ResearchPad - guinea-pigs https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Methamphetamine administration increases hepatic CYP1A2 but not CYP3A activity in female guinea pigs]]> https://www.researchpad.co/article/elastic_article_7848 Methamphetamine use has increased over the past decade and the first use of methamphetamine is most often when women are of reproductive age. Methamphetamine accumulates in the liver; however, little is known about the effect of methamphetamine use on hepatic drug metabolism. Methamphetamine was administered on 3 occassions to female Dunkin Hartley guinea pigs of reproductive age, mimicking recreational drug use. Low doses of test drugs caffeine and midazolam were administered after the third dose of methamphetamine to assess the functional activity of cytochrome P450 1A2 and 3A, respectively. Real-time quantitative polymerase chain reaction was used to quantify the mRNA expression of factors involved in glucocorticoid signalling, inflammation, oxidative stress and drug transporters. This study showed that methamphetamine administration decreased hepatic CYP1A2 mRNA expression, but increased CYP1A2 enzyme activity. Methamphetamine had no effect on CYP3A enzyme activity. In addition, we found that methamphetamine may also result in changes in glucocorticoid bioavailability, as we found a decrease in 11β-hydroxysteroid dehydrogenase 1 mRNA expression, which converts inactive cortisone into active cortisol. This study has shown that methamphetamine administration has the potential to alter drug metabolism via the CYP1A2 metabolic pathway in female guinea pigs. This may have clinical implications for drug dosing in female methamphetamine users of reproductive age.

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<![CDATA[Characterization of an intratracheal aerosol challenge model of Brucella melitensis in guinea pigs]]> https://www.researchpad.co/article/5c8823ccd5eed0c48463903c

B. melitensis is considered the most virulent of the Brucella species, and a need exists for an improved laboratory animal model of infection that mimics natural transmission and disease. Guinea pigs are highly susceptible to infection with Brucella spp. and develop a disease syndrome that mimics natural disease after aerosol inoculation. Intratracheal inoculation is a targeted means of generating aerosols that offer advantages over aerosol chamber delivery. To establish this delivery method, female, Hartley guinea pigs were infected via intratracheal inoculation with PBS or 16M B. melitensis at low dose (101 to 103) or high dose (106 to 108) and monitored for 30 days for signs of disease. Guinea pigs in the high dose groups developed fever between 12–17 days post-inoculation. Bacteria were recovered from the spleen, liver, lymph nodes, lung, and uterus at 30-days post-inoculation and demonstrated dose dependent mean increases in colonization and pathologic changes consistent with human brucellosis. To study the kinetics of extrapulmonary dissemination, guinea pigs were inoculated with 107 CFU and euthanized at 2-hours post inoculation and at weekly intervals for 3 weeks. 5.8x105 to 4.2x106 CFU were recovered from the lung 2 hours post-inoculation indicating intratracheal inoculation is an efficient means of infecting guinea pigs. Starting at 1-week post inoculation bacteria were recovered from the aforementioned organs with time dependent mean increases in colonization. This data demonstrates that guinea pigs develop a disease syndrome that models the human manifestation of brucellosis, which makes the guinea pig a valuable model for pathogenesis studies.

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<![CDATA[Efficacy of equine botulism antitoxin in botulism poisoning in a guinea pig model]]> https://www.researchpad.co/article/5c424393d5eed0c4845e067e

Background

Botulism is a disease caused by neurogenic toxins that block acetylcholine release, resulting in potentially life threatening neuroparalysis. Seven distinct serotypes of botulinum neurotoxins (BoNTs) have been described and are found in nature world-wide. This, combined with ease of production, make BoNTs a significant bioweapon threat. An essential countermeasure to this threat is an antitoxin to remove circulating toxin. An antitoxin, tradename BAT (Botulism Antitoxin Heptavalent (A, B, C, D, E, F, G)–(Equine)), has been developed and its efficacy evaluated against all seven serotypes in guinea pigs.

Methods and findings

Studies were conducted to establish the lethal dose and clinical course of intoxication for all seven toxins, and post-exposure prophylactic efficacy of BAT product. Animals were monitored for signs of intoxication and mortality for 14 days. Guinea pig intramuscular LD50s (GPIMLD50) for all BoNTs ranged from 2.0 (serotype C) to 73.2 (serotype E) of mouse intraperitoneal LD50 units. A dose of 4x GPIMLD50 was identified as the appropriate toxin dose for use in subsequent efficacy and post-exposure prophylaxis studies. The main clinical signs observed included hind limb paralysis, weak limb, change in breathing rate/pattern, and forced abdominal respiration. Mean time to onset of clinical signs ranged from 12 hours (serotype E) to 39 hours (serotype G). Twelve hours post-intoxication was selected as the appropriate time point for intervention for all serotypes apart from E where 6 hours was selected because of the rapid onset and progression of clinical signs. Post-exposure treatment with BAT product resulted in a significantly (p<0.0001) higher survival at >0.008 scaled human dose for serotypes A, B, C, F and G, at >0.2x for serotype D and >0.04x for serotype E.

Conclusions

These studies confirm the efficacy of BAT as a post-exposure prophylactic therapy against all seven known BoNT serotypes.

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<![CDATA[Roles of GP33, a guinea pig cytomegalovirus-encoded G protein-coupled receptor homolog, in cellular signaling, viral growth and inflammation in vitro and in vivo]]> https://www.researchpad.co/article/5c25453fd5eed0c48442c35a

Cytomegaloviruses (CMVs) encode cellular homologs to evade host immune functions. In this study, we analyzed the roles of GP33, a guinea pig CMV (GPCMV)-encoded G protein-coupled receptor (GPCR) homolog, in cellular signaling, viral growth and pathogenesis. The cDNA structure of GP33 was determined by RACE. The effects of GP33 on some signaling pathways were analyzed in transient transfection assays. The redET two-step recombination system for a BAC containing the GPCMV genome was used to construct a mutant GPCMV containing an early stop codon in the GP33 gene (Δ33) and a rescued GPCMV (r33). We found the following: 1) GP33 activated the CRE- and NFAT-, but not the NFκB-mediated signaling pathway. 2) GP33 was dispensable for infection in tissue cultures and in normal animals. 3) In pregnant animals, viral loads of r33 in the livers, lungs, spleens, and placentas at 6 days post-infection were higher than those of Δ33, although the viruses were cleared by 3 weeks post-infection. 4) The presence of GP33 was associated with frequent lesions, including alveolar hemorrhage in the lungs, and inflammation in the lungs, livers, and spleens of the dams. Our findings suggest that GP33 has critical roles in the pathogenesis of GPCMV during pregnancy. We hypothesize that GP33-mediated signaling activates cytokine secretion from the infected cells, which results in inflammation in some of the maternal organs and the placentas. Alternatively, GP33 may facilitate transient inflammation that is induced by the chemokine network specific to the pregnancy.

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<![CDATA[Guinea pig immunoglobulin VH and VL naïve repertoire analysis]]> https://www.researchpad.co/article/5c1c0af3d5eed0c484426f9b

The guinea pig has been used as a model to study various human infectious diseases because of its similarity to humans regarding symptoms and immune response, but little is known about the humoral immune response. To better understand the mechanism underlying the generation of the antibody repertoire in guinea pigs, we performed deep sequencing of full-length immunoglobulin variable chains from naïve B and plasma cells. We gathered and analyzed nearly 16,000 full-length VH, Vκ and Vλ genes and analyzed V and J gene segment usage profiles and mutation statuses by annotating recently reported genome data of guinea pig immunoglobulin genes. We found that approximately 70% of heavy, 73% of kappa and 81% of lambda functional germline V gene segments are integrated into the actual V(D)J recombination events. We also found preferential use of a particular V gene segment and accumulated mutation in CDRs 1 and 2 in antigen-specific plasma cells. Our study represents the first attempt to characterize sequence diversity in the expressed guinea pig antibody repertoire and provides significant insight into antibody repertoire generation and Ig-based immunity of guinea pigs.

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<![CDATA[Determinants of early afterdepolarization properties in ventricular myocyte models]]> https://www.researchpad.co/article/5c059df8d5eed0c4849c996b

Early afterdepolarizations (EADs) are spontaneous depolarizations during the repolarization phase of an action potential in cardiac myocytes. It is widely known that EADs are promoted by increasing inward currents and/or decreasing outward currents, a condition called reduced repolarization reserve. Recent studies based on bifurcation theories show that EADs are caused by a dual Hopf-homoclinic bifurcation, bringing in further mechanistic insights into the genesis and dynamics of EADs. In this study, we investigated the EAD properties, such as the EAD amplitude, the inter-EAD interval, and the latency of the first EAD, and their major determinants. We first made predictions based on the bifurcation theory and then validated them in physiologically more detailed action potential models. These properties were investigated by varying one parameter at a time or using parameter sets randomly drawn from assigned intervals. The theoretical and simulation results were compared with experimental data from the literature. Our major findings are that the EAD amplitude and takeoff potential exhibit a negative linear correlation; the inter-EAD interval is insensitive to the maximum ionic current conductance but mainly determined by the kinetics of ICa,L and the dual Hopf-homoclinic bifurcation; and both inter-EAD interval and latency vary largely from model to model. Most of the model results generally agree with experimental observations in isolated ventricular myocytes. However, a major discrepancy between modeling results and experimental observations is that the inter-EAD intervals observed in experiments are mainly between 200 and 500 ms, irrespective of species, while those of the mathematical models exhibit a much wider range with some models exhibiting inter-EAD intervals less than 100 ms. Our simulations show that the cause of this discrepancy is likely due to the difference in ICa,L recovery properties in different mathematical models, which needs to be addressed in future action potential model development.

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<![CDATA[Standardized guinea pig model for Q fever vaccine reactogenicity]]> https://www.researchpad.co/article/5bca48ef40307c0516656419

Historically, vaccination with Coxiella burnetii whole cell vaccines has induced hypersensitivity reactions in humans and animals that have had prior exposure to the pathogen as a result of infection or vaccination. Intradermal skin testing is routinely used to evaluate exposure in humans, and guinea pig hypersensitivity models have been developed to characterize the potential for reactogenicity in vaccine candidates. Here we describe a refinement of the guinea pig model using an alternate vaccine for positive controls. An initial comparative study used viable C. burnetii to compare the routes of sensitizing exposure of guinea pigs (intranasal vs intraperitoneal), evaluation of two time points for antigen challenge (21 and 42 days) and an assessment of two routes (intradermal and subcutaneous) of challenge using the ruminant vaccine Coxevac as the antigenic control. Animals sensitized by intraperitoneal exposure exhibited slightly larger gross reactions than did those sensitized by intranasal exposure, and reactions were more pronounced when skin challenge was performed at 42 days compared to 21 days post-sensitization. The intradermal route proved to be the optimal route of reactogenicity challenge. Histopathological changes at injection sites were similar to those previously reported and a scoring system was developed to compare reactions between groups receiving vaccine by intradermal versus subcutaneous routes. Based on the comparative study, a standardized protocol for assessment of vaccine reactogenicity in intranasally-sensitized animals was tested in a larger confirmatory study. Results suggest that screens utilizing a group size of n = 3 would achieve 90% power for detecting exposure-related reactogenic responses of the magnitude induced by Coxevac using either of two outcome measures.

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<![CDATA[Truncated Bovine Integrin Alpha-v/Beta-6 as a Universal Capture Ligand for FMD Diagnosis]]> https://www.researchpad.co/article/5989da6eab0ee8fa60b93c4d

Foot-and-mouth disease (FMD) is endemic in many regions of the world and is one of the most prevalent epizootic animal diseases. FMD affects livestock, such as cattle, sheep, goats and pigs, and causes enormous economic losses due to reduced productivity and trade restrictions. Preparedness and early diagnosis are essential for effective control of FMD. Many diagnostic assays are dependent on raising high-affinity, anti-FMD virus (FMDV) serotype-specific antibodies in small animals (rabbits and guinea pigs) that give broad virus coverage. Here we show that soluble, truncated forms of bovine αvβ6 bind FMDV in an authentic RGD and divalent cation dependent interaction and can be used as the trapping reagent in a FMDV sandwich ELISA. In addition, inclusion of FLAG or His tags facilitates simple purification without the loss of virus binding. We also provide evidence that when combined with a guinea pig polyclonal serum, or serotype-specific monoclonal antibodies, the integrin can be used to detect viruses representative of all FMDV serotypes. We also show that recombinant FMDV empty capsids, with stabilising disulphide bonds, can serve as an antigen in the ELISA and can therefore replace inactivated virus antigen as a positive control for the assay. Our results demonstrate the potential use of bovine αvβ6 and FMDV empty capsids in FMD diagnostic assays.

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<![CDATA[Identification of Eight Different Isoforms of the Glucocorticoid Receptor in Guinea Pig Placenta: Relationship to Preterm Delivery, Sex and Betamethasone Exposure]]> https://www.researchpad.co/article/5989db42ab0ee8fa60bd71ec

The placental glucocorticoid receptor (GR) is central to glucocorticoid signalling and for mediating steroid effects on pathways associated with fetal growth and lung maturation but the GR has not been examined in the guinea pig placenta even though this animal is regularly used as a model of preterm birth and excess glucocorticoid exposure. Guinea pig dams received subcutaneous injections of either vehicle or betamethasone at 24 and 12 hours prior to preterm or term caesarean-section delivery. At delivery pup and organ weights were recorded. Placentae were dissected, weighed and analysed using Western blot to examine GR isoform expression in nuclear and cytoplasmic extracts. A comparative examination of the guinea pig GR gene identified it is capable of producing seven of the eight translational GR isoforms which include GRα-A, C1, C2, C3, D1, D2, and D3. GRα-B is not produced in the Guinea Pig. Total GR antibody identified 10 specific bands from term (n = 29) and preterm pregnancies (n = 27). Known isoforms included GRγ, GRα A, GRβ, GRP, GRA and GRα D1-3. There were sex and gestational age differences in placental GR isoform expression. Placental GRα A was detected in the cytoplasm of all groups but was significantly increased in the cytoplasm and nucleus of preterm males and females exposed to betamethasone and untreated term males (KW-ANOVA, P = 0.0001, P = 0.001). Cytoplasmic expression of GRβ was increased in female preterm placentae and preterm and term male placentae exposed to betamethasone (P = 0.01). Nuclear expression of GRβ was increased in all placentae exposed to betamethasone (P = 0.0001). GRα D2 and GRα D3 were increased in male preterm placentae when exposed to betamethasone (P = 0.01, P = 0.02). The current data suggests the sex-specific placental response to maternal betamethasone may be dependent on the expression of a combination of GR isoforms.

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<![CDATA[T140 blocks the SDF-1/CXCR4 signaling pathway and prevents cartilage degeneration in an osteoarthritis disease model]]> https://www.researchpad.co/article/5989db52ab0ee8fa60bdc569

Osteoarthritis (OA) is one of the most common diseases affecting older people; however, there remains no effective targeted drug to combat OA. The aims of this study were (1) to explore the effect of T140 in regulating degeneration of articular cartilage in vivo by targeted blocking of the SDF-1/CXCR4 signaling pathway, and (2) to provide experimental evidence for the development of a novel OA-targeted pharmacotherapy. Thirty-six healthy Hartley guinea pigs were randomly divided into three groups: a T140-treated group (n = 12), a phosphate buffer saline control group (n = 12) and an untreated control group (n = 12). At 2, 4, 6, 8, 10 and 12 weeks of treatment, SDF-1 in serum was quantified by enzyme-linked immunosorbent assay. At 12 weeks of treatment, the cartilage from knee tibial plateau in the knee joint was collected for H&E, Safranin-O staining and Mankin grading; measurement for mRNA levels of matrix metalloproteinases (MMP-3, MMP-9 and MMP-13), aggrecan (ACAN) and collagen II (Col II) using RT-PCR; and measurement for Col II protein levels by western blot. Results showed that SDF-1 in serum increased in the T140 group and increased in the control groups. H&E and Safranin-O staining revealed less cartilage loss in T140-treated animals compared to controls. The mRNA levels of MMP-3, MMP-9 and MMP-13 in cartilage were much lower in the T140 group than other groups, but mRNA levels of ACAN and Col II in cartilage were higher in the T140-treated group. Col II protein levels in the T140 group and control groups were different. T140 can downregulate the expression of matrix-degrading enzyme and lessen the degeneration of cartilage by blocking the SDF-1/CRCR4 signaling pathway in vivo. This mechanism may present a pharmacological target for the treatment of OA.

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<![CDATA[Improved Auditory Nerve Survival with Nanoengineered Supraparticles for Neurotrophin Delivery into the Deafened Cochlea]]> https://www.researchpad.co/article/5989dad6ab0ee8fa60bb81fe

Cochlear implants electrically stimulate spiral ganglion neurons (SGNs) in order to provide speech cues to severe-profoundly deaf patients. In normal hearing cochleae the SGNs depend on endogenous neurotrophins secreted by sensory cells in the organ of Corti for survival. SGNs gradually degenerate following deafness and consequently there is considerable interest in developing clinically relevant strategies to provide exogenous neurotrophins to preserve SGN survival. The present study investigated the safety and efficacy of a drug delivery system for the cochlea using nanoengineered silica supraparticles. In the present study we delivered Brain-derived neurotrophic factor (BDNF) over a period of four weeks and evaluated SGN survival as a measure of efficacy. Supraparticles were bilaterally implanted into the basal turn of cochleae in profoundly deafened guinea pigs. One ear received BDNF-loaded supraparticles and the other ear control (unloaded) supraparticles. After one month of treatment the cochleae were examined histologically. There was significantly greater survival of SGNs in cochleae that received BDNF supraparticles compared to the contralateral control cochleae (repeated measures ANOVA, p = 0.009). SGN survival was observed over a wide extent of the cochlea. The supraparticles were well tolerated within the cochlea with a tissue response that was localised to the site of implantation in the cochlear base. Although mild, the tissue response was significantly greater in cochleae treated with BDNF supraparticles compared to the controls (repeated measures ANOVA, p = 0.003). These data support the clinical potential of this technology particularly as the supraparticles can be loaded with a variety of therapeutic drugs.

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<![CDATA[Deoxyarbutin Possesses a Potent Skin-Lightening Capacity with No Discernible Cytotoxicity against Melanosomes]]> https://www.researchpad.co/article/5989dae9ab0ee8fa60bbe85e

Safe and effective ingredients capable of removing undesired hyperpigmentation from facial skin are urgently needed for both pharmaceutical and cosmetic purposes. Deoxyarbutin (4-[(tetrahydro-2H-pyran-2-yl) oxy] phenol, D-Arb) is a glucoside derivative of hydroquinone. Here, we investigated the toxicity and efficacy of D-Arb at the sub-cellular level (directly on melanosomes) and skin pigmentation using in vivo and in vitro models to compare with its parent compound hydroquinone (1,4-benzenediol, HQ). At first, we examined the ultrastructural changes of melanosomes in hyperpigmented guinea pig skin induced by 308-nm monochromatic excimer lightand/or treated with HQ and D-Arb using transmission electron microscopy. The results showed that prominent changes in the melanosomal membrane, such as bulb-like structure and even complete rupture of the outer membranes, were found in the skin after topical application of 5% HQ for 10 days. These changes were barely observed in the skin treated with D-Arb. To further clarify whether membrane toxicity of HQ was a direct result of the compound treatment, we also examinedultrastructural changes of individual melanosomes purified from MNT1 human melanoma cells. Similar observations were obtained from the naked melanosome model in vitro. Finally, we determined the effects of melanosomal fractions exposed to HQ or D-Arb on hydroxyl radical generation in the Fenton reaction utilizing an electron spin resonance assay. D-Arb-treated melanosomesexhibit a moderate hydroxyl radical-scavenging activity, whereas HQ-treated melanosomessignificantly generate more hydroxyl free radicals. This study suggests that D-Arb possesses a potent ability in skin lightening and antioxidation with less melanosome cytotoxicity.

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<![CDATA[Virus-Specific Immune Memory at Peripheral Sites of Herpes Simplex Virus Type 2 (HSV-2) Infection in Guinea Pigs]]> https://www.researchpad.co/article/5989dae7ab0ee8fa60bbdf69

Despite its importance in modulating HSV-2 pathogenesis, the nature of tissue-resident immune memory to HSV-2 is not completely understood. We used genital HSV-2 infection of guinea pigs to assess the type and location of HSV-specific memory cells at peripheral sites of HSV-2 infection. HSV-specific antibody-secreting cells were readily detected in the spleen, bone marrow, vagina/cervix, lumbosacral sensory ganglia, and spinal cord of previously-infected animals. Memory B cells were detected primarily in the spleen and to a lesser extent in bone marrow but not in the genital tract or neural tissues suggesting that the HSV-specific antibody-secreting cells present at peripheral sites of HSV-2 infection represented persisting populations of plasma cells. The antibody produced by these cells isolated from neural tissues of infected animals was functionally relevant and included antibodies specific for HSV-2 glycoproteins and HSV-2 neutralizing antibodies. A vigorous IFN-γ-secreting T cell response developed in the spleen as well as the sites of HSV-2 infection in the genital tract, lumbosacral ganglia and spinal cord following acute HSV-2 infection. Additionally, populations of HSV-specific tissue-resident memory T cells were maintained at these sites and were readily detected up to 150 days post HSV-2 infection. Unlike the persisting plasma cells, HSV-specific memory T cells were also detected in uterine tissue and cervicothoracic region of the spinal cord and at low levels in the cervicothoracic ganglia. Both HSV-specific CD4+ and CD8+ resident memory cell subsets were maintained long-term in the genital tract and sensory ganglia/spinal cord following HSV-2 infection. Together these data demonstrate the long-term maintenance of both humoral and cellular arms of the adaptive immune response at the sites of HSV-2 latency and virus shedding and highlight the utility of the guinea pig infection model to investigate tissue-resident memory in the setting of HSV-2 latency and spontaneous reactivation.

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<![CDATA[A Novel Class of Small Molecule Agonists with Preference for Human over Mouse TLR4 Activation]]> https://www.researchpad.co/article/5989da74ab0ee8fa60b962ba

The best-characterized Toll-like receptor 4 (TLR4) ligands are lipopolysaccharide (LPS) and its chemically modified and detoxified variant, monophosphoryl lipid A (MPL). Although both molecules are active for human TLR4, they demonstrate a potency preference for mouse TLR4 based on data from transfected cell lines and primary cells of both species. After a high throughput screening process of small molecule libraries, we have discovered a new class of TLR4 agonist with a species preference profile differing from MPL. Products of the 4-component Ugi synthesis reaction were demonstrated to potently trigger human TLR4-transfected HEK cells but not mouse TLR4, although inclusion of the human MD2 with mTLR4 was able to partially recover activity. Co-expression of CD14 was not required for optimal activity of Ugi compounds on transfected cells, as it is for LPS. The species preference profile for the panel of Ugi compounds was found to be strongly active for human and cynomolgus monkey primary cells, with reduced but still substantial activity for most Ugi compounds on guinea pig cells. Mouse, rat, rabbit, ferret, and cotton rat cells displayed little or no activity when exposed to Ugi compounds. However, engineering the human versions of TLR4 and MD2 to be expressed in mTLR4/MD2 deficient mice allowed for robust activity by Ugi compounds both in vitro and in vivo. These findings extend the range of compounds available for development as agonists of TLR4 and identify novel molecules which reverse the TLR4 triggering preference of MPL for mouse TLR4 over human TLR4. Such compounds may be amenable to formulation as more potent human-specific TLR4L-based adjuvants than typical MPL-based adjuvants.

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<![CDATA[Two Isoforms of Yersinia pestis Plasminogen Activator Pla: Intraspecies Distribution, Intrinsic Disorder Propensity, and Contribution to Virulence]]> https://www.researchpad.co/article/5989db10ab0ee8fa60bcbebc

It has been shown previously that several endemic Y. pestis isolates with limited virulence contained the I259 isoform of the outer membrane protease Pla, while the epidemic highly virulent strains possessed only the T259 Pla isoform. Our sequence analysis of the pla gene from 118 Y. pestis subsp. microtus strains revealed that the I259 isoform was present exclusively in the endemic strains providing a convictive evidence of more ancestral origin of this isoform. Analysis of the effects of the I259T polymorphism on the intrinsic disorder propensity of Pla revealed that the I259T mutation slightly increases the intrinsic disorder propensity of the C-terminal tail of Pla and makes this protein slightly more prone for disorder-based protein-protein interactions, suggesting that the T259 Pla could be functionally more active than the I259 Pla. This assumption was proven experimentally by assessing the coagulase and fibrinolytic activities of the two Pla isoforms in human plasma, as well as in a direct fluorometric assay with the Pla peptide substrate. The virulence testing of Pla-negative or expressing the I259 and T259 Pla isoforms Y. pestis subsp. microtus and subsp. pestis strains did not reveal any significant difference in LD50 values and dose-dependent survival assays between them by using a subcutaneous route of challenge of mice and guinea pigs or intradermal challenge of mice. However, a significant decrease in time-to-death was observed in animals infected with the epidemic T259 Pla-producing strains as compared to the parent Pla-negative variants. Survival curves of the endemic I259 Pla+ strains fit between them, but significant difference in mean time to death post infection between the Plastrains and their I259 Pla+ variants could be seen only in the isogenic set of subsp. pestis strains. These findings suggest an essential role for the outer membrane protease Pla evolution in Y. pestis bubonic infection exacerbation that is necessary for intensification of epidemic process from endemic natural focality with sporadic cases in men to rapidly expanding epizootics followed by human epidemic outbreaks, local epidemics or even pandemics.

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<![CDATA[Repeated anaesthesia with isoflurane and medetomidine-midazolam-fentanyl in guinea pigs and its influence on physiological parameters]]> https://www.researchpad.co/article/5989db50ab0ee8fa60bdbfff

Repeated anaesthesia may be required in experimental protocols and in daily veterinary practice, but anaesthesia is known to alter physiological parameters in GPs (Cavia porcellus, GPs). This study investigated the effects of repeated anaesthesia with either medetomidine-midazolam-fentanyl (MMF) or isoflurane (Iso) on physiological parameters in the GP. Twelve GPs were repeatedly administered with MMF or Iso in two anaesthesia sets. One set consisted of six 40-min anaesthesias, performed over 3 weeks (2 per week); the anaesthetic used first was randomized. Prior to Iso anaesthesia, atropine was injected. MMF anaesthesia was antagonized with AFN (atipamezole-flumazenil-naloxone). Abdominally implanted radio-telemetry devices recorded the mean arterial blood pressure (MAP), heart rate (HR) and core body temperature continuously. Additionally, respiratory rate, blood glucose and body weight were assessed. An operable state could be achieved and maintained for 40 min in all GPs. During the surgical tolerance with MMF, the GPs showed a large MAP range between the individuals. In the MMF wake- up phase, the time was shortened until the righting reflex (RR) returned and that occurred at lower MAP and HR values. Repeated Iso anaesthesia led to an increasing HR during induction (anaesthesias 2–6), non-surgical tolerance (anaesthesias 3–6) and surgical tolerance (anaesthesias 4, 6). Both anaesthetics may be used repeatedly, as repeating the anaesthesias resulted in only slightly different physiological parameters, compared to those seen with single anaesthesias. The regular atropine premedication induced HR increases and repeated MMF anaesthesia resulted in a metabolism increase which led to the faster return of RR. Nevertheless, Iso’s anaesthesia effects of strong respiratory depression and severe hypotension remained. Based on this increased anaesthesia risk with Iso, MMF anaesthesia is preferable for repeated use in GPs.

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<![CDATA[Ebolavirus Glycoprotein Fc Fusion Protein Protects Guinea Pigs against Lethal Challenge]]> https://www.researchpad.co/article/5989da4eab0ee8fa60b8d4b7

Ebola virus (EBOV), a member of the Filoviridae that can cause severe hemorrhagic fever in humans and nonhuman primates, poses a significant threat to the public health. Currently, there are no licensed vaccines or therapeutics to prevent and treat EBOV infection. Several vaccines based on the EBOV glycoprotein (GP) are under development, including vectored, virus-like particles, and protein-based subunit vaccines. We previously demonstrated that a subunit vaccine containing the extracellular domain of the Ebola ebolavirus (EBOV) GP fused to the Fc fragment of human IgG1 (EBOVgp-Fc) protected mice against EBOV lethal challenge. Here, we show that the EBOVgp-Fc vaccine formulated with QS-21, alum, or polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly-ICLC) adjuvants induced strong humoral immune responses in guinea pigs. The vaccinated animals developed anti-GP total antibody titers of approximately 105−106 and neutralizing antibody titers of approximately 103 as assessed by a BSL-2 neutralization assay based on vesicular stomatitis virus (VSV) pseudotypes. The poly-ICLC formulated EBOVgp-Fc vaccine protected all the guinea pigs against EBOV lethal challenge performed under BSL-4 conditions whereas the same vaccine formulated with QS-21 or alum only induced partial protection. Vaccination with a mucin-deleted EBOVgp-Fc construct formulated with QS-21 adjuvant did not have a significant effect in anti-GP antibody levels and protection against EBOV lethal challenge compared to the full-length GP construct. The bulk of the humoral response induced by the EBOVgp-Fc vaccine was directed against epitopes outside the EBOV mucin region. Our findings indicate that different adjuvants can eliciting varying levels of protection against lethal EBOV challenge in guinea pigs vaccinated with EBOVgp-Fc, and suggest that levels of total anti-GP antibodies elicit by protein-based GP subunit vaccines do not correlate with protection. Our data further support the development of Fc fusions of GP as a candidate vaccine for human use.

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<![CDATA[Leishmania enriettii (Muniz & Medina, 1948): A highly diverse parasite is here to stay]]> https://www.researchpad.co/article/5989db5cab0ee8fa60be01b5 ]]> <![CDATA[Myocardial KChIP2 Expression in Guinea Pig Resolves an Expanded Electrophysiologic Role]]> https://www.researchpad.co/article/5989dacfab0ee8fa60bb5b40

Cardiac ion channels and their respective accessory subunits are critical in maintaining proper electrical activity of the heart. Studies have indicated that the K+ channel interacting protein 2 (KChIP2), originally identified as an auxiliary subunit for the channel Kv4, a component of the transient outward K+ channel (Ito), is a Ca2+ binding protein whose regulatory function does not appear restricted to Kv4 modulation. Indeed, the guinea pig myocardium does not express Kv4, yet we show that it still maintains expression of KChIP2, suggesting roles for KChIP2 beyond this canonical auxiliary interaction with Kv4 to modulate Ito. In this study, we capitalize on the guinea pig as a system for investigating how KChIP2 influences the cardiac action potential, independent of effects otherwise attributed to Ito, given the endogenous absence of the current in this species. By performing whole cell patch clamp recordings on isolated adult guinea pig myocytes, we observe that knock down of KChIP2 significantly prolongs the cardiac action potential. This prolongation was not attributed to compromised repolarizing currents, as IKr and IKs were unchanged, but was the result of enhanced L-type Ca2+ current due to an increase in Cav1.2 protein. In addition, cells with reduced KChIP2 also displayed lowered INa from reduced Nav1.5 protein. Historically, rodent models have been used to investigate the role of KChIP2, where dramatic changes to the primary repolarizing current Ito may mask more subtle effects of KChIP2. Evaluation in the guinea pig where Ito is absent, has unveiled additional functions for KChIP2 beyond its canonical regulation of Ito, which defines KChIP2 as a master regulator of cardiac repolarization and depolarization.

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<![CDATA[KChIP2 regulates the cardiac Ca2+ transient and myocyte contractility by targeting ryanodine receptor activity]]> https://www.researchpad.co/article/5989db52ab0ee8fa60bdc606

Pathologic electrical remodeling and attenuated cardiac contractility are featured characteristics of heart failure. Coinciding with these remodeling events is a loss of the K+ channel interacting protein, KChIP2. While, KChIP2 enhances the expression and stability of the Kv4 family of potassium channels, leading to a more pronounced transient outward K+ current, Ito,f, the guinea pig myocardium is unique in that Kv4 expression is absent, while KChIP2 expression is preserved, suggesting alternative consequences to KChIP2 loss. Therefore, KChIP2 was acutely silenced in isolated guinea pig myocytes, which led to significant reductions in the Ca2+ transient amplitude and prolongation of the transient duration. This change was reinforced by a decline in sarcomeric shortening. Notably, these results were unexpected when considering previous observations showing enhanced ICa,L and prolonged action potential duration following KChIP2 loss, suggesting a disruption of fundamental Ca2+ handling proteins. Evaluation of SERCA2a, phospholamban, RyR, and sodium calcium exchanger identified no change in protein expression. However, assessment of Ca2+ spark activity showed reduced spark frequency and prolonged Ca2+ decay following KChIP2 loss, suggesting an altered state of RyR activity. These changes were associated with a delocalization of the ryanodine receptor activator, presenilin, away from sarcomeric banding to more diffuse distribution, suggesting that RyR open probability are a target of KChIP2 loss mediated by a dissociation of presenilin. Typically, prolonged action potential duration and enhanced Ca2+ entry would augment cardiac contractility, but here we see KChIP2 fundamentally disrupts Ca2+ release events and compromises myocyte contraction. This novel role targeting presenilin localization and RyR activity reveals a significance for KChIP2 loss that reflects adverse remodeling observed in cardiac disease settings.

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