Early emergence of PNH-like T cells after allogeneic stem cell transplants utilising CAMPATH-1H for T cell depletion.

CAMPATH-1H (C-1H) is widely used in vivo and / or in vitro for T cell depletion in hematopoietic SCT. This humanised monoclonal antibody is specific for CD52, a marker coexpressed on the majority of human lymphocytes with CD48 and other glycosylphosphatidyl-inositol (GPI) anchored proteins. We detected CD52 / CD48 dual expression on >99% of CD3(+) lymphocytes from normal individuals and all 15 post-SCT patients whose transplants did not utilise C-1H. By contrast, 23 / 26 patients with transplants involving C-1H (in vivo, in vitro or both) exhibited populations lacking CD52 expression that accounted for 49.7% (4.2-86.2%) of the CD3+ lymphocytes (median and range) in samples evaluated at a median of 2 months post-SCT. Most CD52- cells also lacked CD48 expression. These GPI- T cells were of either donor or mixed donor / recipient origin. They were predominant in the early months after SCT at times of profound lymphopenia and inversely correlated with the recovery of the absolute lymphocyte count (r= - 0.663, P<0.0001). The presence of CD52- cells has been correlated previously with clinical outcome after CAMPATH therapy for both malignant and nonmalignant diseases.

A DNA delivery system targeting dendritic cells for use in immunization against malaria: a rodent model.

DNA-based vaccination has emerged as a promising method of immunisation since the first demonstration of this technology. Improving the antibody responses is desirable for the protective efficacy and hence broad application of these vaccines. We examined the immunogenicity of a Plasmodium-based DNA vaccine that was targeted to antigen presenting cells by fusion to CTLA4. Fusion proteins comprising the extra-cellular domain of CTLA4, the hinge, CH2 and CH3 domains of human IgG1 and MSP-1 gene fragments were expressed in COS-7 cells. Three of the secreted proteins containing the mouse homologue of CTLA4 were shown to bind differently to the human B7-1 molecule expressed on THP-1 cells. Competition binding assays for two fusion proteins showed that binding was specific. When C57BL/6 mice were immunized with plasmids encoding the fusion proteins, antibodies against two denatured and one non-denatured MSP-1 gene fragments were successfully induced. The usefulness of this strategy in future studies of immunisaton against human malaria is discussed.

Construction of an Actinobacillus pleuropneumoniae-Escherichia coli cosmid cloning vector [Gene 160 (1995) 81-86].

We constructed a cosmid vector, pSW206, which should be useful for the construction of genomic libraries of Actinobacillus pleuropneumoniae (Apl) DNA. pSW206 is based on the broad-host-range plasmid RK2 and can be introduced into Apl by conjugation.

Construction of Actinobacillus pleuropneumoniae-Escherichia coli shuttle vectors: expression of antibiotic-resistance genes.

We constructed several cloning vectors, designated pGZRS-18/19 and pGZRS-38/39, which were based on an endogenous Actinobacillus pleuropneumoniae (Apl) 4.3-kb plasmid. They carry the lacZ alpha-complementation fragment and MCS from pUC18/19, and either the bla gene under the control of a putative Apl promoter or the KmR gene from Tn903. These vectors replicate in representative strains of Apl serotypes 1 and 7, Escherichia coli, Pasteurella haemolytica (Ph) and Haemophilus (Actinobacillus) actinomycetemcomitans. We also found that Apl and Ph did not express genes under the control of the lacZ or bla promoters, suggesting that their RNA polymerases may not utilize these promoters.

Construction of improved Escherichia-Pseudomonas shuttle vectors derived from pUC18/19 and sequence of the region required for their replication in Pseudomonas aeruginosa.

The nucleotide sequence of the 1.9-kb PstI fragment from pRO1614, that allows stable maintenance of pMB1 (ColE1)-based cloning vectors in Pseudomonas, was determined. This fragment encodes a putative origin of replication (ori), a replication-controlling protein, and the C terminus of the Tn3 beta-lactamase-encoding gene. Improved versions of the broad-host-range plasmid vectors, pUCP18 and pUCP19, were constructed by deletion of nonessential DNA or replacement of nonessential DNA with an antibiotic-resistance cassette.

Acceleration of lung disease in children with cystic fibrosis after Pseudomonas aeruginosa acquisition.

As part of the ongoing Wisconsin Cystic Fibrosis (CF) Neonatal Screening Project, we had the unique opportunity to study the longitudinal relationship between Pseudomonas aeruginosa (Pa) acquisition and infection and developing lung disease in children with CF. The primary objective was to determine whether acquisition of Pa was associated with a measurable change in the progression of lung disease. Two outcome measures were used to study 56 patients who were diagnosed through newborn screening: 1) Wisconsin additive chest radiograph score (WCXR), based on the average of scores from a pulmonologist and a radiologist, and 2) the highest forced expired volume in 1 sec (FEV(1))/forced vital capacity (FVC) ratio. We used two measures of Pa acquisition: 1) time of first positive protocol-determined oropharyngeal (with cough) culture, and 2) the magnitude of antibody titer detected by ELISA assays, using as antigen a crude cell lysate, purified exotoxin A, or an elastase toxoid prepared from three Pa strains. Other predictor variables included age, pancreatic status, height-for age, and weight-for-age-percentiles. The best regression model for predicting changes in the WCXR included time to first positive culture and antibody titer for Pa elastase. Prior to Pa acquisition, WCXR worsened by 0.45 points/year (P > 0.25); after Pa acquisition, the rate of worsening increased significantly (P < 0.001) to 1.40 points/year. Each antibody titer level (log base 2) increased the score by 0.48 points (P < 0.001). The best regression model for predicting change in the FEV(1)/FVC included only time to first positive culture. Prior to Pa acquisition, the FEV(1)/FVC ratio declined by 1.29%/year; after Pa infection, the rate of decrease significantly accelerated to 1.81%/year (P = 0.001). Our data show that Pa acquisition is associated with declining pulmonary status in children with CF, and that this effect is probably gradual rather than precipitous. Because these patients were diagnosed and treated aggressively, our estimates of the effects of Pa acquisition may be conservative. We also conclude that the WCXR appears to be more sensitive than FEV(1)/FVC in detecting early changes in lung disease associated with CF.

In vitro production of human fecal mutagen.

The feces of some normal humans were previously shown to be mutagenic by the Salmonella mutagenicity assay with strain TA100. In the present study, the mutagenicity of feces of certain donors can be increased by anaerobic incubation for 96 h. The increase in mutagenicity did not occur upon incubation in the cold or in air, in the presence of antimicrobial agents or if the feces were sterilized by heat. On thin-layer chromatographs, the relative mobility of fecal mutagen for all donors after incubation was the same in any one of 4 different solvent systems. The major mutagenicity appears to be due to a single type of compound which may be produced by anaerobic bacteria.

Laryngeal adductory pressure as a measure of post-reinnervation synkinesis.

Laryngeal adductory pressure (LAP) is the pressure induced as the vocal folds squeeze on a balloon while the recurrent laryngeal nerve (RLN) is stimulated. The LAP has been shown to vary with the frequency of stimulation, with a characteristic slope. The RLN was divided and reanastomosed 4 different ways in 12 canine hemilaryngeal preparations; the 4 subgroups represented a range of expected post-reinnervation synkinesis recovery patterns. The LAP frequency-response curve was measured before surgery and at monthly intervals for 6 months after surgery. In the "best-case" group (RLN adductor and abductor trunks each divided and reanastomosed), the slope was found to return to normal. The 2 whole RLN division-reanastomosis groups (precise realignment or 180 degrees rotation) both gave results similar to those of the "worst-case" group (RLN adductor and abductor trunks divided and transposed); these 3 subgroups were all significantly different from baseline. The slope of the LAP frequency-response curve may be a useful means of indirectly quantifying laryngeal synkinesis.

Laryngeal reinnervation with the hypoglossal nerve. I. Physiology, histochemistry, electromyography, and retrograde labeling in a canine model.

This study was performed to determine whether the hypoglossal nerve (cranial nerve XI [XII]) would serve as a useful donor for laryngeal reinnervation by anastomosis to the recurrent laryngeal nerve (RLN). Twenty hemilarynges in 10 dogs were studied prospectively after XII-RLN anastomosis (group A; n = 5), split XII-RLN anastomosis (group B; n = 3), XII-RLN anastomosis with a 2-cm interposition graft (group C; n = 2), no treatment (group D; n = 5), RLN section (group E; n = 2), or ansa cervicalis-RLN anastomosis (group F; n = 3). Spontaneous activity was observed monthly by infraglottic examination through permanent tracheostomies and was recorded by electromyography. Laryngeal adductory pressure and induced phonation were obtained by stimulating the RLN while passing a pressure transducer balloon or humidified air through the glottis. At sacrifice, the laryngeal muscles were stained for adenosine triphosphatase to determine the ratio of type I to type II fibers. Retrograde labeling of the brain stem was performed with horseradish peroxidase. Infraglottic examination at 6 months showed a full range of adductory motion in groups A and B during the swallow reflex, comparable with that in group D. Groups C and F showed good bulk and tone, but little spontaneous motion. Group E remained paralyzed. Stimulation of the transferred nerves caused more activity in groups A and B than in the other groups; groups C and F partially adducted at high levels. The laryngeal adductory pressure responses of groups A and B were similar to those of group D. The XII-reinnervated larynges were capable of producing normal induced phonation. Retrograde labeling of the RLN showed that the reinnervating axons originated only in the hypoglossal nucleus. Electromyography of the reinnervated adductor muscles confirmed spontaneous activity in the dogs (awake). Histochemical analysis confirmed slow-to-fast transformation of both the posterior and lateral cricoarytenoid muscles, indicating that significant reinnervation occurred. We conclude that the hypoglossal nerve functions well as a donor for adductory reinnervation of the larynx.

Utilization of both phenotypic and molecular analyses to investigate an outbreak of multidrug-resistant Salmonella anatum in horses.

Phenotypic and molecular techniques, including antimicrobial susceptibility testing, plasmid analysis, and pulsed-field gel electrophoresis (PFGE) were used to characterize 15 isolates of multidrug-resistant (MDR) Salmonella anatum cultured during a 16 mo period from horses and a veterinary clinic environment. The isolates were resistant to multiple antimicrobial agents and could be placed into 4 groups based on their antimicrobial resistance patterns. The isolates contained multiple plasmids ranging in size from 2 to > 100 kb that could be grouped into 3 different plasmid profile patterns; these patterns did not correlate with the antimicrobial resistance groupings. Furthermore, antimicrobial resistance was conjugatively transferable. Digestion of genomic DNA from the 15 isolates with 3 different restriction endonucleases, SfiI, SpeI, and XbaI followed by PFGE revealed a highly conserved restriction endonuclease digestion pattern. In contrast, diverse banding patterns were observed with S. anatum obtained from other sources. These observations suggest that the MDR S. anatum isolates represent a common outbreak strain even though they possess different, albeit similar, antibiograms and plasmid profiles. The study showed that PFGE is a useful epidemiological tool for discriminating between unrelated and outbreak-related strains of S. anatum. In conclusion, epidemiological studies of outbreaks caused by MDR isolates of S. anatum should consist of both genotypic and phenotypic methods of analysis.

Control of an outbreak of salmonellosis caused by drug-resistant Salmonella anatum in horses at a veterinary hospital and measures to prevent future infections.

Salmonella anatum was isolated from horses treated at a private veterinary clinic or at a university veterinary medical teaching hospital. All isolates were resistant to most commonly used antibiotics. Because of the severity of disease resulting from outbreaks of infections with drug-resistant strains of S anatum, an epidemiologic investigation was conducted. Enteric bacteria, including S anatum, that were resistant to most antibiotics were isolated from the private veterinary clinic environment. Salmonella anatum was not isolated from the university teaching hospital environment. To prevent transmission, disinfection and isolation protocols were reviewed, and changes were implemented, including discontinuing use of power sprayers for cleaning, improving a two-step disinfection process, restricting movement of horses, and enhancing awareness of Salmonella spp transmission. Communication and prompt action are pivotal in preventing dissemination of resistant strains of Salmonella spp in a clinic or hospital environment.

Nutrient database improvement project: the influence of USDA quality and yield grade on the separable components and proximate composition of raw and cooked retail cuts from the beef chuck.

This study was designed to provide updated information on the separable components, cooking yields, and proximate composition of retail cuts from the beef chuck. Additionally, the impact the United States Department of Agriculture (USDA) Quality and Yield Grade may have on such factors was investigated. Ultimately, these data will be used in the USDA - Nutrient Data Laboratory's (NDL) National Nutrient Database for Standard Reference (SR). To represent the current United States beef supply, seventy-two carcasses were selected from six regions of the country based on USDA Yield Grade, USDA Quality Grade, gender, and genetic type. Whole beef chuck primals from selected carcasses were shipped to three university laboratories for subsequent retail cut fabrication, raw and cooked cut dissection, and proximate analyses. The incorporation of these data into the SR will improve dietary education, product labeling, and other applications both domestically and abroad, thus emphasizing the importance of accurate and relevant beef nutrient data.

Innovative retail merchandising strategies to accommodate for the growing trend of heavier carcass weights in the United States.

Three subprimals from beef carcasses, Average (mean=340.6kg) and Heavy weight (mean=461.6kg), were cut using Innovative versus Conventional cutting styles. Longer (P<0.05) processing times were required for the Heavy compared to Average and Innovative compared to Conventional. Total saleable yields were lower for the Innovative compared to Conventional for the top sirloin butt (P=0.0025) and ribeye (P<0.0001), but not for the strip loin (P=0.1416). However, yields were higher for the Heavy compared to Average for the ribeye (P=0.0054) and strip loin (P=0.0017), but not for the top sirloin butt (P=0.6797). Retail pricing increases for the Innovative compared to Conventional were 11.6% for top sirloin butt, 26.9% for ribeye, and 2.6% for strip loin. Retailers adopting innovative cutting styles to more effectively merchandise heavyweight beef must account for the decreased primary saleable yields and increased labor requirements through increased retail pricing.

Pseudomonas aeruginosa serological analysis in young children with cystic fibrosis diagnosed through newborn screening.

BACKGROUND: With newborn screening (NBS) for cystic fibrosis (CF), eradication of Pseudomonas aeruginosa (PA) is possible if PA detection occurs early. A serological response to infection likely precedes culture positivity in CF patients, so PA serological testing is very appealing in this population. However, controversies continue to exist about serology testing, titer cutoffs for enzyme-linked immunosorbent assay (ELISA) antibody tests, and their value in children with CF. METHODS: This longitudinal, prospective study collected respiratory secretions as oropharyngeal swabs or expectorated sputum for culture and also sera over 6 years in 69 patients diagnosed by NBS. Serology assessed PA antibody titers against cell lysate, exotoxin A, and elastase. A novel statistical approach with weighted receiver operating characteristic (ROC) curves was used to determine best antibody titer cutoff values to predict subsequent PA positive cultures. RESULTS: Using these weighted ROC curves, the order of sensitivity was found to be cell lysate, exotoxin A, and then elastase while age-specific cutoffs were better than fixed cutoffs previously used. Age-specific serological cutoffs both predict and detect PA respiratory infections with a higher sensitivity and specificity. Serological responses to the PA antigens determined that a response to cell lysate occurs significantly earlier than culture positivity. CONCLUSIONS: Age-specific serological cutoffs rather than fixed values against common PA antigens improve early PA identification in infants and young children diagnosed with NBS. Regular serological assessment with age-specific cutoffs in these children appears to be a worthy diagnostic tool.

Problems associated with counterimmunoelectrophoresis assays for detecting Clostridium difficile toxin.

The antitoxins currently used for the detection of Clostridium difficile by counterimmunoelectrophoresis react with other C. difficile antigens in addition to the toxins produced by the bacterium.

Vaspar broth-disk procedure for antibiotic susceptibility testing of anaerobic bacteria.

A modification of the Wilkins-Thiel broth-disk procedure for antibiotic susceptibility testing of anaerobic bacteria is described. This method utilizes an aerobically prepared medium overlaid with molten vaspar. Specialized anaerobic techniques or prereduced media are not required.

Influence of epilepsy and temporal lobe resection on olfactory function.

Olfactory auras accompany some cases of epilepsy. Several aspects of olfactory function, including sensitivity, also may be altered. We reviewed the literature on these topics, as well as studies evaluating the influences of temporal lobe resection and other seizure management procedures on olfactory function. We concluded that: (a) despite several studies, the prevalence of olfactory auras in epilepsy is unknown, with estimates ranging from < 1% to > 30%; (b) epilepsy appears to cause a generalized decrease in olfactory functioning, although increased sensitivity may occur in some epileptic patients at some time in the preictal period; (c) other sensory modalities are also affected by the epileptic process which, in some cases, involve limbic-related temporal lobe structures; (d) many of the olfactory deficits previously attributed to temporal lobe resection actually exist preoperatively; (e) a taste/flavor confusion exists in the reporting of taste auras; (f) unpleasant auras are associated with hyperresponsiveness of neurons, which may explain why most epilepsy-related olfactory auras are described as "bad"; and (g) interesting parallels exist between the effects of the neuroendocrine system on seizure activity and olfactory function.

Response of Neisseria gonorrhoeae to iron limitation: alterations in expression of membrane proteins without apparent siderophore production.

For acquisition of iron, an essential nutrient, most microorganisms produce siderophores (low-molecular-weight iron-chelating compounds) and membrane proteins to serve as receptors for the iron-siderophore complexes. The gonococcus does not appear to produce a siderophore, since the quantity of siderophore detected by bioassays of culture supernatants from strains F62 and FA19 was never greater than the amount present in the uninoculated medium. Iron limitation of the laboratory strains F62 and FA19 and 12 recent clinical isolates resulted in the expression of several iron-repressible membrane proteins. The expression of proteins in the apparent molecular weight range of 70,000 to 100,000 was strain dependent. All strains expressed 36,000-dalton (36K) and 19.5K proteins. FA19 and F62 were also grown in medium containing iron sources commonly encountered in vivo (i.e., transferrin, lactoferrin, hemoglobin, or hemin). Comparison of growth rates indicates that transferrin and lactoferrin were more readily utilized as iron sources than hemin and hemoglobin were. Expression of the iron-repressible proteins varied depending upon the iron source. Fewer iron-repressible proteins were observed when cells were supplied with transferrin or lactoferrin than when the cultures were grown with either hemin or hemoglobin. The 36K protein was expressed with all four iron sources.