MUC1 epithelial mucin (CD227) is expressed by activated dendritic cells.

The MUC1 mucin (CD227) is a cell surface mucin originally thought to be restricted to epithelial tissues. We report that CD227 is expressed on human blood dendritic cells (DC) and monocyte-derived DC following in vitro activation. Freshly isolated murine splenic DC had very low levels of CD227; however, all DC expressed CD227 following in vitro culture. In the mouse spleen, CD227 was seen on clusters within the red pulp and surrounding the marginal zone in the white pulp. Additionally, we confirm CD227 expression by activated human T cells and show for the first time that the CD227 cytoplasmic domain is tyrosine-phosphorylated in activated T cells and DC and is associated with other phosphoproteins, indicating a role in signaling. The function of CD227 on DC and T cells requires further elucidation.

Phosphorylation of the cytoplasmic domain of the MUC1 mucin correlates with changes in cell-cell adhesion.

The MUC1 epithelial mucin is expressed by glandular epithelial cells and is often highly expressed and associated with poor prognosis in adenocarcinomas. Tyrosine phosphorylation of the highly conserved cytoplasmic tail of MUC1 (MUC1-CT) has been demonstrated in MUC1 transfected cells and in one breast cancer cell line. In addition, associations between MUC1 and secondary signalling molecules have been demonstrated in breast cancer cell lines. MUC1 clearly plays a role in intracellular signalling, since we were able to demonstrate tyrosine phosphorylation of the MUC1-CT in breast and ovarian cancer cell lines and in primary cultures of serous ovarian cancers. We were unable to modulate MUC1-CT phosphorylation using conditioned media from cell lines showing the highest levels of signalling. However, in several breast and ovarian cancer cell lines, we clearly showed the highest levels of MUC1-CT tyrosine phosphorylation occurred during recolonisation of culture dishes or in low-density adherent cultures. We now hypothesise that phosphorylation changes may reflect either involvement of MUC1 in cell motility or a redistribution of MUC1 in the membrane during the course of cell-cell adhesion.

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.

Monoclonal antibody recognizing a core epitope on mucin.

Monoclonal antibody TH1 (IgM) was prepared by immunizing mice with deglycosylated (TFMSA-treated) cystic fibrosis mucin. TH1 reacted strongly with TFMSA treated cystic fibrosis mucin but not with the fully glycosylated mucin, indicating reactivity with a core mucin epitope. TH1 showed no reactivity with ovine mucin (98% of glycans as sialyl-Tn) but reacted strongly with desialylated ovine mucin, indicating the epitope for this mab was the Tn-antigen (O-linked GalNAc). However, TH1 showed no reactivity with Tn-positive red blood cells, and the binding of TH1 was not inhibited by GalNAc at 2.5 mg/ml, illustrating the importance of the peptide sequence to which the GalNAc is attached. TH1 stained the majority of cancers of the colon, lung, stomach, ovary, breast, and cervix, and the cellular distribution of this antigen in normal tissue suggested reactivity with immature mucin. This antibody appears to be a useful reagent for the detection of immature mucin.

Antibodies reactive with the protein core of MUC1 mucin are present in ovarian cancer patients and healthy women.

Antibodies reactive with peptide epitopes on the core protein of MUC1 epithelial mucin have been demonstrated in some patients with adenocarcinomas. Because these epitopes can be exposed on MUC1 in the serum of healthy women, we measured concentrations of MUC1-reactive antibodies in the serum of healthy pregnant and non-pregnant women, and in patients with benign and malignant ovarian tumours. Antibodies were measured in an enzyme-linked immunosorbent assay utilising a synthetic peptide corresponding to a 105-amino-acid segment of the MUC1 tandem repeat region (5.25 repeats). MUC1-reactive antibodies were always of an IgM isotype and concentrations were highest in young healthy women and declined progressively with age (P=0.0006) concomitantly with increasing serum MUC1 levels (P=0.003). Regardless of age, antibody levels were lower in cancer patients than in healthy women (P<0.0001), but MUC1 levels were much higher in cancer patients (P<0.0001). Although high antibody levels were associated with greater survival in ovarian cancer (P=0.015), multivariate regression analysis showed that this was not a significant independent prognostic indicator after consideration of the International Federation of Gynaecology and Obstetrics (FIGO) stage, histological type, serum MUC1 levels and age. Serial measurement of MUC1 and MUC1 antibodies during treatment in 18 patients with ovarian cancer and throughout pregnancy in 10 women showed a negative correlation between alterations in MUC1 and MUC1 antibodies. These results show that MUC1-peptide-reactive antibodies are present in the serum of healthy women and women with cancer and that they probably form immune complexes with MUC1, but provide no evidence for an augmentation of the humoral immune response to MUC1 in ovarian cancer

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.

Progesterone stimulates production and secretion of MUC1 epithelial mucin in steroid-responsive breast cancer cell lines.

The effects of oestrogen and progesterone on expression of the MUC1 epithelial mucin were examined in MCF7 and ZR-75-1 breast cancer cells grown in steroid-free medium. Oestrogen caused an increase in cell growth and both cellular and secreted MUC1 in both cell lines. However, after correcting for increases in cellular protein, there was no clear changes. Progesterone, in combination with oestrogen, caused significant increases (over 2-fold, P<0.01) in cellular and secreted MUC1 when compared with oestrogen alone in both cell lines despite no or small increases in cellular protein. Growth of ZR-75-1 cells in a competitive inhibitor of O-glycosylation demonstrated that the increased detection by ELISA was not due to altered glycosylation. Progesterone may modulate expression of MUC1 in steroid-responsive breast cancer cells.

Serum mucin antigen (CASA) as a marker of amiodarone-induced pulmonary toxicity.

Amiodarone is used to treat life-threatening cardiac arrhythmias. Amiodarone-induced pulmonary toxicity (APT) can be difficult to diagnose. APT may result in increased mucus production and mucin expression. Thus, serum mucin-1 was evaluated as a marker for amiodarone-induced pulmonary toxicity. Concentrations of mucin-1 in peripheral blood were determined using cancer-associated serum antigen (CASA) assay in patients taking amiodarone. Eight of ten patients who developed major amiodarone toxicity had high serum CASA levels. Patients with toxicity had a significantly higher mean rank CASA concentration compared with those without major toxicity. CASA shows potential as a marker for amiodarone-induced toxicity, particularly pulmonary toxicity.

Prostate-specific antigen (PSA) and cancer-associated serum antigen (CASA) in distinguishing benign and malignant prostate disease.

The Prostate-Specific Antigen (PSA) and the Cancer-Associated Serum Antigen (CASA) assay for the MUC1 mucin were compared in the serum of 303 patients with malignant or benign prostatic disease. Using cutpoints of 4, 10, and 20 micrograms/l, PSA was elevated in 93%, 81%, and 64% of patients with prostate cancer (n = 113), with corresponding specificities of 55%, 84%, and 96% in benign prostate disease (prostatic hyperplasia or prostatitis, n = 190). Using the recommended cutpoint of 4 Units/ml, CASA was elevated in 38% of patients with prostate cancer, with a specificity of 91% in benign disease. PSA and CASA showed a poor correlation in prostate cancer (r = 0.367) and benign disease (r = 0.158), and CASA was elevated in some PSA negative samples. Used together, PSA > or = 20 micrograms/l and CASA > or = 4 kU/l gave perfect specificity in benign disease, with a corresponding sensitivity of 29% (positive and negative predictive values of 100% and 70%, respectively). However, this combination gave no improvement over the use of PSA alone, with sensitivity 47% when the cutpoint was raised to give perfect specificity. These data suggest that CASA is of little use as an adjunct to PSA in the differentiation of benign and malignant prostate disease.

Monoclonal antibodies recognising sialyl-Tn: production and application to immunochemistry.

In order to develop reagents that can detect the exposed sialyl-Tn antigen (NeuAc alpha 2,6GalNAc alpha 1-O-Ser/Thr) on tumour-associated mucins, we have prepared monoclonal antibodies (mabs 3C2 and 3D1, both IgM) against ovine submaxillary mucin (OSM; > 98% of glycans as sialyl-Tn). These mabs showed strong reactivity with OSM and bovine submaxillary mucin (BSM; 50% of glycans as sialyl-Tn) but did not react with desialylated OSM or BSM. Sialic acid at 1 mg/ml did not significantly inhibit mab binding to OSM, suggesting that the linkage to GalNAc may be important for mab binding. 3C2 and 3D1 also showed similar reactivity to sialyl-Tn reactive mab B72.3, and detected B72.3 captured OSM in a sandwich ELISA. In Western blotting of mucus from a patient with a mucinous ovarian tumour, the mabs reacted with high molecular weight (> 200 kDa) species. In immunohistochemistry, these mabs showed strong reactivity with most cancers of the colon, lung, and stomach, and also some tumours of the ovary and breast. There was only limited reactivity in normal tissue from these sites. The antibodies should be useful reagents for the detection of the sialyl-Tn antigen in human cancers.

CA15-3, CASA, MSA, and TPS as diagnostic serum markers in breast cancer.

This is the first comparison of the three mucin based tests CA15-3, CASA, and MSA, and the cytokeratin-related TPS assay in breast cancer. The mucin markers were superior to TPS in receiver-operator analysis, though no marker was of use in the diagnosis of malignancy due to low sensitivity. Using cutpoints that gave 95% specificity in benign disease (n = 83), corresponding sensitivities in pre-treatment breast cancer (n = 123: 13 in situ, 54 stage I, 45 stage II, 4 stage III, 7 stage IV) were 17% (CA15-3), 16% (CASA), 13% (MSA), and 8% (TPS), with a strong relationship between marker levels and disease stage. These assays did not always detect the same patients, and the use of CA15-3 combined with CASA gave the highest sensitivity (23%), though this was not significantly better than the use of CA15-3 alone. Despite detecting similar antigens, these assays can show markedly different responses in some patients, indicating that one mucin-based test cannot be substituted for another.

Predictive value of the combination of serum markers, CA125, CASA and TPS in ovarian cancer.

The serum markers CA125, CASA and TPS were compared, with particular reference to the clinical applications of these tumor markers in the management of patients with ovarian cancer (discrimination of benign and malignant disease; indicating prognosis; predicting preclinical recurrence), (i) Using recommended cut-off points, CASA (>/=4 U ml-1 and TPS (>/=80 U l-1 showed similar sensitivities in ovarian carcinoma (56% and 57% respectively), though these were lower than with CA125 (85% >/= 35 U ml-1). The combined use of CA125 with either CASA or TPS at higher cut-off points excluding benign disease (CA125> 345 U ml-1; CASA> 6 U ml-1; TPS> 359 U l-1) improved the discrimination of ovarian cancer from benign adnexal masses (100% positive predictive value with 65% of ovarian cancers detected with CA125-CASA, 61% with CA125-TPS vs 46% with CA125 alone). The combined preoperative use of these markers may therefore assist the general gynecologist in avoiding potentially difficult oncologic surgery. (ii) TPS was the best preoperative indicator of prognosis, possibly due to its association with cell proliferation, while CASA was superior as a postoperative prechemotherapeutic prognostic indicator, possibly due to it being a more accurate indicator of residual disease than the other markers, or the surgeons' assessment. Similarly, CASA gave the best differentiation of patients with minimal residual disease (<1 cm) into those with a good or poor prognosis, (iii) CA125 and CASA each detected preclinical recurrence after surgery and adjuvant therapy in seven of 11 patients (mean lead times 4.6 and 3.1 months respectively) while TPS detected four of these patients (mean lead time 2.4 months). The combined use of CA125 with either assay led to the preclinical detection of eight of 11 patients, with the mean lead time increased to 5.3 months with the CA125-CASA combination.

Monoclonal antibodies reacting with the mucin-specific lectin from Sambucus sieboldiana (SSA-M).

Monoclonal antibodies (MAbs) were prepared against the mucin-specific lectin from Sambucus sieboldiana (SSA-M). The majority of MAbs reacted with both free SSA-M and SSA-M bound to porcine mucin. However, MAbs SS16, SS18, and SS19 did not react with mucin-bound lectin, suggesting that these MAbs may react at or near the lectin binding site. Some MAbs also showed reactivity with lectins other than SSA-M (e.g. ConA, Pisum sativum, and Saphora japonica lectins) suggesting possible sequence homology between SSA-M and other lectins. These MAbs should be useful reagents for immunoaffinity purification of SSA-M, characterizing SSA-M structure and binding, and metabolic studies on mucins.

Serum markers CASA and CA 15-3 in ovarian cancer: all MUC1 assays are not the same.

The serum MUC1 markers CASA and CA 15-3 were compared with CA 125 in the serum of patients with ovarian cancer and in pregnant women. Used individually, CASA and CA 15-3 gave sensitivities of 54 and 56% in pre-operative ovarian carcinoma (n = 50), though these were lower than with CA 125 (84%). CASA levels were elevated in 3 women with a negative CA 125, while CA 15-3 was elevated in 2 of these women. The combined use of CA 125 with CASA or CA 15-3 led to the preclinical detection of recurrence in 4/5 patients, with mean lead times of 3.6 and 4.3 months, respectively. Of particular interest was the marked difference in reactivity observed with CASA and CA 15-3 in some patients, despite both assays utilising monoclonal antibodies (MAbs) that react with the MUC1 mucin. CA 15-3 and CASA showed a lower than expected correlation in patients with ovarian cancer (r = 0.70), with some patients having high concentrations of one mucin marker and low concentrations of another. Furthermore, different marker profiles were observed when monitoring the progress of patients with these markers. Marked differences between CA 15-3 and CASA were also observed in the serum of pregnant women (n = 10), where CASA showed marked elevation (mean 33.6 times cutpoint) and CA 15-3 did not (mean 0.88 times cutpoint). These data suggest that the specificities of the MAbs used in these assays affect the glycoform of MUC1 detected, and that it should not be assumed that all MUC1 assays will behave in the same manner.

Transmission of airway pressure to pleural space during lung edema and chest wall restriction.

To investigate the influence of positive end-expiratory pressure (PEEP) on hemodynamic measurements we examined the transmission of airway pressure to the pleural space during varying conditions of lung and chest wall compliance. Eight ventilated anesthetized dogs were studied in the supine position with the chest closed. Increases in pleural pressure were similar for both small and large PEEP increments (5-20 cmH2O), whether measured in the esophagus (Pes) or in the juxtacardiac space by a wafer sensor (Pj). Increments in Pj exceeded the increments in Pes at all levels of PEEP and under each condition of altered lung and chest wall compliance. When chest wall compliance was reduced by thoracic and abdominal binding, the fraction of PEEP sensed in the pleural space increased as theoretically predicted. Acute edematous lung injury produced by oleic acid (OA) did not alter the deflation limb pressure-volume characteristics of the lung, provided that end-inspiratory volume was adequate. With the chest and abdomen restricted OA was associated with less than normal transmission of airway pressure to the pleural space, most likely because the end-inspiratory volume required to restore normal deflation characteristics was not attained. Together these results indicate that the influence of acute edematous lung injury on the transmission of airway pressure to the pleural space depends importantly on the peak volume achieved during inspiration.

Regional blood flow during cardiopulmonary resuscitation in dogs using simultaneous and nonsimultaneous compression and ventilation.

We studied regional blood flow (QR) using radiolabeled microspheres and measured hemodynamic variables in 20 anesthetized dogs in normal sinus rhythm and during ventricular fibrillation treated with cardiopulmonary resuscitation (CPR). Nonsimultaneous compression and ventilation CPR (NSCV-CPR) was performed in seven dogs with a pneumatic piston that gave 50 chest compressions/min with an open airway with 10 ventilations at an airway pressure of 33 mm Hg interposed between each fifth and sixth compression. Simultaneous compression and ventilation (SCV-CPR) was performed in seven dogs with the piston and in six other dogs with a circumferential pneumatic vest. Both devices gave 30 compressions/min simultaneously with 30 ventilations that elevated airway pressure to 80 mm Hg., The abdomen was bound during SCV-CPR. Regional blood flow (mean +/- SD) to the cerebral hemispheres, cardiac ventricles, and kidneys, expressed as ml/min/100 g tissue, was 3.1 +/- 4.0, 3.4 +/- 3.3 and 1.5 +/- 1.5, respectively, during NSCV-CPR; 11.5 +/- 5.9, 4.9 +/- 4.7 and 2.7 +/- 2.7 during SCV-CPR (vest); and 16.2 +/- 7.2, 11.0 +/- 4.0 and 20.1 +/- 20.2 during SCV-CPR (piston) (all p less than 0.05 compared with NSCV-CPR). These results indicate that QR to all organs studied is reduced below normal sinus rhythm levels during CPR for ventricular fibrillation, QR to the brain is proportionately greater than QR to the heart and kidneys, and QR to the brain is greater with both forms of SCV-CPR than with NSCV-CPR.

Venous air embolism.

Venous air embolism causes injury primarily by obstruction of blood flow from the right side of the heart to the left. This is due to mechanical obstruction of the right ventricular pulmonary outflow tract and pulmonary vasculature and to poorly understood pulmonary vasoconstrictive mechanisms. Venous air embolism can result in considerable hypoxemia from ventilation-perfusion maldistribution and shunt. With large emboli, systemic hypotension, myocardial ischemia, and arrhythmias can occur and result in death. One should be familiar with the clinical setting where embolism occurs, as prevention is the best treatment. When air embolism is suspected, placement of the patient in the left lateral decubitus position, initiating closed chest massage or, if possible, aspiration of air through a right atrial or Swan-Ganz catheter are all acceptable forms of treatment. The patient should also be given 100% oxygen.