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Aims Cerebral amyloid angiopathy ( CAA) is a key pathological hallmark of Alzheimer's disease ( AD) characterized by accumulation of amyloid-beta ( Aβ) protein in blood vessel walls. CAA impairs vessel functioning, affects blood brain barrier integrity and accelerates cognitive decline of AD patients. Unfortunately, mechanisms underlying Aβ deposition in the vessel wall remain largely unknown. Factor XIIIa ( FXIIIa) is a blood-derived transglutaminase crucial in blood coagulation by cross-linking fibrin molecules. Evidence is mounting that blood-derived factors are present in CAA and may play a role in protein deposition in the vessel wall. We therefore investigated whether FXIIIa is present in CAA and if FXIIIa cross-link activity affects Aβ aggregation. Methods Using immunohistochemistry, we investigated the distribution of FXIIIa, its activator thrombin and in situ FXIIIa activity in CAA in post-mortem AD tissue. We used surface plasmon resonance and Western blot analysis to study binding of FXIIIa to Aβ and the formation of FXIIIa- Aβ complexes, respectively. In addition, we studied cytotoxicity of FXIIIa- Aβ complexes to cerebrovascular cells. Results FXIIIa, thrombin and in situ FXIIIa activity colocalize with the Aβ deposition in CAA. Furthermore, FXIIIa binds to Aβ with a higher binding affinity for Aβ1-42 compared with Aβ1-40. Moreover, highly stable FXIIIa- Aβ complexes are formed independently of FXIIIa cross-linking activity that protected cerebrovascular cells from Aβ-induced toxicity in vitro. Conclusions Our data showed that FXIIIa colocalizes with Aβ in CAA and that FXIIIa forms unique protein complexes with Aβ that might play an important role in Aβ deposition and persistence in the vessel wall. [ABSTRACT FROM AUTHOR]

The effects of antigenic stimulation on the expansion of T-cell dependent areas in lymph nodes of mice were studied in relation to the effects on high endothelial venules (HEV) located in this area. Lymph nodes regional to areas of skin that had been treated with solutions of oxazolone were studied at several time-points after stimulation. The following measurements were made relative to lymph nodes of untreated animals: (i) the expansion of the T-cell dependent area in combination with the increase of HEV in this area, as detected by the HEV-specific mAb MECA-325, using morphometric analysis; (ii) the influx of FITC-labelled lymphocytes from the blood into the lymph node by FACS; (iii) the capacity of HEV to bind lymphocytes using an in vitro binding assay. Morphometry showed that T-cell dependent areas increased rapidly after stimulation with oxazolone and although the mean area of MECA-325-positive HEV had also increased, this increase lagged behind the expansion of the T-cell area. Therefore the amount of HEV per T-cell area in an antigen-stimulated lymph node was smaller than in an untreated lymph node and correlated with the percentage of FITC-labelled cells that had entered it. In a lymph node from an oxazolone-treated animal this percentage was decreased to the same order of magnitude as the area of HEV per T-cell area, but the overall binding capacity of HEV was not affected by oxazolone treatment. Antigenic stimulation therefore leads to a rapid expansion of the potential sites of lymphocyte entry into a lymph node, but the efficiency of the HEV does not change. [ABSTRACT FROM AUTHOR]

The oral mucosa is an important site to induce immunological tolerance to protein antigens. Previously we have established that oral contacts to allergen can lead to systemic tolerance in both humans and experimental animals. Because of the importance of tolerance induction as a possible way to modulate allergic reactivity, we wished to study the mechanisms involved in efficient tolerance induction via the oral mucosa. Dendritic Langerhans' cells in both skin and oral epithelium are the first cells to encounter antigen. Therefore, possible functional differences between Langerhans' cells from skin and oral mucosa were studied by migration and transfer experiments. It was found that dendritic cells derived from the oral mucosa were not able to transfer tolerance, but that they acted as antigen-presenting cells in sensu stricto irrespective of the source and route of antigen administration. [ABSTRACT FROM AUTHOR]

The role of alveolar macrophages in the pulmonary immune response against various antigens was studied after elimination of alveolar macrophages by intratracheal administration of liposomeencapsulated dichloromethylene diphosphanate. When the responses against T-cell-independent type 1 and type 2 antigens were compared, it was found that elimination of alveolar macrophages had no effect on T-cell-independent antigens. Intratracheal antigen administration resulted in Iow lung associated, local responses, although some response was observed in the spleen. In contrast, elimination of alveolar macrophages resulted in an increase in local pulmonary immune response against T-cell-dependent antigens. We conclude from these experiments that alveolar macrophages play an important role in controlling the local pulmonary immune response against T-cell-dependent antigens by down-regulation of local T-cell populations. The alveolar macrophages do not downregulate the response against intratracheally administered T-cell-independent antigens, although they are important in the protection against inflammatory damage caused by bacterial endotoxins. [ABSTRACT FROM AUTHOR]

Cerebral amyloid angiopathy (CAA) is a pathological hallmark of Alzheimer's disease (AD) and characterized by deposition of amyloid-β (Aβ) protein and smooth muscle cell (SMC) death in cerebral vessel walls. Apolipoprotein E (ApoE) is of importance in both Aβ accumulation and Aβ-mediated toxicity towards SMCs in the cerebral vessel wall, although its exact role in CAA pathogenesis remains unclear. Tissue transglutaminase (tTG) is an enzyme capable of inducing both protein complexes and altered protein bioactivity via post-translational cross-linking. In CAA, tTG and its catalytic activity are associated with deposited Aβ. Furthermore, several apolipoproteins are known substrates of tTG. We therefore investigated whether ApoE is a substrate for tTG and if this affects ApoE's bioactivity. We found strong binding of different ApoE isoforms with tTG and demonstrated tTG-catalysed ApoE multimers. In post-mortem human AD cases, ApoE colocalized with in situ active tTG in CAA. Moreover, human brain SMCs treated with Aβ demonstrated enhanced secretion of both ApoE and tTG, and of TG cross-links in the extracellular matrix. Interestingly, tTG-catalysed cross-linked ApoE failed to protect SMCs against Aβ-mediated cytotoxicity. Together, our data demonstrate a novel tTG-driven post-translational modification of ApoE that might play an important role in CAA. Cerebral amyloid angiopathy (CAA) is a pathological hallmark of Alzheimer's disease (AD) and characterized by amyloid-β (Aβ) protein deposition and cerebral smooth muscle cell (SMC) death. We found that, in contrast to normal vessels, in CAA apolipoprotein E (ApoE) is cross-linked by tissue transglutaminase (tTG) resulting in stable ApoE complexes. These complexes no longer protect cerebral SMC from Aβ-mediated toxicity. Our findings demonstrate a novel mechanism explaining the Aβ-mediated cerebral SMC cell death characteristic of CAA in AD cases., (© 2015 International Society for Neurochemistry.)

Alzheimer's disease (AD), progressive supranuclear palsy (PSP), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), and Pick's disease (PiD) are commonly known as tauopathies. Neurodegeneration observed in these diseases is linked to neuronal fibrillary hyperphosphorylated tau protein inclusions. Transglutaminases (TGs) are inducible enzymes, capable of modifying conformational and/or structural properties of proteins by inducing molecular cross-links. Both transglutaminase 1 (TG1) and transglutaminase 2 (TG2) are abundantly expressed in the brain and are associated with fibrillary hyperphosphorylated tau protein inclusions in neurons of AD, so-called neurofibrillary tangles (NFTs). However, other data obtained by our group suggested that tau pathology in the brain may be primarily related to TG1 and not to TG2 activity. To obtain more information on this issue, we set out to investigate the association of TG1, TG2, and TG-catalysed cross-links with fibrillary hyperphosphorylated tau inclusions in tauopathies other than AD, using immunohistochemistry. We found strong TG1 and TG-catalysed cross-link staining in neuronal tau inclusions characteristic of PSP, FTDP-17 with mutations in the tau gene (FTDP-17T), and PiD brain, whereas, in contrast to AD, TG2 was only rarely observed in these inclusions. Furthermore, using a biochemical approach, we demonstrated that tau is a substrate for TG1-mediated cross-linking. Interestingly, we found co-localization of the TG1 activator, tazarotene-induced gene 3 (TIG3), in the neuronal tau inclusions of PSP, FTDP-17T, and PiD, but not in NFTs of AD cases, indicating that these tau-containing protein aggregates are not identical. We conclude that TG1-catalysed cross-linking, regulated by TIG3, might play an important role in the formation of neuronal tau inclusions in PSP, FTDP-17T, and PiD brain., (Copyright © 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Pro-inflammatory cytokines, e.g. interleukin-1beta (IL-1beta), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNFalpha) as well as neurotoxic molecules such as nitric oxide (NO), that are produced and released by activated glial cells, play an important role in inflammation and oxidative stress occurring during Multiple Sclerosis (MS). Reduction of these processes could therefore be of therapeutic interest. Dimethylfumarate (DMF) and sulforaphane (SP) are well known for their detoxicating properties. Furthermore, they have anti-inflammatory effects as shown clinically by the treatment of inflammatory skin diseases. However, their detoxication and anti-inflammatory action on brain-derived cells is unknown. In the present study we have studied, within the same concentration range, the anti-inflammatory and detoxicating effects of DMF and SP on the production and release of mediators of inflammation and detoxication from lipopolysaccharide (LPS) activated primary co-cultures of rat microglial and astroglial cells. DMF and SP attenuated the LPS-induced production and release of TNFalpha, IL-1beta, IL-6 and NO. In addition, DMF and SP increase both mRNA level and activity of NAD(P)H:quinone reductase (NQO-1), a detoxication enzyme, as well as the cellular glutathione content. We conclude that DMF or SP simultaneously can (1) reduce mediators of inflammation and (2) enhance detoxication enzymes in LPS stimulated co-cultures of astroglial and microglial cells. This double-sided effect could potentially be of therapeutic interest.

The pro-inflammatory cytokines interleukin-1beta (IL-1beta), IL-6, tumor necrosis factor-alpha (TNF-alpha), and nitric oxide (NO) can be produced by activated glial cells and play a critical role in various neurological diseases. Using primary co-cultures of rat microglial and astroglial cells, we investigated the effects of the anti-inflammatory cytokines transforming growth factor-beta1 (TGF-beta1)/beta2, IL-4, and IL-10 on the production of (pro-) inflammatory mediators after stimulation of the cells with lipopolysaccharide (LPS; 0.1 micrograms/ml, 24 h). IL-10 (10 and 100 ng/ml) and IL-4 (5 and 50 U/ml) suppressed the LPS-induced production of NO, IL-6, and TNF-alpha in a dose-dependent manner, whereas TGF-beta1/beta2 (2 and 20 ng/ml) only suppressed NO production. LPS-induced levels of IL-1beta were suppressed by IL-10, but not by IL-4 and TGF-beta1/beta2. Conversely, co-incubation of the glial cells with LPS and antibodies to TGF-beta1/beta2 selectively enhanced LPS-induced NO production, whereas co-incubation with antibody to IL-10 enhanced LPS-induced production of all pro-inflammatory cytokines and NO. This finding strongly suggests that effective concentrations of TGF-beta1/beta2 and IL-10 are produced by LPS-stimulated glial cell co-cultures. Production of IL-10 in these co-cultures was confirmed by measurement of rat IL-10 by radioimmunoassay. We conclude that anti-inflammatory cytokines affect the production of inflammatory mediators in LPS-activated co-cultures of microglial and astroglial cells differentially., (Copyright 2000 Wiley-Liss, Inc.)

Application of the fluorescent contact sensitizer Rhodamin B on mouse epidermis was used to study the migration kinetics of Langerhans cells into the draining lymph nodes. The expression of the dendritic cell markers NLDC-145 and MIDC-8 was followed over time to determine the correlation between these markers and Langerhans cell migration. In contrast with its high expression on intraepidermal Langerhans cells, the expression of NLDC-145 on dendritic cells in the draining lymph node was low at 24 h but increased at later times; in contrast, MIDC-8 expression on dendritic cells decreased. Ten days after Rhodamin B application, antigen-bearing Langerhans cells were still present in the epidermis; application of another unrelated contact sensitizer to the epidermis at this time did not lead to migration of these residual Langerhans cells. These results indicate that not all antigen-bearing Langerhans cells migrate from the skin after application of a contact sensitizer, indicating that signals in addition to simple antigen binding are necessary for migration. During this migration from epidermis to lymph nodes Langerhans cells undergo phenotypic changes. The decreased expression of the endosomal antigens MIDC-8 and MOMA-2 correlates with differentiation from predominantly antigen-processing cells to predominantly antigen-presenting cells. The reduced expression of NLDC-145 is discussed in light of a Langerhans cell-independent pathway of antigen transportation from skin to lymph node.

The role of carbohydrate structures in the interaction of lymphocytes and endothelial cells is well established. Here the influence of sialic acid in the entrance and localization of lymphocytes in the lymphoid white pulp area of the spleen was studied by injecting sialidase in vivo. A role for sialic acid molecules on stromal elements of the spleen was determined. Although the identity of the cells that bear sialidase sensitive receptors could not be established, a role for marginal zone macrophages could be ruled out by macrophage depletion studies.

Tissue selective traffic of lymphocytes into different lymphoid organs is mediated by adherence of blood borne lymphocytes to specialized endothelial cells lining the high endothelial venules (HEV) in lymphoid organs. Lymphocytes discriminate between HEV in peripheral lymph nodes and in mucosal lymphoid tissues by means of membrane associated lymphocyte homing receptors adhering to their putative HEV ligands, the vascular addressins. The expression of particular vascular addressins on HEV is site- or tissue-selective and may be directed by factors unique to a specific location or lymph node environment. In this study we investigated the impact of regional environments on lymph node HEV differentiation and function. Experimentally, this problem was approached by the transplantation of lymph nodes from one region to a second region. The sites selected for receipt of tissues were the mesentery, a mucosal site, and the popliteal fossa, a peripheral site. We found that the phenotype of lymph node HEV following transplantation was influenced by both donor age and transplantation site. The transplantation site could influence vascular addressin expression, when tissues were obtained from late fetal or early neonatal donors and not when obtained from adult donors. Transplanted adult tissues retained their pre-transplantation vascular addressin expression phenotype regardless of transplantation site. Thus the endothelium within adult lymph nodes may be committed to expression of a particular addressin or addressins during lymph node development. It is also possible that regulatory cells or structures present within lymph nodes at the time of transplantation direct vascular addressin expression following tissue engraftment.(ABSTRACT TRUNCATED AT 250 WORDS)

Langerhans cells can be induced to migrate into draining lymph nodes after topical application of antigen. During this migration the cells undergo phenotypical changes which are directly related to the function they exert at the different locations. Here the results of kinetic studies as well as phenotypic characterization of Langerhans cells during migration are described and discussed in view of the apparent differences between Langerhans cells in the skin and dendritic cells in lymph nodes.

In this report we studied the translocation of fluorescent particulate antigens to the draining lymph node, and the migration of fluorescent labeled alveolar macrophages (AM) and peritoneal macrophages (PM) in mice. The results show that intratracheally (IT) instilled particulate antigens translocate to the paracortical T cell area of the draining lymph node. When labeled AM were injected IT, they were found to migrate from the alveolar space into the paracortical T cell area of the draining lymph node. An identical localisation was found after IT injection of labeled PM. When either labeled AM, or PM were injected into the peritoneal cavity, a different migration pattern was observed. Via this route the labeled macrophages migrated to the subcapsular sinus and medulla of the draining lymph nodes. It is shown that the migrated cells are not dendritic cells (DC) present in the cell preparations. A possible role for the micro-environment of the injection site, and the significance of the specific migration pattern of AM is discussed.

Popliteal lymph nodes (LN) of mice were depleted of their macrophage (M phi) populations in the subcapsular sinus and medulla by subcutaneous injection of dichloromethylene diphosphonate (Cl2MDP)-containing liposomes into the footpads. Complete restoration of both M phi populations could be observed as late as 5 months after liposome administration. This relatively long repopulation time could be due to a depot of liposomes, directly killing all M phi precursors after extravasation into the interstitial tissue of the footpad. On the other hand, local interstitial precursors with very low turnover rates may have been depleted in the interstitial tissue of the hind leg. Therefore, two different types of experiments were performed; one in which M phi-depleted LN were replaced by control LN at various time points after liposome treatment, and another whereby M phi-depleted LN were transplanted into control animals. When liposome-treated, M phi-depleted LN were transplanted into control animals, a complete restoration of both populations in the subcapsular sinus and medulla could be observed within 5 weeks. Control LN transplanted into a Cl2MDP-liposome-treated leg showed a rapid disappearance of M phi from the subcapsular sinus and medulla and these cell populations remained absent for at least 7-8 weeks after liposome treatment, when the first cells started to reappear. Complete repopulation of these areas by M phi took as long as 15 weeks. Using labeled liposomes the presence of a continuous liposome depot was found to be very unlikely. These results suggest that the population of precursor cells that will give rise to M phi in the subcapsular sinus and medulla of a LN is probably contained within the interstitial tissue and is almost independent of precursor supply from the blood compartment.

Adherence of lymphocytes to high endothelial venules (HEV3) is the first step in normal lymphocyte emigration and recirculation. At sites of chronic inflammation, venules often become high-walled and may also be a site for leukocytes to leave the bloodstream. The immunologic and inflammatory mediators, responsible for these effects on endothelial cells, may be important for the maintenance and function of HEV in physiological conditions. It is reported here that the morphological and functional aspects of HEV can be studied by organ cultures of lymph nodes (LN). At 24 h of culture, the appearance of the node was still quite normal, whereas the HEV became flat-walled, with a 45-50% reduction in the capacity to bind lymphocytes. This decrease in function of HEV could be reduced when LN were cultured in the presence of lipopolysaccharides (LPS). The effect of LPS on the function of HEV was presumably mediated by macrophages in the subcapsular sinus, because HEV in LN, which were depleted of subcapsular sinus and medullary macrophages previous to culture, could not be stimulated by addition of LPS to the cultures.

Interruption of the afferent lymphatic vessels of rat popliteal lymph nodes led to the disappearance of the monoclonal antibody ED3-reactive subcapsular sinus macrophages within 3 weeks, but had no effect on the ED1+ macrophages in the paracortical area. This disconnection of the afferent lymph flow to the popliteal lymph node also reduced the capacity of high endothelial venules (HEV) to bind lymphocytes and led to a flattening of the HEV. Activating factors or cells in the incoming lymph might be responsible for the maintenance and function of several different cell populations and we therefore wished to determine if the effects of interruption could be restored by injection of recombinant rat interferon-gamma (rIFN-gamma). Injection of rIFN-gamma directly into operated lymph nodes could mediate an apparent increase of ED1+ cells within 24 h but rIFN-gamma could not restore the macrophage subpopulation in the subcapsular sinus, as recognized by monoclonal antibody ED3. Restoration of the decreased binding capacity of HEV could not be observed with the doses and time points tested, suggesting that HEV are a distinct type of endothelium.

The expression of the low molecular weight cytokeratins (K) 7, 8, 18, 19 and the high molecular weight cytokeratin 10 in 21 basal cell carcinoma (BCC) was studied using seven different monoclonal antibodies (MoAbs) with specific anti-cytokeratin activity. MoAbs RCK 105 (anti-K7), RPN 1164 (anti-low molecular weight cytokeratins of basic group), Ks 19.1 (anti-K19) and Cam 5.2 (anti-K8, K18, K19) reacted positively but inconsistently in the BCC that were examined. MoAbs 1166 (anti-K8) and RGE53 (anti-K18) did not react at all. MoAb RKSE 60 (anti-K10) did not react with the tumor cells. From the results of this study, it can be concluded that cytokeratins 7 and 19 are expressed in BCC (43% and 71%, respectively), whereas cytokeratin 8 is not expressed.

We have developed a panel of six monoclonal antibodies, ED10-ED15, directed against reticular cells in peripheral lymphoid organs. Immunohistochemistry revealed prominent differences between these antibodies with regard to their tissue distribution in lymphoid and non-lymphoid organs. Furthermore, the determinants recognized by ED10-ED13 were found to be differentially expressed by reticular cells occupying the various specialized compartments present in peripheral lymphoid organs. The reactivity patterns of these antibodies observed during the ontogenetic development of the spleen suggest that they recognize differentiation antigens expressed by reticular cells. In contrast, ED14 and ED15 were found to have a relatively ubiquitous tissue distribution recognizing reticular cells in each compartment, with a constitutive reactivity during splenic ontogeny. The present results indicate that reticular cells form a heterogeneous population within the lymphoid organs.

Ultrastructural investigations into the location and the expression of the cytokeratins 7, 8, 10 and 19 were undertaken on ultrathin cryosections of 8 basal cell carcinomas (BCC) using a high resolution immunogold labeling method and five different monoclonal antibodies against specific cytokeratins. The results showed that cytokeratin 10 was expressed only in the differentiated keratinocytes. In contrast to the previously reported biochemical and immunohistochemical studies, in this study cytokeratin 19 was expressed not only in the tumor cells of BCC but also in the normal epidermal keratinocytes. The expression of cytokeratin 7 in BCC could not be confirmed but the lack of expression of cytokeratin 8 was confirmed, excluding its potential role as a specific histopathological marker for BCC.