Immune response, ciprofloxacin activity, and gender differences after human experimental challenge by two strains of enterotoxigenic Escherichia coli.

In order to test vaccines against enterotoxigenic Escherichia coli (ETEC)-induced diarrhea, challenge models are needed. In this study we compared clinical and immunological responses after North American volunteers were orally challenged by two ETEC strains. Groups of approximately eight volunteers received 10(9) or 10(10) CFU of E. coli B7A (LT+ ST+ CS6+) or 10(8) or 10(9) CFU of E. coli H10407 (LT+ ST+ CFA/I+). About 75% of the volunteers developed diarrhea after challenge with 10(10) CFU B7A or either dose of H10407. B7A had a shorter incubation period than H10407 (P = 0.001) and caused milder illness; the mean diarrheal output after H10407 challenge was nearly twice that after B7A challenge (P = 0.01). Females had more abdominal complaints, and males had a higher incidence of fever. Ciprofloxacin generally diminished or stopped symptoms and shedding by the second day of antibiotic treatment, but four subjects shed for one to four additional days. The immune responses to colonization factors CS6 and colonization factor antigen I (CFA/I) and to heat-labile toxin (LT) were measured. The responses to CFA/I were the most robust responses; all volunteers who received H10407 had serum immunoglobulin A (IgA) and IgG responses, and all but one volunteer had antibody-secreting cell (ASC) responses. One-half the volunteers who received B7A had an ASC response to CS6, and about one-third had serum IgA or IgG responses. Despite the differences in clinical illness and immune responses to colonization factors, the immune responses to LT were similar in all groups and were intermediate between the CFA/I and CS6 responses. These results provide standards for immune responses after ETEC vaccination.

Educating the next generation of physicians about stroke: incorporating stroke prevention into the medical school curriculum.

BACKGROUND AND PURPOSE: In response to the need to educate physicians about stroke, we have implemented an educational program on stroke prevention for undergraduate medical students within the first-year neuroscience course. This study investigated whether first-year students learned and retained key information about stroke, and used students' feedback both to identify effective curricular components and to explore their attitudes regarding stroke prevention. METHODS: Stroke knowledge and self-assessed confidence in that knowledge before, immediately after, and 8 months after participation in the stroke curriculum were analyzed and compared for 3 classes, using paired t tests and repeated-measures ANOVA. Student feedback about the effectiveness of specific parts of the curriculum and about the importance of stroke prevention was solicited and evaluated. RESULTS: First-year medical students in 3 classes more than doubled their overall stroke knowledge scores (pretest total mean of 8.2; posttest mean 18.0), and retained significant improvement 8 months later (mean 15.7). Subscores in all 4 areas of stroke knowledge tested significantly increased (P<0.001). Students' confidence in their knowledge of stroke risk factors and warning signs, as well as in their knowledge itself, increased (P<0.001). Each of the 3 cohorts demonstrated similar improvements. Feedback indicated heightened awareness and interest in stroke prevention, which was maintained after completion of the curriculum. CONCLUSIONS: These results demonstrate that when instruction on stroke prevention is incorporated into the first-year curriculum, students learn and retain key information. Because entire classes of medical students are involved, this type of approach has the potential to reach all future physicians and therefore to meaningfully impact future stroke care.

Towards the reconstruction of central nervous system white matter using neural precursor cells.

Epidermal growth factor-responsive neural precursor cells were used as donor cells for transplantation into wild-type and myelin-deficient shiverer (shi) mice. The cells engrafted robustly within the CNS following intracerebroventricular and cisternal transplantation in neonatal mice. The cells adopted glial phenotypes, and some functioned as oligodendrocytes, producing myelin basic protein and morphologically normal internodal myelin sheaths. When individual shi mice received two transplants (on post-natal days 1 and 3), donor-derived cells disseminated widely and expressed myelin basic protein in central white matter tracts throughout the brain.

Enteropathogenic Escherichia coli strain RDEC-1 produces a novel electrogenic factor active on rabbit ileum in vitro.

BACKGROUND: Attaching and effacing Escherichia coli demonstrate marked species specificity in inducing diarrhea, although its mechanism remains largely unclear. The purpose of this study was to investigate the existence of a soluble, species-specific factor that induces diarrhea in an in vitro model. METHODS: Stripped rabbit ileum was mounted in Ussing chambers, and changes in potential difference and short-circuit current were monitored after the addition of bacterial culture supernatant. RESULTS: The culture supernatant from rabbit-specific strain RDEC-1, but not from human-specific enteropathogenic Escherichia coli strain E2348/69, induced an increase in potential difference and short-circuit current in rabbit ileum mounted in Ussing chambers. This electrical signal was related to chloride ion secretion, was absent in colonic tissue, and was retained in the 30 to 100-KDa fraction of the supernatant. Preliminary experiments failed to show an involvement of calcium or cyclic nucleotides as intracellular messengers. RDEC-1 cured of a 42-MDa plasmid lost the enterotoxicity whereas conjugation of the plasmid into the negative E. coli recipient HB101 resulted in the expression of toxicity. CONCLUSIONS: The authors describe a novel, species-specific factor that helps to explain RDEC-1 diarrhea, which may be relevant to the pathogenesis of enteropathogenic Escherichia coli infection.

Rumpshaker behaves like juvenile-lethal Plp mutations when combined with shiverer in double mutant mice.

The phenotypes of double mutant mice whose genomes are homozygous for an Mbp (myelin basic protein) mutation and hemizygous for a juvenile-lethal Plp (proteolipid protein) mutation were compared in earlier studies. The results suggested that the shiverer Mpb mutation might have some unexplained ability to partially rescue oligodendrocytes (OLs) from the 'death sentence' that is imposed by the Plp mutations. Conversely, they also indicated that the juvenile-lethal Plp mutations may normalize shiverer OL morphology by reducing the numbers of microprocesses. The Plp mutation rumpshaker produces a mild hypomyelination without reduction in OL numbers and a normal lifespan. This report describes double mutant mice combining two Mbp mutations with rumpshaker, utilizing a common B6C3F1 hybrid-based genetic background. Initial studies on B6C3F1 rumpshaker optic nerve and spinal cord white matter showed unanticipated signs of OL death, with morphologic criteria suggestive of an apoptotic mechanism. In shiverer*rumpshaker double mutant mice, this small class of dying cells could not be identified. White matter morphology was similar to that of mice expressing only the shiverer mutation, except that OL microprocesses were far less abundant. This evidence suggests that, despite their distinctive phenotypic differences, rumpshaker may share more characteristics with the juvenile-lethal Plp mutations than has previously been recognized.

Bacterial infections of the small intestine and colon.

Bacterial infections of the small and large intestine are widespread and continue to be topics of active research. Surveys document the importance of diarrheal disease in many settings. Major breakthroughs in the understanding of pathogenic mechanisms (especially the interactions of bacteria and intestinal cells) continue, particularly with respect to shigella, salmonella, Yersinia species, and enteropathogenic Escherichia coli. Pathogenic mechanisms of other bacteria, such as campylobacter and entero-aggregative E. coli, are not well defined. Vaccines for cholera and typhoid fever are available, and new vaccines are in various stages of development ranging from synthesis of novel constructs to large-scale field trials. Several candidate vaccines are being exploited as carriers of antigens from other pathogens. Extraintestinal complications from salmonella, shigella, campylobacter, Yersinia species, and Shiga toxin-expressing E. coli are receiving much attention. Genomic sequencing of several of these pathogens is underway. The impact of this work is hard to predict, but expectations are high.

Evidence that CNS hypomyelination does not cause death of jimpy-msd mutant mice.

Mice expressing three of the proteolipid protein (Plp) mutations in the mouse (jimpy, jimpy-msd, and jimpy-4J) all have a severe deficiency of CNS myelin and oligodendrocytes (OLs), and die sometime in their 4th postnatal week. The prevailing view has been that the animals' shortened life span and lack of myelin are causally related. Here we describe the survival of jimpy-msd males for as long as postnatal day (P) 210. Although these spontaneously occurring longer-lived jimpy-msd males show a 2- to 8-fold increase in numbers of myelinated axons in many CNS regions, this does not protect them from a later but still premature death. Investigating the cause of premature death may reveal previously undiscovered properties of the myelin genes or the cells that express them, or perhaps additional unsuspected cellular responses that contribute to the disease. This study identifies small accumulations of inflammatory cells in the brain parenchyma of jimpy-msd mice as young as P14 and as old as P60, suggesting that the pathology of the disease produced by at least this Plp mutation may be far more complex than has been previously recognized.

Quaking*shiverer double-mutant mice survive for at least 100 days with no CNS myelin.

Mice expressing mutations that produce CNS hypomyelination often die prematurely: the more severe the hypomyelination, the shorter the life span. However, we have previously described jimpy-msd mice that survive twice as long as usual; although they acquire significantly increased amounts of myelin, they still succumb long before their unaffected littermates. This result contradicts any postulated causal relationship between extent of CNS hypomyelination and premature death of the animal. Here we have addressed this question in another way, by using an animal model that does not involve a proteolipid protein (Plp) gene mutation. We demonstrate that quaking*shiverer double-mutant mice can survive for at least 100 days without any CNS myelin whatsoever. Therefore, at least for a mouse, absence of CNS myelin is not lethal per se.

Occurrence, distribution, and associations of O and H serogroups, colonization factor antigens, and toxins of enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of infectious diarrhea worldwide. Four categories of antigens have been commonly studied: O serogroup, H serogroup, colonization factor antigens (CFA), and toxins. A database has been complied from published reports of nearly 1,000 ETEC isolates from 18 locations and analyzed to determine the occurrence, distribution, and associations of O serogroup, H serogroup, CFA, and toxin type. Tables listing the associations of antigens are presented. This analysis documents the widespread nature and variety of ETEC. Even the most common combination of antigens, O6:H16 CFA/II LTST, accounted for only 11% of the ETEC isolates in the database. It was isolated from 12 locations. Many phenotypes occurred only once. CFA detection based on enzyme-linked antibodies with polyclonal sera is suggested as the preferred assay. A combination of CFA and toxin-based antigens is suggested as the most practical vaccine.

Continuous-infusion 5-fluorouracil and cisplatin for advanced/recurrent transitional cell cancer of the bladder: a Southwest Oncology Group trial.

Significant toxicities result from the use of MVAC (methotrexate, vinblastine, adriamycin, cisplatin) for advanced/ recurrent transitional cell carcinoma of the bladder (ARTCCB). An alternative regimen of 5-fluorouracil (5-FU) and cisplatin was evaluated by Southwest Oncology Group (SWOG). Thirty-eight patients with ARTCCB were treated with continuous infusion 5-FU 1,000 mg/m2/days 1-5 and cisplatin 100 mg/day 1, on a every-21-days schedule. There were two complete responses (CR) and eight partial responses (PR) among 36 eligible patients, for an overall response rate of 28% [95% confidence interval (CI) 14-45%]. Median duration of response was 6 months, and median duration of survival was 9 months. No toxic deaths occurred. Grade 4 leukopenia occurred in 5 patients. Other toxicities were mild. Only two documented infections occurred in 5 patients with neutropenia. The response rate of 28% is better than that achieved with cisplatin alone and not dissimilar to the range of response for MVAC. Toxicities were less and tolerable. This regimen will need further evaluation.

The CS6 colonization factor of human enterotoxigenic Escherichia coli contains two heterologous major subunits.

The genes encoding the CS6 colonization factor were cloned from two human enterotoxigenic Escherichia coli strains of different serotypes. The DNA sequences from both clones were nearly identical and contained four open reading frames. Two of them have homology to genes encoding molecular chaperones and ushers found in many other operons encoding colonization factors. The two remaining open reading frames encode two heterologous major subunit proteins which makes CS6 unique because other colonization factors have only one major subunit. Upstream and downstream of the CS6 operon the DNA sequences of the clones diverged abruptly.

Jimpy-4J mouse has a missense mutation in exon 2 of the Plp gene.

We previously showed that the jimpy-4J mouse mutation is located on the X chromosome, in or closely linked to the proteolipid protein (Plp) gene. The phenotype is characterized by the most severe hypomyelination of any of the naturally occurring myelin mutant mice, sharp reduction in oligodendrocyte number, and virtual absence of PLP protein. Affected animals show tremor, seizures, and die at about 24 postnatal days. We now report that sequencing of Plp genomic and cDNAs identifies a single nucleotide substitution in exon 2 that predicts an Ala38Ser substitutions in a hydrophilic region of PLP/DM20 protein close to a transmembrane domain. This mutation occurs in a very different region of the mouse Plp gene than that jimpy-msd mutations, yet all three produce qualitatively similar phenotypes.

Identification of a family of intimins common to Escherichia coli causing attaching-effacing lesions in rabbits, humans, and swine.

Intimin, an outer membrane protein encoded by eaeA that mediates close attachment of enteropathogenic bacteria to apical surfaces of epithelial cells, is required for formation of the attaching-effacing lesions and for full pathogenesis of the bacteria. Analysis of the eaeA sequence indicates that there is a high degree of homology at the N termini but less at the C termini of intimins. Antisera specific for the C-terminal third of RDEC-1 intimin, used to screen outer membrane proteins from 50 rabbit enteropathogenic Escherichia coli (EPEC), human EPEC, and human enterohemorrhagic E. coli (EHEC) strains, identified cross-reactive intimins from 24 isolates. Sequence analysis of the eaeA genes from human EPEC O111 and EHEC O26 isolates indicates that their intimins have C termini nearly identical to that of RDEC-1 intimin. Our results suggest that there are at least three families of related intimins and that the presence of intimin similar to that of RDEC-1 is not restricted by serogroup or host specificity.

Regulation of cytoskeleton by myelin components: studies on shiverer oligodendrocytes carrying an Mbp transgene.

Oligodendrocytes from the shiverer mutant mouse are missing most of the myelin basic protein (Mbp) gene. In axon-free cultures, they produce membrane sheets with abnormally assembled microtubule and actin-based structures. This suggests that an Mbp gene product may have an important role in regulating the organization and stability of the wild-type oligodendrocyte cytoskeleton. We now present evidence extending these observations, using cultured oligodendrocytes that carry both the shiverer mutation and the Mbp1 transgene which partially corrects their deficit. Shiverer oligodendrocytes that carry one dose of the Mbp1 transgene abnormally express MBP along major cytoskeletal vein-like structures in processes and sheets. Shiverer oligodendrocytes that carry two doses of the Mbp1 transgene contain two types of membrane sheet regions, i.e. regions filled with aberrant punctate foci of MBP, and regions with normal domains of MBP. Immunocytochemical staining data show that the distribution of cytoskeleton and associated 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) is dependent upon how MBP is organized. Bundling of actin filaments occurs only around MBP domains, and the colocalization of CNPase along microtubular structures also appears to be regulated by MBP domains in sheets. Multinucleated oligodendrocytes are observed, a likely result of the inability of dividing pro-oligodendrocytes to bundle actin filaments. In addition, the ability of MBP to mediate extracellular signals that modulate cytoskeleton appears to be dependent upon MBP's organization. Transduction of the galactocerebroside signaling pathway, which results in the destabilization of microtubules but not actin filaments, occurs only in sheets containing MBP domains. The distribution of MBP, however, does not affect the myelin/oligodendrocyte-specific protein signaling pathway, which results in growth of microtubular structures and extensive destabilization of the actin cytoskeleton.

Characterization of the eaeA gene from rabbit enteropathogenic Escherichia coli strain RDEC-1 and comparison to other eaeA genes from bacteria that cause attaching-effacing lesions.

A number of enteric pathogens, including enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli, Hafnia alvei, a strain of Citrobacter freundii, and rabbit EPEC strain RDEC-1 cause attaching-effacing (AE) lesions in the gut mucosa. These bacteria have a pathogenicity cassette (locus of enterocyte effacement or LEE) containing the eaeA gene. This gene encodes intimin, an outer membrane protein required for production of AE lesions. RDEC-1, a non-invasive enteropathogen in young rabbits, produces AE lesions morphologically indistinguishable from lesions caused by human AE bacterial strains. The RDEC-1 example of E. coli diarrhea in rabbits is an important model for studying the pathogenesis of AE bacteria in a natural infection and for analyzing specific roles of the components of LEE. In order to better understand the role of intimin in the development of AE lesions, a portion of DNA within RDEC-1 LEE, containing the eaeA gene and an upstream open reading frame (ORF), was sequenced. The RDEC-1 eaeA gene shared 87%, 92%, and 93% DNA sequence identity and > 80% amino acid sequence identity with the eaeA genes of C. freundii biotype 4280, EHEC O157:H7, and EPEC O127:116, respectively. The carboxy-terminal 280 amino acid residues of intimin has 80%, 56%, and 54% identity with C. freundii, EHEC O157:H7, and EPEC O127:H6 intimins, respectively. The predicted protein encoded by the upstream ORF (156 amino acids) shares 95%, 97%, and 99% amino acid identity with predicted proteins from C. freundii. EHEC O157:H7, and EPEC O127:H6, respectively. The high degree of sequence homology of the ORF and the eueA gene of RDEC-1 with those of other AE bacteria suggests an evolutionary relationship of LEE and supports and facilitates the use of the RDEC-1 model for studying the role of LEE in pathogenesis.

Phase II evaluation of ifosfamide/mesna in metastatic prostate cancer. A Southwest Oncology Group study.

The combination of ifosfamide and mesna was evaluated in a phase II trial in the treatment of metastatic prostate cancer. Two separate groups of patients were to be evaluated: patients with no prior hormonal therapy and hormonally refractory patients. Patients were treated with ifosfamide 1.5 g/M2, and mesna at 30% of the ifosfamide dose was administered immediately before and 4 and 8 h after ifosfamide treatment. Both drugs were given i.v. daily for 5 days every 21 days. Response was assessed every 6 weeks. Of 29 eligible and evaluable patients with hormonally refractory disease, there were two partial responders for a response rate of 7% (95% confidence interval, of 0.1-23%). Of nine eligible patients with no prior hormone treatment, there was one partial response, for a response rate of 11% (95% confidence interval, 0.3-48%). Unfortunately, the target accrual goal for this arm of the study was never achieved. The most common toxicities were myelosuppression and neurologic toxicity. These drugs do not warrant further evaluation in the disease.

Phase II trial of CHIP for the treatment of advanced, hormonally refractory carcinoma of the prostate. A Southwest Oncology Group Study.

CHIP, a second generation analogue of cisplatin, was subjected to a Phase II trial for the treatment of advanced, hormonally refractory carcinoma of the prostate. Forty-six patients were treated with CHIP 300 mg/m2 intravenously every 4 weeks. Evaluations for tumor response were done after three cycles of therapy. Among 40 evaluable patients there were no complete responses, but there were 6 partial responses for an overall response rate of 15% (95% confidence interval of 5.7 to 29.8%). The median time to response was 2.4 months and the median progression-free survival was 4.1 months. Median survival was 8.4 months. The most common toxicities were hematologic and gastrointestinal. While CHIP can be administered on an outpatient basis, its response rate for prostatic carcinoma appears to be similar to that of cisplatin.