Distinct cytokine profiles define clinical immune response to falciparum malaria in regions of high or low disease transmission

The immune effector response to Plasmodium falciparum infection involves a finely-tuned interplay between different cell types and cytokines. However, the processes by which they mediate the development of clinical immunity, in areas of different endemicity, are poorly understood. We analyzed circulating levels of pro-inflammatory (TNF, IFN-γ, IL-12, IL-16) and anti-inflammatory (IL-4, IL-10, IL-13) cytokines in control and patient groups drawn from a P. falciparum-endemic and a non-endemic region of India. The endemic region control population exhibited a lower pro- to anti-inflammatory cytokine ratio, indicating a shift towards a high basal Th2 response. Levels of IL-10 contributed most towards the region-specific difference in basal cytokine response. IL-10 was also the strongest predictor of disease in the endemic region, while IL-12, along with IL-10 and IL-6, contributed most to disease outcome in the non-endemic region. A low, mean IFN-γ/IL-10 ratio was associated with disease severity in the endemic region (p < 0.0001). In contrast, a low mean IL-12/IL-10 ratio correlated with disease outcome in the non-endemic region (p < 0.0001). In the endemic region, IL-13 correlated negatively with IFN-γ in severe patients (Spearman's ρ: -0.49; p : 0.013), while in the non-endemic region, IL-13 correlated negatively with IL-6 in severe malaria patients (Spearman's ρ: -0.485; p : 0.001). In conclusion, levels of pro- and anti-inflammatory cytokines and the relative balance between the Th1 and Th2 response, illustrates how populations residing in areas of varying disease endemicity may respond to P. falciparum-induced immune challenge.     

Effects of mGST A4 transfection on 4-hydroxynonenal-mediated apoptosis and differentiation of K562 human erythroleukemia cells

Cellular levels of downstream products of membrane lipid oxidation appear to regulate differentiation in K562 human erythroleukemia cells. 4-Hydroxynonenal (4-HNE) is a diffusible and relatively stable product of peroxidation of arachidonic and linoleic acids, cellular levels of which are regulated through metabolism to glutathione (GSH) conjugate by glutathione S-transferases (GSTs). A group of immunologically related alpha-class mammalian GSTs expressed in mice (mGST A4-4), rat (rGST A4-4), human (hGST A5.8), and other species, as well as the more distantly related human hGST A4-4, preferentially utilize 4-HNE as a substrate and are suggested to be major determinants of intracellular levels of 4-HNE. Present studies were designed to examine the effects of 4-HNE on K562 cells and to study the effect of transfection of mGSTA4-4 in these cells. Exposure of K562 cells to 20 microM 4-HNE for 2 h resulted in a rapid erythroid differentiation of K562 cells, as well as apoptosis evidenced by characteristic DNA laddering. Stable transfection of cells with mGST A4-4 resulted in a fivefold increase in GST-specific activity toward 4-HNE compared with wild-type or vector-only transfected cells. The mGST A4-4-transfected cells were resistant to the cytotoxic, apoptotic, and differentiating effects of 4-HNE. The mGST A4 transfection also conferred resistance to direct oxidative stress (IC(50) of H(2)O(2) 22, 23, and 35 microM for wild-type, vector-transfected, and mGST A4-transfected cells, respectively). mGST A4-4-transfected cells also showed a higher rate of proliferation compared with wild-type or vector-transfected K562 cells (doubling time 22.1 +/- 0.7, 31 +/- 1.2, and 29 +/- 0.6 h, respectively). Cellular 4-HNE levels determined by mass spectrometry were lower in mGST A4-4-transfected cells compared to cells transfected with vector alone (5.9 pmol/5 x 10(7) cells and 62.9 pmol/5 x 10(7) cells, respectively). Our studies show that 4-HNE can induce erythroid differentiation in K562 cells and that overexpression of mGST A4 suppresses 4-HNE levels and inhibits erythroid differentiation and apoptosis.     

Resistance reversal action of ketoconazole against mefloquine resistance of Plasmodium yoelii nigeriensis

Ketoconazole at 200 mg/kg dose has been found to exert marginal antimalarial action against multidrug resistant (MDR) Plasmodium yoelii nigeriensis (P. yoelii nigeriensis) in Swiss mice with 25% protection (2/8 mice) while at lower Ketoconazole dose i.e., 75-100 mg/kg, 14.28% mice were protected. Mefloquine (MFQ) (at 8 and 16 mg/kg) exerted suppressive action against MDR P. yoelii nigeriensis resulting in 25 and 14.28% protection of mice respectively. Combined treatment with Ketoconazole and mefloquine resulted in protection of 5/6 mice (83.33%) at MFQ 4 mg/kg + Ketoconazole 100 mg/kg dose, 7/8 (87.5%) mice at MFQ 8 mg/kg + Ketoconazole 20 mg/kg dose and 5/7 (71.42%) mice at MFQ 16 mg/kg + Ketoconazole 25 mg/kg dose and 5/6 (83.33%) mice at MFQ 16 mg/kg + Ketoconazole 100 mg/kg dose. Ketoconazole has been found to enhance the protective effect of mefloquine against MFQ resistant P. yoelii nigeriensis resulting in 66-88% protection of the mice treated with the appropriate combinations. The combination also increased suppression of parasitaemia at different times. The Ketoconazole combination with MFQ significantly increased the mean survival time of the treated mice compared to individual drugs alone. The study shows that Ketoconazole when administered with MFQ exerts bio-enhancing action against mefloquine resistance of MDR P. yoelii nigeriensis.     

Genetic analysis of Indian mulberry varieties through molecular markers

India is one of the countries where sericulture is being practiced traditionally. Due to the higher economic return and the greater employment potential, attempts are being made to increase the productivity by developing high yielding mulberry varieties. At the present, Mysore local, Bomaypiasbari, Kanva-2, Bilidevalaya, Kajli, S1, BC(2)59, C776, RFS-175, S-36 and Victory-1 are being cultivated extensively in different parts of India for rearing the silkworm Bombyx mori L. Using 17 random amplified polymorphic DNA (RAPD) and 11 inter-simple sequence repeat (ISSR) primers the genetic relationships among these varieties were analyzed. The RAPD and ISSR primers revealed more than 75% polymorphism among the varieties. The genetic similarity estimated from RAPD markers varied from 0.645, between Kajli and Victory-1 to 0.887, between Kanva-2 and Bilidevalaya. Similarly, the genetic similarity estimated from the ISSR markers ranged from 0.600, between Kajli and Victory-1, to 0.873 between Kanva-2 and BC(2)59. The dendrogram constructed from these markers grouped the varieties into three major groups comprising the low yielding, medium yielding and high yielding. The low genetic similarity between the group of varieties originating from the eastern regions with that of the southern region encourages formation of extensive breeding programs between these groups as to transfer the high yield potential of the southern varieties to the low yielding but highly adaptive eastern varieties.     

Nitric oxide protects against chloroquine resistant Plasmodium yoelii nigeriensis parasites in vitro

Malaria is a life-threatening disease of global concern. The role of nitric oxide in the clearance of malarial parasites is still under debate. Several reports suggest a possible role for nitric oxide in the protection during initial stages of malarial infection. In the present study, we demonstrate that the nitric oxide in combination with low concentrations of chloroquine controls the parasitaemia in vitro. Activated peritoneal macrophages co-cultured with lipopolysaccharide+interferon-gamma or extracts from Tenospora cordifolia as an immunomodulator promoted nitric oxide production by macrophages. The high concentration of nitric oxide in combination with sub-optimal chloroquine suppressed the parasitaemia in the chloroquine resistant malarial infection. Further, the nitric oxide synthase inhibitor, N(G)-mono-methyl-l-arginine, downregulated nitric oxide production by peritoneal macrophages and the resulting levels of parasitaemia were higher, similar to those of untreated controls. These findings support the proposition that nitric oxide has a crucial role in the control of parasitaemia at the initial periods of blood stage malarial infection.     

Transfection of murine dendritic cell line (JAWS II) by a nonviral transfection reagent

Dendritic cells are the most potent antigen-presenting cells that initiate and modulate the host immune system. Based on their immunostimulatory activity, a variety of strategies have been developed to use dendritic cells as vaccines and immunotherapeutic agents against infection and cancer. Genetically modified dendritic cells are useful for immunotherapeutic purposes because of their sustained activity in vivo. However, transfection of dendritic cells with plasmid DNA has been very difficult. While the viral transfection is associated with nonspecific activation of dendritic cells, commonly used nonviral transfection reagents have a low efficiency of transfection. Here we describe an improved, simple, less time-consuming transfection protocol using the nonviral nonliposomal lipid polymer, TransIT-TKO transfection reagent, for transfecting murine dendritic cells (JAWS II) with the gene that encodes Coccidioides immitis antigen 2 (Ag2). The JAWS II cells were cotransfected with pHYG-enhanced green fluorescent protein (EGFP) and pVR1012-C. immitis Ag2 plasmid DNAs using TransIT-TKO reagent. We reproducibly obtained 30%-50% transfection efficiency. The transfected cells maintained their immature phenotype and were functionally active. In addition, the flexibility of this agent for expressing multiple antigens (GFP and C. immitis Ag2) offers an advantage of delivering multiple immunogens.     

Eight-residue Abeta peptides inhibit the aggregation and enzymatic activity of Abeta42

Insoluble Aβ1–42 is the main component of the amyloid plaque. We have previously demonstrated that exposure to low pH can confer the molten globule state on soluble Aβ1–42 in vitro [Biochem. J. 361 (2000) 547] and unfolding experiments with guadinine hydrochloride (GdnHCl) have now confirmed this observation. The molten globule state of the protein has many biological properties and understanding the mechanisms of its formation is an important step in devising a therapeutic strategy for Alzheimer's disease (AD). We therefore investigated the ability of a series of synthetic eight-residue peptides derived from Aβ1–42 to inhibit the acid-induced aggregation of Aβ1–42 and identified the potent peptides to be Aβ15–22, Aβ16–23 and Aβ17–24. A1-antichymotrypsin, a member of the serine proteinase inhibitor (serpin) family is another major component of the amyloid plaque. In the present study, we investigated the proteolytic activity of Aβ1–42 against casein at different pHs. Chemical modification of amino acid residues in Aβ1–42 indicated that serine and histidine residues, but not aspartic acid, are necessary for enzymatic activity, suggesting that it is a serine proteinase. Amino acid substitution studies indicate that glutamic acids at positions 11 and 22 participate indirectly in proteolysis and we surmise that amino acid residues 29–42 are required to stabilize the conformer. A study of metal ions suggested that Cu²⁺ affected the enzymatic activity, but Zn²⁺ and Fe²⁺ did not. Interestingly, Aβ14–21 and Aβ15–22 were the only peptides that inhibited the proteolytic activity of Aβ42. Therefore, Aβ15–22 may control both aggregation of Aβ1–42 at acidic pH and its proteolytic activity at neutral pH. Consequently, we suggest that it may be of use in the therapy of Alzheimer's disease.     

Association of polyadenylation cleavage factor I with U1 snRNP

Splicing and polyadenylation factors interact for the control of polyadenylation and the coupling of splicing and polyadenylation. We document an interaction between the U1 snRNP and mammalian polyadenylation cleavage factor I (CF Im), one of several polyadenylation factors needed for the cleavage of the pre-mRNA at the polyadenylation site. Sucrose density gradient centrifugation demonstrated that CF Im separated into two fractions, a light fraction which contained the known CF Im subunits (72, 68, 59, and 25 kD), and a heavy fraction, rich in snRNPs, which contained predominately the 68- and 25-kD CF Im subunits. Using specific antibodies we found that the heavy fraction contains U1 snRNP/CF Im coprecipitable complexes. These complexes were insensitive to RNase treatment, suggesting that the coprecipitation is not due to RNA tethering. In vitro binding experiments show that both the 68- and 25-kD subunits bind to and comigrate with U1 snRNP. In addition, the 25-kD CF Im subunit binds specifically to the 70K protein of U1 snRNP (U1 70K). This binding may account for the CF Im/U1 snRNP interaction. During these studies we found that mAb 2.73 (mAb 2.73), an established U1 70K antibody, efficiently precipitates the bulk of the CF Im from cellular extracts. Because mAb 2.73 has been used in a number of previous studies related to the U1 snRNP and the U1 70K protein, the precipitation of CF Im must be considered in evaluating past and future data based on the use of mAb 2.73.     

Membrane association of glutathione S-transferase mGSTA4-4, an enzyme that metabolizes lipid peroxidation products.

Lipid peroxidation products have signaling functions and at higher concentrations are toxic and may trigger cell death. The compounds are metabolized predominantly by glutathione S-transferases exemplified by mGSTA4-4, an enzyme highly efficient in glutathione conjugation of 4-hydroxyalkenals, and possessing glutathione peroxidase activity toward phospholipid hydroperoxides. mGSTA4-4 belongs to the predominant group of "canonical" glutathione S-transferases that are soluble and generally localized in the cytoplasm. The intracellular localization of mGSTA4-4 was examined in hepatocytes of normal mouse liver and in transfected HepG2 cells by fluorescence microscopy and digital deconvolution. mGSTA4-4 was found to be predominantly localized at or near the plasma membrane in transfected HepG2 cells, as well as in hepatocytes endogenously expressing the protein. In vitro, mGSTA4-4 associated with liposomes, and this interaction was potentiated when the liposomes contained negatively charged phospholipids. Mutating lysine 115 to glutamic acid resulted in a loss of the plasma membrane targeting of mGSTA4-4 as well as in a significant reduction of its binding to liposomes in vitro. These data suggest preferential targeting of mGSTA4-4 to the plasma membrane that may contain the major substrate(s) for this enzyme. Lysine 115 is critically important for the membrane association of mGSTA4-4, most likely by entering into an electrostatic interaction with negatively charged phospholipid headgroups.     

Light meromyosin paracrystal formation

Studies of paracrystal formation by column purified light meromyosin (LMM) prepared in a variety of ways led to the following conclusions: (a) different portions of the myosin rod may be coded for different stagger relationships. This was concluded from observations that paracrystals with different axial repeat periodicities could be obtained either with LMM framents of different lengths prepared with the same enzyme, or with LMM fragments of identical lengths but prepared with different enzymes. (b) Paracrystals with a 14-nm axial repeat periodicity are most likely formed by the aggregation of sheets with a 44-nm axial repeat within the sheets which are staggered by 14 nm. All of the axial repeat patterns expected from one sheet or aggregates of more than one sheet, on this basis, were observed in the same electron micrograph. (c) C-protein binding probably occurs preferentially to LMM molecules related in some specific way. This was concluded from the observation that the same axial repeat pattern was obtained in paracrystals formed from different LMM preparations in the presence of C-protein, regardless of differences in the axial repeat obtained in the absence of C-protein. (d) Nucleic acid is responsible for the 43-nm axial repeat patterns observed in paracrystals formed by the ethanol-resistant fraction of LMM. In the absence of nuclei acid, paracrystals with a 14nm axial repeat are obtained. (e) The 43-nm axial repeat pattern observed with the ethanol-resistant fraction of LMM is different for LMM preparations obtained by trypsin and papain digestions.