Clinical significance of bone marrow metastases as detected using the polymerase chain reaction in patients with breast cancer undergoing high-dose chemotherapy and autologous bone marrow transplantation.

PURPOSE: The present study evaluates the clinical significance of detection of cytokeratin 19 (K19) in the bone marrow of patients with breast cancer undergoing high-dose chemotherapy (HDCT) and autologous bone marrow transplantation (ABMT). PATIENTS AND METHODS: We studied retrospectively cryopreserved bone marrow aspirates from 83 patients with high-risk stage II, III, and IV breast cancer obtained before bone marrow harvest but after induction chemotherapy. All samples were histologically negative for metastases. Polymerase chain reaction (PCR) for K19 was performed according to methods described previously and results were correlated with the probability of relapse following HDCT and ABMT. RESULTS: The incidence of occult metastases as defined by PCR for K19 message was 52% for 19 stage II, 57% for 14 stage III, and 82% for 50 stage IV patients (two-tailed P = .0075, chi 2 test). The probability of relapse at 3 years after ABMT was 32% and 94% for K19-positive stage II/III and stage IV patients, respectively, versus 10% and 14% for K19-negative stage II/III and stage IV patients, respectively. The difference was significant for stage IV patients (two-tailed P = .0002). CONCLUSION: It has been shown that PCR is a highly sensitive method to detect K19 message in the bone marrow. The incidence of K19 positivity in bone marrow increases significantly with advancing stage. In patients with breast cancer, especially metastatic breast cancer, undergoing HDCT and ABMT, the presence of K19 is associated with a poor prognosis.

The mechanism of action of insulin on phospholipid metabolism in rat adipose tissue. Requirement for protein synthesis and a carbohydrate source, and relationship to activation of pyruvate dehydrogenase.

We studied certain metabolic requirements for insulin-induced increases in phospholipids, and the relationship of phospholipid changes to the insulin-induced activation of pyruvate dehydrogenase, in rat adipocytes and fat pads in vitro. Increases in the contents of phosphatidylinositol and phosphatidylserine mass were maximal in rat fat pads within 10 min of incubation with insulin, and preceded or accompanied measurable increases in pyruvate dehydrogenase activity. In dose-response studies, the contents of these phospholipids and pyruvate dehydrogenase activity increased in parallel in response to increasing concentrations of insulin. Cycloheximide and puromycin inhibited insulin-induced increases in the mass of both of these phospholipids, as well as (in confirmation of previous reports) pyruvate dehydrogenase activity. Effects of insulin on phospholipid metabolism and pyruvate dehydrogenase were found to require an exogenous carbohydrate source, and fructose was nearly as effective as glucose in this regard. Insulin-induced increases in phosphatidylinositol and phosphatidylserine were demonstrated in the mitochondrial fraction, which is also the subcellular locus of pyruvate dehydrogenase. The present findings suggest that there is a relationship between insulin-induced increases in phospholipids and pyruvate dehydrogenase activity, but the nature of this relationship remains to be defined.

Comparison of effects of adrenocorticotropin and Lys-gamma 3-melanocyte-stimulating hormone on steroidogenesis, adenosine 3',5'-monophosphate production, and phospholipid metabolism in rat adrenal fasciculata-reticularis cells in vitro.

Synthetic bovine Lys-gamma 3MSH was found to potentiate the steroidogenic action of ACTH during incubation of rat adrenal fasciculata-reticularis cells in vitro. On the other hand, Lys-gamma 3MSH did not increase basal levels of or ACTH-induced (submaximal) increases in cellular concentrations of cAMP or phosphatidylinositol. Thus, Lys-gamma 3MSH does not appear to simply increase the overall action of ACTH, and moreover, it appears to potentiate steroidogenesis by a mechanism that is considerably different from that employed by ACTH. Observance of an effect of ACTH and failure to observe an effect of gamma 3MSH on adrenal phosphatidylinositol are in keeping with our previous postulation that phospholipids in the phosphatidate-inositide cycle play an important role in promoting cholesterol side-chain cleavage, since ACTH, but not gamma 3MSH, reportedly increases cholesterol side-chain cleavage. In addition, we also presently observed that Lys-gamma 3MSH can markedly increase corticosterone production in the face of a fixed, relatively small, submaximal, ACTH-induced increase in phosphatidylinositol. Thus, if gamma 3MSH enhances steroidogenesis by increasing free cholesterol availability, the latter may require another factor to initiate steroidogenesis, but is, nevertheless, an important determinant of the rate of steroidogenesis.

Validation of in vivo receptor measurements via in vitro radioassay: technetium-99m-galactosyl-neoglycoalbumin as prototype model.

Hepatic binding protein (HBP) is a hepatocyte-specific receptor for serum asialoglycoproteins. The receptor also recognizes a synthetic glycoprotein that has been developed as a radiopharmaceutical, technetium-99m-galactosyl-neoglycoalbumin (99mTc-NGA). This report describes the correlation between receptor parameters measured in vivo via kinetic modeling of 99mTc-NGA and those measured by in vitro radioassay of biopsied liver tissue. Eleven patients with diffuse hepatic disease underwent percutaneous liver biopsy followed by a 99mTc-NGA functional imaging study. In vivo measurements of HBP quantity Ro and forward binding rate constant kb obtained from the kinetic analysis of 99mTc-NGA liver and blood time-activity data were compared to total receptor quantity and the HBP-99mTc-NGA association constant KA as measured by Scatchard binding assay of the biopsied tissue. The correlation coefficients between in vivo and in vitro measurements were 0.73 (df = 8, p = 0.015) and 0.98 (df = 8, p less than 0.01) for Ro and kb, respectively. The in vivo measurements of HBP biochemistry via kinetic analysis of the radiopharmaceutical time-activity data reflect the average concentration and affinity of the receptor. This study further substantiates the validity of 99mTc-NGA as a quantitative probe for the HBP receptor.

Measurement of receptor concentration and forward-binding rate constant via radiopharmacokinetic modeling of technetium-99m-galactosyl-neoglycoalbumin.

Technetium-99m-galactosyl-neoglycoalbumin (99mTc-NGA) is a synthetic ligand to the hepatocyte receptor, hepatic binding protein (HBP). A five-state mathematical model containing a bimolecular chemical reaction was utilized for quantitative estimation of the following physiologic and biochemical parameters: extrahepatic plasma volume Ve; hepatic plasma flow F and volume Vh; receptor-ligand forward-binding rate constant kb and reaction volume Vr; and receptor concentration [R]o. Nine normal subjects were studied. Given (a) liver and heart time-activity data, (b) the patient's weight, height, and hematocrit, (c) the fraction of injected dose in a 3-min blood sample, and (d) the amount and galactose density of the NGA dose, a computer program executed a curve-fit to the kinetic model. Systematic error, as measured by reduced chi-square, ranged from 1.43 to 2.56. Based on the nine imaging studies, the mean and relative error of each parameter were: [R]o, 0.813 +/- (0.11) microM; kb, 2.25 +/- (0.15) microM-1 min-1; F, 0.896 +/- (0.20) liter/min; Ve, 1.67 +/- (0.27) liter; and Vh, 0.228 +/- (0.22) liter. Two unique features of 99mTc-NGA radiopharmacokinetic systems permit the simultaneous estimates of receptor quantity, ligand affinity, and hepatic plasma flow. The first is the ability to administer a quantity of ligand capable of occupying a significant fraction of receptor; and the second is a simple model structure that conserves mass.

Technetium-99m NGA functional hepatic imaging: preliminary clinical experience.

Technetium-99m galactosyl-neoglycoalbumin ( [Tc]NGA) is a radiolabeled ligand to hepatic binding protein, a receptor which resides at the plasma membrane of hepatocytes. This receptor-binding radiopharmaceutical and its kinetic model provide a noninvasive method for the assessment of liver function. Eighteen patients were studied: seven with hepatoma, eight with liver metastases, four with cirrhosis (two had concurrent hepatoma and one chronic active hepatitis), and one patient with acute fulminant non-A, non-B hepatitis. Technetium-99m NGA liver imaging provided anatomic information of diagnostic quality comparable to that obtained with other routine imaging modalities, including computed tomography, angiography, ultrasound, and [Tc]sulfur colloid scintigraphy. Kinetic modeling of dynamic [Tc]NGA data produced estimates of standardized hepatic blood flow, Q (hepatic blood flow divided by total blood volume), and hepatic binding protein concentration, [HBP]. Clinical correlation was by classical Child-Turcotte criteria (CTC). Significant rank correlation was obtained between [HBP] estimates and CTC scores (rs = -0.72, p = 0.001). This correlation supports the hypothesis that [HBP] is a measure of functional hepatocyte mass. The combination of decreased Q and markedly reduced [HBP] may have prognostic significance; all three patients with this combination died of hepatic failure within 6 wk of imaging.

Strongyloides stercoarlis--hyperinfection.

A case of severe Strongyloides stercoralis infection is described in a patient with renal failure due to bilateral cortical necrosis. Because thiabendazole, the drug of choice in strongyloidiasis, is primarily excreted by the kidneys, alternative therapy with a mebendazole was instituted. Although the patient eventually died as a result of the renal failure, successful therapy with mebendazole was well demonstrated.

High-sensitivity detection of minimal residual breast carcinoma using the polymerase chain reaction and primers for cytokeratin 19.

We have developed a reverse transcriptase-polymerase chain reaction (RT-PCR) assay to identify breast carcinoma cells in bone marrow aspirates with high sensitivity and specificity. This assay relies on the detection of cytokeratin 19 (K19) RNA by nested primer PCR followed by annealing to a (32P)-labeled internal sequence probe and autoradiography. In reconstitution experiments, this assay is capable of detecting 10 fg of admixed mammary tumor RNA in 1 microgram of normal marrow RNA (a dilution of 1:10(7)). Thirty of 30 primary breast tumor specimens, 19 of 19 cytologically positive bone marrow aspirate specimens, and three of 11 aspirate negative/biopsy positive specimens showed detectable K19 transcript. This assay shows high specificity, with 50 of 52 negative control aspirates showing no detectable amplification product. False-positive amplification was noted in two of 18 aspirates obtained from patients with active chronic myelogenous leukemia. Of stage II and III postsurgical breast carcinoma patients with histologically negative bone marrows and no radiographic bone disease, 14 of 30 were K19 positive by PCR. RT-PCR analysis of K19 transcript is a highly sensitive and specific method of detecting and monitoring low-level metastatic disease in patients with primary carcinoma of the breast. The presence of K19 RNA in histologically negative bone marrows suggests that this assay may prove a powerful monitor for patients undergoing curative therapy as well as a novel prognostic indicator.

Pseudomembranous colitis without diarrhea presenting clinically as acute intestinal pseudo-obstruction.

Pseudomembranous colitis usually presents with diarrhea in a clinical setting of recent antibiotic use. It is uncommon to see it as a cause of obstipation and colonic pseudo-obstruction. We report an unusual case of an elderly woman with hypertension, congestive heart failure, chronic obstructive pulmonary disease, chronic renal insufficiency, and diabetes mellitus, who was admitted with fever, abdominal pain, and distension without diarrhea. She presented with decreased stool frequency and obstipation. She did not respond to conservative management. Colonoscopy revealed a picture of pseudomembranous colitis, and Clostridium difficile toxin was positive. She responded well to metronidazole therapy.

Spetial and temporal distribution of house dust mite (Astigmata:Pyroglyphidae) allergens Der p 1 and Der f 1 in Barbadian homes.

House features contribute to house dust mite abundance and, therefore, exposure to mite allergens. Our study assessed the hypothesis that modernization of the domestic environment in a tropical setting may lead to a level of allergen from the house dust mites Dermatophagoides pteronyssinus (Trouessart) and D. farinae Hughes that previously has been defined clinically as at risk for people who suffer from allergic disease. Allergen (Der p 1 and Der f 1) levels were measured at 4 sites (mattress, bedroom floor, living room floor, and furniture) in 17 houses in Barbados during dry and rainy seasons. Der p 1(17 of 17 homes) at all 4 sites did not vary significantly from the dry to rainy season. Allergen levels varied according to site, and were highest in living room furniture in both seasons (geometric mean 40.37 and 64.17 micrograms/g, respectively). Concentration of Der p 1 allergens were higher in concrete than in wood or mixed concrete and wood houses. Der f 1(9 of 17 homes) levels were lower than Der p 1 by 1/1,000 (both seasons). Results indicate that season is less important in regard to levels of Der p 1 than house construction and confirm other studies that implicate D. pteronyssinus as a more abundant source of allergen than D. farinae in this tropical setting.

Goodness-of-fit and local identifiability of a receptor-binding radiopharmacokinetic system.

A four-state nonlinear model describing a radiopharmacokinetic system for a hepatic receptor-binding radiopharmaceutical, [99mTc]-galactosyl-neoglycoalbumin (TcNGA), was tested for goodness-of-fit and local identifiability using scanning data from nine healthy subjects and seven patients with severe liver disease. Based on standard deviations of liver and heart imaging data at equilibria as a measure of observational error, the reduced chi-square ranged from 0.5 to 2.6. Values above 1.2 occurred when the subject moved during the 30 min study. Relative standard errors for each parameter were: TcNGA-receptor forward binding rate constant kb, 13-54%; extra-hepatic plasma volume Ve, 0.8-15.0%; hepatic plasma volume Vh, 0.2-6.5%; hepatic plasma flow F, 54----greater than 1000%; and receptor concentration [R]o, 0.3-13%. The highest standard errors occurred when the amount of TcNGA injected exceeded the total amount of receptor. Therefore, when TcNGA functional imaging was performed without excess patient motion and receptor saturation, the kinetic model provided data fits of low systematic error and yielded high precision estimates of receptor concentration and forward binding rate constant. In summary, optimal performance of the kinetic model occurred when the amount of injected TcNGA resulted in the nonlinear operation of the pharmacokinetic system.

Mite sensitivity and exposure in the city of Santa Fe, Argentina.

Little information is available on mite allergen levels and rates of sensitization to domestic mites in South America. The purpose of this study was to establish the rate of sensitization to domestic mites in asthmatics and measure mite allergen levels in pillows and mattresses of mite allergic individuals in Santa Fe, Argentina. Dust samples were collected from the pillows and mattresses of 56 asthmatics (24 females and 32 males, mean age 17.6 +/- 11.5 years), who had been previously skin tested with standardized extracts of Dermatophagoides pteronyssinus, Dermatophagoides farinae and (1:50 w/v) extracts of Aleuroglyphus ovatus, Blomia tropicalis, Chortoglyphus arcuatus. A wheal > or = 3 mm was considered positive. Sensitization to Lepidoglyphus destructor and Euroglyphus maynei was determined by RAST and considered positive when a serum bound > or = 1% of the total counts added. Forty six individuals had a positive skin test to D. pteronyssinus, 43 to D. farinae, 27 to A. ovatus, 38 to B. tropicalis, 27 to C. arcuatus; 38 of 54 individuals had a positive RAST to E. maynei and 22 of 54 to L. destructor. The mean Group 1 (Der p 1 + Derf 1) level in pillows (n = 48) was 6.7 micrograms/g (1-55.4) and in mattresses (n = 52) 4.3 micrograms/g (1-38.8). There is a high degree of sensitization to domestic mites among asthmatics in Santa Fe. High mite allergen levels (> 2 micrograms/g) can be detected very often in pillows and mattresses.

Serial determinations of Der p 1 and Der f 1 show predominance of one Dermatophagoides species.

One dust sample each month was collected for an average of 11 months throughout the year from the bedroom carpet of 39 mite allergic, asthmatic individuals living in the Tampa Bay area of Florida. Dust samples were collected from a square meter area from the bedroom carpet for 2 min using a portable vacuum cleaner and analyzed using two-site monoclonal antibody assays. The geometric mean of Dermatophagoides allergens Der p 1 and Der f 1 levels measured in each individual home was used in the statistical analysis of the results and reflects the average allergen level per year per home. Der p 1 levels were greater than Der f 1 in 21 homes; five homes had between 1.1 and 5 times more; one home had between 5 and 10; and 15 had between 10 and 175. Der f 1 levels were greater than Der p 1 in 18 homes; seven homes had between 1.1 and 5 times more; four had between 5 and 10; and seven homes had between 10 and 44. Spearman rank analysis of the results yielded a negative correlation coefficient (Rho) = -0.213 (p = 0.19) between Der p 1 and Der f 1 levels. These data suggest that when mite allergen levels reach a geometric mean value of approximately 2 micrograms/gram of dust throughout a year, one mite species predominates over the other.

The value of radionuclide scintigraphy in patients with non-visualized gallbladders by oral cholecystography.

This study evaluated the clinical usefulness of Tc-99m-PyG cholescintigraphy in patients who had nonvisualization of the gallbladder after OCG. Nineteen patients with non-visualized gallbladder with OCG subsequently had Tc-99m-PyG cholescintigraphy performed. In nine patients the gallbladder visualized, whereas in ten it did not. Statistical analysis of this limited number of patients showed that Tc-99m-PyG cholescintigraphy was able to separate those patients with clinically significant gallbladder disease from those patients without significant gallbladder disease (p less than 0.05).

[In vivo measurement of hepatic binding protein in chronic liver disease--validation as a measure of hepatic functional reserve].

[Tc-99m] Galactosyl-neoglycoalbumin (TcNGA) is a synthetic radiolabeled ligand specific to the hepatocyte receptor, hepatic binding protein (HBP), a specific receptor to serum asialoglycoprotein. A TcNGA study was performed on 34 humans: normal volunteers (7) chronic hepatitis (6), hepatic cirrhosis (8), and hepatocellular carcinoma superimposed on cirrhosis (13). Heart and liver time activity curves were obtained following intravenous injection of TcNGA (5 mCi, 1.82 x 10(-9) mol/kg). HBP concentration ([HBP]) was calculated by curve-fitting techniques using the nonlinear three compartment model, which includes biomolecular reaction between HBP and TcNGA. [HBP] values were compared with conventional liver function tests. [HBP] had a good correlation with prothrombin time (n = 34, r = 0.694, p = 0.0001) thrombotest (n = 34, r = 0.692, p = 0.0001), hepaplastin test (n = 26, r = 0.787, p = 0.0001), albumin (n = 34, r = 0.712, p = 0.0001), cholinesterase (n = 34, r = 0.801, p = 0.0001), ICGR15 (n = 33, r = 0.761, p = 0.0001), KICG (n = 30, r = 0.709, p = 0.0001), ICG Rmax (n = 12, r = 0.735, p = 0.0064) and Child-Turcotte classification score (n = 34, r = 0.819, p = 0.0001). We concluded that excellent correlations of [HBP] to conventional liver function tests suggest that in vivo receptor measurement via TcNGA kinetic analysis is a sensitive and promising method in the estimation of hepatic functional reserve in patients with chronic liver disease.

Bronchodilator effects on gastric acid secretion.

Nine patients with chronic obstructive pulmonary disease were given oral aminophylline, intravenous aminophylline, and various inhaled and oral adrenergic bronchodilators to determine the effect of these agents on gastric acid secretion and gastrin release. Inhaled epinephrine hydrochloride resulted in an increase in basal acid output of borderline significance (.05 less than P less than .10). Oral aminophylline caused a significant increase in basal acid output from 2.43 to 4.06 mEq (P less than .05). Intravenous aminophylline also caused a significant increase in basal acid output from 0.66 to 2.19 mEq (P less than .01). There were no statistically significant changes in serum gastrin levels after administration of any of the bronchodilators. Aminophylline should be used with caution, if at all, in patients with peptic ulcer disease. In these patients, a beta agonist should be used for initial therapy. If the addition of aminophylline is necessary, antacids should be given.

Hepatic uptake of [99mTc]galactosyl-neoglycoalbumin is sensitive to receptor quantity.

[Technetium-99m]galactosyl-neoglycoalbumin (TcNGA) is a synthetic radioligand specific to the receptor, hepatic binding protein (HBP), which resides exclusively at the cell surface of hepatocytes. If the TcNGA time-activity data are to provide valid estimates of receptor biochemistry via pharmacokinetic modeling, the shape of the hepatic uptake curve must change with alterations in receptor concentration or affinity. An index of TcNGA hepatic uptake, T90, was obtained by calculating the time at which the liver time-activity curve reached 90% of its maximum. Receptor quantity was measured by Scatchard binding assay of liver biopsy samples obtained prior to each TcNGA study. The range of T90 was 3.7-16.8 min; HBP quantity ranged from 0.022 to 0.100 mumol. Significant correlation (r) and slope (b) was observed between T90 and HBP quantity (r = 0.82, P = 0.003; tb = 3.98, P = 0.003). We concluded that the hepatic uptake of TcNGA is sensitive to HBP quantity.

House dust mite, cat, and cockroach allergen concentrations in daycare centers in Tampa, Florida.

BACKGROUND: Allergen exposure in early childhood is a risk factor for sensitization and the development of asthma. Studies performed in Europe, New Zealand, and Singapore indicated the presence of indoor allergens in childcare centers and schools. However, the importance of indoor allergens in daycare centers in humid and warm regions of the world is not known. OBJECTIVE: To measure total mite counts, Der p 1, Der f 1, Fel d 1, and Per a 1 allergens in dust samples and mite allergen airborne concentrations in daycare centers in Tampa, Florida, United States. METHODS: Twenty daycare centers were surveyed for mite, cat, and cockroach allergens in Tampa, FL. One dust and two air samples (one during the day and one during the night) were collected in each center. Dust samples were extracted and analyzed for mite (Der p 1 and Der f 1), cat (Fel d 1), and cockroach (Per a 1) allergens. Mite airborne allergen concentrations were analyzed by RAST inhibition and expressed in standardized mite allergen units per m3 of air (AU/m3). RESULTS: Mites were identified in 15 samples, and concentrations ranged from 10 to 1,200 mites/g (298 +/- 355.2). The most prevalent mite species was Dermatophagoides pteronyssinus ( Der p 1). Der p 1 and/or Der f 1 were detected in 10 daycare centers. Der p 1 was detected in eight centers and ranged from I to 21.8 microg/g of dust (5.4 +/- 6.9); Der f 1 was detected in 3 centers and ranged from 0.2 to 2.1 microg/g of dust (1.3 +/- 0.9). Per a 1 and Fel d 1 were detected in all centers in small quantities; Per a 1 ranged from 8 to 1,806 ng/g (263.1 +/- 449.7) and Fel d 1 from 0.2 to 120 U/g of dust (16.6 +/- 31.7), respectively. Airborne mite allergen was detected in 18 centers and ranged from 0.01 to 2.7 AU/m3 during the day (0.2 +/- 0.6) and from 0.01 to 0.12 AU/m3 during the night (0.06 +/- 0.03), P = 0.001. CONCLUSIONS: Mite, cat, and cockroach allergens are present in daycare centers in Tampa, FL. Mite allergen concentrations exceeded levels that have been associated with sensitization and symptoms in allergic subjects in 40% of these centers.