Management of steroid-resistant late acute cellular rejection following face transplantation: a case report.

Face transplants have been clinically established, and early acute rejections have been reported. Late acute rejections have been less common. Immediate and accurate diagnosis along with successful treatment is critical to prevent graft damage. This case report describes the successful treatment of a severe, steroid-resistant rejection 2 years after a full face transplant.

Cardiopulmonary exercise testing before and after blood transfusion: a prospective clinical study.

BACKGROUND: Cardiopulmonary exercise testing (CPET) is used to risk-stratify patients undergoing major elective surgery, with a poor exercise capacity being associated with an increased risk of complications and death. Patients with anaemia have a decreased exercise capacity and an increased risk of morbidity and mortality after major surgery. Blood transfusion is often used to correct anaemia in the perioperative period but the effect of this intervention on exercise capacity is not well described. We sought to measure the effect of blood transfusion on exercise capacity measured objectively with CPET. METHODS: Patients with stable haematological conditions requiring blood transfusion underwent CPET before and 2-6 days after transfusion. RESULTS: Twenty patients were enrolled and completed both pre- and post-transfusion tests. The mean (sd) haemoglobin (Hb) concentration increased from 8.3 (1.2) to 11.2 (1.4) g dl(-1) after transfusion of a median (range) of 3 (1-4) units of packed red cells. The anaerobic threshold increased from a mean (sd) of 10.4 (2.4) to 11.6 (2.5) ml kg(-1) min(-1) (P=0.018), a mean difference of 1.2 ml kg(-1) min(-1) (95% confidence interval (CI)=0.2-2.2). When corrected for the change in Hb concentration, the anaerobic threshold increased by a mean (sd) of 0.39 (0.74) ml kg(-1) min(-1) per g dl(-1) Hb. CONCLUSIONS: Transfusion of allogeneic packed red cells in anaemic adults led to a significant increase in their capacity to exercise. This increase was seen in the anaerobic threshold, and other CPET variables.

Evidence of the three main clonal Toxoplasma gondii lineages from wild mammalian carnivores in the UK.

Toxoplasma gondii is a zoonotic pathogen defined by three main clonal lineages (types I, II, III), of which type II is most common in Europe. Very few data exist on the prevalence and genotypes of T. gondii in the UK. Wildlife can act as sentinel species for T. gondii genotypes present in the environment, which may subsequently be transmitted to livestock and humans. DNA was extracted from tissue samples of wild British carnivores, including 99 ferrets, 83 red foxes, 70 polecats, 65 mink, 64 badgers and 9 stoats. Parasite DNA was detected using a nested ITS1 PCR specific for T. gondii, PCR positive samples were subsequently genotyped using five PCR-RFLP markers. Toxoplasma gondii DNA was detected within all these mammal species and prevalence varied from 6·0 to 44·4% depending on the host. PCR-RFLP genotyping identified type II as the predominant lineage, but type III and type I alleles were also identified. No atypical or mixed genotypes were identified within these animals. This study demonstrates the presence of alleles for all three clonal lineages with potential for transmission to cats and livestock. This is the first DNA-based study of T. gondii prevalence and genotypes across a broad range of wild British carnivores.

Detection of Neospora caninum in wild carnivorans in Great Britain.

Samples of brain and other tissues were collected from 99 ferrets (Mustela furo), 83 red foxes (Vulpes vulpes), 70 European polecats (Mustela putorius), 65 American mink (Neovison vison), 64 Eurasian badgers (Meles meles) and 9 stoats (Mustela erminea), from around Great Britain. DNA was extracted from approximately 1g of tissue and tested by specific nested ITS1 PCR for Neospora caninum. The results from the PCR demonstrated that Neospora specific DNA was detected in all species of wild carnivorans with the exception of the stoats (0/9). Neospora DNA positive samples were detected in: polecats 18.6% (13/70), badgers 10.9% (7/64), ferrets 10.1% (10/99), foxes 4.8% (4/83) and mink 4.6% (3/65). In the badgers N. caninum DNA positive samples were found in brain (n=2), liver (n=2) and neck muscle (n=3). Selected positive ITS1 DNA sequences were submitted to Genbank. Sequence UKwildlife1 (accession number JX857862) was found in two badgers, whilst UKwildlife2 and UKwildlife3 (accession numbers JX857863 and JX857864 respectively) were found in ferrets, all three sequences demonstrated point mutations at a single base, while sequence UKwildlife4 (accession number JX857865) was found in all the species that tested positive and showed complete identity when compared against published reference sequences for: N. caninum (Nc Liverpool isolate, EU564166). Our data shows that almost all the wild carnivoran mammal species tested are intermediate hosts for N. caninum and are therefore capable of acting as reservoirs of infection for other species. These species could also act as useful sentinel species, demonstrating the presence of the parasite in particular geographical and environmental locations.

Estimating infectivity rates and attack windows for two viruses.

Cells exist in an environment in which they are simultaneously exposed to a number of viral challenges. In some cases, infection by one virus may preclude infection by other viruses. Under the assumption of independent times until infection by two viruses, a procedure is presented to estimate the infectivity rates along with the time window during which a cell might be susceptible to infection by multiple viruses. A test for equal infectivity rates is proposed and interval estimates of parameters are derived. Additional hypothesis tests of potential interest are also presented. The operating characteristics of these tests and the estimation procedure are explored in simulation studies.

The development of immune responses in Balb/c mice following inoculation with attenuated or virulent Neospora caninum tachyzoites.

Balb/c mice were inoculated intraperitoneally (i.p.) with either 5 x 10(6) live virulent (group 1) or 5 x 10(6) live attenuated (group 2) tachyzoites, or Vero cells (group 3). Animals were killed at 0, 14, 28 and 42 days post-inoculation (p.i.), with the remaining mice receiving a lethal challenge on day 48 p.i. Serum, spleen and brain samples were collected post-mortem to examine humoral and cell-mediated immune responses as well as pathological lesions and to quantify parasite loads. On day 14 p.i. group 2 (attenuated) demonstrated statistically significant (P < 0.001) lower levels of mean morbidity and weight loss, while also showing significantly (P = 0.01) higher levels of splenocyte proliferation and IFN-gamma production (P = 0.003), compared to group 1 (virulent). Histology of brain samples showed milder lesions and a lower incidence of positive immunohistochemistry, demonstrating tachyzoites and tissue cysts, and statistically significant (P = 0.03) lower mean burdens of parasite DNA in group 2 (attenuated) compared to group 1 (virulent). All mice in group 2 were protected following challenge on day 48 p.i. whereas naïve control mice succumbed to the challenge. No mice from group 1 (virulent) survived beyond day 24 p.i. so they were not included in the challenge.

Correlation of iron and colon adenomas.

BACKGROUND AND OBJECTIVE: Better colon cancer screening guidelines are needed. This study was conducted to explore the relationship between serum transferrin saturation (as iron is a potential carcinogen) and presence of colon adenomas. This may aid to evolve better colon cancer screening guidelines. METHODS: This study is a retrospective review of computer records. Patients who had colonoscopy and iron studies done between May 1996 and December 2003 were included in the study. The adjusted odds ratio, derived from multiple logistic regression analysis, was used to measure the association between transferrin saturation and colon adenomas. RESULTS: Complete data were available for 124 subjects. The adjusted odds ratio, for predicting the presence of polyp in those patients with transferrin saturation above the median was 10.9 (CI 4.0-29.5, P<0.001). A one percent increase in transferrin saturation was associated with a 1.07 increase the odds of adenoma (CI 1.03-1.11, P<0.001). CONCLUSIONS: Iron levels are directly linked to presence of colon polyps, and might help in evolving better screening guidelines.

High-dose intravenous immunoglobulin and rituximab treatment for antibody-mediated rejection after kidney transplantation: a cost analysis.

Antibody-mediated rejection (AMR) generally occurs in highly sensitized patients. A pilot study was performed on 7 consecutive patients with AMR to assess the efficacy of high-dose intravenous immunoglobulin (IVIG; 2 g/kg) + rituximab (RTX; 375 mg/m(2)) without plasmapheresis. After a 24-month follow-up, 1- and 2-year allograft survivals were 86% and 58%, respectively. C4d became negative in 1 patient posttreatment. Donor-specific antibody (DSA) titers decreased to less than 1:4 in 2 cases. There were 4 infectious complications and 1 case of aseptic meningitis followed by cranial nerve VI palsy. The average hospital charge for 1 administration of IVIG + RTX, including hospital stay and renal biopsy expenses, was approximately $49,000. A combination of IVIG + RTX in late AMR may be beneficial but is an expensive treatment approach for selected renal transplant patients.

Patient flow from critical care to renal services: a year-long survey in a critical care network.

BACKGROUND: The NSF for Renal Services stresses the importance of collaboration between renal services and critical care networks in managing patients with acute renal failure in the most clinically appropriate setting. Anecdotal evidence in our region suggested that some patients were remaining on critical care inappropriately because of a lack of capacity for step-down care in local renal units. AIM: To determine the number of extra days patients spend on critical care receiving single-organ renal support before transfer to a renal unit. DESIGN: Prospective, multi-centre, service evaluation. METHODS: Prospective data were collected over a one-year period by either daily telephone calls or bedside review. Follow-up data were retrieved from electronic and patient records. RESULTS: Five hundred and forty-two patients received renal replacement therapy (RRT) in critical care. With 68 (12.5%) patients already receiving RRT for end-stage renal failure, this gave an incidence of new RRT on critical care of 234 per million population per year. The median duration of RRT on critical care was 4 days (range 1-30). One hundred and twenty-seven patients (23%) were discharged from critical care still requiring RRT. A period of single-organ renal support (median 2 days, range 1-8) was provided to 74 of these patients (58%) using 113 critical care bed days. DISCUSSION: Over half of patients receiving RRT on discharge from critical care in our network received a short period of single-organ renal support before step-down. This may represent either delayed discharge from critical care or a potential opportunity for care in an alternative high-dependency facility.

Generation of MUC1-stimulated mononuclear cells using optimized conditions.

Mucin is a glycoprotein found on the surface of cell membranes of adenocarcinomas. The purpose of these studies was to generate MUC1 multiple tandem repeat (VNTR)-stimulated mononuclear cells (M1SMC). We first determined the optimal conditions to influence the immune response. In these studies, peripheral blood mononuclear cells (PBMC), from patients with adenocarcinomas, were stimulated by different numbers of M1SMC stimulations, various concentrations of MUC1 peptide, washing of PBMC prior to stimulation and days in culture, to determine the optimal conditions to influence the immune response. The results of this study indicate that the mononuclear cells (MC) stimulated twice 1 week apart with MUC1 VNTR1 produced a greater specific killing of the breast cancer cell line MCF-7 than the 0, 1, 3 or 4 weekly stimulations. The optimal molarity for inducing cytotoxicity and cytokines (granulocyte macrophage colony-stimulating factor, gamma-interferon and interleukin-10) was 45 x 10(-8) M (1 microg/ml); except for tumour necrosis factor (TNF)-alpha which was 22 x 10(-8) M (0.5 microg/ml). The unwashed MC were superior to washing them with Ficoll-Hypaque. The optimal number of days in culture for cytotoxicity and cytokine production was after two stimulations (i.e. after day 7). Optimum conditions for generation of M1SMC identified in these studies were two stimulations with peptide, concentration of 45 x 10(-8) M (1 microg/ml) peptide, unwashed cells, and after two stimulations or after 8 days in culture. M1SMC were generated from multiple patients with breast cancer which lysed adenocarcinoma cells.

Aseptic meningitis and abducens nerve palsy as a serious side effect of high dose intravenous immunoglobulin used in a patient with renal transplantation.

Immune globulin intravenous (human) (IGIV) is effective in the treatment of various autoimmune and inflammatory disorders. Recently, high-dose IGIV 2 g/kg has been utilized in the treatment of antibody-mediated rejection in solid organ transplantation. We report a renal transplant recipient who developed aseptic meningitis and diplopia from abducens nerve (cranial nerve VI) palsy following IGIV administration for antibody-mediated rejection. Potential mechanisms of the IGIV-related aseptic meningitis are elaborated. Clinicians should be aware of aseptic meningitis and cranial nerve palsy as an adverse reaction to IGIV exposure and monitor for its signs and symptoms.

Context of MUC1 epitope: immunogenicity.

MUC1 is a glycoprotein found at the secretory poles of normal cells but is hypoglycosylated on the entire surface of cell membranes of adenocarcinomas. In order to determine the influence on the immune response of peptide context for epitope presentation, peripheral blood mononuclear cells (PBMC) from patients with adenocarcinomas, were stimulated with MUC1 peptides derived from the 20 amino acids (aa) long sequence that is characteristic of the MUC1 Variable Number of Tandem Repeats (VNTR). In the seven peptides tested, the T-cell tumor-specific epitope (cTSE) was surrounded by variable numbers of aa and repeated up to 5 times in the same peptide. The results of this study indicate that cultures stimulated with peptide 610 (GSTAPPAHGVTS APDTRPAP) showed the highest specific killing of the MUC1-expressing breast cancer MCF-7 cells. Peptide 610 is also superior to the other peptides in inducing better production of the type 1 cytokines, tissue necrosis factor alpha and interferon gamma. In conclusion, context of the epitope and not sequence alone determines immunogenicity.

Ovine toxoplasmosis: transmission, clinical outcome and control.

Toxoplasma gondii is a significant cause of abortion in sheep. Infection is picked up from the environment and if initiated during pregnancy may cause fetal mortality. Infected sheep remain persistently infected with tissue cysts in brain and muscle (meat), and are also immune and would not be expected to abort again. The live tachyzoite vaccine (Toxovax) protects against abortion and this allows the suggestion that it may also reduce or prevent tissue cyst development in muscle. If this were so it raises the question of whether the vaccine could be used to make meat safer for human consumption.

Role of endogenous transplacental transmission in toxoplasmosis in sheep.

To investigate the potential role of endogenous transplacental transmission of Toxoplasma gondii, 31 seropositive ewes presumed to be persistently infected with the parasite and 15 seronegative ewes were mated and monitored throughout pregnancy and lambing. Antibody titres were determined in precolostral sera from the liveborn lambs and in thoracic fluid from the dead lambs. A PCR for the B1 gene of T gondii was applied to the placentas from all the ewes and to the brains of the stillborn lambs. Samples of brain, lung, liver, spleen and heart from the dead lambs were examined by histopathology. No evidence of toxoplasmosis was detected by histopathology or PCR in any of the samples, but low titres of antibody to T gondii were detected in two liveborn, healthy offspring of a seropositive ewe by the immunofluorescent antibody test (3.2 per cent of pregnancies and 4.1 per cent of lambs in the seropositive group). Antibody to specific antigens of T gondii was demonstrated in sera from these two lambs by Western blotting.

Characterization of the immune response in the placenta of cattle experimentally infected with Neospora caninum in early gestation.

A serial examination of three groups of cattle infected intravenously (iv) (Group 1, n=8) or subcutaneously (sc) (Group 2, n=8) with live Neospora caninum tachyzoites or with VERO cells (Group 3, n=8) at 70 days' gestation was carried out and the nature of the inflammatory responses in the placenta and the presence of parasite antigen were analysed. Immune cells expressing CD3, CD4, CD8, gamma delta (gammadelta) T-cell receptors (TCR), CD79alpha cytoplasmic (cy) (B cells) and NKp46 [natural killer (NK) cells] antigens were identified immunohistochemically and cells expressing mRNA for interferon-gamma (IFN-gamma) were labelled by in-situ hybridization. Intravenous inoculation caused mortality in all fetuses from 28 days post-inoculation (dpi) onwards. Subcutaneous inoculation caused mortality in 50% of the animals by 28dpi. Pathological changes in the placenta consisted of necrosis of fetal placental villi, necrosis and inflammation in adjacent areas of the maternal septum and inflammation at the base of the maternal caruncle. The inflammatory infiltrate consisted mainly of CD3(+) lymphocytes, dominated by CD4(+) and gammadelta TCR(+) cells, with CD8(+) cells present to a lesser extent. The results from the control group indicated fewer NK cells than those occurring in the placenta of human beings or mice. Infiltration of CD4(+) cells and NKp46(+) cells was observed in the caruncular base and septa 14 days after infection, whereas infiltration of gammadelta TCR(+) cells was observed from 28 dpi onwards. To our knowledge this is the first report on the presence and distribution of NK cells in the bovine placenta. Maternal inflammatory cells expressing mRNA for IFN-gamma were identified in animals inoculated with parasites iv or sc at 14 and 28 dpi, respectively. In the sc-inoculated dams with live fetuses at 28, 42 and 56dpi, there was no evidence of parasite antigen, infiltration of immune cells or production of IFN-gamma, suggesting that the parasite had not reached the placenta. The exact cause of fetal death was not established. Tissue destruction by the parasite may have occurred; in addition, there may have been a T helper 1 (Th-1) immune response to the neospora infection at the materno-fetal interface, resulting in infiltrations of CD4T cells, gammadelta T cells and NK cells and the subsequent production of IFN-gamma. It is possible that a pro-inflammatory Th-1 response early in gestation protects the dam by eliminating the parasite; however, it may lead to destruction of the placental tissues themselves and thus be incompatible with fetal survival.

Computer three-dimensional reconstruction of the sinoatrial node.

BACKGROUND: There is an effort to build an anatomically and biophysically detailed virtual heart, and, although there are models for the atria and ventricles, there is no model for the sinoatrial node (SAN). For the SAN to show pacemaking and drive atrial muscle, theoretically, there should be a gradient in electrical coupling from the center to the periphery of the SAN and an interdigitation of SAN and atrial cells at the periphery. Any model should include such features. METHODS AND RESULTS: Staining of rabbit SAN preparations for histology, middle neurofilament, atrial natriuretic peptide, and connexin (Cx) 43 revealed multiple cell types within and around the SAN (SAN and atrial cells, fibroblasts, and adipocytes). In contrast to atrial cells, all SAN cells expressed middle neurofilament (but not atrial natriuretic peptide) mRNA and protein. However, 2 distinct SAN cell types were observed: cells in the center (leading pacemaker site) were small, were organized in a mesh, and did not express Cx43. In contrast, cells in the periphery (exit pathway from the SAN) were large, were arranged predominantly in parallel, often expressed Cx43, and were mixed with atrial cells. An approximately 2.5-million-element array model of the SAN and surrounding atrium, incorporating all cell types, was constructed. CONCLUSIONS: For the first time, a 3D anatomically detailed mathematical model of the SAN has been constructed, and this shows the presence of a specialized interface between the SAN and atrial muscle.

The provision and practice of renal replacement therapy on adult intensive care units in the United Kingdom.

This study surveyed the availability and current practice of renal replacement therapy on adult general intensive care units in the United Kingdom. Questionnaires were returned from 236 units (89%). Renal replacement therapy was provided by 212 (90%) of responding units, treating 9442 patients per year. Renal physicians were involved in the initiation and prescription of treatment in 22 (11%) units. Ninety-one units (43%) had no step down facility on-site for those patients still requiring renal replacement therapy but no longer requiring intensive care. Continuous techniques are used by the majority of units, most commonly, continuous veno-venous haemofiltration, with an ultrafiltration rate of 2000 ml.h-1. Fifty-eight units (28%) use haemofiltration as adjuvant treatment in septic shock. The provision and practice of renal replacement therapy is now an integral part of intensive care medicine in the United Kingdom.

The pathogenesis of neosporosis in pregnant cattle: inoculation at mid-gestation.

To investigate the pathogenesis of bovine neosporosis, 14 pregnant cattle were each inoculated subcutaneously with either 10(7) or 5 x 10(8) Neospora caninum (strain NC1) tachyzoites at 140 days' gestation. Serial necropsies were then carried out over an 8-week period. In the placenta, Neospora DNA and histopathological changes were observed in samples taken 14 days post-inoculation (dpi), with focal necrosis of maternal caruncular septa and fetal placental villi, serum leakage, and a maternal and fetal inflammatory response. At subsequent samplings, pathological changes in the placenta showed signs of resolution. No parasitaemia was detected in the dams in the two weeks following inoculation. In the fetus, Neospora DNA was detected at 14 dpi, and histopathological changes in the fetal central nervous system at 28 and 42 dpi consisted of small foci of necrosis and inflammation. Resolution of placental lesions during the experiment indicated that the disease was being controlled, and fetal infection, although established, did not appear to be progressing to a fatal outcome. The two doses of tachyzoites produced similar results, but the higher dose elicited earlier and more extensive lesions in the placenta and fetus. Control animals remained negative for all parameters recorded. It is concluded that in bovine neosporosis the placenta plays a central role in the pathogenesis and epidemiology of the infection, and that while primary tissue destruction by the parasite may endanger the fetus, the maternal and fetal inflammatory responses may also be damaging.