Granule Glycoprotein Research Articles

The Olfactomedin-4-Defined Human Neutrophil Subsets Differ in Proteomic Profile in Healthy Individuals and Patients with Septic Shock

The specific granule glycoprotein olfactomedin-4 (Olfm4) marks a subset (1–70%) of human neutrophils and the Olfm4-high (Olfm4-H) proportion has been found to correlate with septic shock severity. The aim of this study was to decipher proteomic differences between the subsets in healthy individuals, hypothesizing that Olfm4-H neutrophils have a proteomic profile distinct from that of Olfm4 low (Olfm4-L) neutrophils. We then extended the investigation to septic shock. A novel protocol for the preparation of fixed, antibody-stained, and sorted neutrophils for LC-MS/MS was developed. In healthy individuals, 39 proteins showed increased abundance in Olfm4-H, including the small GTPases Rab3d and Rab11a. In Olfm4-L, 52 proteins including neutrophil defensin alpha 4, CXCR1, Rab3a, and S100-A7 were more abundant. The data suggest differences in important neutrophil proteins that might impact immunological processes. However, in vitro experiments revealed no apparent difference in the ability to control bacteria nor produce oxygen radicals. In subsets isolated from patients with septic shock, 24 proteins including cytochrome b-245 chaperone 1 had significantly higher abundance in Olfm4-H and 30 in Olfm4-L, including Fc receptor proteins. There was no correlation between Olfm4-H proportion and septic shock severity, but plasma Olfm4 concentration was elevated in septic shock. Thus, the Olfm4-H and Olfm4-L neutrophils have different proteomic profiles, but there was no evident functional significance of the differences in septic shock.

Neutrophil azurophilic granule glycoproteins are distinctively decorated by atypical pauci- and phosphomannose glycans

While neutrophils are critical first-responders of the immune system, they also cause tissue damage and act in a variety of autoimmune diseases. Many neutrophil proteins are N-glycosylated, a post-translational modification that may affect, among others, enzymatic activity, receptor interaction, and protein backbone accessibility. So far, a handful neutrophil proteins were reported to be decorated with atypical small glycans (paucimannose and smaller) and phosphomannosylated glycans. To elucidate the occurrence of these atypical glycoforms across the neutrophil proteome, we performed LC-MS/MS-based (glyco)proteomics of pooled neutrophils from healthy donors, obtaining site-specific N-glycan characterisation of >200 glycoproteins. We found that glycoproteins that are typically membrane-bound to be mostly decorated with high-mannose/complex N-glycans, while secreted proteins mainly harboured complex N-glycans. In contrast, proteins inferred to originate from azurophilic granules carried distinct and abundant paucimannosylation, asymmetric/hybrid glycans, and glycan phosphomannosylation. As these same proteins are often autoantigenic, uncovering their atypical glycosylation characteristics is an important step towards understanding autoimmune disease and improving treatment.

Loss of tolerance to glycoprotein 2 isoforms 1 and 4 is associated with Crohn's disease of the pouch.

Zymogen granule glycoprotein 2 (GP2) is a major autoantigen of Crohn's disease-specific pancreatic autoantibodies. To test a link between loss of tolerance to isoforms of GP2 and pouch disorders in a cross-sectional study in ulcerative colitis patients with ileal pouch-anal anastomosis (IPAA). Serum samples of 117 consecutive ulcerative colitis patients after IPAA were tested for presence of Anti-GP2 isoforms 1 (GP21 ) & 4 (GP24 ) IgG and IgA as well as anti-Saccaromyces cervisiae (ASCA) IgG and IgA antibodies in a blinded fashion via enzyme-linked immunosorbent assay. Pouch disorders were diagnosed based on clinical, endoscopic, histological and radiographic criteria. Crohn's disease of the pouch was defined as involvement of the small bowel mucosa proximal to the ileal pouch with Crohn's disease, development of perianal complications or pouch fistula more than 3months after ileostomy closure. Positivity and level of Anti-GP21 IgG (AUC 0.77; P<0.001 & P=0.02, respectively), Anti-GP24 IgG (AUC 0.74; P<0.001 & P=0.025, respectively) and Anti-GP24 IgA (AUC 0.77; P<0.001 to P=0.018, respectively) were specifically associated with Crohn's disease of the pouch. Anti-GP2 was not associated with endoscopic or histological pouch disease activity index. Neither positivity nor levels of ASCA IgG (AUC 0.63; P=0.12 & P=0.35, respectively) or ASCA IgA (AUC 0.67; P=0.38 & P=0.53) were associated with pouch phenotypes. The novel anti-GP21 and GP24 antibodies are associated with Crohn's disease of the pouch in ulcerative colitis patients after IPAA. Serological anti-GP2 antibodies could aid in diagnosis of Crohn's disease of the pouch.

Prevention of venous thromboembolism after resection of primary liver cancer with low molecular weight heparin and its association with P-selectin, lysosomal granule glycoprotein, platelet activating factor and plasma D-dimer.

We aimed at analyzing the efficacy of low molecular heparin in the prevention of venous thromboembolism (VTE) after resection of primary liver cancer and at exploring the correlation of VTE with P-selectin (CD62P), lysosomal granule glycoprotein (CD63), platelet activating factor (PAF) and plasma D-dimer (D-D). A total of 233 patients treated with primary liver cancer resection in our hospital from February 2014 to October 2016 were enrolled in this study. The patients were randomly divided into the observation group (n=117) and the control group (n=116). The observation group received a subcutaneous injection of low molecular weight heparin at 2-7 days after surgery, and the control group was not treated with anticoagulation. The prevalence of VTE and the changes of CD62P, CD63, PAF, and D-D before and after treatment were compared between the two groups. The VTE prevalence after surgery, the changes of CD62P, CD63, PAF, D-D before and after surgery and the correlation of the above indexes with VTE were analyzed. The prevalence of VTE in the observation group was 0.85% (1/117), which was lower than that of the control group (13.79%) (16/116); the difference was statistically significant (p<0.05). There was no significant difference in blood coagulation function between the two groups before and after operation (p>0.05). The CD62P, CD63, PAF, and D-D of the two groups were continuously decreased after the operation, and the serum CD62P, CD63 and plasma D-D of the observation group 6 d and 10 d after operation were lower than that of the control group; the difference was statistically significant (p<0.05). The serum CD62P, CD63 and plasma D-D in the VTE group 6 d and 10 d after operation were lower than those in the non-VTE group; the differences were statistically significant (p<0.05). Low molecular weight heparin can effectively prevent VTE after primary liver cancer resection. Regularly monitoring CD62P, CD63, PAF, and D-D in patients after the operation is pivotal for early diagnosis, evaluation and treatment of VTE.

Antibodies against glycoprotein 2 display diagnostic advantages over ASCA in distinguishing CD from intestinal tuberculosis and intestinal Behçet's disease.

Objectives:There is an increasing need to identify reliable biomarkers for distinguishing Crohn’s disease (CD) from other gastrointestinal disorders sharing similar clinical and pathological features. This study aimed at evaluating the diagnostic potential of antibodies to zymogen granule glycoprotein GP2 (aGP2) in a large, well-defined Chinese cohort with a special focus on their role in discriminating CD from intestinal Behçet's disease (BD) and intestinal tubercolosis (ITB).Methods:A total of 577 subjects were prospectively enrolled, including 171 patients with CD, 208 patients with ulcerative colitis (UC), 71 with BD, 57 with ITB and 70 healthy controls (HC). aGP2 and anti-Saccharomyces cerevisiae antibodies (ASCA) were determined by ELISA. Perinuclear antineutrophil cytoplasmic antibodies were tested by indirect immunofluorescent assay.Results:aGP2 IgG and IgA levels were significantly elevated in patients with CD compared with those in patients with UC, intestinal BD, and ITB and HC. Conversely, ASCA IgG levels were not different between CD and intestinal BD patients, whereas ASCA IgA levels did not discriminate CD from intestinal BD and ITB patients. aGP2 IgA and IgG displayed a better assay performance (larger areas under the curve) over ASCA IgA and IgG in differentiating CD from disease controls (P<0.05). ASCA IgA did not discriminate CD from disease controls. aGP2 IgA and/or IgG was significantly associated with penetrating disease (B3) and ileal CD (L1) (P<0.05), whereas ASCA IgA and/or IgG was not.Conclusions:In comparison with ASCA, aGP2 distinguishes CD from intestinal BD or ITB as disease controls more efficiently, aiding in the differential diagnosis of IBD.

Preliminary findings on the expression of plasma CD63, CD62P, and PAI 1 in patients with acute cerebral infarction

To investigate the expression and clinical significance of lysosomal granule glycoprotein 63 (CD63), P-selectin (CD62P) and endothelial cell plasminogen activator inhibitor (PAI-1) in patients with acute cerebral infarction. A total of 106 patients with acute cerebral infarction (ACI) admitted to our hospital from January to July in 2017 were selected as the patient group; 80 healthy subjects for physical examination in our hospital were selected as the control group. The expression levels of serum CD63, CD62P, and PAI-1 of the subjects were detected. The levels of CD63, CD62P, and PAI-1 in the serum of the patient group were significantly higher than those in the control group. There was a positive correlation between serum CD63 and CD62P (r = 0.672, P &lt; 0.05) in the patient group. There was a positive correlation between serum CD63 and PAI-1 (r = 0.643, P &lt; 0.05) in the patient group. There was also a positive correlation between serum CD62P and PAI-1 (r = 0.601, P &lt; 0.05) in the patient group. Moreover, in other subtypes of cerebral infarction, the expression of CD63, CD62P, and PAI-1 was significantly higher than that of lacunar infarction. CD63, CD62P, and PAI-1 are highly expressed in peripheral blood mononuclear cells (PBMC) and serum of patients with ACI, which may be closely related to the occurrence and development of patients with ACI. These indices may be used as indicators of clinical diagnosis and prognosis in patients with ACI.

Serologic Anti-GP2 Antibodies Are Associated with Genetic Polymorphisms, Fibrostenosis, and Need for Surgical Resection in Crohn's Disease.

The presentation of Crohn's disease (CD) is heterogeneous and often leads to serious complications and need for surgery. We tested serum anti-zymogen granule glycoprotein 2 (GP2) antibodies, including its novel isoform alpha, for association with genetic variants, diagnosis, disease stratification, and prediction of CD courses in a combined cross-sectional and cohort study. Serum samples of 303 CD, 108 ulcerative colitis, 72 other inflammatory gastrointestinal diseases, and 206 controls without predominant gastrointestinal diseases controls (HC) were tested for the presence of Anti-GP2 and Anti-Saccharomyces cervisiae (ASCA) by enzyme-linked immunosorbent assay. Genetic analysis was performed using the Illumina Immunochip. GP2 IgA and IgG had the highest discriminatory capability for CD versus ulcerative colitis and CD versus inflammatory gastrointestinal diseases. We identified an association of GP2 IgA and IgG each with 5 distinct single-nucleotide polymorphisms. Levels of anti-GP2 IgG were moderately associated with ileal disease location. Interestingly, both, anti-GP2 IgA and IgG were exclusively associated with the occurrence of stenosis and need for surgery, independently of disease location, but not with fistulizing CD, early disease onset or disease activity. ASCA IgG and IgA were qualitatively and quantitatively linked to CD, CD complications, and need for surgery. Increased levels of ASCA IgG and IgA and positivity for ASCA IgG, but neither levels nor positivity for GP2 IgG or IgA were predictive of the earlier occurrence of complications or surgery. Anti-GP2 antibodies may aid as a tool for diagnosis and differentiation of CD and could indicate a more complicated CD course.

Serum Anti-GP2 Antibody Isoforms 1 and 4 Are associated With Crohnʼs Disease of the Pouch in Patients With Ileal-Pouch Anal Anastomosis (IPAA)

Introduction: IPAA is the standard therapy for ulcerative colitis (UC) patients requiring colectomy. Inflammatory and non-inflammatory complications of the ileal pouch are common. Serologic antimicrobial antibodies, such as pANCA, cBir1 or anti-glycan antibodies have been linked to pouchitis in IPAA subjects, but limited accuracy restricts their clinical use. Zymogen granule glycoprotein 2 (GP2) is the major autoantigen of CD-specific pancreatic autoantibodies. We tested two novel serologic anti-GP2 isoforms for association with pouch disorders in a cross-sectional analysis in IPAA patients. Methods: 157 serum samples of 143 consecutive and eligible pouch patients (mean age 43.6 +/- 13.4 years, 45.5% female) were obtained from the pouch clinic of a tertiary academic center. Serial samples were available for 13 subjects. Pouch disorders were diagnosed based on clinical, endoscopic, histologic and radiographic criteria. Presence of Anti-GP2 IgG and IgA Isoforms 1 & 4 was tested in serum samples using enzyme-linked immunosorbent assay (ELISA). Clinical data was recorded prospectively (pre-operative diagnosis, indication for colectomy, pouch type, pouch complications). Analysis was performed using Kruskal-Wallis, Pearson's chi square, Wilcoxon signed rank or Fisher's exact tests. Results: Median interval from construction of IPAA to sample procurement was 7.1 years (25th percentile 2.1; 75th percentile 13.2). 89.5% of participants had a J-pouch. Upon sample procurement, patient phenotypes included: normal pouch n=66, irritable pouch syndrome n=5, acute pouchitis n=17, refractory pouchitis n=24, CD of the pouch n=16, cuffitis n=30, other n=22. Presence of Anti-GP2 IgG Isoform 1 (p < 0.001) & 4 (p < 0.001) and Anti-GP2 IgA Isoform 1 (p=0.022) & 4 (p < 0.001) was associated with CD of the pouch. Presence of Anti-GP2 IgG Isoform 4 was linked to cuffitis (p=0.006). No marker was associated with pre-operative CD. Median interval time between serum procurement in IPAA subjects with serial samples was 3 months. There was no significant change in serum levels of Anti-GP2 antibodies over time. Conclusion: This study demonstrates a link between the two isoforms of Anti-GP2 antibodies with CD of the pouch in IPAA patients. Anti-GP2 was not associated with a pre-operative diagnosis of CD and anti-GP2 levels were stable over time. Serologic anti-GP2 antibodies could aid in diagnosis of CD of the pouch after IPAA.

Diagnostic Potential of Zymogen Granule Glycoprotein 2 Antibodies as Serologic Biomarkers in Chinese Patients With Crohn Disease.

The need for reliable biomarkers for distinguishing Crohn disease (CD) from ulcerative colitis (UC) is increasing. This study aimed at evaluating the diagnostic potential of anti-GP2 antibodies as a biomarker in Chinese patients with CD. In addition, a variety of autoantibodies, including anti-saccharomyces cerevisiae antibodies (ASCA), perinuclear anti-neutrophil cytoplasmic antibodies (PANCA), anti-intestinal goblet cell autoantibodies (GAB), and anti-pancreatic autoantibodies (PAB), were evaluated.A total of 91 subjects were prospectively enrolled in this study, including 35 patients with CD, 35 patients with UC, 13 patients with non-IBD gastrointestinal diseases as disease controls (non-IBD DC), and 8 healthy controls (HC). The diagnosis of IBD was determined based on the Lennard-Jones criteria, and the clinical phenotypes of the IBD patients were determined based on the Montreal Classification.Anti-GP2 IgG antibodies were significantly elevated in patients with CD, compared with patients with UC (P = 0.0038), HC (P = 0.0055), and non-IBD DC (P = 0.0063). The prevalence of anti-GP2 IgG, anti-GP2 IgA and anti-GP2 IgA, or IgG antibodies in patients with CD was 40.0%, 37.1%, and 54.3%, respectively, which were higher than those in non-IBD DC (anti-GP2 IgG, 15.4%; anti-GP2 IgA, 7.7%; and anti-GP2 IgA or IgG, 23.1%) and those in patients with UC (anti-GP2 IgG, 11.4%; anti-GP2 IgA, 2.9%; and anti-GP2 IgA or IgG, 14.3%). For distinguishing CD from UC, the sensitivity, specificity, positive predictive value (PPV) and positive likelihood ratios (LR+) were 40%, 88.6%, 77.8%, and 3.51 for anti-GP2 IgG, 37.1%, 97.1%, 92.9%, and 13.0 for anti-GP2 IgA, and 54.3%, 85.3%, 79.2%, and 3.69 for anti-GP2 IgA or IgG. For CD diagnosis, the combination of anti-GP2 antibodies with ASCA IgA increased the sensitivity to 68.6% with moderate loss of specificity to 74.3%. Spearman's rank of order revealed a significantly positive correlation of anti-GP2 IgG with ileocolonic location of disease (L3) (P = 0.043) and a negative correlation of anti-GP2 IgA with biologic therapy (P = 0.012).Our findings suggest that anti-GP2 antibodies could serve as a biomarker for distinguishing patients with CD from patients with UC, and the combination of anti-GP2 antibodies with ASCA IgA may improve the predictive power.

Pro-thrombotic effect of exercise in a polluted environment: a P-selectin- and CD63-related platelet activation effect.

Exposure to diesel exhaust is an important cardiovascular risk factor and may promote atherothrombotic events. Some data suggest that polluted air exposure could affect haemostasis through platelet activation. The aim of the study was to investigate the effects of acute exposure to diesel exhaust on platelet activation and platelet function. We tested the hypothesis in a randomised, crossover study in 25 healthy men exposed to ambient and polluted air; 11 of the subjects also performed exercise during exposure sessions. Platelet activation was evaluated by surface expression of CD62P (P-selectin) and CD63 (dense granule glycoprotein) using flow cytometry of labelled platelets. Platelet function was measured using the PFA-100 platelet function analyser and by Multiplate whole blood impedance platelet aggregometry. Acute diesel exhaust exposure had no effect on platelet activation at rest, but exercise in polluted air increased the collagen-induced expression of CD62P and CD63 (both p< 0.05). The increase in the expression of CD62P and CD63 was related to the total amount of PM2.5 inhaled during the exercise sessions (r=+0.58 and +0.60, respectively, both p< 0.05). Platelet aggregation was not impaired after polluted air exposure at rest or during exercise. In conclusion, in healthy subjects, diesel exhaust exposure induces platelet activation as illustrated by a dose-response increase in the release of CD62P and CD63. This platelet priming effect could be a contributor to the triggering of atherothrombotic events related to air pollution exposure.

Mo1248 Weekend Hospitalizations Are Associated With Increased Post-Operative Complications Following Urgent Surgery for Ulcerative Colitis

Background: Crohǹs disease (CD) often leads to serious complications, such as fistulae and strictures as well as need for surgery. Early and prolonged immunosuppressive therapy has the potential to alter the natural history of CD. Several serum markers have been tested for the stratification of CD, but their use in clinical practice is still limited. Recently zymogen granule glycoprotein 2 (GP2)was identified as themajor autoantigen of CD-specific pancreatic autoantibodies. Aims: We tested serologic anti-GP2 antibodies for their association with complications and surgery, disease location, disease activity and age at disease onset in patients with CD in a cross-sectional analysis. Methods: Serum samples from 303 CD patients of a tertiary university hospital (mean age 36.1 years, 52.8% female) were tested for the presence of Anti-GP2 IgA and Anti-GP2 IgG by ELISA. Clinical data on disease location, age at disease onset, clinical disease activity, serum CRP, complications (strictures and fistulae) and surgery were available in a prospectively maintained database. Analysis was performed using Wilcoxon rank sum test and logistic regression analysis. Reported odds ratios (ORs) correspond to 10 unit (U) increase in marker levels. Results: After a median follow-up time from diagnosis of 104.6 [interquartile range (IQR) 58.1,168.1] months 77.6% of the CD patients had a complication (49.2% fistulae, 58.4% stenosis), 72.3% underwent surgery and 85.5% had ileal disease involvement. At time of sample procurement 36.5% had clinically active disease and 51.6% had elevated CRP. Levels of anti-GP2 IgG were linked to complicated CD (p=0.002, area under the curve (AUC) 0.62) and ileal disease location (p=0.004, AUC 0.64). Interestingly, both, anti-GP2 IgA and IgG were associated with the occurrence of stenosis and need for surgery (p ,0.001 p=0.003, AUC 0.62 0.64), but not with fistulizing CD, early disease onset, clinically active disease or elevated CRP. After correction for early disease onset and ileal involvement anti-GP2 IgG levels remained significantly linked to stenosis (OR for 10 U increase in GP2 IgG: 1.07 (95% confidence interval (CI): 1.01, 1.1)) and need for surgery (OR for 10 U increase in GP2 IgG: 1.1 (95% CI: 1.01, 1.2)). If patients had both, strictures and fistulae, the association of anti-GP2 IgG with stricturing CD remained significant after correction for the presence of fistulas (OR for 10 U increase in GP2 IgG: 1.07 (95% CI 1.02, 1.1)). Conclusions: Specific levels of anti-GP2 antibodies are independently associated with stricturing CD and need for surgical intervention. Ongoing studies evaluate anti-GP2 levels for their predictive capabilities, the stability of these markers over time and the combination with genetic markers to assess their use in clinical practice.

Mo1247 Serologic Anti-GP2 Antibodies Are Associated With Strictures and Need for Surgical Resection in Crohn's Disease

Background: Crohǹs disease (CD) often leads to serious complications, such as fistulae and strictures as well as need for surgery. Early and prolonged immunosuppressive therapy has the potential to alter the natural history of CD. Several serum markers have been tested for the stratification of CD, but their use in clinical practice is still limited. Recently zymogen granule glycoprotein 2 (GP2)was identified as themajor autoantigen of CD-specific pancreatic autoantibodies. Aims: We tested serologic anti-GP2 antibodies for their association with complications and surgery, disease location, disease activity and age at disease onset in patients with CD in a cross-sectional analysis. Methods: Serum samples from 303 CD patients of a tertiary university hospital (mean age 36.1 years, 52.8% female) were tested for the presence of Anti-GP2 IgA and Anti-GP2 IgG by ELISA. Clinical data on disease location, age at disease onset, clinical disease activity, serum CRP, complications (strictures and fistulae) and surgery were available in a prospectively maintained database. Analysis was performed using Wilcoxon rank sum test and logistic regression analysis. Reported odds ratios (ORs) correspond to 10 unit (U) increase in marker levels. Results: After a median follow-up time from diagnosis of 104.6 [interquartile range (IQR) 58.1,168.1] months 77.6% of the CD patients had a complication (49.2% fistulae, 58.4% stenosis), 72.3% underwent surgery and 85.5% had ileal disease involvement. At time of sample procurement 36.5% had clinically active disease and 51.6% had elevated CRP. Levels of anti-GP2 IgG were linked to complicated CD (p=0.002, area under the curve (AUC) 0.62) and ileal disease location (p=0.004, AUC 0.64). Interestingly, both, anti-GP2 IgA and IgG were associated with the occurrence of stenosis and need for surgery (p ,0.001 p=0.003, AUC 0.62 0.64), but not with fistulizing CD, early disease onset, clinically active disease or elevated CRP. After correction for early disease onset and ileal involvement anti-GP2 IgG levels remained significantly linked to stenosis (OR for 10 U increase in GP2 IgG: 1.07 (95% confidence interval (CI): 1.01, 1.1)) and need for surgery (OR for 10 U increase in GP2 IgG: 1.1 (95% CI: 1.01, 1.2)). If patients had both, strictures and fistulae, the association of anti-GP2 IgG with stricturing CD remained significant after correction for the presence of fistulas (OR for 10 U increase in GP2 IgG: 1.07 (95% CI 1.02, 1.1)). Conclusions: Specific levels of anti-GP2 antibodies are independently associated with stricturing CD and need for surgical intervention. Ongoing studies evaluate anti-GP2 levels for their predictive capabilities, the stability of these markers over time and the combination with genetic markers to assess their use in clinical practice.

Identification of Neutrophil Granule Glycoproteins as Lewisx-containing Ligands Cleared by the Scavenger Receptor C-type Lectin

The scavenger receptor C-type lectin (SRCL) is a glycan-binding receptor that has the capacity to mediate endocytosis of glycoproteins carrying terminal Lewisx groups (Galβ1–4(Fucα1–3)GlcNAc). A screen for glycoprotein ligands for SRCL using affinity chromatography on immobilized SRCL followed by mass spectrometry-based proteomic analysis revealed that soluble glycoproteins from secondary granules of neutrophils, including lactoferrin and matrix metalloproteinases 8 and 9, are major ligands. Binding competition and surface plasmon resonance analysis showed affinities in the low micromolar range. Comparison of SRCL binding to neutrophil and milk lactoferrin indicates that the binding is dependent on cell-specific glycosylation in the neutrophils, as the milk form of the glycoprotein is a much poorer ligand. Binding to neutrophil glycoproteins is fucose-dependent, and mass spectrometry-based glycomic analysis of neutrophil and milk lactoferrin was used to establish a correlation between high affinity binding to SRCL and the presence of multiple clustered terminal Lewisx groups on a heterogeneous mixture of branched glycans, some with poly N-acetyllactosamine extensions. The ability of SRCL to mediate uptake of neutrophil lactoferrin was confirmed using fibroblasts transfected with SRCL. The common presence of Lewisx groups in granule protein glycans can thus target granule proteins for clearance by SRCL. PCR and immunohistochemical analysis confirm that SRCL is widely expressed on endothelial cells and thus represents a distributed system that could scavenge released neutrophil glycoproteins both locally at sites of inflammation or systemically when they are released in the circulation.

Autoantibodies to GP2, the major zymogen granule membrane glycoprotein, are new markers in Crohn's disease

Background Crohn's disease (CD) is an inflammatory bowel disease (IBD) characterized by reactivity against microbial and self antigens. Zymogen granule glycoprotein 2 (GP2) was identified as the major autoantigen of CD-specific pancreatic autoantibodies (PAB). Methods Human GP2 was expressed in the Spodoptera frugiperda 9 (Sf9) cell line using the baculovirus system, purified by Ni-chelate chromatography, and used as antigen for anti-GP2 IgA and IgG assessment by enzyme-linked immunosorbent assays (ELISA). Antibodies to mannan of Saccharomyces cerevisiae (ASCA), PAB, and anti-GP2 were investigated in sera of 178 CD patients, 100 ulcerative colitis (UC) patients, and 162 blood donors (BD). Results Anti-GP2 IgG and IgA were found in 48/72 (66.7%) and 23/72 (31.9%) PAB positive and 5/106 (4.7%) and 1/106 (0.9%) PAB negative CD patients (p < 0.0001), respectively. CD patients displayed significantly higher reactivity to GP2 than UC patients and BD (p < 0.0001), respectively. Occurrence of anti-GP2 antibodies correlated with PAB reactivity (Spearmen's rho = 0.493, p < 0.00001). There was a significant relationship between the occurrence of ASCA IgG and anti-GP2 IgG (p = 0.0307). Conclusions Anti-GP2 IgG and IgA constitute novel CD specific autoantibodies, the quantification of which could improve the serological diagnosis of IBD.

Complex carbohydrate histochemistry and ultracytochemistry of the sheep lacrimal gland.

The chemical content of the secretion of the sheep lacrimal gland was analysed at the light and electron microscope levels by applying histochemical techniques and an ultrastructural histochemical method (periodic acid, thiocarbohydrazide and silver proteinate). Mucosubstance histochemistry demonstrated acidic glycoconjugates, mainly sulphated, in the mucous and seromucous glandular cells and in the apical portion of the cells lining the terminal ducts. Moreover, secretory granules, stained with PA-TCH-SP, showed a different localization of the reaction product. The presence of lysozyme was also found in the glandular serous cells. These histochemical studies demonstrate that the secretion of sheep lacrimal glands is mixed, having serous, mucous and seromucous components, and that an excellent correlation exists between the secretory granule substructure and glycoprotein localization.

Changes in neutrophil granule protein and cytoplasmic fibrils in human acute myeloid leukemias.

Granule protein deficiencies in morphologically mature neutrophil cells of peripheral blood from human patients with acute myeloid leukemia was demonstrated using post-embedding immunocytochemistry. Abnormal immunoreactivity of granule proteins was detected in seven of nine patients. Decreased immunoreactivity patterns were found more for the primary granule markers elastase and myeloperoxidase than for the secondary granule marker lactoferrin. Leukemias with a predominant myeloid component, in contrast to those with a predominant monocytoid component, had more neutrophil cells showing immunodeficiencies for one or more granule markers. The proportion of neutrophil cells showing immunodeficiencies varied greatly for each granule marker; more variation was obtained for elastase, lactoferrin and myeloperoxidase than for lysozyme, possibly because lysozyme is a marker for both granule types. In addition, no correlation could be found between any of the immunoreactivity deficiencies for the neutrophil granule glycoproteins elastase, lactoferrin, lysozyme and myeloperoxidase and the abundance of a particular set of ultrastructural features in the circulating leukemic cells from any of the nine patients. Nonetheless, most of the immature myeloid cells from peripheral blood of leukemic patients showing neutrophil protein immunoreactivity abnormalities in one or more granule markers often and randomly displayed one or more unusual ultrastructural features. The clinical and pathological significance of neutrophil granule protein deficiencies and the abundance of fibrillar structures in malignant myeloid cells presently is uncertain.

Thrombospondin as a Marker for Leukemic Megakaryoblasts.

Four cases of acute leukemia are reported in which the blast cells were reactive with a monoclonal antibody to the platelet alpha granule glycoprotein thrombospondin (TSP). Blasts in all four cases were also terminal deoxynucleotidyl transferase (TdT)-positive. Two cases demonstrated the presence of the Philadelphia chromosome, t(9;22). The only cells in normal bone marrow which reacted with antibody to TSP were megakaryocytes. Blasts in nine cases of acute myelogenous leukemia, six cases of acute lymphocytic leukemia, four cases of undifferentiated leukemia, and three cases of myelodysplastic syndrome were negative for TSP. Thus, TSP is useful in the identification of normal megakaryocytes and a subset of acute leukemia cells. The presence of both TSP and TdT in the leukemic blasts of our four patients suggests evolution from a pluripotent stem cell capable of multi-lineage differentiation.

Characterization of human neutrophil glycoproteins expressing the CD15 differentiation antigen (3‐fucosyl‐N‐acetyllactosamine)

The expression of the CD15 antigen, 3-fucosyl N-acetyllactosamine, on neutrophil glycoproteins has been studied by SDS gel electrophoresis and immunoblotting. The antigen is expressed on several glycoproteins, both intracellularly and on the cell surface. Each subcellular compartment appears to contain a specific antigen. A soluble, granule glycoprotein (Mr 80-90K) probably accounts for most of the intracellular staining detected immunohistochemically. Membrane glycoproteins of Mr, 85-90K and 25K are associated with granule membranes, the latter being an integral membrane protein. The CD15 antigen is expressed on several cell surface glycoproteins with Mr in the range of 165K and 105K. These antigens are also contained in an intracellular pool which is brought to the surface on activation of the cells with chemotactic peptides. The 165K and 105K antigens show identical electrophoretic mobility to two of the major glycoproteins detectable by PAS or protein staining of gels of detergent extracts of cell membranes. These glycoproteins include the complement receptor, CR3. The beta chain of CR3 (105K) and to a lesser extent the alpha chain (165K) express CD15; however, most of the CD15 antigen is associated with other glycoproteins of these molecular masses.

Bovine membranous dopamine beta-hydroxylase is not anchored via covalently attached phosphatidylinositol.

The mode of attachment of the membrane-bound form of the chromaffin granule glycoprotein dopamine beta-hydroxylase has been investigated; specifically, the possibility of a covalently attached glycosyl phosphatidylinositol anchor has been examined in detail. Incubation of fragmented chromaffin granule membranes with phosphatidylinositol-specific phospholipase C led to no solubilization of dopamine beta-hydroxylase activity. Further, chemical analyses of the inositol and phosphate content of the soluble form of dopamine beta-hydroxylase indicate that the enzyme contains no covalently attached phosphate or inositol. The results of these studies allow elimination of phosphatidylinositol as a source of membrane anchoring for dopamine beta-hydroxylase and, coupled with previously available data, argue against a post-translational covalent modification of the enzyme as the mechanism of membrane attachment. Possible alternative models for the anchoring of membranous dopamine beta-hydroxylase are discussed.

Isolation of thrombospondin released from thrombinstimulated human platelets by fast protein liquid chromatography on an anion-exchange mono-q column

Thrombospondin, a glycoprotein found in human platelet alpha granules, is thought to play a major role in platelet haemostatic functions. A rapid method to isolate thrombospondin for functional and structural studies was developed. Freshly prepared supernatants from thrombin-stimulated platelets were separated on an anion-exchange Mono-Q column on a fast protein liquid chromatography system. Detection of thrombospondin in the eluted peaks was performed using sodium dodecyl sulphate—polyacrylamide gel electrophoresis combined with silver staining and a solid-phase radioimmunoassay with monoclonal antibodies directed against thrombospondin and other platelet granule glycoproteins. Thrombospondin was isolated rapidly to a high degree of purity using the fast protein liquid chromatography Mono-Q system (20 min), compared with the time taken with other techniques.