System and method for ultrasound modulation of blood cells

Abstract

Embodiments of the invention provide apparatus, systems, and methods of reducing inflammation. A method of reducing inflammation includes: directing ultrasound at a plurality of blood cells, wherein directing ultrasound at the plurality of blood cells causes a reduction of inflammation associated with the plurality of blood cells. A system configured to reduce inflammation includes: an ultrasound transducer configured to direct ultrasound at a plurality of blood cells to cause a reduction of inflammation due to or associated with the plurality of blood cells.

Description

SYSTEM AND METHOD FOR ULTRASOUND MODULATION OF BLOOD CELLSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application is based on and claims priority from U.S. Patent Application Ser. No. 63 / 537,752, filed on September 11, 2023, the entire disclosure of which is incorporated herein by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under 140D0419C0092 awarded by the Defense Advanced Research Projects Agency. The government has certain rights in the invention.BACKGROUND

[0003] Recent research efforts have sought to suppress inflammatory responses by application of ultrasound stimulation to the nervous system, for example by stimulating the vagus and splenic nerves. The stimulation is thought to drive a neural effect in the spleen that causes a cascade of physiological effects that drive an anti-inflammatory effect. However, the mechanism and cellular target are still not fully understood. Thus, there is a need to further study and understand this anti-inflammatory response.SUMMARY

[0004] Embodiments of the invention provide apparatus, systems, and methods of reducing inflammation. In one embodiment, a method of reducing inflammation includes: directing ultrasound at a plurality of blood cells, wherein directing ultrasound at the plurality of blood cells causes a reduction of inflammation associated with the plurality of blood cells.

[0005] In another embodiment, a system of reducing inflammation is provided. The system includes an ultrasound transducer configured to direct ultrasound at a plurality of blood cells to cause a reduction of inflammation associated with the plurality of blood cells.BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Various objects, features, and advantages of the disclosed subject matter can be more fully appreciated with reference to the following detailed description of the disclosed subject matter when considered in connection with the following drawings, in which like reference numerals identify like elements.

[0007] FIG. 1A is a flowchart illustrating an example process for applying ultrasound stimulation of blood cells in accordance with some aspects of the present disclosure.

[0008] FIG. IB is a flowchart illustrating an example process for applying ultrasound stimulation of blood cells in an ex vivo environment in accordance with some aspects of the present disclosure.

[0009] FIG. 1C is a flowchart illustrating another example process for applying ultrasound stimulation of blood cells in an ex vivo environment in accordance with some aspects of the present disclosure.

[0010] FIG. 2 is an example ultrasound stimulation system that may be used in accordance with the systems and methods of the present disclosure.

[0011] FIG. 3 is a block diagram of an ultrasound system that may be used in accordance with the systems and methods of the present disclosure.

[0012] FIG. 4A is a block diagram of a non-limiting example of an ultrasound device for providing stimulation.

[0013] FIG. 4B is a profile view of a non-limiting example therapy module housing.

[0014] FIG. 4C is a profile view of a non-limiting example therapy module base.

[0015] FIG. 4D is a profile view of a non-limiting example assembled therapy module.

[0016] FIG. 5 is a block diagram for a non-limiting example ultrasound system configuration.

[0017] FIG. 6A shows example data from a first example of experimental results obtained using the systems and methods of the present disclosure.

[0018] FIG. 6B shows additional example data from the first example of experimental results obtained using the systems and methods of the present disclosure.

[0019] FIG. 6C shows additional example data from the first example of experimental results obtained using the systems and methods of the present disclosure.

[0020] FIG. 7A shows example data from a second example of experimental results obtained using the systems and methods of the present disclosure.

[0021] FIG. 7B shows additional example data from the second example of experimental results obtained using the systems and methods of the present disclosure.

[0022] FIG. 7C shows additional example data from the second example of experimental results obtained using the systems and methods of the present disclosure.

[0023] FIG. 8A shows example data from a third example of experimental results obtained using the systems and methods of the present disclosure.

[0024] FIG. 8B shows additional example data from the third example of experimental results obtained using the systems and methods of the present disclosure.

[0025] FIG. 8C shows additional example data from the third example of experimental results obtained using the systems and methods of the present disclosure.DETAILED DESCRIPTION

[0026] Before any embodiments of the invention are explained in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the following drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. Unless specified or limited otherwise, the terms “mounted,” “connected,” “supported,” and “coupled” and variations thereof are used broadly and encompass both direct and indirectmountings, connections, supports, and couplings. Further, “connected” and “coupled” are not restricted to physical or mechanical connections or couplings.

[0027] The following discussion is presented to enable a person skilled in the art to make and use embodiments of the invention. Various modifications to the illustrated embodiments will be readily apparent to those skilled in the art, and the generic principles herein can be applied to other embodiments and applications without departing from embodiments of the invention. Thus, embodiments of the invention are not intended to be limited to embodiments shown but are to be accorded the widest scope consistent with the principles and features disclosed herein. The following detailed description is to be read with reference to the figures, in which like elements in different figures have like reference numerals. The figures, which are not necessarily to scale, depict selected embodiments and are not intended to limit the scope of embodiments of the invention. Skilled artisans will recognize the examples provided herein have many useful alternatives and fall within the scope of embodiments of the invention.

[0028] The present disclosure provides systems and methods for targeting blood cells with ultrasound (US) energy to directly apply ultrasound stimulation to blood cells, such as immune cells. For example, the systems and methods provide direct immunomodulation of immune cells, which may be referred to as white blood cells. Directing low-intensity ultrasound stimulation to a group of blood cells can cause a reduction in inflammation, measured by inflammatory markers or other clinical symptoms like swelling or redness, without requiring stimulation of nerves, such as the vagus or splenic nerves, or axon terminals. Such an anti-inflammatory response may have a wide range of clinical, research, and consumer applications. For example clinical applications can include treatment of one or more of: autoimmune disorders (e.g., rheumatoid arthritis, lupus, multiple sclerosis), inflammatory conditions (e.g., inflammatory bowel disease, Crohn’s disease, acute SARS- CoV-2 infection, post-acute sequelae SARS-CoV-2 infection, sepsis, viral infections), dampening of inflammatory response pre- or post-surgery, protection from ischemiareperfusion injury to organs (i.e., kidneys, lungs, heart, gut, brain), pancreatitis, cancer, joint or tissue swelling, infection sites, healing of sites of bleeding or wounds, neurodegenerative diseases, metabolic disorders, digestive disorders, cardiovascular disorders, lung or breathing disorders, stroke, nerve or neural damage issues such as spinal cord damage, etc. Thediscussed method of directing therapeutic ultrasound at blood cells is much simpler than the previously implemented method of directing ultrasound at nerves non-invasively in vivo. It removes the complications that come with targeting areas as small as a nerve, such as the required accuracy and precision needed to target specific nerves. Targeting nerves with ultrasound can also be complicated due to shielding caused by variety in patient anatomy, such as bone or adipose tissue. Instead, ultrasound can be directed at larger targets of surplus blood like lymph nodes, the spleen, or large veins and arteries. By diversifying the ultrasound target, treatment is not limited based on access to nerves, but can be utilized in multiple areas of the body, possibly substituting location based on accessibility. The system and methods may be applied in vivo in a subject, such as a research volunteer, patient, or animal. The system and methods may also be applied for a subject in an ex vivo environment, after removing the blood cells, applying ultrasound stimulation, and returning the blood cells to the subject or to another subject. The system and methods may also be used in an in vitro environment for research or clinical applications. In this way, time cost to subjects can also be minimized to include only the period of time where sample collection occurs, while parameters can be optimized downstream in vitro. This in vitro approach additionally can be used to alter natural or artificial cells and blood samples in a dish or experiment for other research purposes or to be put back into animals.

[0029] In some configurations, it may be desirable to target blood pools or blood reserves or reservoirs. Such targets may include stationary, nearly stationary, or slow-moving blood cells, allowing for repeated stimulation of the blood cells over the period of treatment time. For example, the target location may include pools of blood or fluid located in the spleen, liver, kidney, pancreas, lymph nodes, lymphatic tissue, glymphatic tissue, heart, brain, gut, blood vessels, veins, arteries, capillaries, swelling in joints, swelling in tissue, muscle, tumors, eyeballs, bruising or hematomas, ear region or inner ear region, gums, nasal cavity or regions, etc. In some configurations, the target may be in the brain, for example in the meninges of the subject’s brain, which may contain high density of immune cells. In some configurations, pools of blood or fluid that are stationary or nearly stationary may be targeted. In other aspects, the system and methods may be applied in an ex vivo environment. For example, the blood, bone marrow, lymph, etc., may be removed from the subject or patient, treated with ultrasound stimulation, and returned to the body of the subject or to another subject or even to an animal. In other configurations, the biofluid may be removed from ananimal subject, treated, and returned to a human subject. In another example, donated blood that has received ultrasound treatment ex vivo may be transfused into a blood type matched recipient, such as a human or animal subject. In other configurations, flowing blood may be targeted, which will be described in further detail below.

[0030] FIG. 1A shows a flowchart that demonstrates an example process 100 for applying ultrasound stimulation to immune cells. The process 100 can be applied in vivo using an ultrasound device. For example, the ultrasound device may be a portable or wearable device that can apply ultrasound stimulation intermittently over a period of time, such as hours, days, weeks, etc. The process 100 may also be applied in an ex vivo environment, as shown in FIG. IB, which will be described in further detail below.

[0031] The process 100 begins in block 104 in which patient data or subject data may be acquired. The patient data may include imaging data, which may allow for identification of target blood cells and guide the ultrasound stimulation target determined further in the pipeline (e.g., block 106). For example, block 104 may include medical imaging, such as US, magnetic resonance imaging (MRI), x-ray, computed tomography (CT), fluoroscopy, positron emission tomography (PET), single-photon emission computed tomography (SPECT) etc. In some configurations, the imaging may preferably utilize ultrasound. When using ultrasound imaging, the imaging ultrasound transducers may be integrated with those used for ultrasound stimulation or dual-mode transducers may be used to simultaneously or alternatingly produce images and stimulation. Such images may guide the ultrasound stimulation target. For example, the image may be used to identify pools of blood that contain large quantities of immune cells. Imaging may also identify areas of high density of blood vessels, which may be targeted.

[0032] Acquiring imaging data in block 104 may also include dynamic imaging, which may indicate the speed at which the target blood moves. For example, imaging tracers may be used to track blood flow. Additionally or alternatively, MRI may be used with flow imaging techniques, such as phase-contrast methods or gradient echo techniques, to produce dynamic angiographs. Doppler ultrasound may also be used to estimate blood flow through vessels and may be enhanced with the use of microbubble contrast agents. Such dynamic blood flow data may inform the target location or stimulation timing parameters in blocks 106 and 108, respectively. For example, such dynamic imaging can indicate areas where the blood flow isstationary or nearly stationary, which may be targeted with a stationary ultrasound device. Alternatively, blood flow speeds may guide the use of a moveable ultrasound device or steerable ultrasound beam that may track the flow of blood through the body to target moving blood cells.

[0033] The patient data may also include clinical data that may guide the calibration of stimulation parameters further in the pipeline (e.g., block 108). The clinical patient data may include patient health history, demographic data, family history, historical biomarker data, current biomarker data, dynamic biomarker data, genetic expression data, metabolomic data, immune data, body dimension data etc. Biomarkers may include measurements of cytokines, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), ferritin / d-dimer, etc. For example, cytokine biomarkers may include interleukin- ip (IL-ip), interferon-gamma (IFN- y), monocyte chemoattractant protein- 1 (MCP-1), interleukin 6 (IL-6), interleukin 8 (IL-8), interleukin 10 (IL- 10), tumor necrosis factor alpha (TNF-a), and nuclear factor kappa-light- chain-enhancer of activated B cells (NF-KB), and others. Other data about the patient’s body, such as body mass index (BMI) or the dimensions of the body in the area of interest, may be used to determine the optimal parameters and site for ultrasound stimulation. Various types of patient data may be used alone or in combination to determine the inflammation state of the patient.

[0034] The patient data acquired in block 104 may include a measure of the patient’s inflammation state. In some configurations, such an inflammation state may be determined from a blood draw used to measure cytokine information or other biomarkers. Blood samples may be taken, and the results may be inputted into a data server. Such a server may connect wirelessly or otherwise to the ultrasound stimulation device. The patient’s inflammation state may also be stored from previous clinical tests or patient history on a computer system and accessed from the computer memory. The patient data may alternatively be acquired using external sensors. For example, a cytokine sensor may be implanted into the vascular system of the subject or through a wearable patch or device that is minimally invasive with microneedles into the skin, oral cavity area or ear region (e.g., ear canal, tympanic membrane or other inner ear membranes with access to blood vessels near the membrane surface).

[0035] The ultrasound stimulation target may be set in block 106. The stimulation target may be informed by the patient data acquired in block 104. For example, the target may bedetermined based on the patient’s condition, health history, or cytokine levels. Additionally, imaging data may be used to target immune cells. For example, the imaging data may be used to identify and locate pools of blood that contain high levels of immune or blood cells. Imaging data may also be used to determine areas of high blood vessel density.

[0036] The ultrasound stimulation parameters may be set in block 108. The ultrasound parameters may include the stimulation time or duration, the frequency of the ultrasound wave, the peak pressure of the ultrasound energy, and the pulse width and repetition frequency. These parameters may be set as constant over the treatment time or may change dynamically over the treatment period. Treatment parameters may also include a duty cycle, in which the pipeline 100 may be repeated one time over the course of each duty cycle, separated by a desired treatment break.

[0037] The treatment parameters may be determined, at least in part, based on the patient data acquired in block 104. For example, the inflammation state of the patient may guide the treatment parameters. For example, the ultrasound energy used may be minimized in a case in which the patient exhibits a lower inflammatory response. Conversely, if a patient is experiencing a greater than average inflammatory condition, then the amount of ultrasound treatment delivered could be increased by lengthening the amount of time the device is used or increasing the frequency or duty cycle of treatments. Also, based on the inflammatory response measured by the various biomarkers or clinical symptoms, which can be different for each individual, the stimulation parameters can be adjusted to drive greater inflammatory response or even reduce the anti-inflammatory effect if the response passes below a certain threshold. The ultrasound stimulation parameters may also be determined, in part, by the imaging data optionally acquired in block 104. For example, the imaging data may determine the time available to treat blood at a specified target location, which may inform the stimulation duration and affect the intensity parameters required to achieve a desired response in the limited time available. The ultrasound parameters could also be adjusted based on the patient’s body metrics. For example, if there is a significant layer of adipose tissue between the target and the ultrasound transducer that might attenuate and weaken the therapy, the intensity of ultrasound could be increased.

[0038] Non-limiting example stimulation parameters include stimulation between 50 kHz to 7 MHz, such as between 500 kHz to 1.5 MHz, and pressures in a range of 100 kPa to 1MPa or 25 kPa to 10 MPa depending on the stimulation frequency. The stimulation duration may occur over sessions between 1 minute and 1 hour or more specifically between 5 minutes to 20 minutes; and with pulse widths between 1 ps and 5 ms or more specifically 100 ps to 500 ps. The pulse repetition frequencies can span between 100 Hz and 10 kHz or more specifically 1 or 2 kHz. The number of repetitions may be pre-determined between a single repetition to hundreds of repetitions of these sessions or more specifically one or two sessions per day. Alternatively, these parameters can be adjusted based on evaluation of the immune response, for example in blocks 112 and 114.

[0039] The treatment parameters set in block 108 may also indicate whether or not ultrasound stimulation is required. For example, if the patient’s immune response is in a desired range, as indicated in block 104, the ultrasound stimulation parameters may indicate that no ultrasound stimulation will be applied to the patient. The immune response can be reevaluated further in the pipeline (e.g., blocks 112 and 114) or by repeating process 100.

[0040] Block 110 may include applying ultrasound stimulation, if indicated in block 108. The location targeted and the stimulation parameters used by the ultrasound stimulation in block 110 may be set in blocks 106 and 108, as previously described. Such ultrasound stimulation may be applied directly to the blood cells or other immune cells, without the expressed need to stimulate the nervous system. Thus, the ultrasound stimulation may have a direct effect on the immune cells, causing an anti-inflammatory response. For example, the ultrasound stimulation may be applied to peripheral blood mononuclear cells (PBMCs), which may include lymphocytes (e.g., T cells, B cells, NK cells), monocytes, and dendritic cells.

[0041] In some configurations, applying ultrasound stimulation in block 110 can target blood or immune cells without requiring the stimulation of the nervous system, such as nerves, axons, terminals, other neural tissue, etc. Thus, the stimulation may be directed towards the blood cells without directing the stimulation towards the subject’s neural tissues. In this way, the anti-inflammatory response may be targeted directly, bypassing possible intermediate steps of modulating complex neuroimmune signaling pathways. Directly targeting immune cells may reduce the required stimulation frequency, repetition, intensity, etc., increasing treatment safety and reducing costs and burden on the healthcare system and patient. Directly targeting immune cells also expands the target locations within the body thatcan receive ultrasound immunomodulation and removes the need to target small and difficult to isolate neural structures. Further, target patient demographic can be expanded to be non- exclusionary of individuals lacking end-organs and / or the associated target nerves. By targeting the immune cells directly, instead of altering and avoiding the alteration of neural and organ function through ultrasonic stimulation, the risk of unintended side effects caused by downstream neural circuits being activated is reduced.

[0042] In some configurations, applying ultrasound stimulation in block 110 may include directing stimulation to two or more locations. For example, the ultrasound stimulation may be applied to blood cells at various positions as the blood flows through vessels. The ultrasound can be applied along various points along the flow path using several methods. For example, the ultrasound array may dynamically direct the ultrasound wave along the blood path. Additionally or alternatively, several ultrasound devices may be used, each positioned along various points along the blood flow path. Each device may be triggered based on the speed of blood flow. In some configurations, the ultrasound stimulation device may be dynamically moved along the path of a blood vessel with a speed similar or equal to that of the blood flow. In any case, the target position and trigger timing may be determined based on the speed of blood flow. For example, the vessel path and flow speed may be determined in block 104. Blood tracers may also be used to verify the position and flow speed of target blood cells, which may be imaged simultaneously or nearly simultaneously with ultrasound stimulation. The ultrasound can also be applied to multiple pools of blood across the body, such as different organs or clusters of blood vessels.

[0043] Applying ultrasound stimulation to blood cells in block 110 may cause an antiinflammatory immune response. For example, the immune cells may reduce the production of cytokines or may reduce the signaling effects, reducing cytokines in the blood. Thus, the immune response can be measured or monitored in block 112. For example, the treated blood or nearby blood may be monitored for inflammation markers to indicate the antiinflammatory effect. The effect can be measured using standard or other known clinical or experimental methods for measuring inflammatory markers or biomarkers. For example, the presence of various cytokines can be measured. As a non-limiting example, the cytokines may include interleukin- ip (IL-ip), interferon-gamma (IFN-y), monocyte chemoattractant protein- 1 (MCP-1), interleukin 6 (IL-6), interleukin 8 (IL-8), interleukin 10 (IL- 10), tumornecrosis factor alpha (TNF-a), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-KB), and others. For example, the cytokines may be measured by a cytokine sensor implanted in the patient and / or by analyzing a blood draw by direct protein or RNA transcript quantification. In a non-limiting example, cytokines can be measured in the blood using reverse transcription quantitative real-time PCR (RT-qPCR). Other technologies can be used, such as ELISA, for measuring cytokine levels. Other biomarkers may include erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), ferritin / d-dimer, etc. Other biomarkers may be determined from genetic expression data or other quantification methods based on samples collected from users. Such biomarkers may be used alone or in combination with other biomarkers or other patient data (e.g., age, health condition, metabolomics, etc.). Ratios of various biomarkers or changes of biomarkers over time may also be used as a measure of inflammation state.

[0044] After measuring an immune response in block 112, the measured inflammatory state can be assessed in block 114. For example, a threshold may be set for a biomarker or combination of biomarkers of inflammation of interest (e.g., IL-ip, IFN-y, MCP-1, IL-8, TNF-a, NF-KB, ESR, CRP, IL-6, IL-10, MAPK14, etc.). Other thresholds may be set for other clinically relevant inflammatory biomarkers such as erythrocyte sedimentation rate (ESR) and ferritin / d-dimer. If the measured biomarker cytokine level is adjusted to a clinically significant target range, the immune response to ultrasound stimulation can be deemed sufficient. Additionally or alternatively, the immune response can be evaluated based on the change in inflammation markers between block 112 and those measured in block 104. If reduction in inflammation markers is deemed sufficient, based on a desired threshold or change, the process can end. However, if the anti-inflammatory response is insufficient, the process can iteratively repeat. The ultrasound stimulation parameters may be optionally adjusted in block 116 based on the measured immune response. For example, the intensity, repetition frequency, or duty cycle frequency of the ultrasound stimulation may be increased. The target location may also be adjusted based on the measured inflammation state. A delay time may also be set in block 116, in which the repeated treatment is paused to provide a treatment break and maintain stimulation safety limits, or to accommodate motion of the target. Ultrasound stimulation may be applied repetitively in block 110 with a desired duty cycle. This process can iteratively continue until a desired response is achieved in block 114.

[0045] The process 100 can be repeated with a desired frequency, including all or some of the blocks of process 100. For example, the process 100 may repeat once a day to assess the immune status of a patient. If the immunomodulation is deemed necessary in block 108, the process can continue with ultrasound stimulation in block 110. However, if the patient data acquired in block 104 indicates that the immune response is sufficiently suppressed, the process can continue without modulation (i.e., the ultrasound stimulation parameters are set for no ultrasound stimulation). In this way, a patient’s biomarkers can be periodically monitored, applying ultrasound stimulation when required based on the biomarker measurements.

[0046] In some configurations, it may be desirable to apply ultrasound stimulation in an ex vivo environment with respect to a subject, such as in an in vitro setup or in an in vivo animal model. FIGS. 1B-1C show non-limiting example processes 120 and 130 that can be used to apply ultrasound stimulation to immune cells in an ex vivo environment. For example, FIG. IB provides a general process 120 for treating biofluids for various applications. FIG. 1C provides a more detailed example process 130 for removing a subject’s biofluid or blood, treating the biofluid outside of the body, and returning the subject’s biofluid back to the subject’s body.

[0047] Referring to FIG. IB, an example process 120 is provided for treating a biofluid by ultrasound stimulation. The process 120 may be used for various clinical or research applications, some of which will be described. A biofluid may be accessed in block 122. For example, the biofluid may include blood, plasma, serum, lymph, bone marrow, intestinal fluids, urine, etc. As a non-limiting example, accessing a biofluid may include removing it from a subject or donor, accessing biofluid from an animal (e.g., non-human primate, mouse), accessing artificially made biofluid, accessing blood from a blood bank, or otherwise obtaining a desired biofluid. Block 122 may also include treating or preparing the biofluid. For example, the biofluid may be separated (e.g., by centrifuge) into individual components, such as plasma, red blood cells, platelets, cryoprecipitate, or others. For example, blood cells may be separated from the blood and suspended in a cell culture, agar, or otherwise stored for treatment. The biofluid may be cleaned, tested, or otherwise treated (e.g., leuko-reduced, stained, cooled, etc.) in preparation for ultrasound treatment, storage, or transplant. In someconfigurations, preparing the biofluid may include transplanting the blood into an animal model for ultrasound treatment or experimental purposes.

[0048] Once prepared, the biofluid or cells of interest can be treated by ultrasound stimulation in block 124. For example, ultrasound stimulation may be applied in an in vitro environment (e.g., petri dish). As another non-limiting example, the ultrasound stimulation may be applied in an in vivo animal model. Treating the biofluid or cells in block 124 may include all of some of the steps of process 100 (FIG. 1A), for example, applied in the proper environment for the desired application. Once treated, the biofluid or cells may be utilized for a desired application in block 126. For example, block 126 may include replacing the treated biofluid back into the donor. As another example, block 126 may include transplanting the treated blood into another recipient, such as a patient recovering from trauma or suffering from infection. As another non-limiting example, block 126 may include performing research using the treated blood (e.g., testing inflammation markers over time, transplanting the blood to diseased animal models, etc.). Block 126 may also include replacing or transplanting the treated biofluid into a donor or recipient animal. Block 126 may also include storing the treated biofluid for future use. In this way, anti-inflammatory blood or other biofluids can be prepared and stored for use in clinical or hospital settings, such as in an emergency room for trauma victims.

[0049] Referring to FIG. 1C, another non-limiting example process 130 is provided for treating a biofluid in an ex vivo environment. A biofluid can be removed from a subject in block 132. For example, the biofluid may include blood, plasma, serum, lymph, bone marrow, intestinal fluids, urine, etc. The biofluid may be removed, for example, by IV, blood draw, surgery, bone marrow aspiration, or another known method. In block 124, the fluid can be treated by an ex vivo ultrasound stimulation process that induces an anti-inflammatory response. For example, block 124 may include all or some of the steps presented in process 100 (FIG. 1A) applied in the ex vivo environment. After the cells are modulated with ultrasound energy in the ex vivo environment, the biofluid can be returned to the body of the subject in block 136. Alternatively, the biofluid can be transplanted to another subject in block 136.

[0050] FIG. 2 shows a block diagram of an ultrasound stimulation system 200, which provides a non-limiting example of a system that can be used to apply ultrasound stimulationto blood or immune cells and receive external signals. As a non-limiting example, system 200 may carry out process 100. The system includes a controller 202, which may generally include an ultrasound stimulation pattern module 210, an ultrasound target module 212, a control module 216, a processor 220, a power source 230, a memory 232, a clock module 234, and a communication module 236. The system may also include an ultrasound device 246, which is capable of applying ultrasound stimulation with a specified waveform to a target. The ultrasound device 246 may also be capable of ultrasound imaging. The ultrasound device 246 will be described in further detail below.

[0051] The system 200 may also include external devices 242. For example, the external devices 242 may include a server that may store patient data, system instructions, imaging data, etc. The external devices 242 may also include a user interface that may display system parameters or accept user inputs or operational instructions. Specifically, the input may include various user interface elements, such as a mouse, keyboard, touchpad, touch screen, buttons, and the like. The input may also include various drives and receptacles, such as flash-drives, USB drives, CD / DVD drives, and other computer-readable medium receptacles, for receiving various data and information. To this end, the input may also include various communication ports and modules, such as Ethernet, Bluetooth, WiFi, etc. for exchanging data and information with various external computers, systems, devices, machines, mainframes, servers or networks. The external devices 242 may also include various other sensors, such as external imaging devices, cytokine sensors, localization sensors, etc. The ultrasound device 246 and external devices 242 may be connected to the controller 202 via proper connections, such as data cables, Wi-Fi, Bluetooth, etc.

[0052] The external devices 242 may also be controlled by the processor 220, which may determine instructions and provide them to the external device 242 via the control module 216. For example, the external device 242 may include a cytokine sensor, and the control module 216 may trigger a cytokine measurement at various times throughout the stimulation process.

[0053] The controller 202 may also include various connections, terminals, or wireless communication connections for transmitting signals generated by the ultrasound stimulation pattern module 210, the ultrasound target module 212, or control module 216, or signals measured by external devices 242. Any or all of the elements may be housed in one or morewearable devices, in one or more portable devices carried externally to the body, or on an external server with proper wired or wireless connections between elements.

[0054] The processor 220 can be configured or programmed to perform a variety of functions for operating the controller 202 using instructions stored in memory 232, in the form of a non-transitory computer readable medium, or instructions received via input. In some implementations, the processor 220 may control the sending and receiving of instructions and operational parameters (for example, via a wireless transcutaneous link in the communication module 236), the storage of the operational or stimulation parameters and instructions in memory 232, the transmission of the operational parameters and selective triggering to the ultrasound device 246, as well as synchronizing various functions using the clock module 234. For instance, the processor 220 may communicate with the clock module 234 to determine the timing and synchronization of various stimulations. The processor 220 may also communicate with the clock module 234 ultrasound stimulation pattern module 210, as well as other hardware and digital logic circuitry, to accurately store activation times and parameters in memory 232. By way of example, the processor 220 can be a programmable microprocessor or microcomputer.

[0055] The ultrasound stimulation pattern module 210 may determine ultrasound stimulation parameters and provide instructions to the ultrasound device 246. For example, the ultrasound stimulation pattern module 210 can provide the ultrasound device with waveform parameters, such as ultrasound intensity, ultrasound frequency, pulse width, pulse timing, duty cycle, etc. The ultrasound stimulation pattern module 210 may determine such ultrasound stimulation parameters based on input data from external devices 242. For example, the ultrasound parameters may be informed by measured cytokine levels or other patient data.

[0056] The ultrasound target module 212 can determine the target location for ultrasound stimulation. For example, the ultrasound target module 212 can use data provided by external devices 242, such as imaging data or a user interface, to determine the target location for ultrasound stimulation. For example, the ultrasound target module 212 can automatically identify a target location based on imaging data. For example, the ultrasound target module 212 may automatically analyze imaging data to identify regions of high blood vessel density or stationary blood pools. Alternatively, a user can input a target location using a userinterface. The ultrasound target module 212 may also localize the ultrasound device based on imaging data acquired by the ultrasound device along or using other imaging data provided by external imaging devices or servers.

[0057] The ultrasound target module 212 can also provide localization instructions to the ultrasound device 246 or a user. For example, the ultrasound target module 212 can determine settings for a phased array to stimulate the target location with high localization precision. For example, the phased array may achieve 1 mm x 1 mm x 1 mm spatial precision. The ultrasound target module 212 may also provide instructions to the device user via an external device 242, such as a user interface or display. Such instructions may direct the user or patient how to position the device on the patient to target the desired location.

[0058] In some implementations, the controller 202, along with the ultrasound device 246, may be part of a standalone stimulation system. Alternatively, the controller 202 may be a portable, wearable, or implantable unit that is programmable or configurable using an external device, computer or system. To this end, the communication module 236 may be configured to send and receive various signals, as well as receive power. Specifically, the communication module 236 may include an antenna, or an input-output wire coil, a receiver and transmitter, data converters, as well as other hardware components. As a non-limiting example, the receiver and transmitter may be configured to receive and transmit radiofrequency (RF) signals. In some implementations, the antenna may be configured for transcutaneous wireless two-way communication with an external wearable device, sending and receiving signals when the external wearable device is placed in close proximity. The communication signals may be transmitted through magnetic induction and include information for operating and / or programming the processor 220. For instance, the communication signals may include triggers or command signals for generating stimulations or for communicating patient data. In some configurations, transmitted signals may also be configured to power or recharge battery components powering the controller 202. The antenna may be connected to a receiver and transmitter, which in turn may be connected to serial-to-parallel and parallel-to-serial data convertors, respectively. Any information sent or received, as described, may then be processed by the processor 220.

[0059] The controller 202 may be powered by an internal and / or external power source 230. For example, an internal source may include a standard rechargeable battery,comparable to batteries used in implantable devices (e.g., pacemakers). Alternatively or additionally, the internal power source may include a capacitor in combination with a regulator, such as a single ended primary inductor converter or dc-dc converter, that together can generate a constant current or voltage output for short periods of time. In some implementations, the capacitor may be charged by an external wearable device. As such, the controller 202 may include an induction coil, or thin, tightly wound wire that allows for RF telemetry and / or battery recharge by an external wearable or portable device, configured either as part of the communication module 236, or as separate hardware. Other methods of charging may also be utilized.

[0060] FIG. 3 shows an example ultrasound system that may be used to apply ultrasound modulation of blood cells according to the present disclosure. As a non-limiting example, the ultrasound system 300 may be a part of the stimulation system 200 as the ultrasound device 246 in FIG. 2. The ultrasound system 300 includes a transducer. The transducer may be a transducer array 302 that includes a plurality of separately-driven transducer elements 304. The transducer array 302 can include any suitable ultrasound transducer array, including linear arrays, curved arrays, phased arrays, and so on. Similarly, the transducer array 302 can include a ID transducer, a 1.5D transducer, a 1.75D transducer, a 2D transducer, a 3D transducer, and so on.

[0061] When energized by a transmitter 306, a given transducer element 304 produces a burst of ultrasonic energy. The ultrasonic energy reflected back to the transducer array 302 (e.g., an echo) from the object or subject under study is converted to an electrical signal (e.g., an echo signal) by each transducer element 304 and can be applied separately to a receiver 308 through a set of switches 310. The transmitter 306, receiver 308, and switches 310 are operated under the control of a controller 312, which may include one or more processors. As one example, the controller 312 can include a computer system.

[0062] The transmitter 306 can be programmed to transmit unfocused or focused ultrasound waves. In some configurations, the transmitter 306 can also be programmed to transmit diverged waves, spherical waves, cylindrical waves, plane waves, or combinations thereof. Furthermore, the transmitter 306 can be programmed to transmit spatially or temporally encoded pulses.

[0063] The receiver 308 can be programmed to implement a suitable detection sequence for the imaging task at hand. In some embodiments, the detection sequence can include one or more of line-by-line scanning, compounding plane wave imaging, synthetic aperture imaging, and compounding diverging beam imaging.

[0064] In some configurations, the transmitter 306 and the receiver 308 can be programmed to implement a high frame rate. For instance, a frame rate associated with an acquisition pulse repetition frequency (“PRF”) of at least 100 Hz can be implemented. In some configurations, the ultrasound system 300 can sample and store at least one hundred ensembles of echo signals in the temporal direction.

[0065] The controller 312 can be programmed to implement an imaging or stimulation sequence using the techniques described in the present disclosure, or as otherwise known in the art. In some embodiments, the controller 312 receives user inputs defining various factors used in the design of the imaging or stimulation sequence.

[0066] A scan or stimulation can be performed by setting the switches 310 to their transmit position, thereby directing the transmitter 306 to be turned on momentarily to energize transducer elements 304 during a single transmission event according to the designed imaging or stimulation sequence. The switches 310 can then be set to their receive position and the subsequent echo signals produced by the transducer elements 304 in response to one or more detected echoes can be measured and applied to the receiver 308, for example, during imaging sequences. The separate echo signals from the transducer elements 304 can be combined in the receiver 308 to produce a single echo signal.

[0067] In some configurations, the ultrasound system 300 may include more than one ultrasound array 302. At least one of the arrays may be used for transmitting ultrasound for therapeutic treatment, and at least one other array may be used to transmit and / or receive ultrasound for imaging. In one non-limiting example, the frequency used for treatment may be lower than the frequency used to produce high-quality images. In this way, the ultrasound system 300 can provide an integrated imaging and stimulation system, such that the imaging may inform the localization of the stimulation target. Additional ultrasound arrays 302 may be used to provide stimulation at multiple targets within a subject.

[0068] The echo signals are communicated to a processing unit (not shown), which may be implemented by a hardware processor and memory, to process echo signals or images generated from echo signals. As a non-limiting example, the processing unit can target blood cells for ultrasound stimulation to treat inflammation using the methods described in the present disclosure. Images produced from the echo signals by the processing unit can be displayed on a display system 314.

[0069] In one non-limiting example, a wearable / miniaturized phased array ultrasound device may be used for ultrasound system 300, although other types of ultrasound stimulation devices (e.g., a conventional / cart-based ultrasound system, a portable system, etc.) may also be used. The wearable device may be placed on a subject and worn for extended periods of time. In some configurations, a reprogrammed smartphone or tablet device may be used to control and / or monitor the stimulation. In one non-limiting example, the reprogrammed smartphone or tablet device may allow for Bluetooth wireless control of the stimulation device. For example, the smartphone or tablet device may provide a user interface to input patient data, desired stimulation parameters, or a target location. In some configurations, the control and / or monitor system may be an application installed on a smartphone, tablet device, and the like. In another non-limiting example, a number of devices distributed around a subject may be used to provide the ultrasound stimulation. The overall size of a wearable device may be similar to that of other consumer electronic wearable devices, such as smart watches, when using miniaturized ultrasound array technology. In one non-limiting example, the total footprint of a device may be less than 1.2 cm x 1.2 cm. In another non-limiting example, the footprint of a device may be less than 40 mm x 40 mm. In some non-limiting examples, the total footprint of the device may be larger, such as to cover a larger region of the spleen, liver, kidney, gut, target blood vessel, lymphatic tissue, or other organ. In one non-limiting example, the footprint of the device is the same size as a spleen, which may range from 8-16 cm, or another target organ.

[0070] In some configurations, the ultrasound transducer may be mobile. For example, the ultrasound transducer may be manually moveable or automatically moveable by a motorized system. In this way, the ultrasound stimulation may track blood flow.

[0071] In some configurations, the ultrasound transducer elements 304 can be activated in unison to form a plane wave front. In other configurations, the ultrasound transducer elements 304 can be activated sequentially to form an angled or focused wave front.

[0072] In some configurations, beam steering may be used to target selected locations, for example blood pools or areas of high blood vessel density, with ultrasound stimulation. Locations may be selected to optimize the effects of treatment, such as by targeting immune cells.

[0073] In some configurations, a wearable energy delivery device will have the capability to image and transmit ultrasound energy to a specified target. Imaging and / or beam steering may also be used to compensate for motion of the blood, such as blood flow, or for motion of the target, such as changes in depth due to subject motion. Target organs or blood vessels may naturally move. For example, the spleen moves within the abdominal cavity during heavy breathing and changes in body position. With imaging feedback, the ultrasound beam focus point can be realigned as the distance to the surface of the target changes relative to the transducer array. This provides the potential for closed-loop device operation with the implementation of automated re-targeting algorithms. The ultrasound beam focus point may also track blood flow, guided by imaging data.

[0074] To accomplish a beam steered scan, the transmitter 306 imparts a time delay, 7 / , to the respective pulses 316 that are applied to successive transducer elements 304. If the time delay is zero, Tt= 0, all of the transducer elements 304 will be energized simultaneously and the resulting ultrasonic beam will be directed along an axis 318 normal to the face of the transducer 302 and originating from the center of the transducer array 302. As the time delay increment, 7 , is increased, the ultrasonic beam is directed away from the central axis 318 by an angle, 0. The relationship between the time delay increment, 7j, added successively to■ th * 1 • each Z signal from one end of the transducer array 302, Z = I , to the other end, l = n , is given by the following relationship:

[0076] where S is an equal spacing between centers of adjacent transducer elements 304; c is the velocity of sound in the object under study; R is a range, or depth, at which the transmit beam is to be focused; and Tois a delay offset that ensures that all calculated time delay increment values, Tt, are positive values.

[0077] The second term in Eqn. (1) steers the beam to the desired angle, 3, and the third term is employed when the transmitted beam is to be focused at a fixed range, R . A sector scan is performed by progressively changing the time delays, 7 , in successive excitations. In this manner, the angle, 3, is changed in increments to steer the transmitted beam in a succession of directions. When the direction of the beam is above the central axis 318, the timing of the pulses is reversed; however, Eqn. (1) still applies in this situation.

[0078] The echo signals produced by each burst of ultrasonic energy emanate from reflecting objects located at successive ranges, or depths, R , along the ultrasonic beam. These are sensed separately by each transducer element 304 in the transducer array 302, and a sample of the magnitude of each echo signal at a particular point in time represents the amount of reflection occurring at a specific range, R . Due to the differences in the propagation paths between a focal point, P , and each transducer element 304, however, these echo signals will not occur simultaneously and their amplitudes will not be equal. A function of the receiver 308 is to amplify and demodulate these separate echo signals, impart the proper time delay to each, and sum them together to provide a single echo signal that accurately indicates the total ultrasonic energy reflected from each focal point, P , located at successive ranges, R , along the ultrasonic beam oriented at the angle, 3.

[0079] Under the direction of the digital controller 312, the receiver 308 provides delays during the scan such that the steering of the receiver 308 tracks with the direction of the beam steered by the transmitter 306, and such that the receiver 308 samples the echo signals at a succession of ranges, R , and provides the proper delays to dynamically focus at points, P , along the beam. Thus, each emission of an ultrasonic pulse results in the acquisition of a series of data points that represent the amount of reflected sound from a corresponding series of points, P , located along the ultrasonic beam.

[0080] In some configurations, a physical device, such as a cone, may be used to steer the ultrasound beam to the target location. It will be appreciated by one skilled in the art that other options for directing ultrasound to a target location may be used with the present disclosure.

[0081] Referring to FIG. 4A, a block diagram of a non-limiting example device for providing stimulation is shown. Other ultrasound or stimulation devices may also be used. A therapy module 400 may be included and is capable of providing ultrasound stimulation to a subject. A control module 410 may be used to control the therapy module 400. Control module 410 may include instructions stored for controlling the therapy module 400. Biomarker feedback sensor 440 may provide for feedback data, such as a measure of cytokine levels, and may be in contact with a subject such as through skin layer, through contact with blood via an IV or implanted sensor, or through another contact with the subject. Biomarker feedback sensor 440 may be in wireless communication 460 with control module 410. Power for the system may be provided by power supply / adapter 420. Power supply / adapter 420 may be an AC power adapter that plugs into the wall for standard power input. The therapy module 400 may be wired to the control module 410 using cable 430.Alternatively, the therapy module 400 may be in wireless communication with control module 410.

[0082] The control module may be used for device operation and power transformation to the transmitting transducer. In a non-limiting example, the control module 410 may generate + / - 90 V and 20 V DC outputs. These DC outputs are supplied to the therapy module 400 to power beam forming ICs, MCU and other secondary components. Based on pre-programmed parameters, the beam forming ICs may generate tone-burst pulses with maximum Peak to Peak voltage of approximately 180 V (+ / - 90 V). Using these tone-burst pulses, the phase array transducer may activate piezoelectric elements (such as 128 piezoelectric elements) in a pre-defined manner and produces ultrasound pressure waves. For each device, the ultrasound pressure outputs may be measured and tuned to ensured that the appropriate intensities are below safety thresholds.

[0083] Referring to FIGS. 4B-4D, a non-limiting example therapy module 400 is shown in greater detail. Referring to FIG. 4B, the therapy module 400 may include an outer housing 470, and a transmission array 472. Referring to FIG. 4C, a base 474 may include a cut-outportion 476 for accommodating the transmission array 472. Referring to FIG. 4D, a profile view of therapy module 400 is shown assembled with outer housing 470 and base 474. In some configurations, therapy module 400 may be a small, lightweight, wearable component. In a non-limiting example, the therapy module 400 houses the beam forming integrated circuits (ICs), phase array ultrasonic transducers, and other auxiliary circuits (Bluetooth, memory, and the like).

[0084] The therapy module 400 may be adhered to the body of a subject with a disposable adhesive coupling pad. In a non-limiting example placement, the therapy module 400 may be placed on the torso or head of a subject or otherwise near the target blood cells.

[0085] Referring to FIG. 5, a block diagram is shown for a non-limiting example ultrasound system configuration 500. A power generator 510, which in some configurations may be a DC power supply, may provide power for processor and beam forming integrated circuits 520, which control a stimulation delivered by phased array transducers 530. A clock generator 540 may provide a signal for triggering the beam forming integrated circuits 520. A wireless communication module 550 may be coupled to the beam forming integrated circuits 520, and may provide for diagnostic access, data input / output, user interface access, and the like.

[0086] The system interface may allow for toggling of select features by an authorized user, such as a designated technician, professional, clinician or nurse, for administrate control via a custom tablet app. During therapeutic operation, use of the device may be limited to on / off operation with an automatic shutoff feature and may not require the use of a separate tablet device. Internal to the device, phasing algorithms may be provided for individual channel control to allow focusing and steering of the ultrasonic beam. This may allow the ultrasonic beam to be optimized for high intensity and pressure at the target site of the blood cells. Additional parameters include the pulse duration and repetition frequency, the values of which may be optimized to achieve the desired therapeutic effect.

[0087] In one configuration, a system may be configured with 128-channel or 256- channel beam forming capability for high resolution steering and focusing. Peak frequency may be 400 to 600 kHz and channel spacing at 1.5 mm, which is equivalent to less than half of the ultrasonic wavelength (X / 2) in water at the 500 kHz ultrasonic frequency, optimizingthe ability to steer and focus the ultrasound beam by avoiding energy loss due to side lobe generation. Peak frequency may also be between 600 kHz to 1 MHz or 1 MHz to 2 MHz. An ultra-compact design may be used with the wearable system being smaller than a standard business card.

[0088] In one configuration, a transducer for the system may include 128 transducer elements. Each element may be electrically and physically isolated, allowing for individual phasing and increasing the efficiency by nearly eliminating all acoustic cross-talk between channels. The 128-element array may be separated into four identical sections of 32 elements each. Each of the 32 elements in its respective section may be controlled in tandem with the three equivalent elements from the other sections. This allows the system to effectively operate in an equivalent manner as a 128-channel system for center beam focusing for blood cell stimulation.

[0089] In one configuration, a transducer may include 32 channels tied to 32 elements for full steering and phasing in both X and Y axes. The transducer system can produce over 1 MPa of peak negative acoustic pressure and deliver energy to various depths, depending on the target location. For example, target depths may range between 1 mm to 30 cm. In some configurations, transducers may be provided with as high as 512 elements and channels for higher pressure and larger acoustic ranges.

[0090] For blood cell targeting, the systems have the ability to steer and focus the beam as necessary to achieve the desired therapeutic effect. In one configuration, by phasing the 64 channels of the system and transducer, the beam is able to steer efficiently towards the desired location. For the wearable device, a large number of programs may be stored into the internal memory for very fast rastering between steered and focused conditions. The phasing conditions may be preprogrammed based upon both the achievable targets of the ultrasonic device as well as the desired targets for therapy. For example, in the case of blood stimulation within the spleen, kidney, liver, heart or gut, the device may be configured for targeting a wide range of people with varying organ or tissue depths, and other bodily interferences such as ribs. For certain therapies this can include hundreds or thousands of phasing algorithms that may be used in a raster pattern (e.g., switched) very quickly, possibly hundreds of programs within 1 or a few seconds.

[0091] The phased array transducer can be designed to produce a customized ultrasonic beam profile with optimum energy or pressure profile (i.e., peak pressure, depth, beam area etc.).

[0092] The electrical system may include multi-channel beam forming microprocessor chips for 32 individual channel control for phasing and focusing. The wearable device may be wired to a power adapter that can accommodate standard electrical power inputs (i.e. 100- 240 V, 50 / 60 Hz).

[0093] In some configurations, the system includes a small wearable form factor, low cost, and amenability to scalable manufacturing. Existing instruments used for focused ultrasound medical applications are significantly more expensive; standard multi-channel systems routinely cost between $50,000 to $200,000 per unit, with large cart-based or desktop form factors. Conventional wearable ultrasound energy emission devices are designed for other purposes, such as wound-healing, and operate at inappropriate ultrasound frequencies or with only single or few channel operation, incapable of adequate beamforming for focusing. Hand-held medical ultrasound devices are designed for imaging purposes and are also inadequate for the energy delivery requirements of stimulating specific targets in the body or blood cell stimulation, due to the linear array design which can beam form in only one axis or the tendency to operate at high frequencies, such as more than 3 to 10 MHz.

[0094] In some configurations, hybrid systems and transducers may be provided for ultrasonic imaging feedback. As targeting of blood, immune cells, organs, and obstructions is performed, a beam steering mechanism to avoid artifacts in a resulting image can be used in the process.

[0095] The systems and methods in accordance with the present disclosure may be configured to use feedback provided by biomarker sensors, some of which provide immediate or near-term information regarding cytokine levels that may be relevant to the therapy delivered by the system.

[0096] A non-limiting example biomarker sensor includes a portable biosensor system capable of detecting cytokines (e.g., IL-ip, IFN-y, MCP-1, IL-8, TNF-a, NF-KB, IL-6, IL-10, MAPK14, MCP-1) in unprocessed whole blood using filter paper-based immunosensors and smartphone imaging. This biosensor may detect small variations in cytokine levels in under20 minutes. The paper-based immunosensor generates a colorimetric signal and the pixel intensity of the colorimetric signal may be evaluated using the real-time densitometry enabled by the user-guided smartphone application. The biosensor could also be electrochemical aptamer-based sensing platform that measures concentrations of specific molecules directly in blood and even in the living body, or could also be label-free electrochemical impedance immunosensing platform with various types of electrodes. The ultrasound stimulation system can be controlled via Bluetooth using a smartphone app that could also receive information from this biosensing app for cytokines that is housed on the same smartphone device; in some embodiments the biosensing app may be integrated into the ultrasound stimulation system app. Levels of ultrasound stimulation could be adjusted in response to detected increases or decreases in cytokines based on a predetermined algorithm and thresholds, incorporating data from past hours or days of cytokine monitoring.

[0097] A non-limiting example biomarker sensor includes a miniaturized sensor that uses a fluid such as saliva or sweat to measure cytokine or other biomarkers relevant to inflammation levels. These devices may use various technologies, such as light waves (i.e., ultraviolet or infrared).

[0098] Communication with the system may be provided in real-time or delayed using the communication protocols Bluetooth, RFID, near-field, or WiFi. In some configurations, the biomarker sensors may be physically detached from the rest of the system while communicating wirelessly. Direct integration of a biomarker sensor into the system may also be provided. In this configuration, the biomarker sensor may be integrated directly into the device.

[0099] Communication to the system via an intermediary system may also be provided, such as a networked server. The biomarker sensor may be internet-enabled and provide data to a managed server that will then push the data back through the internet to system, which may also be networked accordingly. The data may be processed by the intermediary server or passed to the system directly.

[0100] Examples

[0101] EDTA tubes of whole human blood were collected from healthy volunteers at a commercial blood bank. The blood was mixed 1 : 1 with phosphate buffered solution (PBS)containing 2% fetal bovine serum (FBS). Peripheral Blood Mononuclear Cells (PBMCs) were isolated with centrifugation by floating the Blood + PBS mixture on top of lymphoprep in a 50 mL conical tube then centrifuging. The PBMCs were removed from the resulting layered mixture, put into an additional PBS + 2% FBS solution to wash them, and then centrifuged into a pellet. The pellet was isolated and resuspended in RPMI-1640 culture medium. At this point, the cell suspension was split into 3 conditions: a control, which was not treated with ultrasound (US) or Lipopolysaccharide (LPS), an LPS control, and the experimental LPS with ultrasound (US) therapy (LPS+US). The LPS+US condition was excited with varying levels of ultrasound depending on the experiment, as described below, in a 3D-printed tube resting in a water bath.

[0102] The 3D-printed tube was designed to be compatible with our ultrasound transducer focusing cones, reduce temperature changes, and minimize ultrasound reflections at the tube interface. After ultrasound treatment, all cells were put into RPMI-1640 in a 12- well plate at a dilution of about 0.5 x 106cells / mL. LPS was added to the appropriate wells at varying concentrations and the plate was incubated at 37°C and 5% CO2 for 4 hours. Next, the cell suspensions were transferred to tubes and pelleted. The pellets were lysed with a buffer from the Qiagen RNeasy Mini kit and RNA extraction was completed using the kit. RNA concentrations were quantified with a fluorometric qubit. RNA was then reverse transcribed at the appropriate concentration into cDNA, which is tested using reverse transcription quantitative real-time PCR (RT-qPCR).

[0103] The qPCR data provided was run in technical duplicate. Primers were purchased from the company Integrated DNA Technologies. The primers were mixed with a Sybr Green Master Mix. The gene RPLP0 was used as a housekeeping gene. The cycle threshold (Ct) was measured for each gene of interest, indicating the number of cycles of amplification required for detection. Thus, higher values of Ct indicated fewer copies of a gene transcript present. Ct values were averaged across technical duplicates. A ACt value was calculated by subtracting the housekeeping average Ct from the gene of interest average Ct. This corrects the Ct values relative to a gene (RPLP0) that does not change due to the experimental intervention. Next, the ACt of the control condition (no LPS nor US) was subtracted from the ACt of both the LPS and LPS+US conditions, which provides AACt values for both conditions. The relative fold change of gene expression can be defined as: 2A(-AACt) for both conditions. This foldchange represents a proportion of change from the control condition to the condition of interest. For example, a fold change of 2 for the LPS condition indicates that the LPS sample produced 200% more RNA transcript for the gene of interest than the control condition. Thus, if the ultrasound therapy was to reduce inflammation, the fold change should be lower for the inflammatory cytokines in the LPS+US condition than in the LPS condition. The fold change of LPS+US compared to the control is plotted in FIGS. 6A, 7A, and 8A for three experimental conditions, as described below.

[0104] To visualize the data in another way, we also calculated the fold change of the LPS+US conditions compared to the LPS condition, meaning we subtracted the ACt of the LPS condition from the ACt of the LPS+US condition, then took 2A(-AACt). This fold change shows the change in expression from LPS to LPS+US. In this case, a fold change less than one indicates that the LPS+US condition produced less transcript than the LPS condition did. The fold change of the LPS+US condition compared to the LPS condition is plotted in FIGS. 6B, 7B, and 8B for three experimental conditions, as described below.

[0105] To visualize the data further, we plotted the percent change from LPS to LPS+US. This percent change was calculated by taking the inverse of (1-fold change)* 100% to yield a percentage. For example, a result of -50% indicates that the amount of RNA transcript produced in the LPS+US case was 50% less than the amount produced in the LPS case. The percentage change from LPS to LPS+US is plotted in FIGS. 6C, 7C, and 8C for three experimental conditions, as described below.

[0106] Example 1

[0107] For the US experimental condition, stimulation was applied for 10 minutes using a 1 MHz transducer with approximately 550 kPa negative peak pressure. The pulse width was set to 136 ps with a 2 kHz triggering frequency. For the LPS control and US+LPS experimental condition, inflammation was induced by an LPS concentration of 10 pg / mL in culture. IL-ip, IFN-y, IL-8, TNF-a, and NF-KB cytokine transcripts were measured for all three experimental conditions.

[0108] FIG. 6 A shows the fold change of five inflammatory cytokines (IL-ip, IFN-y, IL- 8, TNF-a, and NF-KB) relative to a negative control for both LPS and LPS+US conditions using RPLP0 as a housekeeping gene. Ultrasound stimulation reduced the upregulation of allfive inflammatory cytokines after an LPS insult. FIG. 6B shows the fold change of each cytokine relative to the LPS-only condition. The application of ultrasound stimulation reduced the upregulation of each inflammatory cytokine after an LPS insult when compared to an LPS-only condition. Similarly, FIG. 6C shows the results as a percentage change of cytokine transcript of LPS+US stimulation relative to LPS alone. All five inflammatory cytokines showed a reduction after ultrasound stimulation.

[0109] Example 2

[0110] For the US experimental condition, ultrasound stimulation was applied for 20 minutes using a 1 MHz transducer with approximately 780 kPa negative peak pressure. The pulse width was set to 136 ps with a 1 kHz triggering frequency. For the LPS control and US+LPS experimental condition, inflammation was induced by an LPS concentration of 10 pg / mL in culture. IL-ip, IFN-y, IL-8, and TNF-a cytokine transcripts were measured for all three experimental conditions.

[0111] FIG. 7A shows the fold change of four inflammatory cytokines (IL- 1 P, IFN-y, IL- 8, and TNF-a) relative to a negative control for both LPS and LPS+US conditions using RPLP0 as a housekeeping gene. Ultrasound stimulation reduced the upregulation of all four inflammatory cytokines after an LPS insult. FIG. 7B shows the fold change of each cytokine relative to the LPS-only condition. The application of ultrasound stimulation reduced the upregulation of each inflammatory cytokine after an LPS insult when compared to an LPS- only condition. Similarly, FIG. 7C shows the results as a percentage change of cytokine transcript of LPS+US stimulation relative to LPS alone. All four inflammatory cytokines showed a reduction after ultrasound stimulation.

[0112] Example 3

[0113] For the US experimental condition, ultrasound stimulation was applied for 10 minutes using a 1 MHz transducer with approximately 550 kPa negative peak pressure. The pulse width was set to 136 ps with a 2 kHz triggering frequency. For the LPS control and US+LPS experimental condition, inflammation was induced by an LPS concentration of 0.01 pg / mL in culture. IL-ip, IFN-y, MCP-1, IL-8, TNF-a, and NF-KB cytokine transcripts were measured for all three experimental conditions.

[0114] FIG. 8 A shows the fold change of six inflammatory cytokines (IL-ip, IFN-y, MCP-1, IL-8, TNF-a, and NF-KB) relative to a negative control for both LPS and LPS+US conditions using RPLPO as a housekeeping gene. Ultrasound stimulation reduced the upregulation of all six inflammatory cytokines after an LPS insult. FIG. 6B shows the fold change of each cytokine relative to the LPS-only condition. The application of ultrasound stimulation reduced the upregulation of each inflammatory cytokine after an LPS insult when compared to an LPS-only condition. Similarly, FIG. 6C shows the results as a percentage change of cytokine transcript of LPS+US stimulation relative to LPS alone. All six inflammatory cytokines showed a reduction after ultrasound stimulation.

[0115] It will be appreciated by those skilled in the art that while the invention has been described above in connection with particular embodiments and examples, the invention is not necessarily so limited, and that numerous other embodiments, examples, uses, modifications and departures from the embodiments, examples and uses are intended to be encompassed by the claims attached hereto. The entire disclosure of each patent and publication cited herein is incorporated by reference, as if each such patent or publication were individually incorporated by reference herein. Various features and advantages of the invention are set forth in the following claims.

Claims

CLAIMSWhat is claimed is:

1. A method of reducing inflammation, comprising: directing ultrasound at a plurality of blood cells, wherein directing ultrasound at the plurality of blood cells causes a reduction of inflammation due to or associated with the plurality of blood cells.

2. The method of claim 1, further comprising: monitoring at least a portion of the plurality of blood cells for a marker of inflammation.

3. The method of claim 1, wherein directing ultrasound at a plurality of blood cells further comprises: directing ultrasound at the plurality of blood cells in a subject.

4. The method of claim 1, wherein directing ultrasound at a plurality of blood cells further comprises: directing ultrasound at the plurality of blood cells in a subject, wherein the plurality of blood cells is disposed within a region of pooled or slow-moving blood reserves.

5. The method of claim 3, wherein directing ultrasound at the plurality of blood cells in a subject further comprises: directing ultrasound at the plurality of blood cells in the subject, wherein the plurality of blood cells is disposed within at least one of the subject’s spleen, liver, kidney, pancreas, lymph nodes, lymphatic tissue, glymphatic tissue, heart, brain, gut, blood vessels, veins, arteries, capillaries, swelling in joints, swelling in tissue, muscle, tumors, eyeballs, bruising or hematomas, outer ear region, inner ear region, gums, nasal cavity regions.

6. The method of claim 3, wherein directing ultrasound at the plurality of blood cells in a subject further comprises: directing ultrasound at the plurality of blood cells in the subject, wherein the plurality of blood cells is disposed within the meninges of the subject’s brain.

7. The method of claim 3, wherein directing ultrasound at the plurality of blood cells in a subject further comprises: directing ultrasound at the plurality of blood cells in the subject without directing ultrasound at neural tissue in the subject.

8. The method of any one of the preceding claims, wherein the plurality of blood cells comprises immune cells.

9. The method of any one of the preceding claims, wherein the plurality of blood cells comprises peripheral blood mononuclear cells (PBMCs).

10. The method of claim 9, wherein the PBMCs comprise at least one of T cells, B cells, NK cells, monocytes, or dendritic cells.

11. The method of claim 2, wherein the marker of inflammation comprises at least one of ESR, CRP, ferritin / d-dimer, IL-1 , IFN-y, MCP-1, IL-8, TNF-a, NF-KB, IL-6, IL- 10, MAPK14, and MCP-1.

12. The method of any one of the preceding claims, wherein directing ultrasound at a plurality of blood cells further comprises: directing ultrasound at the plurality of blood cells using a 1 MHz transducer.

13. The method of claim 12, wherein directing ultrasound at a plurality of blood cells further comprises: directing ultrasound at the plurality of blood cells using the 1 MHz transducer at 780 kPa pressure, 136 ps pulse width, and 1 or 2 kHz repetition frequency for 20 minutes.

14. The method of claim 12, wherein directing ultrasound at a plurality of blood cells further comprises: directing ultrasound at the plurality of blood cells using the 1 MHz transducer at 550 kPa pressure, 136 ps pulse width, and 1 or 2 kHz repetition frequency for 10 minutes.

15. The method of any one of the preceding claims, wherein directing ultrasound at a plurality of blood cells further comprises: directing ultrasound at the plurality of blood cells using a wearable transducer.

16. The method of any one of the preceding claims, wherein, prior to directing ultrasound at a plurality of blood cells, the method comprises: performing imaging of a subject to identify the plurality of blood cells.

17. The method of claim 16, wherein performing imaging of a subject to identify the plurality of blood cells further comprises: performing imaging of the subject using ultrasound.

18. The method of any one of the preceding claims, wherein directing ultrasound at a plurality of blood cells further comprises: directing ultrasound at the plurality of blood cells in a subject, wherein the plurality of blood cells is disposed in at least two different locations within the subject.

19. The method of claim 18, wherein directing ultrasound at a plurality of blood cells further comprises: directing ultrasound at the plurality of blood cells in a subject in at least two different locations of a blood vessel, wherein the ultrasound is directed at the at least two different locations in the blood vessel based on a rate of flow of blood through the blood vessel.

20. The method of claim 19, wherein directing ultrasound at the plurality of blood cells in a subject in at least two different locations of a blood vessel further comprises: directing ultrasound at the plurality of blood cells in the subject in the at least two different locations of the blood vessel based on adding a tracer substance to the blood.

21. The method of claim 1, wherein directing ultrasound at a plurality of blood cells further comprises: removing the plurality of blood cells from a subject, directing ultrasound at the plurality of blood cells outside of the subject, and returning the plurality of blood cells to the subject.

22. The method of claim 1, wherein directing ultrasound at a plurality of blood cells further comprises: removing the plurality of blood cells from a first subject, directing ultrasound at the plurality of blood cells outside of the first subject to produce a treated plurality of blood cells, and administering the treated plurality of blood cells to a second subject.

23. The method of claim 22, wherein the first subject is one of a first human subject or a first animal subject, and wherein the second subject is one of a second human subject or a second animal subject.

24. The method of claim 1, wherein directing ultrasound at a plurality of blood cells further comprises: directing ultrasound at the plurality of blood cells in an in vitro environment.

25. The method of claim 1, wherein directing ultrasound at a plurality of blood cells further comprises: directing ultrasound at the plurality of blood cells in an in vivo animal model.

26. A system configured to reduce inflammation, comprising:an ultrasound transducer configured to direct ultrasound at a plurality of blood cells to cause a reduction of inflammation due to or associated with the plurality of blood cells.

27. The system of claim 26, further comprising: a sensor configured to monitor at least a portion of the plurality of blood cells for a marker of inflammation.

28. The system of claim 26, wherein the transducer, when directing ultrasound at a plurality of blood cells, is further configured to: direct ultrasound at the plurality of blood cells in a subject.

29. The system of claim 26, wherein the transducer, when directing ultrasound at a plurality of blood cells, is further configured to: direct ultrasound at the plurality of blood cells in a subject, wherein the plurality of blood cells is disposed within a region of pooled or slow-moving blood reserves.

30. The system of claim 28, wherein the plurality of blood cells is disposed within at least one of the subject’s spleen, liver, kidney, pancreas, lymph nodes, lymphatic tissue, glymphatic tissue, heart, brain, gut, blood vessels, veins, arteries, capillaries, swelling in joints, swelling in tissue, muscle, tumors, eyeballs, bruising or hematomas, outer ear region, inner ear region, gums, nasal cavity regions.

31. The system of claim 28, wherein the plurality of blood cells is disposed within the meninges of the subject’s brain.

32. The system of claim 28, wherein the transducer, when directing ultrasound at the plurality of blood cells in a subject, is further configured to: direct ultrasound at the plurality of blood cells in the subject without directing ultrasound at neural tissue in the subject.

33. The system of any one of the preceding claims, wherein the plurality of blood cells comprises immune cells.

34. The system of any one of the preceding claims, wherein the plurality of blood cells comprises peripheral blood mononuclear cells (PBMCs).

35. The system of claim 34, wherein the PBMCs comprise at least one of T cells, B cells, NK cells, monocytes, or dendritic cells.

36. The system of claim 27, wherein the marker of inflammation comprises at least one of ESR, CRP, ferritin / d-dimer, IL-1 , IFN-y, MCP-1, IL-8, TNF-a, NF-KB, IL-6, IL- 10, MAPK14, and MCP-1.

37. The system of any one of the preceding claims, wherein the ultrasound transducer comprises: a 1 MHz transducer.

38. The system of claim 37, wherein the transducer, when directing ultrasound at a plurality of blood cells, is configured to use the 1 MHz transducer at 780 kPa pressure, 136 ps pulse width, and 1 or 2 kHz repetition frequency for 20 minutes.

39. The system of claim 37, wherein the transducer, when directing ultrasound at a plurality of blood cells, is configured to use the 1 MHz transducer at 550 kPa pressure, 136 ps pulse width, and 1 or 2 kHz repetition frequency for 10 minutes.

40. The system of any one of the preceding claims, wherein the ultrasound transducer comprises a wearable transducer.

41. The system of any one of the preceding claims, further comprising an imaging system configured to perform imaging of a subject to identify the plurality of blood cells.

42. The system of claim 41, wherein the imaging system comprises an ultrasound imaging system.

43. The system of any one of the preceding claims, wherein the plurality of blood cells is disposed in at least two different locations within the subject.

44. The system of claim 43, wherein the transducer, when directing ultrasound at a plurality of blood cells, is further configured to: direct ultrasound at the plurality of blood cells in a subject in at least two different locations of a blood vessel, wherein the ultrasound is directed at the at least two different locations in the blood vessel based on a rate of flow of blood through the blood vessel.

45. The system of claim 44, wherein the transducer, when directing ultrasound at the plurality of blood cells in a subject in at least two different locations of a blood vessel, is further configured to: direct ultrasound at the plurality of blood cells in the subject in the at least two different locations of the blood vessel based on adding a tracer substance to the blood.

46. The system of claim 26, wherein the transducer, when directing ultrasound at a plurality of blood cells, is further configured to: direct ultrasound at the plurality of blood cells outside of the subject after the plurality of blood cells has been removed from the subject.

47. The system of claim 26, wherein the transducer, when directing ultrasound at a plurality of blood cells, is further configured to: direct ultrasound at the plurality of blood cells in an in vitro environment.

48. The system of claim 26, wherein the transducer, when directing ultrasound at a plurality of blood cells, is further configured to: direct ultrasound at the plurality of blood cells in an in vivo animal model.

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