ABSTRACT
Purpose
1-phenyl piperazine (PPZ) emerged from a Caco-2 monolayer screen as having high enhancement potential due to a capacity to increase permeation without significant toxicity. Our aim ...was to further explore the efficacy and toxicity of PPZ in rat ileal and colonic mucosae in order to assess its true translation potential.
Methods
Intestinal mucosae were mounted in Ussing chambers and apparent permeability coefficient (Papp) values of
14
C-mannitol and FITC-dextran 4 kDa (FD-4) and transepithelial electrical resistance (TEER) values were obtained following apical addition of PPZ (0.6–60 mM). Exposed issues were assessed for toxicity by histopathology and lactate dehydrogenase (LDH) release. Mucosal recovery after exposure was also assessed using TEER readings.
Results
PPZ reversibly increased the Papp of both agents across rat ileal and distal colonic mucosae in concentration–dependent fashion, accompanied by TEER reduction, with acceptable levels of tissue damage. The complex mechanism of tight junction opening was part mediated by myosin light chain kinase, stimulation of transepithelial electrogenic chloride secretion, and involved activation of 5-HT
4
receptors.
Conclusions
PPZ is an efficacious and benign intestinal permeation enhancer in tissue mucosae. However, its active pharmacology suggest that potential for further development in an oral formulation for poorly permeable molecules will be difficult.
Toxoplasma gondii is a ubiquitous protozoan parasite that is responsible for severe congenital birth defects and fatal toxoplasmic encephalitis in immunocompromized people. Fundamental aspects of ...obligate intracellular replication and pathogenesis are only now beginning to emerge for protozoan parasites. T. gondii has a fragmented pathway for salvaging pyrimidine nucleobases derived from the parasite or host cell, and this limited pyrimidine salvage capacity is funnelled exclusively through uracil phosphoribosyltransferase. Disrupting the function of this enzyme does not affect the growth of T. gondii tachyzoites, which suggests that the de novo pyrimidine biosynthesis pathway may be necessary for growth. We have examined the virulence of T. gondii mutants that lack carbamoyl phosphate synthetase II (uracil auxotrophs) to determine whether de novo pyrimidine biosynthesis is required in vivo. Here we show that T. gondii uracil auxotrophs are completely avirulent not only in immune-competent BALB/c mice but also in mice that lack interferon-γ. A single injection of the uracil auxotroph into BALB/c mice induces long-term protective immunity to toxoplasmosis. Our findings indicate the significance of the de novo pyrimidine biosynthesis pathway for the virulence of parasitic protozoa, and suggest routes for developing vaccines and chemotherapy.
Two separate carbamoyl phosphate synthetase activities are required for the de novo synthesis of pyrimidines and arginine in most eukaryotes.
Toxoplasma gondii is novel in possessing a single ...carbamoyl phosphate synthetase II gene that corresponds to a glutamine-dependent form required for pyrimidine biosynthesis. We therefore examined arginine acquisition in
T. gondii to determine whether the single carbamoyl phosphate synthetase II activity could provide both pyrimidine and arginine biosynthesis. We found that arginine deprivation efficiently blocks the replication of intracellular
T. gondii, yet has little effect on long-term parasite viability. Addition of citrulline, but not ornithine, rescues the growth defect observed in the absence of exogenous arginine. This rescue with citrulline is ablated when parasites are cultured in a human citrullinemia fibroblast cell line that is deficient in argininosuccinate synthetase activity. These results reveal the absence of genes and activities of the arginine biosynthetic pathway and demonstrate that
T. gondii is an arginine auxotroph. Arginine starvation was also found to efficiently trigger differentiation of replicative tachyzoites into bradyzoites contained within stable cyst-like structures. These same parasites expressing bradyzoite antigens can be efficiently switched back to rapidly proliferating tachyzoites several weeks after arginine starvation. We hypothesise that the absence of gene activities that are essential for the biosynthesis of arginine from carbamoyl phosphate confers a selective advantage by increasing bradyzoite switching during the host response to
T. gondii infection. These findings are consistent with a model of host–parasite evolution that allowed host control of bradyzoite induction by trading off virulence for increased transmission.
Purpose
CriticalSorb™ is a novel absorption enhancer based on Solutol
®
HS15, one that has been found to enhance the nasal transport. It is in clinical trials for nasal delivery of human growth ...hormone. The hypothesis was that permeating enhancement effects of the Solutol
®
HS15 component would translate to the intestine.
Methods
Rat colonic mucosae were mounted in Ussing chambers and Papp values of
14
C-mannitol,
14
C-antipyrine, FITC-dextran 4000 (FD-4), and TEER values were calculated in the presence of CriticalSorb™. Tissues were fixed for H & E staining. Caco-2 monolayers were grown on Transwells™ for similar experiments.
Results
CriticalSorb™(0.01% v/v) significantly increased the Papp of
14
C-mannitol, FD-4
14
C-antipyrine across ileal and colonic mucosae, accompanied by a decrease in TEER. In Caco-2 monolayers, it also increased the Papp of
14
C-mannitol FD-4 and
14
C-antipyrine over 120 min. In both monolayers and tissues, it acted as a moderately effective P-glycoprotein inhibitor. There was no evidence of cytotoxicity in Caco-2 at concentrations of 0.01% for up to 24 h and histology of tissues showed intact epithelia at 120 min.
Conclusions
Solutol
®
HS15 is the key component in CriticalSorb™ that enables non-cytotoxic
in vitro
intestinal permeation and its mechanism of action is a combination of increased paracellular and transcellular flux.
Healthy individuals have soluble (extracellular) DNA in their blood, and increased amounts are present in cancer patients. Here we report the detection of specific sequences of the cystic fibrosis ...and K-ras genes in plasma DNA from normal donors by amplification with the polymerase chain reaction. In addition, mutated K-ras sequences are identified by polymerase chain reaction utilizing allele-specific primers in the plasma or serum from three patients with pancreatic carcinoma that contain mutated K-ras genes. The mutations are confirmed by direct sequencing. These results indicate that sequences of single-copy genes can be identified in normal plasma and that the sequences of mutated oncogenes can be detected and identified with allele-specific amplification by polymerase chain reaction in plasma or serum from patients with malignant tumors containing identical mutated genes. Mutated oncogenes in plasma and serum may represent tumor markers that could be useful for diagnosis, determining response to treatment, and predicting prognosis.
IFN-γ-producing CD4⁺ T cells, although important for protection against acute Toxoplasma gondii infection, can cause gut pathology, which may prove to be detrimental for host survival. Here we show ...that mice lacking IL-15 gene develop a down-regulated IFN-※-producing CD4⁺ T cell response against the parasite, which leads to a reduction in gut necrosis and increased level of survival against infection. Moreover, transfer of immune CD4⁺ T cells from WT to$IL-15^{-/-}$mice reversed inhibition of gut pathology and caused mortality equivalent to levels of parental WT mice. Downregulated CD4⁺ T cell response in the absence of IL-15, manifested as reduced antigen-specific proliferation, was due to defective priming of the T cell subset by dendritic cells (DCs) of these animals. When stimulated with antigen-pulsed DCs from WT mice, CD4⁺ T cells from$IL-15^{-/-}$mice were primed optimally, and robust proliferation of these cells was observed. A defect in the DCs of knockout mice was further confirmed by their reduced ability to produce IL-12 upon stimulation with Toxoplasma lysate antigen. Addition of exogenous IL-15 to DC cultures from knockout mice led to increased IL-12 production by these cells and restored their ability to prime an optimal parasite-specific CD4⁺ T cell response. To our knowledge, this is the first demonstration of the role of IL-15 in the development of CD4⁺ T cell immunity against an intracellular pathogen. Furthermore, based on these observations, targeting of IL-15 should have a beneficial effect on individuals suffering from CD4⁺ T cell-mediated autoimmune diseases.
The serine repeat antigen (SERA) of
Plasmodium falciparum is a blood stage malaria vaccine candidate. It has been shown that 120 kDa SERA was proteolytically processed into N-terminal 47 kDa fragment ...(P47), central 56 kDa fragment (P56) that was further converted to 50 kDa (P50), and C-terminal 18 kDa fragment (P18). Here, we have examined the processing of SERA and the localization of its processed fragments by using mouse antibodies directed against recombinant proteins corresponding to different domains of SERA. Western blot analysis showed that all the processing events occurred inside parasitized erythrocytes at the stage just prior to the schizont rupture, that P47 was further processed into two 25 kDa fragments and that the two fragments, which were linked to P18 through disulfide bonds, were associated with the merozoite. In contrast, P50 was completely shed into culture medium and absent from the merozoite. This observation was further supported by the results of indirect immunofluorescence assay. These results could account for the findings that antibodies against P47 were inhibitory to the parasite growth in vitro but those against P50 were not. Finally, we demonstrated that the further processing of P47 is allelic type-dependent. The results of the present study would help in vaccine designing based on SERA.
Genomic DNA clones that coded for the bifunctional dihydrofolate reductase (DHFR) and thymidylate synthase (TS) (DHFR-TS) activities from a pyrimethamine-sensitive strain of Plasmodium falciparum ...were isolated and sequenced. The deduced DHFR-TS protein contained 608 amino acids (71,682 Da). The coding region for DHFR-TS contained no intervening sequences and had a high A+T content (75%). The DHFR domain, in the amino-terminal portion of the protein, was joined by a 94-amino acid junction sequence to the TS domain in the carboxyl-terminal portion of the protein. The TS domain was more conserved than the DHFR domain and both P. falciparum domains were more homologous to eukaryotic than to prokaryotic forms of the enzymes. Predicted secondary structures of the DHFR and TS domains were nearly identical to the structures identified in other DHFR and TS enzymes.
Carbamoyl phosphate synthetase II encodes the first enzymic step of de novo pyrimidine biosynthesis. Carbamoyl phosphate synthetase II is essential for
Toxoplasma gondii replication and virulence. In ...this study, we characterised the primary structure of a 28
kb gene encoding
Toxoplasma gondii carbamoyl phosphate synthetase II. The carbamoyl phosphate synthetase II gene was interrupted by 36 introns. The predicted protein encoded by the 37 carbamoyl phosphate synthetase II exons was a 1,687 amino acid polypeptide with an N-terminal glutamine amidotransferase domain fused with C-terminal carbamoyl phosphate synthetase domains. This bifunctional organisation of carbamoyl phosphate synthetase II is unique, so far, to protozoan parasites from the phylum Apicomplexa (
Plasmodium,
Babesia,
Toxoplasma) or zoomastigina (
Trypanosoma,
Leishmania). Apicomplexan parasites possessed the largest carbamoyl phosphate synthetase II enzymes due to insertions in the glutamine amidotransferase and carbamoyl phosphate synthetase domains that were not present in the corresponding gene segments from bacteria, plants, fungi and mammals. The C-terminal allosteric regulatory domain, the carbamoyl phosphate synthetase linker domain and the oligomerisation domain were also distinct from the corresponding domains in other species. The novel C-terminal regulatory domain may explain the lack of activation of
Toxoplasma gondii carbamoyl phosphate synthetase II by the allosteric effector 5-phosphoribosyl 1-pyrophosphate.
Toxoplasma gondii growth in vitro was markedly inhibited by the glutamine antagonist acivicin, an inhibitor of glutamine amidotransferase activity typically associated with carbamoyl phosphate synthetase II, guanosine monophosphate synthetase, or CTP synthetase.
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