Establishment of a Monoclonal Antibody That Recognizes Cysteine-Rich Domain 1 of Human CD271.

CD271 is a common receptor for all neurotrophins that is localized to neurons, endothelial cells, and the basal layer of the epithelium in normal tissue. Recently, we and others reported that CD271 plays essential roles in the development of squamous cell carcinoma, especially in tumor-initiating cells. Since little is known about how CD271 regulates cancer cell initiation and proliferation, antibodies that recognize different domains of CD271 are needed to enable investigation. Therefore, this study aimed to develop an antihuman CD271 antibody by immunizing mice with a CD271 antigen produced by a baculovirus. The antibody was named hCD271mAb#13, and it recognized cysteine-rich domain 1 with a higher affinity than the commercially available antibody ME20.4. We determined that hCD271mAb#13 is suitable for flow cytometry, Western blotting, immunocytochemistry, and immunohistochemistry of formalin-fixed paraffin-embedded tissue. Use of hCD271mAb#13 for CD271 labeling could enable detailed analyses of cancer cell regulation and other biological processes.

CD271+ Human Mesenchymal Stem Cells Show Antiarrhythmic Effects in a Novel Murine Infarction Model.

BACKGROUND: Ventricular arrhythmias (VA) are a common cause of sudden death after myocardial infarction (MI). Therefore, developing new therapeutic methods for the prevention and treatment of VA is of prime importance. METHODS: Human bone marrow derived CD271+ mesenchymal stem cells (MSC) were tested for their antiarrhythmic effect. This was done through the development of a novel mouse model using an immunocompromised Rag2-/- γc-/- mouse strain subjected to myocardial "infarction-reinfarction". The mice underwent a first ischemia-reperfusion through the left anterior descending (LAD) artery closure for 45 minutes with a subsequent second permanent LAD ligation after seven days from the first infarct. RESULTS: This mouse model induced various types of VA detected with continuous electrocardiogram (ECG) monitoring via implanted telemetry device. The immediate intramyocardial delivery of CD271+ MSC after the first MI significantly reduced VA induced after the second MI. CONCLUSIONS: In addition to the clinical relevance, more closely reflecting patients who suffer from severe ischemic heart disease and related arrhythmias, our new mouse model bearing reinfarction warrants the time required for stem cell engraftment and for the first time enables us to analyze and verify significant antiarrhythmic effects of human CD271+ stem cells in vivo.

Anatomy, immunohistochemistry, and numerical distribution of human splenic microvessels.

The microvascular architecture of the spleen plays an important role in the immunological function of this organ. The different types of vessels are related to different reticular cells each with their own immunomodulatory functions. The present study describes an immunohistochemical and morphometric analysis of the various types of vessels in 21 human autopsy non-pathological splenic samples. On an area of 785,656.37 µm2 for each sample, we classified and quantified the type and number of vascular structures, each according to their morphology and immunohistochemical profile, and obtained the ratios between them. The distribution of trabecular vessels and the characteristics of the venules are reviewed. In our material the so-called "cavernous perimarginal sinus" (anatomical structure previously described by Schmidt et al., 1988) was observed and interpreted as a curvilinear venule shaped by the follicle in contact with the trabecular vein. Our material comprised 261 trabeculae (containing 269 arterial sections and 508 venous sections), 30,621 CD34+ capillaries, 7739 CD271+ sheathed capillaries, 2588 CD169+ sheathed capillaries, and 31,124 CD8+ sinusoids. The total area (TA) (14,765,714.88 µm2) occupied by the sinusoidal sections of the 21 cases was much higher than the TA of the capillary sections (1,700,269.83 µm2). Similarly, the TA (651,985 µm2) occupied by the sections of the trabecular veins was much higher than the TA of the trabecular arteries (88,594 µm2). The total number of CD34+ capillaries and of sinusoids CD8+ was similar for the sum of the 21 cases, nevertheless there were large differences in each case. Statistically the hypothesis that the number of capillaries and sinusoids are present with the same frequency is discarded. In view of the absence of a numerical correlation between capillaries and sinusoids, we postulate that very possibly the arterial and the venous vascular trees are two anatomically independent structures separated by the splenic cords. We believe that this is the first work where splenic microvascularization is simultaneously approached from a morphometric and immunohistochemical point of view.

Dermal CD271+ Cells are Closely Associated with Regeneration of the Dermis in the Wound Healing Process.

Stem cells have recently been shown to play important roles in wound healing. The aim of this study was to investigate the role of dermal CD271+ cells in wound healing. Full-thickness wounds were produced on the backs of 5-year-old and 24-week-old mice, and time-course of wound closure, CD271+ cell counts, and gene expression levels were compared. Delayed wound healing was observed in 24-week-old mice. The peak of CD271+ cell increase was delayed in 24-week-old mice, and gene expression levels of growth factors in wounded tissue were significantly increased in 5-year-old mice. Dermal CD271+ cells purified by fluorescence-activated cell sorting (FACS) expressed higher growth factors than CD271- cells, suggesting that CD271+ cells play important roles by producing growth factors. This study also investigated dermal CD271+ cells in patients with chronic skin ulcers. Dermal CD271+ cells in patients were significantly reduced compared with in healthy controls. Thus, dermal CD271+ cells are closely associated with wound healing.

Antibody Therapy Targeting CD47 and CD271 Effectively Suppresses Melanoma Metastasis in Patient-Derived Xenografts.

The high rate of metastasis and recurrence among melanoma patients indicates the existence of cells within melanoma that have the ability to both initiate metastatic programs and bypass immune recognition. Here, we identify CD47 as a regulator of melanoma tumor metastasis and immune evasion. Protein and gene expression analysis of clinical melanoma samples reveals that CD47, an anti-phagocytic signal, correlates with melanoma metastasis. Antibody-mediated blockade of CD47 coupled with targeting of CD271(+) melanoma cells strongly inhibits tumor metastasis in patient-derived xenografts. This therapeutic effect is mediated by drastic changes in the tumor and metastatic site immune microenvironments, both of whichwhich exhibit greatly increased density of differentiated macrophages and significantly fewer inflammatory monocytes, pro-metastatic macrophages (CCR2(+)/VEGFR1(+)), and neutrophils, all of which are associated with disease progression. Thus, antibody therapy that activates the innate immune response in combination with selective targeting of CD271(+) melanoma cells represents a powerful therapeutic approach against metastatic melanoma.

Expression of natural killer cell regulatory microRNA by uveal melanoma cancer stem cells.

Natural killer (NK) cells are implicated in the control of metastasis in uveal melanoma, a process that has been ascribed to its cancer stem cell subpopulation. NK cell activation is regulated by specific microRNA (miR). The NK cell sensitivity and regulatory miR production of uveal melanoma cancer stem cells was examined. Cancer stem cells enriched from aggressively metastatic MUM2B uveal melanoma cells by selecting CD271+ cells or propagating as non-adherent spheres in stem-cell supportive were more resistant to NK cell cytolysis than cancer stem cells enriched from less aggressively metastatic OCM1 uveal melanoma cells. Both MUM2B and OCM1 cells expressed and secreted NK cell regulatory miRs, including miR 146a, 181a, 20a, and 223. MUM2B cells expressed and secreted miR-155; OCM1 cells did not. Transfecting MUM2B cells with anti-miR-155 increased NK cell sensitivity. CD271+ cells were identified in the blood of patients with metastatic uveal melanoma and were characterized by low expression of melanocyte differentiation determinants and by the ability to form non-adherent spheres in stem-cell supportive media. These cells also expressed NK cell regulatory miRs, including miR-155. These results indicate that uveal melanoma cancer stem cells can vary in their sensitivity to NK cell lysis and their expression of NK cell regulatory miRs. Circulating CD271+ cells from patients with metastatic uveal melanoma manifest cancer stem cell features and express miRs associated with NK cell suppression, including miR-155, that may contribute to metastatic progression.

Preclinical and Clinical Evidence of Mycobacterium tuberculosis Persistence in the Hypoxic Niche of Bone Marrow Mesenchymal Stem Cells after Therapy.

Mycobacterium tuberculosis (MTB), the causative agent of pulmonary tuberculosis, is difficult to eliminate by antibiotic therapy. We recently identified CD271(+) bone marrow-mesenchymal stem cells (BM-MSCs) as a potential site of MTB persistence after therapy. Herein, we have characterized the potential hypoxic localization of the post-therapy MTB-infected CD271(+) BM-MSCs in both mice and human subjects. We first demonstrate that in a Cornell model of MTB persistence in mice, green fluorescent protein-labeled virulent MTB-strain H37Rv was localized to pimonidazole (an in vivo hypoxia marker) positive CD271(+) BM-MSCs after 90 days of isoniazid and pyrazinamide therapy that rendered animal's lung noninfectious. The recovered CD271(+) BM-MSCs from post-therapy mice, when injected into healthy mice, caused active tuberculosis infection in the animal's lung. Moreover, MTB infection significantly increased the hypoxic phenotype of CD271(+) BM-MSCs. Next, in human subjects, previously treated for pulmonary tuberculosis, the MTB-containing CD271(+) BM-MSCs exhibited high expression of hypoxia-inducible factor 1α and low expression of CD146, a hypoxia down-regulated cell surface marker of human BM-MSCs. These data collectively demonstrate the potential localization of MTB harboring CD271(+) BM-MSCs in the hypoxic niche, a critical microenvironmental factor that is well known to induce the MTB dormancy phenotype.