Disrupted NOS signaling in lymphatic endothelial cells exposed to chronically increased pulmonary lymph flow.

Associated abnormalities of the lymphatic circulation are well described in congenital heart disease. However, their mechanisms remain poorly elucidated. Using a clinically relevant ovine model of a congenital cardiac defect with chronically increased pulmonary blood flow (shunt), we previously demonstrated that exposure to chronically elevated pulmonary lymph flow is associated with: 1) decreased bioavailable nitric oxide (NO) in pulmonary lymph; and 2) attenuated endothelium-dependent relaxation of thoracic duct rings, suggesting disrupted lymphatic endothelial NO signaling in shunt lambs. To further elucidate the mechanisms responsible for this altered NO signaling, primary lymphatic endothelial cells (LECs) were isolated from the efferent lymphatic of the caudal mediastinal node in 4-wk-old control and shunt lambs. We found that shunt LECs (n = 3) had decreased bioavailable NO and decreased endothelial nitric oxide synthase (eNOS) mRNA and protein expression compared with control LECs (n = 3). eNOS activity was also low in shunt LECs, but, interestingly, inducible nitric oxide synthase (iNOS) expression and activity were increased in shunt LECs, as were total cellular nitration, including eNOS-specific nitration, and accumulation of reactive oxygen species (ROS). Pharmacological inhibition of iNOS reduced ROS in shunt LECs to levels measured in control LECs. These data support the conclusion that NOS signaling is disrupted in the lymphatic endothelium of lambs exposed to chronically increased pulmonary blood and lymph flow and may contribute to decreased pulmonary lymphatic bioavailable NO.

Caspase-8 deficiency in T cells leads to a lethal lymphoinfiltrative immune disorder.

Caspase-8 is best known for its cell death function via death receptors. Recent evidence indicates that caspase-8 also has nonapoptotic functions. Caspase-8 deficiency is associated with pathologies that are unexpected for a proapoptotic molecule, such as abrogation of activation-induced lymphocyte proliferation, perturbed immune homeostasis, and immunodeficiency. In this study, we report the long-term physiological consequences of T cell-specific deletion of caspase-8 (tcasp8-/-). We show that tcasp8-/- mice develop an age-dependent lethal lymphoproliferative and lymphoinfiltrative immune disorder characterized by lymphoadenopathy, splenomegaly, and accumulation of T cell infiltrates in the lungs, liver, and kidneys. Peripheral casp8-/- T cells manifest activation marker up-regulation and are proliferating in the absence of any infection or stimulation. We also provide evidence suggesting that this immune disorder is different from the autoimmune lymphoproliferative syndrome. Interestingly, the condition described in tcasp8-/- mice manifests features consistent with the disorder described in humans with Caspase-8 deficiency. These findings suggest that tcasp8-/- mice may serve as an animal model to evaluate Caspase-8-deficient patient prognosis and therapy. Overall, our study uncovers novel in vivo functions for caspase-8 in immune regulation.

Benign TdT-positive cells in pediatric and adult lymph nodes: a potential diagnostic pitfall.

Benign terminal deoxynucleotidyl transferase (TdT)-positive cells have been documented in a variety of nonhematopoietic tissues. Scant data are, however, available on their presence in nonneoplastic lymph nodes. This study is aimed to (1) characterize the presence/distribution of benign TdT-positive cells in pediatric and adult reactive lymph nodes and (2) define the phenotype and nature of such elements. This retrospective study considered 141 reactive lymph nodes from pediatric and adult patients without history of neoplastic disease. TdT-positive cells were characterized by immunohistochemical and morphometric analyses, and their presence was correlated with the clinical-pathological features. The nature of TdT-positive cells was investigated by (1) double immunostaining for early lymphoid cell markers and (2) assessment of TdT expression in fetal lymph nodes. Sparse TdT-positive cells were documented in all pediatric cases and in most (76%) adult lymph nodes. TdT-positive cell density was higher in children than adults (15.9/mm2 versus 8.6/mm2; P < .05). TdT positivity did not correlate with any clinical or histological parameter, and double immunostaining disclosed a phenotype compatible with early lymphoid precursors (positivity for CD34 and CD10, and variable expression of CD7). A very high TdT-positive cell density (802.4/mm2) was reported in all fetal lymph nodes. In conclusion, TdT-positive cells are a common finding in pediatric and adult lymph nodes. The interstitial distribution and low number of such cells allow for the differential diagnosis with precursor lymphoid neoplasms. The high density in fetal lymph nodes and the phenotype of such cells suggest their belonging to an immature lymphoid subset gradually decreasing with age.

Isozyme pattern in serially xenotransplanted childhood tumors.

Growth rate, histological course, and polymorphic enzyme pattern (glucose 6-phosphate dehydrogenase, glucose phosphate isomerase, and phosphofructokinase) were studied in eight childhood tumors xenotransplanted serially to nude mice. The growth rate of these tumors (three nephroblastomas, one hypercalcemic renal tumor, three rhabdomyosarcomas, and one malignant histiocytosis) appeared stable for any one particular tumor line. The time interval between two grafts varied from 1 to 3 weeks to 1 to 2 months in correlation with the clinical course of each malignant process. Histological changes were mostly in relation with a progressive dedifferentiation of the grafts. Immunoneutralization of glucose-6-phosphate dehydrogenase and glucose phosphate isomerase made possible the quantification of the stroma reaction in the grafts. A series of ten passages showed the amount of stroma to be constant for a given tumor type but variable from one tumor type to another, except for the malignant histiocytosis which showed an increase in stroma constituent after the sixth passage. One nephroblastoma tumor line showed, during the third passage, a sudden acceleration in the growth rate and complete transformation of the histological and isozymic patterns, which were interpreted as being the result of a murine lymphoma. The fibroblastic form of phosphofructokinase increased in every tumor line, whatever the tumor type. This change may be linked to a progressive dedifferentiation during the passage.

Serum lactate dehydrogenase levels in adults and children with acquired immune deficiency syndrome (AIDS) and AIDS-related complex: possible indicator of B cell lymphoproliferation and disease activity. Effect of intravenous gammaglobulin on enzyme levels.

Twenty-seven of 33 patients with the acquired immune deficiency syndrome (AIDS) or AIDS-related complex (16 adults and 17 children) demonstrated significant elevation of serum lactate dehydrogenase activity, occurring in the isomorphic distribution. Serum lactate dehydrogenase activity was the highest in all nine patients with acute Pneumocystis carinii pneumonitis, in seven of whom extensive interstitial pulmonary infiltrates with lymphocytes and plasma cells were documented. Lactate dehydrogenase activity was also significantly elevated on a long-term basis in all 17 pediatric patients with non-Pneumocystis lymphoid interstitial pneumonitis. Clinical resolution of Pneumocystis carinii pneumonitis was associated with a decline in lactate dehydrogenase activity. Periodic intravenous gammaglobulin was more effective than conventional therapy (trimethoprim/sulfamethoxazole and pentamidine) in achieving clinical and immunologic improvement and reduction of serum lactate dehydrogenase activity in patients with Pneumocystis carinii pneumonitis. Intravenous gammaglobulin was also more effective in patients with AIDS and non-Pneumocystis carinii pneumonitis and lymphoid interstitial pneumonitis. Lactate dehydrogenase activity declined to normal, at least temporarily, in nine of 12 intravenous gammaglobulin-treated patients as compared with only two of 12 untreated patients. Six adult patients with AIDS or AIDS-related complex and no interstitial pneumonitis exhibited normal lactate dehydrogenase levels. These findings suggest that serum lactate dehydrogenase activity in patients with AIDS or AIDS-related complex may be a useful indicator of pulmonary interstitial inflammation. As such, it may be utilized to predict disease course and monitor response to intravenous gammaglobulin treatment.

Histiocytic malignancies. Morphologic, immunologic, and enzymatic heterogeneity.

We have studied 14 hematopoietic malignancies with histologic features of histiocytic differentiation, using frozen section immunologic stains, plastic section enzyme histochemistry, and paraffin section immunocytochemistry. There was morphologic, immunologic, and enzymatic heterogeneity, including findings in seven cases that suggested differentiation toward specialized subsets of histiocytes. Four cases expressed a mature monocyte/macrophage phenotype by frozen section monoclonal antibody staining and three of these had histologic patterns diagnostic of malignant histiocytosis; two other cases had ATPase and S100 protein reactivity and morphologic features consistent with interdigitating (reticulum) cell proliferations; and one case was alkaline phosphatase positive, suggestive of differentiation toward fibroblastic reticulum cells. Four cases had histologic findings consistent with malignant histiocytosis, but weak or unreactive staining patterns and were considered poorly differentiated histiocytic or primitive hematopoietic malignancies. Three other cases, also morphologically consistent with malignant histiocytosis, were identified as probably T-cell lymphomas. The morphologic and phenotypic characteristics of non-neoplastic histiocytes and dendritic cell types and their related neoplasms are discussed. Histiocytic malignancies comprise a diverse group that can be identified and subclassified by immunologic and enzymatic techniques.

Inflammatory pseudotumor of lymph node and spleen: an entity biologically distinct from inflammatory myofibroblastic tumor.

Inflammatory pseudotumors (IPTs) of the lymph node and spleen are an uncommon, benign cause of lymphadenopathy and/or splenomegaly that often bear striking clinicopathologic similarities to the inflammatory myofibroblastic tumors (IMTs) found in soft tissues. These tumors have classically been grouped together under the umbrella category of "inflammatory pseudotumor." Recent evidence shows that IMTs are in fact neoplastic processes that often harbor balanced chromosomal translocations involving the ALK kinase gene. These translocations result in expression of ALK kinase in IMTs as assessed by immunohistochemical studies. However, the relationship between IMT and IPT of the lymph node and spleen is uncertain. To determine if ALK tyrosine kinase expression is also present in IPT, 13 cases of IPT (9 involving lymph nodes, 4 splenic lesions) were examined for the presence of ALK tyrosine kinase by immunohistochemical staining on paraffin-embedded tissue. In addition, in situ hybridization studies for Epstein-Barr virus--encoded RNAs (EBER) and immunoperoxidase studies for human herpesvirus-8 (HHV8)--specific proteins were performed. All cases had clinical, morphologic, and immunophenotypic findings typical of IPT and had varying proportions of fibroblastic and inflammatory components. Age ranged from 11 to 75 (median, 40) years; 8 subjects were male, and 5 were female. None of the cases (0 of 13) had positive staining for ALK kinase or HHV8, and in 1 a lymph node (1 of 13) was focally positive for EBV (EBER) by in situ hybridization. The absence of ALK kinase as detected by immunohistochemical studies in IPT of the lymph node and spleen suggests that this entity is biologically distinct from the histologically similar IMT.

Terminal deoxynucleotidyl transferase-positive lymphadenopathy in acute-phase chronic granulocytic leukemia.

A patient who had Philadelphia chromosome-positive chronic granulocytic leukemia had generalized lymphadenopathy. The lymph node biopsy revealed blast cells with small numbers of eosinophilic myelocytes indicative of granulocytic differentiation. In addition, the blast cells were found to have Philadelphia (Ph1) chromosome and extremely high levels of terminal deoxynucleotidyl transferase (TdT). The patient's peripheral blood and bone marrow reverted to the chronic phase, and the lymphadenopathy disappeared on two occasions with vincristine and prednisone therapy. The extramedullary proliferation of blastic chronic granulocytic leukemia, therefore, seems to share the histologic, cytogenetic biochemical, and chemotherapeutic sensitivity features of the basic disease process. TdT assay of enlarged lymph nodes in acute-phase chronic granulocytic leukemia might be used to identify the patients responsive to vincristine and prednisone despite the granulocytic histologic features of their lymph nodes.

Enzyme histochemistry on normal and pathologic paraffin-embedded lymphoid tissues.

So far, enzyme histochemical examination has been applied, with few exceptions, either to tissue imprints or to cryostat sections of freshly collected samples. This procedure is not easily applicable in routine histopathologic examination. In this study, a simplified tissue embedding procedure is presented which can be performed using an automatic tissue changer. The paraffin embedded samples can be used for both conventional histopathologic examination and for demonstrating enzymes in sections. The enzymes studied (alkaline phosphatase, alpha-naphthyl acetate esterase, acid phosphatase, tartrate resistant acid phosphatase, ATPase, peroxidase, and chloroacetate esterase) gave comparable results in formalin-fixed cryostat sections and paraffin sections in both normal and pathologic lymphoid samples. The only exception was ATPase, which could not be demonstrated on paraffin-embedded material. The technic described has broad application in the analysis of lymphoid diseases.

Serum and lymph-node collagenase in sarcoidosis. Comparison with angiotensin-converting enzyme.

Serum collagenase was significantly elevated in stage II, but not stage I sarcoidosis, in contrast to elevation of serum angiotensin-converting enzyme in stages I and II. Neither serum collagenase nor angiotensin-converting enzyme was elevated in active tuberculosis. Elevated serum collagenase and angiotensin-converting enzyme levels were decreased with steroid therapy. Serum collagenase and angiotensin-converting enzyme were significantly correlated but did not vary identically. Collagenase was not elevated in lymph nodes in sarcoidosis, in contrast to marked elevation of angiotensin-converting enzyme. The results suggest that serum angiotensin-converting enzyme is a more sensitive index of sarcoidosis activity than serum collagenase, which may have an ancillary role in assessment of the disease.

Detection of intracellular immunoglobulin in nodular lymphomas.

In lymph node tissue sections, six of 11 human cases of nodular lymphoma showed immunoglobulin within malignant nodules, and seven of nine cases of benign follicular hyperplasia showed immunoglobulin within follicles. In addition, distributions of lymphocyte cell membrane markers for T cells and B cells were determined in ten of 11 cases of nodular lymphoma. Lymphocyte suspensions in five cases contained monoclonal immunoglobulins and in three cases neoplastic cells showed a lack of surface membrane immunoglobulins. In two cases, the distribution of lymphocyte surface markers could not be distinguished from cells of benign lymph nodes. Combined data from intracytoplasmic immunoglobulin studies and lymphocyte surface marker assays indicated that eight of ten cases are of B cell lineage. Thus, the detection of intracellular immunoglobulin is not helpful in differentiating benign follicular hyperplasia from nodular lymphoma, but is complementary to lymphocyte surface marker assays in the determination of the origin of neoplastic cells in lymphoreticular malignancies.

Immunohistochemical identification of lysozyme in cutaneous lesions of alleged histiocytic nature.

Histiocytosis X, multicentric reticulohistiocytosis, juvenile xanthogranuloma, the "fibrous" type of dermatofibroma, dermatofibrosarcoma protuberans, and malignant fibrous histiocytoma are all characterized by dermal and/or subcutaneous infiltrates composed at least partially of cells having morphologic features suggestive of histiocytes. Paraffin-embedded tissues representing these conditions were stained for lysozyme (muramidase) with a peroxidase-antiperoxidase technic. The cells of juvenile xanthogranuloma were rich in lysozyme. Some of the cells of histiocytosis X showed a positive pattern, and the cells of the other three conditions were essentially negative. This study confirmed the histiocytic nature of juvenile xanthogranuloma and multicentric reticulohistiocytosis, supported the interpretation that there is a histiocytic component in the lesions of histiocytosis X, and cast some doubt on the alleged histiocytic nature of "fibrous" dermatofibroma, dermatofibrosarcoma protuberans, and malignant fibrous histiocytoma.

Use of the bone marrow imprint in the diagnosis of leukemic reticuloendotheliosis ("hairy cell leukemia").

Leukemic reticuloendotheliosis is a distinct entity, often misdiagnosed as chronic lymphocytic leukemia or lymphoma. The neoplastic cells have a specific tartrate-resistant acid phosphatase isoenzyme by which the diagnosis may be secured. Most individuals are leukopenic, and when, as in our case, rare or no circulating cells with tartrate-resistant acid phosphatase activity are found in the peripheral blood, an alternate site must be sought. Bone marrow aspirations often result in "dry taps," however, and cryostat sections of the bone marrow biopsy necessary to demonstrate tartrate-resistant acid phosphatase activity are involved and impractical to obtain for most laboratories. This report illustrates and recommends the simple technic of imprinting the core biopsy, which yields a satisfactory sample with which the specific cytochemical activity can be demonstrated.

Na-K-ATPase activity in the guinea pig stria vascularis in experimentally-induced endolymphatic hydrops.

The effect of endolymphatic hydrops on the Na-K-ATPase activity in the guinea pig stria vascularis was electron microscopically and enzyme cytochemically investigated one year after experimental induction. The morphological observations revealed intercellular dropsy in the basal infoldings of the marginal cells, and shrinkage and disappearance of intermediate cells. Moreover, shrinkage of the marginal cells, especially of the basal infoldings, was occasionally observed. In spite of these morphological alterations, the Na-K-ATPase activity was still detected on the plasma membrane of the basal infoldings of most marginal cells. No remarkable differences were found among the cochlear turns of the specimens examined. However, no reaction product was detected on the basolateral plasma membrane of severely degenerated marginal cells. The present results indicate that the Na-K-ATPase of the plasma membrane of the basal infoldings of the marginal cells plays an important role in the maintenance of the unique ion concentration of the endolymph even in the endolymphatic hydropic condition, and that the Na-K-ATPase activity is attenuated in severely atrophic cells.

Telomerase activity and in situ telomerase RNA expression in malignant and non-malignant lymph nodes.

AIMS/BACKGROUND: Telomerase, an enzyme associated with cellular immortality, is expressed by most malignant tumours, but is inactive in normal somatic cells except for male germ cells and proliferating stem cells. Thus, the measurement of telomerase activity in tissue samples may provide useful diagnostic and prognostic information. The aim of this study was to determine whether telomerase expression is useful for the detection of occult malignant cells in lymph nodes. METHODS: Telomerase activity was compared with histological findings in 123 surgically removed lymph nodes submitted for routine or frozen section diagnosis. Telomerase activity was measured using a modified, semi-quantitative PCR-based telomeric repeat amplification protocol (TRAP). The assay was adapted for single 5 microns OCT embedded cryostat sections. In either fresh tissues or cryostat sections, normalised activity was linear when compared with protein concentration. Furthermore, using an in situ hybridisation method, the human telomerase RNA (hTR) component was measured in a subset of negative and positive nodes. RESULTS: Most (96%) of the 97 histologically negative nodes expressed low levels of activity (mean value of positive samples = 3.0 units/microgram protein) which may be derived from activated lymphocytes that express telomerase activity. All 26 malignant nodes (17 metastases, nine lymphomas) expressed telomerase (mean value = 17.8 units/microgram protein). The rank order levels between the two groups differed significantly (p = 0.0002). In situ results showed clearly that the hTR was expressed relatively highly in metastatic cancer cells and at lower levels in germinal centres of secondary follicles. CONCLUSIONS: Although expression of telomerase by activated lymphocytes may limit its usefulness, measurement of enzyme activity combined with detection of hTR using in situ hybridisation may assist in the histopathological diagnosis of lymph nodes.

Elevation of angiotensin-converting enzyme in granulomatous lymph nodes and serum in sarcoidosis: clinical and possible pathogenic significance.

A statistically highly significant elevation of serum ACE was found in a group of 58 patients with sarcoidosis (serum ACE was elevated in 34% of patients), as compared with normal controls and patients with tuberculosis and various other common diseases. The results suggest that serum ACE is a useful aid for the diagnosis of sarcoidosis when elevated, but that a normal value does not rule out the condition and may occur in more than one-half of monitored patients. There is a trend to diminution of serum ACE with increasing duration of disease with or without steroid therapy, perhaps correlating with the total body mass of active granulomas, as indirectly suggested in preliminary data by correlation of serum ACE with serum globulin in 16 sarcoidosis patients. It is not yet clear whether there is any significant steroid effect on serum ACE, but a significant number of patients on steroid therapy for more than 2-4 yr have elevated serum ACE values, which in some instances are extremely high. There was a 12-fold elevation in ACE to specific activities generally exceeding those of normal lung in granulomatous lymph nodes of 14 patients with sarcoidosis, suggesting that sarcoid granulomas may be actively synthesizing ACE and resulting in elevation of serum ACE. Extensively fibrotic sarcoid lymph nodes had normal or slightly elevated ACE, suggesting that obliteration of granulomas in sarcoid lymph nodes diminishes their ACE content and that this obliteration may be related to the tendency to diminution of serum ACE with time. ACE was not elevated in one tuberculous lymph node or in experimental granulomas, suggesting that elevation of ACE may have some specificity for the granuloma of sarcoidosis rather than being a characteristic of all granulomas. The catalytic and physical properties of ACE in serum and lymph nodes in sarcoidosis were generally similar to normal ACE with respect to pH activity, modulators, polyacrylamide-gel electrophoresis, and Sephadex G-200 gel filtration. However, sarcoid lymph node ACE appeared to be more heat labile than normal lung or lymph node ACE, suggesting the possibility that an abnormal ACE may be present in sarcoidosis. If an abnormal enzyme is indeed present, it might be coded for by a host gene that is not normally expressed or a nonhost gene or it might be a normal ACE that has been altered. No ACE activity was found in circulating white blood cells in sarcoidosis or in control subjects, suggesting that circulating white blood cells may not contain the epithelioid cell precursor or that ACE synthesis (or less likely, uptake) may be turned on at a later stage in the transformation. Lysozyme activity was also elevated in sarcoid lymph nodes. Serum ACE and serum lysozyme were significantly positively correlated in 16 sarcoidosis patients, suggesting a relationship between the two...

Pagetoid reticulosis. Histiocyte marker studies.

Epidermal mononuclear cell infiltrate from three patients with pagetoid reticulosis was examined for the presence of the cytoplasmic markers lysozyme, alpha 1-antitrypsin and alpha 1-antichymotrypsin, using specific antisera and a peroxidase-antiperoxidase technique. Many of the infiltrating cells possessed these markers, indicating that they belonged to the monocyte-macrophage-histiocyte series.

Studies of intracellular distribution of acid phosphatase 5 in the spleen cells of leukemic reticuloendotheliosis by isopycnic gradient centrifugation.

A fresh spleen sample obtained from a patient with leukemic reticuloendotheliosis was homogenized and subjected to centrifugation on a sucrose density gradient. A major portion of acid phosphatase band 5 was observed in the lysosome, confirming that the elevated phosphatase activity in the neoplastic spleen is a lysosomal enzyme. However, a significant amount of brand 5 was also observed in the microsome. The microsomal and lysosomal enzymes have different affinity to CM-cellulose. The relationship between lysosomal and microsomal enzymes has not been established.

Immunohistochemical demonstration of lysozyme in normal, reactive and neoplastic cells of the mononuclear phagocyte system.

Using the peroxidase antiperoxidase (PAP) method, lysozyme (LZM) was shown to exist in normal, reactive and neoplastic cells belonging to the mononuclear phagocyte system (MPS), but was not detected in histiocytosis X cells. Immunostaining for cytoplasmic LZM by the PAP method is useful for identification of mononuclear phagocytes and for diagnosis of the diseases in which these cells participate.

Cytomorphologic and cytochemical aspects of malignant histiocytosis with spindle-cell differentiation. A case report.

Spindle-cell differentiation and tumor formation have been observed rarely in cases of malignant histiocytosis. We describe below one case of malignant histiocytosis with spindle-cell differentiation. Touch preparation of autopsy material revealed cytologic features that correlated with the histologic appearance of atypical components of this entity, such as spindle and nonspindle histiocytes and abundant erythrophagocytosis. Cytochemical properties indicated the histiocytic nature of the cells that composed this tumor. A morphologic account of the different types of cells is presented.