Crosstalk between estrogen, dendritic cells, and SARS-CoV-2 infection.

The novel coronavirus disease 2019 (Covid-19) first appeared in Wuhan and has so far killed more than four million people worldwide. Men are more affected than women by Covid-19, but the cellular and molecular mechanisms behind these differences are largely unknown. One plausible explanation is that differences in sex hormones could partially account for this distinct prevalence in both sexes. Accordingly, several papers have reported a protective role of 17ß-estradiol during Covid-19, which might help explain why women appear less likely to die from Covid-19 than men. 17ß-estradiol is the predominant and most biologically active endogenous estrogen, which signals through estrogen receptor α, estrogen receptor ß, and G protein-coupled estrogen receptor 1. These receptors are expressed in mature cells from the innate and the adaptive immune system, particularly on dendritic cells (DCs), suggesting that estrogens could modulate their effector functions. DCs are the most specialized and proficient antigen-presenting cells, acting at the interface of innate and adaptive immunity with a powerful capacity to prime antigen-specific naive CD8+ T cells. DCs are richly abundant in the lung where they respond to viral infection. A relative increase of mature DCs in broncho-alveolar lavage fluids from Covid-19 patients has already been reported. Here we will describe how SARS-CoV-2 acts on DCs, the role of estrogen on DC immunobiology, summarise the impact of sex hormones on the immune response against Covid-19, and explore clinical trials regarding Covid-19.

ER-mitochondria communication is involved in NLRP3 inflammasome activation under stress conditions in the innate immune system.

Endoplasmic reticulum (ER) stress and mitochondrial dysfunction, which are key events in the initiation and/or progression of several diseases, are correlated with alterations at ER-mitochondria contact sites, the so-called "Mitochondria-Associated Membranes" (MAMs). These intracellular structures are also implicated in NLRP3 inflammasome activation which is an important driver of sterile inflammation, however, the underlying molecular basis remains unclear. This work aimed to investigate the role of ER-mitochondria communication during ER stress-induced NLRP3 inflammasome activation in both peripheral and central innate immune systems, by using THP-1 human monocytes and BV2 microglia cells, respectively, as in vitro models. Markers of ER stress, mitochondrial dynamics and mass, as well as NLRP3 inflammasome activation were evaluated by Western Blot, IL-1ß secretion was measured by ELISA, and ER-mitochondria contacts were quantified by transmission electron microscopy. Mitochondrial Ca2+ uptake and polarization were analyzed with fluorescent probes, and measurement of aconitase and SOD2 activities monitored mitochondrial ROS accumulation. ER stress was demonstrated to activate the NLRP3 inflammasome in both peripheral and central immune cells. Studies in monocytes indicate that ER stress-induced NLRP3 inflammasome activation occurs by a Ca2+-dependent and ROS-independent mechanism, which is coupled with upregulation of MAMs-resident chaperones, closer ER-mitochondria contacts, as well as mitochondrial depolarization and impaired dynamics. Moreover, enhanced ER stress-induced NLRP3 inflammasome activation in the immune system was found associated with pathological conditions since it was observed in monocytes derived from bipolar disorder (BD) patients, supporting a pro-inflammatory status in BD. In conclusion, by demonstrating that ER-mitochondria communication plays a key role in the response of the innate immune cells to ER stress, this work contributes to elucidate the molecular mechanisms underlying NLRP3 inflammasome activation under stress conditions, and to disclose novel potential therapeutic targets for diseases associated with sterile inflammation.

Cellular and molecular mechanisms of glioblastoma malignancy: Implications in resistance and therapeutic strategies.

Glioblastoma (GB) is the more frequent and malignant brain tumour. In spite of all efforts, the median overall survival of GB patients remains approximately 15 months under therapy. The molecular biology underlying GB is complex, which highlight the need of specific treatment strategies. In fact, the deregulation of several molecular signalling pathways, the existence of the blood-brain barrier (BBB), that makes almost all the chemotherapeutic agents inaccessible to the tumour site, and the existence of a population of stem-like cells known to be responsible for tumour recurrence after therapy, can contribute to GB chemoresistance. In the present review, we summarize the reliable factors responsible for the failure of the most important chemotherapeutic agents in GB. Specifically, we describe the utmost important characteristics of the BBB, as well as the genetic, molecular and transcription factors alterations that lead to tumour malignancy, and ultimately their impact on stem-like cell plasticity modulation. Recently, nanocarriers have attracted increasing attention in brain- and tumour-targeted drug-delivery systems, owing to their potential ability to target cell surface specific molecules and to cross the BBB delivering the drug specifically to the tumour cells, improving efficacy and thus reducing non-specific toxicity. In this sense, we will lastly highlight the therapeutic challenges and improvements regarding GB treatment.

Clearance and persistence of SARS-CoV-2 RNA in patients with COVID-19.

Patients with coronavirus disease-2019 may be discharged based on clinical resolution of symptoms, and evidence for viral RNA clearance from the upper respiratory tract. Understanding the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral clearance profile is crucial to establish a re-testing plan on discharge and ending isolation of patients. We aimed to evaluate the number of days that a patient needed to achieve undetectable levels of SARS-CoV-2 in upper respiratory tract specimens (nasopharyngeal swab and/or an oropharyngeal swab). The clearance and persistence of viral RNA was evaluated in two groups of positive patients: those who achieved two negative reverse transcription-polymerase chain reaction (RT-PCR) tests and those who kept testing positive. Patients were organized thereafter in two subgroups, mild illness patients discharged home and inpatients who had moderate to severe illness. Results from RT-PCR tests were then correlated with results from the evaluation of the immune response. The study evidenced that most patients tested positive for more than 2 weeks and that persistence of viral RNA is not necessarily associated with severe disease but may result from a weaker immune response instead.

Nucleolin is expressed in patient-derived samples and glioblastoma cells, enabling improved intracellular drug delivery and cytotoxicity.

One of the major challenges in Glioblastoma (GBM) therapy relates with the existence of glioma stem-like cells (GSCs), known to be chemo- and radio-resistant. GSCs and non-stem GBM cells have the ability to interchange, emphasizing the importance of identifying common molecular targets among those cell sub-populations. Nucleolin overexpression has been recently associated with breast cancer sub-populations with different stem-like phenotype. The goal of this work was to evaluate the potential of cell surface nucleolin as a target in GBM cells. Different levels of nucleolin expression resulted in a 3.4-fold higher association of liposomes targeting nucleolin (functionalized with the nucleolin-binding F3 peptide) in U87, relative to GBM11 glioblastoma cells. Moreover, nucleolin was suggested as a potential marker in OCT4-, NANOG-positive GSC, and in the corresponding non-stem GBM cells, as well as in SOX2-positive GSC. Doxorubicin delivered by liposomes targeting nucleolin enabled a level of cytotoxicity that was 2.5- or 4.6-fold higher compared to the non-targeted counterparts. Importantly, an overexpression of nucleolin was also observed in cells of patient-derived samples, as compared with normal brain. Overall, these results suggested nucleolin as a therapeutic target in GBM.

Choroidal thickness changes stratified by outcome in real-world treatment of diabetic macular edema.

PURPOSE: The aim of this study was to evaluate subfoveal choroidal thickness (SFCT) as a marker of outcome in real-world treatment of diabetic macular edema (DME) and to correlate it with choroidal thicknesses (CT) collected around the fovea. METHODS: Prospective interventional case series included a total of 126 eyes from 126 patients with recently diagnosed DME treated with a 3-monthly loading dose of ranibizumab or aflibercept and PRN thereafter until 24 months (M). CT was manually measured in the central 3500 µm area, subfoveally (SFCT), at 1750 µm right and left from the center in the horizontal plane and at 1750 µm up and down from the center in the vertical plane, by OCT. Anatomic (10% decrease in central retinal thickness) and functional (gain ≥ 5 letters) responses were assessed using univariate and multivariate analyses. The areas under ROC curves were used to assess whether baseline SFCT was a predictor of outcome. RESULTS: CT significantly decreased in all follow-ups (3 months after the 3 injections' loading dose (3M), 6 months (6M), 12 months (12M), 18 months (18M), 24 months (24M)). SFCT and other CT parameters are correlated. SFCT decrease from baseline was related with treatment (p = 0.003 to p < 0.001) but not with anatomic (3M, p = 0.858; 6M p = 0.762) or functional response (3M, p = 0.746; 6M, p = 0.156). SFCT was not found to be predictive of anatomic (AUC = 0.575, p = 0.172) or functional (AUC = 0.515, p = 0.779) outcome. CONCLUSIONS: SFCT is a reliable marker of choroidal thickness. Baseline SFCT decreased with anti-VEGF treatment but did not predict DME outcome.

Urolithins impair cell proliferation, arrest the cell cycle and induce apoptosis in UMUC3 bladder cancer cells.

Ellagitannins have been gaining attention as potential anticancer molecules. However, the low bioavailability of ellagitannins and their extensive metabolization in the gastrointestinal tract into ellagic acid and urolithins suggest that the health benefits of consuming ellagitannins rely on the direct effects of their metabolites. Recently, chemopreventive and chemotherapeutic activities were ascribed to urolithins. Nonetheless, there is still a need to screen and evaluate the selectivity of these molecules and to elucidate their cellular mechanisms of action. Therefore, this work focused on the antiproliferative effects of urolithins A, B and C and ellagic acid on different human tumor cell lines. The evaluation of cell viability and the determination of the half-maximal inhibitory concentrations indicated that the sensitivity to the studied urolithins varied markedly between the different cell lines, with the bladder cancer cells (UMUC3) being the most susceptible. In UMUC3 cells, urolithin A was the most active molecule, promoting cell cycle arrest at the G2/M checkpoint, increasing apoptotic cell death and inhibiting PI3K/Akt and MAPK signaling. Overall, the present study emphasizes the chemopreventive/chemotherapeutic potential of urolithins, highlighting the stronger effects of urolithin A and its potential to target transitional bladder cancer cells.

Tamoxifen in combination with temozolomide induce a synergistic inhibition of PKC-pan in GBM cell lines.

BACKGROUND: Glioblastoma (GBM) is a highly proliferative, angiogenic grade IV astrocytoma that develops resistance to the alkylating agents used in chemotherapy, such as temozolomide (TMZ), which is considered the gold standard. The mean survival time for GBM patients is approximately 12 months, increasing to 14.6 months after TMZ treatment. The resistance of GBM to chemotherapy seems to be associated to genetic alterations and to the constitutive activation of several signaling pathways. Therefore, the combination of different drugs with different mechanisms of action may contribute to circumvent the chemoresistance of glioma cells. Here we describe the potential synergistic behavior of the therapeutic combination of tamoxifen (TMX), a known inhibitor of PKC, and TMZ in GBM. METHODS: We used two GBM cell lines incubated in absence and presence of TMX and/or TMZ and measured cell viability, proliferation, apoptosis, cell cycle, migration ability, cytoskeletal organization and the phosphorylated amount of the p-PKC-pan. RESULTS: The combination of low doses of TMX with increasing doses of TMZ shows an increased antiproliferative and apoptotic effect compared to the effect with TMX alone. CONCLUSIONS: The combination of TMX and TMZ seems to potentiate the effect of each other. These alterations seem to be associated to a decrease in the phosphorylation status of PKC. GENERAL SIGNIFICANCE: We emphasize that TMX is an inhibitor of the p-PKC-pan and that these combination is more effective in the reduction of proliferation and in the increase of apoptosis than each drug alone, which presents a new therapeutic strategy in GBM treatment.

Fusarium dimerum Species Complex (Fusarium penzigii) Keratitis After Corneal Trauma.

We report a case of a keratitis associated with a Fusarium penzigii-a Fusarium dimerum species complex (FDSC)-in a 81-year-old woman after a corneal trauma with a tree branch. At patient admittance, slit lamp biomicroscopy revealed an exuberant chemosis, an inferior corneal ulcer with an associated inflammatory infiltrate, a central corneal abscess, bullous keratopathy and posterior synechiae. Corneal scrapes were obtained for identification of bacteria and fungi, and the patient started antibiotic treatment on empirical basis. Few days later, the situation worsened with the development of hypopyon. By that time, Fusarium was identified in cultures obtained from corneal scrapes and the patient started topical amphotericin B 0.15 %. Upon the morphological identification of the Fusarium as a FDSC, and since there was no clinical improvement, the treatment with amphotericin B was suspended and the patient started voriconazole 10 mg/ml, eye drops, hourly and voriconazole 200 mg iv, every 12 h for 1 month. The hypopyon resolved and the inflammatory infiltrate improved, but the abscess persisted at the last follow-up visit. The molecular identification revealed that the FDSC was a F. penzigii.

Antifungals: From Pharmacokinetics to Clinical Practice.

The use of antifungal drugs started in the 1950s with polyenes nystatin, natamycin and amphotericin B-deoxycholate (AmB). Until the present day, AmB has been considered to be a hallmark in the treatment of invasive systemic fungal infections. Nevertheless, the success and the use of AmB were associated with severe adverse effects which stimulated the development of new antifungal drugs such as azoles, pyrimidine antimetabolite, mitotic inhibitors, allylamines and echinochandins. However, all of these drugs presented one or more limitations associated with adverse reactions, administration route and more recently the development of resistance. To worsen this scenario, there has been an increase in fungal infections, especially in invasive systemic fungal infections that are particularly difficult to diagnose and treat. In 2022, the World Health Organization (WHO) published the first fungal priority pathogens list, alerting people to the increased incidence of invasive systemic fungal infections and to the associated risk of mortality/morbidity. The report also emphasized the need to rationally use existing drugs and develop new drugs. In this review, we performed an overview of the history of antifungals and their classification, mechanism of action, pharmacokinetic/pharmacodynamic (PK/PD) characteristics and clinical applications. In parallel, we also addressed the contribution of fungi biology and genetics to the development of resistance to antifungal drugs. Considering that drug effectiveness also depends on the mammalian host, we provide an overview on the roles of therapeutic drug monitoring and pharmacogenomics as means to improve the outcome, prevent/reduce antifungal toxicity and prevent the emergence of antifungal resistance. Finally, we present the new antifungals and their main characteristics.

CuMV VLPs Containing the RBM from SARS-CoV-2 Spike Protein Drive Dendritic Cell Activation and Th1 Polarization.

Dendritic cells (DCs) are the most specialized and proficient antigen-presenting cells. They bridge innate and adaptive immunity and display a powerful capacity to prime antigen-specific T cells. The interaction of DCs with the receptor-binding domain of the spike (S) protein from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a pivotal step to induce effective immunity against the S protein-based vaccination protocols, as well as the SARS-CoV-2 virus. Herein, we describe the cellular and molecular events triggered by virus-like particles (VLPs) containing the receptor-binding motif from the SARS-CoV-2 spike protein in human monocyte-derived dendritic cells, or, as controls, in the presence of the Toll-like receptors (TLR)3 and TLR7/8 agonists, comprehending the events of dendritic cell maturation and their crosstalk with T cells. The results demonstrated that VLPs boosted the expression of major histocompatibility complex molecules and co-stimulatory receptors of DCs, indicating their maturation. Furthermore, DCs' interaction with VLPs promoted the activation of the NF-kB pathway, a very important intracellular signalling pathway responsible for triggering the expression and secretion of proinflammatory cytokines. Additionally, co-culture of DCs with T cells triggered CD4+ (mainly CD4+Tbet+) and CD8+ T cell proliferation. Our results suggested that VLPs increase cellular immunity, involving DC maturation and T cell polarization towards a type 1 T cells profile. By providing deeper insight into the mechanisms of activation and regulation of the immune system by DCs, these findings will enable the design of effective vaccines against SARS-CoV-2.

Therapeutic implications of an enriched cancer stem-like cell population in a human osteosarcoma cell line.

BACKGROUND: Osteosarcoma is a bone-forming tumor of mesenchymal origin that presents a clinical pattern that is consistent with the cancer stem cell model. Cells with stem-like properties (CSCs) have been identified in several tumors and hypothesized as the responsible for the relative resistance to therapy and tumor relapses. In this study, we aimed to identify and characterize CSCs populations in a human osteosarcoma cell line and to explore their role in the responsiveness to conventional therapies. METHODS: CSCs were isolated from the human MNNG/HOS cell line using the sphere formation assay and characterized in terms of self-renewal, mesenchymal stem cell properties, expression of pluripotency markers and ABC transporters, metabolic activity and tumorigenicity. Cell's sensitivity to conventional chemotherapeutic agents and to irradiation was analyzed and related with cell cycle-induced alterations and apoptosis. RESULTS: The isolated CSCs were found to possess self-renewal and multipotential differentiation capabilities, express markers of pluripotent embryonic stem cells Oct4 and Nanog and the ABC transporters P-glycoprotein and BCRP, exhibit low metabolic activity and induce tumors in athymic mice. Compared with parental MNNG/HOS cells, CSCs were relatively more resistant to both chemotherapy and irradiation. None of the treatments have induced significant cell-cycle alterations and apoptosis in CSCs. CONCLUSIONS: MNNG/HOS osteosarcoma cells contain a stem-like cell population relatively resistant to conventional chemotherapeutic agents and irradiation. This resistant phenotype appears to be related with some stem features, namely the high expression of the drug efflux transporters P-glycoprotein and BCRP and their quiescent nature, which may provide a biological basis for resistance to therapy and recurrence commonly observed in osteosarcoma.

An ultra-sensitive electrochemical biosensor using the Spike protein for capturing antibodies against SARS-CoV-2 in point-of-care.

This work presents an innovative ultra-sensitive biosensor having the Spike protein on carbon-based screen-printed electrodes (SPEs), for monitoring in point-of-care antibodies against SARS-CoV-2, a very important tool for epidemiological monitoring of COVID-19 infection and establishing vaccination schemes. In an innovative and simple approach, a highly conductive support is combined with the direct adsorption of Spike protein to enable an extensive antibody capture. The high conductivity was ensured by using carboxylated carbon nanotubes on the carbon electrode, by means of a simple and quick approach, which also increased the surface area. These were then modified with EDC/NHS chemistry to produce an amine layer and undergo Spike protein adsorption, to generate a stable layer capable of capturing the antibodies against SARS-CoV-2 in serum with great sensitivity. Electrochemical impedance spectroscopy was used to evaluate the analytical performance of this biosensor in serum. It displayed a linear response between 1.0 â€‹pg/mL and 10 â€‹ng/mL, with a detection limit of ∼0.7 â€‹pg/mL. The analysis of human positive sera containing antibody in a wide range of concentrations yielded accurate data, correlating well with the reference method. It also offered the unique ability of discriminating antibody concentrations in sera below 2.3 â€‹µg/mL, the lowest value detected by the commercial method. In addition, a proof-of-concept study was performed by labelling anti-IgG antibodies with quantum dots to explore a new electrochemical readout based on the signal generated upon binding to the anti-S protein antibodies recognised on the surface of the biosensor. Overall, the alternative serologic assay presented is a promising tool for assessing protective immunity to SARS-CoV-2 and a potential guide for revaccination.

Knocking out of CD38 accelerates development of a lupus-like disease in lpr mice.

OBJECTIVES: CD38 participates in lymphocyte ontogeny and function and may be involved in autoimmunity. Absence of CD38 accelerates development of non-obese diabetic (NOD) mice diabetes and anti-CD38 antibodies are good markers of human disease. Little is known regarding systemic autoimmunity. Active SLE patients have higher numbers of CD38(+) T and B cells. CD38 is a candidate gene for the murine Lmb2 lupus locus. We aimed to investigate whether CD38 was involved in lupus development. METHODS: We developed Cd38(-/-)-Fas(lpr)/Fas(lpr) mice and monitored them for development of a lupus-like disease through measurement of protein excretion in urine, histological assessment of the kidneys, quantification of circulating immunoglobulins and autoantibodies. We have also immunophenotyped 2- and 6-month old Cd38(-/-)-Fas(lpr)/Fas(lpr) mice. RESULTS: We found that absence of CD38 accelerated disease development: female Cd38(-/-)-Fas(lpr)/Fas(lpr) mice presented severe proteinuria, GN, deposition of ICs in the renal medulla and increased amounts of circulating immunoglobulin G (IgG), although anti-dsDNA autoantibodies and RF were not significantly increased at disease onset. We have found that Cd38(-/-)-Fas(lpr)/Fas(lpr) male mice, similarly to other murine models of lupus, were able to control disease. Absence of CD38 in lpr mice altered differentiation of T cells and dendritic cells (DC). CONCLUSION: Although the role of CD38 in tolerance is still to be elucidated, we provide evidence that it may play an active role in the control of a murine lupus-like disease.

Nitric oxide stimulates the proliferation of neural stem cells bypassing the epidermal growth factor receptor.

Nitric oxide (NO) was described to inhibit the proliferation of neural stem cells. Some evidence suggests that NO, under certain conditions, can also promote cell proliferation, although the mechanisms responsible for a potential proliferative effect of NO in neural stem cells have remained unaddressed. In this work, we investigated and characterized the proliferative effect of NO in cell cultures obtained from the mouse subventricular zone. We found that the NO donor NOC-18 (10 microM) increased cell proliferation, whereas higher concentrations (100 microM) inhibited cell proliferation. Increased cell proliferation was detected rapidly following exposure to NO and was prevented by blocking the mitogen-activated kinase (MAPK) pathway, independently of the epidermal growth factor (EGF) receptor. Downstream of the EGF receptor, NO activated p21Ras and the MAPK pathway, resulting in a decrease in the nuclear presence of the cyclin-dependent kinase inhibitor 1, p27(KIP1), allowing for cell cycle progression. Furthermore, in a mouse model that shows increased proliferation of neural stem cells in the hippocampus following seizure injury, we observed that the absence of inducible nitric oxide synthase (iNOS(-/-) mice) prevented the increase in cell proliferation observed following seizures in wild-type mice, showing that NO from iNOS origin is important for increased cell proliferation following a brain insult. Overall, we show that NO is able to stimulate the proliferation of neural stem cells bypassing the EGF receptor and promoting cell division. Moreover, under pathophysiological conditions in vivo, NO from iNOS origin also promotes proliferation in the hippocampus.

Rituximab use for lymphoplasmacytic lymphoma during continuous renal replacement therapy. / Uso de rituximabe no tratamento de linfoma linfoplasmocítico durante terapia de substituição renal contínua.

Rituximab safety and efficacy in patients with renal impairment have not been established, nor have the effects of hemodialysis on serum rituximab level. There are only a few published case reports assessing serum rituximab level pre- and postdialysis. No data have been published regarding the usage of rituximab in patients with continuous renal replacement therapy. The authors present a case of a 59-year-old female patient who presented with paraneoplastic tetraparesis. She was admitted to the intensive care unit due to alveolar hemorrhage with respiratory failure and acute kidney injury requiring continuous renal replacement therapy. After a diagnostic workup, the diagnosis of lymphoplasmacytic lymphoma was established. Therapy with rituximab and cyclophosphamide was started. Rituximab levels were determined in serum and dialysate. No rituximab was found in the dialysate. The patient died after 2 months in the intensive care unit from nosocomial pneumonia due to multidrug-resistant Pseudomonas aeruginosa.

A segurança e a eficácia do rituximabe em pacientes com comprometimento renal não foram estabelecidas, e o mesmo ocorre com os efeitos da hemodiálise nos níveis séricos de rituximabe. Atualmente, apenas alguns relatos de caso avaliaram o nível sérico de rituximabe antes e após a diálise. Não foram até aqui publicados dados relativos ao uso de rituximabe em pacientes sob terapia de substituição renal contínua. Os autores apresentam um caso referente a uma mulher com 59 anos de idade atendida com quadro de tetraparesia paraneoplásica. Ela foi admitida no serviço de medicina intensiva devido a hemorragia alveolar com insuficiência respiratória e lesão renal aguda, que necessitou da utilização de terapia de substituição renal contínua. Após os procedimentos diagnósticos, estabeleceu-se o diagnóstico de linfoma linfoplasmocítico. Deu-se início ao tratamento com rituximabe e ciclofosfamida. Os níveis de rituximabe foram determinados no soro e no dialisato. Não se encontrou qualquer nível de rituximabe no dialisato. A paciente faleceu após 2 meses no serviço de medicina intensiva por pneumonia nosocomial causada por Pseudomonas aeruginosa resistente a múltiplos fármacos.

Posaconazole in the treatment of refractory Purpureocillium lilacinum (former Paecilomyces lilacinus) keratitis: the salvation when nothing works.

We report a case of a 41-year-old woman, wearer of contact lenses who was presented to the emergency room with a 2-month history of pain and red eye. She presented with a severe keratitis refractory to quinolones, fortified antibiotics and clotrimazole. Due to the risk of perforation, a tectonic penetrating keratoplasty (PK) was performed. Clinical signs of keratitis recurrence were observed and cultures were positive for Purpureocillium lilacinum (former Paecilomyces lilacinus) The patient did not improve on topical amphotericin B and intracameral voriconazole. Worsening of clinical condition required a new PK. Oral posaconazole was initiated postoperatively and suspended at the fourth postoperative month. The cornea remains clear until the last follow-up visit, 12 months after the second graft. To our knowledge, this is the second case report that documents the effectiveness of oral posaconazole in a refractory P. lilacinus keratitis, resistant to other second-generation triazoles and conventional antifungals.

Thrombophilic risk factors for retinal vein occlusion.

The aim is to study risk factors for retinal vein occlusion (RVO), such as thrombophilic and cardiovascular risk factors (CRF). A retrospective consecutive case series of 60 patients with RVO was made, tested for CRF, hyperhomocysteinemia, lupic anticoagulant, antiphospholipid antibody and 5 gene variants: factor V (FV) Leiden (G1691A), factor II (PT G20210A), 5,1-methylenetetra-hydrofolate reductase (MTHFR; 677 C > T and 1298 A > C), plasminogen activator inhibitor 1 (PAI-1; 4 G/5 G). More than 1 CRF were present in 36 patients (60%), which had a significantly higher mean age at diagnosis (66.7 ± 12.9 versus 59.5 ± 13.7 with ≤1 CRF, [t(57) = -2.05, p = 0.045, d = 0.54). Patients with thermolabile MTHFR forms with decreased enzyme activity (T677T or C677T/A1298C) had a significant lower mean age [57.6 ± 15.1; t (58) = 3.32; p = 0.002; d = 0.846] than patients with normal MTHFR enzyme activity (68.5 ± 10.2). Regarding CRF and thermolabile forms of MTHFR, the mean age at diagnosis could be significantly predicted [F(2,56) = 7.18; p = 0.002] by the equation: 64.8 - 10.3 × (thermolabile MTHFR) - 5.31 × ( ≤ 1CRF). Screening of MTHFR polymorphisms may be useful in younger RVO patients, particularly when multiple CRF are absent.

Evaluation of markers of outcome in real-world treatment of diabetic macular edema.

OBJECTIVE: To evaluate short-term markers of outcome in diabetic macular edema (DME). METHODS: Prospective interventional case series included 122 eyes of 122 patients with recently diagnosed DME. Eyes were treated with a 3-monthly loading dose of ranibizumab or aflibercept and pro re nata thereafter. Serial enhanced deep imaging SD-OCT high resolution scans were used to measure subfoveal choroidal thickness (SFCT) and central retinal thickness (CRT). Anatomic (10% CRT decrease) and functional responses (best corrected visual acuity, BCVA gain ≥5 letters) were assessed at 3 months and 6 months using univariate and multivariate analyses. Parameters tested were gender, duration of diabetes, HbA1c, hypertension, CRT, SFCT, BCVA, ellipsoid zone (EZ) status, subfoveal neuroretinal detachment (SND), anti-VEGF used and laser naivety. A logistic regression model was applied to find independent markers outcome. RESULTS: BCVA increased, CRT and SFCT decreased at 3 months and 6 months. Good metabolic control (p = 0.003), intact baseline EZ (p = 0.030), EZ re-grading at 3 M (p < 0.001) and laser naivety (p = 0.001) were associated with better functional outcome. The multivariate linear regression model showed that baseline SND and CRT are predictors of anatomic response, while lower baseline BCVA and intact EZ are predictors of functional response. CONCLUSION: The presence of SND predicts anatomic response only, while an intact EZ is critical to achieve a good functional outcome in DME.