuclear cells and their subpopulations, and the proliferation of T cell subsets was detected when stimulated by norepinephrine and its antagonists. RESULTS: B16-F10 tumor growth was associated with infiltrating CD8+ T cells. Combinatorial sympathetic and CTLA-4 blockade inhibited tumor growth and enhanced CD8+ infiltration. Meanwhile, all ß1, ß2 and ß3 adrenergic receptors were found to be expressed in human peripheral blood mononuclear cells, activated T cells, monocytes, and monocyte-induced dendritic cells. ß2-adrenergic receptors were expressed in most CD4+ T cells with increased expression in activated CD8+ T cells. Moreover, norepinephrine was able to prevent CD4+ T cell proliferation and ß2-adrenergic receptor antagonists could reverse the inhibition of CD4+, but not CD8+ cell proliferation. CONCLUSIONS: We conclude that the combination of sympathetic and CTLA-4 inhibitors is more effective for inhibiting melanoma progression than a single treatment and might enhance the infiltration of T cells in the tumor site, offering a novel therapeutic approach for immune checkpoint targeting.

Enhancement of specific T-lymphocyte responses by monocyte-derived dendritic cells pulsed with E2 protein of human papillomavirus 16 and human p16INK4A.

INTRODUCTION: Prophylactic vaccines are already available for prevention of human papillomavirus (HPV) infection. However, we still await development of therapeutic vaccines with high efficiency for stimulating specific T lymphocytes to clear HPV infection. OBJECTIVE: This study investigates the potential for subunits of human p16INK4a protein and E2 protein of HPV16 to stimulate dendritic cells and enhance the specific response of T lymphocytes against HPV-infected cells. METHODOLOGY: Immunogenic epitopes of HPV16 E2 and p16INK4a proteins were predicted through the common HLA class I and II alleles present in the Thai population. Then, monocyte-derived dendritic cells (MDCs) were pulsed with HPV16 E2 and/or p16INK4a protein s and their maturity assessed. MDCs pulsed with either or both of these proteins at optimal concentrations were used for activation of autologous T lymphocytes and IFN-γ production was measured for specific response function. RESULTS: HPV16 E2 and p16INK4a proteins contain various immunogenic epitopes which can be presented by antigen-presenting cells via both HLA class I and II molecules. The stimulation of MDCs with either HPV16 E2 or p16INK4a proteins increased percentages and mean fluorescence intensity (MFI) of CD83+ MDCs in a dose-dependent manner. An optimum concentration of 250 ng/mL and 150 ng/mL of HPV16 E2 and p16INK4a proteins, respectively, stimulated MDCs via the MAPK pathway (confirmed by use of MAPK inhibitors). T lymphocytes could be activated by MDCs pulsed with these proteins, leading to high percentages of both CD4+ IFN-γ+ T lymphocytes and CD8+ IFN-γ+ T lymphocytes. The production of IFN-γ was higher in co-cultures containing MDCs pulsed with HPV16 E2 protein than those pulsed with p16INK4a. Interestingly, MDCs pulsed with a combination of HPV16 E2 and p16INK4a significantly increased IFN-γ production of T lymphocytes. The IFN-γ production was inhibited by both HLA class I and II blockade, particularly in co-cultures with MDCs pulsed with a combination of HPV16 E2 and p16INK4a. CONCLUSIONS: This suggests that MDCs pulsed with both proteins enhances specific response of both CD4+ and CD8+ T lymphocytes. This study might provide a strategy for further in vivo study of stimulation of T lymphocytes for therapy of HPV-associated cancer.

Aberrant gene promoter methylation of E-cadherin, p16 INK4a , p14 ARF , and MGMT in Epstein-Barr virus-associated oral squamous cell carcinomas.

The etiology of oral carcinogenesis appears to be multifactorial. There is emerging evidence of the presence of Epstein-Barr virus (EBV) in epithelial oral squamous cell carcinoma (OSCC), but an association of EBV with oral carcinogenesis has not yet been established. Although epigenetic alterations, such as aberrant DNA methylation, are known to contribute to the pathogenesis of oral cancer, the relationship of such alterations with EBV infection is little known. This study aimed to investigate the association between EBV infection and promoter methylation patterns of tumor-associated genes in OSCC tissues. A total of 165 of formalin-fixed paraffin-embedded OSCC tissues were studied (68 of EBV positive and 97 of EBV negative). The promoter methylation patterns were investigated for four tumor-associated genes, E-cadherin, p16 INK4a , p14 ARF , and MGMT, by using methylation-specific polymerase chain reaction (MSP). The frequencies of gene promoter hypermethylation in all cases were 47.3% for E-cadherin, 92.7% for p16 INK4a , 74.5% for p14 ARF , and 35.8% for MGMT. Interestingly, most of the analyzed gene promoters were more frequently hypermethylated in EBV-positive than EBV-negative cases, in particular the E-cadherin (56/22) and MGMT (38/21) gene promoters (p < 0.05). Concomitantly, hypermethylation of multiple gene promoters (≥3) was encountered more frequently in EBV-positive samples. Hypermethylation of the E-cadherin promoter associated with EBV was more frequently observed in moderately and poorly differentiated OSCC tissues. These results indicate that epigenetic changes frequently occur in OSCCs and may partly be induced by EBV infection, therefore, EBV may involve in development and progression of the OSCCs.

Correlation of Circulating CD64+/CD163+ Monocyte Ratio and stroma/peri-tumoral CD163+ Monocyte Density with Human Papillomavirus Infected Cervical Lesion Severity.

HPV infected cervical cells secrete mediators that are gradually changed and have influence on infiltrating M2 phenotypic monocytes in cervical lesions. However, profiles of circulating immune cells in women with cervical lesions and M2 phenotypic monocyte activity in HPV infected cervical lesions are limited. This study aimed to investigate circulating monocyte populations correlated with M2 phenotype density and its activity in HPV infected cervical lesions. HPV DNA was investigated in cervical tissues using PCR. High risk HPV E6/E7 mRNA was detected using in situ hybridization. CD163 immunohistochemical staining was performed for M2 macrophage. CD163 and Arg1 mRNA expression were detected using real-time PCR. Circulating monocyte subpopulations were analyzed using flow cytometry. CD163 and Arg1 mRNA expression were increased according to cervical lesion severity and corresponding with density of M2 macrophage in HSIL and SCC in stroma and peri-tumoral areas. Additionally, the relationship between M2 macrophage infiltration and high risk HPV E6/E7 mRNA expression was found and corresponded with cervical lesion severity. Circulating CD14+CD16+ and CD14+CD163+ monocytes were elevated in No-SIL and cervical lesions. Interestingly, CD14+CD64+ monocyte was greatly elevated in HSIL and SCC, whereas intracellular IL-10+ monocytes were not significantly different between cervical lesions. The correlation between increasing ratio of circulating CD64+/CD163+ monocyte and density of infiltrating CD163+ monocytes was associated with severity of HPV infected cervical lesions. The elevated circulating CD64+/CD163+ monocyte ratio correlates to severity of HPV infected cervical lesions and might be a prognostic marker in cervical cancer progression.

Genome-wide analysis of high risk human papillomavirus E2 proteins in human primary keratinocytes.

The E2 protein is expressed in the early stage of human papillomavirus (HPV) infection that is associated with cervical lesions. This protein plays important roles in regulation of viral replication and transcription. To characterize the role of E2 protein in modulation of cellular gene expression in HPV infected cells, genome-wide expression profiling of human primary keratinocytes (HPK) harboring HPV16 E2 and HPV18 E2 was investigated using microarray. The Principle Components Analysis (PCA) revealed that the expression data of HPV16 E2 and HPV18 E2-transduced HPKs were rather closely clustered. The Venn diagram of modulated genes showed an overlap of 10 common genes in HPV16 E2 expressing HPK and HPV18 E2 expressing HPK. These genes were expressed with significant difference by comparison with control cells. In addition, the distinct sets of modulated genes were detected 14 and 34 genes in HPV16 E2 and HPV18 E2 expressing HPKs, respectively.

E2 proteins of high risk human papillomaviruses down-modulate STING and IFN-κ transcription in keratinocytes.

In the early stages of human papillomavirus (HPV) infection, the viral proteins elicit specific immune responses that can participate to regression of ano-genital lesions. HPV E6 protein for instance can reduce type I interferon (IFN) including IFN-κ that is involved in immune evasion and HPV persistence. To evaluate the role of E2 protein in innate immunity in HPV16-associated cervical lesions, genome-wide expression profiling of human primary keratinocytes (HPK) transduced by HPV16 E2 was investigated using microarrays and innate immunity associated genes were specifically analyzed. The analyses showed that the expression of 779 genes was modulated by HPV16E2 and 92 of them were genes associated with innate immunity. Notably IFN-κ and STING were suppressed in HPK expressing the E2 proteins of HPV16 or HPV18 and the trans-activation amino-terminal domain of E2 was involved in the suppressive effect. The relationship between STING, IFN-κ and interferon stimulated genes (ISGs) in HPK was confirmed by gene silencing and real time PCR. The expression of STING and IFN-κ were further determined in clinical specimens by real time PCR. STING and IFN-κ were down-modulated in HPV positive low grade squamous intraepithelial lesions compared with HPV negative controls. This study demonstrates that E2 proteins of high risk HPV reduce STING and IFN-κ transcription and its downstream target genes that might be an immune evasion mechanism involved in HPV persistence and cervical cancer development.