The testis is considered an immunologically privileged site where germ cell antigens are protected from autoimmune attack. Yet in response to infections, inflammatory diseases, or trauma, there is an influx of leukocytes to testicular interstitium. Interactions between endothelial cells (EC) and circulating leukocytes are implicated in the initiation and evolution of inflammatory processes. Chemokines are a family of chemoattractant cytokines characterized by their ability to both recruit and activate cells. Thus, we investigated the expression of CCL3, its receptors, and adhesion molecules CD31 and CD106 in an in vivo model of experimental autoimmune orchitis (EAO). In EAO, the highest content of CCL3 in testicular fluid coincides with onset of the disease. However, CCL3 released in vitro by testicular macrophages is higher during the immunization period. The specific chemokine receptors, CCR1 and CCR5, were expressed by testicular monocytes/macrophages and an increased number of CCR5+ cells was associated with the degree of testicular lesion. EC also play an essential role by facilitating leukocyte recruitment via their ability to express cell surface adhesion molecules that mediate interactions with leukocytes in the bloodstream. Rats with EAO showed a significant increase in the percentage of CD31+ EC that upregulate the expression of CD106. The percentage of leukocytes isolated from peripheral blood and lymph nodes expressing CD49d (CD106 ligand) also increases during orchitis. These data suggest that cell adhesion molecules, in conjunction with chemokines, contribute to the formation of a chemotactic gradient within the testis, causing the leukocyte infiltration characteristic of EAO histopathology.

Organ transplantation in older people is increasing, but how aging impacts B-cell responses to organ transplantation is still unknown. Here, we show that the depletion of B cells with anti-CD20 antibodies has disparate effects depending on recipient age. In young murine recipients, anti-CD20 treatment impaired the ability of immune modulation to extend skin allograft survival. In contrast, anti-CD20 treatment extended allograft survival in aged recipients treated with immune modulation. Although regulatory B-cell function and the numbers of marginal and follicular B cells were similar between age groups, a subpopulation of B cells, termed age-associated B cells (ABCs), accumulated upon aging. ABCs isolated from aged mice exhibited upregulation of CD73, CD80, CD106, and TLR2 and an increased capacity to augment T-cell alloimmunity compared to ABCs from young mice. Importantly, ABCs from aged, but not young, mice impaired the ability of immune modulation to enhance allograft survival after adoptive transfer into young transplant recipients. Our study indicates that ABCs impair the immune regulation of allografts. Thus, recipient age needs to be considered when proposing B-cell-depleting immune therapy.

A human monoclonal antibody (mAb), designated mNKES, was generated by fusing B cells isolated from an enlarged cervical lymph node of a patient with a carotid body tumor (CBT), with human myeloma cell line KR-12 (6TG). The reactivity of mNKES was tested by the indirect immunofluorescence method. The antigen defined by mNKES was expressed on Burkitt's lymphoma cell lines Raji, Daudi, and Ramos and on B lymphoblastoid cell line IM-9. In addition, mNKES reacted with T cells stimulated with recombinant interleukin-2 (rIL-2) obtained from normal healthy donors. However, mNKES did not react with normal resting human T, B, or adherent cells (monocytes/macrophages). When the reactivity of mNKES and mouse mAbs recognizing the human adhesion-associated antigen (CD10, CD11a, CD11b, CD11c, CD14, CD16, CD18, CD23, CD28, CD29, CD31, CD43, CD44, CD45RA, CD50, CD54, CD58, CD80, CD102, CD106, and HLA class I, and HLA class II antigen) with various cell lines was compared, mNKES reactivity was found to be unique, not resembling that of any of these mouse mAbs. Interestingly, mNKES specifically and rapidly (within 2 hr) induced homotypic cell aggregation of IM-9 cells. This mNKES-induced cell aggregation was completely blocked by the addition of EDTA and when incubated at 4 degrees C. The mAbs reactive with CD11a/CD18 (leukocyte function-associated antigen-1; LFA-1) and CD54 (intercellular adhesion molecule-1; ICAM-1) completely blocked the IM-9 cell aggregation induced by mNKES, and induction of IM-9 cell aggregation by mNKES was significantly blocked in the presence of the protein kinase C inhibitors sphingosine and H-7 and completely blocked by cytochalasin B and cytochalasin D, which inhibit microfilament formation. Regarding biological function, IM-9 cells bearing surface IgG (sIgG) effectively promoted IgG-secreting activity underlying the homotypic cell aggregation induced by mNKES. The surface antigen recognized by mNKES has a molecular size of about 55 kDa, as determined by immunoblotting analysis. These findings indicate that mNKES recognizes a novel adhesion-associated antigen distinct from any previously reported adhesion-associated antigens in terms of pattern of cellular distribution and biological function and that mNKES is the first human mAb found that rapidly induces homotypic cell aggregation and effectively promotes the IgG-secreting activity of human B lymphoblastoid cell line IM-9.

Using the model of hypogonadism in C57Bl/6 male mice, we showed that injection of streptozotocin to newborn animals and high-fat diet induced serum IFN-γ and IL-17 elevation, glucose metabolism disturbances, insulin resistance, destructive changes of the Langerhans islets (deficit of PDX1+β cells), while the number of oligopotent β cell precursors (CD45-TER119-CD133+CD49flow) increased. Diabetes played the role of an inducer of testicular tissue inflammation (pan-hemopoietic cell infiltration, increase of IL-2, IL-17, and IL-23 content) and reproductive system disturbances in mice (decrease in free testosterone concentration, suppression of spermatogenesis, and infertility). The development of hypogonadism was paralleled by an increase in the count of spermatogonial stem cells (CD117+CD29+CD90+), multipotent mesenchymal stromal cells (CD45-CD31-CD90+CD106+), hemangiogenesis precursors (CD45-CD117+Flk1+), and epithelial cells (CD45-CD31-CD49f+CD326+).

BACKGROUND

Anti-neutrophil cytoplasmic autoantibodies (ANCA)-associated disease among other manifestations can underlie rapidly progressive glomerulonephritis (RPGN), with crescentic and necrotizing GN. Differences in pathogenic immune mechanisms in RPGN may provide differences in the renal expression of adhesion molecules mediating these lesions.

METHODS

Renal intercellular adhesion molecule 1 (ICAM-1; CD54) and vascular cell adhesion molecule 1 (VCAM-1; CD106) were assessed in 40 patients with type I RPGN (anti-glomerular basement membrane antibodies, n = 4), type II (immune complexes, n = 17), and type III (ANCA, n = 19). Enzyme-linked immunosorbent assay (ELISA) for detection of immunoglobulin G antibodies against the Goodpasture's antigen and indirect immunofluorescence and ELISA for myeloperoxidase (MPO) and proteinase 3 (PR3) were performed for ANCA testing. Ten normal renal tissues were used as controls. Relationships between ICAM-1 and VCAM-1, histopathologic features, and CD18, CD14, and CD3 cells were analyzed.

RESULTS

Abnormal ICAM-1 and VCAM-1 in tubule was seen in >80% of biopsies with RPGN. Abnormal VCAM-1 in glomerular tuft was seen in >60% of biopsies with RPGN. Glomerular ICAM-1 was associated with less glomerulosclerosis (chi (2) = 6.719, p = 0.01), less interstitial fibrosis (chi (2) = 4.322, p < 0.05), and less tubular atrophy (chi (2) = 8.547, p < 0.005). Glomerular VCAM-1 was associated with glomerular leukocyte infiltration (chi (2) = 4.698, p < 0.05). Glomerular tuft stains of ++/+++ for VCAM-1 was observed in 10% from MPO-ANCA-GN patients but in 60% from PR3-ANCA-GN (Fi = 8.538, p = 0.03).

CONCLUSIONS

The following conclusions can be made from this study. (1) The renal expression of ICAM-1 and VCAM-1 is upregulated in RPGN, and this is associated with the histological activity. (2) De novo expression of VCAM-1 on glomerular tuft suggests that endothelial cells play a role in RPGN. (3) De novo tubular expression of ICAM-1 and VCAM-1 suggests that epithelial cells may participate in adhesive interactions in RPGN. (4) De novo expression of VCAM-1 at the glomerular tuft in PR3-ANCA positive patients seems greater than in MPO-ANCA positive patients, which suggests that testing specific immune activation mechanisms may play a role in ANCA-associated GN.

Tepoxalin, a dual enzyme inhibitor of cyclooxygenase and 5-lipoxygenase has been shown to inhibit T-cell activation. Its immunosuppressive property is distinct from cyclosporin because only tepoxalin, but not cyclosporin, suppresses NF-kappa B activation. Here we report that tepoxalin selectively inhibits intercellular adhesion molecule-1 (ICAM-1, CD54)/MAC-1 (CD11b/CD18) dependent adhesion of polymorphonuclear cells to IL-1 activated human umbilical vein endothelial cells. The mechanism of inhibition is related to the surface expression of several cell adhesion molecules. Flow cytometry analyses on cultured cells that were treated with tepoxalin or antisense oligonucleotides to the P65/p50 subunit of NF-kappa B, and then stimulated with PMA, revealed a reduced expression of CD11b/CD18 on monocytic HL60 cells, and endothelial adhesion molecule-1 (CD62E) and vascular adhesion molecule-1 (CD106) on human umbilical vein endothelial cells. Expression of other adhesion molecules such as lymphocyte function associated-antigen-1 (CD11a/CD18) and CD54 were unaffected. Tepoxalin also inhibited the secretion of a NF-kappa B regulated chemokine, IL-8, a known inducer of CD11b/CD18 expression. Thus the suppression of CD11b/CD18 expression by tepoxalin may involve IL-8. Our results suggest that by inhibiting NF-kappa B activation, surface expression of several adhesion molecules can be modulated and that tepoxalin may be useful in treating selected adhesion mediated events such as leukocyte migration or atherosclerotic plaque formation.

BACKGROUND

Healing of articular cartilage has remained in question with the use of conventional treatment modalities such as subchondral drilling and microfracture. As demonstrated in the past, adult stem cells retain promising clonogenicity. Therefore, we conducted this study to elucidate the effects of cultured autologous chondrogenic satellite cells (CACSCs) compared with subchondral drilling (SCD) for the repair of full-thickness articular cartilage defects.

METHODS

We examined CACSCs isolated from the knee of rabbits using flow cytometry for the expression of stemness and chondrocyte-specific factors. Subsequently, we created a full-thickness cartilage defect model with a diameter of 3 mm and depth of 2 mm on the articular surface of trochlear grooves in the left knee of 24 New Zealand white rabbits. Then we drilled subchondrally through the defect in all animals and stuffed the defects with 10-μg/cm(2) collagen scaffolds. In the treatment group, we instilled CACSCs at 5 × 10(6) cells/mL in the collagen scaffold and collected samples on days 15, 30, and 45.

RESULTS

The CACSCs revealed significant expression of CD106, CD44, collagen type 2, and aggrecan. In conjunction with SCD, CACSCs improved healing of the articular cartilage defect, as evidenced by the formation of hyaline-like tissue grossly and histologically. The healed tissue also revealed a significant (P < 0.05) increase in the expression of collagen type 2 and aggrecan (by real-time polymerase chain reaction) during the experiment.

CONCLUSIONS

In conjunction with SCD, CACSCs may be considered to improve articular cartilage damage.

In this manuscript we describe a potentially new mechanism by which unstimulated human monocytes activate endothelial cells (EC) through the secondary induction of endothelial tumour necrosis factor alpha (TNF-alpha). Serum free supernatants (SN) of peripheral blood mononuclear cells (PBMC) strongly induce the expression of intercellular adhesion molecule 1 (ICAM-1, CD54), vascular cell adhesion molecule 1 (VCAM-1, CD106), and endothelial-leukocyte adhesion molecule 1 (ELAM-1, CD62E) on human EC 24 and 4 h post treatment, respectively. Further characterization of the responsible subpopulation revealed the CD14+ monocytes and a monocytic cell line (MM6) to produce an endothelial activating factor (EAF). The EAF also triggers an adhesion and a transendothelial migration (TEM) of peripheral blood cells. Using neutralization with an anti TNF-alpha MoAb MAK195, EAF is not identical with TNF-alpha, but induces the expression of endothelial TNF-alpha, since MAK195 blocked TEM only when coincubated with EC, not with monocytes. Furthermore, intracellular TNF-alpha was significantly upregulated in EC after treatment with SN-MM6. Another evidence for a secondary autocrine mechanism was provided by culturing the EC with a conditioned medium of SN-MM6 treated EC. This conditioned medium induces an adhesion molecule expression and TEM in a similar way to SN-MM6 and can completely be inactivated by anti TNF-alpha. Taken together, these data may have an impact for, e.g. transplantational settings that donor monocytes may trigger an inflammatory response in the absence of further activation signals by eliciting an endogenous TNF-alpha response in the host.

BACKGROUND

The prevalence of food allergies and other allergic reactions is increasing worldwide, particularly in highly-urbanized populations. Cell adhesion molecules are expressed in response to various pro-inflammatory cytokines. The expression of intercellular adhesion molecule 1 - ICAM-1 (CD54), ICAM-1 (CD106), P-selectin (CD62P), and E-selectin (CD62E) on vascular endothelial cells is induced by such pro-inflammatory cytokines as tumor necrosis factor α (TNF-α) and interleukin-1 (IL-1).

OBJECTIVE

To analyze concentrations of E-selectin and platelet endothelial cell adhesion molecule-1 (PECAM-1) in patients with an allergic type of food sensitivity co-existing with gastritis and to compare them to the values determined in individuals with dyspeptic symptoms not associated with allergic disorders.

METHODS

The study included 80 patients, among them 50 individuals with food sensitivity confirmed based on compulsory standards, and 30 subjects with dyspeptic symptoms not accompanied by allergic conditions. Venous blood samples were taken from each patient and concentrations of E-selectin and PECAM-1 were determined by means of ELISA.

RESULTS

Mean concentrations of sE-selectin and sPECAM-1 in patients with food allergy amounted to 54.0 ±21.6 ng/ml and 132.8 ±31.4 ng/ml, respectively. In individuals without food allergy, mean concentrations of sE-selectin and sPECAM-1 were 57.7 ±17.9 ng/ml and 139.6 ±31.1 ng/ml, respectively. Patients with food allergy and individuals with dyspeptic symptoms not associated with food allergy did not differ significantly in terms of sE-selectin concentrations (Mann-Whitney U-test, p = 0.453028). Similarly, no significant intergroup differences were observed with regard to sPECAM-1 concentrations (Mann-Whitney U-test, p = 0.231054).

CONCLUSIONS

Adhesion molecules play an important role in the development of inflammation. This study did not find significant differences in the concentrations of such molecules as sE-selectin and sPECAM-1 between patients with food allergy and gastritis, and subjects in whom gastritis was not accompanied by atopic disorders. A positive correlation between the concentrations of sPECAM-1 and E-selectin was observed in food allergy patients. Consequently, it can be concluded that these molecules participate in the pathogenesis of the inflammatory process independently of the etiopathogenesis of gastritis.

Acquired aplastic anemia (AA) is characterized by deficiency or dysfunction of the bone marrow (BM) microenvironment. However, little is known about the impairment of BM-derived mesenchymal stem cells (MSCs) in AA patients.

We used Illumina HiSeqTM 2000 sequencing, quantitative real-time polymerase chain reaction (qRT-PCR), flow cytometry (FCM), and Western blotting to test the expression of CD106 gene (vascular cell adhesion molecule 1 (VCAM1)) and CD106 protein of BM-MSCs. Furthermore, we used hematoxylin and eosin (H&E) and histochemical staining analysis, immunofluorescence, and the formation of capillary-like structures to analyze capillary tube-like formation in vitro; we also used the Matrigel plug assay to test in vivo vasculogenesis, and an assay of colony forming units (CFUs) and colony-forming unit-megakaryocyte (CFU-MK) to detect the support function of MSCs in vitro. The in vivo engraftment of CD34+ cells and MSCs in NOD/SCID mice was tested by FACS and survival assay; the expression of NF-κB was tested by NanoPro analysis and immunofluorescence. NF-κB-regulated CD106 gene (VCAM1) was confirmed by tumor necrosis factor alpha (TNF-α)-stimulated and lipopolysaccharide (LPS)-stimulated MSCs, blockade assay, and immunofluorescence.

Here, we report that BM-MSCs from AA patients exhibited downregulation of the CD06 gene (VCAM1) and low expression of CD106 in vitro. Further analysis revealed that CD106+ MSCs from both AA patients and healthy controls had increased potential for in vitro capillary tube-like formation and in vivo vasculogenesis compared with CD106- MSCs, and the results were similar when healthy MSCs were compared with AA MSCs. CD106+ MSCs from both AA patients and healthy controls more strongly supported in vitro growth and in vivo engraftment of CD34+ cells in NOD/SCID mice than CD106- MSCs, and similar results were obtained when healthy MSCs and AA MSCs were compared. The expression of NF-κB was decreased in AA MSCs, and NF-κB regulated the CD106 gene (VCAM1) which supported hematopoiesis.

These results revealed the effect of CD106 and NF-κB in BM failure of AA.

Oral lichen planus is a chronic inflammatory immune-mediated disease. ICAM-1 and VCAM-1 are vascular adhesion molecules that their receptors are located on endothelial cells and leukocytes. The aim of this study is the immunohistochemical evaluation of VCAM1 and ICAM1 in oral lichen planus and to compare these two markers with normal mucosa for evaluation of angiogenesis. This descriptive-analytical study was performed on 70 paraffined blocks of oral lichen planus and 30 normal mucosa samples taken from around the lesions. Samples were stained with H & E and then with Immunohistochemistry using monoclonal mouse anti human VCAM1 (CD106), & monoclonal mouse anti human ICAM1(CD54) for confirmation of diagnosis. Slides were evaluated under light microscope and VCAM1 and ICAM1 positive cells (endothelial cells and leukocytes) were counted. Data were analyzed with Mann-Whitney test, Wilcoxon and Chi-Square and p<0.001 was declared significant. VCAM1 and ICAM1 expression significantly increased compared to normal mucosa in oral lichen planus according to the percentage of stained cells (p=0.000& p=0.000, Mann-Whitney test). Thirty cases of oral normal mucosa associated with lichen planus showed that the VCAM1 has increased significantly in comparison to normal mucosa (p<0.001). Also, ICAM1 expression between lichen planus and normal mucosa, showed a significantly difference (p<0.001). A significant difference between VCAM1 and ICAM1 expression and type of lichen planus was not observed (p>0.05). Regarding the results, it seems that high expression of VCAM1 and ICAM1 is related to oral lichen planus.

Recent in vivo studies suggest that tumor necrosis factor-alpha (TNF-alpha) is involved in the development of the thymus. We postulated that this inflammatory mediator could regulate the influx of progenitor T cells into the thymus. Using an in vitro static adhesion system, we found that TNF-alpha increases the adhesion of a murine progenitor T cell line (FTF1) to a bovine aortic endothelial cell line (1F8), human umbilical vein endothelial (HUVE) cells, and a murine arterial endothelial (MAE) cell line. TNF-alpha treatment of the 1F8 cells resulted in a time- and dose-dependent increase in the adherence of FTF1 cells. Adherence increased during the first 6 hr of treatment with TNF-alpha concentrations ranging from 10(-11) to 10(-9) M. Maximal adherence (6 hr treatment with 10(-10) M of TNF-alpha) was approximately 4.5-fold larger than that of untreated monolayers. A slow decrease in adherence, down to approximately 2-fold at 48 hr, was observed beyond 12 hr of TNF-alpha treatment; in contrast, removal of TNF-alpha after 6 hr of continued stimulation caused the adherence to return to pre-stimulation levels within 24-30 hr. Adhesion of FTF1 cells to TNF-alpha treated 1F8 cells was almost completely blocked by a monoclonal antibody against murine CD49d (very late antigen-4) expressed on FTF1 cells. TNF-alpha-induced adhesion of FTF1 cells to MAE cells was also blocked by monoclonal antibodies against murine CD49d and CD106 (vascular cell adhesion molecule-1). These results support the notion that local secretion of TNF-alpha could modulate the dynamics of adhesion of progenitor T cells to the thymic endothelium.

In order to identify the factors that control the binding of blood leucocytes to cerebral blood vessels we have modified and applied the frozen section assay of Stamper and Woodruff to the study of human brain. Cryostat sections of brain tissue obtained at post mortem were overlaid with blood lymphocytes and experimental conditions were defined which permitted optimum binding of the cells to transected blood vessel walls. The maximal binding of lymphocytes to cerebral vessels occurred when 6 x 10(6) lymphocytes were overlaid onto brain sections for 30 min at 7 degrees C with gentle agitation. Only a small proportion (0.01%) of the added lymphocytes bound to exposed cerebral vessels. However, lymphocytes were far more adherent than monocytes and polymorphonuclear cells (7-fold and 11-fold respectively: p < 0.001) and activation of lymphocytes with IL-2 enhanced their binding to blood vessel walls (mean 130% increase; p < 0.03). Further analysis revealed that CD4-positive T lymphocytes were the predominant cell population binding to the blood vessels. Antibody blocking studies showed that lymphocyte binding to cerebral blood vessels was inhibited by pretreating the lymphocytes with anti-CD11a, anti-CD18 or anti-CD49d (p < or = 0.02) and immunohistochemical studies revealed the presence of the counter-receptors ICAM-1 (CD54) and VCAM-1 (CD106) for these adhesion molecules in addition to the presence of E-selectin (CD62E) and P-selectin (CD62P) on the cerebral blood vessels. The establishment of a technique in situ which measures selective binding of CD4-positive peripheral lymphocytes to sections of cerebral blood vessels will assist in the molecular characterization of factors that control the interaction of leucocytes with the blood-brain barrier in health and disease.

The interaction of normal human B cell precursors with the bone marrow (BM) stromal cells that support their growth and maturation is mediated by adhesion of B cell CD49d/CD29 (alpha 4 beta 1 integrin or VLA-4) to stromal cell signal transduction, we generated CD49d-deficient variants of the RAMOS early b Burkitt's lymphoma cell line. RAMOS was chosen due to its lack of expression of CD49e (alpha 5 integrin) and CD44, which could potentially interact with stromal cells or their extracellular matrix. In contrast to the parental RAMOS cell line, CD49d-deficient RAMOS cell lines did not adhere to primary human BM stromal cells. Tyrosine phosphorylation of several stromal cell proteins was stimulated within 5 to 10 min of contact with either CD49d-expressing or CD49d-deficient RAMOS. These proteins included a 110-kDa substrate tentatively identified as pp125FAK. In contrast, mAb cross-linking of stromal cell CD106 had no significant effect on protein tyrosine phosphorylation. Il-6 production by stromal cells was enhanced up to fourfold over 3 days when stromal cells were cultured in contact with RAMOS cells, regardless of CD49d expression. Stromal cell IL-6 production was not significantly enhanced by RAMOS cells cultured above the stromal cells in Transwell inserts. Thus, we have demonstrated through the use of paired CD49d-expressing and CD49d-deficient RAMOS cell lines that protein tyrosine kinase activity and IL-6 production by primary human BM stromal cells can be enhanced in a manner that is dependent on B cell contact but independent of CD49d-mediated adhesion. These results indicate the potential for B cells to interact with cells in their microenvironment and activate feedback mechanisms mediated by stromal cells, which may regulate lymphopoiesis.

Follicular dendritic cells (FDCs) reside within germinal centers of secondary lymphoid tissue where they play a critical role in antigen-driven immune responses. FDCs express numerous adhesion molecules that facilitate cellular interactions with B and T cells within the germinal center microenvironment. Although human FDCs have been shown to influence B-cell development, very little is known about the ability of FDCs to regulate T-cell responses. To investigate this functional aspect of FDCs, highly enriched preparations were isolated by magnetic cell separation using the FDC-restricted monoclonal antibody HJ2. We found that isolated human FDCs inhibited proliferation of both autologous and allogeneic T cells, and were dependent on the number of FDCs present. Inhibition by FDCs was observed using two serologically distinct superantigens at multiple concentrations (Staphylococcus enterotoxin A and B). In contrast, B cells failed to inhibit, and often augmented superantigen-induced T-cell proliferation. Antibody-blocking studies showed that CD54 and CD106 were involved in the ability of FDC to inhibit T-cell proliferative responses. When FDCs and T cells were separated by a semipermeable membrane, the inhibitory effect was partially abrogated, demonstrating that in addition to cell-cell interactions, a soluble factor(s) was also involved in the process. The addition of indomethicin to cultures improved the proliferative response in the presence of FDCs, indicating that inhibition was mediated, in part, by prostaglandins. These results indicate that FDCs regulate T-cell proliferation by two molecular mechanisms and that FDC:T-cell interactions may play a pivotal role in germinal center development.

We have reported accelerated wound healing induced by intracellular ATP delivery in rabbits, through early massive accumulation, in situ proliferation, and M2 polarization of macrophages. Granulation tissue started to grow within first 24 h of treatment and continued the growth till the wound cavity is completely covered. However, the mechanisms underlying this macrophage response are totally unclear because no one has ever reported this before. In this study, we performed a preliminary exploration of the possible mechanisms by focusing on the roles of cytokines, growth factors, and stem cells in this process. Among the 33 adult rabbits, 18 were used for cytokine measurements and the remaining were used for histological and immunohistochemical studies. Four wounds were created on the ventral side of each ear. Two wounds on one side were treated with ATP-vesicles (10 mM ATP), and the other two were treated with controls (normal saline or Regranex). Dressing changes were made daily and the rabbits were sacrificed at 5 h, 12 h, and 1, 2, 3, 4, 6, 9, 15, and 26 days after wounding. Tissue samples were analyzed for cytokines and growth factors using real-time PCR and immunohistochemical staining. The control wounds showed an immediate increase in proinflammatory cytokines after wound creation but no further increase after this initial spike. The growth factor levels in the control wounds remained unchanged throughout the study. Conversely, the wounds treated with ATP-vesicles showed significantly higher expression of MCP-1 and stem cell markers (CD44, CD106, CD146, and CD34) at day 1, significantly higher IL-1β and TNF-α expression from day 1-4, and significantly higher VEGF-A, VEGF-D, and VEGFR-2 expression from day 4-6 when compared to the controls. The significant upregulation of these factors corresponded to the very early and rapid macrophage accumulation, in situ proliferation, and M2 polarization, resulting in unprecedented rapid granulation tissue generation due to direct macrophage collagen production and neovascularization.

Muscle stem cells (MuSCs) are involved in homeostatic maintenance of skeletal muscle and play a central role in muscle regeneration in response to injury. Thus, understanding MuSC autonomous properties is of fundamental importance for studies of muscle degenerative diseases and muscle plasticity. Rat, as an animal model, has been widely used in the skeletal muscle field, however rat MuSC isolation through fluorescence-activated cell sorting has never been described. This work validates a protocol for effective MuSC isolation from rat skeletal muscles. Tibialis anterior was harvested from female rats and digested for isolation of MuSCs. Three protocols, employing different cell surface markers (CD106, CD56, and CD29), were compared for their ability to isolate a highly enriched MuSC population. Cells isolated using only CD106 as a positive marker showed high expression of Pax7, ability to progress through myogenic lineage while in culture, and complete differentiation in serum-deprived conditions. The protocol was further validated in gastrocnemius, diaphragm, and the individual components of the pelvic floor muscle complex (coccygeus, iliocaudalis, and pubocaudalis), proving to be reproducible. CD106 is an efficient marker for reliable isolation of MuSCs from a variety of rat skeletal muscles.

Under normal homeostatic conditions, the endothelium releases microparticles (MPs), which are known to increase under stressful conditions and in disease states. CD105 (endoglin) and CD106 (vascular cell adhesion molecule-1) are expressed on the surface of endothelial cells and increased expression in response to stress may be observed. A randomised-controlled double-blinded study aimed to examine the use of endothelial MPs as a marker for the state of one's endothelium, as well as whether maintaining acid-base homeostasis affects the release of these MPs. This study tested seven healthy male volunteers, who completed a strenuous cycling protocol, with venous blood analysed for CD105+ and CD106+ MPs by flow cytometry at regular intervals. Prior to each trial participants consumed either 0.3 g·kg(-1) body mass of sodium bicarbonate (NaHCO3), or 0.045 g·kg(-1) body mass of sodium chloride (NaCl). A significant rise in endothelial CD105+ MPs and CD106+ MPs (p<0.05) was observed at 90 min post-exercise. A significant trend was shown for these MPs to return to resting levels 180 min post-exercise in both groups. No significance was found between experimental groups, suggesting that maintaining acid-base variables closer to basal levels has little effect upon the endothelial stress response for this particular exercise mode. In conclusion, strenuous exercise is accompanied by MP release and the endothelium is able to rapidly recover in healthy individuals, whilst maintaining acid-base homeostasis does not attenuate the MP release from the endothelium after exercise.

The expression of several immunoregulatory adhesion proteins and cytokines was studied in the normal epididymis, cryptorchid cryptepididymis, the epididymis of oestrogen-treated mice and the epididymis of non-obese diabetic (NOD) mice at the protein level to see which of these immunoregulatory proteins may be involved in lymphocyte regulation in the normal or pathological epididymis and if cytokine balance in this organ is on the cellular or humoral side. The aim of the study was to characterize the immunological microenvironment of the epididymis to explain the survival of the autoantigenic spermatozoa in this site. In the 6-week-old BALB/c or NOD mouse epididymis there were some CD18 and CD44 expressing cells in the interstitial tissue. There were no differences between these strains in the expression of the studied antigens, except that some CD4 positive cells were present in the interstitial tissue of BALB/c mice. In the cryptorchid cryptepididymis CD4, CD8, CD18, CD44, CD54 and CD106 expressing cells were occasionally present in the connective tissue surrounding the epididymal tubule. In the epididymis of the oestrogen-treated mice these antigens were not expressed. In the cryptorchid cryptepididymis the epithelial cells expressed IL-10 highly and the myoid peritubular cells IL-6. The present results suggest that the epididymal epithelial IL-10 suppressing TH0, TH1 and TH2 immune responses may be involved in the protection of autoantigenic spermatozoa from immune destruction.

BACKGROUND

Mizolastine is a potent, peripherally acting, selective H1-receptor antagonist with potential anti-inflammatory properties. The aim of the study was to evaluate the in vitro effects of mizolastine on the expression of adhesion molecules by primary human airway epithelial and stromal cultures; moreover, the activity of mizolastine on parameters which reflect the immune response efficacy was investigated.

METHODS

Airway epithelial and stromal cells were collected from hypereosinophilic subjects by enzymatic digestion of polyps or turbinates. Cells were stimulated with interferon (IFN)-gamma (500 IU/ml) in the presence of various mizolastine concentrations (6 x 10(-8)-6 x 10(-6) M) for 24 h and the expression of CD106, CD54, CD58 and HLA class I was evaluated. Peripheral blood mononuclear cells from healthy volunteers were incubated with 1% phytohemagglutinin or anti-CD3 monoclonal antibody (20 ng/ml) in the presence of mizolastine, then T lymphocyte proliferation, HLA-DR expression and T cell subpopulations were evaluated.

RESULTS

Both in epithelial and stromal cultures, IFN-gamma significantly upregulated all of the tested surface molecules (p<0.05). The highest dose of mizolastine (6 x 10(-6) M), corresponding to 10-fold the peak plasma level after a single oral administration of 10 mg, was able to act on fibroblasts, significantly downregulating the expression of CD54 (p<0.05). Regarding T lymphocyte proliferation, the addition of mizolastine did not induce any significant change; furthermore, mizolastine was ineffective at all of the tested concentrations on both HLA-DR expression and CD4+/CD8+ ratio.

CONCLUSIONS

This study demonstrated that mizolastine is able to selectively downregulate CD54 expression on stimulated stromal but not epithelial cells without impairing the immune system effectors. The possible clinical significance of these results are an antiallergic property and CD54 modulation on fibroblasts with a good safety profile as far as the lymphocyte response is concerned.

High aldehyde dehydrogenase (ALDH) activity has been associated with stem and progenitor cells in various tissues. Human cord blood and bone marrow ALDH-bright (ALDH(br)) cells have displayed angiogenic activity in preclinical studies and have been shown to be safe in clinical trials in patients with ischemic cardiovascular disease. The presence of ALDH(br) cells in the heart has not been evaluated so far. We have characterized ALDH(br) cells isolated from mouse hearts. One percent of nonmyocytic cells from neonatal and adult hearts were ALDH(br). ALDH(very-br) cells were more frequent in neonatal hearts than adult. ALDH(br) cells were more frequent in atria than ventricles. Expression of ALDH1A1 isozyme transcripts was highest in ALDH(very-br) cells, intermediate in ALDH(br) cells, and lowest in ALDH(dim) cells. ALDH1A2 expression was highest in ALDH(very-br) cells, intermediate in ALDH(dim) cells, and lowest in ALDH(br) cells. ALDH1A3 and ALDH2 expression was detectable in ALDH(very-br) and ALDH(br) cells, unlike ALDH(dim) cells, albeit at lower levels compared with ALDH1A1 and ALDH1A2. Freshly isolated ALDH(br) cells were enriched for cells expressing stem cell antigen-1, CD34, CD90, CD44, and CD106. ALDH(br) cells, unlike ALDH(dim) cells, could be grown in culture for more than 40 passages. They expressed sarcomeric α -actinin and could be differentiated along multiple mesenchymal lineages. However, the proportion of ALDH(br) cells declined with cell passage. In conclusion, the cardiac-derived ALDH(br) population is enriched for progenitor cells that exhibit mesenchymal progenitor-like characteristics and can be expanded in culture. The regenerative potential of cardiac-derived ALDH(br) cells remains to be evaluated.

The study was conducted to know and investigate the mechanism involved during mesenchymal to epithelial transition to unravel questions related to mammary gland development in prepubertal Korean black goat. We, therefore, biopsied mammary fat pad and isolated adipose cells and characterized with stemness factors (CD34, CD13, CD44, CD106, and vimentin) immunologically and through their genetic expression. Furthermore, characterized cells were differentiated to adipogenic (thiazolidinediones and α-linolenic acid) and epithelial (keratinocyte growth factor) lineages. Thiazolidinediones/or in combination with α-linolenic acid demonstrated significant upregulation of adipo-Q, PPAR-γ, CEBP-α, LPL, and resistin. Adipose stem cells in induction mixture (5 μg/ml insulin, 1 μg/ml hydrocortisone, and 10 ng/ml epidermal growth factor) and subsequent treatment with 10 ng/ml keratinocyte growth factor revealed their trans-differentiating ability to epithelial lineage. From 2 d onwards, the cells under keratinocyte growth factor influenced cells to assume rectangular (2-4 d) to cuboidal (8-10 d) shapes. Ayoub-Shklar stain developed brownish-red pigment in the transformed cells. Though, expressions of K8 and K18 were noted to be highly significant (p < 0.01) but expressions of epithelial membrane antigens and epithelial specific antigens were also significant (p < 0.05) compared to 0 d. Conclusively, epithelial transformations of mammary adipose stem cells would add up knowledge to develop therapeutic regimen to deal with mammary tissue injury and diseases.

Fibroblasts play an important role in lung homeostasis and disease. In lung fibrosis, fibroblasts adopt a proliferative and migratory phenotype, with increased expression of α-smooth muscle actin (αSMA) and enhanced secretion of extracellular matrix components. Comprehensive profiling of fibroblast heterogeneity is limited, due to a lack of specific cell-surface markers. We have previously profiled the surface proteome of primary human lung fibroblasts. Here, we sought to define and quantify a panel of cluster of differentiation markers in primary human lung fibroblasts and IPF lung tissue, using immunofluorescence and FACS analysis. Fibroblast function was assessed by analysis of replicative senescence. We observed presence of distinct fibroblast phenotypes in vivo, characterized by various combinations of Desmin, αSMA, CD36, or CD97 expression. Most markers demonstrated stable expression over passages in vitro, but significant changes were observed for CD36, CD54, CD82, CD106, and CD140a. Replicative senescence of fibroblasts was observed from passage 10 onward. CD36- and CD97-positive, but αSMA-negative, cells were present in remodeled areas of IPF lungs. Transforming growth factor-β treatment induced αSMA and collagen I expression, but repressed CD36- and CD97 expression. We identified a panel of stable surface markers in human lung fibroblasts, applicable for positive cell isolation directly from lung tissue. TGF-β exposure represses CD36- and CD97 expression, while increasing αSMA expression; we therefore identified complex surface protein changes during fibroblast-myofibroblast activation. Co-existence of quiescence and activated fibroblast subtypes in the IPF lung suggests dynamic remodeling of fibroblast activation upon subtle changes to growth factor exposure in local microenvironmental niches.

Purpose: Endothelial dysfunction is a known comorbidity in women with polycystic ovary syndrome (PCOS). The aim was to assess if supervised, moderate intensity exercise could potentially impact markers of endothelial disruption; endothelial cell derived microparticles (EMP). Methods: The current study investigated the effects of a supervised 8-week moderate intensity exercise programme on EMP in women with PCOS (n = 11) and control women free from any known disease (n = 10). EMP were enumerated via specific antibody (CD105, CD106) labeling and flow cytometry. Results: CD105+MP significantly reduced in women with PCOS from pre to post-exercise programme, with CD105+ MP reducing from 2114 CD105+ MP per μl platelet free plasma (PFP) to 424 CD105+ MP per μl PFP (p = 0.025). Control women showed no significant change in CD105+ MP (p = 0.25) after completing the same exercise programme. CD106+ MP showed no change in either PCOS (p = 0.95) or control groups (p = 0.99). No significant correlations existed with the changes in EMP compared to body composition changes as a result of exercise. Conclusion: Supervised, moderate intensity exercise independent of substantial weight loss reduced circulating CD105+ MP, likely reflecting an improvement in endothelial function in women with PCOS compared to healthy control women. Additionally, EMP may be a useful marker for physical improvement in exercise programmes for clinical populations.