Soluble interleukin-2 receptor and soluble CD8 antigen in active rheumatoid arthritis.

Concentrations of soluble interleukin-2 receptor (sIL-2R) and of soluble CD8 antigen (sCD8) in sera and in supernatants of phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) derived from patients with active rheumatoid arthritis (RA) were studied. sIL-2R concentrations in sera derived from patients with RA (1484 +/- 382 U/ml) were significantly higher than in sera derived from healthy controls (380 +/- 110 U/ml; P less than 0.0005). In contrast, supernatants of PHA-stimulated PBMC derived from patients with RA contained similar amounts of sIL-2R (727 +/- 467 U/ml) as those derived from healthy control individuals (833 +/- 508 U/ml; P greater than 0.1). When investigated for the presence of sCD8 antigen, sera derived from patients with RA contained significantly lower amounts (30 +/- 28 U/ml) than sera derived from healthy controls (405 +/- 136 U/ml; P less than 0.0005). Similarly, PHA stimulation of PBMC derived from patients with RA resulted in a significantly lower production of sCD8 (35 +/- 46 U/ml) as compared to the one obtained by PHA stimulation of PBMC derived from healthy controls (177 +/- 59 U/ml; P less than 0.0005). This difference could not be explained by a lower proliferative response to PHA by PBMC derived from patients with RA (21,474 +/- 14,022 cpm) as compared to healthy controls (29,549 +/- 11,188 cpm; P greater than 0.05). Our data demonstrate that PBMC derived from patients with active RA differ from PBMC derived from healthy individuals concerning their ability to produce sIL-2R and sCD8.

Effector function of lymphokine-activated killer cells and cytotoxic T lymphocytes in ovarian epithelial carcinoma.

From 20 patients with ovarian carcinoma (4 with stage I and 16 with stage III disease), we obtained peripheral blood mononuclear cells (MNC) from each, tumor-associated MNC from 9, and ascitic MNC from 6. These cells were stimulated with interleukin 2 (IL-2), and radioactive chromium-release cytotoxicity assays were used to evaluate the lymphokine-activated killer (LAK) activity against autologous tumor cells and two natural killer-resistant cell lines. The LAK response was consistently better with control peripheral blood MNC than with patient-derived MNC against all targets. However, within the ascitic MNC population, LAK activity against two cell lines (Daudi and OVCAR) was not statistically different from that of the control MNC. Phenotypic analysis of the ascitic MNC with monoclonal antibodies and flow cytometry revealed an activated population (IL-2 receptor-positive) characterized by predominantly monocytes/macrophages and T cells. In addition, the peripheral blood LAK activity for patients with stage I disease was statistically better against each target than that from patients with stage III disease. These results suggest an immunosuppressive effect directly related to tumor burden. Except perhaps for the ascitic MNC, autologous MNC do not appear to be effective LAK sources. Follow-up of the stage III patients revealed no difference in 2-year survival associated with in vitro LAK response.

Reference values of magnesium and potassium in mononuclear cells and erythrocytes of children.

Reference values for magnesium and potassium contents of mononuclear cells and erythrocytes were estimated in cord blood and in children from infancy through adolescence. No differences were detected between results for boys and girls. The mononuclear magnesium content was independent of age and was within the adult range of values. No significant correlation was shown between magnesium in serum and in mononuclear cells. Mononuclear potassium also showed no age-related differences. The correlation between magnesium and potassium contents in mononuclear cells was significant: however, the correlation was lower when the magnesium and potassium contents were expressed in terms of protein potent: micromoles or millimoles per gram of protein, respectively. The concentration of magnesium in erythrocytes was significantly lower in cord blood and during the first month of life, compared with that at older ages, and showed no significant correlation with serum magnesium. The concentration of erythrocyte potassium was independent of age and showed a low but significant correlation with erythrocyte magnesium content.

T-cell subsets in healthy teenagers: transition to the adult phenotype.

Little is known about the normal range and variability of T-cell subsets in older children. We analyzed peripheral blood mononuclear cell subsets in 112 healthy children, ages 12-19 years (mean +/- SD: 15.4 +/- 1.9 years), using monoclonal antibodies and flow cytometry. The study population included 28 blacks and 84 whites, with 59 boys and 53 girls. The mean +/- SD cell subset values were: CD3+ T cells, 74.0 +/- 7.8%; CD4+ helper-inducer T cells, 46.8 +/- 6.9%; CD8+ suppressor-cytotoxic T cells, 27.3 +/- 5.7%; CD4:CD8 helper:suppressor ratio, 1.81 +/- 0.57; CD16+ natural killer cells, 4.4 +/- 3.1%; CD19+ B cells, 10.0 +/- 5.3%; CD14+ monocytes, 20.0 +/- 6.5%; and HLA-DR cells, 15.4 +/- 4.8%. Overall, boys had a higher proportion of HLA-DR+ cells than girls, attributable to an increase in CD19+ B cells. Blacks tended to have a higher proportion of HLA-DR+ cells than whites, apparently due to an increase in activated T cells. Detailed analysis by age group revealed a striking transition in the pattern of CD4+ and CD8+ cell populations. The CD4:CD8 ratio, higher in boys than girls for ages 12-16, was reversed to the "adult" pattern in 17-19 year olds, with a higher CD4:CD8 ratio in girls. These data provide important baseline values for healthy children and stress the importance of establishing normative ranges for pediatric subjects separately from adults.

Mitogenic effect of alpha 1-microglobulin on mouse lymphocytes. Evidence of T- and B-cell cooperation, B-cell proliferation, and a low-affinity receptor on mononuclear cells.

Human alpha 1-m microglobulin (alpha 1-m), a low molecular weight plasma protein, was found to exert mitogenic effects on mouse lymphocytes from lymph nodes and spleen. The stimulatory effects appeared to be strain-restricted: alpha 1-m induced a varying degree of proliferation of lymphocytes from three strains, whereas one strain responded poorly. Experiments with lymphocyte subpopulations showed only weak stimulatory effects of alpha 1-m on purified T and B lymphocytes cultivated alone. The addition of mitomycin-treated cells of the other subpopulation could not restore the proliferative responses in either T or B lymphocytes. Strong stimulations were recorded only when both T and B lymphocytes were present, indicating that the T and B lymphocytes cooperate to achieve the proliferation. However, FACS studies on cultured splenocytes indicated that the proliferating cells are predominantly B lymphocytes. These data extend our earlier findings of a mitogenic effect of alpha 1-m on guinea pig lymphocytes. Furthermore, results were obtained indicating the presence of a receptor on mononuclear cells. Iodine-labelled alpha 1-m was bound to mononuclear cells prepared from spleens, and the binding could be blocked by an excess of non-labelled alpha 1-m. Scatchard plotting of the data gave an equilibrium constant of 0.7 x 10(5)/M for the binding between alpha 1-m and the receptor. Together with the documented inhibitory activity of alpha 1-m on antigen-driven proliferation of lymphocytes, these results suggest an immunoregulatory role for alpha 1-m.

AIDS-associated progressive multifocal leukoencephalopathy (PML): comparison to non-AIDS PML with in situ hybridization and immunohistochemistry.

We studied brain sections from 10 patients with the acquired immunodeficiency syndrome (AIDS) and progressive multifocal leukoencephalopathy (PML) by in situ hybridization with a biotin-labeled JC virus (JCV) DNA probe and by immunohistochemistry using antibody against the JCV capsid antigen. We compared the results with brain sections studied in the same fashion from 10 PML patients without AIDS. The pathology of JCV infection in AIDS was similar to non-AIDS PML except for minor differences in degree. AIDS-associated pathologic material showed a greater tendency toward necrosis and a higher density of JCV-infected cells. Replication of JCV was restricted to glial cells in all tissue studied. Bizarre astrocytes were less frequent in the AIDS patients, and perivascular inflammatory cells were more frequent. We could not demonstrate JCV in macrophages or microglial cells known to harbor HIV infection. In situ hybridization with nonradioactive probes serves as a useful technique for the confirmation of PML in AIDS.

Emergence and decay of the human Mx homolog in cancer patients during and after interferon-alpha therapy.

The human Mx, an interferon (IFN)-alpha- and IFN-beta-induced 76-kd protein, is a homolog (Mx-homolog) to the murine Mx protein, which is necessary and sufficient to provide adequate resistance against influenza virus in murine cells and in mice. Leukocytes from 36 patients with tumors (chronic myelogenic leukemia, hairy cell leukemia, and malignant melanoma) were monitored for their Mx-homolog content before, during, and after rIFN-alpha-2b therapy. Before therapy, only one patient was slightly positive for Mx-homolog. All 36 patients showed a significant increase of Mx-homolog in their mononuclear cells within the first day of IFN therapy. During therapy, the Mx-homolog levels remained elevated. After cessation of treatment, the Mx-homolog content in the mononuclear cells decreased slowly; within 2 weeks, it was about 20-30% of its value during therapy. However, even after 3 weeks, the Mx-homolog was still detectable. The maximally induced Mx-homolog concentration showed a significant correlation to the IFN dose given in vivo. These data indicate that the Mx-homolog is an excellent marker for monitoring the activity of IFN during IFN therapy. In addition, the in vivo endogenous activation of the IFN system might be detectable by the determination of the Mx-homolog despite the lack of circulating IFN.

Detection of somatotropin receptors on human monocytes.

The expression of somatotropin receptors on human peripheral blood monocytes was investigated. Binding of [125I]somatotropin to human monocytes was found to be specific and saturable. The binding was rapid and time-dependent, and was abolished by pretreatment of monocytes with trypsin. Scatchard plot analysis revealed that each cell bore more than 8 x 10(3) binding sites with an affinity constant of 1 x 10(8) M-1.

Improved detection of the histamine receptor on human peripheral blood mononuclear cells by crosslinking using tritiated as compared with radioiodinated histamine.

In order to detect histamine receptors on the surface of human peripheral blood mononuclear cells, the cells were incubated in the presence of radiolabelled histamine and then the bifunctional crosslinker disuccimidyl suberate was added in various concentrations. They were then solubilized with sodium dodecyl sulphate, boiled, reduced and the lysate separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both 3H and 125I-radiolabelled ligands bound to a 16 kDa band, to be defined although a much clearer and obviously unequivocal signal was obtained with 3H-labelled histamine. This molecule migrated with the same mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 16 kDa subunit which had been purified on a histamine affinity column from Triton X-100 solubilized mononuclear cells, indicating it to be the ligand-binding subunit for the histamine receptor on these cells. For 3H, fluorography with Entensify was required to obtain an autoradiographic signal. Although 3H took much longer to give a signal than 125I, the considerable background, artefacts and heavy lane trailing seen with [125I] histamine were completely abrogated when [3H]histamine was used. In addition, the distinction between specific and nonspecific binding was more clearly seen using [3H]histamine. The modifications reported here which improve signal detection for 3H should encourage the use of tritiated ligands in radioreceptor crosslinking, particularly those of low molecular weight which might otherwise undergo steric modification due to iodination, this having the potential for interfering with receptor ligand binding.

Identification of transfused blood cells in children with severe combined immunodeficiency syndrome by analysis of multiple cell lineages using restriction fragment length polymorphisms.

Four children with severe combined immunodeficiency syndrome (SCID) had evidence of circulating transfused cells detected by restriction fragment length polymorphism (RFLP). In two cases, the source of transfused cells was a single, unirradiated red cell transfusion and the patients developed severe graft-versus-host disease (GVHD). In another instance, engraftment of bone marrow cells, mononuclear cells, and neutrophils was noted 9 weeks following the infusion of 111indium-labeled maternal leukocytes for localization of infection. The patient also developed severe GVHD as a consequence of engraftment of functionally active maternal cells and rejected a paternal T cell-depleted marrow infused at a time when circulating maternal cells were present. In the fourth case, RFLP analysis was performed 2 h following completion of a leukocyte transfusion that had been irradiated with 3000 cGy prior to infusion. Third party mononuclear cells and neutrophils were transiently detected. The third party cells were of no clinical significance. The use of a limited panel of highly informative DNA probes allowed us to identify the affected circulating cell populations and source of foreign leukocytes in children with SCID without requiring a pretransfusion peripheral blood, bone marrow, or fibroblast sample.

Fatty acid composition of phospholipids of plasma and of mononuclear blood cells in children with allergic asthma and the influence of glucocorticoids.

Fatty acid (FA) composition of plasma phospholipids and phospholipids extracted from peripheral mononuclear white blood cells (MNC) was investigated in 11 allergic asthmatic children (age 8.9 +/- 4.6 years), in 10 age-matched non-allergic healthy controls and in 14 allergic and non-allergic children with an acute attack of asthma, who had received prednisolone medication for 2-4 days. In allergic asthmatics eicosapentaenoic acid (20:5n-3) was significantly elevated in both plasma and MNC. The relative amount of 20:5n-3 in MNC as well as in plasma correlated positively with increasing levels of total serum IgE (P less than 0.02). The pattern of the other FAs in plasma and of MNC phospholipids did not differ between allergic asthmatic and non-allergic control children. In children with an acute attack of asthma, who had been treated with glucocorticoids (2 mg prednisolone/kg body weight for 2-4 days), distinct changes of relative FA composition of phospholipids were restricted to plasma, where some very long chain FA (22:4n-6, 22:5n-6) were elevated. No significant changes in FA from MNC phospholipids could be observed after glucocorticoid treatment. These findings may indicate a possible role of 20:5n-3, the precursor of "group 3" eicosanoids, in allergic asthmatic children.

[Glucocorticoid receptors on human peripheral mononuclear and polymorphonuclear leucocytes: changes in patients with yang-deficiency].

It was found that, in former works, the glucocorticoid receptors (GCR) on peripheral mixed leucocytes in patients with Yang-deficiency were decreased. In this work, the mixed leucocytes were further separated into mononuclear (MNL) and polymorphonuclear (PML) leucocytes, and GCR were determined in each part of leucocytes. GCR on MNL and PML in 6 Yang deficient patients were 3473 +/- 413 and 4433 +/- 651 sites/cell respectively, statistically significant from the normal control group (4462 +/- 962 and 5622 +/- 782 sites/cell respectively, P less than 0.05). GCR on MNL, PML and mixed leucocytes in 5 patients were determined simultaneously, and all lowered from the control group. The results were 3369 +/- 370, 4986 +/- 419 and 4524 +/- 852 sites/cell respectively, with the lowest GCR on MNL and highest on PML.

[Effect of quinolones on TNF-alpha production by human monocytes]. / Effet des quinolones sur la production de TNF alpha par les monocytes humains.

Previous studies have shown that in lipopolysaccharide (LPS)--stimulated human monocytes, interleukin-1 (IL-1) production is altered by quinoline derivative antibiotics (quinolones), in a way which depends both on the dose and on the agents used. Given that IL-1 and tumor necrosis factor alpha (TNF) are produced in response to LPS and have some overlapping and synergistic activities, we sought to determine if TNF production was altered under the above-mentioned conditions. We investigated the effects of three quinolones: ciprofloxacin (Cip), pefloxacin (Pef) and ofloxacin (Ofl). These quinolones were found to decrease extracellular TNF production in a dose-dependent manner at concentrations higher than 25 micrograms/ml as previously described by our laboratory with regard to IL-1 production. Moreover, the order of the extracellular decrease in TNF and IL-1 induced by each drug was similar. However, in contrast to IL-1 activity, the quinolones studied also reduced cell-associated TNF. The kinetics of TNF production suggested that the quinolones affected TNF production at a very early step, probably during TNF synthesis rather than during its secretion into the extracellular medium. Furthermore, the quinolone-induced accumulation of intracellular cAMP could explain the extracellular decrease in both IL-1 and TNF production.

High ratio of HTLV-1-infected cells in HTLV-1 associated myelopathy (HAM).

Sixteen patients with HTLV-1 associated myelopathy (HAM) were examined for the presence of HTLV-1 provirus genome by Southern blot analysis of genomic DNA from peripheral blood mononuclear (PBM) cells. Random integration of the provirus was detected in 14 of 16 HAM patients. By contrast, the provirus genome could not be detected in 6 non-HAM HTLV-1 carriers, HAM patients were found to have significantly higher antibody titer to HTLV-1 in the sera compared with carriers. These features of HAM patients, i.e., detectable levels of provirus integration in PBM cells and high antibody titer to HTLV-1 in the sera, were noted in 2 wives of HAM patients with neurological signs and abnormalities. High anti-HTLV-1 antibody titer and detection of the provirus genome by Southern hybridizations may be useful for screening subclinical HAM cases and elucidating pathogenesis.

Pseudohypoaldosteronism in eight families: different forms of inheritance are evidence for various genetic defects.

Pseudohypoaldosteronism is a rare hereditary disorder presenting in early infancy with renal salt loss leading to hyponatremia and hyperkalemia despite high levels of plasma aldosterone. The patients are insensitive to mineralocorticoids; however, sodium supplementation is able to correct electrolyte abnormalities. Absent or greatly diminished type I aldosterone receptors in peripheral mononuclear leucocytes have been recently demonstrated and explain the lack of response to mineralocorticoids. We have studied the mode of inheritance in eight families with a total of nine patients. There was evidence for an autosomal recessive form of inheritance in four families, while the other four families appeared to have an autosomal dominant mode of transmission. In three families the autosomal recessive form was characterized by normal receptor as well as hormone data in both parents, while in one family receptor levels in both parents were greatly reduced, but hormone levels were normal. In the four families with an autosomal dominant mode of transmission there was always one parent with reduced receptor binding in peripheral mononuclear leucocytes and elevated serum hormone levels. These parents were entirely asymptomatic. In an extended family we were able to study an aunt and her newborn daughter, who were both also biochemically affected but clinically asymptomatic. It, therefore, appears that this dual pattern of genetic transmission may indicate differing genetic defects which cause the same clinical picture of pseudohypoaldosteronism.

Type II estrogen binding sites in human peripheral blood mononuclear cells: variations during the menstrual cycle.

We have previously reported that human peripheral blood mononuclear cells (PBMC) contain type II estrogen binding sites (type II EBS). In this study, the fluctuations of type II EBS during the menstrual cycle were analyzed in 6 normally menstruating women. Approximately 3 times higher levels of type II EBS were found in the periovulatory period with respect to both follicular and luteal phases. In postmenopausal women the mean type II EBS levels were similar to those observed in the follicular phase of the cycle. However, in 3 postmenopausal patients a short course of estrogen or tamoxifen resulted in a marked increase of type II EBS levels. Tamoxifen was also found to compete with 17 beta-estradiol for type II EBS in PBMC, although to a lesser extent than diethylstilbestrol.

Cell surface markers, inositol phosphate levels and membrane potential of lymphocytes from young and old human patients.

It is well known that most physiological functions change with aging, including the immune response. Data concerning the aging of lymphocyte subpopulations are conflicting. The antigen density of peripheral blood lymphocytes has been determined by fluorescently tagged OKT-3, OKT-4, OKT-8, OKT-11 and OKM1 monoclonal antibodies in a carefully selected aged (over 87 years) population, and compared to that of young subjects. A substantial difference was found in the percentage distribution of OKT8 and OKM1 subsets. The volume of lymphocytes of the elderly population was significantly less than that of the young. The effect of various monoclonal antibodies on phosphatidylinositol breakdown has also been studied. It was found that only OKT3, acting through the CD3 antigen receptor, was able to induce inositol phosphate formation in both young and elderly, although in the latter population this occurred at a lower level. Because the plasma membrane plays a regulatory role in this process, an important and sensitive functional parameter, the membrane potential, was also monitored and influenced by changing the extracellular K+ concentration. The lymphocytes of the elderly population responded less sensitively to changes in extracellular potassium concentration.

Low intracellular magnesium in patients with acute pancreatitis and hypocalcemia.

To determine the role of magnesium deficiency in the pathogenesis of hypocalcemia in acute pancreatitis, we measured magnesium levels in serum and in peripheral blood mononuclear cells in 29 patients with acute pancreatitis, 14 of whom had hypocalcemia and 15 of whom had normal calcium levels. Only six patients had overt hypomagnesemia (serum magnesium less than 0.70 mmol per liter [1.7 mg per dl]). The mean serum magnesium concentration in hypocalcemic patients was not significantly lower than in normocalcemic patients, but the mononuclear cell magnesium content in hypocalcemic patients with pancreatitis was significantly lower than in normocalcemic patients with pancreatitis (P less than .01). The serum magnesium level did not correlate with that of serum calcium or the mononuclear cell magnesium content, but the latter did significantly correlate with the serum calcium concentration (r = .81, P less than .001). Most patients with hypocalcemia had a low intracellular magnesium content. Three normomagnesemic, hypocalcemic patients with alcoholic pancreatitis also underwent low-dose parenteral magnesium tolerance testing and showed increased retention of the magnesium load. We conclude that patients with acute pancreatitis and hypocalcemia commonly have magnesium deficiency despite normal serum magnesium concentrations. Magnesium deficiency may play a significant role in the pathogenesis of hypocalcemia in patients with acute pancreatitis.