Coldzyme® Mouth Spray reduces duration of upper respiratory tract infection symptoms in endurance athletes under free living conditions.

Upper respiratory tract infection (URTI) can compromise athlete preparation and performance, so countermeasures are desirable. The aim of this study was to assess the effects of ColdZyme® Mouth Spray (ColdZyme) on self-reported upper respiratory tract infection in competitive endurance athletes under free-living conditions. One hundred and twenty-three endurance-trained, competitive athletes (recruited across 4 sites in England, UK) were randomised to control (no treatment, n = 61) or ColdZyme (n = 62) for a 3-month study period (between December 2017 and March 2018; or December 2018 and April 2019). They recorded daily training and illness symptoms (Jackson common cold questionnaire) during the study period. A total of 130 illness episodes were reported during the study with no difference in incidence between groups (episodes per person: 1.1 ± 0.9 Control, 1.0 ± 0.8 ColdZyme, P = 0.290). Episode duration was significantly shorter in ColdZyme compared to Control: Control 10.4 ± 8.5 days vs. ColdZyme 7.7 ± 4.0 days, P = 0.016). Further analysis to compare episodes with poor vs. good compliance with ColdZyme instructions for use (IFU) within the ColdZyme group showed a greater reduction in duration of URTI when compliance was good (9.3 ± 4.5 days in ColdZyme poor IFU compliance vs. 6.9 ± 3.5 days in ColdZyme good IFU compliance, P = 0.040). ColdZyme may be an effective countermeasure to reduce URTI duration, which was significantly lower (by 26-34%) in the ColdZyme treatment group (with no influence on incidence). This may have implications for athlete performance.

Assessment of hemagglutination activity of porcine deltacoronavirus.

Porcine deltacoronavirus (PDCoV) is an emerging swine enteric coronavirus that causes diarrhea in piglets. However, the biological characteristics of PDCoV are unclear. In this study, the hemagglutination (HA) abilities of two PDCoV strains (CH-01 and HNZK-04) were investigated. Our results showed that PDCoV has the ability to agglutinate rabbit erythrocytes after virion pretreatment with trypsin or neuraminidase. Additionally, the HA assay results showed a significant positive correlation with the infectious viral titer. Our results suggest that assessing the HA activity of PDCoV may be a useful diagnostic method for investigating and surveilling PDCoV infections.

Trypsin Pre-Treatment Combined With Growth Factor Functionalized Self-Assembling Peptide Hydrogel Improves Cartilage Repair in Rabbit Model.

The objective of this study was to improve cartilage repair and integration using self-assembling KLD hydrogel functionalized with platelet-derived growth factor-BB and heparin-binding insulin-like growth factor-1 with associated enzymatic trypsin pre-treatment of the native cartilage. Bilateral osteochondral defects were created at the central portion of the femoral trochlear groove of 48 skeletally mature, white New Zealand rabbits. One limb received a randomly assigned treatment and the contralateral limb served as the control. Treated defects were exposed to trypsin for 2 min and filled with self-assembling KLD hydrogel only, or associated to growth factors. All control limbs received KLD hydrogel alone or received only trypsin but not hydrogel. Ninety days post-defect creation, the rabbits were euthanized and magnetic resonance imaging, radiography, macroscopic evaluation, histology, and immunohistochemistry of the joint and repaired tissue were performed. Mixed model analyses of variance were utilized to assess the outcome parameters and individual comparisons were performed using Least Square Means procedure and differences with p-value < 0.05 were considered significant. Trypsin enzymatic pre-treatment improved cellular morphology, cluster formation and subchondral bone reconstitution. Platelet-derived growth factor-BB improved subchondral bone healing and basal integration. Heparin-binding insulin-like growth factor-1 associated with platelet-derived growth factor improved tissue and cell morphology. The authors conclude that self-assembling KLD hydrogel functionalized with platelet-derived growth factor and heparin-binding insulin-like growth factor-1 with associated enzymatic pre-treatment of the native cartilage with trypsin resulted in an improvement on the cartilage repair process. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2307-2315, 2019.

Melanocyte spheroids are formed by repetitive long-term trypsinization.

BACKGROUND: Autologous melanocyte transplantation plays an important role in the treatment of vitiligo. OBJECTIVE: Previous studies have indicated that, compared with melanocytes growing in monolayers, melanocyte spheroids have a better survival in growth factor- and serum-deprived conditions. METHODS: Melanocyte spheroids were obtained from human epidermis by repetitive long-term trypsinization and maintained an aggregated morphology for a short period in certain conditions. RESULTS: Melanocyte spheroids were capable of growing into normal dendritic melanocytes in monolayer when they were harvested and reinoculated in 24-well plates. Immunohistochemical analysis of the melanocyte spheroids revealed that they were positive for HMB45, a melanosome-specific marker. No melanomas occurred when melanocyte spheroids were transplanted into mice. CONCLUSION: Our study provides a promising approach for melanocyte transplantation to treat vitiligo.

Electro-responsive graphene oxide hydrogels for skin bandages: The outcome of gelatin and trypsin immobilization.

A free radical polymerization method was adopted for the fabrication of hybrid hydrogel films based on acrylamide and polyethylene glycol dimethacrylate as plasticizing and crosslinking agents, respectively, to be employed as smart skin bandages. Electro-sensitivity, biocompatibility and proteolytic properties were conferred to the final polymer networks by introducing graphene oxide (0.5% w/w), gelatin or trypsin (10% w/w) in the polymerization feed. The physical chemical and mechanical characterization of hybrid materials was performed by means of determination of protein content, Raman spectroscopy, thermogravimetric analysis and measurement of tensile strength. The evaluation of both water affinity and curcumin release profiles (analyzed by suitable mathematical modelling) upon application of an external electric stimulation in the 0-48 voltage range, confirmed the possibility to modulate the release kinetics. Proper proteolytic tests showed that the trypsin enzymatic activity was retained by 80% upon immobilization. Moreover, for all samples, we observed a viability higher than 94% in normal human fibroblast cells (MRC-5), while a reduction of methicillin-resistant Staphylococcus aureus CFU mL-1 (90%) was obtained with curcumin loaded samples.

Modulation of functional pendant chains within poly(ethylene glycol) hydrogels for refined control of protein release.

Hydrogels are highly attractive delivery vehicles for therapeutic proteins. Their innate biocompatibility, hydrophilicity and aqueous permeability allow stable encapsulation and release of proteins. The release rates also can be controlled simply by altering the crosslinking density of the polymeric network. However, the crosslinking density also influences the mechanical properties of hydrogels, generally opposite to the permeability. In addition, the release of larger proteins may be hindered below critically diminished porosity determined by the crosslinking density. Herein, the physical properties of the hydrogels are tuned by presenting functional pendant chains, independent of crosslinking density. Heterobifunctional poly(ethylene glycol) monomethacrylate (PEGMA) with various end functional groups is synthesized and copolymerized with PEG dimethacrylate (PEGDA) to engineer PEG hydrogels with pendant PEG chains. The pendant chains of the PEG hydrogels consisting of sulfonate, trimethylammonium chloride, and phenyl groups are utilized to provide negative charge, positive charge and hydrophobicity, respectively, to the hydrogels. The release rates of proteins with different isoelectric points are controlled in a wide range by the type and the density of functional pendant chains via electrostatic and hydrophobic interactions.

Quantitative proteomics and SWATH-MS to elucidate peri-receptor mechanisms in human salt taste sensitivity.

Recently, studies on human salt taste sensitivity demonstrated that sodium chloride (NaCl) sensitive and non-sensitive subjects differed in their salivary proteome and, in particular, in endopeptidase activity. In order to investigate individual's NaCl sensitivity and the role of endoprotease activity in salt taste perception, 20 panellists were classified according to NaCl sensitivity and saliva samples collected. A targeted protein quantitation by means of selected-reaction-monitoring (SRM) mass spectrometry and stable-isotope incorporation revealed the joint abundance of lysozyme C and lipocalin-1 to be indicative for non-sensitive subjects. Sensory studies performed after oral challenge with the serine-type endopeptidase trypsin demonstrated a salt enhancing effect which was assumed to be due to an in-vivo generation of salt-modulating peptides as shown by LC-SWATH-MS. Amongst those, the tetrapeptide PLWR was found to elicit salty taste enhancing activity above an extraordinarily low taste threshold concentration of 6.5 µmol/L.

The Role of Trypsin:Chymotrypsin in Tissue Repair.

Tissue damage of all types, such as surgical or accidental injuries, fractures, and burns, stimulates a well-orchestrated, physiological process of healing, which ultimately leads to structural and functional restoration of the damaged tissues. The tissue repair process can be broadly divided into four continuous and overlapping phases-hemostasis and coagulation, inflammation, proliferation, and remodeling. If the process is interrupted or halted during any stage, it leads to impaired healing and formation of a chronic wound. Chronic wounds are associated with significant morbidity, mortality, and poor quality of life. Therefore, prompt and effective management of acute tissue injury is necessary to prevent it from progressing to a chronic wound. Proteolytic enzymes have been used to facilitate tissue repair since ancient times. Trypsin:chymotrypsin is an oral proteolytic enzyme preparation which has been in clinical use since the 1960s. It provides better resolution of inflammatory symptoms and promotes speedier recovery of acute tissue injury than several of the other existing enzyme preparations. This review article revisits the role and clinical utility of trypsin:chymotrypsin combination in tissue repair. FUNDING: Torrent Pharmaceuticals Limited.

Mesenchymal stem cell detachment with trace trypsin is superior to EDTA for in vitro chemotaxis and adhesion assays.

Trypsin is frequently used to dissociate mesenchymal stem cells (MSCs) for in vitro adhesion and chemotaxis assays. However, its potential impact on surface receptor degradation is poorly understood. The purpose of this study was to evaluate the effect of trypsin-EDTA exposure versus PBS-EDTA on MSC surface receptor integrity and function. Primary human MSCs were detached with PBS-EDTA alone, or Cell Dissociation Buffer followed by 30 s exposure to 0.05% w/v trypsin-EDTA (trace trypsin method, TT), or 0.25% w/v trypsin exposure for 2 or 5 min. Cells were characterized for surface integrity of ß1 integrin (CD29) and PDGF Receptor (PDGF-R), and assessed in vitro for adhesion to atelocollagen-coated surfaces and migration to PDGF-BB. PBS-EDTA detachment fully preserved receptor integrity but routinely detached only half of the adherent cells and led to cell aggregates that failed to adhere evenly across the Transwell migration insert. Both CD29 and PDGF-R were significantly degraded by 0.25% trypsin detachment for 2 or 5 min compared to the TT method or PBS-EDTA (p < 0.05). Cells migrated optimally to PDGF-BB when detached with the TT method (3.1-fold vs α-MEM, p = 0.01). Cells attached optimally to atelocollagen when detached using the TT method or PBS-EDTA (6- to 10-fold vs 0.25% trypsin, p < 0.01). CDB followed by trace trypsin-EDTA exposure is recommended over PBS-EDTA to produce a single-cell MSC suspension that preserves receptor integrity and more reproducible receptor-mediated responses.

A Randomized, Clinical Trial to Evaluate Efficacy and Tolerability of Trypsin:Chymotrypsin as Compared to Serratiopeptidase and Trypsin:Bromelain:Rutoside in Wound Management.

INTRODUCTION: Systemic enzyme therapy can play an important role in maintaining normal inflammatory processes within the body and thereby helps support and speed up healing. In the course of the anti-inflammatory action, enzymes degrade damaged cells and necrotic material and, through the inactivation of mediators and toxic products, they restrict the edema and pain. METHOD: The study conducted at Grant Medical College, Mumbai, India was a clinical trial comparing the efficacy and tolerability of three oral enzyme treatment groups-oral tablets containing trypsin:chymotrypsin (TC) (Chymoral Forte®), serratiopeptidase (S) 5 mg oral tablets, and oral enzyme tablets containing trypsin 48 mg, bromelain 90 mg, and rutoside 100 mg (TBR)-to evaluate their healing potential in surgical wounds after orthopedic surgery. RESULTS: A total of 75 patients were screened, randomized, and divided into three groups in 1:1:1 ratio receiving either of the three treatments. In the TC group, erythema was significantly reduced from 3.44 on day 3 to 1.16 on day 10 (p < 0.01). There was significantly better reduction in erythema scores in the TC group as compared to S and TBR groups (p < 0.05) at each follow-up visit. Similarly reduction in the local irritation, wound discharge, edema, induration, and tenderness score with TC treatment at the end of the study was significantly higher than that observed in the other two groups. In addition TC showed significant reduction in pain on the VAS scale (p < 0.01). Global assessment of response to therapy for efficacy and tolerability was reported to be good to excellent in 88% and 92% of the patients on TC as compared to 12% and 8% with S and 12% and 8% with TBR. CONCLUSION: TC provides a better resolution of symptoms of inflammation after orthopedic surgery as compared to S and TBR, thus facilitating better wound healing. Further studies are warranted to confirm the findings. TRIAL REGISTRATION: Clinical Trial Registry of India (Reg. No. CTRI/2011/07/001920).

Rutoside and Hydrolytic Enzymes Do Not Attenuate Marathon-Induced Inflammation.

INTRODUCTION: Vigorous and prolonged exercise such as marathon running increases inflammatory markers and the risk of upper respiratory illness (URI) in athletes. Nutritional supplements are being tested as countermeasures of exercise-induced inflammation and immune dysfunction. METHODS: In this prospective randomized, double-blind, placebo-controlled phase I trial, healthy male runners (N = 138, age 42 ± 11 yr) were supplemented with rutoside (600-1200 mg·d) and hydrolytic enzymes (540-1080 mg·d bromelain, 288-576 mg·d trypsin) (WOB) or placebo (PL) for 1 wk before and 2 wk after the Munich Marathon 2013. Blood samples were collected 5 wk prerace and immediately, 24 h, and 72 h postrace and analyzed for inflammation biomarkers (interleukins [IL] 6 and 10, high-sensitivity C-reactive protein, and leukocytes). URI rates, assessed by the Wisconsin Upper Respiratory Symptom Survey, were compared between the study groups during the 2-wk period after the marathon race. URI was defined if the Wisconsin Upper Respiratory Symptom Survey score was equal or greater than seven, representing either one severe symptom or seven mild symptoms. RESULTS: Immediately postrace, the increase of IL-6 was not significantly different between the WOB and the PL groups (median [interquartile range]: WOB, 33.8 [22.5-58.8] ng·L; PL, 35.6 [24.8-61.29] ng·L; P = 0.758). No significant group differences were observed for increases of IL-10, high-sensitivity C-reactive protein, or leukocytes pre- to postrace (all P > 0.05). From race day until 2 wk after the marathon race, the percentage of individuals with at least one URI did not significantly differ between the groups (WOB, 50.0%; PL, 51.5%; P = 0.859). CONCLUSION: Supplementation with rutoside and hydrolytic enzymes before and after a marathon race did not attenuate postrace inflammation or decrease URI incidence in nonelite male marathon runners.

Evaluation of ColdZyme® Mouth Spray on prevention of upper respiratory tract infections in a boy with primary immunodeficiency: a case report.

BACKGROUND: Primary immunodeficiencies include a variety of disorders that render patients more susceptible to infections. If left untreated, these infections may be fatal. Patients with primary antibody deficiencies are therefore given prophylactic immunoglobulin G replacement therapy. ColdZyme® Mouth Spray is a medical device intended to reduce the probability of catching a cold and/or can help shorten the duration of a cold, if used at an early stage of the infection, by forming a thin protective barrier on the pharyngeal mucous membrane. This is the first report of this kind in the literature. CASE PRESENTATION: The parents of a 12-year-old white boy diagnosed as having common variable immunodeficiency voluntarily started to let their son use ColdZyme® Mouth Spray to reduce common cold infections if possible. Prior to using ColdZyme® Mouth Spray, he had recurrent microbial infections of his ears, sinuses, nose, bronchi, and lungs. He also frequently exhibited continuous rhinorrhea, fungal growth in his oral cavity, and gingivitis with wounds in his gums. As a consequence, his and his family's health-related quality of life was severely compromised. He commenced a twice-daily treatment (morning and evening) with ColdZyme® Mouth Spray; the weekly administration of immunoglobulin G (Hizentra®) for replacement therapy was continued throughout this period. Data were retrieved by using a daily diary about infections and symptoms. His guardians had recorded infection symptoms since he was diagnosed as having common variable immunodeficiency 10 years earlier to follow the effect of the immunoglobulin G treatment. Shortly after commencement of ColdZyme® Mouth Spray treatment, he experienced a marked improvement in symptoms and health-related quality of life. His continuous rhinorrhea disappeared, breathing through his nose was easier, oral fungal infection decreased, and wounds in his gum tissue healed for the first time in several years. CONCLUSIONS: We observed that when ColdZyme® Mouth Spray was used to reduce common cold viral infection in a patient with common variable immunodeficiency on immunoglobulin G replacement therapy, secondary microbial and fungal infections in his oral cavity and oropharynx were also reduced. A controlled study is warranted to confirm the observed results.

Analgesic and anti-edematogenic effects of oral trypsin were abolished after subdiaphragmatic vagotomy and spinal monoaminergic inhibition in rats.

AIMS: Rheumatoid arthritis brings great burdens to the patients. In addition to the highly expensive treatment, they are commonly associated with severe side effects. In such context, the research for safe and affordable treatments is needed. MAIN METHODS: Arthritis was induced by CFA (0.5mg/mL) in female wistar rats. Trypsin was given p.o. (2.95mg/kg; 2mL) 24h after the intra-articular CFA injection. Articular incapacitation was measured daily by counting the paw elevation time (PET; s) during 1-min periods of stimulated walk, throughout the 7-days after intra-articular CFA injection. Articular diameter (AD) was accessed just after each PET measurement, taken the difference between naïve and diseased knee-joint diameter (cm). KEY FINDINGS: The present study showed that orally administered trypsin was able to reduce nociception and edema, effects that could be observed throughout the evaluation period. These effect, however, were not observed in animals underwent subdiaphragmatic vagotomy, suggesting a vagal mediation for trypsin effects. Likewise, these effects were blocked in rats which received intrathecal injection of the neurotoxins 5,7-dihydroxytryptamine or 6-hydroxydopamine, suggesting the involvement of spinal amines from axon terminals. SIGNIFICANCE: The present study proposes that oral trypsin may cause vagal activation, followed by the activation of descending inhibitory pathways and such mechanism may lead to a novel approach for the treatment of arthritis.

Chitosan nanoencapsulated exogenous trypsin biomimics zymogen-like enzyme in fish gastrointestinal tract.

Exogenous proteolytic enzyme supplementation is required in certain disease conditions in humans and animals and due to compelling reasons on use of more plant protein ingredients and profitability in animal feed industry. However, limitations on their utility in diet are imposed by their pH specificity, thermolabile nature, inhibition due to a variety of factors and the possibility of intestinal damage. For enhancing the efficacy and safety of exogenous trypsin, an efficient chitosan (0.04%) nanoencapsulation-based controlled delivery system was developed. An experiment was conducted for 45 days to evaluate nanoencapsulated trypsin (0.01% and 0.02%) along with 0.02% bare trypsin and 0.4% chitosan nanoparticles against a control diet on productive efficiency (growth rate, feed conversion and protein efficiency ratio), organo-somatic indices, nutrient digestibility, tissue enzyme activities, hematic parameters and intestinal histology of the fish Labeo rohita. All the synthesized nanoparticles were of desired characteristics. Enhanced fish productive efficiency using nanoencapsulated trypsin over its bare form was noticed, which corresponded with enhanced (P<0.01) nutrient digestibility, activity of intestinal protease, liver and muscle tissue transaminases (alanine and aspartate) and dehydrogenases (lactate and malate), serum blood urea nitrogen and serum protein profile. Intestinal tissues of fish fed with 0.02% bare trypsin showed broadened, marked foamy cells with lipid vacuoles. However, villi were healthier in appearance with improved morphological features in fish fed with nanoencapsulated trypsin than with bare trypsin, and the villi were longer in fish fed with 0.01% nanoencapsulated trypsin than with 0.02% nanoencapsulated trypsin. The result of this premier experiment shows that nanoencapsulated trypsin mimics zymogen-like proteolytic activity via controlled release, and hence the use of 0.01% nanoencapsulated trypsin (in chitosan nanoparticles) over bare trypsin can be favored as a dietary supplement in animals and humans.

Esophageal barrier function and tight junction expression in healthy subjects and patients with gastroesophageal reflux disease: functionality of esophageal mucosa exposed to bile salt and trypsin in vitro.

BACKGROUND AND AIMS. Gastroesophageal reflux disease (GERD) is associated with impaired epithelial barrier function. However, the influence of acid and/or bile acids on human esophageal epithelial barrier function and the tight junction (TJ) proteins has not been fully elucidated. The aim of the study is to investigate the esophageal barrier function and TJ expression in healthy subjects and patients with GERD. The functionality of esophageal mucosa exposed to bile salt deoxycholic acid (DCA) and trypsin has been studied in vitro. MATERIAL AND METHODS. Endoscopic biopsies from healthy controls and patients with GERD-related symptom with endoscopic erosive signs, as well as esophageal mucosa taken from patients undergoing esophagectomy were evaluated in Ussing chambers and by western blot and immunohistochemistry. RESULTS. The esophageal epithelium from GERD patients had lower electrical resistance and higher epithelial currents than controls. Claudin-1 and -4 were significantly decreased in GERD patients. The bile salt DCA in the low concentration of 1.5 mM and trypsin increased the resistance and claudin-1 expression, while the higher concentration of 2.5 mM DCA and trypsin decreased the resistance and the claudin-3, -4 and E-cadherin expressions. CONCLUSION. In addition to acidic reflux, duodenal reflux components, such as bile salts and trypsin, have the potential to disrupt the esophageal barrier function, partly by modulating the TJ proteins. However, the expression of TJ is dependent on both the refluxed material as well as the concentration of the bile salt.

Potential use of Atlantic cod trypsin in biomedicine.

Surface proteins of viruses and bacteria used for cell attachment and invasion are candidates for degradation by proteases. Trypsin from Atlantic cod (Gadus morhua) was previously demonstrated to have efficacy against influenza viruses in vitro and on skin. In this paper, cod trypsin is shown to be 3-12 times more effective in degrading large native proteins than its mesophilic analogue, bovine trypsin. This is in agreement with previous findings where cod trypsin was found to be the most active among twelve different proteases in cleaving various cytokines and pathological proteins. Furthermore, our results show that cod trypsin has high efficacy against herpes simplex virus type 1 (HSV-1) and the respiratory syncytial virus (RSV) in vitro. The results on the antipathogenic properties of cod trypsin are important because rhinovirus, RSV, and influenza are the most predominant pathogenic viruses in upper respiratory tract infections. Results from a clinical study presented in this paper show that a specific formulation containing cod trypsin was preferred for wound healing over other methods used in the study. Apparently, the high digestive ability of the cold-adapted cod trypsin towards large native proteins plays a role in its efficacy against pathogens and its positive effects on wounds.

Spray drying from organic solvents to prepare nanoporous/nanoparticulate microparticles of protein: excipient composites designed for oral inhalation.

OBJECTIVES: The aim of this study was to determine if spray-drying could successfully produce microparticles containing the model protein trypsin in a form suitable for inhalation. METHODS: Trypsin was spray-dried with raffinose from a methanol : n-butyl acetate solvent system (MeOH : BA). The solvent system was then adjusted to include water, and trypsin was co-spray-dried with raffinose, trehalose or hydroxpropyl-ß-cyclodextrin. The spray-dried products were characterised by SEM, XRD, DSC, TGA and FTIR. Protein biological activity and in-vitro deposition of trypsin : excipient nanoporous/nanoparticulate microparticles (NPMPs) was also assessed. KEY FINDINGS: The inclusion of water in a MeOH : BA solvent system allowed for the successful production of NPMPs of trypsin : excipient by spray-drying. Trypsin formulated as trypsin : excipient NPMPs retained biological activity on processing and showed no deterioration in activity or morphological characteristics when stored with desiccant at either 4 or 25°C. Hydroxpropyl-ß-cyclodextrin showed advantages over the sugars in terms of producing powders with appropriate density and with greater physical stability under high-humidity conditions. Fine particle fractions of between 41 and 45% were determined for trypsin : excipient NPMPs. CONCLUSIONS: NPMPs of trypsin : excipient systems can be produced by spray-drying by adjustment of the solvent system to allow for adequate solubility of trypsin.

Erythrocyte invasions and receptor heterogeneity in field isolates of Nanay river basin Iquitos.

OBJECTIVE: To determine whether the requirements for sialic acid varies and whether several types of silaic acid independent receptors utilized for invasion mechanisms of fresh filed isolates collected around Nanay river basin, Iquitos. METHODS: The field isolates were cultured as described previously by Jensen and Trager and MR4 protocol with little modifications. The erythrocytes preparation and subsequent enzyme treatment was done as described previously by Sharma. with little modification. Invasion assay was performed as described previously by Sharma et al with little modification. RESULTS: The Nanay river basin isolates showed five types of invasion mechanisms or types of receptors-ligand interactions. Here we observed that an equal numbers of neuraminidase sensitive and resistant invasion receptor-ligand interaction profiles as the most common receptor-ligand invasion profiles. Neuraminidase resistance trypsin sensitive chymotrypsin sensitive (NM(R)T(S)CT(S)) invasion of receptor-ligand interaction profile was found in seven isolates, Five field isolates and one reference strain showed neuraminidase sensitive, trypsin sensitive and chymotrypsin resistant (NM(S)T(S)CT(R)) invasion of receptor-ligand interactions, six isolates including one reference strains dd2 showed neuraminidase sensitive, trypsin and chymotrypsin resistance (NM(S)T(R)CT(R)) indicating its dependence on sialic acids and independence of trypsin and chymotrypsin sensitive proteins. Four isolates showed neuraminidase sensitive, trypsin sensitive and chymotrypsin sensitive (NM(S)T(S)CT(S)) invasion of receptor-ligand interactions, seven isolates were neuraminidase resistant, trypsin sensitive and chymotrypsin resistance (NM(R)T(S)CT(R)) invasion of receptor-ligand interactions, indicating its dependence on trypsin sensitive proteins. CONCLUSIONS: The Nanay river basin isolates showed five types of invasion mechanisms or types of receptors-ligand interactions. A full understanding of theses invasion mechanisms may allow the development of novel prophylactic and therapeutic strategies that block erythrocyte receptor-ligand invasion mechanisms.

Effect of increasing trypsin concentrations on seminal coagulum dissolution and sperm parameters in spider monkeys (Ateles geoffroyi).

Seminal coagulum formation in spider monkeys (Ateles geoffroyi) interferes with the efficient recovery and evaluation of spermatozoa. The main objective was to assess the effect of increasing concentrations of trypsin on dissolution of seminal coagulum and spermatic parameters. Seminal coagulum was incubated at 37 °C without trypsin or in the presence of increasing trypsin concentrations (0.1%, 1.0%, and 5.0%). For each sample, coagulum dissolution time was measured, and sperm concentration, viability, motility, and morphology were evaluated using light microscopy and/or transmission electronic microscopy (TEM). Trypsin concentrations of 1.0% and 5.0% more rapidly liquefied seminal coagulum, averaging 32 and 21 min, respectively, compared with nontrypsinized controls, with maintenance of greater sperm viability (70.8% and 72.5%, respectively). Coagulum treated with 1.0% trypsin and the liquid ejaculate fraction averaged higher sperm motility (40.1% and 55.6%, respectively) than control samples, and both 1.0% and 5.0% trypsin treatment allowed recovery of increased numbers of motile spermatozoa. There was greater sperm fragmentation at the head and midpiece level after treatment with 1.0% and 5.0% trypsin (55.8% and 55.9%); however, the percentage of normal morphology in structurally intact spermatozoa did not differ relative to controls. With transmission electronic microscopy imaging, there were similar percentages of spermatozoa with plasma membrane swelling in the midpiece and acrosomal regions in trypsin-treated samples and controls. In conclusion, trypsin treatment of spider monkey seminal coagulum exerted a concentration-dependent effect on dissolution time and various spermatic parameters. Higher trypsin concentrations caused more rapid liquefaction of coagulum and recovery of greater numbers of motile spermatozoa, but may adversely affect fragmentation of spermatozoa and could compromise sperm function and cryopreservation potential.