Myeloproliferative and lymphoproliferative malignancies occurring in the same patient: a nationwide discovery cohort.

Myeloid and lymphoid malignancies are postulated to have distinct pathogenetic mechanisms. The recent observation that patients with a myeloproliferative neoplasm have an increased risk of developing lymphoproliferative malignancy has challenged this assumption. We collected a nationwide cohort of patients with both malignancies. Patients diagnosed in 1990-2015 were identified through the national Danish Pathology Registry. We identified 599 patients with myeloproliferative neoplasm and a concomitant or subsequent diagnosis of lymphoma. Histopathological review of the diagnostic samples from each patient led to a final cohort of 97 individuals with confirmed dual diagnoses of myeloproliferative neoplasm and lymphoma. The age range at diagnosis was 19-94 years (median: 71 years). To avoid the inclusion of cases of therapy-induced myeloproliferative neoplasm occurring in patients previously treated for lymphoma, only patients with myeloproliferative neoplasm diagnosed unequivocally before the development of lymphoma were included. The average time interval between the diagnoses of the two malignancies was 1.5 years. In the majority of patients (90%) both diagnoses were established within 5 years from each other. Among the lymphoma entities, the frequency of peripheral T-cell lymphomas was markedly increased. Interestingly, all but one of the T-cell lymphomas were of angioimmunoblastic type. These findings suggest that myeloproliferative neoplasm and lymphoproliferative malignancy developing in the same patient may have common pathogenetic events, possibly already at progenitor level. We believe that the molecular characterization of the newly developed biorepository will help to highlight the mechanisms driving the genesis and clonal evolution of these hematopoietic malignancies.

Development of a milk-based medium for the selection of urease-defective mutants of Streptococcus thermophilus.

Streptococcus thermophilus strains are used in fermented dairy products for their capacity to metabolize lactose into lactic acid. The rate of lactic acid production in milk is of major economic importance, as rapid acidification prevents growth of undesirable microorganisms. It is also of paramount significance for aroma, texture and flavor of the end product. Besides achieving customer satisfaction, improvement of production rate and operational costs incite industrials into selecting fast acidifying strains. Another important trait of S. thermophilus influencing acidification is the urease, which catabolizes urea into ammonia and has a detrimental effect on acidification. Unfortunately, most of the S. thermophilus strains possess the urease, and the urease-negative ones are necessary for industrial applications. Urease activity is a widely distributed activity in S. thermophilus species, and urease-negative strains are rare. The later are however interesting from an industrial point of view, as they may give faster acidification in dairy applications, because lactic acid is not buffered by urea-derived ammonia. Nowadays, the efforts to improve the characteristics of strains for industrial applications are based on natural strategies such as random mutagenesis. This implies the need of a screening method that is efficient in terms of time and success. In this context, the aim of this study was the development of a new medium that allows selection of urease-defective mutants based on S. thermophilus colony morphology. Discrimination capacity of the new medium was verified using previously characterized urease-negative recombinant strains. The new milk-based medium, applied to industrial S. thermophilus strains subjected to UV mutagenesis, allowed the selection of 3 mutants, partially or completely defective in urease activity. Genetic characterization of urease-defective mutants highlighted the presence of nonsense or missense mutations in the ureA, ureC and ureG genes, thus supporting their phenotype. Evaluation of milk acidification revealed increased performance for one out of three urease-defective mutants compared to wild-type strains.

Task Distribution between Acetate and Acetoin Pathways To Prolong Growth in Lactococcus lactis under Respiration Conditions.

Lactococcus lactis is the main bacterium used for food fermentation and is a candidate for probiotic development. In addition to fermentation growth, supplementation with heme under aerobic conditions activates a cytochrome oxidase, which promotes respiration metabolism. In contrast to fermentation, in which cells consume energy to produce mainly lactic acid, respiration metabolism dramatically changes energy metabolism, such that massive amounts of acetic acid and acetoin are produced at the expense of lactic acid. Our goal was to investigate the metabolic changes that correlate with significantly improved growth and survival during respiration growth. Using transcriptional time course analyses, mutational analyses, and promoter-reporter fusions, we uncover two main pathways that can explain the robust growth and stability of respiration cultures. First, the acetate pathway contributes to biomass yield in respiration without affecting medium pH. Second, the acetoin pathway allows cells to cope with internal acidification, which directly affects cell density and survival in stationary phase. Our results suggest that manipulation of these pathways will lead to fine-tuning respiration growth, with improved yield and stability.IMPORTANCELactococcus lactis is used in food and biotechnology industries for its capacity to produce lactic acid, aroma, and proteins. This species grows by fermentation or by an aerobic respiration metabolism when heme is added. Whereas fermentation leads mostly to lactic acid production, respiration produces acetate and acetoin. Respiration growth leads to greatly improved bacterial growth and survival. Our study aims at deciphering mechanisms of respiration metabolism that have a major impact on bacterial physiology. Our results showed that two metabolic pathways (acetate and acetoin) are key elements of respiration. The acetate pathway contributes to biomass yield. The acetoin pathway is needed for pH homeostasis, which affects metabolic activities and bacterial viability in stationary phase. This study clarifies key metabolic elements that are required to maintain the growth advantage conferred by respiration metabolism and has potential uses in strain optimization for industrial and biomedical applications.

Digital pathology for the validation of tissue microarrays in peripheral T-cell lymphomas.

Systematic validation of construction and analysis parameters when using tissue microarray (TMA) in rare, morphologically heterogenous entities such as peripheral T-cell lymphoma (PTCL) is not reported. We describe a tissue-saving virtual TMA to predetermine the number of cores needed to represent whole tissue sections (WTS) from the same biopsies, using automated and traditional manual methods for the quantification of immunohistochemical stains. Whole paraffin hematoxylin and eosin- and immunohistochemical (CD2, CD30, and Ki-67)-stained sections from 30 PTCLs were digitalized. A virtual TMA with six 1-mm cores per slide was designed to compare agreements in the immunohistochemical scoring. Using digital image analysis and manual stereological counting, immunohistochemical positivity was quantified. Associations were analyzed using the Bland-Altman and correlation plots. In PTCL, we report that 4 cores are required to represent WTS results (ie, agreement within ±10%). High concordance was demonstrated between digital results obtained with WTS compared with 4-core virtual TMA (correlation coefficients: 0.89-0.98), and in the comparative evaluation of 4-core virtual TMA by digital image analysis versus manual stereology (correlation coefficients: 0.91 to 0.99). Virtual TMAs provide an efficient tool for optimizing and validating TMA construction parameters when planning a study. The method can be applied to the same tissues used in a subsequent formal study, without wasting scarce tissue resources. In PTCL, TMAs constructed with four 1-mm cores are representative of WTS. In parallel tests using TMAs and WTS from PTCLs, there is a high level of agreement comparing automated digital with manual stereological methods for the quantification of immunohistochemical biomarker staining.

High intratumoral macrophage content is an adverse prognostic feature in anaplastic large cell lymphoma.

AIMS: Macrophage infiltration has been associated with prognosis in several cancers, including lymphoma, but has not been assessed systematically in anaplastic large cell lymphoma (ALCL). The aim of the study was to correlate expression of the macrophage-associated antigens CD68 and CD163 with pre-therapeutic parameters and outcome in a cohort of treatment-naive ALCL patients. METHODS AND RESULTS: Pre-therapeutic tumour specimens from 52 patients with ALCL were included in a tissue microarray. The intratumoral macrophage content was assessed by immunohistochemical staining for CD68 and CD163, and quantified using digital image analysis. Anaplastic lymphoma kinase (ALK)-positive patients were significantly younger and had a favourable outcome compared with ALK-negative ALCL patients (median age: 42 versus 59 years; P = 0.008). However, ALK expression was not a significant predictor when adjusting for age. Although classical risk factors were distributed evenly between the compared groups, high intratumoral content of CD68 and/or CD163 correlated with poor outcome, in both univariate and multivariate analyses. High intratumoral CD163 content showed the strongest adverse association with both overall and progression-free survival in ALK-negative patients (P < 0.001). CONCLUSIONS: A high content of intratumoral CD68- and/or CD163-positive macrophages correlates with an adverse outcome in ALK-negative ALCL.

Increasing the heme-dependent respiratory efficiency of Lactococcus lactis by inhibition of lactate dehydrogenase.

The discovery of heme-induced respiration in Lactococcus lactis has radically improved the industrial processes used for the biomass production of this species. Here, we show that inhibition of the lactate dehydrogenase activity of L. lactis during growth under respiration-permissive conditions can stimulate aerobic respiration, thereby increasing not only growth efficiency but also the robustness of this organism.

Aerobic respiration metabolism in lactic acid bacteria and uses in biotechnology.

The lactic acid bacteria (LAB) are essential for food fermentations and their impact on gut physiology and health is under active exploration. In addition to their well-studied fermentation metabolism, many species belonging to this heterogeneous group are genetically equipped for respiration metabolism. In LAB, respiration is activated by exogenous heme, and for some species, heme and menaquinone. Respiration metabolism increases growth yield and improves fitness. In this review, we aim to present the basics of respiration metabolism in LAB, its genetic requirements, and the dramatic physiological changes it engenders. We address the question of how LAB acquired the genetic equipment for respiration. We present at length how respiration can be used advantageously in an industrial setting, both in the context of food-related technologies and in novel potential applications.

Complete genome sequence of Bifidobacterium animalis subsp. lactis BB-12, a widely consumed probiotic strain.

Bifidobacterium animalis subsp. lactis BB-12 is a commercially available probiotic strain used throughout the world in a variety of functional foods and dietary supplements. The benefits of BB-12 have been documented in a number of independent clinical trials. Determination of the complete genome sequence reveals a single circular chromosome of 1,942,198 bp with 1,642 predicted protein-encoding genes, 4 rRNA operons, and 52 tRNA genes. Knowledge of this sequence will lead to insight into the specific features which give this strain its probiotic properties.

Increasing acidification of nonreplicating Lactococcus lactis deltathyA mutants by incorporating ATPase activity.

Lactococcus lactis MBP71 deltathyA (thymidylate synthase) cannot synthesize dTTP de novo, and DNA replication is dependent on thymidine in the growth medium. In the nonreplicating state acidification by MBP71 was completely insensitive to bacteriophages (M. B. Pedersen, P. R. Jensen, T. Janzen, and D. Nilsson, Appl. Environ. Microbiol. 68:3010-3023, 2002). For nonreplicating MBP71 the biomass increased 3.3-fold over the first 3.5 h, and then the increase stopped. The rate of acidification increased 2.3-fold and then started to decrease. Shortly after inoculation the lactic acid flux was 60% of that of exponentially growing MBP71. However, when nonspecific ATPase activity was incorporated into MBP71, the lactic acid flux was restored to 100% but not above that point, indicating that control over the flux switched from ATP demand to ATP supply (i.e., to sugar transport and glycolysis). As determined by growing nonreplicating cells with high ATPase activity on various sugar sources, it appeared that glycolysis exerted the majority of the control. ATPase activity also stimulated the rate of acidification by nonreplicating MBP71 growing in milk, and pH 5.2 was reached 40% faster than it was without ATPase activity. We concluded that ATPase activity is a functional means of increasing acidification by nonreplicating L. lactis.

The extent to which ATP demand controls the glycolytic flux depends strongly on the organism and conditions for growth.

Using molecular genetics we have introduced uncoupled ATPase activity in two different bacterial species, Escherichia coli and Lactococcus lactis, and determined the elasticities of the growth rate and glycolytic flux towards the intracellular [ATP]/[ADP] ratio. During balanced growth in batch cultures of E. coli the ATP demand was found to have almost full control on the glycolytic flux (FCC=0.96) and the flux could be stimulated by 70%. In contrast to this, in L. lactis the control by ATP demand on the glycolytic flux was close to zero. However, when we used non-growing cells of L. lactis (which have a low glycolytic flux) the ATP demand had a high flux control and the flux could be stimulated more than two fold. We suggest that the extent to which ATP demand controls the glycolytic flux depends on how much excess capacity of glycolysis is present in the cells.

Expression of genes encoding F(1)-ATPase results in uncoupling of glycolysis from biomass production in Lactococcus lactis.

We studied how the introduction of an additional ATP-consuming reaction affects the metabolic fluxes in Lactococcus lactis. Genes encoding the hydrolytic part of the F(1) domain of the membrane-bound (F(1)F(0)) H(+)-ATPase were expressed from a range of synthetic constitutive promoters. Expression of the genes encoding F(1)-ATPase was found to decrease the intracellular energy level and resulted in a decrease in the growth rate. The yield of biomass also decreased, which showed that the incorporated F(1)-ATPase activity caused glycolysis to be uncoupled from biomass production. The increase in ATPase activity did not shift metabolism from homolactic to mixed-acid fermentation, which indicated that a low energy state is not the signal for such a change. The effect of uncoupled ATPase activity on the glycolytic flux depended on the growth conditions. The uncoupling stimulated the glycolytic flux threefold in nongrowing cells resuspended in buffer, but in steadily growing cells no increase in flux was observed. The latter result shows that glycolysis occurs close to its maximal capacity and indicates that control of the glycolytic flux under these conditions resides in the glycolytic reactions or in sugar transport.

Bacteriophage resistance of a deltathyA mutant of Lactococcus lactis blocked in DNA replication.

The thyA gene, which encodes thymidylate synthase (TS), of Lactococcus lactis CHCC373 was sequenced, including the upstream and downstream regions. We then deleted part of thyA by gene replacement. The resulting strain, MBP71 deltathyA, was devoid of TS activity, and in media without thymidine, such as milk, there was no detectable dTTP pool in the cells. Hence, DNA replication was abolished, and acidification by MBP71 was completely unaffected by the presence of nine different phages tested at a multiplicity of infection (MOI) of 0.1. Nonreplicating MBP71 must be inoculated at a higher level than CHCC373 to achieve a certain pH within a specified time. For a pH of 5.2 to be reached in 6 h, the inoculation level of MBP71 must be 17-fold higher than for CHCC373. However, by adding a limiting amount of thymidine this could be lowered to just 5-fold the normal amount, while acidification was unaffected with MBP71 up to an MOI of 0.01. It was found that nonreplicating MBP71 produced largely the same products as CHCC373, though the acetaldehyde production of the former was higher.