1

Journal of Medical & Biological Sciences Volume 2, Issue 1, 2008 Bioelectromagnetic Energy Fields Accelerate Wound Healing and Activate Immune Cell Function Lisanne D’Andrea-Winslow, Professor, Northwestern College, ldwinslow@nwc.edu Don F. Johnson, Professsor, Northwestern College, dfjohnson@nwc.edu Amy K. Novitski, Research Associate, Northwestern College, aknovitski@nwc.edu Abstract Bioelectromagnetic energy treatment of dermal wounds in the sea urchin Lytechinus variegates showed a 75.7% rate of wound closure in treatment groups compared to 41.9% in controls (t = 3.603 (df=18), p<.01). Cohen’s d yielded a strong effect on size, d = 1.14. SDS-PAGE revealed an increase in 101.8, 63 and 35kDa polypeptides and attenuation in 56, 43 and 32 kDa polypeptides from wounds in treated animals compared to controls. Immune cell activation in treatment groups showed a 59.7% increase over controls. Taken together, the data indicate that biologically generated electromagnetic energy enhances and accelerates an invertebrate wound healing pathway. Introduction The invertebrate immune system found in echinoderms provides a simple, elegant system in which to study immune cell function in the wound healing pathway. The immune system of the sea urchin is comprised of effector cells called coelomocytes which reside in the body cavity (coelom) of the sea urchin and are believed to travel freely into and out of the water vascular system of the animal conferring innate immunity (Coffaro and Hinegardner, 1977; Endean and Boolootian, 1966). Four unique types of coelomocytes have been described in the literature; the red spherule, colorless spherule, petalloid and vibritile coelomocytes, which, in concert, confer a complex, innate immunity (D'Andrea-Winslow et al., 2001; D'Andrea et al., 1994; Edds, 1980; Smith and Davidson, 1992). Investigations of coelomocyte function during wound healing in sea urchins has revealed a complex pathway of tissue regeneration possibly involving activation by immune cytokines such as interleukin (Smith and Davidson, 1992). The role of these primitive immune cells may be quite complex since allograft experiments revealed tissue rejection and even long-term memory, immune functions previously thought not to occur in invertebrate animals (Coffaro and Hinegardner, 1977). Preliminary studies generated from our laboratory have revealed at least the preliminary steps in the pathway of invertebrate tissue regeneration. We have shown that after receiving an external dermal-osseous wound, red spherule granulocytes along with colorless spherule coelomocytes appear to actively migrate into surface wounds of Lytechinus variegatus indicating their role in the wound healing pathway (Schott et al., 2005; Sturycz and D'Andrea-Winslow, 2005). In order for wound closure to occur, CaCO3 spicules are secreted by coelomocytes which become part of the bony framework of the skeletal test (calcium carbonate endoskeleton). Coelomocytes may also secrete important immunochemicals and cytokines at the site of the wound to activate the formation of dermal epithelia for wound closure. Dermal epithelia are stimulated into active repair forming the initial eschar (scab) which heals completely with little or no trace of the initial wound. In the present study of the wound healing pathway in sea urchins, we investigated the effects of bioelectromagnetic energy on wound closure and immune coelomocyte activation. There has been a growing interest in scientific studies investigating the effects of biologically derived electromagnetic energy on biological tissue (Warber et al., 2003). In the literature, these

2

anomalous phenomena have been referred in a variety of ways including, “therapeutic touch,” “healing with intent,” “the laying on of hands,” “electromagnetic energy” just to name a few (Bengston and Krinsley, 2000; Bunnell, 1999; O'Laoire, 1997; Wirth, 1990). Frequently, these terms are used interchangeably even though actual techniques of application might vary among them. One common thread that runs through much of the theoretical considerations of these anomalous phenomena is the idea that electromagnetic energy emitted from the human body interacts with biological tissue at the molecular level, particularly the immune cells, effectuating accelerated or enhanced healing. For example, experimental evidence has shown that the electromagnetic fields generated by humans may be used in a similar manner to the artificially generated pulsed electromagnetic fields (PEMFs) used clinically for stimulating or enhancing a healing response. Bioelectromagnetic fields (BEMFs) were analyzed in a study by Zimmerman (Zimmerman, 1990) where the BEMF emission from the hands of therapeutic touch practitioners could be measured (Green et al., 1991; Waechter and Sergio, 2002; Zimmerman, 1990). The application of BEMFs have been shown in numerous animal and human models to enhance and/or accelerate wound healing in bone and soft tissue as well as enhancing the healing process in a variety of other diseases including cancer (Chen et al., 2002; Nindl et al., 2004; Rosch and Markov, 2004). To date, no studies have been conducted on the effects of biological energy treatment on any invertebrate animal species. However, artificially generated magnetic fields have been shown to accelerate events of early sea urchin development (Falugi et al., 1987; Levin and Ernst, 1995). Our current hypothesis, tested in this study, is whether sea urchin immune cells have the capacity to receive signaling information from biological electromagnetic energy. Our studies indicate that wounds from treatment groups receiving treatment healed statistically faster and more completely than wounds from non-treated control groups. Analysis at the cellular level indicated an increased level of immune cell activation in treated isolated immune cells compared to untreated controls. This is a first report indicating that invertebrate tissue is sensitive to bioelectromagnetic energy, perhaps indicating a universal biological phenomenon. The first in the deuterostome ancestral lineage, echinoderms such as sea urchins are thought to have given rise to vertebrates, including humans. An analysis of the effects of BEMFs in sea urchins may provide important phylogenetic information about primitive immune cell functions involved in active wound healing and other immune pathways. Methods and Materials Wounding of L. variegatus and Bioelectromagnetic Energy Treatment Lytechinus variegatus (Gulf Specimens, Inc., FLA) of comparable size were selected and maintained in a 120 gallon salt water aquarium (25oC) until use. For wound healing studies, two separate twenty-five gallon salt water aquaria were used. The two aquaria were kept in separate laboratories, one serving as a control group and the other as an experimental group. Researchers conducting measurements were blinded to which tank was control and experimental to avoid experimenter bias. Ten sea urchins were placed in each aquarium and allowed to equilibrate for five days prior to wounding. All sea urchins received an identical incision on day one of the study. Sea urchins were wounded by making a longitudinal 2cm x 0.5cm incision using a research grade scalpel along a vertical seam on the aboral aspect of the test. The incision extended ~2-3mm into the test but did not penetrate through the test. All sea urchins used in this study received humane care in handling and maintenance. Bioelectromagnetic energy treatment was applied for ten minute intervals by an experienced practitioner trained in energy medicine each day for up to five consecutive days. Briefly, sea urchins were removed from the aquarium and placed on an adjacent laboratory bench. Bioelectromagnetic energy treatment was applied by the trained practitioner using the “hands near” application technique (for additional references on this technique see Bengston and Krinsley, 2000; Bunnell, 1999; Chen et al., 2002) approximately 3-5 inches above the animal. Laboratory room temperature was measured daily at 25oC. Each animal was given equal

3

treatment during the ten minute application. Concurrent control groups, designated the “no treatment group,” were maintained in a separate laboratory and wounded in an identical manner to experimental groups. Briefly, wounded sea urchins were removed from the aquaria by a blinded, untrained assistant who set the sea urchins on an adjacent laboratory bench in a similar manner to experimental groups. The untrained assistant attended the sea urchins by standing approximately 2 feet away from the control animals for ten minutes each day over the course of the experiment. Wound Measurements and Statistical Analyses Wounds on control and experimental animals were measured daily for longitudinal wound length. Individual sea urchins were marked by natural variations in spine coloration to ensure that the same wounds were carefully being followed for closure. Measurements were taken at the same time each day for up to six consecutive days. Researchers were blinded regarding which tank was control and experimental. Data were averaged over ten sea urchins, analyzed and plotted directly as wound length vs time or normalized with reference to total healing and plotted as percent healing vs time. Additional analysis was conducted by taking the wound measurements of each sea urchin on day 5 of the experiment since healing was complete at this time. These data were submitted to statistical analyses to verify significance. Student’s t test analysis was used to compare mean wound length on day 5 of the treatment and control groups. Effect size was calculated using Cohen’s d to determine the extent to which the null hypothesis is false. Histological Analysis of Immune Cells in Wounds Six sea urchins were placed in each aquarium, one experimental and one designated as “no treatment” control. All sea urchins were wounded in an identical fashion and maintained as control vs experimental groups as described above. Experimental groups received ten minutes of bioelectromagnetic energy treatment daily; controls received no treatment as described above, for up to six consecutive days. Cellular material was removed from healing wounds by a blinded experimenter at the same time each day after initial wounding. Briefly, one sea urchin was removed from the control and experimental group (designated as tank 1 or tank 2 to ensure that each experimenter was blinded) each day, eschar tissue was scraped from the wound and prepared for histology (see below). A new sea urchin was used each day to ensure accurate analysis of the progression of wound healing from day one through day six of the healing process. Tissue was gently scraped from the wound and placed into an isotonic anticoagulant buffer (ACG) containing 0.1M Tris pH 8.0, 50mM EGTA, 0.5M NaCl 26mM KCl. Tissue was gently disrupted by repeated pipetting and immediately placed onto clean glass microscope slides. Wet mounts were sealed with nail polish and viewed on a Zeiss AxioMot microscope using a 63x oil emersion lens equipped with DIC optics. Micrographs were taken and analyzed with Zeiss AxioVision software (Carl Zeiss, Germany). Protein Analysis in Wounds of Control and Treated Animals Total protein in wounds from control and treated animals was determined as follows. Five sea urchins in a control aquarium and five sea urchins in an experimental aquarium were wounded as described above and treated with bioelectromagnetic energy for ten minutes daily for three consecutive days. Control animals were kept in separate aquaria and treated as described above. This experiment was typically conducted on day three after wounding since wounds in treatment groups were nearly 100% healed by day five and comparisons of protein content with control animals was not possible. Briefly, after day three, five sea urchins were removed from each aquaria and total tissue in the wound was carefully scraped from either control or experimental wounds into 1ml of an ice cold homogenization buffer containing 20mM HEPES pH 7.4, 75mM

4

KCl, 250mM sucrose, 2mM MgCl2, 1mM EGTA, 1%Tween-20, 8�g/ml aprotinin, 10mM TAME, 10mM benzamidine, 0.4mg/ml soy bean trypsin inhibitor, 0.01% sodium azide. Control and experimental samples were kept unknown to researchers to ensure a blinded experiment. Tissue was vortexed for approximately ten seconds to disrupt membranes and immediately frozen at -80oC. Samples were subsequently thawed and pulse microcentrifuged at 12,000 rpm in a tabletop Eppindorf microcentrifuge to remove nuclei and debris. The resulting supernatant was used either for Bradford protein analysis or SDS-PAGE. Bradford Assay: Total protein concentrations in control and treated sea urchins were analyzed by the method of Bradford (Bradford, 1976). Total protein samples were compared for protein concentration to a bovine serum albumen protein standard curve using commercially available Bradford Protein Dye Reagent (BioRad, Inc) and analyzed on a KODAK Imaging Station (Ithica, NY). SDS-PAGE: SDS polyacrylamide gel electrophoresis was preformed according to the method of Laemelli (Laemmli, 1970) as modified by Matsudaira and Burgess (Matsudaira and Burgess, 1978). Control and experimental protein samples prepared as above and electrophoresed on a 7.5-20% gradient gel using mini slab gels (IDEA Scientific, St. Paul, MN). Gels were stained with coomassie blue, dried, and bands were analyzed for densitometry on a KODAK Imaging Station (Ithica, NY). Protein molecular weights were determined by comparison of relative mobility compared to prestained high molecular weight range and kaleidoscope molecular weight markers (BioRad, Inc). Densitometry analysis of individual bands as well as protein molecular weight determination was accomplished using Kodak Image Station software according to manufacturer’s instructions. Immune Cell Motility Assay Coelomic fluid from one non-gravid, healthy sea urchin was mixed in a 1:1 ratio with ACG buffer (see above) resulting in approximately 80ml of fluid. The coelomic fluid-ACG mixture was divided equally into two separate beakers, one becoming the control and the other the experimental group. The control sample was kept in a separate laboratory while bioelectromagnetic energy treatment was performed on the experimental sample for 20min. Experimenters were blinded regarding which beaker was the control and which was the experimental group. Beakers were thus labeled Beaker 1 and Beaker 2 to ensure blindedness. After 20 minutes, both samples were taken into the laboratory and prepared for microscopy as described above. All microscopy was conducted on a Zeiss Axiomot microscope as described above, using the AutoMeasure cellular motility analysis program within the AxioVision software module. Motility of the red spherule immune cells was measured by observing bleb formations, which is the mode of motility for this type of cell. Time-lapse video recordings were broken down into increments of 0.250 second frames for each bleb to be analyzed. The blebs that are analyzed have to meet several criteria before measurements are taken. The blebs have to form at the periphery of the cell, lie exclusively in the x/y plane, and be in-focus for the entirety of its formation. Once a bleb formation has met these requirements, the point at which it originates at the periphery of the cell is marked using an event marker. A second mark is made where the bleb formation ends by cycling through each frame of the time-lapse video. Once these points are marked, a linear measuring tool is used to determine the distance between the two points using microns as the unit of distance. The distance between the two points are then divided by the time it took for complete bleb formation. The end result is a velocity measurement in microns/sec. The rate of bleb motility from red spherule cells in control and experimental groups was determined and graphed. Results The Process of Wound Healing in L. variegatus The mechanism of wound closure in Lytechinus variegatus follows a distinct pattern of events leading to complete closure and healing of wounds with no apparent scarring (personal observations). This process has not been described in the literature so we would like to describe

5

the stages of active wound repair that we have repeatedly observed under our laboratory conditions. After an incision into, but not penetrating, the sea urchin test the first observation is a recruitment of two specific types of immune coelomocytes into the wound. Red spherule granulocytes and colorless spherule cells appear in the wound typically 24 hours after incision (Fig. 1, Day 1). Microscopic observations indicated the appearance of dumbbell or sickle-shaped CaCO3 rods (Fig. 1) by day 3-4 after wounding that get laid down into the bony matrix of the test. Gross observation of the wound at this stage reveals the change in appearance of the wound from a creamy white color to a tan color (personal observations). The next phase of healing involves multi-layer epithelial sheets forming over the newly formed bony matrix to form the eschar (scab) and fibrous bundles are clearly observable (Fig.1, Day 6). On gross observation there is a noticeable change to a reddish-brown color with obvious overlying tissue. Complete wound closure typically occurs 6-8 days after the initial wounding with little or no trace of the initial incision. On occasion, due to individual variations, individual animals became weak and sickly after wounding and never successfully healed from incisions. Non-healing rates were dependent on seasonal spawning cycles, with less abundant healing when animals were gravid. Therefore, animals for wound healing studies were used between spawning cycles which occur twice annually in November and March.

Figure 1 Progression of wound healing in the sea urchin L. variegatus. Day 1 shows recruitment of numerous red spherule and colorless spherule coelomocytes into the wound. Day 3 shows the beginning of CaCO3 spicule formation (arrows) in the eschar. Day 5 shows the beginning of epithelial sheet formation with enlarged spicules (arrowheads). By day 6 multilayer sheet formation occurs with collagen-like fibers and bundles.

6

Bioelectromagnetic Energy Accelerates Wound Closure Our aim was to determine whether bioelectromagnetic energy treatment could affect the rate of wound closure observed in L. variegatus. Figure 2 shows an experiment where measurements were taken daily for six days. Control groups showed the partial closure of 2cm wound after six days to a final measurement of 1.3cm while treated animals showed wound closure on the same day to be 0.5cm (Fig 2, panel A). This indicates a rate of healing of approximately 41.9% in controls compared to 75.7% healing in treated animals (Fig 2, panel B). Additional analysis was conducted by taking the wound measurements of each sea urchin on day 5 of the experiment. These data were submitted to statistical analyses to verify significance. Student’s t test analysis applied to the data showed statistical significance, t = 3.603 (df=18), p<.01. The mean wound length for the experimental group (M = .515, SD = .342) was significantly less than the mean wound length of the control group (M = 1.18, SD = .435). Cohen’s d yielded a strong effect size, d = 1.14. Gross observation of wounds over the course of the experiment showed a visible difference in wounds from control vs. experimental groups (Fig 3). All wounds were administered in a similar fashion in control and experimental groups. Wounds on control animals showed visible signs of the wound on day six (Fig 3, panel B, arrow). Wounds on animals treated with bioelectromagnetic energy appeared to heal faster with little trace of the wound by day six (Fig 3, panel D, arrow). In some studies, wounds were completely healed by day four and even day three owing to individual variations in immune potency within individual sea urchins. Treated animals consistently showed greater vitality and longevity compared to controls (personal observations).

Figure 2 Analyzed data showing the rate of wound healing in control groups vs. bioelectromagnetic energy treated experimental groups. Panel A shows the decrease in wound length over the duration of bioelectromagnetic energy treatment. Panel B graphically shows the percent healing as 75.7% percent in bioelectromagnetic energy treated animals vs 41.9% in the control group. Legend: squares= control, diamonds= treated.

7

Figure 3 Representative photographs of wounds from control animals on days 1 and 6 (Panels A, B respectively) and treated animals on days 1 and 6 (Panel C, D respectively). Control animals showed wound remnants on day 6 while in treated animals wound closure was nearly complete (see arrows).

Histological Analysis: Accelerated Immune Cell Recruitment into Wounds of Animals Treated with Bioelectromagnetic Energy Our next study involved an analysis of histology during the course of the wound closure process in the presence and absence of bioelectromagnetic energy treatment. L. variegatus sea urchins were wounded in the same manner as above. Control and experimental aquaria were arranged in the same manner as above and treatment administered in a non-biased, blinded manner as described in the previous experiment. In this experiment, tissue from the eschar was carefully excised and observed microscopically. DIC images revealed red spherule and colorless spherule immune coelomocytes in the wounds of the control on day 2 after wounding, but no CaCO3 spicules were observed (Fig 4, Day 2, control panel). Eschar tissue from treated animals revealed extensive red spherule and colorless spherule coelomocytes and a striking abundance of CaCO3 spicules (Fig 4, Day 2 Treated panel). Spicules with both a dumbbell morphology and crescent or sickle-shaped were observed in abundance (Fig 4 see arrows). Attempts were made to conduct cell counts of red spherule coelomocytes and colorless spherule coelomocytes in wounds of control and treated sea urchins. These studies were difficult to quantify due to the sticky nature of the eschar tissue after it was excised from the wound. Eschar tissue from day three after wounding revealed numerous spicule formation in controls. These spicules seemed most abundantly to be in the crescent or sickle morphologically with few in the dumbbell shape (Fig 4, Day 3 control panel, arrowheads). Eschars from treated animals showed no trace of spicules, but did show the formation of unified sheets of epithelium (Fig 4, Day 3 Treated panel). By day five, control animals showed epithelial growth with scattered red and colorless immune cells with no appearance of spicules (Fig 4, Day 5 control). Histology of eschar tissue from

8

treated animals showed multi-layer tissue sheets containing fibrous material, with little or no trace of immune cells (Fig 4, Day 5 Treated panel).

Figure 4 Histological analysis of cell recruitment into the wound in control vs bioelectromagnetic energy treated animals. Left-hand panels represent tissue from controls, right hand panels from treated animals. Day 2: control wounds contain red spherule and colorless spherule coelomocytes while treated animals show abundant CaC03 spicule formation (arrows). Day 3: tissue from wounds on day 3 of the experiment shows the beginning of spicule formation (arrow heads) in controls while treated animals showed multi-layered sheets of tissue overlying spicules. Day 5: tissue from wounds on the fifth day of the experiment shows the beginning of multi-layered cell sheets in controls and intact healed tissue in treated animals. Bar=15µ

Protein Expression Changes in Treated Wounds vs. Controls Tissue from treated and control wounds was scraped, solubilized and prepared for protein analysis using both the Bradford method for protein concentration determination and SDS-PAGE. Data from one representative study showed total cellular protein levels of 3.5mg/ml for controls and treated preparations (Figure 5, panel A). All repeated experiments gave comparable results with protein levels from control wounds at nearly the same or slightly less total protein than bioelectromagnetic energy treated preparations. Total protein from wounds on treated animals vs controls was equivalently loaded onto 7.5-20% polyacrylamide gels and stained with coomassie blue (Fig. 5, panel B). Bands were identified by Kodak Image Station software analyzed for band density and molecular weight. The polypeptide bands that were identified in both control and bioelectromagnetic energy treated groups were 193kDa, 101.8kDa, 92kDa, 70kDa, 63kDa, 56kDa, 43kDa, 35kDa and 32kDa (Fig 5 Panel B; control in lane 2, treated in lane 3). Histogram analysis of relative band densities of these polypeptides revealed an increase in the levels of

9

some bands with a decrease in the levels of others in treated animals when compared to controls (Fig 6). The levels of the 101.8kDa, 63kDa and 35kDa bands showed an increase in bioelectromagnetic energy treated tissue, while the 56kDa, 43kDa and 32kDa bands were attenuated compared to controls. All other bands remained the same (Fig. 6). Figure 5 Analysis of protein content in bioelectromagnetic energy treated wounds (Exp)

vs. controls (Ctl). Panel A shows no detectable difference in total protein content in treated wounds compared with control. Panel B: SDS-PAGE analysis of proteins in treated vs. control wounds. Tissue from control (Lane 2) and treated (Lane 3) wounds were scraped, solubilized and electrophoresed on 7.5-20% polyacrylamide gels. Molecular weight markers in Lane 1 were 205kDa, 116kDa, 95kDa, 45kDa; in Lane 4 were 200kDa, 130kDa, 78kDa, 39kDa and 31kDa.

Figure 6 Histogram analysis of gel in Figure 5. Relative band densities of the 101.8kDa, 63kDa and 35kDa bands showed an increase in bioelectromagnetic energy treated tissue, while the 56kDa, 43kDa and 32kDa bands were attenuated compared to controls. All other bands remained the same. red bars= control, green bars= treated.

10

Isolated Red Spherule Immune Cells Increase Cellular Activity After Bioelectromagnetic Energy Treatment Since we observed an accelerated accumulation of both red spherule and colorless spherule coelomocytes into the wounds of treated animals we examined activation on the cellular level in the presence and absence of treatment. Video data was analyzed for relative motility of the colorless and red spherule coelomocytes. Colorless spherule cell motility consists of a highly erratic, rapid flagellum-driven movement where the cell bobs into and out of the plane of focus (Schott et al., 2005). It was not possible to obtain accurate measurements of colorless spherule cell motility, although modifications in cell motility measurements are being explored in our lab currently. Red spherule cells, on the other hand, exhibit a distinct motility that results from the formation of numerous cellular extensions, called blebs that continuously extend out of the cell proper and retract. In coelomic fluid taken from healthy sea urchins, formation of blebs occurs at an average measured rate of ~0.4µ/sec. This motility can decrease significantly or even stop altogether in stressed, unhealthy or dying animals (personal observations). Thus, motility can be a useful indicator of immune cell vitality and immune cell activation. Figure 7 shows the rate of bleb motility in control samples vs treated samples. Control red spherule cells showed an average rate of bleb formation motility to be 0.46 µ /sec compared with 0.77 µ /sec in bioelectromagnetic energy treated cells. This represents a 59.7% increase in the rate of cellular motility after treatment with the bioelectromagnetic energy technique.

Figure 7 Rate of active bleb formation in red spherule coelomocytes. The rate of active bleb formation was observed to be 0.46 µ /sec in controls compared with 0.77 µ /sec in bioelectromagnetic energy treated cells.

11

Discussion Recent experiments investigating the immune system in sea urchins have revealed a highly complex innate immune system capable of great diversity in fighting infection (Matsudaira and Burgess, 1978; Nair et al., 2005; Smith and Davidson, 1992). First in the deuterostome lineage, the sea urchin contains a simple, yet rich coelomic fluid that may contain adaptive-like immune pathways, previously unrecognized, that may also actively function in vertebrates and perhaps humans. Coelomocytes challenged with bacterial coat lipopolysaccharide (LPS) showed a strikingly diverse array of anti-microbial proteins appearing in coelomic fluid, far more protein diversity than can be explained by genomic sequence analysis (Nair et al., 2005). These striking findings indicate that the immune system of the sea urchin is capable of highly advanced immunodiversity, perhaps by a previously undescribed mechanism that could affect the way we view the phylogeny of more complex deuterstome immune pathways. Along those lines, we sought to determine if the immune cells of the sea urchin, Lytechinus variegatus, were sensitive to external bioelectromagnetic energy treatment and if this treatment could affect wound healing pathways. We found a 75.7% rate of wound closure in treatment groups compared to 41.9% in controls. In our study, a technique was used that is similar to other techniques described in the literature, such as “non-contact therapeutic touch” (Wirth, 1990) which involves application of treatment from the hands of a trained practitioner without physical contact to the subject. Wirth et al, conducted this type of study on dermal wounds in human subjects showing an accelerated rate of wound closure in treated groups over sham controls (Wirth, 1990). In another study by Bengston and Krinsley (Bengston and Krinsley, 2000) a similar technique, here called the “laying on of hands,” was used to treat mice with induced breast adenocarcinoma. In that study, researchers showed an 87.9% cure rate in mice after 10 days of treatment compared to untreated controls. The most striking element of this study was that mice that had gone into full remission were re-injected with the same mammary adenocarcinoma strain several months later and showed complete immunity to the disease never presenting with any signs of tumor growth. Similar to our study, these studies use a technique where the hands are placed near the subject while the practitioner directs intention of healing. In our study, we have observed a direct and significant correlation between applied bioelectromagnetic energy treatment and accelerated wound closure in the sea urchin, Lytechinus variegatus, thus supporting our hypothesis. Since we do not know the mechanistic effects of bioelectromagnetic energy we cannot rule out other effects that could influence the rate and extent of wound healing in our study. For example, although the temperature was maintained at 25oC in each laboratory, we cannot rule out the possibility of thermal effects from the practitioner’s hands influencing the rate of wound closure. Future experiments investigating bioelectromagnetic energy on sea urchin wounds could include measurement of temperature changes between the practitioner’s hands and the sea urchin wounds during treatment. In these and other similar studies, the anomalous effect has been attributed to biological electromagnetic energy, qi gong energy, subtle energy etc. However, irrespective of the implied energy, it is postulated that the mechanism may involve a stimulation of immune cells through an activation pathway that could enhance or activate healing. In our hands, analysis of eschars from treatment and control wounds revealed acceleration in the appearance of red spherule and colorless spherule coelomocytes. This observation is interesting in light of data observed by Fukushima (Fukushima, 2001) that indicated an increased level of phagocytic activity of human neutrophils in groups treated with bioelectromagnetic energy over controls. Interestingly, the type of control group in these studies and others vary significantly. In some studies, control groups receive a sham treatment by an untrained individual, while in other studies control groups are left alone and receive no treatment whatsoever. In our study, the control group was attended by an untrained, blinded assistant. Since the assistant did not provide a sham control, we deem our control a “no treatment group.” Future studies on the sea urchin system could include a sham control in order to control for unknown variables that may be intrinsic by the presence of the practitioner.

12

Although the mechanisms involved in coupling anomalous effects to cellular activity are not clear, cellular responses are beginning to be identified and understood. It is thought that electrical connections of the cell, perhaps even the negatively charged phospholipids of the plasma membrane, function like antennae that are able to receive a signal and thereby conduct an activating signal transduction pathway (Luben, 1991; Lubin, 1995; Oschman, 1997). In trying to elucidate the underlying molecular mechanism, Levin (Levin, 2003) suggests that endogenous electromagnetic fields, DC electric fields, and very weak photon emission are carriers of morphogenetic information within biological systems. Some studies suggest that electromagnetic fields stimulate a stress response pathway that is similar to the one used by cells in response to heat shock. Endogenous electromagnetic fields are plausible sources of information within biological organisms; however, research also suggests these fields may establish communication between living organisms (Goodman and Blank, 1995; Oschman, 1997). Evidence exists that the biological electromagnetic fields generated by humans (BEMFs) may be used similarly to the artificially generated fields for stimulating or enhancing immune cell function and the healing response (Quinn, 1984; Seto et al., 1992; Tiller, 2004). One speculation is that BEMFs interact directly with immune cells, perhaps through a plasma membrane linkage, and initiate a cascade of second messenger events that lead to enhanced motility and chemotaxis. Along these lines, in vitro studies have shown that BEMFs have an effect on phosphorylation of the motility protein myosin in cell free assays as well as pepsin enzyme activity (Bunnell, 1999; Fukushima, 2001; Meuhsam et al., 1994). Our experiments presented in this report, using bioelectromagnetic energy followed by an analysis of protein levels from sea urchin eschars revealed no significant difference in total protein levels between tissue taken from treated and control animals. However, SDS-PAGE followed by relative band densitometry analysis revealed that on the protein level, changes in the levels of individual proteins in treated and control groups were observed. In tissue extracted from BEMF treated animals, relative band densities of the 101.8kDa, 63kDa and 35kDa bands showed an upregulation, while the 56kDa, 43kDa and 32kDa bands were attenuated compared to controls (Fig. 6). The eschar tissue represents a mixture of several components including, immune cells, epithelial cells, matrix material, etc. Thus, changes in the protein expression between bioelectromagnetic energy treated tissue and controls reflect an effect on cells at the molecular level. Further investigation on these proteins may provide essential information into the molecular mechanisms at work after bioelectromagnetic energy treatment. To begin to investigate sea urchin immune activity at the cell and molecular levels, we conducted studies on activation of the red spherule coelomocytes. These cells in the species Lytechinus variegatus have a unique motility consisting of active undulating cell surface bleb formations within which flow numerous dense granules (Sturycz and D'Andrea-Winslow, 2005). Bleb formation is a useful tool to assess red spherule viability and activation. When sea urchins are seriously sick or dying, red spherule cells slow their motility, stop moving, and in extreme cases will round up and undergo apoptosis. In vitro studies of treated samples in our study showed a significant increase in the motility rate of red spherule coelomocytes. Red spherule coelomocyte activation in treatment groups showed a 59.7% increase over controls. Since bleb formation is involved in red spherule cell motility and immune activation, we postulate the following possible model. Upon wounding, immune red spherule coelomocytes become activated and the healing pathway is initiated. In treatment groups, bioelectromagnetic energy treatment further enhances immune activation by a currently obscure activation pathway. This cellular activation pathway (possibly second messenger) in turn could activate cytoskeletal mechanoenzymes, such as myosin, involved in motility to increase the rate of bleb formation. In a previous study from our lab, we have shown that bleb formation is dependent on F-actin and myosin (Sturycz and D'Andrea-Winslow, 2005). Enhanced activation of the acto-myosin cytoskeleton by BEMFs may be responsible for the increased rate of bleb formation we have observed in our experiments. Increased bleb formation may result in increased motility of the cells, and thus immune cells can arrive at the wound faster and more efficiently. Our experiments have shown that indeed, red spherule coelomocytes in BEMF treated animals arrive at the site of the wound faster than that observed in controls (see Figure 4). Thus, an increased rate of red spherule motility would allow

13

for an accelerated migration to the site of the wound resulting in an overall increase in the rate of wound closure. The sea urchin provides a unique model system to investigate the subcellular activation pathways initiated by healing with biologically derived electromagnetic energy fields. To our knowledge, this study represents the first report of bioelectromagnetic energy treatment having an effect on immune cell activation and wound healing in an invertebrate species. Hopefully, an understanding of these pathways will provide a greater understanding of the underlying molecular mechanisms involved in accelerated tissue repair in invertebrate species and in higher organisms as well. Understanding of the molecular mechanisms of BEMFs in tissue repair and active wound healing processes could provide a reliable, non-invasive alternative for wound treatment and therapies. Acknowledgements We would like to thank John McKeague, Katie Kedrowitz, Mark Patenode, Heather Anholt, Brandon and Heidi Johnson, Elliott Johnson, Kristin Papik and Hollie Niebert for contributing to this work. Special thanks to the members of the Winslow Laboratory for stimulating discussions regarding this work. Special thanks to Amanda Kaiser for aquarium maintenance and animal care. This work was supported by a grant from the Discovery Institute (LDW) and the Northwestern College Faculty Development Award (LDW, DFJ). References Bengston, W.F., and D. Krinsley. 2000. The effect of the "laying on of hands" on transplanted

breast cancer in mice. Journal of Scientific Exploration. 14:353-364. Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of

protein using the principle of dye-binding. Analytical Biochemistry. 72:248-254. Bunnell, T. 1999. The effect of "healing with intent" on pepsin enzyme activity. Journal of

Scientific Exploration. 13:139-148. Chen, K., S.C. Shiflett, N.M. Ponzio, and B. He. 2002. A preliminary study of the effect of external

qu-gong on lymphoma growth in mice. J Altern Complemen Med. 8:615-621. Coffaro, K.A., and R. Hinegardner. 1977. Immune response in the sea urchin Lytechinus pictus.

Science. 197:1389-1390. D'Andrea-Winslow, L., G.R. Strohmeier, B. Rossi, and P. Hofman. 2001. Identification of a sea

urchin Na(+)/K(+)/2Cl(-) cotransporter (NKCC): microfilament-dependent surface expression is mediated by hypotonic shock and cyclic AMP. J Exp Biol. 204:147-56.

D'Andrea, L., M. Danon, A., G.P. Sgourdas, and E.M. Bonder. 1994. Identification of coelomocyte

unconventional myosin and its association with in vivo particle movement. Journal of Cell Science. 107:2081-2094.

Edds, K. 1980. The formation of filopodia during transformation of sea urchin coelomocytes. Cell

Motil. 1:131-140. Endean, R., and R.A. Boolootian. 1966. Physiology of echinodermata: the coelomocytes. Wiley &

Sons, New York. pp. 301-328.

14

Falugi, C., M. Grattarola, and G. Prestipino. 1987. Effects of low-intensity pulsed electromagnetic fields on the early development of sea urchins. Biophysical Journal. 51:999-1003.

Fukushima, M. 2001. Evidence of qi-gong energy and its biological effect on the enhancement of

the phagocytic activity of human polymophonuclear leukocytes. American Journal of Chinese Medicine.

Goodman, R., and M. Blank. 1995. Biosynthetic stress response in cells exposed to

electromagnetic fields. Electromagnetic fields: biological interactions and mechanisms. American Chemical Society, Washington DC. pp. 423-436.

Green, E.E., P.A. Parks, P.M. Guyer, S.L. Fahrion, and L. Coyne. 1991. Anomalous electrostatic

phenomena in exceptional subjects. Subtle Energies. 2:69-94. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of the

bacteriophage T4. Nature. 314:472-474. Levin, M. 2003. Bioelectromagnetics in morphogenesis. Bioelectromagnetics:295-315. Levin, M., and S.G. Ernst. 1995. AC magnetic field effects on early sea urchin development.

Bioelectromagnetics. 16:231-240. Luben, R.A. 1991. Effects of low-energy electromagnetic fields (pulsed and dc) on membrane

signal transduction processes in biological systems. Health Physics. 61:15-28. Lubin, R.A. 1995. Membrane signal transduction mechanisms and biological effects of low-energy

electromagnetic fields. Electromagnetic fields: biological interactions and mechanisms. American Chemical Society, Washington DC. pp438-450 .

Matsudaira, P.T., and D.R. Burgess. 1978. SDS micro-slab linear gradient polyacrylamide gel

electrophoresis. Analytical Biochemistry. 8:386-396. Meuhsam, D.J., M.S. Markov, P.A. Muehsam, A.A. Pilla, R. Shen, and Y. Wu. 1994. Effects of

quigong on cell-free myosin phosphorylation: preliminary experiments. Subtle Energies 5 93-108. Nair, S.V., H. Del Valle, P.S. Gross, D.P. Terwilliger, and L.C. Smith. 2005. Macroarray analysis

of coelomocyte gene expression in response to LPS in the sea urchin. Identification of unexpected immune diversity in an invertebrate. Physiol Genomics. 22:33-47.

Nindl, G., M.T. Johnson, and W.X. Balcavage. 2004. Low-frequency electromagnetic field effects

on lymphocytes: potential for treatment of inflammatory diseases. Marcel Dekker, Inc. New York. O'Laoire, S. 1997. An experimental study of the effects of distant, intercessory prayer on self-

esteem, anxiety, and depression. Alternative Therapies. 3:38-53. Oschman, J.L. 1997. What is healing energy? Part 3: silent pulses. Journal of Bodywork and

Movement Therapies. 1:179-194. Quinn, J. 1984. Therapeutic touch as energy exchange. Adv Nursing Sci. 6:42-49. Rosch, P.J., and M.S. Markov. 2004. Bioelectromagnetic medicine. Marcel Deker, Inc., New York.

15

Schott, M., T.J. Givens, A. Sturycz, and L. D'Andrea-Winslow. 2005. Structural characterization of sea urchin colorless spherule coelomocytes: uniquely motile cells that resemble neutrophils. Molecular Biology of the Cell. 16:758a.

Seto, A., C. Kiusaka, and Nakazato. 1992. Detection of extraordinary large bio-magnetic field

strength from human hands Acupuncture and Electro-Therapeutics Research International Journal 17:75-94.

Smith, L.C., and E.H. Davidson. 1992. The echinoid immune system and the phylogenetic

occurrence of immune mechanisms in deuterostomes. Immunology Today. 13:356-361. Sturycz, A., and L. D'Andrea-Winslow. 2005. Active bleb formation is agated in Lytechinus

variagatus red spherule coelomocytes upon disruption of acto-myosin contractility. Molecular Biology of the Cell. 16:68a.

Tiller, W. 2004. Subtle energies and their role in the bioelectromagnetic phenomena. In

Bioelectromagnetic Medicine. Marcel Dekker, Inc. Waechter, R.L., and L. Sergio. 2002. Manipulation of the electromagnetic spectrum via fields

projected from human hands: a qi energy connection. Subtle Energies & Energy Medicine. 13:233-250.

Warber, S.L., G.L.M. Kile, and B.W. Gillespie. 2003. Energy healing research, healing intention

and energy medicine. Churchill Livingstone, New York. pp.83-99 Wirth, D. 1990. The effect of non-contact therapeutic touch on the healing rate of full thickness

dermal wounds. Subtle Energies. 1:1-19. Zimmerman, J. 1990. Laying-on-of-hands healing and therapeutic touch: a testable theory.

Journal of the Bio-Electromagnetics Institute. 2:8-17.