Radioimmunodetection of gastrointestinal neoplasms with antibodies to carcinoembryonic antigen.

Primary and secondary gastrointestinal tumors have been identified using sheep immunoglobulin G antibody to carcinoembryonic antigen radiolabeled with 131I. 99mTc-pertechnetate and 99mTc-human serum albumin were used to identify tissue spaces and blood pool and to facilitate external substraction imaging. In 13 patients with tumors, 4 of 5 primary sites and 8 of 11 secondary sites were successfully demonstrated. Two patients with benign disease had negative scans. Comparison with conventional methods of scanning showed good correlation.

Effect of dose, molecular size, affinity, and protein binding on tumor uptake of antibody or ligand: a biomathematical model.

A mathematical model has been developed to determine the best approach to improving tumor targeting with antibody. The amount of antibody in the tumor (tumor content) and the tumor:normal tissue antibody concentration ratio (uptake ratio) were calculated over 12 days from injection, using the computer program FACSIMILE to solve the stiff nonlinear differential equations describing the system. Results indicate that success requires an optimal combination of dose, size, and binding affinity of antibody. Increasing the dose to 100 times that presently used for scanning increased both the percentage of injected antibody in the tumor and the uptake ratio by up to 2 orders of magnitude to maximal values determined by affinity. This result could be achieved by coinjecting unlabeled antibody. Increasing affinity from Keq = 10(9) to 10(13)M-1 increased the uptake ratio from 5 to 100 for whole antibody and to 550 for a small ligand, at the calculated optimal dose, but had no effect at the current scanning dose. With decreasing molecular size at average affinity, the same maximum tumor content and uptake ratio were achieved but progressively earlier. At high affinity there was a substantial advantage for a small ligand compared with whole antibody in terms of uptake ratio (550 versus 100) and tumor:normal tissue integral dose ratio (330 versus 60). The uptake of a small ligand was not increased by binding to plasma protein but with increasing time the tumor content was higher than without protein binding.

Estimation of the Stokes radii of serum proteins for a study of protein movement from blood to amniotic fluid.

The effective hydrodynamic (Stokes) radii of fifteen serum proteins were estimated by Sephadex G 200 gel filtration and immunochemical methods. The Waldmann-Meyer and Birch equation (Protides Biol. Fluids, Proc. 21st Colloq. (1974) (Peeters, H., ed.), Vol. 21, pp. 653-656, Pergamon Press, Oxford) was used for maximum accuracy. Three replicate column runs were made to assess the precision for the size determinations (coefficients of variation 0.6-2.7(). Quantive two-dimensional immunoelectrophoresis was used to measure eleven proteins in specimens of serum and amniotic fluid collected from twelve normal pregnancies. There was a close inverse linear relationship between the amniotic fluid/serum ratios of the proteins and their Stokes radii. This indicates that the movement of a protein from the blood to amniotic fluid is determined by the size of the protein. The linear correlation of protein amniotic fluid/serum ratio with Stokes radius was better than that with molecular weight. This demonstrates that, as an expression of protein size, Stokes radius should be used in preference to molecular weight when protein filtration systems are being investigated.

Studies on the expression of Cu,Zn superoxide dismutase in human tissues during development.

The developmental expression of Cu,Zn superoxide dismutase in human lung and erythrocytes has been studied using activity measurements, immunoblotting and immunohistochemistry. Enzyme activity in erythrocytes increased significantly during gestation but no developmental trend was seen in lung. Immunoblotting identified a single enzyme form that was present in a variety of tissues and immunohistochemistry showed the enzyme to have widespread distribution in lung tissue. These data indicate that Cu,Zn superoxide dismutase is consistently expressed during human development and that, unlike in other species, no late-fetal surge in expression occurs.

Studies on the developmental expression of glutathione S-transferase isoenzymes in human heart and diaphragm.

The developmental expression of the basic, near-neutral and acidic isoenzymes of glutathione S-transferase (RX:glutathione R-transferase, EC 2.5.1.18) has been studied in heart and diaphragm. Neither these enzymes nor the putative muscle-specific GST4 isoenzyme demonstrated any developmental trends in expression. In vitro hybridisation and SDS-discontinuous polyacrylamide gel electrophoresis were used to show that the GST4 isoenzyme is a homodimer composed of monomers that have a slightly larger molecular weight than the near-neutral isoenzyme. The sensitivity of GST4 to inhibitors also appeared similar to that of the GST1 2 isoenzyme. Immunodiffusion and immunoblotting techniques were used to show that the acidic enzyme in muscle is immunologically identical to that in other tissues.

Structure, function and properties of antibody binding sites.

Do antibody combining sites possess general properties that enable them to bind different antigens with varying affinities and to bind novel antigens? Here, we address this question by examining the physical and chemical characteristics most favourable for residues involved in antigen accommodation and binding. Amphipathic amino acids could readily tolerate the change of environment from hydrophilic to hydrophobic that occurs upon antibody-antigen complex formation. Residues that are large and can participate in a wide variety of van der Waals' and electrostatic interactions would permit binding to a range of antigens. Amino acids with flexible side-chains could generate a structurally plastic region, i.e. a binding site possessing the ability to mould itself around the antigen to improve complementarity of the interacting surfaces. Hence, antibodies could bind to an array of novel antigens using a limited set of residues interspersed with more unique residues to which greater binding specificity can be attributed. An individual antibody molecule could thus be cross-reactive and have the capacity to bind structurally similar ligands. The accommodation of variations in antigenic structure by modest combining site flexibility could make an important contribution to immune defence by allowing antibody binding to distinct but closely related pathogens. Tyr and Trp most readily fulfil these catholic physicochemical requirements and thus would be expected to be common in combining sites on theoretical grounds. Experimental support for this comes from three sources, (1) the high frequency of participation by these amino acids in the antigen binding observed in six crystallographically determined antibody-antigen complexes, (2) their frequent occurrence in the putative binding regions of antibodies as determined from structural and sequence data and (3) the potential for movement of their side-chains in known antibody binding sites and model systems. The six bound antigens comprise two small different haptens, non-overlapping regions of the same large protein and a 19 amino acid residue peptide. Out of a total of 85 complementarity determining region positions, only 37 locations (plus 3 framework) are directly involved in antigen interaction. Of these, light chain residue 91 is utilized by all the complexes examined, whilst light chain 32, light chain 96 and heavy chain 33 are employed by five out of the six. The binding sites in known antibody-antigen complexes as well as the postulated combining sites in free Fab fragments show similar characteristics with regard to the types of amino acids present. The possible role of other amino acids is also assessed.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of exercise on cerebral perfusion in humans at high altitude.

The effects of submaximal and maximal exercise on cerebral perfusion were assessed using a portable, recumbent cycle ergometer in nine unacclimatized subjects ascending to 5,260 m. At 150 m, mean (SD) cerebral oxygenation (rSO2%) increased during submaximal exercise from 68.4 (SD 2.1) to 70.9 (SD 3.8) (P < 0.0001) and at maximal oxygen uptake (.VO2(max)) to 69.8 (SD 3.1) (P < 0.02). In contrast, at each of the high altitudes studied, rSO2 was reduced during submaximal exercise from 66.2 (SD 2.5) to 62.6 (SD 2.1) at 3,610 m (P < 0.0001), 63.0 (SD 2.1) to 58.9 (SD 2.1) at 4,750 m (P < 0.0001), and 62.4 (SD 3.6) to 61.2 (SD 3.9) at 5,260 m (P < 0.01), and at .VO2(max) to 61.2 (SD 3.3) at 3,610 m (P < 0.0001), to 59.4 (SD 2.6) at 4,750 m (P < 0.0001), and to 58.0 (SD 3.0) at 5,260 m (P < 0.0001). Cerebrovascular resistance tended to fall during submaximal exercise (P = not significant) and rise at .VO2(max), following the changes in arterial oxygen saturation and end-tidal CO(2). Cerebral oxygen delivery was maintained during submaximal exercise at 150 m with a nonsignificant fall at .VO2(max), but at high altitude peaked at 30% of .VO2(max) and then fell progressively at higher levels of exercise. The fall in rSO2 and oxygen delivery during exercise may limit exercise at altitude and is likely to contribute to the problems of acute mountain sickness and high-altitude cerebral edema.

HLA-DR and DQ DNA polymorphisms in subjects of Asian Indian and white Caucasian origin.

There is a close correspondence between serologically defined DR types and DR beta chain restriction fragment length polymorphisms (RFLPs). There is also an association between DR types and DQ alpha and DQ beta RFLPs because of linkage disequilibrium. We present the results of an analysis of DR beta, DQ alpha and DQ beta RFLPs in Asian Indians and white Caucasian subjects. DR beta RFLPs were similar in the two groups. Clearly distinguishable DR beta patterns were observed for DR1, 2, 3, 4, 5, 7 and w10. The DR beta patterns associated with DR3 were, however, also found with w6. The DR7 DR beta patterns were also found with w9. For DR specificities 1, 3, 4, 5, 7 and w10, the associated DQ alpha and DQ beta RFLPs were similar in both racial groups, but for DR2, however, marked differences were found. The DR2-positive white Caucasian subjects all possessed a single DQ alpha/DQ beta combination whereas the DQ alpha/DQ beta patterns in DR2-positive Asian Indians showed considerable heterogeneity. The pattern seen in white subjects was present in only a minority of Asians. DR-DQ relationships clearly vary in different racial groups. RFLP analysis of HLA-linked diseases in different populations should prove to be an important technique in identifying the primary genetic factor(s) in these disorders.

Analysis of MHC class II DNA polymorphisms in Negroid subjects.

Study of the MHC class II region is complicated by strong linkage disequilibrium between DR and DQ. Comparison of DR-DQ haplotypes between different races partly resolves this problem. We present the results of an analysis of DRB, DQA and DQB restriction fragment length polymorphisms in serologically DR-typed subjects of Negroid origin. Clearly distinguishable DRB RFLPs were observed for DR1,2,5,7 and w8. DR4,9 and w10 were uncommon in this group. DR3 was associated with two extended haplotypes, one characterised by the DQw4 allele, the other by the DQw2 allele. A recently recognised DQB RFLP (DQB 2c) was associated with DR7 and also occurred on DR5 and DR9 haplotypes. Both DR5 and DRw6 were heterogeneous in their DR-DQ relationships. Negroid subjects exhibit DR-DQ relationships distinct from other races. These provide scope for further study of MHC class II associations with disease.

Immunodetection of 11 beta-hydroxysteroid dehydrogenase type 2 in human mineralocorticoid target tissues: evidence for nuclear localization.

11 beta-Hydroxysteroid dehydrogenase (11 beta HSI) is an enzyme complex responsible for the conversion of hormonally active cortisol to inactive cortisone; two isoforms of the enzyme have been cloned and characterized. Clinical observations from patients with the hypertensive syndrome apparent mineralocorticoid excess, recently explained on the basis of mutations in the human 11 beta HSD2 gene, suggest that it is the 11 beta HSD2 isoform that serves a vital role in dictating specificity upon the mineralocorticoid receptor (MR). We have raised a novel antibody in sheep against human 11 beta HSD2 using synthetic multiantigenic peptides and have examined the localization and subcellular distribution of 11 beta HSD2 in mineralocorticoid target tissues. The immunopurified antibody recognized a single band of approximately 44 kDa in placenta, trophoblast, and distal colon. In kidney tissue, two bands of approximately 44 and 48 kDa were consistently observed. No signal was seen in decidua, adrenal, or liver. Immunoperoxidase studies on the mineralocorticoid target tissues, kidney, colon, and parotid gland indicated positive staining in epithelial cells known to express the MR: respectively, renal collecting ducts, surface and crypt colonic epithelial cells, and parotid duct epithelial cells. No staining was seen in these tissues in other sites. The intracellular localization of 11 beta HSD2 in kidney and colon epithelial cells was addressed using confocal laser microscopy. Parallel measurements of 11 beta HSD2 and nuclear propidium iodide fluorescence on sections scanned through an optical section of approximately 0.1 micron indicated significant 11 beta HSD2 immunofluorescence in the nucleus. In human kidney, colon, and salivary gland, 11 beta HSD2 protects the MR from glucocorticoid excess in an autocrine fashion. Furthermore, within these tissues, 11 beta HSD2, which had been considered to be a microsomal enzyme, is also found in the nucleus, suggesting that the interaction between the MR and aldosterone or cortisol is in part a nuclear event.

Hormonal and biochemical normalization and tumor shrinkage induced by anti-parathyroid hormone immunotherapy in a patient with metastatic parathyroid carcinoma.

Parathyroid carcinoma is a rare cause of primary hyperparathyroidism, and the efficacy of medical therapy and chemo- and radiotherapy is poor in recurrent or metastatic disease. We report the first case of PTH immunization in which tumor shrinkage accompanied hormonal, biochemical, and clinical improvements in a patient with metastatic parathyroid carcinoma.A 50-yr-old woman with refractory parathyroid carcinoma and pulmonary metastases was immunized eight times between February 2001 and December 2003 with bovine and modified human PTH fragments and intact human PTH, mixed with Freund's adjuvant. Total and ionized calcium and PTH levels were assayed weekly for 6 months and regularly thereafter. Thoracic computed tomography scans were performed regularly. Antibodies to all PTH fragments were detected after two immunizations. Baseline PTH and total calcium were 213.0 ng/liter and 13.96 mg/dl, respectively, and remained elevated during the first three immunizations. From the fourth immunization onward, PTH and calcium decreased, and the patient's clinical condition improved markedly. PTH and calcium levels have remained controlled for more than 24 months, and the sizes (surface area) of pulmonary metastases decreased from baseline by 39-71%. This is the first evidence that PTH immunization not only can improve clinical, hormonal, and biochemical measures in parathyroid carcinoma but also has an antitumor effect.

Placental iodothyronine deiodinase expression in normal and growth-restricted human pregnancies.

We have described the expression of specific iodothyronine deiodinase mRNAs (using quantitative RT-PCR) and activities in normal human placentas throughout gestation and compared our findings to those in placentas from pregnancies affected by intrauterine growth restriction (IUGR). The predominant deiodinase expressed in placenta was type III (D3); type II (D2) was also present. In general terms, the activities of the enzymes D2 and D3 (and mRNAs encoding these enzymes) were higher earlier in gestation (<28 wk) than at term and displayed an inverse relationship with the duration of gestation (P < 0.05). Comparison of the relative expressions of mRNAs encoding D2 and D3 as well as their activities in placentas associated with IUGR (early and late gestational groups) with findings from normal placentas of similar gestational ages revealed no significant differences. Immunolocalization of D2 and D3 in syncytiotrophoblast (including syncytial sprouts) and cytotrophoblast of human placentas was demonstrated at both early and late gestation. Treatment of primary cultures of term cytotrophoblast cells in vitro with increasing doses of T(3) (1, 10, and 100 nM) resulted in increased expression of mRNAs encoding both D2 and D3 at 100-nM concentrations (P < 0.01) compared with control. Experiments with JEG-3 choriocarcinoma cells demonstrated a similar effect on D3 mRNA at 10 and 100 nM T(3) (P < 0.01). The demonstrated changes in iodothyronine deiodinase expression in the placenta across pregnancy are likely to contribute to regulation of the thyroid hormone supply to the developing fetus. The lack of difference in deiodinase expression in normal placentas and those found in IUGR argues against placental deiodinases being responsible for the hypothyroxemia in circulating fetal thyroid hormones observed in this condition.

Pituitary tumor transforming gene and fibroblast growth factor-2 expression: potential prognostic indicators in differentiated thyroid cancer.

Differentiated thyroid cancers are the most common endocrine cancers, but there are no reliable molecular markers of prognosis. Pituitary tumor transforming gene (PTTG) plays several potential roles in tumor initiation and progression, including regulating mitosis and stimulating expression of fibroblast growth factor (FGF)-2. Increased expression of PTTG has been demonstrated in follicular thyroid lesions, and expression of this oncogene has been identified as a potential prognostic marker in pituitary adenomas and colon carcinomas. We assessed the expression of PTTG and FGF-2 and its receptor FGF-R-1 in 27 differentiated thyroid cancers, and we compared this with expression in 11 normal thyroids, 25 multinodular goiters, and 13 Graves' disease specimens. We also examined the relationship between gene expression and clinical markers of tumor behavior. PTTG and FGF-2 were overexpressed in thyroid carcinomas (9.5-fold increase, P = 0.003, and 5.0-fold increase, P < 0.001, respectively) compared with normal thyroid. Increased FGF-2 mRNA expression was independently associated with the findings of lymph node invasion (R(2) = 0.71; P < 0.001) and distant metastasis (R(2) = 0.55; P = 0.009) at tumor presentation, after taking into account known prognostic factors such as age and gender of the patient and size and type of the tumor. High PTTG expression was independently associated with tumor recurrence (R(2) = 0.64; P = 0.003). We conclude that PTTG and FGF-2 expression are potential prognostic markers (and perhaps therapeutic targets) for differentiated thyroid cancer.

The use of a multiwire camera for the detection of 3H labelled cDNA/mRNA hybrids.

We have investigated the use of a multiwire camera for the rapid direct quantitation of 3H labelled cDNA/mRNA hybrids. Growth hormone (GH) complementary DNA (cDNA) probes were labelled with either 3H or 32P and used in dot-blot hybridization assays of pituitary cytoplasmic samples. The results were assessed either by counting in the multiwire camera (3H) or by autoradiography and scanning densitometry (32P). Results obtained by both methods were comparable over a similar range of pituitary cytoplasmic dilutions. The use of a multiwire camera may have several advantages over conventional methods for the detection of radioisotopically labelled cDNA/mRNA hybrids and has great potential for use in this field of endocrine research.

Altitude insomnia: studies during an expedition to the Himalayas.

During an expedition to the Himalayas, we studied the sleep and respiration of six climbers. Three ingested acetazolamide (500 mg) daily throughout the climb and the other three ingested placebo. At high altitude (4,150-4,846 m), each subject ingested temazepam (10 mg) for one night and placebo for another. Acetazolamide improved sleep above 2,750 m, but it is uncertain whether this was due to sedation or to improvements in arterial oxygen saturation. Sleep was markedly disturbed in all subjects above 4,000 m. Temazepam improved sleep, and in subjects taking acetazolamide, it reduced sleep-onset latencies and increased sleep efficiency close to that of sea level values. These observations suggest that the prophylactic use of acetazolamide is likely to improve sleep in climbers and that a low dose of a benzodiazepine such as temazepam (10 mg) may be beneficial at high altitude. Studies are now needed to exclude any possibility of respiratory impairment at altitude before a firm recommendation can be made regarding the routine use of this hypnotic.

Quantitative analysis of serum free light chains. A new marker for the diagnostic evaluation of primary systemic amyloidosis.

Primary systemic amyloidosis is a plasma cell dyscrasia characterized by the accumulation of excess free immunoglobulin light chains (FLCs) as amyloid. One of the diagnostic features of amyloidosis is the presence of circulating monoclonal FLCs in the serum and urine of the patients. The FLC usually is present in small amounts, and immunofixation is required for detection. A nephelometric method for quantitating FLCs in serum has been described using antibodies that recognize only FLC not bound to heavy chain. We describe a retrospective study using this quantitative FLC method for assessing monoclonal FLCs in 95 patients with amyloidosis. The sensitivity of nephelometric serum FLC measurements is particularly useful in patients with negative immunofixation results for serum, urine, or both. In addition, the FLC assay can be used for follow-up of patients with amyloidosis who have undergone stem cell transplantation.

Quantitation of vitronectin in serum: evaluation of its usefulness in routine clinical practice.

AIMS: To make a preliminary assessment of the clinical relevance of serum vitronectin concentrations in various disease groups, using a recently available commercial radial immunodiffusion kit. METHODS: Serum vitronectin concentrations were measured in 80 control subjects and 144 patients with various diseases. The following characteristics were used to evaluate the test procedures: linearity of method, inter- and intrabatch precision, effect of storage, temperature and in vitro activation of the classical and alternative complement pathways on vitronectin concentrations. RESULTS: Significantly reduced serum vitronectin concentrations were found in patients with liver disease, renal disease, and systemic lupus erythematosus (SLE) (normal C3 and C4 concentrations, when compared with normal subjects. This particular method was suitable for measuring vitronectin concentrations in serum samples provided they were stored at -20 degrees C. CONCLUSIONS: The clinical value of measuring serum vitronectin seems to be limited, but a larger study may be justified to ascertain the clinical importance of reduced serum vitronectin concentrations in liver diseases, and the possible role of vitronectin in other disease processes.

Cancer-associated proteins in effusion fluids.

Two cancer-associated proteins, carcinoembryonic antigen (CEA) and pregnancy-associated alpha2 glycoprotein (PAG), together with 13 normal serum proteins were measured in the serum and effusion fluid of patients with ascites and pleural effusions. The results indicate that CEA measurement in effusion fluid is more effective than serum measurement in distinguishing cancerous from congestive or inflammatory effusions. Comparisons with the results of cytological examination suggest that fluid CEA estimation may prove a useful clinical tool. Serum PAG levels were higher in patients with cancer, but fluid determination offers no advantage in separating the disease groups. Similarly, the estimation of individual normal serum proteins in effusion fluids is unlikely to be of diagnostic value.

Cerebral blood flow in acute mountain sickness.

Changes in cerebral blood flow (CBF) were measured using the radioactive xenon technique and were related to the development of acute mountain sickness (AMS). In 12 subjects, ascending from 150 to 3,475 m, CBF was 24% increased at 24 h [45.1 to 55.9 initial slope index (ISI) units] and 4% increased at 6 days (47.1 ISI units). Four subjects had similar increases of CBF when ascending to 3,200 m 3 mo later, indicating the reproducibility of the measurements. In nine subjects, ascending from 3,200 to 4,785-5,430 m, CBF increased to 76.4 ISI units, 53% above estimated sea-level values. CBF and increases in CBF were similar in subjects with or without AMS. In six subjects, CBF was measured before and after therapeutic intervention. At 2 h CBF increased 22% (71.3 to 87.3 ISI units) above pretreatment values in three subjects given 1.5 g acetazolamide, while three subjects given placebo showed no change. Symptoms remained unaltered in all subjects during the 2 h of the study. Overall, the results indicated that increases in CBF were similar in subjects with or without AMS while acetazolamide-provoked increases of CBF in AMS subjects caused no acute change in symptoms. Alterations in CBF cannot be directly implicated in the pathogenesis of AMS.