Inflammation is characterized by an influx of leukocytes into a site of infection or tissue distress which is directed by a cascade of biochemical and cell adhesion events. Inflammation is a natural process which results from your body’s immune defense against pathogens such as bacterial and viruses. Symptoms can include swelling, joint pain, and redness.

Sometimes the body’s defense system (immune system) inappropriately triggers an inflammatory response when there are no foreign substances to fight off. In these diseases, called autoimmune diseases, the body’s normally protective immune system causes damage to its own tissues. The body responds as if normal tissues are infected or somehow abnormal.

Inflammatory diseases refer to diseases that are caused or contributed to by a complicated set of functional and cellular adjustments involving acute or chronic changes in microcirculation, movement of fluids, and influx and activation of inflammatory cells (e.g., leukocytes) and complement, and included autoimmune diseases. Examples include reperfusion injury, ishemia injury, strock, transplant rejection, hepatitis, thyroiditis periodontal disease, arthritis, psoriasis, multiple sclerosis and sepsis.

Inflammation of the central nervous system (CNS) (neuroinflammation) is now recognized to be a feature of all neurological disorders. In multiple sclerosis, there is prominent infiltration of various leukocyte subsets into the CNS. Even when there is no significant inflammatory infiltrates, such as in Parkinson or Alzheimer disease, there is intense activation of microglia with resultant elevation of many inflammatory mediators within the CNS.

Specific Types of Diseases

Alpha-gal syndrome (AGS): is an emerging tick bite-associated immunoglobulin E-mediated allergic condition characterized by reaction to the oligosaccharide galacose-alpha-1,3-galactose (alpha-gal) which is found in mammalin meat and products derived form mammals, including milk and other diary products. Symptoms range from mild (e.g., rash or gastrointestinal upset) to severe (anaphylaxis). Onset typically occurs 2 or mroe hours after exposrue to alpha-gal. No treatment is currently available. Evidence suggests that the reaction is primarily associated with the bite of the lone star tick (Ambylomma americanum) in the United States with cases most prevalent in the sourthern, midwester and mid-Atlantic United States, overlapping the range of the lone star tick. See CDC

Meningitis: inflammation of the membranes covering the brain upon bacterial and viral infection. Most prominent in the cae of bacterial meningitis, clinical symptoms include a horrible headache, fever, malaise and pain when moving the neck.

Polyarteritis nodosa: is a disease in which sigments of medium sized arteries become inflamed and damaged, reducing the blood supply to the organs. It is often fatal if not treated adequately. It usually develops at 40-50 but can occur at any age. Men are three times more likely than women to develop it. Its cause is unknown, but reactions to some drugs and vaccines may cause it.

Polymyositis: is a chronic connective tissue disease characterized by painful inflammation and degeneration of the muscles; dermatomyositis is polymyositis accompanied by skin inflammation. These diseases result in disabling muscle weakness and deterioration. The weakness typically occurs in the shoulders and hips but can affect muscles symmetrically throught the body. Polymyositis and dermatomyositis usually occur in adults from ages 40-60 or in children from ages 5-15. Women are twice as likely as men to develop either disease. The cause is unknown although viruses or autoimmune reactions may play a role. Cancer may also trigger the diases. Symptoms, which may begin during or just after an infection, include muscle weakness (particularly in the upper arms, hips, and thighs), muscle and joint paint, Raynaud’s phenomenon, a rash, difficulty in swallowing, a fever, fatigue, and weight loss. In dermatomyositis, rashes tend to appear at the same time as periods of muscle weakness and other symtpoms.

Psoriasis: is a chronic and recurring disease recognized by its raised red silvery scaled eruptions and plaques of various siezes which can appear anywhere on the skin. The disease is common, affecting from 2-4% of the Caucasian poulation. Psoriasis is a T-cell-mediated inflammatory disease in which the activation of the immune system in focal skin regions, mediated by CD8+ and CD4+ T lymphocytes, results in epidermal hyperplasiia. The cause of psoriatic lesions has been suggested to be related to antigens/superantigens or autoantigens provided by non-dermal inducing factors (PCT/US2006/030281). No known therapeutic method can cure psoriasis, but the majority of cases can be controlled. In less severe cases, treatment with pomades or emollient creams that keep the skin hydrated can be sufficient. In many of the moderate cases of psoriasis, topical formulations (pomades, creams, gels and lotions) containing corticosteroids are used by applying them underneath an occlusive covering made of cellophane or polyehtylene, or incorporating them into an adhesive bandage. In the more severe cases, the antineoplastics methotrexate or cyclosporin, both of which provike serious side effects, can be used.

Applying a topical agent is typically the first approach to treating psoriasis. All topical steroids have anti-inflammatory, anti-pruritic and vasoconstrictive effects. Topical agents include corticosteroids, coal tar, anthralin, calcipotriene and tazarotene. Coal tar treatment involves almost a month of messy topical treatments at a day treatment center. Although this method has a high rate of success in clearing skin, it is reltaively expensive and time-consuming. Anthralin is a synthetic derivative of a tree bark extract and is a cellular antiproliferative agent that decreases the rate of epidermal cell growth. Although anthralin is considered one of the most effective agents available for treating psoriasis, it is not in widespread use because of its high potential to cause irritation and staining of the skin. Calcipotriene is a synthetic vitamin D-3 analog that regulates skin cell production. It is not in widespread use, however, because it is expensive and dosage is limited due to the risk of iritation and vitamin D toxicity. Tazarotene is a retinoid derivative but can also cause irritation.

Phototherpay can also be used for extensive, widespread disease. There are two main forms of phototherapy, UVB and PUVA phototherpay. UVB, or Ultraviolet B, phototherapy uses light having a wavelenght in the range of 290-320 nm. Such phototherpay is usually combined with one or more topical treatment including topically applying coal tar, followed by using UVB, using a coal tar bath, followed by UVB, and then followed by topically applying anthraline, or using UVB in combination with topically applying corticosteroids, calcipotriene, tazarotene, or simply bland emollients.

Sjogren’s syndrome: is a chronic inflammatory disorder characterized by excessive dryness of the eyes, mouth, and other mucous membranes. The syndrome is often associated with other symptoms more characteristic of rheumatoid arthritis of lups. The cause of Sjogren’s syndrome is not known although it is thought to be an autoimmune disease. It is less common than RA and more prevalent in women. Lympoma, a cancer of the lymphatic system, is 44 times mroe common in people who have Sjogren’s syndrome. No cure is available, but symptoms can be relieved.

Systemic Inflammatory Response Syndrome (SIRS): Several infective and non-infective causes of SIRS are recognized. Infective causes of SIRS include sepsis and spetic chock, infection caused bybacterial pathogens, viruses, fungi, and parasites. Non-infective causes of SIRS include haemorrhagic shock, acute pancreatitis, and burns. Systemic leukocyte activation (cytokine-mediated) is a direct consequence of SIRS and if excessive, can lead to MODS and multiple organ failure. As a consequence of overactive SIRS response, leukocytes become activated within the general circulation and some then lodge within the pulmonary microcirculation. As the condition develops, leukocytes migrate into the pulmonary interstitium and increased endothelia permeability leads to tissue oedema. The leukocytes in the lungs both respond and contribute to the infalmmatory process in ARDS.

Vasculitis: is an inflammation of blood vessels. It is not a disease but rather a disease process that occurs in a number of autoimmune connective tissue diseases, such as RA and lupus. No cause is known but in some cases hepatitis viruses are involved. Cells of the immune system, which cause inflammation, surround and infiltrate the affected blood vessels, destroying them and possibly damaging the tissues they supply.

–Microscopic polyangiitis: is a systemic pauci-immune necrotizing vasculitis that affects mainly small vessels. Pathogenesis is unkown.

Mechanism of Inflammation: 

Inflammation involves (but is not limited to) the generation of a chemokine gradient produced by various cells in response to inflammatory stimuli. Migrating leukocytes respond to this gradient and are directed to the site of tissue distress. This migration is controlled by a series of adhesive events occuring between the circulating leukocyte and the endothelial cell. Adhesion molecules expressed by both cell types as as molecular addresses, allowing the appropriate cell type into the site of tissue distress.

Persistent inflammation is a central feature of many diseases and invariably involves the cruitment and activaiton of inflammatory cells that lead to structural changes in the affected organs. In diseases of the lung, for example, over 100 inflammatory mediators have been implicated, and the blocking of a single mediator is unlikely to be effective. In fact, antoagonists for these molecules have so far proved less effective than drugs with a broad spectrum of anti-inflammatory effects, such as glucocorticoids (Dip, Vet. J. 179, 2009, 38-49, p. 39 1st ¶).

Adenosine: is an endogenous nucleoside consisting of the purine base adenine in glycosidic linkage with the sugar ribose. It is present at low concentrations in the extracellular space but is greatly increased under stressful conditions as the result of enzymatic cleavage of the nucleotide adenosine 5′-monophosphate (AMP) by the 5′-nucleotidase. Intracellular concnetrations of adenosine are kept low principally by its conversion to AMP by adenosine kinase, but it may also be degraded to inosine by adenosine deaminase (Dip, Id, p. 38).

Complement system: Studies implicate an important role for natural antibody and the classical pathway of complement in the inflammatory response. For example, ischemia-reperfusion injury can be initiated by clonally specific natural IgM that activates the classical pathway of complement. For example, US Pat Nos: 7,442,783 and 7,442,783 described conserved reion with type II NMHC proteins (corresponding to amino acids 592-603 of Mouse NMHC-IIB (the “N2 self-peptide) as representing a major epitope for bidning of natural IgM following ischmia in an intestinal model.  Carroll,  (US 8,324,352 and 13/668515) described isolated antibodies that bind the N2 self peptide and inhibit such inflammation. 

Treatment Strategies:

Corticosteroids (cortisone-like medicine or steroid):

–Methylprednisolone provides relief for inflamed areas of the body. It is used to treat a number of different conditions, such as inflammation (swelling), severe allergies, adrenal problems, arthritis, blood or bone marrow problems, eye or vision problems, lung or breathing problems (eg, asthma), lupus, skin conditions, kidney problems, ulcerative colitis, and flare-ups of multiple sclerosis. Methylprednisolone is a corticosteroid (cortisone-like medicine or steroid). It works on the immune system to help relieve swelling, redness, itching, and allergic reactions.

Methylprednisolone, a corticosteroid, is similar to a natural hormone produced by your adrenal glands. It is often used to replace this chemical when your body does not make enough of it. It relieves inflammation (swelling, heat, redness, and pain) and is used to treat certain forms of arthritis; skin, blood, kidney, eye, thyroid, and intestinal disorders (e.g., colitis); severe allergies; and asthma. Methylprednisolone is also used to treat certain types of cancer. 

Methylprednisolone is a systemic synthetic corticosteroid that exerts a wide range of physiologic effects similar to naturally occurring glucocorticoids. See Correa, Methylprednisolone

The term “macular degeneration-related disorder” refers to any of a number of conditions in which the retinal maculal degenerates or becomes dysfunctional as a consequence of decreased growth of cells of the macular, increased death or rearrangement of the cells of the macula, loss of normal biological function, or a combination of these events. Macular Degeneration-related Disordersinclude age-related macular disorder (AMD), North Carolina macular dystrophy, Sorsby’s fundus dystrophy, Stargardt’s disease, pattern dysterophy, Best disease, dorminant drusen and radial drusen (“malattia leventinese”).

Age-Related Macular Degeneration (AMD):

AMD is the major cause of irreversible blindness worldwide. AMD is characterized by a progressive loss of vision in the critical central portion of the visual field.  This visual loss is caused by degeneration of the macula, a small circular region of the neural retina that is responsible for one’s ability to appreciate detail and read fine print. Individuals with AMD find images in the important central porition of the visual field greatly blurred or non-existent.

AMD is the elading cause of visual loss in people over 55. Two major clinical phenotypes of AMD are recognized –a nonexudative (dry) type and an exudative (wt) type. (Bora, J. Immunology, 174, 2005, pp. 491-497)

Types of AMD: 

1. Wet AMD: The  more severe form of AMD is typically diagnosed in the late stages of the disease and is associated with the ingrowth of blood vessels into the retina. Wet AMD is characterized by growth of abnormal blood vessels behind the retina under the macula. This “neovascular” form of AMD is responsible for about 90% of the severe visual loss (20/200 or worse). Choroidal neovascularization (CNV) which refers to abnormal or excessive formation of new blood vessels in the chorid layer of the eye. These new blood vessels are fragile and often leak blood and fluid. The blood and fluid raise the macula from its normal place at the back of the eye, causing loss of central vision.

2. Dry AMD: An earlier atrophic, stage of the disease is manifested by abnormal deposition of cellular and extracellular materials in association with Bruch’s membrane and the nearby vascular bed (the choriocapillaris), especially in the macular region. Some of these deposits are termed drusen, based on the German word for nodule. Drusen are concentrated between the retinal pigmented epithelium (RPE) basal lamina and the choriocapillaris. A number of studies have concluded that the presence of macular drusen is a strong risk factor for the development of both atrophic and neovascular AMD (Hageman, Molecular Vision, 1999, 5:28). Dry AMD is characterized by slow breakdown of light sensitive cells in the macula, gradually blurring central vision in the affeced eye. Over time, less of the macula funcitons and central vision is gradually lost.

Etiology:

–Presence of complement moleules:

Inhibitors of complement, such as factor H and CD59, have ben shown to impact the development of choroidal neovascularization in response to laser induced damage of Bruch’s membrane (US8324182).

Various complement components have been found to be associated with AMD Zeng “Lack of association of CFD polymorphisms with advanced age-related macular degeneration” Molecular Vision 2010)

–Drusen and Vitronectin

Vitronectin (also termed “complement S-protein” is a complement inhibitor which binds to C5b-7 and C9. (Milis, Clin Exp Immunol. 92: 114-9, 1993)

Drusen and Vitronectin has been identified as a principal molecular constituent of drusen and other abnormal deposits associated with AMD. On the basis of this identification, WO 95/17673 describes an invention for diagnostics for early AMD based on the presence of abnormal concentrations of vitronectin as well as agents which dissolve or remove drusen and blcok the deposition of vitronectin which comprise drusen.

Treatment: 

1. Phtodynamic therapy: Wet AMD is treated with laser surgery, photodynamic therapy and injections into the eye. These treatments slow progression but do not cure.There is no known form of treatment for advanced stage dry AMD and vision loss is inevitable (US8324182).

2. Anti-angiogenic agents:

–Anti-VEGF: Current treatment for AMD and other types of choroidal neovascularization involve administration of anti-angiogenic agents. Current evidence supports the use of ranibizumab (Roche) which binds to certain subtypes of VEGF as first-line therapy (harding, Eye, 2010, 24 497-505). (see also Cimerli (ranibizumab-eqrn) by Coherus Biosciences). 

—

3. Anti-oxidants: Some reports suggest that orally administered antioxidants (e.g., vitamins E and C, selenium, beta-caroteine) may reduce its incidence and/or severity. Based on this information, AMD patients are advised by some opthalmologists to limit their exposure to intense sunlight, and/or to consider supplementing their diets with multivitamins and trace minerals. A specific high dose formulation of antioxidants and zinc has been shown to prevent intermediate stage AMD from progression to advanced AMD.

4. Complement Inhibition:

Bora discloses that for AMD of the exudative (wet) type the complement system such as the membrane attack complex (MAC) is involved in the etiology of the disease. Bora ruptured Bruch’s membrane as a mouse model for choroidal neovascularization (CNV) of the wet type AMD and found that anti-murine C6 Abs inhibited MAC formationand also resulted in the inhibition of CNV.

Hageman teaches methods for treating ocular diseases such as macular degeneration by administering an effective amount of a therapeutic agent which modulates a complement pathway assocaited moelecule (US Patent Applicaiton 15/897,609, published as US 2018/0335436) such as antibodies which modulate the level of at least one complement pathway assocaited molecule (US 2003/0207309A1).

Fung (US 12/092346) also teaches methods for the treatment of ocular related diseases such as AMD, diabetic retinopathy, ocular angiogenesis by adminsitering complement inhibitors such as those to the alternative complement pathwaqy such as Factor D. See also therapeutic application of antibodies 

6. Difficulties in treatment

Kovesdi (US2007/0098692) states that disorders associated with both neovascular and atrophic components, such as age-related macular degeneration and diabetic retinopathy, are particularly difficult to treat due to the emergence of a wide variety of complications (§3). Giordano (US 2007/0077233) also states that “age-related macular degeneration is clinically difficult to treat (§51).

Zeng “Lack of association of CFD polymorphisms with advanced age-related macular degeneration” Molecular Vision 2010) discloses that AMg is a complex disease cuased by the combination of genetic prediposition and environmental factors and that none of 6 single nucleotide polymorphisms (SNps) of complement factor D were found to be significanly assocaited with advanced AMD. 

Massachusetts Eye and Ear. Flaum Eye Institute

 Definitions:

Retina: the layer of nerve cells lining the back wall inside the eye that senses light. The retina communicates with your brain to help you see. The retina, the inside surface of the eye, is made up of three layers of cells. The layer closest to the external surface of the eyeball consists of two kinds of photoreceptor cells; the rods and cones. Rods, which get their name from the shape of their outer segment, are responsible for black and white vision when the illumination is dim. In contrast, cones are responsible for high visual acuity (sharpenss) and oclar vision. Cones have a cone shaped outer segment. The next layer contains bipolar cells and the layer closest to the cavity of the eye is composed of ganglion cells. Thus, light must first pass through the ganglion cells and bipolar cells in order to reach the photoreceptors. The rods and cones synapse with the bipoplar cells, and the bipolar cells synapse with the ganglion cells, which transmit impulses to the brain via the optic nerve. Ganglion cells are the only neurons of the retina capable of sending action potentials to the brain. The flow of sensory information in the retina is thus opposite to the path of light through the retina.

Macula: An area in the center of the retina that helps you see objects in front of your clearly.

Cornea: the window in the front of your eye that focuses light.

The cornea is a transparent tissue that works as a “lens” within the eye to focus light onto the retina. Consequently, it must retain its transparency if it is to serve this function. This transparency is maintained by the corneal endothelium, which regulates water flow between the aqueous humor and the corneal stroma by pump-and-leak barrier functions. However, the corneal endothelia cells (CECs) that perform this function have severely limited proliferative capacity, so any severe damage to the corneal endothelium, such as that arising from pathological conditions like Fuchs endothelia corneal dystrophy or from iatrongenic damage during cataract surgery, cuases irreversible cell loss. (Koizumi, “Feasibility of a cryopreservation of cultured human corenal endothelial cells, PLOS One, June 21, 2019).

Cornea endothelium (CE):

The CE plays a critical role in the regulation of corneal hydration, maintaining corneal thickness and keeping the cornea transparent. The human CE has very limited propensity to proliferate in vivo. Accordingly, in order to replace dead of damaged corneal endothelial cells (CECs), the existing cells spread out to maintain functional integrity and sustain corneal deturgenscence. In a situation where an individual experiences accelerated or acute corneal endothelail cell loss due to either accidental or surgical trauma, endothelial dysfunction of the CE layer may occur. This results in their inability to pump fluid out of the stroma, causing stromal and epitheilial edema, loss of corneal clarity and visual acuity and will eventually lead to the clinical condition of bullous keratopathy. (Mehta, “Cultivation of human corneal endothelial cells isolated form paired donor corneas” 2011).

Optic nerve: the nerve in the back of your eye that sends signals to your brain.

Dilation: a procedure in which a doctor uses eye drops to widen your pupil, allowing for a close look inside your eye.

Legal blindness: when vision can not be made better than 20/200, meaning that you have to be 20 feet or lower to see an object that someone with normal vision could see 200 feet away.

Sensory Transduction in Photoreceptors:

The transduction of light energy into nerve impulses follows a sequence that is the opposite of the usual way that sensory stimuli are detected. In the dark, the photoreceptor cells release an inhibitory neurotransmitter that hyperpolirizes the bipolar neurons. This prevents the bipolar neurons from releasing excitatory neurotransmitter to the ganglion cells that signal to the brain. The production of inhibitory neurotransmitter by photoreceptor cells is due to the presence of ligand gated Na+ channels. In the dark, many of these channels are open, allowing an influx of Na+ which depolarizes the membrane of photoreceptor cells. In this state, the cells produce inhibitory neurotrasmitter that hyperpolarizes the membrane of bipolar cells.

In the light, the process works in the opposite way. When a photopigment absorbs light, cis-retinal isomerizes and dissociates from the receptor protein, opsin, in which is known as the bleaching reaction. As a result of this dissociation, the opsin receptor protein changes shape, activating its associated G protein. The activated G protein then activates its effector protein, phosphodiesterase, which cleaves cGMP to GMP. The loss of cGMP causes the cGMP gated Na+ channels to close, reducing the dark current. Each opsin is associated with over 100 regulatory G proteins, which, when activated, release subunits that activate hundreds of molecules of the phosphodiesterase enyme. Each enzyme molecule can ocvert thousands of cGMP to GMP, closing the Na+ channels at a rate of about 1000 per soenc and inhibiting the dark current. The absorption of a single photon of light can block the entry of mroe than a million Na+, whichout changing K+ permeability. The photoreceptor becomes hyperpolirzed and releases less inhibitory neurotransmitter. Freed from inhibition, the bipolar cells release excitatory neurotransmitter to the ganglion cells, which send impulses ot the brain.

Evolution of the Eye:

In the early 19902, biologists studied the development of the eye in both vertebrates and insects. In each case, a gene was discovered that codes for a transcription factor important in lens formation; the mouse gene was called Pax6 and the fly gene was termed eyeless. A mutation in the eless gene led to a lack of production of the transcription factor and compelte absence of eye development, giving the gene its name. When the genes were sequenced, it became apparent that they were highly similar. In fact, insertion of the mouse version of the Pax6 into the genome of a fuit fy created a transgene fly. In this fly, the Pax6 gene was turned on by regulatory factors in the fly’s leg and an eye formed ont he leg of the fly.

Common Vision Disorders

The incidence of vision related disorders increases significantly with advancing age. The most serious age related disorders include cataract, glaucoma, and age-related macular degeneration (AMD). Collectively, these three diseases account for about 65% of new cases of legal blindness in the U.S. 8.4 million office visits and 426k hospitalizations annually are attributable to these and related conditions (WO 95/17673).

Age-Related Macular Degeneration (AMD)

Cataract is associated with an increase in the pressure inside of the eye “intraocular pressure or IOP” which eventually results in the degeneration of a portion or the retina. Once detected, glaucoma usually can be arrested by treatment with pressure lowering drugs. Delayed detection and treatment, however, leads to impaired vision and eventual blindess. Glaucoma can be effectively treated by replacing diseased lens with a plastic intraocular lens or IOL.

The number one reason you have cataracts is age. By 50, we can usually start to see some changes in the transparency of the lens that are an indication of early cataracts.

Corneal endothelia decompensation:

In the past the only treatment for corneal endothelial decompensation was transplantation of a donor corea. However, tissue engineering technology is now receiving increased attention as a way to overcome problems of corneal transplantations, which include a shortage of donor corneas, late graft failure due to continuous cell loss, graft rejection and the learning curve involved in performing corneal transplant procedures. (Koizumi, “Feasibility of a cryopreservation of cultured human corenal endothelial cells, PLOS One, June 21, 2019).

Koizumi, (“Feasibility of a cryopreservation of cultured human corneal endothelial cells, PLOS One, June 21, 2019) disclosed in 2013 cell based therapy involving injection of a suspension of cultured human corneal endothelial cells (HCECs) in combination with a Rho kinase inhibitor, into the anterior chamber. All 11 cases recovered corneal transparency and none experience any severe adverse effects. The HCECs were obtained form donor corneas and expanded in vitro culture. The HCECs were harvested form a culture plate, placed in a tube in the form of a cell suspension and immediately transported to the operating room in the same facility. (Koizumi, “Feasibility of a cryopreservation of cultured human corenal endothelial cells, PLOS One, June 21, 2019).

–Cell Culture of human corneal endothelial cells (HCECs): 

Mehta, (“Cultivation of human corneal endothelial cells isolated form paired donor corneas” 2011) discloses culturing isolated primary hCECs in 4 different medium, expanding the cells for two passages and anlyzing them for their propensity to adhere and proliferate., their expression of characteristic corneal endothelium markers; Na+K+/ATPase and ZO-1 and (3) their cellular morphology. hCECs established in the four media exhibited different proliferation profiles with striking norphological differences. Corneal endothelial cells cultured in M1 and M3 could not be propagated beyond the frist and secone passage, respectively. The hCECs cultured in M2 and M4 were significant more proliferative and epxressed markers characteristic of human coneal endothelium.

–Cryopreservation of human corneal endothelial cells (HCECs): 

Koizumi, “Feasibility of a cryopreservation of cultured human corenal endothelial cells, PLOS One, June 21, 2019) further screened several cyroperservation reagents for their effectiveness in preserving HCECs. Bambanker hRM enabled the cryopreservation of HCECs by maintaing cell viability and cell density.

Glaucomas are a group of neuropathic eye diseases characterized by increased intraocular pressure (IOP) and damage to the optic nerve.  Glaucoma can lead to reduction or loss of vision, and is the second leading cause of blindness. Current treatments involve reduction of intraocular pressure by chemical or mechanical means. For example, the most common treatment is trabeculectomy, a surgical procedure in which part of the trabecular meshwork of the eye is removed to allow drainage of the aqueous humor out of the eye into the conjunctiva, thereby reducing intraocular pressure.  However, trabeculectomy is generally followed by inflammation and fibrosis.

A comprehensive dilated-eye exam is the best way to detect glaucoma. Every adult over 50 should get this test every year, but if you have a fmaily history of glaucoma, you may need the test more often. If glacoma is caught early, it is a very treatable disease. When diagnosied late or not at all, glaucoma is extremely aggressive because the optic nerve is already damaged, and a damanged nerve is more vulnerable. Later-stage glaucoma attacks central vision and can cause irreversible blindess.

Corneal Neovascularization (KNV)

The cornea bears an “angiogenic privilege” and is avascular allowing maximal entry of incident light. This angiogenic privilege is maintained by a fine balance between anti-angiogenic factors and angiogenic factors in the cornea. Insults of chemical, mechanical or infectious nature can trigger inflammatory and immune-mediated pathways which upregulate expression of VEGF (vascular endothelial growth factor), the key player in KNV, and its signalling cascades. Common infectious diseases associated with KNV are the following:

1. Aspergillosis, Candidiasis, Chamydia trachomatis, Fusarium, Herpes simplex keratisis:

Herpes stromal keratitis (recurrent infection on the cornea by herpes simplex virus) is the most common cause of infectious corneal blindness in the western world. In the US an estimated 400,000 persons are affected, with 20,000 new cases occurring annually. Each episode increases the risk of future episodes. Current treatment consists of topical steroids in addiiton to prophylactice oral (acyclovir or valacyclovir) or topical (trifuridine) anti-viral drug therapy. Despite this treatment, patients develop severe corneal scarring due to repeated episodes of the disease, which often require corneal transplantaiton.

2. Onchocerciasis, Pseudomonas, Syphillis:

Common inflammatory conditions assocaited with KNV are graft rejection, acne rosacea, Stevens-Johnson syndrome, GVHD, Pemphigoid and Atopic conjunctivitis.

Diabetic Retinopathy:

Uncontrolled, elevated blood sugar damages blood vessels in the retina, cuasing a leakage of fluid into these tissues, a conditions called diabetic retinopathy. The early stage of the disease can be controlled with lifestyle changes by keeping A1c (a diabetes test result) below 6.5 with diet and exercise, as well as maintaining healthy blood pressue and lipid levels.

Diabetic retinopathy is the top cause of blindness in working age adults in the U.S. The later, more symptomatic stage of DR is called profliferative, in which new, but weeak, blood vessels grow in the retina to compensate for those that were damanged by blood sugar. When caught at this later stage, when symptoms like burriness or glare arise, you may need additional eye injections or even surgery.

Ocular Fibrosis:

Treatments for Ocular Fibrosis:

A proposed method of treatment of ocular fibrosis is inhibition of certain lysyl oxidase-type enzymes which have been found to occur in parallel with the fibrotic damage that can follow trabeculectomy.  A lysyl oxidase-type enzyme refers to a member of a family of proteins that catalyzes formation of aldehydes from lysine residues in collagen and elastin precursors. The aldehyde residues of allysine are reactive and can spontaneously condense with other allysine and lysine residues, resulting in crosslinking of collagen molecules to form collagen fibrils. The first member of this family to be isolated was lysyl oxidase, also known as protein-lysine 6-oxidase, protein-L lysine or LoX. Additional lysyl oxidase type enzymes were subsequently discovered and termed LOX-like or LOXL Although all lysyl oxidase-tyep enzymes share a common catalytic domain, they differ, particularly in their amino terminal regions.

Chemical Injiries of the eye: are opthalmic emergiceis that require immediate evaluation adn management. These in juries often result in significant occular morbidity. Injireis caused by alkali agents are more common and generally more serious than those casued by acids. There are three main goals in manageing a chemcial injury,: enhance recovery of the corneal epithelium; augment collagen synthesis while minimizing collagen breakdown and sterile ulceration and control inflammation. (US 2020/0289580).

Rare Eye Diseases

Purtscher’s retinopathy: is a rare retinal disorder characterized by acute visual loss and retinal findings such as cotton-wool spots, intraretinal hemorrhages and retinal whitening following head or chest trauma. When the etiology is not a trauma, the disease is called Purtscher-like retinopathy. Nuermous conditions such as acute pancreatitis, connetive tissue disorders, autoimmune diseases, pregnancy-related diseases, and thrombotic microangiopathic diseases can cause Purtscher-like retinopathy. It has also been associated with aHUS (Turk J. Ophthalmol 47; 6: 2017).

A staggering number of people (over 15%) of the population suffer from hearing loss. the prevalence increases with age, with half the population over the age of 60 experiencing hearing loss. Despite being the third most common chronic condition after arthritis, hearing loss is largely managed (e.g., hearing aids) rather than treated.

The inner ear is an organ capable of rapidly generating an active immune response, although, like the brain, it has a blood labyrinthine barrier and few resident macrophages. Alhtough once considered an immunologically privileged organ, it has connections to the lymphatic system through cervical lymph nodes. Hearing loss in some invidiuals is thought to be mediated by local autoimmune resposnes, and treatment with immunosupressive drugs can restore some cochlear function. A number of otic drugs are in clinical trial stages. For example, SPI-1005 developed by Sound Pharmaceuticals and designed to protect sensory hair cells form noise induced hearing lss is currently in Phase II trials with the US Navy and Marine Corps. Merz Pharmaceuticals is also currently recuriting for Phase III trials with neramexane, which has shown promise in the treatment of tinnitus. Auris Medical also have Phase IIb trials with AM-101 for the treatment of acute inner ear tinnitus and AM-111 to treat acute sensorineural hearing loss. Both Adherex and Sound Pharmaceuticals also have drugs in clinical trials that protect against the adverse side effects of the chemotherapeutic cisplatin.

Common Agents Used in the Treatment of Hearing Disorders:

–Antiboiotics For example azalid antibiotics have been used for topical administration to the ear for the treatment and/or prevention of otic infections (US 2006/0046970). Clinical studies for treating Meniere’s disease using aminoglycoside antiotics such as gentamicin sulfate have been performed with some sucess (WO 97/38698). The local devliery of vancomycin has been shown to have clincial applicaiton for treating chronic otitis media caused by methicillin reistant Staphylococcus aureus (MRSA) (Lee, J. Controlled Release 96 (2004)).

–Glucocorticoids have been tranditionally used to reverse hearing loss in a variety of cochlear disorders. These include autoimmmune and other systemic immune diseases. However, their side effects prevent long term therapy. Most glucocorticoids have three physiological functions: anti-inflammation, immune suppression and increased sodium transport/reabsorption.

–Anti-Cytokine Therapy: Inhibitors of TNF? such as TNF soluble receptor type I has been shown to prevetn experimental otitis media with effusion (OME). The anti-TNF? antibody, Etanercept, has also been shown to reduce inflammatory response which leads to cochlear disease.

–Mineralocorticoid fludrocortisone has also been proposed for reversal of autoimmune hearing loss (US 2006/0013858).

Local Delivery

In recent years there has been increasing interest in the treatment of inner-ear disorders by local rather than systemic application of drugs. Substances are applied intratypanically, which means they are inected into the middle-ear cavity. This procedure is based on the premise that the drug will contact the round window membane (RWM) of the ochlea, enter the scala tympani (ST) and spread throught the ear. The target tissues might include the sensory hair cells, the afferent never fibers and supporintg cells of the cochlea (hearing) or vestibular (balance) portions of the inner ear. Several drugs that are applied directly to the inenr ear are in widespead clinical use for the treatment of inner-ear disorders.

However, the pharmacoknetics of drugs in the inner ear is not well defined and the field is plagued by technical problems in obtaining pure samples of the inenr ear fluids for analysis. Although in theory the local application of drugs to the inner ear has great potentail, in practice there are numerous technical difficulties to overcome. Important issues include (i) which parts of the ear do drugs reach, in what concentration and with what time coure? (ii) how do different delviery methods of application protocols influence the drug levels at each time point at the different locations in the ear? (iii) how variable are the drug levels achieved with different delivery protocols and what are the major sources of variation? (Salt, DDT, 10(19), 2005).

When substances are applied intratympanically, the assumption is that they will enter the scala tympani through the RWM and then be distibuted throughout the cochlear fluids. However, it is very difficult to predict the amounts odf drugs that will actually reach the cochlear fluid space (Nakagawa, Current Drug Therapy, 3(2), 2008).

–Implantable mini-pumps have been used for local drug delivery to the cochlea in animal experiments. The use of a local viral gene transfer a(adenoviral vectors)s a sustained treamtnet of the inner ear can also provide suffiicent proteiction form noise, drug toxicity and re-perfusion injury. There has also been great interest in the use of biomaterials for local drug delviery. Biodegradable polymers containing therapeutic molecules are placed on the RWM, with the therapeutic molecules released into the cochlear fluids form the polymers in a controlled manner via the RWM.

–Viral and Non-Viral Gene transfer: is being used for the sustained treatment of inner ear disorders. A gene known as Math1 and a key regulator of hair cell development which induces regeneration of hair cells and substantially improves hearing thresholds in the deaf inner ear after delivery to nonsensory cells through adenoviral vectors has been shown (Izumikawa, “Auditory hair cell replacement and hearing improvement by Atoh1 gene therapy in deaf mammals. Nat Med 2005; 11: 271-276).

–Penetration enhancers: have been used to deliver medicaments across the tympanic membrane which is a formidable barrier against inroduciton of drugs into the middle ear (this is why antibiotics are typically taken orally). WO 2007/037874 describes enhances such as propylene glycol.

Related Links:       Action on Hearing Loss

Common Ear Disorders

When a foregin antigen enters the inner ear, it is first processed by immunocompetent cells which reside in and around the endolymphatic sac. Once the foreign antigen has been processed by these immunocompetent cells, these cells secrete various cytokines which modulate the immune response of the inner ear. For example, TNF-alpha may play a key role in the initiation and amplification of the immune response. One result of this cytokine release is to facilitate the influx of inflammatory cells which are recrutied from the systemic circulation. These systemic inflammatory cells enter the cochlea via diapedesis through the spiral modiolar vein and its tributaries, and begin to participate in antigen uptake.

Autoimmune inner ear disease (AIED): is a rare disorder appearing in both adults and children that often involves a bilateral disturbance of the audio and vestibular functions of the auris interna. Up to one-third of pateints with AIED also suffer from a systemic autimmune illness, such as inflammatory bowel disease, and rheumatoid arthritis. In AIED the immune system itself begis to damage the delcate inner ear tissues.

Transtympanic administration of the monoclonal antibody against TNF alpha (infliximab) has been shown to result in hearing improvement and reduced disease relapses in patients suffering from autoimmune inner ear disease (AIED) (Wijk, Audiol Neurotol 2006).

Meniere’s Disease: is an idiopathic condition characterized by sudden attacks of vertigo, nausea and vomiting that may last for 3-24 hours, and may subside gradually. Progressive hearing loss, tinnitus and a sensation of pressure in the ears accompanies the disease through time. The cause of Menier’es disease is unknown but is probably related to an imbalance of inner ear fluid homeostasis, including an increase in production or a decrease in reabsorption of innear ear fluid.

The aetiology and pathogenesis of Meniere’s disease remains unknown (Greco, Autoimmunity Reviews, 11, 2012). Intratympanic injection of gentamicin into the middle ear has been used to treat Meniere’s disease. Gentamicin is toxic to the sensory cells of the balance system and thus supresses the vertigo in these patients by partically ablating their vestibular system. There are also cinical reports related to the local applicaiton of glucocorticoids for Menieres disease.

Meniere’s Syndrome: displays similar symptoms as Meniere’s disease and is attributed as a secondary affliction to another disease process such as thyroid disease or inner ear inflammation due to syphillis infection.

Otitits media (OM) (infection of the middle ear): is an inflammatory disorder of the auris media which includes actue otitis media (AOM), otitis media with effusion (OME) and chronic otitis media. Alhtough adults are susceptible to OM (middle ear infections) children are particularly at risk because their relatively short auditory canals can more easily be closed by inflammation. Fluid can then become trapped behind the tympanic membrane (eardrum), which can cause severe pain as well as provide microbes with an inviting environment. Acute otitis media is linked to the activity of pathogenic bacteria commonly found in the indigenous microbiota of the naso-pharyngeal cavity. Quantitatively, the most important pathogens are Streptococcus pneumonia (35%), non-typeable Haemophilus influenzae (30%) and Moraxella catarrhalis (10%). For this reason, acute otitis media is commonly treated by the adminsitration of antibiotics especially in infants. But this has led to development of resistance to commonly prescribed antibiotics. It has been known for some time that human milk has a protective effect against otitis media and this is thought to be due to specific immunoglobulins in the milk such as seretory IgA antibodies in the milk. Bruessow teaches a composition suitable for treatmnet of otitis media by IgA derived from mature bovine milk.  

OM susceptibility is multifactorial and complex, including environmental, microbila and host factors. Bacteria infection accounts for a large percentage of OM cases, with more than 40% of cases attributed to Streptococcus pneumoniae infection. Regardless of the causative agent, increases in cytokine production, including interleukins and TNF, have been observed in the effluent media of individuals afflicted with OM. Because OM can be cuased by a virus, bacteria or both, it is often difficult to identify the exact cause and thus the most appropriate treatment. Treatment opicns in the auris media include treatment with antiboitocis. Pre-treatment with TNF-alpha inhibitors in experimental LPS induced OM animal models have been shown to suppress development of OM.

Sensorineural hearing loss (SNHL): is one of the most prevalent disabilities. Sound stimuli are received by auditory hair cells (HCs) in the bony, snail-shaped cochlea, followed by transduction of the sound stimuli by the HCs to neural signals. Spiral ganglion neurons (SGNs) which are auditory primary neurons, are located in the central bony axis of the cochlea and responsible for transmitting auditory signals to the central auditory system. The cochlea is connected to the tympanic cavity by the round window membrane (RWM). Excessive noise, ototoxic drugs, genetic disorders and aging all contribute to the causes of SNHL.

Vestibular Neuronitis: is characterized by sudden vertigo attacks and may be cause by inflammation of the nerve to the semiicircular canals likely cause by a virus.

Sensorineural Hearing Loss (SHL): occurs when the components of the inner ear or accompanying neural components are affected and/or inflamed. Sensory hearing loss may be hereditary, or it may be caused by acoustic trauma (e.g., very loud nocies), a viral infection, drug-induced or Meniere’s disease. Many studies have focused on finding novel therapeutic molecules that can be used in treatment of SNHL. Despite such research, the translation into useful therapeutic clinical agents has yet to be achieved. However, clinical trials are in progress. For example, gelatin hydrogels are being used for sustained delivery of insulin-like growth factor 1 (IGF-1). Hepatocyte growth factor (HGF) also has been shown to significantly reduce noise exposure.  (Inaoka, Acta Oto-Laryngologica, 2009).

Presbycusis: or age-related hearing loss, occurs as a part of normal agining, and occurs as a result of degeneration of the receptor cells in the spiral organ of Corti in the inner ear. Other causes may also be attributed to a decrease in a number of nerve fibers in the vestibulocochlear nerve, as well as a loss of flexibility of the basilar membrane in the cochlea. There is currently no known cure for permanent hearing damage as a result of presbycusis or excessive noise.

Itinnitus: is the perception of sound without external acoustic timulation and is a very common inner ear disorder.

Links of interest: American Diabetes Association

Introduction/Definitions:

Diabetes is one of the leading causes of death and disability in the US and can lead to renal disease (nepthropathy), micro vascular problems, blindness (retionopathy), extremity amputation and hypertension. In healthy people, when there is too much glucose in the bood, the glucose stimulates the pancreas to produce insulin. Insulin targets are fat, muscle cells and fat cells. Insulin binds to insulin receptor on the cells and activates cascade pathway leading to the entrance of glucose into the cells. Moreover, insulin also binds receptors in the liver and stops the liver from producing glucose.There are two major forms of diabetes mellitus: insulin-dependent (type I) and noninsulin-dependent diabes mellitus (type II). Type I diabestes, also called juvenile-onset diabetes mellitus, most often strikes suddently in childhoold. In contrast, type II daibetes, also called maturity-onset diabetes mellitus, usually develops rather gradually after the age of 40.

Glucagon: is a hormone released by the pancreas that acts as a check on insulin. Whereas insulin lowers blood glucose, glucagon raises it. Scientists thought suppressing glucagon might help treat type 2 diabetes. In looking at hormones, including glucagon, in pancreases from anglerfish, which scientists pulled from Boston harbor, they froze the fish’s hormone-producing pancreatic islet cells in search of uncharted DNA inside, ultimately cloning a gene called proglucagon. The fish gene encoded a large precursor protein that the body chops apart to form glucagon. Also embedded in the proglucagon protein was a stretch of amino acids that resembled glucagon and would come to be called GLP-1. Subsequent looks at the amino acid sequence of proglucagon in mammals, including work in hamsters and humans revealed a second glucagon-like-peptide GLP-2.

GLP-1’s amino acid sequence also shared some features with gastric inhibitory peptide, or GIP, which was then the only known member of a fabled category of hormones called incretins. But GIP was a disappointment. Giving it to people with diabetes had had little effect on their insulin levels. “GIP was a complete bust.” Based on its similarity to glucagon, and the way biologically active glucagon is produced, it was hypothesized that a stretch of 31 amino acids between spots 7 to 37 within the larger GLP-1 peptide might be an incretin. When scientists injected rat pancreas with GLP-1 that had been synthesized, insulin output ticked up. Injection of the peptide into healthy people and those with diabetes. GLP-1 prompted insulin release when glucose levels rose—after eating, for example.  It was latter found that GLP-1 could normalize blood sugars in people with diabetes and caused appetite loss. See Science

Incretins are produced by the gut and spur the pancreas to release insulin—a function scientists thought could make them useful for studying and even treating type 2 diabetes.

Insulin: Vertebrate insulin, synthesized in pancreatic beta-cells, is the key hormone regulator of carbohydrate and fat metabolism; in the brain, it functions as a neuromodulator of energy homeostasis and cognition. Insulin is initially synthesized as a precursor comprising three regions (A, B, and C) from which proteolytic cleavage of the C peptide in the Golgi releases the mature insulin heterodimer with an A and B chain connected by two disulfide bonds. The A chain contains an additional intromolecular disulfide. The primary sequence and arrangement of cysteins that form disulfides are highly conserved in all vertebrates. In contrast, invertebrate insulin family members are more variable and serve in neuronal signaling, memory, reproduction, growth and metabolism. In molluscs, insulins are primarily expressed in neuroendocrine cells, including neurons and cerebral ganglia. Insulin is an essential hormone, but overdoses cause hypoglycemia, and in extreme cases, fatality. Remarkably, evolution has developed a a parallel story line, with normal physiological function of insulin being subverted by a lineage of cone snails as part of their strategy to overcome their fish prey. Gastridium clade, one of 5-6 clades of fish hunting cone snails, use insulin as an offensive w3apon. (Olivera, “Specialized insulin is used for chemical warfare by fish-hunting cone snails” 112(6), 2015, 1743-1748)

Type I diabetes:  

Type 1 diabetes is a devasting autoimmune disease where one’s own T lymphocytes recognize and destroy pancereatic beta cells. Those cells make the insulin that regulates blood sugar. Without insulin, the sugar cannot enter the cells.

TID results from T lymphocyte attack on the insjulin producing beta cells of the islets of Langerhands of the pancreas. The disease unfolds over a number of years, resulting in clinically detectable hyperglycemia and ultimately diagnosis of diabetes. Disease unfolds through two main stages; an occult phase, termed insulities, when a mixed population of luekocytes invades the islets promoting beta cell death and the overt phase, diabetes, when the bulk of beta cells have been destroyed and the pancreas can no longer produce sufficient insulin to control blood glucose levels.

Diagnosis:

There has been effort to identify markers that indirectly signal TID progression. So far the best indicator if islet inflammation has been serum titers of autoantiboides directed against a defined set of B cell antigens. At present, the islet inflilgration, termed insulitis, is only detectable by biopsy, usually at autopsy.

Denis (PNAS, 101(34) 2004) discloses using long circulating mangetofluorescent nanoparticles (CMFN) as probes of microvascular changes accompanying inflammation. The nanopartciles contan a small, monocyrstyalline, superparamagnetic iron oxide core, which exhibts strong mangetic behanvior detectable by high resolution MRI. The 3 nm core is surrounded by a dense modified dextran coating that diminishes the immunogenicity of the assembled partciles and substantially enhances their half life in ciruclation. See drug delivery and magnetic particles for more information 

Treatment:

Patients depend on insulin injections several times per day and have to monitor blood glucase leves clesely at all times. Companies like Eli Lilly mkae special versions of insulin to control potency and limit side effects by minimizing the amount needed.

–transplantation: Islets of the pacrease which contain beta cells can be transplanted.

–stem cells: which can differential into functional beta cells can be an option.

–CD3 antibodies: The monocloanl antibody Teplizumab, binds to CD3 on T cells. The Fc region of this antibody has been modified so that it has non-binding properties. Because CD3 is a co-receptor for T cell activaiton, drugs which target it may result in immunosuppression. Immunomodulatory agents such as anti-CD3-antibodies may restore normal glucose control if provided in very early stages of the disease, such as stage 2 T1DM, when there are still enough beta cells to maintain euglycemia. The mechanisms of action of teplizumab appear to involve weak agonistic activity on signaling via the T cell receptor-CD3 complex associated with the development of anergy, unresponsiveness, and/or apoptosis, particularly of unwanted activated Teff cells. In addition, regulatory cytokines are released and regulatory T cells are expanded that may lead to the reestablishment of immune tolerance.

–Tregs: 

Tregs are a subset of T cells that are not as interested in killing as they are in preventing other T cells form killing and represent a new class of therapy.

—-anti-HLA-A2 CAR Tregs:

Without a targeting mechanism, adminsitered Tregs disappear quickly. But when CAR Tregs are injected intravenously, they will enter the spleen and then the islt transplant, where they stay and accumulate because the transplanted material provides the necessary activaiton signals for Treg survival. HLA seriotype A2 (HLA-A2) is a specific allele in 30% of the population. In a transplant setting, one can have a donor who is HLA-A2* and a recipient who is HLA-A2-. In most clinical settings, one would not want such a HLA mismatch, but in this case, the mismatch would raise a vlaid target that is present only in a trransplanted oran. If one makes anti-HLA-A2 CAR Tregs, they will recognized only HLA-A2* islets or beta cells. That specificity gives them an advantage over blanket treatmetns. CDRs have been taken from an antibody against human leukocyte antigen-A2 (HLA-A2) and grafting those into a different single chain fragment variable (scFv) backbone in what is basically a transplant of specificity. (you have a new DNA sequence that used to recognize x (HER2) and now is going to see y (HLA-A2); if your specific antibody based CAR is not working, this is a good strategy to use an existing antibody that you know works and then try to change the specificity of that antibody.) The procedure is as follows: (1) First, one needs to get the Tregs for CAR Treg expansion, usually by isolating them form periopheral blood using FACS. One can first use magnetic enrichmetn of CD4 T cells, which can raise the percentage of Tregs from 1-5%. Then one can use CD25 enrichment, which could raise that percentage to 70%. One can also filter out cells based on what they do not have. Tregs are CD25 high, but they are also CD127 low. Thus one can increase purity by excluding some activated T cells which could contaimate the sample. (2) Second, expand the T reg culture. One can then activate Tregs using magnetic beads coated with anti-CD3 and anti-CD28 mAbs. Putting those beads into cells simulates an infection. Tregs will begin to cluster in response to a perceived infection. The activation levels of Tregs in a culture will go up and down and one must let them go back to a resting state before forcing them to activate and divide. If one activates Tregs too quickly in succession, they will experience activation induced cell death. IL-2 is mandatory for Tregs to live, but they do not make it. So it must be added. Adding IL-6 and TNF-alpha to a pure Treg cultures can also allow for better Treg growth and expansion. (3) Next clustered regularly interspaced palindromic repeats and associated protein 9 (CRISPR-9) was used to knock in sequences for a CAR that works in Tregs. By replacing a TCR with a CAR, one ensures one CAR copy per cell. The TCR promoter and enhancer are there, so when a T cell is activated, oen can acheive maximum CAR expression. A T cell receptor (TCR) locus is inserted and a grafted anti-HLA-A2 CAR is put in. The HLA-A2 CAR Tregs suppress immune responses in vivo in an HLA-A2 dependent way. The anti-HLA-A2 CAR Tregs suppresed human immune responses when they recognized HLA-A2 in the injected PBMCs, the mouse tissue or both. (Abbot and Ferreria “using Regulatory T cells for treatment of Type 1 diabetes, Part 1, BioProcess international, April 2023 and (Ferriera “using Regulatory T cells for treatment of Type 1 diabetes, Part 2” BioProcess International 21(5) March 2023)).

Tang “Precision Engineering of an Anti-HLA-A2 Chimeric Antigen Receptor in Regulatory T Cells for Transplant Immune Tolerance” Frontiers in Immunology, (2021)) discloses an anti-HLA-A2 CAR (A2-CAR) generated by grafting the complementarity-determining regions (CDRs) of a human monoclonal anti-HLA-A2 antibody into the framework regions of the Herceptin 4D5 single-chain variable fragment and fusing it with a CD28-z signaling domain. The CDRgrafted A2-CAR maintained the specificity of the original antibody. We then generated HLA-A2 mono-specific human CAR Tregs either by deleting the endogenous T-cell receptor (TCR) via CRISPR/Cas9 and introducing the A2-CAR using lentiviral transduction or by directly integrating the CAR construct into the TCR alpha constant locus using homology-directed repair.

Type II diabetes: (see outline)

Numerous epidemiologic studies have evaluated several inflammatory markers, including C-reactive protein, various cytokines, adhesion molecules, and white blood cell (WBC) count for their clinical usefulness in predicting risk of cardiovascular disease. (Zhang, “Association between Myeloperoxidase levels and risk of coronary artery disease” JAMA, 286(17), 2001).

Carotid intima-media thickness (IMT)

IMT increase predicts the risk of cardiovascular evetns, with relatively stronger prognostic power for crebral as compared with coronary vascular events. Inc reased IMT is considered to represetn a manifestation of subclinical atherosclerosis and has been incuded in the lsit of organ damage conditions in European hypertension guidlines. The lack of invasiveness and reepatability makes IMT measurement an attractive biomarker, potentially useful as a therapeutic target in subjects at increased cardiovascular risk. (J American Colelge of Cardiology, 56(24) 2010)

Above average C-reactive prtoein with statin therapy predicts failure of IMT regression in those with optimal LDL cholesterol (<100 mg/dl) but not if LDL cholesterol is <70 mg/dL (ultra-low levels). (Kent, American J. of Cardiology, 92(15), 2003). 

Cholesterol

A lipid test (chloresterol) is often performed to evaluate risks for heart disease. Cholesterol is an important normal body constituent, used in the structure of cell membranes, syntehsis of bile acids and synthesis of steroid hormones. Since cholesterol is water insoluble, most serum cholesterol is carried by lipoprotines (chylomicrons, VLDLC, LDLC, and HDLC). Excess cholesterol in the blood has been correlated with cardiovascular events. LDL is soemtimes referred to as “bad” cholestero, because elevated levels of LDL correlate most directly with cardiovascular events sucha s coronary heart disease. HDL is sometimes referred to as “good” cholesterol since high levels of HDL are correlated with a reduced risk of cardiovascular events sucha s coronary heart disease. The term chloesterol means “total” chloesterol, i.e., VLDLC+LDLC+HDLC. (US 14/886626, published as US 2016/026146). 

Preferably, cholesterol levels are measrued after fasting. The cholesterol measurement is typically reported in milligrams per deciliter (mg/dL). Typically, the higher the total cholesterol, the more at risk a human subject is for a cardiovascular event. A value of total cholesterol of less than 200 mg/dL is a “desirable” level. Levels over 240 mg/dL may put one at almost twice the risk of a cardiovascular vent such as coronary heart disease. (US 14/886626, published as US 2016/026146).

Since cholesterol is water insoluble, most serum cholesterol is carried by lipoproteins (chylomicrons, VLDLC, LDLC and HDLC). The term cholesterol measn “total” cholesterol, i.e., VLDLC + LDLC + HDLC. 

C reactive protein: 

CRP even in the absence of hyperlipidemia, is associated with an increased risk of coronary events. In terms of clinical application, CRP seems to be a stronger predictor of cardiovascular events than LDL cholesterol, and it adds prognostic information at all levels of calculated Framingham Risk and at all levels of the metabolic syndrome, Ridkey, Circulation, 107, 2003, 363-369. The rates of coronary events increase significantly with increases in levels of CRP (Ridker, N Engl J Med, 344(26), 2001).

The usefulness of CRP as a clinical biomarker of as a predicttor of cardiovascular risk is important because because almost half of all myocardial infarctions and strokes occur in persons without elevated levels of low-density lipoprotein cholesteroal (Clin. Cardiol. 26pp. III-39-III-44 (2003).

Herrmann (Cellular and Molecular Biology 50(8) 895-901, 2004) discloses that homocysteine and high sensitivity C-reactive protein (hs-CRP) are were independently associated with the risk of myocardial infarction, stroke and cardiovascular death. 

A CRP level of ≥1 is considered an elevated level. CRP levels of <1m 1 ti 3m abd >3 ng.K cirresoibd ti kiw0 niderate abd gugg risk groups for future cardiovascular vents. (Ridker, Circulation, 2003, 107: 363-369).

Ridker (US7,030,152 and US7,964,614) diclsoes methods for characterizing an individual’s risk profile of developing a future CVD disorder as well as evaluating the likelihood that an individual will benefit from treatment with an agent such as a statin for reducing the risk of future CVD by obtaining a level of a marker of systemic inflammation such as CRP.

Ridker (US11244716) disclose that CRP levels can also be used to determine whether or not statin treament should be continued or changed.

High-Density Lipoprotein (HDLs) (“good” cholesterol)

High-density lipoprotein (HDL): HDL cholesterol concentration are inversely associated with occurrence of acrdiovascular events. Although measurement of HDL cholesterol concentration is useful as part of initial cardiovascular risk assessment, HDL cholesterol concentrations are not predictive of reisdual vascular risk among patients treated with statins who attain very low concentration of LDL (Ridker, Lancel 2010, 376, 333-339).

Low-density lipoprotein (LDL): (“Bad” cholesterol) 

LDLC levels are predictors of risk of cardiovascular event. Typically, the igher the LDLC, the mosre at risk . Levels of LDLC over 160 mg/dL may put a subject at higher risks as compared to levels less than 160 mg/dL. Levels of LDLC over 130 mg/dL with one or more risk factors for a future cardiovascular event may put a subject at higher risks of a cardiovascular event as compared to someone with a level less than 130 mg/dL. A level of LDLC less than 100 mg/dL is desirable in one with a prior cariovascualr event and is on therapy to reduce to risk of a future cardiovascualr vent. A elvel of LDLC less than 70 mg/dL is even more desirable. (US 14/886626, published as US 2016/026146).

An optimal low-density lipoprotein (LDL) cholesterol is less than 100 mg/dl for patients at high cardiovascular risk (Kent, “usefulness of lowering low-density lipoprotein cholesterol to <70 mg/dl and usefulness of C-reactive Protein in Pateint selection” American J. of Cardiology, 92(15), 2003). 

However, almost half of all myocardial infarctions and strokes occur in persons without elevated levels of LDL, showing that other factors than hyperlipidemia contribut to the development of atherosclerosis (Ridker, Clin. Cardiol. 26(III) 2003. 

CRP and LDLC:  

Kent, (“usefulness of lowering low-density lipoprotein cholesterol to <70 mg/dl and usefulness of C-reactive Protein in Pateint selection” American J. of Cardiology, 92(15), 2003) discloses that the likehood of carotid intima-media thickness (CIMT) was related to on therapy CRP value. Among patients with an LDL cholesteraol between 70 and 100 mg/dl, those with a CRP below the median value (0.17 mg/dl) had a more than threefold greater likelihood of CIMT regression than those with a CRP above the median. In contrast the likelihood of CIMT regression if LDL cholesterol was ultra low (<70 mg/dl) or above optimal (>100 mg/dl) was unrelated to CRP levels. 

Ridker, US 2007-0292960 discloses a method of obaining the level of CRP and LCLC from a patient in the course of treatment with a stain  and based on the level of CRP compraed to a control, continuing therapy with the stain when the level of CRP is below the control and changing therapy when the CRP level is above the control, when the level of LDLC is below 70 mg/dL or is above 100 mg/DL.

Because CRP and LDL chloresterol measurements tend to identify different high risk groups, screening for both markers provides a better prognostic information than screening for either alone for cardiovascular events. (Ridker “comparison of C-reactive protein and low-density lipoprotein cholesterol levels in the prediction of first cardiovascular events” N Engl J Med 2002: 347: 1557-1565).

Myeloperoxidase (MPO): 

MPO is an enzyme secreted from activated neutrophils, monocytes and certain tissue macrophages which may also be involved in the development of coronary artery disease (CAD). MPO synthesis occurs during myeloid differentiation in bone marrow and is completed within granulocytes prior to their entry into the circulation. The enzyme is stored within primary granules of neutrophils and monocytes and is not released until leukocyte activation and degranulation. MPO forms free radicals and diffusible oxidants with antimimicrobial activity. However, MPO also promotes oxidative damage of host tissues at sites of inflammation, including atherosclerotic lesions. (Zhang, “Association between Myeloperoxidase levels and risk of coronary artery disease” JAMA, 286(17), 2001). Stimulated phagocytes secrete this enzyme at inflammatory sites, where it generates a powerful reactive oxygen species, hypochlorous acid (HOCl), at physiological chloride concentrations (Sugiyama (American J. Pathology, 158(3), 2001). Hypochlorous acid/hypochlorite (HOCL/OCl-) is formed from H2O2 and chloride ions by MPO. HOCL reacts with a wide range of biological substrates and is a powerful oxidant. The oxidative modification of low density lipoprotein (LDL) is thought to be an important step in the formation of an atherosclerotic lesion (Malle, Eur. J. Biochm. 2, 4495-4503 (2000).  

Daugherty J Clin Invest, 94, 1994, 437-444 also identified MPO in human vasclular lesions and proposed that MPO may contribute to atherogenesis by catalyzing oxidative reactions in the vasclular wall.

Terletskaya (Vrach Delo, 1989, 3: 13-14 (in Russian)) note that during the necrosis of myocardial muscle in MI pateints, MPO apparently gets secreted into the intercellular medium and then to the blood, which causes the reduction of the MPO activity of nuetrophilic granuloctyes. Hazen (US11/753799) discloses a method for administering a therapeutic agent targeted to cardiovascular disease to a patient based on elevated levels of MPO by determining levels of MPO in blood or serum or plasma. eloperoxidase (MPO) is a naturall-occurring heme protein associated with some types of white loo cells. It functions as an oxidant converting inert substrates to reactive oxygen species toxic to pathogens, to aid in phagocytosis. Atherosclerosis (the major cause of coronary artery disease) was known to be a chronic inflammatory disorder and high blood levels of other metabolites had been correlated to CVC. While MPO had been found to be present at elevated levels in atherosclerotic lesions, it had not been shown that MPO was present at elevated levels in blood samples form pateints with atherosclerotic CVC. The patents dislcosed several methods of measuring a patient’s blood MPO level. 

Cleveland Clinic held US Patent Nos: 7,223,552, 9,575,065, 9,581,597 which were held invalid under 35 USC 101 as directed to an ineligible natural law. (Cclevaland Clinic v. True Health Diagnostics LLC, 859 F.3d 1352 (Fed. Cir. 2017) and Cleveland Clinic v. True Health Diagnostics, US Fed. Cir. 2019). At the time of the invention, cardiovascular disease (CVD) was understood to be multifactorial and physicians were developing predictive algorithms based on genetic, environmental and lifestyle factors. However, these factors alone did not fully predict an individual’s risk of developing CVD, in particular, a large number of cardiovascular disorders occured in individuals with apparently low to moderate risk profiles. The Court held that the patents starts and ends with observation of naturally occurring phenomena as in Ariosa Diagnostics v. Sequenom, 788 F.3d 1371 (Fed. Cir. 2015) and becasue the patents did not purport to have invented any of the biological technqiues used to detect MPO or the statistical methods used to compare a patient’s MPO levels to the control goup, the claims recited no further inventive concept sufficient to transform the nature of the claims into a patent-eligible application of the natural law. (the claim in Ariosa’s US Patent 6,258,540 was held ineligible because it was directed to the disovery that paternally inherited cffDNA exists in maternal blood plasma and the amplificaiton and detection techniques were concededly known in the art). 

Oxidation Products

Oxidation may play a role in the initiation and progression of atherosclerosis. Although it is not known how lipoprotein oxidation is initated, diverse oxidants produced by endothelial and inflammatory cells may oxidze lipoproteins. Nitric oxide (NO) and super-oxide radical (O2) are rleeased by these cells and can react with each other to form the strong oxidant and nitrating species peroxynitrite (ONOO-) which is capable of oxidizing lipoproteins. The peroxynitrite reaction with proteins yield nitrotyrosine athat is a stable product formed by the addition of a nitro group to the ortho position of tyrosine. Nitrotyrosine has been determeined in many pathologies and is a useful marker for peroxynitrite detection in human atherosclerosis (Moriel, Biochemical & Biophysical Research Communicatios 232, 332-335 (1997). 

Nitroytyrosine: Hazen (US11/313/012) teaches detecting protein bound nitrotyrosine by an anti-nitrotyrosine antibody on a serum or plasma sample, comparing the levels to a control and adminsitering a lipid lowering agent such as a statin if the level is greater than the control. 

FDA statin drugs

Angioplasty: 

Percutaneous transluminal coronary angioplasty (PTCA) is a widely used and effective method to restor coronary flow in patients with symptomatic cornary artery disease. 

Statins: 

Stations are a class of medications that have been shown to be effective in lowering human total cholesterol, LDLC and triglyceride levels. Statins act at the step of cholesterol synthesis. By reducing the amount of cholesterol synthesized by the cell, thorugh inhibition of the HMG-CoA reductase gene, statins initiate a cycle of events that culminates in the increase of LDLC uptake by liver cells. As LDLC uptake is increased, total cholesterol and LDLC levels in the blood decrease. Lower blood levels of both factors are associated with lower risk of atheroscleorsis and heart-disease, and the statins are widely used to reduce atherosclerotic mobidity and mortality. Examples of statins include simvastatin (Zocor0, lovastatin (Mevacor), pravastatin (Pravachol), fluvastatin (Lescol), atorvastatin (Lipitor), cerivastatin (Baycol), rosuvastatin (Crestor),  and pitivastatin. (Ridker, US Patent Application No: 14/886626, published as US 2016/026146). 

Statin therapy lowers the risk of cardiovascular evetns by reducign plasma cholesterol and practice guidelines for patients with known cardiovascular disease emphasize the important of reaching target goals of LDLC. (US 14/886626, published as US 2016/026146).

Statin therapy has been shown to reduce the level of C reactive protein independently of its effects on lipid levels. Ridker, N England J Medicine 344 No. 26, 2001. See as Ridker (European Heart J. (2001) 22, 2135-2137). In a double blind trial of mean and women with no prior history of CVD (primary prevention cohort) and open label study of people with known CVD (secondary prevention cohort), pravastatin reduced CRP levels at both 12 and 24 weeks in a largely LDL C independent manner (Albert, JAMA, 286(1), 2001).

However, Ridker (“Reduction in C-reactive protein and LDL cholesterol and cardiovascular event rates after initiation of rosuvastatin: a prospective study of the JUPITER trial” (lancet.com, vol 373, April 2009) showed that although LDL cholesterol and hsCRP reductions were only weakly correlelated in individual patients, there was a 65% reduction in vascular events in participants allocated to rosuvastatin who achieved both LDL cholesterol less than 1.8 mmol/L and hsCRP less than 2 mg/L versus a 33% reduction in those who acheived one or neither target. 

Effect of Statins on CRP Levels

Hypolipidaemic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reducatase inhibitor (statin) treatment reduces cardiovascular risk and is also assocatied with the reduction of C-reactive protein (CRP) concentration. (Strandberg, Ann Med, 32(8), pp. 579-83 (2000) Statins are often prescribed to reduce the risk of a future cardivascular event in human subjects with abnormally high cholesterol and-or LDLC levels. It is known that lipid lowering statin therapy lowers not only cholesterol but also lowers the level of CRP.  Ridker US2007-0292960

Effects of Statin Treatment on Lipid levels (e.g., LDL, HDL, triglycerides)

Hypolipidaemic 3-hyroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor (statin) treatment reduces cardiovascular risk and is also associated with the reduction of CRP concentrations. In addition, low-desnity lipoprotein (LDL) cholesterol was substantially decreased and HDL cholesterol increased during statin treatment. The changes of CRP were significantly associated with changes in HDL and apolipoprotein A1 but not with changes in LDL cholesterol or triglycerides. (Strandberg, “Associations between change in C-teactive protein and serum lipids during statin treatment” Ann Med 2000, 32: 579-583)

Satins + Ezetimibe: 

The addition of ezetimibe to statin treatment provides significantly enhanced CRP reductions over and above those achieved with statin monotherpay. (Pearson, Am J. Cardiol. 2009, 103(3), 369-74).

PPAR-alpha

Fibrates (activators of peroxisome proliferators-activated receptor-alpha (PPAR-alpha): have been available for the treamtnet of dyslipidemia (lipid disorders) since the 1970s and are currently widely used behind statins. Fibrates have been well documented to reduce the risk of coronary events through the beneficial effects on lipoproteins such as lowering serum triglyceride and raising HDL. Fibrates can also reduce CRP concentrations and this change in CRP is correlated with change in HDL but not with triglyceride. (Hao, Clin Chem Lab Med 2012, 50(2) 391-397, 2012). 

PSC9 Inhibitors

RNA inhbitors:

Inclisiran (The Medicines Company which has been aquired by Merck): is a small interfering RNA that inhibitors PSC9. It has shown positive results in a phase III clinical study. It treats familial hypercholesteroleaoma. 

PS

Myocardial infarction (MI): 

MI is the most common cause of mortality in developed countries. It is a multifactorial disease that involves atherogenesis, thrombus formuation and propagation. Thrombosis can result in complete or partial occulsion of coronary arteries. The luminal narrowing or blockage of coronary arteries reduces oxygen and nutrient supply to the caridac muscle (cardiac ischemia), leading to myoardial necrosis and/or stunning.

Causes of MI: 

Atherosclerosis is the leading cause of heart disease as well as stroke in the developed world. Atherosclerosis is characterized by the accumulation of lipid particles and cells of the immune system in subendothelial regions, leading to narrowing of the arterial lumen and, following plaque rupture, to thrombosis. Many components of the immune system are involved in the pathologic processes including macrophages that develop into foam cells, T cells, autantibodies, autoantigens which are components of vessel walls and cholesterol partciles and cytokiens that are secreted by cells within atherosclerotic plaques.

Arrhythmias often occur due to slowing of conduction of the electrical impulse through the heart. Rapid impulse conduction is needed for the heart to beat in a steady rhythm. When this is disturbed, the patient may experience a life-threatening cardiac arrhythmia. Among others, conduction slowing and arrhythmias can occur in patients who suffer from a heart attack, heart failure, or from a genetic cause.

The gene, named SCN10a-short (S10s), was identified in the search for a one-off gene therapy that could improve heart function and prevent cardiac arrhythmias.

Risks: 

Genetics plays an important role in MI risk. Families with a positive family history of MI account for 14% of teh general population, 72% of premature MIs, and 48% of all MIs.

Diagnosis and Assays Used to Determine the Risk of Coronary Artery (CAD) and/or Coronary Heart Disease:

Ultrasound: Carotid atherosclerosis can be measured non-invasively by ultrasound and is closely related to all major cardiovascular risk factors and generally accepted to be a strong predictor of clinical cardiovascular disease.

Nuclear Magnetic Resonance (NMR): NMR uses the characteristic signals broadcast by lipoprotein subclasses of different size as the basis of their quantification. Each subclass signal emanates form the aggregate number of terminal methyl groups on the lipids contained within the particle, with the cholesterol esters and triglycerides in the particle core each contributing 3 methyl groups and the phospholipids and unesterified cholesterol in the surface shell each contributing 2 methy groups. The total number of methyl groups contained within a subclass partcile is, to a close approximation, dependent only on the partice’s diameter and is substantially uanffected by differences in lipid composition arising from such sources as varaibility in the rleative amounts of cholesterol ester and triglyceride in the particle core, varying degress of unsaturation of the lipd fatty acyl chains, or varying phospholipd composition. For this reason, the methy NMR signal emitted by each subclass serves as a direct measure of the particle concentraiton of that subclass.

Mechanisms of Action and Pathogenesis:

Mathematical formuals: CHD risk is also known to be contributed to independently by high number of “bad” LDL particles and low numbers of “good” HDL particles. LDL particles can create atherosclerosis by entering the artery wall, becoming oxidized, and then being ingested by macrophages to create cholesterol-rich foam cells, which grow into the atherosclerotic plaque. HDL particles can enter the artery wall and prevent or reverse this process by 1) inhibiting the oxidation of LDL particles and 2) removing cholesterol from the foam cells and delivering it back to the liver – a process called reverse cholesterol transport.

The overal risk of CHD depends on the balance between the bad and good particles. CHD and CAD has conventionally been assed based on measurements of cholesterol content of a patient’s LDL and HDL particles (LDL-C, HDL-C). Treatment decisions may thereafter be made to reduce the “bad” cholesterol (LDL-C) or increase the “good” cholesterol (HDL-C) A convenient combined risk factor is the ratio of LDL-C/HDL-C or the ratio of Total Cholesterol/HDL-C which is almost the same thing.

Treatment Options:

Protein Targets:

–-Propertein covertase substilisini Kexin type 9 (PCSK9): has been implicated as a mjaor regulator of plasma LDL-c and has emerged as a promising target for prevention and treatment of coronary heart disease. Human genetic studies identified gain of function mutations, which were associated with elevated serum levels of LDL-C and premature incidences of caronary heart diseases, whereas loss of function mutaitons were associated with low LDL-C and reduced risk of coronary heart disease. Antiboides to PCSK9prevent the degradation of LDLP receptor, thus lowering serum levels of LDL cholesterol and potently reducing serum cholesteroal in mice and monkeys (Chaparro-Riggers, J. Biological Chemistry, 287(14), 2012, 11090-11097.

Foods to Lower Cholesterol:

Oats: combats contain a fiber called beta-glucan which is known for its cholesterol lowering properties.

Fish Oils (omega-3 fatty acids):

–Vascepa (Amarin) is an icosapent, ethyl, an ethyl ester of an omega-3 fatty acid commonly found i fish oils under the brand name Vascepa. In 2012, the FDA approved Vascepa for the treatment of severe hypertriglyceridemica, a condition in which a patient’s blood triglyceride level is at least 500 mg/dL. In 2019, following the success of Amerin’s additional research, the FDA approved Vascepa for a second use: as a treatment to reduce cardiovascular risk (i.e., mycardial infarction, stroke, coronary revascularization and unstable angina requing hospitalization in patients having blood triglyceride leves of at elast 150 mg/dL. (See US Patent Nos: 9,700,537, 10,568,861).

Links of interest  Vascular Research Foundation

Introduction:

Arherosclerosis (also known as arterioslerotic vascular disease or ASVD) is an inflammatory disease characterized by intense immunological activity. It involves the formation in the arteries of lesions that are characterized by inflammation, lipid accumulation, cell death and fibrosis. Over time, these lesions, which are known as atherosclerotic plaques, mature and gain new characteristics. The atherosclerotic lesion typically comprises fatty streaks that later develop into fibrous plaques. The initial fatty streaks are characterized by the presence of lipid laden foam cells, mainly macrophage in origin. Fatty streaks may regress or progress via a transitional lesion to fibrous plaques. 

Atherosclerosis is a complex, multifactorial, and chronic disease influenced by a wide variety of genetic, environmental, and behavioral activites. (Witztum, Circulation, 1998, 98, 2785-2787). The most severe clincial events follow the ruptures of a plaque which exposes the prothrombotic material in the plaque to the blood and causes sudden thrombotic occlusion of the artery at the site of disruption. In the heart, atherosclerosis can lead to myocardial infarction and heart failues, whereas in the arteries that perfuse the brain, it can cause ischaemic stroke and transient ischaemic attacks.

Atherosclerosis is the most common pathologic process leading to cardiovascular disease (CVD), including myocardial infarction (MI) and Stroke.

Several autoimmune rheumatic conditions, including rheumatoid arthritis, systemic lupus erythematosus and antiphospholipid syndrome, are characterized by enhanced atherosclerosis (Sherer, Nature Clinical Practice Rheumatology, 2(2), 2006).

Definitions:

Cholesterol: is a waxy, fat-like substance found in the walls of cells in all parts of the body, form the nervous system to the liver to the heart. The body uses cholesterol to make hormones, bile acids, vitamin D and other substances. As with oil and water, cholesterol (which is fatty) and blood (which is watery) do not mix. So cholesterol travels in packages called lipoprotines, which have fat (lipid) inside and prtoein outside.. The two main kinds of lipoproteins which carry chaolesterol in the blood are LDL and HDL. If there is too much cholesterol in the bood, some of the excess can become trapped in artery walls. Over time, this builds up and is called plaque. The plaque can narrow vessels and make them less flexible, a condition called artherosclerosis or “hardening of the arteries”. All adults age 20 and older should have their cholesterol levels checked at least once every 5 years. The recommended cholesterol test is called a “liporotein profile” which measures levels of total choelsterol (which includes the cholesterol in all lipoproteins, LDL, HDL and triglycerides. The lipoprotein profile is odne after a 9-12 hour fast. The levels are measrued as milligrams of cholesterol per deciliter of blood, or mg/dL. (“Lowering your Cholesterol with TLC” US Deparmtent of Health and Human Services, December 2005).

Less than 200 mg/dL is a desirable cholesterol level. 200-239 mg/dL is boerderline high and 240 mg/dL and above is high. (“Lowering your Cholesterol with TLC” US Deparmtent of Health and Human Services, December 2005)

Low density lipoprotein (LDL): is also called the “bad” cholesterol because it carries cholesterol to tissues, including the arteries. Most of the chorlesterol in the blood is the LDL form. The higher the level of LDL cholesterol in the blood, the greater your risk for heart disease. (The amin gol in treating high choelsterol is to lower your LDL level. Studies have proven that lowering LDL can prevent heart attaccks and reduce deaths from heart disease. “Lowering your Cholesterol with TLC” US Deparmtent of Health and Human Services, December 2005)

Less than 100 mg/dL is an optimal LDL level. 100-129 mg/dL is abvoe optimal, 130-159 mg/dL is borderline high, 160-189 mg/dL is high and 190 mg/dL and above is very high. (“Lowering your Cholesterol with TLC” US Deparmtent of Health and Human Services, December 2005)

High density lipoprotein (HDL): is called the “good” cholesterol because it takes cholesterol form tissues to the liver, which removes it from the body. A low level of HDL incrases your risk for heart disease. (“Lowering your Cholesterol with TLC” US Deparmtent of Health and Human Services, December 2005)

Less than 40 mg/dL is a major heart disease risk factor. 60 mg/dL and above gives some protein agaisnt heart disease. (“Lowering your Cholesterol with TLC” US Deparmtent of Health and Human Services, December 2005)

Triglycerides: are produced in the liver and are naother type of fat found in the blood and in food. Causes of raised triglycerides are overweight/obesity, physical inactivity, cigarette smoking, excess alcohol intake and a diet very hihg in carbohydrates (60% of caloreis or higher). Recent research indicates that triglyceride levels that are borderline high (150-199 mg/dL) or high (200-499 mg/dL) may increase your risk for heart disease. (lelves of 500 mg/dL or mroe need to be lowered with medication to prvent the pancrease form becoming inflamed. A triglyceride level of 150 mg/dL or higher also is one of the risk factors of the metabolic syndrome. (“Lowering your Cholesterol with TLC” US Deparmtent of Health and Human Services, December 2005)

Risk Factors

The most important risk factors for atheroslerosis invlude smoking, hypertension, dyslipidemia (increased concentration of LDL and decreased concentration of HDL), diabetes, aging, and a family history of premature atherosclerosis. 

Hypercholesterolemia is one of the most important risk factors for atherosclerosis. The mechanisms by which elevated levels of liporproteins, chiefly the apoB containing liporoteins such as LDL, cause an acceleration of atherogenesis are only incompletely understood (Witztum, Circulation , 1998, 98, 2785-2787.

Saturated or trans fat raises your LDL cholesterol level more than anything else in your diet. (“Lowering your Cholesterol with TLC” US Deparmtent of Health and Human Services, December 2005)

Mechanisms of Pathogenesis

The mechanism of atherosclerosis is not well understood (US 11/753799). However, atherosclerosis is a chronic inflammatory disorder. Acute phase reactants (e.g., C-reactive protein, complement proteins) are enriched in fatty streaks and later stages of atherosclerotic lesions. (US 6040147)

High Blood Pressure (Hypertension): can damage the endothelium of blood vessels by stretching them and hardening over time. An inujured endothelium allows LDL cholestoral (“bad cholesteroal”) to enter the lining of the artery which can then build up and cause plaques. Plaques can latter rupture and cause blood clots, resulting in stroke or heart attacks. High blood pressure typically has no symptoms and only about 30% of people have it under control. About 50% of people will have hypertension by the age of 60. Blood pressure higher than 130/80 is seen in a large percentage of people who have their first heart attack and stroke. (normal blood pressure is less than 120 over less than 80). 

C Reactive Protein: In one clinical trial, base line plasma levels of C-reactive protein independently predicted risk of first time myocardial infarction and stroke in apparently healthy individuals (US 6040147). 

Role of Chemokines: Data obtained using knockout mice show a key role for CC-chemokine ligand 2 (CCL2; also known as MCP1) and its receptor, CC-chemokine receptor-2 (CCR), in the initiation of atheroslerosis. Absence of CCL2 or CCR2 limits the entry of monocytes and T cells into the arterial intima and inhibts atherogenesis.

Macrophages and vascular cells of the forming plaques also product the T-cell attractants CCL5 (also known as RANTES), CXC-chemoine ligand 10 (CXCL10; also known as IP10) and CXCL11 (also known as ITAC).

Role of Cell Adhesion Molecules;  Adhesion of leukocytes to the endothelium represents a fundamental, early event in a wide variety of inflammatory conditions such as atherosclerosis. Leukocyte recruitment to the endothelium is started when inducible adhesion molecule receptors on the surface of endothelial cells interact with counterreceptors on immune cells. Polyunstaurated fatty acids and their hydroperoxides which are important components of oxidatively modified LDLs (see below) induce the expression of VCAM-1. US 5846959 discloses a treatment for atheroscelrosis and other inflammatory diseases that are mediated by VCAM-1 which includes the inhiition of the formation of oxidized polyunsaturated fatty acids which involves administering an effective amount of a substace that prevents the oxidation of a polyunsaturated fatty acid. 

Elevated LDL: is a major risk factor for premature coronary artery disease. The onset of this disorder is marked by the appearance of lipid laden macrophages in the intima of the arterial wall. 

LDL oxidation: It has been hypothesized that modification of low-density lipoprotein (LDL) into oxidatively modified LDL (ox-LDL) by reactive oxygen species is a central event that initiates and propagates atherosclerosis. This hypothesis is supported by the fact that lipoprotein like particles with properties consistent with oxidative damage have been isolated from animal and human aortic lesions. Oxidized LDL is a chemoatttractant for monoctyes and T lymphocytes and also inhibits macrophage motility, thereby promoting retention of macrophages in the arterial wall. 

1. Mechanism of LDL oxidation: The mechanisms that oxidatively damage lipoproteins remain poorly understand but cultured endothelial cells, smooth muscle cells and monocyte derived macrophages modify LDL by reactions that require iron or copper implicating cellular mechanisms and free metal ions in lipoprotein oxidation. Activated monocytes oxidatively modify LDL in vitro by a mechanism that is inhibited by superoxide dismutase, catalase, and metal chelators, suggesting the involvement of O2.- and H2O2. H2O2 is formed by dismutation of O2.- produced by a membrane associated NADPH oxidase. Activated monocytes also secrete a heme enzyme myeloperoxidase, that uses H2O2 as a substrate to generate products that can oxidize lipids and proteins. One product is hyperochlorous acid (HOCL), which is formed from CL-. This potent cytotoxin plays a critical role in host defenses against invading bacteria, viruses, and tumor cells, but it may also injure normal tissue. Another product of the myeloperoxidase-H2O2 system at plasma concentrations of CL- and amino acids is tyrosyl radical.  (Daugherty, “Myeloperoxidase, a catalyst for lipoprotein oxidation, is expressed in human atherosclerotic lesions” J. C.in. Invest, 94 1994, 437-444). 

2. Measurement of LDL oxidation: Direct methods for oxidation include F2 isoprostanes, antibody to oxidized LDL, antioxidant status, and breath volatile hydrocarbons.

F2 isoprostanes elevation: F2 isoprostanes are prostaglandin-like compounds formed in vivo from free radical catalyzed peroxidation of arachidonic acid. The F2 isoprostanes are formed by oxidation of arachidonic acid esterified to phosphlipids in cellular membranes. They can also form when LDL undergoes either cell or copper-meidated oxidation. They are released by a phospolipase actvity and circulate in the palsama in free form or as phospholipid esters and can be excreted in urine. ((Witztum, Circulation, 98, 2785-2787, 1998). They are generally measured by gas chromatography-mass spectrometry. F2 isoporstanes can be detected both in body tissues and biological fluids such as plasma and urine. Increased concentrations have been detected in oxidized LDL. Isoprostanes are present in human atherosclerotic plaque ((Lawson, J. Biological Chemistry, 274(35), pp. 2441-24444 1999).  Practico (J. Clin. Invest., 100(8), 1997, 2028-2034) discloses that two F2-isoprostanes, 8-epi PGF2 and IPF2alpha-I, are markers of oxidative stress in vivo and are present in human atherosclerotic plaque. Mehrabi (Cardiovascular Research , 43, 1999, 492-499) also disclose that the isoprostane 8-epi-PGF2 is enriched in islolated coronary arteries of patients suffering from cornoary heart disease (CHD). Davi reports that F2-isoprostane 8-epi-PGF2alpha is enhanced in the vast majority of patients with hypercholesterolemia and that vitamin E supplemental was associated with dose dependent reductions.

Lipoprotein(a): is an additional lipoprotein risk factor. It is distinguished form LDL in that, in Lp (a), attached to the LDL partcile is another apoprotein, termed apoprotein(a) (apo(a). This is linked to the LDL particle by a disulfide linkage. It is a highly glycosylated protein. 

Bacterial infection:  has been associated with increased progression of atherosclerosis in rodent model. Invasive P. gingivalis simulates pro-inflammatory cytokines and CAMs in human endothelial cells.

Toll Like Receptors: TLR2 plays a critical role in the profession of arteroscleorisis.

Main Cholesterol Lowering Drugs:

The major types of choelsterol lowering drugs are the following:

Satins (lovastain, pravastatin, simvastatin, fluvastatin, atorvastatin and rosuvastatin):. Statins top an enzyme that controls the rate at which the body produces cholesterol. They lower LDL levels mroe than other types of drugs -about 20-55% and also meorderately lower triglycerdies and raise HDL. (“Lowering your Cholesterol with TLC” US Deparmtent of Health and Human Services, December 2005)

Ezetimibe: reduces the amount of cholesterol absorbed by the body. Ezetimibe can be combined with a stain to get more lowering of LDL. Exteimibe lowers LDL by about 18-25%.

Bile acid resins: bind with cholesterol containing bil acids in the intestine and are then eliminated form teh body in the stool. They lower LDL by about 15-30%. 

Nicotinic acid (niacin): is a water soluble B vitamin that should be taken only under physician superivision. It improved as lipoproteins -total cholesterol, LDL, triglycerides and HDL. LDL levels are usually reduced by about 5-15%. (“Lowering your Cholesterol with TLC” US Deparmtent of Health and Human Services, December 2005)

Fibrates: mostly lower triglycerides and to a lesser degree, raise HDL levels. They are less effective in lowering LDL levels. (“Lowering your Cholesterol with TLC” US Deparmtent of Health and Human Services, December 2005)

Other Therapies

Angiotensin II receptor Inhibitors: help to relax arteries and veins to reduce high blood pressure (a cuase of atherosclerosis). Angiotension is a chemical in the body that narrows arteries. Commo angiotension II receptor inhibitors include Losartan.  

Antioxidants: such as ?-tocopheroal, probucol, butylated hydroxytoluene and dipehenyl phenylenediamine have been shown to decrease LDL oxidation and atheroxlerosis lesion proression in various animal models of atherosclerosis. Although utylated hydroxytoluene and diphenyl phenylenediamine are effective in the prevention of atheroxclerosis in animals, toxicity limits their utility in humans. Thus, supplementaiton with antioxidant nutrients may be a better approach in the prevention of atherosclerosis. Doses of vitamins E and C in excells of the recommended dietary allowances are well tolerated. Alpha Tocopherol is the most prevalent and biologically active form of vitamin E. 

HSD1 inhibitors: Macrophages in the atherosclerotic lesion express HSDA1. This enzyme appears to amplify the effects of gluccorticoids on atherosclerosis since blockage of HSDA decreases inflammatory tone. HSD1 inhibitors counter regulates inflammatory genes.