Outbreak of Clostridium histolyticum infections in injecting drug users in England and Scotland.

Clostridial infections in injecting drug users in the United Kingdom are a relatively new phenomenon that came to light in 2000 when cases of serious illness and deaths due to Clostridium novyi were recorded. In the period December 2003 to April 2004, the Anaerobe Reference Laboratory received twelve referrals of an extremely rare isolate, Clostridium histolyticum, from cases of infection in injecting drug users submitted from nine different hospitals in England and Scotland. Molecular typing of these isolates by two different methods of pulsed-field gel electrophoresis and PCR ribotyping revealed they are all indistinguishable, indicating a common source of the infections, most probably a batch of heroin that was recently distributed across the UK.

Antibiotic susceptibilities of Gram-positive anaerobic cocci: results of a sentinel study in England and Wales.

OBJECTIVE: A sentinel study was carried out to determine the antimicrobial susceptibilities of Gram-positive anaerobic cocci (GPAC) freshly isolated from clinical material in diagnostic laboratories in England and Wales. METHODS: A total of 113 GPAC isolates consisting predominantly of current or former members of the genus Peptostreptococcus was obtained from 17 sentinel laboratories in England and one in Wales. Minimum inhibitory concentrations (MICs) of 10 antimicrobial agents were determined by the Etest method. The agents tested were: penicillin, tetracycline, erythromycin, cefoxitin, clindamycin, chloramphenicol, imipenem, co-amoxiclav, piperacillin/tazobactam and metronidazole. MIC50 and MIC90 values for each drug-species combination were calculated whenever suitable numbers of each species were obtained. RESULTS: Excellent spectra of activity (0% resistance) against GPAC were seen for metronidazole, piperacillin/tazobactam, cefoxitin, imipenem and chloramphenicol. Low degrees of resistance to co-amoxiclav (3.5%), clindamycin (7.1%), penicillin (7.1%) and significant degrees of resistance to tetracycline (41.6%) and erythromycin (27.4%) were detected. Some examples of putative macrolide-lincosamide linked resistance were noted in seven (6.2%) isolates of GPAC. CONCLUSION: This study is one of the largest susceptibility studies specifically on GPAC carried out to date and the resulting data may be of value to those involved in the empirical treatment of infections involving Gram-positive anaerobic cocci.

Heat and acid tolerance of Clostridium novyi Type A spores and their survival prior to preparation of heroin for injection.

Clostridium novyi Type A was implicated as a cause of an outbreak of serious illness and deaths among drug users in the United Kingdom who injected heroin intramuscularly. A contaminated batch of heroin was believed to be the source of infection. To test the ability of the outbreak strain to survive certain processes associated with heroin use, it was tested for its ability to survive a range of temperature and pH and the process used in preparation of "street" heroin for injection. C. novyi spores survived temperatures of up to 100 degrees C in aqueous solution for 5 min and survived pH 2.0 at ambient temperatures for a similar time. However, a combination of low pH and raised temperatures reduced survival times. An experiment reconstructing the "street" preparation of heroin demonstrated that any C. novyi spores present would survive this process and thus be capable of initiating infection under the right conditions.

Phosphorylation of inhibitory subunit of troponin and phospholamban in rat cardiomyocytes: modulation by exposure of cardiomyocytes to hydroxyl radicals and sulfhydryl group reagents.

Myocytes were isolated from rat heart ventricles and then incubated with [32P]-sodium phosphate to label intracellular ATP stores. Incubations of the [32P]-labelled cardiomyocytes with a beta-adrenoceptor agonist isoproterenol (10 microM) and with a plant diterpene forskolin (100 microM) which directly stimulates adenylyl cyclase increased the phosphorylation of an inhibitory subunit of troponin (TN-I) and phospholamban (PLN). Brief exposure (1 min) of labelled myocytes to the hydroxyl radical generating system (H2O2 plus FeCl2) decreased markedly the stimulatory action of isoproterenol and forskolin on TN-I and PLN phosphorylation. Similar exposure of myocytes to 5-5'-dithiobis-nitrobenzoic acid (DTNB) a sulfhydryl oxidizing reagent exerted little inhibitory effect on the isoproterenol or forskolin stimulated TN-I and PLN phosphorylation. In contrast exposure of myocytes to low concentrations (< 50 microM) of N-ethylmaleimide (NEM) a sulfhydryl alkylating reagent augmented the stimulatory effect of isoproterenol on TN-I and PLN phosphorylation. The results further showed that brief treatment of myocytes to H2O2 plus FeCl2 markedly decreased isoproterenol-, but not forskolin-, stimulated cyclic AMP accumulation in the myocytes. The stimulatory action of NEM on the isoproterenol-stimulated TN-I and PLN phosphorylation appeared related to greater increase in the isoproterenol-stimulated cyclic AMP accumulation in the NEM-treated cardiomyocytes. The results are consistent with the postulate that hydroxyl radical exposure of cardiomyocytes blunts the beta-adrenoceptor-mediated stimulation of adenylyl cyclase leading to decreased phosphorylation of TN-I and PLN and imply that such alterations account in part the reported depressed rate of relaxation of the myocardium exposed to oxygen free radicals.

Protein phosphorylation in rat cardiac microsomes: effects of inhibitors of protein kinase A and of phosphatases.

The phosphorylation of rat cardiac microsomal proteins was investigated with special attention to the effects of okadaic acid (an inhibitor of protein phosphatases), inhibitor 2 of protein phosphatase 1 and inhibitor of cyclic AMP-dependent protein kinase (protein kinase A). The results showed that okadaic acid (5 microM) modestly but reproducibly augmented the protein kinase A-catalyzed phospholamban (PLN) phosphorylation, although exerted little effect on the calcium/calmodulin kinase-catalyzed PLN phosphorylation. Microsomes contained three other substrates (M(r) 23, 19 and 17 kDa) that were phosphorylated by protein kinase A but not by calcium/calmodulin kinase. The protein kinase A-catalyzed phosphorylation of these three substrates was markedly (2-3 fold) increased by 5 microM okadaic acid. Calmodulin was found to antagonize the action of okadaic acid on such phosphorylation. Protein kinase A inhibitor was found to decrease the protein kinase A-catalyzed phosphorylation of microsomal polypeptides. Unexpectedly, inhibitor 2 was also found to markedly decrease protein kinase A-catalyzed phosphorylation of phospholamban as well these other microsomal substrates. These results are consistent with the views that protein phosphatase 1 is capable of dephosphorylating membrane-associated phospholamban when it is phosphorylated by protein kinase A, but not by calcium/calmodulin kinase, and that under certain conditions, calcium/calmodulin-stimulated protein phosphatase (protein phosphatase 2B) is also able to dephosphorylate PLN phosphorylated by protein kinase A. Additionally, the observations show that protein phosphatase 1 is extremely active against the three protein kinase A substrates (M(r) 23, 19 and 17 kDa) that were present in the isolated microsomes and whose state of phosphorylation was particularly affected in the presence of dimethylsulfoxide. Protein phosphatase 2B is also capable of dephosphorylating these three substrates.

Sarcoplasmic reticulum Ca(2+)-pump dysfunction in rat cardiomyocytes briefly exposed to hydroxyl radicals.

The effects of hydroxyl radical exposure of intact cardiomyocytes on sarcoplasmic reticulum (SR) function were investigated. For this purpose, isolated rat heart myocytes were exposed briefly (1 min) to the hydroxyl radical generating system (H2O2/FeCl2 or FeSO4) or 5-5'-dithiobis-nitrobenzoic acid (DTNB), a sulfhydryl oxidizing reagent, and following this a SR-enriched fraction was isolated. Marked decreases in the SR calcium uptake activities were seen in the myocytes exposed to either the hydroxyl radical-generating system or DTNB. The exposure of myocytes to the hydroxyl radical, but not DTNB, markedly increased the amount of malonyldialdehyde (MDA) in the subsequently isolated SR. Total sulfhydryl group content in SR was decreased by exposure of myocytes to DTNB. Further, there was a significant decrease in [3H]-NEM binding to SR isolated from the hydoxyl radical-treated myocytes indicating that sulfhydryl groups are affected (oxidized). Both mannitol and catalase were found to offer complete protection against the inhibitory effect of peroxide +/- iron on calcium uptake. Also the above-mentioned alterations in both MDA and sulfhydryl group content were prevented by mannitol and catalase. Exogenously added cyclic AMP-dependent protein kinase (A-PK) or calmodulin (CAM) increased SR calcium uptake activity. In the SR isolated from the treated myocytes, the stimulatory effects of A-PK and CAM were also seen, although under all assay conditions calcium uptakes were of lower magnitude. The findings are consistent with the view that the damaging effect of the hydroxyl radical and DTNB on the functioning of SR occurs rapidly in the intact cardiomyocytes. The hydroxyl radical-provoked damage involves both protein sulfhydryl and lipid oxidation.

Cardiac allograft vasculopathy. Preferential regulation of endothelial cell-derived mesenchymal growth factors in response to a donor-specific cell-mediated allogeneic response.

We have previously reported that cell-mediated immunity to vascular endothelium is associated with the development of cardiac allograft vasculopathy (CAV). The mechanism by which a cell-mediated immune response to the coronary vascular is translated into the development of CAV is, however unknown. Peripheral blood mononuclear cells (PBMCs) obtained serially following cardiac transplantation were cocultured with donor-specific human aortic endothelial cells (HAECs) in 47 allograft recipients, 9 of whom had CAV (CAV+) at 1 year by angiography. At 20 hr following coculture, HAEC poly (A+) RNA was isolated, reverse-transcribed, and the cDNA-amplified (PCR) for a panel of growth factors (GFs) known to alter smooth muscle cell proliferation or migration. Relative quantitation of PCR product was performed using high-pressure liquid chromatography (HPLC). Three patterns of GF regulation were observed depending on the GF, the time posttransplant, and whether the patient had CAV: (1) no regulation (TGF-beta, PDGF-A early post-tx); (2) upregulation irrespective of CAV (bFGF, PDGF-B, TGF-alpha early post-tx); and (3) preferential or exclusive upregulation by CAV+ patients (PDGF-A and TGF-alpha late post-tx, HB-EGF early and late post-tx). For example, using PBMCs as stimulators, obtained 6 months posttransplant from CAV+ patients, increases in HAEC-derived PDGF-A chain (31 +/- 7 to 69 +/- 11), TGF-alpha (97 +/- 27 to 201 +/- 23), and HB-EGF (78 +/- 16 to 173 +/- 27) mRNA were demonstrated (all P<0.05 or greater using HPLC peak area as units). These data demonstrate that cell-mediated activation of vascular endothelial cells in patients with CAV results in preferential upregulation of certain endothelial-derived mesenchymal growth factors capable of stimulating smooth muscle cell proliferation and migration.

Temporal reduction in acute rejection after heart transplantation is not associated with a reduction in cell-mediated responses to donor-specific vascular endothelium.

BACKGROUND: Over the first 6 months after clinical transplantation, the incidence of rejection falls despite typically substantial decreases in maintenance immunosuppression. Despite this, chronic vascular rejection, manifested by an accelerated form of coronary artery disease is usually evident by the first annual angiogram and continues to progress over subsequent years. METHODS: To investigate this phenomenon further, peripheral blood mononuclear cells were prepared from blood samples obtained from 42 cardiac allograft recipients at 1 week, 3 months, and 6 months after transplantation and co-cultured with endothelial cells isolated and cultured from the aortas of their specific cardiac allograft donors. Donor-specific alloreactivity was assessed by (1) peripheral blood mononuclear cell proliferation (3H-thymidine incorporation) and (2) up-regulation of endothelial cell major histocompatibility complex class I and class II antigens and ICAM-1 expression (flow cytometry) at all three time points. RESULTS: Over this 6-month period, rejection incidence fell from 0.68 rejections/patient to 0.12 rejection/patient. Cyclosporine dose was reduced from 5.6 +/- 0.3 mg/kg (mean +/- standard error of the mean) to 4.5 +/- 0.2 mg/kg, prednisone dose was reduced from 0.58 +/- 0.08 mg/kg to 0.17 +/- 0.02 mg/kg, and azathioprine remained constant at approximately 2 mg/kg over the 6-month period. Despite this reduction in rejection and immunosuppression, no measure of in vitro donor-specific cell-mediated response to endothelial cells decreased over the 6-month time period. Peripheral blood mononuclear cell proliferation in response to donor-specific endothelial cells was unchanged between 1 week (916 +/- 139 counts/min [cpm]) and 3 months (896 +/- 135 cpm) and increased at 6 months (1738 +/- 243 cpm, p < 0.01). The increase in endothelial cell major histocompatibility complex class II expression in response to recipient peripheral blood mononuclear cells likewise was unchanged between 1 week (42.5 +/- 7.8 mean channel shift [mcs]) and 3 months (34.7 +/- 6.6 mcs) and increased substantially at 6 months (95.4 +/- 17.2 mcs, p < 0.02). The magnitude of the increase in endothelial cell major histocompatibility complex class I antigen and ICAM-1 expression in response to co-culture with recipient peripheral blood mononuclear cells did not change over the 6-month period. CONCLUSIONS: These data suggest an important dichotomy between cell-mediated responses to allograft parenchyma versus those to allograft vasculature and may provide an explanation for progressive vascular disease despite the absence of acute rejection.

Cardiac allograft vasculopathy. Association with cell-mediated but not humoral alloimmunity to donor-specific vascular endothelium.

BACKGROUND: Cardiac allograft vasculopathy (CAV) is an accelerated form of coronary artery disease responsible for the majority of late deaths after cardiac transplantation. Although most consider this complication a manifestation of chronic allograft rejection, it has not been established whether this disease is a consequence of humoral or cell-mediated alloreactivity. METHODS AND RESULTS: Human aortic endothelial cells (HAECs) were isolated from donor aortas obtained at the time of organ acquisition for 52 cardiac allograft recipients. Serum and peripheral blood mononuclear cells were obtained from these 52 allograft recipients at several time points during the first year after transplantation. Lymphocyte proliferation (LP) in response to donor-specific HAECs and alloantibody binding to interferon-gamma-treated donor-specific HAECs were performed and correlated with clinical parameters, including HLA matching, acute cellular rejection, and coronary artery disease on surveillance angiography. Ten of the 52 patients studied had angiographic or autopsy evidence of coronary artery disease in the first posttransplantation year (CAV+ group). The CAV+ group had higher LP responses to their donor HAECs at 1 week, 3 months, and 6 months after transplantation compared with the CAV- group (1 week: 1439 +/- 222 versus 824 +/- 141 counts per minute [cpm], P = .026; 3 months: 1282 +/- 388 versus 884 +/- 94 cpm, P = .07; 6 months: 2504 +/- 635 versus 1540 +/- 209 cpm, P = .036; CAV+ versus CAV-, respectively). Only 8 of the 52 patients had donor-specific alloantibodies, and there was no relation between antibody presence and CAV. Other clinical parameters that correlated with CAV included the level of HLA-DR mismatch and the presence of late acute rejection. CONCLUSIONS: CAV is associated with donor-specific cell-mediated alloreactivity to vascular endothelium. Humoral immunity does not appear to have a major role in this disease.

Time course and contact dependence of allogeneic lymphocyte-induced human aortic endothelial cell-derived interleukin-6.

Vascular endothelial cells secrete the pluripotent cytokine interleukin-6, and the induction of this secretion can be regulated by a number of other immune-related cytokines. To determine whether a cellular alloimmunologic response to vascular endothelial cells alters the expression of interleukin-6 production by endothelial cells, we cocultured peripheral blood lymphocytes with a pool of human aortic endothelial cells. In response to the pool of allogeneic human aortic endothelial cells, lymphocytes from 10 separate donors proliferated to varying degrees after 5 days of coculturing. After 20 hours, human aortic endothelial cell-derived messenger RNA coding for interleukin-6 increased an average of 96% after exposure to allogeneic lymphocytes and the amount of biologically active interleukin-6 released into the media increased 69%. The kinetics of human aortic endothelial cell interleukin-6 messenger RNA expression in response to lymphocytes from an additional three donors was determined over a 48-hour period. Human aortic endothelial cell interleukin-6 messenger RNA increased approximately threefold over control, as early as 2 hours after exposure to allogeneic lymphocytes and returned toward control levels by 48 hours. Activation of six additional isolates of lymphocytes with phorbol myristate acetate before exposure to human aortic endothelial cells resulted in an increase in human aortic endothelial cell-derived interleukin-6 bioactivity regardless of whether the cells were in direct contact with the human aortic endothelial cells, but the interleukin-6 level increase was approximately twofold higher in those cocultures where there was direct contact. These data show that allogeneic lymphocytes have the potential of regulating vascular endothelial cell-derived interleukin-6, and direct lymphocyte-endothelial cell contact appears to be required for optimal interleukin-6 induction in this in vitro system.

Phenotypic variations in resting and activated levels of ICAM-1 expression by cultured human aortic endothelial cells.

ICAM-1 is an inducible glycoprotein important in the adhesion, activation, and transmigration of circulating leukocytes across the vascular endothelial monolayer, and it likely plays a key role in the allogeneic response. To determine the reproducibility and significance of variations in resting levels of cell surface ICAM-1, 3 individual measurements of ICAM-1 levels were performed on 26 individual isolates of human aortic endothelial cells (HAECs) both at rest and following activation by allogeneic lymphocytes, using flow cytometry. Resting HAEC ICAM-1 levels varied 10-fold (range 6-60 mean fluorescence channels) depending on the isolate studied. There were strong correlations (r = 0.71 to 0.77, P < 0.0001) between the three measurements (performed no closer than weekly intervals on separate cultures), attesting to the consistency of the phenotypic expression. Constitutive expression of ICAM-1 was not affected by cell age, based upon comparing a subset of these isolates across 3 population doublings. Levels of HAEC ICAM-1 following allogeneic lymphocyte activation varied 15-fold (range 20-300 mean fluorescent channels) and, more important, correlated with resting ICAM-1 levels (r = 0.58, P = 0.002). Finally, constitutive ICAM-1 expression was related to TNF-alpha-induced ICAM-1 levels based upon a subset of the isolates studied. These data suggest that phenotypic, and likely genetic, differences in quiescent endothelial cell adhesion molecule expression can influence inflammatory responses including alloresponsiveness to the vasculature.

Regulation of human aortic endothelial cell-derived mesenchymal growth factors by allogeneic lymphocytes in vitro. A potential mechanism for cardiac allograft vasculopathy.

Cardiac allograft vasculopathy is thought to be triggered by an alloreactive response to the donor coronary vasculature, resulting in smooth muscle cell proliferation and ultimate occlusion of the donor coronary arteries. To determine whether allogeneic lymphocytes are capable of regulating endothelial-derived smooth muscle cell (SMC) growth factors, human aortic endothelial cells (HAECs) were exposed to allogeneic lymphocytes. The HAEC-lymphocyte co-cultures were assessed for (a) lymphocyte proliferation in response to the allogeneic HAECs; (b) release of soluble factors that stimulate human aortic SMC proliferation; and (c) alteration of HAEC mRNA levels for a panel of known SMC growth factors. Co-culture conditioned medium increased SMC proliferation, compared to medium conditioned by HAECs alone. HAECs exposed to allogeneic lymphocytes increased their expression of mRNA for basic fibroblast growth factor, transforming growth factors alpha and beta, and platelet derived growth factor A and B chains. These results demonstrate that allogeneic lymphocytes are capable of inducing HAECs to increase mRNA levels for several mesenchymal growth factors and to release bioactive products capable of stimulating SMC cell proliferation in vitro. Additionally, the data support the hypothesis that alloreactive lymphocytes can stimulate allogeneic donor endothelial cells to produce growth factors that may contribute to the intimal thickening seen in cardiac allograft vasculopathy.

The modulation of human aortic endothelial cell ICAM-1 (CD-54) expression by serum containing high titers of anti-HLA antibodies.

Allograft recipients who have preformed antibodies to MHC determinants or develop these antibodies post-transplantation have a higher incidence of cellular rejection and graft loss. It is unclear whether this association is an etiologic one or whether the presence of these antibodies solely identifies individuals with a more pronounced alloimmunologic response. To determine whether antibodies to MHC determinants have a direct role in enhancing cell-mediated immunity, specifically in altering effector-target cell adhesion, the expression of endothelial cell surface intercellular adhesion molecule-1 (ICAM-1) in response to serum with high-titer anti-HLA antibodies was investigated. The target cells used were a pool of blood group O human aortic endothelial cells (HAECs) representing a wide range of HLA-A, B, C, and DR phenotypes. The test serum was serum pooled from 30 highly sensitized individuals (panel-reactive antibody 80%). Antibody binding to HAECs, and HAEC expression of class I and class II major histocompatibility (MHC) antigens and ICAM-1 were assessed by flow cytometry. General HAEC metabolic changes were assessed by 3H-uridine incorporation as a measure of RNA synthesis. Test serum resulted in almost a 14-fold increase in HAEC surface ICAM-1 expression compared with control serum, and titrations of test serum yielded a strong correlation between IgG bound to HAECs and HAEC ICAM-1 expression (r = 0.92). Test serum induced no change in expression of HAEC class I or class II MHC antigens, or 3H-uridine incorporation. The HAEC ICAM-1-inducing ability of the test serum was retained by concentrating the high molecular weight (> 100 kilodaltons) fraction of the test serum, isolation and purification of IgG from the test serum, and lost by absorbing this fraction with pooled platelets, suggesting that the activity was mediated by antibodies directed against MHC class I determinants. These data suggest that the presence of anti-HLA antibodies is more than a marker for individuals with greater alloreactive responsiveness. Anti-HLA antibodies may directly and specifically alter adhesion of effector cells to the allograft.

The pattern of cytokine messenger RNA expression in human aortic endothelial cells is different from that of human umbilical vein endothelial cells.

Endothelial cells most readily available and most frequently used in investigations of alloimmunity and cytokine expression and function are derived from human umbilical veins. It is unclear whether cells derived from fetal venous tissue are relevant to phenomena related to the adult allograft, especially in areas such as cardiac allograft vasculopathy, a chronic rejection process directed against the coronary arteries. Human aortic endothelial cells (HAECs) were compared to human umbilical vein endothelial cells (HUVECs) for their constitutive expression of poly (A)+ RNA coding for a group of cytokines known to stimulate smooth muscle cell proliferation, including acidic fibroblast growth factor, basic fibroblast growth factor, transforming growth factor alpha, transforming growth factor beta, platelet-derived growth factor A-chain, platelet-derived growth factor B-chain and amphiregulin. Poly (A)+ RNA coding for basic fibroblast growth factor and transforming factor-beta was consistently expressed by all nine isolates of HAECs, but platelet-derived growth factor A- and B-chain were expressed in only six of the nine isolates. In most cases this was related to the presence of transforming growth factor alpha expression. In contrast, HUVECs consistently expressed basic fibroblast growth factor, transforming growth factor-beta, and both platelet-derived growth factor chains. Transforming growth factor alpha expression was never seen in the HUVEC isolates. No endothelial cell isolate expressed mRNA coding for acidic fibroblast growth factor or amphiregulin. There appear to be differences between cytokine gene expression patterns by endothelial cells from different vascular beds.

Tanning salon exposure suppression of DNA repair capacity and mitogen-induced DNA synthesis.

Mitogen responsiveness and the capacity to repair genetic damage were measured in lymphocytes collected from young, healthy, adult Caucasians immediately before exposure in commercial tanning salons and again 24 h after exposure. For every individual studied, tanning exposure produced significant inhibition of phytohemagglutinin-induced mitogenesis or of the ability to repair DNA lesions by unscheduled DNA synthesis. The results imply that such exposure could: (1) pose a significant hazard for individuals who are already immunosuppressed (e.g. cancer patients, AIDS patients or carriers of latent HIV) and (2) increase the carcinogenic effects of environmental mutagens.