Characterization of the interleukin 2 receptors (IL-2R) expressed on human natural killer cells activated in vivo by IL-2: association of the p64 IL-2R gamma chain with the IL-2R beta chain in functional intermediate-affinity IL-2R.

Interleukin 2 (IL-2) receptors expressed on the surface of activated T cells and natural killer (NK) cells exhibit a variety of affinity states depending on their subunit composition. Low-affinity binding is associated with a 55-kDa alpha chain, intermediate-affinity binding with a 70-75-kD beta chain, and high-affinity binding with a bimolecular complex of the alpha and beta subunits. In a previous study of the IL-2 receptors expressed on NK cells obtained from cancer patients after in vivo IL-2 therapy, we documented a discrepancy between the level of beta chain and the level of intermediate-affinity IL-2 binding sites expressed on the cell surface. Based on this result, we postulated that formation of intermediate-affinity receptor sites required a component in addition to the beta chain, and that this component was present at limiting levels in the patient NK cells. In the present study we have examined the structure of the intermediate-affinity receptor complex using monoclonal antibodies that recognize the beta chain, but that do not interfere with its ability to bind IL-2. Evidence is presented establishing the physical association of a novel protein of 64 kD with the beta chain in intermediate-affinity IL-2 binding sites. This molecule, termed IL-2R gamma chain, coprecipitated with beta chains prepared from cells that had been incubated with IL-2, but was undetectable in immunoprecipitates prepared in the absence of IL-2. Examination of gamma chain expression in post-IL-2 therapy NK cells, where only low levels of intermediate-affinity IL-2 binding were detectable, revealed that the gamma chain was associated with, on average, only 10-12% of the beta chains expressed on such cells. This contrasted with approximately equal levels of beta and gamma chain expression on YT cells, a cell line that has both high levels of cell surface beta chain expression and high levels of IL-2 binding. Thus, the ratio of gamma chain to beta chain present in the immunoprecipitates roughly correlated with the proportion of beta chain involved in intermediate-affinity receptor sites. This result suggests that the 64-kD gamma chain is the component responsible for regulating the affinity of IL-2 association with the beta subunit. By further defining the structural components necessary for IL-2 receptor formation, these studies provide additional insight into mechanisms whereby lymphocytes might regulate their responsiveness to IL-2.

Increased expression of the interleukin 2 (IL-2) receptor beta chain (p70) on CD56+ natural killer cells after in vivo IL-2 therapy: p70 expression does not alone predict the level of intermediate affinity IL-2 binding.

The expression of the 70-kD beta subunit of the interleukin 2 receptor (IL-2R) has been examined on peripheral blood lymphocytes (PBL) obtained from patients receiving systemic infusions of IL-2. Using monoclonal antibodies directed against p70, flow cytometric analyses revealed a greater than threefold increase in expression of the IL-2R beta chain on CD56+ natural killer (NK) cells from post-IL-2 therapy PBL relative to pre-therapy cells. The level of p70 expression on the post-therapy cells was three- to fourfold greater (based on fluorescence intensity) than the level of p70 expression on YT cells, an NK-like cell line that expresses approximately 12,000 intermediate affinity IL-2 binding sites/cell. Despite the high level of p70 expression, in 125I-IL-2 binding assays only 790-1,290 intermediate affinity IL-2 binding sites/cell were detected on post-therapy cells from six patients. These data represent the first report of increased p70 expression after in vivo IL-2 administration and suggest a requirement for at least one additional subunit for the formation of functional intermediate affinity IL-2Rs. Furthermore, the presence on the surface of post-therapy NK cells of excess p70 that does not bind IL-2 with intermediate affinity implies that the formation of intermediate affinity IL-2Rs is not solely determined by the level of p70 expression, and that the response of NK cells to IL-2 might be regulated by altering the expression of p70 or some other IL-2R subunit.

Recognition by human V gamma 9/V delta 2 T cells of a GroEL homolog on Daudi Burkitt's lymphoma cells.

All human gamma delta T cells coexpressing the products of the variable (V) region T cell receptor (TCR) gene segments V gamma 9 and V delta 2 recognize antigens from mycobacterial extracts and Daudi cells. Exogenous and endogenous ligands on the cell surface, homologous to the groEL heat shock family, induced reactivities that resembled superantigen responses in this major subset of human peripheral blood gamma delta T cells. Stimulation of human V gamma 9/V delta 2 T cells is not restricted by human leukocyte antigens (HLA), including nonpolymorphic beta 2-microglobulin (beta 2M)-associated class Ib molecules. These data may be important for understanding the role of gamma delta T cells in autoimmunity and in responses to microorganisms and tumors.

Natural killer cells activated by interleukin 2 treatment in vivo respond to interleukin 2 primarily through the p75 receptor and maintain the p55 (TAC) negative phenotype.

Interleukin 2 (IL-2) induced activation of unstimulated resting natural killer (NK) cells or resting T-cells initially occurs following binding of IL-2 through the p75 receptor that is expressed primarily by these cells. However, this IL-2/p75 interaction induces TAC chain synthesis and formation of high affinity IL-2 receptor required for the proliferation of resting peripheral blood lymphocytes. In this study, we present data indicating that NK cells activated by in vivo IL-2 treatment, in contrast to resting NK cells, respond and proliferate to further IL-2 in vitro using primarily the p75 receptor with only a minor component of cells responding through the high affinity receptor. These in vivo activated NK cells minimally expressed the TAC chain and maintained this TAC negative phenotype while proliferating in response to IL-2. The primary involvement of the p75 receptor in the proliferative response of these cells to IL-2 was demonstrated by the need for concentrations of IL-2 higher than 44 pM to obtain a significant response and by the dramatic inhibition of this response by anti-p75 monoclonal antibody. Anti-TAC monoclonal antibody inhibited only the poor proliferation obtained at low doses of IL-2 suggesting a minor role for TAC and high affinity IL-2 receptors. This was in contrast to the partial inhibition of proliferation by anti-p75 or anti-TAC observed in unstimulated pretherapy peripheral blood lymphocytes suggesting that these cells respond to IL-2 through both high affinity receptors and intermediate affinity p75 receptors. The T-cells isolated from in vivo activated peripheral blood lymphocytes, despite expressing TAC, were not responsive to IL-2, suggesting that these cells express predominantly nonfunctional low affinity TAC receptors. NK cells activated by IL-2 in vivo represent a unique model system of IL-2 dependent cells that respond and proliferate to IL-2 essentially through the p75 IL-2 receptor.

Biological and clinical effects of intravenous tumor necrosis factor-alpha administered three times weekly.

Tumor necrosis factor (TNF) is a cytokine with pleiotropic biological and antitumor effects in vitro and in mouse models. The immunological effects of the molecule as a single agent, however, have not been well studied clinically. We conducted a Phase I trial of TNF in 53 patients with advanced malignancies in order to determine the biological and clinical effects of TNF when administered as a 30-min i.v. infusion three times/week. Dose levels of TNF ranged from 5 to 275 micrograms/m2; doses of TNF were escalated between patient groups. The most common clinical toxicities of TNF consisted of rigors, anorexia, headache, and fatigue. Dose-limiting toxicity consisted of hypotension, fatigue, and nausea. Four patients treated at the maximally tolerated dose of 225 micrograms/m2 received dexamethasone to determine whether the toxicities of TNF could be ameliorated. No significant differences in hypotension or subjective symptomatology were observed in those patients receiving dexamethasone and those who did not or between injections in which dexamethasone was administered and when it was not. One patient with colorectal carcinoma treated with 50 micrograms/m2 had a partial response lasting about 9 months. Biological responses were evaluated in 8 patients treated at the maximally tolerated dose before therapy and 24 h afterward. TNF significantly (P less than 0.05 for all) enhanced serum beta 2-microglobulin, serum neopterin, and serum interleukin-2 receptor (Tac antigen) levels. Indoleamine 2,3-dioxygenase activity was also increased 24 h following the administration of TNF, although this increase was only of borderline statistical significance (P = 0.07). TNF did not enhance granulocyte bactericidal activity. The expression of cell surface proteins on monocytes, including HLA-DR, HLA-DQ, beta 2-microglobulin, and the Fc receptor, and serum interleukin-1 activity also were not significantly increased by the administration of TNF. Thus, in humans TNF caused biological response modulation with evidence of HLA Class I (beta 2-microglobulin) increase and T-cell (Tac antigen) and monocyte (neopterin) activation.

Serum CD25 levels during interleukin-2 therapy: dose dependence and correlations with clinical toxicity and lymphocyte surface sCD25 expression.

Using an enzyme-linked immunosorbent assay (ELISA), we have measured serum levels of a soluble form of the p55 subunit of the interleukin-2 receptor complex, soluble CD25 (sCD25), at regular intervals in the sera of 51 pediatric and adult cancer patients receiving recombinant human interleukin-2 (IL-2). The IL-2 was administered in repetitive weekly cycles alone or in combination with lymphokine-activated killer (LAK) cells. Levels of CD25 correlated with clinical toxicities reflected by nadir blood pressures, percentages of weight gained, and minimum Karnofsky performances during IL-2 therapy. Coadministration of autologous in vitro activated LAK cells together with IL-2 did not significantly affect the pattern of sCD25 release relative to administration of IL-2 alone. Examination of sCD25 release in response to different doses of IL-2 revealed a statistically significant dose effect of IL-2 on the sCD25 levels in patient sera. In addition, the level of sCD25 in patient sera also correlated strongly with expression of CD25 on the surface of peripheral blood lymphocytes (PBL) obtained from patients following IL-2 therapy. These studies demonstrate the utility of the sCD25 ELISA as a clinical tool for monitoring patients on treatment regimens that include IL-2.

Reliable assessment of perfusivity and diffusivity from diffusion imaging of the body.

Diffusion-weighted MRI of the body has the potential to provide important new insights into physiological and microstructural properties. The intra-voxel incoherent motion (IVIM) model relates the observed DW-MRI signal decay to parameters that reflect perfusivity (D*) and its volume fraction (f), and diffusivity (D). However, the commonly used voxel-wise fitting of the IVIM model leads to parameter estimates with poor precision, which has hampered their practical usage. In this work, we increase the estimates' precision by introducing a model of spatial homogeneity, through which we obtain estimates of model parameters for all of the voxels at once, instead of solving for each voxel independently. Furthermore, we introduce an efficient iterative solver which utilizes a model-based bootstrap estimate of the distribution of residuals and a binary graph cut to generate optimal model parameter updates. Simulation experiments show that our approach reduces the relative root mean square error of the estimated parameters by 80% for the D* parameter and by 50% for the f and D parameters. We demonstrated the clinical impact of our model in distinguishing between enhancing and nonenhancing ileum segments in 24 Crohn's disease patients. Our model detected the enhanced segments with 91%/92% sensitivity/specificity which is better than the 81%/85% obtained by the voxel-independent approach.

Quantitative body DW-MRI biomarkers uncertainty estimation using unscented wild-bootstrap.

We present a new method for the uncertainty estimation of diffusion parameters for quantitative body DW-MRI assessment. Diffusion parameters uncertainty estimation from DW-MRI is necessary for clinical applications that use these parameters to assess pathology. However, uncertainty estimation using traditional techniques requires repeated acquisitions, which is undesirable in routine clinical use. Model-based bootstrap techniques, for example, assume an underlying linear model for residuals rescaling and cannot be utilized directly for body diffusion parameters uncertainty estimation due to the non-linearity of the body diffusion model. To offset this limitation, our method uses the Unscented transform to compute the residuals rescaling parameters from the non-linear body diffusion model, and then applies the wild-bootstrap method to infer the body diffusion parameters uncertainty. Validation through phantom and human subject experiments shows that our method identify the regions with higher uncertainty in body DWI-MRI model parameters correctly with realtive error of -36% in the uncertainty values.

3D multidetector CT angiographic evaluation of extralobar pulmonary sequestration with anomalous venous drainage into the left internal mammary vein in a paediatric patient.

Pulmonary sequestration is a congenital lung malformation, defined by dysplastic and non-functioning lung tissue lacking normal tracheobronchial connections and accompanied by an anomalous systemic blood supply. Recognition of anomalous arteries and veins in pulmonary sequestration is paramount to making the correct diagnosis. In contrast to intralobar pulmonary sequestration, where anomalous venous drainage is usually into the pulmonary venous system, the pattern of anomalous venous drainage is more varied in extralobar pulmonary sequestration. To the best of our knowledge, anomalous venous drainage to the internal mammary vein in extralobar sequestrations has not been reported. We report an anomalous venous drainage into the internal mammary vein in an extralobar sequestration which was evaluated with 3D multidetector CT angiography.

Gene therapy: a primer for radiologists.

Gene therapy is one of the most rapidly evolving areas in medicine. Radiologists should have an understanding of basic techniques used to identify and clone a gene and insert it into a vector capable of directing expression in mammalian tissues. DNA delivery systems include retroviral vectors (RNA viruses), adenoviral vectors (DNA viruses), and cationic liposomes, along with strategies that involve ultrasound-directed gene transfer, computed tomography-guided gene transfer, and transcatheter gene delivery, in particular via the hepatic artery. Genes being evaluated in preclinical and clinical trials include oncogenes, antioncogenes (tumor suppressor genes), suicide genes, conventional antimetabolites, antiangiogenesis factors, secreted immunostimulatory cytokines such as interleukins and interferons, and immunomodulatory cell surface proteins, including foreign HLA proteins and costimulatory molecules. A foundation in molecular biology is needed for the practicing radiologist interested in but unfamiliar with current gene therapy terminology and experimental strategies. Such a foundation will encourage the dissemination of basic biologic, diagnostic imaging, and interventional oncoradiologic developments and should facilitate integration of the radiologist into the gene therapy team.

Solitary osteosclerotic plasmacytoma: association with demyelinating polyneuropathy and amyloid deposition.

A 51-year-old man presented with a 1-year history of polyneuropathy necessitating the use of a wheelchair. Initial diagnosis was idiopathic chronic inflammatory demyelinating polyneuropathy (CIDP) and associated monoclonal gammopathy. Investigations for multiple myeloma, including bone marrow aspiration and biopsy, were negative. What was initially felt to be an incidental osteosclerotic focus noted on the radiographic bone survey was eventually shown to be a solitary osteosclereotic plasmacytoma with associated amyloid. This dramatically altered treatment. This case emphasizes the importance of including osteosclerotic plasmacytoma in the differential diagnosis of a focal sclerotic bone lesion in the clinical setting of polyneuropathy. These lesions are less likely to progress to multiple myeloma than lytic plasma cell neoplasms, and the presence of polyneuropathy often results in earlier diagnosis and treatment with enhanced prospect of cure. The finding of amyloid deposition within the osteosclerotic lesion may be of prognostic importance.

Severe combined immunodeficiency, interleukin-2 (IL-2), and the IL-2 receptor: experiments of nature continue to point the way.

The recent discovery of molecular defects in three forms of X-linked immunodeficiency has quickly transformed the study of immunodeficiency into one of the most exciting in basic and clinical immunology. The identification of defects in the IL-2R gamma chain in the etiology of X-linked SCID has suggested a heretofore unanticipated functional role of the gamma chain in immunologic development. While new and novel cytokines and cytokine receptors continue to be identified, it has become clear that our knowledge of IL-2, one of the best understood cytokine/receptor systems, is far from complete. Clarifying the molecular interactions between IL-2 and its receptor complex will improve the sophistication with which these interactions are manipulated in the clinic for the treatment of autoimmune disorders and allograft rejection, treatment of lymphoid malignancies, and cytokine-based therapies for immunotherapeutic treatment of nonlymphoid cancers. Recent gene therapy approaches to the treatment of children with the ADA-deficient form of SCID offers yet another exciting path for investigation. The use of retrovirally infected cord blood hematopoietic progenitor cells in attempts to reconstitute the immune system of ADA-deficient SCID children with ADA-producing cells raises the possibility of similarly "correcting" the defect in X-linked SCID. Such approaches almost certainly loom on the near horizon for other diseases. However, in view of the complexity and potentially pleiomorphic nature of defects in the IL-2R gamma chain, both in terms of their identification and correction, gene therapy for treatment of X-linked SCID will require a thorough understanding of the molecular nature of the respective defects. Effective therapy will require precise knowledge of the defects, in terms of their influence on the ligand, receptor, and signaling apparatus, as well as their potential effects on cells of multiple lineages. However, these caveats aside, the potential for understanding and correcting a disease that robs infants at so early an age of the potential for a normal life will continue to make these exciting and extraordinarily rewarding pursuits.

Chronic inflammatory pseudotumor arising in the hepatobiliary-pancreatic system: progressive multisystemic organ involvement in four patients.

OBJECTIVE: Inflammatory pseudotumor is a benign process that can involve most organ systems. The purpose of this report was to evaluate the imaging features of multifocal chronic inflammatory pseudotumor in four patients who presented with clinical, radiographic, and biopsy evidence of pancreatic or biliary malignancy. CONCLUSION: To our knowledge, this is the first report in the radiology literature describing the imaging features of progressive multifocal inflammatory pseudotumor originating from a primary pancreatic or biliary focus. Even on retrospective review, no distinguishing imaging features were identified that could discriminate benign from malignant disease. These findings emphasize the importance of histopathologic analysis in the diagnosis of malignancy, particularly in patients with previously diagnosed inflammatory pseudotumor.

Effects of dietary aroclor 1254 and cyclopropene fatty acids on hepatic enzymes in rainbow trout.

Diets containing Aroclor 1254 (PCB); cyclopropene fatty acids (CPFA), and PCB plus CPFA were fed to rainbow trout (Salmo gairdneri) for 15 weeks to determine the effects on hepatic microsomal activities of ethoxyresorufin-O-deethylase, ethoxycoumarin-O-deethylase, and benzo(a)pyrene monooxygenase. Ethoxyresorufin-O-deethylase activity continued to increase to a level 77-fold higher than control at week 15. Ethoxycoumarin O-deethylase and benzo(a)pyrene monooxygenase activities increased to 7.1-fold and 47-fold over control at week 9, respectively. Cytochrome P-450 values remained approximately 2-fold above controls from week 5 through week 15. At weeks 1 and 3, cytochrome P-450 levels were not significantly different from control. Dietary CPFA significantly depressed ethoresorufin-O-deethylase and ethoxycoumarin-O-deethylase activities, but had no effect on benzo(a)pyrene monooxygenase activity. Ethoxyresorufin-O-deethylase, ethoxycoumarin-O-deethylase, nd benzo(a)pyrene monooxygenase activities in the combined PCB-CPFA-fed trout were significantly higher than in control- or CPFA-fed trout, and significantly lower than in PCB-fed trout. This is the first time dietary PCBs have been shown to induce the MFO system in rainbow trout. These results provide a possible explanation for the effects of dietary PCBs on the metabolism and expression of other chemical carcinogens.

Identification of a direct interaction between interleukin 2 and the p64 interleukin 2 receptor gamma chain.

The interleukin 2 receptor (IL-2R) consists of at least two subunits, alpha and beta, both of which can bind interleukin 2 (IL-2). Recent studies have demonstrated the existence of a third subunit, a 64-kDa molecule termed IL-2R gamma chain, and have suggested that gamma chain functions to regulate the rate of IL-2 dissociation from the receptor. In the present report we have addressed whether the gamma chain modulates IL-2R affinity by contributing contact sites for IL-2 binding. Using reagents that allow the IL-2R complex to be immunoprecipitated through the IL-2 molecule itself, we demonstrate the existence of a stable IL-2-IL-2R gamma-chain complex. These studies thus establish that the IL-2R gamma chain directly contributes to the IL-2-binding site, consistent with the hypothesis that gamma chain influences IL-2R affinity through its direct interaction with IL-2.

Post-transcriptional regulation of a murine homeobox gene transcript in F9 embryonal carcinoma cells.

A 2.4 kb RNA encoded by the murine Hox 1.1 (m6) homeobox gene is induced when F9 stem cells are differentiated with retinoic acid and dibutyryl cyclic AMP. The regulation of Hox 1.1 expression was probed by using cycloheximide, an inhibitor of protein synthesis. Production of the Hox 1.1 RNA in differentiating F9 cells was not blocked by treatment with cycloheximide, indicating that new protein synthesis is not required for its induction. On the contrary, this transcript was detected in F9 stem cells treated with cycloheximide, anisomycin, or emetine alone. Nuclear transcription assays indicated that the Hox 1.1 gene was transcribed in F9 stem cells and that the rate of transcription did not change early in the differentiation of F9 cells. These observations indicate that the induction of Hox 1.1 transcripts in F9 stem cells during differentiation is not regulated at the level of transcription initiation but results from stabilization of the transcript.

Structural analysis of murine genes containing homoeo box sequences and their expression in embryonal carcinoma cells.

The presence of homoeo box sequences in the genomes of vertebrates has suggested that these metazoans possess the equivalent of the homoeotic genes that have a key role in regulating the development of the fruitfly Drosophila melanogaster. We report here that a novel murine homoeo box-containing gene is expressed in embryonal carcinoma stem cells. Transcripts of the sequences that flank the homoeo box of this gene are found in these cells before and after induced differentiation, whereas a specific transcript that seems to contain the homoeo sequence is only present after differentiation.

Participation of the CD94 receptor complex in costimulation of human natural killer cells.

Optimal proliferation and expansion of human NK cells require mitogenic cytokines together with cell contact-dependent co-stimulation. Production of mAb that can modulate human NK cell proliferation yielded NKH3, which recognizes the CD94 Ag. NKH3 immunoprecipitates contain approximately 70-kDa heterodimeric complexes consisting of a approximately 25-kDa glycoprotein and approximately 40- to 45-kDa molecules. Analysis by two-dimensional isoelectric focusing/SDS-PAGE suggests that several different 40- to 45-kDa species are present in the CD94 receptor complex in human NK cells. NKH3 reacted with essentially all resting NK cells, although CD94 is expressed at higher levels on the CD56(bright) (i.e., high level of CD56) CD16(dim/neg) (i.e., low level of or absent CD16) subpopulation than on the more abundant CD56(dim)CD16(bright) NK cell subset. Moreover, the Z199 mAb, which appears to recognize NKG2-A species that can form heterodimers with CD94, stained virtually all CD56(bright) NK cells, but only a subset of CD56(dim) NK cells. Ligation of CD94 augmented the proliferation of CD56(bright) NK cells in response to IL-2 or IL-15 by as much as 10-fold. Secretion of IFN-gamma by CD56(bright) NK cells stimulated with IL-2 or IL-15 was also enhanced up to 10-fold after CD94 ligation. CD94 mAb did not consistently costimulate proliferation of or IFN-gamma production by CD56(dim) NK cells cultured with IL-2 or IL-15. In contrast, irradiated K562 cells costimulated proliferation of both CD56(bright) and CD56(dim) NK cells. These results indicate that CD56(bright) and CD56(dim) NK cells can be costimulated through different receptors, which may allow these distinct NK cell subsets to be independently regulated in vivo.

Human peripheral gamma delta T cells recognize hsp60 molecules on Daudi Burkitt's lymphoma cells.

Studies with the use of polyclonal rabbit antiserum reactive with 60-kDa heat shock proteins (hsp60) suggested that hsp60-related molecules could be found on the surface of Daudi cells and were involved in their recognition by certain human gamma delta T cells. The present study confirms this finding by using a mAb specifically recognizing hsp60. This mAb can block outgrowth of human gamma delta T cells in response to stimulation with Daudi and in response to an extract of the mycobacteria H37Ra. This anti-hsp60 mAb stains the surface of Daudi cells, but does not stain either Raji or EBV-transformed B cells, cells which do not stimulate gamma delta T cell outgrowth. Anti-hsp60 mAb could immunoprecipitate a 60-kDa molecule from the H37Ra extract but was unable to precipitate this 60-kDa molecule if the mAb was first absorbed on Daudi cells. This mAb also precipitated a 60-kDa molecule from the surface of Daudi cells which shows an electrophoretic mobility pattern consistent with hsp60. These experiments demonstrate that human gamma delta T cells recognize hsp60-related epitopes on the surface of Daudi cells and within mycobacterial extracts.