Spatial mapping of proteoglycan content in articular cartilage using near-infrared (NIR) spectroscopy

Diagnosis of articular cartilage pathology in the early disease stages using current clinical diagnostic imaging modalities is challenging, particularly because there is often no visible change in the tissue surface and matrix content, such as proteoglycans (PG). In this study, we propose the use of near infrared (NIR) spectroscopy to spatially map PG content in articular cartilage. The relationship between NIR spectra and reference data (PG content) obtained from histology of normal and artificially induced PG-depleted cartilage samples was investigated using principal component (PC) and partial least squares (PLS) regression analyses. Significant correlation was obtained between both data (R² = 91.40%, p<0.0001). The resulting correlation was used to predict PG content from spectra acquired from whole joint sample, this was then employed to spatially map this component of cartilage across the intact sample. We conclude that NIR spectroscopy is a feasible tool for evaluating cartilage contents and mapping their distribution across mammalian joint.OCIS codes: (170.6510) Spectroscopy, tissue diagnostics; (170.6935) Tissue characterization; (170.3880) Medical and biological imaging     

Benzofuran sulfonates and small self-lipid antigens activate type II NKT cells via CD1d

Natural killer T (NKT) cells detect lipids presented by CD1d. Most studies focus on type I NKT cells that express semi-invariant αβ T cell receptors (TCR) and recognize α-galactosylceramides. However, CD1d also presents structurally distinct lipids to NKT cells expressing diverse TCRs (type II NKT cells), but our knowledge of the antigens for type II NKT cells is limited. An early study identified a nonlipidic NKT cell agonist, phenyl pentamethyldihydrobenzofuransulfonate (PPBF), which is notable for its similarity to common sulfa drugs, but its mechanism of NKT cell activation remained unknown. Here, we demonstrate that a range of pentamethylbenzofuransulfonates (PBFs), including PPBF, activate polyclonal type II NKT cells from human donors. Whereas these sulfa drug–like molecules might have acted pharmacologically on cells, here we demonstrate direct contact between TCRs and PBF-treated CD1d complexes. Further, PBF-treated CD1d tetramers identified type II NKT cell populations expressing αβTCRs and γδTCRs, including those with variable and joining region gene usage (TRAV12-1–TRAJ6) that was conserved across donors. By trapping a CD1d–type II NKT TCR complex for direct mass-spectrometric analysis, we detected molecules that allow the binding of CD1d to TCRs, finding that both selected PBF family members and short-chain sphingomyelin lipids are present in these complexes. Furthermore, the combination of PPBF and short-chain sphingomyelin enhances CD1d tetramer staining of PPBF-reactive T cell lines over either molecule alone. This study demonstrates that nonlipidic small molecules, which resemble sulfa drugs implicated in systemic hypersensitivity and drug allergy reactions, are targeted by a polyclonal population of type II NKT cells in a CD1d-restricted manner.

Natural killer T (NKT) cells are defined as T cells that are restricted to the lipid antigen-presenting molecule, CD1d. The most extensively studied are type I NKT cells, which typically express an invariant T cell receptor (TCR)-α chain consisting of TRAV10–TRAJ18 in humans (TRAV11–TRAJ18 in mice) paired with a constrained repertoire of TCR-β chains, enriched for TRBV25 in humans (TRBV13, 29, 1 in mice) (reviewed in ref. 1). Type I NKT cells are defined by their strong responses to α-galactosylceramide (α-GalCer) and structurally related hexosylceramides presented by CD1d, while in contrast, type II NKT cells are defined as CD1d-restricted T cells that express diverse TCRs and do not recognize α-GalCer (reviewed in refs. 1 and 2). Very little is known about the chemical identity of antigens for type II NKT cells; however, some studies suggest that these cells are abundant in humans (3–5), and, by virtue of their greater TCR diversity, they can interact with a broader range of antigens compared to type I NKT cells (2, 6–14).In 2004, a nonlipidic molecule, phenyl pentamethyldihydrobenzofuransulfonate (PPBF), was described that stimulated a human TRAV10⁻ (type II) NKT cell line (clone ABd) in a CD1d-dependent manner (15). These observations were notable because PPBF resembles various sulfonamide drugs: furosemide (diuretic), sulfasalazine (disease-modifying antirheumatic), and celecoxib (anti-inflammatory) as well as “sulfa” antibiotics such as sulfonamide, sulfapyridine, sulfamethoxazole, sulfadiazine, and sulfadoxine (16). These drugs can cause systemic delayed-type hypersensitivity reactions, which are thought to be mediated by T cells (17–20). Most of our limited understanding of drug hypersensitivity reactions comes from work focusing on Human Leukocyte Antigen (HLA)-restricted conventional T cells, which has led to the proposal of four main mechanisms for small-drug immune activity as reviewed in ref. 21: 1) hapten/prohapten formation, whereby the drug reacts with self-Ags to generate a neo-product that undergoes processing and presentation to T cells; 2) noncovalent/labile pharmacological interaction with immune receptors on the cell surface; 3) superantigen mediating direct linkage of TCRs and Ag-presenting molecules; and 4) anchor site occupation by small molecules in Ag-presenting molecules inducing an altered self-Ag repertoire (22). Whether the CD1d-NKT cell axis is implicated in drug hypersensitivity remains unclear (23). Whereas most antigens in the CD1d system are lipids that use their alkyl chains to bind to CD1d, PPBF is a polycyclic small molecule and so would have to act through an atypical display mechanism. As previous attempts to stain the ABd clone with CD1d-PPBF tetramers were unsuccessful, the atypical drug-like structure of PPBF raised the possibility of direct pharmacological action on T cells rather than the presentation of CD1d-PPBF complexes to TCRs. However, the mechanism of PPBF-mediated type II NKT cell activation remains undefined (15).Here, using TCR-transduced cell lines, CD1d tetramers treated with PPBF, and new analogs in the pentamethylbenzofuransulfonate (PBF) family, we discovered that several molecules stimulate polyclonal NKT cells. Using CD1d tetramers treated with a newly identified and more potent analog of PPBF, we identified populations of type II NKT cells that comprise a polyclonal repertoire of both αβ and γδ T cells, including those with conserved TCR sequences. This enigmatic nature of T cell responses to PBF molecules was resolved using TCR trap technology (24, 25). Mass-spectrometric analysis of all molecules present in CD1d-PBF-TCR complexes indicates that CD1d binds PBFs and small self-lipids that promote CD1d-TCR binding. These data support a model of type II NKT cell recognition of small sulfa drug–like compounds in association with CD1d and flag a possible mechanism in which such cells may be involved in sulfa drug hypersensitivity.     

Smad5 is dispensable for adult murine hematopoiesis

Smad5 is known to transduce intracellular signals from bone morphogenetic proteins (BMPs), which belong to the transforming growth factor-beta (TGF-beta) superfamily and are involved in the regulation of hematopoiesis. Recent findings suggest that BMP4 stimulates proliferation of human primitive hematopoietic progenitors in vitro, while early progenitors from mice deficient in Smad5 display increased self-renewal capacity in murine embryonic hematopoiesis. Here, we evaluate the role of Smad5 in the regulation of hematopoietic stem cell (HSC) fate decisions in adult mice by using an inducible MxCre-mediated conditional knockout model. Surprisingly, analysis of induced animals revealed unperturbed cell numbers and lineage distribution in peripheral blood (PB), bone marrow (BM), and the spleen. Furthermore, phenotypic characterization of the stem cell compartment revealed normal numbers of primitive lin(-)Sca-1(+)c-Kit(+) (LSK) cells in Smad5(-)(/)(-) BM. When transplanted in a competitive fashion into lethally irradiated primary and secondary recipients, Smad5-deficient BM cells competed normally with wild-type (wt) cells, were able to provide long-term reconstitution for the hosts, and displayed normal lineage distribution. Taken together, Smad5-deficient HSCs from adult mice show unaltered differentiation, proliferation, and repopulating capacity. Therefore, in contrast to its role in embryonic hematopoiesis, Smad5 is dispensable for hematopoiesis in the adult mouse.     

Dyspnea assessment and management in hospice patients with pulmonary disorders

Accurate assessments and appropriate management of dyspnea are essential to provide improved quality of life for hospice patients. This study describes methods of assessing dyspnea and interventions used to manage dyspnea in 72 hospice patients with end-stage lung disease or lung cancer. The mean age of the sample was 72.46 years old and the majority was white (80 percent) and male (62 percent). Paired t-tests were used to compare mean scores on admission and near death for dyspnea severity, Karnofsky functional status, pain, and Mini-Mental Status scores. Results showed significant decline in functional and cognitive status, but no significant changes in dyspnea severity and pain. Dyspnea was often assessed subjectively with observational methods only. Use of inhalants, oxygen, positioning, steroids, and oral opioids were the most frequent therapies for dyspnea. Relaxation, guided imagery, and other complementary therapies were rarely used (five percent or less). Measurement of dyspnea needs to be done frequently by using standardized instruments to assess severity and degree of symptom distress as well as the effects of treatment. Clinical trials are needed to determine which dyspnea interventions are most effective in terminally ill patients. Guidelines such as those developed for pain management are needed for effectively managing dyspnea.     

Neurotensin causes tyrosine phosphorylation of focal adhesion kinase in lung cancer cells

The effects of neurotensin on focal adhesion kinase were investigated using lung cancer cells. Neurotensin bound with high affinity to large cell carcinoma cell line NCI-H1299 as did neurotensin-(8-13), but not neurotensin-(1-7) or levocabastine. Addition of 100 nM neurotensin to NCI-H1299 cells caused transient tyrosine phosphorylation of focal adhesion kinase which was maximal after 1-2.5 min. Also, neurotensin-(8-13), but not neurotensin-(1-8) or levocabastine, caused tyrosine phosphorylation of focal adhesion kinase after addition to NCI-H1299 cells. Focal adhesion kinase tyrosine phosphorylation caused by neurotensin was inhibited by the nonpeptide neurotensin receptor antagonist (2-(1-(7-chloroquinolin-4-yl)-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-carbonyl)amino)-adamantane-2-carboxylic acid) (SR48692). SR48692 inhibited the clonal growth of NCI-H1299 cells, whereas neurotensin-stimulated proliferation and levocabastine had no effect. These results indicate that lung cancer cells have functional neurotensin receptors which regulate focal adhesion kinase tyrosine phosphorylation. It remains to be determined if neurotensin receptors and focal adhesion kinase plays a role in lung cancer cellular adhesion and migration.     

Dermoid,teratoma or choristoma? A rare lesion of the tongue in an adult

The nomenclature of developmental lesions in the tongue is confusing. We present a rare case in an adult and describe the clinical and histopathological features. Terminology of these lesions is also discussed.     

AH6809 antagonizes non-small cell lung cancer prostaglandin receptors

The effects of prostaglandin (PG)E2 on lung cancer cells were investigated. 3H-PGE2 bound with high affinity to membranes derived from small cell lung cancer (SCLC) and non-SCLC (NSCLC) cell lines. Using NSCLC NCI-H1299 membranes, specific 3H-PGE2 binding to NCI-H1299 membranes was inhibited with moderate affinity by PGE2, PGE1, PGF2alpha and 6-isopropoxy-9-xanthone-2-carboxylic acid (AH6809) but not PGD2, LTB4 or 5-HETE. By RT-PCR, EP2 receptor PCR products were detected in extracts derived from lung cancer cells. PGE2 caused cAMP elevation in a concentration-dependent manner using NCI-H1299 cells and the increase in cAMP caused by PGE2 was antagonized by AH6809. PGE2 had no effect on cytosolic Ca2+ but PGE2 caused increased c-fos mRNA in NCI-H1299 cells. AH6809 inhibited the proliferation of NCI-H1299 cells using MTT and clonogenic assays. These results indicate that functional PG receptors are present on NSCLC cells which are antagonized by AH6809.