Renal Infiltration of Macrophages in Canine and Feline Chronic Kidney Disease.

During the progression of chronic kidney disease (CKD), macrophage infiltration is a crucial event leading to tubulointerstitial fibrosis. In the present study, macrophages infiltrating renal tissue in dogs and cats with CKD were analysed immunohistochemically. Iba-1 was used as a pan-macrophage marker, CD204 was used as a marker of M2 macrophages and tumour necrosis factor (TNF)-α was used as a marker of M1 macrophages. Signals for Iba1 and CD204 were observed in the interstitium of all tested kidney samples. In dogs, the signals were diffusely scattered. In cats, both diffuse and focal signals were observed. Cells that were positive for Iba1 and CD204 were also observed in the tubular lumina in cats. Co-expression of Iba1 and CD204 was also observed in the infiltrating cells by immunofluorescence labelling, and these cells were negative for TNF-α. By quantitative analysis, the indices for Iba1- and CD204-positive cells were significantly correlated with the concentrations of plasma creatinine and/or urea and the extent of interstitial fibrosis in both dogs and cats. These results demonstrated that renal infiltration of M2 macrophages plays an important role in the progression of CKD in dogs and cats. The distribution pattern of the kidney-infiltrating macrophages was unique in cats and may be associated with a cat-specific renal fibrotic process.

T-cell-replete haploidentical stem cell transplantation is highly efficacious for relapsed and refractory childhood acute leukaemia.

BACKGROUND: Despite improvements in first-line therapies, the outcomes of relapsed or refractory childhood acute leukaemia that has not achieved complete remission after relapse, has relapsed after stem cell transplantation (SCT), has primary induction failure and has relapsed with a very unfavourable cytogenetic risk profile, are dismal. OBJECTIVES AND METHODS: We evaluated the feasibility and efficacy of T-cell-replete haploidentical peripheral blood stem cell transplantation (haplo-SCT) with low-dose anti-human thymocyte immunoglobulin (ATG), tacrolimus, methotrexate and prednisolone (PSL) in 14 paediatric patients with high-risk childhood acute leukaemia. RESULTS: All patients achieved complete engraftment. The median time to reaching an absolute neutrophil count of more than 0.5 × 10(9) L(-1) was 14 days. Acute graft-vs-host disease (aGVHD) of grades II-IV and III-IV developed in 10 (71%) and 2 (14%) patients, respectively. Treatment-related mortality and relapse occurred in one (7%) patient and six (43%) patients, respectively. Eleven patients were alive and seven of them were disease-free with a median follow-up of 36 months (range: 30-159 months). The probability of event-free survival after 2 years was 50%. CONCLUSION: These findings indicate that T-cell-replete haplo-SCT, with low-dose ATG and PSL, provides sustained remission with an acceptable risk of GVHD in paediatric patients with advanced haematologic malignancies.

Anticancer effects of gemcitabine are enhanced by co-administered iRGD peptide in murine pancreatic cancer models that overexpressed neuropilin-1.

BACKGROUND: Impaired drug transport is an important factor that reduces the efficacy of anticancer agents against pancreatic cancer. Here, we report a novel combination chemotherapy using gemcitabine (GEM) and internalised-RGD (iRGD) peptide, which enhances tumour-specific drug penetration by binding neuropilin-1 (NRP1) receptor. METHODS: A total of five pancreatic cancer murine models (two cell line-based xenografts (CXs) and three tumour grafts (TGs)) were treated with either GEM (100 mg kg(-1), q3d × 4) alone or GEM plus iRGD peptide (8 µmol kg(-1)). Evaluation of NRP1 expression in xenografts and 48 clinical cancer specimens was performed by immunohistochemistry (IHC). RESULTS: We identified a subset of pancreatic cancer models that showed NRP1 overexpression sensitive to iRGD co-administration. Treatment with GEM plus iRGD peptide resulted in a significant tumour reduction compared with GEM monotherapy in CXs, but not remarkable in TGs. Potential targets of iRGD were characterised as cases showing NRP1 overexpression (IHC-2+/3+), and these accounted for 45.8% of the clinical specimens. CONCLUSIONS: Internalised RGD peptide enhances the effects of co-administered drugs in pancreatic cancer models, its efficacy is however only appreciable in those employing cell lines. Therefore, the clinical application needs to be given careful consideration.

Protective effect of sivelestat sodium (Eraspol) on postoperative lung dysfunction in patients with type A acute aortic dissection: a pilot study.

AIM: The authors evaluated the protective effect of sivelestat sodium on postoperative lung dysfunction in patients with type A acute aortic dissection who underwent aortic arch surgery with cardiopulmonary bypass (CPB) under deep hypothermia with circulatory arrest (DHCA). METHODS: Twelve patients with type A acute aortic dissection who underwent aortic arch replacement under CPB with DHCA and were pretreated with or without sivelestat sodium (sivelestat group, N.=7 patients; control group, N.=5 patients) were observed. The ratio of arterial oxygen tension to inspired oxygen fraction (P/F ratio) was measured as a parameter of pulmonary function before and after operation. The number of white blood cells was also counted as an index of inflammatory reaction before and after the operation. RESULTS: The P/F ratio decreased significantly after operation in the control group. However, the P/F ratio was unchanged between before and after operation in the sivelestat group. The number of white blood cells tended to increase after operation in the control group, whereas it decreased significantly after operation in the sivelestat group. CONCLUSION: The present study demonstrated the protective effect of sivelestat sodium on postoperative lung injury in patients with acute type A aortic dissection undergoing aortic arch surgery under CPB with DHCA.

Demonstration of inter- and intracellular distribution of boron and gadolinium using micro-proton-induced X-ray emission (Micro-PIXE).

Micro-proton-induced X-ray emission (Micro-PIXE) was applied to determine inter- and intracellular distribution of boron (10B) and gadolinium (157Gd), the capture atoms used to kill tumor cells in neutron capture therapy (NCT). Cultured 9L gliosarcoma cells on Mylar film were exposed to sodium borocaptate (BSH) and gadobenate dimeglumine (Gd-BOPTA). To analyze the inter- and intracellular distribution of 10B and 157Gd in 9L gliosarcoma cells, the cells were irradiated using a proton beam of 1.7 or 3 MeV energy collimated to 1 microm diameter and emission X-ray was detected. The distribution of 10B and 157Gd in 9L gliosarcoma cells was then examined. In this study, we could directly analyze the inter- and intracellular distribution of 10B and 157Gd elements in 9L gliosarcoma cells directly using Micro-PIXE. This is the first report on the distribution of 10B employing a method to detect gamma-rays resulting from the nuclear reaction of 10B using particle-induced gamma-ray emission (PIGE). These results show that the distribution of 157Gd elements was correctly measured using micro-PIXE. 157Gd should have the same tendency as 10B in cultured 9L gliosarcoma cells and agree with the distribution in 9L gliosarcoma cells. Further investigation is necessary for a higher spatial resolution and optimization of the measurement time or improvement of the sampling method. In the future, it will be possible to employ this method to analyze the intracellular microdistribution of the capture element and in the development of new drugs for NCT.

[Surgery for acute aortic dissection concomitant with preceding axillofemoral bypass to prevent malperfusion of visceral organs and limb ischemia].

A 52-year-old man was admitted with sudden onset of epigastralgia. Abdominal X-ray showed dilated intestine and computed tomography (CT) revealed extended type A aortic dissection. Marked abdominal distention and weak pulse of right femoral artery were recognized so malperfusion of visceral organs due to narrowing true lumen compressed by thrombosed false lumen was suggested. In the operation, right axillo-right femoral bypass was established preceding to median sternotomy. This graft was used as an arterial perfusion site of cardiopulmonary bypass, and replacement of the ascending aorta was performed under hypothermic circulatory arrest and retrograde cerebral perfusion. Sign of malperfusion of visceral organs was showed for several days after the operation but it disappeared without further intervention. Axillofemoral inflow of cardiopulmonary bypass may be effective procedure in these cases.

Genetic organization of the Francisella plasmid pFNL10.

We report here the molecular characterization of pFNL10, a 3990-bp cryptic plasmid of Francisella novicida-like F6168. The plasmid was maintained in F. novicida Utah 112 and F. tularensis LVS strains. We sequenced the entire plasmid and found six open reading frames (ORFs)-ORF1, ORF2, ORF3, ORF4, ORF5, and ORFm. ORF3, ORF4, ORF5, and ORFm are located on the same strand, and we designated it the plus strand. ORF1 and ORF2 are on the complementary strand. The ORFs appear to be arranged in two operons, one comprising ORF5 and ORF4 and the other ORF1 and ORF2. There exist two distinct promoters similar to the Escherichia coli sigma(70) promoter, one 5' to ORF1-ORF2 operon and the other 5' to ORF5-ORF4 operon. We found that in both promoters the transcriptional start is an adenosine. ORF3 is positioned in tandem with ORF5-ORF4, but has its own transcriptional start, a thymidine. However, sequence analysis revealed no recognizable promoter in physical proximity to ORF3. Sequence analysis revealed transcriptional terminators immediately downstream of the two operons. Experimental results showed that the ORF1-ORF2 terminator is authentic. But we could not definitively confirm the ORF5-ORF4 terminator. Two sets of direct repeats, one 31 and the other 13 bp, characteristic of ori are positioned between the two promoters. ORF1 encodes a protein that bears homology to the replication initiation protein RepA of various bacteria, and disruption of this ORF indeed blocked pFNL10 replication. In contrast, ORF2 disruption caused formation of plasmid multimers, suggesting aberrant replication. Our analysis also suggests that pFNL10 replicates by the theta mode. The ORF5-ORF4 operon resembles the phd-doc operon of Escherichia coli bacteriophage P1, but the significance of this similarity is unclear.

Association of L* protein of Theiler's murine encephalomyelitis virus with microtubules in infected cells.

We used an antibody raised against a synthetic peptide corresponding to amino acid residues 70-88 for characterizing the L* protein of Theiler's murine encephalomyelitis virus (TMEV), which is only synthesized in DA subgroup strains from an alternative AUG and is out of frame with the viral polyprotein; evidence suggests that L* protein is critical to viral persistence, demyelination, and growth in murine macrophage cell lines. It was synthesized with kinetics similar to that of other viral proteins, although less in amount. After synthesis, it remained stable in the cytoplasm and was not incorporated into virions. Immunofluorescent staining and immunoblotting of microtubule preparations demonstrated that it is associated with microtubules. Expression of L* protein also demonstrated that the 5' one third of the coding region may be responsible for the association. The association of L* protein with microtubules may be important in the disease-inducing and in vitro characters of L* protein.

Clinical results of implantation of an endovascular covered stent-graft via midsternotomy for distal aortic arch aneurysm.

UNLABELLED: We have been implanting endovascular stent grafts (EVG) via midsternotomy for distal aortic arch surgery since February 1997. The early clinical results are evaluated. METHODS: There were 11 true aneurysms (8 fusiform, 3 saccular) and one chronic type B dissection. The average age was 68 yr (63-81). EVGs were PTFE-covered two-8 bend Z stents in the first eight cases and made with the same stents and ultrathin woven Dacron grafts in the last four cases. RESULTS: Total arch replacement and aortocoronary bypass grafting were combined in one and two patients, respectively. The average retrograde cerebral perfusion time was 42+/-8 min. The cardiopulmonary bypass time averaged 211+/-26 min. All patients awoke early after operation (4.5+/-1.2 h). All but one case was extubated within 24h. There was no operative mortality, but paraplegia and cerebral infarction were complicated in one case each. Their maximum diameter (73.9+/-21.2mm) decreased significantly after operation (68.7+/-20.1mm) and one year thereafter (63.1+/-16.0 mm). True aneurysms were thrombosed completely. A chronic type B dissection revealed impending rupture due to false lumen infection one year after operation. The whole descending aorta replacement was performed but the patient died 6 months thereafter due to cerebro-vascular complication. CONCLUSION: Implantation of EVGs reduces operative invasion for distal arch surgery. This procedure should improve mortality, while long-term results have not been clarified.

Repair of leaking postinfarction ventricular septal defect under circulatory arrest via left thoracotomy.

We treated a 60-year-old woman for postinfarction ventricular septal defect (VSD) and closed it by the infarction exclusion method. Postoperatively she was complicated by Candida sternal mediastinitis and residual shunt of VSD. After her sternal infection came under control we repaired the leaking VSD via left thoracotomy under hypothermic circulatory arrest. She recovered well and repair of the leaking VSD under circulatory arrest via left thoracotomy seemed to be a safe and promising alternative for VSD repair.

L* protein of Theiler's murine encephalomyelitis virus is required for virus growth in a murine macrophage-like cell line.

We sought to confirm the importance of L* protein for growth of Theiler's murine encephalomyelitis virus (TMEV) in a macrophage-like cell line, J774-1. The protein is out of frame with the polyprotein and synthesized in DA but not GDVII subgroup strains of TMEV. A recombinant virus, DANCL*/GD, which substitutes the DA 5' noncoding and L* coding regions for the corresponding regions of GDVII and synthesizes L* protein, grew with little restriction in J774-1 cells. In contrast, another recombinant virus, DANCL*-1/GD, which has an ACG rather than an AUG as the starting codon of L* protein at nucleotide 1079, resulting in no synthesis of L* protein, did not grow well. No significant difference between the rates of adsorption to J774-1 cells of these viruses was observed. RNase protection assay demonstrated that DANCL*/GD viral RNA significantly increased, whereas only a minimal increase was observed for DANCL*-1/GD. The present study suggests that L* protein is required for virus growth in macrophages.

Theoretical analysis of right gastroepiploic artery grafting to right coronary artery.

BACKGROUND: The right gastroepiploic artery (GEA) has been used as the second reliable arterial graft for coronary artery bypass grafting (CABG). However, concern regarding the flow competition with the recipient coronary artery has remained. METHODS: An application of in situ GEA grafting to the right coronary artery (RCA) was studied by using a theoretical model. The theoretical model of CABG was given variables; ie, the diameters and the lengths of both in situ GEA and proximal segment of the RCA, and the degree of proximal stenosis in the RCA. According to the range of these variables obtained from clinical data, the ratio of the GEA flow to the flow of the RCA distal to the anastomosis was calculated. RESULTS: Main factors to determine the flows in the two parallel paths were the inner diameters of both vessels, and the degree of the proximal stenosis. When the inner diameters of the GEA were 0.5 mm larger than that of the RCA, the GEA carried more than 50% of the total flow of the RCA distal to the anastomosis despite a moderate stenosis in the RCA. When the inner diameter of the GEA was equal to, or 0.5 mm smaller than, that of the RCA, the GEA flow was dominated by the native RCA flow unless the proximal stenosis was critical. CONCLUSIONS: If the inner diameter of the GEA is 0.5 mm larger than that of the RCA, CABG with the GEA can be applied more widely. If not, the application would basically be limited.

Sarpogrelate reduces mechanical hemolysis in patients with heart valve prostheses.

OBJECTIVE: We evaluate the clinical efficacy of sarpogrelate, an antiplatelet drug that improves red blood cell deformability, to reduce the intravascular hemolysis problems suffered frequently by patients implanted with heart valve prostheses. SUBJECTS: Subjects were 34 patients undergoing mechanical heart valve replacement and having serum lactate dehydrogenase concentrations 20% above the maximum normal range. METHOD: Sarpogrelate was given daily, 100 mg orally for the first 6 months and 200 mg thereafter. RESULTS: Average serum lactate dehydrogenase decreased significantly from 423 +/- 108 IU/l, to 391 +/- 83 IU/l with the 100 mg dose, and to 361 +/- 86 IU/l with the 200 mg dose. The percentage of reticulocytes decreased from 15.5 +/- 5.3/1000 to 15.3 +/- 5.7/1000 at the 100 mg dose and 13.1 +/- 4.0/1000 at the 200 mg dose. Serum iron concentrations increased significantly from 63.2 +/- 24.8 micrograms/dl to 76.2 +/- 16.2 micrograms/dl at the 100 mg dose, and to 70.9 +/- 26.2 micrograms/dl with the 200 mg dose. CONCLUSION: Sarpogrelate is a useful drug for patients with implanted heart valve prostheses and subsequent high serum lactate dehydrogenase because it works as an antiplatelet drug and reduces mechanical hemolysis.

Adrenergic mediation of TNF alpha-stimulated ICAM-1 expression on human brain microvascular endothelial cells.

Adrenergic innervation derived from locus ceruleus has been implicated in regulating BBB permeability and inflammatory responses associated with neurological disorders. This report demonstrates that adrenergic agents attenuate the tumor necrosis factor-alpha (TNF alpha)-induced expression of intercellular adhesion molecule-1 (ICAM-1) on cerebral microvascular endothelial cells (HBMEC) derived from human brains. HBMEC were incubated with isoproterenol (1-10 microM) alone or in the presence of propranolol (10 microM) for 30 min followed by the addition of various concentrations of TNF alpha. ICAM-1 expression on cultured HBMEC was dose-dependently upregulated by TNF alpha. Incubation with isoproterenol significantly reduced levels of ICAM-1 expression indicating the possible involvement of adrenergic agents on ICAM-1 expression. Treatment with propranolol (beta 1/beta 2-adrenergic antagonist) and butoxamine (beta 2-adrenergic antagonist), but not atenolol (beta 1-adrenergic antagonist) reversed this inhibitory effect. Isoproterenol also dose-dependently stimulated cAMP production (assayed by RIA) by HBMEC; propranolol treatment abolished this effect. These data show that the beta 2-adrenergic receptor/cAMP pathway may be partly involved in TNF alpha-stimulated ICAM-1 expression and indicate the possible involvement of adrenergic mediation of capillary function including BBB integrity.

Theiler's murine encephalomyelitis virus (TMEV) subgroup strain-specific infection in neural and non-neural cell lines.

GDVII subgroup strains of Theiler's murine encephalomyelitis virus (TMEV) are highly virulent and produce acute polioencephalomyelitis in mice. Neither viral persistence nor demyelination is demonstrated in the few surviving mice. In contrast, DA subgroup strains are less virulent and establish a persistent central nervous system infection which results in demyelinating disease. We previously reported a subgroup-specific infection in a macrophage-like cell line, J774-1 cells; i.e., GDVII strain does not replicate in J774-1 cells, whereas the DA strain actively replicates in these cells. In addition, this subgroup-specific virus growth is shown to be related to the presence of L* protein, a 17 kDa protein translated out-of-frame of the viral polyprotein from an AUG located 13 nucleotides downstream from the polyprotein's AUG. The present paper demonstrated that this subgroup-specific infection is observed in murine monocyte/macrophage lineage cell lines, but not in other murine cell lines including neural cells. An RNase protection assay also suggested that L* protein-related virus growth is regulated at the step of viral RNA replication. As macrophages are reported to be the major cell harboring virus during the chronic demyelinating stage, the activity of L* protein with respect to virus growth in macrophages may be a key factor in clarifying the mechanism(s) of TMEV persistence, which is probably a trigger to spinal cord demyelination.

Expression of lymphotoxin gene inserted into Theiler's murine encephalomyelitis virus.

Theiler's murine encephalomyelitis virus (TMEV) belongs to the Picornaviridae genus. DA subgroup strains of this virus induce early, non-fatal polioencephalomyelitis followed by demyelination in the spinal cord, with virus persistence. We investigated the use of DA strain as a vector for the introduction of a foreign gene into the central nervous system. Human lymphotoxin (LT) gene was inserted in the L region, the most upstream part of the polyprotein coding region of DA genome. Expression of LT was demonstrated by an immunoblot and an enzyme-linked immunosorbent assay on BHK-21 cells that were infected with the recombinant virus. In addition, the expressed LT showed cytotoxicity against L-929 cells.

Theiler's murine encephalomyelitis virus (TMEV): the role of a small out-of-frame protein in viral persistence and demyelination.

Theiler's murine encephalomyelitis virus (TMEV) belongs to the genus Cardiovirus of the family Picornaviridae and is divided into two subgroups on the basis of different biological activities. GDVII subgroup strains produce acute and fatal polioencephalomyelitis in mice with no virus persistence. In contrast, DA or TO subgroup strains cause an early nonfatal polioencephalomyelitis. TMEV is thought to be an excellent animal model for the human demyelinating disease, multiple sclerosis. Data suggest that macrophages are a major reservoir harboring the virus. A small out-of-frame protein designated L* is synthesized in DA subgroup strains from an alternative, out-of-frame, initiation site. Studies of a DA mutant virus, having an ACG rather than an AUG and therefore does not synthesize L* protein, demonstrate that this protein is important for virus growth in particular cell types and is critical for DA-induced demyelinating disease and virus persistence. In addition, TMEV can be used as a vector for delivering foreign sequences into the central nervous system.

Partial maze procedure is effective treatment for chronic atrial fibrillation associated with valve disease.

The maze procedure may be performed in combination with valve operations to treat chronic atrial fibrillation associated with valve dysfunction. Although we initially used the modified Cox maze III procedure, a more limited partial maze procedure is now preferred because the left atrium might be considered as the electrical impetues for atrial fibrillation. In this study we compared the results of 30 patients (group I) who underwent the full biatrial modified Cox maze III and 20 (group II) patients the partial maze procedure. While the rates of restored sinus rhythm were the same in both groups at 6-month follow-up (I: 83.3%, vs II: 80%), the following advantages were noted in the patients undergoing the partial maze procedure: shorter operative times, lesser elevations of creatine phosphokinase, lower rate of blood transfusion, lower rate of junctional rhythm soon after the operation, and a higher P wave in those patients with restored sinus rhythm. The effectiveness of the partial maze procedure seems equal to that of the biatrial modified Cox maze III procedure for atrial fibrillation associated with valve disease. The partial maze procedure is simple and less invasive, and thus might be applied more frequently as an additional procedure to valve operations without additional risk.