Pertussis toxin and lipopolysaccharide influence phagocytosis of Bordetella pertussis by human monocytes.

The potential of human monocytes to mediate the clearance of Bordetella pertussis infection was examined. Bacteria expressing green fluorescent protein were incubated with adherent peripheral blood monocytes, and phagocytosis was quantified by using fluorescence microscopy. Monocytes internalized only a small percentage of the adherent bacteria. Surface-associated Bvg-regulated virulence factors, including adenylate cyclase toxin and filamentous hemagglutinin, did not affect attachment or phagocytosis. However, 1-h pretreatment with purified pertussis toxin inhibited the ability of monocytes to internalize wild-type bacteria. Mutations affecting the terminal trisaccharide of lipopolysaccharide resulted in reduced internalization without affecting adherence of bacteria to monocytes. Opsonization with human serum played only a modest role in promoting phagocytosis. The viability of internalized bacteria was determined by colony counts following treatment with polymyxin B and gentamicin. Less than 1% of internalized bacteria remained viable. These results suggest that pertussis toxin plays a role in the evasion of monocyte phagocytosis and that these cells represent a potential mediator of the clearance of B. pertussis infection.

BrkA protein of Bordetella pertussis inhibits the classical pathway of complement after C1 deposition.

Bordetella pertussis produces a 73-kDa protein, BrkA (Bordetella resistance to killing), which inhibits the bactericidal activity of complement. In this study we characterized the step in the complement cascade where BrkA acts, using three strains: a wild-type strain, a strain containing an insertional disruption of brkA, and a strain containing two copies of the brkA locus. Following incubation with 10% human serum, killing was greatest for the BrkA mutant, followed by that for the wild-type strain, while the strain with two copies of brkA was the most resistant. Complement activation was monitored by enzyme-linked immunosorbent assay (ELISA) or Western blotting. ELISAs for SC5b-9, the soluble membrane attack complex, showed that production of SC5b-9 was greatest with the brkA mutant, less with the wild type, and least with the strain containing two copies of brkA. Deposition of complement proteins on the bacteria was monitored by Western blotting. A decrease in deposition on the bacteria of C4, C3, and C9 corresponded with decreased complement sensitivity. Deposition of C1, however, was not affected by the presence of BrkA. These studies show that BrkA inhibits the classical pathway of complement activation and prevents accumulation of deposited C4.

Neutralizing antibodies to adenylate cyclase toxin promote phagocytosis of Bordetella pertussis by human neutrophils.

A previous study showed that opsonization with human immune serum could either promote or antagonize phagocytosis of Bordetella pertussis by human neutrophils depending on whether the bacteria expressed adenylate cyclase toxin. Opsonization of the wild-type strain inhibited phagocytosis relative to unopsonized controls. In contrast, mutants lacking adenylate cyclase toxin were efficiently phagocytosed when opsonized with human immune serum. In this study, we examined opsonization in the presence or absence of monoclonal antibodies to adenylate cyclase toxin. Addition of neutralizing monoclonal antibodies to adenylate cyclase toxin converted a serum that previously inhibited both attachment and phagocytosis of the wild-type strain to one that increased both attachment and phagocytosis compared to the no-serum control. Monoclonal antibodies that recognize the adenylate cyclase toxin but fail to neutralize activity were without effect. These results suggest that adenylate cyclase toxin inhibits both Fc receptor-mediated attachment and phagocytosis of B. pertussis by neutrophils.

Characterization of bactericidal immune responses following vaccination with acellular pertussis vaccines in adults.

Sera from six adults, collected before and after acellular pertussis vaccination, and from a placebo control were examined for the ability to elicit two bactericidal immune defenses, (i) antibody-dependent complement-mediated bacterial lysis and (ii) opsonization and phagocytosis by human neutrophils. The samples were chosen based on low preimmunization titers and strong postimmunization responses to various combinations of vaccine antigens. All but two prevaccination samples demonstrated activity indicative of complement-mediated lysis. Preimmunization activity could have been due to prior infection or childhood immunization. Immunization did not result in improved bactericidal activity for any of the individuals, and in two cases immunization caused a statistically significant decrease in complement-mediated lysis. Similarly, opsonization with the postimmunization sera failed to enhance attachment or phagocytosis of bacteria by neutrophils, and one postimmunization sample with a strong response to filamentous hemagglutinin caused an inhibition of phagocytosis that was statistically significant compared to that observed for the no-serum control. In summary, booster immunization of adults with acellular pertussis vaccines was not found to increase bactericidal activity over preimmunization levels. Identifying ways to promote bactericidal immune responses might improve the efficacy of acellular pertussis vaccines.

Antibacterial agents and release of periplasmic pertussis toxin from Bordetella pertussis.

Pertussis toxin accumulates in the periplasm of Bordetella pertussis prior to secretion, and we examined its fate following treatment with antimicrobial agents. Both antibiotics that inhibit protein synthesis (erythromycin and chloramphenicol), transcription (rifampin), or cell wall biosynthesis (cefoperazone and piperacillin) and magnesium sulfate (which inhibits transcription of pertussis toxin, but not bacterial growth) did not prevent release of preformed toxin. In contrast, agents that affect bacterial membranes, such as polymyxin B, lidocaine, procaine, and ethanol, inhibited release of preformed pertussis toxin. These results suggest new protein synthesis is not required for pertussis toxin secretion, but a functional membrane complex is required.

Mutations in the S1 subunit of pertussis toxin that affect secretion.

Pertussis toxin is a member of the AB(5) family of toxins and is composed of five subunits (S1 to S5) present in a 1:1:1:2:1 ratio. Secretion is a complex process. Each subunit has a secretion signal that mediates transport to the periplasm, where processing and assembly occur. Secretion of the assembled 105-kDa toxin past the outer membrane is mediated by the nine proteins encoded in the ptl operon. Previous studies have shown that S1, the catalytically active A subunit of pertussis toxin, is necessary for efficient secretion, suggesting that a domain on S1 may be required for interaction with the secretion apparatus. Previously, recombinant S1 from four different mutants (serine 54 to glycine, serine 55 to glycine, serine 56 to glycine, and arginine 57 to lysine) was shown to retain catalytic activity. We introduced these mutations into Bordetella pertussis and monitored pertussis toxin production and secretion. No pertussis toxin was detected in the serine 54-to-glycine mutant. The other S1 mutants produced periplasmic pertussis toxin, but little pertussis toxin secretion was observed. The arginine 57-to-lysine mutant had the most dramatic secretion defect. It produced wild-type levels of periplasmic pertussis toxin but secreted only 8% as much toxin as the wild-type strain. This phenotype was similar to that observed for strains with mutations in the ptl genes, suggesting that this region may have a role in pertussis toxin secretion.

Bordetella pertussis virulence factors affect phagocytosis by human neutrophils.

The interaction between human neutrophils and wild-type Bordetella pertussis or mutants expressing altered lipopolysaccharide or lacking virulence factors-pertussis toxin, adenylate cyclase toxin, dermonecrotic toxin, filamentous hemagglutinin (FHA), pertactin, or BrkA-was examined. In the absence of antibodies, the wild-type strain and the mutants, with the exception of mutants lacking FHA, attached efficiently to neutrophils. The addition of opsonizing antibodies caused a significant reduction (approximately 50%) in attachment of the wild-type strain and most of the mutants expressing FHA, suggesting that bacterium-mediated attachment is more efficient than Fc-mediated attachment. Phagocytosis was also examined. In the absence of antibodies, about 12% of the wild-type bacteria were phagocytosed. Opsonization caused a statistically significant reduction in phagocytosis (to 3%), possibly a consequence of reduced attachment. Phagocytosis of most of the mutants was similar to that of the wild type, with the exception of the mutants lacking adenylate cyclase toxin. About 70% of the adenylate cyclase toxin mutants were phagocytosed, but only in the presence of opsonizing antibody, suggesting that Fc receptor-mediated signaling may be needed for phagocytosis. These studies indicate that FHA mediates attachment of B. pertussis to neutrophils, but adenylate cyclase toxin blocks phagocytosis.

Phagocytosed Bordetella pertussis fails to survive in human neutrophils.

Previous studies have reported that phagocytosed Bordetella pertussis survives in human neutrophils. This issue has been reexamined. Opsonized or unopsonized bacteria expressing green fluorescent protein (GFP) were incubated with adherent human neutrophils. Phagocytosis was quantified by fluorescence microscopy, and the viability of phagocytosed bacteria was determined by colony counts following treatment with polymyxin B to kill extracellular bacteria. Only 1 to 2% of the phagocytosed bacteria remained viable. Opsonization with heat-inactivated immune serum reduced the amount of attachment and phagocytosis of the bacteria but did not alter survival rates. In contrast to previous reports, these data suggest that phagocytosed B. pertussis bacteria are killed by human neutrophils.

Mutants in the ptlA-H genes of Bordetella pertussis are deficient for pertussis toxin secretion.

The nine ptl genes (A-I) are required for efficient secretion of pertussis toxin past the outer membrane. Mutations were made in ptlA-H by filling in unique restriction sites, generating in-frame deletions, or inserting a FLAG epitope tag. The mutations were cloned into a suicide shuttle plasmid containing the ptxptl operon and introduced into the adenylate cyclase locus of the chromosome of a Bordetella pertussis strain deleted for ptx. The wild-type ptxptl operon restored pertussis toxin expression and secretion. The ptl mutant constructs also restored expression of periplasmic pertussis toxin to the ptx deletion strain but the mutants had a statistically significant decrease in secretion of pertussis toxin of between 5- to 35-fold, suggesting all of the ptl genes must be intact for efficient pertussis toxin secretion. The mutations were also introduced into the adenylate cyclase locus of a wild-type ptxptl strain, resulting in a ptl diploid strain. The PtlC, PtlD, PtlE, PtlF, PtlG and PtlH mutants exerted dominance over the wild-type allele.

Fluorescent labels influence phagocytosis of Bordetella pertussis by human neutrophils.

To explore the role of neutrophil phagocytosis in host defense against Bordetella pertussis, bacteria were labeled extrinsically with fluorescein isothiocyanate (FITC) or genetically with green fluorescent protein (GFP) and incubated with adherent human neutrophils in the presence or absence of heat-inactivated human immune serum. In the absence of antibodies, FITC-labeled bacteria were located primarily on the surface of the neutrophils with few bacteria ingested. However, after opsonization, about seven times more bacteria were located intracellularly, indicating that antibodies promoted phagocytosis. In contrast, bacteria labeled intrinsically with GFP were not efficiently phagocytosed even in the presence of opsonizing antibodies, suggesting that FITC interfered with a bacterial defense. Because FITC covalently modifies proteins and could affect their function, we tested the effect of FITC on adenylate cyclase toxin activity, an important extracellular virulence factor. FITC-labeled bacteria had fivefold-less adenylate cyclase toxin activity than did unlabeled wild-type bacteria or GFP-expressing bacteria, suggesting that FITC compromised adenylate cyclase toxin activity. These data demonstrated that at least one extracellular virulence factor was affected by FITC labeling and that GFP is a more appropriate label for B. pertussis.

A prospective evaluation of upper extremity tendon transfers in children with cervical spinal cord injury.

Three children (five hands) between 6 and 11 years of age with cervical level spinal cord injuries underwent tendon transfers to restore voluntary lateral pinch. Repeated measures of pinch force and the Grasp and Release Test (GRT) were obtained before surgery and at regular intervals after tendon transfers. The Functional Independence Measure (FIM) was administered before surgery and at 12 months after surgery. Responses to open-ended questions were used to supplement the FIM data at 1 year after surgery. Before surgery, no measurable force was obtained in any hand; after tendon transfers, pinch and finger flexion forces increased throughout the follow-up period. On the GRT, manipulation of the heavy objects was possible only after tendon transfers. Improvements were realized in feeding, grooming, bladder management, play, and school tasks. Each child requested surgery to restore pinch in the nondominant hand. Two hands required tenolysis procedures. Despite capsulotomies and aggressive therapy, three hands continued to have range limitations at the metacarpophalangeal joints.

Redesigning the surgery clerkship at MCP Hahnemann School of Medicine to address the educational needs of generalists.

This article describes how the surgery clerkship at MCP Hahnemann School of Medicine was redesigned to provide all students a well-rounded general professional education and to address the specific educational needs of generalists. During the 12-week clerkship, students spend eight weeks on two different general surgery rotations, which include significant experiences in outpatient settings. The evaluation and management of common general surgical problems, as well as the holistic approach to patient care, are emphasized. A nurse educator, recruited through funding obtained from The Robert Wood Johnson Foundation's Generalist Physician Initiative, provides formal instruction in holistic care and teaches bedside procedures. Two weeks are devoted to focused surgical subspecialty experiences addressing common conditions and are conducted primarily in outpatient settings. The remaining two weeks include an integrated musculoskeletal disease rotation, including orthopaedic surgery, rheumatology, physiatry, and radiology. Didactic teaching includes criteria for referral of patients from generalists to specialists. The new clerkship model has been well received by the students. Review of student logs for the first six months indicates the breadth of surgical experience has been maintained and appropriate balance achieved between simple and complex surgical cases. Further evaluation of the model will continue through longitudinal follow-up.

Characterization of human bactericidal antibodies to Bordetella pertussis.

The Bordetella pertussis BrkA protein protects against the bactericidal activity of complement and antibody; however, some individuals mount an immune response that overcomes this bacterial defense. To further characterize this process, the bactericidal activities of sera from 13 adults with different modes of exposure to B. pertussis (infected as adults, occupational exposure, immunized with an acellular vaccine, or no identified exposure) against a wild-type strain and a BrkA complement-sensitive mutant were evaluated. All of the sera killed the BrkA mutant, suggesting past exposure to B. pertussis or cross-reactive organisms. Several samples had no or minimal activity against the wild type. All of the sera collected from the infected and occupationally exposed individuals but not all of the sera from vaccinated individuals had bactericidal activity against the wild-type strain, suggesting that some types of exposure can induce an immune response that can overcome the BrkA resistance mechanism. Adsorbing serum with the wild-type strain removed the bactericidal antibodies; however, adsorbing the serum with a lipopolysaccharide (LPS) mutant or an avirulent (bvg mutant) strain did not always result in loss of bactericidal activity, suggesting that antibodies to either LPS or bvg-regulated proteins could be bactericidal. All the samples, including those that lacked bactericidal activity, contained antibodies that recognized the LPS of B. pertussis. Bactericidal activity correlated best with the presence of the immunoglobulin G3 (IgG3) antibodies to LPS, the IgG subtype that is most effective at fixing complement.

Post-surgical shunt hepatopulmonary syndrome in a case of non-cirrhotic portal hypertension: lack of efficacy of shunt reversal.

Hepatopulmonary syndrome, a consequence of significant liver disease and portal hypertension, is thought to be secondary to the effects of vasoactive substances, normally inactivated in the liver, on the pulmonary vasculature. We report a patient with preserved hepatic function who underwent a decompressive surgical porto-systemic shunt for non-cirrhotic portal hypertension. This patient developed hepatopulmonary syndrome with dyspnoea and oxygen desaturation 2 years post-surgical shunt. Over the next 7 years, the patient's respiratory function became increasingly impaired although hepatic function remained preserved. Because of the hypothesized role of porto-systemic shunting in the aetiology of this syndrome, the surgical shunt was successfully reversed angiographically. No improvement in dyspnoea or oxygen saturation occurred and liver transplantation was undertaken. Six months post-transplant, the patient has decreased his oxygen requirements and is free of dyspnoea. Our experience supports the causal role of porto-systemic shunting in the pathogenesis of hepatopulmonary syndrome but suggests that merely decreasing the extent of porto-systemic shunting is not beneficial. Liver transplantation remains the only reliable therapeutic modality available to these patients.

Bordetella avium virulence measured in vivo and in vitro.

Bordetella avium causes an upper-respiratory-tract disease called bordetellosis in birds. Bordetellosis shares many of the clinical and histopathological features of disease caused in mammals by Bordetella pertussis and Bordetella bronchiseptica. In this study we determined several parameters of infection in the domestic turkey, Meleagris galapavo, and compared these in vivo findings with an in vitro measure of adherence using turkey tracheal rings. In the in vivo experiments, we determined the effects of age, group size, infection duration, and interindividual spread of B. avium. Also, the effect of host genetic background on susceptibility was tested in the five major commercial turkey lines by infecting each with the parental B. avium strain and three B. avium insertion mutants. The mutant strains lacked either motility, the ability to agglutinate guinea pig erythrocytes, or the ability to produce dermonecrotic toxin. The susceptibilities of 1-day-old and 1-week-old turkeys to B. avium were the same, and challenge group size (5, 8, or 10 birds) had no effect upon the 50% infectious dose. Two weeks between inoculation and tracheal culture was optimal, since an avirulent mutant (unable to produce dermonecrotic toxin) persisted for a shorter time. Communicability of the B. avium parental strain between confined birds was modest, but a nonmotile mutant was less able to spread between birds. There were no host-associated differences in susceptibility to the parental strain and the three B. avium mutant strains just mentioned: in all turkey lines tested, the dermonecrotic toxin- and hemagglutination-negative mutants were avirulent whereas the nonmotile mutants showed no loss of virulence. Interestingly, the ability of a strain to cause disease in vivo correlated completely with its ability to adhere to ciliated tracheal cells in vitro.

Characterization of BrkA expression in Bordetella bronchiseptica.

BrkA confers resistance to killing by complement in Bordetella pertussis. Complement resistance in Bordetella bronchiseptica was examined. Four B. bronchiseptica strains possessed the brkA gene; however, only three expressed the protein. Only the strain lacking BrkA was susceptible to complement. Introduction of the B. pertussis brkA gene restored BrkA expression to this strain but did not confer resistance. brkA was mutated in the strains that naturally expressed BrkA, and loss of BrkA did not confer sensitivity to complement. As a species, B. bronchiseptica is more resistant to complement than B. pertussis, and BrkA does not mediate resistance.

Serum resistance in bvg-regulated mutants of Bordetella pertussis.

Serum resistance, or resistance to killing by antibody dependent pathway of complement, in Bordetella pertussis is bvg-regulated and the Bordetella resistance to killing (brk) locus mediates much of the resistance. Here we examined whether other bvg-regulated proteins contribute to serum resistance. We found that neither pertussis toxin, adenylate cyclase toxin, filamentous hemagglutinin, dermonecrotic toxin, tracheal colonization factor, nor Vag8 mutants were sensitive to serum killing compared to the wild-type. Filamentous hemagglutinin has been reported to bind C4 binding protein, an inhibitor of complement, but this activity does not appear to contribute to serum resistance, as evidenced by the resistant phenotype of FHA mutants. Clinical isolates were serum resistant and wild-type strains possessing an additional copy of the brk locus were 2-5-fold more resistant to serum killing.

Massive bleeding from multiple gastric ulcerations in a patient with lymphocytic gastritis and celiac sprue.

Uncontrolled hemorrhage and multisystem organ failure developed in a patient with celiac sprue, lymphocytic gastritis, and diffuse gastric ulceration. A proximal small bowel biopsy showed villous atrophy and lymphoplasmacytic infiltration consistent with celiac sprue. At autopsy, there were no gross or histologic findings to suggest lymphoma. The intestinal lymphocytic infiltrate was not monoclonal, and gene rearrangements were not detected. Lymphocytic gastritis is a rare cause of upper gastrointestinal hemorrhage, which may be the result of sensitivity to gluten or other luminal antigens. This diagnosis should be considered in cases of diffuse gastric ulceration with bleeding in which the endoscopic appearances are not typical of peptic ulcer disease or drug-induced erosions. Ideally, biopsies of gastric and duodenal mucosa should be performed to establish the diagnosis.

Implanted functional electrical stimulation hand system in adolescents with spinal injuries: an evaluation.

OBJECTIVE: To study the utility and functional benefits of an implanted functional electrical stimulation (FES) system for hand grasp and release in adolescents with tetraplegia secondary to spinal cord injuries. DESIGN: Intervention study with before-after trial measurement with each subject as his or her own control. SETTING: Nonprofit pediatric orthopedic rehabilitation facility specializing in spinal cord injury. PARTICIPANTS: A convenience sample of five adolescents between 16 and 18 years of age with C5 or C6 level tetraplegia at least 1 year after traumatic spinal cord injury. Key muscles for palmar and lateral grasp and release were excitable by electrical stimulation. INTERVENTIONS: A multichannel stimulator/receiver and eight electrodes were surgically implanted to provide stimulated palmar and lateral grasp and release. In conjunction with implantation of the FES hand system, surgical reconstruction in the form of tendon transfers, tendon lengthenings and releases, and joint arthrodeses was performed to augment stimulated hand function. Rehabilitation of the tendon transfers and training in the use of the FES hand system were provided. MAIN OUTCOME MEASURES: Measurements of pinch and grasp force, the Grasp and Release Test (GRT), and an assessment of six activities of daily living (ADL) were administered before implantation of the FES hand system and at regular follow-up intervals. Results of the stimulated response of individual muscles and surgical reconstruction were evaluated using standard and stimulated muscle testing techniques and standard assessment of joint range of motion. All subjects completed followup testing. RESULTS: Lateral and palmar forces were significantly greater than baseline forces (p = .043). Heavy objects on the GRT could only be manipulated with FES, and FES increased the level of independence in 25 of 30 ADL comparisons (5 subjects, 6 activities) as compared to baseline. After training, FES was preferred in 21 of 30 comparisons over the typical means of task completion. Of the 40 electrodes implanted, 37 continue to provide excellent stimulated responses and all of the implanted stimulators have functioned without problems. The surgical reconstruction procedures greatly enhanced FES hand function by either expanding the workspace in which to utilize FES (deltoid to triceps transfer), stabilizing the wrist (brachioradialis to wrist extensor transfer), or stabilizing joints (intrinsic tenodesis transfer, FPL split transfer). CONCLUSION: For five adolescents with tetraplegia, the combination of FES and surgical reconstruction provided active palmar and lateral grasp and release. Laboratory-based assessments demonstrated that the FES system increased pinch force, improved the manipulation of objects, and typically increased independence in six standard ADL as compared to pre-FES hand function. The study also showed that the five adolescents generally preferred FES for most of the ADL tested. Data on the benefits of the implanted FES hand system outside of the laboratory are needed to understand the full potential of FES.

Does patient self-reported data have a role in the longitudinal study of Parkinson's disease? An analysis of the PROPATHtrade mark database.

Patient-reported data can be a valuable, adjunctive tool in helping physicians assess the quality-of-life and functional changes in patients with Parkinson's disease (PD). We analyzed and evaluated the largest PD patient-reported database (PROPATHtrade mark) relative to prescribing habits and changes in reported outcomes by therapy. Our analysis included 1436 patients, followed for 1 yr, who completed a series of questionnaires assessing medication therapy, daily activity scores adapted from the Unified Parkinson's Disease Rating Scale (UPDRS), global disease visual analogue scale, and health resources consumption. Our results indicated that physicians are prescribing Eldepryl(R) with increased frequency in patients with early and mild PD. Patients receiving Eldepryl alone or in combination with Sinemet(R) reported better outcomes than those receiving Sinemet monotherapy. There was a great deal of variability in the reported utilization of healthcare resources by patients in the PROPATH program and, thus, no statistical differences were noted for patients treated with different regimens. We conclude that the adjunctive use of longitudinal patient self-reported data programs such as PROPATH can help assess and improve overall patient outcomes. Future controlled studies should be conducted to further evaluate the roles of alternative therapies and patient-reported data in improving quality-of-life and outcomes for PD patients.