'Iron-saturated' lactoferrin is a potent natural adjuvant for augmenting cancer chemotherapy

Bovine lactoferrin (bLf), an iron-containing natural defence protein found in bodily secretions, has been reported to inhibit carcinogenesis and the growth of tumours. Here, we investigated whether natural bLf and iron-saturated forms of bLf differ in their ability to augment cancer chemotherapy. bLf was supplemented into the diet of C57BL/6 mice that were subsequently challenged subcutaneously with tumour cells, and treated by chemotherapy. Chemotherapy eradicated large (0.6 cm diameter) EL-4 lymphomas in mice that had been fed iron-saturated bLf (here designated Lf(+)) for 6 weeks prior to chemotherapy, but surprisingly not in mice that were fed lesser iron-saturated forms of bLf, including apo-bLf (4% iron saturated), natural bLf (approximately 15% iron saturated) and 50% iron-saturated bLf. Lf(+)-fed mice bearing either EL-4, Lewis lung carcinoma or B16 melanoma tumours completely rejected their tumours within 3 weeks following a single injection of either paclitaxel, doxorubicin, epirubicin or fluorouracil, whereas mice fed the control diet were resistant to chemotherapy. Lf(+) had to be fed to mice for more than 2 weeks prior to chemotherapy to be wholly effective in eradicating tumours from all mice, suggesting that it acts as a competence factor. It significantly reduced tumour vascularity and blood flow, and increased antitumour cytotoxicity, tumour apoptosis and the infiltration of tumours by leukocytes. Lf(+) bound to the intestinal epithelium and was preferentially taken up within Peyer's patches. It increased the production of Th1 and Th2 cytokines within the intestine and tumour, including TNF, IFN-gamma, as well as nitric oxide that have been reported to sensitize tumours to chemotherapy. Importantly, it restored both red and white peripheral blood cell numbers depleted by chemotherapy, potentially fortifying the mice against cancer. In summary, bLf is a potent natural adjuvant and fortifying agent for augmenting cancer chemotherapy, but needs to be saturated with iron to be effective.

Adamts5, the gene encoding a proteoglycan-degrading metalloprotease, is expressed by specific cell lineages during mouse embryonic development and in adult tissues.

The secreted metalloprotease ADAMTS5 is implicated in destruction of the cartilage proteoglycan aggrecan in arthritis, but its physiological functions are unknown. Its expression profile during embryogenesis and in adult tissues is therefore of considerable interest. β-Galactosidase (β-gal) histochemistry, enabled by a LacZ cassette inserted in the Adamts5 locus, and validated by in situ hybridization with an Adamts5 cRNA probe and ADAMTS5 immunohistochemistry, was used to profile Adamts5 expression during mouse embryogenesis and in adult mouse tissues. Embryonic expression was scarce prior to 11.5 days of gestation (E11.5) and noted only in the floor plate of the developing brain at E9.5. After E11.5 there was continued expression in brain, especially in the choroid plexus, peripheral nerves, dorsal root ganglia, cranial nerve ganglia, spinal and cranial nerves, and neural plexuses of the gut. In addition to nerves, developing limbs have Adamts5 expression in skeletal muscle (from E13.5), tendons (from E16.5), and inter-digital mesenchyme of the developing autopod (E13.5–15.5). In adult tissues, there is constitutive Adamts5 expression in arterial smooth muscle cells, mesothelium lining the peritoneal, pericardial and pleural cavities, smooth muscle cells in bronchi and pancreatic ducts, glomerular mesangial cells in the kidney, dorsal root ganglia, and in Schwann cells of the peripheral and autonomic nervous system. Expression of Adamts5 during neuromuscular development and in smooth muscle cells coincides with the broadly distributed proteoglycan versican, an ADAMTS5 substrate. These observations suggest the major contexts in which developmental and physiological roles could be sought for this protease.

Methods for the analysis of histone H3 and H4 acetylation in blood

LBH589 is one of the many histone deacetylase inhibitors (HDACi) that are currently in clinical trial. Despite their wide-spread use, there is little literature available describing the typical levels of histone acetylation in untreated peripheral blood, the treatment and storage of samples to retain optimal measurement of histone acetylation nor methods by which histone acetylation analysis may be monitored and measured during the course of a patient’s treatment. In this study, we have used cord or peripheral blood as a source of human leukocytes, performed a comparative analysis of sample processing methods and developed a flow cytometric method suitable for monitoring histone acetylation in isolated lymphocytes and liquid tumors. Western blotting and immunohistochemistry techniques have also been addressed. We have tested these methods on blood samples collected from four patients treated with LBH589 as part of an Australian Children’s Cancer Clinical Trial (CLBH589AAU03T) and show comparable results when comparing in vitro and in vivo data. This paper does not seek to correlate histone acetylation levels in peripheral blood with clinical outcome but describes methods of analysis that will be of interest to clinicians and scientists monitoring the effects of HDACi on histone acetylation in blood samples in clinical trials or in related research studies.

Resistive exercises, with or without whole body vibration, prevent vertebral marrow fat accumulation during 60 days of head-down tilt bed rest in men

Fat accumulates in the bone marrow of lumbar vertebrae with bed rest. Exercise with or without whole body vibration may counter this effect. Our objectives were to measure 1) the vertebral fat fraction (VFF) of men subjected to bed rest who performed resistive exercises with (RVE, n = 7) or without whole body vibration(RE, n = 8) or no exercise (CTR, n = 9) using three MRI techniques; and 2) changes in peripheral blood counts. Twenty-four healthy men (age: 20-45 yr) underwent -6° head-down tilt (HDT) bed rest for 60 days. MRI was performed using three techniques (fat saturation, proton spectroscopy, and in and out of phase) to measure the fat fraction of L(3), L(4), and/or L(5) at baseline, mid-HDT, and end-HDT. Erythrocytes and leukocytes were counted at HDT days 19, 33, 47, 54, and 60. The mean absolute VFF was increased in the CTR group at mid-HDT and end-HDT (+3.9 ± 1.3 and +3.6 ± 1.2%, respectively, both P < 0.05). The RE group had a smaller VFF change than the CTR group at mid-HDT (-0.9 ± 1.2 vs. +3.9 ± 1.3%, P < 0.05). The RVE group had a smaller VFF change than the CTR group at end-HDT (-2.6 ± 1.9 vs. +3.5 ± 1.2%, P < 0.05). Erythrocyte counts were increased in all groups at HDT day 19 and HDT day 33 and in the RE group at HDT day 54 (all P < 0.05). Bed rest for 60 days at -6° HDT increased lumbar VFF in men beyond natural involution. RVE and RE regimens effectively prevented VFF accumulation. Higher erythrocyte counts were not altered by RVE or RE. Whole body vibration, along with RE administered to people with prolonged immobility, may prevent fat accumulation in their bone marrow.

In-vivo cell mediated immunity in elite swimmers in response to training

This study investigated in-vivo cell-mediated immune (CMI) responses in elite swimmers over a 5-month training season, to assess the impact of intense training on changes in T-lymphocyte function. The CMI Multitest was performed early in the season after a period of rest, during peak high-intensity training, and late in the season during the precompetition taper period. The CMI tests were performed at rest prior to a morning training session. There were no significant differences between the swimmers and a control group for any of the seven CMI antigen responses at any of the test points during the season. In the swimmers, there were no significant differences in the number of positive responses to the CMI antigens between the three test points (Friedman's test = 9.6364, p = 0.47) and no significant differences for the CMI cumulative scores (Friedman's test = 11.98, p = 0.29) at each test point. There was no consistent pattern for changes in CMI cumulative scores for individual swimmers over the training season. The findings of this study indicate that, despite reported transient T-lymphocyte immunosuppression immediately after intense exercise, probably associated with acute redistribution and temporary pooling of blood T cell subsets in extremities, the T-lymphocyte function involved in CMI responses is not compromised by extended periods of training at an elite level.

Vitamins D and A can be successfully measured by LC-MS/MS in cord blood diluted plasma

OBJECTIVES: In widely used protocols for the collection and isolation of cord blood mononuclear cells, investigators are left with substantial volumes of diluted plasma which could be used for other measurements. The aim of this study was to ascertain the validity of umbilical cord blood (UCB) diluted plasma samples for vitamin D, A and E analysis compared to UCB serum samples. DESIGN & METHODS: Twenty UCB matched samples of diluted plasma and serum were collected. The samples were analysed by two liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods on two separate occasions. RESULTS: The results of 25(OH)D3 obtained by the two laboratories demonstrated close agreement with a mean difference of 0.14nmol/L [95% confidence interval (95% CI), -6.8 to 7.1]. Both methods demonstrate close agreement for 25(OH)D3 in UCB serum versus diluted UCB plasma; mean difference 2.2nmol/L [95% CI, -9.5 to 13.9] and 4.1nmol/L [95% CI, -14.5 to 6.1] for the results from Lab A and Lab B, respectively. Vitamin A was quantified by Lab A in UCB serum and diluted UCB plasma; mean difference 0.07μmol/L [95% CI, -0.41 to 0.28]. Results of 25(OH)D3 epimer and vitamin E in the diluted UCB plasma were below the limit of quantification, and could not be compared with UCB serum. CONCLUSIONS: Diluted UCB plasma can be used for the quantification of retinol and 25(OH)D3 by LC-MS/MS. By contrast, quantification of 25(OH)D3 epimer and vitamin E in diluted UCB plasma is not supported by this study due to limitations in analytical sensitivity.