Extracardiac 18F-florbetapir imaging in patients with systemic amyloidosis: more than hearts and minds.

PURPOSE: 18F-Florbetapir has been reported to show cardiac uptake in patients with systemic light-chain amyloidosis (AL). This study systematically assessed uptake of 18F-florbetapir in patients with proven systemic amyloidosis at sites outside the heart. METHODS: Seventeen patients with proven cardiac amyloidosis underwent 18F-florbetapir PET/CT imaging, 15 with AL and 2 with transthyretin amyloidosis (ATTR). Three patients had repeat scans. All patients had protocolized assessment at the UK National Amyloidosis Centre including imaging with 123I-serum amyloid P component (SAP). 18F-Florbetapir images were assessed for areas of increased tracer accumulation and time-uptake curves in terms of standardized uptake values (SUVmean) were produced. RESULTS: All 17 patients showed 18F-florbetapir uptake at one or more extracardiac sites. Uptake was seen in the spleen in 6 patients (35%; 6 of 9, 67%, with splenic involvement on 123I-SAP scintigraphy), in the fat in 11 (65%), in the tongue in 8 (47%), in the parotids in 8 (47%), in the masticatory muscles in 7 (41%), in the lungs in 3 (18%), and in the kidney in 2 (12%) on the late half-body images. The 18F-florbetapir spleen retention index (SRI) was calculated. SRI >0.045 had 100% sensitivity/sensitivity (in relation to 123I-SAP splenic uptake, the current standard) in detecting splenic amyloid on dynamic imaging and a sensitivity of 66.7% and a specificity of 100% on the late half-body images. Intense lung uptake was seen in three patients, one of whom had lung interstitial infiltration suggestive of amyloid deposition on previous high-resolution CT. Repeat imaging showed a stable appearance in all three patients suggesting no early impact of treatment response. CONCLUSION: 18F-Florbetapir PET/CT is a promising tool for the detection of extracardiac sites of amyloid deposition. The combination of uptake in the heart and uptake in the spleen on 18F-florbetapir PET/CT, a hallmark of AL, suggests that this tracer holds promise as a screening tool for AL.

Domiciliary humidification improves lung mucociliary clearance in patients with bronchiectasis.

Inspired air humidification has been reported to show some benefit in bronchiectatic patients. We have investigated the possibility that one effect might be to enhance mucociliary clearance. Such enhancement might, if it occurs, help to lessen the risks of recurrent infective episodes. Using a radioaerosol technique, we measured lung mucociliary clearance before and after 7 days of domiciliary humidification. Patients inhaled high flow saturated air at 37 degrees C via a patient-operated humidification nasal inhalation system for 3 h per day. We assessed tracheobronchial mucociliary clearance from the retention of (99m)Tc-labelled polystyrene tracer particles monitored for 6 h, with a follow-up 24-h reading. Ten out of 14 initially recruited patients (age 37-75 years; seven females) completed the study (two withdrew after their initial screening and two prior to the initial clearance test). Seven patients studied were non-smokers; three were ex-smokers (1-9 pack-years). Initial tracer radioaerosol distribution was closely similar between pre- and post-treatment. Following humidification, lung mucociliary clearance significantly improved, the area under the tracheobronchial retention curve decreased from 319 +/- 50 to 271 +/- 46%h (p < 0.07). Warm air humidification treatment improved lung mucociliary clearance in our bronchiectatic patients. Given this finding plus increasing laboratory and clinical interest in humidification mechanisms and effects, we believe further clinical trials of humidification therapy are desirable, coupled with analysis of humidification effects on mucus properties and transport.

Training of staff for the delivery of PET/CT services in the UK.

Evidence for the cost effectiveness of PET/CT imaging is now driving the widespread introduction of PET/CT services throughout the UK. The provision of PET/CT facilities will require a workforce of medical, scientific, technical and engineering staff who are adequately trained and fit for purpose. Suitably trained staff in this speciality are scarce. The development and accreditation of training courses and other educational resources for training programmes in all disciplines will therefore be required at a national and regional level. The implementation of PET/CT training can be achieved more cost-effectively by developing multi-professional learning resources whenever possible. It is intended that the recommendations would be implemented by close co-operation of both public and private healthcare providers together with educational establishments.

The cationic C-terminus of rat Muc2 facilitates dimer formation post translationally and is subsequently removed by furin.

Earlier immunolocalization experiments showed that the extreme cationic C-terminus of the rat intestinal mucin Muc2 (RMC) was present at the base of intestinal goblet cells in the vicinity of ER and golgi compartments, but was not found with the rest of the mucin in apical storage granules. This prompted us to investigate the possibility that an early proteolytic cleavage reaction occurs post-translationally. A plasmid pRMC, encoding the C-terminal 534 amino acids of the mucin, was expressed in COS-7 cells and was shown to undergo cleavage at an R-T-R-R sequence located within the C-terminal 14 amino acids. Cleavage did not occur with the construct RMCfH, a furin site-mutated (A-T-A-A) counterpart of pRMCH (poly His6 tagged RMC). Addition of a furin inhibitor to COS-7 cell incubations also prevented cleavage of RMC and RMCH products. 35S pulse-chase kinetic experiments revealed that a truncated mutant lacking the C-terminal 14 amino acids (pRMCDeltaCT) forms faulty (doublet) dimers in the ER. These were not secreted as efficiently as the normal dimer of wild-type (pRMC) constructs. Thus the cationic C-terminus of rMuc2 apppears to facilitate the correct formation of normal Muc2 domain dimers.

Roles of calreticulin and calnexin during mucin synthesis in LS180 and HT29/A1 human colonic adenocarcinoma cells.

Molecular chaperones are presumed to associate with large secretory mucin glycoproteins during their synthesis in the endoplasmic reticulum (ER), but have not been identified to date. We decided to look for possible involvement of the chaperones calreticulin (CRT) and calnexin (CLN) during synthesis of two similar gastrointestinal mucins, MUC2 and MUC5AC. Pulse-chase labelling of MUC2 and MUC5AC with [(35)S]methionine/cysteine ([(35)S]Promix) was performed using LS180 and HT29/A1 colonic carcinoma cell lines and was followed by immunoprecipitation with anti-mucin and anti-chaperone antibodies. The precipitated labelled mucin precursors were analysed by SDS/PAGE and autoradiography. Using antibodies specific for each mucin, newly synthesized monomeric precursors of both MUC2 and MUC5AC were detected after a 15 min pulse and then disappeared as oligomers were formed during a 2 h chase period. Only homo-oligomers of MUC2 and MUC5AC were present in the cells. Using anti-CRT, the MUC2 monomeric precursor and oligomer were co-precipitated from both cell lines after a 15 min pulse and the oligomer less strongly after a 0.5 h chase, but there was little co-precipitation after a 2 h chase. At this time, MUC2 immunoprecipitated by anti-MUC2 was completely oligomerized and was endo-beta-N-acetylglucosaminidase-resistant, indicating that the mucin had reached the Golgi region. MUC2 co-precipitated with CRT at zero time and 0.5 h was endo-beta-N-acetylglucosaminidase-sensitive; therefore CRT must have associated with MUC2 in the ER. Treatment with tunicamycin (TUN) diminished the binding of MUC2 to CRT, suggesting a requirement for initial N-glycan addition during this process. Using anti-CLN, only a weak co-precipitation of MUC2, compared with that seen with anti-CRT, was detected in LS180 cells. In contrast with the findings for MUC2, there was no co-precipitation of MUC5AC with CRT or CLN from either cell line at the various time points. In conclusion, CRT and CLN appear to be involved in MUC2 synthesis at the stage of folding and oligomerization in the ER. Since no interaction of the chaperones with MUC5AC was detected at a similar stage of synthesis, these two structurally similar secretory mucins seem to have different chaperone requirements in the ER.

SPECT imaging of feet using uniplanar fad-beam collimators.

BACKGROUND: This study was performed to assess the utility of bone SPECT in the feet using a new commercially available uniplanar fan-beam collimator originally designed for cardiac imaging. METHODS: 18 patients with symptoms or signs of probable skeletal pathology in either the foot or ankle were imaged using a two headed gamma camera fitted with uniplanar fan-beam collimators. All patients were imaged 2.5-4 h after administration of 500-750 MBq 99mTc MDP. If indicated planar dynamic and blood pool images were also obtained. The SPECT acquisition was performed in a 128 x 128 matrix, giving a pixel size of 2.00-2.30 mm depending on the radius of orbit. Images were displayed as transaxial, coronal and sagittal slices and a three dimensional volume rendered image and displayed for reading by three readers blind to the clinical results. Sites of abnormal uptake on the foot SPECT scan were then compared with the site of known or suspected pathology and in 17 patients with planar radiology. RESULTS: The SPECT images produced using the uniplanar fan-beam collimators were of good quality in all patients with all three readers finding localisation easiest on the sagittal and three-dimensional images. In 10 patients abnormalities were found which could explain the patient's symptoms or signs and at the site expected from the patient's clinical history. In 5 patients there were abnormalities on the bone scan in the ipsilateral foot but at a different site, all were interpreted as degenerative disease. 2 patients had contralateral degenerative disease to side suggested by the clinical history and no abnormality in the bones of the foot with symptoms. One patient had bilateral degenerative disease. Planar radiology was normal or unhelpful in 13 of the 17 patients in which it was performed. CONCLUSION: SPECT imaging of feet is possible and provides accurate localisation of abnormal uptake when performed using uniplanar fan-beam collimators with a standard acquisition time of 15 min for a double headed gamma camera.

Regulated and unregulated pathways for MUC2 mucin secretion in human colonic LS180 adenocarcinoma cells are distinct.

We have shown previously [McCool, Forstner and Forstner (1994) Biochem. J. 302, 111-118] using pulse-chase labelling of mucin with [3H]threonine that LS180 colonic tumour cells synthesize and secrete MUC2 without the addition of secretagogues. Treatment of the LS180 cells with monensin to disrupt Golgi function was also found to inhibit baseline secretion almost completely. In this paper we show that addition of nocodazole to inhibit microtubule assembly reduced baseline secretion by 53% over a 6 h chase period. In contrast, cytochalasin D did not affect the rate of unstimulated mucin synthesis or secretion, suggesting that baseline secretion is not influenced by disruption of actin microfilaments. In addition, regulated mucin secretion by LS180 cells was studied in response to carbachol, phorbol 12-myristate 13-acetate and A23187. Mucin released in response to secretagogues behaved identically on SDS/PAGE to that secreted under baseline conditions. T84 cells and the B6 subclone of the HT29 cell line responded in a similar manner to LS180 cells and secreted high-molecular-mass mucin which included MUC2 and behaved like LS180 mucin on SDS/PAGE. Neither monensin nor nocodazole significantly affected secretagogue-stimulated mucin secretion. Since these compounds inhibited secretion of labelled mucin under baseline conditions, mucin released by secretagogues must have come from a separate, unlabelled mucin pool in stored granules. Cytochalasin D, on the other hand, caused the release of small amounts of stored mucin, suggesting that actin microfilaments participate in regulated exocytosis. Thus two kinds of mucin secretion occur in LS180 cells. Unregulated secretion depends upon continuous transport of mucin granules from Golgi vesicles to the cell surface and does not utilize stored mucin, whereas regulated secretion involves the release of mucin from storage granules and is not affected by microtubule or Golgi disruption.

Photoreceptor cells in the vitiligo mouse die by apoptosis. TRPM-2/clusterin expression is increased in the neural retina and in the retinal pigment epithelium.

PURPOSE: To determine the mechanism of photoreceptor cell death in the vitiligo mouse, a model of retinal degeneration in which the genetic defect is not retina specific but is instead caused by single point mutation in the microphthalmia (mi) gene that codes for a basic helix-loop-helix DNA transcription factor. METHODS: Detection of apoptotic cells was performed in fixed retinal tissue using the TUNEL assay in animals 1, 2, 4, 6, 8, 16, 32, 40, and 52 weeks. Electron microscopic analysis was used to confirm the morphologic hallmarks of apoptosis, and Southern blot analysis was used to detect internucleosomal DNA fragmentation. Additionally, the expression of a gene associated with apoptosis, TRPM-2/clusterin, was examined. RESULTS: At ages beyond the time of normal retinal programmed cell death, vitiligo retinas had significantly more TUNEL-positive photoreceptor cells and more photoreceptor cells with condensed chromatin than controls. DNA internucleosomal fragmentation ladders were present in vitiligo retinas even as late as 15 weeks, a time well beyond developmental apoptosis in controls. TRPM-2/clusterin mRNA levels in vitiligo neural retinas were similar to controls initially but were two times greater than controls by 12 weeks. Surprisingly, TRPM-2/clusterin mRNA levels were elevated in the retinal pigment epithelium in the mutant; the expression at one week was two times greater than normals and remained elevated for many months, even though retinal pigment epithelial cells showed no morphologic evidence of apoptosis. CONCLUSIONS: The morphologic and biochemical data suggest that photoreceptor cells die by apoptosis in vitiligo mice. The increased retinal TRPM-2/clusterin mRNA levels may be a direct response to these events. The increased expression of this gene in the retinal pigment epithelium, however, may reflect its role in tissue regression and membrane remodeling. Mechanisms by which the mi gene defect might result in the vitiligo retinopathy are proposed.

Reduction of phagosomes in the vitiligo (C57BL/6-mivit/mivit) mouse model of retinal degeneration.

PURPOSE: The vitiligo (C57BL/6-mivit/mivit) mouse has a slowly progressing retinal degeneration, in which photoreceptor cell nuclei are gradually lost and the retinal pigment epithelium (RPE) is unevenly pigmented. The purpose of the present study was to assess the phagocytic ability of the RPE in the vitiligo mouse by determining whether and when a phagocytic burst occurs in affected mice and whether the number of phagosomes varies between control and affected animals. METHODS: Eyes of control and vitiligo mice 4 to 20 weeks of age were embedded in Spurr. Thin sections were cut and examined by electron microscopy to confirm the presence of phagosomes, particularly in the affected animals. Thick (1 micron) sections were cut, and quantitative morphometry was performed at the light microscope level. The length of RPE was determined, and phagosomes were counted in RPE cytoplasmic and microvillous areas. Data were expressed as phagosomes per 1000 microns. RESULTS: The vitiligo mouse has a peak phagocytic episode approximately 2 hours after light onset. The number of phagosomes in 4-week-old affected mice was significantly less than that in controls (13 phagosomes per 1000 microns compared to 30 phagosomes per 1000 microns). By week 8, the number was reduced to approximately 5 per 1000 microns. Phagosome number was not reduced further between weeks 8 and 20 in the affected animal. Macrophage-like cells containing pigment granules and phagosomes were observed in the subretinal space in areas where the rod outer segments had been separated from the RPE. CONCLUSIONS: The vitiligo mouse RPE contains phagosomes, but there are significantly fewer than in controls. It is not known whether a defect in RPE phagocytosis is the direct cause of the retinal defect in this model.

Synthesis and secretion of mucin by the human colonic tumour cell line LS180.

Pulse-chase labelling experiments were performed using the mucin-producing colonic carcinoma cell line LS180. Cells were pulsed with [3H]threonine or [3H]glucosamine and chased in complete media without radiolabel for various lengths of time. From cell and media extracts obtained at each time point, mucin proteins were immunoprecipitated with specific anti-mucin antibodies and analysed by SDS/PAGE and fluorography. At short labelling times with [3H]threonine, without chase, a monomeric thiol-reduction-resistant mucin precursor of apparent molecular mass > 670 kDa was identified. The precursor, in contrast to oligomeric species, was not labelled by [3H]glucosamine but exhibited binding to Vicia villosa isolectin B4, suggesting the presence of some core GalNAc residues. Treatment with tunicamycin to inhibit N-glycosylation had no effect on the apparent mass of the precursor. Identity of the mucin antigen with MUC2 mucin was established by immunoprecipitation with antibodies specific for a MUC2 tandem repeat and C-terminal regions. With increasing chase time the precursor was replaced by thiol-reduction-sensitive mucin oligomers that reached peak intracellular radiolabelling with [3H]threonine by 2 h of chase, and then declined. Only oligomeric mucin was secreted into the medium. Secretion of [3H]threonine-labelled mucin was detectable after 2 h of chase and increased as the cytoplasmic mucin label declined. Monensin inhibited [3H]glucosamine incorporation, sialylation and baseline (non-regulated) mucin secretion without affecting initial [3H]threonine incorporation or oligomerization. Oligomerization and Golgi transport are therefore essential early steps in MUC2 mucin secretion. Oligomerization may follow some core O-glycosylation with GalNAc, but precedes elongation of oligosaccharide chains.

Regulation of mucin secretion in T84 adenocarcinoma cells by forskolin: relationship to Ca2+ and PKC.

The relationship between the adenosine 3',5'-cyclic monophosphate-mediated protein kinase A (PKA)-dependent stimulatory pathway for mucin secretion and Ca(2+)-mediated and protein kinase C (PKC)-mediated secretion was studied in T84 cells, using the postreceptor secretagogues forskolin, A-23187, and phorbol 12-myristate 13-acetate (PMA), the protein kinase inhibitors staurosporine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), high- and low-Ca2+ media, and the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Staurosporine (10(-5) M) inhibited both PMA and forskolin at their maximally effective concentrations, whereas H-7 (5 x 10(-5) M) inhibited only PMA. Stimulation of mucin secretion by forskolin (5 x 10(-5) M) was not significantly affected by the reduction of medium Ca2+ to 47 and 129 nM, equivalent to published values for intracellular Ca2+ concentration ([Ca2+]i). Stimulation by forskolin was reduced by preloading cells with BAPTA, but to a much smaller extent than Ca(2+)-dependent stimulation by A-23187. A-23187-mediated mucin secretion from BAPTA-loaded cells was augmented by high doses of forskolin. Similar concentrations of forskolin had no effect on A-23187-stimulated secretion in calcium-replete cells. Our results indicate that forskolin does not stimulate mucin secretion by increasing Ca2+ entry or releasing Ca2+ from intracellular stores. Forskolin can stimulate mucin secretion in a Ca(2+)-independent manner but is apparently inhibited by high levels of intracellular Ca2+ induced by Ca2+ ionophores in 1.0 mM Ca2+ media.(ABSTRACT TRUNCATED AT 250 WORDS)

Mucin secretion by T84 cells: stimulation by PKC, Ca2+, and a protein kinase activated by Ca2+ ionophore.

T84 adenocarcinoma cells were stimulated to secrete mucin by the phorbol ester phorbol 12-myristate 13-acetate (PMA) and Ca2+ ionophores A23187 and ionomycin. In Ca(2+)-containing media, maximal stimulation by PMA was significantly inhibited by staurosporine, but maximal A23187-stimulated secretion was not affected. Downregulation of protein kinase C (PKC) reduced maximal PMA-stimulated secretion without affecting the response to A23187. Thus PKC activation is not required for maximal Ca(2+)-mediated mucin secretion. PMA stimulated secretion in low-Ca2+ media, with and without intracellular chelation of Ca2+ by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Surprisingly, Ca2+ ionophores also stimulated secretion under the same circumstances. Persistent A23187-stimulated secretion was strongly inhibited by the protein kinase inhibitors staurosporine and H-7. Secretion in Ca(2+)-containing media was also inhibited at submaximal levels of Ca(2+)-ionophore stimulation. These results indicate that PKC and Ca2+ stimulate mucin exocytosis independently. Ca2+ ionophores also stimulate secretion via a protein-kinase dependent pathway. Enhancement of protein kinase inhibition at lower Ca2+ concentrations suggests that the response could be mediated by a Ca2+ ionophore-induced depletion of an intracellular Ca2+ pool.

Human intestinal mucin-like protein (MLP) is homologous with rat MLP in the C-terminal region, and is encoded by a gene on chromosome 11 p 15.5.

A cDNA specific for a human intestinal mucin (MLP) was amplified by PCR from cDNA of cultured human colonic adenocarcinoma cells, LS174T. The human cDNA shared high sequence homology with a corresponding rat intestinal mucin (MLP) cDNA in the 3' terminal region, and hybridized to the same mRNA (approximately 9.0 Kb) that was recognized by a probe for the MUC-2 human intestinal mucin gene. The gene encoding our human mucin peptide also mapped to chromosome 11 p 15.5, the known locus of MUC-2. Our findings suggest that human MLP and MUC-2 are encoded by the same gene and that rat and human intestinal mucin share a common C-terminal amino acid structure.

The T84 human colonic adenocarcinoma cell line produces mucin in culture and releases it in response to various secretagogues.

The T84 colonic adenocarcinoma cell line, which has been used extensively as a model for studies of epithelial chloride secretion, also produces mucin and secretes it in culture. Electron microscopy of fixed sections of cultured cells, along with Immunogold labelling with an antibody to human small intestine (SI) mucin, revealed the presence of goblet-like cells with mucin-containing secretory granules. The mucin was of high molecular mass, had an amino acid composition similar to that of purified human SI and colonic mucins, and competed effectively with SI mucin for binding to the anti-(SI mucin) antibody. A sensitive solid-phase immunoassay specific for intestinal mucins was developed and used to measure mucin secretion by T84 cells. Cultures were treated for 30 min at 37 degrees C with a number of agents known to cause chloride secretion by T84 cell monolayers and the amount of mucin appearing in the medium was measured. Carbachol (1 mM), A23187 (10 microM), prostaglandin E1 (PGE1) (1 microM) and vasoactive intestinal polypeptide (VIP) (0.1 microM) all stimulated mucin release, but histamine (1 mM) had no effect. Whereas VIP is reported to stimulate chloride secretion more strongly than carbachol, it was less effective than carbachol in stimulating mucin secretion. Phorbol 12-myristate 13-acetate (PMA) (0.1-10 microM) also stimulated mucin release strongly, implicating a responsive protein-kinase C-dependent pathway. Additive secretory responses were obtained with combined stimulation by VIP (10 nM-1 microM) and carbachol (1 mM). Responses to stimulation with A23187 (1-10 microM) together with PMA (10 nM-10 microM) suggest that cytosolic Ca2+ concentration is a modulator of PMA activity.

Inhibition of mucin secretion in a colonic adenocarcinoma cell line by DIDS and potassium channel blockers.

The factors which influence the exocytosis of mucins are not well characterized. Since the physical properties of mucins may be affected significantly by the co-secretion of electrolytes and water, we studied the relationship between ion movement and mucin secretion in T84 cells, a human colonic adenocarcinoma cell line which has been well characterized with respect to apical chloride secretion. Secretion of mucin was assessed by immunoassay of mucin appearing in the medium within 30 min of stimulation. Cells were grown on plastic in DMEM/Ham's F12 medium and experiments were carried out at 70% confluence. Mucin secretion was stimulated by the calcium ionophore A23187, or A23187 plus vasoactive intestinal polypeptide. Stimulated mucin secretion was not affected by loop diuretics (furosemide (1 x 10(-3) M) or bumetanide (1 x 10(-4) M)), with or without the addition of ouabain (5 x 10(-5) M) and amiloride (1 x 10(-5) M), making it unlikely that transcellular chloride movements in necessary for mucin secretion. However, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; (1 x 10(-5) and 5 x 10(-5) M) and three potassium channel blockers BaCl2 (1 x 10(-3) and 5 x 10(-3) M), tetraethylammonium chloride (1 x 10(-2) M) and quinine (5 x 10(-4) M) inhibited mucin secretion. A DIDS-sensitive chloride channel or chloride/bicarbonate exchanger and a Ca2(+)-dependent potassium channel may play important roles in mucin secretion. Since plasma membranes are sparingly permeable to DIDS, the DIDS-sensitive site is likely to be on the apical plasma membrane, perhaps at an initiation locus for exocytosis.

Structural requirements of immunoglobulin G for binding to the Fc gamma receptors of the human tumor cell lines U937, HL-60, ML-1, and K562.

Fc receptors (FcR) on U937, HL-60, ML-1, and K562 cells were characterized by using competitive binding assays and EA rosette inhibition studies. Like human monocytes, U937, HL-60, and ML-1 cells bound monomeric human IgG Fc with high affinity and mildly reduced and alkylated human IgG and Fc with a somewhat diminished affinity. In contrast, K562 cells had a much lower affinity for monomeric human IgG or Fc. Concentrations of these proteins as high as 10(-5) M were needed to completely block EA rosette formation. There was no binding of reduced and alkylated IgG and Fc. We assessed the influence of various segments of IgG on FcR interactions by using human pFc', rabbit Facb, mouse IgG2b-IgG2a hybrid Ig, and also studied the effect of reduction of the interchain disulfide bonds. The FcR on all four cell types bound rabbit IgG but not rabbit Facb or human pFc', suggesting that rabbit C gamma 3 domains are required for FcR interaction and that isolated human C gamma 3 domains do not have a human FcR binding site. Murine IgG2a, but not IgG2b, was cytophilic for U937, HL-60, and ML-1 cells. Binding studies with the use of several mouse myeloma variant Ig molecules having hybrid gamma 2b-gamma 2a heavy chains showed that variants with a complete gamma 2a Fc region bound to these FcR-like IgG2a, whereas those having gamma 2a sequences only in the C gamma 3 region and in a short adjacent segment of the C gamma 2 region behaved like IgG2b and did not bind. These results suggested that additional murine gamma 2a sequences are required for FcR binding. Interestingly, the Fc fragments from murine proteins with a complete gamma 2a Fc region bound only to a limited extent. These fragments are shorter than the human IgG1 Fc fragments, and they lack that segment of the hinge region that includes the interchain disulfide bonds. Cleavage of the interchain disulfide bonds of murine Ig having a complete gamma 2a Fc region diminished binding to a similar extent as that observed with human IgG. Together, these findings provide additional insight into the roles of the hinge, C gamma 2, and C gamma 3 regions of human, rabbit, and mouse IgG in their interaction with the FcR of human tumor cells.

Human IgG1 and its Fc fragment bind with different affinities to the Fc receptors on the human U937, HL-60 and ML-1 cell lines.

Measurements were made of the binding of human monomeric 125I-IgG1 and 125I-Fc to U937 cells at room temp. Analyses of the binding data showed that these cells possessed a single class of receptor (FcR) for Fc or IgG and, although both ligands were found to bind to the same number of sites per cell, Fc was found to bind with about twice the affinity of IgG. At 20 degrees C estimates of the forward rate constants and the dissociation rate constants for IgG and Fe were 1.13 and 3.65 X 10(7) M-1 min-1 and 0.33 and 0.57 X 10(-2) min-1 respectively. Independent determinations of the association constants (Ka) under the same experimental conditions gave values of 0.98 X 10(9) M-1 for IgG and 3.1 X 10(9) M-1 for Fc. Thus the Fc fragment of IgG appears to bind to U937 FcR at 3-4 times the rate of IgG and to dissociate at about twice the rate, resulting in higher values of Ka for the Fc-FcR than for the IgG-FcR interaction. Also, in competitive-binding experiments and in EA rosette inhibition assay the Fc fragment was consistently found to be more efficient in FcR binding than IgG. Similar results were obtained using HL-60 and ML-1 cells which possess FcR like those on U937 cells and with IgG1 and Fc prepared from other myelomas. IgG and Fe which had undergone mild reduction and alkylation bound to the same number of FcR per U937 cell as the non-reduced ligands but the affinity of binding was diminished to a similar degree with both ligands, suggesting that the major effect of cleavage of the interchain disulfide bonds on cytophilic binding is due to alteration of the native quaternary relationships of the C gamma 2 and C gamma 3 domains.

Where were you in '82?

1982 was a pivotal year for the US healthcare delivery system. With it came the reality of a new era, an unexplored environment or competition and market reform. The decade ahead means troubled times for some healthcare providers, but it also offers unique opportunities to design and shape a rapidly changing healthcare system. By recognizing trends we are currently experiencing and their impact on group practices, and by identifying what administrators can do to prepare personally and organizationally for the future, medical group practices can place themselves in excellent position for entering the system of the '80s and '90s. The survivors are destined to reap unprecedented economic and professional rewards.