ALBERT

Description: Mesenchymal stem cells (MSC) mostly surround the vasculature system of bone marrow (BM). MSC have been shown to exhibit immune suppressive properties. Since MSC express MHC Class II antigen, the question is whether these cells can act as APC. To this end, we hypothesize that MSC have the ability to present non-self antigens while acting as immune modulators. These dual roles of MSC prevent exacerbated inflammatory responses in the BM, thereby preventing hematopoietic dysfunction. A ‘dampened’ immune response in BM during insults by foreign agents could cause protection of the barrier that separates BM cavity with the periphery. The phagocytic role of MSC was shown by confocal microscopy and fluoresbrite plain YG 1.0-micron microspheres. APC property was demonstrated by challenging MSC with C. albicans (pulsed MSC), followed by exposure to CD4+ cells. The latter was obtained by immunoselection from peripheral blood mononuclear cells (PBMC) cultured for 5 days with C. albicans (10 mg/ml). Proliferation of the CD4+ cells (3H-thymidine incorporation and cell counts) proved APC properties of MSC, at efficiency comparable to macrophages. Overall, the studies show that the window between APC function and the period at which MSC could become immune suppressive is critical, since activated T-cells could destroy the endothelial barrier between BM and lymphatics/peripheral circulation. These studies show that MSC could be key cells in regulating immune responses in BM, and thereby protect BM from failure.

Description: Abstract 5009 Background: The physiological form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25D), has well-established anti-cancer activity, but its potential clinical use is limited by hypercalcemia. Efforts to overcome this difficulty include structural modifications of 1,25D, as well as enhancement of its activity by other compounds such as antioxidants. The PRI analogs (termed “deltanoids”) have been shown to have low calcemic activity in animals, and while the PRI-1906 (24-ene-1,25-dihydroxyvitamin D2) and PRI-2191 ([24R]-1,24-dihydroxyvitamin D3) have greater activity on established Acute Myeloid Leukemia (AML) cell lines than their congeners, their relative potency on differentiation of AML blasts is not entirely clear. We therefore studied the effects of these analogs on leukemic blasts obtained from AML patients with different FAB subtypes, and combined them in some cases with Carnosic Acid (CA), a natural benzenediol abietane diterpene found in rosemary (Rosemarinus officinalis) and common sage (Salvia offinalis). Methods: Leukemia blasts from patients with newly diagnosed, untreated AML were obtained from the peripheral blood after formal consent on an IRB approved protocol. Cells were isolated by Histopaque centrifugation, washed, and placed in tissue culture. 1,25D, PRI-1906, or PRI-2191 (each 10 nM), and CA (10 uM) were added, and cells cultured for the times shown, and then harvested for analysis. Results: Cells from 8 patients were studied (1 patient with FAB-M1, 1 patient with FAB-M2, 1 patient with FAB-M2 with mixed myeloid and lymphoid lineage; 1 patient with FAB-M3; 1 patient with FAB-M4; 3 patients with FAB-M5). Using expression of CD11b/CD14 by immunophenotyping as markers of differentiation, the 1,25D and the deltanoids each induced modest differentiation in AML FAB-M1, with greater differentiation in response to the combination of a deltanoid plus CA. Similar differentiation effects were seen in AML FAB-M2, and more dramatic effects in AML FAB-M3. Differentiation was also seen in AML FAB-M4. In AML FAB-M5, 1,25D and deltanoids induced differentiation, but CA appeared to add little to this process in 2 of the patient's samples. One patient with AML FAB-M5 did show significant differentiation in response to CA as well as 1,25D; of note, that patient had a t(11;19) involving the MLL-1 gene, with normal FLT-3 genotype). In 6 out of the 8 cases, the G1 arrest increased when AML cells were treated with CA alone, but the effect was not enhanced by CA combined with 1,25D or its analogs. MAP Kinase mRNA array analysis showed upregulation of MAP Kinase pathway molecules in response to 1,25D and the deltanoids. Conclusions: 1 The analogs of 1,25D, PRI-1906 and PRI-2191 induce cell cycle arrest and differentiation of AML cells ex vivo, and carnosic acid can enhance this effect. 2. As previously shown for 1,25D, MAPK signaling network is an important regulator of this process. 3.Since the PRI analogs are less calcemic than 1,25D, and carnosic acid has no effect on calcium homeostasis in AML cells, the potential therapeutic ratio is higher for the analogs combined with carnosic acid enhancer. Thus, these compounds may be considered as candidates for chemoprevention/ differentiation therapy of AML. (Supported by NIH Grant 1RO1-CA044722-21 to GPS). Disclosures: No relevant conflicts of interest to declare.

Description: Bone marrow (BM) fibrosis may occur in myeloproliferative diseases, lymphoma, myelodysplastic syndrome, myeloma, and infectious diseases. In this study, the role of substance P (SP), a peptide with pleiotropic functions, was examined. Some of its functions—angiogenesis, fibroblast proliferation, and stimulation of BM progenitors—are amenable to inducing BM fibrosis. Indeed, a significant increase was found in SP-immunoreactivity (SP-IR) in the sera of patients with BM fibrosis (n = 44) compared with the sera of patients with hematologic disorders and no histologic evidence of fibrosis (n = 46) (140 ±12 vs 18 ±3; P 

Description: Hematopoietic stem cells (HSC) are difficult to expand in vitro since expansion media mostly include exogenous factor, e.g., cytokines. Growth factors cause lineage commitment with the HSC exhibiting short-term immune reconstitution. The recently cloned HGFIN (or nmb) gene has been detected in CD34+/CD38− and differentiated immune cells. HGFIN is a single transmembrane protein with multiple consensus regions for p53. In malignant cells, HGFIN mediate tumor progression and in normal cells, HGFIN maintain cell cycle quiescence. We hypothesize that HGFIN negatively regulates proliferation of HSC by prolonging the G0 phase. Short-term knockdown of HGFIN in HSC could cause the cells to exit G0/G1 phase so as to accommodate the insertion of a replacement gene, without changing the pluripotent property of HSC. RT-PCR detected HGFIN mRNA in CD34+/CD38− cells, but not in CD34+/CD38+ cells from bone marrow (BM) aspirates. Treatment of CD34+/CD38− cells with double stranded HGFIN siRNA oligos for 2 h led to their exit from G1 to G2/S (flow cytometry). During this time, adenovirus-LacZ (ß-galactosidase assay) was incorporated in 〉80% CD34+/CD38− cells, compared to 10–20% exposed to mutant siRNA, or untreated. After 24 h exposure to siRNA oligos, the CD34+/CD38− cells reverted to G0/1 phase. The latter cells (with adenovirus-LacZ) were studied in 12-wk long-term culture initiating assay. The results (ß-gal positive progenies) from the LTC-IC assay indicate that despite siRNA treatment, the CD34+/CD38− cells retained multipotential properties. Future investigations with specific HGFIN antibody will map pathways among HGFIN and other molecules that regulate cell cycle progression in CD34+/CD38− cells. In summary, the results show that HGFIN regulate cell cycle quiescence of CD34+/CD38− cells. Transient knock down of HGFIN in these cells allow adenovirus to be inserted without loss of multipotential properties. Understanding the biology of HSC at the molecular level will be relevant to HSC expansion and restoration of immune competence via gene therapy.

Description: Although life expectancy for patients with sickling hemoglobinopathies has been improving in recent years, these patients continue to suffer significant morbidity throughout their lives, and pain is a prominent feature of these disorders. It has been shown that, in the primary care population, patients with the highest risk for sleep disturbances include those with pain (Alattar M, et al., J Am Board Fam Med, 2007, Vol 20, pp 365–74). It has also been reported that patients with sickle cell diseases have clinical features which resemble fibromyalgia syndrome (Schlessinger N, J Rheum, 2004, Vol 31, pp 598–600); fibromyalgia syndrome is characterized both pain and impaired sleep, among other features. In order to further investigate sleep disorders in sickle cell disease patients, patients in a sickle cell disease clinic were asked to complete the Pittsburgh Sleep Quality Index (PSQI) questionnaire. Methods: Using a form IRB-approved specifically for this study, the PSQI was offered to patients in a sickle cell disease clinic, and completed anonymously. Results: 20 patients completed the PSQI with responses meaningful for analysis; they ranged in age from 21 to 50 years. On average, patients reported very poor overall quality of sleep; the mean Global PSQI score was 11.8 (S.D.4.8); this is as compared to historical, healthy control subjects who had a subjective mean Global PSQI score of 2.6. (Buysse D et al, Psychiatry Research, 1989, Vol 28, pp 193–213). All respondents reported that pain contributed to poor sleep at least once during the month prior to the survey, and half of the patients reported that pain contributed to poor sleep at least three times in the week prior to the survey. Fifty percent of those surveyed reported no sense of nocturnal dyspnea whatsoever; only 3 individuals reported that they felt that dyspnea contributed to poor sleep three or more times in the week prior to the survey, and frequent daytime somnolence was reported by only 10 percent of sickle cell patients responding to the survey. Habitual sleep efficiency appears poor in these patients, and subjective sleep quality is also reported as generally poor. Conclusions: Sleep disturbances appear to be very common in sickle cell disease patients, and some, but not all, aspects of these sleep disturbances resemble the characteristics of sleep impairment seen in patients with fibromyalgia syndrome (Osorio C, et al., J Rheum, 2006, Vol 33, pp 1863–65). Although sleep impairment in patients with sickle cell diseases has been attributed to sleep apnea syndromes in a subset of patients with sickle cell diseases, sleep disturbance associated with pain in sickle cell disease appears to be more widespread than could be explained by sleep apnea syndromes. Sleep disturbances associated with pain in sickle cell diseases may in turn lead to further exacerbation of symptoms arising from poor sleep. Interventions to improve sleep may reduce overall pain in these patients, as appears to be the case for patients with fibromyalgia syndrome (Rooks D, Curr Opin Rheum, 2007, Vol 2, pp 111–17).

Description: Background Acquired coagulopathies are a common problem in Hematology, and are most often due either to medication effect, liver disease, or consumption. Among the uncommon causes of acquired coagulopathy, inhibitory auto-antibodies may develop, either in the setting of autoimmune diseases, in the setting of lymphoproliferative disorders, or as isolated inhibitory immunoglobulins. Uncommonly, the adsorption of coagulation factors from the circulation into the tissues by extracellular deposition of pathologic amyloid results in an acquired factor deficiency, due to clearance of factor from the circulation that exceeds the body's ability to produce factor. When amyoidosis does cause a coagulapathy, it is most often the result of the adsorption of Factor X by the amyloid protein, resulting in an acquired Factor X deficiency. However, there are rare reports of amyloidosis being associated with other factor deficiencies. We report a case of amyloidosis that was associated with a severe bleeding diathesis, with the etiology of the bleeding disorder being due to both acquired Factor V deficiency and concomitant acquired von Willebrand Disease. Case Report A previously healthy 51-year-old gentleman presented to an outside medical center for evaluation and management of recurrent bleeding episodes. The patient had a prior medical history significant only for right ankle trauma in the year 2005, following which he underwent a total of 4 surgical procedures; there was no excessive bleeding complicating the patient's surgeries. He was then in his usual state of health until September, 2012 when he developed onset of severe abdominal pain and was admitted to the outside facility. Following hospitalization for several months at the outside facility, he was admitted to our institution. Physical examination was remarkable for extensive ecchymoses, and for splenomegaly to 18 cm. span by exam, confirmed by imaging. CT scan showed multiple peri-caval and periaortic nodes present up to 1.7 cm in size, with shotty inguinal lymph nodes. A complete blood count showed White blood count 21,600, hemoglobin 8.0 g/dL, hematocrit 24%, platelet count 370,000, Hepatic function studies and renal function studies, as well as electrolytes, were normal on admission. Coagulation studies revealed Prothrombin Time prolonged at 16.8 seconds (normal 〈 12.7), aPTT prolonged at 44. Mixing patient plasma with equal volume normal plasma corrected both the PT and aPTT. Detailed factor assays showed markedly decreased Factor V activity of 27%; Ristocetin Cofactor activity was markedly decreased at 49%, but von Willebrand antigen was elevated at 213%. Multimer analysis was consistent with Type II vWD (see figure 1). The patient received fresh frozen plasma and Humate P, with transient correction of the bleeding diathesis. This permitted inguinal lymph node biopsy, which documented AL amyloidosis. Extraction of the protein from the lymph node documented AL lambda light chain amyloid (see figure 2). Marrow biopsy documented IgG lambda multiple myeloma. The patient was treated using Bortezumib plus Dexamethasone, and achieved a complete remission, with normalization of the coagulation parameters and factor levels over the following several months. His bleeding diathesis has fully resolved, and Karnofsky performance status improved to 100%. Conclusion Although there are several case reports of acquired von Willebrand disease on the basis of amyloidosis, and several case reports of acquired Factor V deficiency on the basis of amyloidosis, this appears to be the first reported case of both acquired vWD and acquired Factor V deficiency on the basis of amyloidosis. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 5090 Introduction The evaluation of a patient with new onset hemolytic anemia is among the more common clinical scenarios prompting formal input by a hematologist. Although there are both common and uncommon etiologies, the presentation of Wilson's Disease, itself an uncommon disorder, as a fulminant hemolytic anemia, is very uncommon, and for this reason may not be readily considered within the differential diagnosis of new onset hemolytic anemia. Wilson's Disease occurs worldwide with an average prevalence of only 30 affected individuals per million population. In order to illustrate hemolytic anemia as an initial presentation of Wilson's Disease, a case is reported. Case Report A 28-year-old female patient presented with a recent diarrheal infection and new onset jaundice. Examination revealed an afebrile patient with gross jaundice, but no other abnormal findings. Initial significant laboratory findings included a hemoglobin of 7.1 gm/dL, white blood cell count of 30,900 per microliter, platelet count of 181,000 per microliter, creatinine of 2 mg/dL, and total bilirubin of approximately 10 mg/dL. Within two days, laboratory studies worsened to a serum creatinine of 3.5 mg/dL, total bilirubin 19.5 mg/dL, direct bilirubin 13.1 mg/dL, AST 130 IU/L, ALT 16 IU/L, and alkaline phosphatase 〈 5 IU/L. The serum LDH was markedly elevated at 1258 IU/L. Review of the peripheral blood film showed 87% segmented neutrophils with no schistocytosis. However, a striking stomatocytosis was present, comprising about 20% of the red blood cells (see Figure 1). In the next two days, the clinical condition dramatically worsened. The hemoglobin dropped to 4.4 gm/dL, and the prothrombin time increased from within normal limits to 52.7 seconds. Disseminated intravascular coagulation was not present. Haptoglobin and LDH remained consistent with a brisk intravascular hemolysis, and Coombs testing was negative. In addition, the total bilirubin rose to 52.5 mg/dL, with a direct bilirubin of 38.7 mg/dL. The patient's sensorium was becoming gradually impaired, consistent with a hepatic encephalopathy. Based on this constellation of findings, the possibility of a fulminant manifestation of Wilson's Disease, featuring severe hemolysis, was identified (see Table 1). This led to definitive testing for Wilson's Disease, which included a liver biopsy with qualitative and quantitative measurements for copper (copper dry weight = 966.7 micrograms/gm). With the diagnosis established, and using support from published literature, daily plasmapheresis was utilized to stabilize the patient in anticipation of orthotopic liver transplantation (Jhang JS et al. Journal of Clinical Apheresis 22(1):10-14, 2007). Subsequently, the patient was successfully transplanted, and continues to do well three months afterwards. Conclusion The present case highlights the value of understanding the possible etiologies of stomatocytosis, as observed in the peripheral smear, in the setting of a hemolytic anemia (see Table 2). In addition, as illustrated by this case, plasmapheresis can be an effective bridge to stabilize patients for liver transplantation in patients with Wilson's Disease who present with fulminant hemolytic anemia. Disclosures No relevant conflicts of interest to declare.