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Objective: The occurrence of chickenpox in rapidly developing areas poses substantial seasonal risk to children. However, certain factors influencing local chickenpox outbreaks have not been studied. Here, we examined the relationship between spatial clustering, heterogeneity of chickenpox outbreaks, and socioeconomic factors in Southern China., Methods: We assessed chickenpox outbreak data from Southern China between 2006 and 2021, comprising both relatively fast-growing parts and slower sub-regions, and provides a representative sample of many developing regions. We analyzed the spatial clustering attributes associated with chickenpox outbreaks using Moran's I and local indicators of spatial association and quantified their socioeconomic determinants using Geodetector q statistics., Results: There were significant spatial heterogeneity in the risk of chickenpox outbreaks, with strong correlations between chickenpox risk and various factors, particularly demographics and living environment. Furthermore, interactive effects among specific are factors, such as population density and per capita residential building area, percentage of households with toilets, percentage of rental housing, exhibited q statistics of 0.28, 0.25, and 0.24, respectively., Conclusion: This study provides valuable insights into the spatial dynamics of chickenpox outbreaks in rapidly developing regions, revealing the socioeconomic factors affecting disease transmission. These implications extend the formulation of effective public health strategies and interventions to prevent and control chickenpox outbreaks in similar global contexts., (Copyright © 2024 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.)

The current study tested the expression and potential functions of Gαi1 in nasopharyngeal carcinoma (NPC). The Cancer Genome Atlas (TCGA) database results demonstrate that Gαi1 transcripts' number in NPC tissues is significantly higher than that in the normal nasal epithelial tissues. Its overexpression correlates with poor survival in certain NPC patients. Moreover, Gαi1 is significantly upregulated in NPC tissues of local primary patients and in different primary human NPC cells. Whereas its expression is relatively low in cancer-surrounding normal tissues and in primary nasal epithelial cells. Genetic silencing (via shRNA strategy) or knockout (via CRISPR-sgRNA method) of Gαi1 substantially suppressed viability, proliferation, cell cycle progression, and migration in primary NPC cells, causing significant caspase-apoptosis activation. Contrarily, ectopic Gαi1 expression exerted pro-tumorigenic activity and strengthened cell proliferation and migration in primary NPC cells. Gαi1 is important for Akt-mTOR activation in NPC cells. Akt-S6K phosphorylation was downregulated after Gαi1 shRNA or KO in primary NPC cells, but strengthened following Gαi1 overexpression. In Gαi1-silenced primary NPC cells, a S473D constitutively-active mutant Akt1 (caAkt1) restored Akt-S6K phosphorylation and ameliorated Gαi1 shRNA-induced proliferation inhibition, migration reduction and apoptosis. Bioinformatics analyses proposed zinc finger protein 384 (ZNF384) as a potential transcription factor of Gαi1. In primary NPC cells, ZNF384 shRNA or knockout (via CRISPR-sgRNA method) decreased Gαi1 mRNA and protein expression, whereas ZNF384 overexpression upregulated it. Importantly, there was an increased binding between ZNF384 protein and the Gαi1 promoter in human NPC tissues and different NPC cells. In vivo studies showed that intratumoral injection of Gαi1-shRNA-expressing adeno-associated virus (AAV) impeded subcutaneous NPC xenograft growth in nude mice. Gαi1 downregulation, Akt-mTOR inactivation, and apoptosis induction were detected in Gαi1-silenced NPC xenograft tissues. Gαi1 KO also effectively inhibited the growth of NPC xenografts in nude mice. Together, overexpressed Gαi1 exerts pro-tumorigenic activity in NPC possibly by promoting Akt-mTOR activation., (© 2023. The Author(s).)

Here we studied expression and potential functions of Gαi3 in cervical cancer. The bioinformatics analysis together with the results from local patients' tissues revealed that Gαi3 expression was remarkably elevated in human cervical cancer tissues and different cervical cancer cells, and was associated with poor overall survival and poor disease-specific survival of patients. Gαi3 depletion resulted in profound anti-cervical cancer activity. In primary or immortalized cervical cancer cells, Gαi3 shRNA or CRISPR/Cas9-caused Gαi3 knockout/KO largely hindered cell proliferation and migration, and provoked apoptosis. On the contrast, ectopic Gαi3 overexpression further enhanced cervical cancer proliferation and migration. Akt-mTOR activation in primary cervical cancer cells was significantly reduced after Gαi3 silencing or KO, but was augmented following Gαi3 overexpression. Further studies revealed that the transcription factor GATA4 binding to Gαi3 promoter region was significantly enhanced in cervical cancer tissues and cells. Gαi3 expression was decreased by GATA4 shRNA, but upregulated following GATA4 overexpression. In vivo , the growth of cervical cancer xenografts was robustly suppressed after Gαi3 silencing or KO. Gαi3 depletion and Akt-mTOR inactivation were detected in Gαi3-silenced/-KO cervical cancer xenograft tissues. Together, upregulated Gαi3 is a valuable oncotarget of cervical cancer., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Introduction: Obesity and diabetes are characterized by chronic inflammatory responses. Roux-en-Y gastric bypass (RYGB) is increasingly regarded as an effective approach for the improvement of glucose homeostasis. In this study, we examined the effects of RYGB on the regulation of early inflammatory responses in the liver and adipose tissue in high-fat diet (HFD)-induced obese (DIO) mice., Materials and Methods: RYGB was performed in DIO mice followed by analyses of adiposity, insulin sensitivity, plasma and tissue cytokines and adipokines, tissue NF-κB and JNK/c-Jun activation, and tissue macrophage and T-cell subsets., Results: We found that RYGB resulted in sustained improvement of adiposity and insulin sensitivity. Plasma insulin and leptin levels were increased in untreated DIO mice and reduced in RYGB mice. RYGB maintained plasma adiponectin levels and inhibited monocyte chemoattractant protein-1 and interleukin 6 in white adipose tissue (WAT) and liver. RYGB inhibited NF-κB activation in WAT and muscle, but not in the liver. However, RYGB attenuated the JNK/c-Jun signaling pathway in the liver and WAT at 1 wk after surgery, suggesting that RYGB regulates the tissue-specific inflammatory pathway. RYGB reduced M1-like (F4/80 + /CD11c + ) differentiation and enhanced M2-like population (F4/80 + /CD206c + ). RYGB also regulated CD4 + and CD8 + T-cell infiltration and increased T reg cells in the liver and WAT at the same time point., Conclusions: Our findings demonstrate that RYGB improves obesity and insulin resistance, which are associated with the regulation of early inflammatory reactions in the liver and WAT., (Copyright © 2022 Elsevier Inc. All rights reserved.)

It is urgent to identify and validate biomarkers for early diagnosis and efficient treatment of nasopharyngeal carcinoma (NPC). Recent studies have proposed p38 gamma (p38γ) as a cyclin-dependent kinase (CDK)-like kinase that phosphorylates retinoblastoma (Rb) to promote cyclins expression and tumorigenesis. Here the Gene Expression Profiling Interactive Analysis (GEPIA) database and results from the local NPC tissues demonstrate that p38γ is significantly upregulated in NPC tissues, correlating with poor overall survival. Furthermore, p38γ mRNA and protein expression is elevated in established NPC cell lines (CNE-1 HONE-1 and CNE-2) and primary human NPC cells, but low expression detected in human nasal epithelial cells. In established and primary NPC cells, p38γ depletion, using the shRNA strategy or the CRISPR/Cas9 gene-editing method, largely inhibited cell growth, proliferation and migration, and induced significant apoptosis activation. Contrarily, ectopic p38γ overexpression exerted opposite activity and promoted NPC cell proliferation and migration. Retinoblastoma (Rb) phosphorylation and cyclin E1/A expression were decreased in NPC cells with p38γ silencing or knockout, but increased after p38γ overexpression. Moreover, mitochondrial subcellular p38γ localization was detected in NPC cells. Significantly, p38γ depletion disrupted mitochondrial functions, causing mitochondrial depolarization, reactive oxygen species production, oxidative injury and ATP depletion in NPC cells. In vivo, intratumoral injection of adeno-associated virus-packed p38γ shRNA potently inhibited primary human NPC xenograft growth in nude mice. In p38γ shRNA virus-injected NPC xenograft tissues, p38γ expression, Rb phosphorylation, cyclin E1/A expression and ATP levels were dramatically decreased. Taken together, we conclude that p38γ overexpression is required for NPC cell growth, acting as a promising therapeutic target of NPC., (© 2022. The Author(s).)

Obese subjects have increase probabilities of developing type 2 diabetes (T2D). In this study, we sought to determine whether gastric bypass prevents the progression of prediabetes to overt diabetes in genetically modified mice and chemically induced diabetic mice. Roux-en-Y gastric bypass (RYGB) was performed in C57BL/KsJ-db/db null (BKS-db/db,) mice, high-fat diet (HFD)-fed NONcNZO10/LtJ (NZO) mice, C57BL/6 db/db null (B6-db/db) mice and streptozotocin (STZ)-induced diabetic mice. Food consumption, body weight, fat mass, fast blood glucose level, circulating insulin and adiponectin and glucose tolerance test were analyzed. The liver and pancreatic tissues were subjected to H&E and immunohistochemistry staining and islet cells to flow cytometry for apoptotic analysis. RYGB resulted in sustained normoglycemia and improved glucose tolerance in young prediabetic BKS-db/db mice (at the age of 6 weeks with hyperglycemia and normal insulinemia) and HFD-fed NZO and B6-db/db mice. Remarkably, RYGB improved liver steatosis, preserved the pancreatic β-cells and reduced β-cell apoptosis with increases in circulating insulin and adiponectin in young prediabetic BKS-db/db mice. However, RYGB neither reversed hyperglycemia in adult diabetic BKS-db/db mice (12 weeks old) nor attenuated hyperglycemia in STZ-induced diabetic mice. These results demonstrate that gastric bypass improves hyperglycemia in genetically modified prediabetic mice; however, it should be performed prior to β-cells exhaustion., Competing Interests: The authors have declared that no competing interests exist.

Objective: To examine increases in average height among Chinese children and adolescents., Methods: The data were obtained from the China Health and Nutrition Survey conducted during the period 1989-2015. A stratified multistage cluster sampling method was utilized to select participants aged 2-22 years in each province. Linear regression was used to examine the effects of age, birth cohort, and survey period on height., Results: A total of 15,227 males and 13,737 females were included in the final analysis. Age (A) showed a continuous effect on height. The average heights of the investigated groups increased continuously during the investigation period. By 2015, the average height of the overall group increased by 7.87 cm compared to the average height during the 1989 survey. Moreover, birth year (cohort, C) also had a stable effect on height. Using the height of individuals born in or before 1975 as a reference, the average height of each birth cohort increased in comparison to the previous birth cohort., Conclusions: The height of Chinese children and adolescents was affected by age, period, and cohort effects, and this effect is governed by certain rules. The age-period-cohort model can be used to analyze the trends of children's and adolescent's heights. The findings provide a scientific basis for the formulation of children's and adolescents' growth and development policies in China., (Copyright © 2021 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.)

Three novel Zr-substituted polyoxotungstate aggregates [H 2 N(CH 3 ) 2 ] 7 NaH 2 [Zr 2 Sb 2 O 3 ( A -α-PW 9 O 34 ) 2 ]·16H 2 O ( 1 ), [H 2 N(CH 3 ) 2 ] 6 H 12 [ZrSb 4 (OH)O 2 ( A -α-PW 8 O 32 )( A -α-PW 9 O 34 )] 2 ·33H 2 O ( 2 ), and [H 2 N(CH 3 ) 2 ] 4 Na 11.5 H 4.5 [Zr 4 W 8 Sb 4 P 5 O 49 (OH) 5 ( B -α-SbW 9 O 33 ) 2 ]·53H 2 O ( 3 ) have been made in hydrothermal reactions of the [ B -α-SbW 9 O 33 ] 9- precursor with Zr 4+ cations and PO 4 3- anions in the presence of dimethylamine hydrochloride and sodium acetate buffer (pH = 4.8) and structurally characterized. Different pH values induce structural transformation from tungstoantimonate (TA) to tungstophosphate (TP). 1 is a di-Zr-substituted sandwich-type TP, the tetranuclear heterometallic [Zr 2 Sb 2 O 3 ] 8+ entity sandwiched by two [ A -α-PW 9 O 34 ] 9- moieties. 2 is a double sandwich-type structure, which can be perceived as two equivalent sandwiched [Sb 3 (PW 8 O 32 )(PW 9 O 34 )] 11- further sandwiching one [Sb 2 Zr 2 (OH) 2 O 4 ] 4+ core to form a novel large-size sandwich-type architecture. Different from 1 and 2 , 3 is a tetra-Zr-substituted sandwiched configuration, in which two [ B -α-SbW 9 O 33 ] 9- fragments sandwich a unique 21-core Sb-P-W-Zr oxo cluster ({Zr 4 W 8 Sb 4 P 5 }). Furthermore, the catalytic oxidation of aromatic thioethers by 3 as the heterogeneous catalyst has been investigated, showing high conversion and remarkable selectivity as well as excellent recyclability.

A new nonanuclear heterometal five-layer sandwich silicotungstate [H 2 N(CH 3 ) 2 ] 6 H 5 NaK{[NaMn 8 O 6 (OAc) 6 ][ A-α- SiW 9 O 34 ] 2 }·34H 2 O ( 1 ) has been made under hydrothermal reactions of MnCl 2 ·4H 2 O, KMnO 4 , trivacant [ A -α-SiW 9 O 34 ] 10- {SiW 9 } fragment, and [H 2 N(CH 3 ) 2 ]·Cl in NaOAc-HOAc buffer (pH = 4.8). The polyoxoanion of 1 is a sandwich architecture, in which the nonanuclear heterometal cluster [NaMn 8 O 6 (OAc) 6 ] 7+ made of two triangular pyramid clusters [Mn 4 O 3 (OAc) 3 ] 3+ {Mn 4 } via a hexacoordinate antitrigonal prism Na + linkage is encapsulated by two trivacant fragments of {SiW 9 }. Furthermore, magnetic measurements of compound 1 reveal the existence of antiferromagnetic exchange interactions between the metal centers.

Purpose: We investigated whether metabolic phenotype improvements following gastric bypass are associated with gastric resection strategy in high-fat diet-induced obese (DIO) mice., Materials and Methods: We developed the mouse Roux-en-Y gastric bypass (RYGB) model with different gastric pouch sizes: (i) RYGB with a large gastric pouch (RYGB-LP), where the stomach was transected, and the jejunum was anastomosed to the residual forestomach, in which 30% of the stomach is retained. (ii) RYGB with a small remnant gastric pouch (RYGB-SP), where the stomach was transected 0.8 cm distal to the esophagogastric junction, and the jejunum is attached to a small remnant of the forestomach (~ 10% of the stomach). (iii) RYGB without gastric pouch (RYGB-NP), where the jejunum is anastomosed to the lower portion of the esophagus., Results: Surgical success rate (or 4-week mouse survival rate) of the RYGB-LP, RYGB-SP, and RYGB-NP procedures was 50, 75, and 85%, respectively. Our data demonstrate that all RYGB procedures improved body weight, glucose tolerance, and liver steatosis, compared with untreated DIO mice at 8-week post-surgery. Major surgical complication, such as obstruction at the forestomach, occurred predominantly in RYGB-LP mice, resulting in a higher mortality. Pre- and post-prandial plasma ghrelin levels did not correlate with improved metabolic phenotype after gastric bypass., Conclusions: We conclude that RYGB with different gastric pouch equally improves obesity and glucose tolerance independent of gastric pouch size and total plasma ghrelin levels in the mouse model of RYGB surgery.

A novel octa-Mn-substituted silicotungstate (ST) [H 2 N(CH 3 ) 2 ] 8 H 19 Na 4 {[K(H 2 O) 4 WO 4 ][SiMnW 10 O 38 ] 3 }[SiMnW 10 O 38 ]·27H 2 O (1) has been made by the hydrothermal reaction of the trivacant [A-α-SiW 9 O 34 ] 10- ({SiW 9 }) building block with Mn 2+ and Mn 7+ cations in the presence of dimethylamine hydrochloride and systematically characterized by IR spectroscopy, elemental analysis, thermogravimetric analysis (TGA), powder X-ray diffraction (PXRD) and single crystal X-ray diffraction. 1 is a tetrameric cluster formed by a hexa-Mn II substituted cyclic trimeric {[K(H 2 O) 4 WO 4 ][SiMnW 10 O 38 ] 3 } unit and a {SiMnW 10 O 38 } fragment via two W-O-Mn III linkers. Furthermore, its electrochemistry, thermal stability and magnetic properties have been studied.

Objective: The current study investigated whether bile diversion (BD) improves metabolic phenotype under farnesoid X receptor (FXR) deficiency., Methods: BD was performed in high-fat diet (HFD)-fed FXR knockout (FXRko) and wild-type (WT) animals. Metabolic phenotypes, circulating enteroendocrine hormones, total bile acids (BAs) and BA composition, and cecal gut microbiota were analyzed., Results: FXR-deficient mice were resistant to HFD-induced obesity; however, FXR-deficient mice also developed hyperglycemia and exhibited increased liver weight, liver steatosis, and circulating triglycerides. BD increased circulating total BAs and taurine-b-muricholic acid, which were in line with normalized hyperglycemia and improved glucose tolerance in HFD-fed WT mice. FXR deficiency also increased total BAs and taurine-b-muricholic acid, but these animals remained hyperglycemic. While BD improved metabolic phenotype in HFD-fed FXRko mice, these improvements were not as effective as in WT mice. BD increased liver expression of fibroblast growth factor 21 and peroxisome proliferator-activated receptor γ coactivator-1β and elevated circulating glucagon-like peptide-1 levels in WT mice but not in FXRko mice. FXR deficiency altered gut microbiota composition with a specific increase in phylum Proteobacteria that may act as a possible microbial signature of some diseases. These cellular and molecular changes in FXRko mice may contribute to resistance toward improved metabolism., Conclusions: FXR signaling plays a pivotal role in improved metabolic phenotype following BD surgery., (© 2019 The Obesity Society.)

Traumatic brain injury (TBI) can result in poor functional outcomes and death, and overall outcomes are varied. Growth factors, such as angiopoietin-1 (Ang-1), vascular endothelial growth factor (VEGF), and granulocyte-colony stimulating factor (G-CSF), play important roles in the neurological functions. This study investigated the relationship between serum growth factor levels and long-term outcomes after TBI. Blood samples from 55 patients were collected at 1, 3 and 7 days after TBI. Blood samples from 39 healthy controls were collected as a control group. Serum Ang-1, G-CSF, and VEGF levels were measured using ELISA. Patients were monitored for 3 months using the Glasgow Outcome Scale-Extended (GOSE). Patients having a GOSE score of > 5 at 3 months were categorized as a good outcome, and patients with a GOSE score of 1-5 were categorized as a bad outcome. Our data demonstrated that TBI patients showed significantly increased growth factor levels within 7 days compared with healthy controls. Serum levels of Ang-1 at 1 and 7 days and G-CSF levels at 7 days were significantly higher in patients with good outcomes than in patients with poor outcomes. VEGF levels at 7 days were remarkably higher in patients with poor outcomes than in patients with good outcomes. Receiver operating characteristic analysis showed that the best cut-off points of serum growth factor levels at 7 days to predict functional outcome were 1,333 pg/mL for VEGF, 447.2 pg/mL for G-CSF, and 90.6 ng/mL for Ang-1. These data suggest that patients with elevated levels of serum Ang-1, G-CSF, and decreased VEGF levels had a better prognosis in the acute phase of TBI (within 7 days). This study was registered with the Chinese Clinical Trial Registry (registration number: ChiCTR1800018251) on September 7, 2018., Competing Interests: None

Primary pancreatic α, β, δ, and pancreatic polypeptide (PP) cells are reliable cell models for diabetes research. However, the separation and purification of these cells in living conditions remains an obstacle for researchers. The interaction of visible light with cellular molecules can produce Raman scattering, which can be analyzed to obtain cellular intrinsic molecular fingerprints. It has been speculated that primary pancreatic α, β, δ, and PP cells can be identified and separated from each other according to their spectral differences. To test this hypothesis, Raman spectra detection was performed on rat islet cells. Single islet cells identified by Raman scattering under living conditions were verified using immunohistochemistry. Thus, Raman data were acquired from a pure line of islet cells as a training sample and then used to establish the discriminant function. Then, using the principal component analysis-linear discriminate analysis (PCA-LDA) method, the four types of islet cells could be identified and discriminated by Raman spectroscopy. This study provides a label-free and noninvasive method for discriminating islet cell types in a randomly distributed mixed islet cell population via their physical properties rather than by using antibodies or fluorescence labeling.

Background: Rotavirus is the leading cause of severe diarrhea among young children worldwide. Rotavirus vaccines have demonstrated substantial benefits in many countries that have introduced vaccine nationally. In China, where rotavirus vaccines are not available through the national immunization program, it will be important to review relevant local and global information to determine the potential value of national introduction. Therefore, we reviewed evidence of rotavirus disease burden among Chinese children younger than 5 years to help inform rotavirus vaccine introduction decisions., Methods: We reviewed scientific literature on rotavirus disease burden in China from 1994 through 2014 in China National Knowledge Infrastructure, Wanfang and PubMed. Studies were selected if they were conducted for periods of 12 month increments, had more than 100 patients enrolled and used an accepted diagnostic test., Results: Overall, 45 reports were included and indicate that rotavirus causes ~40% and ~30% of diarrhea-related hospitalizations and outpatient visits, respectively, among children younger than 5 years in China. Over 50% of rotavirus-related hospitalizations occur by age 1 year; ~90% occur by age 2 years. Regarding circulating rotavirus strains in China, there has been natural, temporal variation, but the predominant local strains are the same as those that are globally dominant., Conclusions: These findings affirm that rotavirus is a major cause of childhood diarrheal disease in China and suggest that a vaccination program with doses given early in infancy has the potential to prevent the majority of the burden of severe rotavirus disease.

The metabolic benefits induced by gastric bypass, currently the most effective treatment for morbid obesity, are associated with bile acid (BA) delivery to the distal intestine. However, mechanistic insights into BA signaling in the mediation of metabolic benefits remain an area of study. The bile diversion () mouse model, in which the gallbladder is anastomosed to the distal jejunum, was used to test the specific role of BA in the regulation of glucose and lipid homeostasis. Metabolic phenotype, including body weight and composition, glucose tolerance, energy expenditure, thermogenesis genes, total BA and BA composition in the circulation and portal vein, and gut microbiota were examined. BD improves the metabolic phenotype, which is in accord with increased circulating primary BAs and regulation of enterohormones. BD-induced hypertrophy of the proximal intestine in the absence of BA was reversed by BA oral gavage, but without influencing BD metabolic benefits. BD-enhanced energy expenditure was associated with elevated TGR5, D2, and thermogenic genes, including UCP1, PRDM16, PGC-1α, PGC-1β, and PDGFRα in epididymal white adipose tissue (WAT) and inguinal WAT, but not in brown adipose tissue. BD resulted in an altered gut microbiota profile (i.e., Firmicutes bacteria were decreased, Bacteroidetes were increased, and Akkermansia was positively correlated with higher levels of circulating primary BAs). Our study demonstrates that enhancement of BA signaling regulates glucose and lipid homeostasis, promotes thermogenesis, and modulates the gut microbiota, which collectively resulted in an improved metabolic phenotype., (Copyright © 2016 the American Physiological Society.)

At present, there is no effective treatment for the repair of the optic nerve after injury, or improvement of its microenvironment for regeneration. Intravitreally injected ciliary neurotrophic factor (CNTF) and olfactory ensheathing cells (OECs) promote the long-distance regrowth of severed optic nerve fibers after intracranial injury. Here, we examined the efficacy of these techniques alone and in combination, in a rat model of optic nerve injury. We injected condensed OEC suspension at the site of injury, or CNTF into the vitreous body, or both simultaneously. Retrograde tracing techniques showed that 4 weeks postoperatively, the number of surviving retinal ganglion cells and their axonal density in the optic nerve were greater in rats subjected to OEC injection only than in those receiving CNTF injection only. Furthermore, combined OEC + CNTF injection achieved better results than either monotherapy. These findings confirm that OECs are better than CNTF at protecting injured neurons in the eye, but that combined OEC and CNTF therapy is notably more effective than either treatment alone.

Background: The last case of infection with wild-type poliovirus indigenous to China was reported in 1994, and China was certified as a poliomyelitis-free region in 2000. In 2011, an outbreak of infection with imported wild-type poliovirus occurred in the province of Xinjiang., Methods: We conducted an investigation to guide the response to the outbreak, performed sequence analysis of the poliovirus type 1 capsid protein VP1 to determine the source, and carried out serologic and coverage surveys to assess the risk of viral propagation. Surveillance for acute flaccid paralysis was intensified to enhance case ascertainment., Results: Between July 3 and October 9, 2011, investigators identified 21 cases of infection with wild-type poliovirus and 23 clinically compatible cases in southern Xinjiang. Wild-type poliovirus type 1 was isolated from 14 of 673 contacts of patients with acute flaccid paralysis (2.1%) and from 13 of 491 healthy persons who were not in contact with affected persons (2.6%). Sequence analysis implicated an imported wild-type poliovirus that originated in Pakistan as the cause of the outbreak. A public health emergency was declared in Xinjiang after the outbreak was confirmed. Surveillance for acute flaccid paralysis was enhanced, with daily reporting from all public and private hospitals. Five rounds of vaccination with live, attenuated oral poliovirus vaccine (OPV) were conducted among children and adults, and 43 million doses of OPV were administered. Trivalent OPV was used in three rounds, and monovalent OPV type 1 was used in two rounds. The outbreak was stopped 1.5 months after laboratory confirmation of the index case., Conclusions: The 2011 outbreak in China showed that poliomyelitis-free countries remain at risk for outbreaks while the poliovirus circulates anywhere in the world. Global eradication of poliomyelitis will benefit all countries, even those that are currently free of poliomyelitis.

We have previously shown that mice deficient in protein kinase C theta (PKCθ) have the ability to reject cardiac allografts, but are susceptible to tolerance induction. Here we tested role of B cells in assisting alloimmune responses in the absence of PKCθ. Mouse cardiac allograft transplantations were performed from Balb/c (H-2d) to PKCθ knockout (PKCθ(-/-)), PKCθ and B cell double-knockout (PBDK, H-2b) mice and wild-type (WT) C57BL/6 (H-2b) mice. PBDK mice spontaneously accepted the allografts with the inhibition of NF-κB activation in the donor cardiac allograft. Anti-B cell antibody (rituximab) significantly delayed allograft rejection in PKCθ(-/-), but not in WT mice. Co-transfer of PKCθ(-/-) T plus PKCθ(-/-) B cells or primed sera triggered allograft rejection in Rag1(-/-) mice, and only major histocompatibility complex class II-enriched B cells, but not class I-enriched B cells, were able to promote rejection. This, together with the inability of PKCθ(-/-) and CD28(-/-) double-deficient (PCDK) mice to acutely reject allografts, suggested that an effective cognate interaction between PKCθ(-/-) T and B cells for acute rejection is CD28 molecule dependent. We conclude that T-B cell interactions synergize with PKCθ(-/-) T cells to mediate acute allograft rejection., (© 2013 Steunstichting ESOT. Published by John Wiley & Sons Ltd.)

Morbid obesity is linked to increased incidences of glucose intolerance, Type 2 diabetes mellitus, cardiovascular diseases, various forms of liver disease, and specific forms of cancer. Treatment of obesity by lifestyle modifications (i.e. changes in diet and exercise) and drug therapy is generally ineffective. Bariatric surgery is currently the most effective means of treating obesity and related disorders. We as well as others have developed surgical procedures for application to genetic mouse models that mimic an array of human bariatric surgical procedures used in the treatment of obesity. The application of bariatric surgery to genetic mouse models will broaden our understanding of the role of the gut in metabolic disease. Models that have been developed include gastric banding, sleeve gastrectomy (SG), Roux-en-Y gastric bypass (RYGB) with a complete exclusion of the stomach, duodenal-jejunal bypass (DJB) and biliopancreatic diversion (BPD). The detailed methods of these procedures are provided.

Morbid obesity is linked to increased incidence of glucose intolerance, Type 2 diabetes mellitus, cardiovascular diseases, various forms of liver disease, and specific forms of cancer. Treatment of obesity by lifestyle modifications (i.e., changes in diet and exercise) and drug therapy is generally ineffective. Bariatric surgery is currently the most effective means of treating obesity and related disorders. We as well as others have developed surgical procedures for application to genetic mouse models that mimic an array of human bariatric surgical procedures used in the treatment of obesity. The application of bariatric surgery to genetic mouse models will broaden our understanding of the role of the gut in metabolic disease. Models that have been developed include gastric banding, sleeve gastrectomy (SG), Roux-en-Y gastric bypass (RYGB) with a complete exclusion of the stomach, duodenal-jejunal bypass (DJB), and biliopancreatic diversion (BPD). The detailed methods of these procedures are provided. Curr. Protoc. Mouse Biol. 2:295-306 © 2012 by John Wiley & Sons, Inc., (Copyright © 2012 John Wiley & Sons, Inc.)

Context: Roux-en-Y gastric bypass (RYGB) is one of the most effective long-term therapies for the treatment of severe obesity. Recent evidence indicates that RYGB effects weight loss through multiple physiological mechanisms, including changes in energy expenditure, food intake, food preference, and reward pathways., Objective: Because central melanocortin signaling plays an important role in the regulation of energy homeostasis, we investigated whether genetic disruption of the melanocortin-4 receptor (MC4R) in rodents and humans affects weight loss after RYGB., Methods and Results: Here we report that MC4R(-/-) mice lost substantially less weight after surgery than wild-type animals, indicating that MC4R signaling is necessary for the weight loss effects of RYGB in this model. Mice heterozygous for MC4R remain fully responsive to gastric bypass. To determine whether mutations affect surgically induced weight loss in humans, we sequenced the MC4R gene in 972 patients undergoing RYGB. Patients heterozygous for MC4R mutations exhibited the same magnitude and distribution of postoperative weight loss as patients without such mutations, suggesting that although two normal copies of the MC4R gene are necessary for normal weight regulation, a single normal copy of the MC4R gene is sufficient to mediate the weight loss effects of RYGB., Conclusions: MC4R is the first gene identified that is required for the sustained effects of bariatric surgery. The need for MC4R signaling for the weight loss effects of RYGB in mice underscores the physiological mechanisms of action of this procedure and demonstrates that RYGB both influences and is dependent on the normal pathways that regulate energy balance.

Objective: To evaluate the effect of the aluminum hydroxide (Al-OH) adjuvant on the 2009 pandemic influenza A/H1N1 (pH1N1) vaccine., Methods: In a multicenter, double-blind, randomized, placebo-controlled trial, participants received two doses of split-virion formulation containing 15 μg hemagglutinin antigen, with or without aluminum hydroxide (Al-OH). We classified the participants into six age categories (>61 years, 41-60 years, 19-40 years, 13-18 years, 8-12 years, and 3-7 years) and obtained four blood samples from each participant on days 0, 21, 35, and 42 following the first dose of immunization. We assessed vaccine immunogenicity by measuring the geometric mean titer (GMT) of hemagglutination inhibiting antibody. We used a two-level model to evaluate the fixed effect of aluminum Al-OH and other factors, accounting for repeated measures., Results: The predictions of repeated measurement on GMTs of formulations with or without Al-OH, were 80.35 and 112.72, respectively. Al-OH significantly reduced immunogenicity after controlling for time post immunization, age-group and gender., Conclusion: The Al-OH adjuvant does not increase but actually reduces the immunogenicity of the split-virion pH1N1 vaccine., (Copyright © 2011 The Editorial Board of Biomedical and Environmental Sciences. Published by Elsevier B.V. All rights reserved.)

Objective: To assess the effects of different bariatric surgical procedures on the treatment of obesity and insulin resistance in high fat diet-induced obese (DIO) mice., Background: Bariatric surgery is currently considered the most effective treatment for morbid obesity and its comorbidities; however, a systematic study of their mechanisms is still lacking., Methods: We developed bariatric surgery models, including gastric banding, sleeve gastrectomy, Roux-en-Y gastric bypass (RYGB), modified RYGB (mRYGB) and biliopancreatic diversion (BPD), in DIO mice. Body weight, body fat and lean mass, liver steatosis, glucose tolerance and pancreatic beta cell function were examined., Results: All bariatric surgeries resulted in significant weight loss, reduced body fat and improved glucose tolerance in the short term (4 weeks), compared with mice with sham surgery. Of the bariatric surgery models, sleeve gastrectomy and mRYGB had higher success rates and lower mortalities and represent reliable restrictive and gastrointestinal (GI) bypass mouse bariatric surgery models, respectively. In the long term, the GI bypass procedure produced more profound weight loss, significant improvement of glucose tolerance and liver steatosis than the restrictive procedure. DIO mice had increased insulin promoter activity, suggesting overactivation of pancreatic beta cells, which was regulated by the mRYGB procedure. Compared with the restrictive procedure, the GI bypass procedure showed more severe symptoms of malnutrition following bariatric surgery., Discussions: Both restrictive and GI bypass procedures provide positive effects on weight loss, fat composition, liver steatosis and glucose tolerance; however, in the long term, the GI bypass shows better results than restrictive procedures.

Background: On September 21, 2009, China began administering vaccines, obtained from 10 different manufacturers, against 2009 pandemic influenza A (H1N1) virus infection in priority populations. We aimed to assess the safety of this vaccination program., Methods: We designed a plan for passive surveillance for adverse events after immunization with the influenza A (H1N1) vaccine. Physicians or vaccination providers were required to report the numbers of vaccinees and all adverse events to their local Center for Disease Control and Prevention (CDC), which then reported the data to the Chinese CDC through the online National Immunization Information System's National Adverse Event Following Immunization Surveillance System. Data were collected through March 21, 2010, and were verified and analyzed by the Chinese CDC., Results: A total of 89.6 million doses of vaccine were administered from September 21, 2009, through March 21, 2010, and 8067 vaccinees reported having an adverse event, for a rate of 90.0 per 1 million doses. The age-specific rates of adverse events ranged from 31.4 per 1 million doses among persons 60 years of age or older to 130.6 per 1 million doses among persons 9 years of age or younger, and the manufacturer-specific rates ranged from 4.6 to 185.4 per 1 million doses. A total of 6552 of the 8067 adverse events (81.2%; rate, 73.1 per 1 million doses) were verified as vaccine reactions; 1083 of the 8067 (13.4%; rate, 12.1 per 1 million doses) were rare and more serious (vs. common, minor events), most of which (1050) were allergic reactions. Eleven cases of the Guillain-Barré syndrome were reported, for a rate of 0.1 per 1 million doses, which is lower than the background rate in China., Conclusions: No pattern of adverse events that would be of concern was observed after the administration of influenza A (H1N1) vaccine, nor was there evidence of an increased risk of the Guillain-Barré syndrome.

We have previously shown that islet emboli in the portal vein block blood flow and induce local inflammatory reaction, resulting in functional loss of islet grafts following intraportal transplantation. This study was designed to test whether Toll-like receptor (TLR) activation mediates early islet graft failure. Syngeneic islet grafts were transplanted into chemically induced diabetic mice, and TLR deficient mice were used as donors and/or recipients of islet grafts. Islet viability, proinflammatory cytokines, high-mobility group box-1 (HMGB1) and NF-kappaB activation were analyzed by bioluminesce imaging (BLI), quantitative RT-PCR (qRT-PCR) and histology. Early islet graft failure was observed in mice with intraportal islet engrafts with increased proinflammatory cytokines, HMGB1 expression, NF-kappaB activation, caspase-3 and TUNEL positive cells. Deficiency of TLR4 in donor, but not in recipient, inhibited NF-kappaB activation, reduced proinflammatory cytokines and improved viability of islet grafts. Blockade of HMGB1 with anti-HMGB1 monoclonal antibody (mAb, 2g7) inhibited inflammatory reactions, as evidenced by reduced TNFalpha and IL-1ss production, and improved islet viability. We conclude that TLR4 activation mediates early graft failure following intraportal islet transplantation. Inhibition of TLR4 activation represents a novel strategy to attenuate early graft failure following intraportal islet transplantation.

Liver allografts are spontaneously accepted in the liver transplantation mouse model; however, the basis for this tolerance and the conditions that abrogate spontaneous tolerance to liver allografts are incompletely understood. We examined the role of CpG oligodeoxynucleotide (ODN) in triggering the liver inflammatory reaction and allograft rejection. Bioluminescence imaging quantified the activation of nuclear transcriptional factor kappaB (NF-kappaB) at different time points post-transplantation. Intrahepatic lymphocyte subsets were analyzed by immunofluorescence assay and flow cytometry. The results showed that liver allografts survived for more than 100 days without a requirement for any immunosuppressive therapy. Donor-matched cardiac allografts were permanently accepted, whereas third-party cardiac grafts were rejected with delayed kinetics; this confirmed donor-specific tolerance. NF-kappaB activation in the liver allografts was transiently increased on day 1 and diminished by day 4; in comparison, it was elevated up to 10 days post-transplantation in the cardiac allografts. When CpG ODN was administered at a high dose (50 microg per mouse x 1) to the recipients on day 7 post-transplantation, it induced an acute liver inflammatory reaction with elevated NF-kappaB activation in both allogeneic and syngeneic liver grafts. Multiple doses of CpG ODN (10 microg per mouse x 3) elicited acute rejection of the liver allografts with significant T cell infiltration in the liver allografts, reduced T regulatory cells, and enhanced interferon gamma-producing cells in the intrahepatic infiltrating lymphocytes. These data demonstrate that CpG ODN initiates an inflammatory reaction and abrogates spontaneous tolerance in the liver transplantation mouse model. Liver Transpl 15:915-923, 2009. (c) 2009 AASLD.

Background: Protein kinase C-theta (PKCtheta) mediates critical T-cell receptor signals required for T-cell activation. We have recently shown that PKCtheta knockout (PKCtheta, H-2b) T cells, when transferred into T/B cell-deficient mice, failed to reject fully allogeneic (H-2d) cardiac grafts and that transgenic expression of antiapoptotic Bcl-xL gene in PKCtheta T cells restored allograft rejection., Methods: We used PKCtheta mice as recipients of cardiac allografts, compared with wild-type (WT) cardiac allograft transplantation. Anti-CD154 monoclonal antibody (MR1) and human CTLA4Ig were sued to induce donor-specific tolerance. T-cell proliferation, T-cell subsests, nuclear factor kappa B (NF-kappaB) activation, and Bax and Bcl-xL were analyzed., Results: Although suboptimal anti-CD154 monoclonal antibody or human CTLA4Ig failed to delay cardiac allograft rejection in WT mice, the same therapy induced long-term survival of cardiac allografts in PKCtheta mice. Donor-type second cardiac allografts (H-2d) were accepted, and third-party heart allografts (H-2k) were rejected by tolerant PKCtheta mice. However, tolerance state could not be effectively transferred with T cells from tolerance PKCtheta mice. Compared with WT mice, reduced NF-kappaB activation, T-cell proliferation, and T-cell infiltration in PKCtheta spleens were observed. PKCtheta mice reveal reduced CD4/CD25/FoxP3, Th1/Th17 subsets, and mouse MHC class II (IE)-reactive CD4Vbeta11 T cells. Apoptotic molecule, Bax, was increased and antiapoptotic molecule, Bcl-xL, was reduced in PKCtheta spleen cells., Conclusion: We concluded that PKCtheta mice have a defected alloimmune response and are susceptible to tolerance induction, which is associated with a clonal deletion of T-cell subsets.

Background: We characterized the role of T cell subsets and major histocompatibility complex molecules in allograft rejection and recurrence of autoimmune diabetes., Methods: Adoptive cell transfer and vascularized segmental pancreas transplantation were performed in mice., Results: In an alloimmune response model, transfer of nondiabetic CD4, but not CD8 T cells, elicited pancreas allograft rejection in streptozotocin (STZ)-induced diabetic NOD/scid mice. Pancreas allografts were acutely rejected in STZ-induced diabetic NOD/beta2m mice (confirmed the absence of major histocompatibility complex [MHC] class I and CD8 T cells) and permanently accepted in NOD/CIIT mice (confirmed the absence of MHC class II and CD4 T cells). The results suggest that rejection of pancreas allograft is CD4-dependent and MHC class I-independent. In the autoimmune diabetes model, whole spleen cells obtained from diabetic NOD mice induced autoimmune diabetes in NOD/scid and NOD/CIIT mice, but the onset of diabetes was delayed in NOD/beta2m mice. However, the purified diabetic T cells failed to elicit autoimmune diabetes in NOD/beta2m mice. NOD/scid and NOD/CIIT pancreas grafts were acutely destroyed whereas four of six NOD/beta2m pancreas grafts were permanently accepted in autoimmune diabetic NOD mice., Conclusion: CD4 T cells are sufficient for the induction of allograft rejection, and MHC class I molecule is required to induce recurrent autoimmune diabetes after pancreas transplantation in mice.

Background: The purpose of the current study was to evaluate the role of bioluminescence imaging (BLI) in the determination of nuclear factor (NF)-kappaB activation in cardiac allograft rejection and ischemia-reperfusion injury., Methods: To visualize NF-kappaB activation, luciferase transgenic mice under the control of a mouse NF-kappaB promoter (NF-kappaB-Luc) were used as donors or recipients of cardiac grafts. Alternatively, NF-kappaB-Luc spleen cells were adoptively transferred into Rag2 -/- mice with or without cardiac allografts. BLI was performed posttransplantation to detect luciferase activity that represents NF-kappaB activation., Results: The results show that luciferase activity was significantly increased in the cardiac allografts when NF-kappaB-Luc mice were used as recipients as well as donors. Luciferase activity was also elevated in the wild-type cardiac allografts in Rag2 -/- mice that were transferred with NF-kappaB-Luc spleen cells. CD154 monoclonal antibody (mAb) therapy inhibited luciferase activity and induced long-term survival of cardiac allografts. toll-like receptor-9 ligand, CpG DNA, enhanced luciferase activity and abrogated tolerance induction by CD154 mAb. Luciferase activity was also increased in ischemia-reperfusion injury of the cardiac grafts., Conclusion: BLI using Luc-NF-kappaB mice is a noninvasive approach to visualize the activation of NF-kappaB signaling in mouse cardiac allograft rejection and ischemia-reperfusion injury. CD154 mAb can inhibit NF-kappaB activation, which is reversed by toll-like receptor engagement.

E2F-1 mediates apoptosis through transcriptional regulation of its targets. We report here that E2F-1 acts as a direct transcriptional regulator of dual specificity phosphatase 1 (DUSP1; CL100), a threonine and tyrosine phosphatase that inhibits mitogen-activated protein (MAP) kinases. We found that DUSP1 is transcriptionally induced by ectopic E2F-1 expression and that extracellular signal-regulated kinase 1/2 are dephosphorylated in the presence of E2F-1 and DUSP1. E2F-1 mediates apoptosis in the cellular response to oxidative stress. DUSP1 levels are significantly increased in an E2F-1-dependent manner following oxidative stress but not other stresses examined. DUSP1 mediates the cellular response to oxidative stress. We found that E2F-1 binds to chromatin encompassing the DUSP1 promoter and greatly stimulates the promoter activity of the DUSP1 gene. In particular, E2F-1 physically binds to an E2F-1 consensus sequence and a palindromic motif in the DUSP1 promoter. Interestingly, E2F-1 is acetylated following oxidative stress. Our findings show that E2F-1 is a transcriptional activator of DUSP1 and that DUSP1 is a link between E2F-1 and MAP kinases.

Background: FK778 is a malononitrilamide, a class of immune suppressive compounds with antiviral features and experimental activity in chronic rejection, a potentially interesting combination for organ transplantation. The goal of this project was to study the tolerability, immune suppressive efficacy, and anti-cytomegalovirus (CMV) activity of FK778 and to assess the in vivo relevance of its previously described inhibition of de novo pyrimidine synthesis., Methods: Heart transplants were performed in rats (Brown Norway [BN] to Lewis) and treated with varying doses of FK778 or leflunomide for 28 days. At 28 days, at the time of rejection or at the death of the animal, the allograft and other vital organs were obtained for study by light microscopy and immunohistochemistry. In separate experiments, Lewis rats were given sublethal irradiation, inoculated with rat CMV (Maastricht strain), and treated with varying doses of FK778 and leflunomide. In both the transplant and CMV studies, IP uridine was given at 250 mg/kg to cohorts or animals receiving FK778 and leflunomide., Results: FK778 controls acute rejection and inhibits CMV replication at 20 mg/kg but is toxic at 25 mg/kg. Toxicity is manifested as anemia, changes in hepatic and intestinal histology, and mortality. The toxicity but not the immune suppressive or antiviral efficacy, is reduced significantly by exogenous uridine administration., Conclusion: FK778 has both immune suppressive and antiviral activities, neither of which is entirely dependent on inhibition of pyrimidine synthesis. These, and other published observations, suggest that the antiviral activity and a considerable part of the efficacy of the malononitrilamide family of drugs is attributable to activities other than drug induced pyrimidine deficiency.

Background: Immune response to incompatible ABO antigens on allografts may result in rejection, accommodation, or immune tolerance. Our objective has been to develop a model for studying these three types of immune response to incompatible carbohydrate antigen in alpha1,3-galactosyltransferase knockout (KO) mice. KO mice lack the alpha-gal epitope and can produce the anti-Gal antibody against it after immunization with pig kidney membranes (PKM) that express this epitope., Methods: KO mice were transplanted with syngeneic wild-type (WT) heart expressing alpha-gal epitopes. Subsequently, the mice were lethally irradiated and received lymphocytes including memory anti-Gal B cells from PKM immunized KO mice. Immune response to incompatible alpha-gal epitopes on the graft was determined by transplanted-heart function and by production of anti-Gal after PKM immunizations., Results: Anti-Gal B cells exposed for 1 to 2 weeks to alpha-gal epitopes of WT hearts differentiate into cells producing noncytolytic accommodating antibodies. Exposure for longer periods (2-4 weeks) induces a transition from accommodation into tolerance, indicated by the inability of mice to produce anti-Gal antibodies despite repeated PKM immunizations. WT hearts in accommodating and in tolerized mice continue to function for months., Conclusions: In the absence of T-cell help, anticarbohydrate B cells exposed to incompatible carbohydrate antigens of transplanted organs differentiate first into cells capable of producing accommodating antibodies, but, after prolonged exposure, these B cells gradually become tolerized. These findings suggest that prolonged T-cell suppression in recipients of ABO-incompatible allografts may result in a similar induction of tolerance to incompatible blood-group antigens.

Induction of tolerance to transplantation carbohydrate antigens is of clinical significance in recipients of ABO-incompatible allografts, or of xenografts. The experimental animal model used for studying such tolerance was that of alpha1,3galactosyltransferase (alpha1,3GT) knockout (KO) mice, which lacks the alpha-gal epitope (Galalpha1-3Galbeta1-4GlcNAc-R) and which can produce the anti-Gal antibody against it. In contrast, wild-type (WT) mice synthesize the alpha-gal epitope and are immunotolerant to it. KO lymphocytes transduced in vitro with adenovirus containing the alpha1,3GT gene (AdalphaGT) express alpha-gal epitopes. Administration of such lymphocytes into KO mice resulted in tolerization of naïve and memory anti-Gal B cells. Mice tolerized by AdalphaGT transduced lymphocytes failed to produce anti-Gal following immunizations with pig kidney membranes (PKM) expressing multiple alpha-gal epitopes. This tolerance was perpetuated by transplanted syngeneic WT mouse hearts expressing alpha-gal epitopes. Transplanted WT hearts survived in the tolerized KO mice for at least 100 days, despite repeated PKM immunizations. Control mice receiving lymphocytes transduced with adenovirus lacking the alpha1,3GT gene were not tolerized, but produced anti-Gal and rejected transplanted WT hearts. This study suggests that autologous lymphocytes transduced with adenovirus containing A or B transferase genes may induce a similar tolerance to blood group antigens in humans.

Induction of immune tolerance on memory B cells specific to transplantation carbohydrate antigens was studied in the experimental animal model of alpha1,3galactosyltransferase knockout (KO) mice, which lack the alpha-gal epitope (Galalpha1-3Galbeta1-4GlcNAc-R) and can produce the anti-Gal antibody against it. Memory anti-Gal B cells were generated by immunization of KO mice with pig kidney membranes (ie, xenogeneic cell membranes expressing an abundance of alpha-gal epitopes). Lymphocytes including memory anti-Gal B cells were administered into lethally irradiated KO mice, together with syngeneic wild-type (WT) lymphocytes expressing alpha-gal epitopes. Memory anti-Gal B cells were completely tolerized after being in vivo for 14 days with WT lymphocytes. This was indicated by the lack of anti-Gal immunoglobulin G (IgG) response following immunization with pig kidney membranes vs the extensive anti-Gal response in mice that did not receive WT lymphocytes. Tolerance induction was prevented if T cells were activated by alloantigens. This tolerance was highly specific to anti-Gal B cells and did not affect memory B cells with closely related specificity, such as B cells with anti-blood group A specificity. Tolerance induction on anti-Gal B cells was found to be time dependent and required more than 10 days of in vivo exposure of these B cells to WT lymphocytes. These observations suggest a novel method for induction of tolerance to transplantation carbohydrate antigens in humans, by in vitro transduction of autologous blood lymphocytes with an adenovirus containing the corresponding glycosyltransferase gene and administration of the transduced cells into the circulation after removal of natural antibodies to the antigen.

Tolerance induction to transplantation-associated carbohydrate antigens, such as blood group A or B and the alpha-gal epitope (Gal(alpha)1-3Gal(beta)1-4GlcNAc-R), is of clinical significance. This study demonstrates tolerance induction to the alpha-gal epitope in the experimental animal model of alpha1,3galactosyltransferanse knockout mice (KO mice) lacking alpha-gal epitopes by administering syngeneic lymphocytes expressing alpha-gal epitopes. Repeated immunization of control KO mice with pig kidney membranes (PKM) expressing many alpha-gal epitopes induces an extensive anti-Gal antibody response against this epitope. In contrast, KO mice that received as few as 2 x 10(6) wild-type (WT) lymphocytes were tolerized and failed to produce anti-Gal following PKM immunizations. Accordingly, control mice producing anti-Gal rapidly rejected transplanted WT hearts, whereas tolerized mice did not reject WT hearts. These findings suggest that autologous blood lymphocytes processed to express a carbohydrate antigen may induce a similar tolerance to such an antigen upon administration into humans.

Background: Accommodation in patients transplanted with ABO incompatible allografts describes a state in which antibodies are produced against the incompatible blood group carbohydrate antigen; however, the graft is not rejected. The present study describes an experimental model for antibody-mediated accommodation of organs expressing incompatible carbohydrate antigens., Methods: The model includes alpha1,3galactosyltransferase knockout mice that lack the alpha-gal epitope (Galalpha1-3Galbeta1-4GlcNAc-R), transplanted heterotopically with wild-type (WT) hearts expressing this epitope. The mice are irradiated and receive memory anti-Gal B cells by adoptive transfer. Immunization of these mice with pig-kidney membranes induces the production of large amounts of anti-Gal, which binds specifically to alpha-gal epitopes., Results: Under the described accommodation protocol, transplanted mice produce anti-Gal that binds to alpha-gal epitopes on endothelial cells of the grafted WT heart; however, the WT hearts continued to function for months. Second WT hearts transplanted into accommodating, anti-Gal producing mice, were not rejected. Anti-Gal in accommodating mice was not cytolytic, whereas anti-Gal in rejecting mice readily induced complement-mediated lysis of cells expressing alpha-gal epitopes. In addition, accommodating mice displayed a preferential increase in the anti-Gal immunoglobulin (Ig)G2b subclass., Conclusions: The immune system may be manipulated to accommodate grafts expressing incompatible carbohydrate antigens by preferential production of noncytolytic anticarbohydrate antibodies.

Background: We studied the ability of CD4 and CD8 T cells to induce rejection of pancreas xenografts in a concordant combination using rat pancreas xenografts as donors and chemically induced diabetic mice as recipients., Methods: Lewis rat (2 to 3 weeks old) pancreas xenografts were transplanted into streptozotocin (STZ)-induced diabetic mice. Lymphocyte proliferation and cytokine production were analyzed in vitro. All pancreas xenografts were assessed by functional (blood glucose) and histopathologic examinations., Results: Lewis rat pancreas grafts were rejected within 10 to 13 days, with mononuclear cell infiltrate and tissue necrosis in STZ-induced diabetic mice. A predominant T cell receptor alphabeta -CD4 cell (on day 4) and T cell receptor alphabeta -CD8 cell (on day 8) infiltrate and IgM deposition were found in the pancreas xenografts after transplantation. Anti-CD4 (GK1.5), but not anti-CD8 (YTS169.4), monoclonal antibodies resulted in a prolonged survival of Lewis rat pancreas xenografts. Lewis pancreas xenografts were permanently accepted by CD4 knockout mice but not by CD8 knockout mice. The pancreas xenografts were acutely rejected with a mean survival time of 15.3 days in B cell-deficient mice (microMT/microMT). Transfer of CD4 but not CD8 spleen cells from naïve C57BL/6 mice into Rag2 mice led to acute rejection of transplanted pancreas xenografts. However, activated CD8 spleen cells elicited rejection of Lewis rat pancreas xenografts in SZT-induced diabetic mice., Conclusion: The current results show that CD4 T cells are necessary and sufficient for mediating the rejection of Lewis rat pancreas xenografts in STZ-induced diabetic mice. However, CD8 cells, when activated, can also induce acute rejection of concordant pancreas xenografts.

Background: A major barrier in pig to human organ transplantation is the binding of human anti-Gal to alpha-gal epitopes (Gal alpha 1-3Gal beta 1-4GlcNAc-R) on pig cells, resulting in hyperacute and acute vascular rejection of pig xenografts. Moreover, the immune system in xenograft recipients is activated by these epitopes to produce high affinity anti-Gal, which is also detrimental to xenografts. Production of anti-Gal can be prevented by specific elimination of anti-Gal B cells. This was achieved with the toxin ricin A, coupled to human alpha1-acid glycoprotein modified to carry alpha-gal epitopes. This complex, designated alpha-gal ricin, is targeted in vivo to anti-Gal B cells by interaction with the immunoglobulin molecules (i.e., B cell receptors) on these cells., Methods: Carbohydrate chains on alpha 1-acid glycoprotein were converted to carry alpha-gal epitopes by enzymatic treatment with recombinant alpha 1,3 galactosyltransferase (alpha 1,3GT). This molecule and ricin A were biotinylated and coupled by avidin to generate alpha-gal ricin. The efficacy of alpha-gal ricin in eliminating anti-Gal B cells was studied in the experimental model of alpha 1,3GT knockout (KO) mice. These mice produce large amounts of anti-Gal immunoglobulin G when immunized with pig kidney membranes, as measured by ELISA with alpha-gal epitopes linked to bovine serum albumin (BSA). In the absence of anti-Gal B cells, these mice lack the ability to produce anti-Gal., Results: Repeated administration of alpha-gal ricin into alpha1,3GT KO mice resulted in elimination of anti-Gal B cells, thereby preventing production of anti-Gal immunoglobulin G after immunization with pig kidney membranes. This prevention of anti-Gal production occurred with doses of alpha-gal ricin that were not toxic to the mice and did not affect production of antibodies with other specificities., Conclusions: Administration of alpha-gal ricin results in specific elimination of anti-Gal B cells in alpha 1,3GT KO mice. The elimination of these B cells may prove to be helpful in attempts to achieve immune tolerance to alpha-gal epitopes in primates.

Background: The objective of this study was to evaluate the role of anti-Gal Abs and non-anti-Gal Abs in hyperacute rejection (HAR) of concordant pancreas xenografts compared with heart xenografts. In addition, we tested whether rejection of Lewis rat pancreas grafts was T-cell dependent and could be prevented by anti-T-cell treatment., Methods: To determine the role of anti-Gal Abs in the induction of HAR, Lewis rat pancreas and heart xenografts were transplanted into alpha1,3Galactosyltransferase knockout (GT-Ko) mice treated with normal human serum (NHS) or hyperimmune serum, or into presensitized GT-Ko mice. To investigate whether rejection of pancreas xenograft was mediated by a T-cell dependent response, Lewis rat pancreas grafts were transplanted into streptozotocin (STZ)-induced diabetic GT-Ko mice treated with FK506, anti-CD4 mAbs (GK1.5), and thymectomy. Antidonor-specific IgM and IgG and anti-Gal Abs were analyzed by flow cytometry. Rejected and long-term surviving pancreas xenografts were assessed by functional (blood glucose) and histopathological examination., Results: HAR of Lewis rat pancreas xenografts could not be induced by NHS (0.4 ml), whereas NHS (0.2 ml) resulted in HAR of Lewis heart xenografts. Infusion of Lewis rat-specific hyperimmune serum (0.2 ml) resulted in HAR of Lewis rat pancreas xenografts. In addition, second Lewis rat pancreas grafts were hyperacutely rejected by presensitized GT-Ko mice. Immunohistochemical staining showed a low expression of Galalpha1,3Gal antigen in the endocrine tissue compared with that in the cardiac grafts. The levels of anti-Gal Abs in pancreas xenograft transplantation did not increase in GT-Ko mice after pancreas xenograft transplantation that was significantly increased after heart transplantation. FK506 treatment induced long-term survival of Lewis pancreas xenografts (mean survival time (MST) >90 days). Anti-CD4 treatment delayed rejection of Lewis rat pancreas xenografts with MST of 34.3 days, whereas anti-CD4, in combination with thymectomy, synergistically prolonged survival of pancreas xenograft (MST=70.4 days)., Conclusion: Pancreas xenograft is resistant to anti-Gal Abs-induced HAR but is susceptible to anti-donor specific Abs. Rejection of Lewis pancreas xenograft in STZ-induced, diabetic, GT-Ko mice is T-cell dependent.

Background: The goal of this study was to characterize the importance of splanchnic viscera in liver ischemic reperfusion injury and to enhance the tolerance of liver to warm ischemia injury with portosystemic shunt., Methods: The hepatic blood flow of male Sprague Dawley rats was subjected to 45, 60, 120, and 150 min liver warm ischemia with or without portosystemic shunt (splenic-caval shunt). The production of tumor necrosis factor a (TNFa), nuclear factor-kappaB activation, inducible NO synthase (iNOS) expression, and apoptosis were examined., Results: A total of 67% of rats with 45 min liver warm ischemia (n=6) and 100% of rats with 60 min liver warm ischemia (n=6) died within 1 day. However, all rats with 120 min (n=8) liver warm ischemia in splenic-caval shunt group survived for over 1 day, 6/8 for over 3 days, and 5/8 for over 5 days without significant histological changes of the liver. Serum tumor necrosis factor levels in liver warm ischemic rats were increased, This increase was significantly reversed after portosystemic shunt. After challenge with lipopolysaccharide (1 mg/kg, p.v.), naive rats survived for over 5 days (n=4) with the peak value of rat tumor necrosis factor (240 pg/ml) at 90 min. In contrast, all rats died within one day (n=5) with the peak value of rat tumor necrosis factor a (465 pg/ml) at 45 min after administration of lipopolysaccharide in the rats with liver warm ischemia plus splenic-caval shunt. iNOS expression and nuclear factor-kappaB activation were very strongly increased in the hepatocytes after liver warm ischemia with portosystemic shunt, compared with liver ischemia without portosytemic shunt., Conclusions: We conclude that the splanchnic viscera can contribute to liver ischemic reperfusion injury. Portosystemic shunt enhances the tolerance of liver to warm ischemia through the protective role of iNOS and nuclear factor-kappaB (NF-kappaB).

Background: In the experiment described here, we investigated the effects of the immunosuppressants FK506 and leflunomide (Lef) on the survival of hamster hearts and liver xenografts in Lewis rats., Methods: Lewis rats were used as recipients of hamster heart or liver grafts using different regimens of FK506 and Lef. Donor-matched heart grafts were transplanted into long-term surviving Lewis rat recipients of hamster xenografts to test donor-specific prolongation of xenograft survival. Hyperimmune, late xenograft rejection, and naive sera were transferred into long-term surviving Lewis rat recipients of hamster heart xenografts to determine whether these sera could inhibit the efficacy of donor-specific long-term survival. Anti-donor-specific antibodies were analyzed by flow cytometry., Results: After a short induction with FK506 plus Lef, maintenance treatment with FK506 alone was sufficient to prolong survival of hamster xenografts. All hamster heart and four of six hamster liver xenografts survived for more than 3 months. Second hamster hearts were permanently accepted by Lewis rats bearing long-term surviving hamster heart xenografts when rats were treated with FK506 monotherapy (mean survival time >60 days, n=4). Long-term surviving hamster heart grafts were rejected after transfer of hyperimmune serum but not late xenograft rejection serum or naive serum. Lef and FK506 significantly reduced the production of anti-donor-specific antibodies in Lewis rats transplanted with hamster liver and heart xenografts., Conclusion: Long-term survival of hamster liver and heart xenografts in Lewis rats could be induced by a regimen of short-term FK506 in combination with Lef followed by FK506 monotherapy. The acquired sensitivity of late xenoreactivity to FK506 reflects primarily a modification in the host immune response to the hamster graft.

Background: Ischemic-preconditioning is a process whereby a brief ischemic episode confers a state of protection against subsequent long-term ischemia-reperfusion injury. Ischemic preconditioning has been studied in heart and liver ischemia-reperfusion injury; however, few studies have been performed in the model of preservation-reperfusion injury in liver transplantation. The current study was designed to evaluate the ability of ischemic preconditioning to protect liver grafts from long-term preservation-reperfusion injury., Methods: Male Sprague Dawley rats were used as donors and recipients of orthotopic liver transplantation. Ischemic preconditioning was done by interruption of the portal vein and hepatic artery for 5, 10, and 20 min (5-10, 10-10, and 20-10 groups). Reflow was initiated by removal of the clamp for another 10 min in all groups. The liver was removed and placed in a bath with Euro-Collins solution for different preservation times. Tolerance of the transplanted liver to cold ischemia was determined by survival time and liver function tests. Rat tumor necrosis factor was analyzed by a bioassay. Nomega-Nitro-L-arginine methyl ester, L-arginine, or adenosine was administered to block or stimulate the synthesis of nitric oxide (NO) in the rats that received long-term-preserved liver grafts., Results: Twenty percent of syngeneic rats (n=10) that received a liver graft with a 16-hr cold ischemia time in Euro-Collins solution survived for more than 1 day and 10% survived for more than 5 days. In contrast, 87.5% of rats (n=8) that received a liver graft with ischemic preconditioning (10-10 group) and 16 hr of cold ischemia survived for more than 1 day and 75% for more than 5 days. Recipients of liver grafts with ischemic preconditioning had significantly reduced levels of serum aspartate transaminase and tumor necrosis factor-alpha, as well as increased bile flow, compared with recipients of liver grafts without ischemic preconditioning. Blockage of the NO pathway using Nomega-nitro-L-arginine methyl ester, a stereospecific competitive inhibitor of NO formation, attenuated the protective effect of ischemic preconditioning. Administration of one of two precursors of NO synthesis, L-arginine or adenosine, prolonged the survival of rats that received 16-hr-preserved liver grafts. In addition, L-arginine synergized with short-term ischemic pre conditioning (5-10 group) to increase the survival of rats that received a liver graft with a 16-hr cold ischemia time, and the survival rate was 83% after 5 days. Finally, prolonged ischemic preconditioning (> or = 20 min; 20-10 group) resulted in liver damage and loss of function., Conclusion: The current results show that ischemic preconditioning protects the liver graft from subsequent long-term cold preservation-reperfusion injury in a rat liver transplantation model. The protective role of ischemic preconditioning may be mediated by the endogenous production of NO.

Background: In these experiments, we studied the role of anti-CD4 (Ox38) monoclonal antibody in the induction of allograft unresponsiveness in high-responder Lewis rats in the single liver, kidney, small bowel, and heart versus the combined heart-kidney, heart-liver, and heart-small bowel transplantation models., Methods: ACI heart, kidney, liver, and small bowel allografts were transplanted into untreated and anti-CD4 treated Lewis rats. In selected animals bearing long-surviving ACI liver or kidney allografts for over 3 months, donor-matched second heart or third-party (Brown Norway) heart allografts were transplanted. Simultaneously, heart-liver, heart-kidney, and heart-small bowel transplants were performed on the day of operation. Rejected allografts were verified by autopsy and pathology., Results: ACI liver allografts were permanently accepted by Lewis recipients treated with either regular-dose (5 mg/kg for 4 days) or low-dose (5 mg/kg for 2 days) of anti-CD4 monoclonal antibody. Pretransplant anti-CD4 therapy (5 mg/kg for 4 days but not 5 mg/kg for 2 days) resulted in a long-term survival of kidney allografts (mean survival time [MST] > 100.0 days, n=5). Pretransplant anti-CD4 treatment (5 mg/kg for 4 days) could not induce tolerance when single ACI hearts were transplanted; however, long-term survival of ACI heart allografts could be induced when heart transplants were combined with liver (n=7) or kidney (n=8) transplants. The survival of both ACI heart allografts (MST=25.0 days, n=4) and small bowel allografts (MST=28.0 days, n=4) was also prolonged when simultaneous heart and small bowel transplantation was performed in anti-CD4-treated recipients. The second ACI heart allograft was permanently accepted by tolerant Lewis recipients of ACI liver or kidney allografts induced by anti-CD4 treatment, and third-party heart grafts were acutely rejected without affecting survival of the primary allografts., Conclusion: Our current results show that: (1) there is a vigorous rejection of heart > or = small bowel > kidney > liver in high-responder Lewis rats after pretransplant anti-CD4 therapy; and (2) simultaneous or metachronous combined liver-heart and kidney-heart transplants may protect heart allografts from rejection.

Intracellular pyrimidine nucleotides (PyN) can be synthesized de novo from glutamine, CO2, and ATP, or they can be salvaged from preformed pyrimidine nucleosides. The antiproliferative and immunosuppressive activities of brequinar sodium (BQR) are thought to be due to the inhibition of the activity of dihydroorotate dehydrogenase, which results in a suppression of de novo pyrimidine synthesis. Here we describe the effects of the pyrimidine nucleoSide, uridine, on the antiproliferative and immunosuppressive activities of BQR. In vitro reduction of PyN levels in Con A-stimulated T cells and inhibition of cell proliferation by low concentrations of BQR (< or =65 microM) are reversed by uridine. However, uridine is unable to reverse the effects of high concentrations of BQR (> or =65 microM). The ability of BQR to induce anemia in BALB/c mice is prevented by the coadministration of uridine. In contrast, the immunosuppressive activity of BQR is unaffected by similar doses of uridine. PyN levels in the bone marrow, but not in the spleen, are depressed in mice treated with BQR. These observations suggest that the induction of anemia by BQR is due to depletion of intracellular PyN in hemopoietic stem cells located in the bone marrow. They also suggest that the mechanism of immunosuppression by BQR may be only marginally dependent on depletion of intracellular PyN in lymphocytes located in the periphery. We report a novel activity of BQR: inhibition of tyrosine phosphorylation, and hypothesize that the immunosuppressive activity may be due, in part, to this unsuspected ability of BQR to inhibit tyrosine phosphorylation in lymphocytes.

Background: Vigorous antibody-mediated responses prevent the successful engraftment of hamster hearts transplanted into Lewis rats. Early antibody responses mediating acute rejection of the xenograft are T cell-independent and resistant to the T-cell immunosuppressant, cyclosporine (CsA). Immunosuppression with the combination of leflunomide plus CsA completely prevents xenograft rejection, but when such immunosuppression is stopped the hamster heart is rejected by a process that we term late xenograft rejection. We report here on some of the immunological features of late xenograft rejection., Methods: Lewis rats transplanted with hamster hearts were treated with leflunomide (5 mg/kg/day by gavage) for 14-21 days and CsA (20 mg/kg/day by gavage) continuously from the day of transplant. Serum was harvested and the functional activities of the xenoreactive antibodies were quantitated by in vivo passive transfer of sera, flow cytometry, in vitro C3 deposition assays, and Western blotting., Results: CsA alone prevented late xenograft rejection and the accompanying production of xenoreactive antibodies. The xenoreactive antibodies accompanying acute or late xenograft rejection were predominantly IgM, but only serum from rats undergoing acute xenograft rejection was able to induce hyperacute rejection. The ability of serum to induce hyperacute rejection correlated with its ability to induce C3 deposition on hamster lymphocytes in vitro. The repertoire of hamster antigens recognized by IgM in the serum of rats undergoing late xenograft rejection is more restricted than that of IgM in the serum of rats undergoing acute xenograft rejection. We additionally demonstrate that long-term graft survival is not dependent on graft accommodation., Conclusions: These studies demonstrate that a brief treatment with the combination of leflunomide and CsA profoundly modifies the humoral xenoreactivity in the recipient, converting it from a T-independent into a T cell-dependent response. Differences in functional activity of sera from acute or late xenograft rejection suggest that antigenic specificity defines the ability of IgM to induce complement activation and hyperacute rejection.

We investigated the effects of pretransplant anti-CD4 monoclonal antibody (mAb) combined with human (h) CTLA4Ig on the survival of hamster heart and liver xenografts. Pretransplant anti-CD4 mAb (5 mg/kg x 4 days) or hCTLA4Ig (0.5 mg/rat on days 1, 3, and 5 after transplantation) treatment alone prolonged the survival of hamster liver xenografts in Lewis rats (mean survival time [MST]=10.5 days, n=6, and MST=9.0 days, n=5, respectively, compared with untreated Lewis recipients of hamster liver grafts, MST=6.0 days, n=6). The same regimen could not prevent hamster heart xenorejection. Pretransplant anti-CD4 mAb (5 mg/kg x 4 days) combined with hCTLA4Ig (0.5 mg/rat x 4) treatments increased survival of hamster liver xenograft fourfold (MST=24.2 days, n=5). The current results also show that IgG in the sera from Lewis recipients of hamster liver grafts treated with anti-CD4 mAb and hCTLA4Ig was threefold reduced at 6 days after transplantation compared with untreated Lewis rats. These results suggest a synergistic effect of anti-CD4 mAb combined with hCTLA4Ig in a liver xenograft transplantation model.

This study was designed to investigate the effectiveness of combined perioperative anti-CD4 and human (h)CTLA4Ig therapy in preventing allorejection of small bowel transplantation in high-responder Lewis rat recipients of ACI grafts. Anti-CD4 (5 mg/kg x 4 days) or hCTLA4Ig (0.5 mg/rat x 2 days) therapy alone delayed, but did not prevent, allograft rejection after small bowel transplantation of ACI into Lewis rats. All grafts were rejected in 18 and 10 days, respectively. However, a regimen of anti-CD4 (5 mg/kg x 4 days) combined with hCTLA4Ig (0.5 mg/rat x 2 days) allowed indefinite survival of ACI small bowel allografts. Second donor-matched heart grafts were permanently accepted, whereas third-party (Sprague-Dawley) heart allografts were rejected by the tolerant recipients. These data suggest that these two reagents produced a synergistic effect in preventing allorejection of small bowel transplantation.

The generation of knockout mice with targeted gene disruption has provided a valuable tool for studying the immune response. Here we describe the use of CD4 and CD8 knockout mice to examine the role of CD4+ and CD8+ cells in initiating allotransplantation rejection. Pretreatment with a brief course of depletive anti-CD4 monoclonal antibody therapy allowed permanent survival of heart, but not skin, allografts transplanted across a major histocompatibility barrier. However, skin as well as heart grafts were permanently accepted in the CD4 knockout mice. Transfer of CD4+ cells into CD4 knockout recipient mice 1 d before skin engraftment reconstituted rejection, demonstrating that CD4+ cells are necessary for initiating rejection of allogeneic transplants. Major histocompatibility complex disparate heart and skin allografts transplanted into CD8 knockout recipients were rejected within 10 d. This study demonstrates that CD4+ but not CD8+ T cells are absolutely required to initiate allograft rejection.