Generation and characterization of an IGFBP-7 antibody: identification of 31kD IGFBP-7 in human biological fluids and Hs578T human breast cancer conditioned media

The cDNA encoding mac25 (IGFBP-7) was firsr derived from mRNA isolated from leptomeningial and senescent human mammary epithelial cells (1,2). The open reading frame was shown to predict a protein with homology to the amino terminus of the IGF binding proteins, (IGFBP)1-6. Studies in our laboratory have shown that baculovirus generated mac25 binds IGF-I and-II in a specific manner, leading to the renaming of mac25 as IGFBP-7 (3). Further studies at the cellular level, to identify the involvement of IGFBP-7 in IGF regulation and cell growth, require a specific antibody against the protein, which has yet to be identified in either cultured cells or in vivo. We have now generated three polyclonal antibodies against the purified baculovirus peptide and, by western immunoblots and immunoprecipitation, demonstrated the existence of a specific 31,000 dalton protein. It is a secreted protein, and can be identified in the conditioned media of Hs578T breast cancer cells, as well as in normal human urine, cerebrospinal fluid and amniotic fluid. Subsequent studies with these antibodies should help elucidate the physiological role(s) of this protein.     

Thrombocytopenia following liver transplantation is associated with platelet consumption and thrombin generation

PSA-based screening substantially increases the prostate cancer detection rate and the percentage of organ-confined tumors. It appears that there is some benefit from screening for prostate cancer because of the increased amount of potentially curable disease discovered and the fact that 96% of the pathologically staged tumors detected have histologic features associated with aggressive cancer. Additional evidence that nearly all tumors detected on the basis of initial PSA screening are apt to be clinically significant may be derived from the information that PSA-based screening decreases the incidence of incidental A1 grade III and A2 tumors but does not increase the detection of clinically insignificant A1 grade I and II tumors. At this time, PSA represents the most effective and valuable tool to detect early prostate cancer; therefore, PSA should be used to improve early diagnosis of prostate cancer. Some advances have been made with the introduction of age-specific reference ranges and the ability to measure free to total PSA ratios. The data presented support the clinical usefulness of age-specific reference ranges for serum PSA. Calculation of the free to total PSA ratio is valuable in deciding which screening volunteers require further evaluation, increases the specificity of PSA screening, and as demonstrated may be useful in deciding which patients with isolated PIN should undergo repeat biopsies. Based on these facts, PSA truly can be described as the most important and useful marker for adenocarcinoma of the prostate. Based on these encouraging results and the obligingness of the social insurances, we will be able to continue PSA screening for early detection of prostate cancer for all concerned Tyrolean men in the future.     

Hereditary hemochromatosis

Genetic (hereditary) hemochromatosis is probably the most common autosomal recessive disorder found in white Americans, of whom about 5/1,000 (0.5 percent) are homozygous for the associated gene. The hemochromatosis gene is probably located close to the HLA-A locus on the short arm of chromosome 6. Homozygous individuals may develop severe and potentially lethal hemochromatosis, especially after age 39. Hereditary hemochromatosis involves an increased rate of iron absorption from the gut with subsequent progressive storage of iron in soft organs of the body. Excess iron storage eventually produces pituitary, pancreatic, cardiac, and liver dysfunction and death may result from cardiac arrhythmias, congestive heart failure, and/or hepatic failure or cancer. Early diagnosis can prevent these excess iron-induced problems. Iron overload owing to HLA-linked hereditary hemochromatosis can be distinguished from other causes of hemochromatosis by liver biopsies and interpretations. Patients at risk for genetic hemochromatosis should be screened, identified, and treated as early as age 20 to prevent or minimize the deadly complications of hemochromatosis. Population screening should include measurements of serum iron concentration, total iron binding capacity (TIBC), percent saturation of transferrin, and serum ferritin concentrations. Family members of hereditary hemochromatosis patients are at increased risk and should be tested. Screening, identification and early treatment (phlebotomies, sometimes in combination with the use of Desferal or other iron-chelating agents) may help prevent or reduce iron-related organ damage and premature deaths. Early diagnosis and treatment will reduce the population of aging individuals with severe, complicated hemochromatosis and dramatically reduce medical costs (billions of U.S. dollars per annum) associated with the management of this disease.     

Difference between the measured and ordered dose of catecholamine infusions

OBJECTIVE: To measure the actual concentrations of dopamine, dobutamine, and epinephrine in infusates prepared for patients, and to compare these concentrations with those of the dopamine HCl, dobutamine, and epinephrine HCl infusates that had been prescribed to evaluate drug preparation accuracy. DESIGN: Prospective, unblind study. SETTING: Pediatric intensive care unit in a tertiary-care teaching hospital. PARTICIPANTS: All dopamine, dobutamine, and epinephrine infusions ordered for patients during the 2-month study period were eligible for inclusion in the study. MEASUREMENTS: Daily samples of dopamine, dobutamine, and epinephrine infusates that were prepared for 41 pediatric patients were obtained; the infusate catecholamine concentration was measured by HPLC and compared with the ordered concentration. The concentration than was multiplied by the rate of infusion to determine the catecholamine dose. MAIN RESULTS: There were significant differences between the measured doses of dopamine, dobutamine, and epinephrine and the dopamine HCl, dobutamine, and epinephrine HCl doses (p = 0.0001, p = 0.039, and p = 0.0009, respectively) that had been ordered because of preparation inaccuracies. Failure to account for the HCl salt in the stock drug accounted for some, but not all, of the inaccuracy of the dopamine HCl and epinephrine HCl infusates. There was a wide interday variability in the measured catecholamine dosage in patients receiving the same dose for 3 days or more. CONCLUSIONS: There are daily fluctuations in the preparation of dopamine, dobutamine, and epinephrine infusates that could alter the amount of drug actually delivered to critically ill patients and potentially contribute to their hemodynamic instability.     

Inhibition of experimental proliferative vitreoretinopathy by retroviral vector-mediated transfer of suicide gene. Can proliferative vitreoretinopathy be a target of gene therapy?

PURPOSE: To determine the potential of somatic gene transfer as a treatment for proliferative vitreoretinopathy (PVR), experimental PVR was induced in rabbits by intraocular injection of fibroblasts bearing the herpes simplex virus thymidine kinase (HStk) gene. These transduced cells should be susceptible to cytotoxicity by exposure to ganciclovir (GCV). MATERIALS AND METHODS: Rabbit fibroblasts were transduced with retroviral vectors bearing an HStk gene. Proliferative vitreoretinopathy was induced by injection of 5 x 10(4) normal or HStk gene-transduced fibroblasts (HStk fibroblasts) into rabbit eyes. Ganciclovir (100 micrograms per eye) or saline was injected into the vitreous on days 0 and 4. Experimental animals were divided into three groups: group A received HStk fibroblasts with GCV; group B, normal fibroblasts with GCV; group C, HStk fibroblasts with saline. Proliferative vitreoretinopathy also was induced in several other groups of eyes, some receiving GCV and different proportions of HStk fibroblasts to normal fibroblasts, others receiving only normal fibroblasts and GCV. The eyes were examined by indirect ophthalmoscopy on days 4, 7, 14, and 28, and PVR was classified into six stages (0-5). RESULTS: Proliferative vitreoretinopathy was induced and progressed over time in each group. On day 28, PVR was most severe in animals in group B (average stage, 4.6) and group C (average stage, 4.4). Proliferative vitreoretinopathy was inhibited in group A (average stage, 1.0). The groups that received mixed injection of HStk fibroblasts and normal fibroblasts had intermediate PVR. Results of histologic study showed no apparent toxic or pathologic reaction in the retinochoroidal tissue of group A animals. CONCLUSIONS: Severity of experimental PVR clearly was reduced by transfer of the HStk gene and administration of GCV. This inhibitory effect also was produced by a combination of 10% HStk fibroblasts and 90% normal fibroblasts, indicating a significant bystander effect. These data suggest the potential of somatic gene therapy for the treatment of PVR.     

Ocular neuromyotonia: clinical features, physiological mechanisms, and response to therapy

Ocular neuromyotonia (ONM) is a rare disorder characterized by episodic diplopia, occurring either spontaneously or following sustained eccentric gaze. Most patients have had prior radiation therapy to the sellar and parasellar region. ONM is thought to reflect impaired muscle relaxation due to inappropriate discharges from oculomotor, trochlear, or abducens neurons or axons with unstable cell membranes. Patients with ONM often benefit from membrane stabilizing agents such as carbamazepine. Here we describe a 71-year-old man, with no history of radiation therapy, who for 18 months had had transient episodic diplopia that occurred after down gaze. Clinical examination indicated ONM in muscles supplied by the right oculomotor nerve. Binocular scleral search coil eye movement recordings revealed a defect not only of muscle relaxation but also of maximal muscle contraction. The patient was treated with carbamazepine 200 mg per day with complete resolution of his symptoms. ONM may be more common than previously recognized, and patients with unexplained transient episodic diplopia should be specifically tested for diplopia and ocular misalignment following sustained eccentric gaze.     

Analytical and clinical performance characteristics of Tandem-MP Ostase, a new immunoassay for serum bone alkaline phosphatase

The performance characteristics of the Tandem-MP Ostase assay, a new microplate immunoassay for bone-specific alkaline phosphatase (bone ALP; EC 3.1.3.1) in human sera, are described. Bone ALP is bound to streptavidin-coated microwells by a single biotinylated anti-bone ALP monoclonal antibody. Antigen is detected by the addition of p-nitrophenyl phosphate. The assay is performed at room temperature in <90 min. Imprecision was 2.3-6.1% with a detection limit of 0.6 microg/L. Method comparison of bone ALP measurements with the Tandem-MP Ostase assay and the mass-based Tandem-R Ostase assay (n = 285) indicated regression statistics of Tandem-MP Ostase = 1.03 Tandem-R Ostase + 0.22 microg/L, S(y/x) = 4.0 microg/L, r = 0.97. Serum bone ALP values in apparently healthy men and in pre- and postmenopausal women were also similar between the two Ostase assay formats. Liver ALP reactivity determined using the slope and heat inactivation methods was similar in both Ostase assays. Liver ALP reactivity ranged from 3 microg/L (heat inactivation) to 6 microg/L (slope method) per 100 U/L of liver ALP activity, whereas bone ALP reactivity was 37 microg/L per 100 U/L of bone ALP activity, indicating a liver ALP relative reactivity of 8.1-16.2%. Similar results were obtained with the Alkphase-B bone ALP immunoassay. The Tandem-MP Ostase bone ALP assay demonstrated increased concentrations of serum bone ALP in conditions where bone metabolism is increased and showed a rapid, temporal decrease in serum bone ALP in Paget disease patients on bisphosphonate therapy. In conclusion, the Tandem-MP Ostase assay for serum bone ALP is a rapid, simple, robust nonisotopic alternative to the Tandem-R Ostase immunoradiometric assay that provides an accurate and sensitive assessment of bone turnover.