Intra-articular nerve growth factor regulates development, but not maintenance, of injury-induced facet joint pain & spinal neuronal hypersensitivity.

OBJECTIVE: The objective of the current study is to define whether intra-articular nerve growth factor (NGF), an inflammatory mediator that contributes to osteoarthritic pain, is necessary and sufficient for the development or maintenance of injury-induced facet joint pain and its concomitant spinal neuronal hyperexcitability. METHOD: Male Holtzman rats underwent painful cervical facet joint distraction (FJD) or sham procedures. Mechanical hyperalgesia was assessed in the forepaws, and NGF expression was quantified in the C6/C7 facet joint. An anti-NGF antibody was administered intra-articularly in additional rats immediately or 1 day following facet distraction or sham procedures to block intra-articular NGF and test its contribution to initiation and/or maintenance of facet joint pain and spinal neuronal hyperexcitability. NGF was injected into the bilateral C6/C7 facet joints in separate rats to determine if NGF alone is sufficient to induce these behavioral and neuronal responses. RESULTS: NGF expression increases in the cervical facet joint in association with behavioral sensitivity after that joint's mechanical injury. Intra-articular application of anti-NGF immediately after a joint distraction prevents the development of both injury-induced pain and hyperexcitability of spinal neurons. Yet, intra-articular anti-NGF applied after pain has developed does not attenuate either behavioral or neuronal hyperexcitability. Intra-articular NGF administered to the facet in naïve rats also induces behavioral hypersensitivity and spinal neuronal hyperexcitability. CONCLUSION: Findings demonstrate that NGF in the facet joint contributes to the development of injury-induced joint pain. Localized blocking of NGF signaling in the joint may provide potential treatment for joint pain.

Experimental Methods to Inform Diagnostic Approaches for Painful TMJ Osteoarthritis.

Temporomandibular joint (TMJ) osteoarthritis (OA) is a degenerative disease of the joint that can produce persistent orofacial pain as well as functional and structural changes to its bone, cartilage, and ligaments. Despite advances in the clinical utility and reliability of the Diagnostic Criteria for Temporomandibular Disorders, clinical tools inadequately predict which patients will develop chronic TMJ pain and degeneration, limiting clinical management. The challenges of managing and treating TMJ OA are due, in part, to a limited understanding of the mechanisms contributing to the development and maintenance of TMJ pain. OA is initiated by multiple factors, including injury, aging, abnormal joint mechanics, and atypical joint shape, which can produce microtrauma, remodeling of joint tissues, and synovial inflammation. TMJ microtrauma and remodeling can increase expression of cytokines, chemokines, and catabolic factors that damage synovial tissues and can activate free nerve endings in the joint. Although studies have separately investigated inflammation-driven orofacial pain, acute activity of the trigeminal nerve, or TMJ tissue degeneration and/or damage, the temporal mechanistic factors leading to chronic TMJ pain are undefined. Limited understanding of the interaction between degeneration, intra-articular chemical factors, and pain has further restricted the development of targeted, disease-modifying drugs to help patients avoid long-term pain and invasive procedures, like TMJ replacement. A range of animal models captures features of intra-articular inflammation, joint overloading, and tissue damage. Although those models traditionally measure peripheral sensitivity as a surrogate for pain, recent studies recognize the brain's role in integrating, modulating, and interpreting nociceptive inputs in the TMJ, particularly in light of psychosocial influences on TMJ pain. The articular and neural contributors to TMJ pain, imaging modalities with clinical potential to identify TMJ OA early, and future directions for clinical management of TMJ OA are reviewed in the context of evidence in the field.

Adenine nucleotides stimulate migration in wounded cultures of kidney epithelial cells.

Adenine nucleotides speed structural and functional recovery when administered after experimental renal injury in the rat and stimulate proliferation of kidney epithelial cells. As cell migration is a component of renal regeneration after acute tubular necrosis, we have used an in vitro model of wound healing to study this process. High density, quiescent monkey kidney epithelial cultures were wounded by mechanically scraping away defined regions of the monolayer to simulate the effect of cell loss after tubular necrosis and the number of cells that migrated into the denuded area was counted. Migration was independent of cell proliferation. Provision of adenosine, adenine nucleotides, or cyclic AMP increased the number of migrating cells and accelerated repair of the wound. Other purine and pyrimidine nucleotides were not effective. Arginine-glycine-aspartic acid-serine peptide, which blocks the binding of extracellular fibronectin to its cell surface receptor, completely inhibited migration in the presence or absence of ADP. Very low concentrations of epidermal growth factor (K0.5 approximately 0.3 ng/ml) stimulated migration, whereas transforming growth factor-beta 2 was inhibitory (Ki approximately 0.2 ng/ml). Thus, adenosine and/or adenine nucleotides released from injured or dying renal cells, or administered exogenously, may stimulate surviving cells in the wounded nephron to migrate along the basement membrane, thereby rapidly restoring tubular structure and function.

A human early response gene homologous to murine nur77 and rat NGFI-B, and related to the nuclear receptor superfamily.

When a human fetal muscle cDNA library was screened with the human thyroid hormone receptor alpha 2 cDNA at low stringency, we found a weakly hybridizing cDNA. The sequence of the insert was 2498 basepairs, with an open reading frame of 1794 basepairs encoding a protein of 598 amino acids and a predicted molecular mass of 64 kDa. The DNA-binding domain and the ligand-binding domain are similar to those of steroid and thyroid hormone receptors. Moreover, this cDNA is highly homologous to mouse nur77 and rat NGFI-B, which are early response genes induced by nerve growth factor and other serum growth factors. We designated this gene NAK1. The modulation of expression of NAK1 during stimulation of cell growth was studied. The mRNA of NAK1 was induced rapidly and transiently by growth-stimulating agents, such as adenosine diphosphate, in monkey kidney cells (BSC-1), by phytohemagglutinin in human lymphocytes, and by serum stimulation of arrested fibroblasts. It is expressed in human fetal muscle and adult liver, brain, and thyroid. NAK1 could be a nuclear receptor. It will be of great interest to determine the ligand for NAK1 and the genes that are regulated by it.

An alternate method utilizing small quantities of ligand for affinity purification of monospecific antibodies.

An alternate method was designed to couple a limited quantity of protein to an affinity support when a conventional technique was unsuccessful. This was achieved through the introduction of a small number of sulfhydryl groups to the ligand by reaction with 2-iminothiolane which resulted in a limited number of reactive sites on the protein. Amino groups on an AH-Sepharose 4B matrix were linked to sulfhydryl groups on the ligand using the heterobifunctional agent m-maleimidobenzoyl sulfosuccinimide ester (sulfo-MBS). This method was employed to prepare an affinity support using a cytosolic protein that activates glyceraldehyde-3-phosphate dehydrogenase as a ligand. Monospecific antibody purified from the affinity column recognized only this protein on a Western blot of a cytosolic extract of kidney epithelial cells.

Outcomes following total laryngectomy for squamous cell carcinoma: one centre experience.

OBJECTIVES: To evaluate the clinical outcomes of total laryngectomy (TL), complications and factors affecting survival. DESIGN: Retrospective review of hospital electronic database for head and neck squamous cell carcinoma (SCCa). SETTING: Large district general hospital in England, United Kingdom. PARTICIPANTS: Patients who had TL between January 1994 and January 2008. MAIN OUTCOME MEASURES: 5-year disease specific survival (DSS) and disease-free survival (DFS). RESULTS AND CONCLUSIONS: Seventy-one patients were reviewed, of whom 38 (54%) had laryngeal SCCa and 33 (46%) hypopharyngeal SCCa. The overall mean survival period following TL was 42.4 months. The 5-year DSS and DFS was better for laryngeal SCCa compared to hypopharyngeal SCCa, although not statistically significant (P=0.090, P=0.54 respectively). Patients treated for laryngeal SCCa had a mean survival period of 47.5 months compared to 36.5 months for hypopharyngeal disease. Those who had laryngeal recurrence after primary radiotherapy (RT) demonstrated statistically better survival probability than those who had hypopharyngeal recurrence (P=0.011). Patients without cervical lymphadenopathy had statistically better survival (P=0.049). The most common early complication was related to the cardiorespiratory system. One fatal complication of erosion of the brachiocephalic artery due to the laryngectomy tube was noted. The most common late complication was neopharyngeal stenosis. The commonest cause of death was due to locoregional recurrence, followed by medical co-morbidities. Patients referred to specialised head and neck clinic had a better survival probability than those referred to a general ENT clinic (P=0.37). While there is increasing tendency towards laryngeal conservation, total laryngectomy remains a robust treatment option in selected patients.

Purine nucleotides stimulate DNA synthesis in kidney epithelial cells in culture.

Adenine nucleotides infused into animals with acute renal failure appear to enhance recovery of kidney function and structure. To determine whether these compounds could act by a direct effect on renal cell metabolism, their capacity to stimulate DNA synthesis was evaluated in cultures of monkey kidney epithelial cells (BSC-1 line). AMP and ADP enhanced DNA synthesis by threefold more than was previously observed with other mitogens for these cells. Guanosine and inosine and their nucleotides and adenosine and ATP were also mitogenic but to a lesser extent, whereas pyrimidine derivatives were ineffective. In the presence of AMP, autoradiography of [3H]thymidine-labeled cells indicated that a greater number of cells entered the S phase of the cell cycle, and assessment of cell number revealed increased multiplication. The mitogenic effect of adenine nucleotides was not reproduced by agents that raise the cellular content of cAMP and was serum independent. Adenine nucleotides did not alter DNA synthesis when added to cultures of mouse fibroblasts. These results indicate that provision of exogenous purine nucleosides and nucleotides stimulate DNA synthesis in renal epithelial cells in culture.

Aggregation-deficient embryonal carcinoma cells: defects in peanut agglutinin (PNA) receptors.

The components involved in cell adhesion were studied using the H6 line of embryonal carcinoma cells. H6 cells are especially suitable for studies on cell interactions, since genetic mutants can be selected, and various processes of cell adhesion can be controlled by regulating the calcium concentration in the medium. Three aggregation-defective variants of H6 were isolated, all of which showed reduced binding of the lectin, peanut agglutinin (PNA). Quantitation of PNA receptors on the cell surface by immunoprecipitation of iodinated surface proteins indicated that these receptors were reduced on the variants by one-half to one-quarter. The separation of immunoprecipitated PNA receptors on sodium-dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that one type of receptor, with an apparent molecular weight of 94 kilodaltons, was reduced. Parental and variant cells bind similar quantities of concanavalin A and soybean agglutinin, suggesting that there is no generalized effect on major glycoproteins. Thus, the defect in aggregation and the defect in the 94-kilodalton protein may be correlated, and this glycoprotein may have a role in the mediation of H6 cell-cell adhesion.

ADP activates protooncogene expression in renal epithelial cells.

Purine nucleotides, particularly adenosine diphosphate (ADP), are the most potent mitogens known for monkey kidney epithelial cells of the BSC-1 line. To explore the molecular mechanisms by which ADP exerts its mitogenic effect, we tested the hypothesis that stimulation of DNA synthesis in these renal cells is mediated by activation of protooncogenes. Transcripts of the c-Ha-ras protooncogene were identified in quiescent, high density cells. Addition of ADP to the culture medium stimulated protooncogene expression fourfold. Maximal expression of c-ras was observed by 12 h after exposure to ADP, and preceded the initiation of DNA synthesis. Expression of the c-myc protooncogene was not detected in unstimulated cells, but accumulated maximally after 1 h of exposure to ADP. As with ADP-stimulated expression of the c-ras protooncogene, transcripts of the transferrin receptor gene reached a maximal value at 12 h, whereas the abundance of gamma-actin mRNA was not altered for up to 24 h. The results indicate that exogenous ADP stimulates protooncogene expression before initiation of DNA synthesis in renal epithelial cells in culture. These findings suggest that some physiological effects of this adenine nucleotide could be mediated by proteins specified by protooncogenes.

Signals that release growth factors from renal epithelial cells.

Monkey kidney epithelial cells of the nontransformed BSC-1 line have been used as a model system to investigate growth control. Renal growth in K depletion nephropathy was studied in culture by reducing the K concentration of the medium, which accelerated cell proliferation. This response appeared to be mediated by release of a growth-promoting activity that has an apparent molecular weight of 12,000 to 30,000. Growth stimulation was also observed when the Na concentration of the medium was reduced and was associated with the appearance of two growth-promoting factors (apparent molecular weight, 6,200 and 9,000) that exhibited cell-type specificity. Thus, modest reductions in the extracellular concentration of K or Na result in rapid appearance of autocrine factors that could modulate cell function along the nephron. The most powerful mitogen for BSC-1 cells is adenosine diphosphate (ADP). This nucleotide stimulates expression of several cell cycle-specific genes and proto-oncogenes, and induces secretion of a platelet-derived growth factor-like protein that is not mitogenic for BSC-1 cells. Release of this growth factor by renal epithelial cells in vivo would represent a paracrine mechanism to initiate proliferation of neighboring stromal or vascular cells.

Differential gene expression in migrating renal epithelial cells after wounding.

An in vitro model of wound healing was used to study cell migration that is independent of proliferation during renal regeneration after acute tubular necrosis. Monolayer cultures of high-density, quiescent renal epithelial cells of the BSC-1 line were subjected to scrape wounding and then Northern blot analysis was employed to identify genes that mediate cell migration. After wounding the monolayer, there is maximal induction of the immediate-early genes Egr-1, c-fos, NAK-1, and gro at 1 hour, followed by peak induction of connective tissue growth factor (CTGF) and c-myc at 4 hours. Message levels of urokinase-type plasminogen activator (u-PA) and its inhibitor (PAI-1) and heat shock protein (HSP)-70 are markedly raised 4-8 hours after wounding. Constitutive expression is repressed at 1 hour for transcripts that encode receptors for fibronectin (FN), epidermal growth factor, and hepatocyte growth factor (c-met), and the secreted proteins FN and osteopontin. Expression of genes encoding transforming growth factor (TGF)-beta 1 and -beta 2, retinoic acid receptor alpha, int-1, int-2, and gap junction protein which can play a role in cell movement, appeared unchanged after wounding. Differential expression of genes was a function of cell location relative to the wound; NAK-1, PAI-1, and HSP-70 were induced or stimulated only in cells at the wound edge, u-PA was stimulated in cells away from the wound, and CTGF was induced in each of these populations suggesting that cell-to-cell communication may regulate gene expression after wounding. Adenosine diphosphate, a potent stimulator of cell migration which enhances expression of u-PA and PAI-1 in nonwounded cultures, additively stimulates these genes after wounding and may thereby potentiate wound healing. Thus scrape wounding of renal epithelial cells is followed by induction, stimulation, or repression of specific genes with distinct responses in different populations of cells.

Calcium-induced compaction and its inhibition in embryonal carcinoma cell aggregates.

H6 embryonal carcinoma cells form aggregates of cells in culture medium which contains 2 mM calcium. These aggregates are described as uncompacted, indicating that the individual cells of the aggregate are spherical and are in limited contact with each other. In contrast, compaction of the aggregate, induced by increasing the calcium concentration, results in a tight mass of cells flattened against one another and connected by intercellular junctions. At least 85-97% of the aggregates undergo compaction in 7 mM calcium and are subsequently decompacted if removed to 2 mM calcium. Since calcium ionophore A23187 does not induce compaction, extracellular rather than intracellular calcium seems to be the limiting factor. We have demonstrated that this calcium-induced morphogenetic change is sensitive to inhibition by agents which also prevent the calcium-dependent compaction of the 8-cell mouse embryo. The cytoskeletal-binding drugs tetracaine HCl, colcemid, vinblastine, colchicine, and cytochalasin B each inhibit compaction of H6 aggregates. Interference at surface molecule sites by exposure to the lectins wheat germ agglutinin or concanavalin A or by interruption of glycosylation with exposure to tunicamycin, or by reaction with anti-H6 Fab or anti-F9, also prevent compaction. Since the mouse embryo and embryonal carcinoma cells share certain processes which are involved in initiating and maintaining compaction, these processes and their subsequent roles in differentiation may be examined using embryonal carcinoma cell aggregates.

New vibrational bands in nitrogen laser emission spectra.

Nitrogen laser emission spectra was analyzed using a 0.5-m Jarrell-Ash monochromator. In addition to the (0,0) 337.1-nm band, the laser spectra were found to contain (0,1) 357.69-nm, (2,4) 371.05-nm, (1,0) 315.93-nm, (0,2) 380.49-nm, (0,3) 405.94-nm, (1,3) 375.54-nm, (1,4) 399.84-nm, and (1,2) 353.67-nm bands belonging to the second positive system of the N2 molecule. A new transition from one of the mixed vibrational levels of C and C' states of the N2 molecule was observed in the laser spectra at 331.83 nm with a relative intensity of approximately 29%. Two bands, at 340.85 and 303.49 nm, belonging to the beta system of the NO molecule were also observed. The intensity variation of the prominent bands observed was studied with respect to change in operating pressure and voltage for deriving the optimum conditions for emission at these wavelengths. Variations in the intensity of the bands were also studied with change in distance between the laser head and the monochromator; it was observed that an amplification effect had taken place at 331.83 and 340.85 nm, in addition to the (0,0) 337.1-nm band, in the nitrogen laser discharge.

Junction formation in aggregated embryonal carcinoma cells.

Under the action of supplemental calcium, H6 mouse embryonal carcinoma cell aggregates undergo compaction, a morphological phenomenon similar to mouse embryonic compaction. Formation of various types of cell junctions, especially gap junctions, is associated with compaction of the embryo and we sought to analyze the pattern of junction formation during aggregation and compaction of H6 cells. At 24 hr of aggregation, gap junctions were abundant in both uncompacted and compacted aggregates but quantitative analysis of freeze fracture replicas of these junctions showed a 20-fold increase in the size of the largest gap junctions in compacted aggregates. Such a difference in size could even be detected at 12 hr of aggregation. Tight junctions were not normally formed in 12 hr aggregates but initial stages of tight junction formation could be noticed in 12 hr compacted aggregates. More definitive tight junctions and desmosomes were evident only after 48 hr of aggregation. Thus we have observed that both uncompacted and compacted aggregates can form gap junctions at similar frequencies, suggesting that cell flattening, which contributes to the compacted morphology, is not a requisite for gap junctions. Likewise, generation of the compacted morphology seems to be independent of gap junction formation. This supports the idea that compaction in embryonal carcinoma cells results from calcium-induced cell flattening, probably through the mobilization of cytoskeletal elements. Calcium-dependent features of H6 cell aggregation and compaction enables the independent analysis of separate steps in compaction.

Kidney epithelial cells release growth factors in response to extracellular signals.

The growth of nontransformed monkey kidney epithelial cells in culture appears to be regulated by the interplay of positive and negative autocrine growth factors. Reduction of the potassium or sodium concentration of the medium induces rapid release of novel growth-promoting activities, whereas addition of the mitogen adenosine diphosphate stimulates the appearance of a platelet-derived growth factor-like protein which could function in a paracrine manner. These observations suggest that autocrine and paracrine growth factors could play an important role in physiological and pathological states in the kidney.