Prasanthi Chappa

Extracellular hemin crisis triggers acute chest syndrome in sickle mice

The prevention and treatment of acute chest syndrome (ACS) is a major clinical concern in sickle cell disease (SCD). However, the mechanism underlying the pathogenesis of ACS remains elusive. We tested the hypothesis that the hemolysis byproduct hemin elicits events that induce ACS. Infusion of a low dose of hemin caused acute intravascular hemolysis and autoamplification of extracellular hemin in transgenic sickle mice, but not in sickle-trait littermates. The sickle mice developed multiple symptoms typical of ACS and succumbed rapidly. Pharmacologic inhibition of TLR4 and hemopexin replacement therapy prior to hemin infusion protected sickle mice from developing ACS. Replication of the ACS-like phenotype in nonsickle mice revealed that the mechanism of lung injury due to extracellular hemin is independent of SCD. Using genetic and bone marrow chimeric tools, we confirmed that TLR4 expressed in nonhematopoietic vascular tissues mediated this lethal type of acute lung injury. Respiratory failure was averted after the onset of ACS-like symptoms in sickle mice by treating them with recombinant hemopexin. Our results reveal a mechanism that helps to explain the pathogenesis of ACS, and we provide proof of principle for therapeutic strategies to prevent and treat this condition in mice.

Lectins Identify Glycan Biomarkers on Glioblastoma-Derived Cancer Stem Cells

Glioblastoma (GBM) is a highly aggressive primary brain tumor with a poor prognosis. Despite aggressive therapy with surgery, radiotherapy, and chemotherapy, nearly all patients succumb to disease within 2 years. Several studies have supported the presence of stem-like cells in brain tumor cultures that are CD133-positive, are capable of self-renewal, and give rise to all cell types found within the tumor, potentially perpetuating growth. CD133 is a widely accepted marker for glioma-derived cancer stem cells; however, its reliability has been questioned, creating a need for other identifiers of this biologically important subpopulation. We used a panel of 20 lectins to identify differences in glycan expression found in the glycocalyx of undifferentiated glioma-derived stem cells and differentiated cells that arise from them. Fluorescently labeled lectins that specifically recognize α-N-acetylgalactosamine (GalNAc) and α-N-acetylglucosamine (GlcNAc) differentially bound to the cell surface based on the state of cellular differentiation. GalNAc and GlcNAc were highly expressed on the surface of undifferentiated cells and showed markedly reduced expression over a 12-day duration of differentiation. Additionally, the GalNAc-recognizing lectin Dolichos biflorus agglutinin was capable of specifically selecting and sorting glioma-derived stem cell populations from an unsorted tumor stock and this subpopulation had proliferative properties similar to CD133(+) cells in vitro and also had tumor-forming capability in vivo. Our preliminary results on a single cerebellar GBM suggest that GalNAc and GlcNAc are novel biomarkers for identifying glioma-derived stem cells and can be used to isolate cancer stem cells from unsorted cell populations, thereby creating new cell lines for research or clinical testing.

Alloimmunization to transfused HOD red blood cells is not increased in mice with sickle cell disease

BACKGROUND: Increased rates of red blood cell (RBC) alloimmunization in patients with sickle cell disease may be due to transfusion frequency, genetic predisposition, or immune dysregulation. To test the hypothesis that sickle cell pathophysiology influences RBC alloimmunization, we utilized two transgenic mouse models of sickle cell disease.

Body composition and grip strength are improved in transgenic sickle mice fed a high-protein diet.

Key pathophysiology of sickle cell anaemia includes compensatory erythropoiesis, vascular injury and chronic inflammation, which divert amino acids from tissue deposition for growth/weight gain and muscle formation. We hypothesised that sickle mice maintained on an isoenergetic diet with a high percentage of energy derived from protein (35 %), as opposed to a standard diet with 20 % of energy derived from protein, would improve body composition, bone mass and grip strength. Male Berkeley transgenic sickle mice (S; n 8–12) were fed either 20 % (S20) or 35 % (S35) diets for 3 months. Grip strength (BIOSEB meter) and body composition (dual-energy X-ray absorptiometry scan) were measured. After 3 months, control mice had the highest bone mineral density (BMD) and bone mineral content (BMC) (P < 0·005). S35 mice had the largest increase in grip strength. A two-way ANOVA of change in grip strength (P = 0·043) attributed this difference to genotype (P = 0·025) and a trend in type of diet (P = 0·067). l-Arginine (l-Arg) supplementation of the 20 % diet was explored, as a possible mechanism for improvement obtained with the 35 % diet. Townes transgenic sickle mice (TS; n 6–9) received 0·8, 1·6, 3·2 or 6·4 % l-Arg based on the same protocol and outcome measures used for the S mice. TS mice fed 1·6 % l-Arg for 3 months (TS1.6) had the highest weight gain, BMD, BMC and lean body mass compared with other groups. TS3.2 mice showed significantly more improvement in grip strength than TS0·8 and TS1.6 mice (P < 0·05). In conclusion, the high-protein diet improved body composition and grip strength. Outcomes observed with TS1.6 and TS3.2 mice, respectively, confirm the hypothesis and reveal l-Arg as part of the mechanism.

Renal protection by atorvastatin in a murine model of sickle cell nephropathy

Recent studies have demonstrated pleiotropic effects of statins in various mouse models of kidney disease. In this study, Townes humanized sickle cell mice were treated for 8 weeks with atorvastatin at a dose of 10 mg/kg/day starting at 10 weeks of age. Treatment with atorvastatin significantly reduced albuminuria, and improved both urine concentrating ability and glomerular filtration rate. Atorvastatin also decreased markers of kidney injury and endothelial activation, and ameliorated oxidant stress in renal tissues and peripheral macrophages. Atorvastatin downregulated the expression of mRNA levels of the NADPH oxidases, Cybb (also termed Nox2) and Nox4, which are major sources of oxidant stress in the kidney. These findings highlight the pleiotropic effects of atorvastatin and suggest that it may provide beneficial effects in sickle cell nephropathy.

Alloimmunization to transfused HOD red blood cells is not increased in mice with sickle cell disease: ALLOIMMUNIZATION IN SICKLE CELL MICE

BACKGROUND: Increased rates of red blood cell (RBC) alloimmunization in patients with sickle cell disease may be due to transfusion frequency, genetic predisposition, or immune dysregulation. To test the hypothesis that sickle cell pathophysiology influences RBC alloimmunization, we utilized two transgenic mouse models of sickle cell disease.

Alloimmunization to transfused HOD RBCs is not increased in mice with sickle cell disease

BACKGROUND: Increased rates of red blood cell (RBC) alloimmunization in patients with sickle cell disease may be due to transfusion frequency, genetic predisposition, or immune dysregulation. To test the hypothesis that sickle cell pathophysiology influences RBC alloimmunization, we utilized two transgenic mouse models of sickle cell disease.