ALS phenotypes with mutations in CHMP2B (charged multivesicular body protein 2B).

Mutation in the CHMP2B gene has been implicated in frontotemporal dementia. The authors screened CHMP2B in patients with ALS and several cohorts of control samples. They identified mutations (Q206H; I29V) in two patients with non-SOD1 ALS. Neuropathology of the Q206H case showed lower motor neuron predominant disease with ubiquitylated inclusions in motor neurons. Antibodies to p62 (sequestosome 1) showed novel oligodendroglial inclusions in the motor cortex.

Non-bronchoscopic sampling and culture of bronchial epithelial cells in children.

BACKGROUND: The bronchial epithelium is likely to play a vital role in airway diseases in children, such as asthma and viral-associated wheeze. In adults, studies with primary bronchial epithelial cells cultured from samples obtained by fibre-optic bronchoscopy have provided key insights into the role of the epithelial cell. However, it is difficult to justify bronchoscopy in children to obtain epithelial cells for research purposes. OBJECTIVE: To examine the possibility of retrieving and culturing viable epithelial cells using a blind non-bronchoscopic method from children undergoing elective surgery. METHODS: Subjects were children undergoing elective surgery under general anaesthesia. Following intubation, non-bronchoscopic bronchoalveolar lavage and non-bronchoscopic bronchial brushing were performed. A sheathed bronchial cytology brush was advanced through the endotracheal tube, wedged and then withdrawn 2-3 cm before gentle sampling was used to collect bronchial epithelial cells. Initial samples were used to characterize the number, type and viability of epithelial cells recovered compared to a control group of adults undergoing standard bronchoscopic sampling. Subsequent samples were used to establish primary bronchial epithelial cell cultures in children both with and without wheezing illness. RESULTS: A total of 63 children underwent bronchial brushing [38 male; median age 7.1 years (1.0-14.2 years]. Initial samples (n=30) showed recovery of viable epithelial cells comparable to that from a single brush obtained via a bronchoscope in an adult control group (n=11). In 27 (82%) of the subsequent 33 samples obtained non-bronchoscopically from children, primary bronchial epithelial cell cultures were successfully established. There were no adverse effects attributable to sampling. CONCLUSION: We have shown that non-bronchoscopic bronchial brushing is a safe and effective technique for recovering viable bronchial epithelial cells that consistently yield primary cultures. This method will facilitate examination of the role of the epithelium in paediatric disease.

Hepatocyte growth factor (HGF) protects c-met-expressing Burkitt's lymphoma cell lines from apoptotic death induced by DNA damaging agents.

The relative sensitivity of neoplastic cells to DNA damaging agents is a key factor in cancer therapy. In this paper, we show that pretreatment of Burkitt's lymphoma cell lines expressing the c-met protooncogene with hepatocyte growth factor (HGF) protects them from death induced by DNA damaging agents commonly used in tumour therapy. This protection was observed in assays based on morphological assessment of apoptotic cells and DNA fragmentation assays. The protection was dose- and time-dependent -- maximal protection requiring pre-incubation with 100 ng/ml HGF for 48 h. Western blotting analysis and flow cytometric studies revealed that HGF inhibited doxorubicin- and etoposide-induced decreases in the levels of the anti-apoptotic proteins Bcl-X(L), and to a lesser extent Bcl-2, without inducing changes in the pro-apoptotic Bax protein. Overall, these studies suggest that the accumulation of HGF within the microenvironment of neoplastic cells may contribute to the development of a chemoresistant phenotype.

The role of hepatocyte growth factor and its receptor c-met in interactions between lymphocytes and stromal cells in secondary human lymphoid organs.

Secondary lymphoid tissue consists of two major populations of cells: lymphoid cells and stromal cells. It is generally accepted that these two cell populations influence each other however, factors mediating these processes are poorly understood. In this paper we characterize one of the possible means of communication between stroma and lymphocytes namely through hepatocyte growth factor/c-met receptor interactions. Hepatocyte growth factor (HGF) is a pleiotropic factor that is mainly produced by mesenchymal cells and acts on cells of epithelial origin which express the HGF receptor c-met. Here we demonstrate that biologically active HGF is constitutively produced by fibroblast-like stromal cells from human lymphoid tissues. HGF secretion from stromal cells was increased by direct contact with activated T cells. This increase was abrogated when activated T cells were separated physically from stromal cells. Using neutralizing antibody or cytokine inhibitors we provide evidence that enhancement of HGF production was due to additive effects of T-cell membrane-associated interleukin-1 (IL-1) and CD40 ligand. Finally, we also show that B lymphocytes activated with CD40L/anti-mu or phorbol 12-myristate 13-acetate (PMA) express c-met receptor. Co-culture of activated B cells with stromal cells from spleen leads to enhanced production of immunoglobulins. This can be partially inhibited by introduction of anti-HGF neutralizing antibodies to the culture system. Substitution of stromal cells with recombinant HGF did not produce enhancement of immunoglobulin secretion. On the other hand stimulation of c-met receptor with HGF leads to enhanced integrin-mediated adhesion of activated B cells to vascular cell adhesion molecule (VCAM-1) and fibronectin. On the basis of the above experiments we conclude that HGF production by fibroblast-like stromal cells can be modulated by activated T cells, thus providing signals for the regulation of adhesion of c-met expressing B cells to extracellular matrix proteins. In this way HGF may indirectly influence immunoglobulin secretion by B cells.

Preliminary studies on the effect of dehydroepiandrosterone (DHEA) on both constitutive and phytohaemagglutinin (PHA)-inducible IL-6 and IL-2 mRNA expression and cytokine production in human spleen mononuclear cell suspensions in vitro.

In order to gain further insight into the potential immunological benefits of oral administration of DHEA we have examined its effects on the constitutive and PHA-inducible expression by human spleen cell suspensions in vitro of IL-6 and IL-2. This was studied at both the mRNA and protein levels. The quantification of specific mRNA was undertaken using commercially available quantitative polymerase chain reaction kits. These studies, which were performed on suspensions from six individual spleens, revealed that 10(-5) M DHEA did not impair the expression of IL-6 at either the mRNA or protein level, but may have slightly enhanced the latter. In contrast, IL-2 mRNA levels were increased on most occasions, whilst IL-2 secretion was decreased, albeit slightly. Additional studies revealed that cyclosporin (approx. 10(-5) M) and dexamethasone (10(-7) M) readily inhibited these responses and the production of other cytokines, including interferon-gamma and tumour necrosis factor-alpha. These preliminary studies suggest that high doses of DHEA do not readily inhibit the production of IL-6, and indeed other cytokines, by PHA-stimulated secondary human lymphoid tissue suspensions in vitro. They may also partially explain the meagre immunomodulatory effects noted in some DHEA replacement studies in humans.

Disruption of auxin transport is associated with aberrant leaf development in maize

Despite recent progress, the mechanisms governing shoot morphogenesis in higher plants are only partially understood. Classical physiological studies have suggested that gradients of the plant growth regulator auxin may play a role in controlling tissue differentiation in shoots. More recent molecular genetic studies have also identified knotted1 like homeobox (knox) genes as important regulators of shoot development. The maize (Zea mays L.) mutant rough sheath2 (rs2) displays ectopic expression of at least three knox genes and consequently conditions a range of shoot and leaf phenotypes, including aberrant vascular development, ligular displacements, and dwarfism (R. Schneeberger, M. Tsiantis, M. Freeling, J.A. Langdale [1998] Development 125: 2857-2865). In this report, we show that rs2 mutants also display decreased polar auxin transport in the shoot. We also demonstrate that germination of wild-type maize seedlings on agents known to inhibit polar auxin transport mimics aspects of the rs2 mutant phenotype. The phenotype elaborated in inhibitor-treated plants is not correlated with ectopic KNOX protein accumulation.

The influence of age and gender on serum dehydroepiandrosterone sulphate (DHEA-S), IL-6, IL-6 soluble receptor (IL-6 sR) and transforming growth factor beta 1 (TGF-beta1) levels in normal healthy blood donors.

Dysregulation of IL-6 synthesis is thought to play a role in the development of a number of age-related conditions, such as rheumatoid arthritis, osteoporosis, atherosclerosis, Alzheimer's disease and B cell malignancies. Recently it has been suggested that the production of IL-6 is influenced by the adrenal hormone dehydroepiandrosterone (DHEA) and its sulphated derivative DHEA-S. In humans we investigated the relationship between DHEA-S, IL-6, IL-6 sR and TGF-beta1 in the serum of normal healthy male and female blood donors. Using immunoassay techniques we found that the serum levels of DHEA-S significantly (P = 0.0001) decreased with age in both males and females. Furthermore, mean DHEA-S levels in all age groups were significantly (P = 0.0001) higher in males. Such correlations were not apparent for IL-6 using a standard assay, but a high sensitivity assay revealed that serum IL-6 was significantly (P = 0.0018) positively correlated with age in males only. In addition, serum levels of DHEA-S were significantly (P = 0.048) negatively correlated with serum IL-6, again in male subjects only. In contrast, serum IL-6 sR and TGF-beta1 levels were not correlated with age in either males or females and were not significantly different between the sexes. However, a significant (P = 0.024) negative correlation between DHEA-S and IL-6 sR was found in males. These studies clearly highlight the complex nature of the relationship between these molecules in the ageing process in normal healthy blood donors and demonstrate the need to use high sensitivity assays when measuring IL-6 in apparently healthy individuals under the age of 70 years.

Enhancement of terminal B lymphocyte differentiation in vitro by fibroblast-like stromal cells from human spleen.

Stromal elements are major components of lymphoid tissues contributing to both tissue architecture and function. In this study we report on the phenotype and function of fibroblast-like stromal cells obtained from human spleen. These cells express high levels of CD44 and ICAM-1 and moderate levels of VLA-4, VCAM, CD40 and CD21. They fail to express endothelial, epithelial, lymphocyte and monocyte/macrophage markers. We show that these cells interact with B cell blasts induced in vitro by anti-CD40 and anti-mu stimulation. As a result of these interactions both IL-6 and IgG secretion into culture medium is increased. The enhanced secretion of IgG is partly inhibited by abolishing B cell blaststromal cell contact or by anti-IL-6, anti-VCAM or anti-CD49d antibodies. Our studies also suggest that the ability of stromal cells to promote B cell survival is most likely the underlying mechanism of the enhanced immunoglobulin secretion. Comparison of stromal cells from different lymphoid and non-lymphoid organs revealed that bone marrow- and spleen-derived stromal cells are the most effective in promoting B cell blast differentiation.

Tonsil stromal-cell lines expressing FDC-like properties: isolation, characterization, and interaction with B lymphocytes.

The microenvironment of secondary lymphoid organs consists of two major populations of cells, the lymphoid cells and a population of stromal cells that contribute to both tissue architecture and function. Interactions of both populations are essential for the development and control of humoral immune responses. In this study, stromal-cell preparations were obtained by a multistage process. This involved culturing 300-400-microm slices of human tonsil for 6-8 days at 25 degrees C, trypsin digestion of the residual explant, followed by CD45-positive-cell depletion using magnetic beads, and a final period of culture for 4 days to remove remaining nonadherent cells. Phenotyping with a panel of monoclonal antibodies revealed that the cells express HLA-DR, CD54 (ICAM-1), CD44, but no CD45 nor a range of other markers for epithelial and endothelial cells. Immunoassays of supernatants from stromal cells revealed that IL-6 was produced constitutively, and its production was increased by treatment with TNF-alpha and IFN-gamma. In contrast IL-1, IL-2, IL-4, IL-7, IL-8, IL-10, IL-12, TNF-alpha, and IFNgamma were not produced. Functional tests showed that these cells express follicular dendritic cell-like properties. Coculturing of tonsilar B cells with stromal cells resulted in enhanced proliferation and also led to increased production of immunoglobulins and IL-6, suggesting crucial signaling between these populations.

Organ culture of human lymphoid tissue. II. Marked differences in cytokine production and proliferation between slice and suspension cultures of human spleen.

Recently, we have established a method for the culture of human spleen slices in vitro. The procedure allows thin slices (200-350 microns) of human spleen to be cultured for up to 7 days. Using this method, we have previously established that unstimulated spleen slices spontaneously synthesize and secrete considerably higher levels of immunoglobulin than suspension cultures of the same tissue run in parallel. In this study, we report that there are also marked differences in the cytokine secretion profile between slices and suspensions and in their proliferative response. In brief, control and PHA-stimulated spleen slices secrete high levels of IL-1 beta, IL-6, IL-8 and IL-11 while the levels found in suspension supernatants are appreciably lower. By way of contrast, high levels of IL-2, IL-4, IL-10 and TNF alpha are found in suspension culture supernatants following PHA stimulation while the response in slice cultures is extremely low. These differences are also reflected in the results obtained at the cellular (intracellular cytokine) level. Additional studies reveal that spontaneous immunoglobulin production observed in spleen slices can be inhibited by the addition of specific antibodies to IL-1 beta, IL-6 and TNF alpha and that the bulk of the IL-6 and IL-1 beta detected in culture supernatants represents de novo synthesis. Finally, the background and mitogen-stimulated proliferative response of tissue slices is meagre compared with that observed in spleen suspensions suggesting that proliferation in the former is held under strict control. Collectively, we believe that the tissue slice procedure described provides us with a system for studying integrated events in lymphoid tissues in vitro and evaluating immunomodulatory substances of potential clinical importance.

IL-6, DHEA and the ageing process.

The age-related increase in circulating IL-6 levels in humans which has been attributed to a decline in DHEA production by the adrenal gland is currently attracting attention because of its possible relevance to the aetiology and management of a number of age-related clinical disorders. The potential importance of these observations and suggestions has prompted us to perform more detailed studies on the relationship between IL-6 and DHEA. Using immunoassay techniques we have found in normal healthy individuals over the age of 40 an inverse relationship between plasma DHEA levels and the presence of detectable levels of IL-6 (more than 1 pg/ml). In vitro, studies also revealed that low dose (10(-6)-10(-8) M) of DHEA and DHEAS inhibited the production of IL-6 in unstimulated human spleen cell suspension cultures whilst enhancing its release by explant cultures of the same tissue. In contrast they had no effect on immunoglobulin production. These studies suggest that there is a real, but complex relationship between IL-6 production and DHEA levels which warrants further investigation.

The use of tissue slices in immunological investigations.

Our knowledge of cellular and molecular events taking place during various immune responses comes mainly from in vitro studies where isolated cells are exposed to defined stimuli. This reductionist approach has greatly advanced our understanding of the immune system. However, these studies do not truly reflect the complexity of the integrative events which take place in both primary and secondary lymphoid tissue in vivo. In order to address this problem we have developed a tissue culture procedure which involves the cultivating of precision cut human spleen slices at gas/liquid interface. We have shown, that cells in this culture system show marked differences in cytokine and immunoglobulin production in comparison with conventional single cell suspension cultures, obtained from the same spleen and run in parallel. In this review article we describe the basic technique, results which we have obtained in this system and discuss the possible basis of the observed differences.

An exploratory study of sex differences in orientation to clinical practice.

Few statistically significant sex differences were found in orientation to clinical practice of a randomly selected sample of 300 licensed clinical social workers. 28 male and 69 female licensed clinical social workers had very similar attitudes, philosophies, and behaviors toward clinical practice. These findings add to the limited research available.

The effects of human seminal plasma and PGE2 on mitogen induced proliferation and cytokine production of human splenic lymphocytes and peripheral blood mononuclear cells.

The effects of human seminal plasma (HSP) and prostaglandin E2 (PGE2) on the proliferative responses of human splenic lymphocytes and peripheral blood mononuclear cells (PBMCs) to phytohaemagglutinin (PHA), anti-CD3 and anti-CD3 plus anti-CD28 mAb have been studied. Th1 and Th2 cytokines were also measured in the supernatants of selected cultures. Both HSP and PGE2 reproducibly inhibit the proliferative response to PHA and anti-CD3 mAb in a dose dependent manner. These effects were observed with both fresh and frozen human PBMCs and splenic lymphocytes. HSP and PGE2 however were less effective in inhibiting the co-stimulatory response induced by anti-CD3 plus anti-CD28 mAb. In addition, the HSP and PGE2 treatment used inhibited the production of the Th1 cytokines IL-2 and IFNg but had a differential modulatory effect on Th2 cytokine production, namely enhancing the production of IL-6 whilst simultaneously impairing the synthesis of IL-4 and IL-10.

A comparison of the performance in vitro of precision cut tissue slices and suspensions of human spleen with special reference to immunoglobulin and cytokine production.

During the past decade our knowledge of the cellular and molecular events associated with key immunological responses has been greatly advanced by the use of isolated subpopulations of immunocompetent cells, cloned cell lines and recombinant derived cytokines. Valuable as these studies have been they do not truly reflect the complex integrative events which take place in both primary and secondary lymphoid tissue both in vivo and in vitro. In order to address this problem we have developed a tissue culture procedure which is a modification of that previously used by others to study T cell maturation in the thymus. This involves culturing precision cut slices of human lymphoid tissue in a sponge culture system. Using this technique we have observed marked differences in both immunoglobulin and cytokine secretion between slices and suspensions of human spleen. In brief, cultured slices (mitogen stimulated or otherwise) consistently secrete higher levels of immunoglobulin, IL-1 beta, IL-6, IL-8 and IL-11 and exhibit much lower proliferation than suspensions of the same tissue. Mitogen stimulated suspensions on the other hand secrete higher levels of IL-2, IL-4, IL-10 and TNF alpha than do slices. These differences are also observed at the intracellular cytokine level. Additional studies reveal that the immunoglobulin and cytokine secretion observed is largely due to the de novo synthesis of these molecules and not as a result of spontaneous secretion of preformed products. Furthermore immunoglobulin secretion in both slices and suspensions can be inhibited by the addition of specific antibodies to IL-1 beta, IL-6 and TNF alpha while IL-6 production can be differentially modulated by a variety of substances. Preliminary studies indicate that close interaction between B cells and stromal cells within explants accounts for some of the observed differences. This review article describes the basic technique, summarises the results we have obtained in this system and outlines the possible basis of the observed differences.

The influence of the family preservation model on child sexual abuse intervention strategies: changes in child welfare worker tasks.

There is no easy solution for child sexual abuse. Various innovations have been tried. Most are based on the belief that punishment without treatment is counterproductive in cases of intrafamilial child sexual abuse because it further disrupts the family. Many counties have developed strategies that divert offenders into treatment rather than prison. Professionals in those counties apparently believe that treatment with the threat of prosecution or imprisonment is faster, cheaper, less traumatic for the child, and more effective in reducing recidivism. Ideally, it will also help preserve families. To make such strategies work, child sexual abuse intervention professionals have adapted their activities through all phases of contact with the victim, offender, and the family. They must be collaborators, consultants, liaisons, counselors, and advocates. Finally, there is little research on the effectiveness of the innovative intervention strategies. To discern if those strategies are more effective than traditional approaches in reducing child sexual abuse, in helping the family to cope with the problem, and in minimizing system-induced trauma to the child, continued research is necessary.

Immunomodulatory effects of extracellular secretory vesicles isolated from bovine semen.

We demonstrate that extracellular secretory vesicles isolated from bovine seminal plasma have immunomodulatory properties. They inhibit mitogen induced proliferation of bovine and human peripheral blood lymphocytes in a dose dependent fashion. They also inhibit phagocytosis of latex particles by bovine neutrophils. Phagocytosis of opsonised Staphylococcus aureus however was not affected. Furthermore phorbol ester and chemotactic peptide induced superoxide production was decreased especially when a suboptimal dose of stimulants was used. We suggest that extracellular secretory vesicles may preserve sperm survival in the female reproductive tract.

Organ culture of human lymphoid tissue. I. Characteristics of the system.

The major aim of three-dimensional tissue culture is to preserve the natural architecture of the tissue and thereby allow the cells to retain their original functions during in vitro cultivation. Here we describe a method for the rapid preparation of three-dimensional tissue explants from human lymphoid organs. The precision-cut tissue slices are of uniform size and thickness and can be cryopreserved and stored in liquid nitrogen without substantial loss of viability or functionality of the cells. Upon in vitro culture, cells within the explants survived as well as their counterparts cultured in single cell suspension. However, spontaneous immunoglobulin (Ig) production in explants started more promptly and often reached considerably higher levels than that in suspension cultures run in parallel. Lymphocytes within the slices could be activated by polyclonal stimuli such as PHA, as shown by the upregulation of the activation markers CD23 and CD25 on B and T cells, respectively. However, approximately five-fold higher concentrations of mitogen than those used for suspension cultures were needed. Taken together, the system presented here constitutes a potent tool for the investigation of the complex interactions leading to activation and differentiation of B and T cells in lymphoid organs.

Expression of a common secretory granule specific protein as a marker for the extracellular organelles (prostasomes) in human semen.

OBJECTIVE: To further characterize prostasomes, trilamellar to multilamellar vesicles that are thought to originate from acinar cells of the human prostate and present in appreciable amounts in normal human semen. Purified prostasomes were shown to have immunosuppressive activity in vitro as measured by inhibition of mitogen-induced lymphocyte proliferation and inhibition of superoxide generation by neutrophils. A granule membrane protein, called granulophysin, has recently been identified in the membranes of platelet dense granules. Antibodies that recognize granulophysin also stain granules in different cell types including leukocytes, melanocytes, neurones, endothelial cells, and epithelial cells. DESIGN: The presence of epitopes recognized by antigranulophysin monoclonal antibody in prostasomes was investigated using indirect immunofluorescence and subsequent cytofluorimetric analysis. The protein was also analyzed by Western blotting. Reactivity of antigranulophysin antibody with the prostate tissue was studied by immunoperoxidase staining. RESULTS: A majority of prostasome particles specifically reacted with antigranulophysin antibody. In lysates prepared from prostasomes, a broad band of 32 to 37 kd was detected by Western blotting. CONCLUSION: This report defines granulophysin as a constituent membrane molecule of prostasomes that may serve as a useful marker in elucidation of prostasome function.