HEAT ANTIVIRAL TREATMENT
The invention refers to the field of medicine. More specifically, it relates to a method for treating viral infections, which comprises applying heat to an organ or part of the body comprising a viral load, apparatuses capable of applying heat to said organ or body part and uses thereof. In particular, the invention is applicable to the combat or treatment of viral infections of organs such as the skin and internal organs and parts of the body such as those of the respiratory system (lungs), the female genital tract (e.g., cervix), limbs of the body such as the hands, feet, and male and female sexual organs such as the penis, the scrotal sac, the vulva, and pubic areas. Viruses are obligate intracellular parasites and require the biochemical machinery of the host cell to replicate and spread. The genetic matepal of the virus is typically enclosed in a protein capsid, which in some cases may be surrounded by a lipid envelope. Viruses are extremely diverse in terms of their genetic structures and complexities; some have RNA genomes that encode only a few genes while others have DNA genomes that encode up to 200 genes. Viruses bind to host cells via specific receptors. Immediately after entry to the host cell, the viruses are stripped, nucleic acid released and expressed to give viral proteins. The viral genome replicates and new virus particles (virions) of progeny are assembled and released and they continue to infect neighboring cells. Viral entry into cells is commonly done at mucosal sites. Replication typically occurs on epithelial surfaces, although this may occur in the case of certain viral infections by viremia, ie virus particles enter the systemic circulation and spread through it to infect other tissues. The eukaryotes have developed a number of defense antimicrobial mechanisms based on the recognition of conserved molecular patterns that are shared by large groups of microorganisms, including viruses. The double-stranded RNA (dsRNA) represents one such molecular pattern. In mammalian cells, a defense response that is induced by dsRNA is the induction of interferons, a class of proteins that can reduce viral spread by inhibiting gene expression and causing apoptosis of infected cells. Another antiviral response that is induced by dsRNA is termed RNA interference (RNAi) in animals and the silencing of post-transcriptional gene (PTGS) or RNA silencing in plants (Fire et al., Nature 1998, 391, 806-81 1; Ding, Curr, Opin, Biotechnology 2000, 1 1, 152-156; Hutvánger and Zamore Curr, Opin. Gen. Dev. 2002, 12, 225-232). The key feature of RNAi is its remarkable sequence specificity: the infected organism responds to dsRNA by selectively degrading mRNAs that are homologous in sequence to the dsRNA inducer. Therefore, RNAi can be used to specifically block the expression of not only viral genes, but also of the host cell by the introduction of a homologous dsRNA. The central initiator molecules in RNA silencing are (long) molecules of dsRNA, which are degraded by a target nuclease of dsRNA, indicated for fragments 21 to 23 nt. These short interfering RNAs (siRNAs) subsequently search for homologous cognate RNAs for degradation mediated by the RNA initiated silencing complex (RISC), resulting in a low amount of target cytoplasmic RNA. If the target RNA is either a viral genome, an antigenome or a mRNA, RNA degradation induced by RNAi can specifically inhibit, or even completely block, viral replication. Conversely, if the target RNA is of cellular origin, then the RNA can post-translationally block the expression of the cognate gene. To overcome RNA silencing, it has been found that a number of plant viruses encode RNA silencing suppressors (RSS). The published patent applications having the international publication numbers WO 98/44097, WO 01/38512 and WO 02/057467 describe RNA silencing suppressors derived from plant viruses and their use in plants. WO 02/057301 describes an RSS derived from plant viruses and its use in animal cells, while Li et al. , Science 296: 1319-1321, 2002, describes an RSS derived from insect virus and its activity in plant and insect cells. The use of RSS derived from vertebrates and their use in animal cells was described in the international publication WO 04/035796. A study by Li et al. (Proc. Nati, Acad. Sci. USA 2004, 101, 1350-1355) showed that the NS1 and E3L proteins encoded by the influenza and vaccine viruses, respectively, of mammals were able to suppress the silencing of antiviral RNA in cells of Drosophilae, which strongly supports a role for RNAi as a natural antiviral response, not only in plant cells but also in other non-plant eukaryotic cells. An additional defense of the host against viruses is fever.
It is commonly thought that fever takes advantage of the fact that many invading organisms tolerate a narrower temperature range than the tissues of the body and, therefore, are more susceptible to temperature increases, that is, they can die from the Overheating before bathing human tissues. Fever also stimulates the immune system by increasing the production of antibodies and interferon. Several studies have shown that in vitro viral replication can be suppressed by increases in temperature. For example, Sweet et al. , (Br. J. Exp. Path. 1938, 59, 373-380) described that influenza A virus developed in cultures of nasal turbinate organ (turbinate) was inactivated at pyrexial temperatures. The findings of Sweet et al., Were supported by the results of studies in ferrets that were infected with the virus. In some animals, the fever induced by the viral infection was suppressed by shaving the ferrets or by treatment with an antipyretic drug. It was observed that the mean viral titre in nasal washes decreased less rapidly in animals with a reduced temperature compared to control animals. who developed fever (Husseini et al., (1982) J. of Infectious Diseases, Voí. 145, 520-524). Thus, a correlation between body temperature and viral titers of Influenza A virus was observed. Conti et al. (Antimicr Agents and Chemoth, 1999, Vol 43, 822-829) studied the effect of a brief hyperthermic (HT) treatment (20 minutes, 45 ° C) on the replication of human rhinovirus in HeLa cells. It was observed that HT suppressed viral replication in vitro by more than 90% when applied to specific stages of the virus replication cycle. The replication of the viruses that cause the common cold, including rhinovirus and coxsack? Evirus, occurs mainly in the epithelial cells that line the upper respiratory tract. Vapor inhalation is well known in home remedies for upper respiratory tract infections. This typically involves keeping the head under a towel over a bowl of very hot water for a period of time that typically ranges from 5 to 20 minutes. A nasal decongestant or other aromatic compound can be added to the water. Aromatic oils gently irritate the mucous membranes of the respiratory tract, increasing the secretion of mucus and saliva, which can relieve sore throat and nasal congestion. An apparatus for these applications is known as Rhipoíherm ™. The invention described in the published patent application, publication number WO 03/97143 discloses an alternative apparatus for relieving common colds, which blows a negative ionized stream of heated air to the nasal cavity. While vapor inhalation is commonly used to relieve some of the symptoms of common colds, the evidence that vapor inhalation has some beneficial effect on the course of the disease is very limited. A review of the use of heated humidified air (involving one or more treatments of 20 to 30 minutes) for the common cold concluded that although there was evidence that the symptoms were relieved, there was no objective improvement in the measures produced, such as viral spill or viral titers in nasal washes (Singh M. Hot Air, Humidified for the Common Cold (Cochrane Review), In: The Cochrane Library, Number 2, 2004). Thus, while an inhibitory effect of hyperthermia on viral replication in vitro has been convincingly shown, the therapeutic value of heat in combating viral infections in vivo has thus remained very elusive. The present inventors have surprisingly observed that at increased temperatures, i.e. temperatures above normal body temperature, the activity of RNAi in a population of animal host cells increases compared to that in a similar population of cells. Hosts animals at normal body temperature. Based on this knowledge, the inventors hereby assumed that a heat treatment of an organ or part of the body infected with virus should have an inhibitory effect on the replication of the virus in vivo, which may be sufficient to combat or treat infection with the virus. A sufficient level of inhibition of virus replication requires heating to at least 2 ° C above the normal physiological temperature of the organ or part of the body for a period sufficient to induce protein synthesis of heat-induced genes. These conditions led to a prolonged increase of RNAi in the tissue, which implies that the ability to silence genes via RNAi remains elevated for up to about 96 hours or more, and generally for between about 6 hours to about 96 hours. Typically, the higher the temperature, the shorter the time interval required for the temperature, provided that the higher temperature is not so high as to be detrimental to the viability of the tissue exposed to the heating temperature. (in comparison with the pre-traffic situation). According to the present invention, a method is provided for irradiating a viral disease in a mammal that requires irradiation, which comprises calenating virus-infected cells of said mammal with a heating apparatus to a temperature period during the millennium. of at least 2 ° C higher than that normal physiological femperaíura of the body for said íejido. Preferably, the period is carried out at a femperairy from 2 ° C to approximately 13 ° C above the normal temperature of the material to be treated. The types of drug to which the method of the invention can be applied include virus-infected tissues comprising cells harboring virus particles from organs and body parts, such as epithelial cells of the lungs, nasal passages, trachea, alveoli and cells. similar, and body parts, such as the feet, hands, skin, sexual organs and parts thereof, such as the penis and scrotum, the vulva and other sexual organ arquifera, as appropriate. Also included are the cells of organs such as the kidneys, the liver, the heart, and the uterus, including parts of the cells, such as the cervix, the clitoris, the vulva. both labia majora and labia minora), the surrounding perineum and infected skin. In accordance with the present invention, there is provided a method for inhibiting the replication of a virus in cells infected with tissue virus from a mammalian subject, in vivo, which comprises heating said virus infected tissue at a temperature of at least 2. ° C, but not more than 13 ° C, preferably from at least 2 ° C to about 5 ° C above the normal physiological temperature of said tissue for a duration of at least 1 hour, preferably 1 to 4 hours , more preferably from 1 to 2 hours, most preferably from 1 to 1.5 hours. In a further preferred embodiment of the method of the invention there is provided a method for inhibiting virus replication in vivo in a tissue infected with virus from a mammalian subject, comprising heating said tissue to a temperature of at least 6 ° C, but not more than 9 ° C above its normal physiological temperature for up to 45 minutes so that apoptosis is not induced in the tissue, preferably from about 10 minutes to about 45 minutes. In a further preferred embodiment of the method of the invention, the heating temperature employed can be from at least 10 ° C, but not more than 13 ° C above the normal physiological temperature of the tissue being treated for a duration of less than 10 minutes. minutes so that apoptosis is not substantially induced in the heated tissue. It is thought that this time interval is sufficient to induce the synthesis of proieins of genes induced by heat. Infections with viruses occur more frequently in the respiratory tract than in any other organ. This could be expected when considering the intense and constant direct contact that these tissues have with the physical environment and thus the exposure to microorganisms, such as viruses. In accordance with the concept of the present invention, it is thought that the magnitude of the antiviral RNAi response correlates with the increase in temperature: the increased temperatures increase the RNAi, while the RNAi is suppressed relatively at lower temperatures. The temperature in the cells of the respiratory tract is usually lower than that of other parts of the body, except the temperature of specialized body parts, such as the skin and testicles.
Respiration also results in local cooling of the respiratory tract (upper and lower respiratory tract) to a temperature below the central body temperature. This is thought to be the reason why the respiratory tract seems more sensitive to viral infections than other body parts. Due to the lower temperatures of the cell in cells of the upper respiratory tract and the skin, the response of antiviral RNAi is reduced in comparison with that of cells in other body parts, so that the chance of establishing a infection with virus. In a preferred embodiment of the invention, the mammalian subject that is brought is a human being suffering from a viral infection. As the normal (basic) physiological temperature of a human being is about 37 ° C, the heat treatment of the invention typically comprises heating the tissue to a temperature of at least 39 ° C. However, higher temperatures are preferred that they will inhibit viral replication more efficiently. On the other hand, very high temperatures of human tissues (eg, more than 42 ° C) for a long time, such as more than about 1 hour can prevent the increase of RNAi or can even induce cell death. The induction of proteins by heat shock by heat treatment (in vivo) is well known in the art. The induction of a protein by heat shock (HSP) can then be taken as an indicator for a heat treatment according to the invention that increases the RNAi adequately without causing the death of the cell. Thus, very severe heat treatments that do not induce proteins by heat shock (eg, HSP70) should be avoided in the application of this invention. For example, in a calorie protocol of the invention a human tissue can be heated to a temperature that falls within the range of 37 to 42 ° C, preferably 39 to 42 ° C, for a time interval of at least one hour, or in a heating protocol different from the invention, a human tissue can be heated to a temperature falling within the range from 43 to 46 ° C, during a time interval of not more than one hour, preferably not more than about 15 to 45 minutes. The temperature of the fabric to be treated with the method of the present invention is typically less than that temperature of the heated gas or heated liquid that provides heat to the fabric. The normal physiological temperature of a tissue to be treated can deviate significantly from the normal physiological body temperature of a subject. In these cases, the heating temperature can be adjusted accordingly. For example, the temperature of the nasal passage in humans is 33 ° C. In addition, a study of the thermal map of the respiratory tract in humans showed that during calm breathing of ambient air, the average temperature in the respiratory tract fluctuates from 32 ° C in the upper trachea to 35.5 ° C in the sub-segmental bronchi (McFadden et al., J. Appl. Physiol. 1985; 58 (2): 564- \.
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na ro toloc taurlnc ncir ai-smr \ lno "57 ° (" * "" &> 55i 0 r * on ol f »p cn? jo MG¡- g¡j! o ^ human ^ to be suficiept0" or thus, to reach the inhibition of the viral turnover in a tissue with a normal temperature below the basic body temperature The method of the invention for increasing the antiviral response of the host cell by increasing the degradation of viral nucleic acids via the RNAi machinery of the host cell, it is not limited to any specific type of virus.In fact, any virus that is susceptible to RNAi-mediated degradation of its mRNA or genome or a part thereof will be sensitive to the heat-induced or heat-enhanced RNAi according to the invention., a method as described herein, is used to inhibit the replication of a virus that can enter mucosal sites and / or replicate in epithelial cells. Examples of such viruses include respiratory viruses and herpes viruses. Respiratory viruses are those that cause upper and / or lower respiratory tract infections, including interstitial pneumonia. Upper respiratory tract infections include the common cold (rhinitis), influenza, laryngitis (inflammation of the oral cavity), pharyngitis (sore throat), sinusitis, íonsilifis, measles, and croup (in children). There are more than 20Ó viruses that can cause upper respiratory tract infections. These types of viruses are extremely l?
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Parainfluenzavirus and the Syncyfial Respiralorio Virus (RSV). Both are RNA viruses, which are significant causes of respiratory infection in infants and young children. The initial lesion is bronchiolysis, sometimes necrotic, and less frequently, interstitial pneumonia. The Ebola virus is perfect for Filoviridae and can be sprayed by aerosols. It has been shown that aerogenic infected animals show Ebola virus amphigens associated with the cell present in the epidermis of the respiratory tract, alveolar pneumocytes, and macrophages in the lung and pulmonary lymph nodes; The extracellular anigen was present on the mucosal surfaces of the nose, oropharynx and airways. Coronaviruses are more strand RNA viruses and include uai co | J U CU C I I? Ouoai 111 ic? I? I ico
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R nano / ins a n a l o n o n o n o n o n o n o n o n o r n a n d C h a n a l a r a n a n a n a n a l a n i n a r i n a r i n a r i n a r i n a r i n a tive, it causes pain or an itching in the area supplied nor the affected sensory nerves. These areas are known as dermatomas. Slowly replicating beta herpes viruses are represented by cytomegalovirus (CMV) and human herpesvirus 6 0 and 7. CMV and mainly infect epithelial and endothelial cells. It causes sub-clinical infection in the lung of immunocompetent hosts. and clinical infection in immunocompromised patients. Infection of the lung can result in focal or diffuse inters- sential pneumonia with or without hyaline membrane formation, in-alveolar exudates, hemorrhage, and fibrosis. Human papilloma viruses (HPVs) are members of the genus Papilomavirus of the family Papovaviridae. The HPV genome consists of double-stranded circular DNA conformed by an icosahedral capsid. They are recognized more than 70 HPV types. 0 HPV infections of the genital tract can cause the development of cervical cancer, and when they are irradiated into the respiratory tract of a newborn child, it can be resulfed in papillor alosis. HPV infections can also cause warts on the skin and in the genital, digestive and respiratory tracts. Examples of viruses whose replication is most conveniently deleted using a method of the invention include orlhomyxovirus, influencer virus, paramyxovirus, parainfluence virus, respiratory syncyfial virus (RSV), filovirus such as Ebola virus, coropavirus such as hMPV virus and SARS, picornaviruses such as rhinoviruses, Coxsakie A and B viruses, herpesvirus isolates such as HSV-1, HSV-2, VZV, CMV and HPV. In a preferred embodiment, especially in those cases where the tissue resides in the respiratory system, said tissue is heated by exposing the tissue to a heated and humidified gas, capable of heating the tissue. Thus, in a method of the invention, the duration of the heat transfer is at least two times longer compared to known vapor inhalation treatments, for example those included in the Cochrane study supra, where the subject typically receives a For 20 to 30 minutes, optionally followed by additional days with a 1 to 2 hour interval. The present invention demonstrates that a single heat treatment using a humidified humidified air mixture at 43 ° C for one and a half hours is sufficient to induce the inhibition of viral replication in vivo. The fraiamienfo, of course, can be repeated after some time, for example after one or more days or a week. A humidified gas refers to a physiologically acceptable gas with a relative humidity (RH) of at least 70%, preferably at least 85%, more preferably at least 90%, such as 95, 96, 98, 99 or 100% Preferred gases include air, I /
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so that the texture of the said tissue is raised by at least 2 ° G not above its normal physiological temperature. In a preferred embodiment, the tissue is a mucosal tissue heated by contact with a hot gas and humidified tissue The mucous tissue (or mucous membrane) covers some organs and body cavities (such as the nose, mouth, lungs) and secretes mucus (a thick fluid) to prevent it from drying out. Tissue types, mucous membranes are more susceptible to infection with infectious microbes, including viruses, because they lack the physical barrier of an intact foot.For example, many microbes have access through the epithelium of the gastrointestinal, urogenital or respiratory tract. Because the heated gas is humidified the condensation of water vapor from the heated gas on the surface of the mucosal tissue aids in the transfer of heat to the tissue, in addition, the evaporation of moisture (mucus) of the mucosal tissue surface during hyper-thermal rash, and the loss of heat from the tissue associated with it, it is thought that both are minimized within a period of the present invention. In a preferred embodiment, the gas heated is 100% saturated with water. The temperature of the heated and humidified gas for use in a method of the invention will, of course, depend on several fc = tcrse, including? At the desired temperature of! tissue to be subjected to the hyperthermic treatment. the mode and route of administration! gas and the thermal sensitivity of! (of the) fabric (s) that are exposed to! gas. So, at the temperature of! Humidified gas to be delivered to! Target object can be from 40 ° G to 50 ° C- from "reference from 42 ° C up to 48 ° C, more preferably from 42 ° G up to 46 ° C, most preferably from 43 ° G up to 44 ° C. Inhalation of hot air from 43 ° G to 44 ° G and 100% saturated with water for 1.5 hours seems especially suitable for increasing tissue temperature of the respiratory tract by at least 2 ° C in such a way that the RNAi increases. At the same time, minimize the risk of avoiding the suppression of RNAi and / or cause cell death induced by a trauma with very severe temperature (for example, heating above 42 ° C for a substantial period of time, such as However, other types of heat treatment according to the invention may require a higher or lower temperature of the introduced gas depending on the viral infection to be treated and the tissue in which the virus resides. , a method of the invention n inhibits viral replication in tissue located within the respiratory tract. The respiratory tract consists of an upper and a lower part. The upper respiratory tract (airway or upper airway) consists of the nose, ears, mouth, sinuses, and throat. The lower respiratory arm consists of the trachea, the bronchial tubes and the structures within the lungs. When breathing, air is introduced through the nose or mouth and into the rachis (trachea). The trachea is divided in its bottom end into two bronchi, one for each lung. The mucus in the bronchi serves to prevent the tissue from drying out and traps and is coated with dust particles so that P.Q will pick or infect the delicate tissues of the lungs. The bronchi are divided in the lungs into smaller branches called bronchioles. The bronchioles branch into alveoli which are tiny, muitiiobuted air sacs made of simple squamous cells. The thin wall of the alveoli allows the exchange of air with the equally thin-walled capillaries of the circulatory system. In order to function properly, the alveoli must always remain moist. Although most respiratory infections are in the upper airways, several types of microbial agents also damage the lung. Viral infections predominate in the upper airways. In a preferred embodiment, a method of the invention is used to combat a viral infection in the respiratory tract of a subject in need of such treatment. For this purpose, the infected tissue of the respiratory tract (including the trachea, bronchi, bronchioles and / or alveoli) is put in contact with a heated and humidified gas, such as air or oxygen, for a period of at least one hour. In a method of the invention for inhibiting viral replication in the respiratory tract, a heated, humidified gas is conveniently administered to a subject by inhalation, which can be by spontaneous or mechanical ventilation. Any type of device or apparatus known in the art that is suitable for providing a person with hot moist gas (usually air or oxygen) at a controlled temperature can be used. These include invasive devices, such as an endolracheal tube. Of course, a non-invasive device is preferred. Parficular use in a method of the invention for inhibiting viral replication in the respiratory tract is a respiratory rewarming apparatus typically employed to reheat inhalation of hypothermia. See for example Morrison et al., 1979 J. Appl. Physiol. 1979, 46 (6): 1061 or the RES-Q-AIR non-invasive rewarming system from RESQ Products Inc., in Sooke, B.C., Canada at www.hypothermia-ca.com. Generally speaking, a reheating apparatus for inhalation comprises a water bath, a humidifier and heat exchanger unit, a temperature control and a display unit, a gas intake port and an outlet for the heated, humidified gas. The subject can aspirate the gas through a mouth and nose piece or facial mask. The flow regime may vary, depending on the subject and other conditions. In a preferred embodiment, the subject inhales heated and humidified gas at a flow rate of 10 to 80 l / minute, preferably 15 to 50 l / min, more preferably 20 to 35 l / minute. In one embodiment, the subject inhales the hot and humidified gas with an increased ventilation regime. The ventilation regime of a subject at rest is typically around 10 l / min or even less. To that regime the heating of the tissues, especially the tissue that is deeper (lung) may be insufficient to reach the desired temperature. In a preferred embodiment, the subject inhales the hot and humidified gas while hyperventilating. Hyperventilation is a state in which there is an increased amount of air entering the pulmonary alveoli. The inhalation of hot and humidified gas in a method of the invention in a hyper-venilation sifuation that maximizes respiratory heat exchange. The heat can penetrate the tissue that is deeper (lung). The subject can be asked to spontaneously hype / entile, however this can lead to dizziness. Alternatively, a situation of controlled hyperventilation can be created (for example, aided with CO2). For example, the apparatus may comprise a recirculation circuit so that the expired gas can be recirculated through the heat exchanger. In a preferred embodiment of the invention, the subject first inhales the hot and humidified gas without added CO2 to heat the upper respiratory tract. After some time, for example 10 to 30 minutes, the CO2 can be added to the gas (for example via the recirculation of expired air or a gas cylinder) to help hyperventilate the subject and maximize the heat transfer of the subject. gs to the tissue (preheated). The pressure of the aspirated CO2 (pCO2) is preferably in the range of 5 to 7.5 kPa, more preferably from 6 to 7 kPa. The venililation regime of the subject can be increased by performing the subject's exercise. The use of exercise equipment, such as a training bike or homemade roller band, can help the subject increase its ventilation regime. In a preferred embodiment, a method of the invention comprises the hyperthermal treatment of the respiratory tract using a hot gas which is 100% saturated with water or H2O pressure (pH2O) of at least 6.5 kPa, more preferably at least 7.5 kPa . This maximizes the heat capacity of the gas, delivers heat to the fabrics through condensation and minimizes heat loss through evaporation. The gas saturated with water may be in the form of a nebulized spray or a vapor. Sprays are distinguished from gases containing water vapor by the presence of liquid particles other than small diameter, typically 1 to 5 microns. Preferably, a gas comprising steam is used in the method of the invention. For a maximum effect of the inhaled hot gas, it is preferred that the subject minimize the inhalation of cold (ambient) air following the heat inhalation procedure. Preferably, warm, humidified air is inhaled before going to bed. In one embodiment, cold sores (fires) can be treated using the method of the invention by heating the infected tissue (i.e., the lip / mouth region) of a subject in need of tracing, at a temperature of At least 2 ° C above the normal temperature of said tissue last a period of at least one hour using a hot, humidified gas. This is easily accomplished by using an overheating apparatus for inhalation, as described herein, which is equipped with a face mask large enough to cover the infected area. In a further aspect of the invention, there is provided the use of an apparatus of the invention, as described herein, to deliver a hot and humidified gas to a tissue or body part to inhibit viral replication in vivo. The device is capable of producing humidified gas (for example air) at temperatures that fluctuate from 37 ° C to 50 ° C, and with a relative humidity of up to 100%, depending on the viral treatment that is carried out. More preferably, the apparatus provides a heated gas containing water vapor. In a preferred embodiment, an apparatus is provided for heating the epithelial tissue of the respiratory tract of a subject suffering from a viral infection, at an temperature of at least 2 ° C above the normal physiological temperature of said tissue. for a period of time of at least one hour. Preferably, an apparatus equipped with an air or gas recirculation circuit is used to allow hyperventilation to occur and thereby maximize heat exchange between the hot gas and the epithelial tissues of the respiratory tract. Alternatively, an apparatus of the invention is equipped with a simple CO2 cylinder which in operation allows the controlled addition of CO2 to the inhaled gas, such as air or O2, thus causing the subject to hyperventilate in response to the increased level of CO2 in the inhaled gas
In a specific embodiment of the invention, there is provided a method for inhibiting the replication of herpes virus in tissues of a mammalian subject, such as a human being, that are infected with herpes virus. According to the teachings of the invention, viral replication in the affected tissues can be inhibited by heating such tissues to at least 2 ° C above their normal temperature (for example 39 to 42 ° C) during at least one hour This is easily achieved by heating (locally) the affected tissues, for example, by using heated pads or plasters placed in or very close to the affected tissues. Preferably, these pads or plasters contain a gel material that is rapidly shaped into a shape that allows close contact with the affected area with virus. Such gel pads or plasters comprising gels can be maintained at a suitable temperature (for example 40 to 43 ° C) with a thermostat regulated heater. This regulated thermostatic heater can be used in these gel pads or plasters. Alternatively, exothermic patches such as ThermaCare Air-Activated Heat Wraps (available from Procter &Gamble) can be used. In a further aspect of the invention, there is provided the use of heated elements, such as pads, to heat the virus infected tissue of a subject at a temperature of at least 2 ° C above the normal physiological temperature of said tissue during a period of at least one hour. Preferably, an apparatus is used that is equipped with a thermostatically regulated heater which can be built into the pads. It is preferred to start the antiviral treatment with heat, as provided herein, as sas possible following the initial signs and symptoms of virus infection. If the rep is deleted. Viral infection at an early stage after infection increases the likelihood that the virus will be successfully eradicated by the body's immune system. In the case of a respiratory infection, an irritated nose or a scratchy throat is typically the first sign of infection, followed by hours of sneezing and a liquid nasal discharge. Other symptoms of viral infection (such as herpes) include tingling, itching, burning or redness of the skin at the site of infection. It is further preferred to initiate antiviral trafficking with heat, as provided herein, as sas possible following a known or suspected exposure to infection with a virus. If viral replication is suppressed at an early stage after infection, the likelihood that the virus will be successfully eradicated by the body's immune system will increase and the infection will not progress beyond the sub-clinical stage. Thus, the treatment can be applied as a prophylactic treatment. The concept of the present invention is based on the inhibition of viral replication through prolonged increased RNAi in the tissue, which means that the ability to silence genes via RNAi remains elevated for more than 6 and usually less than 96 hours (compared to the pretreatment situation) after the appropriate heat treatment is stopped. Thus, for the treatment of chronic viral infections preferably the treatment is repeated at regular intervals. In a specific embodiment of the invention, there is provided a method of treating cervical dysplasia which comprises applying heat to an area of the cervix using a heating apparatus of the invention, as described herein. Cervical dysplasia is an abnormal cell growth in the cervix. Cell growth is considered abnormal when some cervical cells appear immature compared to their neighbors that appear normal. Immature cervical cells also divide faster than expected, and their nuclei show specific types of microscopic change. Depending on the nomenclature used for different dysplastic conditions, cervical dysplasia can also be referred to as squamous intraepithelial lesion (SIL) or cervical intraepithelial neoplasia (CIN). Cervical dysplasia is a major health problem because it can progress to cervical cancer. Cervical dysplasia has also been linked to a very common virus called human papillomavirus (HPV). There are more than 70 strains of HPV and more than a third of them can be transmitted sexually. Some strains cause warts, including genital warts, while others can lead to cancer. In particular, HPV-16 and 18 are involved in cervical dysplasia and cells infected with viruses can progress slowly to malignancies under the weight of episomal HPV DNA expression (Meisheimer et al., Clin Cancer Res. 2004, 10 : 3059-3063). Cervical dysplasia can be diagnosed with Pap smears and when diagnosed at an early stage, cervical asia / can be treated to prevent it from developing into cancer. When a Pap smear is examined under a microscope and the abnormal cervical cells involve the lower third of the specimen, the condition is deciphered as "soft" cervical dysplasia (SIL or low grade CIN 1). If the abnormal cervical cells are found in larger portions of the specimen, the condition is deciphered as "moderate" or "severe" cervical splasia (SIL or high grade 2 or 3 CIN). Treatment for cervical dysplasia varies from one woman to another, depending on the location and size of the lesion, and whether it is low-grade or high-grade. Known therapies include destruction of the lesion by cryotherapy or laser treatment and removal of the lesion by a loop electrosurgical excision procedure (LEEP) or by a cone biopsy, which removes a piece of tissue in the form of cone of the opening of the cervix. The present invention now provides a method for inhibiting replication of HPV in cervical cells by heating the affected area to at least 2 ° C above its normal temperature (eg, at 39-42 ° C) for at least one hour. This is achieved by rapidly heating the affected area locally, for example, by the use of a hot probe regulated by thermostat or by other methods known in the art. Treating the affected area of the cervix daily, or every third day or twice a week for two, three or more weeks, significantly reduces the burden of infection with HPV so that the progression toward malignancy stops or even reverses. It is of particular importance to treat the cervix in the pre-malignant stages, preferably SIL or low-grade C1N1. In these stages the cell growth usually still depends on the presence of the virus, whereas in the malignant stages the growth of the tumor cells can be made independent of the virus. The heat treatment according to the invention can be combined with known therapies for cervical dysplasia. Regional hypothermia is known in the art to treat cancers, including cervical cancer (de Wit et al., Br. J. Cancer, 1999, 80 (9): 1387-1391). However, this anti-cancer treatment is based on the observation that tumor cells are more sensitive to heat-induced apoptosis, particularly in combination with cytotoxic agents or radiation. The goal of the anti-cancer hyper-thermal route is to apply sufficiently high temperatures (for example more than 42 ° C) to promote the cell death of tumor cells. In contrast, the present invention involves a heat treatment of pre-malignant, non-tumor cells to increase the mechanism of RNAi in the cells that inhibits the replication of the virus and thus prevented, poinentially if not really, reaching the malignant stage. further, a temperature of more than 42 ° C is typically required for the induction of cell death (tumor) while the preferred temperatures for inhibiting viral replication are 39 to 42 ° C, more preferably 39 to 40 ° C. Naturally, The skilled person will appreciate that a very high temperature that can induce cell death should be avoided in the application of the present invention. The concept of the present invention is based on the inhibition of viral replication by increasing RNAi, an antiviral defense mechanism of the infected host cell that leads to the silencing of viral genes. However, the application of heat treatment in vivo, according to the invention, is not necessarily limited to the increased silencing of the antiviral gene. It is also advantageously used if the RNAi-mediated silencing of an endogenous or exogenous gene of interest is desired by the introduction of homologous dsRNA to the gene in the host cell. Because an RNAi sequence seeks and destroys its target without affecting other genes, the effect of RNAi can be highly selective. There is much speculation in the medical community that the clinical applications of RNAi are potentially infinite: any gene whose expression contributes to the disease (disease gene) is a potential target, from viral genes to oncogenes to genes responsible for heart disease, Alzheimer's, diabetes and many more. In order to increase the effect of RNAi from a dsRNA that is introduced to a cell in situ (either by injection, genetic engineering techniques and / or by using a viral or non-viral delivery vehicle), heat treatment can be contemplated. according to the invention.
In addition, the invention provides a method for increasing the silencing of RNAi-mediated gene in vivo in a tissue, which comprises stably increasing the RNAi in said tissue by providing heat to said tissue. In a preferred embodiment, the fabric is heated to a temperature of at least 2 ° O above its normal physiological temperature for 1 to 4 hours, preferably 1 to 2 hours, more preferably 1 to 1.5 hours. A method of the invention is advantageously used to increase the in vivo silencing of a disease gene. In accordance with the present invention there is also provided an apparatus for inhibiting the replication of a virus in the tissue of the respiratory tract of a subject, comprising: a gas inlet for receiving a gas in said apparatus, a heating device for heating said gas, a humidification device for humidifying said gas, gas guiding means for guiding said gas from the gas inlet to the heating device and to the humidification device and then to a gas outlet to release said heated and humidified gas, a device for increasing the ventilation rate to increase the rate of ventilation of the subject, and a temperature controller that is connected to said heating device and controls the tissue temperature of the subject's respiratory tract, so that in use at least part of said fabric is heated to a temperature of at least 2 and not more than 9 ° C above its temp normal physiological eratura. As discussed above, the ventilation enhancement device may comprise means for adding CO2 to introduce C02 to said gas before it is released from said gas outlet or exercise equipment to be used by the subject. The apparatus may further comprise an oxygen aggregation device for introducing oxygen to said gas before it is released from the gas outlet. The gas outlet may be provided with a mouth and nose piece or face mask. According to the invention there is further provided an apparatus for inhibiting the replication of a virus in the tissue of the rectal tract, the female reproductive tract, the cervix or vagina of a subject, comprising: a penetration element for penetrating the rectal tract, The female reproductive tract, cervix or vagina of the subject, comprising an external wall, a second heating device in use for heating the tissue surrounding said penetration element, a second temperature controller that is connected to said second heating device and controls the temperature of the surrounding tissue so that in use at least part of said tissue is heated at an interval of at least 2 and hasfa
9o C above his normal physiological lemperafura. The present invention can also be used to irradiate the excrements of the body, thus, according to another aspect of the present invention, there is also provided an apparatus for inhibiting the replication of a virus in the tissue of an extremity of a subject. apparatus comprising: a receiving element for receiving the extremity of the subject, said receiving element comprises an internal wall and, in use, at least partially surrounds the extremity of the subject, a third heating device for heating the tissue that is surrounded by the receiving element, and a third temperature controller that is connected to said third heating device and controls the temperature of said tissue of the extremity of a subject, so that in use at least part of said tissue is heated to a temperature of at least 2 and up to 9 ° C above their normal physiological temperature. In the different apparatuses discussed above, the second and third heating devices may be similar in shape and operation, and what is described with respect to one may apply to the ear. In a similar way, you will hear words, as the second and third temperature controllers can be equivalent. The heating devices may comprise liquid heating means for heating a liquid and liquid guiding means for guiding said liquid between said liquid heating means and the penetrating or receiving member. The penetrating and receiving elements may each include a duct through which hot liquid is pumped by means of pumping means to heat the outer wall or the infernal wall, respectively. The outer wall of the penetrating element and the inner wall of the receiving element may comprise an expandable or flexible material, which is preferably elastic. Such expandable or flexible material can be expanded so that substantially complete contact is achieved with the surface of the fabric to be treated with the method of the invention. Of course, the skilled person will appreciate that the expansion of said flexible material can be effected by regulating the flow of liquid and / or gas by means of a pump. The external and internal walls may also comprise protuberances extending towards the tissue. The penetrating element and the receiving element can be provided with a vibrating device to make them vibrate, since this improves the thermal contact with the tissue. The temperature controllers of each apparatus mode may also include time controllers to control the period of time during which the output is heated for an effective time interval that depends on the effect being effected. Now, certain aspects of the invention will be described in detail with reference to the accompanying drawings, in which: Figure 1 shows a schematic view of an embodiment of the method for inhibiting the replication of a virus in the respiratory tract of a subject. Figure 2 shows a schematic view, partially in cross section, of a second embodiment of the apparatus for inhibiting the replication of a virus in the tissue of the rectal tract or female reproductive tract, such as the cervix or vagina of a subject. Figure 3 shows a schematic view, partly in cross section, of a third embodiment of the apparatus for inhibiting the replication of a virus in the tissue of the limb of a subject. Figure 1 shows an embodiment of the apparatus for inhibiting the replication of a virus in the tissue of the respiratory tract of a subject, such an apparatus comprising a gas inlet 2 for receiving a gas. In the embodiment of Figure 1 air is received through the gas inlet 2 from the environment, but it is also possible to connect the gas inlet 2 to a gas container containing a specific gas (preferably oxygen) or a mixture of specific gases. The flow of air to the gas inlet 2 is shown by the attached arrows, as is the subsequent flow of air through the apparatus. The received air is guided by the gas guiding means 5 to the heating device 3 which heats the air. The heating device 3 is connected via the gas guide means 5 to the humidification device 4, which humidifies the air. Through the gas lighter 2 and the heating device 3, the gas guiding means 5 are connected to a device 6 for increasing the ventilation rate comprising a unit 8 of CO2 gas. The unit 8 of CO2 gas can be used to add CO2 to the air passed to the subject so that in use it will increase the ventilation regime of the subject. Instead of altering the gas component to increase the ventilation regime, the ventilation regime increase device 6 may comprise exercise equipment for the subject, whose use causes the respiration rate to increase. The heating device 3 heats the gas before it passes to the humidifier to ensure that adequate moisture is achieved in the air passing through it. The air that passes to the humidifier can be at the temperature required for delivery to the tissue, or it can be at a higher temperature. The gas guide means 5 for the air leaving the heating device and / or the humidification device may be adapted to maintain the gas at the required temperature or may allow heat to dissipate to reach the desired temperature. The gas guiding means extends to the gas outlet 1 1. The air flow coming out of the gas outlet 1 1 is shown by the arrows. The apparatus further comprises a temperature controller 9, which is connected to the heating device 3 for controlling the heating device 3. The temperature controller 9 comprises a time controller 10 for controlling the period of time during which the temperature controller 9 activates the heating device 3. The temperature controller can also be connected to the last part of the gas guide means 5 before the exit 1 1, so that it can monitor the temperature of the gas before it passes to the subject. This can be particularly important if the gas is supplied to the humidification device 4 at a temperature higher than that to which it is to be provided to the subject, to ensure that it has been allowed to cool sufficiently. The gas outlet 1 1 includes a face mask 13 with an elastic retaining band 12 so that the face mask 13 can be held in place on the face of the subject. In use the subject will breathe the heated and humidified mixture of air and C02 so that the tissue temperature of the respiratory tract will rise to a specific temperature and for a specific period of time. In this way, the replication of a virus in the tissue of the subject's respiratory tract will be inhibited. Figure 2 shows a second embodiment of the apparatus for inhibiting viral replication in the tissue of the rectal tract or the female reproductive tract, including the cervix or vagina, of a subject. The apparatus comprises a penetrating element 21, which in use penetrates the rectal tract or the female reproductive organ of the subject. The penetration element 21 comprises an external wall 22 which is flexible and elastic. Within the external wall 22 of the penetration element 21 there is a hollow core 30 with a duct 25 formed therein. In use liquid flows through this duct 25 and then out through the space between the hollow core 30 and the outer wall 22. The flexible outer wall 22 allows the shape of the peeling member 21 to be adjusted in use to the shape of the surrounding shaft. The elastic nature of the outer wall allows it to inflate, so that the size of the wall can also be adjusted. The outer wall can also have protuberances (not mossy) which can provide a larger conjoint surface between the penetrating element 21 and the surrounding tissue without elongating the penetration element 21. The embodiment shown in Figure 2 further comprises a second heating device 23. The second heating device 23 comprises liquid heating means 26 and pumping means 27 for driving heated liquid through the liquid guiding means 28 and towards the penetrating element 21. The second heat-holding device 23 is connected to the penetrating element 21 by the liquid guiding means 28 so that the heated liquid can be passed to and from the penetrating element 21. Part of the diagram including the complete penetration element 21 is shown in a cross-sectional view. The arrows in the diagram show the flow of liquid inside the device. Jiquid guide means 28 transfers the heated liquid from the second heating device 23 to the duct 25. The heated liquid then passes through the penetration element 21 and returns to the second heating device 23 through the return line. of the liquid guide means 28. The liquid flow control means (not shown) are arranged in order to ensure a smooth circulation of the liquid through the liquid 25. The apparatus of the second embodiment also comprises a second 24-year-old confrigator., which is connected to and con fi rms the second calendaring device. The second temperature driver 24 includes a second time controller 29 to control the period of time in which the second temperature controller 24 activates the second heating device 23. In use, the penetration element 21 is placed in the rectal tract or the female reproductive tract of the subject. The flow of the heated liquid through the penetration element 21 causes the external wall 22 to be heated to the desired temperature. In this way, the penetration element 21 heats the surrounding surrounding tissue for a specific period of time so that viral replication in said tissue is inhibited. The apparatus may also comprise a vibration device for vibrating the penetration element 21. In this way, a better thermal contact is achieved between the penetration element 21 and the surrounding tissue. Figure 3 shows a third embodiment of the apparatus for inhibiting viral replication in the tissue of a limb of a subject's body. The apparatus comprises a receiving element 41 for receiving an extremity of the subject. The tip of the body part can be a hand, a foot, the scrotum and / or a phallus. In use, the extremity of the body part is inserted into the receiving element 41, which at least partially surrounds the extremity. The receiving element 41 comprises an outer sleeve 51, defining an opening 50 that gives access to the interior thereof. An inner wall 42, which is flexible and elastic, is placed inside the outer sleeve 51, such that a second duct 45 is formed between them, through which liquid can flow. Within the second conduit 45 the liquid flow control means (not shown) are arranged to ensure a complete circulation of the liquid through the receiving element 41 and therefore around the extremity. The flexible internal wall 42 will be adjusted, in use, to the shape or contour of the received body, and as it is elastic it can be inflated, so that the size thereof can be adjusted to accommodate smaller extremities. The inner wall may comprise inwardly directed protuberances which allow better grip or better contact between the receiving element 41 and the tissue of the extremity of the received body. The apparatus of this third embodiment further comprises a mechanism for supplying calenic liquid which is the same as that described with reference to Figure 2. Similar reference numbers have been used to describe similar paris. The calendaring device 23 pumps the heated liquid through the liquid guiding means 28 to and from the receiving element 41. The arrows, in the part of the diagram that is shown in cross section, will show the flow of liquid. In use, the extention of the fastener is placed in the reception element 41 through the opening 50. The heating device pumps calendered liquid through the second drain 45 into the receiving element 41, which causes the wall 42 to degenerate. of it warms up. In this manner, the reception element 41 warmed the body of the surrounded environment during a specific period of time so that viral replication in said tissue is inhibited. The apparatus may also comprise a vibration device for vibrating the receiving element 41 as this improves the thermal contact between the receiving element 41 and the surrounding tissue. The liquid used in the second and third moralities can be any suitable type, with appropriate viscosity, pelvic capacity and toxicity. Water or light mineral oil are suitable. The thermal contact can be further improved by providing a heat conducting medium between the fabric and the contact wall (eg, the external wall of the penetrating member or the internal wall of the receiving element) of the apparatus. The improved thermal condition between the penetration element or the receiving element and the adjacent tissue improves the efficiency of the present invention. This means of heat conduction can also improve the comfort of use, and it can be gel, saliva, water, lubricant, eic.