Fraud-and-abuse enforcement in Medicare: finding middle ground.

Medicare fraud and abuse cost billions of dollars each year. Yet Congress is considering legislation to hamper enforcement. Providers' anger over enforcement led to a congressional compromise several years ago to limit excesses. If providers and their advocates were to hobble enforcement, this could provoke a backlash. Instead, the existing compromise should be strengthened to accommodate legitimate provider concerns while allowing enforcement against major fraud and abuse. Government should further confine, structure, and check its discretion in applying the False Claims Act. Enhancing the Health Care Financing Administration's capacity to ensure that contractors pay claims properly would remove additional points of friction.

Weak bases increase surface IgM expression in 70Z/3 B lymphoid cell line without increasing kappa gene expression.

A murine B lymphoid cell line, 70Z/3, has proven to be a useful model to study the developmental transition from membrane immunoglobulin (mIgM)-negative to mIgM-positive B cells. 70Z/3 cells have normal intracellular levels of mu heavy chain but kappa mRNA is transcribed at a very low rate. Because both kappa and mu chains are required for IgM to localize on the external membrane the cells remain essentially mIgM negative. A number of biologic agents can stimulate kappa mRNA transcription leading to increased surface IgM expression. Pharmacologic agents which cause cytoplasmic alkalinization have also been shown to increase the level of surface IgM expression when assessed by FITC-labeled anti-IgM antibodies. This increase has been used to argue that cytoplasmic alkalinization is sufficient to stimulate 70Z/3 differentiation but previous studies did not measure kappa mRNA levels or rate of synthesis. In this study we set out to determine whether the increased FITC staining that follows cytoplasmic alkalinization results from increased surface IgM expression and whether that increase results from activation of kappa mRNA transcription. When cells were treated with methylamine, NH4Cl, or monensin there was transient cytoplasmic alkalinization. The levels of surface IgM expression were measured by flow cytometry of cells stained with FITC-labeled anti-mouse kappa and mu. Using this criterion, a 24-hr treatment with either weak bases or monensin increased the level of surface IgM. The increase in fluorescence was not the result of nonspecific binding or uptake by the cell because there was no increase in fluorescence when methylamine-treated cells were stained with FITC-labeled antibody directed against an antigen not found on these cells. Iodination of surface proteins confirmed that the increase in fluorescence was the result of increased levels of IgM protein on the cell surface. However, exposure of the cells to weak bases or monensin caused no increase in either the steady-state level of kappa light chain mRNA, or in the level of kappa protein. We conclude that the transient alkalinization is not sufficient to induce differentiation of the 70Z/3 cells.

Molecular heterogeneity of interleukin-1 receptors.

Previous studies have shown that binding of IL-1 to its receptor and intracellular processing of the IL-1/IL-1 receptor complex appear to be different in B- and T-cells. The current report summarizes recent studies from our laboratory that show that the murine and human IL-1 receptors present on T-cells and fibroblasts are identical in primary sequence within each species, and highly similar even when the two species are compared. At present no cDNA clones have been isolated for IL-1 receptors present on B-cells. However, a monoclonal antibody raised to the receptor on murine T-lineage cells did not bind to a pre-B-lymphoma cell line that displays IL-1 binding sites, nor would cDNA probes derived from a T-cell IL-1 receptor clone cross hybridize at high stringency to mRNA prepared from these cells. In addition the two receptors differ substantially in size, as determined by affinity crosslinking with radiolabeled IL-1 alpha. Taken together, these observations show that major structural differences exist between the IL-1 receptors on B and T lymphocytes, while the receptors on T-cells and fibroblasts are identical polypeptides. We propose that the T-cell/fibroblast receptor be called IL-1RI and the B-cell type IL-1RII.

Evidence for different interleukin 1 receptors in murine B- and T-cell lines.

Previous studies have shown that binding of interleukin 1 (IL-1) to its receptor and intracellular processing of the IL-1/IL-1 receptor complex appear to be different in B- and T-lymphocyte cell lines. In this study we used a B-lymphoid cell line, 70Z/3, and T-lymphoid cell line, EL-4 6.1 C10, to explore further the differences that exist between IL-1 receptors on cells of B and T lineage. We show that a monoclonal antibody against the IL-1 receptor on EL-4 cells does not bind to the IL-1 receptor on 70Z/3 cells. This finding suggests that there are structural differences in the extracellular domains of the IL-1 receptors on the two cell lines. Furthermore, affinity crosslinking showed that the molecular mass of the IL-1 receptor on EL-4 is 87 kDa, whereas that of 70Z/3 is significantly lower (66 kDa). Activation of phospholipid/Ca2+-dependent protein kinase, protein kinase C, by phorbol 12-myristate 13-acetate (PMA) greatly reduced the number of IL-1 binding sites on 70Z/3. But, in sharp contrast, PMA had no effect on surface IL-1 receptor expression on EL-4 cells despite having an equally potent effect in activating protein kinase C. The different effects of protein kinase C suggest that the cytoplasmic domains of the IL-1 receptors in 70Z/3 and EL-4 may also be different. Lastly, a probe containing the entire coding region of the murine T-cell IL-1 receptor hybridized under high stringency conditions with mRNA from EL-4 cells but not with mRNA from 70Z/3 cells. Taken together, the observations made in this study suggest that major structural differences exist between the IL-1 receptors on B and T lymphocytes.

Properties of interleukin-1 and interferon-gamma receptors in B lymphoid cell line.

We have previously shown that interleukin-1 alpha (IL-1 alpha) and interferon-gamma (IFN-gamma) induce surface IgM expression, stimulate Na+/H+ exchange, and activate protein kinase C in the murine pre-B lymphocyte cell line, 70Z/3. Because the two structurally different lymphokines induce similar effects, in this study we set out to compare the properties of the IL-1 and IFN-gamma surface receptors. In contrast to their similar cellular effects, we found that IL-1 alpha and IFN-gamma receptors have different properties. 70Z/3 have high (100 sites/cell) and low (900 sites/cell) affinity IL-1 receptors with dissociation constants (KD) 6 x 10(-11) and 10(-9) M, respectively. In contrast, IFN-gamma receptors are of one class with a KD of 3 x 10(-10) M and are at a higher number, 8000 sites/cell. After binding to their receptors both IL-1 alpha and IFN-gamma are internalized and intracellularly degraded, but the rate of internalization of IFN-gamma is greater than IL-1 alpha. The effective median concentrations (EC50) of IL-1 alpha- or IFN-gamma-induced surface IgM expression are similar (4-5 x 10(-12) M). However, at this concentration 10-fold more of IFN-gamma than IL-1 alpha molecules are bound per cell. Our studies indicate that structurally different lymphokines can induce similar biological events even though their signaling is mediated by surface receptors whose properties are different.

Interferon-gamma and interleukin-1 alpha induce transient translocation of protein kinase C activity to membranes in a B lymphoid cell line. Evidence for a protein kinase C-independent pathway in lymphokine-induced cytoplasmic alkalinization.

We have previously shown that recombinant murine interferon-gamma, rIFN-gamma, and recombinant human interleukin-1 alpha, rIL-1 alpha, induce differentiation of murine pre-B-like cell line 70Z/3, a finding associated with stimulation of Na+/H+ exchange across the plasma membrane. The present study was designed to test whether the enhanced Na+/H+ exchange is mediated by Ca2+/phospholipid-dependent protein kinase C. The results show that two structurally different peptides, rIFN-gamma and rIL-1 alpha, induce identical patterns of transient translocation of protein kinase C from the cytosol to the membranes. The increase in membrane-associated protein kinase C activity was first detected 20 min after exposure to the lymphokines. This activity peaked at 30 min and was back to baseline by 2 h. At each time point, the increase in membrane-associated protein kinase C activity corresponded to a decrease in the activity of protein kinase C in the cytoplasmic fraction. The total cellular activity (cytosol + membrane) remained the same. Two series of experiments were carried out to test the role of protein kinase C in mediating the lymphokine-stimulated Na+/H+ exchange. In the first, the effects of rIFN-gamma and rIL-1 alpha on cytoplasmic pH were measured in the presence of a protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, H-7. In the second, rIFN-gamma- and rIL-1 alpha-induced cytoplasmic alkalinization was determined in cells containing decreased protein kinase C activity. Under both experimental conditions, lymphokine-induced cytoplasmic alkalinization was not attenuated. These results indicate that, although both rIFN-gamma and rIL-1 alpha cause association of protein kinase C with membranes, activation of protein kinase C is not required for rIFN-gamma or rIL-1 alpha to stimulate Na+/H+ exchange across the plasma membrane.

Recombinant murine interferon-gamma-induced differentiation of pre-B lymphocytes is associated with Na+/H+ exchange-dependent and -independent cytoplasmic alkalinization.

We have previously shown that in a murine pre-B lymphocyte cell line, 70Z/3, interleukin-1-induced IgM expression (differentiation) was associated with Na+/H+-mediated cytoplasmic alkalinization. Because interferon-gamma also induces 70Z/3 differentiation, in this study we examined the effects of recombinant murine interferon-gamma (rIFN-gamma) on cell pH. We found that rIFN-gamma (50 units/ml) induced a sustained increase in cell pH, averaging 0.09 pH units above the base line at 30 min and 0.08 at 4 h. Because rIFN-gamma also induced increases in cell Na+ concentration, the data suggests stimulation of Na+/H+ exchange across the cell membrane. Amiloride inhibited the rIFN-gamma-mediated pH rise by only 31% at 30 min, but at 4 h the inhibition was more complete (89%). In contrast to pHi, amiloride totally blocked the Na+ rise at both 30 min and 4 h. This indicates that at the earlier time point the pH rise had a Na+/H+ exchange-dependent and -independent component while at the later time most of the pH rise was due to the Na+/H+ exchanger. The presence of a Na+ independent amiloride-insensitive component was further confirmed by the 0.05 pH rise induced by rIFN-gamma in Na+-free media. Failure to block the Na+-independent rIFN-gamma-mediated pHi rise by anion exchange inhibitors or removal of Ca2+ indicate that this component was not mediated by Cl-/OH- or Ca2+/H+ exchange. The nature of the Na+-independent cell alkalinization process or its role in rIFN-gamma-mediated 70Z/3 differentiation remains to be determined. Because amiloride did not have an effect on rIFN-gamma-induced surface Ig expression or the rIFN-gamma-mediated increase in new kappa light chain-specific mRNA the results indicate that rIFN-gamma-stimulated Na+/H+ is not required for 70Z/3 differentiation.

A microelectrometric titration method for measurement of total intracellular Cl- concentration.

A microelectrometric titration method is described to measure picomole amounts of Cl- present in solutions at micromolar concentrations. This method was used to measure total intracellular chloride concentration ([Cl-]i) in leukocytes. Through the use of submicroliter samples, [Cl-] can be measured in the range of 5-500 microM. There is no measurable interference from other ions normally present in the cell and no intracellular ion is falsely measured as Cl-. [Cl-]i determined by the conventional coulometric titration and the new microelectrometric titration method was the same. Among the commonly used substituting anions, thiocyanate was the only one falsely measured as Cl- and should be avoided in experiments using this method. Because only picomole amounts of Cl- are required, measurements of intracellular pH and total concentrations of other intracellular ions can be done in the same lysate of as few as 5 X 10(5) cells. This feature should make it possible to study the time course of changes in [Cl-]i together with measurements of other intracellular ions following various physiological or experimental maneuvers.

Recombinant human interleukin 1-stimulated Na+/H+ exchange is not required for differentiation in pre-B lymphocyte cell line, 70Z/3.

Interleukin-1 a polypeptide hormone produced by activated macrophages is a mixture of at least two proteins, interleukin-1 alpha (IL-1 alpha) and interleukin-1 beta (IL-1 beta). We have previously shown that macrophage-derived interleukin-1 induced new kappa light chain synthesis for surface IgM expression in a murine pre-B like cell line 70Z/3, a finding associated with an early amiloride-sensitive rise in the total intracellular sodium concentration. Because IL-1 alpha and IL-1 beta are structurally quite different, in this study their effect on 70Z/3 was examined separately. The results show that both human rIL-1 alpha and rIL-1 beta induce the differentiation of 70Z/3, but a higher concentration of rIL-1 beta compared to rIL-1 alpha is needed for a maximal response. At saturating concentrations, both rIL-1 alpha and rIL-1 beta induce a simultaneous rise in intracellular pH and sodium concentration. Because rIL-1 mediated intracellular alkalinization and sodium rise are amiloride sensitive, they likely occur through stimulation of the Na+/H+ exchanger across the cell membrane. Inhibition of the Na+/H+ antiport with an amiloride analog did not have an effect on rIL-1 induced surface IgM expression or the rIL-1-mediated increase in kappa light chain specific mRNA level. Therefore, these results indicate that an increase in pHi or [Na]i is not required for IL-1 induced 70Z/3 differentiation.

Effect of interleukin-1 on intracellular concentration of sodium, calcium, and potassium in 70Z/3 cells.

The cytokine interleukin-1 (IL-1) has been shown to induce the differentiation of a murine pre-B like cell line 70Z/3. An early intracellular event caused by exposure to IL-1 is an amiloride-sensitive progressive rise in the total concentration of intracellular sodium ([Na]i), caused by influx of Na+ from outside, and a transient fall in total intracellular calcium. The results suggest that IL-1-induced influx of Na+ into 70Z/3 occurs via stimulation of the Na+/H+ antiport. IL-1-induced differentiation is also blocked by amiloride suggesting that stimulation of Na+/H+ exchange may play a role in IL-1-induced differentiation.

Cytokine production and utilization by the motheaten mouse.

Splenocytes from the motheaten mouse, after stimulation with alloantigen, lack the ability to utilize exogenous interleukin 1 (IL 1) or interleukin 2 (IL 2), express receptors for IL 2, or produce (IL 2). However, in contrast to other models of autoimmunity and immunodeficiency, after mitogen stimulation, motheaten splenocytes produced as much IL 1 or IL 2 as their normal littermates. In addition, these splenocytes expressed functional IL 2 receptors in the same quantity as normal littermate or wild-type splenocytes. Furthermore, motheaten thymocytes and splenocytes, like their normal littermates, respond synergistically to IL 1 on co-stimulation with mitogen, suggesting expression of an IL 1 receptor. Thus, motheaten mouse splenocytes are unable to utilize an antigen-delivered signal and convert it into cytokine production or IL 2 receptor expression. If the antigen signal is bypassed with mitogen, cytokine production and receptor expression appear normal.

Gene(s) affecting the expression of Qa-1.

Lymphocytes from different strains vary in their expression of antigenic determinants encoded by the Qa1 locus. Thus, a lower percentage of cells from strains bearing H-2Dk are lysed in antibody-mediated cytotoxic tests. These cells fail to completely absorb anti-Qa1 activity from antisera. The unexpressed determinants are present in unactivated cells when subcellular fractions are tested and are detectable on the membrane of mitogen-activated lymphocytes. The gene(s) controlling this phenomenon are dominant and map to a region between H-2S and Tla.

Recognition of alternate alleles and mapping of the Qa1 locus.

Cytotoxicity testing of a new antiserum, B10.A anti-A. TIab, indicates that we have identified products of the Qa1b locus. The strain distribution is antithetical to Qa1a except for strain B10.M, which is reactive with both anti-Qa1a and anti-Qa1b sera, and defines a third allele, Qa1d. Two new recombinants that separate Qa1 and TIa have been established and indicate that Qa1 maps telomeric to TIa. In addition, we have found evidence that other loci, also on chromosome 17, modify the level of detectability of Qa1 antigens by cytotoxic testing.

Suppressor pathways in tumor immunity: a requirement for Qa-1 positive tumor-bearer spleen T cells in suppression of the afferent immune response to tumor antigens.

Spleen cells from mice bearing large methylcholanthrene (MCA)-induced sarcomas were injected intravenously into mice challenged in the hind footpads with heavily-irradiated cells from the same or a different sarcoma. As measured by [3H]-thymidine incorporation on day 5 or 6, cellular proliferation in the draining popliteal lymph nodes of these mice was significantly depressed as compared to control animals receiving normal spleen cells or medium intravenously. The suppression was found to be mediated by a Qa-1-positive, Thy-1 positive cell. It was relatively resistant to cyclophosphamide treatment (100 mg/kg). Furthermore, it had both antigen-specific and non-specific components. The findings are discussed in relation to a suppressor activator cell-suppressor acceptor cell pathway in the immunoregulation of tumor immunity.

The differentiation of cytotoxic T cells in vitro. IV. Interleukin-2 production in primary mixed lymphocyte cultures involves cooperation between Qa-1+ and Qa-1- "helper" T cells.

Studies with C57BL/6-TIaa mice have established that both Qa-1+ and Qa-1- helper T cells are required for the optimal production of Interleukin 2(IL-2) activity in primary MLC. This was established both by depletion of Qa-1 bearing cells by treatment of responder cells with anti-Qa-1 serum in the presence of complement and by positive selection (using FACS II analysis) of those cells displaying Qa-1. Furthermore, microfluoremetry revealed that the great majority of C57BL/6-TIaa and of A/J splenic T cells bore Qa-1 alloantigen but that only those with the highest antigen density were susceptible to complement-mediated lysis.

The Qa-1 antigenic system. Relation of Qa-1 phenotypes to lymphocyte sets, mitogen responses, and immune functions.

The antiserum (B6 X A-Tlab) anti-A (Tlaa) defines several TL antigens expressed exclusively on thymocytes. When reacted with peripheral lymphocytes, the same antiserum defines another antigenic system, provisionally termed Qa-1. The genotypic disparity distinguishing the recipients and donors in this immunization comprises a section of chromosome 17 extending from a crossover point between H-2D and Tla to a presently unmarked point beyond Tla. Therefore although Qa-1 may constitute a single cell surface component, it is equally probable that the Qa-1 system defines two or more cell surface components determined by genes in this region, each of which may be expressed on a different cell set. Cytotoxicity assays indicate that Qa-1 antigen is expressed on Lyt-1 cells and Lyt-123 cells, and may serve to subclassify these two cell sets; it is not known whether Qa-1+ cells may occur within the small Lyt-23 set. There may be also be a cell set with the phenotype Thy-1--:Qa-1+. Another distinctive feature of the Qa-1 system is the characteristic profile of responses to mitogens exhibited by spleen cell populations from which Qa-1+ cells have been eliminated; in conventional assay of [3H]thymidine incorporation the response to lipopolysaccharide was essentially unchanged, the response to phytohemagglutinin M (PHA-M) was virtually abolished, and the response to concanavalin A (Con A) was reduced by 40%. The third distinctive feature of the Qa-1 system is the characteristic profile of changes which elimination of Qa-1+ cells produces in tests of immune function in vitro: (a) proliferation, measured by [3H]thymidine incorporation, in mixed lymphocyte culture (MLC) with major histocompatibility complex (MHC)-incompatible stimulator cells, was not affected. (b) in tests of cell-mediated cytotoxicity (CMC) of MHC-incompatible target cells, neither the generation nor the effector functions of cytotoxic lymphocytes was affected, implying that Lyt-23 prekiller and killer cells are Qa-1--. (c) primary and secondary responses to SRBC were considerably augmented, suggesting that Qa-1+ cells may be responsible for suppression in this test system. (d) accordingly the suppression of the anti-sheep erythrocyte (SRBC) response normally engendered in spleen cells by culture with SRBC was profoundly reduced by elimination of Qa-1+ cells, either before or after culture. (e) the suppression of the anti-SRBC response normally engendered in spleen cells cultured with Con A was reduced by removal of Qa-1+ cells before but not after culture with Con A. Although analysis is as yet far from complete, the Qa-1 system should already be of considerable value because it distinguishes a population of lymphocytes that is not defined by any other antigenic system, according to three criteria: (a) representation of Qa-1 cells among T-cell sets defined by Lyt phenotypes, (b) the profile of responses to mitogens exhibited by lymphocyte populations depleted of Qa-1+ cells, and (c) the profile of immune responses of lymphocyte populations depleted of Qa-1+ cells.

Quantitative isolation of mouse thymus leukemia antigen, TL.

This report describes the purification of TL from papain digests of a tumor line (ASL1) and of an established cell line (L251A). Through the use of gel filtration and ion-exchange chromatography, the TL was purified approximately 100-fold with respect to the original digest. It was noted that the TL isolated from ASL1 had a specific activity 1.4 times higher than that isolated from L251A. The reason for this anomaly is unexplained. However, this work indicates that classical methods of protein chemistry can be used in the purification of these membrane components which are present in only small amounts on the cell surface.