Morphological Changes in Dorsal Root Ganglia Macrophages Associated with Neuropathic Pain Mechanisms Suggest a Novel Target for Chronic Pain Therapy

The present study examined morphological changes in the dorsal root ganglia (DRG) following an innate immune stimulus. The importance of the DRG has increasingly become recognized in pain processing as more than just the home of primary afferent cell bodies. All sensory information passes through the DRG via the primary afferents, and on to the spinal cord. The primary afferents synapse with second-order neurons in the spinal cord that ascend towards the brain, where they transmit the pain signal to the limbic forebrain and/or the somatosensory cortex for processing. The DRG is an interesting niche to study at as it lies outside the blood- brain-barrier (BBB) but projects to the CNS. Therefore, neurons in the DRG are exposed to large circulating molecules (such as LPS) that normally would not be able to act on second-order neurons in the spinal cord, due to the BBB, and thus can indirectly act on second-order neurons by sensitizing their primary afferent inputs, which increases primary afferent output into the spinal cord. Of particular interest, the DRG constitutively possesses a significant population of macrophages. Mice injected with lipopolysaccharide (LPS) will lead to an enhanced pain response that has been speculated to result from the activation of innate immune receptors (TLR4 expressed on the macrophage which leads to the release of active factors that sensitize the sensory neuron). To enable study of these DRG macrophages, we employed a novel 3D imaging technique to visualize morphological changes in whole cells, as opposed to previous imaging methods that only captured slices of cells, which greatly improved our ability to depict the dense and complex cellular environment of the DRG. A machine learning network was employed to train 3D stacks of DRG tissue images at different depths and intensities. After running thousands of replicates (epochs), through curated images, the machine learning program became very accurate and enabled us to characterize, in 3D reconstructions, the DRG macrophage population. 3D imaging of DRG macrophages 5 This approach enabled several observations. 1) Two distinct sub-populations of macrophages in the DRG were found: non-vascular and peri-vascular macrophages. 2) Spinal delivery of LPS which activated TLR4 receptors was found to increase the size of the individual macrophages, but in contrast to previous studies no the number. 3) This change in macrophage size vs. number was confirmed by the lack of change in the macrophage response in CXC3R (-) mutants which do not have circulating macrophages. The macrophage production of many spiny, amorphic processes were likely “double counted” in previous studies done on tissue slices (as opposed to the 3D, whole-cell modeling employed in these studies). By visualizing whole 3-D reconstructed cells, we were able to differentiate between increases in macrophage number versus increased macrophage volume that manifested. These techniques were validated by comparing our data analysis pipeline with commercially-available software, which yielded similar results. Further application, of our techniques and pipeline will enable the 3D imaging of different cell types and their interactions within the DRG

Immunophenotypic signatures of benign and dysplastic granulopoiesis by cytomic profiling

Background: The role of flow cytometry (FCM) in diagnosing myelodysplastic syndromes (MDS) remains controversial, because analysis of myeloid maturation may involve subjective interpretation of sometimes subtle patterns on multiparameter FCM. Methods: Using six‐parameter marker combinations known to be useful in evaluating the myeloid compartment in MDS, we measured objective immunophenotypic differences between non‐neoplastic ( n = 25) and dysplastic ( n = 17) granulopoiesis using a novel method, called Fisher information nonparametric embedding (FINE), that measures information distances among FCM datasets modeled as individual high‐dimensional probability density functions, rather than as sets of two‐dimensional histograms. Information‐preserving component analysis (IPCA) was used to create information‐optimized “rotated” two‐dimensional histograms for visualizing myelopoietic immunophenotypes for each individual sample. Results: There was a consistent trend of segregation of higher‐grade MDS (RAEB and RCMD) from benign by FINE analysis. This difference was accentuated in cases with morphologic dysgranulopoiesis and in cases with clonal cytogenetic abnormalities. However, lower grades of MDS or cases that lacked morphologic dysgranulopoiesis showed much greater overlap with non‐neoplastic cases. Two cases of reactive left shift were consistently embedded within the higher‐grade MDS group. IPCA yielded two‐dimensional histogram projections for each individual case by relative weighting of measured cellular characteristics, optimized for preserving information distances derived through FINE. Conclusions: Objective analysis by information geometry supports the conclusions of previous studies that there are immunophenotypic differences in the maturation patterns of benign granulopoiesis and high grade MDS, but also reinforces the known pitfalls of overlap between low‐grade MDS and benign granulopoiesis and overlap between reactive granulocytic left shifts and dysplastic granulopoiesis. © 2011 International Clinical Cytometry SocietyPeer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/87051/1/20592_ftp.pd

Elevated frequencies of leukemic myeloid and plasmacytoid dendritic cells in acute myeloid leukemia with the FLT3 internal tandem duplication

Some 30% of acute myeloid leukemia (AML) patients display an internal tandem duplication (ITD) mutation in the FMS-like tyrosine kinase 3 (FLT3) gene. FLT3-ITDs are known to drive hematopoietic stem cells towards FLT3 ligand independent growth, but the effects on dendritic cell (DC) differentiation during leukemogenesis are not clear. We compared the frequency of cells with immunophenotype of myeloid DC (mDC: Lin−, HLA-DR+, CD11c+, CD86+) and plasmacytoid DC (pDC: Lin−, HLA-DR+, CD123+, CD86+) in diagnostic samples of 47 FLT3-ITD− and 40 FLT3-ITD+ AML patients. The majority of ITD+ AML samples showed high frequencies of mDCs or pDCs, with significantly decreased HLA-DR expression compared with DCs detectable in ITD− AML samples. Interestingly, mDCs and pDCs sorted out from ITD+ AML samples contained the ITD insert revealing their leukemic origin and, upon ex vivo culture with cytokines, they acquired DC morphology. Notably, mDC/pDCs were detectable concurrently with single lineage mDCs and pDCs in all ITD+ AML (n = 11) and ITD− AML (n = 12) samples analyzed for mixed lineage DCs (Lin−, HLA-DR+, CD11c+, CD123+). ITD+ AML mDCs/pDCs could be only partially activated with CD40L and CpG for production of IFN-α, TNF-α, and IL-1α, which may affect the anti-leukemia immune surveillance in the course of disease progression

Raf Activation Is Regulated by Tyrosine 510 Phosphorylation in Drosophila

The proto-oncoprotein Raf is pivotal for mitogen-activated protein kinase (MAPK) signaling, and its aberrant activation has been implicated in multiple human cancers. However, the precise molecular mechanism of Raf activation, especially for B-Raf, remains unresolved. By genetic and biochemical studies, we demonstrate that phosphorylation of tyrosine 510 is essential for activation of Drosophila Raf (Draf), which is an ortholog of mammalian B-Raf. Y510 of Draf is phosphorylated by the c-src homolog Src64B. Acidic substitution of Y510 promotes and phenylalanine substitution impairs Draf activation without affecting its enzymatic activity, suggesting that Y510 plays a purely regulatory role. We further show that Y510 regulates Draf activation by affecting the autoinhibitory interaction between the N- and C-terminal fragments of the protein. Finally, we show that Src64B is required for Draf activation in several developmental processes. Together, these results suggest a novel mechanism of Raf activation via Src-mediated tyrosine phosphorylation. Since Y510 is a conserved residue in the kinase domain of all Raf proteins, this mechanism is likely evolutionarily conserved

Childhood rhabdomyosarcoma metastatic to bone marrow presenting with disseminated intravascular coagulation and acute tumour lysis syndrome: review of the literature apropos of two cases

The paper presents diagnostic and therapeutic difficulties in two adolescents with widespread rhabdomyosarcoma (RMS) presenting with severe haemorrhages resulting from disseminated intravascular coagulation (DIC) and with laboratory features of acute tumour lysis syndrome (ATLS). Other published cases of childhood RMS with DIC at admission have been listed and reviewed. It has been concluded that the clinical picture of a widespread RMS in children may resemble acute hematologic malignancy and pose a big diagnostic problem. That is why the presence of small blue round cells morphologically similar to lymphoblasts and/or myeloblasts in bone marrow (BM), lacking hematopoietic makers, should prompt the pathologist to consider possible diagnosis of RMS. Inclusion of desmin, MyoD1 and myogenin Myf4 to the immunohistochemical panel is obligatory in such cases. When the representative histopathological tumour specimens are difficult to obtain, the flow cytometric immunophenotyping of BM metastases could help the standard morphological/immunohistological diagnostic procedures and advance the diagnosis. Recently, the flow cytometric CD45− CD56+ immunophenotype together with Myf4 transcript has been assigned to RMS cells infiltrating BM. In children with disseminated RMS complicated with DIC rapid polychemotherapy aimed at diminishing the malignancy-triggered procoagulant activity should be initiated. However, in cases with concomitant ATLS the initial doses of chemotherapy should be reduced and the metabolic disorders and renal function monitored. The prognosis in children with RMS metastatic to BM with signs of DIC or ATLS at admission depends on the response to chemotherapy, however generally it is highly disappointing