Subzero Nonfreezing Hypothermia with Insect Antifreeze Protein Dramatically Improves Survival Rate of Mammalian Cells.

Cells for therapeutic use are often preserved at +4 °C, and the storage period is generally limited to 2-3 days. Here, we report that the survival rate (%) of mammalian cells is improved to 10-20 days when they are preserved with a subzero supercooled solution containing the antifreeze protein (AFP), for which an ability to stabilize both supercooled water and cell membrane integrity has been postulated. We chose adherent rat insulinoma (RIN-5F) cells as the preservation target, which were immersed into -5 °C-, -2 °C-, or +4 °C-chilled "unfrozen" solution of Euro-Collins or University of Washington (UW) containing the AFP sample obtained from insect or fish. Our results show that the survival rate of the cells preserved with the solution containing insect AFP was always higher than that of the fish AFP solution. A combination of the -5 °C-supercooling and insect AFP gave the best preservation result, namely, UW solution containing insect AFP kept 53% of the cells alive, even after 20 days of preservation at -5 °C. The insect AFP locates highly organized ice-like waters on its molecular surface. Such waters may bind to semiclathrate waters constructing both embryonic ice crystals and a membrane-water interface in the supercooled solution, thereby protecting the cells from damage due to chilling.

Naturally-derived protein extract from Gryllus bimaculatus improves antioxidant properties and promotes osteogenic differentiation of hBMSCs.

Naturally-derived proteins or peptides are promising biopolymers for tissue engineering applications owing to their health-promoting activity. Herein, we extracted proteins (~90%) from two-spotted cricket (Gryllus bimaculatus) and evaluated their osteoinductive potential in human bone marrow-derived mesenchymal stem cells (hBMSCs) under in vitro conditions. The extracted protein isolate was analyzed for the amino acid composition and the mass distribution of the constituent peptide fraction. Fourier transform infrared (FTIR) spectroscopy was used to determine the presence of biologically significant functional groups. The cricket protein isolate (CPI) exhibited characteristic protein peaks in the FTIR spectrum. Notably, an enhanced cell viability was observed in the presence of the extracted proteins, showing their biocompatibility. The CPI also exhibited antioxidant properties in a concentration-dependent manner. More significant mineralization was observed in the CPI-treated cells than in the control, suggesting their osteoinductive potential. The upregulation of the osteogenic marker genes (Runx2, ALP, OCN, and BSP) in CPI treated media compared with the control supports their osteoinductive nature. Therefore, cricket-derived protein isolates could be used as functional protein isolate for tissue engineering applications, especially for bone regeneration.

Alternative Protein and Iron Sources from Edible Insects but Not Solanum torvum Improved Body Composition and Iron Status in Malnourished Rats.

Solanum torvum (STO) and edible insects are potential dietary approaches to prevent malnutrition. Hence, we determined the effect of STO and insect powders on improving nutritional status in malnourished rats. Malnutrition was induced in rats by feeding 5% protein, ~2 ppm Fe (LPI) diet for 21 days. During the 14 day repletion, five groups of rats (n = 8) were fed diets supplemented with Acheta domesticus (cricket, ADO), Rhynchophorus phoenicis fabricius (palm weevil larvae, RFA), STO, ADO + STO (TAD), and casein + ferrous sulfate (PIS, positive control), as well as a non-supplemented group (negative control, LPI). A normal (NOM) group was fed protein-Fe sufficient (PIS) diet throughout the study. Body composition was measured by Dual-energy X-ray absorptiometry. The hemoglobin (Hb) repletion method was used to assess relative biological value (RBV, compared to PIS) of the supplemented groups. No differences were found in weight gain, bone mineral content, lean and fat mass, and organ weights among the edible insects and PIS groups, but these results differed from STO and the LPI groups. An increase in Hb Fe and RBV with ADO and RFA was comparable to PIS. ADO and RFA could be excellent sources of protein and bioavailable Fe, making it a sustainable, low-cost food source to prevent malnutrition in humans.

Antiepileptic Effects of Protein-Rich Extract from Bombyx batryticatus on Mice and Its Protective Effects against H2O2-Induced Oxidative Damage in PC12 Cells via Regulating PI3K/Akt Signaling Pathways.

Bombyx batryticatus is a known traditional Chinese medicine (TCM) utilized to treat convulsions, epilepsy, cough, asthma, headaches, and purpura in China for thousands of years. This study is aimed at investigating the antiepileptic effects of protein-rich extracts from Bombyx batryticatus (BBPs) on seizure in mice and exploring the protective effects of BBPs against H2O2-induced oxidative stress in PC12 cells and their underlying mechanisms. Maximal electroshock-induced seizure (MES) and pentylenetetrazole- (PTZ-) induced seizure in mice and the histological analysis were carried out to evaluate the antiepileptic effects of BBPs. The cell viability of PC12 cells stimulated by H2O2 was determined by MTT assay. The apoptosis and ROS levels of H2O2-stimulated PC12 cells were determined by flow cytometry analysis. Furthermore, the levels of malondialdehyde (MDA), superoxide dismutase (SOD), lactate dehydrogenase (LDH), and glutathione (GSH) in PC12 cells were assayed by ELISA and expressions of caspase-3, caspase-9, Bax, Bcl-2, PI3K, Akt, and p-Akt were evaluated by Western blotting and quantitative real-time polymerase chain reaction (RT-qPCR) assays. The results revealed that BBPs exerted significant antiepileptic effects on mice. In addition, BBPs increased the cell viability of H2O2-stimulated PC12 cells and reduced apoptotic cells and ROS levels in H2O2-stimulated PC12 cells. By BBPs treatments, the levels of MDA and LDH were reduced and the levels of SOD and GSH-Px were increased in H2O2-stimulated PC12 cells. Moreover, BBPs upregulated the expressions of PI3K, Akt, p-Akt, and Bcl-2, whereas they downregulated the expressions of caspase-9, caspase-3, and Bax in H2O2-stimulated PC12 cells. These findings suggested that BBPs possessed potential antiepileptic effects on MES and PTZ-induced seizure in mice and protective effects on H2O2-induced oxidative stress in PC12 cells by exerting antioxidative and antiapoptotic effects via PI3K/Akt signaling pathways.

Lung-Infiltrating Foxp3+ Regulatory T Cells Are Quantitatively and Qualitatively Different during Eosinophilic and Neutrophilic Allergic Airway Inflammation but Essential To Control the Inflammation.

Understanding functions of Foxp3+ regulatory T cells (Tregs) during allergic airway inflammation remains incomplete. In this study, we report that, during cockroach Ag-induced allergic airway inflammation, Foxp3+ Tregs are rapidly mobilized into the inflamed lung tissues. However, the level of Treg accumulation in the lung was different depending on the type of inflammation. During eosinophilic airway inflammation, ∼30% of lung-infiltrating CD4 T cells express Foxp3, indicative of Tregs. On the contrary, only ∼10% of infiltrating CD4 T cells express Foxp3 during neutrophilic airway inflammation. Despite the different accumulation, the lung inflammation and inflammatory T cell responses were aggravated following Treg depletion, regardless of the type of inflammation, suggesting regulatory roles for Tregs. Interestingly, however, the extent to which inflammatory responses are aggravated by Treg depletion was significantly greater during eosinophilic airway inflammation. Indeed, lung-infiltrating Tregs exhibit phenotypic and functional features associated with potent suppression. Our results demonstrate that Tregs are essential regulators of inflammation, regardless of the type of inflammation, although the mechanisms used by Tregs to control inflammation may be shaped by environmental cues available to them.

Immunization against full-length protein and peptides from the Lutzomyia longipalpis sand fly salivary component maxadilan protects against Leishmania major infection in a murine model.

Leishmaniasis is an arthropod vectored disease causing considerable human morbidity and mortality. Vaccination remains the most realistic and practical means to interrupt the growing number and diversity of sand fly vectors and reservoirs of Leishmania. Since transmission of Leishmania is achieved exclusively by sand fly vectors via immune-modulating salivary substances, conventional vaccination requiring an unmodified host immune response for success are potentially destined to fail unless immunomodulatory factors are somehow neutralized. Using cationic liposome DNA complexes (CLDC) as an adjuvant system along with Lu. longipalpis sand fly salivary component maxadilan (MAX) as antigen (Ag), we show that mice are protected from the MAX-induced exacerbation of infection with Leishmania major (Lm). The CLDC adjuvant and alum were comparable in terms of lesion induration and decreased parasite burden, however the alum adjuvant imposed more inflammation at the injection site. BALB/c, C3H and C57BL/6 mice vaccinated with MAX-CLDC containing either the full-length MAX or peptides spanning the N- and C-terminal regions of MAX are protected against footpad challenges with Lm co-injected with MAX. When compared to unvaccinated controls, all strains of mice immunized with CLDC containing either peptides encompassing the first 20 N-terminal AA or those spanning the last 15 AA of the C-terminal domain of MAX demonstrated decreased parasite burden after 9 or 18 weeks post challenge with Lm + MAX. MAX-CLDC immunized mice showed increased IFNγ-secreting and decreased IL-4-secreting CD4+ cells in footpad-draining lymph nodes. Antisera from C-terminal peptide (P11) MAX-CLDC-vaccinated animals was capable of recognizing FL-MAX and its C-terminal domain and also blocked MAX-mediated reprogramming of bone marrow-derived dendritic cells (BM-DC) in vitro. This peptide vaccine targeting sand fly MAX, improves host immunity against MAX-mediated immunomodulation.

Insulin-like peptides regulate vitellogenesis and oviposition in the green lacewing, Chrysopa septempunctata.

Insulin-like peptides (ILPs) act through a conserved insulin signaling pathway and play crucial roles in insect metabolism, growth, reproduction, and aging. Application of bovine insulin is able to increase vitellogenin (Vg) mRNA and protein levels in female insects. Here, we first show that injection of bovine insulin into previtellogenic Chrysopa septempunctata female adults promoted ovarian growth, increased Vg protein abundance, elevated reproductive performance, and enhanced protease activity. These data suggested that ILPs play crucial roles in reproductive regulation of the green lacewing, C. septempunctata.

The Protective Role of PAC1-Receptor Agonist Maxadilan in BCCAO-Induced Retinal Degeneration.

A number of studies have proven that pituitary adenylate cyclase activating polypeptide (PACAP) is protective in neurodegenerative diseases. Permanent bilateral common carotid artery occlusion (BCCAO) causes severe degeneration in the rat retina. In our previous studies, protective effects were observed with PACAP1-38, PACAP1-27, and VIP but not with their related peptides, glucagon, or secretin in BCCAO. All three PACAP receptors (PAC1, VPAC1, VPAC2) appear in the retina. Molecular and immunohistochemical analysis demonstrated that the retinoprotective effects are most probably mainly mediated by the PAC1 receptor. The aim of the present study was to investigate the retinoprotective effects of a selective PAC1-receptor agonist maxadilan in BCCAO-induced retinopathy. Wistar rats were used in the experiment. After performing BCCAO, the right eye was treated with intravitreal maxadilan (0.1 or 1 µM), while the left eye was injected with vehicle. Sham-operated rats received the same treatment. Two weeks after the operation, retinas were processed for standard morphometric and molecular analysis. Intravitreal injection of 0.1 or 1 µM maxadilan caused significant protection in the thickness of most retinal layers and the number of cells in the GCL compared to the BCCAO-operated eyes. In addition, 1 µM maxadilan application was more effective than 0.1 µM maxadilan treatment in the ONL, INL, IPL, and the entire retina (OLM-ILM). Maxadilan treatment significantly decreased cytokine expression (CINC-1, IL-1α, and L-selectin) in ischemia. In summary, our histological and molecular analysis showed that maxadilan, a selective PAC1 receptor agonist, has a protective role in BCCAO-induced retinal degeneration, further supporting the role of PAC1 receptor conveying the retinoprotective effects of PACAP.

Resilin-PEG Hybrid Hydrogels Yield Degradable Elastomeric Scaffolds with Heterogeneous Microstructure.

Hydrogels derived from resilin-like polypeptides (RLPs) have shown outstanding mechanical resilience and cytocompatibility; expanding the versatility of RLP-based materials via conjugation with other polypeptides and polymers would offer great promise in the design of a range of materials. Here, we present an investigation of the biochemical and mechanical properties of hybrid hydrogels composed of a recombinant RLP and a multiarm PEG macromer. These hybrid hydrogels can be rapidly cross-linked through a Michael-type addition reaction between the thiols of cysteine residues on the RLP and vinyl sulfone groups on the multiarm PEG. Oscillatory rheology and tensile testing confirmed the formation of elastomeric hydrogels with mechanical resilience comparable to aortic elastin; hydrogel stiffness was easily modulated through the cross-linking ratio. Macromolecular phase separation of the RLP-PEG hydrogels offers the unique advantage of imparting a heterogeneous microstructure, which can be used to localize cells, through simple mixing and cross-linking. Assessment of degradation of the RLP by matrix metalloproteinases (MMPs) illustrated the specific proteolysis of the polypeptide in both its soluble form and when cross-linked into hydrogels. Finally, the successful encapsulation and viable three-dimensional culture of human mesenchymal stem cells (hMSCs) demonstrated the cytocompatibility of the RLP-PEG gels. Overall, the cytocompatibility, elastomeric mechanical properties, microheterogeneity, and degradability of the RLP-PEG hybrid hydrogels offer a suite of promising properties for the development of cell-instructive, structured tissue engineering scaffolds.

Aryl Hydrocarbon Receptor Protects Lungs from Cockroach Allergen-Induced Inflammation by Modulating Mesenchymal Stem Cells.

Exposure to cockroach allergen leads to allergic sensitization and increased risk of developing asthma. Aryl hydrocarbon receptor (AhR), a receptor for many common environmental contaminants, can sense not only environmental pollutants but also microbial insults. Mesenchymal stem cells (MSCs) are multipotent progenitor cells with the capacity to modulate immune responses. In this study, we investigated whether AhR can sense cockroach allergens and modulate allergen-induced lung inflammation through MSCs. We found that cockroach allergen-treated AhR-deficient (AhR(-/-)) mice showed exacerbation of lung inflammation when compared with wild-type (WT) mice. In contrast, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an AhR agonist, significantly suppressed allergen-induced mouse lung inflammation. MSCs were significantly reduced in cockroach allergen-challenged AhR(-/-) mice as compared with WT mice, but increased in cockroach allergen-challenged WT mice when treated with TCDD. Moreover, MSCs express AhR, and AhR signaling can be activated by cockroach allergen with increased expression of its downstream genes cyp1a1 and cyp1b1. Furthermore, we tracked the migration of i.v.-injected GFP(+) MSCs and found that cockroach allergen-challenged AhR(-/-) mice displayed less migration of MSCs to the lungs compared with WT. The AhR-mediated MSC migration was further verified by an in vitro Transwell migration assay. Epithelial conditioned medium prepared from cockroach extract-challenged epithelial cells significantly induced MSC migration, which was further enhanced by TCDD. The administration of MSCs significantly attenuated cockroach allergen-induced inflammation, which was abolished by TGF-ß1-neutralizing Ab. These results suggest that AhR plays an important role in protecting lungs from allergen-induced inflammation by modulating MSC recruitment and their immune-suppressive activity.

Neuropeptidergic input pathways to the circadian pacemaker center of the Madeira cockroach analysed with an improved injection technique.

Light entrainment pathways synchronize the circadian clock of almost all species of the animal and plant kingdom to the daily light dark cycle. In the Madeira cockroach Rhyparobia (Leucophaea) maderae, the circadian clock is located in the accessory medulla of the brain's optic lobes. The clock has abundant neuropeptides with unknown functions. Previous studies suggested that myoinhibitory peptides (MIPs), orcokinins (ORCs), and allatotropin (AT) take part in light input pathways to the circadian clock. As the sequences of AT and ORCs of R. maderae have not yet been determined, with matrix-assisted laser desorption/ionization-time of flight mass spectrometry, the respective Rhyparobia peptides were characterized. To search for light-like phase-shifting inputs to the circadian clock, Rhyparobia-MIP-1, Rhyparobia-AT, and Rhyparobia-ORC were injected at different circadian times, combined with locomotor activity assays. An improved, less invasive injection method was developed that allowed for the analysis of peptide effects within <2 weeks after injection. Rhyparobia-MIP-1 and Rhyparobia-AT injections resulted in dose-dependent monophasic phase response curves with maximum delays at the beginning of the subjective night, similar to light-dependent phase delays. In contrast to Manduca sexta-AT, Rhyparobia-AT did not phase advance locomotor activity rhythms. Only injections of Rhyparobia-ORCs resulted in a biphasic light-like phase response curve. Thus, it is hypothesized that Rhyparobia-MIP-1 and -AT are candidates for relaying light-dependent delays and/or non-photic inputs to the clock, whereas Rhyparobia-ORCs might be part of the light-entrainment pathways relaying phase delays and advances to the circadian clock of the Madeira cockroach.

Immunization with Culex tarsalis mosquito salivary gland extract modulates West Nile virus infection and disease in mice.

Mosquito salivary proteins inoculated during blood feeding modulate the host immune response, which can contribute to the pathogenesis of viruses transmitted by mosquito bites. Previous studies with mosquito bite-naïve mice indicated that exposure to arthropod salivary proteins resulted in a shift toward a Th2-type immune response in flavivirus-susceptible mice but not flavivirus-resistant animals. In the study presented here, we tested the hypothesis that immunization with high doses of Culex tarsalis salivary gland extracts (SGE) with an adjuvant would prevent Th2 polarization after mosquito bite and enhance resistance to mosquito-transmitted West Nile virus (WNV). Our results indicate that mice immunized with Cx. tarsalis SGE produced increased levels of Th1-type cytokines (IFNγ and TNFα) after challenge with mosquito-transmitted WNV and exhibited both a delay in infection of the central nervous system (CNS) and significantly lower WNV brain titers compared to mock-immunized mice. Moreover, mortality was significantly reduced in the SGE-immunized mice, as none of these mice died, compared to mortality of 37.5% of mock-vaccinated mice by 8 days after infected mosquito bite. These results suggest that development of a mosquito salivary protein vaccine might be a strategy to control arthropod-borne viral pathogens such as WNV.

Effects of the antimicrobial peptide cecropin AD on performance and intestinal health in weaned piglets challenged with Escherichia coli.

This study was conducted to determine the effects of the antimicrobial peptide cecropin on performance and intestinal health in piglets. Newly weaned barrows were randomly assigned to one of three treatments (n=8), including a corn-soybean basal diet or similar diets supplemented with antibiotics (100 mg/kg kitasamycin plus 800 mg/kg colistin sulfate) or 400 mg/kg cecropin AD. On day 13, all piglets were orally challenged with 10(9)CFU/mL of Escherichia coli K88. On day 19, all piglets were euthanized and sampled. Before challenge, piglets fed antibiotics had greater weight gain, feed efficiency, nitrogen and energy retention than the control (P<0.05). E. coli challenge decreased weight gain, feed intake and feed efficiency for the control piglets (P<0.05) but not for the antibiotic or cecropin AD treated piglets. The incidence of diarrhea post-challenge in the antibiotic and cecropin AD treatments decreased compared with the control piglets. The total viable counts of cecal E. coli were lower while the Lactobacilli counts were higher in the antibiotic and cecropin AD treatments compared with the control (P<0.05). Cecropin AD treatment decreased total aerobes while increasing total anaerobes in the ileum (P<0.05). A higher villus height to crypt depth ratio in the jejunum and ileum as well as a deeper crypt depth in the jejunum and higher villus height in the ileum were observed in piglets fed antibiotics or cecropin AD compared with control piglets (P<0.05). Piglets fed the control diet had lower levels of secretory IgA in their jejunum and lower serum IgA, IgG, interleukin-1ß and interleukin-6 compared with the other treatments (P<0.05). Overall, these data suggest that cecropin AD enhances pig performance through increasing immune status and nitrogen and energy retention as well as reducing intestinal pathogens in weaned piglets.

In situ imaging of honeybee (Apis mellifera) venom components from aqueous and aluminum hydroxide-adsorbed venom immunotherapy preparations.

BACKGROUND: Treatment with aqueous and aluminum hydroxide (Al[OH](3))-adsorbed purified honeybee (Apis mellifera) venom (HBV) preparations can reduce the incidence of side effects associated with venom immunotherapy. OBJECTIVE: The aim of the present study was to assess these purified HBV immunotherapy preparations in situ. METHODS: Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was used to visualize the distribution of HBV components. The preparations were administered on the back legs of naive Wistar rats. The rats were killed, and cryosectioned tissue sections were subjected to hematoxylin and eosin staining and MALDI-MSI analyses. RESULTS: Low-density maps of tissue distribution of HBV peptides, such as secapin, mast cell degranulating peptide, and melittin (Api m 4) were detected in the tissue after administration of HBV immunotherapy preparations. In addition, release of biogenic amines, cytokines, and leukotrienes was observed, and the distribution of HBV allergens, such as Api m 1 and Api m 2, was shown. At the 24-hour time point, the major HBV allergen Api m 1 was still detected at the site of Al(OH)(3)-adsorbed HVB injection, whereas in the case of aqueous HBV preparation, all the allergens, as well as most of the biogenic amines, were cleared at the 24-hour time point. CONCLUSION: The present study shows that the majority of low-molecular-weight HBV components are rapidly removed from the site of venom immunotherapy administration. Furthermore, Al(OH)(3)-adsorbed HBV preparation demonstrated a depot effect, prolonging the availability of bee venom allergens at the site of administration.

Mucosal sensitization to German cockroach involves protease-activated receptor-2.

BACKGROUND: Allergic asthma is on the rise in developed countries. A common characteristic of allergens is that they contain intrinsic protease activity, and many have been shown to activate protease-activated receptor (PAR)-2 in vitro. The role for PAR-2 in mediating allergic airway inflammation has not been assessed using a real world allergen. METHODS: Mice (wild type or PAR-2-deficient) were sensitized to German cockroach (GC) feces (frass) or protease-depleted GC frass by either mucosal exposure or intraperitoneal injection and measurements of airway inflammation (IL-5, IL-13, IL-17A, and IFNgamma levels in the lung, serum IgE levels, cellular infiltration, mucin production) and airway hyperresponsiveness were performed. RESULTS: Following systemic sensitization, GC frass increased airway hyperresponsiveness, Th2 cytokine release, serum IgE levels, cellular infiltration and mucin production in wild type mice. Interestingly, PAR-2-deficient mice had similar responses as wild type mice. Since these data were in direct contrast to our finding that mucosal sensitization with GC frass proteases regulated airway hyperresponsiveness and mucin production in BALB/c mice (Page et. al. 2007 Resp Res 8:91), we backcrossed the PAR-2-deficient mice into the BALB/c strain. Sensitization to GC frass could now occur via the more physiologically relevant method of intratracheal inhalation. PAR-2-deficient mice had significantly reduced airway hyperresponsiveness, Th2 and Th17 cytokine release, serum IgE levels, and cellular infiltration compared to wild type mice when sensitization to GC frass occurred through the mucosa. To confirm the importance of mucosal exposure, mice were systemically sensitized to GC frass or protease-depleted GC frass via intraperitoneal injection. We found that removal of proteases from GC frass had no effect on airway inflammation when administered systemically. CONCLUSIONS: We showed for the first time that allergen-derived proteases in GC frass elicit allergic airway inflammation via PAR-2, but only when allergen was administered through the mucosa. Importantly, our data suggest the importance of resident airway cells in the initiation of allergic airway disease, and could make allergen-derived proteases attractive therapeutic targets.

In vitro and in vivo evaluation of recombinant silk-elastinlike hydrogels for cancer gene therapy.

The objectives of this study were to evaluate: (i). the influences of hydrogel geometry, DNA molecular weight, and DNA conformation on DNA release from a silk-elastinlike protein polymer (SELP) hydrogel, (ii). the bioactivity and transfection efficiency of encapsulated DNA over time in vitro, (iii). the delivery and transfection of a reporter gene in a murine model of human breast cancer in vivo, and (iv). the in vitro release and bioactivity of adenovirus containing the green fluorescent protein (gfp) gene as a marker of gene transfer. Plasmid DNA was released from SELP hydrogels in a size-dependent manner, with the average effective diffusivity ranging from 1.70+/-0.52 x 10(-12) cm(2)/s for a larger plasmid (11 kbp) to 2.55+/-0.51 x 10(-10) cm(2)/s for a smaller plasmid (2.6 kbp). Plasmid conformation also influenced the rate of release, with the rank order linear>supercoiled>open-circular. DNA retained bioactivity in vitro, after encapsulation in a SELP hydrogel for up to 28 days. Delivery of pRL-CMV from a SELP hydrogel resulted in increased transfection in a murine model of human breast cancer by 1-3 orders of magnitude, as compared to naked DNA. The release of a bioactive adenoviral vector was related to the concentration of the polymer in the hydrogel. These studies indicate that genetically engineered SELP hydrogels have potential as matrices for controlled nonviral and viral gene delivery.

Cecropin B enhances betalactams activities in experimental rat models of gram-negative septic shock.

OBJECTIVE: To investigate the efficacy of imipenem, piperacillin combined with cecropin B in the prevention of lethality in 2 rat models of septic shock. Main outcome measures were bacterial growth in blood and intra-abdominal fluid, endotoxin and TNF-alpha concentrations in plasma, and lethality. BACKGROUND: Sepsis remains a serious clinical problem despite intense efforts to improve survival. It is a major cause of morbidity and mortality in hospitalized patients. The primary cause of Gram-negative shock results from activation of host effector cells by endotoxin, the lipopolysaccharide (LPS) associated with cell membranes of Gram-negative bacteria. METHODS: Adult male Wistar rats were given (1). an intraperitoneal injection of 1 mg of Escherichia coli 0111:B4 LPS or (2). 2 x 1010 CFU of E. coli ATCC 25922. All animals were randomized to receive intraperitoneally isotonic sodium chloride solution, 1 mg/kg cecropin B, 20 mg/kg imipenem, and 120 mg/kg piperacillin alone and combined with 1 mg/kg cecropin B. Each group included 20 animals. RESULTS All compounds reduced the lethality when compared with controls. Piperacillin and imipenem significantly reduced the lethality and the number of E. coli in abdominal fluid compared with saline treatment. On the other hand, each betalactam determined an increase of plasma endotoxin and TNF-alpha concentration. Combination between cecropin B and betalactams showed to be the most effective treatment in reducing all variables measured. CONCLUSION: Cecropin B enhances betalactams activities in Gram-negative sepic shock rat models.

Mosquito larvae proteins modulate luteinizing hormone and prolactin release in pituitary cells of normal and estrogenized rats.

Whilst looking for vertebrate growth factor homologues in insects, we found that a soluble fraction of a 12-80 kDa molecular weight band peaking at 25 kDa, isolated from mosquito larvae extracts by gel permeation chromatography, had a modulatory effect on mouse hepatocytes and adult human mononuclear cell proliferation. The effect disappeared after heating the extract at 90 degrees C for 30 min, suggesting that the active factor may be a protein. In order to determine the activity of the extract on cell function, we assessed the effect of the extract on pituitary hormone secretion in vitro. We assayed a dialyzed fraction (MW greater than 12 kDa) of mosquito larvae for its effect on the release of luteinizing hormone (LH) and prolactin (PRL) from dispersed rat pituitary cells. In normal anterior pituitary (AP) cells we found that the extract had a stimulatory effect on LH release but an inhibitory action on prolactin secretion. In AP cells obtained from estrogen-induced hyperplasia, the extract had an inhibitory effect on prolactin secretion. In all cases the effects were time- and dose-dependent. Interference of the mosquito proteins with the radioimmunoassay was checked and found to be negligible. After a 60 min incubation, cell viability was comparable in control and treated cells. Furthermore, the biological effect of the extract was thermally unstable. Our results suggest that mosquito larvae may share common factors with mammals, probably peptidic in nature, which are able to modulate cell function.

Prevention of cerebral vasospasm by vasodilatory peptide maxadilan following subarachnoid hemorrhage in rabbits.

Maxadilan is a vasodilatory peptide isolated from the blood-feeding sand fly Lutzomyia longipalpis. Its vasodilatory activity, estimated by the formation of erythema on rabbit skin, is greater than those of calcitonin gene-related peptide, vasoactive intestinal polypeptide and pituitary adenylyl cyclase activating polypeptide (PACAP). We have recently demonstrated that maxadilan is a specific agonist for the PACAP type I receptor, which is widely distributed in brain. Therefore, we were interested in the vasodilatory effect of maxadilan on cerebral arteries and the possibility of its clinical use for the delayed cerebral vasospasm following subarachnoid (SAH). In the first experiment, 10(-10) mol/kg of maxadilan (in sterile water) was injected into the cisterna magna three days after the induction of experimental SAH in rabbits (n = 6). Maxadilan dilated spastic basilar arteries within 30 min of the injection, but not at 6 h. In the second experiment, to prolong the vasodilatory effect of maxadilan, tablets containing stearic acid, hydrogenated oil, lactose, hydroxypropylcellulose and 15 mg of maxadilan were prepared. In vitro testing showed that 60% of maxadilan could be released slowly within the initial five days. In vivo experiments were performed to implant the maxadilan tablet (n = 7) and the placebo tablet (n = 6) into the cisterna magna after the induction of experimental SAH in rabbits. The spastic response of the basilar artery was maximum on day three in the placebo-treated groups. In contrast, we observed no significant change in the arterial diameter until day five in the rabbits treated with maxadilan tablet. These data suggest that maxadilan may have therapeutic potency in treating cerebral vasospasm.