Characterization of Haemophilus ducreyi cdtA, cdtB, and cdtC mutants in in vitro and in vivo systems.

Haemophilus ducreyi expresses a soluble cytolethal distending toxin (CDT) that is encoded by the cdtABC gene cluster and can be detected in culture supernatant fluid by its ability to kill HeLa cells. The cdtA, cdtB, and cdtC genes of H. ducreyi were cloned independently into plasmid vectors, and their encoded proteins expressed singly or in various combinations in an Escherichia coli background. All three gene products had to be expressed in order for E. coli-derived culture supernatant fluids to demonstrate cytotoxicity for HeLa cells. Isogenic H. ducreyi cdtA and cdtB mutants were constructed and used in combination with the wild-type parent strain and a previously described H. ducreyi cdtC mutant (M. K. Stevens, J. L. Latimer, S. R. Lumbley, C. K. Ward, L. D. Cope, T. Lagergard, and E. J. Hansen, Infect. Immun. 67:3900-3908, 1999) to determine the relative contributions of the CdtA, CdtB, and CdtC proteins to CDT activity. Expression of CdtA, CdtB, and CdtC appeared necessary for H. ducreyi-derived culture supernatant fluid to exhibit cytotoxicity for HeLa cells. Whole-cell sonicates and periplasmic extracts from the cdtB and cdtC mutants had no effect on HeLa cells, whereas these same fractions from a cdtA mutant had a very modest cytotoxic effect on these same human cells. CdtA appeared to be primarily associated with the H. ducreyi cell envelope, whereas both CdtB and CdtC were present primarily in the soluble fraction from sonicated cells. Both the cdtA mutant and the cdtB mutant were found to be fully virulent in the temperature-dependent rabbit model for experimental chancroid.

Isolated large third-trimester intracranial cyst on fetal ultrasound: fact or fiction?

OBJECTIVE: To distinguish the fact from artifact of an isolated, large, intracranial cyst on prenatal sonography (PSG). BACKGROUND: The use of PSG is rapidly increasing with most obstetric ultrasounds occurring in general community settings like small hospitals and clinics with personnel who have variable training, experience, and interest levels. In contrast, most PSG articles and books are produced in large subspecialty centers with concentrated referral bases plus both highly-trained and experienced personnel. DESIGN/METHODS: We report a series of 2 normal newborn patients who had a large prenatal unilateral intracranial cyst diagnosed by PSG in the 10 years between July of 1989 and 1999 at a rural community hospital. The newborns had imaging studies at birth and their neurodevelopmental progress was followed for several years. Textbook, bibliography and computerized Medline (1966-present) searches including prenatal ultrasound, observer variation, diagnostic errors, reproducibility of results, sensitivity and specificity, accuracy, central nervous system, false-positive, prenatal diagnosis, and brain were examined starting in August 1996 for reports. RESULTS: There were 4079 obstetric ultrasounds performed in 3.5 years, January 1996 through July 1999 at this rural community facility. This rate extrapolates to a total of 11 654 obstetric ultrasounds over the 10-year study period in which the 2 cases of intracranial cyst artifact occurred. Thus, the incidence of 2 intracranial cyst artifacts was estimated as 2/11 654 PSG, a .0002% false-positive rate. CONCLUSIONS: This is the first report of the occurrence of PSG artifacts in a community facility. Artifact is a real problem and needs to be specified in differential diagnoses. There are ways to decrease sonographic artifact-or at least to recognize it-so our estimates at a community hospital for its occurrence are presented with the relevant technical and ethical issues. None of these issues have been previously reported in the pediatric literature. Our false-positive rate for large intracranial cyst compares favorably with other reports. Our estimate may inflate our denominator by reporting scans rather than the number of fetuses scanned, and our numerator may miss cases that moved from the community. Confusion differentiating PSG artifact from reality often occurs when interpreting static or frozen real-time images. The signs that sonogram images may be artifacts include defects that: extend outside the fetal body; change shape, size and echogenecity with different scan planes; are not seen on all examinations; and are isolated in an otherwise normal fetus. Failure to offer quality PSG in clinical settings where it is available restricts access of pregnant women to the diagnosis of fetal anomalies, and therefore restricts access to the options of pregnancy termination, fetal therapy like fetal surgery, and delivery options of timing, setting, and mode. We suggest a multidisciplinary approach to prenatal abnormalities like isolated third trimester unilateral intracranial cyst in both primary and tertiary care settings aids interpretation followed by expectant conservative management without elaborate, risky, or terminal interventions.

Characterization of a Haemophilus ducreyi mutant deficient in expression of cytolethal distending toxin.

Haemophilus ducreyi expresses a soluble cytolethal distending toxin (CDT) that kills HeLa, HEp-2, and other human epithelial cells in vitro. H. ducreyi CDT activity is encoded by a three-gene cluster (cdtABC), and antibody to the cdtC gene product can neutralize CDT activity in vitro (L. D. Cope, S. R. Lumbley, J. L. Latimer, J. Klesney-Tait, M. K. Stevens, L. S. Johnson, M. Purven, R. S. Munson, Jr., T. Lagergard, J. D. Radolf, and E. J. Hansen, Proc. Natl. Acad. Sci. USA 94:4056-4061, 1997). Culture supernatant fluid from a recombinant Escherichia coli strain containing the H. ducreyi cdtABC gene cluster readily killed both HeLa cells and HaCaT keratinocytes and had a modest inhibitory effect on the growth of human foreskin fibroblasts. Insertional inactivation of the cdtC gene in this recombinant E. coli strain eliminated the ability of this strain to kill HeLa cells and HaCaT keratinocytes. This mutated H. ducreyi cdtABC gene cluster was used to construct an isogenic H. ducreyi cdtC mutant. Monoclonal antibodies against the H. ducreyi CdtA, CdtB, and CdtC proteins were used to characterize protein expression by this cdtC mutant. Culture supernatant fluid from this H. ducreyi cdtC mutant did not detectably affect any of the human cells used in this study. The presence of the wild-type H. ducreyi cdtC gene in trans in this H. ducreyi mutant restored its ability to express a CDT that killed both HeLa cells and HaCaT keratinocytes. The isogenic H. ducreyi cdtC mutant was shown to be as virulent as its wild-type parent strain in the temperature-dependent rabbit model for experimental chancroid. Lack of expression of the H. ducreyi CdtC protein also did not affect the ability of this H. ducreyi mutant to survive in the skin of rabbits.

Involvement of the Haemophilus ducreyi gmhA gene product in lipooligosaccharide expression and virulence.

The lipooligosaccharide (LOS) present in the outer membrane of Haemophilus ducreyi is likely a virulence factor for this sexually transmitted pathogen. An open reading frame in H. ducreyi 35000 was found to encode a predicted protein that had 87% identity with the protein product of the gmhA (isn) gene of Haemophilus influenzae. In H. influenzae type b, inactivation of the gmhA gene caused the synthesis of a significantly truncated LOS which possessed only lipid A and a single 2-keto-3-deoxyoctulosonic acid molecule (A. Preston, D. J. Maskell, A. Johnson, and E. R. Moxon, J. Bacteriol. 178:396-402, 1996). The H. ducreyi gmhA gene was able to complement a gmhA-deficient Escherichia coli strain, a result which confirmed the identity of this gene. When the gmhA gene of H. ducreyi was inactivated by insertion of a cat cartridge, the resultant H. ducreyi gmhA mutant, 35000.252, expressed a LOS that migrated much faster than wild-type LOS in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When the wild-type H. ducreyi strain and its isogenic gmhA mutant were used in the temperature-dependent rabbit model for dermal lesion production by H. ducreyi, the gmhA mutant was found to be substantially less virulent than the wild-type parent strain. The H. ducreyi gmhA gene was amplified by PCR from the H. ducreyi chromosome and cloned into the pLS88 vector. When the H. ducreyi gmhA gene was present in trans in gmhA mutant 35000.252, expression of the gmhA gene product restored the virulence of this mutant to wild-type levels. These results indicate that the gmhA gene product of H. ducreyi is essential for the expression of wild-type LOS by this pathogen.

A protective epitope of Moraxella catarrhalis is encoded by two different genes.

The high-molecular-weight UspA protein of Moraxella catarrhalis has been described as being both present on the surface of all M. catarrhalis disease isolates examined to date and a target for a monoclonal antibody (MAb 17C7) which enhanced pulmonary clearance of this organism in a mouse model system (M. E. Helminen et al., J. Infect. Dis. 170:867-872, 1994). A recombinant bacteriophage that formed plaques which bound MAb 17C7 was shown to contain a M. catarrhalis gene, designated uspA1, that encoded a protein with a calculated molecular weight of 88,271. Characterization of an isogenic uspA1 mutant revealed that elimination of expression of UspA1 did not eliminate the reactivity of M. catarrhalis with MAb 17C7. In addition, N-terminal amino acid analysis of internal peptides derived from native UspA protein and Southern blot analysis of M. catarrhalis chromosomal DNA suggested the existence of a second UspA-like protein. A combination of epitope mapping and ligation-based PCR methods identified a second M. catarrhalis gene, designated uspA2, which also encoded the MAb 17C7-reactive epitope. The UspA2 protein had a calculated molecular weight of 62,483. Both the isogenic uspA1 mutant and an isogenic uspA2 mutant possessed the ability to express a very-high-molecular-weight antigen that bound MAb 17C7. Southern blot analysis indicated that disease isolates of M. catarrhalis likely possess both uspA1 and uspA2 genes. Both UspA1 and UspA2 most closely resembled adhesins produced by other bacterial pathogens.

A diffusible cytotoxin of Haemophilus ducreyi.

Little is known about the virulence mechanisms employed by Haemophilus ducreyi in the production of genital ulcers. This Gram-negative bacterium previously has been shown to produce a soluble cytotoxic activity that kills HeLa and HEp-2 cells. We have now identified a cluster of three H. ducreyi genes that encode this cytotoxic activity. The predicted proteins encoded by these genes are most similar to the products of the Escherichia coli cdtABC genes that comprise the cytolethal distending toxin (CDT) of this enteric pathogen. Eleven of 12 H. ducreyi strains were shown to possess this gene cluster and culture supernatants from these strains readily killed HeLa cells. The culture supernatant from a single strain of H. ducreyi that lacked these genes was unable to kill HeLa cells. When the H. ducreyi cdtABC gene cluster was cloned into E. coli, culture supernatant from the recombinant E. coli clone killed HeLa cells. A monoclonal antibody that neutralized this soluble cytotoxic activity of H. ducreyi was shown to bind to the H. ducreyi cdtC gene product. This soluble H. ducreyi cytotoxin may play a role in the development or persistence of the ulcerative lesions characteristic of chancroid.

Identification of tandem genes involved in lipooligosaccharide expression by Haemophilus ducreyi.

A transposon insertion mutant of Haemophilus ducreyi 35000 possessing a truncated lipooligosaccharide (LOS) failed to bind the LOS-specific monoclonal antibody 3E6 (M. K. Stevens, L. D. Cope, J. D. Radolf, and E. J. Hansen, Infect. Immun. 63:2976-2982, 1995). This transposon was found to have inserted into the first of two tandem genes and also caused a deletion of chromosomal DNA upstream of this gene. These two genes, designated lbgA and lbgB, encoded predicted proteins with molecular masses of 25,788 and 40,236 Da which showed homology with proteins which function in lipopolysaccharide biosynthetic in other gram-negative bacteria. The tandem arrangement of the lbgA and lbgB genes was found to be conserved among H. ducreyi strains. Isogenic LOS mutants, constructed by the insertion of a cat cartridge into either the lbgA or the lbgB gene, expressed an LOS phenotype indistinguishable from that of the original transposon-derived LOS mutant. The wild-type LOS phenotype could be restored by complementation with the appropriate wild-type allele. These two LOS mutants proved to be as virulent as the wild-type parent strain in an animal model. A double mutant with a deletion of the lbgA and lbgB genes yielded equivocal results when its virulence was tested in an animal model.

Cloning and sequencing of the gene encoding the Cu,Zn-superoxide dismutase of Haemophilus ducreyi.

The sodC gene of Haemophilus ducreyi was cloned and sequenced. The deduced amino acid sequence of this protein exhibited 71.6% identity and 81.8% similarity to the H. influenzae and H. parainfluenzae copper (Cu), zinc (Zn)-superoxide dismutase (SOD) enzymes. This gene was localized to a 2.2-kb H. ducreyi chromosomal DNA insert in plasmid pHdSOD. SOD activity was expressed in cell-free extracts of Escherichia coli containing the recombinant plasmid pHdSOD and was localized to the periplasmic space. The Cu,Zn-SOD produced by the H. ducreyi sodC gene did not complement the aerobic growth defect of an E. coli SOD-deficient mutant.

A hemoglobin-binding outer membrane protein is involved in virulence expression by Haemophilus ducreyi in an animal model.

Haemophilus ducreyi exhibits a requirement for exogenously supplied heme for aerobic growth in vitro. Nine of ten wild-type isolates of H. ducreyi were shown to contain a readily detectable hemoglobin-binding activity. Spontaneous hemoglobin-binding-negative mutants of two of these wild-type isolates lost the ability to express an outer membrane protein with an apparent molecular mass of approximately 100 kDa. Similarly, the single wild-type isolate that lacked the ability to bind hemoglobin also appeared to lack expression of this same 100-kDa protein. A monoclonal antibody (5A9) to this 100-kDa protein was used to identify a recombinant clone which possessed an H. ducreyi chromosomal fragment containing the gene encoding the 100-kDa protein; this protein was designated hemoglobin utilization protein A (HupA). Nucleotide sequence analysis of the hupA gene revealed that the predicted protein, with a calculated molecular mass of 108 kDa, was similar to TonB-dependent outer membrane proteins of other bacteria. Increasing the concentration of heme in the growth medium resulted in decreased expression of the HupA protein. Mutant analysis was used to prove that the HupA protein was essential for the utilization by H. ducreyi of both hemoglobin and hemoglobin-haptoglobin as sources of heme in vitro. In addition, it was found that an isogenic hupA mutant was less virulent than the wild-type parent strain in the temperature-dependent rabbit model for dermal lesion production by H. ducreyi.

A system for generalized mutagenesis of Haemophilus ducreyi.

The lack of a generalized mutagenesis system for Haemophilus ducreyi has hampered efforts to identify virulence factors expressed by this sexually transmitted pathogen. To address this issue, the transposable element Tn1545-delta 3, which encodes resistance to kanamycin, was evaluated for its ability to insert randomly into the H. ducreyi chromosome and produce stable, isogenic mutants. Electroporation of H. ducreyi with 1 microgram of plasmid pMS1 carrying Tn1545-delta 3 resulted in the production of 10(4) kanamycin-resistant transformants; Southern blot analysis of a number of these transformants indicated that insertion of the transposon into the chromosome occurred at a number of different sites. This pMS1-based transposon delivery system was used to produce an H. ducreyi mutant that expressed an altered lipooligosaccharide (LOS). Passage of this mutant in vitro in the presence or absence of kanamycin did not affect the stability of the transposon insertion. To confirm that the observed mutant phenotype was the result of the transposon insertion, a chromosomal fragment containing Tn1545-delta 3 was cloned from this H. ducreyi LOS mutant. Electroporation of the wild-type H. ducreyi strain with this DNA fragment yielded numerous kanamycin-resistant transformants, the majority of which had the same altered LOS phenotype as the original mutant. Southern blot analysis confirmed the occurrence of proper allelic exchange in the LOS-deficient transformants obtained in this backcross experiment. The ability of Tn1545-delta 3 to produce insertion mutations in H. ducreyi should facilitate genetic analysis of this pathogen.

Use of tissue culture and animal models to identify virulence-associated traits of Haemophilus ducreyi.

To identify virulence-associated properties of Haemophilus ducreyi, 34 strains of this sexually transmitted pathogen were evaluated for in vitro phenotypic characteristics of potential relevance to chancroid pathogenesis and for their ability to produce lesions in the temperature-dependent animal model for chancroid. Of the 34 strains tested, all but three produced a cytopathic effect on human foreskin fibroblasts (HFF) and all but six strains formed large microcolonies on HFF monolayers. A subset of 12 selected strains underwent more extensive analyses and, when evaluated for both their cytadherence kinetics and growth in the presence of HFF monolayers, it was found that several of these strains had a very limited ability to attach to HFF cells. When the same 12 strains were tested in the temperature-dependent rabbit model, only the seven strains which were positive in all of these in vitro-based tests readily produced lesions. In contrast, the five strains that were noted to be deficient in one or more of the phenotypic characteristics scored in the in vitro systems did not produce lesions. This association between the traits measured in vitro and the ability to produce dermal lesions was significant (P = 0.0012). These results suggest that in vitro behavior may be used to predict the virulence potential of H. ducreyi strains. Moreover, the phenotypic characteristics described in this study are appropriate focal points for efforts to determine the molecular basis of the virulence of this pathogen.

Induction of protective immunity to Haemophilus ducreyi in the temperature-dependent rabbit model of experimental chancroid.

The temperature-dependent rabbit model for chancroid, a sexually transmitted disease caused by the fastidious Gram-negative bacterium Haemophilus ducreyi, was used to investigate the abilities of previous infection and immunization with an acellular preparation of H. ducreyi to induce protective immunity. In the first set of experiments, animals were infected intradermally with either the 35000 or Cha-1 strains of H. ducreyi and then rechallenged 30 days later with both the homologous and heterologous strains. In animals infected with the 35000 strain, statistically significant protective immunity occurred only against the homologous strain, whereas protection against both homologous and heterologous challenge was obtained in rabbits previously infected with strain Cha-1. In a separate series of experiments, rabbits were immunized with cell envelopes from either strain 35000 or strain Cha-1 and then challenged with both the homologous and heterologous strains. In rabbits immunized with strain 35000 cell envelopes, significant protective immunity was observed only against challenge with the homologous strain. In animals immunized with strain Cha-1 cell envelopes, protection was obtained against both homologous and heterologous challenge. Histopathologic analysis of sites inoculated with strain 35000 (10(5) CFU) demonstrated that the inflammatory response in control animals was predominantly suppurative (i.e., heterophilic), whereas that of immunized animals was predominantly mononuclear and, at later time points, largely histiocytic. ELISA and Western blot analyses revealed that immunization produced a better humoral immune response than did infection and provided evidence for antigenic cross-reactivity between these two strains. These results provide the experimental basis for continued efforts to identify potential H. ducreyi vaccinogens.

Mycoplasma pneumoniae cytadherence phase-variable protein HMW3 is a component of the attachment organelle.

The subcellular location of the phase-variable cytadherence-accessory protein HMW3 in Mycoplasma pneumoniae has been examined by biochemical and immunoelectron microscopic techniques. Analysis by Western blot (immunoblot) with HMW3-specific antiserum established the presence of this protein within the M. pneumoniae Triton X-100-insoluble fraction or triton shell. Immunogold labeling of Triton-extracted mycoplasmas with affinity-purified antibodies localized HMW3 to the terminal knob on the rodlike extensions of the triton shell, a location that would correspond to the adherence organelle in whole mycoplasmas. Treatment of triton shells with KI resulted in the selective removal of the adherence-accessory proteins HMW1 to HMW4. Analysis of these triton shells by transmission electron microscopy revealed dramatic ultrastructural changes in the filamentous network and core structure. Immunogold labeling of KI-extracted shells reflected the removal of HMW3 from the disrupted tip structure. An examination of ultrathin sections of wild-type cells by transmission electron microscopy following labeling with HMW3-specific antibodies provided further evidence for the nonrandom distribution of HMW3 and its localization to the terminal portion of filamentous cell extensions. Most colloidal gold molecules were associated with the cell interior, but limited peripheral labeling of the terminal region was also observed. Postfixation antibody labeling of whole cells suggested limited exposure of HMW3 on the mycoplasma surface at the tip structure. However, prefixation antibody labeling failed to indicate surface exposure, raising some uncertainty regarding the relationship of HMW3 with the mycoplasma membrane.

Localization of the Mycoplasma pneumoniae cytadherence-accessory proteins HMW1 and HMW4 in the cytoskeletonlike Triton shell.

The location of the cytadherence-accessory high-molecular weight proteins 1 and 4 (HMW1/4) within Mycoplasma pneumoniae cells has been studied by both biochemical and electron microscopic techniques. Peptide mapping studies demonstrated that HMW1/4 share almost identical peptide profiles, suggesting that the two proteins are structurally related. Examination of thin sections of M. pneumoniae with antibodies to HMW1/4 and colloidal gold particles revealed distinct labeling of the filamentous extensions of the mycoplasma cells. Labeling was absent on thin sections of a cytadherence-deficient variant lacking HMW1/4. HMW1/4 partitioned in the detergent-insoluble fraction following Triton X-100 extraction, and analysis by sucrose density gradient centrifugation suggested that HMW1/4 are part of a high-molecular-weight, multiprotein complex. These results were confirmed by immunogold labeling of Triton X-100-extracted M. pneumoniae cells incubated with antibodies to HMW1/4: gold particles bound in specific clusters to detergent-insoluble filaments. Finally, immunogold labeling of whole cells revealed that HMW1/4 are exposed on the cell surface, although to a lesser degree than on the cell interior. These findings indicate that HMW1/4 are membrane proteins associated with the cytoskeletonlike triton shell of M. pneumoniae and localized primarily in the filamentous extensions of the mycoplasma cells.

Disulfide-linked protein associated with Mycoplasma pneumoniae cytadherence phase variation.

Wild-type Mycoplasma pneumoniae possessed a protein with a very high molecular weight under nonreducing conditions (greater than 340,000; designated HMW5); this protein was absent from a noncytadhering phase variant lacking HMW1, 2, 3, and 4. When examined by two-dimensional nonreducing-reducing gel electrophoresis, HMW5 dissociated to yield a single polypeptide spot of molecular weight 190,000 that comigrated with cytadherence phase-variable protein HMW2. Extraction of wild-type mycoplasmas with Triton X-100 revealed the exclusive partitioning of HMW5 with the detergent-insoluble cytoskeletonlike triton shell.

Systematic studies on the effects of the NMDA receptor antagonist MK-801 on cerebral blood flow and responsivity, EEG, and blood-brain barrier following complete reversible cerebral ischemia.

The dose-dependent effects of MK-801, a glutamate receptor antagonist, on changes in CBF, CBF-PaCO2 responsiveness (133Xe clearance), EEG, and blood-brain barrier (methylene blue) were examined after a 15-min period of reversible complete global ischemia induced in halothane-anesthetized cats by occlusion of the vertebral and carotid arteries. Pretreatment with doses of MK-801 of greater than or equal to 0.5 mg/kg had no effect on resting CBF measures and produced a dose-dependent slowing of the dominant EEG frequency. In animals receiving this agent, there was an almost immediate return of baseline EEG patterns upon reinstitution of flow, no hypoperfusion after 2 h of reflow, preservation of CBF and CBF-PaCO2 responsiveness, and maintenance of blood-brain barrier integrity. In contrast, parallel control animals and animals treated with MK-801 at a dose of 0.1 mg/kg exhibited poor recovery based on the above parameters. MK-801 also diminished in a dose-dependent manner the CSF levels of 6-keto-prostaglandin (PG) F1 alpha (stable metabolite of PGI2) and thromboxane (Tx) B2 (stable metabolite of TxA2), which were otherwise elevated in vehicle-treated animals 2 h after reflow. Of particular interest, the CSF TxB2/6-keto-PGF1 alpha ratio in vehicle-treated animals was near 2. In animals pretreated with MK-801, at doses of greater than or equal to 0.5 mg/kg, this ratio was nearly 1. These observations are consistent with a possible triggering role of glutamate release in initiating at least part of the acute sequelae of ischemia. Such release in an electrically silent cell would increase Ca2+ influx and activate free fatty acid metabolism, leading to probable changes in vascular function and changes in blood-brain barrier permeability.

Time course of release in vivo of PGE2, PGF2 alpha, 6-keto-PGF1 alpha, and TxB2 into the brain extracellular space after 15 min of complete global ischemia in the presence and absence of cyclooxygenase inhibition.

The time-dependent release of prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2 alpha), thromboxane (Tx) B2, and 6-keto-PGF1 alpha (6-keto) from brain was measured before, during, and after a 15-min interval of total ischemia (four-vessel occlusion) in halothane-anesthetized cats using the technique of cerebroventricular perfusion. Resting levels of PGE2, PGF2 alpha, 6-keto, and Tx were: 253 +/- 75, 953 +/- 300, 650 +/- 200, and 550 +/- 170 pg/ml, respectively. During the 15-min ischemia, all prostanoids rose significantly, yet the highest levels were not observed until the first 15-60 min of the reflow at which time levels of PGE2, PGF2 alpha, 6-keto, and Tx, as compared with the preischemic baseline, rose approximately 8, 3.4, 3, and 55-fold, respectively. Significantly, although all prostanoids showed increases relative to baseline, the ratios of PGF2 alpha/6-keto and PGE2/6-keto remained stable throughout the experiment in both groups of animals. In contrast, the Tx/6-keto ratio rose from approximately 1 to approximately 30 during the 60 min after reflow in untreated cats. Treatment with zomepirac sodium (5 mg/kg, i.v.), a cyclooxygenase inhibitor, resulted in highly significant reductions in the levels of all prostanoids during the preischemic period. In zomepirac sodium-treated animals, there were also highly significant reductions in the prostanoid response to ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of preischemia cyclooxygenase inhibition by zomepirac sodium on reflow, cerebral autoregulation, and EEG recovery in the cat after global ischemia.

Zomepirac sodium (ZS) (5 mg/kg i.v.) was used to evaluate the effects of preischemia cyclooxygenase inhibition on CBF (as assessed by 133Xe clearance), CBF-PaCO2 responsiveness, and electrophysiologic (EEG) parameters before and after a 15-min period of complete global ischemia produced by four-vessel occlusion and mild hypotension. During the 15-min period of ischemia, CBF was essentially zero. Following reflow all groups displayed an initial hyperemia as compared with control (92 +/- 11 vs. 141-146 ml/100 g/min). Saline-treated animals during reflow displayed a delayed hypoperfusion (26 +/- 3 ml/100 g/min), which showed no improvement during the 2-h reflow period prior to death. In contrast, ZS-treated animals during reflow displayed significantly higher flows during the hypoperfusion phase (72 +/- 9 ml/100 g/min). The CBF-PaCO2 response displayed an approximately sevenfold reduction in slope at 2 h after reflow in saline-treated animals. This decrease in PaCO2 reactivity was not observed in the ZS-pretreated animals. With regard to EEG, all animals showed a total flattening during the 15 min of ischemia. In saline-treated animals only one of seven showed any sign of even marginal recovery. In ZS-treated animals EEG activity showed prominent recovery in seven of seven. Brainstem auditory evoked potentials were monitored and showed prominent recovery of amplitude and latency in ZS but not saline-treated animals during reflow.