Levels of Free Granulocyte Elastase in Bronchial Secretions from Patients with Cystic Fibrosis: Effect of Antimicrobial Treatment Against Pseudomonas aeruginosa

Large amounts of free granulocyte elastase (GE), an enzyme capable of mediating airway damage, have been found in bronchial secretions of patients with cystic fibrosis who are infected with Pseudomonas aeruginosa. This finding indicates an imbalance between GE and its antiproteases, α1-proteinase inhibitor (α1-PI) and bronchial mucosal inhibitor (BMI), in the airways of these individuals. The effect of intravenous antimicrobial treatment against P. aeruginosa on activity and concentration of GE, BMI, and α1-PI was evaluated in 30 treatment courses of 20 patients with cystic fibrosis. Although sputum volume and level of immunoreactive GE decreased and concentrations of α1-PI and BMI increased significantly (P < .05), a high level of free GE persisted. No active α1-PI and BMI were detectable after treatment. High levels of GE correlated with a poor pulmonary condition (rs = .98, P < .001). In vitro, elastolytic activity of bronchial secretions from patients with cystic fibrosis was significantly inhibited by eglin C and an oxidation-resistant variant of α1-PI, both compounds currently produced by recombinant DNA technolog

The immune-checkpoint HLA-G/ILT4 is involved in the regulation of VEGF expression in clear cell renal cell carcinoma

Background: Clear cell renal cell carcinoma (ccRCC), the most aggressive renal cancer, is characterized by early lymph node metastases and bad prognosis. Most therapies targeting advanced or metastatic ccRCC are based, as first-line treatment, on the administration of the vascular endothelial growth factor (VEGF) neutralizing antibody termed Bevacizumab. Despite proven benefits, the expected results were not obtained for the majority of patients. The possibility that an intricate interplay between angiogenesis and immune-checkpoints might exist lead us to evaluate tumor angiogenesis, by means of VEGF expression together with the immune checkpoint HLA-G/ILT4. Methods: Tumor specimens were obtained from patients from two separate cohorts: One from “Evita Pueblo” Hospital from Berazategui, (Buenos Aires, Argentina) and the second includes patients surgically operated at the Urology Department of Saint-Louis Hospital (Paris, France) with a confirmed ccRCC diagnosis. Immunohistochemistry was performed with specific antibodies directed against HLA-G, VEGF-A, VEGF-C, D240, CD34, ILT4 and Ca-IX. In addition, gene expression levels were measured in a cell line derived from a ccRCC patient by semi-quantitative RT-PCR. Results: Our results show that the highly vascularized tumors of ccRCC patients express high levels of VEGF and the immune-checkpoint HLA-G. In addition, ILT4, one of the HLA-G receptors, was detected on macrophages surrounding tumor cells, suggesting the generation of an immune-tolerant microenvironment that might favor tumorigenesis. Notably, RT-qPCR analysis provided the first evidence on the transcriptional relationship between HLA-G/ILT4 and the VEGF family. Namely, in the presence of HLA-G or ILT4, the levels of VEGF-A are diminished whereas those of VEGF-C are increased. Conclusions: In an effort to find new therapeutic molecules and fight against metastasis dissemination associated with the poor survival rates of ccRCC patients, these findings provide the rationale for co-targeting angiogenesis and the immune checkpoint HLA-G.Facultad de Ciencias Médica

Methods for Establishing a Renal Cell Carcinoma Tumor Spheroid Model With Immune Infiltration for Immunotherapeutic Studies

Tumor spheroids play an increasingly important role in cancer research. Their ability to recapitulate crucial features of tumor biology that are lost in the classically used 2D models along with their relative simplicity and handiness have made them the most studied 3D tumor model. Their application as a theranostic tool or as a means to study tumor-host interaction is now well-established in various cancers. However, their use in the field of Renal Cell Carcinoma (RCC) remains very limited. The aim of this work is to present methods to implement a basic RCC spheroid model. These methods cover the steps from RCC tumor dissociation to spheroid infiltration by immune cells. We present a protocol for RCC dissociation using Liberase TM and introduce a culture medium containing Epithelial Growth Factor and Hydrocortisone allowing for faster growth of RCC primary cells. We show that the liquid overlay technique allows for the formation of spheroids from cell lines and from primary cultures. We present a method using morphological criteria to select a homogeneous spheroid population based on a Fiji macro. We then show that spheroids can be infiltrated by PBMCs after activation with OKT3 or IL-15. Finally, we provide an example of application by implementing an immune spheroid killing assay allowing observing increased spheroid destruction after treatment with PD-1 inhibitors. Thus the straightforward methods presented here allow for efficient spheroid formation for a simple RCC 3D model that can be standardized and infused with immune cells to study immunotherapies

The effects of transurethral resection and cystoprostatectomy on dissemination of epithelial cells in the circulation of patients with bladder cancer

This study was undertaken to evaluate the risk of haematogenous dissemination of epithelial cells induced by endoscopic resection and/or cystoprostatectomy for transitional cell carcinoma of the bladder. Thirty-three patients were studied. Thirty-one had different stages and grades of bladder cancer and two patients had benign bladder conditions. Twenty-five cancer patients required transurethral resection of their bladder tumour. Of those, 20 had superficial disease (pTaG1–G2: n = 19; pT1G2: n = 1) and five had muscle invasive tumours (pT2G3: n = 2; pT3aG3: n = 1; pT4G3: n = 2). Five patients underwent radical cystoprostatectomy for muscle invasive cancers (pT2G3: n = 3; pT3bG3: n = 1; pT4G3: n = 1) and one man received chemotherapy for metastatic disease. Venous blood (10 ml) was obtained from the antecubital fossa in each patient, before and 1–2 h after completion of surgery, and prior to treatment in the metastatic patient. An indirect immunocytochemical technique was used to detect circulating epithelial cells after centrifugation on Ficoll gradient and fixation of mononuclear cells on slides, using a monoclonal antibody directed against three cytokeratins: CK8, CK18 and CK19. Circulating epithelial cells were detected only in the patient with metastatic disease. None of the other patients had evidence of epithelial circulating cells before or after surgery. The results suggest that irrespective of disease stage and grade, neither endoscopic nor open bladder surgery leads to detectable dissemination of urothelial cells in the peripheral circulation. These procedures are therefore unlikely to increase the risk of progression and metastasis in transitional cell carcinoma of the bladder. © 1999 Cancer Research Campaig

Does clinical examination aid in the diagnosis of urinary tract infections in women? A systematic review and meta-analysis

<p>Abstract</p> <p>Background</p> <p>Clinicians should be aware of the diagnostic values of various symptoms, signs and antecedents. This information is particularly important in primary care settings, where sophisticated diagnostic approaches are not always feasible. The aim of the study is to determine the probability that various symptoms, signs, antecedents and tests predict urinary tract infection (UTI) in women.</p> <p>Methods</p> <p>We conducted a systematic search of the MEDLINE and EMBASE databases to identify articles published in all languages through until December 2008. We particularly focused on studies that examined the diagnostic accuracy of at least one symptom, sign or patient antecedent related to the urinary tract. We included studies where urine culture, a gold standard, was preformed by primary care providers on female subjects aged at least 14 years. A meta-analysis of the likelihood ratio was performed to assess variables related to the urinary tract symptoms.</p> <p>Results</p> <p>Of the 1, 212 articles identified, 11 met the selection criteria. Dysuria, urgency, nocturia, sexual activity and urgency with dysuria were weak predictors of urinary tract infection, whereas increases in vaginal discharge and suprapubic pain were weak predictors of the absence of infection. Nitrites or leukocytes in the dipstick test are the only findings that clearly favored a diagnosis of UTI.</p> <p>Conclusions</p> <p>Clinical findings do not aid in the diagnosis of UTI among women who present with urinary symptoms. Vaginal discharge is a weak indicator of the absence of infection. The urine dipstick test was the most reliable tool for detecting UTI.</p