Immune escape of BA.2.86 is comparable to XBB subvariants from the plasma of BA.5- and BA.5-XBB-convalescent subpopulations.

The EG.5.1 variant of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been prevalent since mid-July 2023 in the United States and China. The variant BA.2.86 has become a major concern because it is 34 mutations away from the parental variant BA.2 and >30 mutations from XBB.1.5. There is an urgent need to evaluate whether the immunity of the population and current vaccines are protective against EG.5.1 and BA.2.86. Based on a cohort of two breakthrough-infected groups, the levels of neutralizing antibodies (NAbs) against different subvariants were measured using pseudovirus-based neutralization assays. XBB.1.5, EG.5.1, and BA.2.86 are comparably immune-evasive from neutralization by the plasma of individuals recovered from BA.5 infection (BA.5-convalescent) or XBB.1.9.2/XBB.1.5 infection following BA.5 infection (BA.5-XBB-convalescent). NAb levels against EG.5.1 and BA.2.86 subvariants remained >120 geometric mean titers (GMTs) in BA.5-XBB-convalescent individuals 2 months postinfection but were <40 GMTs in BA.5-convalescent individuals. Furthermore, the XBB-targeting messenger RNA (mRNA) vaccine RQ3033 induced higher levels of NAbs against XBB.1.5, EG.5.1, and BA.2.86 than against BA.5-XBB infection. The results suggest that BA.2.86 and EG.5.1 are unlikely to cause more severe concerns than the currently circulating XBB subvariants and that the XBB.1.5-targeting mRNA vaccine tested has promising protection against EG.5.1 and BA.2.86.

Development of SARS-CoV-2 animal vaccines using a stable and efficient NDV expression system.

With the continuation of the coronavirus disease 2019 pandemic and the emergence of new severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants, the control of the spread of the virus remains urgent. Various animals, including cats, ferrets, hamsters, nonhuman primates, minks, tree shrews, fruit bats, and rabbits, are susceptible to SARS-CoV-2 infection naturally or experimentally. Therefore, to avoid animals from becoming mixing vessels of the virus, vaccination of animals should be considered. In the present study, we report the establishment of an efficient and stable system using Newcastle disease virus (NDV) as a vector to express SARS-CoV-2 spike protein/subunit for the rapid generation of vaccines against SARS-CoV-2 in animals. Our data showed that the S and S1 protein was sufficiently expressed in rNDV-S and rNDV-S1-infected cells, respectively. The S protein was incorporated into and displayed on the surface of rNDV-S viral particles. Intramuscular immunization with rNDV-S was found to induce the highest level of binding and neutralizing antibodies, as well as strong S-specific T-cell response in mice. Intranasal immunization with rNDV-S1 provoked a robust T-cell response but barely any detectable antibodies. Overall, the NDV-vectored vaccine candidates were able to induce profound humoral and cellular immunity, which will provide a good system for developing vaccines targeting both T-cell and antibody responses.

Dispersal route of the Asian house rat (Rattus tanezumi) on mainland China: insights from microsatellite and mitochondrial DNA.

BACKGROUND: Rattus tanezumi is a common commensal rat and an important host animal of bubonic plague in South China and Southeast Asia. The northward dispersal of this species in mainland China has been reported in recent decades, along with more recent intercontinental expansion. Population genetics of R. tanezumi in mainland China were studied to explain the relationship between dispersal history and the ancient and modern transportation networks of China. RESULTS: In total, 502 individuals belonging to 18 populations were collected from 13 provincial areas. Nine microsatellite loci and two mtDNA sequences were analyzed. The results indicate that R. tanezumi populations from Yunnan have highest genetic diversity and populations from Tibet with lowest genetic diversity. 18 populations can be divided into four clusters, the first cluster including populations from southwest Yunnan, the second including two populations of Tibet, the third for populations from middle and east of mainland China, and the forth for two populations from north Yunnan. Both microsatellite and mtDNA data reveal that the populations from coastal areas are closely related to populations from Yunnan, whereas populations from Tibet are closely related with populations from Sichuan. CONCLUSIONS: The results suggest that early dispersal of R. tanezumi in mainland China depended on shipping transportation, with subsequent expansion from coastal areas into Central China occurring along the Yangzi River. Further, the linkages between populations in Tibet and Sichuan point to a modern era introduction via the Chuan-Zang highway, rather than along the Tea Horse Ancient Road.

Niche modeling predictions of the potential distribution of Marmota himalayana, the host animal of plague in Yushu County of Qinghai.

BACKGROUND: After the earthquake on 14, April 2010 at Yushu in China, a plague epidemic hosted by Himalayan marmot (Marmota himalayana) became a major public health concern during the reconstruction period. A rapid assessment of the distribution of Himalayan marmot in the area was urgent. The aims of this study were to analyze the relationship between environmental factors and the distribution of burrow systems of the marmot and to predict the distribution of marmots. METHODS: Two types of marmot burrows (hibernation and temporary) in Yushu County were investigated from June to September in 2011. The location of every burrow was recorded with a global positioning system receiver. An ecological niche model was used to determine the relationship between the burrow occurrence data and environmental variables, such as land surface temperature (LST) in winter and summer, normalized difference vegetation index (NDVI) in winter and summer, elevation, and soil type. The predictive accuracies of the models were assessed by the area under the curve of the receiving operator curve. RESULTS: The models for hibernation and temporary burrows both performed well. The contribution orders of the variables were LST in winter and soil type, NDVI in winter and elevation for the hibernation burrow model, and LST in summer, NDVI in summer, soil type and elevation in the temporary burrow model. There were non-linear relationships between the probability of burrow presence and LST, NDVI and elevation. LST of 14 and 23 °C, NDVI of 0.22 and 0.60, and 4100 m were inflection points. A substantially higher probability of burrow presence was observed in swamp soil and dark felty soil than in other soil types. The potential area for hibernation burrows was 5696 km(2) (37.7% of Yushu County), and the area for temporary burrows was 7711 km(2) (51.0% of Yushu County). CONCLUSIONS: The results suggested that marmots preferred warm areas with relatively low altitudes and good vegetation conditions in Yushu County. Based on these results, the present research is useful in understanding the niche selection and distribution pattern of marmots in this region.

High prevalence and genetic heterogeneity of rodent-borne Bartonella species on Heixiazi Island, China.

We performed genetic analysis of Bartonella isolates from rodent populations from Heixiazi Island in northeast China. Animals were captured at four sites representing grassland and brushwood habitats in 2011 and examined for the prevalence and genetic diversity of Bartonella species, their relationship to their hosts, and geographic distribution. A high prevalence (57.7%) and a high diversity (14 unique genotypes which belonged to 8 clades) of Bartonella spp. were detected from 71 rodents comprising 5 species and 4 genera from 3 rodent families. Forty-one Bartonella isolates were recovered and identified, including B. taylorii, B. japonica, B. coopersplainsensis, B. grahamii, B. washoensis subsp. cynomysii, B. doshiae, and two novel Bartonella species, by sequencing of four genes (gltA, the 16S rRNA gene, ftsZ, and rpoB). The isolates of B. taylorii and B. grahamii were the most prevalent and exhibited genetic difference from isolates identified elsewhere. Several isolates clustered with strains from Japan and far-eastern Russia; strains isolated from the same host typically were found within the same cluster. Species descriptions are provided for Bartonella heixiaziensis sp. nov. and B. fuyuanensis sp. nov.

Neutral Forces and Balancing Selection Interplay to Shape the Major Histocompatibility Complex Spatial Patterns in the Striped Hamster in Inner Mongolia: Suggestive of Broad-Scale Local Adaptation.

BACKGROUND: The major histocompatibility complex (MHC) plays a key role in the adaptive immune response to pathogens due to its extraordinary polymorphism. However, the spatial patterns of MHC variation in the striped hamster remain unclear, particularly regarding the relative contribution of the balancing selection in shaping MHC spatial variation and diversity compared to neutral forces. METHODS: In this study, we investigated the immunogenic variation of the striped hamster in four wild populations in Inner Mongolia which experience a heterogeneous parasitic burden. Our goal was to identify local adaptation by comparing the genetic structure at the MHC with that at seven microsatellite loci, taking into account neutral processes. RESULTS: We observed significant variation in parasite pressure among sites, with parasite burden showing a correlation with temperature and precipitation. Molecular analysis revealed a similar co-structure between MHC and microsatellite loci. We observed lower genetic differentiation at MHC loci compared to microsatellite loci, and no correlation was found between the two. CONCLUSIONS: Overall, these results suggest a complex interplay between neutral evolutionary forces and balancing selection in shaping the spatial patterns of MHC variation. Local adaptation was not detected on a small scale but may be applicable on a larger scale.

Climate heterogeneity, season variation, and sexual dimorphism modulate the association between MHC II diversity and parasite variation in striped hamster.

Parasite-mediated selection is widely believed to play a crucial role in maintaining the diversity of the major histocompatibility complex (MHC) genes, which is thought to be maintained through heterozygote advantage, rare-allele advantage, and fluctuating selection. However, the relationship between parasite pressure and MHC diversity has yielded inconsistent findings. These inconsistencies may arise from the influence of environmental factors and individual variations in traits on host-parasite interactions. To address these issues, our study extensively investigated populations of striped hamsters inhabiting regions characterized by environmental heterogeneity. The primary objective was to examine the universality of parasite-mediated selection mechanisms. Our observations revealed the presence of multiple parasite infections, accompanied by spatial and temporal variations in parasite communities and infection patterns among individual hamsters. Specifically, the temperature was found to influence all four parasite indices, while the presence of gamasid mites and parasite richness decreased with increasing precipitation. We also noted significant seasonal variation in parasite dynamics. Moreover, a significant sexual dimorphism was observed with males exhibiting a considerably higher parasite burden compared to their female counterparts. Lastly, we identified the maintenance of MHC polymorphism in striped hamsters as being driven by the heterozygote advantage and fluctuating selection mechanisms. This study underscores the significance of ecological processes in comprehending host-parasite systems and highlights the necessity of considering environmental factors and individual traits when elucidating the mechanisms underlying MHC diversity mediated by parasites.

SARS-CoV-2-specific CD4+ T cells are associated with long-term persistence of neutralizing antibodies.

Understanding the decay and maintenance of long-term SARS-CoV-2 neutralizing antibodies in infected or vaccinated people and how vaccines protect against other SARS-CoV-2 variants is critical for assessing public vaccination plans. Here, we measured different plasm antibody levels 2 and 12 months after disease onset, including anti-RBD, anti-N, total neutralizing antibodies, and two neutralizing-antibody clusters. We found that total neutralizing antibodies declined more slowly than total anti-RBD and anti-N IgG, and the two neutralizing-antibody clusters decayed even more slowly than total neutralizing antibodies. Interestingly, the level of neutralizing antibodies at 12 months after disease onset was significantly lower than that at 2 months but more broadly neutralized SARS-CoV-2 variants, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Lambda (C.37). Significant immune escape by the Omicron variant (B.1.1.529) was also observed 2 months post-recovery. Furthermore, we revealed that a high percentage of virus-specific CD4+ T cells and cTfh1 were associated with a slower decline in humoral immunity, accompanied by higher levels of CXCR3 ligands such as CXCL9 and CXCL10, higher frequency of cTfh1, and lower levels of cTfh2 and cTfh17. Our data highlight the importance of coordinating T-cell and humoral immunity to achieve long-term protective immunity.

Heterologous boosting with third dose of coronavirus disease recombinant subunit vaccine increases neutralizing antibodies and T cell immunity against different severe acute respiratory syndrome coronavirus 2 variants.

Waned vaccine-induced immunity and emerging severe acute respiratory syndrome coronavirus 2 variants with potential for immune escape pose a major threat to the coronavirus disease (COVID-19) pandemic. Here, we showed that humoral immunity components, including anti-S + N, anti-RBD IgG, and neutralizing antibodies (NAbs), gradually waned and decreased the neutralizing capacity against emerging Omicron variants at 3 and 6 months after two inactivated COVID-19 vaccinations. We evaluated two boosting strategies with either a third dose of inactivated vaccine (homologous, I-I-I) or a recombinant subunit vaccine (heterologous, I-I-S). Both strategies induced the production of high levels of NAbs with a broad neutralizing capacity and longer retention. Interestingly, I-I-S induced 3.5-fold to 6.8-fold higher NAb titres than I-I-I, with a broader neutralizing capacity against six variants of concern, including Omicron. Further immunological analysis revealed that the two immunization strategies differ considerably, not only in the magnitude of total NAbs produced, but also in the composite pattern of NAbs and the population of virus-specific CD4+ T cells produced. Additionally, in some cases, heterologous boosted immunity induced the production of more effective epitopes than natural infection. The level of I-I-S-induced NAbs decreased to 48% and 18% at 1 and 3 months after booster vaccination, respectively. Overall, our data provide important evidence for vaccination strategies based on available vaccines and may help guide future global vaccination plans.

SARS-CoV-2 vaccination-infection pattern imprints and diversifies T cell differentiation and neutralizing response against Omicron subvariants.

The effects of different SARS-CoV-2 vaccinations and variant infection histories on imprinting population immunity and their influence on emerging escape mutants remain unclear. We found that Omicron (BA.1) breakthrough infection, regardless of vaccination with two-dose mRNA vaccines (M-M-o) or two-dose inactivated vaccines (I-I-o), led to higher neutralizing antibody levels against different variants and stronger T-cell responses than Delta breakthrough infection after two-dose inactivated vaccine vaccination (I-I-δ). Furthermore, different vaccination-infection patterns imprinted virus-specific T-cell differentiation; M-M-ο showed higher S/M/N/E-specific CD4+ T cells and less portion of virus-specific CD45RA+CD27-CD8+ T cells by ex vivo assay. Breakthrough infection groups showed higher proliferation and multi-function capacity by in vitro assay than three-dose inactivated vaccine inoculated group (I-I-I). Thus, under wide vaccination coverage, the higher immunogenicity with the Omicron variant may have helped to eliminate the population of Delta variant. Overall, our data contribute to our understanding of immune imprinting in different sub-populations and may guide future vaccination programs.

Spatiotemporal Dynamics of Scrub Typhus in Jiangxi Province, China, from 2006 to 2018.

Background: Scrub typhus (ST) has become a significant potential threat to public health in Jiangxi. Further investigation is essential for the control and management of the spatiotemporal patterns of the disease. Methods: Time-series analyses, spatial distribution analyses, spatial autocorrelation analysis, and space-time scan statistics were performed to detect spatiotemporal dynamics distribution of the incidence of ST. Results: From 2006 to 2018, a total of 5508 ST cases occurred in Jiangxi, covering 79 counties. The number of ST cases increased continuously from 2006 to 2018, and there was obvious seasonality during the variation process in each year, with a primary peak in autumn (September to October) and a smaller peak in summer (June to August). From 2007 to 2018, the spatial distribution of the ST epidemic was significant heterogeneity, and Nanfeng, Huichang, Xunwu, Anyuan, Longnan, and Xinfeng were hotspots. Seven spatiotemporal clusters were observed using Kulldorff's space-time scan statistic, and the most likely cluster only included one county, Nanfeng county. The high-risk areas of the disease were in the mountainous, hilly region of Wuyi and the southern mountainous region of Jiangxi. Conclusions: Targeted interventions should be executed in high-risk regions for the precise prevention and control of ST.

Scrub typhus in previously unrecognized areas of endemicity in China.

Scrub typhus, caused by Orientia tsutsugamushi, has emerged recently in areas of northern China where the disease had not been known to exist. We analyzed epidemiological, clinical, and laboratory data for 104 patients who were admitted to a hospital in Fuyang City between 26 September and 1 November 2008. We showed that the major clinical manifestations of the patients were fever (100%), headache (82%), myalgias (77%), eschar (67%), rash (52%), and unusual facial flushing (62%). Among the 104 patients, the sera of 98% contained IgM antibodies to O. tsutsugamushi detected by indirect immunofluorescence assays (IFA), and DNA of the O. tsutsugamushi 56-kDa gene was amplified by PCR from the blood of 36 patients. We conclude that 104 patients were infected with scrub typhus in Fuyang City, Anhui Province. Our study indicates that physicians need to consider the diagnosis of scrub typhus for febrile patients living in northern China, where scrub typhus had not been considered to exist in the past.

Spatio-temporal patterns of scrub typhus in mainland China, 2006-2017.

BACKGROUND: Scrub typhus, a serious public health problem in the Asia-Pacific area, is endemic in the "tsutsugamushi triangle" area. Scrub typhus has been widespread and has become a significant health concern in China. However, spatiotemporal patterns need to be investigated further. OBJECTIVE: This study aims to explore spatiotemporal patterns, diffusion characteristics and regional distribution differences of scrub typhus cases in mainland China from January 2006 to December 2017. METHOD: Monthly cases of scrub typhus reported at the county level during 2006-2017 were obtained. Time-series analyses, spatial distribution analyses, spatial diffusion analyses, spatial autocorrelation analyses and space-time scan statistic analyses were used to explore spatiotemporal characteristics of scrub typhus. RESULTS: A total of 121 251 scrub typhus cases were reported in 30 provinces (or municipalities) of mainland China during 2006-2017, which rose exponentially. There were seasonal characteristics from June to November for scrub typhus. Scrub typhus had been diffused from south, southwest, southeast and eastern coasts to center, north, northeast and northwest in mainland China. Scrub typhus occurrences were from point to surrounding regions, and from south to north every year. The peak periods of scrub typhus became longer and longer from north to southwest to south in mainland China. There existed a single peak in Southwest region and North region, respectively, but existed a bimodal peak for South region. Scrub typhus cases were clustered in Yunnan, Guangdong, Guangxi, Fujian and Anhui among June to November. The scrub typhus epidemics in Guangdong and Yunnan were the most serious. CONCLUSIONS: The results in this study can be guide targeted public health interventions against scrub typhus at the county level.

Mosquito population dynamics during the construction of Three Gorges Dam in Yangtze River, China.

BACKGROUND: Mosquitoes are responsible for spreading many diseases and their populations are susceptible to environmental changes. The ecosystems in the Three Gorges Region were probably altered because of changes to the environment during the construction of the Three Gorges Dam (TGD), the world's largest hydroelectric dam by generating capacity. METHODS: We selected three sites at which to monitor the mosquitoes from 1997 to 2009. We captured adult mosquitoes with battery-powered aspirators fortnightly between May and September of each year in dwellings and sheds. We identified the mosquito species, and examined changes in the species density during the TGD construction. We monitored changes in the species and density of mosquitoes in this area for 13 years during the TGD construction and collected information that could be used to support the control and prevention of mosquito-borne infections. RESULTS: We found that the mosquito species composition around the residential areas remained the same, and the density changed gradually during the TGD construction. The changes in the populations tended to be consistent over the years, and the densities were highest in July, and were between 3 and 5 times greater in the sheds than in the dwellings. CONCLUSIONS: The mosquito species and populations remained stable during the construction of the TGD. The mosquito density may have increased as the reservoir filled, and may have decreased during the clean-up work. Clean-up work may be an effective way to control mosquitoes and prevent mosquito-borne diseases.

Governmental supervision and rapid detection on dengue vectors: An important role for dengue control in China.

BACKGROUND: China experienced an unprecedented outbreak of dengue fever in 2014, the National Health and Family Planning Commission of the People's Republic of China (NHFPC) carried out a series of supervision work on integrated vector management (IVM), and Chinese Center for Disease Control and Prevention (China CDC) conducted a rapid detection on vector density in some areas with high dengue incidence. The goal of this study was to explain the effect of these actions, which play an important role for dengue control, and we wish to give a good example for dengue control in China, even in the world. METHODS: Compare mosquito vector density with Breteau Index (BI) and dengue incidence after or along with control work vs. before. Data was entered and analyzed by Microsoft Excel 2007 and SPSS19.0. RESULTS: Average value of BI from 22.82 in September dropped to 3.93 along with supervision and rapid detection. BI showed a significant decrease (paired sample t-test, t=3.061, P=0.018≺0.05). Dengue incidence decreased gradually along with supervision and rapid detection. CONCLUSIONS: Supervised work on IVM by NHFPC and the rapid detection on dengue vector Aedes by China CDC promoted to cut down the dengue vector density, then reduced dengue incidence; both played an important role for dengue control throughout China in 2014.

[Roles of mitochondria in vitamin E succinate-induced apoptosis in human gastric cancer SGC-7901 cells].

OBJECTIVE: In order to investigate the roles of mitochondria in vitamin E succinate (VES)-induced apoptosis in human gastric cancer SGC-7901 cells. METHODS: Mitochondrial transmembrane potential (deltapsi(m)) was observed by confocal laser scanning microscopy, while the expression of cytochrome c in cytosol and caspase-3 was measured by western blotting after the cells were treated with VES at 5, 10, 20 microg/ml. RESULTS: VES obviously decreased deltapsi(m) with dose- and time-dependent relationship, increased the expression of cytochrome c in cytosol and caspase-3 and activated caspase-3 as well. CONCLUSION: VES-induced apoptosis in SGC-7901 cells might involve mitochondrial permeability transition, release of cytochrome c and activation of caspase-3 downstream.

c-Jun N-terminal kinase is required for vitamin E succinate-induced apoptosis in human gastric cancer cells.

AIM: To investigate the roles of c-Jun N-terminal kinase (JNK) signaling pathway in vitamin E succinate-induced apoptosis in human gastric cancer SGC-7901 cells. METHODS: Human gastric cancer cell lines (SGC-7901) were treated with vitamin E succinate (VES) at 5, 10, 20 mg/L. Succinic acid and vitamin E were used as vehicle controls and condition medium only as an untreated (UT) control. Apoptosis was observed by 4', 6-diamidine-2'-phenylindole dihydrochloride (DAPI) staining for morphological changes and by DNA fragmentation for biochemical alterations. Western blot analysis was applied to measure the expression of JNK and phosphorylated JNK. After the cells were transiently transfected with dominant negative mutant of JNK (DN-JNK) followed by treatment of VES, the expression of JNK and c-Jun protein was determined. RESULTS: The apoptotic changes were observed after VES treatment by DNA fragmentation. DNA ladder in the 20 mg/L VES group was more clearly seen than that in 10 mg/L VES group and was not detected following treatment of UT control, succinate and vitamin E. VES at 5, 10 and 20 mg/L increased the expression of p-JNK by 2.5-, 2.8- and 4.2-fold, respectively. VES induced the phosphorylation of JNK beginning at 1.5 h and produced a sustained increase for 24 h with the peak level at 12 h. Transient transfection of DN-JNK blocked VES-triggered apoptosis by 52%. DN-JNK significantly increased the level of JNK, while decreasing the expression of VES-induced c-Jun protein. CONCLUSION: VES-induced apoptosis in human gastric cancer SGC-7901 cells involves JNK signaling pathway via c-Jun and its downstream transcription factor.

[Roles of ERK1/2 MAPK in vitamin E succinate-induced apoptosis in human gastric cancer SGC-7901 cells].

In order to investigate the roles of ERK1/2 mitogen-activated protein kinase in vitamin E succinate (VES)-induced apoptosis in human gastric cancer SGC-7901 cells, apoptosis was observed by DAPI staining and the phosphorylation of ERK1/2 by VES at different doses and different time points was measured by western blot. The results showed that VES obviously induced cells to undergo apoptosis and apoptotic rate after 24 h and 48 h of treatment with VES at 20 micrograms/ml. VES was 14.2% and 89.4%, respectively. The expression of p-ERK was evidently reduced by VES at 5, 10 and 20 micrograms/ml for 24 h. ERK1/2 was immediately activated by VES at 20 micrograms/ml, but the expression was decreased for 2 h, then increased again and reached the top level for 12 h. The data implicated that ERK1/2 pathway might be involved in VES-induced apoptosis, but in the proliferation eventually.

Differentiation-associated genes regulated by c-Jun and decreased in the progression of esophageal squamous cell carcinoma.

Transcription factor c-Jun plays a key role in controlling epithelium cell proliferation, apoptosis and differentiation. However, molecular mechanism and biological functions of c-Jun in squamous differentiation and the progression of esophageal squamous cell carcinoma (ESCC) remain elusive. In this study, we found that c-Jun bound directly to the promoter region, and activated the transcription of differentiation-associated genes including cystatin A, involucrin and SPRR3 in vivo. Ectopic expression of c-Jun enhanced SPRR3 transactivation in KYSE450 cells. Conversely, TAM67, a dominant negative mutant of c-Jun, inhibited SPRR3 transactivation. c-Jun increased expression of SPPR3 mainly via a PKC/JNK pathway in response to TPA in KYSE450 cells. Furthermore, c-Jun was remarkably reduced in esophageal cancer. Interestingly, cystatin A, involucrin and SPRR3 were significantly downregulated as well, and associated with differentiation grade. Expression of c-Jun was correlated with the expression of these genes in normal epithelium and ESCC. Importantly, the expression of these genes was remarkably decreased during the malignant transformation from normal epithelium to low-grade intraepithelial neoplasia (LGIN) or high-grade intraepithelial neoplasia (HGIN). The expression of cystatin A and involucrin was significantly reduced from LGIN to HGIN. These results suggest c-Jun was involved in the regulation of differentiation-associated genes in ESCC. These genes might serve as the potential markers in distinguishing normal epithelium from esophageal squamous intraepithelial neoplasia.