Loss Of Epitopes Research Articles

Occult hepatitis B virus infection in hepatitis B vaccinated children in Taiwan

Presence of occult HBV infection in HBV vaccinated children remains largely unknown. The aim of this study was to investigate the prevalence of occult HBV infection among HBV vaccinated children in Taiwan. Forty-six HBsAg negative sera from vaccinated children were enrolled randomly. HBV serum markers were detected by ELISA, and the titers of HBV DNA were determined by quantitative real-time PCR. Pre-S, S and pre-core/core genes were amplified by nested PCR and analyzed. Anti-HBs was detected in 23 (50%) children, and the positivity decreased according to age. Five (10.9%) children were classified into occult HBV infection by positivity of nested PCR in at least two regions; they had a low titer (mean titer 1.60x10(4)copies/ml). Sequence analyses of S gene showed occult isolates were variants; no G145R but C139S vaccine escape mutant was found. Variation and deletion were found in pre-S region; pre-S deletion was more frequent in 3' terminus of pre-S1 which leads to loss of immune epitopes and function sites. This pilot study indicates that the prevalence of occult HBV infection is 10.9% in HBV vaccinated children. Since this is a small study, a study of a large population is needed to confirm the findings herein.

HLA-DM Mediates Epitope Selection by a “Compare-Exchange” Mechanism when a Potential Peptide Pool Is Available

BackgroundHLA-DM (DM) mediates exchange of peptides bound to MHC class II (MHCII) during the epitope selection process. Although DM has been shown to have two activities, peptide release and MHC class II refolding, a clear characterization of the mechanism by which DM facilitates peptide exchange has remained elusive.Methodology/Principal FindingsWe have previously demonstrated that peptide binding to and dissociation from MHCII in the absence of DM are cooperative processes, likely related to conformational changes in the peptide-MHCII complex. Here we show that DM promotes peptide release by a non-cooperative process, whereas it enhances cooperative folding of the exchange peptide. Through electron paramagnetic resonance (EPR) and fluorescence polarization (FP) we show that DM releases prebound peptide very poorly in the absence of a candidate peptide for the exchange process. The affinity and concentration of the candidate peptide are also important for the release of the prebound peptide. Increased fluorescence energy transfer between the prebound and exchange peptides in the presence of DM is evidence for a tetramolecular complex which resolves in favor of the peptide that has superior folding properties.Conclusion/SignificanceThis study shows that both the peptide releasing activity on loaded MHCII and the facilitating of MHCII binding by a candidate exchange peptide are integral to DM mediated epitope selection. The exchange process is initiated only in the presence of candidate peptides, avoiding possible release of a prebound peptide and loss of a potential epitope. In a tetramolecular transitional complex, the candidate peptides are checked for their ability to replace the pre-bound peptide with a geometry that allows the rebinding of the original peptide. Thus, DM promotes a “compare-exchange” sorting algorithm on an available peptide pool. Such a “third party”-mediated mechanism may be generally applicable for diverse ligand recognition in other biological systems.

A Single CD8+ T Cell Epitope Sets the Long-Term Latent Load of a Murid Herpesvirus

The pathogenesis of persistent viral infections depends critically on long-term viral loads. Yet what determines these loads is largely unknown. Here, we show that a single CD8+ T cell epitope sets the long-term latent load of a lymphotropic gamma-herpesvirus, Murid herpesvirus-4 (MuHV-4). The MuHV-4 M2 latency gene contains an H2-Kd -restricted T cell epitope, and wild-type but not M2− MuHV-4 was limited to very low level persistence in H2d mice. Mutating the epitope anchor residues increased viral loads and re-introducing the epitope reduced them again. Like the Kaposi's sarcoma–associated herpesvirus K1, M2 shows a high frequency of non-synonymous mutations, suggesting that it has been selected for epitope loss. In vivo competition experiments demonstrated directly that epitope presentation has a major impact on viral fitness. Thus, host MHC class I and viral epitope expression interact to set the long-term virus load.

Cleavage of Type II Collagen by Cathepsin K in Human Osteoarthritic Cartilage

Cathepsin K is a cysteine protease of the papain family that cleaves triple-helical type II collagen, the major structural component of the extracellular matrix of articular cartilage. In osteoarthritis (OA), the anabolic/catabolic balance of articular cartilage is disrupted with the excessive cleavage of collagen II by collagenases or matrix metalloproteinases. A polyclonal antibody against a C-terminal neoepitope (C2K) generated in triple-helical type II collagen by the proteolytic action of cathepsin K was prepared and used to develop an enzyme-linked immunosorbent assay to study the generation of this epitope and the effects of its presence in normal adult and osteoarthritic femoral condylar articular cartilage. The generation of the C2K epitope in explant culture and the effect of a specific cathepsin K inhibitor were studied. The neoepitope, which is not generated by the collagenase matrix metalloproteinase-13, increased with age in articular cartilage and was significantly elevated in osteoarthritic cartilage compared with adult nonarthritic cartilage. Moreover, in explants from three of eight OA patients, the generation of the neoepitope in culture was significantly reduced by a specific, nontoxic inhibitor of cathepsin K. These data suggest that cathepsin K is involved in the cleavage of type II collagen in human articular cartilage in certain OA patients and that it may play a role in both OA pathophysiology and the aging process.

Molecular Evolution of Human Immunodeficiency Virus Type 1 upon Transmission between Human Leukocyte Antigen Disparate Donor-Recipient Pairs

BackgroundTo address evolution of HIV-1 after transmission, we studied sequence dynamics in and outside predicted epitopes of cytotoxic T lymphocytes (CTL) in subtype B HIV-1 variants that were isolated from 5 therapy-naive horizontal HLA-disparate donor-recipient pairs from the Amsterdam Cohort Studies on HIV-1 infection and AIDS.Methodology/Principal FindingsIn the first weeks after transmission, the majority of donor-derived mutations in and outside donor-HLA-restricted epitopes in Gag, Env, and Nef, were preserved in the recipient. Reversion to the HIV-1 subtype B consensus sequence of mutations in- and outside donor-HLA-restricted CTL epitopes, and new mutations away from the consensus B sequence mostly within recipient-HLA-restricted epitopes, contributed equally to the early sequence changes. In the subsequent period (1–2 years) after transmission, still only a low number of both reverting and forward mutations had occurred. During subsequent long-term follow-up, sequence dynamics were dominated by forward mutations, mostly (50–85%) in recipient-HLA-restricted CTL epitopes. At the end of long-term follow-up, on average 43% of the transmitted CTL escape mutations in donor-HLA-restricted epitopes had reverted to the subtype B consensus sequence.Conclusions/SignificanceThe relatively high proportion of long-term preserved mutations after transmission points to a lack of back selection even in the absence of CTL pressure, which may lead to an accumulating loss of critical CTL epitopes. Our data are supportive for a continuous adaptation of HIV-1 to host immune pressures which may have implications for vaccine design.

High-throughput microarray analysis of pectic polymers by enzymatic epitope deletion

Pectic polysaccharides are abundant plant cell wall glycan polymers and are also widely used as functional food ingredients. The fine structure of pectins, and especially the degree and pattern of methyl-esterification of the homogalacturonan (HG) backbone, play a major part in determining functional properties. We have developed a high-throughput method of analysing pectic polymers by combining microarray-technology, pectin lyase (PL) and anti-HG monoclonal antibodies (mAbs). The technique demonstrates that pectins immobilised in a microarray format are effective substrates for PL, and that epitope loss or degradation can be tracked by the deletion of mAb binding.

The loss of immunodominant epitopes affects interferon-γ production and lytic activity of the human influenza virus-specific cytotoxic T lymphocyte responsein vitro

In the present study, we examined the effect of the loss of the human leucocyte antigen (HLA)-B*3501-restricted nucleoprotein (NP)(418-426) epitope on interferon (IFN)-gamma-production and lytic activity of the human cytotoxic T lymphocyte (CTL) response in vitro. Extensive amino acid variation at T cell receptor contact residues of the NP(418-426) epitope has led to repeated evasion from specific CTL. We generated recombinant influenza viruses with variants of the NP(418-426) epitope, which were used to stimulate peripheral blood mononuclear cells obtained from six HLA-B*3501-positive study subjects in order to expand virus-specific CTL. Loss of the NP(418-426) epitope resulted in a significant reduction of IFN-gamma-expressing CD8+ T cells, similar to that observed previously after the loss of the HLA-B*2705-restricted NP(383-391) epitope. In addition, the effect of the loss of the NP(418-426) epitope on the lytic activity of the virus-specific CTL response was assessed. Also this functional property of the virus-specific CTL response was affected significantly by the loss of this and the NP(383-391) epitope, as determined using the newly developed fluorescent antigen-transfected target cell (FATT)-CTL assay. These findings indicate that the loss of single immunodominant epitopes affects the functionality of the virus-specific CTL response significantly.

Effects of Fibrillin-1 Degradation on Microfibril Ultrastructure

Current models of the elastic properties and structural organization of fibrillin-containing microfibrils are based primarily on microscopic analyses of microfibrils liberated from connective tissues after digestion with crude collagenase. Results presented here demonstrate that this digestion resulted in the cleavage of fibrillin-1 and loss of specific immunoreactive epitopes. The proline-rich region and regions near the second 8-cysteine domain in fibrillin-1 were easily cleaved by crude collagenase. Other sites that may also be cleaved during microfibril digestion and extraction were identified. In contrast to collagenase-digested microfibrils, guanidine-extracted microfibrils contained all fibrillin-1 epitopes recognized by available antibodies. The ultrastructure of guanidine-extracted microfibrils differed markedly from that of collagenase-digested microfibrils. Fibrillin-1 filaments splayed out, extending beyond the width of the periodic globular beads. Both guanidine-extracted and collagenase-digested microfibrils were subjected to extensive digestion by crude collagenase. Collagenase digestion of guanidine-extracted microfibrils removed the outer filaments, revealing a core structure. In contrast to microfibrils extracted from tissues, cell culture microfibrils could be digested into short units containing just a few beads. These data suggest that additional cross-links stabilize the long beaded microfibrils in tissues. Based on the microfibril morphologies observed after these experiments, on the crude collagenase cleavage sites identified in fibrillin-1, and on known antibody binding sites in fibrillin-1, a model is proposed in which fibrillin-1 molecules are staggered in microfibrils. This model further suggests that the N-terminal half of fibrillin-1 is asymmetrically exposed in the outer filaments, whereas the C-terminal half of fibrillin-1 is present in the interior of the microfibril.

The Lectin Dolichos Biflorus Agglutinin Recognizes Glycan Epitopes on the Surface of Murine Embryonic Stem Cells: A New Tool for Characterizing Pluripotent Cells and Early Differentiation

Cell surface markers are key tools that are frequently used to characterize and separate mixed cell populations. Existing cell surface markers used to define murine embryonic stem cells (mESCs) such as stage-specific embryonic antigen 1 (SSEA1), Forssman antigen (FA), alkaline phosphatase (AP), and CD9 are limiting, however, because they do not unambiguously define the pluripotent state and are not reliable indicators of differentiation commitment. To identify glycan cell surface markers that would circumvent this problem, we used a panel of 18 lectins to identify epitopes specifically elevated on the surface of mESCs, which, during differentiation, decrease with kinetics that precede currently used markers such as CD9, SSEA1, FA, and AP. The anticipated outcome of this analysis was to identify glycans that have utility as reliable mESC markers and high-resolution readouts for early differentiation commitment. Here, we show that the lectin Dolichos biflorus agglutinin (DBA) recognizes alpha-N-acetylgalactosamine (GalNAc) cell surface epitopes on mESCs (CD9(high) SSEA1(high) AP(high) DBA(high)). These glycan epitopes decline markedly in cells undergoing the first definable step of differentiation, the transition from mESCs to primitive ectoderm (CD9(high) SSEA1(high) AP(high) DBA(low)). Loss of GalNAc epitopes is, therefore, the earliest cell surface change that can be assigned to differentiating cells, and the only cell surface marker known to be tightly associated with the pluripotent state. The lectin DBA is, therefore, a useful tool to characterize mESC cultures by nondestructive approaches, an indicator of differentiation commitment, and a predictor of developmental potency.

Thrice-Weekly Low-Dose Rituximab Decreases CD20 Loss via Shaving and Promotes Enhanced Targeting in Chronic Lymphocytic Leukemia

Treatment of chronic lymphocytic leukemia (CLL) patients with standard dose infusion of rituximab (RTX), 375 mg/m2, induces clearance of malignant cells from peripheral blood after infusion of 30 mg of RTX. After completion of the full RTX infusion, substantial recrudescence of CLL cells occurs, and these cells have lost > 90% of CD20. To gain insight into mechanism(s) of CD20 loss, we investigated the hypothesis that thrice-weekly low-dose RTX (20 or 60 mg/m2) treatment for CLL over 4 wk would preserve CD20 and enhance leukemic cell clearance. During initial infusions in all 12 patients, the first 30 mg of RTX promoted clearance of > 75% leukemic cells. Four of six patients receiving 20 mg/m2 RTX retained > or = 50% CD20, and additional RTX infusions promoted further cell clearance. However, four of six patients receiving 60 mg/m2 had CD20 levels < 20% baseline 2 days after initial infusions, and additional RTX infusions were less effective, presumably due to epitope loss. Our results suggest that when a threshold RTX dose is exceeded, recrudesced RTX-opsonized cells are not cleared, due to saturation of the mononuclear phagocytic system, but instead are shaved of RTX-CD20 complexes by acceptor cells. Thrice-weekly low-dose RTX may promote enhanced clearance of circulating CLL cells by preserving CD20.

Increased frequencies of CD8+ T lymphocytes recognizing wild‐type p53‐derived epitopes in peripheral blood correlate with presence of epitope loss tumor variants in patients with hepatocellular carcinoma

Wild-type (WT) sequence p53 peptides are attractive candidates for broadly applicable cancer vaccines. The aim of this study was to evaluate the potential of a WT p53-based immunotherapeutic approach for patients with hepatocellular carcinoma (HCC). Circulating CD8+ T cells specific for WT p53(149-157) and WT p53(264-272) HLA-A*0201 restricted epitopes were directly identified in the peripheral blood by the use of peptide/HLA-A2.1 tetramers in 24 HCC patients. Cytotoxic T lymphocyte (CTL) activity after WT p53 peptide-specific stimulation was assessed by analysis of granzyme B and interferon-gamma mRNA transcription, using a quantitative real-time polymerase chain reaction assay. Tumor immunophenotyping was performed to evaluate the p53 status, the expression of major histocompatibility complex (MHC) and costimulatory molecules in freshly isolated tumor cells. HCC patients exhibited significantly higher frequencies of WT p53-specific memory CD8+ T cells and stronger WT p53-specific CTL activity, when compared with healthy controls. Increased frequencies of p53-specific CD8+ T cells and their activity correlated with selective HLA-A2 allele loss and reduced costimulatory molecule expression of tumor cells. Moreover, augmented numbers of p53-specific T cells coincided with high MHC class II expression in tumor cells but were inversely related to the T status of the tumor node metastasis staging system. Our results indicate the existence of natural immunosurveillance and tumor immune evasion, involving a T cell response against WT p53 tumor antigen in patients with HCC. These findings may have important implications for the future development of cancer vaccines.

Induction of protective and therapeutic antitumour immunity using a novel tumour‐associated antigen‐specific DNA vaccine

DNA vaccination has become an attractive immunization strategy against cancer. However, a major problem of DNA vaccination is its limited potency to be taken up by the antigen-presenting cells. In contrast, loss of immunogenic epitopes of tumour cells has urged the development of vaccines against multiple epitopes. In this study, we developed a novel strategy for the APC to efficiently cross-present a fusion tumour antigen, which contains both MHC class I-restricted and class II-restricted T-cell epitopes from Her-2/neu and p53 in a cognate manner. The N-terminus of the fusion Her-2/neu, p53 protein was linked to the sequence encoding for human secondary lymphoid-tissue chemokine for secretion and chemokinesis, and the C-terminus of the fusion protein was linked to a cell-binding domain of IgG (Fc portion, the cell-binding domain of IgG) for receptor-mediated internalization. Here, we show that the introduction of fused-gene DNA vaccine by gene gun reduced the size of established tumours and prolonged the lifespan of tumour-bearing mice. Results show that this DNA vaccination strategy can broadly enhance the antigen-specific cellular and humoral immune responses. This vaccine is capable of inducing adaptive immunity and may provide a novel, generic design for the development of therapeutic and preventive DNA vaccines.

Evidence of a selective epitope loss of anti-transglutaminase immunoreactivity in gluten-free diet celiac sera: A new tool to distinguish disease-specific immunoreactivities

The aim of the present study was to evaluate the epitope specific humoral human tissue transglutaminase (tTG) immunoreactivity against 3 different human recombinant tTG constructs [(full-length tTG (a.a. 1–687), tTG (a.a. 227–687); tTG (a.a. 473–687)] before and after the introduction of a gluten-free diet (GFD). To this end, sera from 64 celiac disease (CD) subjects on a gluten-containing diet (44 f, 20 m) and after 0.6 ± 0.3 years and 2.1 ± 1.3 years of GFD were studied using a quantitative radioimmunoprecipitation assay. All 64 CD patients at diagnosis were full-length anti-tTG (a.a. 1–687)Ab positive. These Abs significantly decreased in frequency and titer after 6 months and 2 years of GFD. However, at low titers, 64.1% (41/64) of CD patients were still fl-tTG (a.a. 1–687)Ab positive after 2 years of GFD. At disease diagnosis, 70.3% (45/64) of the CD patients had Abs directed against fragments (227–687) and/or (473–687) of the tTG protein. This percentage, after 2 years of GFD, significantly decreased to 18.7%, whereas almost 50% of GFD patients had no tTG (227–687) and tTG (473–687) fragment reactivity, but only persistent, low-titer full-length tTG (1–687)Abs. We suggest that the selective loss of immunoreactivity against tTG (227–687) and tTG (473–687) fragments in CD patients with a GFD, could be due to quantitative decrease of autoreactivity driven by tTG-gliadin interaction underlying celiac disease pathogenesis.

Reactive oxygen species deglycosilate glomerular α-dystroglycan

In the kidney, dystroglycan (DG) has been shown to cover the basolateral and apical membranes of the podocyte. alpha-DG is heavily glycosilated, which is important for its binding to laminin and agrin in the glomerular basement membrane. Furthermore, alpha-DG is negatively charged, which maintains the filtration slit open. Reactive oxygen species (ROS) are known to degrade and depolymerize carbohydrates, and to play a role in several glomerular diseases. Therefore, we evaluated the effect of ROS on the glycosilation of glomerular alpha-DG. By using specific antibodies directed against the core protein or glyco-epitopes of alpha-DG, this was studied in a solid-phase assay, in situ on kidney sections, and in vivo in adriamycin nephropathy. A ligand overlay assay was used to study binding of alpha-DG to its ligands. Exposure to ROS leads to a loss of carbohydrate epitopes on alpha-DG both in vitro and on kidney sections. In the in vitro assays, a decreased binding of deglycosilated alpha-DG to laminin and agrin was found. In adriamycin nephropathy, where radicals play a role, we observed a loss of alpha-DG carbohydrate epitopes. We conclude that deglycosilation of glomerular alpha-DG by ROS leads to disruption of the agrin-DG complex, which in vivo may lead to the detachment of podocytes. Furthermore, loss of negative charge in the filtration slit may lead to foot process effacement of podocytes.

RHD(T201R, F223V) cluster analysis in five different ethnic groups and serologic characterization of a new Ethiopian variant DARE, the DIII type 6, and the RHD(F223V)

The RHD phylogeny in humans shows four main clusters of which three are predominantly observed in (African) black persons. Each of the African clusters is characterized by specific amino acid substitutions relative to the Eurasian RHD allele. RH phylogeny defines the framework for identification of clinically relevant aberrant alleles. This study focuses on the weak D type 4 cluster (characterized by RHD(T201R, F223V) (602C>G 667T>G)) in five ethnic groups. A total of 1702 samples were screened for the presence of 602C>G and 667T>G by sequence-specific polymerase chain reaction (PCR-SSP). Eighty samples were assigned to the weak D type 4 cluster and were molecularly characterized by PCR-SSP and RHD sequencing. Antigens of aberrant alleles were characterized with monoclonal anti-D according to the 37-epitope model when possible. Five new aberrant alleles, DIII type 6, DIII type 7, DARE, RHD(T201R, F223V) (without 819G>A), and RHD(F223V), were identified and DIII type 6, DARE, and RHD(F223V) were serologically characterized with monoclonal anti-D. Both the DARE and RHD(F223V) showed epitope loss. It is postulated that the 1136C>T nucleotide substitution (characteristic for the DAU allele cluster) is present on the DVa(KOU) allele. Identification of the new variant alleles refines the phylogeny of RHD in humans. The proposed DVa(KOU) allele with 1136C>T (DVa(KOU)T379M) is probably caused by conversion of the DAU0 allele and the DVa(KOU) allele, forming a phylogenetic link between the DV allele and the DAU cluster. By describing the RHD(F223V) (602C>G) and RHD(T201R, F223V) (602C>G and 667T>G) alleles formal proof is given for the origin of the non-Eurasian cluster.

Evaluation of zinc salt based fixatives for preserving antigenic determinants for immunohistochemical demonstration of murine immune system cell markers

Conventional aldehyde based fixatives produce good morphological preservation. However, owing to their cross-linking mechanism of action, epitope loss may occur during fixation compromising the tissue for subsequent immunohistochemical (IHC) analysis. IHC is an important tool for characterizing antigen, cytokine and cytomorphological markers. The increasing use of mouse models for study of pathogenesis has highlighted the need to investigate alternative fixatives. In the study reported here, tissue samples from RIII mice with immune mediated lesions, Mycobacterium bovis infected mice, and uninfected control mice were fixed in either zinc salt fixative or buffered formalin, then tested for IHC using a panel of antibodies (CD3, CD4, CD8, CD45, CD54, F4/80, Interferon-gamma and MIP2). Zinc salt fixation preserved processing-sensitive murine cell markers (CD4, CD8 and CD54) and improved the intensity of immunolabeling for CD45, F4/80 and CD3. Buffered formalin failed to preserve any of the processing-sensitive murine epitopes for demonstration by subsequent IHC.

Novel weak D types 31 and 32: adsorption‐elution–supported D antigen analysis and comparison to prevalent weak D types

Weak D types are thought to express rather quantitative than qualitative D antigen variants. Distinct type-specific phenotypes and weak D cases with anti-D alloimmunization, however, suggest a variable degree of D antigen alteration. Variant D types were investigated by use of molecular typing, RHD sequencing, extended serologic D antigen investigations, and flow cytometric D antigen density determination. Two novel weak D types were discovered, termed weak D type 31 and 32 with single RHD nucleotide substitutions coding for amino acid exchanges in predicted intracellular RhD polypeptide stretches, with antigen densities of approximately 130 and 50 D sites per red blood cell, respectively. Adsorption-elution technique-supported D epitope mapping of these two weak D types, the recently described weak D type 26, and of the most common Central European weak D types (weak D types 1, 2, 3, 4.0, and 4.1) demonstrated the expression of all tested D epitopes. In contrast, a distinct D epitope loss was detected in weak D type 15 and partial D control samples. All novel and prevalent weak D types expressed all tested D epitopes. Our results indicate that adsorption-elution techniques may be of advantage whenever D epitope loss is suspected in extremely weak D variants.

A comprehensive analysis of DEL types: partial DEL individuals are prone to anti‐D alloimmunization

The D antigen of the polymorphic Rh blood group system is of particular clinical importance regarding transfusion- and pregnancy-induced alloimmunization. Different RhD variants with specific clinical implications have been characterized. The least expressed D variants collectively called DEL are serologically detectable only by adsorption-elution techniques, with so far only poorly defined antigenic properties. A comprehensive immunohematologic analysis of five of the six currently known DEL genotypes was performed. DEL phenotypes associated with the RHD(M295I), RHD(IVS3+1g>a), RHD(K409K), RHD(X418L), or RHD(IVS5-38del4) allele were characterized with extended serology and flow cytometry. Epitope mapping with adsorption-elution revealed a prominent D epitope loss in the RHD(IVS3+1g>a)-associated DEL phenotype, whereas in the other four DEL types no signs of qualitative D antigen alteration were detected. The observation of alloanti-D in two RHD(IVS3+1g>a) cases confirmed the partial nature of this DEL phenotype. The RHD(M295I) phenotype exhibited the highest D antigen expression among all investigated DEL types, as determined by a semiquantitative adsorption-elution approach and flow cytometry. In conclusion, evidence is provided that different DEL genotypes code either for partial or complete D antigen expression and that this finding is clinically relevant.

WZsGreen/+: a new green fluorescent protein knock-in mouse model for the study of KIT-expressing cells in gut and cerebellum

In the small intestine, interstitial cells of Cajal (ICC) surrounding the myenteric plexus generate the pacemaking slow waves that are essential for an efficient intestinal transit. The underlying molecular mechanisms of the slow wave are poorly known. KIT is currently the sole practical marker for ICC. Attempts to purify living ICC have so far largely failed, due to the loss of the KIT epitope during enzymatic dissociation. Aiming to identify and isolate living ICC, we designed a knock-in strategy to express a fluorescent tag in KIT-expressing cells by inserting the sequence of the novel green fluorescent protein ZsGreen into the first exon of the c-Kit gene, creating a null allele called W(ZsGreen). In the gastrointestinal tract of heterozygous W(ZsGreen)/+ mice, tiny ZsGreen fluorescent dots were observed in all KIT-expressing ICC populations, with exception of ICC at the deep muscular plexus in small intestine. During development of the gastrointestinal tract, ZsGreen expression followed KIT expression in a spatiotemporal way. Stellate and basket KIT-expressing cells in the molecular layer of the cerebellum also exhibited ZsGreen dots, whereas no ZsGreen was detected in skin, testis, and bone marrow. ZsGreen dot-containing intestinal cells could be isolated from jejunum and maintained alive in culture for at least 3 days. ZsGreen is a suitable alternative to EGFP in transgenic animals. The novel W(ZsGreen)/+ model reported here appears to be a promising tool for live studies of KIT-expressing cells in the gastrointestinal tract and cerebellum and for the further analysis of pacemaker mechanisms.

An Immunologically Cryptic Epitope of Plasmodium falciparum Circumsporozoite Protein Facilitates Liver Cell Recognition and Induces Protective Antibodies That Block Liver Cell Invasion

Circumsporozoite, a predominant surface protein, is involved in invasion of liver cells by Plasmodium sporozoites, which leads to malaria. We have previously reported that the amino terminus region (amino acids 27-117) of P. falciparum circumsporozoite protein plays a critical role in the invasion of liver cells by the parasite. Here we show that invasion-blocking antibodies are induced by a polypeptide encoding these 91 amino acids, only when it is presented in the absence of the rest of the protein. This suggests that when present in the whole protein, the amino terminus remains immunologically cryptic. A single reactive epitope was identified and mapped to a stretch of 21 amino acids from position 93 to 113. The epitope is configurational in nature, since its recognition was affected by deleting as little as 3 amino acids from either end of the 21-residue peptide. Lysine 104, the only known polymorphic position in the epitope, affected its recognition by the antibodies, and its conversion to leucine in the protein led to a substantial loss of binding activity of the protein to the hepatocytes. This indicated that in the protein, the epitope serves as a binding ligand and facilitates the interaction between sporozoite and hepatic cells. When considered along with the observation that in its native state this motif is immunologically unresponsive, we suggest that hiding functional moieties of the protein from the immune system is an evasion strategy to preserve liver cell binding function and may be of importance in designing anti-sporozoite vaccines.