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Biomedical subjects

M R John

Publications and source records attributed to M R John.

13 recordsLinked to original sources
PubMed2002

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.

Animals↗
PubMed2001

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.

Alanine↗
PubMed2001

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.

Female↗
PubMed2001

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.

Amino Acid Sequence↗
PubMed2001

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.

Adult↗
PubMed1999

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.

Adenocarcinoma↗
PubMed1999

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.

Adolescent↗
PubMed1998

Screening for colorectal neoplasms with a new immunological human faecal haemoglobin and albumin test.

In Germany, screening for colorectal cancer shows low efficiency, which is partly due to demographic changes with a rising mean age of the population, a low participation rate and an unsatisfactory sensitivity of guaiac tests for detecting faecal occult-blood. Therefore, a pilot screening study with a new immunological faecal haemoglobin and albumin test was performed in Ostringen, Germany to assess its compliance, performance characteristics and cost-effectiveness. Two thousand, seven hundred and eighty-five persons (1,498 women and 1,287 men) collected 1 ml samples from two different sites of one stool. The upper limit of normal was 10 micrograms/g stool for haemoglobin and 100 micrograms/g stool for albumin. The compliance was 82%; 224 persons (8%) had a positive test result. Of these, 184 underwent full colonoscopy. We detected 14 colorectal cancers, 10 of which were Dukes' stage A carcinomas removed by endoscopic polypectomy, 34 large adenomas and 43 small adenomas. The detection rate for colorectal neoplasms was above the rate described for other immunological haemoglobin tests and for Haemoccult tests. The specificity of the test--defined with false-positive results if a normal colon mucosa and no other reasons for upper or lower gastrointestinal bleeding were found--was 99.5%. The cost-effectiveness was assessed by comparing the diagnostic costs with the savings resulting from prevention of colorectal carcinomas by endoscopic polypectomy of malignant polyps (Dukes' stage A). The savings in our screening study exceeded the diagnostic costs by approximately 2.3 times. The combined immunological faecal haemoglobin and albumin test should substitute the Haemoccult test in colorectal cancer screening because of its higher sensitivity and specificity combined with cost-effectiveness and good patient compliance.

Adolescent↗
PubMed1998

Validity of new immunological human fecal hemoglobin and albumin tests in detecting colorectal neoplasms--an endoscopy-controlled study.

BACKGROUND: Screening for occult blood by means of guaiac tests has an unsatisfactory sensitivity for the detection of colorectal neoplasms. To increase sensitivity and specificity the immunological determination of human hemoglobin and albumin in feces has been developed. The validity of analyzing only two samples from one bowel movement of either test is not known. METHODS: An immunological determination of human fecal hemoglobin and albumin using luminescence immunoassays (LIA) was performed in 739 patients with gastrointestinal complaints before scheduled colonoscopy. Each patient collected two 1 ml samples from one stool. There were no dietary restrictions. RESULTS: The sensitivity for detecting colorectal carcinomas was 95.3% (95% confidence interval 84.2-99.4%) with hemoglobin and 67.4% (95% confidence interval 51.2-80.9%) with albumin. The sensitivity for detecting large adenomatous polyps was 62.9% (95% confidence interval 50.5-74.1%) with hemoglobin and 32.9% (95% confidence interval 22.1-45.1%) with albumin. The specificity was 97% for hemoglobin, 96% for albumin and 94% for the combined test. CONCLUSIONS: The immunological determination of fecal hemoglobin is superior to albumin and has a better sensitivity for the detection of colorectal neoplasms than that reported for guaiac tests, even if two samples from one bowel movement are examined. The immunological determination of fecal hemoglobin should therefore be evaluated for use in colorectal cancer screening.

Colonoscopy↗
PubMed1997

Solid phase competitive luminescence immunoassay for immunoglobulin A in faeces: development and clinical validation.

We describe a new simple solid-phase competitive luminescence immunoassay (LIA) for the determination of immunoglobulin A (IgA) in faeces. The assay utilizes an anti-alpha-chain IgA antibody which is coated to polystyrene beads and acridinium ester-labelled human IgA as tracer and, therefore, measures both monomeric and polymeric IgA. Dilution recovery of an internal standard was 96, 100 and 103%. Interassay and intra-assay coefficients of variation (C.V.) ranged from 4.5 to 12.9%. The upper limit of normal of faecal IgA in 122 healthy controls was found to be 300 mg/l IgA (mean 73 mg/l, specificity of 99.2%). Patients with inactive Crohn's disease (Crohn's disease activity index (CDAI < 150, n = 14) had faecal IgA values up to 3317 mg/l (mean 1073 mg/l; P < 0.0001). In the active group (CDAI > 150, n = 26) faecal IgA values ranged from 49 to 4094 mg/l (mean 1253 mg/l; P < 0.0001). Patients with ulcerative colitis were divided into a group with active disease (n = 18) and a remission group (n = 16) with values up to 1843 mg/l faecal IgA (man 486 mg/l; P < 0.0032) and up to 602 mg/l faecal IgA (mean 176 mg/l; P < 0.4833), respectively. We also studied patients with non-inflammatory diseases of the gut with this assay. This LIA has proved to be a reliable method for the determination of elevated faecal IgA concentrations and for the detection of pathological findings in the gastrointestinal tract, especially in Crohn's disease.

Adolescent↗
PubMed1995

Solid-phase competitive luminescence immunoassay for lysozyme in faeces.

We have developed a new simple solid-phase luminescence immunoassay (LIA) for the determination of faecal lysozyme. The assay utilises a polyclonal capture antibody coated to polystyrene beads and acridinium ester-labelled human lysozyme as tracer. Samples are incubated with polystyrene beads and tracer overnight at 4 degrees C. After a thorough washing step, emitted light is measured by an automated luminometer for 2 seconds. The standard curve uses five standards ranging from 0.025 to 6.4 mg/l. The method has a sensitivity of 0.02 mg/l. Dilution recoveries for three samples were 88, 104 and 108%. Intraassay coefficients of variation (CV, n = 24) were 10.1% and 11.7% for a healthy control and a patient sample; interassay CV (n = 16) were 6.7% and 13.1% for the same healthy control but another patient sample. The normal range of faecal lysozyme in 80 healthy controls was found to be 0.02-1 mg/l (97.5 percentile) with a median of 0.28 mg/l. Fifty-three patients with Crohn's disease had faecal lysozyme values ranging from 0.16 to 100.7 mg/l with a median of 1.75 mg/l, and 30 patients with ulcerative colitis showed levels between 0.09 and 118 mg/l with a median of 1.11 mg/l. The assay has proved useful for differentiating healthy individuals from those with inflammatory bowel disease and might be a valuable tool for diagnosing or evaluating inflammatory bowel disease.

Adolescent↗