Detection of Toxoplasma gondii DNA in primary intraocular B-cell lymphoma.

Primary intraocular lymphoma, a variant of primary central nervous system lymphoma with ocular involvement, is a large B-cell non-Hodgkin's lymphoma. Some cases of primary intraocular lymphoma have been reported to be associated with microorganisms including Epstein-Barr virus (EBV) and human herpes virus-8 (HHV-8), but not parasites. We analyzed 10 cases of primary intraocular lymphoma using microdissection and PCR. Tumor and normal cells were microdissected from ocular tissue on slides and subjected to PCR for genes from Toxoplasma gondii, EBV, and HHV-8. We detected Toxoplasma gondii, not HHV-8 or EBV, DNA in the lymphoma but not in normal cells of two cases that resembled ocular toxoplasmosis clinically. We speculate that Toxoplasma gondii may play a role in some forms of primary intraocular B-cell lymphoma.     

刚地弓形虫培养上清对THP-1增殖的影响及可能机制

 利用弓形虫速殖子细胞内寄生的特性和对宿主细胞生物学行为的影响,提取弓形虫体外细胞共培养上清,并研究上清对人急性单核细胞白血病细胞THP-1增殖及细胞周期的影响。方法 取对数生长期的THP-1细胞（浓度为5×105）分别接种于不同细胞培养瓶,对照组加入含10%胎牛血清RPMI-1640,试验组加入相同体积不同数量(2×107mL-1、 4×107mL-1、 8×107 mL-1 )弓形虫速殖子培养上清孵育不同时间后, MTT法检测THP-1细胞增殖抑制率;流式细胞仪检测细胞周期改变;以Western印迹方法分析上清作用48h后细胞核转录因子NF-κB/p65与周期蛋白cyclinD1表达或活性的变化。结果 MTT法检测结果显示弓形虫培养上清抑制THP-1细胞株增殖,且呈时间剂量依赖性。流式细胞仪检测显示处理组细胞周期在G0/G1期产生阻滞; Western印迹方法分析THP-1细胞株的NF-κB/p65、cyclin D1 蛋白表达量下降,结论 刚地弓形虫培养上清能够抑制人急性单核细胞白血病细胞THP-1细胞株增殖并可通过细胞株的NF-κB信号途径来下调cyclin D1蛋白表达引起人急性单核细胞白血病细胞THP-1细胞株G0/G1期阻滞。     

Pituitary pathology in acquired immunodeficiency syndrome

Pituitary morphology was studied in 49 autopsied patients with acquired immunodeficiency syndrome. Direct infectious involvement was noted in six adenohypophyses (12%), including five cases by cytomegalovirus and one by Pneumocystis carinii. Two cases with neurohypophysial lesions presumably caused by cytomegalovirus and one questionable case of Toxoplasma gondii were also observed. In all instances these changes were associated with generalized and/or cerebral infection by these same agents. Neither Kaposi's sarcoma nor malignant lymphoma was encountered in the pituitary glands. Acute necrotic foci, presumably due to infarction, were noted in four cases. Four pituitary microadenomas (8%) and four hyperplastic nodules were identified. The incidence of such noninfectious lesions, as well as the prevalence and distribution of the various immunoreactive adenohypophysial cell types, were similar to those seen in the pituitary glands of age-matched male control patients.     

Interactions between normal and tumoral tissues at the boundary of human anterior pituitary adenomas

We studied the boundary between adenoma and peritumoral anterior pituitary tissues in order to understand their mutual interactions during tumour progression. We selected 18 adenomas of different secretory type, grade and invasiveness in which fragments of peritumoral anterior pituitary were still attached to the adenoma. Immunohistochemistry was performed on serial sections with markers of the basement membranes (type IV collagen), the hormone-producing cells of the normal and neoplastic anterior pituitary, and the folliculo-stellate cells (S-100 protein). In passing from tumour to gland, localized areas of passive compression of the normal gland were seen in only 3 cases. In all the tumours, the boundary consisted partly or solely of a transitional zone characterized by the presence of enlarged cell-cords. Openings in the basement membrane of these enlarged cell-cords were seen in contact with the tumour tissue. Normal and neoplastic cells intermingled in the transitional zone. Normal residual cells could be seen in the central area of the tumour but no adenomatous cells were observed in the gland around the tumour. Folliculo-stellate cells were concentrated in the vicinity of the transition zone. These findings favour the existence of an active process of adenoma expansion within the normal parenchyma, without noticeable infiltration of tumour cells into surrounding gland.     

Collision Tumors of the Sella: Craniopharyngioma and Silent Pituitary Adenoma Subtype 3: Case Report

Collision tumors of the sella turcica are rare and consist mainly of more than one type of pituitary adenoma, usually a corticotropin- and a prolactin-producing adenoma. The association of a craniopharyngioma and a pituitary adenoma is rare. Herein, we report the first case of an association between craniopharyngioma and silent pituitary adenoma subtype 3. It involved a 12-year-old boy who underwent a frontal craniotomy with surgical removal of a calcified sellar tumor. Histology revealed an adamantinomatous craniopharyngioma; however, by electron microscopy, there was conclusive evidence of adenoma cells showing the ultrastructural features of silent pituitary adenoma subtype 3. Endocrine and neuroimaging as well as detailed immunohistochemical and ultrastructural studies were undertaken. The literature is also reviewed.     

刚地弓形虫培养上清抑制肺癌A549细胞系增殖

目的研究刚地弓形虫(Toxoplasma gondii)培养上清对体外培养的肺癌细胞A549增殖及细胞周期的影响。方法取对数生长期的A549细胞(浓度为5×104mL-1)分别接种于不同细胞培养板,对照组加入RPMI-1640孵育,试验组加入相同体积不同数量(4×107mL-1、8×107mL-1、16×107mL-1)弓形虫速殖子培养上清孵育不同时间后,四甲基氮噻唑蓝(MTT)法检测吸光度(A490值);PI染色后检测细胞周期;以Western blot检测细胞周期蛋白cyclinB1、cdc2表达或活性。结果弓形虫培养上清呈时间剂量依赖性抑制A549细胞系增殖,处理组细胞周期在G2/M期产生阻滞;A549细胞系的cyclinB1、cdc2蛋白表达量下降。结论刚地弓形虫培养上清能够抑制肺癌A549细胞系增殖,并通过调节cyclinB1、cdc2等蛋白表达或活性改变引起肺癌A549细胞系G2/M期阻滞。     

Localization of prolactin in chromophobe pituitary adenomas: study of human necropsy material by immunoperoxidase technique.

In order to identify prolactin-producing tumours in human pituitary glands, 45 chromophobe adenomas, obtained from unselected necropsies, have been studied by various staining procedures including the immunoperoxidase technique for the demonstration of prolactin. The presence of immunoreactive prolactin was revealed in the cytoplasm of the tumour cells in six cases (13%), indicating that the occurrence of prolactin-producing adenomas is not rare. No correlations were established between tumours and clinical history. Two adenomas were detected in female and four in male patients. The age of the patients at necropsy ranged from 28 to 75 years. Three adenomas were associated with disseminated carcinoma, two with fatal liver disease, and one with diabetes mellitus, atherosclerosis, and pyelonephritis. Manifest endocrine symptoms were not disclosed, and endocrine investigations, including measurements of blood prolactin levels, were not undertaken. Thus, direct evidence is lacking as to whether or not these tumours were actively secreting prolactin. In the non-tumorous parts of the anterior lobes the number of prolactin cells was decreased in two cases, suggesting that prolactin released from the adenoma cells suppressed prolactin production in the non-tumorous pituitary. However, the number of prolactin cells of the non-tumorous adenohypophysis seemed to be unchanged in two and increased in another two cases. The present findings conclusively proved the existence of the prolactin-producing adenomas as a distinct entity. These tumours do not stain with acid or basic dyes, they are PAS or thionin negative, and do not contain immunoreactive growth hormone. Thus, by conventional staining procedures they are indistinguishable from other chromophobe adenoma types. Herlant's erythrosin and Brookes' carmoisine methods, claimed spedifically to stain prolactin cells, failed to provide reliable results, hence their use cannot be recommended in tumour identification. Immunoperoxidase staining of prolactin is the only technique which conclusively reveals the presence of immunoreactive prolactin in the cytoplasm of the tumour cells and permits diagnosis. It is proposed that this technique be introduced in pituitary morphological studies. Its application may lead to a better understanding of problems related to prolactin-producing tumours and their secretory activity.     

Combined sellar fibrosarcoma and prolactinoma with neuronal metaplasia: Report of a case unassociated with radiotherapy

We report the occurrence of a primary pituitary fibrosarcoma causally unrelated to radiotherapy, admixed in association with a prolactin cell pituitary adenoma showing neuronal metaplasia. These unique findings were associated with multiple endocrine neoplasia type 1 (MEN 1). Primary fibrosarcoma involving the sella is a very rare tumor. The majority of cases have been associated with prior irradiation of either a pituitary adenoma or a craniopharyngioma. Pituitary adenoma with neuronal metaplasia is also rare and usually occurs in the setting of acromegaly. Despite the intimate association of both elements in our lesion, no transition of adenoma to sarcoma was demonstrable by immunohistochemistry or in situ hybridization studies.     

Rapid prenatal diagnosis of congenital Toxoplasma infection by using polymerase chain reaction and amniotic fluid.

Infection of pregnant women with Toxoplasma gondii places the developing fetus at risk for congenital infection. We report a prospective study of 43 documented cases of acute maternal Toxoplasma infections acquired during gestation in which the polymerase chain reaction (PCR) was evaluated for diagnosis of fetal infection and compared with the current standard methods. On the basis of direct lysis of pelleted amniotic fluid cells followed by amplification of a gene sequence specific for T. gondii, PCR correctly identified the presence of T. gondii in five of five samples of amniotic fluid from four proven cases of congenital infection. PCR also detected three of five positive cases from a nonprospective group. The two diagnostic methods of comparable speed, detection of specific immunoglobulin M from fetal blood and and inoculation of amniotic fluid into tissue culture, correctly identified only 3 and 4 of the 10 positive samples, respectively. The considerably more time-consuming methods of mouse inoculation of amniotic fluid and fetal blood both detected 7 of 10 positive samples. There were no false-positive diagnoses by any of the methods. Therefore, detection of T. gondii by PCR appears to be the most promising method for prenatal diagnosis of congenital Toxoplasma infection, since it is both extremely rapid and highly sensitive.     

DNA聚合酶链反应检测畸形儿心肌组织中弓形虫的研究

孕妇感染弓形虫是胎儿先天性畸形的危险因素。本文运用被称之为ＤＮＡ聚合酶链反应的体外基因扩增技术检测３０例先天性畸形儿尸检石蜡包埋心肌组织弓形虫。结果表明，３０例先天性畸形儿石蜡心肌组织切片经体外扩增后出现阳性条带者１３例，而对照组（１０例）无一例阳性（０．０５＞Ｐ＞０．０１）。研究结果进一步支持弓形虫的感染与胎儿先天性畸形有密切关系，本文还讨论了聚合酶链反应技术与其它最新技术比较，聚合酶链反应技术具有微量、特异性强、敏感度高、反应周期短、操作简便，对长期保存并已消失感染性的样品也有效等优点，适应于临床推广。     

The immunohistochemistry of pituitary adenomas

A great deal of progress has been achieved in recent years in the field of immunohistochemistry of pituitary adenoma. Continued use of more new antisera and monoclonal antibodies against numerous hormones in the adenohypophysis have resulted in new approaches to classification of pituitary adenoma. However, new problems have been discovered, on the other hand, by large-scale studies in recent years. The great number of multihormonal pituitary adenomas and possible change of the immunohistochemically detectable hormone status in cases of recurrent tumours have particularly re-emphasised the need for new thinking about patterns of classification. It would appear somewhat problematic, in this context, to uncritically accept terms, such as ACTH cellular adenoma or GH cellular adenoma. Reference is also made to the distribution pattern of cell and tissue markers in pituitary adenomas. The paper is based on thorough literature screening as well as on experience obtained by the authors from 450 cases of pituitary adenoma of which 260 had been analysed by immunohistochemistry, 131 by morphometry, and 80 by electron microscopy.     

High prevalence rate of pituitary incidentaloma: is it associated with the age-related decline of the sex hormones levels?

Incidental pituitary adenoma is the common finding during brain imaging. According to multistep model of pituitary tumourigenesis genetic alterations provide the initiating event that transforms cells while hormones play a role in promoting cell proliferation. Development of pituitary adenoma in a case of excessive hypophysiotrophic hormones production or reduced feedback suppression by target gland hormones emphasizes the importance of hormonal stimulation in pituitary tumourigenesis. Pituitary hyperplasia has been reported in pregnancy, hypothyroidism and conditions such as CRH or GHRH hypersecretion. Moreover, recent study reported one case of gonadotroph macroadenoma and two cases of gonadotroph cells hyperplasia in patients with Klinefelter syndrome probably due to protracted stimulation of gonadotroph cells because of lack of androgen feedback. Significant changes of the hypothalamic-pituitary-gonadal axis occurred with aging. In females, after menopause, estradiol level decreases by 35-fold and estrone level by 20-fold that results in increased gonadotropins levels. Similarly, FSH, but not LH, level is increased with advancing age in men, too, although the age-related difference in the level is less in comparison with women. Regarding these data, we hypothesised that high prevalence rate of pituitary incidentaloma in the elderly is associated with age-related decline in sex hormones levels and subsequent lack of feedback suppression leading to permanent gonadotrophs stimulation which is the crucial step in the pituitary tumour development. According to previously mentioned multistep model of pituitary tumourigenesis, incidentaloma will develop only in persons with already present intrinsic pituitary cell defects. However, further studies have to answer the questions of whether the incidence of pituitary tumours is more frequent in elderly, whether women with late onset menopause or those taking long-term hormone replacement therapy have lower rate of pituitary incidentaloma, and finally, is there any correlation between pituitary tumours incidence and serum concentrations of LH, FSH, bioavailable testosterone or estradiol.     

Plurihormonal pituitary adenomas: immunostaining of all pituitary hormones is mandatory for correct classification

AIMS: We studied the clinicopathological characteristics of plurihormonal pituitary adenomas. METHODS AND RESULTS: The study material included 167 plurihormonal adenomas, which consisted of 31% of the surgically removed pituitary adenomas that we collected during a 12-year period. The mean age of patients with plurihormonal adenoma was 45.7 years (range 13-75 years). There were 86 men and 81 women. All tumours were fully classified by immunohistochemical staining for seven pituitary hormones or subunits. Thirty immunohistochemical subtypes of plurihormonal adenomas were recognized. Hormonal symptoms were present in 70% of patients, while serum hormonal levels were increased in 89% of patients. Most patients had symptoms related to only one of the hormones and only 7% of patients had symptoms related to two hormones. The most common hormonal symptom was acromegaly (50%); symptoms related to hyperprolactinaemia ranked second (20%). Double immunostaining of all the possible combinations of the hormones was performed in 30 selected tumours, and they all showed mixtures of hormones in individual adenoma cells in any hormonal combinations studied. The latter finding supported the view that plurihormonal adenomas are monomorphous adenomas. CONCLUSIONS: Plurihormonal adenomas are common pituitary adenomas. Immunohistochemical staining of all pituitary hormones is mandatory for correct classification.     

免疫组织化学ABC法观察扁桃酸对小鼠急性弓形虫病肝脏病变的作用

目的用免疫组织化学ABC法观察扁桃酸治疗小鼠急性弓形虫病肝脏的病变，探讨扁桃酸对病变肝脏的保护作用。方法扁桃酸200mg／kg，2次／d，口服和静脉注射治疗弓形虫感染小鼠，同时设立乙胺嘧啶及扁桃酸药物对照组，治疗不同时间后取肝脏组织，常规制作病理切片，免疫组织化学ABC法常规染色。结果阳性对照组可见大量肝细胞坏死区，其内可见形态典型的弓形虫速殖子，扁桃酸治疗组仅见少量散在的黄染肝细胞，乙胺嘧啶对照组小鼠肝组织内也仅见少量散在的黄染肝细胞，但肝细胞受到明显的毒性损害。因而扁桃酸能有效地抑制弓形虫速殖子入侵肝细胞，减轻弓形虫速殖子对肝细胞的破坏程度，且扁桃酸对肝细胞的毒副作用极少。结论扁桃酸对弓形虫感染小鼠的肝脏有明显的保护作用。     

A pleiomorphic GH pituitary adenoma from a Carney complex patient displays universal allelic loss at the protein kinase A regulatory subunit 1A (PRKARIA) locus

Carney complex (CNC) is a familial multiple endocrine neoplasia syndrome associated with GH-producing pituitary tumours and transmitted as an autosomal dominant trait. Mutations of the PRKAR1A gene are responsible for approximately half the known CNC cases but have never found in sporadic pituitary tumours. Pituitary tissue was obtained from an acromegalic CNC patient heterozygote for a common (PRKARIA)_i-inactivating mutation. Both immunohistochemistry and electron microscopy showed a highly pleiomorphic pituitary adenoma. The cell culture population appeared morphologically heterogeneous and remained so after more than 30 passages. The mixture was comprised of cells strongly immunostained for GH, spindle-shaped myofibroblast-like cells, and cuboid cells with large axonal projections (negative for GH). The population appeared to have both epithelial and mesenchymal cells. Both at baseline and at passage 30, cytogenetic analysis indicated the presence of normal 46, XY diploid karyotype, whereas losses of the PRKARIA_i locus were demonstrated in more than 98% of the cells by fluorescent in situ hybridisation, supporting this gene's involvement in pituitary tumorigenesis. Allelic loss may have occurred in a single precursor cell type that differentiated and clonally expanded into several phenotypes. Epithelial-to-mesenchymal transition may also occur in CNC-associated pleiomorphic pituitary adenomas.     

Early prenatal diagnosis of congenital toxoplamosis using amniotic fluid samples and tissue culture

The presence of Toxoplasma gondii in amniotic fluid was demonstrated using tissue culture in four of nine cases of congenital toxoplasmosis, whereas Toxoplasma gondii antigen was undetectable using a sensitive enzyme immunoassay technique. Parasites were identified in monolayers four days after inoculation using an indirect immunofluorescence assay. Since tissue culture may provide evidence of infection within a few days, this method is proposed for early prenatal diagnosis of congenital toxoplasmosis.     

Direct genotyping of Toxoplasma gondii in ocular fluid samples from 20 patients with ocular toxoplasmosis: predominance of type II in France

We report the direct genotyping analysis of Toxoplasma gondii in ocular samples collected from 20 patients, as well as associated clinical and epidemiological data. This work was aimed at better understanding the impact of genotypes of Toxoplasma gondii strains on toxoplasmic retinochoroiditis. For this purpose, we studied the aqueous humor (AH) or vitreous humor (VH) of 20 patients presenting with ocular toxoplasmosis (OT) in 2 hospitals in France. Genetic characterization was obtained with microsatellite markers in a multiplex PCR assay. In contrast to the results of previous studies, we found no association between atypical Toxoplasma gondii genotypes and the occurrence of OT. Considering the local epidemiological data, our OT patients seemed to be infected more frequently by ordinary type II strains found in the environment. In conclusion, direct genotyping of Toxoplasma gondii strains from aqueous or vitreous humor showed a predominance of the type II genotype in ocular toxoplasmosis; this may be due to a high exposure rate of this genotype in humans.     

Toxoplasmosis--morphologic diagnosis options for the ever topical disease

It has been estimated that 60 percent of people in Croatia will be infected with Toxoplasma gondii until the age of 40. The infection is most frequently asymptomatic, or presented with an lymphadenitis, acute infection during pregnancy that can cause serious damage to the fetus in 10-30 percent, depending on the gestation phase. After acute infection Toxoplasma gondii resides in the body for life, being controlled by cellular immunity. In case of the immune system compromise, reactivation of the infection and serious inflammatory changes, mostly in the central nervous system, occur. Prenatal infection and serious clinical picture in immunocompromised patients classify toxoplasmosis among ever topical infections, thus all research toward better understanding of its pathogenesis in immunocompetent patients is of utmost importance. The aim of the study was to systematically analyze the morphology and changes in cytologic smears of lymph nodes from patients acutely infected with Toxoplasma gondii, and to morphologically visualize Toxoplasma gondii in the lymph nodes by use of specific monoclonal antibodies. For this purpose, 30 aspirates of lymph nodes of patients who had definite serologic evidence for and clinical picture of acute toxoplasma infection were analyzed by use of classic cytology and immunocytochemistry methods. Results confirmed a recognizable cytologic picture of reactive hyperplasia of follicular center cells and granulomatous inflammation, with a unique finding of Toxoplasma gondii tachyzoite in lymphatic cells.     

垂体生长激素细胞腺瘤的电镜及免疫电镜观察

24 cases of growth hormone(GH)-producing pituitary adenomas were studied with electron microscopy and immunoelectron microscopy by protein A-gold complex, 6 cases were identified as densely granulated GH adenoma and 15 cases as sparsely granulated GH adenoma, among which 4 cases were proved by immunoelectron microscopy to be containing granules with prolactin(PRL) activity simultaneously. Intracytoplasmic fibrous bodies were often seen in the sparsely granulated cells anyhow, not all those cells with fibrous bodies possess the secretory granules with GH activity, and fibrous bodies were also detected in some PRL cells of certain mixed type adenoma. This suggests that fibrous bodies might not be the specific morphological feature of pituitary growth hormone cell adenomas.     

Method of polymerase chain reaction in toxoplasmosis diagnosis

Since introduction of polymerase chain reaction for the detection of DNA Toxoplasma gondii 398 biological samples from 301 patients were examined from 2000 to 2003. Positive finding of toxoplasmosis DNA we noted in 23 cases. Polymerase chain reaction enables exact and fast diagnosis of the actual parasitemia Toxoplasma gondii, which is important especially in the pregnant patients, new-born with suspicion on congenital toxoplasmosis and in the patients with immunosupression.