Immunotolerance induction effectivity in hemophilia A children and neutralizing alloantibodies. / Efectividad de inducción de tolerancia inmune en niños con hemofilia A y aloanticuerpos neutralizantes.

INTRODUCTION: The development of anti-factor VIII neutralizing antibodies in hemophilia A is the most severe com plication related to treatment. Immune tolerance induction (ITI) is the only known treatment for eradicating inhibitors. A successful ITI allows using factor VIII (FVIII) again for the treatment or prophylaxis of hemorrhagic events. OBJECTIVE: To report the experience of pediatric patients who underwent ITI in the country's public health care network. PATIENTS AND METHOD: Retrospective and descriptive analysis of 13 pediatric patients with severe Hemophilia A and high-titer inhibitors persis tence who underwent ITI and complete follow-up. Plasma-derived FVIII concentrate was used at 70 180 IU/kg/day doses. The success of the treatment is defined by achieving a negative titer and a half life recovery of the FVIII. The results were expressed in median (range). RESULTS: In 13 patients, the inhibitor was identified at an average age of 17.6 months, after 35.2 days of exposure to the FVIII. 11 patients (84.6%) recovered the half-life of FVIII after 49.6 months of treatment. In the patients who responded to treatment, the inhibitor titer was negative at 6 months on average. CONCLUSIONS: ITI is the treatment of choice for patients with hemophilia A and inhibitors persistence. ITI must be perso nalized since the time response is variable in each patient.

Enabling a bioinspired N,N,N-copper coordination motif through spatial control in UiO-67: synthesis and reactivity.

Metal-organic frameworks (MOFs) featuring zirconium-based clusters are widely used for the development of functionalized materials due to their exceptional stability. In this study, we report the synthesis of a novel N,N,N-ligand compatible with a biphenyl dicarboxylic acid-based MOF. However, the resulting copper(I) complex exhibited unexpected coordination behaviour, lacking the intended trifold coordination motif. Herein, we demonstrate the successful immobilization of a bioinspired ligand within the MOF, which preserved its crystalline and porous nature while generating a well-defined copper site. Comprehensive spectroscopic analyses, including X-ray absorption, UV/Vis, and infrared spectroscopy, were conducted to investigate the copper site and its thermal behaviour. The immobilized ligand exhibited the desired tridentate coordination to copper, providing access to a coordination motif otherwise unattainable. Notably, water molecules were also found to coordinate to copper. Upon heating, the copper centre within the MOF exhibited reversible dehydration, suggesting facile creation of open coordination sites. Furthermore, the copper site displayed reduction at elevated temperatures and subsequent susceptibility to oxidation by molecular oxygen. Lastly, both the molecular complexes and the MOF were evaluated as catalysts for the oxidation of cyclohexane using hydrogen peroxide. This work highlights the successful immobilization of a bioinspired ligand in a zirconium-based MOF, shedding light on the structural features, thermal behaviour, and catalytic potential of the resulting copper sites.

Gene transfer using bovine adeno-associated virus in the guinea pig cochlea.

Gene transfer into the cells of the cochlea is useful for both research and therapy. Bovine adeno-associated virus (BAAV) is a new viral vector with potential for long-term gene expression with little or no side effects. In this study, we assessed transgene expression using BAAV with beta-actin-GFP as a reporter gene, in the cochleae of normal and deafened guinea pigs. We used two different routes to inoculate the cochlea: scala media (SM) or scala tympani (ST). Auditory brainstem response assessments were carried out before inoculation, 7 days after inoculation and immediately before killing, to assess the functional consequences of the treatment. We observed threshold shifts because of the surgical invasion, but no apparent pathology associated with the virus. Fourteen days after the injection, animals were killed and cochleae assessed histologically. Epi-fluorescence showed that BAAV transduced the supporting cells of both normal and deafened animals through SM and ST inoculations. Transgene expression in cells of the membranous labyrinth after ST inoculation is an important outcome because of the greater feasibility of this route for future clinical application. BAAV facilitates efficient transduction of the membranous labyrinth epithelium with minimum pathogenicity and may become clinically applicable for inner ear gene therapy.

Single-Center Long-Term Follow-Up of Kidney Donors in Argentina (Hospital Italiano de Buenos Aires).

INTRODUCTION: Living kidney donor (LKD) transplantation is increasing due to organ shortage. Clinical studies have shown that the risk of developing end-stage renal disease (ESRD) in donors is similar to that in the general population. Our goal was to evaluate postdonation renal outcomes assessed by glomerular filtration rate (GFR), proteinuria, and blood pressure. METHODS: A total of 210 LKD transplants were performed at Hospital Italiano de Buenos Aires between 2000 and 2014. Postdonation outcomes were analyzed in 109 donors. GFR was assessed by 24-hour creatinine clearance (as 24-hour ClCr) and estimated using the Modification of Diet in Renal Disease (MDRD) and Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equations. Additionally, we correlated the predonation renal functional reserve (RFR) with postdonation GFR. Donor results were compared to the expected GFR (adjusted to age and single kidney). Other renal outcome indicators measured were albuminuria and blood pressure, and they were compared (predonation and postdonation) using univariate analysis. RESULTS: A total of 109 patients were followed up for 47 ± 34 months (range, 12-168): 70% were female, age at donation was 48.58 years (range, 25-70), and predonation serum creatinine was 0.85 ± 0.17 mg/dL. Postnephrectomy GFR (24-hour ClCr) was significantly lower compared to predonation GFR (105.38 ± 21.78 mL/min/1.73 m2 vs 90.14 ± 17.78 mL/min/1.73 m2). However, postdonation GFR was not significantly different compared to the expected GFR. No differences were found for blood pressure or albuminuria. Age >50 and an RFR (<20%) was associated with a lower GFR. CONCLUSIONS: In this population of LKD, renal outcome (24-hour CrCl, albuminuria, and blood pressure) was within the expected outcome for healthy individuals after uninephrectomy.

Utilisation of controlled pore topology for the separation of bioparticles in a mixed-glass beads column.

To study the flow of shaped particles in porous media, elution of spherical and rod-like micro-organisms was performed through beds of spherical glass beads. A 0.04 cm/s constant flow rate was used with 5 microm yeast suspensions, 1 microm latex micro-spheres and rod-like bacilli Lactobacillus bulgaricus 6 microm long and 0.5 microm in diameter. Yeast cells' diameter is close to the bacilli length and micro-spheres have the same diameter as bacilli. All particle types have similar density. To make the different packing beds, 1.125 mm coarse beads and 0.1115 mm fine beads were used. Experiments were carried out using a column loaded with the binary packing (volume fraction of coarse particles in the mixture 0.7) or a monosize packing with the same amount of coarse or fine particles as used in the binary packing. Analysis of experimental results was based on two models: pure exclusion effect and hydrodynamic separation model [hydrodynamic chromatography (HDC)]. Results for spheres show that the classic HDC model fits to the experimental data whenever the ratio of particle size to the pathway bend scale is high ( approximately 1/100, micro-spheres). However, if this ratio increases and becomes approximately 1/20, the HDC model needs to be corrected due to the effect of channel wall curvature on exclusion. This led to a modified HDC equation of the form R=B/(1+2lambda-2.8lambda(2)), where R is the retention, lambda is the aspect ratio and constant B>or=1. Bacillus separation follows an exclusion mechanism, since pore topology is important in the separation of shaped particles when the aspect ratio approaches lambda=0.1. In the case of a binary packing bed, rod-like particles display a different behaviour than the one exhibited by the spherical particles of the same scale as bacilli, either in length or in diameter. This may be explained by the interaction between rod-like bacilli and the bed's pore topology. A generalised exclusion model for particles was proposed to be R=A/(1-lambda)(z), where A is the coefficient proportional to the tortuosity and the parameter z=1, 2 or 3 depends mainly on pore shape. Controlled pore topology opens interesting applications for bio-separation (in porous micro-fluidic devices, deep bed filtration) and might be especially important for macromolecules and micro-organisms separation with different shapes.

Surface-enhanced Raman scattering on colloidal nanostructures.

Surface-enhanced Raman scattering combines extremely high sensitivity, due to enhanced Raman cross-sections comparable or even better than fluorescence, with the observation of vibrational spectra of adsorbed species, providing one of the most incisive analytical methods for chemical and biochemical detection and analysis. SERS spectra are observed from a molecule-nanostructure enhancing system. This symbiosis molecule-nanostructure is a fertile ground for theoretical developments and a realm of applications from single molecule detection to biomedical diagnostic and techniques for nanostructure characterization.

Efectividad de inducción de tolerancia inmune en niños con hemofilia A y aloanticuerpos neutralizantes / Immunotolerance induction effectivity in hemophilia A children and neutralizing alloantibodies

Resumen: Introducción: El desarrollo de aloanticuerpos neutralizantes anti-factor VIII en hemofilia A es la complicación más seria relacionada al tratamiento. La inducción de tolerancia inmune (ITI) o inmunotolerancia es el único tratamiento que erradica inhibidores, permitiendo utilizar nuevamente factor VIII para el tratamiento o profilaxis de eventos hemorrágicos. Objetivo: reportar la experiencia en niños sometidos a inmunotolerancia en la red pública del país. Pacientes y Método: Análisis retrospectivo y descriptivo de 13 niños con Hemofilia A severa e inhibidores persistentes de alto título, que recibieron ITI y seguimiento completo. Se utilizó concentrado de FVIII plasmático en dosis de 70-180 UI/Kg/diarias, definiendo éxito como la negativización del inhibidor y recu peración de la vida media del FVIII. Resultados expresados en media (rango). Resultados: En 13 pacientes se identificó el inhibidor, a una edad de 17,6 meses (2-48), tras 35,2 días (9-112) de exposición a FVIII. Once pacientes (84,6%) recuperaron la vida media del FVIII, tras 49,6 meses (26-70) de tratamiento. En los pacientes que respondieron, el título del inhibidor se negativizó en 7,3 meses (1-20). Conclusiones: En niños con hemofilia A e inhibidores persistentes de alto título, la ITI tiene un elevado éxito. Dado que el tiempo de respuesta es variable, la inmunotolerancia debe ser personalizada.

Abstract: Introduction: The development of anti-factor VIII neutralizing antibodies in hemophilia A is the most severe com plication related to treatment. Immune tolerance induction (ITI) is the only known treatment for eradicating inhibitors. A successful ITI allows using factor VIII (FVIII) again for the treatment or prophylaxis of hemorrhagic events. Objective: To report the experience of pediatric patients who underwent ITI in the country's public health care network. Patients and Method: Retrospective and descriptive analysis of 13 pediatric patients with severe Hemophilia A and high-titer inhibitors persis tence who underwent ITI and complete follow-up. Plasma-derived FVIII concentrate was used at 70 180 IU/kg/day doses. The success of the treatment is defined by achieving a negative titer and a half life recovery of the FVIII. The results were expressed in median (range). Results: In 13 patients, the inhibitor was identified at an average age of 17.6 months, after 35.2 days of exposure to the FVIII. 11 patients (84.6%) recovered the half-life of FVIII after 49.6 months of treatment. In the patients who responded to treatment, the inhibitor titer was negative at 6 months on average. Conclusions: ITI is the treatment of choice for patients with hemophilia A and inhibitors persistence. ITI must be perso nalized since the time response is variable in each patient.

Brainstem tegmental necrosis and olivary hypoplasia: a lethal entity associated with congenital apnea.

Previously, we reported an isolated case of a newborn with central apnea at birth, ventilator-dependence, and combined malformative and destructive brainstem lesions (1). We now report 2 additional cases with similar clinicopathologic features. All 3 patients were male (XY karyotype) and required immediate ventilatory support in the delivery room. Perinatal complications included polyhydramnios and breech presentation. Variable cranial nerve palsies and orofacial and limb anomalies were present. The patients dies within minutes of withdrawal of ventilatory support at 2 to 11 weeks after birth. Significant neuropathologic findings were localized to the caudal pons and medulla, and included tegmental necrosis (neuronal loss, gliosis, mineralization) with involvement of respiratory-related nuclei, and anomalies of rhombic lip derivatives (olivary hypoplasia, arcuate nucleus hyperplasia). Three-dimensional computer reconstructions facilitated clinicoanatomic correlations, and underscored the restriction of the lesions to pontine and medullary rhombomeres and rhombic lip. The histopathology of these cases suggests a malformative process occurring at the end of the first trimester (time of rhombic lip migrations), and a superimposed destructive process (tegmental necrosis) in the second half of gestation. Although the etiology is unknown, the segmental nature of the lesions suggests the possibility of an abnormality in homeobox gene regulation. These cases likely represent a distinct clinicopathologic entity that should be considered in the differential diagnosis of Moebius syndrome and failure to breathe at birth.

Third ventricular chordoid glioma: a distinct clinicopathologic entity.

We have encountered a series of 8 third ventricular neoplasms with a distinctive chordoid appearance that appear to represent a clinicopathologic entity. The tumors occurred in 7 females and 1 male, ranging in age from 31 to 70 years. In all cases, imaging studies showed a large well-circumscribed third ventricular mass; a cystic component was noted in 2. The tumors consisted of cords and clusters of cohesive, oval-to-polygonal epithelioid cells with abundant eosinophilic cytoplasm, relatively uniform round-to-oval nuclei, and inconspicuous nucleoli. Mitotic activity was absent. The stroma consisted of scant, coarse fibrillar processes, as well as prominent, slightly basophilic, extracellular mucin resembling that in chordomas. Throughout the tumor, and surrounding its well-defined borders, were infiltrates of mature lymphocytes and plasma cells. Russell bodies were prominent in the latter. Adjacent brain tissue showed reactive changes with gliosis and numerous Rosenthal fibers. Immunohistochemically, tumor cells were strongly reactive for GFAP and vimentin, but negative or only weakly staining for EMA. The MIB-1 labeling index was approximately 1%. Ultrastructural examination of 4 cases revealed focal microvilli, scattered "intermediate" junctions, and focal basal lamina formation. Neither desmosomes nor cilia were seen. Total resections were achieved in 2 cases; only subtotal removals were achieved in 6. Subsequent tumor enlargement was noted in 3 of the 6 patients with incomplete resection, and of these, two died at post-operative intervals of 8 months and 3 years. The other patient survives 4 years post-operatively with stable residual disease. Of the 2 patients with total resection, 1 was lost to follow-up; the other, during a brief follow-up period, did well without evidence of recurrence.

Endogenous peroxidatic activity in astrocytes after spinal cord injury.

After spinal cord injury, endogenous peroxidatic-like activity develops along the axis of the cord. At 2 weeks postinjury, this activity appears in cells whose processes are intimately associated with microvessels. The objectives of this study were to further characterize this response and to identify the cellular localization of endogenous peroxidatic-like activity. After traumatic injury to the rat spinal cord, adjacent sections of spinal cord were processed in medium to visualize antiglial fibrillary acidic protein, endogenous peroxidatic activity, or cytochrome oxidase activity. In addition, certain sections, stained for endogenous peroxidatic-like activity, were prepared for electron microscopy. To identify the nature of the activity, some sections were exposed to an incubation medium that included inhibitors of either catalase or heme protein activity. The distribution of prominent glial fibrillary acidic protein immunoreactivity in the dorsal columns corresponded to that of marked staining for endogenous peroxidatic-like activity and cytochrome oxidase. At the ultrastructural level, endogenous peroxidatic-like activity was identified in the cytoplasmic compartment of the astrocyte. This activity was abolished when potassium cyanide (an inhibitor of heme protein) was added to the incubation medium. Spinal cord injury elicited a pronounced cellular response along the axis of the cord that was characterized by enhanced staining for antiglial fibrillary acidic protein, cytochrome oxidase activity, and endogenous peroxidatic-like activity. It is not clear whether pronounced cytochrome oxidase activity corresponded to astrocytes that also expressed prominent endogenous peroxidatic-like activity. However, according to both light and ultrastructural findings, endogenous peroxidatic-like activity was prominently associated with the astrocytic cytoplasm. The biochemical nature of the peroxidatic activity is unknown, but these results suggest that it is related to a heme-containing protein.

Liquid formula diet for human metabolic studies.

A liquid formula diet for use in studies with human subjects has been developed. All of the components can be obtained readily from commercial sources. For preparation of the diet, only equipment that customarily is found in institutional kitchens is needed. Acceptability over periods of several months is manifested by a large number of patients who consumed the liquid formula diet as their sole source of food.

SALL1 expression in the human pituitary-adrenal/gonadal axis.

SALL1 was originally identified on the basis of its DNA sequence homology to the region-specific homeotic gene Sal, in Drosophila melanogaster, which acts as a downstream target of hedgehog/tumor growth factor-beta-like decapentaplegic signals. The SALL1 gene has been associated with the Townes-Brocks Syndrome (TBS), a disorder characterized by multiorgan dysgenesis including renal and genital malformations. In this study, SALL1 message production was evaluated in association with the tissue localization of the protein product of SALL1, p140. SALL1 protein expression was observed in various adult and fetal tissues which elaborate reproductive endocrine hormones. The p140 was localized in specific microanatomic sites of the pituitary, adrenal cortex and the placenta. In the human pituitary, SALL1 protein expression was limited to the adenohypophysis, where it colocalized to those cells producing GH and the gonadotropins, LH and FSH. SALL1 expression was also found in most of the fetal and adult adrenal cortex in addition to the trophoblastic cells of the placenta. This pattern of expression complements prior studies demonstrating p140 in testicular fetal Leydig cells, adult Leydig and Sertoli cells, and granulosa cells of the ovary. The SALL1 protein was also shown here to be highly expressed in trophoblast tumors, which overproduce sex hormones. The expression patterns of SALL1 at multiple levels of the reproductive endocrine axis and the phenotypic effects associated with TBS suggest that SALL1 may have an important role in the interaction of the pituitary-adrenal/gonadal axis during reproduction.

Cardiomyopathy and myocyte intranuclear inclusions in neuronal intranuclear inclusion disease: a case report.

Neuronal intranuclear inclusion disease (NIID) is a progressive, usually fatal degenerative neurologic condition characterized by eosinophilic, intranuclear inclusions in neurons of the central and peripheral nervous system. We report a boy with onset of disease manifestations at age 3 and death at age 9, who showed clinical and pathologic findings characteristic of NIID. The case is unique because of cardiomyopathy manifested 1 year prior to death. Postmortem findings confirmed the presence of cardiomyopathy and revealed intranuclear inclusions in myocytes. Neither nuclear inclusions in the myocardium nor cardiac involvement have previously been reported in NIID.

Experimental fluid percussion brain injury: vascular disruption and neuronal and glial alterations.

Because of the potential relationship between vascular disturbances and secondary tissue damage, we identified areas of brain which exhibited hemorrhage and leakage of protein during the acute stage after experimental brain injury and subsequently studied the development of pathologic changes, including cavity formation, neuronal necrosis, and gliosis within these regions. The development of pathologic changes was evaluated at 1, 6, and 24 h and 1, 2, and 4 weeks after lateral, fluid percussion (FP) brain injury of moderate severity in the rat. Vascular disruption in the acute stages, as evidenced by hemorrhage and leakage of Evans blue albumin, was most prominent 6 h postinjury and was maximal in the parieto-occipital cortex. From 1 to 24 h after injury, regions of the injured hemisphere, including the cortex and hippocampus, exhibited abnormal neurons which stained with acid fuchsin and Alizarin red, histochemical markers for injured neurons and calcium, respectively. These same regions suffered significant neuronal cell loss from 1 to 4 weeks after injury. The distribution of reactive astrocytes was also evaluated by immunocytochemical localization of glial fibrillary acidic protein (GFAP). By 2 weeks postinjury, a prominent cavity was present in the frontoparietal and occipital cortices. Although astrogliosis was most pronounced in the cortex surrounding the cavity, prominent reactive astrocytes were widely distributed throughout the injured hemisphere. This study characterized the pathological changes which occur after experimental traumatic brain injury. In particular, we propose that neuronal cell injury in the hippocampus serves as a useful 'window' to assess beneficial efficacy of pharmacological intervention in the treatment of brain injury.

The temporal profile and morphologic features of neuronal death in human stroke resemble those observed in experimental forebrain ischemia: the potential role of apoptosis.

Although neuronal death has been studied in experimental models of ischemia, the precise mechanisms regulating cell death remain unclear. Furthermore, the timing and pattern of neuronal death in human stroke has not been extensively studied. To further our understanding of ischemia-induced neuronal death, we examined the temporal profile of histochemical and morphologic characteristics of hippocampal neuronal death following experimental forebrain ischemia and compared these findings to human brain specimens obtained from subjects suffering cerebral infarction. Transient forebrain ischemia (TFI) was induced in normothermic adult rats by bilateral carotid artery occlusion combined with hypotension. Animals were sacrificed at 6, 12, 18, 24, 48, and 72 h and 7, 14, and 28 days following ischemia (n = 4 at each time point). Experimental tissue was analyzed using light and electron microscopy as well as TUNEL histochemistry. A total of 27 human brain specimens with neuropathological confirmation of ischemic damage and appropriate controls were also examined using light microscopy and TUNEL histochemistry. Dense TUNEL staining in hippocampal CA-1 neurons was present at 48 and 72 h following experimental ischemia. Prior to these times, little or no nuclear staining was noted and after 72 h nuclear staining diminished rapidly. Ultrastructural findings at these time points demonstrated many features similar to those seen in cells undergoing apoptosis, such as cell shrinkage with increased electron density, chromatin condensation with formation of heterochromatin, intact plasma membranes, and intact intracellular organelles. In a similar fashion, human stroke specimens during the subacute period showed dense nuclear TUNEL staining in penumbral neurons, whereas in the acute or chronic stages little or no staining was noted. Our results demonstrate that the timing of morphologic changes and TUNEL histochemistry following human stroke resembles that observed in experimental TFI. Furthermore, neuronal death in both experimental ischemia and human stroke share several features characteristic of apoptotic cell death.

Spinal subdural abscess: successful treatment with limited drainage and antibiotics in a patient with AIDS.

The authors present a case of Staphylococcus aureus spinal subdural abscess in a patient with AIDS. Although complete surgical drainage has been strongly advocated in the literature, this patient made a complete neurologic and symptomatic recovery and radiographically demonstrated resolution of the abscess with only limited surgical drainage and parenteral antibiotics. Magnetic resonance imaging findings of this unusual lesion are discussed. Relevant literature in the management of spinal subdural abscesses is reviewed.

Early pregnancy diagnosis in alpaca (Lama pacos) and llama (Lama glama) by ultrasound.

An ultrasonography study of early pregnancy diagnosis was carried out in 19 alpacas and 12 llamas, after controlled matings. The aim was to determine the earliest gestational age at which pregnancy diagnosis by transrectal ultrasonography could be achieved, and to generate an empirical formula for gestational sac diameter (GSD) growth as a function of gestational age (GA), allowing an estimate of GA during the first month of pregnancy. We found that pregnancy diagnosis may be carried out as early as 9 days after mating in alpacas and 7 days in llamas. This diagnosis was found to be accurate at 23 days in alpacas and 34 days in llamas. The empirical relations that best describe the relationship between GSD and GA were GA = logGSD + 1.2339/0.0585 r = 0.85; P < 0.001 in alpacas, and GA = logGSD + 1.2649/0.0546 r = 0.77, P < 0.001 in llamas, where GA is measured in days and GSD in centimeters. Our results also indicate that ultrasonography is a reliable technique for early pregnancy diagnosis. Furthermore, the empirical formulae reliably make it possible to estimate GA from GSD during the first month of pregnancy and their use might improve the efficiency of camelid breeders.

[Sulfonamide and toltrazuril therapy of experimental turkey coccidiosis]. / Zur Sulfonamid- und Toltrazuriltherapie der experimentellen Putenkokzidiose.

Following experimental infection of caged turkeys with a mixed oocyst suspension of E. meleagrimitis, E. adenoeides and E. gallopavonis, the efficacy of sulfachlorpyrazine, sulfaquinoxaline + pyrimethamine, sulfaquinoxaline + diaveridine, and toltrazuril was investigated based on clinical and parasitological parameters. Infected turkeys developed severe clinical symptoms. Only weak symptoms were observed in those groups which were treated with the sulfa- and sulfa-DHFR-inhibitors. The toltrazuril-treated group remained free of clinical symptoms. At the 8th day after infection, the clinical lesions coincided with the intensity of the gut lesions together with parasitological findings based on microscopical examination of mucosal smears. The same coincidence was observed for parameters such as oocyst excretion and body weight development.

Role of plasmin and gelatinase in extracellular matrix degradation by cultured rat mesangial cells.

We have examined the ability of mesangial cells (MCs) to degrade extracellular matrix (ECM) using cultured rat MCs grown on thin films of radiolabeled Matrigel. ECM degradation by cultured MCs was observed only in presence of exogenously added plasminogen and was a function of plasminogen concentration (0-50 mU), cell number (0-50,000 cells), and length of incubation (0-72 h). A high positive correlation (r > 0.93) was observed between ECM degradation and plasmin activity in medium, suggesting an important role for plasmin in ECM degradation by cultured MCs. This suggestion was confirmed by ability of plasmin inhibitors, alpha 2-antiplasmin (40 micrograms/ml) and aprotinin (216 kallikrein inhibition units/ml), to inhibit (> 90%) ECM degradation. Inhibitors of cysteine proteinases [trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane, 10 microM] and aspartic proteinases (pepstatin, 5.0 micrograms/ml) had no effect on ECM degradation. However, in presence of plasminogen, inhibitors of matrix metalloproteinases, TIMP-1 (40 micrograms/ml) and o-phenanthroline (100 microM), inhibited ECM degradation -42 +/- 4% and -43 +/- 3% (SE), respectively (n = 8-10). Thus, in addition to plasmin, a matrix metalloproteinase(s) is also involved in ECM degradation by cultured rat MCs. Zymography of culture medium obtained from MCs grown on radiolabeled Matrigel films in absence of plasminogen revealed only two closely migrating bands of gelatinase activity, relative mol wt of approximately 70,000-72,000. MCs grown in absence of plasminogen failed to degrade ECM despite presence of gelatinase in medium, indicating that, in absence of plasmin, gelatinase is present in an inactive form, either as a latent proenzyme or as a gelatinase-inhibitor complex.(ABSTRACT TRUNCATED AT 250 WORDS)