Post-keratoplasty Endophthalmitis by Multidrug-resistant Pseudomonas Aeruginosa With Positive Culture of the Contralateral Donor Cornea: A Case Report.

PURPOSE: The aim of this paper is to present the case report of a patient developing endophthalmitis after penetrating keratoplasty caused by a multidrug-resistant Pseudomonas aeruginosa, detected only in the contralateral donor tissue. CASE REPORT: A 77-year-old man underwent an uneventful penetrating keratoplasty with a preoperative culture-negative donor cornea; however, the fellow cornea grew multidrug-resistant Pseudomonas aeruginosa. The patient developed and was treated for endophthalmitis after penetrating keratoplasty, and aqueous and vitreous taps grew P. aeruginosa with antibiotic resistance identical to the isolate from the mate cornea. Sequence analysis of the 16S ribosomal gene from the two isolates and confirmation analyzing the sequence of P. aeruginosa heat shock protein gene (groES) were performed showing the same strain for both organisms. CONCLUSION: This case report documents the presence of the same multidrug-resistant P. aeruginosa causing endophthalmitis after penetrating keratoplasty and in the contralateral donor tissue, suggesting that we must be cautious in deciding to transplant tissues with positive culture in the contralateral donor cornea.

Overexpression of peroxiredoxin 2 in pterygium. A proteomic approach.

Pterygium is one of the most frequent pathologies in ophthalmology, and is a benign, fibrovascular lesion originating from the bulbar conjunctiva. It is composed of an epithelium and highly vascular, subepithelial, loose connective tissue. The etiology of pterygium is not clearly understood; the most widely recognized originating factor is ultraviolet radiation. It has been proposed that pterygium and neoplasia have common features, raising the possibility that pterygium is a neoplastic-like growth disorder. In this study, proteomic analysis was performed to show that peroxiredoxin 2 is overexpressed in pterygia compared to healthy conjunctivas. Twelve pterygium specimens were obtained together with healthy conjunctival tissue from the same eyes. Total proteins of pterygia and healthy conjunctivas were analyzed in SDS-PAGE. This analysis showed protein bands expressed exclusively in pterygium samples at the range of 20-25 kDa. After this, 2D electrophoresis was performed for the separation of total proteins; differential spots expressed in pterygium were excised and sequenced. Mass spectrometry (MS) data were searched in the NCBInr and EST databases using the MASCOT program. The spot was identified as peroxiredoxin 2. Real-time PCR, western blot and immunohistochemistry showed that peroxiredoxin 2 was increased in pterygium compared to healthy conjunctiva. Although, these results suggest that overexpression of peroxiredoxin 2 in pterygium could protect the cell against oxidative stress-induced apoptosis, further studies are required to establish the functional role of peroxiredoxin 2 in pterygium to determine its role in peroxidation and apoptosis in this pathology.

Two different PABPN1 expanded alleles in a Mexican population with oculopharyngeal muscular dystrophy arising from independent founder effects.

BACKGROUND: Oculopharyngeal muscular dystrophy (OPMD) is a late onset hereditary myopathy of autosomal dominant transmission characterised by ptosis, dysphagia and limb weakness. The disease is caused by short heterozygous expansions of a (GCN)(10) triplet located in the first exon of the PABPN1 gene at chromosome 14q11.1. Most affected individuals from North America and Europe carry a mutant (GCN)(13) allele. Although evidence for a founder mutation effect has been shown in several populations with OPMD, analysis of large groups of patients from different ethnic backgrounds will help to identify the relative contribution of each allele to the disease and a possible genotype-phenotype correlation. METHODS: 22 unrelated patients with OPMD from Mexico, a previously uncharacterised population, were clinically and molecularly analysed. Detailed ophthalmological and clinical examinations were performed in each proband and molecular analysis of the PABPN1 gene was carried out by PCR amplification and allele-specific cloning/sequencing. Two single nucleotide polymorphisms (SNPs) linked to PABPN1 were determined in each individual and in a number of affected first-degree relatives. RESULTS: 15 subjects (68%) carried a mutant (GCN)(15) or (GCG)(11)(GCA)(3)(GCG) PABPN1 allele; the remaining 7 (32%) exhibited an abnormal (GCN)(13) or (GCG)(9)(GCA)(3)(GCG) allele. Analysis of two SNPs linked to PABPN1 strongly suggests that both expanded alleles originate from two independent founder effects. In addition, in this particular population the (GCN)(15) allele was associated with an earlier onset of the disease (mean 46.5 years) compared with the (GCN)(13) allele (mean 54.7 years). CONCLUSION: The results of this study suggest that OPMD in the Mexican population is mostly due to (GCG)(11) or (GCG)(9) PABPN1 expanded alleles arising from two independent founder effect mutations. These findings add to the definition of the genetic features of the disease and to the establishment of a probable genotype-phenotype correlation.

[Genetic characterization of adenovirus isolated from follicular conjunctivitis and epidemic keratoconjunctivitis in a group of Mexican patients]. / Caracterización genética de adenovirus aislados de conjuntivitis folicular y queratoconjuntivitis epidémica en un grupo de pacientes mexicanos.

OBJECTIVE: To determine the adenovirus serotype in Mexican patients with folicular conjunctivitis and keratoconjunctivitis. METHODS: Adenovirus-specific PCR was used to analyze sample scrapings from the inferior fornix of patients with follicular conjunctivitis and clinical suspicion of adenovirus from January 2005 to December 2006. Identification of the serotype was made by automated sequencing. The nucleotide sequences obtained were compared with the reported sequences in GenBank. Descriptive statistical analyses were performed on the results. RESULTS: Of the 77 samples with clinical data of follicular conjunctivitis that were analyzed, 43 (56%) presented adenovirus. The sequencing of each positive sample allowed the identification of Ad1, Ad2, Ad3 and Ad8; the sequences of the serotype were identical those reported in GenBank with accession numbers: AF 534906 and AY 224420 for a sequence of the gene coding for the filament of Ad1 and Ad2 respectively, and AY 854180 and DQ 149614 for a sequence of the gene that codes for the Hexon protein of Ad3 and Ad8 respectively. From the statistical analysis it was possible to determine that a preferential seasonality of the serotype does not exist. CONCLUSION: In this work the Ad1, Ad2 and Ad3 serotypes were identified in patients with clinical diagnosis of follicular conjunctivitis in 2005. Ad2 was the predominant serotype. Ad8 was also detected in an outbreak of epidemic keratoconjunctivitis. From an epidemiological point of view, no serotype found seems to have a preferred seasonality.

Caracterización genética de adenovirus aislados de conjuntivitis folicular y queratoconjuntivitis epidémica en un grupo de pacientes mexicanos / Genetic characterization of adenovirus isolated from follicular conjunctivitis and epidemic keratoconjuntivitis in a group of mexican patients

Objetivo: Conocer los serotipos de adenovirus en pacientes mexicanos con conjuntivitis folicular y queratoconjuntivitis. Métodos: Se analizaron por PCR específica para adenovirus, muestras de fondo de saco conjuntival inferior de 77 pacientes diagnosticados con conjuntivitis folicular por sospecha clínica de infección por adenovirus, de enero de 2005 a diciembre de 2006. La identificación de los serotipos se realizó por secuenciación automatizada. Las secuencias de nucleótidos obtenidos fueron comparados con las secuencias reportadas en el GenBank. En el análisis de los resultados se utilizó la estadística descriptiva. Resultados: Se analizaron 77 muestras de las cuales el 56% presentaron adenovirus. La secuenciación de cada muestra positiva permitió la identificación de Ad1, Ad2, Ad3 y Ad8; las secuencias de los serotipos fueron idénticas a las reportadas en el GenBank con las direcciones: AF 534906 y AY 224420 para una secuencia del gen que codifica el filamento de Ad1 y Ad2 respectivamente y AY 854180 y DQ 149614 para una secuencia del gen que codifica la proteína Hexón de Ad3 y Ad8 respectivamente. En el análisis estadístico, se pudo observar que no existe, en apariencia, una estacionalidad preferencial de los serotipos identificados. Conclusiones: En este trabajo se identificaron los serotipos Ad1, Ad2 y Ad3 en pacientes con diagnóstico clínico de conjuntivitis folicular en el 2005, Ad2 fue el serotipo predominante. También se detectó en un brote de queratoconjuntivitis epidémica por Ad8. Desde el punto de vista epidemiológico, ningún serotipo encontrado parece tener estacionalidad preferente

Objetive: To determine the adenovirus serotype in Mexican patients with folicular conjunctivitis and keratoconjunctivitis. Methods: Adenovirus-specific PCR was used to analyze sample scrapings from the inferior fornix of patients with follicular conjunctivitis and clinical suspicion of adenovirus from January 2005 to December 2006. Identification of the serotype was made by automated sequencing. The nucleotide sequences obtained were compared with the reported sequences in GenBank. Descriptive statistical analyses were performed on the results. Results: Of the 77 samples with clinical data of follicular conjunctivitis that were analyzed, 43 (56%) presented adenovirus. The sequencing of each positive sample allowed the identification of Ad1, Ad2, Ad3 and Ad8; the sequences of the serotype were identical those reported in GenBank with accession numbers: AF 534906 and AY 224420 for a sequence of the gene coding for the filament of Ad1 and Ad2 respectively, and AY 854180 and DQ 149614 for a sequence of the gene that codes for the Hexon protein of Ad3 and Ad8 respectively. From the statistical analysis it was possible to determine that a preferential seasonality of the serotype does not exist. Conclusion: In this work the Ad1, Ad2 and Ad3 serotypes were identified in patients with clinical diagnosis of follicular conjunctivitis in 2005. Ad2 was the predominant serotype. Ad8 was also detected in an outbreak of epidemic keratoconjunctivitis. From an epidemiological point of view, no serotype found seems to have a preferred seasonality (Arch Soc Esp Oftalmol 2008; 83: 161-168)

[Identification of adenovirus associated with conjunctivitis by molecular methodology]. / Identificación por métodos moleculares de adenovirus asociados a conjuntivitis.

OBJECTIVE: To identify adenovirus in patients with conjunctivitis by molecular methods such as the Polymerase Chain Reaction (PCR) and DNA sequencing. METHODS: Samples of scrapings from the inferior fornix of 51 patients (39 diagnosed with Follicular Conjunctivitis and 12 diagnosed with Vernal Conjunctivitis) were processed by generic PCR to identify adenovirus. All the samples that were PCR positive were cultured on VERO cells for virus isolation, with this being demonstrated by immunofluorescence. For the identification of the isolated serotype, the multiplex PCR was utilized and DNA automated sequencing was employed to identify the genetic variants. RESULTS: Twenty-eight of the individuals diagnosed with Follicular Conjunctivitis and six of those diagnosed with Vernal Conjunctivitis, had positive results to adenovirus (67%). The cultures in VERO cells allowed the isolation of eight samples. Only three of the isolated viruses (one Ad1 and two Ad2) were identified by the multiplex PCR used to identify the subgenus C adenovirus. An Ad1 genetic variant was identified by automated sequencing while the Ad2 serotypes were identical to the ones reported by Genbank. CONCLUSIONS: The Polymerase Chain Reaction and DNA sequencing are useful tools to identify and characterize microorganisms responsible for diseases such as conjunctivitis caused by adenoviruses.

Identificación por métodos moleculares de adenovirus asociados a conjuntivitis / Identification of adenovirus associated with conjunctivitis by molecular methodology

Objetivo: Por medio de métodos moleculares comola Reacción en Cadena de la Polimerasa (PCR) y lasecuenciación del material genético, identificar adenovirusen pacientes con conjuntivitis.Métodos: Se procesaron muestras de raspado delsaco conjuntival inferior de 51 pacientes (39 diagnosticadoscon conjuntivitis folicular y 12 diagnosticadoscon conjuntivitis vernal) para identificaradenovirus por medio de la PCR genérica. Todas lasmuestras positivas en la PCR genérica, fueron cultivadasen células VERO para el aislamiento delvirus, éste se demostró por inmunofluorescencia. Seutilizó la PCR múltiple para la caracterización delos serotipos aislados y las variantes genéticas seidentificaron mediante la secuenciación automatizada.Resultados: Veintiocho de los pacientes diagnosticadoscon conjuntivitis folicular y seis de los diagnosticadoscon conjuntivitis vernal resultaron positivosa adenovirus (67%). El cultivo en célulasVERO permitió el aislamiento del virus de 8 muestras.Sólo tres de los aislados fueron identificados(un serotipo Ad1 y dos Ad2) por la PCR múltipleque identifica adenovirus del subgénero C. Por estreptomicisecuenciaciónautomatizada se identificó unavariante genética correspondiente al Ad1, mientrasque los aislados de Ad2 fueron idénticos a losreportados en el Banco de Genes.Conclusiones: La Reacción en Cadena de la Polimerasay la Secuenciación son herramientas útilespara la identificación y caracterización de agentescausantes de enfermedades tales como la Conjuntivitispor Adenovirus

Objective: To identify adenovirus in patients with conjunctivitis by molecular methods such as the Polymerase Chain Reaction (PCR) and DNA sequencing. Methods: Samples of scrapings from the inferior fornix of 51 patients (39 diagnosed with Follicular Conjunctivitis and 12 diagnosed with Vernal Conjunctivitis) were processed by generic PCR to identify adenovirus. All the samples that were PCR positive were cultured on VERO cells for virus isolation, with this being demonstrated by immunofluorescence. For the identification of the isolated serotype, the multiplex PCR was utilized and DNA automated sequencing was employed to identify the genetic variants. Results: Twenty-eight of the individuals diagnosed with Follicular Conjunctivitis and six of those diagnosed with Vernal Conjunctivitis, had positive results to adenovirus (67%). The cultures in VERO cells allowed the isolation of eight samples. Only three of the isolated viruses (one Ad1 and two Ad2) were identified by the multiplex PCR used to identify the subgenus C adenovirus. An Ad1 genetic variant was identified by automated sequencing while the Ad2 serotypes were identical to the ones reported by Genbank. Conclusions: The Polymerase Chain Reaction and DNA sequencing are useful tools to identify and characterize microorganisms responsible for diseases such as conjunctivitis caused by adenoviruses

BCG specificity of an inhibitory seric factor from tuberculosis anergic patients that acts on non-adherent PPD reactive cells.

The effect of tuberculosis anergic immune sera adsorbed with BCG was studied on cocultures of adherent and non-adherent cells from PPD+ tuberculosis patient (TBP PPD+). This effect on the cocultures was quantified by lymphocyte transformation (LT) test using PPD as antigen. Only those cocultures with non-adherent cells from TBP PPD+ patients treated with anergic sera, inhibited the LT response induced by PPD, whereas sera adsorption with BCG eliminated the inhibitory effect.

Characterization of an inhibitory seric factor from tuberculosis anergic patients that acts on non-adherent PPD reactive cells.

Non-adherent cells from PPD+ tuberculosis patients (TBP PPD+) and from healthy individuals treated with whole tuberculosis anergic immune sera or with its protein A-Sepharose IgG fraction, or with sera fraction separated by PPD-Sepharose chromatography, were submitted to immunofluorescence assays. Anti-human IgG or IgM FITC-conjugate were used to reveal the assays, and results were expressed by a fluorescence percentage or fluorescence index. The presence of IgG over the surface of PPD+ non-adherent cells was detected. High fluorescence percentages were observed only in those PPD+ cells treated with whole anergic serum or with its IgG fraction. Positive fluorescence index values were obtained only in those PPD+ cells treated with anergic serum, meanwhile fluorescence index was always negative when non-bound fractions from PPD-Sepharose were used. Results suggest that non-adherent population are the cell targets for the serum inhibitory factor, which previously has been detected to inhibit antigen response in PPD reactive cells and, point out the specific behavior of this factor, since it was eliminate by PPD-Sepharose chromatography. The IgG nature of the factor was demonstrated by SDS-PAGE and immunoelectrophoresis.

Differential effects of Trypanosoma cruzi on the transcription of the p55IL-2R, c-fos, c-myc and CD69 genes in activated human lymphocytes.

Mitogen-activated lymphocytes co-cultured with either purified Trypanosoma cruzi trypomastigotes or the filtrate of trypomastigote suspensions in culture medium manifest a significant decrease in their capacities to express p55 interleukin-2 receptor molecules (p55IL-2R) on their membrane and proliferate. In this study we found that the cytoplasmic levels of p55IL-2R are also markedly reduced under these conditions. This inhibition appeared to result from altered gene transcription since the levels of p55IL-2R mRNA in phytohaemagglutinin (PHA)-stimulated human peripheral blood mononuclear cells (PBMC) dropped substantially in the presence of parasite suspension filtrate. The rates of decay for p55IL-2R mRNA determined in cultures lacking and containing the parasite filtrate after addition of actinomycin D to inhibit further RNA synthesis were comparable. These results indicated that decreased p55IL-2R mRNA was not due to decreased stability of this mRNA under our conditions and pointed to a transcriptional or pre-transcriptional modification as the likely mechanism by which T. cruzi affects activated lymphocytes. The parasite filtrate did not appear to affect transcription of c-fos or c-myc (known to occur in the very early stages of lymphocyte activation) or that of CD69 (which is concomitant with p55IL-2R transcription). Thus, decreased p55IL-2R gene transcription appears to be a somewhat selective effect of a T. cruzi-derived molecule(s) rather than the consequence of an overall shutdown of gene transcription.

Trypanosoma cruzi-induced decrease in the level of interferon-gamma receptor expression by resting and activated human blood lymphocytes.

A substantial proportion of human peripheral blood mononuclear cells (PBMC) manifested a decreased capacity to express membrane interferon-gamma receptors (IFN-gamma R) when co-cultured with Trypanosoma cruzi. Among the lymphocytes, B cells accounted for the bulk of this effect, evidenced by a marked drop in the proportion of CD19+ or CD20+ cells expressing IFN-gamma R. Decreased IFN-gamma R expression by B lymphocytes was seen as early as 3 h after co-culture with T. cruzi and persisted for at least 24 h. The parasite had no detectable effect on CD19, CD20 or DR antigen expression by B lymphocytes. Neither the proportion of B cells expressing these markers nor the membrane density of these molecules varied significantly in the presence of T. cruzi. In PBMC cultures stimulated with Staphlyococcus aureus Cowan I (SACI), T. cruzi decreased the percentages of both IFN-gamma R+ and IFN-R+bright (cells expressing above-normal levels of surface IFN-gamma R) B lymphocytes. Cell-free filtrates of T. cruzi suspensions reproduced the suppressive effects of living parasites on IFN-gamma R expression by B cells. When T. cruzi present, the intracellular levels of IFN-gamma R molecules in resting or SACI-activated B lymphocytes, represented by fluorescence intensity, were well below control values, suggesting that decreased surface expression resulted from suppressed IFN-gamma R synthesis. Among T (CD3+) cells, 10.8% to 39.6% (7 donors) expressed surface IFN-gamma R and did so at a very low level. These percentages were also reduced by T. cruzi.(ABSTRACT TRUNCATED AT 250 WORDS)

Does interleukin-2 restore lymphocyte responses suppressed by Trypanosoma cruzi?

There has been disagreement about the ability of exogenous interleukin-2 (IL-2) to restore responsiveness to lymphocytes from either Trypanosoma cruzi-infected animals or normal individuals co-cultured with this parasite. The discrepancy has been attributed to the use of different strains of mice or T. cruzi isolates, or to the use of lymphoid cells from different organs. As T. cruzi inhibits the expression of IL-2 receptors by activated lymphocytes in vitro, we were able to test whether restoration of responsiveness by exogenous IL-2 might depend on the level of suppression present in the system. Human or mouse lymphocytes stimulated with phytohaemagglutinin (PHA) exhibited gradual decreases in IL-2 receptor expression, [3H]thymidine incorporation and IL-2 secretion as the concentration of T. cruzi in the culture increased. Exogenous IL-2 afforded a degree of restoration of both IL-2 receptor expression and [3H]thymidine uptake which was substantial at the lower, but very small--if any--at the higher, parasite concentrations tested. Trypanosoma cruzi could not have competed with the lymphocytes for IL-2 because it did not bind significant amounts of this cytokine. These results suggested that the controversy about the corrective effects of IL-2 may be more apparent than real, reflecting variations in the extent of immunosuppression present in different model systems of T. cruzi-associated immunosuppression.

Tuberculous anergic sera or purified protein derivative treatment induces modification in lymphocyte transformation of cells from patients with tuberculosis.

Peripheral blood mononuclear cells from 20 purified protein derivative (PPD)-reactive (PPD+) tuberculous patients were cultured in autologous, tuberculous anergic, or normal serum. After 12 h of incubation, the serum was eliminated and lymphocyte transformation with PPD was performed. Transformation was inhibited only in cells incubated with anergic serum. In contrast, cells from 11 anergic tuberculous (PPD-) individuals recovered the ability to respond to an optimal PPD dose after treatment with high PPD concentrations followed by several washings. The cells which recovered returned to their initial anergic state when incubated with sera from anergic patients. Under both conditions, incubation with sera did not abolish the response to the mitogen phytohemagglutinin. Cells from healthy PPD+ or PPD- individuals were used as controls. The most important finding derived from serum analysis was the increased levels of specific immunoglobulins G and A in anergic patients.