The plant-based immunomodulator curcumin as a potential candidate for the development of an adjunctive therapy for cerebral malaria

The clinical manifestations of cerebral malaria (CM) are well correlated with underlying major pathophysiological events occurring during an acute malaria infection, the most important of which, is the adherence of parasitized erythrocytes to endothelial cells ultimately leading to sequestration and obstruction of brain capillaries. The consequent reduction in blood flow, leads to cerebral hypoxia, localized inflammation and release of neurotoxic molecules and inflammatory cytokines by the endothelium. The pharmacological regulation of these immunopathological processes by immunomodulatory molecules may potentially benefit the management of this severe complication. Adjunctive therapy of CM patients with an appropriate immunomodulatory compound possessing even moderate anti-malarial activity with the capacity to down regulate excess production of proinflammatory cytokines and expression of adhesion molecules, could potentially reverse cytoadherence, improve survival and prevent neurological sequelae. Current major drug discovery programmes are mainly focused on novel parasite targets and mechanisms of action. However, the discovery of compounds targeting the host remains a largely unexplored but attractive area of drug discovery research for the treatment of CM. This review discusses the properties of the plant immune-modifier curcumin and its potential as an adjunctive therapy for the management of this complication.     

Withanone as an inhibitor of survivin: A potential drug candidate for cancer therapy

Survivin, the smallest inhibitor of apoptosis protein, which has been reported to be highly expressed in almost all known cancers, plays a dual role in survival as well as the proliferation of cancer cells. It inhibits apoptosis by inhibiting caspases as well as facilitating mitosis by becoming a part of chromosomal passenger complex through its BIR5 domain. Docking studies carried out with herbal ligand withanone derived from roots of Withania somnifera have shown strong binding affinity of −19.1088 kJ/mol with BIR5 domain of survivin and in turn interferes with inhibitory action against caspases and may lead to apoptosis. Binding of withanone at BIR5 domain of survivin may also interfere with chromosomal passenger complex and lead to halt the mitotic process within the cancer cell. Docking studies support various experimental outcomes and suggest withanone as a potential candidate molecule in cancer therapy.     

Trichuris suis: a secretory chymotrypsin/elastase inhibitor with potential as an immunomodulator

A serine protease inhibitor, termed TsCEI, was purified from adult-stage Trichuris suis by acid precipitation, affinity chromatography (elastase-agarose), and reverse-phase HPLC. The molecular weight of TsCEI was estimated at 6.437 kDa by laser desorption mass spectrometry. TsCEI potently inhibited both chymotrypsin (K(i) = 33.4 pM) and pancreatic elastase (K(i) = 8.32 nM). Neutrophil elastase, chymase (mouse mast cell protease-1, mMCP-1), and cathepsin G were also inhibited by TsCEI, whereas trypsin, thrombin, and factor Xa were not. The cDNA-derived amino acid sequence of the mature TsCEI consisted of 58 residues including 9 cysteine residues with a molecular mass of 6.196 kDa. TsCEI displayed 48% sequence identity to a previously characterized trypsin/chymotrypsin inhibitor of T. suis, TsTCI. TsCEI showed 36% sequence identity to a protease inhibitor from the hemolymph of the honeybee Apis mellifera. Sequence similarity was also detected with the trypsin/thrombin inhibitor of the European frog Bombina bombina, the elastase isoinhibitors of the nematode Anisakis simplex, and the chymotrypsin/elastase and trypsin inhibitors of the nematode Ascaris suum. The inhibitors of T. suis, an intestinal parasite of swine, may function as components of a parasite defense mechanism by modulating intestinal mucosal mast cell-associated, protease-mediated, host immune responses.     

Antigenicity studies in humans and immunogenicity studies in mice: an MSP1P subdomain as a candidate for malaria vaccine development

The newly identified GPI-anchored Plasmodium vivax merozoite surface protein 1 paralog (MSP1P) has a highly antigenic C-terminus that binds erythrocytes. To characterize the antigenicity and immunogenicity of two regions (PvMSP1P-19 and -33) of the highly conserved C-terminus of MSP1P relative to PvMSP1-19, 30 P. vivax malaria-infected patients and two groups of mice (immunized with PvMSP1P-19 or -33) were tested for IgG subclass antibodies against PvMSP1P-19 and -33 antigens. In the patients infected with P. vivax, IgG1 and IgG3 levels were significantly higher than those levels in healthy individuals, and were the predominant response to the two C-terminal fragments of PvMSP1P (p < 0.05). In mice immunized with PvMSP1P-19, IgG1 levels were the highest while IgG2b levels were similar to IgG1 levels. The levels of Th1 cytokines in mice immunized with PvMSP1P-19 or -33 were significantly higher than those in mice immunized with PvMSP1-19 (p < 0.05). Our results indicate that: (i) IgG1 and IgG3 (IgG2b in mice) are predominant IgG subclasses in both patients infected with P. vivax and mice immunized with PvMSP1P-19 or -33; (ii) the C-terminus of MSP1P induces a Th1-cytokine response. This immune profiling study provides evidence that MSP1P may be a potential candidate for vivax vaccine.     

Electrostatic potential as a tool to understand interactions between malaria vaccine candidate peptides and MHC II molecules

One of the most important problems in vaccine development consists in understanding receptor–ligand interactions between Class II Major Histocompatibility Complex molecules (MHC II) and antigenic peptides involved in inducing an appropriate immune response. In this study, we used X-ray crystallography structural data provided by the HLA-DRβ1*0301–CLIP peptide interaction to compare native non-immunogenic and specifically-modified immunogenic peptides derived from the malarial SALSA protein, by analyzing molecular electrostatic potential surfaces on the most important regions of the peptide binding groove (Pockets 1, 4, 6 and 9). Important differences were found on the electrostatic potential induced by these peptides, particularly in MHC II conserved residues: Qα9, Sα53, Nα62, Nα69, Yβ30, Yβ60, Wβ61, Qβ70, Kβ71 and Vβ86, the same ones involved in establishing hydrogen bonds between Class II molecule-peptide and the recognition by T cell receptor, it correlating well with the change in their immunological properties.The results clearly suggest that modifications done on the electrostatic potential of these amino acids could favor the induction of different immune responses and therefore, their identification could allow modifying peptides a priori and in silico, so as to render them into immunogenic and protection-inducers and hence suitable components of a chemically-synthesized, multi-antigenic, minimal subunit based vaccine.     

Pregnenolone as a potential candidate for hormone therapy for female reproductive disorders targeting ERβ

Many steroid hormones such as estrogen (E2) bind to their receptors for the regulation of biological processes. Pregnenolone (P5) is the precursor form of almost all steroid hormones and is often used to treat skin disorders and neurological complications. However, the mechanism and physiological function of P5 in reproductive organs are not well established. In this study, we investigated the effects of P5 on activation and expression of E2 receptor (ER) in the uteri and ovaries. To study the mechanism of P5 directly, Ishikawa cells were transfected with E2 response element (ERE)‐luciferase plasmid and isoforms of ER. ERE‐luciferase activity induced by P5 was similar to that induced by E2, and P5 showed high activity for ERβ without any relevance to P5‐metabolizing hormones such as progesterone (P4) and E2. In an animal study, immature female rats treated with P5 showed upregulation of ERα and downregulation of ERβ in the uteri, which is the main organ expressing ERα. In ERβ‐expressing organ ovaries, estrogen receptor 1, estrogen receptor 2, and P4 receptor were all downregulated by P5 and E2. Also, a decrease of ovarian cell proliferation and viability was observed in response to P5 relative to the control, suggesting that P5 may be a candidate for antiproliferative hormone of ovarian cancer. These findings suggest that P5 stimulates ERE promoter by ERβ‐mediated signaling in the uteri and ovaries. Activation of ERβ by P5 may help in understanding the mechanism of ER‐related female reproductive diseases such as endometriosis and ovarian cancer.     

Two decades of plant-based candidate vaccines: a review of the chimeric protein approaches

Genetic engineering revolutionized the concept of traditional vaccines since subunit vaccines became reality. Additionally, over the past two decades plant-derived antigens have been studied as potential vaccines with several advantages, including low cost and convenient administration. More specifically, genetic fusions allowed the expression of fusion proteins carrying two or more components with the aim to elicit immune responses against different targets, including antigens from distinct pathogens or strains. This review aims to provide an update in the field of the production of plant-based vaccine, focusing on those approaches based on the production of chimeric proteins comprising antigens from human pathogens, emphasizing the case of cholera toxin/E. coli enterotoxin fusions, chimeric viruses like particles approaches as well as the possible use of adjuvant-producing plants as expression hosts. Challenges for the near future in this field are also discussed.     

Use of a human immunodeficiency virus type 1 Rev mutant without nucleolar dysfunction as a candidate for potential AIDS therapy.

Applications of transdominant mutants of human immunodeficiency virus type 1 (HIV-1) regulatory proteins, especially Rev mutant, have been attempted for gene therapy against AIDS, because the Rev protein is essential for viral replication. We have previously reported that a mutant Rev protein (dRev) lacking its nucleolar targeting signal remained out of nuclei in expressed cells and strongly inhibited the function of Rev. To investigate the effects of dRev on HIV-1 replication, we established several dRev-expressing human cell lines with two different vector systems and examined virus production in these cells. An HIV-1-derived vector containing drev cDNA was constructed and introduced into CD4-positive HeLa cells and cells of the human T-cell line CCRF-CEM (CEM). In dRev-expressing HeLa cells, virus replication, syncytium formation, and cell death caused by HIV-1 infection were remarkably suppressed, and the same vector also conferred a resistant phenotype on CEM cells. The production was also suppressed in CEM cells containing the drev gene driven by a cytomegalovirus promoter. In addition, we found that dRev did not cause nucleolar dysfunction in a transient assay, in contrast to other transdominant mutants and wild-type Rev. Since dRev cannot migrate into the nuclei, it is expected not to interfere with nuclear/nucleolar functions of the host cell. We conclude that dRev is one promising candidate as an antiviral molecule for gene therapy against AIDS.     

Elamipretide as a potential candidate for relieving cryodamage to human spermatozoa during cryopreservation

As a technique widely used in assisted reproduction, human spermatozoa cryopreservation makes it possible to conserve functional sperm for a long time, but the impact of cryodamage on sperm during the process could not be ignored. The objective of the present study was to investigate the efficacy of Elamipretide, a novel small mitochondrial targeting short cytoprotective peptide, in attenuating cryodamage during spermatozoa cryopreservation. Semen samples were collected and cryopreserved in freeze solution containing different concentrations (0.0, 0.1, 1, and 10 μM) of Elamipretide. Sperm motility, viability, membrane integrity, mitochondrial membrane potential, DNA fragmentation, antioxidant profiles, and acrosome reaction were measured and analyzed. The results showed that supplementation of the freeze media with Elamipretide (1 and 10 μM) significantly improved post-thaw sperm parameters including motility and viability, stability of the plasma membrane, and mitochondria and chromosomes. In addition, by adding Elamipretide, excessive oxidation and acrosome dysfunction in sperm cells undergoing freeze-thaw were also significantly attenuated. Therefore, Elamipretide may be a potential candidate for relieving cryodamage to human spermatozoa during cryopreservation.     

Modulation of gut mucosal microbiota as a mechanism of probiotics-based adjunctive therapy for ulcerative colitis

This was a pilot study aiming to evaluate the effects of probiotics as adjunctive treatment for ulcerative colitis (UC). Twenty-five active patients with UC were assigned to the probiotic (n = 12) and placebo (n = 13) groups. The probiotic group received mesalazine (60 mg kg⁻¹ day⁻¹) and oral probiotics (containing Lactobacillus casei Zhang, Lactobacillus plantarum P-8 and Bifidobacterium animalis subsp. lactis V9) twice daily for 12 weeks, while the placebo group received the same amounts of mesalazine and placebo. The clinical outcomes were assessed. The gut mucosal microbiota was profiled by PacBio single-molecule, real-time (SMRT) sequencing of the full-length 16S rRNA of biopsy samples obtained by colonoscopy. A significantly greater magnitude of reduction was observed in the UC disease activity index (UCDAI) in the probiotic group compared with the placebo group (P = 0.043), accompanying by a higher remission rate (91.67% for probiotic-receivers versus 69.23% for placebo-receivers, P = 0.034). The probiotics could protect from diminishing of the microbiota diversity and richness. Moreover, the gut mucosal microbiota of the probiotic-receivers had significantly more beneficial bacteria like Eubacterium ramulus (P < 0.05), Pediococcus pentosaceus (P < 0.05), Bacteroides fragilis (P = 0.02) and Weissella cibaria (P = 0.04). Additionally, the relative abundances of the beneficial bacteria correlated significantly but negatively with the UCDAI score, suggesting that the probiotics might alleviate UC symptoms by modulating the gut mucosal microbiota. Our research has provided new insights into the mechanism of symptom alleviation in UC by applying probiotic-based adjunctive treatment.     

NEDD4-2 as a potential candidate susceptibility gene for epileptic photosensitivity

Photosensitive seizures occur most commonly in childhood and adolescence, usually as a manifestation of complex idiopathic generalized epilepsies (IGEs). Molecular mechanisms underlying this condition are yet to be determined because no susceptibility genes have been identified. The NEDD4-2 (Neuronally Expressed Developmentally Downregulated 4) gene encodes a ubiquitin protein ligase proposed to regulate cell surface levels of several ion channels, receptors and transporters involved in regulating neuronal excitability, including voltage-gated sodium channels (VGSCs), the most clinically relevant of the epilepsy genes. The regulation of NEDD4-2 in vivo involves complex interactions with accessory proteins in a cell type specific manner. We screened NEDD4-2 for mutations in a cohort of 253 families with IGEs. We identified three NEDD4-2 missense changes in highly conserved residues; S233L, E271A and H515P in families with photosensitive generalized epilepsy. The NEDD4-2 variants were as effective as wild-type NEDD4-2 in downregulating the VGSC subtype Na(v)1.2 when assessed in the Xenopus oocyte heterologous expression system showing that the direct interaction with the ion channel was not altered by these variants. These data raise the possibility that photosensitive epilepsy may arise from defective interaction of NEDD4-2 with as yet unidentified accessory or target proteins.     

Oral curcumin mitigates the clinical and neuropathologic phenotype of the Trembler-J mouse: a potential therapy for inherited neuropathy

Mutations in myelin genes cause inherited peripheral neuropathies that range in severity from adult-onset Charcot-Marie-Tooth disease type 1 to childhood-onset Dejerine-Sottas neuropathy and congenital hypomyelinating neuropathy. Many myelin gene mutants that cause severe disease, such as those in the myelin protein zero gene (MPZ) and the peripheral myelin protein 22 gene (PMP22), appear to make aberrant proteins that accumulate primarily within the endoplasmic reticulum (ER), resulting in Schwann cell death by apoptosis and, subsequently, peripheral neuropathy. We previously showed that curcumin supplementation could abrogate ER retention and aggregation-induced apoptosis associated with neuropathy-causing MPZ mutants. We now show reduced apoptosis after curcumin treatment of cells in tissue culture that express PMP22 mutants. Furthermore, we demonstrate that oral administration of curcumin partially mitigates the severe neuropathy phenotype of the Trembler-J mouse model in a dose-dependent manner. Administration of curcumin significantly decreases the percentage of apoptotic Schwann cells and results in increased number and size of myelinated axons in sciatic nerves, leading to improved motor performance. Our findings indicate that curcumin treatment is sufficient to relieve the toxic effect of mutant aggregation-induced apoptosis and improves the neuropathologic phenotype in an animal model of human neuropathy, suggesting a potential therapeutic role in selected forms of inherited peripheral neuropathies.     

Integrins as a potential target for targeted anticancer therapy

The review briefly summarizes information of structure of integrins and their involvement in the development and malignant progression of tumors. Special attention is paid to approaches based on modification of functional properties of integrins that prevent/antagonize tumor growth and progression; these approaches developed in modern experimental biology have certain perspective in clinical application.     

VU6005806/AZN-00016130, an advanced M4 positive allosteric modulator (PAM) profiled as a potential preclinical development candidate

This letter describes progress towards an M₄ PAM preclinical candidate that resulted in the discovery of VU6005806/AZN-00016130. While the thieno[2,3-c]pyridazine core has been a consistent feature of key M₄ PAMs, no work had previously been reported with respect to alternate functionality at the C3 position of the pyridazine ring. Here, we detail new chemistry and analogs that explored this region, and quickly led to VU6005806/AZN-00016130, which was profiled as a putative candidate. While, the β-amino carboxamide moiety engendered solubility limited absorption in higher species precluding advancement (or requiring extensive pharmaceutical sciences formulation), VU6005806/AZN-00016130 represents a new, high quality preclinical in vivo probe.     

Prevention of malaria using ITNs: potential for achieving the millennium development goals

The use of insecticide treated nets is effective in reducing all cause malaria mortality and morbidity between 17 and 43% in children under five years and provides protection to pregnant women who are most susceptible to malaria. ITNs (Insecticide Treated Nets) are easy to use and require less technical and capital outlay to implement compared with other vector control methods. They are cost-effective, which has led to widespread implementation of ITNs by countries on a large scale. ITN use has however been limited due to the cost outlay households require to make towards the purchase of nets, households' inability to associate the effectiveness of the net with the insecticide leading to low re-treatment rates in most settings and the seasonality associated with the spread of malaria. This chapter provides a review of research on ITN, strategies of improving the availability and effectiveness of the nets and a comparison of ITNs and other malaria preventive methods. The review highlights inequity in ITN use among various socio-economic groups with the poorest being the least to benefit from ITNs even where they are highly subsidized. It discusses the break through in the production of PermaNet to resolve the problem of low re-treatment of nets.     

Identification of an outer membrane protein of Salmonella enterica serovar Typhimurium as a potential vaccine candidate for Salmonellosis in mice

We report our investigation of the functions of PagN in Salmonella pathogenesis and its potential as a vaccine candidate. Further investigation conducted in this study indicates that the outer membrane protein PagN is important for Salmonella adhesion/invasion of epithelial cells as well as bacterial virulence. When pagN was deleted from Salmonella enterica serovar Typhimurium (S. Typhimurium), the adhesion and invasion of HT-29 epithelial cells was significantly decreased compared with the wild type strain. Mice infected with the pagN mutant strain exhibited less pathological signs in the intestine and survived longer than the wild-type-infected mice. PagN is widely distributed and conserved among clinical isolates of different Salmonella serovars, making PagN a potential vaccine candidate for Salmonella infection. To elucidate the potential of PagN as a vaccine, we expressed and purified recombinant PagN (rPagN). When rPagN was tested in mice, it provided significant protection against Salmonella infection in vivo. In vitro, anti-PagN serum enhanced clearance of Salmonella, indicating a contribution of PagN-specific antibodies to the killing process. This correlates well with the observed protection of mice immunized with rPagN. Our preliminary results indicate more functions of PagN in S. Typhimurium virulence as well as its potential as a protective vaccine.     

Rheumatoid arthritis: A study of relaxation and temperature biofeedback training as an adjunctive therapy

Rheumatoid arthritis (RA) is a painful systemic disease and is believed to be exacerbated by stress. Relaxation and biofeedback strategies have demonstrated utility in alleviating both pain and stress-related symptomatology, and therefore were tested for efficacy with this disease in a two-phase study. First, 24 patients were taught a relaxation technique and then trained in either temperature elevation or reduction. Second, a group of 15 patients thus trained was compared with 8 others who received traditional physiotherapy modalities. Psychological tests, functional/physical evaluations, as well as measurements related to pain, sleep, and other activities were carried out. Results of the first study revealed significant and positive changes following treatment that were primarily related to pain, tension, and sleep patterns for both groups, but no differential effects were noted between temperature elevation or reduction conditions. This was attributed to both groups having maintained temperature above baseline during biofeedback training. The results of the second study consistently favored the relaxation and biofeedback over the physiotherapy group on the physical/functional indices. The psychological measures tended to remain constant throughout both studies, leading to the conclusion that the effectiveness of treatment was specific to physical functioning rather than to a psychological enhancement of well-being.This work was conducted in partial fulfillment of the doctoral requirements at North Texas State University by Phillip McGraw. This work was also performed pursuant to Institutional Grant No. 5-S07-RR05426-12 and NIAMDD No. 5 P60 AM20628-02.