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Remission of Congenital Nephrotic Syndrome from Diffuse Mesangial Sclerosis with IVIG Therapy
319
Interleukin-2 Directly Enhances Natural Killer Cell F-actin Reorganization in a Wiskott-Aldrich Syndrome in-vitro Model S. Roy-Ghanta, S. Maru, O. Isacof, J. S. Orange; University of Pennsylvania, Children’s Hospital of Philadelphia, Philadelphia, PA. RATIONALE: Patients with Wiskott-Aldrich Syndrome (WAS) have defects in natural killer (NK) cells which can be rescued by IL-2. We have recapitulated the WAS NK phenotype by utilizing a selective, reversible inhibitor of Wiskott-Aldrich Syndrome Protein (WASP) called Wiskostatin. This study was devised to evaluate the effects of IL-2 on filamentous actin (F-actin) content in Wiskostatin treated and untreated NK cells. METHODS: NK cells were control-treated or treated with Wiskostatin in the absence or presence of exogenous IL-2. F-actin content was determined by flow cytometry after staining with Alexafluor 647-conjugated phalloidin. Distribution of actin in GFP actin transduced NK cells was also observed using real time fluorescence microscopy. Cytotoxicity against EBV-transformed MHC-class I negative B cells was evaluated using 51Cr release assays. RESULTS: We were surprised to find via flow cytometry that IL-2 alone increased F-actin content of NK cells. This demonstrates a direct effect of IL-2 on the cytoskeleton in absence of other stimulatory signals. Similarly, microscopy revealed a shift of GFP-actin to the periphery of the NK cell after treatment with IL-2 consistent with increased F-actin accumulation. Wiskostatin alone decreased F-actin content and cytotoxicity consistent with a blockade of WASP. This effect of Wiskostatin was mostly reversed when IL-2 was added to Wiskostatin treated NK cells. CONCLUSIONS: IL-2 directly stimulates cytoskeleton reorganization alone and in the presence of Wiskostatin. Ongoing studies will elucidate how IL-2 bypasses this blockade. Funding: AAAAI Altana Award
320
Remission of Congenital Nephrotic Syndrome from Diffuse Mesangial Sclerosis with IVIG Therapy J. E. Gentner1, E. G. Wood1, C. A. Vogler1, A. M. Beck2, A. P. Knutsen1; 1 Saint Louis University, St. Louis, MO, 2Washington University, St. Louis, MO. RATIONALE: Patients with congenital nephrotic syndrome have hypogammaglobulinemia secondary to proteinuria, which results in increased susceptibility to infection. IVIG, used until definitive treatment of kidney transplantation could be performed, appears to have altered the natural course of renal disease in three patients with congenital nephrotic syndrome with diffuse mesangial sclerosis. METHODS: Three patients with congenital nephrotic syndrome were given regular infusions of IVIG over a span of several years. Patient 1 underwent repeat kidney biopsy at age 8 months, 4 years, and 14 years. RESULTS: Although there is often significant morbidity and mortality associated with congenital nephrotic syndrome, all three of these patients have undergone clinical remission. In patient 1, attempts to wean the frequency of IVIG infusion resulted in significant proteinuria and hypoalbuminemia which subsequently resolved when monthly IVIG was resumed. Biopsies at presentation and at age 8 months showed diffuse mesangial sclerosis. Repeat biopsies at 4 years and 14 years of age have shown remarkable reduction in mesangial sclerosis. Patient 1 is still receiving IVIG. Patient 2 was on peritoneal dialysis at the time of IVIG initiation. The patient’s proteinuria resolved over a period of several months, and dialysis was eventually discontinued. Patient 3 also had improvement in proteinuria and hypoalbuminemia while on IVIG therapy. CONCLUSION: Administration of IVIG in these three patients with congenital nephrotic syndrome with diffuse mesangial sclerosis led to resolution of proteinuria and improvement in renal function, making renal transplant unnecessary. Funding: Saint Louis University
321
Recombinant Human Hyaluronidase Facilitates Dispersion of Subcutaneously Administered Gammagard Liquid and Enables Administration of a Full Monthly Dose in a Single Site to Patients with Immunodeficiency Diseases I. R. Melamed1, M. R. Stein2, R. L. Wasserman3, H. Leibl4, W. Engl4,
R. C. Yocum5, R. I. Schiff6; 1First Allergy and Clinical Research Center, Centennial, CO, 2Allergy Associates of the Palm Beaches PA, N Palm Beach, FL, 3Dallas Allergy, Dallas, TX, 4Baxter, Vienna, AUSTRIA, 5 Halozyme, San Diego, CA, 6Baxter, Westlake Village, CA. RATIONALE: Compared to intravenous infusions, subcutaneous administration of gammaglobulin reduces the incidence of systemic reactions, does not require sometimes-difficult iv access, improves trough levels, and gives patients more independence. The main drawback is the need for multiple sites weekly due to the limited ability of tissue to accept large volumes. Recombinant human hyaluronidase (rHuPH20, Halozyme) transiently cleaves hyaluronic acid enzymatically in the subcutaneous tissue, facilitating dispersion of solutions. METHODS: This pilot study determined the amount of enzyme required to enable a monthly dose of Gammagard Liquid 10% (GGL, Baxter BioScience) to be infused in a single site at rates equivalent to iv infusions. Also evaluated was the effect of rHuPH20 on bioavailability of GGL given subcutaneously compared to intravenously. Eleven immunodeficient patients were infused with varying amounts of rHuPH20 with one, two, three, and then four week doses of GGL. RESULTS: Ten of the patients achieved monthly doses of 25.5 to 61.2 grams (255 to 612 ml) in a single site, at rates of 120 to 300 ml/hr. The maximum rate was determined by pump characteristics, not patient tolerability. A minimum of 50 U rHuPH20/gm GGL was required to achieve these rates. One patient withdrew from the study citing moderate discomfort with the one-week dose. The ten patients completing the study experienced only mild local reactions, such as swelling and redness; no drug-related allergic reactions occurred. The effect of rHuPH20 on bioavailability is being evaluated. CONCLUSIONS: rHuPH20-enabled single site subcutaneous administration of a monthly dose of more than 400 mg/kg is feasible. Funding: Baxter/Halozyme
322
Asthma and Steroid use in 462 Survey Respondents with Primary Immune Deficiency on IVIg L. Estrella, C. S. Patel, L. R. Forbes, C. Cunningham-Rundles; Mt. Sinai School of Medicine, New York, NY. RATIONALE: Primary immune deficiency (PID) leads to recurrent sinopulmonary tract infections. Delay in diagnosis leads to increased morbidity. Recent work indicates that subjects with clinical illnesses characteristic of PID have often been given the diagnosis of asthma; this study investigates if this is true for a cohort of subjects diagnosed and treated for specific PID conditions. METHODS: Between 9/19/06 and 1/2/07 subscribers of I.G. Living!, a free publication with a national circulation of 25,000 sent to people receiving IVIg, were directed to an online 59 question survey. RESULTS: Respondents included 596 subjects; excluding those with malignancies or autoimmune disease, 462 remained. Age ranged from infancy to 601 years with 20% between 41-50 years old. Reported PIDs included CVID, IgG subclass deficiency, antibody deficiency, hypogammaglobulinemia, IgA deficiency, XLA, complement deficiency, SCID, neutropenia, DiGeorge syndrome, Hyper-IgE, Hyper-IgM, NK defect, Specific polysaccharide antibody deficiency, IgM deficiency, NEMO and T-cell defects. 21 patients (4%) could not recall their specific diagnosis and 3 have not yet been given a diagnosis. Of the group, 90% had sinusitis, 71% bronchitis, 61% otitis, 55% pneumonia and 26% meningitis or sepsis. Almost all (93%) reported at least one hospitalization. Surprisingly, asthma had been diagnosed in 270 patients (58%) and of those, 87% had been given oral and/or inhaled glucocorticoids. CONCLUSIONS: More than half of these patients diagnosed with PID have also been diagnosed with asthma and the great majority has been given corticosteroids. The benefit or ill effects of this therapy in subjects with diagnosed PID has not been previously examined. Funding: National Institutes of Health AI-48693
SUNDAY
Abstracts S83
J ALLERGY CLIN IMMUNOL VOLUME 121, NUMBER 2
Interleukin-2 Directly Enhances Natural Killer Cell F-actin Reorganization in a Wiskott-Aldrich Syndrome in-vitro Model S. Roy-Ghanta, S. Maru, O. Isacof, J. S. Orange; University of Pennsylvania, Children’s Hospital of Philadelphia, Philadelphia, PA. RATIONALE: Patients with Wiskott-Aldrich Syndrome (WAS) have defects in natural killer (NK) cells which can be rescued by IL-2. We have recapitulated the WAS NK phenotype by utilizing a selective, reversible inhibitor of Wiskott-Aldrich Syndrome Protein (WASP) called Wiskostatin. This study was devised to evaluate the effects of IL-2 on filamentous actin (F-actin) content in Wiskostatin treated and untreated NK cells. METHODS: NK cells were control-treated or treated with Wiskostatin in the absence or presence of exogenous IL-2. F-actin content was determined by flow cytometry after staining with Alexafluor 647-conjugated phalloidin. Distribution of actin in GFP actin transduced NK cells was also observed using real time fluorescence microscopy. Cytotoxicity against EBV-transformed MHC-class I negative B cells was evaluated using 51Cr release assays. RESULTS: We were surprised to find via flow cytometry that IL-2 alone increased F-actin content of NK cells. This demonstrates a direct effect of IL-2 on the cytoskeleton in absence of other stimulatory signals. Similarly, microscopy revealed a shift of GFP-actin to the periphery of the NK cell after treatment with IL-2 consistent with increased F-actin accumulation. Wiskostatin alone decreased F-actin content and cytotoxicity consistent with a blockade of WASP. This effect of Wiskostatin was mostly reversed when IL-2 was added to Wiskostatin treated NK cells. CONCLUSIONS: IL-2 directly stimulates cytoskeleton reorganization alone and in the presence of Wiskostatin. Ongoing studies will elucidate how IL-2 bypasses this blockade. Funding: AAAAI Altana Award
320
Remission of Congenital Nephrotic Syndrome from Diffuse Mesangial Sclerosis with IVIG Therapy J. E. Gentner1, E. G. Wood1, C. A. Vogler1, A. M. Beck2, A. P. Knutsen1; 1 Saint Louis University, St. Louis, MO, 2Washington University, St. Louis, MO. RATIONALE: Patients with congenital nephrotic syndrome have hypogammaglobulinemia secondary to proteinuria, which results in increased susceptibility to infection. IVIG, used until definitive treatment of kidney transplantation could be performed, appears to have altered the natural course of renal disease in three patients with congenital nephrotic syndrome with diffuse mesangial sclerosis. METHODS: Three patients with congenital nephrotic syndrome were given regular infusions of IVIG over a span of several years. Patient 1 underwent repeat kidney biopsy at age 8 months, 4 years, and 14 years. RESULTS: Although there is often significant morbidity and mortality associated with congenital nephrotic syndrome, all three of these patients have undergone clinical remission. In patient 1, attempts to wean the frequency of IVIG infusion resulted in significant proteinuria and hypoalbuminemia which subsequently resolved when monthly IVIG was resumed. Biopsies at presentation and at age 8 months showed diffuse mesangial sclerosis. Repeat biopsies at 4 years and 14 years of age have shown remarkable reduction in mesangial sclerosis. Patient 1 is still receiving IVIG. Patient 2 was on peritoneal dialysis at the time of IVIG initiation. The patient’s proteinuria resolved over a period of several months, and dialysis was eventually discontinued. Patient 3 also had improvement in proteinuria and hypoalbuminemia while on IVIG therapy. CONCLUSION: Administration of IVIG in these three patients with congenital nephrotic syndrome with diffuse mesangial sclerosis led to resolution of proteinuria and improvement in renal function, making renal transplant unnecessary. Funding: Saint Louis University
321
Recombinant Human Hyaluronidase Facilitates Dispersion of Subcutaneously Administered Gammagard Liquid and Enables Administration of a Full Monthly Dose in a Single Site to Patients with Immunodeficiency Diseases I. R. Melamed1, M. R. Stein2, R. L. Wasserman3, H. Leibl4, W. Engl4,
R. C. Yocum5, R. I. Schiff6; 1First Allergy and Clinical Research Center, Centennial, CO, 2Allergy Associates of the Palm Beaches PA, N Palm Beach, FL, 3Dallas Allergy, Dallas, TX, 4Baxter, Vienna, AUSTRIA, 5 Halozyme, San Diego, CA, 6Baxter, Westlake Village, CA. RATIONALE: Compared to intravenous infusions, subcutaneous administration of gammaglobulin reduces the incidence of systemic reactions, does not require sometimes-difficult iv access, improves trough levels, and gives patients more independence. The main drawback is the need for multiple sites weekly due to the limited ability of tissue to accept large volumes. Recombinant human hyaluronidase (rHuPH20, Halozyme) transiently cleaves hyaluronic acid enzymatically in the subcutaneous tissue, facilitating dispersion of solutions. METHODS: This pilot study determined the amount of enzyme required to enable a monthly dose of Gammagard Liquid 10% (GGL, Baxter BioScience) to be infused in a single site at rates equivalent to iv infusions. Also evaluated was the effect of rHuPH20 on bioavailability of GGL given subcutaneously compared to intravenously. Eleven immunodeficient patients were infused with varying amounts of rHuPH20 with one, two, three, and then four week doses of GGL. RESULTS: Ten of the patients achieved monthly doses of 25.5 to 61.2 grams (255 to 612 ml) in a single site, at rates of 120 to 300 ml/hr. The maximum rate was determined by pump characteristics, not patient tolerability. A minimum of 50 U rHuPH20/gm GGL was required to achieve these rates. One patient withdrew from the study citing moderate discomfort with the one-week dose. The ten patients completing the study experienced only mild local reactions, such as swelling and redness; no drug-related allergic reactions occurred. The effect of rHuPH20 on bioavailability is being evaluated. CONCLUSIONS: rHuPH20-enabled single site subcutaneous administration of a monthly dose of more than 400 mg/kg is feasible. Funding: Baxter/Halozyme
322
Asthma and Steroid use in 462 Survey Respondents with Primary Immune Deficiency on IVIg L. Estrella, C. S. Patel, L. R. Forbes, C. Cunningham-Rundles; Mt. Sinai School of Medicine, New York, NY. RATIONALE: Primary immune deficiency (PID) leads to recurrent sinopulmonary tract infections. Delay in diagnosis leads to increased morbidity. Recent work indicates that subjects with clinical illnesses characteristic of PID have often been given the diagnosis of asthma; this study investigates if this is true for a cohort of subjects diagnosed and treated for specific PID conditions. METHODS: Between 9/19/06 and 1/2/07 subscribers of I.G. Living!, a free publication with a national circulation of 25,000 sent to people receiving IVIg, were directed to an online 59 question survey. RESULTS: Respondents included 596 subjects; excluding those with malignancies or autoimmune disease, 462 remained. Age ranged from infancy to 601 years with 20% between 41-50 years old. Reported PIDs included CVID, IgG subclass deficiency, antibody deficiency, hypogammaglobulinemia, IgA deficiency, XLA, complement deficiency, SCID, neutropenia, DiGeorge syndrome, Hyper-IgE, Hyper-IgM, NK defect, Specific polysaccharide antibody deficiency, IgM deficiency, NEMO and T-cell defects. 21 patients (4%) could not recall their specific diagnosis and 3 have not yet been given a diagnosis. Of the group, 90% had sinusitis, 71% bronchitis, 61% otitis, 55% pneumonia and 26% meningitis or sepsis. Almost all (93%) reported at least one hospitalization. Surprisingly, asthma had been diagnosed in 270 patients (58%) and of those, 87% had been given oral and/or inhaled glucocorticoids. CONCLUSIONS: More than half of these patients diagnosed with PID have also been diagnosed with asthma and the great majority has been given corticosteroids. The benefit or ill effects of this therapy in subjects with diagnosed PID has not been previously examined. Funding: National Institutes of Health AI-48693
SUNDAY
Abstracts S83
J ALLERGY CLIN IMMUNOL VOLUME 121, NUMBER 2