VEGFA165 can rescue excess steroid secretion, inflammatory markers, and follicle arrest in the ovarian cortex of High A4 cows†.

A population of cows with excess androstenedione (A4; High A4) in follicular fluid, with follicular arrest, granulosa cell dysfunction, and a 17% reduction in calving rate was previously identified. We hypothesized that excess A4 in the ovarian microenvironment caused the follicular arrest in High A4 cows and that vascular endothelial growth factor A would rescue the High A4 phenotype. In trial 1, prior to culture, High A4 ovarian cortex (n = 9) had greater numbers of early stage follicles (primordial) and fewer later-stage follicles compared to controls (n = 11). Culture for 7 days did not relieve this follicular arrest; instead, High A4 ovarian cortex had increased indicators of inflammation, anti-Mullerian hormone, and A4 secretion compared to controls. In trial 2, we tested if vascular endothelial growth factor A isoforms could rescue the High A4 phenotype. High A4 (n = 5) and control (n = 5) ovarian cortex was cultured with (1) PBS, (2) VEGFA165 (50 ng/mL), (3) VEGFA165B (50 ng/mL), or (4) VEGFA165 + VEGFA165B (50 ng/mL each) for 7 days. Follicular progression increased with VEGFA165 in High A4 cows with greater early primary, primary, and secondary follicles than controls. Similar to trial 1, High A4 ovarian cortex secreted greater concentrations of A4 and other steroids and had greater indicators of inflammation compared to controls. However, VEGFA165 rescued steroidogenesis, oxidative stress, and fibrosis. The VEGFA165 and VEGFA165b both reduced IL-13, INFα, and INFß secretion in High A4 cows to control levels. Thus, VEGFA165 may be a potential therapeutic to restore the ovarian steroidogenic microenvironment and may promote folliculogenesis.

Dimerization of Dengue Virus E Subunits Impacts Antibody Function and Domain Focus.

Dengue virus (DENV) is responsible for the most prevalent and significant arthropod-borne viral infection of humans. The leading DENV vaccines are based on tetravalent live-attenuated virus platforms. In practice, it has been challenging to induce balanced and effective responses to each of the four DENV serotypes because of differences in the replication efficiency and immunogenicity of individual vaccine components. Unlike live vaccines, tetravalent DENV envelope (E) protein subunit vaccines are likely to stimulate balanced immune responses, because immunogenicity is replication independent. However, E protein subunit vaccines have historically performed poorly, in part because the antigens utilized were mainly monomers that did not display quaternary-structure epitopes found on E dimers and higher-order structures that form the viral envelope. In this study, we compared the immunogenicity of DENV2 E homodimers and DENV2 E monomers. The stabilized DENV2 homodimers, but not monomers, were efficiently recognized by virus-specific and flavivirus cross-reactive potently neutralizing antibodies that have been mapped to quaternary-structure epitopes displayed on the viral surface. In mice, the dimers stimulated 3-fold-higher levels of virus-specific neutralizing IgG that recognized epitopes different from those recognized by lower-level neutralizing antibodies induced by monomers. The dimer induced a stronger E domain I (EDI)- and EDII-targeted response, while the monomer antigens stimulated an EDIII epitope response and induced fusion loop epitope antibodies that are known to facilitate antibody-dependent enhancement (ADE). This study shows that DENV E subunit antigens that have been designed to mimic the structural organization of the viral surface are better vaccine antigens than E protein monomers.IMPORTANCE Dengue virus vaccine development is particularly challenging because vaccines have to provide protection against four different dengue virus stereotypes. The leading dengue virus vaccine candidates in clinical testing are all based on live-virus vaccine platforms and struggle to induce balanced immunity. Envelope subunit antigens have the potential to overcome these limitations but have historically performed poorly as vaccine antigens, because the versions tested previously were presented as monomers and not in their natural dimer configuration. This study shows that the authentic presentation of DENV2 E-based subunits has a strong impact on antibody responses, underscoring the importance of mimicking the complex protein structures that are found on DENV particle surfaces when designing subunit vaccines.

The effect of amyloid-ß peptide on synaptic plasticity and memory is influenced by different isoforms, concentrations, and aggregation status.

The increase of oligomeric amyloid-beta (oAß) has been related to synaptic dysfunction, thought to be the earliest event in Alzheimer's disease pathophysiology. Conversely, the suppression of endogenous Aß impaired synaptic plasticity and memory, suggesting that the peptide is needed in the healthy brain. However, different species, aggregation forms and concentrations of Aß might differently influence synaptic function/dysfunction. Here, we have tested the contribution of monomeric and oligomeric Aß42 and Aß40 at 200 nM and 200 pM concentrations on hippocampal long-term potentiation and spatial memory. We found that, when at 200 nM, oAß40, oAß42, and monomeric Aß42 impaired long-term potentiation and memory, whereas only oAß42 200 pM enhanced synaptic plasticity and memory and rescued the detrimental effect due to depletion of endogenous Aß. Interestingly, quantification of monomer-like and oligomer-like species carried out by transmission electron microscopy revealed an increase of the monomer/oligomer ratio in the oAß42 200 pM preparation, suggesting that the content of monomers and oligomers depends on the final concentration of the solution.

Phoenixin participated in regulation of food intake and growth in spotted scat, Scatophagus argus.

Phoenixin (Pnx) is an endogenous peptide known to be involved in reproduction and food intake in rats, with two active isoforms, phoenixin-14 (Pnx-14) and phoenixin-20 (Pnx-20). However, little is known about the functions of Pnx in teleost. Here, pnx was cloned and was detected in all tissues of both male and female in spotted scat (Scatophagus argus), including growth axis, hypothalamus, pituitary, and liver. Real-time PCR analysis showed that pnx in the hypothalamus increased significantly after 2 d and 7 d fasting, while reduced significantly after re-feeding (P < 0.05). When pituitary and liver fragments were cultured in vitro with Pnx-14 and Pnx-20 (10 nM and 100 nM) for 6 h, the expression of ghrhr (growth hormone-releasing hormone receptor) and gh (growth hormone) in the pituitary, and ghr1 (growth hormone receptor 1) in the liver increased significantly, except ghr2 (growth hormone receptor 2) incubated with 10 nM and 100 nM Pnx-20 and ghr1 incubated with 10 nM Pnx-20. Similarly, the expression of ghrhr and gh in the pituitary, as well as ghr1 and ghr2 in the liver, increased significantly after injecting S. argus with Pnx-14 and Pnx-20 (10 ng/g and 100 ng/g body weight). These results indicate that Pnx is likely to be involved in the regulation of food intake, and also regulates the growth of S. argus by increasing ghrhr and gh expression in the pituitary, ghr1 and ghr2 in the liver, and ghr1 directly in the liver.

Immunogenicity of a Staphylococcus aureus-cholera toxin A2/B vaccine for bovine mastitis.

Staphylococcus aureus causes a chronic, contagious disease of the udder, or mastitis, in dairy cows. This infection is often refractory to antibiotic treatment, and has a significant economic impact on milk production worldwide. An effective vaccine to prevent S. aureus mastitis would improve animal health, reduce antibiotic dependence and inform human vaccine approaches. The iron-regulated surface determinant A (IsdA) and clumping factor A (ClfA) are conserved S. aureus extracellular-matrix adhesins and target vaccine antigens. Here we report the results of two bovine immunogenicity trials using purified IsdA and ClfA-cholera toxin A2/B chimeras (IsdA-CTA2/B and ClfA-CTA2/B). Cows were intranasally inoculated with IsdA-CTA2/B + ClfA-CTA2/B at dry off and followed for 70 days. Trial 1 utilized three groups with one or two booster doses at a total concentration of 600 or 900 µg. Trial 2 utilized two groups with one booster at a total concentration of 1200 µg. Humoral immune responses in serum and milk were examined by ELISA. Responses in serum were significant between groups and provide evidence of antigen-specific IgG induction after vaccination in both trials. Cellular proliferation was detected by flow cytometry using antigen-stimulated PBMCs from day 60 of Trial 2 and revealed an increase in CD4+ T cells from vaccinated cows. IsdA and ClfA stimulation induced IL-4 expression, but not IFN-γ or IL-17, in PBMCs from day 60 as determined by cytokine expression analysis. Opsonophagocytosis of S. aureus confirmed the functional in vitro activity of anti-IsdA antibodies from Trial 2 serum and milk. The vaccine was well tolerated and safe, and results support the potential of mucosally-delivered CTA2/B chimeras to protect cows from mastitis caused by S. aureus.

Co-delivery of a laminin-111 supplemented hyaluronic acid based hydrogel with minced muscle graft in the treatment of volumetric muscle loss injury.

Minced muscle autografting mediates de novo myofiber regeneration and promotes partial recovery of neuromuscular strength after volumetric muscle loss injury (VML). A major limitation of this approach is the availability of sufficient donor tissue for the treatment of relatively large VMLs without inducing donor site morbidity. This study evaluated a laminin-111 supplemented hyaluronic acid based hydrogel (HA+LMN) as a putative myoconductive scaffolding to be co-delivered with minced muscle grafts. In a rat tibialis anterior muscle VML model, delivery of a reduced dose of minced muscle graft (50% of VML defect) within HA+LMN resulted in a 42% improvement of peak tetanic torque production over unrepaired VML affected limbs. However, the improvement in strength was not improved compared to a 50% minced graft-only control group. Moreover, histological analysis revealed that the improvement in in vivo functional capacity mediated by minced grafts in HA+LMN was not accompanied by a particularly robust graft mediated regenerative response as determined through donor cell tracking of the GFP+ grafting material. Characterization of the spatial distribution and density of macrophage and satellite cell populations indicated that the combination therapy damps the heightened macrophage response while re-establishing satellite content 14 days after VML to a level consistent with an endogenously healing ischemia-reperfusion induced muscle injury. Moreover, regional analysis revealed that the combination therapy increased satellite cell density mostly in the remaining musculature, as opposed to the defect area. Based on the results, the following salient conclusions were drawn: 1) functional recovery mediated by the combination therapy is likely due to a superposition of de novo muscle fiber regeneration and augmented repair of muscle fibers within the remaining musculature, and 2) The capacity for VML therapies to augment regeneration and repair within the remaining musculature may have significant clinical impact and warrants further exploration.

Immune protection efficacy of FAdV-4 surface proteins fiber-1, fiber-2, hexon and penton base.

The spread of hydropericardium syndrome has recently become serious in China since 2015. There is, therefore, an urgent need for new, safe and effective vaccines that prevent the disease. Here, the immune protection induced by Escherichia coli-expressed capsid proteins of fowl adenovirus serotype 4, including fiber-1, fiber-2, penton base and hexon (loop-1 region) were compared in chickens at different inoculation amounts. According to challenge mortalities and tissue gross/micro lesion results, fiber-2 induced the best protection, followed by fiber-1 and hexon. Fiber-1 and fiber-2 provided complete protection against 105.5 TCID50 viral load challenge with 100 or 50µg doses per chicken, respectively. Penton could induce effective protection only at the high dosage of 200µg per chicken. The immunoprotective characteristics of these FAdV-4 capsid proteins may prove useful for developing subunit vaccines to control hydropericardium syndrome.

Current Insights Into Inositol Isoforms, Mediterranean and Ketogenic Diets for Polycystic Ovary Syndrome: From Bench to Bedside.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a complex syndrome characterized by reproductive and metabolic implications. Lifestyle changes, such as diet and exercise, are considered first-line treatment for women affected by PCOS. Pharmacologic treatments target the hormonal and metabolic dysregulations associated to the disease such as insulin resistance, anovulation, hirsutism and menstrual irregularities. OBJECTIVE: To focus on the role of inositol isoforms, as well as Mediterranean and ketogenic diets, as possible therapeutic strategies in PCOS women. METHOD: Narrative overview, synthesizing the findings of literature retrieved from searches of computerized databases. RESULTS: Accumulating evidence suggests that two inositol isoforms, myo- and D-chiro-, may play a pivotal role in re-addressing both hormonal and metabolic parameters toward homeostasis, counteracting the symptoms and signs typical of this syndrome. In addition, studies focused on Mediterranean and ketogenic diet provided positive results in patients affected by obesity and type 2 diabetes, so these dietetic regimens could represent a fascinating dietetic treatment for the management of PCOS. CONCLUSION: Both the isoforms of inositol are effective in improving ovarian function and metabolism in patients with PCOS. In spite of accumulating evidence, it is currently not possible to draw firm conclusion(s) about the efficacy of these interventions considering the severe bias due to different samples size, dose, and duration of intervention among the published studies on this topic. Furthermore, future longitudinal cohort studies along with prospective interventional trials may contribute to better clarify the role of Mediterranean and ketogenic diets in the treatment of PCOS.

Calcitonin gene-related peptide increases acetylcholine quantal size in neuromuscular junctions of mice.

We used an intracellular microelectrode technique to study the mechanisms of action of two isoforms (human and rat) of calcitonin gene-related peptide (CGRP) on the evoked and spontaneous quantal secretion of acetylcholine (ACh) in mouse diaphragm motor synapses. Recordings of miniature endplate potentials (MEPPs) and evoked multiquantal endplate potentials (EPPs) in a cut neuromuscular preparation showed that CGRP increased the amplitude of EPPs without influencing their quantal content. Both isoforms of CGRP in a wide range of concentrations (1nM-1µM) provoked a similar considerable increase in MEPPs amplitude in a dose-dependent manner (up to 150-160% compared to control) without changing their frequency, rise-time, and decay. Inhibition of CGRP-receptors by truncated CGRP (CGRP8-37) completely prevented the potentiating effect of CGRP on the MEPPs amplitude. The effect of CGRP was not accompanied by changes in input resistance of muscle fiber membrane but was fully prevented by inhibition of vesicular ACh transport by vesamicol. Inhibition of protein kinase A (PKA) by H-89 also prevented CGRP action on the MEPPs amplitude. It is concluded that, in mammalian neuromuscular junctions, different isoforms of exogenously applied CGRP uniformly potentiate amplitudes of evoked and spontaneous postsynaptic potentials acting presynaptically via an increase in ACh quantal size.

EV71 virus-like particles produced by co-expression of capsid proteins in yeast cells elicit humoral protective response against EV71 lethal challenge.

BACKGROUND: Enterovirus 71 (EV71) is the most common causative pathogens of hand, foot and mouth disease (HFMD) associated with severe neurological complications. There is a great need to develop prophylactic vaccine against EV71 infection. RESULTS: EV71 virus-like particle (VLP) was produced in yeast expression system by the co-expression of four EV71 structural proteins VP1-VP4. Immunization with the recombinant VLPs elicited potent anti-EV71 antibody responses in adult mice and anti-VLP sera were able to neutralize EV71 virus in vitro. Neonatal mice model demonstrated VLP immunization conferred protection to suckling mice against the lethal viral challenge. CONCLUSIONS: Co-expression of four EV71 structural proteins VP1-VP4 in yeast expression systems is an effective method to produce EV71 VLPs. VLP-based vaccine shows great potential to prevent EV71 infection.

Low-Dose IL-17 Therapy Prevents and Reverses Diabetic Nephropathy, Metabolic Syndrome, and Associated Organ Fibrosis.

Diabetes is the leading cause of kidney failure, accounting for >45% of new cases of dialysis. Diabetic nephropathy is characterized by inflammation, fibrosis, and oxidant stress, pathologic features that are shared by many other chronic inflammatory diseases. The cytokine IL-17A was initially implicated as a mediator of chronic inflammatory diseases, but recent studies dispute these findings and suggest that IL-17A can favorably modulate inflammation. Here, we examined the role of IL-17A in diabetic nephropathy. We observed that IL-17A levels in plasma and urine were reduced in patients with advanced diabetic nephropathy. Type 1 diabetic mice that are genetically deficient in IL-17A developed more severe nephropathy, whereas administration of low-dose IL-17A prevented diabetic nephropathy in models of type 1 and type 2 diabetes. Moreover, IL-17A administration effectively treated, prevented, and reversed established nephropathy in genetic models of diabetes. Protective effects were also observed after administration of IL-17F but not IL-17C or IL-17E. Notably, tubular epithelial cell-specific overexpression of IL-17A was sufficient to suppress diabetic nephropathy. Mechanistically, IL-17A administration suppressed phosphorylation of signal transducer and activator of transcription 3, a central mediator of fibrosis, upregulated anti-inflammatory microglia/macrophage WAP domain protein in an AMP-activated protein kinase-dependent manner and favorably modulated renal oxidative stress and AMP-activated protein kinase activation. Administration of recombinant microglia/macrophage WAP domain protein suppressed diabetes-induced albuminuria and enhanced M2 marker expression. These observations suggest that the beneficial effects of IL-17 are isoform-specific and identify low-dose IL-17A administration as a promising therapeutic approach in diabetic kidney disease.

Intranasal Delivery of RGD Motif-Containing Osteopontin Icosamer Confers Neuroprotection in the Postischemic Brain via αvß3 Integrin Binding.

Osteopontin (OPN) is a phosphorylated glycoprotein possessing an arginine-glycine-aspartate (RGD)-motif, which binds to several cell surface integrins and mediates a wide range of cellular processes. Inductions of OPN have been reported in the postischemic brain, and the neuroprotective effects of OPN have been demonstrated in animal models of stroke. In the present study, we showed a robust neuroprotective effect of RGD-containing icosamer OPN peptide (OPNpt20) in a rat model of focal cerebral ischemia (middle cerebral artery occlusion, MCAO). Intranasally administered OPNpt20 reduced mean infarct volume by 79.7 % compared to the treatment-naïve MCAO control animals and markedly ameliorated neurological deficits. In addition, OPNpt20 significantly suppressed the inductions of iNOS and of inflammatory markers in postischemic brains and in primary microglial cultures, demonstrating anti-inflammatory effects. Administration of a mutant peptide, in which RGD was replaced by arginine-alanine-alanine (RAA), failed to suppress infarct volumes in MCAO animals and co-administration of OPNpt20 with anti-αvß3 integrin antibody failed to suppress iNOS induction in primary microglia culture, indicating that the RGD motif in OPNpt20 and endogenous αvß3 integrin play critical roles. Furthermore, pull-down assay revealed a direct binding between OPNpt20 and αvß3 integrin in primary microglia culture. Together, these results indicate that RGD-containing OPN icosamer has therapeutic potential in the postischemic brain and αvß3 integrin-mediated anti-inflammatory effect might be an underlying mechanism.

Full-length human placental sFlt-1-e15a isoform induces distinct maternal phenotypes of preeclampsia in mice.

OBJECTIVE: Most anti-angiogenic preeclampsia models in rodents utilized the overexpression of a truncated soluble fms-like tyrosine kinase-1 (sFlt-1) not expressed in any species. Other limitations of mouse preeclampsia models included stressful blood pressure measurements and the lack of postpartum monitoring. We aimed to 1) develop a mouse model of preeclampsia by administering the most abundant human placental sFlt-1 isoform (hsFlt-1-e15a) in preeclampsia; 2) determine blood pressures in non-stressed conditions; and 3) develop a survival surgery that enables the collection of fetuses and placentas and postpartum (PP) monitoring. METHODS: Pregnancy status of CD-1 mice was evaluated with high-frequency ultrasound on gestational days (GD) 6 and 7. Telemetry catheters were implanted in the carotid artery on GD7, and their positions were verified by ultrasound on GD13. Mice were injected through tail-vein with adenoviruses expressing hsFlt-1-e15a (n = 11) or green fluorescent protein (GFP; n = 9) on GD8/GD11. Placentas and pups were delivered by cesarean section on GD18 allowing PP monitoring. Urine samples were collected with cystocentesis on GD6/GD7, GD13, GD18, and PPD8, and albumin/creatinine ratios were determined. GFP and hsFlt-1-e15a expression profiles were determined by qRT-PCR. Aortic ring assays were performed to assess the effect of hsFlt-1-e15a on endothelia. RESULTS: Ultrasound predicted pregnancy on GD7 in 97% of cases. Cesarean section survival rate was 100%. Mean arterial blood pressure was higher in hsFlt-1-e15a-treated than in GFP-treated mice (∆MAP = 13.2 mmHg, p = 0.00107; GD18). Focal glomerular changes were found in hsFlt-1-e15a -treated mice, which had higher urine albumin/creatinine ratios than controls (109.3 ± 51.7 µg/mg vs. 19.3 ± 5.6 µg/mg, p = 4.4 x 10(-2); GD18). Aortic ring assays showed a 46% lesser microvessel outgrowth in hsFlt-1-e15a-treated than in GFP-treated mice (p = 1.2 x 10(-2)). Placental and fetal weights did not differ between the groups. One mouse with liver disease developed early-onset preeclampsia-like symptoms with intrauterine growth restriction (IUGR). CONCLUSIONS: A mouse model of late-onset preeclampsia was developed with the overexpression of hsFlt-1-e15a, verifying the in vivo pathologic effects of this primate-specific, predominant placental sFlt-1 isoform. HsFlt-1-e15a induced early-onset preeclampsia-like symptoms associated with IUGR in a mouse with a liver disease. Our findings support that hsFlt-1-e15a is central to the terminal pathway of preeclampsia, and it can induce the full spectrum of symptoms in this obstetrical syndrome.

IL-33 isoforms: their future as vaccine adjuvants?

The identification and characterization of cytokine isoforms is likely to provide critical important new insight into immunobiology. Cytokine isoforms can provide additional diversity to their complex biological effects that participate in control and protection against different foreign pathogens. Recently, IL-33 has been identified as a proinflammatory cytokine having several different biologically active isoform products. Originally associated with Th2 immunity, new evidence now supports the role of two IL-33 isoforms to facilitate the generation of protective Th1 and CD8 T cell immunity against specific pathogens. Therefore, a better understanding of the IL-33 isoforms will inform us on how to utilize them to facilitate their development as tools as vaccine adjuvants for immune therapy.

Delta-lactoferrin induces cell death via the mitochondrial death signaling pathway by upregulating bax expression.

Delta-lactoferrin (∆Lf) is a transcription factor belonging to the lactoferrin family, the expression of which inhibits cell proliferation and leads to Skp1 and DcpS gene transactivation. In this study, we showed that ∆Lf expression also induces cell death via apoptosis in HEK 293 and MCF7 cells using a cell viability assay and DNA fragmentation. Western blot analyses showed that apoptosis was caspase-9, 7 and 8 dependent. Proteolytic cleavage of the endonuclease PARP was significantly increased. The levels of expression of Bcl family members were detected by immunochemistry and showed that the Bcl-xl/Bax and Bcl-2/Bax protein ratios were decreased. We determined that the pro-apoptotic effects of ∆Lf are mainly mediated by the activation of the mitochondria-dependent death-signaling pathway. Apoptosis induction by ∆Lf is concomitant with increased cellular levels of Bax protein. Analysis of the Bax promoter region detected a ∆Lf response element located at -155 bp from the transcription start site. Both luciferase reporter gene and chromatin immunoprecipitation assays confirmed that ∆Lf interacts in vitro and in vivo specifically with this sequence. Its deletion, realized using directed mutagenesis, totally abolished ∆Lf transcriptional activity, identifying it as a ∆Lf-responsive element. These results indicate that the Bax gene is a novel ∆Lf target. Moreover we also showed that the O-GlcNAc/P interplay, which controls ∆Lf transcriptional activity, modulates Bax transactivation.

Neuregulin 1 role in Schwann cell regulation and potential applications to promote peripheral nerve regeneration.

Neuregulin 1 (NRG1) is a multifunctional and versatile protein: its numerous isoforms can signal in a paracrine, autocrine, or juxtacrine manner, playing a fundamental role during the development of the peripheral nervous system and during the process of nerve repair, suggesting that the treatment with NRG1 could improve functional outcome following injury. Accordingly, the use of NRG1 in vivo has already yielded encouraging results. The aim of this review is to focus on the role played by the different NRG1 isoforms during peripheral nerve regeneration and remyelination and to identify good candidates to be used for the development of tissue engineered medical devices delivering NRG1, with the objective of promoting better nerve repair.

Functional genomics lead to new therapies in follicular lymphoma.

Recent technological advances allow analysis of genomic changes in cancer in unprecedented detail. The next challenge is to prioritize the multitude of genetic aberrations found and identify therapeutic opportunities. We recently completed a study that illustrates the use of unbiased genetic screens and murine cancer models to find therapeutic targets among complex genomic data. We genetically dissected the common deletion of chromosome 6q and identified the ephrin receptor A7 (EPHA7) as a tumor suppressor in lymphoma. Notably, EPHA7 encodes a soluble splice variant that acts as an extrinsic tumor suppressor. Accordingly, we developed an antibody-based strategy to specifically deliver EPHA7 back to tumors that have lost this gene. Recent sequencing studies have implicated EPHA7 in lung cancer and other tumors, suggesting a broader therapeutic potential for antibody-mediated delivery of this tumor suppressor for cancer therapy. Together, our comprehensive approach provides new insights into cancer biology and may directly lead to the development of new cancer therapies.

Rhesus macaque theta defensins suppress inflammatory cytokines and enhance survival in mouse models of bacteremic sepsis.

Theta-defensins (θ-defensins) are macrocyclic antimicrobial peptides expressed in leukocytes of Old World monkeys. The peptides are broad spectrum microbicides in vitro and numerous θ-defensin isoforms have been identified in granulocytes of rhesus macaques and Olive baboons. Several mammalian α- and ß-defensins, genetically related to θ-defensins, have proinflammatory and immune-activating properties that bridge innate and acquired immunity. In the current study we analyzed the immunoregulatory properties of rhesus θ-defensins 1-5 (RTDs 1-5). RTD-1, the most abundant θ-defensin in macaques, reduced the levels of TNF, IL-1α, IL-1ß, IL-6, and IL-8 secreted by blood leukocytes stimulated by several TLR agonists. RTDs 1-5 suppressed levels of soluble TNF released by bacteria- or LPS-stimulated blood leukocytes and THP-1 monocytes. Despite their highly conserved conformation and amino acid sequences, the anti-TNF activities of RTDs 1-5 varied by as much as 10-fold. Systemically administered RTD-1 was non-toxic for BALB/c mice, and escalating intravenous doses were well tolerated and non-immunogenic in adult chimpanzees. The peptide was highly stable in serum and plasma. Single dose administration of RTD-1 at 5 mg/kg significantly improved survival of BALB/c mice with E. coli peritonitis and cecal ligation-and-puncture induced polymicrobial sepsis. Peptide treatment reduced serum levels of several inflammatory cytokines/chemokines in bacteremic animals. Collectively, these results indicate that the anti-inflammatory properties of θ-defensins in vitro and in vivo are mediated by the suppression of numerous proinflammatory cytokines and blockade of TNF release may be a primary effect.

Monoclonal antibodies against Actinobacillus pleuropneumoniae TonB2 protein expressed in Escherichia coli.

TonB is known to be a bacterial periplasmic protein that transduces proton from the inner membrane to the outer membrane receptor in complex with the ExbB and ExbD proteins. Actinobacillus pleuropneumoniae TonB2 protein is the second TonB protein that is important for iron acquisition and virulence. The TonB2 protein was verified to be immunogenic and could afford partial protection for animals from lethal infection. In the present study, the recombinant TonB2 (rTonB2) was overexpressed in Escherichia coli BL21(DE3) and purified. The rTonB2 was then used as antigen to immunize BALB/c mice for the production of monoclonal antibodies (MAb). Four clones of TonB2-specific MAb secretion hybridomas--2F2, 2G8, 3D2, and 6F10--were selected. The MAbs 2F2, 3D2, and 6F10 were classified as IgG1 isotype and 2G8 was of IgG2a isotype. Western blot and ELISA results indicated that MAbs had specific binding activity to rTonB2. The MAbs generated here will be used for further functional analyses of the TonB2 protein.