Sulfide oxidation by members of the Sulfolobales.

The oxidation of sulfur compounds drives the acidification of geothermal waters. At high temperatures (>80°C) and in acidic conditions (pH <6.0), oxidation of sulfide has historically been considered an abiotic process that generates elemental sulfur (S0) that, in turn, is oxidized by thermoacidophiles of the model archaeal order Sulfolobales to generate sulfuric acid (i.e. sulfate and protons). Here, we describe five new aerobic and autotrophic strains of Sulfolobales comprising two species that were isolated from acidic hot springs in Yellowstone National Park (YNP) and that can use sulfide as an electron donor. These strains significantly accelerated the rate and extent of sulfide oxidation to sulfate relative to abiotic controls, concomitant with production of cells. Yields of sulfide-grown cultures were ∼2-fold greater than those of S0-grown cultures, consistent with thermodynamic calculations indicating more available energy in the former condition than the latter. Homologs of sulfide:quinone oxidoreductase (Sqr) were identified in nearly all Sulfolobales genomes from YNP metagenomes as well as those from other reference Sulfolobales, suggesting a widespread ability to accelerate sulfide oxidation. These observations expand the role of Sulfolobales in the oxidative sulfur cycle, the geobiological feedbacks that drive the formation of acidic hot springs, and landscape evolution.

Morphology of nares associated with stereo-olfaction in baleen whales.

The sensory mechanisms used by baleen whales (Mysticeti) for locating ephemeral, dense prey patches in vast marine habitats are poorly understood. Baleen whales have a functional olfactory system with paired rather than single blowholes (nares), potentially enabling stereo-olfaction. Dimethyl sulfide (DMS) is an odorous gas emitted by phytoplankton in response to grazing by zooplankton. Some seabirds use DMS to locate prey, but this ability has not been demonstrated in whales. For 14 extant species of baleen whale, nares morphometrics (imagery from unoccupied aerial systems, UAS) was related to published trophic level indices using Bayesian phylogenetic mixed modelling. A significant negative relationship was found between nares width and whale trophic level (ß = -0.08, lower 95% CI = -0.13, upper 95% CI = -0.03), corresponding with a 39% increase in nares width from highest to lowest trophic level. Thus, species with nasal morphology best suited to stereo-olfaction are more zooplanktivorous. These findings provide evidence that some baleen whale species may be able to localize odorants e.g. DMS. Our results help direct future behavioural trials of olfaction in baleen whales, by highlighting the most appropriate species to study. This is a research priority, given the potential for DMS-mediated plastic ingestion by whales.

The flavodiiron protein from Syntrophomonas wolfei has five domains and acts both as an NADH:O2 or an NADH:H2 O2 oxidoreductase.

Flavodiiron proteins (FDPs) are a family of enzymes with a significant role in O2 /H2 O2 and/or NO detoxification through the reduction of these species to H2 O or N2 O, respectively. All FDPs contain a minimal catalytic unit of two identical subunits, each one having a metallo-ß-lactamase-like domain harboring the catalytic diiron site, and a flavodoxin-like domain. However, more complex and diverse arrangements in terms of domains are found in this family, of which the class H enzymes are among the most complex. One of such FDPs is encoded in the genome of the anaerobic bacterium Syntrophomonas wolfei subsp. wolfei str. Goettingen G311. Besides the core domains, this protein is predicted to have three additional ones after the flavodoxin core domain: two short-chain rubredoxins and a NAD(P)H:rubredoxin oxidoreductase-like domain. This enzyme, FDP_H, was produced and characterized and the presence of the predicted cofactors was investigated by a set of biochemical and spectroscopic methodologies. Syntrophomonas wolfei FDP_H exhibited a remarkable O2 reduction activity with a kcat = 52.0 ± 1.2 s-1 and a negligible NO reduction activity (~ 100 times lower than with O2 ), with NADH as an electron donor, that is, it is an oxygen-selective FDP. In addition, this enzyme showed the highest turnover value for H2 O2 reduction (kcat = 19.1 ± 2.2 s-1 ) ever observed among FDPs. Kinetic studies of site-directed mutants of iron-binding cysteines at the two rubredoxin domains demonstrated the essential role of these centers since their absence leads to a significant decrease or even abolishment of O2 and H2 O2 reduction activities.

Relationships between fluid mixing, biodiversity, and chemosynthetic primary productivity in Yellowstone hot springs.

The factors that influence biodiversity and productivity of hydrothermal ecosystems are not well understood. Here we investigate the relationship between fluid mixing, biodiversity, and chemosynthetic primary productivity in three co-localized hot springs (RSW, RSN, and RSE) in Yellowstone National Park that have different geochemistry. All three springs are sourced by reduced hydrothermal fluid, but RSE and RSN receive input of vapour phase gas and oxidized groundwaters, with input of both being substantially higher in RSN. Metagenomic sequencing revealed that communities in RSN were more biodiverse than those of RSE and RSW in all dimensions evaluated. Microcosm activity assays indicate that rates of dissolved inorganic carbon (DIC) uptake were also higher in RSN than in RSE and RSW. Together, these results suggest that increased mixing of reduced volcanic fluid with oxidized fluids generates additional niche space capable of supporting increasingly biodiverse communities that are more productive. These results provide insight into the factors that generate and maintain chemosynthetic biodiversity in hydrothermal systems and that influence the distribution, abundance, and diversity of microbial life in communities supported by chemosynthesis. These factors may also extend to other ecosystems not supported by photosynthesis, including the vast subterranean biosphere and biospheres beneath ice sheets and glaciers.

Ecological Dichotomies Arise in Microbial Communities Due to Mixing of Deep Hydrothermal Waters and Atmospheric Gas in a Circumneutral Hot Spring.

Little is known of how the confluence of subsurface and surface processes influences the assembly and habitability of hydrothermal ecosystems. To address this knowledge gap, the geochemical and microbial composition of a high-temperature, circumneutral hot spring in Yellowstone National Park was examined to identify the sources of solutes and their effect on the ecology of microbial inhabitants. Metagenomic analysis showed that populations comprising planktonic and sediment communities are archaeal dominated, are dependent on chemical energy (chemosynthetic), share little overlap in their taxonomic composition, and are differentiated by their inferred use of/tolerance to oxygen and mode of carbon metabolism. The planktonic community is dominated by putative aerobic/aerotolerant autotrophs, while the taxonomic composition of the sediment community is more evenly distributed and comprised of anaerobic heterotrophs. These observations are interpreted to reflect sourcing of the spring by anoxic, organic carbon-limited subsurface hydrothermal fluids and ingassing of atmospheric oxygen that selects for aerobic/aerotolerant organisms that have autotrophic capabilities in the water column. Autotrophy and consumption of oxygen by the planktonic community may influence the assembly of the anaerobic and heterotrophic sediment community. Support for this inference comes from higher estimated rates of genome replication in planktonic populations than sediment populations, indicating faster growth in planktonic populations. Collectively, these observations provide new insight into how mixing of subsurface waters and atmospheric oxygen create dichotomy in the ecology of hot spring communities and suggest that planktonic and sediment communities may have been less differentiated taxonomically and functionally prior to the rise of oxygen at ∼2.4 billion years ago (Gya). IMPORTANCE Understanding the source and availability of energy capable of supporting life in hydrothermal environments is central to predicting the ecology of microbial life on early Earth when volcanic activity was more widespread. Little is known of the substrates supporting microbial life in circumneutral to alkaline springs, despite their relevance to early Earth habitats. Using metagenomic and informatics approaches, water column and sediment habitats in a representative circumneutral hot spring in Yellowstone were shown to be dichotomous, with the former largely hosting aerobic/aerotolerant autotrophs and the latter primarily hosting anaerobic heterotrophs. This dichotomy is attributed to influx of atmospheric oxygen into anoxic deep hydrothermal spring waters. These results indicate that the ecology of microorganisms in circumneutral alkaline springs sourced by deep hydrothermal fluids was different prior to the rise of atmospheric oxygen ∼2.4 Gya, with planktonic and sediment communities likely to be less differentiated than contemporary circumneutral hot springs.

How the Anaerobic Enteropathogen Clostridioides difficile Tolerates Low O2 Tensions.

Clostridioides difficile is a major cause of diarrhea associated with antibiotherapy. After germination of C. difficile spores in the small intestine, vegetative cells are exposed to low oxygen (O2) tensions. While considered strictly anaerobic, C. difficile is able to grow in nonstrict anaerobic conditions (1 to 3% O2) and tolerates brief air exposure indicating that this bacterium harbors an arsenal of proteins involved in O2 detoxification and/or protection. Tolerance of C. difficile to low O2 tensions requires the presence of the alternative sigma factor, σB, involved in the general stress response. Among the genes positively controlled by σB, four encode proteins likely involved in O2 detoxification: two flavodiiron proteins (FdpA and FdpF) and two reverse rubrerythrins (revRbr1 and revRbr2). As previously observed for FdpF, we showed that both purified revRbr1 and revRbr2 harbor NADH-linked O2- and H2O2-reductase activities in vitro, while purified FdpA mainly acts as an O2-reductase. The growth of a fdpA mutant is affected at 0.4% O2, while inactivation of both revRbrs leads to a growth defect above 0.1% O2 O2-reductase activities of these different proteins are additive since the quadruple mutant displays a stronger phenotype when exposed to low O2 tensions compared to the triple mutants. Our results demonstrate a key role for revRbrs, FdpF, and FdpA proteins in the ability of C. difficile to grow in the presence of physiological O2 tensions such as those encountered in the colon.IMPORTANCE Although the gastrointestinal tract is regarded as mainly anoxic, low O2 tension is present in the gut and tends to increase following antibiotic-induced disruption of the host microbiota. Two decreasing O2 gradients are observed, a longitudinal one from the small to the large intestine and a second one from the intestinal epithelium toward the colon lumen. Thus, O2 concentration fluctuations within the gastrointestinal tract are a challenge for anaerobic bacteria such as C. difficile This enteropathogen has developed efficient strategies to detoxify O2 In this work, we identified reverse rubrerythrins and flavodiiron proteins as key actors for O2 tolerance in C. difficile These enzymes are responsible for the reduction of O2 protecting C. difficile vegetative cells from associated damages. Original and complex detoxification pathways involving O2-reductases are crucial in the ability of C. difficile to tolerate O2 and survive to O2 concentrations encountered in the gastrointestinal tract.

Phylogenomic analysis of novel Diaforarchaea is consistent with sulfite but not sulfate reduction in volcanic environments on early Earth.

The origin(s) of dissimilatory sulfate and/or (bi)sulfite reducing organisms (SRO) remains enigmatic despite their importance in global carbon and sulfur cycling since at least 3.4 Ga. Here, we describe novel, deep-branching archaeal SRO populations distantly related to other Diaforarchaea from two moderately acidic thermal springs. Dissimilatory (bi)sulfite reductase homologs, DsrABC, encoded in metagenome assembled genomes (MAGs) from spring sediments comprise one of the earliest evolving Dsr lineages. DsrA homologs were expressed in situ under moderately acidic conditions. MAGs lacked genes encoding proteins that activate sulfate prior to (bi)sulfite reduction. This is consistent with sulfide production in enrichment cultures provided sulfite but not sulfate. We suggest input of volcanic sulfur dioxide to anoxic spring-water yields (bi)sulfite and moderately acidic conditions that favor its stability and bioavailability. The presence of similar volcanic springs at the time SRO are thought to have originated (>3.4 Ga) may have supplied (bi)sulfite that supported ancestral SRO. These observations coincide with the lack of inferred SO42- reduction capacity in nearly all organisms with early-branching DsrAB and which are near universally found in hydrothermal environments.

Plasmid Copy Number of pTRKH3 in Lactococcus lactis is Increased by Modification of the repDE Ribosome-Binding Site.

Plasmids for DNA vaccination are exclusively produced in the Gram-negative Escherichia coli. One important drawback of this system is the presence of lipopolysaccharides. The generally recognized as safe Lactococcus lactis (L. lactis) would constitute a safer alternative for plasmid production. A key requirement for the establishment of a cost-effective L. lactis-based plasmid manufacturing is the availability of high-copy number plasmids. Unfortunately, the highest copy number reported in Gram-positive bacteria for the pAMß1 replicon is around 100 copies. The purpose of this work is to engineer the repDE ribosome-binding site (RBS) of the pTRKH3 plasmid by site-directed mutagenesis in order to increase the plasmid copy number in L. lactis LMG19460 cells. The pTRKH3-b mutant is the most promising candidate, achieving 215 copies of plasmid per chromosome, a 3.5-fold increase when compared to the nonmodified pTRKH3, probably due to a stronger RBS sequence, a messenger RNA secondary structure that promotes the RepDE expression, an ideal intermediate amount of transcriptional repressors and the presence of a duplicated region that added an additional RBS sequence and one new in-frame start codon. pTRKH3-b is a promising high-copy number shuttle plasmid that will contribute to turn lactic acid bacteria into a safer and economically viable alternative as DNA vaccines producers.

How superoxide reductases and flavodiiron proteins combat oxidative stress in anaerobes.

Microbial anaerobes are exposed in the natural environment and in their hosts, even if transiently, to fluctuating concentrations of oxygen and its derived reactive species, which pose a considerable threat to their anoxygenic lifestyle. To counteract these stressful conditions, they contain a multifaceted array of detoxifying systems that, in conjugation with cellular repairing mechanisms and in close crosstalk with metal homeostasis, allow them to survive in the presence of O2 and reactive oxygen species. Some of these systems are shared with aerobes, but two families of enzymes emerged more recently that, although not restricted to anaerobes, are predominant in anaerobic microbes. These are the iron-containing superoxide reductases, and the flavodiiron proteins, endowed with O2 and/or NO reductase activities, which are the subject of this Review. A detailed account of their physicochemical, physiological and molecular mechanisms will be presented, highlighting their unique properties in allowing survival of anaerobes in oxidative stress conditions, and comparing their properties with the most well-known detoxifying systems.

The multidomain flavodiiron protein from Clostridium difficile 630 is an NADH:oxygen oxidoreductase.

Flavodiiron proteins (FDPs) are enzymes with a minimal core of two domains: a metallo-ß-lactamase-like, harbouring a diiron center, and a flavodoxin, FMN containing, domains. FDPs are O2 or NO reducing enzymes; for many pathogens, they help mitigate the NO produced by the immune system of the host, and aid survival during fluctuating concentrations concentrations of oxygen. FDPs have a mosaic structure, being predicted to contain multiple extra domains. Clostridium difficile, a threatening human pathogen, encodes two FDPs: one with the two canonical domains, and another with a larger polypeptide chain of 843 amino acids, CD1623, with two extra domains, predicted to be a short-rubredoxin-like and an NAD(P)H:rubredoxin oxidoreductase. This multi-domain protein is the most complex FDP characterized thus far. Each of the predicted domains was characterized and the presence of the predicted cofactors confirmed by biochemical and spectroscopic analysis. Results show that this protein operates as a standalone FDP, receiving electrons directly from NADH, and reducing oxygen to water, precluding the need for extra partners. CD1623 displayed negligible NO reductase activity, and is thus considered an oxygen selective FDP, that may contribute to the survival of C. difficile in the human gut and in the environment.

Subsurface processes influence oxidant availability and chemoautotrophic hydrogen metabolism in Yellowstone hot springs.

The geochemistry of hot springs and the availability of oxidants capable of supporting microbial metabolisms are influenced by subsurface processes including the separation of hydrothermal fluids into vapor and liquid phases. Here, we characterized the influence of geochemical variation and oxidant availability on the abundance, composition, and activity of hydrogen (H2 )-dependent chemoautotrophs along the outflow channels of two-paired hot springs in Yellowstone National Park. The hydrothermal fluid at Roadside East (RSE; 82.4°C, pH 3.0) is acidic due to vapor-phase input while the fluid at Roadside West (RSW; 68.1°C, pH 7.0) is circumneutral due to liquid-phase input. Most chemotrophic communities exhibited net rates of H2 oxidation, consistent with H2 support of primary productivity, with one chemotrophic community exhibiting a net rate of H2 production. Abundant H2 -oxidizing chemoautotrophs were supported by reduction in oxygen, elemental sulfur, sulfate, and nitrate in RSW and oxygen and ferric iron in RSE; O2 utilizing hydrogenotrophs increased in abundance down both outflow channels. Sequencing of 16S rRNA transcripts or genes from native sediments and dilution series incubations, respectively, suggests that members of the archaeal orders Sulfolobales, Desulfurococcales, and Thermoproteales are likely responsible for H2 oxidation in RSE, whereas members of the bacterial order Thermoflexales and the archaeal order Thermoproteales are likely responsible for H2 oxidation in RSW. These observations suggest that subsurface processes strongly influence spring chemistry and oxidant availability, which in turn select for unique assemblages of H2 oxidizing microorganisms. Therefore, these data point to the role of oxidant availability in shaping the ecology and evolution of hydrogenotrophic organisms.

Diversity and complexity of flavodiiron NO/O2 reductases.

Flavodiiron proteins (FDPs) are a family of enzymes endowed with nitric oxide (NO) or oxygen reductase activities, forming the innocuous nitrous oxide (N2O) or water molecules, respectively. FDPs are widespread in the three life kingdoms, and have a modular nature, being each monomer minimally constituted by a metallo-ß-lactamase-like domain containing a catalytic diiron centre, followed by a flavodoxin one, with a flavin mononucleotide. Since their discovery, additional domains have been found in FDPs, attached to the C-terminus, and containing either extra metal (iron) centers or extra flavin binding modules. Following an extensive analysis of genomic databases, we identified novel domain compositions, and proposed a new classification of FDPs in eight classes based on the nature and number of extra domains.

Androgenic-anabolic steroids inhibited post-exercise hypotension: a case control study.

BACKGROUND: There is evidence of hypertensive effects caused by anabolic androgenic steroids (AAS). A single exercise session promotes the acute reduction of blood pressure, but the effects of AAS on this phenomenon are unknown. OBJECTIVES: To investigate the post-exercise blood pressure response in androgenic-anabolic steroid users. METHODS: Thirteen AAS users (23.9±4.3 years old) and sixteen controls (22.1±4.5 years old) performed a session of aerobic exercise. Heart rate and blood pressure were assessed before exercise and during a 60min post-exercise resting period. Repeated ANOVA measures were used to determine differences between the groups. RESULTS: While the control group had a significant reduction in post-exercise systolic blood pressure of up to 13.9±11.6mmHg at 40min, this phenomenon was limited among AAS users who reached a maximum of 6.2±11.5mmHg at 60min. The between groups comparison revealed significant higher post-exercise hypotension (PEH) for the control group at 30min (-12.9±14.1mmHg versus -2.9±7.6mmHg), 40min (-13.9±11.6mmHg versus -2.5±8.3mmHg), 50min (-13.9±13.9mmHg versus -5.0±7.9mmHg) and 60min (-12.5±12.8mmHg versus -6.2±11.5mmHg). There was no significant diastolic PEH in any of the groups. CONCLUSIONS: This study demonstrated impaired systolic post-exercise hypotension as a new adverse effect of AAS usage.

The use of 1% toluidine blue eye drops in the diagnosis of ocular surface squamous neoplasia.

PURPOSE: To evaluate the use of in vivo toluidine blue (TB) dye in the diagnosis of ocular surface squamous neoplasia and to correlate staining intensity with the histopathological diagnosis. METHODS: A prospective study was performed at the Federal University of Sao Paulo, Brazil. Patients with conjunctival epithelial lesions were examined by slit-lamp biomicroscopy, with and without 1% TB eye drops, and the results were photo documented. Before the instillation of the dye, 1% proxymetacaine HCl eye drops were used. All patients were submitted to surgery and histopathological analysis to confirm the diagnosis. The patients were grouped according to the histopathological aspects of the lesions into 3 groups: group 1-patients with ocular surface squamous neoplasia; group 2-patients with premalignant lesions; and group 3-patients with pterygium. The digital images were analyzed by 2 masked examiners who had no previous access to the histopathological results. The photographs were classified according to the positivity and intensity of the staining. The statistical analysis method chosen depended on the type of data, with the level of significance set as P < 0.05. RESULTS: Forty-seven patients were included in the study: 10 had benign lesions (pterygium), 10 had premalignant lesions (actinic keratosis), and 27 had malignant lesions (conjunctival intraepithelial neoplasia and conjunctival squamous cell carcinoma). Agreement between observers regarding the analysis of the digital photographs was 100% for positivity and 82.9% for intensity of staining (κ = 0.938). Ninety percent of patients with premalignant lesions and all patients with malignant lesions showed positive staining with 1% TB. One patient had positive staining, but histopathological examination revealed a benign lesion (false positive). CONCLUSIONS: The use of 1% TB eye drops is an efficient method for the clinical diagnosis of ocular surface squamous neoplasia and premalignant lesions. Nevertheless, the intensity of the staining does not correlate with the degree of malignancy of these tumors.

Impaired glucose tolerance plus hyperlipidaemia induced by diet promotes retina microaneurysms in New Zealand rabbits.

UNLABELLED: With the increasing prevalence of diabetes mellitus and metabolic syndrome worldwide, experimental models are required to better understand the pathophysiology and therapeutic approaches to preserve pancreatic beta cells, attenuate atherosclerosis and protect target organs. The aims of this study were to develop an experimental model of impaired glucose tolerance combined with hypercholesterolaemia induced by diet and assess metabolic alterations and target organ lesions. New Zealand male rabbits were fed high-fat/high-sucrose (10/40%) and cholesterol-enriched diet for 24 weeks, when they were sacrificed. Biochemistry, fundus photographs with fluorescein angiography and pathological analyses were performed. Cholesterol-fed and normal animals of same age were compared. RESULTS: The animals with diet-induced impaired glucose tolerance combined with hypercholesterolaemia gained weight, increased blood glucose, total cholesterol, LDL-C and triglycerides and decreased HDL-C (P < 0.05 vs. baseline). Fructosamine levels and the homeostasis model assessment of insulin resistance (HOMA-IR) index were increased, while there was a reduction in the HOMA-ß (P < 0.05 for all vs. baseline). Histomorphologic findings of this model were aortic atherosclerosis, hepatic steatofibrosis and glomerular macrophage infiltration. Early clinical features of diabetic retinopathy with hyperfluorescent dots consistent with presence of retina microaneurysms were seen since week 12, progressing up to the end of the experiment (P < 0.0005 vs. baseline and 12 weeks). Our model reproduced several metabolic characteristics of human diabetes mellitus and promoted early signs of retinopathy. This non-expensive model is suitable for studying mechanistic pathways and allowing novel strategic approaches.

Extramedullary plasmacytoma of the third eyelid gland in a dog.

A case of extramedullary plasmacytoma of the third eyelid gland in a 7-year-old American Cocker Spaniel is reported. An enlargement of the third eyelid gland, abundant mucopurulent discharge, mild hyperemia and corneal pigmentation in the OD was present. Excisional biopsy of the mass revealed the gland was infiltrated and partially destroyed by a uniform population of neoplastic plasma cells. The neoplastic cells were positive for CD138, Ki-67 and lambda light chain. CD20, CD3, kappa light chain and cytokeratin were negative. Twelve months following surgery, no recurrence was observed. To the authors' knowledge, this is the first extramedullary plasmacytoma of the third eyelid gland reported in dogs.

The expression of cyclooxygenase 2 in retinoblastoma: primary enucleated eyes and enucleation after conservative treatment.

PURPOSE: To provide insight into the relationship between cyclooxygenase-2 (COX-2) expression and histopathologic features of retinoblastoma specimens treated either by primary or secondary enucleation. DESIGN: Laboratory investigation. METHODS: Twenty-five retinoblastoma specimens received between 1994 and 2003 were retrieved for this study from the Ocular Pathology Registry, Federal University of São Paulo, Brazil and the Henry C. Witelson Ocular Pathology Laboratory and Registry, McGill University, Montreal, Canada. The specimens retrieved were divided into two groups: Group I, enucleation was performed as a form of primary treatment (n = 15) and Group II, enucleation after failure of conservative treatment (n = 10). Patient information and formalin-fixed, paraffin-embedded specimens were obtained. New sections of these blocks were used for hematoxylin and eosin stain and for immunoassay using a monoclonal mouse anti-COX-2 antibody. Two independent ophthalmic pathologists reviewed all of the microslides. RESULTS: Twenty-three specimens (92%) presented a high expression of COX-2 (15 in Group I; eight in Group II) and there was no statistical difference in COX-2 expression between the two groups (P = .07). However, all specimens expressed COX-2 to a different degree. The areas of tumor invasion were positive for COX-2 in 87.5% of the two groups. CONCLUSION: Most retinoblastoma specimens revealed a high COX-2 expression. Future studies will be necessary to correlate the high expression of COX-2 in retinoblastoma and a possible applicability of anti-COX-2 medications in the treatment of these tumors.

Relationship between histopathological features of chemotherapy treated retinoblastoma and P-glycoprotein expression.

BACKGROUND: P-glycoprotein (P-gp) has been identified as a possible mediator of chemoresistance in retinoblastoma. The aim of this study was to determine the expression of P-gp in retinoblastoma treated with chemotherapy prior to enucleation. METHODS: Seventeen enucleated specimens of retinoblastoma from 16 patients were studied. Nine had been treated with chemotherapy alone, and eight had been treated with chemotherapy and other forms of local treatment. Tumour differentiation as well as choroidal and optic nerve invasion were assessed. P-gp immunohistochemical staining was performed and evaluated as negative, low or high. RESULTS: Histopathological assessment of the cases showed that 14 of 17 eyes (82.3%) had viable retinoblastoma cells. Nine retinoblastomas were considered regressed with a well-differentiated component, five regressed retinoblastomas had viable cells with poor differentiation and three retinoblastomas had regressed leaving no viable cells. Sixteen of 17 retinoblastomas were P-gp positive. In the one case with optic nerve invasion and the three cases with massive choroidal invasion, P-gp expression was found in invading retinoblastoma cells. CONCLUSION: Almost all retinoblastomas expressed P-gp. High levels of P-gp expression might play a role in chemotherapy resistance of retinoblastoma or, conversely, chemotherapy might induce P-gp expression. These results might have an impact on management of bilateral retinoblastoma.

Immunohistochemical profile of lymphoid lesions of the orbit.

BACKGROUND: Orbital idiopathic inflammation, lymphoid hyperplasia, and lymphoma may all present clinically in the same manner. Histopathology and especially immunohistochemistry play a major role in the differential diagnosis. The purpose of this study was to determine the immunophenotypic features of these lesions. METHODS: Fifty-five orbital lymphoid lesions were retrieved from the ophthalmic pathology registries at McGill University, Montreal, Canada, and the Federal University of São Paulo, São Paulo, Brazil. Formalin-fixed, paraffin-embedded, histopathologic sections were stained with hematoxylin and eosin and periodic acid-Schiff. The sections were also immunostained for B-cell (CD20) and T-cell (CD43) markers and for immunoglobulin light chains kappa and lambda. Two pathologists determined the histopathologic and immunohistochemical pattern of each lesion in a masked fashion. RESULTS: Of the 55 lesions, 11 (20%) were idiopathic chronic inflammations, 22 (40%) were lymphoid hyperplasias and 22 (40%) were lymphomas. Idiopathic inflammation displayed a predominance of T cells and all lesions expressed polyclonal light chains. Lymphoid hyperplasia displayed a mixture of B cells and T cells, with a slight predominance of the former and all lesions expressed polyclonal light chains. Lymphoma showed a striking predominance of B cells and all lesions expressed monoclonal light chains, usually kappa (63.7%). The differences in the mean percentages of B cells among the orbital lymphoid lesions (inflammation, 35%; hyperplasia, 65.9%; lymphoma, 87.3%) were statistically significant (p < 0.001). INTERPRETATION: Orbital lymphoid lesions can be differentiated based on the percentages of B cells and T cells and the monoclonal or polyclonal expression of immunoglobulin light chains.