Encapsulation of Recombinant MOMP in Extended-Releasing PLGA 85:15 Nanoparticles Confer Protective Immunity Against a Chlamydia muridarum Genital Challenge and Re-Challenge

Recently we reported the immune-potentiating capacity of a Chlamydia nanovaccine (PLGA-rMOMP) comprising rMOMP (recombinant major outer membrane protein) encapsulated in extended-releasing PLGA [poly (D, L-lactide-co-glycolide) (85:15)] nanoparticles. Here we hypothesized that PLGA-rMOMP would bolster immune-effector mechanisms to confer protective efficacy in mice against a Chlamydia muridarum genital challenge and re-challenge. Female BALB/c mice received three immunizations, either subcutaneously (SC) or intranasally (IN), before receiving an intravaginal challenge with C. muridarum on day 49 and a re-challenge on day 170. Both the SC and IN immunization routes protected mice against genital challenge with enhanced protection after a re-challenge, especially in the SC mice. The nanovaccine induced robust antigen-specific Th1 (IFN-γ, IL-2) and IL-17 cytokines plus CD4+ proliferating T-cells and memory (CD44high CD62Lhigh) and effector (CD44high CD62Llow) phenotypes in immunized mice. Parallel induction of antigen-specific systemic and mucosal Th1 (IgG2a, IgG2b), Th2 (IgG1), and IgA antibodies were also noted. Importantly, immunized mice produced highly functional Th1 avidity and serum antibodies that neutralized C. muridarum infectivity of McCoy fibroblasts in-vitro that correlated with their respective protection levels. The SC, rather than the IN immunization route, triggered higher cellular and humoral immune effectors that improved mice protection against genital C. muridarum. We report for the first time that the extended-releasing PLGA 85:15 encapsulated rMOMP nanovaccine confers protective immunity in mice against genital Chlamydia and advances the potential towards acquiring a nano-based Chlamydia vaccine.Fil: Sahu, Rajnish. University of Alabama at Birmingahm; Estados UnidosFil: Dixit, Saurabh. University of Alabama at Birmingahm; Estados UnidosFil: Verma, Richa. University of Alabama at Birmingahm; Estados UnidosFil: Duncan, Skyla A.. University of Alabama at Birmingahm; Estados UnidosFil: Smith, Lula. University of Alabama at Birmingahm; Estados UnidosFil: Giambartolomei, Guillermo Hernan. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Singh, Shree R.. University of Alabama at Birmingahm; Estados UnidosFil: Dennis, Vida A.. University of Alabama at Birmingahm; Estados Unido

The Chlamydia M278 Major Outer Membrane Peptide Encapsulated in the Poly(lactic acid)-Poly(ethylene glycol) Nanoparticulate Self-Adjuvanting Delivery System Protects Mice Against a Chlamydia muridarum Genital Tract Challenge by Stimulating Robust Systemic and Local Mucosal Immune Responses

Recently, we reported that our PPM chlamydial nanovaccine [a biodegradable co-polymeric PLA-PEG (poly(lactic acid)-poly(ethylene glycol))-encapsulated M278 peptide (derived from the major outer membrane protein (MOMP) of Chlamydia)] exploits the caveolin-mediated endocytosis pathway for endosomal processing and MHC class II presentation to immune-potentiate Chlamydia-specific CD4+ T-cell immune effector responses. In the present study, we employed the Chlamydia muridarum mouse infection model to evaluate the protective efficacy of PPM against a genital tract challenge. Our results show that mice immunized with PPM were significantly protected against a homologous genital tract challenge evidently by reduced vaginal bacterial loads. Protection of mice correlated with enhanced Chlamydia-specific adaptive immune responses predominated by IFN-γ along with CD4+ T-cells proliferation and their differentiation to CD4+ memory (CD44high CD62Lhigh) and effector (CD44high CD62Llow) T-cell phenotypes. We observed the elevation of M278- and MOMP-specific serum antibodies with high avidity in the ascending order IgG1 > IgG2b > IgG2a. A key finding was the elevated mucosal IgG1 and IgA antibody titers followed by an increase in MOMP-specific IgA after the challenge. The Th1/Th2 antibody titer ratios (IgG2a/IgG1 and IgG2b/IgG1) revealed that PPM evoked a Th2-directed response, which skewed to a Th1-dominated antibody response after the bacterial challenge of mice. In addition, PPM immune sera neutralized the infectivity of C. muridarum in McCoy cells, suggesting the triggering of functional neutralizing antibodies. Herein, we reveal for the first time that subcutaneous immunization with the self-adjuvanting biodegradable co-polymeric PPM nanovaccine immune-potentiated robust CD4+ T cell-mediated immune effector responses; a mixed Th1 and Th2 antibody response and local mucosal IgA to protect mice against a chlamydial genital tract challenge

CYP450 phenotyping and metabolite identification of quinine by accurate mass UPLC-MS analysis: a possible metabolic link to blackwater fever

BACKGROUND: The naturally occurring alkaloid drug, quinine is commonly used for the treatment of severe malaria. Despite centuries of use, its metabolism is still not fully understood, and may play a role in the haemolytic disorders associated with the drug. METHODS: Incubations of quinine with CYPs 1A2, 2C9, 2C19, 2D6, and 3A4 were conducted, and the metabolites were characterized by accurate mass UPLC-MS(E) analysis. Reactive oxygen species generation was also measured in human erythrocytes incubated in the presence of quinine with and without microsomes. RESULTS: The metabolites 3-hydroxyquinine, 2’-oxoquininone, and O-desmethylquinine were observed after incubation with CYPs 3A4 (3-hydroxyquinine and 2’-oxoquininone) and 2D6 (O-desmethylquinine). In addition, multiple hydroxylations were observed both on the quinoline core and the quinuclidine ring system. Of the five primary abundance CYPs tested, 3A4, 2D6, 2C9, and 2C19 all demonstrated activity toward quinine, while 1A2 did not. Further, quinine produced robust dose-dependent oxidative stress in human erythrocytes in the presence of microsomes. CONCLUSIONS: Taken in context, these data suggest a CYP-mediated link between quinine metabolism and the poorly understood haemolytic condition known as blackwater fever, often associated with quinine ingestion

Scalable Preparation and Differential Pharmacologic and Toxicologic Profiles of Primaquine Enantiomers

Hematotoxicity in individuals genetically deficient in glucose-6-phosphate dehydrogenase (G6PD) activity is the major limitation of primaquine (PQ), the only antimalarial drug in clinical use for treatment of relapsing Plasmodium vivax malaria. PQ is currently clinically used in its racemic form. A scalable procedure was developed to resolve racemic PQ, thus providing pure enantiomers for the first time for detailed preclinical evaluation and potentially for clinical use. These enantiomers were compared for antiparasitic activity using several mouse models and also for general and hematological toxicities in mice and dogs. (+)-(S)-PQ showed better suppressive and causal prophylactic activity than (−)-(R)-PQ in mice infected with Plasmodium berghei. Similarly, (+)-(S)-PQ was a more potent suppressive agent than (−)-(R)-PQ in a mouse model of Pneumocystis carinii pneumonia. However, at higher doses, (+)-(S)-PQ also showed more systemic toxicity for mice. In beagle dogs, (+)-(S)-PQ caused more methemoglobinemia and was toxic at 5 mg/kg of body weight/day given orally for 3 days, while (−)-(R)-PQ was well tolerated. In a novel mouse model of hemolytic anemia associated with human G6PD deficiency, it was also demonstrated that (−)-(R)-PQ was less hemolytic than (+)-(S)-PQ for the G6PD-deficient human red cells engrafted in the NOD-SCID mice. All these data suggest that while (+)-(S)-PQ shows greater potency in terms of antiparasitic efficacy in rodents, it is also more hematotoxic than (−)-(R)-PQ in mice and dogs. Activity and toxicity differences of PQ enantiomers in different species can be attributed to their different pharmacokinetic and metabolic profiles. Taken together, these studies suggest that (−)-(R)-PQ may have a better safety margin than the racemate in human

Thermosonication an Alternative to Thermal Pasteurization for Nagpur Mandarin Juice

Nagpur mandarin is well known globally for its excellent nutritional benefits and flavour. These all characteristics get affected due to a lack of knowledge & processing techniques. Thermal pasteurization is a processing technique mostly used for juice processing before storage. Thermal pasteurization treatment leads to a loss in various physicochemical qualities of juice viz. ascorbic acid content, and carotenoid content. Thermal pasteurization treatment also leads to an increase in limonin content and the browning value of the juice. Therefore, an experiment was carried out to check the effect fascorbic acid and carotenoid content using the ultrasound combined with higher temperatures known as ensthermosensation on Nagpur mandarin (Citrus reticulata Blanco) juice as compared. Treatments were also used to observe its effect on limonin, browning, total soluble solids, pH & acidity as compared to thermal pasteurization. Juice samples were thermosonicated at temperatures of 50 ℃, 55 ℃, 60 ℃, and 63 ℃ for 25, 20, 15 and 10 min respectively at a constant frequency of 40 kHz. Juice samples were also thermally pasteurized at 65 ℃, 75 ℃, 85 ℃, and 95 ℃ for 80, 60, 40, and 20 sec. Samples were analyzed after treatment. Thermosonication treatments showed better ascorbic acid content and carotenoid content in comparison to thermally pasteurized juice samples. Limonin value and browning value were also less in thermosonicated juice samples. Both treatments do not have any significant effect on Physicochemical characteristics like TSS, pH & acidity. fromThe result indicated that ensthermosensation treatments performed better than thermal pasteurization treatments and can be used as an alternative technique for Nagpur mandarin juice processing

Impact of Thermo-sonication on Pectin Methylesterase (PME) Inactivation & Cloud Stability of Nagpur Mandarin Juice

Nagpur mandarin is well known globally for its excellent nutritional benefits and flavour. These all characteristics get affected during storage due to a lack of knowledge & processing techniques. One of the most important parameters which decide the quality of fruit juice during storage is cloud stability, which gets affected during storage due to the action of an enzyme named pectin methylesterase. Pectin methylesterase enzyme (PME) is a heat-resistant enzyme that impacts the juice quality by decreasing the cloud stability. Thermosonication is a technique to reduce the PME activity in juice by utilizing the principle of heat and ultrasound. Therefore, an experiment was carried out to inactivate the PME utilizing the ultrasound combined with higher temperature known as thermosensation on Nagpur mandarin (Citrus reticulata Blanco) juice. This study was initiated to calculate the effect of thermosensation treatment on the pectin methylesterase enzyme (PME) activity along with the effect of thermosensation on cloud stability in comparison with various thermal pasteurization treatments. Juice samples were thermosonicated at temperatures of 50 ℃, 55 ℃, 60 ℃, and 63 ℃ for 25, 20, 15 and 10 min respectively at a constant frequency of 40 kHz. Juice samples were also thermally pasteurized at 65 ℃, 75 ℃, 85 ℃, and 95 ℃ for 80, 60, 40, and 20 sec. Samples were analyzed after treatment at 0 days and 7 days of refrigerated storage at 4 ℃. Result indicated that thermosensation at 63 ℃ for 10 min at 40 kHz frequency showed much reduced PME activity with inactivation of 72.56% over control whereas thermal pasteurization at 95 ℃ showed inactivation of 69.31%. The same treatment also showed maximum cloud activity as compared to control and thermally pasteurized juice samples. Hence thermosensation can be used as an alternative processing technique for the inactivation of PME and cloud stability

Nanofiller-Enhanced Soft Non-Gelatin Alginate Capsules for Modified Drug Delivery

Capsules are one of the major solid dosage forms available in a variety of compositions and shapes. Developments in this dosage form are not new, but the production of non-gelatin capsules is a recent trend. In pharmaceutical as well as other biomedical research, alginate has great versatility. On the other hand, the use of inorganic material to enhance material strength is a common research topic in tissue engineering. The research presented here is a combination of qualities of alginate and montmorillonite (MMT). These two materials were used in this research to produce a soft non-gelatin modified-release capsule. Moreover, the research describes a facile benchtop production of these capsules. The produced capsules were critically analyzed for their appearance confirming resemblance with marketed capsules, functionality in terms of drug encapsulation, as well as release and durability

Effect of Thermosonication on Total Soluble Solid, pH and Titrable Acidity of Nagpur Mandarin Juice

Nagpur mandarin is well known globally for its excellent nutritional benefits and flavor. Various physicochemical characteristics get affected during storage due to lack of knowledge & processing techniques. One of the most parameters which decide the quality and taste of fruit juice during storage is total soluble solid, pH & titrable acidity. These parameters get affected during storage and shows a decreasing value for total soluble solid, pH & titrable acidity. Therefore, an experiment was carried out to understand the effect of thermosonication and thermal pasteurization on total soluble solid, pH and titrable acidity of Nagpur mandarin (Citrus reticulata Blanco) juice. Samples were analyzed after treatment at 0 day and 7 days of refrigerated storage at 4 ℃. Result indicated that thermosonication and thermal pasteurization treatments on total soluble solid, pH and titrable acidity showed non-significant effect. However, heat pasteurization was found to be superior over thermosonication. Heat pasteurization at 95℃ for 20 seconds recorded better performance in total soluble solid, pH & acidity compared to other treatment

Caveolin-mediated endocytosis of the Chlamydia M278 outer membrane peptide encapsulated in poly(lactic acid)-Poly(ethylene glycol) nanoparticles by mouse primary dendritic cells enhances specific immune effectors mediated by MHC class II and CD4+ T cells

We previously developed a Chlamydia trachomatis nanovaccine (PPM) by encapsulating a chlamydial M278 peptide within poly(lactic acid)-poly(ethylene glycol) biodegradable nanoparticles that immunopotentiated Chlamydia-specific immune effector responses in mice. Herein, we investigated the mechanistic interactions of PPM with mouse bone marrow-derived dendritic cells (DCs) for its uptake, trafficking, and T cell activation. Our results reveal that PPM triggered enhanced expression of effector cytokines and chemokines, surface activation markers (Cd1d2, Fcgr1), pathogen-sensing receptors (TLR2, Nod1), co-stimulatory (CD40, CD80, CD86) and MHC class I and II molecules. Co-culturing of PPM-primed DCs with T cells from C. muridarum vaccinated mice yielded an increase in Chlamydia-specific immune effector responses including CD3+ lymphoproliferation, CD3+CD4+ IFN-γ-secreting cells along with CD3+CD4+ memory (CD44high and CD62Lhigh) and effector (CD44high and CD62Llow) phenotypes. Intracellular trafficking analyses revealed an intense expression and colocalization of PPM predominantly in endosomes. PPM also upregulated the transcriptional and protein expression of the endocytic mediator, caveolin-1 in DCs. More importantly, the specific inhibition of caveolin-1 led to decreased expression of PPM-induced cytokines and co-stimulatory molecules. Our investigation shows that PPM provided enhancement of uptake, probably by exploiting the caveolin-mediated endocytosis pathway, endosomal processing, and MHC II presentation to immunopotentiate Chlamydia-specific immune effector responses mediated by CD4+ T cells.Fil: Dixit, Saurabh. Alabama State University; Estados UnidosFil: Sahu, Rajnish. Alabama State University; Estados UnidosFil: Verma, Richa. Alabama State University; Estados UnidosFil: Duncan, Skyla. Alabama State University; Estados UnidosFil: Giambartolomei, Guillermo Hernan. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; ArgentinaFil: Singh, Shree R.. Alabama State University; Estados UnidosFil: Dennis, Vida A.. Alabama State University; Estados Unido

Effect of Different Growing Media on Growth and Flowering of Petunia (Petunia hybrida L.)

The Department of Floriculture and Landscape Architecture, COH, Mandsaur, RVSKVV, Gwalior undertook the current study to assess the effectiveness of various growing media on the growth and flowering of petunia (Petunia hybrida L.) (M.P.). The experiment had fourteen treatments, three replications, and was set up using a Completely Randomized Design (CRD). Different combinations of cocopeat, FYM, sand, soil and vermicompost were used to create the growth media. In terms of number of branches, flowers per plant, days to blooming, length of flowering, fresh weight of root mass, and dried weight of root mass, data showed that T14 [Cocopeat + Sand + Soil + Vermicompost (1:1:1:1)] produced the greatest results. In contrast, T8 [Sand + Vermicompost (1:1)] outperformed all other treatments in terms of floral diameter, fresh above-ground mass weight, and dry above-ground mass weight. Regarding plant length, number of leaf per plant, and stem diameter, treatment T2 [vermicompost] has been shown to be superior to other treatments