Trophoblast Glycoprotein (TPGB/5T4) in Human Placenta: Expression, Regulation, and Presence in Extracellular Microvesicles and Exosomes.

BACKGROUND: Many parallels exist between growth and development of the placenta and that of cancer. One parallel is shared expression of antigens that may have functional importance and may be recognized by the immune system. Here, we characterize expression and regulation of one such antigen, Trophoblast glycoprotein (TPGB; also called 5T4), in the placenta across gestation, in placentas of preeclamptic (PE) pregnancies, and in purified microvesicles and exosomes. METHODS: Trophoblast glycoprotein expression was analyzed by real-time reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunohistochemistry. Regulation of 5T4 in cytotrophoblast cells was examined under either differentiating conditions of epidermal growth factor or under varying oxygen conditions. Microvesicles and exosomes were purified from supernatant of cultured and perfused placentas. RESULTS: Trophoblast glycoprotein expression was prominent at the microvillus surface of syncytiotrophoblast and on the extravillous trophoblast cells, with minimal expression in undifferentiated cytotrophoblasts and normal tissues. Trophoblast glycoprotein expression was elevated in malignant tumors. In cytotrophoblasts, 5T4 was induced by in vitro differentiation, and its messenger RNA (mRNA) was increased under conditions of low oxygen. PE placentas expressed higher 5T4 mRNA than matched control placentas. Trophoblast glycoprotein was prominent within shed placental microvesicles and exosomes. CONCLUSION: Given the potential functional and known immunological importance of 5T4 in cancer, these studies reveal a class of proteins that may influence placental development and/or sensitize the maternal immune system. In extravillous trophoblasts, 5T4 may function in epithelial-to-mesenchymal transition during placentation. The role of syncytiotrophoblast 5T4 is unknown, but its abundance in shed syncytial vesicles may signify route of sensitization of the maternal immune system.

A phase II trial of huperzine A in mild to moderate Alzheimer disease.

OBJECTIVE: Huperzine A is a natural cholinesterase inhibitor derived from the Chinese herb Huperzia serrata that may compare favorably in symptomatic efficacy to cholinesterase inhibitors currently in use for Alzheimer disease (AD). METHODS: We assessed the safety, tolerability, and efficacy of huperzine A in mild to moderate AD in a multicenter trial in which 210 individuals were randomized to receive placebo (n = 70) or huperzine A (200 µg BID [n = 70] or 400 µg BID [n = 70]), for at least 16 weeks, with 177 subjects completing the treatment phase. The primary analysis assessed the cognitive effects of huperzine A 200 µg BID (change in Alzheimer's Disease Assessment Scale-cognitive subscale [ADAS-Cog] at week 16 at 200 µg BID compared to placebo). Secondary analyses assessed the effect of huperzine A 400 µg BID, as well as effect on other outcomes including Mini-Mental State Examination, Alzheimer's Disease Cooperative Study-Clinical Global Impression of Change scale, Alzheimer's Disease Cooperative Study Activities of Daily Living scale, and Neuropsychiatric Inventory (NPI). RESULTS: Huperzine A 200 µg BID did not influence change in ADAS-Cog at 16 weeks. In secondary analyses, huperzine A 400 µg BID showed a 2.27-point improvement in ADAS-Cog at 11 weeks vs 0.29-point decline in the placebo group (p = 0.001), and a 1.92-point improvement vs 0.34-point improvement in the placebo arm (p = 0.07) at week 16. Changes in clinical global impression of change, NPI, and activities of daily living were not significant at either dose. CONCLUSION: The primary efficacy analysis did not show cognitive benefit with huperzine A 200 µg BID. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that huperzine A 200 µg BID has no demonstrable cognitive effect in patients with mild to moderate AD.

Motivations and perceived influences on rural and urban general practitioners when prescribing conventional non-steroidal anti-inflammatory drugs or COX-2 inhibitors.

BACKGROUND AND OBJECTIVE: Prescribers in rural and remote locations perceive that there are different influences on their prescribing compared with those experienced by urban prescribers. The aim of this study was to compare the motivations and perceived influences on general practitioners (GPs) when prescribing COX-2 inhibitors rather than conventional non-steroidal anti-inflammatory drugs (NSAIDs) between rural and urban-based GPs in Queensland, Australia. METHODS: A questionnaire was administered to two geographically distinct groups of GPs, one urban (n=67) and one rural (n=67), investigating the reasons that the GP would prescribe a COX-2 inhibitor rather than a conventional NSAID or vice versa and also focusing on patients requesting a prescription for a COX-2 inhibitor. RESULTS AND DISCUSSION: A 51% response rate (n=68) was achieved. The difference between the rural and the urban GPs was that the urban GPs were more likely to perceive that they were influenced to prescribe COX-2 inhibitors by their patients' knowledge of these new (at the time) drugs. GPs in both the rural and urban areas perceived the COX-2 selective inhibitors to be safer than conventional NSAIDs, and that there was little difference in terms of efficacy between the two drug classes. However, GPs from both of the study areas stated that conventional NSAIDs were preferred over COX-2 selective inhibitors, primarily due to their expense, if their patients were not at risk for developing a GI bleed. CONCLUSION: The motivations and perceived influences to prescribe a COX-2 inhibitor in rural and in urban areas of Queensland, Australia were very similar. Almost all surveyed GPs in rural and urban areas had patients request a prescription, or enquire about the COX-2 inhibitors. Urban GPs were more likely to feel pressured to prescribe a COX-2 inhibitor than their rural counterparts, agreeing with other research which found that patient pressure to prescribe appears to be greater in urban general practice.

Uptake of new drugs in rural and urban areas of Queensland, Australia: the example of COX-2 inhibitors.

OBJECTIVE: This study compared the changes over time in the volume of prescriptions of COX-2 selective inhibitors between rural and urban Queensland to reveal any difference in the uptake of the prescribing of these new drugs between two geographically distinct areas. METHODS: This study used data from an administrative claims database. Dispensing data were obtained for celecoxib and rofecoxib in two areas, one rural and one urban, defined by postcodes. The numbers of consumers in these areas were similar and they were served by similar numbers of general practitioners. The number of defined daily doses (DDDs) of celecoxib and rofecoxib dispensed at specific times was calculated. RESULTS: Statistical analysis revealed no significant difference between the total numbers of DDDs of COX-2-selective non-steroidal anti-inflammatory drugs dispensed in the rural and urban groups over the period August 2000 to December 2002 (P=0.81). The rate of uptake of usage was also clearly similar between the urban and the rural groups. Total usage peaked in August 2000 in both groups (urban 39 DDD/1,000 people per day; rural 37 DDD/1,000 people per day), coinciding with the pharmaceutical benefits scheme (subsidized) listing of celecoxib. The number of DDDs declined dramatically in the following month, and then peaked again in May 2002 (urban 34, rural 36). The number of DDDs then steadily decreased in both areas after October 2002. CONCLUSION: The results suggest that the marketing of the new COX-2 inhibitors and the patients' anticipation of a safe and effective treatment have overcome the geographical boundaries of Queensland. Both areas had very high rates of uptake of the prescribing of these new drugs.

Thymidine kinase, thymidylate synthase, and dihydropyrimidine dehydrogenase profiles of cell lines of the National Cancer Institute's Anticancer Drug Screen.

PURPOSE: To determine the expression of three targets of 5-fluorouracil (5-FU) and 5-fluoro-2'-deoxyuridine (FdUrd) in human tumor cell lines and to compare these with the 50% growth inhibition concentrations (GI(50)) from the National Cancer Institute database. EXPERIMENTAL DESIGN: Thymidine kinase (TK) activity was assessed by conversion of [(3)H]thymidine to [(3)H]TMP. Thymidylate synthase (TS) protein expression was determined by Western analysis. TS and dihydropyrimidine dehydrogenase (DPD) mRNA expression were measured by quantitative reverse transcription-PCR. RESULTS: The median (range) for the targets were as follows: 5-FU GI(50), 20.8 microM (0.8-536); FdUrd GI(50), 0.75 microM (0.25-237); TK, 0.93 nmol/min/mg (0.16-5.7); in arbitrary units: TS protein, 0.41 (0.05-2.95); TS mRNA, 1.05 (0.12-6.41); and DPD mRNA, 1.09 (0.00-24.4). A moderately strong correlation was noted between 5-FU and FdUrd GI(50)s (r = 0.60), whereas a weak-moderate correlation was seen between TS mRNA and protein expression (r = 0.45). Neither TS expression nor TK activity correlated with 5-FU or FdUrd GI(50)s, whereas lines with lower DPD expression tended to be more sensitive to 5-FU. Cell lines with faster doubling times and wild-type p53 were significantly more sensitive to 5-FU and FDURD: CONCLUSIONS: The lack of correlation may in part be attributable to the influence of downstream factors such as p53, the observation that the more sensitive cell lines with faster doubling times also had higher TS levels, and the standard procedure of the screen that uses a relatively short (48-h) drug exposure.

A pilot study of interferon alpha-2a, fluorouracil, and leucovorin given with granulocyte-macrophage colony stimulating factor in advanced gastrointestinal adenocarcinoma.

We reported previously that the addition of recombinant Escherichia coli human granulocyte-macrophage colony stimulating factor (GM-CSF) to a 5-fluorouracil (5-FU) and leucovorin (LV) regimen seemed to ameliorate diarrhea and permit increased 5-FU dose intensity (J. L. Grem et al., J. Clin. Oncol., 12: 560-568, 1994). We then tested the effect of GM-CSF given with a more toxic regimen of 5-FU/LV/IFN-alpha (IFN alpha-2a). Thirty-one patients with a good performance status and no prior chemotherapy for systemic disease received IFN alpha(-2a (5 MU/m2 s.c., days 1-7), 5-FU (370 mg/m2 i.v., days 2-6), LV (500 mg/m2 i.v., days 2-6), and GM-CSF (Saccharomyces cerevisiae 250 microg/m2 s.c., days 7-18) every 3 weeks. Toxicities and 5-FU dose intensity were compared with that observed in our prior Phase II trial with 5-FU/LV/IFN alpha-2a (J. L. Grem et al., J. Clin. Oncol., 11: 1737-1745, 1993). In comparison with the prior Phase II study, the WBC and granulocyte nadirs in the present trial were significantly higher. When trends in toxicity grades for all cycles were compared, stratifying for 5-FU dose, the incidence and severity of mucositis, skin rash, WBC toxicity, and granulocyte toxicity were significantly lower in the present trial, whereas nausea/vomiting and fatigue were significantly worse. The delivered 5-FU dose intensity for all cycles of therapy appeared to be significantly higher in the present trial. Six of 28 evaluable patients had a partial response (21.4%), and 13 (46%) had stable disease for > or =12 weeks. Despite treatment-related toxicity, patient quality of life did not worsen during the study. No correlation was observed between thymidylate synthase content in primary tumor specimens and response, time to treatment failure, or survival. The addition of GM-CSF appeared to decrease the severity of leukopenia, granulocytopenia, mucositis, and skin rash when compared with our prior experience with this regimen of 5-FU/LV/IFN alpha-2a, at the cost of greater nausea/vomiting and fatigue. The potential impact of increased 5-FU dose intensity on clinical response, however, remains to be determined.

Epitope mapping of a series of human thymidylate synthase monoclonal antibodies.

We have reported previously the development and application of several monoclonal antibodies to thymidylate synthase (TS). In this study, we used a series of overlapping 17-mer peptides that spanned the entire TS protein to map the epitope recognized by three TS monoclonal antibodies (TS 106, TS 109, and TS 110). Using an ELISA, we identified two peptides (R126-F142 and L131-R147) that bound all three antibodies, which suggests that each antibody recognized a similar epitope on TS. A second set of peptides, representing sequential single-residue truncations from either the amino terminus or the carboxyl terminus starting with a G129-E145 17-mer, was synthesized. A 10-amino acid sequence P133-F142 (PVYGFQWRHF) was identified as the binding epitope for all three antibodies. Further investigation via substitution mutational analysis of each residue within this epitope revealed that residues F137, W139, R140, H141, and F142 were critical for maximal binding of TS 106 and TS 110. TS 109 showed a similar pattern except in regard to R140, with which there was no apparent loss of binding. In addition to the utility of the three antibodies in detecting and measuring TS levels, identification of the binding locus permits the potential application of these antibodies in the investigation of TS enzymatic and regulatory function.

Thymidylate synthase expression and response to neoadjuvant chemotherapy in patients with advanced head and neck cancer.

BACKGROUND: Thymidylate synthase (TS), an essential enzyme in DNA synthesis, is a target for the fluoropyrimidines, an important group of antineoplastic agents used widely in the treatment of head and neck cancer. PURPOSE: We evaluated relationships between the level and/or pattern of tumor TS expression and response to fluorouracil (5-FU)-based neoadjuvant chemotherapy in patients with advanced head and neck cancer. METHODS: Tumor specimens from 86 patients were available for this retrospective analysis. The patients were enrolled in four consecutive phase II studies that tested combinations of 5-FU, leucovorin, and cisplatin with or without added methotrexate plus piritrexim or interferon alfa-2b (IFN alpha-2b). TS protein expression in the tumors was assessed by use of the TS 106 monoclonal antibody and standard immunohistochemical staining techniques. TS immunostaining was classified according to its level of intensity (TS 0-1 = low, TS 2 = intermediate, and TS 3 = high) and according to its extent (focal pattern = less than 25% of tumor cells positive; diffuse pattern = greater than or equal to 25% of tumor cells positive). Data from 79 patients were available for an analysis of tumor TS expression and patient/tumor characteristics; 70 patients were assessable for their response to neoadjuvant chemotherapy. RESULTS: There was a statistically significant association between the level of tumor TS expression and the degree of tumor differentiation; a higher proportion of patients whose tumors exhibited TS 0-1 immunostaining had undifferentiated or poorly differentiated tumors than patients whose tumors exhibited TS 2 or TS 3 immunostaining (P = .04, Jonckheere-Terpstra trend test). Among the 70 patients who were assessable for response to neoadjuvant chemotherapy, TS 3 tumor immunostaining was associated with a lower rate of complete response (i.e., complete disappearance of clinically detectable disease for a minimum of 4 weeks from time of initial determination) than was TS 2 or TS 0-1 immunostaining, but this association was not statistically significant (P = .09, exact trend test); among the 39 patients who were treated with regimens that included 5-FU, leucovorin, cisplatin, and IFN alpha-2b, this inverse association between TS immunostaining intensity and response was statistically significant (P = .02, exact trend test). Tumor TS immunostaining intensity and overall survival were not found to be associated. Patients with tumors exhibiting a focal pattern of TS immunostaining have experienced significantly longer survival than patients with tumors exhibiting a diffuse pattern; for the 53 patients with diffuse tumor TS immunostaining, the median survival was 24.7 months, whereas the median survival has not yet been reached for the 22 patients with focal tumor TS immunostaining (P = .04, two-tailed logrank test). However, the survival advantage for the focal versus diffuse TS immunostaining pattern was limited to patients whose tumors also exhibited a TS 3 level of immunostaining intensity. CONCLUSIONS AND IMPLICATIONS: Characterization of tumor TS expression may be of value in identifying patients with advanced head and neck cancer who would benefit from fluoropyrimidine-based neoadjuvant chemotherapy.

Recent advances in the biology of colorectal cancer.

The identification of the precise molecular defects responsible for the common forms of inherited colorectal cancer has significantly advanced our understanding of both inherited and sporadic disease. These advances coupled with a rapid accumulation of information on the molecular genotype and biological phenotype of colorectal cancer have identified potential markers that may prove to be not only of prognostic value but also important as screening tools and therapeutic targets. These molecular and biological features include replication errors, mutations of oncogenes and tumor suppressor genes and expression of tumor specific antigens and cytokeratins. This review highlights important recent advances that further our understanding of the biology and genetics of colorectal cancer.

Reduction of Brucella species and Francisella tularensis cross-reacting agglutinins by dithiothreitol.

Cross-reaction agglutinin titers to Brucella abortus antigen were found in 42 of 128 tularemia serum specimens, and cross-reaction titers to Francisella tularensis antigen were found in 8 of 34 brucellosis serum specimens. The cross-reaction titers were reduced to 10 or less by dithiothreitol, suggesting that the titers are due to immunoglobulin M antibody.

Effect of centrifugation and microagglutination techniques on Brucella agglutinin titers.

The microagglutination technique without centrifugation was more effective than centrifugation of the standard tube test for increasing Brucella agglutinin titers of specimens with a titer greater than or equal to 160 but was less effective than centrifugation of the standard tube test for specimens with a titer less than 160.

Determination of brucella immunoglobulin G agglutinating antibody titer with dithiothreitol.

The routine brucella agglutination test measures both immunoglobulin M (IgM) and IgG brucella antibody titers; however, only an elevated IgG titer is significant for differentiating active from inactive disease in patients with symptoms lasting 3 or more weeks. The IgG antibody titer can be determined by treating the serum wih 2-mercaptoethanol to inactivate the IgM brucella antibodies while leaving the IgG brucella antibodies intact. Dithiothreitol, which also inactivates IgM, was compared with 2-mercaptoethanol for the determination of IgG brucella agglutination titers. The dithiothreitol and 2-mercaptoethanol test results agreed within +/- 1 dilution step in 103 of 105 serum specimens tested, for a 98.1% rate of agreement. The results indicate that dithiothreitol can be used in place of 2-mercaptoethanol for determining IgG brucella agglutination titers. Dithiothreitol does not have the offensive odor or the irritant properties of 2-mercaptoethanol.