Immunodominant tuberculosis CD8 antigens preferentially restricted by HLA-B

CD8(+) T cells are essential for host defense to intracellular bacterial pathogens such as Mycobacterium tuberculosis (Mtb), Salmonella species, and Listeria monocytogenes, yet the repertoire and dominance pattern of human CD8 antigens for these pathogens remains poorly characterized. Tuberculosis (TB), the disease caused by Mtb infection, remains one of the leading causes of infectious morbidity and mortality worldwide and is the most frequent opportunistic infection in individuals with HIV/AIDS. Therefore, we undertook this study to define immunodominant CD8 Mtb antigens. First, using IFN-gamma ELISPOT and synthetic peptide arrays as a source of antigen, we measured ex vivo frequencies of CD8(+) T cells recognizing known immunodominant CD4(+) T cell antigens in persons with latent tuberculosis infection. In addition, limiting dilution was used to generate panels of Mtb-specific T cell clones. Using the peptide arrays, we identified the antigenic specificity of the majority of T cell clones, defining several new epitopes. In all cases, peptide representing the minimal epitope bound to the major histocompatibility complex (MHC)-restricting allele with high affinity, and in all but one case the restricting allele was an HLA-B allele. Furthermore, individuals from whom the T cell clone was isolated harbored high ex vivo frequency CD8(+) T cell responses specific for the epitope, and in individuals tested, the epitope represented the single immunodominant response within the CD8 antigen. We conclude that Mtb-specific CD8(+) T cells are found in high frequency in infected individuals and are restricted predominantly by HLA-B alleles, and that synthetic peptide arrays can be used to define epitope specificities without prior bias as to MHC binding affinity. These findings provide an improved understanding of immunodominance in humans and may contribute to a development of an effective TB vaccine and improved immunodiagnostics.     

Assessment of developmental toxicity of vorinostat, a histone deacetylase inhibitor, in Sprague-Dawley rats and Dutch Belted rabbits

BACKGROUND: The developmental toxicity potential of vorinostat (suberoylanilide hydroxamic acid [SAHA], ZOLINZA), a potent inhibitor of histone deacetylase (HDAC), was assessed in Sprague-Dawley rats and Dutch Belted rabbits. HDAC inhibitors have been shown to mediate the regulation of gene expression, induce cell growth, cell differentiation, and apoptosis of tumor cells. Range-finding studies established oral dose levels of 5, 15, or 50 mg/kg/day and 20, 50, or 150 mg/kg/day in rats and rabbits, respectively. METHODS: Animals were dosed on Gestation Days 6-20 or 7-20, respectively, with litter/fetal parameters evaluated on GD 21 and 28, respectively. Separate studies evaluated toxicokinetic parameters at the mid- and high-dose levels. RESULTS: There was no maternal toxicity observed at the highest dose levels; however, hematology and serum biochemistry changes were characterized in the range-finding studies. Vorinostat did not induce morphological malformations in either rat or rabbit fetuses. In rats, drug-related developmental toxicity was observed only in the high-dose group and consisted of markedly decreased fetal weight and increases in fetuses with a limited number of skeletal variations. In rabbits, drug-related developmental toxicity was also observed only in the high-dose group and consisted of slightly decreased fetal weight and increases in fetuses with a short 13th rib and incomplete ossification of metacarpals. Maternal exposures to vorinostat based on AUC and Cmax values were comparable at the high-dose levels of both species. Rabbits tolerated higher dosages probably due to more extensive metabolism. Maternal concentrations of vorinostat were approximately 1,000-fold above the known in vitro HDAC inhibitory concentration. CONCLUSIONS: Review of previous work with valproic acid, another HDAC inhibitor, suggest that the developmental toxicity profiles of these 2 compounds are not the result of HDAC inhibition but involve other mechanisms.     

Computational models evaluating the impact of sieve plates and radial water exchange on phloem pressure gradients

The sugar conducting phloem in angiosperms is a high resistance pathway made up of sieve elements bounded by sieve plates. The high resistance generated by sieve plates may be a trade‐off for promoting quick sealing in the event of injury. However, previous modeling efforts have demonstrated a wide variation in the contribution of sieve plates towards total sieve tube resistance. In the current study, we generated high resolution scanning electron microscope images of sieve plates from balsam poplar and integrated them into a mathematical model using Comsol Multiphysics software. We found that sieve plates contribute upwards of 85% towards total sieve tube resistance. Utilizing the Navier–Stokes equations, we found that oblong pores may create over 50% more resistance in comparison with round pores of the same area. Although radial water flows in phloem sieve tubes have been previously considered, their impact on alleviating pressure gradients has not been fully studied. Our novel simulations find that radial water flow can reduce pressure requirements by half in comparison with modeled sieve tubes with no radial permeability. We discuss the implication that sieve tubes may alleviate pressure requirements to overcome high resistances by regulating their membrane permeability along the entire transport pathway.     

Epidermal stem cells: interactions in developmental environments

Homeostasis of continuously renewing adult tissues, such as the epidermis of the skin, is maintained by epidermal stem cells (EpiSC), which are a small population of undifferentiated, self-renewing basal keratinocyte cells that produce daughter transit amplifying (TA) cells to make up the majority of the proliferative basal cell population in the epidermis. We have isolated EpiSC from neonatal and adult skin, and shown that these cells can regenerate an epidermis that lasts long term in vitro and in vivo, and that permanently expresses a recombinant gene in the regenerated tissue (Bickenbach and Dunnwald, 2000; Dunnwald et al., 2001). When we injected murine EpiSC into the developing blastocyst environment of the mouse, we found that both neonatal and adult EpiSC retained some ability to participate in the formation of tissues from all three germ layers (Liang and Bickenbach, 2002; Bickenbach and Chinnathambi, 2004; Liang et al., 2004). Although it appears evident that EpiSC act as pluripotent stem cells, how this reprogramming takes place is not understood. EpiSC might directly transdifferentiate into other cell types or they might first dedifferentiate into a more primitive cell type, and then proceed to develop along a cell lineage pathway. To begin to unravel this, we co-cultured EpiSC with embryonic stem (ES) cells, and found that EpiSC could alter their cell lineage protein expression to that of a more primitive cell type. We also placed EpiSC in a wounded environment and found that EpiSC interacted with the mesenchymal cells repopulating the wound bed. Our findings indicate that the population of cells that we isolate as EpiSC has a pluripotent capability. This has led us to postulate a paradigm shift for somatic stem cells. We propose that tissues maintain a sequestered population of uncommitted stem cells that retain a regenerative response which is enhanced when the cells are exposed to developmental or stress influences.     

Dopamine modulates the plasticity of mechanosensory responses in Caenorhabditis elegans

Dopamine-modulated behaviors, including information processing and reward, are subject to behavioral plasticity. Disruption of these behaviors is thought to support drug addictions and psychoses. The plasticity of dopamine-mediated behaviors, for example, habituation and sensitization, are not well understood at the molecular level. We show that in the nematode Caenorhabditis elegans, a D1-like dopamine receptor gene (dop-1) modulates the plasticity of mechanosensory behaviors in which dopamine had not been implicated previously. A mutant of dop-1 displayed faster habituation to nonlocalized mechanical stimulation. This phenotype was rescued by the introduction of a wild-type copy of the gene. The dop-1 gene is expressed in mechanosensory neurons, particularly the ALM and PLM neurons. Selective expression of the dop-1 gene in mechanosensory neurons using the mec-7 promoter rescues the mechanosensory deficit in dop-1 mutant animals. The tyrosine hydroxylase-deficient C. elegans mutant (cat-2) also displays these specific behavioral deficits. These observations provide genetic evidence that dopamine signaling modulates behavioral plasticity in C. elegans.     

Cathepsin L and Arg/Lys aminopeptidase: a distinct prohormone processing pathway for the biosynthesis of peptide neurotransmitters and hormones

Peptide neurotransmitters and hormones are synthesized as protein precursors that require proteolytic processing to generate smaller, biologically active peptides that are secreted to mediate neurotransmission and hormone actions. Neuropeptides within their precursors are typically flanked by pairs of basic residues, as well as by monobasic residues. In this review, evidence for secretory vesicle cathepsin L and Arg/Lys aminopeptidase as a distinct proteolytic pathway for processing the prohormone proenkephalin is presented. Cleavage of prohormone processing sites by secretory vesicle cathepsin L occurs at the NH2-terminal side of dibasic residues, as well as between the dibasic residues, resulting in peptide intermediates with Arg or Lys extensions at their NH2-termini. A subsequent Arg/Lys aminopeptidase step is then required to remove NH2-terminal basic residues to generate the final enkephalin neuropeptide. The cathepsin L and Arg/Lys aminopeptidase prohormone processing pathway is distinct from the proteolytic pathway mediated by the subtilisin-like prohormone convertases 1/3 and 2 (PC1/3 and PC2) with carboxypeptidase E/H. Differences in specific cleavage sites at paired basic residue sites distinguish these two pathways. These two proteolytic pathways demonstrate the increasing complexity of regulatory mechanisms for the production of peptide neurotransmitters and hormones.     

Observation of sequence specificity in the seeding of protein amyloid fibrils

It is well established that the rate of formation of fibrils by amyloidogenic proteins is enhanced by the addition of preformed fibrils, a phenomenon known as seeding. We show that the efficiency of seeding fibril formation from solutions of hen lysozyme by a series of other proteins depends strongly on the similarity of their sequences. This observation is consistent with the importance of long-range interactions in stabilizing the core structure of amyloid fibrils and may be associated with the existence of a species barrier observed in the transmissible spongiform encephalopathies. In addition, it is consistent with the observation of a single dominant type of protein in the deposits associated with each form of amyloid disease.     

Transient neutrophil infiltration after allergen challenge is dependent on specific antibodies and Fc gamma III receptors

Following allergen challenge of sensitized mice, neutrophils are the first inflammatory cells found in bronchoalveolar lavage (BAL) fluid. To determine the underlying mechanism for their accumulation, mice were sensitized to OVA on days 0 and 14, and received, on day 28, a single intranasal challenge (s.i.n.) with either OVA or ragweed. Eight hours after the s.i.n., BAL fluid was obtained. BALB/c mice sensitized and challenged with OVA showed significantly higher total cell counts and numbers of neutrophils in BAL fluid compared to the OVA-sensitized and ragweed-challenged or nonsensitized mice. Levels of neutrophil chemokines in BAL fluid supernatants were markedly elevated in the sensitized and OVA-challenged mice; Fc epsilon RI-deficient mice showed comparable numbers of neutrophils and neutrophil chemokines in BAL fluid after s.i.n. But in sensitized mice lacking the Fc common gamma-chain and B cell-deficient mice, the number of neutrophils and levels of neutrophil chemokines in BAL fluid were significantly lower. Further, mice lacking the FcgammaRIII did not develop this early neutrophil influx. Neutrophil infiltration could be induced in naive mice following intranasal instillation of allergen combined with allergen-specific IgG1. In addition, macrophages from sensitized mice were stimulated with allergen and activated to produce neutrophil chemokines. These results demonstrate that neutrophil influx after allergen challenge requires prior sensitization, is allergen-specific, is mediated through FcgammaRIII, and is dependent on the presence of Ab.