Discordance between HbA1c and fructosamine: evidence for a glycosylation gap and its relation to diabetic nephropathy.

OBJECTIVE: Discordances between HbA1c and other measures of glycemic control are common in clinical practice and remain unexplained. We developed a measure of discordance between HbA1c and fructosamine (FA) (glycosylated serum proteins) to conduct a systematic evaluation. We termed this the glycosylation gap (GG) and sought to determine its relationship to diabetic nephropathy. RESEARCH DESIGN AND METHODS: Measurements of HbA1c and FA on the same sample in 153 people were used to calculate GG, defined as the difference between measured HbA1c and HbA1c predicted from FA based on the population regression of HbA1c on FA. RESULTS: GG had a broad distribution (range, -3.2% to 5.5%); 40% of samples had values indicating major differences in prediction of complications risk by the measured versus predicted HbA1c. GG was highly correlated (r = 0.81) between measurements repeated in 65 patients 23 +/- 2 weeks apart, indicating that the discordances are reliable and not explained by differences in turnover of underlying proteins. In 40 patients with type 1 diabetes of >or = 15 years' duration, an increase in GG by 1% was associated with a 2.9-fold greater frequency of increasing nephropathy stage (P = 0.0014). GG was -0.8 +/- 0.2% in subjects with no nephropathy, -0.3 +/- 0.2% with microalbuminuria/hypertension, and 0.7 +/- 0.3% in subjects with proteinuria or renal dysfunction (P < 0.05). GG correlated better with nephropathy than did either HbA1c or FA alone in this population. CONCLUSIONS: The glycosylation gap may be a useful clinical research tool for evaluating physiologic sources of variation in diabetic complications beyond glycemic control.

Development of olfactory receptor neuron selectivity in the rat fetus.

Olfactory receptor neurons begin to differentiate from stem cells on day E10 of embryonic life in the rat. By day E16, the receptor epithelium is well populated. On this day single neuron action potentials could be recorded with some ease and the electro-olfactogram was well developed. The receptor neurons were functional in that they responded to the vapors of odorous substances. However, they were not selective. Each cell responded to nearly all of the substances in the stimulus set. The first synaptic connections between receptors and mitral cells are established on day E18. The olfactory marker protein is reported to appear first in the receptors on the same day. By day E21, single unit responses changed dramatically. The cells became selective, responding to about half of the substances in our set. The electro-olfactogram reached its limiting amplitude well before this time.

Characterization of flaA- and flaB- mutants of Serpulina hyodysenteriae: both flagellin subunits, FlaA and FlaB, are necessary for full motility and intestinal colonization.

Motility of Serpulina hyodysenterlae is thought to play a pivotal role in the enteropathogenicity of this spirochete. To test this, a series of isogenic mutants containing specifically disrupted flagellar alleles (flaA1 and flaB1) were constructed and examined for virulence and ability to colonize the intestinal tract of mice. Mice challenged with the wild-type, parent strain showed a dose-related response to the challenge organism. In contrast, all flagellar mutant strains demonstrated aberrant motility in vitro and a significantly reduced ability to colonize and infect mice. To some extent, this degree of reduction in colonizing ability was dependent on the wild-type background strain used for mutant construction. A flaB1- strain generated from a 'laboratory isolate' was unable to colonize the mouse gut even at high challenge doses, although its parent was virulent for mice. However, when the same parent strain was 'animal-passed' prior to disruption of flaB1, the resulting flaB1- strain was able to transiently colonize the mouse gut and induce intestinal lesions. A comparison of a series of flagellar mutants constructed using the animal-passed parent strain further revealed that specific inactivation of flaB1 resulted in a more pronounced reduction in virulence and colonizing ability than that which occurred with two flaA1 mutants. Taken together, these data suggest that motility is an essential virulence factor of S. hyodysenteriae and that both sheath and core flagellin subunits, FlaA and FlaB, are necessary for full motility and intestinal colonization.

PauA: a novel plasminogen activator from Streptococcus uberis.

Chromosomal DNA from two geographically distinct isolates of Streptococcus uberis was used to clone the plasminogen activator in an active form in Escherichia coli. The cloned fragments from each strain contained four potential open reading frames (ORFs). That for the plasminogen activator encoded a protein of 286 amino acids (33.4 kDa) which is cleaved between residues 25 and 26 during secretion by S. uberis. The amino acid sequence of the mature protein showed only weak homology (23.5-28%) to streptokinase. The plasminogen activator gene, pauA, in S. uberis was located between two ORFs with high homology to the DNA mismatch repair genes, hexA and hexB, and not on a DNA fragment between the genes encoding an ATP binding cassette transporter protein (abc) and a protein involved in the formation and degradation of guanosine polyphosphates (rel) as is the case for streptokinase in other streptococci.

Establishment of an experimental model of a Staphylococcus aureus abscess in mice by use of dextran and gelatin microcarriers.

Staphylococcus aureus UC 9271 mixed with dextran or gelatin microcarrier beads and injected subcutaneously into mice resulted in the formation of reproducible, sustained abscesses with as few as 2 x 10(3) cfu. Without microcarrier beads, 4 x 10(7) cfu were required to produce an abscess. The abscesses that developed with microcarriers attained a diameter of up to 1.5 cm and persisted for several days before discharging through the skin. The pH of the abscesses fell from 7.1 to 6.6 within 24 h. Histological and microscopic examination of the abscesses revealed an influx of phagocytic cells, mostly polymorphonuclear leucocytes, within 1-2 h after injection. Cell debris accumulated and the abscess became encapsulated 24-48 h after infection. Enzymatic digestion of the abscess contents allowed analysis of the host and bacterial cell populations and treatment with lysostaphin permitted differentiation between phagocytosed and free bacterial populations of S. aureus. Phagocytosed but viable S. aureus comprised c. 50% of the total bacterial population after 24 h; however, by 96 h the phagocytosed population was only 1-5% of the total population, primarily because of an increase in extracellular bacterial numbers. Prevention of abscess formation by antibiotic treatment based upon the minimal inhibitory concentration (MIC) of an antibiotic for S. aureus was not always predictable. Tetracycline did not prevent abscess formation even though it possessed a low MIC for S. aureus; methicillin had a borderline MIC value but was quite active. However, the MIC values were quite predictive of antibiotic cures in a systemic-lethal S. aureus infection in mice.

Virulence and adhesive properties of serotypes A and B of Candida albicans isolated from paediatric burn patients.

The virulence and adhesive properties of 50 isolates of Candida albicans serotypes A and B collected over 6 years from 48 paediatric burn patients were examined to provide more detailed information about candidal pathogenesis in burn patients and to examine the relevance of the commonly used epithelial cell adhesion assay for determining fungal virulence. The isolates represented a fair distribution of serotypes (29 isolates were serotype A and 21 isolates were serotype B) and a total of 28 serotype-biotype combinations were found; 32% of the serotype-biotype combinations appeared only once, while 44% of the isolates showed similar biotype tests for two of three digits. Adhesion of the isolates to plastic and to buccal epithelial cells (BECs) was examined and compared after growth in a chemically defined medium. There were significant differences in the adhesion of individual isolates to plastic or BECs, but no correlation was found between biotype and adhesiveness. Serotype B isolates were found to be more adhesive to BECs (p less than 0.05) but not to plastic. There was no apparent correlation between candidal adhesiveness and site of isolation from these patients (autografts, blood, faeces, throat swabs, tracheal aspirates, wounds and intravenous catheters), although isolates from catheters were generally less adhesive to epithelial cells. Virulence in a systemic infection mouse model revealed that there were significant differences in virulence between isolates, but no correlation was found between virulence and the biotype, serotype or site of isolation. Similarly, no correlation was found between virulence and adhesiveness or cell-surface hydrophobicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanisms of association of Candida albicans with intestinal mucosa.

The association of Candida albicans with gastrointestinal (GI) mucosal surfaces was studied in vitro and in vivo. The caecal mucosal surfaces from antibiotic-treated and untreated control mice challenged orally with C. albicans revealed that large numbers of C. albicans were associated with the intestinal epithelium of antibiotic-treated mice but not with that of the control mice that possessed an indigenous wall-associated bacterial flora. Moreover, Candida cells only penetrated deep into the mucosa of animals in which the ecology of the intestinal microflora had been disrupted. In mice given antibiotics, C. albicans was associated with the mucosa of all areas of the GI tract; the caecal mucosa had the most associated Candida, whereas the stomach and small intestine had very few associated yeasts. Further examination of caecal mucosa from antibiotic-treated mice showed that C. albicans associated with the mucosa by at least five distinct mechanisms. These included: adhesion to epithelium, adhesion to mucus, co-adhesion to adherent fungi, co-adhesion to adherent bacteria, and entrapment in the mucous gel overlying the epithelium. The cell-surface hydrophobicity of C. albicans also was examined and found not to play a role in Candida adhesion to intestinal mucosa. The predominant association mechanisms appeared to be entrapment in the mucous gel, and adhesion to mucus and the epithelium. The ecological and pathological significance of co-adhesion by C. albicans to attached organisms is unclear but it may be important in the initiation of mucosal lesions.

A randomized, prospective study measuring outcomes after antibiotic therapy intervention by a multidisciplinary consult team.

Our aim was to identify financial and outcome benefits of therapeutic intervention by a multidisciplinary antimicrobial treatment team composed of pharmacists, a clinical microbiologist, and an infectious disease specialist. Of 252 consecutive inpatients receiving suboptimal intravenous antibiotics identified by the clinical pharmacist, 127 were prospectively randomized to intervention and 125 to a control group. The groups were similar with regard to severity of illness, infection type, and time from admission to randomization. Physicians received timely, detailed reviews of relevant microbiologic and clinical data with recommendations of possible optimal antibiotic choices, dosages, and rationales. Median length of stay after randomization for control and intervention groups was 9.0 days and 5.7 days, respectively (3.3-day difference, p=0.0001). Fifteen (12.0%) and eight patients (6.3%), respectively, died, although the time-specific mortality risk was not significantly different when length of postrandomization follow-up and time to death were taken into account. Physician acceptance of suggestions was 89%. Median patient charges for radiology, laboratory, pharmacy, and room were reduced by $4404/intervention, and median hospital costs were reduced by $2642/intervention. A multidisciplinary antimicrobial therapy team can be a useful information source for physicians, improve outcomes in hospitalized patients receiving intravenous antimicrobials, and result in substantial cost savings.

Motility and chemotaxis in Serpulina hyodysenteriae.

Chemotactic- or motility-regulated mucus association appears to be the predominant mechanism of mucosal association by the causative agent of swine dysentery, Serpulina hyodysenteriae. In the present study, a modification of the Adler capillary assay was used to evaluate the chemotactic responses of S. hyodysenteriae to a variety of potential stimuli. First, however, it became necessary to study factors that influenced motility of the spirochete in vitro, since standard cultivation methods produced motility inferior to that observed for in vivo grown cells. A number of factors were found to influence S. hyodysenteriae motility, but of these growth medium and growth phase appeared to be the most important. The type and even batch of culture medium also were found to have a significant influence on S. hyodysenteriae motility. Optimal motility and chemotaxis for S. hyodysenteriae was observed when the cells were harvested in mid- to late-log phase, and in vivo-like motility could be induced by suspending the cells in physiologic saline. S. hyodysenteriae was strongly attracted to hog gastric mucin, certain concentrations of blood, L-fucose, L-serine and other compounds. Selected sugars and other amino acids did not serve as chemoattractants for S. hyodysenteriae. The chemotactic response of S. hyodysenteriae toward L-fucose and L-serine, constituents of mucin, may be important factors in the affinity of the spirochete for the mucus in the intestinal tract of swine.

In vitro activity of ceftiofur and its primary metabolite, desfuroylceftiofur, against organisms of veterinary importance.

Ceftiofur (XNL) and its primary metabolite, desfuroylceftiofur (DXNL), were evaluated for in vitro activity against 539 isolates from veterinary sources. Actinobacillus pleuropneumoniae, Pasteurella spp., Haemophilus somnus, Salmonella spp., Escherichia coli, staphylococci, and streptococci were tested. Overall, XNL and DXNL were equivalent in activity against the gram-negative organisms with all minimum inhibitory concentrations (MICs) within 1 serial dilution. Against the staphylococci, MIC difference of 2-3 serial dilutions were detected with an MIC90 for XNL and DXNL of 1.0 and 4.0-8.0 micrograms/ml, respectively. Although the MIC90 obtained for Streptococcus suis for each compound was within 1 dilution, the MIC values against individual strains were 2-3 dilutions greater for DXNL than for XNL. The MICs obtained with the bovine and equine streptococci for DXNL (MIC90 = 0.03 microgram/ml) were 5 serial dilutions higher than those obtained for XNL (MIC90 < or = 0.0019). Although DXNL was less active than XNL against the streptococci, these differences were not clinically important because both XNL and DXNL were highly active for these bacteria. Although these differences are of little importance with the streptococci, they may have important implications for susceptibility testing of the staphylococci. In conclusion, with the exception of the staphylococci, both XNL and DXNL were highly active against the organisms tested, with MICs for both compounds several fold lower than plasma levels achieved during dosing of XNL.

Interpretive criteria for antimicrobial susceptibility testing of ceftiofur against bacteria associated with swine respiratory disease.

Ceftiofur, an extended-spectrum cephalosporin, is active against a variety of animal pathogens, including organisms associated with swine respiratory disease. However, minimum inhibitory concentration (MIC) breakpoint and disk diffusion interpretive criteria have not been established for swine pathogens. Susceptibility tests were performed by broth microdilution MIC and disk diffusion methods on 246 bacterial species that cause swine respiratory disease. Ceftiofur was active against Salmonella sp., Pasteurella multocida, Actinobacillus pleuropneumoniae, Streptococcus suis, and Escherichia coli but was not active against Bordetella bronchiseptica measured by MIC. Based on pharmacokinetic studies of ceftiofur in swine after a single intramuscular injection of 3 or 5 mg/kg body weight of ceftiofur and on the MIC and disk diffusion data, we recommend MIC breakpoints and disk diffusion distances, respectively, of < or = 2 micrograms/ml and > or = 21 mm for susceptible, 4 micrograms/ml and 18-20 mm for intermediate, and > or = 8 micrograms/ml and > or = 17 mm for resistant classification for swine pathogens. When these breakpoints were applied to data from a previous study using bovine pathogens, only 1 minor interpretive error occurred.

Motility of the pathogen and intestinal immunity of the host in experimental cholera.

1) Motility seems to be required for an intimate association with the intestinal mucosa which is necessary for efficient toxin delivery in the pathogenicity of V. cholerae. 2) A vaccine of semipurified flagella (CF) afforded a high degree of active and passive immunity in the rabbit ileum to both homologous and heterologous challenge strains. 3) The nature of the immunoglobulin type involved in the active immunity elicited by CF was not clear but preliminary indications implicate both s-IgA and IgG.

A vaccine strain of pseudorabies virus with deletions in the thymidine kinase and glycoprotein X genes.

A pseudorabies virus (PRV) mutant with deletions in genes for glycoprotein X (gX) and thymidine kinase, designated delta GX delta TK, was constructed and evaluated as a vaccine for protecting swine against PRV-induced mortality. Doses greater than or equal to 10(3) plaque-forming units (PFU) of this strain given to mice provided protection from challenge exposure with virulent PRV. Sera tested from mice inoculated with delta GX delta TK had high titers of neutralizing antibody to PRV, but reactivity in the same sera was not significantly different from that in sera from noninoculated mice (controls) when sera from both groups were evaluated by use of an ELISA with gX antigen produced in Escherichia coli. Compared with noninoculated pigs (controls), those given delta GX delta TK (greater than or equal to 10(2) PFU) were protected completely from lethal challenge exposure, without experiencing adverse effects on weight gain and with reduction of shedding of virulent challenge virus. Serotest results indicated that, although inoculated pigs responded with strong neutralizing antibody titers, the response of delta GX delta TK-inoculated pigs to gX, as determined by ELISA before challenge exposure, was not significantly greater than the ELISA values obtained from control pigs. The ELISA values from a group of pigs inoculated with a commercially available vaccine were significantly (P less than 0.05) higher than those of control pigs. The experimental vaccine, delta GX delta TK, was avirulent for mice, swine, and sheep, but was mildly virulent for calves (mortality, 1 of 12) and more virulent for dogs (mortality, 3 of 6) and cats (mortality, 2 of 6).(ABSTRACT TRUNCATED AT 250 WORDS)

Efficacy of ceftiofur hydrochloride for treatment of experimentally induced colibacillosis in neonatal swine.

Ceftiofur hydrochloride was tested for effectiveness against induced colibacillosis in neonatal swine. In this model, pigs less than 12 hours old were inoculated via stomach tube with a virulent, K99+, nalidixic acid-resistant strain of Escherichia coli. Six hours after challenge exposure, 1 dose of ceftiofur was administered either IM or orally in experiment 1 and orally only in experiment 2. Mortality, shedding of bacteria, fecal consistency scores, and body weight changes were monitored for 10 days. In experiment 1 (n = 383 pigs), all treatments at dosage that ranged between 0.5 and 64.0 mg of ceftiofur/kg of body weight significantly (P less than 0.001) reduced mortality, bacterial shedding, and diarrhea and increased weight gain, compared with findings in untreated controls. There were no detectable differences between oral and IM routes, except that there was greater reduction in bacteria shedding associated with the oral route of administration. In experiment 2 (n = 505 pigs), ceftiofur was administered orally either once at 6 hours after challenge exposure or twice at 6 and at 48 hours after the first dose. Dosage of ceftiofur was 0, 5, 10, 20, 30, or 60 mg/kg administered once, or half the same dose was administered at each of 2 times. At the optimal dosage (10 mg/kg), a single dose was as effective as 2 doses. The single administration at all dosages reduced mortality, bacterial shedding, and diarrhea scores and increased body weight gain, compared with findings in untreated pigs (P less than 0.01). In this induced infection model, the optimal treatment dosage was determined to be 10 mg/kg administered once.

Protection of mice and swine from pseudorabies virus-induced mortality by administration of pseudorabies virus-specific mouse monoclonal antibodies.

Hybridomas were selected for secretion of monoclonal antibodies directed against pseudorabies virus (PRV) glycoproteins. Each monoclonal antibody was capable of neutralizing PRV in vitro in the presence of complement. This panel of antibodies was used in passive immunization studies to protect mice and swine from PRV-induced mortality. The most protective antibody in mice was 3A4, specific for PRV glycoprotein gp50, which afforded as high as 100% protection. Although antibody 3A4 was partially protective in swine, antibody 3D11, which is specific for PRV glycoprotein glll, afforded greater protection--83% protection when ascitic fluid was used and 100% protection when immunoglobulin concentrated from cell cultures was used at a dose of 150 mg/pig. These studies demonstrated that monoclonal antibodies may be useful for short-term prophylaxis against PRV-induced disease and that antibody directed against either PRV glycoprotein glll or gp50 is sufficient to protect animals from PRV-induced mortality.

Ceftiofur sodium, a broad-spectrum cephalosporin: evaluation in vitro and in vivo in mice.

Ceftiofur sodium, a broad-spectrum beta-lactamase-resistant cephalosporin, was evaluated in vitro and in vivo in mice. Ceftiofur is the sodium salt of (6R, 7R)-7[( 2-amino-4-thiazolyl)-Z- (methoxyimino)acetyl]amino)-3-[( (2-furanylcarbonyl)thio]methyl)-8-oxo-5- thia-1-azabicyclo-[4.2.0]oct-2-ene-2-carboxylate. Minimal inhibitory concentration values were obtained with 264 strains representing 9 genera and 17 species of bacterial pathogens from cattle, swine, sheep, horses, poultry, dogs, cats, and human beings. Ceftiofur was more active than was ampicillin against all strains tested including beta-lactamase-producing organisms. In mice with systemic infections, ceftiofur was more active than or equivalent to ampicillin, cephalothin, cefamandole, cloxacillin, cefoperazone, or pirlimycin. These protection tests included infections with Escherichia coli, Haemophilus pleuropneumoniae, H somnus, Pasteurella haemolytica, P multocida, Salmonella typhimurium, or Staphylococcus aureus. In infant mice with E coli-induced lethal diarrhea and in mice with S aureus and E coli-induced mastitis, ceftiofur was comparable or more active than was ampicillin.

Therapeutic effect of optimal lincomycin concentration in drinking water on necrotic enteritis in broilers.

The efficacy of 16.9 mg lincomycin/liter of drinking water was evaluated for the treatment of necrotic enteritis (NE) in 743 broiler-type chickens. Birds were raised in a facility containing a built-up litter obtained from a source that experienced NE. They were commingled from 1 day of age until NE was observed. Two groups of 6 pens each were given 0 or 16.9 mg lincomycin/liter of drinking water. Water medication was offered fresh daily for 7 days and the study was terminated 3 weeks after initiation of therapy. The susceptibility of Clostridium perfringens to lincomycin was determined in vitro. The test organism was susceptible to lincomycin as reflected by minimal inhibitory concentration and minimal lethal concentration of .156 microgram/ml. Mortality attributed to NE was 0% in lincomycin treated birds and 14% in nonmedicated control birds (P less than .01). Lincomycin water medication was highly effective for the treatment of NE in broilers.

Host-response patterns of intramammary infections in dairy cows.

Many different bacterial species have the ability to cause an infection of the bovine mammary gland and the host response to these infections is what we recognize as mastitis. In this review we evaluate the pathogen specific response to the three main bacterial species causing bovine mastitis: Escherichia coli, Streptococcus uberis and Staphylococcus aureus. In this paper we will review the bacterial growth patterns, host immune response and clinical response that results from the intramammary infections. Clear differences in bacterial growth pattern are shown between bacterial species. The dominant pattern in E. coli infections is a short duration high bacteria count infection, in S. aureus this is more commonly a persistent infection with relative low bacteria counts and in S. uberis a long duration high bacteria count infection is often observed. The host immune response differs significantly depending on the invading bacterial species. The underlying reasons for the differences and the resulting host response are described. Finally we discuss the clinical response pattern for each of the three bacterial species. The largest contrast is between E. coli and S. aureus where a larger proportion of E. coli infections cause potentially severe clinical symptoms, whereas the majority of S. aureus infections go clinically unnoticed. The relevance of fully understanding the bovine host response to intramammary infection is discussed, some major gaps in our knowledge are highlighted and directions for future research are indicated.