Multifunctional fluorescent probes for high-throughput characterization of hexosaminidase enzyme activity.

Microbial polysaccharides composed of N-acetylglucosamine (GlcNAc), such as chitin, peptidoglycan and poly-ß-(1 â†’ 6)-GlcNAc (dPNAG), play a critical role in maintaining cell integrity or in facilitating biofilm formation in numerous fungal and bacterial pathogens. Glycosyl hydrolase enzymes that catalyze the degradation of these ß-GlcNAc containing polysaccharides play important roles in normal microbial cell physiology and can also be exploited as biocatalysts with applications as anti-fungal, anti-bacterial, or biofilm dispersal agents. Assays to rapidly detect and characterize the activity of such glycosyl hydrolase enzymes can facilitate their development as biocatalyst, however, currently available probes such as 4-methylumbelliferyl-ß-GlcNAc (4MU-GlcNAc) are not universally accepted as substrates, and their fluorescent signal is sensitive to changes in pH. Here, we present the development of a new multifunctional fluorescent substrate analog for the detection and characterization of hexosaminidase enzyme activity containing a 7-amino-4-methyl coumarin (AMC) carbamate aglycone. This probe is widely tolerated as a substrate for exo-acting ß-hexosaminidase, family 19 endo-chitinase, and the dPNAG hydrolase enzyme Dispersin B (DspB) and enables detection of hexosaminidase enzyme activity via either single wavelength fluorescent measurements or ratiometric fluorescent detection. We demonstrate the utility of this probe to screen for recombinant DspB activity in Escherichia coli cell lysates, and for the development of a high-throughput assay to screen for DspB inhibitors.

Investigation of seminal plasma chitotriosidase-1 and leukocyte elastase as potential markers for 'silent' inflammation of the reproductive tract of the infertile male - a pilot study.

Inflammatory mediators - chitotriosidase-1 (CHIT1) and leukocyte elastase (LE) - were analyzed in human seminal plasma in relation to total antioxidative status (TAS) and pro-inflammatory markers IL-1ß and IL-6. Samples collected from 34 males who were part of infertile couples were divided into normozoospermic (N; n = 12, without symptoms of inflammation), oligozoospermic (O; n = 11) and teratozoospermic (T; n = 11) groups. significant differences were observed only in CHIT1 concentration between N and O samples. However, a higher mean LE concentration was also observed in O and T patients (3.7-times and 900-times, respectively) compared with the N group. in IL-1ß and IL-6 concentrations, an upward trend was observed from N, through O, up to the T group. The positive correlation between the concentration of IL-1ß and the activity and specific activity of CHIT11 as well as the moderate negative correlation between concentrations of IL-1ß and CHIT1 may suggest that elevated CHIT11 levels appeared in early stages of inflammation before the increase in IL-1ß concentrations, or remained stable even after the levels of cytokine decreased. The above seem to confirm the role of CHIT1 in the manifestation of 'silent' inflammation at a very early stage. To conclude, CHIT1 concentration appears to be an interesting biomarker that signals the presence of possible 'silent' inflammation accompanying oligozoospermia. We cannot draw such conclusions regarding LE concentration, because, although we observed differences in the mean values and medians between analyzed groups, they were not significant. The utility of CHIT1 in the follow-up of oligozoospermia-associated 'silent' subclinical inflammation is promising, but further studies on a larger patient test set are required.

Sheep and goats raised in mixed flocks have diverse immune status around parturition.

Several physiological and metabolic changes take place in dairy ruminants around parturition (late pregnancy, parturition, and early lactation). Dairy species are genetically selected for their higher milk production compared with non-dairy species. This fact causes a constant stress that impairs the immune status of the animal, with consequences for its welfare and performance. In the present study, we assessed the immune status of high-yield dairy sheep and goats by quantifying IgG and IgM concentrations, as well as chitotriosidase (ChT) and complement system [total complement system (TC) and alternative complement pathway (AC)] activity in blood plasma around parturition. We also measured IgG and IgM concentrations and ChT activity in colostrum and milk during the first 40 d postpartum. The lowest blood IgG concentration was at parturition in both species. We detected no differences in blood IgG concentrations between species. Blood IgM concentrations were constant in both species throughout the study period. However, blood IgM concentrations were greater in sheep than in goats. Blood ChT activity was greater in goats than in sheep, and both species showed constant activity of this enzyme throughout the study period. We observed no differences in complement system (TC and AC) activity between sheep and goats. In addition, both TC and AC activity were constant in both species throughout the experiment. In general, IgG and IgM concentrations were greater in sheep colostrum than in goat colostrum, but these differences disappeared after d 4 (IgG) and d 3 (IgM) postpartum. In both species, the highest IgG and IgM concentrations were measured in colostrum, gradually decreasing during the first days postpartum. Chitotriosidase activity decreased in both species from colostrum to milk, although goats always showed greater ChT activity than sheep. Both sheep and goats seemed to be more susceptible to infectious diseases around parturition. As well, goats showed greater ChT activity in blood, colostrum, and milk than sheep. This fact may give these animals additional protection against parasite and fungal infections.

Evaluation of chitotriosidase as a biomarker for adipose tissue inflammation in overweight individuals and type 2 diabetic patients.

BACKGROUND: Overweight and obesity can lead to adipose tissue inflammation, which causes insulin resistance and on the long-term type 2 diabetes mellitus (T2D). The inflammatory changes of obese-adipose tissue are characterized by macrophage infiltration and activation, but validated circulating biomarkers for adipose tissue inflammation for clinical use are still lacking. One of the most secreted enzymes by activated macrophages is chitotriosidase (CHIT1). OBJECTIVE: To test whether circulating CHIT1 enzymatic activity levels reflect adipose tissue inflammation. METHODS: Plasma and adipose tissue samples of 105 subjects (35 lean, 37 overweight, and 33 T2D patients) were investigated. CHIT1 mRNA levels were determined in adipose tissue-resident innate immune cells. CHIT1 mRNA levels, protein abundance, and plasma enzymatic activity were subsequently measured in adipose tissue biopsies and plasma of control subjects with varying levels of obesity and adipose tissue inflammation as well as in T2D patients. RESULTS: In adipose tissue, CHIT1 mRNA levels were higher in stromal vascular cells compared to adipocytes, and higher in adipose tissue-residing macrophages compared to circulating monocytes (p < 0.001). CHIT1 mRNA levels in adipose tissue were enhanced in overweightcompared to lean subjects and even more in T2D patients (p < 0.05). In contrast, plasma CHIT1 enzymatic activity did not differ between lean, overweight subjects and T2D patients. A mutation of the CHIT1 gene decreases plasma CHIT1 activity. CONCLUSIONS: CHIT1 is expressed by adipose tissue macrophages and expression is higher in overweight subjects and T2D patients, indicating its potential as tissue biomarker for adipose tissue inflammation. However, these differences do not translate into different plasma CHIT1 activity levels. Moreover, a common CHIT1 gene mutation causing loss of plasma CHIT1 activity interferes with its use as a biomarker of adipose tissue inflammation. These results indicate that plasma CHIT1 activity is of limited value as a circulating biomarker for adipose tissue inflammation in human subjects.

Senescence mirrors the extent of liver fibrosis in chronic hepatitis C virus infection.

BACKGROUND: Chronic viral hepatitis is linked to fibrotic liver injury that can progress to liver cirrhosis with its associated complications. Recent evidence suggests a role of senescence in liver fibrosis, although the senescence regulators contributing to fibrosis progression remain unclear. AIM: To investigate the role of senescence and different senescence markers for fibrosis progression in patients with chronic hepatitis C virus (HCV) infection. METHODS: The expression of the cell cycle inhibitors p21, p27 and p16 as well as the senescence markers p-HP1γ and γ-H2AX was analysed in liver tissue with different fibrosis stages. Senescence-associated chitotriosidase activity was measured in sera of HCV patients (n = 61) and age-matched healthy individuals (n = 22). RESULTS: We found a remarkable up-regulation of the cell cycle inhibitors and senescence markers in chronic HCV infection compared to healthy liver tissue. Liver tissue with relevant fibrosis stages (F2-3) or cirrhosis (F4) revealed a significant increase in senescent cells compared to livers with no or minimal fibrosis (F0-1). In cirrhotic livers, a significantly higher number of p-HP1γ, p21 and p27 positive cells was detected compared to liver tissue with F2-3 fibrosis. Importantly, we identified T-cells as the dominant cell type contributing to increased senescence during fibrosis progression. Compared to healthy individuals, serum chitotriosidase was significantly elevated and correlated with histological fibrosis stages and liver stiffness as assessed by transient elastography. CONCLUSIONS: Senescence of hepatic T-cells is enhanced in chronic viral hepatitis and increases with fibrosis progression. Serological detection of senescence-associated chitotriosidase might allow for the non-invasive detection of relevant fibrosis stages.

Chitotriosidase inhibits allergic asthmatic airways via regulation of TGF-ß expression and Foxp3+ Treg cells.

BACKGROUND: Chitotriosidase (chitinase 1, Chit1), a major true chitinase in humans, is induced in childhood asthma and has been implicated in the pathogenesis of a variety of inflammatory and tissue remodeling responses. However, the role and the mechanisms that underlie these contributions to the diseases have not been defined. We hypothesized that Chit1 plays a significant role in the pathogenesis of allergic asthma. METHODS: Wild-type and Chit1-deficient mice and cells in culture were used to define the roles of Chit1 in models of allergic adaptive Th2 inflammation. In addition, the levels of sputum Chit1 were evaluated in pediatric asthma patients and compared to control. RESULTS: The levels of sputum Chit1 were significantly increased in the patients with childhood asthma. Mice with Chit1 null mutation demonstrated enhanced allergic Th2 inflammatory and cytokine and IgE responses to OVA or house dust mite allergen sensitization and challenge. However, the expression levels of TGF-ß1 were significantly decreased with a diminished number of Foxp3+ regulatory T cells (Treg) in the lungs of Chit1-/- mice compared to WT controls. In vitro, the absence of Chit1 significantly reduced TGF-ß-stimulated conversion of CD4+ CD25- naïve T cells to CD4+ Foxp3+ Treg cells, suggesting Chit1 is required for optimal effect of TGF-ß1 in Treg cell differentiation. CONCLUSION: Chit1 plays a protective role in the pathogenesis of allergic inflammation and asthmatic airway responses via regulation of TGF-ß expression and Foxp3+ Treg cells.

Factors affecting the incidence and outcome of Trueperella pyogenes mastitis in cows.

The main factors affecting the outcome of Trueperella pyogenes (T. pyogenes) mastitis were examined through a survey of diagnostic data and interviews relating to the occurrence of T. pyogenes mastitis in 83 quarters from 82 Holstein cows between August 2012 and April 2014. Ultimately, one cow was sold during the examination, and 82 quarters from 81 cows were used for analysis on prognosis. T. pyogenes mastitis occurred year round in both lactating and dry cows. The incidence of T. pyogenes mastitis did not significantly differ by month or show seasonality in either lactating or dry cows. Therefore, the occurrence of T. pyogenes mastitis also differed from that of summer mastitis. The 1-month survival rate of infected cows was 64.6% (53/82), and the recovery rate of quarters with T. pyogenes mastitis was 14.6% (12/82). Bivariate logistic regression analysis was performed with survival and culling of infected cows as objective variables and with recovery and non-recovery of quarters with T. pyogenes mastitis as objective variables. The severe cases were significantly culled (odds ratio, 16.30) compared to mild cases, and the status of quarters didn't recover (odds ratio, 6.50). The results suggest that mild to moderate symptom severity at the time of onset are the main factors affecting outcomes in cows and recovery of quarters infected with T. pyogenes mastitis. Further, high level of NAGase activity also suggested the potential use as an indicator of culling of cows with T. pyogenes mastitis.

Treatment with rhDNase in patients with cystic fibrosis alters in-vitro CHIT-1 activity of isolated leucocytes.

Recent data suggest a possible relationship between cystic fibrosis (CF) pharmacotherapy, Aspergillus fumigatus colonization (AC) and/or allergic bronchopulmonary aspergillosis (ABPA). The aim of this study was to determine if anti-fungal defence mechanisms are influenced by CF pharmacotherapy, i.e. if (1) neutrophils form CF and non-CF donors differ in their ability to produce chitotriosidase (CHIT-1); (2) if incubation of isolated neutrophils with azithromycin, salbutamol, prednisolone or rhDNase might influence the CHIT-1 activity; and (3) if NETosis and neutrophil killing efficiency is influenced by rhDNase. Neutrophils were isolated from the blood of CF patients (n = 19; mean age 26·8 years or healthy, non-CF donors (n = 20; 38·7 years) and stimulated with phorbol-12-myristate-13-acetate (PMA), azithromycin, salbutamol, prednisolone or rhDNase. CHIT-1 enzyme activity was measured with a fluorescent substrate. NETosis was induced by PMA and neutrophil killing efficiency was assessed by a hyphae recovery assay. Neutrophil CHIT-1 activity was comparable in the presence or absence of PMA stimulation in both CF and non-CF donors. PMA stimulation and preincubation with rhDNase increased CHIT-1 activity in culture supernatants from non-CF and CF donors. However, this increase was significant in non-CF donors but not in CF patients (P < 0·05). RhDNase reduced the number of NETs in PMA-stimulated neutrophils and decreased the killing efficiency of leucocytes in our in-vitro model. Azithromycin, salbutamol or prednisolone had no effect on CHIT-1 activity. Stimulation of isolated leucocytes with PMA and treatment with rhDNase interfered with anti-fungal defence mechanisms. However, the impact of our findings for treatment in CF patients needs to be proved in a clinical cohort.

Bronchoalveolar lavage chitotriosidase activity as a biomarker of sarcoidosis.

BACKGROUND: Chitotriosidase (CTO) was shown to be a good biomarker of sarcoidosis. Increased levels in bronchoalveolar lavage fluid (BALF) were reported and associated with more severe forms of the disease. OBJECTIVES: The aim of the study was to evaluate the value of CTO in BALF as a routine biomarker of sarcoidosis. METHODS: The study included 85 patients in 9 control subjects in whom serum and BALF CTO were measured. RESULTS: Significantly higher CTO levels were detected in BALF of sarcoidosis patients than in control subjects (p < 0.001). There was good correlation between serum and BALF CTO levels in sarcoidosis patients (Spearman's Rho 0.481, p < 0.001). Serum but not BALF CTO had good correlation with clinical parameters. Only in a group of patients with BALF CTO above upper normal range there was association of BALF CTO with impaired FVC (p = 0.020) and chest radiograph score (0-2 vs. 3-4, p = 0.016). CONCLUSIONS: In comparison to serum CTO no additional benefit of determining CTO in BAL for routine sarcoidosis workup was shown.

Stereoselective synthesis of 2-acetamido-1,2-dideoxyallonojirimycin (DAJNAc), a new potent hexosaminidase inhibitor.

A practical synthesis of the previously unreported N-acetyl-D-allosamine glycomimetic DAJNAc is described. The reaction sequence involves Pd-catalyzed allylic substitution by phthalimide in an azaheterobicyclic scaffold as the key step. The new iminosugar resulted in being a stronger ß-N-acetylglucosaminidase (human placenta) competitive inhibitor than the D-gluco (DNJNAc) and D-galacto (DGJNAc) stereoisomers.

Airborne fungal cell fragments in homes in relation to total fungal biomass.

Fungal exposure may induce respiratory symptoms. The causative agents are compounds in the fungal cell wall. Fragments of microbes may be present in air samples but are not measurable using conventional spore counting or by the determination of viable organisms. This study assesses the proportion of fungal cell biomass and endotoxin in different particle size fractions in air samples from homes. Air samples were collected from 15 homes using a cyclone sampler, collecting particles in three aerodynamic size fractions: <1.0, 1.0-1.8, and >1.8 µm. N-Acetylhexosaminidase (NAHA) was determined as a marker of fungal cell biomass. Endotoxin was determined using the Limulus amebocyte lysate method. NAHA and endotoxin in the size range <1.0 µm comprised up to 63% (mean 22.7%) and 96.3% (mean 22.6%) of the total concentrations, respectively. There were significant relationships between the amounts of NAHA and endotoxin in the total amount and in the size fraction >1.8 µm but not in the smaller fractions. The results demonstrate significant amounts of fungal cell biomass and endotoxin in particles <1.0 µm. Homes with reported mold damage had a lower concentration of NAHA in particles <1.0 µm than homes without mold damage. To assess airborne exposure for diagnostic and preventive purposes, measurement techniques that include this fraction should be considered.

Salivary hexosaminidase in smoking alcoholics with bad periodontal and dental states.

BACKGROUND: A sensitive alcohol marker, ß-hexosaminidase (HEX), in the saliva of alcoholics, is investigated for the first time. METHODS: The activity, specific-activity and output of total HEX and its isoenzymes HEX A and HEX B were measured in the saliva of healthy controls (C), alcohol-dependent non-smokers (ANS), and alcohol-dependent smokers (AS). RESULTS: We observed a significantly increased activity/specific-activity and output of HEX A in the ANS and AS groups, due to the inflammatory state of the oral-cavity/salivary-glands. Significantly increased activity of HEX A contributed to an increase in the salivary activity of the total HEX in the ANS group. A significant decrease in the activity/specific-activity of HEX B in AS seemed to be due to HEX B inactivation by cigarette smoke. We noticed a tendency for deteriorated dental state (lower decayed-missing-filled-teeth index - DMFT), worse periodontal state (higher gingival index - GI and papilla-bleeding index - PBI) in AS, and worse periodontal state (higher GI) in ANS, as compared to the controls. We found no differences in the salivary protein concentrations between all groups and decreased salivary flow in both alcoholic groups as compared to the controls. In alcoholics, the area under the curve (AUC) for HEX A activity/specific-activity was significantly greater than for HEX and HEX B. The salivary HEX A activity/specific-activity had good/excellent sensitivity and specificity in smoking and non-smoking alcoholics, whereas salivary HEX and HEX B had poor/fair sensitivity and specificity. CONCLUSIONS: Salivary HEX A may be helpful in the diagnosis of chronic alcohol intoxication, even in smokers.

Chitotriosidase levels in patients with severe endometriosis.

OBJECTIVE: To study the levels of chitotriosidase activity in the peritoneal fluid and the plasma of patients with severe endometriosis and control subjects. MATERIALS AND METHODS: Twenty-five women with laparoscopically and histopathologically confirmed endometriosis (study group) and 27 control patients who had undergone laparoscopic surgery were included. Peritoneal fluid and peripheral blood were obtained from all the patients before the surgery. Chitotriosidase activities were measured. RESULTS: Analysis of chitotriosidase activity in the peritoneal fluid of patients with endometriosis showed that there was no significant difference between endometriosis and control group, respectively (32.04 ± 64.20 vs. 15.25 ± 31.17 nmol/mL/h; p > 0.05). Analysis of chitotriosidase activity in plasma of patients with endometriosis showed significantly increased levels of chitotriosidase levels compared with the control group (74.81 ± 60.54 vs. 14.10 ± 26.17; p < 0.001), respectively. CONCLUSION: We found that the activity of chitotriosidase in plasma was statistically higher in severe endometriosis patients than women without endometriosis.

In vitro and in vivo reactivity to fungal cell wall agents in sarcoidosis.

Sarcoidosis is an inflammatory disease. Epidemiological and treatment studies suggest that fungi play a part in the pathogenesis. The aim of this work was to study the effect of fungal cell wall agents (FCWA) on the in vitro secretion of cytokines from peripheral blood monocytes from subjects with sarcoidosis and relate the results to fungal exposure at home and clinical findings. Subjects with sarcoidosis (n=22) and controls (n=20) participated. Peripheral blood mononuclear cells were stimulated with soluble or particulate ß-glucan (S-glucan, P-glucan), chitin or lipopolysaccharide (LPS), whereafter tumour necrosis factor (TNF)-α, interleukin (IL)-6, IL-10 and IL-12 were measured. The severity of sarcoidosis was determined using a chest X-ray-based score. Serum cytokines (IL-2R, IL-6, IL-10 and IL-12) were determined. To measure domestic fungal exposure, air in the bedrooms was sampled on filters. N-acetylhexosaminidase (NAHA) on the filters was measured as a marker of fungal cell biomass. The induced secretion of cytokines was higher from peripheral blood mononuclear cells (PBMC) from subjects with sarcoidosis. P-glucan was more potent than S-glucan inducing a secretion. Chitin had a small effect. Among subjects with sarcoidosis there was a significant relation between the spontaneous PBMC production of IL-6, IL-10 and IL-12 and the NAHA levels at home. The P-glucan induced secretion of IL-12 was related to the duration of symptoms at the time of diagnosis. Their X-ray scores were related to an increased secretion of cytokines after stimulation with LPS or P-glucan. Subjects with sarcoidosis have a higher reactivity to FCWA in vitro and to home exposure. The influence of FCWA on inflammatory cells and their interference with the inflammatory defense mechanisms in terms of cytokine secretion could be important factors for the development of sarcoidosis.

Effects of induced parturition in goats on immunoglobulin G and chitotriosidase activity in colostrum and plasma and on plasma concentrations of prolactin.

The effect of induction of parturition with a PGF(2)α analog on plasma concentration of prolactin (PRL) and its effects on colostrum concentration of IgG and chitotriosidase (ChT) activity were studied in 16 pregnant Majorera goats. Treated goats, those in which parturition was induced, had greater concentrations of PRL than control goats 24 h before parturition (P < 0.05) and 48 h after parturition (P < 0.05). Control goats had greater concentrations of PRL than treated goats 96 h after parturition (P < 0.05). Plasma concentration of IgG did not differ between groups during the experimental period, but colostrum concentrations of IgG were greater in control goats than in treated goats at parturition (P < 0.05). Plasma ChT activity decreased during the period 72 h before parturition to 24 h after parturition in control and treated goats. Time evolution after partum affected the colostrum ChT activity, being greater at parturition than after parturition in both groups (P < 0.05). In summary, concentration of IgG in colostrum is slightly diminished if parturition is induced. Induction of parturition causes an early increase in PRL, which is most likely responsible for preterm suppression of IgG transport into mammary secretions.

Osseous manifestations of adult Gaucher disease in the era of enzyme replacement therapy.

Enzyme replacement therapy (ERT) for Gaucher disease with mannose-terminated glucocerebrosidase has proved its therapeutic position with salutary effects on hematologic abnormalities, visceral infiltration, and quality of life. The frequency of new bone complications is reduced but not eliminated. Established osteonecrosis is beyond salvage. A systematic description of the burden of bone manifestations, persisting despite ERT, should inform future remedial strategies. Thus, we conducted this study to quantify the burden of residual skeletal disease and to explore putative relationships between clinical, radiologic, and biochemical factors and bone sequelae associated with disability.Consecutive adult patients attending 3 referral centers in the United Kingdom were invited to participate. A representative group of 100 patients agreed to a structured interview, clinical examination, radiologic review, and completion of questionnaires. Osteonecrosis was evident in 43%, Erlenmeyer flask deformity in 59%, fragility fracture in 28%, osteomyelitis in 6%, and lytic lesions in 4%. Mobility was impaired in 32% of patients, while 15% experienced significant pain. The EuroQol 5D (EQ5D) quality of life summary measure was reduced and was associated with osteonecrosis and fragility fracture. Eight patients experienced new osteonecrosis after the start of ERT, though the presentation and evolution were often atypical. Nine patients had been treated from childhood and had an excellent outcome. Osteonecrosis was associated with age of presentation and with splenectomy-indeed, we observed a strong temporal association between splenectomy and incidence of osteonecrosis.The biomarkers PARC/CCL18 and chitotriosidase were associated with prevalent osteonecrosis, and, in particular, with osteonecrosis occurring despite treatment. This study documents significant residual skeletal pathology and disability in patients in the mature phase of their treatment in a developed region. The temporal association between splenectomy and osteonecrosis implies causation. The relationship between clinical and biochemical markers and existing bone complications sets the scene for future prospective studies that will focus on management strategies informed by credible assessment of risk.

[Diagnosis, biomarkers and biochemical alterations in Gaucher's disease]. / Diagnóstico, biomarcadores y alteraciones bioquímicasde la enfermedad de Gaucher.

Gaucher's disease (GD) is an inherited metabolic disease due to lack of activity of the enzyme betaglucocerebrosidase. The diagnosis of GD is usually performed by fluorimetric analysis of the enzyme betaglucocerebrosidase. It can also be done by measuring the activity of tandem mass spectrometry. This enzyme can be analyzed in different samples such as leukocytes, fibroblasts, blood dried on paper and in case of prenatal diagnosis in chorionic villi or culture of amniocytes. Various biomarkers have been described for monitoring GD once diagnosed, to evaluate the response to potential treatments. Chitotriosidase activity is the most widely used biomarker for the assessment of GD, and for patients homozygous for the null CHIT1 gene variants, in general, is replaced by the analysis of the biomarker CCL18.

Increased expression of acidic mammalian chitinase and chitotriosidase in the nasal mucosa of patients with allergic rhinitis.

OBJECTIVES/HYPOTHESIS: Acidic mammalian chitinase (AMCase) has emerged as an important mediator of allergic asthma in both animal models and in humans. Recently, chitotriosidase has been suggested to play a role in innate immunity because of its phagocytic-specific expression. Thus, AMCase and chitotriosidase may play a role in the pathogenesis of allergic nasal mucosa. The expression and pattern of distribution of AMCase and chitotriosidase were, therefore, determined in normal and allergic nasal mucosa. STUDY DESIGN: Controlled, prospective study. METHODS: Normal inferior turbinate mucosa was obtained in patients who were admitted for augmentation rhinoplasty. Allergic turbinate mucosa was obtained from patients who had perennial allergic rhinitis during septo-turbinate surgery. Reverse transcriptase-polymerase chain reaction (RT-PCR), immunohistochemistry, and Western blotting were applied to the normal and allergic nasal mucosa. RESULTS: The expression of AMCase and chitotriosidase mRNAs and proteins analyzed by RT-PCR and Western blot were detected in all normal and allergic turbinate mucosa tested. The levels of expression of AMCase and chitotriosidase mRNAs and proteins were increased in allergic turbinate mucosa compared with normal turbinate mucosa. In both normal and allergic turbinate mucosa, AMCase and chitotriosidase were detected in the epithelium, inflammatory cells, and submucosal glands. The staining intensity for AMCase and chitotriosidase was stronger in allergic nasal mucosa than normal nasal mucosa. CONCLUSIONS: AMCase and chitotriosidase are constitutively expressed in normal turbinate mucosa, suggesting involvement in defense against chitin-containing pathogens. Upregulation of these chitinases in allergic condition suggests that they may play a role in the nasal allergic reaction like other inflammatory mediators in allergic rhinitis. Laryngoscope, 2010.

Chitotriosidase is the primary active chitinase in the human lung and is modulated by genotype and smoking habit.

BACKGROUND: Chitinolytic enzymes play important roles in the pathophysiology of allergic airway responses in mouse models of asthma. Acidic mammalian chitinase (AMCase) and chitotriosidase (CHIT1) have chitinolytic activity, but relatively little is known about their expression in human asthma. OBJECTIVE: We sought to determine the expression and activity of AMCase and CHIT1 in healthy subjects, subjects with asthma, and habitual smokers, taking account of the null 24-bp duplication in the CHIT1 gene. METHODS: We measured chitinase activity in bronchoalveolar lavage (BAL) fluid at multiple pHs by using a synthetic chitin substrate. We also determined AMCase and CHIT1 gene expression in epithelial brushings and BAL fluid macrophages by means of real time RT-PCR. Paired DNA samples were genotyped for the CHIT1 duplication. RESULTS: In all subgroups the pH profile of chitinase activity in BAL fluid matched that of CHIT1, but not AMCase, and chitinase activity was absent in subjects genetically deficient in active CHIT1. Although AMCase protein was detectable in lavage fluid, AMCase transcripts in macrophages were consistent with an isoform lacking enzymatic activity. Median chitinase activity in BAL fluid tended to be lower than normal in asthmatic subjects but was increased 7-fold in habitual smokers, where CHIT1 gene expression in macrophages was increased. CONCLUSIONS: Chitinase activity in the lung is the result of CHIT1 activity. Although AMCase protein is detectable in the lung, our data indicate that it is inactive. Chitinase activity is not increased in subjects with asthma and in fact tends to be decreased. The high levels of chitinase activity in habitual smokers result from upregulation of CHIT1 gene expression, especially in macrophages.

Safety of HES 130/0.4 (Voluven(R)) in patients with preoperative renal dysfunction undergoing abdominal aortic surgery: a prospective, randomized, controlled, parallel-group multicentre trial.

BACKGROUND AND OBJECTIVE: Patients with impaired renal function are at risk of developing renal dysfunction after abdominal aortic surgery. This study investigated the safety profile of a recent medium-molecular-weight hydroxyethyl starch (HES) preparation with a low molar substitution (HES 130/0.4) in this sensitive patient group. METHODS: Sixty-five patients were randomly allocated to receive either 6% hydroxyethyl starch (Voluven); n = 32) or 3% gelatin (Plasmion); n = 33) for perioperative volume substitution. At baseline, renal function was impaired in all study patients as indicated by a measured creatinine clearance < 80 mL min(-1). The main renal safety parameter was the peak increase in serum creatinine up to day 6 after surgery. RESULTS: Both treatment groups were compared for non-inferiority (pre-defined non-inferiority range hydroxyethyl starch < gelatin + 17.68 micromol L(-1) or 0.2 mg dL(-1). Other renal safety parameters included minimum postoperative creatinine clearance, incidence of oliguria and adverse events of the renal system. Baseline characteristics, surgical procedures and the mean total infusion volume were comparable. Non-inferiority of hydroxyethyl starch vs. gelatin could be shown by means of the appropriate non-parametric one-sided 95% CI for the difference hydroxyethyl starch-gelatin [-infinity, 11 micromol L(-1)]. Oliguria was encountered in three patients of the hydroxyethyl starch and four of the gelatin treatment group. One patient receiving gelatin required dialysis secondary to surgical complications. Two patients of each treatment group died. CONCLUSION: As we found no drug-related adverse effects of hydroxyethyl starch on renal function, we conclude that the choice of the colloid had no impact on renal safety parameters and outcome in patients with decreased renal function undergoing elective abdominal aortic surgery.