First experience of the use of a generic of plerixafor in peripheral blood stem cell mobilization in multiple myeloma and lymphoma patients.

Mobilization failure in patients is a major therapeutic concern which makes subsequent ASCT impossible. A new growth factor called Plerixafor (Mozobil®) developed by the pharmaceutical industry (Sanofi-aventis, France), is a chemoreceptor antagonist, CXCR4 type, which disrupts the interaction of SDFI and CXCR4, thereby enhancing the effect of G-CSF mobilization and is especially indicated for mobilization failure. Currently, there is a generic of plerixafor developed by the pharmaceutical industry (Hetero Drugs Ltd, India). The brand name of this medicine is Mozifor®. The objective of this study was to evaluate if generic plerixafor has the same efficacy and safety as originator plerixafor when used with G-CSF in the mobilization of PBSCs for autologous ASCT in multiple myeloma (MM) and lymphoma failure patients. The 32 patients received plerixafor were divided in two groups. The first group concerns the 11 consecutive patients prospectively received generic plerixafor (Mozifor®) in the period between January to July 2020. These were compared with a retrospective control cohort (second group n = 21) who had been treated between 2009 and 2019 with originator plerixafor (Mozobil®). For the Mozifor® group, the mean CD34+ was 4.54x106/kg(1.56-6.79), the median time to achieve an absolute neutrophil count >0.5 G/L was 13 days (range: 8-21). The median time to self-sustained platelet count >20 G/L was 15 days (range: 8-24). For the Mozobil® group, the mean CD34+ was 3.1x106/kg (0.56-8.91) (p=0.86), the median time to achieve an absolute neutrophil count >0.5 G/L was 10 days (range 7-23). The median time to self-sustained platelet count >20 G/L was 13 days (range: 7-29). Our study showed that the generic of plerixafor was practically identical to that of the originator (Mozobil®) with no significant difference (p = 0.52). This study demonstrates the safety and feasibility of mobilization PBSC with generic plerixafor in ASCT in MM and lymphoma. Although these outcomes are encouraging, prospective comparison with other traditional auto-HCT regimens used for patients with MM and lymphoma is warranted.

A simplified method for autologous stem cell transplantation in multiple myeloma.

BACKGROUND AND OBJECTIVES: We evaluated the efficacy and safety of non-cryopreserved storage of autologous hematopoietic stem cells with no post-transplant granulocyte colony-stimulating factor (G-CSF) support in adult patients undergoing autologous stem cell transplantation (ASCT) for multiple myeloma (MM). DESIGN AND SETTING: Retrospective review of patients undergoing ASCT from May 2009 to July 2011. PATIENTS AND METHODS: Autologous stem cell were mobilized using G-CSF. Leukapheresis to harvest stem cells was performed on day -2 and -1. The grafts were kept in a conventional blood bank refrigerator at 4°C until reinfusion on day 0. The conditioning regimen consisted of melphalan 200 mg/m2 in all patients. The post-chemotherapy myeloablative phase was managed without growth factors. RESULTS: Between May 2009 to July 2011, 54 adults with MM were treated in our center in Oran. The median age at ASCT was 55 years (range, 35-65). There were 37 males and 17 females. The median harvested CD34+ cell count was 3.60X106/kg (range, 1.90 to 10.52). All patients had neutrophil engraftment on the median of day 10 (range, 6-17) and platelet transfusion independence on the median of day 13 (range 9-24). In the 47 evaluable patients the median post-transplant overall survival had not been reached; the estimated overall survival at 30 months was 93.8% (0.05%) , and the estimated disease-free survival at 27 months was 93.6% (0.05%). CONCLUSION: High-dose chemotherapy and ASCT using non-cryopreserved stem cells and no G-CSF support is safe and feasible in the treatment of MM under our work conditions in developing countries.

How we assess adequacy of fine-needle aspiration materials intended for flow cytometric analysis.

Many articles have been published on the subject of FNAFNA, highlighting the usefulness of flow cytometry in the diagnosis and classification of lymphomas. But occasionally, flow cytometric evaluation fails to detect an abnormal population in a FNAFNA specimen involved by lymphoid neoplasm. Sampling errors (poor viability, peripheral blood contamination and hypocellular specimens) are the major reasons of this failure. In our laboratory we use a simple, fast and cost-effective approach to assess adequacy of FNAFNA materials and in this paper, we describe this procedure with giving some examples of interpretations of our results.

Childhood leprosy in an urban clinic, Hyderabad, India: clinical presentation and the role of household contacts.

A retrospective case note study was done of children below the age of 14 years who attended Dhoolpet Leprosy Research Centre (DLRC) over the decade 1990-1999. The aim of the study was to describe the pattern of clinical presentation, the role of household or near neighbour contacts and the incidence of neuritis and reactions. In all, 3118 leprosy patients were registered during this period, of whom 306 were children [182 (60%) male]; 95 children had a single patch, 159 had five or fewer than five patches and 37 had multiple patches. The youngest case detected was 9 months old. The spectrum of leprosy in these children was: TT 62 (20.3%); BT 203 (66.3%); BB 3 (1%); BL 23 (7.5%); LL 5 (1.6%) and PNL 10 (3.3%). Twenty-nine cases (9.4%) were smear positive. Ninety-one children (29.7%) developed a reaction, 86 type I and five type II. A history of contact was present in 119 (38.8%) cases, family contact in 113 (95%) and other than family in six (5%). Classification of the contact was available in only 60 patients. Among the contacts of the index case, 21 (35%) suffered from PB leprosy and 39 (65%) from MB leprosy. All contacts were from the immediate family. This study shows that childhood leprosy cases continue to present in significant numbers to this outpatient clinic. There is a high level of family contact with leprosy in these cases, strengthening the strategy of screening children in leprosy-affected households. The high incidence of reactions and nerve damage in children emphasizes the importance of early detection and treatment.

The human homologue of the Aspergillus nuclear migration gene nudC is preferentially expressed in dividing cells and ciliated epithelia.

We recently identified a novel human gene, HnudC, homologous to an Aspergillus nidulans gene coding for a protein crucial to nuclear migration, cell wall morphogenesis, and cell growth. While mRNA for this gene is expressed in most tissues, HNUDC protein expression is highly regulated. To provide insight into the function of this protein, we performed immunohistochemical analysis of the distribution of HNUDC in 19 different human tissues. Intense immunolabeling was observed in proliferating cells, including spermatocytes at all stages, early hematopoietic cells, cortical thymocytes, immunoblasts, and basal colonic and esophageal mucosa. Within a given tissue, cells with different proliferative capacities demonstrated different levels of HNUDC expression. HNUDC was also highly expressed in ciliated epithelia including those found in ependyma, bronchial mucosa, and fallopian tubes. Immunolabeling was moderate in several non-proliferating tissues, but little or no labeling was observed in most other tissues examined. We also demonstrated by western blotting that most cell lines express extremely high levels of HNUDC compared to their normal counterparts. While this supports a role for HnudC in cell proliferation, these data indicate that cell lines are not a reliable measure of HNUDC protein expression in normal tissues. We conclude that HNUDC is highly expressed in cell lines and the proliferating cells of normal tissues, consistent with our hypothesis that HNUDC is conserved throughout evolution for a crucial function in cell division. In addition, the high level in ciliated cells suggests an important role in ciliary motility or assembly, analogous to its role in A. nidulans nuclear movement.

Mitotic histone H3 phosphorylation by the NIMA kinase in Aspergillus nidulans.

Phosphorylation of histone H3 serine 10 correlates with chromosome condensation and is required for normal chromosome segregation in Tetrahymena. This phosphorylation is dependent upon activation of the NIMA kinase in Aspergillus nidulans. NIMA expression also induces Ser-10 phosphorylation inappropriately in S phase-arrested cells and in the absence of NIMX(cdc2) activity. At mitosis, NIMA becomes enriched on chromatin and subsequently localizes to the mitotic spindle and spindle pole bodies. The chromatin-like localization of NIMA early in mitosis is tightly correlated with histone H3 phosphorylation. Finally, NIMA can phosphorylate histone H3 Ser-10 in vitro, suggesting that NIMA is a mitotic histone H3 kinase, perhaps helping to explain how NIMA promotes chromatin condensation in A. nidulans and when expressed in other eukaryotes.

The nuclear migration gene NudC and human hematopoiesis.

The filamentous fungus Aspergillus nidulans nudC (nuclear distribution C) gene is required for movement of nuclei following mitosis and for normal colony growth. It is highly conserved, structurally and functionally, throughout most of evolution. The human homolog, called HnudC, has been cloned and has an important role in cell proliferation. In hematopoiesis, HNUDC is highly expressed in early hematopoietic precursors and declines during normal differentiation. Stimulation of proliferation of the erythroleukemia cell line TF-1 with GM-CSF enhances HnudC protein and mRNA expression and treatment with antisense (but not sense) oligonucleotides to HnudC mRNA significantly reduces cell division. A significant increase in HNUDC is present in bone marrow aspirates from patients with acute lymphoblastic leukemia (ALL) and acute myelogenous leukemia (AML) compared to the level in normal cellular counterparts, demonstrating dysregulated expression in leukemia. These data support the conclusion that HnudC plays a functional role in promoting hematopoietic cell growth and that it is involved in leukemogenesis.

Interaction between developmental and cell cycle regulators is required for morphogenesis in Aspergillus nidulans.

In Aspergillus nidulans, mutation of the transcriptional regulator brlA arrests formation of asexual spore-forming structures called conidiophores but does not hinder vegetative hyphal growth. During conidiophore development a 6-fold, brlA-dependent increase in the kinase activities of NIMX(cdc2) and NIMA occurs. A similar level of kinase induction was promoted by ectopic expression of brlA. Northern and Western analysis revealed marked induction of nimX(cdc2) mRNA after ectopic expression of brlA and increased amounts of NIMX(cdc2). Therefore, nimX(cdc2) is developmentally regulated by brlA indicating a direct role for brlA in the regulation of cell cycle genes. That correct regulation of nimX(cdc2) is important for normal development was further supported by analysis of conidiophore development and septation in cell cycle specific mutants. Most noticeably, the nimX(cdc2AF) mutation promoted inappropriate septation and hindered the switch from filamentous growth to budding growth seen during conidiophore development. Therefore, in contrast to the situation previously reported for other multicellular eukaryotes, interaction between developmental regulators and cell cycle regulators is essential for normal morphogenesis in A.nidulans.

Checkpoint defects leading to premature mitosis also cause endoreplication of DNA in Aspergillus nidulans.

The G2 DNA damage and slowing of S-phase checkpoints over mitosis function through tyrosine phosphorylation of NIMX(cdc2) in Aspergillus nidulans. We demonstrate that breaking these checkpoints leads to a defective premature mitosis followed by dramatic rereplication of genomic DNA. Two additional checkpoint functions, uvsB and uvsD, also cause the rereplication phenotype after their mutation allows premature mitosis in the presence of low concentrations of hydroxyurea. uvsB is shown to encode a rad3/ATR homologue, whereas uvsD displays homology to rad26, which has only previously been identified in Schizosaccharomyces pombe. uvsB(rad3) and uvsD(rad26) have G2 checkpoint functions over mitosis and another function essential for surviving DNA damage. The rereplication phenotype is accompanied by lack of NIME(cyclinB), but ectopic expression of active nondegradable NIME(cyclinB) does not arrest DNA rereplication. DNA rereplication can also be induced in cells that enter mitosis prematurely because of lack of tyrosine phosphorylation of NIMX(cdc2) and impaired anaphase-promoting complex function. The data demonstrate that lack of checkpoint control over mitosis can secondarily cause defects in the checkpoint system that prevents DNA rereplication in the absence of mitosis. This defines a new mechanism by which endoreplication of DNA can be triggered and maintained in eukaryotic cells.

The extremely conserved pyroA gene of Aspergillus nidulans is required for pyridoxine synthesis and is required indirectly for resistance to photosensitizers.

Numerous disparate studies in plants, filamentous fungi, yeast, Archaea, and bacteria have identified one of the most highly conserved proteins (SNZ family) for which no function was previously defined. Members have been implicated in the stress response of plants and yeast and resistance to singlet oxygen toxicity in the plant pathogen Cercospora. However, it is found in some anaerobic bacteria and is absent in some aerobic bacteria. We have cloned the Aspergillus nidulans homologue (pyroA) of this highly conserved gene and define this gene family as encoding an enzyme specifically required for pyridoxine biosynthesis. This realization has enabled us to define a second pathway for pyridoxine biosynthesis. Some bacteria utilize the pdx pyridoxine biosynthetic pathway defined in Escherichia coli and others utilize the pyroA pathway. However, Eukarya and Archaea exclusively use the pyroA pathway. We also found that pyridoxine is destroyed in the presence of singlet oxygen, helping to explain the connection to singlet oxygen sensitivity defined in Cercospora. These data bring clarity to the previously confusing data on this gene family. However, a new conundrum now exists; why have highly related bacteria evolved with different pathways for pyridoxine biosynthesis?

A homolog of the fungal nuclear migration gene nudC is involved in normal and malignant human hematopoiesis.

The filamentous fungus Aspergillus nidulans nudC gene has an essential function in movement of nuclei following mitosis and is required for normal colony growth. Here, the molecular cloning and role in hematopoiesis of a human gene (designated HnudC) homologous to A. nidulans nudC is reported. The amino terminus of the larger human protein (HNUDC = 45 kDa) does not overlap with A. nidulans NUDC (22 kDa). However, NUDC and the C-terminal 94 amino acids of HNUDC are 67% identical. The C-terminal region of the HnudC gene fully complements the A. nidulans temperature-sensitive nudC3 mutation, suggesting that nudC has an essential function in cell growth that is conserved from filamentous fungi to humans. In initial studies, HNUDC levels were much higher in erythroid precursors compared to most other human tissues. Therefore, the potential role of HnudC in hematopoiesis was explored. In normal human bone marrow, HNUDC protein and mRNA are highly expressed in early myeloid and erythroid precursors and decline as these cells terminally differentiate. To determine whether hematopoietic growth factors induce HnudC expression, TF-1 cells were stimulated by granulocyte-macrophage colony-stimulating factor. This induced a significant increase in HNUDC protein and HnudC mRNA, suggesting that enhancement of HnudC expression in response to growth factor stimulation may be mediated at the transcription level. Furthermore, HNUDC was significantly enhanced in lysates of bone marrow aspirates from patients with acute myelogenous and acute lymphoblastic leukemia compared to aspirates from normal controls, suggesting that HnudC is involved in malignant hematopoietic cell growth as well. These data demonstrate that HNUDC is highly expressed in normal and malignant human hematopoietic precursors and suggest it is of functional importance in the proliferation of these cells.

A mitogen-activated protein kinase (MPKA) is involved in polarized growth in the filamentous fungus, Aspergillus nidulans.

An Aspergillus nidulans kinase gene, which encodes a protein kinase with high similarity to mitogen-activated protein kinases involved in cell wall construction and morphogenesis in yeast species, was cloned and sequenced. Targeted deletion of the Aspergillus nidulans kinase gene indicates that this kinase is involved in germination of conidial spores and polarized growth. These defects were largely remedied on complex high osmolarity media, although abnormal swellings of hyphal tips were still observed. Glycerol (1 M) only supported the growth of compact colonies. The Aspergillus nidulans kinase gene is, thus, required for normal polarized growth at several stages of colony formation in the filamentous fungus A. nidulans.

Regulation of the anaphase-promoting complex/cyclosome by bimAAPC3 and proteolysis of NIMA.

Surprisingly, although highly temperature-sensitive, the bimA1(APC3) anaphase-promoting complex/cyclosome (APC/C) mutation does not cause arrest of mitotic exit. Instead, rapid inactivation of bimA1(APC3) is shown to promote repeating oscillations of chromosome condensation and decondensation, activation and inactivation of NIMA and p34(cdc2) kinases, and accumulation and degradation of NIMA, which all coordinately cycle multiple times without causing nuclear division. These bimA1(APC3)-induced cell cycle oscillations require active NIMA, because a nimA5 + bimA1(APC3) double mutant arrests in a mitotic state with very high p34(cdc2) H1 kinase activity. NIMA protein instability during S phase and G2 was also found to be controlled by the APC/C. The bimA1(APC3) mutation therefore first inactivates the APC/C but then allows its activation in a cyclic manner; these cycles depend on NIMA. We hypothesize that bimAAPC3 could be part of a cell cycle clock mechanism that is reset after inactivation of bimA1(APC3). The bimA1(APC3) mutation may also make the APC/C resistant to activation by mitotic substrates of the APC/C, such as cyclin B, Polo, and NIMA, causing mitotic delay. Once these regulators accumulate, they activate the APC/C, and cells exit from mitosis, which then allows this cycle to repeat. The data indicate that bimAAPC3 regulates the APC/C in a NIMA-dependent manner.

A role for NIMA in the nuclear localization of cyclin B in Aspergillus nidulans.

NIMA promotes entry into mitosis in late G2 by some mechanism that is after activation of the Aspergillus nidulans G2 cyclin-dependent kinase, NIMXCDC2/NIMECyclin B. Here we present two independent lines of evidence which indicate that this mechanism involves control of NIMXCDC2/NIMECyclin B localization. First, we found that NIMECyclin B localized to the nucleus and the nucleus-associated organelle, the spindle pole body, in a NIMA-dependent manner. Analysis of cells from asynchronous cultures, synchronous cultures, and cultures arrested in S or G2 showed that NIMECyclin B was predominantly nuclear during interphase, with maximal nuclear accumulation in late G2. NIMXCDC2 colocalized with NIMECyclin B in G2 cells. Although inactivation of NIMA using either the nimA1 or nimA5 temperature-sensitive mutations blocked cells in G2, NIMXCDC2/NIMECyclin B localization was predominantly cytoplasmic rather than nuclear. Second, we found that nimA interacts genetically with sonA, which is a homologue of the yeast nucleocytoplasmic transporter GLE2/RAE1. Mutations in sonA were identified as allele-specific suppressors of nimA1. The sonA1 suppressor alleviated the nuclear division and NIMECyclin B localization defects of nimA1 cells without markedly increasing NIMXCDC2 or NIMA kinase activity. These results indicate that NIMA promotes the nuclear localization of the NIMXCDC2/ NIMECyclin B complex, by a process involving SONA. This mechanism may be involved in coordinating the functions of NIMXCDC2 and NIMA in the regulation of mitosis.

A cyclin-dependent kinase family member (PHOA) is required to link developmental fate to environmental conditions in Aspergillus nidulans.

We addressed the question of whether Aspergillus nidulans has more than one cyclin-dependent kinase gene and identified such a gene, phoA, encoding two PSTAIRE-containing kinases (PHOAM1 and PHOAM47) that probably result from alternative pre-mRNA splicing. PHOAM47 is 66% identical to Saccharomyces cerevisiae Pho85. The function of this gene was studied using phoA null mutants. It functions in a developmental response to phosphorus-limited growth but has no effect on the regulation of enzymes involved in phosphorus acquisition. Aspergillus nidulans shows both asexual and sexual reproduction involving temporal elaboration of different specific cell types. We demonstrate that developmental decisions in confluent cultures depend upon both the initial phosphorus concentration and the inoculation density and that these factors influence development through phoA. In the most impressive cases, absence of phoA resulted in a switch from asexual to sexual development (at pH 8), or the absence of development altogether (at pH 6). The phenotype of phoA deletion strains appears to be specific for phosphorus limitation. We propose that PHOA functions to help integrate environmental signals with developmental decisions to allow ordered differentiation of specific cell types in A.nidulans under varying growth conditions. The results implicate a putative cyclin-dependent kinase in the control of development.