Immune responses against a single CD8+-T-cell epitope induced by virus vector vaccination can successfully control Trypanosoma cruzi infection

In order to develop CD8+-T-cell-mediated immunotherapy against intracellular infectious agents, vaccination using recombinant virus vectors has become a promising strategy. In this study, we generated recombinant adenoviral and vaccinia virus vectors expressing a single CD8+-T-cell epitope, ANYNFTLV, which is derived from a Trypanosoma cruzi antigen. Immunogenicity of these two recombinant virus vectors was confirmed by the detection of ANYNFTLV-specific CD8+ T cells in the spleens of immunized mice. Priming/boosting immunization using combinations of these two recombinant virus vectors revealed that the adenovirus vector was efficient for priming and the vaccinia virus vector was effective for boosting the CD8+-T-cell responses. Moreover, we also demonstrated that the ANYNFTLV-specific CD8+-T-cell responses were further augmented by coadministration of recombinant vaccinia virus vector expressing the receptor activator of NFkappaB (RANK) ligand as an adjuvant. By priming with the adenovirus vector expressing ANYNFTLV and boosting with the vaccinia virus vectors expressing ANYNFTLV and RANK ligand, the immunized mice were efficiently protected from subsequent challenge with lethal doses of T. cruzi. These results indicated, for the first time, that the induction of immune responses against a single CD8+-T-cell epitope derived from an intrinsic T. cruzi antigen was sufficient to control lethal T. cruzi infection.     

Pathological role of large intestinal IL-12p40 for the induction of Th2-type allergic diarrhea

IL-12 consists of two disulfide-linked subunits, p40 and p35, that form functionally active heterodimers for the induction of Th1 cells. In contrast to IL-12 heterodimers, p40 monomers and homodimers possess inhibitory effects on Th1 cells leading to the creation of a Th2 environment. Although it has been shown that IL-12p40 acts as antagonist of IL-12p70 in vitro, no evidence is currently available whether IL-12p40 is functional in vivo. We now report that IL-12p40 plays an important pathological role in anintestinal allergic disease. A high expression of IL-12p40 protein was demonstrated in epithelial cells, dendritic cells, and macrophages in large but not small intestine of allergic diarrhea-induced mice. Interestingly, neutralization with anti-IL-12p40 mAbs reduced the incidence and delayed the onset of disease development. Lower levels of ovalbumin (OVA)-specific IgE Abs in serum were detected in anti-IL-12p40 mAb-treated mice than in control Ab-treated mice. The secretion of Th2 cytokines and eotaxin by the mononuclear cells isolated from the large intestine of anti-IL-12p40 mAb-treated mice was significantly decreased. Finally, the removal of the IL-12p40 gene resulted in complete inhibition of disease development. These results show that over-expression of IL-12p40 is an important contributing factor for the generation of the dominant Th2-type environment in the large intestine of mice with allergic diarrhea.     

Probiotic bifidobacteria protect mice from lethal infection with Shiga toxin-producing Escherichia coli O157:H7

The anti-infectious activity of probiotic Bifidobacteria against Shiga toxin-producing Escherichia coli (STEC) O157:H7 was examined in a fatal mouse STEC infection model. Stable colonization of the murine intestines was achieved by the oral administration of Bifidobacterium breve strain Yakult (naturally resistant to streptomycin sulfate) as long as the mice were treated with streptomycin in their drinking water (5 mg/ml). The pathogenicity of STEC infection, characterized by marked body weight loss and subsequent death, observed in the infected controls was dramatically inhibited in the B. breve-colonized group. Moreover, Stx production by STEC cells in the intestine was almost completely inhibited in the B. breve-colonized group. A comparison of anti-STEC activity among several Bifidobacterium strains with natural resistance to streptomycin revealed that strains such as Bifidobacterium bifidum ATCC 15696 and Bifidobacterium catenulatum ATCC 27539(T) did not confer an anti-infectious activity, despite achieving high population levels similar to those of effective strains, such as B. breve strain Yakult and Bifidobacterium pseudocatenulatum DSM 20439. The effective strains produced a high concentration of acetic acid (56 mM) and lowered the pH of the intestine (to pH 6.75) compared to the infected control group (acetic acid concentration, 28 mM; pH, 7.15); these effects were thought to be related to the anti-infectious activity of these strains because the combination of a high concentration of acetic acid and a low pH was found to inhibit Stx production during STEC growth in vitro.     

Clinical heterogeneity of neonatal intrahepatic cholestasis caused by citrin deficiency: case reports from 16 patients

A deficiency of citrin, which is encoded by the SLC25A13 gene, causes both adult-onset type II citrullinemia (CTLN2) and neonatal intrahepatic cholestasis (NICCD). We analyzed 16 patients with NICCD to clarify the clinical features of the disease. Severe intrahepatic cholestasis with fatty liver was the most common symptom, but the accompanying clinical features were variable, namely; suspected cases of neonatal hepatitis or biliary atresia, positive results from newborn screening, tyrosinemia, failure to thrive, hemolytic anemia, bleeding tendencies and ketotic hypoglycemia. Laboratory data showed elevated serum bile acid levels, hypoproteinemia, low levels of vitamin K-dependent coagulation factors, and hypergalactosemia. Hypercitrullinemia was detected in 11 out of 15 patients examined. Most of the patients were given a lactose-free and/or medium chain triglycerides-enriched formula and lipid-soluble vitamins. The prognosis of the 16 patients is going fairy well at present, but we should observe these patients carefully to see if they manifest any symptom of CTLN2 in the future.     

Oncocytic renal cell carcinoma having papillotubular growth: rare morphological variant of papillary renal cell carcinoma

Herein is described a unique renal cell tumor with previously unreported morphological and immunohistochemical features. The patient was a 78-year-old Japanese man. A huge left renal tumor was found on ultrasound during evaluation of left abdominal distention. The tumor was macroscopically characterized by a non-infiltrative border, pale yellow to grayish color, foci of hemorrhage and partial edematous change. Histologically the tumor had an extensive small tubular growth pattern often with papillary fronds mainly composed of oncocytic cells with deeply eosinophilic granular cytoplasm. Clear vacuolated cells were scattered among the oncocytic cells. The present case had an unusual immunohistochemical profile for all known types of renal cell tumors, but both the oncocytic cells and the clear vacuolated cells were strongly immunoreactive for α-methylacyl-coenzyme A racemase. It is concluded that the tumor may be a candidate for a rare variant of papillary renal cell carcinoma. Further cases having similar features are awaited for a definitive classification of this tumor as a previously undescribed tumor type.     

β(1)-Adrenoceptor stimulation enhances the differentiation of mouse induced pluripotent stem cells into neural progenitor cells

The cyclic AMP/protein kinase A signaling pathway is thought to be involved in neural differentiation of mesenchymal stem cells. In the present study, we examined the involvement of β-adrenoceptor signaling on the differentiation of mouse induced pluripotent stem (iPS) cells into neural progenitor cells. Mouse iPS cells were cultured on ultra-low-attachment dishes to induce embryoid body (EB) formation. All-trans retinoic acid (ATRA, 1μM) and/or the β-adrenoceptor agonist l-isoproterenol (0.3 or 1μM) were added to the EB cultures for 4 days, then EBs were plated on gelatin-coated plates and cultured for 7 or 14 days. Subtype-specific antibody staining revealed that mouse iPS cells express β(1)-adrenoceptors predominantly. Although treatment with l-isoproterenol alone did not affect the expression of Nestin (a specific marker for neural progenitor cells), l-isoproterenol significantly enhanced ATRA-induced Nestin expression. Pretreatment of EBs with either atenolol (a selective β(1)-adrenoceptor antagonist) or H89 (a protein kinase A inhibitor) significantly inhibited the l-isoproterenol-enhancement of ATRA-induced Nestin expression. In addition, the l-isoproterenol treatment significantly enhanced ATRA-induced expression of NeuN (a neuron-specific nuclear protein). These findings suggest that β(1)-adrenoceptor stimulation enhances ATRA-induced neural differentiation of mouse iPS cells.     

Vaccine vectors derived from a large collection of simian adenoviruses induce potent cellular immunity across multiple species

Replication-defective adenovirus vectors based on human serotype 5 (Ad5) induce protective immune responses against diverse pathogens and cancer in animal models, as well as elicit robust and sustained cellular immunity in humans. However, most humans have neutralizing antibodies to Ad5, which can impair the immunological potency of such vaccines. Here, we show that rare serotypes of human adenoviruses, which should not be neutralized in most humans, are far less potent as vaccine vectors than Ad5 in mice and nonhuman primates, casting doubt on their potential efficacy in humans. To identify novel vaccine carriers suitable for vaccine delivery in humans, we isolated and sequenced more than 1000 adenovirus strains from chimpanzees (ChAd). Replication-defective vectors were generated from a subset of these ChAd serotypes and screened to determine whether they were neutralized by human sera and able to grow in human cell lines. We then ranked these ChAd vectors by immunological potency and found up to a thousandfold variation in potency for CD8+ T cell induction in mice. These ChAd vectors were safe and immunologically potent in phase 1 clinical trials, thereby validating our screening approach. These data suggest that the ChAd vectors developed here represent a large collection of non-cross-reactive, potent vectors that may be exploited for the development of new vaccines.     

Age-related dysfunction of the lacrimal gland and oxidative stress: evidence from the Cu,Zn-superoxide dismutase-1 (Sod1) knockout mice

An imbalance between free radical generation and radical scavenging antioxidant systems results in oxidative stress, which has been associated with cell injury observed in many age-related diseases. The superoxide dismutase (SOD) family is a major antioxidant system, and deficiency of Cu,Zn-superoxide dismutase-1 (Sod1) in mice leads to many different phenotypes that resemble accelerated aging. In this study we examined the morphologic features and the secretory functions of the lacrimal glands in Sod1(-/-) mice. Lacrimal glands showed atrophy of acinar units; fibrosis; infiltration with CD4(+) T cells, monocytes, and neutrophils; increased staining with both 4-hydroxy-2-nonenal and 8-hydroxy-2'-deoxyguanosine; increases in apoptotic cells; and the presence of the epithelial-mesenchymal transition in senescent Sod1(-/-) mice. Electron microscopy findings revealed evidence of epithelial-mesenchymal transition, presence of swollen and degenerated mitochondria, and the presence of apoptotic cell death in the lacrimal glands of senescent Sod1(-/-) mice. These alterations were also associated with the accumulation of secretory vesicles in acinar epithelial cells, decreased production of both stimulated and nonstimulated tears, and a decline in total protein secretion from the lacrimal glands. Our results suggest that Sod1(-/-) mice may be a good model system in which to study the mechanism of reactive oxygen species-mediated lacrimal gland alterations.