Serum Protease Inhibitors Research Articles

Détection des différentes formes du prostate-specific antigen et autres kallicréines dans le cancer de la prostate

Prostate cancer is the most common cancer in men over 50 years old and the second leading cause of cancer death. Prostate-specific antigen (PSA) is widely used for the diagnosis and follow-up of prostate cancer. PSA, also called kallikrein 3, is a member of the human kallikrein-type serine protease family and circulates in the blood stream in the form of complexes with serum protease inhibitors and in free form. However, free PSA is also a heterogeneous mixture of different molecular PSA forms: proPSA, intact and clived mature forms. The clinical significance of these different forms is still unclear but their specific measurement in serum could improve the specificity of PSA for detecting cancer or predicting treatment outcome. Others kallikreins including kallikrein 2, 4, 11, 14 and 15 are also emerging as complementary markers to PSA for prostate cancer. Multiple detection of the different molecular forms of PSA, as well as of these kallikreins, in addition to total PSA, could significantly increase the diagnostic utility of PSA and may add prognostic value by bringing clinical information on the cancer progression.

A Segment of γ ENaC Mediates Elastase Activation of Na+ Transport

The epithelial Na+ channel (ENaC) that mediates regulated Na+ reabsorption by epithelial cells in the kidney and lungs can be activated by endogenous proteases such as channel activating protease 1 and exogenous proteases such as trypsin and neutrophil elastase (NE). The mechanism by which exogenous proteases activate the channel is unknown. To test the hypothesis that residues on ENaC mediate protease-dependent channel activation wild-type and mutant ENaC were stably expressed in the FRT epithelial cell line using a tripromoter human ENaC construct, and protease-induced short-circuit current activation was measured in aprotinin-treated cells. The amiloride-sensitive short circuit current (INa) was stimulated by aldosterone (1.5-fold) and dexamethasone (8-fold). Dexamethasone-treated cells were used for all subsequent studies. The serum protease inhibitor aprotinin decreased baseline INa by approximately 50% and INa could be restored to baseline control values by the exogenous addition of trypsin, NE, and porcine pancreatic elastase (PE) but not by thrombin. All protease experiments were thus performed after exposure to aprotinin. Because NE recognition of substrates occurs with a preference for binding valines at the active site, several valines in the extracellular loops of α and γ ENaC were sequentially substituted with glycines. This scan yielded two valine residues in γ ENaC at positions 182 and 193 that resulted in inhibited responses to NE when simultaneously changed to other amino acids. The mutations resulted in decreased rates of activation and decreased activated steady-state current levels. There was an ∼20-fold difference in activation efficiency of NE against wild-type ENaC compared to a mutant with glycine substitutions at positions 182 and 193. However, the mutants remain susceptible to activation by trypsin and the related elastase, PE. Alanine is the preferred P1 position residue for PE and substitution of alanine 190 in the γ subunit eliminated INa activation by PE. Further, substitution with a novel thrombin consensus sequence (LVPRG) beginning at residue 186 in the γ subunit (γTh) allowed for INa activation by thrombin, whereas wild-type ENaC was unresponsive. MALDI-TOF mass spectrometric evaluation of proteolytic digests of a 23-mer peptide encompassing the identified residues (T176-S198) showed that hydrolysis occurred between residues V193 and M194 for NE and between A190 and S191 for PE. In vitro translation studies demonstrated thrombin cleaved the γTh but not the wild-type γ subunit. These results demonstrate that γ subunit valines 182 and 193 are critical for channel activation by NE, alanine 190 is critical for channel activation by PE, and that channel activation can be achieved by inserting a novel thrombin consensus sequence. These results support the conclusion that protease binding and perhaps cleavage of the γ subunit results in ENaC activation.

A prostate-specific antigen–activated N-(2-hydroxypropyl) methacrylamide copolymer prodrug as dual-targeted therapy for prostate cancer

Prostate cancer targeted peptide prodrugs that are activated by the serine protease activity of prostate-specific antigen (PSA) are under development in our laboratory. To enhance delivery and solubility of these prodrugs, macromolecular carriers consisting of N-(2-hydroxypropyl) methacrylamide (HPMA)-based copolymers were covalently coupled to a PSA-activated peptide prodrug. HPMA copolymers are water-soluble, nonimmunogenic synthetic carriers that exhibit promise for drug delivery applications. These macromolecular copolymers enter the interstitium of solid tumors by the enhanced permeability and retention effect. The PSA-activated peptide substrate imparts selectivity because it is specifically hydrolyzed to release a cytotoxin at the site of prostate tumor. Enzymatically active PSA is present in high amounts in the extracellular fluid of a tumor, but PSA is inactivated in blood by binding to serum protease inhibitors. As an initial proof of concept, the HPMA copolymer was synthesized with a peptide substrate (HSSKLQ) bound to a fluorophore, 7-amino-4-methylcoumarin (AMC). PSA cleavage of the HPMA-HSSKLQ-AMC copolymer was observed, which led to the synthesis of an HPMA-based copolymer with the prodrug SSKYQ-L12ADT [HPMA-morpholinocarbonyl-Ser-Ser-Lys-Tyr-Gln-Leu-12-aminododecanoyl thapsigargin (JHPD)]. L12ADT is a potent analogue of the highly cytotoxic natural product thapsigargin. HPMA-JHPD was hydrolyzed by PSA in vitro and was toxic to prostate cancer cells in the presence of active PSA. The HPMA-JHPD produced no systemic toxicity when given at a 500 micromol/L L12ADT equivalent dose. Analysis of tumor tissue from mice treated with a single or multiple dose of the HPMA-JHPD copolymer showed release and accumulation of the L12ADT toxin within the tumor tissue.

ALPHA1-ANTITRYPSIN DEFICIENCY DIAGNOSED IN PATIENTS OVER THE AGE OF 45

PURPOSE: Alpha One Antitrypsin Deficiency (AATD) is a hereditary disorder characterized by deficiency in a serum protease inhibitor. This deficiency predisposes patients to early onset emphysema through uncontrolled proteolytic attack in the lung. There is great variability in the age of onset of clinically recognizable lung disease amongst AATD patients. Estimates of the frequency of deficiency phenotypes in the United States population predict anywhere from 40,000 to 100,000 patients should have AATD.

Inter-α-Trypsin Inhibitor Attenuates Complement Activation and Complement-Induced Lung Injury

Complement activation is a central component of inflammation and sepsis and can lead to significant tissue injury. Complement factors are serum proteins that work through a cascade of proteolytic reactions to amplify proinflammatory signals. Inter-alpha-trypsin inhibitor (IaI) is an abundant serum protease inhibitor that contains potential complement-binding domains, and has been shown to improve survival in animal sepsis models. We hypothesized that IaI can bind complement and inhibit complement activation, thus ameliorating complement-dependent inflammation. We evaluated this hypothesis with in vitro complement activation assays and in vivo in a murine model of complement-dependent lung injury. We found that IaI inhibited complement activation through the classical and alternative pathways, inhibited complement-dependent phagocytosis in vitro, and reduced complement-dependent lung injury in vivo. This novel function of IaI provides a mechanistic explanation for its observed salutary effects in sepsis and opens new possibilities for its use as a treatment agent in inflammatory diseases.

Site of Blood Vessel Damage and Relevance of CD18 in a Murine Model of Immune Complex-Mediated Vasculitis

How neutrophils (polymorphonuclear neutrophils, PMNs) damage vessels in leukocytoclastic vasculitis (LcV) mediated by immune complexes (ICs) is unclear. If degradative enzymes and oxygen radicals are released from PMNs while adhering to the inner side of the vessel wall, they could be washed away by the blood stream or neutralized by serum protease inhibitors. We investigated if in LcV PMNs could damage vessels from the tissue side after transmigration. We used CD18-deficient (CD18-/-) mice because the absence of CD18 excludes transmigration of PMNs. When eliciting the Arthus reaction in ears of CD18-/- mice, deposition of ICs was not sufficient to recruit PMNs or to induce IC-mediated LcV. Injection of PMNs intradermally in CD18-/- mice allowed us to investigate if bypassing diapedesis and placing PMNs exclusively on the abluminal side leads to vascular destruction. We found that injected PMNs gathered around perivascular ICs, but did not cause vessel damage. Only intravenous injection of wild-type PMNs could re-establish the Arthus reaction in CD18-/- mice. Thus, PMNs cause vessel damage during diapedesis from the luminal side, but not from the perivascular space. We suggest that in order to shield the cytotoxic products from the blood stream, ICs induce particularly tight interactions between them, PMNs and endothelial cells.

Serum protease inhibitor concentrations and total antitrypsin activity in diabetic and non-diabetic children during adolescence

The aim of the study was the assessment of the concentrations and establishment of mutual relationships between three main protease inhibitors: alpha-1-antitrypsin (AAT), alpha-2-macroglobulin (alpha-2-M) and antithrombin-III (AT-III), and of the total trypsin inhibitory capacity (TIC) in the serum of diabetic and non-diabetic children during adolescence. Forty-nine children (24 girls and 25 boys) with type 1 diabetes mellitus and 24 non-diabetic children (13 girls and 11 boys) were divided according to the Tanner scale into three groups: pre-, peri- and post-pubertal. The concentrations of AAT, alpha-2-M and AT-III were determined by the radial immunodiffusion method on NOR-Partigen plates (Dade-Behring), while TIC was determined by the method using BAPNA as substrate. Means and medians of serum AAT [1.55 g/l, 1.40 (95% confidence interval, 1.42-1.68), respectively] and TIC [10.6 mg trypsin/100 ml, 10.3 (95% CI, 9.5-11.7)] in diabetic children were lower than means and medians of AAT [1.81 g/l, 1.60 (95% CI 1.55-2.07), respectively] and TIC [12.5 mg trypsin/100 ml, 13.2 (95% CI, 10.9-14.1)] in non-diabetic children. A comparison of variables between Tanner groups shows an increasing trend of AAT concentration in diabetic children and a decreasing trend of TIC in non-diabetic subjects. In contrast to pre- and peri-puberty, no correlations were found in the postpubertal period between the studied parameters, either in diabetic or non-diabetic patients. Hyperglycaemia and the duration of diabetes were found to have a significant association with alpha-2-M and AT-III concentrations, but not with AAT serum concentrations. The concentrations and correlations between serum protease inhibitors in diabetic children during adolescence are disrupted compared with non-diabetic children. Taking into account the unfavourable consequences of vascular complications resulting from serum trypsin inhibitor changes and protease- antiprotease imbalance, diabetic children are at greater risk of this occurring during adolescence.

Prohormone Convertase 1/3 Is Essential for Processing of the Glucose-dependent Insulinotropic Polypeptide Precursor

The physiology of the incretin hormones, glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), and their role in type 2 diabetes currently attract great interest. Recently we reported an essential role for prohormone convertase (PC) 1/3 in the cleavage of intestinal proglucagon, resulting in formation of GLP-1, as demonstrated in PC1/3-deficient mice. However, little is known about the endoproteolytic processing of the GIP precursor. This study investigates the processing of proGIP in PC1/3 and PC2 null mice and in cell lines using adenovirus-mediated overexpression. Supporting a role for PC1/3 in proGIP processing, we found co-localization of GIP and PC1/3 but not PC2 in intestinal sections by immunohistochemistry, and analysis of intestinal extracts from PC1/3-deficient animals demonstrated severely impaired processing to GIP, whereas processing to GIP was unaltered in PC2-deficient mice. Accordingly, overexpression of preproGIP in the neuroendocrine AtT-20 cell line that expresses high levels of endogenous PC1/3 and negligible levels of PC2 resulted in production of GIP. Similar results were obtained after co-expression of preproGIP and PC1/3 in GH4 cells that express no PC2 and only low levels of PC1/3. In addition, studies in GH4 cells and the alpha-TC1.9 cell line, expressing PC2 but not PC1/3, indicate that PC2 can mediate processing to GIP but also to other fragments not found in intestinal extracts. Taken together, our data indicate that PC1/3 is essential and sufficient for the production of the intestinal incretin hormone GIP, whereas PC2, although capable of cleaving proGIP, does not participate in intestinal proGIP processing and is not found in intestinal GIP-expressing cells.

Accouchement des patientes enceintes infectées par le VIH : étude rétrospective de 358 grossesses suivies entre 2000 et 2004

Evaluate the mode of delivery of HIV-infected women and the risk of mother-to-child transmission. A retrospective study conducted on HIV-infected women who delivered at the maternity ward of Bichat Hospital in Paris between 1st January 2000 and 31(st) December 2004. Pregnancy care, antiretroviral therapy, decision of the mode of delivery and neonate treatment were conformable to the French recommendations. The analysis was performed on 332 cases out of 358 pregnancies followed during this period. 75% received a Highly Active Anti Retroviral Therapy (HAART), 24% an AZT monotherapy and 1% did not receive any antiretroviral treatment. Plasmatic HIV viral load was under the level of detectability (50 copies/ml) for 64,6% of women under HAART and 28,7% of women under AZT monotherapy. Only 31,7% of women under HAART delivered vaginally. 44,7% of women under HAART with undetectable viral load at the moment of delivery delivered vaginally. 59,5% of women who were allowed to deliver vaginally had finally a vaginal delivery. 332 women gave birth to 341 babies with 9 twin pregnancies and one still-birth at 22 WA. Out of these 340 babies, 3 babies whose mother received HAART were HIV infected (2 in utero and 1 per-partum). The reasons why only one third of HIV-infected women could deliver vaginally in this study are primarily the persistence of a detectable HIV viral load under HAART. Women's choice of the mode of delivery comes next, which depends on the quality of the counselling about the benefits and risks of the cesarean section in the context of HIV infection. The third reason is obstetrical contra indications to vaginal delivery in the context of HIV infection. In the future, it is possible to reduce the incidence of cesarean section in HIV-infected women by elevating the level of HIV plasmatic viral load which allowed vaginal delivery (1000 copies/ml), by improving the observance to antiretroviral treatment, by adaptating antiretroviral medications posology using determination of serum protease inhibitors concentration and by modifying obstetrical management with less restrictive contra indications to vaginal delivery. However the impact of prophylactic cesarean section when plasmatic HIV viral load is undetectable must still be evaluated.

Interaction of Mannan Binding Lectin with α2 Macroglobulin via Exposed Oligomannose Glycans: A CONSERVED FEATURE OF THE THIOL ESTER PROTEIN FAMILY?

The serum collectin mannan-binding lectin (MBL) binds to oligomannose and GlcNAc-terminating glycans present on microorganisms. Using a commercial affinity chromatography resin containing immobilized MBL we screened human and mouse serum for endogenous MBL-binding targets. We isolated the serum protease inhibitor alpha(2) macroglobulin (alpha2M), a heavily glycosylated thiol ester protein (TEP) composed of four identical 180-kDa subunits, each of which has eight N-linked glycosylation sites. alpha2M has previously been reported to interact with MBL; however, the interaction was not characterized. We investigated the mechanism of formation of complexes between alpha2M and MBL and concluded that they form by the direct binding of oligomannose glycans Man(5-7) occupying Asn-846 on alpha2M to the lectin domains (carbohydrate recognition domains) of MBL. The oligomannose glycans are accessible for lectin binding on both active alpha2M (thiol ester intact) and protease-cleaved alpha2M (thiol ester cleaved). We demonstrate that MBL is able to interact with alpha2M in the fluid phase, but the interaction does not inhibit the binding of MBL to mannan-coated surfaces. In addition to alpha2M, two other members of the TEP family, C3 and C4, which also contain oligomannose glycans, were captured from human serum using the MBL resin. MBL binding may be a conserved feature of the TEPs, dating from their ancestral origins. We suggest that the inhibition of proteases on the surface of microorganisms by an ancestral alpha2M-like TEP may generate "arrays" of oligomannose glycans to which MBL or other lectins can bind. Binding would lead to opsonization or activation of enzyme systems such as complement.

Correlation of the Serum Antithrombin III to Injury Severity in Patients with Severe Trauma

Study Objectives: Antithrombin III (AT-III) is a serum protease inhibitor that inhibits the blood coagulation protease thrombin and is seen to be present in low levels in cases of shock, sepsis, or major trauma. Coagulopathy and hemorrhage are known contributors to trauma prognosis but the actual relationships of AT-III to mortality and to injury severity are unknown. The purpose of this study was to determine the correlation between AT-III and injury severity.

Characteristics of the amylase of Arthrobacter psychrolactophilus

Properties of the extracellular amylase produced by the psychrotrophic bacterium, Arthrobacter psychrolactophilus, were determined for crude preparations and purified enzyme. The hydrolysis of soluble starch by concentrated crude preparations was found to be a nonlinear function of time at 30 and 40 degrees C. Concentrates of supernatant fractions incubated without substrate exhibited poor stability at 30, 40, or 50 degrees C, with 87% inactivation after 21 h at 30 degrees C, 45% inactivation after 40 min at 40 degrees C and 90% inactivation after 10 min at 50 degrees C. Proteases known to be present in crude preparations had a temperature optimum of 50 degrees C, but accounted for a small fraction of thermal instability. Inactivation at 30, 40, or 50 degrees C was not slowed by adding 20 mg/ml bovine serum albumin or protease inhibitor cocktail to the preparations or the assays to protect against proteases. Purified amylase preparations were almost as thermally sensitive in the absence of substrate as crude preparations. The temperature optimum of the amylase in short incubations with Sigma Infinity Amylase Reagent was about 50 degrees C, and the amylase required Ca(+2) for activity. The optimal pH for activity was 5.0-9.0 on soluble starch (30 degrees C), and the amylase exhibited a K (m) with 4-nitrophenyl-alpha-D-maltoheptaoside-4,6-O-ethylidene of 120 microM at 22 degrees C. The amylase in crude concentrates initially hydrolyzed raw starch at 30 degrees C at about the same rate as an equal number of units of barley alpha-amylase, but lost most of its activity after only a few hours.

Artifactual Isoform Profile Modification Following Treatment of Human Plasma or Serum with Protease Inhibitor, Monitored by 2-Dimensional Electrophoresis and Mass Spectrometry

The inclusion of protease inhibitors in serum or plasma samples has been found to significantly impact the isoform profile of selected plasma proteins as seen on 2-dimensional electrophoresis (2-DE) gels. With the addition of a protease inhibitor cocktail, several human plasma protein trains [depleted of albumin and immunoglobulin G (IgG)] exhibited higher isoelectric point (pI) isoforms. This shift was especially apparent for apolipoprotein A1 (apo A1), a relatively high abundance protein. The six protease inhibitor components of the cocktail were individually investigated with albumin and IgG depleted human plasma, and it was shown that the observed effects were caused by 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), a serine protease inhibitor that covalently modifies proteins and/or peptides. Several serine-and/or tyrosine-containing peptides of apo A1 were modified with a concomitant mass increase of 183 Da, which is consistent with the mass increase expected following reaction with AEBSF. These modifications were observed with increasing propensity in the higher pI spots. An increase in both the number and proportion of modified peptides with increasing pI was also observed. A model is proposed for the random or stochastic coupling of AEBSF-derived moieties to serine and/or tyrosine residues throughout apo A1 and potentially other plasma proteins.

Protein Kinase A-dependent Phosphorylation of Lutheran/Basal Cell Adhesion Molecule Glycoprotein Regulates Cell Adhesion to Laminin α5

Lutheran (Lu) blood group and basal cell adhesion molecule (B-CAM) antigens reside on two glycoprotein (gp) isoforms Lu and Lu(v13) that belong to the Ig superfamily and differ only by the size of their cytoplasmic tail. Lu/B-CAM gps have been recognized as laminin alpha5 receptors on red blood cells and epithelial cells in multiple tissues. It has been shown that sickle red cells exhibit enhanced adhesion to laminin alpha5 when intracellular cAMP is up-regulated by physiological stimuli such as epinephrine and that this signaling pathway is protein kinase A- and Lu/B-CAM-dependent. In this study, we analyzed the relationship between the phosphorylation status of Lu/B-CAM gps and their adhesion function to laminin alpha5. We showed that Lu isoform was phosphorylated in sickle red cells as well as in erythroleukemic K562 and epithelial Madin-Darby canine kidney cells and that this phosphorylation is enhanced by different stimuli of the PKA pathway. Lu gp is phosphorylated by glycogen synthase kinase 3 beta, casein kinase II, and PKA at serines 596, 598, and 621, respectively. Alanine substitutions of serines 596 and 598 abolished phosphorylation by glycogen synthase kinase 3 beta and casein kinase II, respectively, but had no effect on adhesion of K562 cells to laminin under flow conditions. Conversely, mutation of serine 621 prevented phosphorylation by PKA and dramatically reduced cell adhesion. Furthermore, stimulation of K562 cells by epinephrine increased Lu gp phosphorylation by PKA and enhanced adhesion to laminin. It is postulated that modulation of the phosphorylation state of Lu gp might be a critical factor for the sickle red cells adhesiveness to laminin alpha5 in sickle cell disease.

Stimulation of immunity in Indian major carp Catla catla with herbal feed ingredients

Catla catla, catla (150 ± 20 g) were fed a diet containing seed of Achyranthes aspera (0.5%) and control diet without A. aspera for four weeks prior to and after ip injection with chicken erythrocytes. Fish were sampled for four consecutive weeks after immunization. Hemagglutination antibody titers were significantly higher in the test group of fishes compared with the control group. Serum globulin levels were significantly ( P t -test < 0.05) higher in the test group than control group on days 14 and 21. Anti-trypsin activity due to total serum protease inhibitors and α1-antiprotease was also significantly ( P t -test < 0.05) higher in the test group of fishes than the control. RNA/DNA ratio of spleen and kidney was also significantly ( P t -test < 0.05) higher in test group than the control group. All these results confirm that A. aspera enhances the immunity of catla.

Microglia-derived Pronerve Growth Factor Promotes Photoreceptor Cell Death via p75 Neurotrophin Receptor

Reports implicating microglia-derived nerve growth factor (NGF) during programmed cell death in the developing chick retina led us to investigate its possible role in degenerative retinal disease. Freshly isolated activated retinal microglia expressed high molecular weight forms of neurotrophins including that of nerve growth factor (NGF), brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4. Conditioned media from cultured retinal microglia (MGCM) consistently yielded a approximately 32-kDa NGF-reactive band when supplemented with bovine serum albumin (BSA) or protease inhibitors (PI); and promoted cell death that was suppressed by NGF immunodepletion in a mouse photoreceptor cell line (661w). The approximately 32 kDa protein was partially purified (MGCM/p32) and was highly immunoreactive with a polyclonal anti-pro-NGF antibody. Both MGCM/p32 and recombinant pro-NGF protein promoted cell death in 661w cultures. Increased levels of pro-NGF mRNA and protein were observed in the RCS rat model of retinal dystrophy. MGCM-mediated cell death was reversed by p75NTR antiserum in p75NTR(+)/trkA(-) 661w cells. Our study shows that a approximately 32 kDa pro-NGF protein released by activated retinal microglia promoted degeneration of cultured photoreceptor cells. Moreover, our study suggests that defective post-translational processing of NGF might be involved in photoreceptor cell loss in retinal dystrophy.

101. Toxicology and Biodistribution Studies of a Recombinant Adeno-Associated Virus 2 (rAAV2) Alpha-1 Antitrypsin (AAT) Vector

A series of safety and biodistribution studies were undertaken to support the development of a muscle-based gene therapy system for AAT deficiency. AAT is the primary circulating serum proteinase inhibitor, and its deficiency can result in emphysema. The test article that was used in this toxicology study was rAAV serotype 2 with the CMV/Beta Actin promoter driving expression of the human AAT gene. There were 4 dosing arms in the primary toxicology study (PTS) with sacrifice time points being at 21 days and 90 days post vector administration. In the biodistribution study (BDS), there were 5 dosing arms with duplicate doses given intramuscularly and intravenously. The sacrifice time point for this study was at 60 days post vector administration. The PTS was performed in 48-C57/Bl6 mice (24M/24F) with the primary safety endpoint being the histopathologic examination of organs collected at necropsy. CBC and serum chemistry analyses were also performed. Gene transfer efficiency was evaluated from gDNA isolated from organs and whole blood by real-time PCR. The BDS was performed in 36-C57/Bl6 mice (18M/18F) to determine the potential for vector dissemination following test article administration IV and IM. In the PTS, no abnormalities were noted in the CBC and serum chemistry data among test article and control article study arms. Histopathology results were essentially normal with no vector related findings. Real Time PCR indicated that vector DNA was present in the peripheral blood 24 hours post administration, but decreased to near baseline levels at the time of sacrifice. Vector DNA was not detectable in gonad tissue. Real Time PCR results in the BDS also indicated vector DNA present in peripheral blood at 24 hours post vector administration. At sacrifice, IV injected mice vector levels were higher than IM injected mice. Vector DNA was found in skeletal muscle, lung, heart, liver, diaphragm, kidney and pancreas at the time of necropsy via IM administration at dose dependent levels. No vector DNA was detected in the skeletal muscle following IV administration, but substantial amounts were found in other organs. Gonads were negative for vector DNA with both IM and IV administration indicating that the risk of germline transmission with our gene therapy vector would be relatively low.

Protein-losing enteropathy in dogs is associated with decreased fecal proteolytic activity.

Measurement of proteolytic activity in feces is a traditional method for the diagnosis of exocrine pancreatic insufficiency (EPI). A drawback of this method is the occurrence of falsely low results that may lead to a false-positive diagnosis of EPI. We hypothesized that intestinal loss of serum proteinase inhibitors in protein-losing enteropathy (PLE) may inhibit fecal proteolytic activity and be a potential source of false low results. The objective of this study was to determine the effect of PLE on fecal proteolytic activity in dogs. Fecal proteolytic activity was measured using a radial diffusion casein digestion assay in 12 samples from 4 clinically healthy control dogs and 30 samples from 16 dogs with PLE. Gastrointestinal protein loss was assessed using an ELISA to determine fecal canine alpha 1-proteinase inhibitor concentration. The relationship between the concentration of canine alpha 1-proteinase inhibitor in the feces and the diameter cleared in the casein digestion assay was determined. The mean clearing diameter was compared between control dogs and dogs with PLE. A significant negative correlation was observed between fecal canine alpha1-proteinase inhibitor concentration and casein clearing diameter (P <.001, Pearson r =.6317, r2 =.3999). Mean clearing diameter was significantly lower in dogs with PLE than in control dogs (12.63 vs 16.83 mm, P <.001, two-tailed Student's t-test). Increased fecal loss of alpha1-proteinase inhibitor in dogs with PLE is associated with a significant decrease in fecal proteolytic activity and may result in a false positive diagnosis of EPI.

Deposition of IgD, alpha-1-antitrypsin and alpha-1-antichymotrypsin on Demodex folliculorum and D. brevis infesting the pilosebaceous unit.

A total of seven routinely processed biopsy specimens of facial skin lesions with infestation of Demodex folliculorum or D. brevis were immunostained for plasma proteins and secretory proteins. The cuticular layer of the mites located within the pilosebaceous unit was selectively immunoreactive for IgD (delta chain), alpha-1-antitrypsin and alpha-1-antichymotrypsin. Negative results were obtained for IgG, IgA, IgM, IgE, albumin, fibrinogen, C3, amyloid P component, prealbumin, lysozyme and lactoferrin. These findings suggest a novel function of IgD and serum protease inhibitors as a protective host response to the mite.

Complex formation between human kallikrein 13 and serum protease inhibitors

Background: The kallikrein family is a group of 15 serine protease genes clustered on chromosome 19q13.4. Human kallikrein gene 13 ( KLK13) is a member of this family and encodes for a trypsin-like, secreted serine protease (hK13). Given that other kallikreins are sequestered by serum protease inhibitors, we hypothesized that hK13 may also interact with similar inhibitors. Our objective was to identify serum protease inhibitors that interact with human hK13. Methods: Recombinant hK13 produced in yeast was added to male and female sera and various biological fluids and the spiked samples were analyzed with an hK13 ELISA assay. Enzymatically active hK13 was 125I-labeled and used in in vitro reactions with candidate protease inhibitors and serum samples. The mixtures were then subjected to gel filtration and SDS-PAGE analysis. Candidate inhibitors were also tested in enzymatic assays of hK13 activity. Results: The recovery of recombinant hK13 from male and female sera, measured by three versions of the hK13-ELISA, ranged from 5% to 10%. The same recovery was obtained when serum samples from males and females were spiked with hK13 from amniotic fluid and seminal plasma. However, when hK13 was added to other biological fluids, such as amniotic fluid and breast milk, recovery ranged from 70% to 98%. In vitro analysis indicated that enzymatically active 125I-labeled hK13 forms SDS-stable complexes with α 2-antiplasmin, α 2-macroglobulin and α 1-antichymotrypsin. When added to serum, active hK13 formed stable complexes with molecular masses corresponding to hK13 and the inhibitors mentioned above. Conclusions: hK13 interacts and forms complexes with serum protease inhibitors, including α 2-macroglobulin, α 1-antichymotrypsin and α 2-antiplasmin.