The challenges of recruiting never-smokers with chronic obstructive pulmonary disease from the large population-based Swedish CArdiopulmonary bioImage study (SCAPIS) cohort.

Background: A substantial proportion of individuals with COPD have never smoked, and it is implied to be more common than previously anticipated but poorly studied. Aim: To describe the process of recruitment of never-smokers with COPD from a population-based cohort (n = 30 154). Methods: We recruited never-smokers with COPD, aged 50-75 years, from six University Hospitals, based on: 1) post broncho-dilator forced expiratory volume in 1 second/forced vital capacity (FEV1/FVC) < 0.70 and 2) FEV1 50-100% of predicted value and 3) being never-smokers (self-reported). In total 862 SCAPIS participants were identified, of which 652 were reachable and agreed to a first screening by telephone. Altogether 128 (20%) were excluded due to previous smoking or declined participation. We also applied a lower limit of normal (LLN) of FEV1/FVC (z-score<-1.64) according to the Global Lung Initiative to ensure a stricter definition of airflow obstruction. Results: Data on respiratory symptoms, health status, and medical history were collected from 492 individuals, since 32 were excluded at a second data review (declined or previous smoking), prior to the first visit. Due to not matching the required lung function criteria at a second spirometry, an additional 334 (68%) were excluded. These exclusions were by reason of: FEV1/FVC ≥0.7 (49%), FEV1 > 100% of predicted (26%) or z-score ≥ -1,64 (24%). Finally, 154 never-smokers with COPD were included: 56 (36%) women, (mean) age 60 years, FEV1 84% of predicted, FEV1/FVC: 0.6, z-score: -2.2, Oxygen saturation: 97% and BMI: 26.8 kg/m2. Conclusions: The challenges of a recruitment process of never-smokers with COPD were shown, including the importance of correct spirometry testing and strict inclusion criteria. Our findings highlight the importance of repeated spirometry assessments for improved accuracy in diagnosing COPD.

Development of in vitro methods to model the impact of vaginal lactobacilli on Staphylococcus aureus biofilm formation on menstrual cups as well as validation of recommended cleaning directions.

Introduction: Menstrual cups (MC) are a reusable feminine hygiene product. A recent publication suggested that Staphylococcus aureus (S. aureus) biofilms can form on MCs which may lead to increased risk of menstrual Toxic Shock Syndrome (mTSS). Additionally, there is concern that buildup of residual menses may contribute to microbial growth and biofilm formation further increasing mTSS risk. Quantitative and qualitative analysis of in vitro tests were utilized to determine if S. aureus biofilm could form on MC in the presence of the keystone species Lactobacillus after 12 h of incubation. The methodology was based on a modification of an anaerobic in vitro method that harnesses the keystone species hypothesis by including a representative of vaginal lactic acid bacteria. Methods: MCs were incubated anaerobically for 12 h in Vaginal Defined Media (VDM) with the two morphologically distinct bacteria, Lactobacillus gasseri (L. gasseri) and S. aureus. Colony Forming Units (CFU) for each organism from the VDM broth and sonicated MC were estimated. In addition, a separate experiment was conducted where S. aureus was grown for 12 h in the absence of L. gasseri. Qualitative analysis for biofilm formation utilized micro-CT (µ-CT) and cryogenic scanning electron microscopy (Cryo-SEM). Results: Samples collected from the media control had expected growth of both organisms after 12 h of incubation. Samples collected from VDM broth were similar to media control at the end of the 12-h study. Total S. aureus cell density on MC following sonication/rinsing was minimal. Results when using a monoculture of S. aureus demonstrated that there was a significant growth of the organism in the media control and broth as well as the sonicated cups indicating that the presence of L. gasseri was important for controlling growth and adherence of S. aureus. Few rod-shaped bacteria (L. gasseri) and cocci (S. aureus) could be identified on the MCs when grown in a dual species culture inoculum and no biofilm was noted via µ-CT and cryo-SEM. Additionally, efforts to model and understand the validity of the current labeled recommendations for MC cleaning in-between uses are supported. Discussion: The data support continued safe use of the Tampax® cup when used and maintained as recommended.

Reduced bronchoalveolar macrophage phagocytosis and cytotoxic effects after controlled short-term exposure to wood smoke in healthy humans.

BACKGROUND: Exposure to wood smoke has been shown to contribute to adverse respiratory health effects including airway infections, but the underlying mechanisms are unclear. A preceding study failed to confirm any acute inflammation or cell influx in bronchial wash (BW) or bronchoalveolar lavage (BAL) 24 h after wood smoke exposure but showed unexpected reductions in leukocyte numbers. The present study was performed to investigate responses at an earlier phase, regarding potential development of acute inflammation, as well as indications of cytotoxicity. METHODS: In a double-blind, randomised crossover study, 14 healthy participants were exposed for 2 h to filtered air and diluted wood smoke from incomplete wood log combustion in a common wood stove with a mean particulate matter concentration of 409 µg/m3. Bronchoscopy with BW and BAL was performed 6 h after exposure. Differential cell counts, assessment of DNA-damage and ex vivo analysis of phagocytic function of phagocytosing BAL cells were performed. Wood smoke particles were also collected for in vitro toxicological analyses using bronchial epithelial cells (BEAS-2B) and alveolar type II-like cells (A549). RESULTS: Exposure to wood smoke increased BAL lactate dehydrogenase (LDH) (p = 0.04) and reduced the ex vivo alveolar macrophage phagocytic capacity (p = 0.03) and viability (p = 0.02) vs. filtered air. BAL eosinophil numbers were increased after wood smoke (p = 0.02), while other cell types were unaffected in BW and BAL. In vitro exposure to wood smoke particles confirmed increased DNA-damage, decreased metabolic activity and cell cycle disturbances. CONCLUSIONS: Exposure to wood smoke from incomplete combustion did not induce any acute airway inflammatory cell influx at 6 h, apart from eosinophils. However, there were indications of a cytotoxic reaction with increased LDH, reduced cell viability and impaired alveolar macrophage phagocytic capacity. These findings are in accordance with earlier bronchoscopy findings at 24 h and may provide evidence for the increased susceptibility to infections by biomass smoke exposure, reported in population-based studies.

Safety assessment scheme for menstrual cups and application for the evaluation of a menstrual cup comprised of medical grade silicone.

BACKGROUND: Ensuring menstrual cup safety is paramount, yet a menstrual cup safety assessment scheme is lacking. This paper presents a quadripartite scheme, showing how it can be applied. METHODS: The Tampax Menstrual Cup was evaluated in the safety assessment scheme: (1) Biocompatibility and chemical safety of cup constituents. Extractables were obtained under different use condition; exposure-based risk assessments (EBRA) were conducted for extractables exceeding thresholds of toxicological concern. (2) Physical impact to vaginal mucosa. After physical evaluations, the Tampax Cup and another cup were assessed in a randomised double-blinded, two-product, two-period cross-over clinical trial (65 women, mean age 34.2 years). (3) Impact to vaginal microbiota (in vitro mixed microflora assay and evaluation of vaginal swabs). (4) In vitro growth of Staphylococcus aureus and toxic shock syndrome toxin-1 (TSST-1) production. FINDINGS: Biocompatibility assessments and EBRA of cup constituents showed no safety concerns. In the randomised clinical trial, all potentially product-related adverse effects were mild, vaginal exams were unremarkable, no clinically relevant pH changes occurred, post-void residual urine volume with and without cup were similar, and self-reported measures of comfort along with reports of burning, itching and stinging between cups were comparable. Cup use had no effect on microbial growth in vitro or in the 62 subjects who completed the trial or on in vitro TSST-1 production. INTERPRETATION: The quadripartite safety assessment scheme allows evaluation of menstrual cup safety. The Tampax Cup is safe and well-tolerated upon intended use. As with all feminine hygiene products, post-market safety surveillance confirmed this conclusion. FUNDING: By Procter & Gamble.

Design of a PDZbody, a bivalent binder of the E6 protein from human papillomavirus.

Chronic infection by high risk human papillomavirus (HPV) strains may lead to cancer. Expression of the two viral oncoproteins E6 and E7 is largely responsible for immortalization of infected cells. The HPV E6 is a small (approximately 150 residues) two domain protein that interacts with a number of cellular proteins including the ubiquitin ligase E6-associated protein (E6AP) and several PDZ-domain containing proteins. Our aim was to design a high-affinity binder for HPV E6 by linking two of its cellular targets. First, we improved the affinity of the second PDZ domain from SAP97 for the C-terminus of HPV E6 from the high-risk strain HPV18 using phage display. Second, we added a helix from E6AP to the N-terminus of the optimized PDZ variant, creating a chimeric bivalent binder, denoted PDZbody. Full-length HPV E6 proteins are difficult to express and purify. Nevertheless, we could measure the affinity of the PDZbody for E6 from another high-risk strain, HPV16 (Kd = 65 nM). Finally, the PDZbody was used to co-immunoprecipitate E6 protein from HPV18-immortalized HeLa cells, confirming the interaction between PDZbody and HPV18 E6 in a cellular context.

Rapid detection of Salmonella in pet food: design and evaluation of integrated methods based on real-time PCR detection.

Reducing the risk of Salmonella contamination in pet food is critical for both companion animals and humans, and its importance is reflected by the substantial increase in the demand for pathogen testing. Accurate and rapid detection of foodborne pathogens improves food safety, protects the public health, and benefits food producers by assuring product quality while facilitating product release in a timely manner. Traditional culture-based methods for Salmonella screening are laborious and can take 5 to 7 days to obtain definitive results. In this study, we developed two methods for the detection of low levels of Salmonella in pet food using real-time PCR: (i) detection of Salmonella in 25 g of dried pet food in less than 14 h with an automated magnetic bead-based nucleic acid extraction method and (ii) detection of Salmonella in 375 g of composite dry pet food matrix in less than 24 h with a manual centrifugation-based nucleic acid preparation method. Both methods included a preclarification step using a novel protocol that removes food matrix-associated debris and PCR inhibitors and improves the sensitivity of detection. Validation studies revealed no significant differences between the two real-time PCR methods and the standard U.S. Food and Drug Administration Bacteriological Analytical Manual (chapter 5) culture confirmation method.

Indoleamine 2,3-dioxygenase contributes to tumor cell evasion of T cell-mediated rejection.

The priming of an appropriate anti-tumor T cell response rarely results in the rejection of established tumors. The characteristics of tumors that allow them to evade a T cell-mediated rejection are unknown for many tumors. We report on evidence that the expression of the immunosuppressive enzyme, indoleamine 2,3-dioxygenase (IDO) by mononuclear cells that invade tumors and tumor-draining lymph nodes, is 1 mechanism that may account for this observation. Lewis lung carcinoma (LLC) cells stimulated a more robust allogeneic T cell response in vitro in the presence of a competitive inhibitor of IDO, 1-methyl tryptophan. When administered in vivo this inhibitor also resulted in delayed LLC tumor growth in syngeneic mice. Our study provides evidence for a novel mechanism whereby tumors evade rejection by the immune system, and suggests the possibility that inhibiting IDO may be developed as an anti-cancer immunotherapeutic strategy.

Phase I trial of a B7-1 (CD80) gene modified autologous tumor cell vaccine in combination with systemic interleukin-2 in patients with metastatic renal cell carcinoma.

PURPOSE: A reason that the immune system may fail to reject tumors is that T cells encounter tumor antigen derived peptides on the surface of tumor cells in a tolerizing rather than activating context since tumor cells do not express T cell co-stimulatory molecules such as B7-1 (CD80). In preclinical models over expression of B7-1 on the surface of tumor cells has been shown to activate T cells which kill tumor cells. We conducted a phase I clinical trial testing this approach in patients with metastatic renal cell carcinoma. MATERIALS AND METHODS: Resected tumors from 15 patients were disaggregated and adapted to tissue culture, transduced with the B7-1 gene and injected subcutaneously as a vaccine. The dose of the vaccine was escalated in 3 separate cohorts of patients, and systemic interleukin-2 (IL-2) was administered as an adjuvant designed to enhance the proliferation of the vaccine activated T cells. RESULTS: Of the 15 patients 9 had measurable disease, 2 had a partial response and 2 had stable disease. Perivascular T cell infiltrates at autologous tumor delayed type hypersensitivity skin test sites developed in 3 of the 4 patients with stable disease or partial response. Although the patients experienced the usual and expected toxicity from the IL-2, there was no significant toxicity observed with the vaccine. CONCLUSIONS: The B7-1 gene modified autologous tumor cell vaccine is safe and can be combined with systemic IL-2 with acceptable toxicity. Immunological and clinical responses were observed in some of the patients. A phase II trial is reasonable to determine the efficacy of this approach.