Direct detection of Mycobacterium bovis by a dry loop-mediated isothermal amplification assay in cattle samples collected during routine abattoir examination in Malawi.

The lack of quick, accurate, and low-cost detection methods has hindered the active control strategies for bovine tuberculosis (bTB) in resource-limited countries with a high burden of disease. We developed a dry loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of Mycobacterium bovis, the principal causative agent of bTB, and evaluated the efficacy of the assay using suspected bTB samples collected during routine meat inspection at major regional abattoirs in Malawi. Template genomic DNA was extracted directly from the granulomatous bTB-like lesion (crude extracted DNA), as well as growth from the incubated mycobacterial growth indicator tubes (MGIT). Field results were visualized by the naked eye within 40 min following a color change of the amplified products. The sensitivity and specificity of the dry LAMP assay while using 152 DNA samples extracted from MGIT with confirmed M. bovis results were 98% and 88%, respectively. When 43 randomly selected crude DNA samples from lesions were used, the sensitivity and specificity of the dry LAMP assay were 100% and 75%, respectively. Our LAMP assay offers the potential to meet the demands for a low-cost and rapid field detection tool for bTB in resource-limited countries in which bTB is endemic.

Genetic Diversity of African Trypanosomes in Tsetse Flies and Cattle From the Kafue Ecosystem.

We clarified the genetic diversity of Trypanosoma spp. within the Kafue ecosystem, using PCR targeting the internal transcribed spacer 1 and the cathepsin L-like cysteine protease (CatL) sequences. The overall prevalence of Trypanosoma spp. in cattle and tsetse flies was 12.65 and 26.85%, respectively. Cattle positive for Trypanosoma vivax had a significantly lower packed cell volume, suggesting that T. vivax is the dominant Trypanosoma spp. causing anemia in this area. Among the 12 operational taxonomic units (OTUs) of T. vivax CatL sequences detected, one was from a known T. vivax lineage, two OTUs were from known T. vivax-like lineages, and nine OTUs were considered novel T. vivax-like lineages. These findings support previous reports that indicated the extensive diversity of T. vivax-like lineages. The findings also indicate that combining CatL PCR with next generation sequencing is useful in assessing Trypanosoma spp. diversity, especially for T. vivax and T. vivax-like lineages. In addition, the 5.42% prevalence of Trypanosoma brucei rhodesiense found in cattle raises concern in the community and requires careful monitoring of human African trypanosomiasis.

Transfection of live, tick derived sporozoites of the protozoan Apicomplexan parasite Theileria parva.

Theileria parva is an important veterinary protozoan causing the tick-borne disease East Coast fever. Transfection of Theileria parasites will facilitate the investigation of many aspects of this apicomplexan infection and its unique host-parasite interaction. The pathogen has the extraordinary capacity of transforming B and T cells into clonally dividing cancerous cell lines in a reversible way. Sequence data of the entire T. parva genome are available and in vitro infected cell lines can easily be generated, thereby eliminating the use of animals in the evaluation of the evolution of the transfected sporozoites. Here we report, for the first time, on experiments towards successful transient transfection of T. parva sporozoites, making use of a new generation transfection device. Plasmid DNA containing the strong EF-1α promoter and an Azami Green reporter gene were integrated by nucleofection into freshly purified T. parva sporozoites. Expression of Azami Green was detected with a fluorescence microscope and confirmed by counter staining with a monoclonal directed against a sporozoite protein. Despite the fact that transfection efficiencies are still low, this is the first step towards a stable infection method of T. parva parasites. In the long run, transfected parasites might become an alternative way to induce immunity without clinical signs.

Right atrial obstruction due to cardiac extension of hepatocellular carcinoma.

Hepatocellular carcinoma is fatal and has a marked propensity for vascular invasion. However, tumor thrombi rarely occur in the right atrium. A 72-year-old man was admitted with dyspnea and disturbed consciousness. Abdominal computed tomography and echocardiography showed a huge tumor thrombus in the inferior vena cava and the right atrium. The tumor thrombus was surgically removed to treat the hemodynamic compromise. The patient underwent transcatheter arterial chemoembolization postoperatively and survived for 6 months.

A novel chest packing technique for intractable bleeding after open heart surgical procedures.

Intractable bleeding after cardiac surgical procedures is a life-threatening complication. In most cases, the main bleeding site is present in the retrosternal space, not the pericardial space. Packing the chest may be a useful technique for achieving hemostasis. Herein, we describe a novel and effective procedure for the treatment of intractable bleeding in the retrosternal space using a sheet of oxidized regenerated cellulose and sponges after cardiac surgical procedures.

MDM2 regulates a novel form of incomplete neoplastic transformation of Theileria parva infected lymphocytes.

Our efforts are concerned with identifying features of incomplete malignant transformation caused by non viral pathogens. Theileria parva (T. parva) is a tick-transmitted protozoan parasite that can cause a fatal lymphoproliferative disease in cattle. The T. parva-infected lymphocytes display a transformed phenotype and proliferate in culture media like the other tumor cells, however those cells will return to normal after antiprotozoal treatment reflecting the incomplete nature of transformation. To identify signaling pathways involved in this form of transformation of T. parva-infected cells, we screened a library of anticancer compounds. Among these, TIBC, a specific inhibitor of MDM2, markedly inhibited proliferation of T. parva-infected lymphocytes and promoted apoptosis. Therefore we analyzed MDM2 function in T. parva-infected cells. Several T. parva-infected cell lines showed increased expression level of MDM2 with alternatively spliced isoforms compared to the lymphoma cells or ConA blasts. In addition, buparvaquone affected MDM2 expression in T. parva transformed cells. Moreover, p53 protein accumulation and function were impaired in T. parva-infected cells after cisplatin induced DNA damage despite the increased p53 transcription level. Finally, the treatment of T. parva-infected cells with boronic-chalcone derivatives TIBC restored p53 protein accumulation and induced Bax expression. These results suggest that the overexpression of MDM2 is closely linked to the inhibition of p53-dependent apoptosis of T. parva-infected lymphocytes. Aberrant expression of host lymphocyte MDM2 induced by cytoplasmic existence of T. parva, directly and/or indirectly, is associated with aspects of this type of transformation of T. parva-infected lymphocytes. This form of transformation shares features of oncogene induced malignant phenotype acquisition.

Occlusion of left coronary ostium with a rudimentary aortic cusp.

A 74-year-old woman with exertional angina was diagnosed with occlusion of the left coronary ostium associated with a rudimentary aortic valve cusp. A transesophageal echocardiogram, a multi-detector computed tomographic scan, and a coronary angiographic scan revealed the rudimentary aortic cusp covering the small left coronary aortic sinus leading to occlusion of the ostium of the left coronary artery, despite the intact coronary arteries. After excision of the rudimentary left coronary cusp, the left coronary ostium appeared intact. An aortic valve replacement with annular enlargement using a bioprosthetic valve was performed. The patient uneventfully recovered without angina.

A schizont-derived protein, TpSCOP, is involved in the activation of NF-kappaB in Theileria parva-infected lymphocytes.

Theileria parva is a tick-transmitted intracellular protozoan parasite that causes East Coast fever, a fatal bovine lymphoproliferative disease. The molecular mechanisms that underlie host cell transformation by T. parva schizonts have been studied extensively, and it is known that the nuclear factor-kappa B (NF-kappaB) is activated in schizont-infected cells, making T. parva-transformed cells resistant to apoptosis. However, the mechanism by which the parasite triggers the activation of NF-kappaB remains enigmatic. In the present study, we biochemically characterized a novel protein, which we termed TpSCOP (T. parvaschizont-derived cytoskeleton-binding protein), which is expressed in the schizont stage of T. parva. TpSCOP was shown to interact with F-actin in vitro. Expression of TpSCOP in a murine lymphocytic cell line resulted in the activation of NF-kappaB signaling pathways, leading to apoptosis resistance. The activation of mitogen-activated protein kinase (MAPK), including extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), was also detected. Furthermore, the introduction of TpSCOP into T. parva-infected cells also enhanced the activation of NF-kappaB. This is the first report to demonstrate that a parasite-derived molecule has the ability to activate the host NF-kappaB pathway. Based on these results, TpSCOP likely plays an important role in apoptosis inhibition during Theileria infection.

Development of an in vivo tissue-engineered, autologous heart valve (the biovalve): preparation of a prototype model.

OBJECTIVE: This study aimed to develop an autologous heart valve without using traditional in vitro tissue-engineering methods, which necessitate complicated cell management protocols under exceptionally clean laboratory facilities. METHODS: An autologous heart valve construct composed of trileaflets was prepared using a specially designed mold. The mold was prepared by covering a silicone rod with a crown-shaped tubular polyurethane scaffold containing 3 horns. The mold was implanted in the dorsal subcutaneous space in Japan White rabbits for 4 weeks. After harvesting, the implanted trileaflet valve-shaped structure with an internal diameter of either 5 or 20 mm was obtained by trimming the membranous tissue formed between the horns located around the silicone rod. The valve substitute was examined both macroscopically and histologically. The tensile strength of the leaflets was measured to rupture. The degree of regurgitation in valve function was evaluated using a flow circuit by calculating the ratio of the regurgitation volume to the forward flow volume. RESULTS: After implantation, the mold was completely covered with connective tissue consisting mostly of collagen and fibroblasts. Harvesting of the mold was straightforward, because there was little adhesion between the formed tissue and the native skin tissue. The trileaflet heart valve construct was obtained after withdrawing the inserted rods and trimming the membranous tissues formed between the horns of the scaffold. It was firmly attached to the scaffold, the interstices and surface of which revealed connective tissues composed of components similar to those of the leaflet tissue. Although the mechanical properties of the leaflet tissue were less efficient than those of the native porcine aortic valve leaflets, satisfactory valvular functions were demonstrated under pulsatile conditions using a flow circuit. No regurgitation was observed under retrograde hydrostatic pressures of up to 60 mm Hg, the physiologic pressure acting on the aortic valves during retrograde aortic flow. CONCLUSIONS: The biovalve, an autologous, in vivo tissue-engineered, trileaflet, valve-shaped construct, was developed using our novel in-body tissue architecture technology. The biovalve has the potential to be an ideal prosthetic heart valve, with excellent biocompatibility to the growth of the recipient's heart.

Does off-pump coronary artery bypass grafting beneficially affect renal function?

BACKGROUND: Off-pump coronary artery bypass grafting (CABG) has been reported to beneficially affect renal function, but this remains to be confirmed. The purpose of the present paper was to study the effects of off-pump CABG on renal function and analyse predictors of postoperative renal impairment in patients who received off-pump CABG. METHODS: A total of 451 patients who underwent isolated CABG between January 1999 and August 2003 were retrospectively studied. No patient was receiving dialysis. A total of 300 patients (228 men) underwent off-pump CABG (off-pump group) and 151 patients (104 men) underwent on-pump CABG (on-pump group). Perioperative serum creatinine levels and creatinine ratios (peak postoperative creatinine level/preoperative creatinine level) were compared between the groups. RESULTS: Renal impairment (serum creatinine >1.5 mg/dL) developed postoperatively in 12.7% of the off-pump group and 18.5% of the on-pump group (P = 0.1). The creatinine ratio was significantly lower in the off-pump group (1.2 +/- 0.4) than in the on-pump group (1.4 +/- 0.7, P = 0.003). Logistic regression analysis demonstrated that the strongest predictors of postoperative renal impairment in off-pump CABG were left ventricular dysfunction (odds ratio 10.8) and multivessel grafting (odds ratio 4.3). CONCLUSIONS: Off-pump CABG provides better renal protection than on-pump CABG. However, perioperative renal function should be closely monitored in patients who have left ventricular dysfunction or who undergo multivessel grafting, even when off-pump CABG is performed.

One-stage unifocalization and palliative right ventricular outflow tract reconstruction.

We report a successful case of one-stage unifocalization concomitant with palliative right ventricular outflow tract reconstruction for pulmonary atresia and ventricular septal defect with major aortopulmonary collateral arteries and central pulmonary arterial absence. Through a median sternotomy, one-stage unifocalization was accomplished using autologous pericardial conduit and no prosthetic material. To achieve an adequate pulmonary blood flow, the right ventricular outflow tract was reconstructed so that it would be 70% of the normal pulmonary annular diameter, while the ventricular septal defect was left open. One-stage unifocalization concomitant with palliative right ventricular outflow tract reconstruction is considered to be a good surgical option for selected patients with these diseases.

Partial translocation for repair of left ventricular rupture after mitral valve replacement.

A new partial translocation technique to repair left ventricular rupture after mitral valve replacement and reimplant a mitral prosthesis is described. We repaired a tear from the interior by using mattress stitches buttressed with strips of Dacron felt, constructed a new annulus with a crescent-shaped piece of bovine pericardium on the left atrial wall above the repaired mitral annulus, and implanted a new prosthesis by using the newly constructed mitral annulus. We consider avoidance of mechanical stress by the prosthesis on the repaired site crucial to a successful outcome, and this new method is useful.

Impact of myocardial angiotensin-converting enzyme activity on coronary vascular resistance and serum brain natriuretic peptide concentration in coronary bypass surgery.

Angiotensin-converting enzyme (ACE) inhibitors have cardioprotective effects in animals, but whether that occurs in humans is still controversial. The effect of myocardial ACE activity on coronary vascular resistance during coronary artery bypass surgery and on serum brain natriuretic peptide (BNP) concentration after surgery was studied in myocardial tissue sampled from the right atrium of patients during cardiac surgery (n=20). Tissue enzyme activity (nmol/min per mg protein) was measured using a photometric technique, and the flow rate and pressure upon antegrade infusion of a crystalloid cardioplegic solution was measured for calculating the coronary vascular resistance (mmHg. ml(-1). min(-1)). Serum BNP concentration (pg/ml) was measured on days 0 and 5 after the surgery. Linear regression between tissue ACE activity and coronary vascular resistance (y = 0.46x + 0.56, r=0.85) as well as serum BNP concentration on days 0 (y = 129x + 30, r=0.59) and 5 (y = 347x + 180, r=0.73) after the surgery was significant (x: ACE activity; y: coronary vascular resistance/serum BNP concentration). The results indicate that inhibition of myocardial ACE activity might improve coronary circulation during surgery and hence, cardiac function after surgery.

An operative case of inferior vena cava stenosis due to fibrosis around permanent pacemaker leads.

A 56-year-old woman presented with general fatigue, dyspnea on exercise, and hepatomegaly subsequent to secondary implantation of a pacemaker. On admission, cardiac echo examination showed tricuspid valve regurgitation due to a migrated looped lead. At surgery, there was evidence of severe stenosis at both orifices of the superior and inferior vena cavae due to fibrous tissue around the leads. We removed the fibrous tissue, pacing leads, and generator. New leads were anchored onto the epicardium and a generator was inserted under the rectus muscle. The postoperative course was satisfactory and the symptoms disappeared.

Extracardiac total cavopulmonary connection using a Y-shaped graft.

We report on 17-year-old Fontan candidate with a severely distorted central pulmonary artery (PA) who underwent a successful extracardiac total cavopulmonary connection using a Y-shaped bifurcated graft. A nonanatomic pathway from the inferior vena cava to the left PA was constructed and positioned anterior to the ascending aorta. The other arm was used as a conduit between the inferior vena cava and the right PA. All procedures were performed under temporary venous bypass without cardiopulmonary bypass.