Dopachrome tautomerase is a retinoblastoma-specific gene, and its proximal promoter is preferentially active in human retinoblastoma cells.

Purpose: Retinoblastoma (RB) is a malignant childhood intraocular tumor. Current treatment options for RB have undesirable side effects. A comprehensive understanding of gene expression in human RB is essential for the development of safe and effective new therapies. Methods: We reviewed published microarray and RNA sequencing studies in which gene expression profiles were compared between human RB and normal retina tissues. We investigated the expression of genes of interest using quantitative reverse transcription PCR. We examined the activities of cloned promoter DNA fragments with luciferase assay. Results: Dopachrome tautomerase (DCT) was among the most overexpressed genes in RB in published studies. We found that DCT was highly expressed in six of 13 samples microdissected from Thai RB tissues. Expression of DCT was absent or barely detected in retina tissues, various human ocular cells, and major organs. We also demonstrated that the -657 to +411 DCT promoter fragment efficiently directs RB cell-specific transcription of the luciferase reporter gene in cell lines. Conclusions: The present work highlights that DCT is one of the most RB-specific genes. The regulatory elements required for this cell-specific gene expression are likely located within its proximal promoter.

Comparative analysis of a Thai congenital-Zika-syndrome-associated virus with a Thai Zika-fever-associated virus.

In this study, we compared the characteristics of two strains of Zika virus (ZIKV) isolated in Thailand, one isolated from a febrile patient and one isolated from tissues of a fetus medically terminated due to congenital Zika syndrome (CZS). Replication profiles showed that the isolate from the fetal tissues replicated significantly more slowly than the fever-associated isolate in human lung A549 cells during the first 24 hours postinfection but showed a similar growth profile over longer-term infection. A much smaller difference was observed in Aedes albopictus C6/36 cells. In a quasispecies analysis, a high proportion (approximately 20%) of nonfunctional genomes was identified, caused by an adenine insertion in the prM gene. This insertion was found to be present in two Thai fever strains and as such may represent a common feature of Thai endemic ZIKV. Comparison between viral RNA copy number and viral titer showed that the isolate from fetal tissues was produced more efficiently than the fever-associated isolate. Together, these results suggest that different ZIKV isolates differ in their replication capacity, and this might contribute to the fetotropic potential of a particular strain.

Unusual non-nanophthalmic uveal effusion syndrome with histologically normal scleral architecture: a case report.

BACKGROUND: To report an unusual case of non-nanophthalmic uveal effusion syndrome (UES) with histologically normal sclera but responsive to scleral resection. CASE PRESENTATION: A73-year-old man presented with a bullous retinal detachment without ciliochoroidal detachment on funduscopic examination of the right eye. The axial length of both eyes was normal. Extensive investigations for possible causes of exudative retinal detachment were performed with unremarkable results except for choroidal hyperpermeability on indocyanine green angiography (ICGA). Ultrasound biomicroscopy (UBM) revealed scleral thickening with peripheral choroidal elevation leading to the diagnosis of UES. Partial thickness sclerectomy and sclerotomy was performed resulting in complete retinal reattachment, reduction of choroidal hyperpermeability on ICGA and improvement of visual acuity. However, histological studies of the excised sclera revealed no scleral architectural changes or abnormal deposits. CONCLUSIONS: The diagnosis of UES in non-nanophthalmic eyes is challenging. Thorough systemic and ocular investigations are critical to rule out other etiologies. UBM can be helpful to evaluate scleral thickness and anterior choroid in equivocal cases. Our case was unique in that, although the sclera was thick, no abnormal microscopic scleral architecture could be identified. Misdiagnosis may lead to different surgical procedures such as vitrectomy resulting in unfavorable outcomes.

Neuraminidase Activity and Resistance of 2009 Pandemic H1N1 Influenza Virus to Antiviral Activity in Bronchoalveolar Fluid.

UNLABELLED: Human bronchoalveolar fluid is known to have anti-influenza activity. It is believed to be a frontline innate defense against the virus. Several antiviral factors, including surfactant protein D, are believed to contribute to the activity. The 2009 pandemic H1N1 influenza virus was previously shown to be less sensitive to surfactant protein D. Nevertheless, whether different influenza virus strains have different sensitivities to the overall anti-influenza activity of human bronchoalveolar fluid was not known. We compared the sensitivities of 2009 pandemic H1N1, seasonal H1N1, and seasonal H3N2 influenza virus strains to inhibition by human bronchoalveolar lavage (BAL) fluid. The pandemic and seasonal H1N1 strains showed lower sensitivity to human BAL fluid than the H3N2 strains. The BAL fluid anti-influenza activity could be enhanced by oseltamivir, indicating that the viral neuraminidase (NA) activity could provide resistance to the antiviral defense. In accordance with this finding, the BAL fluid anti-influenza activity was found to be sensitive to sialidase. The oseltamivir resistance mutation H275Y rendered the pandemic H1N1 virus but not the seasonal H1N1 virus more sensitive to BAL fluid. Since only the seasonal H1N1 but not the pandemic H1N1 had compensatory mutations that allowed oseltamivir-resistant strains to maintain NA enzymatic activity and transmission fitness, the resistance to BAL fluid of the drug-resistant seasonal H1N1 virus might play a role in viral fitness. IMPORTANCE: Human airway secretion contains anti-influenza activity. Different influenza strains may vary in their susceptibilities to this antiviral activity. Here we show that the 2009 pandemic and seasonal H1N1 influenza viruses were less sensitive to human bronchoalveolar lavage (BAL) fluid than H3N2 seasonal influenza virus. The resistance to the pulmonary innate antiviral activity of the pandemic virus was determined by its neuraminidase (NA) gene, and it was shown that the NA inhibitor resistance mutation H275Y abolished this resistance of the pandemic H1N1 but not the seasonal H1N1 virus, which had compensatory mutations that maintained the fitness of drug-resistant strains. Therefore, the innate respiratory tract defense may be a barrier against NA inhibitor-resistant mutants, and evasion of this defense may play a role in the emergence and spread of drug-resistant strains.

Expression of importin-α isoforms in human nasal mucosa: implication for adaptation of avian influenza A viruses to human host.

BACKGROUND: Transportation into the host cell nucleus is crucial for replication and transcription of influenza virus. The classical nuclear import is regulated by specific cellular factor, importin-α. Seven isoforms of importin-α have been identified in human. The preference of importin-α3 of avian influenza virus and -α7 isoform of human strains during replication in human cells was previously identified. In addition, both avian and human influenza viruses were shown to use importin-α1 isoform for their replication. FINDING: The mRNA levels of importin-α1, -α3, and -α7 isoforms in human respiratory tract was determined by real-time RT-PCR. The results indicate that mRNA level of importin-α7 was significantly higher than that of importin-α1 (p-value < 0.0001) and importin-α3 (p-value < 0.0001) isoforms in human nasal mucosa while importin-α1 was detected as the highest expression importin-α isoform in lung tissues. CONCLUSIONS: These results may explain the preference of importin-α7 isoforms in seasonal influenza viruses in human upper respiratory tract and may suggest a selective pressure toward importin-α7 in human respiratory tract infection of an avian virus.

Long-term result of autologous cultivated oral mucosal epithelial transplantation for severe ocular surface disease.

The present study aimed to investigate the clinical outcomes of autologous cultivated oral mucosal epithelial transplantation (COMET) on human amniotic membrane (AM) for corneal limbal stem cell deficiency (LSCD). In this prospective, noncomparative case series, 20 eyes (18 patients) with bilateral severe ocular surface disease were chosen to undergo COMET on human AM. The primary outcome was clinical success, and the secondary outcomes were the best-corrected visual acuity difference, corneal opacification, symblepharon formation, and complications. The mean patient age was 48.2 ± 15.5 years. The mean follow-up time was 31.9 ± 12.1 months (range 8-50 months). All except one eye exhibited complete epithelialization within the first postoperative week. A successful clinical outcome, defined as a stable ocular surface without epithelial defects, a clear cornea without fibrovascular tissue invasion at the pupillary area, and no or mild ocular surface inflammation, was obtained in 15 of 20 eyes (75 %). The clinical success rate at 1 year was 79.3 %, and that at 4 years (end of follow-up) was 70.5 %. Fourteen of 20 (70 %) eyes exhibited improvement in visual acuity after COMET, and some required subsequent cataract surgery (2 eyes), penetrating keratoplasty (3 eyes), or keratoprosthesis implantation (1 eye). Preoperative symblepharon was eliminated in most eyes (8 of 13, 61.5 %) after COMET combined with eyelid reconstruction when needed. The only complication was corneal perforation (1 eye) induced by a severe eyelid abnormality; treatment with a tectonic corneal graft was successful. COMET can successfully restore ocular surface damage in most eyes with corneal LSCD.

The N-linked glycosylation site at position 158 on the head of hemagglutinin and the virulence of H5N1 avian influenza virus in mice.

N-linked glycosylation of the influenza virus hemagglutinin (HA) protein plays crucial roles in HA structure and function, evasion of neutralizing antibodies, and susceptibility to innate soluble antiviral factors. The N-linked glycosylation site at position 158 of highly pathogenic H5N1 virus was previously shown to affect viral receptor-binding preference. H5N1 viruses show heterogeneity with respect to the presence of this glycosylation site. Clade 1 viruses that caused outbreaks in Southeast Asia in 2004 contained this glycosylation site, while the site is absent in the more recent clade 2 viruses. Here, we show that elimination of this glycosylation site increases viral virulence in mice. The mutant lacking the glycosylation site at position 158 showed unaltered growth kinetics in vitro and a comparable level of sensitivity to a major antiviral protein found in respiratory secretions, surfactant protein D (SP-D).

Evaluation of nested pcr technique for detection of Pythium insidiosum in pathological specimens from patients with suspected fungal keratitis.

Diagnosis of Pythium keratitis is problematic due to the difficulty in obtaining a culture report resulting in unnecessarily prolonged usage of antimicrobial medication due to misdiagnosis. This study evaluated and compared nested PCR technique with culture and immunoperoxidase staining assays of Pythium insidiosum in paraffin-embedded corneal tissues from patients with suspected fungal keratitis. Six of 51 pathological reports compatible with fungal infection and 6 of 48 culture-proven fungal keratitis were identified as Pythium. Twenty-seven specimens were PCR-positive for Pythium insidiosum. In comparison with fungal culture for P. insidiosum, PCR had 83% sensitivity and 77% specificity with fair agreement (Kappa score of 0.227, p = 0.001). The mean age of PCR-positive is younger than PCR-negative group and there is a female preponderance in Pythium-infected group (p = 0.002 and p = 0.004, respectively). Nineteen specimens had positive results using immunoperoxidase staining assay with fair agreement to culture method (Kappa 0.340, p < 0.001), and 83% sensitivity, 85% specificity and 85% accuracy (95% CI: 76.7-90.7). PCR-based technique compared with culture and/or immunoperoxidase staining assay had 91.7% sensitivity, 81.8% specificity and 83% accuracy (95% CI: 74.5-89.1) with moderate agreement (Kappa 0.477, p < 0.001). Thus nested PCR detection of P. insidiosum should be employed in preliminary diagnosis of Pythium keratitis in order to initiate proper management.

NK/T-cell lymphoma of the nasal cavity causing contralateral dacryoadenitis.

PURPOSE: To report a case of NK/T-cell lymphoma of the nasal cavity with contralateral lacrimal gland involvement. METHODS: Observational case report. RESULTS: A 39-year-old woman with a 5-month history of right fungal rhinosinusitis was referred to our hospital. A nasal mucosal biopsy performed before referral was consistent with Aspergillus sp. Despite surgical and parenteral antifungal treatment, the symptoms continued to deteriorate. On admission, the ophthalmic evaluation showed inflammation over the left lacrimal gland area. The fundus examination revealed bilateral subretinal infiltration. Computed tomography scans of the orbits and sinuses showed mucosal thickening of the right nasal mucosa and sinuses. There was heterogeneous enhancement and infiltration of the left lacrimal gland. Lacrimal gland biopsy and repeated biopsies of the nasal cavity and sinus tissue were performed. Immunohistopathology of the lacrimal glands and nasal mucosa showed extranodal nasal-type NK/T-cell lymphoma. The patient was treated with cyclophosphamide, vincristine, adriamycin, prednisolone (3 cycles), and intrathecal methotrexate. The patient developed sepsis and died 2 months after initiation of chemotherapy. CONCLUSION: Dacryoadenitis can be a clinical manifestation of NK/T-cell lymphoma. To our knowledge, this is the first reported case of nasal NK/T-cell lymphoma with contralateral dacryoadenitis.

Well-Circumscribed Intramuscular Lipoma of Superior Rectus Muscle.

Extraocular muscle lipoma is a rare benign mesenchymal tumor of the orbit. We report a case of a 37-year-old woman who presented with chronic progressive proptosis and inferior globe displacement of left eye. External eye examination revealed a yellowish mass at the superior bulbar conjunctiva. Magnetic resonance imaging showed a well-circumscribed mass confined in the superior rectus muscle belly and tendon with a fat signal. Debulking surgery was performed using the transconjunctival and vertical lid split approach. A pathological study demonstrated matured adipose tissue cells encapsulated by a thin layer of fibrous tissue, in addition to the chronic non-specific inflammation of the tenon capsule tissue sample. Histopathological findings of the mass were consistent with a well-circumscribed intramuscular lipoma. The symptoms of the patient were significantly improved 3 months after surgical and short-course systemic steroid treatments. However, long-term surveillance is needed.

Genetic analysis of allogenic donor cells after successful allo-limbal epithelial transplantation in simple and cultivated limbal epithelial transplantation procedures.

This non-comparative cohort study investigated long-term donor cell survival after allogenic simple/cultivated limbal epithelial transplantations (allo-SLET/allo-CLET, respectively) by genetic analysis. Transplanted corneal epithelial cells, which underwent impression cytology and/or corneal-button biopsy, were examined for personal identities of autosomal short-tandem repeats; the percentages of donor cells were calculated based on matching recipient or donor buccal-DNA references. Twelve patients were included; 4 underwent allo-CLET, 8 underwent allo-SLET. Eight patients (67%) had total limbal stem cell deficiency (LSCD). Genetic analysis was performed postoperatively (mean, 55.3 months). Donor cells were detected in 4 of 12 patients (25%), all of whom underwent allo-SLET; 1 patient had a donor genotype and 3 patients had a mixed donor/recipient genotype. The longest time of donor cell detection was 30 months. Seven patients (58%) used systemic immunosuppressives at the time of genetic analysis (mean use, 22.5 months). Allogenic donor cells survived in both procedures for the long term postoperatively, which encourages the long-term use of systemic immunosuppressives. Donor cells may not be the only factor in graft survival, in that most successful cases had a recipient profile. Their presence for a specific time may promote niches for the patients' own cells to repopulate, especially for partial LSCD.

Case report: Increased viral receptor expression associated with high viral load and severe pneumonia in a young patient infected with 2009 H1N1 influenza a with no pre-existing conditions.

A case of unusually high severity of influenza pneumonia leading to acute respiratory distress syndrome and death was investigated. This was a previously a healthy 28-year-old man with no underlying conditions, admitted to a hospital during the first wave of influenza pandemic in Thailand in July 2009. He had experienced high fever and influenza-like illness for 5 days before coming to the hospital. He developed acute respiratory distress syndrome and expired on day 7 after admission. In comparison to three other cases of influenza pneumonia in the same outbreak with known risk factors for severe influenza, such as pregnancy and diabetes mellitus, a much higher viral load was detected in the lungs of this patient despite antiviral treatment. In agreement with the high viral load, the lung specimens from this patient, but not the other three patients, showed a high expression of α-2,6-linked sialic acid by lectin staining. The gene responsible for the synthesis of this sialic acid was also found to be upregulated. The data indicated overexpression of the viral receptor as a potential mechanism for severe disease in some patients.

The effect of coenzyme Q10 and curcumin on chronic methanol intoxication induced retinopathy in rats.

BACKGROUND: The retinal pathophysiology of methanol intoxication is that formate inhibits retinal mitochondrial function and increases oxidative stress. OBJECTIVE: To investigate the effect of coenzyme Q10 and curcumin on chronic methanol intoxication causing retinopathy in rats. MATERIAL AND METHOD: The authors designed an experimental study of chronic methanol intoxication in rats depleted of folate with methotrexate. The studied group received methanol (2 mg/kg body weight in saline by intraperitoneal injection) and methotrexate (0.1 mg/kg body weight in saline by subcutaneous injection) every other day for ten weeks to induce chronic methanol intoxication, while another group received saline as vehicle and served as control group. The studied rats were confirmed to develop significant retinopathy after 10 weeks and then assigned to three treatment arms: either corn oil (as control) or coenzyme Q10 (20 mg/kg/day) or Curcuma longa extract (2.5 mg/kg/day) for four weeks. Eyes were enucleated and the retinal tissue was prepared for histological examination. The sections were evaluated by an experienced pathologist and blinded to the experimental conditions. RESULTS: Histological analysis revealed that animals treated with both methanol and methotrexate showed vacuolation of photoreceptor inner segment and disaggregation of cells in the inner and outer nuclear layers of the retina compared to a normal histological appearance in control animals. The retinal histology in the experimental animals with administration of Coenzyme Q10 or Curcuma longa extract appeared essentially normal and this was not found in the experimental animals which received corn oil. CONCLUSION: Coenzyme Q10 and curcumin administration improves retinal histology by reversing the pathological changes due to chronic methanol and establish a morphologically normal retina.

Chronic recalcitrant bacterial infection in steroid modified interstitial (stromal) keratitis: presentation and management.

OBJECTIVE: To report histopathologically proven bacterial infection manifested multifocal interstitial (stromal) keratitis (IK) with definite previous history of prolong topical steroid use. Standard managements of bacterial keratitis did not provoke enough benefit. MATERIAL AND METHOD: A retrospective analysis of 19 eyes in 15 patients referred to Siriraj Hospital between 2004 and 2010. RESULTS: Multifocal intrastromal infiltration, with relatively quiet ocular reaction and mild inflammation were initially presented in all eyes. They all previously had been diagnosed of presumed viral keratitis, and had been given topical corticosteroid treatment for a prolonged period of time without healing. Autoimmune disease workups were all negative. Corneal scrapings showed negative culture results in all eyes. However, bacteria within stromal lamellae with absent or minimal inflammatory cells were demonstrated in all eyes by corneal biopsies. In addition, cytology results obtained from 16S rDNA sequencing revealed Stenotrophomonas maltophilia in one eye and coagulase-negative staphylococci in two eyes. No case responded well to intensive topical and systemic antibiotics. However they were successfully treated with penetrating keratoplasty (11 eyes, 57.9%) or intrastromal antibiotic injections (8 eyes, 42.1%). CONCLUSION: Bacterial infection should be a concern in prolonged chronic IK. This was considered as primary bacterial IK or bacterial superinfection in immunocompromised cornea. Early recognition and appropriately aggressive managements contribute to successful outcome. Corneal biopsy is always essential and 16S rDNA sequencing is useful in this distinct clinical entity.

Viral load of the highly pathogenic avian influenza H5N1 virus in infected human tissues.

The highly pathogenic avian influenza A (H5N1) virus is a virulent virus that causes an acute febrile respiratory disease with high mortality in humans. To gain a better insight of H5N1 viral distributions in infected human tissues, the levels of viral RNA were determined in the autopsy tissues from two patients who were infected with H5N1 virus by using real-time reverse transcription-polymerase chain reaction. In one patient who died on day 6 of the illness, the viral load in the lung was extremely high, whereas the levels of viral RNA in the other organs were more than 6 log lower. In the other patient who died on day 17 of the illness, the viral load was similar in the lung and other organs, and was comparable to the viral load in the extra-pulmonary tissues of the first patient. These results suggested that while the H5N1 virus can cause disseminated infection in humans, the lung is still the major site of viral replication, and viral replication in the lung in the later stages may decrease as a result of the depletion of the available target cells. In addition, the mRNA levels of the tumor necrosis factor-α (TNF-α) were found to be associated with the viral titers.

Decreased expression of surfactant protein D mRNA in human lungs in fatal cases of H5N1 avian influenza.

Microarray analysis of gene expression profile of lungs from two fatal H5N1 influenza cases identified 3,435 genes with higher than twofold changes in mRNA levels as compared to those of normal lung. One thousand nineteen genes and 2,416 genes were up-regulated and down-regulated commonly, respectively. Gene ontology analysis identified several ontology terms with significant association with these genes, most of which are related to cellular metabolism and regulation of cellular process including apoptosis and chemotaxis. Pulmonary surfactant protein D (SP-D) was found to be down-regulated. Quantitative RT-PCR confirmed the levels of SP-D mRNA in the lungs infected with H5N1 to be lower than those of normal lungs and lungs from patients with acute respiratory distress syndrome. SP-D plays multiple roles in respiratory innate defense against various pathogens, regulation of inflammatory responses, and maintenance of alveolar integrity. Reduction of SP-D in H5N1 influenza may play important roles in the pathogenesis of the disease.

Entomophthoramycosis: a rare fungal orbital infection presenting with dacryocystitis.

We report a case of a rare fungal orbital infection in an infant presenting with dacryocystitis. The causative organism was Conidiobolus sp. of the order Entomophthorales. There is no standard treatment for entomophthoramycosis. Our patient responded well to combined antifungal therapy without aggressive surgical débridement.

Full Genomic Sequences of H5N1 Highly Pathogenic Avian Influenza Virus in Human Autopsy Specimens Reveal Genetic Variability and Adaptive Changes for Growth in MDCK Cell Cultures.

The entire H5N1 highly pathogenic avian influenza viral genomes were identified in the frozen autopsy specimens: the trachea, lung, colon, and intestinal feces from a patient who died of the disease in 2006. Phylogenetic analysis of the viral genomes showed that these viruses belonged to clade 1 and were the reassortants generated from the reassortment of the viruses within the same clade. The sequencing data from the autopsy specimens revealed at least 8 quasispecies of the H5N1 viruses across all 4 specimen types. These sequences were compared to those derived from the virus isolates grown in Madin Darby canine kidney (MDCK) cells. The virus isolates from the trachea, lung, and fecal specimens showed 27 nucleotide substitutions, leading to the changes of 18 amino acid residues. However, there was no change in the amino acid residues that determined the viral virulence. The changes were more commonly observed in the lung, particularly in the HA and NA genes. Our study suggested that the adaptation changes for the viral fitness to survive in a new host species (MDCK cells) might involve many genes, for example, the amino acid substitution 177G or 177W adjacent to the receptor-binding residues in the HA1 globular head and the substitution M315I in PB2. However, a mutation changes near the receptor binding domain may play an important role in determining the cell tropism and is needed to be further explored.

Phenotypic Characterization of Corneal Epithelium in Long-Term Follow-Up of Patients Post-Autologous Cultivated Oral Mucosal Epithelial Transplantation.

PURPOSE: To analyze the phenotype of the corneal epithelium in patients with long-term follow-up who underwent autologous cultivated oral mucosal epithelial transplantation (COMET) using in vivo confocal microscopy (IVCM) and impression cytology with immunofluorescence staining (ICIF). METHODS: Thirteen eyes from patients with severe limbal stem cell deficiency, who underwent COMET at least 48 months before, were recruited in this noncomparative cohort study. After eye examination, IVCM and ICIF were performed. Clinical manifestations of the cornea were evaluated and compared with epithelial findings detected by IVCM and ICIF [cytokeratin (CK) 3, CK7, and CK12]. Two corneal buttons derived from patients receiving the corneal transplantation post-COMET were sent for immunohistochemistry (CK3, CK6, CK7, CK12, paired box gene 6, p63, zonula occludens-1, and integrin ß -1). RESULTS: The mean age of patients was 51.2 ± 20.6 years, and the mean follow-up time since COMET was 78.7 ± 16.3 months. Six of 13 eyes showed clinically successful COMET. In these eyes, IVCM demonstrated predominant cornea-like epithelium and ICIF reported positivity for CK3 and CK12, confirming the presence of oral mucosal and corneal epithelium. Meanwhile, 7 eyes showed total conjunctivalization, corresponding with substantial conjunctival epithelium detected by IVCM and positivity for conjunctival (CK7) and oral mucosal epithelial (CK3) markers detected by ICIF. The immunohistochemistry of corneal buttons stained positive for oral mucosal, corneal epithelial, and stem cell markers (CK3, CK12, and p63). CONCLUSIONS: In long-term follow-up of COMET, epithelium of successful patients demonstrated cornea-like phenotype, whereas failed cases revealed mainly conjunctival phenotype. However, there were evidences that oral mucosal epithelial cells remained across the cornea in both successful and failed COMET as detected by IVCM and ICIF.