Up- and down-regulation of granulocyte/macrophage-colony stimulating factor activity in murine skin increase susceptibility to skin carcinogenesis by independent mechanisms.

The role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in tumorigenesis is complex. On the one hand, GM-CSF can promote tumor cell growth, survival, and even metastasis. On the other hand, it can stimulate tumor cell rejection. In skin, it is early expressed after topic application of tumor-promoting agents and therefore may be responsible for changes that correlate with skin tumor promotion (e.g., epidermal hyperproliferation and inflammation). To analyze GM-CSF function in skin tumorigenesis, we generated transgenic mice epidermally overexpressing either GM-CSF or a GM-CSF antagonist. Both types of transgenic mice exhibited significantly increased numbers of benign tumors in a two-step skin carcinogenesis experiment using 7',12'-dimethylbenz[a]anthracene (DMBA) as initiator and 12-O-tetradecanoylphorbol-CSF displayed a significantly elevated carcinoma burden following a single-step carcinogenesis protocol consisting of tumor initiation only. Therefore, endogenous promotion is responsible for elevated tumor development in GM-CSF-overexpressing mice. In antagonist transgenic animals, an increased tumorigenicity of modified B16 tumor cells after cutaneous transplantation as compared with nontransgenic or GM-CSF transgenic mice was observed. Thus, the antitumor activity leading to the repression of tumor cell growth in control mice is GM-CSF dependent and is compromised in mice expressing the antagonist. We suggest that both, up-regulation and down-regulation of GM-CSF activity in skin, increase the incidence and growth of tumors via two independent mechanisms: endogenous tumor promotion in the case of increased GM-CSF activity and compromised tumor cell rejection in the case of decreased GM-CSF activity.

Implementation and refinement of the emergency severity index.

OBJECTIVES: To implement a new five-level emergency department (ED) triage algorithm, the Emergency Severity Index (ESI), into nursing practice, and validate the instrument with a population-based cohort using hospitalization and ED length of stay as outcome measures. METHODS: The five-level ESI algorithm was introduced to triage nurses at two university hospital EDs, and implemented into practice with reinforcement and change management strategies. Interrater reliability was assessed by a posttest and by a series of independent paired patient triage assignments, and a staff survey was performed. A cohort validation study of all adult patients registered during a one-month period immediately following implementation was performed. RESULTS: Eight thousand two hundred fifty-one ED patients were studied. Weighted kappa for reproducibility of triage assignments was 0.80 for the posttest (n = 62 nurses), and 0.73 for patient triages (n = 219). Hospitalization was 28% overall and was strongly associated with triage level, decreasing from 58/63 (92%) of patients in triage category 1, to 12/739 (2%) in triage category 5. Median lengths of stay were two hours shorter at either triage extreme (high and low acuity) than in intermediate categories. Outcomes followed a-priori predictions. Staff nurses rated the new program easier to use, and more useful as a triage instrument than previous three-level triage. They provided feedback, which resulted in significant revisions to the algorithm and educational materials. CONCLUSIONS: Triage nurses at these two hospitals successfully implemented the ESI algorithm and provided useful feedback for further refinement of the instrument. Emergency Severity Index triage reproducibly stratifies patients into five groups with distinct clinical outcomes.

Capnocytophaga ochracea: characterization of a plasmid-encoded extended-spectrum TEM-17 beta-lactamase in the phylum Flavobacter-bacteroides.

A plasmid-encoded extended-spectrum TEM beta-lactamase with a pI of 5.5 was detected in a Capnocytophaga ochracea clinical isolate. The bla gene was associated with a strong TEM-2 promoter and was derived from bla(TEM-1a) with a single-amino-acid substitution: Glu(104)-->Lys, previously assigned to TEM-17, which is thus the first TEM beta-lactamase to be reported in the phylum Flavobacter-Bacteroides.

Nucleotide sequences of genes coding for fimbrial proteins in a cryptic genospecies of Haemophilus spp. isolated from neonatal and genital tract infections.

Nineteen isolates belonging to a cryptic genospecies of Haemophilus (referred to here as genital strains) isolated from genital tract infections (6 strains) and from neonatal infections (13 strains) were studied for fimbrial genes. Sixteen strains exhibit peritrichous fimbriae observed by electron microscopy. By PCR with primers corresponding to the extreme ends of the Haemophilus influenzae type b (Hib) hifA and hifD genes and Southern blotting, a hifA-like gene (named ghfA) and a hifD-like gene (named ghfD) were identified in 6 of the 19 strains. Five of these six strains were from the genital tracts of adults, and one was from a neonate. For each gene, the nucleotide sequence was identical for the six strains. A hifE-like gene (named ghfE) was amplified from only one of the 19 genital strains of Haemophilus, but the ghfE probe gave a signal in Southern hybridization with the five other strains positive for ghfA and ghfD. Therefore, these strains may carry a ghfE-like gene. The Hib fimbrial gene cluster is located between the purE and pepN genes as previously described. For the 13 genital Haemophilus strains that lack fimbrial genes, this region corresponds to a noncoding sequence. Another major fimbrial gene designated the fimbrin gene was previously identified in a nontypeable H. influenzae strain. A fimbrin-like gene was identified for all of our 19 genital strains. This gene is similar to the ompP5 gene of many Haemophilus strains. Therefore, other, unidentified genes may explain the piliation observed in electron microscopy on genital Haemophilus strains which do not possess LKP-like fimbrial genes. Fimbrial genes were significantly associated with strains isolated from the genital tract. They may confer on the strain the ability to survive in the genital tract.

Characterization of Streptococcus agalactiae strains by randomly amplified polymorphic DNA analysis.

A collection of 54 unrelated Streptococcus agalactiae strains isolated from cerebrospinal fluid samples from neonates and 60 unrelated strains isolated from carriers that had been previously studied by multilocus enzyme electrophoresis (R. Quentin, H. Huet, F.-S. Wang, P. Geslin, A. Goudeau, and R. K. Selander, J. Clin. Microbiol. 33:2576-2581, 1995) were characterized by randomly amplified polymorphic DNA (RAPD) assay. Four primers, 5'AGGGGGTTCC3', 5'AACGCGCAAC3', 5'GCATCAATCT3', and 5'AGTCGGGTGG3', named OPS16, AP42, A4, and OPS11, respectively, were selected from 29 primers tested. This investigation identified 71 RAPD types. The three families of strains defined by multilocus enzyme electrophoresis analysis, which contain most of the cerebrospinal fluid isolates, were also identified by clustering analysis of RAPD data. Each of these three groups exhibits specific RAPD patterns or fragments. The discriminatory power of the RAPD typing method was also evaluated. The simplest typing scheme was obtained by the combination of RAPD typing done with primers AP42 and OPS11 and serotyping (index of discrimination, 0.97).

Paramedic interpretation of prehospital lead-II ST-segments.

OBJECTIVE: To determine the reliability of ST-segment interpretation by paramedics from lead-II rhythm strips obtained in the prehospital setting. DESIGN: Prospective, blinded study of 127 patients transported by an urban/rural emergency medical services system with complaints consistent with ischemic heart disease. METHODS: Emergency department physicians asked emergency medical technician-paramedics (EMT-P) via radio to evaluate ST-segments for elevation or depression and grade it as "mild," "moderate," or "severe." Then, this rhythm strip was interpreted blindly by emergency physicians who also interpreted the lead-II obtained from a 12-lead electrocardiogram (ECG) obtained in the emergency department (ED). The field interpretation was compared with the subsequent readings and the final in-patient diagnosis using positive predictive value (PPV), negative predictive value (NPV), and the Kappa statistic. Markedly discrepant interpretations were analyzed separately. RESULTS: Using physician interpretation as the reference standard, paramedic interpretation of the lead-II ST-segments obtained in the prehospital setting was correct (within +/- 1 gradation) in 113 out of 127 total cases (89%). Of 105 patients for whom final hospital diagnosis was available, the ST-segment on the rhythm strip obtained in the prehospital setting, had a positive predictive value of 74% and a negative predictive value of 85% for myocardial ischemia or myocardial infarction (MI) (p < 0.001, Kappa = 0.59). Discordant interpretations between the paramedics and emergency physicians often were related to a basic misunderstanding of rhythm strip morphology. CONCLUSION: Field interpretation of ST-segments by paramedics is fairly accurate as judged both by emergency physicians and correlation with final patient outcome, but its clinical utility is unproved. A small but clinically significant number of outliers, consisting of markedly discrepant false positives, reflects paramedic uncertainty in identifying the deviations of the ST-segment.

Genetic diversity of rRNA operons of unrelated Streptococcus agalactiae strains isolated from cerebrospinal fluid of neonates suffering from meningitis.

The genetic diversity of a collection of 54 unrelated Streptococcus agalactiae strains isolated from the cerebrospinal fluid of neonates and of 60 unrelated carrier strains was evaluated by investigating the restriction fragment length polymorphism of the rRNA gene region. Three restriction enzymes were selected for use: PstI, HindIII, and CfoI. Clustering analysis revealed two phylogenetic groups of strains with 40% divergence. Group I contained two clusters, A and B, and group II contained three clusters, C, D, and E. Strains of serotype Ia were mostly distributed in cluster A, and strains of serotype Ib were mostly distributed in cluster E. Serotype III isolates did not cluster. Nevertheless, 37 of 39 isolates belonging to cluster B were serotype III. With HindIII, two rRNA gene banding patterns characterized 38 of the 39 strains of cluster B, which represents a high-virulence group. In addition, two rRNA gene banding patterns with each enzyme and/or a pair of CfoI fragments of 905 and 990 bp identified 81% of the invasive strains. On account of the genetic homogeneity of the cerebrospinal fluid strains, ribotyping is a powerful typing method for investigation of nosocomial or epidemic invasive infections only when all three enzymes are used or when PstI and HindIII or PstI and CfoI are combined with serotyping (index of discrimination, > 0.95).

Genetic identification of cryptic genospecies of Haemophilus causing urogenital and neonatal infections by PCR using specific primers targeting genes coding for 16S rRNA.

Previous genetic analysis of Haemophilus influenzae strains isolated from genital and neonatal infections identified a group of biotype IV that constitutes a cryptic genospecies only distantly related to H. influenzae and H. Haemolyticus. Small-subunit rRNA genes of two representative strains of this genital Haemophilus genospecies (strains 16N and 2406) were sequenced. The analysis indicated that these strains form a monophyletic unit with H. haemolyticus and H. influenzae biogroups Influenzae and Aegyptius and are more closely related to H. haemolyticus than to H. influenzae biogroups Influenzae and Aegyptius. 16S rRNA gene sequences were used to formulate primers for PCR-based identification of cryptic genital Haemophilus organisms. A 242-bp fragment was amplified from strains belonging to the genital Haemophilus genospecies but not from strains of 12 other Haemophilus species, including strains of H. influenzae biotype IV sensu stricto.

Adherence to human cells of a cryptic Haemophilus genospecies responsible for genital and neonatal infections.

Haemophilus strains usually identified as Haemophilus influenzae biotype IV belonging to a cryptic genospecies are responsible for genital and neonatal infections. As a first approach to identifying the bacterial factors involved in the pathogenesis of these unusual diseases, we studied the piliation, adherence, and invasion properties of 17 strains assigned to this cryptic genospecies. Twelve strains spontaneously displayed abundant peritrichous piliation, and two strains expressed peritrichous pili after enrichment procedures. For virtually all strains, piliation correlated with adhesion to cultured HeLa cells of genital origin and to a lesser extent with adhesion to HEp-2 cells of laryngeal origin. A variation in the adherence properties of the various strains was observed: all piliated strains except one adhered to 50 to 100% of HeLa cells, with a mean number of bacteria per cell varying from 4 to 50. Adherence was not dependent on the state of growth for most strains, was more pronounced with HeLa cells than with HEp-2 cells for 10 of the 12 highly adherent strains, was time and inoculum dependent, and was not followed by significant invasion of cells. Most of the strains belonging to this unusual Haemophilus clone possess adhesins that do not recognize erythrocyte receptors, since agglutination of human erythrocytes was observed with only 3 of the 14 piliated strains.

Plasmid-mediated ROB-1 beta-lactamase in Pasteurella multocida from a human specimen.

A Pasteurella multocida human isolate was resistant to beta-lactams because of production of ROB-1 beta-lactamase. The beta-lactamase was encoded by a 4.3-kb plasmid closely related to that of a Pasteurella bovine strain, as shown by Sau3A restriction profile and hybridization with a plasmid probe containing the blaROB-1 gene.

Prevalence of virulence markers of enteric Campylobacter in France and Tunisia.

Forty-nine strains of Campylobacter jejuni and C. coli were isolated from the stools of 49 patients clinically documented for diarrhoea and fever, and living either in the Paris metropolitan area (30) or in the Tunis area (19). The strains were identified biotyped, serotyped and studied for association with HeLa cells and the ability to elongate Chinese ovary cells (CHO). The C. jejuni biotype I was more frequent among Tunisian strains and the C. jejuni biotype II was more frequent among French strains. Twenty-four strains associated with HeLa cells (A phenotype) and 21 elongated CHO (E phenotype). These 2 phenotypes were independently distributed in individual strains and were not related to the biotypes. We defined 4 pathovars according to the presence (A and E) or absence (a and e) of these 2 markers. The prevalence of the 4 pathovars was not correlated with the origin of the strain. The lack of a virulence marker (phenotype a/e) was correlated with the lack of clinical signs of diarrhoea and fever (p = 4 x 10(-5)). We concluded that at least 1 of the 2 in vitro virulence markers is related to the pathogenicity of the strains in the humans.

Adhesion to HeLa cells of Campylobacter jejuni and C. coli outer membrane components.

Adhesion of Campylobacter jejuni and C. coli to epithelial cells is thought to be a decisive step in enteritis. In this work, we tried to determine which bacterial components are responsible for this phenomenon. Outer membrane (OM) extracts were prepared from strains of C. jejuni (3 strains) and C. coli (2 strains). These strains had been isolated from stools of febrile patients with diarrhoea and were able to adhere to HeLa cells in culture. After incubation of bacterial OM extracts with HeLa cells in culture, bacterial adherent material was recovered, subjected to electrophoresis and immunoblotted. Bacterial adherent antigens were revealed by a rabbit antiserum raised against whole bacterial cells. Antigenic fractions, ranging from 26 to 30 kDa, were found to preferentially bind to HeLa cells (cell-binding fractions; CBF). These antigens were proteins and were distinct from flagellin and lipopolysaccharide. Bacteria incubated with a rabbit antiserum raised against homologous CBF, were unable to bind to HeLa cells. Moreover, the inhibitory effect decreased when the antiserum was diluted. Under the same conditions, a rabbit antiserum raised against a non-adherent OM fraction of 92 kDa did not prevent bacteria from binding to HeLa cells.

[Campylobacter pyloridis and gastroduodenal pathology]. / Campylobacter pyloridis et pathologie gastroduodénale.

After the first report by Marshall and Warren of the presence of gastritis associated Campylobacter pyloridis in antral mucosa, many groups have found the same association in many countries of Europe, USA, Japan. In France, too C.P. is found in antral mucosa. We have studied 119 dyspeptic patients; during endoscopy 3 biopsy specimens were taken, one for microaerophilic culture and 2 for pathologic examination. We found the germ in 27 p. 100 of 22 normal subjects but no culture was positive. In chronic interstitial gastritis it is found in 90 p. 100 of cases and in 98 p. 100 if a duodenal ulcer is associated. Ten patients with healed duodenal ulcer have been treated by V Penicillin: 3 millions unit/day during 2 weeks. In five cases C.P. disappeared but 2 of them relapse 2 months later.

Association with HeLa cells of Campylobacter jejuni and Campylobacter coli isolated from human feces.

We developed a rapid in vitro test for determining the association of Campylobacter jejuni and C. coli with HeLa cells. Association was expressed as a weighted mean of the number of bacteria associated with one cell in an association index (AI). The reproducibility of the AI was checked by repeating the test six times, using four strains chosen at random. Means and standard deviations of the means were 7.3 +/- 1.2, 6.8 +/- 0.9, 1.8 +/- 1.2, and 0.1 +/- 0.2. The experimental conditions for which the results are reliable have been standardized. Among 42 strains from human feces, two groups appeared: for 22 nonassociative strains (52%), AI values ranged from 0.0 to 2.1 (mean +/- SD, 0.5 +/- 0.6); for 20 associative strains (48%), AI values ranged from 3.5 to 8.3 (mean +/- SD, 6.2 +/- 1.4). Of these 42 strains, 17 were clinically documented. Diarrhea occurred more frequently in patients infected with associative strains than in those infected with noninvasive strains (7/7 versus 3/10, P = 0.01). Fever also occurred more frequently in patients infected with associative strains (6/7 versus 2/10, P = 0.03). Transmission electron microscopy and viable counts made after killing of extracellular bacteria by gentamicin support the fact that associated Campylobacter spp. are adherent to the cell membrane and are internalized into cytoplasmic vacuoles. The described test seems to be a convenient and rapid method for estimating the pathogenicity of a given strain.

[Evaluation of the sensitivity of Haemophilus to antibiotics by the modified API-ATB system]. / Evaluation de la sensibilité des haemophilus aux antibiotiques par le système API-ATB modifié.

A simple micromethod in a liquid medium using the API-ATB system was developed for testing the susceptibility of Haemophilus to antibiotics. To evaluate this method, 50 strains, including 12 beta-lactamase producers, were studied. Results were compared to those obtained using MIC determination in a liquid medium (reference) and an agar diffusion method (routine). For all three techniques, a Mueller-Hinton medium enriched in hemoglobin and NAD was used, and cultures were incubated at 37 degrees C for 24 hours in normal atmosphere. Influence of the inoculum on results was evaluated using the API-ATB method for all antibiotics and MIC determination for ampicillin; the optimal inoculum was found to be 8.10(5) CFU/ml. Beta-lactamase was looked for using the chromogen cephalosporin test associated with the API-ATB system. Values of MICs for the various antibiotics were consistent with previous reports. Paired comparison of techniques showed a 5.3% disagreement rate between API-ATB and MIC, with only 0.5% major discrepancies; in contrast, the disagreement rate exceeded 10% when disk diffusion was compared with the two other techniques. We conclude to the reliability and reproducibility of the API-ATB method which seems capable of improving current routine evaluations of the susceptibility of Haemophilus to antibiotics.

[Assay of total serum bile acids. Reference values in children]. / Dosage des acides biliaires totaux sériques. Valeurs de référence chez l'enfant.

Serum total bile acids were measured using an enzyme micromethod involving 3-hydroxysteroid dehydrogenase (Sterognost 3 Fluorometry) without prior extraction. The test specimen was 50 microliters of serum, with reading by spectrofluorometry. The degree of accuracy assessed at several concentration levels was acceptable for values greater than 5 mumol/l. The calibration function was linear between 0 and 80 mumol/l. Lower limit of detection was 0.88 mumol/l. Results given by this method were compared with those obtained using the Schwarz technique involving extraction of bile acids and their enzyme estimation. The correlation between the two methods was satisfactory. Absence of interference by bilirubin was confirmed. Total bile acids were measured in the serum of fasting children, aged from 1 day to 4 years. High values were seen at birth (m = 8.33 mumol/l, SD = 3.46, n = 10) as well as during the first month of life. Serum total bile acid concentration decreased up to the age of 10 months (m = 2.13 mumol/l, SD = 1.91, n = 31). After one year, they increased progressively, reaching at the age of 4 values similar to those in the adult (m = 3.80 mumol/l, SD = 2.17, n = 30). Particularly high values were found in children with delayed growth or in premature infants (m = 19.66 mumol/l, SD = 10.4, n = 15). Serum total bile acid levels were lower in umbilical cord blood and in the newborn. The increase in serum bile acids found in the newborn may be due to slight cholestasis or to a reduction in the hepatic clearance, showing the special type of hepatic function in the young child.