Identifying hypothermia death in a mouse model by ATR-FTIR.

The identification of hypothermia death (HD) is difficult for cadavers, especially the distinction from death due to alternative causes. A large number of studies have shown that brown adipose tissue (BAT) plays critical roles in thermoregulation of mammals. In this study, BAT of mice was used for the discrimination of HD using attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR). A modified mouse HD model conducted by Feeney DM was used in this study to obtain infrared spectra of BAT. Principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to establish discrimination models. The PLS-DA and OPLS-DA models exhibit prominent discriminative efficiency, and the accuracy of HD identification using fingerprint regions and ratios of absorption intensity is near 100% in both the calibration and validation sets. Our preliminary study suggests that BAT may be an extremely effective target tissue for identification of cadavers of HD, and ATR-FTIR spectra combined with chemometrics have also shown potential for cadaver identification in forensic practice in a fast and accurate manner.

One-Pot Cu/SAPO-34 for Continuous Methane Selective Oxidation to Methanol.

Cu/SAPO-34 synthesized via a one-pot method with relatively low silicon content and copper loading at around 2 wt.% facilitated continuous oxidation of methane to methanol with a methanol space time yield of 504 µmolCH3OH/gcat/h. Remarkably, the methanol yield exceeded 1800 mmolCH3OH/molCu/h at 623 K. Typically, the presence of trace oxygen in the system was the key to maintaining the high selectivity to methanol. Characterization results from a series of techniques, including XRD, SEM, TEM, H2-TPR, NH3-TPD, UV-vis, and FTIR, indicated that Cu2+ existed in the position where it moves from hexagonal rings to elliptical cages as the active center.

Clinical and biological heterogeneities in triple-negative breast cancer reveals a non-negligible role of HER2-low.

BACKGROUND: HER2-low could be found in some patients with triple-negative breast cancer (TNBC). However, its potential impacts on clinical features and tumor biological characteristics in TNBC remain unclear. METHODS: We enrolled 251 consecutive TNBC patients retrospectively, including 157 HER2-low (HER2low) and 94 HER2-negtive (HER2neg) patients to investigate the clinical and prognostic features. Then, we performed single-cell RNA sequencing (scRNA-seq) with another seven TNBC samples (HER2neg vs. HER2low, 4 vs. 3) prospectively to further explore the differences of tumor biological properties between the two TNBC phenotypes. The underlying molecular distinctions were also explored and then verified in the additional TNBC samples. RESULTS: Compared with HER2neg TNBC, HER2low TNBC patients exhibited malignant clinical features with larger tumor size (P = 0.04), more lymph nodes involvement (P = 0.02), higher histological grade of lesions (P < 0.001), higher Ki67 status (P < 0.01), and a worse prognosis (P < 0.001; HR [CI 95%] = 3.44 [2.10-5.62]). Cox proportional hazards analysis showed that neoadjuvant systemic therapy, lymph nodes involvement and Ki67 levels were prognostic factors in HER2low TNBC but not in HER2neg TNBC patients. ScRNA-seq revealed that HER2low TNBC which showed more metabolically active and aggressive hallmarks, while HER2neg TNBC exhibited signatures more involved in immune activities with higher expressions of immunoglobulin-related genes (IGHG1, IGHG4, IGKC, IGLC2); this was further confirmed by immunofluorescence in clinical TNBC samples. Furthermore, HER2low and HER2neg TNBC exhibited distinct tumor evolutionary characteristics. Moreover, HER2neg TNBC revealed a potentially more active immune microenvironment than HER2low TNBC, as evidenced by positively active regulation of macrophage polarization, abundant CD8+ effector T cells, enriched diversity of T-cell receptors and higher levels of immunotherapy-targeted markers, which contributed to achieve immunotherapeutic response. CONCLUSIONS: This study suggests that HER2low TNBC patients harbor more malignant clinical behavior and aggressive tumor biological properties than the HER2neg phenotype. The heterogeneity of HER2 may be a non-negligible factor in the clinical management of TNBC patients. Our data provide new insights into the development of a more refined classification and tailored therapeutic strategies for TNBC patients.

Conventional papillary thyroid carcinoma with intraglandular lymphatic dissemination shows more aggressive features.

OBJECTIVE: To investigate the invasive capability and other clinicopathological features of conventional papillary thyroid carcinoma (CVPTC) with intraglandular lymphatic dissemination. METHODS: Seventy-three conventional papillary thyroid carcinoma patients receiving total thyroidectomy were analyzed in this study. The expression of BRAF-V600E, D2-40 and CD31 in all thyroid samples was detected by immunohistochemical staining. The results were evaluated by two pathologists and were statistically analyzed. The rate of positive BRAF-V600E expression and the clinical invasiveness of CVPTC with intraglandular dissemination, multifocal non-intraglandular dissemination-CVPTC and single focus-CVPTC were evaluated. The correlation between BRAF-V600E expression, lymphatic vessel density, microvessel density and the clinicopathological characteristics of conventional papillary thyroid carcinoma were assessed. RESULTS: Twenty-five intraglandular dissemination-CVPTC, 17 multifocal non-intraglandular dissemination-CVPTC and 31 single focus-CVPTC cases were included in this study. The results showed that BRAF-V600E expression was independently correlated with intraglandular dissemination, age and pN staging (P < 0.05). The lymphatic vessel density in the intraglandular dissemination-CVPTC group was higher than that in the non-intraglandular dissemination-CVPTC group (P < 0.05). Compared with cases without intraglandular dissemination, intraglandular dissemination-CVPTC was associated with a younger age, higher lymph node metastasis rate, pN staging, the expression of BRAF-V600E and increased Capsule invasion and lymphovascular tumor thrombus (P < 0.05). During the follow-up of 30 months (median 15 months), two patients in the intraglandular dissemination-CVPTC group had cervical lymph node metastasis after the first operation. CONCLUSIONS: Intraglandular dissemination-CVPTC shows more aggressive features, and intraglandular lymphatic dissemination may be a potential biological indicator of poor prognosis.

Lipid emulsion-induced hypertension post resection of pediatric neuroblastoma: a case report and literature review.

BACKGROUND: Parenteral Nutrition (PN) is preferred when patient is unable to eat. Most clinically widely used lipid emulsion is now attracting more attention in its stability and adverse reactions. We report here the first case of lipid emulsions caused hypertension. CASE PRESENTATION: A 1.5 years old girl was diagnosed with neuroblastoma and underwent chemotherapy subsequently followed by resection surgery. She received PN for nutritional support after surgery. with the initiation of PN, this patient developed hypertension. Possible causes of hypertension were evaluated. After the discontinuation of lipid emulsions in PN, her hypertensive symptoms ceased. The lipid emulsion was therefore considered as the cause of her hypertension. CONCLUSIONS: The pathogenesis of hypertension caused by fatty milk is possibly associated with increased production of reactive oxygen species, increased oxidative stress and vasoconstriction.

Turkey adenovirus 3, a siadenovirus, uses sialic acid on N-linked glycoproteins as a cellular receptor.

Turkey adenovirus 3 (TAdV-3) is the causative agent of an immune-mediated disease in turkeys, haemorrhagic enteritis, through targeting B lymphocytes. In the present study, we investigated the role of sialic acid in TAdV-3 entry and characterized the structural components of TAdV-3 receptor(s) on RP19, B lymphoblastoid cells. Removal of the cell-surface sialic acids by neuraminidases or blocking of sialic acids by wheat germ agglutinin lectin reduced virus infection. Pre-incubation of cells with Maackia amurensis lectin or Sambucus nigra agglutinin resulted in virus reduction, suggesting that TAdV-3 uses both α2,3-linked and α2,6-linked sialic acids as attachment receptor. Virus infectivity data from RP19 cells treated with sodium periodate, proteases (trypsin or bromelain) or metabolic inhibitors (dl-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, tunicamycin, or benzyl N-acetyl-α-d-galactosaminide) indicated that N-linked, but not O-linked, carbohydrates are part of the sialylated receptor and they are likely based on a membrane glycoprotein, rather than a glycolipid. Furthermore, our data, in conjunction with previous findings, implies that the secondary receptor for TAdV-3 is a protein molecule since the inhibition of glycolipid biosynthesis did not affect the virus infection, which was rather reduced by protease treatment. We can conclude that terminal sialic acids attached to N-linked membrane glycoproteins on B cells are used for virus attachment and are essential for successful virus infection.

Pig model mimicking chronic hepatitis E virus infection in immunocompromised patients to assess immune correlates during chronicity.

Chronic hepatitis E virus (HEV) infection is a significant clinical problem in immunocompromised individuals such as organ transplant recipients, although the mechanism remains unknown because of the lack of an animal model. We successfully developed a pig model of chronic HEV infection and examined immune correlates leading to chronicity. The conditions of immunocompromised patients were mimicked by treating pigs with an immunosuppressive regimen including cyclosporine, azathioprine, and prednisolone. Immunocompromised pigs infected with HEV progressed to chronicity, because 8/10 drug-treated HEV-infected pigs continued fecal virus shedding beyond the acute phase of infection, whereas the majority (7/10) of mock-treated HEV-infected pigs cleared fecal viral shedding at 8 wk postinfection. During chronic infection, serum levels of the liver enzyme γ-glutamyl transferase and fecal virus shedding were significantly higher in immunocompromised HEV-infected pigs. To identify potential immune correlates of chronic infection, we determined serum levels of cytokines and cell-mediated immune responses in pigs. Results showed that HEV infection of immunocompromised pigs reduced the serum levels of Th1 cytokines IL-2 and IL-12, and Th2 cytokines IL-4 and IL-10, particularly during the acute phase of infection. Furthermore IFN-γ-specific CD4+ T-cell responses were reduced in immunocompromised pigs during the acute phase of infection, but TNF-α-specific CD8+ T-cell responses increased during the chronic phase of infection. Thus, active suppression of cell-mediated immune responses under immunocompromised conditions may facilitate the establishment of chronic HEV infection. This pig model will aid in delineating the mechanisms of chronic HEV infection and in developing effective therapeutics against chronic hepatitis E.

Enhanced GII.4 human norovirus infection in gnotobiotic pigs transplanted with a human gut microbiota.

The role of commensal microbiota in enteric viral infections has been explored extensively, but the interaction between human gut microbiota (HGM) and human norovirus (HuNoV) is poorly understood. In this study, we established an HGM-Transplanted gnotobiotic (Gn) pig model of HuNoV infection and disease, using an infant stool as HGM transplant and a HuNoV GII.4/2006b strain for virus inoculation. Compared to germ-free Gn pigs, HuNoV inoculation in HGMT Gn pigs resulted in increased HuNoV shedding, characterized by significantly higher shedding titres on post inoculation day (PID) 3, 4, 6, 8 and 9, and significantly longer mean duration of virus shedding. In addition, virus titres were significantly higher in duodenum and distal ileum of HGMT Gn pigs on PID10, while comparable and transient HuNoV viremia was detected in both groups. 16S rRNA gene sequencing demonstrated that HuNoV infection dramatically altered intestinal microbiota in HGMT Gn pigs at the phylum (Proteobacteria, Firmicutes and Bacteroidetes) and genus (Enterococcus, Bifidobacterium, Clostridium, Ruminococcus, Anaerococcus, Bacteroides and Lactobacillus) levels. In summary, enhanced GII.4 HuNoV infection was observed in the presence of HGM, and host microbiota was susceptible to disruption upon HuNoV infection.

Infection Dynamics of Hepatitis E Virus in Wild-Type and Immunoglobulin Heavy Chain Knockout JH-/- Gnotobiotic Piglets.

Hepatitis E virus (HEV), the causative agent of hepatitis E, is an important but incompletely understood pathogen causing high mortality during pregnancy and leading to chronic hepatitis in immunocompromised individuals. The underlying mechanisms leading to hepatic damage remain unknown; however, the humoral immune response is implicated. In this study, immunoglobulin (Ig) heavy chain JH-/- knockout gnotobiotic pigs were generated using CRISPR/Cas9 technology to deplete the B-lymphocyte population, resulting in an inability to generate a humoral immune response to genotype 3 HEV infection. Compared to wild-type gnotobiotic piglets, the frequencies of B lymphocytes in the Ig heavy chain JH-/- knockouts were significantly lower, despite similar levels of other innate and adaptive T-lymphocyte cell populations. The dynamic of acute HEV infection was subsequently determined in heavy chain JH-/- knockout and wild-type gnotobiotic pigs. The data showed that wild-type piglets had higher viral RNA loads in feces and sera compared to the JH-/- knockout pigs, suggesting that the Ig heavy chain JH-/- knockout in pigs actually decreased the level of HEV replication. Both HEV-infected wild-type and JH-/- knockout gnotobiotic piglets developed more pronounced lymphoplasmacytic hepatitis and hepatocellular necrosis lesions than other studies with conventional pigs. The HEV-infected JH-/- knockout pigs also had significantly enlarged livers both grossly and as a ratio of liver/body weight compared to phosphate-buffered saline-inoculated groups. This novel gnotobiotic pig model will aid in future studies into HEV pathogenicity, an aspect which has thus far been difficult to reproduce in the available animal model systems.IMPORTANCE According to the World Health Organization, approximately 20 million HEV infections occur annually, resulting in 3.3 million cases of hepatitis E and >44,000 deaths. The lack of an efficient animal model that can mimic the full-spectrum of infection outcomes hinders our ability to delineate the mechanism of HEV pathogenesis. Here, we successfully generated immunoglobulin heavy chain JH-/- knockout gnotobiotic pigs using CRISPR/Cas9 technology, established a novel JH-/- knockout and wild-type gnotobiotic pig model for HEV, and systematically determined the dynamic of acute HEV infection in gnotobiotic pigs. It was demonstrated that knockout of the Ig heavy chain in pigs decreased the level of HEV replication. Infected wild-type and JH-/- knockout gnotobiotic piglets developed more pronounced HEV-specific lesions than other studies using conventional pigs, and the infected JH-/- knockout pigs had significantly enlarged livers. The availability of this novel model will facilitate future studies of HEV pathogenicity.

Identification and Characterization of a Luteinizing Hormone Receptor (LHR) Homolog from the Chinese Mitten Crab Eriocheir sinensis.

Luteinizing hormone (LH), a pituitary gonadotropin, coupled with LH receptor (LHR) is essential for the regulation of the gonadal maturation in vertebrates. Although LH homolog has been detected by immunocytochemical analysis, and its possible role in ovarian maturation was revealed in decapod crustacean, so far there is no molecular evidence for the existence of LHR. In this study, we cloned a novel LHR homolog (named EsLHR) from the Chinese mitten crab Eriocheir sinensis. The complete sequence of the EsLHR cDNA was 2775bp, encoding a protein of 924 amino acids, sharing 71% amino acids identity with the ant Zootermopsis nevadensis LHR. EsLHR expression was found to be high in the ovary, while low in testis, gill, brain, and heart, and no expression in the thoracic ganglion, eye stalk, muscle, and hepatopancreas. Quantitative PCR revealed that the expression level of EsLHR mRNA was significantly higher in the ovaries in previtellogenic (Pvt), late vitellogenic (Lvt), and germinal vesicle breakdown (GVBD) stages than that in the vitellogenic (Mvt) and early vitellogenic (Evt) stages (P < 0.05), and, the highest and the lowest expression were in Lvt, and Evt, respectively. The strong signal was mainly localized in the ooplasm of Pvt oocyte as detected by in situ hybridization. The crab GnRH homolog can significantly induce the expression of EsLHR mRNA at 36 hours post injection in vivo (P < 0.01), suggesting that EsLHR may be involved in regulating ovarian development through GnRH signaling pathway in the mitten crab.

[Detection and analysis of an ATP2A2 mutation in a family with Darier-White disease].

OBJECTIVE: To explore the molecular basis for a pedigree affected with Darier-White disease. METHODS: Genomic DNA was isolated from 3 patients and 1 unaffected member from the pedigree, as well as 80 healthy controls. Targeted sequence capture and next-generation sequencing were used to screen mutations of skin disease-related genes. Candidate mutations were verified by Sanger sequencing, and co-segregation analysis was carried out to confirm the pathogenicity of mutation. Conservation analysis and protein structure and function were also predicted with Bioinformatic tools. RESULTS: A heterozygous mutation c.2246G>T (p.G749V) was identified in exon 15 of ATP2A2 gene in all 3 patients from the pedigree, but not in the unaffected member or 80 healthy controls. The corresponding amino acid was highly conserved, and mutation of which can lead to structural and functional changes of the protein. CONCLUSION: The c.2246G>T missense mutation of the ATP2A2 gene probably underlies the Darier-White disease in this pedigree by causing damages to the structure and function of sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2).

Two novel CPS1 mutations in a case of carbamoyl phosphate synthetase 1 deficiency causing hyperammonemia and leukodystrophy.

BACKGROUND: Carbamoyl phosphate synthetase 1 deficiency (CPS1D) is a rare autosomal recessive disorder of the urea cycle, mostly characterized by hyperammonemia and the concomitant leukodystrophy. The onset of CPS1D can be at any age, and the clinical manifestations are variable and atypical. Genetic tests are indispensable for accurate diagnosis of CPS1D on the basis of biochemical tests. METHODS: Blood tandem mass spectrometric analysis and urea organic acidemia screening were performed on a Chinese neonatal patient with low activity, recurrent seizures, and hyperammonemia. Next-generation sequencing and Sanger sequencing were followed up for making a definite diagnosis. Bioinformatics tools were used for the conservation analysis and pathogenicity predictions of the identified mutations. RESULTS: Increased lactate in urea and decreased citrulline in blood were detected in the patient. Two novel mutations (c.173G>T, p.G58V in exon 2 and c.796G>A, p.G266R in exon 8) in CPS1 identified in the neonatal patient were found through coseparation verification. Both of the two mutations were predicted to be deleterious, and the two relevant amino acids exerted highly evolutionarily conserved. The final diagnosis of the patient was compound heterozygous CPS1D. CONCLUSION: This study described the specific clinical characteristics and the variations of physiological and biochemical indices in a Chinese neonatal patient with CPS1D, which facilitated the diagnosis and mechanism research of the disease. Two novel causative missense mutations were identified, which enriched the mutation spectrum of CPS1D in China and worldwide. Advice of prenatal diagnosis was given to the family for a new pregnancy.

Lactobacillus rhamnosus GG modulates innate signaling pathway and cytokine responses to rotavirus vaccine in intestinal mononuclear cells of gnotobiotic pigs transplanted with human gut microbiota.

BACKGROUND: A better understanding of mechanisms underlying dose-effects of probiotics in their applications as treatments of intestinal infectious or inflammatory diseases and as vaccine adjuvant is needed. In this study, we evaluated the modulatory effects of Lactobacillus rhamnosus GG (LGG) on transplanted human gut microbiota (HGM) and on small intestinal immune cell signaling pathways in gnotobiotic pigs vaccinated with an oral attenuated human rotavirus (AttHRV) vaccine. RESULTS: Neonatal HGM transplanted pigs were given two doses of AttHRV on 5 and 15 days of age and were divided into three groups: none-LGG (AttHRV), 9-doses LGG (AttHRV + LGG9X), and 14-doses LGG (AttHRV + LGG14X) (n = 3-4). At post-AttHRV-inoculation day 28, all pigs were euthanized and intestinal contents and ileal tissue and mononuclear cells (MNC) were collected. AttHRV + LGG14X pigs had significantly increased LGG titers in the large intestinal contents and shifted structure of the microbiota as indicated by the formation of a cluster that is separated from the cluster formed by the AttHRV and AttHRV + LGG9X pigs. The increase in LGG titers concurred with significantly increased ileal HRV-specific IFN-γ producing T cell responses to the AttHRV vaccine reported in our previous publication, suggesting pro-Th1 adjuvant effects of the LGG. Both 9- and 14-doses LGG fed pig groups had significantly higher IkBα level and p-p38/p38 ratio, while significantly lower p-ERK/ERK ratio than the AttHRV pigs, suggesting activation of regulatory signals during immune activation. However, 9-doses, but not 14-doses LGG fed pigs had enhanced IL-6, IL-10, TNF-α, TLR9 mRNA levels, and p38 MAPK and ERK expressions in ileal MNC. Increased TLR9 mRNA was in parallel with higher mRNA levels of cytokines, p-NF-kB and higher p-p38/p38 ratio in MNC of the AttHRV + LGG9X pigs. CONCLUSIONS: The relationship between modulation of gut microbiota and regulation of host immunity by different doses of probiotics is complex. LGG exerted divergent dose-dependent effects on the intestinal immune cell signaling pathway responses, with 9-doses LGG being more effective in activating the innate immunostimulating TLR9 signaling pathway than 14-doses in the HGM pigs vaccinated with AttHRV.

Intranasal P particle vaccine provided partial cross-variant protection against human GII.4 norovirus diarrhea in gnotobiotic pigs.

UNLABELLED: Noroviruses (NoVs) are the leading cause of nonbacterial acute gastroenteritis worldwide in people of all ages. The P particle is a novel vaccine candidate derived from the protruding (P) domain of the NoV VP1 capsid protein. This study utilized the neonatal gnotobiotic pig model to evaluate the protective efficacies of primary infection, P particles, and virus-like particles (VLPs) against NoV infection and disease and the T cell responses to these treatments. Pigs either were vaccinated intranasally with GII.4/1997 NoV (VA387)-derived P particles or VLPs or were inoculated orally with a GII.4/2006b NoV variant. At postinoculation day (PID) 28, pigs either were euthanized or were challenged with the GII.4/2006b variant and monitored for diarrhea and virus shedding for 7 days. The T cell responses in intestinal and systemic lymphoid tissues were examined. Primary NoV infection provided 83% homologous protection against diarrhea and 49% homologous protection against virus shedding, while the P particle and VLP vaccines provided cross-variant protection (47% and 60%, respectively) against diarrhea. The protection rates against diarrhea are significantly inversely correlated with T cell expansion in the duodenum and are positively correlated with T cell expansion in the ileum and spleen. The P particle vaccine primed for stronger immune responses than VLPs, including significantly higher numbers of activated CD4+ T cells in all tissues, gamma interferon-producing (IFN-γ+) CD8+ T cells in the duodenum, regulatory T cells (Tregs) in the blood, and transforming growth factor ß (TGF-ß)-producing CD4+ CD25- FoxP3+ Tregs in the spleen postchallenge, indicating that P particles are more immunogenic than VLPs at the same dose. In conclusion, the P particle vaccine is a promising vaccine candidate worthy of further development. IMPORTANCE: The norovirus (NoV) P particle is a vaccine candidate derived from the protruding (P) domain of the NoV VP1 capsid protein. P particles can be easily produced in Escherichia coli at high yields and thus may be more economically viable than the virus-like particle (VLP) vaccine. This study demonstrated, for the first time, the cross-variant protection (46.7%) of the intranasal P particle vaccine against human NoV diarrhea and revealed in detail the intestinal and systemic T cell responses by using the gnotobiotic pig model. The cross-variant protective efficacy of the P particle vaccine was comparable to that of the VLP vaccine in pigs (60%) and to the homologous protective efficacy of the VLP vaccine in humans (47%). NoV is now the leading cause of pediatric dehydrating diarrhea, responsible for approximately 1 million hospital visits for U.S. children and 218,000 deaths in developing countries. The P particle vaccine holds promise for reducing the disease burden and mortality.

Highly selective colorimetric bacteria sensing based on protein-capped nanoparticles.

A rapid and cost-effective colorimetric sensor has been developed for the detection of bacteria (Bacillus subtilis was selected as an example). The sensor was designed to rely on lysozyme-capped AuNPs with the advantages of effective amplification and high specificity. In the sensing system, lysozyme was able to bind strongly to Bacillus subtilis, which effectively induced a color change of the solution from light purple to purplish red. The lowest concentration of Bacillus subtilis detectable by the naked eye was 4.5 × 10(3) colony-forming units (CFU) mL(-1). Similar results were discernable from UV-Vis absorption measurements. A good specificity was observed through a statistical analysis method using the SPSS software (version 17.0). This simple colorimetric sensor may therefore be a rapid and specific method for a bacterial detection assay in complex samples.

Lactobacillus rhamnosus GG Dosage Affects the Adjuvanticity and Protection Against Rotavirus Diarrhea in Gnotobiotic Pigs.

OBJECTIVES: The use of immunostimulatory strains of probiotics as adjuvants has been increasingly recognized as a promising approach in enhancing vaccine immunogenicity; however, dose effects of probiotic adjuvants are not well defined. In the present study, we examined dose effects of a commonly used probiotic strain, Lactobacillus rhamnosus GG (LGG), on immunomodulation with 2 different dosages. METHODS: Neonatal gnotobiotic pigs were inoculated with 2 oral doses of attenuated human rotavirus (AttHRV) vaccines and fed with 5 doses (LGG5X; total 2.1 × 10(6) colony-forming units) or 9 doses (LGG9X; total 3.2 × 10(6) colony-forming units) of LGG, starting at 3 days of age. RESULTS: Both LGG feeding regimens enhanced the protection rate of AttHRV vaccine against diarrhea on virulent human rotavirus challenge. LGG5X, but not LGG9X, significantly enhanced rotavirus-specific intestinal memory B-cell responses to AttHRV; LGG5X also significantly enhanced virus-specific intestinal immunoglobulin A (IgA) antibody-secreting cell responses. Both regimens significantly enhanced rotavirus-specific serum IgA antibody responses to AttHRV. They also enhanced rotavirus-specific interferon-γ-producing effector/memory T-cell responses to AttHRV vaccine, with LGG9X being more effective than LGG5X, and both regimens downregulated CD4+CD25-FoxP3+ regulatory T (Treg) cell responses in most lymphoid tissues examined prechallenge and postchallenge and maintained the CD4+CD25+FoxP3+ Treg population in the ileum and intraepithelial lymphocyte postchallenge. LGG9X, however, did not significantly reduce total CD4+CD25-FoxP3+ Treg frequencies in the intestine and transforming growth factor-ß-producing and interleukin (IL)-10-producing Treg frequencies in the blood. CONCLUSIONS: These results indicate that LGG at both dosages functioned as effective probiotic adjuvant for AttHRV vaccine, but different dosages differentially modulated immune responses to favor either the mucosal IgA response (LGG5X) or the T-cell response (LGG9X).

Administration of hydrogen-rich saline in mice with allogeneic hematopoietic stem-cell transplantation.

BACKGROUND: Hydrogen, as a novel antioxidant, has been shown to selectively reduce the level of hydroxyl radicals and alleviate acute oxidative stress in many animal experiments. Hydrogen-rich saline provides a high concentration of hydrogen that can be easily and safely applied. Allogeneic hematopoietic stem-cell transplantation (HSCT) has been the most curative therapy for hematological malignancies. However, acute graft-versus-host disease (aGVHD) is the main cause of death in post-transplantation patients. In this study, we examined whether hydrogen-rich saline would show favorable effects on acute GVHD in mice. MATERIAL AND METHODS: After lethal irradiation, BALB/c mice received bone marrow transplantation from C57BL/6 mice. Hydrogen-rich saline (5 ml/kg) was given to recipient mice in the hydrogen group once a day by intraperitoneal injection, and saline (5 ml/kg) was given to recipient mice in the saline group. Survival rates were monitored, clinical and pathological scores of aGVHD were determined after bone marrow transplantation (BMT), and the serum cytokine levels were examined on the 7th day after BMT. RESULTS: This study proves that hydrogen-rich saline increased the survival rate, reduced clinical and histopathological scores of aGVHD, promoted the recovery of white blood cells, reduced the serum cytokine levels, and reversed tissue damage after transplantation in mice. CONCLUSIONS: Hydrogen has potential as an effective and safe therapeutic agent in aGVHD.

Development of enzyme-linked immunosorbent assay for determination of polybrominated diphenyl ether BDE-121.

Our interests are in the development of immunoassay-based fast scanning methods for persistent organic pollutants. To develop the immunoassay method of polybrominated diphenyl ether (PBDE), a model compound of PBDE, 2,3',4,5',6-pentabromodiphenylether (BDE-121), has been chosen to develop its antibody and the competitive indirect enzyme-linked immunosorbent assay (ELISA) is developed. The hapten of BDE-121 containing reactive carboxylic acid was synthesized and conjugated to carrier proteins (bovine serum albumin [BSA] and ovalbumin [OVA]). Anti-BDE-121 polyclonal antibody was then developed in rabbits as a result of immunization with the BDE-121-BSA conjugate. The optimal amount of coating antigen BDE-121-OVA conjugate and the dilution of antiserum needed in the ELISA were determined with the checkerboard method, and the effects of the properties of PBST (phosphate-buffered saline and Tween 20) buffer (pH and salt concentration) and chemical solvent (types and concentrations) on the ELISA were investigated to achieve a rapid robust assay with high sensitivity. Under the optimized conditions, the developed indirect ELISA shows a linear detection range from 1.74 to 84.1 ng/ml, with an IC50 value of 8.07 ng/ml and a detection limit of 0.644 ng/ml. In total, 11 kinds of compounds were tested for calculating the cross-reactivity, which was less than 8% for nearly all of them. Real samples were analyzed by the proposed immunoassay and gas chromatography/mass spectrometry (GC/MS).

Dual functions of Lactobacillus acidophilus NCFM as protection against rotavirus diarrhea.

OBJECTIVE: The aim of the study was to examine the dose effects of Lactobacillus acidophilus (LA) NCFM strain on rotavirus-specific antibody and B-cell responses in gnotobiotic pigs vaccinated with an oral attenuated human rotavirus (AttHRV). METHODS: Pigs were inoculated with AttHRV vaccine in conjunction with high-dose LA (14 doses, total 2.2 × 10(6) colony-forming units [CFU]), intermediate-dose LA (MidLA) (9 doses, total 3.2 × 10(9) CFU), low-dose LA (LoLA) (5 doses, total 2.1 × 10(6) CFU), or without LA feeding. Protection against rotavirus shedding and diarrhea was assessed upon challenge with a virulent HRV. Rotavirus-specific immunoglobulin A (IgA) and IgG antibodies in serum and rotavirus-specific IgA and IgG antibody-secreting cells (ASCs) and memory B cells in ileum, spleen, and blood of the pigs were measured and compared among treatment groups. RESULTS: The MidLA, but not high-dose LA or LoLA, significantly reduced rotavirus diarrhea (MidLA-only group) and significantly improved the protection conferred by AttHRV vaccine (MidLA + AttHRV group). Associated with the increased protection, MidLA significantly enhanced rotavirus-specific antibody, ASCs, and memory B-cell responses to AttHRV vaccine. High-dose LA or LoLA did not enhance virus-specific antibody and ASC responses, and hence did not improve the vaccine efficacy. CONCLUSIONS: These findings highlight the importance of dose selection and indicate that certain specific lactobacilli strains at the appropriate dose have the dual function of reducing rotavirus diarrhea and enhancing the immunogenicity and protective efficacy of rotavirus vaccines.

Serum LINC00339 is a promising biomarker for prognosis prediction of nasopharyngeal carcinoma.

BACKGROUND: Long non-coding RNAs (lncRNAs) in serum were useful and promising biomarkers for diagnostic and prognostic application. Herein, we investigated the serum lncRNA LINC00339 expression and its role in nasopharyngeal carcinoma. METHODS: In this study, we recruited a cohort of 129 nasopharyngeal carcinoma patients, 68 patients with nasopharyngitis, and 80 healthy controls. Serum LINC00339 levels were measured by reverse transcription-quantitative polymerase chain reaction. Receiver operating characteristic (ROC) and Kaplan-Meier curve analyses were conducted to evaluate th e clinical role of LINC00339. The effects of linc00339 on cellular activities were measured using CCK-8 and Transwell assays. RESULTS: We observed that serum LINC00339 expression was upregulated in nasopharyngeal carcinoma patients and closely associated with tumor node metastasis stage, lymph node metastasis, and overall survival rate. Meanwhile, ROC analysis showed serum LINC00339 had diagnostic value to distinguish nasopharyngeal carcinoma patients from healthy individuals and nasopharyngitis patients. Silencing of LINC00339 could repress cellular behaviors by targeting miR-152. CONCLUSION: This study clarified that LINC00339 was upregulated in nasopharyngeal carcinoma, and that serum LINC00339 may act as a diagnostic or prognostic marker, and a hopeful therapeutic target for patients with nasopharyngeal carcinoma.