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Biomedical subjects

R T Parmley

Publications and source records attributed to R T Parmley.

At least 37 records · Page 2Linked to original sources
PubMed1990

Postoperative chemotherapy for primary intracranial germ cell tumor.

Two children with primary intracranial mixed germ cell tumors are described who were successfully treated by partial resection of the tumor followed by sequential combination chemotherapy without radiation therapy. The chemotherapy consisting of VP-16 and cisplatin alternating with vincristine, methotrexate, and bleomycin resulted in apparent complete response after 6 to 7 months of treatment. Disease-free remission has continued 30-34 months off therapy. A small residual mass in one patient continues to decrease in size on magnetic resonance imaging and is presumed to represent postsurgical change rather than malignant tumor. This report demonstrates that chemotherapy may be effective in primary germ cell tumors of the suprasellar and pineal regions and could be considered for primary treatment instead of radiotherapy.

Adolescent↗
PubMed1989

Disseminated primitive neuroectodermal tumor: diagnosis using immunocytochemistry, electron microscopy, and molecular probes.

A patient with a disseminated small cell tumor presented with hyperuricemia, gingival hypertrophy, lymphadenopathy, and bone marrow replacement with tumor cells. Initial histologic examination and clinical presentation were consistent with presumed marker silent lymphoma/leukemia. Despite initial treatment with and response to lymphoma/leukemia therapy the patient relapsed in the testis, bone marrow, pancreas, and skin whereupon subsequent and retrospective immunocytochemical, ultrastructural, cytogenetic, and molecular analysis led to the diagnosis of primitive neuroectodermal tumor (PNET). Despite extensive investigation and autopsy no primary site of tumor could be found demonstrating that PNET should be considered in the differential diagnosis of disseminated small cell tumors without an apparent primary.

Adolescent↗
PubMed1989

Abnormal peroxidase-positive granules in "specific granule" deficiency.

"Specific granule" deficiency (SGD) has been previously associated with lactoferrin deficiency. The antimicrobial peptides termed defensins, comprising 30% of normal primary granule proteins, have also been shown to be markedly deficient in SGD. The present study was undertaken to correlate these findings with ultrastructural morphometric analysis and peroxidase cytochemistry. Peroxidase-positive, rim-stained, large, defensin-rich dense granules, previously described as a subpopulation of azurophil or primary granules in normal neutrophils, were markedly decreased in a patient with SGD. Morphometric studies of peroxidase-positive granules indicated an average peroxidase-positive granule area (all profiles) in the patient of 0.019 +/- 0.017 micron 2 (mean +/- SD, n = 941) compared to control values from normal neutrophils of two volunteers of 0.049 +/- 0.033 micron 2 (n = 896) and 0.050 +/- 0.039 micron 2 (n = 873) (P less than 0.001 between patient and control samples). Granule histograms showed a single peak of small peroxidase-positive granules, whereas control samples contained more prominent subpopulations of larger peroxidase-positive granules. The total number of peroxidase-positive granules per 100 micron 2 of cytoplasm in the patient was 255 +/- 124 (mean +/- SD, n = 15 cell profiles), which was similar to control values of 266 +/- 63 and 212 +/- 109. Thus, the defensin deficiency in SGD is associated with a decrease in size rather than number of peroxidase-positive granules; suggesting that defensins contribute to normal peroxidase-positive granule size and that SGD is a more global granule deficiency than originally thought.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Proteins↗
PubMed1988

Ultrastructural cytochemistry of complex carbohydrates in developing feline neutrophils.

The feline species provides animal models for at least six congenital lysosomal disorders. Since knowledge of normal feline neutrophils is a prerequisite for studies of their abnormalities, the present report describes the morphology and cytochemistry of normal feline neutrophils and compares the subcellular distribution of sulfate- and vicinal-glycol-containing complex carbohydrates to that of peroxidase and acid phosphatase. Immature feline primary granules, formed in promyelocytes, were stained for peroxidase, acid phosphatase, sulfate, and vicinal glycols. During maturation, primary granules retained strong staining for peroxidase, but staining for vicinal glycols decreased, and acid phosphatase and sulfate reactivity was lost. Secondary granules formed in myelocytes lacked peroxidase, acid phosphatase, and sulfate staining, but stained intensely for vicinal-glycol-containing complex carbohydrates. No analogues of tertiary granules previously described in rabbits and humans were demonstrated in feline neutrophils. However, a new sequential staining technique for peroxidase and vicinal glycols has suggested the formation in myelocytes and late neutrophils of a third granule type that contained peroxidase, acid phosphatase, and vicinal glycols but lacked sulfate staining. Thus, the staining characteristics of primary and secondary granules in cats closely resembled those in humans and rabbits. The third (late-forming) type of granule has not previously been described in other species.

Acid Phosphatase↗
PubMed1988

Morphometry of feline neutrophil granule genesis.

During neutrophil granule genesis, the formation of primary granules is generally thought to be limited to the promyelocyte stage; whereas synthesis of secondary granules is thought to occur only at the myelocyte stage. This hypothesis was tested morphometrically in feline neutrophils that are known to contain both granule types. Marrow specimens obtained from six cats were stained with peroxidase for identification of neutrophil primary granules and counterstained with periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) for identification of secondary granules. By regression analysis using arithmetic models, numbers of cytoplasmic granules in 311 cells were correlated with the degree of nuclear chromatin condensation, which was shown to be an adequate parameter for cell maturation. Promyelocytes and myelocytes had similar mean numbers of peroxidase-positive granules per unit area. A significant increase (p less than or equal to 0.0001) in the numbers of peroxidase-positive granules was noted between the metamyelocyte and the mature neutrophil stage, despite the lack of peroxidase activity in endoplasmic reticulum and Golgi lamellae. By contrast, a significant increase of peroxidase-negative granules between the metamyelocyte and the mature neutrophil stage was not clearly established with these methods. The increase in peroxidase-positive granules may indicate continued production of peroxidase-containing granules and/or redistribution of peroxidase among lysosomal organelles in late feline neutrophils.

Animals↗
PubMed1988

Neutrophil lactoferrin content: variation among mammals.

Lactoferrin (Lf) in blood and/or marrow neutrophils was semiquantified using indirect immunofluorescence technique in nine mammalian species. Neutrophil iron-binding reactivity (NFeBR), which corresponds primarily to Lf, was also visualized and semiquantified using functional cytochemical (FeNTA-AF) technique at the light microscopic level in these nine and in an additional fifteen mammalian species, and in selected species at the ultrastructural level. Neutrophil immunoreactive Lf was positively correlated with total cellular and granule content of NFeBR among these nine species, and with previously reported concentrations of neutrophil Lf quantified by radioimmunoassay. Relative levels of Lf in neutrophil extracts from rat, hamster, and human were confirmed using SDS-polyacrylamide gel electrophoresis and immunoblotting. Relatively high levels of immunoreactive neutrophil Lf and/or NFeBR were observed in carnivores (ten species) and primates (six species). Among rodents (five species), the levels were variable, and the artiodactyls (four species) studied had low levels. These results demonstrate that neutrophil Lf levels vary widely among mammalian species. In addition, FeNTA-AF technique provides a rapid means of evaluating animals for relative quantities of neutrophil Lf.

Animals↗
PubMed1988

Differential staining of neutrophils and monocytes: surface and cytoplasmic iron-binding proteins.

Lactoferrin, transferrin, and ferritin were systematically visualized and semiquantified in neutrophils and monocytes/macrophages using indirect immunofluorescence and functional cytochemical techniques. They localized on cell surfaces and within the cytoplasm at the light and electron microscopical levels. In normal subjects, subpopulations of blood neutrophils and monocytes had surface lactoferrin, but little surface transferrin or ferritin was observed on these cells. Most neutrophils had brilliant granular cytoplasmic positivity for lactoferrin; variable fractions of monocytes had weak to moderate diffuse cytoplasmic lactoferrin staining localized most prominently to the cytoplasmic matrix. Most neutrophils had cytoplasmic ferritin, but few had cytoplasmic transferrin, whereas larger subpopulations of monocytes had cytoplasmic staining reactions for both proteins. To analyse maturing cells, the iron nitrilotriacetate-acid ferrocyanide method was adapted for the light microscopical analysis of neutrophils and monocytes/macrophages in soft agar culture. Further, a combined stain that visualizes iron nitrilotriacetate-acid ferrocyanide reactivity and alpha-naphthyl butyrate esterase activity in cells in blood and marrow smears was developed. The relative quantities and subcellular distribution of iron-binding proteins in neutrophils and monocytes/macrophages defined by the present methods can be correlated with biochemical, maturational, and functional properties of these cells.

Bone Marrow↗
PubMed1988

Ultrastructural silver enhancement of Prussian blue-reactive iron in hematopoietic and intestinal cells.

Prussian blue has been widely used to localize iron in a variety of tissues at the light and electron microscopic level. In the present study, thin sections of human marrow and blood cells and rat duodenal cells were exposed to silver proteinate (SP) after staining en bloc with acid ferrocyanide (AF), with and without prior iron saturation using iron nitrilotriacetate (FeNTA). Silver deposition was observed over Prussian blue-reactive sites and significantly enhanced sites of minimal AF and FeNTA-AF staining. AF-SP stain deposits were present in the cytoplasmic matrix, granules, and occasionally on the surfaces of macrophages, monocytes, and erythroblasts. FeNTA-AF-SP stained additional cytoplasmic and surface sites in erythroblasts and stained neutrophil granules intensely. Duodenal epithelium from iron-loaded rats demonstrated strong AF-SP staining of ferric iron in microvilli, apical cytoplasmic matrix, and lateral membranes. Similar preparations from iron-replete rats stained sparsely; however, intense AF-SP staining was observed after iron saturation with FeNTA. SP similarly enhanced luminal ferrous iron deposits stained with acid ferricyanide in rats given intraluminal ferrous iron. AF-SP stain deposits were removed by exposure of thin sections to NH4OH, KCN, or HNO3 but were not affected by prior exposure to HIO4 or NaBH4, consistent with a silver cyanide or complex stain precipitate rather than reduced silver or silver ferriferrocyanide. SP enhancement of Prussian blue allows identification of reactive sites not readily visualized with AF or FeNTA-AF alone, and offers the potential for differentiating AF staining from other deposits or organelles of comparable density.

Animals↗
PubMed1988

Iron-binding reactivity in mature neutrophils: relative cell content quantification by cytochemical scoring.

We have developed a technique that permits evaluation and semi-quantification of iron-binding function in mature neutrophils. Neutrophil iron-binding reactivity (NFeBR) visualized using the iron nitrilotriacetate-acid ferrocyanide technique was rated 0 to 5+ in 100 segmented cells; the ratings were totaled to yield a score (NFeBRS). Males and post-menopausal females had significantly higher NFeBRS than pre-menopausal females. Neonates had low values, and a homogeneous distribution of NFeBR among neutrophils. In pregnancy and acute infection, NFeBRS were significantly increased. In a patient with congenital lactoferrin (Lf) deficiency, the NFeBRS was very low. In Ph1-positive chronic myelogenous leukemia, 13 of 17 patients had low NFeBRS due to decreased NFeBR, which was heterogeneously distributed among mature neutrophils. By ultrastructural analysis of mature neutrophils in two such patients, the stain deposits in FeBR-positive granules were of normal intensity, but the numbers of positive granules were decreased in many cells. NFeBRS were also low in 12 of 23 patients with other myeloproliferative disorders, and in seven of 15 patients with acute non-lymphoblastic leukemia, but in only seven of 63 patients with other neoplasms. NFeBRS were significantly correlated (p less than 0.008) with values of neutrophil Lf content quantified by immunologic assays in a wide variety of conditions and over a broad range of values. These results augment observations of neutrophil Lf made using immunological methods.

Adult↗
PubMed1988

Cell culture studies and oncogene expression in juvenile chronic myelogenous leukemia.

Juvenile chronic myelogenous leukemia (JCML) may be distinguished from adult CML based upon in vitro cell growth characteristics. We studied four untreated children with JCML and report additional unique findings. Peripheral blood (PB) and bone marrow (BM) cells were grown in soft agar. Without exogenous colony-stimulating activity (CSA) there was exuberant "spontaneous" colony formation in both PB and BM cultures. In the absence of exogenous stimulus, PB colony morphology was predominantly, but not exclusively, monocyte/macrophage. When PB was depleted of adherent cells, "spontaneous" colony formation was nearly completely abrogated. Cultures were also performed in the presence of various sources of CSA including giant cell tumor-conditioned medium (GCT-CM), a melanoma cell line-CM (LD1-CM), human placenta-CM (HPCM), and normal PB mononuclear cell (PBMC) feeder layers. Colony formation was typically increased with HPCM and PBMC, whereas in two patients GCT-CM and LD1-CM failed to stimulate additional colony growth when compared to cultures without exogenous CSA and, in fact, appeared to inhibit baseline "spontaneous" growth. The morphology of colonies in the presence of exogenous stimuli was highly variable. Because of the recent association between the c-fms protooncogene product and the receptor for the monocyte growth factor CSF-1, we analyzed the PB cells from two JCML patients for c-fms expression. Although expressed, c-fms levels were less than that in an adult with Ph1-positive CML in chronic phase. These studies indicate that in JCML, there are dramatic increases in both PB and BM colony-forming cells and that "spontaneous" growth is dependent on an accessory adherent cell fraction. Furthermore, patterns of responsiveness to various sources of CSA suggest that the colony-forming cells may not be a uniform population of malignant cells.

Animals↗
PubMed1988

Monensin disruption of neutrophil granule genesis.

The Na+/H+ ionophore monensin (M) has been used widely to study intracellular pH gradients and acidic subcellular compartments. In the present study, cultured myeloid leukemia HL60 cells, directly sampled bone marrow cells, and peripheral blood neutrophils were exposed to 1-5 microM monensin for 0.5-20 hours. The effects were evaluated using ultrastructural, cytochemical, and biochemical methods. In HL60 cells and marrow promyelocytes treated with monensin, progressive vacuolation of the trans then the cis Golgi was observed. These vacuoles lacked diaminobenzidine (DAB) reactive peroxidase, high iron diamine (HID) reactive sulfated glycoconjugates, and periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) reactive vicinal glycol containing complex carbohydrates, but some cis Golgi elements retained osmium zinc iodide reactive reducing groups. The number of normal intensely stained HID reactive granules decreased and an incomplete granule that was DAB-positive/HID-negative, PA-TCH-SP-negative with flocculent matrix density increased in frequency as a function of time and concentration of monensin. Treatment of HL60 cells with monensin markedly reduced 35SO4 incorporation but myeloperoxidase labeling and activity per cell remained constant, although it shifted to lower density granule fractions consistent with the persistent DAB staining of endoplasmic reticulum and synthesis of a DAB-positive, HID-negative granule in intact HL60 cells. The Golgi complex of monensin-treated myelocytes and segmented neutrophils was also vacuolated. A subpopulation of preformed primary granules in promyelocytes, myelocytes, and segmented neutrophils appeared to increase in size and peripheral or central electron lucency. These selective effects of monensin indicate that granule components may be packaged into DAB-positive organelles that are deficient in trans Golgi-derived elements (HID- and PA-TCH-SP-negative) and that some preformed primary granules contain a monensin sensitive Na+/H+ gradient.

Bone Marrow↗
PubMed1987

Cytochemistry and ultrastructural morphometry of cultured HL60 myeloid leukemia cells.

Cultured human myeloid leukemia (HL60) cells were characterized using ultrastructural cytochemical methods and differences identified when cells were compared for low (17 to 47), middle (69 to 100), and high (214 to 244) passages or to normal promyelocytes aspirated from bone marrow. Endoplasmic reticulum and transition structures (pre-Golgi compartment) of HL60 cells stained positively for peroxidase using diaminobenzidine but stained sparsely for reducing groups with osmium-zinc iodide. Staining of Golgi elements was relatively indistinct with diaminobenzidine and strong with osmium-zinc iodide, in comparison to freshly harvested promyelocytes which have intense diaminobenzidine and osmium-zinc iodide staining of the pre-Golgi and Golgi compartments. Cytoplasmic polyribosomes were more numerous in middle and high passage cells, whereas dilatation of endoplasmic reticulum was less prominent in these cells. The mean granule size was significantly increased in low passage cells, and staining of peroxidase was more prominent by light and electron microscopy when compared to high passage cells. Cytoplasmic granules demonstrated strong complex carbohydrate staining, indicating a lack of granule maturation in HL60 cells. Terminally differentiated myeloid cells were more frequent in low passage samples, and some neutrophil granule maturation appeared to occur within these cells, whereas all eosinophil granules consistently remained immature with intense complex carbohydrate staining and lack of crystalloid formation. These studies demonstrate significant differences between HL60 cells and normal promyelocytes, and also passage-dependent maturational differences in HL60 cells. These differences should be considered in evaluating parameters of cell growth and maturation and in the biochemical and enzymatic characterization of these cells.

3,3'-Diaminobenzidine↗
PubMed1987

Biochemical and ultrastructural effects of monensin on the processing, intracellular transport, and packaging of myeloperoxidase into low and high density compartments of human leukemia (HL-60) cells.

The biosynthesis of myeloperoxidase in human promyelocytic leukemia HL-60 cells was studied by pulse-chase and immunoprecipitation methods and separation of subcellular organelles using Percoll density gradient fractionation. These studies revealed that in control and monensin (1 microM) treated cells, more than 85% of the total immunoprecipitable radiolabeled myeloperoxidase was present predominantly in precursor form (Mr 91,000) and resided in lower density compartments after an initial 3-h labeling period. Using biochemical and ultrastructural techniques, the lower density regions of the gradient were found to contain elements of the endoplasmic reticulum and the Golgi complex. Following a 16-h chase period, about 70% of the radiolabeled myeloperoxidase in untreated cells was found predominantly in denser regions of the gradient and was present mainly in the form of the mature large subunit (Mr 63,000). These dense regions were shown to contain azurophilic granules by means of the distribution of beta-glucuronidase and myeloperoxidase activities and by electron microscopy. Processing of myeloperoxidase and its deposition into dense granules were blocked by monensin treatment. Following a 16-h chase period in the presence of monensin, approximately 80% of the radiolabeled myeloperoxidase continued to reside in lower density compartments and was predominantly in precursor (Mr 91,000) and intermediate (Mr 81,000 and 74,000) forms. Only about 10% of the radiolabeled myeloperoxidase was associated with dense azurophilic granules. Monensin treatment produced large, Golgi-derived vacuoles which were isolated using Percoll density centrifugation and identified by electron microscopy. These vacuoles were found to be essentially devoid of peroxidase activity and pulse-labeled, newly synthesized radiolabeled myeloperoxidase species. The effects of monensin on transport and processing were reversible after a 3-h exposure and 16-h chase period in the absence of monensin. Taken together, these data indicate that maturation of myeloperoxidase is closely linked to its deposition into dense azurophilic granules via a monensin-sensitive process(es). The lower density compartments within which immature myeloperoxidase species accumulate in the presence of monensin appear to be functionally related to or associated with Golgi or endoplasmic reticulum structures distinct from the large monensin-induced vacuoles.

Cell Line↗
PubMed1987

Loss of hepatitis B antibody in human immunodeficiency virus-positive hemophilia patients.

The presence of human immunodeficiency virus (HIV) antibody in hemophilia patients was correlated with the loss of existing antibody to hepatitis B surface antigen (HBsAb) or the inability to develop an antibody to hepatitis B after receiving commercially available hepatitis B vaccine. Of the 137 patients studied 66 were HIV-positive and 71 were HIV-negative. Evidence of HBsAb (n = 44) or exposure to hepatitis vaccine (n = 12) was found in 85% of HIV positive patients at some time during their care in our clinic. However, 20% demonstrated subsequent antibody loss and/or did not respond to hepatitis vaccine. Loss of HBsAb or vaccine nonresponse was restricted to patients less than 21 years of age (72% of all patients). This result contrasted to only a 3% loss of HBsAb or vaccine nonresponse in the HIV-negative patients who had acquired the HBsAb (n = 23) or were given the hepatitis vaccine (n = 29). This result suggests that loss or alterations of hepatitis B immunity occur in association with HIV infection or exposure.

Child↗
PubMed1987

Defensin-rich dense granules of human neutrophils.

Defensins are a newly recognized class of small, cationic polypeptides that have in vitro microbicidal activity toward certain bacteria, fungi, and viruses. Human neutrophil granules were separated into 13 density fractions by using a high-resolution Percoll gradient centrifugation procedure, and the distribution of the three defensin polypeptides in these fractions was determined. Levels of defensins and several granule marker proteins were estimated in each fraction from relative staining intensities of bands following acid-urea and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of total acid-extractable proteins. These results were confirmed by enzyme immunoassay measurements of defensins and quantitative determinations of the typical azurophil granule components, myeloperoxidase, beta-glucuronidase, lysozyme, and elastase. The five higher density granule fractions (H1 through H5) contained fourfold higher relative amounts of defensins as compared with the eight lower density fractions (L1 through L8), accounting for approximately 50% of the total protein. In particular, fraction H5 was especially enriched in defensins but was relatively deficient in myeloperoxidase, beta-glucuronidase, lysozyme, and elastase. Ultrastructural morphology showed that fraction H5 contained the largest granules. Seventy percent of these granules exhibited electron-dense rims and electron-lucent central regions when stained with methanolic uranyl acetate-lead citrate, and 70% showed this same characteristic rim-staining pattern after limited reaction (30 minutes) for peroxidase with diaminobenzidine. These distinctively large, rim-stained granules were identified in intact, mature peripheral blood neutrophils as well as in human bone marrow promyelocytes, indicating that their synthesis occurs during early myeloid development. This unusual granule type may play a specialized role in the microbicidal functions of the neutrophil, distinct from that of typical azurophil granules.

Blood Proteins↗
PubMed1987

Preleukemia in Fanconi's anemia: hematopoietic cell multinuclearity, membrane duplication, and dysgranulogenesis.

A 25-year-old male had Fanconi's anemia characterized by small stature, the onset of pancytopenia in the first decade of life, a high spontaneous breakage rate of the chromosomes, and the development of acute myeloblastic leukemia. In the preleukemic phase, marrow erythroblasts were multinucleated, and had duplicated nuclear and cytoplasmic membranes with frequent nuclear pockets and cytoplasmic vacuoles, respectively. All neutrophilic and eosinophilic granulocytes had severe quantitative and qualitative defects of granulogenesis; autophagy and nuclear pockets were also observed in the majority of granulocytic cells. Platelets had decreased granulation and extremely dilated canaliculi. Decreased titration scores with anti-I and anti-i were observed with the patient's erythrocytes and those of his clinically normal mother. The unusual morphologic and serologic findings in this patient appear to have resulted from a membrane abnormality affecting the cells of several hematopoietic lines and their organelles.

Adult↗
PubMed1987

Small intestinal regulation of iron absorption in the rat.

Ultrastructural, biochemical, and immunologic studies of the small intestinal mucosa of rats were undertaken to investigate factors associated with the regulation of iron absorption. The quantity of iron within mucosal cells was proportionate to the degree of iron repletion. Although the quantity of iron-binding substances was similar in iron-deficient and iron-loaded animals, the unsaturated iron-binding capacity of mucosal cells varied inversely with the state of iron repletion of animals in ultrastructural, biochemical, and immunologic observations. Although only certain mucosal cells contained iron-binding substances, their number was not increased in iron-deficient animals. Greater quantities of iron and iron-binding substances were observed in duodenal mucosa than in ileal mucosa. These findings are consistent with the hypothesis that the quantity of unsaturated iron-binding substances within intestinal mucosal cells regulates iron absorption. These findings are only partially explained by changes observed in the concentration of ferritin and transferrin within intestinal mucosa and suggest that other iron-binding substances may also participate in the regulation of iron absorption.

Animals↗