Case Report: Primary Tuberculosis of the Bilateral Breast.

Primary breast tuberculosis is rare. We report a case of bilateral primary breast tuberculosis. The patient received incisional drainage and debridement of both breasts. Histopathology of the breast tissues revealed chronic granulomatous inflammation and positive acid-fast stain. The patient received antitubercular therapy for 18 months, and she achieved complete resolution.

Proteomic analysis reveals significant elevation of heat shock protein 70 in patients with chronic heart failure due to arrhythmogenic right ventricular cardiomyopathy.

As proteins are the ultimate biological determinants of phenotype of disease, we screened altered proteins associated with heart failure due to arrhythmogenic right ventricular cardiomyopathy (ARVC) to identify biomarkers potential for rapid diagnosis of heart failure. By 2-dimensional gel electrophoresis and mass spectrometry, we identified five commonly altered proteins with more than 1.5 fold changes in eight ARVC failing hearts using eight non-failing hearts as reference. Noticeably, one of the altered proteins, heat shock protein 70 (HSP70), was increased by 1.64 fold in ARVC failing hearts compared with non-failing hearts. The increase of cardiac HSP70 was further validated by Western blot, immunochemistry, and enzyme-linked immunosorbent assay (ELISA) in failing hearts due to not only ARVC, but also dilated (DCM, n = 18) and ischemic cardiomyopathy (ICM, n = 8). Serum HSP70 was also observed to be significantly increased in heart failure patients derived from the three forms of cardiomyopathies. In addition, we observed hypoxia/serum depletion stimulation induced significantly elevation of intracellular and extracellular HSP70 in cultured neonatal rat cardiomyocytes. For the first time to our knowledge, we revealed and clearly demonstrated significant up-regulation of cardiac and serum HSP70 in ARVC heart failure patients. Our results indicate that elevated HSP70 is the common feature of heart failure due to ARVC, DCM, and ICM, which suggests that HSP70 may be used as a biomarker for the presence of heart failure due to cardiomyopathies of different etiologies and may hold diagnostic/prognostic potential in clinical practice.

Upregulated expression of cardiac ankyrin repeat protein in human failing hearts due to arrhythmogenic right ventricular cardiomyopathy.

AIMS: Expression of cardiac ankyrin repeat protein (CARP) is augmented in heart failure due to dilated or ischaemic cardiomyopathy. It is unclear whether CARP is upregulated in heart failure due to arrhythmogenic right ventricular cardiomyopathy (ARVC). In the present study, we investigated the expression pattern of CARP and the correlation between CARP and the well-known heart failure marker pro-atrial natriuretic peptide (proANP) in ARVC failing hearts. METHODS AND RESULTS: Gene microarray analysis demonstrated increased CARP expression in ARVC failing hearts compared with non-failing control hearts, which was further validated by real-time RT-PCR, western blot, and ELISA at the mRNA and protein levels. Fractionation experiments revealed that the upregulation of CARP expression is restricted to the nuclei of residual cardiac cells in ARVC failing hearts. Regression analysis showed a positive correlation between CARP and proANP in ARVC failing hearts. CONCLUSION: Augmented CARP expression may be a common molecular event in failing hearts regardless of cardiomyopathic aetiology. The upregulation of nuclear CARP expression and positive correlation between cardiac CARP and proANP suggests that CARP may be used as a genetic marker existing in the nuclei in contrast to proANP existing in the cytosol of cardiac cells in heart failure patients.

Apolipoprotein D as a novel marker in human end-stage heart failure: a preliminary study.

Apolipoprotein D (Apo D) is reported to be in close association with developing and mature blood vessels, and involved in enhanced smooth muscle cell migration after injury. This study was designed to clarify the expression pattern of Apo D and the possibility of Apo D as a new marker in human end-stage heart failure. Individual RNA samples obtained from independent left ventricular tissue of six heart failure patients derived from cardiomyopathies of different aetiologies during cardiac transplantation and six non-failing control subjects were hybridized to the gene microarray containing, in total, 35 000 well-characterized Homo sapiens genes. Apo D was one of the highly expressed genes (3.3-fold upregulated) detected by microarray, which was further confirmed by quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) (5.88-fold upregulated) in failing hearts compared with non-failing hearts. Both Western blotting and immunohistochemistry analyses also demonstrated the higher levels of Apo D protein in failing hearts. Importantly, we observed elevated levels of plasma Apo D in heart failure patients compared with non-failing control subjects. We demonstrated, for the first time to our knowledge, that Apo D was highly expressed in the mRNA and protein levels in human failing hearts compared with non-failing hearts. Furthermore, our finding of elevated plasma Apo D levels in patients with heart failure provides clues that Apo D may act not only as a cardiac molecular marker but also as a circulating biomarker in patients with heart failure.

[Establishment of an assay for PrP(Sc) detection based on streptomycin precipitation].

To establish a new Western blotting assay for PrP(Sc) detection, we optimized the Western blotting assay with a precipitation procedure of streptomycin sulfate. After digestion with PK, 10% scrapie infected hamster brain homogenates were incubated with 60 mmol/L streptomycin and the precipitated PrP(Sc) was recovered by centrifugation. The enrichment of PrP(Sc) by streptomycin sulfate precipitation was evaluated using Western blotting assay. The results showed streptomycin could bind to PK-treated PrP(Sc), forming high molecular masses, but not influence the glycosylated patterns on SDS-PAGE. Western blot assay revealed that the detective sensitivity of the streptomycin-precipitation PrP(Sc) was remarkably improved. As a sensitive, specific, rapid and flexible protocol for PrP(Sc), the protocol in this study has the potential utility, alone or combined with other techniques, for the detection of low level PrP(Sc) in the specimens from central nerve system, or from peripheral organs or body fluids.

Human tau protein forms complex with PrP and some GSS- and fCJD-related PrP mutants possess stronger binding activities with tau in vitro.

Microtubule associated protein tau is considered to play roles in some types of human transmissible spongiform encephalopathies (TSE). In this study, the full-length and several truncated human tau proteins were expressed from E. coli and purified. Using GST pull down, co-immunoprecipitation assay and tau-coated ELISA, the molecular interaction between tau protein and PrP was confirmed in the context of the full-length human tau. The N terminus (amino acids 1-91) and tandem repeats region (amino acids 186-283) of tau protein were responsible for the interaction with PrP. The octapeptide repeats within PrP directly affected the binding activity of PrP with tau. GSS-related mutant PrP102L and fCJD- related mutants with two and seven extra octarepeats showed more active binding capacity with tau than wild-type PrP. The molecular interactions between PrP and tau protein highlight a potential role of tau in the biological function of PrP and the pathogenesis of TSE.

[Preliminary analyses for influence of mutant PrPs with different number of octapeptide].

OBJECTIVE: The present study was conducted to understand the effects of PrP in different octapeptide repeats on proliferation of HeLa cells. METHODS AND RESULTS: Mutant PrPs with octapeptide repeat insertion were transiently expressed in HeLa cells and their results of MTT assay showed stronger cytotoxic effect on the proliferation of cells than wild-type PrP. Annexin V/PI assay also demonstrated that the expression of mutant PrPs was much easier to induce apoptosis than wild-type in HeLa cells. The percentage of both early and late stage apoptosis in mutant groups were significantly higher than that of wild type. CONCLUSION: These data suggest that the expression of mutant PrPs associated with familial CJD is much easier to induce apoptosis in cultured cells than expression of wild type PrP.

[Generation and identification of phage engineering antibodies library against hamster prion protein].

OBJECTIVE: Generation and Identification of Phage Engineering Antibodies Library against Hamster Prion Protein. METHODS: Fab antibodies were identified and confirmed. BALB/c mice were immuned with PrP proteins. After the third immunization, the total RNA was extracted from the mice spleens. The genes of heavy Fd Fragments and light chain of antibodies amplified by a series of specific primers of human IgG Fab fragment were cloned into phagemid vector pComb3. RESULTS: The combinatorial Fab library were constructed successfully and the cloning efficiencies both of light chain and Fd fragments were about near 10(6). The Fab library were panned by four cycles and screened with purified haPrP23-231 antigen on microtiter plates. 12 mAbs were isolated after four cycles of panning, five of which were sequenced and resulted sequence data were analyzed by alignment with GenBank immunoglobulin genes. Two strain of new heavy and light chain genes of Fab antibodies were identified and confirmed. CONCLUSION: The research in this article will provide foundation for study of diagnosis and therapy of prion.

Generation of genetic engineering monoclonal antibodies against prion protein.

Two strains of Fab monoclonal antibodies (mAbs) against prion protein, designated as IV-66 and IV-78, were selected from the phage display libraries. The gene sequences encoding the light kappa chain and heavy Fd chain of IV-78 were inserted into a baculovirus expression cassette vector for mouse IgG expression. Western blot, Dot-ELISA and immunoprecipitation confirmed that these Fab and IgG mAbs reacted well with the recombinant hamster and human PrP proteins expressed in prokaryotic and in mammalian cells and PrP(Sc) from scrapie-infected hamsters. It demonstrates that mAbs against prion protein are successfully generated by phage-display technique.

Preparation of monoclonal antibodies against prion proteins with full-length hamster PrP.

OBJECTIVE: To prepare the PrP specific monoclonal antibodies (mAbs) that can be used for the detection of mammalian prions and study of pathogenesis of prion diseases. METHODS: Several BALB/c mice were immunized with recombinant hamster prion protein (HaPrP). Three hybridoma cell lines designated as B7, B9, and B10, secreting monoclonal antibodies against HaPrP, were established by hybridoma technique. The mAbs reactivities were evaluated with ELISA, Western blot, and immunohistochemistry. RESULTS: The mAbs produced by these cell lines reacted well with different recombinant hamster PrP proteins. Western blot analyses showed that mAbs B7 and B9 reacted with PrPSc from the scrapie-infected animals after proteinase K digestion with three glycosylated forms. The mAbs exhibited cross-reactivity with various PrPC from several other mammalian species, including humans and cattles. Immunohistochemistry assays confirmed that mAbs B7 and B9 could recognize not only extracellular but also intracellular PrPsSc. CONCLUSION: The mAbs of prion protein are successfully generated by hybridoma technique and can be applied for the diagnosis of prion associated diseases.