RESUMEN

ETHNOPHARMACOLOGICAL RELEVANCE: Panax notoginseng flowers, which are the buds of the traditional Chinese medicinal herb Sanqi, are widely used in China for their cough-ameliorating properties, with demonstrated therapeutic effects in the treatment of both acute and chronic coughs. However, both the antitussive mechanism and active compound basis of P. notoginseng flowers remain poorly understood. AIM OF THE STUDY: We investigated the antitussive effects of P. notoginseng flowers, identified the bioactive constituents responsible for alleviating cough symptoms, and elucidated the underlying pharmacological mechanisms. MATERIALS AND METHODS: We analyzed the major chemical constituents of aqueous extracts of P. notoginseng flowers using liquid chromatography-mass spectrometry and quantitatively analyzed the key component, 20S-ginsenoside Rh2, using high-performance liquid chromatography. Using a cough reflex model in healthy mice and an ovalbumin-induced, highly sensitive guinea pig cough model, we verified the suppressive effects of P. notoginseng flowers and their saponin constituents on coughing. Furthermore, we explored the mechanisms of action of the key ion channels, NaV1.7 and TRPV1, using whole-cell patch-clamp techniques and molecular docking. Finally, the therapeutic mechanisms of P. notoginseng flowers on pathological cough were revealed using hematoxylin and eosin staining, immunohistochemistry, and western blotting. RESULTS: The active components of P. notoginseng flowers were primarily protopanaxadiol-type saponins, among which 20S-ginsenoside Rh2 had the highest content (51.46 mg/g). In the mouse model, P. notoginseng flowers exhibited antitussive effects comparable to those of pentoxyverine citrate. Although its main saponin component, 20S-ginsenoside Rh2, showed slightly weaker effects, it still demonstrated concentration-dependent inhibition of channel activity. The whole-cell patch-clamp technique and virtual molecular docking showed that Rh2 might exert its effects by directly binding to the NaV1.7 and TRPV1 channels. In the guinea pig model, P. notoginseng flowers and their saponin components not only reduced cough frequency and prolonged the latency period before cough onset, but also significantly inhibited tracheal and pulmonary inflammation and the overexpression of TRPV1. CONCLUSIONS: 20S-Ginsenoside Rh2, the major bioactive saponin in P. notoginseng flowers, exhibits potent antitussive effects. The potential mechanism of action of 20S-Ginsenoside Rh2 in the treatment of cough may involve inhibiting NaV1.7 and TRPV1 channel currents through direct binding to core protein active sites and downregulating TRPV1 expression.

Asunto(s)

Antitusígenos , Tos , Regulación hacia Abajo , Flores , Ginsenósidos , Canal de Sodio Activado por Voltaje NAV1.7 , Panax notoginseng , Canales Catiónicos TRPV , Animales , Canales Catiónicos TRPV/metabolismo , Cobayas , Flores/química , Tos/tratamiento farmacológico , Ginsenósidos/farmacología , Antitusígenos/farmacología , Masculino , Ratones , Panax notoginseng/química , Regulación hacia Abajo/efectos de los fármacos , Humanos , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Canal de Sodio Activado por Voltaje NAV1.7/efectos de los fármacos , Células HEK293 , Simulación del Acoplamiento Molecular , Cricetulus , Modelos Animales de Enfermedad , Células CHO , Saponinas/farmacología , Ovalbúmina

RESUMEN

Trixacarus caviae is a sarcoptic mange mite infesting guinea pigs. Infestation in immunosuppressed animals produces severe dermatological problems, including alopecia, intense pruritus, hyperkeratosis and non-dermatological issues (e.g., seizures). Treatment options are limited and include topical application of macrocyclic lactones or amitraz or injectable administration of ivermectin or doramectin. Considering the severity of the disease and the challenging treatment, the present paper aimed to determine the efficacy of oral afoxolaner in a severe case of infestation with T. caviae in a pet guinea pig. One female guinea pig was referred to the New Companion Animal Clinic due to severe dermatological problems. A clinical evaluation was done, and skin scrapings were collected and examined under the microscope. Small mites were detected and morphologically identified as T. caviae. The animal was treated with a single oral dose of 2.50 mg/kg afoxolaner, and the lesions, presence/absence of mites and intensity of pruritus were evaluated periodically until 2 months post-treatment. A week after the medication, the lesions were milder, but pruritus was still present and was attributed to the healing process. Further examinations showed significant improvement with the complete remission of clinical signs and no mites at the microscopic examination after 4 weeks. Afoxolaner was safe and effective in this guinea pig for the treatment of T. caviae mange with no repetition needed.

Asunto(s)

Naftalenos , Animales , Cobayas , Femenino , Naftalenos/administración & dosificación , Naftalenos/uso terapéutico , Infestaciones por Ácaros/veterinaria , Infestaciones por Ácaros/tratamiento farmacológico , Infestaciones por Ácaros/parasitología , Acaricidas/uso terapéutico , Acaricidas/administración & dosificación , Mascotas , Pirimidinas/administración & dosificación , Pirimidinas/uso terapéutico , Enfermedades de los Roedores/tratamiento farmacológico , Enfermedades de los Roedores/parasitología , Isoxazoles

RESUMEN

BACKGROUND: Preterm birth is associated with brain injury and long-term behavioral abnormalities, for which there are limited prevention options. When born preterm, infants prematurely lose placental neurosteroid (allopregnanolone) support. This increases the risk of excitotoxic damage to the brain, which increases the risk of injury, causing long-term deficits in behavior, myelination, and alterations to neurotransmitter pathways. We propose that postnatal restoration of neurosteroid action through zuranolone therapy will reduce neurological impairments following preterm birth. METHODS: Guinea pig dams underwent survival cesarean section surgery to deliver pups prematurely (GA64) or at term (GA69). Between birth and term equivalence age, preterm pups received vehicle (15% ß-cyclodextrin) or the allopregnanolone analogue zuranolone (1 mg/kg/day). Behavioral analysis was performed at postnatal day (PND) 7 and 40, before tissue collection at PND 42. Immunostaining for myelin basic protein (MBP), as well as real-time polymerase chain reaction to characterize oligodendrocyte lineage and neurotransmitter pathways, was performed in frontal cortex tissues. RESULTS: Zuranolone treatment prevented the hyperactive phenotype in preterm-born offspring, most markedly in males. Additionally, preterm-related reductions in MBP were ameliorated. Several preterm-related alterations in mRNA expression of dopaminergic, glutamatergic, and GABAergic pathways were also restored back to that of a term control level. CONCLUSION: This is the first study to assess zuranolone treatment as a neuroprotective therapy following preterm birth. Zuranolone treatment improved behavioral outcomes and structural changes in the preterm offspring, which continued long term until at least a late childhood timepoint. Clinical studies are warranted for further exploring the neuroprotective possibilities of this treatment following preterm birth.

Asunto(s)

Lóbulo Frontal , Pregnanolona , Nacimiento Prematuro , Animales , Pregnanolona/farmacología , Femenino , Cobayas , Masculino , Nacimiento Prematuro/prevención & control , Nacimiento Prematuro/tratamiento farmacológico , Lóbulo Frontal/efectos de los fármacos , Lóbulo Frontal/metabolismo , Animales Recién Nacidos , Embarazo , Conducta Animal/efectos de los fármacos , Conducta Animal/fisiología , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/administración & dosificación , Oligodendroglía/efectos de los fármacos , Oligodendroglía/metabolismo , Proteína Básica de Mielina/metabolismo

RESUMEN

ABSTRACT: The effects of the calcium sensitizer levosimendan on hemodynamics and survival in guinea pigs intoxicated with the calcium blockers verapamil or diltiazem were evaluated in a randomized controlled study. One hundred four animals were randomized to be intoxicated with either verapamil (2.0 mg/kg) or diltiazem (4.5 mg/kg) and thereafter further randomized into 6 groups which received either saline (control), 3 different regimes of levosimendan, calcium chloride, and levosimendan combined with calcium chloride. The hemodynamics and survival of the animals were followed for 60 minutes after intoxication.The negative inotropic effect of calcium blockers was seen as a decrease by over 70% of the positive derivative of the left ventricular pressure. This was reversed by levosimendan. Moreover, both verapamil and diltiazem-induced marked hypotension (-69% and -63% of the baseline value, respectively) which was also reversed by levosimendan. The combined levosimendan and calcium chloride treatment had a synergistic effect in reversing verapamil or diltiazem-induced deterioration in hemodynamics.Both verapamil and diltiazem intoxications decreased the survival rate of guinea pigs to 13%. Levosimendan addition improved survival dose-dependently up to a survival rate of 75% and 88% in the verapamil and diltiazem groups, respectively. Low dose of levosimendan combined with calcium chloride improved survival in verapamil and diltiazem group to 88% and 100%, respectively.In conclusion, the administration of levosimendan improved hemodynamics and survival in calcium channel blocker intoxicated guinea pigs. The synergistic effect of levosimendan and calcium chloride suggests that this combination could be an effective antidote in calcium channel blocker intoxications.

Asunto(s)

Antídotos , Bloqueadores de los Canales de Calcio , Diltiazem , Hidrazonas , Piridazinas , Simendán , Verapamilo , Animales , Simendán/farmacología , Cobayas , Bloqueadores de los Canales de Calcio/farmacología , Hidrazonas/farmacología , Piridazinas/farmacología , Diltiazem/farmacología , Verapamilo/farmacología , Antídotos/farmacología , Masculino , Hemodinámica/efectos de los fármacos , Cloruro de Calcio , Cardiotónicos/farmacología , Sinergismo Farmacológico , Modelos Animales de Enfermedad , Quimioterapia Combinada , Tasa de Supervivencia

RESUMEN

To evaluate the changes of choroidal thickness (CT) and blood flow related to myopia, and its effects of vascular endothelial growth factor (VEGFA) on choroidal vessels in myopia. Subjects were included and divided into emmetropia (EM), non-high myopia (Non-HM) and high myopia (HM) groups. we measured choroidal thickness (CT), choriocapillaris vessel density (VD), and VEGFA content in tears in humans (137 subjects for CT, VD and 84 for tear) and detected the role of VEGFA in the choroid in form-deprivation myopia (FDM) in guinea pigs. Twenty-four guinea pigs were divided into control and FDM groups, and the expression changes of choroidal vessels and VEGFA were observed and compared using immunohistochemistry and Western blotting. Twenty-one guinea pigs were divided into control, FDM + Vehicle and FDM + Conbercept groups. The changes of diopter, axis length and choroidal vessels after intravitreal injection of Conbercept were observed. There were significant differences in CT and VD among the three groups (p < 0.05). VEGFA levels in tears were significantly lower in the myopic groups, with a decreasing trend from EM to Non-HM to HM. The choroidal vascular area fraction of FDM decreased compared to the control group. FDM guinea pigs exhibited reduced choroidal vasculature and significant downregulation of VEGFA expression. Following intravitreal injection of conbercept, the FDM + Conbercept group showed greater myopia, longer axial length, and lower choroidal vascular area fraction compared to the control group. VEGFA may participate in the regulation of choroidal blood vessels and blood flow in the progression of myopia. The reduction in VEGFA may accelerates the progression of myopia.

Asunto(s)

Coroides , Miopía , Factor A de Crecimiento Endotelial Vascular , Coroides/metabolismo , Coroides/irrigación sanguínea , Coroides/patología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Cobayas , Miopía/metabolismo , Miopía/patología , Masculino , Humanos , Femenino , Adulto , Biomarcadores/metabolismo , Tomografía de Coherencia Óptica , Densidad Microvascular , Modelos Animales de Enfermedad , Proteínas Recombinantes de Fusión

RESUMEN

Repeated exposure of animals to Ixodes scapularis ticks can result in acquired tick resistance (ATR). The first manifestation of ATR is erythema at the tick bite site, however, the specific peptide targets and mechanisms associated with this early aspect of ATR are not understood. In this study, we immunized guinea pigs with a lipid nanoparticle containing the mRNA encoding 25 amino acids in the carboxyl terminus of Salp14 (Salp14-C mRNA-LNP), an I. scapularis salivary protein. The animals produced high titers of IgG directed at the carboxyl terminus of Salp14. Guinea pigs immunized with Salp14-C mRNA-LNP and then exposed to I. scapularis, developed erythema at the tick bite site. Transcriptomics of the skin of guinea pigs at the I. scapularis bite sites elucidated selected pathways, including histamine activation, that are associated with the development of erythema. The study demonstrates that an mRNA vaccine encoding a small peptide can induce the initial phase of ATR in guinea pigs.

Asunto(s)

Ixodes , Mordeduras de Garrapatas , Animales , Cobayas , Mordeduras de Garrapatas/inmunología , Ixodes/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Vacunación/métodos , Proteínas y Péptidos Salivales/inmunología , Proteínas y Péptidos Salivales/genética , Epítopos/inmunología , Femenino , ARN Mensajero/inmunología , ARN Mensajero/genética , Nanopartículas/química , Eritema/inmunología , Eritema/etiología , Vacunas de ARNm , Liposomas

RESUMEN

Pulmonary surfactant serves as a barrier to respiratory epithelium but can also regulate airway smooth muscle (ASM) tone. Surfactant (SF) relaxes contracted ASM, similar to ß2-agonists, anticholinergics, nitric oxide, and prostanoids. The exact mechanism of surfactant relaxation and whether surfactant relaxes hyperresponsive ASM remains unknown. Based on previous research, relaxation requires an intact epithelium and prostanoid synthesis. We sought to examine the mechanisms by which surfactant causes ASM relaxation. Organ bath measurements of isometric tension of ASM of guinea pigs in response to exogenous surfactant revealed that surfactant reduces tension of healthy and hyperresponsive tracheal tissue. The relaxant effect of surfactant was reduced if prostanoid synthesis was inhibited and/or if prostaglandin E2-related EP2 receptors were antagonized. Atomic force microscopy revealed that human ASM cells stiffen during contraction and soften during relaxation. Surfactant softened ASM cells, similarly to the known bronchodilator prostaglandin E2 (PGE2) and the cell softening was abolished when EP4 receptors for PGE2 were antagonized. Elevated levels of PGE2 were found in cultures of normal human bronchial epithelial cells exposed to pulmonary surfactant. We conclude that prostaglandin E2 and its EP2 and EP4 receptors are likely involved in the relaxant effect of pulmonary surfactant in airways.

Asunto(s)

Dinoprostona , Relajación Muscular , Músculo Liso , Surfactantes Pulmonares , Tráquea , Cobayas , Animales , Humanos , Masculino , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , Músculo Liso/metabolismo , Relajación Muscular/efectos de los fármacos , Dinoprostona/farmacología , Dinoprostona/metabolismo , Surfactantes Pulmonares/metabolismo , Surfactantes Pulmonares/farmacología , Tráquea/efectos de los fármacos , Tráquea/fisiología , Tráquea/metabolismo , Subtipo EP2 de Receptores de Prostaglandina E/metabolismo , Células Cultivadas , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo

RESUMEN

The auditory brainstem response (ABR) can be used to evaluate hearing sensitivity of animals. However, typical measurement protocols are time-consuming. Here, an adaptive algorithm is proposed for efficient ABR threshold estimation. The algorithm relies on the update of the predicted hearing threshold from a Gaussian process model as ABR data are collected using iteratively optimized stimuli. To validate the algorithm, ABR threshold estimation is simulated by adaptively subsampling pre-collected ABR datasets. The simulated experiment is performed on 5 datasets of mouse, budgerigar, gerbil, and guinea pig ABRs (27 ears). The datasets contain 68-106 stimuli conditions, and the adaptive algorithm is configured to terminate after 20 stimuli conditions. The algorithm threshold estimate is compared against human rater estimates who visually inspected the full waveform stacks. The algorithm threshold matches the human estimates within 10 dB, averaged over frequency, for 15 of the 27 ears while reducing the number of stimuli conditions by a factor of 3-5 compared to standard practice. The intraclass correlation coefficient is 0.81 with 95% upper and lower bounds at 0.74 and 0.86, indicating moderate to good reliability between human and algorithm threshold estimates. The results demonstrate the feasibility of a Bayesian adaptive procedure for rapid ABR threshold estimation.

Asunto(s)

Algoritmos , Umbral Auditivo , Potenciales Evocados Auditivos del Tronco Encefálico , Animales , Potenciales Evocados Auditivos del Tronco Encefálico/fisiología , Umbral Auditivo/fisiología , Cobayas , Ratones , Humanos , Gerbillinae/fisiología , Estimulación Acústica/métodos , Reproducibilidad de los Resultados , Audición/fisiología

RESUMEN

Human hair is increasingly employed as a non-invasive biomonitoring matrix for exposure to organic contaminants (OCs). Decontamination procedures are generally needed to remove external contamination from hair prior to analysis of OCs. Despite various existing decontamination protocols, their impacts on internally incorporated (endogenous) OCs in hair remain poorly understood. This study aims to quantitatively assess the impact of decontamination procedures on endogenous OCs in hair, and investigate optimal decontamination processes and factors influencing the removal of endogenous OCs. In this study, guinea pig was exposed to 6 OCs (triphenyl phosphate (TPHP), tris(1,3-dichloro-2-propyl) phosphate (TDCPP), and tri-n-butyl phosphate (TNBP), bisphenol A (BPA), perfluorooctanoic acid (PFOA), and phenanthrene (PHE)), and 6 decontamination procedures with different solvents (methanol, n-hexane, acetone, ultrapure water, Triton X-100, and sodium dodecyl sulfate) were used to rinse exposed guinea pig hair. All OCs and three metabolites (diphenyl phosphate (DPHP), dibutyl phosphate (DBP), and bis(1,3-dichloro-2-propyl) phosphate (BDCPP)) were detected in the majority of washing solutions. The decontamination procedures apparently resulted in the release of endogenous OCs from hair. The percentages of residual OCs in hair exhibited a linear or exponential decrease with more washing cycles. Furthermore, the residuals of OCs in hair washed with organic and aqueous solvents showed negative correlations with molecular weight, polarizability, and their initial concentrations. Although these findings need to be validated with a broader range of OCs, the results obtained in this study provide compelling evidence that current hair decontamination procedures have significant impacts on the analysis of endogenous OCs in hair. Therefore, it is important to interpret quantitative data on hair OC concentrations with caution and to thoroughly consider each decontamination procedure during analysis.

Asunto(s)

Monitoreo Biológico , Descontaminación , Cabello , Descontaminación/métodos , Cabello/química , Cobayas , Animales , Fluorocarburos/metabolismo , Fluorocarburos/análisis , Contaminantes Orgánicos Persistentes/metabolismo , Compuestos de Bencidrilo , Fenoles/análisis , Caprilatos , Organofosfatos/metabolismo , Fenantrenos/metabolismo , Monitoreo del Ambiente/métodos

RESUMEN

AIMS: Cancer-related thrombosis (CAT) is a common complication in cancer patients, significantly impacting their quality of life and survival prospects. Nattokinase (NK) has potent thrombolytic properties, however, its efficacy is limited by low oral bioavailability and the risk of severe allergic reactions with intravenous use. Heparin (HP) is a widely used anticoagulant in clinical settings. This study aimed to overcome the intravenous toxicity of NK and explore its effect on CAT in advanced tumors. MAIN METHODS: In this study, NK-HP electrostatic complexes were constructed, and their safety and thrombolytic efficacy were verified through guinea pig allergy tests, mouse tail vein tests, and both in vivo and in vitro thrombolysis experiments. Additionally, an S180 advanced tumor model was developed and combined with sialic acid-modified doxorubicin liposomes (DOX-SAL) to investigate the impact of NK-HP on CAT and its antitumor effects in advanced tumors. KEY FINDINGS: We observed that NK-HP can eliminate the intravenous injection toxicity of NK, has strong thrombolytic performance, and can prevent thrombosis formation. Intravenous injection of NK-HP can enhance the antitumor effect of DOX-SAL by reducing the fibrin content in advanced tumors and increasing the levels of the cross-linked protein degradation product D-dimer. SIGNIFICANCE: This study developed a method to eliminate the intravenous injection toxicity of NK, proposing a promising therapeutic strategy for CAT treatment, particularly for CAT in advanced tumors, and improving the efficacy of nano-formulations in anti-tumor therapy.

Asunto(s)

Heparina , Neoplasias , Subtilisinas , Trombosis , Animales , Subtilisinas/administración & dosificación , Ratones , Trombosis/tratamiento farmacológico , Inyecciones Intravenosas , Heparina/administración & dosificación , Neoplasias/tratamiento farmacológico , Fibrinolíticos/administración & dosificación , Fibrinolíticos/farmacología , Electricidad Estática , Cobayas , Masculino , Doxorrubicina/administración & dosificación , Doxorrubicina/farmacología , Liposomas , Humanos

RESUMEN

The ciliary muscle constitutes a crucial element in refractive regulation. Investigating the pathophysiological mechanisms within the ciliary muscle during excessive contraction holds significance in treating ciliary muscle dysfunction. A guinea pig model of excessive contraction of the ciliary muscle induced by drops pilocarpine was employed, alongside the primary ciliary muscle cells was employed in in vitro experiments. The results of the ophthalmic examination showed that pilocarpine did not significantly change refraction and axial length during the experiment, but had adverse effects on the regulatory power of the ciliary muscle. The current data reveal notable alterations in the expression profiles of hypoxia inducible factor 1 (HIF-1α), ATP2A2, P53, α-SMA, Caspase-3, and BAX within the ciliary muscle of animals subjected to pilocarpine exposure, alongside corresponding changes observed in cultured cells treated with pilocarpine. Augmented levels of ROS were detected in both tissue specimens and cells, culminating in a significant increase in cell apoptosis in in vivo and in vitro experiments. Further examination revealed that pilocarpine induced an increase in intracellular Ca2+ levels and disrupted MMP, as evidenced by mitochondrial swelling and diminished cristae density compared to control conditions, concomitant with a noteworthy decline in antioxidant enzyme activity. However, subsequent blockade of Ca2+ channels in cells resulted in downregulation of HIF-1α, ATP2A2, P53, α-SMA, Caspase-3, and BAX expression, alongside ameliorated mitochondrial function and morphology. The inhibition of Ca2+ channels presents a viable approach to mitigate ciliary cells damage and sustain proper ciliary muscle function by curtailing the mitochondrial damage induced by excessive contractions.

Asunto(s)

Apoptosis , Calcio , Senescencia Celular , Pilocarpina , Animales , Pilocarpina/farmacología , Cobayas , Apoptosis/efectos de los fármacos , Calcio/metabolismo , Senescencia Celular/efectos de los fármacos , Cuerpo Ciliar/metabolismo , Masculino , Células Cultivadas , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

Intact capsids of foot-and-mouth disease virus (FMDV) play a vital role in eliciting a protective immune response. Any change in the physico-chemical environment of the capsids results in dissociation and poor immunogenicity. Structural bioinfomatics studies have been carried out to predict the amino acids at the interpentameric region that resulted in the identification of mutant virus-like particles(VLPs) of FMDV serotype Asia1/IND/63/1972. The insect cell expressed VLPs were evaluated for their stability by sandwich ELISA. Among 10 mutants, S93H showed maximum retention of antigenicity at different temperatures, indicating its higher thermal stability as revealed by the in-silico analysis and retained the antigenic sites of the virus demonstrated by Sandwich ELISA. The concordant results of the liquid phase blocking ELISA for estimation of antibody titre of known sera with stable mutant VLP as antigen in place of virus antigen demonstrate its diagnostic potential. The stable mutant VLP elicited a robust immune response with 85.6 % protection in guinea pigs against virus challenge. The stabilized VLP based antigen requires minimum biosafety and cold storage for production and transit besides, complying with differentiation of infected from vaccinated animals. It can effectively replace the conventional virus handling during antigen production for prophylactic and diagnostic use.

Asunto(s)

Virus de la Fiebre Aftosa , Fiebre Aftosa , Serogrupo , Virus de la Fiebre Aftosa/inmunología , Virus de la Fiebre Aftosa/genética , Animales , Fiebre Aftosa/prevención & control , Fiebre Aftosa/diagnóstico , Fiebre Aftosa/inmunología , Cobayas , Vacunas de Partículas Similares a Virus/inmunología , Vacunas de Partículas Similares a Virus/genética , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/sangre , Antígenos Virales/inmunología , Antígenos Virales/genética , Proteínas de la Cápside/inmunología , Proteínas de la Cápside/genética , Proteínas de la Cápside/química , Vacunas Virales/inmunología , Vacunas Virales/genética , Mutación

RESUMEN

BACKGROUND AND OBJECTIVES: Fractional ablative resurfacing techniques are preferred treatments for facial rejuvenation of aged skin. This study was performed to investigate the cutaneous effects of using a fractional picosecond laser at 1064 nm with a diffractive lens. METHODS: The penetration depth according to the location of the handpiece tip was evaluated using an acrylic panel. Laser induced optical breakdown (LIOB) and cutaneous damage were observed after hematoxylin and eosin staining in guinea pigs. Collagen formation was evaluated using Victoria staining, Masson's trichrome (MT) staining, and immunohistochemical staining for collagen type III. RESULTS: The penetration depth for LEVEL 1 was 499.98-935.23 µm (average: 668.75 ± 182.84 µm); the LIOB cavity area was 1664.17 ± 650.52 µm2. The penetration depth of LEVEL 2 was 257.12-287.38 µm (average: 269.77 ± 14.55 µm) with an LIOB cavity area of 1335.85 ± 214.41 µm2. At LEVEL 3, that was 36.17-53.69 µm (average: 52.15 ± 20.81 µm) and the LIOB cavity area was 1312.67 ± 1069.12 µm2. No epidermal tissue damage was observed and collagen formation was observed from day 14 under all conditions. CONCLUSION: Diffractive optical element (DOE) lens arranged laser treatment system controlled the position of LIOB occurrence and an irradiating area.

Asunto(s)

Terapia por Luz de Baja Intensidad , Animales , Cobayas , Terapia por Luz de Baja Intensidad/métodos , Terapia por Luz de Baja Intensidad/instrumentación , Terapia por Luz de Baja Intensidad/efectos adversos , Rejuvenecimiento , Piel/efectos de la radiación , Envejecimiento de la Piel/efectos de la radiación

RESUMEN

Small mammals are very popular companion animals, and the incidence of particular tumour types in these animals is the subject of extensive research. We carried out a retrospective and comparative analysis of the incidence of reproductive tract and mammary tumours and tumour-like lesions collected from 103 pet rabbits, 75 pet rats, 71 guinea pigs, 12 mice, 11 hamsters, eight African pygmy hedgehogs, four ferrets and two chinchillas. The results indicate that uterine tumours and tumour-like lesions are common in pet rabbits, guinea pigs and African pygmy hedgehogs. In pet rabbits, the most common uterine tumour was endometrial adenocarcinoma, while in guinea pigs benign lesions predominated (ie, leiomyoma, endometrial adenoma, cystic endometrial hyperplasia and deciduoma). Uterine tumours in African pygmy hedgehogs included adenosarcomas and endometrial polyps. Ovarian lesions were found only in guinea pigs (ovarian rete adenomas, rete cysts) and African pygmy hedgehogs (mostly granulosa cell tumours), while testicular tumours were diagnosed in pet rabbits, one pet rat and one guinea pig. Mammary tumours were common in pet rabbits, pet rats, guinea pigs, mice, hamsters and African pygmy hedgehogs. In pet rats, the most common mammary tumour was fibroadenoma, while in other animals carcinomas predominated. In guinea pigs and, to a lesser extent, in pet rats, a significant percentage of mammary tumours occurred in males. Guinea pigs seem to be predisposed to mammary tumours of ductal origin. This study describes for the first time uterine angioleiomyoma in the pet rabbit and mammary spindle cell carcinoma in the Djungarian hamster and chinchilla.

Asunto(s)

Neoplasias de los Genitales Femeninos , Neoplasias Mamarias Animales , Animales , Femenino , Conejos , Ratas , Estudios Retrospectivos , Cobayas , Neoplasias Mamarias Animales/patología , Neoplasias de los Genitales Femeninos/veterinaria , Neoplasias de los Genitales Femeninos/patología , Ratones , Masculino , Cricetinae , Hurones , Mascotas , Chinchilla

RESUMEN

Glutathione (GSH) exhibits considerable potential in the cosmetic industry for reducing intracellular tyrosinase activity and inhibiting melanin synthesis. However, its efficacy is hindered by limited permeability, restricting its ability to reach the basal layer of the skin where melanin production occurs. The transdermal enhancer peptide TD1 has emerged as a promising strategy to facilitate the transdermal transfer of proteins or peptides by creating intercellular gaps in keratinocytes, providing access to the basal layer. The primary objective of this study is to enhance the transdermal absorption capacity of GSH while augmenting its inhibitory effect on melanin. Two coupling structures were designed for investigation: linear (TD1-linker-GSH) and branched (TD1-GSH). The study examined the impact of the peptide skeleton on melanin inhibition ability. Our findings revealed that the linear structure not only inhibited synthetic melanin production in B16F10 cells through a direct pathway but also through a paracrine pathway, demonstrating a significant tyrosinase inhibition of nearly 70 %, attributed to the paracrine effect of human keratinocyte HaCaT. In pigmentation models of guinea pigs and zebrafish, the application of TD1-linker-GSH significantly reduced pigmentation. Notably, electric two-photon microscopy demonstrated that TD1-linker-GSH exhibited significant transdermal ability, penetrating 158.67 ± 9.28 µm into the skin of living guinea pigs. Molecular docking analysis of the binding activity with tyrosinase revealed that both TD1-linker-GSH and TD1-GSH occupy the same active pocket, with TD1-linker-GSH binding more tightly to tyrosinase. These results provide a potential foundation for therapeutic approaches aimed at enriched pigmentation and advance our understanding of the mechanisms underlying melanogenesis inhibition.

Asunto(s)

Administración Cutánea , Glutatión , Melaninas , Monofenol Monooxigenasa , Pez Cebra , Melaninas/metabolismo , Animales , Humanos , Cobayas , Glutatión/metabolismo , Glutatión/química , Monofenol Monooxigenasa/metabolismo , Monofenol Monooxigenasa/antagonistas & inhibidores , Péptidos/química , Péptidos/farmacología , Péptidos/síntesis química , Péptidos/administración & dosificación , Ratones , Estructura Molecular , Relación Estructura-Actividad , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/administración & dosificación , Melanogénesis

RESUMEN

OBJECTIVE: Flagellin protein, an integral component of flagella, provides motility to several bacterial species and also acts as a candidate antigen in diagnostics and subunit vaccines. The bulk production of flagellin with retention of all conformational epitopes using recombinant protein technology is of paramount importance in the development of pathogen-specific immuno-assays and vaccines. We describe the production of highly soluble and immuno-reactive rFliA(C) protein of Clostridium chauvoei, a causative agent of blackleg or black quarter (BQ) affecting cattle and small ruminants worldwide. The bacterium is known to possess peritrichous flagella that provide motility and also act as a virulence factor with high protective antigenicity. METHODS: Upon sequence and structural analysis, a partial fliA(C) gene from Clostridium chauvoei was cloned and the recombinant mature protein with N- and C- terminal truncation was over-expressed as a His-tagged fusion protein (∼25 kDa) in Escherichia coli. Subsequently, rFliA(C) protein was purified by single-step affinity chromatography and characterized for its immuno-reactivity in laboratory animals, Western blot, and indirect-ELISA format. RESULTS: rFliA(C) was highly soluble and was purified in high quantity and quality. rFliA(C) elicited antigen-specific conformational polyclonal antibodies in rabbit and guinea pig models, as well as anti-Clostridium chauvoei-specific antibodies being specifically detected in BQ-vaccinated and convalescent sera of bovines in Western blot and in indirect-ELISA format. Further, no cross reactivity was noted with antibodies against major bovine diseases (e.g., foot-and-mouth disease, IBR, LSDV, hemorrhagic septicaemia, brucellosis, and leptospirosis). CONCLUSION: The study indicated the production of conformational recombinant flagellin-rFliA(C)-antigen and its potential utility in development of diagnostics for detection of Clostridium chauvoei-specific antibodies in BQ-recovered and/or vaccinated animals.

Asunto(s)

Anticuerpos Antibacterianos , Clostridium chauvoei , Flagelina , Proteínas Recombinantes , Flagelina/inmunología , Flagelina/genética , Animales , Clostridium chauvoei/inmunología , Clostridium chauvoei/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/genética , Conejos , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Cobayas , Vacunas Bacterianas/inmunología , Vacunas Bacterianas/genética , Infecciones por Clostridium/veterinaria , Infecciones por Clostridium/inmunología , Infecciones por Clostridium/microbiología , Bovinos , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/genética , Enfermedades de los Bovinos/inmunología , Enfermedades de los Bovinos/microbiología , Escherichia coli/genética , Escherichia coli/metabolismo , Ensayo de Inmunoadsorción Enzimática , Clonación Molecular

RESUMEN

HYPOTHESIS: Microneedle-mediated intracochlear injection of siRNA-Lipofectamine through the round window membrane (RWM) can be used to transfect cells within the cochlea. BACKGROUND: Our laboratory has developed 100-µm diameter hollow microneedles for intracochlear injection through the guinea pig RWM. In this study, we test the feasibility of microneedle-mediated injection of siRNA and Lipofectamine, a commonly used reagent with known cellular toxicity, through the RWM for cochlear transfection. METHODS: Fluorescently labeled scramble siRNA was diluted into Lipofectamine RNAiMax and OptiMEM. One microliter of 5 µM siRNA was injected through the RWM of Hartley guinea pigs at a rate of 1 µl/min (n = 22). In a control group, 1.0 µl of Lipofectamine, with no siRNA, was diluted into OptiMEM and injected in a similar fashion (n = 5). Hearing tests were performed before and either at 24 hours, 48 hours, or 5 days after injection. Afterward, animals were euthanized, and cochleae were harvested for imaging. Control cochleae were processed in parallel to untreated guinea pigs. RESULTS: Fluorescence, indicating successful transfection, was observed within the basal and middle turns of the cochlea with limited distribution in the apex at 24 and 48 hours. Signal was most intense in the organ of Corti, spiral ligament, and spiral ganglion. Little to no fluorescence was observed at 5 days post-injection. No significant changes in auditory brainstem response (ABR) were noted post-perforation at 5 days, suggesting that siRNA-Lipofectamine at low doses does not cause cochlear toxicity. CONCLUSIONS: Small volumes of siRNA and Lipofectamine can be effectively delivered to cochlear structures using microneedles, paving the way for atraumatic cochlear gene therapy.

Asunto(s)

Terapia Genética , Liposomas , ARN Interferente Pequeño , Transfección , Animales , Cobayas , ARN Interferente Pequeño/administración & dosificación , Transfección/métodos , Terapia Genética/métodos , Lípidos/administración & dosificación , Lípidos/química , Cóclea , Ventana Redonda , Agujas , Potenciales Evocados Auditivos del Tronco Encefálico/efectos de los fármacos , Oído Interno , Microinyecciones/métodos

RESUMEN

There are four genogroups and 18 genotypes of human sapoviruses (HuSaVs) responsible for acute gastroenteritis. To comprehend their antigenic and virological differences, it is crucial to obtain viral stocks of the different strains. Previously, we utilized the human duodenum-derived cell line HuTu80, and glycocholate, a conjugated bile acid, to replicate and propagate GI.1, GI.2, and GII.3 HuSaVs (H. Takagi et al., Proc Natl Acad Sci U S A 117:32078-32085, 2020, https://10.1073/pnas.2007310117). First, we investigated the impact of HuTu80 passage number on HuSaV propagation. Second, we demonstrated that taurocholate improved the initial replication success rate and viral RNA levels in fecal specimens relative to glycocholate. By propagating 15 HuSaV genotypes (GI.1-7, GII.1-5, -8, and GV.1-2) and accomplishing preparation of viral stocks containing 1.0 × 109 to 3.4 × 1011 viral genomic copies/mL, we found that all strains required bile acids for replication, with GII.4 showing strict requirements for taurocholate. The deduced VP1 sequences of the viruses during the scale-up of serial passaged virus cultures were either identical or differed by only two amino acids from the original sequences in feces. In addition, we purified virions from nine strains of different genotypes and used them as immunogens for antiserum production. Enzyme-linked immunosorbent assays (ELISAs) using rabbit and guinea pig antisera for each of the 15 strains of different genotypes revealed distinct antigenicity among the propagating viruses across genogroups and differences between genotypes. Acquisition of biobanked viral resources and determination of key culture conditions will be valuable to gain insights into the common mechanisms of HuSaV infection. IMPORTANCE: The control of human sapovirus, which causes acute gastroenteritis in individuals of all ages, is challenging because of its association with outbreaks similar to those caused by human norovirus. The establishment of conditions for efficient viral propagation of various viral strains is essential for understanding the infection mechanism and identifying potential control methods. In this study, two critical factors for human sapovirus propagation in a conventional human duodenal cell line were identified, and 15 strains of different genotypes that differed at the genetic and antigenic levels were isolated and used to prepare virus stocks. The preparation of virus stocks has not been successful for noroviruses, which belong to the same family as sapoviruses. Securing virus stocks of multiple human sapovirus strains represents a significant advance toward establishing a reliable experimental system that does not depend on limited virus-positive fecal material.

Asunto(s)

Infecciones por Caliciviridae , Duodeno , Genotipo , Sapovirus , Replicación Viral , Sapovirus/genética , Humanos , Duodeno/virología , Duodeno/inmunología , Línea Celular , Animales , Infecciones por Caliciviridae/virología , Infecciones por Caliciviridae/inmunología , Gastroenteritis/virología , Antígenos Virales/inmunología , Antígenos Virales/genética , Heces/virología , Conejos , Cobayas , Variación Genética , ARN Viral/genética , Cultivo de Virus , Ácidos y Sales Biliares

RESUMEN

This study aimed to investigate the effects of the intravenous administration of lidocaine in the auditory cortex after the systemic administration of salicylate. Healthy male albino Hartley guinea pigs were divided into two groups. The control group received only lidocaine, whereas the experimental group received lidocaine after checking for the effects of salicylate. Extracellular recordings of spikes in the primary auditory cortex and dorsocaudal areas in healthy albino Hartley guinea pigs were continuously documented (pre- and post-lidocaine, pre- and post-salicylate, and post-salicylate after adding lidocaine to post-salicylate). We recorded 160 single units in the primary auditory cortex from five guinea pigs and 155 single units in the dorsocaudal area from another five guinea pigs to confirm the effects of lidocaine on untreated animals. No significant change was detected in either the threshold or Q10dB value after lidocaine administration in the primary auditory cortex and dorsocaudal areas. Spontaneous firing activity significantly decreased after lidocaine administration in the primary auditory cortex and dorsocaudal areas. Next, we recorded 160 single units in the primary auditory cortex from five guinea pigs and 137 single units in the dorsocaudal area from another five guinea pigs to determine the effects of lidocaine on salicylate-treated animals. The threshold was significantly elevated after salicylate administration; however, no additional change was detected after adding lidocaine to the primary auditory cortex and dorsocaudal areas. Regarding the Q10dB value, lidocaine negated the significant changes induced by salicylate in the primary auditory cortex and dorsocaudal areas. Moreover, lidocaine negated the significant changes in spontaneous firing activities induced by salicylate in the primary auditory cortex and dorsocaudal areas. In conclusion, changes in the Q10dB value and spontaneous firing activities induced by salicylate administration are abolished by lidocaine administration, suggesting that these changes are related to the presence of tinnitus.

Asunto(s)

Corteza Auditiva , Lidocaína , Salicilatos , Acúfeno , Animales , Cobayas , Corteza Auditiva/efectos de los fármacos , Corteza Auditiva/fisiopatología , Lidocaína/farmacología , Acúfeno/inducido químicamente , Masculino , Salicilatos/farmacología , Anestésicos Locales/farmacología

RESUMEN

Tetanus vaccines for human and veterinary use are produced by formaldehyde-induced inactivation of tetanus neurotoxin (TeNT) purified from Clostridium tetani cultures. Due to the high morbidity caused by exposure to TeNT it is essential that the quality control of tetanus vaccines includes testing for absence of tetanus toxin as prescribed by European Pharmacopoeia monographs 0452 and 0697. Currently this test is carried out in guinea pigs for each bulk of tetanus toxoid. To test the applicability of the in vitro BINACLE ("binding and cleavage") assay as an alternative method for the quality control of tetanus vaccines, two collaborative studies were run by the European Directorate for the Quality of Medicines & HealthCare under the aegis of the Biological Standardisation Programme. The first collaborative study indicated that the method allows sensitive TeNT detection. However, a clear conclusion could not be drawn due to the high variability of the results. To address the variability, the protocol was optimised and further standardised for the second study. The study results demonstrated good assay precision, both with respect to repeatability and reproducibility. Importantly, the limit of detection was 0.11 ng/mL TeNT in five out of nine laboratories and 0.33 ng/mL in four out of nine laboratories, suggesting that the BINACLE assay can detect TeNT with similar sensitivity as in vivo toxicity tests and can thus be taken into consideration as an alternative method to the current compendial in vivo test.

Asunto(s)

Toxina Tetánica , Toxoide Tetánico , Toxoide Tetánico/normas , Animales , Reproducibilidad de los Resultados , Toxina Tetánica/toxicidad , Cobayas , Tétanos , Control de Calidad , Bioensayo/normas , Bioensayo/métodos , Límite de Detección , Humanos