Journal of Translational Medicine - Latest Comments

correction

Hajime Isomoto — 2011-04-11T09:15:41Z

Please change as follows.
B.21
miR-205 induces an epithelial-mesenchymal transition (EMT)-like phenotype through regulating zinc finger E-box binding homeobox 2 (ZEB2) expression
should be corrected into
'Inhibition of miR-205 induces an epithelial-mesenchymal transition (EMT)-like phenotype through regulating zinc finger E-box binding homeobox 2 (ZEB2) expression'

B.23
The miR-205 _expression levels did not differ among the histological subclasses of ESCC differentiation (Figure 5C), albeit those in invasive ESCC with poor differentiation were significantly lower than in intraepithelial ESCC (Figure 5D).
should be corrected into
'The miR-205 _expression levels did not differ among the histological subclasses of ESCC differentiation (Figure 5C), albeit those in invasive ESCC with poor differentiation were significantly higher than in intraepithelial ESCC (Figure 5D)'

Transfection tutorial

Rachel Oliver — 2011-03-18T11:56:49Z

Here’s a video that will help increase transfection efficiency. http://www.americanbiotechnologist.com/blog/primary-cell-transfection/

Neutrophil ATP related parameters in chronic fatigue syndrome

Norman Booth — 2011-02-28T01:26:01Z

Norman E Booth, Mansfield College, University of Oxford
Sarah Myhill, Sarah Myhill Ltd
John McLaren-Howard, Acumen Medical Ltd

It is gratifying to see experts in the biomedical field turn their attention to the devastating illness Chronic Fatigue Syndrome (CFS/ME), as exemplified in the above paper by Vermeulen et al [1]. Further studies of the postexertional malaise following exercise, either physical or mental, are extremely important because this malaise is the most characteristic and disabling symptom of CFS/ME. Also, in spite of many studies, there is inadequate understanding of the biochemical processes which lead to this malaise.

In 2009, we published our findings from an audit where we compared deficiencies in the provision of ATP in neutrophils with the degree of disability of patients with CFS [2]. Here we use data from 53 controls and 61 CFS patients (in the same age range 18-65 years as the controls). The patient group spanned a wide range of CFS Ability from 0 to 7 on the Bell CFS Ability scale [2-3].

The paper by Vermeulen et al [1] compares exercise performance in CFS patients and controls. These authors conclude that the decrease in mitochondrial ATP production with work rate detected in their tests on peripheral blood mononuclear cells (PBMCs) is a secondary phenomenon. They focus attention on differences in transport capacity of oxygen. While agreeing about the importance of this and supporting the premise that more research is needed to understand oxygen transport-related issues in CFS, a number of ATP-related issues concern us greatly.

Vermeulen et al quote the work by Maianski et al [4] on neutrophils in support of their dismissal of the importance of ATP-related parameters in CFS. The work in [4] concentrates on aspects restricted to apoptosis. The work of van Raam et al [5] further defines these issues and concludes that neutrophils retain some respiratory chain complex activity but this activity is limited to certain complexes in the respiratory chain and exists mainly for the maintenance of mitochondrial membrane potential. These papers raise critical doubt about the validity of our previous findings and we feel that it is important to explore these issues in terms of the ATP-related tests we have done and the results we have published.

With regard to the exercise procedures given to the 15 CFS patients in the Vermeulen et al study [1], we are confident that many of the patients in our study [2], including all of those in the severe and very severe categories would not have been able to carry out the exercise procedures. Although the patients in both studies met the Centers for Disease Control criteria for CFS [6], we are well aware that there is enormous patient-to-patient variability in the degree of physical and mental incapacity. We felt it a strength of our paper that the measured biochemical parameters were compared individually and collectively with the CFS Ability [2-3] of the individual patients. This was much simpler with the higher number of patients in our study. We now discuss the issues relating to neutrophil ATP-related parameters in the three parts of the ATP profile test that we used.

Whole cell ATP and ATP complexed with Mg
ATP often functions as a complex with magnesium (Mg) and in many reactions Mg is an essential cofactor. Due to the fact that intracellular Mg deficiencies are common in CFS, we measured the whole cell ATP in neutrophils in the presence of excess Mg and again with endogenous Mg only. The ATP concentrations given in Figure 1 of the Maianski et al paper [4] and in Figure 4 of van Raam et al [5] lie between our results with and without added Mg. These may simply relate to methodological differences in the test procedures. We found that 87% of the patients were below the minimum value of the controls in the ATP with only endogenous Mg, i.e. the product ATP x (ATP Ratio). However, the value of the product was not a strong indicator of CFS Ability with a correlation coefficient of only r = 0.086.

We did not compare baseline whole cell ATP levels in neutrophils and PBMCs. It is clear from van Raam et al [5] that although their ATP levels are similar to ours, there are markedly differing effects of some known inhibitors of oxidative phosphorylation. As a result, Vermeulen et al [1] understandably criticize our use of neutrophils to explore ATP function in CFS patients. It is appropriate that we should proceed with further studies comparing neutrophils with PBMCs and we are doing so. Until such results are available, we wish to explain further aspects of the work already done and the tests we have used.

Inhibition study and ADP to ATP re-conversion - the Ox Phos parameter
Following the measurement of the whole cell ATP, we used sodium azide to inhibit ATP production prior to a two-stage re-measurement of ATP. The azide ion is an inhibitor of cytochrome c oxidase, a component of Complex IV in the mitochondrial respiratory electron transfer chain. It is in the initial step that our results differ from those of Maianski et al [4]. We saw a rapid (in less than 3 minutes) fall in measured ATP, usually to just a few percent (7.5 +/- 3.4 % for the controls) of the starting value. After removal of the inhibitor, in our control group we saw the total ATP levels return to between 60% and 90% of their original values as shown in the right-hand histogram in Figure 2C of our paper [2]. These results appear to be inconsistent with the findings shown in Figure 1 of Maianski et al [4]. However, they measured after 6 hours with and without inhibitor and also there may have been some compensatory process involved.

For the 38% of the patients who had similar recoveries to the controls, the degree of recovery was independent of CFS Ability, while for the other group (62% of the patients) with recoveries below 60% (and as low as less than 10%) there was a strong correlation with CFS Ability, as shown in Figure 2C of our paper [2]. We attribute these low recovery values to an inability of the mitochondria to reliably reconvert ADP to ATP. In contrast, Vermeulen et al [1] conclude that mitochondrial ATP production shows no defect.

In order to make a comparison of the two studies we will have to ignore the fact that in our study the values of the Ox Phos parameter for the patients definitely fall into two groups, and we just take the averages. However we can do this for each value of CFS Ability. Vermeulen et al [1] made 4 measurements of ATP synthesis and 1 of CK in plasma for CPET1 and CPET2 and for patients and for controls. The only convenient way to compare the two studies is to compare ratios of the mean value of each parameter for patients with the corresponding mean for the controls, Patients/Controls. Because we are just comparing mean values we can use the standard error of the mean (SEM) for each mean value rather than the larger standard deviation (SD).

The Ox Phos ratio for the moderate patients (CFS Ability = 4 to 7) is 0.851 +/- 0.065 (SEM, n=21) while there is a strong decrease for the more severely ill patients (0.62 +/- 0.09, 0.62 +/- 0.12 and 0.21 +/- 0.25 respectively for CFS Ability = 3, 2 and 1). This shows that mitochondrial ADP-ATP recycling does occur in mitochondria of neutrophils and is strongly correlated with CFS illness severity.

The 8 Complex I and II ATP synthesis ratios, Patients/Controls, computed from the data of Table 4 [1], are all less than unity but the errors are such that they are also consistent with no decrease from unity. The weighted average ATP synthesis ratio is 0.905 +/- 0.062 which is consistent with the above Ox Phos ratio of the moderate group of patients. Thus the ATP synthesis data [1] cannot support the statement that mitochondrial ATP production shows no defect.

ADP - ATP translocator (TL) study
The third part of our ATP profile test explored the functionality of the translocator protein (TL or ANT for adenine nucleotide translocator), the electrogenic antiport which plays the essential role of transporting ADP into mitochondria for recycling. It also transports the ATP synthesized by recycling back into the cytosol where it is used.

Neutrophils contain fewer mitochondria than PBMCs but there are sufficient for study purposes. We separated mitochondria from neutrophils and made 3 aliquots. The first was used to measure ATP within the mitochondria. The second aliquot was provided with excess ADP and the third aliquot deprived of ADP. The exact analysis conditions were decided experimentally and designed to maximize the production of mitochondrial ATP from ADP (parameter TL OUT), and the provision of ATP to the external artificial cytoplasm (parameter TL IN). In this way we explored the efficiency of ADP-ATP translocator sites in the mitochondrial inner membrane.

In our control group the mitochondrial ATP increase was greater than 35% in the TL OUT part of the test with excess ADP present, while 60% of the CFS patients failed to exceed this minimum. In the TL IN part of the test where ADP access was restricted, the mitochondrial ATP decrease was between 50 and 75% for the control group. For 30/61 (or 50%) of the patients the decrease in the mitochondrial ATP was less than for the controls indicating a blocking of ATP transfer to the artificial cytosol. Both TL parameters show strong correlation with CFS Ability, and their product (TL OUT) x (TL IN) gives r = 0.683. One of us (JMH) has proceeded with further studies of TL function and has found chemical blocking in such cases. The results of molecular level fluorescence microscopy and the identification of the blocking agents by Micro Raman Spectroscopy and Fourier Transform Infrared Spectroscopy will be the subject of a further paper.

Conclusions
Our study [2] compared ATP-related parameters in CFS patients and in a similar number of controls. In retrospect, we can see that this work using neutrophils should have been paralleled by similar studies using PBMCs and we have already begun that task. In the interim, we commend the results of our paper as a significant contribution to understanding the energy depletion in CFS patients. The laboratory findings closely paralleled the functional ability of the patients. This in no way belittles the contribution made by Vermeulen et al [1] who conclude that transport capacity of oxygen is limited in CFS patients. We wish to see both of these avenues of investigation explored more fully.

REFERENCES
1. Vermeulen RCW, Kurk RM, Visser FC, Sluiter W, Scholte HR: Patients with chronic fatigue syndrome performed worse than controls in a controlled repeated exercise study despite a normal oxidative phosphorylation capacity. J Transl Med 2010, 8:93.
2. Myhill S, Booth NE, McLaren-Howard J: Chronic fatigue syndrome and mitochondrial dysfunction. Int J Clin Exp Med 2009, 2:1-16.
3. Bell DS: The Doctor's Guide to Chronic Fatigue Syndrome. New York: Da Capo Press; 1994.
4. Maianski NA, Geissler J, Srinivasula SM, Alnemri ES, Roos D, Kuijpers TW: Functional characterization of mitochondria in neutrophils: a role restricted to apoptosis. Cell Death and Differentiation 2004, 11:143-153.
5. van Raam BJ, Sluiter W, de Wit E, Roos D, Verhoeven AJ, Kuijpers TW: Mitochondrial Membrane Potential in Human Neutrophils Is Maintained by Complex III Activity in the Absence of Supercomplex Organisation.
PLoS ONE 2008, 3:e2013.
6. Fukuda K, Straus SE, Hickie I, Sharpe MC, Dobbins JG, Komaroff A: The chronic fatigue syndrome: a comprehensive approach to its definition and study. Ann Intern Med 1994, 121:953-959.

Additional information on the collaborative groups involved in the Taskforce

Lisa Butterfield — 2009-02-25T17:05:17Z

The iSBTc has brought together experts from multiple stakeholders who have been focusing significant effort in this area including the United States Food and Drug Administration (FDA); the National Cancer Institute (NCI); the National Institutes of Health (NIH); members from industry; and partnering organizations such as the Biotherapy Development Association (BDA), Association for Immunotherapy of Cancer (CIMT), Cancer Vaccine Consortium (CVC), Nordic Center for Development of Antitumour Vaccines (NCV-network), the Italian Network for Tumor Biotherapy (NIBIT) as well as academic experts in the field of immunologic biomarker development and standardization. The planned October 2009 workshop is unique in that it is meant to be an interactive exchange rather than merely a didactic exercise, as it is organized around lively discussion and sharing of data on biomarker development. Ultimately, the workshop will define the “state of the art” in assay and cellular product development and standardization.

"iSBTc-FDA-NCI Workshop on Prognostic and Predictive Immunologic Biomarkers in Cancer", to take place October 28, 2009, Washington, D.C.
Website: www.isbtc.org

Further development of ADRS for TSE investigation

George Robin Barclay — 2007-11-26T13:02:56Z

In the competing interests section of this paper we state that "Radar World Ltd participated on a voluntary basis out of scientific curiosity and is not currently developing a commercial interest in this field". For clarification, this does not mean that ADROK Ltd (formerly Radar World Ltd) have no further interest in this - far from it. They would be very interested in developing this technology further with suitable collaborators, and would invite an approach by anyone who might be interested in developing this for clinical (and/or veterinary) application.

HIFU and potential immune response in cancer therapy

George Suarez — 2007-11-05T12:43:01Z

We have been using HIFU in combination with autologous patient's Dendrite cell therapy for advanced prostate cancer with excellent outcomes. There is no doubt that the combination therapy holds great promise for advanced stage desease in prostate cancer, as well as potential other types of cancer. For more information visit www.internationalhifucenters.com

Thank you for choosing an Open Access Journal

Graham Steel — 2007-09-16T16:55:46Z

I wish to publicly thank the Authors of this important Paper for choosing to publish this in an Open Access Journal.

By making such Papers freely and widely available, I firmly believe will vastly speed up science/research in this regard.

The speed in which this Paper was submitted, Peer Reviewed and published on the internet is a far cry from traditional business models.

Moreover, with this Paper being immediately archived in PubMed and PubMed Central, this is extremely important and the way forward.

I have already alerted many colleagues to this Paper. Without Open Access, I would simply not have been able to do so !!

I sincerely hope that more and more Authors like these ones publish via Open Access and ensure that their work is deposited in an Institutional Repository.

Kind regards,

Graham

Graham Steel

Co-Founder,CJD Alliance

Our findings conclude that immune suppression is not needed for a successful transplant of cord blood

Norman Ende — 2007-05-02T13:08:52Z

In essence, we strongly agree with Dr. Thomas Ichim’s January 30, 2007 review article “Cord Blood in regenerative medicine: do we need immune suppression?”. Since 2000, our laboratory has produced positive clinical responses after administering unaltered cord blood mononuclear cells to animal models including Amyotrophic Lateral Sclerosis 1, 2, Huntington’s Disease3, Diabetes I and II4-6, Alzheimer’s 7and Parkinson’s8, Lupus9, Prostate Cancer12, and Atherosclerosis13 all without the use of immunosuppressant.

In our initial research, outlined in our 2000 ALS paper2, we used radiation, but found immunosuppressant was unnecessary10. All the animal models of human diseases, treated only with cord blood mononuclear cells, without immunosuppressant, have since had appropriate clinical improvement. Only one of several hundred HUCB treated animals has developed evidence of GVHD.

The first cord blood transplantation, nearly 40 years ago on a leukemia patient, administered multiple units and only minimal immunosuppressant for that time11. We believe that cord blood has some stem cells that are so immature they are not recognized by the host as foreign, yet are totipotent 9,10 .

Sincerely,

Norman Ende, MD

Reference:

1. Chen R, Ende N. The potential for the use of mononuclear cells from human umbilical cord blood in the treatment of amyotrophic lateral sclerosis in SOD1 mice. J Med 2000;31(1-2):21-30.

2. Ende N, Weinstein F, Chen R, Ende M. Human umbilical cord blood effect on sod mice (amyotrophic lateral sclerosis). Life Sci 2000;67(1):53-9.

Abstract

Ende N, Chen R, Weinstein F, Bagtas-Ricafort L, Ende M: Human umbilical cord blood (HUCB) and effect on sod mice (amyotrophic lateral sclerosis) Modern Pathology, Vol.13, 2000.

3. Ende N, Chen R. Human umbilical cord blood cells ameliorate Huntington's disease in transgenic mice. J Med 2001;32(3-4):231-40.

Abstract

Ende N, Chen R. The effect of a megadose of human umbilical cord blood mononuclear cells on Huntington disease mice. Am. J Clin Path 114 (4), 2000.

4. Ende N, Chen R, Mack R. NOD/LtJ type I diabetes in mice and the effect of stem cells (Berashis) derived from human umbilical cord blood. J Med 2002;33(1-4):181-7.

Abstract

Ende N, Chen R. Human umbilical cord blood mononuclear cells and mice with type I diabetes. Am. Jr. of Clin Path. #37:629, 2002.

5. Ende N, Chen R, Reddi AS. Effect of human umbilical cord blood cells on glycemia and insulitis in type 1 diabetic mice. Biochem Biophys Res Commun 2004;325(3):665-9.

6. Ende N, Chen R, Reddi AS. Transplantation of human umbilical cord blood cells improves glycemia and glomerular hypertrophy in type 2 diabetic mice. Biochem Biophys Res Commun 2004;321(1):168-71.

Abstract

Ende, N., Chen R., Reddi A.S. Transplantation of Umbilical Cord Blood Mononuclear Cells on Mouse Animal Model B6.V-Lepob Type 2 Diabetes. Am J Cln Patho, 2004; 112:625-661.

7. Ende N, Chen R, Ende-Harris D. Human umbilical cord blood cells ameliorate Alzheimer's disease in transgenic mice. J Med 2001;32(3-4):241-7.

Abstract

Ende N, Chen R, Ende-Harris D: The effect of megadose of human umbilical cord blood mononuclear cells on Alzheimer’s disease mice. Mod Path, 14(1):207A, 2001.

8. Ende N, Chen R. Parkinson's disease mice and human umbilical cord blood. J Med 2002;33(1-4):173-80.

Abstract

Ende N, Chen R. Effect of human umbilical cord blood on mice with Parkinson's disease. Am Jr. of Clin. Path. Vol 36:629, 2002.

9. Ende N, Czarneski J, Raveche E. Effect of human cord blood transfer on survival and disease activity in MRL-lpr/lpr mice. Clin Immunol Immunopathol 1995;75(2):190-5.

Abstract

Czarneski J, Ende N, Smith I and Raveche ES: The hematopoietic effects of human cord blood and irradiation in MRL-lpr/lpr mice. Acta Haematologica, 10th Symposium on Molecular Bio. Of Hematopoiesis and Treatment of Leukemias and Lymphomas 98. Supplement 1 (122), 1997.

10. Ende N. The Berashis cell: a review--is it similar to the embryonic stem cell? J Med 2000;31(3-4):113-30.

11. Ende M, Ende N. Hematopoietic transplantation by means of fetal (cord) blood. A new method. Va Med Mon (1918) 1972;99(3):276-80.

12. Ende N, Chen R, Reddi AS. Administration of human umbilical cord blood cells delays the onset of prostate cancer and increases the lifespan of the TRAMP mouse. Cancer Lett 2006;231(1):123-8.

Abstract

Ende N., and Chen R. “Umbilical cord blood mononuclear cells effect prostate cancer in TRAMP mice” Am J Cln Patho; 112:625-661, 2004

Abstract

13. Ende N, Ende M., Chen R., Coakley K., Reddi A. Human Umbilical Cord Blood Cell Administration’s Reduction of Atherosclerotic Plaques in the Ldlr tm1Her Mouse without Immunospuression. Arch Pathol Lab Med— Vol 130, September 2006.

Lyme Disease and co infections/CFS/antibiotics

kim marott — 2006-09-19T19:10:58Z

Our daughter presented at 14 with severe headaches, joint pain, muscle and tendon pain,severe fatigue, mental disturbances, depression for 5 years. Was diagnosed with CFS and Fibromyalgia with no relief...eventually diagnosed with bipolar disorder. After The Western Blot blood test given at age 19 she showed accutely positive for Lyme Disease with Bartonella and Mycoplasma bacterial co-infections...One of the antibiotics that is recommended is Azithromycin. COULD CFS BE CAUSED BY THE LYME SPIROCHETTE AND NO ONE IS TESTING FOR IT?

Modeling Niche construction behaviour in Ovarian cancer

Peter R Main — 2006-01-02T08:28:07Z

Wang et al provide an extensive and logical review of inflammatory and thrombotic controls possibly operant via angiogensis and other systems, evident in ovarian cancer and its peritoneal response.

Such a system has features of "niche construction" in complex adaptive systems (CAS) useful in ecosystems modeling. In such systems there appears a major problem of dealing with dynamic adaptive diversity (ie rapid mutation that alters the crosstalk being used by the cancer to control host cell lines). Adaptation of tumour to therapeutic challenges is a well established mode of failure in therapies.

This diversity of a CAS subject to control intervention, needs to be formally considered, when planning the early clinical extension of their work to interventions of thrombotic or inflammatory pathways.

Wang et al's report does not consider diversity as central to their topic, yet diversity is central to both the underlying inflammatory /thrombotic / angiogenic systems, and their modulation by tumour. Diversity of these systems may be central to future clinical failure to translate theory to therapy -we do not yet know.

A significant further sampling of the 402 genes differentially expressed in malign vs benign peritoneum, and 663 genes differentially expressed in malign vs benign stroma, may reflect expression diversity, of immediate relevance to planning clinical interventions.

Finally, In my view, for a progressive epublication paradigm, I suggest a minimum additional requirement for such published analysis, is the online deposition of the genetic data background to their report. That at least would enable future cross-checking of relevant involved genes in meta-analysis of new reports with past reports.

Fragmentation of isolated bits of data, across time, geography and language, is surely a shamefully obsolete practice belonging to horse and buggy days, not modern worldwide BMC epublication.