Proteomic and Transcriptomic Analyses in the Slipper Snail Crepidula fornicata Uncover Shell Matrix Genes Expressed During Adult and Larval Biomineralization.

The gastropod shell is a composite composed of minerals and shell matrix proteins (SMPs). SMPs have been identified by proteomics in many molluscs, but few have been studied in detail. Open questions include (1) what gene regulatory networks regulate SMP expression, (2) what roles individual SMPs play in biomineralization, and (3) how the complement of SMPs changes over development. These questions are best addressed in a species in which gene perturbation studies are available; one such species is the slipper snail, Crepidula fornicata. Here, SEM and pXRD analysis demonstrated that the adult shell of C. fornicata exhibits crossed lamellar microstructure and is composed of aragonite. Using high-throughput proteomics we identified 185 SMPs occluded within the adult shell. Over half of the proteins in the shell proteome have known biomineralization domains, while at least 10% have no homologs in public databases. Differential gene expression analysis identified 20 SMP genes that are up-regulated in the shell-producing mantle tissue. Over half of these 20 SMPs are expressed during development with two, CfSMP1 and CfSMP2, expressed exclusively in the shell gland. Together, the description of the shell microstructure and a list of SMPs now sets the stage for studying the consequences of SMP gene knockdowns in molluscs.

Cell interactions in invertebrate development.

By utilizing both genetics and physical manipulations, studies on invertebrate embryos have provided substantial information on how cell interactions influence cell fate during development. The picture emerging from these studies suggest that while there may be varied and intricate patterns of cell interactions during the development of different embryos, the signaling molecules that convey positional information across cell membranes have been highly conserved.

Differences in clinical manifestations between childhood-onset lupus and adult-onset lupus: a meta-analysis.

OBJECTIVE: It is known that age at disease onset has an impact on the clinical course and outcome of systemic lupus erythematosus (SLE); however, the precise differences in the prevalence of SLE manifestations are debated. Our objective was to conduct a systematic literature review and meta-analysis of all studies that directly compare childhood-onset lupus with adult-onset lupus to determine which clinical manifestations vary with age at disease onset. METHODS: A comprehensive literature search of the MEDLINE/PubMed,EMBASE, CINAHL, and SCOPUS databases was conducted to identify relevant articles. Study quality was assessed using the STROBE checklist. Study sample characteristics and clinical manifestation event rates were extracted from each study. Pooled odds ratios (ORs) were calculated using the random effects method, and between-study heterogeneity was quantified using the I (2) statistic. RESULTS: Of the 484 studies identified by the search strategy, 16 were included in this review. The total number of patients was 5993 adults and 905 children with SLE. Study quality was on average 16/32, ranging from 8 to 29. Several statistically significant differences were found: malar rash, ulcers/mucocutaneous involvement, renal involvement, proteinuria, urinary cellular casts, seizures, thrombocytopenia, hemolytic anemia, fever, and lymphadenopathy were more common in childhood-onset SLE with ORs ranging from 1.3 to 3.7; however, Raynaud's, pleuritis, and sicca were more common in adult-onset SLE (twice as common). CONCLUSIONS: The results of this meta-analysis suggest that some clinical manifestations of lupus are different in childhood-onset SLE and adult-onset SLE.

Phorbol esters alter cell fate during development of sea urchin embryos.

Protein kinase C (PKC) has been implicated as important in controlling cell differentiation during embryonic development. We have examined the ability of 12-O-tetradecanoyl phorbol-13-acetate (TPA), an activator of PKC, to alter the differentiation of cells during sea urchin development. Addition of TPA to embryos for 10-15 min during early cleavage caused dramatic changes in their development during gastrulation. Using tissue-specific antibodies, we have shown that TPA causes the number of cells that differentiate as endoderm and mesoderm to increase relative to the number that differentiate as ectoderm. cDNA probes show that treatment with TPA causes an increase in accumulation of RNAs specific to endoderm and mesoderm with a concomitant decrease in RNAs specific to ectoderm. Treatment of isolated prospective ectodermal cells with TPA causes them to differentiate into endoderm and mesoderm. The critical period for TPA to alter development is during early to mid cleavage, and treatment of embryos with TPA after that time has little effect. These results indicate that PKC may play a key role in determining the fate of cells during sea urchin development.

Identification of potential CTL epitopes of tumor-associated antigen MAGE-1 for five common HLA-A alleles.

Identification of CTL epitopes for tumor-specific responses is important for the development of immunotherapies to treat cancer patients. We have developed a strategy to identify potential CTL epitopes based on screening of sequences of target proteins for presence of specific motifs recognized by the most common HLA-A alleles, and identification of high affinity binding peptides using in vitro quantitative assays. A systematic analysis using the sequence of the product of the tumor-associated MAGE-1 gene has been carried out. All possible peptides of nine and ten residues, containing binding motifs for HLA-A1, -A2.1, A-3.2, -A11 and -A24 were synthesized and tested for binding using a quantitative assay. Out of 237 possible peptide/MHC combinations, 47 cases demonstrated good binding affinity (Kd < or = 500 nM). Several peptides were identified as good MHC binders for each one of the five HLA-A alleles studied (five for HLA-A1, 11 for HLA-A2.1, 10 for HLA-A3.2, 16 for HLA-A11 and five for HLA-A24. Furthermore, eight of these peptides were found to bind well to more than one HLA-A allele. These results have important implications for the development of immunotherapeutic vaccines to treat malignant melanoma.

In vitro induction of primary, antigen-specific CTL from human peripheral blood mononuclear cells stimulated with synthetic peptides.

A protocol for in vitro induction of primary, antigen-specific CTL from human peripheral blood mononuclear cells (PBMCs) was developed. Antigen presenting cells (APCs) consisted of Staphylococcus aureus Cowan-I (SAC-I) activated PBMCs treated with a citrate-phosphate buffer at pH 3 to release endogenous peptides bound to surface MHC. This treatment resulted in transient expression of empty class I molecules which could be subsequently stabilized with peptide and beta 2-microglobulin (beta 2m). SAC-I activated PBMCs from HLA-A2.1 normal donors loaded with HBV core 18-27 peptide following acid treatment were used to stimulate PBMCs depleted of CD4+ T cells, in the presence of recombinant interleukin-7 (rIL-7). After 12 days, cells were restimulated with autologous, peptide-pulsed, adherent cells and tested for CTL activity 7 days later. In 23 independent experiments from 13 different HLA-A2.1 donors, this protocol resulted in induction of primary CTL more than 90% of the time. As indicated by both the frequency and magnitude of the response against peptide-sensitized target cells, SAC-I activated PBMCs treated with acid were the most efficient stimulator APC. Thirteen per cent of the cultures generated were capable of lysing target cells transfected with the HBV core antigen and, in general, these CTL cultures exhibited high avidity for the HBV core peptide. This protocol is generally applicable to different antigens and class I alleles, and thus, may be utilized to screen large numbers of peptides to identify human CTL epitopes.

Characterization of a gene encoding a developmentally regulated winged helix transcription factor of the sea urchin Strongylocentrotus purpuratus.

Spfkh1 is a Strongylocentrotus purpuratus transcription factor that contains a winged helix DNA binding domain. Both the gene and overlapping cDNAs encoding this factor have been cloned and completely sequenced. We have mapped the start of transcription by primer extension to a site 600 base pairs 5' to the start of translation. Spfkh1 is transcribed in one open reading frame that contains the DNA binding domain, nuclear localization signal and transactivation domain. The deduced amino acid sequence encodes a 40. 7kDa protein with a pI of 9.96. Alignments of the DNA binding domain with other forkhead domains reveal that this gene falls into Class II of the winged helix transcription factors. We have identified a unique carboxyl-terminal motif of unknown function that is present in all winged helix Class II transcription factors. A phylogenetic analysis of the DNA binding domains shows that, within the Class II, Spfkh1 groups with the deuterostomes as opposed to the protostomes. Analysis of the sequence 5' to the start of translation revealed binding sites for a large number of different transcription factors, many of which are present in multiple copies. The constellation of binding sites in the cis-regulatory region indicates that Spfkh1 is regulated by a complex set of factors, some of which are known to be endoderm specific. Included among these are binding sites for factors downstream of the Wnt/beta-catenin and hedgehog signaling pathways, implicating these pathways in both regulation of Spfkh1 and specification of endoderm.

The optimization of helper T lymphocyte (HTL) function in vaccine development.

Helper T lymphocyte (HTL) responses play an important role in the induction of both humoral and cellular immune responses. Therefore, HTL epitopes are likely to be a crucial component of prophylactic and immunotherapeutic vaccines. For this reason, Pan DR helper T cell epitopes (PADRE), engineered to bind most common HLA-DR molecules with high affinity and act as powerful immunogens, were developed. Short linear peptide constructs comprising PADRE and Plasmodium-derived B cell epitopes induced antibody responses comparable to more complex multiple antigen peptides (MAP) constructs in mice. These antibody responses were composed mostly of the IgG subclass, reactive against intact sporozoites, inhibitory of schizont formation in liver invasion assays, and protective against sporozoite challenge in vivo. The PADRE HTL epitope has also been shown to augment the potency of vaccines designed to stimulate a cellular immune response. Using a HBV transgenic murine model, it was found that CTL tolerance was broken by PADRE-CTL epitope lipopeptide, but not by a similar construct containing a conventional HTL epitope. There are a number of prophylactic vaccines that are of limited efficacy, require multiple boosts, and/or confer protection to only a fraction of the immunized population. Also, in the case of virally infected or cancerous cells, new immunotherapeutic vaccines that induce strong cellular immune responses are desirable. Therefore, optimization of HTL function by use of synthetic epitopes such as PADRE or pathogen-derived, broadly crossreactive epitopes holds promise for a new generation of highly efficacious vaccines.

Immunization with the HBV core 18-27 epitope elicits CTL responses in humans expressing different HLA-A2 supertype molecules.

The human leukocyte antigen (HLA)-A2 restricted HBV core 18-27 epitope is immunodominant in the context of HLA-A2.1 and subdominant in the context of the other HLA-A2 supertype molecules, as defined by frequency of recognition by memory cytotoxic T lymphocyte (CTL) responses from acute hepatitis B virus (HBV) patients, and on the basis of its binding affinity to purified HLA molecules in vitro. Herein, we show that immunization with a lipopeptide containing HBV core 18-27 epitope induces CTL responses in patients expressing different HLA-A2 supertype molecules, with indistinguishable frequency and magnitude. No difference in responses was noted between patients expressing either one or two different HLA-A2 supertype molecules. Thus, complexes of HBV core 18-27 bound to different HLA-A2 supertype alleles do not appear to act as altered peptide ligands, and do not cross antagonize CTL responses. These results substantiate the immunological relevance of the HLA supertypes concept, and illustrate its potential usefulness for the development of vaccines.

Spreading of HIV-specific CD8+ T-cell repertoire in long-term nonprogressors and its role in the control of viral load and disease activity.

Long-term non-progressors (LTNP) represent a minority of human immunodeficiency virus (HIV) infected individuals characterized by stable or even increasing CD4+ T-cell count and by stronger immune responses against HIV than progressors. In this study, HIV-specific effector CD8+ T cells, as detected by both a sensitive ex vivo enzyme-linked immunospot (ELISPOT) assay and specific major histocompatibility complex (MHC) peptide tetramers, were at a low frequency in the peripheral blood of LTNP, and recognized a lower number of HIV peptides than their memory resting cell counterparts. Both factors may account for the lack of complete HIV clearance by LTNP, who could control the viral spread, and displayed a higher magnitude of cytotoxic T lymphocyte (CTL) responses than progressors. By combining cell purification and ELISPOT assays this study demonstrates that both effector and memory resting cells were confined to a CD8+ population with memory CD45RO+ phenotype, with the former being CD28- and the latter CD28+. Longitudinal studies highlighted a relatively stable HIV-specific effector repertoire, viremia, and CD4+ T-cell counts, which were all correlated with maintenance of nonprogressor status. In conclusion, the analysis of HIV-specific cellular responses in these individuals may help define clear correlates of protective immunity in HIV infection.

Complications of total hip arthroplasty associated with the use of an acetabular component with a Hylamer liner.

We observed early failure and radiographic signs of accelerated wear as early as one to three years after insertion, without cement, of a metal-backed acetabular component with a liner made of Hylamer (DePuy). This finding prompted us to review a larger cohort of patients in whom that liner had been used. Two hundred and thirty-three components with a Hylamer liner, evaluated at a minimum of two years, had a mean rate of wear of 0.27 millimeter per year compared with 0.12 millimeter per year for a contemporaneous group of fifty acetabular cups with a conventional ultra-high molecular weight polyethylene liner made by another manufacturer. At a mean of 3.2 years, we found a significant difference (p < 0.000000006) between the mean rate of wear (0.20 millimeter per year) when the Hylamer liner articulated with a DePuy modular cobalt-chromium femoral head and the mean rate (0.29 millimeter per year) when the liner articulated with an Osteonics modular cobalt-chromium femoral head. Radiographic evaluation revealed a significant correlation between the total linear wear and the prevalence of osteolytic lesions (r2 = 0.76, linear regression analysis). We found that wear of 1.5 millimeters or more could be detected by the unaided eye. Because of the positive correlation between osteolysis and wear of 1.5 millimeters or more, we defined a hip with a liner that had that amount of wear as a hip at risk. We concluded that the wear characteristics of a Hylamer liner in vivo are inferior to those of a conventional ultra-high molecular weight polyethylene liner. Also, the rate of wear of the liner is greater when the femoral head is from a manufacturer other than DePuy. A patient who has a total hip replacement that includes a Hylamer liner should be monitored frequently for signs of wear and osteolytic changes. Additional investigations, with longer durations of follow-up and larger populations, are needed to understand fully the importance of our findings.

HLA-binding peptides as a therapeutic approach for chronic HIV infection.

Herein, we describe the Epimmune approach to prophylaxis and development of a multi-epitope vaccine for immunotherapy of HIV-1 infection. The central strategy of our program is to induce cellular immune responses, cytotoxic T-lymphocytes (CTL) and helper Tlymphocytes (HTL), specific for conserved epitopes from both structural and regulatory proteins of HIV-1. The HIV-1 derived and HLA-restricted CTL and HTL epitopes needed to design and construct the experimental vaccines are now known and allow for broad and non-ethnically biased coverage of the human population. The design optimization of an epitope-based DNA vaccine and evaluating methods for various DNA vaccine delivery technologies for possible use in clinical trials are addressed.

Shoulder range of motion in elite tennis players. Effect of age and years of tournament play.

We studied glenohumeral rotational range of motion in 39 members of the United States Tennis Association National Tennis Team and touring professional program. We took goniometric measurements of internal and external rotation of dominant and nondominant shoulders at the glenohumeral joint with the humerus at 90 degrees of abduction. We categorized the tennis players by age and by years of tournament play. Results were analyzed by total rotation, internal rotation, external rotation, and dominant-to-nondominant shoulder differences. In our results, dominant internal rotation of the shoulder declined and the difference between dominant and nondominant internal rotation increased with both age and years of tournament play. Men and women tennis players showed the same degree of deficits in range of motion. Significant analysis of variance statistics were calculated for dominant internal rotation with years of total play, dominant total rotation with years of total play, and nondominant total rotation with age. Moderate negative correlations were found between dominant internal rotation and years of total play and dominant total rotation and years of total play. These results indicate a loss of internal rotation that seems progressive with longer periods of play. This loss of internal rotation of the shoulder is an absolute loss of motion because total rotation also decreases. Early detection and a corrective training program should be considered because adaptations may result in deleterious biomechanics affecting both performance and risk of injury.

Closed-chain rehabilitation for upper and lower extremities.

Closed-chain exercise protocols are used extensively in rehabilitation of knee injuries and are increasingly used in rehabilitation of shoulder injuries. They are felt to be preferable to other exercise programs in that they simulate normal physiologic and biomechanical functions, create little shear stress across injured or healing joints, and reproduce proprioceptive stimuli. Because of these advantages, they may be used early in rehabilitation and have been integral parts of "accelerated" rehabilitation programs. The authors review the important components of a closed-chain rehabilitation program and provide examples of specific exercises that are used for rehabilitation of knee and shoulder injuries.

Shoulder rehabilitation: clinical application, evaluation, and rehabilitation protocols.

This chapter has discussed and illustrated a shoulder rehabilitation protocol based on restoration of the anatomy, biomechanics, physiology, and motor firing organization that is responsible for normal shoulder function. Many protocols exist in the literature, most of which appear to achieve good results. There are no outcomes data to suggest the most efficacious protocols. Adherence to the basic principles outlined in these chapters appears to allow the best framework for constructing a good program.