Molecular cloning of the guinea-pig IL-5 receptor alpha and beta subunits and reconstitution of a high affinity receptor.

The functional IL-5 receptor is a heteromeric complex consisting of an alpha and beta subunit. The cloning, sequencing and expression of guinea-pig IL-5Ralpha and beta subunits is described. The guinea-pig IL-5Ralpha subunit cDNA encodes a protein of M(r)47 kDa, which is 72 and 66% homologous to the human and murine orthologs, respectively. Three guinea-pig IL-5Rbeta subunit cDNA clones were isolated, which differ in the N-terminus and are 56-64% homologous to the human and murine IL-5Rbeta subunits. Expressing human IL-5Ralphabeta and guinea-pig IL-5Ralphabeta(1)in the baculovirus-insect cell system resulted in recombinant receptors which bound hIL-5 with high affinity (K(d)=0.19 and 0.11 nM, respectively). Expressing just gpIL-5Ralpha was not sufficient to demonstrate binding. This contrasts with the human receptor, where hIL-5Ralpha alone can bind hIL-5 with high affinity. gpIL-5Ralphabeta(1)bound both hIL-5 and mIL-5 with comparable affinity (K(i)=0.10 and 0.06 nM), similar to that seen with hIL-5Ralphabeta. Thus, both the heteromeric hIL-5R and gpIL-5Ralphabeta(1)can bind multiple IL-5 orthologs with high affinity whereas the murine IL-5R is selective for the murine ligand.

Expression and in vitro properties of guinea pig IL-5: comparison to human and murine orthologs.

Interleukin-5 (IL-5) is a key mediator of eosinophilic inflammation. The biological role of this cytokine in an allergic airway inflammatory response has been widely demonstrated in guinea pigs, yet the interaction of guinea pig IL-5 (gpIL-5) with its receptor has not been studied. Experiments were performed to quantitate the interaction of gpIL-5 with gpIL-5r and to compare this affinity with that of hIL-5 and mIL-5 and their cognate receptors. The cross-species affinity and agonist efficacy were evaluated to see if gpIL-5r had a restricted species reactivity (as is the case with mIL-5r) or did not distinguish between IL-5 orthologs (similar to hIL-5r). gpIL-5 was cloned using mRNA isolated from cells obtained by bronchoalveolar lavage. Recombinant gpIL-5 was expressed in T. ni insect cells and purified from spent media. Binding assays were performed using insect cells expressing hIL-5ralphabeta or gpIL-5ralphabeta1 as previously described (Cytokine, 12:858-866, 2000) or using B13 cells which express mIL-5r. The agonist potency and efficacy properties of each IL-5 ortholog were evaluated by quantitating the proliferative response of human TF-1 cells and murine B13 cells. gpIL-5 bound with high affinity to recombinant gpIL-5r as demonstrated by displacing [125I]hIL-5 (Ki = 160 pM). gpIL-5 also bound to hIL-5r with high affinity (Ki = 750 pM). hIL-5 and mIL-5 showed similar, high-affinity binding profiles to both gpIL-5r and hIL-5r. In contrast, gpIL-5 and hIL-5 did not bind to the mIL-5r as demonstrated by an inability to displace [125I]mIL-5, even at 1000-fold molar excess. These differences in affinity for IL-5r orthologs correlated with bioassay results: human TF-1 cells showed roughly comparable proliferative responses to guinea pig, human and murine IL-5 whereas murine B13 cells showed a strong preference for murine over guinea pig and human IL-5 (EC50 = 1.9, 2200 and 720 pM, respectively). Recombinant gpIL-5 binds to the gpIL-5r with high affinity, similar to that seen with the human ligand-receptor pair. gpIL-5r and hIL-5r do not distinguish between the three IL-5 orthologs whereas mIL-5r has restricted specificity for its cognate ligand.

Evaluation of cross-resistance between responses to cisplatin, hyperthermia, and radiation in human glioma cells and eight clones selected for cisplatin resistance.

Human glioma cells were exposed to stepwise increasing concentrations of cisplatin and given a final, acute, high concentration treatment of cisplatin. From the surviving cells, eight cisplatin resistant clones were selected. These clones demonstrated a range of cisplatin sensitivities that were retained in the absence of cisplatin when cells were continually passaged. These cells were tested for cross-resistance to radiation and hyperthermia at 42 and 45 degrees C. The data showed that seven of the eight clones were also more radioresistant than the parental line, while one was more radiosensitive. The degree of cisplatin resistance was not related to the degree of radiation resistance. For hyperthermia at 42 and 45 degrees C, some of the clones were slightly more resistant than the parental line, while one clone was much more sensitive. This was not the same clone that was radiosensitive. In conclusion, there was no direct correlation between cisplatin resistance, radiation resistance, and hyperthermia response, although some of the clones were resistant to all three treatments.

Cloning, sequencing and functional expression of a guinea pig lung bradykinin B2 receptor.

Kinin receptors are classified as B1 and B2 based upon agonist and antagonist potencies and cloning and expression studies. Using sequences from human and rat bradykinin B2 receptors, polymerase chain reaction (PCR) was utilized to isolate cDNA from guinea pig lung. The receptor obtained is predicted to have 372 amino acids and shares > 80% sequence homology with human, rat, rabbit and mouse B2 receptors. In competition binding experiments in Chinese hamster ovary (CHO-K1) cells in which the guinea pig cDNA was expressed, [3H]bradykinin was displaced by kinin receptor ligands with an order of potency consistent with a B2 subtype. In CHO cells expressing the guinea pig receptor, bradykinin caused a concentration 45Ca2+ efflux. A B1 receptor agonist, desArg9-bradykinin, also caused 45Ca2+ efflux but with a potency several orders of magnitude lower than bradykinin. Curiously, several B1 and B2 receptor antagonists induced 45Ca2+ efflux, indicating that this receptor may be coupled differently in CHO cells than in native tissues.

Cell cycle perturbations in cisplatin-sensitive and resistant human ovarian carcinoma cells following treatment with cisplatin and low dose rate irradiation.

PURPOSE: To investigate cell cycle pertubations in plateau-phase human ovarian carcinoma cells following treatment with cisplatin, low dose-rate irradiation (LDRI), or combined cisplatin and LDRI, in order to understand cell cycle mechanisms by which these two treatment modalities interact. METHODS: Human ovarian carcinoma cells sensitive (A2780) and resistant (2780CP) to cisplatin were grown to plateau phase and given protracted cisplatin treatments (A2780 0.7 and 2 microg/ml; 2780CP 5 and 15 microg/ml) and/or LDRI (0.41 cGy/min). Cell cycle distribution following treatment was determined by two-parameter flow cytometry, based on bromodeoxyuridine (BrdU) uptake and DNA content using propidium iodide staining. RESULTS: The cisplatin-sensitive A2780 cells exposed to cisplatin alone for up to 28 h showed depletion of the G1 fraction and accumulation in S-phase, although the percentage of S-phase cells actively incorporating BrdU dropped to almost zero. The cisplatin-resistant 2780CP cells exposed to cisplatin alone showed a G1 arrest when exposed to 15 microg/ml, but not when exposed to 5 microg/ml. LDRI alone caused little cell cycle redistribution different from controls in either cell line. When LDRI was combined with cisplatin, no significant cell cycle redistribution was observed, apart from a decline in the actively incorporating S-phase fraction. CONCLUSIONS: Cisplatin caused A2780 cells to accumulate in nonincorporating S-phase, with no evidence of G1 arrest. Cisplatin-resistant 2780CP cells showed a G1 block when exposed to a high enough cisplatin concentration. This could indicate a mechanism of cisplatin resistance in these cells. LDRI alone or in combination with cisplatin did not result in significant cell cycle redistribution.

Cisplatin, hyperthermia and radiation treatment in human cisplatin-sensitive and resistant glioma cell lines.

In this study, the effects of mild protracted hyperthermia, combined with prolonged exposure to cisplatin and low dose-rate irradiation (LDRI), were examined in two human cell lines. The cell lines are human glioma parental and cisplatin-resistant variant cells. The results show that mild hyperthermia at 40 degrees C was able to sensitize both the parental and the variant cisplatin-resistant cells to cisplatin treatments (1 microgram/ml for up to 20 h) when the two treatments were given concurrently. When mild hyperthermia and cisplatin were given with LDRI concurrently, additional enhanced cell killing was observed in both the parental and the cisplatin-resistant variant cells. Further analysis of the results showed that when the effects of the trimodality treatment were normalized to the effects of the combined treatment of mild hyperthermia with cisplatin, the residual cell killing was still greater than that observed for radiation alone, indicating a synergistic interaction. This synergistic interaction was greater for the parental line compared to the cisplatin-resistant line. Thus, these data show that the concurrent application of mild hyperthermia, low concentration, long duration, cisplatin and low-dose rate irradiation may be an effective form of treatment in both normally responding and cisplatin-resistant variant human tumour cell lines.

Cisplatin and low dose rate irradiation in cisplatin resistant and sensitive human glioma cells.

PURPOSE: Human glioma cell lines resistant (U373MGCP) and sensitive (U373MG) to cisplatin were used to evaluate the effect of cisplatin as a sensitizer to low dose rate irradiation (LDRI). METHODS AND MATERIALS: A cisplatin resistant glioma cell line U373MGCP was developed by chronic exposure of parental U373MG cells to cisplatin. Plateau phase cells were treated with cisplatin, high dose rate (HDR) irradiation (1.12 Gy/min), LDRI (0.0088 Gy/min), or cisplatin concurrent with LDRI. Cell survival was determined by the colony forming assay. RESULTS: Both cell lines showed increased resistance to radiation at LDR compared with HDR, with Dose Modifying Factors (DMF at 10% survival level) of 1.7 for U373MG and 2.5 for U373MGCP. The increased LDR sparing effect in the cisplatin resistant U373MGCP cells indicates increased repair proficiency. The resistant cell line showed a fourfold increase in resistance to cisplatin cytotoxicity at the 10% survival level compared with the parental U373MG cells. Cisplatin enhanced the response of both cell lines to LDRI. The DMFs were 1.2, 1.2, and 1.7, respectively, for the sensitive U373MG cell line given 1 microgram/ml, and the resistant cell line given 3 or 6 micrograms/ml cisplatin treatments concurrent with LDRI. CONCLUSIONS: These data show that cisplatin can be an effective sensitizer to LDRI in both cisplatin resistant and sensitive glioma cell lines. However, in the resistant cell line, higher concentrations of cisplatin were necessary to achieve the same level of sensitization as in the sensitive cell line.

Correlation between Gd-enhanced MR imaging and histopathology in treated and untreated 9L rat brain tumors.

Gadolinium-based MR imaging contrast agents cause signal enhancement of intracerebral tumors on T1-weighted MR images because they cross the compromised blood-brain barrier, increasing the T1 relaxation rate of extracellular water. Tumor extent measured by Gd-enhanced MR imaging often does not agree with T2-weighted MRI or biopsy measurements, due to possible peritumoral barrier defects or infiltration of tumor cells beyond the region of enhancement. In this study, the 9L rat brain tumor model was used to measure the correlation between tumor size measured by Gd-enhanced and unenhanced MRI and histological measurements. There was excellent agreement between Gd-enhanced MRI measurements and histology. Morphological features of untreated 9L intracranial tumors and those treated with radiation and/or cisplatin were compared with histological features. No significant change in intracranial tumor size was detected in the treated animals up to 7 days following treatment, despite substantial tumor necrosis evident on histological analysis.

Literature review of the usefulness of nerve conduction studies and electromyography for the evaluation of patients with carpal tunnel syndrome. AAEM Quality Assurance Committee.

The sensitivity and specificity of nerve conduction studies (NCS's) and electromyography (EMG) for the diagnosis of carpal tunnel syndrome (CTS) were evaluated by a critical review of the literature. With a search of the medical literature in English through May 1991, 165 articles were identified and reviewed on the basis of six criteria of scientific methodology. The findings of 11 articles that met all six criteria and the results of 48 additional studies that met four or five criteria are presented. We concluded that median sensory and motor NCS's are valid and reproducible clinical laboratory studies that confirm a clinical diagnosis of CTS with a high degree of sensitivity and specificity. Clinical practice recommendations are made based on a comparison of the sensitivities of the several different median nerve conduction study (NCS) techniques.

Inhibition of potentially lethal damage recovery by cisplatin in a brain tumor cell line.

Radiation resistant tumours such as gliomas show enhanced capacity for potentially lethal damage recovery (PLDR), which can be inhibited by cisplatin. The 9L rat brain tumour cell line, like human glioma cell lines, shows a large capacity for PLDR. Cisplatin administered at 6 micrograms/ml for 1 hour immediately following acute irradiation (18 Gy) is shown to cause significant inhibition of PLDR, while 3 micrograms/ml causes little inhibition. Cisplatin-radiation treatment sequence affects PLDR inhibition, with maximum effect seen when cisplatin is administered immediately after irradiation, during the period of rapid cellular recovery. These data suggest that optimum interaction between radiation and cisplatin treatments can be achieved by maximizing intratumoural cisplatin levels during the post-irradiation recovery period.

Effects of bradykinin receptor antagonists on antigen-induced respiratory distress, airway hyperresponsiveness and eosinophilia in guinea-pigs.

1. We examined effects of bradykinin (BK) receptor antagonists on airway hyperresponsiveness and eosinophilia in sensitized guinea-pigs that had been administered single, as well as repeated (chronic) challenges with inhaled ovalbumin. In addition, the effects of BK antagonists on antigen-induced respiratory distress during the chronic study were noted. 2. At 24 h following single antigen challenge, guinea-pigs exhibited airway hyperresponsiveness to the bronchoconstrictor effect of i.v. histamine, characterized by a left shift in the dose-response curve. In addition, responses to the maximum dose of histamine that could be used were significantly increased in hyperresponsive guinea-pigs. The percentages of bronchoalveolar fluid, eosinophil and neutrophils also increased. 3. A BK B1 receptor antagonist, desArg9-[Leu8]-BK, significantly inhibited airway hyperresponsiveness induced by single antigen challenge. A B2 receptor antagonist, D-Arg-[Hyp3, Thi5,8,D-Phe7]-BK (NPC 349) had a small, but statistically significant inhibitory effect on responsiveness to the highest histamine dose in challenged animals. DesArg9-[Leu8]-BK significantly inhibited the neutrophilia, whereas NPC 349 inhibited infiltration by both cell types. 4. Chronic antigen challenge also caused airway hyperresponsiveness to i.v. acetylcholine (ACh), distinguished by an increase in the slope of the dose-response curve. Thus, the magnitude of the bronchoconstrictor responses to the maximum dose of ACh that could be used was significantly increased. No change in sensitivity to ACh was evident. Marked eosinophilia was also noted in the trachea, bronchi and lung parenchyma. 5. Airway hyperresponsiveness and eosinophilia, induced by chronic antigen challenge, were markedly inhibited by the B2 antagonists, D-Arg-[Hyp3,D-Phe7]-BK (NPC 567) or D-Arg-[Hyp3,Thi5d-Tic7,Tic8]-BK (NPC 16731).NPC 16731 also abolished antigen-induced cyanosis, and delayed the onset of dyspnoea,doubling the time taken for animals to exhibit respiratory distress.6. The ability of BK receptor antagonists to inhibit antigen-induced airway hyperresponsiveness, in addition to eosinophilia, indicates an important role for endogenous kinins. Moreover, the abrogation of eosinophil infiltration suggests that BK has a significant function in maintaining allergic inflammation of the airways.

A symmetrical 1,4-dihydropyridine with opposite actions on K(+)-induced contractions in guinea-pig ileum.

The effects of 1,4-dihydro-2,6-dimethyl-4-phenyl-pyridine-3,5-dicarboxylic acid dimethyl ester (DHMP) on Ca2(+)-evoked contractions in potassium-depolarized guinea-pig ileum longitudinal muscle were evaluated. DHMP (1-10 nM) potentiated Ca2(+)-evoked contractions; maximum enhancement was seen at 1 nM. A concentration-dependent inhibition of Ca2(+)-induced contractility was observed at higher concentrations. Racemic BAY K 8644 (1 nM - 1 microM), on the other hand, enhanced ileum responses to Ca2+ with a maximum effect at 3 nM. BAY K 8644, but not DHMP, directly elicited concentration-dependent contractions. The results provide further support for the hypothesis that dihydropyridines can act as both Ca2+ agonists and antagonists.

Evidence for a pulmonary B3 bradykinin receptor.

We have examined pulmonary effects of bradykinin (Bk) in vivo and in vitro in guinea pigs and their potential inhibition by antagonists of Bk B1 and B2 receptors. Bk was a potent bronchoconstrictor in vivo and caused contractions of isolated, epithelium-denuded trachealis. D-Arg[Hyp3,D-Phe7]-Bk (NPC567) and D-arg[Hyp3,Thi5,8,D-Phe7]-Bk (NPC349), B2 receptor antagonists, were weak inhibitors of Bk-induced bronchoconstriction in vivo and were virtually inactive as antagonists of Bk-induced airway smooth muscle contraction. Several other B2 antagonists as well as B1 antagonist, des-Arg9-[Leu8]-Bk, did not inhibit Bk-induced tracheal contraction. The B1 receptor agonist des-Arg9-Bk was without effect on tracheal tone. Tracheal responses to Bk were unaffected by antagonists of muscarinic, histamine, serotonin, and catecholamine receptors. The inability of the antagonists to inhibit Bk is unlikely to be due to their degradation, because NPC567 was only weakly active in the presence of inhibitors of kininase I (EC 3.4.11.2), kininase II (EC 3.4.15.1), and neutral endopeptidase (EC 3.4.24.11). These studies were corroborated by ligand binding experiments in guinea pig and ovine airways. In [3H]Bk binding, the Bk antagonists had no effect in guinea pig trachea, slightly displaced [3H]Bk in ovine trachea, and inhibited approximately 60% of total specific binding in lung. des-Arg9-[Leu8]-Bk and several other agents, including atropine, neurokinin A, substance P, and vasoactive intestinal peptide, had no effect on lung Bk binding. Bk and its analogs were not degraded during the binding assay. These data suggest that pulmonary tissue, particularly in the large airways, contains a novel Bk binding site, a B3 receptor, which may be involved in Bk-induced bronchoconstriction.