Quercetin inhibits Toxoplasma gondii tachyzoite proliferation and acts synergically with azithromycin.

Quercetin (QUE) is a natural polyphenol known to have numerous pharmacological properties against infectious and non-infectious diseases. Azithromycin (AZ) is an antibiotic that belongs to the azalide class of antimicrobials and an antiparasitic that is known to be effective in combination with clindamycin against pyrimethamine/sulfadiazine-resistant Toxoplasma gondii tachyzoites in clinical settings. Both compounds are known to target protein synthesis and have anti-inflammatory properties. However, little is known about QUE and AZ synergistic interaction against T. gondii growth. Here, we report for the first time the effects of the combination of QUE and AZ on T. gondii growth. The 50% inhibitory concentration (IC50) for QUE at 72 h of interaction was determined to be 0.50 µM, whereas AZ gave an IC50 value of 0.66 µM at 72 h of interaction with parasites. Combination testing of QUE and AZ in a ratio of 2:1 (QUE:AZ) showed an IC50 value of 0.081 µM. Interestingly, a fractional inhibitory index value of 0.28 was observed, indicating a strong synergy. QUE was also found to upregulate the generation of reactive oxygen species and cause dysfunction of the mitochondria membrane of both intracellular and extracellular T. gondii tachyzoites. Overall, the results indicate that QUE is a novel lead capable of synergizing with AZ for inhibiting T. gondii growth and may merit future investigation in vivo for possible combination drug development.

Anti-Toxoplasma gondii activity of Trametes versicolor (Turkey tail) mushroom extract.

Toxoplasma gondii (T. gondii) infection continues to rise globally in humans and animals with high socioeconomic and public health challenges. Current medications used against T. gondii infection are limited in efficacy, safety, and affordability. This research was conducted to assess the higher fungi extract effect on T. gondii tachyzoites growth in vitro and possibly decipher its mechanism of action. Furthermore, we evaluated the extract's effect on human foreskin fibroblast viability. The methanol extracts of Turkey tail (TT) mushroom was tested against T. gondii tachyzoites growth using an RH-RFP type I strain that expresses red fluorescent protein throughout culture in a dose-dependent manner using a fluorescent plate reader. Similarly, we tested the effect of the extract on host cell viability. We observed that TT extract inhibited tachyzoites growth with a 50% minimum inhibitory concentration (IC50s), IC50 = 5.98 ± 1.22 µg/mL, and 50% cytotoxic concentration (CC50s), CC50 ≥ 100 µg/mL. It was discovered that TT extract induced strong mitochondria superoxide and  reactive oxygen species production and disrupted mitochondria membrane potential in T. gondii tachyzoites. Additionally, scanning electron microscopy depicted that TT extract and pyrimethamine (PY) caused a morphological deformation of tachyzoites in vitro. In conclusion, TT methanol extract made up of phytosterols, bioactive sphingolipids, peptides, phenolic acids, and lactones could be a promising source of new compounds for the future development of anti-Toxoplasma gondii drugs. Extracts were non-cytotoxic, even at higher concentrations.

Inhibition of Toxoplasma gondii Growth by Dihydroquinine and Its Mechanisms of Action.

Toxoplasma gondii is a zoonotic parasite that infects the brain of humans and causes cerebral toxoplasmosis. The recommended drugs for the treatment or prophylaxis of toxoplasmosis are pyrimethamine (PY) and sulfadiazine (SZ), which have serious side effects. Other drugs available for toxoplasmosis are poorly tolerated. Dihydroquinine (DHQ) is a compound closely related to quinine-based drugs that have been shown to inhibit Plasmodium falciparum and Plasmodium berghei in addition to its anti-arrhythmia properties. However, little is known about the effect of DHQ in T. gondii growth and its mechanism of action in vitro. In this study, we report the anti-Toxoplasma and anti-invasion properties of DHQ. DHQ significantly inhibited T. gondii tachyzoite growth with IC50s values of 0.63, 0.67, and 0.00137 µM at 24, 48, and 72 h, respectively. Under similar conditions, SZ and PY, considered as the gold standard drugs for the treatment of toxoplasmosis, had IC50s values of 1.29, 1.55, and 0.95 and 3.19, 3.52, and 2.42 µM, respectively. The rapid dose-dependent inhibition of T. gondii tachyzoites by DHQ compared to the standard drugs (SZ and PY) indicates that DHQ has high selective parasiticidal effects against tachyzoite proliferation. Remarkably, DHQ had an excellent selectivity index (SI) of 149- and 357-fold compared to 24- and 143-fold for PY and SZ, respectively, using fibroblast cells. In addition, DHQ disrupted T. gondii tachyzoite mitochondrial membrane potential and adenosine triphosphate (ATP) production and elicited high reactive oxygen species (ROS) generation. Taking all these findings together, DHQ promises to be an effective and safe lead for the treatment of toxoplasmosis.

Extracts from salivary glands stimulate aggression and inositol-1, 4, 5-triphosphate (IP3) production in the vomeronasal organ of mice.

Mammals use chemical cues to coordinate social and reproductive behaviors. Chemical cues are detected by the VNO organ (VNO), which is a cartilage-encased elongated organ associated with the vomer bone in the rostral nasal cavity. The resident intruder paradigm was utilized to examine the ability of saliva and its feeder exocrine glands, the submaxillary, parotid, and sublingual glands to mediate aggression in mice. Saliva and extracts from submaxillary and parotid glands, but not extracts from sublingual glands of male CD-1 mice, induced a greater number of attacks and lower latencies to sniff and attack (p<0.05) and significantly increased IP(3) production (p<0.05) versus vehicle (PBS) in CD-1 male mice VNO. We further show that CD-1 male mouse saliva and submaxillary gland extract induced significantly more attacks and a lower latency to attack in lactating female CD-1 mice and produced significantly more inositol triphosphate (IP(3)), indicative of phospholipase C(beta) signaling which mediates pheromonal activity, in CD-1 female VNO compared to PBS. Castrated CD-1 male mouse saliva, and exocrine gland extracts induced significantly less IP(3) production in male VNO and less aggression by CD-1 males and lactating females compared to responses to normal CD-1 male mouse saliva and gland extracts. Thus, chemical cues present in saliva, submaxillary and parotid glands of CD-1 male mice are capable of stimulating aggression in male and female congenic mice which are correlated with significant production of IP(3) in the VNO. Additionally, these stimulations of aggression and IP(3) production are shown to be androgen-dependent.

Pregnancy block by MHC class I peptides is mediated via the production of inositol 1,4,5-trisphosphate in the mouse vomeronasal organ.

The vomeronasal organ (VNO) has evolved to link an animal's behavior to its environment in a highly species-specific fashion. In mice, it is thought to be the primary sensory system responsible for the detection of pheromones. Pheromones regulate a variety of responses including mate recognition in the context of selective pregnancy failure. MHC (major histocompatibility complex) class I peptides have been identified as compounds that elicit the pregnancy block effect via the VNO. However, the transduction cascade of these molecules is unknown and it is not known if the production of these compounds are androgen dependent. By using male urine and MHC peptides, we show that female mice treated with MHC peptides (in urine or PBS) and urine from castrated males or juvenile mice of different haplotypes respond to the Bruce Effect paradigm in a manner equivalent to female mice exposed to whole urine. In addition to providing new evidence that urine from castrated or juvenile males and MHC peptides can induce pregnancy block, we show correlation of the effect with an increase in inositol 1,4,5-trisphosphate.

Chemosensory cues from the lacrimal and preputial glands stimulate production of IP3 in the vomeronasal organ and aggression in male mice.

The social and reproductive behaviors of most mammals are modulated by chemosensory cues. The perception of some of these cues is mediated by the vomeronasal organ, which is a cartilage-encased elongated organ associated with the vomer bone in the rostral nasal cavity. Several studies have shown that chemosensory cues are present in urine, seminal fluid or vaginal secretions but only a few studies have focused on exocrine glands as a source of chemosensory cues. Here we show that chemosensory cues present in two exocrine glands, i.e., the preputial gland located at the caudal region and the lacrimal gland located at the rostral region, are capable of stimulating aggression in male mice. We further show that these extracts can stimulate the production of inositol-(1,4,5)-trisphosphate in the vomeronasal organ.

Attenuation of the production of inositol 1,4,5-trisphosphate in the mouse vomeronasal organ by antibodies against the alphaq/11 subfamily of G-proteins.

The social and reproductive behaviors of most mammals are modulated by pheromones, which are perceived by the vomeronasal organ (VNO). Vomeronasal transduction in vertebrates is activated through G-protein-coupled receptors, which in turn leads to the generation of inositol 1,4,5-trisphosphate (IP(3)) and diacylglycerol (DAG) by the activity of phospholipase C. DAG has been shown to gate the transient receptor potential channel 2, whereas IP(3) may play a role in stimulating the release of calcium from the endoplasmic reticulum store. To investigate the role of the alpha subunits of G(q/11) in the transduction process, microvillar membranes from female mice VNO were preincubated with a selective C-terminal peptide antibody against Galpha(q/11) and then stimulated with adult male urine. Incubation of VNO membranes with antibodies against Galpha(q/11) blocked the production of IP(3) in a dose-dependent manner. We were also able to impair the production of IP(3) when we stimulated with 2-heptanone or 2,5-dimethylpyrazine in the presence of antibodies against the alpha subunit of G(q/11). 2-Heptanone is a known pheromone that has been linked to VIR receptors. Thus, our observations indicate that the alpha subunits of G(q/11) play a role in pheromonal signaling in the VNO.

Sex-specific responses to urinary chemicals by the mouse vomeronasal organ.

Social behaviors of most mammals are affected by chemical signals, pheromones, exchanged between conspecifics. Previous experiments have shown that behavioral responses to the same pheromone differ depending on the sex and endocrine status of the respondent. Although the exact mechanism of this dimorphism is not known, one possible contributor may be due to sexually dimorphic receptors or due to differences in central processing within the brain. In order to investigate the differences in response between male and female mice to the same pheromonal stimulus two urinary compounds (2-heptanone and 2,5-dimethylpyrazine) were used to stimulate the production of Inositol (1,4,5)-trisphosphate (IP(3)) in microvillar membrane preparations of the vomeronasal organ as an indirect measurement of pheromonal stimulation. Incubation of such membranes from prepubertal mice with urine from the same sex or opposite sex, results in an increase in production of IP(3). This stimulation is mimicked by GTPgammaS and blocked by GDPbetaS. Furthermore we found that 2-heptanone present in both male and female urine was capable of stimulating increased production of IP(3) in the female VNO but not the male VNO. Finally, 2,5-dimethylpyrazine present only in female urine was also only capable of stimulating increased production of IP(3) in the female VNO.

Involvement of G(q/11) in signal transduction in the mammalian vomeronasal organ.

Social behaviors of most mammals are profoundly affected by pheromones. Pheromones are detected by G-protein coupled receptors in the vomeronasal organ (VNO). To investigate the role of G alpha(q/11) in vomeronasal signal transduction pathways, microvillar membranes from murine VNO were prepared. Incubation of such membranes from prepubertal females with adult male urine results in an increase in production of inositol-(1,4,5)-trisphosphate (IP(3)). This stimulation is mimicked by GTP gamma S, blocked by GDP beta S and is tissue specific. Furthermore, use of bacterial toxins such as pertussis that lead to ADP-ribosylation of the G-protein alpha subunits of G(o) and G(i2) do not block the increase in IP(3) levels but U-73122, a PLC inhibitor, blocks the production of IP(3). Studies with monospecific antibodies revealed the presence of three G-proteins, G alpha(o), G alpha(i2) and G alpha(q/11)-related protein, in vomeronasal neurons, concentrated on their microvilli. Our observations indicate that pheromones in male urine act on vomeronasal neurons in the female VNO via a receptor-mediated, G alpha(q/11)-protein-dependent increase in IP(3) levels.