Levitt's CO breath test in the differential diagnosis of chronic isolated hyperbilirubinemia.

A key component of the differential diagnosis of isolated hyperbilirubinemia (HB) is distinguishing between hemolytic and non-hemolytic types. Routine hemolysis screening markers have unsatisfactory sensitivity and specificity. Erythrocyte (RBC) lifespan shortening, the gold standard marker of hemolysis, is seldomly measured due to the cumbersome and protracted nature of standard methods. A new Levitt's CO breath test method may enable simple, rapid RBC lifespan measurement. In this pilot prospective diagnostic study, Levitt's CO breath test was evaluated to discriminate hemolytic from non-hemolytic HB in adults. One hundred and thirty eligible non-smoking adult patients who were aged 18 or older, referred for chronic (>6 months) isolated HB or had a known diagnosis of isolated HB of a rare cause, were recruited, including 77 with non-hemolytic HB and 53 with hemolytic HB. ROC curve analysis was applied to determine the optimal cutoff for discriminating between hemolytic and non-hemolytic HB, and the performance was calculated. Results showed that the mean RBC lifespan in non-hemolytic HB (93 ± 26 d) was reduced (p= 0.001 vs. normal reference value of 126 d), but longer than that in hemolytic HB (36 ± 17 d;p= 0.001). RBC lifespans did not differ significantly between 26 patients with simple hemolytic HB (32 ± 14 d) and 27 patients with a Gilbert syndrome comorbidity (40 ± 18 d). ROC curve analysis revealed an optimal lifespan cutoff for discriminating between hemolytic and non-hemolytic HB of 60 d (AUC = 0.982), with a diagnostic accuracy of 95.4%, 94.3% sensitivity and 96.1% specificity respectively. These results indicate that Levitt's CO breath test seems to be very sensitive and specific for detecting hemolysis in adult patients with chronic isolated HB, and could enable simple, rapid, and reliable differential diagnosis of isolated HB. A large-scale validation study of the method is warranted.

Human erythrocyte lifespan measured by Levitt's CO breath test with newly developed automatic instrument.

Existing standard techniques for erythrocyte (RBC) lifespan measurement, such as quantitation of labeling with isotopes or biotin, are cumbersome and time-consuming. Given that endogenous CO originates mainly from degraded RBCs, a team lead by Levitt developed a CO breath test to enable more efficient RBC lifespan estimation. The purpose of this study was to evaluate the reliability of Levitt's CO breath test method with our newly developed automatic instrument. RBC lifespan measurements conducted by Levitt's CO breath test method were conducted in 109 healthy subjects and 91 patients with chronic hemolytic anemia. In healthy subjects, the RBC lifespan was 126 ± 26 days, similar to values obtained with classical standard labeling methods. RBC lifespan did not differ significantly between males and females or between juveniles and adults, and did not correlate with age. To our knowledge, this datum represents an RBC lifespan average for the largest sample to date. In subjects with hemolytic anemia, RBC lifespan was 29 ± 14 days, which is significantly shorter than that of the healthy subjects (p = 0.001). Using 75 days as a cut-off, diagnostic accuracy for hemolytic anemia in the present study sample was 100%. In conclusion, the present results indicate that Levitt's CO breath test is an ideal method for human RBC lifespan measurement, and the newly developed automatic instrument is reliable and convenient for clinical practice.

Rapid CO breath test screening of drugs for protective effects on ribavirin-induced hemolysis in a rabbit model: a pilot study.

Hemolytic anemia is a major side effect of ribavirin antiviral treatment for chronic hepatitis C. Ribavirin dose reduction may compromise the antiviral response and erythropoietin can take several weeks to alleviate anemia. The purpose of the present study was to screen potentially protective drugs against ribavirin-induced hemolytic anemia in a rabbit model, using our modified CO breath test for measuring erythrocyte (RBC) lifespan, the gold standard diagnostic index of hemolysis. Fifteen rabbits were divided randomly into five groups (N = 3/group): one vehicle control group, one ribavirin (only)-treated (RBV) group, and three groups initially treated with ribavirin only, followed by a combination of ribavirin with prednisone (RBV + Pred), polyene phosphatidyl choline (RBV + PPC), or reduced glutathione (RBV + GSH). RBC lifespan was calculated from accumulated CO measured in a closed rebreath apparatus, blood volume measured by the Evan's blue dye (EBD) dilution test, and hemoglobin concentration data. The RBC lifespan was normal in the vehicle control group (44-60 d), but reduced significantly in all of the ribavirin-treated groups before the addition of screened drugs (17-35 d). RBC lifespan rebounded significantly with the addition of glutathione, but not with the addition of prednisone or polyene phosphatidyl choline. A similar overall drug effect pattern was seen in the hemoglobin concentration and reticulocyte count data. In conclusion, the results of this pilot study indicate that reduced glutathione can attenuate ribavirin-induced hemolytic anemia, and that the RBC lifespan measured with our modified rapid CO breath test is feasible and reliable for use in animal studies.

The draft genome, transcriptome, and microbiome of Dermatophagoides farinae reveal a broad spectrum of dust mite allergens.

BACKGROUND: A sequenced house dust mite (HDM) genome would advance our understanding of HDM allergens, a common cause of human allergies. OBJECTIVE: We sought to produce an annotated Dermatophagoides farinae draft genome and develop a combined genomic-transcriptomic-proteomic approach for elucidation of HDM allergens. METHODS: A D farinae draft genome and transcriptome were assembled with high-throughput sequencing, accommodating microbiome sequences. The allergen gene structures were validated by means of Sanger sequencing. The mite's microbiome composition was determined, and the predominant genus was validated immunohistochemically. The allergenicity of a ubiquinol-cytochrome c reductase binding protein homologue was evaluated with immunoblotting, immunosorbent assays, and skin prick tests. RESULTS: The full gene structures of 20 canonical allergens and 7 noncanonical allergen homologues were produced. A novel major allergen, ubiquinol-cytochrome c reductase binding protein-like protein, was found and designated Der f 24. All 40 sera samples from patients with mite allergy had IgE antibodies against rDer f 24. Of 10 patients tested, 5 had positive skin reactions. The predominant bacterial genus among 100 identified species was Enterobacter (63.4%). An intron was found in the 13.8-kDa D farinae bacteriolytic enzyme gene, indicating that it is of HDM origin. The Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed a phototransduction pathway in D farinae, as well as thiamine and amino acid synthesis pathways, which is suggestive of an endosymbiotic relationship between D farinae and its microbiome. CONCLUSION: An HDM genome draft produced from genomic, transcriptomic, and proteomic experiments revealed allergen genes and a diverse endosymbiotic microbiome, providing a tool for further identification and characterization of HDM allergens and development of diagnostics and immunotherapeutic vaccines.

[Cloning, expression and identification of Der f7 gene from Dermatophagoides farinae and its immunological characteristics].

OBJECTIVE: To clone and express Der f7 gene of Dermatophagoides farinae, and identify its immunogenicity. METHODS: Total RNA was extracted from D. farinae mites. A reference sequence (Accession No. AY283292) was used to design specific primers. The Der f7 gene fragment was amplified by RT-PCR, and cloned into pET-32a vector. The recombinant plasmid was transformed into E. coli BL21 (DE3) and induced with IPTG for protein expression. The recombinant protein was purified by Ni2+ chelating affinity chromatography and analyzed by SDS-PAGE and Western blotting. RESULTS: The Der f7 gene fragment was about 650 bp, and shared 99% homology with the published one (Accession No. FJ436108). SDS-PAGE result showed its relative molecular weight (M(r)) of 23 000. The recombinant protein showed appropriate combination ability with IgE in sera of mite allergic patients. CONCLUSION: Der f 7 gene has been expressed in prokaryotic expression system and shows allergenicity.

A two-site monoclonal antibody immunochromatography assay for rapid detection of peanut allergen Ara h1 in Chinese imported and exported foods.

Food allergen labeling has not yet been implemented in China. Therefore, a gold immunochromatography assay (GICA) was developed using two monoclonal antibodies (mAb) against the peanut allergen Ara h1. The GICA was specific for standard peanut samples with a sensitivity of 10ng/ml. Peanut protein traces extracted from 124 food products imported and exported by China Customs were easily and rapidly detected by GICA. 68 food samples originally labeled as containing peanuts were positive for Ara h1 and 54 food samples labeled as not containing peanuts were negative for Ara h1, indicating that the labels from the manufacturers were accurate. However, 2 food samples labeled as not containing peanuts tested positive for Ara h1. The present GICA provides a fast, simple, semi-quantitative method for the determination of peanut allergens in foods. This detection system can be used to ensure the safety of food imported and exported by China Customs.

Monoclonal antibodies against recombinant Der f 3 reveal localization of Der f 3 in the gut and faecal pellets of Dermatophagoides farinae.

Home dust mite derived materials are known to be a major source of problematic inhalant allergens. The aim of this study was to determine the localization of the group 3 allergen, Der f 3, within Dermatophagoides farinae, in order to assess the relative importance of excreted materials and nonexcreted body components as allergen sources. Recombinant Der f 3 (rDer f 3) was expressed in bacteria and purified as an immunogen for production of monoclonal antibodies (mAb) against it. Dermatophagoides farinae mites and their faecal pellets were embedded in paraffin, and serial sections were immunoprobed with mAb clone 3D3 against Der f 3. D. farinae midgut mucosa, gut contents and faecal pellets were strongly immunopositive for Der f 3. Der f 3 immunoreactive products were not detected in any other internal organs of the mite. These results suggest that Der f 3 allergen may be synthesized in and secreted from the digestive tract and excreted from the mite's body in the faecal pellets.

Artesunate induces oncosis-like cell death in vitro and has antitumor activity against pancreatic cancer xenografts in vivo.

Pancreatic cancer is highly resistant to the currently available chemotherapeutic agents. Less than 5% of patients diagnosed with this disease could survive beyond 5 years. Thus, there is an urgent need for the development of novel, efficacious drugs that can treat pancreatic cancer. Herein we report the identification of artesunate (ART), a derivative of artemisinin, as a potent and selective antitumor agent against human pancreatic cancer cells in vitro and in vivo. ART exhibits selective cytotoxic activity against Panc-1, BxPC-3 and CFPAC-1 pancreatic cancer cells with IC(50) values that are 2.3- to 24-fold less than that of the normal human hepatic cells (HL-7702). The pan caspase inhibitor zVAD-fmk did not inhibit the cytotoxic activity of ART. Electron microscopy of ART-treated cells revealed severe cytoplasmic swelling and vacuolization, swollen and internally disorganized mitochondria, dilation (but not fragmentation) of the nuclei without chromatin condensation, and cell lysis, yielding a morphotype that is typical of oncosis. The ART-treated cells exhibited a loss of mitochondrial membrane potential (DeltaPsim) and ART-induced cell death was inhibited in the presence of the reactive oxygen species (ROS) scavenger N-acetyl-cysteine (NAC). Importantly, ART produced a dose-dependent tumor regression in an in vivo pancreatic cancer xenografts model. The in vivo antitumor activity of ART was similar to that of gemcitabine. Taken together, our study suggests that ART exhibits antitumor activity against human pancreatic cancer via a novel form of oncosis-like cell death, and that ART should be considered a potential therapeutic candidate for treating pancreatic cancer.

Sublingual immunotherapy efficacy of Dermatophagoides farinae vaccine in a murine asthma model.

BACKGROUND: Allergen-specific sublingual immunotherapy is a potential treatment for allergic diseases. Its effective dose and underlying mechanism are still to be explored. Here, we investigated the efficacy and mechanism of sublingually administered Dermatophagoides farinae (Der f) vaccine in a murine asthma model. METHODS: BALB/c mice were sensitized intraperitoneally with Der f extract absorbed to alum, followed by sublingual treatment with Der f vaccine for 6 weeks. The mice were subsequently challenged intranasally with Der f extract for 1 week. We analyzed their clinical symptoms, antibody levels, cytokine levels, T-cell proliferation and the regulatory T-cell numbers. RESULTS: Mice treated with high-dose Der f sublingual vaccine prior to challenge displayed alleviated symptoms such as airway hyperreactivity, lung inflammation and mucus production, as well as less eosinophilic cells in bronchoalveolar lavage fluid. Interestingly, reduced responses of Der-f-specific IgE and increased responses of Der-f-specific IgA and IgG1 were aroused in the high-dose Der f sublingual vaccine group. We also observed that interleukin-4 was reduced and interferon-gamma and interleukin-10 were increased among splenocytes and in bronchoalveolar lavage fluid, which inhibited Der-f-specific T-cell proliferation of the spleen and increased CD4+CD25+Foxp3+regulatory T cells in the spleen. However, mice treated with low-dose Der f sublingual vaccine developed allergic asthma. CONCLUSION: Our results illustrate that high-dose Der f sublingual vaccine may play a role in immunologic protection in murine allergic asthma, possibly by inducing regulatory T cells and Th1 reaction.

Conservative management of pediatric nephrolithiasis caused by melamine-contaminated milk powder.

OBJECTIVE: In this article we report our experience with the diagnostic screening and management of children with melamine-induced nephrolithiasis. METHODS: A total of 1091 children younger than 4 years who had been exposed to melamine-contaminated formula from September 17 to October 12, 2008, were screened for nephrolithiasis at the department of pediatrics at Shenzhen Nanshan Hospital in China. During the clinical examination, each patient's demographic characteristics were recorded together with the details of his or her milk-consumption profile during the contamination scare and any clinical signs of poisoning. Urinary stones were detected by B-ultrasonography, and renal status was examined by a routine urine test panel and a renal function test. When urinary stones were detected, patients were ordered to cease consumption of the suspected formula, and a conservative treatment course was adopted, including infusion of fluids, urinary alkalinization, increased water consumption, and diuresis. RESULTS: Of the 1091 children screened, 12 (1.1%) were diagnosed with kidney stones. They had been exposed to the contaminated milk from 1 to 24 months. Eleven (91.7%) of these 12 patients had consumed milk with a high level of melamine content (955-2563 ppm); 1 patient (8.3%) had consumed milk with a low-level melamine content (6.2-17.0 ppm). Six patients exhibited dysuria; the remaining 6 patients were asymptomatic. All 12 patients had normal renal function, although 4 had proteinuria, and 1 had hematuria. The kidney stones were resolved within 3 to 5 days of commencing treatment in all 12 cases. CONCLUSIONS: Nephrolithiasis was associated with high melamine-exposure levels. A combination of B-ultrasonography and urinalysis is suitable for screening for pediatric nephrolithiasis caused by melamine poisoning. The condition can be resolved with a conservative treatment approach in patients without serious clinical symptoms who have normal kidney function.

New BZLF1 sequence variations in EBV-associated undifferentiated nasopharyngeal carcinoma in southern China.

The viral lytic gene BZLF1 triggers replication of the Epstein-Barr virus (EBV), which is commonly found in nasopharyngeal carcinoma (NPC). Here, RT-PCR revealed five new BZLF1 variants in 8 of 12 NPC and 4 of 12 non-NPC nasopharyngeal biopsies from an NPC-endemic area in southern China. The deduced peptide sequence of the dominant BZLF1 variant differed by 11 amino acids from that of the prototypical strain B95.8 (V01555). Anti-ZEBRA antibody levels were higher in NPC than that in non-NPC patients (P < 0.001). These findings demonstrated a dominant BZLF1 variant in southern Chinese EBV-associated NPC and non-NPC patients.

[Cloning, expression and purification of allergen arginine kinase from Periplaneta americana and its allergic activity].

OBJECTIVE: To clone the gene of arginine kinase (AK) from Periplaneta americana, produce its recombinant protein and investigate its allergenicity. METHOD: The cDNA of AK was cloned using specific primers from the total RNA of P. americana. The cloned gene was inserted into pMD18-T vector and digested by BamHI and HindIII. The cDNA was sequenced and subcloned into pET-28a expression vector. The cloned AK cDNA gene was expressed in Escherichia coli BL21 (DE3) by IPTG induction. The recombinant AK (rAK) was purified by metal (Ni2+) chelating affinity chromatography. Its allergenicity was examined by both Western blotting and enzyme-linked immunosorbent assay (ELISA). RESULT: The cloned cDNA ORF sequence (Accession no. EU429466) contained 1068 bp and encoded 365 amino acids. Its sequence homology with the published one (Accession no. AY563004) was 99.9% at nucleotide level. The allergen rAK was highly expressed in E. coli BL21 (DE3) as a soluble protein mainly with the molecular weight of about Mr 45000 under induction of IPTG and purified by 6-His-tag purification system. Both in the non-denaturalization and denaturalization conditions, the recombinant allergen was identified as its affinity to IgE antibodies from the cockroach-allergic patient sera by Western blotting and ELISA. CONCLUSION: The recombinant cockroach arginine kinase has been obtained with proper allergenicity.

[Cloning, expression and purification of dust mite allergen Der f 3 and identification of its allergic activity].

OBJECTIVE: To clone, express and identify Der f 3 gene. METHODS: Live mites were collected from southern China region, identified as Dermatophagoides farinae, and cultured. The total RNA was extracted. The Der f 3 gene fragment was amplified by RT-PCR and sequenced. The Der f 3 gene fragment encoding a serine protease mature peptide was sub-cloned into the expression vector pET-His. The recombinant pET-Der f 3 plasmid was inserted into E.coli BL21 and induced to express Der f 3 coding protein by IPTG. The recombinant Der f 3 with 6 his-tag was then purified by chelating resin and its allergic activity was identified by Western blotting. RESULTS: The Der f 3 gene fragment with 840 bases was determined. Its sequence homology with the published one (GenBank No.D63858) was 99.5% at nucleotide level. It was sub-cloned into expressing vector pET-His and the recombinant allergen rDer f 3 was highly expressed in E.coli BL21 (DE3) under induction of IPTG, and purified by 6-His-tag purification system. Using Western blotting method, the allergic activity of the purified recombinant allergen was identified as its affinity to IgE antibodies from the mite-allergic patient sera. CONCLUSION: Der f3 gene has been successfully cloned and its prokaryotic expression vector is constructed.

[Detection of Periplaneta americana sIgE with chemiluminescent immunoassay (CLIA)].

Crude extract of Periplaneta americana was prepared by liquid nitrogen grinding. After being purified with DEAE Sephadex A-50 ion exchange chromatography, the protein content of the extract was determined and the extract solution was prepared at gradient concentrations. The crude extract and purified allergen at different concentrations were dotted respectively on nitrocellulose (NC) membrane. Patient serum, bio-IgE, sa-HRP, luminal regents were added to the membrane. The chemiluminescence was displayed by exposing to X-film. The result revealed that the minimum protein content of crude Periplaneta americana extract detected by CLIA is 0.87 microg/ml, with 90% accordance to skin test positive patients, and 100% accordance to those with negative skin test and ELISA detection.