Osteopetrotic (op/op) mutant mice suffer from congenital osteopetrosis due to a severe deficiency of osteoclasts. Furthermore, the total number of mononuclear phagocytes is extremely low in affected ...mice. Serum, 11 tissues, and different cell and organ conditioned media from op/op mice were shown to be devoid of biologically active colony-stimulating factor 1 (CSF-1), whereas all of these preparations from littermate control +/+ and +/op mice contained the growth factor. The deficiency was specific for CSF-1 in that serum or conditioned media from op/op mice possessed elevated levels of at least three other macrophage growth factors. Partial correction of the op/op defect was observed following intraperitoneal implantation of diffusion chambers containing L929 cells, which in culture produce CSF-1 as their sole macrophage growth factor. No rearrangement of the CSF-1 gene in op/op mice was detected by Southern analysis. However, in contrast to control lung fibroblasts, which contained 4.6- and 2.3-kilobase CSF-1 mRNAs, only the 4.6-kilobase species was detected in op/op cells. An alteration in the CSF-1 gene is strongly implicated as the primary defect in op/op mice because they do not contain detectable CSF-1, their defect is correctable by administration of CSF-1, the op locus and the CSF-1 gene map within the same region of mouse chromosome 3, their CSF-1 mRNA biosynthesis is altered, and the op/op phenotype is consistent with the phenotype expected in a CSF-1 deficient mouse.
Background. Treatment with interleukin‐2 (IL‐2) and lymphokine‐activated killer cells (LAK) resulted in responses in some patients with advanced renal cell carcinoma (RCC). However, the relative ...therapeutic benefit of the addition of LAK to IL‐2 was unknown.
Methods. A randomized Phase III trial was conducted in patients with RCC comparing continuous intravenous infusion (CI) IL‐2 alone with CI IL‐2 plus LAK. Interleukin‐2 was administered at 3 × 106 U/m2/day on days 1–5, 13–17, 21–24, and 28–31. Patients on the LAK treatment arm underwent leukapheresis on days 8–10 and LAK cell reinfusion on days 13–15. The results are reported with long‐term follow‐up. The published experience with IL‐2 alone or with the addition of LAK was investigated in a quantitative literature survey. The response proportions were studied by schedule (high dose bolus, moderate dose, low dose) and by concomitant administration of LAK.
Results. Seventy‐one patients were treated, 36 on the IL‐2 arm and 35 on the IL‐2 plus LAK arm. Four patients (6%) had major responses (two complete, two partial). The median survival of all patients was 13 months (95% confidence interval CI, 9–18 months). There were no differences between treatment arms with regard to response (P=0.61) and survival (P=0.67). More patients on the LAK arm experienced pulmonary toxicity (P=0.008).
The overall weighted response proportion was 16% (95% CI, 8%‐24%) for the 39 published series of 1291 patients treated with IL‐2. The 95% confidence intervals for response proportion overlapped when compared by schedule and by administration of LAK.
Conclusions. The dose and schedule of IL‐2 used in this study resulted in a low level of antitumor activity and the addition of LAK did not improve the response rate against RCC. Given the infrequent, but reproducible, responses with IL‐2 and interferon‐based regimens, continued investigation of these agents is warranted as is the study of new cytokines. Alternative treatment strategies should be studied in RCC and new agents and treatment regimens that appear promising in Phase II studies must be studied in randomized tria.
Modification of tumor cells with one or more costimulatory adhesion molecules has been proposed as a means to develop therapeutic cancer vaccines for use in human immunotherapy. Expression of B7-1 ...(CD80) in tumors by gene transfer creates an immunogenic tumor cell that induces antitumor immunity and protects mice from further challenge with wild-type tumor cells. In this report, we demonstrate that protein transfer of glycosyl-phosphatidylinositol (GPI)-anchored costimulatory molecules into tumor cell membranes could be used as an alternative to gene transfer for tumor immunotherapy. Incubation of isolated tumor membranes with purified GPI-anchored B7-1 results in stable incorporation of B7-1 on tumor cell membranes within a few hours. Immunization of C57BL/6 mice with EG7 tumor membranes modified to express GPI-B7-1 by protein transfer induces tumor-specific T-cell proliferation and CTLs. In addition, immunization with these EG7 membranes protects mice from parental tumor challenge. The protein transfer approach used here does not require foreign vectors or live tumor cells and is completed within a matter of hours. Irradiated cells or membrane preparations from fresh or frozen tumor tissue can be used. Therefore, protein transfer of glycolipid-anchored molecules provides an efficient and novel approach to modify tumor membranes for human immunotherapy. This approach is not limited to costimulatory molecules because any cell surface protein can be converted to a GPI-anchored form by recombinant techniques.
To generate a potent cell-mediated immune response, at least two signals are required by T cells. One is engagement of the T-cell receptor with peptide-bearing major histocompatibility complex ...molecules. The other signal can be delivered by various molecules on the antigen-presenting cell, such as B7-1 (CD80). Many tumor cells escape immune recognition by failing to express these costimulatory molecules. Transfection of the B7 gene into some murine tumor cells allows for immune recognition and subsequent rejection of the parental tumor. We have studied an alternative approach for the introduction of B7-1 onto the surface of tumor cells. This method involves purified glycosyl-phosphatidylinositol (GPI)-anchored proteins which can spontaneously incorporate their lipid tail into cell membranes. We have created and purified a GPI-anchored B7-1 molecule (called GPI-B7) which is able to bind its cognate ligand, CD28, and incorporate itself into tumor cell membranes after a short incubation. Tumor cells that have been reconstituted with GPI-B7 can provide the costimulatory signal needed to stimulate T cells. These findings suggest an approach for the introduction of new proteins onto cell membranes to create an effective tumor vaccine for potential use in human immunotherapy.
Mice that are mutant at the op locus have a severe deficiency of mononuclear phagocytes due to an inactivating mutation in the CSF-1 (macrophage colony-stimulating factor, M-CSF) gene. op/op mice are ...toothless, possessing skeletal abnormalities, a low body weight, and compromised fertility; they are osteopetrotic due to a deficiency of osteoclasts. The congenital osteopetrosis, toothless phenotype, osteoclast deficit, and the defects in splenic and femoral macrophages were corrected by routes of administration of human recombinant CSF-1 that maintained normal circulating CSF-1 concentrations. Early restoration of circulating CSF-1 was required for rescue of the toothless phenotype, but only partially restored body weight. In contrast, the deficiencies of pleural and peritoneal cavity macrophages and the reduced female fertility were not corrected by restoration of circulating CSF-1. These results suggest that although circulating CSF-1 is required for osteoclast and macrophage production, local synthesis and action of the growth factor are important for certain target cell populations.
SF were prepared, expanded, and characterised as described previously. 4 To examine the IL16 encoding transcript amounts, SF were incubated in complete medium for 24 or 48 hours in the presence of ...one of the following chemicals: 1 ng/ml phorbol-12-myristate-13-acetate (PMA), an activator of protein kinase C (PKC); 200 ng/ml ionomycin (Iono), a calcium ionophor; 10 μM of adenosine-3',5'-cyclic monophosphate (cAMP), which stimulates protein kinase A (PKA); 10 nM okadaic acid (Oka), a phosphatase inhibitor; 10 μM MAS-7, which activates G-proteins; 100 μM H-7 dihydrochloride (H-7), an inhibitor of protein kinases; and 10 nM staurosporine (Stauro), a protein kinase inhibitor (all from Calbiochem or Biomol). RA SF are resistant to induction apoptosis by overexpression of sentrin, Bcl-2, and mutant forms of p53. 6- 9 Therefore the RA SF, especially, may be able to respond to a staurosporine induced pathway with enhanced IL16 transcript amounts. Because protein kinase C activator PMA reduced IL16 transcripts in SF, the data suggest that in SF the transcription of IL16 might be regulated through protein kinase C dependent pathways.
Osteopetrosis and the absence of colony-stimulating factor 1 (CSF-1) in op/op mice are associated with decreased cellularity of the bone marrow (to one tenth of the normal), a very significant ...reduction in the number of cells recovered from peritoneal, pleural, and alveolar lavages, moderate leukopenia, and a slight decrease in the number of cells per spleen and thymus. Furthermore, op/op mice possess deficiencies in the number of macrophages in various organs. These cells are apparently absent in the bone marrow, severely reduced (5%-15% of the normal number) in peritoneal and pleural cavities and in the lungs. In addition, a marked decrease in the frequency and total number of circulating monocytes is present (5% of the normal). The deficiency of macrophages is less severe in the liver, spleen, and thymus of op/op mice (approximately 30% of those seen in normal). There is a concomitant redistribution of macrophage progenitor cells (granulocyte-macrophage colony-forming units, CFU-GM) in op/op mice from the marrow to the spleen and liver, associated with an increased sensitivity to interleukin 3 (IL-3). Their total number is decreased at least threefold compared to control mice. Moreover, op/op mice have at least a fivefold reduction in the total number of day-11 spleen colony-forming units (CFU-S) associated with their redistribution to the spleen and liver. These data suggest that the macrophage system in op/op mice is reduced at all levels tested, that is, at the level of mature macrophages, the level of progenitors, and the level of stem cells, whereas the redistribution of progenitor and stem cells could be viewed as a secondary consequence of osteopetrosis. Furthermore, these data suggest that macrophage dependency in vivo on CSF-1 is limited and different in various organs. Particularly in the liver, spleen, and thymus, other growth factors may significantly compensate for CSF-1 deficiency. Based on the relative decrease in the number of CFU-GM in the op/op mice, it appears that the population size of these progenitors is less dependent on CSF-1 than the hematopoietic stem cell population size as evidenced by the day-11 CFU-S assay. The day-11 CFU-S population is severely reduced in op/op mice, suggesting a physiological involvement of CSF-1 in expanding its size. These data provide evidence that CSF-1, besides acting on the final and intermediate stages of macrophage maturation, may also play a role in early stages of hematopoiesis.
The histologic diagnosis of active myocarditis is frequently difficult to establish. A nonhistologic marker of immune activation would be clinically useful in identifying cases of immune-mediated ...myocarditis. A viral etiology with subsequent autoimmunity to cardiac antigens has been implicated in human myocarditis. Because autoimmunity and viral disease are commonly associated with increased expression of major histocompatibility complex (MHC) antigens on targeted tissue, we examined endomyocardial biopsy samples from patients with active myocarditis for abnormal levels of MHC antigen expression. Thirteen patients with active myocarditis and eight control patients with other well-defined cardiac diagnoses (coronary disease, amyloidosis or neoplasm) were studied.
A sensitive radioimmunoassay was developed that utilized monoclonal antibodies to human MHC class I and class II antigens in order to quantitate the expression of both of these antigens within each biopsy. Abnormal MHC class I and class II antigen expression was present in 11 of 13 myocarditis specimens and 1 of 8 control samples (specificity 88%, sensitivity 84.6%). Active myocarditis samples had approximately a 10-fold increase in MHC class I and class II expression. Immunoperoxidase staining localized abnormal MHC expression primarily within microvascular endothelium and along myocyte surfaces (11 of 13).
This study is the first to demonstrate a marked increase in major histocompatibility complex antigen expression within the myocardium of patients with active myocarditis. The identification of abnormal histocompatibility antigen expression within an endomyocardial biopsy may prove a useful adjunct to the histologic diagnosis of myocarditis.
Normal human peripheral blood lymphocytes were tested for their susceptibility to infection with retroviruses isolated from patients with the acquired immunodeficiency syndrome (AIDS) or AIDS-related ...complex. Of 10 normal individuals tested, lymphocytes from all subjects became infected and produced virus as detected by assay for Mg+2-dependent reverse transcriptase. Lymphocytes from different individuals were demonstrated to be either high or low producers of reverse transcriptase after infection. The kinetics of virus production were similar in cells from both high- and low-producing individuals. A significant correlation was observed between high and low viral-producing lymphocytes and expression of the Leu-3/T4 (CD4) surface molecule. Mitogen-stimulated peripheral blood lymphocytes exposed to HTLV-III/LAV manifested productive viral infection, as reflected by the appearance of early syncytia, followed by reverse transcriptase. Unstimulated peripheral blood lymphocyte cultures displayed late syncytia but no detectable reverse transcriptase upon exposure to virus. The addition of anti-human interferon-alpha did not appear to have an appreciable effect on viral production in normal peripheral blood lymphocytes exposed to the virus.
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