The human alpha defensin HD5 neutralizes JC polyomavirus infection by reducing endoplasmic reticulum traffic and stabilizing the viral capsid.

Progressive multifocal leukoencephalopathy (PML) is a fatal disease with limited treatment options, both clinically and in the research pipeline. Potential therapies would target and neutralize its etiologic agent, JC polyomavirus (JCPyV). The innate immune response to JCPyV infection has not been studied, and little is known about the initial host response to polyomavirus infection. This study examined the ability of a human alpha defensin, HD5, to neutralize JCPyV infection in human fetal glial cells. We show that HD5, by binding to the virion, blocks infection. The JCPyV-HD5 complexes bind to and enter host cells but are reduced in their ability to reach the endoplasmic reticulum (ER), where virions are normally uncoated. Furthermore, HD5 binding to the virion stabilizes the capsid and prevents genome release. Our results show that HD5 neutralizes JCPyV infection at an early postentry step in the viral life cycle by stabilizing the viral capsid and disrupting JCPyV trafficking. This study provides a naturally occurring platform for developing antivirals to treat PML and also expands on the known capabilities of human defensins.

Infusion of Lin- bone marrow cells results in multilineage macrochimerism and skin allograft tolerance in minimally conditioned recipient mice.

Donor-specific immunological tolerance using high doses of donor bone marrow cells (BMC) has been demonstrated in mixed chimerism-based tolerance induction protocols; however, the development of graft versus host disease (GVHD) remains a risk. In the present study, we demonstrate that the infusion of low numbers of donor Lin(-) bone marrow cells (Lin(-) BMC) 7 days post allograft transplantation facilitates high level macrochimerism induction and graft tolerance. Full-thickness BALB/c skin allografts were transplanted onto C57BL/6 mice. Mice were treated with anti-CD4 and anti-CD8 mAbs on day 0, +2, +5, +7 and +14 along with low dose busulfan on day +5. A low dose of highly purified Lin(-) BMC from BALB/c donor mice was infused on day +7. Chimerism and clonal cell deletion were evaluated using flow cytometry. Donor-specific tolerance was tested by donor and third-party skin grafting and mixed leukocyte reaction (MLR). Lin(-) BMC infusion with minimal immunosuppression led to stable, mixed, multilineage macrochimerism and long-term allograft survival (>300 days). Mixed donor-recipient macrochimerism was observed. Donor-reactive T cells were clonally deleted and a 130% increase in CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) was observed in the spleen. Tolerant mice subsequently accepted second donor, but not third-party (C3H), skin grafts and recipient splenocytes failed to react with allogeneic donor cells indicating donor-specific immunological tolerance was achieved. We conclude that the infusion of donor Lin(-) BMC without cytoreductive recipient conditioning can induce indefinite survival of skin allografts via mechanisms involving the establishment of a multilineage macrochimeric state principally through clonal deletion of alloreactive T cells and peripherally induced CD4(+)Foxp3(+) Tregs.

Comparative analysis of angiogenic gene expression in normal and impaired wound healing in diabetic mice: effects of extracorporeal shock wave therapy.

Impaired wound healing is a persistent clinical problem which has been treated with mixed results. Studies aimed at elucidating the mechanism of impaired wound healing have focused on small cohorts of genes which leave an incomplete picture of the wound healing process. We aimed to investigate impaired wound healing via a comprehensive panel of angiogenic/inflammation-related genes and wound closure kinetics with and without the application of extracorporeal shock wave therapy (ESWT), which has been demonstrated to improve wound healing. Full-thickness skin from the dorsal surface of "normal" (BALB/c) and "impaired" (db (+)/db (+)) mice was excised, and wound margin tissue was harvested 2, 7, and 10 days post injury. A separate, but identical wound model was established over 40 days in order to measure wound closure kinetics. Over time, the normal non-ESWT treated wounds exhibited varying patterns of elevated expression of 25-30 genes, whereas wounds with impaired healing displayed prolonged elevated expression of only a few genes (CXCL2, CXCL5, CSF3, MMP9, TGF-α). In response to ESWT, gene expression was augmented in both types of wounds, especially in the expression of PECAM-1; however, ESWT had no effect on wound closure in either model. In addition, multiple doses of ESWT exacerbated the delayed wound healing, and actually caused the wounds to initially increase in size. These data provide a more complete picture of impaired wound healing, and a way to evaluate various promising treatments.

Regulation of kidney development by Shp2: an unbiased stereological analysis.

Genes that regulate renal branching morphogenesis are likely to indirectly regulate nephron endowment, but few have been validated to do so in vivo. PTPN11, which encodes the nonreceptor protein tyrosine phosphatase Shp2, acts downstream of receptor tyrosine kinases to modulate the Ras-MAPK pathway and has been implicated in branching morphogenesis in vitro and in invertebrates, and is therefore a candidate in vivo regulator of nephron number. In this work, heterozygous null mutant Shp2(+/-) mice at postnatal days 30-35 were compared with their wild-type (WT) littermates using unbiased stereology to determine if, indeed, the former had decreased nephron number due to their 50% decrease in gene/protein dosage. Although there was a trend toward decreases in total glomerular (nephron) number and kidney volume in Shp2(+/-) mice compared with WT, neither difference was statistically significant (11310 vs. 12198 glomeruli, P = 0.22; 62.8 mm(3) vs. 66.0 mm(3) renal volume; P = 0.40). We conclude that loss of 50% gene/protein dosage of PTPN11/Shp2 is insufficient to affect glomerular (and thereby nephron) number in mouse kidneys in vivo.

Wound trauma mediated inflammatory signaling attenuates a tissue regenerative response in MRL/MpJ mice.

BACKGROUND: Severe trauma can induce pathophysiological responses that have marked inflammatory components. The development of systemic inflammation following severe thermal injury has been implicated in immune dysfunction, delayed wound healing, multi-system organ failure and increased mortality. METHODS: In this study, we examined the impact of thermal injury-induced systemic inflammation on the healing response of a secondary wound in the MRL/MpJ mouse model, which was anatomically remote from the primary site of trauma, a wound that typically undergoes scarless healing in this specific strain. Ear-hole wounds in MRL/MpJ mice have previously displayed accelerated healing and tissue regeneration in the absence of a secondary insult. RESULTS: Severe thermal injury in addition to distal ear-hole wounds induced marked local and systemic inflammatory responses in the lungs and significantly augmented the expression of inflammatory mediators in the ear tissue. By day 14, 61% of the ear-hole wounds from thermally injured mice demonstrated extensive inflammation with marked inflammatory cell infiltration, extensive ulceration, and various level of necrosis to the point where a large percentage (38%) had to be euthanized early during the study due to extensive necrosis, inflammation and ear deformation. By day 35, ear-hole wounds in mice not subjected to thermal injury were completely closed, while the ear-hole wounds in thermally injured mice exhibited less inflammation and necrosis and only closed partially (62%). Thermal injury resulted in marked increases in serum levels of IL-6, TNFalpha, KC (CXCL1), and MIP-2alpha (CXCL2). Interestingly, attenuated early ear wound healing in the thermally injured mouse resulted in incomplete tissue regeneration in addition to a marked inflammatory response, as evidenced by the histological appearance of the wound and increased transcription of potent inflammatory mediators. CONCLUSION: These findings suggest that the observed systemic inflammatory response of a severe thermal injury undoubtedly has an adverse effect on wound healing and tissue regeneration.

Timing of captopril administration determines radiation protection or radiation sensitization in a murine model of total body irradiation.

OBJECTIVE: Angiotensin II (Ang II), a potent vasoconstrictor, affects the growth and development of hematopoietic cells. Mixed findings have been reported for the effects of angiotensin-converting enzyme (ACE) inhibitors on radiation-induced injury to the hematopoietic system. We investigated the consequences of different regimens of the ACE inhibitor captopril on radiation-induced hematopoietic injury. MATERIALS AND METHODS: C57BL/6 mice were either sham-irradiated or exposed to (60)Co total body irradiation (0.6 Gy/min). Captopril was provided in the water for different time periods relative to irradiation. RESULTS: In untreated mice, the survival rate from 7.5 Gy was 50% at 30 days postirradiation. Captopril treatment for 7 days prior to irradiation resulted in radiosensitization with 100% lethality and a rapid decline in mature blood cells. In contrast, captopril treatment beginning 1 hour postirradiation and continuing for 30 days resulted in 100% survival, with improved recovery of mature blood cells and multilineage hematopoietic progenitors. In nonirradiated control mice, captopril biphasically modulated Lin(-) marrow progenitor cell cycling. After 2 days, captopril suppressed G(0)-G(1) transition and a greater number of cells entered a quiescent state. However, after 7 days of captopril treatment Lin(-) progenitor cell cycling increased compared to untreated control mice. CONCLUSION: These findings suggest that ACE inhibition affects hematopoietic recovery following radiation by modulating the hematopoietic progenitor cell cycle. The timing of captopril treatment relative to radiation exposure differentially affects the viability and repopulation capacity of spared hematopoietic stem cells and, therefore, can result in either radiation protection or radiation sensitization.

Angiogenic response to extracorporeal shock wave treatment in murine skin isografts.

Skin grafts are commonly utilized and proven effective methods of open wound coverage. Revascularization through neoangiogenesis is a pivotal mechanism for skin graft integration and durability. Extracorporeal shock-wave treatment (ESWT) has been demonstrated to accelerate wound repair; however, its mechanism-of-action is unclear. We investigated the role of ESWT in early revascularization of full-thickness skin isografts in a murine model. Cohorts of mice were euthanized and skin grafts were harvested 6 h, 2, 4, and 7 days post grafting +/- ESWT. Various aspects of graft neovascularization were measured including gross morphology, quantitative microscopy (vessel number, density), immunohistochemistry (CD31), cDNA SuperArrays for 84 angiogenesis-specific genes, and custom-designed 'Wound Repair' TaqMan Low Density Array (TLDA) cards to assess expression of 188 wound repair genes. We demonstrate that a single administration of ESWT immediately following skin grafting significantly enhances recipient graft revascularization (increased vessel number, size, and density). An augmented early pro-angiogenic and suppressed delayed pro-inflammatory response to ESWT was accompanied by significantly increased expression of both skin graft CD31 and angiogenesis pathway-specific genes, including ELR-CXC chemokines (CXCL1, CXCL2, CXCL5), CC chemokines (CCL2, CCL3, CCL4), cytokines (IL-1 beta, IL-6, G-CSF, VEGF-A), matrix metalloproteinases (MMP3, MMP9, MMP13), hypoxia-inducible factors (HIF-1 alpha), and vascular remodeling kinase (Mst1), as early as 6 h and up to 7 days post grafting and treatment. These findings suggest that early pro-angiogenic and anti-inflammatory effects of ESWT promote tissue revascularization and wound healing by augmenting angiogenesis and dampening inflammation.

Genistein induces radioprotection by hematopoietic stem cell quiescence.

PURPOSE: In this study we addressed whether genistein-induced radioprotection in mice is associated with alterations of the cell cycle of hematopoietic stem and progenitor cells. MATERIALS AND METHODS: C57BL/6J female mice received a single subcutaneous injection of genistein (200 mg/kg) 24 h prior to a lethal dose (7.75 Gy, (60)Co) of total body irradiation. Proliferation-associated Ki-67 protein/7-aminoactinomycin-D (Ki67/7AAD) cell cycle staining was used to differentiate between G(0), G(1), and S/G(2)/M in bone marrow cell populations negative for expression of mature hematopoietic lineage marker cells but positive for expression of stem cell antigen-1 and tyrosine kinase receptor for stem cell factor (Lin(-)Sca-1(+)cKit(+), LSK(+)). Quantitative real-time polymerase chain reaction (qRT-PCR) microarrays were utilized to examine cell cycle specific genes. RESULTS: At 24 h following radiation exposure, a greater percentage of LSK(+) in genistein-treated mice accumulated in the G(0) phase of the cell cycle, whereas a large percentage of LSK(+) bone marrow cells from untreated and vehicle (PEG-400)-treated mice progressed into the G(1) and S/G(2)/M phases. Moreover, the absolute number of marrow total LSK(+), long-term LSK(+), and short-term LSK(+) increased 2.8, 12.1, and 4.2-fold, respectively, at 7 days post-irradiation in genistein-treated vs. untreated irradiated mice. Lin(-) cells from genistein-treated mice expressed fewer DNA damage responsive and cell cycle checkpoint genes than LSK(+) from untreated or vehicle-treated mice. CONCLUSION: Pretreatment with genistein provides in vivo protection from acute myelotoxicity through extended quiescence followed by reduced senescence of marrow repopulating LSK(+).