Mongkol Uiprasertkul

Apoptosis and Pathogenesis of Avian Influenza A (H5N1) Virus in Humans

Apoptosis may play a crucial role in the pathogenesis of pneumonia and lymphopenia caused by this virus in humans.

Mouse DNA contamination in human tissue tested for XMRV

BackgroundWe used a PCR-based approach to study the prevalence of genetic sequences related to a gammaretrovirus, xenotropic murine leukemia virus-related virus, XMRV, in human prostate cancer. This virus has been identified in the US in prostate cancer patients and in those with chronic fatigue syndrome. However, with the exception of two patients in Germany, XMRV has not been identified in prostate cancer tissue in Europe. Most putative associations of new or old human retroviruses with diseases have turned out to be due to contamination. We have looked for XMRV sequences in DNA extracted from formalin-fixed paraffin- embedded prostate tissues. To control for contamination, PCR assays to detect either mouse mitochondrial DNA (mtDNA) or intracisternal A particle (IAP) long terminal repeat DNA were run on all samples, owing to their very high copy number in mouse cells.ResultsIn general agreement with the US prevalence, XMRV-like sequences were found in 4.8% of prostate cancers. However, these were also positive, as were 21.5% of XMRV-negative cases, for IAP sequences, and many, but not all were positive for mtDNA sequences.ConclusionsThese results show that contamination with mouse DNA is widespread and detectable by the highly sensitive IAP assay, but not always with less sensitive assays, such as murine mtDNA PCR. This study highlights the ubiquitous presence of mouse DNA in laboratory specimens and offers a means of rigorous validation for future studies of murine retroviruses in human disease.

Hyperinduction of Cyclooxygenase-2-Mediated Proinflammatory Cascade: A Mechanism for the Pathogenesis of Avian Influenza H5N1 Infection

The mechanism for the pathogenesis of H5N1 infection in humans remains unclear. This study reveals that cyclooxygenase-2 (COX-2) was strongly induced in H5N1-infected macrophages in vitro and in epithelial cells of lung tissue samples obtained during autopsy of patients who died of H5N1 disease. Novel findings demonstrated that COX-2, along with tumor necrosis factor alpha and other proinflammatory cytokines were hyperinduced in epithelial cells by secretory factors from H5N1-infected macrophages in vitro. This amplification of the proinflammatory response is rapid, and the effects elicited by the H5N1-triggered proinflammatory cascade are broader than those arising from direct viral infection. Furthermore, selective COX-2 inhibitors suppress the hyperinduction of cytokines in the proinflammatory cascade, indicating a regulatory role for COX-2 in the H5N1-hyperinduced host proinflammatory cascade. These data provide a basis for the possible development of novel therapeutic interventions for the treatment of H5N1 disease, as adjuncts to antiviral drugs.

Topical 0.002% Mitomycin C for the Treatment of Conjunctival-corneal Intraepithelial Neoplasia and Squamous Cell Carcinoma

PURPOSE To demonstrate the efficacy of topical 0.002% mitomycin C (MMC) as an adjunctive and alternative treatment in primary and recurrent conjunctival-corneal intraepithelial neoplasia (CCIN) and squamous cell carcinoma (SCC). METHODS The medical records of 7 patients with histopathologically confirmed CCIN and conjunctival SCC were retrospectively reviewed. All cases were treated with topical 0.002% MMC 4 times daily. The tumor size pre- and post-treatment, clinical response, and ocular complications were evaluated. RESULTS The mean age of the patients was 56 +/- 13.4 years. The most common presenting symptom was foreign body sensation (57.1%) with a mean duration of 2.3 +/- 3.8 months. Six patients had pathologically proven CCIN (85.7%) and 1 had SCC (14.3%). Before MMC treatment, 6 eyes (85.7%) had recurrences after surgical excision. The tumor-free period ranged from 2 to 19 months. Two patients had multiple recurrences. MMC 0.002% 4 times daily was applied for a period of 5.4 +/- 4.4 weeks (range, 2-14). All had complete tumor regression as observed clinically and confirmed by impression cytology. Side effects of MMC therapy included ocular irritation, mild conjunctival hyperemia, and punctate keratopathy. There were no serious complications detected. The mean follow-up time was 30.7 +/- 15 months (range, 2-52) with no evidence of clinical recurrence in any case. CONCLUSIONS Topical 0.002% MMC showed a favorable outcome as an adjunctive and alternative treatment of CCIN and SCC with regression of primary and recurrent tumors.

Positive selection at the receptor-binding site of haemagglutinin H5 in viral sequences derived from human tissues

Highly pathogenic H5N1 avian influenza virus has spread through at least 45 countries in three continents. Despite the ability to infect and cause severe disease in humans, the virus cannot transmit efficiently from human to human. The lack of efficient transmission indicates the incompletion of the adaptation of the avian virus to the new host species. The required mutations for the complete adaptation and the emergence of a potential pandemic virus are likely to originate and be selected within infected human tissues. Differential receptor preference plays an important role in the species-tropism of avian influenza. We have analysed quasispecies of sequences covering the receptor-binding domain of the haemagglutinin gene of H5N1 viruses derived from fatal human cases. We employed a likelihood ratio test to identify positive-selection sites within the quasispecies. Nine of seventeen positive-selection sites identified in our analyses were found to be located within or flanking the receptor-binding domain. Some of these mutations are known to alter receptor-binding specificity. This suggests that our approach could be used to screen for mutations with significant functional impact. Our data provide new candidate mutations for the viral adaptation to a human host, and a new approach to search for new genetic markers of potential pandemic viruses.

Efficacy of cultivated corneal epithelial stem cells for ocular surface reconstruction

Purpose To investigate the clinical outcomes of cultivated corneal limbal epithelial transplantation (CLET) using human amniotic membrane for corneal limbal stem-cell deficiency. Methods Prospective, noncomparative case series. Eighteen patients (19 eyes) with severe ocular surface diseases were chosen to undergo CLET using human amniotic membrane. Twelve eyes received auto-CLET, and seven eyes received allo-CLET. Clinical outcomes of corneal surface epithelialization, conjunctivalization, inflammation, visual acuity, graft status, and complications were observed. Results Corneal epithelium cultivated on amniotic membrane (two to four layers) was positive for molecular markers p63, ABCG2, CK3, and CK12. The mean patient age was 44.7 ± 15.2 years. A successful clinical outcome, defined as corneal epithelialization without central conjunctivalization or severe inflammation, was obtained in 14 (73.7%) of 19 eyes (mean follow-up 26.1 ± 13.5 months; range 6–47). A histopathologic success, defined as absence of goblet cells at the central cornea, was achieved in 12 (63.2%) eyes. Clinical failures occurred in five (26.3%) of 19 eyes, and histopathologic failures occurred in seven (36.8%) of 19 eyes. Survival analysis at 1 year showed that the clinical success rate was 77.9% and the pathological success rate was 72.3%. Fourteen of 19 (73.7%) eyes had visual acuity improvements after CLET. Six cases underwent penetrating keratoplasty; five of these grafts remained clear after 20.4 ± 6.9 months (range, 12–31) of follow-up. Complications included infectious keratitis (three cases) and recurrent symblepharon (one case). All complicated cases had lid abnormalities. Factors affecting the final clinical outcomes were lid abnormalities, abnormal corneal stromal beds, and complications. Conclusion CLET can successfully restore ocular surface damage in most cases with corneal limbal stem cell deficiency.

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. J. Med. Virol. 83:1418–1423, 2011.

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. J. Med. Virol. 83:1410–1417, 2011.

Enhanced Susceptibility of Nasal Polyp Tissues to Avian and Human Influenza Viruses

Background Influenza viruses bind and infect respiratory epithelial cells through sialic acid on cell surface. Differential preference to sialic acid types contributes to host- and tissue-tropism of avian and seasonal influenza viruses. Although the highly pathogenic avian influenza virus H5N1 can infect and cause severe diseases in humans, it is not efficient in infecting human upper respiratory tract. This is because of the scarcity of its receptor, α2,3-linked sialic acid, in human upper airway. Expression of sialic acid can be influenced by various factors including inflammatory process. Allergic rhinitis and nasal polyp are common inflammatory conditions of nasal mucosa and may affect expression of the sialic acid and susceptibility to influenza infection. Methodology/Principal Finding To test this hypothesis, we detected α2,3- and α2,6-linked sialic acid in human nasal polyp and normal nasal mucosal tissues by lectin staining and infected explants of those tissues with avian influenza viruses H5N1 and seasonal influenza viruses. We show here that mucosal surface of nasal polyp expressed higher level of α2,3- and α2,6-linked sialic acid than normal nasal mucosa. Accordingly, both H5N1 avian influenza viruses and seasonal influenza viruses replicated more efficiently in nasal polyp tissues explants. Conclusions/Significance Our data suggest a role of nasal inflammatory conditions in susceptibility to influenza infection, especially by avian influenza viruses, which is generally inefficient in infecting human upper airway. The increased receptor expression may contribute to increased susceptibility in some individuals. This may contribute to the gradual adaptation of the virus to human population.

Prognostic factors and treatment outcomes of retinoblastoma in pediatric patients: a single-institution study

PurposeSince 1997, our institute has used neoadjuvant chemotherapy for intraocular retinoblastoma. However, some of the patients showed signs of recurrence months to years later. We therefore attempted to determine the prognostic factors of treatment outcomes and survival in our patients.MethodsWe reviewed 90 patients treated from 1997 to 2006. The following information was recorded: demographic and ophthalmological data, clinical classification, histopathological data, and treatment methods and outcomes.ResultsEnucleation was avoided in two of 57 eyes in the unilateral group. Sixteen of 54 eyes in the bilateral group were salvaged by systemic chemotherapy with local treatment. There was no difference in histopathological findings between the two groups. Nine of 57 patients in the unilateral group demonstrated poor outcomes, compared with four of 27 in the bilateral group. Significant poor prognostic factors for survival were optic nerve head invasion, orbital involvement, abnormal results on bone marrow aspiration, lumbar puncture, bone scan, and group E or F classification.ConclusionsThe 15% mortality rate in our patients is slightly higher than that reported in developed countries. Delayed diagnosis and treatment is the main cause of avoidable deaths. Improving education of both clinicians and parents would increase survival rates in this potentially fatal disease.