A1 beta-casein milk protein and other environmental pre-disposing factors for type 1 diabetes.

Globally type 1 diabetes incidence is increasing. It is widely accepted that the pathophysiology of type 1 diabetes is influenced by environmental factors in people with specific human leukocyte antigen haplotypes. We propose that a complex interplay between dietary triggers, permissive gut factors and potentially other influencing factors underpins disease progression. We present evidence that A1 ß-casein cows' milk protein is a primary causal trigger of type 1 diabetes in individuals with genetic risk factors. Permissive gut factors (for example, aberrant mucosal immunity), intervene by impacting the gut's environment and the mucosal barrier. Various influencing factors (for example, breastfeeding duration, exposure to other dietary triggers and vitamin D) modify the impact of triggers and permissive gut factors on disease. The power of the dominant trigger and permissive gut factors on disease is influenced by timing, magnitude and/or duration of exposure. Within this framework, removal of a dominant dietary trigger may profoundly affect type 1 diabetes incidence. We present epidemiological, animal-based, in vitro and theoretical evidence for A1 ß-casein and its ß-casomorphin-7 derivative as dominant causal triggers of type 1 diabetes. The effects of ordinary milk containing A1 and A2 ß-casein and milk containing only the A2 ß-casein warrant comparison in prospective trials.

Clinical porcine islet xenotransplantation under comprehensive regulation.

BACKGROUND: Xenotransplantation with porcine islets is a promising approach to overcome the shortage of human donors. This is the first report of phase 1/2a xenotransplantation study of encapsulated neonatal porcine islets under the current framework of regulations for xenotransplantation in New Zealand. METHODS: Newborn piglets were anesthetized and bled, and the pancreata were removed with the use of sterile technique and processed. Encapsulated neonatal porcine islets were implanted with the use of laparoscopy into the peritoneal cavity of 14 patients with unstable type 1 diabetes without any immunosuppressive drugs. The patients received encapsulated islets of 5,000 (n = 4; group 1), 10,000 (n = 4; group 2), 15,000 (n = 4; group 3), or 20,000 (n = 2; group 4) islet equivalents per kg body weight. Outcome was determined from adverse event reports, HbA1c, total daily insulin dose, and frequency of unaware hypoglycemic events. To assess graft function, transplant estimated function (TEF) scores were calculated. Sufficient or marginal numbers of encapsulated neonatal porcine islets were transplanted into streptozotocin-induced diabetic B6 mice as an in vivo functional assay. RESULTS: There were 4 serious adverse events, of which 3 were considered to be possibly related to the procedure. Tests for porcine endogenous retrovirus DNA and RNA were all negative. The numbers of unaware hypoglycemia events were reduced after transplantation in all groups. Four of 14 patients attained HbA1c <7% compared with 1 at baseline. The average TEF scores were 0.17, 0.02, -0.01, and 0.08 in groups 1, 2, 3, and 4 respectively. The in vivo study demonstrated that a sufficient number of the transplanted group reversed diabetes with positive porcine C-peptide. CONCLUSIONS: Transplantation of encapsulated neonatal porcine islets was safe and was followed by a reduction in unaware hypoglycemia events in unstable type 1 diabetic patients. The mouse in vivo assessment data demonstrated certain graft function.

Encapsulated living choroid plexus cells: potential long-term treatments for central nervous system disease and trauma.

In neurodegenerative disease and in acute brain injury, there is often local up-regulation of neurotrophin production close to the site of the lesion. Treatment by direct injection of neurotrophins and growth factors close to these lesion sites has repeatedly been demonstrated to improve recovery. It has therefore been proposed that transplanting viable neurotrophin-producing cells close to the trauma lesion, or site of degenerative disease, might provide a novel means for continuous delivery of these molecules directly to the site of injury or to a degenerative region. The aim of this paper is to summarize recent published information and present new experimental data that indicate that long-lasting therapeutic implants of choroid plexus (CP) neuroepithelium may be used to treat brain disease. CP produces and secretes numerous biologically active neurotrophic factors (NT). New gene microarray and proteomics data presented here indicate that many other anti-oxidant, anti-toxin and neuronal support proteins are also produced and secreted by CP cells. In the healthy brain, these circulate in the cerebrospinal fluid through the brain and spinal cord, maintaining neuronal networks and associated cells. Recent publications describe how transplanted CP cells and tissue, either free or in an immunoprotected encapsulated form, can effectively deliver therapeutic molecules when placed near the lesion or site of degenerative disease in animal models. Using simple techniques, CP neuroepithelial cell clusters in suspension culture were very durable, remaining viable for 6 months or more in vitro. The cell culture conditions had little effect on the wide range and activity of genes expressed and proteins secreted. Recently, completed experiments show that implanting CP within alginate-poly-ornithine capsules effectively protected these xenogeneic cells from the host immune system and allowed their survival for 6 months or more in the brains of rats, causing no adverse effects. Previously reported evidence demonstrated that CP cells support the survival and differentiation of neuronal cells in vitro and effectively treat acute brain injury and disease in rodents and non-human primates in vivo. The accumulated preclinical data together with the long-term survival of implanted encapsulated cells in vivo provide a sound base for the investigation of these treatments for chronic inherited and established neurodegenerative conditions.

Encapsulated porcine islet transplantation: an evolving therapy for the treatment of type I diabetes.

BACKGROUND: Allogeneic tissue-based therapies for Type I diabetes have demonstrated efficacy but are limited due to tissue-sourcing constraints, as the number of patients exceeds that of tissue donors. Porcine islets derived from designated pathogen-free sources could be an alternative, particularly if delivered in a way that evades the host immune system's rejection. METHODS: This review focuses on approaches designed to protect xenogeneic islets from immune rejection by provision of perm-selective barriers. RESULTS: Designated pathogen-free herds could provide a supply of wild-type porcine islets that are well tolerated when administered in a suitable protective delivery vehicle. Such barrier systems have enabled amelioration of diabetes in a variety of animal models and preliminary evidence suggests that similar results could be attained in humans. CONCLUSION: With advances in biomaterial design, source tissue selection, and the evolution of critical cell processing techniques, contemporary encapsulated porcine islet therapies offer a new level of clinical promise.

Diabetes--a man made disease.

The recent increase in both forms of diabetes must be caused by a modern change in the environment. Candidate agents must satisfy at least three criteria. Firstly, the agent must have increased in the environment recently, secondly that it causes diabetes in appropriate animal models, and thirdly that there is a plausible diabetogenic mechanism. Modern food processing can produce glycation end products, oxidised ascorbic acid and lipoic acid, all of which may cause diabetes. Infant formula in particular has high levels of glycation products, and added ascorbic acid. A casomorphin released from A1 beta-casein (but not the A2 variant) can become glycated and have adverse immune effects. Food processing and additives can be posited as a man made cause of the increase in both forms of diabetes. This hypothesis does not exclude other environmental agents which meet the above three criteria.

Intraperitoneal alginate-encapsulated neonatal porcine islets in a placebo-controlled study with 16 diabetic cynomolgus primates.

BACKGROUND: A nonhuman primate model of diabetes is valuable for assessing porcine pancreatic islet transplants that might have clinical benefits in humans. METHODS: Neonatal porcine islets, microencapsulated in alginate-polyornithine-alginate, were injected intraperitoneally (10,000 IEQs/kg islets) into eight adult male cynomolgus monkeys rendered diabetic with streptozotocin. Eight diabetic controls were given an equivalent dose of empty placebo capsules. All subjects received a repeat transplant 3 months after the first. RESULTS: The transplant was well tolerated and no adverse or hypoglycemic events occurred. There were two deaths from nontransplant treatment or diabetic complications unrelated to the transplants. After transplantation, the average insulin dose was reduced in the islet-treated group and increased in the control group. At 12 weeks after the first transplant there was a mean 36% (95% CI: 6% to 65%, P = .02) drop in daily insulin dose compared with the control group. After 24 weeks the difference increased to a mean of 43% (95% CI: 12% to 75%, P = .01) without significant differences in blood glucose values between the two groups. Individual responses after islet transplant varied and one monkey was weaned off insulin by 36 weeks. At terminal autopsy, organs appeared normal and there was no visible peritoneal reaction. No animal had polymerase chain reaction (PCR)-amplified signals of porcine endogenous retrovirus or exogenous virus infections in blood or tissues. CONCLUSION: Repeated intraperitoneal transplantation of microencapsulated neonatal porcine islets is a safe procedure in diabetic primates. It was shown to result in a significant reduction in insulin dose requirement in the majority of animals studied, whereas insulin requirement increased in controls.

Transplantation of micro- and macroencapsulated piglet islets into mice and monkeys.

Neonatal porcine islets within alginate microcapsules transplanted intraperitoneally (IP) or within semi-permeable macrocapsules (TheraCyte) and transplanted subcutaneously (SC) survive and reverse diabetes for up to 16 weeks in diabetic autoimmune nonobese diabetic (NOD) mice. The islets in microcapsules transplanted IP into nondiabetic cynomolgus monkeys survived for 8 weeks. Similar results were shown with islets transplanted in TheraCytes. Neither species showed adverse effects or evidence of infection with porcine endogenous retroviruses or other endemic pig viruses. Proof of principle is illustrated for successful xenotransplantation in humans.

Xenotransplantation of neonatal porcine liver cells.

Xenotransplantation of porcine liver cell types may provide a means of overcoming the shortage of suitable donor tissues to treat hepatic diseases characterized by inherited inborn errors of metabolism or protein production. Here we report the successful isolation, culture, and xenotransplantation of liver cells harvested from 7- to 10-day-old piglets. Liver cells were isolated and cultured immediately after harvesting. Cell viability was excellent (>90%) over the duration of the in vitro studies (3 weeks) and the cultured cells continued to significantly proliferate. These cells also retained their normal secretory and metabolic capabilities as determined by continued release of albumin, factor 8, and indocyanin green (ICG) uptake. After 3 weeks in culture, porcine liver cells were loaded into immunoisolatory macro devices (Theracyte devices) and placed into the intraperitoneal cavity of immunocompetant CD1 mice. Eight weeks later, the devices were retrieved and the cells analyzed for posttransplant determinations of survival and function. Post mortem analysis confirmed that the cell-loaded devices were biocompatible, and were well-tolerated without inducing any notable inflammatory reaction in the tissues immediately surrounding the encapsulated cells. Finally, the encapsulated liver cells remained viable and functional as determined by histologic analyses and ICG uptake/release. The successful harvesting, culturing, and xenotransplantation of functional neonatal pig liver cells support the continued development of this approach for treating a range of currently undertreated or intractable hepatic diseases.

Identification of pig circovirus type 2 in New Zealand pigs.

Interest in porcine circovirus has been stimulated by the recent emergence of postweaning multisystemic wasting syndrome (PMWS) in pigs and the potential use of pig organs for xenotransplantation in humans. Porcine circovirus type 1 (PCV1) is considered to be widespread in pigs but nonpathogenic. Circovirus type 2 (PCV2) is a similar virus but has been differentiated only recently as a separate type. High tissue concentrations of PCV2 are associated with lesions in PMWS cases, but the etiological role of this agent in the disease remains unclear. The presence of PCV1 in New Zealand pigs has been previously reported based on serological data. PMWS has been recently recorded in New Zealand pigs. The epidemiology of PCV2 in New Zealand pigs has not been examined. The purpose of the study was to look for evidence of circoviruses in New Zealand pig herds. Pig circovirus DNA was sought in various tissues using the polymerase chain reaction. Circovirus type 2 was found in New Zealand pig herds, without any evidence that PMWS has ever occurred in these herds. Newborn piglets were shown to have infection, suggesting vertical transmission of the virus.

Monitoring for presence of potentially xenotic viruses in recipients of pig islet xenotransplantation.

This study represents a long-term follow-up of human patients receiving pig islet xenotransplantation. Eighteen patients had been monitored for up to 9 years for potentially xenotic pig viruses: pig endogenous retrovirus, pig cytomegalovirus, pig lymphotropic herpesvirus, and pig circovirus type 2. No evidence of viral infection was found.

CNS grafts of rat choroid plexus protect against cerebral ischemia in adult rats.

The present study examined the neuroprotective effects of choroid plexus isolated from adult rats and encapsulated within alginate microcapsules. In vitro, conditioned media from cultured choroid plexus produced a marked, dose-dependent protection of embryonic cortical neurons against serum deprivation-induced cell death. In vivo studies demonstrated that a one-hour middle cerebral artery occlusion in adult Wistar rats produced profound motor and neurological impairments 1-3 days after stroke. In contrast, stroke animals transplanted with encapsulated choroid plexus cells displayed a significant reduction in both motor and neurological abnormalities. Histological analysis 3 days post-transplantation revealed that choroid plexus transplants significantly decreased the volume of striatal infarction. This is the first report demonstrating the therapeutic potential of transplanted choroid plexus for stroke.

A wheat-based, diabetes-promoting diet induces a Th1-type cytokine bias in the gut of NOD mice.

Dietary antigens are candidate environmental factors in the pathogenesis of type 1 diabetes. In the non-obese diabetic (NOD) mouse, an animal model of type 1 diabetes, cereal-based diets promote disease development, whereas the diets based on hydrolysed proteins or non-diabetogenic proteins are protective. The hypothesis that diabetogenic diets modulate the cytokine balance in the gut was tested. NOD mice were fed with NTP-2000 (mainly a wheat-based milk-free diet) or Prosobee (a semi-purified hypoallergenic diet based on soy protein isolate) or Prosobee plus casein (milk protein fraction). The mRNA levels of IFN-gamma, IL-10, TNF-alpha, TGF-beta, and inducible NO synthase in the small intestine and the Peyer's patches were determined by semi-quantitative RT-PCR. Mice fed on the cereal-based NTP-2000 diet expressed higher levels of the Th1-type and pro-inflammatory markers IFN-gamma, TNF-alpha, and inducible NO synthase mRNA compared to the Prosobee-fed animals. The expression of the counterregulatory cytokines IL-10 and TGF-beta was unaffected. This resulted in a significant bias of the intestinal cytokine balance towards T helper cell type 1 after feeding NTP-2000. The cytokine mRNA levels in the gut-associated Peyer's patches were not affected. Thus, modulation of gut immunoreactivity by diet may contribute to disease development in NOD mice.

A multi-centre, blinded international trial of the effect of A(1) and A(2) beta-casein variants on diabetes incidence in two rodent models of spontaneous Type I diabetes.

AIMS/HYPOTHESIS: The diabetes-inducing potential of cows' milk is still debated and there is no consensus on the diabetogenicity of individual milk proteins. A(1)-beta-casein has been associated with increased diabetes frequency in ecological studies and in NOD mice. Our aim was to ascertain whether A(1)-beta-casein was more diabetogenic than A(2) and to test the diabetogenicity of a milk-free diet in animals representing different forms of spontaneous Type I (insulin-dependent) diabetes mellitus. METHODS: Defined diets were coded and shipped to laboratories in New Zealand (NOD/NZ), Canada (BB) and the UK (NOD/Ba). Base diets were Pregestimil (PG) and ProSobee (PS). Purified fractions of whole casein (WC), A(1)or A(2)-beta-casein were added at 10%. A milk-free, wheat-predominant, NTP-2000 diet was the control. Animals were fed from weaning up to 150 or 250 days, and insulitis, diabetes frequency and expression of pancreatic cytokines were assessed. RESULTS: Diabetes incidence was highest in three locations in animals fed NTP-2000. PG and PS diets were protective except for NOD/Ba mice fed PG+WC where incidence was similar to NTP-2000. A(1) and A(2) diets were protective in both models, but A(1) beta-casein was slightly more diabetogenic in PS-fed BB rats. The New Zealand study was confounded by an infection. CONCLUSION/INTERPRETATION: A milk-free, wheat-predominant diet was highly diabetogenic in three widely separate locations in both animal models. A previous result that A(1) beta-casein was more diabetogenic than A(2) beta-casein in NOD mice was not confirmed; both beta-casein variants were protective in BB rats and NOD mice. Whole Casein promoted diabetes in NOD/Ba but protected BB showing that unique diabetes haplotypes react differently to dietary proteins. A(1)- was more diabetogenic than A(2)-beta-casein only in PS-fed BB rats. Neither the analysis of insulitis nor of pancreatic cytokine gene expression showed a difference between A(1) or A(2) beta-casein fed animals. Milk caseins are unlikely to be exclusive promoters of Type I diabetes, but could enhance the outcome of diabetes in some cases. Other diet components such as wheat could be more important promoters of Type I diabetes.

No evidence of infection with porcine endogenous retrovirus in recipients of encapsulated porcine islet xenografts.

Transplantation of pig tissues into humans has the potential for cotransferring pig infections. Knowledge of the epidemiology of pig infections transmissible to humans allows the development of risk limitation strategies at the source herd level, but potentially infectious pig endogenous retrovirus (PERV) is ubiquitous in all domestic pigs and therefore is not avoidable. Using a specific and sensitive RT-PCR and nested PCR for PERV nucleic acids with primers, the screening of pigs from New Zealand herds for the presence and expression of the PERV was conducted. The presence of PERV proviral DNA (pol and env region) and viral RNA was demonstrated in all tested pig tissues including pancreas, liver, spleen, brain, heart, and PBMC. Using the same assays it was established that different tissues (liver, spleen, and heart) of nude and nonobese diabetic (NOD) mice previously transplanted with nonencapsulated pig islets were PERV DNA and RNA negative. Alginate polylysine capsules prepared with encapsulated pig islets were tested for possible leakage of viral particles or viral nucleic acids. RNA was extracted from the supernatant of viable encapsulated pig islet cells grown in culture for 2 months. No evidence of PERV RNA or of cellular nucleic acids could be found. Two adult type I diabetic subjects were transplanted with 1 x 10(6) neonatal pig islets encased in alginate capsules into the peritoneal cavity. One patient was immunosuppressed. Both showed evidence of graft function (up to 34% reduction in insulin dose, corresponding increase in serum pig C-peptide) for up to 2 years. DNA and RNA were extracted from PBMC and blood plasma of both patients at 19 months posttransplant. No evidence of PERV proviral DNA or RNA could be detected. Piglet islets contain PERV DNA and RNA, but this does not traverse the capsules used or produce any evidence of infection in nude and nonobese diabetic (NOD) mice or humans.

The gut cytokine balance as a target of lead toxicity.

The impact of exposure to lead on gut cytokine gene expression and oral tolerance was analyzed. Oral tolerization with ovalbumin (OVA) increased levels of IL-10 and TGF-beta in gut tissue while IFN-gamma mRNA levels remained unchanged in both autoimmune diabetes prone NOD and normal C57BL/6 mice. This shift towards Th2/Th3 type cytokine gene expression was completely abolished by concomitant treatment with PbCl2 (6 x 0.5 mg/kg) in NOD mice while the cytokine balance in C57BL/6 mice was unaffected. Suppression of Th2/Th3 type cytokine expression was associated with a dampened oral tolerance response to OVA as determined by T cell proliferation assays. We conclude that in autoimmunity prone NOD mice environmental toxicants may disturb immune homeostasis by targeting the gut immune system.

Type I (insulin-dependent) diabetes mellitus and cow milk: casein variant consumption.

Previously published Type I (insulin-dependent) diabetes mellitus incidence in 0 to 14-year-old children from 10 countries or areas was compared with the national annual cow milk protein consumption. Countries which were selected for study had appropriate milk protein polymorphism studies, herd breed composition information and low dairy imports from other countries. Total protein consumption did not correlate with diabetes incidence (r = +0.402), but consumption of the beta-casein A1 variant did (r = +0.726). Even more pronounced was the relation between beta-casein (A1+B) consumption and diabetes (r = +0.982). These latter two cow caseins yield a bioactive peptide beta-casomorphin-7 after in vitro digestion with intestinal enzymes whereas the common A2 variant or the corresponding human or goat caseins do not. beta-casomorphin-7 has opioid properties including immunosuppression, which could account for the specificity of the relation between the consumption of some but not all beta-casein variants and diabetes incidence.

Genetic determination of islet cell autoimmunity in monozygotic twin, dizygotic twin, and non-twin siblings of patients with type 1 diabetes: prospective twin study.

OBJECTIVE: To test the hypothesis that non-diabetic dizygotic and monozygotic twin siblings of patients with type 1 diabetes have a similar high prevalence of islet cell autoantibodies, thus suggesting that islet cell autoimmunity is mainly environmentally determined. DESIGN: Prospective twin study. SETTING: Two specialist centres for diabetes in the United States. PARTICIPANTS: Non-diabetic monozygotic twin (n=53), dizygotic twin (n=30), and non-twin (n=149) siblings of patients with type 1 diabetes; 101 controls. MAIN OUTCOME MEASURES: Analysis of progression to diabetes and expression of anti-islet autoantibodies. RESULTS: Monozygotic twin siblings had a higher risk of progression to diabetes (12/53) than dizygotic twin siblings (0/30; P<0.005). At the last follow up 22 (41.5%) monozygotic twin siblings expressed autoantibodies compared with 6 (20%) dizygotic twin siblings (P<0.05), 16 (10.7%) non-twin siblings (P<0.0001), and 6 (5.9%) controls (P<0.0001). Monozygotic twin siblings expressed multiple (>/=2) antibodies more often than dizygotic twin siblings (10/38 v 1/23; P<0.05). By life table analysis the probability of developing positive autoantibodies was higher among the monozygotic twin siblings bearing the diabetes associated HLA DQ8/DQ2 genotype than in those without this genotype (64.2% (95% confidence interval 32.5% to 96%) v 23.5% (7% to 40%) at 10 years of discordance; P<0.05). CONCLUSION: Monozygotic and dizygotic twins differ in progression to diabetes and expression of islet cell autoantibodies. Dizygotic twin siblings are similar to non-twin siblings. These two observations suggest that genetic factors play an important part in determination of islet cell autoimmunity, thus rejecting the hypothesis. In addition, there is a high penetrance of islet cell autoimmunity in DQ8/DQ2 monozygotic twin siblings.

Double-label immunofluorescence study of glutamic acid decarboxylase in the fetal and adult ovine pancreas by light and confocal microscopy: evidence for predominant beta-cell coexpression.

Glutamic acid decarboxylase (GAD) is present in the central nervous system and in several nonneuronal tissues including the pancreatic islets. There are two isoforms with molecular weights of 65 kDa (GAD65) and 67 kDa (GAD67). The cellular specificity of the two molecular forms of GAD and their levels within the mammalian islets may be species-dependent, being coexpressed in both beta and in non-beta cells. We have examined the ovine pancreas, from the adult and fetal stages of late gestation, for the expression of GAD65 within the islet cells by double-label immunofluorescence light and confocal microscopy. In the adult tissue, GAD65 was colocalized in a majority of the beta cells (> 95%), with only a few glucagon and somatostatin cells (< 5%) showing immunolocalization. During the fetal stages GAD65 also showed a similar predominant beta-cell coexpression. The enzyme was also detected in a few fetal glucagon (< 5%) but not somatostatin cells. In the degenerating large fetal islets, GAD65 was also observed in the majority of the residual beta cells. These results demonstrate that in the ovine pancreas GAD65 is expressed during fetal development and is predominantly beta-cell-restricted. This pattern of expression is maintained during adult life. However, the physiological role of pancreatic GAD and/or its biosynthetic product, gamma-aminobutyric acid, in islet function in the sheep and in other ruminants remains unclear.

A population based strategy to prevent insulin-dependent diabetes using nicotinamide.

It has been postulated that treatment with nicotinamide may prevent or delay the onset of insulin dependent diabetes mellitus. We report the findings of a population based diabetes prevention trial which tests this hypothesis. 33,658 school children aged 5-7.9 years were randomly selected (by school) from a total population of 81,993 of such children in the Auckland (New Zealand) region. They were offered testing for islet cell antibodies. 20,195 (60%) consented to testing. Of these 185 had islet cell antibodies and met the criteria for treatment with nicotinamide. 173 received this treatment. The study population has an average follow up time of 7.1 years. The diabetes incidence of the untested controls was: 16.07 (12.4-20.5 95% CI) /100,000 person years at risk; in the group who were tested and treated when deemed appropriate: 7.14 (3.1-14.1 95% CI); and in the group offered testing but who did not consent ("refusers'): 18.48 (10.1-31.0 95% CI). The tested group had a rate of diabetes of 41% (20-85 95% CI) of the other groups combined after an age adjustment, which is significant (p = 0.008). The tested group combined with the "refuser' group (i.e. "intention to treat') also has a lower incidence than the control group (p = 0.12). Nicotinamide has a protective effect against the development of insulin dependent diabetes in this setting but the size of the effect has a wide confidence interval. Further follow up may define the magnitude of the protective effect within narrower limits.