The metabolism of lactulose by intestinal bacteria

About 60 strains of intestinal bacteria were cultured in lactulose-containing media to quantitate both sugar fermentation and non-gaseous end-products. Certain species of Clostridia (especially C. perfringens), lactobacilli and bacteroides utilised the disaccharide extensively, while other organisms were unable to metabolise this substrate, beta-Galactosidase activity did not parallel growth on lactulose in all cases. The major fermentation products were acetic, lactic and butyric acids. Hydrogen and carbon dioxide were the only gases qualitatively detected. Clostridial strains exhibited a butyric type fermentation, and most lactobacilli were homofermentative. Fermentation products were estimated for selected species throughout their growth cycles. The products of lactulose fermentation by mixed bacterial cultures varied with incubation conditions such as pH, but correlated well with those produced by pure cultures. Studies on lactulose transport by C. perfringens indicated 14 methodological limitations in assaying (C) lactose uptake and in the use of NADH-based procedures. o-Nitrophenyl O-D-galactopyranoside uptake by lactulose grown whole cells and an absence of phospho-beta-D-galactosidase suggested an active transport of the disaccharide. The inducible beta-galactosidase was partially purified and characterised fructose 1,6-bisphosphate inhibited enzyme activity by 26%, and lactulose or lactose hydrolysis required K ions. Galactokinase was inducible in galactose, lactulose or lactose grown cells. Fructose 1-phosphate kinase (FIPK) and fructose 6-phosphate kinase were detected in fructose grown cell-free extracts; FIPK was partially purified five-fold by affinity chromatography. The glucose effect was observed in C. perfringens grown on lactulose, and could not be eleviated by external cyclic AMP or dibutyryl cAMP. Assays for the cyclic nucleotide in lactulose grown cells and extracellular fluid were in the negative. This inhibition was not observed during growth on a mixture of lactulose with fructose, and co-utilisation of lactulose with galactose and lactose respectively was apparent.<p

Immune defects in the risk of infection and response to vaccination in monoclonal gammopathy of undetermined significance and multiple myeloma

The plasma cell proliferative disorders monoclonal gammopathy of undetermined significance (MGUS) and malignant multiple myeloma (MM) are characterized by an accumulation of transformed clonal plasma cells in the bone marrow and production of monoclonal immunoglobulin. They typically affect an older population, with median age of diagnosis of approximately 70 years. In both disorders, there is an increased risk of infection due to the immunosuppressive effects of disease and conjointly of therapy in MM, and response to vaccination to counter infection is compromised. The underlying factors in a weakened immune response in MGUS and MM are as yet not fully understood. A confounding factor is the onset of normal aging, which quantitatively and qualitatively hampers humoral immunity to affect response to infection and vaccination. In this review, we examine the status of immune alterations in MGUS and MM and set these against normal aging immune responses. We focus primarily on quantitative and functional aspects of B-cell immunity. Furthermore, we review the current knowledge relating to susceptibility to infectious disease in MGUS and MM, and how efficacy of conventional vaccination is affected by proliferative disease-related and therapy-related factors

Exome sequencing in classic hairy cell leukaemia reveals widespread variation in acquired somatic mutations between individual tumours apart from the signature BRAF V(600)E lesion

In classic Hairy cell leukaemia (HCLc), a single case has thus far been interrogated by whole exome sequencing (WES) in a treatment naive patient, in which BRAF V(600)E was identified as an acquired somatic mutation and confirmed as occurring near-universally in this form of disease by conventional PCR-based cohort screens. It left open however the question whether other genome-wide mutations may also commonly occur at high frequency in presentation HCLc disease. To address this, we have carried out WES of 5 such typical HCLc cases, using highly purified splenic tumour cells paired with autologous T cells for germline. Apart from BRAF V(600)E, no other recurrent somatic mutation was identified in these HCLc exomes, thereby excluding additional acquired mutations as also prevalent at a near-universal frequency in this form of the disease. These data then place mutant BRAF at the centre of the neoplastic drive in HCLc. A comparison of our exome data with emerging genetic findings in HCL indicates that additional somatic mutations may however occur recurrently in smaller subsets of disease. As mutant BRAF alone is insufficient to drive malignant transformation in other histological cancers, it suggests that individual tumours utilise largely differing patterns of genetic somatic mutations to coalesce with BRAF V(600)E to drive pathogenesis of malignant HCLc disease

Tumour infiltrating lymphocytes correlate with improved survival in patients with oesophageal adenocarcinoma

BACKGROUND: Oesophageal adenocarcinoma (OAC) is increasingly common in the west, and survival remains poor at 10-15 % at 5 years. Immune responses are increasingly implicated as a determining factor of tumour progression. The ability of lymphocytes to recognise tumour antigens provides a mechanism for a host immune attack against cancer providing a potential treatment strategy.MATERIALS AND METHODS: Tumour infiltrating lymphocytes (TILs: CD3+, CD4+, CD8+ and FOXp3+) were assessed by immunohistochemistry using tissue microarrays in a contemporary and homogeneous cohort of OAC patients (n = 128) undergoing curative treatment.RESULTS: Multivariate analysis identified three independent prognostic factors for improved cancer-specific survival (CSS): increased CD8+ TILs (p = 0.003), completeness of resection (p &lt; 0.0001) and lower pathological N stage (p &lt; 0.0001). Independent prognostic factors for favourable disease-free survival included surgery-only treatment (p = 0.015), completeness of resection (p = 0.001), increased CD8+ TILs (p &lt; 0.0001) and reduced pathological N stage (p &lt; 0.0001). Higher levels of TILs in the pathological specimen were associated with significant pathological response to neoadjuvant chemotherapy (NAC). On multivariate analysis increased levels of CD4+ (p = 0.017) and CD8+ TILs (p = 0.005) were associated with significant local tumour regression and lymph node downstaging, respectively.DISCUSSION: Our results establish an association of TILs and survival in OAC, as seen in other solid tumours, and identify particular TIL subsets that are present at higher levels in patients who responded to NAC compared to non-responders. These findings highlight potential therapeutic strategies in EAC based on utilising the host immunological response and highlight the immune responses biomarker potential

Targeted somatic mutation of the BCL6 proto-oncogene and its impact on lymphomagenesis

Cloning translocation breakpoints which cluster suspiciously to specific chromosomal loci has proved fruitful, leading to the identification of genes implicated in the onset of hematological malignancy. One of the most notable is BCL6, located on chromosome 3q27. The BCL6 is now known to encode a nuclear transcriptional repressor, with pivotal roles in germinal center (GC) formation and regulation of lymphocyte function, differentiation and survival. Unusually, the BCL6 gene locus is also actively targeted by the somatic mutation (SM) mechanism, at a rate indicative of specific, regulated events in both normal and malignant B-cells. These mutations occur in ~30% of normal centrocytes and centroblasts, but not in naive or pre-GC B-cells. They are also observed in ~70% of diffuse large B-cells lymphomas, ~30% of follicular lymphomas (FL) and at various frequencies in many lymphoma subtypes. Mutations are generated in the 5' proximity of the BCL6 promoter, including the first intron and are mainly single nucleotide substitutions, but with insertions and deletions also observed. Mutations in BCL6 occur independently of translocations, although mutational levels can be dramatically influenced by aberrantly translocated chromosomal elements, which map in the vicinity of the gene. Indeed, SMs are directly implicated in the generation of chromosomal translocations, as suggested by the overlap of the breakpoint cluster region and the mutational cluster domain. The prognostic value of the overall level of BCL6 mutations in specific lymphoma populations is, in the main, not as yet fully resolved. The accumulation of mutations in BCL6 during high grade transformation of FL, a mutational clustering and specific recurrent mutations suggest that some mutations may be selected for by their effect on the survival of the tumoral clone. In fact, it is now clear that SM can target and disrupt regulatory motifs in BCL6 to result in upregulated gene expression. Exogenous factors can also perturbate SM in BCL6. Viral infection elevates BCL6 mutational activity, suggesting a potential link with onset of virus-associated lymphoma. These findings to date reveal several mechanisms which can influence specific mutations targeting BCL6, and which may contribute to lymphomagenesis by dysregulating control of BCL6 expression

Hairy cell leukemia cell lines lack signature BRAF mutations of typical disease and MAP2K1 mutations of atypical disease

Tumor-derived cell lines provide important models of disease but require validation with regard to parental disease. Hairy cell leukemia (HCL) is a rare B cell leukemia with characteristic hair-like cytoplasmic projections on tumor cells and they uniquely express multiple surface immunoglobulin (sIg) isotypes. A mutation [V(600)E] in exon 15 of BRAF, a signal transduction protein kinase, has recently been identified as near universal in typical or classic HCL (HCLc), but absent in atypical variant HCL (HCLv) and HCLc expressing the rearranged IGHV4-34 gene. In a few HCLc cases lacking the [V(600)E] mutation, alternative mutations in [D(449)E] and [F(468)C] have been reported in BRAF. We previously examined 5 HCL cell lines used as relevant disease models, Hair-M, HCLL-7876, EH, Eskol, HC-1, and HCLv-07 (the latter of known HCLv origin) and found that while they recapitulated phenotypic and functional characteristics of HCLc, they lacked the definitive BRAF [V(600)E] mutation, as did HCLv-07. More recently, whole exome next-generation sequencing studies in HCLv and HCLc/IGHV4-34 disease subsets have identified recurrent mutations in the MAP2K1 gene in ∼50% of atypical cases, all lacking the BRAF [V(600)E] mutation. MAP2K1 encodes MEK1, a dual-specificity kinase that is a direct effector of BRAF. A mutant mitogen-activated protein kinase (MAPK) pathway emerges as an important driver of neoplastic transformation in HCLc and in HCLv and HCLc/IGHV4-34 disease. We examined the reported MAP2K1 mutations and alternative BRAF mutations in each of the HCL cells lines. In 5/5 lines, no MAP2K1 or alternative BRAF mutations were found, including in the HCLv cell line. These data indicate derivation of known HCL cell lines from atypical HCL disease that lack MAP2K1 mutations, and suggest caution in their experimental use to represent specific HCL subsets. They further substantiate the importance of whole genome sequencing in providing robust genetic markers to validate disease models, in addition to identifying driver mutations relevant to understanding disease origins

Variant B cell receptor isotype functions differ in hairy cell leukemia with mutated BRAF and IGHV genes

A functional B-cell receptor (BCR) is critical for survival of normal B-cells, but whether it plays a comparable role in B-cell malignancy is as yet not fully delineated. Typical Hairy Cell Leukemia (HCL) is a rare B-cell tumor, and unique in expressing multiple surface immunoglobulin (sIg) isotypes on individual tumor cells (mult-HCL), to raise questions as to their functional relevance. Typical mult-HCL also displays a mutated BRAF V(600)E lesion. Since wild type BRAF is a primary conduit for transducing normal BCR signals, as revealed by deletion modelling studies, it is as yet not apparent if mutated BRAF alters BCR signal transduction in mult-HCL. To address these questions, we examined BCR signalling in mult-HCL cases uniformly displaying mutated BRAF and IGHV genes. Two apparent functional sets were delineated by IgD co-expression. In sIgD(+ve) mult-HCL, IgD mediated persistent Ca(2+) flux, also evident via &gt;1 sIgH isotype, linked to increased ERK activation and BCR endocytosis. In sIgD(-ve) mult-HCL however, BCR-mediated signals and downstream effects were restricted to a single sIgH isotype, with sIgM notably dysfunctional and remaining immobilised on the cell surface. These observations reveal discordance between expression and function of individual isotypes in mult-HCL. In dual sIgL expressing cases, only a single sIgL was fully functional. We examined effects of anti-BCR stimuli on mult-HCL survival ex-vivo. Significantly, all functional non-IgD isotypes increased ERK1/2 phosphorylation but triggered apoptosis of tumor cells, in both subsets. IgD stimuli, in marked contrast retained tumor viability. Despite mutant BRAF, BCR signals augment ERK1/2 phosphorylation, but isotype dictates functional downstream outcomes. In mult-HCL, sIgD retains a potential to transduce BCR signals for tumor survival in-vivo. The BCR in mult-HCL emerges as subject to complex regulation, with apparent conflicting signalling by individual isotypes when co-expressed with sIgD. This suggests the possibility that mutant BRAF by-passes BCR constraints in mult-HCL