Rapid T cell receptor delineation reveals clonal expansion limitation of the magnitude of the HIV-1-specific CD8+ T cell response

TCRs mediate CTL specificity, but TCRs recognizing the same epitope often differ between persons due to their stochastic derivation. The role of this variability in the pathogenesis of virus infections and malignancies has been technically difficult to study. We apply an adaptation of TCR spectratyping to study HIV-specific CTLs, defining the clonal breadth and sequences of epitope-specific TCRs from PBMCs without cellular sorting or molecular cloning. Examining 48 CTL responses in 12 persons reveals a mean of 4.5 ± 2.7 clones per response, of both public and private clonotypes. The number of identified epitope-specific TCRs correlates with CTL frequency across epitopes, suggesting that clonal breadth limits the magnitude of the CTL response against HIV-1 in vivo. HLA A- and B-restricted CTLs are similar in their TCR breadth in this small cohort, preliminarily suggesting that qualitative differences may account for their disparate impacts on pathogenesis. Overall, these findings demonstrate that the magnitude of the CTL response in chronic HIV-1 infection is constrained by TCR clonal breadth, suggesting maximal expansion of CTLs in response to chronic antigenic stimulation.     

Role of cadherin-17 in oncogenesis and potential therapeutic implications in hepatocellular carcinoma

Cadherin is an important cell adhesion molecule that plays paramount roles in organ development and the maintenance of tissue integrity. Dysregulation of cadherin expression is often associated with disease pathology including tissue dysplasia, tumor formation, and metastasis. Cadherin-17 (CDH17), belonging to a subclass of 7D-cadherin superfamily, is present in fetal liver and gastrointestinal tract during embryogenesis, but the gene becomes silenced in healthy adult liver and stomach tissues. It functions as a peptide transporter and a cell adhesion molecule to maintain tissue integrity in epithelia. However, recent findings from our group and others have reported aberrant expression of CDH17 in major gastrointestinal malignancies including hepatocellular carcinoma (HCC), stomach and colorectal cancers, and its clinical association with tumor metastasis and advanced tumor stages. Furthermore, alternative splice isoforms and genetic polymorphisms of CDH17 gene have been identified in HCC and linked to an increased risk of HCC. CDH17 is an attractive target for HCC therapy. Targeting CDH17 in HCC can inhibit tumor growth and inactivate Wnt signaling pathway in concomitance with activation of tumor suppressor genes. Further investigation on CDH17-mediated oncogenic signaling and cognate molecular mechanisms would shed light on new targeting therapy on HCC and potentially other gastrointestinal malignancies.     

Senescent skeletal muscle fibroadipogenic progenitors recruit and promote M2 polarization of macrophages

Senescent cells compromise tissue structure and function in older organisms. We recently identified senescent fibroadipogenic progenitors (FAPs) with activated chemokine signaling pathways in the skeletal muscle of old mice, and hypothesized these cells may contribute to the age-associated accumulation of immune cells in skeletal muscle. In this study, through cell–cell communication analysis of skeletal muscle single-cell RNA-sequencing data, we identified unique interactions between senescent FAPs and macrophages, including those mediated by Ccl2 and Spp1. Using mouse primary FAPs in vitro, we verified increased expression of Ccl2 and Spp1 and secretion of their respective proteins in the context of both irradiation- and etoposide-induced senescence. Compared to non-senescent FAPs, the medium of senescent FAPs markedly increased the recruitment of macrophages in an in vitro migration assay, an effect that was mitigated by preincubation with antibodies to either CCL2 or osteopontin (encoded by Spp1). Further studies demonstrated that the secretome of senescent FAPs promotes polarization of macrophages toward an M2 subtype. These data suggest the unique secretome of senescent FAPs may compromise skeletal muscle homeostasis by recruiting and directing the behavior of macrophages.     

The Macrophage Galactose-Type Lectin Can Function as an Attachment and Entry Receptor for Influenza Virus

Specific protein receptors that mediate internalization and entry of influenza A virus (IAV) have not been identified for any cell type. Sialic acid (SIA), the primary attachment factor for IAV hemagglutinin, is expressed by numerous cell surface glycoproteins and glycolipids, confounding efforts to identify specific receptors involved in virus infection. Lec1 Chinese hamster ovary (CHO) epithelial cells express cell surface SIA and bind IAV yet are largely resistant to infection. Here, we demonstrate that expression of the murine macrophage galactose-type lectin 1 (MGL1) by Lec1 cells enhanced Ca²⁺-dependent IAV binding and restored permissivity to infection. Lec1 cells expressing MGL1 were infected in the presence or absence of cell surface SIA, indicating that MGL1 can act as a primary receptor or as a coreceptor with SIA. Lec1 cells expressing endocytosis-deficient MGL1 mediated Ca²⁺-dependent IAV binding but were less sensitive to IAV infection, indicating that direct internalization via MGL1 can result in cellular infection. Together, these studies identify MGL1 as a cell surface glycoprotein that can act as an authentic receptor for both attachment and infectious entry of IAV.     

Exome sequencing as a tool for Mendelian disease gene discovery

Exome sequencing - the targeted sequencing of the subset of the human genome that is protein coding - is a powerful and cost-effective new tool for dissecting the genetic basis of diseases and traits that have proved to be intractable to conventional gene-discovery strategies. Over the past 2 years, experimental and analytical approaches relating to exome sequencing have established a rich framework for discovering the genes underlying unsolved Mendelian disorders. Additionally, exome sequencing is being adapted to explore the extent to which rare alleles explain the heritability of complex diseases and health-related traits. These advances also set the stage for applying exome and whole-genome sequencing to facilitate clinical diagnosis and personalized disease-risk profiling.     

Isolation and characterization of a novel lectin from the wild mushroom Xerocomus spadiceus

A lectin was isolated from extracts of fruiting bodies of the mushroom Xerocomus spadiceus using a procedure that involved ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-cellulose, and gel filtration by fast protein liquid chromatography on Superdex 75. The lectin was unadsorbed on DEAE-cellulose and Affi-gel blue gel and adsorbed on CM-Sepharose. The lectin was capable of eliciting an approximately four-fold stimulation of mitogenic response in murine splenocytes. The hemagglutinating activity was stable up to 60 degrees C, halved at 70 degrees C, reduced to 25% at 75 degrees C and dwindled to an undetectable level at 80 degrees C. The activity remained unaltered in the presence of various divalent (Ca2+, Mg2+, Zn2+ and Mn2+) chlorides up to a salt concentration of 10 mM except in the case of ZnCl2. FeCl3 up to a concentration of 10 mM did not affect the hemagglutinating activity of the lectin. The hemagglutinating activity of the lectin was doubled in the presence of 5 mM AlCl3 or 10 mM ZnCl2 and quadrupled in the presence of 10 mM AlCl3. The activity was reduced in the presence of HCl and NaOH. Among the large number of carbohydrates tested, only inulin was able to inhibit the hemagglutinating activity of the lectin.     

Specific strength and voluntary muscle activation in young and elderly women and men.

The extents to which decreased muscle size or activation are responsible for the decrease in strength commonly observed with aging remain unclear. Our purpose was to compare muscle isometric strength [maximum voluntary contraction (MVC)], cross-sectional area (CSA), specific strength (MVC/CSA), and voluntary activation in the ankle dorsiflexor muscles of 24 young (32 +/- 1 yr) and 24 elderly (72 +/- 1 yr) healthy men and women of similar physical activity level. Three measures of voluntary muscle activation were used: the central activation ratio [MVC/(MVC + superimposed force)], the maximal rate of voluntary isometric force development, and foot tap speed. Men had higher MVC and CSA than did women. Young men had higher MVC compared with elderly men [262 +/- 19 (SE) vs. 197 +/- 22 N, respectively], whereas MVC was similar in young and elderly women (136 +/- 15 vs. 149 +/- 16 N, respectively). CSA was greater in young compared with elderly subjects. There was no age-related impairment of specific strength, central activation ratio, or the rate of voluntary force development. Foot tap speed was reduced in elderly (34 +/- 1 taps/10 s) compared with young subjects (47 +/- 1 taps/10 s). These results suggest that isometric specific strength and the ability to fully and rapidly activate the dorsiflexor muscles during a single isometric contraction were unimpaired by aging. However, there was an age-related deficit in the ability to perform rapid repetitive dynamic contractions.     

Comprehensive Evaluation of Peripheral Nerve Regeneration in the Acute Healing Phase Using Tissue Clearing and Optical Microscopy in a Rodent Model

Peripheral nerve injury (PNI), a common injury in both the civilian and military arenas, is usually associated with high healthcare costs and with patients enduring slow recovery times, diminished quality of life, and potential long-term disability. Patients with PNI typically undergo complex interventions but the factors that govern optimal response are not fully characterized. A fundamental understanding of the cellular and tissue-level events in the immediate postoperative period is essential for improving treatment and optimizing repair. Here, we demonstrate a comprehensive imaging approach to evaluate peripheral nerve axonal regeneration in a rodent PNI model using a tissue clearing method to improve depth penetration while preserving neural architecture. Sciatic nerve transaction and end-to-end repair were performed in both wild type and thy-1 GFP rats. The nerves were harvested at time points after repair before undergoing whole mount immunofluorescence staining and tissue clearing. By increasing the optic depth penetration, tissue clearing allowed the visualization and evaluation of Wallerian degeneration and nerve regrowth throughout entire sciatic nerves with subcellular resolution. The tissue clearing protocol did not affect immunofluorescence labeling and no observable decrease in the fluorescence signal was observed. Large-area, high-resolution tissue volumes could be quantified to provide structural and connectivity information not available from current gold-standard approaches for evaluating axonal regeneration following PNI. The results are suggestive of observed behavioral recovery in vivo after neurorrhaphy, providing a method of evaluating axonal regeneration following repair that can serve as an adjunct to current standard outcomes measurements. This study demonstrates that tissue clearing following whole mount immunofluorescence staining enables the complete visualization and quantitative evaluation of axons throughout nerves in a PNI model. The methods developed in this study could advance PNI research allowing both researchers and clinicians to further understand the individual events of axonal degeneration and regeneration on a multifaceted level.     

Histone H2A.F/Z mRNA is stored in the egg cytoplasm and basally regulated in the sea urchin embryo.

The sea urchin H2A.F/Z histone is a member of a subclass of highly conserved H2A variants. Sequence analysis confirms that H2A.F/Z mRNA is polyadenylated. In situ hybridization studies demonstrate that maternal H2A.F/Z message is stored in the egg cytoplasm and present at equal levels in all cells of the mesenchyme blastula-stage embryo, suggesting that H2A.F/Z is not coordinately regulated with DNA synthesis. When blastula-stage embryos were exposed to DNA synthesis inhibitors, no effect on the steady-state level of H2A.F/Z mRNA was observed, while the level of late class H2B mRNA decreased substantially. These results provide evidence that the basal mode of regulation of this unusual histone variant is conserved evolutionarily.