Entrapment of subscapular artery by the splitting of radial nerve- putative clinical implications.

The third part of the axillary artery has an intimate relationship with the cords of the brachial plexus. The subscapular artery, the largest branch of the axillary artery, arises from its third part. The radial nerve is a branch of the posterior cord of the brachial plexus and its supplies the extensors of the arm, forearm and dorsum of the hand. During routine undergraduate dissection of the axilla of a formalin-fixed cadaver of about 70 years, the subscapular artery was found sandwiched between two divisions of the radial nerve. These anterior and posterior divisions of the radial nerve arose immediately after the formation of the radial nerve and encircled the subscapular artery and fused to form a single nerve subsequently. This variant anatomy can lead to conditions like subscapular entrapment causing ischemia of the scapular region and radial nerve compression causing weakness of the extensors of the upper limb. Injury to the nerve and vessel can occur while performing diagnostic and therapeutic procedures in the area. Knowledge of these variations provides a precautious approach by surgeons and other interventionists while working on this area.

Subjective Global Nutritional Assessment [SGNA] in Children on Chronic Dialysis- A Prospective Observational Study.

Introduction: Nutritional assessment in children undergoing chronic dialysis is challenging as no single objective reference tool is available. There is a need to explore the application of the subjective global nutritional assessment (SGNA) tool in these children. This study assessed the nutritional status of children on chronic dialysis using SGNA, evaluated the utility of SGNA parameters in the longitudinal assessment of nutrition, and compared the SGNA tool with other nutritional measures. Methods: Children 2-18 years of age on chronic dialysis for at least 1 month were prospectively studied over a period of 18 months with two follow-up visits at least 3 months apart. Malnutrition was diagnosed by SGNA (well-nourished, moderately, and severely malnourished), mid-arm circumference <5th centile for age and gender, and serum albumin <3.8 g/l at baseline and follow-up. Results: In 41 children on dialysis (age: 124.8 ± 32 months), 73% had moderate or severe malnutrition by SGNA. Height for age (P = 0.008), weight for height (P = 0.004), dietary intake (P = 0.025) functional capacity (P = 0.001), loss of subcutaneous fat (P < 0.001), and muscle wasting (P < 0.001) were significantly associated with the presence and severity of malnutrition. SGNA showed a poor agreement with MUAC and serum albumin. On follow-up, there was no significant change in the category of nutritional status (P = 0.63) and no individual SGNA parameter was associated with the presence or severity of malnutrition. Conclusion: Two-thirds of the children on chronic dialysis were diagnosed with moderate to severe malnutrition by SGNA, while the majority remained in the same category of nutritional status on follow-up. Only half of the parameters used for assessment were strongly associated with the presence and severity of malnutrition. SGNA showed a poor agreement with objective nutritional measures and was not responsive in identifying a change in the nutritional status on follow-up.

Trajectory of cartilage loss within 4 years of knee replacement--a nested case-control study from the osteoarthritis initiative.

OBJECTIVE: Knee replacement (KR) represents a clinically important endpoint of knee osteoarthritis (KOA). Here we examine the 4-year trajectory of femoro-tibial cartilage thickness loss prior to KR vs non-replaced controls. METHODS: A nested case-control study was performed in Osteoarthritis Initiative (OAI) participants: Cases with KR between 12 and 60 month (M) follow-up were each matched with one control (without KR through 60M) by age, sex, and baseline radiographic stage. Femoro-tibial cartilage thickness was measured quantitatively using magnetic resonance imaging (MRI) at the annual visit prior to KR occurrence (T0), and at 1-4 years prior to T0 (T-1 to T-4). Cartilage loss between cases and controls was compared using paired t-tests and conditional logistic regression. RESULTS: One hundred and eighty-nine knees of 164 OAI participants [55% women; age 64 ± 8.7; body mass index (BMI) 29 ± 4.5] had KR and longitudinal cartilage data. Comparison of annualized slopes of change across all time points revealed greater loss in the central medial tibia (primary outcome) in KRs than in controls [94 ± 137 vs 55 ± 104 µm; P = 0.0017 (paired t); odds ratio (OR) 1.36 (95% confidence interval (CI): 1.08-1.70)]. The discrimination was stronger for T-2 → T0 [OR 1.61 (1.33-1.95), n = 127] than for T-1 → T0, and was not statistically significant for intervals prior to T-2 [i.e., T-4 → T-2, OR 0.97 (0.67-1.41), n = 60]. Results were similar for total medial femoro-tibial cartilage loss (secondary outcome), and when adjusting for pain and BMI. CONCLUSIONS: In knees with subsequent replacement, cartilage loss accelerates in the 2 years, and particularly in the year prior to surgery, compared with controls. Whether slowing this cartilage loss can delay KR remains to be determined.

Direct comparison of fixed flexion, radiography and MRI in knee osteoarthritis: responsiveness data from the Osteoarthritis Initiative.

OBJECTIVE: Minimum radiographic joint space width (mJSW) represents the Food and Drug Administration (FDA) standard for demonstrating structural therapeutic benefits for knee osteoarthritis (KOA), but only shows moderate responsiveness (sensitivity to change). We directly compare the responsiveness of magnetic resonance imaging (MRI)-based cartilage thickness and JSW measures from fixed-flexion radiography (FFR) and explore the correlation of region-matched changes between both methods. METHODS: Nine hundred and sixty-seven knees of Osteoarthritis Initiative participants with radiographic KOA were studied: 445 over 1 year with coronal FLASH MRI and FFR, and 375/522 over 1/2 years with sagittal DESS MRI and FFR. Standardized response means (SRM) of cartilage thickness and mJSW were compared using the sign-test. RESULTS: With FLASH MRI, SRM was -0.28 for medial femorotibial compartment (MFTC) cartilage loss vs -0.15 for mJSW, and -0.32 vs -0.22 for the most sensitive MRI subregion (central MFTC) vs the most sensitive fixed-location JSW(x = 0.25). With DESS MRI, 1-year SRM was -0.34 for MFTC vs -0.22 for mJSW and -0.44 vs -0.28 for central MFTC vs JSW(x = 0.225). Over 2 years, the SRM was significantly greater for MFTC than for mJSW (-0.43 vs -0.31, P = 0.017) and for central MFTC than for JSW(x = 0.225) (-0.51 vs -0.44, P < 0.001). Correlations between changes in spatially matched MRI subregions and fixed-location JSW were not consistently higher (r = 0.10-0.51) than those between non-matched locations (r = 0.15-0.50). CONCLUSIONS: MRI displays greater responsiveness in KOA than JSW FFR-based JSW, with the greatest SRM observed in the central medial femorotibial compartment. Fixed-location radiographic measures appear not capable of determining the spatial distribution of femorotibial cartilage loss.

Lessons learned from the development of oral calcitonin: the first tablet formulation of a protein in phase III clinical trials.

Oral delivery of proteins has been hampered by an array of difficulties. However, promising novel oral delivery systems have been developed. 5-CNAC, formulated with the peptide salmon calcitonin, is in phase III clinical trials for the treatment of osteoporosis or osteoarthritis and could become the first marketed oral peptide. This article reviews key findings and implications from studies undertaken to date with this oral formulation. Findings include these: (1) the optimal calcitonin tablet dose is 0.8 mg; (2) 0.8 mg of oral calcitonin is rapidly absorbed, reaching maximum concentration in 15 to 30 minutes, and is eliminated from plasma with a short half-life-9 to 15 minutes; (3) the 0.8-mg tablet is more highly absorbed than the marketed nasal formulation, with biomarker levels indicating significantly greater efficacy in suppression of bone resorption; (4) drug absorption is increased with dosing at least 10 minutes before a meal rather than postprandially and also with 50 mL of water; (5) the optimal timing of dosing for osteoporosis therapy is in the evening to mitigate the circadian peak in bone resorption; and (6) the oral formulations of synthetic and recombinant calcitonin have similar pharmacokinetic and pharmacodynamic properties. These key findings may aid researchers in the development of other oral formulations.

Parallel scanning near-field photolithography: the snomipede.

The "Millipede", developed by Binnig and co-workers (Bining, G. K.; et al. IBM J. Res. Devel. 2000, 44, 323.), elegantly solves the problem of the serial nature of scanning probe lithography processes, by deploying massive parallelism. Here we fuse the "Millipede" concept with scanning near-field photolithography to yield a "Snomipede" that is capable of executing parallel chemical transformations at high resolution over macroscopic areas. Our prototype has sixteen probes that are separately controllable using a methodology that is, in principle, scalable to much larger arrays. Light beams generated by a spatial modulator or a zone plate array are coupled to arrays of cantilever probes with hollow, pyramidal tips. We demonstrate selective photodeprotection of nitrophenylpropyloxycarbonyl-protected aminosiloxane monolayers on silicon dioxide and subsequent growth of nanostructured polymer brushes by atom-transfer radical polymerization, and the fabrication of 70 nm structures in photoresist by a Snomipede probe array immersed under water. Such approaches offer a powerful means of integrating the top-down and bottom-up fabrication paradigms, facilitating the reactive processing of materials at nanometer resolution over macroscopic areas.

Electron beam lithographically-defined scanning electrochemical-atomic force microscopy probes: fabrication method and application to high resolution imaging on heterogeneously active surfaces.

This paper describes in detail the use of electron beam lithography (EBL) to successfully batch microfabricate combined scanning electrochemical-atomic force microscopy (SECM-AFM) probes. At present, the process produces sixty probes at a time, on a 1/4 of a three-inch wafer. Using EBL, gold triangular-shaped electrodes can be defined at the tip apex, with plasma enhanced chemical vapor deposited silicon nitride serving as an effective insulating layer, at a thickness of 75 nm. The key features of the fabrication technique and the critical steps are discussed. The capability of these probes for SECM-AFM imaging in both tapping and constant distance mode is illustrated with dual topographical-electrochemical scans over an array of closely-spaced 1 microm diameter Pt disc electrodes, held at a suitable potential to generate an electroactive species at a transport-limited rate. As highlighted herein, understanding diffusion to heterogeneous electrode surfaces, including array electrodes, is currently topical and we present preliminary data highlighting the use of SECM-AFM as a valuable tool for the investigation of diffusion and reactivity at high spatial resolution.

Nanowire probes for high resolution combined scanning electrochemical microscopy - atomic force microscopy.

We describe a method for the production of nanoelectrodes at the apex of atomic force microscopy (AFM) probes. The nanoelectrodes are formed from single-walled carbon nanotube AFM tips which act as the template for the formation of nanowire tips through sputter coating with metal. Subsequent deposition of a conformal insulating coating, and cutting of the probe end, yields a disk-shaped nanoelectrode at the AFM tip apex whose diameter is defined by the amount of metal deposited. We demonstrate that these probes are capable of high-resolution combined electrochemical and topographical imaging. The flexibility of this approach will allow the fabrication of nanoelectrodes of controllable size and composition, enabling the study of electrochemical activity at the nanoscale.

Characterization of batch-microfabricated scanning electrochemical-atomic force microscopy probes.

A procedure for the batch microfabrication of scanning electrochemical-atomic force microscopy (SECM-AFM) probes is described. The process yields sharp AFM tips, incorporating a triangular-shaped electrode (base width 1 microm, height 0.65 microm) at the apex. Microfabrication was typically carried out on (1)/(4) 3-in. wafers, yielding 60 probes in each run. The measured spring constant of the probes was in the range 1-1.5 N m(-1). To date, processing has been carried out twice successfully, with an estimated success rate for the fabrication process in excess of 80%, based on field emission-scanning electron microscopy imaging of all probes and current-voltage measurements on a random selection of approximately 30 probes. Steady-state voltammetric measurements for the reduction of Ru(NH(3))(6)(3+) in aqueous solution indicate that the electrode response is well-defined, reproducible, and quantitative, based on a comparison of the experimental diffusion-limited current with finite element simulations of the corresponding mass transport (diffusion) problem. Topographical imaging of a sputtered Au film with the SECM-AFM probes demonstrates lateral resolution comparable to that of conventional Si(3)N(4) AFM probes. Combined electrochemical-topographical imaging studies have been carried out on two model substrates: a 10-microm-diameter disk ultramicroelectrode (UME) and an array of 1-microm-diameter UMEs, spaced 12.5 microm apart (center to center). In both cases, an SECM-AFM probe was first employed to image the topography of the substrates. The tip was then moved back a defined distance from the surface and use to detect Ru(NH(3))(6)(2+) produced at the substrate, biased at a potential to reduce Ru(NH(3))(6)(3+), present in bulk solution, at a diffusion-controlled rate (substrate generation-tip collection mode). These studies establish the success of the batch process for the mass microfabrication of SECM-AFM tips.

Micromachined nanocalorimetric sensor for ultra-low-volume cell-based assays.

Current strategies for cell-based screening generally focus on the development of highly specific assays, which require an understanding of the nature of the signaling molecules and cellular pathways involved. In contrast, changes in temperature of cells provides a measure of altered cellular metabolism that is not stimulus specific and hence could have widespread applications in cell-based screening of receptor agonists and antagonists, as well as in the assessment of toxicity of new lead compounds. Consequently, we have developed a micromachined nanocalorimetric biological sensor using a small number of isolated living cells integrated within a subnanoliter format, which is capable of detecting 13 nW of generated power from the cells, upon exposure to a chemical or pharmaceutical stimulus. The sensor comprises a 10-junction gold and nickel thermopile, integrated on a silicon chip which was back-etched to span a 800-nm-thick membrane of silicon nitride. The thin-film membrane, which supported the sensing junctions of the thermoelectric transducer, gave the system a temperature resolution of 0.125 mK, a low heat capacity of 1.2 nJ mK(-1), and a rapid (unfiltered) response time of 12 ms. The application of the system in ultra-low-volume cell-based assays could provide a rapid endogenous screen. It offers important additional advantages over existing methods in that it is generic in nature, it does not require the use of recombinant cell lines or of detailed assay development, and finally, it can enable the use of primary cell lines or tissue biopsies.

A suspended membrane nanocalorimeter for ultralow volume bioanalysis.

A nanocalorimetric suspended membrane sensor for pL volumes of aqueous media was fabricated by bulk silicon micromachining using anisotropic wet etching and photo and electron beam lithographic techniques. A high-temperature sensitivity of 125 microK and a rapid unfiltered time constant of 12 ms have been achieved by integrating a miniaturized reaction vessel of 0.7-nL volume on a 800-nm-thick and 300 x 300- microm2-large silicon nitride membrane, thermally insulated from the surrounding bulk silicon. The combination of a ten-junction gold and nickel thermoelectric sensor with an integrated ultralow noise preamplifier has enabled the resolution of 15-nW power in a single measurement, a result confirmed by electrical calibration. The combination of a high sensitivity and rapid response time is a consequence of miniaturization. The choice of gold and nickel as sensor material provided the maximum thermal sensitivity in the context of ease of fabrication and cost. The nanocalorimetric sensor has the potential for integration in an ultralow-volume high-density array format for the characterization of processes in which there is an exchange of heat.

Human PTH-(7-84) inhibits bone resorption in vitro via actions independent of the type 1 PTH/PTHrP receptor.

The linear sequence of intact mammalian PTH consists of 84 amino acids, of which only the most amino(N)-terminal portion, i.e. PTH-(1-34), is required for the classical actions of the hormone on mineral ion homeostasis mediated by the type 1 PTH/PTHrP receptor (PTH1R). Like the N-terminus, the carboxyl (C)-terminal sequence of PTH is highly conserved among species, and various circulating PTH C-fragments are generated by peripheral metabolism of intact PTH or are directly secreted, in a calcium-dependent manner, by the parathyroid glands. Certain synthetic PTH C-fragments exert actions on bone and cartilage cells that are not shared by PTH-(1-34), and specific binding of PTH C-peptides has been demonstrated in bone cells in which PTH1R expression was eliminated by gene targeting. The peptide human (h) PTH-(7-84) recently was shown to inhibit the calcemic actions of hPTH-(1-34) or hPTH-(1-84) in parathyroidectomized animals. To determine whether this anticalcemic effect of hPTH-(7-84) in vivo might result from direct actions on bone, we studied its effects on both resorption of intact bone in vitro and formation of osteoclasts in primary cultures of murine bone marrow. Human (h) PTH-(7-84) (300 nM) reduced basal 72-h release of preincorporated (45)Ca from neonatal mouse calvariae by 50% (9.6 +/- 1.9% vs. 17.8 +/- 5.7%; P < 0.001) and similarly inhibited resorption induced by hPTH-(1-84), hPTH-(1-34), 1,25-dihydroxyvitamin D(3) (VitD), PGE(2), or IL-11. In 12-d murine marrow cultures, both hPTH-(7-84) (300 nM) and hPTH-(39-84) (3000 nM) lowered VitD-dependent formation of osteoclast-like cells by 70%. On the contrary, these actions of hPTH-(7-84) were not observed with the PTH1R antagonists hPTH-(3-34)NH(2) and [L(11),D-W(12),W(23),Y(36)]hPTHrP-(7-36)NH(2), which, unlike hPTH-(7-84), did inhibit PTH1R-dependent cAMP accumulation in ROS 17/2.8 cells. We conclude that hPTH-(7-84), acting via receptors distinct from the PTH1R and presumably specific for PTH C-fragments, exerts a direct antiresorptive effect on bone that may be partly due to impaired osteoclast differentiation.

A case of neuroendocrine oncogenic osteomalacia associated with a PHEX and fibroblast growth factor-23 expressing sinusidal malignant schwannoma.

Oncogenic osteomalacia is a rare paraneoplastic syndrome that is characterized biochemically by hypophosphatemia and low plasma 1,25-dihydroxyvitamin D3, and clinically by osteomalacia, pseudofractures, bone pain, fatigue, and muscle weakness. We present a patient with a malignant schwannoma as the underlying cause of this disorder. A permanent cell line (HMS-97) derived from this tumor showed evidence of neuroendocrine differentiation by immunohistochemistry and of neurosecretory activity by electron microscopy. The cell line did express PHEX (phosphate-regulating gene with homologies to endopeptidases located on the X-chromosome) and FGF-23 (fibroblast growth factor-23) transcripts on northern hybridization; however, none of the known mutations from the related mendelian disorders of X-linked hypophosphatemic rickets or autosomal-dominant hypophosphatemic rickets could be detected. Tumor cell (HMS-97)-derived conditioned medium did not inhibit phosphate transport in a standard opossum kidney cell assay and in animal experiments. The medium also showed no PTH1- or PTH2-receptor-stimulating bioactivity. HMS-97 cells might be useful for further studies that aim to determine the genetic mechanism that leads to the observed PHEX and FGF-23 expression, both of which might have a direct role in the pathogenesis of oncogenic osteomalacia. In addition, these cells might be a useful tool for the investigation of neuroendocrine Schwann cell function and autoimmune peripheral nerve disease.

Development of a novel immunoradiometric assay exclusively for biologically active whole parathyroid hormone 1-84: implications for improvement of accurate assessment of parathyroid function.

We developed a novel immunoradiometric assay (IRMA; whole parathyroid hormone [PTH] IRMA) for PTH, which specifically measures biologically active whole PTH(1-84). The assay is based on a solid phase coated with anti-PTH(39-84) antibody, a tracer of 125I-labeled antibody with a unique specificity to the first N-terminal amino acid of PTH(1-84), and calibrators of diluted synthetic PTH(1-84). In contrast to the Nichols intact PTH IRMA, this new assay does not detect PTH(7-84) fragments and only detects one immunoreactive peak in chromatographically fractionated patient samples. The assay was shown to have an analytical sensitivity of 1.0 pg/ml with a linear measurement range up to 2,300 pg/ml. With this assay, we further identified that the previously described non-(1-84)PTH fragments are aminoterminally truncated with similar hydrophobicity as PTH(7-84), and these PTH fragments are present not only in patients with secondary hyperparathyroidism (2 degrees -HPT) of uremia, but also in patients with primary hyperparathyroidism (1 degrees -HPT) and normal persons. The plasma normal range of the whole PTH(1-84) was 7-36 pg/ml (mean +/- SD: 22.7 +/- 7.2 pg/ml, n = 135), whereas over 93.9% (155/165) of patients with 1 degrees -HPT had whole PTH(1-84) values above the normal cut-off. The percentage of biologically active whole PTH(1-84) (pB%) in the pool of total immunoreactive "intact" PTH is higher in the normal population (median: 67.3%; SD: 15.8%; n = 56) than in uremic patients (median:53.8%; SD: 15.5%; n = 318; p < 0.001), although the whole PTH(1-84) values from uremic patients displayed a more significant heterogeneous distribution when compared with that of 1 degrees -HPT patients and normals. Moreover, the pB% displayed a nearly Gaussian distribution pattern from 20% to over 90% in patients with either 1 degrees-HPT or uremia. The specificity of this newly developed whole PTH(1-84) IRMA is the assurance, for the first time, of being able to measure only the biologically active whole PTH(1-84) without cross-reaction to the high concentrations of the aminoterminally truncated PTH fragments found in both normal subjects and patients. Because of the significant variations of pB% in patients, it is necessary to use the whole PTH assay to determine biologically active PTH levels clinically and, thus, to avoid overestimating the concentration of the true biologically active hormone. This new assay could provide a more meaningful standardization of future PTH measurements with improved accuracy in the clinical assessment of parathyroid function.

Changing serine-485 to alanine in the opossum parathyroid hormone (PTH)/PTH-related peptide receptor enhances PTH stimulation of phospholipase C in a stably transfected human kidney cell line: a useful model for PTH-analog screening?

Using site-directed mutagenesis, we have introduced a serine-485-to-alanine mutation in the opossum parathyroid hormone (PTH) receptor. This amino acid is considered to be phosphorylated by protein kinase A upon ligand binding. Both wild-type (WT) and mutant receptor were stably expressed in 293-EBNA HEK cells. The mutant receptor showed comparable binding characteristics and only a slight increase in cAMP production compared with WT. However, the PTH dose-dependent increase in inositol phosphate production was 24-fold for the mutant receptor vs. 6-fold for the WT receptor. This mutant might prove useful in the sensitive detection of phospholipase C activation through various ligands, as the PTH receptor becomes a target of therapeutic intervention in osteoporosis.

Tuberoinfundibular peptide 39 binds to the parathyroid hormone (PTH)/PTH-related peptide receptor, but functions as an antagonist.

The tuberoinfundibular peptide TIP39 [TIP-(1-39)], which exhibits only limited amino acid sequence homology with PTH and PTH-related peptide (PTHrP), stimulates cAMP accumulation in cells expressing the PTH2 receptor (PTH2R), but it is inactive at the PTH/PTHrP receptor (PTH1R). However, when using either (125)I-labeled rat [Nle(8,21),Tyr(34)]PTH-(1-34)amide (rPTH) or (125)I-labeled human [Tyr(36)]PTHrP-(1-36)amide [PTHrP-(1-36)] for radioreceptor studies, TIP-(1-39) bound to LLCPK(1) cells stably expressing the PTH1R (HKrk-B7 cells), albeit with weak apparent affinity (243 +/- 52 and 210 +/- 64 nM, respectively). In comparison to the parent peptide, the apparent binding affinity of TIP-(3-39) was about 3-fold higher, and that of TIP-(9-39) was about 5.5-fold higher. However, despite their improved IC(50) values at the PTH1R, both truncated peptides failed to stimulate cAMP accumulation in HKrk-B7 cells. In contrast, the chimeric peptide PTHrP-(1-20)/TIP-(23-39) bound to HKrk-B7 cells with affinities of 31 +/- 8.2 and 11 +/- 4.0 nM when using radiolabeled rPTH and PTHrP-(1-36), respectively, and it stimulated cAMP accumulation in HKrk-B7 and SaOS-2 cells with potencies (EC(50), 1.40 +/- 0.3 and 0.38 +/- 0.12 nM, respectively) and efficacies (maximum levels, 39 +/- 8 and 31 +/- 3 pmol/well, respectively) similar to those of PTH-(1-34) and PTHrP-(1-36). In both cell lines, TIP(9-39) and, to a lesser extent, TIP-(1-39) inhibited the actions of the three agonists with efficiencies similar to those of [Leu(11),D-Trp(12),Trp(23),Tyr(36)]PTHrP-(7-36)amide, an established PTH1R antagonist. Taken together, the currently available data suggest that the carboxyl-terminal portion of TIP-(1-39) interacts efficiently with the PTH1R, at sites identical to or closely overlapping those used by PTH-(1-34) and PTHrP-(1-36). The amino-terminal residues of TIP-(1-39), however, are unable to interact productively with the PTH1R, thus enabling TIP-(1-39) and some of its truncated analogs to function as an antagonist at this receptor.

A novel immunoradiometric assay detects full-length human PTH but not amino-terminally truncated fragments: implications for PTH measurements in renal failure.

In 8 adolescents with end-stage renal disease (ESRD), basal PTH concentrations measured with a novel immunoradiometric assay (IRMA) (Scantibodies Laboratory, Inc.; S-IRMA) were invariably lower than those estimated with an established assay (Nichols Institute; N-IRMA) (263 +/- 228 versus 645 +/- 442 pg/ml, respectively; p<0.00001). During in vivo dynamic testing, set points for calcium-regulated PTH release were indistinguishable for both IRMAs (1.21 +/- 0.05 versus 1.22 +/- 0.06). However, maximal PTH concentrations were significantly lower when measured by S-IRMA then by N-IRMA (557 +/- 448 and 1114 +/- 606 pg/ml, respectively); minimum PTH concentrations were 41 +/- 65 pg/ml (5.0 +/- 4.2% of maximum) and 189 +/- 137 pg/ml (13.6 +/- 7.2% of maximum), respectively. Correlation between PTH and blood ionized calcium indicated that PTH measured by S-IRMA decreased more readily than the concentrations determined by N-IRMA. The N-IRMA showed indistinguishable cross-reactivity with hPTH(1-84) and hPTH(7-84), while the S-IRMA detected only the full-length peptide. Furthermore, the radiolabeled detection antibody of the N-IRMA interacted equivalently with hPTH(1-34) and hPTH(2-34), while the S-IRMA showed crossreactivity only with hPTH(1-34). These differences in assay specificity could explain the observed differences in ESRD, and suggest that PTH concentrations estimated by the S-IRMA reflect more accurately the amount of biologically active PTH in the circulation. Since low concentrations of PTH are frequently associated with adynamic bone disease, our findings may have significant implications for the treatment of renal osteodystrophy with calcium and/or biologically active vitamin D analogs.

Endothelial cell DNA transfer and expression using petri dish electroporation and the nonreplicating vaccinia virus/T7 RNA polymerase hybrid system.

The nonreplicating vaccinia virus MVA/T7 RNA polymerase hybrid system was tested with Petri dish electroporation for ectopic gene expression in human umbilical vein endothelial cells (HUVECs). A range of voltages (150-450 V), pulse times (10-40 ms), DNA concentrations (0-20 microg/ml) and infection levels (0-15 multiplicities of infection) were tested for effects on T7 promoter-directed chloramphenicol acetyltransferase (CAT) activity after MVA/T7RP infection. MVA/T7RP-directed expression was transient and at least 10 000-fold in excess of nonviral, cytomegalovirus enhancer-directed expression. Use of a Petri dish electrode with the MVA/T7RP system showed increased viability compared with a cuvette electrode. Overexpression of interleukin-2 alpha subunit (IL2Ralpha) pro- tein followed by anti-IL2Ralpha-directed magnetic immunoaffinity cell sorting allowed isolation of the transfected population. The high fidelity of cellular sorting was shown by segregation of CAT activity in the IL2Ralpha-sorted population after transfection of T7 promoter-directed bicistronic IL2Ralpha/CAT DNA. Expression of a panel of proteins including the fluorophore green fluorescent protein as detected by fluorescence microscopy and p21cip1, p27kip1, pp60c-src, FGF-1, pRb, p107 and pRb2/p130 proteins was also achieved. Thus, use of the nonreplicating vaccinia virus/T7 RNA polymerase expression system with Petri dish electroporation is feasible for certain applications for the manipulation of HUVECs by gene transfer.

Detection of colorectal neoplasms by the highly sensitive hemoglobin-haptoglobin complex in feces.

Screening for fecal occult blood by means of guaiac tests has an unsatisfactory sensitivity for the detection of colorectal neoplasms. The immunological determination of human hemoglobin in feces has a higher sensitivity and specificity, but hemoglobin is degraded during its transport through the gastrointestinal tract. We compared the hemoglobin test to a newly developed immuno-chemiluminometric (ILMA) assay for quantifying the hemoglobin-haptoglobin complex in feces which shows high stability against degradation. From each of 621 patients with gastrointestinal complaints before scheduled colonoscopy we collected two 1-ml samples from a single stool; there were no dietary restrictions. The sensitivity for detecting colorectal carcinomas proved 87% with hemoglobin. With the hemoglobin-haptoglobin complex it was 87% at a cutoff level of 1.5 microg/g feces, 83% at 2.0 microg/g feces, and 78% at 2.5 and 3.0 microg/g feces. The sensitivity for detecting large adenomatous polyps was 54% with hemoglobin, 76% with the hemoglobin-haptoglobin complex at a cutoff point of 1.5 microg/g feces, 73% with the hemoglobin-haptoglobin complex at 2.0 and 2.5 microg/g feces, and 65% with the hemoglobin-haptoglobin complex at 3.0 microg/g feces. The optimal cutoff point for the hemoglobin-haptoglobin complex was estimated to be 2.0 microg/g stool. The specificity for hemoglobin (99%) was significantly higher than that for the hemoglobin-haptoglobin complex at 2.0 microg/g feces (96%). Immunological determination of the hemoglobin-haptoglobin complex in feces has a comparable sensitivity as the fecal hemoglobin assay for colorectal carcinomas and a significantly higher sensitivity for adenomatous polyps but a significantly lower specificity. Its use for colorectal cancer prevention is currently being evaluated in a screening study.