Water uptake in barley grain: Physiology; genetics and industrial applications.

Water uptake by mature barley grains initiates germination and is the first stage in the malting process. Here we have investigated the effects of starchy endosperm cell wall thickness on water uptake, together with the effects of varying amounts of the wall polysaccharide, (1,3;1,4)-ß-glucan. In the latter case, we examined mutant barley lines from a mutant library and transgenic barley lines in which the (1,3;1,4)-ß-glucan synthase gene, HvCslF6, was down-regulated by RNA interference. Neither cell wall thickness nor the levels of grain (1,3;1,4)-ß-glucan were significantly correlated with water uptake but are likely to influence modification during malting. However, when a barley mapping population was phenotyped for rate of water uptake into grain, quantitative trait locus (QTL) analysis identified specific regions of chromosomes 4H, 5H and 7H that accounted for approximately 17%, 18% and 11%, respectively, of the phenotypic variation. These data indicate that variation in water uptake rates by elite malting cultivars of barley is genetically controlled and a number of candidate genes that might control the trait were identified under the QTL. The genomics data raise the possibility that the genetic variation in water uptake rates might be exploited by breeders for the benefit of the malting and brewing industries.

The fungal community structure of barley malts from diverse geographical regions correlates with malt quality parameters.

Malt is a preferred base for fermentations that produce beer or whisky. Barley for malt is grown under diverse environments in different geographical locations. Malt provides an ecological niche for a varied range of microorganisms with both positive and negative effects on its quality for brewing. Little information exists in the literature on the microbial community structure of Australian malt as well as broader global geographical differences in the associated fungal and bacterial communities. The aims of the present study were to compare the bacterial and fungal community structures of Australian commercial malt with its international counterparts originating from different geographical regions using terminal restriction fragment length polymorphism (TRFLP) fingerprinting and clone library analyses of ribosomal RNA genes. Further, the relationship between malt associated microbial communities and conventional malt quality parameters was also compared. Results showed that differences in fungal communities of malts from different geographical location were more pronounced than bacterial communities. TRFLP analysis discriminated high quality commercial malts with low fungal loads from malts deliberately infected with fungal inocula (Fusarium/Penicillium). Malt moisture, beta-amylase, α-amylase and limit dextrinase contents showed significant correlations with fungal community structure. This investigation concluded that fungal community structure was more important to subsequent malt quality outcomes than bacteria.

A Canadian evidence-based guideline for the first-line treatment of follicular lymphoma: joint consensus of the Lymphoma Canada Scientific Advisory Board.

Follicular lymphoma (FL) is the most common indolent non-Hodgkin lymphoma (NHL) in North America. Because of the heterogeneity of the disease, treatment options vary from observation to aggressive therapies or stem cell transplantation, or both. Although advances in treatment have improved outcomes, the disease remains largely incurable. In Canada, no unified national guideline exists for the front-line treatment of FL; provincial guidelines vary and are largely based on funding. There is therefore a need for evidence-based national treatment guidelines that are supported by Canadian hematologists to ensure that patients with FL have equitable access to the best available care. A group of experts from across Canada developed a national evidence-based treatment guideline to provide health care professionals with clear guidance on the first-line management of FL. Results of a systematic review of the literature are presented with consensus recommendations based on available evidence.

Final report of a phase 2 clinical trial of lenalidomide monotherapy for patients with T-cell lymphoma.

BACKGROUND: Patients with T-cell lymphomas face a poorer prognosis compared with patients with B-cell lymphomas. New therapeutic approaches need to be developed to improve outcomes for these patients. METHODS: Forty patients with recurrent and refractory T-cell lymphomas other than mycosis fungoides and patients with untreated T-cell lymphoma who were not candidates for combination chemotherapy were prescribed oral lenalidomide at a dose of 25 mg daily on days 1 to 21 of each 28-day cycle, with standardized dose reductions for toxicity. The primary endpoint was overall response rate (ORR), and secondary endpoints were complete and partial response rates, progression-free survival (PFS), overall survival (OS), and safety. The authors also determined duration of response (DoR). RESULTS: A total of 40 patients were enrolled in the current study; 1 patient was subsequently deemed ineligible. The ORR was 10 of 39 patients (26%); 3 patients (8%) achieved complete responses and 7 patients achieved partial responses. Three patients had stable disease for ≥5 cycles. The median OS was 12 months (range <1 month to ≥69 months), the median PFS was 4 months (range, <1 month to ≥50 months), and the median DoR was 13 months (range 2 months to ≥37 months), including 5 responses that lasted >1 year. Toxicity was in keeping with the known safety profile of lenalidomide. Among the patients who had recurrent/refractory peripheral T-cell lymphoma (29 patients), the ORR was 24%, the median OS was 12 months, the median PFS was 4 months, and the median DoR was 5 months (range, 2 months to ≥37 months). CONCLUSIONS: In the current study, the use of oral lenalidomide monotherapy demonstrated clinically relevant efficacy among patients with systemic T-cell lymphomas. It appears to have excellent potential as an agent in combination therapy for patients with T-cell lymphoma.

Reovirus modulates autophagy during oncolysis of multiple myeloma.

Multiple myeloma (MM) is a clonal plasma cell malignancy that accounts for 10-15% of newly diagnosed hematological cancers. Although significant advances have been made in the treatment of MM the disease still remains incurable. The oncolytic potential of reovirus has previously been demonstrated by others and us and is currently in phase III clinical trials for solid tumors. In addition a phase I clinical trial has recently been initiated for MM. Despite the clinical activity, the mechanism(s) of cell death caused by reovirus in MM is yet not yet well elucidated. A comprehensive understanding of reovirus-mediated histology-specific cell death mechanisms is imperative if this therapeutic is to become a standard of care for patients. Previously we have shown that reovirus-mediated cell death of breast and prostate cancer is orchestrated via apoptosis. The present study demonstrates for the first time that in addition to inducing apoptosis reovirus also upregulates autophagy during oncolysis of MM.

TRFLP analysis reveals that fungi rather than bacteria are associated with premature yeast flocculation in brewing.

Premature yeast flocculation (PYF) is a sporadic fermentation problem in the brewing industry that results in incomplete yeast utilization of fermentable sugars in wort. Culture-independent, PCR-based fingerprinting techniques were applied in this study to identify the associations between the occurrence of the PYF problem during brewery fermentation with barley malt-associated microbial communities (both bacteria and fungi). Striking differences in the microbial DNA fingerprint patterns for fungi between PYF positive (PYF +ve) and negative (PYF -ve) barley malts were observed using the terminal restriction fragment length polymorphism (TRFLP) technique. The presence of terminal restriction fragments (TRFs) of 360-460 bp size range, for fungal HaeIII restriction enzyme-derived TRFLP profiles appeared to vary substantially between PYF +ve and PYF -ve samples. The source of the barley malt did not influence the fungal taxa implicated in PYF. TRFLP analysis indicates bacterial taxa are unlikely to be important in causing PYF. Virtual digestion of fungal sequences tentatively linked HaeIII TRFs in the 360-460 bp size range to a diverse range of yeast/yeast-like species. Findings from this study suggest that direct monitoring of barley malt samples using molecular methods could potentially be an efficient and viable alternative for monitoring PYF during brewery fermentations.

Interim report of a phase 2 clinical trial of lenalidomide for T-cell non-Hodgkin lymphoma.

BACKGROUND: Novel therapies are needed to improve outcomes in T-cell lymphomas. The authors report the interim results of a prospective multicenter trial evaluating lenalidomide in T-cell lymphomas. METHODS: Patients with recurrent and refractory T-cell lymphomas other than mycosis fungoides and untreated patients ineligible for combination chemotherapy were prescribed oral lenalidomide (25 mg daily) on Days 1 to 21 of each 28-day cycle until disease progression, death, or unacceptable toxicity. The primary endpoint was overall response rate. Secondary endpoints were progression-free survival (PFS), overall survival (OS), and safety. The 2-stage design allows for up to 40 patients. RESULTS: At the time of this interim analysis, 24 patients were enrolled in this study, and 23 were evaluable for response. The median age was 65 years. The overall response rate was 7 (30%) of 23; all were partial responses. Two patients had stable disease for ≥5 cycles. Responses were seen in anaplastic, angioimmunoblastic, and peripheral T-cell unspecified histologies. Median PFS was 96 days (range, 8-696+ days). Median OS was 241 days (range, 8-696+ days). The most common grade 4 adverse event was thrombocytopenia (33%). The most common grade 3 adverse events were neutropenia (21%), febrile neutropenia (17%), and pain not otherwise specified (17%). Rash correlated with response to therapy (P=.003). CONCLUSIONS: In patients with recurrent and refractory T-cell lymphomas, oral lenalidomide monotherapy has clinical activity, and toxicity is consistent with the known safety profile of lenalidomide. Further study of lenalidomide in these diseases is warranted.

Mutation or polymorphism of the CD20 gene is not associated with the response to R-CHOP in diffuse large B cell lymphoma patients.

BACKGROUND: Rituximab, a chimeric antibody targeted to the human CD20 molecule, is a major component of the R-CHOP protocol used to treat patients with diffuse large B cell lymphoma (DLBCL). It is also a very expensive drug. Though the response rate of R-CHOP is higher than that of CHOP alone, patients may still experience a poor response. One possible mechanism that may mediate the poor response to R-CHOP is poor binding of the drug to the CD20 molecule, perhaps due to mutations or polymorphisms of the CD20 gene that affect its structure. To test this hypothesis and perhaps define a new predictor of R-CHOP response, our pilot study evaluated the CD20 gene sequence from patients exhibiting a poor outcome to R-CHOP. METHODS: Eleven patients with DLBCL who exhibited a recurrence of their disease and/or died within 24 months of R-CHOP treatment were categorized as showing poor progression-free survival (poor outcomes). Paraffin-embedded tissue specimens from the original tumors were evaluated for mutations or polymorphisms of the CD20 gene. Furthermore, sections from these patients and as well as from matched control patients who were categorized as good outcome patients (no progression of their disease within 36 months) were stained with anti-CD20 antibody and compared for CD20 protein expression. RESULTS: DNA sequence analyses revealed that no mutations were observed in DNA from the coding region of the CD20 gene in any of the initial tumors from 11 patients who showed poor outcomes with R-CHOP therapy. One case showed a synonymous single nucleotide polymorphism in exon 2 (C216T; rs2070770; Ile>>Ile). No significant differences in CD20 expression was observed between good and poor outcome patients with R-CHOP. CONCLUSIONS: Our results do not support association of CD20 mutations in specimens of the initial tumor or structural polymorphisms of the CD20 gene with patients who exhibited poor outcomes to R-CHOP.

Randomized trial of high-dose chemotherapy with autologous peripheral-blood stem-cell support compared with standard-dose chemotherapy in women with metastatic breast cancer: NCIC MA.16.

PURPOSE: We conducted a multicenter, randomized trial to compare progression-free survival (PFS), overall survival (OS), and quality of life in women with metastatic breast cancer (MBC) receiving high-dose chemotherapy plus autologous stem-cell transplantation (ASCT; HDCT) compared with standard-dose therapy. PATIENT AND METHODS: Between April 1997 and December 2000, 386 women with MBC and no prior chemotherapy for metastatic disease were registered. After initial response to anthracycline- or taxane-based induction chemotherapy, 224 patients were randomly assigned: 112 to high-dose cyclophosphamide, mitoxantrone, and carboplatin chemotherapy and ASCT (HDCT), and 112 to standard therapy (ST). Median age was 47 years (range, 25 to 67 years). Thirty two percent of women randomly assigned had estrogen and progesterone receptor-negative breast cancer, 42% had visceral metastases, and 58% had bone metastases. Complete remission rates before random assignment were 11% for those receiving HDCT and 12% for those receiving ST. RESULTS: After a median follow-up of 48 months, 79 deaths were observed in the HDCT arm and 77 deaths were observed in the ST arm; seven patients (6%) in the HDCT arm died as a result of toxicity. The median OS was 24 months for the HDCT arm (95% CI, 21 to 35 months) and 28 months for ST (95% CI, 22 to 33 months; hazard ratio [HR], 0.9; 95% CI, 0.6 to 1.2; P = .43). PFS was 11 months for HDCT and 9 months for ST (HR, 0.6 in favor of HDCT; 95% CI, 0.5 to 0.9; P = .006). CONCLUSION: HDCT did not improve OS in women with MBC when used as consolidation after response to induction chemotherapy.

Comparison of CD34 and monocyte-derived dendritic cells from mobilized peripheral blood from cancer patients.

Dendritic cells (DCs) are potent antigen-presenting cells that are integral to the initiation of T-cell immunity. Two cell types can be used as a source for generating DCs: monocytes and CD34(+) stem cells. Despite many investigations characterizing DCs, none have performed a direct paired comparison of monocyte and stem cell-derived DCs. Therefore, it is unclear whether one cell source has particular advantages over the other, or whether inherent differences exist between the two populations. We undertook the following study to determine if there were any differences in DCs generated from monocytes or CD34(+) cells from mobilized peripheral blood. DCs were generated by culturing the adherent cells (monocytes) in interleukin-4 and GM-CSF for 7 days, or by culturing nonadherent cells (CD34(+)) in the presence of GM-CSF and tumor necrosis factor alpha for 14 days. The resulting DCs were compared morphologically, phenotypically, functionally, and by yield. We could generate morphologically and phenotypically similar DCs. Differences were encountered when expression levels of some cell surface markers were examined (CD86, HLA-DR). There was no difference in how the DCs performed in a mixed lymphocyte reaction (p = .3). Further, no statistical difference was discovered when we examined cellular (DC) yield (p = .1); however, there was a significant difference when yield was normalized to the starting number of monocytes or CD34(+) cells (p = .016). Together, these data demonstrate that differences do exist between monocyte-derived DCs and CD34-derived DCs from the same cellular product (apheresis) from the same individual.

Efficacy and safety evaluation of human reovirus type 3 in immunocompetent animals: racine and nonhuman primates.

PURPOSE: Human reovirus type 3 has been proposed to kill cancer cells with an activated Ras signaling pathway. The purpose of this study was to investigate the efficacy of reovirus in immunocompetent glioma animal models and safety/toxicity in immunocompetent animals, including nonhuman primates. EXPERIMENTAL DESIGN: Racine glioma cells 9L and RG2 were implanted s.c. or intracranially in Fisher 344 rats with or without reovirus antibodies, followed by treatment of reovirus. To study whether reovirus kills contralateral tumors in the brain and to determine viral distribution, we established an in situ dual tumor model followed by reovirus intratumoral inoculation only into the ipsilateral tumor. To evaluate neurotoxicity/safety of reovirus, Cynomolgus monkeys and immunocompetent rats were given intracranially with reovirus, and pathological examination and/or behavioral studies were done. Viral shedding and clinical biochemistry were systematically studied in monkeys. RESULTS: Intratumorally given reovirus significantly suppressed the growth of both s.c. and intracranially tumors and significantly prolonged survival. The presence of reovirus-neutralizing antibodies did not abort the reovirus' antitumor effect. Reovirus inhibited glioma growth intracranially in the ipsilateral but not the contralateral tumors; viral load in ipsilateral tumors was 15 to 330-fold higher than the contralateral tumors. No encephalitis or behavioral abnormalities were found in monkeys and rats given reovirus intracranially. No treatment-related clinical biochemistry changes or diffuse histopathological abnormality were found in monkeys inoculated intracranially with Good Manufacturing Practice prepared reovirus. Microscopic changes were confined to the region of viral inoculation and were dose related, suggesting reovirus intracranially was well tolerated in nonhuman primates. CONCLUSIONS: These data show the efficacy and safety of reovirus when it is used in the treatment of gliomas in immunocompetent hosts. Inoculation of reovirus into the brain of nonhuman primates did not produce significant toxicities.

A case of an accessory testicular artery.

Dissection of an adult male cadaver revealed the presence of an accessory left testicular artery in addition to the normal right and left testicular arteries. In this case the accessory left testicular artery originated from the ventrolateral wall of the descending aorta. The origin was located between the superior mesenteric artery and the left renal vein. The accessory artery continued to course from the aorta laterally toward the superior ventral portion of the left kidney and then passed ventrally to the kidney on its course inferiorly to the pelvic region. Communication was observed between the accessory left testicular artery and the left renal artery. This variation of gonadal vasculature is of interest from the point of view of its embryogenesis, and possible clinical significance.

Priming with dendritic cells can generate strong cytotoxic T cell responses to chronic myelogenous leukemia cells in vitro.

Dendritic cells (DC) are antigen-presenting cells that can elicit potent antigen-specific responses. Since the development of techniques to cultivate these cells from peripheral blood, there has been a great deal of interest in their use in immunotherapeutic strategies. Here we show that morphologically, phenotypically, and functionally characteristic DC can be generated in vitro from peripheral blood mononuclear cells (PBMC) isolated from frozen apheresis product (AP) of cancer patients. These DC, when pulsed with whole-tumor lysate, protein, or RNA from a chronic myelogenous leukemia (CML) cell line, can induce anti-CML specific cytotoxicity in vitro by autologous cytotoxic T lymphocytes (CTL). RNA and protein-pulsed DC were more effective than lysate-pulsed DC at inducing cytotoxicity at low effector:target (E:T) ratios. These results were comparable to those obtained when fresh healthy peripheral blood was used as the source of PBMC, indicating that neither the malignant state of the patient nor the storage period detrimentally affected the generation or functionality of DC. CML cells were found to increase their level of MHC class I expression after exposure to CTL and pulsed DC thereby becoming better targets. These investigations lend support for the utilization of DC to generate anti-tumor responses in CML.

Prophylactic anticonvulsants in patients with brain tumour.

OBJECTIVE: We conducted a clinical trial to determine if prophylactic anticonvulsants in brain tumour patients (without prior seizures) reduced seizure frequency. We stopped accrual at 100 patients on the basis of the interim analysis. METHODS: One hundred newly diagnosed brain tumour patients received anticonvulsants (AC Group) or not (No AC Group) in this prospective randomized unblinded study. Sixty patients had metastatic, and 40 had primary brain tumours. Forty-six (46%) patients were randomized to the AC Group and 54 (54%) to the No AC Group. Median follow-up was 5.44 months (range 0.13-30.1 months). RESULTS: Seizures occurred in 26 (26%) patients, eleven in the AC Group and 15 in the No AC Group. Seizure-free survivals were not different; at three months 87% of the AC Group and 90% of the No AC Group were seizure-free (log rank test, p = 0.98). Seventy patients died (unrelated to seizures) and survival rates were equivalent in both groups (median survival = 6.8 months versus 5.6 months, respectively; log rank test, p = 0.50). We then terminated accrual at 100 patients because seizure and survival rates were much lower than expected; we would need > or = 900 patients to have a suitably powered study. CONCLUSIONS: These data should be used by individuals contemplating a clinical trial to determine if prophylactic anticonvulsants are effective in subsets of brain tumour patients (e.g. only anaplastic astrocytomas). When taken together with the results of a similar randomized trial, prophylactic anticonvulsants are unlikely to be effective or useful in brain tumour patients who have not had a seizure.