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Biomedical subjects

M A Alvarez

Publications and source records attributed to M A Alvarez.

At least 37 records · Page 2Linked to original sources
PubMed2000

Molecular basis for therapeutic decisions in chronic myeloid leukemia patients after allogeneic bone marrow transplantation.

BACKGROUND AND OBJECTIVES: Recent progress in the development of diagnostic techniques has greatly facilitated the monitoring of minimal residual disease (MRD) in patients with chronic myeloid leukemia (CML) after allogeneic bone marrow transplantation (BMT), the only curative treatment for this disease. The presence of the P210(bcr-abl) rearrangement in CML cells has allowed highly sensitive detection of MRD by polymerase chain reaction (PCR). However, complete eradication of the leukemic clone may not be a necessary prerequisite for long-term remission or cure. This observation limits the value of qualitative PCR analysis for prediction of progressive disease and highlights the need to monitor the proliferative activity of the malignant clone in order to permit timely detection of impending relapse and, thus, early therapy. This article discusses the applicability of several molecular methods to the monitoring of treatment efficacy and early assessment of clonal expansion in patients with CML after BMT. It also presents guidelines for clinical use of PCR analyses and the most effective approaches to treat relapsed patients. INFORMATION SOURCES: The authors have been working in this field, both experimentally and at a clinical level, contributing original papers to peer-reviewed journals. The material examined in this review includes articles published in journals covered by MedLine and reviews from journals with a high impact factor. STATE OF THE ART AND PERSPECTIVES: In view of the very limited value of qualitative PCR in detecting CML patients destined to relapse after BMT, several investigators have developed molecular assays that enable the kinetics of MRD to be monitored over time (e.g. quantitative PCR for P210(bcr-abl), PCR analysis of whole blood/lineage-specific chimerism and qualitative PCR for P190(bcr-abl)). These molecular strategies closely trace the kinetics of leukemic regrowth. Disease evolution in relapsed patients is consistently characterized by the sequential detection of increasing P210(bcr-abl) transcript levels, increasing myeloid mixed chimerism and finally, P190(bcr-abl) positivity preceding cytogenetic relapse. A 10-fold or greater increase in the expression of P210(bcr-abl) confirmed by a minimum of three independent quantitative PCR analyses and/or a progressive increase in the percentage of host myeloid cells in three consecutive chimerism analyses and/or P190(bcr-abl) mRNA detection must be regarded as an indication of incipient disease progression and should provide a rationale for initiation of treatment. There are various approaches to the management of the patient who relapses. The first step, if possible, is to reduce or terminate immune suppression. If the patient is not receiving this therapy, he or she can be treated with hydroxyurea or interferon or can be offered a second transplant. However, infusion to the patient of lymphoid cells (DLI) collected from the original donor has the capacity to restore complete remission in 70-80% of cases. Currently, several strategies are being used to minimize the severity of graft-versus-host disease after DLI (optimization of transfused lymphocyte doses, modification of the transfused lymphocyte subsets, administration of lymphocytes in escalating doses or lymphocyte transfection with a suicide gene), to reduce the incidence of marrow aplasia (stem cell support) and to increase the rate of complete responses (cytokines associated with DLI, leukemia-reactive cytotoxic lymphocytes, tyrosine kinase inhibitors or pre-emptive DLI).

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1999

Prevention of graft-versus-host disease in high risk patients by depletion of CD4+ and reduction of CD8+ lymphocytes in the marrow graft.

From March 1994 to September 1997, 30 patients with hematological malignancies (12 ANLL, 10 CML, four ALL and four multiple myeloma) received HLA-identical allogeneic bone marrow transplants with the marrow graft selectively depleted of CD4+ lymphocytes and the CD8+ cell content adjusted to 1x10(6)/kg. Total depletion of CD4+ and partial depletion of CD8+ lymphocytes was carried out by an immunomagnetical method. All patients were considered as having high risk for developing GVHD by at least one of the following criteria: patient age >35 years; donor age >35 years; donor multiparity or marrow from an unrelated donor. Twenty-four cases received marrow from an identical sibling and six from an unrelated donor. In order to assess the role of methotrexate (MTX) in addition to cyclosporin A (CsA) after transplant, patients were randomly assigned to received either CsA alone (n = 15) or CsA plus a short course of MTX (n = 15). No case of primary graft failure was observed, but two patients developed late graft failure. Six patients presented grade II acute GVHD and no case of severe III-IV GVHD was seen. The actuarial probability of developing grade II-IV acute GVHD was 25.9+/-9.6% for the entire population. Patients receiving post-transplant CsA + MTX had significantly less probability of acute GVHD than those receiving CsA exclusively (6.7+/-6.4% vs. 50.5+/-17.8%, P = 0.03) and the schedule of post-transplant immunosuppression was the only factor associated with the incidence of acute GVHD in a multivariate analysis. The actuarial incidence of chronic GVHD for the entire population was 31.8+/-12.5, and there was no significant difference between both groups with additional prophylaxis. Four patients with CML and three with ANLL relapsed: the actuarial probability of remaining in complete remission for all patients was 53.6+/-17.3%. For patients with acute leukemia, the probability of remaining in complete remission did not differ significantly between those transplanted in first complete remission and those receiving a transplant in more advanced phases of the disease (87.5+/-11.6% vs. 72.9+/-16.5%; P = 0.44). The incidence of mixed chimerism assessed by PCR was 34%. Nineteen patients are alive between 2 and 43 months post-transplant, the probability of overall survival being 57.8+/-10.4%. Our data indicate that this method of selective T cell depletion is very effective in preventing acute GVHD in high risk patients, particularly when used in combination with post-transplant CsA + MTX.

Adolescent↗
PubMed1999

Allogeneic bone marrow transplantation vs chemotherapy for the treatment of childhood acute lymphoblastic leukaemia in second complete remission (revisited 10 years on).

In 1989 we carried out a trial comparing allogeneic BMT to chemotherapy (CT) in 76 children with relapsed acute lymphoblastic leukaemia (ALL). Ten years on we have clinically revised outcome to firmly establish the role of each treatment, to analyse the importance of length of first remission and to provide long-term actuarial results for disease-free survival (DFS) and relapse rate in each group. For 21 patients within the transplantation group, probability of DFS and relapse are 42.8 +/- 10.8% and 40.2 +/- 11.7% (s.e.), respectively. In the chemotherapy group, probability of DFS is 10.0 +/- 4.74% (P = 0.001) and probability of relapse 87.5 +/- 5.2% (P = 0.0004). These results strongly reflect those at initial analysis, confirming a key role of BMT in the management of ALL in second remission. Moreover, on univariate analysis only two factors influenced DFS: treatment group and length of first complete remission (less or more than 30 months from first CR). Thus, it seems clear that the best therapeutic option in early relapse is BMT, whereas DFS in late relapse is at the limit of significance (P = 0.07), with a higher relapse rate in the CT group. Although encouraging results using intensified rotational combination chemotherapy have been published, prospective randomised studies are needed to assess with certainty the best therapeutic option in these patients.

Adolescent↗
PubMed1999

Stable expression of the Lactobacillus casei bacteriophage A2 repressor blocks phage propagation during milk fermentation.

A general strategy was applied to implement resistance against temperate bacteriophages that infect food fermentation starters through cloning and expression of the phage repressor. Lactobacillus casei ATCC 393 and phage A2 were used to demonstrate its feasibility as milk fermentation is drastically inhibited when the strain is infected by this phage. The engineered strain Lact. casei EM40::cI, which has the A2 repressor gene (cI) integrated into the genome, was completely resistant and able to ferment milk whether phage was present or not. In addition, viable phages were eliminated from the milk, probably through adsorption to the cell wall. Finally, the integration of cI in the genome resulted in a stable resistance phenotype, being unnecessary selective pressure during milk fermentation.

Animals↗
PubMed1998

The site-specific recombination system of the Lactobacillus species bacteriophage A2 integrates in gram-positive and gram-negative bacteria.

The region of the bacteriophage A2 genome involved in site-specific recombination with the DNA of Lactobacillus spp. has been identified. Two orfs, transcribed from the same strand, have been found immediately upstream of the phage attachment site (attP). The orf adjacent to attP predicts a 385-amino-acid protein that presents significant similarity with site-specific recombinases of the integrase family. The other orf encodes a basic polypeptide of 76 amino acid residues. The junctions of the prophage with the genomes of its hosts have been determined, allowing the identification of the host attachment site (attB), which has a common 19-nucleotide core region with attP. The attB site is located at the 3' end of the transfer RNALeu gene (anticodon CAA). Nonreplicative plasmids containing the A2-specific recombination cassette integrate into different lactobacilli but also into unrelated Gram-positive bacteria such as Lactococcus lactis and even into Escherichia coli. In Lc. lactis, integration occurs in a previously unknown intergenic region, whereas in E. coli, it maps within the rrnD operon, 5' of rrsD gene. Comparison of the integration sites in the different hosts indicates that some flexibility is permitted in the attB sequence, since Lc. lactis and E. coli only share 13 and 11 nucleotides, respectively, with the 19-nucleotide core sequence of the lactobacilli.

Amino Acid Sequence↗
PubMed1998

Autologous peripheral blood stem cell transplantation (PBSCT) mobilized with G-CSF in AML in first complete remission. Role of intensification therapy in outcome.

In order to determine if peripheral blood stem cells (PBSC) collected after priming with G-CSF in AML in first complete remission (CR) can be used for autologous transplantation and to evaluate the efficacy of early intensification therapy as in vivo purging, we studied 35 consecutive patients with AML in first CR. After standard induction and consolidation chemotherapy, 24 of them were treated with one (10 patients) or two (14 patients) cycles of high-dose cytarabine plus etoposide prior to PBSC collection. G-CSF was used as the priming agent. Of the 35 patients scheduled for peripheral blood stem cell transplantation (PBSCT), three relapsed before transplantation, and the 32 remaining underwent PBSCT. High-dose therapy consisted of either total body irradiation plus cyclophosphamide or busulphan plus cyclophosphamide. The median number of CD34+ cells infused was 3.24 x 10(6)/kg (range 0.15-14). The median times to reach a PMN count of 0.5 x 10(9)/l and a platelet count of 50 x 10(9)/l were 12 (8-28) and 30 (11-345) days, respectively. There was no transplant-related mortality. Twelve patients relapsed between 2 and 21 months post-PBSCT. With a median follow-up of 28 months, actuarial disease-free survival (DFS) is 52.41 +/- 9% in the intent-to-treat group and 57.4 +/- 9.8% in patients who underwent PBSCT. The probability of DFS is significantly higher for patients who receive early intensification therapy prior to both PBSC collection and PBSCT as compared with patients that do not: 68.8 +/- 10.27% vs 35.5 +/- 12.6%, P = 0.0418. These results indicate the feasibility of PBSCT in AML using G-CSF-mobilized PBSC. The use of intensification treatment as 'purging in vivo' prior both to collection of PBSC and PBSCT significantly reduces the risk of relapse in this group of patients.

Adolescent↗
PubMed1998

Identification of the repressor-encoding gene of the Lactobacillus bacteriophage A2.

The repressor gene of the Lactobacillus phage A2 has the following properties: it (i) encodes a 224-residue polypeptide with DNA binding and RecA cleavage motifs, (ii) is expressed in lysogenic cultures, and (iii) confers superinfection immunity on the host. Adjacent, but divergently transcribed, lies another open reading frame whose product resembles the lambda Cro protein. In the 161-bp intergenic segment, putative promoters and operators have been detected.

Amino Acid Sequence↗
PubMed1998

Peroxidase production in vitro by Armoracia lapathifolia (horseradish)-transformed root cultures: effect of elicitation on level and profile of isoenzymes.

Transformed roots of Armoracia lapathifolia (horseradish) were established by infection with Agrobacterium rhizogenes LBA 9402. They were used as a culture system in vitro for peroxidase production in vitro, to avoid many of the problems that affect the traditional production from field-grown species of Armoracia sp. The time course of growth of these cultures showed that total peroxidase attained maximum levels at the end of the exponential growth phase. At this stage of culture, elicitation assays were performed with AgNO3 and CuSO4 as abiotic elicitors and with fungal extracts of Verticillum sp., Monodyctis cataneae and Aspergillus niger as biotic elicitors. The best results were obtained with Verticillum sp., 24 h after elicitation, with an increase of approx. 100% in peroxidase activity. The isoenzyme pattern analysed by isoelectric focusing revealed predominantly basic and acidic isoenzymes in both plant roots and transformed root cultures. Elicited samples showed a similar isoenzyme pattern with a slight increase in basic isoenzymes.

Biomass↗
PubMed1997

Lymphokine-activated killer (LAK) cell generation from peripheral blood stem cells by in vitro incubation with low-dose interleukin-2 plus granulocyte-macrophage colony-stimulating factor.

Previous reports have demonstrated granulocyte-macrophage colony-stimulating factor (GM-CSF)-mediated enhancement of lymphokine activated killer (LAK) cell function. Based on these studies we have developed a model of LAK cell generation from peripheral blood stem cells (PBSC) from cancer patients by in vitro incubation with low-dose interleukin-2 (IL-2) + GM-CSF. PBSC from seven patients were incubated for 48 h at 37 degrees C in serum-free culture medium supplemented with IL-2 at increasing concentrations (10, 100 or 1000 IU/ml) in the presence or absence of 10 IU/ml GM-CSF. LAK activity generated in cultures with 10 IU/ml IL-2 + GM-CSF was significantly higher than that generated by 10 IU/ml IL-2 and did not differ from LAK generation at optimal concentrations of IL-2 (100 and 1000 IU/ml). PBSC from five additional patients were incubated with low-dose IL-2 + GM-CSF after sequential depletion of the CD4+ and CD8+ T cell subsets. LAK activity was significantly reduced by depletion of both CD4+ and CD8+ T cells and almost completely abolished after depletion of both subsets, suggesting that T cells and not NK cells are the main LAK precursors in this model. Six patients have received two courses of LAK cells generated in vitro by low-dose IL-2 + GM-CSF on day +1 and +8 after PBSC transplant in combination with GM-CSF and IL-2 administration in vivo. The mean LAK activity in peripheral blood of these patients dramatically increased immediately after transplant from a mean of 10% to 43.2% on day +2 and remained increased during the period studied. These results are encouraging and suggest that the administration of in vitro generated LAK cells early after transplant may have a role in the control of minimal residual disease.

Adolescent↗
PubMed1997

[Gestational diabetes: the use of an intensive treatment program at the regional level].

In this study the results are shown of the application of an intensified treatment program of Gestational Diabetes (GD) in a regional setting. From January 1991 to June 1995, 115 women with GD were attended at Mútua Hospital in Terrassa; 62.6% of them required insulin to optimize the metabolic control. When insulin dependent women were compared with non insulin dependent women, diagnosis was earlier; age, progestational age, serum glucose in the O'Sullivan test and diagnostic basal serum glucose in oral overload (GOO) were higher. Regarding perinatal results, the incidence of gestational hypertensive disturbances, hydramnios, and urinary tract infections were 7%, 1.7%, and 8%, respectively. Perinatal mortality was 0.86%. Delivery occurred after 37 weeks in 96.5% of cases and the cesarean section was performed in 26%; nevertheless, this rate was similar to that in the obstetric population attended at this institution (25%). Birth weight was high for gestational age in 7.8% of newborns (NB) and low in 2.5%. Factors associated with fetal weight included progestational maternal weight, ponderal increase during pregnancy and metabolic control (HbA1c). The present study suggests that the application of an intensified treatment program for GD can be carried out in a regional setting and with overall successful results.

Adult↗
PubMed1996

Comparative analysis of expression of the SalI restriction-modification system in Escherichia coli and Streptomyces.

The salIR and salIM genes encode the endonuclease and methyltransferase components of the SalI restriction-modification system from Streptomyces albus G. Expression of the salI genes in Escherichia coli was investigated and major differences with Streptomyces were found. In E. coli there is no detectable expression of the salI R gene due to inactivity of the sal-pR promoter region. In the natural host of the system this region directs transcription of the salI genes as a bicistronic message. In contrast to salIR, salIM is transcribed in the heterologous host from a promoter within the salI DNA. Since sal-pR is not active, the gene cannot be expressed as part of the salI operon. It is probably transcribed from sal-pM, a promoter internal to the operon which allows independent expression of the modification gene in Streptomyces. Replacement of sal-pR by the strong pLac promoter allows expression of salIR in E. coli and enhances expression of salIM. The resulting strain produces about 10 times more endonuclease than a Streptomyces clone containing the SalI system under the control of sal-pR.

Catechol 2,3-Dioxygenase↗
PubMed1996

Engineered biosynthesis of novel polyketides: regiospecific methylation of an unnatural substrate by the tcmO O-methyltransferase.

TcmO is an O-methyltransferase that methylates the C-8 hydroxyl to Tcm B3, a four-ring aromatic intermediate in the tetracenomycin biosynthetic pathway of Streptomyces glaucescens. The gene encoding this enzyme was expressed in Streptomyces coelicolor CH999 together with the actinorhodin polyketide synthase (PKS) gene cluster, which is responsible for the biosynthesis of 3,8-dihydroxy-methylanthraquinone carboxylic acid (DMAC) and its decarboxylated analog, aloesaponarin. The resulting recombinant strain produced approximately equal quantities of aloesaponarin and a new product but no DMAC. Spectroscopic analysis revealed that the novel polyketide was the 3-O-methylated analog of DMAC. An in vitro radioisotopic assay was developed for tcmO. The enzyme requires S-adenosylmethionine as a co-substrate. It has a Km of 3 microM and a kcat of 2.7 min-1 for DMAC. A series of monocyclic, bicyclic, and tricyclic aromatic compounds were also tested as candidate substrates in vitro. Remarkably, none was modified by tcmO within detectable limits of the assay. Together, these results highlight the interesting molecular recognition features of this enzyme. On one hand, there appears to be some flexibility in the number and structures of unreactive rings, since both Tcm and B3 and DMAC are good substrates. However, 6-methylsalicylic acid, a monocyclic analog of the reactive ring, is not recognized by the enzyme. Likewise, neither aloesaponarin (which only differs from DMAC in the reactive ring) nor carminic acid (which only differs in the distal nonreactive ring) is modified. Thus, the binding energy for the tcmO-catalyzed methyl transfer appears to involve significant contributions from both the aromaticity and the functionality of polycyclic substrates.

Anthraquinones↗
PubMed1996

Engineered biosynthesis of novel polyketides: properties of the whiE aromatase/cyclase.

The ORFVI from the cluster of genes, which is responsible for the biosynthesis of the Streptomyces coelicolor spore pigment, the whiE cluster, has been described as a bifunctional aromatase/cyclase. In order to evaluate its potential use for generating novel polyketides, combinations of this gene with those encoding minimal polyketide synthase enzymes with or without a ketoreductase from S. coelicolor A3(2) were constructed and analyzed in vivo. Analysis of the polyketide products generated from these constructs indicates that the whiE-ORFVI enzyme has properties similar to those of TcmN, although the whiE aromatase/cyclase normally acts on a polyketide intermediate that is four carbons longer than the TcmN substrate. The whiE aromatase/cyclase can influence the regiospecificity of the first cyclization of unreduced, but not reduced, backbones and is also responsible for the second ring aromatization. An unusual new polyketide, EM18, was identified which is not seen in equivalent strains expressing the tcmN aromatase/cyclase or the act aromatase genes. The structure of EM18 suggests that the WhiE-ORFVI product might have some unique properties within this family of polyketide synthase subunits, and may therefore be useful in the design of combinatorial biosynthetic strategies.

Anti-Bacterial Agents↗
PubMed1995

Expression of the SalI restriction-modification system of Streptomyces albus G in Escherichia coli.

The salIR and salIM genes of Streptomyces albus G encode the restriction endonuclease (ENase) and DNA methyltransferase (MTase) of the SalI restriction-modification (R-M) system. In S. albus G, the genes constitute an operon that is mainly transcribed from a promoter located upstream from salIR, the first gene of the operon. In addition, a second promoter, at the 3' end of salIR, allows independent transcription of the MTase gene. Expression of salIR and salIM in Escherichia coli was investigated. The ENase gene was not expressed in the heterologous host, probably due to inactivity of the main promoter of the salI operon. In contrast to salIR, salIM was functional in E. coli. Preliminary S1 nuclease mapping experiments suggest that the alternative promoter of the MTase gene can initiate transcription in the heterologous, as well as in the homologous host.

Cloning, Molecular↗
PubMed1995

Optimal timing of granulocyte colony-stimulating factor (G-CSF) administration after bone marrow transplantation. A prospective randomized study.

The positive role of G-CSF in hastening the myeloid recovery of patients undergoing allogeneic bone marrow transplantation (ALLO-BMT) or autologous bone marrow transplantation (ABMT) has recently been established. Considerable knowledge about adequate doses and route of administration has been accumulated in the past few years. Nonetheless, the optimal time to start growth-factor administration remains undetermined. We have performed a stratified study according to the source of hematopoietic progenitors (ALLO-BMT or ABMT), underlying disease and its stage, and acute graft-versus-host disease (GVHD) prophylaxis regimen and randomized patients in two arms: group A, which started G-CSF on day 0 (36 patients), and group B, which started on day +7 post-BMT (39 patients). The same dose (5 micrograms/kg/day) and route of administration were employed in both groups. We found no significant differences in the time to reach an absolute neutrophil count (ANC) of 0.1, 0.5, and 1 x 10(9)/l and 50 x 10(9) platelets/l (medians: 10 and 11, 14.5 and 14, 17 and 16, 23 and 24 days, respectively, in groups A and B). We did not find differences in the days of fever or days on antibiotic treatment with less than 1 x 10(9)/l ANC, rate of bacteriemia, or days of hospitalization in both groups. In contrast, a considerable saving of G-CSF in B group was found (mean days of infusion in group A, 18, versus 11 in group B) (p < 0.0001). This is equivalent to a saving of 1120 $US per patient.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1995

Pseudomonas aeruginosa strain MA01 aerobically metabolizes the aminodinitrotoluenes produced by 2,4,6-trinitrotoluene nitro group reduction.

Many microbes reduce the nitro substituents of 2,4,6-trinitrotoluene (TNT), producing aminodinitrotoluenes (ADNTs). These compounds are recalcitrant to further breakdown and are acutely toxic. In a search for organisms capable of metabolizing ADNTs, a bacterial strain was isolated for the ability to use 2-aminobenzoate (anthranilate) as sole C-source. This isolate, Pseudomonas aeruginosa MA01, metabolized TNT by first reducing one nitro group to form either 2-amino-4,6-dinitrotoluene (2ADNT) or 4-amino-2,6-dinitrotoluene (4ADNT). However, strain MA01 was distinct from other TNT-reducing organisms in that it transformed these compounds into highly polar metabolites through an O2-dependent process. Strain MA01 was able to cometabolize TNT, 2ADNT, and 4ADNT in the presence of a variety of carbon and energy sources. During aerobic cometabolism with succinate, 45% of uniformly ring-labeled [14C]TNT was transformed to highly polar compounds. Aerobic cometabolism of purified [14C]2ADNT and [14C]4ADNT with succinate as C-source produced similar amounts of these polar metabolites. During O2-limited cometabolism with succinate as C-source and nitrate as electron acceptor, less than 8% of the [14C]TNT was transformed to polar metabolites. Purified 2,6-diamino-4-nitrotoluene was not metabolized, and while 2,4-diamino-6-nitrotoluene was acetylated, the product (N-acetyl-2,4-diamino-6-nitrotoluene) was not further metabolized. Therefore, strain MA01 metabolized TNT by oxidation of the ADNTs and not by reduction the remaining nitro groups on the ADNTs.

Aerobiosis↗
PubMed1995

Murine splenic proliferative response to human recombinant erythropoietin along hypoxia.

Hypoxia is the best physiological stress to disturb the erythropoietic steady state. The present study has been undertaken in the aim to analize the splenic erythropoietic proliferative response with different doses of recombinant human erythropoietin under hypoxic conditions along 18 days using the DNA synthesis assay. Normoxic splenic progenitors failed to show significative erythroid replication at 0 days. A clearly rh Epo response was noticed from 2 to 8 days of hypoxia. Splenic proliferation returned to basal pattern from 10 days to the end of the experience. The highest proliferative activity, 25 fold increase over control (p < 0.001), was found at 6 days from 62.5 to 250 mU/ml rh Epo. These results support suggestions that hypoxia induces a transiently erythroid splenic proliferative response changing its quantitative parameters in the Epo dose-response relationship during the physiological adaptation.

Animals↗