Screening and Structural Characterization of Heat Shock Response Elements (HSEs) in Entamoeba histolytica Promoters.

Entamoeba histolytica (E. histolytica) exhibits a remarkable capacity to respond to thermal shock stress through a sophisticated genetic regulation mechanism. This process is carried out via Heat Shock Response Elements (HSEs), which are recognized by Heat Shock Transcription Factors (EhHSTFs), enabling fine and precise control of gene expression. Our study focused on screening for HSEs in the promoters of the E. histolytica genome, specifically analyzing six HSEs, including Ehpgp5, EhrabB1, EhrabB4, EhrabB5, Ehmlbp, and Ehhsp100. We discovered 2578 HSEs, with 1412 in promoters of hypothetical genes and 1166 in coding genes. We observed that a single promoter could contain anywhere from one to five HSEs. Gene ontology analysis revealed the presence of HSEs in essential genes for the amoeba, including cysteine proteinases, ribosomal genes, Myb family DNA-binding proteins, and Rab GTPases, among others. Complementarily, our molecular docking analyses indicate that these HSEs are potentially recognized by EhHSTF5, EhHSTF6, and EhHSTF7 factors in their trimeric conformation. These findings suggest that E. histolytica has the capability to regulate a wide range of critical genes via HSE-EhHSTFs, not only for thermal stress response but also for vital functions of the parasite. This is the first comprehensive study of HSEs in the genome of E. histolytica, significantly contributing to the understanding of its genetic regulation and highlighting the complexity and precision of this mechanism in the parasite's survival.

Self-regulation predicts companionship in children with autism.

Self-regulation is associated with many positive outcomes in children with and without autism, including increased mental health and academic achievement, and decreased problem behavior. Less is known regarding whether and how self-regulation and symptoms of mental health challenges (internalizing and externalizing problems) relate to social outcomes, such as friendship quality and loneliness. Parents and teachers of 106 children with autism aged 5-12 reported on children's self-regulation difficulties and externalizing and internalizing symptoms. Four-to-five months later, children reported on the quality of their friendship with their best friend (companionship, conflict, helpfulness, sense of relationship security, closeness), and their feelings of loneliness. Linear regression was used to examine the effects of self-regulation and symptoms of mental health challenges on friendship quality and loneliness. Less self-regulation difficulties predicted stronger companionship and girls had better quality friendships with their best friend than did boys, in terms of companionship, helpfulness, security and closeness, confirming that they have a protective advantage in friendship development. Autism symptoms, IQ, and age were not associated with friendship quality or loneliness. Results highlight the importance of self-regulation and mental health interventions for school-aged children with autism.

The novel EhHSTF7 transcription factor displays an oligomer state and recognizes a heat shock element in the Entamoeba histolytica parasite.

The heat shock response is a conserved mechanism that allows cells to respond and survive stress damage and is transcriptionally regulated by the heat shock factors and heat shock elements. The P-glycoprotein confer the multidrug resistance phenotype; Entamoeba histolytica has the largest multidrug resistance gene family described so far; one of these genes, the EhPgp5 gene, has an emetine-inducible expression. A functional heat shock element was localized in the EhPgp5 gene promoter, indicating transcriptional regulation by heat shock factors. In this work, we determined the oligomer state of EhHSTF7 and the recognition of the heat shock element of the EhPgp5 gene. The EhHSTF7 recombinant protein was obtained as monomer and oligomer. In silico molecular docking predicts protein-DNA binding between EhHSTF7 and 5'-GAA-3' complementary bases. The rEhHSTF7 protein specifically binds to the heat shock element of the EhPgp5 gene in gel shift assays. The competition assays with heat shock element mutants indicate that 5'-GAA-3' complementary bases are necessary for the rEhHSTF7 binding. Finally, the siRNA-mediated knockdown of Ehhstf7 expression causes downregulation of EhPgp5 expression, suggesting that EhHSTF7 is likely to play a key role in the E. histolytica multidrug resistance. This is the first report of a transcription factor that recognizes a heat shock element from a gene involved in drug resistance in parasites. However, further analysis needs to demonstrate the biological relevance of the EhHSTF7 and the rest of the heat shock factors of E. histolytica, to understand the underlying regulation of transcriptional control in response to stress.

A Calpain-Like Protein Is Involved in the Execution Phase of Programmed Cell Death of Entamoeba histolytica.

Oxygen or nitrogen oxidative species and chemical stress induce the programmed cell death (PCD) of Entamoeba histolytica trophozoites. PCD caused by the aminoglycoside G418 is reduced by incubation with the cysteine protease inhibitor E-64; however, no typical caspases or metacaspases have been detected in this parasite. Calpain, a cysteine protease activated by calcium, has been suggested to be part of a specific PCD pathway in this parasite because the specific calpain inhibitor Z-Leu-Leu-Leu-al diminishes the PCD of trophozoites. Here, we predicted the hypothetical 3D structure of a calpain-like protein of E. histolytica and produced specific antibodies against it. We detected the protein in the cytoplasm and near the nucleus. Its expression gradually increased during incubation with G418, with the highest level after 9 h of treatment. In addition, a specific calpain-like siRNA sequence reduced the cell death rate by 65%. All these results support the hypothesis that the calpain-like protein is one of the proteases involved in the execution phase of PCD in E. histolytica. The hypothetical interactome of the calpain-like protein suggests that it may activate or regulate other proteins that probably participate in PCD, including those with EF-hand domains or other calcium-binding sites.

Frequency of the L1014F Mutation in the Sodium Channel Gene, in Culex quinquefasciatus (Diptera: Culicidae) Populations From Rural and Urban Areas of Yucatan State, Mexico.

Culex quinquefasciatus Say (Diptera: Culicidae) is a mosquito species that has attracted a lot of attention from a medical and veterinary point of view; however, little is known about the frequency of L1014F mutations that have been found in the sodium channel gene, with this being a target for DDT and pyrethroid insecticides. The distribution and frequency of the L1014F mutation in Cx. quinquefasciatus populations was determined in rural and urban areas of Yucatan, Mexico from January 2015 to March 2016. Nine hundred fifty adult females out of 17,727 immature states were collected and analyzed in all sites sampled (n = 10). Susceptible homozygotes were identified (L1014/L1014) in 12% (114/950), heterozygous individuals (F1014/L1014) in 34% (323/950), and mutated homozygotes (F1014/F1014) in 54% (513/950) during the dry and rainy seasons. In this work, study areas with a high frequency of L1014F mutation were identified. These findings may help guarantee a more effective and efficient use of the resources available for the control of this vector.

Synthesis and self-assembling properties of 4,6-O-benzylidene acetal protected D-glucose and D-glucosamine ß-1,2,3-triazole derivatives.

Sugar based low molecular weight gelators (LMWGs) are useful small molecules that can form reversible supramolecular gels with many applications. Selective functionalization of common monosaccharides has resulted in several classes of effective LMWGs. Recently we found that certain peracetylated sugars containing anomeric triazole functional groups were effective gelators. In this study we synthesized two series of 4,6-O-benzylidene acetal protected ß-1,2,3-triazolyl glycoside of D-glucose and N-acetyl D-glucosamine derivatives and evaluated their self-assembling properties in a few solvents. Several gelators were obtained and the gelation properties of these compounds rely on the structures of the 4-triazolyl substituents. Typically, alkyl derivatives resulted in effective gelation in organic solvents and aqueous mixtures of ethanol and dimethyl sulfoxide. But further acetylation of these compounds resulted in loss of gelation properties. The gels were characterized using optical microscopy, rheology, and FTIR spectroscopy. We also analyzed the molecular assemblies, using 1H NMR spectroscopy to probe the influences of the hydroxyl, amide, and triazole functional groups. Naproxen was used as a model drug and it formed co-gels with compound 25 in DMSO water mixtures. Using UV spectroscopy, we found that naproxen was slowly released from the gel to aqueous solution. The general structure and gelation trend obtained here can be useful in designing sugar based biomaterials. We expect that further structural optimization can lead to more effective gelators that are compatible with different drug molecules for encapsulation and sustained release.

A Novel Heat Shock Element (HSE) in Entamoeba histolytica that Regulates the Transcriptional Activation of the EhPgp5 Gene in the Presence of Emetine Drug.

Transcriptional regulation of the multidrug resistance EhPgp5 gene in Entamoeba histolytica is induced by emetine stress. EhPgp5 overexpression alters the chloride-dependent currents that cause trophozoite swelling, diminishing induced programmed cell death (PCD) susceptibility. In contrast, antisense inhibition of P-glycoprotein (PGP) expression produces synchronous death of trophozoites and the enhancement of the biochemical and morphological characteristics of PCD induced by G418. Transcriptional gene regulation analysis identified a 59 bp region at position -170 to -111 bp promoter as putative emetine response elements (EREs). However, insights into transcription factors controlling EhPgp5 gene transcription are missing; to fill this knowledge gap, we used deletion studies and transient CAT activity assays. Our findings suggested an activating motif (-151 to -136 bp) that corresponds to a heat shock element (HSE). Gel-shift assays, UV-crosslinking, binding protein purification, and western blotting assays revealed proteins of 94, 66, 62, and 51 kDa binding to the EhPgp5 HSE that could be heat shock-like transcription factors that regulate the transcriptional activation of the EhPgp5 gene in the presence of emetine drug.

Soybean agglutinin-conjugated silver nanoparticles nanocarriers in the treatment of breast cancer cells.

Silver nanoparticles (AgNPs) induce diverse cell-death mechanisms, similar to those promoted by anticancer chemotherapeutics; however, they have not been tested in vivo because their action is not limited to cancer cells. Therefore, in vivo evaluations of their effectiveness should be developed with targeting systems. Breast cancer shows changes in the sugar expression patterns on cell surfaces, related to cancer progression and metastases; those changes have been identified previously by the specific binding of soybean agglutinin (SBA). Here is proposed the use of SBA to target the AgNP activity in breast cancer. For that, the present work reports the synthesis of AgNPs (3.89 ± 0.90 nm) through the polyol method, the generation of AgNP nanocarriers, and the bioconjugation protocol of the nanocarrier with SBA. The free AgNPs, the AgNP nanocarriers, and the SBA-bioconjugated AgNP nanocarriers were tested for cytotoxicity in breast cancerous (MDA-MB-231and MCF7) and non cancerous (MCF 10A) cells, using the MTT assay. AgNPs demonstrated cytotoxic activity in vitro, the non cancerous cells (MCF 10A) being more sensible than the cancerous cells (MDA-MB-231 and MCF7) showing LD(50) values of 128, 205, and 319 µM Ag, respectively; the nanoencapsulation decreased the cytotoxic effect of AgNPs in non cancerous cells, maintaining or increasing the effect on the cancer-derived cells, whereas the SBA-bioconjugation allowed AgNP cytotoxic activity with a similar behavior to the nanocarriers. Future experiments need to be developed to evaluate the targeting effect of the SBA-bioconjugated AgNP nanocarriers to study their functionality in vivo.

Inhibition of Pten deficient Castration Resistant Prostate Cancer by Targeting of the SET - PP2A Signaling axis.

The PP2A signaling axis regulates multiple oncogenic drivers of castration resistant prostate cancer (CRPC). We show that targeting the endogenous PP2A regulator, SET (I2PP2A), is a viable strategy to inhibit prostate cancers that are resistant to androgen deprivation therapy. Our data is corroborated by analysis of prostate cancer patient cohorts showing significant elevation of SET transcripts. Tissue microarray analysis reveals that elevated SET expression correlates with clinical cancer grading, duration of neoadjuvant hormone therapy (NHT) and time to biochemical recurrence. Using prostate regeneration assays, we show that in vivo SET overexpression is sufficient to induce hyperplasia and prostatic intraepithelial neoplasia. Knockdown of SET induced significant reductions in tumorgenesis both in murine and human xenograft models. To further validate SET as a therapeutic target, we conducted in vitro and in vivo treatments using OP449 - a recently characterized PP2A-activating drug (PAD). OP449 elicits robust anti-cancer effects inhibiting growth in a panel of enzalutamide resistant prostate cancer cell lines. Using the Pten conditional deletion mouse model of prostate cancer, OP449 potently inhibited PI3K-Akt signaling and impeded CRPC progression. Collectively, our data supports a critical role for the SET-PP2A signaling axis in CRPC progression and hormone resistant disease.

Calpain-like: A Ca(2+) dependent cystein protease in Entamoeba histolytica cell death.

Entamoeba histolytica programmed cell death (PCD) induced by G418 is characterized by the release of important amounts of intracellular calcium from reservoirs. Nevertheless, no typical caspases have been detected in the parasite, the PCD phenotype is inhibited by the cysteine protease inhibitor E-64. These results strongly suggest that Ca(2+)-dependent proteases could be involved in PCD. In this study, we evaluate the expression and activity of a specific dependent Ca(2+) protease, the calpain-like protease, by real-time quantitative PCR (RTq-PCR), Western blot assays and a enzymatic method during the induction of PCD by G418. Alternatively, using cell viability and TUNEL assays, we also demonstrated that the Z-Leu-Leu-Leu-al calpain inhibitor reduced the rate of cell death. The results demonstrated 4.9-fold overexpression of calpain-like gene 1.5 h after G418 PCD induction, while calpain-like protein increased almost two-fold with respect to basal calpain-like expression after 3 h of induction, and calpain activity was found to be approximately three-fold higher 6 h after treatment compared with untreated trophozoites. Taken together, these results suggest that this Ca(2+)-dependent protease could be involved in the executory phase of PCD.

Targeting the MYC and PI3K pathways eliminates leukemia-initiating cells in T-cell acute lymphoblastic leukemia.

Disease relapse remains the major clinical challenge in treating T-cell acute lymphoblastic leukemia (T-ALL), particularly those with PTEN loss. We hypothesized that leukemia-initiating cells (LIC) are responsible for T-ALL development and treatment relapse. In this study, we used a genetically engineered mouse model of Pten(-/-) T-ALL with defined blast and LIC-enriched cell populations to demonstrate that LICs are responsible for therapeutic resistance. Unlike acute and chronic myelogenous leukemia, LICs in T-ALL were actively cycling, were distinct biologically, and responded differently to targeted therapies in comparison with their differentiated blast cell progeny. Notably, we found that T-ALL LICs could be eliminated by cotargeting the deregulated pathways driven by PI3K and Myc, which are altered commonly in human T-ALL and are associated with LIC formation. Our findings define critical events that may be targeted to eliminate LICs in T-ALL as a new strategy to treat the most aggressive relapsed forms of this disease.

A salicylic acid-induced lectin-like protein plays a positive role in the effector-triggered immunity response of Arabidopsis thaliana to Pseudomonas syringae Avr-Rpm1.

Salicylic acid (SA) is one of the key hormones that orchestrate the pathogen-induced immune response in plants. This response is often characterized by the activation of a local hypersensitive reaction involving programmed cell death, which constrains proliferation of biotrophic pathogens. Here, we report the identification and functional characterization of an SA-induced legume lectin-like protein 1 (SAI-LLP1), which is coded by a gene that belongs to the group of early SA-activated Arabidopsis genes. SAI-LLP1 expression is induced upon inoculation with avirulent strains of Pseudomonas syringae pv. tomato via an SA-dependent mechanism. Constitutive expression of SAI-LLP1 restrains proliferation of P. syringae pv. tomato Avr-Rpm1 and triggers more cell death in inoculated leaves. Cellular and biochemical evidence indicates that SAI-LLP1 is a glycoprotein located primarily at the apoplastic side of the plasma membrane. This work indicates that SAI-LLP1 is involved in resistance to P. syringae pv. tomato Avr-Rpm1 in Arabidopsis, as a component of the SA-mediated defense processes associated with the effector-triggered immunity response.

Entamoeba histolytica P-glycoprotein (EhPgp) inhibition, induce trophozoite acidification and enhance programmed cell death.

Programmed cell death (PCD) is induced in Entamoeba histolytica by a variety of stimuli in vitro and in vivo. In mammals, intracellular acidification serves as a global switch for inactivating cellular processes and initiates molecular mechanisms implicated in the destruction of the genome. In contrast, intracellular alkalinization produced by P-glycoprotein overexpression in multidrug-resistant cells has been related to apoptosis resistance. Our previous studies showed that overexpression of E. histolytica P-glycoprotein (PGP) altered chloride-dependent currents and triggered trophozoite swelling, the reverse process of cell shrinkage produced during PCD. Here we showed that antisense inhibition of PGP expression produced a synchronous death of trophozoites and the enhancement of biochemical and morphological characteristics of PCD induced by G418. The nucleus was contracted, and the nuclear membrane was disrupted. Moreover, chromatin was extensively fragmented. Ca(2+) concentration was increased, while the intracellular pH (ipH) was acidified. In contrast, PGP overexpression prevented intracellular acidification and circumvented the apoptotic effect of G418.

Influence of physical properties of solid biomass fuels on the design and cost of storage installations.

The aim of this work consists on determining biomass fuels properties and studying their relation with fixed and variable costs of stores and handling systems. To do that, dimensions (length and diameter), bulk density, particle density and durability of several brands and batches of wood pellets and briquettes were tested, according to international standards. Obtained results were compared with those in literature. Bulk density tests were applied for several other biomass fuels too, and later used to determinate which ones of all the biomass-fuels tested are economically more profitable for a typical transport/store system made of a screw conveyor and a concrete bunker silo.

Human and mouse DOCK10 splicing isoforms with alternative first coding exon usage are differentially expressed in T and B lymphocytes.

DOCK10 is a member of the dedicator of cytokinesis (DOCK) family of Rho GTPase activators preferentially expressed in lymphocytes. In this paper, we analyzed DOCK10 mRNA diversity produced because of alternative splicing. Alternative first coding exon usage led to 2 main protein-coding transcripts, DOCK10.1 and DOCK10.2. Full-length cDNA clones of both isoforms were obtained from both normal human peripheral blood mononuclear cells and mouse spleen for the first time for human DOCK10.1, mouse DOCK10.1, and mouse DOCK10.2. Human and mouse DOCK10.1 clones corresponded to the protein coding assemblies provided by the National Center for Biotechnology Information as Reference Sequences for DOCK10. Our analysis especially focused on human cDNA clones, of which 63% were alternatively spliced forms involving diverse exons and introns. DOCK10.1 expression was enriched in normal T cells, and DOCK10.2 expression was enriched in normal B cells and chronic lymphocytic leukemia (CLL) B cells. Both isoforms were upregulated in response to interleukin-4 in B cells, both normal and CLL, but not in T cells. Our data suggest that cell-specific mechanisms regulate expression of the alternative first exon variants of DOCK10 in vertebrates.

Suppression of leukemia development caused by PTEN loss.

Multiple genetic or molecular alterations are known to be associated with cancer stem cell formation and cancer development. Targeting such alterations, therefore, may lead to cancer prevention. By crossing our previously established phosphatase and tensin homolog (Pten)-null acute T-lymphoblastic leukemia (T-ALL) model onto the recombination-activating gene 1(-/-) background, we show that the lack of variable, diversity and joining [V(D)J] recombination completely abolishes the Tcrα/δ-c-myc translocation and T-ALL development, regardless of ß-catenin activation. We identify mammalian target of rapamycin (mTOR) as a regulator of ß-selection. Rapamycin, an mTOR-specific inhibitor, alters nutrient sensing and blocks T-cell differentiation from CD4(-)CD8(-) to CD4(+)CD8(+), the stage where the Tcrα/δ-c-myc translocation occurs. Long-term rapamycin treatment of preleukemic Pten-null mice prevents Tcrα/δ-c-myc translocation and leukemia stem cell (LSC) formation, and it halts T-ALL development. However, rapamycin alone fails to inhibit mTOR signaling in the c-Kit(mid)CD3(+)Lin(-) population enriched for LSCs and eliminate these cells. Our results support the idea that preventing LSC formation and selectively targeting LSCs are promising approaches for antileukemia therapies.

Cryopreservation impact on blood progenitor cells: influence of diagnoses, mobilization treatments, and cell concentration.

BACKGROUND: The aim of this study was to analyze the impact of cryopreservation in series of peripheral blood progenitor cells stratified by diagnosis, mobilization treatments, and cell concentration, as well as the accuracy of the control aliquots. STUDY DESIGN AND METHODS: Viability and colony-forming unit-granulocyte-macrophage (CFU-GM), CD34+ cell, lymphocyte, monocyte, and granulocyte counts and recovery were analyzed in 397 leukapheresis procedures before freezing and after thawing. Data from control cryotubes were compared to those from infusing bags. RESULTS: Cell viability decreased after thawing. Viability recovery was lower in cryotubes than in bags in non-Hodgkin's lymphoma (NHL), in cyclophosphamide plus granulocyte-colony-stimulating factor (Cy+G-CSF) mobilization, and in cell concentration of median or greater. Viability recovery in cryotube was higher in NHL (92.1%) than in Hodgkin's disease (HD; 87.3%) and in G-CSF (95.9%) than Cy+G-CSF mobilization (91.3%). The number of CD34+ cells decreased after thawing in total group, Cy+G-CSF mobilization, and cell concentration less than median subgroups. CD34+ cell recovery was higher in cryotubes (111.3%) than in bags (99.6%) in multiple myeloma (MM; p = 0.015). CFU-GM decreased after thawing in all groups. CFU-GM recovery was lower in cryotubes than in bags in MM (26.0% vs. 59.3%) and in Cy+G-CSF mobilization (49.8% vs. 76.3%). CFU-GM recovery in cryotubes was lower in MM compared with NHL (61.5%), HD (45.1%), and breast cancer (84.0%). Lymphocytes, monocytes, and granulocytes showed differences in the subgroups. CONCLUSION: Cryopreservation negatively impacts in cell viability, CD34+ cell recovery, granulocytes, and CFU-GM, although slight differences between the groups were observed. Cryotubes satisfactorily reflected the quality of the infused cells.

Splenic rupture in a plasma cell leukemia, mobilized with G-CSF for autologous stem cell transplant.

Splenic rupture (SR) is a rare adverse event observed in patients treated with G-CSF as a peripheral hematopoietic stem cell (PHSC) mobilizing agent, mostly in myeloma multiple and amiloidosis; to date, to our knowledge, it has not been previously described in plasma-cell leukemia (PCL). We report a case of a woman with PCL, who presented a SR after PHSC mobilization with Cyclophosphamide+G-CSF. The spleen removed showed hematopoietic foci and amiloid material. In the course of a second mobilization, 2 months after, the patient died from sepsis. We considered it important to report this case, in order to keep in mind the possibility of SR in patients with malignant gammopathy.

Entamoeba histolytica: differential gene expression during programmed cell death and identification of early pro- and anti-apoptotic signals.

We have demonstrated that programmed cell death (PCD) in Entamoeba histolytica is induced in vitro by G418 aminoglycoside antibiotic. To ascertain if biochemical and morphological changes previously observed are paired to molecular changes that reflect a genetic program, we looked here for early differential gene expression during the induction of PCD. Using cDNA-amplified fragment length polymorphisms (AFLPs) and in silico derived analysis we showed in E. histolytica a differential gene expression during PCD induced by G418. The genes identified encoded for proteins homologous to Glutaminyl-tRNA synthase, Ribosomal Subunit Proteins 40S and 18S, Saposin-like, Silent Information Regulator-2 (Sir-2), and Grainins 1 and 2. Using real-time quantitative PCR (RT Q-PCR), we found that glutaminyl-tRNA synthetase, sir-2, grainins and saposin-like genes were strongly overexpressed after 30min of PCD induction, while its expression dramatically decreased up to 60min. On the other hand, overexpression of ribosomal genes increased only 7-fold of basal expression, showing a progressive down-regulation up to 90min. glutaminyl-tRNA synthetase, sir-2 and grainins could act as negative regulators of PCD, trying to control the biochemical changes related to PCD activation. Overexpression of saposin-like gene could act as up-regulator of some cell death pathways. Our results give evidence of the first genes identified during the early stage of PCD in E. histolytica that could be implicated in regulation of apoptotic pathways.

Large-volume-apheresis facilitates autologous transplantation of hematopoietic progenitors in poor mobilizer patients.

Given that pre-apheresis CD34(+) cell count (PA-CD34) predicts the apheresis' yield, a minimum of 5 to 20 PA-CD34/microl is required in many institutions to initiate cell collection. The aim of this study was to clarify whether large-volume-apheresis (LVA) could facilitate progenitor cell transplantation in patients with low PA-CD34. Apheresis was initiated in 226 patients, disregarding PA-CD34, at days: +5 in G-CSF, +10 in cyclophosphamide+G-CSF, and +15 to +20 in other chemotherapy+G-CSF mobilization, when leucocytes >2.5 x 10(9)/L. Four times the blood volume was processed. Patients were grouped according to their PA-CD34: >or=10/microl (group-A, n = 143); <10/microl but >or=5/microl (group-B, n = 40) and <5/microl (group-C, n = 43). No differences were found in diagnoses, gender, age, previous treatments or mobilization regimen between groups. Enough CD34(+) cells (>1.9 x 10(6)/kg) were obtained in 31 patients (72%) from group-C, although in this group two mobilizations were needed in 20 patients (46.5%), compared to 5 (3.5%) and 1 (2.5%) in groups A and B, respectively (P < 0.01). Evenly three apheresis or more were required in 28 patients (65.1%) from group-C, compared to 8 (5.6%) and 6 (15.0%) in groups A and B, respectively (P < 0.01). In conclusion LVA can facilitate autologous transplantation in poor-mobilizer-patients, low PA-CD34 should not be an inflexible exclusion factor.