Towards PET imaging of the dynamic phenotypes of microglia.

There is increasing evidence showing the heterogeneity of microglia activation in neuroinflammatory and neurodegenerative diseases. It has been hypothesized that pro-inflammatory microglia are detrimental and contribute to disease progression, while anti-inflammatory microglia play a role in damage repair and remission. The development of therapeutics targeting the deleterious glial activity and modulating it into a regenerative phenotype relies heavily upon a clearer understanding of the microglia dynamics during disease progression and the ability to monitor therapeutic outcome in vivo. To that end, molecular imaging techniques are required to assess microglia dynamics and study their role in disease progression as well as to evaluate the outcome of therapeutic interventions. Positron emission tomography (PET) is such a molecular imaging technique, and provides unique capabilities for non-invasive quantification of neuroinflammation and has the potential to discriminate between microglia phenotypes and define their role in the disease process. However, several obstacles limit the possibility for selective in vivo imaging of microglia phenotypes mainly related to the poor characterization of specific targets that distinguish the two ends of the microglia activation spectrum and lack of suitable tracers. PET tracers targeting translocator protein 18 kDa (TSPO) have been extensively explored, but despite the success in evaluating neuroinflammation they failed to discriminate between microglia activation statuses. In this review, we highlight the current knowledge on the microglia phenotypes in the major neuroinflammatory and neurodegenerative diseases. We also discuss the current and emerging PET imaging targets, the tracers and their potential in discriminating between the pro- and anti-inflammatory microglia activation states.

Saliva suppresses osteoclastogenesis in murine bone marrow cultures.

Saliva can reach mineralized surfaces in the oral cavity; however, the relationship between saliva and bone resorption is unclear. Herein, we examined whether saliva affects the process of osteoclastogenesis in vitro. We used murine bone marrow cultures to study osteoclast formation. The addition of fresh sterile saliva eliminated the formation of multinucleated cells that stained positive for tartrate-resistant acid phosphatase (TRAP). In line with the histochemical staining, saliva substantially reduced gene expression of cathepsin K, calcitonin receptor, and TRAP. Addition of saliva led to considerably decreased gene expression of receptor activator of nuclear factor kappa-B (RANK) and, to a lesser extent, that of c-fms. The respective master regulators of osteoclastogenesis (c-fos and NFATc1) and the downstream cell fusion genes (DC-STAMP and Atp6v0d2) showed decreased expression after the addition of saliva. Among the costimulatory molecules for osteoclastogenesis, only OSCAR showed decreased expression. In contrast, CD40, CD80, and CD86-all costimulatory molecules of phagocytic cells-were increasingly expressed with saliva. The phagocytic capacity of the cells was confirmed by latex bead ingestion. Based on these in vitro results, it can be concluded that saliva suppresses osteoclastogenesis and leads to the development of a phagocytic cell phenotype.

LPS converts Gr-1(+)CD115(+) myeloid-derived suppressor cells from M2 to M1 via P38 MAPK.

Myeloid-derived suppressor cells (MDSCs) are heterogeneous populations of immature myeloid cells with strong immunosuppressive function, and play a critical role in the immune evasion of cancer. A subset of MDSCs share many similar characteristics with tumor-associated macrophages (TAMs), but it is largely unclear whether MDSCs also have M1/M2 type polarization in tumor microenvironments. In the present study, we found that Gr-1(+)CD115(+) monocytes in tumor-bearing mice exhibited M2 characteristics with significantly lower expression of iNOS and higher expression of Arginase I. Immunofluorescence staining showed that Gr-1(+)CD115(+) monocytes in tumor sites from LPS-injected mice had a higher expression of iNOS. Similarly, in vitro experiments displayed that LPS-treated Gr-1(+)CD115(+) cells expressed higher levels of iNOS, IL-6, TNF, IL-12, and IL-10 compared with those in non-treated Gr-1(+)CD115(+) monocytes. Extensive study showed that LPS-treated Gr-1(+)CD115(+) monocytes had less ability to convert the CD4(+)CD25(-)cells into CD4(+)CD25(+) Tregs, and also had less suppressive function on CD4(+)CD25(-) conventional T cells. LLC tumors in LPS-injected mice grew significantly slower than those in non-LPS-injected mice. Further experiments suggested that LPS may function through the P38 MAPK signaling pathway to increase the expression of iNOS, and of MyD88 independently. Thus, we can get conclusion that Gr-1(+)CD115(+) monocytes in tumor-bearing mice show M2 type characteristics and LPS can skew this M2 type cells into M1 type through the P38 MAPK pathway and lead to inhibition of the suppressive function of Gr-1(+)CD115(+) monocytes. It suggests that LPS or its analogs may be potential drugs for tumor treatment, inflammation induced by LPS or other components of bacterium or virus may be benefit to the inhibition of tumor cell growth in vivo.

Aeroallergen challenge promotes dendritic cell proliferation in the airways.

Aeroallergen provocation induces the rapid accumulation of CD11c(+)MHC class II (MHC II)(+) dendritic cells (DCs) in the lungs, which is driven by an increased recruitment of blood-derived DC precursors. Recent data show, however, that well-differentiated DCs proliferate in situ in various tissues. This may also contribute to their allergen-induced expansion; therefore, we studied DC proliferation in the airways of mice in the steady state and after local aeroallergen provocation. Confocal whole-mount microscopy was used to visualize proliferating DCs in different microanatomical compartments of the lung. We demonstrate that in the steady state, CD11c(+)MHC II(+) DCs proliferate in both the epithelial and subepithelial layers of the airway mucosa as well as in the lung parenchyma. A 1-h pulse of the nucleotide 5-ethynyl-2'-deoxyuridine was sufficient to label 5% of DCs in both layers of the airway mucosa. On the level of whole-lung tissue, 3-5% of both CD11b(+) and CD11b(-) DC populations and 0.3% of CD11c(+)MHC II(low) lung macrophages incorporated 5-ethynyl-2'-deoxyuridine. Aeroallergen provocation caused a 3-fold increase in the frequency of locally proliferating DCs in the airway mucosa. This increase in mucosal DC proliferation was later followed by an elevation in the number of DCs. The recruitment of monocyte-derived inflammatory DCs contributed to the increasing number of DCs in the lung parenchyma, but not in the airway mucosa. We conclude that local proliferation significantly contributes to airway DC homeostasis in the steady state and that it is the major mechanism underlying the expansion of the mucosal epithelial/subepithelial DC network in allergic inflammation.

The spermatogonial stem cell niche in the collared peccary (Tayassu tajacu).

In the seminiferous epithelium, spermatogonial stem cells (SSCs) are located in a particular environment called the "niche" that is controlled by the basement membrane, key testis somatic cells, and factors originating from the vascular network. However, the role of Leydig cells (LCs) as a niche component is not yet clearly elucidated. Recent studies showed that peccaries (Tayassu tajacu) present a peculiar LC cytoarchitecture in which these cells are located around the seminiferous tubule lobes, making the peccary a unique model for investigating the SSC niche. This peculiarity allowed us to subdivide the seminiferous tubule cross-sections in three different testis parenchyma regions (tubule-tubule, tubule-interstitium, and tubule-LC contact). Our aims were to characterize the different spermatogonial cell types and to determine the location and/or distribution of the SSCs along the seminiferous tubules. Compared to differentiating spermatogonia, undifferentiated spermatogonia (A(und)) presented a noticeably higher nuclear volume (P < 0.05), allowing an accurate evaluation of their distribution. Immunostaining analysis demonstrated that approximately 93% of A(und) were GDNF receptor alpha 1 positive (GFRA1(+)), and these cells were preferentially located adjacent to the interstitial compartment without LCs (P < 0.05). The expression of colony-stimulating factor 1 was observed in LCs and peritubular myoid cells (PMCs), whereas its receptor was present in LCs and in GFRA1(+) A(und). Taken together, our findings strongly suggest that LCs, different from PMCs, might play a minor role in the SSC niche and physiology and that these steroidogenic cells are probably involved in the differentiation of A(und) toward type A(1) spermatogonia.

Absence of colony stimulation factor-1 receptor results in loss of microglia, disrupted brain development and olfactory deficits.

The brain contains numerous mononuclear phagocytes called microglia. These cells express the transmembrane tyrosine kinase receptor for the macrophage growth factor colony stimulating factor-1 (CSF-1R). Using a CSF-1R-GFP reporter mouse strain combined with lineage defining antibody staining we show in the postnatal mouse brain that CSF-1R is expressed only in microglia and not neurons, astrocytes or glial cells. To study CSF-1R function we used mice homozygous for a null mutation in the Csflr gene. In these mice microglia are >99% depleted at embryonic day 16 and day 1 post-partum brain. At three weeks of age this microglial depletion continues in most regions of the brain although some contain clusters of rounded microglia. Despite the loss of microglia, embryonic brain development appears normal but during the post-natal period the brain architecture becomes perturbed with enlarged ventricles and regionally compressed parenchyma, phenotypes most prominent in the olfactory bulb and cortex. In the cortex there is increased neuronal density, elevated numbers of astrocytes but reduced numbers of oligodendrocytes. Csf1r nulls rarely survive to adulthood and therefore to study the role of CSF-1R in olfaction we used the viable null mutants in the Csf1 (Csf1(op)) gene that encodes one of the two known CSF-1R ligands. Food-finding experiments indicate that olfactory capacity is significantly impaired in the absence of CSF-1. CSF-1R is therefore required for the development of microglia, for a fully functional olfactory system and the maintenance of normal brain structure.

Defining the anatomical localisation of subsets of the murine mononuclear phagocyte system using integrin alpha X (Itgax, CD11c) and colony stimulating factor 1 receptor (Csf1r, CD115) expression fails to discriminate dendritic cells from macrophages.

The murine mononuclear phagocyte (MNP) system comprises a diverse population of cells, including monocytes, dendritic cells (DC) and macrophages. Derived from the myeloid haematopoietic lineage, this group of cells express a variety of well characterized surface markers. Expression of the integrin alpha X (Itgax, CD11c) is commonly used to identify classical DC, and similarly expression of colony stimulating factor 1 receptor (Csf1r, CD115) to identify macrophages. We have characterized the expression of these markers using a variety of transgenic mouse models. We confirmed previous observations of Itgax expression in anatomically defined subsets of MNPs in secondary lymphoid organs, including all MNPs identified within the germinal centres. The majority of MNPs in the intestinal lamina propria and lung express Itgax. All mucosal Itgax expressing cells also express Csf1r suggesting Csf1-dependent haematopoietic derivation. This double-positive population included germinal centre MNPs. These data reveal that Itgax expression alone does not specifically define classical DC. These results suggest more cautious interpretation of Itgax-dependent experimentation and direct equation with uniquely DC-mediated activities, particularly in the functioning of non-lymphoid MNPs within the intestinal lamina propria.

Measurement of macrophage growth and differentiation.

This unit provides protocols for measuring the abundance and growth of macrophage precursors in agar cultures and the proliferation of isolated mature macrophages in vitro, by either direct cell counting or by DNA measurement. Methods for the immunohistochemical identification of macrophages and the determination of their proliferative status in vivo by immunofluorescence are also included. It also describes methods for characterization of macrophage differentiation through the immunofluorescence analysis of cell-surface expression of CSF-1 receptor.

Expressions of RANKL/RANK and M-CSF/c-fms in root resorption lacunae in rat molar by heavy orthodontic force.

The differentiation and functions of osteoclasts are regulated by receptor activator of nuclear factor-κB (RANK)/receptor activator of nuclear factor-κB ligand (RANKL) system that stimulates osteoclasts formation. Macrophage colony-stimulating factor (M-CSF) is also essential for osteoclastogenesis. A recent immunocytochemical study reported that RANKL/RANK and M-CSF/c-fms were localized in the periodontal ligament of rat molars during experimental orthodontic tooth movement. The present study focused on the expressions of RANKL/RANK and M-CSF/c-fms in root resorption area during experimental tooth movement in rats. Forty 6-week-old male Wistar rats were subjected to an orthodontic force of 10 or 50 g with a closed coil spring (wire size: 0.005 inch, diameter: 1/12 inch) ligated to the maxillary first molar cleat by a 0.008 inch stainless steel ligature wire to induce a mesial tipping movement of the upper first molars. Experimental tooth movement was undertaken for 10 days. Each sample was sliced into 6 µm continuous sections in a horizontal direction and prepared for haematoxylin and eosin (H and E) and immunohistochemistry staining for tartrate-resistant acid phosphatase (TRAP), RANK, RANKL M-CSF, and c-fms in root resorption area. Statistical analysis was carried out using a Mann-Whitney U-test with a significance level of P<0.01. On days 7 and 10, immunoreactivity for RANKL/RANK and M-CSF/c-fms was detected in odontoclasts with an orthodontic force of 50 g, but not 10 g. Therefore, RANKL/RANK and M-CSF/c-fms systems may be involved in the process of root resorption by heavy orthodontic force.

Compressive mechanical force augments osteoclastogenesis by bone marrow macrophages through activation of c-Fms-mediated signaling.

Little is known about the effects of mechanical forces on osteoclastogenesis by bone marrow macrophages (BMMs) in the absence of mechanosensitive cells, including osteoblasts and fibroblasts. In this study, we examined the effects of mechanical force on osteoclastogenesis by applying centrifugal force to BMMs using a horizontal microplate rotor. Our findings, as measured by an in vitro model system, show that tumor necrosis factor (TNF)-α is capable of inducing osteoclast differentiation from BMMs and bone resorption in the presence of macrophage-colony stimulating factor (M-CSF) and is further facilitated by receptor activator of nuclear factor-kappaB (NF-κB) ligand (RANKL). Application of force to BMMs accelerated TNF-α-induced osteoclastogenesis; this was inhibited either by anti-TNF-α or anti-TNF-α receptor but not by OPG. TNF-α also increased c-Fms expression at both mRNA and protein levels in BMMs. An anti-c-Fms antibody completely inhibited osteoclast differentiation and bone resorption induced by TNF-α but partially blocked osteoclastogenesis stimulated in combination with RANKL. These results suggest that TNF-α (in the presence of M-CSF) is capable of inducing osteoclastogenesis from BMMs, and that osteoclastogenesis is significantly stimulated by force application through the activation of c-Fms-mediated signaling. Overall, the present study reveals the facilitating effect of mechanical force on osteoclastic differentiation from BMMs without the addition of mechanosensitive cells.

The origin and development of nonlymphoid tissue CD103+ DCs.

CD103(+) dendritic cells (DCs) in nonlymphoid tissues are specialized in the cross-presentation of cell-associated antigens. However, little is known about the mechanisms that regulate the development of these cells. We show that two populations of CD11c(+)MHCII(+) cells separated on the basis of CD103 and CD11b expression coexist in most nonlymphoid tissues with the exception of the lamina propria. CD103(+) DCs are related to lymphoid organ CD8(+) DCs in that they are derived exclusively from pre-DCs under the control of fms-like tyrosine kinase 3 (Flt3) ligand, inhibitor of DNA protein 2 (Id2), and IFN regulatory protein 8 (IRF8). In contrast, lamina propria CD103(+) DCs express CD11b and develop independently of Id2 and IRF8. The other population of CD11c(+)MHCII(+) cells in tissues, which is CD103(-)CD11b(+), is heterogenous and depends on both Flt3 and MCSF-R. Our results reveal that nonlymphoid tissue CD103(+) DCs and lymphoid organ CD8(+) DCs derive from the same precursor and follow a related differentiation program.

GM-CSF regulates intimal cell proliferation in nascent atherosclerotic lesions.

The contribution of intimal cell proliferation to the formation of early atherosclerotic lesions is poorly understood. We combined 5-bromo-2'-deoxyuridine pulse labeling with sensitive en face immunoconfocal microscopy analysis, and quantified intimal cell proliferation and Ly-6C(high) monocyte recruitment in low density lipoprotein receptor-null mice. Cell proliferation begins in nascent lesions preferentially at their periphery, and proliferating cells accumulate in lesions over time. Although intimal cell proliferation increases in parallel to monocyte recruitment as lesions grow, proliferation continues when monocyte recruitment is inhibited. The majority of proliferating intimal cells are dendritic cells expressing CD11c and major histocompatibility complex class II and 33D1, but not CD11b. Systemic injection of granulocyte/macrophage colony-stimulating factor (GM-CSF) markedly increased cell proliferation in early lesions, whereas function-blocking anti-GM-CSF antibody inhibited proliferation. These findings establish GM-CSF as a key regulator of intimal cell proliferation in lesions, and demonstrate that both proliferation and monocyte recruitment contribute to the inception of atherosclerosis.

Bone marrow angiotensin AT1 receptor regulates differentiation of monocyte lineage progenitors from hematopoietic stem cells.

BACKGROUND: The angiotensin II (Ang II) type 1 (AT(1)) receptor is expressed in bone marrow (BM) cells, whereas it remains poorly defined how Ang II regulates differentiation/proliferation of monocyte-lineage cells to exert proatherogenic actions. METHODS AND RESULTS: We generated BM chimeric apoE(-/-) mice repopulated with AT(1)-deficient (Agtr1(-/-)) or wild-type (Agtr1(+/+)) BM cells. The atherosclerotic development was significantly reduced in apoE(-/-)/BM-Agtr1(-/-) mice compared with apoE(-/-)/BM-Agtr1(+/+) mice, accompanied by decreased numbers of BM granulocyte/macrophage progenitors (GMP:c-Kit(+)Sca-1(-)Lin(-)CD34(+)CD16/32(+)) and peripheral blood monocytes. Macrophage-colony-stimulating factor (M-CSF)-induced differentiation from hematopoietic stem cells (HSCs:c-Kit(+)Sca-1(+)Lin(-)) to promonocytes (CD11b(high)Ly-6G(low)) was markedly reduced in HSCs from Agtr1(-/-) mice. The expression of M-CSF receptor c-Fms was decreased in HSCs/promonocytes from Agtr1(-/-) mice, accompanied by a marked inhibition in M-CSF-induced phosphorylation of PKC-delta and JAK2. c-Fms expression in HSCs/promonocytes was mainly regulated by TNF-alpha derived from BM CD45(-)CD34(-) stromal cells, and Ang II specifically regulated the TNF-alpha synthesis and release from BM stromal cells. CONCLUSIONS: Ang II regulates the expression of c-Fms in HSCs and monocyte-lineage cells through BM stromal cell-derived TNF-alpha to promote M-CSF-induced differentiation/proliferation of monocyte-lineage cells and contributes to the proatherogenic action.

Global expression profiling of sex cord stromal tumors from Men1 heterozygous mice identifies altered TGF-beta signaling, decreased Gata6 and increased Csf1r expression.

Heterozygous disruption of the Men1 gene predisposes mice to the development of multiple endocrine tumors, accurately mimicking the human MEN1 cancer predisposition syndrome. Additionally, Men1(+/-) mice frequently develop sex cord adenomas. The mechanism underlying the susceptibility of these mice to sex cord tumor development has not been fully determined, but data suggest it may involve transcriptional regulation of key growth promoting/repressing genes. To identify potential menin-regulated genes that may be important for tumor suppression in sex cord cells, we compared the global gene expression profiles of testis and ovary adenomas with other endocrine tumors of the pancreas and pituitary from Men1 heterozygous mice and with control tissues. Gonadal tumors clustered separately from pancreas and pituitary tumors with only a few genes (e.g., Cdkn2c) commonly dysregulated in all tumor types. Testis and ovary tumors displayed a higher level of transcriptional similarity to each other than they did to their respective control tissues. Among genes that had decreased expression in tumors was significant over-representation of genes associated with the TGF-beta, hedgehog and Wnt signaling, indicating that loss of menin function affects these pathways at the level of transcription. Aberrant protein expression in Leydig and granulosa cells of 2 transcriptionally dysregulated gene products, Gata6 and Csf1r were confirmed by immunohistochemistry. We propose that sex cord tumor susceptibility in Men1(+/-) mice involves deregulated cell proliferation due to dysregulation of multiple cell growth regulating genes including: reduced Cdkn2c transcription, loss of TGF-beta pathway tumor suppressor function (e.g., Gata6) and transcriptional activation of Csf1r.

Osteoclast differentiation during experimental tooth movement by a short-term force application: an immunohistochemical study in rats.

OBJECTIVE: The origin of osteoclasts responsible for bone resorption during orthodontic tooth movement is not yet clear. Their precursors may reside within the periodontal ligament (PDL) or could be recruited from the circulation or the bone marrow. The aim of this study was to investigate the spatial and sequential distribution of osteoclast precursors during experimental tooth movement by using three differentiation markers: receptor for macrophage colony stimulating factor (c-Fms), receptor activator of nuclear factor-kappaB (RANK), and calcitonin receptor (CTR). MATERIAL AND METHODS: Six-week-old Wistar rats were used. Elastic bands were inserted between the upper 1st and 2nd molars for 1, 2, 3, and 6 days. Immunohistochemical staining for c-Fms, RANK, or CTR was performed on parasagittal sections and positive cells were counted. RESULTS: Before force application, many c-Fms+ and a few RANK+ precursors were present in the bone marrow. No c-Fms+ osteoclast precursors were observed in the PDL. After force application, the number of RANK+ but not c-Fms+ precursors increased rapidly in the PDL. In bone marrow, the number of c-Fms+ and RANK+ precursors also increased rapidly, as did multinuclear c-Fms+, RANK+, and CTR+ cells. Subsequently, the number of c-Fms+, RANK+, and CTR+ multinuclear cells in the PDL increased. After 6 days, the expression profiles tended to return to baseline levels. CONCLUSION: Osteoclast precursors differentiate within the bone marrow and then migrate into the PDL during early tooth movement.

Increased expression of activating factors in large osteoclasts could explain their excessive activity in osteolytic diseases.

Large osteoclasts (>or=10 nuclei) predominate at sites of pathological bone resorption. We hypothesized this was related to increased resorptive activity of large osteoclasts and have demonstrated previously that larger osteoclasts are 8-fold more likely to be resorbing than small osteoclasts (2-5 nuclei). Here we ask whether these differences in resorptive activity can be explained by differences in expression of factors involved in osteoclast signaling, fusion, attachment, and matrix degradation. Authentic rabbit osteoclasts and osteoclasts derived from RAW264.7 cells showed similar increases in c-fms expression (1.7- to 1.8-fold) in large osteoclasts suggesting that RAW cells are a viable system for further analysis. We found 2- to 4.5-fold increases in the expression of the integrins alpha(v) and beta(3), the proteases proMMP9, matMMP9 and pro-cathepsinK, and in activating receptors RANK, IL-1R1, and TNFR1 in large osteoclasts. In contrast, small osteoclasts had higher expression of the fusion protein SIRPalpha1 and the decoy receptor IL-1R2. The higher expression of activation receptors and lower expression of IL-1R2 in large osteoclasts suggest they are hyperresponsive to extracellular factors. This is supported by the observation that the resorptive activity in large osteoclasts was more responsive to IL-1beta, and that this increased activity was inhibited by the IL-1 receptor antagonist, IL-1ra. This increased responsiveness of large osteoclasts to IL-1 may, in part, explain the pathological bone loss noted in inflammatory diseases. The heterogeneity in receptor expression and the differential response to cytokines and their antagonists could prove useful for selective inhibition of large osteoclasts actively engaged in pathological bone loss.

Targeted expression of csCSF-1 in op/op mice ameliorates tooth defects.

OBJECTIVE: The aim of this study was to characterize the tooth phenotype of CSF-1-deficient op/op mice and determine whether expression of csCSF-1 in these mice has a role in primary tooth matrix formation. DESIGN: Ameloblasts and odontoblasts, isolated from wt/wt frozen sections using laser capture microdissection, were analysed for csCSF-1, sCSF-1 and CSF-1R mRNA by RT-PCR. Mandibles, excised from 8 days op/op and wt/wt littermates, were examined for tooth morphology as well as amelogenin and DMP1 expression using in situ hybridisation. op/opCS transgenic mice, expressing csCSF-1 in teeth and bone using the osteocalcin promoter, were generated. Skeletal X-rays and histomorphometry were performed; teeth were analysed for morphology and matrix proteins. RESULTS: Normal dental cells in vivo express both CSF-1 isoforms and CSF-1R. Compared to wt/wt, op/op teeth prior to eruption showed altered dental cell morphology and dramatic reduction in DMP1 transcripts. op/opCS mice showed marked resolution of osteopetrosis, tooth eruption and teeth that resembled amelogenesis imperfecta-like phenotype. At 3 weeks, op/op teeth showed severe enamel and dentin defects and barely detectable amelogenin and DMP1. In op/opCS mice, DMP1 in odontoblasts increased to near normal and dentin morphology was restored; amelogenin also increased. Enamel integrity improved in op/opCS, although it was thinner than wt enamel. CONCLUSIONS: Results demonstrate that ameloblasts and odontoblasts are a source and potential target of CSF-1 isoforms in vivo. Expression of csCSF-1 within the tooth microenvironment is essential for normal tooth morphogenesis and may provide a mechanism for coordinating the process of tooth eruption with endogenous matrix formation.

Analysis of immune cell infiltrates during squamous carcinoma development.

Infiltration of leukocytes into tissue is a common feature of many physiological and pathological conditions. Histopathologically, the diversity of leukocytes that infiltrate a tissue associated with a pathophysiologic response cannot be appreciated and/or examined unless highly selective immunologic detection methods are utilized. Specific populations of infiltrating leukocytes into squamous tissues harboring pre-malignant and/or malignant keratinocytes have recently been demonstrated to play a functionally significant role in the pathogenesis of squamous carcinomas. To evaluate immune cell types and quantify changes in their relative presence and localization during multi-stage neoplastic progression, we performed flow cytometry and histochemical detection using lineage-selective markers. Herein, we provide detailed methodology facilitating these analyses.

A transient microenvironment loaded mainly with macrophages in the early developing human pancreas.

We have previously shown that the human developing pancreas, as a tissue under construction and remodeling, is composed of epithelial ducts and differentiated endocrine cells surrounded by mesenchyme. The physiologic importance of resident tissue leukocytes, expected to enter through the mesenchyme in remodeling functions, prompted us to investigate human developing pancreases for the presence of leukocyte lineages and for expression of cytokines and receptors involved in their recruitment and differentiation. Immunohistochemistry studies were performed on 69 human, paraffin-embedded pancreases at 6-12 weeks of development (WD). Cytokines and receptor transcripts were monitored by reverse transcription (RT)-PCR, by immunohistochemistry when antibodies were available or by in situ hybridization (ISH). We show that numerous cells expressing CD45RA, HLADR and CD68 antigens are cellular components of the mesenchyme of all the pancreases at 6-12 WD. So-called constitutive chemokines (SLC (CCL19), stromal-derived factor 1 (SDF1) (CXCL12)) and a macrophage-specific growth/survival factor, colony-stimulating factor 1 (CSF1), were detected in epithelial duct cells. Both epithelial and mesenchymal cells expressed chemokine receptors, suggesting a role in leukocyte recruitment and possibly in early pancreatic development. In conclusion, we demonstrated the presence of CD45RA resident leukocyte-derived lineages, mostly macrophages, in the early human pancreatic mesenchyme. These cells may have migrated in the tissue through the vascular system, attracted by constitutively expressed chemokines, and locally surviving through CSF1 signaling. The role of macrophages in epithelium/mesenchyme interaction-mediated pancreatic development remains to be demonstrated.

[Clinical characteristics and immunophenotype of aged patients with acute leukemia].

In order to analyze the clinical characteristics and biological features of acute leukemia in elderly, 104 acute leukemia patients in elderly were retrospectively analyzed and compared with 71 acute leukemia patients below 60 years old. The results showed that: (1) the proportion of AML in the aged group (73%) was higher than that in the young group (54.9%), and the difference was statistically significant (P < 0.05), but AML (M3) was absent in the aged group; (2) the median of bone marrow blast cell in the aged group was significantly lower than that in the young group (P < 0.05); (3) in AML, the frequently of CD14 expression was higher in the aged group (18.8%) than that in the young group (2.6%), while the expression frequencies of CD15 (37.5%), CD117 (62.5%), and CD38 (59.4%) were respectively lower in the aged group than that in the young group which were (69.2%) for CD15, (89.7%) for CD17, and (84.6%) for CD38 respectively, and the difference was also statistically significant (P < 0.05). (4) CD19 was most frequently expressed in ALL of the aged group and the positive rate was 100%; (5) there was no significant difference in expression of special lineage antigens and overlapping lineage antigens between the aged group and the young group (P > 0.05); (6) the expression frequency of unfavorable karyotypes in the aged group was higher than that in the young group, and the difference was statistically very significant (P < 0.01); (7) the complete remission rate (CR rate) in the aged group was 42.9%, 2-year survival rate in the aged group was 5.4%, and treatment-related mortality rate in the aged group was 26.8%, while the CR rate in the young group was 76.6%, the difference was statistically significant (P < 0.05). It is concluded that the expression frequency of CD14 associated with unfavorable prognosis is higher in the aged group than that in the young group, while the expression frequency of CD15 associated with favorable prognosis is lower in the aged group than that in the young group. The expression frequency of unfavorable karyotypes in the aged group is higher than that in the young group. The CR rate of acute leukemia in elderly is low, thus the patients in elderly often have unfavorable prognosis.