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.

Characterization of an interleukin 6 cytokine family antagonist protein from a marine sponge, Callyspongia sp.

An inhibitor of IL-6 binding to the human hepatoma line HepG2 and myeloma cell line U266 was identified in a saline extract of the marine sponge, Callyspongia sp. Functional activity, measured through the increase in haptoglobin production by HepG2 cells stimulated with IL-6, could be strongly inhibited by the extract. Similarly, IL-6-induced production of IgM by the B cell line SKW6.4 was substantially reduced. In neither cell line was there evidence of toxicity produced by the extract. Other sponges of the Callyspongia species were found to contain analogous activity. The activity was destroyed by trypsin treatment or boiling of the extract, suggesting that the inhibition is due to a protein. When the binding of IL-6 to its receptor complex was dissected in vitro, inhibition of binding of IL-6 to soluble receptor by the extract was not detected, but binding of the IL-6-sIL-6R complex to soluble gp130 was inhibited in a dose-dependent fashion. This was borne out in cellular assays since the extract inhibited activation of HepG2 cells stimulated with oncostatin M or leukemia inhibitory factor, cytokines which also use gp130 for signal transduction. These results suggest that the Callyspongia extract contains a protein which blocks the interaction of the IL-6 family of cytokines with their signal transduction moiety, gp130. Elucidation of the structure and mode of action of such a protein would be helpful in designing gp130 antagonists to inhibit the functions of this cytokine family, overproduction of which has been associated with cancer and pathologies of autoimmune disease and AIDS.

The heterologous expression and characterization of human prostaglandin G/H synthase-2 (COX-2).

The open reading frame of human cyclooxygenase-2 was cloned by pcr amplification of IL-1 beta stimulated human dermal fibroblast cDNA. The coding region was used to construct a recombinant baculovirus which when used to infect Sf9 cells directed the expression of recombinant human cyclooxygenase-2. The heterologously expressed enzyme was characterized and found to display all salient features of cyclooxygenase. Large-scale microsomal preparations of infected cells yielded more than 20 units of enzyme with a specific activity of 240 nmoles prostaglandin product/mg protein.

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.

Identification of formyl Met-Leu-Phe in culture filtrates of Helicobacter pylori.

Helicobacter pylori synthesizes and secretes a substance which co-chromatographs and is antigenically cross-reactive with the bacterial chemotactic peptide fMet-Leu-Phe. Using reverse phase and affinity chromatography this substance has now been purified. Carboxypeptidase Y microsequencing has verified that this material is fMet-Leu-Phe. The infiltration of polymorphonuclear leucocytes to sites of H. pylori infection may be a response to mucosal permeation of soluble, diffusable bioreactive substances such as fMet-Leu-Phe.

Neutrophil activation by Helicobacter pylori.

Helicobacter pylori infection of the stomach is accompanied by a persistent polymorphonuclear leukocyte (PMNL) infiltrate of the mucosa. The aim of this work was to study the activation of human PMNL by substances produced by H pylori. Filtered H pylori conditioned media stimulated a significant PMNL oxidative burst (p less than 0.002). This was equal to 26% of the maximal response stimulated by the PMNL chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP, 1 mumol/l). The response to FMLP was prolonged by the combined presence of complement inactivated human anti-H pylori plasma and conditioned medium (p less than 0.002). High pressure liquid chromatography of an extract of conditioned medium showed a fraction that stimulated PMNL, eluted, and antigenically cross reacted with FMLP. Washed H pylori cells, and those opsonised with complement inactivated human anti-H pylori plasma, did not induce a significant oxidative burst. Opsonized H pylori, however, prolonged the oxidative burst induced by FMLP (p less than 0.02). In conclusion, H pylori synthesizes and secretes a substance, probably FMLP, that may account for the PMNL accumulation that accompanies H pylori infections. Immune complexes composed of H pylori antigen and specific antibody potentiate the PMNL oxidative burst. This combination of H pylori derived products, and host PMNL and antibodies, may be involved in the mucosal damage observed in H pylori associated gastritis.

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.

Nonspecific esterases in normal human blood cells: characterization of isoenzymes by isoelectric focusing in polyacrylamide gel.

Using the high resolution technique of isoelectric focusing in polyacrylamide gel, isoenzymatic components of nonspecific esterase were resolved in esterase-rich extracts obtained from normal human monocytes, lymphocytes, granulocytes and platelets. In the case of each cell type, different isoenzymatic patterns of nonspecific esterase activity could be visualized. These studies extend the cytochemistry of nonspecific esterases by detection of previously undescribed isoenzymatic components, and suggest that the elaboration of nonspecific esterase isoenzymes may be a function of cellular differentiation.