Cytokines, chemokines and growth factors profile in human aqueous humor in idiopathic uveitis.

To investigate which cytokines, chemokines and growth factors are involved in the immunopathogenesis of idiopathic uveitis, and whether cytokine profiles are associated with. Serum and aqueous humor (AH) samples of 75 patients with idiopathic uveitis were analyzed by multiplex immunoassay. Infectious controls consisted of 16 patients with ocular toxoplasmosis all confirmed by intraocular fluid analyses. Noninfectious controls consisted of 7 patients with Behçet disease related uveitis and 15 patients with sarcoidosis related uveitis. The control group consisted of AH and serum samples from 47 noninflammatory control patients with age-related cataract. In each sample, 27 immune mediators ± IL-21 and IL-23 were measured. In idiopathic uveitis, 13 of the 29 mediators, including most proinflammatory and vascular mediators such as IL-6, IL-8, IL-12, G-CSF, GM-CSF, MCP-1, IP-10, TNF-α and VEGF, were significantly elevated in the aqueous humor when compared to all controls. Moreover, IL-17, IP-10, and IL-21, were significantly elevated in the serum when compared to all controls. We clustered 4 subgroups of idiopathic uveitis using a statistical analysis of hierarchical unsupervised classification, characterized by the order of magnitude of concentrations of intraocular cytokines. The pathogenesis of idiopathic uveitis is characterized by the presence of predominantly proinflammatory cytokines and chemokines and vascular endothelial growth factor with high expression levels as compared to other causes of uveitis. There are indications for obvious Th-1/ IL21-Th17 pathways but also IL9-Th9 and increased IFN-γ-inducing cytokine (IL12) and IFN-γ-inducible CXC chemokine (IP-10). The combined data suggest that immune mediator expression is different among idiopathic uveitis. This study suggests various clusters among the idiopathic uveitis group rather than one specific uveitis entity.

Dificultades en la detección de genomas del nuevo coronavirus 2 (SARS CoV-2) / Pandemic and social inequiality: the popular neighborhoods of the Buenos Aires suburbs in preventive social isolation

RESUMEN El manejo de las infecciones virales respiratorias, tanto a nivel nacional como a nivel mundial, requiere resultados científicos de calidad. La reacción en cadena de la polimerasa de transcriptasa inversa (rRT-PCR, por su sigla en inglés) es considerada el "patrón de oro" para detectar el genoma del nuevo coronavirus 2 (SARS-CoV-2), agente causal de la enfermedad por el nuevo coronavirus (COVID-19) sobre todo en la fase aguda de la infección. Su uso es controvertido fuera de un contexto de exposición viral. El objetivo del presente trabajo es analizar escollos encontrados durante la detección del genoma del SARS-CoV-2 que pueden producir resultados falsos. Los falsos negativos de rRT-PCR pueden deberse al momento y la eficacia de la toma de la muestra, la congelación, el almacenamiento y la descongelación, y a la inactivación térmica de la virulencia. Además, las señales retardadas de los controles internos invalidan la negatividad. Por otra parte, las muestras con escaso material biológico llevan a conclusiones negativas falsas, por lo que determinar un umbral (número mínimo de células epiteliales) contribuirá a reducirlas. Sin embargo, la mayoría de los kits detectan ADN humano, pero no fueron calibrados para cuantificar carga celular. Los ácidos ribonucleicos nucleares (ARN) virales adheridos a guantes, tubos y gorros, -entre otros elementos-, son fuente de falsos positivos. Las farmacopeas sugieren que la contaminación externa se controle en series de 100 muestras con al menos una representatividad del 10%. Si se extrapola esta aproximación al laboratorio de análisis clínicos, en lugar de uno se deberían procesar al menos 10 controles negativos contiguos a 10 positivos cada 100 pruebas. Mejorar la detección por rRT-PCR implica un aumento de al menos 20% en el costo de los reactivos, por lo que se necesitan recursos adicionales.

ABSTRACT Emerging respiratory viral infections like the severe coronavirus disease (CoVID 19) caused by novel coronavirus 2 (SARS-CoV-2) require quality results for science-based responses. The reverse transcriptase polymerase chain reaction (rRT-PCR) is considered the gold standard for detecting SARS-CoV-2 (particularly in the acute phase of infection). The aim of the present work was to analyze pitfalls during the search of viral genomes. False negative conclusions are result of sampling timing, performances of swabbing, storage, and thawing and heat-infectivity inactivation. Samples with low biologic material also lead to false negatives. Qualitative controls to detect the presence of human DNA are available in several kits but they were not calibrated for quantification of human cell loads. Moreover, negativity cannot be reported for samples with delayed signals for the internal control (due to deficiency in extraction and/or retro transcription and/or or to the presence of rRT-PCR inhibitors). The viral RNA that may have stick on gloves, on tubes, caps, etc. may produce false positives. The International Pharmacopoeias recommend for external contamination to test at least 10% of the samples. Couples of 10 negative contiguous to 10 positive controls randomly distributed should be therefore included in each series of 100 rRT-PCR tests. These improvements increase the cost of each determination (at least by 20% only for the reactants) and require additional resources.

Dificultades en la detección de genomas del Nuevo Coronavirus 2 (SARS COV-2) / SARS Coronavirus 2 (SARS CoV 2) genome detection pitfalls

El manejo de las infecciones virales respiratorias, tanto a nivel nacional como a nivel mundial, requiere resultados científicos de calidad. La reacción en cadena de la polimerasa de transcriptasa inversa (rRT-PCR, por su sigla en inglés) es considerada el "patrón de oro" para detectar el genoma del nuevo coronavirus 2 (SARS-CoV-2), agente causal de la enfermedad por el nuevo coronavirus (COVID-19) sobre todo en la fase aguda de la infección. Su uso es controvertido fuera de un contexto de exposición viral. El objetivo del presente trabajo es analizar escollos encontrados durante la detección del genoma del SARS-CoV-2 que pueden producir resultados falsos. Los falsos negativos de rRT-PCR pueden deberse al momento y la eficacia de la toma de la muestra, la congelación, el almacenamiento y la descongelación, y a la inactivación térmica de la virulencia. Además, las señales retardadas de los controles internos invalidan la negatividad. Por otra parte, las muestras con escaso material biológico llevan a conclusiones negativas falsas, por lo que determinar un umbral (número mínimo de células epiteliales) contribuirá a reducirlas. Sin embargo, la mayoría de los kits detectan ADN humano, pero no fueron calibrados para cuantificar carga celular. Los ácidos ribonucleicos nucleares (ARN) virales adheridos a guantes, tubos y gorros, -entre otros elementos-, son fuente de falsos positivos. Las farmacopeas sugieren que la contaminación externa se controle en series de 100 muestras con al menos una representatividad del 10%. Si se extrapola esta aproximación al laboratorio de análisis clínicos, en lugar de uno se deberían procesar al menos 10 controles negativos contiguos a 10 positivos cada 100 pruebas. Mejorar la detección por rRT-PCR implica un aumento de al menos 20% en el costo de los reactivos, por lo que se necesitan recursos adicionales.

Cytoprotective Effects of Natural Highly Bio-Available Vegetable Derivatives on Human-Derived Retinal Cells.

Retinal pigment epithelial cells are crucial for retina maintenance, making their cytoprotection an excellent way to prevent or slow down retinal degeneration. In addition, oxidative stress, inflammation, apoptosis, neovascularization, and/or autophagy are key pathways involved in degenerative mechanisms. Therefore, here we studied the effects of curcumin, lutein, and/or resveratrol on human retinal pigment epithelial cells (ARPE-19). Cells were incubated with individual or combined agent(s) before induction of (a) H2O2-induced oxidative stress, (b) staurosporin-induced apoptosis, (c) CoCl2-induced hypoxia, or (d) a LED-autophagy perturbator. Metabolic activity, cellular survival, caspase 3/7 activity (casp3/7), cell morphology, VEGF levels, and autophagy process were assessed. H2O2 provoked a reduction in cell survival, whereas curcumin reduced metabolic activity which was not associated with cell death. Cell death induced by H2O2 was significantly reduced after pre-treatment with curcumin and lutein, but not resveratrol. Staurosporin increased caspase-3/7 activity (689%) and decreased cell survival by 32%. Curcumin or lutein protected cells from death induced by staurosporin. Curcumin, lutein, and resveratrol were ineffective on the increase of caspase 3/7 induced by staurosporin. Pre-treatment with curcumin or lutein prevented LED-induced blockage of autophagy flux. Basal-VEGF release was significantly reduced by lutein. Therefore, lutein and curcumin showed beneficial protective effects on human-derived retinal cells against several insults.

A Comparison of the Dexamethasone Implant (Ozurdex®) and Inferior Fornix-Based Sub-Tenon Triamcinolone Acetonide for Treatment of Inflammatory Ocular Diseases.

PURPOSE: To evaluate the efficacy and safety of dexamethasone (DEX) implant compared with inferior fornix-based sub-Tenon triamcinolone injection (PSTA) for treatment of uveitis. METHODS: A total of 48 eyes received DEX and 49 eyes received PSTA as the first treatment. RESULTS: A total of 31 eyes were implanted with DEX relapsed (64.5%) after the first injection, while 32 eyes were injected with PSTA as the first treatment relapsed (65.3%). Kaplan-Meier estimated survival to overall relapse after the first injection was a mean 20 months± 3.6 months for DEX (median,7) and 14 months± 1.9 months (median,9) for the PSTA (P = 0.505). Of 49 eyes receiving the PSTA implant as the first treatment, inflammation persisted in 14.3% after the first injection but persisted in none after the DEX injection (P = 0.005). CONCLUSIONS: DEX implantation achieved a higher rate of disease control in the initial 12 weeks postinjection with a relative equivalence in the duration of effect and relapse rates when compared with PSTA.

Increase in Ocular Syphilis Cases at Ophthalmologic Reference Center, France, 2012-2015.

We describe the frequency, demographic and clinical features, and visual outcomes of ocular syphilis infections observed during 2012-2015 at a tertiary reference center in Paris, France. Twenty-one cases (29 eyes) were identified. The occurrence of ocular syphilis increased from 1 case in 2012 to 5 cases in 2013, 6 cases in 2014, and 9 cases in 2015 (2.22-25.21/1,000 individual patients/year for the period). Among case-patients, an annual 20%-33% were co-infected with HIV. Seventy-six percent of ocular syphilis infections occurred in men who have sex with men. Seventy-five percent of case-patients had a good final visual outcome (best-corrected visual acuity >0.3 logMAR score). Visual outcome was worse for HIV-positive patients than for HIV-negative patients (p = 0.0139). At follow-up, the best visual outcomes were observed in patients whose mean time from first ocular symptom to consultation was 15 days (SD +19 days).

Social Sciences for the Prevention of Blindness.

Organizations working for the elimination of Chlamydia-triggered blindness (trachoma) follow the WHO SAFE strategy (surgery for trichiasis, antibiotics, face washing and environmental changes) with the aim to achieve a minimum of 80% of children with clean faces in endemic communities, mass treatment covering the whole district with trachoma rates of 10% or more and surveillance plans. Trachoma recurrence that is common after implementing the SAFE strategy 3, 5 or even 7 times evidence that the cognitive processes requiring assimilation and integration of knowledge did not register with parents, caretakers and children. Moreover, repeated awareness campaigns to improve hygiene did not systematically produce irreversible changes of behavior in neglected populations. In view of this evidence, the rational behind mass drug administration as the mainstay of preventable blindness elimination demands a wider scope than simple mathematical models. The reluctance to see disappointing outcomes that leads to repeated interventions may suggest from a sociologic point of view that the strategies are products of those evaluating the activities of those who fund them and vice versa. A similar articulation emerges for reciprocal interactions between researchers and those judging the pertinence and quality of their work. So far, the lack of autocritic elimination strategy approaches may expose inbred circles that did not properly grasp the fact that antibiotics, trichiasis surgery and education limited to improvement of hygiene are inefficient if not associated with long-term basic educational actions in schools.

Enzymatic treatment of specimens before DNA extraction directly influences molecular detection of infectious agents.

INTRODUCTION: Biological samples, pharmaceuticals or food contain proteins, lipids, polymers, ammoniums and macromolecules that alter the detection of infectious agents by DNA amplification techniques (PCR). Moreover the targeted DNA has to be released from the complex cell walls and the compact nucleoprotein matrixes and cleared from potential inhibitors. The goal of the present work was to assess the efficiency of enzymatic pretreatments on infectious agents to make DNA available for further extraction and amplification. METHODS: Staphylococcus epidermidis, Streptococcus mitis, Propionibacterium acnes, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Aspergillus niger and Fusarium solani were mixed with an internal control virus and treated with: 1) proteinase K; 2) lyticase and 3) lyticase followed by proteinase K. DNAs was manually extracted using the QIAmp DNA Mini kit or the MagNA Pure Compact automate. DNA extraction yields and the inhibitors were assessed with a phocid Herpesvirus. Bacterial detection was performed using TaqMan real-time PCR and yeasts and filamentous Fungi with HRM (real-time PCR followed by high-resolution melting analysis). RESULTS: Viral DNA was released, extracted and detected using manual and automatic methods without pre enzymatic treatments. Either the manual or the automatic DNA extraction systems did not meet the sensitivity expectations if enzymatic treatments were not performed before: lyticase for Fungi and Proteinase K for Bacteria. The addition of lyticase and proteinase K did not improve results. For Fungi the detection after lyticase was higher than for Proteinase K, for which melting analysis did not allow fungal specification. DISCUSSION: Columns and magnetic beads allowed collecting DNA and separate PCR inhibitors. Detection rates cannot be related to DNA-avidity of beads or to elution but to the lack of proteolysis.

The limits of medical interventions for the elimination of preventable blindness.

BACKGROUND: Health authorities are working toward the global elimination of trachoma by the year 2020 with actions focused on the World Health Organization SAFE strategy (surgery of trichiasis, antibiotics, face washing and environmental changes) with emphasis on hygienist approaches for education. OBJECTIVES: The present survey was performed to assess the sustainability of the SAFE strategy 3 years after trachoma was eliminated from 6 villages. METHODS: In February 2013 a rapid trachoma assessment was conducted in 6 villages of Kolofata's district, Extreme north Region, Cameroon, where trachoma was eliminated in 2010. A total of 300 children (1-10 years) from 6 villages were examined by trained staff. RESULTS: The prevalence of active trachoma (children aged > 1 and < 10 years) in 2013 was 15% and in at least 25% was observed absence of face washing and flies in their eyes and nose. Income level, quality of roads, hygiene, and illiteracy were similar in all the villages; they did not change between 2010 and 2013 and could not be analyzed as independent risk factors. DISCUSSION: The heterogeneity of methods described for clinical trials makes it inappropriate to conduct meta-analysis for the present and for other SAFE-related trials. The results obtained after implementation the SAFE strategy (recurrence) reveal that the causes (infectious agents and dirtiness) and effects (illness) were not connected by illiterate people living under conditions of extreme poverty. So far, antibiotics, surgery and hygiene education are insufficient for the sustainability of trachoma elimination and highlight that hypothetic-deductive processes seem not operational after implementing the awareness campaigns. Trachoma recurrence detected in 2013 in sedentary populations of Kolofata receiving efficacious treatments against Chlamydia sp. suggest that the elimination goals will be delayed if strategies are limited to medical actions. Restricting efforts to repeated pharmacological and surgical interventions for people infected with susceptible bacteria could be understood as the hidden side of a passive attitude toward basic education actions.

Kinetics of expansion of human limbal epithelial progenitor cells in primary culture of explants without feeders.

The aims of this study were to determine whether human limbal explant cultures without feeder cells result in expansion of epithelial progenitors and to estimate the optimal expansion time for progenitor cells. Limbal explants from ten human corneas were cultured for 7, 9, 11, 14, 18, and 21 days. Limbal explants from two corneas were enzymatically dissociated or directly cultured for 14 days. Progenitor cells were characterized by their ability to form colonies, by immunocytochemistry, and by quantitative real-time polymerase chain reaction. Colonies were identified after 9, 11, 14, and 18 days of culture, but not after 21 days. The number of colonies per explant was significantly higher after 14 days than after 9 and 21 days. The mean percentage of seeded cells giving rise to clones was 4.03% after 14 days of culture and 0.36% for non-cultured dissociated limbal epithelial cells. The number of cells giving rise to clones per cornea significantly increased from an average of 2275 for non-cultured cells to 24266 for cells cultured for 14 days. Immunocytochemical analysis detected positive staining for cytokeratin (CK) 3, CK5/6/8/10/13/18, CK19, vimentin, p63, and p63α, in both cultures and clones. CK3 expression increased significantly with culture time. Transcript expression was observed for CK3, CK19, vimentin, and Delta N p63α at each culture time point, both in cultures and clones. The optimal culture time for limbal explants in cholera toxin-free Green medium without feeder cells was 14 days leading to the expansion of progenitors.

Donor tissue selection for anterior lamellar keratoplasty.

PURPOSE: To assess the influence of donor characteristics on the outcome of anterior lamellar keratoplasty (ALK) and to evaluate whether corneal donor tissue considered unsuitable for penetrating or posterior lamellar keratoplasty due to poor endothelial condition may be safely used for ALK. METHODS: Institutional setting. One hundred sixty-six consecutive ALK (166 patients) performed for optical indication in eyes with corneal diseases not involving the corneal endothelium. The main outcome measures were graft survival, early (0-12 months postoperatively) and late (after 12 months) annual endothelial cell loss, and postoperative logarithm of the minimum angle of resolution visual acuity. RESULTS: The average and extreme values of donor tissue characteristics were: donor age, 70.6 years (range, 28-88 years); organ culture time, 20.9 days (range, 12-35 days); graft endothelial cell density before transplantation, 2047 cells per millimeters (range, 100-3300 cells/mm2); and deswelling time, 2.0 days (range, 1-4 days). The average follow-up time of patients was 48.1 ± 24.8 months (mean ± SD). None of the donor characteristics significantly influenced graft survival or postoperative endothelial cell loss (early and late phase). Donor age >80 years was associated with lower postoperative visual acuity at all postoperative points in time (P < 0.05). At 3 years, the mean logarithm of the minimum angle of resolution visual acuity was 0.44 (20/55) for grafts from donors older than 80 years and 0.25 (20/35) for younger donors. This result was shown to be significant both in univariate and in multivariate analysis. CONCLUSIONS: Grafts from elderly donors should be discarded before ALK. Conversely, donor tissue with poor endothelial cell density (<2000 cells/mm2) is suitable for ALK.

Findings in detection of Herpesviridae by polymerase chain reaction and intraocular antibody production in a case series of anterior uveitis.

PURPOSE: To target the use of two biologic tests in the diagnostic of viral Herpesviridae anterior uveitis (AC) by the consideration of clinical behavior and delay of intraocular sampling. METHODS: Aqueous humor samples were collected from 42 patients suspected of having AU of infectious origin at presentation. The diagnosis of infectious uveitis was confirmed by quantification of antibodies with the Goldmann-Witmer coefficient (GWC) and/or detection of Herpesviridae genomes with PCR. The data were compared with data of 16 uveitis control samples used to calculate the specificity of the tests. RESULTS: Sixteen out of 42 eyes (38%) had a final diagnosis of anterior segment infectious uveitis of viral origin (Herpesviridae) confirmed by PCR positive result (5/14 eyes; 14 of the 16 eyes were tested by PCR) and/or specific intraocular antibody synthesis (14/16 eyes). CONCLUSIONS: While the GWC is progressively less often performed, these findings suggest that it still has a role in AU suspected of herpesvirus etiology.

Contributions to the Improvement of Living Conditions among Neglected Populations with Trachoma.

OBJECTIVE: Trachoma (Chlamydia-triggered blinding infection) provoked irreversible visual impairment in about 8 million people in 2011, and the prevalence among children with dirty faces is more than three fold that among children with clean faces. In 250 villages with a high prevalence of trachoma (Kolofata district, Far North Region, Cameroon), the lack of water for facial cleanliness was reported during trachoma awareness campaigns. The objective of this study was to determine if the lack of water was linked with the absence of means to dig wells. METHODS: Wells, waterholes, motorcycles, irrigation pumps, electricity, goats and oxen, cell phones and distance from waterholes were recorded in January 2011 in 50 randomized villages of Kolofata's district. RESULTS: The number of villages with <25 goats and <5 oxen was 0 and the number of adults owning <1 goat was 0. The cost of a pail of water was 0.01 USD. Motorcycles, cell phones and televisions have been reported in more than 66% of villages. The cost for the construction of lined shaft wells ranged between 15-35 goats and 0.5-3 oxen; the cost for drinking water wells ranged between 50-200 goats and 3-30 oxen. DISCUSSION: No link between the means for digging wells at the village level and access to water was found. Social solidarity, which refers to a social debt owed by each person to his/her group, should be added to training guides to gauge its ability to release people from the dead end of having to wait for external assistance to gain access to water.

Clinical and microbiological assessment of trachoma in the kolofata health district, far north region, cameroon.

BACKGROUND AND AIMS: Trachoma is a sight-threatening process triggered by the infection of the conjunctiva with Chlamydiae. Blindness associated with trachoma was reported in Sahelian areas of Cameroon. However, data on the prevalence of this neglected infection in the Far North Region are not available. The aim of this study was a) to assess clinical trachoma and b) to detect Chlamydia in the conjunctiva of trachomatous populations living in the Far North Regions of Cameroon. METHODS: A total of 2,423 randomly selected children (1-10 years) and 1,590 women over 14 from randomly selected villages from the Kolofata Health District (115,000 inhabitants) were included in a cross-sectional study in February 2009. Trained staff examined and obtained conjunctival swabs from trachomatous subjects. DNA was extracted and amplified to detect Chlamydia DNA by real-time PCR. The quality of sampling was assessed by quantifying the number of epithelial cells. RESULTS: Children (2,397 or 98.9% of the predicted number) and women (1,543; 97.0%) were examined. The prevalence of follicular trachoma (TF) in children was 21% (95% CI 17.8-24.5) and of intense inflammatory trachoma (TI) 5.2% (95% CI 3.6-7.3). Among the women, trichiasis (TT) was observed in 3.4% (95% CI 2.4-4.7), corneal opacities (CO) in 1.4% (95% CI 0.8-2.3) and trachoma-related blindness in 0.9% (95% CI 0.4-1.8). Conditions related to income, illiteracy, latrines, water supply and animals wandering close to dwellings were similar in all the villages. PCR was positive in 35% of children with active trachoma and in 6% of adult females presenting TT and/or related corneal opacities. CONCLUSION: The prevalence of trachoma and the severe trachoma sequelae found during this survey underline the urgent need to implement efficient blindness prevention interventions to improve the visual future of the people in the Sahelian region.

Rapid detection and simultaneous molecular profile characterization of Acanthamoeba infections.

Diagnosis of Acanthamoeba by microscopic examination, culture, and polymerase chain reactions (PCRs) has several limitations (sensitivity, specificity, lack of detection of several strains, cost of testing for discrimination among strains). We developed a new high-resolution melting real-time PCR (HRM) to detect and characterize Acanthamoeba infections. HRM performances were evaluated with strains from the American Type Culture Collection (ATCC) and with 20 corneal scrapings. The DNA extracted from specimens were amplified, detected, and characterized in 1 run using 2 original primers diluted in a solution containing an intercalating dye. Detection and molecular characterization of Acanthamoeba infections could be achieved in less than 2.5 h with a dramatic reduction in cost of reactants (postamplification procedures and radioactive or fluorescent-labeled molecular probes were unnecessary). HRM detection limits were 0.1 cyst/µL or less (including genotypes T5 and T11), and its sensitivity and specificity were higher than other molecular tests. For the tested strains from the ATCC, the HRM drafted 4 different profiles: Type I (genotypes T2 and T4), Type II (T5 and T7), Type III (T8), and Type IV (T1, T3, T6, T9, T11, T12, and T13).

Addition of an induction regimen of antiangiogenesis and antitumor immunity to standard chemotherapy improves survival in advanced malignancies.

Studies have shown that cancer requires two conditions for tumor progression: cancer cell proliferation and an environment permissive to and conditioned by malignancy. Chemotherapy aims to control the number and proliferation of cancer cells, but it does not effectively control the two best-known conditions of the tumor-permissive environment: neoangiogenesis and tolerogenic immunity. Many malignant diseases exhibit poor outcomes after treatment with chemotherapy. Therefore, we investigated the potential benefits of adding an induction regimen of antiangiogenesis and antitumor immunity to chemotherapy in poor outcome disease. In a prospective, randomized trial, we included patients with advanced, unresectable pancreatic adenocarcinomas, non-small cell lung cancer, or prostate cancer. Two groups of each primary condition were compared: group 1 (G1), n = 30, was treated with the standard chemotherapy and used as a control, and group 2 (G2), n = 30, was treated with chemotherapy plus an induction regimen of antiangiogenesis and antitumor immunity. This induction regimen included a low dose of metronomic cyclophosphamide, a high dose of Cox-2 inhibitor, granulocyte colony-stimulating factor, a sulfhydryl (SH) donor, and a hemoderivative that contained autologous tumor antigens released from patient tumors into the blood. After treatment, the G2 group demonstrated significantly longer survival, lower blood level of neoangiogenesis and immune-tolerance mediators, and higher blood levels of antiangiogenesis and antitumor immunity mediators compared with the G1 group. Toxicity and quality of life were not significantly different between the groups. In conclusion, in several advanced malignancies of different primary localizations, an increase in survival was observed by adding an induction regimen of antiangiogenesis and antitumor immunity to standard chemotherapy.

New strategy for rapid diagnosis and characterization of fungal infections: the example of corneal scrapings.

PURPOSE: The prognosis of people infected with Fungi especially immunocompromised depends on rapid and accurate diagnosis to capitalize on time administration of specific treatments. However, cultures produce false negative results and nucleic-acid amplification techniques require complex post-amplification procedures to differentiate relevant fungal types. The objective of this work was to develop a new diagnostic strategy based on real-time polymerase-chain reaction high-resolution melting analysis (PCR-HRM) that a) detects yeasts and filamentous Fungi, b) differentiates yeasts from filamentous Fungi, and c) discriminates among relevant species of yeasts. METHODS: PCR-HRM detection limits and specificity were assessed with a) isolated strains; b) human blood samples experimentally infected with Fungi; c) blood experimentally infected with other infectious agents; d) corneal scrapings from patients with suspected fungal keratitis (culture positive and negative) and e) scrapings from patients with suspected bacterial, viral or Acanthamoeba infections. The DNAs were extracted and mixed with primers diluted in the MeltDoctor® HRM Master Mix in 2 tubes, the first for yeasts, containing the forward primer CandUn (5'CATGCCTGTTTGAGCGTC) and the reverse primer FungUn (5'TCCTCCGCTT ATTGATATGCT) and the second for filamentous Fungi, containing the forward primer FilamUn (5'TGCCTGTCCGAGCGTCAT) and FungUn. Molecular probes were not necessary. The yields of DNA extraction and the PCR inhibitors were systematically monitored. RESULTS: PCR-HRM detected 0.1 Colony Forming Units (CFU)/µl of yeasts and filamentous Fungi, differentiated filamentous Fungi from yeasts and discriminated among relevant species of yeasts. PCR-HRM performances were higher than haemoculture and sensitivity and specificity was 100% for culture positive samples, detecting and characterizing Fungi in 7 out 10 culture negative suspected fungal keratitis. CONCLUSIONS: PCR-HRM appears as a new, sensitive, specific and inexpensive test that detects Fungi and differentiates filamentous Fungi from yeasts. It allows direct fungal detection from clinical samples and experimentally infected blood in less than 2.30 h after DNA extraction.

Effects of isoproterenol and cholera toxin on human limbal epithelial cell cultures.

PURPOSE: Cholera toxin and isoproterenol (ß-adrenergic receptor agonist) are largely used to enhance cell proliferation. The aim of the study was to assess the effects of cholera toxin and isoproterenol on growth and differentiation of cells cultured from human superficial limbal explants. METHODS: Limbal epithelial cells were cultured from superficial limbal explantsin basal medium either supplemented with cholera toxin or isoproterenol for 3 weeks. Growth kinetics and morphometry were studied by light and confocal microscopy. Progenitor and differentiated epithelial cell markers were studied by immunocytochemistry, flow cytometry, Colony Formation Assay, and reverse transcription and polymerase chain reaction. RESULTS: Cell proliferation was significantly higher with 0.5 µg/ml (p = 0.049), 1 µg/ml (p = 0.005), and 2 µg/ml (p = 0.008) isoproterenol whereas, cholera toxin and 4 µg/ml isoproterenol did not significantly increase cell proliferation. Multilayered epithelial cell sheets were obtained in all culture conditions. Addition of isoproterenol resulted in smaller cell size (p < 0.05) 14 days after cells were cultured, whereas cholera toxin had no effects. Strong expression of cytokeratins 3 and 4/5/6/8/10/13/18 and lower expression of cytokeratin 19, vimentin, and Delta N p63α were observed after 3 weeks of culture with no significant differences in the percentage of positive cells according to culture medium. Colony-forming efficiencies were observed after 2 weeks in all culture condition but not after 3 weeks. CONCLUSION: Isoproterenol was more efficient than cholera toxin for enhancing cell proliferation and resulted in smaller cell size. It appears to be useful and safe for growing human limbal epithelial progenitors from limbal explants with no feeders before transplantation to patients with limbal deficiency.

Modeling approach to characterize intraocular doripenem pharmacokinetics after intravenous administration to rabbits, with tentative extrapolation to humans.

The aim of this study was to determine the penetration of doripenem administered intravenously into the rabbit aqueous and vitreous humors. Nineteen New Zealand White rabbits received a 20-mg dose of doripenem intravenously over 60 min. Specimens of aqueous humor, vitreous humor, and blood were obtained 30 min (n = 5), 1 h (n = 5), 2 h (n = 5), and 3 h (n = 4) after the beginning of the infusion and analyzed by high-performance liquid chromatography (HPLC). A pharmacokinetic (PK) model was developed to fit the experimental data. Doripenem concentrations in aqueous humor were lower than those in plasma ultrafiltrates at all sampling times, with an average aqueous humor-to-plasma ultrafiltrate area under the concentration-time curve ratio estimated as 8.3%. A pharmacokinetic model with peripheral elimination described the data adequately and was tentatively used to predict concentration-versus-time profiles and pharmacokinetic-pharmacodynamic (PK-PD) target attainment in patients under various dosing regimens. In conclusion, systematically administered doripenem does not seem to be a promising approach for the treatment of intraocular infections, especially since it could not be detected in the vitreous humor. However, this study has provided an opportunity to develop a new PK modeling approach to characterize the intraocular distribution of doripenem administered intravenously to rabbits, with tentative extrapolation to humans.

Infectious keratitis in severe limbal stem cell deficiency: characteristics and risk factors.

PURPOSE: To evaluate the incidence, clinical and microbiological characteristics and risk factors of infectious keratitis in patients with limbal stem cell deficiency (LSCD). METHODS: Retrospective comparative case series of 35 patients with severe LSCD. RESULTS: The mean follow-up time was 46 months. Infectious keratitis were mainly caused by Gram positive bacteria (94%). Only 7 infections (37%) healed under fortified adapted antibiotics. In 8 cases (42%), amniotic membrane transplantation was required and in 4 cases (21%) «à chaud¼ keratoplasty was performed. Significant risk factors associated with infectious keratitis were: soft contact lens extended wear, history of persistent epithelial defects, number of quadrants of corneal vascularization, re-epithelialization time after amniotic membrane or corneal transplantation, and use of corticosteroid or cyclosporin eye drops. CONCLUSION: Infectious keratitis in LSCD is frequent and severe. The restoration of the epithelial barrier integrity and a careful use of therapeutic contact lenses may help to prevent infection.