Novel bifidobacteria strains isolated from nonconventional sources. Technological, antimicrobial and biological characterization for their use as probiotics.

AIM: To characterize four novel autochthonous bifidobacteria isolated from monkey faeces and a Bifidobacterium lactis strain isolated from chicken faeces by evaluating their technological and biological/functional potential to be used as probiotics. Different stressors, including food process parameters and storage, can affect their viability and functionality. METHODS AND RESULTS: The resistance to frozen storage, tolerance to lyophilization and viability during storage, thermal, acidic and simulated gastric resistance, surface hydrophobicity and antimicrobial activity against pathogens were studied. Bifidobacterium lactis Bb12 and INL1 were used as reference strains. The results obtained demonstrated that the new isolates presented strain-dependent behaviour. Good results were obtained for thermal resistance, frozen storage at -80°C and lyophilized powders maintained at 5°C. Cell viability during refrigerated storage was higher when the strains were resuspended in milk at pH 5·0 than at 4·5. The surface hydrophobicity ranged between 7 and 98% depending on the strain. The simulated gastric resistance was improved for the strains incorporated in cheese. Regarding antimicrobial activity, bifidobacteria isolated from monkey presented higher inhibitory capacity than the reference strains. CONCLUSION: This research provides a deeper insight into new strains of bifidobacteria isolated from primates and chicken that have not been previously characterized for their potential use in dairy products and confirm the most robust stress tolerance of B. lactis. SIGNIFICANCE AND IMPACT OF THE STUDY: The possibility of expanding the available bifidobacteria with the potential to be added to a probiotic food necessarily implies characterizing them from different points of view, especially when considering unknown species. For monkey isolates (which showed higher antimicrobial activity against pathogens), more in-depth knowledge is needed before applying strategies to improve their performance. On the contrary, the chicken isolate B. lactis P32/1 showed similar behaviour to the references B. lactis strains; therefore, it could be considered as a potential probiotic candidate.

Effect of alkyl groups on the cellular hydrolysis of stavudine phosphoramidates.

We examined the effect of cellular metabolism of three alkyl-substituted amino acid ester phosphoramidate derivatives of stavudine in different cell lines. Marked cell-to-cell differences were found in both the rate of hydrolysis and chiral selectivity. This selectivity implies that different enzymes may be involved in the metabolism of these compounds depending on the cell type involved. Notably, both the methyl and ethyl substituted derivatives underwent hydrolysis in presence of various cell lines, whereas the tert-butyl substituted compound was resistant to hydrolysis implying that steric hindrance associated with this group along with electron density may play a key role in the hydrolysis profile of these compounds. Additionally we found this mimicked the hydrolysis profiles obtained for bacterial enzymes. Furthermore, our results suggest that the site of attack of the cellular enzymes is confined to the ester side chain of the molecule. This result is also consistent with our earlier observation using bacterial enzymes as well as using 'd' isomers.

Large germline deletions of mitochondrial complex II subunits SDHB and SDHD in hereditary paraganglioma.

More than 30% of adrenal pheochromocytomas are hereditary. These neuroendocrine tumors are major components of three inherited cancer syndromes: multiple endocrine neoplasia type 2, von Hippel-Lindau disease (VHL), and pheochromocytoma/paraganglioma syndrome (PC/PGL). Germline mutations in RET; VHL; and SDHB, SDHC, and SDHD are associated with multiple endocrine neoplasia type 2, VHL, and PC/PGL, respectively. The majority (>70%) of hereditary extraadrenal PCs [catecholamine-secreting paragangliomas (PGL)] are accounted for by germline intragenic mutations in SDHB, SDHC, or SDHD. Therefore, a subset of hereditary PGL is not accounted for. Here we report two unrelated hereditary PGL families, one with a germline whole-gene deletion of SDHD (family 4194), the other a partial deletion of SDHB (family BRZ01). Although they were initially designated mutation negative for all of the PC-associated genes after PCR-based analysis, we suspected that a large deletion or rearrangement might be present. Genotyping around the PC-associated genes demonstrated that both families were consistent with linkage with one of these genes. Using fine structure genotyping and semiquantitative duplex PCR analysis, we identified an approximately 96-kb deletion spanning SDHD in family 4194 and an approximately 1-kb deletion involving the 5' end of SDHB in family BRZ01. Thus, including SDHB and SDHD deletion analysis could increase gene-testing sensitivity for PGL patients, which would aid in genetic counseling and management of patients and families.

Isolation of Beauveria bassiana and Metarhizium anisopliae var. anisopliae from Boophilus microplus tick (Canestrini, 1887), in Rio de Janeiro State, Brazil.

The objective of this work were to isolate and identify strains of entomopathogenic fungi from ingurgitated female Boophilus microplus ticks, collected from the soil in the municipality of Paracambi, Rio de Janeiro State, Brazil. The ingurgitated females were inoculated in the selective medium oat dodine agar (oda), where 49 colonies of Beauveria bassiana (71%) and 20 of Metarhizium anisopliae var. anisopliae (29%) were isolated. These isolated strains characterize for the first time in Brazil the natural occurrence of these species of fungi in this tick, and will be used to conduct bioassays to evaluate the pathogenicity and virulence of these strains for ticks of the genus Boophilus microplus.

Clonal composition of human adamantinomatous craniopharyngiomas and somatic mutation analyses of the patched (PTCH), Gsalpha and Gi2alpha genes.

Craniopharyngioma is the most common childhood tumor and thought to arise from embryonic remnants of Rathke's pouch. The paucity of published data on the molecular basis of these tumors prompted us to examine 22 adamantinomatous craniopharyngiomas looking for genetic abnormalities. Using the X-linked polymorphic androgen receptor gene as a tool for X-chromosome inactivating analysis, we found that a subset of craniopharyngiomas are monoclonal and therefore are probably due to acquired somatic genetic defects. Thus, we investigated these tumours for mutations within three candidate genes, Gsalpha, Gi2alpha and patched (PTCH). Using single stranded conformational polymorphism (SSCP), denaturing gradient gel electrophoresis and direct sequencing, the presence of somatic mutations in these genes could not be demonstrated in any tumor. Our data indicate that a subset of craniopharyngiomas are monoclonal and the mutations in the PTCH, Gsalpha, and Gi2alpha contribute little if any to craniopharyngioma development.

Bone marrow leukemic progenitor cell content in pediatric T-lineage acute lymphoblastic leukemia patients with an isolated extramedullary first relapse.

Isolated extramedullary relapse in childhood acute lymphoblastic leukemia (ALL) is associated frequently with the T-lineage immunophenotype and may be accompanied by occult bone marrow disease. We employed highly sensitive multiparameter flow cytometry and blast colony assays to quantify the leukemic progenitor cell (LPC) burden in the pretreatment bone marrows of 15 pediatric T-lineage ALL patients with an isolated extramedullary first relapse. Sites of extramedullary relapse were CNS (11 patients), testes (3 patients), and both CNS and testes (1 patient). Bone marrow LPC were detectable in 8 patients (53%) and undetectable in 7 patients (47%) at day 0 of post-relapse induction therapy, with LPC counts ranging from 0/10(6) mononuclear cells (MNC) to 518/10(6) MNC (mean +/- SEM, 50+/-34/10(6) MNC). Five of 9 patients with an early relapse (< 18 months after achieving a first complete remission [CR1]) and 3 of 6 patients with a late relapse (> or = 18 months from CR1) had detectable bone marrow LPC at day 0. Five of 8 patients with NCI-defined poor risk ALL and 3 of 7 patients with NCI-defined standard risk ALL had detectable LPC at day 0. Following post-relapse induction chemotherapy. LPC counts were detectable in bone marrows of 4 of 6 evaluated patients. Thus, approximately half of the extramedullary relapse T-lineage ALL patients studied had substantial occult involvement of the bone marrow. These findings may partly explain the previously observed poor prognosis of T-lineage patients following a CNS relapse.

Chemosensitivity of TEL-AML1 fusion transcript positive acute lymphoblastic leukemia cells.

The TEL-AML1 fusion which results from a cryptic t(12;21) translocation is the most frequently occurring genetic abnormality in childhood acute lymphoblastic leukemia (ALL) and has been associated with an excellent treatment outcome. In the present study, we examined the FAS/BCL-2 expression profiles and chemosensitivity of primary leukemic cells from children with newly diagnosed t(12;21)TEL-AML1 fusion transcript-positive versus t(12;21)TEL-AML1 fusion transcript-negative standard risk ALL. TEL-AML1(+) ALL cells expressed higher levels of the pro-apoptotic protein Fas and lower levels of the anti-apoptotic protein Bcl2 than TEL-AML1(-) ALL cells, as determined by confocal laser scanning microscopy. TEL-AML1(+) ALL cells were more sensitive to the apoptosis-inducing effects of serum deprivation, dexamethasone and vincristine than TEL-AML1(-) ALL cells. This study provides novel mechanistic insights regarding the chemosensitivity of TEL-AML1(+) ALL cells and provides a cogent explanation for the excellent leukemia-free survival outcome of children with TEL-AML1(+) ALL treated on contemporary chemotherapy programs.

Residual bone marrow leukemic progenitor cell burden after induction chemotherapy in pediatric patients with acute lymphoblastic leukemia.

We used highly sensitive multiparameter flow cytometry and blast colony assays to quantify the leukemic progenitor cell (LPC) burden of postinduction chemotherapy bone marrows from newly diagnosed and relapsed pediatric patients with acute lymphoblastic leukemia (ALL). Of 890 newly diagnosed patients, 243 (27%) had detectable LPC in the postinduction bone marrow samples with an average (mean +/- SE) LPC content of 22+/-9 LPC/10(6) mononuclear cell (MNC; range, 0-7199/10(6) MNC; median, 0/10(6) MNC). By comparison, 24 of 50 (48%) patients with relapsed ALL had detectable LPC in their postinduction bone marrow specimens (P = 0.003), and their average LPC content was 202+/-139 LPC/10(6) MNC. Fewer patients with B-lineage ALL (170 of 786; 22%) than patients with T-lineage ALL (73 of 104; 70%) harbored residual LPC in their postinduction bone marrow specimens (P < 0.0001). This correlation with immunophenotype was independent of the National Cancer Institute risk classification. Similarly, 19 of 44 (43%) patients with relapsed B-lineage ALL versus 5 of 6 (83%) patients with relapsed T-lineage ALL harbored residual LPC in their postinduction bone marrow specimens (P = 0.09). Among newly diagnosed patients, those with high-risk ALL seemed to have larger numbers of residual LPC in their bone marrow after induction chemotherapy than those with standard risk ALL (53+/-26, n = 286 versus 7+/-1, n = 604, P = 0.04). LPC of patients with standard risk ALL who had a slow early marrow response at day 7 seemed to be more resistant to the three-drug induction chemotherapy than patients who had a rapid early marrow response. Overall, the order of chemosensitivity of LPC was: newly diagnosed standard risk B-lineage > newly diagnosed higher risk B-lineage > newly diagnosed standard risk T-lineage > newly diagnosed higher risk T-lineage > relapsed B-lineage > relapsed T-lineage. Notably, LPC- patients whose end-of-induction remission bone marrow specimens had zero LPC had an excellent early event-free survival outcome. Within the standard and high-risk subsets, LPC- patients had a 2.6-fold lower and 2.4-fold lower incidence of events, respectively, than LPC+ patients. At 6 months, 12 months, as well as 24 months, the ranking order for better event-free survival was: standard risk, LPC- > high risk, LPC- > standard risk, LPC+ > high risk, and LPC+.

Microsatellite instability in sporadic parathyroid adenoma.

Parathyroid adenomas are usually benign uniglandular tumors, and inactivation of several tumor suppressor genes, notably the MEN 1 gene, or activation of oncogenes have been implicated in the tumorigenesis. Genomic instability, indicative of the involvement of DNA mismatch repair genes, has not been previously described in parathyroid adenomas. A single large parathyroid adenoma was resected from an 8.5-yr-old Brazilian patient with no personal or family history of other endocrinopathies. Analysis of paired tumor-nontumor DNA using 23 microsatellite markers, located on chromosomes 1, 10, and 11 was carried out. Microsatellite instability was detected in nine markers (D1S191, D1S212, D1S413, D1S2848, RET, D11S901, D11S903, INSR, and INT2), whereas no allelic loss was detected with any of the analyzed markers. Immunohistochemical analysis of retinoblastoma protein expression revealed low levels of expression, but no histopathological signs of malignancy. We conclude that in this single, apparently sporadic parathyroid adenoma, DNA mismatch repair genes might be involved in parathyroid tumorigenesis.

Paucity of leukemic progenitor cells in the bone marrow of pediatric B-lineage acute lymphoblastic leukemia patients with an isolated extramedullary first relapse.

Isolated extramedullary relapse in childhood acute lymphoblastic leukemia (ALL) may be accompanied by occult bone marrow disease. We used a highly sensitive assay to quantify leukemic progenitor cells (LPCs) in the bone marrow of such patients. Multiparameter flow cytometry and blast colony assays were used to detect LPCs in the bone marrow of 31 pediatric B-lineage ALL patients with an isolated extramedullary first relapse. Sites of relapse were central nervous system (22 patients), testes (7 patients), and eye (2 patients). Bone marrow (BM) LPC counts ranged from 0/10(6) mononuclear cells (MNCs) to 356/10(6) MNCs (mean +/- SE, 27.8+/-13.1/10(6) MNCs). LPCs were undetectable in 19 patients (61%). The BM LPC burden at the time of extramedullary relapse was similar, regardless of site (Wilcoxon P = 0.77) or time of relapse (Wilcoxon P = 0.80). Compared with higher risk, standard risk at initial diagnosis showed a trend for increased BM LPC burden (mean +/- SE, 44.6+/-17.1 versus 7.5+/-3.3; Wilcoxon P = 0.22). After successful postrelapse induction chemotherapy, LPC counts in 21 evaluated patients ranged from 0/10(6) to 175/10(6) MNCs (mean +/- SE, 15.9+/-9.6/10(6) MNCs). By comparison, LPC burden was higher after successful induction chemotherapy among children with an early BM relapse (range, 0 to 3262/ 106 MNC; mean +/- SE, 166+/-107; Wilcoxon P = 0.11). Thus, not all patients with an extramedullary relapse have occult systemic failure with substantial involvement of the bone marrow, and after reinduction therapy, LPC counts were lower in these patients than in patients treated for an overt BM first relapse.

Prognostic significance of T-lineage leukemic cell growth in SCID mice: a Children's Cancer Group study.

Contemporary intensive therapies are effective for the majority of pediatric T-lineage acute lymphoblastic leukemia (ALL) patients, thus current challenge is to identify patients who may benefit from alternative treatment modalities. Previously, we demonstrated that human leukemic cell growth in the severe combined immunodeficiency (SCID) mouse was a significant prognostic factor for very high risk B-lineage ALL patients. In the current report we show that primary leukemic cells from 24 of 88 (27%) T-lineage ALL patients (SCID+) caused histopathologically detectable leukemia in SCID mice. These SCID+ patients were similar to SCID- (n = 64) patients with respect to virtually all presenting features, including age, sex, race, and leukocyte count. Growth of primary leukemic cells in SCID mice was not a significant predictor of outcome for the aggregate population of T-lineage ALL patients. Two-year event-free survival (EFS) outcomes for SCID+ patient and SCID- patients were 76.2% (SD = 5.6%) and a 64.0% (SD = 10.4%; p = 0.20). Overall survival also was similar between the two groups (p = 0.36). Among the subset of patients with M1 or M2 marrow status by day 7 of induction chemotherapy (rapid early responders), those who were SCID+ had poorer outcomes than those who were SCID-, with a 2-year EFS of 68.4% (SD = 11.9%) vs. 85.7% (SD = 6.0%) and relative hazard rate of 3.06 (p = 0.06). These data suggest that leukemic cell growth in SCID mice may identify a subset of T-lineage ALL patients who are at higher risk for relapse despite achieving a rapid early response to induction chemotherapy.

Prognostic significance of B-lineage leukemic cell growth in SCID mice: a Children's Cancer Group Study.

Primary leukemic cells isolated from children (N = 681 ) with newly diagnosed B-lineage ALL enrolled on risk-adjusted treatment protocols of the Children's Cancer Group (CCG) were injected via the tail vein into 7-10 week old SCID mice. Leukemic cells from 104 of 681 patients (15.3%) were able to engraft and proliferate in one or more SCID mouse organs. These SCID+ patients were somewhat more likely than SCID patients to be older than 10 years of age (p = 0.03) and have WBC counts >20,000/microL (p = 0.04), but the groups were similar with respect to all other presenting features. Event-free survival (EFS) outcome at 3 years of follow-up was similar for SCID+ patients compared with SCID- patients (79.2%, SD = 5. 1% vs. 84.8%, SD = 2.8%; p = 0.20). Overall survival also was similar between the two groups (p = 0.93). This result was maintained within the subgroups of lower risk (N = 448) and higher risk (N = 233) patients. However, there were trends for poorer outcome among patients whose cells caused overt leukemia in SCID mice and infiltrated either 6 or more organs (p = 0.03), skeletal muscle (p = 0.0003), kidney (p = 0.05), or spleen (p = 0.06). Thus, engraftment of primary leukemic cells in SCID mice was not a significant predictor of outcome for the aggregate population of B-lineage ALL patients, the majority of whom were low risk, treated according to contemporary intensive chemotherapy programs of the CCG. However, development of disseminated overt leukemia and infiltration of SCID mouse skeletal muscle by primary leukemic cells from adjacent bone marrow may reflect a biologically more aggressive disease and identify patients at higher risk for treatment failure.

Clinical significance of MLL-AF4 fusion transcript expression in the absence of a cytogenetically detectable t(4;11)(q21;q23) chromosomal translocation.

Leukemic cells from bone marrow (BM) of 17 infants and 127 children with newly diagnosed ALL, as well as fetal liver and BM and normal infant BM samples, were analyzed for presence of a t(4;11) translocation using standard cytogenetic techniques and expression of an MLL-AF4 fusion transcript using standard reverse transcriptase-polymerase chain reaction (RT-PCR) assays as well as nested RT-PCR that is 100-fold more sensitive than standard RT-PCR. Overall, 9 of 17 infants and 17 of 127 noninfant pediatric ALL patients were positive for expression of MLL-AF4 fusion transcripts, as determined by standard and/or nested RT-PCR assays. None of the MLL-AF4(+) cases were positive for E2A-PBX1 or BCR-ABL fusion transcript expression. Although 8 of 9 MLL-AF4(+) infants had cytogenetically detectable t(4;11)(q21;q23), 15 of the 17 MLL-AF4(+) noninfants were t(4;11)-. Infants with MLL-AF4(+) ALL had poor outcomes, whereas non-infant MLL-AF4(+)/t(4;11)- patients had favorable outcomes similar to MLL-AF4(-) patients. Notably, MLL-AF4 transcripts also were detected by nested RT-PCR in 4 of 16 fetal BMs, 5 of 13 fetal livers, and 1 of 6 normal infant BMs, but not in any of the 44 remission BM specimens from pediatric ALL patients. Our results provide unprecedented evidence that MLL-AF4 fusion transcripts can be present in normal hematopoietic cells, indicating that their expression is insufficient for leukemic transformation of normal lymphocyte precursors.

Large scale manufacturing of TXU(anti-CD7)-pokeweed antiviral protein (PAP) immunoconjugate for clinical trials.

We have conjugated the murine monoclonal anti-CD7 antibody TXU to the plant hemitoxin pokeweed antiviral protein (PAP) to construct an effective immunotoxin against CD7 antigen positive hematologic malignancies. The scaled-up production and purification of TXU antibody, PAP toxin, and TXU-PAP immunotoxin permitted the manufacturing of a highly purified clinical-grade TXU-PAP preparation. In clonogenic assays, TXU-PAP elicited selective and potent cytotoxicity against CD7 antigen positive human leukemia cells and killed primary clonogenic leukemic cells from T-lineage acute lymphoblastic leukemia (ALL) patients. To our knowledge, this pre-IND work represents the first effort of producing a clinical-grade PAP immunotoxin for treatment of T-lineage ALL. Since the CD7 antigen is also expressed on AML cells, TXU-PAP could also be useful for the treatment of CD7 positive acute myeloid leukemia (AML) patients.

Expression of BCR-ABL, E2A-PBX1, and MLL-AF4 fusion transcripts in newly diagnosed children with acute lymphoblastic leukemia: a Children's Cancer Group initiative.

We used reverse transcriptase polymerase chain reaction (RT-PCR) assays to examine primary leukemic cells in on-study diagnostic bone marrow specimens from 642 children with newly diagnosed acute lymphoblastic leukemia (ALL) for the expression of MLL-AF4, E2A-PBX1, and BCR-ABL fusion transcripts. All PCR assays were performed centrally in the Children's Cancer Group ALL Biology Reference Laboratory. MLL-AF4 transcript was found in only 0.7% of the study population which excluded infants. E2A-PBX1 transcript was found in 2.5% of the study population and 3.3% of B-precursor cases. Expression was associated with massive hepatomegaly. BCR-ABL transcript was found in 2.3% of cases and correlated with older age, induction failure, and inferior event-free survival (EFS). RT-PCR assays allow rapid identification of patients with MLL-AF4 and BCR-ABL positive ALL. These patients have a poor outcome with contemporary therapy and rapid identification facilitates timely allocation to innovative treatment programs.

Cellular expression of antiapoptotic BCL-2 oncoprotein in newly diagnosed childhood acute lymphoblastic leukemia: a Children's Cancer Group Study.

We found a marked variation in BCL-2 oncoprotein expression levels of primary leukemic cells from 338 children with newly diagnosed acute lymphoblastic leukemia (ALL). None of the high-risk features predictive of poor treatment outcome in childhood ALL, such as older age, high white blood cell (WBC) count, organomegaly, T-lineage immunophenotype, ability of leukemic cells to cause overt leukemia in severe combined immunodeficient (SCID) mice, presence of MLL-AF4, and BCR-ABL fusion transcripts were associated with high levels of BCL-2 expression. Overall, high BCL-2 levels were not associated with slow early response, failure to achieve complete remission, or poor event-free survival. High BCL-2 levels in primary leukemic cells predicted slow early response only in T-lineage ALL patients, which comprised approximately 15% of the total patient population. Even for this small subset of patients, the level of BCL-2 expression did not have a significant impact on the short-term event-free survival.

Diversity of microfungi in the sandy soil of Ipanema Beach, Rio de Janeiro, Brazil.

The mycobiota of the sandy soil of Ipanema Beach, Rio de Janeiro, Brazil, was investigated in 144 sand samples collected at four different sites along the sea coast, divided into three subsites, for a period of one year. A total of 4285 yeast colonies and of 6956 of colonies filamentous fungi were isolated using conventional media and techniques. Representatives of the filamentous fungi corresponding to a total of 1334 colonies were identified and assigned to 34 genera and 170 species. The genera of highest incidence and their respective numbers of species were as follows: Aspergillus, 30.4%, 32 spp.; Penicillium, 16.2%, 35 spp.; Fusarium, 12,6%, 33 spp.; Trichoderma, 6.4%, 7 spp.; Paecilomyces, 3.7%, 10 spp.; Cladosporium, 3.1%, 8 spp. and Acremonium, 1.0%, 8 spp. Several other genera and species were detected at quite low occurrences. Non-sporulating fungi (18.3%) and Coelomycetes (Sphaeropsidales) (1.9%) were also detected. Most of the genera detected belonged to the Deuteromycotina, with fewer proportions belonging to the Ascomycotina and Zygomycotina. Moniliaceae was represented by the largest number of species and Dematiaceae was represented by the largest number of genera. In terms of seasonal distribution, the largest number of filamentous fungi was isolated during the winter and the smallest number the Summer.