The use of flow cytometry to examine calcium signalling by TRPV1 in mixed cell populations.

Flow cytometric analysis of calcium mobilisation has been in use for many years in the study of specific receptor engagement or isolated cell:cell communication. However, calcium mobilisation/signaling is key to many cell functions including apoptosis, mobility and immune responses. Here we combine multiplex surface staining of whole spleen with Indo-1 AM to visualise calcium mobilisation and examine calcium signaling in a mixed immune cell culture over time. We demonstrate responses to a TRPV1 agonist in distinct cell subtypes without the need for cell separation. Multi parameter staining alongside Indo-1 AM to demonstrate calcium mobilization allows the study of real time calcium signaling in a complex environment.

Anti-inflammatory effects of infliximab in mice are independent of tumour necrosis factor α neutralization.

Infliximab (IFX) has been used repeatedly in mouse preclinical models with associated claims that anti-inflammatory effects are due to inhibition of mouse tumour necrosis factor (TNF)-α. However, the mechanism of action in mice remains unclear. In this study, the binding specificity of IFX for mouse TNF-α was investigated ex vivo using enzyme-linked immunosorbent assay (ELISA), flow cytometry and Western blot. Infliximab (IFX) did not bind directly to soluble or membrane-bound mouse TNF-α nor did it have any effect on TNF-α-induced nuclear factor kappa B (NF-κB) stimulation in mouse fibroblasts. The efficacy of IFX treatment was then investigated in vivo using a TNF-α-independent Trichuris muris-induced infection model of chronic colitis. Infection provoked severe transmural colonic inflammation by day 35 post-infection. Colonic pathology, macrophage phenotype and cell death were determined. As predicted from the in-vitro data, in-vivo treatment of T. muris-infected mice with IFX had no effect on clinical outcome, nor did it affect macrophage cell phenotype or number. IFX enhanced apoptosis of colonic immune cells significantly, likely to be driven by a direct effect of the humanized antibody itself. We have demonstrated that although IFX does not bind directly to TNF-α, observed anti-inflammatory effects in other mouse models may be through host cell apoptosis. We suggest that more careful consideration of xenogeneic responses should be made when utilizing IFX in preclinical models.

Cross-talk between neural and immune receptors provides a potential mechanism of homeostatic regulation in the gut mucosa.

The relationship between elements of the immune system and the nervous system in the presence of bacteria has been addressed recently. In particular, the sensory vanilloid receptor 1 (transient receptor potential cation channel subfamily V member 1 (TRPV1)) and the neuropeptide calcitonin gene-related peptide (CGRP) have been found to modulate cytokine response to lipopolysaccharide (LPS) independently of adaptive immunity. In this review we discuss mucosal homeostasis in the gastrointestinal tract where bacterial concentration is high. We propose that the Gram-negative bacterial receptor Toll-like receptor 4 (TLR4) can activate TRPV1 via intracellular signaling, and thereby induce the subsequent release of anti-inflammatory CGRP to maintain mucosal homeostasis.

Colonic transcriptional profiling in resistance and susceptibility to trichuriasis: phenotyping a chronic colitis and lessons for iatrogenic helminthosis.

BACKGROUND: Helminth therapy is advocated to restore and maintain control of inflammatory responses, particularly chronic colitis. However, helminths can induce chronic colitis in susceptible individuals. Susceptibility has an immunogenetic basis: defining this is essential if nematode therapy is to be successfully and safely targeted in inflammatory bowel disease (IBD). To validate a preclinical mouse model we phenotyped the response to Trichuris muris in mice. We determined colonic transcriptional activity in naïve and infected mice and linked differential gene expression to mechanistic pathways. METHODS: T. muris-infected resistant (BALB/c) and susceptible (AKR) mice were studied to a chronic colitic timepoint (day 35). Colonic genome-wide expression was performed by microarray. Significant transcriptional changes were analyzed by cluster and gene ontology filtering and KEGG pathway mapping. RESULTS: Day 35 infected AKR displayed chronic diarrhea, weight loss, and transmural colonic inflammation; BALB/c remained asymptomatic, cleared the infection, and demonstrated normal histology. Compared to BALB/c mice, infected AKR upregulated gene expression clusters were overrepresented by immune response, chemotaxis, and apoptosis pathways. Cellular/tissue homeostasis and tight junction pathways dominated downregulated AKR expression clusters. Infected AKR T-helper cell development/polarization markers demonstrated predominant T(H) 1/T(H) 17 transcriptional activity. Colitic AKR data mirrored established murine models and human colitis. CONCLUSIONS: T. muris infection in the mouse shows striking phenotypic and transcriptional similarities to widely used models of IBD and human IBD. This preclinical mouse model presents a platform to examine biological commonalities among chronic colitides. However, these data urge caution in untargeted therapeutic helminth use until risk/benefit in susceptible individuals is more fully understood.

Rapid purification and characterization of L-dopachrome-methyl ester tautomerase (macrophage-migration-inhibitory factor) from Trichinella spiralis, Trichuris muris and Brugia pahangi.

Macrophage-migration-inhibition factor (MIF) is an essential stimulator of mammalian T-lymphocyte-dependent adaptive immunity, hence MIF orthologues might be expressed by infectious organisms as an immunosubversive stratagem. Since MIF actively catalyses the tautomerization of the methyl ester of l-dopachrome (using dopachrome tautomerase), the occurrence of MIF orthologues in several parasitic helminths was investigated by assaying and characterizing such activity. Evidence of MIF orthologues (dopachrome tautomerase) was found in the soluble fraction of the nematodes Trichinella spiralis (stage 4 larvae) and Trichuris muris (adults), and the filarial nematode Brugia pahangi (adults). The MIF orthologues of Tr. muris (TmMIF) and B. pahangi (BpMIF) were purified to homogeneity using phenyl-agarose chromatography, that of T. spiralis (TsMIF) required a further step: cation-exchange FPLC. Retention time on reverse-phase HPLC and Mr on SDS/PAGE of the nematode MIFs were similar to those of human MIF. N-terminal sequences (19 residues) of TsMIF and TmMIF showed 47 and 36% identity, respectively, with human MIF. The N-terminal sequence of BpMIF (14 residues) was identical to that of an MIF orthologue in the genome of B. malayi (Swiss-Prot, P91850) and showed 43% identity to either human or TsMIF. TsMIF had 10-fold higher dopachrome tautomerase activity than MIF from the other sources. The enzyme activities of TsMIF, BpMIF and TmMIF were less sensitive to inhibition by haematin (I50: >15 microM, >15 microM and 2.6 microM, respectively) than that of human MIF (I50 0.2 microM). Significant dopachrome tautomerase or phenyl-agarose-purifiable MIF-like protein was not detected in the soluble fraction of the nematodes Heligmosomoides polygyrus and Nippostrongylus brasiliensis, the cestode Hymenolepis diminuta, or the trematodes Schistosoma mansoni, S. japonicum and S. haematobium, or the free-living nematode, Caenorhabditis elegans, which does contain an MIF-related gene.

Interaction of macrophage-migration-inhibitory factor with haematin.

Macrophage-migration-inhibitory factor (MIF) is retained by S-hexylglutathione-agarose but is not specifically eluted in high yield. Human liver MIF was purified in high yield using retention by phenyl-agarose at low ionic strength and cation-exchange FPLC as described for bovine lens MIF [Rosengren, Bucala, Aman, Jacobsson, Odh, Metz and Rorsman (1996) Mol. Med. 2, 143-149]. The l-dopachrome methyl ester tautomerase activity of human liver MIF was not inhibited by a variety of glutathione S-conjugates, eicosanoids or glucocorticoids but was very sensitive to inhibition by haematin (IC50 100-200 nM). The inhibition was non-competitive and showed positive co-operativity (h=5.8). Similar sensitivity to haematin was obtained with purified recombinant human MIF. The sensitivity of MIF to haematin is approx. 1000-fold greater than for any previously described ligands, and is within its physiological range. Therefore the interaction is likely to be important in modulating the function of MIF in the initiation of immune responses.

Results of mechanical circulatory assistance before heart transplantation.

Between February 1984 and December 1987, 63 patients were accepted as candidates for heart transplantation. Eighteen patients (29%) required some form of mechanical circulatory support before transplantation; eight patients received an intraaortic balloon pump, five patients had left ventricular assist devices, two patients received biventricular assist devices, and in three patients the total artificial heart was implanted. Fourteen of the 18 patients underwent transplantation with seven longterm survivors.

Delayed reversal of impaired vasodilation in congestive heart failure after heart transplantation.

The effects of changes in central cardiovascular function on peripheral vasodilation were investigated. Strain gauge plethysmography was used to measure the maximal blood flow response following release of forearm arterial occlusion and the peak reactive hyperemic blood flow response (ml/min.100 ml) before and twice after orthotopic heart transplantation in 10 subjects with severe congestive heart failure. The 2 posttransplantation studies were done before hospital discharge (mean 18 days after transplantation) and again after discharge (mean 114 days after transplantation). Transplantation led to a significant but delayed increase in maximal vasodilation (reactive hyperemic blood flow: pretransplant 21 +/- 3; predischarge 25 +/- 2; postdischarge 43 +/- 5) and a concurrent significant reduction in minimal forearm resistance. Although the improvement in peripheral vasodilator function may be linked to improvement in cardiac function, this linkage is not direct, nor is it immediate. If the normalization of maximal metabolic blood flow is related to resumption of normal physical activity postdischarge, then much of the basic abnormality in vasodilator capacity in congestive heart failure may be related to physical deconditioning.

Aortic rupture caused by fungal aortitis: successful management after heart transplantation.

A 26-year-old man with end-stage idiopathic cardiomyopathy was supported with a Pierce-Donachy left ventricular assist device for 31 days before orthotopic heart transplantation. Fungal endocarditis was discovered at the time of recipient cardiectomy, and antifungal therapy was begun. Fungal mediastinitis developed 4 days after transplantation and was treated with mediastinal irrigation. Massive mediastinal hemorrhage caused by fungal aortitis occurred on two occasions and was successfully treated with a bovine pericardial patch. The patient is well 9 months after transplantation.

Atrial natriuretic peptide in recipients of human orthotopic heart transplants.

Atrial natriuretic peptide (ANP) has been proposed to play a role in the maintenance of fluid and electrolyte homeostasis. ANP can be secreted in response to atrial stretch, and right atrial pressure has been proposed to control its secretion in vivo. This study was designed to characterize plasma immunoreactive (IR)-ANP in heart transplant recipients and to determine if IR-ANP levels in these patients correlate with cardiac filling pressures. Nineteen heart transplant recipients were studied during endomyocardial biopsy (group 1), and 20 patients with symptoms of coronary artery disease were studied during cardiac catheterization (group 2). Central venous blood samples and right heart catheterization data were obtained in each patient. IR-ANP levels were measured with a radioimmunoassay for alpha human ANP in extracted plasma samples. There were no differences between groups 1 and 2 in right atrial pressure (6.5 +/- 0.8 versus 6.1 +/- 0.6), pulmonary capillary wedge pressure (13.3 +/- 1.2 versus 11.2 +/- 1.3), mean pulmonary artery pressure (18.0 +/- 1.2 versus 18.2 +/- 1.3), or cardiac index (2.8 +/- 0.0 versus 2.8 +/- 0.1), but patients in group 1 had a higher mean blood pressure (110.6 +/- 2.4 versus 98.3 +/- 3.1, p less than 0.001). Plasma IR-ANP levels in heart transplant recipients were higher than normal (217.0 +/- 28.1 versus 19.2 +/- 2.1 pg/ml, p less than 0.001) and higher than in cardiac catheterization patients (217.0 +/- 28.1 verus 71.1 +/- 11.5 pg/ml, p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

The use of a paracorporeal ventricular assist device as a bridge to cardiac transplantation.

Our experience with six patients has demonstrated that the Pierce-Donachy VAD is capable of providing adequate circulatory support with acceptable morbidity and mortality in patients awaiting cardiac transplantation. Four of the six patients received cardiac allografts and three patients have been discharged.

Long-term results of ventricular assist pumping in postcardiotomy cardiogenic shock.

Short-term survival of patients who require ventricular assist pumping for postcardiotomy cardiogenic shock has been encouraging and has provided enough survivors to allow evaluation of long-term results. We observed nine survivors, 39 to 69 years of age (mean = 54.6) for 10 to 53 months (mean = 31) after their discharge from the hospital. In three patients, the angiographic ejection fraction was normal before the operation, whereas in six patients it was moderately to severely impaired. All nine patients were in New York Heart Association Functional Class IV before operation. After operation, eight of the nine patients were in Functional Class I or II. In the remaining patient, the ejection fraction fell markedly and the postoperative functional class remained unchanged. Two patients were employed full-time and one, part-time. Five patients were retired but active and had no cardiac disability, although two of the five did have residual partial noncardiac disability. The remaining patient, a housewife, continued to have compensated congestive heart failure but was relieved of angina. During the follow-up period, five of the nine patients died suddenly after 12, 18, 25, 30, and 38 months from causes that included pulmonary embolus and probable arrhythmias. Survivors who have had ventricular assist pumping for postcardiotomy cardiogenic shock have a good chance of resuming active lives. The risk of being a "cardiac cripple" is small, as is the risk of significant noncardiac disability.

Aortic valve replacement and combined aortic valve replacement and coronary artery bypass grafting: predicting high risk groups.

To determine which groups of patients are at highest risk for operative or late mortality, 259 consecutive patients who underwent operation between 1978 and 1984 were studied; 170 underwent aortic valve replacement and 89 underwent aortic valve replacement combined with coronary artery bypass grafting. Multivariate analysis of risk factors selected emergency operation and patient age older than 70 years as the strongest predictors for operative death. Although patients having aortic valve replacement and coronary artery bypass grafting had a higher operative mortality rate (13.5 versus 3.5%), the combined operation had no independent predictive effect on early or late results. At a mean follow-up time of 48 months after surgery, 72% of the survivors of operation were living, 10% were lost to follow-up and 18% were dead. Seventy-seven percent of long-term survivors were in New York Heart Association functional class I or II. The incidence of thromboembolism, paravalvular leak, bacterial endocarditis and hemorrhage each occurred at a rate of less than 1% per patient-year. The factors associated with late death were preoperative age, male sex, left ventricular end-diastolic pressure, cardiac index and functional class. Despite an increase in operative mortality, patients undergoing emergency operation were not at higher risk of late death. Operative mortality is concentrated among several high risk groups. For patients undergoing elective operation, operative mortality is low, especially if the patient is less than 70 years old. Late results are good for all groups of patients undergoing operation, including those who are at greater risk of dying at operation.

Disseminated Trichosporon beigelii (cutaneum) infection in an artificial heart recipient.

A 44-year-old man with end-stage ischemic cardiomyopathy was supported with an intra-aortic balloon and The Penn State Heart (artificial) prior to orthotopic cardiac transplantation on the 14th hospital day. At the time of transplantation, intraoperative cultures of pericardial and mediastinal fluid showed growth of Trichosporon beigelii (cutaneum). Shortly thereafter the patient developed visceral dissemination of T beigelii with no associated skin lesions. He was treated with amphotericin B and rifampin, but postmortem examination showed persistent, disseminated infection.

Mechanical support of the circulation followed by cardiac transplantation.

Improvements in both mechanical circulatory support devices and immune therapy promise a wider use of sequential mechanical support as a bridge to orthotopic cardiac transplantation. The intra-aortic balloon pump, the left and right ventricular assist pumps, and the pneumatic artificial heart represent the range of devices capable of keeping a patient alive who is awaiting a donor organ. The major difficulty in using circulatory support devices is infection, which is caused by their required percutaneous tubes. We report here our experiences with mechanical circulatory support devices as a bridge to cardiac transplantation. In a series of 31 consecutive transplant procedures, six patients have required preoperative mechanical circulatory support. The intra-aortic balloon pump was used in two patients for 2 and 14 days, respectively, before transplantation. Both patients are well 10 and 11 months after the transplant procedure. Two patients required the left ventricular assist device for 11 and 21 days and are alive 3 weeks and 8 months, respectively, after transplantation. One patient was supported by the pneumatic artificial heart for 10 days before a donor heart became available but died of septic shock 17 days after transplantation. A second patient received a pneumatic artificial heart 7 days after transplantation when the heart transplant failed. He has been in stable condition for 45 days but is recovering from renal failure. Our early experiences indicate that either partial or total mechanical support as a bridge to transplantation is successful if overwhelming sepsis or renal failure can be avoided.

Bridge to heart transplantation: the Penn State experience.

At The Pennsylvania State University, we have performed four bridge-to-heart transplantation procedures in the past year. Two patients were supported with an LVAD, and two patients were supported with a TAH. One patient had a successful transplantation and remains well 10 months after operation; two patients died of sepsis after transplantation. A fourth patient has had a TAH in place for greater than 110 days and is currently awaiting transplantation. The problems that remain to be solved are developing strategies to minimize the risk of infection, defining the indications for use of an LVAD vs a BVAD vs a TAH, developing an effective regimen to prevent thromboembolism, and determining the proper interval for transplantation after the insertion of a mechanical pump as a bridge.

Thyroid hormone levels in heart and kidney cadaver donors.

Experimental animal work has shown that thyroid hormone levels become undetectable 9 hours after brain death. It is unknown whether such an acutely hypothyroid state contributes to the hemodynamic instability of brain-dead donors or whether these donors should be resuscitated with thyroid hormone. No previous clinical study has examined thyroid hormone levels in human brain-dead organ donors. We retrospectively examined the thyroid hormone levels as measured by triiodothyronine and thyroxine in 22 human cadaver donors. Eight donors provided heart and kidney allografts, and the remaining 12 were kidney donors only. No donor had a normal triiodothyronine level and 10 were below normal, with undetectable levels in 12. Thyroxine levels were normal in 10 and below normal in 12. In comparing donors with below normal to undetectable triiodothyronine levels and donors with normal to below normal thyroxine levels, no statistically significant differences were found regarding blood pressure during harvest, duration of harvest, or dopamine requirements during harvest. Donors with a closed-head injury plus multiple injuries had statistically lower thyroxine values than donors with only a closed-head injury. For the heart donors, no correlation was found between thyroid hormone levels and the duration or dose of dopamine required for the heart allograft recipients after transplant. The incidence of acute tubular necrosis in the kidney transplants did not correlate with the donor thyroid hormone levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Survival following rupture of a pancreatic abscess in a heart transplant recipient.

A 43-year-old man underwent orthotopic heart transplantation for end-stage ischemic cardiomyopathy. Immunosuppressive therapy consisted of cyclosporine and corticosteroids. The diagnosis of acute pancreatitis was made on the ninth postoperative day and was based on clinical symptoms and an upper gastrointestinal barium study. Both serum and urine amylase values were normal. Abdominal ultrasound examination was nondiagnostic. Two weeks postoperatively, the patient's clinical condition deteriorated sharply. Chest and abdominal roentgenograms revealed free intraperitoneal air, as well as air in the lesser sac. Diagnosis of a ruptured pancreatic abscess was made, and he underwent immediate exploratory laparotomy. Four liters of purulent fluid were present in the peritoneal cavity. A ruptured pancreatic abscess was found, and it had dissected above the superior mesenteric vessels and down the right gutter over the inferior vena cava. After extensive retroperitoneal debridement and copious irrigation, multiple surgical drains were placed. The patient is now well and is performing normal daily activities 16 months after the transplantation procedure. The incidence and proposed causes of pancreatitis occurring after heart transplant are reviewed, and we discuss our management of this complication.