Eliminación de huevos de los pequeños estróngilos en la materia fecal de los equinos y su importancia práctica en la posibilidad de tratamientos selectivos / Fecal shedding of equine small strongyle eggs and its practical importance for selective treatment

Resumen Los pequeños estróngilos son los parásitos de mayor prevalencia e importancia en los equinos de todo el mundo, y el desarrollo generalizado de la resistencia de estos nematodos a los antihelmínticos está impulsando la aplicación de tratamientos selectivos (TS) para disminuir o demorar el desarrollo de estos fenómenos. Esta estrategia se basa en conteos de huevos de nematodos en la materia fecal de todos los integrantes del grupo animal para tratar solamente aquellos que sobrepasan un determinado umbral o cut off. Este principio de TS se ve favorecido por la consistencia de los conteos de huevos por gramo de heces (hpg) en los equinos y está siendo empleada en algunos países del hemisferio norte, especialmente en los animales adultos. En la Argentina, existe una ausencia de información básica en relación a las condiciones climáticas, de pastoreo y manejo que podrían resultar en cargas parasitarias y transmisión de nematodos diferentes a las observadas en el hemisferio norte. En este contexto, el objetivo de este estudio fue caracterizar la eliminación de huevos de pequeños estróngilos en 436 equinos de diferentes edades pertenecientes a 19 establecimientos del área central de la Argentina. El 90% de los equinos mostró huevos de estróngilos en las heces y los valores de hpg fueron menores a 200 y mayores a 1.000 en el 32% y 22% de los equinos respectivamente. La distribución de los huevos de estróngilos entre individuos de todas las edades fue sobre-dispersa o agregada, con una media aritmética general de 671,59 ± 789,76 y un valor de k o parámetro de agregación inversa de 0,72 (distribución de la binomial negativa). La edad de los animales (potrillos vs adultos) y los biotipos (deportivo vs trabajo) en la categoría adultos influenciaron la magnitud de los valores del hpg (p<0,001). En esta última categoría el 35% de los animales fue responsable de la excreción de aproximadamente el 80% de los huevos eliminados en las pasturas. Si bien existen algunas diferencias con lo observado en países del hemisferio norte con poblaciones parasitarias más agregadas o sobre-dispersas, los TS para el control de los pequeños estróngilos en equinos adultos podrían también ser una estrategia válida bajo nuestras condiciones para disminuir la presión de selección sobre el genoma parasitario y permitir el desarrollo de poblaciones en refugio.

Abstract Small strongyles are the most prevalent and important parasites in grazing horses throughout the world and the widespread development of antihelminthic resistance has prompted in some countries of the northern hemisphere the application of selective treatments (ST) to reduce or delay the development of these phenomena. This strategy is based on determining strongyle fecal egg counts of all herd members and treating only those exceeding a certain threshold or cut off. However, there is an absence of basic information on horses maintained under climatic, grazing or management conditions different from those observed in the northern hemisphere. In this context, the aim of this study was to characterize the elimination of eggs of small strongyles in horses of different ages from 19 farms in the central area of Argentina. Out of a total of 436 horses, 90% showed positive strongyles egg counts and in these animals the count of eggs per gram of feces (epg) was under 200 and over 1,000 in 32% and 22% of the horses respectively. The distribution of strongyle eggs among individuals of all categories was over-dispersed or aggregated with a general epg arithmetic mean of 671.59 ± 789.76 and a value of the inverse aggregation parameter of 0.72 (negative binomial distribution). The age of the animals (foals vs. adults) and the biotypes in the adult category (sports vs. work) influenced the magnitude of the epg values (P <0.001). In the latter category, approximately 35% of the animals were responsible for the excretion of approximately 80% of the eggs in the pastures. Although some differences on magnitude and distribution of strongyle fecal egg counts were observed in countries of the northern hemisphere, ST of small strongyles in adult equines could also be a valid strategy under Argentinian conditions to reduce the selection pressure on the parasitic genome and allow the development of refugia populations.

Resistencia antihelmíntica en equinos: Observaciones sobre el período de reaparición de huevos luego del tratamiento con ivermectina en potrancas y yeguas madres

En los equinos parasitados por los pequeños estróngilos, un menor período requerido luego del tratamiento con antihelmínticos para la reaparición de huevos (PRH) de los nematodes en la materia fecal, puede ser utilizado como un indicador de la presencia de resistencia o pérdida de eficacia a estas drogas. En el presente trabajo se evaluó la eficacia clínica y el PRH luego de tratamientos con ivermectina (0,2 mgr/kg) en equinos adultos (yeguas madres) y jóvenes (potrancas) naturalmente parasitados por pequeños estróngilos en un establecimiento de la provincia de Tucumán. La eficacia clínica determinada en la segunda semana post-tratamiento y utilizando un test de reducción en el conteo de huevos, osciló entre el 99,8 y el 100% en yeguas y potrancas respectivamente. Por su parte el PRH (considerado como el período post- tratamiento en que se alcanza el 10% del hpg pre-tratamiento o una reducción del mismo < 90%) fue de seis y siete semanas para las potrancas y yeguas madres respectivamente (p=0,001). En la sexta semana post- tratamiento, las potrancas mostraron poseer 11,74 veces más riesgo (IC 95% = 2,58-53,38) de reaparición de huevos que las yeguas madres. Estas diferencias ocasionadas por la edad de los animales deberían considerarse cuando se realizan estudios para establecer el status de susceptibilidad o resistencia a los antihelmínticos basados en el PRH para evitar asunciones incorrectas sobre el mismo.

In horses parasitized by small strongyles, a shortened strongyle egg reappearance period (ERP) after treatment with anthelmintics is considered an early indicator of the presence of resistance or loss of efficacy to these drugs. In the present work, clinical efficacy and ERP were evaluated after treatments with ivermectin in adult horses (broodmares) and juveniles (its fillies) naturally parasitized by small strongyles in an farm of Tucumán province. The clinical efficacy determined in the second week after treatment, and using a test of reduction in the egg count, ranged between 99.8 and 100% in mares and fillies respectively. The ERP (considered as the post- treatment period in which 10% of the pre-treatment epg was reached or a reduction of it < 90%) was six and seven weeks for the fillies and mother mares respectively (p= 0.001). In the sixth week after treatment, the fillies showed 11.74 times more risk (95% CI = 2.58-53.38) of reappearance of eggs than the brood mares. These differences caused by the age of the animals should be considered when conducting studies to establish the status of susceptibility or resistance to anthelmintics based on the ERP to avoid incorrect assumptions.

Pequeños estróngilos de los equinos: Eficacia clínica y periodo de reaparición de huevos luego del tratamiento con moxidectina y pirantel

El control de los pequeños estróngilos de los equinos (grupo Ciatostoma) se basa casi exclusivamente en la aplicación de antihelmínticos. En nuestro país, el desarrollo de resistencia generalizada a los bencimidazoles, está limitando las alternativas químicas disponibles a las lactonas macrocíclicas (ivermectina y moxidectina) y al pirantel, consideradas como drogas de larga y corta acción respectivamente. La información actualizada sobre la actividad de estas drogas en el campo es crítica para determinar su eficacia y detectar el desarrollo de fenómenos de resistencia a los antihelmínticos. En los equinos el período de reaparición de huevos (PRH) luego del tratamiento es considerado como un indicador temprano de la presencia de resistencia. En el presente trabajo se evaluó la eficacia clínica y el PRH luego de tratamientos con moxidectina y pirantel en equinos adultos de cinco establecimientos de las provincias de Santa Fe y Córdoba naturalmente parasitados por pequeños estróngilos. La eficacia clínica determinada al día 15 pos tratamiento utilizando un test de reducción en el conteo de huevos, osciló entre el 99,8 y el 100% para la moxidectina y entre el 98,9 y el 98,8% para el pirantel. Por su parte en el presente estudio el PRH fue de al menos 100 días para la moxidectina y de 35 días para el pirantel. Estos resultados indican que ambas drogas se muestran activas para el control de estos nematodes y que las poblaciones estudiadas (alguna de ellas resistentes a bencimidazoles) permanecen actualmente susceptibles a la moxidectina así como al pirantel. Esta última droga es de uso limitado en nuestro país, pero su inclusión en los programas de control contra los pequeños estróngilos podría reducir la dependencia y la presión de selección sobre las lactonas macrocíclicas y contribuir a mantener la vida útil de las mismas.

The control of the small strongyles (Ciathostoma group) in horses is based on the application of anthelmintics. In our country, the development of generalized resistance to benzimidazoles is limiting the chemical alternatives available to macrocyclic lactones (ivermectin and moxidectin) and to pyrantel, considered as long-acting and short-acting drugs respectively. Updated information on the activity of these drugs in the field, is critical for determining its efficacy and detecting the development of anthelmintic resistance. In these horse nematodes the period of egg reappearance (ERP) after treatment is considered as an early indicator of the presence of resistance. The present study evaluated the clinical efficacy and ERP after moxidectin and pirantel treatments in adult horses naturally parasitized by small strongyles from five farms from Santa Fe and Córdoba provinces. Clinical efficacy determined at day 14 or 15 post treatment using a test of reduction in the egg count ranged from 98.9 to 98.8% for the pirantel and 99.8 to 100% for moxidectin. The ERP was at least 100 days for moxidectin and 35 days for the pirantel. These results indicate that both drugs are active for the control of these nematodes and that the populations studied (some of them resistant to benzimidazoles) remain currently susceptible to moxidectin as well as to pyrantel. This last drug is of limited use in Argentina, but its inclusion in the control programs against the small strongyls could reduce the dependence and the selection pressure on the macrocyclic lactones and contribute to maintain the useful life of the same ones.

Aislamiento y caracterización de Corynebacterium pseudotuberculosis biotipo ovis en Lama glama en Córdoba, Argentina / Isolation and characterization of Corynebacterium pseudotuberculosis biotype ovis in Lama glama in Córdoba, Argentina

Corynebacterium pseudotuberculosis es el agente causal de la linfadenitis caseosa en distintas especies animales. No existen reportes en Argentina de casos clínicos en camélidos. Esta comunicación describe el aislamiento y tipificación de Corynebacterium pseudotuberculosis biotipo ovis en una llama (Lama glama) en Córdoba, Argentina. (AU)

Corynebacterium pseudotuberculosis is the causal agent of caseous lymphadenitis in different animal species. There are no reports in Argentina of clinical cases in camelids. This communication describes the isolation and typing of Corynebacterium pseudotuberculosis biotype ovis in llama (Lama glama) in Córdoba, Argentina. (AU)

Eficacia del monepantel para el control de aislamientos de Haemonchus contortus y Trichostrongylus spp. con resistencia múltiple (ivermectina y febendazole) en caprinos

El monepantel es un nuevo antihelmíntico registrado en nuestro país exclusivamente para el control de los nematodes gastrointestinales de los ovinos y su uso ha sido direccionado mayormente hacia el control de parásitos resistentes a las clases de antihelmínticos disponibles actualmente. Estos mismos nematodes también parasitan a los caprinos, pero en estos rumiantes la patofisiología y la respuesta a los antihelmínticos es diferente, lo cual resulta en un mayor parasitismo y complejidad en el manejo de la resistencia. La presente comunicación informa sobre la eficacia del monepantel en dos hatos caprinos mantenidos bajo condiciones de campo y parasitados naturalmente por los géneros de nematodes gastrointestinales más comunes del área central de la Argentina (Haemonchus y Trichostrongylus) y portando alelos de resistencia múltiple (ivermectina y febendazole). El test de reducción en el conteo de huevos post tratamiento comparando diversas fórmulas, indicaron que en todos los hatos el monepantel por vía oral y a la dosis de 3,75 mg/kg de peso (1,5 veces mayor a la dosis ovina), resultó en eficacias del 99% al 100 %. Se realizan breves consideraciones sobre el uso potencial de esta droga en caprinos.

Monepantel is a new anthelmintic registered in Argentina exclusively for control of gastrointestinal nematodes of sheep and mostly directed toward controlling resistant parasites to current available classes of anthelmintics. The same nematodes also parasitize goats but pathophysiology and response are different in these hosts resulting in more severe parasitism and complexity in handling anthelmintic resistance. This report assess the efficacy of monepantel in goats maintained under field conditions and naturally parasitized by most common gastrointestinal nematodes from central Argentina (Haemonchus and Trichostrongylus) carrying alleles of multiple resistance (ivermectin and febendazole). According to the egg count reduction test, efficacies of 99% to 100% were observed after monepantel treatment at 3.75 mg/kg orally (1.5 ovine dose).

Polymorphisms of cell cycle control genes influence the development of sporadic medullary thyroid carcinoma.

BACKGROUND: The role of key cell cycle regulation genes such as, CDKN1B, CDKN2A, CDKN2B, and CDKN2C in sporadic medullary thyroid carcinoma (s-MTC) is still largely unknown. METHODS: In order to evaluate the influence of inherited polymorphisms of these genes on the pathogenesis of s-MTC, we used TaqMan SNP genotyping to examine 45 s-MTC patients carefully matched with 98 controls. RESULTS: A multivariate logistic regression analysis demonstrated that CDKN1B and CDKN2A genes were related to s-MTC susceptibility. The rs2066827*GT+GG CDKN1B genotype was more frequent in s-MTC patients (62.22%) than in controls (40.21%), increasing the susceptibility to s-MTC (OR=2.47; 95% CI=1.048-5.833; P=0.038). By contrast, the rs11515*CG+GG of CDKN2A gene was more frequent in the controls (32.65%) than in patients (15.56%), reducing the risk for s-MTC (OR=0.174; 95% CI=0.048-0.627; P=0.0075). A stepwise regression analysis indicated that two genotypes together could explain 11% of the total s-MTC risk. In addition, a relationship was found between disease progression and the presence of alterations in the CDKN1A (rs1801270), CDKN2C (rs12885), and CDKN2B (rs1063192) genes. WT rs1801270 CDKN1A patients presented extrathyroidal tumor extension more frequently (92%) than polymorphic CDKN1A rs1801270 patients (50%; P=0.0376). Patients with the WT CDKN2C gene (rs12885) presented larger tumors (2.9±1.8 cm) than polymorphic patients (1.5±0.7 cm; P=0.0324). On the other hand, patients with the polymorphic CDKN2B gene (rs1063192) presented distant metastases (36.3%; P=0.0261). CONCLUSION: In summary, we demonstrated that CDKN1B and CDKN2A genes are associated with susceptibility, whereas the inherited genetic profile of CDKN1A, CDKN2B, and CDKN2C is associated with aggressive features of tumors. This study suggests that profiling cell cycle genes may help define the risk and characterize s-MTC aggressiveness.

Comprehensive analysis of RET gene should be performed in patients with multiple endocrine neoplasia type 2 (MEN 2) syndrome and no apparent genotype-phenotype correlation: an appraisal of p.Y791F and p.C634Y RET mutations in five unrelated Brazilian families.

BACKGROUND: We previously identified a four-generation family with medullary thyroid cancer (MTC) and a germline p.Y791F RET mutation whose cancer lacked a strong genotype-phenotype correlation. The entire gene coding region of the RET gene should be sequenced when genotype-phenotype discrepancies are observed in patients with multiple endocrine neoplasia type 2 (MEN 2), even if a RET hotspot mutation has been identified. METHODS: A new genetic test was performed in the index case of this family with the p.Y791F RET germline mutation. The entire coding region of the RET gene was investigated by direct sequencing of PCR products. Once a mutation was identified, the target exon was sequenced in all at-risk relatives. RESULTS: An additional p.C634Y germline mutation in the RET gene was identified in the reported family. The double mutation occurred in cis and segregated with the phenotype. Through the Brazilian Genetic Screening Program developed at our institution, we additionally report the combination of these two mutations (p.C634Y/p.Y791F) in the RET gene in four other unrelated families. The overall penetrance of MTC and pheochromocytoma in patients with the p.C634Y/p.Y791F mutations was 79% and 13%, respectively. CONCLUSION: Our data emphasises that a comprehensive analysis of the RET gene may reveal multiple germline mutations in MEN 2 patients who exhibit an atypical clinical course of the disease.

Genes of detoxification are important modulators of hereditary medullary thyroid carcinoma risk.

CONTEXT: Different inherited profiles of genes involved in cellular mechanisms of activation and detoxification of carcinogenic products can provide specific protection or determine the risk for cancer. Low-penetrance polymorphic genes related to the biotransformation of environmental toxins have been associated with susceptibility to and the phenotype of, human tumours. OBJECTIVE: To investigate the role of germline inheritance of polymorphisms in CYP1A2*F, CYP1A1 m1, GSTP1, NAT2 and TP53 genes in hereditary medullary thyroid carcinoma (HMTC) patients. DESIGN: This study was developed in University of Campinas (Unicamp). PATIENTS: We studied 132 patients with HMTC, 88 first-degree relatives of HMTC patients and 575 control individuals. MEASUREMENTS: All patients with MTC and their relatives were sequenced for the RET gene and five genes were genotyped using TaqMan(®) system. RESULTS: We observed that the inheritance of CYP1A2*F (OR = 2·10; 95% CI = 1·11-3·97; P = 0·022), GSTP1 (OR = 4·41; 95% CI = 2·47-7·88; P < 0·001) and NAT2 (OR = 2·54; 95% CI = 1·16-5·58; P = 0·020) variants increased the risk for HMTC. In addition, multiple regression analysis showed that the inheritance of GSTP1 polymorphisms was associated with the diagnosis in older patients (B = 8·0229; 95% IC = ± 5·5735; P = 0·0054). Concerning the group of HTMC relatives, CYP1A2*F (OR = 2:40; 95% CI = 1·19-4·86; P = 0·015), CYP1A1 m1 (OR = 2·79; 95% CI = 1:04-7·51; P = 0·042), GSTP1 (OR = 2·86; 95% IC = 1·53-5·32; P < 0·001) and NAT2 (OR = 2·25; 95% IC = 1·20-4·22; P = 0·012) were associated with HMTC risk. CONCLUSIONS: We have demonstrated that the inheritance of specific genes determining the individual response to environmental toxins may contribute to the risk and phenotypic variability that exists in patients with HMTC. Moreover, we identified a group at risk in relatives of HMTC patients.

Long-term treatment with standardized extract of Ginkgo biloba L. enhances the conditioned suppression of licking in rats by the modulation of neuronal and glial cell function in the dorsal hippocampus and central amygdala.

Our group previously demonstrated that short-term treatment with a standardized extract of Ginkgo biloba (EGb) changed fear-conditioned memory by modulating gene expression in the hippocampus, amygdaloid complex and prefrontal cortex. Although there are few controlled studies that support the long-term use of EGb for the prevention and/or treatment of memory impairment, the chronic use of Ginkgo is common. This study evaluated the effects of chronic treatment with EGb on the conditioned emotional response, assessed by the suppression of ongoing behavior and in the modulation of gene and protein expression. Male adult Wistar rats were treated over 28days and assigned to five groups (n=10) as follows: positive control (4mgkg(-1) Diazepam), negative control (12% Tween 80), EGb groups (0.5 and 1.0gkg(-1)) and the naïve group. The suppression of the licking response was calculated for each rat in six trials. Our results provide further evidence for the efficacy of EGb on memory. For the first time, we show that long-term treatment with the highest dose of EGb improves the fear memory and suggests that increased cAMP-responsive element-binding protein (CREB)-1 and glial fibrillary acidic protein (GFAP) mRNA and protein (P<0.001) in the dorsal hippocampus and amygdaloid complex and reduced growth and plasticity-associated protein 43 (GAP-43) (P<0.01) in the hippocampus are involved in this process. The fear memory/treatment-dependent changes observed in our study suggest that EGb might be effective for memory enhancement through its effect on the dorsal hippocampus and amygdaloid complex.

Evidence that polymorphisms in detoxification genes modulate the susceptibility for sporadic medullary thyroid carcinoma.

AIM: Polymorphic low-penetrance genes have been consistently associated with the susceptibility to a series of human tumors, including differentiated thyroid cancer. METHODS: To determine their role in medullary thyroid cancer (MTC), we used TaqMan SNP method to genotype 47 sporadic MTC (s-MTC) and a control group of 578 healthy individuals for CYP1A2*F, CYP1A1m1, GSTP1, NAT2 and 72TP53. A logistic regression analysis showed that NAT2C/C (OR=3.87; 95% CI=2.11-7.10; P=2.2×10(-5)) and TP53C/C genotypes (OR=3.87; 95% CI=1.78-6.10; P=2.8×10(-4)) inheritance increased the risk of s-MTC. A stepwise regression analysis indicated that TP53C/C genotype contributes with 8.07% of the s-MTC risk. RESULTS: We were unable to identify any relationship between NAT2 and TP53 polymorphisms suggesting they are independent factors of risk to s-MTC. In addition, there was no association between the investigated genes and clinical or pathological features of aggressiveness of the tumors or the outcome of MTC patients. CONCLUSION: In conclusion, we demonstrated that detoxification genes and apoptotic and cell cycle control genes are involved in the susceptibility of s-MTC and may modulate the susceptibility to the disease.

Gene expression profiles reveal that DCN, DIO1, and DIO2 are underexpressed in benign and malignant thyroid tumors.

To investigate the molecular events involved in the pathogenesis and/or progression of thyroid tumors, we compared the gene expression profiles of three thyroid carcinoma cell lines, which represent major tumor subtypes of thyroid cancer and normal thyroid tissue. Using cDNA array methodology, we investigated the expression of 1807 open reading frame expressed sequence tags (ORESTES), selected from head and neck tumor libraries generated through the Brazilian Human Cancer Project-LICR/FAPESP. We found that 505 transcripts were differentially expressed in the thyroid carcinoma cell lines. Using a more stringent criterion, transcripts underexpressed or overexpressed more than fivefold in 1 of 3 or 3 of 3 carcinoma cell lines, a list of 55 ESTs were detected. Five candidate genes were further validated by quantitative polymerase chain reaction (qPCR) in an independent set of 52 thyroid tumors and 22 matched normal thyroid tissues. DCN was found underexpressed in a high percentage of the follicular thyroid adenomas, follicular thyroid carcinomas, and follicular variant of papillary thyroid carcinomas. DIO1 and DIO2 were underexpressed in nearly all papillary thyroid carcinomas. These genes not only could help to better define a tumor signature for thyroid tumors, but may, in part, also become useful as potential targets for thyroid tumor treatment.

Expression of Smad4 and Smad7 in human thyroid follicular carcinoma cell lines.

Smad proteins have been shown tomediate the signal transduction pathway downstream of the transforming growth factor beta (TGFbeta). TGFbeta induces the phosphorylation of Smad2 and Smad3 which associate with Smad4 and translocate to the nucleus where they regulate gene transcription; besides these stimulatory Smads, the inhibitory Smads, Smad6 and Smad7, oppose signaling by blocking receptors and interrupting the phosphorylation of Smads2/3. The loss of TGFbeta-sensitivity, caused by inactivation of components of TGFbeta signaling, as Smad4, underlies a wide variety of human disorders, including cancer. In addition, the overexpression of the inhibitory Smad7, which prevents the phosphorylation of Smad2/3 and consequently inhibits TGFbeta signaling pathways, was observed in some diseases. In the present study we investigated the expression of Smad4 and Smad7 in thyroid cell lines (NPA papillary carcinoma, WRO follicular carcinoma and ARO anaplastic carcinoma) by RT-PCR and immunocytochemistry. Our results show that Smad4 was expressed in all thyroid cell lines and controls analyzed, differently from other classes of tumors where Smad4 expression was deleted. On the other hand, Smad7 was overexpressed in ARO anaplastic cell line, the most malignant follicular thyroid carcinoma. Our data suggest that the abrogation of the TGFbeta response by Smad7 overexpression may be a mechanism for the tumor aggressiveness observed in undifferentiated thyroid tumors.

What can digital transcript profiling reveal about human cancers?

Important biological and clinical features of malignancy are reflected in its transcript pattern. Recent advances in gene expression technology and informatics have provided a powerful new means to obtain and interpret these expression patterns. A comprehensive approach to expression profiling is serial analysis of gene expression (SAGE), which provides digital information on transcript levels. SAGE works by counting transcripts and storing these digital values electronically, providing absolute gene expression levels that make historical comparisons possible. SAGE produces a comprehensive profile of gene expression and can be used to search for candidate tumor markers or antigens in a limited number of samples. The Cancer Genome Anatomy Project has created a SAGE database of human gene expression levels for many different tumors and normal reference tissues and provides online tools for viewing, comparing, and downloading expression profiles. Digital expression profiling using SAGE and informatics have been useful for identifying genes that have a role in tumor invasion and other aspects of tumor progression

What can digital transcript profiling reveal about human cancers?

Important biological and clinical features of malignancy are reflected in its transcript pattern. Recent advances in gene expression technology and informatics have provided a powerful new means to obtain and interpret these expression patterns. A comprehensive approach to expression profiling is serial analysis of gene expression (SAGE), which provides digital information on transcript levels. SAGE works by counting transcripts and storing these digital values electronically, providing absolute gene expression levels that make historical comparisons possible. SAGE produces a comprehensive profile of gene expression and can be used to search for candidate tumor markers or antigens in a limited number of samples. The Cancer Genome Anatomy Project has created a SAGE database of human gene expression levels for many different tumors and normal reference tissues and provides online tools for viewing, comparing, and downloading expression profiles. Digital expression profiling using SAGE and informatics have been useful for identifying genes that have a role in tumor invasion and other aspects of tumor progression.

TRAP-silver staining, a highly sensitive assay for measuring telomerase activity in tumor tissue and cell lines

Measurement of telomerase activity in clinically obtained tumor samples may provide important information for use as both a diagnostic marker and a prognostic indicator for patient outcome. In order to evaluate telomerase activity in tumor tissue without radiolabeling the product, we developed a simple telomeric repeat amplification protocol-silver-staining assay that is less time-consuming, is safe and requires minimal equipment. In addition, we determined the sensitivity of the silver-staining method by using extracts of telomerase-positive thyroid carcinoma cell lines which were serially diluted from 5,000 to 10 cells. Telomerase activity was also assayed in 19 thyroid tumors, 2 normal controls and 27 bone marrow aspirates. The results indicate that the technique permits the detection of telomerase activity from 5000 to as few as 10 cells. We propose that it could be immediately applicable in many laboratories due to the minimal amount of equipment required

TRAP-silver staining, a highly sensitive assay for measuring telomerase activity in tumor tissue and cell lines.

Measurement of telomerase activity in clinically obtained tumor samples may provide important information for use as both a diagnostic marker and a prognostic indicator for patient outcome. In order to evaluate telomerase activity in tumor tissue without radiolabeling the product, we developed a simple telomeric repeat amplification protocol-silver-staining assay that is less time-consuming, is safe and requires minimal equipment. In addition, we determined the sensitivity of the silver-staining method by using extracts of telomerase-positive thyroid carcinoma cell lines which were serially diluted from 5,000 to 10 cells. Telomerase activity was also assayed in 19 thyroid tumors, 2 normal controls and 27 bone marrow aspirates. The results indicate that the technique permits the detection of telomerase activity from 5000 to as few as 10 cells. We propose that it could be immediately applicable in many laboratories due to the minimal amount of equipment required.

The contribution of 700,000 ORF sequence tags to the definition of the human transcriptome.

Open reading frame expressed sequences tags (ORESTES) differ from conventional ESTs by providing sequence data from the central protein coding portion of transcripts. We generated a total of 696,745 ORESTES sequences from 24 human tissues and used a subset of the data that correspond to a set of 15,095 full-length mRNAs as a means of assessing the efficiency of the strategy and its potential contribution to the definition of the human transcriptome. We estimate that ORESTES sampled over 80% of all highly and moderately expressed, and between 40% and 50% of rarely expressed, human genes. In our most thoroughly sequenced tissue, the breast, the 130,000 ORESTES generated are derived from transcripts from an estimated 70% of all genes expressed in that tissue, with an equally efficient representation of both highly and poorly expressed genes. In this respect, we find that the capacity of the ORESTES strategy both for gene discovery and shotgun transcript sequence generation significantly exceeds that of conventional ESTs. The distribution of ORESTES is such that many human transcripts are now represented by a scaffold of partial sequences distributed along the length of each gene product. The experimental joining of the scaffold components, by reverse transcription-PCR, represents a direct route to transcript finishing that may represent a useful alternative to full-length cDNA cloning.

Detection of recurrent thyroid cancer by sensitive nested reverse transcription-polymerase chain reaction of thyroglobulin and sodium/iodide symporter messenger ribonucleic acid transcripts in peripheral blood.

To investigate whether circulating thyroglobulin (Tg) messenger ribonucleic acid (mRNA) and sodium/iodide symporter (NIS) mRNA transcripts in peripheral blood are valuable in the follow-up of patients with thyroid cancer, we developed highly sensitive nested Tg and NIS mRNA detection assays and compared their accuracy with serum thyroglobulin (sTg) and whole body scan with 131I during the monitoring of 34 patients with well differentiated thyroid carcinoma who had undergone total thyroidectomy (17 of 34 also submitted to thyroid ablation with radioiodine) and were taking T4. Circulating Tg mRNA was found in 13 of 34 patients, 5 of 13 with detectable and 8 of 13 with undetectable sTg. From these 8 patients with undetectable Tg, 6 showed no cervical radioiodine uptake, and 3 presented proven metastatic disease (2 of them positive for antithyroglobulin antibodies). NIS mRNA was detected in 11 of 34 patients, but its measurement did not improve the ability to detect patients with metastases. Overall, identification of metastatic thyroid cancer was better associated with Tg mRNA than with NIS mRNA, sTg, or whole body scan (83% vs. 16.6% vs. 50% vs. 50%; P < 0.001). These data showed that circulating Tg mRNA is not only a more sensitive marker of residual thyroid tissue or thyroid cancer than sTg, particularly in patients during T4 therapy and with positive antithyroglobulin antibodies, but also was more sensitive than NIS mRNA in all patients.

Identification of human chromosome 22 transcribed sequences with ORF expressed sequence tags.

Transcribed sequences in the human genome can be identified with confidence only by alignment with sequences derived from cDNAs synthesized from naturally occurring mRNAs. We constructed a set of 250,000 cDNAs that represent partial expressed gene sequences and that are biased toward the central coding regions of the resulting transcripts. They are termed ORF expressed sequence tags (ORESTES). The 250,000 ORESTES were assembled into 81,429 contigs. Of these, 1, 181 (1.45%) were found to match sequences in chromosome 22 with at least one ORESTES contig for 162 (65.6%) of the 247 known genes, for 67 (44.6%) of the 150 related genes, and for 45 of the 148 (30.4%) EST-predicted genes on this chromosome. Using a set of stringent criteria to validate our sequences, we identified a further 219 previously unannotated transcribed sequences on chromosome 22. Of these, 171 were in fact also defined by EST or full length cDNA sequences available in GenBank but not utilized in the initial annotation of the first human chromosome sequence. Thus despite representing less than 15% of all expressed human sequences in the public databases at the time of the present analysis, ORESTES sequences defined 48 transcribed sequences on chromosome 22 not defined by other sequences. All of the transcribed sequences defined by ORESTES coincided with DNA regions predicted as encoding exons by genscan. (http://genes.mit.edu/GENSCAN.html).