Evaluation of spleen cell population and effect of splenectomy on granuloma modulation in BALB/c mice infected with Schistosoma mansoni

A kinetic study of the cells present in the spleen of BALB/c mice infected with Schistosoma mansoni was carried out. The lymphocytes were evaluated phenotypically with monoclonal antibodies and the effect of splenectomy on the modulation of periovular granuloma was also investigated. The infected mice had proportional increases in the numbers of neutophils, plasma cells, macrophages and eosinophils in the spleen. The largest number of neutrophil, plasma cells and macrophage were found between the 8th and the 12th week of infection, while the amount of eosinophils were higher later on, around the 20th week. The lymphocytes phenotipically characterized as Thy 1.2, Lyt 1.2 (CD4) increased mildly in proportional numbers. However, the percentage of lymphocytes with the Lyt 2.2 (CD8) phenotype, which is characteristic of supressor and cytotoxic T cells, increased significantly with the progress of the disease. The numbers of B lymphocytes, which comprise 50% of the mononuclear cells present in the spleen, increased significantly till the 16th week they began to decrease. The mean diameters of periovular granulomas were comparatively similar in both experimental groups (splenectomized and non-splenectomized mice). However, the evolutive types of granuloma (exudative, intermediate and fibrous) in splenectomized mice were proprtionally different from those of non splenectomized mice in the 16th and 24th week of infection. It is inferred that lymphonodes or other secondary lymphoide organs, in the abscence of the spleen, assume a modulating action on periovular granulomas, although the evolution of the granulomas is somehow delayed in splenectomized mice

Determinaçao de valores referenciais de subpopulaçao linfocitaria em nossa populaçao normal / Establishing a reference range for lymphocytes subset value in our normal population

Foram processadas 62 amostras de sangue heparinizado, com a finalidade de se determinar valores de normalidade encontrados em nosso meio para a subpopulaçao linfocitaria T4 E T8, expressos em percentuais e em numeros absolutos. Estabeleceu-se tambem uma relaçao numerica entre T4/T8

Poblaciones linfoides en hepatitis crónica B tratadas con interferón recombinante / Lymphoid populations in chronic hepatitis B treated with recombinant interferon

Se estudiaron 43 pacientes con hepatitis crónica tipo B y AgeHB positivo, tratados con interferón alfa recombinante. A 21 de ellos se les realizó inmunosupresión previa con prednisona durante 2 semanas. Se evaluó su respuesta inmune celular mediante la cuantificación de subpoblaciones linfoides y los anticuerpos monoclonales T3 (CD3), T4 (CD4), T8 (CD8) antes y después del tratamiento. Se compararon estos resultados con los de un grupo control compuesto por 24 sujetos supuestamente sanos. Para la comparación se utilizó la prueba t de Student con un nivel de significación del 1 %. Hubo incremento de T4 en el grupo que recibió prednisona y disminución significativa de T8 en ambos grupos respecto al control, después del tratamiento, aunque sin diferencias entre los grupos de tratamiento. En el que respondió al tratamiento existía una disminución de T4 antes de iniciar la terapéutica lo cual serviría como factor pronóstico de la terapia antiviral de estos pacientes

Alteraciones cuantitativas de las subpoblaciones linfocitarias en la retinosis pigmentaria / Quantitative changes in lymphocytic subpopulation in pigmentary retinosis

Se determinan los valores relativos de las células CD3+, CD4+ y CD8+ periféricas, los niveles de inmunocomplejos circulantes y las concentraciones séricas de las proteinas C3 y C4 del sistema de complemento en 27 pacientes con el objetivo de investigar la posible presencia de alteraciones inmunológicas en la retinosis pigmentaria. Se encontraron valores disminuidos de las células CD3+ y CD4+ en los pacientes, lo que se asocia con un índice CD4/CD8 bajo en relación con el grupo control. No se demostró la presencia de inmunocomplejos circulantes en ninguno de los enfermos. Las cifras séricas medias del C3 y C4 fueron inferiores a las de los controles. El descenso de las células CD4+ encargadas de inducir y cooperar en una respuesta inmune indica un defecto en los mecanismos de competencia inmunológica en la retinosis pigmentaria

Immunohistochemical localization of nuclear 3,5,3'-triiodothyronine receptor proteins in rat tissues studied with antiserum against C-ERB A/T3 receptor.

We have previously raised an anti-c-erb A peptide antibody (designated 4B II) which immunoprecipitated in vitro transcription/translation products of c-erb A alpha 1 and beta. 4B II could recognize nuclear T3 receptor (NT3R) without distinction between difference in species and tissues. Using 4B II, we studied immunohistochemical localization of NT3R proteins in various tissues of the rat. Cryostat sections (4-6 microns) of selected rat tissues were incubated with 4B II at 4C overnight, followed by fluorescein-isothyocianate-conjugated anti-rabbit immunoglobulin G for 60 min at 25 C. The cellular localization of fluorescence in all tissues examined was exclusively nuclear. Under the same conditions, control sections stained with antiserum which had previously absorbed with c-erb A peptide or inactive serum showed no specific staining. In the brain the large nuclei, supposed to be neuronal, were strongly stained in the cerebral cortex and the granular layer of the cerebellum. In the kidney, cells in the glomerulus, the distal, but not the proximal, tubules, and the collecting ducts exhibited nuclear staining. Nuclear fluorescence was observed homogeneously in the heart and liver, but the intensity was much weaker in the latter. Less intense fluorescence was seen in the testis and spleen, although specific immunostaining was clearly observed in the nuclei of spermatocytes, Leydig cells, and the heads of the sperms in the testis, and many lymphocytes in the spleen. Nuclei of follicular cells of the thyroid exhibited very strong fluorescence, suggesting existence of plenty of NT3R proteins. The anterior pituitary showed strong immunostaining in most nuclei, and clear nuclear fluorescence was also detected in the intermediate lobe of the pituitary. The present study showed that NT3R distributes selectively in certain types of cells in many tissues and that the content of NT3R proteins seems to correlate with the concentration of c-erb A mRNA alpha 1 and beta among many organs.

Estudio citogenético, bioquímico y de la función reproductiva en personas expuestas a plaguicidas / Cytogenetic, biochemist and reproductive function studie in exposed biocides

El intercambio de cromátides hermanas (ICH) (sensible indicador de mutágenos ambientales), el hemograma, la transaminasa glutámico oxalacética (TGO), la transaminasa glutámico pirúvica (TGP), la colinesterasa, los organoclorados y organofosforados en sangre periférica y el esperma, fueron estudiados en 44 individuos expuestos a plaguicidas y 36 controles no expuestos. El grupo de individuos expuestos estaba constituido por trabajadores agropecuarios en contacto directo o indirecto con los plaguicidas (organoclorados y organofosforados). Los estudios a este grupo se realizaron en 3 épocas distintas. Para cada una de las variables en consideración, se realizaron comparaciones expuestos y controles mediante el test "T" de Student. Se encontró significativo incremento en los eosinófilos y organoclorados, disminución en los glóbulos blancos del grupo expuesto con respecto al control; en el esperma se observaron alteraciones en la movilidad. En los ICH, niveles de TGO, de TGP, organoclorados y colinesterasa, no se detectaron diferencias significativas con respecto a los controles. Este trabajo es un enfoque global al problema y de ninguna manera pretende considerarse definitivo, se continuará profundizando en los diversos aspectos que lo constituyen.

Satellite association and nucleolar organizer regions in the elderly

A expressäo dos genes ribossômicos representada pelas freqüências de associaçöes de satélites e de regiöes organizadoras de nucléolo (RONs) ativas foi avaliada em culturas de linfócitos de um grupo de recém-nascidos e um grupo de indivíduos com idades entre 70-90 anos, através de técnica de coloraçäo pela prata. Observamos freqüências semelhantes de RONs ativas em ambos os grupos ao lado de uma freqüência mais elevada de associaçöes de satélites no grupo dos idosos

Presence of cell lineage-specific hypomethylated sites in the major breakpoint cluster region.

To examine the role of DNA methylation in breakpoint location of chromosomal translocation, HpaII sites in and flanking the M-bcr on chromosome 22 were mapped in DNA from blood granulocytes and lymphocytes, bone marrow cells, thymic tissue, and spermatozoa from normal individuals. Allelic HpaII sites were identified clustered in a 600-base pair genomic area of the M-bcr. Bone marrow cells and blood granulocyte DNA showed identical allelic patterns. Thymic tissue and blood lymphocytes showed identical allelic patterns distinct from bone marrow cells and blood granulocytes. Spermatozoa showed a third methylation pattern. In all individuals, the HpaII sites were present within the BamHI/BglII fragment of the M-bcr, the same area associated with high breakpoint frequency in chronic myelogenous leukemia (CML). Three of 15 patients with chronic phase CML showed fully methylated rearranged BglII/BglII M-bcr restriction fragments not seen in normal bone marrow cells. These methylation patterns of the M-bcr may be important in CML breakpoint location and may be a marker for tissue differentiation.

Interpretation of microscopic image analysis of cell nuclei.

This study shows that microscopic image analyses of nuclear DNA have common characteristics among fixation methods and tissue types. We find that microscopic imaging measurements require both nuclear area and DNA concentration to properly convey diagnostic information. Algorithms are developed which enable infiltrating lymphocytes to act as internal DNA controls for each sample. The DNA content and patterns measured by microscopic imaging were found to be related to patient survival and to cytologic diagnosis.

Compensatory increase in calcium extrusion activity of untreated lymphocytes from swine susceptible to malignant hyperthermia.

We tested the hypothesis that lymphocytes from swine with susceptibility to malignant hyperthermia (MH) had calcium extrusion activity higher than unaffected swine. Cytoplasmic concentration of ionized calicum was determined by use of dual emission spectrofluorometry and measurement of the ratio of free to calcium-bound form of the fluorescent calcium dye indo-1. Net calcium accumulation and unidirectional calcium extrusion rate were dependent on intracellular calcium concentration. Calcium extrusion from calcium-loaded lymphocytes was monitored while calcium influx was inhibited by suspending the cells in calcium-free medium with a calcium chelator. Net calcium accumulation of untreated lymphocytes was monitored in calcium-replete medium. A novel method of calculation of ionized calcium was used. This method confirmed our previous findings of lower ionized calcium concentration (86 +/- 40 and 370 +/- 216 nmol/L; P less than 0.01) and slower rates of calcium accumulation 39 +/- 16 and 127 +/- 52 nmol/L/min) in untreated lymphocytes from MH-susceptible swine compared with controls. These changes were attributable to calcium extrusion activity two- to three-fold higher in lymphocytes of MH-susceptible swine (154 +/- 36 and 408 +/- 47 nmol/L/min at 175 nmol/L; 972 +/- 111 and 1,690 +/- 505 nmol/L/min at 425 nmol/L). These data were compatible with our model of higher calcium extrusion activity being a compensatory adaptation of MH-susceptible swine lymphocytes to their hypersensitivity to stimuli that increase cytoplasmic calcium concentration.

Whole cell preparation of squamous cell carcinoma for cytometric analysis of DNA.

To date, analysis of the DNA content of head and neck squamous cell carcinomas has relied on the homogenation of the entire tissue specimen and subsequent staining and quantitation of the naked nuclei. This methodology does not make allowance for the extremely variable nature of these tumors with respect to their cellular composition. Further, by destroying the cytoplasm and cell membranes, this methodology makes it impossible to distinguish the DNA content of the tumor cells from that of the background stromal and inflammatory cells. The authors present a methodology for the selective exclusion of inflammatory cell infiltrates from the DNA analysis of these tumors. Using this technique, it has been found that exclusion of the inflammatory cells allows the investigator to look more specifically at the malignant cell population. This has been most helpful in those samples in which the tumor cells have been diploid or near-diploid. With this technical refinement, the relationship between DNA ploidy and clinical prognosis may be more accurately assessed.

Mixed gonadal dysgenesis. A review of 15 patients reporting single cases of malignant intratubular germ cell neoplasia of the testis, endometrial adenocarcinoma, and a complex vascular anomaly.

The clinicopathologic features of 15 patients with mixed gonadal dysgenesis are presented with special regard to cardiovascular and neoplastic disease. Seven (47%) cases, all phenotypic females, had gonadal tumors: gonadoblastoma (5), germinoma (4), malignant intratubular germ cell neoplasia (1), and a unique gonadal stromal tumor (1). Gonadoblastoma was found in 4 of 10 testes and 4 of 17 streak gonads, and associated with germinoma in 4 cases. One patient developed grade 1 endometrial adenocarcinoma after estrogen therapy. Cardiovascular diseases (ie, bicuspid aortic valve, and a unique right aortic arch with a retroesophageal arch segment, aberrant left subclavian artery, coarctation, and dissection) are documented in our series. At the time of diagnosis of mixed gonadal dysgenesis, removal of streak gonads or testes will prevent further gonadal tumor development Cardiovascular examination may identify treatable and potentially lethal disease.

Chromosome breakage in lymphocytes from members of cancer families showing autosomal dominant inheritance.

We have conducted investigations on members of three families with increased predisposition to cancer which appears to be inherited as an autosomal dominant trait. The aim of our studies overall is to provide markers for the mutant genes involved, so that gene carriers may be monitored closely for signs of malignant disease. This paper reports on studies of chromosome breakage in lymphocytes from affected and at-risk family members and control subjects. No increase in spontaneous chromosome breakage was observed in family members compared with controls. An increased sensitivity to chromosome damage induced by the alkylating agent, N-methyl-N1-nitro-N-nitrosoguanidine (MNNG), was observed in three members from two families; one person was affected, the others at risk. These families included cases of osteosarcoma, in addition to various types of cancer of epithelial origin. Two members (one affected, one at-risk) of a third family showed increased sensitivity to the radio-mimetic agent, bleomycin. This family appeared to represent the cancer family syndrome. Whilst not conclusive at present, our results appear to justify investigation of members of cancer families with respect to sensitivity to chromosome breaking agents.

Binding activity and autophosphorylation of the insulin receptor from patients with myotonic dystrophy.

To clarify a mechanism of insulin resistance associated with myotonic dystrophy, we studied the insulin receptor by using three different types of cells--circulating erythrocytes, cultured skin fibroblasts, and Epstein-Barr virus(EBV)-transformed lymphocytes. In myotonic dystrophy, insulin binding to erythrocytes and fibroblasts was significantly decreased as a result of a reduction of the binding affinity. Insulin binding to EBV-transformed lymphocytes was normal. When the receptors were purified from fibroblasts with wheat germ agglutinin, we could not find a decrease in the binding affinity seen in the intact cells. No difference was observed in the magnitude of basal and insulin-stimulated autophosphorylation of insulin receptors from EBV-transformed lymphocytes between the control and myotonic dystrophy. Southern blot analysis of the insulin receptor gene revealed no restriction fragment length polymorphism associated with myotonic dystrophy. These findings suggest that there is no primary defect of the insulin receptor per se in terms of insulin binding and autophosphorylation in myotonic dystrophy. The reduction of the insulin binding to erythrocytes and fibroblasts may be caused by the plasma membrane abnormality that affects the binding affinity of the receptor.

Lymphocyte subset alterations in nodes regional to human melanoma.

The lymphocyte subpopulations in tumor-draining lymph nodes of melanoma patients were determined using two-color flow cytometry. Data were analyzed according to: (a) the staging of the melanoma; (b) whether or not the nodes contained tumor; and (c) their distance from the primary tumor. Compared with Stage I patients (without metastasis), uninvolved nodes of stage II patients (with nodal metastases) had a significant decrease in helper/inducer (CD4+) T-cells (P less than 0.001), with a corresponding increase in cytotoxic/suppressor (CD8+) cells (P less than 0.001) and Leu 19+ natural killer (CD56+) cells (P less than 0.01). In some patients the presence of tumor within a node was associated with a large decrease in CD3+ total T-cells, whereas in others tumor involvement had little influence on lymphocyte phenotypes. When analyzed by distance from the primary tumor, nodes closest to tumor in Stage I patients contained a smaller percentage of CD19+ B-cells. In Stage II, tumor-free nodes nearest to tumor showed an increase in CD19+ cells, but statistical significance was not reached. CD56+ natural killer cells increased progressively in nodes near tumor and were more numerous in Stage II uninvolved nodes compared with Stage I nodes. Alterations in phenotypically defined lymph node lymphocytes occur in nodes regional to melanoma as the disease progresses, as growth of metastases occurs, and in tumor-free nodes nearest to tumor. These alterations may be essential to the establishment and progression of metastases.

The 56-59-kilodalton protein identified in untransformed steroid receptor complexes is a unique protein that exists in cytosol in a complex with both the 70- and 90-kilodalton heat shock proteins.

It has previously been shown that 9S, untransformed progestin, estrogen, androgen, and glucocorticoid receptor complexes in rabbit uterine and liver cytosols contain a 59-kDa protein [Tai, P. K., Maeda, Y., Nakao, K., Wakim, N. G., Duhring, J. L., & Faber, L. E. (1986) Biochemistry 25, 5269-5275]. In this work we show that the monoclonal antibody KN 382/EC1 raised against the rabbit 59-kDa protein reacts with 9S, untransformed glucocorticoid receptor complexes in cytosol prepared from human IM-9 lymphocytes but not with 4S salt-transformed receptors. The human protein recognized by the EC1 antibody is a 56-kDa protein (p56) of moderate abundance located predominantly in the cytoplasm by indirect immunofluorescence. There are at least six isomorphs of p56 by two-dimensional gel analysis. N-Terminal sequencing (20 amino acids) shows that p56 is a unique human protein. When p56 is immunoadsorbed from IM-9 cell cytosol, both the 70- and 90-kDa heat shock proteins are coadsorbed in an immune-specific manner. Neither heat shock protein reacts directly with the EC1 antibody. We conclude that p56 exists in cytosol in a higher order complex containing hsp70 and hsp90, both of which in turn have been found to be associated with untransformed steroid receptors.

Rat spleen lymphocytes contain an immunoactive and bioactive luteinizing hormone-releasing hormone.

An interaction between the immune and endocrine systems has been long known. This association is further strengthened by the finding that splenic lymphocytes have the capacity to produce molecules similar to if not the same as classical hormones, including several members of the opiate family, PRL, GH, and neuropeptide Y. Because of such findings and because of information from other laboratories suggesting that LHRH might have direct effects upon the immune system, we hypothesized that immune cells themselves might contain LHRH. Lymphocytes were purified from spleens of intact adult male Sprague-Dawley rats and the cells were lysed with sodium hydroxide. The concentration of immunoreactive LHRH was 403 +/- 184 pg/20 X 10(6) lymphocytes. Increasing amounts of lymphocyte lysate displaced [125-I]LHRH from LHRH antibody in a manner parallel to that produced by synthetic hypothalamic LHRH, suggesting immunologic similarity between lymphocyte and hypothalamic LHRH. Lymphocyte LHRH-like immunoactivity coeluted from Nova-Pak C18 columns with synthetic hypothalamic LHRH. When lymphocyte lysates were applied to rat anterior pituitary cells in monolayer culture, significant stimulation of LHRH secretion was seen, from 2,144 +/- 54 pg LH/ml.4 h to 15,364 +/- 587 pg LH/ml.4 h (P less than 0.001), a finding verified in five additional experiments. In other studies, this LH response evoked by lymphocyte lysates was found to be dose dependent and could be significantly inhibited by an LHRH-antagonist. Furthermore, when lymphocyte lysate and identically treated synthetic LHRH were HPLC fractionated, there was coelution of lysate and hypothalamic LHRH bioactivity. The lysate itself contained no substantial LH immunoreactivity. Thus, lymphocytes from spleens of adult male rats contain an immunoactive and bioactive LHRH, a finding further strengthening an association between the endocrine and immune systems.

Increase of CALLA-positive stimulated lymphoid cells in transient erythroblastopenia of childhood.

In 1986 and 1987 11 children with TEC (transient erythroblastopenia of childhood) were referred to our hospital. Bone marrow aspirations were performed to exclude haematological malignancy. There was a marked reduction of erythropoiesis in 9 cases (1%-8%), two children had already recovered (33% and 44% erythropoiesis). Eight patients exhibited high percentages of stimulated lymphoid cells. The subsequent immunotyping revealed the expression of CALLA (common acute lymphoblastic leukaemia antigen) on these cells but there was no other sign for malignancy. The patients recovered without any specific treatment except transfusions of packed red cells. Eight patients were followed up 11-18 months after initial presentation and were all found to be in good health. A prominent increase of CALLA-positive stimulated lymphoid cells has also been found in other haematological diseases such as neutropenia and immune thrombocytopenia. The expression of CALLA in bone marrow lymphocytes is a general reactive change to various alterations.

Relationship of one form of human histamine-releasing factor to connective tissue activating peptide-III.

We have previously reported purification of three forms of histamine-releasing factors (HRFs) from mixtures of streptokinase-streptodornase stimulated human mononuclear cells and platelets with apparent molecular masses of 10-12, 15-17, and 40-41 kD (1989. J. Clin. Invest. 83:1204-1210). We have also prepared mouse MAbs against the 10-12-kD HRF (1989. J. Allergy Clin. Immunol. 83:281). Affinity-purified 10-12-kD HRF appears as a broad band upon polyacrylamide gel electrophoresis in the presence of SDS. We determined the NH2-terminal amino acid sequence of the top and bottom halves of this broad band. Sequence analysis revealed striking homology between this HRF and connective tissue activating peptide-III (CTAP-III), a platelet-derived 8-10-kD protein known to cause mitogenesis and extracellular matrix formation in fibroblast cultures. 19 of 21 NH2-terminal residues in the top half of the HRF band were identical to the NH2-terminal sequence of CTAP-III. 20 of 21 NH2-terminal residues in the bottom half were identical to the NH2-terminal sequence of neutrophil-activating peptide-2, which is derived from CTAP-III by proteolytic cleavage between residues 15 and 16. Purified CTAP-III also released histamine from basophils. Rabbit antiserum raised against either native or recombinant CTAP-III recognized affinity-purified HRF in immunodot blot assays, and MAb against HRF recognized CTAP-III in both dot blot and microtiter plate based immunoassays. These data demonstrate the first structural, functional, and immunologic relationship between one form of human HRF and a previously described cell product.