Normal Peripheral Leukocytes Research Articles

Expressions of four major protein Ser/Thr phosphatases in human primary leukemic cells.

Activity and expression of four major protein serine/threonine (Ser/Thr) phosphatases, protein phosphatase type 1 (PP1), protein phosphatase type 2A (PP2A), protein phosphatase type 2B (PP2B) and protein phosphatase type 2C (PP2C) were evaluated in normal peripheral leukocytes, and in various leukemic cells from patients with acute myelogenous leukemia (AML), common acute lymphocytic leukemia (cALL), or chronic lymphocytic leukemia (CLL). PP1 was the most abundant phosphatase in blood cells, and relative abundance of each phosphatase was: PP1 > PP2A > PP2B approximately = PP2C. PP1 activity and its expressions were higher in blasts of AML-M4 and -M5 than in cells of AML-M1, cALL and CLL. PP2A activity and its expression were higher in blasts of AML-M3, -M4 and -M5 than in cells of AML-M1, cALL and CLL. Activity and expression of both PP1 and PP2A in normal monocytes were highest, and PP2A activity in normal neutrophils was lowest among normal leukocytes. PP2B activity and its expression were higher in blasts of AML-M2, -M3 and normal lymphocytes. PP2C activity and its expression were relatively constant in various leukemic cell types. Activities of PP1 and PP2A of AML blasts correlated positively with the expression of CD11b, whereas activities of PP1 and PP2B correlated negatively with the expression of CD7. Thus, each phosphatase was ubiquitously but differently expressed in various leukemic cell types and in normal leukocytes. These data also suggest that expressions of PP1, PP2A and PP2B are relatively low in leukemic blasts arresting at the stage of early pluripotent stem cells, and are differently modulated during the course of myelomonocytic commitment and maturation.

Clonal X-inactivation analysis of human tumours using the human androgen receptor gene (HUMARA) polymorphism: a non-radioactive and semiquantitative strategy applicable to fresh and archival tissue

Assessment of clonality of cellular proliferations is important in experimental and clinical cancer research. X-chromosome inactivation studies are widely used to assess clonality, but most assays require relatively large amounts of high molecular weight DNA. Two PCR-based strategies, the phosphoglycerate kinase (PGK) and the human androgen receptor (HUMARA) clonality assays allow studies of small tissue samples. The HUMARA assay was adapted to non-radioactive analysis taking advantage of an automated sequencer providing high resolution of alleles and immediate quantitation. This assay was validated by comparison with X-inactivation patterns obtained by Southern analysis with the probes M27β and PGK. Fifteen gastrointestinal carcinomas, 25 benign goiter nodules and normal peripheral leukocytes of 27 individuals (12 who were under 15 years and 15 over 80 years) were analysed. Furthermore, DNA extracted from formalin-fixed paraffin-embedded tissue (FPT) was analysed with the two PCR-based methods and compared with X-inactivation patterns determined by Southern analysis of high molecular weight (HMW) DNA. This modified HUMARA assay is reliable in most patients; as with other clonality assays, constitutive skewing in normal tissue precludes clonal analysis in some individuals. Extremely skewed X-inactivation patterns were found in normal peripheral leukocytes of 7 out of 15 old females (over 80 years) and in 1 of 12 of the young females tested (under 15 years). Comparison of results obtained with HMW and FPT DNA yielded consistent results for the HUMARA assay whereas the PGK PCR assay was much less reliable. The HUMARA assay thus permits studies of selected areas of tissue sections without significant stromal components, allowing correlation of histological and genotype findings in fresh and archival specimens.

Septic arthritis following arthroscopy: Clinical syndromes and analysis of risk factors

During a 4-year study of 4,256 knee arthroscopies, eighteen patients became infected (infection rate 0.42%). Occurrence of infection was strongly associated with use of long-acting intraarticular intraoperative corticosteroids. Infection was more common among patients with longer surgery operating times, increased numbers of procedures during surgery, prior procedures, and performance of chondroplasty or soft tissue debridement. Subsequent to the study, the infection rate fell to 0.1%. Twenty-four infections were studied (our eighteen plus six other concurrent community cases); twelve were due to Staphylococcus aureus, eleven to coagulase-negative staphylococci, and one to Enterobacter cloacae. Seventy percent of the patients had onset of symptoms within 3 days of surgery. Most patients with coagulase-negative staphylococcal infections had fevers <38.3°C (101°F), negative Gram stains on synovial fluid, normal peripheral leukocyte counts, and somewhat indolent, mild clinical syndromes, while most patients with S. aureus infections had higher fevers, positive synovial Gram stains, peripheral leukocytosis, and more acute and severe clinical syndromes. Knee pain, swelling, and warmth always occurred, but erythema was noted in only 30% of patients. Treatment with 2 weeks of intravenous antibiotics was successful in all but one patient. Long-term results were excellent in sixteen of twenty-two patients.

Decreased Levels of Steroid 21-Hydroxylase (P450(c21)) and Its mRNA in an Adrenocortical Adenoma Associated with 21-Hydroxylase Deficiency.

Adrenocortical adenoma incidentally found in a 37-yr-old female patient, with simple virilizing form of 21-hydroxylase deficiency, was studied. Cultured adenoma cells revealed excessive secretion of 17 alpha-hydroxyprogesterone in response to 10(-8) M ACTH, compared with those of 11-deoxycortisol and cortisol, which indicated impaired activity of the 21-hydroxylase. To elucidate the molecular mechanisms of this defective 21-hydroxylase in the adenoma, we analyzed the gene encoding specific cytochrome P450 (P450c21) for steroid 21-hydroxylation and its expression. DNA and RNA were extracted from the adrenal adenoma and were hybridized with a probe of human P450c21 gene, by Southern and Northern blot analysis. In Southern blot analysis with Taq I, Bgl II or Bam HI, there was no difference between the pattern of restriction fragments in DNA from the adenoma and normal peripheral leucocytes. Northern blot analysis of the adenoma showed the same size of P450c21 mRNA as in the normal adrenal gland, but the amount was low--about a half that of the normal adrenal. In Western blot analysis with polyclonal antibody to P450c21, only a small amount of P450c21 protein was detected in the adenoma, although it was found to be of the same molecular weight as that in the normal adrenal gland. In view of these findings it is conceivable as one of possibilities that a mild and small mutation in the structural or promotor region of the P450c21 gene may cause the decreased 21-hydroxylase activity in this adenoma.

Monoclonal Antibody GB24 Recognizes a Trophoblast‐Lymphocyte Cross‐Reactive Antigen

GB24 is a mouse monoclonal antibody (IgG1) raised against human term placental microvilli. This antibody displayed pan-trophoblast reactivity pattern. In addition, GB24 recognized normal peripheral leukocytes and transformed cell lines (Daudi, HL-60, Jurkat, AV3, BeWo, HT-29) by using membrane immunofluorescence and flow cytometry. By SDS-polyacrylamide gel electrophoresis, this antibody immunoprecipitated two proteins of 62 kilodaltons (kDa) and 75 kDa from placental microvilli. Isoelectrofocusing analysis revealed that these two bands exhibited different isoelectric points: 4.7 for the 62 kDa protein, 4.6 and 4.4 for the two spots corresponding to the 75 kDa protein. Microvilli from different placentae revealed substantial differences regarding the intensity of the labeling of these two bands, suggesting that the antigens recognized by GB24 are probably allotypic.

Studies on hematopoietic stem cell

The ability of marrow cells from uremic patients to form granulocyte-colonies in an agar gel system was studied using the method of Robinson et al. The seven patients (in 4 patients under hemodialysis) studied here were markedly uremic and anemic. In our system colonies appeared rapidly on culture plates after four days of incubation and grew as a colony of mononuclear cells which were definitely positive for peroxidase indicating granulocytes morphologically.The number of colonies formed in cultures of patients with anemia of chronic renal failure was slightly decreased due to the decrease in marrow cellularity in uremia, but was not significant to that of normal subjects. Interestingly, the number of colonies formed per plate was directly propotional to the number of nucleated cells in marrows obtained from subjects. In addition, peripheral leukocytes obtained from uremic patients as a colony stimulating factor (CSF) in cultures stimulated colony forming cells (CFC) with normal activity to form colonies in vitro when compared to that of normal peripheral leukocytes. These findings suggest that the ability of marrow cells from uremic patients to form colonies in vitro might not be affected even in uremic condition, indicating that the number and function of CFC in uremia may be intact with normal granulopoiesis.

Mixed leukocyte stimulation of normal peripheral leukocytes by autologous lymphoblastoid cells.

Lymphocyte culture lines can be established by using buffy coat cells from normal or diseased individuals. Established lymphocytoblasts from a normal donor were used in the one-way mixed leukocyte culture test and were found to stimulate the peripheral lymphocytes of that donor. These lymphocytoblasts were also capable of stimulating allogeneic lymphocyte populations.

Deficiency of pneumococcal serum opsonizing activity in sickle-cell disease.

Abstract Serum opsonizing activity for the pneumococcus was studied with a phagocytic test using normal peripheral leukocytes incubated in normal serum and in serum from patients with sickle-cell disease. Heat-labile serum opsonizing activity for the pneumococcus was markedly deficient in serum from the patients, with a mean phagocytosis of 6.5 per cent whereas control children with AA2 hemoglobin showed a mean value of 35.1 per cent. Serum opsonizing activity for salmonella was comparable in patients with sickle-cell disease and controls, although much of the activity was heat stable. Hemolytic complement activity was similar in both groups of children. Pathologic changes in the spleen in sickle-cell disease may result in functional autosplenectomy. Consequently, these observations may reflect the impairment of splenic clearance and heat-labile opsonin synthesis.

Herpes-type virus and chromosome marker in normal leukocytes after growth with irradiated Burkitt cells.

Cultured cells derived from male patients with Burkitt's lymphoma and harboring herpes-type virus particles were lethally irradiated. These irradiated cells induced normal peripheral leukocytes of female infants to grow within 2 to 4 weeks after mixed cultivation. Cells of a line free of this agent failed to stimulate growth. If either type of cell was cultured separately, it did not survive under the experimental conditions. Herpes-type viral antigen and C-group chromosomal marker previously described in cultured Burkitt cells were found in all of the female cell cultures that were obtained.

Complement-fixation tests with cell lines derived from Burkitt's lymphoma and acute leukemias.

Armstrong, Donald (Children's Hospital of Philadelphia, Philadelphia, Pa.), Gertrude Henle, and Werner Henle. Complement-fixation tests with cell lines derived from Burkitt's lymphoma and acute leukemias. J. Bacteriol. 91:1257-1262. 1966.-Cells of various lines isolated from Burkitt's lymphomas and acute leukemias and disintegrated by freezing and thawing or sonic treatment were found to react in complement-fixation tests with a considerable proportion of human sera. At least 10(7) cells per milliliter were required for antigenic activity. All but one of 13 sera from Burkitt lymphoma patients were positive, with titers ranging from 1:8 to 1:320. About 20% of sera from American children and 60% of sera from adults, regardless of diagnosis, showed titers in a similar range. Sera giving positive tests with one of the neoplastic white cell antigens usually reacted also with many if not all of the others, but rarely with antigens derived from normal peripheral leukocyte cultures and not at all with HeLa or other human nonleukocytic cells. Various observations indicate that the complement-fixation test measures mainly antigens which are different from those detected by immunofluorescence. The nature of the reactions described remains obscure.

The use of isoimmune sera for identification of hemopoietic cells from blood-injected radiation chimeras.

Use has been made of the antigenic differences among mice with respect to both erythrocytes and nucleated cells to identify hemopoietic cells of radiation chimeras. By employing isoimmune sera in agglutination and cytotoxic techniques, it has been shown that long-term leukemoid blood chimeras contain lymphocytes, erythrocytes, and nucleated bone marrow cells of the blood-donor type; blood from normal mice can repopulate lymphoid tissues of radiationbone marrow chimeras, and the blood-derived cells might take part in graft-versus-host immune reactions; and normal peripheral leukocytes from F/sub 1/ hybrid donors can reestablish lymphopoiesis, granulopoiesis, and erythropoiesis when injected into lethally irradiated parent strain mice. Cytotoxic typing of the nucleated hemopoietic cells shows that they are of donor origin. (auth)