RNA-seq analysis of the rat placentation site reveals maternal obesity-associated changes in placental and offspring thyroid hormone signaling.

INTRODUCTION: In animal models, maternal obesity (OB) leads to augmented risk of offspring OB. While placental function is influenced by maternal habitus, the effect of maternal obesity on the interacting zones of the placenta [the labyrinth (LZ), junctional (JZ) and metrial gland (MG)] remains unknown. METHODS: Using a rat maternal obesity model, we conducted transcriptomic profiling of the utero-placental compartments and fetal liver (FL) at dpc 18.5, in conjunction with analyses of mRNA expression of key thyroid hormone (TH) signaling genes in the placenta, fetus and weanling offspring. RESULTS AND DISCUSSION: Gene expression analysis of placenta and offspring revealed that each utero-placental compartment responds distinctly to maternal OB with changes in inflammatory signaling, lipid metabolism and hormone stimulus being the predominant effects. OB-induced alterations in 17 genes were confirmed by qPCR, including reductions in thyrotropin-releasing hormone (Trh) in JZ. We further characterized mRNA and protein expression of TH signaling regulators including deiodinases (Dio), TH receptors (Tr), and downstream targets (uncoupling proteins (Ucp)). A concerted down-regulation of multiple facets of thyroid hormone signaling in the JZ and FL was observed. JZ expression of thyroid hormone signaling components Trh, Dio2, Trα, and Ucp2 were negatively associated with maternal leptin. mRNA expression of TRH, TRß and UCP1 were also decreased in term placenta from OB women. Finally, our studies identified persistent impairments in expression of TH related genes in tissues from offspring of obese dams. CONCLUSIONS: The role of lower placental thyroid expression is worthy of further study as a possible pathway that leads to low energy metabolism and obesity in animals born to obese mothers.

Maternal obesity is associated with a lipotoxic placental environment.

Maternal obesity is associated with placental lipotoxicity, oxidative stress, and inflammation, where MAPK activity may play a central role. Accordingly, we have previously shown that placenta from obese women have increased activation of MAPK-JNK. Here, we performed RNA-sequencing on term placenta from twenty-two subjects who were dichotomized based on pre-pregnancy BMI into lean (BMI 19-24 kg/m(2); n = 12) and obese groups (BMI, 32-43 kg/m(2); n = 12). RNA-seq revealed 288 genes to be significantly different in placenta from obese women by ≥ 1.4-fold. GO analysis identified genes related to lipid metabolism, angiogenesis, hormone activity, and cytokine activity to be altered in placenta from obese women. Indicative of a lipotoxic environment, increased placental lipid and CIDEA protein were associated with decreased AMPK and increased activation of NF-κB (p65) in placenta from obese women. Furthermore, we observed a 25% decrease in total antioxidant capacity and increased nuclear FOXO4 localization in placenta from obese women that was significantly associated with JNK activation, suggesting that maternal obesity may also be associated with increased oxidative stress in placenta. Maternal obesity was also associated with decreased HIF-1α protein expression, suggesting a potential link between increased inflammation/oxidative stress and decreased angiogenic factors. Together, these findings indicate that maternal obesity leads to a lipotoxic placental environment that is associated with decreased regulators of angiogenesis and increased markers of inflammation and oxidative stress.

A comprehensive analysis of the human placenta transcriptome.

As the conduit for nutrients and growth signals, the placenta is critical to establishing an environment sufficient for fetal growth and development. To better understand the mechanisms regulating placental development and gene expression, we characterized the transcriptome of term placenta from 20 healthy women with uncomplicated pregnancies using RNA-seq. To identify genes that were highly expressed and unique to the placenta we compared placental RNA-seq data to data from 7 other tissues (adipose, breast, hear, kidney, liver, lung, and smooth muscle) and identified several genes novel to placental biology (QSOX1, DLG5, and SEMA7A). Semi-quantitative RT-PCR confirmed the RNA-seq results and immunohistochemistry indicated these proteins were highly expressed in the placental syncytium. Additionally, we mined our RNA-seq data to map the relative expression of key developmental gene families (Fox, Sox, Gata, Tead, and Wnt) within the placenta. We identified FOXO4, GATA3, and WNT7A to be amongst the highest expressed members of these families. Overall, these findings provide a new reference for understanding of placental transcriptome and can aid in the identification of novel pathways regulating placenta physiology that may be dysregulated in placental disease.

Effect of preharvest anti-fungal compounds on Aspergillus steynii and A. carbonarius under fluctuating and extreme environmental conditions.

Ochratoxin A (OTA) has been found in pre-harvest and freshly harvested wheat. Spanish climatic conditions point to Aspergillus species as probably responsible for this OTA. In this study the effectiveness of 5 non-specific antifungal chemicals used on wheat fields (25.9% tebuconazole+60.0% N,N-capramide dimethyl; 12.70% tebuconazole+12.7% prothioconazole+59.5% N,N-amide dimethyldecane; 12.5% epoxiconazole; 12.5% tetraconazole; and 70% thiophanate methyl) and an extract from Equisetum arvense were investigated in vitro on wheat by recording growth (colony size, fungal growth and DNA concentration) and OTA production of two ochratoxigenic isolates of Aspergillus carbonarius and three of A. steynii, simulating current and extreme climatic conditions. Inoculated wheat was incubated under two alternating temperature cycles (20/30°C and 25/35°C) with photoperiod (14/10h lightness/darkness), and two moisture levels (40 and 25%). The Aspergillus species tested seemed to be able to persist in predicted future climatic conditions, in particular, A. steynii, a high OTA producer. Azoles were effective in controlling the growth of A. carbonarius and A. steynii, and this effectiveness may not be compromised by the increase in temperature and decrease of humidity. However, azoles are not useful for the prevention of OTA accumulation, which could be only reduced in A. carbonarius under non-extreme conditions. Although some adjustment will probably be required, further studies should be conducted in the field, since the antifungals used in this study are applied at flowering and not directly on the grain. Moreover, timing of antifungal application may need to be optimized. Finally, Equisetum extract showed promising results as an antifungal, however further work to adjust the applied concentrations is required.

Dietary fat source alters hepatic gene expression profile and determines the type of liver pathology in rats overfed via total enteral nutrition.

To determine if dietary fat composition affects the progression of nonalcoholic fatty liver disease (NAFLD), we overfed male Sprague-Dawley rats low (5%) or high (70%) fat diets with different fat sources: olive oil (OO), corn oil (CO), or echium oil (EO), with total enteral nutrition (TEN) for 21 days. Overfeeding of the 5% CO or 5% EO diets resulted in less steatosis than 5% OO (P < 0.05). Affymetrix array analysis revealed significant differences in hepatic gene expression signatures associated with greater fatty acid synthesis, ChREBP, and SREBP-1c signaling and increased fatty acid transport (P < 0.05) in the 5% OO compared with 5% CO group. The OO groups had macrosteatosis, but no evidence of oxidative stress or necrosis. The 70% CO and 70% EO groups had a mixture of micro- and macrosteatosis or only microsteatosis, respectively; increased oxidative stress; and increased necrotic injury relative to their respective 5% groups (P < 0.05). Oxidative stress and necrosis correlated with increasing peroxidizability of the accumulated triglycerides. Affymetrix array analysis comparing the 70% OO and 70% CO groups revealed increased antioxidant pathways and lower expression of genes linked to inflammation and fibrosis in the 70% OO group. A second study in which 70% OO diet was overfed for 50 days produced no evidence of progression of injury beyond simple steatosis. These data suggest that dietary fat type strongly influences the progression of NAFLD and that a Mediterranean diet high in olive oil may reduce the risk of NAFLD progressing to nonalcoholic steatohepatitis.

Tratamiento quirúrgico de fractura cervical en el Hospital Regional Rancagua: análisis de un periodo de 4 años / Surgical treatment of cervical fracture in the Rancagua Regional Hospital: analysis of a period of 4 years

Introducción: La gravedad, las implicancias neurológicas y el alto costo del tratamiento de la fractura cervical, hacen de esta lesión un tema de suma importancia. Esta injuria provoca graves limitaciones e invalidez a los afectados, en su mayoría en plena actividad laboral, impactando en lo médico, social y económico. Objetivos: Describir los pacientes tratados quirúrgicamente de fractura cervical traumática en el Hospital Regional Rancagua, por el equipo de Neurocirugía dentro de un periodo de 4 años. Materiales y Métodos: Se evaluó - 24 pacientes con antecedente de fractura cervical el mecanismo del trauma, el tiempo transcurrido desde el ingreso hospitalario hasta la cirugía, nivel y compromiso de la lesión medular, tipo de abordaje quirúrgico, complicaciones médicas, quirúrgicas y seguimiento post-operatorio, entre otros. Resultados: 5 mujeres y 19 hombres, edad promedio 39 años (rango de edad 14 a 75 años), principales mecanismos de trauma fueron los accidentes automovilísticos-atropello (58 por ciento). El nivel medular más frecuentemente lesionado fue C4-C5 (25 por ciento). En el 50 por ciento de los pacientes se clasificó de entrada como Frankel A, por lo que un 50 por ciento de todos los pacientes ingresaron tetrapléjicos, y de estos, un 33 por ciento egresó tetraparéticos. Del ingreso a cirugía hubo un tiempo de espera promedio 5 días. Dentro de las complicaciones médicas la causa respiratoria (46 por ciento) fue la más frecuente, necesitando 64 por ciento de estos pacientes ventilación mecánica. Se registró 1 infección de herida operatoria donde el abordaje fue posterior. El Índice de Barthel promedio de 14 pacientes fue de 49 puntos. De 6 pacientes con puntaje Cero, 85 por ciento de estos ingresaron como Frankel A y 50 por ciento egresaron tetrapléjicos. Los pacientes se rehabilitaron en promedio de 2.5 meses. Discusión: Logramos objetivar nuestra realidad. La técnica quirúrgica fue prácticamente uniforme entre los pacientes, sin complicaciones...

Introduction: Gravity, neurological implications and high costs of treating cervical fracture, makes of this injury an issue of most importance. This injury causes severe limitations and disability to those affected, mostly in full working activity, impacting on the medical, social and economic. Objectives: To describe patients surgically treated for traumatic cervical fracture at the Rancagua Regional Hospital, by the team of Neurosurgery Department, within a period of 4 years. Materials and Methods: We evaluated 24 patients with an history of cervical fracture mechanism of trauma, the time from hospital admission to surgery, and commitment level of spinal cord injury, type of surgery, medical and surgical complications and follow-up post -operation, among others. Results: 5 women and 19 men, average age 39 years (ranging age from 14 to 75 years), major trauma mechanisms were motor vehicle accidents (58 percent). The most frequently injured spinal level was C4-C5 (25 percent). 50 percent of patients were classified as Frankel A at the admission, so that 50 percent of all patients admitted were quadriplegics, and of these, 33 percent were discharged as tetraparétic. Admission to the surgery were an average of 5 days. Within cause respiratory medical complications (46 percent) was the most frequent, requiring 64 percent of these patients mechanical ventilation. There was only 1 wound infection where the approach was posterior. The average Barthel Index of 14 patients was 49 points. In 6 patients with zero score, 85 percent were admitted as Frankel A and 50 percent egressed as tetraplejic. Patients were rehabilitated an average of 2.5 months. Conclusions: We were able to objectify our reality. The surgical technique was nearly uniform among patients without severe postoperative surgical complications...

Environmental service payments: evaluating biodiversity conservation trade-offs and cost-efficiency in the Osa Conservation Area, Costa Rica.

The cost-efficiency of payments for environmental services (PES) to private landowners in the Osa Conservation Area, Costa Rica, is evaluated in terms of the trade-off between biodiversity representation and opportunity costs of conservation to agricultural and forestry land-use. Using available GIS data and an 'off-the-shelf' software application called TARGET, we find that the PES allocation criteria applied by authorities in 2002-2003 were more than twice as cost-efficient as criteria applied during 1999-2001. Results show that a policy relevant assessment of the cost-effectiveness of PES relative to other conservation policies can be carried out at regional level using available studies and GIS data. However, there are a number of data and conceptual limitations to using heuristic optimisation algorithms in the analysis of the cost-efficiency of PES. Site specific data on probabilities of land-use change, and a detailed specification of opportunity costs of farm land, labour and capital are required to use algorithms such as TARGET for ranking individual sites based on cost-efficiency. Despite its conceptual soundness for regional conservation analysis, biodiversity complementarity presents a practical challenge as a criterion for PES eligibility at farm level because it varies depending on the set of areas under PES contracts at any one time.

Detección in vivo mediante RAPD de alteraciones en el ADN producidas por benzo(a)pireno / In vivo detection of DNA alterations produced by Benzo(a)pyrene using the RAPD technique

L a técnica de RAPD (Amplificación al Azar de ADN Polimórfico) permite detectar alteraciones inespecíficas en el ADN procedente de células que poseen una dotación genética idéntica, como son las líneas celulares establecidas, mediante la comparación del patrón de bandas de las células expuestas y no expuestas a la acción de genotóxicos.En los últimos años hemos desarrollado una metodología sensible y reproducible utilizando la línea celular RTG-2, derivada de trucha arco iris (Oncorhynchus mykiss). Sin embargo, es preciso comprobar la capacidad predictiva de este ensayo mediante estudios in vivo. La línea celular RTG-2, como se ha evidenciado en trabajos anteriores, presenta una gran similitud genética con la especie de la que procede. Por ello, en este trabajo, se ha llevado a cabo -una -exposición -subletal a benzo(a)pireno mediante inyección intraperitoneal de 69 mg/g de p.c. en alevines de trucha arco iris, valorando la aparición de mutaciones mediante la comparación del patrón de bandas obtenido a partir del ADN de células de sangre periférica, a diferentes tiempos (1 - 3 meses). Debido a que la presencia de bandas polimórficas dificulta el análisis entre los grupos de individuos tratados y no tratados, las comparaciones se realizaron en un mismo individuo antes y después del tratamiento. Los análisis cualitativos y cuantitativos mostraron tanto la aparición de nuevas bandas, como alteraciones en Su intensidad confirmando, de esta manera, los resultados que previamente habíamos obtenido in vitro tras exposiciones a este mismo genotóxico (AU)

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DNA fingerprint comparison of rainbow trout and RTG-2 cell line using random amplified polymorphic DNA.

The detection of genotoxic effects using in vitro cell systems can be extremely useful in risk assessment procedures. However, care should be taken in the extrapolation of in vitro results since, amongst other factors, established cell lines may deviate from the genetic characteristics of their species. In this work, the genetic similarities between the RTG-2 cell line and rainbow trout individuals (Oncorhynchus mykiss) from several fish farms have been studied by the RAPD technique. Results show a significant analogy in the band patterns obtained for both systems, up to 73% of the bands composing the fingerprint of the RTG-2 cell line were found in all the individuals analysed. The inter-population similarity index (Lynch, 1990), considering the RTG-2 cell line as a population, gives a value of 0.931 between both systems. The dendrogram constructed from all the individuals, considering the RTG-2 cell line as just another individual of a single population, showed that the genetic structure of the cell line was not different from those of the other individuals tested. The strong genetic similarity of both systems, together with the previously proven capability of the RAPD technique to detect genetic alterations caused in vitro by genotoxic agents, can be very useful in genetic ecotoxicological studies.

Demonstration of dose dependent cytotoxic activity in cancer cells by specific human chorionic gonadotropin monoclonal antibodies.

BACKGROUND: Previous studies in which monoclonal antibodies (MoAbs) were used against different epitopes of human chorionic gonadotropin (hCG) demonstrated the presence of membrane-associated hCG and its subunits by cancer cells of different types and origins and by human embryonic and fetal cells. To elucidate the mechanism of action of a synthetic vaccine against hCG, experiments were conducted to determine the presence or absence of direct dose dependent cytolytic activity by hCG MoAbs, including those elicited by the vaccine. METHODS: Human adenocarcinoma cells from the uterine cervix (ATCC HeLa CCL 2.0) grown in defined media at 37 degrees C were treated for 2-3 days with different selected doses of each of 12 MoAbs directed against different epitopes of hCG. Three of these MoAbs were against three different epitopes of the synthetic hCGbeta vaccine. RESULTS: There was a direct dose dependent effect by a MoAb directed against the natural hCGbeta carboxy terminal peptide (CTP), by a MoAb directed against hCGalpha, and by one of the three MoAbs produced by the synthetic hCGbeta-CTP, which is the main component of the World Health Organization (WHO) vaccine being tested for fertility control and for cancer treatment or prevention. CONCLUSIONS: For the first time (to the authors' knowledge), these results show the existence of hCG MoAbs that have direct dose related cytotoxicity at 37 degrees C and explain the mechanism of action of the WHO anti-hCG vaccine.

Detection of membrane-associated human chorionic gonadotropin and its subunits on human cultured cancer cells of the nervous system.

Cultured human cancer cells from the nervous system, which included brain cancers, neuroblastomas, medulloblastomas, and retinoblastomas, were analyzed by analytical flow cytometry for the presence of membrane-associated human chorionic gonadotropin (hCG), its subunits, and fragments. Live cells and a panel of monoclonal antibodies directed to epitopes located in three different sites of the hCG molecule were used in the analysis. For in vivo studies, the cultured human glioma cells were grown in athymic (nude) mice, and their tumors were excised and fixed in Bouin's fixative, and embedded in paraffin for subsequent immunocytochemical analysis of tissue sections. Cells from a benign uterine leiomyoma were used as a negative control. Membrane-associated and cytoplasmic hCG, its subunits, and its fragments were present in cells from all the cancers studied. These results correlate with our in vitro and in vivo studies which showed the presence of translatable levels of hCG beta mRNA in all cancers, including the cancers of the nervous system, proving that these malignant neoplasms are no different from carcinomas, sarcomas, malignant lymphomas, or leukemias in that they all have the same biochemical denominator. Our findings give the scientific basis for the use of active and/or passive immunization against hCG for prevention or as a primary adjuvant therapy for these types of cancers.

Metastatic phenotype correlates with high expression of membrane-associated complete beta-human chorionic gonadotropin in vivo.

BACKGROUND: Investigations using living human cancer cells and the nude mouse model were conducted to evaluate the expression of human chorionic gonadotropin (hCG) in various cancers grown in vitro and in vivo. The aim was to determine whether membrane-associated hCG in any of its forms is a characteristic metastatic marker, and at what levels or ratios. METHODS: Human cancer cell lines known to produce tumors that metastasize spontaneously when grown in nude mice (n = 4) were compared with those that do not produce such tumors (n = 4) using analytical (quantitative) flow cytometry. Monoclonal antibodies directed to epitopes of intact hCG (hCG-holo) and its subunits, including beta-human chorionic gonadotropin with its carboxy-terminal peptide (hCG beta-CTP), allowed for the determination of hCG beta-CTP/hCG-holo ratios. RESULTS: No significant difference in hCG beta-CTP/hCG-holo ratios was found between the cultured human cancer cells that do not metastasize spontaneously (ratio = 2.39) and those that do (ratio = 2.13), and no difference was seen in their growth rate in nude mice. However, the cells isolated from tumors that do not metastasize spontaneously showed a decrease in their ratios to values less than 1. They reverted to their original values after reestablishment in culture and subsequent passages. In contrast, the ratios shown by cells isolated from tumors that metastasize spontaneously increased to 3 to 6 times their original values in culture, then reverted to their original values after reestablishment in culture and subsequent passages. CONCLUSIONS: To our knowledge, these data demonstrate the following for the first time: 1) There is a direct in vivo correlation between human cancer cells that metastasize spontaneously in nude mice and the expression of membrane-associated complete hCG beta (hCG beta-CTP); and the correlation identifies this molecule as a characteristic metastatic phenotype marker. 2) The marked ratio variations under different conditions indicate that the metastatic phenotype is an unstable event. 3) Growth and local invasion in vivo correlates with the expression of hCG-holo.

Human chorionic gonadotropin-beta subunit gene expression in cultured human fetal and cancer cells of different types and origins.

BACKGROUND: The authors' previous investigations using living cultured human cancer cells and cells isolated from cancer tissues, analytical flow cytometry, and monoclonal antibodies directed to epitopes located in five different sites of the human chorionic gonadotropin (hCG) molecule, identified the presence of membrane-associated hCG, its subunits and fragments, by cells from all cancers, irrespective of type and origin, indicating that the expression of these sialoglycoproteins is a common phenotypic characteristic of cancer. Although benign neoplasms do not express these compounds, cultured human embryonic and fetal cells also express the same materials. To corroborate these findings, five fetal cell lines and 28 cancer cell lines were randomly selected from those previously studied, to determine the presence of translatable levels of hCG-beta (hCG beta) mRNA. METHODS: All cell lines were grown under identical conditions. Determination of hCG beta mRNA was made by extracting the total RNA from the cells, followed by synthesis of cDNA with RNase H- reverse transcriptase and polymerase chain reaction amplification using specific hCG beta-luteinizing hormone-beta (hLH beta) primers. The presence of amplified hCG beta cDNA was corroborated by hybridization of the product with an hCG beta-specific oligonucleotide and Southern blot analyses of the hybridization products. Gestational choriocarcinoma cells and HeLa adenocarcinoma of cervical cells, known producers of biologically active hCG, were positive control subjects, and human pituitary cells were used as negative control subjects. RESULTS: The results showed single and multiple hCG beta gene activation by the fetal cells and the different types of cancer, indicating that at any given time, there is the possibility of activation of as many as four genes of the six genes of the hCG beta-hLH beta gene cluster, even though alternative gene splicing cannot be ruled out. CONCLUSIONS: In addition to the authors' previous findings, the results of these studies support the concept that cancer is a problem of development and differentiation, and, to the authors' knowledge, prove definitively for the first time that synthesis and expression of hCG, its subunits, and its fragments, is a common biochemical denominator of cancer, providing the scientific basis for studies of its prevention and/or control by active and/or passive immunization against these sialoglycoproteins.

Immunological detection of membrane-associated human luteinizing hormone correlates with gene expression in cultured human cancer and fetal cells.

We have demonstrated the expression of membrane-associated hCG and its subunits and fragments by cells from 78 human cancer cell lines of different types and origins, indicating that such expression is a common phenotypic characteristic of cultured human malignant cells. Because human (h) LH beta has 80% homology with hCG beta and is coded by one of the seven genes in the gene cluster located in chromosome 19, it was important to determine whether hLH and its beta-subunit are also expressed as membrane-associated proteins by cells from human cancer cell lines. Thus, 11 cancer cell lines of different types and origins were adapted to grow in serumless medium, with Nutridoma-HU or SP as serum substitute, and analyzed by flow cytometry using two monoclonal antibodies directed to different conformational epitopes of intact hLH and a monoclonal antibody reacting with an epitope of hLH beta-free. The cells were also analyzed simultaneously for the expression of hCG and its subunits and fragments. Determination of translatable levels of hLH beta and hCG beta messenger RNAs (mRNAs) was performed in cells from some of the cancer cell lines, including the JEG-3 choriocarcinoma cell line, and in cells from a human fetal lung cell line. The analytical flow cytometry studies showed that in addition to the expression of membrane-associated hCG in all of its forms, expression of membrane-associated intact (holo) hLH and its free beta-subunit occurred in every case. These findings were corroborated by the presence of translatable levels of hLH beta and hCG beta mRNAs in all of the cancer cell lines analyzed, indicating that the expression of these membrane-associated glycoproteins is a phenotypic characteristic of human cancer cells and that the activation of the hCG beta-hLH beta gene cluster is nonselective. The presence of translatable levels of hCG beta-hLH beta mRNAs in the cultured human fetal lung cells punctuates once more the in vivo and in vitro biochemical similarities between fetal and cancer cells.

Development, characterization, and application of monoclonal antibodies to the native and synthetic beta COOH-terminal portion of human chorionic gonadotropin (hCG) that distinguish between the native and desialylated forms of hCG.

Although the pregnancy hormone hCG has been extensively mapped immunochemically, few monoclonal antibodies have been produced to the unique COOH-terminal region of its beta-subunit (beta CTP). We now report the development and characterization of five such monoclonal antibodies. Three of these antibodies were developed to the synthetic peptide analog of the hCG beta-(109-145) region coupled to diphtheria toxoid, and two antibodies to a conjugate of bovine thyroglobulin and the peptide hCG beta-(115-145) prepared from hCG with its carbohydrate moieties intact. The monoclonal antibodies raised against the synthetic peptide bound hCG, desialylated hCG, and synthetic peptide to a similar extent, whereas antibodies generated to the natural hCG peptide did not bind to the synthetic peptide analog of the COOH-terminal peptide (beta CTP) region or to desialyated hCG. These new monoclonal antibodies could distinguish between native and desialyated hCG in liquid phase immunoassays as well as by Western blots. They are highly specific reagents for such Western blotting and were used for studies of a crude human pituitary gonadotropin preparation to demonstrate that it contained intact hCG beta without the internal peptide bond cleavages found in the subunit present in human blood and urine. Competition experiments using combinations of monoclonal antibodies and rabbit anti-beta CTP antiserum demonstrated that two epitopes exist within the beta-(115-145) region of hCG, one of which depends on the presence of carbohydrate. In summary, the new monoclonal hCG beta CTP antibodies reported here can 1) discriminate between native and desialylated hCG, 2) identify hCG and nicked hCG on Western blots, 3) provide an immunoaffinity purification tool for hCG, and 4) bind to two distinct epitopes on the beta CTP.

Flow cytometry method for the analysis of membrane-associated human chorionic gonadotropin, its subunits, and fragments on human cancer cells.

A quantitative flow cytometry method for the analysis of membrane-associated human chorionic gonadotropin (hCG), its subunits, and fragments on human cancer cells was developed using a double-antibody reaction; a flow cytometry with a 2-W argon laser, standard settings, and filters for fluorescein isothiocyanate use; commercially available software; and the ectopic hCG producer CCL 2 HeLa cells from the American Type Culture Collection (ATCC) as a cell control to standardize the reagents and for overall quality control. Twenty-two monoclonal antibodies (MoAb) and immunoglobulin G fractions from three rabbit polyclonal antisera were tested for effects of antibody concentration (titration), reproducibility at different levels of epitope expression, and variability of epitope expression to select appropriate primary antibodies. Based on the results of the various tests, three polyclonal immunoglobulin G antibodies and a panel of nine MoAb directed to epitopes located in five different regions on the hCG molecule were selected as first antibodies. Their specificity was determined by using two unrelated MoAb of the same isotype at the same concentration to replace the primary MoAb and by a competition experiment. The unrelated MoAb also were used for the selection of the appropriate control fluorescence profile needed for the software. The unique characteristics of this method were: the use of living cells, standardized reagents, internal and external quality control, and the highest sensitivity, which could detect as few as 10(3) molecules of fluorochrome per cell. Serial analyses of the ATCC CCL 2 HeLa cells and two of its variants and of the eutopic hCG producer JEG-3 choriocarcinoma cells revealed the expression of membrane-associated epitopes of intact hCG, its subunits, and fragments by a high percentage of the cells, indicating that the expression of these sialoglycoproteins by these two different types of cancer cells is a common phenotypic characteristic.

Expression of membrane-associated human chorionic gonadotropin, its subunits, and fragments by cultured human cancer cells.

The expression of human chorionic gonadotropin (hCG), its subunits, and fragments on the cell membrane of cultured human cancer cells was investigated using a flow cytometric method. This method uses living cells; a double-antibody reaction; a flow cytometer with an argon laser, standard settings, and filters for fluorescein isothiocyanate; commercially available software; the American Type Culture Collection (ATCC) CCL 2 HeLa cell line as cell control and overall quality control; polyclonal rabbit antisera raised against the hCG dimer, its alpha subunit (hCG alpha), and its beta subunit (hCG beta); and a panel of monoclonal antibodies (MoAb) recognizing different epitopes on the intact hCG molecule, its subunits, and fragments. The purified immunoglobulin G fractions from the polyclonal antisera were used to estimate the total expression of the membrane-associated glycoproteins; the MoAb were used to detect the expression of epitopes of the hCG dimer, its subunits, and fragments. The results of the analyses done on cells from 74 established cancer cell lines of different types and origins (including 52 carcinomas, 10 sarcomas, 4 leukemias, 6 lymphomas, and 2 retinoblastomas) showed variable degrees of reactivity in a great percentage of cells in all cell lines studied with MoAb directed against different conformational epitopes of intact hCG (hCG-holo), hCG beta, hCG beta-free, the carboxy terminal peptide (CTP) of hCG beta, and an epitope of hCG alpha. The expression of the membrane-associated epitopes of hCG and its subunits was found to be a phenotypic marker characteristic of all evaluated cultured human cancer cell lines, irrespective of their type or origin. There were, however, quantitative and qualitative differences in the expression of the different epitopes. Thus, hCG beta, free and as part of hCG-holo, recognized by the MoAb against hCG beta-CTP, was expressed by a high percentage of cells of most cell lines. There was great variability in the expression of hCG-holo, recognized by MoAb B109. For this reason some groups of cancers expressed larger amounts of incompetent hCG alpha and/or hCG beta than others. Cell lines derived from adenocarcinomas of the lung were the only exception to this general finding; the expression of small amounts of hCG-holo was caused by a low degree of hCG alpha synthesis.

Human choriogonadotropin-like material in bacteria of different species: electron microscopy and immunocytochemical studies with monoclonal and polyclonal antibodies.

Immunocytochemical studies using antisera to whole human choriogonadotropin (hCG), to its alpha- and beta-subunits and to the COOH-terminal peptide of hCG beta, and two monoclonal antibodies to hCG beta, demonstrated expression of hCG-like material, its individual subunits and/or fragments in nine bacterial strains. Seven of these were isolated from patients with cancer and were definitely identified as Streptococcus faecalis (three strains), Staphylococcus haemolyticus (two strains) and Staphylococcus epidermidis and Escherichia coli (single strains). The other two strains were cell-wall-deficient (CWD) variants, one identified as Streptococcus bovis, isolated from the blood of a patient with a fever of unknown origin and a possible brain abscess. The other was a Gram-negative diphtheroid isolated from the urine of a pregnant woman, which during the period of study reverted to a Gram-positive Corynebacterium identified as a 'C. ulcerans' strain and expressed the hCG-like factor only during its phase as Gram-negative diphtheroid. Electron microscopy of these nine strains (including negative controls of strains of the same species subjected to the same immunocytochemical analyses and under identical cultural conditions) revealed morphological alterations in the bacterial cell walls and cytoplasmic material and/or bizarre forms of reproduction in six of the nine strains expressing hCG-like material including the two CWD variants. Collectively, these results provided evidence that (1) hCG-producing bacteria isolated from patients with overt cancer are not a new and unique species as claimed by others, and (2) there is a close resemblance between the bacterial protein and the human trophoblastic hormone, based on immunochemical recognition of different parts of the hCG molecule.(ABSTRACT TRUNCATED AT 250 WORDS)