Complementation of a nonmotile flaB mutant of Borrelia burgdorferi by chromosomal integration of a plasmid containing a wild-type flaB allele.

With the recent identification of antibiotic resistance phenotypes, the use of reporter genes, the isolation of null mutants by insertional inactivation, and the development of extrachromosomal cloning vectors, genetic analysis of Borrelia burgdorferi is becoming a reality. A previously described nonmotile, rod-shaped, kanamycin-resistant B. burgdorferi flaB::Km null mutant was complemented by electroporation with the erythromycin resistance plasmid pED3 (a pGK12 derivative) containing the wild-type flaB sequence and 366 bp upstream from its initiation codon. The resulting MS17 clone possessed erythromycin and kanamycin resistance, flat-wave morphology, and microscopic and macroscopic motility. Several other electroporations with plasmids containing wild-type flaB and various lengths (198, 366, or 762 bp) of sequence upstream from the flaB gene starting codon did not lead to functional restoration of the nonmotile flaB null mutant. DNA hybridization, PCR analysis, and sequencing indicated that the wild-type flaB gene in nonmotile clones was present in the introduced extrachromosomal plasmids, while the motile MS17 clone was a merodiploid containing single tandem chromosomal copies of mutated flaB::Km and wild-type flaB with a 366-bp sequence upstream from its starting codon. Complementation was thus achieved only when wild-type flaB was inserted into the borrelial chromosome. Several possible mechanisms for the failure of complementation for extrachromosomally located flaB are discussed.

Utility of a demographic equation in detecting impaired NART performance after TBI.

OBJECTIVES: This study applies a demographic regression equation devised by Crawford, Allan, Cochrane, and Parker (1990) to determine its utility in New Zealand, and to determine the proportion of persons with traumatic brain injury (TBI) with impaired performance on the National Adult Reading Test (NART). METHOD: The NART was administered to 80 community participants, 65 people with traumatic brain injury (TBI), and 27 orthopaedic controls. The Crawford et al. (1990) equation was applied to all three samples. RESULTS: The Crawford et al. (1990) equation was applicable in the New Zealand community sample. It was found that 30% of the TBI sample had impaired performance on the NART. Using corrected NART scores, a significantly larger proportion of the TBI sample was detected as intellectually impaired than in the control sample. CONCLUSION: Nearly one-third of the TBI sample evidenced impaired performance on the NART. Consequently, clinicians that use the NART in assessing people with TBI should use the demographic equation to check the accuracy of NART performances, and correct the NART scores if this is indicated.

Combinatorial use of antibodies to secreted mycobacterial proteins in a host immune system-independent test for tuberculosis.

Laboratory diagnosis of tuberculosis is often difficult. Immunodetection of circulating Mycobacterium tuberculosis proteins shed during active infection would not depend on an intact host immune response and could take advantage of the speed and low costs afforded by antibody-based assays. We previously showed that patients with active tuberculosis had increased levels of circulating antigen 85 (Ag85) proteins independent of their tuberculin skin test status (S. I. Bentley-Hibbert, X. Quan, T. Newman, K. Huygen, and H. P. Godfrey, Infect. Immun. 67:581-588, 1999). To extend these observations to a Mycobacterium bovis BCG-vaccinated population and to another secreted mycobacterial protein, Ag85 and PstS-1 (protein antigen B, p38 antigen) were quantified in sera from 97 Chilean tuberculosis patients and healthy controls (many of whom had received BCG as children) using dot immunobinding, mouse monoclonal anti-BCG Ag85 complex antibody, and chicken antipeptide antibodies reactive with M. tuberculosis Ag85B and PstS-1. The latter antibodies had been raised to peptide-derived immunogens expressed on a novel proprietary protein carrier in Escherichia coli. Median serum Ag85 levels measured by using either anti-Ag85 antibody were significantly higher in patients with active tuberculosis than in healthy controls (P, <0.001 to 0.01); the median serum PstS-1 levels were similar in patients and controls. The sensitivity of significantly elevated circulating Ag85 levels in patients with pulmonary tuberculosis measured by anti-Ag85 complex or anti-Ag85B antibodies was 60 and 55%, respectively, but increased to 77% when results obtained with both anti-Ag85 antibodies were considered jointly (P < 0.02). The corresponding specificities for individual and joint consideration were 95, 85, and 80%, respectively. These results indicate that elevated Ag85 levels can be detected in patients with active tuberculosis even after BCG vaccination and suggest that combinatorial use of antibodies directed at different epitopes of this protein could provide a viable strategy for developing new host immune response-independent diagnostic tests for tuberculosis.

The use of hairless (IAF/HA-HO) guinea pigs for the determination of delayed-type hypersensitivity to tuberculin.

Guinea pigs are a classic animal model for studying delayed-type hypersensitivity (DTH) reactions. However, skin irritation due to hair removal can interfere with the evaluation of the modulation of these responses by various mediators. A DTH model using hairless (IAF/HA-HO) guinea pigs, sensitized with complete Freund's adjuvant and repeatedly skin tested with tuberculin, purified protein derivative, (PPD) has therefore been developed. At 10 weeks after sensitization, intradermal PPD elicited minimal erythema at 6 h, which increased over the next 18 h to a maximum at 24 h, and declined by 48 h. The response could be quantified by bioassay using graded doses of PPD. Reactions at 24 h were characterized by predominantly mononuclear cell deep and superficial dermal infiltrates. Dermal DTH in hairless guinea pigs is thus, grossly and histologically similar to that seen in Hartley guinea pigs.

Caregiver depression following traumatic brain injury (TBI): a consequence of adverse effects on family members?

Many studies have demonstrated that the behaviour of individuals with traumatic brain injury (TBI) predicts the emotional adjustment of their caregivers. The primary objective of the present study was to obtain an understanding of potential moderating and mediating variables between carer depression and analogous stressors. Seven sets of predictor variables (demographic variables, concurrent stressful life events, behavioural problems, social role problems, extent of adverse effects on family members, appraisal, and support) and the criterion variable of depression in caregivers were examined. Fifty-eight carers participated in the study at 6 months, 1 year, 2 years, or 3 years following injury. The number of adverse effects on family members (other than the informant) was the only stressor significantly related to carer depression. However, carer appraisal of adverse family effects was found to mediate the relationship between stressor and depression, and carer perception of support effectiveness was found to moderate the effect of adverse family effects on depression. Forty-six per cent of the variance in caregiver depression was accounted for by carers appraisal of adverse family effects and the interaction of adverse family effects and support effectiveness. These findings highlight the importance of supporting families as a whole in the rehabilitation of persons with TBI.

Restricted growth of ent(-) and tonB mutants of Salmonella enterica serovar Typhi in human Mono Mac 6 monocytic cells.

Monocytes and macrophages are an important host defense in humans infected with Salmonella enterica serovar Typhi. Bacterial ability to survive in these cells is therefore a crucial virulence characteristic of this pathogen. In this study, we demonstrate that growth of a Salmonella enterica serovar Typhi enterochelin synthesis mutant and a tonB mutant in the human monocyte cell line Mono Mac 6 is restricted compared to that of the parental wild-type Ty2 strain. These results suggest that enterochelin- and TonB-mediated iron uptake plays a role in S. enterica serovar Typhi pathogenesis, and also suggest that mutations in iron uptake may attenuate S. enterica serovar Typhi strains for human beings.

Serum antigen 85 levels in adjunct testing for active mycobacterial infections in orangutans.

Diagnosis of active mycobacterial disease in orangutans (Pongo pygmaeus) has been impeded by high levels of non-specific intradermal skin test reactivity to mycobacterial antigens. This may be due in part to cross reactivity between antigens, tuberculin concentrations used or other species-specific factors. Antigen 85 (Ag85) complex proteins are major secretory products of actively growing mycobacteria, and measurement of serum Ag85 could provide a method for determining active mycobacterial infections that was not dependent on host immunity. Serum Ag85 was measured by dot-immunobinding assay using monoclonal anti-Ag85, purified Ag85 standard and enhanced chemiluminescence technology in coded serum samples from 14 captive orangutans from a zoo in Colorado, 15 semi-captive orangutans in Malaysia, and 19 free-ranging wild orangutans in Malaysia. Orangutans from Colorado (USA) were culture negative for Mycobacterium tuberculosis and M. avium, although all had laboratory suspicion or evidence of mycobacterial infection; median serum Ag85 was 10 microU/ml (range, <0.25-630 microU/ml). Of the semi-captive orangutans, six were skin test reactive and two were culture positive for M. avium on necropsy. Median serum Ag85 for this group was 1,880 microU/ml (0.75-7,000 microU/ml), significantly higher than that of Colorado zoo or free-ranging Malaysian orangutans. Median serum Ag85 in the latter group was 125 microU/ml (range, 0.75-2,500 microU/ml). These data suggest that suggest that additional studies using more specific reagents and more samples from animals of known status are appropriate.

The impact of coping on emotional adjustment to spinal cord injury (SCI): review of the literature and application of a stress appraisal and coping formulation.

OBJECTIVES: This article reviews literature examining the psychological adjustment to Spinal Cord Injury (SCI) and illustrates the applicability of a Stress Appraisal and Coping model (SAC) for rehabilitation of this population. METHOD: Articles concerning psychological adjustment to SCI from the previous three decades have been reviewed and critiqued. When possible the articles have been discussed in a SAC framework. RESULTS: The literature indicates that psychological adjustment to SCI is largely predictable from psychological variables including coping, appraisal and psychosocial resources. Recent literature has suggested psychological intervention can promote positive psychological adjustment following SCI for those individuals at risk of developing clinical levels of depression. CONCLUSION: The SAC model provides a comprehensive formulation to incorporate the heterogeneity of populations with SCI. Suggestions for future research include developing assessment and treatment regimes specifically tailored to the strengths and weaknesses of an individual as highlighted in the model.

Analysis of the bmp gene family in Borrelia burgdorferi sensu lato.

BmpA, BmpB, BmpC, and BmpD are homologous Borrelia burgdorferi lipoproteins of unknown functions, encoded by the bmp genes of paralogous chromosomal gene family 36. At least some of the Bmp proteins are immunogens in infected vertebrate hosts. The genetic organization of the bmp region has been characterized for a variety of B. burgdorferi sensu lato strains by Southern hybridization, PCR amplification, and DNA sequencing. All four bmp genes were present in the same relative order in all B. burgdorferi sensu lato low- and high-passage-number isolates. While there were no differences in the relative orders of the bmp genes in these species, variations in DNA sequence in the bmpD-bmpC and bmpC-bmpA intergenic regions were significantly more common than in the corresponding 3' bmpD and bmpC coding regions. The genetic structure of the chromosomal region containing the bmp genes thus appears to be well conserved across different species of B. burgdorferi, but variations in DNA fine structure that prevent PCR primer annealing may occur in this region and make Southern hybridization much more reliable than PCR for detection of the presence of these genes. Our results also suggest that bmp gene products may be used as reagents in the preparation of vaccines and diagnostic assays to protect against and diagnose Lyme disease produced by B. burgdorferi sensu lato.

Determining clinically meaningful cognitive impairment following traumatic brain injury.

AIMS: To study the reliability and validity of ratings of: neuropsychological impairment with test data from traumatically brain-injured patients. METHODS: Neuropsychological test results from 66 traumatically brain-injured adults and 27 orthopaedic controls were rated for neuropsychological impairment by an experienced neuropsychologist and three undergraduate students provided with brief training in test interpretation. Ratings were based on the discrepancy between a patient's current cognitive level and their estimated premorbid cognitive level. Decision making rules were utilised in making the ratings. The raters, who were blind with respect to the patients diagnostic group membership, independently rated test results. Test results were rerated using the same method approximately two weeks later. RESULTS: The ratings of novice raters were in good to very good agreement with the ratings of an expert. All raters evidenced very good to excellent test-retest reliability. Higher rates of neuropsychological impairment were found in the traumatically brain-injured group than in the orthopaedic control group. Discriminant function analysis suggested that raters employed information from all neuropsychological measures (with the exception of current intellectual level), in making their overall ratings of neuropsychological impairment. CONCLUSION: These findings provide encouraging preliminary evidence in support of the reliability and validity of individual case-based ratings of neuropsychological impairment. The high false positive rate in the control sample may reflect the relatively low specificity of neuropsychological impairment.

Pathophysiology of antigen 85 in patients with active tuberculosis: antigen 85 circulates as complexes with fibronectin and immunoglobulin G.

Antigen 85 (Ag85) complex proteins are major secretory products of Mycobacterium tuberculosis and induce strong cellular and humoral immune responses in infected experimental animals and human beings. We have previously shown that nanogram doses of these 30- to 32-kDa fibronectin-binding proteins inhibit local expression of delayed hypersensitivity by a T-cell fibronectin-dependent mechanism. Circulating levels of Ag85 might be expected to be elevated in patients with active tuberculosis and possibly to play a role in systemic anergy in these patients. To test this hypothesis, Ag85 was measured in serum and urine by a monoclonal antibody-based dot immunobinding assay in 56 patients and controls with known skin test reactivity. Median serum Ag85 levels were 50- to 150-fold higher in patients with active tuberculosis than in patients with active M. avium-intracellulare disease or other nontuberculous pulmonary disease or in healthy controls (P < 0.001). The median and range of serum Ag85 in patients with active tuberculosis was not significantly different between skin test-positive and -negative subjects. Patients with active M. avium disease could be distinguished from those with disease due to M. tuberculosis by monoclonal anti-Ag85 antibodies of appropriate specificities. No increases in urinary Ag85 were detected in any patient, regardless of the Ag85 level in serum. Chromatographic analysis and immunoprecipitation studies of serum revealed that Ag85 existed in the serum of these patients complexed to either fibronectin or immunoglobulin G (IgG). Uncomplexed circulating Ag85 was demonstrable in serum from fewer than 20% of patients with active tuberculosis. In patients with active tuberculosis, Ag85 is therefore likely to circulate primarily as complexes with plasma fibronectin and IgG rather than in unbound form. The existence of Ag85 complexes with plasma proteins would account for its lack of urinary clearance.

Detection of elevated levels of circulating antigen 85 by dot immunobinding assay in captive wild animals with tuberculosis.

Antemortem diagnosis of tuberculosis in captive wild animals is often difficult. In addition to the variability of host cellular immune response, which does not always indicate current active infection, reactivity to saprophytic or other mycobacteria is common and may interfere with the interpretation of the intradermal tuberculin skin test. Furthermore, the immobilization required for administering the test and evaluating skin reactions in these animals may result in unacceptable levels of morbidity and mortality, of particular concern in individuals of rare or endangered species. Proteins of the antigen 85 (Ag85) complex are major secretory products of actively metabolizing mycobacteria in vitro. Production of these proteins by mycobacteria during growth in vivo could result in increases in circulating levels of Ag85 in hosts with active tuberculosis. A dot blot immunoassay has been used to detect and quantify circulating Ag85 in captive wild animals with tuberculosis. Elevated levels of Ag85 were observed in animals with active tuberculosis as compared with uninfected animals. Study populations included a herd of nyala (Tragelaphus angasi) (n = 9) with no history of exposure to Mycobacterium bovis. Serum Ag85 levels ranged from <5 to 15 microU/ ml (median, 5 microU/ml). The other group included 11 animals from a mixed collection with a documented history of an M. bovis outbreak. Animals with pulmonary granulomatous lesions (n = 3) had serum Ag85 levels ranging from 320 to 1,280 microU/ml (median, 320 microU/ml). Animals with only chronic mediastinal or mesenteric lymphadenitis (n = 4) had serum Ag85 levels ranging from <5 to 320 microU/ml (median, 52.5 microU/ml). Animals with no lesions present on necropsy (n = 4) had serum Ag85 levels ranging from <5 to 80 microU/ml (median, <5 microU/ml). This assay could provide an important adjunct to intradermal skin testing for antemortem diagnosis of tuberculosis in nondomestic species.

Cognitive dysfunction in patients with pituitary tumour who have been treated with transfrontal or transsphenoidal surgery or medication.

OBJECTIVE: This study was carried out to examine the neuropsychological status of patients treated for pituitary tumour by transfrontal surgery, transphenoidal surgery or medical treatment only, with or without radiotherapy. DESIGN AND MEASUREMENTS: Three groups of 23 patients who had been treated for pituitary tumour were compared with 23 healthy controls on a range of neuropsychological measures. The surgical patients were also subdivided into two groups and compared. The neuropsychological measures were standardized psychological tests designed to assess aspects of attention, memory and executive function. PATIENTS: The patients were those who had been treated with transfrontal surgery (n = 23), transsphenoidal surgery (n = 23) and medication only (n = 23). The groups did not differ with respect to age, education or premorbid ability level as assessed by the National Adult Reading Test. All participants were free of known sources of cognitive impairment other than pituitary tumour. RESULTS: Comparison of the four groups revealed that nearly half of the transfrontal, one-third of the transsphenoidal and one-quarter of the non-surgical group had three or more neuropsychological tests scores below the 10th percentile compared to less than 5% of the controls. Impairments in memory and executive function were found in both surgical groups. The non-surgical patients appeared to have problems only on tasks requiring high levels of cognitive processing. Differences were found between the two surgical groups with respect to the severity of the cognitive impairment, the transfrontal patients having more severe impairment than the transsphenoidal. No significant negative effects on cognitive functioning were associated with radiotherapy; however, transfrontal surgery patients who had not been treated with radiotherapy were found to be more impaired than other patients. This was thought to be related to radical surgery. CONCLUSIONS: Many patients with treated pituitary tumour suffer significant cognitive impairment. The severity and nature of impairment differs between treatment groups, although the cause of this could not be addressed by this study. Recommendations are made for future research and clinical practice.

An animal model of autism: behavioural studies in the GS guinea-pig.

Autism is a human behavioural pathology marked by major difficulties in abnormal socialization, language comprehension and stereotypic motor patterns. These behavioural abnormalities have been associated with corticocerebral and cerebellar abnormalities in autistic patients, particularly in vermal folia VI and VII. Progress in understanding this disease has been hindered by the absence of a non-primate animal model. GS guinea-pigs are a partially inbred, non-ataxic guinea-pig strain with cerebellar and corticocerebral abnormalities similar to those reported to exist in human patients with autism. In order to determine if GS guinea-pigs represent an animal model of autism, their behaviour was compared with that of Hartley strain guinea-pigs. GS animals learned a motor task significantly more rapidly than Hartley guinea-pigs, but performed it in a more stereotypic manner and were less influenced by environmental stimuli than Hartleys. GS animals exhibited significantly less exploratory behaviour in a novel environment and were significantly less responsive to 50-95 dBA pure tones than Hartley guinea-pigs. In a social interaction assay, GS guinea-pigs interacted significantly less frequently with each other or with Hartley guinea-pigs than Hartleys did under the same conditions. GS behaviour thus exhibits autistic-like behaviour patterns: motor stereotypy, lack of exploration and response to environment and poor social interaction. Coupled with the neuropathological findings, this abnormal behaviour suggests that GS guinea-pigs could be a useful animal model of autism.

Caring for a family member with a traumatic brain injury.

The responses to a questionnaire on subjective burden are reported for 52 primary caregivers of a group of persons with traumatic brain injuries sustained an average of 6 years previously. The aim of the study was to examine satisfaction with social support, perception of coping skills, and appraisal of symptoms as predictors of strain in the carers. A range of responses, both positive and negative, to the work of caring for a relative with a head injury was reported. A high prevalence rate of emotional and behavioural changes in the persons with head injuries was found and the amount of distress caused by these symptoms was found to be predictive of burden. The other factor important in predicting burden was the carers' ratings of their satisfaction with their ability to cope with the work of caregiving. Social support, injury severity, and the demographic characteristics of the persons with head injury and their carers were not significant predictors. Depression in the carers was also investigated and the variable most predictive of elevated depression scores was coping satisfaction. These findings reinforce the importance of strengthening carers coping resources in rehabilitation work with head injured persons and their families.

Conversation with traumatically brain-injured individuals: a controlled study of behavioural changes and their impact.

The conversations of 62 traumatically brain-injured (TBI) patients, assessed between 6 months and 3 years post-injury, were compared with those of an orthopaedic control (OC) group (n = 25). Conversations involving TBI subjects were rated as significantly less interesting, less appropriate, less rewarding and more effortful than interactions involving OC subjects, and were characterized by differences in the frequency of prompt usage and turn duration. Furthermore, measures of turn duration and prompt frequency were significantly associated with the perceived quality of conversation. These findings provide a microbehavioural description of the social process through which TBI individuals fail to adequately reinforce others.

Psychosocial consequences of caring for a spouse with multiple sclerosis.

Fifty-five spousal caregivers of persons with multiple sclerosis completed a questionnaire assessing the stress associated with caregiving. They were found to experience a range of negative effects, similar to those reported by other groups of carers of persons with degenerative neurological diseases. The behaviors causing most distress to the carers were associated with motor problems, sudden mood changes, partner upsetting other people, incontinence and pain. Overall, wives had higher burden scores than husbands. This was accounted for primarily by their higher levels of physical distress such as tiredness. Following the stress-appraisal-coping model of Lazarus and Folkman (1984) it was hypothesized that individual differences in burden would be accounted for primarily by the carers' appraisal of the symptoms, perceived social support, and satisfaction with coping. Regression analyses supported the importance of satisfaction with coping and social support as predictors of perceived burden. In addition, life satisfaction was found to be predicted by burden independently of other predictors.