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Rubens Camargo Siqueira1,2 1Department of Retina, Rubens Siqueira Research Center, São José do Rio Preto, São Paulo, Brazil; 2Postgraduate Department, Faculty of Medicine of São José do Rio Preto (FAMERP), São José do Rio Preto, São Paulo, BrazilCorrespondence: Rubens Camargo Siqueira, Email siqueiraretina@gmail.comAbstract: Photobiomodulation (PBM) is a type of phototherapy that employs light-emitting diodes (LEDs) or low-power lasers to selectively administer specific wavelengths of visible light, ranging from 500 to 1000 nm, including near-infrared (NIR) wavelengths. LEDs are advantageous compared to lasers due to their ability to treat large areas at a lower cost, lack of tissue damage potential in humans, and reduced risk of eye-related accidents. The ophthalmology community has recently taken interest in PBM as a promising novel approach for managing various retinal conditions such as age-related macular degeneration, retinopathy of prematurity, retinitis pigmentosa, diabetic retinopathy, Leber’s hereditary optic neuropathy, amblyopia, methanol-induced retinal damage, and potentially others. This review critically assesses the existing body of research on PBM applications in the retina, focusing on elucidating the underlying mechanisms of action and evaluating the clinical outcomes associated with this therapeutic modality.Keywords: photobiomodulation, near-infrared, light emitting diode, age-related macular degeneration

Carina Costa Cotrim, Luiza Toscano, André Messias, Rodrigo Jorge, Rubens Camargo Siqueira Department of Ophthalmology, Otorhinolaryngology and Head and Neck Surgery, Ribeirao Preto School of Medicine, University of Sao Paulo, Sao Paulo, Brazil Purpose: To evaluate the therapeutic potential and safety of intravitreal injections of bone marrow mononuclear fraction (BMMF) containing CD34+ cells in patients with atrophic age-related macular degeneration (AMD).Methods: Ten patients with atrophic AMD and best-corrected visual acuity (BCVA) in the worse-seeing eye of ≤20/100 were enrolled in this study. The bone marrow from all patients was ­aspirated and processed for mononuclear cell separation. A 0.1 mL suspension of BMMF CD34+ cells was injected into the vitreous cavity of the worse-seeing eye. Patients were evaluated at Baseline and 1,3,6,9 and 12 months after injection. Ophthalmic evaluation included BCVA measurement, microperimetry, infrared imaging, fundus autofluorescence and SD-optical coherence tomography at all study visits. Fluorescein angiography was performed at Baseline and at 6 and 12 months after intravitreal therapy.Results: All patients completed the 6-month follow-up, and six completed the 12-month follow-up. Prior to the injection, mean BCVA was 1.18 logMAR (20/320-1), ranging from 20/125 to 20/640-2, and improved significantly at every follow-up visit, including the 12-month one, when BCVA was 1.0 logMAR (20/200) (P

Rubens Camargo Siqueira,1,2 Roberto Luiz Kaiser Junior,2 Lilian Piron Ruiz,2 Milton Arthur Ruiz2 1Department of Ophthalmology, Otorhinolaryngology and Head and Neck Surgery, School of Medicine of Ribeirão Preto, University of Sao Paulo, Ribeirão Preto, Sao Paulo, Brazil; 2Department of Bone Marrow Transplantation, Beneficencia Portuguesa Hospital, Sao José do Rio Preto, Sao Paulo, Brazil Purpose: To report a case of a patient with ischemic retinopathy associated with Crohn’s disease.Case report: This report presents a case of a 28-year-old female patient with Crohn’s disease and sudden decrease of visual acuity in the right eye. Fluorescein angiography, optical coherence tomography, and multifocal electroretinography confirmed the clinical features of ischemic retinopathy. After systemic corticosteroid treatment, the patient developed epiretinal membrane without significant improvement in visual acuity.Discussion: The patient presented with ischemic retinopathy associated with Crohn’s disease with deficiency of central visual acuity. Periodic examination by a retina specialist is recommended for patients being treated for Crohn’s disease. Keywords: Crohn, ischemic retinopathy, epiretinal membrane, OCT, ERG

Rubens Camargo Siqueira1,2, Rodrigo Jorge21Rubens Siqueira Research Center, São José do Rio Preto, São Paulo, Brazil; 2Retina and Vitreous Section, Department of Ophthalmology, School of Medicine of Ribeirão Preto, University of São Paulo, BrazilAbstract: Clinical laboratories are strong, integral partners in personalized health care. Laboratory databases hold a vast amount of data on human phenotypes, genotypes, biomarkers, progression of disease, and response to therapy. These structured and unstructured free text data are critical for patient care and a resource for personalized medicine and translational research. Laboratory data are integrated into many electronic medical records that provide “summary reports” and “trending” to visualize longitudinal patient data. Recent advances in ophthalmology such as gene therapy, cell therapy using stem cells, and also retinal prosthesis explore the potential of translational research marking a new era in research into the diagnosis and treatment of eye diseases.Keywords: translational, retinal diseases, stem cell, gene therapy

Ocular toxoplasmosis (OT) is characterised by intraocular inflammation due to Toxoplasma gondii infection. Studies have found that interleukin 17 (IL-17) plays a central role in the pathology of OT. However, nucleotide variability in IL17 and interleukin 17 receptor (IL17R) genes has not been characterised in OT. As cytokine gene polymorphisms may influence the expression of these molecules, the aim of this study was to verify whether IL17A (rs2275913), IL17F (rs763780), IL17RA (rs4819554) and IL17RC (rs708567) polymorphisms are associated with OT in a Brazilian population. This study enrolled 214 patients seropositive for T. gondii (110 with OT and 104 without) and 107 controls. Polymorphisms were identified by PCR-restriction fragment length polymorphism analysis, validated by DNA sequencing with chi-square and multivariate analyses being used to assess possible associations between polymorphisms and OT. Logistic regression under the dominant model revealed a protection factor against OT of the C mutant allele of the IL17F (rs763780) polymorphism. The T/C-C/C genotypes were significantly more common in patients without OT compared to those with OT (p value = 0.0066) and controls (p value = 0.014). Findings from this study suggest that the IL17F polymorphism may have an influence in the immunopathology of OT in Brazilian individuals., (© 2024 John Wiley & Sons Ltd.)

Dowling Degos disease (DDD) is a rare autosomal dominant genodermatosis characterized by acquired, slowly progressive reticulated pigmented lesions primarily involving flexural skin areas. Mutations in KRT5, POGLUT-1 and POFUT-1 genes have been associated with DDD, and loss-of-function mutations in PSENEN, a subunit of the gamma-secretase complex, were found in patients presenting with DDD or DDD comorbid with hidradenitis suppurativa (HS). A nonsense mutation in NCSTN, another subunit of the gamma-secretase, was already described in a patient suffering from HS and DDD but whether NCSTN could be considered a novel gene for DDD is still debated. Here, we enrolled a four-generation family with HS and DDD. Through Whole Exome Sequencing (WES) we identified a novel nonsense mutation in the NCSTN gene in all the affected family members. To study the impact of this variant, we isolated outer root sheath cells from patients' hair follicles. We showed that this variant leads to a premature stop codon, activates a nonsense-mediated mRNA decay, and causes NCSTN haploinsufficiency in affected individuals. In fact, cells treated with gentamicin, a readthrough agent, had the NCSTN levels corrected. Moreover, we observed that this haploinsufficiency also affects other subunits of the gamma-secretase complex, possibly causing DDD. Our findings clearly support NCSTN as a novel DDD gene and suggest carefully investigating this co-occurrence in HS patients carrying a mutation in the NCSTN gene., (© 2023 The Authors. Experimental Dermatology published by John Wiley & Sons Ltd.)

Introduction: Ocular toxoplasmosis (OT) is an intraocular inflammation caused by Toxoplasma gondii infection that affects the retina and choroid, giving rise to posterior uveitis. Genetic polymorphisms in cytokine genes may exert influence in the expression of these molecules and play a significant role in inflammatory responses and susceptibility to OT. The aim of this study was to evaluate the role of polymorphisms rs16944 (-511 C > T) of the interleukin (IL) 1β gene and rs1800896 (-1082 G > A) of the IL10 gene on OT in Brazilian individuals with a serologic diagnosis of T. gondii and after conducting fundoscopic exams., Methods: Participants with a positive serology were classified into two distinct groups according to the presence (G1; n  = 110) or absence (G2; n  = 104) of OT. The control group (G3) consisted of individuals without the infection ( n  = 108)., Results: It was observed that the C/C genotype of the IL1β gene polymorphism was a protective factor for OT ( p  = 0.02, OR = 0.28, 95% CI 0.08-0.78 for G1 vs. G2; p  = 0.03; OR = 0.29, 95% CI 0.09-0.82 for G1 vs. G3), according to the recessive inheritance model., Conclusions: The -511C.T polymorphisms of the IL1β gene seems to play an important role in the pathogenesis of OT in Brazilian individuals., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Araujo, Ayo, Previato, de Faria, Frederico, Siqueira, de Almeida, Pereira-Chioccola, de Mattos and Brandão.)

Purpose: The purpose of this study was to investigate the vascular effects of photobiomodulation using a light-emitting diode on the chorioallantoic embryonic membrane of chicken eggs grouped into different times of exposure and to detect the morphological changes induced by the light on the vascular network architecture using quantitative metrics., Methods: We used a phototherapy device with light-emitting diode (670 nm wavelength) as the source of photobiomodulation. We applied the red light at a distance of 2.5 cm to the surface of the chorioallantoic embryonic membrane of chicken eggs in 2, 4, or 8 sessions for 90 s and analyzed the vascular network architecture using AngioTool software (National Cancer Institute, USA). We treated the negative control group with 50 μl phosphate-buffered-saline (pH 7.4) and the positive control group (Beva) with 50 μl bevacizumab solution (Avastin, Produtos Roche Químicos e Farmacêuticos, S.A., Brazil)., Results: We found a decrease in total vessel length in the Beva group (24.96% ± 12.85%) and in all the groups that received 670 nm red light therapy (2× group, 34.66% ± 8.66%; 4× group, 42.42% ± 5.26%; 8× group, 38.48% ± 6.96%), compared with the negative control group. The fluence of 5.4 J/cm2 in 4 sessions (4×) showed more regular vessels. The number of junctions in the groups that received a higher incidence of 670 nm red light (4× and 8×) significantly decreased (p<0.0001)., Conclusion: Photo-biomodulation helps reduce vascularization in chorioallantoic embryonic membrane of chicken eggs and changes in the network architecture. Our results open the possibility of future clinical studies on using this therapy in patients with retinal diseases with neovascular components, especially age-related macular degeneration.

Objective:  The aim of this study was to evaluate the influence of near-cortical over-drilling holes on the mechanical behaviour of locking plate constructs applied in maned wolf's femur by using mechanical testing and finite element method (FEM)., Study Design:  Seven pairs of adult maned wolves ( Chrysocyon brachyurus ) femur bones were randomly distributed into four groups. In all groups, a 3.5 mm locking compression plate, designed with 12 combi-holes and one locked, was applied to the lateral surface of the femur. G1 ( n  = 4) received bicortical locking screws placed in holes 1, 3, 5, 8, 10 and 12. In G2 ( n  = 5), the plate was applied as used in G1, but the application of the locked screws involved the near-cortical over-drilling technique. In G3 ( n  = 4), the plate was applied as used in G2, but the size of the near-cortical over-drilling was larger. The combi-holes 6 and 7 were maintained over a 10 mm fracture gap without screws. All constructs were tested for failure in the axial load. The axial load was applied eccentrically to the femoral head., Results:  Statistical differences were observed in the maximum load with G3 > G1 and G3 > G2, and in the deflection with G2 > G1 and G2 > G3. The FEM showed the lowest total displacement of the bone-plate constructs as well as of the plate in G1 compared with G2 and G3., Conclusion:  The near-cortical over-drilling technique used in unstable fractures induced in the maned wolf's femur showed by static axial compression test that maximum load and deflection are dependent on drill hole size induced in the near-cortex. Based on FEM, the lowest total displacement of the bone-plate constructs was observed in Group 1., Competing Interests: None declared., (Thieme. All rights reserved.)

Purpose: To evaluate in-vivo degradation of two bioabsorbable interference screws., Methods: Twenty-two crossbred Santa Inês ewes were used. A poly-DL-lactide (PDLLA) screw (70%/30%) was inserted in the right pelvic limb, and a PDLLA screw (70%) + β-tri-calcium phosphate (β-TCP) (30%) in the left pelvic limb. Animals were euthanized at one, four, seven and a half and 18 months after surgery. Plain radiography, computed tomography (CT), microCT, and histological analysis were accomplished., Results: PDLLA screw was hypodense at all evaluation moments, but with progressive density increase along the central axis, whereas PDLLA/β-TCP was initially hyperdense and progressively lost this characteristic. No adverse reactions were observed on histological evaluation., Conclusions: The inclusion of β-TCP favors screw degradation since the PDLLA/β-TCP screws evidenced a more intense degradation process than the PDLLA screws at the last evaluation. PDLLA screws showed higher bone production, evident around the screw thread, inside the lateral perforations, and in the central canal, whereas the PDLLA/β-TCP screws presented less bone tissue at the implantation site.

Objective: To evaluate the short-term result of retinal functional behavior in patients with dry age-related macular degeneration (AMD) corrected by photobiomodulation (PBM) with 670 nm light-emitting diode (LED) light. Materials and methods: Ten patients with dry AMD underwent a treatment consisting of nine PBM sessions with LED light of 670 nm with two cycles of 50 mW/cm 2 , producing 4 J/cm 2 per dose in 88 sec. The studied eye was compared with the baseline (before therapy), and after nine PBM sessions, the following aspects were evaluated: best-corrected visual acuity (VA), retinal sensitivity, and characteristics of the correction area by the fundus automated perimetry using the Compass system. A functional and structural assessment of the retina was also performed using the multifocal electroretinography (ERG), optical coherence tomography (OCT), fluorescence retinography (FR), and autofluorescence (AF). All examinations were performed 1, 4, and 16 weeks after the therapy. The Chi-square and Student's t -tests were used for comparisons. The analyses followed the 95% confidence level ( p -value ≤0.05). Results: The BCVA significantly improved, from an average of 1.1 to 0.98 LogMAR ( p  = 0.01). The visual field examination, according to the parameters of mean deviation, standard deviation, and index of deviation of background perimeter, showed a significant improvement of -12.6% to -10.6%, 10.54% to 9.89%, and 56% to 60%, respectively ( p  = 0.02, 0.03, and 0.02, respectively). No participant had an adverse effect during the follow-up period; neither did any participant experience abnormalities in OCT, ERG, FR, and AF findings. Conclusions: In this short-term study, the PBM technique in patients with dry AMD showed the potential to improve VA and macular perimetry without causing significant adverse events. A larger number of patients and a longer follow-up will be necessary to further assess the success of this technique in these patients.

Purpose: To report electroretinographic (ERG) findings in advanced glaucoma treated with a single intravitreal injection of bone marrow-derived mesenchymal stem cells (MSCs)., Methods: Intravitreal injection of autologous MSCs (1 × 10 6 cells) was performed in 2 eyes from 2 patients with open-angle glaucoma in advanced stage of optic neuropathy (ClinicalTrials.gov, NCT02330978, 01.05.2015): cup/disk ratio worse than 0.9, visual field mean deviation index lower than - 15 dB, visual acuity of light perception, but controlled intraocular pressure. ERG tests were recorded at baseline and week 1, 4 and 48 after injection, using DTL electrodes following the ISCEV standard: After dark adaptation, ERG was elicited using white flashes of 0.01 cd.s/m 2 and 3.0 cd.s/m 2 , followed by 10-min light adaptation (30 cd/m 2 ) and stimuli of 3.0 cd.s/m 2 and 30 Hz flicker., Results: Patients did not show improvement on visual acuity or visual field after treatment. At baseline, ERG responses showed typical findings for advanced glaucoma, with a- and b-wave amplitude and latency within normative range, but reduced photopic negative responses. No noteworthy changes were observed on ERG responses for both cases up to 1 week after treatment, but at day 15, one patient showed retinal detachment with proliferative vitreoretinopathy and was removed from the trial. The other patient kept ERG responses stable throughout study period., Conclusion: Although no ERG response changes were observed after MSCs injection in one case, the complication observed on the second one, along with the lack of visual function improvement, warrants further studies involving modified MSCs to treat ocular disorders, including glaucoma., Trial Registration: ClinicalTrials.gov, NCT02330978- missed in pdf.

To assess the therapeutic potential and the safety of intravitreous use of a bone marrow mononuclear fraction (BMMF) containing CD34+ cells in patients with Stargardt type macular dystrophy. The study was conducted on 10 patients with Stargardt dystrophy with worse eye visual acuity ≤ 20/125. A bone marrow aspirate was obtained from all patients, and after processing in the cell therapy center (CTC), 0.1 ml of the intravitreous BMMF suspension was injected into the eye with worse visual acuity. A sham injection was performed in the contralateral eye. The patients were evaluated at baseline and one, three, and six months after the injection. All of them were submitted to measurement of best corrected visual acuity (BCVA), microperimetry, multifocal electroretinography (mfERG) and full field electroretinography (ffERG), autofluorescence (AF), and optical coherence tomography (OCT). Fluorescein angiography was also performed before and six months after the injection. All patients completed the six-month period of evaluation. Mean visual acuity of the treated eye was 1.1 logMAR (20/250) before intravitreous (IV) injection, 0.96 logMAR (20/200+2) one month after injection, and 0.92 logMAR (20/160-1) 3 months after injection. In the untreated eye, mean VA was 1.0 logMAR (20/200) at baseline and 0.96 logMAR (20/200+2) and 0.94 logMAR (20/160-2) one and three months after injection, respectively. In the treated group, VA at baseline ranged from best acuity of 20/125-1 to worst acuity of 20/640+2, going through 20/100+2 and 20/400 during the first month. In the untreated group, BCVA ranged from 20/100+2 to 20/400 at baseline and from 20/100 to 20/400 after one month. The results for the treated group differed significantly at all follow-up times, whereas no significant difference was observed in the untreated group. Regarding the mean sensitivity of microperimetry, although there was improvement throughout all months, a significant difference occurred only during the first month. In the untreated eye, there was no significant difference in any analysis. Angiofluoresceinography did not reveal neovessel formation or tumor growth. The remaining exams were used in order to aid the diagnosis. The results indicate that the use of intravitreous BMMF in patients with Stargardt dystrophy is safe and is associated with a discrete improvement of BCVA and microperimetry in the treated eye compared to the untreated one., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2020 Carina Costa Cotrim et al.)

Duffy blood group phenotypes [Fy(a + b-), Fy(a-b+), Fy(a + b+), Fy(a-b-)], characterized by the expression of Fy a , and Fy b antigens, are present in red blood cells. Therefore, we hypothesize that the non-hematopoietic expression of these antigens might influence cell invasion by T. gondii. 576 consecutive patients from both genders were enrolled. The presumed OT clinical diagnosis was performed. Duffy phenotyping was performed by hemagglutination in gel columns and for the correct molecular characterization Fy(a-b-) phenotype, using PCR-RFLP. Anti-T. gondii IgG antibodies were detected by ELISA. Chi-square, Fisher's exact tests were used to compare the proportions. OT was present in 22.9% (n = 132) and absent in 77.1% (n = 444) of patients. The frequencies of anti-T. gondii IgG antibodies were higher in OT (127/132, 96.2%) than those without this disease (321/444, 72.3%) (p < .0001). None of the Duffy antigens or phenotypes were associated with T. gondii infection (χ2: 2.222, GL: 3, p = .5276) as well as the risk of OT (χ2: 0.771, GL: 3, p = .8566). Duffy blood group system phenotypes and their antigens do not constitute risk factors for infection by T. gondii infection and the development of OT., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Purpose: To evaluate 24-week visual acuity and anatomic outcomes of two "pro re nata" (prn) treatment strategies (intravitreal bevacizumab [IVB] prn versus intravitreal triamcinolone acetonide [IVT] prn) in patients with persistent diabetic macular edema (pDME) after 24 weeks of prn-IVB., Methods: One hundred eyes with center-involving DME were enrolled and treated with prn-IVB for 24 weeks; at week 24, eyes with pDME (central subfield thickness [CST] on spectral domain optical coherence tomography > 300 μm) were randomized to IVB monthly prn (group I; prn-IVB) or IVT every 3 months prn (group II; prn-IVT) and eyes in which the CST was ≤ 300 μm were assigned to continue prn-IVB (group III)., Results: Seventy-four eyes completed a 48-week study period. At week 24, 65 (79.3%) eyes still had DME with CST > 300 μm and, therefore, were randomized to prn-IVB (group I, n = 33) or prn-IVT (group II, n = 32); the remaining 17 (20.7%) eyes had CST ≤ 300 μm and were assigned to continued treatment with prn-IVB (group III). At baseline, mean CST (μm) ± standard error of the mean (SEM) was 447.2 ± 24.4, 478.0 ± 19.7, and 386.0 ± 21.0 in groups I, II, and III, respectively (p > 0.05). At week 48, there was no significant difference in mean CST between groups I and II (369.9 ± 23.3 and 426.0 ± 26.1, respectively; p = 0.9995). A significant reduction in mean CST, compared with baseline, was noted at weeks 28 (p = 0.0002) and 44 (p = 0.0002) in group II. Group I did not show a significant reduction in mean CST compared with baseline at any study visit. There were no significant differences in mean CST between groups I and II at any study visit. At baseline, mean ± SEM best-corrected visual acuity (BCVA) (logMAR) was 0.50 ± 0.00, 0.60 ± 0.10, and 0.50 ± 0.10 in groups I, II, and III, respectively (p > 0.05). At week 48, there was no statistically significant difference in mean BCVA between groups I and II (0.50 ± 0.10 and 0.80 ± 0.10, respectively; p = 0.4473). There was no significant improvement in mean BCVA, as compared with baseline, at any study follow-up visit in any of the groups. Group II demonstrated significantly lower BCVA after 24 weeks of IVT (at week 48) compared with baseline (p = 0.0435). There was no significant difference in mean BCVA between groups I and II at any time-point., Conclusion: In eyes with pDME after 24 weeks of treatment with prn-IVB, there was no difference between continued treatment with prn-IVB versus a treatment switch to prn-IVT with respect to mean BCVA or mean CST at week 48. However, BCVA was stable in the prn-IVB group, while prn-IVT was associated with BCVA reduction from baseline and a higher risk of IOP elevation.

Essential oils (EOs) are considered a new class of ecological products aimed at the control of insects for industrial and domestic use; however, there still is a lack of studies involving the control of fleas. Ctenocephalides felis felis, the most observed parasite in dogs and cats, is associated with several diseases. The aim of this study was to evaluate the in vitro activity, the establishment of LC50 and toxicity of EOs from Alpinia zerumbet (Pers.) B. L. Burtt & R. M. Sm, Cinnamomum spp., Laurus nobilis L., Mentha spicata L., Ocimum gratissimum L. and Cymbopogon nardus (L.) Rendle against immature stages and adults of C. felis felis. Bioassay results suggest that the method of evaluation was able to perform a pre-screening of the activity of several EOs, including the discriminatory evaluation of flea stages by their LC50. Ocimum gratissimum EO was the most effective in the in vitro assays against all flea stages, presenting adulticide (LC50 = 5.85 μg cm-2), ovicidal (LC50 = 1.79 μg cm-2) and larvicidal (LC50 = 1.21 μg cm-2) mortality at low doses. It also presented an excellent profile in a toxicological eukaryotic model. These findings may support studies involving the development of non-toxic products for the control of fleas in dogs and cats.

Degenerative retinal diseases such as retinitis pigmentosa, Stargardt's macular dystrophy, and age-related macular degeneration are characterized by irreversible loss of vision due to direct or indirect photoreceptor damage. No effective treatments exist, but stem cell studies have shown promising results. Our aim with this review was to describe the types of stem cells that are under study, their effects, and the main clinical trials involving them.

Ocular toxoplasmosis (OT) is one of the most common manifestations of Toxoplasma gondii infection and can be related with congenital or acquired infections. OT cause posterior uveitis that cause serious sequelae as complete loss of vision. microRNAs (miRNAs) are small non-coding RNAs, which have regulatory roles in cells by silencing messenger RNA. This study evaluated gene expression of miR-155-5p, miR-146a-5p, miR-21-5p, miR-29c-3p and miR-125b-5p in plasma of 51 patients with ocular toxoplasmosis (OT Group), 26 individuals with asymptomatic toxoplasmosis (AT Group), and 25 healthy individuals seronegative for toxoplasmosis (NC Group). Peripherical blood samples were collected in tube with EDTA for plasma isolation, laboratorial diagnosis for toxoplasmosis and RNA extraction. miRNA expression of each sample was performed by qPCR and values were expressed in Relative Quantification (RQ). Results showed that miR-155-5p and miR-29c-3p were up-expressed in OT patients than AT individuals. On the other hand, miR-21-5p and miR-125b-5p were down-expressed in OT patients. Differences were statistically significant. miR-146a-5p expression was similar in OT patients and AT individuals, without significant difference. In addition, comparative analysis for miRNA levels between AT and OT groups confirms these results. So far, this is the first study to evaluate circulating miRNA levels in ocular toxoplasmosis. These findings may contribute to further studies evaluating the exact role of these miRNAs in the course of infection, which may help in understanding the complex parasite-host interaction and future use in diagnosis, prognosis and therapeutic control in ocular toxoplasmosis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Ocular toxoplasmosis is one of the most common complications caused by the infection with the parasite Toxoplasma gondii . The risk of developing eye lesions and impaired vision is considered higher in Brazil than other countries. The clinical diagnosis is difficult and the use of sensitive and specific laboratorial methods can aid to the correct diagnosis of this infection. We compared serological methods ELISA and ELFA, and molecular cPCR, Nested PCR and qPCR for the diagnosis of T. gondii infection in groups of patients clinically evaluated with ocular diseases non-toxoplasma related (G1 = 185) and with lesions caused by toxoplasmosis (G2 = 164) in an Ophthalmology clinic in Brazil. Results were compared by the Kappa index, and sensitivity (S), specificity (E), positive predictive value (PPV), and negative (NPV) were calculated. Serologic methods were in agreement with ELISA more sensitive and ELFA more specific to characterize the acute and chronic infections while molecular methods were discrepant where qPCR presented higher sensitivity, however, lower specificity when compared to cPCR and Nested PCR., (Copyright © 2020 Murata, Previato, Frederico, Barbosa, Nakashima, Faria, Silveira Carvalho, Meira Strejevitch, Pereira-Chioccola, Castiglioni, de Mattos, Siqueira and Brandão de Mattos.)

Cancer is one of the leading causes of death worldwide, and the number of patients has only increased each year, despite the considerable efforts and investments in scientific research. Since natural products (NPs) may serve as suitable sources for drug development, the cytotoxicity against cancer cells of 2221 compounds from the Nuclei of Bioassays, Ecophysiology, and Biosynthesis of Natural Products Database (NuBBE DB ) was predicted using CDRUG algorithm. Molecular modeling, chemoinformatics, and chemometric tools were then used to analyze the structural and physicochemical properties of these compounds. We compared the positive NPs with FDA-approved anticancer drugs and predicted the molecular targets involved in the anticancer activity. In the present study, 46 families comprising potential anticancer compounds and at least 19 molecular targets involved in oncogenesis. To the best of our knowledge, this is the first large-scale study conducted to evaluate the potentiality of NPs sourced from Brazilian biodiversity as anticancer agents, using in silico approaches. Our results provided interesting insights about the mechanism of action of these compounds, and also suggested that their structural diversity may aid structure-based optimization strategies for developing novel drugs for cancer therapy., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The aim of this study was to determine some radiographic reference values for hip joint, femur and patella in maned wolves. Ten maned wolves (Chrysocyon brachyurus), five live (G1) and five cadavers (G2), were used. There were no statistically significant differences in the radiological measurements between right and left pelvic limbs of the G1 and G2, except for mechanical lateral proximal femoral angle (mLPFA) in both Groups. The comparison of the measurements between Groups showed difference only for mechanical lateral distal femoral angle (mLDFA). Norberg angle values were 113.1° and 112.9° for G1 and G2. The values of femoral angle of inclination by Hauptman A and Hauptman B methods were 139.7° and 128.6° for G1, and 139.3° and 128.7° for G2. Femoral varus angle values were 5.5° for G1 and 3.9° for G2. The values of anatomic lateral distal femoral angle and anatomic lateral proximal femoral angle were 96.8° and 95.2° for G1, and 95.0° and 95.6° for G2. The values of mLDFA for G1 and G2 were 96.5° and 95.7°. The values of mLPFA were 95.8 (right) and 93.5 (left) for G1, and 95.7 (right) and 93.8 (left) for G2. The values of patellar ligament length, patellar length and L:P ratio were 4.6, 2.4 cm and 1.9 for G1, and 4.4, 2.3 cm and 1.8 for G2. In conclusion, the radiographic reference values described for the hip joint, the femur and the patella, may be useful in the diagnosis of diseases and deformities in maned wolves., (© 2018 Blackwell Verlag GmbH.)

Background: Bone marrow-derived mesenchymal stromal cell (MSC) therapy is a promising treatment for several degenerative ocular diseases; however, no reproducible method of monitoring these cells into the eye has been established. The aim of this study was to describe successful bioluminescence imaging (BLI) to detect viable luciferase-expressing MSC in the eye., Methods: Human donor MSC in culture were transduced with 50 μl luciferase lentiviral vector (three viral particles/cell) prior to intraocular injection. Twenty-one right eyes of 21 rabbits were evaluated through BLI after receiving 1 × 10 6 luciferase-expressing MSC intravitreally. Contralateral eyes were injected with vehicle (phosphate-buffered saline (PBS)) and were used as controls. At seven different time points (1 h to 60 days), D-luciferin (40 mg/ml, 300 μl PBS) was injected in subsets of six enucleated eyes for evaluation of radiance decay through BLI analysis. CD90 and CD73 immunofluorescence was studied in selected eyes., Results: Eyes injected with MSC showed high BLI radiance immediately after D-luciferin injection and progressive decay until 60 days. Mean BLI radiance measures from eyes with luciferase-expressing MSC were significantly higher than controls from 8 h to 30 days. At the thirtieth day, positive CD90- and CD73-expressing cells were observed only in the vitreous cavity of eyes injected with MSC., Conclusions: Viable MSC were identified in the vitreous cavity 1 month after a single injection. Our results confirmed BLI as a useful and reliable method to detect MSC injected into the eye globe.

CC chemokine receptor type 5 (CCR5) is a chemokine receptor that influences the immune response to infectious and parasitic diseases. This study aimed to determine whether the CCR5Δ32 and CCR5 59029 A/G polymorphisms are associated with the development of ocular toxoplasmosis in humans. Patients with positive serology for Toxoplasma gondii were analyzed and grouped as 'with ocular toxoplasmosis' (G1: n=160) or 'without ocular toxoplasmosis' (G2: n=160). A control group (G3) consisted of 160 individuals with negative serology. The characterization of the CCR5Δ32 and CCR5 59029 A/G polymorphisms was by PCR and by PCR-RFLP, respectively. The difference between the groups with respect to the mean age (G1: mean age: 47.3, SD±19.3, median: 46 [range: 18-95]; G2: mean age: 61.3, SD±13.7, median: 61 [range: 21-87]; G3: mean age: 38.8, SD±17.9, median: 34 [range: 18-80]) was statistically significant (G1 vs.G2: p-value <0.0001; t=7.21; DF=318; G1 vs.G3: p-value <0.0001; t=4.32; DF=318; G2 vs. G3: p-value <0.0001; t=9.62; DF=318). The Nagelkerke r 2 value was 0.040. There were statistically significant differences for the CCR5/CCR5 (p-value=0.008; OR=0.261), AA (p-value=0.007; OR=2.974) and AG genotypes (p-value=0.018; OR=2.447) between G1 and G2. Individuals with the CCR5/CCR5 genotype and simultaneously the CCR5-59029 AA or AG genotypes have a greater risk of developing ocular toxoplasmosis (4% greater), which may be associated with a strong and persistent inflammatory response in ocular tissue., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Purpose: To evaluate the safety and feasibility of a 25-gauge biodegradable implant containing 350 μg of dexamethasone (DDS-25) for the treatment of decreased vision due to macular edema associated with central or branch retinal vein occlusion., Methods: Prospective, nonrandomized, open-label, Phase I clinical trial, including 10 patients with decreased vision (best-corrected early treatment diabetic retinopathy study visual acuity of 20/40 or worse) due to macular edema associated with central retinal vein occlusion (n = 4) or branch retinal vein occlusion (n = 6) for more than 4 months. Comprehensive ophthalmic evaluation, including best-corrected visual acuity, spectral domain optical coherence tomography (Spectralis Heidelberg Engineering) for determination of central subfield thickness, full-field electroretinography (ISCEV standard ERG), and fluorescein angiography, was performed at baseline, and 1, 4, 12, and 24 weeks after intravitreal DDS-25 insertion., Results: Mean best-corrected visual acuity was 0.72 ± 0.1 logMAR (20/100) at baseline and improved by 7 early treatment diabetic retinopathy study letters to 0.58 ± 0.08 logMAR (20/80 + 1) at 24 weeks (P = 0.049), with 3 central retinal vein occlusion and 3 branch retinal vein occlusion patients improving between 1 and 4 early treatment diabetic retinopathy study lines. Significant central subfield thickness reduction was observed at 24 weeks compared with baseline (P = 0.011); mean ± standard error (range) central subfield thickness (μm) was 461.2 ± 41.3 (288-701) at baseline, and 439.6 ± 40.4 (259-631), 442.5 ± 44.6 (255-632), 354.6 ± 31.2 (228-537), and 316.5 ± 26.4 (226-441) at 1, 4, 12, and 24 weeks, respectively. No significant changes in electroretinography responses or area of retinal nonperfusion were observed during 24 weeks of follow-up. There was no significant change in mean intraocular pressure at any of the study visits compared with baseline. One patient had mild anterior chamber inflammation (1-5 cells) at one week after DDS-25 insertion., Conclusion: In this Phase I study demonstrating the feasibility of intravitreal DDS-25 insertion for the treatment of decreased vision due to macular edema associated with retinal vein occlusion, no safety concerns were observed. A larger prospective randomized study with longer follow-up is warranted to confirm these findings.

This study describes the osteology and radiology of the pelvic limb in maned wolves. Ten (five live and five dead) maned wolves (Chrysocyon brachyurus), five males and five females, aged from 2 to 7 years old were used. Digital radiographs were taken and recorded for both pelvic limbs in all animals. Osteology was correlated with the radiographic images. The pelvis had a rectangular shape, and the obturator foramen (foramen obturatum) was oval. The femoral neck (collum femoris) was short and thick. The greater trochanter (trochanter major) extended proximally to near the dorsum of the femoral head (caput ossis femoris). The lateral femoral condyle (condylus lateralis) was larger than the medial condyle (condylus medialis), and the intercondylar fossa (fossa intercondylaris) had a slightly oblique orientation. The proximal tibia displayed medial and lateral condyles with the medial larger. The femur was slightly shorter than the tibia. Seven tarsal bones (ossa tarsi) were present, four long metatarsal bones (ossa metatarsalia II - V) and a short first metatarsal bone (os metatarsal I)., (© 2017 Blackwell Verlag GmbH.)

Objective: The incidence of adverse events in myeloablative transplant protocols is high in refractory Crohn's disease; this study used low doses of cyclophosphamide. Fourteen patients were submitted to non-myeloablative autologous hematopoietic stem cell transplantation., Results: The average number of days of anemia (hemoglobin < 10 g/dL) was 5.4 ± 4.2 and 14 ± 2.4 in the mobilization and conditioning phases, respectively. The mean number of days of neutropenia (neutrophils < 0.5 × 10 9 /L) in the mobilization phase was 1.7 ± 1.5 while it was 7.6 ± 1.4 in the conditioning phase. When comparing the conditioning and mobilization phases, there was an increased number days of leukopenia (white blood cells < 1.0 × 10 9 /L), lymphocytopenia (lymphocytes < 0.5 × 10 9 /L) and thrombocytopenia (platelets < 25 × 10 9 /L). Crohn's Disease Activity Index values before the transplant ranged from 155 to 450.5 (mean 281.2 ± 79.0) and at 30 days after the procedures they ranged from 45.4 to 177 (mean 95.8 ± 35.4). Moreover, the procedure improved in overall quality of life of patients. Non-myeloablative autologous hematopoietic stem cell transplantation with lower doses of cyclophosphamide leads to lower rates of hematological toxicity and adverse events compared to protocols described in the literature. Trial registration NCT 03000296: Date 9 December 2016.

This study evaluated levels for mRNA expression of 7 cytokines in ocular toxoplasmosis. Peripheral blood mononuclear cells (PBMC) of patients with ocular toxoplasmosis (OT Group, n = 23) and chronic toxoplasmosis individuals (CHR Group, n = 9) were isolated and stimulated in vitro with T. gondii antigen. Negative controls (NC) were constituted of 7 PBMC samples from individuals seronegative for toxoplasmosis. mRNA expression for cytokines was determined by qPCR. Results showed a significant increase in mRNA levels from antigen stimulated PBMCs derived from OT Group for expressing IL-6 (at P < .005 and P < .0005 for CHR and NC groups, respectively), IL-10 (at P < .0005 and P < .005 for CHR and NC groups, respectively) and TGF-β (at P < .005) for NC group. mRNA levels for TNF-α and IL-12 were also upregulated in patients with OT compared to CHR and NC individuals, although without statistical significance. Additionally, mRNA levels for IL-27 and IFN-γ in PBMC of patients with OT were upregulated in comparison with NC individuals. Differences between OT and NC groups were statistically significant at P < .05 and P < .0005, respectively., (© 2017 John Wiley & Sons Ltd.)

Background: The aim of this study is to report a case of cystoid macular oedema (CME) associated with Rosai-Dorfman Disease (RDD)., Case Presentation: A 52-year-old male initially presented with a two-month history of a congested left eye. At presentation, visual acuity was 20/20 in the right eye and 20/80 in the left eye. Biomicroscopy showed conjunctival hyperaemia in the left eye with a slight elevation, suggesting a subconjunctival mass. Retinal fluorescein angiography and optical coherence tomography (OCT) revealed the presence of CME in the left eye. A clinical examination revealed nodular lesions in the ears and a lump in the subcutaneous tissue of the left arm. A biopsy of the subcutaneous lesion showed histological and immunohistochemical characteristics of RDD. The patient was treated with intravitreal triamcinolone (0.1 mL/4 mg). One month after treatment, there was complete regression of the oedema with a significant improvement in visual acuity to 20/20., Conclusions: This is the first reported case of RDD associated with cystoid macular oedema. Macular oedema responded to intravitreal treatment with triamcinolone.

Purpose: To evaluate the therapeutic potential and safety of intravitreal injections of bone marrow mononuclear fraction (BMMF) containing CD34 + cells in patients with atrophic age-related macular degeneration (AMD)., Methods: Ten patients with atrophic AMD and best-corrected visual acuity (BCVA) in the worse-seeing eye of ≤20/100 were enrolled in this study. The bone marrow from all patients was aspirated and processed for mononuclear cell separation. A 0.1 mL suspension of BMMF CD34 + cells was injected into the vitreous cavity of the worse-seeing eye. Patients were evaluated at Baseline and 1,3,6,9 and 12 months after injection. Ophthalmic evaluation included BCVA measurement, microperimetry, infrared imaging, fundus autofluorescence and SD-optical coherence tomography at all study visits. Fluorescein angiography was performed at Baseline and at 6 and 12 months after intravitreal therapy., Results: All patients completed the 6-month follow-up, and six completed the 12-month follow-up. Prior to the injection, mean BCVA was 1.18 logMAR (20/320 -1 ), ranging from 20/125 to 20/640 -2 , and improved significantly at every follow-up visit, including the 12-month one, when BCVA was 1.0 logMAR (20/200) ( P <0.05). Mean sensitivity threshold also improved significantly at 6, 9 and 12 months after treatment ( P <0.05). Considering the area of atrophy identified by fundus autofluorescence, significant mean BCVA and mean sensitivity threshold improvement were observed in patients with the smallest areas of atrophy. Fluorescein angiography did not identify choroidal new vessels or tumor growth., Conclusion: The use of intravitreal BMMF injections in patients with AMD is safe and is associated with significant improvement in BCVA and macular sensitivity threshold. Patients with small areas of atrophy have a better response. The paracrine effect of CD34 + cells may explain the functional improvement observed; however, larger series of patients are necessary to confirm these preliminary findings., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

The objective of this study was to investigate the influence of the genes encoding the KIR receptors and their HLA ligands in the susceptibility of ocular toxoplasmosis. A total of 297 patients serologically-diagnosed with toxoplasmosis were selected and stratified according to the presence (n = 148) or absence (n = 149) of ocular scars/lesions due to toxoplasmosis. The group of patients with scars/lesions was further subdivided into two groups according to the type of ocular manifestation observed: primary (n = 120) or recurrent (n = 28). Genotyping was performed by PCR-SSOP. Statistical analyses were conducted using the Chi-square test, and odds ratio with a 95% confidence interval was also calculated to evaluate the risk association. The activating KIR3DS1 gene was associated with increased susceptibility for ocular toxoplasmosis. The activating KIR together with their HLA ligands (KIR3DS1-Bw4-80Ile and KIR2DS1 + /C2 ++ KIR3DS1 + /Bw4-80Ile + ) were associated with increased susceptibility for ocular toxoplasmosis and its clinical manifestations. KIR-HLA inhibitory pairs -KIR2DL3/2DL3-C1/C1 and KIR2DL3/2DL3-C1- were associated with decreased susceptibility for ocular toxoplasmosis and its clinical forms, while the KIR3DS1 - /KIR3DL1 + /Bw4-80Ile + combination was associated as a protective factor against the development of ocular toxoplasmosis and, in particular, against recurrent manifestations. Our data demonstrate that activating and inhibitory KIR genes may influence the development of ocular toxoplasmosis.

Importance: Although mostly asymptomatic, patients with choroidal nevi carry a moderate risk for malignant transformation and visual loss. A novel noninvasive imaging assessment could change the current clinical evaluation of choroidal nevi., Observation: Three patients with a recent diagnosis of choroidal nevi underwent a novel adaptive optical assessment that detected potential photoreceptor abnormalities in the retina overlying the choroidal nevi., Conclusions and Relevance: Adaptive optics imaging may provide high-resolution en face images of retinal structural changes in the photoreceptor mosaic overlying the choroidal nevi. Cone attenuation may be an important component of structural damage in choroidal nevi and may correlate and possibly predict functional visual loss.

This study investigated whether polymorphisms of the MICA (major histocompatibility complex class I chain-related gene A) gene are associated with eye lesions due to Toxoplasma gondii infection in a group of immunocompetent patients from southeastern Brazil. The study enrolled 297 patients with serological diagnosis of toxoplasmosis. Participants were classified into two distinct groups after conducting fundoscopic exams according to the presence (n = 148) or absence (n = 149) of ocular scars/lesions due to toxoplasmosis. The group of patients with scars/lesions was further subdivided into two groups according to the type of the ocular manifestation observed: primary (n = 120) or recurrent (n = 28). Genotyping of the MICA and HLA alleles was performed by the polymerase chain reaction-sequence specific oligonucleotide technique (PCR-SSO; One Lambda®) and the MICA-129 polymorphism (rs1051792) was identified by nested polymerase chain reaction (PCR-RFLP). Significant associations involving MICA polymorphisms were not found. Although the MICA*002~HLA-B*35 haplotype was associated with increased risk of developing ocular toxoplasmosis (P-value = 0.04; OR = 2.20; 95% CI = 1.05-4.60), and the MICA*008~HLA-C*07 haplotype was associated with protection against the development of manifestations of ocular toxoplasmosis (P-value = 0.009; OR: 0.44; 95% CI: 0.22-0.76), these associations were not statistically significant after adjusting for multiple comparisons. MICA polymorphisms do not appear to influence the development of ocular lesions in patients diagnosed with toxoplasmosis in this study population.

Background: Toxoplasmosis was recently included as a neglected disease by the Center for Disease Control. Ocular toxoplasmosis is one clinical presentation of congenital or acquired infection. The laboratory diagnosis is being used worldwide to support the clinical diagnosis and imaging. The aim of this study was to evaluate the use of serology and molecular methods to monitor acute OT in immunocompetent patients during treatment., Methods: Five immunocompetent patients were clinically diagnosed with acute OT. The clinical evaluation was performed by ophthalmologic examination using the Early Treatment Diabetic Retinopathy Study, best-corrected visual acuity, slit lamp biomicroscopy, fundoscopic examination with indirect binocular ophthalmoscopy color fundus photography, fluorescein angiography and spectral optical coherence tomography (OCT). Serology were performed by ELISA (IgA, IgM, IgG) and confirmed by ELFA (IgG, IgM). Molecular diagnoses were performed in peripheral blood by cPCR using the Toxoplasma gondii B1 gene as the marker. Follow-up exams were performed on day +15 and day +45., Results: Only five non-immunocompromised male patients completed the follow up and their data were used for analysis. The mean age was 41.2 ± 11.3 years (median: 35; range 31-54 years). All of them were positive for IgG antibodies but with different profiles for IgM and IgA, as well as PCR. For all patients the OCT exam showed active lesions with the inner retinal layers being abnormally hyper-reflective with full-thickness disorganization of the retinal reflective layers, which assumed a blurred reflective appearance and the retina was thickened., Conclusions: The presence of IgA and IgM confirmed the acute infection and thus was in agreement with the clinical evaluation. Our results show the adopted treatment modified the serological profile of IgM antibodies and the PCR results, but not the IgG and IgA antibodies and that imaging is a good tool to follow-up patients.

Purpose: To determine the in vivo release profile and retinal safety of cyclosporine A (CsA) delivered from a biodegradable poly-lactide-co-glycolide (PLGA) device in the vitreous cavity of rabbits' eyes., Methods: A total of 60 animals (60 eyes) divided into two groups were used. For the in vivo release study, 32 eyes received PLGA implants containing 350 µg of CsA, and 16 eyes received the implants without drug (control). Four animals of CsA group and two of the control group were killed weekly until 8 weeks; the vitreous was removed, and CsA concentration was evaluated. Ophthalmological examination was performed in the animals prior to implant placement and weekly during the study period. Electroretinography (ERG) was performed in other six animals for each group, treated and control, at the beginning and at the end of the study (8 weeks) when they were killed and had their eyes processed for histology., Results: No sign of inflammation was noticed on slit lamp examinations and the IOP maintained stable during the study period in CsA and control groups. CsA concentration in the vitreous (ng/ml) was 257.07 ± 117.23, 271.15 ± 98.96, 296.66 ± 86.25, 256.27 ± 99.22, 304.50 ± 88.18, 326.35 ± 105.24, 491.25 ± 119.90 and 589.93 ± 132.55 after 1, 2, 3, 4, 5, 6, 7 and 8 weeks of implantation, respectively. At the end of the study, 21.67 % of mass loss was found. The retina did not show any histological alteration in either group, but a significant reduction in dark-adapted b-wave amplitude was observed in the CsA group, with no changes in a-wave amplitude., Conclusions: These data show that the PLGA system is safe, but the selective reduction in ERG b-wave amplitude indicates that the PLGA with 350 µg CsA causes retinal function impairment, specifically on the rod postreceptor pathway, 8 weeks after implantation. These ERG changes were not associated with any histological damage as seen at the light microscopy level.

Purpose: To evaluate the association between the VEGF-C936T polymorphism and serum vascular endothelial growth factor (VEGF) levels, lifestyle, and demographic parameters in patients with age-related macular degeneration (AMD)., Methods: A total of 183 individuals were enrolled in the present study, including 88 patients with AMD receiving clinical and pharmacological treatment (study group, SG) and 95 individuals without AMD as controls (control group, CG). The presence of the VEGF-C936T polymorphism and serum VEGF levels were determined using polymerase chain reaction/restriction fragment length polymorphism and enzyme-linked immunosorbent assay, respectively. Significance was set at P<0.05 for all statistical analyses., Results: The homozygous wild-type genotype (CC) and the C allele were predominant in both groups (P=0.934 and P=0.938, respectively). Serum VEGF levels (assessed in 57% and 31% of patients in the SG and CG, respectively) were comparable between groups (SG, 307.9 ± 223.6 pg/mL; CG, 305.1 ± 212.3 pg/mL; P=0.955). A significantly higher prevalence of smoking (44% vs 25%; P=0.01) and hypertension (66% vs 48%; P=0.025) was observed in the SG than in the CG. The distribution of alcohol consumption and dyslipidemia was similar between groups (P>0.05)., Conclusions: In the present study group of Brazilian patients, the VEGF-C936T polymorphism was not found to be associated with age-related macular degeneration. However, smoking and systemic arterial hypertension (SAH) were found to be potential independent risk factors for the development of age-related macular degeneration. Comparable serum VEGF levels in both study groups may reflect the efficacy of pharmacological treatment of AMD.