Browsing by Author "Novitzky, Nicolas"
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- ItemOpen AccessCancer/Testis Antigen Expression Panel Incorporating MAGEC1 and BAGE2 Predicts Multiple Myeloma Disease Stage and Severity(OMICS publishing group, 2016-04-07) Shires, Karen; Teuchert, Andrea; Wienand, Kirsty; Shankland, Iva; Novitzky, NicolasTo provide a single molecular assay that could be used to easily stage Multiple Myeloma patients at diagnosis, we investigated the association between the simultaneous expression of 7 Multiple Myeloma-associated Cancer/Testis Antigens and biochemical parameters that are currently used for disease staging. We analysed the mRNA expression of MAGEC1, MAGEA3/A6, BAGE2, PRAME, NYESO1, SSX2 and PAGE by qualitative reverse transcription PCR using RNA extracted from diagnostic bone marrow samples from 39 patients covering the Multiple Myeloma disease continuum and compared this to levels of key biochemical parameters at diagnosis. We found that the Cancer/Testis Antigen panel was expressed in a specific order that was specifically associated with the severity of disease. This allowed the Cancer/Testis Antigens expression profile to successfully place the patient clearly into either stage I or stage III of the disease, with further sub-stratification in the stage III grouping. In addition, we putatively identified MAGEC1 expression as a confirmatory diagnostic marker for symptomatic Multiple Myeloma and clearly associated BAGE2 expression exclusively with stage III disease. We also demonstrated the novel finding of PAGE expression in Multiple Myeloma, with an association with more advanced disease. We suggest that this particular molecular Cancer/Testis Antigen panel can be used at diagnosis as a single test to clearly stage patients
- ItemOpen AccessEffects of bFGF (Basic Fibroblast growth factor) on the Haematopoietic Sequelae that follow transplantation(2000) Makgoka, Seretloane Japhtaline; Novitzky, NicolasRecovery following bone marrow transplantation is associated with the reduction in the clonogenic potential of stroma-adherent CD34⁺ progenitors. The bone marrow stroma is also affected, resulting in poor support for the growth of stroma-adherent multipotent progenitors that form blastic colonies (CFU-bl). To determine the possible mechanisms of marrow damage in patients treated with peripheral blood stem cells (PBSCs) and bone marrow transplantation (BMT), we studied the effects of bFGF, a cytokine known to stimulate the survival and proliferation of fibroblasts, on the propagation of clonogenic progenitors and the marrow stroma. METHODS: In order to obtain the optimal bFGF concentrations to be used on patients' haematopoietic progenitors and bone marrow rnicroenvironment, haematologically normal individuals were studied in dose response studies. Control mononuclear cells from the Ficoll-Histopaque interface layer were divided into two aliquots: one to establish a stromal layer and to culture fibroblastic progenitors (CFU-Fs) in the presence of 0, 2 and 20 ng/ml bFGF with and without 20 ng/ml heparan sulphate (HS). The second aliquot was for the selection of the progenitor population. Stroma was quantitated by placing culture dishes on a grid containing 1mm squares and scoring the number of squares occupied by stromal layers as the percentage area covered. After 3 weeks of culture, a single cell suspension was prepared by incubation of stroma with 5% trypsin solution, and number of cells in the dishes enumerated with a particle counter. CFU-Fs were terminated on the 9th day of culture, stained with May-Grilnwald-Giemsa and scored using an inverted microscope. From the second aliquot, CD34⁺ cells were incubated with paramagnetic beads and target cells isolated with a magnet. Selected l x l 04/ml cells were cultured on prefom1ed controls' stroma that has been treated with 0, 2 and 20 ng/ml bFGF. Stroma-adhered cells were covered with 0.3% agar and cultured for 6 days. Aggregates of more than 20 cells were counted as CFU-bl. RESULTS: In normal individuals, the median surface area of the petri dish covered by stroma at 3 weeks of culture was 55% (range 30-65) and was significantly improved upon the addition of 2 ng/ml (median 70%; range 50-95 ; p= <0.05) and 20 ng/ml bFGF (median 80%; range 65-99; p= 0.004). Stromal cell numbers were 0.61 x 10⁶/2ml (range 0. 15-1.66), and they increased significantly with the addition of 2 and 20 ng/ml bFGF (p=0.03). The median colony forming unit-blasts (CFU-bl) scores were 121.8 x 10⁴/ml (range 43-271), and they expanded significantly with the addition of 20 ng/ml bFGF with and without heparan sulphate (p=0.03 and 0.01). It was then concluded that the 2 and 20 ng/ml bFGF with heparan sulphate be used on patients' cells in vitro. The median surface area covered by stromal layers in patients' samples at 3 weeks was 40% (range 0-55 vs. normal 50%), and it was in1proved significantly upon the addition of bFGF (median 78 vs. 50%; p= 0.001). Supplementation of stromal layers with bFGF accounted for a significant increase in patients stromal cell numbers (2.11 vs. normal 0.61 x 106/2ml; p< 0.05). Patients' CD34⁺ cells panned on normal stromal layers resulted in significantly fewer CFU-bl (median 40 vs. normal 90 x 10⁴/ml CFU-bl; p=0.009), but CFU-bl numbers were corrected following the addition of bFGF, matching the scores achieved by normal individuals (85 vs. normal 90 x l 04/ml). Normal CD34⁺ cells proliferated poorly on patients' stromal layers in the absence of bFGF (3 9 vs. normal 90 x 10⁴/ml), but colony numbers increased significantly upon addition of bFGF (91 vs. normal 90 x 10⁴/ml CFU-bl). Thirteen patients receiving peripheral blood stem cells (PBSCs) and nine patients undergoing bone marrow transplantation (BMT) were compared. These two stem cell sources were then compared to the normal population. Stromal layers in patients receiving PBSC grafts covered a greater surface area than the area covered in patients undergoing BMT (median 65 vs. 30%). They also had higher blastic colony than scores from BMT recipients (47 vs. 12 x 10⁴/ml CFU-bl; p= 0.2). Results can be summarized that bone marrow stroma from patients receiving mobilized progenitor cells proliferates better than those receiving bone marrow grafts. It can be concluded that poor stromal layer and CD34⁺ cell proliferation following peripheral blood stem cell transplantation can be corrected by addition of basic Fibroblastic Growth Factor.
- ItemOpen AccessHIV-associated Hodgkin lymphoma at Groote Schuur Hospital, Western Cape, South Africa(2017) Swart, Luhan; Opie, Jessica; Novitzky, NicolasBackground: Human immunodeficiency virus (HIV) is associated with an increased risk of developing Hodgkin lymphoma (HL). South Africa (SA) has the highest HIV prevalence rate in the world. There is currently no 5-year overall survival (OS) outcome based data for HIV-associated HL from SA. Methods: A bone marrow database was compiled of all bone marrow biopsies (BMB) reported at National Health Laboratory Service (NHLS) Groote Schuur Hospital (GSH) between January 2005 and December 2012. Patients who had a BMB performed for staging of HL or where HL was diagnosed on the BMB were included for further analysis. Clinical and laboratory data was extracted from medical and laboratory records. Primary outcome measures included histological subtype, bone marrow infiltration (BMI) by HL, CD4 count, HIV-viral load (HIV-VL), tuberculosis (TB) data, treatment with chemotherapy and 5-year overall survival (OS). Results: The database included 6569 BMB and 219 patients of these had HL and were included for analysis. The median age at presentation (32 years) was similar in the HIV+ and HIV-populations. While males predominated in the HIV-group, females predominated in the HIV+ group (male:female ratio of 1.5:1 vs 0.7:1, respectively). The majority of patients (71%) were HIV negative (HIV-) and 29% were HIV positive (HIV+). The diagnosis of HL was made on BMB in 17% of cases. BMI was seen in 37%(82/219) overall, and was found in more HIV+ patients (61%; 39/64) than HIV-patients (28%; 43/155; p= 0.03). The histological subtype varied according to HIV status with nodular sclerosis classical Hodgkin lymphoma (NSCHL) being most frequent in the HIV-group and classical Hodgkin lymphoma (CHL)-unclassifiable the most frequent in the HIV+ group. HIV+ patients had a median CD4 count of 149 x106/L and 39% were anti-retroviral therapy (cART) naive at HL diagnosis. HIV+ patients had received anti-TB therapy more frequently than HIV-patients (72% vs 17%; p= 0.007). More HIV+ patients did not receive chemotherapy than HIV-patients (31% vs 3%; p= 0.001). The 5-year OS was 56%. HIV+ patients with BMI had a 5-year OS of 18%. BMI, HIV status, low CD4 count, histological subtype and TB therapy had a statistical significant impact on 5-year OS (p< 0.01). Conclusion: BMB provided the diagnosis of HL in 17% of cases, confirming its diagnostic utility in our setting. BMI by HL was more common in HIV+ patients and was associated with significantly worse survival. Our cohort showed similar survival outcomes to other countries in Africa, Asia and Central America with comparable socio-economic constraints to SA.
- ItemOpen AccessInteractions between the haematopoietic stem cell and the myeloid microenvironment in aplastic anaemia(1993) Novitzky, Nicolas; Jacobs, PeterIn patients with aplastic anaemia that respond to immunosuppressive therapy, quantitative, morphological and functional haematologic derangement have been reported. To explain these findings, abnormalities in the marrow stroma or the stem cell have been postulated. To define the relative contribution of each of the latter, the integrity of the bone marrow from sixteen patients that responded to anti-lymphocyte globulin and high dose methyl prednisolone was compared to normal individuals. Bone marrow mononuclear cells were divided into two fractions. From the first, stroma was cultured in aMEM containing 12.5% of both horse and foetal calf serum and 10-5 M hydrocortisone at 37° C in 5% CO2 in 90% humidity. The medium was changed weekly. Upon confluence, these stromal layers were studied morphologically and with cytospin preparations stained with Sudan black, 0 red oil, alkaline and acid phosphatases. The remainder was monocyte and lymphocyte depleted, CD 34+ progenitors were selected with paramagnetic beads and the population morphologically and immunophenotypically defined. To determine the functional status, control or patient CD 34+ progenitors, were suspended for two hours on normal or aplastic stroma for adherence to take place. The non-adhesive fraction was decanted by standardised washing and cultured for fourteen days in the presence of PHA-conditioned medium in the CFU-gm assay. Strama-adherent progenitors were covered with 0.3% agar and cultured for five days. Aggregates with more than twenty cells were scored (CFU-bl). The remaining CD 34+ cells were cultured in the mixed colony assay with combinations of recombinant cytokines belonging to the G protein super-family and the tyrosine kinase group in dose response studies. Light density cells from patients with treated aplasia contained significantly fewer CD 34+ cells than those present in the control suspensions (mean 0.65%, SD 0.35% vs 1.62%, SD 1.4%; p= 0.002). Normal and aplastic stroma became confluent at three and four weeks. There was no difference on the morphology or the cytochemical stains between the two groups. Functionally, aplastic bone marrow stroma supported CFU-bl formation no differently from normal layers. However, CD 34+ precursors from the patients cultured on control stroma resulted in significantly fewer CFU-bl (p= 0.0002,) and CFU-gm (p= 0.0009). This work provides original evidence supporting the reduced clonogenicity of the corresponding populations of CFU-bl from patients with aplasia is unrelated to attachment to the stroma, but intrinsic to the CD 34+ cells. Moreover, this study shows for the first time that exposure of these progenitors to growth factors belonging to the G protein and tyrosine kinase receptor families have defective responses, correctable only at supra physiological concentrations, while effects on combinations containing c-kit ligand, appear preserved. Following immunosuppressive therapy, the bone marrow is repopulated by a hypoproliferative progenitor cell population which responds suboptimally to physiological cytokine stimulation. This suggests that abnormal interactions between receptors and their ligands or alterations in the signal transduction for cell division by the cytokines belonging to the G superfamily lead to suboptimal growth.
- ItemOpen AccessSplenectomy for immune thrombocytopenia : our 11-year experience(2015) Antel, Katherine; Novitzky, NicolasSplenectomy has been practiced for the treatment of ITP for the past few decades. Currently it is utilised when a patient is either dependent or resistant to steroid treatment and the platelet count remains less than 30×109/L. Recently new agents have been added to the armamentarium used to treat ITP, including immune-suppressants such as rituximab and the new thrombopoetin-receptor agonists. This has brought into question the role of surgery for the treatment of ITP, and the need to compare the response and complication rates of splenectomy to these newer agents. Historic studies done on splenectomy for the treatment of ITP have been performed in the setting of low HIV prevalence. There is a relative paucity of data on the response rate in HIV-associated thrombocytopenia to splenectomy and the durability of response to splenectomy is unclear in this patient population. We retrospectively analysed 73 consecutive patients who underwent splenectomy for ITP from 2001 to 2011. The primary objective was to determine the rate of complete response, this was defined as a platelet count greater than 100×109/L at one year post splenectomy. Results were compared between HIV positive and HIV negative patients. The secondary objectives were: to evaluate the intra-operative and post†operative complications and mortality in the HIV positive and HIV negative groups, and to investigate for associations between co-morbidities, pre-operative treatment and response to splenectomy.
- ItemOpen AccessStudy of genetic modifiers of fetal hemoglobin and mechanisms of hydroxyurea-induced γ-globin expression in sickle cell disease(2016) Pule, Gift Dineo; Wonkam, Ambroise; Mowla, Shaheen; Novitzky, NicolasSickle Cell Disease (SCD) is a growing global problem with firm roots in sub-Saharan Africa (SSA) representing over 3/4 of the global burden of the disease. The prevalence of the sickle mutation (HbS) in SSA has been amplified by the partial resistance to Plasmodium falciparum malaria, which is endemic along tropical equatorial Africa. Several genetic variants have since been associated with fetal hemoglobin (HbF), the disease-ameliorating globin protein, including variants at three principal loci; BCL11A, HBS1L-MYB intergenic polymorphisms (HMIP1/2) and the β-globin gene cluster, which together account for 10 - 20% HbF variance in SCD patients. Similarly, numerous signalling pathways have been implicated in the regulation of γ-globin expression, however, a complete understanding of the regulation of HbF remains elusive. The overall aims of this project were: 1a) to investigate the known variants in key HbF-promoting loci such as BCL11A erythroid-specific enhancer, BCL11A, HBS1L-MYB intergenic polymorphism (HMIP1/2), the β-globin gene cluster, as well as the influence of the co-inheritance of 3.7kb alpha globin gene deletion in a cohort of SCD patients from Cameroon; and 1b) to validate novel HbF-promoting loci reported in 2 genome-wide association studies (GWAS) carried out in a population of Sardinians (Italy) and SCD patients from Tanzania and explore the influence of known promoter variants in SAR1 associated with HbF in African American patients amongst Cameroonian SCD patients; 2) to investigate the molecular mechanisms of hydroxyurea (HU)-induced production of HbF using a primary erythroid cell model from hematopoietic stem cells (HSCs) derived from umbilical cord blood and lastly, 3) to investigate the prevalence of SCD-related polymorphisms; β-globin gene haplotype, HbS mutation and malaria-resistance variants in 3 SCD-unaffected (HbAA) cohorts from South Africa, Zimbabwe and Malawi.