Volumetric breast density is essential for predicting cosmetic outcome at the late stage after breast-conserving surgery.

BACKGROUND: The critical issue related to breast-conserving therapy (BCT) is that cosmetic outcomes deteriorate with long-term follow-up. There is little research for breast density as a predictor of cosmetic outcomes at the late stage after BCT. To improve the long-term quality of life after BCT of breast cancer patients, the correlation of volumetric breast density (VBD) and cosmetic outcome at the late stage after BCT was evaluated. STUDY DESIGN: Breast volume, fibroglandular tissue volume, adipose tissue volume, and VBD were calculated on mammography using image analysis software (Volpara(®)) in 151 patients with BCT. Furthermore, the correlation of breast density and the change of breast volume over time was analyzed on mammography in 99 patients who were followed-up long-term after BCT. RESULTS: On multivariate analysis, VBD was a predictor of cosmetic outcome after BCT with percent breast volume excised (PBVE). Decreased adipose tissue volume and increased fibrosis were more common in patients with VBD < 15%. Furthermore, remnant breast volume continued to decrease over time in low breast density patients during long-term follow-up. 93% of patients with VBD ≥ 15% and PBVE < 10% had a better cosmetic outcome, while 60% of patients with VBD < 15% and PBVE ≥ 10% had a worse cosmetic outcome after BCT. CONCLUSIONS: While PBVE was involved in cosmetic outcome at the early stage after BCT, VBD was associated with cosmetic outcome at the late stage after BCT. Thus, a combination of VBD and PBVE could predict cosmetic outcome after BCT and contribute to the selection for the appropriate BCT.

Functional analysis of recombinant 2-Cys peroxiredoxin from the hard tick Haemaphysalis longicornis.

Ticks are obligate haematophagous arthropods that feed on vertebrate blood containing high levels of iron. The host-derived iron reacts to oxygen in the tick's body, and then high levels of reactive oxygen species, including hydrogen peroxide (H(2)O(2)), may be generated. High levels of H(2)O(2) cause oxidative stress to aerobic organisms. Therefore, antioxidant responses are necessary to control H(2)O(2). We focused on peroxiredoxins (Prxs), H(2)O(2) -scavenging enzymes. The sequence of Haemaphysalis longicornis 2-Cys Prx (HlPrx2) was identified from fat body cDNA libraries of this tick and recombinant HlPrx2 was then prepared using Escherichia coli. By comparison with the 2-Cys Prxs of other organisms, we found two conserved cysteines in HlPrx2, Cys51 and Cys172. We examined the antioxidant activity of HlPrx2 and mutant proteins produced by a single base substitution, converting one or both of these cysteines into serines. The assays revealed that proteins containing Cys51 showed antioxidant activity when H(2)O(2) was removed. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and size-exclusion chromatography demonstrated that only the wild-type HlPrx2 formed homodimers and that all of the proteins that we made had a high molecular weight peak. These results indicate that both Cys51 and Cys172 are essential for the dimerization of HlPrx2, whereas only the Cys51 residue is necessary for antioxidant activity.

Rapid deterioration of renal insufficiency after magnetic resonance imaging with gadolinium-based contrast agent.

Gadolinium (Gd)-based contrast media were introduced as alternatives to iodinated media for magnetic resonance imaging (MRI). Although originally thought to be non-nephrotoxic, Gd-based contrast media have recently been reported to be associated with acute kidney injury. The underlying mechanism of Gd-induced renal injury is not completely understood. We report an 80-year-old patient with buccal mucosa cancer for whom MRI with Gd-based contrast agent was conducted 3 times within 3 weeks. The patient developed rapid deterioration of preexisting renal insufficiency, and developed uremic symptoms and pulmonary edema. The patient was hemodialyzed 3 times. This resulted in improvement of renal function and clinical symptoms. This case emphasizes the potential nephrotoxicity of Gd-based contrast media and suggests that renal insufficiency, diabetes mellitus, old age and high dose of Gd-based contrast medium are risk factors for acute kidney injury.

Observation of three-dimensional internal structure of steel materials by means of serial sectioning with ultrasonic elliptical vibration cutting.

A three-dimensional (3D) internal structure observation system based on serial sectioning was developed from an ultrasonic elliptical vibration cutting device and an optical microscope combined with a high-precision positioning device. For bearing steel samples, the cutting device created mirrored surfaces suitable for optical metallography, even for long-cutting distances during serial sectioning of these ferrous materials. Serial sectioning progressed automatically by means of numerical control. The system was used to observe inclusions in steel materials on a scale of several tens of micrometers. Three specimens containing inclusions were prepared from bearing steels. These inclusions could be detected as two-dimensional (2D) sectional images with resolution better than 1 mum. A three-dimensional (3D) model of each inclusion was reconstructed from the 2D serial images. The microscopic 3D models had sharp edges and complicated surfaces.

Nitric oxide attenuates vascular endothelial cadherin-mediated vascular integrity in human chronic inflammation.

In this study, we examined the role of nitric oxide (NO) in controlling vascular integrity mediated by vascular endothelial (VE)-cadherin in chronic inflammation. Periapical granulomas were analysed for the expression of inducible NO synthase (iNOS) and VE-cadherin, and more iNOS expression than VE-cadherin was shown. Human umbilical vein endothelial cells (HUVECs) were stimulated with proinflammatory cytokines and lipopolysaccharide extracted from Porphyromonas gingivalis and it induced iNOS expression, whereas it reduced VE-cadherin expression, compared with negative controls. On the other hand, pre-incubation with 1400W, an iNOS-specific inhibitor, markedly reduced iNOS expression in stimulated HUVECs and restored VE-cadherin expression to its control level, suggesting that vascular integrity was modulated in conjunction with the reduction of NO. Immunocytochemistry confirmed the functional role of NO in cultured HUVEC monolayers with or without 1400W. These data are consistent with a hypothesis suggesting that NO could attenuate VE-cadherin-mediated vascular integrity in human chronic inflammation.

Molecular characterization of a novel 32-kDa merozoite antigen of Babesia gibsoni with a better diagnostic performance by enzyme-linked immunosorbent assay.

We cloned and expressed a novel gene encoding a 32-kDa merozoite protein of Babesia gibsoni (BgP32). The length of nucleotide sequence of the cDNA was 1464 bp with an open reading frame of 969 bp. The truncated recombinant BgP32 (rBgP32) without a signal peptide and C-terminal hydrophobic sequence was expressed in Escherichia coli as a soluble glutathione-S-transferase (GST) fusion protein. Western blotting demonstrated that the native protein was 32-kDa, consistent with molecular weight of the predicted mature polypeptide. Enzyme-linked immunosorbent assay (ELISA) using rBgP32 detected specific antibodies from 8 days to 541 days post-infection in the sequential sera from a dog experimentally infected with B. gibsoni. Moreover, the antigen did not cross-react with B. canis subspecies and closely related protozoan parasites, indicating that rBgP32 is a specific diagnostic antigen. Analysis of 47 sera taken from dogs with anaemic signs revealed that rBgP32 detected a higher proportion of B. gibsoni seropositive samples (77%) than its previously identified rBgP50 (68%) homologue. These results indicate that the BgP32 is a novel immunodominant antigen of B. gibsoni, and rBgP32 might be useful for diagnosis of B. gibsoni infection.

Acute renal infarction in a patient with left atrial myxoma.

A 24-year-old male first attended our hospital with acute onset of right flank pain radiating to the right lower quadrant of the abdomen. A contrast-enhanced computer tomography (CT) scan showed renal infarction, and he was admitted immediately for treatment. On admission, the right lower abdominal pain diminished gradually. On the second day in hospital, a left atrial echogenic mass was detected which filled the left atrial cavity; it appeared to be a left atrial myxoma measuring 3.9+/-4.9 cm. The patient was immediately transferred and underwent emergency surgery. Histologic examination confirmed the diagnosis of myxoma. Post-operatively, he recovered well and was discharged from hospital without any further specific treatment.

Thrombotic thrombocytopenic purpura associated with polyarteritis nodosa.

We present a case of classical polyarteritis nodosa (PN) overlapping thrombotic thrombocytopenic purpura (TTP). A 70-year-old woman was transferred to our hospital because of general fatigue and fever. On admission, laboratory findings revealed leukocytosis, normochromic normocytic anemia and renal dysfunction. About one week later, she developed disturbance of consciousness, and laboratory findings revealed rapidly progressive thrombocytopenia and renal dysfunction. We suspected the presence of microscopic polyangiitis (MPA), based on mild elevation of myeloperoxidase (MPO) anti-neutrophil cytoplasmic antibody (ANCA). On post-admission Day 11, renal biopsy was performed but the diagnosis of MPA could not be confirmed because of the absence of glomerular crescent formation or vasculitis. However, the biopsy specimen showed many collapsed glomeruli and interstitial inflammation, indicating the presence of occlusive lesions, such as vasculitis in larger arteries. We instituted methylprednisolone pulse therapy, cyclophosphamide and plasma exchange, because the clinical symptoms also satisfied the criteria of TTP. Despite the intensive treatment, the patient died on 43rd day of hospitalization due to thalamic hemorrhage. Autopsy showed typical findings of classical PN including disruption of arterial walls and fibrinoid necrosis in the medium-sized arteries of the kidneys and colon. We detected reduced activity of von Willebrand factor-cleaving protease (VWF-CP) and the presence of plasma inhibitory IgG against VWF-CP. A better understanding of the mechanisms would be useful.

Fenofibrate induces apoptotic injury in cultured human hepatocytes by inhibiting phosphorylation of Akt.

Fibric acid derivatives have a potent and effective lipid-lowering action, however, the use of these compounds is sometimes limited due to the occurrence of hepatic injury. In the present study, we characterized cell injury induced by fenofibrate in cultured human hepatocytes. Fenofibrate caused a loss of cell viability and nuclear damage as assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling or by DNA electrophoresis, in which caspase activation is involved. The cell injury was accompanied by the shrinkage and the translocation of phosphatidyl serine from inner membrane to the outer membrane as determined by annexin V stain. The mRNA expression for bcl-2 was reduced by fenofibrate. An immunofluorescent stain with antiserum raised against phosphorylated Akt revealed that fenofibrate inhibited insulin-stimulated phosphorylation of Akt. Like fenofibrate, several compounds that inhibit the phosphorylation of Akt, including wortmannin, SH-6 and a high concentration (100 microM) of SB203580, reduced the viability of cultured human hepatocytes. Both nuclear damage and cell injury induced by fenofibrate were reversed by insulin in a concentration-dependent manner. In contrast, bezafibrate or 8(S)-hydroxyeicosatetraenoic acid had no hepatotoxic action. These findings suggest that fenofibrate causes caspase-dependent apoptosis in human hepatocytes by inhibiting phosphorylation of Akt, in which PPARalpha is not involved.

Synthesis of infectious in vitro transcripts from Cassia yellow blotch bromovirus cDNA clones and a reassortment analysis with other bromoviruses in protoplasts.

Cassia yellow blotch virus (CYBV), genus Bromovirus, was isolated from the Australian native legume, Cassia pleurocarpa, in western Queensland, and its host range was found to be distinct from other bromoviruses. In this study, CYBV was shown to infect systemically and efficiently a model plant species, Arabidopsis thaliana, as we recently reported for another bromovirus, Spring beauty latent virus (SBLV). We constructed full-length cDNA clones of CYBV genomic RNAs from which infectious in vitro transcripts can be transcribed, and determined their complete nucleotide sequences. CYBV RNA3 contains the box B motif in the intercistronic region, but lacks the subgenomic promoter-like sequence in the 5' noncoding region, as does Brome mosaic virus (BMV). To understand relationships among bromoviruses, we generated reassortants between CYBV and three other bromoviruses, BMV, SBLV and Cowpea chlorotic mottle virus. We found that all reassortants between BMV and CYBV accumulated viral RNAs to detectable levels in protoplasts of Nicotiana benthamiana, even when RNAs 1 and 2, which encode the replication proteins 1a and 2a, respectively, were heterologous. Sequence comparison and reassortment experiments of CYBV and other bromoviruses demonstrated that CYBV is closely related to BMV.

Characterisation of the gene encoding a protective antigen from Babesia microti identified it as eta subunit of chaperonin containing T-complex protein 1.

Passive immunisations with a monoclonal antibody termed 1-5H showed a partial but significant inhibition of parasitaemia against Babesia microti challenge infection. By immunoscreening with 1-5H, a clone (termed p58 gene) was obtained from a cDNA expression library of B. microti and the complete nucleotide sequence was determined. A protein homology search showed significant amino acid identities to the eta subunit of the chaperonin containing T-complex protein 1 (CCT) of human (59%), mouse (58%) and Plasmodium falciparum (62%). Genomic analyses indicated that the p58 gene is present as a single copy gene and contains a total of approximately 400-bp introns in the genome of B. microti. The mAb 1-5H recognised a 58-kDa protein of B. microti and was found to cross-react with a 60-kDa protein of Babesia rodhaini. These results suggest the possibility that the p58 protein is the CCT eta subunit of B. microti and functions as a chaperonin.

Interferon-gamma-induced apoptosis in host cells infected with Neospora caninum.

Interferon-gamma (IFN-gamma) has a crucial role for host defence against parasite infection. It is not clear, however, how IFN-gamma affects the parasite-infected host cells. The effect of IFN-gamma on Neospora caninum-infected cells was investigated in murine fibroblasts and canine kidney cells in vitro. In the presence of IFN-gamma, the viability of the infected host cell was decreased and apoptotic cell death occurred, as analysed by DNA stainings with propidium iodide and a terminal deoxy-nucleotidyltransferase-mediated dUTP-biotin nick end labelling (TUNEL) and DNA fragmentation. The percentage of apoptotic cells depended on the dose of IFN-gamma. Flow cytometric analysis indicated a significant increase of FasL expression on the IFN-gamma treated cells following N. caninum infection. Moreover, IFN-gamma treatment down-regulated Bcl-2 expression in the cells cultured with N. caninum while parasite infection up-regulated Bcl-2 expression. The present study suggests that the IFN-gamma induced increases of FasL expression and down-regulated Bcl-2 expression in N. caninum-infected cells are associated with apoptosis in vitro.

Molecular characterization of a peroxiredoxin from the hard tick Haemaphysalis longicornis.

Antioxidant enzymes in eukaryotes play an important role in protection against the oxygen radicals generated during aerobic metabolism. Here we report the cloning and characterization of a cDNA encoding the antioxidant enzyme peroxiredoxin from the hard tick Haemaphysalis longicornis (HlPrx). HlPrx is 939 bp long and contains a 101 bp non-translated sequence at the 5' end and a polyadenylation singnal followed by a poly(A) tail at the 3' end. HlPrx encodes a full-length protein with a predicted molecular mass of 26 kDa that possesses one cysteine residue at amino acid 49 that is conserved among Prx proteins of various species. GenBanktrade mark analysis showed that the deduced amino acid sequence had significant similarity to mammalian and plant Prxs at the amino acid level. A DNA-nicking assay revealed that Escherichia coli-expressed recombinant HlPrx (rHlPrx) inhibited oxidative-nicking of supercoiled plasmid DNA. Two-dimensional immunoblot analysis with mouse antirHlPrx serum showed reaction with a major constituent protein spot in extracts of adult ticks. In addition, immunoblot analysis showed that rHlPrx was immunoreacted with serum from rabbits repeatedly infested with H. longicornis. Localization analysis using mouse antirHlPrx serum revealed that native HlPrx was highly expressed in the salivary gland of the tick. Moreover, Northern blot analysis showed that the level of HlPrx transcripts was increased during blood sucking. The present results indicate that HlPrx may be an important detoxifying enzyme during the normal life span as well as during blood sucking in ticks.

Modified protection against Toxoplasma gondii lethal infection and brain cyst formation by vaccination with SAG2 and SRS1.

Numerous studies have supported the importance of immunity to SAG1, the most predominant antigen of Toxoplasma tachyzoite, in protection against Toxoplasma gondii infection. Nevertheless, vaccination with SAGI provides insufficient protection when compared with that of Toxoplasma lysate (TL). In order to screen the Toxoplasma antigens for immunogenic potential shown by modified protection or induction of specific immune response after infection, recombinant antigens were prepared in Eschericha coli using DNA fragments corresponding to SAG1, SAG2, SAG3, SRS1 and P54 of T. gondii RH strain maintained in our laboratory. Each of the recombinant antigen products or a mixture of the five antigens (Mix) was used to vaccinate mice. Mice then received a lethal dose of T. gondii. Up to 25% of the mice vaccinated with SAG2, SRS1, P54 and Mix survived, whereas there were no survivors in gene 10- (negative control), SAG1- and SAG3- vaccinated groups. In all the survivors, brain cysts were not observed. Conversely, vaccination with TL almost completely protected mice in the acute phase but permitted brain cyst formation and resulted in gradual decrease of survivors to 33% during 4 months of experiments. Western blot analysis on convalescent sera showed an extensive IgG induction to a 30 kDa antigen in TL-vaccinated mice, a 22 kDa in SAG2-vaccinated mice and a 55 kDa in P54-vaccinated mice. The protection modified by boost in specific antibody is suggestive of the immunogenic potential of SAG2, SRS1 and possibly P54 against T. gondii infection.

Expression of canine interferon-gamma by a recombinant vaccinia virus and its antiviral effect.

A recombinant vaccinia virus-expressing canine interferon (IFN)-gamma (vv/cIFN-gamma) was constructed. In rabbit kidney (RK13) and canine A72 cells infected with vv/cIFN-gamma, IFN activity was detected in the culture supernatants of both cell types. Canine IFN-gamma was also detected in both cell extracts by Western blot. The activity of the recombinant canine IFN-gamma in RK13 cells was higher than that in A72 cells. The vv/cIFN-gamma could not grow in A72 cells at a low multiplicity of infection, probably due to the antiviral activity of the canine IFN-gamma produced. Although exogenous IFN-gamma did not inhibit the growth of vaccinia virus, addition of anti-canine IFN-gamma serum recovered the growth of the vv/cIFN-gamma on A72 cells in a dose-dependent manner. These results suggest that the growth of vv/cIFN-gamma was inhibited by IFN-gamma produced in a paracrine and autocrine manner. In addition, the recombinant canine IFN-gamma inhibited the multiplication of canine herpesvirus, pseudorabies virus and canine adenovirus type 1 in Madin-Darby canine kidney cells. The antiviral effect of canine IFN-gamma was more effective than that of canine IFN-beta. From the present studies, we concluded the recombinant virus may be a useful suicide viral vector.

Increased susceptibility to Toxoplasma gondii infection in SAG-1 transgenic mice.

SAG-1, one of the major surface proteins of Toxoplasma gondii, has been reported to play an important role in immune and pathogenic mechanisms of the parasites but its exact function is still unclear. We investigated the time courses of T. gondii infection in B6C3F1 transgenic mice carrying the SAG-1 gene. SAG-1 transgenic mice were infected intraperitoneally with a high virulent RH strain or a low virulent Beverley strain of T. gondii. When infected with RH strain tachyzoites, no significant differences in time courses of survivals between SAG-1 transgenic and wild-type mice were observed. Both groups succumbed to an acute infection within 8 days after infection. However, a lower survival rate (20%) was observed in SAG-1 transgenic mice than in wild-type (80%), when infected with Beverley strain cysts. This result indicates that SAG-1 transgenic mice are more susceptible to T. gondii infection as compared with their wild-type counterpart. ELISA using recombinant SAG-1 protein indicates that SAG-1 transgenic mice do not produce antibodies to the SAG-1 molecule. These findings may provide a critical tool for analysing the molecular mechanisms of pathogenesis and host immune responses during toxoplasmosis.

Phylogenetic relationships of the benign Theileria species in cattle and Asian buffalo based on the major piroplasm surface protein (p33/34) gene sequences.

In order to examine the taxonomic relationship of Theileria sp. of Asian buffalo to the benign Theileria spp. of cattle, we sequenced and compared the major piroplasm protein (p33/34) genes of these parasites. The two consensus sequences determined for the buffalo parasite were of the same length (852 bp) and showed >80% identity with the sequences of the homologous genes (849 bp) in the cattle parasites. Alignment of the inferred aa sequences with those of Theileria sergenti and Theileria buffeli predicted that there is an insertion of a single residue at the N-terminus in the inferred polypeptide of the buffalo parasite. Phylogenetic analyses based on the aa sequences suggested that Theileria sp. of the Asian buffalo should be classified within the benign Theileria parasite group as a separate species from the cattle parasites. Based on this, we propose a rearrangement of the currently used classification for the benign Theileria species in cattle and Asian buffalo.

Usefulness of the high-frequency ultrasound probe in pretherapeutic staging of superficial-type colorectal tumors.

We evaluated the usefulness of the high-frequency ultrasound probe (HFUP, 20 MHz) to determine the depth of tumor invasion in 45 patients with superficial colorectal tumors. The correct diagnostic rate was 66% (30/45) when the depth of tumor invasion was classified into the following 6 layers: mucosa (m), upper 1/3 (sm1), middle 1/3 (sm2), and lower 1/3 (sm3) areas of the submucosa, muscularis propria (mp), and the subserosa or deeper areas (s). However, when the depth of tumor invasion was evaluated in 3 layers (m-sm1, sm2-sm3, and mp-deeper layer), which is the classification used to select cases for endoscopic mucosal resection, the correct diagnostic rate was 88.9% (40/45). These results suggest that the HFUP is useful to determine the depth of invasion to select treatment for superficial colorectal tumors.