Potential application of green extracts rich in phenolics for innovative functional foods: natural deep eutectic solvents as media for isolation of biocompounds from berries.

The health-promoting effects of berries have attracted attention due to the possible application of their extracts as functional ingredients in food products. Natural deep eutectic solvents (NADESs) are a new generation of environmentally friendly solvents for the extraction of natural products, and they are green alternatives to organic solvents, and they can improve the solubility, stability, and bioavailability of isolated biocompounds. In this study, an efficient eco-friendly method was used for the extraction of phenolic compounds from different berries: chokeberries, blueberries, and black goji berries with a range of eutectic solvents consisting of hydrogen bond acceptors (HBAs) such as choline chloride, L-proline, L-glycine, and L-lysine and hydrogen bond donors (HBDs) such as malic, citric, tartaric, lactic and succinic acids, glucose and glycerol. The obtained results indicated the ability of NADESs towards selective extraction of phenolics; the eutectic system choline chloride : malic acid showed selective extraction of anthocyanins, while choline chloride : glycerol and choline chloride : urea showed selectivity towards flavonoids and phenolic acids. The methodology for screening of the NADES extraction performance, which included chromatographic profiling via high-performance thin layer chromatography combined with chemometrics and spectrophotometric essays, allowed effective assessment of optimal eutectic solvents for isolation of different groups of phenolics. Great antioxidant and antimicrobial activities of extracts, along with the green nature of eutectic solvents, enable NADES berry extracts to be used as "green-labelled" functional foods or ingredients.

Facilitation of Symplastic Effector Protein Mobility by Paired Effectors Is Conserved in Different Classes of Fungal Pathogens.

It has been discovered that plant pathogens produce effectors that spread via plasmodesmata (PD) to allow modulation of host processes in distal uninfected cells. Fusarium oxysporum f. sp. lycopersici (Fol) facilitates effector translocation by expansion of the size-exclusion limit of PD using the Six5/Avr2 effector pair. How other fungal pathogens manipulate PD is unknown. We recently reported that many fungal pathogens belonging to different families carry effector pairs that resemble the SIX5/AVR2 gene pair from Fol. Here, we performed structural predictions of three of these effector pairs from Leptosphaeria maculans (Lm) and tested their ability to manipulate PD and to complement the virulence defect of a Fol SIX5 knockout mutant. We show that the AvrLm10A homologs are structurally related to FolSix5 and localize at PD when they are expressed with their paired effectors. Furthermore, these effectors were found to complement FolSix5 function in cell-to-cell mobility assays and in fungal virulence. We conclude that distantly related fungal species rely on structurally related paired effector proteins to manipulate PD and facilitate effector mobility. The wide distribution of these effector pairs implies Six5-mediated effector translocation to be a conserved propensity among fungal plant pathogens. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The PTI-suppressing Avr2 effector from Fusarium oxysporum suppresses mono-ubiquitination and plasma membrane dissociation of BIK1.

Plant pathogens use effector proteins to target host processes involved in pathogen perception, immune signalling, or defence outputs. Unlike foliar pathogens, it is poorly understood how root-invading pathogens suppress immunity. The Avr2 effector from the tomato root- and xylem-colonizing pathogen Fusarium oxysporum suppresses immune signalling induced by various pathogen-associated molecular patterns (PAMPs). It is unknown how Avr2 targets the immune system. Transgenic AVR2 Arabidopsis thaliana phenocopies mutants in which the pattern recognition receptor (PRR) co-receptor BRI1-ASSOCIATED RECEPTOR KINASE (BAK1) or its downstream signalling kinase BOTRYTIS-INDUCED KINASE 1 (BIK1) are knocked out. We therefore tested whether these kinases are Avr2 targets. Flg22-induced complex formation of the PRR FLAGELLIN SENSITIVE 2 and BAK1 occurred in the presence and absence of Avr2, indicating that Avr2 does not affect BAK1 function or PRR complex formation. Bimolecular fluorescence complementation assays showed that Avr2 and BIK1 co-localize in planta. Although Avr2 did not affect flg22-induced BIK1 phosphorylation, mono-ubiquitination was compromised. Furthermore, Avr2 affected BIK1 abundance and shifted its localization from nucleocytoplasmic to the cell periphery/plasma membrane. Together, these data imply that Avr2 may retain BIK1 at the plasma membrane, thereby suppressing its ability to activate immune signalling. Because mono-ubiquitination of BIK1 is required for its internalization, interference with this process by Avr2 could provide a mechanistic explanation for the compromised BIK1 mobility upon flg22 treatment. The identification of BIK1 as an effector target of a root-invading vascular pathogen identifies this kinase as a conserved signalling component for both root and shoot immunity.

The primary function of Six5 of Fusarium oxysporum is to facilitate Avr2 activity by together manipulating the size exclusion limit of plasmodesmata.

Pathogens produce effector proteins to manipulate their hosts. While most effectors act autonomously, some fungal effectors act in pairs and rely on each other for function. During the colonization of the plant vasculature, the root-infecting fungus Fusarium oxysporum (Fo) produces 14 so-called Secreted in Xylem (SIX) effectors. Two of these effector genes, Avr2 (Six3) and Six5, form a gene pair on the pathogenicity chromosome of the tomato-infecting Fo strain. Avr2 has been shown to suppress plant defense responses and is required for full pathogenicity. Although Six5 and Avr2 together manipulate the size exclusion limit of plasmodesmata to facilitate cell-to-cell movement of Avr2, it is unclear whether Six5 has additional functions as well. To investigate the role of Six5, we generated transgenic Arabidopsis lines expressing Six5. Notably, increased susceptibility during the early stages of infection was observed in these Six5 lines, but only to Fo strains expressing Avr2 and not to wild-type Arabidopsis-infecting Fo strains lacking this effector gene. Furthermore, neither PAMP-triggered defense responses, such as ROS accumulation and callose deposition upon treatment with Flg22, necrosis and ethylene-inducing peptide 1-like protein (NLP), or chitosan, nor susceptibility to other plant pathogens, such as the bacterium Pseudomonas syringae or the fungus Verticilium dahlia, were affected by Six5 expression. Further investigation of the ability of the Avr2/Six5 effector pair to manipulate plasmodesmata (PD) revealed that it not only permits cell-to-cell movement of Avr2, but also facilitates the movement of two additional effectors, Six6 and Six8. Moreover, although Avr2/Six5 expands the size exclusion limit of plasmodesmata (i.e., gating) to permit the movement of a 2xFP fusion protein (53 kDa), a larger variant, 3xFP protein (80 kDa), did not move to the neighboring cells. The PD manipulation mechanism employed by Avr2/Six5 did not involve alteration of callose homeostasis in these structures. In conclusion, the primary function of Six5 appears to function together with Avr2 to increase the size exclusion limit of plasmodesmata by an unknown mechanism to facilitate cell-to-cell movement of Fo effectors.

Air pollution and surrounding greenness in relation to ischemic stroke: A population-based cohort study.

BACKGROUND: Evidence for the association between environmental exposures and ischemic stroke (IS) is limited and inconsistent. We aimed to assess the relationship between exposure to air pollutants, residential surrounding greenness, and incident IS, and to identify population subgroups particularly sensitive to these exposures. METHODS: We used data from administrative health registries of the public healthcare system in Catalonia, Spain to construct a cohort of individuals aged 18 years and older without a previous stroke diagnosis at 1st January 2016 (n = 3 521 274). We collected data on sociodemographic characteristics and cerebrovascular risk factors, and derived exposure at the participant's residence to ambient levels of fine particulate matter (PM2.5), black carbon (BC), nitrogen dioxide (NO2), and Normalized Difference Vegetation Index (NDVI) in a 300 m buffer as an indicator of greenness. The primary outcome was IS diagnosis at any point during the follow-up. We used Cox proportional hazards models to estimate associations between environmental exposures and incident IS and stratified analyses to investigate effect modification. RESULTS: Between 1st January 2016 and 31st December 2017, 10 865 individuals were admitted to public hospitals with an IS diagnosis. Median exposure levels were: 17 µg/m3 PM2.5, 35 µg/m3 NO2, 2.28 µg/m3 BC and 0.27 NDVI. Individuals with higher residential exposure to air pollution were at greater risk of IS: HR 1·04 (95% CI:0·99-1·10) per 5 µg/m3 of PM2.5; HR 1.05 (95% CI:1·00-1·10) per 1 µg/m3 of BC; HR 1·04 (95% CI:1·03-1·06) per 10 µg/m3 of NO2. Conversely, individuals with higher residential surrounding green space, had lower risk of IS (HR 0·84; CI 95%:0·7-1.0). There was no evidence of effect modification by individual characteristics. CONCLUSIONS: Higher incidence of IS was observed in relation to long-term exposures to air pollution, particularly NO2, in a region that meets European health-based air quality standards. Residential surrounding greenness was associated with lower incidence of IS.

Digital phenotyping and the COVID-19 pandemic: Capturing behavioral change in patients with psychiatric disorders.

The COVID-19 pandemic has led to unprecedented societal changes limiting us in our mobility and our ability to connect with others in person. These unusual but widespread changes provide a unique opportunity for studies using digital phenotyping tools. Digital phenotyping tools, such as mobile passive monitoring platforms (MPM), provide a new perspective on human behavior and hold promise to improve human behavioral research. However, there is currently little evidence that these tools can reliably detect changes in behavior. Considering the Considering the COVID-19 pandemic as a high impact common environmental factor we studied potential impact on behavior of participants using our mobile passive monitoring platform BEHAPP that was ambulatory tracking them during the COVID-19 pandemic. We pooled data from three MPM studies involving Schizophrenia (SZ), Major Depressive Disorder (MDD) and Bipolar Disorder (BD) patients (N = 12). We compared the data collected on weekdays during three weeks prior and three weeks subsequent to the start of the quarantine. We hypothesized an increase in communication and a decrease in mobility. We observed a significant increase in the total time spent on communication applications (median 179 and 243 min per week respectively, p = 0.005), and a significant decrease in the number of unique places visited (median 6 and 3 visits per week respectively, p = 0.007), while the total time spent at home did not change significantly (median 64 and 77 h per week, respectively, p = 0.594). The data provides a proof of principle that digital phenotyping tools can identify changes in human behavior incited by a common external environmental factor.

Development and Characterization of a Model for Inducing Fetal Hemoglobin Production in Cynomolgus Macaques (Macaca fasicularis).

Hydroxyurea induces production of fetal hemoglobin (HbF), a tetramer of α and γ globin proteins and corresponding heme molecules, normally found in less than 1% of adult RBC. Increases in circulating HbF are correlated with clinical improvement of patients with hemoglobinopathies, and hydroxyurea, as a daily medication, is the standard treatment for sickle cell anemia. Although olive baboons (Papio anubis) are considered a key model species for HbF induction, cynomolgus macaques (Macaca fasicularis) are another species that conserves the ability to produce HbF into maturity. In this study, moderate anemia was experimentally induced in cynomolgus macaques by phlebotomy, to stimulate accelerated erythropoiesis and HbF production. In contrast to previous studies, vascular access ports were implanted for phlebotomy of conscious monkeys, followed by fluid replacement. As total Hgb levels dropped, reticulocyte counts and the percentage of HbF-expressing cells increased. Once total Hgb levels declined to less than 8 g/dL, 2 courses of oral hydroxyurea (once daily for 5 d) were completed, with a 9-d interval between courses. After hydroxyurea dosing, the percentage of HbF-expressing cells and total HbF were increased significantly. In addition, a significant but transient decrease in reticulocyte count and a transient increase in MCV occurred, replicating the characteristic response of patients receiving hydroxyurea. Daily clinical observations revealed no serious health issues or decreases in food consumption or activity levels. Methods were established for assessing the patency of vascular access ports. This study details a new protocol for the safe and routine induction of moderate anemia in cynomolgus macaques and validates its use in the investigation of novel pharmacologic entities to induce the production of HbF.

The Fusarium oxysporum Avr2-Six5 Effector Pair Alters Plasmodesmatal Exclusion Selectivity to Facilitate Cell-to-Cell Movement of Avr2.

Pathogens use effector proteins to manipulate their hosts. During infection of tomato, the fungus Fusarium oxysporum secretes the effectors Avr2 and Six5. Whereas Avr2 suffices to trigger I-2-mediated cell death in heterologous systems, both effectors are required for I-2-mediated disease resistance in tomato. How Six5 participates in triggering resistance is unknown. Using bimolecular fluorescence complementation assays we found that Avr2 and Six5 interact at plasmodesmata. Single-cell transformation revealed that a 2xRFP marker protein and Avr2-GFP only move to neighboring cells in the presence of Six5. Six5 alone does not alter plasmodesmatal transduction as 2xRFP was only translocated in the presence of both effectors. In SIX5-expressing transgenic plants, the distribution of virally expressed Avr2-GFP, and subsequent onset of I-2-mediated cell death, differed from that in wild-type tomato. Taken together, our data show that in the presence of Six5, Avr2 moves from cell to cell, which in susceptible plants contributes to virulence, but in I-2 containing plants induces resistance.

Visualization and Quantification of Cell-to-cell Movement of Proteins in Nicotiana benthamiana.

Cell-to-cell movement of proteins through plasmodesmata is a widely-established mechanism for intercellular signaling in plants. Current techniques to study intercellular protein translocation rely on single-cell transformation using particle bombardment or transgenic lines expressing photo-inducible fluorophores. The method presented here allows visualization and objective quantification of (effector) protein movement between N. benthamiana leaf cells. Agroinfiltration is performed using a single binary vector encoding a GFP-tagged protein of interest that is either mobile or non-mobile (MP; non-MP), together with an ER-anchored mCherry. Upon creation of mosaic-like transformation patterns, cell-to-cell movement of the MP can be followed by monitoring translocation of the GFP signal from mCherry labeled transformed cells into neighboring non-transformed cells. This process can be visualized using confocal microscopy and quantified following protoplast isolation and flow cytometric cell analysis. This method overcomes the limitations of existing methods as it allows rapid and objective quantification of protein translocation without the need of creating transgenic plants.

Knowledge Management Framework for Emerging Infectious Diseases Preparedness and Response: Design and Development of Public Health Document Ontology.

BACKGROUND: There are increasing concerns about our preparedness and timely coordinated response across the globe to cope with emerging infectious diseases (EIDs). This poses practical challenges that require exploiting novel knowledge management approaches effectively. OBJECTIVE: This work aims to develop an ontology-driven knowledge management framework that addresses the existing challenges in sharing and reusing public health knowledge. METHODS: We propose a systems engineering-inspired ontology-driven knowledge management approach. It decomposes public health knowledge into concepts and relations and organizes the elements of knowledge based on the teleological functions. Both knowledge and semantic rules are stored in an ontology and retrieved to answer queries regarding EID preparedness and response. RESULTS: A hybrid concept extraction was implemented in this work. The quality of the ontology was evaluated using the formal evaluation method Ontology Quality Evaluation Framework. CONCLUSIONS: Our approach is a potentially effective methodology for managing public health knowledge. Accuracy and comprehensiveness of the ontology can be improved as more knowledge is stored. In the future, a survey will be conducted to collect queries from public health practitioners. The reasoning capacity of the ontology will be evaluated using the queries and hypothetical outbreaks. We suggest the importance of developing a knowledge sharing standard like the Gene Ontology for the public health domain.

Coccidioidomycosis in an Indoor-housed Rhesus Macaque (Macaca mulatta).

Coccidioides spp. are saprophytic, dimorphic fungi that are endemic to arid climates, are capable of infecting many species, and result in diverse clinical presentations. An indoor-housed laboratory rhesus macaque presented with weight loss and decreased activity and appetite. During the diagnostic evaluation, a bronchiolar-alveolar pattern in the cranial lung lobes, consistent with bronchopneumonia, was noted on radiographs. Given the poor prognosis, the macaque was euthanized. Confirming the radiographic assessment, gross necropsy findings included multifocal to coalescing areas of consolidation in the right and left cranial lung lobes. Microscopically, the consolidated regions were consistent with a pyogranulomatous bronchopneumonia and contained round, nonbudding, fungal yeast structures considered to be morphologically consistent with Coccidioides immitis. Culture and colony morphology results were confirmed through additional diagnostic testing. Sequencing of the D1-D2 domain of the 28S large ribosomal subunit positively matched with a known sequence specific to C. immitis. Serology for Coccidioides spp. by both latex agglutination (IgM) and immunodiffusion (IgG) was positive. In this rhesus macaque, the concordant results from histology, culture, DNA sequencing, and serology were collectively used to confirm the diagnosis of coccidioidomycosis. This animal likely acquired a latent pulmonary infection with Coccidioides months prior to arrival, when housed outdoors in a Coccidioides-endemic area. The nonspecific clinical presentation in this macaque, coupled with the recent history of indoor housing and lag between clinical presentation and outdoor housing, can make similar diagnostic cases challenging and highlights the need for awareness regarding animal source when making an accurate diagnosis in an institutional laboratory setting.

Low Ankle-Brachial Index is a Simple Physical Exam Sign Predicting Intracranial Atherosclerotic Stenosis in Ischemic Stroke Patients.

BACKGROUND: The investigation of ischemic stroke etiology is commonly limited to the heart and extracranial vessels. Nevertheless, the diagnosis of intracranial stenosis may carry important therapeutic implications. The aims of this study were to determine the prevalence and clinical predictors of intracranial atherosclerotic stenosis (ICAS) in a sample of patients with ischemic stroke. METHODS: Consecutive patients admitted to a university-based outpatient stroke clinic underwent CT angiography of the intracranial and extracranial brain vessels. Clinical, demographic, and laboratory characteristics were compared between patients with increasing levels of stenosis. Ankle-brachial index (ABI) was measured to quantify peripheral arterial disease, defined as an ABI less than or equal to .9. Multivariable ordinal logistic regression was constructed to predict increasing stenosis grades (none, 1%-49%-mild, 50%-69%-moderate, 70%-100%-severe). RESULTS: We studied 106 subjects, mean age 62 ± 15 years, 54% female. ICAS was present in 38 (36%) patients: 19 (50%) mild, 7 (18%) moderate, and 12 (32%) severe. Of 74 patients where ABI was measured, low ABI was found more frequently with increasing ICAS severity (26%, 42%, 67%, and 89% of patients with none, mild, moderate, and severe ICAS, respectively). In univariable analysis, higher age, presence of diabetes, abdominal obesity, and low ABI correlated with increasing stenosis grades. In multivariable analysis, only low ABI remained independently associated with increasing stenosis grades. CONCLUSIONS: The ABI is independently associated with increasing severity of ICAS, making it a potentially useful triaging tool for more invasive test selection.

Monitoring changes in anti-tuberculosis treatment: associated factors determined at the time of diagnosis.

OBJECTIVES: To determine predictive factors for changes in standard anti-tuberculosis chemotherapy at the time of diagnosis. METHODS: A prospective study was performed among tuberculosis (TB) patients treated at specialised centres during 2008-2009. Treatment outcome was monitored per standard guidelines. Treatment was considered successful if the patient was cured or completed treatment. Factors associated with treatment modification were analysed at the bivariate and multivariate levels using logistic regression. RESULTS: A total of 427 patients were included in the study. The initial standard treatment regimen was retained for 249 patients (58.3%), extended to 9 months for 36 (8.4%) and changed for 142 (33.3%). Factors associated with a change of regimen at the multivariate level were female sex, age ≥ 50 years, human immunodeficiency virus infection, comorbidities, alcoholism, hospitalisation and culture-positive sputum. Drug resistance and toxicity were analysed independently. Treatment outcome was successful in 97.2% of cases without a regimen change and in 87.3% of those with a changed regimen (P < 0.001). CONCLUSION: Factors associated with changes in the initial anti-tuberculosis regimen should be considered for rigorous follow-up. Results obtained through individualised treatment provided by specialists were good despite the complexity of the cases treated.

Assessment of luteal function in the vervet monkey as a means to develop a model for obesity-related reproductive phenotype.

The objective of the current study was to characterize luteal function in vervet monkeys. Urine from 12 adult female vervets housed at an academic research center was collected for 10 weeks from single-caged monkeys in order to assess evidence of luteal activity (ELA) as determined by urinary excretion of pregnanediol glucuronide (Pdg) and estrone conjugates (E1c). Dual energy X-ray absorptiometry (DXA) was performed on the monkeys to assess body composition, bone density, and fat mass. Menstrual cyclicity was determined using records of vaginal bleeding. ELA was observed in 9 monkeys and was characterized by a late follicular rise in E1c followed by a progressive increase in Pdg excretion. Mean menstrual cycle length was 26.7 ± 3.8 days and the average day of luteal transition was 14 ± 1.8. Three monkeys without ELA had a clearly defined E1c rise (mean 12-fold from nadir) followed by an E1c drop that was not accompanied by Pdg rise and coincided with vaginal bleeding. Among the 9 ELA monkeys, excretion of E1c tended to negatively associate with fat mass, although this finding did not reach statistical significance (r = -0.61, p = 0.08). Similar to women, vervet monkeys experience an increase in E1c late in the follicular phase of the menstrual cycle which is followed by a subsequent luteal Pdg peak. Assessment of urinary reproductive hormones allows for identification of cardinal menstrual cycle events; thus, the similarity of vervet cycles to human menstrual cycles makes them a useful model for obesity-related human reproductive impairment.

Expression and regulation of Kit ligand in the ovary of the hen.

The Kit system, composed of Kit ligand (KL) and its tyrosine kinase receptor, cKit, has been well characterized in mammals. Studies have shown that it is involved in signaling between the oocyte and somatic cells during the process of follicle maturation. We characterized KL mRNA expression during follicle maturation in the domestic hen, examined regulation of KL and a possible function of the Kit system. KL mRNA expression was assessed using quantitative PCR (n=4 replicates) in follicles of various sizes (1, 3, 5, 6-12 mm, F1). Expression of KL mRNA decreased significantly (p<0.01) with follicle development and was highest in <1 mm follicles, which contained the theca as well as granulosa layers, with high levels also found in the granulosa layer of 3 mm follicles and ovarian stroma. To study regulation of KL mRNA, granulosa cells from 6-8 mm follicles (n=4 replicates) were plated in M199 plus 0.1% BSA in the presence of various treatments including: oocyte conditioned medium (OCM), Vitamin D(3), FSH, estradiol, progesterone and testosterone. OCM caused a dose-related increase (p<0.05) in expression of KL mRNA; Vitamin D(3) increased and FSH decreased expression of KL mRNA. cKit was detected (at the expected size) in the theca layer of 3-5 mm follicles and in a lysate of whole <1mm follicles. Culture of granulosa cells in the presence of OCM resulted in a decrease of P4 secretion, an effect blocked by pre-incubation of OCM with cKit antibody. Although OCM caused a dose-related increase in E2 secretion from theca, this was not blocked by cKit antibody.

Diagnosing TB infection in children: analysis of discordances using in vitro tests and the tuberculin skin test.

The aim of the present study was to compare the performance of the interferon (IFN)-γ tests (QuantiFERON®-TB Gold In-Tube (QFT-G-IT) and T-SPOT®.TB) with the tuberculin skin test (TST) in diagnosing tuberculosis (TB) infection in children, and to analyse discordant results. This was a prospective study including 98 children from contact-tracing studies and 68 children with TST indurations ≥ 5 mm recruited during public health screenings. Positive IFN-γ tests results were associated with risk of exposure (p<0.0001). T-SPOT.TB was positive in 11 (78.6%) out of 14 cases with active TB and QFT-G-IT in nine (64.3%) out of 14 cases. Sensitised T-cells against Mycobacterium avium were detected in six out of 12 children not vaccinated with bacille Calmette-Guérin (BCG), a TST induration 5-9 mm in diameter and both IFN-γ tests negative. In concordant IFN-γ tests results, a positive correlation was found (p = 0.0001) between the number of responding cells and the amount of IFN-γ released. However, in discordant IFN-γ tests results this correlation was negative (p = 0.371): an increase in the number of spot-forming cells correlated with a decrease in the amount of IFN-γ released. The use of IFN-γ tests is helpful for the diagnosis of TB infection, avoiding cross-reactions with BCG immunisation and nontuberculous mycobacterial infections. The analysis of highly discordant results requires further investigation to elucidate possible clinical implications.

Does the presence of unwanted dermal fibroblasts limit the usefulness of autologous epidermal keratinocyte grafts?

OBJECTIVES: Fibroblasts sometimes occur after enzymatic isolation of epidermis. They proliferate quickly, overgrowing the culture. A pure epithelial culture is essential for therapy using a keratinocyte graft. The aim of this study was to determine the possibility of fibroblast elimination from culture to prevent fibroblast overgrowth and obtain a pure monolayer of keratinocytes. MATERIAL AND METHODS: We analyzed three epidermal-derived cultures. Cells were cultured in medium contained Dulbecco's Modified Eagle Medium (DMEM) and Ham's F-12 at a 3:1 ratio with 5% autologous serum and additives. The epithelial culture was confirmed using pancytokeratin MMF. If fibroblast like cells were present, they were removed using 0.01% edetate disodium dihydrate (Na2EDDA). This procedure was repeated until we obtained pure primary keratinocyte cultures. RESULTS: Fibroblast detachment was observed after Na2EDDA treatment. The procedure was performed twice and pure primary cultures of keratinocyte were achieved in two cases. These two cultures maintained their epithelial-like morphology and cytokeratin expression. One culture was treated four times with Na2EDDA with no effect; the morphology of the cultures became fibroblast-like with no observed cytokeratin expression. CONCLUSIONS: Unwanted dermal fibroblasts can be separated from primary keratinocyte cultures during the first few days after the isolation. Cocultures of unwanted dermal fibroblasts and epidermal keratinocytes can be reverted to pure keratinocyte monolayers suitable as grafts for transplantation.

Tuberculosis screening among immigrants holding a hunger strike in churches.

SETTING: In January 2001, approximately 600 immigrants held a sit-down and hunger strike in several churches in Barcelona to force the Spanish government to comply with demands to regulate their immigration status. Following the diagnosis of a case of smear-positive pulmonary tuberculosis (TB) in one of the immigrants, we performed a large contact investigation. OBJECTIVES: To describe contact investigation procedures used in this setting and to evaluate contact investigation results. METHODS: Demographic variables were collected, and tuberculin skin tests (TST) and chest radiograph examinations were performed. Odds ratios (OR) with 95% confidence intervals (CI) were calculated and logistic regression was used for multivariate analyses. RESULTS: A total of 541 TSTs were performed. Of these, 86% were read and 40.5% yielded a positive reaction with an induration >14 mm. In a multivariate analysis, the risk of presenting a TST induration >14 mm was found to be three times higher among those aged >35 years compared to those <24 years (OR 3.40; 95%CI 1.76-6.59), and for immigrants from Bangladesh (OR 3.14; 95%CI 1.16-6.10) and Pakistan (OR 2.04; 95%CI 1.11-3.73) compared to those from India. A total of 314 chest radiographs examinations were performed and three additional cases of TB were identified, yielding a TB prevalence of 0.7%. CONCLUSIONS: By focusing efforts and conducting targeted TB screening in this high-risk population, it was possible to complete the intervention in only 3 days. A high prevalence of TB infection and TB disease was found.