scholarly journals Impression cytology is a non-invasive and effective method for ocular cell obtention from babies with Congenital Zika Syndrome: perspectives in OMIC studies

2019 ◽  
Author(s):  
Raquel Hora Barbosa ◽  
Maria Luiza B. dos Santos ◽  
Thiago P. Silva ◽  
Liva Rosa-Fernandes ◽  
Ana Pinto ◽  
...  
Keyword(s):  
Zika Virus ◽  
Ocular Surface ◽  
Clinical Signs ◽  
Impression Cytology ◽  
Morphological Alterations ◽  
Non Invasive ◽  
Viable Cells ◽  
Congenital Zika Syndrome ◽  
Microscopic Features ◽  
Conjunctival Epithelial Cells

AbstractIMPORTANCENoninvasive techniques for obtaining ocular surface cells (neuroepithelial) from babies with Congenital Zika Syndrome CZS - resulting from infection by zika virus (ZIKV) during gestational period (malformations include ocular abnormalities and microcephaly) - remain to be determined.OBJECTIVESThe aim of this study was to describe an optimized impression cytology method for the isolation of viable cells from babies with CZS in satisfactory amounts and quality to enable the application in the context of genome approaches well as morphological and molecular evaluations.DESIGN, SETTINGS AND PARTICIPANTSIn this observational study, ocular surface samples were obtained with a hydrophilic nitrocellulose membrane (through optimized impression cytology method) from twelve babies referred to the Pediatric Service of the Antonio Pedro Hospital, Universidade Federal Fluminense (UFF), Niteroi, Rio de Janeiro, Brazil. Samples were collected with an authorized informed consent from both eyes of eight ZIKV infected babies according to the CZS diagnostic criteria (4 babies with positive PCR for Zika virus in gestation and presence of clinical signs which included ocular abnormalities and microcephaly and 4 babies with positive PCR for Zika virus during gestation but no clinical signs identified) and four unaffected babies (control samples / negative PCR, without clinical signs). Cells were used for microscopy analyses, transcriptomic and proteomic experiments and molecular procedure.MAIN OUTCOMES AND MEASURESThe microscopic features of the conjunctival epithelial cells were described by both direct analysis of the membrane-attached cells and analysis of cytospinned captured cells using several staining procedures, including viability evaluation. In parallel, molecular approaches were performed.RESULTSOn impression cytology, a considerable amount of viable cells were captured. Epithelial basal, polyhedral and goblet cells were clearly identified in all groups. All cases of ZIKV infected babies showed clear morphological alterations (cell keratinization, piknosis, karyolysis, anucleation and vacuolization). Genomic DNA and RNA were successfully isolated from all samples and allowed the establishment of transcriptomic and proteomic studies. Transcriptome analysis showed 8582 transcripts quantified in all samples and 63 differentially expressed genes in ocular cells from the exposed babies. Proteomics analysis allowed the identification of 2080, 2085 and 2086 high confident and unique proteins with at least one unique peptide in the unaffected, exposed to ZIKV and asymptomatic and CZS babies, respectively, being 2062 in common. Multivariate supervised analysis using the total quantitative protein features revealed a clear discrimination between the groups.CONCLUSIONS AND RELEVANCEOur method proved to be a suitable, fast, and non-invasive tool for detailed and precise morphological analyses with a perspective of application in OMIC studies for clinical and research studies of CZS.Key pointsQuestionAre the ocular surface cells of babies with Congenital Zika Syndrome viable to investigate the association between Zika virus infection during embryogenesis and ocular impairment?FindingsTo this date, this is the first study using an approach with perspectives in morphological, molecular and “OMICs” research from ocular samples captured by impression cytology of ZIKV infected babies during embryogenesis. The microscopic features of the conjunctival epithelial cells from all ZIKV infected babies showed clear morphological alterations.MeaningOcular cell surface capture offers a powerful model for studying the pathways involved in ocular diseases associated with ZIKV.

scholarly journals CImP: Cellular Imprinting Proteomics applied to ocular disorders elicited by Congenital Zika virus Syndrome

2019 ◽  
Author(s):  
Livia Rosa-Fernandes ◽  
Raquel Hora Barbosa ◽  
Maria Luiza B. dos Santos ◽  
Claudia B. Angeli ◽  
Thiago P. Silva ◽  
...  
Keyword(s):  
Zika Virus ◽  
Ocular Surface ◽  
Molecular Mechanisms ◽  
Membrane Filter ◽  
Neurological Diseases ◽  
Specific Protein ◽  
Protein Markers ◽  
Impression Cytology ◽  
Zikv Infection ◽  
Molecular Alterations

AbstractIMPORTANCEOcular complications in infants with Congenital Zika Syndrome (CZS) have been reported. However, the molecular mechanisms underlying of eye dysfunctions are presently unknown.OBJECTIVEA method (termed Cellular Imprinting Proteomics, CImP) for the identification and quantification of the ocular surface proteome using a minimally invasive membrane filter device is described. Moreover, The CImP method was applied to profile the molecular alterations in the eyes of infants exposed to Zika virus (ZIKV) infection during gestation.DESIGN, SETTINGS AND PARTICIMPANTSThe CImP method was applied to a cohort divided into three conditions: 1) Ctrl (infants with no infectious diseases, n=5). 2) Zikv (infants exposed to ZIKV gestation, with no microcephaly, n=5). 3) ZikvCZS(infants exposed to ZIKV, with microcephaly, n=3). All conditions were age and sex-matched. An improved impression cytology method was used to capture the outermost ocular surface cells. The number of impression cytology membrane collected was: Ctrl (12), Zikv (14) and ZikvCZS(8). Proteins were extracted and analysed using mass spectrometry-based proteomics technology followed by statistical analysis. Parallel reaction monitoring was performed to validate the expression of specific protein markers.RESULTSUsing the CImP method, 2209 proteins were identified on the membrane-captured conjunctiva epithelial cells. Modulation of neutrophil degranulation, cell death, ocular and neurodevelopment pathways are reported in infants with CZS compared to matched controls. Moreover, the molecular pattern of ocular surface cells retrieved from infants infected during the gestation but with no CZS was different from matched controls.CONCLUSIONS AND PERSPECTIVESMolecular alterations in the ocular cell surface associated to ZIKV infection with and without CZS complications are reported for the first time. We predict that this method will be introduced successfully in the study of several neurological diseases with the aim to identify novel diagnostic and therapeutic biomarkers.

Impression cytology for detection of clinically suspected ocular surface disorders: A cross-sectional study

2020 ◽  
pp. 112067212093209
Author(s):  
Seyed Mohamadmehdi Moshtaghion ◽  
Mohammad Abolhosseini ◽  
Mozhgan Rezaei Kanavi ◽  
Seyed Bagher Hosseini ◽  
Mahnoush Rezaei Kanavi
Keyword(s):  
Ocular Surface ◽  
Acanthamoeba Keratitis ◽  
Cross Sectional Study ◽  
Impression Cytology ◽  
Diagnostic Modality ◽  
Cross Sectional ◽  
Non Invasive ◽  
Pigmented Lesions ◽  
Ocular Surface Diseases ◽  
Proper Diagnosis

Purpose: Investigating impression cytology (IC) results of various types of clinically suspected ocular surface lesions over a 14-year period in a referral center in Iran. Methods: IC findings obtained from patients with different types of ocular surface disorders between 2005 and 2018 were reviewed. Agreement between clinical suspicions and IC results was evaluated by calculating Cohen’s Kappa coefficient (CKC). Results: Clinical suspicions in 688 surveyed eyes were ocular surface squamous neoplasia (OSSN, 42.0%), limbal stem cell deficiency (LSCD, 36.3%), dry eye-related disorders (DERD, 11.5%), Acanthamoeba keratitis (AK, 7.2%), benign pigmented lesions (BPL, 1.9%), immune-related conjunctivitis (IRC, 0.7%), and malignant pigmented lesions (MPL, 0.4%). General agreement between clinical suspicions and IC results was 0.68 for all groups. This agreement was almost perfect in AK (CKC = 0.966) and BPLs (CKC = 0.843), and was substantial in MPLs (CKC = 0.749), OSSNs (CKC = 0.684), and LSCD (CKC = 0.612). CKC in IRC (0.567) and DERDs (0.443) was moderate. Histopathologic results were available in 22 eyes and were well-correlated with corresponding IC results (CKC = 0.86). Multiple post-treatment follow-up sessions of IC were performed in 51 eyes (11.4%) that had diagnosis of LSCD (31), OSSN (17), and MPL (3) at the first IC session. Conclusion: Our survey not only demonstrated an overall substantial agreement between IC results and primary clinical suspicions, but also showed an almost perfect correlation between IC results and existent histopathologic data. Therefore, IC as a non-invasive diagnostic modality can be of great importance in proper diagnosis of various ocular surface diseases especially when distinguishing malignant from benign lesions is required.

scholarly journals Role of Conjunctival Ultraviolet Autofluorescence in Ocular Surface Squamous Neoplasia

2020 ◽  
Vol 6 (6) ◽  
pp. 422-429
Author(s):  
Saumya Yadav ◽  
Noopur Gupta ◽  
Rashmi Singh ◽  
Mukesh Patil ◽  
Rachna Meel ◽  
...  
Keyword(s):  
Clinical Diagnosis ◽  
Outcome Measures ◽  
Ocular Surface ◽  
Surgical Intervention ◽  
Morphological Characteristics ◽  
Impression Cytology ◽  
Ocular Surface Squamous Neoplasia ◽  
Non Invasive ◽  
The Mean

<b><i>Objective:</i></b> To evaluate the adjunctive role of conjunctival autofluorescence in the management of ocular surface squamous neoplasia (OSSN). <b><i>Materials and Methods:</i></b> Seventeen patients with clinically diagnosed OSSN were included. Morphological characteristics, type of OSSN, and autofluorescence photographs of the lesion were captured. Presence and area of conjunctival ultraviolet autofluorescence (CUVAF) were the main outcome measures. <b><i>Results:</i></b> Overall, 17 patients with 15 (88%) primary and 2 (12%) recurrent OSSN were included. Common locations were temporal (<i>n</i> = 10), nasal (<i>n</i> = 5), and diffuse variety (<i>n</i> = 2). Morphologically, there were 4 (22.2%) nodular, 4 (22.2%) leucoplakic, 3 (16.7%) gelatinous, and 1 (5.5%) each of papillary, nodulo-ulcerative, and diffuse variety. Mixed morphology was present in 4 eyes (22.2%). Sixteen of 18 eyes (88.9%) with OSSN displayed autofluorescence on CUVAF images. The mean area of CUVAF was 15.82 mm<sup>2</sup> (10.77–19.59 mm<sup>2</sup>). Autofluorescence was reported in 8 eyes (44.4%) which had negative reports on impression cytology. <b><i>Conclusions:</i></b> Conjunctival autofluorescence was seen in the majority of cases with OSSN, in spite of negative cytology reports. Our study demonstrates that CUVAF may serve as an effective ancillary, non-invasive, and resource-friendly tool for supplementing the clinical diagnosis of OSSN, especially in diffuse and recurrent lesions that are not amenable to surgical intervention.

Clinical and in vivo confocal microscopic features of neuropathic corneal pain

2019 ◽  
Vol 104 (6) ◽  
pp. 768-775 ◽  
Author(s):  
Andrew R Ross ◽  
Mouhamed Ali Al-Aqaba ◽  
Amna Almaazmi ◽  
Marco Messina ◽  
Mario Nubile ◽  
...  
Keyword(s):  
Ocular Surface ◽  
Clinical Signs ◽  
Topical Anaesthesia ◽  
Ocular Surface Disease Index ◽  
Corneal Epithelial ◽  
Epithelial Thickness ◽  
Normal Limits ◽  
Microscopic Features ◽  
Corneal Epithelial Thickness

AimsTo describe clinical and in vivo confocal microscopy (IVCM) features of neuropathic corneal pain (NCP) without clinically visible signs.MethodsProspective, observational study of 27 eyes of 14 patients who had continuous severe ocular pain for one or more years, with minimal or no ocular surface signs and were non-responsive to topical lubricants, steroids and/or ciclosporin. All patients were evaluated using Ocular Surface Disease Index, Oxford grading scale, Schirmer test 1, Cochet Bonnet esthesiometry and response to topical anaesthesia. Central and paracentral regions of the cornea of patients and seven healthy controls were studied by IVCM. Corneal epithelial thickness and sub-basal nerve density were measured in patients and controls.ResultsFour patients responded to topical anaesthesia (responsive group (RG)), indicating peripheral NCP while 10 patients did not show any improvement (non-responsive group (NRG)), indicating central NCP. Schirmer-1 test was within normal limits in the RG but significantly greater in the NRG (p<0.001). None of the other clinical parameters nor corneal epithelial thickness were statistically significantly different. The sub-basal nerve density was significantly reduced (p<0.008) in patients compared with controls. Stroma of all patients demonstrated activated keratocytes and spindle, lateral and stump microneuromas. There was a statistically significant greater number of microneuromas (p<0.0001) and activated keratocytes in RG compared with NRG.ConclusionNCP without visible clinical signs does not represent typical dry eye disease. Distinct signs demonstrated on IVCM suggest that peripheral NCP, which responds to topical anaesthesia, and central NCP, which does not, are separate entities.

scholarly journals Global RNAseq of ocular cells reveals gene dysregulation in both asymptomatic and with Congenital Zika Syndrome infants exposed prenatally to Zika virus

2020 ◽  
Author(s):  
Livia Rosa-Fernandes ◽  
Amina Bedrat ◽  
Maria Luiza B. dos Santos ◽  
Ana Pinto ◽  
E Lucena ◽  
...  
Keyword(s):  
Gene Expression ◽  
Zika Virus ◽  
Clinical Signs ◽  
Neuronal Cell ◽  
Embryonic Cells ◽  
Zikv Infection ◽  
Gene Dysregulation ◽  
Congenital Zika Syndrome ◽  
Invasive Method ◽  
And Control

AbstractIn 2015, Brazil reported an outbreak identified as Zika virus (ZIKV) infection associated with congenital abnormalities. To date, a total of 86 countries and territories have described evidence of Zika infection and recently the appearance of the African ZIKV lineage in Brazil highlights the risk of a new epidemic. The spectrum of ZIKV infection-induced alterations at both cellular and molecular levels is not completely elucidated. Here, we present for the first time the gene expression responses associated with prenatal ZIKV infection from ocular cells. We applied a recently developed non-invasive method (impression cytology) which use eye cells as a model for ZIKV studies. The ocular profiling revealed significant differences between exposed and control groups, as well as a different pattern in ocular transcripts from Congenital Zika Syndrome (CZS) compared to ZIKV-exposed but asymptomatic infants. Our data showed pathways related to mismatch repair, cancer, and PI3K/AKT/mTOR signaling and genes probably causative or protective in the modulation of ZIKV infection. Ocular cells revealed the effects of ZIKV infection on primordial neuronal cell genes, evidenced by changes in genes associated with embryonic cells. The changes in gene expression support an association with the gestational period of the infection and provide evidence for the resulting clinical and ophthalmological pathologies. Additionally, the findings of cell death- and cancer-associated deregulated genes raise concerns about the early onset of other potential pathologies including the need for tumor surveillance. Our results thus provide direct evidence that infants exposed prenatally to the Zika virus, not only with CZS but also without clinical signs (asymptomatic) express cellular and molecular changes with potential clinical implications.

scholarly journals Non-Invasive versus Invasive Samples for Zika Virus Surveillance: A Comparative Study in New Caledonia and French Guiana in 2015–2016

2021 ◽  
Vol 9 (6) ◽  
pp. 1312
Author(s):  
Marie-Alice Fraiture ◽  
Wim Coucke ◽  
Morgane Pol ◽  
Dominique Rousset ◽  
Ann-Claire Gourinat ◽  
...  
Keyword(s):  
Zika Virus ◽  
French Guiana ◽  
New Caledonia ◽  
Early Stage ◽  
Simultaneous Detection ◽  
Clinical Signs ◽  
Added Value ◽  
Serum Samples ◽  
Presenting Symptoms ◽  
Non Invasive

Zika virus, an arbovirus responsible for major outbreaks, can cause serious health issues, such as neurological diseases. In the present study, different types of samples (serum, saliva, and urine), collected in 2015–2016 in New Caledonia and French Guiana from 53 patients presenting symptoms and clinical signs triggered by arbovirus infections, were analyzed using a recently developed, and in-house validated, 4-plex RT-qPCR TaqMan method for simultaneous detection and discrimination of the Zika and Chikungunya viruses. Subsequently, statistical analyses were performed in order to potentially establish recommendations regarding the choice of samples type to use for an efficient and early stage Zika infection diagnosis. On this basis, the use of only urine samples presented the highest probability to detect viral RNA from Zika virus. Moreover, such a probability was improved using both urine and saliva samples. Consequently, the added value of non-invasive samples, associated with a higher acceptance level for collection among patients, instead of serum samples, for the detection of Zika infections was illustrated.

scholarly journals Association between Genetic Variants in NOS2 and TNF Genes with Congenital Zika Syndrome and Severe Microcephaly

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 325
Author(s):  
Julia A. Gomes ◽  
Eduarda Sgarioni ◽  
Juliano A. Boquett ◽  
Ana Cláudia P. Terças-Trettel ◽  
Juliana H. da Silva ◽  
...  
Keyword(s):  
Risk Factors ◽  
Zika Virus ◽  
Genetic Variants ◽  
Educational Level ◽  
Congenital Anomalies ◽  
Family Income ◽  
First Trimester ◽  
In Utero ◽  
Zikv Infection ◽  
Congenital Zika Syndrome

Zika virus (ZIKV) causes Congenital Zika Syndrome (CZS) in individuals exposed prenatally. Here, we investigated polymorphisms in VEGFA, PTGS2, NOS3, TNF, and NOS2 genes as risk factors to CZS. Forty children with CZS and forty-eight children who were in utero exposed to ZIKV infection, but born without congenital anomalies, were evaluated. Children with CZS were predominantly infected by ZIKV in the first trimester (p < 0.001) and had mothers with lower educational level (p < 0.001) and family income (p < 0.001). We found higher risk of CZS due the allele rs2297518[A] of NOS2 (OR = 2.28, CI 95% 1.17–4.50, p = 0.015). T allele and TT/CT genotypes of the TNF rs1799724 and haplotypes associated with higher expression of TNF were more prevalent in children with CZS and severe microcephaly (p = 0.029, p = 0.041 and p = 0.030, respectively). Our findings showed higher risk of CZS due ZIKV infection in the first trimester and suggested that polymorphisms in NOS2 and TNF genes affect the risk of CZS and severe microcephaly.

Proteomics and metabolomics studies exploring the pathophysiology of renal dysfunction in autosomal dominant polycystic kidney disease and other ciliopathies

2019 ◽  
Vol 35 (11) ◽  
pp. 1853-1861 ◽  
Author(s):  
Miriam Zacchia ◽  
Emanuela Marchese ◽  
Elena Martina Trani ◽  
Marianna Caterino ◽  
Giovanna Capolongo ◽  
...  
Keyword(s):  
Kidney Disease ◽  
Clinical Signs ◽  
Research Area ◽  
Clinical Proteomics ◽  
Clinical Feature ◽  
Physiological Importance ◽  
Non Invasive ◽  
Human Disorders ◽  
Autosomal Dominant Polycystic Kidney

Abstract The primary cilium (PC) was considered as a vestigial organelle with no significant physiological importance, until the discovery that PC perturbation disturbs several signalling pathways and results in the dysfunction of a variety of organs. Genetic studies have demonstrated that mutations affecting PC proteins or its anchoring structure, the basal body, underlie a class of human disorders (known as ciliopathies) characterized by a constellation of clinical signs. Further investigations have demonstrated that the PC is involved in a broad range of biological processes, in both developing and mature tissues. Kidney disease is a common clinical feature of cilia disorders, supporting the hypothesis of a crucial role of the PC in kidney homoeostasis. Clinical proteomics and metabolomics are an expanding research area. Interestingly, the application of these methodologies to the analysis of urine, a biological sample that can be collected in a non-invasive fashion and possibly in large amounts, makes these studies feasible also in patients. The present article describes the most recent proteomic and metabolomic studies exploring kidney dysfunction in the setting of ciliopathies, showing the potential of these methodologies in the elucidation of disease pathophysiology and in the discovery of biomarkers.

scholarly journals Transitional Cell Carcinomas of the Urinary Tract in a Colony of Beagle Dogs

1989 ◽  
Vol 26 (6) ◽  
pp. 455-461 ◽  
Author(s):  
K. J. Nikula ◽  
S. A. Benjamin ◽  
G. M. Angleton ◽  
A. C. Lee
Keyword(s):  
Urinary Tract ◽  
Transitional Cell ◽  
Clinical Signs ◽  
Conclusive Evidence ◽  
Whole Body ◽  
Beagle Dogs ◽  
Long Term Effects ◽  
Significant Difference ◽  
Microscopic Features

Gross and light microscopic features of transitional cell carcinomas (TCC) of the urinary tract were examined in Beagle dogs used for the study of the long-term effects of low-dose, whole-body, 60Co gamma radiation. Thirty-eight cases of TCC occurred among 990 dogs that were from 0 to 14 years of age. There was no conclusive evidence of a radiation effect. The 38 TCC were equally divided between male and female dogs, but there was a significant difference in the sex distribution of urethra-origin TCC. Eleven males had a primary urethral TCC compared to only two females. There was no significant difference between the urethra-origin and bladder-origin TCCs in the number of tumors that caused clinical signs, metastasized, or that contributed to the death of the dog. All cases of urethral TCC in male dogs occurred in the prostatic urethra. The majority of these cases were not recognized to be neoplasms at gross necropsy, but microscopic examination revealed the TCC. Our findings differ from previous reports stating that TCC occurs more frequently in female than male dogs, and they especially differ from reports claiming that urethra-origin TCC is predominately a disease of female dogs.

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