Lymphocyte subsets in relapsing-remitting multiple sclerosis: a longitudinal study of B lymphocytes and T lymphocytes.

Changes in lymphocyte subset populations may provide clues to the dysimmune mechanisms involved in relapsing remitting multiple sclerosis (RRMS). The lymphocyte subgroup CD4+ CD45RA+, thought to be responsible for the induction of suppression is decreased in some patients with MS compared to controls. A possible role for another lymphocyte subset, CD19+CD5+ lymphocytes, has been proposed in autoimmune diseases and multiple sclerosis (MS). To expand this we studied CD4+CD45RA+ (T) lymphocytes and CD19+CD5+ (B) lymphocytes in nine patients with relapsing-remitting MS (RRMS) and nine controls. The patients were examined monthly for an average of ten months and nine relapses were observed in seven patients. One patient underwent monthly gadolinium enhanced magnetic resonance imaging (MRI). Normal percentages CD4+CD45RA+ lymphocytes were found in patients with RRMS. No significant abnormalities in the CD19+CD5+ lymphocyte subpopulation were noted, although a tendency for higher percentages of this subset (approaching statistical significance, P = 0.056) was detected.

In vitro enhancement of natural killer cell activity against herpesvirus-infected targets in patients with acute lymphocytic leukemia.

The present study was carried out to determine whether natural killer (NK) activity against virus-infected target cells could be enhanced in peripheral blood lymphocytes (PBL) taken from patients with acute lymphocytic leukemia. Nonspecific cell-mediated killing of heterologous herpesvirus-infected B lymphoblastoid targets could be enhanced by pretreating PBL with interferons-alpha or -gamma, or with interleukin-2. NK activity was substantially enhanced over that of untreated cells, and virtually all cytolytic activity was attributable to CD16+ NK cells. Pretreatment of PBL with a combination of cytokines resulted in additive, but never synergistic enhancement of NK activity. The results suggest that cytokine therapy may provide a useful means for treating severe varicella zoster infections in immunocompromised children.

Pharmacokinetics of recombinant interleukin-2 in children with malignancies: a Pediatric Oncology Group study.

To develop effective interleukin-2 (IL-2) protocols for pediatric malignancies, it is important to define IL-2 pharmacokinetics in children. In a phase I trial, we studied IL-2 pharmacokinetics in seven children, aged 6-18, five with leukemia, one with neuroblastoma, and one with rhabdomyosarcoma. IL-2 was administered as a 15-min i.v. infusion of either 1 X 10(6) CU/m2/dose or 3 X 10(6) CU/m2/dose (every Monday, Wednesday, and Friday for 3 weeks). IL-2 levels were determined using an IL-2-dependent murine T lymphocyte cell line bioassay. Peak IL-2 levels of 120-426 and 330-740 CU/ml were achieved after the lower and higher doses, respectively. Pediatric IL-2 kinetics resembled data reported for adults, fitting a two-compartment model (least-squares-regression technique), with an alpha half-life of 14.0 +/- 5.6 min (range, 6.3-23.1) and a beta half-life of 51.4 +/- 10.7 min (range, 33.0-66.0). The volume of distribution approximated total extracellular fluid (mean, 0.18 L/kg). Further clinical trials are needed to identify which pediatric malignancies are sensitive to immunotherapy and to establish the optimal treatment regimens.

A phase I study of interleukin-2 in children with cancer and evaluation of clinical and immunologic status during therapy. A Pediatric Oncology Group Study.

The authors performed a Phase I study to assess the toxicity and hematologic effect of recombinant human interleukin-2 (rIL-2) in seven children with advanced malignancies. The rIL-2 was given as a bolus injection of 1 or 3 X 10(6) U/m2/dose three times a week (Monday, Wednesday, and Friday) for 3 weeks. No life-threatening toxicity occurred with the dose of 1 X 10(6) U/m2 of rIL-2. At a dose of 3 X 10(6) U/m2, therapy had to be terminated due to cardiovascular toxicity in two patients. Toxic effects at low-dose rIL-2 included fever, nausea, vomiting, and mild hypotension. High-dose rIL-2 toxicity included fluid retention, increased creatinine, oliguria, elevated liver enzymes, and significant hypotension. Immunologic studies showed that rIL-2 caused a drop in the number of circulating peripheral blood mononuclear cells, T-cells, and natural killer cells which returned to pretherapy levels or above by 24 to 48 hours. The rIL-2 exerted no growth or stimulatory activity on the leukemic cell population. To the authors' knowledge, this is the first report of a Phase I study of IL-2 therapy in children.

Production and characterization of monoclonal antibodies to human myeloperoxidase.

Monoclonal antibodies (mAbs) against human myeloperoxidase (MPO) have been derived for immunopurifying MPO and immunophenotyping acute leukemias. Eight antibodies were obtained from a fusion of the P3 plasmacytoma cell line with splenic lymphocytes from mice immunized with purified human MPO. Hybridoma supernatant culture fluids were screened for antibody to MPO by an enzyme-linked immunosorbent assay. The specificity of the mAbs was characterized by immunoblotting, immunoprecipitation, and reactivity against various hematopoietic cells, cell lines, and leukemic blast cells. The eight mAbs generated were of the IgG1 isotype. One of these mAbs was successfully used to develop a one-step immunoaffinity chromatographic purification procedure for MPO. Immunoblotting and immunoprecipitation experiments suggested that the eight mAbs were reactive with at least three antigenic determinants on the MPO molecule. Immunofluorescent studies showed that these mAbs reacted specifically with cells of granulocytic-monocytic lineage and were negative with lymphoid cells. Our results suggest that these mAbs should be useful reagents for immunophenotyping hematopoietic cells.

Effect of retinoic acid on myeloid antigen expression and clonal growth of leukemic cells from children with acute non lymphocytic leukemia--a Pediatric Oncology Group Study.

We have used a panel of 5 monoclonal antibodies against normal myeloid-differentiation antigens to determine retinoic acid-induced changes in cell surface antigens on ANLL bone marrow cells from 24 children at the time of diagnosis. Two of these antibodies (T5A7 and 5F1) detect antigens expressed on normal mature granulocytes and on all monocytes, respectively. The percentage of positive cells for each monoclonal antibody was determined by indirect immunofluorescence. After 5 days incubation with 1 microM RA in liquid culture, cells from 11 of 24 patients showed substantially increased expression of one or both antigens detected by T5A7 and 5F1. Leukemic bone marrow cells from these patients were also cultured in methylcellulose medium with and without 1 microM RA for one week, and cells from 16 of 24 patients showed clonal growth. Cultures from 10 of these 16 patients showed RA-induced inhibition of colony growth; of these 10 patients, cultures from six patients showed RA-induced increases in antigens associated with maturing myeloid cells. This suggests that the RA-induced inhibition of clonal growth observed with leukemic cells from these patients may be accompanied by the increased expression of maturation-associated myeloid antigens by these cells in the presence of RA.

Antibody response to a solubilized tumor-associated membrane antigen (TAMA) from the murine Lewis lung tumor.

A tumor-associated membrane antigen (TAMA) has been solubilized from the C57B1/6 murine Lewis lung tumor using the detergent Triton X100. Xenoantiserum prepared in rabbits was used to identify this tumor antigen in Triton extracts by double immunodiffusion in agar. The antigen was partially purified using a combination of DEAE and Sephadex G200 chromatography followed by sucrose gradient isoelectric focusing. Isoelectric focusing also showed the antigen to have a pI at pH 4.7. Sodium dodecylsulfate polyacrylamide gel electrophoresis indicated the antigen to be of 37,000 molecular weight. The 125I protein A antibody-binding assay was used to further characterize the specificity of the antigen using rabbit antisera to the Triton extract. The same assay system also detected a specific antitumor humoral response in syngeneic mice immunized with the Triton extract.

Protein-A antibody-binding TPAAB) technique for detection of tumor-associated membrane antigen (TAMA) of Lewis lung carcinoma.

Xenoantisera were prepared in rabbits against intact paraformaldehyde-fixed Lewis lung carcinoma (LLCa) cells and membrane solubilized proteins extracted with Triton X-100 and 3M KCI. The antisera were analyzed for antibodies to tumor-associated membrane antigens (TAMA) by means of a solid-phase radioimmunoassay (RIA). THe immunoassay employed 125-labelled Staphylococcus aureus protein A (SPA) to detect specific antibody binding to glutaraldehyde-fixed Lewis lung tumor target cells. The protein-A antibody-binding (PAAB) method is rapid, sensitive and reproducible. The PAAB revealed a possible LLCa-TAMA as evidenced by the reactivity of xenoantisera raised to LL paraformaldehyde tumor cell vaccine (TCV) and LL tumor Triton X-100 (TTri) and 3M KCI (TKCI) extracts of membrane preparations. The reactivity of our antisera followed linear dose-response kinetics but the degree of reactivity decreased from anti-TCV to anti-TTri to anti-TKCI respectively. The LL-TAMA demonstrated specificity since our antisera did not significantly cross-react with normal C56BL/6 cellular antigens or other known murine tumor-associated surface antigens of virally and chemically-induced tumor systems.