Biomedical subjects

P Graber

Publications and source records attributed to P Graber.

At least 19 recordsLinked to original sources

PubMed1998

Cytokine-like factor-1, a novel soluble protein, shares homology with members of the cytokine type I receptor family.

In this report we describe the identification, cloning, and expression pattern of human cytokine-like factor 1 (hCLF-1) and the identification and cloning of its murine homologue. They were identified from expressed sequence tags using amino acid sequences from conserved regions of the cytokine type I receptor family. Human CLF-1 and murine CLF-1 shared 96% amino acid identity and significant homology with many cytokine type I receptors. CLF-1 is a secreted protein, suggesting that it is either a soluble subunit within a cytokine receptor complex, like the soluble form of the IL-6R alpha-chain, or a subunit of a multimeric cytokine, e.g., IL-12 p40. The highest levels of hCLF-1 mRNA were observed in lymph node, spleen, thymus, appendix, placenta, stomach, bone marrow, and fetal lung, with constitutive expression of CLF-1 mRNA detected in a human kidney fibroblastic cell line. In fibroblast primary cell cultures, CLF-1 mRNA was up-regulated by TNF-alpha, IL-6, and IFN-gamma. Western blot analysis of recombinant forms of hCLF-1 showed that the protein has the tendency to form covalently linked di- and tetramers. These results suggest that CLF-1 is a novel soluble cytokine receptor subunit or part of a novel cytokine complex, possibly playing a regulatory role in the immune system and during fetal development.

Amino Acid Sequence↗

PubMed1998

The nitric oxide synthase/nitric oxide and heme oxygenase/carbon monoxide pathways in the human ureter.

OBJECTIVE: To investigate the nitric oxide synthase (NOS)/nitric oxide (NO) and heme oxygenase (HO)/carbon monoxide (CO) pathways in the human isolated ureter. METHODS: Immunohistochemical studies were performed. NOS activity was measured by monitoring the conversion of [3H]-arginine to [3H]-citrulline. Functional inhibitory effects mediated by NO and CO were assessed, and correlated with cyclic nucleotide levels. RESULTS: The overall innervation of the ureter was moderate, however more prominent in the distal segment. Relative to overall innervation, neuronal NOS-immunoreactive (-IR) nerves were few. In the submucosa, neuronal NOS-IR varicose nerves were found closely together with varicose nerves containing calcitonin gene-related peptide immunoreactivity. In the distal ureter, nerve trunks were demonstrated, expressing immunoreactivity for HO-2. Ca(2+)-dependent NOS activity was 53 +/- 13 pM/mg protein/h. In isolated preparations, NO decreased endothelin-1-induced contraction in a concentration-dependent manner. In strips exposed to NO, there was a 6-fold increase of the cyclic GMP levels in comparison to control preparations (p < 0.001). CO exerted no effect on induced ureteral tone. CONCLUSIONS: Neuronal NOS- and HO-2-IR nerves can be demonstrated in the human ureter, where NO, but probably not CO, may contribute to the regulation of tone. Although the physiological roles for NO and CO remain to be established, the NOS/NO/cyclic GMP pathway may be a target for drugs producing relaxation of the human ureter. The richer innervation of the distal ureter may be of importance for the coordination of ureteral peristalsis and the motility of the ureterovesical junction.

Adolescent↗

PubMed1998

The interleukin-4/interleukin-13 receptor of human synovial fibroblasts: overexpression of the nonsignaling interleukin-13 receptor alpha2.

Interleukin (IL)-4 and IL-13 are known to bind to shared heteromultimeric receptor complexes of variable composition. Given the many regulatory effects of IL-4 and IL-13 on synovial cells, we aimed to characterize their IL-4/IL-13 receptor (R). Cultivated synovial fibroblasts expressed transcripts for IL-4Ralpha and IL-13Ralpha1, the human homolog of the recently cloned mouse IL-13R, but not the common gamma-chain of the IL-2R. In particular, IL-13Ralpha2 mRNA, encoding a different IL-13R recently cloned from human renal carcinoma cells, was expressed at a strikingly high level. Correspondingly, a predominant protein migrating at 65 to 75 kd was cross-linked by iodinated IL-13 and was not cross-competed by an excess of unlabeled IL-4. However, by flow cytofluorometry, IL-13Ralpha1 (detected by the anti-lL-13Ralpha1 mAb 65) and IL-4Ralpha (detected by the mAb S697) were expressed at similar low density. Radioligand binding studies revealed for both cytokines approximately 300 receptors/cell with similar high affinity. An additional class of IL-13Rs was identified after occupation of the shared high-affinity receptors by the nonsignaling, double-mutant IL-4121R-->D, 124Y-->D (RY-IL-4). In these experiments, 1251-IL-13 bound to a single receptor population with a Kd of approximately 300 pM and approximately 5000 sites/cell, matching the published affinity of monomeric IL-13Ralpha2 when expressed in COS7 cells. RY-IL-4 blocked the IL-4- and IL-13-mediated vascular cell adhesion molecule (VCAM)-1 expression and Stat6 activation, suggesting that the large number of high-affinity IL-13Ralpha2 monomers are silent receptors, likely representing a decoy target for IL-13.

Carrier Proteins↗

PubMed1997

[Do results of surgical treatment of cancer clinically localized to the prostate justify early screening?].

Prostate cancer is the most common malignancy and represents the second leading cause of cancer death in men of western countries. Mortality of this tumor is particularly high beyond ten years. For patients with more than 10 years of life expectancy, curative treatment, if available, is mandatory. For the others, observation and differed hormonal palliative treatment is a reasonable alternative. Radical prostatectomy is the gold standard for treatment of clinically localized prostatic cancer. Cure rate is very high when the tumor histologically is strictly confined to the prostate. Morbidity has been reduced with the development of the anatomical approach. Our experience suggests that this reduced morbidity can also be achieved in urology clinics with relatively low recruitment. In addition, our results show that chances for cure are still very high for tumors reaching or even penetrating the prostatic capsule, as long as the seminal vesicles are not invaded. Early screening of the Prostate diagnoses a vast majority of tumors before the stage of seminal vesicle invasion. Early screening may thus be recommended for patients with 10 years or more of life expectancy.

Adenocarcinoma↗

PubMed1997

Spatial orientation of the alpha and betac receptor chain binding sites on monomeric human interleukin-5 constructs.

Interleukin-5 (IL-5), a disulfide-linked homodimer, can be induced to fold as a biological active monomer by extending the loop between its third and fourth helices (Dickason, R. R., and Huston, D. P. (1996) Nature 379, 652-655). We have designed eight monomeric IL-5 proteins to optimize biological activity and stability of the monomer. This was achieved by (i) inserting the joining loop at three different positions, (ii) by introducing an additional intramolecular disulfide bridge onto these backbones, and (iii) by creating circular permutations to fix the position of the carboxyl-terminal helix relative to the three other helices. The proteins dimerize with Kd values ranging from 20 to 200 microM and are therefore monomeric at the picomolar concentrations where they are biologically active. Introduction of a second disulfide confers increased stability, but this increased rigidity results in lower activity of the protein. Contrary to wild type IL-5, mutation of the betac contact residue on the first helix, Glu12, to Lys, into the circularly permutated constructs, did not abolish TF-1 proliferative and eosinophil activation activities. These results indicate that activation of the IL-5 receptor complex is not mediated solely by Glu12 on the first helix, and alternative mechanisms are discussed.

Binding Sites↗

PubMed1997

The 25-kDa soluble CD23 activates type III constitutive nitric oxide-synthase activity via CD11b and CD11c expressed by human monocytes.

CD23, a low-affinity receptor for IgE, was recently shown to bind to CD11b and CD11c molecules on human monocytes. The 25-kDa soluble fragment of CD23 (sCD23), was tested for its capacity to elicit various signaling pathways in human monocytes. sCD23 was found to trigger an early increase in cGMP accumulation, through the generation of nitric oxide. This was a result of activating the L-arginine pathway, since the sCD23-mediated effect was inhibited in the presence of substituted nonmetabolizable L-arginine analogues. In addition, the increase in cGMP was suppressed by calcium chelators and inhibitors of the calcium/calmodulin complex, suggesting the involvement of a constitutive, calcium-dependent nitric oxide synthase (NOS). Indeed, the presence of an endothelial constitutive type III NOS mRNA was detected in nonactivated human monocytes, and the corresponding protein has been detected by flow cytometry. Moreover, sCD23 was shown to induce a calcium influx in monocytes, in accordance with an activation of a constitutive NOS through a transient increase in [Ca2+]i. As expected, these events were mimicked by mAbs against CD11b and CD11c, the macrophage receptors for CD23. In addition to these early events, sCD23 and the agonistic anti-CD11b and CD11c mAbs, which all trigger the release of proinflammatory mediators such as TNF-alpha, were shown to act through an NO-dependent process.

Cell Line↗

PubMed1997

An interleukin 5 mutant distinguishes between two functional responses in human eosinophils.

Interleukin 5 (IL-5) is the key cytokine involved in regulating the production and many of the specialized functions of mature eosinophils including priming, adhesion, and survival. We have generated a point mutant of human IL-5, IL-5 (E12K), which is devoid of agonist activity in both a TF-1 cell proliferation assay and a human eosinophil adhesion assay. However, IL-5 (E12K) is a potent and specific antagonist of both these IL-5-dependent functional responses. In both receptor binding and cross-linking studies the wild-type and IL-5 (E12K) mutant exhibit virtually identical properties. This mutant protein was unable to stimulate tyrosine phosphorylation in human eosinophils, and blocked the phosphorylation stimulated by IL-5. In contrast, IL-5 (E12K) is a full agonist in a human eosinophil survival assay, although with reduced potency compared to the wild-type protein. This IL-5 mutant enables us to clearly distinguish between two IL-5-dependent functional responses and reveals distinct mechanisms of receptor/cellular activation.

Cells, Cultured↗

PubMed1997

Inhibition of apoptosis in a human pre-B-cell line by CD23 is mediated via a novel receptor.

Human CD23 is a 45-kD type II membrane glycoprotein, which functions as a low-affinity receptor for IgE and as a ligand for the CD21 and CD11b/CD11c differentiation antigens. CD23 is released from the surface of cells as soluble fragments, and a 25-kD species of soluble CD23 (sCD23) appears to act as a multifunctional cytokine. In this report, sCD23 is shown to sustain the growth of low cell density cultures of a human pre-B-acute lymphocytic leukemia cell line, SMS-SB: no other cytokine tested was able to induce this effect. Flow cytometric analysis indicates that sCD23 acts to prevent apoptosis of SMS-SB cells. SMS-SB cells cultured at low cell density possess low levels of bcl-2 protein. Addition of sCD23 to cells at low cell density maintained bcl-2 expression at levels equivalent to those observed in SMS-SB cells cultured at higher cell densities. No CD23 mRNA was found in SMS-SB cells, ruling out an autocrine function for CD23 in this cell line model. Although SMS-SB cells do not express the known receptors for CD23, namely CD21, CD11b-CD18, or CD11c-CD18, the cells specifically bind CD23-containing liposomes, but not glycophorin-containing liposomes. Binding of CD23-containing liposomes is inhibited by anti-CD23 but not by anti-CD21 or anti-CD11b/c monoclonal antibodies. The data show that sCD23 prevents apoptosis of the SMS-SB cell line by acting through a novel receptor.

Apoptosis↗

PubMed1997

Clinical evaluation of a method for detecting superficial surgical transitional cell carcinoma of the bladder by light-induced fluorescence of protoporphyrin IX following the topical application of 5-aminolevulinic acid: preliminary results.

BACKGROUND AND OBJECTIVE: In bladder cancer, conventional white light endoscopic examination of the bladder does not provide adequate information about the presence of "flat" urothelial lesions such as carcinoma in situ. In the present investigation, we examine a new technique for the photodetection of such lesions by the imaging of protoporphyrin IX (PpIX) fluorescence following topical application of 5-aminolevulinic acid (ALA). STUDY DESIGN/MATERIALS AND METHODS: Several hours after bladder instillation of an aqueous solution of ALA in 34 patients, a Krypton ion laser or a filtered Xenon arc-lamp was used to excite PpIX fluorescence. Tissue samples for histological analysis were taken while observing the bladder wall either by means of a video camera, or by direct endoscopic observation. RESULTS: A good correlation was found between the PpIX fluorescence and the histopathological diagnosis. On a total of 215 biopsies, 143 in fluorescent and 72 in nonfluorescent areas, all visible tumors on white light cytoscopy appeared in a bright red fluorescence with the photodetection technique. In addition, this method permitted to discover 47 unsuspected carcinomatous lesions on white light observation, among which 40% were carcinoma in situ. CONCLUSION: PpIX fluorescence induced by instillation into the bladder of 5-ALA is an efficient method of mapping the mucosa in bladder carcinoma.

Administration, Cutaneous↗

PubMed1997

Localization of nitric oxide synthase and haemoxygenase, and functional effects of nitric oxide and carbon monoxide in the pig and human intravesical ureter.

The distribution of nitric oxide synthase (NOS)-immunoreactive (IR) and haemoxygenase (HO)-IR nerves was investigated in the pig and human intravesical ureter (IVU). NOS activity was measured by monitoring the conversion of [3H]-arginine to [3H]-citrulline. Effects of NO and resulting changes in cyclic nucleotide concentrations were assessed in vitro. The effects of carbon monoxide (CO) on IVU motility was also tested. Immunohistochemistry revealed an abundant overall innervation of the IVU and numerous NOS-IR nerves. Nerve trunks were also found expressing immunoreactivity for HO-1, one of the enzymes synthetising CO. Similar profiles of nerve structures expressing immunoreactivities for NOS and tyrosine-hydroxylase (TH), as well as NOS and vasoactive intestinal peptide (VIP) were demonstrated. In the pig IVU, measurement of NOS activity revealed a moderate calcium-dependent catalytic activity, NO and the NO-donor SIN-1 reduced in a concentration-dependent manner serotonin-induced contractions of pig and human IVU, and the spontaneous contractions of pig IVU. In pig IVU strips precontracted with the thromboxane analogue U-46619, tetrodotoxin-sensitive relaxations were abolished by the NOS inhibitor NG-nitro-L-arginine. CO exerted no significant effect on spontaneous or induced contractions in the pig and human IVU. In precontracted strips of the pig and human IVU exposed to SIN-1 or NO, significant increases of cyclic GMP levels were measured in comparison to control preparations. The results suggest that the L-arginine/NO/cyclic GMP pathway may play a role in the regulation of the valve function in the uretero-vesical junction (UVJ). A role for CO in the UVJ has yet to be established.

Adult↗

PubMed1997

Mouse CD23 regulates monocyte activation through an interaction with the adhesion molecule CD11b/CD18.

CD23 is expressed on a variety of hemopoietic cells. Recently, we have reported that blocking CD23 interactions in a murine model of arthritis resulted in a marked improvement of disease severity. Here, we demonstrate that CD11b, the alpha chain of the beta 2 integrin adhesion molecule complex CD11b/CD18 expressed on monocytes interacts with CD23. Using a recombinant fusion protein (ZZ-CD23), murine CD23 was shown to bind to peritoneal macrophages and peripheral blood cells isolated from mice as well as the murine macrophage cell line, RAW. The interactions between mouse ZZ-CD23 and CD11b/CD18-expressing cells were significantly inhibited by anti-CD11b monoclonal antibodies. A functional consequence was then demonstrated by inducing an up-regulation of interleukin-6 (IL-6) production following ZZ-CD23 incubation with monocytes. The addition of Fab fragments generated from the monoclonal antibody CD11b impaired this cytokine production by 50%. Interestingly, a positive autocrine loop was identified as IL-6 was shown to increase CD23 binding to macrophages. These results demonstrate that similar to findings using human cells, murine CD23 binds to the surface adhesion molecule, CD11b, and these interactions regulate biological activities of murine myeloid cells.

Animals↗

PubMed1997

A bacterial signal peptide directs efficient secretion of eukaryotic proteins in the baculovirus expression system.

Escherichia coli remains an organism of choice for the production of recombinant proteins required in large quantities. Whenever possible, secretion is the preferred strategy since it permits easy and efficient purification from the extracellular medium. Our efforts to use E. coli to secrete a human CD23 soluble variant fused to a pair of IgG binding domains via the Staphylococcal protein A signal peptide were unsuccessful. Surprisingly, when the same construct was expressed in the baculovirus system, efficient secretion was observed and cleavage of the signal peptide occurred at the expected site. Varying the genes in the fusions or the tags, or the topology of the gene and the tag, did not affect the high-level secretion and cleavage at the correct site. We envision that fusion of the bacterial signal sequence to eukaryotic recombinant genes will prove to be a tool of value for efficient protein secretion in insect cells using the baculovirus expression system.

Amino Acid Sequence↗

PubMed1997

Clinical assessment of fluorescence cytoscopy during transurethral bladder resection in superficial bladder cancer.

The prognosis of superficial bladder cancer in terms of recurrence and disease progression is related to bladder tumor multiplicity and the presence of concomitant "plane" tumors such as high-grade dysplasia and carcinoma in situ. This study in 33 patients aimed to demonstrate the role of fluorescence cystoscopy in transurethral resection of superficial bladder cancer. The method is based on the detection of protoporphyrin-IX-induced fluorescence in urothelial cancer cells by topical administration of 5-aminolevulinic acid. The sensitivity and the specificity of this procedure on apparently normal mucosa in superficial bladder cancer are estimated to be 82.9% and 81.3%, respectively. Thus, fluorescence cytoscopy is a simple and reliable method for mapping the bladder mucosa, especially in the case of multifocal bladder disease, and it facilitates the screening of occult dysplasia.

Adult↗

PubMed1997

Oxybutynin in the treatment of early detrusor instability after transurethral resection of the prostate.

OBJECTIVE: To evaluate the symptomatic and urodynamic effects of oxybutynin in the control of irritative micturitional symptoms during the first week after transurethral resection of benign prostatic hyperplasia (BPH). PATIENTS AND METHODS: Fifty-three patients (median age 67 years, interquartile range 62-72) were included prospectively in a double-blind placebo-controlled study. Pre-operatively, uroflowmetry and cystometrography (CMG) were performed, and the post-void residual volume (PVR) measured; symptoms were rated according to the Boyarski score. CMG was repeated on the first post-operative day and medication was started on the third day. Before withdrawing the catheter on the fifth day. CMG was repeated. Three days later, symptoms were evaluated according to the Boyarski score and uroflowmetry and the estimate of PVR reassessed. RESULTS: In comparison with placebo, oxybutynin significantly decreased frequency, urgency and detrusor pressure at first sensation of filling. However, oxybutynin did not lower the rate of pre-operative detrusor instability and exerted no effect on the maximal capacity of the bladder and corresponding detrusor pressure. Dryness of mouth was reported in 13% and 65% of patients receiving placebo and oxybutynin, respectively. CONCLUSION: Oxybutynin alleviates early irritative symptoms after transurethral resection of BPH, without consistently modifying bladder urodynamics.

Aged↗

PubMed1997

Safety and efficiency of laparoscopic varicocelectomy in one hundred consecutive cases.

The purpose of this study is to report the safety and efficiency of laparoscopic varicocelectomy in 100 consecutive patients operated at a single teaching institution. There were three indications: (1) infertility associated with oligo-astheno-teratospermia (n = 52); (2) chronic dragging sensation of the left testicle (n = 42), and (3) incidental finding of a large varicocele in young adolescents (n = 6). In group 1, the postoperative pregnancy rate was 47% and sperm quantity, concentration and mobility were significantly increased. The efficiency in pain control and clinical outcome amounted to 80 and 100% in groups 2 and 3, respectively. Complications were few and minor. There was no intraperitoneal organ or major vessel injury. The mean length of hospital stay (0.9 days) and median total recovery time (5 days) were remarkably short. In conclusion, laparoscopic varicocelectomy is a safe procedure. It is as efficient as open spermatic vein ligation, and provides the patient with a short hospital stay and quick full recovery.

Adult↗

PubMed1997

Structure and functions of CD23.

This review summarizes recent data on CD23, a low affinity receptor for IgE (Fc epsilon RII). CD23 is the only FcR which does not belong to the immunoglobulin gene superfamily. The CD23 molecule was discovered independently as an IgE receptor on human lymphoblastoid B cells [1], as a cell surface marker expressed on Epstein-Barr-Virus-transformed B cells (EBVCS) [2] and as a B-cell activation antigen (Blast 2) [3]. CD23 was shown to be a low affinity receptor for IgE [4,5]. Similar to most FcR, soluble forms of CD23 (sCD23) are released into extracellular fluids. The soluble fragments formed by proteolytic cleavage of surface CD23 are not only capable of binding IgE (IgE binding factors) but also exhibit multiple functions that are not IgE related. These observations together with the finding that CD23 displays significant homology with Ca(2+)-dependent (C-type) animal lectins, suggested the existence of natural ligands other than IgE. The recent finding that CD23 interacts with CD21, CD11b and CD11c indicates that CD23 should be viewed not only as a low affinity IgE receptor but also as an adhesion molecule involved in cell-cell interaction. After a brief overview of the molecular structure, there follows a discussion of the biological activities ascribed to human CD23.

Animals↗

PubMed1997

[Vaporization of urethral stenosis using the KTP 532 laser].

The authors treated 16 patients presenting with a total of 20 anterior urethral strictures using the KTP 16 Laser. The aetiology was iatrogenic in 50% of cases, infectious in 20% of cases, traumatic in 20% of cases and unknown in 10% of cases. The stricture was situated in the bulbous urethra (80%), membranous urethra (10%) or penil urethra (10%). Laser vaporization of the urethral stricture was performed over the entire circumference of the urethra when necessary, followed by bladder drainage by urethral catheter for 24 hours. All patients were prospectively reviewed at 3 weeks, 3 months and 6 months (clinical symptoms, uroflowmetry, cystourethrography). A complete symptom and urodynamic success was obtained in 13 patients (81%) at 3 and 6 months. The stricture recurred in 4 patients, but only three of them (19%) required treatment (reoperation of repeat dilatations). The mean maximum flow rate increased from 6 mL/s to 20 mL/s at 3 months and was maintained at 19 mL/s at 6 months. No intraoperative or postoperative complications were observed. In conclusion, our results confirm that KTP 532 laser urethral strictures is a reliable and effective method in the medium term. These good results also suggest an advantage in terms of the recurrence rate in comparison with internal urethrotomy. However, our series needs to be evaluated with a longer follow-up and prospective, randomized trials comparing the two methods need to be conducted.

Adult↗

PubMed1997

[The clinical value of fluorescence cystoscopy in the detection of superficial transitional epithelial cell carcinoma of the bladder].

The prognosis of superficial bladder cancer in terms of local recurrence and transformation into invasive cancer is related to the multiplicity of tumor sites and the concomitant presence of "flat" tumours, such as dysplasia and carcinoma in situ. This study of 51 patients emphasizes the value of fluorescence cystoscopy in the early detection of superficial transitional cell carcinomas. This method is based on induction of fluorescence in carcinomatous sites which selectively accumulate an endogenous photosensitizer, protoporphyrin IX, in response to intravesical administration of its precursor, 5-aminolevulinic acid. The sensitivity of the method in the present series was close to 93%. Fluorescence cystoscopy is a simple and reliable method to map the bladder mucosa looking for areas of dysplasia and carcinoma in situ.

Administration, Intravesical↗