Diabetes Mellitus

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In April 1994 issue of Bulletin of Experimental Biology and Medicine, Vol. 117 , an official jounal of the Russian Academy of Medical Scinces, there are two summary articles about 16 years of Soviet, Russian, and International Institute of Biological Medicine in Moscow clinical experience in the treatment of over 3,000 patients with type 1 diabetes mellitus, and type ½, with complications: retinopathy, nephropathy, polyneuropathy, and peripheral arterial disease, as described in detail in the chapter ‘Therapeutic goal of fetal cell xeno-transplantation’. The average success rate was 60 – 80%, depending upon the type of complication and the stage of its development. Duration of the effect was 9 – 18 months, and longer after subsequent treatment. Treatment of diabetic women during pregnancy decreased significantly the rate of complications and has nearly eliminated the fetal death. VI.BIBLIOGRAPHY [146, 147]


Diabetes mellitus (DM) is a disease with enormous variability of its clinical and biochemical features. The much less common type 1 of DM (‘IDDM’) is substantially better clinically defined than the type 2 of DM (‘NIDDM’). The incidence and prevalence of DM, with its life-threatening and crippling complications, has been growing worldwide with such a speed that one could speak of an ‘epidemic’. Several new therapeutic approaches have been developed in an attempt to solve this problem. New methods of intensive insulinotherapy, that attempt to simulate normal functioning of pancreatic ß-cells, improve the physician's ability to maintain the relationship between levels of insulin and blood glucose. For optimal insulinotherapy automatic systems of insulin delivery were introduced, of sophisticated closed type, permitting imitation of physiological feedback (used in situations of ‘clinical crises’), and of simpler open type. But the transplantologic methods of treatment appear the most promising, since only in this way the normal physiology can be restored. VI.BIBLIOGRAPHY [17]


Full correction of carbohydrate metabolism in patients with IDDM can be accomplished by the organ transplantation of pancreas. The great risk of such surgical procedure, i.e. transplant rejection, destruction of transplant by enzymatic activity of the exocrine portion, leading to the creation of vascular shunts, and thrombosis of vascular anastomoses, etc., and the need for life endangering continuous high dosage immunosuppression afterward, has limited the indication of this operation to those instances where also a kidney transplantation is carried out. VI.BIBLIOGRAPHY [120] As a rule, at that stage, the complications of DM, the main cause of death of such patients, have become severe and irreversible. VI.BIBLIOGRAPHY [121]


Fetal precursor cell transplantation permits normalization of carbohydrate metabolism and prevention of development of late complications of DM at much earlier stage of the disease. The advantages of this therapeutic approach are:

1/ from the patient's viewpoint the treatment is simple, and carries a minimal risk, regardless of route of implantation used;

2/ there is a great variety of animal sources of fetal precursor cell transplants available;

3/ immunosuppression can be avoided in every patient. VI.BIBLIOGRAPHY [121, 122]


A comparison of treatment of diabetes mellitus by fetal precursor cell transplantation and insulin can be summarized as follows:

1/ insulin cannot cure diabetes mellitus;

2/ insulin cannot prevent disabling, often life threatening, complications of diabetes mellitus;

3/ even the optimal insulinotherapy cannot stop relentless progress of diabetic complications, only fetal precursor cell transplantation can;

4/ but fetal precursor cell transplantation cannot replace insulinotherapy yet: it must be used simultaneously with insulinotherapy.


As the clinical experience of the past four decades have shown only fetal precursor cell transplantation can stop the relentless progress of the complications of diabetes mellitus once they start. This implies that the transplanted cells of all organs and tissues involved in carbohydrate metabolism trigger, directly or indirectly, the production of those endocrine or paracrine hormones that prevent the microangiopathic changes, the basis of all diabetic complications.


A. Carrel stated that insulin does not cure diabetes, and diabetes cannot be overcome until medical science succeeds to regenerate or replace islet cells, i.e. not only ß-cells. Regeneration of islet cells is a more efficient way of treating diabetes mellitus than giving the patients daily injections of insulin. This statement is still valid today, even after the DCCT trial in the U.S.  Insulin prevents death of a new diabetic but cannot stop the development of diabetic complications, severely disabling, and often fatal after years of suffering.  The cause of all diabetic complications is still unknown but is probably due to the lack of other, still unknown, hormones produced by various cells of Langerhans islets of pancreas, or by different cells of various organs of the regulatory system of carbohydrate and lipid metabolism. 


Only fetal precursor cell transplantation can treat diabetic complications with success. Before the introduction of primary tissue culture into the preparation of cell xeno-transplants the success rate of treatment of diabetes was minimal. It was believed that human fetal cell transplantation solves this problem but U.S. FDA approved clinical trials of late 80-ies by HANA Biologicals failed as well. It is clear that the primary tissue culture method of preparation of fetal precursor cell transplants it the key to the therapeutic success.


Fetal precursor cell transplantation can succesfully treat complications of diabetes mellitus. In 1978 the first IDDM patient with retinopathy and nephropathy was succesfully treated in Moscow: this female patient remained insulin-independent for over 21 years.


In summary, the success rate of treatment by fetal precursor cell transplantation, described in detail in the chapter ‘Therapeutic goal of fetal precursor cell transplantation’, for patients in the


pre- proliferative stage of diabetic retinopathy has been 65%


pre- azotemic stage of diabetic nephropathy 60%


any stage of diabetic poly-neuropathy 95%


pre- obstructive stage of diabetic vasculopathy 70%


clinically un-controllable children’s ‘brittle diabetes’ 90%.


Part of the reason for such high success rate has been that no immunosuppression had to be used for treatment when fetal precursor cell transplants are prepared by the method described in the section ‘Manufacture of fetal precursor cell transplants’. Besides well known side-effects, the specific problem of immunosuppression in diabetics is that it causes an increased metabolic demand on Beta-cells of pancreatic islets so that their capacity to produce insulin may be exhausted. This deleterious effect is much greater for islet cell transplants than for organ transplants of pancreas.


Diabetes mellitus is a heterogenous group of diseases with one common finding , that ofhyperglycemia, responsible for polyuria, polydipsia, loss of weight, polyphagia, and complications, i.e. retinopathy with potential loss of sight, nephropathy leading to kidney failure, polyneuropathy with intractable pain, lower extremity arterial disease with crural ulcers, gangrene requiring amputations, autonomous neuropathy with gastrointestinal, genitourinary, cardiovascular abnormalities, and sexual dysfunction. There is an increased incidence of atherosclerosis in coronary, brain and peripheral arteries, and hypertension. Glucose reacts with hemoglobin HbA to create HbA1c, and increased concentration of HbA1c proves that hyperglycemia has been present for many months. HbA1c has a higher affinity to oxygen than HbA, and does not easily release oxygen at the periphery.


Pathologic anatomy:


Absolute mass of the endocrine portion of pancreas in late human fetus is 300 mg, which represents about 10% of total weight of pancreas. In children the mass of the endocrine portion grows to 450 mg, that represents only 7% of the total weight of pancreas, however. In adulthood the weight of endocrine portion reaches 1,500 mg, but that represents only 2% of the total weight of pancreas. VI.BIBLIOGRAPHY [73, p.103]


At least four endocrine cell types with specific granules are currently recognized in the Langerhans islands of pancreas. A(Alpha) cells produce glucagon, are localized in the periphery of the islets, and appear first during the embryonic development. B (Beta) cells produce insulin, including pro-insulin and C-peptide, and are localized in the center of islets. D(Delta) cells produce somatostatin, are localized paracentrally in association with A cells, and appear second during the embryonic development. PP (or F) cells produce Pancreatic Polypeptide, that possibly suppresses enzyme production and gallbladder contraction, are localized in the periphery of the islets but also scattered outside of islets, and are the last to appear. Additional cell types have been described on the basis of their electron-dense secretory granules but no peptide hormones have been atributed to them yet. VI.BIBLIOGRAPHY [163, pp. 1997-1999]


Two types of islets are recognized: Beta-cell rich, scattered throughout the gland, and PP-cell rich, restricted to the posterior lobe of pancreas.


In an autopsy study the weight of whole pancreas in healthy subjects was 1,395 mg, while in IDDM it was 413 mg, and in NIDDM 1,449 mg. The loss of endocrine tissue observed in IDDM was almost completely restricted to the PPcells-poor anterior lobe of pancreas, where it reached 81%. Here B cells were practically absent, but the ‘atrophic islets’ still contained numerous A, D, and PP cells. In the PP cells-rich posterior lobe the decrease of total endocrine tissue was not significant. Here many islets contained a few B cells. The mass of A, D, and PP cells, and the ratio of D to A cells were the same in IDDM and normal controls. In NIDDM the mass of A cells was increased but, the mass of B, D, and PP cells was the same as in healthy controls. Outside the islets in three out of four cases rare B cells still could be identified, usually between the acinar cells. The proportion of D-cells was increased 3.5 times in IDDM.VI.BIBLIOGRAPHY [168]


In another autopsy study of IIDM patients dying within a year of the onset of symptoms three populations of islets were recognized: the majority of islets were B cell deficient, but contained a normal complement of A, D, and PP cells; some islets were affected by insulinitis, particularly those that still contained B cells; and some islets were totally unaffected by the disease. Aberrant expression of class II MHC was demonstrated on the great majority of B cells in IDDM of recent onset. Such abnormalities of MHC expression are unique to IDDM, and precede the start of the insulinitis in a given islet. VI.BIBLIOGRAPHY [163, pp. 2000-2001].


There is a general agreement that in order to attain insulin-independence after cell transplantation a certain minimum quantity of islets has to be transplanted: about 350,000. VI.BIBLIOGRAPHY [136] This has been proven by cases of auto-transplantation of pancreatic islets. VI.BIBLIOGRAPHY [137] In allo-transplantation such number of islets can be obtained only from adult cadavers, and in these situations the severe rejection reaction is unavoidable, unless massive immunosuppression would be used. The better alternative is a fetal precursor cell xeno-transplantation.


Pathogenetically it is often hard to determine if the development of disease is due to autoimmune destruction of beta cells of Langerhans islands of pancreas with resultant deficit of insulin or resistance to insulin by target cells. Even though there are excellent clinical guides for separating various causes of hyperglycemia, in the case of a specific patient all such processes are at work and it is hard to decide which one, or both working together, are the primary causes.


Absolute majority of diabetics falls into two categories: - type 1 diabetes mellitus, also known as insulin-dependent (IDDM), - type 2 diabetes mellitus, also known as insulin-independent (NIDDM)


Diabetes mellitus type 1, formerly known as juvenile diabetes mellitus, is either due to autoimmune mechanism or is idiopathic. Autoimmunity is the favored way to explain pathogenesis of DM Type 1. Destruction of ß-cells of Langerhans islands of pancreas is due to cell-based autoimmune process. It reaches such a magnitude that there is an absolute paucity of endogenous insuline so that patient’s life is dependent on a continuous supply of exogenous insulin. The autoimmune pathogenesis is proven by a typical infiltration of Langerhans islands by various immunocytes in the early stages and finding of the following markers: - Islet cell auto-antibodies identifiable years before onset of the disease, - Glutamic acid decarboxylase auto-antibodies, - Insulin auto-antibodies, - Tyrosinephosphatases 1A-2 and 1A-2ß auto-antibodies.

Such markers are found in 85 – 90% of newly diagnosed patients, while their incidence in general population is only 5%.


The course of autoimmune destruction of ß-cells of Langerhans islands of pancreas is variable. In some patients, especially infants and children, it can be very fast, in others, such as adults, it can be slow. While in children and adolescents a ketoacidosis can be the first evidence of disease, in adults the remaining still functioning ß-cells respond to the body needs for years before exogenous insulin has to become a part of treatment program.


There can be some genetic and exogenous predispositions to cell-mediated autoimmune destruction of ß-cells. It is believed that only a tendency toward diabetes in inherited, not the disease itself.


Among genetic predispositions the most important is a strong association with HLA-system: 95% of Caucasian patients with IDDM has a haplotype HLA-DR3 or HLA-DR4, while only 50% of general population. These haplotypes are of global nature, and other autoimmune disease are in distinct association with the same haplotypes. Genes coding antigens HLA-DQ, especially DQ2-DQ8, are connected with the highest risk for IDDM.


In summary, although the majority of IDDM patients carries predisposing HLA-alleles, not all such carriers will become diabetics. At the same time, the presence of protective HLA-alleles is a guarantee of protection against diabetes mellitus. It appears that predisposing or protective role of HLA-alleles is becoming less important with the age of patient at the time the first clinical symptoms and signs of the disease appear.


Environmental predispositions are obesity, lack of breastfeeding, viral infections by rubeola, coxsackie, enteroviruses, chemical toxins, i.e. rat poison, nitrosamines from smoked food, etc., increased load on Beta-cells, i.e. prolonged hyperglycemia, hormones, i.e. during pregnancy or puberty, ethnic factors, etc.


Latent autoimmune DM of adults, LADA: Many patients were classified as NIDDM when in reality they had late in life occurring and slowly developing IDDM. This type is now called LADA. Autoantibodies proving autoimmune insulinitis are present except with different frequency of various kinds. There is different frequency and ratio between predisposing and protective HLA-alleles. The course of disease is milder and slower and the same applies to the aggressivity of destructive process of insulinitis. The same applies to about biochemical findings.


Diabetes mellitus type 2, previously known as diabetes of old age, represents 80 – 90 % cases of diabetes mellitus worldwide, and is characterized by a combination of resistance toward the effects of insulin by target cells at the periphery and insufficient compensatory response in terms of its secretion by Beta-cells. Degree of hyperglycemia is sufficient for causing pathologic and functional changes in various target tissues while patient is completely asymptomatic and that applies to micro- and macro-angiopathic complications. These patients do not need insulin to survive. The basis of diabetes is not a destruction of Beta-cells by an autoimmune process, so that there are no such markers to be found in the patient’s body, and there is no association with HLA-system.


The lowered sensitivity to insulin is due to genetic predisposition, that has been proven. Lowered sensitivity to insulin affects predominantly glucose metabolism, while metabolism of fats and proteins functions normally: in NIDDM there is high level hyperglycemia but without disturbed fatty metabolism, i.e. lack of ketoacidosis. There is an increased concentration of fatty acids in blood, and decreased consumption of glucose in muscle and fatty tissues. The insulin resistance leads to increased release of insulin. The subsequent down-regulation of receptor causes further increase of resistance, and a ‘vitious circle’ is closed. Majority of patients are obese, which is the result of a separate genetic predisposition, excessive intake of highly caloric food, lack of physical activity, emotional psychic stress, aging.


Diabetes mellitus can be due to increased secretion of antagonists of insulin: somatotropin, as seen in acromegaly, glucocorticoids, as found in Cushing’s disease, or stress in steroid diabetes, adrenalin, due to stress, progesterons and choriomammotropin as happens in gestation diabetes mellitus, ACTH, thyroid hormones, glucagons. Insulin antagonists gain upper hand when blood glucose levels drop.


The first and most important one, in particular in acute situations, is glucagon made by Alpha-cells of Langerhans islands, released in response to falling levels of blood glucose. It binds glucagon receptors in the hepatocytes, the only glucagons receptors in the body, and stimulates the liver to

1/ increase the rates of glycogenolysis,

2/ decrease the rate of glycogen synthesis,

3/ decrease the rates of glycolysis,

4/ increase the rates of gluconeogenesis,

5/ decrease the rates of fatty acid synthesis.


Adrenaline is released from adrenal medulla in response to signals from the brain triggered by falling blood glucose levels. Adrenaline has different effect after binding to its receptors in different cells:

1/ In muscle, as there is no glucose-6-phosphatase, adrenaline will trigger the use of glucose-6-phosphate for glycolysis, but not as a source of glucose, so that muscle glycogen cannot be a source of glucose for blood, and for brain.

2/ In adipose tissue adrenaline activates lipase, which hydrolyzes triacylglycerides and thereby activates fat, so that fatty acids are released for use as fuel substrates in order to conserve levels of blood glucose.

3/ In liver adrenaline activates breakdown of glycogen and inhibition of fatty acid synthesis, thus shutting down fat anabolism.


Cortisol is also an insulin antagonist.


In type I diabetes mellitus the insulin antagonists have usually an upper hand. In type II diabetes mellitus the defect lies in the insulin signaling pathway so that the binding of insulin cannot elicit the usual anabolic responses, or the rapid clearance of glucose from blood.


Genetic disturbance of insulin effect is rare. Mutations of gene for human insulin receptor cause

- leprechaunism with intrauterine growth retardation, failure to thrive, facial dysmorpia, acanthosis nigricans, lipoatrophy, hyperinsulinism, postprandial hyperglycemia with hypoglycemia on empty stomach, lethal already in infancy;

- Rabson-Mendelhall syndrome with short stature, acanthosis nigricans, dental and nail anomalies, oversized penis, mental retardation, hyperplasia of pineal gland.


Insulin-resistant lipoatrophic diabetes is due to post-receptor signal transmission defects.


Gestation diabetes mellitus occurs in 4% of pregnancies, disappears after delivery, but sometimes it may take years to do so.


Biochemistry and physiology of insulin:


Under normal circumstances insulin is produced as a linear polypeptide pre-proinsulin first, with a signal sequence at N-terminus that facilitates its movement into the lumen of endoplasmic reticulum. In endoplasmic reticulum this signal sequence is removed and a much shorter proinsulin is generated. Proinsulin molecule is bent and disulfide bridges are created between the side chains of neighboring amino acid. Eventually proinsulin is selectively cleaved and its one portion, C-peptide, is released whereby a proinsulin becomes a mature insulin.


Following food ingestion, a glucose concentration begins to rise in blood, glucose enters Beta-cells of Langerhans islands, and triggers a depolarization event, a rise in intracellular calcium, and release of insulin from insulin granules within Beta-cells. Levels of insulin in blood rises. Insulin interacts with insulin receptors on the surface of striated muscle cells and adipose cells, which generally show the strongest responses to insulin of all cells. An increased uptake of glucose by stimulated cells is carried out by a glucose transporter GLUT 4 that enables a facilitated diffusion for glucose. Besides that insulin

1/ increases the rate of glycolysis,

2/ increases the rate of glycogen synthesis,

3/ decreases the rate of glycogen breakdown,

4/ decreases rates of gluconeogenesis,

5/ increases the rate of synthesis of fatty acids and triacylglycerides,

6/ decreases breakdown of triacylglycerides,

7/ increases the rate of protein synthesis.

Overall metabolic effects of insulin is the promotion of anabolism, while suppressing catabolism of glycogen and fat.


In diabetes mellitus the fat is used as a principal fuel with deleterious consequences for cells, and the body in toto. In absence of insulin that stops Beta-oxidation from going into overdrive, an enormous quantity of triacylglycerides breaks down in adipose tissue due to unopposed adrenaline stimulation, and that leads to an excessive Beta-oxidation and enormous rates of ketone bodies synthesis in the liver. Ensuing ketonemia causes metabolic acidosis, and a drop of tissue pH. Hyperglycemia with ketonemia dehydrate cells, with loss of water and electrolytes, hypotension, diabetic coma, and kidney failure.



Treatment of diabetes mellitus and its complications by fetal cell transplantation:


Indications:

VI.BIBLIOGRAPHY Diabetes Mellitus, types 1 and mixed 1/2, particularly with complications, such as

a. VI.BIBLIOGRAPHY Diabetic Retinopathy

b. VI.BIBLIOGRAPHY Diabetic Nephropathy

c. VI.BIBLIOGRAPHY Diabetic Polyneuropathy

d. VI.BIBLIOGRAPHY Diabetic Lower Extremity Arterial Disease.

VI.BIBLIOGRAPHY Brittle Diabetes Mellitus in children; and

VI.BIBLIOGRAPHY Diabetes Mellitus in pregnancy, or diabetes mellitus as a cause of female infertility and VI.BIBLIOGRAPHY habitual pregnancy loss.


The key fetal precursor cell transplant for the treatment of diabetic complications is Langerhans islands of pancreas, the co-transplants used in the described clinical system are fetal precursor cell transplants of liver, adrenal cortex, stomach / intestine (‘peripancreatic block’), placenta and hypothalamus. Sometimes, anterior lobe of pituitary, parathyroid, gonads, may be added. VI.BIBLIOGRAPHY [328]


When patient suffers from a ‘pure’ type 2 DM, or NIDDM, the implantation of islet cells of pancreas, is meaningless, but an addition of cell transplant of intestine may be considered.


The sooner the patient receives fetal precursor cell transplantation after the diagnosis of diabetic complication was established, the better will be the success rate of such therapy.


In U.S. the sole criterion of success of fetal precursor cell transplantation in the treatment of diabetes mellitus has been a complete insulin-independence. Factors such as a stabilization of the course of DM or a prevention of further progression of secondary complications of DM have been disregarded. The first U.S. attempts at fetal allo-transplantation of non-cultured pancretic islets were reported in 1976, and were considered unsuccesful.


In U.S.S.R. the first fetal allo-transplantation of cultured pancreatic islets was carried out at RITAOMH in 1978, and was succesful. The criterion of success was a prevention of further progression of diabetic complications in the treated patient for 2 years. In reality the patient was insulin-independent for the next 21 years.


Since the insulin-independence was obtained only rarely, and even then it was only temporary, the new criteria of success after fetal cell transplantation became the increase of serum C-peptide, or appearance of C-peptide in serum of the initially C-peptide-negative patient, and the lowering of dose of exogenous insulin. Pronounced chronic IDDM, 3 - 5 years after onset, is accompanied by an absolute insulin deficiency, with absence of C-peptide in serum, even after glucose stimulation. The criterion of rejection is a recurrence of hyperglycaemia and lowering of serum C-peptide level. VI.BIBLIOGRAPHY [135] However, the serum C-peptide never goes above the lower level of normal, even after allo-transplantation, and thus cannot explain the beneficial clinical effect of fetal precursor cell transplantation over a long time, sometimes for over one year. On the other side, in the instances where C-peptide level reached normal or near normal level after cell transplantation, patients were clinically not insulin-independent.


In the U.S.S.R. and C.I.S. (‘Commonwealth of Independent States’) the key criterion of success has been an arrest of further progression of microangiopathic complication of IDDM.


In a study of 100 IDDM patients treated by xeno-transplantation of islet cells, in 100 % of patients there was a clinical evidence of damaged structure and function of the capillary network of lower extremities, in 74 % vasomotor abnormalities, and 41.2 % of patients were found to have a diabetic retinopathy. During the follow-up after 3 weeks, and after 3 months - 4 years after the procedure, in 74 % of patients there was a stabilization of the hemodynamic status of lower extremities, in 18 % there was an improvement and in 8 % worsening. Stabilization of retinal pathologic changes was observed in 70.4 %, improvement in 24.7 % and worsening in 4.9 % of patients. In 34.1 % there was an improvement in visual acuity, in 60 % stabilization, and in 5.9 % worsening. VI.BIBLIOGRAPHY [68]


In a study of 55 IDDM patients the vascular condition of lower extremities was evaluated by rheovasography, and capillaroscopy, the eyegrounds by direct and inverted opthalmoscopy, and the kidney function by laboratory tests. In 80% of patients there was a vasomotor abnormality, and in 100% there were abnormal capillaroscopic findings: 30% of patients were in the 1st and 70% in the 2nd stage. After xeno-transplantation with a follow up for 1 year in 73 % a stabilization was observed, in 22% an improvement, and in 5% worsening. Ophthalmologically in 43% of patients an improvement and in 57% a stabilization was observed. In 20% of patients visual acuity improved, in the remaining 80% it stayed the same. 24-hour proteinuria dropped from 1,8 gm/l to 1,2 gm/l. VI.BIBLIOGRAPHY [84]


Of 85 IDDM patients 41% were found to have a diabetic retinopathy: 1st stage in 11, 2nd stage (pre-proliferative) in 21, and 3rd stage (proliferative) in 3 patients. All 3 patients with stage 3 and one patient with stage 2 had to be removed from the study because of increased severity of their diabetic nephropathy. Follow-up examinations were 24 hours, 2 - 3 weeks, 3, 6, 9 months, up to 4 years, after allo- and/or xeno-transplantation of islet cells. In 58 - 86% there was a gradual improvement of retinal pathologic changes over 3 - 9 months, and in 24% of patients in stage 1, there was a complete regression of retinopathy. Following intercurrent diseases in 6% of patients a worsening occurred over the period of 1 - 2 years. Visual acuity increased in 34.1%, stabilization in 59.4% and worsening in 6.5.% of patients. VI.BIBLIOGRAPHY [88]


In another study of 100 of patients with moderate and severe diabetes mellitus from 18 to 60 years of age in 72 there were abnormal rheovasographic findings, and in all 100 there were abnormal capillaroscopic findings: in 25 of the 1st and in 75 of the 2nd stage. Follow up was 3 weeks, 3, 6, 9 12 months up to 3 years after transplantation of islet cells. In 74% of patients there was a gradual stabilization, in 10% an improvement, and in 3 worsening. VI.BIBLIOGRAPHY [89]


In own study 106 patients with IDDM were treated by transplantation of cultured islet cells (‘CT’) of newborn rabbits. All patients selected for CT suffered of severe IIDM, with labile course, quick progression of the disease, and diabetic complications.


All patients were divided into two groups. 73 patients of the 1st group, 48 males and 25 females, underwent intramuscular CT. Their age was from 18 to 57 years, with a median of 35.4 years. Duration of diabetes was from 6 to 35 years, with a median of 17.4 years. Labile course of the disease was noted in 76.8% of patients, i.e. in 41.1% there were frequent (1 - 4 times a day) , and in 35.7% less frequent (1 - 2 times a week) episodes of hypoglycemia. In 69.9 % of patients there was a secondary ketoacidosis. Majority of patients were admitted at Russian Research Institute of Transplantology and Artificial Organs of Russian Academy of Medical Sciences (‘RITAOMH’) in the state of sub- or fully decompensated carbohydrate metabolism, with the median blood sugar of 11.7±1.4 Mmol/l, the daily dose of insulin ranged from 18 to 96 U., with a median of 51.3±15.4 U.


33 patients of the 2nd group, 24 males and 9 females, underwent intraportal CT. Their age was from 7 to 55 years of age, with a median of 32,6 years, duration of diabetes was from 7 to 25 years. Labile course of the disease was noted in 87.9% of patients, in 27.4% there were frequent and in 60.6% less frequent hypoglycaemic episodes. In 81.8% of patients there was ketoacidosis. Majority of patients were admitted in the state of sub- and decompensated carbohydrate metabolism. The median blood sugar was 11.6±1.0 Mmol/l. The daily dose of insulin ranged from 18 to 60 U., with a median of 49±9.7 U.


Frequency of IDDM complications is in the table 1.


Table 1

Frequency of IDDM complications

IDDMComplication Group #1 No. of patients I.M. XT % Group #2 No. of patients IntraportalXT %
Polyneuropathy 68 93.1 31 93.9
Peripheral
Angiopathy 68 93.1 31 93.9
Angioretinopathy of which 57 78 28 84.9
Proliferative 22 30.1 12 36.4
Nephropathy 50 68.5 19 57.8


Cultures of pancreatic islet cells of newborn rabbits were used for the transplantation. The rabbit insulin is close in sequencing of aminoacids to the human insulin. Pancreatic islet cells were prepared by tissue culture of 11 days’ duration. For intramuscular transplantation floating or mixed fraction was used, while for intraportal the attached fraction.


Intramuscular transplantation of cultures of pancreatic islet cells was carried out under local anesthesia into the subaponeurotic space of the right rectus abdominis muscle above umbilicus through a large-bore needle.


Intraportal transplantation of cultures of pacreatic islet cells was carried out under local anesthesia by exposing the umbilical vein, its dilatation, followed by catheterization of the portal vein. Implantation of cultures was carried out after preliminary intraportal injection of 5,000 U. of heparin along with intra-operative control of the intra-portal pressure.


Lowering of hyperglycemia was observed post-transplantation, most commonly after 7 - 10 days. But in 6 recipients of the 1st group (8.2%) and 12 of 2nd group (36.4%), there were minor hypoglycaemic episodes during the first 1 - 2 days. Apparently the cause of hypoglycaemia was the release of insulin from ß-cells damaged during transplantation. In 23 cases of the 1st group (31.5%) and 2 cases of the 2nd group (6.0%) the lowering of hyperglycemia occurred only after 1 - 2 months.


Lowering of glycaemia required to decrease the dose of exogenous insulin.


Changes of glycemia in the recipients of CT are in Table 2


Table 2
Changes of glycemia
Median Glycemia (Mmol / l)
Post XT
Implantation site Pre XT 14 days 1 month 3 months 6 months 10-12 months
Group #1 11.7 10.8 8.5 8.7 9.1 10.9
I.M. XT ±1.4 ±0.9 ±0.9* ±0.9* ±1.1* ±1
Group #2 11.6 10.8 8.9 8.1 8 9.2
Intraportal X ±1.0 ±0.9 ±0.8 ±0.2 ±0.3** ±0.6**


* P < 0.05 in comparison with initial value

** P < in comparison to the 1st group


Another serious criterion for an evaluation of carbohydrate metabolism of the recipients after the CT are the dynamic changes of glycosylated hemogobin HbA1c, seen in the Table 3:


Table 3
Changes of HbA1c


Post XT
Implantation site Pre-XT 3 months 6 months 10-12 months
Group #1 13.8 9.2 9.5 10.2
I.M. XT ±1 ±0.6* ±0.1* ±0.4*
Group #2 12.9 8.3 8.4 8.7
Intraportal XT ±1.5 ±0.2* ±0.3* ±0.9*



* P < 0.05 in comparison with initial value

** P < 0.05 in comparison to the 1st group



The optimal regimen of insulinotherapy was established already before cell transplantation. After transplantation all patient were maintained on the same types of insulin, and as much as possible, with the same frequency. This makes the interpretation of changes in the need for exogenous insulin after CT much easier, as seen in Table 4:


Table 4:
Changes of dosage of exogenous insulin after CT


Post XT
Implantation site Pre-XT 14 days 1 month 3 months 6 months 10-12 months
Group #1 51.3 49.9 41.2 40.1 41.8 47.4
IM ±15.4 ±13.7 ±13.2* ±13.0* ±13.3* ±14.1
Group #2 49.3 40.3 33 26.3 25.7 37.1
Intraportal XT ±9.7 ±9.4 ±9.0* ±6.9** ±6.2** ±10.4**
0% 100 81.7 66.9 53.3 52.1 75.2



* P < 0.05 in comparison with initial value

** P < 0.05 in comparison to the 1st group


The pain syndrome of distal polyneuropathy substantially decreased after CT. In 35 out of 43 recipients (81.4%) of the 1st group, followed up for 3 months after transplantation, the pain in lower extremities either substantially decreased or disappeared. After 6 months such effect was maintained in 74.2% of followed patients and after 10 - 12 months in 50%. In 2nd group the percentage of such patients was higher. So 3 months post-transplantation 87.8% of the recipient had no pain, and after 10 - 12 months 76,1%.


All parameters reflecting status of blood flow in patients with diabetic angiopathies of lower extremities of both groups significantly differed from parameters of the control group (p< 0,01). The half-time of disappearance of the isotope from the tissue depot was increased 1,5 times, tissue circulation was substantially lowered at rest, and in particular was decreased almost 3 times with exercise leading to ischemia. After CT the half-time disappearance of Tc99M from the tissue depot decreased and tissue circulation increased. In comparison with initial parameters the changes were statistically significant (p<0,05). The most pronounced parameters were obtained by measuring blood flow with exercise leading to ischaemia in the recipients of the 1st group. There were no statistically significant differences between the patients of both groups.


Evaluation of concentration of serum C-peptide showed that in 29 out of 56 recipients (51.8%) of the 1st group and 18 out of 33 recipients of the 2nd group (54.5%) the initial level of C-peptide was zero. All measurements below the sensitivity level of the method were so interpreted. In all initially C-peptide-negative recipients of the 1st group the concentration of C-peptide remained below the sensitivity level of the method, and out of 18 initially C-peptide-negative patients of the 2nd group in 5 only did the level of C-peptide not reach over the sensitivity limit of the method - see Table 5:


Table 5:
C-peptide levels


Post XT
Implantation site Initial Value of C-Peptide Pre-XT 14 Days 1 Month 3-6 Months
Control Group of Healthy Donors 0.266
N = 20 ±0.07
Group #1 C-peptide + 0.108 0.163 0.166 0.152
I.M. XT N = 27 ±0.08* ±0.03** ±0.05** ±0.03**
Group #2 C-peptide - - 0.111 0.173 0.158
Intraportal XT N = 13 ±0.03 ±0.02 ±0.04
C-peptide + 0.115 0.281 0.18 0.188
N = 15 ±0.06* ±0.09** ±0.04** ±0.07**



* Pk < 0.01 in comparison with control group

** P < 0.05 in comparison with initial value

^ P < in comparison to the first group



In cases of intraportal CT a one-time determination of of concentration of hormones in the portal and hepatic veins permits objective evaluation of the functional activity of the transplants. In the angiographic operating room the catheterization of hepatic vein was carried out and simultaneous collection of blood specimens from portal and hepatic veins was done. Subsequently 20 ml of 20% glucose was injected into portal vein and blood was collected 1 min., 5 min., 15 min., 30 min. and 60 minutes afterward. The data are in the Table 6:

Table 6
Concentration of hormones in portal and hepatic veins


TIME Portal Vein Hepatic Vein
InsulinU/ml C-peptide pmol/l Glucagon picog/l Insulin U/min C-peptide pmol/l Glucagon picog/l
Start 4.9 0.12 187.3 6.1 0.21 187.7
±0.6 ±0.04 ±18.2 ±1.0^ ±0.08^ ±26.2
1 minute 4.2 0.13 176.9 9.1 1.2 195.4
±0.5 ±0.07 ±22.5 ±1.3*^ ±0.46*^ ±19.8
5 minutes 4.2 0.12 140.1 5.5 0.15 176.9
±0.8 ±0.1 ±18.0* ±0.9^ ±0.08 ±20.9
15 minutes 4.6 0.11 156.8 2.3 0.12 218.4
±0.6 ±0.09 ±15.9 ±0.04* ±0.03 ±32.7
30 minutes 2.6 0.1 202.2 3.9 0.11 226.5
±0.03* ±0.02 ±24.1 ±0.5* ±10.05 ±27.8
60 minutes 3.5 0.11 198.3 1.5 0.12 166.4
±0.5 ±0.03 ±29.3 ±0.1* ±0.07 ±13.8


* P < 0.05 in comparison with starting value

^ P < 0.05 in comparison with portal vein value


In this study there were 27 repeated CT’s, in 19 cases intramuscular and in 3 intraportal, and 5 cases of third intraportal transplantation. Indications were worsening of general condition of patients, with decompensation of carbohydrate metabolism, increased dosage of exogenous insulin, re-appearance of symptoms of secondary complications. In 18 cases the previous transplantation was an allotransplantation of human fetal islet cells. By comparison the results of allo- and xeno-transplantation of cultured islet cells were in principle the same, with the exception of the need in exogenous insulin: 18 - 22% of the initial dose after xeno-transplntation as compared with 20 - 60% after allotransplantation.


The obtained results show that repeated CT’s from newborn rabbits permit prolongation of compensation of diabetes, and that it is preferrable to carry it out prior to the return of unfavorable laboratory findings.


The evaluation of parameters of cellular immunity showed that T lymphocyte count in the recipients of the 1st group was on the first day decreased to the level of parameters of control group of healthy blood donors (1227±260/µl), on the 7 - 10th day further normalization took place, and on 14 - 20th day the T-lymphocyte count again increased to the initial higher level (1668±294/µl), in comparison with the control group. Activation index was higher during the entire study, which was the same situation as at the beginning. The findings in the 2nd group were in principle the same as in the 1st group. The T-lymphocyte count was somewhat higher at 14 - 20 days after transplantation (2115±209/µl), but the difference was not statistically significant.


In the evaluation of humoral immunity, the B-lymphocyte count in the recipients of the 1st group was lower than in the control group during the entire study, but in the recipients of the 2nd group the B-lymphocyte count was higher, reaching 925±59/µl after 14 - 20 days, which was twice as high as in the control group (p<0,05), and more than 4-times as high as before transplantation (p<0,05). Concentration of IgA, IgG, IgM, remained the same in both groups for the duration of the study, and was on the same level as that of the control group.


Besides the standard tests also antigen-specific methods of evaluation of immune status were used. The studied parameters showed that there were no obvious changes of the immune status of the recipients, that proves a satisfactory elimination of class II MHC surface antigen carrying ‘passenger cells’ in the process of the cultivation of pancreatic islet cells of newborn rabbits. No statistically significant differences in the cellular immunity were found between various implantation sites. VI.BIBLIOGRAPHY [71]


Up to 80% of children with therapeutically uncontrollable ‘brittle’ diabetes had already developed typical diabetic complications by the time of their referral for fetal precursor cell transplantation, and such patients benefit from such therapy in 81% of cases. A group of 86 children and juveniles with otherwise clinically uncontrollable ‘brittle’ diabetes was treated by transplantation of human and animal fetal tissues. 13 patients received cell transplantation twice and one patient three times. In summary 81±6% transplantations had good result, and not a single one harmed the patient. The main benefit was the clinical compensation of diabetic state. On admission only 6 children had normal HbA1c, 12 children higher HbA1c, and all others were in the state of decompensation, i.e. HbA1c from 10.2% to 15.9%. Already 7 to 15 days after cell transplantation a median HbA1c was lowered to 8.25±0.43%, and 3 months after fetal cell transplantation to 5.69±0.40%, and then began to rise to 8.10±1.00% at 6 months, to 9.29±0.92% at 9 months, and 11.89±1.56% at 12 months after cell transplantation. In terms of clinical compensation, on admission only 10±2% of patients were compensated, while 3 months after cell transplantation it was 94±3%, at 6 months 75±6%, at 12 months 70±7%, and at 18 months after cell transplantation 54±8% of children were compensated. Most importantly 76% of patients was discharged with one insulin injection a day, which in this age group is extremely important for compliance.


Average level of C-peptide on admission was 0.51±0.32 µgm, two weeks after fetal cell transplantation it was 3 times higher, and three months after cell transplantation 4.4 times higher. At 12 months after cell transplantation the level of C-peptide returned to the original level. The higher the level of C-peptide at the beginning, the better the result of cell transplantation, which proves the high probability that fetal cell transplantation stimulated endogenous production of insulin in some patients.


The effect of cell transplantation disappears 10 – 14 months post-treatment.


A detailed diagnostic effort revealed that 81 out of 86 patients had already a clinical evidence of diabetic complications, such as diabetic microangiopathies, i.e. retinopathy, nephropathy, and hepatopathy, diffuse lipodystrophy, encephalopathy, lipoid necrobiosis, and the incidence and severity of all complications significantly improved during 12 months after cell transplantation.


The clinical status of all 14 patients that served as controls became worse during 6 – 15 months of observation. VI.BIBLIOGRAPHY [222]


Our own study of treatment by cell transplantation of 35 children with insulin-dependent diabetes mellitus, carried out by the 1st Republic Pediatric Hospital of Ministry of Health Care of Russian Federation, was reported at the 1st Symposium on Transplantation of Human Fetal Tissues in Moscow, December 4 – 7, 1995, under the title: “Experience with transplantation of cultured ß-cells to children with insulin-dependent diabetes mellitus at the 1st Republican Pediatric Hospital.” Cell transplantation of solely ß-cells of islets of pancreas was carried out. All 6 children with recently diagnosed IDDM became insulin independent within 3 weeks, and remained so for 4 – 8 months. Remarkably, all basic parameters of cell and humoral immunity were within normal limits during this time. The clinical status of all other patients substantially improved but not to the point of insulin-independence, which was due to low dosage of transplanted ß-cells, and mono-therapy by ß-cell of islets of pancreas alone.


Based on the enormous past experience with human fetal cell transplantation with absence of reactions of any sort there was no need to lower the dosage of cell transplants for safety reasons. Monotherapy by ß-cell of islets of pancreas alone is sufficient only for patients with recent IDDM, but not at the later stage, in particular when the autoimmune process is still active: here cell transplants of various immune organs and tissues are necessary to inhibit the autoimmune destruction of patient’s islet cells.


Unknown number of children with recent onset of diabetes mellitus have been treated successfully with fetal precursor cell transplantation: there have been some cures, and in other patients at least a delay in the onset of diabetic condition. If one could postpone the onset of child's diabetes by one or more years, it would be of tremendous value because of well known deleterious effect of diabetic condition on growth and development of such children.


When a female diabetic has been under treatment for infertility for over a year without a success, fetal precursor cell transplantation should be strongly considered. If a female diabetic has had 2 – 3 miscarriages, fetal precursor cell transplantation is indicated. When a pregnant diabetic delivered a baby with a diabetic fetal distress syndrome, fetal precursor cell transplantation should be carried out before her next pregnancy, or even during her next pregnancy, between 12th and 16th week.


A method of retrobulbar implantation of cell transplants of ß-cells of islets of pancreas developed by Fedorov Ophthalmological Institute in Moscow, was reported on at the 1st Symposium on Human Fetal Tissue Transplantation in Moscow, December 4 – 7, 1995, under the title: “Retrobulbar transplantation of cultures of islet cells to patients with diabetic retinopathy”. Our International Institute of Biological Medicine provided cell transplants for this treatment. Analysis of observations of 40 patients with diabetic retinopathy over 3 years showed in 76% of cases of pre-proliferative retinopathy a remarkable improvement, consisting of resorption and decrease of frequency of recurrent hemorrhage, and in 47% of cases of proliferative diabetic retinopathy an improvement in aggressivity of the proliferative process and diffuse neo-vascularization, all accompanied by a noticeably better visual acuity.


Retrobulbar implantation of cultured cell transplants of retina developed by International Institute of Biological Medicine was carried out by the Fedorov Ophthalmological Institute as treatment for retinitis pigmentosa.

Immunological reactions after fetal precursor cell transplantation

There are a few reports on the changes of immune parameters after xeno-transplantation of cultured islet cells. VI.BIBLIOGRAPHY [48, 66, 69, 92, 93] The fact that IDDM is an autoimmune disease, the basis of which is selective destruction of ß-cells, caused by the development of the cellular and humoral immune reaction, has made the analysis of generally rather subtle laboratory immunological findings after cell xeno-transplantation quite complex. In testing of IDDM patients, changes in ratios and function of immunocompetent cells on one side, and appearance of circulating antibodies to the antigens of patient's own ß-cells on the other side, have been seen. In the early stages of IDDM, autoantibodies, reversal of CD4+/CD8+ ratio, as well as activated T-lymphocytes, are present. In the chronic stage of the disease, as ß-cells are gradually destroyed, autoantibodies to the antigens of patient's own ß-cells disappear, and the main findings during the entire course of the disease are abnormal relationships (and ratios) of various subpopulations of immunocompetent cells. At the same time these abnormal relationships are quite heterogenous, i.e. CD4+/CD8+ ratio is sometimes in favor of T-helpers, sometimes in favor of T-suppressors, probably depending upon the stage of the disease. A possibility cannot be ruled out that immune response to the exogenous insulin, or metabolic crises during the chronic course of IDDM, play part.


After fetal precursor cell transplantation there is an activation of both cellular and humoral immunity. The immune reactions are most pronounced on 7th – 10th day treatment. Some authors claim that cellular immune reactions peak 2 days after, while humoral reactions 14 days post-transplantation. T-helpers dominate the immune response, and the whole picture is that of a delayed hypersensitivity reaction. Increased level of proliferative and metabolic activity of lymhocytes (2.5x) is observed on 7 - 10th day, and lasts 7 days. There is an increased activity of macrophages. Sometimes the T-lymphocyte count is increased, while the B-lymphocyte count remains the same. Occasionally, on 7th day the index of leukocyte migration inhibition is decreased. The changes of immunoglobulin level after cell xeno-transplantation are variable and statistically insignificant.


A group of 55 patients was followed before and after an allotransplantation of islet cells, and total T and B cell count, CD4+ and CD8+, circulating immune complexes, IgG, IgA, IgM, were measured. Before the procedure a decreased phagocyte activity of white blood cells, a generalized immune deficit, and an increased level of circulating immune complexes, were observed. After islet tissue transplantation there was a decrease of CD8+ and increase of CD4+, their ratio < 1, and an increase of IgM. VI.BIBLIOGRAPHY [69]


In a study of 157 xeno-transplantations of islet cells before the procedure 70.1% of IDDM patients had laboratory evidence of immune deficit, while after islet cell xeno-transplantation the immune deficit was only 22.5%. With NIDDM patients the numbers were 79% and 18%, respectively. In patients with normal CD4+ and CD8+ before the procedure, these parameters remained normal even after islet cell xeno-transplantation, but their ratio changed in favor of CD4+. In IDDM patients with low T lymphocytes before the procedure, the T cell count normalized one month after xeno-transplantation, but the ratio was in favor of CD4+. In IDDM patients with low B lymphocytes before the procedure, the B cell count became normal one month after xeno-transplantation as well. All other immune parameters: IgG, IgA, IgM, circulating immune complexes, phagocytosis index, were within limits of normal both before and after the procedure in both IDDM and NIDDM patients. VI.BIBLIOGRAPHY [66]


In another study 7 IDDM patients were treated by allo- (6) or xeno-transplantation (1) of islet cells and the level of islet cell surface antibodies was continuously measured. No association between the level of islet cell surface antibodies and allo- or xeno- transplantation, age, duration of diabetes, was observed. One half of patients had an increase of autoantibodies with a peak two weeks after the procedure, while the second half showed no response. VI.BIBLIOGRAPHY [92]


In 61 diabetics treated with xeno-transplantation of islet cells, levels of IgG, IgA, and IgM, were measured before and 3 - 5 days, 3 months and 10 - 12 months after the procedure. There was an increase of immunoglobulins after xeno-transplantation, but the rises were not statistically significant. In patients with increased immunoglobulins before the procedure, their level decreased after xeno-transplantation. VI.BIBLIOGRAPHY [93]


Cellular immunity: T- lymphocyte count decreased on 1st day post-CT to the level of healthy controls, then on 14 – 20th returned to the initial level, as compared with healthy controls. Activation index of T lymphocyte was higher than in healthy controls initially as well as during the entire follow-up after CT. There were no differences between 1st and 2nd group, where the 1st group had subaponeurotic implantation, while the 2nd group intraportal implantation of islet cell transplants..


Humoral immunity: In 1st group B-lymphocyte count was lower than in healthy controls during the entire study, while in 2nd group it was 14 – 20 days after CT twice as high as in controls (p<0,05), and four times higher than before CT (p<0,05).


Concentration of IgG, IgM, IgA, remained the same in both groups for the duration of follow-up after CT, and was the same as in healthy controls.


Conclusions:


1/ There were no obvious changes of the immune status of patients after cell transplantation when fetal precursor cell transplants were prepared by a BCRO method of primary tissue culture from fetal/newborn rabbits.


2/ There were no obvious changes of the immune status of patients after fetal precursor cell xeno-transplantation as compared with cell allo-transplantation.


3/ No statistically significant differences in cellular immunity were found between I.M. and intra-portal implantation sites after fetal precursor cell xeno-transplantation. VI.BIBLIOGRAPHY [48]



Reactivation of IDDM after SCT


A unique problem with diabetes mellitus is the recurrence of the original disease process in the transplanted islets. IDDM patients have antibodies directed toward several ß-cell products, including insulin. There is a prevalence of insulin-specific T cells in islet cell infiltrates, too. These insulin-specific T cells are capable of adoptive transfer of diabetes mellitus, i.e. they can destroy ß-cells in vivo in two different animal systems. VI.BIBLIOGRAPHY [56, 165] In this respect, the xeno-transplant, particularly a discordant one, could be expected to be relatively immune to recurrence of disease, since the membrane antigens which would form the target of an immune insulinitis are quite dissimilar to those which were the targets of the original disease process. VI.BIBLIOGRAPHY [37]


Theoretically, the xeno-transplant islets are largely protected from autoimmune processes. The xenogeneic islets may have a special utility in fetal precursor cell transplantation to patients with diabetes mellitus suffering from an autoimmune islet destruction. The resistance of the xeno-transplants to autoimmune recurrence as a late cause of graft failure seems well established in a number of models. The mechanism of the long term functional tolerance is not known, but the phenomenon of humoral adaptation, whereby xeno-transplants in residence for prolonged periods of time appear to be resistant to rejection after such a long time may play a part. VI.BIBLIOGRAPHY [63]


Bio-Cellular Research Organization LLC filed in February 1999 four Investigational New Drug applications with U.S. FDA for the treatment of advanced stages of the life threatening and severely disabling complications of IDDM (‘Insulin Dependent Diabetes Mellitus’) - Diabetic retinopathy - Diabetic nephropathy - Diabetic polyneuropathy - Diabetic lower extremity arterial disease

by own method of fetal cell transplantation, described in this text.


Incidence

Since the data are available, it is perhaps interesting to see the magnitude of problem with one serious disease treatable with fetal precursor cell transplantation with a great success, in one country.


According to the National Institute of Health data as of April 1, 1998:


As many as 15.7 million of people in the U.S. (~6%) had diabetes mellitus, of which 7% suffered from insulin-dependent diabetes mellitus (‘IDDM’), i.e. 1.1. million, and the rest from other types, mostly insulin-independent diabetes mellitus (‘NIDDM’);


Diabetic retinopathy is the leading cause of new cases of blindness of adults in U.S., after 15 years of diabetes 97% of IDDM, 80% of insulin treated NIDDM, and 55% of non-insulin treated NIDDM patients will have retinopathy, of which number around one third will develop the stage of proliferative retinopathy leading to blindness;


Diabetic nephropathy, the leading cause of end-stage kidney disease in U.S. requiring artificial kidney treatment (hemodialysis) or kidney transplantation, takes place in 35% of all diabetics, after 15 years 25% of diabetics will have protein in urine, and of those 50% of IDDM and 11% of NIDDM patients will be on hemodialysis within 10 years;


Diabetic polyneuropathy will develop after 15 years in 30 – 70% of diabetics, equally in IDDM and NIDDM patients;


Diabetic lower extremity arterial disease has been the cause of one half of all leg amputations in U.S.;


Diabetes mellitus is 7th leading cause of death in U.S.: 200,000 deaths reported each year;


Diabetics have much higher incidence of heart disease, at an earlier age, and with fatal prognosis, than non-diabetics;


Diabetics have 2.5. times higher risk of stroke than non-diabetics;


Many digestive diseases, infections, dental problems, depressions, are substantially more common in diabetics than non-diabetics.


The above statistics have gotten worse since 1995. 


Clinical protocol for fetal precursor cell transplantation treatment of patients with complications of diabetes mellitus


A proper preparation of the patient for fetal precursor cell transplantation is mandatory. A diabetic patient has to be brought into as good a metabolic state as possible by standard therapeutic means, i.e. to carry out fetal cell transplantation while the patient is in the state of ketosis, hyperosmolality or prolonged hypoglycaemia is probably minimally effective and should be done only as a last resort. Elimination of ketoacidosis, frequent hypoglycaemia or hyperosmolality by hospitalization, with intravenous fluids, frequent doses of regular insulin in accordance with glucometer measurements, etc., so that the patient’s clinical condition will be compensated as well as possible;


Parameters to be followed before and after fetal precursor cell transplantation, and their frequency:

- General:

i/ level of serum C-peptide once a month x3, then every 3 months

ii/ level of HbA1c once a month x3, then every 3 months

iii/ level of serum insulin once a month x3, then every 3 months

iv/ serum cholesterol, total, LDL, HDL, triglycerides, every 3 months

v/ home blood glucose self-monitoring (with diary) several times a day

vi/ avoidance of hypoglycaemia, observe if it happens and how often

vii/ avoidance of ketoacidosis and hyperosmolality, observe if it happens

viii/ decreased requirement of exogenous insulin (with diary)



- Immunological: once a month x 3, then every 3 months

i/ total lymphocytes

ii/ T-lymphocytes (CD3+)

iii/ T-helpers (CD4+)

iv/ T-suppressors (CD8+) and CD4/CD8

v/ NK (CD16)

vi/ B-lymphocytes (CD22 and CD19)

vii/ serum IgG, IgA, IgM

viii/ serum complement (CH50)


Special:


- diabetic retinopathy: - retinal photography: Canon office photographs once a month

- 7 standard special photos every 3 months

- visual acuity and visual fields once a month


- diabetic nephropathy:

- proteinuria/24 hr urine every 3 months

- microalbuminuria every 3 months

- serum creatinine once a month

- creatinine clearance every 3 months

- blood pressure once a month


- diabetic polyneuropathy:

- EMG every 3 months

- nerve conduction studies of Tibial nerve

                             Sural nerve
                             Median nerve
                             Ulnar nerve every 3 months

- pain analog scale once a month

- blood pressure lying, sitting, standing once a month

- orthostatic changes once a month

- EKG R-R variation every 3 months


- diabetic vasculopathy:

- Doppler ultrasound: every month x3, then Q3months

- Doppler probe

- Doppler blood pressure ankle/arm

- Doppler segmental blood pressure

- plethysmograph waveform change


- ‘brittle’ diabetes of children:

- retinal photography every 3 months

- microalbuminuria every 3 months

- serum creatinine once a month


Frequency of office visits: 4 weeks and 48 hours before fetal precursor cell transplantation, 24 hours after, and then once a week for the first month after fetal precursor cell transplantation, once a month thereafter.



Other hormone deficiency disorders, where a re-establishment of normal hormonal balance by hormone replacement therapy was not possible, have been succesfully treated by fetal precursor cell transplantation with increasing frequency.