Unaccustomed, eccentrically biased exercise induces trauma to muscle and/or connective tissue. Tissue damage activates an acute inflammatory response. Inflammation requires the effective interaction of different physiological and biological systems. Much of this is coordinated by the de novo synthesis of families of protein molecules, cytokines. The purpose of the present paper was to determine changes in blood levels of various cytokines in response to exercise-induced muscle damage that was effected using high-intensity eccentric exercise. Six healthy, untrained, college-age male subjects were required to perform the eccentric phase of a bench press and a leg curl (4 sets, 12 repetitions/set) at an intensity equivalent to 100% of their previously determined one-repetition maximum. Samples of blood were drawn at the following times: before exercise and 1.5, 6, 12, 24, 48, 72, 96, 120, and 144 h after exercise. These samples were analyzed for interleukins (IL): IL-1beta, IL-6, and IL-10; tumor necrosis factor-alpha; colony stimulating factors (CSF): granulocyte-CSF, macrophage-CSF, and GM-CSF; for cell adhesion molecules (CAM): P- and E-selectin, and intercellular cell adhesion molecule (ICAM-1), and vascular cell adhesion molecule (VCAM-1). Results were analyzed using a repeated-measures analysis of variance (P = 0.05). Compared to baseline values, IL-1beta was reduced (P = 0.03) at 6, 24, and 96-144 h after exercise; IL-6 was elevated (P = 0.01) at 12, 24, and 72 h after exercise; IL-10 was elevated (P = 0.009) between 72 and 144 h after exercise; M-CSF was elevated (P = 0.005) at 12 and 48-144 h after exercise; and P-selectin was reduced (P = 0.01) between 24 and 144 h after exercise. It is concluded that when high-intensity eccentric exercise is compared to strenuous endurance exercise, post-exercise changes in cytokines do occur, but they are generally of a smaller magnitude, and occur at a later time period after the termination of exercise.

The polypyrimidine tract-binding protein (PTB) functions primarily as an IRES trans-acting factor in the propagation of hepatitis C virus and picornaviruses. PTB interacts with secondary structures within the 3-and 5-untranslated regions of these viral genomes to mediate efficient IRES-mediated viral translation. PTB has also been reported to bind to the untranslated region of the single-stranded RNA dengue virus (DENV), suggesting a similar function for PTB in flaviviruses. Indeed small interfering RNA-mediated PTB knockdown inhibited the production of infectious DENV, and this inhibition was specific to PTB knockdown and not due to a nonspecific anti-viral state. In fact, PTB depletion did not inhibit the production infectious yellow fever virus, another flavivirus. Nevertheless, whereas PTB knockdown led to a significant decrease in the accumulation of DENV viral RNAs, it did not impair translation. Moreover, PTB was shown to interact with the DENV nonstructural 4A protein, a known component of the viral replication complex, and with the DENV genome during infection. These data suggest that PTB interacts with the replication complex of DENV and is acting at the level of viral RNA replication. Dengue virus (DENV)3 is the etiologic agent of dengue fever, currently the most prevalent arthropod-borne viral disease of humans (1). Dengue fever can be caused by any of four closely related but antigenically distinct DENV serotypes (DENV-1, DENV-2, DENV-3, and DENV-4). DENV belongs to the Flaviviridae family, which comprises other medically important pathogens including the Japanese encephalitis, yellow fever (YFV), and hepatitis C (HCV) viruses (2).The Flaviviridae family of viruses has single-stranded positive polarity RNA genomes, which are mRNAs coding viral proteins required for replication. The viral RNA-dependent RNA polymerase, NS5, in conjunction with other viral nonstructural (NS) proteins and host cellular proteins, copies complementary negative strand RNAs from the genomic RNA template, which in turn serve as templates for the synthesis of new positive strand RNAs (2). Replication, transcription, and assembly of virions require cis-acting elements within the 5Ј-and 3Ј-UTRs of the viral genomic RNA (3-6). These elements interact with host cellular RNA-binding proteins including the polypyrimidine tract-binding protein (PTB). PTB, a heterogeneous nuclear ribonucleoprotein (7), is involved in multiple aspects of cellular mRNA metabolism, including the regulation of alternative splicing, RNA stability, and internal ribosomal entry (IRES)-mediated translation of viral and cellular mRNAs (8 -10).The role of PTB in the propagation of the positive singlestranded RNA viruses has largely been studied with HCV and picornaviruses, for which PTB primarily functions as an IRES trans-acting factor (ITAF), activating viral translation initiation (11,12). Its role in RNA replication of these viruses is however still debated (13-16). PTB has also been reported to bind to the UTR of the DENV-4 (17) and Japanese encephalitis ...

Correspondence between the T-cell epitope responses of vaccine immunogens and those of pathogen antigens is critical to vaccine efficacy. In the present study, we analyzed the spectrum of immune responses of mice to three different forms of the SARS coronavirus nucleocapsid (N): (1) exogenous recombinant protein (N-GST) with Freund's adjuvant; (2) DNA encoding unmodified N as an endogenous cytoplasmic protein (pN); and (3) DNA encoding N as a LAMP-1 chimera targeted to the lysosomal MHC II compartment (p-LAMP-N). Lysosomal trafficking of the LAMP/N chimera in transfected cells was documented by both confocal and immunoelectron microscopy. The responses of the immunized mice differed markedly. The strongest T-cell IFN-gamma and CTL responses were to the LAMP-N chimera followed by the pN immunogen. In contrast, N-GST elicited strong T cell IL-4 but minimal IFN-gamma responses and a much greater antibody response. Despite these differences, however, the immunodominant T-cell ELISpot responses to each of the three immunogens were elicited by the same N peptides, with the greatest responses being generated by a cluster of five overlapping peptides, N76-114, each of which contained nonameric H2d binding domains with high binding scores for both class I and, except for N76-93, class II alleles. These results demonstrate that processing and presentation of N, whether exogenously or endogenously derived, resulted in common immunodominant epitopes, supporting the usefulness of modified antigen delivery and trafficking forms and, in particular, LAMP chimeras as vaccine candidates. Nevertheless, the profiles of T-cell responses were distinctly different. The pronounced Th-2 and humoral response to N protein plus adjuvant are in contrast to the balanced IFN-gamma and IL-4 responses and strong memory CTL responses to the LAMP-N chimera.

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An accurate molecular diagnosis for viral pathogens is highly dependent on pre-analytical procedures. The efficiencies of two viral RNA extraction methods (liquid phase partition and silica-based adsorption chromatography) and the effects of handling and storage on the stability of RNA isolated from dengue virus (DENV) were studied. Viral RNA extracted from spiked sera or clinical samples characterized with DENV infection were quantified by TaqMan real-time PCR. The presence of high serum proteins severely affected the recovery of DENV RNA by the liquid phase partition, but not the silica-based method. The recovery with Trizol liquid phase partition method was significantly improved by a concomitant addition of a co-precipitant and the reduction of sera proteins, resulting in recoveries similar to that of the silica-based methods. Repeated freeze-thaw cycles did not affect the recovery of viral RNA. While intact DENV was found to be stable in serum for up to 2 hour at 25°C, recovery of viral RNA from sera stored in the lysis/ binding buffer was stable for up to 5 days. These data indicate that the choice of viral RNA extraction methods , the conditions for handling , and storing of clinical sera critically affect the quantification of viral nucleic acid from clinical samples. This will impact the accuracy and reproducibility of DENV diagnosis by PCR-based assays. Dengue virus (DENV) has emerged as an important vector-borne viral disease, 1 usually afflicting rural areas of endemic countries and posing tremendous health problems in these regions.2 Hence, to prevent and control the progression of dengue disease, the World Health Organization has recommended the augmentation of active and accurate laboratory-based surveillance for early reporting of dengue virus infections to the public health authorities. Pre-analytical variables, including the storage and transport of patient samples, the stability of viral RNA in the samples and methods of isolating viral RNA of high yield and quality, have major impacts on the development and performance of any successful molecular diagnostics. 4,5 Hence, the technical challenges associated with these pre-analytical issues must first be identified and optimized. Various commercially available viral RNA extraction methods are currently in use for clinical diagnostics of viral diseases. These extraction kits are generally based on two methods-liquid phase partition (eg, TRIzol LS) and silica-based nucleic acid adsorption chromatography (eg, High Pure Viral RNA and QIAamp Viral RNA kits). The recoveries of viral RNA by these two methods differ substantially with different viruses, 6 -12 thus making the choice of any particular method for the isolation of viral RNA uncertain. While there were several studies on the isolation and stability of other viruses, few reports have focused on the systematic evaluation of the stability and recovery of DENV RNA from sera. [13][14][15] This study examined the contributions of various pre-analytical variables to the sensitivity of DENV RNA d...