Search PubMed⌕ Search

Biomedical subjects

C R MacKenzie

Publications and source records attributed to C R MacKenzie.

At least 19 recordsLinked to original sources
PubMed2005

Acute hemorrhagic pericarditis in a child with pneumonia due to Chlamydophila pneumoniae.

Chlamydophila pneumoniae is mainly responsible for respiratory tract infections but has also been associated with endocarditis and myocarditis. We report a case of pneumonia in a child with hemorrhagic pericardial effusion with a positive result by a new C. pneumoniae TaqMan PCR, suggesting a pericardial inflammation directly induced by C. pneumoniae. C. pneumoniae should be suspected in patients with community-acquired pneumonia and concurrent pericarditis. Empirical treatment with azithromycin seems feasible.

Acute Disease↗
PubMed2004

Blood culture sampling rates at a German pediatric university hospital and incidence of invasive pneumococcal disease.

BACKGROUND: Recent pediatric surveillance studies suggest the incidence of pneumococcal bacteremia, but not meningitis, is lower in Germany than in most developed countries. Suboptimal case assessment in routine clinical practice has been suspected of contributing to this apparent discrepancy. METHODS: We aimed to assess the blood culture sampling rate at a German pediatric university hospital and the disease burden associated with pneumococcal bacteremia in children under 5 years of age. The study design was retrospective, based on data-linkage and chart review. RESULTS: Blood cultures were frequently obtained in sepsis (96%; CI 78-99%) and meningitis (95%; CI 77-99%), but less commonly in pneumonia (49%; CI 43-54%) and fever without focus (48%; CI 38-59%). Pneumococci were the most common source of clinically significant bacteremia in previously healthy children. CONCLUSION: These blood culture sampling rates may be insufficient for the sensitive detection of pneumococcal bacteremia. Epidemiological surveillance based on poorly standardized diagnostic practices is prone to under-assessment.

Age Distribution↗
PubMed2004

Inhibition of human herpes simplex virus type 2 by interferon gamma and tumor necrosis factor alpha is mediated by indoleamine 2,3-dioxygenase.

Genital herpes simplex virus type 2 (HSV-2) is a significant clinical problem. Infection in pregnancy may result in disseminated infection of the newborn with encephalitis. We analyzed the antiviral effects induced by interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) in cervix carcinoma cells (HeLa) and astrocytoma cells (86HG39). We found that replication of HSV-2 in HeLa cells and in 86HG39 cells is inhibited after stimulation of the cells by IFN-gamma and TNF-alpha. The antiviral effect of IFN-gamma is enhanced in the presence of TNF-alpha, while stimulation by TNF-alpha alone did not induce antiviral activity. We found that IFN-gamma induces a strong activation of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) and in addition, that the IFN-gamma-induced IDO activity was enhanced in the presence of TNF-alpha. Furthermore, we found that the induction of IDO activity is responsible for the inhibition of herpes simplex virus replication, since the presence of excess amounts of l-tryptophan abrogates the antiviral effect induced by IFN-gamma and the combination of IFN-gamma and TNF-alpha. We therefore conclude that the antiviral effect against HSV-2 mediated by type II interferon and TNF-alpha are dependent on IDO activation.

Antiviral Agents↗
PubMed2004

Role of indoleamine-2,3-dioxygenase in alpha/beta and gamma interferon-mediated antiviral effects against herpes simplex virus infections.

Gamma interferon (IFN-gamma)-mediated indoleamine-2,3-dioxygenase (IDO) activity in human astrocytoma cells and in native astrocytes was found to be responsible for the inhibition of herpes simplex virus replication. The effect is abolished in the presence of excess amounts of L-tryptophan. Both IFN-alpha and IFN-beta restricted herpes simplex virus replication in both cell types, but (in contrast to the results seen with IFN-gamma) the addition of an excess amount of L-tryptophan did not inhibit the induced antiviral effect.

Antiviral Agents↗
PubMed2002

Measuring recovery after a hip fracture using the SF-36 and Cummings scales.

The objective of this study was to assess outcomes of traditional treatment of fractures using the SF-36 and the Cummings Hip Scale. In designing randomized clinical trials, it is necessary to determine the timing of assessment either for progress or for the main outcome. We set out to document the recovery of patients after surgery for hip fracture using current standard methods of medical care. This was a prospective study of a cohort of patients. Patients who were receiving standard medical care completed the SF-36 and the Cummings Hip Scale at previously determined times postoperatively. The SF-36 has eight subscales, including assessments of physical function, physical role behaviors, bodily pain, mental health, social role, emotional role, vitality and general health. Thirty-eight patients completed the questionnaires at 1 year postoperatively as well as previous time points. On the Cummings Hip Scale and the physical function, bodily pain, mental health, social function, emotional role, vitality and general health subscales of the SF-36, recovery is near complete at 6 months. Only the physical role subscale differs, with a statistically significant difference between the values at 6 months and 1 year, (p = 0.02). Patients attained over 90% of the 1 year value by 6 months for all except the physical role subscale. The physical role subscale reached 85%. For a hip fracture patient who is on the road to recovery, the majority of the recovery has therefore taken place by 6 months.

Aged↗
PubMed2002

Localization of indoleamine 2,3-dioxygenase in human female reproductive organs and the placenta.

Indoleamine 2,3-dioxygenase (IDO) has been implicated in regulation of feto-maternal tolerance and protection against intracellular and extracellular pathogens. We have studied the expression of IDO in the human female reproductive tract and the placenta by immunohistochemistry. Endometrial glandular and surface epithelial cells showed increasing IDO expression during the course of the menstrual cycle. In term placenta, IDO was irregularly localized to the mesenchymal core and found in isolated areas of the syncytiotrophoblast. In first trimester pregnancy, IDO was not present in placental villi, but was present in glandular epithelium of the decidua, and there were distinctly positive cells scattered in the connective tissue, sometimes in conjunction with lymphoid aggregates. The endothelium of spiral arteries and of capillaries showed some, albeit no generalized, reactivity. IDO was also present in the epithelium of cervical glands and of Fallopian tubes. Specificity of antibody binding was confirmed by Western blot analysis. IDO mRNA was detected in first trimester decidua as determined by RT-PCR. IDO is secreted, as determined by analysis of cervical mucus by high pressure liquid chromatography for the presence of the tryptophan metabolite L-kynurenine, indicating IDO activity. Our results support the concept of IDO providing a mechanism of innate immunity protecting against ascending infections in the female reproductive tract.

Female↗
PubMed2001

Binding of plasminogen and tissue plasminogen activator to plasmin-modulated factor X and factor Xa.

Previous work in our laboratory has suggested that the fibrinolytic enzyme plasmin (Pn) inactivates coagulation factors X (FX) and Xa (FXa) in the presence of Ca(2+) and anionic phospholipid (aPL), producing fragments which bind plasminogen (Pg) and accelerate tissue plasminogen activator (t-PA). Our goals here were to determine if the Pn-mediated fragments of FX or FXa remain associated, whether they directly bind t-PA, and to quantify their interaction with Pg. Binding to aPL, benzamidine-Sepharose, or the active-site inhibitor dansyl-Glu-Gly-Arg-chloromethyl ketone demonstrated that Pn cleavage yielded noncovalent heterodimers of a fragment containing the aPL-binding domain (FXgamma(47) or FXagamma(33)) and a 13-kDa fragment (FXgamma(13) or FXagamma(13)). Both ligand blotting and surface plasmon resonance (SPR) showed that Pn-cleaved FX and FXa bound t-PA directly when Pn-treatment was effected in the presence of aPL and Ca(2+). Using SPR, apparent K(d) values of 1-3 microM and 0.3-0.4 microM were measured directly and by competition for the FXgamma(47/13)-Pg and FXagamma(33/13)-Pg interactions, respectively. For the first time, Pg-binding to a receptor was shown to be Ca(2+) enhanced, although primarily mediated by C-terminal lysine residues. Mathematical modeling of kinetic data suggesting two Pg per FXgamma(47/13) or FXagamma(33/13) was consistent with our conclusion that each subunit of FXgamma(47/13) or FXagamma(33/13) contains a C-terminal lysine. Earlier X-ray structures show that these Lys residues are distal from each other and the membrane, supporting the model where each interacts with a separate Pg. t-PA acceleration by FXgamma(47/13) or FXagamma(33/13) may therefore involve simultaneous presentation of two substrate molecules.

Anions↗
PubMed2001

Optimal design features of camelized human single-domain antibody libraries.

We have constructed a human V(H) library based on a camelized V(H) sequence. The library was constructed with complete randomization of 19 of the 23 CDR3 residues and was panned against two monoclonal antibody targets to generate V(H) sequences for determination of the antigen contact residue positions. Furthermore, the feasibility and desirability of introducing a disulfide bridge between CDR1 and CDR3 was investigated. Sequences derived from the library showed a bias toward the use of C-terminal CDR3 residues as antigen contact residues. Mass spectrometric analyses indicated that CDR1-CDR3 disulfide formation was universal. However, surface plasmon resonance and NMR data showed that the CDR3 constraint imposed by the disulfide bridge was not always desirable. Very high yields of soluble protein products and lack of protein aggregation, as demonstrated by the quality of the (1)H-(15)N HSQC spectra, indicated that the V(H) sequence for library construction was a good choice. These results should be useful in the design of V(H) libraries with optimal features.

Amino Acid Sequence↗
PubMed2001

The detergent octylglucoside neutralizes lipopolysaccharide in a specific manner.

The stimulatory effect of lipopolysaccharide (LPS) on human macrophages was found to be neutralized by the detergent octylglucoside (OG). Both macrophage stimulation and reactivity in a limulus amebocyte lysate test were suppressed by suspension of LPS in OG at concentrations between 0.25 and 2.5 mM, whereas other stimulatory lipopeptides and lipid containing stimulants were unaffected by OG. LPS at concentrations causing maximal stimulation of macrophages could be completely neutralized by non-toxic concentrations of OG. In addition, it was found that the neutralization in complex mixtures of macromolecules, such as bacterial cell lysate, was specific for LPS and that the stimulatory activity of the other substances in the mixture was not affected by the OG.

Adhesins, Bacterial↗
PubMed2000

Quantitative analyses of binding affinity and specificity for glycolipid receptors by surface plasmon resonance.

In recent years, rapid expansion has been seen of the application of SPR to a wide range of biomolecular interactions. For protein-carbohydrate interactions, SPR techniques offer the means of presenting glycolipids, and potentially glycoproteins, in an environment that closely resembles their in vivo situation. It is already clear that the technology can provide much additional insight into these interactions because it monitors the interactions under conditions that approach physiologic ones and can allow for the investigation of parameters that are not accessible by conventional approaches. Strategies for the immobilization of glycolipids on BIACORE sensor chips are not limited to the two approaches described here. For example, liposomes containing biotinylated phospholipid could be captured by avidin or streptavidin surfaces; this strategy has been used for the capture of natural membrane vesicles. Also, Biacore AB has announced plans to introduce a version of the CM5 chip that has been derivatized with an undisclosed group that mediates the capture of large amounts of liposomes.

Animals↗
PubMed2000

Characterization of high affinity monoclonal antibodies specific for chlamydial lipopolysaccharide.

Pathogens belonging to the genus Chlamydia contain lipopolysaccharide with a 3-deoxy-D- manno- oct-2-ulosonic acid (Kdo) trisaccharide of the sequence alpha-Kdo-(2-->8)-alpha-Kdo-(2-->4)-alpha-Kdo. This lipopolysaccharide is recognized in a genus-specific pattern by murine monoclonal antibodies (mAbs), S25-23 and S25-2 (both IgG1kappa), which bind as the minimal structures the trisaccharide and the terminal Kdo-disaccharide, respectively. The variable domains of these mAbs were reverse transcribed from mRNA which was isolated from hybridomas and cloned as single-chain variable fragments (scFvs) in E.coli TG1. The kinetics of binding of whole antibodies, Fab fragments and scFvs to natural and synthetically modified ligands were determined by surface plasmon resonance (SPR) using synthetic neoglycoconjugates. As examples of an antibody-carbohydrate interaction involving anionic carboxyl groups on the ligand, we report that the affinities of these antibodies are higher than usually observed in carbo-hydrate-protein interactions (K(D)of 10(-3)to 10(-5)M). SPR analy-ses of monovalent Fab and scFv binding to the natural trisaccharide epitope gave dissociation constants of 770 nM for S25-2 and 350 nM for S25-23, as determined by global fitting (simultaneous fitting of several measurements at different antibody concentrations) of sensorgram data to a one-to-one interaction model. Local fitting (separate fitting of individual sensorgram data at different antibody concentrations) and Scatchard analysis of the data gave kinetic and affinity constants that were in good agreement with those obtained by global fitting. The SPR data also showed that while S25-2 bound well to several Kdo disaccharides and carboxyl-reduced Kdo ligands, S25-23 did not. Identification of amino acids in the complementarity determining regions revealed the presence of a large number of positively charged amino acids which were located towards the center of the combining site, thus suggesting a different recognition mechanism than that observed for neutral ligands. The latter mainly involves aromatic amino acids for hydrophobic stacking inter-actions and hydrogen bonds.

Amino Acid Sequence↗
PubMed2000

Major life events as antecedents to hip fracture.

BACKGROUND: This study sought to determine whether the number of antecedent life events reported in the year before hip fracture among elderly patients was normal for the population from which these patients derive. Major life events are events such as births, deaths, major financial dealings, and major health changes. METHODS: Life events reported in the year before a fall and hip fracture for 111 hip fracture patients were compared with those of a control sample of 90 nonfracture, community-dwelling ambulatory elderly. RESULTS: The total number of life events was higher in the hip fracture group (p = 0.0001) than in the community control group. Fracture was also associated with the number of events experienced (adjusted OR, 2.1; 95% CI, 1.6-2.7; p < 0.0007), notwithstanding age, marital status, and education. CONCLUSION: Older persons who had sustained a fall-related traumatic hip fracture experienced an increased number of major life events compared with a nonfracture population sample of community-dwelling elderly controls.

Activities of Daily Living↗
PubMed1999

Analysis of receptor binding by the channel-forming toxin aerolysin using surface plasmon resonance.

Aerolysin is a channel-forming bacterial toxin that binds to glycosylphosphatidylinositol (GPI) anchors on host cell-surface structures. The nature of the receptors and the location of the receptor-binding sites on the toxin molecule were investigated using surface plasmon resonance. Aerolysin bound to the GPI-anchored proteins Thy-1, variant surface glycoprotein, and contactin with similar rate constants and affinities. Enzymatic removal of N-linked sugars from Thy-1 did not affect toxin binding, indicating that these sugars are not involved in the high affinity interaction with aerolysin. Aerolysin is a bilobal protein, and both lobes were shown to be required for optimal binding. The large lobe by itself bound Thy-1 with an affinity that was at least 10-fold weaker than that of the whole toxin, whereas the small lobe bound the GPI-anchored protein at least 1000-fold more weakly than the intact toxin. Mutation analyses provided further evidence that both lobes were involved in GPI anchor binding, with certain single amino acid substitutions in either domain leading to reductions in affinity of as much as 100-fold. A variant with single amino acid substitutions in both lobes of the protein was completely unable to bind the receptor. The membrane protein glycophorin, which is heavily glycosylated but not GPI-anchored, bound weakly to immobilized proaerolysin, suggesting that interactions with cell-surface carbohydrate structures other than GPI anchors may partially mediate toxin binding to host cells.

Bacterial Toxins↗
PubMed1999

The role of homophilic binding in anti-tumor antibody R24 recognition of molecular surfaces. Demonstration of an intermolecular beta-sheet interaction between vh domains.

The murine antibody R24 and mouse-human Fv-IgG1(kappa) chimeric antibody chR24 are specific for the cell-surface tumor antigen disialoganglioside GD3. X-ray diffraction and surface plasmon resonance experiments have been employed to study the mechanism of "homophilic binding," in which molecules of R24 recognize and bind to other molecules of R24 though their heavy chain variable domains. R24 exhibits strong binding to liposomes containing disialoganglioside GD3; however, the kinetics are unusual in that saturation of binding is not observed. The binding of chR24 to GD3-bearing liposomes is significantly weaker, suggesting that cooperative interactions involving antibody constant regions contribute to R24 binding of membrane-bound GD3. The crystal structures of the Fabs from R24 and chR24 reveal the mechanism for homophilic binding and confirm that the homophilic and antigen-binding idiotopes are distinct. The homophilic binding idiotope is formed largely by an anti-parallel beta-sheet dimerization between the H2 complementarity determining region (CDR) loops of two Fabs, while the antigen-binding idiotope is a pocket formed by the three CDR loops on the heavy chain. The formation of homophilic dimers requires the presence of a canonical conformation for the H2 CDR in conjunction with participation of side chains. The relative positions of the homophilic and antigen-binding sites allows for a lattice of GD3-specific antibodies to be constructed, which is stabilized by the presence of the cell membrane. This model provides for the selective recognition by R24 of cells that overexpress GD3 on the cell surface.

Animals↗
PubMed1999

Inhibition of indoleamine 2,3-dioxygenase in human macrophages inhibits interferon-gamma-induced bacteriostasis but does not abrogate toxoplasmastasis.

Induction of indoleamine 2,3-dioxygenase (IDO) by IFN-gamma results in growth inhibition of Toxoplasma and Chlamydia spp. as well as tumor cells. This is caused by the degradation, and therefore depletion, of L-tryptophan necessary for cell protein synthesis. Human macrophages stimulated with IFN-gamma express IDO and inhibit the growth of intracellular toxoplasma and chlamydia as well as that of extracellular bacteria such as group B streptococci. Here we describe experiments in which the L-tryptophan analog, 6-chloro-DL-tryptophan (CDLT) caused a dose-dependent inhibition in the IFN-gamma-induced IDO-mediated L-tryptophan degradation in monocyte-derived macrophages and glioblastoma cells. An inhibition of IDO activity of up to 80 % was observed at concentrations of CDLT of 750 microM. Expression of IDO at this concentration, as shown by Northern blot analysis, was unimpaired. This inhibition of IDO was coupled in glioblastoma cells by a complete abrogation of the IFN-gamma-induced toxoplasmastasis in these cells. IDO inhibition by CDLT in human macrophages resulted in a complete abrogation of the IFN-gamma-induced growth inhibition of streptococci and staphylococci. In contrast to this, IFN-gamma-induced toxoplasmastasis was not inhibited in human macrophages by CDLT-mediated IDO inhibition.

Animals↗
PubMed1999

IFN-gamma activated indoleamine 2,3-dioxygenase activity in human cells is an antiparasitic and an antibacterial effector mechanism.

In nearly all human cells IFN-gamma stimulation leads to an activation of indoleamine 2,3-dioxygenase (IDO) activity, which is responsible for anti-toxoplasma and anti-chlamydia effects. We have recently shown that IDO activation is also a defense mechanism against extracellular beta-hemolytic streptococci groups A, B, C and G in human glioblastoma cells, fibroblasts and macrophages. Similar effects were also seen with enterococci and in approximately 65% of staphylococci tested, including multiresistant strains of both species. In addition, we have found that IDO activity is differentially regulated in different cells. For example we have found that TNF-alpha enhances IFN-gamma induced IDO activity and antimicrobial effect in human glioblastoma cells whereas both IFN-gamma mediated effects were blocked by TNF-alpha as well as by IL-1 in a human uroepithelial cell line. We were able to show that the IL-1 and TNF-alpha mediated inhibition of IFN-gamma-induced IDO activity in uroepithelial cells is due to stimulation of inducible nitric oxide synthase. In human astrocytoma cells, IL-1 and TNF-alpha did not inhibit IDO activity and in concordance with this finding these cells did not show a detectable nitric oxide production.

Animals↗