Nuclear localization of lactoferrin in the human granulocyte: Artifact incurred during slide preparation.

Within the blood cells, lactoferrin is found only in the late stage neutrophilic granulocytes. Lactoferrin first appears in these cells during the myelocyte stage of development coincidentally with the specific or secondary granules. Most investigators report a cytoplasmic immunocytochemical localization reaction within the granulocyte. However, others have observed a prominent nuclear localization reaction. Treating the cells with certain fixatives was shown to prevent the relocation of lactoferrin from the cytoplasm to the nucleus when the localization was done on granulocytes prepared by smearing. The present study demonstrated that the relocation of lactoferrin is only a problem when cells were smeared or cytocentrifuged onto slides or fractionated for the purpose of isolating cellular organelles. Under these conditions the selection of fixative is an important consideration. Exposing isolated lactoferrin to a fixative effective in retaining lactoferrin in the cytoplasm of granulocytes smeared on slides did not alter a number of its physical properties. The results suggest that maintenance of the normal cytoarchitecture or effect of fixative on other cellular components prevents the relocation of lactoferrin within the cell during tissue processing and the direct action of fixation on lactoferrin is probably not responsible for this effect.

Lactoferrin: nuclear localization in the human neutrophilic granulocyte?

Conditions were established whereby nuclear or cytoplasmic immunocytochemical localization of lactoferrin was observed in the human peripheral blood granulocyte. A positive nuclear staining reaction was obtained when cells were either not fixed or treated with most fixatives. However, treatment of blood smears with formaldehydeacetone prior to the immunocytochemical localization gave a cytoplasmic staining reaction. A method was developed that allowed us to examine proteins obtained from granulocyte nuclei isolated from fixed cells. Only a trace amount of lactoferrin was detected in the electrophoretically separated nuclear proteins obtained from granulocytes treated for 1 min with formaldehyde-acetone. However, lactoferrin was major protein component in nuclei isolated from untreated cells, cells treated with other fixatives, or cells preincubated in buffered saline prior to formaldehyde-acetone fixation. formaldehyde-acetone treatment of nuclear materials containing lactoferrin did not extract lactoferrin or mask it from detection, thus indicating that lactoferrin found in the mature neutrophilic granulocyte nucleus is most likely acquired from other cellular organelles during tissue processing.

Alport's syndrome associated with macrothrombopathic thrombocytopenia.

The combined occurrence of hereditary nephritis with nerve deafness (Alport's syndrome) and macrothrombocytopathic thrombocytopenia is very rare. The authors have had the opportunity to study such a case in a 20-year-old man who had been followed since birth. The clinical history, renal biopsy, platelet studies, and autopsy findings are presented. The renal pathologic findings are well defined; however, the hemostatic abnormalities and the hearing loss are not well characterized. In this paper, an attept is made to clarify the diverse platelet functional and morphologic abnormalties.

CD20 (pan-B cell antigen) expression on bone marrow-derived T cells.

Antibodies directed against CD20 (L26, Leu 16, and B1) are frequently used to determine the presence of B lymphocytes. However, recent publications describe the unexpected presence of CD20-positive T cells in the peripheral blood of normal subjects and occasional T-cell neoplasms that express CD20. To determine the presence of CD20-positive T cells in bone marrow, flow cytometric analysis was performed on 34 aspirate specimens (14 normal, 5 acute lymphoblastic lymphoma [ALL], 5 acute myelogenous leukemia [AML], 4 HIV positive, 2 myelodysplastic/myeloproliferative, 2 chronic myelogenous leukemia [CML], 1 chronic lymphocytic lymphoma [CLL], 1 multiple myeloma). A small population of cells coexpressing CD3 (Leu 4) and CD20dim (Leu 16) was identified in 94% of the specimens, representing 0% to 11% (mean 1.77%) of marrow mononuclear cells and 0% to 22.2% (mean 6.54%) of marrow lymphoid cells. There was no correlation between the percentage of CD20-positive T cells and the CD4:CD8 ratio, patient age, gender, or diagnosis. CD20dim positive cells included immature B cells and CD20-positive T cells. Although evaluation of CD20 expression is useful in delineating B-cell processes, caution should be exercised in interpreting its expression on bone marrow T-lymphoid cells. CD20 expression on T cells may be seen in either normal, reactive, or neoplastic processes.

Biphenotypic acute leukemia in adults.

Leukemias are characterized by an idiopathic proliferation of a progenitor cell that is committed to a single cell lineage. However, leukemias with dual-lineage differentiation are being described, especially within the pediatric age group. The authors reviewed 118 cases of adult acute leukemia phenotyped by immunofluorescent flow cytometry; 7 cases demonstrated mixed cell lineage. Immunophenotypically these cases were defined by early B-lymphocyte differentiation (TdT, HLA-DR, and CD19) coexpressed with a myeloid receptor (CD13, CD15, or CD33) on the same leukemic cell. Routine cytochemical evaluation demonstrated punctate positivity of the blasts with naphthol AS-D chloroacetate esterase stain in five of seven cases. Cytogenetic analysis revealed structural abnormalities of chromosome 11 in four of the seven cases. Three of these studies showed a break at 11q23-24, the location of the human proto-oncogene ets-1. Clinically, two of these leukemias represented chronic myelogenous leukemia in blast crisis, and all cases behaved aggressively. The authors' data suggest that mixed lineage leukemias are an identifiable subset of adult acute leukemias and are associated with a poor prognosis.

Response of rhesus monkey lymphocytes to short-term administration of THC.

Four Rhesus monkeys were subjected to daily administration of 2.5 mg of tetrahydrocannabinol (THC)/kg body wt, after establishing the norms for complete blood count, T- and B-cell concentrations, and the dose response of thymidine incorporation after PHA stimulation. THC was administered daily for 3 weeks, the treatment was stopped, and then the animals were allowed to recover for 4 weeks. Cellular responses, incorporation studies and fibrinogen levels were determined during the treatment and recovery phases. Compared to 4 vehicle-treated animals, the THC-treated animals experienced significant augmentation of both their total white cell and their neutrophil counts during the recovery phase which returned to normal levels during the recovery phase. There was no alteration in total lymphocyte count or T- or B-cell concentrations. Fibrinogen levels of the THC-treated animals during the treatment phase were also elevated compared to controls, and the levels diminished to the same values as the vehicle-treated animals during recovery phase. Possible mechanisms for the response of Rhesus monkeys to short-term administration of THC are discussed.

Distribution of HLA antigens in a Mexican--American population and a comparison with two similar populations from different geographical locales.

The frequencies of 32 HLA antigens of the A and B series were determined in a previously unstudied population of Mexican-Americans in South Texas. The phenotype and gene frequencies for this group are presented and are contrasted with those determined in studies of Mexican/Mexican--American populations from two different locales. The statistically significant differences observed emphasize the need for indigenous controls when evaluating racially heterogeneous populations for disease-associated HLA antigens.

A study of HLA antigens in adults with acute rheumatic fever.

By use of a microlymphocytotoxic assay to detect 32 HLA antigens of the A and B series, we examined 49 Mexican-American adults with acute rheumatic fever and contrasted the findings with 100 ethnically identical controls. Eighty-nine percent of the total possible antigens were identified. Strong associations between HLA antigens from either locus and acute rheumatic fever were not detected. These findings serve to emphasize the importance of nongenetic factors in the pathogenesis of acute rheumatic fever and help clarify the role of HLA antigens in rheumatic diseases.

Changes in T-lymphocyte subsets during childhood Epstein-Barr virus infectious mononucleosis.

Lymphocyte subsets were measured using monoclonal antibodies in 11 children with Epstein-Barr virus-induced infectious mononucleosis and compared with those of 10 normal children. In acute infectious mononucleosis the percentage of T8+ lymphocytes was greater while the percentage of T4+ lymphocytes and the T4+ to T8+ ratio were less than those measured in normal children. The percentage and absolute number of T lymphocytes, as enumerated by E rosetting, did not differ from the values for normal children. The children with acute infectious mononucleosis had a somewhat lower T8+ response than that observed in four adult infectious mononucleosis patients. With clinical recovery, the T lymphocyte-subset values returned toward normal. T8+ lymphocytes, a phenotype subset with predominantly suppressor activity, presumably reduce normal cellular immune functions transiently and may limit the continued proliferation of Epstein-Barr virus-infected B lymphocytes.

Toxicity of a family from vacuumed mercury.

A family of four developed toxic blood levels of mercury after the mother vacuumed a spilled jar of liquid mercury from a closet in their apartment. The youngest son developed severe thrombocytopenia which was initially diagnosed as idiopathic thrombocytopenic purpura secondary to viral illness. A possible association between acute mercury toxicity and idiopathic thrombocytopenic purpura has not been previously reported. Chelation therapy with penicillamine for the older child was administered soon after toxic blood mercury levels were known by the physician. Because thrombocytopenia has been reported to occur in up to 5% of patients receiving penicillamine therapy, the younger child was treated with dimercaptosuccinic acid. The mother was also treated with dimercaptosuccinic acid. The father received dimercaprol therapy. The toxic effects and rationale for now outdated therapeutic uses of mercury are discussed.

Comparison of chicken erythroid cell nuclear isolation methods using morphological, immunochemical and biochemical criteria.

Chicken erythroid nuclei were prepared using four published methods. Our findings indicate that nuclei prepared by nitrogen cavitation are less likely to be contaminated with plasma membrane fragments than those made by procedures involving cell disruption by hypotonic lysis. However, globin gene sequences were much less sensitive to DNase I digestion in nuclei prepared by nitrogen cavitation. This suggests that the conformation of chromatin was altered by the cavitation procedure. Analysis of the proteins solubilized during limited DNase I digestion of nuclei prepared by both hypotonic lysis and cavitation revealed no appreciable differences in HMG proteins but a notable difference in the RNP-associated proteins and core histones.

Adrenergic responsiveness after abrupt propranolol withdrawal in normal subjects and in patients with angina pectoris.

Adrenergic responsiveness after abrupt propranolol withdrawas during exogenous and esdogenous catecholamine stimulation was assessed in 10 normal subjects and 10 patients with angina pectoris. Propranolol, 160 mg/day, was administered for 2 weeks and then stopped. During an epinephrine infusion, period (p < 0.005). There were no differences from control 96 hours after the drug had been stopped in both groups or at 144 hours in the angina patients who were studied for a longer time. At 48 hours of heart rate and the pressure-rate product were significantly less than control level in the angina patient, but not in the normal subjects. Similar results were observed during exercise in both groups. The epinephrine-induced increase in free fatty acids was blocked by propranolol (p < 0.005), was still attenuated at 48 hours of withdrawals (p < 0.05), but returned to control levels thereafter in both groups. Resting serum triiodothyromine levels decreased with propranolol ( < 0.005) and remaind low throughout the withdrawal period. Measurements of dopamine beta-hydroxylase, plasma platelet factor 4, and platelet aggregation at rest and after exercise did not change significantly during or after propranolol administration. Plasma norepinephrine and epinephrine values were not changed from control during the withdrawal period at rest or after exerise. We conclude that there is no evidence of hypersensitivity to beta-adrenergically mediated responses after abrupt propranolol withdrawal.

Five novel point mutations: two causing haemophilia B and three causing factor X deficiency.

Factors IX and X are plasma glycoproteins important in the middle phase of the coagulation cascade, and a bleeding disorder of variable severity results from abnormalities in the expression of either gene encoding these proteins. Nearly 380 unique molecular mechanisms cause factor IX deficiency, or haemophilia B, but only a limited number of mutations causing congenital factor X deficiency have been characterized to date. In this study enzymatic amplification has been used to examine the molecular basis for factor IX deficiency in two patients and factor X deficiency in two patients. Genomic DNA was isolated from each patient and synthetic oligonucleotide primers were used in the polymerase chain reaction to amplify each exon, splice junction and polyadenylation site. Amplified DNA was then cloned into pUC18 and sequenced. Five novel point mutations were identified, two occurring in the eighth exon of the factor IX gene and three in the eighth exon of the factor X gene. One of the haemophilia B mutations and one of the factor X mutations altered homologous histidine residues near the serine of the catalytic triad.

Human granulocyte-specific nuclear antigen(s). II. Detection of antigens in human proliferative syndromes.

Antisera raised to dehistonized chromatin from isolated normal human granulocytes revealed the presence of chromatin-associated antigens specific for the human neutrophils that appear during late stages of myeloid cellular differentiation. Immunological specificity was demonstrated by C fixation, immunodiffusion, and immunocytochemical reactions. Chromatin prepared from both normal granulocytes and specimens of myeloid leukemia showed immunologic reactivity. Although the normal antigens were detected in a specimen of CML, the position of immunodiffusion precipitin lines was different from that obtained with normal granulocyte chromatin. In addition, chromatin prepared from the myeloid leukemic cell line HL-60 expressed only one of the three precipitin bands normally found in immunodiffusion. The immunocytochemical staining reaction was confined to the nucleus of mature neutrophils in normal peripheral blood smears. Greater than 90% of cells in peripheral blood specimens of CML showed positive immunocytochemical nuclear staining. In other types of leukaemia, the normal mature granulocyte reacted with antiserum, but the nonmyeloid leukemic cells in these specimens did not. The specificity of immunologic reactions described here suggests the usefulness of nuclear antigens as cell markers.

Cigarette smoking, dietary hyperlipidemia, and experimental atherosclerosis in the baboon.

In separate experiments, we fed 30 male and 25 female baboons a diet enriched in cholesterol and saturated fat for periods of 3.3 and 2.6 years. Using operant conditioning with water rewards, we trained the animals to puff on smoking machines in a human-like manner. Half of the animals smoked more than 40 cigarettes per day, while the remaining animals (controls) puffed air. Initially, the diet produced twofold (males) and threefold (females) elevations from baseline levels in serum cholesterol concentrations, but over the course of the experiments, the serum cholesterol decreased to 1.5 (males) and 2.0 (females) times baseline levels in both cigarette smokers and controls. Blood carbon monoxide concentration, plasma thiocyanate concentration, and urine cotinine concentration were significantly greater in smokers than in controls. Responses to smoking in males included lymphocytosis, elevated fasting blood glucose concentration, and decreased seminal vesicle weight. In females, hemoglobin and mean corpuscular hemoglobin concentrations were elevated. The extent of atherosclerosis was examined after 2.8 (males) and 1.6 (females) years of smoking. Among males, the extent of lesions in carotid arteries was significantly greater in smokers than in controls, but there were no significant differences in atherosclerosis in the aorta or the brachial, iliac-femoral, or coronary arteries. Among females, there were no significant differences in atherosclerosis between smokers and controls in any artery. These experiments show little effect of 2 to 3 years of cigarette smoke inhalation and concurrent modest elevation of blood carboxyhemoglobin on experimental atherosclerosis in the presence of moderate hyperlipidemia.

Atherosclerosis-related responses to cigarette smoking in the baboon.

Thirty-six young adult male baboons (Papio cynocephalus) were fed an atherogenic diet (40% calories from lard, 1.5 mg cholesterol/kcal) and taught to puff by operant conditioning with water rewards. Eighteen baboons (smokers) were assigned randomly to smoke 43 cigarettes a day, and 18 baboons (shams) were assigned randomly to puff air under conditions equivalent to those of the experimental group. During months 14-19 of smoking, cigarette-smoking baboons had significantly higher carbon monoxide and thiocyanate concentrations in blood and cotinine concentrations in urine. There were no significant differences in serum total cholesterol, VLDL + LDL cholesterol, HDL cholesterol or triglyceride concentrations of smokers and shams. Smoking baboons had significantly higher fasting blood glucose concentrations and lymphocyte counts. Platelet count, platelet aggregation, food and water intake, and body weight were not significantly different in the two groups.