SPIKENET: An Evidence-Based Therapy for Long COVID.

The COVID-19 pandemic has been one of the most impactful events in our lifetime, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Multiple SARS-CoV-2 variants were reported globally, and a wide range of symptoms existed. Individuals who contract COVID-19 continue to suffer for a long time, known as long COVID or post-acute sequelae of COVID-19 (PASC). While COVID-19 vaccines were widely deployed, both unvaccinated and vaccinated individuals experienced long-term complications. To date, there are no treatments to eradicate long COVID. We recently conceived a new approach to treat COVID in which a 15-amino-acid synthetic peptide (SPIKENET, SPK) is targeted to the ACE2 receptor binding domain of SARS-CoV-2, which prevents the virus from attaching to the host. We also found that SPK precludes the binding of spike glycoproteins with the receptor carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) of a coronavirus, murine hepatitis virus-1 (MHV-1), and with all SARS-CoV-2 variants. Further, SPK reversed the development of severe inflammation, oxidative stress, tissue edema, and animal death post-MHV-1 infection in mice. SPK also protects against multiple organ damage in acute and long-term post-MHV-1 infection. Our findings collectively suggest a potential therapeutic benefit of SPK for treating COVID-19.

Acute and Long COVID Intestinal Changes in an Experimental Model of Coronavirus in Mice.

The COVID-19 pandemic, which emerged in early 2020, has had a profound and lasting impact on global health, resulting in over 7.0 million deaths and persistent challenges. In addition to acute concerns, there is growing attention being given to the long COVID health consequences for survivors of COVID-19 with documented cases of cardiovascular abnormalities, liver disturbances, lung complications, kidney issues, and noticeable cognitive deficits. Recent studies have investigated the physiological changes in various organs following prolonged exposure to murine hepatitis virus-1 (MHV-1), a coronavirus, in mouse models. One significant finding relates to the effects on the gastrointestinal tract, an area previously understudied regarding the long-lasting effects of COVID-19. This research sheds light on important observations in the intestines during both the acute and the prolonged phases following MHV-1 infection, which parallel specific changes seen in humans after exposure to SARS-CoV-2. Our study investigates the histopathological alterations in the small intestine following MHV-1 infection in murine models, revealing significant changes reminiscent of inflammatory bowel disease (IBD), celiac disease. Notable findings include mucosal inflammation, lymphoid hyperplasia, goblet cell hyperplasia, and immune cell infiltration, mirroring pathological features observed in IBD. Additionally, MHV-1 infection induces villous atrophy, altered epithelial integrity, and inflammatory responses akin to celiac disease and IBD. SPIKENET (SPK) treatment effectively mitigates intestinal damage caused by MHV-1 infection, restoring tissue architecture and ameliorating inflammatory responses. Furthermore, investigation into long COVID reveals intricate inflammatory profiles, highlighting the potential of SPK to modulate intestinal responses and restore tissue homeostasis. Understanding these histopathological alterations provides valuable insights into the pathogenesis of COVID-induced gastrointestinal complications and informs the development of targeted therapeutic strategies.

Predictors of lack of glycemic control in persons with type 2 diabetes.

BACKGROUND: Professional guidelines recommend an HbA1c < 7% for most people with diabetes and < 8.5% for those with relaxed glycemic goals. However, many people with type 2 diabetes mellitus (T2DM) are unable to achieve the desired HbA1c goal. This study evaluated factors associated with lack of improvement in HbA1c over 3 years. METHODS: All patients with T2DM treated within a major academic healthcare system during 2015-2020, who had at least one HbA1c value > 8.5% within 3 years from their last HbA1c were included in analysis. Patients were grouped as improved glycemic control (last HbA1c ≤ 8.5%) or lack of improvement (last HbA1c > 8.5%). Multivariate logistic regression analysis was performed to assess independent predictors of lack of improvement in glycemic control. RESULTS: Out of 2,232 patients who met the inclusion criteria, 1,383 had an improvement in HbA1c while 849 did not. In the fully adjusted model, independent predictors of lack of improvement included: younger age (odds ratio, 0.89 per 1-SD [12 years]; 95% CI, 0.79-1.00), female gender (1.30, 1.08-1.56), presence of hypertension (1.29, 1.08-1.55), belonging to Black race (1.32, 1.04-1.68, White as reference), living in low income area (1.86,1.28-2.68, high income area as reference), and insurance coverage other than Medicare (1.32, 1.05-1.66). Presence of current smoking was associated with a paradoxical improvement in HbA1c (0.69, 0.47-0.99). In a subgroup analysis, comparing those with all subsequent HbA1c values > 8.5% (N = 444) to those with all subsequent HbA1c values < 8.5% (N = 341), similar factors were associated with lack of improvement, but smoking was no longer significant. CONCLUSION: We conclude that socioeconomic factors like race, type of insurance coverage and living in low-income areas are associated with lack of improvement in HbA1c over a period of 3-years in people with T2DM. Intervention strategies focused on low-income neighborhoods need to be designed to improve diabetes management.

The anti-CD40L monoclonal antibody AT-1501 promotes islet and kidney allograft survival and function in nonhuman primates.

Prior studies of anti-CD40 ligand (CD40L)-based immunosuppression demonstrated effective prevention of islet and kidney allograft rejection in nonhuman primate models; however, clinical development was halted because of thromboembolic complications. An anti-CD40L-specific monoclonal antibody, AT-1501 (Tegoprubart), was engineered to minimize risk of thromboembolic complications by reducing binding to Fcγ receptors expressed on platelets while preserving binding to CD40L. AT-1501 was tested in both a cynomolgus macaque model of intrahepatic islet allotransplantation and a rhesus macaque model of kidney allotransplantation. AT-1501 monotherapy led to long-term graft survival in both islet and kidney transplant models, confirming its immunosuppressive potential. Furthermore, AT-1501-based regimens after islet transplant resulted in higher C-peptide, greater appetite leading to weight gain, and reduced occurrence of cytomegalovirus reactivation compared with conventional immunosuppression. These data support AT-1501 as a safe and effective agent to promote both islet and kidney allograft survival and function in nonhuman primate models, warranting further testing in clinical trials.

Implantable niche with local immunosuppression for islet allotransplantation achieves type 1 diabetes reversal in rats.

Pancreatic islet transplantation efficacy for type 1 diabetes (T1D) management is limited by hypoxia-related graft attrition and need for systemic immunosuppression. To overcome these challenges, we developed the Neovascularized Implantable Cell Homing and Encapsulation (NICHE) device, which integrates direct vascularization for facile mass transfer and localized immunosuppressant delivery for islet rejection prophylaxis. Here, we investigated NICHE efficacy for allogeneic islet transplantation and long-term diabetes reversal in an immunocompetent, male rat model. We demonstrated that allogeneic islets transplanted within pre-vascularized NICHE were engrafted, revascularized, and functional, reverting diabetes in rats for over 150 days. Notably, we confirmed that localized immunosuppression prevented islet rejection without inducing toxicity or systemic immunosuppression. Moreover, for translatability efforts, we showed NICHE biocompatibility and feasibility of deployment as well as short-term allogeneic islet engraftment in an MHC-mismatched nonhuman primate model. In sum, the NICHE holds promise as a viable approach for safe and effective islet transplantation and long-term T1D management.

Performance of islets of Langerhans conformally coated via an emulsion cross-linking method in diabetic rodents and nonhuman primates.

Polyethylene glycol (PEG)-based conformal coating (CC) encapsulation of transplanted islets is a promising ß cell replacement therapy for the treatment of type 1 diabetes without chronic immunosuppression because it minimizes capsule thickness, graft volume, and insulin secretion delay. However, we show here that our original CC method, the direct method, requiring exposure of islets to low pH levels and inclusion of viscosity enhancers during coating, severely affected the viability, scalability, and biocompatibility of CC islets in nonhuman primate preclinical models of type 1 diabetes. We therefore developed and validated in vitro and in vivo, in several small- and large-animal models of type 1 diabetes, an augmented CC method-emulsion method-that achieves hydrogel CCs around islets at physiological pH for improved cytocompatibility, with PEG hydrogels for increased biocompatibility and with fivefold increase in encapsulation throughput for enhanced scalability.

Extended survival versus accelerated rejection of nonhuman primate islet allografts: Effect of mesenchymal stem cell source and timing.

Mesenchymal stem cells (MSC) have been shown to be immunomodulatory, tissue regenerative, and graft promoting; however, several questions remain with regard to ideal MSC source and timing of administration. In this study, we utilized a rigorous preclinical model of allogeneic islet cell transplantation, incorporating reduced immune suppression and near to complete mismatch of major histocompatibility antigens between the diabetic cynomolgus monkey recipient and the islet donor, to evaluate both the graft promoting impact of MSC source, that is, derived from the islet recipient, the islet donor or an unrelated third party as well as the impact of timing. Co-transplant of MSC and islets on post-operative day 0, followed by additional IV MSC infusions in the first posttransplant month, resulted in prolongation of rejection free and overall islet survival and superior metabolic control for animals treated with recipient as compared to donor or third-party MSC. Immunological analyses demonstrated that infusion of MSC from either source did not prevent alloantibody formation to the islet or MSC donor; however, treatment with recipient MSC resulted in significant downregulation of memory T cells, decreased anti-donor T cell proliferation, and a trend toward increased Tregulatory:Tconventional ratios.

Umbilical cord mesenchymal stem cells for COVID-19 acute respiratory distress syndrome: A double-blind, phase 1/2a, randomized controlled trial.

Acute respiratory distress syndrome (ARDS) in COVID-19 is associated with high mortality. Mesenchymal stem cells are known to exert immunomodulatory and anti-inflammatory effects and could yield beneficial effects in COVID-19 ARDS. The objective of this study was to determine safety and explore efficacy of umbilical cord mesenchymal stem cell (UC-MSC) infusions in subjects with COVID-19 ARDS. A double-blind, phase 1/2a, randomized, controlled trial was performed. Randomization and stratification by ARDS severity was used to foster balance among groups. All subjects were analyzed under intention to treat design. Twenty-four subjects were randomized 1:1 to either UC-MSC treatment (n = 12) or the control group (n = 12). Subjects in the UC-MSC treatment group received two intravenous infusions (at day 0 and 3) of 100 ± 20 × 106 UC-MSCs; controls received two infusions of vehicle solution. Both groups received best standard of care. Primary endpoint was safety (adverse events [AEs]) within 6 hours; cardiac arrest or death within 24 hours postinfusion). Secondary endpoints included patient survival at 31 days after the first infusion and time to recovery. No difference was observed between groups in infusion-associated AEs. No serious adverse events (SAEs) were observed related to UC-MSC infusions. UC-MSC infusions in COVID-19 ARDS were found to be safe. Inflammatory cytokines were significantly decreased in UC-MSC-treated subjects at day 6. Treatment was associated with significantly improved patient survival (91% vs 42%, P = .015), SAE-free survival (P = .008), and time to recovery (P = .03). UC-MSC infusions are safe and could be beneficial in treating subjects with COVID-19 ARDS.

Transplantation of PEGylated islets enhances therapeutic efficacy in a diabetic nonhuman primate model.

Islet cell transplantation can lead to insulin independence, reduced hypoglycemia, and amelioration of diabetes complications in patients with type 1 diabetes. The systemic delivery of anti-inflammatory agents, while considered crucial to limit the early loss of islets associated with intrahepatic infusion, increases the burden of immunosuppression. In an effort to decrease the pharmaceutical load to the patient, we modified the pancreatic islet surface with long-chain poly(ethylene glycol) (PEG) to mitigate detrimental host-implant interactions. The effect of PEGylation on islet engraftment and long-term survival was examined in a robust nonhuman primate model via three paired transplants of dosages 4300, 8300, and 10 000 islet equivalents per kg body weight. A reduced immunosuppressive regimen of anti-thymocyte globulin induction plus tacrolimus in the first posttransplant month followed by maintenance with sirolimus monotherapy was employed. To limit transplant variability, two of the three pairs were closely MHC-matched recipients and received MHC-disparate PEGylated or untreated islets isolated from the same donors. Recipients of PEGylated islets exhibited significantly improved early c-peptide levels, reduced exogenous insulin requirements, and superior glycemic control, as compared to recipients of untreated islets. These results indicate that this simple islet modification procedure may improve islet engraftment and survival in the setting of reduced immunosuppression.

Operational immune tolerance towards transplanted allogeneic pancreatic islets in mice and a non-human primate.

AIMS/HYPOTHESIS: Patients with autoimmune type 1 diabetes transplanted with pancreatic islets to their liver experience significant improvement in quality of life through better control of blood sugar and enhanced awareness of hypoglycaemia. However, long-term survival and efficacy of the intrahepatic islet transplant are limited owing to liver-specific complications, such as immediate blood-mediated immune reaction, hypoxia, a highly enzymatic and inflammatory environment and locally elevated levels of drugs including immunosuppressive agents, all of which are injurious to islets. This has spurred a search for new islet transplant sites and for innovative ways to achieve long-term graft survival and efficacy without life-long systemic immunosuppression and its complications. METHODS: We used our previously established approach of islet transplant in the anterior chamber of the eye in allogeneic recipient mouse models and a baboon model of diabetes, which were treated transiently with anti-CD154/CD40L blocking antibody in the peri-transplant period. Survival of the intraocular islet allografts was assessed by direct visualisation in the eye and metabolic variables (blood glucose and C-peptide measurements). We evaluated longitudinally the cytokine profile in the local microenvironment of the intraocular islet allografts, represented in aqueous humour, under conditions of immune rejection vs tolerance. We also evaluated the recall response in the periphery of the baboon recipient using delayed-type hypersensitivity (DTH) assay, and in mice after repeat transplant in the kidney following initial transplant with allogeneic islets in the eye or kidney. RESULTS: Results in mice showed >300 days immunosuppression-free survival of allogeneic islets transplanted in the eye or kidney. Notably, >70% of tolerant mice, initially transplanted in the eye, exhibited >400 days of graft survival after re-transplant in the kidney without immunosuppression compared with ~30% in mice that were initially transplanted in the kidney. Cytokine and DTH data provided evidence of T helper 2-driven local and peripheral immune regulatory mechanisms in support of operational immune tolerance towards the islet allografts in both models. CONCLUSIONS/INTERPRETATION: We are currently evaluating the safety and efficacy of intraocular islet transplantation in a phase 1 clinical trial. In this study, we demonstrate immunosuppression-free long-term survival of intraocular islet allografts in mice and in a baboon using transient peri-transplant immune intervention. These results highlight the potential for inducing islet transplant immune tolerance through the intraocular route. Therefore, the current findings are conceptually significant and may impact markedly on clinical islet transplantation in the treatment of diabetes.

Steroid-Free Immune Suppression Impairs Glycemic Control in a Healthy Cynomolgus Monkey.

The need for chronic immune suppression (IS) is one of the hurdles precluding widespread use of islet cell transplantation to restore glycemic control in patients with type 1 diabetes. We report the case of a healthy nonhuman primate (NHP) treated on and off for over 2.5 years with steroid-free IS, consisting of daclizumab induction and maintenance therapy with rapamycin and low dose tacrolimus. Treatment for 1 year resulted in a striking destabilization of glycemic control, with concomitant decreases in fasting c-peptide and insulin levels. Although these changes gradually reversed during a wash out period of 7 months, retreatment with the same therapy led to accelerated deterioration in glycemic control. Intravenous glucose tolerance and percentage of glycosylated hemoglobin testing further supported a dramatic effect on metabolic control. IS also led to decreases in weight during treatment. Histological evaluation of the pancreas revealed islet hyperplasia, with varying sizes and endocrine cell ratios that differed from normal islet composition, and parenchymal infiltration with adipose tissue. These deleterious effects of IS on glucose control and endocrine components in the native pancreas of a healthy NHP suggest that IS agents commonly utilized for islet transplantation may contribute to failure in islet allograft function in long-term transplant patients.

MRI-Guided, Noninvasive Delivery of Magneto-Electric Drug Nanocarriers to the Brain in a Nonhuman Primate.

A magnetically guided brain delivery method previously demonstrated in mice has not yet been translated for clinical applications due to the mismatch of available static magnet dimensions in relation to the human brain size and shape. To develop a human-compatible methodology, we explored magnetic resonance imaging (MRI) as a tool for the delivery of magneto-electric nanoparticles (MENPs) into the brain of a baboon, as a proof-of-concept study. MRI brain image analysis showed a reduction in T2* value at the basal ganglia, hemisphere, and vertex, thereby confirming successful MENP delivery to the brain. The observation of well-integrated morphologically healthy tissues and no blood toxicity over the study duration confirmed the biocompatibility of MENPs and the delivery procedure. Outcomes of this research present MRI-assisted delivery of MENPs to the brain as a safe and noninvasive method in larger species such as baboons and one step closer to human translation. This MENP-based nanomedicine delivery method can be used for clinical application in order to investigate effective central nervous system (CNS) therapies.

Microencapsulated adult porcine islets transplanted intraperitoneally in streptozotocin-diabetic non-human primates.

BACKGROUND: Xenogeneic donors would provide an unlimited source of islets for the treatment of type 1 diabetes (T1D). The goal of this study was to assess the function of microencapsulated adult porcine islets (APIs) transplanted ip in streptozotocin (STZ)-diabetic non-human primates (NHPs) given targeted immunosuppression. METHODS: APIs were encapsulated in: (a) single barium-gelled alginate capsules or (b) double alginate capsules with an inner, islet-containing compartment and a durable, biocompatible outer alginate layer. Immunosuppressed, streptozotocin-diabetic NHPs were transplanted ip with encapsulated APIs, and graft function was monitored by measuring blood glucose, %HbA1c, and porcine C-peptide. At graft failure, explanted capsules were assessed for biocompatibility and durability plus islet viability and functionality. Host immune responses were evaluated by phenotyping peritoneal cell populations, quantitation of peritoneal cytokines and chemokines, and measurement of anti-porcine IgG and IgM plus anti-Gal IgG. RESULTS: NHP recipients had reduced hyperglycemia, decreased exogenous insulin requirements, and lower percent hemoglobin A1c (%HbA1c) levels. Porcine C-peptide was detected in plasma of all recipients, but these levels diminished with time. However, relatively high levels of porcine C-peptide were detected locally in the peritoneal graft site of some recipients at sacrifice. IV glucose tolerance tests demonstrated metabolic function, but the grafts eventually failed in all diabetic NHPs regardless of the type of encapsulation or the host immunosuppression regimen. Explanted microcapsules were intact, "clean," and free-floating without evidence of fibrosis at graft failure, and some reversed diabetes when re-implanted ip in diabetic immunoincompetent mice. Histology of explanted capsules showed scant evidence of a host cellular response, and viable islets could be found. Flow cytometric analyses of peritoneal cells and peripheral blood showed similarly minimal evidence of a host immune response. Preformed anti-porcine IgG and IgM antibodies were present in recipient plasma, but these levels did not rise post-transplant. Peritoneal graft site cytokine or chemokine levels were equivalent to normal controls, with the exception of minimal elevation observed for IL-6 or IL-1ß, GRO-α, I-309, IP-10, and MCP-1. However, we found central necrosis in many of the encapsulated islets after graft failure, and explanted islets expressed endogenous markers of hypoxia (HIF-1α, osteopontin, and GLUT-1), suggesting a role for non-immunologic factors, likely hypoxia, in graft failure. CONCLUSIONS: With donor xenoislet microencapsulation and host immunosuppression, APIs corrected hyperglycemia after ip transplantation in STZ-diabetic NHPs in the short term. The islet xenografts lost efficacy gradually, but at graft failure, some viable islets remained, substantial porcine C-peptide was detected in the peritoneal graft site, and there was very little evidence of a host immune response. We postulate that chronic effects of non-immunologic factors, such as in vivo hypoxic and hyperglycemic conditions, damaged the encapsulated islet xenografts. To achieve long-term function, new approaches must be developed to prevent this damage, for example, by increasing the oxygen supply to microencapsulated islets in the ip space.

Paracrine Interactions within the Pancreatic Islet Determine the Glycemic Set Point.

Every animal species has a signature blood glucose level or glycemic set point. These set points are different, and the normal glycemic levels (normoglycemia) of one species would be life threatening for other species. Mouse normoglycemia can be considered diabetic for humans. The biological determinants of the glycemic set point remain unclear. Here we show that the pancreatic islet imposes its glycemic set point on the organism, making it the bona fide glucostat in the body. Moreover, and in contrast to rodent islets, glucagon input from the alpha cell to the insulin-secreting beta cell is necessary to fine-tune the distinctive human set point. These findings affect transplantation and regenerative approaches to treat diabetes because restoring normoglycemia may require more than replacing only the beta cells. Furthermore, therapeutic strategies using glucagon receptor antagonists as hypoglycemic agents need to be reassessed, as they may reset the overall glucostat in the organism.

Bioengineering the Endocrine Pancreas: Intraomental Islet Transplantation Within a Biologic Resorbable Scaffold.

Transplantation of pancreatic islets is a therapeutic option to preserve or restore ß-cell function. Our study was aimed at developing a clinically applicable protocol for extrahepatic transplantation of pancreatic islets. The potency of islets implanted onto the omentum, using an in situ-generated adherent, resorbable plasma-thrombin biologic scaffold, was evaluated in diabetic rat and nonhuman primate (NHP) models. Intraomental islet engraftment in the biologic scaffold was confirmed by achievement of improved metabolic function and preservation of islet cytoarchitecture, with reconstitution of rich intrainsular vascular networks in both species. Long-term nonfasting normoglycemia and adequate glucose clearance (tolerance tests) were achieved in both intrahepatic and intraomental sites in rats. Intraomental graft recipients displayed lower levels of serum biomarkers of islet distress (e.g., acute serum insulin) and inflammation (e.g., leptin and α2-macroglobulin). Importantly, low-purity (30:70% endocrine:exocrine) syngeneic rat islet preparations displayed function equivalent to that of pure (>95% endocrine) preparations after intraomental biologic scaffold implantation. Moreover, the biologic scaffold sustained allogeneic islet engraftment in immunosuppressed recipients. Collectively, our feasibility/efficacy data, along with the simplicity of the procedure and the safety of the biologic scaffold components, represented sufficient preclinical testing to proceed to a pilot phase I/II clinical trial.

Decision-making in diabetes mellitus type 1.

Decreased treatment adherence in patients with diabetes mellitus type 1 (type 1 DM) may reflect impairments in decision-making and underlying associated deficits in working memory and executive functioning. Other factors, including comorbid major depression, may also interfere with decision-making. The authors sought to review the clinically relevant characteristics of decision-making in type 1 DM by surveying the literature on decision-making by patients with type 1 DM. Deficiencies in decision-making in patients with type 1 DM or their caregivers contribute to treatment nonadherence and poorer metabolic control. Animal models of type 1 DM reveal deficits in hippocampal-dependent memory tasks, which are reversible with insulin. Neurocognitive studies of patients with type 1 DM reveal lowered performance on ability to apply knowledge to solve problems in a new situation and acquired scholarly knowledge, psychomotor efficiency, cognitive flexibility, visual perception, speed of information-processing, and sustained attention. Other factors that might contribute to poor decision-making in patients with type 1 DM, include "hypoglycemia unawareness" and comorbid major depression (given its increased prevalence in type 1 DM). Future studies utilizing novel treatment strategies to help patients with type 1 DM make better decisions about their disease may improve their glycemic control and quality of life, while minimizing the impact of end-organ disease.

Macroporous three-dimensional PDMS scaffolds for extrahepatic islet transplantation.

Clinical islet transplantation has demonstrated success in treating type 1 diabetes. A current limitation is the intrahepatic portal vein transplant site, which is prone to mechanical stress and inflammation. Transplantation of pancreatic islets into alternative sites is preferable, but challenging, as it may require a three-dimensional vehicle to confer mechanical protection and to confine islets to a well-defined, retrievable space where islet neovascularization can occur. We have fabricated biostable, macroporous scaffolds from poly(dimethylsiloxane) (PDMS) and investigated islet retention and distribution, metabolic function, and glucose-dependent insulin secretion within these scaffolds. Islets from multiple sources, including rodents, nonhuman primates, and humans, were tested in vitro. We observed high islet retention and distribution within PDMS scaffolds, with retention of small islets (< 100 µm) improved through the postloading addition of fibrin gel. Islets loaded within PDMS scaffolds exhibited viability and function comparable to standard culture conditions when incubated under normal oxygen tensions, but displayed improved viability compared to standard two-dimensional culture controls under low oxygen tensions. In vivo efficacy of scaffolds to support islet grafts was evaluated after transplantation in the omental pouch of chemically induced diabetic syngeneic rats, which promptly achieved normoglycemia. Collectively, these results are promising in that they indicate the potential for transplanting islets into a clinically relevant, extrahepatic site that provides spatial distribution of islets as well as intradevice vascularization.

From biomarkers to a clue of biology: a computation-aided perspective of immune gene expression profiles in human type 1 diabetes.

Dysregulated expression of key immune genes may cause breakdown of immunological tolerance and development of autoimmune disorders such as type 1 diabetes (T1D). General immune insufficiencies have also been implicated as a trigger of autoimmunity, due to their potential impact on immune homeostasis. Recent studies have detected evidence of systemic reduction in immune gene expression in long-term diabetic patients but the changes were not present before or at T1D onset. The changes could not be merely correlated with alteration in metabolic parameters. The studies also identified a dynamic expression pattern of several well-known as well as little-studied, immune-related genes during the course of T1D. An intriguing "ratio profile" of immune regulatory genes, such as CTLA4 and members of the S100 family, versus "baseline" immune genes, such as CD3G, prompted us to further examine immune gene expression relationships for a set of molecules representing T cells, B cells, and myeloid cells. No evidence was found to suggest an overall breach of tolerance equilibrium in T1D. Perplexingly, patients with long-term T1D presented a gene expression profile that was surprisingly more coordinated in analyses of "networking" relationship. Computational analyses of the "ratio profiles" or "relationship profiles" of immune gene expression might provide a clue for further studies of immunobiology in human T1D and other autoimmune diseases, as to how the profiles may be related to the pathogenic cause of the disease, to the effect of the diseases on immune homeostasis, or to an immunological process associated with the course of the diseases but is neither a direct cause nor a direct effect of the diseases.

Immune profiling by multiple gene expression analysis in patients at-risk and with type 1 diabetes.

There is a need for biomarkers to monitor the development and progression of type 1 DM. We analyzed mRNA expression levels for granzyme B, perforin, fas ligand, TNF-α, IFN-γ, Foxp3, IL-10, TGF-ß, IL-4, IL-6, IL-17, Activation-induced cytidine deaminase (AID) and Immunoglobulin G gamma chain (IgG) genes in peripheral blood of at-risk, new-onset and long-term type 1 DM , and healthy controls. The majority of the genes were suppressed in long-term type 1 DM compared to controls and new-onset patients. IFN-γ, IL-4 and IL-10 mRNA levels were significantly higher in new-onset compared to at-risk and long-term groups. There was decreased mRNA expression for AID and IgG and up-regulation of IFN-γ with age in controls. Data suggest an overall depressed immunity in long-term type 1 DM. Increased gene expression levels for IFN-γ, IL-4 and IL-10 in new-onset patients from at-risk patients might be used as potential markers for progression of the disease.

Choice of immunosuppression influences cytomegalovirus DNAemia in cynomolgus monkey (Macaca fascicularis) islet allograft recipients.

This retrospective study reviews the results of our experience with the occurrence of CMV DNAemia in islet cell transplanted cynomolgus monkeys subjected to different immunosuppressive protocols, including induction treatment with thymoglobulin (TMG), with a combination of thymoglobulin and fludarabine (FLUD), with cyclophosphamide, or with daclizumab. CMV DNA in the peripheral blood (CMV DNAemia) of 47 monkeys was quantified by real-time PCR on a weekly to biweekly basis. As compared to other immunosuppressive regimens, and in association with greater decreases in WBC, lymphocyte, CD3+CD4+, and CD3+CD8+ lymphocyte counts, frequent CMV DNAemia occurred earlier (within the first month posttransplant), and was of greater severity and duration in recipients of TMG ± FLUD. Treatment of recipients with alternative induction agents that resulted in less dramatic reductions in WBC and lymphocyte counts, however, resulted in occurrence of CMV DNAemia after postoperative day 60. The frequency, average intensity, duration, and area under the curve (AUC) for CMV DNAemia in animals receiving TMG ± FLUD were 75-100%, 4.02 ± 1.75 copies/ng DNA, 23.0 ± 5.3 days, and 367.0 ± 121.1 days × copies/ng DNA, respectively; corresponding values in animals receiving other treatments (0-44%, 0.19 ± 0.10 copies/ng DNA, 0.5 ± 0.3 days, and 75.4 ± 40.2 days × copies/ng DNA, respectively) were significantly different. The value of WBC, T and B cells at the nadir of cell depletion greatly affects the occurrence of CMV DNAemia. No animals developed CMV DNAemia within the next 3 weeks when the lowest value of WBC, lymphocyte, CD3+, CD3+CD4+, CD3+CD8+, or CD20+ cells was above 4500, 1800, 300, 200, 150, or 300 cells/µl, respectively. Oral valganciclovir prophylaxis did not completely prevent the appearance of CMV DNAemia.