The value of the immunoperoxidase slide assay in the diagnosis of malignant pleural effusions in breast cancer

Whether immunocytochemical studies of malignant pleural effusions due to breast cancer would increase the diagnostic yield as compared with conventional effusion cytology was examined in 30 cases with biopsy-proven metastatic spread to the pleura. Conventional cytology was performed on air-dried smears as well as on cytocentrifuge preparations stained with the May-Grünwald-Giemsa stain. Immunocytochemistry was performed with monoclonal antibodies against carcinoembryonic antigen (CEA), epithelial membrane antigen (EMA) and human leukocyte antigen (HLA) and the peroxidase-antiperoxidase technique on glass slides after Ficoll-Hypaque centrifugation. By conventional cytology, 13 cases (43%) were positive for malignant cells, 6 cases (20%) were suspicious, and 11 cases (37%) were negative. In marked contrast, all 30 cases were immunocytologically positive for malignancy. Tumor cells in all cases demonstrated a positive reaction for EMA. Some mesothelial cells were also positive for EMA, but their reaction pattern was clearly distinguishable from that of the tumor cells. Twenty-one cases (70%) also showed CEA-positive tumor cells; mesothelial cells never reacted with CEA. Some tumor cells showed a loss of HLA expression. In conclusion, this immunocytologic method can be recommended as a routine procedure for greatly increasing the diagnostic yield of cytology in pleural effusions due to breast cancer.     

Theoretical analysis of the effect of cell recycling on recombinant cell fermentation processes.

A cell recycle system is studied for two-stage continuous fermentation. Cell recycle around the second stage provides higher cell concentrations than processes without recycle and a longer residence time of the cell, which is necessary for inducible products, especially in recombinant cell fermentation. Residence time distribution of the cell in the fermentor is important for the optimization of inducible products. The residence time distributions are studied for the cases with and without significant cell growth in the second stage. With cell growth in the second stage, three cases are considered. These are the cases of (1) zero residence time for two daughter cells after the cell division, (2) zero residence time of one daughter cell after the cell division and inherited residence time for the other daughter cell from the mother cell after the cell division, and (3) two daughter cells having the residence time of the mother cell after the cell division.     

Bone marrow stromal culture from patients with myelodysplastic syndromes and patients at different stages of acute nonlymphocytic leukaemia

The haematopoietic microenvironment or stroma plays a decisive role for the proliferation and differentiation of haemopoietic cells. We studied if bone marrow cells from patients with myelodysplastic syndromes (MDS) and acute nonlymphocytic leukaemias (ANLL) are altered in their ability to form adherent stromal layer with active haemopoiesis in the Dexter liquid culture. Bone marrow cells were obtained from 24 normal volunteers, 28 patients with ANLL in different stages of the disease and 9 patients with MDS. There are no differences between the stromal layers of patients with ANLL in complete remission and those of normal volunteers after two weeks of cultivation. However, bone marrow cells from patients with ANLL before treatment and from patients in relapse formed a poor adherent stromal layer in most cases. In 6 of 9 cases we found the normal stromal grade of bone marrow cells from patients with MDS. There were qualitative differences in the nonadherent cell population between normal and ANLL patients in complete remission. In most cases we found morphologically recognizable erythroid cells after two-weeks Dexter liquid culture of bone marrow cells from patients with ANLL in complete remission, which were not seen with normal volunteers. This could be an indication of harmful effects on the balance of haematopoiesis caused by previous infiltration with leukaemic cells or/and high-dose chemotherapy.     

The paracortical area in dermatopathic lymphadenitis and other reactive conditions of the lymph node

We compared the paracortical area in 4 cases of dermatopathic lymphadenitis (DL) with the same area in 11 cases of various other reactive conditions of the lymph node by immuno- and enzymehistochemical techniques. In addition, electron microscopy was performed on three cases of DL. The paracortical nodules in DL proved to be composed of a variable number of dendritic, OKT6+ OKIa + ATPase+ cells, admixed with helper T-lymphocytes. All other lymph nodes studied lacked dendritic OKT6+ cells, whereas OKIa positivity was found in the cortical (follicular centers and mantle zones) and paracortical area (lymphocytes and scattered dendritic cells). Short incubation for ATPase revealed a paracortical, pericellular staining pattern in cases of DL, whereas in all other cases this staining pattern was observed only after long incubation times. On electron microscopy, three types of dendritic cells were found in DL, namely interdigitating reticulum cells ( IDRC ). Langerhans cells (LC) and macrophages. Intermediate forms between IDRC and LC, containing a few Birbeck granules and a well developed rough endoplasmic reticulum, were found. It is suggested that immunoreactivity for the monoclonal antibody OKT6 is restricted to cases of DL, and is due to the appearance of dendritic cells that have LC-characteristics. These cells either arrive from the skin along afferent lymph vessels, or are the result of a local transformation process of IDRC that acquire LC-characteristics, i.e. OKT6 immunoreactivity and Birbeck granules.     

Fine needle aspiration diagnosis of lipoblastoma of the parotid region. A case report

BACKGROUND: Lipoblastomas are rare tumors of embryonal fat that occur in infants and children. They are usually located in the extremities and trunk. Two cases in the parotid region have been described. A diagnosis on fine needle aspiration (FNA) specimens has been reported in six cases. CASE: Lipoblastoma of the parotid region occurred in a 6-year-old boy and was diagnosed by FNA. Cytology showed rare lipoblasts and hibernomalike cells in a myxoid background with spindle and stellate mesenchymal cells, mature adipose cells and plexiform capillaries. A 7.0-cm, well-circumscribed mass with lobulated adipose tissue and delicate fibrous bands was resected. Microscopically, it showed a lobulated myxoid stroma, many capillaries, mesenchymal cells, lipoblasts and mature adipose cells. CONCLUSION: Lipoblastoma has to be differentiated from myxoid and lipomatous soft tissue tumors, especially from myxoid liposarcoma, a malignancy that classically affects older individuals and shows pleomorphism, atypical lipoblasts and chromosome-12 translocation. A lipoblastoma diagnosis must be established only after careful consideration of all available clinical, radiologic, cytogenetic and morphologic data.     

Effect of stress treatments on the detection of Listeria monocytogenes and enterotoxigenic Escherichia coli by the polymerase chain reaction

The relationship between viability assessed by plate counts and detectability by gene probe-polymerase chain reaction (PCR) techniques was examined with cells of Escherichia coli and Listeria monocytogenes previously exposed to a range of stress treatments. In all cases the organisms were detectable by PCR after plate counts had declined to zero. Treatment with acid or hydrogen peroxide caused loss of PCR soon after viability was lost, but strong PCR signals were obtained from starved or desiccated cells long after cells became non-viable. Exposure to temperatures up to 100°C had little effect on detection by PCR and even autoclaving cells at 121°C for 15 min failed to abolish PCR detection completely. There is thus no simple relationship between viability and detectability by PCR. Detection of pathogens by PCR in environmental monitoring requires additional evidence of viability before risk can be properly assessed.     

Restoration of spermatogenesis and male fertility by transplantation of dispersed testicular cells in the chicken

Transplantation of male germ cells into sterilized recipients has been widely used in mammals for conventional breeding and transgenesis purposes. This study presents a workable approach for germ cell transplantation between male chickens. Testicular cells from adult and prepubertal donors were dispersed and transplanted by injection directly into the testes of recipient males sterilized by repeated gamma irradiation. We describe the repopulation of the recipient seminiferous epithelium up to the production of heterologous sperm in about 50% of transplanted males. In comparison to males transplanted with testicular cell preparations from adult donors, in which the first ejaculates with sperm were recovered about 5 wk after transfer, a substantial interval (about 10 wk) was necessary to obtain ejaculates after the transfer of testicular cells from prepubertal donors. However, in both cases, recipient males produced ejaculates capable of fertilizing ova and producing progeny expressing donor genes.     

Microtubule organization in lymphoid malignancies.

The abnormal organization of actin-containing microfilaments and vimentin-containing intermediate filaments in neoplastic lymphocytes of T and B cell origin has been described. We investigated microtubules of pathologic cells from 34 lymphoid malignancies, by immunofluorescence microscopy, using monoclonal tubulin antibody. In most cases, apart from two cases of lymphoma, one T cell lymphoma and one B cell lymphoma, interphase leukemia cells, lymphoma cells, and myeloma cells were shown to contain well-organized microtubules which were associated with a microtubule organization center at one end. In the cells of a patient with T cell lymphoma, although microtubules were not visible in the lymphoma cells from lymph nodes, they became visible after 72 hours in culture with concanavalin A (Con A) and interferon alpha. Cap formation was observed with antitubulin monoclonal antibody in the peripheral blood lymphocytes from a chronic lymphocytic leukemia patient, but well-developed microtubules were observed on other occasions in the same patient. There were no obvious structural differences between microtubules in T and B cell lymphoid malignancies, but leukemia cells and lymphoma cells with irregularly shaped nuclei, such as adult T cell leukemia cells and B cell lymphoma cells with cleaved nuclei, had complicated microtubules surrounding their irregular nuclei. In general, after blastogenic stimuli with phytohemagglutinin-P (PHA-P), Con A, and pokeweed mitogen (PWM), the development of the microtubules was proportional to the incorporation of 3H thymidine (3H-TDR). In most cases, after incubation with granulocyte colony-stimulating factor (G-CSF) and interferon alpha, the number of intact cells decreased and the number of degenerated cells increased, but the intact cells had intact microtubules.     

Initiation of primary cell cultures from human intracranial tumors on extracellular matrix from bovine corneal endothelial cells

Tissue specimens from 105 human gliomas and 57 human meningiomas were obtained at surgery, dissociated into single cells and small cell aggregates and then plated onto plain plastic tissue culture dishes and dishes which had been precoated with an extracellular matrix (ECM) derived from bovine corneal endothelium. In 80% of the glioma cases we observed a marked improvement in initial plating efficiency, colony formation and speed of attachment when cells were plated on ECM. In 5 cases cells attached only to the ECM-coated dishes but remained afloat in the untreated dishes. In addition it could be noted that over the first 2 days, those cells which had been initiated on ECM showed more signs of morphological differentiation, i.e., extension of cytoplasmic processes or formation of fiber networks between cell groups. If adaptation occurred and proliferation began in vitro, either immediately or after a several days' lag phase, both the ECM-cultured cells as well as those which slowly had adapted to culture on plastic could be passed on to untreated culture ware and perpetuated thereon. In the case of well-differentiated low-grade gliomas where no growth in culture took place, the cultures on ECM could at least be used for initial experiments in the primary cultures (P0). Meningiomas usually attached well to both, plastic or ECM. In 50% of our cases the plating efficiency was higher on ECM but after successful initial culture, the delay until the cells on plastic reached confluence in comparison with those on ECM was 1 or 2 days. Again there were 2 cases in which the cells would not plate on plastic. Here the cells which after 1 day were still afloat plated to more than 80% within the first 2 h after transfer to ECM. In all cases the cells from plastic and ECM cultures were indistinguishable and could be passed onto untreated dishes henceforth. In later culture stages ECM offers several advantages: It is easier to shift cells to serum-free defined culture conditions, the cells will grow at a faster rate on ECM when in higher passages and the maximal number of passages possible is higher on ECM.     

Cytology of angiosarcoma in effusions

The cytologic and immunocytochemical findings in pleural effusions from three cases of angiosarcoma are presented. In two of the cases, the primary lesion was on the scalp; in the third case, an angiosarcoma of the small intestine developed after radiotherapy for Hodgkin's disease. Single malignant cells and small clusters of cells were seen in cytologic preparations from two cases while only single cells were seen in preparations from one case. The malignant cells had delicate, finely vacuolated cytoplasm with distinct borders. No specific morphologic features were noted. Immunoperoxidase studies revealed binding of Ulex europaeus and reactivity for vimentin in all three cases and expression of Factor VIII-related protein in two of the cases but no expression of epithelial markers. The clinical history and immunoperoxidase studies are necessary to distinguish angiosarcoma from metastatic adenocarcinoma and other malignancies in effusions.     

脑脊液中转移性腺癌细胞免疫表型的变化

目的探讨脑脊液中转移腺癌细胞在没有血供的条件下的生长特征,是否有血管内皮标记物CD34,CD105,FⅧ,淋巴管内皮标记物D2_40及碱性成纤维生长因子(b-FGF)的表达,并促进肺癌的脑转移及肿瘤细胞自我生存的调控。方法采集109例腺癌脑转移患者的脑脊液为研究对象,其中肺癌脑转移107例(包括49例肺癌术后5年内脑转移,58例无肺癌病史直接经脑脊液穿刺发现肺癌脑转移),乳腺癌2例。以40例主要成分为炎性细胞的脑脊液及40例原发性肺腺癌组织标本为对照,采用免疫化学染色方法检测脑转移腺癌细胞及腺癌组织中CD34,CD105,FⅧ,D2_40,b-FGF,VEGF及vimentin的表达。结果 109例脑脊液标本中,CD34,CD105,FⅧ,D2_40,b-FGF及VEGF在转移癌细胞中的阳性率分别为64.2%,67.9%,66.9%,63.8%,56.8%,70.6%明显高于脑脊液对照组(阳性率均为0)且差异具有统计学意义(P0.05)。vimentin在脑脊液转移腺癌细胞中阳性表达,表达率为88.1%。在原发肺癌标本中,CD34,CD105,FⅧ和VEGF表达较弱或不表达。D2-40在癌中不表达。B-FGF与Vimentin在原发肺腺癌中的表达率分别为50.1%和29.3%。结论在肺癌脑转移过程中,肿瘤细胞能够表达不同的血管生长因子,提示可能具备内皮细胞的生物学特性,可能有助于增强肿瘤细胞的转移能力和对环境的耐受能力。     

Stimulation of adenyl cyclase and fixation of iodine in human cancerous thyroid cells in culture

In primary cultures of cells from human thyroid cancer, functional activity was investigated by adenylate cyclase responsiveness and radioiodine uptake. In cells from well-differentiated follicular carcinomas (2 cases), TSH stimulation of cAMP accumulation is similar to that of normal thyroid cells. In contrast, in papillary carcinomas (11 cases), the mean dose-response curve is much lower than normal. In thyroid cancers, radioiodine uptake by the cells has been detected in some cases after 3 days of 125I incorporation. As previously observed, our results suggest the existence of a relationship between adenylcyclase responsiveness and 125I uptake by the cells.     

Value of the filter imprint technique in aspiration biopsy cytology

In 468 fine needle aspiration (FNA) biopsies, after smearing of the aspirate on glass slides, additional material was obtained by flushing the needle with a fixative. The cells were collected on Millipore filters, from which imprints were prepared. The filter imprints were found to be sufficiently cellular in 60% of the cases, which reduced by 33 (7.3%) the number of cases with unsatisfactory aspirates. Diagnostic specificity and sensitivity were not influenced by the technique. The filter imprint technique was of particular value in breast aspirates.     

Damage to human endothelial cells by cholestane-3 beta,5 alpha,6 beta-triol and the protective effects of preparations that raise the intracellular level of cAMP]

Effect of drugs, which are able to elevate the intracellular level of cAMP, on resistance of human umbilical vein endothelial cells (HUVEC) to cholestane-3 beta,5 alpha, 6 beta-triol (Triol)-induced injury was studied. Triol at a concentration of 62 microM caused death of 50% of cells after a 24 hour incubation. Addition of forskolin (10 microM), methylisobutylxantine (100 microM), or 8-Br-cAMP (100 microM) into the incubation medium prevented injury of HUVEC under these conditions. These findings indicate that endothelial resistance to the injury can be regulated by the adenylate cyclase system. A comparative study on Triol-induced injury of adult human aortic endothelial cells isolated separately from zones of low (LP) and high (HP) probability of atherosclerosis was also performed. In 7 cases endothelial cells isolated from the LP zones were more resistant to Triol-induced injury, in 2 cases the differences were not significant. The development of atherosclerotic lesion in HP zones is likely to be associated with a higher sensitivity of endothelial cells from these zones to different injuring agents.     

Dependence of DNA-histograms on the sampling techniques in fine needle aspirates of the breast.

48 fine needle aspiration biopsy (FNAB) samples from 25 breast cancer cases, originally used for cytodiagnosis were subjected to DNA cytometry. There were air dried smears stained with the MGG method, and samples stained with HE or PAP stain after 50% ethanol fixation and cytocentrifugation. Different sampling strategies were applied. Four methods were tested: method 1: cell groups measured, method 2: all cells measured, method 3: free cells measured, and method 4: atypical free cells measured. Method 4 showed most often DNA aneuploid histogram patterns, sampling method 1 had the highest number of DNA diploid histogram patterns. Diagnostic approaches may benefit from a sampling method detecting the hiding aneuploid cell population. Grading of neoplasm could potentially benefit from other approaches.     

Comparison of tissue disaggregation techniques of transitional cell bladder carcinomas for flow cytometry and chromosomal analysis

DNA index (DI) measurements and chromosomal analysis of 42 transitional cell carcinomas were done after mechanical and enzymatical disaggregation of the tumor specimens. The results obtained with these different disaggregation techniques were compared in the 33 cases (79%) that showed recognizable chromosomes. The enzymatically obtained cell suspensions could not be used for chromosomal analysis after short-term culture of 24 hours. In four cases, the DI after enzymatical treatment could not be estimated. In most cases, the DI obtained from the tumor cells was similar for both aggregation techniques, with the exception of four cases of enzymatically treated cell suspensions in which the DI could not be estimated. The average DI of the aneuploid tumors was 13% higher than the corresponding chromosome count. In 19% of the aneuploid tumors the proportion of aneuploid cells could not be measured after enzymatical treatment. In the remaining suspensions the proportion of diploid cells was higher after enzymatical disaggregation than after mechanical treatment. It is concluded that for flow cytometric and direct chromosomal analysis of bladder tumors, the mechanical disaggregation technique is most suitable.     

Results of a phase I clinical trial of vaccination of glioma patients with fusions of dendritic and glioma cells

Several reports of clinical trials of immunotherapy using dendritic cells have been published to date. In this study, we investigated the safety and clinical response of immunotherapy with fusions of dendritic and glioma cells for the treatment of patients with malignant glioma. Eight patients with malignant glioma, ranging in age from 4 to 63 years old, participated in this study. Dendritic cells were generated from peripheral blood. Cultured autologous glioma cells were established from surgical specimens in each case. Fusion cells of dendritic and glioma cells were prepared with polyethylene glycol, and the fusion efficiency ranged from 9.2 to 35.3% (mean, 21.9%). All patients received the fusion cells every three weeks for a minimum of 3, and a maximum of 7, immunizations. Fusion cells were injected intradermally, close to a cervical lymph node. The percentage of CD16- and CD56-positive cells in peripheral blood lymphocytes slightly increased after immunization in 4 out of 5 cases investigated. Peripheral blood mononuclear cells were incubated with irradiated autologous glioma or U87MG cells and supernatants were harvested. In 6 cases analyzed, the concentration of interferon-gamma in the supernatant increased after immunization. Clinical results showed that there were no serious adverse effects and two partial responses. Although the results of the phase I clinical trial of fusion cells indicated that this treatment safely induced immune responses. we were unable to establish a statistically significant treatment-associated response rate, due to the limited sample population. Therefore, further evaluation of the role of adjuvant cytokines is necessary.     

Characterization of a subpopulation of human T lymphocytes reactive with an heteroantiserum to human brain.

A rabbit antiserum to human fetal brain reacted after suitable absorptions with a subpopulation of human normal T cells. The distribution of reactive T cells varied according to the organ tested: 23% of peripheral blood lymphocytes, 5% of tonsil lymphocytes, and less than 1% of thymocytes were positive. Reactive cells did not transform after phytohemagglutinin or pokeweed stimulation but were at least weakly stimulated by allogeneic cells. T-derived neoplastic cells from one case of T acute lymphoblastic leukemia, two patients with Sezary syndrome, and from three out of five cases of T chronic lymphocytic leukemias (CLL) yielded negative results. In contrast, all the leukemic cells from two other patients with T-derived CLL were positive suggesting a proliferation of homogeneous cells arising from only a subpopulation of T lymphocytes.     

Influence of Culture Conditions on the Length of the Lag Period of Chlorophyll Synthesis in Preilluminated Dark-grown Euglena

Two parameters of the potentiation process, the lengths of preillumination and of the subsequent dark phase have been determined for Euglena cells which had been grown in the dark under two different culture conditions. In both cases a maximum potentiation response is obtained after a preillumination period of about 2 hours. The minimum length of the dark period necessary for maximum potentiation changes from 1 hour for cells which contain, at the moment of illumination, a high paramylum content, to 6 hours for cells which have a low paramylum content.