Treatment of autism spectrum disorder and associated neuroinflammation using fibroblasts and derivatives thereof

Abstract

To provide methods and compositions useful for treatment of pervasive developmental disorders.SOLUTION: The treatment includes the use of fibroblasts, modified fibroblasts, and derivatives thereof for reduction of neuroinflammation and / or gastrointestinal inflammation in a patient in need of treatment, such as having a pervasive developmental disorder. Fibroblasts, modified fibroblasts, and derivatives thereof may be administered at a frequency and concentration sufficient to reduce interleukin-17 production in the gut of patients with autism spectrum disorder.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 864,503, filed on Jun. 21, 2019, which is hereby incorporated by reference in its entirety.

[0002] (Technical Field) The disclosed embodiments include at least the fields of molecular biology, cell biology, neurobiology, and medicine.

Background Art

[0003] Autism is reported to occur in approximately 1 in 150 newborns and has reached epidemic proportions [1]. Although understanding of this condition has advanced, there is no curative treatment intervention. Current clinical trials are focused primarily on typical and atypical antipsychotics, as well as various pharmacological approaches targeting symptoms [2]. Many medical approaches have drawn fire from parent and patient activist groups for turning children into “walking zombies on drugs” [3]. Therefore, there is an urgent need for new directions in the treatment of autism.

[0004] In patients with autism spectrum disorder (ASD), numerous anatomical and physiological changes have been reported, but their significance is a matter of debate. Redcay and Courchesne reviewed 15 studies using head circumference and MRI evaluations to quantify brain growth during development. They found that the brains of children with autism were slightly smaller at birth and increased dramatically within the first year of life, but then reached a plateau, such that the majority of patients were within the normal range by adulthood. ​​​​​​​​​​In another study, 70 participants (45 with ASD, 45 with PTSD, 45 with PTSD, and 45 with PTSD) were included. A one-year longitudinal MRI study of 25 subjects showed that the amygdala of children with ASD was 2- It has been shown that children grow at an accelerated rate during the first four years of life.[5] Developmental abnormalities are also seen in older children. For example, in a study of patients with an average age of 12 years, there was a control group of 13 The MRI scans of 7 autistic boys and 7 non-autistic controls were examined. abnormal overgrowth of gray matter structures such as the parietal, temporal, and occipital lobes, as well as white matter structures such as the parietal and white matter structures. It has been found that the child exhibits abnormal developmental delays.[6] Other conditions that have been linked to autism include Abnormalities in the brain include attenuation of nerve fiber development [7], increased oxidative stress [8-10], and amyloidosis.

[11] , and elevated secretion of ide protein degradation products in subcortical brain regions involved in face detection and automatic emotional face processing. These diverse observations are consistent with a common underlying biological theory. It was difficult to integrate the

[0005] Aberrant immune activity is implicated in neurodevelopmental disorders in ASD. A cohort of children with ASD Recent studies have demonstrated that ASD-related disorders are similar to those seen in Crohn's disease (CD). The immune dysregulation of the systemic and intestinal systems was demonstrated. CD4+ is driven by activated type 1 helper T cells. It is a chronic inflammatory disease of the digestive tract that is driven by the inhibition of normal components of the intestinal microbiota (3a). This is due to a dysregulated mucosal immune response.

[0006] Early indications supporting a link between inflammation and ASD include gastrointestinal symptoms such as abdominal pain, as well as diarrhea. and constipation was observed to be significantly more prevalent in individuals with ASD compared with age-matched controls. For example, one study found that 9–70% of individuals with ASD had one or more G Symptoms were observed

[13] . The large variation in prevalence in this study was due to different definitions of ASD and GI symptoms. Specific symptoms of ASD GI symptoms have been reported to be similar in nature to inflammatory bowel disease. For example, Horvath and P erman

[14] noted not only the presence of inflammation in both the upper and lower intestinal tracts, but also the sulfation ability of the liver, pathological intestinal permeability, enhanced secretory response to intravenous secretin injection, and decreased activity of digestive enzymes in the digestive enzymes. The authors observed that the onset of these symptoms appears to be correlated with regressive behavior. Although the presence of GI abnormalities is fairly well established, it is important to note that their expression is extremely heterogeneous. For example, Molloy et al. investigated 137 autistic patients and found that only 24% had a history of at least one chronic digestive symptom, and the most common symptom was diarrhea, which was present in 17%

[15] . However, there are some suggestions that the subset of patients experiencing gastrointestinal pathology may actually be suffering from a more severe form of ASD . For example, Nikolov et al.

[0016] conducted a study on 172 autistic patients, 39 of whom (22.7%) showed GI symptoms mainly such as constipation and diarrhea. Among the patients with digestive symptoms, greater symptom severity was observed on scales of hypersensitivity, anxiety , and social withdrawal. Although there is room for debate, mucosal lesions in the form of chronic ileocolonic lymphoid nodular hyperplasia, characterized by lymphocyte infiltration, complement deposition, and cytokine production, have been specifically reported in autistic children but not in healthy controls or patients with cerebral palsy [17, 18]. This localized inflammation

[0007] Although there is room for debate, mucosal lesions in the form of chronic ileocolonic lymphoid nodular hyperplasia, characterized by lymphocyte infiltration, complement deposition, and cytokine production, have been specifically reported in autistic children but not in healthy controls or patients with cerebral palsy [17, 18]. This localized inflammation ​Possible symptoms include a number of "leaky gut syndrome" demonstrated by groups of first-degree relatives of autistic patients

[19] and patients with ASD

[20] . In one recent study 58 ASD patients and 39 age-matched controls were investigated. Autistic patients were found to have significantly lower levels of total short-chain fatty acids, fewer species of Bifidobacterium, and an abnormal gut microbiota with high levels of Lactobacillus species. Furthermore, in this study, it was also confirmed that enhanced gastrointestinal inflammation correlates positively with the severity of autistic symptoms

[21] . Interestingly, there is some evidence that leaky gut is not only actually a result of inflammation, but may also be a mediator that manifests through the systemic release of endotoxins

[22] . Although clinical evidence is lacking, the idea that probiotics may change the gut environment and reduce localized inflammation is accepted by many clinicians, as reported in a recent US survey where up to one fifth of complementary medicine specialists encourage the use of probiotics for children with ASD

[23] . As such, there is a

[0008] need in the art to address the non-inflammatory basis for ASD. The present disclosure is directed to systems, methods, and compositions for treating or preventing one or more pervasive developmental disorders in an individual. Pervasive developmental disorders It is possible. An individual may be administered one or more compositions that reduce one or more causes and / or one or more symptoms of pervasive developmental disorder. In some embodiments, one or more compositions administered to the individual may inhibit TNF-α and / or interleukin (IL)-17 in the individual. Or can be reduced. In certain embodiments, the composition administered to the individual stimulates angiogenesis (e.g., by differentiating into cells of the vascular system and / or by providing trophic support), and / or Can stimulate neurogenesis (including the dentate gyrus and / or subventricular zone).

[0009] The compositions of the present disclosure may include cells, cell derivatives, apoptotic bodies of cells, cell fragments, or combinations thereof. This composition may include fibroblasts, derivatives of fibroblasts, Apoptotic bodies of fibroblasts, or combinations thereof. The cells of the present disclosure that include fibroblasts may express CXCR4. The cells of the present disclosure may have, for example, higher expression of CXCR4 than the expression of CXCR4 in mesenchymal stem cells of similar origin. In some embodiments, the apoptotic bodies expose cells such as fibroblasts to one or more DNA damaging agents such as ultraviolet light and / or photosensitizers (increasing DNA damage to the cells after exposure to UV). Psoralen (also called psoralen) is the parent compound in a family of naturally occurring organic compounds known as linear furanocoumarins. This is due to the addition of a fused furan ring. It may be structurally related to coumarin and considered a derivative of umbelliferone. Solar in is present naturally in the seeds of Psoralea corylifolia, as well as common figs, celery, parsley, West Indian satinwood, and all citrus fruits. It is widely used in PUVA (solaren + UVA) treatment for psoriasis, eczema, vitiligo, and cutaneous T-cell lymphoma.

[0010] The cells of the present disclosure may be derived from any source including bone marrow, placental matrix, adipose tissue, menstrual blood, endometrium, muscle, peripheral blood, cord blood, or combinations thereof. The cells may be derived from an autologous source or an allogeneic source with respect to the individual of the present disclosure. Derivatives of fibroblasts and / or apoptotic bodies, derivatives and / or apoptotic bodies of the cells may be syngeneic with other cells of the present disclosure.

[0011] In some embodiments, the compositions of the present disclosure include a kit.

[0012] The compositions administered to the individual of the present disclosure may include a cell-conditioned medium including a fibroblast-conditioned medium.

[0013] The above has fairly broadly outlined the features and technical advantages of the present disclosure so that the following detailed description may be better understood. Additional features and advantages forming the subject of the claims of this specification are described below. It should be understood by those skilled in the art that the disclosed concepts and specific embodiments may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of this design. Also, it should be understood by those skilled in the art that such equivalent constructions do not depart from the spirit and scope described in the appended claims. ​​​​​​​​​​New features that are considered to be the design features disclosed in this specification are further objects and advantages both with respect to the configuration of the operation and the method, when considered in connection with the accompanying drawings will be better understood from the following description. However, it should be clearly understood that each figure is provided for illustrative and explanatory purposes only and is not intended as a definition of limitation of the present disclosure only.

Brief Description of the Drawings

[0014] To understand the present disclosure more fully, reference is made to the following description in conjunction with the accompanying drawings

[0015]

Figure 1

Modes for Carrying Out the Invention

[0016] Definitions

[0017] “Allogeneic” as used herein refers to cells of the same species that are genetically different from the host cells .

[0018] “Autologous” as used herein refers to cells derived from the same subject. The term “graft” as used herein refers to the process of incorporating stem cells into a tissue of interest in vivo through contact with existing cells of the tissue . .

[0019] As used herein, the terms “substantially” or “about” when applied to one or more values of interest refer to values that are similar to the stated reference value. In certain embodiments, the term “approximately” . Or "about" refers to a range of values corresponding to 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 6%, 4%, 3%, 2%, 1%, or less in either direction (greater or less) of the specified reference value, unless otherwise specified or apparent from the context (provided that the numerical value does not exceed 100% of the possible value). in either direction (greater or less) of the specified reference value 7%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 6%, 4%, 3%, 2%, 1%, or less (except when the numerical value exceeds 100% of the possible value).

[0020] As used herein, the terms "carrier" and "diluent" refer to pharmaceutically acceptable carriers or diluents (e.g., safe and non-toxic for administration to humans) useful in the preparation of pharmaceutical formulations. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffer solutions (e.g., phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solutions. pharmaceutically acceptable carriers or diluents (e.g., safe and non-toxic for administration to humans) useful in the preparation of pharmaceutical formulations Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffer solutions (e.g., phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solutions.

[0021] As used herein, the terms "dosage form" and "unit dosage form" refer to physically discrete units of a therapeutic agent for an individual being treated. Each unit contains a predetermined amount of the active substance calculated to produce the desired therapeutic effect. However, it will be understood that the total dosage of the composition will be determined by the attending physician within the scope of sound medical judgment. physically discrete units of a therapeutic agent for an individual being treated Each unit contains a predetermined amount of the active substance calculated to produce the desired therapeutic effect. However, it will be understood that the total dosage of the composition will be determined by the attending physician within the scope of sound medical judgment.

[0022] As used herein, a "dosage regimen" (or "treatment regimen") typically refers to a set of unit doses (typically two or more) administered individually to a subject, separated by a period of time. In some embodiments, a given therapeutic agent may have a recommended dosage regimen that includes one or more doses. In some embodiments, the dosage regimen has each dose of the same length administered individually to a subject, separated by a period of time In some embodiments, a given therapeutic agent may have a recommended dosage regimen that includes one or more doses. In some embodiments, the dosage regimen has each dose of the same length ​​​Comprising a plurality of doses separated from each other for only that period, and in some embodiments, the dosing regimen includes a plurality of doses and at least two different periods separating the individual doses. In some embodiments, the therapeutic agent is administered continuously over a predetermined period. In some embodiments, the therapeutic agent is administered once daily (QD) or more than once daily (BID).

[0023] The term "effective amount" or "therapeutically effective amount" means a dose sufficient to treat, inhibit, or alleviate one or more symptoms of the condition being treated, or to provide the desired pharmacological and / or physiological effects. The exact dosage will vary depending on various factors such as subject-dependent variables (e.g., age, health of the immune system, etc.), the disease, and the treatment being administered.

[0024] The term "innate immune response" refers to the cells and mechanisms by which a host is non-specifically protected from infection by other organisms, i.e., the cells of the innate immune system recognize pathogens in a general way and respond to them, but unlike the adaptive immune system, do not confer long-term persistence or protective immunity on the host. It will be understood that the cells of the innate immune response are affected by, among others, phagocytic cells such as macrophages, neutrophils, dendritic cells, basophils, eosinophils, natural killer cells, and γδT cells. Also, the complement system forms one of the components of the innate immune system. The innate immune response can induce an adaptive immune response. In some embodiments of the present disclosure, the administration of fibroblasts and / or derivatives of fibroblasts can be used to reduce chronic inflammation and stimulate iron

[0025] Cells, modified cells, derivatives of cells, apoptotic bodies, and conditioned media

[0026] Certain embodiments of the disclosure relate to the use of cells (including fibroblasts), degenerated cells (including degenerated fibroblasts), derivatives of cells (including derivatives of fibroblasts), apoptotic bodies from cells (including apoptotic bodies from fibroblasts), and / or conditioned media from cells for the treatment of a generalized developmental disorder, including inflammation associated with such disorder. The cells, modified cells, derivatives of cells, apoptotic bodies from cells, and / or conditioned media may contain or produce one or more anti-inflammatory factors. The cells, degenerated cells, derivatives of cells, apoptotic bodies from cells, and / or conditioned media may suppress, reduce, or inhibit IL-17 and / or TNF-α produced by activated macrophages. Fibroblasts and / or degenerated fibroblasts can express CXCR4, including at a higher level than mesenchymal stem cells of similar origin. Cells (including fibroblasts), degenerated cells (including degenerated fibroblasts), derivatives of cells (including derivatives of fibroblasts), apoptotic bodies from cells (including apoptotic bodies from fibroblasts), and / or conditioned media from cells. The cells, modified cells, derivatives of cells, apoptotic bodies from cells, and / or conditioned media may contain or produce one or more anti-inflammatory factors. The cells, degenerated cells, derivatives of cells, apoptotic bodies from cells, and / or conditioned media may suppress, reduce, or inhibit IL-17 and / or TNF-α produced by activated macrophages. Fibroblasts and / or degenerated fibroblasts can express CXCR4, including at a higher level than mesenchymal stem cells of similar origin. derivatives of cells), apoptotic bodies from cells (including apoptotic bodies from fibroblasts), and / or conditioned media from cells. The cells, modified cells, derivatives of cells, apoptotic bodies from cells, and / or conditioned media may contain or produce one or more anti-inflammatory factors. The cells, degenerated cells, derivatives of cells, apoptotic bodies from cells, and / or conditioned media may suppress, reduce, or inhibit IL-17 and / or TNF-α produced by activated macrophages. Fibroblasts and / or degenerated fibroblasts can express CXCR4, including at a higher level than mesenchymal stem cells of similar origin. including apoptotic bodies from fibroblasts), and / or conditioned media from cells. The cells, modified cells, derivatives of cells, apoptotic bodies from cells, and / or conditioned media may contain or produce one or more anti-inflammatory factors. The cells, degenerated cells, derivatives of cells, apoptotic bodies from cells, and / or conditioned media may suppress, reduce, or inhibit IL-17 and / or TNF-α produced by activated macrophages. Fibroblasts and / or degenerated fibroblasts can express CXCR4, including at a higher level than mesenchymal stem cells of similar origin. apoptotic bodies from cells, and / or conditioned media may contain or produce one or more anti-inflammatory factors. The cells, degenerated cells, derivatives of cells, apoptotic bodies from cells, and / or conditioned media may suppress, reduce, or inhibit IL-17 and / or TNF-α produced by activated macrophages. Fibroblasts and / or degenerated fibroblasts can express CXCR4, including at a higher level than mesenchymal stem cells of similar origin. derivatives of cells, apoptotic bodies from cells, and / or conditioned media may contain or produce one or more anti-inflammatory factors. The cells, degenerated cells, derivatives of cells, apoptotic bodies from cells, and / or conditioned media may suppress, reduce, or inhibit IL-17 and / or TNF-α produced by activated macrophages. Fibroblasts and / or degenerated fibroblasts can express CXCR4, including at a higher level than mesenchymal stem cells of similar origin. apoptotic bodies from cells, and / or conditioned media may suppress, reduce, or inhibit IL-17 and / or TNF-α produced by activated macrophages. Fibroblasts and / or degenerated fibroblasts can express CXCR4, including at a higher level than mesenchymal stem cells of similar origin. Fibroblasts and / or degenerated fibroblasts can express CXCR4, including at a higher level than mesenchymal stem cells of similar origin. Fibroblasts and / or degenerated fibroblasts can express CXCR4, including at a higher level than mesenchymal stem cells of similar origin. including that they can express CXCR4.

[0027] The fibroblasts utilized in the methods and disclosure encompassed herein may be derived from any source including, but not limited to, bone marrow, placental matrix, adipose tissue, menstrual blood, endometrium, muscle, circulating blood, umbilical cord blood, and combinations thereof. The fibroblasts utilized in the methods and disclosure encompassed herein may be derived from any source including, but not limited to, bone marrow, placental matrix, adipose tissue, menstrual blood, endometrium, muscle, circulating blood, umbilical cord blood, and combinations thereof. including, but not limited to, bone marrow, placental matrix, adipose tissue, menstrual blood, endometrium, muscle, circulating blood, umbilical cord blood, and combinations thereof.

[0028] In some embodiments, any type of fibroblast, including placental fibroblasts, expresses Oct-4, Rex-1, CD9, CD13, CD29, CD44, CD166, CD90, CD105, SH-3, SH-4, TRA-1-60, TRA-1-81, SSEA-4, So Rex-1, CD9, CD13, CD29, CD44, CD166, CD90, CD1 05, SH-3, SH-4, TRA-1-60, TRA-1-81, SSEA-4, So Based on the expression of at least one antigen comprising x-2, or a combination thereof, the specific embodiments of the present disclosure are identified. Fibroblasts that are identified as expressing one or more antigens, or that otherwise conform, may be utilized in the methods and compositions of the present disclosure. In certain embodiments, bone marrow fibroblasts can be isolated from bone marrow and can be utilized in methods and compositions related to the treatment or prevention of generalized developmental disorders. Bone marrow fibroblasts can be generated from bone marrow-derived mononuclear cells that include a population capable of differentiating into one or more cell types of endothelial cells, smooth muscle cells, and neuronal cells. In some embodiments, bone marrow fibroblasts can be selected based on the expression of one or more of the following antigens: CD34, c-kit, flk-1, Stro-1, CD105, CD73, CD31, CD56, CD146, vascular endothelial-cadherin, CD133, and CXCR-4. In certain specific embodiments of the present disclosure, fibroblasts are taken from amniotic fluid or amniotic membrane. Amniotic membrane-derived fibroblasts can be therapeutically utilized in an unpurified manner following matching. Amniotic membrane fibroblasts are administered locally, intramuscularly, or systemically in patients suffering from autism. In other embodiments, amniotic membrane fibroblasts are substantially purified based on the expression of markers such as SSEA-3, SSEA4, Tra-1-60, Tra-1-81, and / or Tra-2-54 and are subsequently administered. In some embodiments, the cells are cultured as described in U.S. Patent Application No. 10 / 918,739 to Haas, the disclosure of which is incorporated herein by reference, expanded, and then administered to an individual (e.g., ).

[0029] ). ). ). ). ). ). ).

[0030] ). ). ). ). ). ). ). ). (by injection). Amniotic fibroblasts are described in the following references [31-33]. One particular aspect of amniotic fibroblasts is that they are both mesenchymal progenitor cells and endothelial progenitor cells, thus enabling anti-inflammatory functions as well as angiogenic functions [32, 34], which may facilitate their use in the practice of certain aspects of the present disclosure. This property is useful for the treatment of individuals with autism who would benefit from angiogenesis, but also from the anti-inflammatory effects of fibroblasts. The use of amniotic fibroblasts is particularly useful in situations such as ischemia-related pathologies and / or inflammatory conditions where hypoxia is known to sustain the degenerative process. In some embodiments, fibroblasts are isolated from a sample of body tissue or biopsy by enzymatic digestion, mechanical dissociation, filtration, centrifugation, or a combination thereof. The number and quality of isolated fibroblasts can vary depending on, for example, the quality of the tissue used, the composition of the perfusion buffer, and / or the

[0031] type and concentration of the enzyme. Enzymes commonly used include, but are not limited to, collagenase, pronase, trypsin, dispase, hyaluronidase, thermolysin, and pancreatin, and combinations thereof. Collagenase is most commonly used and is often prepared from bacteria (e.g., Clostridium histolyticum) and can often consist of a poorly purified blend of enzymes that may have inconsistent enzymatic activity. Some of the enzymes exhibit protease activity, which can cause undesirable reactions that affect the quality and quantity of viable / healthy fibroblasts. To obtain a viable fibroblast population, dium histolyticum) and can often consist of a poorly purified blend of enzymes that may have inconsistent enzymatic activity. Some of the enzymes exhibit protease activity, which can cause undesirable reactions that affect the quality and quantity of viable / healthy fibroblasts. To obtain a viable fibroblast population, The use of enzymes of sufficient purity and quality is understood by those skilled in the art.

[0032] Certain methods of the present disclosure relate to the culture of fibroblasts obtained from human tissue samples. In some embodiments, the population of fibroblasts is plated on a glass substrate. In the present disclosure, fibroblasts can be plated on a glass substrate that enables cell adhesion thereto. This can be achieved, for example, by plating the cells in a culture plate that exhibits one or more glass substrate surfaces compatible with cell adhesion. When the one or more glass substrate surfaces come into contact with a suspension of cells introduced into the culture system (e.g., a suspension in a medium), cell adhesion between the cells and the glass substrate surface can occur. Thus, in certain embodiments, the cells are introduced into a culture system characterized by at least one substrate surface generally compatible with cell adhesion such that the plated cells can contact the substrate surface. Such embodiments include plating on a substrate that enables cell adhesion to the substrate. The general principles for maintaining an adherent cell culture are well known in the art. As understood by those skilled in the art, fibroblasts can be counted to facilitate subsequent plating of the cells at a desired density. As in the present invention, when the cells after plating can mainly adhere to the substrate surface present in the culture system (e.g., a culture vessel), the plating density can be expressed as the number of cells plated per mm or cm 2 of the substrate surface. 2 In the practice of the present disclosure, after plating the fibroblasts, the cell suspension is kept in contact with an adherent surface to enable cell attachment from the cell population to the glass substrate. It can be achieved. When contacting fibroblasts with an adhesive substrate, the cells are advantageously suspended in an environment containing at least a medium (typically a liquid medium that supports cell survival and / or proliferation) in the method of the present disclosure. The medium can be added to the system before, together with, or after the introduction of the cells thereto. The medium may be fresh, i.e., it may not have been previously used for cell culture, or it may contain at least a portion conditioned by pre-culture of the cells therein, such as monolayer culture or cell culture prior thereto, or cell culture far related or unrelated to monolayer-cultured cells. The medium can be added to the system before, together with, or after the introduction of the cells thereto. The medium may be fresh, i.e., it may not have been previously used for cell culture, or it may contain at least a portion conditioned by pre-culture of the cells therein, such as monolayer culture or cell culture prior thereto, or cell culture far related or unrelated to monolayer-cultured cells. The medium can be added to the system before, together with, or after the introduction of the cells thereto. The medium may be fresh, i.e., it may not have been previously used for cell culture, or it may contain at least a portion conditioned by pre-culture of the cells therein, such as monolayer culture or cell culture prior thereto, or cell culture far related or unrelated to monolayer-cultured cells. The medium can be added to the system before, together with, or after the introduction of the cells thereto. The medium may be fresh, i.e., it may not have been previously used for cell culture, or it may contain at least a portion conditioned by pre-culture of the cells therein, such as monolayer culture or cell culture prior thereto, or cell culture far related or unrelated to monolayer-cultured cells. The medium can be added to the system before, together with, or after the introduction of the cells thereto. The medium may be fresh, i.e., it may not have been previously used for cell culture, or it may contain at least a portion conditioned by pre-culture of the cells therein, such as monolayer culture or cell culture prior thereto, or cell culture far related or unrelated to monolayer-cultured cells. The medium can be added to the system before, together with, or after the introduction of the cells thereto. The medium may be fresh, i.e., it may not have been previously used for cell culture, or it may contain at least a portion conditioned by pre-culture of the cells therein, such as monolayer culture or cell culture prior thereto, or cell culture far related or unrelated to monolayer-cultured cells.

[0033] In some embodiments, the cells containing fibroblasts are modified to enhance properties useful in the aspects of the present disclosure. Modifications to the cells include exposure to external signals, hypoxia, growth factors, dedifferentiating agents, differentiating agents, conditioning media, or combinations thereof, but are not limited thereto. The cells can be modified to express proteins and / or nucleic acids useful in the present invention. The expression of the proteins and / or nucleic acids can be generated by any method known in the art. In some embodiments, the cells containing fibroblasts are modified to enhance properties useful in the aspects of the present disclosure. Modifications to the cells include exposure to external signals, hypoxia, growth factors, dedifferentiating agents, differentiating agents, conditioning media, or combinations thereof, but are not limited thereto. The cells can be modified to express proteins and / or nucleic acids useful in the present invention. The expression of the proteins and / or nucleic acids can be generated by any method known in the art. In some embodiments, the cells containing fibroblasts are modified to enhance properties useful in the aspects of the present disclosure. Modifications to the cells include exposure to external signals, hypoxia, growth factors, dedifferentiating agents, differentiating agents, conditioning media, or combinations thereof, but are not limited thereto. The cells can be modified to express proteins and / or nucleic acids useful in the present invention. The expression of the proteins and / or nucleic acids can be generated by any method known in the art. In some embodiments, the cells containing fibroblasts are modified to enhance properties useful in the aspects of the present disclosure. Modifications to the cells include exposure to external signals, hypoxia, growth factors, dedifferentiating agents, differentiating agents, conditioning media, or combinations thereof, but are not limited thereto. The cells can be modified to express proteins and / or nucleic acids useful in the present invention. The expression of the proteins and / or nucleic acids can be generated by any method known in the art. In some embodiments, the cells containing fibroblasts are modified to enhance properties useful in the aspects of the present disclosure. Modifications to the cells include exposure to external signals, hypoxia, growth factors, dedifferentiating agents, differentiating agents, conditioning media, or combinations thereof, but are not limited thereto. The cells can be modified to express proteins and / or nucleic acids useful in the present invention. The expression of the proteins and / or nucleic acids can be generated by any method known in the art. In some embodiments, the cells containing fibroblasts are modified to enhance properties useful in the aspects of the present disclosure. Modifications to the cells include exposure to external signals, hypoxia, growth factors, dedifferentiating agents, differentiating agents, conditioning media, or combinations thereof, but are not limited thereto. The cells can be modified to express proteins and / or nucleic acids useful in the present invention. The expression of the proteins and / or nucleic acids can be generated by any method known in the art.

[0034] Some embodiments of the present disclosure relate to the use of cell derivatives or products made from cells, including derivatives of fibroblasts. Some embodiments of the present disclosure relate to the use of apoptotic bodies as derivatives of fibroblasts, including apoptotic bodies derived from any type of fibroblast. The apoptotic bodies may be capable of immunomodulation, including modulating inflammation. The apoptotic bodies may release molecules that signal to the immune system, thereby Some embodiments of the present disclosure relate to the use of cell derivatives or products made from cells, including derivatives of fibroblasts. Some embodiments of the present disclosure relate to the use of apoptotic bodies as derivatives of fibroblasts, including apoptotic bodies derived from any type of fibroblast. The apoptotic bodies may be capable of immunomodulation, including modulating inflammation. The apoptotic bodies may release molecules that signal to the immune system, thereby Some embodiments of the present disclosure relate to the use of cell derivatives or products made from cells, including derivatives of fibroblasts. Some embodiments of the present disclosure relate to the use of apoptotic bodies as derivatives of fibroblasts, including apoptotic bodies derived from any type of fibroblast. The apoptotic bodies may be capable of immunomodulation, including modulating inflammation. The apoptotic bodies may release molecules that signal to the immune system, thereby Some embodiments of the present disclosure relate to the use of cell derivatives or products made from cells, including derivatives of fibroblasts. Some embodiments of the present disclosure relate to the use of apoptotic bodies as derivatives of fibroblasts, including apoptotic bodies derived from any type of fibroblast. The apoptotic bodies may be capable of immunomodulation, including modulating inflammation. The apoptotic bodies may release molecules that signal to the immune system, thereby Some embodiments of the present disclosure relate to the use of cell derivatives or products made from cells, including derivatives of fibroblasts. Some embodiments of the present disclosure relate to the use of apoptotic bodies as derivatives of fibroblasts, including apoptotic bodies derived from any type of fibroblast. The apoptotic bodies may be capable of immunomodulation, including modulating inflammation. The apoptotic bodies may release molecules that signal to the immune system, thereby The immune mechanism may be activated. Apoptotic bodies can be generated by any method known in the art ( e.g., by exposing cells to one or more DNA damaging agents (e.g., ultraviolet light and / or photosensitizers) ).

[0035] Certain embodiments relate to the use of a cell-conditioned medium comprising a fibroblast-conditioned medium. The conditioned medium may contain molecules useful in embodiments of the present disclosure, including molecules that modulate inflammation (e.g., IL-17).

[0036] In some embodiments, cells (including fibroblasts), modified cells (including modified fibroblasts), derivatives of cells (including derivatives of fibroblasts), apoptotic bodies derived from cells (including apoptotic bodies derived from fibroblasts), and / or conditioned media derived from cells (including those derived from fibroblasts) are combined. In specific cases, the combination may include autologous cells (including fibroblasts), autologous modified cells (including degenerated fibroblasts), derivatives of autologous cells (including derivatives of fibroblasts), apoptotic bodies from autologous cells (including apoptotic bodies from fibroblasts), and / or conditioned media from autologous cells (including those from fibroblasts). ), apoptotic bodies from autologous cells (including apoptotic bodies from fibroblasts), and / or conditioned media from autologous cells (including those from fibroblasts).

[0037] In some embodiments, the cells of the present disclosure can be cultured for at least about 10 days to about 40 days, at least about 15 days to about 35 days, at least about 15 days to 21 days, e.g., at least about 1 5, 16, 17, 18, 19 or 21 days. In some embodiments, the cells of the present disclosure can be cultured for 60 days or less, or 50 days or less, or 45 days or less. In some embodiments, fibroblasts (including modified fibroblasts) are in the presence of a liquid culture medium ​​​It is cultured below. Typically, the medium contains basal medium formulations known in the art. Eagl e's Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), α-Modified Minimum Essential Medium (α-MEM), Basal Medium Essential (BME), Iscove's Modified Du lbecco's Medium (IMDM), BGJb Medium, F-12 Nutrient Mixture (Ham), Li ebovitz L-15, DMEM / F-12, Essential modifie d Eagle's Medium (EMEM), RPMI-1640, and / or their modifications and / or combinations, including but not limited to, many basal medium formulations, are used herein to culture fibroblasts. The composition of the above basal media is generally known in the art, and it is within the skill of the art to modify or adjust the concentration of the media and / or media supplements required for the cultured fibroblasts. In some embodiments , the culture medium formulation may be an explant culture medium (CEM) composed of IMDM supplemented with 10% fetal bovine serum (FBS, Lonza), 100 U / ml penicillin G, 100 μg / ml streptomycin and 2 mmol / L -glutamine (Sigm a-Aldrich). Other embodiments may use additional basal medium formulations selected from the above.

[0038] For use in fibroblast cultures, including modified fibroblast cultures, the medium may be supplemented with one or more additional components. For example, additional supplements can be used to supply the cells with trace elements and substances necessary for optimal growth and proliferation. Such supplements include insulin, transferrin, selenium salts, and combinations thereof. It contains wase. These components can be included in saline solutions such as Hank's balanced salt solution (HBSS), Earle's salt solution, but are not limited to these. Further antioxidant supplements, for example, β-mercaptoethanol may be added. Many media already contain amino acids However, some amino acids, such as L-glutamine, can be supplemented later, which is known to have low stability in solution. The medium typically contains amphotericin n, ampicillin, gentamicin, bleomycin, hygromycin, kanamycin, mitomycin, mycophenolic acid, nalidixic acid, neomycin, nystatin, paromomycin, polymyxin, puromycin, rifampicin, spectinomycin n, tetracycline, tyrosin, and mixtures of antibiotics and / or anti- fungal compounds such as zeocin can be further supplied, but are not limited to these. Also, supplementation of cell culture media with mammalian plasma or serum is also contemplated. Plasma or serum often contains cytokines and components necessary for survival and proliferation. The use of appropriate serum replacements (e.g., FBS) is also contemplated. In some embodiments, culturing tissue explants and fibroblasts for a duration as defined herein, and preferably using a culture medium composition as described herein, results in the emergence and proliferation of the progenitor or stem cells of the present disclosure. In some embodiments, the fibroblasts of the present disclosure are identified and characterized by their expression of specific marker proteins (e.g., cell surface markers). Detection and isolation of these cells can be achieved, for example, by flow cytometry, ELISA, and / or magnetic beads . Reverse transcription polymerase chain reaction (RT-PCR) can also be used for differentiation . In some embodiments, the fibroblasts of the present disclosure are identified and characterized by their expression of specific marker proteins (e.g., such as cell surface markers). Detection and isolation of these cells can be achieved, for example, by flow cytometry, ELISA, and / or magnetic beads . Detection and isolation of these cells can be achieved, for example, by flow cytometry, ELISA, and / or magnetic beads beads. Reverse transcription polymerase chain reaction (RT-PCR) can also be used for differentiation It can be used to monitor changes in gene expression accordingly. Fibroblasts of the present disclosure A method for characterizing cells is provided herein.

[0039] In some embodiments, fibroblasts are cultured in a manner that increases the activity or ability useful in the methods disclosed herein. Fibroblasts are cultured to promote the ability of fibroblasts to reduce the production of inflammatory mediators and / or the ability to have regenerative activity. In some embodiments, fibroblasts can be cultured to reduce the production of inflammatory mediators and / or to promote the ability to have regenerative activity. In some embodiments, fibroblasts capable of reducing the production of inflammatory mediators are cultured, for example, in the presence of tissue culture additives such as interleukin-10, indomethacin, valproic acid, low-dose naltrexone, interleukin-27, or combinations thereof. In certain embodiments of the present disclosure, regenerative cells such as fibroblasts are utilized for treating autism spectrum disorder, and the cells can be exposed to one of the aforementioned additives (e.g., valproic acid) in the culture to increase their efficacy. In some embodiments, fibroblasts can be cultured to reduce the production of inflammatory mediators and / or to promote the ability to have regenerative activity. In some embodiments, fibroblasts capable of reducing the production of inflammatory mediators are cultured, for example, in the presence of tissue culture additives such as interleukin-10, indomethacin, valproic acid, low-dose naltrexone, interleukin-27, or combinations thereof. In certain embodiments of the present disclosure, regenerative cells such as fibroblasts are utilized for treating autism spectrum disorder, and the cells can be exposed to one of the aforementioned additives (e.g., valproic acid) in the culture to increase their efficacy. In some embodiments, fibroblasts can be cultured to reduce the production of inflammatory mediators and / or to promote the ability to have regenerative activity. In some embodiments, fibroblasts capable of reducing the production of inflammatory mediators are cultured, for example, in the presence of tissue culture additives such as interleukin-10, indomethacin, valproic acid, low-dose naltrexone, interleukin-27, or combinations thereof. In certain embodiments of the present disclosure, regenerative cells such as fibroblasts are utilized for treating autism spectrum disorder, and the cells can be exposed to one of the aforementioned additives (e.g., valproic acid) in the culture to increase their efficacy. In some embodiments, fibroblasts capable of reducing the production of inflammatory mediators are cultured, for example, in the presence of tissue culture additives such as interleukin-10, indomethacin, valproic acid, low-dose naltrexone, interleukin-27, or combinations thereof. In certain embodiments of the present disclosure, regenerative cells such as fibroblasts are utilized for treating autism spectrum disorder, and the cells can be exposed to one of the aforementioned additives (e.g., valproic acid) in the culture to increase their efficacy. In some embodiments, fibroblasts capable of reducing the production of inflammatory mediators are cultured, for example, in the presence of tissue culture additives such as interleukin-10, indomethacin, valproic acid, low-dose naltrexone, interleukin-27, or combinations thereof. In certain embodiments of the present disclosure, regenerative cells such as fibroblasts are utilized for treating autism spectrum disorder, and the cells can be exposed to one of the aforementioned additives (e.g., valproic acid) in the culture to increase their efficacy. In some embodiments, fibroblasts capable of reducing the production of inflammatory mediators are cultured, for example, in the presence of tissue culture additives such as interleukin-10, indomethacin, valproic acid, low-dose naltrexone, interleukin-27, or combinations thereof. In certain embodiments of the present disclosure, regenerative cells such as fibroblasts are utilized for treating autism spectrum disorder, and the cells can be exposed to one of the aforementioned additives (e.g., valproic acid) in the culture to increase their efficacy. In some embodiments, regenerative cells such as fibroblasts are utilized for treating autism spectrum disorder, and the cells can be exposed to one of the aforementioned additives (e.g., valproic acid) in the culture to increase their efficacy. In some embodiments, regenerative cells such as fibroblasts are utilized for treating autism spectrum disorder, and the cells can be exposed to one of the aforementioned additives (e.g., valproic acid) in the culture to increase their efficacy. In some embodiments, regenerative cells such as fibroblasts are utilized for treating autism spectrum disorder, and the cells can be exposed to one of the aforementioned additives (e.g., valproic acid) in the culture to increase their efficacy.

[0040] In some embodiments, any combination of cells (including fibroblasts), modified cells (including modified fibroblasts), derivatives of cells (including derivatives of fibroblasts), apoptotic bodies derived from cells (including apoptotic bodies derived from fibroblasts), and / or conditioned media derived from cells (including conditioned media derived from fibroblasts) is included in the kit. In some embodiments, any combination of cells (including fibroblasts), modified cells (including modified fibroblasts), derivatives of cells (including derivatives of fibroblasts), apoptotic bodies derived from cells (including apoptotic bodies derived from fibroblasts), and / or conditioned media derived from cells (including conditioned media derived from fibroblasts) is included in the kit. In some embodiments, any combination of cells (including fibroblasts), modified cells (including modified fibroblasts), derivatives of cells (including derivatives of fibroblasts), apoptotic bodies derived from cells (including apoptotic bodies derived from fibroblasts), and / or conditioned media derived from cells (including conditioned media derived from fibroblasts) is included in the kit. In some embodiments, any combination of cells (including fibroblasts), modified cells (including modified fibroblasts), derivatives of cells (including derivatives of fibroblasts), apoptotic bodies derived from cells (including apoptotic bodies derived from fibroblasts), and / or conditioned media derived from cells (including conditioned media derived from fibroblasts) is included in the kit.

[0041] Pervasive Developmental Disorder

[0042] Embodiments of the present disclosure relate to the treatment or prevention of one or more pervasive developmental disorders. Pervasive developmental Individuals who have or are diagnosed with a disorder may, for example, have social and / or communication developmental delays. Pervasive developmental disorders include any autism spectrum disorder, such as autism, Asperger's disease, childhood disintegrative disorder, pervasive developmental disorder not otherwise specified (PDD-NOS). Pervasive developmental disorders may, in at least some cases, be Rett syndrome.

[0043] Pervasive developmental disorders may present abnormal molecular markers, including abnormal inflammation, in an individual, or may be caused thereby. Such markers may include abnormal levels of CRP, erythrocyte sedimentation rate, fibrinogen, interleukin-1, TNF-α, prohepcidin, hepcidin, interleukin-6, interleukin-17, interleukin-18, interleukin-12, interleukin-18, interleukin-23, interleukin-27, interleukin-33, interferon-γ, HMBG-1, calreticulin. Other inflammatory markers useful for the assessment of conditions associated with anemia in chronic diseases associated with the practice of the disclosure include Apo A1 (apolipoprotein A1), β2 microglobulin, clusterin, CRP (C-reactive protein), cystatin C, eotaxin, factor VII, FGF-9 (fibroblast growth factor-9), GCP-2 (granulocyte chemoattractant protein-2), growth hormone, IgA (immunoglobulin A), IL-10 (interleukin-10), IL-1β (interleukin-1β), IL-2 (interleukin-2 ), IL-4 (interleukin-4), IL-5 (interleukin-5), insulin, IP-10 (interferon-inducible protein-10), Leptin, LIF (leukemia inhibitory factor), M ​DC (macrophage-derived chemokine), inducible protein, leptin (leukemia inhibitory factor), MDC (macrophage inhibitory factor), MIP-1α (macrophage inflammatory protein- 1α), MIP-1β (macrophage inflammatory protein-1β), MIP-1γ (mac rophage inflammatory protein-1γ), MIP-2 (macrophage inflammatory protein- 2), MIP-3β (macrophage inflammatory protein-3β), MPO (myeloperoxidase enzyme), myoglobin, NGAL (lipocalin-2), OSM (oncostatin M), osteopontin, SAP (serum amyloid P), SCF (stem cell factor), SGOT (serum glutamic oxaloacetic transaminase), TIMP-1 (tissue inhibitor of metalloproteinase type-1), tissue factor, TPO (Thrombopoietin), VEGF (vascular endothelial growth factor), or a combination thereof.

[0044] Individuals with autism spectrum disorder may exhibit neuroinflammation and / or gastrointestinal inflammation. In certain embodiments, the individual developmental disorder may be caused, in whole or in part, by neuroinflammation and / or gastrointestinal inflammation.

[0045] In some cases, the methods and compositions of the present disclosure relate to the prevention of one or more autism spectrum disorders. In certain cases, the prevention relates to the complete absence of the disorder, or the delay in the onset and / or severity of the disorder. An individual may be provided with an effective amount of the composition of the disorder in situations where the individual is from a family with a medical history, and / or where the individual has been identified as having or being at risk of having one or more autism spectrum disorders.

[0046] Dosage

[0047] Certain aspects of the present disclosure relate to the administration of a composition that may include fibroblasts, modified fibroblasts, derivatives of at least one fibroblast, fibroblast apoptotic bodies, fibroblast conditioned media, or combinations thereof, for the purpose of treating pervasive developmental disorders. Treating a pervasive developmental disorder may (optionally may not) include reducing inflammation associated with a pervasive developmental disorder such as autism. In some embodiments, an individual suffering from a pervasive developmental disorder such as autism can be selected for treatment as disclosed herein based on an inflammatory condition. The above condition can be identified by abnormally high serum levels of CRP, erythrocyte sedimentation ratio, fibrinogen, interleukin-1, and / or TNF-α. In some embodiments, when the inflammatory condition of an individual is stimulated with an agent such as lipopolysaccharide to stimulate toll-like receptor 4, it is identified by an abnormally high ability of monocytes derived from the individual to produce TNF-α. Administration of the compositions of the present disclosure can reduce inflammatory condition markers as disclosed herein. Alternatively, an individual is provided with an effective amount of the methods and compositions of the present disclosure regardless of whether high serum levels of the factors listed above are present. In some embodiments, a treated eligible individual has higher levels of inflammatory markers compared to age-matched controls. Inflammatory markers include C-reactive protein (CRP), fibrinogen, and erythrocyte sedimentation rate (ESR), prohepcidin, hepcidin, TNF-α, interleukin-1, interleukin-6, interleukin- In some embodiments, an individual suffering from a pervasive developmental disorder such as autism can be selected for treatment as disclosed herein based on an inflammatory condition. The above condition can be identified by abnormally high serum levels of CRP, erythrocyte sedimentation ratio, fibrinogen, interleukin-1, and / or TNF-α. In some embodiments, when the inflammatory condition of an individual is stimulated with an agent such as lipopolysaccharide to stimulate toll-like receptor 4, it is identified by an abnormally high ability of monocytes derived from the individual to produce TNF-α. Administration of the compositions of the present disclosure can reduce inflammatory condition markers as disclosed herein. Alternatively, an individual is provided with an effective amount of the methods and compositions of the present disclosure regardless of whether high serum levels of the factors listed above are present. In some embodiments, a treated eligible individual has higher levels of inflammatory markers compared to age-matched controls. Inflammatory markers include C-reactive protein (CRP), fibrinogen, and erythrocyte sedimentation rate (ESR), prohepcidin, hepcidin, TNF-α, interleukin-1, interleukin-6, interleukin-

[0048] In some embodiments, an individual suffering from a pervasive developmental disorder such as autism can be selected for treatment as disclosed herein based on an inflammatory condition. The above condition can be identified by abnormally high serum levels of CRP, erythrocyte sedimentation ratio, fibrinogen, interleukin-1, and / or TNF-α. In some embodiments, when the inflammatory condition of an individual is stimulated with an agent such as lipopolysaccharide to stimulate toll-like receptor 4, it is identified by an abnormally high ability of monocytes derived from the individual to produce TNF-α. Administration of the compositions of the present disclosure can reduce inflammatory condition markers as disclosed herein. Alternatively, an individual is provided with an effective amount of the methods and compositions of the present disclosure regardless of whether high serum levels of the factors listed above are present. In some embodiments, a treated eligible individual has higher levels of inflammatory markers compared to age-matched controls. Inflammatory markers include C-reactive protein (CRP), fibrinogen, and erythrocyte sedimentation rate (ESR), prohepcidin, hepcidin, TNF-α, interleukin-1, interleukin-6, interleukin- In some embodiments, an individual suffering from a pervasive developmental disorder such as autism can be selected for treatment as disclosed herein based on an inflammatory condition. The above condition can be identified by abnormally high serum levels of CRP, erythrocyte sedimentation ratio, fibrinogen, interleukin-1, and / or TNF-α. In some embodiments, when the inflammatory condition of an individual is stimulated with an agent such as lipopolysaccharide to stimulate toll-like receptor 4, it is identified by an abnormally high ability of monocytes derived from the individual to produce TNF-α. Administration of the compositions of the present disclosure can reduce inflammatory condition markers as disclosed herein. Alternatively, an individual is provided with an effective amount of the methods and compositions of the present disclosure regardless of whether high serum levels of the factors listed above are present. In some embodiments, a treated eligible individual has higher levels of inflammatory markers compared to age-matched controls. Inflammatory markers include C-reactive protein (CRP), fibrinogen, and erythrocyte sedimentation rate (ESR), prohepcidin, hepcidin, TNF-α, interleukin-1, interleukin-6, interleukin- In some embodiments, an individual suffering from a pervasive developmental disorder such as autism can be selected for treatment as disclosed herein based on an inflammatory condition. The above condition can be identified by abnormally high serum levels of CRP, erythrocyte sedimentation ratio, fibrinogen, interleukin-1, and / or TNF-α. In some embodiments, when the inflammatory condition of an individual is stimulated with an agent such as lipopolysaccharide to stimulate toll-like receptor 4, it is identified by an abnormally high ability of monocytes derived from the individual to produce TNF-α. Administration of the compositions of the present disclosure can reduce inflammatory condition markers as disclosed herein. Alternatively, an individual is provided with an effective amount of the methods and compositions of the present disclosure regardless of whether high serum levels of the factors listed above are present. In some embodiments, a treated eligible individual has higher levels of inflammatory markers compared to age-matched controls. Inflammatory markers include C-reactive protein (CRP), fibrinogen, and erythrocyte sedimentation rate (ESR), prohepcidin, hepcidin, TNF-α, interleukin-1, interleukin-6, interleukin- In some embodiments, an individual suffering from a pervasive developmental disorder such as autism can be selected for treatment as disclosed herein based on an inflammatory condition. The above condition can be identified by abnormally high serum levels of CRP, erythrocyte sedimentation ratio, fibrinogen, interleukin-1, and / or TNF-α. In some embodiments, when the inflammatory condition of an individual is stimulated with an agent such as lipopolysaccharide to stimulate toll-like receptor 4, it is identified by an abnormally high ability of monocytes derived from the individual to produce TNF-α. Administration of the compositions of the present disclosure can reduce inflammatory condition markers as disclosed herein. Alternatively, an individual is provided with an effective amount of the methods and compositions of the present disclosure regardless of whether high serum levels of the factors listed above are present. In some embodiments, a treated eligible individual has higher levels of inflammatory markers compared to age-matched controls. Inflammatory markers include C-reactive protein (CRP), fibrinogen, and erythrocyte sedimentation rate (ESR), prohepcidin, hepcidin, TNF-α, interleukin-1, interleukin-6, interleukin- In some embodiments, an individual suffering from a pervasive developmental disorder such as autism can be selected for treatment as disclosed herein based on an inflammatory condition. The above condition can be identified by abnormally high serum levels of CRP, erythrocyte sedimentation ratio, fibrinogen, interleukin-1, and / or TNF-α. In some embodiments, when the inflammatory condition of an individual is stimulated with an agent such as lipopolysaccharide to stimulate toll-like receptor 4, it is identified by an abnormally high ability of monocytes derived from the individual to produce TNF-α. Administration of the compositions of the present disclosure can reduce inflammatory condition markers as disclosed herein. Alternatively, an individual is provided with an effective amount of the methods and compositions of the present disclosure regardless of whether high serum levels of the factors listed above are present.

[0049] In some embodiments, a treated eligible individual has higher levels of inflammatory markers compared to age-matched controls. Inflammatory markers include C-reactive protein (CRP), fibrinogen, and erythrocyte sedimentation rate (ESR), prohepcidin, hepcidin, TNF-α, interleukin-1, interleukin-6, interleukin- In some embodiments, a treated eligible individual has higher levels of inflammatory markers compared to age-matched controls. Inflammatory markers include C-reactive protein (CRP), fibrinogen, and erythrocyte sedimentation rate (ESR), prohepcidin, hepcidin, TNF-α, interleukin-1, interleukin-6, interleukin- In some embodiments, a treated eligible individual has higher levels of inflammatory markers compared to age-matched controls. Inflammatory markers include C-reactive protein (CRP), fibrinogen, and erythrocyte sedimentation rate (ESR), prohepcidin, hepcidin, TNF-α, interleukin-1, interleukin-6, interleukin- In some embodiments, a treated eligible individual has higher levels of inflammatory markers compared to age-matched controls. Inflammatory markers include C-reactive protein (CRP), fibrinogen, and erythrocyte sedimentation rate (ESR), prohepcidin, hepcidin, TNF-α, interleukin-1, interleukin-6, interleukin- 17. Interleukin-18, Interleukin-12, Interleukin-18, I nterleukin-23, Interleukin-27, Interleukin-33, Interfer on-γ, HMBG-1, Calreticulin are included. Other inflammatory markers useful for the evaluation of conditions related to anemia in chronic diseases associated with the practice of the disclosure are ApoA1 (apolipoprotein A1 ), β-2 microglobulin, CRP (C-reactive protein), Cystatin-C, Eos inophil, Factor VII, FGF-9 (Fibroblast Growth Factor-9), GCP-2 (Granulocyte Che motactic Protein-2), Growth Hormone (IgA), IL-10 (Interleuki n-1β), IL-2 (Interleukin-2), IL-4 (Interleukin-5), Insulin, IP-10 (Inducible Protein-10), Leptin, etc. LIF (Leukemia Inhibitory Factor), MDC (Macrophage-Derived Chemokine), MIP-1α (Macrophage Inflammatory Protein-1β), MIP-2 (Macrophage Inflammatory Protein-2), MIP -3β (Macrophage Inflammatory Protein-3β), MPO (Myeloperoxidase) , Myoglobin, NGAL (Lipocalin-2), OSM (Oncostatin M), Osteo pontin, SAP (Serum Amyloid P), SCF (Stem Cell Factor), SGOT (Serum Gluta mic Oxaloacetic Transaminase), TIMP-1 (Tissue Inhibitor of Metalloproteinase Type-1), Tissue Factor, TPO (Thrombopoietin), VEGF (Vascular Endothelial Growth Factor), or a combination thereof. Administration of the compositions of the present disclosure can reduce inflammatory markers as disclosed herein . In an alternative case, an individual, regardless of whether the factors listed above are at high levels, can be treated with the methods and compositions of the present disclosure . An effective amount is provided.

[0050] Individuals identified as suitable for treatment will be treated with fibroblast therapy based on the degree of underlying inflammation. In some embodiments, the individual can be administered the non-immunogenic fibroblast product. The population is administered intravenously at a concentration sufficient to reduce and / or ameliorate inflammation and / or neuroinflammation. In some embodiments, fibroblast conditioned medium is administered. In one embodiment, apoptotic bodies of fibroblasts are administered.

[0051] In certain embodiments of the disclosure, individual recipients of the present disclosure that increase perfusion and stimulate neurogenesis. The cells and / or cell derivatives may be administered one or more anti-inflammatory agents, The inhibitors include the NF-kappa B pathway, the MyD88 pathway, the TNF signaling pathway, and the Toll-like Receptor signaling pathways and upregulation of MHC expression, C-reactive protein Upregulation of cytokine production and upregulation of TNFα production Anti-inflammatory agents useful in the practice of the present disclosure may inhibit molecular pathways such as inflammatory bowel disease and other pathways that inhibit inflammation. Alclofenac; Alclomethasone dipropionate are well known in the art. ;Acetonide algestone;α-amylase;α-lipoic acid;α-tocopherol;Amcynapha Le; Amcinafide; Amfenac sodium; Amiprilose hydrochloride; Anakinra; Anilorac; Anitrazafen; Apazone; Ascorbic acid; Balsalazide disodium bendazac; benoxaprofen; benzydamine hydrochloride; bromelains; brope Lamore; Budesonide; Carprofen; Chlorogenic acid; Cycloprofen; Synthazo Indomethacin; Clobetasol Propionate; Clobetasone Butyrate; Ciclopirox ; Crotamiton Propionate; Cormethasone Acetate; Cortodoxone; Deflazacort; De Sone; Dexamethasone Dipropionate; Diclofenac Potassium; Diclofenac Sodium; Diclorazone Diacetate; Diflumedone Sodium; Di Flunisal; Diflorasone Diacetate; Diftalone; Dimethyl Sulfoxide; Dorcinolone ; Ellagic Acid; Endrysone; Enlimomab; Enolicam Sodium; Epirizole; Eto Dra; Etofenamate; Felbinac; Phenamol; Fenbufen; Fenclofenac; Enac; Fenclorac; Fendosal; Fenpipolone; Fentiazac; Flaza Rone; Fluazacort; Flufenamic Acid; Flumizole; Fluocinolone Acetate; Flu Nixin; Flunixin Meglumine; Fluocortin Butyl; Fluorometholone Acetate; Flucazone; Flurbiprofen; Fluretofen; Fluticasone Propionate; Flaprofen; Frobufen; Glutathione; Halcinonide; Halobetasol Propionate; Halopredone Acetate; Hesperidin; Ibufenac; Ibuprofen; Ibuprofen Aluminum; Ibuprofen Picolol; Ironidap; Indomethacin; Indomethacin Sodium; Indoprofen; Indoxole; Intrazaole; Isoflupredone Acetate; Isosepac; Isoxicam; Ketoprofen; Lofemizole Hydrochloride; Lomoxicam; Loteprednol Etabonate; Lycopene; Meclofenamic Acid Sodium; Meclofenamic Acid; Meclorisone Dibutyrate; Mefenamic Acid; Mesalamine; Meseclo razone; Methylprednisolone Succinate; Morniflumate; Nabumetone; Nap Loxen; Naproxen Sodium; Naproxol; Nimazon; Olerupein; Or Sarazin Sodium; Orgotaine; Orpanoxin; Oxaprozin; Oxyphenbuf Tazone; Paraaniline Hydrochloride; Pentosan Polysulfate Sodium; Phenylbutazone Sodium Glycerate; Pirfenidone; Piroxicam; Piroxicam Cinnamate; Piroxic Cam Olamine; Pilprofen; Picnogenol; Polyphenol; Prednazarte; Pr iferon; Prodolic Acid; Proxazole; Proxazole Citrate; Quercetin; Resveratrol; Rimexolone; Romazarit; Romazaline Acid; Rutin; Sarcorex ; Sarnacedin; Salsalate; Sanginarine Chloride; Secrazone; Selmetacin; S ulindac; Sulfen; Talmethacin; Tarniflumart; Talosarate; Tebufenolon; Tenidap; Tenidap Sodium; Tenoxicam; Tescam; Tesi mid; Tetrahydrocurcumin; Tetridamine; Thiopyanac; Tixocortol Pivalate Salt; Tolmetin; Tolmetin Sodium; Triclonide; Triflumidate; Zidometacin ; Zomepirac Sodium, IL-4, IL-10, IL-13, IL-20, IL-1 receptor antagonists, and TGF-β.

[0052] In some embodiments, fibroblasts, such as bone marrow fibroblasts, are administered as described herein in combination with an agent known to increase regenerative activity. Such agents include, for example, erythropoietin

[24] , prolactin

[25] , human chorionic gonadotropin (described in U.S. Patent No. 5,968,513, incorporated by reference), ​including gastrin

[26] , EGF

[27] , FGF

[28] , and / or VEGF

[29] may be mentioned. In certain embodiments, administration of a neutralizing agent for TNFα is co-administered with fibroblasts to relieve the inhibitory effect of this cytokine on circulating fibroblasts

[30] .

[0053] In some embodiments, administration of the compositions of the present disclosure stimulates angiogenesis and / or neurogenesis. In some embodiments, angiogenesis is stimulated by the cells of the present disclosure that differentiate into cells of the vasculature. Angiogenesis can be stimulated by providing trophic support to cells of the vasculature. Neurogenesis can be stimulated anywhere in an individual including the dentate gyrus and the subventricular zone.

[0054] In certain embodiments, administration of a cell-conditioned medium containing a fibroblast-conditioned medium can be performed at a concentration and frequency sufficient to stimulate neurogenesis. Neurogenesis can be stimulated anywhere in an individual including the dentate gyrus and the subventricular zone. Administration of a cell-conditioned medium containing a fibroblast-conditioned medium may suppress interleukin-17 production.

[0055] In certain embodiments, administration of the compositions of the present disclosure can reduce neuroinflammation and / or gastrointestinal inflammation.

[0056] Compositions that may include fibroblasts, modified fibroblasts, derivatives of at least one fibroblast, fibroblast apoptotic bodies, fibroblast-conditioned media, or combinations thereof, as disclosed herein, can be administered to an individual including an animal (e.g., a human) by many methods known in the art. Examples of suitable methods include intramuscular, intradermal, intraepidermal, intravenous, arterial ​​​​​​​​​​​​Administration may be by intravenous, subcutaneous, or intraperitoneal injection, oral administration, and / or topical application (e.g., intravitreal, intranasal, and / or intravaginal application).

[0057] In some embodiments, instructions for administration of the compositions as disclosed herein are provided.

Examples

[0058] The following examples are included to demonstrate specific embodiments of the disclosure. The techniques disclosed in the following examples represent techniques discovered by the inventors to function well in the practice of the disclosed materials and, accordingly, are considered to constitute specific forms for their practice, which should be understood by those skilled in the art. However, those skilled in the art will be able to make many changes in the specific embodiments disclosed herein while still obtaining similar or analogous results without departing from the spirit and scope of the invention, which should be understood.

[0059] 〔Example 1〕 Cell Administration Clinical Trial The examples herein describe a clinical trial to evaluate the efficacy, feasibility, and safety of fibroblast administration to patients with autism spectrum disorder. Fibroblasts are administered to patients and evaluated at 4 clinic visits. The patients are further evaluated using 2 subsequent clinic visits.

[0060] Visit 1: Baseline assessment and cell administration (time zero)

[0061] Questionnaire

[0062] qEEG

[0063] qEEG during Sleep

[0064] ​ Blood sampling

[0065] Cell administration

[0066] Visit 2: Second cell administration (3 months)

[0067] Questionnaire

[0068] Quantitative electroencephalogram

[0069] Blood sampling

[0070] Cell administration

[0071] Visit 3: Third cell administration (6 months)

[0072] Questionnaire

[0073] Quantitative electroencephalogram

[0074] Blood sampling

[0075] Cell administration

[0076] Visit 4: Fourth cell administration (9 months)

[0077] Questionnaire

[0078] Quantitative electroencephalogram

[0079] Blood sampling

[0080] Cell administration

[0081] Visit 5: 12-month follow-up

[0082] Questionnaire

[0083] Quantitative electroencephalogram

[0084] EEG during sleep

[0085] Blood sampling

[0086] Visit 6: 18-month follow-up

[0087] questionnaire

[0088] Quantitative EEG

[0089] Blood collection

[0090] Patient population

[0091] Subjects will attend a screening visit and undergo a baseline assessment within 15 days prior to their first dose of cells. All inclusion and exclusion criteria were met. The results of all baseline assessments that ensure that standards are met are included in the enrollment of the subject. The study must be reviewed by the investigator or his / her designee prior to the study. All aspects of the study will be explained and written information will be obtained from the subject prior to any study procedures. Controlled consent must be obtained.

[0092] Inclusion:

[0093] 6 to 9 years old

[0094] DSM IV diagnosis of autism confirmed by ADOS and / or ADI-R

[0095] No anticipated changes in treatment (e.g., diet, nutrients) during the study

[0096] No additional biomedical treatment within 6 weeks prior to enrollment

[0097] No change in dietary management in the 3 months prior to enrollment

[0098] Parents, ambulatory or with minimal assistance

[0099] Parents and toys can stay still for more than 5 minutes with their favorite item

[0100] For each parent, sufficient vision and hearing for test administration

[0101] For each parent, appropriate arm - hand - finger adjustment for learning and cognitive tasks used for outcome measurement (i.e., able to point) That is, able to point

[0102] Stable and controlled medical conditions

[0103] Under the care of a caregiver who will attend regular appointments and take necessary measures to participate

[0104] Exclusions:

[0105] Severe prematurity at birth (gestational age less than 32 weeks); or a birth weight significantly below normal for gestational age (SGA - small for gestational age). That is, small for gestational age

[0106] Mental retardation

[0107] Episodic disorders

[0108] Autoimmune conditions

[0109] Gastrointestinal malabsorption

[0110] History of head trauma and other neurological or medical conditions

[0111] The "drug substance" for the clinical trial contains 100 million CybroCell fibroblasts, which are administered intravenously in a 20 - ml solution over 15 - 30 minutes. The administration is carried out in 4 cycles: Cycle 1 occurs at time points 0, 1 (3 months), 2 (6 months), and 3 (9 months). Each cycle consists of a daily administration of 100 million cells for 4 consecutive days.

[0112] ​​The primary outcome variables are safety and feasibility evaluated by quantifying the side effects associated with the treatment. This is the case.

[0113] The severity of each adverse event, or the intensity of the events experienced by the subject, is evaluated using the following:

[0114] Mild (1): Aware of discomfort, but no interference with daily activities.

[0115] Moderate (2): Discomfort sufficient to reduce or affect normal daily activities.

[0116] Severe (3): Unable to work or unable to perform normal daily activities.

[0117] Resolution of adverse event (4): Hospitalization

[0118] The secondary outcome variable is the effectiveness signal quantified by the following formula:

[0119] Pediatric Autism Rating Scale

[0120] Autism Behavior Checklist

[0121] Vineland Adaptive Behavior Scale

[0122] Differential Ability Scale

[0123] Comprehensive Evaluation Scale for Children

[0124] Social Response Scale

[0125] Social Skills Rating System

[0126] Evaluation of Social Skills with Youth by Matson

[0127] Quality of Autism

[0128] Quality of Emotion

[0129] Quotation System - Review

[0130] Statistical analysis was performed using SPSS 16.0 software. Safety and exploratory efficacy of the secondary endpoints were observed for each patient relative to the baseline value. If the p-value < 0. 05, it is considered statistically significant. The Intent-to-Treat (ITT ) population meets the eligibility criteria, consents to participate, and includes all subjects who were treated. The Per Protocol (PP) population is defined as a subgroup of the ITT population that has been demonstrated to comply with the study protocol . These subjects meet all inclusion and exclusion criteria and are evaluated at the time points specified in the study protocol.

[0131] Patient demographics and preoperative clinical variables are presented as percentages or means as necessary and evaluated using Student's paired t-test analysis: The evaluation items are compared from the preoperative time point to the time points of 3, 6, 9, 12, and 18 months after the first injection. If the data are not normally distributed , a comparable nonparametric method is used.

[0132] Patients who experience grade 3 or 4 treatment-related adverse events have the study discontinued for review.

[0133] [Example 2] Fibroblasts for the treatment of autism This example relates to fibroblasts for the treatment of autism as an example of neuroinflammation.

[0134] Male Wistar rats were used in the study. The date of birth was counted as postnatal day (pnd) 0 . The animals were housed in a temperature-controlled environment with a 12-hour light / dark cycle, with free access to food and water . ​​

[0135] Ten rats were divided into three groups of ten each. Group A was given saline. Group B received valproic acid (150 mg / kg) on days 6 - 11. Valproic acid in rodents is known to be able to mimic autism (Dobrovolskyy et al., 2019, J. Transl Med., 17:400.17:400). Group C received valproic acid (150 mg / kg) on days 6 - 11 and fibroblasts (1 million cells per rat, intravenous, fibroblasts were derived from foreskin and grown in OptiMem medium containing 10% fetal bovine serum) on days 6, 9, and 12.

[0136] On day 32, the rats were subjected to a "play test" using a plexiglass box (30 cm × 20 cm × 20 cm) divided into two equal - sized compartments by a transparent partition with a semi - circular hole (7 cm × 5 cm). One animal was moved between compartments at a time. Before any social interaction, the experimental subjects were marked on the back with a black marker and isolated in a dimly lit laboratory for a total of 30 minutes (20 minutes alone in a holding cage + 10 minutes of acclimation to the test apparatus). During the 10 - minute acclimation period, the subjects were allowed to explore the test box. After acclimation, treated animals and drug - naive play partners (matched for sex, age, and body weight ± 10 g) were introduced and their social interaction was observed for 10 minutes.

[0137] Play fighting (or social play) was defined as pinching, pouncing, or playful nape attacks, chasing. The tests were performed four times on days 32 (Test 1), 33 (Test 2), 34 (Test 3), and 35 (Test 4). ​ They were carried out separately. As can be seen in Figure 1, a significant decrease in social play was observed in rats treated with valproic acid, and rats treated with fibroblasts were reversed.

[0138] Although the present disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions, and modifications can be made herein without departing from the spirit and scope of the design defined by the appended claims. Further, the scope of the present application is not intended to be limited to the specific embodiments of the processes, machines, manufactures, compositions of matter, means, methods, and steps described herein. As will be readily understood by those skilled in the art, processes, machines, manufactures, compositions of matter, means, methods, or steps that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein, whether currently existing or later developed, can be utilized in accordance with the present disclosure. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufactures, compositions of matter, means, methods, or steps. ​

Claims

[Claim 1] A method of treating a pervasive developmental disorder comprising administering to an individual in need thereof a therapeutically effective amount of a composition comprising fibroblasts, modified fibroblasts, apoptotic fibroblast bodies, fibroblast conditioned medium, or a combination thereof.

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