Perioperative change in CA125 is an independent prognostic factor for improved clinical outcome in advanced ovarian cancer.

OBJECTIVE: Despite being the most important prognostic factor for prolonged overall survival in epithelial ovarian cancer (EOC), the measurement of residual disease is hampered by its subjective character. Additional assessment tools are needed to establish the success of cytoreductive surgery in order to predict patients' prognosis more accurately. The aim of this study is to evaluate the independent prognostic value of perioperative CA125 change in advanced stage EOC patients. STUDY DESIGN: We identified all patients who underwent primary cytoreductive surgery for advanced stage (FIGO IIB-IV) EOC between 2008 and 2015, from the Netherlands Cancer Registry. The relative perioperative change in CA125 was categorized into four groups; increase, <50% decline, 50-79% decline and ≥80% decline. Overall survival (OS) was analyzed using Kaplan-Meier survival curves and multivariable cox regression models. RESULTS: We included 1232 eligible patients with known pre- and postoperative CA125 serum levels. Patients with a decline of ≥80% in CA125 levels experienced improved OS compared to those with a decline of <50% (univariable Hazard Ratio (HR) 0.45, 95%CI 0.36-0.57). The prognostic effect of perioperative CA125 change was independent of patient- and treatment characteristics, such as the extent of residual disease after cytoreductive surgery (multivariable HR≥80% 0.52(0.41-0.66)). CONCLUSIONS: This study shows that the perioperative change in CA125 is an independent prognostic factor for overall survival after primary surgery for EOC patients. This pleads for the use of a combined model, consisting of perioperative CA125 change and the outcome of residual disease, in order to predict the prognosis of EOC patients more accurately.

Association of an intact E2 gene with higher HPV viral load, higher viral oncogene expression, and improved clinical outcome in HPV16 positive head and neck squamous cell carcinoma.

To assess the relationship of E2 gene disruption with viral gene expression and clinical outcome in human papillomavirus (HPV) positive head and neck squamous cell carcinoma, we evaluated 31 oropharyngeal and 17 non-oropharyngeal HPV16 positive carcinomas using two PCR-based methods to test for disruption of E2, followed by Sanger sequencing. Expression of HPV16 E6, E7 and E2 transcripts, along with cellular ARF and INK4A, were also assessed by RT-qPCR. Associations between E2 disruption, E2/E6/E7 expression, and clinical outcome were evaluated by Kaplan-Meier analysis for loco-regional recurrence and disease-specific survival. The majority (n = 21, 68%) of HPV16 positive oropharyngeal carcinomas had an intact E2 gene, whereas the majority of HPV16 positive non-oropharyngeal carcinomas (n = 10, 59%) had a disrupted E2 gene. Three of the oropharyngeal tumors and two of the non-oropharyngeal tumors had deletions within E2. Detection of an intact E2 gene was associated with a higher DNA viral load and increased E2/E6/E7, ARF and INK4A expression in oropharyngeal tumors. Oropharyngeal carcinomas with an intact E2 had a lower risk of loco-regional recurrence (log-rank p = 0.04) and improved disease-specific survival (p = 0.03) compared to tumors with disrupted E2. In addition, high E7 expression was associated with lower risk of loco-regional recurrence (p = 0.004) as was high E6 expression (p = 0.006). In summary, an intact E2 gene is more common in HPV16 positive oropharyngeal than non-oropharyngeal carcinomas; the presence of an intact E2 gene is associated with higher HPV viral load, higher viral oncogene expression, and improved clinical outcome compared to patients with a disrupted E2 gene in oropharyngeal cancer.

Centralisation of epithelial ovarian cancer surgery: results on survival from a peripheral teaching hospital.

OBJECTIVE: The objective of this retrospective descriptive study was to assess overall survival and disease free survival of patients treated for epithelial ovarian cancer by a gynaecologic-oncologist in a single Dutch peripheral teaching hospital and to identify independent prognostic factors. STUDY DESIGN: A retrospective series of 242 patients treated for epithelial ovarian cancer between 1999 and 2011 at Meander Medical Centre was reviewed. Data on patient, tumour and treatment characteristics were collected. Outcomes were overall survival and progression free survival. Data were analysed using the Kaplan-Meier method, log-rank test and Cox regression analysis. RESULTS: Median follow-up was 35 months (range 1-203). Staging procedures were performed in 81 patients of which 63% were complete. 61% of patients had advanced stage disease. In 46%, debulking surgery was complete. Five-year overall survival and progression free survival for all patients was 52% and 47%, respectively. Multivariate analysis identified performance status [HR=1.89 and 1.92 for performance status 2, HR=7.01 and 2.69 for performance status 3], FIGO stage [HR=3.59 for stage II, HR=5.43 and 5.64 for stage III, HR=12.17 and 10.21 for stage IV] and residual disease after debulking surgery [HR=2.01 and 1.72 for incomplete debulking] as independent prognostic factors for overall survival and progression free survival respectively. CONCLUSION: Survival after surgery for epithelial ovarian cancer in this cohort is comparable to survival in centralised clinics presented in literature. Partial concentration of cancer care by recruitment of specialised gynaecologic-oncologists in teaching hospitals might be an alternative to complete centralisation of epithelial ovarian cancer treatment in larger cancer centres.

The diagnostic process of cervical cancer; areas of good practice, and windows of opportunity.

OBJECTIVE: Despite an extensive screening programme in The Netherlands, some cases of cervical cancer are still diagnosed in late stages of disease. The aim of the present study was to investigate which elements in the diagnostic process of cervical cancer may be improved. METHODS: This is a retrospective study of 120 patients with cervical cancer diagnosed between January 1st 2008 and June 1st 2010 at the University Medical Center Utrecht. Patient charts, referral information, and pathology results were analyzed. RESULTS: 39.1% of cancer cases were screen or interval detected; the other 60.9% of patients had not been screened, either due to non-attendance or because they fell outside the age range for screening. The final diagnosis of cervical cancer was established by biopsy in 77 (64.2%) and by excision of the cervical transformation zone in 35 (29.2%) of the patients. Fifteen (43%) of these excisions could have been avoided if biopsies would have been taken at the first examination, and had shown invasive cancer. CONCLUSIONS: Cervical cancer screening aims at early detection of precursor lesions to decrease the incidence of cancer. This in-depth analysis suggests that improvement of quality of care is to be expected from correct recognition of cervical cancer by physicians and adjustments of the screening programme to reach younger women and non-responders.

[The dynamics of feed-forward loop depends on regulator type in indirect pathway].

Gene networks contain a recurring motif, called the feed-forward loop, in which a transcription factor regulates target expression directly and indirectly via the second regulator. Here we present the results of mathematical modeling of feed-forward loops with either the transcription factor or miRNA as a repressor in the indirect pathway. We showed that the substitution of the transcription factor with miRNA changes the dynamic behavior of the feed-forward loop and lends new properties critical for biological system functioning.

Follow-up of women after a first episode of postmenopausal bleeding and endometrial thickness greater than 4 millimeters.

OBJECTIVE: To estimate the incidence of recurrent postmenopausal bleeding among women who were diagnosed with an endometrial thickness greater than 4 mm. METHODS: We designed a prospective cohort study and included consecutive women not using hormone replacement therapy, presenting with a first episode of postmenopausal bleeding. We evaluated patients who had an endometrial thickness greater than 4 mm at transvaginal ultrasonography and benign endometrial sampling; presence of carcinoma was ruled out by office endometrial sampling, hysteroscopy, and/or dilation and curettage. Time until recurrent bleeding was measured, and diagnosis at recurrent bleeding was recorded. RESULTS: Among 318 patients who had an endometrial thickness greater than 4 mm, 222 patients had benign histology results and were available for follow-up. During follow-up, 47 (21%, 95% confidence interval 16-27%) patients had recurrent bleeding, with a median time to recurrent bleeding of 49 weeks (interquartile range 18 to 86 weeks). There was no difference with respect to recurrence rate between patients with polyp removal, patients with a normal hysteroscopy, and patients with office endometrial sampling alone at the initial workup. Two patients were diagnosed with atypical endometrial hyperplasia upon recurrent bleeding. CONCLUSION: The recurrence rate of postmenopausal bleeding in women with endometrial thickness greater than 4 mm is 20%. This recurrence rate is not related to incorporation of hysteroscopy or polyp removal at the initial workup. LEVEL OF EVIDENCE: II.

Inadequate office endometrial sample requires further evaluation in women with postmenopausal bleeding and abnormal ultrasound results.

OBJECTIVE: To determine whether further histologic assessment can be omitted after office sampling produced a nondiagnostic specimen. METHODS: Data were retrieved from a prospective cohort study of 913 women presenting with postmenopausal bleeding. This study was limited to women with an endometrial thickness either 5 mm or greater or that could not be measured, and in whom an endometrial biopsy performed in the office yielded nondiagnostic results. RESULTS: Endometrial thickness was nonreassuring or unknown in 516 women, of whom 403 (78.1%) underwent office endometrial sampling. In 66 women the amount of tissue obtained was not sufficient for pathologic characterization. Further investigation revealed an endometrial malignancy in 3 of these 66 women and atypical hyperplasia in 1. CONCLUSION: In women with postmenopausal bleeding and a nonreassuring transvaginal ultrasound evaluation, a nondiagnostic office endometrial sample does not rule out endometrial cancer and further endometrial sampling is advisable.

The relation between age, time since menopause, and endometrial cancer in women with postmenopausal bleeding.

The objective is to assess among women with postmenopausal bleeding the relationship of age and time since menopause on one hand and the presence of endometrial cancer and atypical hyperplasia on the other hand. In a multicenter prospective cohort study, 614 women presenting with postmenopausal bleeding were included. Women underwent transvaginal sonography and, in cases where the endometrial thickness was >4 mm, endometrial sampling. Splines were used to assess the association between each of the continuous variables and (pre)malignancy of the endometrium. Subsequently, univariate and multivariate analysis were performed. The average age for women without (pre)malignancy was 61.7 years (SD 9.8). As malignant and premalignant cases were found to have similar age, these subgroups were merged in the analyses. Age was an independent predictor of (pre)malignancy. In women younger than 55 years, the odds ratio was 1.9 (95% CI: 1.1-3.3) for each year under 55 years of age and 1.03 (95% CI: 1.00-1.06) for each year over 55 years of age. The risk of (pre)malignancy of the endometrium was 4.9% in women less than 3 years postmenopausal versus 19.7% in women more than 20 years postmenopausal. However, in a multivariate analysis only age contributed to the prediction of risk. This study demonstrates that, in postmenopausal women with vaginal bleeding, the risk of (pre)malignancy of the endometrium is low in women under 50 years of age, increases considerably until 55 years of age, and rises only modestly with further advancing age. Future studies should explore whether these findings can be incorporated in the diagnostic work-up of women with postmenopausal bleeding.

[Severe vitamin-D deficiency in more than half of the immigrant pregnant women of non-western origin and their newborns]. / Ernstige vitamine D-deficiëntie bij ruim de helft van de niet-westerse allochtone zwangeren en hun pasgeborenen.

OBJECTIVE: To determine the prevalence of vitamin-D deficiency in pregnant women and their newborns. DESIGN: Descriptive. METHOD: During the period of one year (April 2004-April 2005) 545 pregnant women of Dutch/European origin and 131 pregnant women of non-Western origin (mainly Turkish and Moroccan) were studied during their 10th and/or 30th week of pregnancy for calcidiol (vitamin-D) and calcium levels. The study took place in the Amersfoort region in the center of the Netherlands. In addition, cord blood samples were taken for vitamin-D and calcium levels from the 442 and 81 Dutch/European and non-Western newborns respectively. RESULTS: A severe deficiency was found (calcidiol < 20 nmol/l) in 55% of non-European women compared to 5% of Dutch/West-European women. From the cord blood samples, a severe vitamin-D deficiency (calcidiol < 13 nmol/l) was found in 54% of the newborns of non-European origin compared to 6% of the Dutch/West-European newborns. Vitamin-D concentrations in pregnant women at term were strongly correlated to the concentrations in the newborns' cord blood (R = 0.84). The calcium levels of pregnant women and newborns did not differ significantly between both population groups. CONCLUSION: More than half of the non-European pregnant women and their newborns had a severe vitamin-D deficiency. Screening for vitamin D deficiency and adequate suppletion for this risk group appears to be necessary. The causes and consequences of vitamin-D deficiency in pregnancy are discussed.

Structure of a neutral glycosphingolipid recognized by human antibodies in polyagglutinable erythrocytes from the rare NOR phenotype.

NOR is a rare inheritable polyagglutination phenomenon that has been described in two families. Our recent studies on these erythrocytes showed they contained at least two unique neutral glycosphingolipids, and based on their reactivity with Griffonia simplicifolia IB4 (GSL-IB4) isolectin (Kusnierz-Alejska, G., Duk, M., Storry, J. R., Reid, M. E., Wiecek, B., Seyfried, H., and Lisowska, E. (1999) Transfusion 39, 32-38), both oligosaccharide chains terminated with an alpha-galactose residue. The reactivity with GSL-IB4 suggested that these oligosaccharide chains terminated with a Galalpha1-->3Gal- sequence and that anti-NOR agglutinins were common human anti-Galalpha1-->3Gal xenoantibodies. In this report we describe the structure of one NOR component (NOR1) that migrated on thin-layer chromatographic plates in the region of pentaglycosylceramides. Treatment of this sample with alpha-galactosidase and beta-N-acetylhexosaminidase was followed by high-performance thin-layer chromatography with product detection by lectins and the anti-Gb4 monoclonal antibody. The results suggested that NOR1 was an alpha-galactosylated Gb4Cer with a beta-N-acetylhexosaminidase-resistant GalNAc residue. Gas phase disassembly by ion trap mass spectrometry analysis showed the sequence to be Hex1-->4HexN1-->3Hex1-->4Hex1-->4Hex linked to a ceramide composed of C18 sphingosine and a C24 monounsaturated fatty acid. Together these data indicate NOR1 to be a novel Galalpha1-->4GalNAcbeta1-->3Galalpha1-->4Galbeta1-->4 Glc-Cer structure. Additionally it has been shown that NOR glycolipids are recognized by human antibodies that were distinct from the known anti-Galalpha1-->3Gal xenoantibodies.

Lectin and anti-carbohydrate antibody assays using chemically modified ligands.

Microtiter plate assays and 'lectinoblotting' with the use of biotinylated lectins are sensitive and easy to perform methods that can be combined with simple procedures of chemical modifications of glycoproteins immobilized on ELISA plates or blots (desialylation by mild acid hydrolysis, Smith degradation, beta-elimination). These modifications are helpful in the determination of lectin and anti-carbohydrate antibody specificities, or in the characterization of glycoconjugates by means of lectins and antibodies.

Red blood cell antigens responsible for inherited types of polyagglutination.

The three described types on inheritable polyagglutination are related to altered carbohydrate structures in glycoproteins or/and glycolipds on the erythrocyte surface. HEMPAS, a condition causing anemia and other pathological symptoms, is characterized by impaired biosynthesis of N-glycans, mostly those carried by band 3 and band 4.5 erythrocyte membrane proteins. Cad erythrocytes have abnormal glycophorin O-glycans, structurally related to the more common human Sd(a) and murine CT determinants, and accumulate an Sd(a)-like ganglioside. NOR erythrocytes express recently detected abnormal alpha-galactose-terminated glycosphingolipids, which strongly react with G. simplicifolia IB4 isolectin, but do not react with human anti-Galalpha1-3Gal antibodies.

Isolation and characterization of glycophorin from nucleated (chicken) erythrocytes.

A sialoglycoprotein fraction was isolated from chicken erythrocytes by two methods based on the phenol extraction or chloroform/2-propanol extraction of differently prepared erythrocyte membranes. Both preparations gave in SDS-PAGE two major PAS-stained bands (GP2 and GP3), which migrated as 60- and 33-kDa species, respectively, compared to reference proteins, or as 44- and 23-kDa molecules, compared to human glycophorins. Some less abundant slower migrating PAS-stained components, antigenically related to GP2 and GP3, also were detected. No evidence for the presence of antigenically distinct glycoproteins of leukosialin type was obtained. Interconversion in SDS-PAGE, similar carbohydrate composition, and similar antigenic properties of GP2 and GP3 indicated that they are a dimer and monomer, respectively, of the same glycoprotein which shows properties that allow it to be classified as a glycophorin. Lectin binding studies and methylation analysis of beta-elimination products of chicken glycophorin preparation showed the presence of O-glycans and N-glycans. The major O-glycans include sialylated Galbeta1-3GalNAc units and more complex GlcNAc-containing chains. Among the N-glycans, there are complex-type biantennary structures with a bisecting GlcNAc residue, accompanied by chains with additional antennas linked to alpha-mannose residues. A characteristic feature of the chicken glycophorin is a relatively high proportion of N-glycans to O-glycans, compared to the glycophorin A from human erythrocytes.

The murine monoclonal antibody NaM26-4C6 identifies a common structure on band 3 and glycophorin C.

The murine monoclonal antibody NaM26-4C6 (IgM class), obtained from the splenocytes of a BALB/c mouse immunized with human umbilical cord red blood cells, was characterized by agglutination test and immunoblotting analysis. The structure of the NaM26-4C6 epitope was further elucidated by using a series of peptides synthesized on pins. The antibody agglutinated untreated and chymotrypsin-treated but not trypsin- or neuraminidase-treated human erythrocytes. Agglutination-inhibition test demonstrated that the antibody recognizes an epitope located on the N-terminal trypsin-sensitive portion of glycophorin C. The antibody bound on immunoblots to glycophorin C, and also to the band 3 protein and its 69-kDa N-terminal fragment but did not bind to desialylated and de-O-glycosylated glycophorin C. Peptide mapping allowed localization of the binding site on the 23-kDa N-terminal intracellular peptide of band 3. The antibody binds to the amino-acid sequences 22EDPDIP27 of band 3 protein and 15SLEPDPGM22 of glycophorin C, and residues D and P were found to be essential. The new epitope identified by NaM26-4C6 corresponds to a linear amino acid sequence located on the N-terminal intracellular portion of band 3 and to a more complex structure involving oligosaccharide chains on the N-terminal extracellular domain of GPC.

NOR polyagglutination and Sta glycophorin in one family: relation of NOR polyagglutination to terminal alpha-galactose residues and abnormal glycolipids.

BACKGROUND: This report describes the characterization of polyagglutinable red cells (RBCs), identified in two generations of a Polish family. CASE REPORT: Untreated and modified RBCs of the proposita (TS) were tested by serologic methods, using human sera, antibodies, lectins, and inhibitors of agglutination. Moreover, glycophorins were characterized by sodium docecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, and glycolipids were purified, fractionated by thin-layer chromatography, and detected with Ricinus communis agglutinin I (RCA-I, specific for galactose residues) and Griffonia simplicifolia IB4 lectin (GSL-IB4, specific for Gal alpha1-3Gal- structure). Some of the experiments were also performed on RBCs of members of TS's family. RESULTS: Polyagglutination, found in four members of TS's family, was identified as the second case of an earlier described NOR polyagglutination. The polyagglutination was decreased by treating the RBCs with alpha-galactosidase and was inhibited by a neutral glycolipid fraction from NOR+ RBCs. Detection of neutral glycolipids of TS's RBCs on the thin-layer plate by RCA-I and GSL-IB4 revealed the presence of components that were not detectable in control RBCs. Moreover, Western blotting of RBC membranes from five family members with glycophorin monoclonal antibodies and agglutination assays with anti-St(a) and anti-Dantu sera identified the presence of St(a) glycophorin in four members of the family, two of whom were NOR+ and two NOR-. CONCLUSION: Our results showed that two rare features of TS's RBCs, NOR polyagglutination and St(a) glycophorin, are inherited independently, and that NOR+ RBCs contain neutral glycolipids with an abnormal oligosaccharide structure, most likely terminated with alpha-galactosyl residues.

Purification of human anti-TF (Thomsen-Friedenreich) and anti-Tn antibodies by affinity chromatography on glycophorin A derivatives and characterization of the antibodies by microtiter plate ELISA.

The TF and Tn antigens were obtained from glycophorin A (GPA) by desialylation under mild acidic conditions and by desialylation followed by Smith degradation, respectively. A method of purification of anti-TF and anti-Tn antibodies from human sera by affinity chromatography on the immobilized asialoGPA (TF antigen) and on asialo-agalactoGPA (Tn antigen), respectively, is described. Purity of the antibodies was demonstrated by SDS-polyacrylamide gel electrophoresis and their specific reactivity with TF or Tn antigens was shown using hemagglutination and the microtiter plate ELISA. A high unspecific binding of human immunoglobulins to the ELISA plates was encountered, therefore optimal conditions for the most specific binding of the antibodies to the target antigens were selected. Problems of the unspecific binding of immunoglobulins were more difficult to overcome when the antibodies were determined in whole sera by their binding to antigen-coated ELISA plates.

Role of sialosyl Lewis(a) in adhesion of colon cancer cells--the antisense RNA approach.

To study whether the adhesion of colon cancer cells to E-selectin can be directly affected by changes in the expression level of sialosyl Le(a) antigen we created a specific loss-of-function phenotype. A stable subclone (CX-1.1) with high expression of sialosyl Le(a) structure, obtained from a heterogenous population of colon carcinoma CX-1 cells, was transfected with an expression vector containing a fragment of cDNA for alpha1,3/4-fucosyltransferase in antisense orientation. After transfection, the cell line was isolated which did not express sialosyl Le(a) antigen and lacked the alpha1,3/4-fucosyltransferase activity, despite an unchanged level of mRNA specific for this enzyme. It was found that the specific lack of expression of sialosyl Le(a) carbohydrate structure on the surface of colon cancer cells completely abolished their adhesion to E-selectin. To evaluate which cellular glycoconjugates carry sialosyl Le(a) antigen, glycoproteins as well as glycolipids of CX-1.1 cells were analysed for the expression of this structure. Anti-sialosyl Le(a) antibodies detected multiple glycoprotein bands with apparent molecular masses of 65-280 kDa on western blots, and an intense band representing sialosyl Le(a)-ganglioside on a thin-layer chromatogram. Using O-sialoglycoprotease from Pasteurella haemolytica and an alkaline beta-elimination procedure, it was shown that protein-linked sialosyl Le(a) structures are carried mostly by mucin-type glycoproteins. However, treatment of CX-1.1 cells with O-sialoglycoprotease did not decrease either their binding to E-selectin-expressing Chinese hamster ovary cells, or binding of anti-sialosyl Le(a) antibodies to the cell surface. These results suggested that cleavage of sialomucins uncovered cryptic sialosyl Le(a)-ganglioside, which was inaccessible for the antibody and E-selectin in untreated cells. This hypothesis was confirmed to some extent by the higher accessibility of gangliosides to galactose oxidase on the surface of O-sialoglycoprotease-treated CX-1.1 cells, comparing to untreated cells. We propose that glycoproteins as well as gangliosides carrying sialosyl Le(a) structures, when properly exposed and present in high density on surface of cancer cells, can effectively support the adhesion of cancer cells to E-selectin.

beta-Elimination of O-glycans from glycoproteins transferred to immobilon P membranes: method and some applications.

Selective beta-elimination of O-glycans from glycoproteins transferred from electrophoretic gels onto Immobilon P membranes is described. The experiments were performed with erythrocyte membrane proteins, in which glycophorins are the major poly-O-glycosylated components, and with lysates of human colon cancer cells CX-1.1. Lectins and monoclonal antibodies against peptidic, glycopeptidic, and carbohydrate epitopes were used to examine the effect of degradation. Experiments with erythrocyte membrane proteins showed that after heating the blots in 0.055 M NaOH for 16 h at 40 degrees C the O-glycans of glycophorins were undetectable, while N-glycans and peptidic epitopes of proteins were detected with unchanged or even increased intensity compared to untreated blots. The method was used to show that most protein-linked sialyl-Lea epitopes present on CX-1.1 cancer cells are located on O-glycosidic chains. Moreover, beta-elimination on the blots allows examination of the dependence of peptidic epitopes on O-glycosylation. This was shown using monoclonal antibodies specific for blood group M- or N-related epitopes of glycophorin A (GPA). Most of these antibodies recognize glycopeptidic epitopes dependent on O-glycosylation and, therefore, they did not detect GPA on NaOH-treated blots. Some less frequent anti-M antibodies cross-reacting with the rare GPA variant of Mg type are specific for a peptidic epitope which is unrelated to the MN blood group-specific amino acid sequence in unglycosylated peptides, but is recognized in GPA-M only in the glycosylated antigen. These antibodies, which showed specificity for GPA-M on untreated blots, detected GPA-M, GPA-N, and glycophorin B on NaOH-treated blots.

Blood group MN-dependent difference in degree of galactosylation of O-glycans of glycophorin A is restricted to the GalNAc residues located on amino acid residues 2-4 of the glycophorin polypeptide chain.

Glycophorin A (GPA) of human erythrocytes contains a minor number of unsubstituted GalNAc residues (Tn receptors) which are recognized by Moluccella laevis lectin (MLL). The lectin reacts better with blood group N- than M-type of GPA which suggests a higher number of Tn receptors in GPA-N than in GPA-M. To find out whether this difference is restricted to a defined domain of GPA, the N-terminal tryptic glycopeptides of GPA-M and GPA-N (a.a. residues 1-39) and their fragments obtained by degradation with CNBr (a.a. residues 1-8 and 9-39) were analyzed. The untreated and desialylated glycopeptides were tested as inhibitors of MLL in ELISA, and the content of GalNAc-ol was determined in the products of beta-elimination of the asialoglycopeptides by gas-liquid chromatography/mass spectrometry. The asialoglycopeptides 1-39 and 1-8 derived from GPA-N showed about 2 and 4 times higher content of non-galactosylated GalNAc residues, respectively, and higher reactivity with MLL than their counterparts derived from GPA-M, while asialoglycopeptides 9-39 of GPA-M and GPA-N did not show such differences. These results demonstrate that higher expression of non-galactosylated GalNAc in GPA-N than in GPA-M is confined to GalNAc residues located in the amino-terminal portion of GPA polypeptide chain, between the blood group M- and N-specific amino acid residues 1 and 5.

Degradation of glycophorin A of human erythrocytes in patients with myelo- or lymphoproliferative disorders: possible role of neutrophil proteases.

We have previously reported that glycophorin A (GPA) of human erythrocytes (carrying blood group M and N determinants) was totally digested by incubation of erythrocytes with human neutrophil elastase (HNE) and cathepsin G (CathG). The membrane-bound GPA fragments fractionated by SDS-PAGE gave characteristic patterns of bands detected by immunoblotting with the monoclonal antibody PEP80. Erythrocytes were incubated with HNE and CathG at low enzyme concentrations, similar to those found in vivo. Characteristic electrophoretic patterns of bands derived from a partial GPA digestion were observed and these patterns were different for both enzymes and different from those obtained after total GPA digestion. GPA was also partially digested by incubation of erythrocytes with granulocytes in the presence of Ca2+ and calcium ionophore and electrophoretic pattern of digestion products was similar to that obtained with low doses of HNE. No GPA digestion products were detected after treatment of erythrocytes with plasmin and kallikrein. Untreated erythrocytes of 21 patients with various myelo- or lymphoproliferative disorders were tested by SDS-PAGE of RBC membranes and immunoblotting with the anti-GPA PEP80 antibody. GPA degradation products, resembling those formed by a mild CathG treatment of control RBC, were detected in nine patients. GPA fragmentation was in some cases accompanied by a reduced expression of blood group MN determinants. No distinct relation was observed between the occurrence of GPA degradation in erythrocytes and increases in plasma concentrations of HNE-alpha1-proteinase inhibitor (alpha1-PI) complex considered to be an indication of a release of neutrophil proteinases in vivo. However, the results suggested that a partial GPA degradation in haematological proliferative disorders may occur due to limited proteolysis by neutrophil proteinases, most likely by CathG.