A quantitative real time PCR method to analyze T cell receptor Vβ subgroup expansion by staphylococcal superantigens

<p>Abstract</p> <p>Background</p> <p>Staphylococcal enterotoxins (SEs), SE-like (SEl) toxins, and toxic shock syndrome toxin-1 (TSST-1), produced by <it>Staphylococcus aureus</it>, belong to the subgroup of microbial superantigens (SAgs). SAgs induce clonal proliferation of T cells bearing specific variable regions of the T cell receptor β chain (Vβ). Quantitative real time PCR (qRT-PCR) has become widely accepted for rapid and reproducible mRNA quantification. Although the quantification of Vβ subgroups using qRT-PCR has been reported, qRT-PCR using both primers annealing to selected Vβ nucleotide sequences and SYBR Green I reporter has not been applied to assess Vβ-dependent expansion of T cells by SAgs.</p> <p>Methods</p> <p>Human peripheral blood mononuclear cells were stimulated with various SAgs or a monoclonal antibody specific to human CD3. Highly specific expansion of Vβ subgroups was assessed by qRT-PCR using SYBR Green I reporter and primers corresponding to selected Vβ nucleotide sequences.</p> <p>Results</p> <p>qRT-PCR specificities were confirmed by sequencing amplified PCR products and melting curve analysis. To assess qRT-PCR efficiencies, standard curves were generated for each primer set. The average slope and R²of standard curves were -3.3764 ± 0.0245 and 0.99856 ± 0.000478, respectively, demonstrating that the qRT-PCR established in this study is highly efficient. With some exceptions, SAg Vβ specificities observed in this study were similar to those reported in previous studies.</p> <p>Conclusions</p> <p>The qRT-PCR method established in this study produced an accurate and reproducible assessment of Vβ-dependent expansion of human T cells by staphylococcal SAgs. This method could be a useful tool in the characterization T cell proliferation by newly discovered SAg and in the investigation of biological effects of SAgs linked to pathogenesis.</p

A case of hepatoblastoma misdiagnosed as combined hepatocellular carcinoma and cholangiocarcinoma in an adult

Hepatoblastoma usually occurs in children under the age of 2 years, with very few cases reported in adults. We experienced a case of adult hepatoblastoma in a 36-year-old female with chronic hepatitis B. She had experienced sudden onset abdominal pain. Her serum alpha-fetoprotein level was markedly elevated, and abdominal CT showed a 9-cm mass with internal hemorrhage in the right hepatic lobe with hemoperitoneum, so an emergency hepatic central bisectionectomy was performed. The initial histologic examination revealed that the mass mimicked combined hepatocellular carcinoma and cholangiocarcinoma with spindle-cell metaplasia of the cholangiocarcinoma element. Follow-up abdominal CT performed 3 months later showed a 5.5-cm metastatic mass in the left subphrenic area. Laparoscopic splenectomy with mass excision was performed, and hepatoblastoma was confirmed histologically. A histologic re-examination of previously obtained surgical specimens also confirmed the presence of hepatoblastoma. Metastatic hepatoblastoma was found at multiple sites of the abdomen during follow-up, and so chemotherapy with cisplatin, 5-fluorouracil (5-FU), and vincristine was applied, followed by carboplatin and doxorubicin. Despite surgery and postoperative chemotherapy, she died 12 months after symptom onset

Production of Hydrogen and Carbon Black by Methane Decomposition Using DC-RF Hybrid Thermal Plasmas

A continuous production of carbon black and hydrogen has been investigated by thermal decomposition of methane using a prototype processing system of DC-RF hybrid thermal plasma, which has great advantage over other thermal sources like combustion or DC plasma torches in synthesizing new nanostructured materials by providing high-temperature environment and longer residence time for reactant gases due to its larger hot core region and lower flow velocity. Appropriate operation conditions and reactor geometries for the effective synthesis process are predicted first from the relevant theoretical bases, such as thermodynamic equilibrium calculations, two-dimensional thermal flow analysis, and chemical kinetic simulation. Based on these derived operation and design parameters, a reaction chamber and a DC-RF hybrid torch are fabricated for the processing system, which is followed by methane decomposition experiments with it. The methane injected into the processing system is converted mostly into hydrogen with a small volume fraction of acetylene, and fine carbon particles of 20-50 nm are identified from their TEM images. Material analyses of BET, DBP and XRD indicate that the synthesized carbon black has excellent properties such as large surface area, high electrical conductivity, and highly graphitized structures with good crystallization

Quantitative agreement of Dzyaloshinskii-Moriya interactions for domain-wall motion and spin-wave propagation

The magnetic exchange interaction is the one of the key factors governing the basic characteristics of magnetic systems. Unlike the symmetric nature of the Heisenberg exchange interaction, the interfacial Dzyaloshinskii-Moriya interaction (DMI) generates an antisymmetric exchange interaction which offers challenging opportunities in spintronics with intriguing antisymmetric phenomena. The role of the DMI, however, is still being debated, largely because distinct strengths of DMI have been measured for different magnetic objects, particularly chiral magnetic domain walls (DWs) and non-reciprocal spin waves (SWs). In this paper, we show that, after careful data analysis, both the DWs and SWs experience the same strength of DMI. This was confirmed by spin-torque efficiency measurement for the DWs, and Brillouin light scattering measurement for the SWs. This observation, therefore, indicates the unique role of the DMI on the magnetic DW and SW dynamics and also guarantees the compatibility of several DMI-measurement schemes recently proposed.Comment: 24 pages, 5 figure

The Group B Streptococcal Adhesin BspC Interacts with Host Cytokeratin 19 To Promote Colonization of the Female Reproductive Tract

Streptococcus agalactiae, otherwise known as Group B Streptococcus (GBS), is an opportunistic pathogen that vaginally colonizes approximately one third of healthy women. During pregnancy, this can lead to in utero infection, resulting in premature rupture of membranes, chorioamnionitis, and stillbirths. Furthermore, GBS causes serious infection in newborns, including sepsis, pneumonia, and meningitis. Previous studies have indicated that GBS antigen (Ag) I/II family proteins promote interaction with vaginal epithelial cells; thus, we hypothesized that the Ag I/II Group B streptococcal surface protein C (BspC) contributes to GBS colonization of the female reproductive tract (FRT). Here, we show that a ΔbspC mutant has decreased bacterial adherence to vaginal, ecto-, and endocervical cells, as well as decreased auto-aggregation and biofilm-like formation on cell monolayers. Using a murine model of vaginal colonization, we observed that the ΔbspC mutant strain exhibited a significant fitness defect compared to wild-type (WT) GBS and was less able to ascend to the cervix and uterus in vivo, resulting in reduced neutrophil chemokine signaling. Furthermore, we determined that BspC interacts directly with the host intermediate filament protein cytokeratin 19 (K19). Surface localization of K19 was increased during GBS infection, and interaction was mediated by the BspC variable (V) domain. Finally, mice treated with a drug that targets the BspC V-domain exhibited reduced bacterial loads in the vaginal lumen and reproductive tissues. These results demonstrate the importance of BspC in promoting GBS colonization of the FRT and that it may be targeted therapeutically to reduce GBS vaginal persistence and ascending infection

Evaluation of two mutants of \u3ci\u3eMycobacterium avium\u3c/i\u3e subsp. \u3ci\u3eparatuberculosis\u3c/i\u3e as candidates for a live attenuated vaccine for Johne’s disease

Control of Johne’s disease, caused by Mycobacterium avium subsp. paratuberculosis, has been difficult because of a lack of an effective vaccine. To address this problem we used targeted gene disruption to develop candidate mutants with impaired capacity to survive ex vivo and in vivo to test as a vaccine. We selected relA and pknG, genes known to be important virulence factors in Mycobacterium tuberculosis and Mycobacterium bovis, for initial studies. Deletion mutants were made in a wild type Map (K10) and its recombinant strain expressing the green fluorescent protein (K10-GFP). Comparison of survival in an ex vivo assay revealed deletion of either gene attenuated survival in monocyte-derived macrophages compared to survival of wild-type K10. In contrast, study in calves revealed survival in vivo was mainly affected by deletion of relA. Bacteria were detected in tissues from wild-type and the pknG mutant infected calves by bacterial culture and PCR at three months post infection. No bacteria were detected in tissues from calves infected with the relA mutant (P \u3c 0.05). Flow cytometric analysis of the immune response to the wild-type K10-GFP and the mutant strains showed deletion of either gene did not affect their capacity to elicit a strong proliferative response to soluble antigen extract or live Map. Quantitative RTPCR revealed genes encoding IFN-ƴ, IL-17, IL-22, T-bet, RORC, and granulysin were up-regulated in PBMC stimulated with live Map three months post infection compared to the response of PBMC pre-infection. A challenge study in kid goats showed deletion of pknG did not interfere with establishment of an infection. As in calves, deletion of relA attenuated survival in vivo. The mutant also elicited an immune response that limited colonization by challenge wild type Map. The findings show the relA mutant is a good candidate for development of a live attenuated vaccine for Johne’s disease

Does swab type matter? Comparing methods for \u3ci\u3eMannheimia haemolytica\u3c/i\u3e recovery and upper respiratory microbiome characterization in feedlot cattle

Background: Bovine respiratory disease (BRD) is caused by interactions among host, environment, and pathogens. One standard method for antemortem pathogen identification in cattle with BRD is deep-guarded nasopharyngeal swabbing, which is challenging, costly, and waste generating. The objective was to compare the ability to recover Mannheimia haemolytica and compare microbial community structure using 29.5 inch (74.9 cm) deep-guarded nasopharyngeal swabs, 16 inch (40.6 cm) unguarded proctology swabs, or 6 inch (15.2 cm) unguarded nasal swabs when characterized using culture, real time-qPCR, and 16S rRNA gene sequencing. Samples for aerobic culture, qPCR, and 16S rRNA gene sequencing were collected from the upper respiratory tract of cattle 2 weeks after feedlot arrival. Results: There was high concordance of culture and qPCR results for all swab types (results for 77% and 81% of sampled animals completely across all 3 swab types for culture and qPCR respectively). Microbial communities were highly similar among samples collected with different swab types, and differences identified relative to treatment for BRD were also similar. Positive qPCR results for M. haemolytica were highly concordant (81% agreed completely), but samples collected by deep-guarded swabbing had lower amounts of Mh DNA identified (Kruskal–Wallis analysis of variance on ranks, P \u3c 0.05; Dunn-test for pairwise comparison with Benjamini–Hochberg correction, P \u3c 0.05) and lower frequency of positive compared to nasal and proctology swabs (McNemar’s Chi-square test, P \u3c 0.05). Conclusions: Though differences existed among different types of swabs collected from individual cattle, nasal swabs and proctology swabs offer comparable results to deep-guarded nasopharyngeal swabs when identifying and characterizing M. haemolytica by culture, 16S rRNA gene sequencing, and qPCR

Bovine Staphylococcus aureus superantigens stimulate the entire T 2 cell repertoire of cattle.

Superantigens (SAgs) represent a diverse family of bacterial toxins that induce Vβ-specific T cell proliferation associated with an array of important diseases in humans and animals, including mastitis of dairy cows. However, an understanding of the diversity and distribution of SAg genes among bovine Staphylococcus aureus and their role in the pathogenesis of mastitis is lacking. Population genomic analysis of 195 bovine S. aureus isolates representing 57 unique sequence types revealed that strains encode 2 to 12 distinct SAgs and that the majority of isolates contain 5 or more SAg genes. A genome-scale analysis of bovine reference strain RF122 revealed a complement of 11 predicted SAg genes, which were all expressed in vitro. Detection of specific antibodies in convalescent cows suggests expression of 7 of 11 SAgs during natural S. aureus infection. We determined the Vβ T cell activation profile for all functional SAgs encoded by RF122 revealing evidence for bovine host-specific activity among recently identified RF122-encoded SAgs SElY and SElZ. Remarkably, we discovered that some strains have evolved the capacity to stimulate the entire T-cell repertoire of cattle through an array of diverse SAgs suggesting a key role in bovine immune evasion

Blastomycosis-like Pyoderma with Good Response to Acitretin

Blastomycosis-like pyoderma is a rare, vegetating skin lesion that is an unusual exaggerated tissue reaction possibly to prolonged primary or secondary bacterial infection. We report a case of blastomycosis-like pyoderma in a man with Down syndrome, diabetes mellitus and hypothyroidism who responsed poorly to conventional therapies, including antibiotics and correction of predisposing factors for a long time, but experienced dramatic improvement after 3 months of acitretin treatment