Sequence variation within botulinum neurotoxin serotypes impacts antibody binding and neutralization

The botulinum neurotoxins (BoNTs) are category A biothreat agents which have been the focus of intensive efforts to develop vaccines and antibody-based prophylaxis and treatment. Such approaches must take into account the extensive BoNT sequence variability; the seven BoNT serotypes differ by up to 70% at the amino acid level. Here, we have analyzed 49 complete published sequences of BoNTs and show that all toxins also exhibit variability within serotypes ranging between 2.6 and 31.6%. To determine the impact of such sequence differences on immune recognition, we studied the binding and neutralization capacity of six BoNT serotype A (BoNT/A) monoclonal antibodies (MAbs) to BoNT/A1 and BoNT/A2, which differ by 10% at the amino acid level. While all six MAbs bound BoNT/A1 with high affinity, three of the six MAbs showed a marked reduction in binding affinity of 500- to more than 1,000-fold to BoNT/A2 toxin. Binding results predicted in vivo toxin neutralization; MAbs or MAb combinations that potently neutralized A1 toxin but did not bind A2 toxin had minimal neutralizing capacity for A2 toxin. This was most striking for a combination of three binding domain MAbs which together neutralized >40,000 mouse 50% lethal doses (LD(50)s) of A1 toxin but less than 500 LD(50)s of A2 toxin. Combining three MAbs which bound both A1 and A2 toxins potently neutralized both toxins. We conclude that sequence variability exists within all toxin serotypes, and this impacts monoclonal antibody binding and neutralization. Such subtype sequence variability must be accounted for when generating and evaluating diagnostic and therapeutic antibodies.     

Improvement of Schwann cell attachment and proliferation on modified hyaluronic acid strands by polylysine

Hyaluronic acid (HyA) has the intrinsic ability to promote cell proliferation and reduce scar formation. However, the clinical use of HyA has so far been limited because of its water solubility and nonadhesive characteristics. Increasing interest in HyA as a clinically useful biomaterial has prompted our study of altering HyA's physical properties to render it a potential component of nerve grafts. In this study, strands of HyA were cross-linked by glutaraldehyde (Glut), coated with polylysine, and then inoculated with Schwann cells (SCs). Results in vivo and in vitro demonstrated that cross-linked HyA strands were water insoluble and thus less biodegradable. Poly-D-lysine-resurfaced strands showed significant SC attachment of 350-400 cells/mm(2), compared to uncoated controls (0-10 cells/mm(2), p < 0.01). Fibroblast control groups showed an attachment of 40-100 cells/mm(2) on coated strands. Immunostaining for proliferating cells showed SCs as and fibroblasts as +. Cells neither adhered to nor proliferated on the modified HyA strands that were not resurfaced. The results suggest that polylysine promotes SC attachment and proliferation to glutaraldehyde-cross-linked HyA strands, the product being a three-dimensional composite with low solubility that may have potential application in nerve grafts.     

Alterations of plasma antioxidants and mitochondrial DNA mutation in hair follicles of smokers

The effects of long-term smoking on mitochondrial DNA (mtDNA) deletions in hair follicles were investigated in subjects with different antioxidant capacity. Twenty-two male smokers with a smoking index of greater than 5 pack-years and without any known systemic diseases were recruited for this study. Forty healthy nonsmoking males were included as controls. We found that the concentrations of ascorbate and alpha-tocopherol and the activities of glutathione S-transferase (GST) and glutathione peroxidase in blood plasma were significantly decreased in smokers. The levels of glutathione and protein thiols in whole blood and the incidence of a 4,977 bp deletion of mtDNA (dmtDNA) in hair follicles were significantly increased in smokers. A significantly higher incidence of the 4,977 bp dmtDNA was found in smokers with plasma GST activity less than 5.66 U/l (OR = 7.2, P = 0.020). Using multiple covariate ANOVA and logistic regression, we found that age and low plasma GST activity were the only two risk factors for the 4,977 bp dmtDNA. These results suggest that smoking depletes antioxidants and causes mtDNA deletions and that plasma GST may play an important role in the preservation of the mitochondrial genome in tissue cells of smokers.     

Dissemination of CTX-M-3 and CMY-2 beta-lactamases among clinical isolates of Escherichia coli in southern Taiwan

A total of 1,210 clinical isolates of Escherichia coli collected from a university hospital in southern Taiwan were screened for production of extended-spectrum beta-lactamases (ESBLs). Expression of classical ESBLs (resistant to extended-spectrum beta-lactam agents and susceptible to beta-lactam inhibitors) was inferred in 18 isolates by the phenotypic confirmatory test. These included 10 isolates producing CTX-M-3, 2 strains carrying SHV-12, 1 strain harboring SHV-5, 1 strain expressing TEM-10, and 4 strains producing unidentifiable ESBLs with a pI of 8.05, 8.0, or 7.4. Eighteen isolates that showed decreased susceptibilities to ceftazidime and/or cefotaxime, negative results for the confirmatory test, and high-level resistance to cefoxitin (MICs of >/=128 microg/ml) were also investigated. Five isolates were found to produce CMY-2 AmpC enzymes, one isolate carried both CTX-M-3 and CMY-2, and the remaining three and nine isolates expressed putative AmpC beta-lactamases with pIs of >9.0 and 8.9, respectively. Thus, together with the isolate producing CTX-M-3 and CMY-2, 19 (1.6%) isolates produced classical ESBLs. Pulsed-field gel electrophoresis revealed that all isolates carrying CTX-M-3 and/or CMY-2 were genetically unrelated, indicating that dissemination of resistance plasmids was responsible for the spread of these two enzymes among E. coli in this area. Among the 16 isolates expressing CTX-M-3 and/or CMY-2, 5 might have colonized outside the hospital environment. Our data indicate that CTX-M-3 and CMY-2, two beta-lactamases initially identified in Europe, have been disseminated to and are prevalent in Taiwan.     

Characterization of the structural elements in lipid A required for binding of a recombinant fragment of bactericidal/permeability-increasing protein rBPI23.

Both human bactericidal/permeability-increasing protein (BPI) and a recombinant amino-terminal fragment of BPI (rBPI23) have been shown to bind with high affinity to the lipid A region of lipopolysaccharide (LPS) (H. Gazzano-Santoro, J. B. Parent, L. Grinna, A. Horwitz, T. Parsons, G. Theofan, P. Elsbach, J. Weiss, and P. J. Conlon, Infect. Immun. 60:4754-4761, 1992). In the present study, lipid A preparations derived from bacterial LPS as well as synthetic lipid A's and various lipid A analogs were used to determine the structural elements required for rBPI23 binding. rBPI23 bound in vitro to a variety of synthetic and natural lipid A preparations (both mono- and diphosphoryl forms), including lipid A's prepared from Escherichia coli and Salmonella, Neisseria, and Rhizobium species. Binding does not require that the origin of negative charge be phosphate, since rBPI23 bound with high affinity to lipid A's isolated from Rhizobium species that contain carboxylate (Rhizobium trifolii) or sulfate (Rhizobium meliloti) anionic groups and lack phosphate. Lipid A acyl chains are important, since rBPI23 did not bind to four synthetic variants of the beta(1-6)-linked D-glucosamine disaccharide lipid A head group, all devoid of acyl chains. rBPI23 also bound weakly to lipid X, a monosaccharide lipid precursor of LPS corresponding to the reducing half of lipid A. Lipid IVA, a precursor identical to E. coli lipid A except that it lacks the 2' and 3' acyl chains, was the simplest structure identified in this study that rBPI23 bound with high affinity. These results demonstrate that rBPI23 has a binding specificity for the lipid A region of LPS and binding involves both electrostatic and hydrophobic components.     

Predominance of methicillin-resistant Staphylococcus aureus in the residents and environments of long-term care facilities in Taiwan

Background/purpose

This study investigated the distribution and persistence of multidrug resistant organisms (MDROs) including methicillin-resistant Staphylococcus aureus (MRSA), carbapenem-resistant Enterobacteriaceae (CRE), carbapenem-resistant Pseudomonas aeruginosa (CRPA), and multidrug-resistant Acinetobacter baumannii (MDRAB) in six long-term care facilities (LTCFs).

Do Treatment Plans Matter? Moving From Recommendations to Action

We investigated whether a service-planning document outlining recommendations for what providers should address in treatment (i.e., targets) and the associated clinical techniques they should employ (i.e., practices) influenced the targets and practices that providers reported actually implementing during the subsequent treatment episode. Participants included 94 youths ages 4 to 17 (M = 13.57, SD = 3.59) who received community-based mental health services from the Hawai'i Child and Adolescent Mental Health Division. Data on targets and practices were compared across initial Mental Health Treatment Plans and Monthly Treatment and Progress Summaries. Data were analyzed using two-level, generalized mixed effects models with two-way cross-classification or linear mixed effects models. Providers were more likely to report the use of targets and practices in treatment if they were included within the treatment plan. In addition, the more closely targets addressed during treatment followed the recommended targets from the treatment plan, the more closely implemented practices followed the recommended practices listed in the treatment plan. Furthermore, as providers shifted their focus to different targets, a shift in their use of practices was also evident over time. Last, practices for which there is demonstrated efficacy for particular targets were more likely to be used. Service planning documents appear to help organize care; however, results also suggest possible limitations to the current system. These findings highlight potential areas for improvement in planning and care delivery.     

Insoluble immune complex-stimulated neutrophil leukotriene B4 production is dependent on Fc gamma RII and Fc gamma RIII and independent of pertussis toxin-sensitive signal transduction pathways.

Leukotriene B4 (LTB4) release initiated by interaction of immune complexes (ICs) with Fc gamma RII and Fc gamma RIII receptors on human neutrophils was studied using well-defined complexes. Immune complexes consisting of polyclonal rabbit antibody to human albumin were prepared at equivalence (insoluble complex) and at five times antigen excess (soluble complex). Incubation of human neutrophils with soluble and insoluble ICs led to the synthesis of LTB4 from endogenous arachidonic acid (AA). LTB4 release induced by ICs was markedly inhibited by monoclonal antibodies against either Fc gamma RII or Fc gamma RIII receptor. Treatment of neutrophils with pertussis toxin significantly inhibited the release of LTB4 induced by soluble ICs. However pertussis toxin treatment minimally inhibited the LTB4 release induced by insoluble ICs. Crosslinking of either Fc gamma RII and Fc gamma RIII receptors on neutrophil surfaces induced LTB4 release. This is the first experimental observation showing that both Fc gamma RII and Fc gamma RIII directly induce neutrophil LTB4 metabolism in the absence of exogenous AA. These studies also suggest the involvement of novel pertussis toxin insensitive signal transduction pathways in insoluble ICs stimulation of neutrophils.     

Interaction of Inflammatory Cells and Oral Microorganisms VII. In Vitro Polymorphonuclear Responses to Viable Bacteria and to Subcellular Components of Avirulent and Virulent Strains of Actinomyces viscosus

Both virulent (V) and avirulent (AV) strains of Actinomyces viscosus T14 are capable of colonizing the oral cavity of gnotobiotic rats, but only T14-V causes destructive periodontal disease. The basis for this difference in in vivo pathogenicity has not been adequately defined. In the present study we compared the capacities of T14-AV and T14-V to provoke in vitro extracellular release of lysosomal constituents from human polymorphonuclear leukocytes (PMNs). In serum-free cultures, viable T14-V but not T14-AV stimulated discharge of PMN lysosomes. The release response was correlated with PMN phagocytic activity; thus, PMNs readily ingested T14-V but not T14-AV. To explain these differences in PMN-bacteria interactions, subcellular fractions of T14-AV or T14-V were incubated with PMNs. A crude, insoluble sonic extract derived from T14-V caused PMN lysosome release, but a similar fraction from T14-AV was inactive. However, following extensive washing and treatment with deoxyribonuclease or sodium dodecyl sulfate, cell wall fractions of T14-AV stimulated lysosome release. These procedures apparently removed an extracellular polysaccharide slime which is synthesized by T14-AV but not by T14-V. There was a significant reduction in the capacities of viable T14-V or cell wall fractions of T14-V or T14-AV to provoke PMN lysosome release when these agents were preincubated with a slime material isolated from T14-AV. This inhibitory influence of slime was overcome by the addition of fresh or heated (56°C, 30 min) serum to the PMN-bacteria cultures. The data suggest a relationship between the abilities of the avirulent and virulent strains of A. viscosus T14 to act as periodontal pathogens in vivo and to serve as stimuli for PMN lysosome release in vitro.