Aerobic high-intensity intervals improve V̇O2max more than supramaximal sprint intervals in females, similar to males.

INTRODUCTION: Maximal oxygen uptake (V̇O2max ) is a pivotal factor for aerobic endurance performance. Recently, aerobic high-intensity interval training (HIIT) was documented to be superior to sprint interval training (SIT) in improving V̇O2max in well-trained males. However, as mounting evidence suggests that physiological responses to training are sex-dependent, examining the effects of HIIT versus SIT on V̇O2max , anaerobic capacity, and endurance performance in females is warranted. METHODS: We randomized 81 aerobically well-trained females (22 ± 2 years, 51.8 ± 3.6 mL∙kg-1 ∙min-1 V̇O2max ), training three times weekly for 8 weeks, to well-established protocols: (1) HIIT 4 × 4 min at ~95% of maximal aerobic speed (MAS), with 3 min active recovery (2) SIT 8 × 20 s at ~150% of MAS, with 10 s passive recovery (3) SIT 10 × 30 s at ~175% of MAS, with 3.5 min active recovery. RESULTS: Only HIIT 4 × 4 min increased V̇O2max (7.3 ± 3.1%), different from both SIT groups (all p < 0.001). Anaerobic capacity (maximal accumulated oxygen deficit) increased following SIT 8 × 20 s (6.5 ± 10.5%, p < 0.05), SIT 10 × 30 s (14.4 ± 13.7%, p < 0.05; different from HIIT 4 × 4 min, p < 0.05). SIT 10 × 30 s resulted in eight training-induced injuries, different from no injuries following HIIT 4 × 4 min and SIT 8 × 20 s (p < 0.001). All groups improved long-distance (3000-meter) and sprint (300-meter) running performance (all p < 0.001). SIT protocols improved sprint performance more than HIIT 4 × 4 min (p < 0.05). Compared to previous male results, no increase in V̇O2max following SIT 8 × 20 s (p < 0.01), and a higher injury rate for SIT 10 × 30 s (p < 0.001), were evident. CONCLUSIONS: In aerobically well-trained women, HIIT is superior to SIT in increasing V̇O2max while all-out treadmill running SIT is potentially more harmful.

Aerobic high-intensity intervals are superior to improve V̇O2max compared with sprint intervals in well-trained men.

Maximal oxygen uptake (V̇O2max ) may be the single most important factor for long-distance running performance. Interval training, enabling high intensity, is forwarded as the format that yields the largest increase in V̇O2max . However, it is uncertain if an optimal outcome on V̇O2max , anaerobic capacity, and running performance is provided by training with a high aerobic intensity or high overall intensity. Thus, we randomized 48 aerobically well-trained men (23 ± 3 years) to three commonly applied interval protocols, one with high aerobic intensity (HIIT) and two with high absolute intensity (sprint interval training; SIT), 3× week for 8 weeks: (1) HIIT: 4 × 4 min at ~95% maximal aerobic speed (MAS) with 3 min active breaks. (2) SIT: 8 × 20 s at ~150% MAS with 10 s passive breaks. (3) SIT: 10 × 30 s at ~175% MAS with 3.5 min active breaks. V̇O2max increased more (p < 0.001) following HIIT, 4 × 4 min (6.5 ± 2.4%, p < 0.001) than SIT, 8 × 20 s (3.3 ± 2.4%, p < 0.001) and SIT, 10 × 30 s (n.s.). This was accompanied by a larger (p < 0.05) increase in stroke volume (O2 -pulse) following HIIT, 4 × 4 min (8.1 ± 4.1%, p < 0.001) compared with SIT, 8 × 20 s (3.8 ± 4.2%, p < 0.01) and SIT, 10 × 30 (n.s.). Anaerobic capacity (maximal accumulated oxygen deficit) increased following SIT, 8 × 20 s (p < 0.05), but not after HIIT, 4 × 4 min, nor SIT, 10 × 30 s. Long-distance (3000-m) endurance performance increased (p < 0.05-p < 0.001) in all groups (HIIT, 4 × 4 min: 5.9 ± 3.2%; SIT, 8 × 20 s: 4.1 ± 3.7%; SIT, 10 × 30 s: 2.2 ± 2.2%), with HIIT increasing more than SIT, 10 × 30 s (p < 0.05). Sprint (300-m) performance exhibited within-group increases in SIT, 8 × 20 s (4.4 ± 2.0%) and SIT, 10 × 30 s (3.3 ± 2.8%). In conclusion, HIIT improves V̇O2max more than SIT. Given the importance of V̇O2max for most endurance performance scenarios, HIIT should typically be the chosen interval format.

Tumor budding score predicts lymph node status in oral tongue squamous cell carcinoma and should be included in the pathology report.

BACKGROUND: The majority of oral cavity cancers arise in the oral tongue. The aim of this study was to evaluate the prognostic value of tumor budding in oral tongue squamous cell carcinoma, both as a separate variable and in combination with depth of invasion. We also assessed the prognostic impact of the 8th edition of the American Joint Committee on Cancer's TNM classification (TNM8), where depth of invasion (DOI) supplements diameter in the tumor size (T) categorization. METHODS: Patients diagnosed with primary oral tongue squamous cell carcinoma were evaluated retrospectively. Spearman bivariate correlation analyses with bootstrapping were used to identify correlation between variables. Prognostic value of clinical and histopathological variables was assessed by Log rank and Cox regression analyses with bootstrapping using 5-year disease specific survival as outcome. The significance level for the hypothesis test was 0.05. RESULTS: One-hundred and fifty patients had available material for microscopic evaluation on Hematoxylin and Eosin-stained slides and were included in the analyses. Reclassification of tumors according to TNM8 caused a shift towards a higher T status compared to the previous classification. The tumor budding score was associated with lymph node metastases where 23% of the patients with low-budding tumors had lymph node metastases, compared with 43% of those with high-budding tumors. T-status, lymph node status, tumor budding, depth of invasion, and the combined tumor budding/depth of invasion score were all significantly associated with survival in univariate analyses. In multivariate analyses only N-status was an independent prognosticator of survival. CONCLUSION: Reclassification according to TNM8 shifted many tumors to a higher T-status, and also increased the prognostic value of the T-status. This supports the implementation of depth of invasion to the T-categorization in TNM8. Tumor budding correlated with lymph node metastases and survival. Therefore, information on tumor budding can aid clinicians in treatment planning and should be included in pathology reports of oral tongue squamous cell carcinomas.

Runaway Train: A Leaky Radiosensitive SCID with Skin Lesions and Multiple Lymphomas.

The nuclease Artemis is essential for the development of T-cell and B-cell receptors and repair of DNA double-strand breaks, and a loss of expression or function will lead to a radiosensitive severe combined immunodeficiency with no functional T-cells or B-cells (T-B-SCID). Hypomorphic mutations in the Artemis gene can lead to a functional, but reduced, T-cell and B-cell repertoire with a more indolent clinical course called "leaky" SCID. Here, we present the case of a young man who had increasingly aggressive lymphoproliferative skin lesions from 2 years of age which developed into multiple EBV+ B-cell lymphomas, where a hypomorphic mutation in the Artemis gene was found in a diagnostic race against time using whole exome sequencing. The patient was given a haploidentical stem cell transplant while in remission for his lymphomas and although the initial course was successful, he succumbed to a serious Pneumocystis jirovecii pneumonia 5 months after the transplant. The case underscores the importance of next-generation sequencing in the diagnosis of patients with suspected severe immunodeficiency.

Phosphatase of regenerating liver-3 (PRL-3) is overexpressed in classical Hodgkin lymphoma and promotes survival and migration.

BACKGROUND: Phosphatase of regenerating liver-3 (PRL-3) is implicated in oncogenesis of hematological and solid cancers. PRL-3 expression increases metastatic potential, invasiveness and is associated with poor prognosis. With this study, we aimed to show a possible oncogenic role of PRL-3 in classical Hodgkin lymphoma (cHL). METHODS: PRL-3 expression was measured in 25 cHL patients by immunohistochemistry and gene expression was analyzed from microdissected malignant cells. We knocked down PRL-3 in the cHL cell lines L1236 and HDLM2 and used small molecular inhibitors against PRL-3 to investigate proliferation, migration and cytokine production. RESULTS: PRL-3 protein was expressed in 16% of patient samples. In three different gene expression datasets, PRL-3 was significantly overexpressed compared to normal controls. PRL-3 knockdown reduced proliferation, viability and Mcl-1 expression in L1236, but not in HDLM2 cells. Thienopyridone, a small molecule inhibitor of PRL-3, reduced proliferation of both L1236 and HDLM2. PRL-3 affected IL-13 secretion and enhanced STAT6 signaling. IL-13 stimulation partially rescued proliferation in L1236 cells after knockdown of PRL-3. PRL-3 knockdown reduced migration in both L1236 and HDLM2 cells. CONCLUSION: PRL-3 was overexpressed in a subset of cHL patients. Inhibition of PRL-3 increased IL-13 cytokine production and reduced migration, proliferation and viability. The effects could be mediated through regulation of the anti-apoptotic molecule Mcl-1 and a feedback loop of IL-13 mediated activation of STAT6. This point to a role for PRL-3 in the pathogenesis of Hodgkin lymphoma, and PRL-3 could be a possible new drug target.

Can spectral-spatial image segmentation be used to discriminate experimental burn wounds?

Hyperspectral imaging (HSI) is a noncontact and noninvasive optical modality emerging the field of medical research. The goal of this study was to determine the ability of HSI and image segmentation to discriminate burn wounds in a preclinical porcine model. A heated brass rod was used to introduce burn wounds of graded severity in a pig model and a sequence of hyperspectral data was recorded up to 8-h postinjury. The hyperspectral images were processed by an unsupervised spectral­spatial segmentation algorithm. Segmentation was validated using results from histology. The proposed algorithm was compared to K-means segmentation and was found superior. The obtained segmentation maps revealed separated zones within the burn sites, indicating a variation in burn severity. The suggested image-processing scheme allowed mapping dynamic changes of spectral properties within the burn wounds over time. The results of this study indicate that unsupervised spectral­spatial segmentation applied on hyperspectral images can discriminate burn injuries of varying severity.

Allelic mutations in noncoding genomic sequences construct novel transcription factor binding sites that promote gene overexpression.

The growth and survival factor hepatocyte growth factor (HGF) is expressed at high levels in multiple myeloma (MM) cells. We report here that elevated HGF transcription in MM was traced to DNA mutations in the promoter alleles of HGF. Sequence analysis revealed a previously undiscovered single-nucleotide polymorphism (SNP) and crucial single-nucleotide variants (SNVs) in the promoters of myeloma cells that produce large amounts of HGF. The allele-specific mutations functionally reassembled wild-type sequences into the motifs that affiliate with endogenous transcription factors NFKB (nuclear factor kappa-B), MZF1 (myeloid zinc finger 1), and NRF-2 (nuclear factor erythroid 2-related factor 2). In vitro, a mutant allele that gained novel NFKB-binding sites directly responded to transcriptional signaling induced by tumor necrosis factor alpha (TNFα) to promote high levels of luciferase reporter. Given the recent discovery by genome-wide sequencing (GWS) of numerous non-coding mutations in myeloma genomes, our data provide evidence that heterogeneous SNVs in the gene regulatory regions may frequently transform wild-type alleles into novel transcription factor binding properties to aberrantly interact with dysregulated transcriptional signals in MM and other cancer cells.

HGF and IGF-1 synergize with SDF-1α in promoting migration of myeloma cells by cooperative activation of p21-activated kinase.

Stromal-derived factor (SDF)-1α, insulin-like growth factor (IGF)-1 and hepatocyte growth factor (HGF) are potent mediators of cell migration. We studied the effect of combinations of these cytokines on the migration of myeloma cells. When SDF-1α was combined with either HGF or IGF-1, we found a striking synergy in the cytokines' ability to guide cells across a transwell membrane. Between HGF and IGF-1 there was no cooperativity. However, the effects of HGF and IGF-1 were not redundant. HGF and SDF-1 caused concentration gradient-directed migration, as opposed to IGF-1, which apparently caused randomly directed cell movement. The SDF-1α-driven migration of JJN-3 cells, a myeloma cell line secreting large amounts of HGF, was reduced when JJN-3 cells were given an inhibitor of the HGF receptor, demonstrating a cooperative activity between autocrine HGF and exogenous SDF-1α. There was a clear positive correlation between the degree of cytokine-induced migration and phosphorylation of p21-activated kinase (PAK) both in primary myeloma cells and in cell lines including INA-6 and IH-1. Downregulation of PAK with small interfering RNA in INA-6 cells resulted in decreased cytokine-driven migration. This study shows synergy between SDF-1α and HGF/IGF-1 in inducing migration of myeloma cells, yet each cytokine has distinct properties in the way it regulates cell migration. These findings are likely to be of clinical relevance because multiple myeloma cells are located in an environment containing HGF and IGF-1 and are exposed to an SDF-1α gradient between the bone marrow and peripheral blood.

Immunohistochemical analysis of hepatocyte growth factor and c-Met in plasma cell disease.

AIMS: Interaction with the bone marrow microenvironment is important for homing and survival of myeloma cells. One cytokine involved in this process is hepatocyte growth factor (HGF). HGF, by binding to the receptor tyrosine kinase c-Met, mediates a broad range of tumour progression activities. Our aims were to investigate whether HGF and c-Met are present in bone marrow and extramedullary tumours from patients with monoclonal plasma cell disease, and whether c-Met is activated. METHODS AND RESULTS: Expression of HGF, c-Met and phospho-c-Met was studied by immunohistochemistry in biopsies from 80 patients with monoclonal plasma cell disease. Cytoplasmic staining for HGF in plasma cells was demonstrated in 58 of 68 biopsies from multiple myeloma patients (85%), but also in biopsies from nine of 10 healthy individuals. Membranous staining for c-Met was found in 25 of 63 multiple myeloma patients (40%) and in none of 10 healthy individuals. Membranous staining for phospho-c-Met was found in biopsies from 15 of 21 c-Met-positive myeloma patients (71%). CONCLUSIONS: Our data point to c-Met expression as one of the factors that distinguishes normal from malignant plasma cells, and indicate that the HGF/c-Met system is activated in multiple myeloma patients.

c-Met signaling promotes IL-6-induced myeloma cell proliferation.

OBJECTIVES: Hepatocyte growth factor (HGF) is a constituent of the myeloma microenvironment and is elevated in sera from myeloma patients compared to healthy individuals. Increased levels of serum HGF predict a poor prognosis. It has previously been shown by us and others HGF can act as a growth factor to myeloma cells in vitro although these effects have been moderate. We therefore wanted to investigate if HGF could influence the effects of interleukin (IL)-6. METHODS: Myeloma cell lines and primary samples were tested for the combined effects of IL-6 and HGF in inducing DNA synthesis and migration. Expression levels of c-Met protein were analysed by Western blotting and flow cytometry. Signaling pathways were examined by Western blotting using phosphospecific antibodies and a Ras-GTP pull down assay. RESULTS: HGF potentiated IL-6-induced growth in human myeloma cell lines and in purified primary myeloma cells. There was also cooperation between HGF and IL-6 in induction of migration. There seemed to be two explanations for this synergy. IL-6-treatment increased the expression of c-Met making cells HGF responsive, and IL-6 was dependent on c-Met signaling in activating both Ras and p44/42 MAPK by a mechanism involving the tyrosine phosphatase Shp2. CONCLUSIONS: The results indicate that besides from being a myeloma growth factor alone, HGF can also potentiate the effects of IL-6 in myeloma proliferation and migration. Thus, c-Met signaling could be a target for therapy of multiple myeloma.

Mn2+ regulates myeloma cell adhesion differently than the proadhesive cytokines HGF, IGF-1, and SDF-1alpha.

Adhesion of multiple myeloma (MM) cells in the bone marrow (BM) is important for the growth and survival of the myeloma cells. Very late antigen-4 (VLA-4) is one of the main adhesion receptors that mediate MM cell binding to fibronectin (FN). In this study we have examined the effect of divalent cations on adhesion of MM cells to FN, and compared this type of adhesion with the adhesion induced by the cytokines HGF, IGF-1 and SDF-1alpha. Mn(2+) induced adhesion in all cell lines tested. Cytokine- and Mn(2+)-induced VLA-4-mediated adhesion were different in many respects, including binding specificity, adhesion kinetics and the activation state of VLA-4. To study a potential role of divalent cations in vivo, we measured the concentrations of divalent cations in BM plasma from 14 MM patients. We also found that Mn(2+)-mediated adhesion to FN activated the MAPK pathway, indicating that the interaction of MM-cells with FN mediated by Mn(2+) could play a critical role for growth and proliferation. In conclusion, this study shows a potential important role of divalent cations in MM cell biology and supports earlier studies pointing to activated VLA-4 as a key for homing of MM cells to the BM.

Hepatocyte growth factor promotes migration of human myeloma cells.

Multiple myeloma is characterized by the accumulation and dissemination of malignant plasma cells in the bone marrow. Cell migration is thought to be important for these events. We studied migration in a Transwell two-chamber assay and tested the motogenic effect of various cytokines. In addition to insulin-like growth factor-1 and stromal cell-derived growth factor-1alpha, previously known as chemoattractants for myeloma cells, we identified hepatocyte growth factor as a potent attractant for myeloma cells. Hepatocyte growth factor-mediated migration was dependent on phosphatidylinositol-3-kinase, involved the MAPK/Erk signaling cascade and VLA-4 integrins, but did not involve Akt, mTOR or G proteins.

A selective c-met inhibitor blocks an autocrine hepatocyte growth factor growth loop in ANBL-6 cells and prevents migration and adhesion of myeloma cells.

PURPOSE: We wanted to examine the role of the hepatocyte growth factor (HGF) receptor c-Met in multiple myeloma by applying a novel selective small molecule tyrosine kinase inhibitor, PHA-665752, directed against the receptor. EXPERIMENTAL DESIGN: Four biological sequels of HGF related to multiple myeloma were studied: (1) proliferation of myeloma cells, (2) secretion of interleukin-11 from osteogenic cells, (3) migration of myeloma cells, and (4) adhesion of myeloma cells to fibronectin. We also examined effects of the c-Met inhibitor on intracellular signaling pathways in myeloma cells. RESULTS: PHA-665752 effectively blocked the biological responses to HGF in all assays, with 50% inhibition at 5 to 15 nmol/L concentration and complete inhibition at around 100 nmol/L. PHA-665752 inhibited phosphorylation of several tyrosine residues in c-Met (Tyr(1003), Tyr(1230/1234/1235), and Tyr(1349)), blocked HGF-mediated activation of Akt and p44/42 mitogen-activated protein kinase, and prevented the adaptor molecule Gab1 from complexing with c-Met. In the HGF-producing myeloma cell line ANBL-6, PHA-665752 revealed an autocrine HGF-c-Met-mediated growth loop. The inhibitor also blocked proliferation of purified primary myeloma cells, suggesting that autocrine HGF-c-Met-driven growth loops are important for progression of multiple myeloma. CONCLUSIONS: Collectively, these findings support the role of c-Met and HGF in the proliferation, migration, and adhesion of myeloma cells and identify c-Met kinase as a therapeutic target for treatment of patients with multiple myeloma.