Development of an antigen ELISA using monoclonal antibodies against recombinant VSG for the detection of active infections of Trypanosoma evansi in animals.

Trypanosoma evansi, a haemo-flagellated protozoan parasite causes chronic wasting disease in a wide range of animals. For its diagnosis, blood smear examination is useful in clinical cases for direct identification of the parasite but in latent infection the carrier animals are difficult to screen out by conventional blood smear test. Harboring low level of parasites and showing no symptom, the carrier animals for surra can act as a source of infection. The level of parasitaemia fluctuates, especially during latent infection; moreover the antibodies which are not found early in the infection may persist even after recovery or chemotherapy. In the present study a double antibody sandwich ELISA exploring, monoclonal antibodies and hyperimmune serum, raised against recombinant variable surface glycoprotein has been developed to detect circulating trypanosome antigens. The developed antigen detection ELISA (Ag-ELISA) was evaluated using 652 blood samples collected from cattle, buffalo, equine and camel. The statistical analysis of the data showed diagnostic sensitivity and specificity at 97.4% and 96.4% respectively, with a positive-negative cut-off OD value >0.28. Furthermore, the detection limit of the assay was found to 7.15 trypanosomes per mL. The present finding revealed that the developed assay can be exploited as a potential diagnostic test in the detection of circulating trypanosome antigens and also can be used as a population screening test for multiple animal species for detection of active infection for further treatment and control of the disease.

Antigen detection ELISA: A sensitive and reliable tool for the detection of active infection of surra.

Trypanosomosis, an endemic disease in Asia, America (central and south) and Africa causes havoc economical loss in livestock industry. The carrier animals which are symptomless and harbours low level of parasites can act as a source of infection. The level of parasitaemia fluctuates, especially during the latent infection; moreover the antibodies which are not found early in the infection may persit even after recovery or chemotherapy. The parasitological and/or serological tests always can not detect current infection or carrier animals. Hence, in the present study double antibody sandwitch antigen detection ELISA (Ag-ELISA) is developed to detect circulating trypanosomes. The new assay has been evaluated using 554 field samples comprising bovine and camel. The diagnostic sensitivity and specificity of the new assay was found to be 97.4% and 99.0% respectively, with a Cohen's kappa value of 0.96. The developed assay could detect 11.5 Trypanosoma evansi per mL from the experimentally infected blood, buffy coat and purified T. evansi samples. The findings revealed that the developed assay can be exploited as a potential diagnostic tool in the detection of active trypanosomal infection.

Development and evaluation of recombinant antigen and monoclonal antibody based competition ELISA for the sero- surveillance of surra in animals.

Trypanosoma evansi, a haemoflagellated protozoan parasite, is responsible for chronic as well as the acute debilitating disease called surra in a wide range of herbivores and carnivores including domestic and wild animals. Since the parasite is having wide host range, there is a need for diagnostic test which can detect the T. evansi specific antibody in different species of animals for generating sero-surveillance data. In the present study we developed and evaluated competitive enzyme immunoassay using monoclonal antibodies (MAbs) raised against recombinant variable surface glycoprotein (rVSG) of T. evansi. The immunoreactivity of the developed MAbs (IgG3-subtype) was evaluated by immunoblot as well as ELISA and subsequently used in the development and standardization of competitive ELISA (C-ELISA). Further, the serological data generated from the C-ELISA using reference samples constituting true positive or surely infected (35), true negative (45), sero-positive (225) and sero-negative (215) samples and was analyzed statistically. The true positivity/negativity was determined by thin blood smear examination and diagnostic PCR assay, While, seropositivity/seronegativity of the reference samples was determined through standard reference tests. The data showed the diagnostic sensitivity of 92.6% and specificity of 96.4% with Cohen's kappa value of 0.88. In order to determine the utility of C-ELISA in detecting T. evansi antibodies in different species of animals, the assay was further evaluated with 1361 field sera sample comprising bovine, horse, donkey and camel. Since the C-ELISA described herein has showed high sensitivity and specificity, this single test can be explored in the sero-surveillance of T. evansi in a wide range of animals.

An inhibition enzyme immuno assay exploring recombinant invariant surface glycoprotein and monoclonal antibodies for surveillance of surra in animals.

The present study is aimed at the development of inhibition ELISA (I-ELISA) exploring monoclonal antibodies (MAbs) and recombinant invariant surface glycoprotein. The extracellular domain (ED) of invariant surface glycoprotein (ISG-75) from Trypanosoma evasni has been heterologously expressed in Pichia pastoris (X-33). The recombinant ISG-75 (rISG-75ED) was characterized by immunoblot and ELISA, followed by the production of MAbs against rISG-75ED. The MAbs were characterized by immunoblot and then explored in the development of I-ELISA for the detection of surra. The diagnostic potential of the developed test has been evaluated using 1192 field sera sample including cattle, buffalo, donkey, horse and camel. The statistical analysis of the data showed optimum combination of diagnostic sensitivity and specificity at 98.8% and 99.2% respectively, with cut-off percentage inhibition (PI) value of >45. The Cohen's kappa coefficient of agreement was found to be 0.98. Hence, the diagnostic test developed in the present study can be exploited as a potential and reliable tool in the serodiagnosis and surveillance of surra in animals.

Cytokine gene expression and pathology in mice experimentally infected with different isolates of Trypanosoma evansi.

Aim of the present study was to assess the cytokine gene expression in liver, kidney and spleen and histopathological changes in mice infected with buffalo and dog isolates of Trypanosoma evansi. Forty-four Swiss albino mice was divided into eleven groups of four mice each and injected subcutaneously with 1 × 105 trypanosomes of buffalo and dog isolate to twenty mice each, four mice served as control. Mice were examined for clinical signs, blood smear for trypanosome counts. Blood for PCR, liver, kidney, spleen, heart, lung, testis and abdominal muscle for histopathology and liver, kidney, spleen for cytokine gene expression studies, were collected. Mice showed dullness, lethargy, hunched back, sluggish movements on D4 and D5 in buffalo and dog isolate, respectively. Parasite count in blood varied between the two isolates of T. evansi. By PCR, trypanosome DNA was detected on D1 and D2 for buffalo and dog isolate, respectively. Splenomegaly was observed in mice infected with buffalo isolate but not with dog isolate. Histopathological changes were observed in liver, kidney, spleen and heart of mice but no changes in testis and abdominal muscles. Blood vessels of liver, heart, lung showed presence of trypanosomes in mice infected with buffalo isolate but not for dog isolate. Cytokine gene expression of IL-2, IL-4, IL-6, IL-12, TNF-α and IFN-γ increased in liver, kidney and spleen in both these isolates. However, the buffalo isolate exhibited pronounced increase in cytokine gene expression when compare to dog isolate of T. evansi. Anti-inflammatory cytokine gene IL-10 showed 50-60 and 10-20 folds increment in buffalo and dog isolates, respectively. This is the first report of IL-4, IL-6, IL-10 and IL-12 cytokine changes in mice infected with T. evansi. A variation in pathogenicity between buffalo and dog isolates was recorded indicating buffalo isolate of T. evansi remained more pathogenic in mice.

Flagellar antigen based CI-ELISA for sero-surveillance of surra.

Trypanosoma evansi causes a disease known as 'surra' in wide range of domesticated and wild animals including cattle, buffaloes, horses, camels etc. The disease is transmitted through the bites of haematophagous tabanid flies and is characterized by undulating fever, chronic progressive weakness, and hypoglycemia leading to low productivity in animals. In the present study, monoclonal antibodies (MAbs) have been produced (IgG3 sub-type) against purified flagellar (FLA) protein of T. evansi and its immunoreactivity was evaluated by serological tests. MAb and purified protein were then exploited in the development of CI-ELISA and the diagnostic potentiality of the new ELISA test has been evaluated using 1230 sera samples from field animals including cattle, buffaloes, camels, horses and donkeys. The statistical analysis of the data showed optimum combination of sensitivity and specificity at 95.8 and 94.4, respectively. The positive-negative cut off percentage inhibition (PI) value was found to be >55, with a Cohen's Kappa value of 0.83. The study showed that the new assay has potential for application in sero-diagnosis as well as sero-surveillance of surra.

Sero-diagnosis of surra exploiting recombinant VSG antigen based ELISA for surveillance.

Trypanosoma evansi, a haemoflagellate, causes "surra" an important chronic wasting disease of a wide range of wild and domestic herbivorous and carnivorous animals including cattle, buffaloes, camels, horses, etc. The untreated recovered animal can act as a carrier without exhibiting the disease symptoms and can be a source of infection to healthy animals. The diagnosis and subsequent treatment of the carrier animals is helpful to curb the disease. As the parasitaemia in carrier animals is very scanty, the conventional blood smear examination, which is widely practiced in the field, cannot detect such condition. For this purpose improved diagnostics are very much useful for mass sero-screening test such as ELISA. In the present study, the VSG of T. evansi was expressed in prokaryotic system (E. coli) and thereafter its immunoreactivity has been evaluated in immuno blot and enzyme immuno assay. The expressed protein showed 95.6% sensitivity, 98.0% specificity and 0.93 Cohen's kappa value, when compared with standard antigens. The developed antigen has also been validated with field serum samples from bovine, camel and horse collected from different states of India. The data showed that the developed recombinant antigen can be a diagnostic tool to detect carrier animals as well as control of the disease.

Inhibition of vascular endothelial growth factor by octreotide in colorectal cancer patients.

Vascular endothelial growth factor (VEGF) seems to be essential for angiogenesis and for the growth of colorectal cancer; thus its inhibition can arrest tumor growth and decrease metastatic potential. Octreotide has been shown to inhibit growth of colorectal tumors in vitro and in vivo. Part of the antiproliferative activity of octreotide could be related to its antiangiogenic properties. Effects of octreotide on VEGF expression were evaluated in 35 patients with operable colorectal cancer receiving octreotide for 2 weeks before surgery. Tissue VEGF expression and serum VEGF concentrations were determined before and after treatment with octreotide. There was a statistically significant reduction in the tissue VEGF expression both considering the percentage of VEGF positive cells (P = 0.006) and the intensity of VEGF staining (P = 0.003). A similar significant reduction was observed in serum values of VEGF (P = 0.03). The present study indicates that octreotide inhibits expression of VEGF in colorectal cancer patients, and, furthermore, that serum VEGF expression correlates with tissue VEGF, representing a safe method to monitor the activity of antiangiogenic agents.

Effects of calcium and vitamin supplementation on colon cell proliferation in colorectal cancer.

Calcium and antioxidant vitamins, such as A, C, and E, have been shown to reduce colorectal epithelial proliferation and thereby to act as possible chemoprotective agents in colorectal cancer. We investigated the effects of an intervention with calcium and vitamins on cell proliferation in the colonic mucosa of patients operated on for colorectal cancer. Patients with resected colorectal cancer Dukes' stage B-C were randomized to receive daily 30,000 IU of axerophthol palmitate (vitamin A) plus 1 g ascorbic acid (vitamin C) plus 70 mg of dl-alpha-tocopherol acetate (vitamin E) and 2 g natural calcium daily or indistinguishable placebo for 6 months. At the time of surgery and after 6 and 12 months of treatment, cell kinetics of normal colonic mucosa were assessed by using proliferating cell nuclear antigen (PCNA). Ninety patients were enrolled and 77 were assessable: 34 in the treatment group and 43 in the placebo group. A significant reduction of mean total PCNA labeling index (PCNALI) was evident in both groups after 6 months (vitamins/calcium, from 16.11 +/- 2.43 to 10.71 +/- 2.81; placebo, from 17.30 +/- 2.63 to 12.53 +/- 3.40). The difference in the percentage of reduction of mean PCNALI between baseline and after 6 months was not statistically significant in the treatment and placebo groups: 34% and 28%, respectively. A second control, 6 months after discontinuation of vitamin and calcium supplementation, showed a further decrease of mean total PCNALI in both groups, but this was not statistically significant. Our randomized trial showed that calcium and vitamin supplementation does not reduce cell kinetics of colon epithelium. Furthermore, this study suggests the need for extreme caution in the interpretation and publication of studies on chemoprotectants in colon cancer without a control group.

Expression of p53 protein and resistance to preoperative chemotherapy in locally advanced gastric carcinoma.

BACKGROUND: Inactivation of the p53 gene has been reported to be associated with resistance to chemotherapy. The authors evaluated the significance of p53 status to the clinical outcomes of patients with locally advanced, unresectable gastric carcinoma (LAGC) who received chemotherapy. METHODS: Thirty chemotherapy-naive patients with LAGC received weekly administration of cisplatin 40 mg/m2, epi-doxorubicin 35 mg/m2, 5-fluorouracil 500 mg/m2, 6S-leucovorin 250 mg/m2, and glutathione 1500 mg/m2. After eight administrations of these agents, patients were assessed for response. Biopsy specimens of primary tumors were analyzed for p53 status using monoclonal antibody Bp53-12. RESULTS: Characteristics of patients were as follows: The median age was 66 years (range, 44-70 years); 18 were males and 12 were females. Eastern Cooperative Oncology Group performance status was 0 for 14 patients and 1 for 16. Histology was intestinal for 13 patients; for 17, it was diffuse. The site of the primary tumor was the cardia in 8 patients, the body of the stomach in 13, and the antrum in 9. The response rate (assessed with CT scan and endoscopy) for patients with p53 negative tumors was significantly higher than for those with overexpression of p53 (71% vs. 12%, P=0.004). CONCLUSIONS: p53 status analyzed before chemotherapy seems to be associated with response to treatment in patients with LAGC. This may provide a useful guide to selecting neoadjuvant chemotherapy for these patients.

S-phase fraction can predict event free survival in patients with pT2-T3N0M0 colorectal carcinoma: implications for adjuvant chemotherapy.

BACKGROUND: Adjuvant chemotherapy for colorectal carcinoma was found to improve survival of patients with American Joint Committee on Cancer/International Union Against Cancer Stage III disease. The usefulness of chemotherapy in patients with Stage II disease continues to be debated, and it is likely that only those patients with a poor prognosis should receive adjuvant chemotherapy. Biologic prognostic factors may allow further insight into the optimal treatment strategy for patients with Stage II or earlier disease. In this study the prognostic role of S-phase fraction (SPF) determined by flow cytometry was assessed in patients with Stage I-II colorectal carcinoma. METHODS: Specimens of surgically resected colorectal carcinoma were examined for SPF by flow cytometric DNA analysis. Consecutive patients referred to the study institution were considered eligible for this study. The main inclusion criteria were a Stage I-II tumor together with sufficient tumor material and adequate follow-up information. For each stage of disease, SPF data were associated with the recurrence rate and the disease free survival (DFS). RESULTS: Analysis was performed on 167 patients (65 with Stage I disease and 102 with Stage II disease). Among Stage I patients, SPF was high in 20 patients and low in 45 patients. In Stage II patients, there were 36 patients with low SPF and 66 patients with high SPF. In both stages, the recurrence rate and DFS were significantly worse for the subgroups of patients with high SPF. CONCLUSIONS: SPF has revealed prognostic differences among patients with surgically resected Stage I-II colorectal carcinoma. These data should be considered for planning future trials in the adjuvant setting because patients with high SPF may benefit from adjuvant chemotherapy.

Different doses of pamidronate in patients with painful osteolytic bone metastases.

Cancer patients with painful osteolytic bone metastases who had failed initial treatment with hormones and/or chemotherapy were each randomized to receive one of three pamidronate doses as outpatients: 45, 60, 90 mg given every 3 weeks for 12 weeks. Seventy patients were enrolled in this study, for a total of 265 infusions. There were 64 patients who completed 12 weeks of therapy. Forty-eight patients took nonsteroidal antinflammatory drugs, while 22 patients received morphine before pamidronate treatment. A reduction in bone pain and mobility scores was observed in all three different dose groups: in 11 of 23 patients (47%) at 45 mg; in 12 of 24 patients (50%) at 60 mg; and in 16 of 23 patients (69%) at 90 mg. However, while for patients receiving pamidronate at 90 mg median changes in pain and mobility were statistically significant at the 6th week, for patients receiving 45 mg they were not significant until the 12th week and for patients receiving 60 mg, until the 9th week. In weeks 0-6, the daily consumption of analgesics was reduced in 3 patients in the 45-mg arm, in 4 patients in the 60-mg arm, and in 7 patients in the 90-mg arm. In weeks 7-12, the daily consumption of analgesics was reduced in 8 patients receiving 45 mg, in 8 patients receiving 60 mg, and in 7 patients receiving 90 mg. No significant toxicity was recorded. In 2 patients (45 and 90 mg) fever (> 38 degrees C) and myalgia were observed after the first administration. In conclusion, our results seem to confirm the utility of higher doses of pamidronate in patients with painful bone metastases, because of the faster symptom relief achieved.

The clinical impact of teniposide in the treatment of elderly patients with small-cell lung cancer.

Teniposide (VM26) has been claimed to be active with a moderate toxicity in elderly patients affected by small-cell lung cancer (SCLC). Twenty-two patients with SCLC older than 65 years received VM26 as first-line chemotherapy at a dose of 60 mg/m2 on 5 consecutive days every 3 weeks. Age distribution ranged from 67 to 80 years (median 72 years). Fourteen patients were men and eight were women. Twelve patients had limited disease (LD) and ten extensive disease (ED). One patient (LD) had a complete response, and four (3 LD, 1 ED) achieved a partial response for an overall response rate of 22.7% (95% CI 6-40%). The most frequent toxicity was myelosuppression: 20 and 15% of patients had grade 3 leukopenia and thrombocytopenia, respectively. Our results seem to suggest that VM26 by this schedule is moderately effective in elderly patients with SCLC, and it cannot be recommended as a routine treatment.

Inhibition of tumor cell kinetics and serum insulin growth factor I levels by octreotide in colorectal cancer patients.

BACKGROUND & AIMS: Octreotide was shown to inhibit the growth of colon cancer and to reduce serum concentrations of tumor growth factors such as insulin-like growth factor I (IGF-I) and epidermal growth factor (EGF) in vitro and in animal models. Effects of octreotide on tumor cell kinetics and serum concentration of IGF-I and EGF in patients with colorectal cancer were evaluated. METHODS: Seventy-five patients with colorectal cancer were randomized to receive octreotide (200 micrograms daily) in the 2 weeks before surgery or the usual medications. Samples of tumor tissue were taken at endoscopy and at surgery. [3H]Thymidine labeling index and flow cytometry were used to assess the S-phase fraction. In octreotide-treated patients, plasma levels of IGF-I, EGF, and growth hormone were assessed before and after treatment. RESULTS: There was a statistically significant reduction in the mean percentage of the S-phase fraction as a result of octreotide treatment measured by both [3H]thymidine labeling index (P = 0.001) and flow cytometry (P = 0.001). No reduction in the percentage of the S-phase fraction was observed in the control group patients. Serum values of IGF-I were significantly reduced by octreotide, whereas EGF and growth hormone levels were not affected. CONCLUSIONS: Octreotide reduces the proliferative activity of tumor cells and the serum IGF-I levels in patients with colorectal cancer. This activity may have a role in the treatment of colorectal cancer.

Evaluation of factors influencing 5-fluorouracil-induced diarrhea in colorectal cancer patients. An Italian Group for the Study of Digestive Tract Cancer (GISCAD) study.

Diarrhea is one of the dose-limiting toxicities for administration of fluorouracil (5FU) in patients with colorectal cancer and can result in severe morbidity and mortality. No well-defined prognostic factors influencing 5FU-associated diarrhea have been identified, which means its occurrence is unforeseeable. The aim of this study was to check whether any characteristics related to patients or chemotherapy could allow the identification of subsets of patients at higher risk of developing diarrhea while receiving a regimen containing 5FU. A logistic regression analysis was performed with age, sex, site of primary tumor, presence of primary tumor, presence of colostomy, time since surgery, number of courses of chemotherapy, diarrhea in previous courses, season of treatment, and chemotherapeutic regimens used as model parameters to predict occurrence of diarrhea in 258 colorectal cancer patients receiving a 5FU-containing regimen. Presence of primary tumor (P = 0.004), previous episodes of chemotherapy-related diarrhea (P = 0.00005) and summer season (P = 0.014) were found to be significant risk factors for developing diarrhea. The other variables examined, such as age, sex, chemotherapeutic regimen, site of primary tumor, presence of colostomy, and time since surgery, were not significantly correlated to diarrhea. Chemotherapeutic regimen was the only parameter that allowed prediction of the severity of diarrhea: 5FU/6S-leucovorin/interferon caused more severe diarrhea, followed by 5FU/leucovorin weekly. Although the analysis of these clinical features does not seem to allow the definition of a well-defined subset of colorectal cancer patients at higher risk of 5FU-induced diarrhea, it can be recommended that patients with primary tumor, or who have experienced diarrhea in earlier courses of chemotherapy or are receiving treatment in summer should be carefully monitored, especially in the first cycles.

Tumor markers in the diagnosis of malignant serous effusions.

To assess the possibility of increasing the detection rates of cytological examination in malignant effusions by the selection of specific tumor markers for a given type of tumor, we measured CEA, CA 19.9, CA 15.3, MCA, PSA, and AFP in malignant effusions from 89 patients with the following primary malignancies: colon, stomach, breast, liver, prostate, lung, and kidney. Cytological examination was positive in only 35 of 89 patients (40%), while the tumor markers were positive in 72 of 89 cases (80%). However, apart from small cell lung and kidney cancers, where the lack of a specific tumor marker resulted in no advantage, in the other types of tumors, the specific marker for each tumor identified correctly malignant effusions in 72 of 74 cases (97%). In fact, CEA was positive in 11 of 11 effusions induced by colorectal cancer; CA 19.9 in 28 of 30 gastric cancer effusions, while MCA and CA 15.3 were positive in breast cancer effusions (16/22 and 20/22). Finally, elevated AFP and PSA indicated hepatocellular and prostate cancer, respectively. In conclusion, in cancer patients with elevated effusion levels of specific tumor markers, the effusions could be considered of a malignant nature even without cytologically demonstrable tumor cells.

Death and Transfiguration of a Triple Junction in the South Atlantic

Three major lithospheric plates-Antarctic, South American, and African-meet in the South Atlantic near Bouvet Island where the Mid-Atlantic Ridge (MAR), the Southwest Indian Ridge (SWIR), and the American Antarctic Ridge converge toward a fast evolving triple junction. A major magmatic pulse has recently built a new, swollen segment of the SWIR (Spiess Ridge) that is propagating toward the MAR at a rate of 4 to 5 centimeters per year, disrupting a former ridge-ridge-ridge (RRR) triple junction. A new triple junction will be established about 70 kilometers to the north when the propagating SWIR/Spiess segment will impact with the MAR, probably within the next 1 million years. The American Antarctic Ridge will take advantage of the MAR/SWIR duel by capturing an approximately 70-kilometer stretch of MAR, whereas the Antarctic plate will increase its size.

Measurements of cellular proliferation by H-3-thymidine-labeling index and flow cytometry in breast cancer patients.

In 50 node negative breast cancer patients, tumor S-phase fraction (SPF) was determined by H-3-thymidine labeling index ((HTLI)-H-3) or flow cytometry (FC). Forty-five patients had tumor cell kinetics measured by both techniques. Twenty-three patients were classified as having high proliferative activity and 22 low by (HTLI)-H-3, while 32 as highly proliferating and 13 low proliferating by FC. In 30 patients only, both indices agreed on identifying high or low proliferative activity. These results suggest the need of more careful attention to standardization and quality control of cell kinetic data before carrying out clinical trials based on these parameters.

S-phase fraction and tumor aneuploidy in colorectal carcinoma of young patients.

BACKGROUND: Young patients with colorectal carcinomas are considered to have a worse prognosis than older patients. It was the goal of this study to assess if biologic characteristics of tumors in young patients differ from those observed in 2 different groups of patients with the same clinical characteristics but ranging in age either from 41 to 60 years or 61 years and older, respectively. METHODS: Colorectal carcinoma tumor samples were obtained from storage from patients age 40 years and younger and examined for tumor ploidy and S-phase fraction. For each younger patient, a control was selected among patients matched for Dukes stage, site of primary tumor, and sex, with the two age groups. RESULTS: Thirty-one of 1361 patients (2.2%) with colorectal carcinoma treated at our institution between 1984 and 1994 age 40 years or younger. Tumor aneuploidy was present in 3 younger patients, in 5 patients in the 41 to 60 years age group, and in 5 patients in the 61 years and older age group. S-phase fraction was 27.67 +/- 13.62 in patients younger than 40 years, 25.35 +/- 11.6 in the 40 to 61 years age group, and 22.45 +/- 8.48 in the 61 years and older age group. These differences were not statistically significant. CONCLUSIONS: It appears that there are no significant differences in S-phase fraction and tumor aneuploidy in patients younger or older than 40 years, suggesting that colorectal tumors arising in young people do not have different biologic properties.