Hydroxychloroquine Diminishes Lymphoproliferation in the Fas Deficient MRL/lpr−/− Murine Model of Autoimmune Lymphoproliferative Syndrome (ALPS).

Abstract Hydroxychloroquine (HCQ) is an anti-malarial drug in clinical use for decades that is finding further use as a steroid sparing agent in the treatment of immune disorders such as chronic GVHD, lupus and rheumatoid arthritis. HCQ is a lysosomotropic agent with more recent evidence showing immunomodulatory anti-TNF activity. It is currently being explored as a cytotoxic antineoplastic/antimicrobial agent. Hence, studies were conducted to determine the efficacy of HCQ to control the lymphoproliferation associated with ALPS. ALPS is an inherited disorder of apoptosis leading to lymphoproliferation and autoimmunity. The majority of ALPS patients are classified as Type Ia (>70%), having germline mutations in Fas. Other ALPS patients are classified as Type Ib, II, IV or III, if they have mutations in FasL, casapases, NRAS, or no identified mutations, respectively. They often present with childhood onset autoimmune cytopenias with lymphadenopathy, splenomegaly, increased circulating double negative T cells (DNT; TCRa/b+CD3+CD4−CD8−), defective apoptosis in vitro, and have an increased risk of lymphoma. Similarly, MRL/lpr−/− mice homozygous for Fas mutations develop an ALPS-like disease with massive lymphadenopathy, splenomegaly, hypergammaglobulinemia, autoimmune glomerulonephritis, and expansion of DNT cells secondary to defective lymphocyte apoptosis leading to lymphomagenesis. Currently, there are no proven therapies for the lymphoproliferation underlying ALPS itself. PBMCs from normal controls and ALPS Type Ia patients were cultured in vitro with 0–120 ug/mL HCQ in the presence or absence of 50 uM of the pan-caspase inhibitor Z-VAD-FMK, the caspase 9 inhibitor Z-LEHD-FMK, or the caspase 8 inhibitor Z-IETD-FMK. A dose response was observed with a high degree of cell death noted at 120 ug/mL after 48 hours, with an LD50 of 40 ug/mL. HCQ induced cell death was through a caspase-independent mechanism based on no inhibition of cell death by Z-VAD-FMK, Z-LEHD-FMK, or Z-IETD-FMK. Further preclinical studies were conducted in the MRL/lpr−/− murine model of ALPS. Forty five, 8-week old female MRL/lpr−/− mice were treated with 30 or 60 mg/kg of HCQ by gavage in sterile PBS or PBS alone three days per week for up to 14 weeks. Reductions of the spleen weight (p = 0.045, 437±85 vs 240±27) and cellularity (p = 0.08, 217±32 vs 148±18) were observed in high dose HCQ treated mice compared to controls at 7 weeks. Reductions in cellularity were also noted in the lymph nodes (p = 0.032, 118±33 vs 38±6). There was a trend towards decreasing DNT percentages in the spleen (21±5 vs 14±2), LN (70±4 vs 67±2) and blood (57±6 vs 51±5). Additionally, the CBC showed only marginal reductions in the WBC count (4.2±0.5 vs 3.5±0.3), hematocrit (49±0.3 vs 46±1.0), polymorphonuclear cells (33±3.7 vs 29±3.5) and monocytes (23±3.2 vs 17±2.5). Future studies are planned to more clearly determine the effect of HCQ on autoimmune kidney disease. Based on our in vitro and in vivo data, HCQ is effective at reducing lymphoproliferative activity in Fas deficient MRL/lpr−/− mice. It is being further explored in early phase clinical trials as a lympholytic agent to shrink lymph nodes and abrogate hypersplenism in ALPS patients. HCQ may also prove beneficial for long term use, especially in children, as a steroid sparing agent for treating refractory autoimmune cytopenias in ALPS.

Download Full-text

Valproic Acid (VPA), a Histone Deacetylase (HDAC) Inhibitor, Diminishes Lymphoproliferation in the Fas Deficient MRL/lpr−/− Murine Model of Autoimmune Lymphoproliferative Syndrome (ALPS).

Blood ◽

10.1182/blood.v108.11.2497.2497 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2497-2497

Author(s):

Kennichi C. Dowdell ◽

Lesley Pesnicak ◽

Victoria Hoffman ◽

Kenneth Steadman ◽

Mark Ruddel ◽

...

Keyword(s):

Valproic Acid ◽

Cell Death ◽

Lymph Nodes ◽

Histone Deacetylase ◽

Murine Model ◽

Hdac Inhibitor ◽

Vitro Assay ◽

Type Ia ◽

Autoimmune Cytopenias

Abstract ALPS is an inherited disorder of apoptosis leading to lymphoproliferation and autoimmunity. ALPS Type Ia, Ib and II are associated with germline mutations in Fas, FasL and Casapase 8 or 10, respectively; patients in whom no mutations have been identified are classified as Type III. The vast majority of patients are ALPS Type Ia (greater than 70%). They often present with childhood onset autoimmune cytopenias associated with lymphadenopathy, splenomegaly, increased double negative T cells (DNT; TCRα/β+CD3+CD4−CD8−), defective apoptosis by in vitro assay, and have an increased risk of lymphoma. Similarly, MRL/lpr−/− mice homozygous for Fas mutations develop an ALPS-like disease with massive lymphadenopathy, splenomegaly, hypergammaglobulinemia, autoimmune glomerulonephritis, and expansion of DNT cells secondary to defective lymphocyte apoptosis leading to lymphomagenesis. Currently, there are no proven therapies for the lymphoproliferation underlying ALPS itself, although complications like autoimmune cytopenias and post-splenectomy sepsis are manageable. Hence, studies were conducted to determine the efficacy of valproic acid (VPA) to control the lymphoproliferation associated with ALPS. VPA is a histone deacetylase (HDAC) inhibitor in clinical use for the last four decades as an anticonvulsant in children and adults, and recently being explored as an anti-neoplastic agent. PBMCs from normal controls and ALPS Type Ia patients were cultured in vitro with 0–4 mM VPA in the presence or absence of 50 uM of the pan-caspase inhibitor Z-VAD-FMK. A dose response was observed with a high degree of cell death noted at 4 mM after 48 hours, with an LD50 of 2 mM. VPA appeared to induce cell death by both caspase-dependent and -independent mechanisms based on partial inhibition of VPA-induced cell death by Z-VAD-FMK. Further preclinical studies were conducted in the MRL/lpr−/− murine model of ALPS. Twenty, 8-week old female MRL/lpr−/− mice were treated intraperitoneally (i.p.) with 500 mg/kg of VPA in sterile PBS or PBS alone five days per week for 8 weeks. Significant reduction of the spleen weight (p=0.034) and cellularity (p=0.0001) was observed in VPA treated (n=10) mice compared to controls (n=10). Reductions in size and cellularity were also observed in the lymph nodes (p=0.09 and 0.0002, respectively). A concomitant decrease (p<0.05) in DNT cells was observed in the spleen (11.2±0.6 vs 8.1±0.4) and blood (9.3±0.96 vs 5.5±0.6). Serum drug levels peaked (462±10 ug/mL) by 2 hours post-i.p. injection of VPA, where-as a 2.5 fold increase in histone acetylation was observed in the spleen at 4 hours, following a single injection. Analysis of the effects of VPA on autoimmune renal disease in these animals is underway. Based on our in vitro and in vivo data, VPA is effective at reducing lymphoproliferative activity in Fas deficient MRL/lpr−/− mice. It is being further explored in early phase clinical trials as a lympholytic agent to shrink lymph nodes and abrogate hypersplenism in ALPS patients.

Download Full-text

Sulfated poly-amido-saccharides (sulPASs) are anticoagulants in vitro and in vivo

Chemical Science ◽

10.1039/d1sc02302k ◽

2021 ◽

Vol 12 (38) ◽

pp. 12719-12725

Author(s):

Maria Varghese ◽

Rae S. Rokosh ◽

Carolyn A. Haller ◽

Stacy L. Chin ◽

Jiaxuan Chen ◽

...

Keyword(s):

Murine Model ◽

Protamine Sulfate ◽

Clotting Time ◽

Risk Of Bleeding ◽

Increased Risk

Heparin mimicking sulfated poly-amido-saccharides (sulPASs) are anticoagulants resistant to heparanases and reversed by protamine sulfate. In an in vivo murine model, sulPASs extend clotting time without the increased risk of bleeding.

Download Full-text

Iron Causes Lipid Oxidation and Inhibits Proteasome Function in Multiple Myeloma Cells: A Proof of Concept for Novel Combination Therapies

Cancers ◽

10.3390/cancers12040970 ◽

2020 ◽

Vol 12 (4) ◽

pp. 970 ◽

Cited By ~ 3

Author(s):

Jessica Bordini ◽

Federica Morisi ◽

Fulvia Cerruti ◽

Paolo Cascio ◽

Clara Camaschella ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Death ◽

Cell Lines ◽

Iron Toxicity ◽

Ammonium Citrate ◽

Ferric Ammonium Citrate ◽

Ferric Carboxymaltose ◽

High Dose ◽

Iron Loading

Adaptation to import iron for proliferation makes cancer cells potentially sensitive to iron toxicity. Iron loading impairs multiple myeloma (MM) cell proliferation and increases the efficacy of the proteasome inhibitor bortezomib. Here, we defined the mechanisms of iron toxicity in MM.1S, U266, H929, and OPM-2 MM cell lines, and validated this strategy in preclinical studies using Vk*MYC mice as MM model. High-dose ferric ammonium citrate triggered cell death in all cell lines tested, increasing malondialdehyde levels, the by-product of lipid peroxidation and index of ferroptosis. In addition, iron exposure caused dose-dependent accumulation of polyubiquitinated proteins in highly iron-sensitive MM.1S and H929 cells, suggesting that proteasome workload contributes to iron sensitivity. Accordingly, high iron concentrations inhibited the proteasomal chymotrypsin-like activity of 26S particles and of MM cellular extracts in vitro. In all MM cells, bortezomib-iron combination induced persistent lipid damage, exacerbated bortezomib-induced polyubiquitinated proteins accumulation, and triggered cell death more efficiently than individual treatments. In Vk*MYC mice, addition of iron dextran or ferric carboxymaltose to the bortezomib-melphalan-prednisone (VMP) regimen increased the therapeutic response and prolonged remission without causing evident toxicity. We conclude that iron loading interferes both with redox and protein homeostasis, a property that can be exploited to design novel combination strategies including iron supplementation, to increase the efficacy of current MM therapies.

Download Full-text

Efficacy of Humanized High-Dose Meropenem, Cefepime, and Levofloxacin against Enterobacteriaceae Isolates Producing Verona Integron-Encoded Metallo-β-Lactamase (VIM) in a Murine Thigh Infection Model

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00794-15 ◽

2015 ◽

Vol 59 (11) ◽

pp. 7145-7147 ◽

Cited By ~ 13

Author(s):

Islam M. Ghazi ◽

Jared L. Crandon ◽

Emil P. Lesho ◽

Patrick McGann ◽

David P. Nicolau

Keyword(s):

Murine Model ◽

Infection Model ◽

High Dose ◽

Content Type ◽

High Mics

ABSTRACTWe aimed to describe thein vivoactivity of humanized pharmacokinetic exposures of meropenem and comparators against Verona integron-encoded metallo-β-lactamase (MBL) (VIM)-producingEnterobacteriaceaein a murine model. Levofloxacin activity was predicted by its MIC, and cefepime activity displayed variability, whereas meropenem produced a >1 log CFU reduction against all isolates despite high MICs indicative of resistance. Our results suggest that despitein vitroresistance, high-dose meropenem may be a possible option against infections caused byEnterobacteriaceaeproducing MBL-type carbapenemases.

Download Full-text

Vitamin D accelerates resolution of inflammatory responses during tuberculosis treatment

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1200072109 ◽

2012 ◽

Vol 109 (38) ◽

pp. 15449-15454 ◽

Cited By ~ 183

Author(s):

Anna K. Coussens ◽

Robert J. Wilkinson ◽

Yasmeen Hanifa ◽

Vladyslav Nikolayevskyy ◽

Paul T. Elkington ◽

...

Keyword(s):

Vitamin D ◽

Antimicrobial Therapy ◽

Potential Role ◽

Inflammatory Responses ◽

Vitamin D Supplementation ◽

Tuberculosis Treatment ◽

Sputum Smear ◽

High Dose ◽

Increased Risk

Calcidiol, the major circulating metabolite of vitamin D, supports induction of pleiotropic antimicrobial responses in vitro. Vitamin D supplementation elevates circulating calcidiol concentrations, and thus has a potential role in the prevention and treatment of infection. The immunomodulatory effects of administering vitamin D to humans with an infectious disease have not previously been reported. To characterize these effects, we conducted a detailed longitudinal study of circulating and antigen-stimulated immune responses in ninety-five patients receiving antimicrobial therapy for pulmonary tuberculosis who were randomized to receive adjunctive high-dose vitamin D or placebo in a clinical trial, and who fulfilled criteria for per-protocol analysis. Vitamin D supplementation accelerated sputum smear conversion and enhanced treatment-induced resolution of lymphopaenia, monocytosis, hypercytokinaemia, and hyperchemokinaemia. Administration of vitamin D also suppressed antigen-stimulated proinflammatory cytokine responses, but attenuated the suppressive effect of antimicrobial therapy on antigen-stimulated secretion of IL-4, CC chemokine ligand 5, and IFN-α. We demonstrate a previously unappreciated role for vitamin D supplementation in accelerating resolution of inflammatory responses during tuberculosis treatment. Our findings suggest a potential role for adjunctive vitamin D supplementation in the treatment of pulmonary infections to accelerate resolution of inflammatory responses associated with increased risk of mortality.

Download Full-text

Somatic Fas Mutations Account for Nearly One Third of Autoimmune Lymphoproliferative Syndrome (ALPS) Cases with Previously Unknown Genetic Mutations.

Blood ◽

10.1182/blood.v114.22.710.710 ◽

2009 ◽

Vol 114 (22) ◽

pp. 710-710

Author(s):

Kennichi Dowdell ◽

Julie Niemela ◽

Susan Price ◽

Joie Davis ◽

Katie Perkins ◽

...

Keyword(s):

Peripheral Blood ◽

Late Onset ◽

Clinical Phenotype ◽

Lymphocyte Apoptosis ◽

Type Iii ◽

Type Ia ◽

Lymphoproliferative Syndrome ◽

Autoimmune Cytopenias ◽

Age Range

Abstract Abstract 710 Background: Autoimmune lymphoproliferative syndrome (ALPS) is characterized by childhood onset of lymphadenopathy, hepatosplenomegaly, autoimmune cytopenias, elevated (>1%) double negative T (DNT; CD3+, TCRalpha-beta+, CD4−, CD8−, B220+) lymphocytes in peripheral blood and an increased risk of lymphoma; primarily due to impaired lymphocyte apoptosis. Most cases (65%; 173 individuals in our cohort), known as ALPS Type Ia, are associated with dominant heterozygous germline mutations in the gene TNFRSF6 encoding the protein for CD95 (Fas, Apo-1). Another 5% of patients have mutations in FasL (Type Ib), caspases (Type II) or NRAS (Type IV). However, approximately 30% of ALPS patients in our cohort have no mutation found upon genomic sequencing of DNA from peripheral blood. We designated these patients as ALPS Type III (16%) if they met all the criteria of ALPS, i.e., elevated DNT cell numbers, nonmalignant chronic lymphadenopathy/splenomegaly and defective lymphocyte apoptosis by in vitro assay; we called them ALPS phenotype (14%) if they had all the features as described above but did not demonstrate an in vitro apoptosis defect. Holzelova et. al. (NEJM 2004; 351:1409) previously identified somatic Fas mutations in DNT cell population in 6 patients with ALPS phenotype. However, there was no information as to the proportion of ALPS patients expressing a somatic Fas mutation or any differences in their clinical phenotype. Methods and Results: Over the last 5 years we sought to determine the proportion of ALPS Type III and ALPS phenotype patients with somatic Fas mutations in their DNT cell population and to further clinically characterize these patients. DNT cells were purified to >50% by magnetic bead separation and DNA was sequenced for Fas. We found somatic Fas mutations in the DNT cells of 11/31 (35.5%) patients; 5/15 with ALPS Type III (5 males; age range = 1-17 years; median = 12 years), and 6/16 with ALPS phenotype (3 males and 3 females; age range = 3 mo–48 years; median age = 1 year). All the mutations clustered to an approximately 150 base region of the intracellular domain of Fas (exons 7, 8 and 9), except for one mutation in exon 3. All mutations resulted in functional loss of normal Fas signaling based on the mutation type or having been previously observed in ALPS Typa Ia. The somatic ALPS patients showed a similar clinical phenotype to that of ALPS Type Ia with increased DNT cell numbers (Median 6%; Range 4-19%), and increased levels of biomarkers like serum vitamin B12, IL-10 and sFasL. All 4 patients with onset of symptoms in infancy have required long-term treatment of their refractory autoimmune cytopenias with mycophenolate mofetil for the last 2-8years, while one of them underwent splenectomy. Inability to demonstrate defective lymphocyte apoptosis by in vitro testing of unseparated lymphocytes in ALPS phenotype patients with somatic Fas mutations is likely due to the relatively small number of affected cells (<20%) in their peripheral blood. Additionally, we hypothesize that the late onset of clinical disease in ALPS Type III patients with somatic Fas mutations may reflect the time required to accumulate a threshold number of cells that are able to confer clinical manifestations. Conclusion: Thus, the majority of somatic ALPS mutations can be detected by sequencing exons 7-9 of isolated DNT cell DNA. Patients with somatic Fas mutations now comprise the second largest group of known genetic mutations in ALPS, followed by patients with caspase 10, NRAS, caspase 8 and FasL germline mutations, respectively. We recommend testing for somatic Fas mutations in ALPS Type III patients (particularly those with late onset) and all ALPS phenotype patients, using ungated DNT purities of >50%. These data also highlight the role of somatic mutations in the pathogenesis of nonmalignant hematological conditions in adults and children, as well as in clonal malignancies. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

SHIP1 Inhibition As Novel Therapeutic Approach in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood-2018-99-117053 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 894-894

Author(s):

Veronika Ecker ◽

Martina Braun ◽

Tanja Neumayer ◽

Markus Muschen ◽

Jürgen Ruland ◽

...

Keyword(s):

T Cells ◽

Cell Death ◽

B Cells ◽

Immune Responses ◽

Lymph Nodes ◽

Peripheral Blood ◽

Immune Cell ◽

Lymphocytic Leukemia ◽

Bcr Signaling

Abstract Chronic lymphocytic leukemia (CLL) is one of the most common B cell malignancies in the Western world. Malignant B cells are blocked from differentiating into immunoglobulin producing-plasma cells and clonally accumulate in the spleen, lymph nodes, bone marrow and peripheral blood. CLL is characterized by immunosuppression throughout all disease stages, which is mediated by increased numbers of myeloid-derived suppressor cells (MDSCs), regulatory T cells (Jitschin and Braun et al., Blood 2014) and direct inhibitory effects of the malignant CLL cells on T cells (Christopoulos etal., Blood 2011). Over the past decade, significant improvement in understanding the pathogenesis of CLL has highlighted the importance of active B cell receptor (BCR) signaling. This has revealed promising targeted treatment options, including the small molecule inhibitors targeting the phosphatidylinositol-3-kinase (PI3K) signaling pathway. Idelalisib and Duvelisib are under clinical investigation for CLL treatment, however, treatment-related toxicities are limiting their application and none of these approaches are curative, highlighting the importance of functional anti-tumor immune responses in CLL for prolonged treatment efficacy. Here, we are testing a novel approach that aims to selectively target CLL B cells and simultaneously restore an appropriate immune cell function. The phosphatase SH2-domain-containing inositol 5ʹ-phosphatase 1 (SHIP1) serves as negative feedback molecule and downregulates PI3K signaling in B cells by dephosphorylating the 5`phosphate of Phosphatidylinositol (3,4,5)-trisphosphate. We hypothesize that CLL cells rely on such negative regulators for optimal survival and can only tolerate a maximum signaling level. We are therefore testing whether SHIP1 inhibition induces hypersignaling and thereby CLL cell death. Furthermore, we are investigating whether SHIP1 inhibition simultanously stimulates immune responses, as it has been shown to induce expansion of murine hematopoietic and mesenchymal stem cell compartments (Brooks et al., Stem cells 2014). 3α-Aminocholestane (3AC) is a small molecule inhibitor of SHIP1 and can be used for pharmacological inhibition. First, we investigated the expression and phosphorylation levels of SHIP1 in CLL. We found SHIP1 to be expressed at various levels in CLL peripheral blood and strongly phosphorylated compared to age-matched healthy donors. Besides, SHIP1 transcription is upregulated in lymph nodes as compared to peripheral blood, which is in line with the assumption of increased BCR signaling in secondary lymphoid organs. We then set out to investigate the consequences of SHIP1 phosphatase inhibition. Similarly, to recent findings in acute lymphoblastic leukemia (Chen et al., Nature 2015), pharmacological inhibition of SHIP1 lead to rapid cell death of CLL cells. We further investigated the mode of cell death and observed specific features of apoptosis, namely caspase 3/7 activation and phosphatidylserine exposure on the outer cell membrane. This has been tested on primary CLL patient samples and T cell leukemia/lymphoma 1 (TCL1)-driven murine CLL cells and was not observed or significantly less pronounced in other lymphoma cell lines or healthy primary B cells. To confirm the specificity of the observed effects, we genetically activated AKT with a GFP reporter in the TCL1-driven mouse model in vivo and in vitro. By tracking GFP-expressing CLL cells, we observed an initial expansion followed by rapid cell death in vitro. When we induced AKT activation in vivo, GFP+ CLL cells were not detectable in the peripheral blood, total CLL count declined upon induction and we found decreased tumor burden in the secondary lymphoid organs, particularly in the lymph nodes. In addition to the direct effects on CLL cells, we sought to investigate the impact of SHIP1 inhibition on other immune cell functions. We observed that SHIP1 inhibition lowers the activity threshold of T cells: When we stimulated a reporter cell line with suboptimal doses of anti-CD3, 3AC treatment significantly enhanced the response rate. Therefore, we propose SHIP1 as a novel interesting target in CLL. In contrast to kinase inhibition and downregulation of the BCR signaling strength, phosphatase inhibition and BCR signaling overaction provides an attractive new treatment strategy for elimination of malignant CLL cells and stimulation of immune responses. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Pyrimethamine Does Not Diminish Lymphoproliferation in MRL/lpr−/− Mice and Patients with Autoimmune Lymphoproliferative Syndrome (ALPS).

Blood ◽

10.1182/blood.v106.11.2396.2396 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2396-2396

Author(s):

V. Koneti Rao ◽

Kennichi C. Dowdell ◽

Faith Dugan ◽

Janet K. Dale ◽

Lesley Pesnicak ◽

...

Keyword(s):

Lymph Node ◽

Aphthous Ulcer ◽

Lymphocyte Apoptosis ◽

Childhood Onset ◽

Massive Lymphadenopathy ◽

Increased Risk ◽

Before And After ◽

Type Ia ◽

Lymphoproliferative Syndrome

Abstract ALPS is an inherited disorder of lymphocyte apoptosis leading to childhood onset of chronic lymphadenopathy, hepatosplenomegaly, autoimmunity, and an increased risk of lymphoma in a subset of patients with mutations in the intracellular domain of Fas (ALPS Type Ia). Similarly, MRL/lpr −/− mice are homozygous for Fas mutations and develop massive lymphadenopathy and splenomegaly associated with hypergammaglobulinemia, glomerulonephritis, and expansion of TCR αβ+, CD4−/CD8−double-negative (DN) T cells that are pathognomonic of ALPS. There remain no proven therapies for the lymphoproliferation underlying ALPS itself, although investigators in Europe reported that some children with ALPS showed reductions in spleen and lymph node sizes while on a weekly regimen of pyrimethamine/sulfadoxine (Fansidar@) (Br J Haematol.2002; 117:176). However, in vitro studies using stimulated human PBMCs, revealed that lymphocyte apoptosis is induced by the antifolate agent, pyrimethamine (Pyr) at an EC50 of 2.5ug/mL, and not sulfadoxine. This suggested that Pyr alone might prove clinically beneficial, thus avoiding risks of allergy and hypersensitivity associated with sulfa drugs. Hence, studies were conducted in the MRL/lpr−/− murine model of ALPS with Pyr alone given 2mg/kg twice weekly by gavage for 8 weeks, and in combination with sulfadoxine. Neither Pyr nor Fansidar yielded significant shrinkage of lymphoid mass in mice. A pilot study of the safety and efficacy of Pyr was conducted under an FDA IND in 6 children with ALPS Type 1a and one with ALPS Type III (with no identifiable mutation). Aged 6 to 17 yrs (median; 13 y), these 6 males and 1 female patient were enrolled following informed consent to be treated with oral Pyr at escalating doses (25–50mg) given twice weekly for 12 weeks. Computerized tomography was done before and after treatment to determine whether these patients would show 40 and 50% reductions in the sizes of their lymphadenopathy and/or splenomegaly, respectively. Lymph node sizes were assessed as sums of the bi-dimensional products of 3 to 5 nodes. Spleen sizes were measured as products of their craniocaudal and anterioposterior dimensions at the superior mesenteric artery level, as well as spleen volumes computed by digital image processing. The effects of treatment on other well-defined laboratory features of ALPS were also assessed. Pyr was discontinued after 11 days in one patient due to skin rash. Dose escalation was interrupted transiently in 3 others because of neutropenia, aphthous ulcer, or leucopenia. There were no significant changes in study outcome measures. The aggregate experience with Pyr and Fansidar in mice, as well as with Pyr alone in ALPS patients (Table) lead us to conclude that these agents do not manifest sufficient effectiveness to warrant larger trials.

Download Full-text

Oxidation of Met1606 in von Willebrand factor is a risk factor for thrombotic and septic complications in chronic renal failure

Biochemical Journal ◽

10.1042/bj20111798 ◽

2012 ◽

Vol 442 (2) ◽

pp. 423-432 ◽

Cited By ~ 11

Author(s):

Vincenzo De Filippis ◽

Stefano Lancellotti ◽

Fabio Maset ◽

Barbara Spolaore ◽

Nicola Pozzi ◽

...

Keyword(s):

Von Willebrand Factor ◽

Bacterial Infections ◽

Vascular Endothelial Cells ◽

Polymorphonuclear Cells ◽

Septic Complications ◽

Bacterial Adhesins ◽

Increased Risk ◽

Von Willebrand ◽

Willebrand Factor

CKD (chronic kidney disease) is a life-threatening pathology, often requiring HD (haemodialysis) and characterized by high OS (oxidative stress), inflammation and perturbation of vascular endothelium. HD patients have increased levels of vWF (von Willebrand factor), a large protein (~240 kDa) released as UL-vWF (ultra large-vWF polymers, molecular mass ~20000–50000 kDa) from vascular endothelial cells and megakaryocytes, and responsible for the initiation of primary haemostasis. The pro-haemostatic potential of vWF increases with its length, which is proteolytically regulated by ADAMTS-13 (a disintegrin and metalloproteinase with thrombospondin motifs 13), a zinc-protease cleaving vWF at the single Tyr1605–Met1606 bond, and by LSPs (leucocyte serine proteases), released by activated PMNs (polymorphonuclear cells) during bacterial infections. Previous studies have shown that in vitro oxidation of Met1606 hinders vWF cleavage by ADAMTS-13, resulting in the accumulation of UL-vWF that are not only more pro-thrombotic than shorter vWF oligomers, but also more efficient in binding to bacterial adhesins during sepsis. Notably, HD patients have increased risk of developing dramatic cardiovascular and septic complications, whose underlying mechanisms are largely unknown. In the present study, we first purified vWF from HD patients and then chemically characterized its oxidative state. Interestingly, HD-vWF contains high carbonyl levels and increased proportion of UL-vWF polymers that are also more resistant to ADAMTS-13. Using TMS (targeted MS) techniques, we estimated that HD-vWF contains >10% of Met1606 in the sulfoxide form. We conclude that oxidation of Met1606, impairing ADAMTS-13 cleavage, results in the accumulation of UL-vWF polymers, which recruit and activate platelets more efficiently and bind more tightly to bacterial adhesins, thus contributing to the development of thrombotic and septic complications in CKD.

Download Full-text

Therapeutic Evaluation of Palbociclib and Its Compatibility with Other Chemotherapies in Primary and Recurrent Nasopharyngeal Carcinoma

10.21203/rs.3.rs-36929/v1 ◽

2020 ◽

Author(s):

Zhichao Xue ◽

Vivian Wai Yan Lui ◽

Yongshu Li ◽

Jia Lin ◽

Chanping You ◽

...

Keyword(s):

Cell Death ◽

Nasopharyngeal Carcinoma ◽

Cell Lines ◽

Therapeutic Option ◽

Substantial Reduction ◽

Inhibitory Effect ◽

High Dose ◽

Ki 67

Abstract Background: Recent genomic analyses revealed that druggable molecule targets could only be detected in around 6% of nasopharyngeal carcinoma (NPC) patients. Yet, an addiction to dysregulated CDK4/6-cyclinD1 signalling pathway is an essential event in the pathogenesis of NPC. Using our newly established xenografts and cell lines derived from primary, recurrent and metastatic NPC, we aimed to evaluate the therapeutic efficacy of a specific CDK4/6 inhibitor, palbociclib, and its compatibility with other chemodrugs in treating NPC.Methods: The efficacy of single treatment of palbociclib on NPC models was first evaluated, followed by concurrent treatment with cisplatin or suberanilohydroxamic acid (SAHA). RNA sequencing was used to profile the related pathways in governing the drug response. Palbociclib-resistant NPC cell lines were also established to demonstrate if cisplatin could be used as a second-line treatment once the cells developed resistance to palbociclib. The efficacy of palbociclib treatment on cisplatin-resistant NPC cells was also examined. Results: Palbociclib single drug treatment was confirmed to have a cell cycle arresting effect of NPC cells in G1 phase in vitro. It also had a significant inhibitory effect in all the 6 NPC tumor models in vivo, with a substantial reduction in total tumor volume and proliferation marker Ki-67. Concurrent use of palbociclib dampened the cytotoxic effect of cisplatin in NPC cells in vitro. Notably, combination of palbociclib with SAHA resulted in synergistic cell death of NPC both in vitro and in vivo. Autophagy-associated cell death was found to be involved in the enhanced tumor growth inhibitory effect in the combined palbociclib+SAHA treatment. NPC cell lines trained to sustain growth in high dose of palbociclib and cisplatin remained sensitive in subsequent treatment of cisplatin or palbociclib respectively.Conclusions: This study provides essential evidences to position palbociclib as an alternative therapeutic option to NPC treatment, and to aware the effective administrative timing of palbociclib with other chemodrugs. The findings give the basis for planning of the first-in-human clinical trials of palbociclib regimens in NPC patients.

Download Full-text