Intravenous injection of an immunoconjugate (anti-PSA-IgG conjugated to 5-fluoro-2'-deoxyuridine) selectively inhibits cell proliferation and induces cell death in human prostate cancer cell tumors grown in nude mice.

Current chemotherapeutic and/or endocrine treatments for adenocarcinoma of the prostate are not delivered selectively to prostate cancer cells, therefore, they are used in very high doses that induce many unpleasant side effects in patients. New approaches are, therefore, needed to deliver drugs directly to prostate cancer cells to improve treatment effects. We hypothesized that antibody immunoglobulin G (IgG) against human prostate specific antigen (PSA) (anti-PSA-IgG) could function as a carrier protein for conjugated chemotherapeutic drugs (such as 5-fluoro-2'-deoxyuridine, doxorubicin, etc.) and that the immunoconjugate could be delivered selectively to PSA-producing neoplastic prostate. Immunoconjugate would then preferentially inhibit cell proliferation and induce cell death in PSA-producing tumor cells, but not in non-PSA-producing prostate cancer cells or other solid organs of the host. The short-term treatment effect could be assessed by measuring cell death and cell proliferation in tumor-bearing animals. We tested our hypothesis by intravenously injecting an immunoconjugate (anti-PSA-IgG-5-fu-2'-d) into nude mice with subcutaneous PSA-producing LNCaP or non-PSA-producing Du-145 prostate tumors. During 5 days of treatment, we observed that immunoconjugate was retained preferentially in PSA-producing LNCaP tumors where it produced cytotoxic effects in neoplastic prostate cells as revealed by decreased cell proliferation and increased cell death, but similar effects were not observed in non-PSA-producing Du-145 tumor cells or mouse organs. Analysis of untreated control mouse with LNCaP tumor, anti-PSA-IgG alone, anti-irrelevant-IgG-drug complex, and drug alone treatments indicated that there was little or no cytotoxic effects of these treatments on LNCaP and Du-145 tumors, and host organs. Our analysis of control and experimental data showed that the immunoconjugate was highly specific in imparting cytotoxic effects on LNCaP prostate tumors, but not on Du-145 tumors and mouse organs. Thus, we have shown that the immunoconjugate selectively delivered a chemotherapeutic drug to PSA-producing prostate tumor cells where it produced measurable cytotoxic effects on cell proliferation and cell death. This is the first report to show a successful delivery of a chemotherapeutic drug in the immunoconjugate to PSA-producing LNCaP prostate tumors in nude mice and without inducing cytotoxic effects on mouse organs.

Selection of Microorganisms for Biological Control of Silver Scurf (Helminthosporium solani) of Potato Tubers.

Few management strategies exist for silver scurf, an important postharvest disease of potatoes. In this study, the microbiota of 47 agricultural soils and 7 tuber samples was screened for biological control agents of silver scurf. Soil or periderm samples were transferred to separate samples of γ irradiation-sterilized field soil enriched with potato periderm. After incubation, the samples were assayed for biological suppressiveness to silver scurf using a whole-tuber/infested soil assay. Over 430 isolates of bacteria, yeasts, and actinomycetes were recovered from tubers and soil associated with the 12 most suppressive soil samples. Thirteen strains were selected for further study on three different strains of Helminthosporium solani, including one that was resistant to thiabendazole. Microbial strains that significantly inhibited H. solani (P ≤ 0.05) in at least one experiment were identified as Pseudomonas putida (PM1), Nocardia globerula (S244), and Xanthomonas campestris (P76). Colonization studies with rifampicin-resistant strains of putative biological control agents revealed that long-term colonization of the tuber surface was not necessary to reduce disease symptoms. Highly variable levels of conidiophore production prevented selection of the single most suppressive strain. Possible sources of variability in biological control are discussed, including physiological age of the tuber, tuber infection in the field, and uneven free moisture in the storage.

Influence of antifungal compounds from a soil-borne actinomycete onFusarium spp. in asparagus.

Asparagus decline syndrome is caused by fungal infection of asparagus roots and crowns byFusarium oxysporum f.sp.asparagi (FOA) andF. moniliforme (FM). Several soil-borne microorganisms have been found inhibitory toFusarium pathogens in other crops. A novelStreptomyces spp. (ME2-27-19A) was isolated from asparagus field soil and found to be inhibitory to FOA and FM in vitro. Solvent extraction of ME2-27-19A and Chromatographic purification of the extract yielded compound(s) that were inhibitory to FOA and FM at 40Μg/ml. ME2-27-19A extract produced variable control of FOA and FM in vitro, and was phytotoxic at 1000 (Μg/ml. In soil, ME2-27-19A extract reduced theFusarium population at 100Μg/ml, but also reduced the asparagus shoot length.

Facilitated method for measurement of biliary secretion rates in healthy humans.

We have developed a facilitated method for determining secretion of constituents into bile. The ratio of constituent/bilirubin was measured in gallbladder bile and multiplied by bilirubin secretion rate, estimated by measuring endogenous production of carbon monoxide (VCO) by breath sampling. Accuracy of this method was assessed by measuring secretion rate of 99mTechnetium-labeled disofenin during steady-state constant intravenous infusion. In nine subjects, mean (+/- SEM) secretion of disofenin by the CO method was 104.2 +/- 7.2% of expected and by standard marker perfusion was 97.8 +/- 13.1% of expected. In ten subjects, secretion rate of cholesterol by the CO method averaged 103 mumol/h by the CO method compared to 113 by marker perfusion (NS). Compared to marker perfusion (which is believed to reflect 24-h secretion rate), the CO method significantly underestimated secretion rate of bile acid (1110 vs. 1332 mumol/h, P = 0.076) and lecithin (295 vs. 413 mumol/h, P = 0.01), probably because gallbladder bile contained a disproportionate amount of fasting versus postprandial bile. Thus, this new method provides an accurate secretion rate for biliary constituents secreted at a relatively constant rate, including cholesterol, with less variability than marker perfusion. However, it can be used to estimate secretion of bile acid and lecithin only when a 20-30% underestimation of 24-h secretion is acceptable.

Dosimetry and pharmacokinetics of monoclonal antibody A6H with human renal cell carcinoma xenografts: single dose study.

Implantable miniature thermoluminescent dosimeters and conventional biodistribution analysis were used to determine the locally absorbed radiation dose delivered to three morphologically distinct human renal cell carcinoma xenografts (TK-39, TK-82 and TK-177C; N = 87) following a 50 microCi infusion of 131iodine-labeled monoclonal antibody A6H. Xenografts were clearly detected by radioimmuno-scintigraphy. Pronounced differences were noted among the three xenografts in MAb pharmacokinetics and in the locally absorbed irradiation doses which ranged from 2 to 5 cGy per injected microCi of 131iodine-labelled A6H.

Monoclonal antibody-targeted radiotherapy of renal cell carcinoma using a nude mouse model.

Radiation dosimetry and monoclonal antibody (MAB)-targeted radiotherapy studies were performed to evaluate the feasibility of using tumor-preferential MAB as targeting agents for internal radiotherapy of renal cell carcinoma (RCC). Two human RCC xenograft lines, TK-177G and TK-82, were established in nude mice and studied using MAB A6H as a targeting agent. This MAB has previously demonstrated excellent in vivo localization to RCC xenografts. Two doses of A6H (13 to 19 micrograms) labeled with iodine 131 (110 to 130 microCi) caused the tumor to regress or arrested the tumor growth in both xenografts. Similar doses (18 to 43 micrograms; 120 microCi) of 131I-labeled control MAB AFP-22 or of unlabeled A6H did not inhibit tumor growth. While most mice in the control groups had tumors greater than 250 mg in weight by day 43, none of the tumors in mice treated with 131I-labeled A6H grew to that size during the 3-month observation period. Sequential computerized scintigraphy was used to calculate the amount of radioisotope localized in tumor versus normal mouse tissue. Therapeutic doses of 131I-labeled A6H delivered a median calculated radiation dose of 38 cGy/microCi (range, 28 to 57) injected dose to RCC xenografts, and a median of 0.9 cGy/microCi to normal mouse tissues. These findings suggest that A6H is able to target radioisotopes highly specifically to RCC and achieve a therapeutic effect in the experimental setting.

Comparison of fresh tissue incubation assay and the in vivo localization of monoclonal antibodies to renal cell carcinoma.

Few in vitro tests currently available are able to accurately predict the in vivo localization of monoclonal antibodies (Mabs) to cancer. We report on a fresh tissue incubation assay (FTIA) and compare the results of this assay to the in vivo localization of renal cell carcinoma (RCC)-reactive Mab A6H and control Mab AFP-22 to RCC and non-RCC xenografts implanted in nude mice. Both the FTIA and in vivo localization study demonstrated highly selective uptake of A6H in RCC but not in non-RCC xenografts. Radioimmunoscintigraphy using A6H clearly visualized RCC xenografts in every attempt, while AFP-22 did not highlight any of the tumor xenografts. The results demonstrate that FTIA may be a useful in vitro assay for selecting Mabs for in vivo application, and that radioimmunoscintigraphy is a potentially useful tool in detecting cancer sites.

Localization of human renal cell carcinoma xenografts with a tumor-preferential monoclonal antibody.

We previously described an immunoglobulin G1 monoclonal antibody (UMVA-RCC-A6H) that is highly reactive with human renal cell carcinoma (RCC) and has little cross-reactivity to other cell types both normal and malignant. In efforts detailed herein, radiolabeled A6H selectively localized to RCC xenografts and provided high resolution images of the xenografts. Also, A6H clearly discriminated between RCC xenografts and other human tumor xenografts. Consistent images of RCC xenografts (greater than 60 mg) were obtained without background subtraction. The amount of radiolabeled A6H in the tumor usually ranged from five to twenty times that of the blood. Normal mouse tissues, abscesses, and other human tumor xenografts contained less radiolabel per mg than did blood. A control monoclonal antibody of the same isotype failed to exhibit any localization in xenografts or normal tissues. Approximately 40% of the radiolabeled A6H dose per g was localized in the RCC xenograft 2 days after injection, although at the time of imaging about 60% of the radiolabel remaining in the mouse was associated with the xenograft. These results demonstrate that a RCC restrictive monoclonal antibody does specifically localize to RCC xenografts and supports the hope that this approach may have clinical value for diagnosis, staging, or treatment.

Relation between in vivo abscess localization and in vitro migration and adherence of neutrophils.

Rabbit peripheral blood and glycogen-stimulated peritoneal neutrophils were labeled with [111In]indium oxine and transfused intravenously into recipient rabbits with experimental abdominal abscesses due to Staphylococcus aureus. Peritoneal neutrophils harvested 4 hr after glycogen infusion localized within the abscesses to a greater extent than did peripheral blood neutrophils (P less than .002). In an in vitro chemotaxis under-agarose assay, peripheral blood neutrophils had greater random migration (P less than .002) and directed migration (P less than .01) than did peritoneal cells. In an in vitro glass slide adherence assay, peritoneal neutrophils were more adherent than were blood neutrophils (P less than .05). The discrepancy between in vivo and in vitro findings may be due to the increased adherence of peritoneal neutrophils. Glycogen-stimulated peritoneal neutrophils have been exposed in vivo to C5a, which is known to decrease migration and increase adherence in vitro of polymorphonuclear neutrophils; consequently, in vivo exposure of neutrophils to C5a may mean in vitro migration data may be misleading in predicting results in vivo.

Monoclonal antibodies in human renal cell carcinoma and their use in radioimmune localization and therapy of tumor xenografts.

A series of monoclonal antibodies (Mabs) to human renal cell carcinoma (RCC) material was developed. Two Mabs (D5D and A6H) that showed especially restrictive reactivities were radiolabeled with iodine 131 and tested in nude mice bearing human tumor xenografts for their ability to specifically localize RCC. Extensive studies of tissue radioactive uptake indicated that these Mabs could specifically localize RCC tumors with some mice achieving high tumor:blood ratios ranging from 15 to 60. Scintigraphic scanning revealed specific and consistent detection of RCC xenografts. Finally, preliminary results indicate that larger intravenous doses of radiolabeled RCC Mabs were effective as radioimmune therapy in inhibiting RCC xenograft growth. Mabs can be produced that are highly restrictive to human RCC and may be useful clinically for radioimmunoscintigraphy or therapy.

Serum tests for pancreatitis in patients with abdominal pain.

Though the serum total amylase test has been used for the diagnosis of pancreatitis for over 50 years, both its clinical sensitivity and specificity are far from perfect. We first present the results of serial serum total amylase, pancreatic isoamylase, lipase, and immunoreactive trypsin tests in nine patients during the week after their admission to the hospital with a diagnosis of acute pancreatitis, and then compare the serum total amylase, lipase, and immunoreactive trypsin levels in the initial serum submitted for amylase analysis from 100 patients because of the clinical suspicion of acute pancreatitis. In the former group of patients, the serum total amylase test was the least sensitive of the tests for pancreatitis after the first hospital day. In the latter group of patients, the largest discordance was found in patients with elevated serum total amylase levels, but normal lipase and immunoreactive trypsin levels. In 90% of these discordant cases, the elevation of serum total amylase was due to salivary amylase, yielding a maximum clinical specificity of only 71% for serum total amylase. Based on these data, we conclude that alternate tests deserve careful consideration as replacements for the serum total amylase test.

Hyperprolactinaemia in male diabetics.

We recently investigated two patients with diabetes and elevated serum prolactin levels in whom no cause of hyperprolactinaemia could be found. For this reason we measured fasting serum prolactin levels in 72 diabetic males and compared the results with those of 63 healthy males and 90 nondiabetic males attending an Impotence Clinic. The diabetic group had significantly higher serum prolactin levels (13.1 +/- 0.9 ng/ml) than the two control groups (9.9 +/- 0.6 ng/ml for normal males and 7.7 +/- 0.3 ng/ml for the non-diabetic impotent group). Eighteen percent of the diabetics studied had serum prolactin levels above the normal range for males (greater than 20 ng/ml). There was no correlation between serum prolactin levels and duration of diabetes, glycosylated haemoglobin level or presence of clinically apparent retinopathy. The correlation between serum prolactin level and fasting plasma glucose was weak though statistically significant (r = 0.26, P less than 0.05).

Effect of cholestasis on hepatic transport of 99mtechnetium p-isopropyl-iminodiacetic acid.

Hepatobiliary imaging with 99mTc-p-isopropyl-iminodiacetic acid (PIPIDA) and other acetanilidoiminodiacetic acid derivatives is a frequently used clinical tool in evaluating patients with jaundice. However, there has been little objective assessment of the effects of cholestasis on hepatic transport of acetanilioiminodiacetic acid derivatives. In our studies, transport of 99mTc-PIPIDA by isolated rat hepatocytes obtained from animals with extrahepatic obstruction secondary to bile duct ligation or intrahepatic cholestasis induced by ethinyl estradiol therapy was determined. Uptake constants for 99mTc-PIPIDA by hepatocytes isolated from livers of animals with ligated bile ducts were significantly decreased compared with uptake by liver cells from sham-operated controls (0.0030 +/- 0.0003 vs. 0.0089 +/- 0.0010 femtomole X 10(6) cells-1 X min-1 X pmol/L-1; p less than 0.001). Hepatocytes isolated from livers of animals given ethinyl estradiol also demonstrated significantly reduced 99mTc-PIPIDA uptake compared with controls given propylene glycol (0.0034 +/- 0.0002 vs. 0.0060 +/- 0.0004 fmol X 10(6) cells-1 X min-1 X pmol/L-1; p less than 0.001). Fractional rates of efflux of the study compound from hepatocytes preincubated with 99mTc-PIPIDA were significantly decreased in experiments using ethinyl estradiol (p less than 0.005) but did not differ significantly from controls in studies of bile duct ligation. 99mTc-PIPIDA uptake was significantly decreased in the presence of high bile salt concentrations (100 to 200 mumol/L), but unconjugated bilirubin concentrations as high as 200 mumol/L (approximately 12 mg/dl) had no effect on hepatocyte uptake of the ligand. The finding that cholestasis significantly impairs hepatocyte uptake of 99mTc-PIPIDA provides a possible explanation for the clinical observation that a patent biliary tree and normal serum bilirubin level are not always sufficient to ensure normal hepatobiliary imaging. These data also suggest that elevation of bile acid levels during cholestasis may either contribute to impaired uptake of hepatobiliary imaging agents or serve as a marker of cholestasis-induced abnormalities in the liver functions responsible for hepatic transport of these compounds.

The role of the endogenous opiates in zinc deficiency anorexia.

Anorexia is a major symptom of zinc deficiency, but the mechanism(s) for this anorexia are poorly defined. Recent studies have suggested an integral role for endogenous opiate peptides in appetite regulation. Dynorphin, a leucine-enkephalin containing opiate peptide, is a potent inducer of spontaneous feeding. In this study we showed that zinc deficient animals were relatively resistant to dynorphin-induced feeding. Measurement of dynorphin levels using a highly sensitive radioimmunoassay showed that zinc deficient animals had lower levels of dynorphin in the hypothalamus than did ad lib fed animals, with weight restricted animals having intermediate values. [3H]-naloxone binding was significantly increased to isolated brain membranes from zinc deficient animals using 1 nM unlabeled naloxone when compared to ad lib fed controls with the weight restricted animals again having intermediate values. These data suggest that abnormalities in endogenous opiate regulation of appetite may well play a role in the anorexia of zinc deficiency. The effects of zinc deficiency on endogenous opiate action appear to include alterations in receptor affinity, a post-receptor defect and alterations in the synthesis and/or release of dynorphin.

Salivary thyroxine as an estimate of free thyroxine: concise communication.

To test the hypothesis that the levels of salivary thyroxine (T4) reflect those of circulating free T4, we developed a radioimmunoassay (RIA) sensitive to low levels of T4. Concurrent saliva and serum samples were obtained from 32 euthyroid volunteers, ages 19-64. Salivary and serum T4 and cortisol levels were measured by RIA. Salivary albumin was measured by nephelometry. Salivary T4 levels were higher than predicted, 4.2-35 ng/dl (normal range 0.6-2.0). No correlation was found between salivary T4 and serum levels of free T4 and total T4 but there was a significant correlation between salivary T4 and albumin (r = 0.82). Salivary cortisol levels agreed with reported results and showed no correlation with salivary albumin. We conclude that salivary levels of drugs and hormones may be strongly affected by protein binding, and caution must be exercised in using salivary levels as an estimate of circulating free levels.

Impotence in medical clinic outpatients.

One thousand one hundred eighty men in a medical outpatient clinic were screened as to the presence of impotence. Four hundred one men (34%) were impotent, and of those, 188 (47%) chose to be examined for their problem. After a comprehensive evaluation the following diagnoses were obtained: medication effect, 25%; psychogenic, 14%; neurological, 7%; urologic, 6%; primary hypogonadism, 10%; secondary hypogonadism, 9%; diabetes mellitus, 9%; hypothyroidism, 5%; hyperthyroidism, 1%; hyperprolactinemia, 4%; miscellaneous, 4%; and unknown causes, 7%. The mean age of the impotent patients was 59.4 years, and the prevalence of alcoholism was 7%. Luteinizing hormone, follicle-stimulating hormone, testosterone, thyroxine, triiodothyronine (T3), T3 resin uptake, and prolactin studies were necessary to diagnose individual cases. We conclude that erectile dysfunction is a common and often overlooked problem in middle-aged men followed in a medical clinic.