HER2-Positive Metaplastic Spindle Cell Carcinoma Associated with Synchronous Bilateral Apocrine Carcinoma of the Breast.

Apocrine carcinoma, which is strictly defined as over 90% of tumor cells showing apocrine differentiation, is a rare variant of breast cancer. Here we report an uncommon case in which apocrine carcinomas developed concurrently in both breasts; in addition, a sarcomatoid spindle cell lesion was coincident in the right breast. Both apocrine carcinomas were immunohistochemically negative for estrogen receptor (ER) and progesterone receptor (PgR), but diffusely positive for androgen receptor (AR), GCDFP-15, and HER2. The presence of intraductal components in bilateral carcinomas and the absence of lymph node metastasis suggested that they were more likely to be individual primary lesions rather than metastatic disease. The spindle cell lesion showed a relatively well-circumscribed nodule contiguous with the apocrine carcinoma. HER2 oncoprotein overexpression was observed not only in the apocrine carcinoma, but also in the spindle cell lesion. Since the spindle cell component was intimately admixed with apocrine carcinoma and had focal cytokeratin expression, we diagnosed it as metaplastic spindle cell carcinoma, which was originated from the apocrine carcinoma. To our knowledge, this is the first case report of a patient with synchronous bilateral apocrine carcinomas coinciding with metaplastic carcinoma.

Clinical features of adult patients with acute hepatitis B virus infection progressing to chronic infection.

Background. Information regarding the progression of acute hepatitis B virus (HBV) infection to chronic infection in adults is scarce. Methods. Twenty-five adult patients with acute HBV infection (14 men and 11 women, 18-84 years old), whose clinical features progressed to those of chronic infection (group A) or did not (group B), were studied retrospectively. Results. There were 3 and 22 patients in groups A and B, respectively. Two of the 3 patients of group A lacked the typical symptoms of acute hepatitis. No differences were found between groups with respect to age, sex, or HBV genotypes. However, total bilirubin and alanine aminotransaminase levels were significantly lower in group A. Conclusions. Three of the 25 adult patients with acute HBV infection progressed to chronic infection. Hepatitis was mild in these patients. Patients with mild acute hepatitis B or unapparent HBV infection may have a higher risk of progressing to chronic infection.

Hepatitis B virus reactivation induced by infliximab administration in a patient with Crohn's disease.

A 47-year-old man diagnosed with Crohn's disease was treated with infliximab. He tested negative for hepatitis B surface antigen (HBsAg) and hepatitis B surface antibody (anti-HBs) but positive for anti-HB core antibody (anti-HBc). He tested positive for hepatitis B virus (HBV-) DNA 3 months after treatment and was administered entecavir. HBV-DNA test showed negative results 1 month later. ALT was persistently within the normal range, and HBV-DNA was persistently negative thereafter despite the continuation of infliximab every 8 weeks. In our hospital, 14 patients with inflammatory bowel disease, who tested negative for HBsAg, were treated with infliximab; 2 of them tested positive for anti-HBs and/or anti-HBc, and HBV reactivation was observed in 1 patient (the present patient). The present case and these findings highlight that careful follow-up is needed in patients with inflammatory bowel disease treated with infliximab who test positive for anti-HBc and/or anti-HBs.

Clinical features and hepatitis B virus (HBV) genotypes in pregnant women chronically infected with HBV.

OBJECTIVE: The purpose of this study was to clarify the clinical features and hepatitis B virus (HBV) genotypes in pregnant women chronically infected with HBV. METHODS: Among 1,489 pregnant women who visited our hospital in 2010, 26 were positive for hepatitis B surface antigens (HBsAg). Of these subjects, 21 from whom informed consent was obtained were included in this study. The clinical features and HBV markers, including genotypes, were investigated. RESULTS: No adverse events were observed in the subjects or the neonates during pregnancy or the perinatal period. The HBV genotypes were C in 14 cases, D in six cases, and undetermined in one case. Hepatitis B e antigens and a high viral load (>7.0 log copies/mL) were found in four and six subjects with genotype C, respectively, and in none of subjects with genotype D. The alanine aminotransferase (ALT) levels and platelet counts were within the normal ranges during pregnancy in all subjects except two and three subjects with genotype C, respectively. Three subjects with genotype C showed transient elevations of ALT after delivery. CONCLUSION: The majority of subjects were anti-HBe-positive with normal ALT levels; however, some subjects with genotype C showed a high viral load, elevated ALT levels and/or low platelet counts. The pregnancies and deliveries were safe; however, transient elevations of ALT after delivery were observed in some subjects with genotype C.

An adult patient with acute infection with hepatitis B virus genotype C that progressed to chronic infection.

In 2008, a 28-year-old woman consulted our hospital due to general fatigue. Her ALT level was within normal range but she was positive for hepatitis B surface antigen (HBsAg). Her ALT level was nearly within normal range thereafter and she was consistently positive for HBeAg. Later, it was proven that she was negative for HBsAg in 1999. She had been a sex worker in 2007-2008. Complete genome sequencing revealed that her HBV was genotype C. The present case may indicate that it is possible for acute infection with HBV genotype C to progress to chronic infection in adults.

Biofilm formation or internalization into epithelial cells enable Streptococcus pyogenes to evade antibiotic eradication in patients with pharyngitis.

Streptococcus pyogenes is the bacterium most frequently isolated from patients with pharyngitis. Although various antibiotics including penicillin are effective, antibiotic treatment failure in cases of streptococcal pharyngitis have been reported. Herein, we investigated mechanisms associated with recurrent streptococcal pharyngitis. Clinically isolated S. pyogenes strains showed serotype-specific features, with emm12 strains most frequently detected and emm6 strains more likely to produce biofilm. The architectures of formed biofilms were observed using a fluorescence microscope with Live/Dead staining. Furthermore, various cationic antimicrobial peptides were tested to evaluate their inhibitory activities toward biofilms formed by S. pyogenes. After treatments with high concentrations of antibiotics, S. pyogenes survived in biofilm even when dead bacterial cells covered the surface. Other findings demonstrated that some antimicrobial peptides have inhibitory effects on forming and formed biofilm. Moreover, emm4, emm6, and emm75 strains showed significantly higher levels of invasion capacity into Detroit 562 cells than strains with other genotypes. Additionally, more than half of the strains temporarily escaped killing by penicillin alone by internalization into epithelial cells, even when the antibiotic concentration used was greater than the 10-fold minimum inhibitory concentration (MIC) for planktonic S. pyogenes. Also, combined administrations of multiple antibiotics were more effective to eradicate strains more likely to be internalized. Finally, flow cytometric analyses demonstrated that emm12 strains with higher invasive capabilities expressed PrtF1 protein on the bacterial surface. These findings suggest that S. pyogenes isolated from patients with recurrent streptococcal pharyngitis have emm type-specific features that allow escape from eradication by antibiotics.

Epidemiological characterization of Streptococcus pyogenes isolated from patients with multiple onsets of pharyngitis.

Streptococcus pyogenes causes a broad spectrum of acute infections and is the bacterium most frequently isolated from patients with pharyngitis. A number of antibiotics including penicillin have been shown to be effective, although antibiotic treatment failure in cases of streptococcal pharyngitis have been reported. Herein, we aimed to elucidate the features of recurrent strains using clinical isolates. Ninety-three S. pyogenes organisms were obtained from Japanese patients with recurrent pharyngitis. Following genetic characterization, M-type isolates from patients with recurrent pharyngitis differed from those obtained at initial onset in 11 of 49 episodes, and pulsed field gel electrophoresis analysis showed different patterns in those cases. Additionally, spe genotyping revealed that the Spe type of the strains obtained at secondary onset corresponded with those from the initial onset in 22 cases. Furthermore, antibiotic susceptibility testing revealed that more than half of the strains were resistant to macrolides and lincosamides, which was a much greater ratio as compared with the strains obtained from initial onsets in previous studies. Our results suggest that recurrence and reinfection are often confused during the diagnosis of repetitive and persistent streptococcal pharyngitis. Moreover, the present S. pyogenes organisms were less susceptible to antibiotics, which raises caution about their appropriate use in clinical practice.

Prevalence and characteristics of cytolethal distending toxin-producing Escherichia coli from children with diarrhea in Japan.

In the present study, we examined the prevalence and characteristics of CTEC among diarrheal children in Japan during a year-long surveillance study. A PCR-RFLP assay for the detection and differentiation of five types of E. coli cdtB gene (types I through V) was developed, and 362 stool specimens collected from patients reporting to pediatric departments in two hospitals were analyzed. Of the 35 samples (9.7%) that were positive for the cdtB gene, 21 were positive for cdt-I, three for cdt-II, four for cdt-III, three for cdt-IV and four samples were positive for cdt-V, as determined by different molecular techniques. The recovery of CTEC having cdt alleles was a little less, which included 19 with cdt-I, one cdt-II, three cdt-III, three cdt-IV and four with cdt-V. Among 30 CTEC strains isolated, the majority of them (43%) belonged to serogroup 02. The other virulence genes such as astA, cnfl, eaeA, cnf2 and bfpA genes were detected in 14 (47%), 11 (37%), four (13%), three (10%) and one (3.3%) strains of CTEC, respectively. However, the other common virulence-associated genes specific for DEC were not detected in these strains. Interestingly, an untypable cdt gene was detected by PCR-RFLP in Providencia alcalifaciens. Our data indicate that CTEC may be associated with diarrheal children in Japan and most of them do not belong to a conventional enteropathogenic pathovar and thus differ from strains isolated in developing countries.

NMR investigation of the electrostatic effect in binding of a neuropeptide, achatin-I, to phosphatidylcholine bilayers.

Achatin-I (Gly1-d-Phe2-Ala3-Asp4), known as a neuropeptide containing a d-amino acid, binds to the surface of a zwitterionic phosphatidylcholine (PC) membrane only when the peptide N-terminal amino group is in the ionized state, NH3+ (Kimura, T.; Okamura, E.; Matubayasi, N.; Asami, K.; Nakahara, M. Biophys. J. 2004, 87, 375-385). To gain mechanistic insights into how the binding equilibrium is delicately controlled by the ionization state of the N-terminal amino group, peptide-lipid binding interactions are investigated by selectively enriched 15N (at the N-terminus) and natural-abundance 13C NMR spectroscopy. Upon binding to the PC membrane, the 15N NMR of the N-terminal NH3(+) shifts upfield. This observation supports a mechanism that the role of the N-terminal NH3(+) in stabilizing the binding state is through electrostatic attraction with a headgroup negative charge, i.e., PO4(-). Interestingly, when the side chain beta-carboxyl group in Asp4 is deionized at acidic pH, the 15N signal of the N-terminal NH3(+) exhibits no significant chemical-shift change upon membrane binding of achatin-I. The Asp4 side chain thus regulates efficiency of the electrostatic binding between the peptide N-terminal NH3(+) and the lipid headgroup PO4(-). 13C chemical shifts in the hydrophobic D-Phe2 residue are largely perturbed upon membrane binding, in the case where the side chain beta-CO2(-) in Asp4 is deionized; the deionization of Asp4 beta-CO2(-) increases the net hydrophobicity of achatin-I with a reduction of both the electrostatic hydration and the electrostatic attraction with the headgroup N(CH3)3(+) in the most superficial region of the PC membrane, resulting in deeper anchoring of the phenyl ring. Hence, the electrostatic effect of the side chain beta-CO2(-) in Asp4 floats achatin-I on the PC membrane surface, and the binding equilibrium is sensitively controlled by the ionization state of the N-terminal NH3(+).

Clinicopathologic features of the severe form of acute type 1 autoimmune hepatitis.

BACKGROUND & AIMS: Acute onset autoimmune hepatitis (acute AIH) is difficult to diagnose by using serologic data. In addition, some patients with the severe form of acute AIH do not respond to immunosuppressive therapy and have a poor outcome. In this study, we analyzed the clinicopathologic features of patients who were diagnosed as having acute AIH. METHODS: Retrospective review of patients with presumed acute AIH at Ehime University Graduate School of Medicine was performed. RESULTS: In liver tissue, infiltrates in the portal area and plasma cell infiltration are more common in acute AIH compared with acute hepatitis caused by other causes. Patients with acute AIH who did not have severe jaundice at the time of diagnosis exhibited a very good response to corticosteroid therapy, despite lower titers of antinuclear antibody (ANA). Most patients with acute AIH with higher levels of bilirubin and titers of ANA in sera respond poorly to corticosteroid therapy. CONCLUSIONS: Histologic findings might be useful for the early diagnosis of acute AIH. Acute AIH patients with high levels of bilirubin and high titers of ANA in sera often do not respond to corticosteroid therapy.

Frequency and effects of bacterial infection in children with influenza under oseltamivir treatment.

BACKGROUND: New antiviral drugs can rapidly improve the symptoms of influenza, but some patients still have prolonged fever and complications. The cause of the prolonged symptoms despite antiviral therapy remains unclear. Recent studies have shown a synergistic effect between influenza viruses and bacteria. This study investigated the frequency of bacterial infection in such patients and its effects on the clinical course to determine the need for antibiotics. METHODS: In two seasons (December 2001 through March 2002, and December 2002 through March 2003), throat cultures were obtained from 387 patients with influenza being treated with oseltamivir, and clinical courses were observed. Control throat cultures were obtained from 109 healthy children. RESULTS: The detection rate of pathogens was higher in patients with influenza (54.3%) than in control (23.9%, p<0.001). The most common pathogen was Streptococcus pneumoniae (49.7%) in patients with influenza and was Haemophilus influenzae (69.2%) in controls. Of the patients with normal flora, 4.1% had fever for 4 or more days and showed pathogens in throat cultures on day 4. Of the patients with pathogen-positive cultures who did not receive antibiotics, 40.3% had fever for 4 or more days. CONCLUSION: Throat cultures obtained on the first or fourth day of treatment with oseltamivir were positive for pathogenic bacteria in all patients with fevers for 4 or more days. Our observations suggest that patients with influenza and prolonged fever despite receiving oseltamivir should be given antibiotics.

Evaluation of the nuclear delivery and intra-nuclear transcription of plasmid DNA condensed with micro (mu) and NLS-micro by cytoplasmic and nuclear microinjection: a comparative study with poly-L-lysine.

BACKGROUND: The efficient nuclear delivery of plasmid DNA (pDNA) is essential for the development of a promising non-viral gene vector. In an attempt to achieve nuclear delivery, NLS-mu, a novel pDNA condenser, was prepared. This consists of mu, a highly potent polypeptide for condensing the pDNA, and a SV40 T antigen-derived nuclear localization signal (NLS(SV40)). METHODS: The utility of NLS-mu was assessed in terms of green fluorescent protein (GFP) expression after cytoplasmic and nuclear microinjection of GFP-encoding pDNA along with the transfection, and compared with mu and poly-L-lysine (PLL). Trans-gene expression after cytoplasmic microinjection was affected by the efficiencies of nuclear transfer and following intra-nuclear transcription. To evaluate the nuclear transfer process separately, we introduced a parameter, a nuclear transfer score (NT score), which was calculated as the trans-gene expression after cytoplasmic microinjection divided by that after nuclear microinjection. RESULTS: As expected, the rank order of trans-gene expression after the transfection and cytoplasmic microinjection was NLS-mu > mu > PLL. However, the calculated NT scores were unexpectedly ranked as mu = NLS-mu > PLL, suggesting that mu, and not NLS(SV40), is responsible for the nuclear delivery of pDNA. In addition, confocal images of rhodamine-labeled pDNA indicated that pDNA condensed with mu and NLS-mu was delivered as a condensed form. In comparing the nuclear transcription, the rank order of trans-gene expression after nuclear microinjection was PLL = NLS-mu > mu, suggesting that intra-nuclear transcription is inhibited by efficient condensation by mu, and is avoided by the attachment of NLS(SV40). CONCLUSIONS: Collectively, NLS-mu, which consists of chimeric functions, is an excellent DNA condenser, and the process is based on mu-derived nuclear transfer and NLS(SV40)-derived efficient intra-nuclear transcription.

Four-base codon/anticodon strategy and non-enzymatic aminoacylation for protein engineering with non-natural amino acids.

Techniques for position-specific incorporation of non-natural amino acids in an in vitro protein synthesizing system are described. First, a PNA-assisted non-enzymatic tRNA aminoacylation with a variety of natural and non-natural amino acids is described. With this technique, one can aminoacylate a specific tRNA simply by adding a preformed amino acid activated ester-PNA conjugate into an in vitro protein biosynthesizing system. Second, the genetic code is expanded by introducing 4-base codons that can be exclusively translated to non-natural amino acids. The most advantageous point of the 4-base codon strategy is to introduce multiple amino acids into specific positions in single proteins by using mutually orthogonal 4-base codons and orthogonal tRNAs. An easy and quick method for preparation of tRNAs possessing 4-base anticodons is also described. Combination of the non-enzymatic aminoacylation and the 4-base codon/anticodon strategy gives an easy and widely applicable technique for incorporating a variety of non-natural amino acids into proteins in vitro.

Simple and quick chemical aminoacylation of tRNA in cationic micellar solution under ultrasonic agitation.

A simple and quick method for direct aminoacylation of a tRNA with a non-natural amino acid was developed by using an N-protected amino acid cyanomethyl ester as a substrate solubilized in CTACl micelle under ultrasonic agitation.

In situ chemical aminoacylation with amino acid thioesters linked to a peptide nucleic acid.

tRNA-specific chemical aminoacylation was achieved with nonnatural amino acids. A nonnatural amino acid was activated as a thioester derivative, and the latter was linked through a spacer to the N-terminal of a 9-mer peptide nucleic acid that is complementary to the 3'-terminal region of yeast phenylalanine tRNA. Efficient aminoacylation was observed when the amino acid thioester-spacer-PNA conjugate was mixed with the tRNA. The PNA-assisted aminoacylation was also successful in an Escherichia coli in vitro protein synthesizing system that contained an orthogonalized tRNA. The in situ aminoacylation/in vitro translation gave a mutant protein in which the nonnatural amino acid was incorporated into the position directed by a CGGG 4-base codon/anticodon pair.

Incorporation of anthraquinonyl group into lambda-Cro repressor protein for strand- and position-specific photocleavage of double-stranded DNA.

lambda-Cro repressor protein incorporated with a 2-anthraquinonylalanine (anqAla) at the 64th position was chemically synthesized by solid-phase method. The 64th position was selected according to previous information on various mutants of Cro incorporated with a single anqAla unit, that were synthesized through an Escherichia coli in vitro protein-synthesizing system. The 64anqAla mutant bound to a dsDNA of consensus operator sequence and underwent strand- and position-specific photocleavage of the dsDNA at the GG sequence after treatment with piperidine. The mutant also underwent position-specific self-photoscission. The self-photoscission was retarded in the presence of the dsDNA.

Facile aminoacylation of pdCpA dinucleotide with a nonnatural amino acid in cationic micelle.

A simple and versatile method for aminoacylation of a dinucleotide (pdCpA) in aqueous micellar solution was developed by using a hydrophobic amino acid derivative, N-pentenoyl-L-2-naphthylalanine cyanomethyl ester (Pen-napAla-OCM), and a CTACl micelle.

Effect of maclorides on duration and resolution of symptoms and complication of pneumonia in children with influenza.

UNLABELLED: We randomly administered cephalosporins or macrolides to 365 pediatric patients with influenza-like symptoms and compared the clinical course and complication rate of pneumonia. One hundred and fifty-four patients received cephalosporins (Group 1) and 211 received macrolides (Group 2). There were no significant differences in age, male/female ratio and body weight between the two groups. Macrolides alleviated fever significantly faster than cephalospoins (3.8plus minus 1.4 days vs 4.3plus minus 1.4 days), though maximum body temperature showed no significant difference between the two groups. Thirty-nine patients underwent laboratory examinations and twenty-nine had high influenza A (H3N2) virus haemagglutinate inhibition (HI) titer, six had high influenza B (B1) virus HI titer and four did not show any elevation of influenza virus HI titer. Thirteen patients in Group 1 and two patients in Group 2 suffered from pneumonia and the complication rate was significantly lower in Group 1 than in Group 2 (8.4% vs 0.9%). All of them recovered within two weeks and did not have any other complications. CONCLUSION: Macrolides are more effective in reducing the time required to alleviate fever and complication rate of pneumonia than cepharosporines in children with influenza and influenza-like illness. These results indicate that macrolides may have therapeutic value for influenza virus infection.