Changes of synapsin I messenger RNA expression during rat brain development

Synapsin I is a synaptic phosphoprotein that is involved in the short-term regulation of neurotransmitter release. In this report we present the first extensive study of the developmental expression of its corresponding messenger ribonucleic acid (mRNA) by in situ hybridization and northern blot analysis in rat brain. Synapsin I mRNA showed pronounced differences in expression in different brain regions during postnatal development. The early expression of synapsin I mRNA in ontogenetically older regions such as the thalamus, the piriform cortex and the hippocampus coincides with the earlier maturation of these regions, in contrast to its later expression in ontogenetically younger areas such as the cerebellum and the neocortex. An intriguing expression pattern was found in the hippocampus. In all hippocampal subregions synapsin I mRNA expression increased from postnatal day (PND) 1 to 17. After PND 17, however, there was a marked dissociation between persisting high expression levels in CA3 and the dentate gyrus and a strong decline in synapsin I mRNA expression in CA1. The persistence of synapsin I in some adult rat brain regions indicates that it plays a part in synapse formation during plastic adaption in neuronal connectivities.     

Axon development in mouse cerebellum: embryonic axon forms and expression of synapsin I

A fundamental question in central nervous system development is the timing of synaptogenesis in relation to invasion of targets by afferent axons. A related question is how growth cones transform into synaptic terminals. These two aspects of axon maturation were examined in developing mouse cerebellum, by labeling single axons with horseradish peroxidase, to study their form and cytology, and by immunocytochemical staining of a synaptic vesicle antigen, synapsin I, a phosphoprotein found on synaptic vesicles in all mature CNS synapses. From embryonic day 16 to postnatal day 3, horseradish peroxidase-labeled afferent axons extend well into the cerebellum and have simple forms. At embryonic day 16, axon growing tips are synapsin I-negative. Synapsin I is first expressed at embryonic day 17, and by embryonic day 18, fibers are stained throughout the cerebellum. Synapsin I expression coincides with a general increase in synaptic specializations, although growing tips continue to have the cytology of growth cones. During the period that axons have primitive shapes, synapsin I is distributed throughout the terminal arbor, corresponding to the presence of small vesicles along neurite lengths, even at non-synaptic sites. After postnatal day 3, when synaptic terminals develop into stereotypic shapes and engage in characteristic synaptic relations, synapsin I is restricted to boutons. Thus, the synapse-specific protein synapsin I is expressed in fetal mouse brain, long before nerve endings have the structure and connections of adult brain. In cerebellar axons, the expression of this protein follows axon arrival, coincides with the appearance of elementary synapses, and accompanies the transformation of growing tips into stereotypic synaptic boutons. The time course of expression of synapsin I, a phosphoprotein that may be involved in synaptic efficacy, suggests that transmitter release may influence early axon-target cell interactions.     

Increased HIF-1alpha expression in tumor cells and lymphocytes of tumor microenvironments predicts unfavorable survival in esophageal squamous cell carcinoma patients

The expression of hypoxia-induced factor (HIF)-1α is up-regulated in tumor microenvironments under hypoxia condition. However, the prognostic significance of HIF-1α in esophageal squamous cell carcinoma (ESCC) is still elusive. We measured the HIF-1α expression by immunochemistry in tumor specimens from 136 resected ESCC; in the current study, the HIF-1α expression in tumor cells was significantly associated with tumor stage (P = 0.003) and lymph node metastasis (P = 0.006); whereas the HIF-1α expression in tumor-infiltrating lymphocytes (TILs) had no relationship with patients’ clinicopathological parameters. Patients with high HIF-1α expression in tumor cells or in TILs showed worse survival related to those with low HIF-1α expression. Multivariate analysis demonstrated that expression of HIF-1α in TILs was an independent factor for DFS (P = 0.007) and OS (P = 0.013). Additionally, the expression of HIF-1α in tumor cells was an independent factor for DFS (P = 0.037) and OS (P = 0.033) in locoregional ESCC patients, whereas the expression of HIF-1α in TILs was an independent factor for DFS (P = 0.048) and OS (P = 0.039) in metastatic ESCC patients. Correlation analysis revealed that expressions of HIF-1α in tumor cells and in TILs were positively correlated, and patients with combined high HIF-1α in both tumor cells and TILs had the worst survivals (P < 0.05). These findings suggest that the HIF-1α expressions in different cell populations of ESCC microenvironments have different clinical relevance and prognostic impact on patients.     

Synapsin regulates vesicle organization and activity-dependent recycling at Drosophila motor boutons

Synapsin is a phosphoprotein reversibly associated with synaptic vesicles. We investigated synapsin function in mediating synaptic activity during intense stimulation at Drosophila motor boutons. Electron microscopy analysis of synapsin(−) boutons demonstrated that synapsin maintains vesicle clustering over the periphery of the bouton. Cyclosporin A pretreatment disrupted peripheral vesicle clustering, presumably due to increasing synapsin phosphorylated state. Labeling recycling vesicles with a fluorescent dye FM1-43 followed by photoconversion of the dye into electron dense product demonstrated that synapsin deficiency does not affect mixing of the reserve and recycling vesicle pools but selectively reduces the size of the reserve pool. Intense stimulation produced a significant increase in vesicle abundance and vesicle redistribution toward the central core of synapsin (+) boutons, while in synapsin (−) boutons the area occupied by vesicles did not change and the increase in vesicle numbers was not as prominent. However, intense stimulation produced an increase in basal release at synapsin(−) but not in synapsin(+) boutons, suggesting that synapsin may direct vesicles to the reserve pool. Finally, synapsin deficiency inhibited an increase in quantal size and formation of endosome-like cisternae, which was activated either by intense electrical stimulation or by high K⁺ application. Taken together, these results elucidate a novel synapsin function, specifically, promoting vesicle reuptake and reserve pool formation upon intense stimulation.     

Host biomarkers and biological pathways that are associated with the expression of experimental cerebral malaria in mice

Cerebral malaria (CM) is a primary cause of malaria-associated deaths among young African children. Yet no diagnostic tools are available that could be used to predict which of the children infected with Plasmodium falciparum malaria will progress to CM. We used the Plasmodium berghei ANKA murine model of experimental cerebral malaria (ECM) and high-density oligonucleotide microarray analyses to identify host molecules that are strongly associated with the clinical symptoms of ECM. Comparative expression analyses were performed with C57BL/6 mice, which have an ECM-susceptible phenotype, and with mice that have ECM-resistant phenotypes: CD8 knockout and perforin knockout mice on the C57BL/6 background and BALB/c mice. These analyses allowed the identification of more than 200 host molecules (a majority of which had not been identified previously) with altered expression patterns in the brain that are strongly associated with the manifestation of ECM. Among these host molecules, brain samples from mice with ECM expressed significantly higher levels of p21, metallothionein, and hemoglobin α1 proteins by Western blot analysis than mice unaffected by ECM, suggesting the possible utility of these molecules as prognostic biomarkers of CM in humans. We suggest that the higher expression of hemoglobin α1 in the brain may be associated with ECM and could be a source of excess heme, a molecule that is considered to trigger the pathogenesis of CM. Our studies greatly enhance the repertoire of host molecules for use as diagnostics and novel therapeutics in CM.     

Embryonic stem cell markers Sox-2 and OCT4 expression and their correlation with WNT signal pathway in cervical squamous cell carcinoma

Background: Both the expression of embryonic stem cells (ESCs) markers (Sox2, Oct4) and the Wnt signal pathway (β-catenin) are crucial for progression of various human malignancies. The purpose of this study was to investigate the clinicopathologic significance of Sox2, Oct4 and β-catenin in cervical squamous cell carcinoma (CSCC) and to study their correlation with the occurrence and prognosis. Methods: Sox2, Oct4 and β-catenin were assessed using immunohistochemistry in normal cervix tissues (n = 28) and invasive cervical squamous cell carcinoma (n = 43). Associations of Sox2, Oct4 and β-catenin levels with clinicopathological characteristics and with overall survival were studied using uni- and multivariate analysis. Results: The expression levels of Sox2, Oct4 and β-catenin were highly increased in CSCC compared with the normal cervix tissues. The ESCs markers expression (Sox2 and Oct4) correlated significantly with β-catenin expression. High expression of Sox2, but not that of Oct4 or β-catenin, was correlated with poorer differentiation (P < 0.05). Furthermore, Sox2 expression was significantly correlated with patients’ status of survival in advanced CSCC (P < 0.05), whereas there was no significant finding in Oct4 or β-catenin expression. Conclusions: These findings provide evidence that both ESCs biomarkers (Sox2, Oct4) and Wnt signal pathway (β-catenin) are activated in CSCC. Sox2 can be regarded as a novel predictor of poor prognosis for CSCC patients.     

Elevated levels of soluble CD14 in serum of patients with acute Plasmodium falciparum malaria

Serum sCD14, tumour necrosis factor-alpha (TNF-α), IL-6, and endotoxin were analysed in 45 patients with complicated malaria, in 14 patients with Gram-negative septicaemia and in 24 healthy subjects by ELISA. Malaria patients with renal failure (n = 16) had higher levels than patients without renal failure (n = 29) (8116+1440 μg/lversus 9453+1017 μg/lP<0.05) and both had higher levels than patients with septicaemia (6155+1635μg/l) and normal subjects (2776+747 μg/l). A significant correlation between sCD14 and IL-6 (r = 0.756) and TNF (r = 0.822) existed. However, no relation between sCD14 and serum endotoxin or indices of clinical disease severity (parasitaemia, fever, parasite or fever clearance time) was seen. Although the role of sCD14 in malaria remains to be determined, elevated levels may participate in the inflammatory response in complicated malaria.     

miR-144 regulates transforming growth factor-β1 iduced hepatic stellate cell activation in human fibrotic liver

Objectives: Activation of hepatic stellate cells (HSCs) into collagen producing myofibroblasts is critical for pathogenesis of liver fibrosis. Transforming growth factor-β1 (TGF-β1) is one of the main profibrogenic mediators for HSC transdifferentiation. Recent studies have shown effect of microRNAs (miRNAs) on regulating TGF-β1-induced HSC activation during liver fibrosis. Here, we aimed to explore the roles of miR-144 and miR-200c in human liver fibrosis. Methods: Expression of TGF-β1 was detected in 42 fibrotic and 18 normal human liver tissues by quantitative real time polymerase chain reaction (qRT-PCR) and immunohistochemistry, and its correlation with α-smooth muscle actin (α-SMA) was calculated. miR-144 and miR-200c expression level in fibrotic liver tissues were also detected by qRT-PCR. The correlation of TGF-β1 expression with miR-200c and miR-144 in the fibrotic liver was analyzed. Results: The results showed that TGF-β1 expression was much higher in fibrotic liver than that in normal liver tissues (P<0.05). TGF-β1 protein high expressing liver fibrosis showed α-SMA positive cells in the liver parenchyma indicating activated HSCs. Expression of TGF-β1 in fibrotic liver was significantly correlated with α-SMA expression (R=0.633, P<0.001). Furthermore, miR-144 was less expressed in liver fibrosis (P<0.05) and was significantly correlated with expression of TGF-β1 in fibrotic liver tissues (R=-0.442, P<0.01). However, miR-200c did not show significant difference between normal and fibrotic liver (P=0.48) and correlation with TGF-β1 expression (R=0.106, P=0.51). Conclusion: All the results indicate that miR-144 can be a novel regulator of TGF-β1-induced HSC activation during liver fibrosis.     

Functional analysis of UMOD gene and its effect on inflammatory cytokines in serum of essential hypertension patients

Objective: The study aimed to investigate the function of uromodulin (UMOD) gene and its effect on inflammatory cytokines in serum of essential hypertension patients. Methods: The online database and software of computer were used for bioinformatics analysis on UMOD gene as well as the structure and function of its encoding proteins. Moreover, radioimmunoassay and enzyme linked immunosorbent assay was adopted to validate the content of urine UMOD protein of essential hypertension patients and their serum inflammatory cytokines. Results: As an alkaline and hydrophilic protein, UMOD has no transmembrane region, but it does have a signal peptide sequence. It is mainly located extracellularly, belonging to a secreted protein, whose secondary structure was based mainly on Random coil which account for 58.44%. According to function prediction, it is found that the UMOD protein has stress response which may be participate in the inflammatory reaction. It has been observed from the experiment which was designed on the basis of the correlation between inflammation reaction and essential hypertension that the content of urine UMOD protein of essential hypertension patients who is in stage I was (28.71±10.53) mg/24 h and when compared with the control group’s content (30.15±14.10 mg/24 h), the difference was not obviously; The content of urine UMOD protein of essential hypertension patients who’s in stage II and III was (18.24±6.12) mg/24 h and (9.43±3.16) mg/24 h, respectively, which were obviously lower than that of the control group (P<0.01). Additionally, the serum inflammatory cytokines, such as TNF-α, IL-6 and IL1-α content of essential hypertension patients were all markedly higher than that of control group (P<0.05). Conclusion: For essential hypertension patients, there’s a close relationship between the expression level of UMOD gene and inflammatory cytokines, which were manifested as the negative correlation between the level of the gene’s expression and inflammatory cytokines. That has certain reference value to realize the targeted treatment for essential hypertension through regulated blood pressure conversely in the view of expression level of inflammatory cytokines.     

Association of CD14 and TLR4 with LPS-stimulated human normal skin fibroblasts in immunophenotype changes and secretion of TGF-β1 and IFN-γ

Objectives: We attempted to explore the association of CD14 and TLR4 with LPS-stimulated human normal skin fibroblasts in immunophenotype changes and secretion of TGF-β1 and IFN-γ, and to expand the current knowledge of the mechanisms that underlie LPS-induced scar formation. Methods: We randomized the human normal skin fibroblasts cultured in vitro into four groups. The expression profile of immune phenotypes was determined by immunohistochemical staining. Ultrastructure of cells was observed by use of transmission electron microscopy. Secretion status of TGF-β1 and IFN-γ was inspected using ELISA assay. Results: Compared with group A, the expressions of α-SMA and α1 (I) procollagen in groups B, C, D were lower, and it in group D were the lowest in all groups. The cells in group A were diversification under the electron microscope, and the ratio of the nuclear to plasma of the fibroblasts was large, with unregular nuclear membrane, more Golgi apparatus, rough endoplasmic reticulum, and microfilament and canaliculus appeared. The ultrastructure of the fibroblasts in group B, C, D was spindle and the nuclear was large, with regular nuclear membrane, more Golgi apparatus, rough endoplasmic reticulum. ELISA assay indicated that the secretion of TGF-β1 markedly lowered in groups B, C, D in comparison to group A, with the most marked decline observed in group D. Interestingly, we found significantly increased IFN-γ secretion in groups B, C, D (P < 0.05), with the latter group showing the most notable increase (P < 0.01). Conclusion: These data suggest that both combined and isolated use of CD14 and TLR4 significantly reduce α-SMA expression levels, the number of α1 (I) pro-collagen positive cells, and TGF-β secretion, while substantially increased IFN-γ secretion. The reduction and increase are especially notable when pretreating with CD14 and TLR4 combined. Here we thus draw a conclusion that both CD14 and TLR4 are associated with the immunophenotype changes and secretion of TGF-β1 and IFN-γ in LPS-stimulated human normal skin fibroblasts.     

Up-regulation of CC chemokine receptor 6 on tonsillar T cells and its induction by in vitro stimulation with α-streptococci in patients with pustulosis palmaris et plantaris

Pustulosis palmaris et plantaris (PPP) is a tonsil-related disease; tonsillectomy is somewhat effective in treating the condition. However, the aetiological association between the tonsils and PPP has not yet been elucidated fully. Recently, some chemokines and chemokine receptors, including CC chemokine receptor (CCR) 4, CCR6 and CX chemokine receptor (CXCR) 3, have been reported to play important roles in the development of psoriasis, a disease related closely to PPP. In this study, we found that CCR6 expression on both tonsillar and peripheral blood T cells was up-regulated more intensively in PPP patients than in non-PPP patients (P < 0·001 for both), but CCR4 and CXCR3 expressions were not. In vitro stimulation with α-streptococcal antigen enhanced CCR6 expression significantly on tonsillar T cells in PPP patients (P < 0·05), but this was not observed in non-PPP patients. The chemotactic response of tonsillar T cells to the CCR6 ligand CC chemokine ligand (CCL) 20 was significantly higher in PPP patients than in non-PPP patients (P < 0·05). The percentage of CCR6-positive peripheral blood T cells decreased after tonsillectomy in PPP patients (P < 0·01); this decrease correlated with an improvement of skin lesions (P < 0·05, r = −0·63). The numbers of CCR6-positive cells and the expression of CCL20 were increased significantly in pathological lesions compared with non-pathological lesions in PPP skin (P < 0·01, P < 0·05 respectively). These results suggest that a novel immune response to α-streptococci may enhance CCR6 expression on T cells in tonsils and that CCR6-positive T cells may move to peripheral blood circulation, resulting in recruitment to target skin lesions expressing CCL20 in PPP patients. This may be one of the key roles in pathogenesis of the tonsil-related disease PPP.     

Downregulated miR-29c correlates with increased BACE1 expression in sporadic Alzheimer’s disease

Background: Beta-site Amyloid precursor protein Cleaving Enzyme 1 (BACE1) is conceived as a potential target for therapies against Alzheimer disease (AD) which is characterized by the accumulation of plaques formed of short β-amyloid (APPβ) peptides. Recently, such microRNAs, as miR-29a, miR-29b-1 have been shown to correlate with abnormally high levels of BACE1 and APPβ in sporadic AD. Methods: In order to confirm whether miR-29c correlates with the BACE1 upregulation in sporadic AD, we firstly evaluated the expression of miR-29c and BACE1, the APPβ accumulation in sporadic AD brain tissues and analyzed the correlation of miR-29c with BACE1. Then we determined the regulation of miR-29c in human heuroblastoma SH-SY5Y cells on the BACE1 expression and APPβ accumulation. And finally we determined the targeting to 3’ UTR of BACE1 by miR-29c by a luciferase reporter. Results: It was demonstrated that miR-29c was downregulated in sporadic AD brains, in an association with an upregulation of BACE1 in both mRNA and protein level of BACE1, and also an elevated APPβ accumulation. And the manipulated high level of miR-29c with miR-29c mimics transfection significantly reduced the protein level of BACE1 and APPβ accumulation in human neuroblastoma SH-SY5Y cells. Further luciferase reporter assay demonstrated that miR-29c targets the 3’ UTR of BACE1 and downregulated the BACE1 in HEK293 cells. Conclusion: Present study indicated that miR-29c was downregulated in sporadic AD brains, and it targeted the 3’ UTR of BACE1, reduced the BACE1 expression, and downregulated the APPβ accumulation in vitro.     

Synapsin I injected presynaptically into goldfish mauthner axons reduces quantal synaptic transmission

1. Synapsin I was injected into a vertebrate presynaptic axon to analyze its action on quantal synaptic transmission. Two microelectrodes were used for simultaneous intracellular recording from pairs of identified neurons in the goldfish brain. The postsynaptic electrode was placed in a cranial relay neuron (CRN) within 100 microns of its synapse with the Mauthner neuron. The presynaptic electrode impaled the Mauthner axon (M-axon) 50-200 microns from the first electrode. 2. Spontaneous miniature excitatory postsynaptic potentials (mEPSPs) and evoked postsynaptic potentials (EPSPs) were recorded at steady states before and after synapsin I was microinjected into the presynaptic M-axon. Responses were digitized and subsequently analyzed by computer for quantal parameters. 3. In 12 experiments, injection of synapsin I resulted in a reduction in transmission. The decrease in EPSP amplitude began approximately 30 s after the injection, reached a plateau within 10 min, and appeared to be reversible and dose dependent. 4. Injection of synapsin I decreased quantal content (m), with no effect on postsynaptic receptor sensitivity or on amount of transmitter per quantum. Further analysis based on the simplest binomial model for quantal release revealed that synapsin I consistently reduced the number of quantal units available for release (n) although the probability of release (p) was either unchanged or slightly increased. Injected synapsin I may thus bind to presynaptic vesicles and prevent transmitter quanta from entering a pool subject to evoked release.     

Inhibition effect of Peg-IFNα-2b and Imatinib alone or combination on imatinib-resistant gastrointestinal stromal tumors cell lines

To investigate the inhibition effect of polyethylene glycol interferon α-2b and imatinib alone or combination on imatinib-resistant GIST cell lines, and to explore the possible mechanism. Imatinib resistant GIST cell lines (GIST-R) were exposured to either Peg-IFNα-2b or imatinib alone or combination. The proliferative inhibition rates and the combination index (CI) values of GIST-R cells were detected by MTT assay. The apoptotic rates of GIST-R cells were detected by flow cytometry. The expression levels of phospho-mammalian target of rapamycin (p-mTOR), and Bcl-2 of GIST-R cells were analyzed by Western blot. GIST-R cells presents remarkable resistance to imatinib, and the resistance index (RI) were (P<0.05). And The proliferative inhibition rate and the apoptotic rate of GIST-R cells in combination of Peg-IFNα2b and Imatinib group were higher than those in Peg-IFNα-2b or imatinib alone group (P<0.05). The CI value of Peg-IFNα-2b and imatinib was less than each alone, which had a synergistic effect (CI=0.63). As compared with the control (GIST-R cells without any treatment), the expression levels of p-mTOR and Bcl-2 proteins of GIST-R cells in combination of Peg-IFNα-2b and imatinib group were decreased (P<0.01). The combination of Peg-IFNα-2b and imatinib generats a synergistic effect in GIST-R cells, and reversal of drug resistance. This effect may be related with apoptosis and down-regulation of the expression of p-mTOR.     

Expression of reactive oxygen species-related proteins in metastatic breast cancer is dependent on the metastatic site

This study was performed to investigate the expression of reactive oxygen species (ROS)-related proteins and to analyze the implications for primary and metastatic breast cancer. We constructed a tissue microarray containing 143 metastatic breast cancers (52 lung metastases, 38 bone metastases, 37 brain metastases, and 16 liver metastases) and performed immunohistochemical staining for ROS-related proteins (catalase, GSTπ, TxNIP, and MnSOD). Analysis of ROS-related protein expression in metastatic breast cancers according to the metastatic sites revealed site-specific expression patterns. The expression of tumoral catalase was lower in bone metastases (P = 0.012), and stromal GSTπ expression was higher in bone and liver metastases (P < 0.001). The highest ROS activation status was observed for lung metastases, while non-activated ROS was observed for bone metastases (P = 0.001). Primary cancers were positive for stromal GSTπ, but a subset of lung metastases were negative (P = 0.021). Univariate analysis revealed that shorter overall survival (OS) was associated with negative catalase expression of the tumor (P = 0.026). Furthermore, univariate analyses according to the metastatic sites revealed that shorter OS was associated with TxNIP-positive tumors (P = 0.032) and the expression of stromal catalase (P = 0.032) in brain metastases. Tumors that were negative for MnSOD expression (P < 0.001) but positive for stromal catalase expression (P = 0.022) were associated with shorter OS in patients with liver metastases. In conclusion, cancer cells and stromal tissues showed different ROS-related protein expression patterns according to the metastatic site. In addition, the expression of ROS-related proteins is associated with patient prognosis.     

Inducible nitric oxide synthase expression is increased in the brain in fatal cerebral malaria

AIMS: Nitric oxide (NO) has been hypothesized to play a major role in the pathogenesis of cerebral malaria caused by P. falciparum infection. NO may act as a local neuroactive mediator contributing to the coma of cerebral malaria (CM). We hypothesized that increased expression of inducible nitric oxide synthase (iNOS) may cause increased release of NO, and examined the expression and distribution of iNOS in the brain during CM. MATERIAL AND RESULTS: Brain tissues from fatal cases of cerebral malaria in Thai adults were examined using immunohistochemical staining to detect iNOS. The distribution and strength of staining was compared between 14 patients with CM, three of whom were recovering from coma, and controls. iNOS expression was found in endothelial cells, neurones, astrocytes and microglial cells in CM cases. There was also strong staining in macrophages surrounding ring haemorrhages. iNOS staining was decreased in recovering malaria cases compared to acute CM, and was low in controls. Quantification showed a significant association between the intensity and number of iNOS positive vessels with the severity of malaria related histopathological changes, although the total number of cells staining was not increased compared to recovering CM cases. CONCLUSIONS: This study indicates that an acute induction of iNOS expression occurs in the brain during CM. This occurs in a number of different cells types, and is increased in the acute phase of CM compared to cases recovering from coma. As NO may activate a number of secondary neuropathological mechanisms in the brain, including modulators of synaptic function, induction of iNOS expression in cerebral malaria may contribute to coma, seizures and death.     

Clinical, parasitological and immunological features of canal cleaners hyper-exposed to Schistosoma mansoni in the Sudan

The present work was a longitudinal study on Schistosoma mansoni infection in occupationally hyperexposed canal cleaners in the Sudan and the influence of therapy on the parasitological and humoral immune parameters. Chronically infected canal cleaners (n = 28) were more resistant to reinfection (Fisher's exact test, P < 0.05) than newly recruited canal cleaners (n = 17). Chronically infected canal cleaners had a significantly higher degree of Symmers' fibrosis (χ² = 19.1, P < 0.0001), significantly larger portal vein diameter (P < 0.05) and enlarged spleen (χ² = 4.2, P < 0.05) than recently infected, newly recruited canal cleaners. ELISA was used to detect IgG, IgA and IgM in response to whole worm homogenate (WWH) and cercarial homogenate (CH). Chronically infected canal cleaners had significantly higher IgG to WWH antigen than newly recruited canal cleaners and normally exposed individuals (P < 0.05), while both chronically infected and newly recruited canal cleaners had higher IgG levels to CH antigen than normally exposed individuals (P < 0.05). The newly recruited canal cleaners had a significantly higher IgM level to CH antigen than chronically infected canal cleaners (P < 0.05). The IgG level to WWH antigen increased significantly after treatment in newly recruited canal cleaners and normally exposed individuals (P < 0.05). The IgA level to CH antigen increased significantly after treatment in the chronically infected group (P < 0.05). Comparison of the serological parameters between the different study groups with regards to infection and treatment is discussed.     

Cytolytic mechanisms and T-cell receptor Vβ usage by ex vivo generated Epstein–Barr virus-specific cytotoxic T lymphocytes

Ex-vivo-generated Epstein–Barr virus (EBV)-specific cytotoxic T lymphocytes (CTL) have been used for cellular adoptive immunotherapy of EBV-associated lymphomas. Here we investigated the phenotypes, cytolytic mechanisms, polyfunctionality and T-cell receptor (TCR) usage in growing and established CTL, generated by weekly stimulation with an EBV-transformed autologous lymphoblastoid cell line (LCL). Our results showed that phenotypically mature CTL developed within the first 4 weeks of culture, with an increase in CD45RO and CD69, and a decrease in CD45RA, CD62L, CD27 and CD28 expression. Spectratyping analysis of the variable β-chain of the TCR revealed that TCR repertoire remained diverse during the course of culture. Cytotoxicity of CTL was significantly inhibited by concanamycin A (P < 0·0001) and ethylene glycol-bis tetraacetic acid (P < 0·0001), indicating that a calcium and perforin-mediated exocytosis pathway with the release of granzyme B was the principal cytotoxic mechanism. The CTL mainly produced interferon-γ (IFN-γ) or tumour necrosis factor-α (TNF-α) upon restimulation with autologous LCL, although there were some polyfunctional cells producing IFN-γ and TNF-α. Granzyme B, perforin and Fas ligand were detected in CD8⁺ and CD4⁺ cells in all CTL; however, a greater proportion of CD8⁺ than CD4⁺ T cells expressed granzyme B (P < 0·0001) and more granzyme B was detected in CD8⁺ T cells than in CD4⁺ T cells (P = 0·001). This difference was not observed with Fas ligand or perforin expression. Our results provide insight into the basic characteristics of ex-vivo-generated CTL.