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)