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Asparagus ( Asparagus officinalis L.) is one of the widely consumed vegetables. To investigate the mechanism underlying the anti-allergic responses of asparagus, we extracted different fractions from asparagus and measured their inhibitory effects on β-hexosaminidase release in RBL-2H3 cells in vitro and an atopic dermatitis NC/Nga mouse model in vivo . The lipid fractions from asparagus were extracted with 50% ethanol, separated using chloroform by liquid-liquid phase separation, and fractionated by solid-phase extraction. Among them, acetone fraction (rich in glycolipid) and MeOH fraction (rich in phospholipid) markedly inhibited β-hexosaminidase release from RBL-2H3 cells. In NC/Nga mice treated with picryl chloride, atopic dermatitis was alleviated following exposure to the 50% EtOH extract, acetone fraction, and methanol fraction. The inhibitory effects of asparagus fractions in vivo were supported by the significant decrease in serum immunoglobulin E (IgE) levels. The phospholipid fractions showed significantly better inhibitory effects, and phosphatidic acid from this fraction showed the best inhibitory effect on β-hexosaminidase release. In mice challenged with ovalbumin (OVA), oral administration of asparagus extract and its fractions decreased the OVA-specific IgE level and total IgE, indicating that these effects may be partly mediated through the downregulation of antigen-specific IgE production. Taken together, the present study shows for the first time that asparagus extract and its lipid fractions could potentially mitigate allergic reactions by decreasing degranulation in granulocytes. Our study provides useful information to develop nutraceuticals and functional foods fortified with asparagus.

Background: Glucosaminidase (Gmd) is known to be a protective antigen in animal models of Staphylococcus aureus osteomyelitis. We compared the endogenous anti-Gmd antibody levels in sera of patients with culture-confirmed S. aureus bone infections to their sera at 1 year after operative treatment of the infection., Methods: A novel global biospecimen registry of 297 patients with deep-wound culture-confirmed S. aureus osteomyelitis was analyzed to assess relationships between baseline anti-Gmd serum titers (via custom Luminex assay), known host risk factors for infection, and 1-year postoperative clinical outcomes (e.g., infection control, inconclusive, refracture, persistent infection, septic nonunion, amputation, and septic death)., Results: All patients had measurable humoral immunity against some S. aureus antigens, but only 20 patients (6.7%; p < 0.0001) had high levels of anti-Gmd antibodies (>10 ng/mL) in serum at baseline. A subset of 194 patients (65.3%) who completed 1 year of follow-up was divided into groups based on anti-Gmd level: low (<1 ng/mL, 54 patients; 27.8%), intermediate (<10 ng/mL, 122 patients; 62.9%), and high (>10 ng/mL, 18 patients; 9.3%), and infection control rates were 40.7%, 50.0%, and 66.7%, respectively. The incidence of adverse outcomes in these groups was 33.3%, 16.4%, and 11.1%, respectively. Assessing anti-Gmd level as a continuous variable showed a 60% reduction in adverse-event odds (p = 0.04) for every tenfold increase in concentration. No differences in patient demographics, body mass index of >40 kg/m, diabetes status, age of ≥70 years, male sex, Charlson Comorbidity Index of >1, or Cierny-Mader host type were observed between groups, and these risk factors were not associated with adverse events. Patients with low anti-Gmd titer demonstrated a significant 2.68-fold increased odds of adverse outcomes (p = 0.008)., Conclusions: Deficiency in circulating anti-Gmd antibodies was associated serious adverse outcomes following operative treatment of S. aureus osteomyelitis. At 1 year, high levels of anti-Gmd antibodies were associated with a nearly 3-fold increase in infection-control odds. Additional prospective studies clarifying Gmd immunization for osteomyelitis are needed., Level of Evidence: Prognostic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Staphylococcus aureus (S. aureus) osteomyelitis remains a major clinical problem. Anti-glucosaminidase (Gmd) antibodies (1C11) are efficacious in prophylactic and therapeutic murine models. Feasibility, safety and pharmacokinetics of 1C11 passive immunisation in sheep and endogenous anti-Gmd levels were quantified in osteomyelitis patients. 3 sheep received a 500 mg intravenous (i.v.) bolus of 1C11 and its levels in sera were determined by enzyme-linked immunosorbent assay (ELISA) over 52 d. A humanised anti-Gmd monoclonal antibody, made by grafting the antigen-binding fragment (Fab) portion of 1C11 onto the fragment crystallisable region (Fc) of human IgG1, was used to make a standard curve of mean fluorescent intensity versus concentration of anti-Gmd. Anti-Gmd serum levels were determined in 297 patients with culture-confirmed S. aureus osteomyelitis and 40 healthy controls. No complications or adverse events were associated with the sheep 1C11 i.v. infusion and the estimated circulating half-life of 1C11 was 23.7 d. Endogenous anti-Gmd antibody levels in sera of osteomyelitis patients ranged from < 1 ng/mL to 300 µg/mL, with a mean concentration of 21.7 µg/mL. The estimated circulating half-life of endogenous anti-Gmd antibodies in sera of 12 patients with cured osteomyelitis was 120.4 d. A clinically relevant administration of anti-Gmd (500 mg i.v. = 7 mg/kg/70 kg human) was safe in sheep. This dose was 8 times more than the endogenous anti-Gmd levels observed in osteomyelitis patients and was predicted to have a half-life of > 3 weeks. Anti-Gmd passive immunisation has potential to prevent and treat S. aureus osteomyelitis. Further clinical development is warranted.

Background: Breast milk Chitotriosidase (Chit 1) shows antifungal effect and has an active role in the natural immune response against certain pathogens. The aim of this study was to compare colostrum Chit 1 levels from mothers of term and preterm infants. Materials and Methods: The study included 72 mothers of 32 preterm and 40 term infants (gestational age; 33.7 ± 1.8 vs. 39.1 ± 1.1 weeks, birth weight; 1931.7 ± 539.8 vs. 3350.9 ± 419.7 g). Breast milk samples were taken at postnatal 24-48 hours. Chit 1 level was evaluated with the quantitative calorimetric method. Results: No significant difference was determined between the term and preterm groups in terms of maternal age, education level, weight gain in pregnancy, and body mass index (BMI). The median colostrum Chit 1 level was higher in the preterm group, but the difference was not statistically significant between two groups ( p  = 0.43). There is no association between colostrum Chit 1 level, maternal age, gravida, BMI, infant gender, income level, and pre-eclampsia. The colostrum Chit 1 level of mothers who had weight gain exceeding the recommended limits was significantly lower than mothers with weight gain within the recommended limits in the term group (4346.2 vs. 4914.2, p  = 0.021). A negative correlation was determined between the birthweight of term infants and the colostrum Chit 1 levels ( p  = 0.045, r  = -0.319). Conclusion: Although the data need to be validated by further investigation, the observations made in this study seem to indicate that colostrum Chit-1 may have role in the protection of preterm infants.

Recent studies suggest that HIV infection is an independent risk factor for the development of chronic obstructive pulmonary disease (COPD). We hypothesized that HIV infection and cigarette smoking synergize to alter the function of alveolar macrophages (AMs). To test this hypothesis, global transcriptome analysis was performed on purified AMs from 20 individuals split evenly between HIV-uninfected nonsmokers and smokers and untreated HIV-infected nonsmokers and smokers. Differential expression analysis identified 143 genes significantly altered by the combination of HIV infection and smoking. Of the differentially expressed genes, chitinase 1 (CHIT1) and cytochrome P450 family 1 subfamily B member 1 (CYP1B1), both previously associated with COPD, were among the most upregulated genes (5- and 26-fold, respectively) in the untreated HIV-infected smoker cohort compared with HIV-uninfected nonsmokers. Expression of CHIT1 and CYP1B1 correlated with the expression of genes involved in extracellular matrix organization, oxidative stress, immune response, and cell death. Using time-of-flight mass cytometry to characterize AMs, a significantly decreased expression of CD163, an M2 marker, was seen in HIV-infected subjects, and CD163 inversely correlated with CYP1B1 expression in AMs. CHIT1 protein levels were significantly upregulated in bronchoalveolar lavage fluid from HIV-infected smokers, and increased CHIT1 levels negatively correlated with lung function measurements. Overall, these findings raise the possibility that elevated CHIT1 and CYP1B1 are early indicators of COPD development in HIV-infected smokers that may serve as biomarkers for determining this risk., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Objective: To investigate the role of neuroinflammation in asymptomatic and symptomatic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) mutation carriers., Methods: The neuroinflammatory markers chitotriosidase 1 (CHIT1), YKL-40 and glial fibrillary acidic protein (GFAP) were measured in cerebrospinal fluid (CSF) and blood samples from asymptomatic and symptomatic ALS/FTD mutation carriers, sporadic cases and controls by ELISA., Results: CSF levels of CHIT1, YKL-40 and GFAP were unaffected in asymptomatic mutation carriers (n=16). CHIT1 and YKL-40 were increased in gALS (p<0.001, n=65) whereas GFAP was not affected. Patients with ALS carrying a CHIT1 polymorphism had lower CHIT1 concentrations in CSF (-80%) whereas this polymorphism had no influence on disease severity. In gFTD (n=23), increased YKL-40 and GFAP were observed (p<0.05), whereas CHIT1 was nearly not affected. The same profile as in gALS and gFTD was observed in sALS (n=64/70) and sFTD (n=20/26). CSF and blood concentrations correlated moderately (CHIT1, r=0.51) to weak (YKL-40, r=0.30, GFAP, r=0.39). Blood concentrations of these three markers were not significantly altered in any of the groups except CHIT1 in gALS of the Ulm cohort (p<0.05)., Conclusion: Our data indicate that neuroinflammation is linked to the symptomatic phase of ALS/FTD and shows a similar pattern in sporadic and genetic cases. ALS and FTD are characterised by a different neuroinflammatory profile, which might be one driver of the diverse presentations of the ALS/FTD syndrome., Competing Interests: Competing interests: DRT received consultancies from Covance Laboratories (UK) and GE Healthcare (UK), received a speaker honorarium from GE Healthcare (UK), and collaborated with Novartis Pharma Basel (Switzerland).PO, PW, PS, SAS, FN, AEV, JDS, PMA, JK, AD, KFa, KFl, HJ, ML, KM, AK, JP, AS, DYH, JHW, ACL and MO report no competing interests., (© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

Immunoglobulin E (IgE) plays a central role in the pathogenesis of Type I hypersensitivity through interaction with a high-affinity receptor (FcεRIα). For therapeutic applications, substantial attention has been focused recently on the blockade of the IgE interaction with FcεRIα. While exploring better options for preventing allergic diseases, we found that the Fab fragment of the rat anti-murine IgE antibody (Fab-6HD5) strongly inhibited passive cutaneous anaphylaxis (PCA) in vivo, as well as spleen tyrosine kinase (Syk) activity and β-hexosaminidase release from basophilic leukemia cells in vitro. The in vivo effects of Fab-6HD5 pre-administration were maintained over a long period of time for at least 10 days. Using flow cytometry analysis, we also found that Fab-6HD5 did not recognize the IgE Cε3 domain containing specific binding sites for FcεRIα. Furthermore, deletion-mapping studies revealed that Fab-6HD5 recognized conformational epitopes on the Cε2 domain of IgE. Given that the Cε2 domain plays a key role in stabilizing the interaction of IgE with FcRIα, our results suggest that the specific binding of Fab-6HD5 to the Cε2 domain prevents allergic reactions through destabilizing the preformed IgE-FcεRIα complex on rat mast cells. Although the present study was performed using animal models, these findings support the idea that a certain antibody directed against IgE CH domains may contribute to preventing allergic diseases through interacting with IgE-FcεRIα complex.

Background: Cancer progression has been associated with altered immune cell function and activation. Neopterin, which is secreted by interferon-γ stimulated macrophages, exhibits an association with multiple cancer types and metastatic disease. Chitotriosidase, which is secreted by chronically activated macrophages and granulocyte-macrophage colony-stimulating factor stimulated neutrophils has not been studied in the setting of cancer., Objective: The goal of this discovery study was to screen chitotriosidase for diagnostic capacity in detecting cancer and compare its operating characteristics with those of neopterin., Methods: Serum from subjects with breast (n= 66) or prostate (n= 70) cancer, and from 204 subjects free of malignant disease were studied. Chitotriosidase was measured by enzyme activity assay, while neopterin was measured by a competitive enzyme immunoassay. Statistical analyses included group comparisons by Mann Whitney U test, diagnostic capacity by receiver operating characteristics (ROC) curve analysis and biomarker associations with physiologic and clinical measures by Spearman correlation., Results: Chitotriosidase activity was significantly higher in both cancer types compared with gender matched controls, though only in breast cancer was the diagnostic capacity significant (area under the ROC curve of 0.97 ± 0.01). In contrast, neopterin was significantly elevated in prostate cancer and exhibited discriminatory capacity (area under the ROC curve of 0.76 ± 0.05). Age, BMI, % body fat and metastasis were variables that correlated with neopterin, but not chitotriosidase levels., Conclusions: The operating characteristics of serum chitotriosidase were different from neopterin and further analysis of chitotriosidase as a biomarker for breast cancer is warranted.

Rationale: Serum chitinases may be novel biomarkers of airway inflammation and remodeling, but less is known about factors regulating their levels., Objectives: To examine serum chitotriosidase activity and YKL-40 levels in patients with asthma and chronic obstructive pulmonary disease (COPD) and evaluate clinically relevant factors that may affect chitinase levels, including genetic variability, corticosteroid treatment, disease exacerbations, and allergen exposure., Methods: Serum chitotriosidase (CHIT1) activity and YKL-40 (CHI3L1) levels, as well as the CHIT1 rs3831317 and CHI3L1 rs4950928 genotypes, were examined in subsets of patients with mild to moderate asthma (n = 76), severe asthma (n = 93), and COPD (n = 64) taking part in the European multicenter BIOAIR (Longitudinal Assessment of Clinical Course and Biomarkers in Severe Chronic Airway Disease) study. Blood was obtained at baseline, before and after a 2-week oral steroid intervention, up to six times during a 1-year period, and during exacerbations. Baseline chitinase levels were also measured in 72 healthy control subjects. The effect of allergen inhalation on blood and sputum YKL-40 levels was measured in two separate groups of patients with mild atopic asthma; one group underwent repeated low-dose allergen challenge (n = 15), and the other underwent high-dose allergen challenge (n = 16)., Measurements and Main Results: Serum chitotriosidase and YKL-40 were significantly elevated in patients with asthma and those with COPD compared with healthy control subjects. Genotype and age strongly affected both YKL-40 and chitotriosidase activity, but associations with disease remained following adjustment for these factors. Correlations were observed with lung function but not with other biomarkers, including exhaled nitric oxide, blood eosinophils, periostin, and IgE. Generally, acute exacerbations, allergen-induced airway obstruction, and corticosteroid treatment did not affect circulating chitinase levels., Conclusions: YKL-40 and chitotriosidase are increased in asthma and more so in COPD. The data in the present study support these substances as being relatively steroid-insensitive, non-T-helper cell type 2-type biomarkers distinctly related to chronic inflammatory disease processes.

Pulmonary mycoses are often associated with type-2 helper T (Th2) cell responses. However, mechanisms of Th2 cell accumulation are multifactorial and incompletely known. To investigate Th2 cell responses to pulmonary fungal infection, we developed a peptide-MHCII tetramer to track antigen-specific CD4+ T cells produced in response to infection with the fungal pathogen Cryptococcus neoformans. We noted massive accruement of pathologic cryptococcal antigen-specific Th2 cells in the lungs following infection that was coordinated by lung-resident CD11b+ IRF4-dependent conventional dendritic cells. Other researchers have demonstrated that this dendritic cell subset is also capable of priming protective Th17 cell responses to another pulmonary fungal infection, Aspergillus fumigatus. Thus, higher order detection of specific features of fungal infection by these dendritic cells must direct Th2 cell lineage commitment. Since chitin-containing parasites commonly elicit Th2 responses, we hypothesized that recognition of fungal chitin is an important determinant of Th2 cell-mediated mycosis. Using C. neoformans mutants or purified chitin, we found that chitin abundance impacted Th2 cell accumulation and disease. Importantly, we determined Th2 cell induction depended on cleavage of chitin via the mammalian chitinase, chitotriosidase, an enzyme that was also prevalent in humans experiencing overt cryptococcosis. The data presented herein offers a new perspective on fungal disease susceptibility, whereby chitin recognition via chitotriosidase leads to the initiation of harmful Th2 cell differentiation by CD11b+ conventional dendritic cells in response to pulmonary fungal infection.

Towards the development of a methicillin-resistant Staphylococcus aureus (MRSA) vaccine we evaluated a neutralizing anti-glucosaminidase (Gmd) monoclonal antibody (1C11) in a murine model of implant-associated osteomyelitis, and compared its effects on LAC USA300 MRSA versus a placebo and a Gmd-deficient isogenic strain (ΔGmd). 1C11 significantly reduced infection severity, as determined by bioluminescent imaging of bacteria, micro-CT assessment of osteolysis, and histomorphometry of abscess numbers (p < 0.05). Histology also revealed infiltrating macrophages, and the complete lack of staphylococcal abscess communities (SAC), in marrow abscesses of 1C11 treated mice. In vitro, 1C11 had no direct effects on proliferation, but electron microscopy demonstrated that 1C11 treatment phenocopies ΔGmd defects in binary fission. Moreover, addition of 1C11 to MRSA cultures induced the formation of large bacterial aggregates (megaclusters) that sedimented out of solution, which was not observed in ΔGmd cultures or 1C11 treated cultures of a protein A-deficient strain (ΔSpa), suggesting that the combined effects of Gmd inhibition and antibody-mediated agglutination are required. Finally, we demonstrated that macrophage opsonophagocytosis of MRSA and megaclusters is significantly increased by 1C11 (p < 0.01). Collectively, these results suggest that the primary mechanism of anti-Gmd humoral immunity against MRSA osteomyelitis is macrophage invasion of Staphylococcal abscess communities (SAC) and opsonophagocytosis of megaclusters. ., (© 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Background: Staphylococcus aureus infections remain a major complication of orthopaedic surgery. Although serum C-reactive protein is useful for diagnosis, there are no specific tests for host immunity that can assess a patient's risk for serious infection. On the basis of the identification of glucosaminidase as a potentially protective antigen in animal models, we tested the hypotheses that anti-glucosaminidase IgG (immunoglobulin G) levels vary in sera of mice and orthopaedic patients with Staphylococcus aureus infections and that physical and neutralizing titers correlate., Methods: In vitro ELISAs (enzyme-linked immunosorbent assays) were developed to quantify binding (physical) and enzyme-neutralizing (functional) anti-glucosaminidase IgG titers. The assays were validated with use of sera from naive, Staphylococcus aureus-challenged, and glucosaminidase-immunized mice. The physical, functional, and isotype titers of anti-glucosaminidase IgG were measured in sera from twenty-four patients with a confirmed Staphylococcus aureus infection following orthopaedic surgery and in sera from twenty noninfected patients. The specificity of the anti-glucosaminidase assay was evaluated by means of linear regression and receiver-operator characteristic curve analysis., Results: In mice, the analytic range of the physical titer assay for anti-glucosaminidase IgG was determined to be 1 ng/mL to 1 μg/mL, and physical titers correlated with functional titers (p &lt; 0.002). Although all patients had measurable anti-glucosaminidase IgG, the physical titers in the infected patients were significantly higher by a factor of two compared with those in the healthy controls (p = 0.015). The physical titers were significantly correlated with the functional titers (p &lt; 0.0001). Receiver-operator characteristic curve analysis demonstrated a diagnostic specificity of 0.72 (p = 0.014) for the assay. The anti-glucosaminidase titer in almost every patient was dominated by the IgG1 isotype., Conclusions: Humoral immunity against glucosaminidase varied in mammals with Staphylococcus aureus osteomyelitis. Anti-glucosaminidase titers in sera were a potential biomarker of infection and have the potential to assess the quality of host immunity against Staphylococcus aureus., Clinical Relevance: Staphylococcus aureus infections can be challenging to diagnose, and there is no diagnostic test for host immunity. We demonstrated a cost-effective assay for determining the anti-glucosaminidase titer, which can be readily combined with conventional serology to improve diagnosis and to assess host immunity against Staphylococcus aureus.

Context: Chitin is the polysaccharide and is found in insects, parasites and fungi. Chitin has shown various immunological effects in in vivo and in vitro models. In this study, crystallinity controlled N-acetyl glucosamine (CCG) which has a high solubility was prepared from the low molecular weight chitosan. However, the effect of CCG in an allergic response is not clear., Objective: To investigate the effect and regulatory mechanism of CCG on allergic responses., Methods: To demonstrate the effect of CCG, we induced systemic anaphylactic shock, ear swelling response, passive cutaneous anaphylaxis (PCA), and inflammatory reaction., Results: CCG inhibited the compound 48/80-induced systemic anaphylactic shock and ear swelling responses. IgE-mediated PCA was inhibited by the oral administration or topical application of CCG. Histamine and β-hexosaminidase release from mast cells was decreased with the treatment of CCG. CCG also inhibited phorbol 12-myristate 13-acetate and calcium ionophore A23187-induced interleukin-1β production and mRNA expression by suppressing NF-κB activation and IκBα phosphorylation. Furthermore, CCG suppressed the activation of caspase-1., Conclusion: These results suggest that CCG may have beneficial applicability as a candidate for an anti-allergic agent.

Several soft-tissue dermal fillers have been reported to provoke immunogenicity and may cause adverse reactions despite claims regarding their safety. This study aimed to assess biomaterial-induced macrophage activation, cell-mediated immune response and oxidative stress in 169 patients with dermal bioimplants. To this end, we analysed plasma concentrations of myeloperoxidase (MPO), the chitinase-like proteins chitotriosidase and YKL-40 and molecular oxidative damage. The present study shows, for the first time, that the components of innate immunity: chitotriosidase and YKL-40, are significantly higher in patients with certain bioimplants and these markers of monocyte/macrophage activation rose progressively as adverse reactions (AR) evolved. Plasma MPO levels increased 4-fold in filler users with AR and 3-fold in those without. Analysis by filler type showed subjects injected with calcium hydroxylapatite, methacrylate, acrylamides and silicone to have values significantly above those of non-filler subjects for at least two plasma biomarkers, probably because the afore-mentioned biomaterials are permanent and prone to trigger AR in the long term. By contrast, hyaluronic acid alone elicited little immune response. Plasma concentrations of markers of oxidative damage to lipids and proteins were found to be significantly higher in users of four of the nine dermal fillers studied. These diffusible products of molecular peroxidation would stem from the reaction catalysed by MPO that generates potent oxidants, leading to cell oxidative damage which, in turn, may exert deleterious effects on the organism. Overall, the results of this study on the effects of a range of dermal fillers point to chronic activation of the immune response mediated by macrophages and PMNs. The increases in plasma of MPO, chitotriosidase and YKL-40 proteins and products of macromolecular peroxidation suggests that these molecules could serve as blood-based biochemical markers and alert to the risk of chronic immune system activation and development of adverse events that may arise from the use of certain bioimplants., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Infections after chemotherapy often cause significant morbidity in patients with acute myeloid leukaemia (AML). Chitotriosidase (CHIT) and mannose-binding lectin (MBL) are part of the innate immune system. Polymorphism in the CHIT-coding gene (CHIT1) may be associated with Gram-negative sepsis in children with AML, and polymorphism in the MBL-coding gene (MBL2) seems to modify the risk of infections in several patient groups. The purpose of this study was to investigate the possible associations between polymorphisms in CHIT1, MBL2 and sepsis in adult patients treated with high-dose chemotherapy for AML. We included 190 patients treated with 526 cycles of chemotherapy. The follow-up period was 6 months from the diagnosis of AML. Prophylactic antibiotics were not used. We identified 604 febrile episodes with 246 episodes of sepsis. Thirty-two patients (17%) either died from infection or infection was a major concomitant factor for death. No significant associations between CHIT1 polymorphism and sepsis (P = 0.85) or death caused by sepsis (P = 0.14) were found. Furthermore, no significant associations between MBL2 polymorphism and sepsis (P = 0.76) or death caused by sepsis (P = 0.24) were observed. The severe and long-lasting neutropenia and mucositis after chemotherapy may explain why the MBL system does not protect against sepsis in patients with AML. Replacement therapy with recombinant MBL is not likely to decrease the risk of sepsis in patients with AML.

Gaucher disease (GD) is associated with upregulation of CD1d and MHC-class II expression by monocytes. While the physiological impact of CD1d upregulation remains uncertain, it has been proposed that MHC-class II upregulation is associated with inflammation. Hereby, we show that the decrease in MHC-class II expression seen in GD patients under therapy correlates positively with chitotriosidase activity, a marker of inflamed macrophages. We also show that retinoic acid (RA) and the beta-glucocerebrosidase inhibitor conduritol-B-epoxide (CBE) lead to upregulation of CD1d expression by THP-1 cells, which correlated with an increase in mRNA expression. In vitro co-culture experiments showed that RA treated THP-1 cells were more stimulatory for CD4(+) than for CD8(+) T cells, as determined by CFSE loss, in comparison to untreated THP-1 cells. Interestingly, even though addition of exogenous isoglobotrihexosylceramide (iGb3), a physiological CD1d ligand, augmented the percentage of dividing CD4(+) T cells, we could not detect a significant expansion of CD4(+)Valpha24(+) invariant Natural Killer T (iNKT) cells. In contrast, addition of alpha-galactosylceramide (alpha-GC) induced expansion of Valpha24(+) iNKT cells as determined by using alpha-GC-loaded human CD1d dimers. These results strengthen the existence of a cross-talk between monocyte lipid accumulation, inflammation and changes in cell surface CD1d and MHC-class II in monocytes, which may result in inappropriate recognition events by immune cells and perpetuate chronic inflammation., (Copyright 2009 Elsevier GmbH. All rights reserved.)

Purpose of Review: The present review provides an overview of the chitinase and chitinase-like proteins, chitotriosidase (CHIT1), YKL-40, and acid mammalian chitinase, and summarizes the genetic studies of asthma and immune-mediated diseases with polymorphisms in the genes encoding these proteins, CHIT1, CHI3L1, and CHIA, respectively., Recent Findings: Polymorphisms in the CHIT1, CHIA, and CHI3L1 genes influence chitotriosidase enzyme activity, acid mammalian chitinase activity, and YKL-40 levels, respectively. Regulatory SNPs in CHI3L1 were also associated with asthma, atopy, and immune-mediated diseases, and nonsynonymous SNPs in CHIA were associated with asthma. No CHIT1 polymorphisms, including a common nonfunctional 24-bp duplication allele, have been associated with asthma., Summary: These genes represent novel asthma susceptibility genes. Variations in CHI3L1 and CHIA have been associated with asthma risk. Polymorphisms in CHIT1 have not yet been associated with asthma, but few studies have been reported. Given that chitotriosidase is the major chitinase in the airways and a common nonfunctional allele is present in many populations, additional studies of this gene are warranted. Lastly, studies of all three genes need to be conducted in populations of diverse ancestries.

Background: Human chitotriosidase is a chitinase selectively expressed by activated macrophages. An increase in chitotriosidase activity was previously described by us in the serum and bronchoalveolar lavage of sarcoidosis patients., Objective: The aim of the present study was to analyze serum chitotriosidase activity in a larger number of sarcoidosis patients to verify the reported increase with respect to controls and to compare serum chitotriosidase levels in patients with sarcoidosis and tuberculosis, two granulomatous disorders of different etiology., Methods: Chitotriosidase activity was measured in the serum of 96 sarcoidosis patients, 15 pulmonary tuberculosis patients and 30 healthy controls., Results: We found significantly higher serum chitotriosidase activity in sarcoidosis patients than controls (p < 0.01) and in sarcoidosis patients than tuberculosis patients (p < 0.01), confirming a striking elevation of chitotriosidase activity (>10 times greater than normal) in pulmonary sarcoidosis patients. This is the first time that chitotriosidase activity has been analyzed in the serum of patients with pulmonary tuberculosis; it was found to be significantly lower than in sarcoidosis patients and not significantly greater than in controls., Conclusion: Although the mechanisms leading to the increase in chitotriosidase activity in sarcoidosis are still unknown, this enzyme may be specifically involved in the pathogenesis of the disease. Further studies with a greater number of patients are needed to confirm these results and to determine whether chitotriosidase could be a marker with diagnostic or prognostic value in sarcoidosis., (Copyright 2007 S. Karger AG, Basel.)

In humans, two types of chitinases have been identified: chitotriosidase I (CHIT1) and acid mammalian chitinase (AMCase). They are enzymes that cleave chitin, a polysaccharide contained in many different human parasites. So far, only little is known about their function in human and especially in human diseases. Recently we have described association of polymorphisms of AMCase with bronchial asthma in a pediatric population. In this study we were interested in whether CHIT1 is also involved in the genetics of asthma. The amino acid variants Gly102Ser and Ala442Gly, as well as a 24 bp duplication within CHIT1, were typed by means of restriction fragment length polymorphisms on 322 children with asthma and 270 randomly chosen adult controls. Statistical analyses made use of the Armitage's trend test; haplotypes were calculated by FAMHAP and FASTEHPLUS. The amino acid variants showed no association with bronchial asthma. The 24 bp duplication, previously shown to completely demolish CHIT1 activity, was also evenly distributed between asthmatics and controls. Finally, the haplotype showed no association with the disease. We conclude from our results that CHIT1 does not play a major role in the development of bronchial asthma in Caucasian children. The results might also imply that the two human chitinases that have been identified so far have quite distinct functions in human diseases even though they have the same substrate.

Man has been found to produce highly conserved chitinases. The most prominent is the phagocyte-derived chitotriosidase, the plasma levels of which are markedly elevated in some pathological conditions. Here, we report that both polymorphonuclear neutrophils (PMNs) and macrophages (m) are a source of chitotriosidase. The enzyme is located in specific granules of human PMNs and secreted following stimulation with granulocyte macrophage colony-stimulating factor (GM-CSF). In addition, GM-CSF induces expression of chitotriosidase in m that constitutively secrete the enzyme and partly accumulate it in their lysosomes. Studies with recombinant human chitotriosidase revealed that the enzyme targets chitin-containing fungi. These findings are consistent with earlier observations concerning anti-fungal activity of homologous plant chitinases and beneficial effects of GM-CSF administration in individuals suffering from invasive fungal infections. In conclusion, chitotriosidase should be viewed as a component of the innate immunity that may play a role in defence against chitin-containing pathogens and the expression and release of which by human phagocytes is highly regulated.

Aim: To provide information on the specificity of induced anti-pig antibodies (Abs) in baboons after exposure and sensitization to pig antigens., Materials and Methods: Baboons (n=7) received either porcine mobilized peripheral blood progenitor cells (n=3), kidney (n=3) or heart (n=1) transplants. After rejection of these cells or organs, pre- and post-rejection sera were analyzed by enzyme-linked immunosorbent assay (ELISA) and flow cytometry to detect and measure anti-Galactosealpha1,3Galactose (Gal) and anti-non-Gal Abs. To study the anti-non-Gal carbohydrate response, the sera were incubated with pig red blood cells pretreated with alpha-galactosidase (to remove Gal) and three other exoglycosidases to remove other potential oligosaccharide epitopes, and studied by flow cytometry. To study the anti-swine leukocyte antigen (SLA) response, non-Gal Abs from two baboons sensitized with kidneys from inbred miniature swine of dd or aa haplotype, respectively, were adsorbed on cells of aa, cc, or dd haplotypes, and binding to aa, cc or dd cells was measured by flow cytometry. Cytotoxicity of anti-non-Gal Abs was tested in vitro by a complement-mediated cytotoxicity assay, using pig cells as targets., Results: In pre-transplant and pre-rejection sera, anti-Gal Abs were detected, but anti-non-Gal Abs were either absent or at minimal levels. After exposure to pig antigens, baboons developed induced anti-Gal and anti-non-Gal Abs. No anti-non-Gal Abs directed to the tested carbohydrate epitopes could be detected. Anti-non-Gal Abs showed minor evidence of specific SLA haplotype reactivity, suggesting that the major Ab response was to pan-pig determinants. Anti-non-Gal Abs showed a low level of complement-mediated lysis of pig cells in vitro., Conclusions: In this limited study, no Ab response to non-Gal carbohydrates was observed, and anti-SLA specificity was minor, indicating that most induced anti-non-Gal Ab was directed against non-specific pig proteins, including SLA-epitopes.

Background: The most prevalent anticarbohydrate antibodies in human serum are anti-Gal interacting specifically with the alpha-gal epitope (Galalpha1-3Galbeta1-4GlcNAc-R) and anti-blood group antibodies interacting with blood group A and B antigens. The alpha-gal epitope, although absent in humans, comprises part of the core of carbohydrate chain in A and B antigens. Therefore, it was of interest to determine whether immunoglobulin (Ig) G antibodies, elicited in patients rejecting ABO-incompatible kidney allografts, can interact with the alpha-gal epitope., Methods: Anti-A and anti-B antibodies were determined by enzyme-linked immunosorbent assay (ELISA) with blood group A or B human red cell membranes, as solid phase antigens. Anti-Gal was determined by ELISA with alpha-gal-bovine serum albumin as solid-phase antigen. Specific removal of anti-Gal was performed by adsorption on fixed rabbit red cells., Results: Blood group O patients who underwent transplantation with either A or B kidney produced an antibody that bound to all three carbohydrate antigens. This multispecific antibody, designated anti-Gal A/B, is specific to the core alpha-gal epitope within A and B antigens. Recipients of allograft expressing incompatible blood group B also produce anti-Gal B antibody, which binds to the core alpha-gal epitope only in the B antigen. Anti-Gal A/B and anti-Gal B constitute most of the elicited anti-blood group antibody response. Allograft recipients also produced pure anti-A, or pure anti-B, which require the complete blood group structure for binding., Conclusions: The findings in this study imply that much of the immune response elicited by incompatible A or B antigens on kidney allografts results in activation of anti-Gal B-cell clones producing antibodies to the core alpha-gal epitope in these blood group antigens. Only less than 25% of the elicited antibodies interact with the complete A or B antigens (i.e., pure anti-A or pure anti-B). These findings suggest that prevention of the anti-Gal response may decrease the immune rejection of ABO-incompatible allografts.

Glutaminase (EC 3.5.1.2) is a key enzyme in rapidly proliferating cells. Using anti-sense technology, an Ehrlich ascites tumor cell line (0.28AS-2) with reduced glutaminase activity has been obtained. We investigated the in vivo growth characteristics of the 0.28AS-2 cells. When injected i.p. into normal Swiss albino mice, the 0.28AS-2 cells were unable to grow. On the contrary, when injected into nude mice, they developed into solid tumors. Mice inoculated with 0.28AS-2 cells kept immunologic memory and rejected a second inoculation with parental Ehrlich ascites tumor cells. Expression of both polymorphic epithelial mucin-1 (MUC-1) and the enzyme N-acetyl-alpha-D-galactosaminidase, proteins implicated in host immune system escape, were markedly diminished in 0.28AS-2 cells. Study of the immune system response in mice inoculated with 0.28AS-2 cells revealed an increase in splenic CD18 cells and the presence of a large number of activated F4/80+ macrophages in the ascites cavity. These features, not observed in mice inoculated with parental Ehrlich ascites tumor cells, indicate that a distinctive, strong immune response occurred in animals inoculated with 0.28AS-2 cells. Our results suggest that inhibition of glutaminase expression using anti-sense technology induces phenotypic changes in Ehrlich ascites tumor cells that allow the development of an effective anti-tumor immune response, which makes the cells unable to develop in vivo tumors.

A serum glycoprotein, Gc protein (vitamin D3-binding protein), can be converted by beta-galactosidase of B cells and sialidase of T cells to a potent macrophage-activating factor (MAF), a protein with N-acetylgalactosamine as the remaining sugar moiety. Thus, Gc protein is the precursor for MAF. Treatment of Gc protein with immobilized beta-galactosidase and sialidase generates a remarkably high titered macrophage-activating factor (GcMAF). When peripheral blood monocytes/ macrophages (designated macrophages) of 33 systemic lupus erythematosus patients were incubated with GcMAF (100 pg/ml), the macrophages of all patients were activated as determined by superoxide generation. However, the precursor activity of patient plasma Gc protein was lost or reduced in these patients. Loss of the precursor activity was the result of deglycosylation of plasma Gc protein by alpha-N-acetylgalactosaminidase activity found in the patient plasma. Levels of plasma alpha-N-acetylgalactosaminidase activity in individual patients had an inverse correlation with the MAF precursor activity of their plasma Gc protein. Deglycosylated Gc protein cannot be converted to macro-phage-activating factor. The resulting defect in macro-phage activation may lead to an inability to clear pathogenic immune complexes. Thus, elevated plasma alpha-N-acetylgalactosaminidase activity resulting in the loss of MAF precursor activity and reduced macro-phage activity may play a role in the pathogenesis of systemic lupus erythematosus.

Hexosaminidases, lysosomal enzymes whose deficiency is responsible for several genetic disorders, exist as two major forms: form A, containing two types of subunits alpha and beta; and form B, containing only beta subunits. We have used a technique involving successively electrophoresis of denatured proteins, transfer (blotting) onto nitrocellulose, and labelling by appropriate antibodies raised against the dissociated forms of hexosaminidases A and B. This technique allows the detection of alpha and beta subunits in crude extracts of normal tissues. The presence of beta chains was demonstrated in the liver of a fetus affected with Sandhoff's disease, deficient in functional hexosaminidases A and B.

The residual enzyme of the fibroblasts of a child with homozygous type 0 GM2 gangliosidosis (Sandhoff-Jatzkewitz disease) has been found to correspond with a minor fraction of enzyme which can be isolated from normal fibroblasts by repeated chromatography. This enzyme is designated as hexosaminidase (hex) S. It reacts with antiserum prepared against homogeneous hex A but not with serum prepared against homogeneous hex B. These findings support our previously described model of the relationship between hex A and hex G: hex A has the structure (alpha beta)3, while hex B is (beta)6. Type B GM2 gangliosidosis (Tay-Sachs disease) is the alpha- mutation, while type 0 GM2 gangliosidosis (Sandhoff-Jatzkewitz disease) is the beta- mutation. In the absence of normal beta subunits there is increased polymerization of alpha subunits forming hex S, which probably has a structure of (alpha)6. A parallel between the thalassemias and GM2 gangliosidosis is evident: deficiency of one of the chains of which the protein is composed leads to an excess of polymers comprised of the other chains. In type B GM2 gangliosidosis, the excess of beta chanis leads to increased amounts of hex B beta)6; in type 0 GM2 gangliosidosis, the excess of alpha chains leads to formation of increased amounts of the alpha chain polymer, hex S.

The immunological characteristics of feline beta-hexosaminidase (beta-D-N-acetylglucosaminidase, EC 3.2.1.30) isoenzymes, Hex A and Hex B, were studied. Immunization of rabbits and goats with either cat Hex A or Hex B produced antibodies which reacted with a common antigenic marker shared by both Hex A and Hex B. With properly absorbed antisera, a unique antigenic marker was demonstrated on cat Hex A, but not on Hex B. This antigenic profile is comparable to that of the human beta-hexosaminidase isozymes, in which both Hex A and Hex B share the antigenic determinant, beta, while only Hex A possesses the antigenic determinant, alpha. Ho cross-reactivity between the two species could be demonstrated using goat or rabbit antisera to either feline of human beta-hexosaminidase. These immunological data validate feline Gm2 gangliosidosis as a model for human Gm2 gangliosidosis type II, and facilitate the investigation of enzyme replacement therapy.

Antisera were raised in rabbits against native and sodium dodecylsulfate denatured forms of human acid alpha-glucosidase and beta-hexosaminidases A and B. Anti-native enzyme antisera were able to precipitate all or nearly all enzyme activity from cell extracts, and to eliminate all stainable activity on electrophoresis. Antisera prepared against denatured enzymes precipitated only a minor part of enzyme activity. Electrophoretic analysis showed that these antisera were able to bind to the enzyme molecule. The result was a slowing down of the anodic migration but not immobilization. The use of variants with hexosaminidase deficiencies helped to clarify the action of the antisera on the various hexosaminidase isozymes.

Antibodies against placental hexosaminidase A and kidney alpha-subunits were raised in rabbits after cross-linking the antigens with glutaraldehyde. Anti-(alpha(n)-subunit) antiserum (anti-alpha(n)) precipitated hexosaminidase A but not hexosaminidase B, whereas anti-(hexosaminidase A) antiserum precipitated both hexosaminidases A and B. Specific anti-(hexosaminidase A) antiserum was prepared by absorbing antiserum with hexosaminidase B. Both anti-alpha(n) and anti-(hexosaminidase A) antisera precipitated the CR (cross-reacting) material from eight unrelated patients with Tay-Sachs disease. Immunotitration, immunoelectrophoresis, double-immunodiffusion and radial-immunodiffusion techniques were used to demonstrate the presence of CR material. The CR-material-antibody complex was enzymically inactive. Antiserum raised against kidney or placental hexosaminidase A, without cross-linking with glutaraldehyde, failed to precipitate the CR material, implying that treatment of the protein with glutaraldehyde exposes antigenic determinants that are hidden in the native protein. Since anti-(hexosaminidase B) antiserum did not precipitate the CR material during the immunoelectrophoresis of Tay-Sachs liver extracts, it is suggested that altered alpha-subunits do not combine with beta-subunits. By using immunotitration we have demonstrated the competition between the hexosaminidase B-free Tay-Sachs liver extract and hexosaminidase A for the common binding sites on monospecific anti-(cross-linked hexosaminidase A) antiserum. The amount of CR material in the liver samples from seven cases of Tay-Sachs desease was found to be in the same range as theoretically calculated alpha-subunits in normal liver samples. Similar results were obtained by the radial-immunodiffusion studies. The present studies therefore suggest that Tay-Sachs disease is caused by a structural-gene mutation.

beta-N-Acetylhexosaminidase I2 was purified from human liver by a combination of concanavalin A chromatography, DEAE-cellulose chromatography, gel filtration and affinity chromatography on 2-acetamido-N-(6-aminohexanoyl)-2-deoxy-beta-D-glucopyranosylamine coupled to CNBr-activated Sepharose 4B. Its specific activity was 130 mumol/min per mg of protein compared with values of 150 and 320 mumol/min/mg of protein for beta-N-acetylhexosaminidases A and B purified from the same tissue. Km values for I2, A and B were 1.0 mM, 0.8 mM and 0.74 mM respectively. On gradient gel electrophoresis under non-denaturing conditions, hexosaminidase I2 behaved similarly to A and appeared to have an Mr between 100 000 and 110 000. beta-N-Acetylhexosaminidase I2 was resolved into two major polypeptides, of Mr 56 000 and 29 000, on SDS/polyacrylamide-gel electrophoresis under denaturing conditions. Immunoblotting with anti-(hexosaminidase alpha-subunit) serum confirmed that the 56 000-Mr component was the alpha-subunit and anti-(hexosaminidase B) serum reacted with the 29 000 Mr component. beta-N-Acetylhexosaminidase I2 more closely resembles form A than B, but the features of its structure that allow it to be separated from A on the basis of net charge have not yet been found.

Antibodies were produced against purified beta-N-acetylhexosaminidase from Dictyostelium discoideum. Ouchterlony tests produced a single precipitin band with both beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase activities. Both activities were co-precipitated in exactly the same proportions in quantitative immunoprecipitation reactions. We conclude that one enzyme protein catalyses the hydrolysis of both N-acetylglucosaminides and N-acetylgalactosaminides.

An immunochemical method has been developed for the quantitative determination of species-specific gene products, for instance alpha-galactosidase and N-acetyl-alpha-galactosaminidase, in man-rodent hybrid cells and in the parental cell lines. Antisera raised against the purified enzymes are covalently coupled to Sepharose 4B. The gene products are specifically removed from a cell lysate by incubating with the appropriate Sepharose-coupled antiserum. After centrifugation followed by washing of the precipitated Sepharose, the enzymic activity can be quantitatively measured on the Sepharose beads. With this technique it has been demonstrated that the ability of human N-acetyl-alpha-galactosaminidase (also known as alpha-galactosidase B) to hydrolyze alpha-galactosidic linkages is lost when the enzyme is expressed in man-Chinese hamster hybrid cells.

Hexasaminidase P, the main isozyme of hexosaminidase in pregnancy serum, was isolated and purified 600--700-fold by a two-step purification procedure--affinity chromatography on Sepharose-bound epsilon-aminocaproyl-N-acetylglucosylamine, followed by ion-exchange chromatography on DEAE-cellulose. The purified enzyme was subjected to biochemical and immunochemical analysis. Its catalytic property, namely, kinetic behavior, is similar to that of the major isozymes of hexosaminidase, A and B. However, it differs from these isozymes in its electrophoretic mobility and in its apparent molecular weight which is around 150 000 compared with 100 000 of the A and B isozymes. Immunochemical analysis indicates that the P isozymes is antigenically cross-reactive with both A and B isozymes, but it does not contain the A-specific antigenic determinants, and exhibits identical antigenic specificity to hexasaminidase B. Two possible structures are suggested that are compatible with the experimental data: (a) a hexosaminidase B like structure with higher extent of glycosylation; (b) a hexameter of beta chain, possibly arranged as three beta2 subunits.

Those proteins of human liver that cross-reacted with antibodies raised to apparently homogenous hexosamindases A and B were detected by immunodiffusion. Cross-reacting proteins with high molecular weights (greater than 2000000) and intermediate molecular weights (70000--200000) were present both in the unadsorbed fraction and in the 0.05--0.2M-NaCl eluate obtained by DEAE-cellulose chromatography at pH7.0. The unadsorbed fraction also contained a cross-reacting protein of low molecular weight (10000--70000). The possible structural and functional relationships between hexosaminidase and the cross-reacting proteins are discussed. An apparently cross-reacting protein present in the 0.05M-NaCl eluate from the DEAE-cellulose column was serologically unrelated to hexosaminidase, but it gave a reaction of immunological identify with one of the apparently cross-reacting proteins having the charge and size characteristics of hexosaminidase A. It is suggested that immunochemical methods may provide criteria for the homogeneity of enzyme preparations superior to those of conventional methods.

Hexosaminidase S (HEX S), the residual isozyme found in tissues and body fluids of children with the O variant of GM2 gangliosidosis, was purified from tissues of variant individuals and biochemically and immunochemically characterized. This enzyme has an apparent molecular weight of 103,000 with an isoelectric point of 4.2, is heat labile to the same extent as HEX A, and loses most of its activity following heating for 30 min at 50 degrees C. HEX S reacts immunologically with the antisera against either HEX A or B, but the reaction is considerably stronger with the anti-A serum or with antibody preparations which react exclusively with the A isozyme. Results obtained by a radioimmunoassay using the various antisera indicated that there is no antigenically cross reacting material which lacks enzymatic activity in the variant tissues. These findings are in accord with a suggested molecular structure of two subunits, each composed of two alpha chains (alpha2 alpha2) for HEX S; it also implies that alpha and beta chains have some structural similarity which is manifested in antigenic cross-reactivity.

Human hexosaminidase A was covalently bound to soluble poly(N-vinylpyrrolidone), and the effect of this binding on the enzyme inactivation by various procedures was investigated. Whereas the polymer-bound hexosaminidase underwent inactivation to the same extent as the free enzyme, when exposed to heat or acidic pH, the conjugation to polymer appeared to protect the enzyme towards proteolysis. Thus, the polymer-bound enzyme exhibited considerably higher resistance to treatment of both pronase and macrophage cathepsins. The clearance rate from rabbit blood, of the polymer-bound enzyme (expressed as enzyme activity), was shown to be significantly slower than that of the free enzyme.

An initial attempt to prepare monoclonal antibodies specific for the Dictyostelium discoideum lysosomal enzyme beta-glucosidase was unsuccessful. All of the antibodies resulting from this fusion recognized an extremely immunogenic epitope that is present on all of the lysosomal enzymes of Dictyostelium. In two succeeding fusions, changes in the immunization schedule intended to increase the immune response to enzyme-specific epitopes were not entirely successful. Although nine hybridomas producing antibodies specific for beta-glucosidase resulted from these two fusions, most (70%) of the cell lines isolated secrete antibodies that recognize the shared, immunodominant epitope. Moreover, the nine beta-glucosidase-specific antibodies proved to be of limited utility since none recognize the native enzyme. Therefore, we attempted to tolerize a BALB/c mouse to the common epitope by injecting the lysosomal enzyme, N-acetylglucosaminidase, within 40 h after birth. As an adult, this animal was immunized with beta-glucosidase. Fusion of the spleen cells from this mouse with myeloma cells resulted in the isolation of nine hybridoma lines that produce antibodies specific for beta-glucosidase. No antibodies reactive with the common epitope were detected. These results suggest that tolerization may provide a means whereby an undesired class of antibody-producing cell lines can be selectively eliminated from the products of a fusion.

beta-Galactosidase purified to apparent homogeneity from human placenta occurred in two separable fractions. A low molecular mass form (relative mass (Mr) 170 000) is composed of a single polypeptide chain (Mr 70 000). This was derived from a larger form by molecular sieve chromatography at both low (10 mM) and high (500 mM) NaCl concentration. The larger form of beta-galactosidase also contains small amounts of two polypeptides with apparent Mr values of 23 000 and 35 000 daltons. Both forms of the enzyme hydrolyze synthetic aryl galactosides and natural glycolipid substrates at comparable rates. Antibodies raised in rabbits against the low Mr beta-galactosidase also cross-reacts with the high Mr enzyme. The antibody preparation also cross-reacted with beta-hexosaminidase even though the latter was found at very low levels in the antigen, as judged by lack of detection of representative protein bands on sodium dodecyl sulfate - polyacrylamide gel electrophoresis and enzyme activity measurements. A portion of this cross-reactivity (35%) against beta-hexosaminidase could not be absorbed from the preparation without the simultaneous loss of beta-galactosidase activity, suggesting that the two enzymes show a degree of antigenic identity.

beta-N-Acetylhexosaminidase from boar epididymis was separated into two forms, A and B, on DEAE-cellulose. Both these forms were excluded from Sepharose S-200 and had apparent Mr values of 510 000 on gradient gel electrophoresis under non-denaturing conditions. Affinity chromatography on 2-acetamido-N-(6-aminohexanoyl)-2-deoxy-beta-D-glucopyranosylam ine coupled to CNBr-activated Sepharose 4B was used to separate and purify beta-N-acetylhexosaminidases A and B that had specific activities of 115 and 380 mumol/min per mg of protein respectively. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of denatured beta-N-acetylhexosaminidase A gave a single major component of Mr 67 000. beta-N-Acetylhexosaminidase B also had this component, and in addition had polypeptides of Mr 29 000 and 26 000. All these polypeptides were glycosylated. Antiserum to the B form precipitated form A from solution and reacted with the 67 000-Mr component or form A after electrophoretic transfer from sodium dodecyl sulphate/polyacrylamide gels to nitrocellulose sheets. The 67 000-Mr components of forms A and B yielded identical peptide maps when digested with Staphylococcus aureus V8 proteinase, and the 29 000-Mr and 26 000-Mr components in form B may be related to the 67 000-Mr polypeptide.