The human interleukin-6 receptor alpha chain gene is localized on chromosome 1 band q21

An overview of efforts to induce neutralizing antibodies in order to develop an effective vaccine against AIDS is presented. The principal Neutralizing Determinant (PND) on the HIV-1 envelope is described. PND variability and the induction of neutralizing antibodies by synthetic peptides representing PND are discussed. The use of a cocktail of different peptides representing the PND sequences of the majority of HIV-1 isolates, as well as the construction of hybrid immunogens containing PND of several viral isolates, could overcome the problems related to PND variability. A different approach based on the possibility of inducing a type of intracellular immunity is also discussed: a cellular clone (F12) obtained in our laboratory from Hut-78 cells infected with supernatant of cultured lymphocytes from an HIV-infected patient, does not release viral particles despite the presence of a full-length HIV-1 provirus. Moreover, F12 cells are fully resistant against superinfection with any HIV-1 or HIV-2 isolates. We are now attempting to reproduce the homologous viral interference by transferring the F12/HIV genome of the clone into HIV-susceptible cells in order to render these cells resistant to HIV infection.     

Cloning of the human IL-13R alpha1 chain and reconstitution with the IL4R alpha of a functional IL-4/IL-13 receptor complex

To study the anti-inflammatory mechanisms of inhaled corticosteroids and beta-agonist therapies, we evaluated basal and stimulus-induced superoxide production by human airway inflammatory cells obtained by bronchoalveolar lavage from normal volunteers before and after 3 weeks of an inhaled corticosteroid (flunisolide) and beta-agonist (metaproterenol). Assay of superoxide production by the bronchoalveolar lavage cells was performed in the presence of media alone or media containing phorbol ester by optical density determination of reduced ferricytochrome c at 550 nm. Interleukin-1 beta released from unstimulated cells and cells stimulated with lipopolysaccharide was quantitated by enzyme immunoassay. Interestingly, phorbol ester-stimulated superoxide production was strikingly inhibited (P < 0.05) by inhaled therapies, while stimulus induced Interleukin-1 beta production was not significantly affected (P = 0.12). Suppression of oxidant production by airway inflammatory cells may be a major mechanism for the beneficial anti-inflammatory effects of inhaled corticosteroids and beta-agonists.     

DNA methylation in the promoter region of the p16 (CDKN2/MTS-1/INK4A) gene in human breast tumours

The p16 (CDKN2/MTS-1/INK4A) gene is one of several tumour-suppressor genes that have been shown to be inactivated by DNA methylation in various human cancers including breast tumours. We have used bisulphite genomic sequencing to examine the detailed sequence specificity of DNA methylation in the CpG island promoter/exon 1 region in the p16 gene in DNA from a series of human breast cancer specimens and normal human breast tissue (from reductive mammaplasty). The p16 region examined was unmethylated in the four normal human breast specimens and in four out of nine breast tumours. In the other five independent breast tumour specimens, a uniform pattern of DNA methylation was observed. Of the nine major sites of DNA methylation in the amplified region from these tumour DNAs, four were in non-CG sequences. This unusual concentration of non-CG methylation sites was not a general phenomenon present throughout the genome of these tumour cells because the methylated CpG island regions of interspersed L1 repeats had a pattern of (almost exclusively) CG methylation similar to that found in normal breast tissue DNA and in DNA from tumours with unmethylated p16 genes. These data suggest that DNA methylation of the p16 gene in some breast tumours could be the result of an active process that generates a discrete methylation pattern and, hence, could ultimately be amenable to therapeutic manipulation.     

Functional expression of soluble human interleukin-11 (IL-11) receptor alpha and stoichiometry of in vitro IL-11 receptor complexes with gp130

The interleukin-6 (IL-6) family of cytokines activates signaling through the formation of either gp130 homodimers, as for IL-6, or gp130-leukemia inhibitory factor receptor (LIFR) heterodimers as for ciliary neurotrophic factor (CNTF), leukemia inhibitory factor, oncostatinM, and cardiotrophin-1. Recent in vitro studies with IL-6 and CNTF have demonstrated that higher order hexameric receptor complexes are assembled in which signaling chain dimerization is accompanied by the dimerization of both the cytokine molecule and its specific receptor alpha subunits (IL-6Ralpha or CNTFRalpha, respectively). IL-11 is a member of the IL-6 family and known to require gp130 but not LIFR for signaling. In this study we investigate the functional and biochemical composition of the IL-11 receptor complex. The human IL-11 receptor alpha-chain was cloned from a human bone marrow cDNA library. IL-11Ralpha was shown to confer IL-11 responsiveness to human hepatoma cells either by cDNA transfection or by adding a soluble form of the receptor (sIL11Ralpha) expressed in the baculovirus system to the culture medium. In vitro immunoprecipitation experiments showed that sIL11Ralpha specifically binds IL-11 and that binding is enhanced by gp130. Similarly to IL-6 and CNTF, gp130 is able to induce dimerization of the IL-11.IL-11Ralpha subcomplex, the result of which is the formation of a pentameric receptor complex. However, in contrast to the other two cytokines, IL-11 was unable to induce either gp130 homodimerization or gp130/LIFR heterodimerization. These results strongly suggest that an as yet unidentified receptor beta-chain is involved in IL-11 signaling.     

X-SCID B cell responses to interleukin-4 and interleukin-13 are mediated by a receptor complex that includes the interleukin-4 receptor alpha chain (p140) but not the gamma c chain

We previously reported that patients with mild to moderate airflow limitation have a lower exercise capacity than age-matched controls with normal lung function, but the mechanism of this reduction remains unclear (1). Although the reduced exercise capacity appeared consistent with deconditioning, the patients had altered breathing mechanics during exercise, which raised the possibility that the reduced exercise capacity and the altered breathing mechanics may have been causally related. Reversal of reduced exercise capacity by an adequate exercise training program is generally accepted as evidence of deconditioning as the cause of the reduced exercise capacity. We studied 11 asymptomatic volunteer subjects (58 +/- 8 yr of age [mean +/- SD]) selected to have a range of lung function (FEV1 from 61 to 114% predicted, with a mean of 90 +/- 18% predicted). Only one subject had an FEV1 of less than 70% predicted. Gas exchange and lung mechanics were measured during both steady-state and maximal exercise before and after training for 30 min/d on 3 d/wk for 10 wk, beginning at the steady-state workload previously determined to be the maximum steady-state exercise level that subjects could sustain for 30 min without exceeding 90% of their observed maximal heart rate (HR). The training workload was increased if the subject's HR decreased during the training period. After 10 wk, subjects performed another steady-state exercise test at the initial pretraining level, and another maximal exercise test. HR decreased significantly between the first and second steady-state exercise tests (p < 0.05), and maximal oxygen uptake (VO2max) and ventilation increased significantly (p < 0.05) during the incremental test, indicating a training effect. However, the training effect did not occur in all subjects. Relationships between exercise parameters and lung function were examined by regression against FEV1 expressed as percent predicted. There was a significant positive correlation between VO2max percent predicted and FEV1 percent predicted (p < 0.02), and a negative correlation between FEV1 and end-expiratory lung volume (EELV) at maximal exercise (p < 0.03). There was no significant correlation between FEV1 and maximal HR achieved during exercise; moreover, all subjects achieved a maximal HR in excess of 80% predicted, suggesting a cardiovascular limitation to exercise. These data do not support the hypothesis that the lower initial VO2max in the subjects with a reduced FEV1 was due to deconditioning. Although increased EELV at maximal exercise, reduced VO2max and a reduced VO2max response with training are all statistically associated with a reduced FEV1, there is no direct evidence of causality.     

A phase I study of recombinant human soluble interleukin-1 receptor (rhu IL-1R) in patients with relapsed and refractory acute myeloid leukemia

PURPOSE: The recombinant human interleukin-1 receptor (rhu IL-1R) is a soluble truncated form of the type 1 full-length membrane-bound receptor that binds IL-1 with identical affinity to that of the membrane form. As such, it may have clinical potential by sequestering IL-1, thereby preventing it from binding to its membrane-bound receptor and eliciting a biological effect. As IL-1 has been shown to regulate leukemic cell proliferation in an autocrine fashion, a phase I trial of rhu IL-1R was conducted in patients with relapsed and refractory acute myeloid leukemia (AML). METHODS: The study group comprised 11 patients who were sequentially treated on one of three dose levels, receiving a single intravenous (i.v.) bolus dose on day 1 followed by 13 days of daily subcutaneous (s.c.) injections with the option of an additional 14 days of treatment if a response of stable disease or better was achieved. Dose level 1 i.v. bolus 500 microg/m2, s.c. dose 250 microg/m2 per day (five patients); dose level 2 i.v. bolus 1000 microg/m2, s.c. dose 500 microg/m2 per day (three patients); dose level 3 i.v. bolus 2000 microg/m2, s.c. dose 1000 microg/m2 per day (three patients). Owing to limited drug availability, the study was designed to only examine these three dose levels. RESULTS: rhu IL-IR was well tolerated. There was no grade 3 or 4 non-hematological toxicity related to the study drug and the maximum tolerated dose was not reached. No IL-1R-blocking antibodies developed during the course of the study. Serum levels of IL-1beta, IL-6 and TNF were undetectable before, during and after rhu IL-IR administration. The terminal half-life after i.v. dosing was at least 7-12 h, and after s.c. dosing 2-4 days. Serum levels of rhu IL-1R up to 360- and 25-fold those of pretreatment levels were achieved after i.v. and s.c. dosing respectively. No patient had a complete, partial or minor response to treatment; four had stable disease and seven had progressive disease. CONCLUSIONS: rhu IL-1R therapy was safe but did not have any apparent antileukemic effect at the doses administered.     

Crystals of a 1:1 complex between human interleukin-4 and the extracellular domain of its receptor alpha chain

The specific high-affinity binding of interleukin-4 (IL-4) to its receptor alpha chain is the crucial primary event during IL-4 signalling. Single crystals, suitable for high resolution diffraction studies, have been obtained from a complex between IL-4 and the ectodomain of the receptor alpha chain, also called IL-4-binding protein (IL-4BP). The orthorhombic crystals are in spacegroup P2(1)2(1)2(1) with cell constants a = 5.038 nm, b = 6.841 nm, c = 10.95 nm and diffract to a resolution of at least 0.25 nm when exposed to synchrotron radiation. The volume of the unit cell suggests the presence of a 1:1 IL-4/IL-4BP complex and HPLC analysis of the crystals confirmed that IL-4 and IL-4BP were present in equimolar amounts. An IL-4 variant comprising a total of four methionine residues was generated, labelled with selenomethionine and crystallised in complex with IL-4BP. The crystals are isomorphous to that of the complex with normal IL-4 and therefore can be used to solve the crystallographic phase problem by the method of multiple anomalous diffraction (MAD). The crystal structure of the IL-4/IL-4BP complex will help to understand how IL-4 and other helical cytokines bind and activate their cognate receptor.     

Generation of monoclonal antibodies to the rabbit interleukin-2 receptor alpha chain (CD25) and its distribution in HTLV-1-transformed rabbit T cells

Rabbits can be infected with human retroviruses such as human T-cell leukemia virus-1 (HTLV-1) and human immunodeficiency virus (HIV), and provide useful animal models to study retroviral diseases such as adult T-cell leukemia and HIV. Previously we have succeeded in generating monoclonal antibodies (mAbs) against rabbit CD4, CD5 and CD11a antigens. To make this animal species more amenable to cellular and molecular studies, we have attempted to extend the panel of mAbs against rabbit CD antigens. Here we report on the generation of three neutralizing mAbs against interleukin-2 receptor alpha chain (IL-2R alpha) (CD25), Kei-alpha 1 (IgG2b), Kei-alpha 2 (IgG2a) and Kei-alpha 3 (IgG1). They specifically recognize the rabbit Mr 55,000 IL-2 binding protein, IL-2R alpha, and completely inhibit both high- and low-affinity IL-2 binding to F648b cells that express IL-2R alpha as well as IL-2R beta. The use of mAb Kei-alpha 1 confirmed that the rabbit IL-2R alpha is not only a low-affinity IL-2R on its own but also an essential component of high-affinity IL-2R as found in other animal species, and that rabbit activated T cells including HTLV-1-transformed cell lines express high levels of the IL-2R alpha. Together with mAbs against various rabbit CD antigens that we reported previously, these neutralizing mAbs to IL-2R alpha will be valuable for studies of human retrovirus infections, such as those induced by HTLV-1 or HIV, in rabbits.     

Analysis of human IL-2/IL-2 receptor beta chain interactions: monoclonal antibody H2-8 and new IL-2 mutants define the critical role of alpha helix-A of IL-2

Interleukin 2 (IL-2) interacts with a receptor (IL-2R) composed of three subunits (IL-2R alpha, IL-2R beta and IL-2R gamma). IL-2R beta plays a critical role in signal transduction. An anti-human IL-2 mAb (H2-8) produced after immunization with peptide 1-30 of IL-2 was found to recognize the region occupied by Asp20, at the exposed interface between alpha-helices A and C. Muteins at position 17 and 20 are not recognized by mAb H2-8. mAb H2-8 specifically inhibits the IL-2 proliferation of TS1beta cells which are dependent on the expression of human IL-2R beta chain for IL-2 proliferation. Substitution at internal position Leu17 demonstrates that this position is essential for IL-2 binding and IL-2 bioactivity. New IL-2 mutants at position Asp20 have been analysed. Substitutions Asp --> Asn, Asp --> Lys, Asp --> Leu, show a correlation between diminished affinity for IL-2 receptor and reduced bioactivity measured on TS1beta cells. Mutein Asp Arg lose affinity for IL-2R and bioactivity simultaneously. Furthermore, during the course of the study we have found that mutein Asp20 --> Leu is an IL-2 antagonist. The biological effects of mAb H2-8 and the properties of new mutants at positions 17 and 20 demonstrate that this region of alpha helix-A is involved in IL-2-IL-2R beta interactions.