Liposomes as adjuvant for combination vaccines

In the present study tetanus toxoid (TT) loaded liposomes and diphtheria toxoid (DT) loaded liposomes were prepared by reverse phase evaporation method and after combining these two vaccines the potential advantages were investigated. Prepared systems were characterized for the size, shape and entrapment efficiency. SDS-PAGE analysis of TT and DT was also performed. The selected liposomal formulations were administered subcutaneously to Balb/c mice and their immune responses were determined using ELISA after 15, 30, 45 days. After boosting the maximum immune response was observed after 45 days and was found to be 0.831 and 0.749 for TT loaded liposome and DT loaded liposomes respectively. When the mice were immunized subcutaneously with the physical mixture of TT loaded liposomes and DT loaded liposomes the immune response for the combination vaccine was found to be 1.44 and 0.741 for the TT and DT respectively. The result showed that the immune response of TT increased when it was combined with DT in liposomes. This confirms adjuvantcity of DT vis-a-vis immunogenicity. Thus, carrier mediated cocktail vaccination holds promise for clinical applications.     

Role of a putative polysaccharide locus in Bordetella biofilm development

Bordetellae are gram-negative bacteria that colonize the respiratory tracts of animals and humans. We and others have recently shown that these bacteria are capable of living as sessile communities known as biofilms on a number of abiotic surfaces. During the biofilm mode of existence, bacteria produce one or more extracellular polymeric substances that function, in part, to hold the cells together and to a surface. There is little information on either the constituents of the biofilm matrix or the genetic basis of biofilm development by Bordetella spp. By utilizing immunoblot assays and by enzymatic hydrolysis using dispersin B (DspB), a glycosyl hydrolase that specifically cleaves the polysaccharide poly-beta-1,6-N-acetyl-D-glucosamine (poly-beta-1,6-GlcNAc), we provide evidence for the production of poly-beta-1,6-GlcNAc by various Bordetella species (Bordetella bronchiseptica, B. pertussis, and B. parapertussis) and its role in their biofilm development. We have investigated the role of a Bordetella locus, here designated bpsABCD, in biofilm formation. The bps (Bordetella polysaccharide) locus is homologous to several bacterial loci that are required for the production of poly-beta-1,6-GlcNAc and have been implicated in bacterial biofilm formation. By utilizing multiple microscopic techniques to analyze biofilm formation under both static and hydrodynamic conditions, we demonstrate that the bps locus, although not essential at the initial stages of biofilm formation, contributes to the stability and the maintenance of the complex architecture of Bordetella biofilms.     

ApoA-I mimetic peptides with differing ability to inhibit atherosclerosis also exhibit differences in their interactions with membrane bilayers

Two homologous apoA-I mimetic peptides, 3F-2 and 3F(14), differ in their in vitro antiatherogenic properties (Epand, R. M., Epand, R. F., Sayer, B. G., Datta, G., Chaddha, M., and Anantharamaiah, G. M. (2004) J. Biol. Chem. 279, 51404-51414). In the present work, we demonstrate that the peptide 3F-2, which has more potent anti-inflammatory activity in vitro when administered intraperitoneally to female apoE null mice (20 microg/mouse/day) for 6 weeks, inhibits atherosclerosis (lesion area 15,800 +/- 1000 microm(2), n = 29), whereas 3F(14) does not (lesion area 20,400 +/- 1000 microm(2), n = 26) compared with control saline administered (19,900 +/- 1400 microm(2), n = 22). Plasma distribution of the peptides differs in that 3F-2 preferentially associates with high density lipoprotein, whereas 3F(14) preferentially associates with apoB-containing particles. After intraperitoneal injection of (14)C-labeled peptides, 3F(14) reaches a higher maximal concentration and has a longer half-time of elimination than 3F-2. A study of the effect of these peptides on the motional and organizational properties of phospholipid bilayers, using several NMR methods, demonstrates that the two peptides insert to different extents into membranes. 3F-2 with aromatic residues at the center of the nonpolar face partitions closer to the phospholipid head group compared with 3F(14). In contrast, only 3F(14) affects the terminal methyl group of the acyl chain, decreasing the (2)H order parameter and at the same time also decreasing the molecular motion of this methyl group. This dual effect of 3F(14) can be explained in terms of the cross-sectional shape of the amphipathic helix. These results support the proposal that the molecular basis for the difference in the biological activities of the two peptides lies with their different interactions with membranes.     

Structure and putative function of dark- and light-adapted as well as UV-exposed eyes of the food store pest Psyllipsocus ramburi Sélys-longchamps (Insecta: Psocoptera: Psyllipsocidae)

The psocopteran Psyllipsocus ramburi Sélys-Longchamps can render food stuffs unpalatable and may serve as an intermediate host for cestodes. Its two circular compound eyes consist of about 26 facets, capped by strongly convexly curved corneae of 10-18 microm in diameter. Corneal nipples or interommatidial hairs are not developed. Beneath each corneal lens a cluster of four cone cells, enveloped by two primary pigment cells, separates an ommatidial group of eight retinula cells from the inner corneal surface. Membrane specializations of the retinula cells, known as the microvilli, measure 60 nm in diameter, and collectively make up the rhabdom, which is columnar in shape and has a distal diameter of 4 or 5 microm, depending on whether it is day- or night-adapted. Cone cell lengths measure 4.5 microm during the day and 8.5 microm at night and retinula cell screening pigments closely approach the edge of the rhabdom during the day. A 1-h exposure to UV-A (lambda(max)=351 nm) of ca. 1200 lx causes an almost total destruction of the photoreceptive membranes of the rhabdom and bleached all retinula cell screening pigments, but not the pigment grains of the primary pigment cells. Calculations, based on the anatomical data, suggest that the eyes are adapted to function under dim light levels, but cannot produce sharp images since their best possible acceptance angles are 22 degrees and 28 degrees in light- and dark-adapted states, respectively. Destruction of vision, likely affecting biorhythm and reproduction, by exposing the insects to UV-A may offer an alternative to the use of chemicals in controlling these insects.     

Isolation of potentially pathogenic Escherichia coli O157:H7 from the Ganges River

Escherichia coli serotype O157:H7 was detected among bacteria collected from the Ganges River. O157:H7 isolates tested positive for stx(1), stx(2), and eae gene sequences. Identification of potentially pathogenic isolates from extensively used source water indicates that O157:H7 may be a significant but as yet underacknowledged public health concern in India.     

KATP channel knockout worsens myocardial calcium stress load in vivo and impairs recovery in stunned heart

Gene knockout of the KCNJ11-encoded Kir6.2 ATP-sensitive K(+) (K(ATP)) channel implicates this stress-response element in the safeguard of cardiac homeostasis under imposed demand. K(ATP) channels are abundant in ventricular sarcolemma, where subunit expression appears to vary between the sexes. A limitation, however, in establishing the full significance of K(ATP) channels in the intact organism has been the inability to monitor in vivo the contribution of the channel to intracellular calcium handling and the superimposed effect of sex that ultimately defines heart function. Here, in vivo manganese-enhanced cardiac magnetic resonance imaging revealed, under dobutamine stress, a significantly greater accumulation of calcium in both male and female K(ATP) channel knockout (Kir6.2-KO) mice compared with sex- and age-matched wild-type (WT) counterparts, with greatest calcium load in Kir6.2-KO females. This translated, poststress, into a sustained contracture manifested by reduced end-diastolic volumes in K(ATP) channel-deficient mice. In response to ischemia-induced stunning, male and female Kir6.2-KO hearts demonstrated accelerated time to contracture and increased peak contracture compared with WT. The outcome on reperfusion, in both male and female Kir6.2-KO hearts, was a transient reduction in systolic performance, measured as rate-pressure product compared with WT, with protracted increase in left ventricular end-diastolic pressure, exaggerated in female knockout hearts, despite comparable leakage of creatine kinase across groups. Kir6.2-KO hearts were rescued from diastolic dysfunction by agents that target alternative pathways of calcium handling. Thus K(ATP) channel deficit confers a greater susceptibility to calcium overload in vivo, accentuated in female hearts, impairing contractile recovery under various conditions of high metabolic demand.     

TGF-beta-regulated collagen type I accumulation: role of Src-based signals

Transforming growth factor- (TGF-) stimulates myofibroblast transdifferentiation, leading to type I collagen accumulation and fibrosis. We investigated the function of Src in TGF--induced collagen I accumulation. In human mesangial cells, PTyr416 Src (activated Src) was 3.3-fold higher in TGF--treated cells than in controls. Src activation by TGF- was blocked by rottlerin and by a dominant negative mutant of protein kinase C (PKC), showing that TGF- activates Src by a PKC-based mechanism. Pharmacological inhibitors and a dominant negative Src mutant prevented the increase in collagen type I secretion in cells exposed to TGF-. Similarly, on-target Src small interference RNA (siRNA) prevented type I collagen secretion in response to TGF-, but off-target siRNA complexes had no effect. It is well established in mesangial cells that upregulation of type I collagen by TGF- requires extracellular signal-regulated kinase 1/2 (ERK1/2), and we found that activation of ERK1/2 by TGF- requires Src. In conclusion, these results suggest that stimulation of collagen type I secretion by TGF- requires a PKC-Src-ERK1/2 signaling motif. mesangial cells; fibrosis; glomerulus; transforming growth factor-