Impact of Vitamin D3 on Carbonyl-Oxidative Stress and Matrix Metalloproteinases after Acute Intracerebral Hemorrhage in Rats with Type 2 Diabetes Mellitus.

INTRODUCTION: Diabetes mellitus is associated with the development of carbonyl-oxidative stress (COS) and an increased risk of a cerebral hemorrhage. Vitamin D3 is considered an additional drug to have an impact on COS and proteolysis in the extracellular matrix. OBJECTIVE: The study aimed to evaluate the impact of D3 on the COS-markers and matrix metalloproteinases MMP2/MMP9 activity after acute intracerebral hemorrhage (ICH) in rats with experimental type 2 diabetes mellitus (Т2DM) compared to metformin (Met). METHODS: T2DM was induced in rats via the intraperitoneal injection of streptozotocin (STZ) and nicotinamide (NA), ICH - by microinjection of bacterial collagenase into the striatum. Rats were randomized into five groups: 1 - intact animals (n = 8), 2 - T2DM (n = 9); 3 - T2DM+ICH (n = 7); 4 - T2DM+ICH+Met (n = 7); 5 - T2DM+ICH+D3 (n = 7). Blood glucose, glycated hemoglobin, and oral glucose tolerance test (OGTT) were assessed using commercial kits. Advanced oxidation protein products (AOPP), protein carbonyls (PC370/430), and ischemia-modified albumin (IMA) were measured by spectrophotometry, advanced glycation end products (AGEs) by quantitative fluorescence, and matrix metalloproteinases MMP2/9 by gelatin zymography. RESULTS: D3 does not significantly affect the glucose level and OGTT in rats with T2DM+ICH. However, it reduces AOPP, PC, and AGEs, thus reducing the COS index. In contrast, the activity of proMMP9 increases after D3 administration. These effects of D3 have been reported to be stronger and sometimes opposite to those of metformin. CONCLUSION: D3 supplementation may decrease the negative consequences of a cerebral hemorrhage in T2DM by reducing COS and preventing the accumulation of COS-modified proteins in the brain by regulating the expression and activity of MMP9.

Tetrandrine Attenuates Podocyte Injury by Inhibiting TRPC6-Mediated RhoA/ROCK1 Pathway.

Tetrandrine (Tet), a compound found in a traditional Chinese medicine, presents the protective effect for kidney function. Our study is aimed at clarifying the efficacy and underlying mechanism of Tet on podocyte injury. In this study, podocyte injury was induced in rats with adriamycin (ADR), and MPC5 podocytes were constructed with TRPC6 overexpression. We found that Tet treatment reduced the levels of proteinuria, serum creatinine, and blood urea nitrogen and increased plasma albumin levels in ADR-induced rats. Tet reduced intracellular Ca2+ influx and apoptosis in MPC5 podocytes overexpressing TRPC6. Tet downregulated the expression of renal TRPC6, RhoA, and ROCK1 and upregulated the expression of synaptopodin; meanwhile, it reduced calcineurin activity in vivo and in vitro. In conclusion, Tet protects against podocyte by affecting TRPC6 and its downstream RhoA/ROCK1 signaling pathway.

Photobiomodulation Mitigates Cerebrovascular Leakage Induced by the Parkinsonian Neurotoxin MPTP.

The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is commonly used to model Parkinson's disease (PD) as it specifically damages the nigrostriatal dopaminergic pathway. Recent studies in mice have, however, provided evidence that MPTP also compromises the integrity of the brain's vasculature. Photobiomodulation (PBM), the irradiation of tissue with low-intensity red light, mitigates MPTP-induced loss of dopaminergic neurons in the midbrain, but whether PBM also mitigates MPTP-induced damage to the cerebrovasculature has not been investigated. This study aimed to characterize the time course of cerebrovascular disruption following MPTP exposure and to determine whether PBM can mitigate this disruption. Young adult male C57BL/6 mice were injected with 80 mg/kg MPTP or isotonic saline and perfused with fluorescein isothiocyanate FITC-labelled albumin at various time points post-injection. By 7 days post-injection, there was substantial and significant leakage of FITC-labelled albumin into both the substantia nigra pars compacta (SNc; p < 0.0001) and the caudate-putamen complex (CPu; p ≤ 0.0003); this leakage partly subsided by 14 days post-injection. Mice that were injected with MPTP and treated with daily transcranial PBM (670 nm, 50 mW/cm2, 3 min/day), commencing 24 hours after MPTP injection, showed significantly less leakage of FITC-labelled albumin in both the SNc (p < 0.0001) and CPu (p = 0.0003) than sham-treated MPTP mice, with levels of leakage that were not significantly different from saline-injected controls. In summary, this study confirms that MPTP damages the brain's vasculature, delineates the time course of leakage induced by MPTP out to 14 days post-injection, and provides the first direct evidence that PBM can mitigate this leakage. These findings provide new understanding of the use of the MPTP mouse model as an experimental tool and highlight the potential of PBM as a therapeutic tool for reducing vascular dysfunction in neurological conditions.

Serum and Serum Albumin Inhibit in vitro Formation of Neutrophil Extracellular Traps (NETs).

The formation of neutrophil extracellular traps (NETs) is an immune defense mechanism of neutrophilic granulocytes. Moreover, it is also involved in the pathogenesis of autoimmune, inflammatory, and neoplastic diseases. For that reason, the process of NET formation (NETosis) is subject of intense ongoing research. In vitro approaches to quantify NET formation are commonly used and involve neutrophil stimulation with various activators such as phorbol 12-myristate 13-acetate (PMA), lipopolysaccharides (LPS), or calcium ionophores (CaI). However, the experimental conditions of these experiments, particularly the media and media supplements employed by different research groups, vary considerably, rendering comparisons of results difficult. Here, we present the first standardized investigation of the influence of different media supplements on NET formation in vitro. The addition of heat-inactivated (hi) fetal calf serum (FCS), 0.5% human serum albumin (HSA), or 0.5% bovine serum albumin (BSA) efficiently prevented NET formation of human neutrophils following stimulation with LPS and CaI, but not after stimulation with PMA. Thus, serum components such as HSA, BSA and hiFCS (at concentrations typically found in the literature) inhibit NET formation to different degrees, depending on the NETosis inducer used. In contrast, in murine neutrophils, NETosis was inhibited by FCS and BSA, regardless of the inducer employed. This shows that mouse and human neutrophils have different susceptibilities toward the inhibition of NETosis by albumin or serum components. Furthermore, we provide experimental evidence that albumin inhibits NETosis by scavenging activators such as LPS. We also put our results into the context of media supplements most commonly used in NET research. In experiments with human neutrophils, either FCS (0.5-10%), heat-inactivated (hiFCS, 0.1-10%) or human serum albumin (HSA, 0.05-2%) was commonly added to the medium. For murine neutrophils, serum-free medium was used in most cases for stimulation with LPS and CaI, reflecting the different sensitivities of human and murine neutrophils to media supplements. Thus, the choice of media supplements greatly determines the outcome of experiments on NET-formation, which must be taken into account in NETosis research.

Entering new areas in known fields: recombinant fusion protein linking recombinant factor VIIa with recombinant albumin (rVIIa-FP) - advancing the journey.

The novel fusion protein linking recombinant factor VIIa with recombinant albumin (rVIIa-FP) is designed to extend the half-life of recombinant factor VIIa (rFVIIa) and improve the care of hemophilia A or B patients with inhibitors. Preclinical studies in various animal models have demonstrated markedly improved pharmacokinetic and pharmacodynamic properties, as well as prolonged retention in the joint tissues, of rVIIa-FP compared with a commercially available rFVIIa (NovoSeven®). A phase I study in healthy volunteers - the first study in the PROLONG-7FP program - confirmed that rVIIa-FP has a good tolerability profile in doses of up to 1,000µg/kg and has demonstrated enhanced pharmacodynamic activity relative to rFVIIa. The half-life of rVIIa-FP at the highest dose investigated in the study was 8.5hours, which represents a 3- to 4-fold half-life extension compared with rFVIIa. Encouraging results from preclinical and phase I studies have led to the initiation of clinical studies of rVIIa-FP in patients with congenital hemophilia A or B and inhibitors, and in patients with confirmed factor VII deficiency. The results from these studies are awaited with interest by clinicians and patients alike.

The Coagulation Factor XIIa Inhibitor rHA-Infestin-4 Improves Outcome after Cerebral Ischemia/Reperfusion Injury in Rats.

BACKGROUND AND PURPOSE: Ischemic stroke provokes severe brain damage and remains a predominant disease in industrialized countries. The coagulation factor XII (FXII)-driven contact activation system plays a central, but not yet fully defined pathogenic role in stroke development. Here, we investigated the efficacy of the FXIIa inhibitor rHA-Infestin-4 in a rat model of ischemic stroke using both a prophylactic and a therapeutic approach. METHODS: For prophylactic treatment, animals were treated intravenously with 100 mg/kg rHA-Infestin-4 or an equal volume of saline 15 min prior to transient middle cerebral artery occlusion (tMCAO) of 90 min. For therapeutic treatment, 100 mg/kg rHA-Infestin-4, or an equal volume of saline, was administered directly after the start of reperfusion. At 24 h after tMCAO, rats were tested for neurological deficits and blood was drawn for coagulation assays. Finally, brains were removed and analyzed for infarct area and edema formation. RESULTS: Within prophylactic rHA-Infestin-4 treatment, infarct areas and brain edema formation were reduced accompanied by better neurological scores and survival compared to controls. Following therapeutic treatment, neurological outcome and survival were still improved although overall effects were less pronounced compared to prophylaxis. CONCLUSIONS: With regard to the central role of the FXII-driven contact activation system in ischemic stroke, inhibition of FXIIa may represent a new and promising treatment approach to prevent cerebral ischemia/reperfusion injury.

Protein-based photothermal theranostics for imaging-guided cancer therapy.

The development of imageable photothermal theranostics has attracted considerable attention for imaging guided photothermal therapy (PTT) with high tumor ablation accuracy. In this study, we strategically constructed a near-infrared (NIR) cyanine dye by introducing a rigid cyclohexenyl ring to the heptamethine chain to obtain a heptamethine dye CySCOOH with high fluorescence intensity and good stability. By covalent conjugation of CySCOOH onto human serum albumin (HSA), the as-prepared HSA@CySCOOH nanoplatform is highly efficient for NIR fluorescence/photoacoustic/thermal multimodality imaging and photothermal tumor ablation. The theranostic capability of HSA@CySCOOH was systematically evaluated both in vitro and in vivo. Most intriguingly, complete tumor elimination was achieved by intravenous injection of HSA@CySCOOH (CySCOOH, 1 mg kg(-1); 808 nm, 1.0 W cm(-2) for 5 min) into 4T1 tumor-bearing mice, with no weight loss, noticeable toxicity, or tumor recurrence being observed. This as-prepared protein-based nanotheranostics exhibits high water dispersibility, no off target cytotoxicity, and good biodegradability and biocompatibility, thus facilitating its clinical translation to cancer photothermal theranostics.

Composition of protein supplements used for human embryo culture.

PURPOSE: To determine the composition of commercially available protein supplements for embryo culture media and test if differences in protein supplement composition are biologically relevant in a murine model. METHODS: Amino acid, organic acid, ion and metal content were determined for 6 protein supplements: recombinant human albumin (AlbIX), human serum albumin (HSA and Buminate), and three complex protein supplements (SSS, SPS, LGPS). To determine if differences in the composition of these supplements are biologically relevant, mouse one-cell embryos were collected and cultured for 120 hours in each protein supplement in Global media at 5 and 20 % oxygen in an EmbryoScope time-lapse incubator. The compositions of six protein supplements were analyzed for concentrations of 39 individual amino acids, organic acids, ions and elements. Blastocyst development and cell cycle timings were calculated at 96-hours of culture and the experiments were repeated in triplicate. Blastocyst gene expression was analyzed. RESULTS: Recombinant albumin had the fewest undefined components , the lowest concentration of elements detected, and resulted in high blastocyst development in both 5 and 20 % oxygen. Buminate, LGPS and SPS had high levels of transition metals whereas SSS had high concentrations of amino acids. Pre-compaction mouse embryo development was delayed relative to embryos in AlbIX for all supplements and blastocyst formation was reduced in Buminate, SPS and SSS. CONCLUSIONS: The composition of protein supplements are variable, consisting of previously undescribed components. High concentrations of pro-oxidant transition metals were most notable. Blastocyst development was protein dependent and showed an interaction with oxygen concentration and pro-oxidant supplements.

Immunotoxicity assessment of rice-derived recombinant human serum albumin using human peripheral blood mononuclear cells.

Human serum albumin (HSA) is extensively used in clinics to treat a variety of diseases, such as hypoproteinemia, hemorrhagic shock, serious burn injuries, cirrhotic ascites and fetal erythroblastosis. To address supply shortages and high safety risks from limited human donors, we recently developed recombinant technology to produce HSA from rice endosperm. To assess the risk potential of HSA derived from Oryza sativa (OsrHSA) before a First-in-human (FIH) trial, we compared OsrHSA and plasma-derived HSA (pHSA), evaluating the potential for an immune reaction and toxicity using human peripheral blood mononuclear cells (PBMCs). The results indicated that neither OsrHSA nor pHSA stimulated T cell proliferation at 1x and 5x dosages. We also found no significant differences in the profiles of the CD4(+) and CD8(+) T cell subsets between OsrHSA- and pHSA-treated cells. Furthermore, the results showed that there were no significant differences between OsrHSA and pHSA in the production of cytokines such as interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin (IL)-10 and IL-4. Our results demonstrated that OsrHSA has equivalent immunotoxicity to pHSA when using the PBMC model. Moreover, this ex vivo system could provide an alternative approach to predict potential risks in novel biopharmaceutical development.

Salivary α-amylase, serum albumin, and myoglobin protect against DNA-damaging activities of ingested dietary agents in vitro.

Potent DNA-damaging activities were seen in vitro from dietary chemicals found in coffee, tea, and liquid smoke. A survey of tea varieties confirmed genotoxic activity to be widespread. Constituent pyrogallol-like polyphenols (PLPs) such as epigallocatechin-3-gallate (EGCG), pyrogallol, and gallic acid were proposed as a major source of DNA-damaging activities, inducing DNA double-strand breaks in the p53R assay, a well characterized assay sensitive to DNA strand breaks, and comet assay. Paradoxically, their consumption does not lead to the kind of widespread cellular toxicity and acute disease that might be expected from genotoxic exposure. Existing physiological mechanisms could limit DNA damage from dietary injurants. Serum albumin and salivary α-amylase are known to bind EGCG. Salivary α-amylase, serum albumin, and myoglobin, but not salivary proline-rich proteins, reduced damage from tea, coffee, and PLPs, but did not inhibit damage from the chemotherapeutics etoposide and camptothecin. This represents a novel function for saliva in addition to its known functions including protection against tannins. Cell populations administered repeated pyrogallol exposures had abatement of measured DNA damage by two weeks, indicating an innate cellular adaptation. We suggest that layers of physiological protections may exist toward natural dietary products to which animals have had high-level exposure over evolution.

In vitro activity of photoactivated disinfection using a diode laser in infected root canals.

OBJECTIVE: To investigate the lethal activity of photoactivated disinfection (PAD) on Enterococcus faecalis (ATCC 29212) and mixed populations of aerobic or anaerobic bacteria in infected root canals using a diode laser after the application of a photosensitizer (PS). MATERIALS AND METHODS: First, the bactericidal activity of a low power diode laser (200 mW) against E. faecalis ATCC 29212 pre-treated with a PS (toluidine blue) for 2 min were examined after different irradiation times (30 s, 60 s and 90 s). The bactericidal activity in the presence of human serum or human serum albumin (HSA) was also examined. Second, root canals were infected with E. faecalis or with mixed aerobic or anaerobic microbial populations for 3 days and then irrigated with 1.5% sodium hypochlorite and exposed to PAD for 60 s. RESULTS: Photosensitization followed by laser irradiation for 60 s was sufficient to kill E. faecalis. Bacteria suspended in human serum (25% v/v) were totally eradicated after 30 s of irradiation. The addition of HSA (25 mg/ml or 50 mg/ml) to bacterial suspensions increased the antimicrobial efficacy of PAD after an irradiation time of 30 s, but no longer. The bactericidal effect of sodium hypochlorite was only enhanced by PAD during the early stages of treatment. PAD did not enhance the activity of sodium hypochlorite against a mixture of anaerobic bacteria. CONCLUSIONS: The bactericidal activity of PAD appears to be enhanced by serum proteins in vitro, but is limited to bacteria present within the root canal.

Development of a neuromedin U-human serum albumin conjugate as a long-acting candidate for the treatment of obesity and diabetes. Comparison with the PEGylated peptide.

Neuromedin U (NMU) is an endogenous peptide implicated in the regulation of feeding, energy homeostasis, and glycemic control, which is being considered for the therapy of obesity and diabetes. A key liability of NMU as a therapeutic is its very short half-life in vivo. We show here that conjugation of NMU to human serum albumin (HSA) yields a compound with long circulatory half-life, which maintains full potency at both the peripheral and central NMU receptors. Initial attempts to conjugate NMU via the prevalent strategy of reacting a maleimide derivative of the peptide with the free thiol of Cys34 of HSA met with limited success, because the resulting conjugate was unstable in vivo. Use of a haloacetyl derivative of the peptide led instead to the formation of a metabolically stable conjugate. HSA-NMU displayed long-lasting, potent anorectic, and glucose-normalizing activity. When compared side by side with a previously described PEG conjugate, HSA-NMU proved superior on a molar basis. Collectively, our results reinforce the notion that NMU-based therapeutics are promising candidates for the treatment of obesity and diabetes.

FGF21 can be mimicked in vitro and in vivo by a novel anti-FGFR1c/ß-Klotho bispecific protein.

The endocrine hormone FGF21 has attracted considerable interest as a potential therapeutic for treating diabetes and obesity. As an alternative to the native cytokine, we generated bispecific Avimer polypeptides that bind with high affinity and specificity to one of the receptor and coreceptor pairs used by FGF21, FGFR1c and ß-Klotho. These Avimers exhibit FGF21-like activity in in vitro assays with potency greater than FGF21. In a study conducted in obese male cynomolgus monkeys, animals treated with an FGFR1c/ß-Klotho bispecific Avimer showed improved metabolic parameters and reduced body weight comparable to the effects seen with FGF21. These results not only demonstrate the essential roles of FGFR1c and ß-Klotho in mediating the metabolic effects of FGF21, they also describe a first bispecific activator of this unique receptor complex and provide validation for a novel therapeutic approach to target this potentially important pathway for treating diabetes and obesity.

Intracranial hyperthermia through local photothermal heating with a fiberoptic microneedle device.

BACKGROUND AND OBJECTIVES: The fiberoptic microneedle device (FMD) seeks to leverage advantages of both laser-induced thermal therapy (LITT) and convection-enhanced delivery (CED) to increase volumetric dispersal of locally infused chemotherapeutics through sub-lethal photothermal heat generation. This study focused on determination of photothermal damage thresholds with 1,064 nm light delivered through the FMD into in vivo rat models. MATERIALS AND METHODS: FMDs capable of co-delivering laser energy and fluid agents were fabricated through a novel off-center splicing technique involving fusion of a multimode fiberoptic to light-guiding capillary tubing. FMDs were positioned at a depth of 2.5 mm within the cerebrum of male rats with fluoroptic temperature probes placed within 1 mm of the FMD tip. Irradiation (without fluid infusion) was conducted at laser powers of 0 (sham), 100, 200, 500, or 750 mW. Evans blue-serum albumin conjugated complex solution (EBA) and laser energy co-delivery were performed in a second set of preliminary experiments. RESULTS: Maximum, steady-state temperatures of 38.7 ± 1.6 and 42.0 ± 0.9 °C were measured for the 100 and 200 mW experimental groups, respectively. Histological investigation demonstrated needle insertion damage alone for sham and 100 mW irradiations. Photothermal damage was detected at 200 mW, although observable thermal damage was limited to a small penumbra of cerebral cortical microcavitation and necrosis that immediately surrounded the region of FMD insertion. Co-delivery of EBA and laser energy presented increased volumetric dispersal relative to infusion-only controls. CONCLUSION: Fluoroptic temperature sensing and histopathological assessments demonstrated that a laser power of 100 mW results in sub-lethal brain hyperthermia, and the optimum, sub-lethal target energy range is likely 100-200 mW. The preliminary FMD-CED experiments confirmed the feasibility of augmenting fluid dispersal using slight photothermal heat generation, demonstrating the FMD's potential as a way to increase the efficacy of CED in treating MG.

The effect of tissue inhibitors on the antibacterial activity of chitosan nanoparticles and photodynamic therapy.

INTRODUCTION: Newer antibacterial alternatives such as chitosan nanoparticles (CSnps) and photodynamic therapy (PDT) have been investigated to achieve effective root canal disinfection. The current study aims to assess the effect of various tissue inhibitors such as dentin, dentin matrix, pulp tissue, bacterial lipopolysaccharides (LPSs), and bovine serum albumin (BSA) on the antibacterial activity of CSnps and PDT. METHODS: The antibacterial effect of CSnps and PDT using photosensitizers, rose bengal (RB), and methylene blue (MB) were tested on planktonic Enterococcus faecalis American Type Culture Collection 29212 with or without pretreatment using different tissue inhibitors for an hour. Bacterial survival was assessed after 1, 8, and 24 hours of incubation with CSnps and after PDT using RB and MB. RESULTS: Pulp and BSA inhibited the antibacterial effect of CSnps significantly (P < .05). The antibacterial effect of CSnps was not affected by dentin, dentin matrix, or LPSs. The antibacterial activity of PDT using MB and RB was inhibited in a decreasing order by dentin matrix, BSA, pulp, dentin, and LPSs (P < .05). The effect of tissue inhibitors was higher in the case of PDT with RB. Depending on the antibacterial mechanism of CSnps and PDT, different inhibitory patterns were observed with different tissue inhibitors. CONCLUSIONS: The tissue inhibitors existing within the root canal affected the antibacterial activity of CSnps and PDT at varying degrees. Further research is required to enhance their antimicrobial efficacy in an endodontic environment.

Neuroprotection for ischaemic stroke: translation from the bench to the bedside.

Neuroprotection seeks to restrict injury to the brain parenchyma following an ischaemic insult by preventing salvageable neurons from dying. The concept of neuroprotection has shown promise in experimental studies, but has failed to translate into clinical success. Many reasons exist for this including the heterogeneity of human stroke and the lack of methodological agreement between preclinical and clinical studies. Even with the proposed Stroke Therapy Academic Industry Roundtable criteria for preclinical development of neuroprotective agents for stroke, we have still seen limited success in the clinic, an example being NXY-059, which fulfilled nearly all the Stroke Therapy Academic Industry Roundtable criteria. There are currently a number of ongoing trials for neuroprotective strategies including hypothermia and albumin, but the outcome of these approaches remains to be seen. Combination therapies with thrombolysis also need to be fully investigated, as restoration of oxygen and glucose will always be the best therapy to protect against cell death from stroke. There are also a number of promising neuroprotectants in preclinical development including haematopoietic growth factors, and inhibitors of the nicotinamide adenine dinucleotide phosphate oxidases, a source of free radical production which is a key step in the pathophysiology of acute ischaemic stroke. For these neuroprotectants to succeed, essential quality standards need to be adhered to; however, these must remain realistic as the evidence that standardization of procedures improves translational success remains absent for stroke.

Hyaluronic acid viscosupplements from avian and non-mammalian sources exhibit biocompatibility profiles with unique, source-specific, antigenic profiles.

The objective of this study was to evaluate and compare the biocompatibility profiles of hyaluronic acid (HA) viscosupplements from avian and non-mammalian sources. Inflammatory and immune reactions were assessed in models of both clinically relevant and stringent immunological exposure conditions. Experiments were conducted to evaluate tissue reactions and immunological responses and assess antibody formation with the capacity to bind directly to and cross-react with the different viscosupplements. Mice were exposed to viscosupplements using the air pouch inflammation model and specific immunization using Freund's complete adjuvant (FCA). Murine pouch membrane tissue reactions revealed generally mild to moderate responses, with cellular infiltration and cytokine profiles of pouch tissue characteristic of predominantly fibroblastic responses rather than marked inflammatory reactions. In vitro testing indicated that pouch injections did not elicit detectable T-cell proliferative responses, while antibody assays revealed that mice immunized with viscosupplements in FCA and subsequently boosted were capable of mounting an antibody response with a range of specificities. High reactivity to avian serum albumin was seen in sera from mice injected with HA from an avian source, while low positive reactivity to the bacterial antigen Staphylococcal Lipotechoic Acid was observed in sera from mice injected with HA from bacterial sources. These specificities did not indicate any propensity to cross-react, suggesting that patients with adverse immune responses to HA from an avian source should be unresponsive to subsequent injection with HA from a non-avian source. Overall, the findings demonstrate that viscosupplements exhibit good biocompatibility profiles in the murine air pouch, but when provoked to elicit immunological reactions exhibit unique antigenic spectra. These findings suggest that an immunologically mediated immune reaction directed against avian proteins should not necessarily be a contraindication for the administration of non-avian viscosupplements.

Sonochemical proteinaceous microspheres for wound healing.

In this work, we report a novel approach using proteinaceous microspheres of bovine serum albumin (BSA), human serum albumin (HSA) and silk fibroin (SF) containing different organic solvents, namely n-dodecane, mineral oil and vegetable oil, to reduce the activity of human neutrophil elastase (HNE) found in high levels on chronic wounds. The ability of these devices to inhibit HNE was evaluated using porcine pancreatic elastase (PPE) solution as a model of wound exudates. The results obtained indicated that the level of PPE activity can be tuned by changing the organic solvent present on different protein microspheres, thus showing an innovative way of controlling the elastase-antielastase imbalance found in chronic wounds. Furthermore, these proteinaceous microspheres were shown to be important carriers of elastase inhibitors causing no cytotoxicity in human skin fibroblasts in vitro, making them suitable for biomedical applications, such as chronic wounds.

Immunization of beef heifers against gonadotropin-releasing hormone prevents luteal activity and pregnancy: Effect of conjugation to different proteins and effectiveness of adjuvants.

Three experiments were conducted to evaluate methods of immunization against GnRH on antibody titer, luteal activity, and pregnancy in beef heifers. Experiment 1 evaluated the efficacy of adjuvants with 30 heifers. Control heifers were immunized against human serum albumin (HSA) emulsified in Freund's complete adjuvant (FCA). The other 4 treatments contained GnRH conjugated to HSA (HSA-GnRH) emulsified in FCA, Freund's incomplete adjuvant (FIA), DEAE dextran (DD) + mineral oil (MO), or DD+FIA. Treatment was in the mammary gland for all experiments. Titers against GnRH for heifers immunized against HSA-GnRH with FCA, DD+MO, or DD+FIA were greater than titers for HSA-GnRH with FIA or control heifers (P < 0.01). Body weight was reduced (P < 0.05) in control and FCA heifers compared with FIA, DD+MO, and DD+FIA heifers. Heifers immunized with DD+MO and DD+FIA had fewer granulomas in mammary glands than heifers treated with FCA (P < 0.01). In Exp. 2, 36 heifers were used to determine the effect of the protein conjugated to GnRH on titers against GnRH. Heifers (6/treatment) received a primary immunization against GnRH conjugated to HSA (HSA-GnRH), ovalbumin (OA-GnRH), or keyhole limpet hemocyanin (KL-GnRH), or heifers were immunized against each carrier protein. Antigens were emulsified in DD+FIA. Immunization of heifers against OA-GnRH, KL-GnRH, or HSA-GnRH suppressed luteal activity (P < 0.01) for 23, 16, and 12 wk, respectively, and antibody titers against GnRH were greater (P < 0.01) for 19, 5, and 7 wk, respectively, compared with heifers immunized against the carrier proteins. In Exp. 3, 90 heifers were used to determine the effect of immunization against GnRH on ovarian activity and pregnancy rate. Heifers (30/treatment) received a primary and 2 or 3 booster immunizations against GnRH conjugated to OA, and controls received a primary and 2 booster immunizations against OA. All antigens were emulsified in DD+FIA. At 8 wk after primary immunization, heifers were exposed to fertile bulls for 24 wk. Pregnancy rate was less (P < 0.01) for 3-booster heifers (13%) compared with control (83%) and 2-booster (62%) heifers. We conclude that immunization against GnRH, conjugated to OA and emulsified in DD+FIA, does not influence ADG and produces sufficient titers against GnRH to prevent estrous cycles with few mammary granulomas. Immunization against GnRH with 3 booster immunizations prevented luteal activity and pregnancy in most beef heifers for more than 4 mo.

Comparison of haemolytic activity of tentacle-only extract from jellyfish Cyanea capillata in diluted whole blood and erythrocyte suspension: diluted whole blood is a valid test system for haemolysis study.

In this paper, we utilized two different test systems to compare the haemolysis of tentacle-only extract (TOE) devoid of nematocysts from jellyfish Cyanea capillata, the 1% whole blood and 0.45% erythrocyte suspension approximately with the same erythrocyte concentration from the blood samples of sheep, rabbit, mouse, rat and human, respectively. Without exception, the haemolytic activity of TOE was dose-dependent in both test systems from all the five kinds of blood samples, while it was generally stronger in erythrocyte suspension than that in diluted whole blood at the relatively high concentration of TOE. When various aliquots of plasma were added into the erythrocyte suspension test system, the haemolytic activity of TOE was declined with the plasma quantity increasing, and dropped to about 20% at the presence of two aliquots of plasma. If serum albumin of 0.5 mg/ml, approximately the same albumin content in 1% whole blood, was added into the erythrocyte suspension test system instead, the haemolysis of TOE was similarly inhibited. The effects of GSH, ascorbic acid and protease inhibitor on the haemolytic activity of TOE were detected in the erythrocyte suspension and diluted whole blood simultaneously, and the test results were coincident between the two systems. These results suggested that the inconsistency of TOE haemolysis between the erythrocyte suspension and diluted whole blood is a universal occurrence in the mammals, and blood plasma plays a dose-dependent protective role against haemolysis which may be due to serum albumin. Diluted whole blood is a valid and convenient test system for haemolysis study in vitro.