Diagnostic accuracy of upper cervical spine instability tests: a systematic review.

BACKGROUND: Patients with neck pain, headache, torticollis, or neurological signs should be screened carefully for upper cervical spine instability, as these conditions are "red flags" for applying physical therapy interventions. However, little is known about the diagnostic accuracy of upper cervical spine instability tests. PURPOSE: The purpose of this study was to evaluate the diagnostic accuracy of upper cervical spine instability screening tests in patients or people who are healthy. DATA SOURCES: PubMed, CINAHL, EMBASE, and RECAL Legacy databases were searched from their inception through October 2012. STUDY SELECTION: Studies were included that assessed the diagnostic accuracy of upper cervical instability screening tests in patients or people who are healthy and in which sensitivity and specificity were reported or could be calculated using a 2 × 2 table. DATA EXTRACTION AND QUALITY ASSESSMENT: Two reviewers independently performed data extraction and the methodological quality assessment using the QUADAS-2. DATA SYNTHESIS: Depending on heterogeneity, statistical pooling was performed. All diagnostic parameters (sensitivity, specificity, predictive values, and likelihood ratios) were recalculated, if possible. RESULTS: Five studies were included in this systematic review. Statistical pooling was not possible due to clinical and statistical heterogeneity. Specificity of 7 tests was sufficient, but sensitivity varied. Predictive values were variable. Likelihood ratios also were variable, and, in most cases, the confidence intervals were large. LIMITATIONS: The included studies suffered from several biases. None of the studies evaluated upper cervical spine instability tests in patients receiving primary care. CONCLUSIONS: The membranes tests had the best diagnostic accuracy, but their applicability as a test for diagnosing upper cervical spine instability in primary care has yet to be confirmed.

Chromodomain proteins in development: lessons from CHARGE syndrome.

In humans, heterozygous mutations in the adenosine triphosphate-dependent chromatin remodeling gene CHD7 cause CHARGE syndrome, a common cause of deaf-blindness, balance disorders, congenital heart malformations, and olfactory dysfunction with an estimated incidence of approximately 1 in 10,000 newborns. The clinical features of CHARGE in humans and mice are highly variable and incompletely penetrant, and most mutations appear to result in haploinsufficiency of functional CHD7 protein. Mice with heterozygous loss of function mutations in Chd7 are a good model for CHARGE syndrome, and analyses of mouse mutant phenotypes have begun to clarify a role for CHD7 during development and into adulthood. Chd7 heterozygous mutant mice have postnatal delayed growth, inner ear malformations, anosmia/hyposmia, and craniofacial defects, and Chd7 homozygous mutants are embryonic lethal. A central question in developmental biology is how chromodomain proteins like CHD7 regulate important developmental processes, and whether they directly activate or repress downstream gene transcription or act more globally to alter chromatin structure and/or function. CHD7 is expressed in a wide variety of tissues during development, suggesting that it has tissue-specific and developmental stage-specific roles. Here, we review recent and ongoing analyses of CHD7 function in mouse models and cell-based systems. These studies explore tissue-specific effects of CHD7 deficiency, known CHD7 interacting proteins, and downstream target sites for CHD7 binding. CHD7 is emerging as a critical regulator of important developmental processes in organs affected by human CHARGE syndrome.

The motor and tail regions of myosin XV are critical for normal structure and function of auditory and vestibular hair cells.

Recessive mutations in myosin 15, a class XV unconventional myosin, cause profound congenital deafness in humans and both deafness and vestibular dysfunction in mice homozygous for the shaker 2 and shaker 2(J) alleles. The shaker 2 allele is a previously described missense mutation of a highly conserved residue in the motor domain of myosin XV. The shaker 2(J) lesion, in contrast, is a 14.7 kb deletion that removes the last six exons from the 3"-terminus of the Myo15 transcript. These exons encode a FERM (F, ezrin, radixin and moesin) domain that may interact with integral membrane proteins. Despite the deletion of six exons, Myo15 mRNA transcripts and protein are present in the post-natal day 1 shaker 2(J) inner ear, which suggests that the FERM domain is critical for the development of normal hearing and balance. Myo15 transcripts are first detectable at embryonic day 13.5 in wild-type mice. Myo15 transcripts in the mouse inner ear are restricted to the sensory epithelium of the developing cristae ampularis, macula utriculi and macula sacculi of the vestibular system as well as to the developing organ of Corti. Both the shaker 2 and shaker 2(J) alleles result in abnormally short hair cell stereocilia in the cochlear and vestibular systems. This suggests that Myo15 may be important for both the structure and function of these sensory epithelia.

Autologous human monocyte-derived dendritic cells genetically modified to express melanoma antigens elicit primary cytotoxic T cell responses in vitro: enhancement by cotransfection of genes encoding the Th1-biasing cytokines IL-12 and IFN-alpha.

DNA-based immunization strategies designed to elicit cellular antitumor immunity offer an attractive alternative to protein- or peptide-based approaches. In the present study we have evaluated the feasibility of DNA vaccination for the induction of CTL reactivity to five different melanoma Ags in vitro. Cultured, monocyte-derived dendritic cells (DC) were transiently transfected with plasmid DNA encoding human MART-1/Melan-A, pMel-17/gp100, tyrosinase, MAGE-1, or MAGE-3 by particle bombardment and used to stimulate autologous PBMC responder T cells. CTL reactivity to these previously identified melanoma Ags was reproducibly generated after two or three stimulations with genetically modified DC. Co-ordinate transfection of two melanoma Ag cDNAs into DC promoted CTL responders capable of recognizing epitopes from both gene products. Coinsertion of genes encoding the Th1-biasing cytokines IL-12 or IFN-alpha consistently enhanced the magnitude of the resulting Ag-specific CTL reactivity. Importantly, DC transfected with a single melanoma Ag cDNA were capable of stimulating Ag-specific CTL reactivity restricted by multiple host MHC alleles, some of which had not been previously identified. These results support the inherent strengths of gene-based vaccine approaches that do not require prior knowledge of responder MHC haplotypes or of relevant MHC-restricted peptide epitopes. Given previous observations of in situ tumor HLA allele-loss variants, DC gene vaccine strategies may elicit a greater diversity of host therapeutic immunity, thereby enhancing the clinical utility and success of such approaches.

Autologous human dendriphages pulsed with synthetic or natural tumor peptides elicit tumor-specific CTLs in vitro.

The recent identification of tumor-associated antigens and tumor-associated antigen-derived peptide epitopes recognized by cytolytic T lymphocytes (CTLs) in the context of major histocompatibility complex (MHC) class I molecules has prompted the development of peptide-based vaccines for the treatment of human cancers, particularly melanoma. The design of such clinical protocols requires an understanding of the inherent immunogenicity of the peptide(s) and a choice of a facilitating adjuvant promoting cellular immunity against these peptides. We have evaluated the abilities of a series of defined synthetic peptide epitopes derived from MART-1/Melan-A, gp100, tyrosinase, and MAGE-3 or unfractionated peptides naturally presented by melanoma MHC molecules to elicit HLA-A2-restricted and melanoma-reactive CTLs from the peripheral blood of normal donors or patients with metastatic melanoma. Autologous peripheral blood dendritic cells (DCs), which were easily generated from all donors when cultured in the presence of recombinant human interleukin-4 and recombinant human granulocyte-macrophage colony-stimulating factor were pulsed with melanoma peptides and used to "prime" and/or "boost" CTL cultures in vitro. Our results suggest that antimelanoma CTLs may be reproducibly generated in short-term in vitro cultures in this manner using either a subset of the defined synthetic peptides (MART-1/Melan-A27-35, MART-1/Melan-A32-40, gp100(280-288), tyrosinase368-376, and MAGE-3(271-279)) or unfractionated peptides (containing both idiotypic and shared melanoma epitopes) derived from freshly isolated autologous melanoma lesions. These in vitro data support the use of autologous DCs prepulsed with such peptides as an appropriate antigen adjuvant delivery system in melanoma peptide-based vaccines.

Host immune response in renal cell cancer: interleukin-4 (IL-4) and IL-10 mRNA are frequently detected in freshly collected tumor-infiltrating lymphocytes.

Human renal cell cancer (RCC) is clearly responsive to immunotherapy. Clinical responses may be mediated by "non-specific" (e.g. natural killer, NK, cells) or "specific" MHC-class-I-restricted tumor-specific CD8+ T lymphocytes. Typically RCC progresses, however, despite significant infiltration of various lymphoid cells. We examined freshly isolated RCC tumor-infiltrating lymphocytes (TIL) derived from seven RCC patients for cytokine expression by the polymerase chain reaction (PCR). Established RCC tumor cell lines derived from these RCC patients were negative for interleukin-2 (IL-2), IL-4, IL-10, and interferon gamma and found to be positive for tumor necrosis factor alpha (TNF alpha), IL-6, IL-1 beta, granulocyte/macrophage-colony-stimulating factor (GM-CSF), and transforming growth factor beta 1 (TGF beta 1) message as detected by PCR. An identical pattern of cytokine mRNA expression was identified in other long-term RCC lines and in normal human kidney cells upon culture, but not in two Wilms tumor cell lines tested. Short-term-, and long-term-established RCC lines, but not Wilms tumor lines, secreted substantial levels of GM-CSF, TNF alpha, IL-1 beta, and IL-6 as detected by enzyme-linked immunosorbent assay. Both RCC lines and Wilms tumor lines secreted TGF beta 1. In comparison, normal kidney cells secreted IL-6 and GM-CSF, but not IL-1 beta, or TFG beta 1 under identical in vitro cell culture conditions. We applied PCR-based methods to characterize the cytokine mRNA expression pattern in immune cells infiltrating into renal cell cancer without the need for expansion of such effector cells in vitro. Examining freshly collected RCC TIL by PCR from patients with primary cell cell cancer, we could demonstrate that such cells, but not lympho-mononuclear cells harvested from normal human kidney tissue, typically exhibit IL-4 and IL-10 mRNA expression.

Endoftalmitis por acremonium kiliense / Fungal endophthalmitis for acremonium kiliense

Se presenta un caso de endoftalmitis micótica del ojo derecho en un paciente de 26 años, que había sufrido una úlcera traumática la cual se perforó a pesar del tratamiento local y general con antimicóticos. Se debieron realizar sucesivos colgajos conjuntivales para lograr curación, también se utilizó como último recurso una lente de contacto terapéutica. Del material obtenido por raspado de la úlcera de córnea y del humor vítreo se aisló Acremonium kiliense. El tratamiento médico-quirúrgico fue prolongado debido a la agresividad del hongo; se logró salvar la integridad del globo ocular

Cytolytic T-cell clones define HLA-A2-restricted human cutaneous melanoma peptide epitopes: correlation with T-cell receptor usage.

PURPOSE: We studied the T-cell receptor alpha chain and beta chain variable region usage in HLA-A2-restricted and melanoma-specific T lymphocytes and correlated such T-cell receptor usage with HLA-A2-presented peptide-specific T-cell recognition. MATERIALS AND METHODS: Tumor-infiltrating lymphocytes were isolated from a metastatic melanoma lesion and cloned by limiting dilution. Clonal and oligoclonal tumor-infiltrating lymphocytes were analyzed for major histocompatibility complex class I--presented melanoma peptide recognition by using acid-eluted and high-performance liquid chromatography--fractionated melanoma peptides presented by HLA-A2 as targets. The T-cell receptor variable regions of the alpha and beta chains of each individual T-cell clone or oligoclonal T-cell population were analyzed by mRNA extraction and reverse transcribed cDNA by polymerase chain reaction using a panel of T-cell receptor alpha and beta variable region specific primers. RESULTS: We demonstrated that individual T-cell clones are capable of recognizing peptides within multiple high-performance liquid chromatography fractions containing melanoma epitopes, and that individual high-performance liquid chromatography fractions containing melanoma epitopes can be recognized by T-cell clones exhibiting limited usage of the T-cell receptor alpha and beta variable region chains. DISCUSSION: These results confirm the heterogeneity of T-cell-defined melanoma antigens in a single individual and suggest the possibility of developing novel antimelanoma therapeutic reagents using either peptides (as immunogens) or the T-cell receptors themselves (as gene therapy when introduced into lymphoid effectors).

Mass spectrometric identification of a naturally processed melanoma peptide recognized by CD8+ cytotoxic T lymphocytes.

We and others have previously reported that melanoma-specific, cytotoxic T lymphocytes (CTL) define a minimum of six class I-presented peptide epitopes common to most HLA-A2+ melanomas. Here we show that three of these peptide epitopes are coordinately recognized by a CTL clone obtained by limiting dilution from the peripheral blood of an HLA-A2+ melanoma patient. Tandem mass spectrometry was used to characterize and sequence one of these three naturally processed melanoma peptides. One of the potential forms of the deduced peptide sequence (XXTVXXGVX, X = I or L) matches positions 32-40 of the recently identified melanoma gene MART-1/Melan-A. This peptide (p939; ILTVILGVL) binds to HLA-A2 with an intermediate-to-low affinity and is capable of sensitizing the HLA-A2+ T2 cell line to lysis by CTL lines and clones derived from five different melanoma patients. A relative high frequency of anti-p939-specific effector cells appear to be present in situ in HLA-A2+ melanoma patients, since p939 is also recognized by freshly isolated tumor infiltrating lymphocytes. p939 represents a good candidate for the development of peptide-based immunotherapies for the treatment of patients with melanoma.

Synthesis and characterization of wild-type and variant gamma-carboxyglutamic acid-containing domains of factor VII.

Synthetic peptides corresponding to portions of the wild-type and variant sequences of the human factor VII gamma-carboxyglutamic acid (Gla)-containing domain have been prepared by direct peptide synthesis using the Fmoc-based protection strategy. Peptides were purified by ion-exchange and reversed-phase chromatography and characterized as the correct products. A peptide comprising residues 1-49 (GP 1-49) inhibited the activation of factor X (FX) by soluble tissue factor (sTF) and recombinant activated factor VII (rFVIIa). In the absence of phospholipid, no inhibition by this peptide was observed. GP 1-49 did not inhibit the hydrolysis of a peptidyl substrate by rFVIIa in the presence of either sTF or relipidated TF apoprotein in the presence or absence of phospholipid. A similar peptide (residues 1-38, GP 1-38) that did not contain the aromatic stack region was also inhibitory. Two variant peptides, one identical to GP 1-49 but lacking the N-terminal alanine residue (GP 2-49) and one identical to GP 1-38 but with an arginine to alanine substitution at position 9 (GP 1-38 R9A), showed substantially reduced inhibitory activity. Kinetic analysis of the inhibition of Xa generation by GP 1-49 revealed a noncompetitive mode of inhibition, probably via a substrate-depletion mechanism. GP 1-49 does not inhibit by preventing FX binding to phospholipid surfaces. This indicates that the N-terminal residues of the FVII Gla domain are important for the structural integrity of the peptide, and implicates the Gla domain per se in a direct interaction with phospholipid-bound FX.

Effects of serum and insulin-like growth factors on human neuroblastoma cell growth.

Insulin-like growth factors (IGF-I and IGF-II) are mitogenic polypeptides expressed in both developing and adult tissues. To examine the effects of IGFs on neuronal growth, we used SH-SY5Y neuroblastoma cells as an in vitro model of nervous system development. In the current study, we found that either IGF-I (0.1 to 10 nM), insulin (0.1 to 5 micrograms/ml) or calf serum (0.1 to 3%) increased SH-SY5Y proliferation over a 3 day period in a dose dependent manner. In each case, treatment with anti-IGF-I receptor antibodies blocked cell proliferation. IGF-II mRNA levels correlated with SH-SY5Y cell density; subconfluent cells expressed high levels of IGF-II mRNA while low levels of IGF-II mRNA were present in confluent cells. Similarly, serum deprivation increased IGF-I receptor mRNA by 4-fold. Collectively, these results support the concept that an IGF/IGF-I receptor system at least partially mediates SH-SY5Y cell proliferation and suggests the importance of IGFs in regulating neuronal growth.

IGF receptor function and regulation in autocrine human neuroblastoma cell growth.

Insulin-like growth factor-II (IGF-II) and its receptors (type I and II IGF receptors) are expressed in the nervous system in a tissue and developmentally specific manner. We have previously shown that SH-SY5Y human neuroblastoma cells synthesize and secrete high levels of IGF-II, and respond to it with increased neuritic outgrowth, DNA synthesis, and cell proliferation. SH-SY5Y cells also produce type I IGF and IGF-II/M6P receptors; however, it is not known whether these receptors mediate the observed growth promoting effects of IGF-II. In this study, we assayed the role of type I IGF receptor and IGF-II/M6P receptor expression in mediating autocrine IGF-II induced growth. Using anti-receptor antibodies, we found that IGF-II stimulates cell proliferation via the type I IGF receptor but not via the IGF-II/M6P receptor. By Northern analysis, we detected increased mRNA expression of both receptors, with more dramatic changes in type I IGF receptor expression. Collectively, our results indicate a role for the type I IGF receptor in mediating IGF-II induced autocrine neuroblastoma cell growth.

PKC activity and PKC-alpha mRNA content are reduced in serum-derived human neuroblastoma cells without concomitant induction of differentiation.

Protein kinase C (PKC) is a serine/threonine kinase which is thought to play an important role in cellular proliferation and differentiation. PKC activity is stimulated physiologically by diacylglycerol and experimentally by phorbol esters. Long-term exposure of human neuroblastoma cells to phorbol esters results in down-regulation of PKC activity and induction of neuronal differentiation. In this study, we explored the hypothesis that reduced PKC expression is necessary for differentiation of the human neuroblastoma cell line SK-N-SH. PKC activity and PKC-alpha mRNA levels were assayed in cultured SK-N-SH cells over a period of several days in the presence or absence of serum. These determinants of PKC expression were compared with several known markers of neuroblastoma differentiation, including neurite outgrowth and steady-state levels of c-myc and GAP43 mRNA. We observed steady losses of PKC activity and PKC-alpha mRNA content after transfer of cells to serum-free or chemically defined media. However, morphological and biochemical differentiation of SK-N-SH cells occurred only in chemically defined medium, perhaps due to the presence of insulin. We conclude that while loss of PKC may be associated with neuroblastoma differentiation, diminished PKC alone is not sufficient to induce or support the differentiation process.

Regulation of insulin-like growth factor-IL expression and its role in autocrine growth of human neuroblastoma cells.

Insulin-like growth factor-II (IGF-II) is highly expressed in fetal tissues and may act as an autocrine growth factor during early embryogenesis. The SH-SY5Y human neuroblastoma cell line also expresses IGF-II and its receptors and responds to exogenous IGF-II with increased DNA synthesis, cell division, and neuritic outgrowth. For this study, we tested the hypothesis that IGF-II mediates autocrine growth of SH-SY5Y cells in serum-free media. SH-SY5Y cells plated at high densities proliferated in serum-free media, whereas sparsely plated cells did not. IGF-II mRNA levels increased within 24 hours of serum deprivation and were associated with increased immunoreactive IGF-II protein. Exogenous addition of IGF-II increased 3H-TdR incorporation and cell number in a dose- and time-dependent fashion. By nuclear labelling experiments using 5-Bromo-2' deoxyuridine (BrdU), we detected a twofold higher percentage of S phase nuclei after a 24-hour incubation in IGF-II. Treatment of SH-SY5Y cells with anti-IGF-II antibodies in serum-free media inhibited cell proliferation, and this inhibition was partially overcome by the addition of increasing concentrations of IGF-II. Collectively, our results indicate that IGF-II mediates an autocrine growth mechanism in SH-SY5Y cells that is associated with increased IGF-II expression.