[Source Appointment and Assessment of Heavy Metal Pollution in Surface Dust in the Main District Bus Stops of Tianshui City].

In this study, a PMF model was used to identify the sources and pollution level of heavy metals in the surface dust of a bus station. On the basis of the traditional heavy metal pollution evaluation methods, the Hakanson toxicity response coefficient was used to modify the traditional weight. The matter-element extension theory was introduced to reflect the toxicological properties and hazard degree of the heavy metals, and the matter-element extension model was established to evaluate the pollution level of heavy metals in the surface dust of the study area. The results were compared with Igeo, PN, and RI. ① Except for Co and V, the other heavy metals were higher than the Gansu soil background values by 1.29-9.30 times. The points of Cu and Pb exceeded the rate by 100%, and Cr, Ni, and As exceeded the rate by 96.15%, 94.23%, and 96.15%, respectively. ② PMF showed that source 1 was a natural source, and its contribution rate to V was 32.12%. Source 2 was natural-traffic pollution sources, contributing 51.50% and 33.37% to Cu and Co, respectively. Source 3 was a construction waste pollution source, with contribution rates of 45.06% and 44.70% for Cr and Ni, respectively, and source 4 was a coal-traffic mixed source, with contribution rates of 49.89% and 75.25% for As and Pb, respectively. ③ The matter-element evaluation results showed that the surface dust of the bus stops was mainly class IV (moderately polluted), and 13% of sample points were still clean, 37% were moderately polluted, and 25% were slightly and heavily polluted. The results of this method were quite different from the PN results and were more consistent with the RI results, indicating that its evaluation results were more sensitive and can be used for heavy metal pollution assessment.

A recurrence quantification analysis-based channel-frequency convolutional neural network for emotion recognition from EEG.

Constructing a reliable and stable emotion recognition system is a critical but challenging issue for realizing an intelligent human-machine interaction. In this study, we contribute a novel channel-frequency convolutional neural network (CFCNN), combined with recurrence quantification analysis (RQA), for the robust recognition of electroencephalogram (EEG) signals collected from different emotion states. We employ movie clips as the stimuli to induce happiness, sadness, and fear emotions and simultaneously measure the corresponding EEG signals. Then the entropy measures, obtained from the RQA operation on EEG signals of different frequency bands, are fed into the novel CFCNN. The results indicate that our system can provide a high emotion recognition accuracy of 92.24% and a relatively excellent stability as well as a satisfactory Kappa value of 0.884, rendering our system particularly useful for the emotion recognition task. Meanwhile, we compare the performance of the entropy measures, extracted from each frequency band, in distinguishing the three emotion states. We mainly find that emotional features extracted from the gamma band present a considerably higher classification accuracy of 90.51% and a Kappa value of 0.858, proving the high relation between emotional process and gamma frequency band.

Effect of gap junctions on RAW264.7 macrophages infected with H37Rv.

BACKGROUND: Apoptosis and inflammation have been shown to play an important role in the mechanisms involved in the pathogenesis of Mycobacterium tuberculosis (MTB) infection. When macrophages undergo apoptosis and polarization, gap junctions (GJs) may be needed to provide conditions for their functions. Connexin 43 (Cx43) and connexin 37 (Cx37) are the main connexins in macrophages that participate in the formation of GJ channels. METHODS: An H37Rv infection RAW264.7 macrophage model was established to investigate the associate between connexins and host macrophage immune defense response after MTB infection. First, Real-time Polymerase Chian Reaction (RT-PCR) was used to detect the mRNA expression of Cx43 and Cx37. Cx43 protein expression and location was detected by western blotting and immunofluorescence. Confocal microscope was used to assay the gap junctional intercellular communication (GJIC). Then, electron microscope used to observe the morphology of macrophages. Finally, RAW264.7 macrophage apoptosis and mitochondrial membrane potential was detected by flow cytometry, and the expression of inflammation factors such as CD86, CD206, and IL-6, IL-10, TNF-α, and TGF-ß were detected by Real-time PCR and enzyme-linked-immunosorbent serologic assay (ELISA). RESULTS: H37Rv infection significantly promoted host macrophage Cx43 mRNA and protein expression (increased 1.6-fold and 0.3-fold respectively), and enhanced host macrophage GJIC. When host macrophage cell-to-cell communication induced by H37Rv infection, the apoptosis rate and inflammatory factors expression also increased. CONCLUSIONS: The results confirm that H37Rv infection can obviously induce host macrophage Cx43 expression and enhance GJIC, which may implicated in host macrophage inflammatory reaction, to regulate the release of inflammatory factors and/or initiate apoptosis to activate host immune defense response.

Biological and Biomechanical Evaluation of Autologous Tendon Combined with Ligament Advanced Reinforcement System Artificial Ligament in a Rabbit Model of Anterior Cruciate Ligament Reconstruction.

OBJECTIVE: To compare the biomechanical and histological changes in a rabbit model after reconstructing the anterior cruciate ligament (ACL) with solely autologous tendon and with autologous tendon combined with the ligament advanced reinforcement system (LARS) artificial ligament. METHODS: Anterior cruciate ligament reconstruction was performed in 72 knees from 36 healthy New Zealand white rabbits (bodyweight, 2500-3000 g). The Achilles tendons were harvested bilaterally. The left ACL were reconstructed solely with autografts (autologous tendon group), while the right ACL were reconstructed with autografts combined with LARS ligaments (combined ligaments group). The gross observation, histological determination, and the tension failure loads in both groups were evaluated at 12 weeks (n = 18) and 24 weeks (n = 18) postoperatively. RESULTS: Gross examination of the knee joints showed that all combined ligaments were obviously covered by a connective tissue layer at 12 weeks, and were completely covered at 24 weeks. Fibrous tissue ingrowth was observed between fascicles and individual fibers in the bone-artificial ligament interface at both time points; this fibrovascular tissue layer localized at the bone-artificial ligament interface tended to be denser in specimens obtained at 24 weeks compared with those obtained at 12 weeks. The tension failure loads of the knees were similar in the autologous tendon group and the combined ligaments group at 12 weeks (144.15 ± 3.92 N vs. 140.88 ± 2.75 N; P > 0.05), and at 24 weeks (184.15 ± 1.96 N vs. 180.88 ± 3.21 N; P > 0.05). CONCLUSION: Reconstructing the ACL in rabbits using autologous tendon combined with the LARS artificial ligament results in satisfactory biointegration, with no obvious immunological rejection between the autologous tendon and the artificial ligament, and is, therefore, a promising ACL reconstruction method.

Improving the immunogenicity of a trivalent Neisseria meningitidis native outer membrane vesicle vaccine by genetic modification.

Trivalent native outer membrane vesicles (nOMVs) derived from three genetically modified Neisseria meningitidis serogroup B strains have been previously evaluated immunologically in mice and rabbits. This nOMV vaccine elicited serum bactericidal activity (SBA) against multiple N. meningitidis serogroup B strains as well as strains from serogroups C, Y, W, and X. In this study, we used trivalent nOMVs isolated from the same vaccine strains and evaluated their immunogenicity in an infant Rhesus macaque (IRM) model whose immune responses to the vaccine are likely to be more predictive of the responses in human infants. IRMs were immunized with trivalent nOMV vaccines and sera were evaluated for exogenous human serum complement-dependent SBA (hSBA). Antibody responses to selected hSBA generating antigens contained within the trivalent nOMVs were also measured and we found that antibody titers against factor H binding protein variant 2 (fHbpv2) were very low in the sera from animals immunized with these original nOMV vaccines. To increase the fHbp content in the nOMVs, the vaccine strains were further genetically altered by addition of another fHbp gene copy into the porB locus. Trivalent nOMVs from the three new vaccine strains had higher fHbp antigen levels and generated higher anti-fHbp antibody responses in immunized mice and IRMs. As expected, fHbp insertion into the porB locus resulted in no PorB expression. Interestingly, higher expression of PorA, an hSBA generating antigen, was observed for all three modified vaccine strains. Compared to the trivalent nOMVs from the original strains, higher PorA levels in the improved nOMVs resulted in higher anti-PorA antibody responses in mice and IRMs. In addition, hSBA titers against other strains with PorA as the only hSBA antigen in common with the vaccine strains also increased.

A small hairpin RNA targeting myeloid cell leukemia-1 enhances apoptosis in host macrophages infected with Mycobacterium tuberculosis.

Myeloid cell leukemia-1 (Mcl-1) plays an important role in various cell survival pathways. Some studies indicated that the expression of Mcl-1 was upregulated in host cells during infection with the virulent Mycobacterium tuberculosis strain, H37Rv. The present study was designed to investigate the effect of inhibiting Mcl-1 expression both in vivo and in vitro on apoptosis of host macrophages infected with M. tuberculosis using a small hairpin (sh)RNA. Mcl-1 expression was detected by the real time-polymerase chain reaction, western blotting, and immunohistochemistry. Flow cytometry and transmission electron microscopy were used to measure host macrophage apoptosis. We found elevated Mcl-1 levels in host macrophages infected with M. tuberculosis H37Rv. The expression of Mcl-1 was downregulated efficiently in H37Rv-infected host macrophages using shRNA. Knockdown of Mcl-1 enhanced the extent of apoptosis in H37Rv-infected host macrophages significantly. The increased apoptosis correlated with a decrease in M. tuberculosis colony forming units recovered from H37Rv-infected cells that were treated with Mcl-1-shRNA. Reducing Mcl-1 accumulation by shRNA also reduced accumulation of the anti-apoptotic gene, Bcl-2, and increased expression of the pro-apoptotic gene, Bax, in H37Rv-infected host macrophages. Our results showed that specific knockdown of Mcl-1 expression increased apoptosis of host macrophages significantly and decreased the intracellular survival of a virulent strain of M. tuberculosis. These data indicate that interference with Mcl-1 expression may provide a new avenue for tuberculosis therapy.

Suppression of Mcl-1 induces apoptosis in mouse peritoneal macrophages infected with Mycobacterium tuberculosis.

The effect of myeloid cell leukemia-1 (Mcl-1) inhibition on apoptosis of peritoneal macrophages in mice infected with Mycobacterium tuberculosis was investigated and the primary signaling pathway associated with the transcriptional regulation of Mcl-1 was identified. Real-time PCR and western blotting indicated that Mcl-1 transcript and protein expression are upregulated during infection with virulent M. tuberculosis H37Rv and Xinjiang strains but not with attenuated M. tuberculosis strain H37Ra or Bacillus Calmette-Guérin. Mcl-1 transcript and protein expression were downregulated by specific inhibitors of the Janus kinase/signal transducer and activator of transcription (JAK/STAT), mitogen-activated protein kinase (MAPK) and phosphoinositol 3-kinase (PI3K) pathways (AG490, PD98059 and LY294002, respectively). The strongest inhibitor of Mcl-1 expression was PD98059, the MAPK inhibitor. Flow cytometry demonstrated that the rate of apoptosis in peritoneal macrophages is significantly higher in mice infected with M. tuberculosis and the rate of apoptosis is correlated with the virulence of the strain of M. tuberculosis. Apoptosis was found to be upregulated by AG490, PD98059 and LY294002, whereas inhibition of the MAPK pathway sensitized the infected macrophages to apoptosis. Taken together, these results suggest that specific downregulation of Mcl-1 significantly increases apoptosis of peritoneal macrophages and that the MAPK signaling pathway is the primary mediator of Mcl-1 expression.

Rapid isolation of dengue-neutralizing antibodies from single cell-sorted human antigen-specific memory B-cell cultures.

Monitoring antigen-specific memory B cells and the antibodies they encode is important for understanding the specificity, breadth and duration of immune response to an infection or vaccination. The antibodies isolated could further help design vaccine antigens for raising relevant protective immune responses. However, developing assays to measure and isolate antigen-specific memory B cells is technically challenging due to the low frequencies of these cells that exist in the circulating blood. Here, we describe a flow cytometry method to identify and isolate dengue envelope-specific memory B cells using a labeled dengue envelope protein. We enumerated dengue-envelope specific memory B cells from a cohort of dengue seropositive donors using this direct flow cytometry assay. A more established and conventional assay, the cultured B ELISPOT, was used as a benchmark comparator. Furthermore, we were able to confirm the single-sorted memory B-cell specificity by culturing B cells and differentiating them into plasma cells using cell lines expressing CD40L. The culture supernatants were assayed for antigen binding and the ability of the antibodies to neutralize the cognate dengue virus. Moreover, we successfully isolated the heavy and light Ig sequences and expressed them as full-length recombinant antibodies to reproduce the activity seen in culture supernatants. Mapping of these antibodies revealed a novel epitope for dengue 2 virus serotype. In conclusion, we established a reproducible methodology to enumerate antigen-specific memory B cells and assay their encoded antibodies for functional characterization.

Effects of Ziziphus jujuba fruit extracts on cytochrome P450 (CYP1A2) activity in rats.

Drug-drug interactions have become a serious problem in the clinic, since plant-based medicines are extensively used. The present study investigated the effects of Ziziphus jujuba fruit (ZJ) extract on the pharmacokinetics of phenacetin, a typical substrate of a cytochrome P450 enzyme CYP 1A2, in rats. The rats were pretreated with the water extract (1.0 g · kg(-1)) or the ethanolic extract (3.6 g · kg(-1)) of ZJ for 10 days, and the pharmacokinetics of phenacetin was investigated after intravenous administration. In an in vitro assay, acetaminophen formation in the hepatic microsomes of ZJ-treated rats was investigated to assess CYP1A2 activity. Our results demonstrated that the treatment with the water and ethanolic extracts of ZJ decreased the plasma concentration of phenacetin and increased the plasma concentration of acetaminophen, resulting in a 43.2% and 15.5% reduction in the AUC0-120 of phenacetin, respectively, and a 53.2% and 64.9% increase in the AUC0-120 of acetaminophen, respectively after intravenous administration. The water or ethanolic extract of ZJ significantly increased the clearance of phenacetin and acetaminophen formation in hepatic microsomes. In conclusion, ZJ extracts displayed effects on the pharmacokinetics of phenacetin and increased the CYP1A2 activity in rats. Therefore, precaution on drug-drug interactions should be taken when ZJ is co-administered with drugs metabolized by CYP1A2, which may result in decreased concentrations of these drugs.

Sporozoite neutralizing antibodies elicited in mice and rhesus macaques immunized with a Plasmodium falciparum repeat peptide conjugated to meningococcal outer membrane protein complex.

Antibodies that neutralize infectivity of malaria sporozoites target the central repeat region of the circumsporozoite (CS) protein, which in Plasmodium falciparum is comprised primarily of 30-40 tandem NANP tetramer repeats. We evaluated immunogenicity of an alum-adsorbed (NANP)(6) peptide conjugated to an outer membrane protein complex (OMPC) derived from Neisseria meningitidis, a carrier protein used in a licensed Haemophilus influenzae pediatric vaccine. Mice immunized with (NANP)(6)-OMPC adsorbed to Merck's alum adjuvant (MAA), with or without Iscomatrix® as co-adjuvant, developed high levels of anti-repeat peptide antibody that inhibited in vitro invasion of human hepatoma cells by transgenic P. berghei sporozoites that express P. falciparum CS repeats (PfPb). Inhibition of sporozoite invasion in vitro correlated with in vivo resistance to challenge by the bites of PfPb-infected mosquitoes. Challenged mice had >90% reduction of hepatic stage parasites as measured by real-time PCR, and either sterile immunity, i.e., no detectable blood stage parasites, or delayed prepatent periods which indicate neutralization of a majority, but not all, sporozoites. Rhesus macaques immunized with two doses of (NANP)(6)-OMPC/MAA formulated with Iscomatrix® developed anti-repeat antibodies that persisted for ~2 years. A third dose of (NANP)(6)-OMPC/MAA+ Iscomatrix® at that time elicited strong anamnestic antibody responses. Rhesus macaque immune sera obtained post second and third dose of vaccine displayed high levels of sporozoite neutralizing activity in vitro that correlated with presence of high anti-repeat antibody titers. These preclinical studies in mice of different MHC haplotypes and a non-human primate support use of CS peptide-OMPC conjugates as a highly immunogenic platform to evaluate CS protective epitopes. Potential pre-erythrocytic vaccines can be combined with sexual blood stage vaccines as a multi-antigen malaria vaccine to block invasion and transmission of Plasmodium parasites.

[Inhibitory effect of dihydroartemisinin on the growth of human prostate cancer PC-3M cells and its mechanism].

OBJECTIVE: To study the effects of dihydroartemisinin on the apoptosis of and the vascular endothelial growth factor (VEGF) expression in prostate cancer cell line PC-3M in androgen-independent prostate cancer. METHODS: PC-3M cells were treated with different doses (0, 25, 50 and 100 micromol/L) of dihydroartemisinin for 48 hours, their growth activity analyzed by MTT colorimetric assay and flow cytometry, and changes in the activities of caspase-3 and -8 detected by colorimetric assay. The expression of VEGF mRNA was determined by semi-quantitative RT-PCR, and that of the VEGF protein by Western blotting. RESULTS: Compared with the 0 micromol/L control group, the 25, 50 and 100 micromol/L dihydroartemisinin groups showed significantly increased apoptosis of PC-3M cells ([2.92 +/- 0.45]% vs [8.85 +/- 0.74]%, [12.83 +/- 0.84]% and [18.65 +/- 1.24]%, P < 0.01), and dose-dependent increase in the activities of caspase-8 ([0.47 +/- 0.05 ] U/microg vs [1.22 +/- 0.15], [1.76 +/- 0.07] and [2.91 +/- 0.24] U/microg, P < 0.01) and caspase-3 ([0.44 +/- 0.07] U/microg vs [0.95 +/- 0.08], [1.48 +/- 0.14] and [2.92 +/- 0.45] U/microg, P < 0.01). The expressions of VEGF mRNA and protein were decreased in a concentration-dependent manner. CONCLUSION: Dihydroartemisinin can significantly suppress the growth of PC-3M cells, promote their apoptosis and reduce the expressions of VEGF mRNA and protein, which may serve to explain its inhibitory effect on tumor and angiogenesis.

Analysis of the bactericidal response to an experimental Neisseria meningitidis vesicle vaccine.

Rabbit immunogenicity studies on an experimental trivalent native outer membrane vesicle vaccine derived from three serogroup B strains were conducted to evaluate the effectiveness of this vaccine at inducing an antibody response with serum bactericidal activity against meningococcal strains of other serogroups in addition to serogroup B strains. The results showed that the vaccine was capable of inducing an effective broad-based bactericidal antibody response in rabbits against a small sample of Neisseria meningitidis strains of serogroups C, W135, and X and, to a lesser extent, serogroups A and Y. Analysis of antibody specificity using a bactericidal depletion assay revealed that antibodies to lipooligosaccharide (LOS), PorA, and NadA induced in rabbits by the experimental trivalent outer membrane vesicle vaccine were responsible for most of the bactericidal activity against strains of the other N. meningitidis serogroups. In the case of serogroup A N. meningitidis strains, the outer membrane antigen NadA was primarily responsible for protection. The outer membrane antigens fHbp and OpcA were also effective in removing some bactericidal activity from the sera.

[Primary non-Hodgkin's lymphoma of the testis and penis: clinical analysis of 5 cases].

OBJECTIVE: To improve the clinical diagnosis and treatment of primary non-Hodgkin's lymphoma of male genitalia. METHODS: We retrospectively reviewed the clinical data of 5 cases of primary non-Hodgkin's lymphoma of male genitalia, 4 in the testis and 1 in the penis, we also analyzed the relevant literature and clinical significance of the disease. RESULTS: All the 5 cases were treated by surgery and pathologically confirmed to be non-Hodgkin's lymphoma. Three of them received chemotherapy, and the other 2 (1 in the testis and 1 in the penis) underwent both chemotherapy and radiotherapy after the operation. Follow-up averaged 25 months, during which 1 of the patients died and the other 4 survived. CONCLUSION: Primary non-Hodgkin's lymphoma of male genitalia is an uncommon disease with atypical clinical presentations and poor prognosis, which occurs mostly in elderly males. Definite diagnosis of the disease mainly depends on histopathology and immunohistochemistry. Surgery with multiagent chemotherapy and radiotherapy is advisable for its treatment.

A fully human monoclonal antibody to Staphylococcus aureus iron regulated surface determinant B (IsdB) with functional activity in vitro and in vivo.

A fully human monoclonal antibody (CS-D7, IgG1) specific for the iron regulated surface determinant B (IsdB) of Staphylococcus aureus was isolated from the Cambridge Antibody Technology (CAT) scFv antibody library. As compared to previously described IsdB specific murine monoclonals, CS-D7 has a unique, non-overlapping binding site on IsdB, and exhibits increased in vivo activity. The antibody recognizes a conformational epitope spanning amino acids 50 to 285 and has a binding affinity of 340 (± 75) pM for IsdB. CS-D7 bound to a wide variety of S. aureus strains, but not to an isdB deletion mutant. The antibody mediated opsonophagocytic (OP) killing in vitro and mediated significant protection in vivo. In a murine lethal sepsis model, the antibody conferred protection from death when dosed prior to challenge, but not when dosed after challenge. Importantly, in a central venous catheter (CVC) model in rats, the antibody reduced bacteremia and prevented colonization of indwelling catheters. Protection was observed when rats were dosed with CS-D7 prior to challenge as well as post challenge. IsdB is currently being investigated for clinical efficacy against S. aureus infection, and the activity of this human IsdB specific antibody supplements the growing body of evidence to support targeting this antigen for vaccine development.

The Starling mechanism of the urinary bladder contractile function and the influence of hyperglycemia on diabetic rats.

UNLABELLED: Diabetic cystopathy is one of the common complications of diabetes. Bladder dysfunction in diabetes is attributable to diabetic neuropathy that induces sensory and autonomic nerve dysfunction. MATERIALS AND METHODS: In the present study, the contractile mechanism of the bladder was evaluated both with and without electrical stimulation in normal rats, streptozotocin (STZ)-induced diabetic rats, and diabetic rats with insulin treatment. RESULTS: Both the normal and diabetic rats had optimal capacity of bladder and optimal length of detrusor muscle strips. The peak values of the volume-pressure curves of the bladder and length-tension curves of detrusor muscle strips as well as the enhanced values after electrical stimulation in 6- and 10-week diabetic groups were lower than in the 6- and 10-week normal groups and insulin-treated groups. However, there was no significant difference in peak and enhanced values between normal rats and diabetic rats treated with insulin, indicating that voiding function was improved after insulin treatment. CONCLUSIONS: The contractile function of rat's bladder including normal rats, diabetic rats, and diabetic rats treated with insulin is similar to the 'Starling mechanism.' It can be impaired by hyperglycemia, and insulin treatment is helpful to restore this function.

Clonal mesenchymal stem cells derived from human bone marrow can differentiate into hepatocyte-like cells in injured livers of SCID mice.

There is increasing evidence that human mesenchymal stem cells (hMSCs) can be a valuable, transplantable source of hepatocytes. Most of the hMSCs preparations used in these studies were likely heterogeneous cell populations, isolated by adherence to plastic surfaces or by density gradient centrifugation. Therefore, the participation of other unknown trace cell populations cannot be rigorously discounted. Here we report the isolation and establishment of a cloned human MSC line (chMSC) from human bone marrow primary culture, through which we confirmed the hepatic differentiation capability of authentic hMSCs. chMSCs expressed markers of mesenchymal cells, but not markers of hematopoietic stem cells. In vitro, chMSCs can differentiate into either mesenchymal cells or cells exhibiting hepatocyte-like phenotypes. When transplanted intrasplentically into carbon tetrachloride-injured livers of SCID mice, EGFP-tagged chMSCs engrafted into the host liver parenchyma, exhibited typical hepatocyte morphology, form a three-dimensional architecture, and differentiate into hepatocyte-like cells expressing human albumin and alpha-1-anti-trypsin. By confocal microscopy, ultrafine intercellular nanotubular structures were visible between adjacent transplanted and host hepatocytes. We postulate that these structures may assist in the phenotype conversion of chMSCs, possibly by exchange of cytoplasmic components between native hepatocytes and transplanted cells. Thus, a clonal pure population of hMSCs, which can be expanded in culture, may have potential as a cellular source for substitution damaged cells in hepatic injury.

Selection and characterization of murine monoclonal antibodies to Staphylococcus aureus iron-regulated surface determinant B with functional activity in vitro and in vivo.

In an effort to characterize important epitopes of Staphylococcus aureus iron-regulated surface determinant B (IsdB), murine IsdB-specific monoclonal antibodies (MAbs) were isolated and characterized. A panel of 12 MAbs was isolated. All 12 MAbs recognized IsdB in enzyme-linked immunosorbent assays and Western blots; 10 recognized native IsdB expressed by S. aureus. The antigen epitope binding of eight of the MAbs was examined further. Three methods were used to assess binding diversity: MAb binding to IsdB muteins, pairwise binding to recombinant IsdB, and pairwise binding to IsdB-expressing bacteria. Data from these analyses indicated that MAbs could be grouped based on distinct or nonoverlapping epitope recognition. Also, MAb binding to recombinant IsdB required a significant portion of intact antigen, implying conformational epitope recognition. Four MAbs with nonoverlapping epitopes were evaluated for in vitro opsonophagocytic killing (OPK) activity and efficacy in murine challenge models. These were isotype switched from immunoglobulin G1 (IgG1) to IgG2b to potentially enhance activity; however, this isotype switch did not appear to enhance functional activity. MAb 2H2 exhibited OPK activity (> or =50% killing in the in vitro OPK assay) and was protective in two lethal challenge models and a sublethal indwelling catheter model. MAb 13C7 did not exhibit OPK (<50% killing in the in vitro assay) and was protective in one lethal challenge model. Neither MAb 13G11 nor MAb 1G3 exhibited OPK activity in vitro or was active in a lethal challenge model. The data suggest that several nonoverlapping epitopes are recognized by the IsdB-specific MAbs, but not all of these epitopes induce protective antibodies.

Staphylococcus aureus capsule type 8 antibodies provide inconsistent efficacy in murine models of staphylococcal infection.

Staphylococcus aureus is a clinically important capsule-forming bacterium. The capsule polysaccharide (CPs) occurs as different chemical structures depending on the serotype of the organism, but one form, capsular polysaccharide type 8 (CPs8) found in clinical isolates, is largely unstudied. The potential of CPs8 as a vaccine target was evaluated using two approaches. The first approach used a conjugate vaccine, made by chemically linking purified CPs8 to the outer membrane protein complex of N. meningitidis serotype B (OMPC). In efficacy studies, the CPs8-OMPC conjugate vaccine was immunogenic in Balb/c mice, however the immune response gave no protection from death after a lethal intravenous (IV) challenge with S. aureus Becker. In the second approach, two monoclonal antibodies were produced against CPs8 (mAbs 8E8 and 1C10). These were found to have functional activity in an opsonophagocytic killing assay (OPA), and provided protection from a lethal challenge when bacteria were pre-opsonized ex vivo before intra-peritoneal (IP) challenge. However, mAb 8E8 was not efficacious in the lethal challenge model, in which antibodies were passively transferred to the peritoneum and the animals were infected via the tail vein 18-24 h later. Additionally, the monoclonal antibodies did not opsonize capsule-expressing S. aureus Becker obtained from in vivo growth conditions. These results indicated that functional capsule antibodies may not be sufficient for protection from S. aureus under all in vivo conditions.

Purification and characterization of alginate lyase from marine Vibrio sp. YWA.

Extracellular alginate lyase secreted by marine Vibrio sp. YWA, isolated from decayed Laminaria japonica, was purified by a combination of ammonium sulfate precipitation and diethylaminoethyl-Sephacel column chromatography. The results show that the molecular mass of alginate lyase was approximately 62.5 kDa, with an optimal pH and temperature at pH 7.0 and 25 degrees C, respectively. K(m) was approximately 72.73 g/L. The activity of the enzyme was enhanced by EDTA and Zn(2+), but inhibited by Ba(2+). The substrates specificity analysis shows that it was specific for hydrolyzing poly-beta-D-1,4-mannuronate in alginate.

A novel Staphylococcus aureus vaccine: iron surface determinant B induces rapid antibody responses in rhesus macaques and specific increased survival in a murine S. aureus sepsis model.

Staphylococcus aureus is a major cause of nosocomial infections worldwide, and the rate of resistance to clinically relevant antibiotics, such as methicillin, is increasing; furthermore, there has been an increase in the number of methicillin-resistant S. aureus community-acquired infections. Effective treatment and prevention strategies are urgently needed. We investigated the potential of the S. aureus surface protein iron surface determinant B (IsdB) as a prophylactic vaccine against S. aureus infection. IsdB is an iron-sequestering protein that is conserved in diverse S. aureus clinical isolates, both methicillin resistant and methicillin sensitive, and it is expressed on the surface of all isolates tested. The vaccine was highly immunogenic in mice when it was formulated with amorphous aluminum hydroxyphosphate sulfate adjuvant, and the resulting antibody responses were associated with reproducible and significant protection in animal models of infection. The specificity of the protective immune responses in mice was demonstrated by using an S. aureus strain deficient for IsdB and HarA, a protein with a high level of identity to IsdB. We also demonstrated that IsdB is highly immunogenic in rhesus macaques, inducing a more-than-fivefold increase in antibody titers after a single immunization. Based on the data presented here, IsdB has excellent prospects for use as a vaccine against S. aureus disease in humans.