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Water skiing is associated with severe injuries to the proximal hamstring muscles. We wanted to define the mechanism of injury, describe the associated pathologic changes, determine the functional limitations of patients, and suggest measures to prevent injury. Twelve patients with water skiing-related hamstring injuries were included. Six patients were experienced skiers and six were novices. The mechanism of injury was identical in five of six novice skiers. Each sustained the injury while attempting to get up on one or two skis from a submerged position. In contrast, the expert skiers all sustained injury secondary to a fall while skiing. Physical examination documented evidence of complete or partial avulsion of the proximal hamstring muscle origins in all patients. In addition, six patients had magnetic resonance imaging or computed tomography scans that confirmed the location and extent of the tear. Convalescence ranged from 3 months to 1.5 years before the patient could return to vigorous activities. Seven patients (58%) returned to most of their preinjury sports, albeit at a lower level. Five patients (42%), all with complete disruptions, were unable to run or participate in sports requiring agility. Two of these patients required delayed surgical repairs because of persistent functional limitations. [ABSTRACT FROM AUTHOR]

An international sampling study yielded 69 samples of extended-release prescription pharmaceuticals for legal sale in the U.S. Samples included 29 lots of innovator and 40 lots of generic solid oral extended-release drugs manufactured at 16 different facilities and containing 6 different active ingredients. Dosage unit uniformity and dissolution were tested for each lot. All samples met the relevant testing criteria for dosage unit uniformity and dissolution. There were no indications that manufacturer or region impacted a product's acceptability for use by patients. The variability of attributes was used to calculate a process performance index (Ppk) for each facility. Higher Ppk values suggest less variability relative to specification limits. Only two manufacturers fell below a 4-sigma manufacturing benchmark Ppk of 1.33 for dosage unit uniformity: a European manufacturer of a brand drug and an Asian manufacturer of a generic drug. Conversely, all but four manufacturers fell below a 4-sigma benchmark for the minimum Ppk across their product's dissolution timepoints: generic drug manufacturers in India (two), the U.S., and Canada. Compared to the immediate-release products of a previous study, Ppks were generally lower for extended-release products. A retrospective analysis found that manufacturers performing below median Ppks submitted more Field Alert Reports after the end of the sampling period., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

The genome of Mycobacterium tuberculosis ( Mtb ) harbors the genetic machinery for assembly of the Fimbrial low-molecular-weight protein (Flp) type IV pilus. Presumably, the Flp pilus is essential for pathogenesis. However, it remains unclear whether the pili genes are transcribed in culture or during infection of host cells. This study aimed to shed light on the expression of the Flp pili-assembly genes ( tadZ, tadA, tadB, tadC, flp, tadE , and tadF ) in Mtb growing under different growth conditions (exponential phase, stationary phase, and dormancy NRP1 and NRP2 phases induced by hypoxia), during biofilm formation, and in contact with macrophages and alveolar epithelial cells. We found that expression of tad/flp genes was significantly higher in the stationary phase than in exponential or NRP1 or NRP2 phases suggesting that the bacteria do not require type IV pili during dormancy. Elevated gene expression levels were recorded when the bacilli were in contact for 4 h with macrophages or epithelial cells, compared to mycobacteria propagated alone in the cultured medium. An antibody raised against a 12-mer peptide derived from the Flp pilin subunit detected the presence of Flp pili on intra- and extracellular bacteria infecting eukaryotic cells. Altogether, these are compelling data showing that the Flp pili genes are expressed during the interaction of Mtb with host cells and highlight a role for Flp pili in colonization and invasion of the host, subsequently promoting bacterial survival during dormancy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Alteri, Rios-Sarabia, De la Cruz, González-y-Merchand, Soria-Bustos, Maldonado-Bernal, Cedillo, Yáñez-Santos, Martínez-Laguna, Torres, Friedman, Girón and Ares.)

Viscera receive innervation from sensory ganglia located adjacent to multiple levels of the brainstem and spinal cord. Here we examined whether molecular profiling could be used to identify functional clusters of colon afferents from thoracolumbar (TL), lumbosacral (LS), and nodose ganglia (NG) in male and female mice. Profiling of TL and LS bladder afferents was also performed. Visceral afferents were back-labeled using retrograde tracers injected into proximal and distal regions of colon or bladder, followed by single-cell qRT-PCR and analysis via an automated hierarchical clustering method. Genes were chosen for assay (32 for bladder; 48 for colon) based on their established role in stimulus detection, regulation of sensitivity/function, or neuroimmune interaction. A total of 132 colon afferents (from NG, TL, and LS ganglia) and 128 bladder afferents (from TL and LS ganglia) were analyzed. Retrograde labeling from the colon showed that NG and TL afferents innervate proximal and distal regions of the colon, whereas 98% of LS afferents only project to distal regions. There were clusters of colon and bladder afferents, defined by mRNA profiling, that localized to either TL or LS ganglia. Mixed TL/LS clustering also was found. In addition, transcriptionally, NG colon afferents were almost completely segregated from colon TL and LS neurons. Furthermore, colon and bladder afferents expressed genes at similar levels, although different gene combinations defined the clusters. These results indicate that genes implicated in both homeostatic regulation and conscious sensations are found at all anatomic levels, suggesting that afferents from different portions of the neuraxis have overlapping functions. SIGNIFICANCE STATEMENT Visceral organs are innervated by sensory neurons whose cell bodies are located in multiple ganglia associated with the brainstem and spinal cord. For the colon, this overlapping innervation is proposed to facilitate visceral sensation and homeostasis, where sensation and pain are mediated by spinal afferents and fear and anxiety (the affective aspects of visceral pain) are the domain of nodose afferents. The transcriptomic analysis performed here reveals that genes implicated in both homeostatic regulation and pain are found in afferents across all ganglia types, suggesting that conscious sensation and homeostatic regulation are the result of convergence, and not segregation, of sensory input., (Copyright © 2020 the authors.)

Importance: Health care practitioners and patients must have information to support their confidence in the quality of prescription pharmaceuticals., Objective: To determine whether there were clear and substantive differences in major quality attributes between difficult-to-make solid oral dosage form pharmaceutical products marketed in the US., Design, Setting, and Participants: This quality improvement study analyzed US Food and Drug Administration-collected samples of 252 drug products marketed in the US and manufactured in the US, Canada, Europe, India, and the rest of Asia. These drug products were immediate-release solid oral dosage forms considered difficult to make on the basis of product quality history. This sampling included 35 innovator and 217 generic drug samples manufactured by 46 different firms containing 17 different active ingredients. Statistical analysis was performed from February to November 2019., Main Outcomes and Measures: All products were tested within their shelf life on the basis of the legally recognized tests of the US Pharmacopeia for the major quality attributes of dosage unit uniformity and dissolution. These tests measure dosage consistency and drug release, respectively. The consistency of either attribute was used to calculate a process performance index to describe the variability in manufacturing., Results: All 252 drug product samples met the US market standards for dosage unit uniformity and dissolution, although the process performance index (Ppk) for dissolution fell below the level of 4-sigma capability (ie, <1 error per 1600) for 11 different manufacturers and for generics in 4 of 5 regions, including the US. As part of a retrospective analysis, manufacturers performing above the median Ppk for either dissolution or dosage unit uniformity submitted fewer product quality defect reports (mean field alert reports of 0.22 and 0.63, respectively) than those falling at or below the median Ppk for these attributes (mean field alert reports of 2.1 and 1.7, respectively)., Conclusions and Relevance: All samples met the US market standards for dosage unit uniformity and dissolution, indicating acceptability for use by patients regardless of manufacturer or region. To our knowledge, this is the largest sampling study of pharmaceutical manufacturers for the US market and these data provide objective insight into the quality of prescription drugs with high manufacturing risks.

Sensory neuron nicotinic acetylcholine receptors (nAChRs) contribute to pain associated with tissue injury. However, there are marked differences between rats and mice with respect to both the properties and distribution of nAChR currents in sensory neurons. Because both species are used to understand pain signaling in humans, we sought to determine whether the currents present in either species was reflective of those present in human sensory neurons. Neurons from the L4/L5 dorsal root ganglia were obtained from adult male and female organ donors. Nicotine evoked currents were detected in 40 of 47 neurons (85%). In contrast with the naïve mouse, in which almost all nAChR currents are transient, or the rat, in which both mouse-like transient and more slowly activating and inactivating currents are detected, all the currents in human DRG neurons were slow, but slower than those in the rat. Currents were blocked by the nAChR antagonists mecamylamine (30 µmol/L), but not by the TRPA1 selective antagonist HC-030031 (10 µmol/L). Single cell polymerase chain reaction analysis of nicotinic receptor subunit expression in human DRG neurons are consistent with functional data indicating that receptor expression is detected 85 ± 2.1% of neurons assessed (n = 48, from 4 donors). The most prevalent coexpression pattern was α3/β2 (95 ± 4% of neurons with subunits), but α7 subunits were detected in 70 ± 3.4% of neurons. These results suggest that there are not only species differences in the sensory neuron distribution of nAChR currents between rodent and human, but that the subunit composition of the channel underlying human nAChR currents may be different from those in the mouse or rat. PERSPECTIVE: The properties and distribution of nicotine evoked currents in human sensory neurons were markedly different from those previously observed in mice and rats. These observations add additional support to the suggestion that human sensory neurons may be an essential screening tool for those considering moving novel therapeutics targeting primary afferents into clinical trials., (Copyright © 2019 the American Pain Society. All rights reserved.)

Primary afferents are known to be inhibited by kappa opioid receptor (KOR) signaling. However, the specific types of somatosensory neurons that express KOR remain unclear. Here, using a newly developed KOR-cre knockin allele, viral tracing, single-cell RT-PCR, and ex vivo recordings, we show that KOR is expressed in several populations of primary afferents: a subset of peptidergic sensory neurons, as well as low-threshold mechanoreceptors that form lanceolate or circumferential endings around hair follicles. We find that KOR acts centrally to inhibit excitatory neurotransmission from KOR-cre afferents in laminae I and III, and this effect is likely due to KOR-mediated inhibition of Ca 2+ influx, which we observed in sensory neurons from both mouse and human. In the periphery, KOR signaling inhibits neurogenic inflammation and nociceptor sensitization by inflammatory mediators. Finally, peripherally restricted KOR agonists selectively reduce pain and itch behaviors, as well as mechanical hypersensitivity associated with a surgical incision. These experiments provide a rationale for the use of peripherally restricted KOR agonists for therapeutic treatment., (Copyright © 2018 Elsevier Inc. All rights reserved.)

From 2005 to 2014, 370 data entries of dissolution failures of solid oral drug products were assessed with respect to the solubility of drug substances, dosage forms [immediate release (IR) vs. modified release (MR)], and manufacturers (brand name vs. generic). The study results show that the solubility of drug substances does not play a significant role in dissolution failures; however, MR drug products fail dissolution tests more frequently than IR drug products. When multiple variables were analyzed simultaneously, poorly water-soluble IR drug products failed the most dissolution tests, followed by poorly soluble MR drug products and very soluble MR drug products. Interestingly, the generic drug products fail dissolution tests at an earlier time point during a stability study than the brand name drug products. Whether the dissolution failure of these solid oral drug products has any in vivo implication will require further pharmacokinetic, pharmacodynamic, clinical, and drug safety evaluation. Food and Drug Administration is currently conducting risk-based assessment using in-house dissolution testing, physiologically based pharmacokinetic modeling and simulation, and post-market surveillance tools. At the meantime, this interim report will outline a general scheme of monitoring dissolution failures of solid oral dosage forms as a pharmaceutical quality indicator., (Published by Elsevier Inc.)

CONFERENCE PROCEEDING Proceedings of the PDA/FDA Adventitious Viruses in Biologics: Detection and Mitigation Strategies Workshop in Bethesda, MD, USA; December 1-3, 2010 Guest Editors: Arifa Khan (Bethesda, MD), Patricia Hughes (Bethesda, MD) and Michael Wiebe (San Francisco, CA).

This is the third in a series of seven articles discussing the Recall Root Cause Research project conducted by the Division of Manufacturing and Product Quality, Center for Drug Evaluation and Research. This paper reviews the regulatory and scientific impact of a common and recurring opportunistic pathogen, Burkholderia cepacia. B. cepacia is comprised of closely related species called Burkholderia cepacia complex, which has contaminated many finished pharmaceutical products and environments used to manufacture pharmaceuticals. This review includes a brief perspective as described in several U.S. Food and Drug Administration (FDA) documents, and assesses root cause using product recall reports and FDA Establishment Inspection Reports. We identify several possible points of origin for microbial contamination. This discussion also includes concern with anomalies in test methods that may influence B. cepacia measurement. The issue of objectionable microorganisms and whether B. cepacia can readily be included in a compendial chapter is briefly discussed. Finally, this paper underscores that drugs contaminated with B. cepacia pose a serious threat to susceptible patients, particularly those with cystic fibrosis or who are otherwise immunocompromised. It is therefore important to prevent B. cepacia from contaminating pharmaceutical manufacturing environments, raw materials, and finished products., Lay Abstract: Burkholderia cepacia is a species of bacterium that is commonly found in natural environments such as soil, water, rhizosphere and agriculture products. The species name represents a group of closely related organisms. These bacteria have contaminated many drug products and can create public health concerns. Pharmaceutical products that are contaminated with B. cepacia may pose serious consequences to vulnerable patients (e.g., compromised immune system). Preventing B. cepacia contamination in drugs by addressing the potential sources of this bacteria in a drug manufacturing operation is an important public health goal. This review highlights potential sources of B. cepacia species as they relate to U.S. Food and Drug Administration findings recorded in data from Establishment Inspection Reports and Warning Letters.

The "enhanced intracellular survival" (eis) gene of Mycobacterium tuberculosis (Mtb) is involved in the intracellular survival of M. smegmatis. However, its exact effects on host cell function remain elusive. We herein report that Mtb Eis plays essential roles in modulating macrophage autophagy, inflammatory responses, and cell death via a reactive oxygen species (ROS)-dependent pathway. Macrophages infected with an Mtb eis-deletion mutant H37Rv (Mtb-Δeis) displayed markedly increased accumulation of massive autophagic vacuoles and formation of autophagosomes in vitro and in vivo. Infection of macrophages with Mtb-Δeis increased the production of tumor necrosis factor-α and interleukin-6 over the levels produced by infection with wild-type or complemented strains. Elevated ROS generation in macrophages infected with Mtb-Δeis (for which NADPH oxidase and mitochondria were largely responsible) rendered the cells highly sensitive to autophagy activation and cytokine production. Despite considerable activation of autophagy and proinflammatory responses, macrophages infected with Mtb-Δeis underwent caspase-independent cell death. This cell death was significantly inhibited by blockade of autophagy and c-Jun N-terminal kinase-ROS signaling, suggesting that excessive autophagy and oxidative stress are detrimental to cell survival. Finally, artificial over-expression of Eis or pretreatment with recombinant Eis abrogated production of both ROS and proinflammatory cytokines, which depends on the N-acetyltransferase domain of the Eis protein. Collectively, these data indicate that Mtb Eis suppresses host innate immune defenses by modulating autophagy, inflammation, and cell death in a redox-dependent manner.

Colonic volvulus is an uncommon disease that predisposes patients to bowel obstruction in both the adult and pediatric population. The international literature offers few reports of synchronous or metachronous volvulus of 2 organs of the gastrointestinal tract. We describe a unique case of a patient who presented with recurrent metachronous volvulus of the sigmoid colon, cecum, and stomach. The patient underwent multiple operations for bowel obstruction, lysis of adhesions, and colon resection. The interesting intraoperative findings were a very long mesentery and peritoneal attachments of the intraabdominal gastrointestinal organs that made the stomach and colon extremely mobile and thus susceptible to volvulus. Prophylactic pexis of the cecum and the stomach during the first operation, in light of the elongated mesentery, may have prevented the subsequent episodes of volvulus.

Bordetella bronchiseptica can establish prolonged airway infection consistent with a highly developed ability to evade mammalian host immune responses. Upon initial interaction with the host upper respiratory tract mucosa, B. bronchiseptica are subjected to antimicrobial reactive nitrogen species (RNS) and reactive oxygen species (ROS), effector molecules of the innate immune system. However, the responses of B. bronchiseptica to redox species at physiologically relevant concentrations (nM-microM) have not been investigated. Using predicted physiological concentrations of nitric oxide (NO), superoxide and hydrogen peroxide (H2O2) on low numbers of CFU of B. bronchiseptica, all redox active species displayed dose-dependent antimicrobial activity. Susceptibility to individual redox active species was significantly increased upon introduction of a second species at subantimicrobial concentrations. An increased bacteriostatic activity of NO was observed relative to H2O2. The understanding of Bordetella responses to physiologically relevant levels of exogenous RNS and ROS will aid in defining the role of endogenous production of these molecules in host innate immunity against Bordetella and other respiratory pathogens.

Objective: To report a case of a severe interaction between simvastatin, amiodarone, and atazanavir resulting in rhabdomyolysis and acute renal failure., Background: A 72-year-old white man with underlying human immunodeficiency virus, atrial fibrillation, coronary artery disease, and hyperlipidemia presented with generalized pain, fatigue, and dark orange urine for 3 days. The patient was taking 80 mg simvastatin at bedtime (initiated 27 days earlier); amiodarone at a dose of 400 mg daily for 7 days, then 200 mg daily (initiated 19 days earlier); and 400 mg atazanavir daily (initiated at least 2 years previously). Laboratory evaluation revealed 66,680 U/L creatine kinase, 93 mg/dL blood urea nitrogen, 4.6 mg/dL creatinine, 1579 U/L aspartate aminotransferase, and 738 U/L alanine aminotransferase. Simvastatin, amiodarone, and the patient's human immunodeficiency virus medications were all temporarily discontinued and the patient was given forced alkaline diuresis and started on dialysis. Nine days later the patient's creatine kinase had dropped to 1695 U/L and creatinine was 3.3 mg/dL. The patient was discharged and continued outpatient dialysis for 1 month until his renal function recovered., Discussion: The risk of rhabdomyolysis is increased in the presence of concomitant drugs that inhibit simvastatin metabolism. Simvastatin is metabolized by CYP3A4. Amiodarone and atazanavir are recognized CYP3A4 inhibitors., Conclusions: Pharmacokinetic differences in statins are an important consideration for assessing the risk of potential drug interactions. In patients requiring the concurrent use of statins and CYP3A4 inhibitors, pravastatin, fluvastatin, and rosuvastatin carry the lowest risk of drug interactions; atorvastatin carries moderate risk, whereas simvastatin and lovastatin have the highest risk and should be avoided in patients taking concomitant CYP3A4 inhibitors.

In the male monkey, luteinising hormone (LH) secretion is regulated by a negative feedback action of testicular testosterone that is exerted indirectly at the hypothalamic level to decelerate pulsatile gonadotrophin-releasing hormone release (GnRH). The purpose of the present experiment was to investigate whether the kisspeptin-G protein-coupled receptor 54 (GPR54) signalling pathway is involved in mediating the action of testosterone to suppress GnRH release in the monkey, as has been indicated by studies of nonprimates. To this end, 12 castrated adult male rhesus monkeys were implanted with either testosterone containing or empty Silastic capsules. Testosterone treatment produced a square wave increment in circulating testosterone levels within the physiologic range. After suppression of LH and follicle-stimulating hormone secretion was established at 5-6 weeks of testosterone exposure, the animals were killed and expression of the genes encoding for kisspeptin, GPR54 and GnRH determined in the mediobasal hypothalamus and preoptic area of both treated and control animals using RNase protection assays. The suppression in pituitary gonadotrophin secretion was associated with a reduction in kisspeptin mRNA levels in the mediobasal hypothalamus, but not the preoptic area. GPR54 mRNA levels, on the other hand, were not influenced by testosterone treatment. These results are consistent with those previously reported for the rodent, and suggest that the neurobiology of the negative feedback action of testicular testosterone on LH secretion in the monkey, a representative higher primate, may be mediated by kisspeptinergic neurones upstream to the GnRH network.

Mycobacterium tuberculosis is responsible for nearly 3 million human deaths worldwide every year. Understanding the mechanisms and bacterial factors responsible for the ability of M. tuberculosis to cause disease in humans is critical for the development of improved treatment strategies. Many bacterial pathogens use pili as adherence factors to colonize the host. We discovered that M. tuberculosis produces fine (2- to 3-nm-wide), aggregative, flexible pili that are recognized by IgG antibodies contained in sera obtained from patients with active tuberculosis, indicating that the bacilli produce pili or pili-associated antigen during human infection. Purified M. tuberculosis pili (MTP) are composed of low-molecular-weight protein subunits encoded by the predicted M. tuberculosis H37Rv ORF, designated Rv3312A. MTP bind to the extracellular matrix protein laminin in vitro, suggesting that MTP possess adhesive properties. Isogenic mtp mutants lost the ability to produce Mtp in vitro and demonstrated decreased laminin-binding capabilities. MTP shares morphological, biochemical, and functional properties attributed to bacterial pili, especially with curli amyloid fibers. Thus, we propose that MTP are previously unidentified host-colonization factors of M. tuberculosis.

The eis gene of Mycobacterium tuberculosis has been shown to play a role in the survival of the avirulent Mycobacterium smegmatis within the macrophage. In vitro and in vivo analysis of Deltaeis deletion mutants and complemented strains showed no effect on survival of M. tuberculosis in U-937 macrophages or in a mouse aerosol infection model, respectively. Further studies were done in an attempt to determine the role of eis in M. tuberculosis intracellular survival and to define a phenotypic difference between wild-type and the Deltaeis deletion mutant. Bioinformatic analysis indicated that Eis is an acetyltransferase of the GCN5-related family of N-acetyltransferases. Immunofluorescence microscopy and Western blot analysis studies demonstrated that Eis is released into the cytoplasm of M. tuberculosis-infected U-937 macrophages. Eis was also found in the extravesicular fraction and culture supernatant of M. tuberculosis-infected macrophages. The effect of Eis on human macrophage cytokine secretion was also examined. Eis modulated the secretion of IL-10 and TNF-alpha by primary human monocytes in response both to infection with M. tuberculosis and to stimulation with recombinant Eis protein. These results suggest that Eis is a mycobacterial effector that is released into the host cell to modulate inflammatory responses, possibly via transcriptional or post-translational means.

Acquired constriction ring syndrome is a clinical condition of infancy characterized by circumferential constriction of a toe or another appendage, such as fingers and genitalia. The foot and ankle specialist should be aware of this condition because vascular obstruction of the affected appendage can rapidly lead to gangrene and autoamputation. Treatment consists of prompt identification and removal of the constricting foreign material. Although this condition is uncommon, it can lead to digital loss. Early treatment yields a good prognosis. A case report is presented of a 9-week-old infant who experienced acquired constriction ring syndrome caused by a strand of hair wrapped around the third and fourth toes that was treated by unwinding the hair under loupe magnification.

This paper summarizes parenteral drug contamination case studies presented at industry conferences and a Food and Drug Administration advisory committee meeting in the period of 2000-2004. CGMP deficiencies associated with each contamination event are discussed. The key role of a well-functioning quality system in contamination prevention is emphasized.

Reporter systems efficient at monitoring temporal gene expression in slow-growing mycobacteria would significantly aid the characterization of gene expression in specific environments. Bacterial luciferase is a reporter that has not been widely used to study gene expression in mycobacteria. This report describes the determination of the degradation of bacterial luciferase in Mycobacterium tuberculosis H37Ra and its utility as a reporter of temporal gene expression in this slow-growing mycobacterium. The inducible/repressible alanine dehydrogenase promoter of M. tuberculosis H37Rv was used to track the decay kinetics of Vibrio harveyi luciferase in both mid-log phase and stationary phase grown M. tuberculosis H37Ra, which proved to be highly similar during both phases of growth. The luciferase reporter was then used to detect changes in expression from the heat-shock promoter, phsp60, of M. bovis BCG during M. tuberculosis H37Ra growth in culture. Quantitative real-time PCR analysis of groEL2, the hsp60 homologue in M. tuberculosis, displayed a similar pattern of expression to phsp60-driven luciferase. These results strongly suggest that the luciferase reporter can be used to monitor temporal changes in gene expression in M. tuberculosis and may serve as a novel system to examine gene expression under specific conditions.

To further understand Mycobacterium tuberculosis pathogenesis, the regulation of potential virulence genes needs to be investigated. The eis gene of M. tuberculosis H37Rv enhances the intracellular survival of Mycobacterium smegmatis, which does not contain eis, within macrophages (J. Wei, J. L. Dahl, J. W. Moulder, E. A. Roberts, P. O'Gaora, D. B. Young, and R. L. Friedman, J. Bacteriol. 182:377-384, 2000). Experiments were done to characterize the eis promoter in M. smegmatis and M. tuberculosis H37Ra. The putative -10 and -35 regions matched the Escherichia coli sigma(70) consensus 67 and 83%, respectively, making it a group A/SigA-like mycobacterial promoter. Expression of site-directed variants of the core promoter region, determined by flow cytometry using gfp as a reporter, showed that the putative -10 region is essential for eis expression. In addition, site-directed alteration of the eis promoter to the consensus E. coli sigma(70) promoter elements increased gfp transcription to levels similar to that driven by the heat shock promoter, phsp60, of Mycobacterium bovis BCG. Upstream promoter deletion analysis showed that a 200- and 412-bp region of the promoter was necessary for maximum expression of gfp in M. smegmatis and M. tuberculosis H37Ra, respectively. Random mutagenesis of the 412-bp eis promoter, using a catechol 2,3-dioxygenase screen and activity assay, defined nucleotides upstream of the core promoter region that are essential to eis expression in both M. smegmatis and M. tuberculosis H37Ra, including a region homologous to a DinR cis element.

Objective: The purpose of this study was to determine the relative influences of the extent of disease present before surgery and completeness of cytoreduction on survival for patients with advanced ovarian cancer., Methods: Patients (408) with stage IIIC epithelial ovarian cancer had cytoreductive surgery before systemic platinum-based combination chemotherapy. A ranking system (0-3) was devised to prospectively quantify the extent of disease involving: (1) right upper quadrant (diaphragm/hepatic, and adjacent peritoneal surfaces), (2) left upper quadrant (omentum/gastro-colic ligament, spleen, stomach, transverse colon, splenic flexure of colon), (3) pelvis (reproductive organs, recto-sigmoid, pelvic peritoneum), (4) retroperitoneum (pelvic/aortic nodes), and (5) central abdomen (small bowel, ascending/descending colon, mesentery, anterior abdominal wall, pericolic gutters). Survival was analyzed (log rank and Cox regression) on the basis of the rankings at these anatomic regions, the sum of intraabdominal rankings, and the cytoreductive outcome., Results: Overall median and estimated 5-year survivals were 58.2 months and 49%. On univariate analysis, the central abdominal (P = 0.008) and left upper quadrant (P = 0.03) rankings, the sum of rankings (P = 0.01), and the cytoreductive outcome (P 1 cm residual, RR 2.98; P = 0.001)., Conclusions: Cytoreduction to a visibly disease-free outcome has a more significant influence on survival than the extent of metastatic disease present before surgery. Operative efforts should not be abbreviated on the hypothesis that extensive disease at specific anatomic regions precludes long-term survival.

This study examined the relative role of FSH and LH in governing testicular inhibin B secretion in the rhesus monkey. Adult male monkeys, rendered hypogonadotropic and hypogonadal by administration of a GnRH receptor antagonist (acyline), were implanted with testosterone (T)-filled or empty capsules. Following T-induced restoration of spermatogenesis, both groups received recombinant human FSH and vehicle for 12 d. Juvenile male monkeys received an 11-d infusion of single-chain recombinant human LH and recombinant human FSH, either alone or in combination. In adults, chronic hypogonadotropism resulted in a modest reduction of circulating inhibin B levels, which was more than fully reversed by FSH. In the presence of T, which exerted a marked suppression in inhibin B secretion, FSH restored inhibin B levels only to those observed before acyline treatment. In juveniles, treatment with single-chain recombinant human LH led to a suppression of inhibin B secretion and curtailed the FSH-induced stimulation of this testicular hormone. The T-induced decrease in inhibin B secretion was associated with suppression in inhibin-beta(B) mRNA levels, but FSH stimulation of inhibin B secretion occurred in the absence of clear changes in expression of this subunit gene. These findings indicate that inhibin B secretion by the monkey testis is governed by the inhibitory and stimulatory action of LH and FSH, respectively. The action of LH is presumably indirect and likely mediated by T inhibition of inhibin-beta(B) gene expression. The molecular basis of the stimulatory action of FSH on inhibin B secretion requires further study.

Background: Laparoscopic cholecystectomy has become the gold standard for the treatment of symptomatic cholelithiasis. Many authors-including investigators at our institution, who reported one of the initial experiences with laparoscopic cholecystectomy in July 1992-have documented a definite learning curve associated with this procedure. We present a follow-up study of our experience with laparoscopic cholecystectomy and compare these data to an earlier study of the initial experience with laparoscopic cholecystectomy at the Beth Israel Medical Center., Methods: We retrospectively reviewed 300 consecutive patients from March 1998 through March 1999. The patient population was epidemiologically similar to that of the original study with regard to age, sex, and American Society of Anesthesia (ASA) classification. However, whereas the initial population included only patients with chronic disease, in our study 13.7% of the patients had been admitted through the emergency room with acute stone disease of the biliary tract., Results: We found a 5.7% conversion rate, a 1% rate of major complication, and a 5.7% rate of minor complication rates, as compared to the initial study's rates of 12%, 4%, and 10%, respectively. Whereas none of the patients in the original study left the hospital on the day of surgery and only 49% were discharged within 1 day, in our group, 29 patients (10%) underwent ambulatory procedures and an additional 186 patients (62%) were discharged on the 1st post-operative day. The average duration of the operation was 90 min, which did not represent a statistical improvement over the time of 93 min reported in the earlier study., Conclusions: Since 1992, both the conversion rate and length of stay have declined at our hospital, but operative time has remained essentially the same. These findings probably reflect a bimodal learning curve, the increase in the number of cholangiograms and additional intraoperative procedures now performed, the greater severity of gallbladder disease currently treated with laparoscopic cholecystectomy, and increases in the number of attending physicians as well as the level of residents who perform this procedure.

Mycobacterium tuberculosis is a facultative intracellular pathogen that has evolved the ability to survive and multiply within human macrophages. It is not clear how M. tuberculosis avoids the destructive action of macrophages, but this ability is fundamental in the pathogenicity of tuberculosis. A gene previously identified in M. tuberculosis, designated eis, was found to enhance intracellular survival of Mycobacterium smegmatis in the human macrophage-like cell line U-937 (J. Wei et al., J. Bacteriol. 182:377-384, 2000). When eis was introduced into M. smegmatis on a multicopy vector, sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the appearance of a unique 42-kDa protein band corresponding to the predicted molecular weight of the eis gene product. This band was electroeluted from the gel with a purity of >90% and subjected to N-terminal amino acid sequencing, which demonstrated that the 42-kDa band was indeed the protein product of eis. The Eis protein produced by M. tuberculosis H37Ra had an identical N-terminal amino acid sequence. A synthetic polypeptide corresponding to a carboxyl-terminal region of the deduced eis protein sequence was used to generate affinity-purified rabbit polyclonal antibodies that reacted with the 42-kDa protein in Western blot analysis. Hydropathy profile analysis showed the Eis protein to be predominantly hydrophilic with a potential hydrophobic amino terminus. Phase separation of M. tuberculosis H37Ra lysates by the nonionic detergent Triton X-114 revealed the Eis protein in both the aqueous and detergent phases. After fractionation of M. tuberculosis by differential centrifugation, Eis protein appeared mainly in the cytoplasmic fraction but also in the membrane, cell wall, and culture supernatant fractions as well. Forty percent of the sera from pulmonary tuberculosis patients tested for anti-Eis antibody gave positive reactions in Western blot analysis. Although the function of Eis remains unknown, evidence presented here suggests it associates with the cell surface and is released into the culture medium. It is produced during human tuberculosis infection and therefore may be an important M. tuberculosis immunogen.

Pertussis toxin expression in the Gram-negative respiratory pathogen, Bordetella pertussis, is regulated by the BvgAS two-component system. Previous studies suggested that an additional gene encoding a Bvg accessory factor (Baf) was required, along with BvgAS, for expression of a ptx-lacZ fusion in Escherichia coli grown in rich medium. However, other studies showed that BvgAS is sufficient for ptx-lacZ expression in minimal medium. Here we show that Baf acts with BvgAS to further increase ptx-lacZ expression in E. coli grown in minimal media and this is concomitant with a two-fold increase in BvgA protein levels. Gene replacement experiments show that baf is essential for viability of B. pertussis, suggesting that Baf affects the expression of other genes in addition to ptx.

Introduction: In previous studies we described mechanisms by which acute elevation of the intraabdominal pressure (IAP) induces intracranial hypertension (ICHTN). Here we sought to define the role of ICHTN in mediating systemic hypertension (HTN) during CO(2) pneumoperitoneum (PNP)., Methods: Six large animals (swine) were hyperventilated to buffer hypercarbia. Intracranial pressure (ICP) was monitored with a Camino intraparenchymal ICP monitoring system. A Foley catheter was introduced intracranially via a separate burr hole. At phase 1, changes in ICP, central venous pressure (CVP), and mean arterial pressure (MAP) were recorded during periods of CO(2) PNP at IAP levels of 15, 20, 25, and 30 mm Hg. At phase 2, ICHTN was produced directly by inflating the intracranial balloon to the same ICP levels that had been measured in phase 1 for each degree of IAP. CVP and MAP were recorded. Repeated measures analysis of variance was applied., Results: At phase 1, the mean DeltaCVP, DeltaICP, and DeltaMAP increased relative to the degree of IAP (P = 0.0001, 0.0004, and 0.024, respectively). At phase 2, the increments in DeltaMAP were significant (P = 0.024) and in the same direction and amplitude as at phase 1., Conclusions: In this study, increasing the IAP with CO(2) PNP with a consequent increase of ICP and direct manipulation of the ICP produced a comparable systemic HTN. We believe that this further supports our hypothesis: Elevated IAP produces an immediate increase in the CVP, which impairs venous drainage from the central nervous system (CNS), increases the ICP, and initiates a CNS-mediated response and systemic HTN., (Copyright 2000 Academic Press.)

Intracellular survival plays a central role in the pathogenesis of Mycobacterium tuberculosis. To identify M. tuberculosis genes required for intracellular survival within macrophages, an M. tuberculosis H37Rv plasmid library was constructed by using the shuttle vector pOLYG. This plasmid library was electroporated into Mycobacterium smegmatis 1-2c, and the transformants were used to infect the human macrophage-like cell line U-937. Because M. smegmatis does not readily survive within macrophages, any increased intracellular survival is likely due to cloned M. tuberculosis H37Rv DNA. After six sequential passages of M. smegmatis transformants through U-937 cells, one clone (p69) was enriched more than 70% as determined by both restriction enzyme and PCR analyses. p69 demonstrated significantly enhanced survival compared to that of the vector control, ranging from 2.4- to 5.3-fold at both 24 and 48 h after infection. DNA sequence analysis revealed three open reading frames (ORFs) in the insert of p69. ORF2 (1.2 kb) was the only one which contained a putative promoter region and a ribosome-binding site. Deletion analysis of the p69 insert DNA showed that disruption of ORF2 resulted in complete loss of the enhanced intracellular survival phenotype. This gene was named the enhanced intracellular survival (eis) gene. By using an internal region of eis as a probe for Southern analysis, eis was found in the genomic DNA of various M. tuberculosis strains and of Mycobacterium bovis BCG but not in that of M. smegmatis or 10 other nonpathogenic mycobacterial species. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis showed that all M. smegmatis eis-containing constructs expressed a unique protein of 42 kDa, the predicted size of Eis. The expression of this 42-kDa protein directly correlated to the enhanced survival of M. smegmatis p69 in U-937 cells. These results suggest a possible role for eis and its protein product in the intracellular survival of M. tuberculosis.

Background: This study examined the impact of secondary cytoreductive surgery on survival of patients with recurrent epithelial ovarian carcinoma., Methods: One hundred six patients with a disease free interval (DFI) > 6 months after primary treatment underwent secondary cytoreductive surgery. Multivariate analysis determined which variables influenced the cytoreductive outcome and survival., Results: Eighty-seven patients (82.1%) underwent removal of all visible tumor. The median and estimated 5-year survival for the entire cohort after recurrence was 35.9 months and 28%, respectively. The probability of complete cytoreduction was influenced by the largest size of recurrent tumor (< 10 cm ¿90.0% vs. > 10 cm ¿66.7%; P = 0.003), use of salvage chemotherapy before secondary surgery (chemotherapy given ¿64.3% vs. chemotherapy not given ¿93.8%; P = 0.001), and preoperative Gynecologic Oncology Group performance status (0 ¿100%, 1 ¿91.4%, 2 ¿82.4%, and 3 ¿47.4%; P = 0.001). Survival was influenced by the DFI after primary treatment (6-12 months ¿median, 25.0 months vs. 13-36 months ¿median, 44.4 months vs. > 36 months ¿median, 56.8 months; P = 0.005), the completeness of cytoreduction (visibly disease free ¿median, 44.4 months vs. any residual disease ¿median, 19.3 months; P = 0.007), the use of salvage chemotherapy before secondary surgery (chemotherapy given ¿median, 24.9 months vs. chemotherapy not given ¿median, 48.4 months; P = 0.005), and the largest size of recurrent tumor (< 10 cm ¿median, 37.3 months vs. > 10 cm ¿median, 35.6 months; P = 0.04)., Conclusions: Complete cytoreduction is possible for the majority of patients with recurrent epithelial ovarian carcinoma and maximizes survival if undertaken before salvage chemotherapy. The authors believe a randomized trial should be initiated to confirm these findings., (Copyright 2000 American Cancer Society.)

In previous studies we reported that an acute elevation in intra-abdominal pressure (IAP) is responsible for the elevation in intracranial pressure (ICP) and mean blood pressure (MBP). Thus far, the reasons for the increased ICP during an acute elevation in IAP and the combined effects of increased IAP and ICP on hemodynamics have not been reported. Five large animals (swine) were studied. Each animal served as its own control. A subarachnoid screw was placed for ICP monitoring. The jugular vein, femoral vein, and femoral artery were cannulated. ICP, MBP, central venous pressure above (CVPA) and below (CVPB) the diaphragm, and PaC02 were monitored after a pneumoperitoneum with C02 was established at 5, 15, and 30 mm Hg of IAP. Cavography was performed to evaluate the morphology of the inferior vena cava at different increments of IAP. Measurements were obtained in reverse Trendelenburg (group 1), supine (group 2), and Trendelenburg (group 3) positions. Multiple regression analysis was used to examine the effects of IAP and positioning in separate models with different blood pressures as dependent variables. Increased IAP significantly increased CVPA, CVPB, ICP, and MBP. There were no changes in cerebral perfusion pressure. The change in position (from group 1 to group 3) significantly increased CVPA and decreased the CVPB. Cavograms performed on animals in the supine position with increased IAP showed a narrowing of the IVC at the level of the diaphragm. Increases in IAP will increase ICP and MBP without altering the cerebral perfusion pressure. A mechanical effect mediated by compression of the inferior vena cava at the level of the diaphragm with increased central venous pressure and decreased drainage from the lumbar plexus and central nervous system is responsible for this effect.

Background: The side effects of acute elevations in intraabdominal pressure (IAP) are related to a multifactorial etiology. Previous studies have reported that acute elevations in IAP produce an immediate increase in intracranial pressure (ICP). This study was designed to analyze the reasons for increased ICP during acute elevations of IAP and to determine the combined effects of IAP and changes in ventilation indices on ICP and hemodynamic indices., Study Design: Five pigs were studied. A subarachnoid screw was placed for ICP monitoring. The jugular vein, femoral vein, and femoral artery were cannulated. Mean arterial pressure (MAP), central venous pressure (CVP), ICP, and arterial pressure of carbon dioxide (PaCO2) were monitored before and after carbon dioxide pneumoperitoneum was established at 0, 10, and 20 mmHg of IAP Effects of hyperventilation and hypoventilation were recorded and compared with baseline ventilation. Cavography was performed to evaluate the morphology of the inferior vena cava (IVC) at different levels of IAP. Multiple regression and Student's t-test were used to examine the effects of IAP and ventilation on dependent variables., Results: The IVC showed a progressive narrowing at the level of the diaphragm as IAP was increased. There was a simultaneous increase in CVP, MAP, and ICP. The mean changes in ICP with hypoventilation were significantly larger than with hyperventilation., Conclusions: Acutely increased IAP displaces the diaphragm cranially, narrowing the IVC and increasing intrathoracic pressure. This increases CVP and increases ICP by venous stasis and increased pressure in the sagittal sinus with decreased resorption of cerebrospinal fluid. Hemodynamic changes are directly related to the rise in ICP. Hypoventilation and hypercarbia significantly increase ICP when compared with hyperventilation and hypocarbia. Hyperventilation does not significantly decrease ICP during acute elevations of IAP.

Objective: Despite correlation between the completeness of surgical cytoreduction and survival for patients with advanced ovarian cancer, relatively few undergo complete cytoreduction. This study was initiated to prospectively determine the ability to surgically eliminate all visible disease in patients with stage IIIC and IV epithelial ovarian cancer and the associated impact on survival., Methods: Between 1990 and 1996, 163 consecutive patients underwent primary cytoreduction. The goal was the excision or ablation of all visible disease prior to initiation of systemic platinum-based combination chemotherapy. A multivariate analysis determined which clinical and pathologic variables influenced the probability of achieving complete cytoreduction (logistic regression) and survival (Cox proportional hazards model)., Results: One hundred thirty-nine patients (85.3%) underwent removal of all visible tumor, 22 (13.5%) had cytoreduction to 75 implants, P = 0.005), and stage (IIIC vs IV, P = 0.006). The probability of survival was independently influenced by age (61 years, P = 0.003), volume of ascites (1 liter, P = 0.01), stage (IIIC vs IV, P = 0.04), histology (clear cell and mucinous vs all other, P = 0.03), and the completeness of cytoreductive operation (complete vs incomplete cytoreduction, P = 0.02)., Conclusions: Complete cytoreduction is possible for the majority of patients and improves survival, even compared to operations with minimal (

Bordetella pertussis is the causative agent of the respiratory disease pertussis or whopoping cough. Btr, an oxygen-responsive transcriptional regulator of B. pertussis, is homologous to the FNR protein of E. coli. Using a murine respiratory model, we observed in the present study that Btr is important in growth and survival of B. pertussis in vivo. A titration assay was developed that identified genes containing Btr binding sites including B. pertussis sodB and btr, E. coli aspA and a new B. pertussis gene, brg1. The brg1 gene encodes a protein similar to the LysR family of transcriptional regulators and its expression is activated threefold by Btr under anaerobic growth conditions but unaffected by Btr aerobically. The nucleotide sequence flanking brg1 encodes proteins with similarity to various metabolic enzymes. Putative overlapping promoters and a Btr binding site (FNR box) were identified in the DNA sequence between brg1 and the adjacent genes. These intervening sequences may represent sites for regulation by Btr and Brg1.

From 1990 to 1995, 120 consecutive patients with stage IIIC and IV ovarian carcinoma underwent surgical cytoreduction to < or = 1-cm residual disease followed by platinum-based chemotherapy. At the conclusion of chemotherapy all patients who were clinically disease free and whose CA-125 was < 35 were offered a second-look operation that obtained at least 100 tissue specimens. Of 107 patients who qualified for second look, 78 underwent the procedure. Forty-three (55.1%) had negative pathology, 20 (25.6%) were microscopically positive, and 15 (19.2%) had gross disease. Patients with positive findings received individualized salvage therapy. Patient age (P = 0.01) and the number of implants at primary surgery (P = 0.004) correlated with second-look results. Twelve (27.9%) of the patients with negative pathology have recurred. Eleven of these patients had metastatic disease > or = 10 cm at primary surgery (P = 0.003). Patients refusing second look had a median survival of 39.1 months. Approximately 60% of patients who underwent second look remain alive. Stepwise logistic regression selected two covariates significantly affecting survival: the number of implants at primary surgery (P = 0.0130) and performance of a second look (P = 0.0103). Using the protocol described in a population of optimally resected patients with advanced-stage ovarian cancer, second-look laparotomy can impact positively on survival. Patients with > 10-cm metastatic disease at primary surgery and negative second-look findings should be the focus of future protocols for consolidation chemotherapy.

Background: This study was undertaken to compare safety, outcome, and costs of laparoscopic (LS) and open splenectomy (OS) for a variety of hematologic diseases., Study Design: The records of 137 patients who underwent splenectomy (63 LS and 74 OS) at a large private teaching hospital between March 1991 and April 1996 were reviewed retrospectively. Diagnosis, age, gender, operative time, blood loss, splenic weight, time to resumption of oral diet, postoperative hospital stay, morbidity, mortality, and costs (direct and operative) were analyzed by multivariate statistical analysis., Results: Laparoscopic splenectomy patients had significantly shorter hospitalization and time to resumption of an oral diet (p < 0.01); although operative costs were higher, total direct costs were not. Idiopathic thrombocytopenic purpura patients had earlier resumption of an oral diet after LS, shorter postoperative stay, and comparable OR time. Five patients (7%) were converted, with outcomes similar to OS except for greater operative time and cost. Grade II complications occurred in three LS and four OS patients; Grade III in three OS patients; and Grade IV in two OS patients. There were two major complications of LS and eight of OS, with two deaths. Multivariate analysis showed that operative time and time to resumption of oral intake were significantly related to age, diagnosis, operative technique, and splenic weight. Duration of postoperative hospitalization was related to operative technique, splenic weight, and major complications. Costs (direct and operative) were related to age, splenic weight, and major complications, but not to operative technique., Conclusions: LS results are influenced by splenic weight, disease, and age. Splenic weight appears to be the crucial determinant of operative time and length of hospitalization. LS is a superior treatment for patients with idiopathic thrombocytopenic purpura and patients with small spleens.

Background: An effort was made to determine whether a policy of routine cholangiography affects the incidence, morbidity, and cost of bile duct injuries., Methods: A retrospective review of consecutive 3,242 laparoscopic cholecystectomies was performed. Most patients had routine intraoperative cholangiography., Results: There were 12 bile duct injuries (0.37%). All injuries were Bismuth levels 1 and 2. Eleven of 12 injuries were recognized intraoperatively. Ten were repaired primarily and one required hepaticojejunostomy. All repairs were successful. Average hospital charges were $26,669. One of 12 patients had delayed recognition of a bile duct injury and underwent primary repair over a T-tube on postoperative day 7. Hospital charges were $43,957., Conclusion: Routine cholangiography did not appear to decrease the absolute incidence of bile duct injuries compared to previously published reports. Injury severity, morbidity, late sequelae, and costs were reduced by a policy of routine cholangiography.

Background: A comparison of safety, efficacy, and cost of laparoscopic splenectomy (LS) vs open splenectomy (OS) for idiopathic thrombocytopenic purpura (ITP) was performed., Methods: The records of 49 consecutive patients who underwent splenectomy for ITP (31 LS and 18 OS) at a large metropolitan teaching hospital between 3/91 and 8/95 were reviewed. Morbidity, mortality, hospital stay, operative time, blood loss, time to oral fluid intake, direct costs, and operating room (OR) costs were analyzed., Results: Age, sex, comorbidity, and spleen size were similar in both groups. LS was successful in 94% of patients in whom it was attempted. Operative times showed a learning curve for LS, with average times for the last ten cases (94 +/- 35 min) significantly shorter than for the first ten (p = 0.01) and also shorter than for OS (103 +/- 45 min). Postsurgical hospital stay was 2.9 +/- 1.3 days for LS and 6.9 +/- 3. 0 days for OS (p < 0.001). Patients tolerated an oral diet 1.2 +/- 0. 5 days after LS and 3.2 + 0.7 days after OS (p < 0.001). Direct hospital cost was $5,509 +/- 3,636 for LS and $9,031 +/- 12,752 for OS. In the LS group, six patients (21%) had accessory spleens identified and removed, compared with two patients (11%) in the OS group. Platelet counts did not respond in two (7%) patients in the LS group, but no accessory spleens were identified by nuclear scan. One major complication occurred in the LS group. There were no cases of splenosis or mortality in either group., Conclusions: LS is a safe and effective treatment for ITP, with significantly shorter postoperative hospital stay than OS.

From October 1992 until September 1995, 859 patients with 1000 inguinal hernias underwent a transabdominal preperitoneal mesh repair (TAPP) in the First Department of Surgery of the Diakonie-Hospital Rotenburg/Wümme, Germany. One hundred and seventeen of these repairs were for recurrences after previous open hernia surgery. There were 76 (7.6%) peri- and postoperative complications, but most of them were minor without necessity for surgical intervention. There were no implant infections or any adverse reaction of the mesh. During the postoperative follow-up period, which ranged from 2-36 months, 11 early recurrences were observed after TAPP, but none were in the recurrent hernia group. Our results support other studies and randomized trials reported in the literature and demonstrate that TAPP is an effective and safe operative procedure especially for the repair of recurrent groin hernias.

Bordetella pertussis produces a catalase and a Fe-superoxide dismutase. The importance of these enzymes in virulence was investigated, in vitro as well as in vivo, by using mutants deficient in their production. The catalase-deficient mutant survived within polymorphonuclear leukocytes, killed J774A.1 macrophages through apoptosis, and behaved as the parental strain in a murine respiratory infection model. These results suggest no direct role for catalase in B. pertussis virulence. The absence of expression of Fe-superoxide dismutase had profound effects on the bacterium including a reduced ability to express adenylate cyclase-hemolysin and pertactin, two factors important for B. pertussis pathogenesis. The Fe-superoxide dismutase-deficient mutant also had decreased abilities to colonize and persist in the murine respiratory infection model.