Interference-free SERS tags for ultrasensitive quantitative detection of tyrosinase in human serum based on magnetic bead separation.

Tyrosinase (TYR) expression and activity determine the rate and yield of melanin production. Studies have shown that TYR is a potential biomarker for melanoma and highly sensitive detection of TYR benefits early diagnosis of melanoma-related diseases. In this study, we developed a method that combines surface-enhanced Raman scattering (SERS) and sandwich-type immunity for sensitive detection of TYR, in which 4-mercaptobenzonitrile (4 MB) embedded between the Au core and Au shell (Au4MB @ Au) core-shell structure was employed as a SERS probe for quantitative detection of TYR while the magnetic bead serves as a capture substrate. Our results demonstrated that under magnetic separation, the specific SERS signal obtained is highly correlated with TYR concentrations. Furthermore, the combination of magnetic beads and Au4MB @ Au core-shell structure significantly improved the sensitivity of the sensing platform, resulting in detection limits of 0.45 ng mL-1. More importantly, the detection and analysis of TYR concentration in human serum samples showed good accuracy and an excellent recovery rate. Accuracy of the system was investigated from % recovery of spiked TYR standard solutions and found to be in the range of 90-104%, which further verified the feasibility and reliability of our method applied in a complex environment. We anticipate this SERS-based immunoassay method to be applied to TYR detection in the clinical setting and to be extended to other promising related fields.

Dietary Protein and Carbohydrates Affect Immune Function and Performance in a Specialist Herbivore Insect (Manduca sexta).

Nutrition structures ecology and evolution across all scales of biological organization. It is well known that nutrition can have direct effects on performance and fitness, but indirect effects on physiological systems that mediate biotic interactions have been studied less frequently. Here, we focus on the interaction between nutrition, performance, and the immune system in a specialist herbivorous insect, Manduca sexta. We used a conceptual framework in nutritional ecology (the geometric framework) to examine how changes in diet quality affect aspects of the immune system used for defense against parasitoids. We raised caterpillars throughout their entire larval development on five different experimental diets that varied in protein and carbohydrate content and measured five aspects of the immune system: encapsulation, phenoloxidase activity, prophenoloxidase activity, total hemolymph protein, and hemocyte density. Overall, different parts of the immune function varied in response to interactions between carbohydrates, protein, and intake, but protein reductions had the largest impacts-mostly detrimental. In addition, our data suggest that diet quality mediates the relationship between performance (growth and survival) and immune function, as well as trade-offs among different components of immune function. Our work is the first to examine the interplay between nutrition, performance, and immune function with the geometric framework in a specialist insect herbivore.

Dosage and frequency effects of the microalgae Dunaliella sp. on the diet of Litopenaeus vannamei challenged with Vibrio parahaemolyticus.

Carotenoid sources in shrimp diets have shown to be effective for improving survival, growth, reproductive capacity, stress resistance, and also for diminishing disease. Dunaliella sp. is known to have high levels of ß-carotenes, which works as pro-vitamin A, enhancing the immune response in shrimp. However, the administration of Dunaliella sp. in shrimp diet needs to be evaluated to determine the appropriate dose and frequency of administration needed to optimize performance in cultured white shrimp. Diets with three different concentrations of Dunaliella sp. flour (1.5, 2 and 3%) were tested, and each one was administered at three different time frequencies: daily, and at 3- and 7-days intervals. Shrimp fed for 20 days were then infected with Vibrio parahaemolyticus (1 × 106 CFU/mL). Hemolymph parameters including protein, glucose, lactate, cholesterol and triglycerides were analyzed to evaluate shrimp stress status. Additionally, L. vannamei innate non-specific immune response was examined by evaluating the activity of prophenoloxidase (proPO), phenoloxidase (PO) and superoxide dismutase (SOD) in hemolymph; shrimp survival was also recorded. Survival after infection with V. parahaemolyticus was higher for shrimp fed with diets consisting of 2% Dunaliella sp. administered every 3 and 7 days. Shrimp fed a diet consisting of 2% or 3% Dunaliella sp. administered every third day showed positive physiological and immune responses to infection. A decrease in lipid oxidation in plasma triglycerides was observed at 48 h post inoculation in shrimp fed at all diets regimes due to Dunaliella sp. antioxidant action. Experimental results suggest the importance of Dunaliella sp. dosage and feeding frequency in L. vannamei diet to improve the survival and immune response.

Functionalized carbon quantum dots with dopamine for tyrosinase activity analysis.

Tyrosinase (TYR) is a key enzyme in melanin biosynthesis and its activity is an important biomarker for dermatological disorders, such as vitiligo, melanoma and actinic damages. Sensitive assay for TYR activity is significant for basic and clinical research. In this work, a facile fluorescent assay for TYR activity based on dopamine functionalized carbon quantum dots (CQDs-Dopa) has been developed. Dopamine (Dopa) was covalently bond to CQDs through a simple one-pot hydrothermal method, and the prepared CQDs-Dopa exhibited a fluorescence emission at 499 nm under exciting wavelength at 310 nm with a quantum yield of approximately 2.1%. When TYR was mixed with CODs-Dopa, the dopamine moiety in CQDs-Dopa conjugate was oxidized to O-dopaquinone, and an intra-particle photo-induced electron transfer (PET) process consequently occurred between CQDs and O-dopaquinone to quench the fluorescence of CQDs-Dopa. TYR activity can be determined based on the fluorescence quenching degree of CQDs-Dopa. This assay covered two broad linear ranges: 44.4-711.1 U L-1 and 711.1-2925.4 U L-1, with detection limit of 17.7 U L-1. The proposed fluorescent assay was applied to TYR activity measurement in human serum samples. It showed promising potential for TYR activity assay in clinical applications.

Two-Photon Semiconducting Polymer Dots with Dual-Emission for Ratiometric Fluorescent Sensing and Bioimaging of Tyrosinase Activity.

Semiconducting polymer dots (Pdots) with one-, two-photon excitation and dual-emission have been synthesized by coprecipitation of two conjugated polymers including poly(9,9-dioctylfluorenyl-2,7-diyl) (PFO) and poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-(1-cyanovinylene-1,4-phenylene)] (CN-PPV) and have been further functionalized with l-tyrosine methyl ester (Tyr-OMe) via electrostatic assembly for ratiometric fluorescent sensing and bioimaging of tyrosinase activity. Tyrosinase-catalyzed oxidation of Tyr-OMe effectively modulate the dual-emission fluorescence of PFO/CN-PPV@Tyr-OMe Pdots from orange to blue through a selective photoinduced electron transfer (PET) process. A two-photon ratiometric sensor at almost zero-background interference and bioimaging of tyrosinase activity have been demonstrated, suggesting the potential biomedical applications of the prepared functionalized Pdots.

The immunomodulation of inducible nitric oxide in scallop Chlamys farreri.

Nitric oxide (NO) is an important signalling molecule which plays an indispensable role in immunity of all vertebrates and invertebrates. In the present study, the immunomodulation of inducible NO in scallop Chlamys farreri was examined by monitoring the alterations of haemocyte behaviours and related immune molecules in response to the stimulations of LPS and/or with S-Methylisothiourea Sulphate (SMT), an inhibitor of inducible NO synthase (NOS). The total activity of NOS and NO concentration in the haemolymph of scallop C. farreri increased significantly at 3, 6 and 12 h after LPS stimulation respectively, whereas their increases were fully repressed when scallops were treated in the collaborating of LPS and SMT. Meanwhile, some cellular and humoral immune parameters were determined after the stimulation of LPS and SMT to investigate the role of inducible NO in innate immunity of scallop. After LPS stimulation, the highest levels of haemocytes apoptosis and phagocytosis were observed at 24 h (38.5 ± 2.5%, P < 0.01) and 12 h (38.6 ± 0.2%, P < 0.01), respectively, and the reactive oxygen species (ROS) level (5.88 ± 0.90%, P < 0.01) of haemocytes and anti-bacterial activity of haemolymph (10.0 ± 2.2%, P < 0.01) all elevated dramatically at 12 h. Although the activity of lysozyme and phenoloxidase (PO) in haemolymph both declined at 48 h (93.0 ± 6.3 U mgprot(-1), 0.40 ± 0.06 U mgprot(-1), P < 0.01), superoxide dismutase (SOD) activity and GSH concentration both increased to the highest level at 24 h post treatment (99.2 ± 8.1 U mgprot(-1), 93.0 ± 6.3 nmol mgprot(-1), P < 0.01). After the collaborating treatment of LPS and SMT, the apoptosis index increased much higher from 48 h, while the increase of haemocytes phagocytosis, ROS level and haemolymph anti-bacteria activities were suppressed completely at 12 h. The declines of lysozyme and PO activity in haemolymph were reversed at 48 h, and the rise of SOD activity and GSH concentration started earlier from 3 h. These results indicated clearly that NO could participate in the scallop immunity and play a crucial role in the modulation of immune response including haemocytes apoptosis and phagocytosis, anti-bacterial activity and redox homeostasis in the haemolymph of scallop.

Age- and density-dependent prophylaxis in the migratory, cannibalistic Mormon cricket Anabrus simplex (Orthoptera: Tettigoniidae).

As a result of the increased potential for disease transmission, insects are predicted to show an increased constitutive immunity when crowded. Cannibalistic aggressive interactions further increase the risk of wounding and pathogen transmission in crowds. Nymphal Mormon crickets Anabrus simplex Haldeman were collected in Montana and reared in the laboratory either solitarily or at densities similar to that experienced by Mormon crickets in migratory bands. As teneral adults, solitarily-reared Mormon crickets tended to have greater phenoloxidase activity than those reared in groups. Sampling enzyme activity a second time when the adults were nearing reproductive maturity, group-reared Mormon crickets had elevated levels of prophenoloxidase and encapsulated foreign objects faster than solitarily-reared insects. Rearing density did not have a significant effect on either the darkness of the cuticle or antibacterial activity. This is the first report of age-related responses of adult insect immunity to crowding.

IL-8 and cathepsin B as melanoma serum biomarkers.

Melanoma accounts for only a small portion of skin cancer but it is associated with high mortality. Melanoma serum biomarkers that may aid early diagnosis or guide therapy are needed clinically. However, studies of serum biomarkers have often been hampered by the serum interference that causes false readouts in immunological tests. Here we show that, after using a special buffer to eliminate the serum interference, IL-8 and cathepsin B levels were significantly elevated in melanoma patients (p < 0.05). More importantly, the combination of IL-8 and cathepsin B were also studied as a prognosis marker for melanoma mortality. Our study provides a novel approach to examine serum biomarkers.

Rapid nested-PCR for tyrosinase gene detection on chip.

The availability of non-invasive, fast and sensitive technologies for detection of circulating cancer cells is still a critical need of clinical oncology, particularly for diagnosis of aggressive and highly metastatic tumors, like malignant melanoma. Here we present the first nested polymerase chain reaction process carried out by a microfabricated, hybrid plastic-glass microfluidic chip on the tyrosinase gene, a predictive marker for melanoma diagnosis. The device is a hybrid system consisting of a glass microchannel embedded in an elastomeric matrix, and operating in flow-oscillating modality on a droplet of biological sample. The convection heat transfer and the temperature distribution inside the carrier fluid in the device are investigated. The oil responds to temperature changes with a characteristic time around 53 s, and exhibits three different thermal gradients along the capillary, with temperature variations below 4°C in correspondence of heater electrodes. The sample heating/cooling rates in the chip are as high as 16°C/s, allowing rapid processes. The nested polymerase chain reaction process is performed in less than 50 min, namely more than four times faster than in a standard thermocycler. The rapidity of the analysis method, combined with the simple and low-cost fabrication, reduced sample evaporation, and flexibility of the overall microfluidic platform, make it promising for the detection of events of tumor spreading.

Tyrosinase mRNA levels in the blood of uveal melanoma patients: correlation with the number of circulating tumor cells and tumor progression.

We measured tyrosinase mRNA levels by real-time quantitative reverse transcription-PCR (qRT-PCR), in the blood of patients with uveal melanoma. Results were correlated with clinical data and, in a subgroup of patients, with the number of circulating tumor cells (CTC) assessed using isolation by size of epithelial tumor cells (ISET). Forty-one patients with uveal melanoma were longitudinally investigated over a period of 5 years. The standard curve of the qRT-PCR method used melanoma cell line SK-MEL-28, added to the blood of normal donors and it was calibrated on a synthetic RNA standard (1 SK-MEL-28 cell corresponding to 18 tyrosinase mRNA copies) to improve the procedural standardization to facilitate the comparison of data collected at different laboratories. Increased tyrosinase mRNA levels were found in at least one of the blood samples in 20 of 41 (49%) uveal melanoma patients (median 0.8 SK-MEL-28 cell equivalents/ml blood; range 0.1-14.4). A significant correlation was found between mRNA tyrosinase levels and tumor dimension (P<0.01), disease-free and overall survival (P<0.05). CTC were isolated by ISET in five of 16 patients (5.8, 2.33, 2.00, 1.25, and 0.75 CTC/ml of blood) and the corresponding tyrosinase mRNA levels were 2.13, 1.37, 0.83, 0.58, and 0.35 SK-MEL-28 cell equivalents/ml of blood. Tyrosinase was undetectable in 11 ISET-negative patients. Tyrosinase assay by qRT-PCR is a noninvasive method for the detection of tumor progression in uveal melanoma patients. The mRNA tyrosinase levels can be taken as an indirect parameter correlated to the number of CTC isolated from blood by ISET.

Tyrosinase expression as a molecular marker for investigating the presence of circulating tumor cells in melanoma patients.

In 1991, Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) was introduced to assess the expression of Tyrosinase in the peripheral blood of melanoma patients, in order to identify the presence of Circulating Melanoma Cells. To date, hundreds of studies, some of which are reviewed here, were performed to assess the clinical value of tyrosinase expression alone, and/or, in addition to other molecular markers. Unfortunately no consensus on the utility of tyrosinase detection exists. In this paper, we underline the presence of too many variables that may interfere with the detection of circulating melanoma cells: from withdrawal and RNA extraction, to Reverse Transcriptase-Polymerase Chain Reaction and the assays used for the analysis of amplification products.

Prognostic value of microphthalmia-associated transcription factor and tyrosinase as markers for circulating tumor cells detection in patients with melanoma.

The aim of this study was to analyze microphthalmia-associated transcription factor (MITF) as a marker for the detection of circulating melanoma cells, determine its prognostic value in melanoma patients, and compare it with tyrosinase. Blood samples from 201 melanoma patients in all stages of the disease and 40 healthy volunteers were analyzed. RNA was isolated from mononuclear cell fraction of the blood and assayed by reverse transcription-PCR for the expression of MITF and tyrosinase. All samples from healthy volunteers were negative for both MITF and tyrosinase. Out of 201 blood samples from melanoma patients 32 were positive for MITF, 20 for tyrosinase, and four for both MITF and tyrosinase. Analysis of MITF as an additional marker to tyrosinase allowed for detection of circulating melanoma cells in a larger number of melanoma patients in comparison to tyrosinase analysis alone (48 vs. 20 positive). A positive value of MITF was associated with shorter progression-free (P=0.005) and overall survival (P=0.042). A positive value of tyrosinase was associated with shorter overall survival (P=0.012), whereas there was no significant association between the value of tyrosinase and progression-free survival. The value of MITF was selected with multivariate analysis as the independent prognostic factor for progression-free survival, whereas the only independent prognostic factor for overall survival was the stage of disease. This study has shown that MITF is a specific marker for detection of circulating melanoma cells that has a prognostic value in melanoma patients. Determination of MITF in addition to tyrosinase improved the detection of circulating melanoma cells in melanoma patients.

Circulating melanoma cells and distant metastasis-free survival in stage III melanoma patients with or without adjuvant interferon treatment (EORTC 18991 side study).

AIM: To evaluate the prognostic and predictive importance of detection of melanoma cells in peripheral blood using reverse transcriptase polymerase chain reaction (RT-PCR) in stage III cutaneous melanoma patients after sentinel or regional lymph node dissection. PATIENTS AND METHODS: Serial testing for tyrosinase and Mart-1/Melan-A transcripts in peripheral blood was performed every 6 months over a maximum period of 60 months in a subset of patients enrolled in EORTC 18991 phase 3 trial, comparing pegylated interferon-alpha-2b with observation. Univariate and multivariate analyses were performed to estimate the role of RT-PCR as prognostic and predictive factor for distant metastasis-free survival (DMFS). RESULTS: Among 299 patients who underwent RT-PCR analyses, 109 (36.5%) had at least one positive sample, either at time of randomisation (N=17) or subsequently (N=92). The cumulative rate of positive results was similar in the two treatment groups, as the DMFS from first RT-PCR positivity. RT-PCR result, positive versus negative, at a given time point, had no prognostic impact on subsequent DMFS. Cox time-dependent analysis indicated a significantly higher risk of developing distant metastasis in patients with a positive sample as compared to those with a negative one: hazard ratio (HR) of 2.23 (95% confidence interval (CI), 1.40-3.55; p<.001). These results were comparable in the 2 treatment groups, indicating that RT-PCR assessment was not predictive for treatment outcome. CONCLUSION: Detection of circulating tumour cells by RT-PCR for tyrosinase and Mart-1/Melan-A was a time-dependent moderate prognostic factor for subsequent development of distant metastasis in stage III melanoma patients.

Impact of molecular staging methods in primary melanoma: reverse-transcriptase polymerase chain reaction (RT-PCR) of ultrasound-guided aspirate of the sentinel node does not improve diagnostic accuracy, but RT-PCR of peripheral blood does predict survival.

PURPOSE: This study analyzes (1) the value of tyrosinase reverse-transcriptase polymerase chain reaction (RT-PCR) of aspirates obtained by ultrasound-guided fine-needle aspiration cytology (US-FNAC) of sentinel nodes (SNs) in patients with melanoma before sentinel lymph node biopsy (SLNB) and (2) the value of RT-PCR of blood samples of all SLNB patients. PATIENTS AND METHODS: Between 2001 and 2003, 127 patients with melanoma (median Breslow depth, 2.1 mm) underwent SLNB. FNAC was performed in all SNs of all patients pre- and post-SLNB. The aspirates were partly shock-frozen for RT-PCR and were partly used for standard cytology. Peripheral blood was collected at the time of SLNB and at every outpatient visit thereafter. RESULTS: Thirty-four (23%) of 120 SNs were positive for melanoma. SN involvement was predicted by US-FNAC with a sensitivity of 82% and a specificity of 72%. Additional tyrosinase RT-PCR revealed the same sensitivity of 82% and a specificity of 72%. At a median follow-up time of 40 months from first blood sample, peripheral-blood RT-PCR was a significant independent predictor of disease-free survival (DFS) and overall survival (OS; P < .001). CONCLUSION: US-FNAC is highly accurate and eliminates the need for SLNB in 16% of all SLNB patients. RT-PCR of the aspirate or excised SN does not improve sensitivity or specificity. RT-PCR of blood samples predicts DFS and OS.

Multimarker real-time reverse transcription-PCR for quantitative detection of melanoma-associated antigens: a novel possible staging method.

BACKGROUND: Detection of melanoma cells in peripheral blood is a promising method for monitoring haematogenous spread of melanoma cells. It enables us to detect early metastasis and to better stratify candidates for adjuvant immunotherapy. Inconsistent data on the sensitivity and clinical relevance of this method have been reported. STUDY DESIGN: We developed a multimarker real-time reverse transcription-PCR (RT-PCR) for quantification of five melanoma markers: Melan-A, gp100, MAGE-3, MIA and tyrosinase. In this prospective study, 65 patients with resected cutaneous melanoma stage IIB-III were screened. Peripheral blood samples were collected every 3 months for the following 18 months, and circulating melanoma cells were examined and compared with clinical staging results. RESULTS: Eighteen patients relapsed during the trial and showed different types of melanoma progression. All these patients experienced statistically significant tumour marker elevation in the period from 0 to 9 months before the disease progression. MAGE-3 was the most sensitive progression marker. In patients with progression, we observed three concordant positive markers in 39% of cases, two concordant positive markers in 28%, and finally one marker in 33%. CONCLUSIONS: This report describes a multiple-marker real-time RT-PCR, which is able to provide quantitative data on melanoma markers in the peripheral blood of melanoma patients. Measurement of the studied molecular markers in our hands represents a prognostic factor and a useful method for early detection of metastasis and treatment response of melanoma patients.

Traditional urinary cytology and tyrosinase RT-PCR in metastatic melanoma patients: correlation with clinical status.

BACKGROUND: The finding of a suspicious urinary cytology is not uncommon in melanoma patients, in as much as morphology alone is often unable to distinguish the variable cytological features of melanoma cells. To date, although tyrosinase reverse transcription (RT)-PCR assay has been used to identify melanoma cells in peripheral blood and tissues, this method has not been applied to the analysis of urine samples. METHODS: RT-PCR mRNA tyrosinase expression was analysed in 79 urine samples from patients with metastatic melanoma and correlated with standard morphology/immunocytology. The results were compared with the disease course and presence of genito-urinary involvement. RESULTS: A positive RT-PCR expression was found in 18/79 urine samples from patients with metastases; four of the 18 patients had positive cytology, nine had atypical cytology, and five had negative cytology. Genito-urinary metastases were demonstrated in 27.8% tyrosinase-positive patients but in only 9.8% of the negative patients. The majority of tyrosinase-positive patients had a progressive disease unresponsive to chemotherapy. Urine samples from 20 patients with non-melanoma cancer and 20 healthy subjects were all negative. CONCLUSIONS: Our data demonstrate the higher sensitivity of RT-PCR compared with standard cytology in detection of urinary melanoma cells, and suggest that this assay could be used as an additional tool in the presence of negative or suspicious cytology.

Developmental modulation of immunity: changes within the feeding period of the fifth larval stage in the defence reactions of Manduca sexta to infection by Photorhabdus.

In insect pathogen interactions, host developmental stage is among several factors that influence the induction of immune responses. Here, we show that the effectiveness of immune reactions to a pathogen can vary markedly within a single larval stage. Pre-wandering fifth-stage (day 5) larvae of the model lepidopteran insect Manduca sexta succumb faster to infection by the insect pathogenic bacterium Photorhabdus luminescens than newly ecdysed fifth-stage (day 0) caterpillars. The decrease in insect survival of the older larvae is associated with a reduction in both humoral and cellular defence reactions compared to less developed larvae. We present evidence that older fifth-stage larvae are less able to over-transcribe microbial pattern recognition protein and antibacterial effector genes in the fat body and hemocytes. Additionally, older larvae show reduced levels of phenoloxidase (PO) activity in the cell-free hemolymph plasma as well as a dramatic decrease in the number of circulating hemocytes, reduced ability to phagocytose bacteria and fewer melanotic nodules in the infected tissues. The decline in overall immune function of older fifth-stage larvae is reflected by higher bacterial growth in the hemolymph and increased colonization of Photorhabdus on the basal surface of the insect gut. We suggest that developmentally programmed variation in immune competence may have important implications for studies of ecological immunity.

[Detection of circulating malignant cells in patients with uveal melanoma]. / Pesquisa de células malignas circulantes em pacientes com melanoma maligno de coróide.

PURPOSE: The purpose of our study was to detect circulating malignant cells (CMCs) in oversea-shipped blood samples of patients with uveal melanoma diagnosed in Brazil. METHODOS: Melan-A and tyrosinase were the two markers used for the detection of CMCs, using reverse transcriptase nested polymerase chain reaction (RT-nested-PCR) in 6 patients with uveal melanoma. The expression of beta-actin and GAPDH were used to assess the quality of the material. RESULTS: Five patients (83.33%) tested positive for the presence of CMCs. The RT-nested-PCR was positive for melan-A in 4 patients (66.7%) and positive for tyrosinase in 4 (66.7%) of the 6 patients. Three (50%) patients were positive for both markers. One (16.7%) patient was negative for both markers. All negative controls were negative. CONCLUSION: The quality of the blood samples shipped overseas, from patients with uveal melanoma, was preserved. The detection of CMCs using RT-nested-PCR was positive in the majority of the patients.

Pesquisa de células malignas circulantes em pacientes com melanoma maligno de coróide / Detection of circulating malignant cells in patients with uveal melanoma

OBJETIVO: Avaliar a possibilidade de identificação de células malignas circulantes nas amostras de sangue periférico de pacientes brasileiros com melanoma maligno de coróide enviadas para análise no exterior. MÉTODOS: Os marcadores melan-A e tirosinase foram usados para detectar a presença de células malignas circulantes, pela transcrição reversa seguida de reação em cadeia da polimerase e análise seqüencial de DNA (RT-nested-PCR), em seis pacientes com melanoma maligno de coróide, diagnosticados no Brasil. RESULTADOS: Cinco pacientes deste grupo (83,33 por cento) foram considerados positivos. A reação de RT-nested-PCR foi positiva para melan-A em quatro (66,7 por cento) e positiva para tirosinase em quatro (66,7 por cento) dos seis pacientes testados. Três (50 por cento) pacientes foram positivos para os dois marcadores. Um (16,7 por cento) paciente foi negativo para ambos marcadores. CONCLUSÃO: A pesquisa de células malignas circulantes usando RT-nested-PCR, foi positiva na maioria dos pacientes estudados. A qualidade das amostras de sangue periférico dos pacientes brasileiros foi mantida no material preservado mesmo após ter sido enviado ao exterior.

PURPOSE: The purpose of our study was to detect circulating malignant cells (CMCs) in oversea-shipped blood samples of patients with uveal melanoma diagnosed in Brazil. METHODOS: Melan-A and tyrosinase were the two markers used for the detection of CMCs, using reverse transcriptase nested polymerase chain reaction (RT-nested-PCR) in 6 patients with uveal melanoma. The expression of beta-actin and GAPDH were used to assess the quality of the material. RESULTS: Five patients (83.33 percent) tested positive for the presence of CMCs. The RT-nested-PCR was positive for melan-A in 4 patients (66.7 percent) and positive for tyrosinase in 4 (66.7 percent) of the 6 patients. Three (50 percent) patients were positive for both markers. One (16.7 percent) patient was negative for both markers. All negative controls were negative. CONCLUSION: The quality of the blood samples shipped overseas, from patients with uveal melanoma, was preserved. The detection of CMCs using RT-nested-PCR was positive in the majority of the patients.

Behavioural and physiological effects of the trophically transmitted cestode parasite, Cyathocephalus truncatus, on its intermediate host, Gammarus pulex.

Some parasites with complex life-cycles are able to manipulate the behaviour of their intermediate hosts in a way that increases their transmission to the next host. Gammarids infected by the tapeworm Cyathocephalus truncatus (Cestoda: Spathebothriidea) are known to be more predated by fish than uninfected ones, but potential behavioural manipulation by the parasite has never been investigated. In this study, we tested the hypothesis that C. truncatus is able to manipulate the behaviour of one of its intermediate hosts, Gammarus pulex (Crustacea: Amphipoda). To assess if any behavioural change was linked to other phenotypic alterations, we also measured the immunity of infected and uninfected individuals and investigated the pathogenic effects of the parasite. Infected gammarids were significantly less photophobic than uninfected ones, but no effect of infection on the level of immune defence was found. The results on survival, swimming activity and oxygen consumption suggest that the parasite also has various pathogenic effects. However, the alteration in host phototaxis was not correlated to some of these pathogenic effects. Therefore, we propose that the modification in host reaction to light is a behavioural manipulation, explaining the previously observed increase of gammarid predation rate.