Bioinspired zwitterionic triblock copolymers designed for colloidal drug delivery: 2 - Biological evaluation.

In this work, poly(lactide) nanoparticles were equipped with a bioinspired coating layer based on poly[2-(methacryloyloxy)ethyl phosphorylcholine] and then evaluated when administered to the lungs and after intravenous injection. Compared to the plain counterparts, the chosen zwitterionic polymer shell prevented the coated colloidal formulation from aggregation and conditioned it for lower cytotoxicity, protein adsorption, complement activation and phagocytic cell uptake. Consequently, no interference with the biophysical function of the lung surfactant system could be detected accompanied by negligible protein and cell influx into the bronchoalveolar space after intratracheal administration. When injected into the central compartment, the coated formulation showed a prolonged circulation half-life and a delayed biodistribution to the liver. Taken together, colloidal drug delivery vehicles would clearly benefit from the investigated poly[2-(methacryloyloxy)ethyl phosphorylcholine]-based polymer coatings.

Comparison study of surface-initiated hydrogel coatings with distinct side-chains for improving biocompatibility of polymeric heart valves.

Polymeric heart valves (PHVs) present a promising alternative for treating valvular heart diseases with satisfactory hydrodynamics and durability against structural degeneration. However, the cascaded coagulation, inflammatory responses, and calcification in the dynamic blood environment pose significant challenges to the surface design of current PHVs. In this study, we employed a surface-initiated polymerization method to modify polystyrene-block-isobutylene-block-styrene (SIBS) by creating three hydrogel coatings: poly(2-methacryloyloxy ethyl phosphorylcholine) (pMPC), poly(2-acrylamido-2-methylpropanesulfonic acid) (pAMPS), and poly(2-hydroxyethyl methacrylate) (pHEMA). These hydrogel coatings dramatically promoted SIBS's hydrophilicity and blood compatibility at the initial state. Notably, the pMPC and pAMPS coatings maintained a considerable platelet resistance performance after 12 h of sonication and 10 000 cycles of stretching and bending. However, the sonication process induced visible damage to the pHEMA coating and attenuated the anti-coagulation property. Furthermore, the in vivo subcutaneous implantation studies demonstrated that the amphiphilic pMPC coating showed superior anti-inflammatory and anti-calcification properties. Considering the remarkable stability and optimal biocompatibility, the amphiphilic pMPC coating constructed by surface-initiated polymerization holds promising potential for modifying PHVs.

Unmasking the Mechanism behind Miltefosine: Revealing the Disruption of Intracellular Ca2+ Homeostasis as a Rational Therapeutic Target in Leishmaniasis and Chagas Disease.

Originally developed as a chemotherapeutic agent, miltefosine (hexadecylphosphocholine) is an inhibitor of phosphatidylcholine synthesis with proven antiparasitic effects. It is the only oral drug approved for the treatment of Leishmaniasis and American Trypanosomiasis (Chagas disease). Although its precise mechanisms are not yet fully understood, miltefosine exhibits broad-spectrum anti-parasitic effects primarily by disrupting the intracellular Ca2+ homeostasis of the parasites while sparing the human hosts. In addition to its inhibitory effects on phosphatidylcholine synthesis and cytochrome c oxidase, miltefosine has been found to affect the unique giant mitochondria and the acidocalcisomes of parasites. Both of these crucial organelles are involved in Ca2+ regulation. Furthermore, miltefosine has the ability to activate a specific parasite Ca2+ channel that responds to sphingosine, which is different to its L-type VGCC human ortholog. Here, we aimed to provide an overview of recent advancements of the anti-parasitic mechanisms of miltefosine. We also explored its multiple molecular targets and investigated how its pleiotropic effects translate into a rational therapeutic approach for patients afflicted by Leishmaniasis and American Trypanosomiasis. Notably, miltefosine's therapeutic effect extends beyond its impact on the parasite to also positively affect the host's immune system. These findings enhance our understanding on its multi-targeted mechanism of action. Overall, this review sheds light on the intricate molecular actions of miltefosine, highlighting its potential as a promising therapeutic option against these debilitating parasitic diseases.

Successful treatment of recurrent visceral leishmaniasis relapse in an immunocompetent adult female with functional hypopituitarism in Bangladesh.

BACKGROUND: Currently available treatment options are mostly effective in achieving long-term cure in visceral leishmaniasis (VL) patients. However, there have been reports of recurrence of this illness in both immunosuppressed and immunocompetent patients. CASE PRESENTATION: We report the first case of recurrent VL relapse in a 19-year-old immunocompetent female with functional hypopituitarism (hypogonadotropic hypogonadism with central hypothyroidism) from Bangladesh, who has been treated three times previously with optimal dosage and duration- liposomal amphotericin B (LAmB) alone and in combination with miltefosine. We treated the patient successfully with a modified treatment regimen of 10 mg/kg body weight LAmB for two consecutive days along with oral miltefosine for seven days as loading dose. For secondary prophylaxis, the patient received 3 mg/kg body weight LAmB along with oral miltefosine for seven days monthly for five doses followed by hormonal replacement. The patient remained relapse free after 12 months of her treatment completion. CONCLUSION: In the absence of protective vaccines against Leishmania species and standard treatment regimen, this modified treatment regimen could help the management of recurrent relapse cases.

ABC transporter inhibition by beauvericin partially overcomes drug resistance in Leishmania tropica.

Leishmaniasis is a neglected tropical disease infecting the world's poorest populations. Miltefosine (ML) remains the primary oral drug against the cutaneous form of leishmaniasis. The ATP-binding cassette (ABC) transporters are key players in the xenobiotic efflux, and their inhibition could enhance the therapeutic index. In this study, the ability of beauvericin (BEA) to overcome ABC transporter-mediated resistance of Leishmania tropica to ML was assessed. In addition, the transcription profile of genes involved in resistance acquisition to ML was inspected. Finally, we explored the efflux mechanism of the drug and inhibitor. The efficacy of ML against all developmental stages of L. tropica in the presence or absence of BEA was evaluated using an absolute quantification assay. The expression of resistance genes was evaluated, comparing susceptible and resistant strains. Finally, the mechanisms governing the interaction between the ABC transporter and its ligands were elucidated using molecular docking and dynamic simulation. Relative quantification showed that the expression of the ABCG sub-family is mostly modulated by ML. In this study, we used BEA to impede resistance of Leishmania tropica. The IC50 values, following BEA treatment, were significantly reduced from 30.83, 48.17, and 16.83 µM using ML to 8.14, 11.1, and 7.18 µM when using a combinatorial treatment (ML + BEA) against promastigotes, axenic amastigotes, and intracellular amastigotes, respectively. We also demonstrated a favorable BEA-binding enthalpy to L. tropica ABC transporter compared to ML. Our study revealed that BEA partially reverses the resistance development of L. tropica to ML by blocking the alternate ATP hydrolysis cycle.

Leishmania blood parasite dynamics during and after treatment of visceral leishmaniasis in Eastern Africa: A pharmacokinetic-pharmacodynamic model.

BACKGROUND: With the current treatment options for visceral leishmaniasis (VL), recrudescence of the parasite is seen in a proportion of patients. Understanding parasite dynamics is crucial to improving treatment efficacy and predicting patient relapse in cases of VL. This study aimed to characterize the kinetics of circulating Leishmania parasites in the blood, during and after different antileishmanial therapies, and to find predictors for clinical relapse of disease. METHODS: Data from three clinical trials, in which Eastern African VL patients received various antileishmanial regimens, were combined in this study. Leishmania kinetoplast DNA was quantified in whole blood with real-time quantitative PCR (qPCR) before, during, and up to six months after treatment. An integrated population pharmacokinetic-pharmacodynamic model was developed using non-linear mixed effects modelling. RESULTS: Parasite proliferation was best described by an exponential growth model, with an in vivo parasite doubling time of 7.8 days (RSE 12%). Parasite killing by fexinidazole, liposomal amphotericin B, sodium stibogluconate, and miltefosine was best described by linear models directly relating drug concentrations to the parasite elimination rate. After treatment, parasite growth was assumed to be suppressed by the host immune system, described by an Emax model driven by the time after treatment. No predictors for the high variability in onset and magnitude of the immune response could be identified. Model-based individual predictions of blood parasite load on Day 28 and Day 56 after start of treatment were predictive for clinical relapse of disease. CONCLUSION: This semi-mechanistic pharmacokinetic-pharmacodynamic model adequately captured the blood parasite dynamics during and after treatment, and revealed that high blood parasite loads on Day 28 and Day 56 after start of treatment are an early indication for VL relapse, which could be a useful biomarker to assess treatment efficacy of a treatment regimen in a clinical trial setting.

Chitosan nanoparticles improve the effectivity of miltefosine against Acanthamoeba.

BACKGROUND: Acanthamoeba keratitis (AK) is a corneal sight-threatening infection caused by the free-living amoebae of the genus Acanthamoeba. Early and appropriate treatment significantly impacts visual outcomes. Mucoadhesive polymers such as chitosan are a potential strategy to prolong the residence time and bioavailability of the encapsulated drugs in the cornea. Regarding the recent administration of miltefosine (MF) for treating resistant AK, in the present study, we synthesized miltefosine-loaded chitosan nanoparticles (MF-CS-NPs) and evaluated them against Acanthamoeba. METHODOLOGY/PRINCIPAL FINDINGS: Chitosan nanoparticles (CNPs) were prepared using the ionic gelation method with negatively charged tripolyphosphate (TPP). The zeta-potential (ZP) and the particle size of MF-CS-NPs were 21.8±3.2 mV and 46.61±18.16 nm, respectively. The release profile of MF-CS-NPs indicated linearity with sustained drug release. The cytotoxicity of MF-CS-NPs on the Vero cell line was 2.67 and 1.64 times lower than free MF at 24 and 48 hours. This formulation exhibited no hemolytic activity in vitro and ocular irritation in rabbit eyes. The IC50 of MF-CS-NPs showed a significant reduction by 2.06 and 1.69-fold in trophozoites at 24 and 48 hours compared to free MF. Also, the MF-CS-NPs IC50 in the cysts form was slightly decreased by 1.26 and 1.21-fold at 24 and 48 hours compared to free MF. CONCLUSIONS: The MF-CS-NPs were more effective against the trophozoites and cysts than free MF. The nano-chitosan formulation was more effective on trophozoites than the cysts form. MF-CS-NPs reduced toxicity and improved the amoebicidal effect of MF. Nano-chitosan could be an ideal carrier that decreases the cytotoxicity of miltefosine. Further analysis in animal settings is needed to evaluate this nano-formulation for clinical ocular drug delivery.

Vascular Endothelial Cell-Derived Exosomal Sphingosylphosphorylcholine Attenuates Myocardial Ischemia-Reperfusion Injury through NR4A2-Mediated Mitophagy.

Cardiomyocyte survival is a critical contributing process of host adaptive responses to cardiovascular diseases (CVD). Cells of the cardiovascular endothelium have recently been reported to promote cardiomyocyte survival through exosome-loading cargos. Sphingosylphosphorylcholine (SPC), an intermediate metabolite of sphingolipids, mediates protection against myocardial infarction (MI). Nevertheless, the mechanism of SPC delivery by vascular endothelial cell (VEC)-derived exosomes (VEC-Exos) remains uncharacterized at the time of this writing. The present study utilized a mice model of ischemia/reperfusion (I/R) to demonstrate that the administration of exosomes via tail vein injection significantly diminished the severity of I/R-induced cardiac damage and prevented apoptosis of cardiomyocytes. Moreover, SPC was here identified as the primary mediator of the observed protective effects of VEC-Exos. In addition, within this investigation, in vitro experiments using cardiomyocytes showed that SPC counteracted myocardial I/R injury by activating the Parkin and nuclear receptor subfamily group A member 2/optineurin (NR4A2/OPTN) pathways, in turn resulting in increased levels of mitophagy within I/R-affected myocardium. The present study highlights the potential therapeutic effects of SPC-rich exosomes secreted by VECs on alleviating I/R-induced apoptosis in cardiomyocytes, thereby providing strong experimental evidence to support the application of SPC as a potential therapeutic target in the prevention and treatment of myocardial infarction.

Plasma metabolomic profile in orthostatic intolerance children with high levels of plasma homocysteine.

BACKGROUND: Orthostatic intolerance, which includes vasovagal syncope and postural orthostatic tachycardia syndrome, is common in children and adolescents. Elevated plasma homocysteine levels might participate in the pathogenesis of orthostatic intolerance. This study was designed to analyze the plasma metabolomic profile in orthostatic intolerance children with high levels of plasma homocysteine. METHODS: Plasma samples from 34 orthostatic intolerance children with a plasma homocysteine concentration > 9 µmol/L and 10 healthy children were subjected to ultra-high-pressure liquid chromatography and quadrupole-time-of-flight mass spectrometry analysis. RESULTS: A total of 875 metabolites were identified, 105 of which were significantly differential metabolites. Choline, 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine, 1-(1Z-octadecenyl)-2-(4Z,7Z,10Z,13Z,16Z,19Z-docosahexaenoyl)-sn-glycero-3-phosphocholine, histidine, isocitric acid, and DL-glutamic acid and its downstream metabolites were upregulated, whereas 1-palmitoyl-sn-glycero-3-phosphocholine, 1-stearoyl-sn-glycerol 3-phosphocholine, sphingomyelin (d18:1/18:0), betaine aldehyde, hydroxyproline, and gamma-aminobutyric acid were downregulated in the orthostatic intolerance group compared with the control group. All these metabolites were related to choline and glutamate. Heatmap analysis demonstrated a common metabolic pattern of higher choline, 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine, and DL-glutamic acid, and lower sphingomyelin (d18:1/18:0), 1-stearoyl-sn-glycerol 3-phosphocholine, and 1-palmitoyl-sn-glycero-3-phosphocholine in patients with certain notable metabolic changes (the special group) than in the other patients (the common group). The maximum upright heart rate, the change in heart rate from the supine to the upright position, and the rate of change in heart rate from the supine to the upright position of vasovagal syncope patients were significantly higher in the special group than in the common group (P < 0.05). Choline, 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine, and DL-glutamic acid were positively correlated with the rate of change in heart rate from the supine to the upright position in vasovagal syncope patients (P < 0.05). CONCLUSIONS: The levels of choline-related metabolites and glutamate-related metabolites changed significantly in orthostatic intolerance children with high levels of plasma homocysteine, and these changes were associated with the severity of illness. These results provided new light on the pathogenesis of orthostatic intolerance.

Monitoring the Long-Term Effectiveness of Miltefosine in Indian Post-Kala-Azar Dermal Leishmaniasis.

Post-kala-azar dermal leishmaniasis (PKDL), the dermal sequel to visceral leishmaniasis (VL), is characterized by hypopigmented macules (macular) and/or papules and nodules (polymorphic). Post-kala-azar dermal leishmaniasis plays a significant role in disease transmission, emphasizing the need for monitoring chemotherapeutic effectiveness. Accordingly, this study aimed to quantify the parasite burden in PKDL patients after treatment with miltefosine by a quantitative polymerase chain reaction (qPCR). A Leishmania kinetoplastid gene-targeted qPCR was undertaken using DNA from skin biopsy specimens of patients with PKDL at three time points, i.e., at disease presentation (week 0, n = 157, group 1), upon completion of treatment (week 12, n = 39, group 2), and at any time point 6 months after completion of treatment (week ≥36, n = 54, group 3). A cycle threshold (Ct) <30 was considered the cutoff for positivity, and load was quantified as the number of parasites/µg genomic DNA (gDNA); cure was considered when samples had a Ct >30. The parasite load at disease presentation (group 1) was 10,769 (1,339-80,441)/µg gDNA (median [interquartile range]). In groups 2 and 3, qPCR results were negative in 35/39 cases (89.7%) and 48/54 cases (88.8%), respectively. In the 10/93 (10.8%) qPCR-positive cases, the parasite burdens in groups 2 and 3 were 2,420 (1,205-5,661)/µg gDNA and 22,195 (5,524-100,106)/µg gDNA, respectively. Serial monitoring was undertaken in 45 randomly selected cases that had completed treatment; all cases in groups 2 or 3 had a Ct >30, indicating cure. Overall, qPCR confirmed an 89.2% cure (as 83/93 cases showed parasite clearance), and the persistent qPCR positivity was attributed to nonadherence to treatment or unresponsiveness to miltefosine and remains to be investigated.

Leishmania and HIV co-infection: first naturally Leishmania strain presenting decreased susceptibility to miltefosine, recovered from a patient in Portugal.

BACKGROUND: In Europe, up to 70% of visceral leishmaniasis (VL) cases occurring in adults living with HIV. People living with HIV with VL co-infection often display persistent parasitemia, requiring chronic intermittent anti-Leishmania therapies. Consequently, frequent VL relapses and higher mortality rates are common in these individuals. As such, it is of paramount importance to understand the reasons for parasite persistence to improve infection management. METHODS: To outline possible causes for treatment failure in the context of HIV-VL, we followed a person living with HIV-VL co-infection for nine years in a 12-month period. We characterized: HIV-related clinicopathological alterations (CD4+ T counts and viremia) and Leishmania-specific seroreactivity, parasitemia, quantification of pro-inflammatory cytokines upon stimulation and studied a Leishmania clinical isolate recovered during this period. RESULTS: The subject presented controlled viremia and low CD4+ counts. The subject remained PCR positive for Leishmania and also seropositive. The cellular response to parasite antigens was erratic. The isolate was identified as the first Leishmania infantum case with evidence of decreased miltefosine susceptibility in Portugal. CONCLUSION: Treatment failure is a multifactorial process driven by host and parasite determinants. Still, the real-time determination of drug susceptibility profiles in clinical isolates is an unexplored resource in the monitoring of VL.

Appraisal of Chitosan-Coated Lipid Nano-Combination with Miltefosine and Albendazole in the Treatment of Murine Trichinellosis: Experimental Study with Evaluation of Immunological and Immunohistochemical Parameters.

PURPOSE: Resistance and adverse consequences of albendazole (ABZ) in treating trichinellosis urged demand for secure and effective new drugs. The current study aimed to assess the effect of chitosan-coated lipid nano-combination with albendazole and miltefosine (MFS) in treating experimental murine trichinellosis and evaluating pathological and immunological changes of trichinellosis. MATERIALS AND METHODS: One hundred twenty Swiss albino mice were divided into six groups. Each group was subdivided into a and b subgroups based on the scarification time, which was 7- and 40-days post-infection (PI), respectively. The treatment efficacy was evaluated using parasitological, histopathological, serological (interleukin (IL)-12 and IL-4 serum levels), immunohistochemical (GATA3, glutathione peroxidase1 (GPX1) and caspase-3), and scanning electron microscopy (SEM) methods. RESULTS: The most effective drug was nanostructured lipid carriers (NLCs) loaded with ABZ (G5), which showed the most significant reduction in adults and larval count (100% and 92.39%, respectively). The greatest amelioration in histopathological changes was reported in G4 treated with MFS. GATA3 and caspase-3 were significantly reduced in all treated groups. GPX1 was significantly increased in G6 treated with MFS + NLCs. The highest degenerative effects on adults and larvae by SEM were documented in G6. CONCLUSION: Loading ABZ or MFS on chitosan-coated NLCs enhanced their efficacy against trichinellosis. Although ABZ was better than MFS, their combination should be considered as MFS caused a significant reduction in the intensity of infection. Furthermore, MFS showed anti-inflammatory (↓GATA3) and antiapoptotic effects (↓caspase-3), especially in the muscular phase. Also, when loaded with NLCS, it showed an antioxidant effect (↑GPX1).

Myotubularin-related protein 6 is an ion channel-associated pro-leishmanial phosphatase.

Residing obligatorily as amastigotes within the mammalian macrophages, the parasite Leishmania donovani inflicts the potentially fatal, globally re-emerging disease visceral leishmaniasis (VL) by altering intracellular signaling through kinases and phosphatases. Because the phosphatases that modulate the VL outcome in humans remained unknown, we screened a human phosphatase siRNA-library for anti-leishmanial functions in THP-1, a human macrophage-like cell line. Of the 251 phosphatases, the screen identified the Ca++-activated K+-channel-associated phosphatase myotubularin-related protein-6 (MTMR6) as the only phosphatase whose silencing reduced parasite load and IL-10 production in human macrophages. Virulent, but not avirulent, L. donovani infection increased MTMR6 expression in macrophages. As virulent L. donovani parasites expressed higher lipophosphoglycan, a TLR2-ligand, we tested the effect of TLR2 stimulation or blockade on MTMR6 expression. TLR1/TLR2-ligand Pam3CSK4 enhanced, but TLR2 blockade reduced, MTMR6 expression. L. donovani infection of macrophages ex vivo increased, but miltefosine treatment reduced, MTMR6 expression. Corroboratively, compared to endemic controls, untreated VL patients had higher, but miltefosine-treated VL patients had reduced, MTMR6 expression. The phosphatase siRNA-library screening thus identified MTMR6 as the first TLR2-modulated ion channel-associated phosphatase with significant implications in VL patients and anti-leishmanial functions.

Miltefosine reduces coxsackievirus B3 lethality of mice with enhanced STAT3 activation.

Coxsackievirus B3 (CVB3), one serotype of enteroviruses, can induce fatal myocarditis and hepatitis in neonates, but both treatment and vaccine are unavailable. Few reports tested antivirals to reduce CVB3. Several antivirals were developed against other enterovirus serotypes, but these antivirals failed in clinical trials due to side effects and drug resistance. Repurposing of clinical drugs targeting cellular factors, which enhance viral replication, may be another option. Parasite and cancer studies showed that the cellular protein kinase B (Akt) decreases interferon (IFN), apoptosis, and interleukin (IL)-6-induced STAT3 responses, which suppress CVB3 replication. Furthermore, miltefosine, the Akt inhibitor used in the clinic for parasite infections, enhances IL-6, IFN, and apoptosis responses in treated patients, suggesting that miltefosine could be the potential antiviral for CVB3. This study was therefore designated to test the antiviral effects of miltefosine against CVB3 in vitro and especially, in mice, as few studies test miltefosine in vitro, but not in vivo. In vitro results showed that miltefosine inhibited viral replication with enhanced activation of the cellular transcription factor, STAT3, which is reported to reduce CVB3 both in vitro and in mice. Notably, STAT3 knockdown abolished the anti-CVB3 activity of miltefosine in vitro. Mouse studies demonstrated that miltefosine pretreatment reduced CVB3 lethality of mice with decreased virus loads, organ damage, and apoptosis, but enhanced STAT3 activation. Miltefosine could be prophylaxis for CVB3 by targeting Akt to enhance STAT3 activation in the mechanism, which is independent of IFN responses and hardly reported in pathogen infections.

Disseminated Leishmaniasis, a Severe Form of Leishmania braziliensis Infection.

Disseminated leishmaniasis (DL) is an emergent severe disease manifesting with multiple lesions. To determine the relationship between immune response and clinical and therapeutic outcomes, we studied 101 DL and 101 cutaneous leishmaniasis (CL) cases and determined cytokines and chemokines in supernatants of mononuclear cells stimulated with leishmania antigen. Patients were treated with meglumine antimoniate (20 mg/kg) for 20 days (CL) or 30 days (DL); 19 DL patients were instead treated with amphotericin B, miltefosine, or miltefosine and meglumine antimoniate. High levels of chemokine ligand 9 were associated with more severe DL. The cure rate for meglumine antimoniate was low for both DL (44%) and CL (60%), but healing time was longer in DL (p = 0.003). The lowest cure rate (22%) was found in DL patients with >100 lesions. However, meglumine antimoniate/miltefosine treatment cured all DL patients who received it; therefore, that combination should be considered as first choice therapy.

Therapeutic success and failure in using miltefosine to treat dogs naturally infected with Leishmania infantum.

In urban environments, domestic dogs (Canis familiaris) are a major reservoir for the parasite Leishmania infantum. Miltefosine has been used as the standard treatment for canine visceral leishmaniasis in Brazil. However, therapeutic failures have been reported. In the present study, two dogs (CG03 and CG06) with a diagnosis of infection by L. infantum underwent two cycles of treatment with miltefosine (Milteforan™ - Virbac®). Analyses showed increases in the parasite load of both CG03 and CG06, even after treatment. The clinical score of CG03 dropped from 1 to 0 (after one round of treatment), such that this dog became asymptomatic. CG06 showed clinical worsening, such that its score increased from 1 to 2. After the second therapeutic round, the parasite load in CG03 was found to have decreased, but it was still higher than before drug treatment even though this dog was physically asymptomatic. There was no decrease in the parasite load in CG06 and there was clinical worsening. The clinical response of these dogs to the treatment differed, but the parasite load remained high in both cases, which poses a risk to public health, making it essential take measures to prevent the sandfly vector from accessing the dog.

Embedding Thiols into Choline Phosphate Polymer Zwitterions.

The compositional scope of polymer zwitterions has grown significantly in recent years and now offers designer synthetic materials that are broadly applicable across numerous areas, including supracolloidal structures, electronic materials interfaces, and macromolecular therapeutics. Among recent developments in polymer zwitterion syntheses are those that allow insertion of reactive functionality directly into the zwitterionic moiety, yielding new monomer and polymer structures that hold potential for maximizing the impact of zwitterions on the macromolecular materials chemistry field. This manuscript describes the preparation of zwitterionic choline phosphate (CP) methacrylates containing either aromatic or aliphatic thiols embedded directly into the zwitterionic moiety. The polymerization of these functional CP methacrylates by reversible addition-fragmentation chain-transfer methodology yields polymeric zwitterionic thiols containing protected thiol functionality in the zwitterionic units. After polymerization, the protected thiols are liberated to yield thiol-rich polymer zwitterions which serve as precursors to subsequent reactions that produce polymer networks as well as polymer-protein bioconjugates.

Paxillin participates in the sphingosylphosphorylcholine-induced abnormal contraction of vascular smooth muscle by regulating Rho-kinase activation.

BACKGROUND: The Ca2+-independent contraction of vascular smooth muscle is a leading cause of cardiovascular and cerebrovascular spasms. In the previous study, we demonstrated the involvement of Src family protein tyrosine kinase Fyn and Rho-kinase in the sphingosylphosphorylcholine (SPC)-induced abnormal and Ca2+-independent contraction of vascular smooth muscle, but the specific mechanism has not been completely clarified. METHODS: Paxillin knockdown human coronary artery smooth muscle cells (CASMCs) and smooth muscle-specific paxillin knockout mice were generated by using paxillin shRNA and the tamoxifen-inducible Cre-LoxP system, respectively. CASMCs contraction was observed by time-lapse recording. The vessel contractility was measured by using a myography assay. Fyn, Rho-kinase, and myosin light chain activation were assessed by immunoprecipitation and western blotting. The paxillin expression and actin stress fibers were visualized by histological analysis and immunofluorescent staining. RESULTS: The SPC-induced abnormal contraction was inhibited in paxillin knockdown CASMCs and arteries of paxillin knockout mice, indicating that paxillin is involved in this abnormal contraction. Further study showed that paxillin knockdown inhibited the SPC-induced Rho-kinase activation without affecting Fyn activation. In addition, paxillin knockdown significantly inhibited the SPC-induced actin stress fiber formation and myosin light chain phosphorylation. These results suggest that paxillin, as an upstream molecule of Rho-kinase, is involved in the SPC-induced abnormal contraction of vascular smooth muscle. CONCLUSIONS: The present study demonstrated that paxillin participates in the SPC-induced abnormal vascular smooth muscle contraction by regulating Rho-kinase activation. Video Abstract.

Leishmania spp. diagnosis and therapeutic management in a cat from urban area in Ibagué (Colombia).

BACKGROUND: Leishmania spp., a protozoan transmitted by sandflies, widely affects humans and dogs in Colombia, nevertheless feline leishmaniasis (FeL) remains understudied. OBJECTIVE: This study reports a case of feline leishmaniasis in Colombia and its therapeutic management. METHODS: Complete blood count, renal and hepatic serum biochemistry, nodular lesion cytology, FeLV/FIV snap test, abdominal ultrasound, and molecular diagnosis of Leishmania spp. 16 s rRNA gene amplification by real-time-PCR (qPCR), ITS-1 and hsp70 gene by endpoint-PCR and Sanger sequencing were performed. RESULTS: The patient was negative for FIV/FeLV and showed leukocytosis, lymphocytosis, thrombocytopenia, neutrophilia, monocytosis, hypergammaglobulinemia, increased gamma-glutamyl-transferase, cortical nephrocalcinosis, diffuse heterogeneous splenic parenchyma, and cholangitis. Nodular lesion cytology, qPCR and Sanger sequencing confirmed the diagnosis of Leishmania spp. The patient was treated with allopurinol and miltefosine. After treatment, clinical signs disappeared. CONCLUSION: Clinical examination, cytology, and molecular tests allowed a rapid and sensitive FeL diagnosis. Allopurinol and miltefosine improved the clinical condition of the cat.