Fc-epsilon-receptor-bearing lymphocytes in patients with clonorchiasis

Expression of Fc-epsilon-receptors (Fc epsilon R) on peripheral blood lymphocytes was examined in patients with clonorchiasis. Mean serum IgE levels in these patients was 2,518 IU/ml. Fc epsilon R was detected by flow cytometry analysis with monoclonal antibody. The frequency of Fc epsilon R+ lymphocytes and the density of Fc epsilon R on the cells in the patients were similar to those in normal subjects. Most of the Fc epsilon R+ lymphocytes were B cells. The number of Fc epsilon R+ cells decreased after incubation in serum-free medium at 37 degrees C for 3 h. The amount of regulatory molecules of IgE production, IgE binding factor, was not significantly different between the patients and normal subjects. The number of Fc gamma R bearing T or B cells in the patients was also similar to that of normal subjects. These results indicate that the mechanisms of elevated serum IgE in patients with clonorchiasis might be different from other diseases with hyperglobulinemia E.     

Detection and preliminary characterization of circulating immune complexes in patients with Lyme disease

To investigate whether circulating immune complexes can be used as a disease marker for assessment of the activity of Lyme disease and for monitoring patients' response to treatment, we tested 104 sera from patients with different stages of Lyme disease using the C1q enzyme-linked immunosorbent assay (ELISA) and a modified Raji cell test. Among 62 sera of patients with clinically active disease 27 sera (43.5%) reacted positively in the C1q-ELISA and 21 sera (33.9%) positively in the Raji cell test. In contrast, serum circulating immune complexes were found in less than 10% of 42 sera after antibiotic treatment. Similar results were obtained by both tests in 35 cerebrospinal fluid samples from patients with neuroborreliosis. Most importantly, dot blot analysis revealed the presence of both Borrelia burgdorferi-specific antigen(s) and host-derived components in the isolated immune complexes from serum samples of patients with active Lyme disease. These results indicate that detection of circulating immune complexes may be an useful parameter for judging the activity of Lyme disease. Moreover, preliminary characterization of spirochete-specific immune complexes implies new pathophysiological aspects of Lyme disease. Received: 31 July 1997     

Detection and characterization of functional T cells in mice with severe combined immune deficiency

CD3+ alloresponsive T cell clones were derived from mice with severe combined immune deficiency (scid mice). T cell receptor beta and gamma gene rearrangements were analyzed to obtain insight into the nature and origin of these clones. We hypothesized that developing scid lymphocytes with an active, impaired recombinase system might generate functional lymphocytes by rare productive rearrangements at two critical antigen receptor loci. One alloresponsive clone showed evidence of both normally rearranged T cell receptor genes and genes with abnormal J region-associated deletions, supporting this hypothesis. Four additional alloresponsive clones, however, showed only conventional gene rearrangements. These data leave open the possibility that the recombinase activity, believed defective in scid mice, may be normalized in rare early B and T lymphoid cells or their precursors, to give rise to functional lymphocytes.     

Detection of T suppressor cells in patients with organ allografts

Specific immunosuppression of host's immune response to donor HLA antigens has been a major goal to clinical transplantation. Recent evidence has been accumulating to show that a distinct population of T cells expressing the CD8(+) CD28(-) phenotype display suppressor function and inhibit Th activation and proliferation by modulating the APC function. To assess the presence of Ts in transplant recipient's circulation, we have developed a flow cytometry method that measures the expression of costimulatory molecules on donor APC exposed to recipient Th and Ts. Our results demonstrate that quantitation of the capacity of CD8(+) CD28(-) T cells from patient circulation to suppress the activation of costimulatory molecules (CD80, CD86) on donor APC offers a reliable tool for monitoring specific immunosuppression against the graft in solid organ transplantation.     

Detection and characterization of human herpesvirus-8-infected cells in bone marrow biopsies of human immunodeficiency virus-positive patients

We studied 15 bone marrow biopsy specimens from patients with human immunodeficiency virus infection for detection of Kaposi sarcoma herpesvirus (KSHV/HHV-8) DNA sequences by a very sensitive and specific polymerase chain reaction (PCR) assay (with 3 different sets of primers). In addition, we used immunohistochemistry with antiviral interleukin-6 (vIL-6) and anti-latent nuclear antigen-1 (LNA-1) antibodies to localize the infected cells on tissue sections. Among the 15 samples, 6 had positive PCR results with the 3 sets of primers (orf26, orf72, orf75). Interestingly, in 2 of these 6 patients (both with Kaposi sarcoma) vIL-6 and LNA-1 were detected in mononuclear lymphoid cells but not in stromal cells of the bone marrow. The detection of vIL-6--positive lymphoid cells in bone marrow suggests a homing for HHV-8--infected elements in this tissue. The local release of vIL-6 may play some role in the plasmacytosis observed in bone marrow in the acquired immunodeficiency syndrome. HUM PATHOL 32:288-291.     

Detection and immunochemical characterization of glutamic acid decarboxylase autoantibodies in patients with autoimmune diabetes mellitus

Since GAD65 undergoes post-translational processing and targeting to subcellular compartments and membranes, it may exhibit different immunochemical properties in the cell context compared with the soluble protein expressed in the cell-free eukaryotic system used in the reference method for GADA assessment (radioligand binding assay (RBA)). In the present work, we detected and characterized GADA in 72 sera from patients with type 1 diabetes mellitus (DM) and 14 sera from adult-onset diabetes patients using analytical systems in which GAD65 is expressed in a cellular context: confocal indirect immunofluorescence (IIF) and electron microscopy after immunogold labeling on monolayers of transfected Chinese hamster ovary (CHO) cells, and immunoprecipitation (IP) of metabolically labeled GAD65. Eighteen serum samples, 16 from type 1 diabetes patients and two from adult-onset diabetes patients, were positive by confocal IIF but scored negative by RBA. All of these 18 sera immunoprecipitated a 65 kDa protein, supporting the existence of the GADA marker in such patients. It may be concluded that GADA negativity by the conventional RBA method using the soluble antigen, as well as negativity for other common markers measured by similar methods, is not enough to rule out the existence of the specific autoimmune component in childhood or adult-onset diabetes. Other analytical methods based in a more physiological presentation of the autoantigen structure, as confocal IIF and IP, bring an extra support to assess the complete repertoire of specific autoantibodies to native-like and membrane-bound, or denatured, beta-cell antigens.     

Ultrastructural and immunocytochemical characterization of circulating mononuclear cells in patients with myelomatosis.

The ultrastructure of blood mononuclear cells from two IgG myeloma patients was studied, and cells reacting with anti-idiotypic serum and polyspecific anti-Ig serum were characterized by immunoperoxidase techniques. Abnormal, mononuclear cells were present in the blood of both patients, which morphologically were classified as atypical small to medium-sized lymphocytes, polymorphic immature lymphocytes (lymphoblasts), predominantly of the lymphoplasmocytic type and atypical, plasmocytic cells or myeloma cells. Immunocytochemical observations showed that most of the abnormal cells, including atypical small to medium-sized lymphocytes, reacted with anti-idiotypic and polyspecific anti-Ig serum. Periods of relapse and remission were correlated with an increase and decrease, respectively, of the number of abnormal cells and cells which reacted with anti-idiotype and anti-Ig serum. The observations indicate that circulating lymphoid cells are part of the myeloma clone.     

Detection of cancer cells in effusions from patients diagnosed with gynaecological malignancies

The detection of malignant cells in pleural, peritoneal, and pericardial fluids of cancer patients marks the presence of metastatic disease and is associated with a grave prognosis. We evaluated five epithelial markers for the detection of cancer cells in 94 fresh pleural, peritoneal and pericardial effusions. Eighty-four of the samples were regarded as adequate for analysis after evaluation of cytological smears, including 61 samples from patients known to have gynaecological neoplasms. The other 23 samples were from patients with various non-gynaecological malignancies or tumours of unknown origin. Our control cases were 10 fallopian tubes not affected by any malignancy and 12 malignant mesotheliomas. Cell blocks from all cases were stained for CA-125, BerEP4, carcinoembryonic antigen (CEA), BG8 (Lewis Y blood antigen), and B72.3 (TAG-72). Fifty-one of 84 cases were diagnosed as malignant or suggestive of malignancy in cytological smears and/or cell block sections. However, staining for epithelial markers highlighted the presence of malignant cells in 7 additional cases. When membrane staining was evaluated, the sensitivity of the markers studied in detecting malignant cells was as follows: CA-125: 88%, BerEP4: 78%, CEA: 26%, BG8: 86%, B72.3: 79%. Membrane positivity for CEA, B72.3 and BerEP4 was not detected in reactive mesothelial cells. However, membranous staining in mesothelial cells was evident in 13% and 31% of cases with the use of BG8 and CA-125, respectively. Weak cytoplasmic staining for CEA was observed in mesothelial cells in 2 cases. When Ber-EP4, B72.3, and BG8 staining results in cancer cells were combined, the following sensitivity levels were observed: BG8+B72.3: 91%; BG8+Ber-EP4: 90%; B72.3+Ber-EP4: 93%; BG8+ Ber-EP4+B72.3: 95%. The detection of malignant cells in effusions is facilitated by the use of immunocytochemistry using a wide panel of antibodies. BerEP4 and B72.3 appear to be the best markers when both sensitivity and specificity are considered, followed by BG8, while CEA and CA-125 have a limited role in the detection of metastases from gynaecological tumours owing to the low sensitivity of the former and the low specificity of the latter. Analysis of all staining results should be based on a thorough morphological examination. Received: 14 December 1998 / Accepted: 23 February 1999     

Detection and characterization of anti-tumour effector cells in Meth-A-bearing mice treated with recombinant human interleukin 2.

When Meth-A fibrosarcoma-bearing BALB/c mice were injected subcutaneously with 10 micrograms of recombinant human interleukin 2(rIL-2) once a day for 10 days, tumour growth inhibition was in the range of 22-31% of that of the control animals. Anti-tumour effector cells against Meth-A were detected in the spleen cells of the tumour-bearing BALB/c mice injected with rIL-2, using a modified Winn-type neutralization assay with the auxiliary injection of rIL-2. To induce the strongest anti-tumour activity in this assay system, the following were necessary: 1) the effector cells were derived from tumour-bearing BALB/c mice; 2) the donors of the effector cells were injected with rIL-2; 3) the recipient mice in the Winn assay were auxiliarily injected with rIL-2 (a modified Winn assay). The anti-tumour effector activity detected in the modified Winn assay was inhibited by treatment with anti-CD8 or anti-asialo GM1 antibodies plus complement (C), but not completely. We supposed that at least two kinds of anti-Meth-A effector cells with different surface antigens, positive for CD8 and asialo GM1 antigens, were induced in the Meth-A-bearing BALB/c mice injected with rIL-2; these populations seemed to function independently and at least partly as anti-tumour effector cells in this tumour-host system. These spleen cells showed in vitro cytotoxicity against Meth-A cells, which are resistant to NK cells, if the activity was measured in a 24 h 51Cr-release assay in the presence of rIL-2.     

急性冠脉综合征患者Th17和Treg细胞的检测及意义

目的:探讨急性冠脉综合征患者Th17细胞和Treg细胞的变化及意义。方法:选取51例急性冠脉综合征患者,25例稳定性心绞痛(SA)患者,27例同期住院的健康对照者,分别采用流式细胞术、实时定量PCR和ELISA法检测外周血Th17细胞和Treg细胞百分率、关键核转录因子RORγt、STAT3和Foxp3 mRNA的表达以及血浆IL-17A和TGF-β1的浓度。结果:①ACS患者外周血中Th17细胞百分率、RORγt和STAT3 mRNA显著高于SA患者和对照组(P<0.01);②与SA组和对照组相比,ACS患者CD4+CD25+Foxp3+Treg和CD4+Foxp3+Treg细胞百分率,Foxp3 mRNA的表达以及血浆TGF-β1的浓度显著降低(P<0.01)。结论:ACS患者外周血中Th17/Treg失衡,Th17/Treg失衡可能与斑块的不稳定密切相关。     

Cord blood B cells are mature in their capacity to switch to IgE-producing cells in response to interleukin-4 in vitro.

Neonatal B cells have been considered immature because of their impaired capacity to produce immunoglobulins in response to polyclonal activators in vitro. Here we demonstrate that cord blood mononuclear cells (MNC) produce normal levels of IgE in vitro when cultured in the presence of interleukin-4 (IL-4), indicating that the B cells are mature in their capacity to switch to IgE-producing cells. However, in contrast to adult peripheral blood T cells, cord blood T cells failed to produce detectable levels of IL-4 upon activation by phytohaemagglutinin (PHA) concanavalin A (Con A) or combinations of PHA and the phorbol ester TPA. Interferon-gamma (IFN-gamma) production by cord blood T cells following activation by Con A or PHA was also strongly reduced. However, high levels of IFN-gamma, significantly higher than those produced by adult T cells, were synthesized in response to combinations of PHA and TPA, indicating that IFN-gamma production by cord blood T cells is not intrinsically defective. In contrast, cord blood T cells produced levels of IL-2 that were significantly higher than those obtained by adult T cells tested in parallel. Collectively, our data indicate that the minimal levels of IgE production measured in cord blood (less than 1 U/ml) are not due to immaturity of the cord blood B cells, but may be associated with the failure of cord blood T cells to produce detectable levels of IL-4, which has been shown to be responsible for induction of IgE synthesis both in vitro and in vivo.     

Clonal isolation and characterization of bone marrow stromal cells from patients with osteoarthritis

The demand for treatment strategies for damaged musculoskeletal tissue is continuously growing, especially with the increasing number of older people with degenerative diseases of the skeletal system such as osteoarthritis (OA). Because depletion of multipotent cells has been implicated in degenerative joint diseases, cell-based therapies have been proposed for tissue regeneration, especially for cartilage repair. The aim of the present study is to focus on the possibility of deriving and expanding multipotential mesenchymal stem cells (MSCs) from bone marrow samples of patients with OA by characterizing MSCs at the single cell level. Single-cell clonal cultures were established in 96-well plates by limiting dilution of bone marrow stromal cells (BMSCs) from three patients with OA. Fourteen clones were established for subsequent characterization. There was a wide variation in cell doubling times, with the time taken to reach 20 population doublings ranging from 37 days to more than 100 days. The clones were grouped into fast-growing and slow-growing clones. All except one of the fast-growing stem cell clones were tripotential. However, the slow-growing clones showed limited differentiation potential and morphological changes associated with cellular senescence with extended duration in culture. Flow cytometric analysis indicated a strong need to investigate for novel cell-surface characteristic markers of BMSCs because there was no obvious difference in the expression of the selected characteristic BMSC cell surface markers CD29, CD44, CD90, CD105, and CD166 between fast-growing and slow-growing clones. This study has demonstrated the existence of a fast-growing multipotential MSC population from bone marrow samples of patients with OA. Therefore, despite a supposedly smaller stem cell compartment in these patients, we demonstrate here that they can still yield a potentially therapeutically useful source of syngeneic MSCs.     

The further characterization of autoantibodies reactive with extra-adrenal steroid-producing cells in patients with adrenal disorders

Antibodies reactive with the steroid-producing cells in the gonads are described in the sera of ten patients, the majority of whom were known to have idiopathic adrenal insufficiency (Addison's disease) associated with premature ovarian failure. The immunofluorescent staining pattern of these antibodies with steroid-producing cells in the ovary, testis, placenta and adrenal cortex are illustrated. The staining patterns and the results of absorption studies indicate that there are a multiplicity of antibodies reacting with different antigens in the ovary and to a lesser extent in the testis. Most of these antigens are also represented in the adrenal cortex, but are not evenly distributed throughout the cortex. Some of these antigens are not represented in the zona glomerulosa while others are not represented in the zona reticularis.     

Molecular characterization and expression of a cysteine protease from Clonorchis sinensis and its application for serodiagnosis of clonorchiasis

Cysteine proteases play essential roles in parasite physiology as well as in host-parasite interactions through their modulation of various biological and pathobiological events. In the present study, a full-length sequence encoding cysteine protease of Clonorchis sinensis (CsCP) was isolated from our adult cDNA library. The open reading frame contains 984 bp encoding 327 amino acids. The present amino acid sequence shared 68% identity with two known CsCP genes and 29-49% identity with that of other species. Bioinformatics analysis showed that conserved domains and characteristic amino acid residues of cysteine proteases were observed in this sequence. Real-time PCR experiments revealed that CsCP was consecutively transcribed in various developmental stages of the parasite, including adult worm, excysted juvenile, metacercaria and egg. Recombinant CsCP (rCsCP) could be probed by rat anti-CsCP serum, rabbit anti-excretory-secretory products (ESP) serum and serum from human infected with Clonorchis sinensis in Western blot. The result of immunolocalization showed that CsCP was mainly located in the oral sucker, excretory bladder and tegument of cercariae and metacercariae, as well as the intestine of adult worm. The rCsCP-based IgG and its isotypes were all detected in sera from human infected with C. sinensis by enzyme-linked immunosorbent assay, and the level of IgG1 is the highest. The receiver-operating characteristic (ROC) analysis was used to determine the most appropriate cut-off value that yielded the high sensitivity (86.96%) and specificity (70.42%). These results revealed that CsCP may play an important role in the biology of C. sinensis and could be a diagnostic candidate for clonorchiasis.     

Detection and characterization of antigens in urine of patients with acute, congenital, and chronic Chagas' disease.

Monoclonal antibodies raised against purified Trypanosoma cruzi urinary antigens were used in an enzyme-linked immunosorbent assay (ELISA) capture test for parasite antigens present in urine specimens of Argentinean and Brazilian patients with Chagas' disease. At diagnosis, antigenuria was demonstrated by ELISA in all acutely and congenitally infected infants studied. Moreover, T. cruzi urinary antigens were detected in samples from three of five patients with acute infections and four of five patients with congenital infections following chemotherapy. At least one ELISA-positive urine specimen from each individual was recorded in a longitudinal survey of 12 chronic chagasic patients. The same parasitic antigens (90 to 80 kDa, pI 5.7 to 6.0; 70 to 65 kDa, pI 4.9 to 4.5; 50 to 45 kDa, pI 5.3 to 5.1; and 40 to 35 kDa, pI 4.8 to 4.5) were identified by immunoprecipitation and two-dimensional polyacrylamide gel electrophoresis analysis of urine samples from patients with different forms of chagasic infection. The 90- to 80-kDa urinary protein resembles a trypomastigote-shed antigen. Determination of antigenuria proved valuable for early diagnosis of Chagas' disease and also for diagnosis of chronic cases with conflicting serology.     

梅毒血清固定患者外周血Th17细胞的检测

目的检测梅毒血清固定患者外周血Th17细胞的细胞比例和相关细胞因子的变化,初步探讨Th17细胞在梅毒血清固定现象中的作用。方法梅毒血清固定患者30例,同时以30例健康体检人作为对照组。采用流式细胞术(Flow cytometry, FCM)检测外周血单个核细胞(peripheral blood mononuclear cel s, PBMC)中Th17细胞在CD4+T细胞中的比例;采用实时定量PCR方法检测Th17细胞特异性转录因子孤独受体γt（orphan nuclear receptor gammat , RORγt）及细胞因子IL-17mRNA的表达。结果梅毒血清固定患者外周血Th17细胞比例及RORγt和IL-17的表达量显著低于健康对照组（P<0.05）。结论 Th17细胞的异常可能是梅毒血清固定现象形成的重要原因之一。