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.     

Analysis of allergenic components of Bermuda grass pollen by monoclonal antibodies

A panel of 16 monoclonal antibodies (MoAbs) directed against Bermuda grass (Cynodon dactylon) pollen (BGP) were generated for identification and purification of the major allergenic components of the eliciting antigen (Ag). Radioimmunoprecipitation (RIP) analysis revealed that there were at least eight antigenic components with molecular weights (MW) ranging from 12 kilodalton (12 kDa) to 200 kDa. Each of these components has distinct biochemical characteristics based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF). Among them, Cyn d Bd67K and Cyn d Bd58K were basic proteins, Cyn d Bd35K consisted of at least four isomeric components with isoelectric points ranging from 6.2 to 7.2. The other antigens (Cyn d Bd68K, 48K, 38K, Cyn d Bd200K, Cyn d Bd46K, Cyn d Bd25K and Cyn d Bd12K) were all acidic proteins. The IgE binding capacity of all these antigens was determined with sera from 11 BGP-allergics by using a modified radioallergosorbent test. All but one of the antigens (Cyn d Bd200K) were found to react with human IgE from sera of BGP-allergic patients. Among those human IgE-binding molecules, Cyn d Bd35K reacted with allergic sera most frequently (10 of 11), followed by Cyn d Bd58K (8 of 11) and Cyn d Bd46K (7 of 11) respectively. Our results suggest that Cyn d Bd35K, Cyn d Bd58K, and Cyn d Bd46K are major allergens of BGP, and the MoAbs we obtained should be valuable tools for further purification of these allergens.     

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.     

Use of PCR to study epidemiology of Serratia marcescens isolates in nosocomial infection.

A method to characterize strains of Serratia marcescens based on the PCR amplification of enterobacterial repetitive intergenic consensus sequences has been developed. The PCR fingerprints were generated from boiled supernatants prepared directly from bacterial colonies without the need for DNA extraction. The technique was applied to isolates obtained during an outbreak of pneumonia from seven mechanically ventilated patients, and its result indicated that the outbreak was due to the spread of two epidemic strains. This technique was validated by comparison with rRNA gene restriction analysis. There was complete concordance between these two techniques in discriminating the outbreak-related strains from epidemiologically unrelated isolates. Typing with both biochemical profile and antibiogram profile, though simple, was found to be less reliable than genotyping. The results show that this enterobacterial repetitive intergenic consensus PCR provides a rapid and simple means of typing S. marcescens isolates for epidemiologic studies.     

Interferon in cerebrospinal fluid

Summary Interferon (IFN) was measured in serum and cerebrospinal fluid (CSF) of dogs after experimental (intranasal) infection with different strains of virulent canine distemper virus (CDV). Viral strains employed produce neurological changes in dogs that range from acute inflammatory to subacute, delayed demyelinating encephalomyelitis. With few exceptions, first appearance of serum-IFN correlated with the first elevated body temperature 4 days post-infection (p.i.). By 16 days p.i. IFN had disappeared from the serum of all infected dogs. In contrast, IFN was constantly detectable in CSF in dogs with CDV infection of the central nervous system (CNS). It was first detected 5 days p.i., was continuously detectable during the variable preclinical phase and into the period when signs of acute or delayed encephalomyelitis were evident. Dogs from which CDV would be retrieved from CNS tissue at necropsy always had CSF-IFN (up to 56 days p.i.). In contrast, dogs that recovered from infection, substantiated at necropsy by minimal, resolving CNS lesions and non-detectable virus, had IFN in CSF demonstrable for only a brief post-inoculation period. CSF-IFN appears to be a valid marker for CDV persistence in the canine CNS and may have broader applications.With 3 Figures     

Ovarian stimulation by clomiphene citrate and hMG in combination with cetrorelix acetate for ICSI cycles

BACKGROUND: The introduction of GnRH antagonists such as cetrorelix acetate has made possible the simplification of ovarian stimulation. However, the most effective protocol for their administration has not yet been clearly defined. METHODS: Forty women with male-factor infertility undergoing 40 ICSI cycles were included in the study. Clomiphene citrate at 100 mg a day was given from cycle day 3 through day 7. hMG at 150 IU was given on cycle days 4, 6 and 8, and was adjusted from day 9 according to the follicular and hormone responses. Cetrorelix acetate at 2.5 mg was administered when the leading follicle reached 14 mm. The remaining 0.5 mg was divided into two 0.25 mg injections for possible later use. Serum FSH, LH, estradiol and progesterone levels were measured daily from the day of cetrorelix acetate injection until hCG was given. RESULTS: Serum LH level was suppressed effectively for 4 days. Four patients (10%) needed one or two additional injections of 0.25 mg cetrorelix acetate. No premature LH surge was detected in any of the women treated. Sixteen women became pregnant (40%), of which 14 pregnancies (35%) were ongoing at the time of writing. CONCLUSIONS: This study demonstrates that this new protocol is feasible for couples with male-factor infertility undergoing ICSI.