Vascular Endothelial Growth Factor Expression Patterns in non- Human Papillomavirus - Related Pterygia: An Experimental Study on Cell Spot Arrays Digital Analysis.

PURPOSE: The role of angiogenic factors -such as vascular endothelial growth factor (VEGF) - in the development and progression of pterygia lesions remains under investigation. In the current study, we analyzed VEGF protein expression in a series of pterygia and normal conjunctiva epithelia. METHODS: Using a liquid-based cytology assay, thirty (n = 30) cell specimens were obtained by applying a smooth scraping on conjunctiva epithelia and fixed accordingly. None of them had a history of Human Papillomavirus (HPV) infection. Similarly, the same process was applied also in normal conjunctiva epithelia (n = 10; control group). We constructed five (n = 5) slides each containing eight (n = 8) cell spots. An immunocytochemistry (ICC) assay was implemented. Digital image analysis was also performed for evaluating objectively the corresponding immunostaining intensity levels. RESULTS: All the examined pterygia cell samples over-expressed the marker. High staining intensity levels were detected in 15/30 (50%), whereas the rest 15/30 (50%) demonstrated moderate expression. Overall VEGF expression was statistically significantly higher in pterygia compared to normal conjunctiva epithelia (p=.0001). Concerning the other parameters, VEGF protein expression did not associate with the gender of the patients (p = 0.518), the presence of a recurrent lesion (p = 0.311), the anatomical location (p = 0.191) or with their morphology (p = 0.316). Interestingly, the recurrent lesions demonstrated the highest levels of VEGF expression. CONCLUSIONS: VEGF overexpression is a frequent event in pterygia playing a potentially central molecular role in the progression of the lesion. Cell spot array analysis -based on liquid cytology- seems to be an innovative, easy-to-use technique for analyzing a broad variety of molecules in multiple specimens on the same slide by applying different ICC assays.

Lack of HPV in pterygium with no evidence of autoinoculation and the role of cytokines in pterygium with dry eye.

This study evaluated human papillomavirus's (HPV) role in pterygium pathogenesis, its autoinoculation from genitalia to ocular surface, potential cytokines involved, and crosstalk cytokines between pterygium and dry eye (DE). This cross-sectional study enrolled 25 healthy controls (HCs) and 116 pterygium patients. Four subgroups of pterygium and DE were used in cytokine evaluations. Conjunctival and pterygium swabs and first-void urine samples (i.e., genitalia samples) were collected for HPV DNA detection using real-time polymerase chain reaction. Tear cytokines interleukin (IL)-6, IL-18, and vascular endothelial growth factor (VEGF) in tears were evaluated. No HPV DNA was detected in conjunctival or pterygium swabs. No association was found between HPV DNA in urine samples and that from conjunctival or pterygium swabs. Tear VEGF levels were significantly higher in pterygium patients than in HCs, with no markedly different levels between primary and recurrent pterygia. Tear IL-6, IL-18, and tear VEGF were significantly higher in participants with DE, regardless of pterygium status. In conclusion, HPV infection was not a pathogenic factor of pterygia. The hypothesis of HPV transmitting from the genitals to ocular surfaces was nullified. Tear VEGF was involved in both pterygia and DE, whereas tear IL-6 and IL-18 played roles only in DE.

Human papillomavirus infection and ocular surface disease (Review).

Human papillomavirus (HPV) infection has been implicated as a primary cause of lesions in the anogenital region, skin, oropharynx and respiratory tract. Additionally, the role of HPV in the pathogenesis of ocular surface disease has also been extensively studied. Conjunctival papilloma development has been strongly associated with the HPV infection of certain subtypes. On the other hand, the role of HPV in conjunctival pterygium, conjunctival intraepithelial neoplasia (CIN) and ocular surface squamous neoplasia (OSSN) remains controversial. Genetic predisposition and environmental factor is important in HPV hosts as regards the pathogenesis of ocular surface disease. Several studies have indicate a synergic role of HPV with ultraviolet radiation in pterygium establishment. A higher recurrence risk rate and more aggressive disease of ophthalmic pterygium is observed in cases of HPV infection. The purpose of this review was to provide a systematic review of the literature and to assist in a better understanding of the role of HPV in ocular surface disease.

Ocular Human Papillomavirus Infections.

CONTEXT: - Human papillomavirus (HPV) has a well-known role in the pathogenesis of squamous cell carcinoma and precursor lesions of the cervix, anogenital region, and head and neck, but its role in the development of squamous neoplasms of the eye, particularly the conjunctiva, remains unclear. OBJECTIVE: - To review recent evidence implicating HPV in the pathophysiology of ocular lesions. DATA SOURCES: - Published articles obtained from a PubMed search of the English literature were the primary sources for this review. CONCLUSIONS: - The low-risk HPV types 6 and 11 appear to play a role in the development of at least a subset of conjunctival squamous papillomas. The role of HPV in the pathogenesis of pterygium and ocular surface squamous neoplasia is less well defined. There is evidence to suggest that HPV may be a cofactor in the development of these lesions, acting in concert with ultraviolet radiation and/or human immunodeficiency virus infection in a subgroup of cases.

Identification of human papillomavirus in pterygium.

PURPOSE: The purpose of this study was to evaluate the involvement of human papillomavirus in the pathogenesis of primary and recurrent pterygium in northern Israel. METHODS: A retrospective study examined 100 randomly chosen pterygium specimens with solar elastosis, from 100 patients who underwent pterygium surgery during 2012-2013 at the Emek Medical Center. All the specimens were analysed for evidence of human papillomavirus infection by immunohistochemistry. RESULTS: Human papillomavirus was not detected in any of the 100 pterygia samples by immunohistochemistry. These used samples were taken from 100 patients with mean age of 51.5 years and a primary: recurrent ratio of 8.09:1. CONCLUSION: We conclude from our study that human papillomavirus infection does not appear to be an important pathogenic factor of pterygium in Israel.

Prevalence and viral load of oncogenic human papillomavirus (HPV) in pterygia in multi-ethnic patients in the Malay Peninsula.

PURPOSE: The aim of the study was to determine the prevalence of human papillomavirus (HPV) in primary and recurrent pterygia samples collected from different ethnic groups in the equatorial Malay Peninsula. METHODS: DNA was extracted from 45 specimens of freshly obtained primary and recurrent pterygia from patients and from 11 normal conjunctival swabs from volunteers with no ocular surface lesion as control. The presence of HPV DNA was detected by nested PCR. PCR-positive samples were subjected to DNA sequencing to determine the HPV genotypes. Real-time PCR with HPV16 and HPV18 type-specific TaqMan probes was employed to determine the viral DNA copy number. RESULTS: Of 45 pterygia samples with acceptable DNA quality, 29 (64.4%) were positive for HPV DNA, whereas all the normal conjunctiva swabs were HPV negative. Type 18 was the most prevalent (41.4% of positive samples) genotype followed by type 16 (27.6%). There was one case each of the less common HPV58 and HPV59. Seven of the samples harboured mixed infections of both HPV16 and HPV18. All the four known recurrent pterygia samples were HPV-positive, whereas the sole early-stage pterygium sample in the study was HPV-negative. There was no significant association between HPV-positive status with gender or age. A high proportion of patients from the Indian ethnic group (five of six) were HPV-positive, whereas the Malay patients were found to have higher HPV positivity than the Chinese. The viral load of HPV18 samples ranged between 2 × 10(2) and 3 × 10(4) copies per µg, whereas the viral load of HPV16 specimen was 4 × 10(1) to 10(2) copies per µg. CONCLUSION: This report describes for the first time the quantitative measurement of HPV viral DNA for pterygium samples. The high prevalence of oncogenic HPVs in our samples suggests a possible role for HPV in the pathogenesis of pterygia. Moreover, the relatively low HPV viral load is concordant with the premalignant nature of this ocular condition.

Detecting human papillomavirus in ocular surface diseases.

PURPOSE: Human papillomavirus (HPV) infection has been implicated as a possible inducing factor for benign and neoplastic ocular surface diseases such as pterygia and ocular-surface squamous neoplasia (OSSN). However, the wide range in HPV prevalence previously reported for both diseases adds controversy to, and highlights the limitations of, this field. The aim of this study was to determine the prevalence of HPV in pterygia and OSSN and to devise a standardized approach for detecting viral DNA in ocular tissue samples. METHODS: DNA was extracted from a variety of specimens (n = 160), including formalin-fixed paraffin-embedded tissue shavings, fresh tissue, and cultured cells. Nested PCR for HPV with consensus and subtype-specific primers was used to detect viral DNA. Confirmatory assays, including molecular sequencing, histology, and immunohistochemistry for HPV E6 protein and p16 were also performed. RESULTS: HPV was not detected in pterygia or normal conjunctiva. However, 6.5% (3/46) of OSSN samples were HPV-positive by PCR, sequencing, and immunohistochemistry. Positive cases were all squamous cell carcinoma of the conjunctiva (SCCC), the most severe form of OSSN, representing 12.5% (3/24) of SCCCs in our cohort. HPV-16 was the genotype identified in each case and this correlated with the presence of koilocytes and intense immunoreactivity for p16. Our study found no association between pterygia and OSSN with other oncogenic viruses, such as EBV or CMV, as they were just as prevalent in normal conjunctiva. CONCLUSIONS: The low prevalence of HPV-16 in ocular surface disease suggests infection is not a cause but a cofactor in disease development.

Progress in the pathogenesis of pterygium.

Pterygium is a type of benign uncontrolled growth of the conjunctive tissue that lays over the sclera. It can significantly alter visual function in advanced cases and become inflamed, leading to redness and irritation in the area. Although the exact etiology of pterygium remains uncertain, recent advances have provided important insight into the pathogenesis of pterygium. These studies indicate that tumor suppressor gene p53 and other genes associated with DNA repair, cell proliferation, migration and angiogenesis are critical for the development of pterygium. In addition, Human papillomavirus infection has been shown to be a risk factor in some populations. In this article, the current understanding of the pathogenesis of pterygium is reviewed.

Viral involvement in the pathogenesis and clinical features of ophthalmic pterygium (Review).

Pterygium is a potentially vision-threatening fibrovascular lesion originating from the conjunctiva that often extends on the corneal surface. Although it has been extensively studied, its pathogenesis has yet to be fully elucidated. Recent evidence on molecular genetic abnormalities in pterygium suggested neoplastic changes of limbal stem cells potentially associated with exposure to ultraviolet (UV) light. Human papillomavirus (HPV) is an oncogenic virus, associated with squamo-proliferative lesions of the anogenital region, skin and oropharynx. Several studies have shown HPV involvement in the pathogenesis of conjunctival neoplastic lesions, including papilloma and squamous cell carcinoma. The involvement of HPV as a co-factor in the pathogenesis of pterygium, although suggested by several studies using PCR and immunohistochemical techniques, remains controversial. Moreover, a marked variation in the prevalence of HPV in ophthalmic pterygium has been reported by different studies. Ethnic susceptibility and methodological differences in the detection of HPV may account for this variation. Surgical excision, often using sophisticated techniques, is the standard current method of therapy for pterygium. However, recurrences are frequent and recurrent lesions tend to be more aggressive. If indeed HPV is involved in pterygium pathogenesis or recurrence, anti-viral medications or vaccination may be new options in pterygium therapy.

Association of human papilloma virus with pterygia and ocular-surface squamous neoplasia.

There are more microorganisms that colonize the human body than resident cells; some are commensal whereas others are pathogenic. Pathogenic microorganisms are sensed by the innate or adaptive immune system, an immune response is initiated, and the infection is often cleared. Some microorganisms have developed strategies to evade immune defenses, ensuring their long-term survival with potentially devastating consequences for the host. Approximately 18% of all cancers can be attributed to infective agents; the most common being Helicobacter pylori, Human papilloma virus (HPV) and Hepatitis B and C virus in causing stomach, cervical and liver carcinoma, respectively. This review focuses on whether HPV infection is necessary for initiating pterygia, a common benign condition and ocular-surface squamous neoplasia (OSSN), a rare disease with metastatic potential. The search engine PubMed was used to identify articles from the literature related to HPV and pterygium or conjunctival neoplasia. From 34 investigations that studied HPV in pterygia and OSSN, a prevalence rate of 18.6% (136/731) and 33.8% (144/426), respectively, was recorded. The variation in HPV prevalence (0-100%) for both disease groups may have arisen from study-design faults and the techniques used to identify the virus. Overall, the data suggest that HPV is not necessary for initiating either condition but may be a co-factor in susceptible hosts. Currently, over 60 million people worldwide have been immunized with HPV vaccines, but any effect on pterygium and OSSN development may not be known for some time as these lesions can evolve over decades or occur in older individuals.

Presence of human papillomavirus in pterygium in Taiwan.

PURPOSE: To evaluate the prevalence and possible role of human papillomavirus (HPV) in the formation of pterygia in patients in Taiwan, a tropical country with high prevalence of pterygium. METHODS: A total of 62 patients with 65 pterygia were retrospectively examined. Ten normal conjunctiva, 8 conjunctival nevi, and 2 malignant conjunctival melanomas served as controls. HPV detection and typing were accomplished using polymerase chain reaction amplification of the viral sequences. HPV-positive specimens underwent further investigation with fluorescence in situ hybridization. Clinical histories were recorded for each patient. RESULTS: Based on polymerase chain reaction analysis, 2 of 65 pterygia harbored HPV type 18, and they were also fluorescence in situ hybridization positive. No conjunctival control had HPV. There was no statistically significant correlation between pterygium and the presence of HPV. The presence of HPV was not significantly different between primary and recurrent pterygia. CONCLUSIONS: The limited presence of HPV DNA in pterygium does not conclude that HPV is necessary or acting alone in the formation of pterygium, but HPV may still be implicated to play a role in some pterygia in Taiwan.

No evidence for a pathogenic role of human papillomavirus infection in ocular surface squamous neoplasia in Germany.

PURPOSE: The etiology of ocular surface squamous neoplasia is unknown. Possible etiologic factors are physical and/or viral damage by human papillomavirus (HPV), especially in Sub-saharian populations. This study focused on the presence of human papillomavirus in ocular surface squamous neoplasia in comparison to pterygia and normal conjunctiva. METHODS: Thirty-one consecutive samples of ocular surface squamous neoplasia from a single institution (24 conjunctival intraepithelial neoplasias of various grades and 7 invasive conjunctival squamous cell carcinomas) were analyzed for evidence of HPV infection by immunohistochemistry and multiplex polymerase chain reaction (PCR). The results were compared to 11 samples of pterygia and 5 of normal conjunctiva. RESULTS: Twenty-one (68%) of 31 ocular surface squamous neoplasia showed solar elastosis, while all cases analyzed were negative for HPV. Six (19%) of 31 ocular surface squamous neoplasia specimens demonstrated overexpression of p53 with a lack of p21 upregulation indicating a functional tumor suppressor gene p53 (TP53) mutation. Carcinomas presented a dysbalance between proliferation and apoptosis possibly contributing to tissue transformation and tumor growth. CONCLUSIONS: In our study, exposition to ultraviolet (UV) appears to be an important risk factor for the development of ocular surface squamous neoplasia, while HPV infection was not detected. TP53 mutations were also rare but may play a role in the progression from conjunctival intraepithelial neoplasia to invasive carcinoma in a subset of cases.

HPV infection and p53 inactivation in pterygium.

PURPOSE: Our recent report indicated that tumor suppressor gene (p53) mutations and protein aberrant expression were detected in pterygium. Inactivation of p53 by Human papillomavirus (HPV) 16/18 E6 plays a crucial role in cervical tumorigenesis. In this study, we further speculate that p53 inactivation may be linked with HPV infection in pterygium pathogenesis. To investigate the involvement of HPV 16/18 E6 in p53 inactivation in pterygium, the association between HPV 16 or HPV 18 infection, the HPV E6 oncoprotein, and p53 protein expression was analyzed in this study. METHODS: HPV 16/18 infection was detected by nested-polymerase chain reaction (nested-PCR), the p53 mutation was detected by direct sequencing, and the p53 and the HPV 16/18 E6 proteins were studied using immunohistochemistry on 129 pterygial specimens and 20 normal conjunctivas. RESULTS: The HPV 16/18 was detected in 24% of the pterygium tissues (31 of 129) but not in the normal conjunctiva, and the HPV16/18 E6 oncoprotein was detected in 48.3% of HPV 16/18 DNA-positive pterygium tissues (15 of 31). In addition, p53 protein negative expression in pterygium was correlated with HPV16/18 E6 oncoprotein expression but not with a p53 mutation. CONCLUSIONS: HPV 16/18 E6 contributes to HPV-mediated pterygium pathogenesis as it is partly involved in p53 inactivation and is expressed in HPV DNA-positive pterygium.

Investigation of human papillomavirus and Epstein-Barr virus DNAs in pterygium tissue.

PURPOSE: Recent studies postulated the presence of a probable relationship between pterygium and neoplasia. This study aimed to investigate the role of two oncogenic viruses, human papillomavirus (HPV) and Epstein-Barr virus (EBV), in the development of conjunctival pterygia. METHODS: Polymerase chain reaction was used to identify the presence of HPV and EBV in 30 primary and 10 recurrent pterygia samples. Twenty conjunctival samples obtained from patients undergoing cataract surgeries were used as the control group. Patient groups had similar sex, race, and age distribution to eliminate bias. For exploration of HPV in groups, two different PCR methods (in-house PCR with two different primer sets and one real-time PCR method) were studied. The presence of EBV was shown by real-time PCR method. RESULTS: HPV was identified in none of the pterygia and control group patients. However, EBV was detected in 3 out of 30 (10%) primary pterygia patients and in none of the recurrent pterygia and control patients. CONCLUSIONS: Up to now, HPV has been blamed as the major viral pathogen in the etiopathogenesis of pterygium. The current results suggest that EBV may also be involved in the pathogenesis of pterygium, but further larger studies with larger cohorts are required to confirm this hypothesis.

Occurrence of human papillomavirus in pterygia.

PURPOSE: The aim of the study was to assess the occurrence of human papillomavirus (HPV) DNA in pterygium. METHODS: The study involved 89 patients undergoing surgical procedures at the Department of Ophthalmology, Medical University of Lublin, Poland. Group 1 included 58 patients with clinically diagnosed pterygium. Group 2 consisted of 31 individuals with normal conjunctiva. The material was collected during elective surgical procedures. The presence of HPV genome was determined using polymerase chain reaction (PCR). Once the presence of HPV DNA was confirmed, 28 HPV genotypes were determined using reverse hybridization. RESULTS: The determinations confirmed the presence of HPV DNA in pterygium. In the material collected from 58 cases of pterygium (group 1), HPV DNA was identified in 16 patients (27.6%). In the material from 31 diagnostic specimens of normal conjunctiva (group 2), the presence of HPV was demonstrated in three cases (9.7%). A statistically significant difference was found in the presence of HPV DNA between the patients from groups 1 and 2 (p = 0.041). HPV type 16 was most common and was demonstrated in 56% of HPV-positive cases of pterygium. HPV 16 and HPV 6 co-infections were found in 19% of cases, while HPV 18 and HPV 6 co-infections were observed in 13%. In group 2, all three patients with HPV showed HPV 18. CONCLUSION: It seems that HPV is not necessary to induce pterygium; however, it might play a synergistic role in the multi-stage process of its development.

TP53 gene expression, codon 72 polymorphism and human papillomavirus DNA associated with pterygium.

Pterygium is a disease of unknown origin and pathogenesis that can be vision threatening. Several researchers believe that pterygium is UV-related and that abnormal expression of p53 protein and infection with human papillomavirus (HPV) are risk factors for pterygium, but their experiments have been inconclusive. We investigated its relation with p53 protein expression, p53 gene codon 72 polymorphism and infection with HPV DNA. Pterygial samples were obtained from 36 patients; 21 normal conjunctival samples were used as controls. Expression of p53 protein was studied by immunohistochemistry, using the antibody DO-7. Analysis for the p53 genotype was made by polymerase chain reaction, using specific primers for the arginine and proline alleles, and an analysis for HPV was made of the pterygium patients and control group. Fourteen of the 36 pterygial specimens were positive for abnormal p53 expression. Thirty-one of the patients were heterozygotic and three were homozygotic for the proline allele; two were homozygotic for the arginine allele; in the control group 12 of 21 were heterozygotic and seven of these 21 were homozygotic for the proline allele; two were homozygotic for the arginine allele. Twenty-one of the pterygium patients were positive for HPV; HPV type 1 was found in nine of these, type 2 in seven and both types in five. Only two of the 21 controls had HPV; both had type 16. We suggest that abnormal expression of p53, p53 codon 72 polymorphisms and HPV DNA are required co-factors for the development of pterygium.

Detection of human papillomavirus in pterygium and conjunctival papilloma by hybrid capture II and PCR assays.

AIM: To elucidate the putative role of human papillomavirus (HPV) infection in pterygium and conjunctival papilloma. METHODS: Hybrid capture II (HC-II) and polymerase chain reaction (PCR) assays were performed to detect HPV in pterygium (42 samples obtained from 40 patients) and conjunctival papilloma (8 samples from 6 patients). The amount of HPV DNA was evaluated by measurement of relative light units (RLUs) on a luminometer. RESULTS: All papilloma samples were positive for HPV DNA by PCR and HC-II. The RLU values for specimens of recurrent and re-recurrent papilloma were markedly higher than those for specimens of primary lesions. HPV was detected by PCR in 2 of 42 (4.8%) beta-globin-positive pterygium specimens, whereas HC-II showed that HPV was negative in all pterygium samples. CONCLUSIONS: Our results support the hypothesis that HPV DNA is associated with the pathogenesis of conjunctival papilloma, but not pterygium. RLU measurement by HC-II may serve as a marker for evaluating the activity of HPV in conjunctival tumours.

Herpes simplex virus and pterygium in Taiwan.

PURPOSE: To evaluate the presence of herpes simplex virus (HSV) in pterygia to study the possible association between HSV and pterygia in Taiwan, a tropical country with a high prevalence of pterygium. METHODS: Sixty-five pterygia, 10 normal conjunctiva, 8 conjunctival nevi, and 2 malignant conjunctival melanomas were obtained. Clinical histories were recorded for each patient. HSV detection was accomplished by polymerase chain reaction amplification of viral sequences. HSV-positive specimens underwent subsequent DNA in situ hybridization. Results were statistically analyzed. RESULTS: By using polymerase chain reaction, HSV was detected in 3 (5%) pterygia, and no conjunctival control displayed HSV. All 3 HSV-positive pterygia studies were DNA in situ hybridization negative. There was no statistically significant correlation between pterygium and the presence of HSV. CONCLUSIONS: HSV is not associated with pterygium formation in Taiwan; the pathogenesis of pterygia is still incompletely understood.

Human papillomavirus and pterygium. Is the virus a risk factor?

BACKGROUND: Pterygium is a disease of unknown origin and pathogenesis that might be vision threatening. It is characterised by a wing-like conjunctival overgrowth of the cornea. Several studies have investigated human papillomavirus (HPV) as a risk factor for the development of pterygia, but the results are inconclusive. AIM: To investigate a large sample of pterygia for the presence of HPV in order to clarify the putative association between pterygia and HPV. METHODS: 100 specimens of pterygium from Danish patients and 20 normal conjunctival biopsy specimens were investigated for the presence of HPV with PCR technique using beta-globin primers to access the quality of the extracted DNA and the HPV primers MY09/11 and GP5+/6+. HPV-positive specimens underwent subsequent HPV typing with type-specific HPV primers and further investigation with DNA in situ hybridisation (ISH). RESULTS: 90 of 100 investigated pterygia proved suitable for HPV analysis by PCR. As beta-globin could not be amplified, 10 specimens were excluded from the study. 4 of 90 pterygia harboured HPV. HPV type 6 was identified in all four HPV-positive pterygia. The 20 normal conjunctival biopsy specimens were beta-globin positive and HPV negative. All four pterygia that were HPV type 6 positive were DNA ISH negative. CONCLUSIONS: The low presence of HPV DNA in pterygia does not support the hypothesis that HPV is involved in the development of pterygia in Denmark.

Lack of papillomavirus (HPV) in pterygia of a Brazilian sample.

PURPOSE: Because the influence of papillomavirus (HPV) in pterygium pathogenesis is controversial, the aim of this study was to identify whether it is present in the pterygia lesions in our region. METHODS: Thirty-six patients with unilateral primary pterygia were submitted to excision of pterygia and a sample of normal conjunctiva. Tissues were submitted to polymerase chain reaction (PCR) evaluation for papillomavirus DNA detection. RESULTS: We were unable to detect any HPV DNA in all studied specimens. CONCLUSION: According to our results papillomarivus is not important for pterygium formation.