Tumor necrosis factor-alpha from macrophages enhances LPS-induced clara cell expression of keratinocyte-derived chemokine

Tumor necrosis factor (TNF)-alpha is a cytokine produced by alveolar macrophages in response to LPS in the lung. Clara cells are bronchiolar epithelial cells that produce a variety of proinflammatory cytokines in response to LPS but not to TNF-alpha. In this study, we examined whether TNF-alpha affects Clara cell cytokine production in the setting of LPS stimulation. Using a transformed murine Clara cell line (C22), we observed that both LPS and TNF-alpha induced production of keratinocyte-derived chemokine (KC) and monocyte chemoattractant protein (MCP)-1. We also found that simultaneous LPS and TNF-alpha stimulation is synergistic for KC production, but additive for MCP-1 production. By using a Transwell coculture system of RAW264.7 macrophages and Clara cells isolated from C57Bl/6 mice, we found that macrophages produce a soluble factor that enhances Clara cell KC production in response to LPS. Cocultures of Clara cells from mice deficient in TNF-alpha receptors with RAW264.7 macrophages demonstrated that the effect of macrophages on Clara cells is mediated primarily via TNF-alpha. To determine whether these findings occur in vivo, we treated wild-type and TNF receptor-deficient mice intratracheally with LPS and examined the expression of KC. LPS-treated, TNF receptor-deficient mice showed much less KC mRNA in airway epithelial cells compared with wild-type mice. In contrast, a similar number of KC-expressing cells was seen in the lung periphery. Thus, upregulation of KC by Clara cells in the setting of LPS stimulation is largely dependent on TNF-alpha originating from alveolar macrophages. These findings shed light on macrophage-Clara cell interactions in regulating the pulmonary inflammatory response to LPS.     

Differential expression of tartrate-resistant acid phosphatase isoforms 5a and 5b by tumor and stromal cells in human metastatic bone disease

Tartrate-resistant acid phosphatase (TRAP) exists in human serum as two major isoforms, monomeric 5a and proteolytically processed enzymatically active 5b. The 5b isoform is secreted by osteoclasts and has recently been advocated as a serum marker for bone metastasis in breast cancer patients. The 5a isoform, on the other hand, is not bone-derived and has been proposed to be a marker of activated macrophages and chronic inflammation. In this study, expression of TRAP protein and enzymatic activity in bone metastases from different primary sites was examined. TRAP activity was high in bone metastases from prostate cancer, intermediate in breast cancer, and low in lung and kidney cancers. The partially purified TRAP from breast cancer bone metastasis samples exhibited the enzymatic characteristics of purple acid phosphatase. Both 5a and 5b isoforms were expressed in bone metastases of different histogenetic origins, i.e. prostate, breast, lung and kidney, and also a novel previously unreported 42 kDa variant of the TRAP 5a isoform was identified in bone metastases. This novel TRAP 5a isoform was absent in human bone, indicating that the 42 kDa variant is specific to metastatic cancer tissue. Immunohistochemistry revealed that metastatic cancer cells were the predominant source of TRAP 5a, whereas tumor-associated macrophages and occasionally multinucleated giant cells in the tumor stroma preferentially expressed the proteolytically processed TRAP 5b variant. Our results indicate the presence of a previously unstudied variant of monomeric TRAP 5a in cancer cells, which may have functional and diagnostic implications. Moreover, the presence of TRAP-positive macrophages in bone metastases could, together with cancer cells and osteoclasts, contribute to the elevated levels of serum TRAP activity observed in patients with bone metastases.     

Role of non-canonical Wnt signaling in osteoblast maturation on microstructured titanium surfaces

The Wnt signaling pathway inhibitor Dickkopf-2 (Dkk2) regulates osteoblast differentiation on microstructured titanium (Ti) surfaces, suggesting involvement of Wnt signaling in this process. To test this, human osteoblast-like MG63 cells were cultured on tissue culture polystyrene or Ti (smooth PT (Ra=0.2 μm), sand-blasted and acid-etched SLA (Ra=3.22 μm), modSLA (hydrophilic SLA)). Expression of Wnt pathway receptors, activators and inhibitors was measured by qPCR. Non-canonical pathway ligands, receptors and intracellular signaling molecules, as well as bone morphogenetic proteins BMP2 and BMP4, were upregulated on SLA and modSLA, whereas canonical pathway members were downregulated. To confirm that non-canonical signaling was involved, cells were cultured daily with exogenous Wnt3a (canonical pathway) or Wnt5a (non-canonical pathway). Alternatively, cells were cultured with antibodies to Wnt3a or Wnt5a to validate that Wnt proteins secreted by the cells were mediating cell responses to the surface. Wnt5a, but not Wnt3a, increased MG63 cell differentiation and BMP2 and BMP4 proteins, suggesting Wnt5a promotes osteogenic differentiation through production of BMPs. Effects of exogenous and endogenous Wnt5a were synergistic with surface microstructure, suggesting the response also depends on cell maturation state. These results indicate a major role for the non-canonical, calcium-dependent Wnt pathway in differentiation of osteoblasts on microstructured titanium surfaces during implant osseointegration.     

Investigation of the human serotonin 6 (5‐HT6) receptor gene in bipolar affective disorder and schizophrenia

Serotonin (5‐hydroxytryptamine, 5‐HT) is a neurotransmitter that mediates a wide range of central nervous functions by activating multiple 5‐HT receptor subtypes. A possible irregularity of serotonergic neurotransmission has been implicated in a variety of neuropsychiatric diseases. In the present study, we performed a systematic mutation scan of the complete coding region and splice junctions of the 5‐HT₆ receptor gene to explore the contribution of this gene to the development of bipolar affective disorder and schizophrenia. Investigating 137 unrelated individuals (including 45 bipolar affective patients, 46 schizophrenic patients, and 46 unrelated controls), we identified six single base substitutions (126G/T, 267C/T, 873+30C/T, 873+128A/C, 1128G/C, 1376T/G). Comparing frequencies between patients and controls, we observed a significant overrepresentation of the 267C allele among bipolar patients (P=0.023 not corrected for multiple testing). This finding was followed up in an independent sample of 105 bipolar family trios using a family‐based association design. Fifty‐one transmissions could be examined. In 30 cases allele 267C and in 21 cases allele 267T were transmitted to the affected offspring. Although this result was far from statistical significance (transmission disequilibrium test=1.59, P=0.208), the limited number of possible transmissions may have prevented detection of smaller effects. Our preliminary data suggest that bipolar affective disorder may be associated with variation in the 5‐HT₆ gene. It will be important to extend the present analysis to larger samples. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:217–221, 2000. © 2000 Wiley‐Liss, Inc.     

Patients' satisfaction with information and experiences with counseling on cardiovascular medication received at the pharmacy

Objective

To assess the extent to which patients feel they have received enough information on cardiovascular drugs and experienced counseling at the pharmacy. In addition, to identify factors that are predictors for patient satisfaction with the information received.

Methods

Fifteen community pharmacies participated. New and chronic users of cardiovascular medication received a questionnaire containing sociodemographic and health questions, a measure of satisfaction with information received (SIMS), beliefs about medication (BMQ), and frequency of pharmacy counseling.

Results

Of the 578 respondents, 335 (58%) indicated to be unsatisfied with the information received on 3 or more SIMS items. Patients’ age, beliefs about medication, duration of cardiovascular treatment and use of antithrombotics predicted patients’ satisfaction with information received. Two-thirds of patients reported ‘never’ to have experienced 5 of 8 counseling activities at the pharmacy.

Conclusions

A considerable proportion of patients are unsatisfied with the information received on cardiovascular medication. The majority of patients have only received a limited scope of medication counseling at the pharmacy.

Practice implications

Information and counseling should be tailored to patients’ needs and concerns about cardiovascular medication and the experience patients already have with treatment. Pharmacists could enhance their role in supporting patients using cardiovascular medication.     

Critical role of B cells in the development of T cell tolerance to aeroallergens

Respiratory exposure to allergen induces the development of allergen-specific CD4(+) T cell tolerance that effectively protects against the development of allergic-sensitization and T(h)2-biased immunity. The establishment of T cell unresponsiveness to aeroallergens is an active process preceded by a transient phase of T cell activation that requires T cell co-stimulation and is critically influenced by the antigen-presenting cell type. In this study we examined the role of B cells in the development of respiratory tolerance following intranasal (i.n.) exposure to a prototypic protein antigen. We found that respiratory exposure of BCR-transgenic (Tg) mice to minute quantities of cognate antigen effectively induced T cell unresponsiveness, indicating that antigen presentation by antigen-specific B cells greatly enhanced the development of respiratory tolerance. In contrast, respiratory T cell unresponsiveness could not be induced in B cell-deficient JHD mice exposed to i.n. antigen, although T cell tolerance developed in JHD mice reconstituted with B cells, suggesting that B cells are required for the induction of respiratory T cell tolerance. Respiratory exposure of BCR-Tg mice to cognate antigen induced activation of antigen-specific T cells and partial activation of antigen-specific B cells, as demonstrated by enhanced expression by B cells of class II MHC and B7 molecules but lack of antibody secretion. Our data indicate that B cells critically influence the immune response to inhaled allergens and are required for the development of allergen-specific T cell unresponsiveness induced by respiratory allergen.     

Construction and in vivo evaluation of a dual layered collagenous scaffold with a radial pore structure for repair of the diaphragm

In each organ the extracellular matrix has a specific architecture and composition, adapted to the functional needs of that organ. As cells are known to respond to matrix organization, biomaterials that take into account the specific architecture of the tissues to be regenerated may have an advantage in regenerative medicine. In this study we focussed on the diaphragm, an organ essential for breathing, and consisting of radial oriented skeletal muscle fibres diverging from a central tendon plate. To mimic this structure dual layered collagenous scaffolds were constructed with a radial pore orientation, prepared by inward out freezing, and reinforced by a layer of compressed collagen. Similar scaffolds with a random round pore structure were taken as controls. Scaffolds were first mildly crosslinked by formaldehyde vapour fixation for initial stabilization (13% and 17% crosslinking for the radial and control scaffolds, respectively), and further crosslinked using aqueous 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide (38% and 37% crosslinking, respectively). Scaffolds were implanted into a surgically created diaphragm defect in rats and explanted after 12 weeks. Macroscopically, integration of the radial scaffolds with the surrounding diaphragm was better compared with the controls. Cells had infiltrated further into the centre of the scaffolds (P = 0.029) and there was a tendency of blood vessels to migrate deeper into the radial scaffolds (P = 0.057, compared with controls). Elongated cells (SMA-positive) were aligned with the radial structures. In conclusion, collagenous scaffolds with a stable radial pore structure can be constructed which facilitate cellular in-growth and alignment in vivo.     

Molecular mechanisms of pulmonary vascular development.

In this era of rapidly advancing vascular biology research, a vast array of growth factors and signaling molecules have been recognized as key players in the mechanisms that control lung vascular development. In the lung, vascular development is a complex, multistep process that includes specialization of primitive cells to vascular progenitors; formation of primitive vascular networks; remodeling with local regression and branching; specialization toward arteries, veins, and lymphatics; stabilization of vessels by matrix production and recruitment of supporting cells; and maintenance of the vascular structure. This complex, highly organized process requires exquisite orchestration of the regulatory activity of multiple molecules in a specific temporospatial order. Most of these molecules are members of 3 major growth factor families that have been recently identified. They are the vascular endothelial growth factor, angiopoietin, and ephrin families. Understanding the functional reach of several members of these growth factor families is integral to an appreciation of the etiology and pathogenesis of developmental lung vascular disorders affecting newborns.This review summarizes recent advances in the molecular bases of lung vascular development and some of the pulmonary diseases resulting from aberrant vascular growth, including bronchopulmonary dysplasia, alveolar capillary dysplasia, congenital cystic pulmonary disorders, congenital pulmonary hemangiomatosis, and lung hypoplasia.     

Event-related potentials (ERPs) to schematic faces in adults and children.

Event-related potentials (ERPs) were recorded from 4-year-old 8-10-year-old children and adults to a schematic face, inverted face and jumbled face. The subjects were instructed to fixate the stimuli and no other response was required. The schematic face and inverted face were shown with a frequency of 20% each and the remaining presentations (60%) were of the jumbled face. P1 and N170 peak latency were measurable in the children and adults. These peaks were at longer latencies in the children. P3 was measurable in the adults and 8-10-year-old children but not the 4-year-olds. The adults had larger and longer latency P1 and smaller amplitude N170 to the inverted face than the other faces. In contrast, the P1 was unaffected by inversion in the children and the N170 was not smaller to the inverted or jumbled face. It is concluded that this result reflects developmental differences in the processing of configuration, with the children relying on an under-specified configuration of the face.