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.

Investigations were undertaken to describe and quantitate the topographical abnormalities which develop in the organ of Corti as a result of acoustic trauma, and to determine their relationship to the associated losses of cochlear function assessed by the compound action Potential (N1) and cochlear microphonics (CM). Thirty guinea pigs were exposed, while anaesthetised, to a tone of 3 KHz at 125 dB SPL for 30 minutes. Both organs of Corti were examined by scanning electron microscopy either immediately or 1,3,7 or 14 days after exposure. In the second study 26 anaesthetised guinea pigs were exposed to 5 KHz at 125 dB SPL for 30 minutes. Cochlear potentials were recorded from the round window of the right cochlea and N1 audiograms (sound Pressure level required to elicit N1 Plotted against frequency) and 10 μv isopotential curves (sound pressure level required to produce 10 μv CM plotted against frequency) were produced for each animal either within one hour or 1,7,14 or 28 days later. The same organ of Corti was examined by scanning electron microscopy. Cochlear potentials were compared to mean values from 16 normal guinea pigs. Most animals developed topographical changes in one or both organs of Corti after exposure to 3 KHz (90%) and in the right organ of Corti after 5 KHz (92%). There was a wide variation in both the length (3 KHz: 0.1-4.15 mm; 5 KHz: 0.5-16.0 mm) of lesions and the number of hair cells affected. Both these indices of damage increased significantly (p <0.01) in the 24 hours following exposure to 5 KHz. Early damage to hair cells included either collapse, fusion or loss of stereocilia and there was an increase in the proportion of affected cells towards the centre of the lesions where supporting cells were damaged also. Subsequent to exposure, collapsed stereocilia appeared to become fused and some hair cells, particularly OHC, with stereocilia abnormalities were lost. However, others, particularly IHC, remained for up to 28 days despite abnormal stereocilia. Early changes occurred around the position of maximum displacement of the basilar membrane and subsequent extension of the lesions occurred equally in both apical and basal directions. Fusion was the only stereocilia change to develop after exposure. Regions of the organ of Corti showing supporting cell damage were replaced within 3-7 days by the proliferation of inner sulcus and Claudius’ cells. The substantial initial loss of both N1 thresholds and CM sensitivity partially recovered during the first 24 hours after exposure, but paradoxically, this was associated with the significant increase in the topographical changes in the organ of Corti. N1 threshold loss occurred over all frequencies and was maximum 1/2 – 1 octave higher than the exposure frequency (5 KHz). All lesions occurred within regions corresponding to changes in N1 thresholds. In the first 24 hours topographical changes occurred over a much smaller area of the organ of Corti than indicated by changes in N1 thresholds. Seven or more days later the longer lesions (30%) reflected the extent of changes in N1 but the remainder were smaller than indicated by this functional loss. This suggests that functionally important damage to the cochlea is more extensive than indicated by hair cell loss, stereocilia abnormality or supporting cell changes in the organ of Corti. Therefore, investigations of the effects of noise should not be based simply on topographical changes in the organ of Corti as these often underestimate the extent of injury to the cochlea.

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.

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.

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.

Investigations were undertaken to describe and quantitate the topographical abnormalities which develop in the organ of Corti as a result of acoustic trauma, and to determine their relationship to the associated losses of cochlear function assessed by the compound action Potential (N1) and cochlear microphonics (CM). Thirty guinea pigs were exposed, while anaesthetised, to a tone of 3 KHz at 125 dB SPL for 30 minutes. Both organs of Corti were examined by scanning electron microscopy either immediately or 1,3,7 or 14 days after exposure. In the second study 26 anaesthetised guinea pigs were exposed to 5 KHz at 125 dB SPL for 30 minutes. Cochlear potentials were recorded from the round window of the right cochlea and N1 audiograms (sound Pressure level required to elicit N1 Plotted against frequency) and 10 μv isopotential curves (sound pressure level required to produce 10 μv CM plotted against frequency) were produced for each animal either within one hour or 1,7,14 or 28 days later. The same organ of Corti was examined by scanning electron microscopy. Cochlear potentials were compared to mean values from 16 normal guinea pigs. Most animals developed topographical changes in one or both organs of Corti after exposure to 3 KHz (90%) and in the right organ of Corti after 5 KHz (92%). There was a wide variation in both the length (3 KHz: 0.1-4.15 mm; 5 KHz: 0.5-16.0 mm) of lesions and the number of hair cells affected. Both these indices of damage increased significantly (p <0.01) in the 24 hours following exposure to 5 KHz. Early damage to hair cells included either collapse, fusion or loss of stereocilia and there was an increase in the proportion of affected cells towards the centre of the lesions where supporting cells were damaged also. Subsequent to exposure, collapsed stereocilia appeared to become fused and some hair cells, particularly OHC, with stereocilia abnormalities were lost. However, others, particularly IHC, remained for up to 28 days despite abnormal stereocilia. Early changes occurred around the position of maximum displacement of the basilar membrane and subsequent extension of the lesions occurred equally in both apical and basal directions. Fusion was the only stereocilia change to develop after exposure. Regions of the organ of Corti showing supporting cell damage were replaced within 3-7 days by the proliferation of inner sulcus and Claudius’ cells. The substantial initial loss of both N1 thresholds and CM sensitivity partially recovered during the first 24 hours after exposure, but paradoxically, this was associated with the significant increase in the topographical changes in the organ of Corti. N1 threshold loss occurred over all frequencies and was maximum 1/2 – 1 octave higher than the exposure frequency (5 KHz). All lesions occurred within regions corresponding to changes in N1 thresholds. In the first 24 hours topographical changes occurred over a much smaller area of the organ of Corti than indicated by changes in N1 thresholds. Seven or more days later the longer lesions (30%) reflected the extent of changes in N1 but the remainder were smaller than indicated by this functional loss. This suggests that functionally important damage to the cochlea is more extensive than indicated by hair cell loss, stereocilia abnormality or supporting cell changes in the organ of Corti. Therefore, investigations of the effects of noise should not be based simply on topographical changes in the organ of Corti as these often underestimate the extent of injury to the cochlea.

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.