The constant battle between pathogen and host has led to substantial diversity and adaptability of the host immune system. Pathogens too, have evolved unique mechanisms to evade their hosts. The production of superantigens is one of these mechanisms. Superantigens are potent T cell mitogens that have the unique ability to bind simultaneously to major histocompatibility complex (MHC) class II molecules and T cell receptors (TCRs). The resulting uncontrolled activation of up to 20% of all T cells and the subsequent cytokine release, can lead to fever, shock and death. Superantigens are not processed intracellularly like conventional antigens but instead bind as intact proteins to MHC class II molecules expressed on the surface of professional antigen presenting cells. On the hypothesis that the unique properties of superantigens may serve useful for vaccine delivery, several bacterial superantigens were selectively mutated at their TCR-binding site with the ultimate goal of creating a safe, non-toxic carrier protein that could target antigen presenting cells by binding to MHC class II. Antigen presenting cells that expressed MHC class II were indeed targeted by the TCR-binding-deficient superantigens. Cellular internalisation of the superantigen into vesicles was observed as early as 30 min. These supcrantigens were also shown to traffic to, and be captured by, the lymph nodes of immunised mice. Using TCR-binding-deficicnt superantigens as vaccine carrier proteins, enhanced antigenicity and immunogenicity of the conjugated MHC class I-restricted peptide antigen. GP33, was observed in a mouse model. In vitro studies revealed up to 200-fold enhancement of antigenicity when GP33 was conjugated to superantigen. Enhanced immunogenicity was also observed in vivo, with conjugates providing protection against Lymphocytic choriomeningitis virus infection after only a single immunisation. These results indicate that modified superantigens are able to safely deliver peptides for cross-presentation, and may serve as a novel mechanism for vaccine delivery.

Perinatal asphyxia is thought to be a major cause of subsequent neurological deficits. Pathological studies suggest that many of these events occur before birth. However, the relationship between specific prenatal events and neurological outcome is not clear. This thesis tested the hypothesis that certain fetal factors play a role in determining the severity and distribution of neuronal loss following in utero asphyxia. Chronically instrumented fetal sheep at three different gestational ages; midgestation (90d), late-gestation (120-130d) or near term (> 135d) were subjected to either a single or repeated insult. The insult consisted of an episode of either systemic asphyxia (umbilical cord occlusion) or cerebral ischaemia (carotid artery occlusion). The fetal parietal cortical electroencephalogram (EEG), cortical impedance (CI) indicating intracellular edema, blood pressure (MAP), electrocardiogram (ECG) and frequent fetal blood gases and metabolites were measured. Three days after the insult, histopathological analysis or immunohistochemistry was performed to determine neuronal loss and specific neurotransmitters respectively. Transient (10min) occlusion of the umbilical cord in late-gestation fetuses, resulted in severe fetal asphyxia, hypotension (24±5mmHg, p<0.01), bradycardia (72±14bpm, p<0.001), depressed EEG activity (-17±2dB, p<0.001) and intracellular edema. The intracellular edema resolved within 27±6min, whereas the EEG activity was depressed for 5±2h, despite rapid recovery of pO2. Neither seizures or infarction were observed. The degree of hypotension, increase in CI, lactate and recovery of post-asphyxial EEG intensity were more marked in 135d fetuses compared with the midgestation fetus (p<0.01). Neuronal loss, which was only observed in the older group, was predominantly in the hippocampus and associated with the severity of hypotension during occlusion. Repeated episodes of cerebral ischaemia, altered the distribution of neuronal loss compared with single insults, inducing damage mainly in the striatum. The frequency of the insults determined the severity of the damage. Similarly, recurrent episodes of fetal asphyxia induced predominantly striatal neuronal loss. Each occlusion resulted in fetal hypoxia and bradycardia accompanied by increased T/QRS ratio as noted on the ECG. Progressively severe hypotension and lactic acidosis developed during successive occlusions. The EEG was depressed and CI increased with each occlusion. After the asphyxial episodes, blood pressure and heart rate returned to normal, while the T wave was inverted for 310±60min. The EEG remained depressed for 90±10min and intermittent seizures developed at 3.3±0.6h after the last occlusion. The extent of neuronal loss correlated with the degree of hypotension, increase in T/QRS ratio, duration of post-asphyxial EEG depression and number of seizures. Immunohistochemical analysis showed loss of striatal GABAergic projection neurons. These findings demonstrate that certain prenatal factors, such as neurological maturation, pattern of the insult and cardiovascular instability can influence neuronal outcome following fetal asphyxia. An isolated brief episode of asphyxia can lead to selective hippocampal neuronal loss, while repeated insults induce predominantly striatal damage. These distributions of neuronal loss may be associated with postnatal sequelae such as learning disorders and cerebral palsy.

The use of mental health legislation to determine involuntary treatment for people suffering from mental illness (civil commitment) is a controversial issue, centred on the ability of civil commitment to be coercive by limiting patients’ choice, autonomy and self-determination The intent of the New Zealand Mental Health (Compulsory Assessment and Treatment) Act 1992 was to limit coercion by emphasising informed consent (even if treatment can be administered without it); recognising the civil rights of patients subject to civil commitment; and encouraging involuntary treatment in the least restrictive environment (the community). However, there is no evaluative research that considers the extent to which patients subjected to the legislation perceive coercion. The aim of this thesis was to consider the extent to which mental health legal status equates with coercion, the factors that impact on patients’ perceptions of coercion and the factors that have the potential to ameliorate such perceptions. Empirical cross-sectional comparison studies, measuring perceived coercion using a validated psychometric measure, were undertaken at three points during the implementation of civil commitment. These involved a comparison between involuntary and voluntary patients admitted to acute inpatient psychiatric services, a comparison between involuntary patients admitted to acute psychiatric inpatient services and involuntary patients admitted to forensic psychiatric services, and a comparison between involuntary and voluntary outpatients. The studies found that legal status is only a broad index of the amount of coercion perceived by patients. Some voluntary patients feel coerced and some involuntary patients found the process non-coercive. Perceptions of coercion cannot be fully explained by socio-demographic and clinical characteristics, or by coercive incidents that occur throughout the process of civil commitment. Rather, the perceptions relate to the total experience of civil commitment, including the interactive processes with clinicians. In this regard, involving patients in proceedings that are experienced as fair and just (procedural justice) has a marked impact on reducing patients’ perceptions of coercion. In conclusion, the findings are underscored by legal requirements and ethics in order to provide clinical guidelines for implementing civil commitment.

The temporal and spatial relationships between altered vascular function and cardiac muscle cell injury at the margins of developing myocardial infarcts were investigated, because such knowledge might provide potential for intervention in the evolution of myocardial infarcts and limitation of their size. Regional myocardial ischaemia was modelled in isolated rabbit hearts subjected to ligation of the ventral interventricular branch of the left coronary artery (0,30,60,120 or 240 minutes duration), the remainder of the heart being continuously perfused with oxygenated Krebs-Henseleit buffer solution. After the experimental ischaemic period, the whole heart was fixed by perfusion with phosphate-buffered 2.5% glutaraldehyde which, in preliminary studies, was shown not only to preserve the morphological appearance of cardiac muscle cells, but also to stabilise the distribution of flow through the myocardial blood vessels in the pattern pertaining immediately prior to fixation. Polymerising acrylic resin (L.R. White) was then injected into the vessels of the ischaemic and non-ischaemic regions simultaneously at identical pressures. Resin injected into the ischaemic region contained lead dioxide whilst that injected into the other vessels contained Fat Red 7B dye to allow identification of the source of supply of blood vessels within the heart. Cryofracture, freeze-drying and imaging by scanning electron microscopy (SEM) with a backscattered electron detector and low vacuum specimen chamber conditions were used. This made possible examination of transmural segments of myocardium spanning the margins of the ischaemic and control ventricular myocardium containing blood vessels filled by resin. SEM showed severe injury of cardiac muscle cells after 60 minutes of ischaemia, as characterised by separation and swelling of some organelles. Earlier ischaemic changes in some cells (focal increase in prominence of t-tubules and sarcoplasmic reticulum) were seen after 30 minutes. There was a transmural progression in development of irreversible injury from the subendocardium to the epicardium between 60 and 120 minutes corresponding to the "wavefront phenonenon". The lateral margins did not show such marked progression and were typically sharply demarcated on a cell-to-cell basis after 60 minutes. An increase in the proportion of functional capillary pathways (from 55% to 85%) in early (30 minutes) ischaemia was succeeded by a profound perfusion defect, corresponding to the "no-reflow" phenomenon, which had a very close temporal and spatial association with severe injury of cardiac muscle cells. Loss of patency was associated with increased proportions of collapsed, compressed capillaries and swollen myocardial cells. This study demonstrated that there is a significant region of myocardium which for a period shows intermediate degrees of myocyte injury (and is thus potentially salvageable) in the subepicardial portion of the developing infarct. Contrary to the claims of various authors similar potentially salvageable lateral "border zones" were neither large nor non-existent. Within 150 microns of the typically abrupt boundary, small discontinuous areas (<20% of this region) showed intermediate degrees of injury, and there was also an increased proportion of non-functional capillaries which were not collapsed or compressed, resulting in a 'low-flow' zone. This narrow lateral zone requires further investigation to determine whether it is static, and thus of negligible size, or whether it moves in advance of infarction and is thus pathogenetically significant.

Testicular function is stimulated from the pituitary gland by the gonadotrophic hormones FSH and LH. The testis in turn regulates gonadotrophin secretion by negative feedback. The agents of feedback are steroids, and in addition, probably a protein hormone, which has been given the name inhibin. A method capable of detecting inhibin-like activity in bull seminal plasma (bSP) was developed. Administration of bSP at the time of castration to five-week old male rats inhibited the post-castration rise of serum concentrations of FSH and LH otherwise seen 24 h later. The degree of inhibition depended on the dose; 0.5 ml bSP or the equivalent amount of bSP-extract always suppressed FSH and LH to levels typical of intact rats. The rats' sensitivity to 0.2 ml bSP varied with the time of year, possibly reflecting seasonal changes in the onset of puberty. The time-course of action of bSP-extract was studied in intact rats. Serum FSH was suppressed 12, 24 and 48 h after a single injection, but not at 3 or 6 h. LH was suppressed at 6, 12, 24 and possibly 48 h.

A procedure was developed to purify ovine placental lactogen (oPL) from fetal cotyledonary tissue of late gestation. A prolactin radioreceptor assay using mammary tissue membranes from the late pregnant rabbit was established and used to monitor lactogenic activity throughout the purification.

Chronic Fatigue Syndrome (CFS) is an illness characterized by persistent debilitating fatigue of uncertain origin. Precipitating and perpetuating factors of this illness are thought to be distinct and the aim of this thesis was to gain greater insight into the role of cognitive factors which may maintain the condition. This work was guided by two central frameworks, the self-regulatory model of illness representations and the cognitive taxonomy of psychopathology. These were used to define the different cognitive constructs and to investigate the way they function as a system to maintain pathological schema and disability in CFS. Three studies using different methodologies were conducted to test the hypotheses. The first employed a descriptive comparative design to ascertain whether CFS patients have unique cognitions which contribute to their disability over time. The sample was comprised of CFS patients without depression (n=39), CFS patients with a concurrent diagnosis of depression (n=14), patients with a primary diagnosis of depression (n=20); and healthy controls (n=38). The groups were matched in aggregate for age, gender, race, and education. Subjects completed the Cognitive Errors Questionnaire-Revised, which measures cognitive distortions relevant to both general and somatic events, and the Illness Perception Questionnaire, which measures the five dimensions of the illness representation in conjunction with other standard measures. Between-group analyses confirmed that the depressed group was distinguished by a low self-esteem, feelings of guilt and self-recriminations, the propensity to make cognitive distortions across all situations, and to attribute their illness to internal, stable and global factors. In contrast, the CFS patients were characterized by low ratings of their current health status, a strong illness identity, external attributions for their illness, and distortion in thinking that were specific to somatic experiences. CFS depressed patients had lower self-esteem than non-depressed patients and had the most pessimistic illness beliefs. A six month follow-up showed that CFS patients' cognitive structures and level of disability remained remarkably stable. Illness identity, serious consequences, somatic errors, and limiting coping accounted for a substantial proportion of the variance in CFS patients’ disability scores over time. These results are discussed in terms of their support for both of the cognitive models. CFS patients appeared to have distinct cognitions which were associated with ongoing disability. The subsequent two quasi-experimental studies were conducted in a single laboratory session. The first of these used standardized neuropsychological tests to determine whether psychological variables, particularly somatic focus, interfere with CFS patients’ performance on high load attention tasks. The discrepancy between CFS patients’ subjective reports of concentration and memory difficulties and objective evidence of these deficits was also investigated. The subjects included 25 CFS patients matched for age, gender, and intelligence with two groups of healthy controls. One of these groups underwent a somatic induction procedure as part of the investigation of the effects of somatic preoccupation on attention tasks. The tests included the verbal memory subscales from the Wechsler Memory Scale-Revised and the Paced Auditory Serial Addition Task (PASAT), a measure of divided attention and speed of information processing. The analyses of the induction data failed to support the validity of this procedure resulting in the somatic control group being dropped from the analysis. Consistent with previous studies the principal deficit in the CFS group appeared to be on the PASAT. The CFS group appeared to be less accurate than healthy controls in their appraisal of their performance, which were related to negative mood rather than objective performance. Depression was also related to high performance expectations in the CFS group, but not the controls. The results did not support the original assumption that somatic preoccupation contributes to neuropsychological difficulties in CFS. However, mood factors were clearly shown to impact on both the objective and subjective experience of symptoms. The aim of the final study was to investigate the concordance between the self-report data collected in study one and information processing biases in CFS. Comparisons of the CFS patients and healthy controls on a modified Stroop attention task and a self-schema memory task, found no evidence of an illness-related bias in CFS patients’ processing of information. Rather, they demonstrated a significant tendency to be distracted by and remember depressed-relevant stimuli. The exception was their propensity to make somatic interpretations. These results are discussed in terms of the defensiveness hypothesis, which proposes that CFS patients’ negative, external illness perceptions and somatic distortions may act as a defence against underlying feelings of low self-esteem. The complex nature of CFS patients’ cognitive structures was revealed and the need to use measures which do not rely on self-reports was clearly demonstrated. These studies provided further support for the central role of cognitive factors and mood in perpetuating CFS.

Several studies have shown that non-cycling cells are resistant to the cytotoxic effects induced by amsacrine (m-AMSA; 4'-(9-acridinylamino)methanesulphon-m-anisidide). This resistance may limit the activity of m-AMSA and related 9-anilinoacridine antitumour agents against solid tumours. The biochemical mechanism(s) for this resistance have been investigated using spontaneously transformed Chinese hamster fibroblast (AA8 cells) in log- and plateau-phase spinner cultures. In early plateau phase most cells entered a growth-arrested state with a G1-G0 DNA content and showed a marked decrease in sensitivity to cytotoxicity after a 1-h exposure to m-AMSA or its solid tumour-active analogue, CI-921. Studies with radiolabelled m-AMSA demonstrated that changes in sensitivity to m-AMSA-induced cell killing were not due to a difference in uptake or retention of drug by log- and plateau-phase cells, and there was no significant metabolism of drug by either log- or plateau-phase cells. Thus, after a 1-h exposure to [3H]-m-AMSA at 37°C, a small proportion (1%) of cell-associated radioactivity was covalently bound to macromolecules, but most of the cell-associated radioactivity represented unchanged m-AMSA. There was no evidence for any oxidative metabolism to reactive quinoidal species in these tumour cells. However, studies with a fluorescence assay for DNA unwinding indicated that plateau-phase cells were 3 to 4 times less sensitive to m-AMSA-induced DNA breakage than log-phase cells, and changes in sensitivity to m-AMSA-induced DNA breakage correlated with changes in sensitivity to cell killing by m-AMSA as cell progressed from log to plateau phase. Further studies showed that the decrease in sensitivity to m-AMSA-induced DNA stand breakage correlated with a decrease in sensitivity to covalent DNA-protein complex formation in plateau-phase cells after m-AMSA treatment. Combined with evidence that the DNA lesions rapidly disappeared from both log- and plateau-phase cells following the removal of m-AMSA (half-time approx. 4 min), this indicated that the lesions detected by the FADU assay probably arose from the stimulation of DNA-topoisomerase II (topo II) cleavable complex formation by m-AMSA. K+/SDS precipitation assays with [32p] 3’-end-labelled pBR322 DNA indicated that nuclear extracts containing topo II activity from plateau-phase cells were 3- to 4-fold less sensitive to stimulation of DNA-topo II complex formation by m-AMSA than nuclear extracts from log-phase cells. This change in sensitivity to m-AMSA-induced DNA-topo II complex formation was therefore similar to that observed with intact cells. However, P4 unknotting assays indicated that topo II activity in nuclear extracts from plateau-phase cells was only 2-fold lower than that in nuclear extracts from log-phase cells. Resistance to treatment with m-AMSA may therefore reflect a decrease in topo II activity and/or a decrease in sensitivity of topo II enzymes to stimulation of cleavable complex formation by m-AMSA in non-cycling cells.

Members of the integrin superfamily of adhesion molecules are involved in cell to cell, cell to ECM, and cell to pathogen interactions, and are of fundamental importance in many biological processes. The β7 integrins α4β7 and αEβ7 have evolved to play specialized roles in gut mucosal immunity. α4β7 mediates the homing of lymphocytes to intestinal Peyer's patches, mesenteric lymph nodes, and the lamina propria by binding to the vascular addressin MAdCAM-1 (Fong et al., 1997). αEβ7 binds epithelial E-cadherin retaining lymphocytes at the intraepithelial compartment of the mucosa. The expression of αEβ7 is induced on migratory lymphocytes by TGF-β secreted by gut enterocytes. The signalling mechanisms responsible for basal and TGF-β-induced expression of β7 integrins are not well understood. Previous studies identified two TGF-β1 response regions in the β7 gene promoter termed TGFBRR-1 and TGFBRR-2 encompassing nucleotides -509 to -398 and -121 to -34. Here, TGF-β1 activation of the β7 gene proximal promoter is shown to be mediated by MAPK family members. TGF-β1 stimulation of TK-1 T cells increased the steady-state mRNA levels of the β7 gene relative to α4 transcripts, and led to enhanced phosphorylation of c-Jun. TGF-β1 stimulation induced rapid increases in the binding of nuclear protein complexes to TGFBRR-1 and -2. Sp1 and Sp3 which mediate TGF-β1 signalling were shown to bind to an Sp1 cis-element encompassing nucleotide positions -67 to -60 within TGFBRR-2. The interaction of Sp1 with its cognate binding site was c-Jun dependent. In contrast, there was no evidence for involvement of the Smad proteins. ATF-2 was identified to bind to a region encompassing nucleotide positions -699 to -689 just upstream of TGFBRR-1. Sp1 and ATF-2 expression vectors co-transfected into Sp1-deficient SL-2 cells synergized to drive the activity of a β7 gene luciferase reporter. Mutation of the ATF-2-binding site modestly reduced β7 gene reporter activity. The involvement of c-Jun in TGF-β signalling and interaction of Sp1 with the β7 gene promoter suggested that MAP kinase pathways might mediate β7 gene transcription. Specific chemical inhibitors were used to ascertain which of the three MAPK pathways namely p38, JNK, and ERK were involved. Results obtained by nuclear run-on transcription analysis which measures nascent RNA synthesis showed that both the p38 and JNK pathways regulate β7 gene expression in TK-1 cells, whereas only the p38 pathway regulates α4 gene expression. Thus, treatment of TK-1 cells with the p38 inhibitor SB203580 and the JNK inhibitor SP600125 inhibited the synthesis of β7 transcripts, whereas only SB203580 inhibited the synthesis of α4 transcripts. Conversely, sodium arsenite which induces JNK and p38 upregulated nascent synthesis of α4 and β7 RNA transcripts. SB203580 blocked the binding of nuclear factors to TGFBRR-1, and ATF-2 binding to nucleotide position -699 to -689. Similarly, SP600125 blocked the binding of Sp1 and Sp3 to TGFBRR-2, whereas unexpectedly SB203580 enhanced their binding. Furthermore, both SB203580 and SP600125 decreased cell-surface expression of the β7 subunit and SB203580 inhibited TK-1 cell adhesion to MAdCAM-1. In contrast, the MEK inhibitor PD98059 had no effect on the expression of nascent β7 RNA transcripts and cell-surface expression of the β7 subunit, suggesting that the ERK pathway is not involved in regulation of β7 gene expression in TK-1 cells. In contrast to the results obtained with TK-1 cells, SB203580, SP600125, and PD98059 each inhibited the nascent synthesis of α4, β7, and αE transcripts in peripheral blood lymphocytes. In conclusion, this study has revealed for the first time that both the p38 and JNK pathways mediate TGF-β1-induced expression of the integrin β7 gene. Expression of the β7 gene is Sp1-dependent, and involves the synergistic cooperation of c-Jun and ATF-2. It is proposed that the p38 and JNK pathways play a role by triggering the activation and translocation of c-Jun and ATF-2 to the nucleus. / Whole document restricted, but available by request, use the feedback form to request access.

Lactose malabsorption (LM) in children was diagnosed by an elevated breath hydrogen (BH) level following a 10g lactose load. A portable gas chromatograph and a semiconductor detector, designed and constructed for use in the Pacific Islands is described. Following verification on known malabsorber and. normal subjects, the technique was used to determine the prevalence of LM in Europeans at Auckland and Rarotonga, and in Samoans at two locations in Auckland and two locations in Western Samoa. The prevalence of LM in Europeans was significantly (p<0.01) higher at Rarotonga than at Auckland. For Samoans, the prevalence of LM was significantly (p<0.01) higher in Western Samoa than at Auckland. The prevalence of LM was very highly significantly (p<0.001) related to race. Each child tested for LM filled in a questionaire to determine attitude, consumption of and perceived intolerance to milk, milk biscuits and ice cream. Lactose malabsorption was significantly (p<0.05) correlated to milk consumption and to attitude to dairy products, but not to sex, age, and perceived intolerance. The consumption of dairy products was very highly significantly (p<0.0001) correlated to attitude, and highly significantly (p<0.001) correlated to location and perceived intolerance. There was no significant correlation between consumption and race, sex or age. Perceived intolerance to individual dairy products was significantly correlated to attitude to milk (p<0.0001), milk biscuits (p<0.02) and ice cream (p<0.001). Perceived intolerance was not related to age, sex, race, location or the actual symptoms following the consumption of 10g lactose. The unexpected finding of increased LM in the Pacific Islands, was investigated further by studying the LM status of the Medical Team during a visit to western Samoa, and by performing a microbiological survey of water quality. It was found that half of the Medical Team 3/6, became malabsorbers during the week spent in Western Samoa. On returning to New Zealand it was shown that lactase levels took 3 months to normalise. The water supply in Western Samoa was shown to contain very high levels of coliform bacteria. The currently held hypothesis that genetic factors are solely involved in the onset of LM, was not supported. The evidence from the survey supported environmental factors are also involved in adult onset LM. The hypothesis suggesting that dietary lactose was a requirement for retaining elevated lactase levels, was tested using Galactosemic and Phenyl Ketonuria patients. None of the patients had developed LM although they had been on a low lactose diet for years, hence the theory was not supported. The BH method proved highly successful in diagnosing LM with many of the children actually enjoying it.