Isoenzyme patterns of Blastocystis hominis patient isolates derived from symptomatic and healthy carriers

Isolates of Blastocystis hominis derived from patients with intestinal symptoms and from healthy carriers were cultured in vitro and isoenzyme patterns of hexokinase (E.C. 2.7.1.1), phosphoglucomutase (E.C. 2.7.5.1) and glucose phosphate isomerase (E.C. 5.3.1.9) were investigated to find evidence for pathogenic and non‐pathogenic subspecies of B. hominis . For this purpose we examined 2000 patients of the out‐patient department of the Institute for Tropical Medicine in Tübingen. From these, we obtained 232 B. hominis patient isolates; 119 isolates could be tested for the three isoenzymes. Blastocystis hominis possesses 5 patterns for hexokinase, 11 for phosphoglucomutase and 35 for glucose phosphate isomerase, showing that B. hominis is highly polymorphic. However, there was no correlation between isoenzyme patterns and disease of patients.     

人芽囊原虫的致病性与遗传多样性研究进展

人芽囊原虫是否具有致病性一直颇有争议,无论是流行病学研究还是实验动物研究均存在相互矛盾的结论。研究发现,人芽囊原虫具有广泛的遗传多样性,近年来,研究者用各种方法对此进行了研究。他们将人芽囊原虫分成不同的种群,并尝试探讨其遗传多样性与致病性的关系。该文就人芽囊原虫的致病性、遗传多样性及两者关系的研究进展进行了综述。     

腹泻患者及培养基中人芽囊原虫的各期形态研究

目的研究光镜下各期人芽囊原虫(Blastocystishominis,B.h)形态,以进一步研究其生活史及致病性,并为临床检验提供形态依据。方法使用洛克氏液-鸡蛋-血清培养基,对腹泻患者粪便的B.h进行体外连续培养,经碘液染色及铁苏木素染色,在光镜下研究其各期形态、结构。结果通过形态学研究观察到空泡型、颗粒型、阿米巴型及包囊型以及各型之间的相互转化。结论B.h形态多样,临床多见空泡型和颗粒型。空泡型可成囊。     

中药牛至油、鸦胆子对人芽囊原虫的体外杀灭作用

目的通过观察牛至油、鸦胆子对人芽囊原虫的体外杀灭作用,筛选治疗人芽囊原虫感染有效药物及浓度。方法从腹泻患者新鲜粪便中分离人芽囊原虫,分别用含牛至油和鸦胆子1640培养基培养,药物设200、400、800、1600、3200和6400μg/ml组。于加药24h和72h作虫体计数,评价药物效果,试验设甲硝唑药物对照组和不加药空白对照组。结果牛至油1600、3200μg/ml作用72h、6400μg/ml作用24h,虫体全部死亡,最适杀虫体浓度为800μg/ml;鸦胆子6400μg/ml24h虫体全部死亡,最适杀虫浓度为1600～3200μg/ml;甲硝唑40μg/ml72h、≥80μg/ml24h虫体全部死亡。结论牛至油和鸦胆子具有体外杀灭人芽囊原虫作用。且牛至油的杀虫效果强于鸦胆子。     

全球人群人芽囊原虫感染情况及基因亚型研究进展

人芽囊原虫(Blastocystis hominis)是一种寄生于人和动物肠道内且与多种胃肠道疾病(如腹痛、腹泻)密切相关的寄生虫.人芽囊原虫呈世界性分布,不同国家间甚至同一国家不同地区间流行率和优势基因亚型差异显著.本文对全球人群人芽囊原虫感染率、基因亚型及其地理分布进行综述,旨在为了解人芽囊原虫在全球流行情况和防控...     

人芽囊原虫形态分类及遗传学多样性的研究进展

通过光镜和电镜已广泛研究了人芽囊原虫的形态学,但它的生物学的许多其他方面仍处于未知领域。现在利用大量的各种分子学手段研究芽囊原虫,已使人们开始理解它的生物学。该综述着眼于人芽囊原虫最近研究的一些热点,包括分类学关系和遗传学的多样性以及物种形成等方面。     

Comparison of N-glycan pattern of recombinant human coagulation factors II and IX expressed in Chinese hamster ovary (CHO) and African green monkey (Vero) cells

The N-glycan patterns of recombinant human coagulation factors II (rF-II) and IX (rF-IX), derived from both transfected Chinese hamster ovary (CHO) cells and African green monkay (Vero) cells produced at industrial scale, were analyzed by binding to carbohydrate-specific lectins and were compared with the glycan structure of human plasma-derived coagulation factors. Human plasma-derived coagulation factors II (hpF-II) and IX (hpF-IX) exhibited complex-type glycan structures with carbohydrate chains capped with (2–6)-sialic acid. Terminal galactose-(1–4)-N-acetylglucosamine units were detected in hpF-IX. Both CHO cell-derived rF-II and rF-IX exhibited complex-type glycosylation and contained (2–3)-sialic acid in addition to terminal galactose-(1–4)-N-acetylglucosamine. Vero cell-derived rF-IX exhibited a complex-type glycan structure similar to that of CHO cell-derived rF-IX. In contrast, rF-II produced by Vero cells exhibited a glycan microheterogeneity composed of hybrid-type glycosylation containing high-mannose structures and complex-type glycosylation containing (2–3)-sialic acid. Galactose-(1–4)-N-acetylglucosamine structures and a low concentration of (2–6)-sialic acid were detected in both microheterogeneity fractions of Vero cell-derived rF-II. Although different in their carbohydrate structures, coagulation factors II and IX obtained recombinantly from both transformed CHO cells and Vero cells exhibited coagulation activities comparable with the plasma-derived proteins.     

The influence of DNA-topoisomerase II inhibitors novobiocin and fostriecin on the induction and repair of DNA damage in Chinese hamster ovary (CHO) cells treated with mitoxantrone.

Novobiocin (NB) at the concentration of 2 mmol/l added to the culture medium together with mitoxantrone (MIT) (0.05-0.2 micrograms/ml) reduced the number of MIT-induced single-strand breaks of DNA to approximately one half measured by alkaline DNA unwinding and hydroxyapatite chromatography of DNA and similarly it reduced also the fraction of DNA linked to proteins measured by the K(+) -SDS precipitation method. Neither repair of the induced DNA breaks nor removal of the DNA-protein cross-links were markedly influenced by NB action. The specific inhibitor of topoisomerase II, fostriecin, exerted no effect on the induction of DNA breaks by MIT or their repair. Measurement of intracellular concentration of MIT has revealed that in the presence of NB the uptake of MIT into cells is reduced similarly as the number of induced DNA breaks to approximately one half. The combination of 0.1 mmol araC + 10 mmol HU slightly reduced the number of induced DNA breaks, but did not affect their repair. The present results suggest that (1) MIT induces DNA damage which is not repaired by excision repair, (2) MIT induces protein associated breaks of DNA, (3) topoisomerase II does not probably participate in the formation of DNA breaks induced by MIT, as the specific inhibitor of topoisomerase II, fostriecin exerts no effect on either the induction or repair of these breaks.     

血管活性肠肽对体外大肠癌 HT29细胞粘附和侵袭作用的影响

目的观察血管活性肠肽(VIP)和双丁酰环磷酸腺苷(db-cAMP)对体外大肠癌 HT29细胞对大鼠血管内皮的粘附和侵袭的影响。方法取出大鼠主动脉,建立体外癌细胞—血管内皮粘附试验模型。用光镜和扫描电镜观察 HT29细胞对大鼠血管内皮粘附作用和其形态学变化,以及 VIP 和 db-cAMP 对细胞粘附的影响并在扫描电镜上计数量化分析。结果在血管内皮上可清晰辨认癌细胞并可观察其对血管内皮的粘附和侵袭的形态学改变.VIP 组的粘附细胞和高活性粘附细胞显著多于对照组(P<0.05),相反 db-cAMP 则无此作用.结论 VIP可促进体外 HT29细胞对血管内皮的粘附和侵袭,其机制可能与激活癌细胞 A 激酶系统有关。     

Mitotic and neurogenic effects of dehydroepiandrosterone (DHEA) on human neural stem cell cultures derived from the fetal cortex

Dehydroepiandrosterone (DHEA) is a neurosteroid with potential effects on neurogenesis and neuronal survival in humans. However, most studies on DHEA have been performed in rodents, and there is little direct evidence for biological effects on the human nervous system. Furthermore, the mechanism of its action is unknown. Here, we show that DHEA significantly increased the growth rates of human neural stem cells derived from the fetal cortex and grown with both epidermal growth factor (EGF) and leukemia inhibitory factor (LIF). However, it had no effect on cultures grown in either factor alone, suggesting a specific action on the EGF/LIF-responsive cell. Precursors of DHEA such as pregnenolone or six of its major metabolites, had no significant effect on proliferation rates. DHEA did not alter the small number (<3%) of newly formed neuroblasts or the large number (>95%) of nestin-positive precursors. However, the number of glial fibrillary acidic protein-positive cells, its mRNA, and protein were significantly increased by DHEA. We found both N-methyl-d-aspartate and sigma 1 antagonists, but not GABA antagonists, could completely eliminate the effects of DHEA on stem cell proliferation. Finally we asked whether the EGF/LIF/DHEA-responsive stem cells had an increased potential for neurogenesis and found a 29% increase in neuronal production when compared to cultures grown in EGF/LIF alone. Together these data suggest that DHEA is involved in the maintenance and division of human neural stem cells. Given the wide availability of this neurosteroid, this finding has important implications for future use.     

Effects of granulocyte-macrophage colony stimulating factor produced in Chinese hamster ovary cells (regramostim), Escherichia coli (molgramostim) and yeast (sargramostim) on priming peripheral blood progenitor cells for use with autologous Done marrow after high-dose chemotherapy

Abstract: Peripheral blood progenitor cells (PBPCs) were collected without prior association with chemotherapy but after the administration of granulocyte-macrophage colony-stimulating factor (GM-CSF) produced in Chinese hamster ovary cells (CHO-GM, regramostim), Escherichia coli (E. coli-GM, molgramostim), or yeast (Yeast-GM, sargramostim) and used in conjunction with autologous bone marrow after high-dose chemotherapy in 69 patients with breast cancer or melanoma. The mean peripheral white blood cell (WBC) counts increased by 2.2 to 2.7-fold after regramostim, 4.5 to 7.3-fold after molgramostim and 4.3-fold after sargramostim. All patients underwent three leukaphereses. The mean (& standard error) total nucleated pheresed cells per kg × 10⁸ were 4.15 & 0.56, 15.10 & 1.77 and 7.24 & 1.00 for patients receiving regramostim, molgramostim or sargramostim respectively. The mean (& standard error) granulocyte-macrophage colony-forming units per kg × 10⁴ mobilized into the PB were 8.75 & 3.63, 71.03 & 17.85, and 65.11 & 18.74 for patients receiving regramostim, molgramostim, or sargramostim respectively. The total mean (& standard error) CD34+ cells per kg × 10⁷ collected by three leukaphereses were 3.28 & 1.62, 1.34 & 0.51 and 2.57 & 1.93, for patients receiving regramostim, molgramostim or sargramostim respectively. The use of either molgramostim- or sargramostim-primed PBPCs led to complete elimination of absolute leukopenia with a WBC count under 100/mm³ in 64% and 77% of patients treated, respectively. Patients receiving molgramostim-primed PBPCs required fewer red blood cells transfusions than patients receiving regramostim-primed PBPCs (p = 0.0062). Our data indicate that PBPCs collected without prior association with chemotherapy but after either molgramostim or sargramostim with autologous bone marrow support and GM-CSF shorten the hematopoietic recovery after myeloablative chemotherapy in patients with breast cancer or melanoma.     

Effects of granulocyte-macrophage colony stimulating factor produced in Chinese hamster ovary cells (regramostim), Escherichia coli (molgramostim) and yeast (sargramostim) on priming peripheral blood progenitor cells for use with autologous bone marrow after high-dose chemotherapy

Abstract: Peripheral blood progenitor cells (PBPCs) were collected without prior association with chemotherapy but after the administration of granulocyte-macrophage colony-stimulating factor (GM-CSF) produced in Chinese hamster ovary cells (CHO-GM, regramostim), Escherichia coli (E. coli-GM, molgramostim), or yeast (Yeast-GM, sargramostim) and used in conjunction with autologous bone marrow after high-dose chemotherapy in 69 patients with breast cancer or melanoma. The mean peripheral white blood cell (WBC) counts increased by 2.2 to 2.7-fold after regramostim, 4.5 to 7.3-fold after molgramostim and 4.3-fold after sargramostim. All patients underwent three leukaphereses. The mean (± standard error) total nucleated pheresed cells per kg × 10⁸ were 4.15 ± 0.56, 15.10 ± 1.77 and 7.24 ± 1.00 for patients receiving regramostim, molgramostim or sargramostim respectively. The mean (± standard error) granulocyte-macrophage colony-forming units per kg × 10⁴ mobilized into the PB were 8.75 ± 3.63, 71.03 ± 17.85, and 65.11 ± 18.74 for patients receiving regramostim, molgramostim, or sargramostim respectively. The total mean (± standard error) CD34+ cells per kg × 10⁷ collected by three leukaphereses were 3.28 ± 1.62, 1.34 ± 0.51 and 2.57 ± 1.93, for patients receiving regramostim, molgramostim or sargramostim respectively. The use of either molgramostim- or sargramostim-primed PBPCs led to complete elimination of absolute leukopenia with a WBC count under 100/mm³ in 64% and 77% of patients treated, respectively. Patients receiving molgramostim-primed PBPCs required fewer red blood cells transfusions than patients receiving regramostim-primed PBPCs (p = 0.0062). Our data indicate that PBPCs collected without prior association with chemotherapy but after either molgramostim or sargramostim with autologous bone marrow support and GM-CSF shorten the hematopoietic recovery after myeloablative chemotherapy in patients with breast cancer or melanoma.     

Effects of recombinant human stem cell factor (rh-SCF) on colony formation and long-term bone marrow cultures (LTBMC) in patients with myelodysplastic syndromes

Abstract: Stem cell factor (SCF), the ligand of the c-kit receptor, is a potent enhancing cytokine for haematopoietic cells in the presence of IL-3, GM-CSF and erythropoietin (Epo). In the clonogenic assays of 63 MDS patients, the addition of rh-SCF + GM-CSF and/or IL-3 induced a significant increase (p<0.001) in the number and size of CFU-GM. Never reaching the levels of controls, this increase was seen in all FAB subtypes, but particularly in RA. There was no significant increase in cluster formation, even in RAEB or RAEBt. Rh-SCF (10 ng/ml) led to mean increases of up to 26 times in the number of Epo-dependent BFU-E colonies, particularly in RA (p<0.001) and RAEB (p<0.05). Individual responses varied widely (especially in RA) from no response to supranormal levels. Added to the weekly refeed of 37 MDS LTBMC, SCF (10 ng/ml) induced only a 7% mean increase in both cell output and the number of clonogenic cells recovered in the supernatant. Immunohistochemical examination of the supernatant showed significant increases in differentiating myeloid cells in all examined cases, and in erythroid cells in 3 cases; blast cells increased in only 3 cases. These data suggest that rh-SCF is capable of at least partially reversing defective MDS myeloid haematopoiesis, and leads no overt risk of leukaemic transformation. Its potent effect on erythroid cells is encouraging for future clinical applications in patients, particulary if they are selected by means of in vitro tests.     

Discovery of amino acid variants in the human glucose-dependent insulinotropic polypeptide (GIP) receptor: the impact on the pancreatic beta cell responses and functional expression studies in Chinese hamster fibroblast cells

Summary The two incretins, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), are insulinotropic factors released from the small intestine to the blood stream in response to oral glucose ingestion. The insulinotropic effect of GLP-1 is maintained in patients with Type II (non-insulin-dependent) diabetes mellitus, whereas, for unknown reasons, the effect of GIP is diminished or lacking. We defined the exon-intron boundaries of the human GIP receptor, made a mutational analysis of the gene and identified two amino acid substitutions, A207 V and E354Q. In an association study of 227 Caucasian Type II diabetic patients and 224 matched glucose tolerant control subjects, the allelic frequency of the A207 V polymorphism was 1.1 % in Type II diabetic patients and 0.7 % in control subjects (p = 0.48), whereas the allelic frequency of the codon 354 polymorphism was 24.9 % in Type II diabetic patients versus 23.2 % in control subjects. Interestingly, the glucose tolerant subjects (6 % of the population) who were homozygous for the codon 354 variant had on average a 14 % decrease in fasting serum C-peptide concentration (p = 0.01) and an 11 % decrease in the same variable 30 min after an oral glucose load (p = 0.03) compared with subjects with the wild-type receptor. Investigation of the function of the two GIP receptor variants in Chinese hamster fibroblasts showed, however, that the GIP-induced cAMP formation and the binding of GIP to cells expressing the variant receptors were not different from the findings in cells expressing the wild-type GIP receptor. In conclusion, amino acid variants in the GIP receptor are not associated with random Type II diabetes in patients of Danish Caucasian origin or with altered GIP binding and GIP-induced cAMP production when stably transfected in Chinese hamster fibroblasts. The finding of an association between homozygosity for the codon 354 variant and reduced fasting and post oral glucose tolerance test (OGTT) serum C-peptide concentrations, however, calls for further investigations and could suggest that GIP even in the fasting state regulates the beta-cell secretory response. [Diabetologia (1998) 41: 1194–1198] Received: 13 February 1998 and in final revised form: 29 May 1998     

In vitro and in vivo effects of 2'-deoxycoformycin (Pentostatin) on tumour cells from human gammadelta+ T-cell malignancies

Hepatosplenic gammadelta+ T-cell lymphoma represents a rare neoplasm of post-thymic phenotype, characterized by an aggressive clinical course and a poor response to conventional chemotherapy. In the present study, we have examined the cytotoxic effects of the purine analogue 2'-deoxycoformycin (dCF) on cultured mononuclear cells and purified gammadelta+ tumour cells from bone marrow or peripheral blood of four patients with hepatosplenic gammadelta+ T-cell lymphoma. At a concentration of 10 microM, dCF, in the presence of 2'-deoxyadenosine (dAdo), displayed an early and selective cytotoxic effect on gammadelta+ tumour T cells. After 48 h of in vitro exposure to dCF, the absolute number of viable CD3+/gammadelta+ tumour T cells was reduced by more than 90% in all samples with respect to control cultures, with absolute counts of viable CD3+/alphabeta+ lymphocytes being reduced only by 6-40% of the initial cell input. Analysis of cultures after 5 d of exposure to dCF plus dAdo revealed the persistence of normal CD3+/alphabeta+ T cells, which accounted, however, for only 20-25% of the initial cell input. Accordingly, the combination of dCF (10-100 microM) plus dAdo was able to induce a dose-dependent inhibition of clonogenic growth and [3H]-thymidine incorporation in purified CD3+/CD4-/CD8- gammadelta+ tumour cells. We also report that one patient with hepatosplenic gammadelta+ T-cell lymphoma in terminal leukaemic phase showed a striking haematological response to single-agent dCF given as fourth-line treatment. In particular, the selective clearance of gammadelta+ tumour T cells in peripheral blood and bone marrow was observed starting after the second course of treatment. Our results suggest that dCF may represent a potentially active drug for the management of this aggressive form of T-cell lymphoma.     

Effect of the pineal gland on circadian rhythmicity of colony forming units for granulocytes and macrophages (CFU-GM) from rat bone marrow cell cultures

The present study provides evidence that the pineal gland has a physiological role in the proliferation of colony forming units for granulocytes and macrophages (CFU-GM). A biphasic circadian rhythm of CFU-GM proliferation in rat bone marrow cell cultures (BMC) from intact animals peaking at 0600 and 1800 was observed. Pinealectomy (Px) at 1600 obliterated the circadian rhythm patterns of CFU-GM. Afternoon injections of melatonin (1630, 20 micrograms/per day for 10 days) to Px animals restored the rhythmicity. When pinealectomy was done at 0800, the morning peak remained unaltered and the colony number at 1800 was higher than that found in the afternoon Px animals. In conclusion, the pineal gland or its main hormone melatonin seems to have a regulatory role in the proliferation of CFU-GM in rat BMC. Further, the expression of the activity of CFU-GM in rat BMC depends on the time when pinealectomy is done or melatonin is substituted.     

Differential effects of cetuximab and AEE 788 on epidermal growth factor receptor (EGF-R) and vascular endothelial growth factor receptor (VEGF-R) in thyroid cancer cell lines

This study evaluated the role of EGF and the effects of EGF-targeting drugs (Cetuximab, AEE 788) on growth, apoptosis, and autocrine VEGF-secretion of thyroid cancer (TC) cells. Autocrine activation of the epidermal growth factor receptor (EGF-R) is commonly regarded to contribute to the malignant phenotype of TC cells and may therefore represent a rational therapeutic target. Out of a number of TC cell lines two anaplastic (Hth74, C643), one follicular (FTC133), and one papillary thyroid cancer cell line (TPC1) were analyzed in depth for VEGF-R-and EGF-R-expression, basal and EGF-stimulated (1-100 ng/ml) VEGF protein secretion and proliferation. Subsequently the antiprolifereative and antiangiogenic effect of cetuximab (Erbitux), a monoclonal antibody that blocks the EGF-R and AEE 788, a novel dual-kinase inhibitor of EGF-R and VEGF-R were assessed, and the downstream EGF-R signal transduction was analyzed by means of detecting phosphorylated pEGF-R, pVEGF-R, pAkt, and p-MAPK. EGF stimulated VEGF-mRNA expression and protein secretion in all TC cell lines. The EGF-R antagonist Cetuximab consistently decreased VEGF secretion in all TC cell lines (min. 15%, n.s. in C643 cells and max. 90% in Hth74 cells, P < 0.05), but did not affect tumor cell proliferation in vitro. In contrast, the EGF-R- and VEGF-R-kinase inhibitor AEE 788 not only reduced VEGF secretion (min. 55%, P < 0.05 in C643 and max. 75%, P < 0.05, in FTC133), but also exhibited a dose-dependent inhibition of tumor cell proliferation (min. 75%, P < 0.05 in C643 and max. 95%, P < 0.05 in Hth74) and was a potent inductor of apoptosis in two of four TC cell lines. These effects were always accompanied by reduced levels of pEGF-R, pVEGF-R, pAkt, and pMAPK. Although inhibition of the EGF-receptor by Cetuximab potently disrupts autocrine secretion of VEGF, only the concurrent inhibition of the VEGF- and EGF receptor, e.g., by AEE 788 induces reduced proliferation and apoptosis in vitro. This suggests a particular rationale for the use of tyrosine kinase inhibitors with dual modes of action such as AEE 788 in thyroid cancer.     

原发性肝癌组织中Kruppel样因子6基因的变异及其抗细胞增殖功能的改变

目的初步探讨基因突变对Kruppel样因子6(KLF6)抗肝癌细胞增殖功能的影响及机制。方法以聚合酶链反应(PCR)扩增原发性肝癌及其癌旁组织KLF6基因外显子2序列,对扩增的PCR产物进行双向序列测定。构建野生型及突变型KLF6真核表达质粒并分别转染人肝癌细胞株HepG2,流式细胞仪及四甲基偶氮唑盐试验观察KLF6基因对细胞周期及细胞增殖活性的影响。以western blot技术检测外源KLF6 基因对HepG2细胞p21WAF1表达的影响。结果 23例原发性肝癌组织中2例(8.7％)出现KLF6基因突变, 其中1例导致氨基酸改变(W162G)。转染野生型KLF6的HepG2细胞G0～G1期所占细胞的比例明显增高,而转染突变型KLF6实验组G0～G1期细胞无显著增加。转染野生型KLF6对HepG2细胞生长的抑制率显著高于突变型KLF6实验组和空载体实验对照组。外源野生型KLF6基因能上调肝癌细胞p21WAF1表达,而突变型KLF6基因上调p21WAF1表达的能力较低。结论 KLF6基因突变可能在原发性肝癌发病机制中起到一定作用,但基因突变可能不是KLF6基因失活的主要原因。上调p21WAF1表达是KLF6基因抑制肝癌细胞增殖的重要途径。