Immune response studies with <Emphasis Type="Italic">Wuchereria bancrofti</Emphasis> vespid allergen homologue (WbVAH) in human lymphatic filariasis

A homologue of Brugia malayi venom allergen (BmVAH) was cloned from the infective stages (L3) of Wuchereria bancrofti. Sequence analysis showed 90% sequence identity between WbVAH and BmVAH. Recombinant WbVAH was then expressed and purified. VAH from other nematode parasites is being evaluated as potential vaccine candidates. Because W. bancrofti infections are more prevalent than B. malayi, it will significantly benefit using W. bancrofti antigens for vaccine development. In this study, we have evaluated the human immune responses to rWbVAH in putatively immune individuals who live in the endemic regions (endemic normal, EN) to determine the vaccine potential of WbVAH. These responses were then compared to those in infected individuals (microfilaraemic, MF and chronic pathology, CP). Results show that EN subjects carry WbVAH-specific IgG1, IgG2, and IgG3 circulating antibodies. It is interesting to note that CP patients also carried antibodies against WbVAH that was mainly of the IgG3 isotype. Peripheral blood mononuclear cells (PBMC) from EN individuals responded strongly to rWbVAH by proliferating and secreting IFN-γ. PBMC from MF patients also proliferated in response to rWbVAH but secreted mainly IL-10. Thus, there was a clear dichotomy in the cytokine production by infected patients vs individuals who are putatively immune (EN). Although vaccine potential of WbVAH has not been established yet, our findings suggest that WbVAH mediated immune responses in EN individuals is primarily Th1-biased. Further vaccination studies are underway in animal models to determine the role of WbVAH in protective immunity against W. bancrofti and B. malayi infections. S.B.A. and M.G. contributed equally to this study and both should be considered first authors.     

An in vitro model to evaluate the cytokine response in<Emphasis Type="Italic"> Echinococcus</Emphasis> infections

With the aim of proposing an alternative model to animal experimentation, we investigated cytokine production in response to antigens in an in vitro system. This is a co-culture system of healthy human leukocytes and enterocyte-like Caco-2 cells. The antigens tested, EgA31, EgTrp, and FABP1, are candidates for vaccines in infections caused by Echinococcus spp. in the gut. All three have previously been described in the protoscolex stage and belong to protein families which confer protective immunity against several helminths. In this study, we evaluate the Th1/Th2 profile (Th1: IL-12, IFN-; Th2: IL-6, IL-10) in response to protoscoleces, EgA31 and the mixture of EgA31, EgTrp and FABP1. No cytokine production was detected in response to protoscoleces. Neither IFN- nor IL-6, but a significant IL-10 and IL-12 concentration was detected in response to both types of antigens. These findings suggest that EgA31 and the mixture EgA31/EgTrp/FABP1 generated an immunogenic response associated with a mixed Th1/Th2 cytokine.     

Immune responses to recombinant <Emphasis Type="Italic">Brugia malayi</Emphasis> pepsin inhibitor homolog (Bm-33) in patients with human lymphatic filariaisis

Immune responses to recombinant Brugia malayi pepsin inhibitor homolog (rBm-33) were investigated in patients with human lymphatic filariasis (microfilaremics (MF) and chronic pathology (CP)) along with endemic normals (EN). Flow cytometric analysis (24 h) revealed CD4⁺ T cell activation in patients (MF and CP) compared to normals (EN), with increased expression of CD69 and diminished levels of CD62L and CD127. This was associated with an elevated expression of CD154 but not CD28 and CTLA4 in CP patients. However, Bm-33-induced cytokine expression profile (IL-1β, IL-12, IL-8, IFN-γ, IL-10 and TGF-β) did not exhibit any significant difference between normals and patients at the same time point. Although CD4⁺ T cell activation was observed initially in filarial patients (24 h), lymphoproliferation studies (96 h) suggested diminished proliferation compared to normals, indicating functional inactivation in the former upon prolonged antigen exposure. This indicates that rBm-33 induces an early T cell activation in MF and CP patients followed by a decreased lymphoproliferation that might contribute to immune suppression in these individuals.     

Dichotomy between<Emphasis Type="Italic"> Lactobacillus rhamnosus</Emphasis> and<Emphasis Type="Italic"> Klebsiella pneumoniae</Emphasis> on dendritic cell phenotype and function

The reaction of the intestinal immune system to intestinal bacteria shows striking differences between various bacterial strains. Whereas Klebsiella pneumoniae induces a fierce proinflammatory reaction, the probiotic strain Lactobacillus rhamnosus has clear anti-inflammatory effect in gastrointestinal disease and allergy. The molecular basis for this dichotomy is poorly understood but is likely to involve different modulation of antigen-presenting dendritic cells (DC) by L. rhamnosus and K. pneumoniae. Hence we evaluated phenotypic and functional characteristics of DC matured in the presence of L. rhamnosus and K. pneumoniae. Monocyte-derived immature DC were cultured in the presence of live bacteria to obtain mature DC. Both micro-organisms induced maturation of immature DC as shown by CD83 and CD86 expression, but receptors involved in activation of Th1 cells were expressed predominantly on DC exposed to K. pneumoniae. In contrast to K. pneumoniae, maturation with L. rhamnosus resulted in lower TNF-, IL-6, and IL-8 production by immature DC and lower IL-12 and IL-18 production by mature DC. Moreover, L. rhamnosus led to the development of T cells without a typical Th phenotype whereas K. pneumoniae induced a Th1 immune response, dependent mainly on IL-12 production. Thus our results strongly support the concept that differential modulation of DC explains the differences in the immune response to various bacterial strains and indicates that K. pneumoniae induces Th1 immune responses via DC.Abbreviations DC Dendritic cell - FACS Fluorescence-activated cell sorter - ICAM Intercellular adhesion molecule - IFN Interferon - IL Interleukin - LPS Lipopolysaccharide - MFI Mean fluorescence intensity - PAMP Pathogen associated molecular pattern - Th T helper cell - TNF Tumor necrosis factor     

Repeat induced point mutation in two asexual fungi, <Emphasis Type="Italic">Aspergillus niger</Emphasis> and <Emphasis Type="Italic">Penicillium </Emphasis><Emphasis Type="Italic">chrysogenum</Emphasis>

Repeat induced point mutation (RIP) is a gene silencing mechanism present in fungal genomes. During RIP, duplicated sequences are efficiently and irreversibly mutated by transitions from C:G to T:A. For the first time, we have identified traces of RIP in transposable elements of Aspergillus niger and Penicillium chrysogenum, two biotechnologically relevant fungi. We found that RIP in P. chrysogenum has affected a large set of sequences, which also contain other mutations. On the other hand, RIP in A. niger is limited to only few sequences, but literally all mutations are RIP-like. Surprisingly, RIP occurred only in transposon sequences that have disrupted open reading frames in A. niger, a phenomenon not yet reported for other fungi. In both fungal species, we identified two sequences with strong sequence similarity to Neurospora crassa RID. RID is a putative DNA methyltransferase and the only known enzyme involved in the RIP process. Our findings suggest that both A. niger and P. chrysogenum either had a sexual past or have a sexual potential. These findings have important implications for future strain development of these fungi.     

<Emphasis Type="Italic">Taenia solium</Emphasis> metacestode antigens which are protective for pigs induce Th1/Th2 mixed responses in mice

The purpose of this study was to determine the Th1 and Th2 cytokine responses induced by Taenia solium metacestode antigens in mice and correlate them with the immune responses elicited in vivo. To assess this aim, mice were inoculated with metacestode antigens. RNA was obtained from spleen cells of immunized or control mice incubated with metacestode antigens and used to determine the cytokine profile. Peripheral blood eosinophilia was measured daily in each mouse and specific serum antibody levels were determined. Results showed that metacestode antigens induce the synthesis of IL-4, IL-5 and IFN-gamma mRNAs in spleen cells. They also induced peripheral blood eosinophilia and elicited specific IgE and IgG antibodies, especially IgG1. Three antigens were recognized by all IgG subclasses and by IgE (104, 88 and 7 kDa), and a 57-kDa protein was recognized by IgG1, IgG2a, IgG2b, and IgE. IgG1 and IgG2b recognized 52, 30 and 20 kDa antigens. Immune responses elicited in vivo and the cytokine profile showed good correlation.     

<Emphasis Type="Italic">Toxoplasma gondii</Emphasis>: expression and characterization of a recombinant protein containing SAG1 and GRA2 in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Toxoplasma gondii is an obligate intracellular protozoan which infects most species of warm-blooded animals and causes toxoplasmosis. Previous immunological and immunization studies have demonstrated the potential role of T. gondii antigens SAG1 and GRA2 as a vaccine candidate. In the present study, we have cloned, expressed, and purified a recombinant protein SAG1–GRA2 in Pichia pastoris. Results showed that P. pastoris was a robust system producing a large amount of highly purified and biological activity protein. BALB/c mice immunized with SAG1–GRA2 elicited stronger humoral and cellular responses in comparison to control groups. This immunization resulted in an enhanced Th1 immune response as measured by IgG2a antibody production and increased splenocyte IFN-γ production, whereas no IL-4 was detected. After a lethal challenge with the highly virulent T. gondii RH strain, a prolonged survival time in SAG1–GRA2-immunized mice was observed in comparison to control groups. Our data demonstrate that SAG1–GRA2 triggered a protective response against toxoplasmosis. Therefore, SAG1–GRA2 protein might be a good candidate for the further development of a multiantigenic vaccine.     

Characterization of <Emphasis Type="Italic">p</Emphasis>24 Gene of <Emphasis Type="Italic">Spodoptera litura</Emphasis> Multicapsid Nucleopolyhedrovirus

Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) p24 gene is 753 bp long, potentially encoding 244 amino acids with a predicted molecular weight of 27.3 kDa. Homology analysis indicated that SpltMNPV P24 has 20–36% amino acid identity with that of other known baculoviruses. RT-PCR results showed that the p24 gene is transcribed actively at the late stage of infection and the mRNA start site was mapped within a consensus baculovirus late promoter sequence (ATAAG). Western blot analysis of extracts from SpltMNPV-infected S. litura cells detected a specific 28 kDa protein, and this protein was not N-glycosylated. Structural localization revealed that SpltMNPV P24 was associated with the nucleocapsid of occlusion-derived virus (ODV) as a complex form of 83 kDa.     

Molecular characterization of a<Emphasis Type="Italic"> Brugia malayi</Emphasis> transglutaminase

Prior studies have demonstrated that transglutaminase (TGase) from the human filarial parasite Brugia malayi is critical for the growth and development of the larval stages. In this report, we describe the cloning and partial characterization of a cDNA encoding the B. malayi TGase (BmTGase). Using RT-PCR and RACE-PCR, the cDNA was amplified from adult worm mRNA. BmTGase is 1,881 bp long and codes for a protein with a predicted molecular mass of 54 kDa. Amino acid sequence analysis of BmTGase revealed significant homology to the protein disulfide isomerase (PDI), particularly, to the PDI-related protein ERp60, a PDI isoform found in the lumen of endoplasmic reticulum. The activity of recombinant B. malayi TGase enzyme (rBmTG) was found to be calcium-dependent and could be inhibited by EDTA. ELISA studies showed that approximately 88% of 48 sera from healthy Indian patients living in a bancroftian filariasis endemic area were reactive with rBmTG. In contrast, only 33% of sera from patients with clinical filariasis were reactive to rBmTG. Non-endemic sera were uniformly non-reactive. Additional studies are needed to elucidate the role, if any, of B. malayi TGase in protective immunity to filariasis.     

Association of positional and functional candidate genes<Emphasis Type="Italic"> FGF1</Emphasis>,<Emphasis Type="Italic"> FBN2</Emphasis>, and<Emphasis Type="Italic"> LOX</Emphasis> on 5q31 with intracranial aneurysm

We previously performed a genome-wide linkage study of intracranial aneurysm (IA) and found positive evidence of linkage at chromosomes 5q22–31, 7q11, and 14q22. In the present study, we focus on 5q31, where three candidate genes, fibroblast growth factor 1 (FGF1), fibrillin 2 (FBN2), and lysyl oxidase gene (LOX) lie, and evaluate associations with IA. Genomic DNAs were obtained from 172 IA patients and 192 controls. Association analysis was performed with ten, five, and four single-nucleotide polymorphisms (SNPs) identified in FGF1, FBN2, and LOX, respectively. A difference in allelic frequency was observed for only the SNP at intron 4 in FGF1 (χ²=4.44, df=1, P=0.035). Although a haplotype association was observed with the combination of ten SNPs in FGF1 (χ²=16.04, df=1, P=0.00006), significant haplotype associations were not observed when haplotypes were constructed with the three, two, and four SNPs in FGF1 according to the linkage disequilibrium structure. No associations of FBN2 and LOX with IA were detected in the present study.     

Occurrence of cystacanths of <Emphasis Type="Italic">Plagiorhynchus cylindraceus</Emphasis> (Acanthocephala) in the terrestrial isopods <Emphasis Type="Italic">Trachelipus squamuliger</Emphasis> and <Emphasis Type="Italic">Armadillidium vulgare</Emphasis> (Oniscidea) in Bulgaria

In total, 2097 individuals of Trachelipus squamuliger and 20 individuals of Armadillidium vulgare from four habitats (three woodland sites and one pasture) in the region of Stara Zagora, Bulgaria, were examined for the presence of cystacanths of Plagiorhynchus cylindraceus, a common acanthocephalan parasite of passerine birds. In T. squamuliger from woodland habitats, cystacanths were found with prevalence 4.0–9.3%, intensity 1–5 (mean 1.22–1.57) and mean abundance 0.057–0.113. No significant differences were observed between infections in males and females of T. squamuliger. None of the T. squamuliger individuals from the pasture examined was infected. Out of 48 infected females of T. squamuliger, only one had developed eggs (in agreement with previous studies revealing the negative effect of the cystacanths on the development of female gonads of woodlice). One individual of A. vulgare was infected with a single cystacanth. The occurrence of P. cylindraceus in T. squamuliger is a new host record.     

An aspartate aminotransferase of<Emphasis Type="Italic"> Wolbachia</Emphasis> endobacteria from<Emphasis Type="Italic"> Onchocerca volvulus</Emphasis> is recognized by IgG1 antibodies from residents of endemic areas

Wolbachia are intracellular alpha-proteobacteria, closely related to Rickettsia, that infect various arthropods and filarial parasites. In the present study, the cDNA encoding the aspartate aminotransferase (AspAT) of Wolbachia from the human pathogenic filarial parasite Onchocerca volvulus (Ov-WolAspAT) was identified. At the amino acid level, the identity of the Ov-WolAspAT was 56% to Rickettsia prowazekii AspAT and 54% to the AspAT of the nitrogen-fixing bacterium Sinorhizobium meliloti, but the highest degree of identity was found to the putative AspAT of Wolbachia from Brugia malayi and Drosophila melanogaster (85%). All of these bacterial AspATs are members of the AspAT subclass Ib. A 35 kDa fragment of the Ov-WolAspAT was expressed in Escherichia coli, and immunolocalization using polyclonal antibodies against this antigen revealed that Ov-WolAspAT is present in a considerable proportion of the Wolbachia from O. volvulus, as well as in the endobacteria of several other filarial parasites. Western blot analysis using recombinant Ov-WolAspAT as antigen showed that IgG1 antibodies were present in 70 (51%) individuals living in areas endemic for O. volvulus, B. malayi or Wuchereria bancrofti and no IgG4 or IgE antibodies were found. Among 40 sera of persons from Uganda and Liberia who were putatively not infected with human filarial parasites, 11 (28%) individuals presented IgG1 antibodies, while none of the 33 sera from healthy Europeans and none of the 14 sera from patients with proven Rickettsia or Brucella infections reacted with the antigen. These results also show that an intracellular protein of Wolbachia endobacteria (WolAspAT) acts as antigen in human filariasis.     

Influence of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> quorum sensing signal molecule <Emphasis Type="Italic">N</Emphasis>-(3-oxododecanoyl) homoserine lactone on mast cells

Quorum sensing system is a cell-to-cell communication system that plays a pivotal role in virulence expression in bacteria. Recent advances have demonstrated that the Pseudomonas aeruginosa quorum sensing molecule, N-3-oxododecanoyl homoserine lactone (3OC₁₂-HSL), exerts effects on mammalian cells and modulates host immune response. Mast cells (MCs) are strategically located in the tissues that are constantly exposed to external stimulus. Therefore, it is very much possible that 3OC₁₂-HSL may interact with MCs. Little is known, however, about specific effects of 3OC₁₂-HSL on MCs. To address this, we investigated the influence of 3OC₁₂-HSL on cell viability, apoptosis, intracellular calcium and cytokine release in MCs. We found that at high concentrations (100 μM), 3OC₁₂-HSL inhibited proliferation and induced apoptosis in P815. The 3OC₁₂-HSL treatment significantly increased intracellular calcium release in both P815 and HMC-1. We also observed that 3OC₁₂-HSL-induced histamine release and degranulation in HMC-1 cells. Furthermore, 3OC₁₂-HSL-induced IL-6 production at lower concentrations (6.25–12.5 μM) but steadily reduced IL-6 production at high concentration (50–100 μM). These data demonstrate that P. aeruginosa 3OC₁₂-HSL affects MCs function.     

Functional analysis of <Emphasis Type="Italic">Kluyveromyces lactis</Emphasis> carboxylic acids permeases: heterologous expression of <Emphasis Type="Italic">KlJEN1</Emphasis> and <Emphasis Type="Italic">KlJEN2</Emphasis> genes

The present work describes a detailed physiological and molecular characterization of the mechanisms of transport of carboxylic acids in Kluyveromyces lactis. This yeast species presents two homologue genes to JEN1 of Saccharomyces cerevisiae: KlJEN1 encodes a monocarboxylate permease and KlJEN2 encodes a dicarboxylic acid permease. In the strain K. lactis GG1888, expression of these genes does not require an inducer and activity for both transport systems was observed in glucose-grown cells. To confirm their key role for carboxylic acids transport in K. lactis, null mutants were analyzed. Heterologous expression in S. cerevisiae has been performed and chimeric fusions with GFP showed their proper localization in the plasma membrane. S. cerevisiae jen1Δ cells transformed with KlJEN1 recovered the capacity to use lactic acid, as well as to transport labeled lactic acid by a mediated mechanism. When KlJEN2 was heterologously expressed, S. cerevisiae transformants gained the ability to transport labeled succinic and malic acids by a mediated mechanism, exhibiting, however, a poor growth in malic acid containing media. The results confirmed the role of KlJen1p and KlJen2p as mono and dicarboxylic acids permeases, respectively, not subjected to glucose repression, being fully functional in S. cerevisiae. O. Queirós and L. Pereira contributed equally to this work.     

Development of a vaccine against <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

A vaccine to prevent infections caused by Staphylococcus aureus would have a tremendously beneficial impact on public health. In contrast to typical encapsulated bacterial pathogens, such as Streptococcus pneumoniae, H. influenzae, and Neisseria meningitides, the capsule of S. aureus is not clearly linked to strain virulence in vivo. Furthermore, it is not clear that natural infection caused by S. aureus induces a protective humoral immune response, as does infection caused by typical encapsulated bacteria. Finally, pure B cell or antibody deficiency, in either animal models or in patients, does not predispose to more frequent or more severe S. aureus infections, as it does for infections caused by typical encapsulated bacteria. Rather, primary immune mechanisms necessary for protection against S. aureus infections include professional phagocytes and T lymphocytes (Th17 cells, in particular) which upregulate phagocytic activity. Thus, it is not clear whether an antibody-mediated neutralization of S. aureus virulence factors should be the goal of vaccination. Rather, the selection of antigenic targets which induce potent T cell immune responses that react to the broadest possible array of S. aureus strains should be the focus of antigen selection. Of particular promise is the potential to select antigens which induce both humoral and T cell-mediated immunity in order to generate immune synergy against S. aureus infections. A single-antigen vaccine may achieve this immune synergy. However, multivalent antigens may be more likely to induce both humoral and T cell immunity and to induce protection against a broader array of S. aureus isolates. A number of candidate vaccines are in development, raising the promise that effective vaccines against S. aureus will become available in the not-so-distant future. Possible development programs for such vaccines are discussed.     

Host Innate Immune Response to <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

This review focuses on recent progress in our understanding of Mycobacterium tuberculosis survival in macrophages, the interaction of M. tuberculosis with Toll-like receptors (TLRs) and the establishment of the link between innate and adaptive immunity, and TLRs and interferon-γ-mediated antimicrobial pathways in macrophages. We also propose a paradigm that TLR2 signaling regulates the magnitude of the host Th1 response leading to either M. tuberculosis persistence and latent infection or replication and disease.     

Spontaneous capture of oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis>) chloroplasts by wild <Emphasis Type="Italic">B</Emphasis>. <Emphasis Type="Italic">rapa</Emphasis>: implications for the use of chloroplast transformation for biocontainment

Environmental concerns over the cultivation of Genetically Modified (GM) crops largely centre on the ecological consequences following gene flow to wild relatives. One attractive solution is to deploy biocontainment measures that prevent hybridization. Chloroplast transformation is the most advanced biocontainment method but is compromised by chloroplast capture (hybridization through the maternal lineage). To date, however, there is a paucity of information on the frequency of chloroplast capture in the wild. Oilseed rape (Brassica napus, AACC) frequently hybridises with wild Brassica rapa (AA, as paternal parent) and yields B. rapa-like introgressed individuals after only two generations. In this study we used chloroplast CAPS markers that differentiate between the two species to survey wild and weedy populations of B. rapa for the capture of B. napus chloroplasts. A total of 464 B. rapa plants belonging to 14 populations growing either in close proximity to B. napus (i.e. sympatric <5 m) or else were allopatric from the crop (>1 km) were assessed for chloroplast capture using PCR (trnL-F) and CAPS (trnT-L-Xba I) markers. The screen revealed that two sympatric B. rapa populations included 53 plants that possessed the chloroplast of B. napus. In order to discount these B. rapa plants as F₁ crop-wild hybrids, we used a C-genome-specific marker and found that 45 out of 53 plants lacked the C-genome and so were at least second generation introgressants. The most plausible explanation is that these individuals represent multiple cases of chloroplast capture following introgressive hybridisation through the female germ line from the crop. The abundance of such plants in sympatric sites thereby questions whether the use of chloroplast transformation would provide a sufficient biocontainment for GM oilseed rape in the United Kingdom.     

<Emphasis Type="Italic">Leishmania</Emphasis> (<Emphasis Type="Italic">Viannia</Emphasis>) <Emphasis Type="Italic">shawi</Emphasis> purified antigens confer protection against murine cutaneous leishmaniasis

Objective  

Leishmania (Viannia) shawi was characterized only recently, and few studies concerning the immunogenic and protective properties of its antigens have been performed. The present study aimed to evaluate the protective potential of the five antigenic fractions isolated from L. (V.) shawi promastigotes in experimental cutaneous leishmaniasis.     

Association analysis of <Emphasis Type="Italic">SLC22A4</Emphasis>, <Emphasis Type="Italic">SLC22A5</Emphasis> and <Emphasis Type="Italic">DLG5</Emphasis> in Japanese patients with Crohn disease

Crohn disease (CD) is an inflammatory bowel disease characterized by chronic transmural, segmental, and typically granulomatous inflammation of the gut. Recently, two novel candidate gene loci associated with CD, SLC22A4 and SLC22A5 on chromosome 5 known as IBD5 and DLG5 on chromosome 10, were identified through association analysis of Caucasian CD patients. We validated these candidate genes in Japanese patients with CD and found a weak but possible association with both SLC22A4 (P=0.028) and DLG5 (P=0.023). However, the reported genetic variants that were indicated to be causative in the Caucasian population were completely absent in or were not associated with Japanese CD patients. These findings imply significant differences in genetic background with CD susceptibility among different ethnic groups and further indicate some difficulty of population-based studies.     

Hypermethylation of<Emphasis Type="Italic"> Chfr</Emphasis> and<Emphasis Type="Italic"> hMLH1</Emphasis> in gastric noninvasive and early invasive neoplasias

Human tumors are genetically unstable, and the instability exists at two distinct levels—the chromosomal level and the nucleotide level. Chfr and hMLH1 hypermethylation, which may lead to chromosomal instability (CIN) and microsatellite instability (MSI), respectively, was analyzed in gastric noninvasive neoplasias (NIN, Padova international classification) and submucosal invasive adenocarcinomas and in their corresponding non-neoplastic gastric epithelia. Results were compared with microsatellite status, p53 immunoreactivity, and cellular phenotype. Hypermethylation of Chfr and hMLH1 was observed in: 10% (1/10) and 0% (0/10) of low-grade NIN (L-NIN); 63% (5/8) and 63% (5/8) of high-grade NIN, including suspicion for carcinoma without invasion (H-NIN); 36% (5/14) and 57% (8/14) of high-grade NIN, including carcinoma without invasion; and 35% (7/20) and 25% (5/20) of submucosal invasive adenocarcinomas, respectively. Hypermethylation was less frequent in L-NIN than H-NIN (P<0.05) for Chfr and was also less frequent in L-NIN than the others (P<0.05) for hMLH1. We failed to find a significant correlation between Chfr hypermethylation and chromosomal loss of heterozygosity, although hypermethylation of hMLH1 was significantly associated with high-frequency MSI (P<0.01). Expression of p53 was not associated with Chfr or hMLH1 methylation. As for cellular phenotype, hypermethylation of Chfr and hMLH1 was frequent in tumors exhibiting the foveolar epithelial phenotype (50%, 2/4 and 75%, 3/4, respectively) and the ordinary phenotype (40%, 16/40 and 38%, 15/40, respectively), but never in those with the complete-type intestinal metaplastic phenotype (0%, 0/8 for both). In addition, hypermethylation of Chfr and hMLH1 occurred concurrently (P<0.01); methylation was more frequent in patients over 70 years of age (P<0.01), and it was also present in some samples of non-neoplastic gastric epithelia from elderly patients. Thus, some gastric tumors with the foveolar or ordinary phenotype may develop as a result of age-related methylation of Chfr and hMLH1, although Chfr methylation was not associated with CIN.