Monoclonal antibody against the poly-γ-d-glutamic acid capsule of Bacillus anthracis protects mice from enhanced lethal toxin activity due to capsule and anthrax spore challenge

Background

The poly-γ-d-glutamic acid (PGA) capsule, a major virulence factor of Bacillus anthracis, protects bacilli from immune surveillance and allows its unimpeded growth in the host. Recently, the importance of the PGA in the pathogenesis of anthrax infection has been reported. The PGA capsule is associated with lethal toxin (LT) in the blood of experimentally infected animals and enhances the cytotoxicity of LT.

Methods

To investigate the role of anti-PGA Abs on progression of anthrax infection, two mouse anti-PGA mAbs with K_d values of 0.8 μM and 2.6 μM respectively were produced and in silico three dimensional (3D) models of mAbs with their cognitive PGA antigen complex were analyzed.

Results

Anti-PGA mAbs specifically bound encapsulated B. anthracis H9401 and showed opsonophagocytosis activity against the bacteria with complement. The enhancement effect of PGA on LT-mediated cytotoxicity was confirmed ex vivo using mouse bone marrow-derived macrophages and was effectively inhibited by anti-PGA mAb. Passive immunization of mAb completely protected mice from PGA-enhanced LT toxicity and partially rescued mice from anthrax spore challenges. 3D structure models of these mAbs and PGA complex support specific interactions between CDR and cognitive PGA. These results indicate that mouse mAb against PGA capsule prevents the progress of anthrax disease not only by eliminating the vegetative form of encapsulated B. anthracis but also by inhibiting the enhanced cytotoxic activity of LT by PGA through specific binding with PGA capsule antigen.

General significance

Our results suggest a potential role for PGA antibodies in preventing and treating anthrax infection.     

Comparison of stress distribution in dental crown with different cusp angles: 3D finite element analysis

The purpose of this study is to measure the failure risk of a crown depending on the cusp angle. Three all-ceramic crown models consisting of C_H (high incline), C_M (middle incline), and C_L (low incline) are designed. Stress is applied to the crown with Loading case-1 (top of cusp tip) and Loading case-2 (middle of cusp ridge) with the use of FEA software. In Loading case-1 and case-2, the C_H showed the highest Maximum Principal Stress (MPS) while the CL showed the lowest MPS. The cusp angle is an influential factor affecting stress distribution in dental crowns.     

Manganese-containing superoxide dismutase and its gene from Candida albicans

Mitochondrial manganese-containing superoxide dismutase was purified around 112-fold with an overall yield of 1.1% to apparent electrophoretic homogeneity from the dimorphic pathogenic fungus, Candida albicans. The molecular mass of the native enzyme was 106 kDa and the enzyme was composed of four identical subunits with a molecular mass of 26 kDa. The enzyme was not sensitive to either cyanide or hydrogen peroxide. The N-terminal amino acid sequence alignments (up to the 18th residue) showed that the enzyme has high similarity to the other eukaryotic manganese-containing superoxide dismutases. The gene sod2 encoding manganese-containing superoxide dismutase has been cloned using a product obtained from polymerase chain reaction. Sequence analysis of the sod2 predicted a manganese-containing superoxide dismutase that contains 234 amino acid residues with a molecular mass of 26173 Da, and displayed 57% sequence identity to the homologue of Saccharomyces cerevisiae. The deduced N-terminal 34 amino acid residues may serve as a signal peptide for mitochondrial translocation. Several regulatory elements such as stress responsive element and haem activator protein 2/3/4/5 complex binding sites were identified in the promoter region of sod2. Northern analysis with a probe derived from the cloned sod2 revealed a 0.94-kb band, which corresponds approximately to the expected size of mRNA deduced from sod2.     

The dynamin-like protein ADL1C is essential for plasma membrane maintenance during pollen maturation

Dynamin-related GTPases regulate a wide variety of dynamic membrane processes in eukaryotes. Here, we investigated the function of ADL1C, a member of the Arabidopsis 68 kDa dynamin-like protein family. Analysis of heterozygous adl1C-1 indicates that the mutation specifically affects post-meiotic male gametogenesis. Fifty percent of the mature pollen from heterozygous adl1C-1 androecia are shriveled and fail to germinate in vitro. During microspore maturation, adl1C-1 pollen grains display defects in the plasma membrane and intine morphology, suggesting that ADL1C is essential for the formation and maintenance of the pollen cell surface and viability during desiccation. Consistent with a role in cell-surface dynamics, immunofluorescence microscopy indicates that ADL1C is localized to the cell plate of dividing somatic cells and to the tip of expanding root hairs. We propose that ADL1C functions in plasma membrane dynamics, and we discuss the role of the ADL1 family in plant growth and development.     

Survival Nomogram for Curatively Resected Korean Gastric Cancer Patients: Multicenter Retrospective Analysis with External Validation

BackgroundA small number of nomograms have been previously developed to predict the individual survival of patients who undergo curative resection for gastric cancer. However, all were derived from single high-volume centers. The aim of this study was to develop and validate a nomogram for gastric cancer patients using a multicenter database.MethodsWe reviewed the clinicopathological and survival data of 2012 patients who underwent curative resection for gastric cancer between 2001 and 2006 at eight centers. Among these centers, six institutions were randomly assigned to the development set, and the other two centers were assigned to the validation set. Multivariate analysis using the Cox proportional hazard regression model was performed, and discrimination and calibration were evaluated by external validation.ResultsMultivariate analyses revealed that age, tumor size, lymphovascular invasion, depth of invasion, and metastatic lymph nodes were significant prognostic factors for overall survival. In the external validation, the concordance index was 0.831 (95% confidence interval, 0.784–0.878), and Hosmer-Lemeshow chi-square statistic was 3.92 (P = 0.917).ConclusionsWe developed and validated a nomogram to predict 5-year overall survival after curative resection for gastric cancer based on a multicenter database. This nomogram can be broadly applied even in general hospitals and is useful for counseling patients, and scheduling follow-up.     

Efficacy and Safety of Sorafenib Therapy on Metastatic Renal Cell Carcinoma in Korean Patients: Results from a Retrospective Multicenter Study

Objective

To evaluate the efficacy and safety of sorafenib for Korean patients with metastatic renal cell carcinoma (mRCC).

Methods

A total of 177 mRCC patients using sorafenib as first- (N = 116), second- (N = 43), and third-line (N = 18) therapies were enrolled from 11 Korean centers between 2006 and 2012. The patient characteristics, therapy duration, tumor response, disease control rate, and tolerability were assessed at baseline and at routine follow-ups, and the progression-free survival (PFS) and overall survival (OS) times and rates were analyzed.

Results

Among all patients, 18 (10.2%) stopped sorafenib treatment for a median of 1.7 weeks, including 15 (8.5%) who discontinued the drug, while 40 (22.6%) and 12 (6.8%) patients required dose reductions and drug interruptions, respectively. Severe adverse events (AEs) or poor compliance was observed in 64 (36.2%) patients, with 118 (7.4%) ≥grade 3 AEs. During the treatment, one myocardial infarction was observed. The number of ≥grade 3 AEs in the first-line sorafenib group was 71 (6.8% of the total 1048 AEs). During a median follow-up of 17.2 months, the radiologically confirmed best objective response rate, disease control rate, median PFS, and median OS were 22.0%, 53.0%, 6.4 months (95% confidence interval [CI], 5.2–8.9), and 32.6 months (95% CI, 27.3–63.8) for the total 177 sorafenib-treated patients, respectively, and 23.2%, 56.0%, 7.4 months (95% CI, 5.5–10.5), and not reached yet (95% CI, 1.0–31.1) for the first-line sorafenib group, respectively.

Conclusions

Sorafenib produced tolerable safety, with a ≥grade 3 AE rate of 7.4% and an acceptable disease control rate (53.0%) in Korean mRCC patients.     

Molecular cloning of Plasmodium vivax calcium-dependent protein kinase 4

A family of calcium-dependent protein kinases (CDPKs) is a unique enzyme which plays crucial roles in intracellular calcium signaling in plants, algae, and protozoa. CDPKs of malaria parasites are known to be key regulators for stage-specific cellular responses to calcium, a widespread secondary messenger that controls the progression of the parasite. In our study, we identified a gene encoding Plasmodium vivax CDPK4 (PvCDPK4) and characterized its molecular property and cellular localization. PvCDPK4 was a typical CDPK which had well-conserved N-terminal kinase domain and C-terminal calmodulin-like structure with 4 EF hand motifs for calcium-binding. The recombinant protein of EF hand domain of PvCDPK4 was expressed in E. coli and a 34 kDa product was obtained. Immunofluorescence assay by confocal laser microscopy revealed that the protein was expressed at the mature schizont of P. vivax. The expression of PvCDPK4-EF in schizont suggests that it may participate in the proliferation or egress process in the life cycle of this parasite.     

Expression and secretion of the protective antigen of Bacillus anthracis in Bacillus brevis

We used the Bacillus brevis-pNU212 system to develop a mass production system for the protective antigen (PA) of Bacillus anthracis. A moderately efficient expression-secretion system for PA was constructed by fusing the PA gene from B. anthracis with the B. brevis cell-wall protein signal-peptide encoding region of pNU212, and by introducing the recombinant plasmid, pNU212-mPA, into B. brevis 47-5Q. The clone producing PA secreted about 300 microg of recombinant PA (rPA) per ml of 5PY-erythromycin medium after 4 days incubation at 30 degrees C. The rPA was fractionated from the culture supernatant of B. brevis 47-5Q carrying pNU212-mPA using ammonium sulfate at 70% saturation followed by anion exchange chromatography on a Hitrap Q, a Hiload 16/60 Superdex 200 gel filtration column and a phenyl sepharose hydrophobic interaction column, yielding 70 mg rPA per liter of culture. The N-terminal sequence of the purified rPA was identical to that of native PA from B. anthracis. The purified rPA exhibited cytotoxicity towards J774A.1 cells when combined with lethal factor. The rPA formulated in either Rehydragel HPA or MPL-TDM-CWS adjuvant (Ribi-Trimix) elicited the expression of a large amount of anti-PA and neutralizing antibodies in guinea pigs and completely protected them against a 100 LD50 challenge with fully virulent B. anthracis spores.     

CED-1, CED-7, and TTR-52 regulate surface phosphatidylserine expression on apoptotic and phagocytic cells

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Highlights? PS is expressed on the surface of apoptotic and phagocytic cells during apoptosis ? CED-7 and TTR-52 mediate time-dependent loss of PS from surface of apoptotic cells ? CED-7 and TTR-52 promote generation of extracellular PS vesicles ? CED-7/TTR-52/CED-1 promote phagocyte PS expression, important for corpse engulfment