Improved enzyme-linked immunosorbent assay using C-terminal truncated recombinant antigens of Babesia bovis rhoptry-associated protein-1 for detection of specific antibodies

An enzyme-linked immunosorbent assay (ELISA) based on a recombinant rhoptry-associated protein-1 (RAP-1) of Babesia bovis has been previously developed, but it was imperfect because some cross-reactions were still present in Babesia bigemina-infected bovine sera. To improve its accuracy for the specific detection of the antibodies to B. bovis, we constructed three C-terminal truncated recombinant antigens of the RAP-1-rCT1 (amino acids [aa] 301 to 408), rCT2 (aa 388 to 490), and rCT3 (aa 466 to 565)-by using a baculovirus expression system and evaluated their diagnostic potentials using ELISA. rCT1 and rCT2 were better diagnostic antigens in their sensitivities and diagnostic efficiencies than rCT3, although none of the recombinant antigens showed any cross-reactivity to B. bigemina-infected bovine sera. These results confirmed that the N-terminal 300-aa region caused cross-reactivity of the entire RAP-1 antigen, and the C-terminal truncated recombinant antigens were shown to be useful reagents for species-specific serodiagnosis.     

Elevated expression of E-cadherin and α-, β-, and γ-catenins in metastatic lesions compared with primary epithelial ovarian carcinomas

E-cadherin and catenins play key roles in cell adhesion and motility. Little is known about the changes in expression of these molecules in the progression of ovarian carcinomas. In the present study, the immunohistochemical expression of E-cadherin and α-, β-, and γ-catenins was examined in 77 cases of ovarian carcinoma. In addition, the expression of these molecules was evaluated in 26 matched pairs of primary and metastatic lesions of advanced ovarian carcinomas. Of the 77 primary lesions, positive staining for E-cadherin and α-, β-, and γ-catenin was observed in 75 (97%), 63 (82%), 71 (92%) and 57 (74%) cases, respectively. Positivity for E-cadherin and α-, β-, and γ-catenin was significantly decreased in stage III and IV tumors compared with stage I and II tumors, suggesting that expression of the cadherin-catenin complex is reduced with the advancing stages of a tumor. Interestingly, expression of E-cadherin and α-, β-, and γ-catenin in the lesions of peritoneal dissemination was significantly increased compared with the primary lesions. These findings suggest that expression of the cadherin-catenin complex changes markedly and that reexpression may occur during the peritoneal dissemination of ovarian carcinoma cells.     

In vivo study of the effects of cryopreservation on heart valve xenotransplantation.

BACKGROUND: Recent reports have suggested that cryopreservation reduces the immunogenicity of donor tissue. The immunomodulation by cryopreservation might influence on the tissue durability after xenotransplantation. We investigated the in vivo morphologic changes in cryopreserved xenograft (CXG) heart valves. MATERIAL AND METHOD: We transplanted a fresh (fresh xenograft; FXG) and a cryopreserved (CXG) porcine aortic root and a cryopreserved canine (cryopreserved allograft; CAG) aortic root into the abdominal aorta of a dog without any immunosuppressive agents. Explanted grafts on the 21st to 49th days after implantation were analyzed morphologically with light microscopy using some special stains, immunohistochemical analysis, and scanning electron microscopy (SEM). RESULT: Light microscopy showed the absence of smooth muscle cells in the media of the aorta in any group after transplantation. FXG valves did not maintain any cellularity after transplantation. CXG valves contained cellular infiltration in themselves. CAG valves contained numerous fibroblasts, which showed the maintenance of tissue integrity without allowing cellular infiltration. The structure of elastic fibers was well maintained, even in the part of CXG valve with cellular infiltration. Immunohistochemical studies documented the infiltration of T lymphocytes in CXG valves that were labeled by anti-CD3 antibodies. SEM demonstrated that no endothelia were seen on the surface of the valves in any group after transplantation. CONCLUSION: We concluded that the cryopreservation method might provide an immunomodulation of xenogeneic heart valves for transplantation.     

Regulation of the type I IFN induction: a current view

The type I IFN-alpha/beta gene family was identified about a quarter of a century ago as a prototype of many cytokine gene families, which led to the subsequent burst of studies on molecular mechanisms underlying cytokine gene expression and signaling. Although originally discovered for their activity to confer an antiviral state on cells, more evidence has recently been emerging regarding IFN-alpha/beta actions on cell growth, differentiation and many immunoregulatory activities, which are of even greater fundamental biological significance. Indeed, much attention has recently been focused on the induction and function of the IFN-alpha/beta system regulated by Toll-like receptors (TLRs), which are critical for linking the innate and adaptive immunities. The understanding of the regulatory mechanisms of IFN-alpha/beta gene induction by TLRs and viruses is an emerging theme, for which much new insight has been gained over the past few years.     

Seeligeriolysin O, a protein toxin of Listeria seeligeri, stimulates macrophage cytokine production via Toll-like receptors in a profile different from that induced by other bacterial ligands

Seeligeriolysin O (LSO), a member of cholesterol-dependent cytolysins of Listeria seeligeri, exhibits cytokine-inducing activity. In this study, we examined the profile of cytokines expressed in macrophages of mice after stimulation with full-length form of recombinant LSO (rLSO530), C-terminal-truncated protein (rLSO483) and two authentic cytokine-inducing Toll-like receptor (TLR) ligands from bacteria, peptidoglycan (PGN) and LPS. Both rLSO530 and rLSO483 were able to induce IL-12 p40 and IL-12 p70 more strongly in macrophages than PGN or LPS. In contrast, IFN-beta and nitric oxide were induced by LPS but not by rLSO530, rLSO483 or PGN. In the presence of exogenously added IFN-beta, IL-12 p40 and IL-12 p70 production was inhibited after LSO stimulation, but IL-12 p70 production was enhanced after PGN stimulation. Although LSO signaling appeared to be associated with both TLR2 and TLR4, the profile of cytokine production by LSO stimulation was distinct from those by stimulation with PGN or LPS. Thus, it was shown that LSO is a unique bacterial ligand that induces macrophage cytokine production in a manner different from PGN or LPS.     

Expression of BRCA1 protein in benign, borderline, and malignant epithelial ovarian neoplasms and its relationship to methylation and allelic loss of the BRCA1 gene

BRCA1 is a putative tumour suppressor gene responsible for a hereditary ovarian cancer syndrome. To clarify the possible involvement of BRCA1 in the development of sporadic ovarian neoplasms, this study analysed the immunohistochemical expression of BRCA1 protein in normal ovarian surface epithelium and 119 epithelial ovarian tumours (19 benign, 24 borderline, and 76 malignant tumours). Loss of heterozygosity (LOH) of BRCA1 was examined using three microsatellite markers to analyse the relationship between BRCA1 expression and alterations of the BRCA1 gene. Methylation of the BRCA1 promoter was also analysed by methylation-specific PCR. In ovarian carcinomas showing heterogeneous expression of BRCA1 protein in the same tumour, LOH and methylation status were analysed using microdissection techniques. Finally, the relationship of BRCA1 expression or its genetic alteration to clinicopathological parameters and patient survival was analysed. Ovarian surface epithelial cells expressed BRCA1 protein. Decreased expression of BRCA1 was found in 16% of benign tumours, 38% of borderline tumours, and 72% of carcinomas. LOH of BRCA1 was demonstrated in no benign tumours, 15% of borderline tumours, and 66% of carcinomas. Methylation of BRCA1 was not detected in benign or borderline tumours, but was present in 31% of carcinomas. Reduced expression of BRCA1 correlated with the presence of gene methylation. The frequency of BRCA1 methylation and LOH was higher in serous carcinomas than in other types. In one of the three serous carcinomas that showed heterogeneous expression of BRCA1, BRCA1-positive borderline-like tumour cells were LOH-positive and methylation-negative, whereas adjacent BRCA1-negative carcinoma cells were LOH-positive and methylation-positive. The prognosis of carcinoma patients did not correlate with BRCA1 expression or genetic status. These findings suggest that reduced expression of BRCA1 protein along with genetic and epigenetic changes of the BRCA1 gene play an important role in the development of sporadic ovarian carcinomas, particularly those of serous histology.     

SR proteins preferentially associate with mRNAs in the nucleus and facilitate their export to the cytoplasm

Different classes of RNA are exported to the cytoplasm by distinct mechanisms. Each class of RNA forms distinct complexes with nuclear proteins prior to its export to the cytoplasm. In our attempt to obtain comprehensive information of protein factors that specifically associate with mRNAs in the nucleus, we performed in vivo UV-crosslinking analysis after microinjection of various RNAs into Xenopus oocyte nucleus. We found a group of proteins preferentially crosslinked to mRNAs. Immunoprecipitation experiments revealed that some of the crosslinked signals corresponded to SR (serine/arginine-rich) proteins, a family of essential RNA-binding proteins involved in pre-mRNA splicing. It was previously suggested that some members of SR protein family are involved in export of a specific intronless mRNA, histone H2A mRNA and some spliced mRNAs. However, it is still to be clarified if SR proteins are involved in export of general mRNAs, especially general intronless mRNAs that do not contain specific RNA export elements. When we microinjected an antibody against SR proteins into the nucleus, export of mRNAs was severely inhibited, regardless of whether the mRNAs were produced via pre-mRNA splicing or not, whereas export of other RNAs was not affected. These results unequivocally showed that SR proteins are involved in export of both general intronless and spliced mRNAs.     

Biotin deficiency in amino acid formula nutrition for an infant with milk protein allergy]

We reported a 4-month-old girl with biotin deficiency caused by amino acid formula. Two weeks after birth, she was diagnosed as having a milk protein allergy. After switching to amino acid formula from usual formula, her symptoms and laboratory findings became normal. About three weeks after the beginning of amino acid formula, she developed intractable skin erosions around the eyes, mouth, neck, and anogenital area. By measuring concentrations of some trace elements, she was diagnosed as having a biotin deficit, because of the organic aciduria and undetectable serum biotin concentration. Her serum biotinidase level was normal. Upon administration of oral biotin supplementation, all her symptoms and laboratory findings were dramatically improved. Since amino acid formula contains very few biotin, we should pay attention to biotin deficiency when infants receiving amino acid formula.