Patent Application: US-201213621702-A

Abstract:
disclosed are tests , correlations and “ theranostic ” interventions aimed at optimizing general health through quantification of cells with regenerative potential in circulation . some embodiments include use of circulating endothelial progenitor cell testing as a means of quantifying general health status in a patient . the combination of epc testing together with a naturopathic intervention , wherein the selection and dosage of said naturopathic intervention is tailored based on said quantitative test is provided . in other embodiments , regenerative cells are selected from the group comprising of very small embryonic like cells , cd34 cells with hematopoietic potential , and circulating mesenchymal stem cells . naturopathic interventions include recommendations of lifestyle modification , dietary supplements , intravenous vitamins , detoxification , acupuncture , and guided imagery .

Description:
embodiments of the present invention are described below . it is , however , expressly noted that the present invention is not limited to these embodiments , but rather the intention is that modifications that are apparent to the person skilled in the art and equivalents thereof are also included . the endothelium is developed embryologically from the dual hematopoietic and endothelial progenitor , the hemangioblast [ 1 ]. it is believed that circulating endothelial progenitor cells ( epc ) are in some ways related to this cell , being an “ adult version ” of it . four decades ago a study supported the possibility of this “ adult hemangioblast ,” whose role is to replenish old / injured endothelium . specifically , it was found that endothelial - like cells , that are non - thrombogenic and morphologically similar to endothelium are found on a plastic graft that was tethered to the thoracic artery of a pig [ 2 ]. more recent studies have performed molecular characterization of epc . specifically , a 1997 paper by asahara et al . described epc as bone marrow derived vegr - 2 positive , cd34 positive monocyte - like cells , having ability to differentiate into endothelial cells in vitro and in vivo based on expression of cd31 , enos , and e - selectin [ 3 ]. one of the first examinations of circulating epc was performed in the hindlimb ischemia model in mice and rabbit models . it was shown that there was an increase in circulation of epc in response to ischemic insult [ 4 ]. furthermore , these studies demonstrated that cytokine - induced augmentation of epc mobilization elicited a therapeutic angiogenic response . using irradiated chimeric systems , it was demonstrated that the ischemia - mobilized epc derive from the bone marrow , and that these cells participate both in sprouting of pre - existing blood vessels as well as the initiation of de novo blood vessel production [ 5 ]. subsequent to the initial phenotypic characterization by asahara et al [ 3 ], more detailed descriptions of the human epc were reported . for example , cd34 cells expressing the markers , vegf - receptor 2 , cd133 , and cxcr - 4 receptor , with migrational ability to vegf and sdf - 1 has been a more refined epc definition [ 6 ]. however there is still some controversy as to the precise phenotype of the epc , since the term implies only ability to differentiate into endothelium . specifically , both cd34 +, vegfr2 +, cd133 +, as well as cd34 +, vegfr2 +, cd133 − have been reported to act as epc [ 7 ]. more recent studies suggest that the subpopulation lacking cd133 and cd45 are phenotypically immature , precursor epc [ 8 ]. other phenotypes have been ascribed to cells with epc activity , one study demonstrated monocyte - like cells that express cd14 , mac - 1 and the dendritic cell marker cd11c have epc activity based on uptake of acetylated ldl and binding to the ulex - lectin [ 9 , 10 ]. evidence supporting a role for epc in vascular endothelial turnover came from studies in the apolipoprotein e knockout ( apoe ko ) mouse , which are genetically predisposed to development of atherosclerosis due to inability to impair catabolism of triglyceride - rich lipoproteins . when these mice are lethally irradiated and reconstituted with labeled bone marrow stem cells , it was found that areas of the vasculature with high endothelial turnover , which were the areas of elevated levels of sheer stress , had incorporated the majority of new endothelial cells derived from the bone marrow epc [ 11 ]. the possibility that endogenous bone marrow derived epc possess such a regenerative function was also tested in a therapeutic setting . atherosclerosis is believed to initiate from endothelial injury with a proliferative neointimal response that leads to formation of plaques . when bone marrow derived epc are administered subsequent to wire injury , a substantial reduction in neointima formation was observed [ 12 ]. the argument can obviously made that wire injury of an artery does not resemble the physiological conditions associated with plaque development . to address this , wassmann et al [ 13 ], used apoe ko mice that were fed a high cholesterol diet and observed reduction in endothelial function as assessed by the flow mediated dilation assay . when epc were administered from wild - type mice restoration of endothelial responsiveness was observed . epc have also been demonstrated to possess an anti - aging function in a series of experiments in which 3 month old syngeneic cardiac grafts were heterotopically implanted into 18 month old recipients . loss of graft viability , associated with poor neovascularization , was observed subsequent to transplanting , as well as subsequent to administration of 18 month old bone marrow mononuclear cells . in contrast , when 3 month old bone marrow mononuclear cells were implanted , grafts survived . antibody depletion experiments demonstrated bone marrow derived pdgf - bb was essential in integration of the young heart cells with the old recipient vasculature [ 14 ]. these experiments suggest that young epc or epc - like cells have ability to integrate and interact with older vasculature . in one aspect of the present technology , epc are quantified as a general measurement of health , and specifically , to assess the response to naturopathic intervention . for example , the food supplement citicholine is used by many naturopaths to “ enhance thinking clarity .” citicoline is a chemical that is found naturally in the body that acts as an intermediate in the generation of phosphatidylcholine from choline . it is sold as a food supplement in the usa . a recent study ( sobrino et al . cdp - choline treatment increases circulating endothelial progenitor cells in acute ischemic stroke . neurol res . 2011 july ; 33 ( 6 ): 572 - 7 ) demonstrated that this food supplement appears to increase numbers of circulating epc . forty eight stroke patients were randomized into treatment ( 26 patients ) and placebo ( 22 patients ). the treatment consisted of daily administration of 2000 mg of citicoline per day for 6 weeks after the stroke . a statistically significant increase in epc was noted at day 7 after treatment with citicoline . patients who received clotbuster drugs together with citicoline also had higher epc numbers . given that citicoline appears to have some therapeutic effects in stroke , traumatic brain injury and cognitive impairment ( as reviewed in secades j j , citicoline : pharmacological and clinical review , 2010 update . rev neurol 2011 mar . 14 ; 52 suppl 2 : s1 - s62 ), it may be possible that some of the effects mediated by this food supplement are associated with its ability to increase circulating epc . one embodiment of the current present technology is to provide epc numbers in patients taking supplements such as citicoline , correlate its epc augmenting effect in specific patients , and determine whether other interventions are needed to obtain an “ optimized ” epc number in circulation . in some embodiments , an optimized epc number can be in reference to levels determined across a general population of patients , or within a specific subpopulation . in some embodiments , optimized epc numbers can vary for an individual patient . for example , where a patent has been previously tested for circulating epc levels , the optimized epc number for that patient can be a threshold known to correlate with improved or improving health for that specific patient . this optimized number may also be correlated with other parameters within the scope of the present technology . for example , inflammatory markers may be assessed in combination . detection of circulating epc can be important in response to therapies such as hyperbaric oxygen , which is commonly used by naturopathic physicians . thom et al [ 15 ] examined diabetic patients who underwent therapy for hyperbaric oxygen . they reported more than a twofold elevation ( p = 0 . 004 ) in circulating stem cells after treatments . interestingly the circulating stem cells , which were quantified based on expression of cd34 , contained two - to threefold higher levels of hypoxia inducible factors - 1 , - 2 , and - 3 , as well as thioredoxin - 1 ( p & lt ; 0 . 003 ), than cells present in blood before hyperbaric oxygen treatment or therapy ( hbo ( 2 ) t ). they further found that nitric oxide synthase activity is acutely increased in patients &# 39 ; platelets following hbo ( 2 ) t and remains elevated for at least 20 hours . mechanistically they proposed hyperbaric oxygen increases circulating stem cells through stimulation of nitric oxide production [ 16 ]. without being bound to theory , it appears that the intervention of hyperbaric oxygen treatment increases stem cells in circulation [ 17 - 20 ], and others have found that increased stem cells in circulation correlate with better prognosis for a variety of indications . however , to our knowledge , the personalized use of circulating regenerative cell testing as a guide for administration of naturopathic interventions has not been practiced . numerous strategies that are used in the context of naturopathic medicine seem to mediate their activities through the inhibition of inflammation . previously it has been shown that inhibitors of inflammation increase epc numbers . various agents are known to decrease inflammation , these include tnf blockers such as remicade [ 21 ], consumption of various dietary supplements [ 22 , 23 ], caloric restriction [ 24 ], exercise [ 25 , 26 ], eating blueberries [ 27 ], green tea [ 28 ], or statin therapy [ 29 ]. the drug crestor has been shown to increase circulating epc levels in vivo [ 30 ], in part through reduction of detrimental effects of asymmetric dimethylarginine on epc [ 31 ]. granulocyte colony stimulating factor ( g - csf ) has been used clinically for mobilization of hematopoietic stem cells ( hsc ) for more than a decade during donor stem cell harvesting . mechanistically g - csf is believed to induce a mmp - dependent alteration of the sdf - 1 gradient in the bone marrow [ 32 , 33 ], as well as function through a complement - dependent remodeling of the bone marrow extracellular matrix [ 34 , 35 ]. it was found that in addition to mobilizing hsc , g - csf stimulates mobilization of epc as well , through mechanisms that are believed to be related [ 7 , 36 ]. several studies have been performed in which g - csf was administered subsequent to infarct . although it is impossible to state whether the mobilization of hsc or epc accounted for the beneficial effects , we will overview some of these studies . the front - integrated revascularization and stem cell liberation in evolving acute myocardial infarction by granulocyte colony - stimulating factor ( firstline - ami ) trial evaluated 30 patients with st - elevation myocardial infarction treated with control or g - csf after successful revascularization [ 37 ]. fifteen patients received 6 days of g - csf at 10 μg / kg body weight , whereas the other 15 received standard care only . four months after the infarct , the group that received g - csf possessed a thicker myocardial wall at the area of infarct , as compared to controls . this was sustained over a year . statistically significant improvements in ejection fraction , as well as inhibition of pathological remodeling was observed in comparison to controls . a larger subsequent study with 114 patients , 56 treated and 58 control demonstrated “ no influence on infarct size , left ventricular function , or coronary restenosis ” [ 38 ]. there may be a variety of reasons to explain the discrepancy between the trials . one most obvious one is that the mobilization was conducted immediately after the heart attack , whereas it may be more beneficial to time the mobilization with the timing of the chemotactic gradient released by the injured myocardium . this has been used to explain discrepancies between similar regenerative medicine trials [ 39 ]. supporting this possibility is a study in which altered dosing was used for the successful improvement in angina [ 40 ]. furthermore , a recent study last year demonstrated that in 41 patients with large anterior wall ami an improvement in lef ventricle ejection fraction ( lvef ) and diminished pathological remodeling was observed [ 41 ]. there is an indication that post - infarct mobilization can have a therapeutic role . other clinically - applicable mobilizers may be evaluated and used in the methods described herein . for example , growth hormone , which is used in “ antiaging medicine ” has been demonstrated to improve endothelial responsiveness in healthy volunteers [ 42 ], and patients with congestive heart failure [ 43 ], this appears to be mediated through mobilization of endothelial progenitor cells [ 44 , 45 ]. thus in one embodiment of the present technology , the “ optimizing ” of epc levels for a particular health situation is performed by being able to monitor epc levels and utilizing various interventions as needed . for example , interventions can include continuing with a course of treatment , discontinuing a course of treatment , increasing or decreasing the frequency or dosage levels of one or more treatments . in certain embodiments , the intervention can be a naturopathic intervention . in some embodiments , the naturopathic interventions is selected from the group consisting of : recommendations of lifestyle modification , dietary supplements , intravenous vitamins , detoxification , acupuncture , and guided imagery . having generally described this technology , a further understanding can be obtained by reference to certain specific examples which are provided herein for purposes of illustration only , and are not intended to be limiting . within the context of the present technology , other cells besides epc may be used as circulating regenerative cells . in one embodiment of the present technology , quantification of circulating very small embryonic like cells ( vsel ) is performed as a marker of general health . specifically , edta - anticoagulated peripheral samples ( 2 × 2 . 7 ml ) are drawn from patients . the absolute numbers of leukocytes and lymphocytes in said peripheral blood are determined at the same time with an automatic cell counter , for example , using the cell - dyn 3500 , abbott diagnostics , santa clara , calif .). in order to purify peripheral blood mononuclear cells ( pbmc ), lysis of red blood cells is performed using a hypotonic lysis buffer , for example , the bd pharm lyse buffer ( bd biosciences pharmingen , san diego , calif .). the pbmc are subsequently used for flow cytometric analysis to determine vsel content . typically , care can be taken to reproducibly utilize the same reagent batches for consistency of testing . the expression of cd34 , cd133 , and cxcr4 on human pbmncs is evaluated by flow cytometry . the staining and analyses of protein marker expression are performed by staining in phosphate - buffered saline ( pbs ; ca2 +- and mg2 +- free ) supplemented with 5 % bovine calf serum ( hyclone , logan , utah ). the following monoclonal antibodies directly conjugated with phycoerythrin ( pe ) or allophycocyanin ( apc ) may be used for quantification : pe - anti - cd34 ( bd biosciences pharmingen ); apc - anti - cxcr4 ( bd biosciences pharmingen ); and apc - anti - cd133 ( miltenyi biotec , auburn , calif .). to determine the proportion of cd34 +/ cd133 +, cxcr4 +/ cd34 +, and cxcr4 −/ cd34 + cells , a dual - color flow cytometric analysis is performed . however , for this to occur , it can be helpful to utilize an appropriate isotype control ( bd biosciences pharmingen ). for staining , pbmncs 10 6 are mixed with antibody stained , and after a 20 - minute incubation on ice , cells were washed twice in pbs . thereafter , the cell pellet is resuspended in 0 . 3 ml pbs and analyzed by facsaria ( bd biosciences , san jose , calif .) and cell quest software ( bd biosciences ). typically , 50 , 000 events are acquired to determine the percentage of the examined subpopulation within the pbmc population . in other experiments , a single - cell suspension is stained for lineage markers ( cd56 , cd235a , cd3 , cd66b , cd24 , cd19 , cd14 , cd16 , cd2 ) conjugated with fluorescein isothiocyanate , cd45 conjugated with pe , and cxcr4 conjugated with apc for 30 minutes on ice . after being washed , cells are analyzed by fluorescence - activated cell sorting ( bd biosciences ). at least 10 5 events were acquired and analyzed by cell quest software ( bd biosciences ). in one embodiment , vsel are quantified as lin − cd45 − cxcr4 + cells of a small ( less than 7 microns ) size . while others have demonstrated that vsel cells increase in circulation after heart attack [ 46 ], and stroke [ 47 ], the surprising discovery of using these cells as a general marker of health represents a key advancement in the field . the above description discloses several methods and systems of the present invention . this invention is susceptible to modifications in the methods and materials , as well as alterations in the fabrication methods and equipment . such modifications will become apparent to those skilled in the art from a consideration of this disclosure or practice of the invention disclosed herein . consequently , it is not intended that this invention be limited to the specific embodiments disclosed herein , but that it cover all modifications and alternatives coming within the true scope and spirit of the invention . all references cited herein including , but not limited to , published and unpublished applications , patents , and literature references , are incorporated herein by reference in their entirety and are hereby made a part of this specification . to the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification , the specification is intended to supersede and / or take precedence over any such contradictory material . the term “ comprising ” as used herein is synonymous with “ including ,” “ containing ,” or “ characterized by ,” and is inclusive or open - ended and does not exclude additional , unrecited elements or method steps . the following references have been referred to in the text . the entire content of each reference is incorporated herein . without limitation all of the cells , proteins , markers , assays , and methods can be used with the technology described herein and / or can be combined with the methods and materials described herein . 1 . basak , g . w ., et al ., human embryonic stem cells hemangioblast express hla - antigens . j transl med , 2009 . 7 : p . 27 . 2 . stump , m . m ., et al ., endothelium grown from circulating blood on isolated intravascular dacron hub . am j pathol , 1963 . 43 : p . 361 - 7 . 3 . asahara , t ., et al ., isolation of putative progenitor endothelial cells for angiogenesis . science , 1997 . 275 ( 5302 ): p . 964 - 7 . 4 . takahashi , t ., et al ., ischemia - and cytokine - induced mobilization of bone marrow - derived endothelial progenitor cells for neovascularization . nat med , 1999 . 5 ( 4 ): p . 434 - 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