Tuberculin-active carbohydrate that induces inhibition of macrophage migration but not lymphocyte transformation.

A tuberculin carbohydrate fraction, GAE, in sensitized animals induced a delayed type of skin reactivity and inhibited the migration of macrophages but failed to stimulate lymphocyte transformation in vitro. Tuberculin protein-containing fractions were active in each test. These results show that in vitro lymphocyte transformation is not necessarily a corollary of delayed type hypersensitivity.

Enhancement of delayed hypersensitivity inflammatory reactions in guinea pig skin by 12(R)-hydroxy-5,8,14-eicosatrienoic acid.

Delayed-type hypersensitivity (DTH) reactions are initiated by sensitized T cells. Their progression is dependent upon the local release of various autacoids, including cytokines and eicosanoids, by T cells, infiltrating inflammatory cells, and resident tissue cells. 12(R)-hydroxy-5,8,14-eicosatrienoic acid [12(R)-HETrE], an eicosanoid produced by skin and cornea, possesses potent proinflammatory properties at picomolar concentrations including vasodilation, increase in membrane permeability, neutrophil chemotaxis, and angiogenesis. Because DTH reactions are associated with many of these same phenomena, we examined the effect of 12(R)-HETrE and related 12-hydroxyeicosanoids on the expression of DTH to purified protein derivative of tuberculin in sensitized guinea pigs. In the absence of purified protein derivative of tuberculin, none of the eicosanoids evoked erythema or edema after intradermal injection at doses up to 100 pmol. When injected together with purified protein derivative of tuberculin, 12(R)-hydroxy-5,8,10,14-eicosatetraenoic acid [12(R)-HETE], but not its enantiomer 12(S)-HETE, significantly inhibited macroscopic expression of delayed reactivity (erythema) only at the highest dose tested, 10 pmol. In contrast, 12(R)-HETrE significantly enhanced expression of DTH at doses between 1 fmol and 1 pmol (50% and 30% increases above control, respectively). Its stereoisomer, 12(S)-HETrE, did not enhance DTH at any tested dose, but was able to block the activity of 12(R)-HETrE when injected simultaneously. Enhancement or inhibition of visible skin responses was not associated with qualitative or quantitative changes in cellular infiltrates at the reaction site. 12(R)-HETrE had no effect on the nonimmunologic inflammatory skin reaction induced by phorbol myristate acetate, suggesting selectivity toward DTH. We conclude that 12(R)-HETrE enhances DTH via a yet to be determined mechanism and that its stereoisomer, 12(S)-HETrE, may be a useful antagonist for studying the inflammatory actions of this eicosanoid.

Induction of specific anti-guinea pig T cell sera in rabbits.

In an attempt to increase the specificity of antisera raised in rabbits against strain 2 guinea pig thymocytes and brain, the rabbits were screened for titres of natural antibodies to thymocytes and other lymphocytes. Although unimmunized rabbits commonly had moderate titres of cytotoxic antibodies to guinea pig thymocytes, occasional animals had low titres to thymocytes and moderate titres to bone marrow cells. Intravenous immunization of this latter group of rabbits with thymocytes led to the production of high titred anti-thymocyte sera which were easily made specific for thymus-derived lymphocytes (T cells) by absorption with L2 C lymphoma, a bone marrow-derived lymphoma of strain 2 guinea pigs. Sera raised against guinea pig brain in complete Freund's adjuvant which had high titres of antibodies to both thymocytes and bone marrow cells could be made specific for T cells only with great difficulty. The cytotoxic activity of the anti-T cell serum could be absorbed by strain 2 thymocytes and brain homogenates, while high dilutions of this serum inhibited the formation of spontaneous rosettes between guinea pig lymphoid cells and normal rabbit erythrocytes.

Clonal analysis of the T-cell response of mice to herpes simplex virus: correlation between lymphokine production in vitro and the induction of delayed-type hypersensitivity and antiviral activity in vivo.

The properties of two morphologically distinct L3T4+, Lyt2- "helper" T-cell clones specific for herpes simplex virus were investigated. Both of the clones produced IL-3 and interferon, but neither produced IL-2. Clone D6.6 produced macrophage agglutinating factor, a fibronectin-like lymphokine, and also a delayed hypersensitivity (DH) response when injected locally into syngeneic mice. Despite the presence of a DH producing clone and a non-DH producing clone, both were able to reduce the local virus titre to an equivalent degree. It is suggested that this protective activity is associated with the production of interferon-gamma. The significance of these results to mechanisms of protection against herpes simplex virus in vivo is discussed.

Murine alpha-2-macroglobulin: localization on a subpopulation of macrophages.

We have previously shown that mouse peritoneal macrophages synthesize and secrete alpha-2-macroglobulin (alpha 2M) in culture. We have now examined whether alpha 2M delineates a subpopulation of murine macrophages. Mouse alpha 2M was purified from plasma by gel filtration and immunoabsorption to remove IgM. Purified alpha 2M was an active proteinase inhibitor, had a pI of 4.8, and a relative molecular mass (Mr) of 765 kD which dropped to 194 kD on reduction. Antisera raised against mouse alpha 2M were not cross-reactive with guinea pig or human alpha 2M, or with antigens in guinea pig, human, bovine or equine serum. One to 18 per cent of freshly isolated bone marrow, resident peritoneal or induced peritoneal macrophages displayed immunoreactive cytoplasmic and surface alpha 2M as detected by single cell immunoperoxidase assay or flow cytometry. alpha 2M was not detected in or on thymocytes or bone marrow lymphocytes. In culture, bone marrow-derived macrophages displayed peak levels of cytoplasmic alpha 2M on day 7 and two peaks of surface alpha 2M on days 2-3 and 5-7. The fact that alpha 2M can function as an anti-proteinase, can modulate lymphokine production and is present on inflammatory macrophages suggests a regulatory role for macrophages bearing and secreting this molecule at tissue sites of inflammation.

Characterization of lymphokine fibronectin from guinea pig lymphoid cell culture supernatants.

Fibronectins (FN) in guinea pig lymphoid cell culture supernatants have been studied using a panel of polyclonal and monoclonal anti-FN antibodies to clarify their relationship with macrophage agglutination factor (MAggF), an inflammatory lymphokine sharing many properties with this family of high molecular weight glycoproteins. MAggF contained cellular FN epitopes, and was reversibly bound by antibodies specific for cellular FN. Enzyme-linked immunoassay and inhibition of MAggF activity by monoclonal anti-plasma FN antibodies revealed immunoreactive FN in guinea pig lymphoid cell culture supernatants to share three epitopes with plasma FN and to lack a fourth epitope present in plasma FN. Immunoreactive FN in gelatin-affinity purified lymph node cell culture supernatants was polydisperse; MAggF activity (Mr 410 kD) was associated with only 13% of total immunoreactive FN. Although a low molecular weight FN fragment (Mr 67 kD) was associated with MAggF activity in salt-fractionated peritoneal exudate culture supernatants, it was not possible to generate MAggF activity by limited proteolysis of MAggF-inactive, high molecular weight FN in lymph node cell culture supernatants. We conclude that MAggF is a cellular FN containing a number of epitopes in common with plasma FN and suggest it may be a unique species of cellular FN produced by T lymphocytes involved in initiating delayed hypersensitivity reactions.

Parlin, a general microcomputer program for parallel-line analysis of bioassays.

Commonly used manual and calculator methods for analysis of clinically important parallel-line bioassays are subject to operator bias and provide neither confidence limits for the results nor any indication of their validity. To remedy this, the authors have written a general program for statistical analysis of these bioassays for the IBM Personal Computer and its compatibles. The program has been used for analysis of bioassays for specific coagulation factors and inflammatory lymphokines and for radioimmunoassays for prostaglandins. The program offers a choice of no transform, logarithmic, or logit transformation of data, which are fitted to parallel lines for standard and unknown. It analyzes the fit for parallelism and linearity with an F test, and calculates the best estimate of the result and its 95% confidence limits. Comparison of results calculated by PARLIN with those previously obtained manually shows excellent correlation (r greater than 0.99). Results obtained using PARLIN are quickly available with current assay technics and provide a complete evaluation of the bioassay at no increase in cost.

Identification and mapping of a chromosomal gene cluster of Borrelia burgdorferi containing genes expressed in vivo.

A clone containing a 6.4 kb Borrelia burgdorferi chromosomal DNA insert reacted only with sera from patients with Lyme disease and not with any normal human or rabbit sera. Restriction enzyme analysis indicated that this DNA fragment was located on the B. burgdorferi chromosomal map between rpoB and p22A; its direction of transcription was towards p22A. Sequence analysis suggests that LA006 encodes six proteins: three previously described immunodominant lipoproteins of the 39 kDa Bmp protein family, BmpA, BmpB and BmpC; a 51 kDa MgtE magnesium transporter protein; a 16 kDa protein kinase C inhibitor; and a 56 kDa protein with similarity to an uncharacterized Escherichia coli chromosomal open reading frame.

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.

Cloning and DNA sequence analysis of bmpC, a gene encoding a potential membrane lipoprotein of Borrelia burgdorferi.

Immunoscreening of a lambda gt11 genomic library of Borrelia burgdorferi expressed in Escherichia coli permitted detection of a clone containing a partial sequence of a B. burgdorferi gene encoding a protein with significant homology to TmpC of Treponema pallidum. Subsequent cloning and DNA sequence analysis revealed an open reading frame encoding a protein with 353 amino acid residues. The open reading frame is preceded by putative promoter sequences and a ribosome binding site, and is initiated with a TTG. The putative protein shares 26% identity with TmpC, contains a signal peptidase II sequence, and is also homologous to the gene products of the recently described bmpA and bmpB of B. burgdorferi. This gene has been designated bmpC. Additional sequencing and restriction analysis indicate that it is located at approximately 400 kbp on the chromosomal map of B. burgdorferi, immediately upstream of bmpA and bmpB.

A novel non-ataxic guinea pig strain with cerebrocortical and cerebellar abnormalities.

This is the first description of GS guinea pigs, a partially inbred, non-ataxic, albino Peruvian (long-hair) strain with abnormal motor behavior and seizures. GS guinea pigs show gross and microscopic cerebellar and microscopic cerebrocortical abnormalities compared to Hartley strain animals. There is little difference between GS and short-hair guinea pig strains in Purkinje cell function, electrically evoked Ca2+ transients or immune responsiveness. The GS strain may prove useful in studying altered functions of the cerebellum in human disease.

The role of alcohol-related expectancies in the prediction of drinking behaviour in a simulated social interaction.

This study investigated the relationship between socially-relevant alcohol-related beliefs and drinking behaviour in a laboratory-based study. Ninety male subjects were told they would be videotaped while completing the Simulated Social Interaction Test; they were then assigned to one of three conditions. In the first, alcohol consumption prior to the interaction was optional, in the second, a standard dose was administered, and in the third, no alcohol was offered. The outcome measures included drinking behaviour and rated level of social skill. In the optional condition, beliefs that alcohol would reduce anxiety in social situations and enhance social competence predicted amount consumed. The decision to continue drinking after the compulsory dose was predicted by anxiety level. In the no alcohol condition, rated social skill was negatively correlated with the beliefs that alcohol reduces tension and reduces fear of negative evaluation. Results are discussed with reference to social learning approaches to understanding drinking behaviour.

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.

Interventions for amnesics: a review.

Research on interventions for amnesics is reviewed which suggests that it is possible to teach amnesics specific information or memory skills but that these are not likely to generalize or to be maintained spontaneously. It is suggested that interventions with clearly defined behavioural goals and which have programmed for generalization and maintenance of treatment gains may prove to be most effective. The purpose of interventions for chronically hospitalized cognitively impaired patients does not appear to have been considered adequately, and it is concluded that future research will be hampered unless greater consideration is given to information processing theory relating to amnesia. Possible directions for future research are discussed.

The assessment of memory impairment: the relationship between different methods of evaluating dysmnesic deficits.

The relationship between three different methods of assessing memory impairment: cognitive tests, questionnaires, and observational rating scales, was assessed in 20 dysmnesic alcoholic in-patients. The cognitive tests were subdivided into those which measured everyday memory skills and those which were traditional experimental-laboratory tasks. All three methods were highly intercorrelated and the correlation between practical and experimental cognitive tests was also high. Memory test scores were not as highly correlated with either performance on perceptual-motor tasks or full-scale IQ.

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.

First impressions count: a controlled investigation of social skill following closed head injury.

This study examined social skills in 14 closed head injured (CHI) patients who were assessed at four months post-injury and compared with 19 orthopaedic control (OC) patients. Social skills deficits were found to be more common in the CHI patients, of whom over half were classified as socially unskilled. CHI patients displayed poorer social skills in the earlier (but not later) part of an extended social interaction, indicating that they make a poor first impression. It is suggested that poor initial impression formation skills may be one reason why CHI patients fail to establish and maintain friendships. Implications of these findings for patient management are discussed. Close others of CHI patients reported higher levels of mood disturbance. Higher levels of close other hostility were associated with social skills deficits in CHI patients.

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.

Cognitive rehabilitation of amnesic alcoholics: a twelve month follow-up study.

The rehabilitation status of amnesic alcoholic patients who previously participated in either a memory training programme, an activation control programme, or an assessment only condition, was determined at a 12 month follow-up. An earlier study found a non-specific improvement in memory functioning for patients in both group programmes. These patients were also found to be more likely to be discharged from hospital at the 12 month follow-up. It is suggested that further attempts to rehabilitate brain-damaged alcoholics may be justified.

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.