Immunosuppressive human anti-CD83 monoclonal antibody depletion of activated dendritic cells in transplantation.

Current immunosuppressive/anti-inflammatory agents target the responding effector arm of the immune response and their nonspecific action increases the risk of infection and malignancy. These effects impact on their use in allogeneic haematopoietic cell transplantation and other forms of transplantation. Interventions that target activated dendritic cells (DCs) have the potential to suppress the induction of undesired immune responses (for example, graft versus host disease (GVHD) or transplant rejection) and to leave protective T-cell immune responses intact (for example, cytomegalovirus (CMV) immunity). We developed a human IgG1 monoclonal antibody (mAb), 3C12, specific for CD83, which is expressed on activated but not resting DC. The 3C12 mAb and an affinity improved version, 3C12C, depleted CD83(+) cells by CD16(+) NK cell-mediated antibody-dependent cellular cytotoxicity, and inhibited allogeneic T-cell proliferation in vitro. A single dose of 3C12C prevented human peripheral blood mononuclear cell-induced acute GVHD in SCID mouse recipients. The mAb 3C12C depleted CMRF-44(+)CD83(bright) activated DC but spared CD83(dim/-) DC in vivo. It reduced human T-cell activation in vivo and maintained the proportion of CD4(+) FoxP3(+) CD25(+) Treg cells and also viral-specific CD8(+) T cells. The anti-CD83 mAb, 3C12C, merits further evaluation as a new immunosuppressive agent in transplantation.

Practical blood dendritic cell vaccination for immunotherapy of multiple myeloma.

Therapeutic vaccination combined with new drugs may cure multiple myeloma (MM). We have developed a bio-process to purify CMRF-56 monoclonal antibody (mAb) and a standard operating procedure to immunoselect blood dendritic cells (BDC). Leucopheresed mononuclear cells were cultured overnight, labelled with CMRF-56 mAb and BDC prepared using a clinical scale immunoselection system. The mean BDC yield from healthy donors was 48% (n = 6, purity 28%). Preparations from MM patients (n = 6, yield 47%, purity 35%) primed cytotoxic T lymphocytes (CTL) to clinically relevant MM antigens. This procedure can be performed readily by clinical cell manufacturing units to facilitate BDC vaccination studies.

The MUC3 gene encodes a transmembrane mucin and is alternatively spliced.

Epithelial mucins are a family of secreted and cell surface glycoproteins expressed by epithelial tissues and implicated in epithelial cell protection, adhesion modulation and signaling. The gene encoding human MUC3 (hMUC3), localised to chromosome 7q22, is most highly expressed in the small intestine. It has previously been reported to be a non-transmembrane mucin with minimal homology to its suggested orthologues from rat (rMuc3) and mouse (mMuc3). RT-PCR was performed to investigate the carboxyl terminus of the published sequence of hMUC3 from normal colon and small intestine tissues and also from a series of 10 colorectal cancer cell lines. Two distinct PCR products were identified. In contrast to the previously published hMUC3 sequence, which terminates shortly after a single cysteine-rich EGF-like domain, conceptual protein translation of the dominant and largest PCR product identified two extracellular cysteine-rich EGF-like domains separated by an N-glycosylation-rich domain and a potential coiled-coil region, followed by a putative transmembrane region and a 75 amino acid cytoplasmic tail. The smaller of the two PCR products was found to be an alternative splice variant of MUC3 including the first EGF-like domain but lacking part of the second EGF-like domain and the transmembrane region. Nine out of 10 colorectal cancer cell lines were found to express MUC3. Interestingly, one of the cell lines, LoVo, expressed predominantly the alternative splice form lacking a transmembrane domain. Structural homology of the new protein sequence of hMUC3 with rMuc3 and mMuc3 indicates it is closely related to the rodent proteins and is likely to be involved in ligand-binding and intracellular signaling. The new finding that MUC3 encodes a transmembrane molecule presents a new paradigm for the structure of this mucin and the manner in which it may function.

Peritoneal fluid from ovarian cancer patients stimulates MUC1 epithelial mucin expression in ovarian cancer cell lines.

The MUC1 epithelial mucin is a transmembrane glycoprotein that is frequently but variably over-expressed by adenocarcinomas. It is used as a diagnostic serum tumour marker and is a candidate target for tumour immunotherapy. Peritoneal fluid (PF) samples from ovarian cancer patients were investigated for their ability to modulate MUC1 expression in 6 ovarian cancer cell lines which showed a range from very low to high endogenous MUC1 expression. Cell lines were cultured in 20% PF for 4 days, fixed in situ and MUC1 assayed by ELISA. MUC1 expression was stimulated by some PF samples in 5 of 6 lines tested. MUC1 expression in the PE04 cell line (very low endogenous expression) was increased by 35 of 36 PFs tested (p < 0.05); stimulation varied between PFs but was greater than with 100 IU/mL hu-r-gamma-interferon. Western blotting confirmed the stimulation of MUC1 in PE04 cells and FACS showed an increase in the proportion of cells expressing MUC1. The active factor was partially purified by gel filtration and was shown to stimulate PE04 cells in a dose-dependent manner. Concentrations of IL1beta, IL4, IL6, IL8, IL10, TNF-alpha, TGF-beta and GM-CSF were often very high in PF and varied substantially between different PF samples but did not correlate with the degree of MUC1 stimulatory activity.

The fate of ingested 14C-urea in the urea breath test for Helicobacter pylori infection.

The metabolic fate of the radioactive carbon in the 14C-urea breath test for Helicobacter pylori was investigated in 18 subjects. After ingestion of labelled urea, breath was sampled for 24 h, and urine was collected for 3 days. Subjects were designated high or low expirers on the basis of their breath counts, and this agreed well with H. pylori serologic analyses. When given 185 or 37 kBq of 14C-urea, 51% (SD = 16%, n = 11) of the label was recovered from the breath of high expirers, and 7% (SD = 3%, n = 7) from the breath of low expirers. The mean combined urinary and breath recovery for high expirers was 86% (SD = 7%), and for low expirers it was 97% (SD = 3%). It is concluded that the long-term retention of 14C from ingested 14C-urea is low. The results enable a more accurate estimation to be made of radiation exposure resulting from the 14C-urea breath test.

Antibacterial action of the urease inhibitor acetohydroxamic acid on Helicobacter pylori.

The urease inhibitor acetohydroxamic acid (AHA) was assessed for its bacteriostatic and bactericidal effects on Helicobacter pylori. For eight isolates of H pylori, the minimum inhibitory concentration (MIC) was either 200 mg/l or 400 mg/l. Interactions between AHA and antimicrobial drugs used to treat H pylori were also determined. For most isolates AHA reduced the MIC for colloidal bismuth subcitrate (CBS), tetracycline, metronidazole, and amoxicillin. In a few isolates, however, AHA increased the minimum bactericidal concentration (MBC) for these antimicrobial treatments. In vitro AHA is active against H pylori and it interacts with other agents directed against H pylori.

Molecular weight of gastric mucus glycoprotein is a determinant of the degree of subsequent aspirin induced chronic gastric ulceration in the rat.

Mucus was sampled from the gastric mucosal surface of anaesthetised rats. Three weeks later these rats were orally dosed each day with aspirin (375 mg/kg) for six months. Then the number and size of the aspirin induced chronic gastric ulcers were assessed. Gel filtration chromatography of the mucus samples showed that mucus glycoprotein was present in both high and low molecular weight forms. There was a natural variation between individual rats in the percentage of glycoprotein in the high molecular weight form (mean = 58.9%; SD = 9.6%; n = 23). This variation correlated strongly with the degree of subsequent aspirin induced chronic gastric ulceration (r = -0.85, p less than 0.001). This is the first time that a pre-existent variability in a mucosal defence factor has been shown to predict susceptibility of the stomach to chronic ulceration.

Peptic erosion of gastric mucus in the rat.

1. The effect of pepsin on the loss of mucus glycoprotein from the gastric epithelial mucus layer was studied in the rat. 2. Pepsin was instilled into the gastric lumen, and luminal contents were subsequently assayed. 3. Glycoprotein loss increased with luminal pepsin, up to a concentration of 1 mg pepsin/ml. 4. Luminal glycoprotein had a molecular size distribution intermediate between subunit, and native mucus glycoprotein of the epithelial mucus layer. 5. Incubation of gastric epithelial scrapings with pepsin demonstrated that insoluble, native mucus glycoprotein was rapidly degraded to soluble glycoprotein of similar molecular size distribution to that found in vivo in the lumen.

Analytical errors in clinical laboratories as assessed by an interlaboratory survey.

Combined errors ar the commonest systematic errors in laboratory results and occur with most tests studied. In these errors, results are reduced (or, less commonly, increased) by a factor, and this effect is compensated by the addition (or subtraction) of a constant amount. The effect is that results are in error in opposite direction at high and low levels. Inconsistency is predominant and is due mainly to imprecision, although for some tests (iron, cholesterol, calcium, and triglycerides among those studied) other factors such as non-specificity are significant. An interlaboratory survey based upon external method assessment using linear regression analysis provided objective information about analytical error in laboratories which is not usually obtained, while at the same time meeting the usual functions of surveys in the quality audit of performance.

Kober reaction kinetics and their influence on the design of assays for oestrogens in urine during pregnancy.

Rates of reaction of different oestrogens in Kober reagent vary greatly. Rate constants were measured between 100 degrees C and 150 degrees C. Oestradiol, oestrone, 16-oxo-oestradiol, 16 alpha hydroxyoestrone, 16-epioestriol and urine pool show two sequential first order reactions at 100 degrees C; oestriol and its conjugates give a single reaction (slower than the other oestrogens except for the very slow oestretrol). Above 120 degrees C differences decrease, all oestrogens having one rate for Kober product formation: the decay reaction, which is also first order, becomes significant. Oestriol and its conjugates have relatively high apparent activation energies in the Kober reaction (120-138 kJmol-1) compared to other oestrogens studied (105-124 kJmol-1). The apparent activation energy for the decay reaction is the same within experimental error (115 +/- 3 kJmol-1). This is consistent with a common product formed from oestrogen reacting with Kober reagent. Analytical methods must respond similarly to major urinary oestrogens. Appropriate conditions include 100 degrees C for at least 20 min or 135 degrees C for 3 to 4 minutes.

PIP: Because rates of reaction of different estrogens in Kober reagent vary greatly, the rate constants were measured between 100 and 150 degrees centrigrade. This temperature dependency was studied in reactions with estradiol, estrone, estriol 16-oxo-estradiol, 16 alpha hydroxyestrone, 16-epiestriol, and urine pool to determine its effects on the outcomes of urine pregnancy tests. Estradiol and estrone and their respective conjugates showed 2 sequential first-order reactions at 100 degrees, whereas estriol and its conjugates showed only a single reaction, which was much slower than that for the other estrogens except for the very slow estretrol rate. When temperatures of assay were raised above 120 degrees centigrade, differences among the reactions decreased, and all estrogens had 1 rate for Kober product formation. At this point, the decay reaction, which is also first order, becomes significant. Estriol and its conjugates had relatively high apparent activation energies in the Kober reaction (120-138 kJmol(1)) compared with other estrogens studied (105-124). The apparent activation energy for the decay reaction was the same within experimental error (115+ or -3), which is consistent with a common product formed from estrogen reacting with Kober reagent. Therefore, appropriate conditions for analyzing major urinary estrogens include 100 degrees centigrade for at least 20 minutes or 135 degrees centigrade fro 3-4 minutes.

Regression analysis in interlaboratory surveys: a case study with cholesterol and triglycerides.

1. A new interlaboratory survey design, that uses regression analysis to compare results from each laboratory with target values, was tested using cholesterol and triglyceride analyses. The fifty New Zealand laboratories involved showed considerable interlaboratory variation (CV = 8% to 27% for cholesterol, 13% to 113% for triglycerides), 30% and 40% of which was associated with systematic differences between laboratories. 2. End-of-period summaries using regression analysis confirmed the presence of systematic errors. These were either simple types caused apparently by incorrect standardisation (regression slope, B not equal to 1.0) or inappropriate blank correction (intercept, A not equal to zero) or complex types presumably due to nonlinearity or nonspecificity. Graphical display of results from each laboratory aided fault diagnosis and allowed the detection of between-run standardisation differences. 3. Method comparison studies were made: the only highly significant result being lower precision achieved by enzymatic cholesterol methods compared with other colorimetric methods.

Routine serum lipid analysis in New Zealand.

For two years pairs of serum specimens with a wide range of cholesterol and triglyceride concentrations were regularly dispatched each month to all New Zealand medical laboratories known to measure blood lipids. Four weeks after specimen dispatch each laboratory received a report which displayed all results along with the overall means, standard deviations, and results from "reference laboratories". Six-monthly summaries were prepared for each laboratory in which the previous 12 results were compared with the corresponding "target values" by regression analysis. This allowed classification of inaccuracy into one or more of three categories. Random error (imprecision) explained most of the discrepancies, but systematic errors also contributed strongly to the observed interlaboratory variation. No single class of laboratories performed significantly differently from any others. Approximately 60 percent of the 16000 cholesterol analyses done each month in New Zealand, and 40 percent of the 10000 triglyceride analyses, are performed with precision thought to be adequate for clinical usage.

Simulation of laboratory errors and their effects on interlaboratory quality-control programs.

The random errors in an analytical method are additive and can be classified into analytical response-dependent and -independent terms. Non-random errors, caused by systematic faults in the analytical procedure, are not always distriguishable from the random errors, but some cases of non-linear assay response and unsuitable standardisation can be studied usefully in models without random error. Interlaboratory quality control programs cannot distinguish systematic and random error until the pattern of results on a number of specimens, or pairs of specimens, can be studied. In this case linear regression analysis is a powerful method for distinguishing different forms of error especially when response-dependent random errors do not predominate. The range of concentrations used for regression whould be as wide as that in which quantitative distinctions are used in clincal diagnosis and treatment. Preliminary reports, of the results on which the regression analysis is based, are most suitably presented on Youden diagrams with paired specimens.

A design for interlaboratory quality-control programs.

A design for interlaboratory quality-control programs is described. Speimens are despatched in pairs to participating laboratories. The results returned by laboratories are compared with each other and with reference results, but the frequent brief reports which are prepared are not regarded as the main laboratory assessments, although these reports can alert laboratories to gross imprecision and inaccuracy. When sufficient results have been returned, a linear regression analysis is carried out between results from each laboratory and the reference results. The statistics obtained from the regression data provide a concise source of information about the form of inaccuracy (imprecision and systematic error) present.

Contributions of other sterols to the estimation of cholesterol.

The responses of 5alpha-cholestan-3beta-ol, 5alpha-cholest-7-ene-3beta-ol and cholesta-5,7-dien-3beta-ol, normally found in human serum, were examined by: (1) the Liebermann-Burchard reaction, (2) the Zak (ferric chloride) reaction, (3) an enzymatic cholesterol method monitored by estimating the amount of hydrogen peroxide produced, (4) an enzymatic cholesterol method monitored by observing the change in absorbance at 240 nm, and (5) gas chromatography. The results show that none of these methods is specific for cholesterol; contributions from the sterols examined range from zero to more than 150% relative to cholesterol. For the first four methods contributions depend on the conditions under which each test is performed.