Testosterone Enhances KV Currents and Airway Smooth Muscle Relaxation Induced by ATP and UTP through P2Y4 Receptors and Adenylyl Cyclase Pathway.

Numerous studies suggest the involvement of adenosine-5'-triphosphate (ATP) and similar nucleotides in the pathophysiology of asthma. Androgens, such as testosterone (TES), are proposed to alleviate asthma symptoms in young men. ATP and uridine-5'-triphosphate (UTP) relax the airway smooth muscle (ASM) via purinergic P2Y2 and P2Y4 receptors and K+ channel opening. We previously demonstrated that TES increased the expression of voltage-dependent K+ (KV) channels in ASM. This study investigates how TES may potentiate ASM relaxation induced by ATP and UTP. Tracheal tissues treated with or without TES (control group) from young male guinea pigs were used. In organ baths, tracheas exposed to TES (40 nM for 48 h) showed enhanced ATP- and UTP-evoked relaxation. Tetraethylammonium, a K+ channel blocker, annulled this effect. Patch-clamp experiments in tracheal myocytes showed that TES also increased ATP- and UTP-induced K+ currents, and this effect was abolished with flutamide (an androgen receptor antagonist). KV channels were involved in this phenomenon, which was demonstrated by inhibition with 4-aminopyridine. RB2 (an antagonist of almost all P2Y receptors except for P2Y2), as well as N-ethylmaleimide and SQ 22,536 (inhibitors of G proteins and adenylyl cyclase, respectively), attenuated the enhancement of the K+ currents induced by TES. Immunofluorescence and immunohistochemistry studies revealed that TES did not modify the expression of P2Y4 receptors or COX-1 and COX-2, while we have demonstrated that this androgen augmented the expression of KV1.2 and KV1.5 channels in ASM. Thus, TES leads to the upregulation of P2Y4 signaling and KV channels in guinea pig ASM, enhancing ATP and UTP relaxation responses, which likely limits the severity of bronchospasm in young males.

Revealing nitrogenous VX metabolites and the whole-molecule VX metabolism in the urine of guinea pigs.

VX, a well-known organophosphorus nerve agent (OPNA), poses a significant threat to public safety if employed by terrorists. Obtaining complete metabolites is critical to unequivocally confirm its alleged use/exposure and elucidate its whole-molecular metabolism. However, the nitrogenous VX metabolites containing 2-diisopropylaminoethyl moiety from urinary excretion remain unknown. Therefore, this study applied a newly developed untargeted workflow platform to discover and identify them using VX-exposed guinea pigs as animal models. 2-(N,N-diisopropylamino)ethanesulfonic acid (DiPSA) was revealed as a novel nitrogenous VX metabolite in urine, and 2-(Diisopropylaminoethyl) methyl sulfide (DAEMS) was confirmed as another in plasma, indicating that VX metabolism differed between urine and plasma. It is the first report of a nitrogenous VX metabolite in urine and a complete elucidation of the VX metabolic pathway. DiPSA was evaluated as an excellent VX exposure biomarker. The whole-molecule VX metabolism in urine was characterized entirely for the first time via the simultaneous quantification of DiPSA and two known P-based biomarkers. About 52.1% and 32.4% of VX were excreted in urine as P-based and nitrogenous biomarkers within 24 h. These findings provide valuable insights into the unambiguous detection of OPNA exposure/intoxication and human and environmental exposure risk assessment.

Effect of exogenous calcitriol on myopia development and axial length in guinea pigs with form deprivation myopia.

The annual increase in myopia prevalence poses a significant economic and health challenge. Our study investigated the effect of calcitriol role in myopia by inducing the condition in guinea pigs through form deprivation for four weeks. Untargeted metabolomics methods were used to analyze the differences in metabolites in the vitreous body, and the expression of vitamin D receptor (VDR) in the retina was detected. Following form deprivation, the guinea pigs received intraperitoneal injections of calcitriol at different concentrations. We assessed myopia progression using diopter measurements and biometric analysis after four weeks. Results indicated that form deprivation led to a pronounced shift towards myopia, characterized by reduced choroidal and scleral thickness, disorganized collagen fibers, and decreased scleral collagen fiber diameter. Notably, a reduction in calcitriol expression in vitreous body, diminished vitamin D and calcitriol levels in the blood, and decreased VDR protein expression in retinal tissues were observed in myopic guinea pigs. Calcitriol administration effectively slowed myopia progression, preserved choroidal and scleral thickness, and prevented the reduction of scleral collagen fiber diameter. Our findings highlight a significant decrease in calcitriol and VDR expressions in myopic guinea pigs and demonstrate that exogenous calcitriol supplementation can halt myopia development, enhancing choroidal and scleral thickness and scleral collagen fiber diameter.

Fast freezing inhibits melanin synthesis of melanocytes by modulating the Wnt/ß-catenin signalling pathway.

Skin hyperpigmentation is mainly caused by excessive synthesis of melanin; however, there is still no safe and effective therapy for its removal. Here, we found that the dermal freezer was able to improve UVB-induced hyperpigmentation of guinea pigs without causing obvious epidermal damage. We also mimic freezing stimulation at the cellular level by rapid freezing and observed that freezing treatments <2.5 min could not decrease cell viability or induce cell apoptosis in B16F10 and Melan-A cells. Critically, melanin content and tyrosinase activity in two cells were greatly reduced after freezing treatments. The dramatic decrease in tyrosinase activity was associated with the downregulation of MITF, TYR, TRP-1 and TRP-2 protein expression in response to freezing treatments for two cells. Furthermore, our results first demonstrated that freezing treatments significantly reduced the levels of p-GSK3ß and ß-catenin and the nuclear accumulation of ß-catenin in B16F10 and Melan-A cells. Together, these data suggest that fast freezing treatments can inhibit melanogenesis-related gene expression in melanocytes by regulating the Wnt/ß-catenin signalling pathway. The inhibition of melanin production eventually contributed to the improvement in skin hyperpigmentation induced by UVB. Therefore, fast freezing treatments may be a new alternative of skin whitening in the clinic in the future.

Noise-induced hearing loss alters potassium-chloride cotransporter KCC2 and GABA inhibition in the auditory centers.

Homeostatic plasticity, the ability of neurons to maintain their averaged activity constant around a set point value, is thought to account for the central hyperactivity after hearing loss. Here, we investigated the putative role of GABAergic neurotransmission in this mechanism after a noise-induced hearing loss larger than 50 dB in high frequencies in guinea pigs. The effect of GABAergic inhibition is linked to the normal functioning of K + -Cl- co-transporter isoform 2 (KCC2) which maintains a low intracellular concentration of chloride. The expression of membrane KCC2 were investigated before and after noise trauma in the ventral and dorsal cochlear nucleus (VCN and DCN, respectively) and in the inferior colliculus (IC). Moreover, the effect of gabazine (GBZ), a GABA antagonist, was also studied on the neural activity in IC. We show that KCC2 is downregulated in VCN, DCN and IC 3 days after noise trauma, and in DCN and IC 30 days after the trauma. As expected, GBZ application in the IC of control animals resulted in an increase of spontaneous and stimulus-evoked activity. In the noise exposed animals, on the other hand, GBZ application decreased the stimulus-evoked activity in IC neurons. The functional implications of these central changes are discussed.

[New drugs for horses and production animals in 2023]. / Neue Arzneimittel für Pferde und landwirtschaftliche Nutztiere 2023.

In 2023, no new active pharmaceutical ingredients were released on the German market for horses and food-producing animals. Two established veterinary active pharmaceutical ingredients became available for additional species: The phosphorus compound butafosfan was also approved for horses, dogs, and cats and the mineral sodium chloride as an isotonic sodium chloride solution was also approved for rabbits and guinea pigs. In addition, for small animals, there were new releases of an agent (pergolidmesilate) in a novel pharmaceutical formulation and a lower content of the active ingredient, one drug (fluralaner) in a smaller package size as well as one drug (oxalic acid dehydrate) with a new route of administration. Furthermore, one combination of active ingredients (diprophylline+heptaminol) is available on the market for horses and food producing animals again.

Effects of cytochalasin D on relaxation process of skinned taenia cecum and carotid artery from guinea pig.

Actin linked regulatory mechanisms are known to contribute contraction/relaxation in smooth muscle. In order to clarify whether modulation of polymerization/depolymerization of actin filaments affects relaxation process, we examined the effects of cytochalasin D on relaxation process by Ca2+ removal after Ca2+-induced contraction of ß-escin skinned (cell membrane permeabilized) taenia cecum and carotid artery preparations from guinea pigs. Cytochalasin D, an inhibitor of actin polymerization, significantly suppressed the force during relaxation both in skinned taenia cecum and carotid artery. The data fitting analysis of the relaxation processes indicates that cytochalasin D accelerates slow (latch-like) bridge dissociation. Cytochalasin D seems to directly disrupts actin filament organization or its length, resulting in modulation of actin filament structure that prevents myosin binding.

Brimonidine as a possible treatment for myopia.

BACKGROUND: Myopia is becoming a huge burden on the world's public health systems. The purpose of this study was to explore the effect of brimonidine in the treatment of form-deprivation myopia (FDM) and the relationship between intraocular pressure (IOP) and myopia development. METHODS: Monocular form deprivation myopia (FDM) was induced in three-week-old pigmented male guinea pigs. They were treated with 3 different methods of brimonidine administration (eye drops, and subconjunctival or intravitreal injections). Four different concentrations of brimonidine were tested for each method (2µg/µL, 4µg/µL, 20µg/µL, and 40µg/µL). All treatments continued for a period of 21 days. Tonometry, retinoscopy, and A-scan ultrasonography were used to monitor intraocular pressure, refractive error and axial length (AL), respectively. RESULTS: Treatment with subconjunctival brimonidine at 40µg/µL, and intravitreal brimonidine at 2µg/µL and 4µg/µL, inhibited the development of FDM. The myopic refraction, excessive axial length, and elevation of IOP were significantly decreased. Brimonidine in eye drops was ineffective. CONCLUSION: Brimonidine at appropriate doses significantly reduced the development of FD myopia in guinea pigs. The IOP may change with FD myopia.

Therapeutic prime/pull vaccination of HSV-2-infected guinea pigs with the ribonucleotide reductase 2 (RR2) protein and CXCL11 chemokine boosts antiviral local tissue-resident and effector memory CD4+ and CD8+ T cells and protects against recurrent genital herpes.

Following acute herpes simplex virus type 2 (HSV-2) infection, the virus undergoes an asymptomatic latent infection of sensory neurons of dorsal root ganglia (DRG). Chemical and physical stress cause intermittent virus reactivation from latently infected DRG and recurrent virus shedding in the genital mucosal epithelium causing genital herpes in symptomatic patients. While T cells appear to play a role in controlling virus reactivation from DRG and reducing the severity of recurrent genital herpes, the mechanisms for recruiting these T cells into DRG and the vaginal mucosa (VM) remain to be fully elucidated. The present study investigates the effect of CXCL9, CXCL10, and CXCL11 T-cell-attracting chemokines on the frequency and function of DRG- and VM-resident CD4+ and CD8+ T cells and its effect on the frequency and severity of recurrent genital herpes in the recurrent herpes guinea pig model. HSV-2 latent-infected guinea pigs were immunized intramuscularly with the HSV-2 ribonucleotide reductase 2 (RR2) protein (Prime) and subsequently treated intravaginally with the neurotropic adeno-associated virus type 8 expressing CXCL9, CXCL10, or CXCL11 chemokines to recruit CD4+ and CD8+ T cells into the infected DRG and VM (Pull). Compared to the RR2 therapeutic vaccine alone, the RR2/CXCL11 prime/pull therapeutic vaccine significantly increased the frequencies of functional tissue-resident and effector memory CD4+ and CD8+ T cells in both DRG and VM tissues. This was associated with less virus in the healed genital mucosal epithelium and reduced frequency and severity of recurrent genital herpes. These findings confirm the role of local DRG- and VM-resident CD4+ and CD8+ T cells in reducing virus shedding at the vaginal site of infection and the severity of recurrent genital herpes and propose the novel prime-pull vaccine strategy to protect against recurrent genital herpes.IMPORTANCEThe present study investigates the novel prime/pull therapeutic vaccine strategy to protect against recurrent genital herpes using the latently infected guinea pig model. In this study, we used the strategy that involves immunization of herpes simplex virus type 2-infected guinea pigs using a recombinantly expressed herpes tegument protein-ribonucleotide reductase 2 (RR2; prime), followed by intravaginal treatment with the neurotropic adeno-associated virus type 8 expressing CXCL9, CXCL10, or CXCL11 T-cell-attracting chemokines to recruit T cells into the infected dorsal root ganglia (DRG) and vaginal mucosa (VM) (pull). We show that the RR2/CXCL11 prime-pull therapeutic vaccine strategy elicited a significant reduction in virus shedding in the vaginal mucosa and decreased the severity and frequency of recurrent genital herpes. This protection was associated with increased frequencies of functional tissue-resident (TRM cells) and effector (TEM cells) memory CD4+ and CD8+ T cells infiltrating latently infected DRG tissues and the healed regions of the vaginal mucosa. These findings shed light on the role of tissue-resident and effector memory CD4+ and CD8+ T cells in DRG tissues and the VM in protection against recurrent genital herpes and propose the prime-pull therapeutic vaccine strategy in combating genital herpes.

Seihaito, a Kampo medicine, attenuates IL-13-induced mucus production and goblet cell metaplasia.

Goblet cell hyperplasia and increased mucus production are features of airway diseases, including asthma, and excess airway mucus often worsens these conditions. Even steroids are not uniformly effective in mucus production in severe asthma, and new therapeutic options are needed. Seihaito is a Japanese traditional medicine that is used clinically as an antitussive and expectorant. In the present study, we examined the effect of Seihaito on goblet cell differentiation and mucus production. In in vitro studies, using air-liquid interface culture of guinea-pig tracheal epithelial cells, Seihaito inhibited IL-13-induced proliferation of goblet cells and MUC5AC, a major component of mucus production. Seihaito suppressed goblet cell-specific gene expression, without changing ciliary cell-specific genes, suggesting that it inhibits goblet cell differentiation. In addition, Seihaito suppressed MUC5AC expression in cells transfected with SPDEF, a transcription factor activated by IL-13. Furthermore, Seihaito attenuated in vivo goblet cell proliferation and MUC5AC mRNA expression in IL-13-treated mouse lungs. Collectively, these findings demonstrated that Seihaito has an inhibitory effect on goblet cell differentiation and mucus production, which is at least partly due to the inhibition of SPDEF.

pH- and Matrix Metalloproteinase-Responsive Multifunctional Bilayer Microneedles Platform for Treatment of Tinea Pedis.

Persistent foot odor and itchiness are common symptoms of tinea pedis, significantly disrupting the daily life of those affected. The cuticular barrier at the site of the tinea pedis is thickened, which impedes the effective penetration of antifungal agents. Additionally, fungi can migrate from the skin surface to deeper tissues, posing challenges in the current clinical treatment for tinea pedis. To effectively treat tinea pedis, we developed a platform of bilayer gelatin methacrylate (GelMA) microneedles (MNs) loaded with salicylic acid (SA) and FK13-a1 (SA/FK13-a1@GelMA MNs). SA/FK13-a1@GelMA MNs exhibit pH- and matrix metalloproteinase (MMP)-responsive properties for efficient drug delivery. The MNs are designed to deliver salicylic acid (SA) deep into the stratum corneum, softening the cuticle and creating microchannels. This process enables the antibacterial peptide FK13-a1 to penetrate through the stratum corneum barrier, facilitating intradermal diffusion and exerting antifungal and anti-inflammatory effects. In severe cases of tinea pedis, heightened local pH levels and MMP activity further accelerate drug release. Our research demonstrates that SA/FK13-a1@GelMA MNs are highly effective against Trichophyton mentagrophytes, Trichophyton rubrum, and Candida albicans. They also reduced stratum corneum thickness, fungal burden, and inflammation in a guinea pig model of tinea pedis induced by T. mentagrophytes. Furthermore, it was discovered that SA/FK13-a1@GelMA MNs exhibit excellent biocompatibility. These findings suggest that SA/FK13-a1@GelMA MNs have significant potential for the clinical treatment of tinea pedis as well as other fungal skin disorders.

Serum Prestin After Ototoxin Exposure Is Not Dependent on Outer Hair Cell Loss.

HYPOTHESIS: Cyclodextrin (CDX)-induced serum prestin burst is not dependent on outer hair cell (OHC) loss. BACKGROUND: Serum prestin has been proposed as a biomarker for ototoxicity. We recently used an automated Western approach to quantify serum prestin changes in a newly introduced model of CDX ototoxicity. To gain insights into prestin as a biomarker, here we further characterize serum prestin in the CDX model. METHODS: Guinea pigs were treated with 750, 3,000, or 4,000 mg/kg CDX, and serum samples were obtained through up to 15 weeks after exposure. Serum prestin levels were quantified using automated Western, and hair cell counts were obtained. RESULTS: All three doses induced an N -glycosylated ~134-kDa prestin burst; however, only the 3,000 and 4,000 mg/kg resulted in robust OHC loss. Prestin levels returned to baseline where they remained up to 15 weeks in the absence of OHCs. CONCLUSION: The ~134-kDa prestin burst induced after CDX administration is N -glycosylated, representing a posttranslational modification of prestin. Serum prestin seems to be a promising biomarker when using therapeutics with ototoxic properties because it is not dependent on OHC loss as a necessary event, thus affording the opportunity for early detection and intervention.

Humanization of a mouse anti-human complement C6 monoclonal antibody as a potential therapeutic for certain complement-mediated diseases.

The assembly of tissue-damaging membrane attack complexes (MACs; C5b-9) is a major mechanism by which excessive complement activation causes diseases. We previously developed a mouse anti-human C6 monoclonal antibody (mAb) 1C9 that selectively inhibits the assembly of MACs in human and non-human primates. In this project, we found that 1C9 also cross-reacted with rat and guinea pig C6, and determined its binding domains on C6 using different truncated C6 proteins. We then humanized the anti-C6 mAb by molecular modeling and complementarity-determining region grafting. After screening a library of 276 humanized variants with different combinations of humanized light and heavy chains in biophysical assays, we identified clone 3713 with the best developability profile, and an increased affinity against C6 when compared with the parental 1C9 mAb. This humanized 3713 mAb inhibited human, monkey, and rat complement-mediated hemolysis in vitro, and more importantly, it significantly reduced complement-mediated hemolysis in vivo in rats. These results demonstrated the successful humanization of the anti-C6 mAb and suggested that the humanized 3713 mAb could be further developed as a new therapeutic that selectively targets MAC for certain complement-mediated pathological conditions.

Effect of isoflurane anaesthetic time on ocular a-scan ultrasonography measures and their relationship to age and OCT measures in the Guinea pig.

A-scan ultrasonography enables precise measurement of internal ocular structures. Historically, its use has underpinned fundamental studies of eye development and aberrant eye growth in animal models of myopia; however, the procedure typically requires anaesthesia. Since anaesthesia affects intra-ocular pressure (IOP), we investigated changes in internal ocular structures with isoflurane exposure and compared measurements with those taken in awake animals using optical coherence tomography (OCT). Continuous A-scan ultrasonography was undertaken in tri-coloured guinea pigs aged 21 (n = 5), 90 (n = 5) or 160 (n = 5) days while anaesthetised (up to 36 min) with isoflurane (5% in 1.5L/min O2). Peaks were selected from ultrasound traces corresponding to the boundaries of the cornea, crystalline lens, retina, choroid and sclera. OCT scans (Zeiss Cirrus Photo 800) of the posterior eye layers were taken in 28-day-old animals (n = 19) and compared with ultrasound traces, with choroid and scleral thickness adjusted for the duration of anaesthesia based on the changes modelled in 21-day-old animals. Ultrasound traces recorded sequentially in left and right eyes in 14-day-old animals (n = 30) were compared, with each adjusted for anaesthesia duration. The thickness of the cornea was measured in enucleated eyes (n = 5) using OCT following the application of ultrasound gel (up to 20 min). Retinal thickness was the only ultrasound internal measure unaffected by anaesthesia. All other internal distances rapidly changed and were well fitted by exponential functions (either rise-to-max or decay). After 10 and 20 min of anaesthesia, the thickness of the cornea, crystalline lens and sclera increased by 17.1% and 23.3%, 0.4% and 0.6%, and 5.2% and 6.5% respectively, whilst the anterior chamber, vitreous chamber and choroid decreased by 4.4% and 6.1%, 0.7% and 1.1%, and 10.7% and 11.8% respectively. In enucleated eyes, prolonged contact of the cornea with ultrasound gel resulted in an increase in thickness of 9.3% after 10 min, accounting for approximately half of the expansion observed in live animals. At the back of the eye, ultrasound measurements of the thickness of the retina, choroid and sclera were highly correlated with those from posterior segment OCT images (R2 = 0.92, p = 1.2 × 10-13, R2 = 0.55, p = 4.0 × 10-4, R2 = 0.72, p = 5.0 × 10-6 respectively). Furthermore, ultrasound measures for all ocular components were highly correlated in left and right eyes measured sequentially, when each was adjusted for anaesthetic depth. This study shows that the depth of ocular components can change dramatically with anaesthesia. Researchers should therefore be wary of these concomitant effects and should employ adjustments to better render 'true' values.

Effective skin decontamination with RSDL® (reactive skin decontamination lotion kit) following dermal exposure to a Novichok class nerve agent.

In recent years, various poisoning incidents have been reported, involving the alleged use of the so-called Novichok agents, resulting in their addition to the Schedule I list of the Organisation for the Prohibition of Chemical Warfare (OPCW). As the physicochemical properties of these agents are different from the 'classical' nerve agents, such as VX, research is needed to evaluate whether and to what extent existing countermeasures are effective. Here, we evaluated the therapeutic potential of RSDL® (Reactive Skin Decontamination Lotion Kit) for the neutralization of percutaneous toxicity caused by Novichok agents, both in vitro and in vivo. Experiments showed the three selected Novichok agents (A230, A232, A234) could be degraded by RSDL lotion, but at a different rate. The half-life of A234, in the presence of an excess of RSDL lotion, was 36 min, as compared to A230 (<5 min) and A232 (18 min). Following dermal exposure of guinea pigs to A234, application of the RSDL kit was highly effective in preventing intoxication, even when applied up until 30 min following exposure. Delayed use of the RSDL kit until the appearance of clinical signs of intoxication (3-4 h) was not able to prevent intoxication progression and deaths. This study determines RSDL decontamination as an effective treatment strategy for dermal exposure to the Novichok agent A234 and underscores the importance of early, forward use of skin decontamination, as rapidly as possible.

Inhibition kinetics of acetylcholinesterase and butyrylcholinesterase from various species by 2-(2-cresyl)-4H-1,3,2-benzodioxaphosphorin-2-oxide (CBDP).

The aerotoxic syndrome has been associated with exposure to tricresyl phosphate (TCP), which is used as additive in hydraulic fluids and engine lubricants. The toxic metabolite 2-(2-cresyl)-4H-1,3,2-benzodioxaphosphorin-2-oxide (CBDP) is formed from the TCP isomer tri-ortho-cresyl phosphate (TOCP) in vivo and is known to react with the active site serine in acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) resulting in the inhibition of the enzymes. Previous in vitro studies showed pronounced species differences in the inhibition kinetics of cholinesterases by organophosphorus compounds (OP), which must be considered in the development of relevant animal models for the investigation of OP poisoning and the aerotoxic syndrome. The present study was designed to investigate the inhibition kinetics of human, Cynomolgus monkey, pig, mini pig, guinea pig, mouse, and rat AChE as well as BChE by CBDP under standardized conditions. There were similar rate constants for the inhibition (ki) of human, Cynomolgus monkey and mouse AChE by CBDP. In contrast, the ki values obtained for guinea pig, mini pig, pig, and rat AChE were 2.8- to 5.9-fold lower than that of human AChE. The results of the present study confirmed CBDP as one of the most potent inhibitors of human BChE, indicating a ki value of 3.24 ± 0.33 ×108M-1min-1, which was about 1,140-fold higher than that of human AChE. Accordingly, a markedly more pronounced inhibition rate of BChE from the species guinea pig, mini pig, pig, rat, Cynomolgus monkey, and mouse by CBDP was found as compared to those of AChE from the respective sources, indicating 2.0- to 89.6-fold higher ki values.

Study on the inhibition activity and mechanism of Tanreqing against Klebsiella pneumoniae biofilm formation in vitro and in vivo.

Objective: To evaluate the antibacterial effect of Tanreqing (TRQ) against K. pneumoniae and its inhibition activity on bacterial biofilm formation in vitro and in vivo, and to explore the mechanism of the inhibitory effects of TRQ on K. pneumoniae biofilm formation. Methods: An in vitro biofilm model of K. pneumoniae was established, and the impact of TRQ on biofilm formation was evaluated using crystal violet staining and scanning electron microscopy (SEM). Furthermore, the clearance effect of TRQ against K. pneumoniae in the biofilm was assessed using the viable plate counting method; q-RT PCR was used to evaluate the inhibitory effect of different concentrations of TRQ on the expression of biofilm-related genes in Klebsiella pneumoniae; The activity of quorum sensing signal molecule AI-2 was detected by Vibrio harveyi bioluminescence assay; Meanwhile, a guinea pig lung infection model of Klebsiella pneumoniae was constructed, and after treated with drugs, pathological analysis of lung tissue and determination of bacterial load in lung tissue were performed. The treatment groups included TRQ group, imipenem(IPM) group, TRQ+IPM group, and sterile saline group as the control. Results: The formation of K. pneumoniae biofilm was significantly inhibited by TRQ in vitro experiments. Furthermore, when combined with IPM, the clearance of K. pneumoniae in the biofilm was notably increased compared to the TRQ group and IPM group alone. q-RT PCR analysis revealed that TRQ down-regulated the expression of genes related to biofilm formation in K. pneumoniae, specifically luxS, wbbm, wzm, and lsrK, and also inhibited the activity of AI-2 molecules in the bacterium. In vivo experiments demonstrated that TRQ effectively treated guinea pig lung infections, resulting in reduced lung inflammation. Additionally, when combined with IPM, there was a significant reduction in the bacterial load in lung tissue. Conclusion: TRQ as a potential therapeutic agent plays a great role in the treatment of K. pneumoniae infections, particularly in combination with conventional antibiotics. And TRQ can enhanced the clearance effect on the bacterium by inhibiting the K. pneumoniae biofilm formation, which provided experimental evidence in support of clinical treatment of TRQ against K. pneumoniae infections.

In Vivo Biocompatibility Study on Functional Nanostructures Containing Bioactive Glass and Plant Extracts for Implantology.

In this paper, the in vivo behavior of orthopedic implants covered with thin films obtained by matrix-assisted pulsed laser evaporation and containing bioactive glass, a polymer, and natural plant extract was evaluated. In vivo testing was performed by carrying out a study on guinea pigs who had coated metallic screws inserted in them and also controls, following the regulations of European laws regarding the use of animals in scientific studies. After 26 weeks from implantation, the guinea pigs were subjected to X-ray analyses to observe the evolution of osteointegration over time; the guinea pigs' blood was collected for the detection of enzymatic activity and to measure values for urea, creatinine, blood glucose, alkaline phosphatase, pancreatic amylase, total protein, and glutamate pyruvate transaminase to see the extent to which the body was affected by the introduction of the implant. Moreover, a histopathological assessment of the following vital organs was carried out: heart, brain, liver, and spleen. We also assessed implanted bone with adjacent tissue. Our studies did not find significant variations in biochemical and histological results compared to the control group or significant adverse effects caused by the implant coating in terms of tissue compatibility, inflammatory reactions, and systemic effects.

False positive findings associated with adenoviral vector-based vaccine underscore the regulatory necessity to eliminate abnormal toxicity test.

Accumulating evidence has shown that the abnormal toxicity test (ATT) is not suitable as a quality control batch release test for biologics and vaccines. The purpose of the current study was to explore the optimal ATT experimental design for an adenoviral vector-based vaccine product to avoid false positive results following the standard test conditions stipulated in the Pharmacopoeias. ATT were conducted in both mice and guinea pigs based on methods in Pharmacopeias, with modifications to assess effects of dose volume and amount of virus particles (VPs). The results showed intraperitoneal (IP) dosing at human relevant dose and volume (i.e., VPs), as required by pharmacopeia study design, resulted in false positive findings not associated with extraneous contaminants of a product. Considering many gene therapy products use adeno associated virus as the platform for transgene delivery, data from this study are highly relevant in providing convincing evidence to show the ATT is inappropriate as batch release test for biologics, vaccine and gene therapy products. In conclusion, ATT, which requires unnecessary animal usage and competes for resources which otherwise can be spent on innovative medicine research, should be deleted permanently as batch release test by regulatory authorities around the world.

A single dose of AC102 restores hearing in a guinea pig model of noise-induced hearing loss to almost prenoise levels.

Although sudden sensorineural hearing loss (SSNHL) is a serious condition, there are currently no approved drugs for its treatment. Nevertheless, there is a growing understanding that the cochlear pathologies that underlie SSNHL include apoptotic death of sensory outer hair cells (OHCs) as well as loss of ribbon synapses connecting sensory inner hair cells (IHCs) and neurites of the auditory nerve, designated synaptopathy. Noise-induced hearing loss (NIHL) is a common subtype of SSNHL and is widely used to model hearing loss preclinically. Here, we demonstrate that a single interventive application of a small pyridoindole molecule (AC102) into the middle ear restored auditory function almost to prenoise levels in a guinea pig model of NIHL. AC102 prevented noise-triggered loss of OHCs and reduced IHC synaptopathy suggesting a role of AC102 in reconnecting auditory neurons to their sensory target cells. Notably, AC102 exerted its therapeutic properties over a wide frequency range. Such strong improvements in hearing have not previously been demonstrated for other therapeutic agents. In vitro experiments of a neuronal damage model revealed that AC102 protected cells from apoptosis and promoted neurite growth. These effects may be explained by increased production of adenosine triphosphate, indicating improved mitochondrial function, and reduced levels of reactive-oxygen species which prevents the apoptotic processes responsible for OHC death. This action profile of AC102 might be causal for the observed hearing recovery in in vivo models.