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Biomedical subjects

M J Hickey

Publications and source records attributed to M J Hickey.

At least 19 recordsLinked to original sources
PubMed1997

Tumor necrosis factor-alpha induces leukocyte recruitment by different mechanisms in vivo and in vitro.

It is well established that E-selectin is the endothelial adhesion molecule that is primarily responsible for mediating leukocyte rolling on TNF-alpha-stimulated cultured endothelial cells. Despite this, few studies in in vivo inflammatory models have observed reduced leukocyte accumulation using mAbs against E-selectin. The objective of this study was to compare the function of E-selectin on endothelial cells in vitro with its role in TNF-alpha-induced leukocyte recruitment in vivo using EL246, a mAb that blocks the function of E-selectin on activated feline endothelial cells. In vitro experiments using feline endothelial cells showed that EL246 functionally inhibits E-selectin-dependent leukocyte recruitment induced by TNF-alpha, without affecting the function of other rolling mechanisms. Intravital microscopy of single 25- to 40-microm venules in the feline mesentery was then used to examine leukocyte rolling and adhesion in response to superfusion with TNF-alpha. TNF-alpha treatment significantly increased the number of both rolling and adherent leukocytes and significantly decreased leukocyte rolling velocity. Treatment with EL246 (1 mg/kg), either i.v. at the start of the TNF-alpha protocol or directly into the superior mesenteric artery after 3 h of TNF-alpha treatment, had no effect on leukocyte rolling, adhesion, or rolling velocity. However, treatment with the selectin-binding carbohydrate, fucoidan, reduced leukocyte rolling to below baseline levels. These results suggest that in contrast to its prominent role on cultured endothelial cells, E-selectin does not contribute to leukocyte recruitment in TNF-alpha-stimulated feline mesenteric venules in vivo.

Animals↗
PubMed1997

The association between alpha4-integrin, P-selectin, and E-selectin in an allergic model of inflammation.

In this study, we examined the relationship between the endothelial selectins (P-selectin and E-selectin) and whether they are critical for alpha4-integrin-dependent leukocyte recruitment in inflamed (late phase response), cremasteric postcapillary venules. Animals were systemically sensitized and 2 wk later challenged intrascrotally with chicken ovalbumin. Leukocyte rolling flux, adhesion, and emigration were assessed at baseline and 4 and 8 h postantigen challenge. There was a significant increase in leukocyte rolling flux, adhesion, and emigration in sensitized and challenged mice at both 4 and 8 h. At 8 h, the increase in leukocyte rolling flux was approximately 50% inhibitable by an anti-alpha4-integrin antibody, 98% inhibitable by fucoidin (a selectin-binding carbohydrate), and 100% inhibitable by an anti-P-selectin antibody. P-selectin-deficient animals displayed no leukocyte rolling or adhesion at 8 h after challenge. However, at 8 h there were many emigrated leukocytes in the perivascular space suggesting P-selectin-independent rolling at an earlier time point. Indeed, at 4 h postantigen challenge in P-selectin-deficient mice, there was increased leukocyte rolling, adhesion, and emigration. The rolling in the P-selectin-deficient mice at 4 h was largely alpha4-integrin dependent. However, there was an essential E-selectin-dependent component inasmuch as an anti-E-selectin antibody completely reversed the rolling, and in E-selectin and P-selectin double deficient mice rolling, adhesion and emigration were completely absent. These results illustrate that P-selectin underlies all of the antigen-induced rolling with a brief transient contribution from E-selectin in the P-selectin-deficient animals. Finally, the antigen-induced alpha4-integrin-mediated leukocyte recruitment is entirely dependent upon endothelial selectins.

Animals↗
PubMed1997

Mild hypothermia protects against ischaemia-reperfusion injury in rabbit skeletal muscle.

In three groups of rabbits, the rectus femoris muscle was subjected to 4 hours of total ischaemia. In Group 1 (normothermia, n = 5) the core temperature was maintained within the range 36-38 degrees C for the duration of ischaemia. In Group 2 (total hypothermia, n = 5) the core temperature was allowed to fall to 31.5-33.5 degrees C. In Group 3 (muscle only hypothermia, n = 5) core temperature was maintained as in Group 1 but the muscle temperature was allowed to fall to 29.5-31.5 degrees C. After 24 hours of reperfusion the muscles were harvested and measurements made of muscle viability, oedema and myeloperoxidase content. The mean (s.e.m.) muscle viability of Group 1, 19.5 (3.8)%, was significantly less than that of both Group 2, 86.0 (2.0)%, and Group 3, 87 (4.1)%, (P < 0.001). Muscle oedema and myeloperoxidase levels were elevated in all experimental groups, but differences were not significant. These findings indicate that ischaemia-reperfusion injury in skeletal muscle in this model is highly temperature-sensitive, small reductions in muscle temperature during ischaemia providing significant protection against ischaemia-reperfusion injury.

Animals↗
PubMed1997

Timing of administration of dexamethasone or the nitric oxide synthase inhibitor, nitro-L-arginine methyl ester, is critical for effective treatment of ischaemia-reperfusion injury to rat skeletal muscle.

1. The effects of the nitric oxide synthase (NOS) inhibitors, NG-nitro-L-arginine-methyl ester (L-NAME), nitroiminoethyl-L-ornithine and S. methylisothiourea on skeletal muscle survival following 2 h of tourniquet ischaemia and 24 h of reperfusion were compared with those of the anti-inflammatory steroid, dexamethasone. 2. Administration of each of the NOS inhibitors or dexamethasone 30 min before reperfusion reduced the degree of skeletal muscle necrosis 24 h after reperfusion. 3. The influence of timing of drug administration was investigated. L-NAME administered 30 min before reperfusion, at 3 h after reperfusion, but not thereafter, significantly improved muscle survival compared with saline-treated controls. Dexamethasone administered 30 min before, or at 3 or 8 h after reperfusion, but not at 16 h, significantly improved muscle survival, but neither agent had protective effects when administered before ischaemia. 4. After 8 h of reperfusion of ischaemic skeletal muscle, cell-free homogenates contained Ca(2+)-independent (inducible) NOS activity which was reduced in dexamethasone-treated (2.5 mg/kg) rats. Furthermore, inducible NOS mRNA levels, as detected by reverse transcriptase-PCR, were increased after 8 h of reperfusion in saline, but not in dexamethasone-treated rats. 5. These data suggest a significant deleterious effect of endogenous NO which may be restricted to the first 3 h of the reperfusion phase of ischaemia-reperfusion injury, and raise the possibility of effective treatment of incipient reperfusion injury, even after several hours of reperfusion.

Animals↗
PubMed1997

Inducible nitric oxide synthase-deficient mice have enhanced leukocyte-endothelium interactions in endotoxemia.

Nitric oxide (NO) from constitutive NO synthase (NOS) has been postulated to be a homeostatic regulator of leukocyte-endothelial cell interactions. By contrast, the inducible NO synthase (iNOS) isoform has been invoked as a potential pathogenic enzyme in numerous inflammatory diseases. The objective of this study was to determine whether the iNOS isoform is also capable of functioning as a regulator of leukocyte recruitment. Mice received endotoxin (LPS, 30 microg/kg, i.v.); 2-4 h later, intravital microscopy was used to examine leukocyte rolling and adhesion in postcapillary venules of the cremaster muscle and the sinusoids and postsinusoidal venules of the hepatic microcirculation. Leukocyte recruitment into the lung was also examined. RT-PCR confirmed that this treatment induced iNOS mRNA expression in wild-type mice as early as 2 h after LPS treatment. Between 2 and 4 h after LPS administration, the number of rolling and adherent leukocytes in cremasteric postcapillary venules and of adherent cells in liver postsinusoidal venules of iNOS-deficient mice were significantly higher than in wild-type mice. Leukocyte accumulation in the lung (measured by myeloperoxidase assay) was also significantly elevated in iNOS-deficient animals. These effects could not be attributed to differences in systemic blood pressure, shear rates, circulating leukocyte numbers, or baseline levels of rolling and adhesion because these parameters were not different between the two groups. To establish whether the differences in leukocyte recruitment were related to the leukocytes per se, perfusion of iNOS+/+ or iNOS-/- septic blood over purified E-selectin (using parallel plate flow chambers) revealed much larger recruitment of iNOS-/- leukocytes. These results suggest that iNOS induced in response to LPS releases NO that is capable of reducing leukocyte accumulation by affecting leukocytes directly and raises the possibility that induction of iNOS is a homeostatic regulator for leukocyte recruitment.

Analysis of Variance↗
PubMed1997

Rapid screening of natural products for antimycobacterial activity by using luciferase-expressing strains of Mycobacterium bovis BCG and Mycobacterium intracellulare.

The object of this study was to investigate the ability of a rapid luciferase assay to detect antimycobacterial activity in plant extracts. Recombinant strains of Mycobacterium bovis BCG (rBCG) and Mycobacterium intracellulare expressing firefly luciferase were used as the test organisms. Assays were conducted in a 96-well minitube format under biosafety level 2 conditions. Control and test wells were sampled immediately after inoculation and after 3 (recombinant M. intracellulare) and 5 (rBCG) days of incubation to measure luminescence with a microplate luminometer, and the relative change in luminescence was calculated as a percentage of control values. As an alternative test method, Alamar blue was added after 12 days of incubation, and changes in color were read visually. A total of 480 extracts were tested. Sixteen extracts were active against rBCG, and of those, seven were also active against recombinant M. intracellulare. With activity defined as a relative change in luminescence of < or = 1% (i.e., > or = 99% inhibition) and a persistence of blue color after addition of Alamar blue, there was 99.0% agreement between the two methods. Our results suggest that the luciferase assay is rapid and accurate and has the potential to greatly accelerate the evaluation of antimycobacterial activity in plant extracts in vitro. With this method, it is possible to screen a large number of samples in a short period of time.

Anti-Bacterial Agents↗
PubMed1997

Endogenous but not exogenous nitric oxide decreases TNF-alpha-induced leukocyte rolling.

Inhibition of nitric oxide (NO) synthesis in mesenteric microvessels increases leukocyte rolling. The objective of this study was to determine whether inducible NO synthase (iNOS) can modulate tumor necrosis factor-alpha (TNF-alpha)-induced leukocyte rolling. Leukocyte rolling was examined using intravital microscopy of TNF-alpha-treated feline mesenteric microvasculature. Leukocyte rolling increased progressively over 3 h of TNF-alpha treatment. Pretreatment with the selective iNOS inhibitor aminoguanidine further doubled TNF-alpha-induced leukocyte rolling. Aminoguanidine alone did not affect baseline blood pressure or leukocyte kinetics. However, in the same animals NG-nitro-L-arginine methyl ester caused a rapid increase in blood pressure, confirming that constitutive NOS activity persisted in aminoguanidine-treated animals. Furthermore, aminoguanidine did not affect leukocyte rolling in an acute model of leukocyte recruitment (ischemia/reperfusion), suggesting that the exacerbated rolling induced by aminoguanidine with TNF-alpha as a stimulus was not a nonspecific effect. Addition of the NO donor spermine-NO had no effect on TNF-alpha-associated leukocyte rolling. These data raise the possibility of a physiological role for the increased production of NO from iNOS, i.e., regulation of leukocyte rolling and potentially the inflammatory response.

Animals↗
PubMed1996

A mutation in the human cyclin-dependent kinase interacting protein, CksHs2, interferes with cyclin-dependent kinase binding and biological function, but preserves protein structure and assembly.

A mutation directing an amino acid substitution in the conserved beta-hinge region of one of the human Cks isoforms, CksHs2, was constructed by site-directed mutagenesis. Replacement of glutamine for glutamate 63 (E63Q) was predicted to stabilize the beta-interchanged dimeric and hexameric assembly of CksHs2. However, such an effect was seen only at high, non-physiological pH. Three-dimensional structures of the E63Q hexameric mutant protein were determined to 2.6 A resolution in a P4(3)2(1)2 space group and 2.1 A in the C2 space group isostructural with wild-type, and both were shown to be virtually identical to the refined 1.7 A wild-type structure. Thus, the E63Q mutation did not alter the wild-type structure and assembly of CksHs2 but, surprisingly, disrupted the essential biological function of the protein and significantly reduced its ability to bind to cyclin-dependent kinases. The Kd of wild-type CksHs2 for CDK2 was 5.05 x 10(-8) M, whereas the affinity of the mutant protein for CDK2 was too low to allow a determination. These data, coupled with the observation that monomeric but not hexameric CksHs2 interacts with cyclin-dependent kinases, suggest that glutamine 63 is likely to be directly involved in cyclin-dependent kinase binding in vitro and in vivo.

Base Sequence↗
PubMed1996

Compensatory ahpC gene expression in isoniazid-resistant Mycobacterium tuberculosis.

Mutations that eliminate KatG catalase-peroxidase activity prevent activation of isoniazid and are a major mechanism of resistance to this principal drug for the treatment of Mycobacterium tuberculosis infections. However, the loss of KatG activity in clinical isolates seemed paradoxical because KatG is considered an important factor for the survival of the organism. Expression of either KatG or the recently identified alkyl hydroperoxidase AhpC was sufficient to protect bacilli against the toxic effects of organic peroxides. To survive during infection, isoniazid-resistant KatG mutants have apparently compensated for the loss of KatG catalase-peroxidase activity by a second mutation, resulting in hyperexpression of AhpC.

Amino Acid Sequence↗
PubMed1996

Human mitochondrial manganese superoxide dismutase polymorphic variant Ile58Thr reduces activity by destabilizing the tetrameric interface.

Human manganese superoxide dismutase (MnSOD) is a homotetrameric enzyme which protects mitochondria against oxygen-mediated free radical damage. Within each subunit, both the N-terminal helical hairpin and C-terminal alpha/beta domains contribute ligands to the catalytic manganese site. Two identical four-helix bundles, symmetrically assembled from the N-terminal helical hairpins, form a novel tetrameric interface that stabilizes the active sites. The 2.5 A crystallographic structure of the naturally occurring polymorphic variant Ile58Thr MnSOD reveals that the helical hairpin mutation Thr58 causes two packing defects in each of the two four-helix bundles of the tetrameric interface. Similar mutations, expected to cause packing defects in the Cu,ZnSOD dimer interface, are associated with the degenerative disease amyotrophic lateral sclerosis. Ile58Thr MnSOD is primarily dimeric in solution and is significantly less thermostable than the normal enzyme, with decreases of 15 degrees C in the main melting temperature and 20 degrees C in the heat-inactivation temperature. Consequently, this mutant MnSOD is compromised at normal body temperatures: thermal inactivation, predicted from the decrease in thermal stability, occurs with a theoretical half-life of only 3.2 h at 37 degrees C (1.4 h at 41 degrees C), compared with 3.1 years for native MnSOD. This prediction is supported by direct measurements: incubation at 41.7 degrees C for 3 h has no effect on the activity of native MnSOD but completely inactivates mutant MnSOD. Rapid inactivation of Ile58Thr MnSOD at the elevated temperatures associated with fever and inflammation could provide an early advantage by killing infected cells, but also would increase superoxide-mediated oxidative damage and perhaps contribute to late-onset diseases.

Binding Sites↗
PubMed1996

Crystal structure and mutational analysis of the human CDK2 kinase complex with cell cycle-regulatory protein CksHs1.

The 2.6 Angstrom crystal structure for human cyclin-dependent kinase 2(CDK2) in complex with CksHs1, a human homolog of essential yeast cell cycle-regulatory proteins suc1 and Cks1, reveals that CksHs1 binds via all four beta strands to the kinase C-terminal lobe. This interface is biologically critical, based upon mutational analysis, but far from the CDK2 N-terminal lobe, cyclin, and regulatory phosphorylation sites. CDK2 binds the Cks single domain conformation and interacts with conserved hydrophobic residues plus His-60 and Glu-63 in their closed beta-hinge motif conformation. The beta hinge opening to form the Cks beta-interchanged dimer sterically precludes CDK2 binding, providing a possible mechanism regulating CDK2-Cks interactions. One face of the complex exposes the sequence-conserved phosphate-binding region on Cks and the ATP-binding site on CDK2, suggesting that CKs may target CDK2 to other phosphoproteins during the cell cycle.

Base Sequence↗
PubMed1996

Effects of low dose intra-arterial monoclonal antibodies to ICAM-1 and CD11/CD18 on local and systemic consequences of ischaemia-reperfusion injury in skeletal muscle.

The aim of this study was to investigate the effects of intra-arterial infusion of low doses of monoclonal antibodies (Mabs) against adhesion molecules (the neutrophil CD18 integrins, and the endothelial adhesion molecule, ICAM-1) on reperfusion injury in skeletal muscle. The rabbit rectus femoris muscle was rendered ischaemic for 2 1/2 hours. Mabs were infused (approximately 0.5 mg/kg) commencing 20 minutes before the end of ischaemia and for the first hour of reperfusion. 24 hours after reperfusion, the muscle was assessed for viability, oedema and neutrophil infiltration (myeloperoxidase (MPO) levels). The results of the viability assessment (control--20.9 (0-47.5)% [median (range)], anti-CD18--30.5 (3.0-89.4)%, anti-ICAM-1--27.9 (7.8-78.1)% and anti-CD18 combined with anti-ICAM-1--45.2 (15.6-92.3)%) showed no significant differences between groups, while analysis of MPO in the postischaemic muscle showed that the anti-ICAM-1 Mab reduced neutrophil infiltration significantly. Furthermore, in contralateral unoperated muscles MPO levels were elevated 24 hours after ischaemia in the contralateral muscle. This increased neutrophil infiltration was prevented by pretreatment with anti-ICAM-1. These results suggest that low doses of anti-CD18 and anti-ICAM-1 Mabs do not reduce reperfusion injury in skeletal muscle but may help to protect against systemic effects of severe trauma. The evidence suggests that reperfusion injury in this skeletal muscle model may be largely independent of neutrophil involvement.

Animals↗
PubMed1996

A morphological study of the long-term repair process in experimentally stretched but unruptured arteries and veins.

As many avulsion amputations are incomplete and the vessels remain intact, the immediate pathology and long-term repair process (to 3 months post-injury) of experimentally stretched but unruptured rabbit femoral arteries and veins were examined. In stretched arteries, circumferential skip lesions involving endothelium, internal elastic lamina (IEL) and media occurred frequently and often up to 3 cm from the point of stretch. Medial smooth muscle cells (SMC) were significantly damaged or lost at lesions. Macrophages and neutrophils were found in lesions 1-4 days post-injury. Between 2-4 days, lesions were covered by endothelium and synthetic state SMC appeared in the media. At 1 week, a thin neointima (which persisted to 3 months) covered many lesions. The media at lesions gradually filled with SMC but generally remained disorganised even at 3 months post-injury. Stretching caused tears in vein walls, particularly close to the point of injury. There was no evidence of venous damage or repair in specimens examined 3 weeks and 3 months post-injury.

Amputation, Traumatic↗
PubMed1996

Orthotopic vascularized osteochondral allografts in an immunosuppressed rat model.

This study examined the survival of orthotopic, vascularized, osteochondral allografts, following 12 weeks of immunosuppression and transfer into a naive, allograft host up to 14 weeks later, and compared the results with those previously reported for similar grafts in a heterotopic position. Knee-joint allografts between DA (donor) and Lewis (recipient) rat strains were assessed by quantitative histology up to 6 months after transplantation, and graft microcirculation was examined by India-ink infusion. Graft repopulation was assessed by re-transferring the graft to a naive, non-suppressed allograft host 12 to 26 weeks after the initial transplantation. Isografts survived for as long as grafts were examined (6 months) and showed good healing of the graft/host bone junction, although long-term isografts showed some deterioration of the growth plate. Non-suppressed allografts rejected within 2 weeks. Allografts in hosts immunosuppressed for 12 weeks remained healthy and healed in a similar manner to the isografts. Following cessation of immunosuppression, allografts progressively deteriorated, with mononuclear cell infiltration apparent in graft bone marrow and muscle in the later stages examined. Transfer to second non-suppressed hosts resulted in rapid rejection of the allografts, indicating that, as shown previously in heterotopic, osteochondral allografts, little or no graft repopulation by host-derived cells had occurred during the protected period in the first host.

Animals↗
PubMed1996

Biochemical and genetic data suggest that InhA is not the primary target for activated isoniazid in Mycobacterium tuberculosis.

An examination of the pattern of lipid biosynthetic responses to isoniazid (INH) treatment of Mycobacterium tuberculosis and Mycobacterium smegmatis suggests that the mode of action of activated INH differs between these 2 organisms. Transformation of M. smegmatis with inhA on a plasmid construct conferred high-level resistance to INH, while the same construct failed to confer resistance upon M. tuberculosis. The inhA region from 2 clinical isolates whose resistance has been attributed to changes in the upstream promoter region has been cloned and was not sufficient to impart INH resistance to the level of the parent strain on sensitive M. tuberculosis. These putative mutant promoter elements appear to elevate expression levels of gene fusion reporter constructs, suggesting some noncausal connection between the observed mutations and the lipid metabolism of drug-resistant organisms. These results suggest that InhA is not the major target for activated INH in M. tuberculosis.

Antitubercular Agents↗
PubMed1996

Luciferase in vivo expression technology: use of recombinant mycobacterial reporter strains to evaluate antimycobacterial activity in mice.

The development of new drugs and vaccines directed against Mycobacterium tuberculosis is severely impeded by the slow growth of this organism and the need to work under stringent biosafety conditions. These difficulties pose considerable obstacles when animal studies with M. tuberculosis are performed. We investigated whether a novel approach termed luciferase in vivo expression, using an enhanced luciferase-expressing mycobacterial strain, could be used to evaluate antimycobacterial activity in mice. Vectors that expressed firefly luciferase (lux gene) at high levels in the bacillus Calmette-Gu-erin (BCG) strain of Mycobacterium bovis were constructed for use in vivo. One recombinant BCG reporter strain (rBCG-lux) was selected for high-level expression of the lux gene product and for its ability to replicate in mice. Methodology to monitor in vivo growth of the rBCG-lux reporter strain in mice by direct assay of luciferase luminescence in organ homogenates was developed. The utility of this approach for assessing the in vivo efficacies of antimycobacterial compounds was evaluated. The activities of standard antimycobacterial drugs were directly apparent in mice infected with the rBCG-lux reporter strain by statistically significant reductions in spleen luminescence. In addition, antimycobacterial immunity was also evident in BCG-immunized mice, in which suppression of rBCG-lux growth in comparison with that in naive mice was clearly observed. The use of luciferase in vivo expression for the in vivo evaluation of antimycobacterial activity compared favorably with standard CFU determinations in terms of time, labor, expense, and statistical significance but permitted the evaluation of antimycobacterial drugs and immunity in mice in 7 days or less. Thus, the use of this technology can greatly accelerate the process of evaluation of antibiotics and immunogens in animal models for the slowly growing pathogenic mycobacteria.

Animals↗
PubMed1996

Bioluminescence screening in vitro (Bio-Siv) assays for high-volume antimycobacterial drug discovery.

Bioluminescence-based assays to indicate antimicrobial susceptibility have been developed and validated for recombinant strains of Mycobacterium tuberculosis, Mycobacterium bovis BCG, Mycobacterium avium, and Mycobacterium intracellulare expressing an integrated eukaryotic luciferase gene. MICs determined with these bioluminescence assays for several antimycobacterial agents, including isoniazid, ethambutol, rifampin, amikacin, streptomycin, ciprofloxacin, and clarithromycin, compared favorably with traditional BACTEC methods and visual estimations of the inhibitory end point. Assay methodology has been optimized for the analysis of large numbers of novel compounds and is simple, inexpensive, and labor efficient. The availability of these four recombinant mycobacteria has permitted a strategy for drug discovery employing the nonpathogenic BCG strain for mass screening purposes with subsequent confirmation of activity against the pathogenic mycobacteria. Furthermore, evidence suggests that the BCG-based screen may allow the direct identification of bactericidal agents.

Anti-Bacterial Agents↗