ATP-Bioluminescence as a method to evaluated microbiological quality of UHT milk / ATP-Bioluminescência como método para avaliação da qualidade microbiológica de leite UAT

New approaches are needed to quickly indicate possible contamination of UHT milk, among them the technique of ATP-Bioluminescence. Therefore, the aim of this study was to compare the results of culture methods with the results of ATP-Bioluminescence technique of 102 UHT whole milk samples incubated at 48, 72, and 168 hours. UHT milk samples were analyzed for the presence of mesophilic and psychrotrophic aerobic microorganisms using Plate Count Agar (PCA), Brain-Heart Infusion (BHI) media and PetrifilmTM Aerobic Count (AC) plates. The ATP-Bioluminescence technique was applied through the Microbial Luminescent Screening (MLS) system. Significant correlations were found between counts of aerobic mesophilic microorganisms on PCA, PetrifilmTM AC, BHI and results of ATP bioluminescence technique (P≤0.05). The ATP-Bioluminescence technique had higher correlation with counting method in PCA than BHI media. At lower pass/fail limits of Relative Light Units (60, 50, 45 and 40 RLU), the number of samples identified as positive increased and statistically agreed with aerobic mesophilic microorganism counts (P>0.05). For the dairy industry, the ATP-Bioluminescence technique may become an important tool that assists the official methods to quickly monitor the microbiological quality of UHT milk though this will likely require a threshold below 150 RLU...

Novos métodos são necessários para detectar de forma rápida a contaminação do leite UAT, entre eles, a técnica de ATP-Bioluminescência. Portanto, objetivou-se comparar os resultados de métodos de cultura tradicionais com os resultados de ATP-Bioluminescência de 102 amostras de leite UAT integral incubadas por 48, 72 e 168 horas. Os leites UAT foram analisados quanto à presença de micro-organismos aeróbicos mesófilos e psicrotróficos usando os ágares PCA, BHI e placas PetrifilmTM AC. A técnica de ATP-Bioluminescência foi aplicada por meio do sistema Microbial Luminescent Screening (MLS). Significantes correlações foram obtidas entre as contagens de micro-organismos mesófilos aeróbios em PCA, PetrifilmTM AC, BHI e os resultados da técnica de ATP-Bioluminescência (p<0,05). A técnica de ATP-Bioluminescência tem maior correlação com o método de contagem em meio PCA que BHI. Quando valores limites de Unidades Relativas de Luz (60, 50, 45 e 40 RLU) foram menores, o número de amostras identificadas como positivas aumentou e concordou estatisticamente com a contagem de micro-organismos mesófilos aeróbios (P>0,05). Para as indústrias de laticínios, a técnica de ATP-Bioluminescência pode se tornar uma ferramenta auxiliar aos métodos oficiais para o monitoramento rápido da qualidade microbiológica do leite UAT, desde que sejam utilizados limites abaixo de 150 RLU...

Bone marrow lesions are associated with altered trabecular morphometry.

OBJECTIVE: Bone marrow lesions (BMLs) are a common magnetic resonance (MR) feature in patients with osteoarthritis, however their pathological basis remains poorly understood and has not been evaluated in vivo. Our aim was to evaluate the trabecular structure associated with the presence and size of BMLs present in the same regions of interest (ROI) using quantitative MR-based trabecular morphometry. DESIGN: 158 participants in the Osteoarthritis Initiative (OAI) were imaged with a coronal 3D fast imaging with steady state precession (FISP) sequence for trabecular morphometry in the same session as the OAI 3 T MR knee evaluation. The proximal medial tibial subchondral bone in the central weight-bearing ROI on these knee 3D FISP images were quantitatively evaluated for apparent bone volume fraction, trabecular number, spacing, and thickness. BMLs were also evaluated in the subchondral bone immediately adjacent to the articular cartilage. BML volume was also evaluated within the same trabecular morphometry ROI and semi-quantitatively classified as none, small, or large. Kruskal-Wallis test was used to determine if mean apparent bone volume fraction, trabecular number, spacing, or thickness differed by BML score. RESULTS: Compared to knees with ROIs containing no BMLs, knees with small or large BMLs had statistically higher apparent bone volume fraction (P < 0.01), trabecular number (P < 0.01), and thickness (P = 0.02), and lower trabecular spacing (P < 0.01). CONCLUSIONS: Compared to knees with ROIs containing no BMLs, knees with ROIs containing small or large BMLs had higher apparent bone volume fraction, trabecular number and thickness, but lower trabecular spacing. These findings may represent areas of locally increased bone remodeling or compression.

Cross-sectional DXA and MR measures of tibial periarticular bone associate with radiographic knee osteoarthritis severity.

OBJECTIVE: We evaluated the relationship of medial proximal tibial periarticular areal bone mineral density (paBMD) and trabecular morphometry and determined whether these bone measures differed across radiographic medial joint space narrowing (JSN) scores. METHODS: 482 participants of the Osteoarthritis Initiative (OAI) Bone Ancillary Study had knee dual X-ray absorptiometry (DXA) and trabecular bone 3T magnetic resonance imaging (MRI) exams assessed at the same visit. Medial proximal tibial paBMD was measured on DXA and apparent trabecular bone volume fraction (aBV/TV), thickness (aTb.Th), number (aTb.N), and spacing (aTb.Sp) were determined from MR images. Radiographs were assessed for medial JSN scores (0-3). We evaluated associations between medial paBMD and trabecular morphometry. Whisker plots with notches of these measures versus medial JSN scores were generated and presented. RESULTS: Mean age was 63.9 (9.2) years, BMI 29.6 (4.8) kg/m(2), and 53% were male. The Spearman correlation coefficients between DXA-measured medial paBMD and aBV/TV was 0.61 [95% confidence interval (CI) 0.55-0.66]; between paBMD and aTb.Th was 0.38 (95%CI 0.30-0.46); paBMD and aTb.N was 0.65 (95%CI 0.60-0.70); paBMD and aTb.Sp was -0.65 (95%CI -0.70 to -0.59). paBMD and the trabecular metrics were associated with medial JSN scores. CONCLUSION: The moderate associations between periarticular trabecular bone density and morphometry and their relationship with greater severity of knee OA support hypotheses of remodeling and/or microscopic compression fractures in the natural history of OA. Longitudinal studies are needed to assess whether knee DXA will be a predictor of OA progression. Further characterization of the periarticular bone in OA utilizing complementary imaging modalities will help clarify OA pathophysiology.

Incidence of Yersinia spp. in food in Sao Paulo, Brazil.

This study examined the occurrence of Y. enterocolitica and other Yersinia species in Brazilian food products. Samples included raw vegetables (lettuce, spinach, watercress and chicory), raw and pasteurized milk as well as meat and meat products. Raw milk samples were obtained in a dairy plant and the other food samples were purchased at the retail level in Sao Paulo city. Yersinia spp. was isolated from raw milk (45.2%), pasteurized milk (14.3%), raw vegetables samples (13.3%) and meat and meat products (40.0%). Most of the strains isolated were Y. enterocolitica followed by Y. intermedia. This high incidence in raw milk and meat and meat products implies that these products are a likely sources of contamination with Yersinia spp.

Molecular diagnosis of Philadelphia chromosome-positive chronic myeloid leukemia.

BACKGROUND: In virtually all Ph1 chromosome-positive CML patients, the breakpoint on chromosome 22 maps in a very restricted area of 5.8 Kb, which has been named "breakpoint cluster region" or "bcr". Several molecular probes of this region are presently available, and this makes the molecular diagnosis of CML a useful approach which can be particularly important in those cases in which cytogenetic analysis does not reveal the presence of a Ph1 chromosome. Here we report the problems and our experience during the molecular analysis of the 478 patients examined so far. METHODS: Molecular analyses were performed after digestion of the DNA with 2 to 4 restriction enzymes and hybridization with different probes. Individual samples were subjected to PCR since no rearrangements had been obtained with Southern blotting. RESULTS: Rearrangement bands were detected in all the samples examined. In 473 cases the breakpoint was located within the bcr. In one of these cases, it was detected only after PCR analysis, and in two cases only after the use of the PHL/BCR probe. In 5 cases the breakpoint was localized either 5' or 3' with respect to the bcr. CONCLUSIONS: In this paper, the criteria for a correct molecular diagnosis of CML are presented. The "PHL/BCR" probe appeared to be very specific and time-saving, since it required only one digestion to evidence the rearrangement. Our results confirm the high specificity of the breakpoint on chromosome 22 in CML and the relatively rare incidence of molecular variants.

Cytogenetic events after bone marrow transplantation for Philadelphia chromosome positive chronic myeloid leukemia.

Allogeneic bone marrow transplantation is the only way to cure patients with Ph1+ chronic myeloid leukemia. It is commonly assumed that, in order to obtain a cure for the patients, the leukemic clone must be completely destroyed by the conditioning treatment and the donor's bone marrow must repopulate the hemopoietic niches leading to a "complete chimera". However, cytogenetic analyses, supported by molecular ones, indicate that Ph1+ cells, far from being completely destroyed by chemo-radiotherapy may persist for a long time, probably in the majority of the patients. As demonstrated by the outcome of patients receiving T-cell depleted marrow, immune mechanisms must be involved in controlling and progressively reducing the size of the residual leukemic clone. Furthermore, immunodulating therapeutic strategies, represented by cyclosporin-A discontinuation or alpha interferon treatment, may successfully reduce the Ph1+ cell population even after a full relapse.

New type of Bcr/Abl junction in Philadelphia chromosome-positive chronic myelogenous leukemia.

A new and rare type of Bcr/Abl junction between exon C3 of the 3' portion of the Bcr gene and Abl exon 2 has been identified in the leukemic cells of two Ph1-positive chronic myelogenous leukemia patients in chronic phase. This is the fourth type of Bcr/Abl junction so far identified in Ph1-positive hematologic malignancies and is a consequence of an unusual breakpoint position on chromosome 22 that falls approximately 20 kb downstream of the major breakpoint cluster region (bcr) of the Bcr gene. The new hybrid mRNA is 540 base pairs (bp) longer than that expressed by the K562 cell line and could codify for a Bcr/Abl protein carrying 180 additional aminoacids with respect to the larger P210 protein so far identified. The hematologic phenotype expressed by the two patients carrying this unusual type of Bcr/Abl rearrangement does not significantly differ from that commonly seen in chronic myelogenous leukemia.

ABL proteins in Philadelphia-positive acute leukaemias and chronic myelogenous leukaemia blast crises.

The Philadelphia chromosome (Ph1) is present in 95% of chronic myelogenous leukaemias (CML) and 15% of acute lymphoblastic leukaemias (ALL). This cytogenetic marker is due to a t(9;22) translocation, which causes a rearrangement of the ABL oncogene. In order to better define the relationship between type of genomic rearrangement, variant ABL protein expressed and haematological phenotype, a series of Ph1-positive acute leukaemias, both myeloblastic (AML) and lymphoblastic, and several CML lymphoid blast crises have been analysed at the DNA and protein level. The results confirm the presence of the ABL protein P210 in all cases of CML, ALL and AML positive for rearrangement in the bcr region of chromosome 22, and, surprisingly, in one AML case apparently negative for bcr rearrangement. The ABL protein P190 was found to be present only in cases of ALL negative for bcr rearrangement. Polymerase chain reaction (PCR) analysis of the types of 9/22 junctions present in the mRNA of CML lymphoid blast crises showed no evidence of 'ALL-type' transcripts.

HTLV-I positive adult T-cell leukaemia-lymphoma: report of a typical case from Italy.

A case of acute adult T-cell leukemia-lymphoma (ATLL) was observed in northeast Italy, presenting with fever, lymphadenomegaly, splenomegaly, hypercalcemia and renal failure. Leukaemic cells were morphologically typical, expressed a T-cell CD4+ phenotype, did not display any helper functions, and grew in vitro under supply of exogenous interleukin-2. Antibodies to human T-cell lymphotropic virus (HTLV-I) were found in the serum, and the virus was isolated from leukaemic cells. The family members who could be tested were seronegative. The patient had never travelled outside Italy, had never received blood transfusions and did not belong to any known categories at risk of viral disease transmission. Present knowledge of the epidemiology of HTLV-I infection warns that other cases of HTLV-I induced disease are expected to occur outside already recognised endemic areas. This case suggests that untraceable, presumably short-term exposures can also account for HTLV-I transmission.

Molecular and cytogenetic studies of a patient with Philadelphia-negative, BCR-positive chronic myeloid leukemia and t(12;12)(q13;p12).

A patient with Philadelphia (Ph1)-negative, breakpoint cluster region (bcr)-positive chronic myeloid leukemia (CML) is reported. Pulsed-field gel electrophoretic analysis demonstrated the comigration of both ABL and BCR sequences on the same BssHII and SacII fragment. Moreover, in situ hybridization studies demonstrated that ABL sequences had been moved from band 9q34 to 22q11 and that the additional t(12;12)(q13;p12) was not involved in the ABUBCR related translocation. Nevertheless, a possible role of oncogenes or regulatory sequences activated or inhibited by the additional translocation cannot be excluded.

Immunoglobulin DNA Analysis as a Marker of Clonality in the Follow-up of Patients with Hairy Cell Leukemia Treated with Alpha-Interferon.

In fourteen patients with hairy cell leukemia (HCL) the configuration of the immunoglobulin (Ig) heavy chain genes was used as a marker of clonality, to monitor the response of the neoplastic population to treatment with alpha-interferon (a-IFN). In agreement with the morphological, hematological and immunological data, twelve of them showed, after a variable length of therapy, a complete disappearance of rearranged bands in peripheral blood cells. In one patient, who was treated less intensively, the molecularly-defined neoplastic population was still present on two consecutive determinations, whilst in the last patient persistence of disease was repeatedly documented despite prolonged A-IFN treatment. Three further cases were analyzed sequentially: in two, no rearranged bands could be found at repeated determinations; the third, who was in complete remission whilst on 3 × 10(6) U of α-IFN every other day, showed recurrence of disease nine months later when on a maintenance protocol with 3 × 10(6) U/weekly. Nine bone marrow specimens were also analyzed following treatment with α-IFN. In four a monoclonally rearranged band could still be detected, while in another four, reversal of fibrosis and hemopoietic recovery wits coupled with the absence of a molecularly recognizable neoplastic clone. In the last (case, persistence of disease paralleled the findings in the peripheral blood cells. These data indicate that α-IFPJ is capable of producing a specific cytolytic effect on the leukemic population in HCL, which in some cases may lead to complete clonal remissions. Analysis at the DNA level may represent a valuable tool towards monitoring the clinical course of HCL patients and for optimal individual therapeutic scheduling.

5q- and t(2;11) in a patient with M2 acute non-lymphocytic leukemia. Case report.

A patient with M2-ANLL and a 46,XX,del(5)(q22q33), t(2;11)(p21;q24) karyotype is described. The diagnosis was made after a short period of myelodysplastic syndrome. After chemotherapy consisting of Daunorubicin and Arabinosylcytosine in continuous infusion, the patient reached a complete remission. The chromosome pattern described here has been observed in two other patients with refractory anemia and refractory anemia with excess of blasts, respectively. The breakpoints on the chromosomes 2, 5 and 11 allow us to hypothesize the involvement of N-myc, c-fms, GM-CSF and IL-3 genes.

Cytogenetic and molecular studies in patients with chronic myeloid leukemia and variant Philadelphia translocations.

Out of 105 Philadelphia (Ph) positive chronic myeloid leukemia patients analyzed, six (5.7%) carried a variant Ph translocation, namely t(6;9;9;10;22)(q24;p13;q34;p15;q11); t(9;13;22)(q34;q21;q11);der(2)(2pter----2q31::9q21---- 9q34::22q11----22qter) and der(9)t(2;9) (9pter----9q21::2q31----2qter);t(7;9;22)(q11;q34 ;q11), 14q + ;t(7;9;22)(q35;q34;q11), and t(9;11;22) (q34;q13;q11), respectively. Five of these patients were analyzed with Southern blotting. Three of them showed an atypical molecular pattern; namely, the patient with t(9;13;22) showed no rearrangement in the breakpoint cluster region (bcr), the patient with t(7;9;22)(q35;q34;q11) showed a 3' deletion, and the patient with t(7;9;22), 14q + showed a bcr rearrangement 3' to the exon 4 of the M-BCR. Chromosome in situ hybridization studies demonstrated that in patient one, a two-step translocation occurred: the first step moved the 3' bcr from chromosome 22 to chromosome 9, and the second moved the terminal part of 22q, carrying the c-sis protooncogene, to 10p. Variant Ph translocations appear to be associated with atypical molecular breakpoints.

Evaluation of complete remission in hairy cell leukemia patients treated with alpha-IFN.

In 10 HCL patients, who following treatment for more than 1 year with alpha-IFN, had achieved a normalization of the splenomegaly and a disappearance of circulating hairy cells together with a complete restoration of all peripheral hematological values, a multiple assessment of the apparent complete remission (CR) was carried out. The presence of residual disease was investigated by bone marrow histology, bone marrow immunohistochemistry, immunoglobulin (Ig) gene analysis and on the basis of the serum levels of the soluble form of the Interleukin-2 receptor (sIL-2R). Examination of bone marrow biopsies showed a pattern of CR in 5 cases; 4 of them revealed no evidence of Ig heavy chain gene rearrangement, as well as a near normalization of the serum levels of sIL-2R. Immunohistochemical studies were carried out on embedded paraffin sections with the monoclonal antibody 4KB5 (CD45R) and were assessable in 4 of the 5 patients considered in CR and in 3 of the 4 cases with no Ig gene rearrangement. In 2 the pattern of CR was confirmed, while in the 3rd a minimal but persistent disease (5%) was suspected.