Release Of Hyaluronidase Research Articles

Programmed Remodeling of the Tumor Milieu to Enhance NK Cell Immunotherapy Combined with Chemotherapy for Pancreatic Cancer.

Natural killer (NK) cell-based adoptive immunotherapy has demonstrated encouraging therapeutic effects in clinical trials for hematological cancers. However, the effectiveness of treatment for solid tumors remains a challenge due to insufficient recruitment and infiltration of NK cells into tumor tissues. Herein, a programmed nanoremodeler (DAS@P/H/pp) is designed to remodel dense physical stromal barriers and for dysregulation of the chemokine of the tumor environment to enhance the recruitment and infiltration of NK cells in tumors. The DAS@P/H/pp is triggered by the acidic tumor environment, resulting in charge reversal and subsequent hyaluronidase (HAase) release. HAase effectively degrades the extracellular matrix, promoting the delivery of immunoregulatory molecules and chemotherapy drugs into deep tumor tissues. In mouse models of pancreatic cancer, this nanomediated strategy for the programmed remodeling of the tumor microenvironment significantly boosts the recruitment of NK92 cells and their tumor cell-killing capabilities under the supervision of multiplexed near-infrared-II fluorescence.

Meniscal Fibrocartilage Repair Based on Developmental Characteristics: A Proof-of-Concept Study

Background: Unlike the adult meniscus, the fetal meniscus possesses robust healing capacity. The dense and stiff matrix of the adult meniscus provides a biophysical barrier for cell migration, which is not present in the fetal meniscus. Inspired by developmental characteristics, modifying the matrix of the adult meniscus into a fetal-like, loose and soft microenvironment holds opportunity to facilitate repair, especially in the avascular zone. Hypothesis: Modifying the dense and stiff matrix of the adult meniscus into a fetal-like, loose and soft microenvironment could enhance cell migration to the tear interface and subsequent robust healing capacity. Study Design: Controlled laboratory study. Methods: Fresh porcine menisci were treated with hyaluronidase or collagenase. The density and arrangement of collagen fibers were assessed. The degradation of proteoglycans and collagen was evaluated histologically. Cell migration within the meniscus or the infiltration of exogenous cells into the meniscus was examined. Dendritic silica nanoparticles with relatively large pores were used to encapsulate hyaluronidase for rapid release. Mesoporous silica nanoparticles with relatively small pores were used to encapsulate transforming growth factor–beta 3 (TGF-β3) for slow release. A total of 24 mature male rabbits were included. A longitudinal vertical tear (0.5 cm in length) was prepared in the avascular zone of the medial meniscus. The tear was repaired with suture, repaired with suture in addition to blank silica nanoparticles, or repaired with suture in addition to silica nanoparticles releasing hyaluronidase and TGF-β3. Animals were sacrificed at 12 months postoperatively. Meniscal repair was evaluated macroscopically and histologically. Results: The gaps between collagen bundles increased after hyaluronidase treatment, while collagenase treatment resulted in collagen disruption. Proteoglycans degraded after hyaluronidase treatment in a dose-dependent manner, but collagen integrity was maintained. Hyaluronidase treatment enhanced the migration and infiltration of cells within meniscal tissue. Last, the application of fibrin gel and the delivery system of silica nanoparticles encapsulating hyaluronidase and TGF-β3 enhanced meniscal repair responses in an orthotopic longitudinal vertical tear model. Conclusion: The gradient release of hyaluronidase and TGF-β3 removed biophysical barriers for cell migration, creating a fetal-like, loose and soft microenvironment, and enhanced the fibrochondrogenic phenotype of reparative cells, facilitating the synthesis of matrix and tissue integration. Clinical Relevance: Modifying the adult matrix into a fetal-like, loose and soft microenvironment via the local gradient release of hyaluronidase and TGF-β3 enhanced the healing capacity of the meniscus.

A universal dual-readout viscosity flow sensor based on biotarget-triggered hyaluronidase release from aptamer-capped metal-organic frameworks

Paper-based sensors are particularly appealing for on-site detection owing to the avoidance of expensive and complex instruments. A dual-readout viscosity flow sensor based on biotarget-triggered hyaluronidase (HAase) release from aptamer-capped metal-organic frameworks (MOFs) was designed for on-site detection. The aptamer-capped MOFs (HAase@MOF) were fabricated using UiO-67-NH2 as a nanocarrier for HAase and the double-stranded DNA (dsDNA) as a gatekeeper. The dsDNA structure of HAase@MOF was unlocked to release HAase in the presence of the target; thus, HAase hydrolyzed the hyaluronic acid hydrogel, changing the viscosity and acidity. The reaction solutions were pipetted onto the pH paper strips and the changes in the diffusion distance and the color signal were used to quantify the biotarget concentration. In this context, cytochrome c (cyt c) was selected as the model biotarget. The proposed sensor exhibited excellent specificity and reproducibility for detection of cyt c in human serum and bacterial biofilms. Overall, this study provides a low-cost, rapid workflow, and simple method for biotarget detection with dual-readout signal. Notably, other biotargets could be detected via directly changing the capping dsDNA structure of the MOFs. Thus, the dual-readout viscosity flow sensor possesses great promising in developing commercial products for on-site detection and sensing.

Sustained degradation of hyaluronic acid using an in situ forming implant.

In pancreatic cancer, excessive hyaluronic acid (HA) in the tumor microenvironment creates a viscous stroma, which reduces systemic drug transport into the tumor and correlates with poor patient prognosis. HA can be degraded through both enzymatic and nonenzymatic methods to improve mass transport properties. Here, we use an in situ forming implant to provide sustained degradation of HA directly at a local, targeted site. We formulated and characterized an implant capable of sustained release of hyaluronidase (HAase) using 15kDa poly(lactic-co-glycolic) acid and bovine testicular HAase. The implant releases bioactive HAase to degrade the HA through enzymatic hydrolysis at early timepoints. In the first 24 h, 17.9% of the HAase is released, which can reduce the viscosity of a 10mg/mL HA solution by 94.1% and deplete the HA content within primary human pancreatic tumor samples and ex vivo murine tumors. At later timepoints, as lower quantities of HAase are released (51.4% released in total over 21 d), the degradation of HA is supplemented by the acidic by-products that accumulate as a result of implant degradation. Acidic conditions degrade HA through nonenzymatic methods. This formulation has potential as an intratumoral injection to allow sustained degradation of HA at the pancreatic tumor site.

Differences in the mechanism of type 1 and type 2 diabetes-induced skin dryness by using model mice.

Diabetes induces dry skin that may cause infective diseases. In this study, we aimed to clarify the mechanism of diabetes-induced skin dryness in animal models. We also examined the difference in the mechanism of skin dryness in type 1 and type 2 diabetes. We examined skin dryness in type 1 diabetes model mice (streptozotocin [STZ] induction), non-obesity type 2 diabetes model mice (newborn STZ injection), and obesity type 2 diabetes model mice (KK-Ay/TaJcl). An increase in transepidermal water loss was observed in the type 1 diabetes model mice, and reduced skin hydration was observed in the type 2 diabetes model mice. In the type 1 diabetes model mice, an increase in advanced glycation end products and matrix metalloproteinase-9 led to a decline in collagen IV level, inducing skin dryness. In the obesity type 2 diabetes model mice, an increase in the release of histamine and hyaluronidase by mast cells resulted in a decline in the level of hyaluronic acid, inducing skin dryness. However, in the non-obesity type 2 diabetes model mice, the main factors of skin dryness could not be clearly identified. Nevertheless, inflammatory cytokine levels increased. We hypothesize that inflammatory cytokines disrupt the collagen of the skin. Diabetes caused skin dryness in each mouse model, and the mechanism of skin dryness differed by diabetes type.

Enzymatic Hydrolysis of Hyaluronic Layer before Drug Dosing

The local therapy is a particularly proper approach in treatment of cancer diseases, at which the drugs are highly toxic. Unfortunately, antitumor drugs diffusion is quite often limited by hyaluronic acid layers abundantly found in a cancer tissue. The undertaken approach assumed relaxation of hyaluronan layer by using a hydrolytic enzyme - hyaluronidase. The considered process corresponds to the heterogenic catalysis with substrate occurred as a layer and a catalyst in a native form. It was shown that enzyme concentration around 4 mg/L kept for 10 minutes in body fluid surrounding HA layers is enough to allow the drug to diffuse. Cyanocobalamin, as a model drug, was applied in diffusion processes testing. The possibility of hyaluronidase releasing in the place close to hyaluronic acid layers was also considered. Hydrogel carriers promoting fast release of hyaluronidase were characterized. The profile of releasing Hase from the tested carriers based on κ-carrageenan or sodium alginate with polyvinyl alcohol corresponded to Higuchi model. An expected rate value (the mass transport coefficient app. 4.0.10-7 m/s) was obtained for blends 0.5% κ-carrageenan, 4% polyvinyl alcohol and 0.5% sodium alginate, 6% polyvinyl alcohol.

Rat Caltrin Protein Modulates the Acrosomal Exocytosis During Sperm Capacitation1

Caltrin is a small and basic protein of the seminal vesicle secretion that inhibits sperm calcium uptake. The influence of rat caltrin on sperm physiological processes related to fertilizing competence was studied by examining its effect on 1) spontaneous acrosomal exocytosis, 2) protein tyrosine phosphorylation, and 3) sperm-egg interaction. Results show that the presence of caltrin during in vitro capacitation both reduced the rate of spontaneous acrosomal exocytosis without altering the pattern of protein tyrosine phosphorylation, and enhanced the sperm ability to bind to the zona pellucida (ZP). The significantly higher proportion of sperm with intact acrosome observed in the presence of caltrin was accompanied by a strong inhibition in the acrosomal hyaluronidase release. Enhancement of sperm-ZP binding was evident by the increase in the percentage of eggs with bound spermatozoa as well as in the number of bound sperm per egg. Similar results were obtained when the assays were performed using spermatozoa preincubated with caltrin and then washed to remove the unbound protein, indicating that the sperm-bound caltrin was the one involved in both acrosomal exocytosis inhibition and sperm-ZP binding enhancement. Caltrin bound to the sperm head was partially released during the acrosomal exocytosis induced by Ca-ionophore A23187. Indirect immunofluorescence and immunoelectron microscopy studies revealed that caltrin molecules distributed on the dorsal sperm surface disappeared after ionophore exposure, whereas those on the ventral region remained in this localization after the treatment. The present data suggest that rat caltrin molecules bound to the sperm head during ejaculation prevent the occurrence of the spontaneous acrosomal exocytosis along the female reproductive tract. Consequently, more competent spermatozoa with intact and functional acrosome would be available in the oviduct to participate in fertilization.

RISUG™ (reversible inhibition of sperm under guidance) – An antimicrobial as male vas deferens implant for HIV free semen

HIV transmission from the male to the female is a major health problem. A hypothesis proposing an intra vas deferens implant of an antimicrobial compound to prevent the infection spread is presented. Mechanisms of action for the inhibition could include inactivating HIV in sperms passing through the vas deferens; drug release from the implant to destroy HIV entering into semen from genital structures distal to the vas deferens; and sperm acrosome released hyaluronidase mediated reabsorption of HIV. A subcomponent of the implant flowing along sperm pathway may have a role in reducing the entry of HIV from a positive female into penile tissue. A new drug RISUG (reversible inhibition of sperm under guidance) presently undergoing clinical trials for its contraceptive effect in the male (because it disrupts the sperm acrosome by an electrical charge and pH lowering effects) has also antimicrobial action. The drug being a combination of styrene maleic anhydride (SMA) and dimethyl sulfoxide (DMSO) on being injected into the lumen of the vas deferens produces styrene maleic acid thereby lowering pH; induces electrochemical action leading to a stable electrical charge generation; releases mandelic acid; and induces acrosome reaction in sperms with consequent release of hyaluronidase and sperm inactivation. Moreover, one time administration into the lumen of the vas gives long term action. All these phenomena very well match with the needs for HIV clearance of semen and hence RISUG is here proposed as a possible candidate material for the HIV inhibiting vas deferens implant when delivered in below contraceptive threshold dosage. For experimental validation, after obtaining data on the semen HIV load under control conditions in the HIV positive males inducted into the study, 30 mg of SMA in 120 microl of DMSO (contraceptive dose being 60 mg SMA+120 microl DMSO) is to be injected into vasa deferens bilaterally. Thereafter at intervals of one month the viral load needs to be determined in semen obtained either by masturbation or in lubricant free condom at intercourse - the method of collection remaining the same throughout for a particular subject. A significant reduction in the semen viral load following RISUG administration will validate the hypothesis. Speculated reduced female to male HIV transmission is more difficult to test. Nonspecific indications will come from a population study of the incidence of RISUG treated men becoming HIV positive as compared to that in the general population.

Happily at Work

Happily at Work

Release of hyaluronidase during in vitro development of Ascaris suum from the third to fourth larval stage.

An enzyme that degraded glycosaminoglycan hyaluronic acid was released during in vitro development of Ascaris suum L3 to L4. The enzyme did not hydrolyze glycosaminoglycan chondroitin sulfate A. One molecular form of hyaluronidase was detected, with a molecular weight estimated at 47.8 +/- 8.6 kDa by sucrose density gradient centrifugation and at 55.0 +/- 1.3 kDa by substrate SDS-PAGE zymography. Activity of the enzyme was optimal between pH 5.0 and 6.0, and was present at neutral pH. Hyaluronidase activity was not affected by 5 mM concentrations of cupric sulfate, zinc chloride, calcium chloride, manganese chloride or EDTA. In addition, NaCl had no effect on enzyme activity at concentrations of 0.2-1.0 M. The highest level of hyaluronidase was present in culture fluid collected between days 4 and 6 of in vitro culture, and this period corresponded with that of the highest rate of increase in the percentage of L4. The presence or absence of hyaluronic acid plays a key role in basic developmental processes of vertebrates and is regulated, in part, by hyaluronidases. Developmental processes occurring during the transition of A. suum L3 to L4 may likewise depend on hyaluronidase. In addition, the infection process of a number of organisms, including some nematodes, depends on hyaluronidase. A. suum may likewise utilize hyaluronidase to facilitate larval migration within the host.

Acceleration of hyaluronidase production in the course of batch cultivation of Clostridium perfringens can be achieved with bacteriolytic enzymes.

As Clostridium perfringens hyaluronidase has cell-bound enzyme features, an enzymatic approach has been designed to facilitate the release of hyaluronidase into culture through increasing the clostridial cell wall permeability. As a result of the application of lytic peptidase from Actinomyces rutgersensis, beta-N-acetylglucosaminidase and beta-N-acetylmuramidase (both from Bacillus subtilis) commercially available preparations at the end of the producer's exponential growth phase, a 5.3-, 4.8- and 4.0-fold acceleration, respectively (but no enhancement), of hyaluronidase production in the course of batch cultivation of Cl. perfringens has been achieved. This also resulted in an approximately 10-fold reduction in undesirable side lecithinase activity irrespective of the bacteriolytic enzyme preparation used.

Lysogenic activity of enhancer caltrin and the influence of phospholipids on its expression.

Enhancer caltrin permeabilizes the plasma membrane of bovine epididymal spermatozoa as indicated by the release of hyaluronidase from the acrosome and lactate dehydrogenase (LDH) from the sperm cytosol. A previously reported increased calcium uptake by the sperm in the presence of enhancer caltrin was apparently due, in part, to calcium entry into the mitochondria, which had become accessible to external calcium. At 37 microM (200 micrograms/ml), enhancer caltrin released about 30% of the total hyaluronidase in the acrosome and 50% of the cytosolic LDH from epididymal sperm (4 x 10(7)/ml). This event was prevented by phosphatidylserine (PS), presumably through caltrin-phospholipid complex formation, whereas phosphatidylcholine (PC) was ineffective. Cardiolipin induced the release of LDH and this too was prevented by enhancer caltrin. Lysophosphatidylserine (LPS), on the other hand, potentiated the lysogenic activity of enhancer caltrin, promoting the release of the full complement of hyaluronidase and LDH even at a molar ratio of only 1:1 with caltrin. The effect of mixtures of LPS and PS on the lysogenic property of enhancer caltrin was investigated, and it was found that PS suppressed the potentiating effect of LPS. Release of hyaluronidase and LDH took place only when the LPS/PS molar ratio was greater than 2. The implications of these findings for the role of caltrin in mammalian fertilization are discussed.

Functional properties of caltrin proteins from seminal vesicle of the guinea pig

Caltrin proteins from seminal vesicle content of the guinea pig bind with great specificity to different regions of the spermatozoa. Indirect immunofluorescence studies with polyclonal antibodies showed that caltrin I binds to the head, on the acrosomal cup, while caltrin II binds on the principal tail and the neck. No fluorescence was detected either in the midpiece or in the post-acrosomal area of the head when sperm were exposed to either of the caltrins. Calcium-induced hyaluronidase release, which occurs during the acrosomal reaction, was dramatically inhibited by caltrin I (approximately 85% inhibition). Caltrin II was less effective in preventing the enzyme release (approximately 50% inhibition). Chemical modification of the structure modified the biological activity of the two caltrins. Reduction and carboxymethylation of the cysteine residues diminished the inhibitory activity on 45Ca2+ uptake and reduced the ability of the proteins to react with their antibodies. Removal of the carbohydrate portion by chemical deglycosylation transformed the inhibitor proteins into enhancers of calcium uptake into the spermatozoa. Caltrin proteins from the guinea pig appear to play the same physiological role as bovine caltrin, regulating specifically calcium transport across the spermatozoal membranes related with the acrosome reaction and hyperactivation process. The dual behavior of caltrins to inhibit or enhance Ca2+ uptake enables them to fulfill this function. Nevertheless, molecular mechanisms different from those described for bovine caltrin seem to be involved in the control of the functional activity of the guinea pig caltrins.

Assessment of heterospecific zona-free ovum penetration under fully defined conditions

The prognostic value of the sperm penetration assay (SPA) using zona-free hamster ova has not been universally accepted. This is partly due to technical problems with the assay, resulting in 5-17% false negative results. The advantage of replacing albumin with defined synthetic polymers in the SPA culture media, increasing the calcium gradient and incorporating potential active tract agents and antioxidants, has been investigated. These studies have resulted in the development of a procedure that has produced no negative results among known fertile donors (n = 55). Application of this technique to a non-specific group of patients with suspected sub-fertility produced a different pattern of sperm penetration compared with the 'standard' SPA in 27% of cases. Improved sperm velocity, motile survival, enhanced hyaluronidase release and rate of acrosome induction were noted using the synthetic media.

Hyaluronidase activity of rabbit skin wound granulation tissue fibroblasts.

The objective of this work was to identify and compare hyaluronidase activities of normal dermal and dermal wound granulation tissue fibroblasts. Direct evidence of the fibroblast as a source of tissue hyaluronidase was obtained. Fourth passage rabbit dermal fibroblasts were harvested on culture days 4, 8, 14, 18, and 22. Hyaluronidase activity and [35S]-sulfate- or [3H]-glucosamine-labeled glycosaminoglycans (GAGs) were monitored. Hyaluronidase assays were performed on medium and cellular fractions at the designated intervals. Enzyme activity of cellular fractions for both normal dermal and 14-day post-wound granulation tissue fibroblasts increased progressively through culture day 8. Thereafter (days 14-22), an eight-fold drop in cellular activity was coupled with cell death and emergence of hyaluronidase activity in medium fractions. Marked increases in degradation of secreted matrix components were concurrent with lysis-induced release of hyaluronidase. In this culture system, hyaluronidase activity was confined exclusively to cellular fractions and was released into the medium only under non-physiological conditions conducive to cellular death and lysis. Accordingly, this work suggests that previously reported skin wound hyaluronidases may be of fibroblastic origin and that susceptible GAGs are not degraded extracellularly, but, rather, must be internalized as a prerequisite to depolymerization.

Semen assessment, fertility and the selection of Hereford bulls for use in AI.

Nineteen young Hereford bulls were used to study the relationship between semen characteristics and fertility in artificial insemination following 15 320 inseminations. Seven measures of sperm motility, morphological abnormalities, the release of hyaluronidase, ATP content and sperm head measurements were examined as predictors of fertility (49-day fixed-interval non-return rate). Two assessments of motility, three categories of abnormal spermatozoa, acrosomal changes and the release of hyaluronidase had predictive power. Multiple regression analysis showed that a combination of sperm motility after dilution in saline, motility after thawing and the proportion of coiled tails and proximal protoplasmic droplets provided the best prediction of fertility and allowed bulls to be ranked in order of observed non-return rate (%) with a Spearman correlation better than +0.80.

Effect of Male Accessory Gland Secretions on Sensitivity of Porcine Sperm Acrosomes to Cold Shock, Initiation of Motility and Loss of Cytoplasmic Droplets

Hyaluronidase release was used as an index of acrosomal membrane damage during cold shock of epididymal boar sperm and ejaculated sperm from intact and vesiculectomized boars. Sperm were also incubated with seminal plasma from intact and vasectomized boars to examine the contributions of male accessory gland secretions. Acrosomal membranes of epididymal sperm were more resistant to cold shock than those of ejaculated sperm. Only 36% of the hyaluronidase released by ejaculated sperm was released by the epididymal sperm in spite of similar hyaluronidase content of the sperm. Preincubation of epididymal sperm in seminal plasma from both intact and vasectomized boars increased resistance to cold shock by 60 to 80%. Initial dilution of epididymal sperm with seminal plasma, rather than Ringer-fructose buffer, was associated with low progressive motility and with retention of cytoplasmic droplets. In contrast, acrosomal membranes of ejaculated sperm from intact and vesiculectomized boars exhibited similar sensitivity to cold shock, releasing hyaluronidase capable of forming .20 and .19 mumol N-acetylglucosamine from hyaluronic acid/10(8) sperm in 8 min. Moreover, seminal plasma from vasectomized boars had no effect on acrosomal sensitivity to cold shock of ejaculated sperm from vesiculectomized boars.

Release of hyaluronidase and beta-N-acetylhexosaminidase during in vitro incubation of hamster sperm.

Previous studies have shown that hamster sperm release a significant amount of hyaluronidase before and independently of the normal acrosome reaction. In this study, we have used improved methods for in vitro incubation to investigate the time course of the release of hyaluronidase and hexosaminidase from hamster sperm. When hamster sperm are incubated in medium which allows capacitation, 34 to 47% of the total mechanically extractable hyaluronidase and 34 to 51% of beta-N-acetylhexosaminidase are released into solution prior to and independently of the normal acrosome reaction (ARx). An additional 40 to 50% of the hyaluronidase and 34 to 51% of the hexosaminidase are released at the time of the normal ARx. Control experiments indicate that the early release is not due to the presence of dead sperm in culture and that the normal ARx is required for the second release. Increasing amounts of TCA-precipitated bovine serum albumin in the culture medium stimulated the early (1 hr) release of both enzymes. The data are consistent with the ideas that a significant amount of both enzymes is released from the sperm surface by 1 hr of incubation and that about the same amount of each enzyme is released during the normal ARx. Hyaluronidase and hexosaminidase release at the time of the acrosome reaction was measured for the first time using hamster sperm. The biphasic release of these enzymes may indicate that they have a dual function in fertilization and may help explain how sperm can penetrate the cumulus and corona radiata without undergoing an acrosome reaction.

Release of acrosomal hyaluronidase follows increased membrane permeability to calcium in the presumptive capacitation sequence for spermatozoa of the bovine and other mammalian species.

Brief exposure to a medium of elevated ionic strength combined with subsequent incubation in dialyzed fluid collected from bovine ovarian follicles results in an enhanced uptake of Ca2+ by bovine ejaculated sperm. An uptake of Ca2 + by either epididymal or ejaculated sperm is also induced by treatment with the divalent cation ionophore A23187 and is followed by release of the aerosomal enzyme hyaluronidase. A similar release of enzyme from ejaculated sperm is produced by hypertonic treatment and follicular fluid, but not by either alone. Uptake of Ca2 + and release of hyaluronidase likewise follow ionophore treatment of epididymal sperm from the guinea pig. rabbit, and hamster. In control experiments, hyaluronidase release is not induced by treatment with the protonophore, carbonylcyanide-in-chlorophenylhydrazone, by the respiratory inhibitor, rotenone, or by treatment with A23 187 in a Ca2 +.free medium. It is concluded that two presumptive components of the capacitation sequence of bovine spermatozoa, enhanced influx of Ca2 + and subsequent release of acrosomal contents, are produced either by ionophore treatment or by hypertonic extraction and exposure to follicular fluid, procedures that produce capacitation in vitro for the sperm of several other mammalian species.

Glycosaminoglycan metabolism following acute myocardial infarction and the effects of intravenous hyaluronidase therapy

A series of thirteen patients with acute myocardial infarction was studied. In the 12 patients heparinised on admission, there was a fall in serum hyaluronidase activity, free amino sugar and uronic acid levels. This contrasted with a rise in creatine kinase activity. In a single unheparinised patient, the hyaluronidase level rose. This suggests that necrotic cardiac tissue releases hyaluronidase. Any beneficial effects of the local release of hyaluronidase may be minimized by heparin therapy. Study of patients on a trial of intravenous bovine testicular hyaluronidase following acute myocardial infarction could detect no rise in hyaluronidase levels. This confirms previous reports that human serum inhibits bovine testicular hyaluronidase.