Predicting the function of islets after transplantation

C H A P T E R

44 Predicting the function of islets after transplantation Carly M. Darden⁎,†, Anne Elizabeth Farrow‡, Shanthini K. Rajan†, Muhaib Lakhani†, Michael C. Lawrence†, Bashoo Naziruddin‡ *Institute of Biomedical Studies, Baylor University, Waco, TX, United States, †Islet Cell Laboratory, Baylor University Medical Center, Baylor Scott and White Research Institute, Dallas, TX, United States, ‡Annette C. and Harold C. Simmons Transplant Institute, Baylor University Medical Center, Dallas, TX, United States

O U T L I N E Introduction

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Glucose tolerance and stimulation tests Oral glucose tolerance test Glucose clamp techniques Intravenous glucose tolerance test Mixed-meal tolerance test

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Indices for solitary transplantation HYPO score and lability index MAGE index SUITO index Clarke score C-peptide-to-glucose ratio β-Score Transplant estimated function Transplanted functional islet mass

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Biomarkers of graft failure Measurements of alloimmune response Soluble CD30 Cytotoxic lymphocyte genes Microparticles in peripheral blood Autoimmune recurrence

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Proteins C-peptide

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Transplantation, Bioengineering, and Regeneration of the Endocrine Pancreas, Volume 1 https://doi.org/10.1016/B978-0-12-814833-4.00044-7

GAD65 Doublecortin PPP1R1A UCH-L1 HMGB1 CXCL10 CCL2

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Nucleic acids Circulating cell-free DNA Ratio of unmethylated to methylated insulin DNA Micro RNAs Long noncoding RNAs Circular RNAs

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Current noninvasive imaging techniques for pancreatic islet transplantation Bioluminescence imaging Fluorescence imaging Ultrasonography Positron emission tomography Single-photon emission computed tomography Magnetic resonance imaging

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Summary

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References

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© 2020 Elsevier Inc. All rights reserved.

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44.  Predicting the function of islets after transplantation

Introduction Diabetes is the seventh leading cause of death in the United States, but it is considered a “silent killer” due to its contributions to further complications and comorbid conditions. Based on the data from 2015, 30.3 million Americans have been diagnosed with diabetes, and 1.25 million Americans have type 1 diabetes (T1D).1 T1D is the chronic autoimmune destruction of insulin-­ producing beta cells within pancreatic islets. No cure currently exists, so patients rely on insulin injections to regulate blood glucose homeostasis. Unfortunately, these injections do not maintain the natural blood glucose profiles of healthy islets. Exogenous insulin injections lessen hyperglycemic spikes, but hypoglycemic events remain a threat. Islet transplantation was introduced in 1990 and was further improved by the Edmonton protocol. The transplantation of donor islets is an effective beta-cell replacement therapy for T1D. In addition, islet transplantation can eliminate the pain of chronic pancreatitis patients; viable islets are isolated before pancreas removal and transplanted back into the patient via the portal vein, where they engraft in the hepatic sinusoids. There is a significant amount of beta-cell loss postinfusion due to an instant blood-mediated inflammatory reaction, hypoxia, and hyperglycemia.2 The stress from islet isolation and transplant upregulates pro-inflammatory genes associated with hypoxic damage, apoptosis, mitochondrial damage, and endoplasmic reticular stress, which leads to poor long-term function of the graft.3 Loss of the islet graft in patients may occur. This sometimes requires exogenous insulin supplementation or additional islet transplants despite immunosuppressive techniques. To avoid complete loss of islets, it is vital to detect early signs of islet graft stress and loss. Since isolated islets are typically infused into the liver via the portal vein, biopsy examination of the engrafted islets is impractical. Furthermore, biopsy examination may not provide an accurate representation of the graft in its entirety. Therefore, various methods of assessing graft function indirectly have been proposed. This chapter discusses a variety of methods for the clinical assessment of islet graft function: glucose tolerance and stimulation tests, indices to measure the degree and severity of hypoglycemia, alloantibody measurements, proteins, nucleic acids, and noninvasive imaging techniques. Currently, islet transplant centers use a combination of islet autoantibodies, genetic markers, and metabolic markers to measure islet damage and predict the success of the islet survival.4 The ideal biomarker should result in consistent and reproducible results, present a quantifiable potential of T1D development at a defined period, and be of favorable molar abundance and tissue specificity.4,5 The current biomarkers are real-­

time indicators of human beta-cell injury. Yet, clinical T1D is preceded by an asymptomatic phase in which autoantibodies begin to form. Researchers need defined biomarkers of this asymptomatic stage in order to be aware of islet stress preceding damage to prevent clinical hyperglycemia.4

Glucose tolerance and stimulation tests Glucose tolerance tests can provide an array of information on graft function. Each type has its own advantages and disadvantages that determine its utilization in a clinical setting.

Oral glucose tolerance test The oral glucose tolerance test (OGTT) is routinely used to diagnose type 2 diabetes mellitus in a clinical setting.6 A bolus of glucose is given to the patient, and blood glucose measurements are taken at intervals of time to monitor the tolerance and response of the islets to glucose.6 In comparison to the mixed-meal tolerance test (MMTT), discussed below, OGTT is much shorter, requiring only three glucose measurements to be drawn over a span of 2 h. The OGTT and MMTT were designed to mimic physiological conditions and to quantify the interrelationship of glucose levels, insulin secretory response, and insulin clearance.7 Patients with abnormal glucose tolerance (>200 mg/dL) may have microvascular or macrovascular complications.6 The patient must endure moderate discomfort due to fasting and blood draws. Overall, exact assessment of glucose tolerance from these measurements alone is difficult, and the test lacks valuable information regarding insulin sensitivity and resistance.

Glucose clamp techniques Glucose clamp techniques make use of intravenous infusions of glucose to raise blood glucose levels 6.9 mmol/L above basal levels.7 Constant infusion adjustments maintain the basal level and are followed by arginine stimulation to measure total insulin secretory capacity.7 These techniques have two types: hyperglycemic and euglycemic. The hyperglycemic clamp technique allows for the quantification of beta-cell sensitivity to glucose. The euglycemic clamp technique allows for the quantification of tissue sensitivity to insulin. The hyperglycemic clamp technique is considered the gold standard in assessing insulin secretion. It offers advantages over traditional glucose tolerance tests because it quantifies the amount of metabolized glucose; it can quantify beta-cell response and examine the early and late phases of insulin secretion.8 Beta-cell insulin sensitivity is better

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Indices for solitary transplantation

assessed using the euglycemic clamp technique because it is an index of whole-body insulin sensitivity.9 The euglycemic clamp technique has a higher correlation to the rate of whole-body glucose disposal.9 In addition, the risk of potentially fatal hypoglycemic events is decreased due to this test. It is important to note that these techniques are very time consuming, labor intensive, and expensive. Experienced personnel must be able to perform and supervise the tests, and human error can lead to misrepresentation of graft function.

Intravenous glucose tolerance test The intravenous glucose tolerance test (IVGTT) is used for very accurate assessment of the first phase of insulin release.6 Abnormal IVGTT results occur prior to diabetes onset.6 IVGTT is rarely used and is never used to diagnose diabetes.10 Glucose is intravenously injected and blood glucose levels are measured within the first 5 min. Compared to OGTT, the glucose sample—usually a 50% glucose solution—does not have to pass through the digestive system, and this allows greater sensitivity in the assay.11 This test is limited by the use of an intravenous line, which can be difficult to start and maintain throughout the procedure. This assay risks hyperglycemia and further beta-cell damage.12 Many institutions are not equipped with the materials and qualified personal to interpret results.7 Similar to OGTT, patients are required to fast beforehand, and both tests take 2 h to complete. In addition, intravenous injection bypasses the full incretin hormone response stimulated by oral ingestion.

Mixed-meal tolerance test MMTT is used to assess the efficiency of insulin production. It is a sensitive, reproducible test that allows

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for precise C-peptide evaluation. MMTT represents the body response to a normal load of mixed nutrients absorbed from the gut at different rates.7 It gives an accurate representation of overall graft function under physiological conditions and an estimate of beta-cell sensitivity.13,14 However, this test is time consuming, labor intensive, and difficult to standardize.7 Samples taken intravenously are preferred due to multiple measurement time points as well as minimized discomfort. Although the test can be completed by performing multiple blood draws, that can cause the patient more irritation and discomfort.

Indices for solitary transplantation Quantifying hypoglycemia has proven to be difficult. A single, overarching system for measuring the degree and severity of hypoglycemia has yet to be determined. This section reviews the various indices proposed thus far (Fig. 1).13

HYPO score and lability index The HYPO score and lability index are based on daily glucose monitoring, the frequency of hypoglycemic episodes, and individuals’ perception of their overall control of diabetes. The lability index measures the change in glucose overtime, whereas the HYPO score tracks the duration and severity of hypoglycemic unawareness.13 The HYPO score is obtained and determined by the patient, but patient compliance is often less than satisfactory, as the HYPO score requires diligent reporting of these variables over 4 weeks.13 Although the HYPO score can identify those who may benefit from a change in insulin regimen, it fails to provide a longitudinal overview of individuals’ glycemic control.

FIG. 1  Brief characteristics of indices using a fasting blood sample to estimate beta cell function after islet transplantation.

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44.  Predicting the function of islets after transplantation

MAGE index Mean amplitude of glycemic excursion (MAGE) is the mean of blood glucose changes when they have exceeded, either positively or negatively, one standard deviation from the original blood glucose concentration in 24 h.15 Thus, it is a measure of blood glucose stability.16 Higher MAGE values correspond to diabetic patients, but are only significant with large amplitudes of glucose excursion. Control patients have MAGE values of 1.0–3.3 mmol/L, but T1D patients have values up to 15 mmol/L.16 This test can be time consuming and error prone since patients measure their own blood glucose levels.

SUITO index The secretory unit of islet transplant objects (SUITO) index is a calculation that utilizes fasting blood glucose and C-peptide levels to predict graft function based on insulin independence. The SUITO equation is fasting C-peptide (ng/mL) [fasting blood glucose—(63  mg/ dL)] × 1500. A SUITO index of 100 corresponds to insulin independence and reduced hypoglycemic events.12 A SUITO index of 0 indicates complete dependence on exogenous insulin sources. This index also has the advantage of using serum C-peptide instead of insulin levels, which avoids overlapped measurement of endogenous and exogenous insulin amounts if the individual is administering exogenous insulin.

Clarke score The Clarke score is a noninvasive, eight-question self-assessment seeking to characterize hypoglycemic awareness. This method categorizes hypoglycemia aware and unaware patients according to their risk.17 Since the scale is self-reported and not clearly defined, the Clarke score is not an ideal method for determining hypoglycemic awareness, as it fails to eliminate bias. The score report ranges from reduced awareness to complete awareness, but leaves a “nonclassifiable” category, which introduces ambiguous answers.17 The Clarke score lacks true predictive quality due to a basis of retrospective knowledge.17

C-peptide-to-glucose ratio C-peptide is a 31-amino-acid peptide released in equimolar amounts with insulin by pancreatic beta cells. C-peptide is lost in circulation due to hepatic retention and is eventually excreted in the urine.18 Currently, C-peptide is measured before and after islet transplant to determine islet graft survival.19 It has low variability and high reproducibility.19 The stability of C-peptide

makes it an advantageous biomarker. Patients can collect samples from home and send them in for analysis. This eliminates hospital visits and time restraints and increases patient convenience. C-peptide measurements are used in conjunction with other diagnostic tests and indices, because alone they fail to include longitudinal glycemic control (i.e., hemoglobin [Hb]A1c), individual metabolism, or the use of exogenous insulin. For example, the C-peptideto-glucose ratio (CP/G) is a simple calculation that predicts graft function, corrects for glycemic values, and correlates with other indices. The CP/G ratio predicts islet graft function and correlates with patient β-score and glucose levels. However, the patient’s exogenous insulin and HbA1c levels are not incorporated into the equation.19 The C-peptide-to-creatinine ratio corrects urinary C-peptide levels by adjusting against urinary creatinine levels.20 The C-peptide to glucose-creatinine ratio (CP/GCr) adjusts for renal function.19 Faradji et al. found that CP/G is more indicative of graft dysfunction than CP/GCr or C-peptide measurements alone.19 CP/G correlates with the mass of transplanted islets and the clinical β-score.19

β-Score The β-score is a reliable method of assessing overall graft function. This measurement is determined from HbA1c, daily insulin requirements, fasting plasma glucose, and stimulated C-peptide response.21 It is advantageous in that it captures multiple aspects of glycemic control (i.e., beta-cell secretion and insulin resistance/ sensitivity). It is of no surprise, then, that the measurements used to calculate the β-score—fasting glucose, HbA1c, daily insulin, and stimulated C-peptide—require more effort than the questionnaire- and calculation-­ based indices previously mentioned. The patient must provide accurate daily blood sugar and insulin logs and take a glucose tolerance test with a stimulated C-peptide. There is a correlation between β-score and 90-min glucose levels after MMTT.20

Transplant estimated function The transplant estimated function (TEF) estimates the amount of endogenous secreted insulin over a 24-h period, effectively estimating beta-cell function.21 Like the β-score, TEF draws on HbA1c and daily exogenous insulin requirements.21 It is simpler, however, in that it is only looking at beta-cell secretion; therefore, no glucose stimulation test is required. TEF is comparative to other beta-cell function indices, but its overall value as an index is limited by its inability to account for insulin sensitivity and possible resistance.20

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Biomarkers of graft failure

Transplanted functional islet mass

Measurements of alloimmune response

Functional beta-cell mass has a good correlation to glucose-stimulated insulin secretion in response to arginine or another stimulant. The above tests are indirect ways to deduce functional islet mass. Transplant of a greater number of islets results in a smaller loss of islets before engraftment due to inflammatory responses and thrombotic mechanisms.22

Alloimmune response can be measured in numerous ways. This section describes the use of soluble CD30, cytotoxic lymphocyte genes, microparticles in peripheral blood, and autoimmune recurrence. The sections that follow review various proteins and nucleic acids. Table 1 summarizes the information on each biomarker and its test, functions, limitations, and time of expression.

Soluble CD30

Biomarkers of graft failure All protein biomarker assays have the general limitation of short serum and ex vivo half-lives. The proteins are present in low molar concentrations, autoreactive antibodies are available only for specific proteins, and there is reliance on the immunodetection method.23 Real-time indicators of human beta-cell stress and damage need to be identified with proteins that have favorable molarity, are specific for beta cells, and are nonphysiologically secreted.24 While there are multiple damage markers for islets, there is a need for stress markers that identify earlier abnormalities.

Soluble CD30 is currently used as a predictive marker of lung, kidney, and heart transplant rejection. This cell membrane protein of the tumor necrosis factor receptor family is expressed on circulating CD4+ and CD8+ T cells.25 CD30+ T-cell activation causes CD30 cleavage and release from the cell into the serum. In the serum, CD30 is important for alloimmune responses.25 In 2010, Hire et al. investigated 19 allograft islet recipient cases of variable immunosuppressive treatments and found that a reduction in soluble CD30 correlated with full graft functioning.26 Soluble CD30 protein expression preceded loss of islet function by 3–4 months. While soluble CD30

TABLE 1  Protein and nucleic acid biomarkers in pancreas and islet transplantation

Early inflammatory response

Humoral immunity

Cellular immunity

Biomarker

Test

Function

Limitations

Time of expression

CCL2

Multiplex immunoassay

Induces migration and infiltration of macrophages to further destroy islets Negative association with long-term graft survival

Lack of tissue specificity

Immediately after infusion

HMGB1

ELISA

Released by dying islet cells

Lack of tissue specificity

During islet infusion, 24 h after infusion

GAD65, IA-2 and ZnT8

IP assay

Posttransplant changes predictive of the outcome

No standardized method of detection

Present for years

Soluble CD30

ELISA serum samples

Expressed on circulating CD4+ and CD8+ T cells Greater levels indicate poor graft function

Needs more trials

Precedes loss of islet function by 3–4 mo

Cytotoxic lymphocyte genes: granzyme B, perforin, Fas ligand

qPCR

Contributes to allograft rejection

No information on the amount of graft affected or the rate of decline; could be upregulated by other sources of infection

Peak cytotoxic lymphocyte genes occur 2 mo prior to rejection

Microparticles

ELISA

Elevated levels due to graft rejection

Need more evidence and reproducibility

Within the first week after transplant and lost when islet function restored Continued

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44.  Predicting the function of islets after transplantation

TABLE 1  Protein and nucleic acid biomarkers in pancreas and islet transplantation—cont’d

Protein biomarkers

Nucleic acids

Biomarker

Test

Function

Limitations

Time of expression

C-peptide

ELISA

Measure of islet durability Lower values associated with islet dysfunction

Provides no information on percent islet graft rejection

Seen in islet isolation procedure

GAD65

ELISA

Recipient serum levels indicate islet damage and poor outcome

Circulating antibodies present Low molar concentration Assay sensitivity

5–10 h after STZ diabetes induction in mouse

Doublecortin

Affinity capture, liquid chromatography, MS, IP assays

Released by injured beta cells

Assay sensitivity

1–3 h after infusion

Protein phosphatase IP assays 1, regulatory (inhibitor) subunit 1A

Released into plasma after damage to beta cells

Short half-life of 15 min Not consistent or reproducible

2–6 h after STZ injection in mouse

UCH-L1

qPCR

Role in ubiquitinproteasome system of insulin production Released due to islet injury

Short half-life of 20 min in rats Low molar concentration in humans Inconsistent detection

2–4 h peak expression after STZ in rat and human beta cells

CXCL10

ELISA, qPCR

High quantities indicate poor long-term transplant outcome Attracts immune cells that further destroy graft

Provides no information on percent islet graft function or rejection

Released in highest quantities after islet perfusion

Cytotoxic lymphocyte genes: granzyme B, perforin, Fas ligand

qPCR

Contributes to allograft rejection

No information on the amount of graft affected or the rate of decline; could be upregulated by other sources of infection

Peak cytotoxic lymphocyte genes occur 2 mo prior to rejection

Circulating cell-free DNA

qPCR

Elevated expression results in greater insulin requirements

Not tissue specific; expression can also be due to damage in nonislet cells

24-h measurement associated with posttransplant outcome

Long noncoding RNA

qPCR

Regulates gene transcription by recruiting chromatin-modifying enzymes

No clinical trials

After 48 h, HFD in mice

Circular RNA

qPCR

CircHIPK3 and ciRS-7/ CDR1 are important in determining islet transplant outcomes

No clinical trials

45-min treatment with high glucose

Unmethylated/ methylated insulin DNA ratio

Droplet digital PCR

Reflects beta-cell death and new-onset type 1 diabetes

Short half-life

Within 2 h of cell injury or death and after transplant

Micro RNA

qPCR

Participates in gene expression, cell-tocell communication, differentiation, immune activation, mRNA regulation, and protein translation

High noise-to-signal ratio in exosomal miRNA

10–20 min after pancreas digestion

ELISA, enzyme-linked immunosorbent assay; HFD, high-fat diet; HMGB1, high-mobility group box 1; IP, immunoprecipitation; MS, mass spectrometry; qPCR, quantitative polymerase chain reaction; STZ, streptozotocin.

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Proteins

is easily quantified from serum samples, no further research has been published, and the reproducibility of the results is questionable.

Cytotoxic lymphocyte genes Cytotoxic lymphocyte (CL) genes include granzyme B, perforin, and Fas ligand. Elevated expression and involvement of these cytotoxic lymphocyte effector molecules contribute to allograft rejection of the kidney, heart, and islets.27 In 2004, a University of Miami study found that patients who restarted insulin therapy after transplant due to hyperglycemia had significant elevation of CL genes.23,27 Since peak elevations of CL genes occurred weeks to months before graft rejection,28 CL genes may prove to be an early indicator of islet allograft loss. These genes are obtained from blood circulation and amplified by polymerase chain reaction (PCR), thus allowing a less invasive measurement and requiring only small amounts of tissue.27 CL gene elevations do not provide information on the amount of graft affected or the rate of decline. These measurements are not specific to the graft itself and thus could be upregulated by other sources of infection.

Microparticles in peripheral blood Microparticles are plasma membrane fragments of apoptotic cells circulating in the peripheral blood.29 Toti et  al. found that the quantity of microparticles in the blood directly relates to the degree of cell death.29 Acute cellular rejection of a transplanted graft is evident by elevated levels of microparticles. Microparticles have a distinct cell origin and are early markers of rejection.29 There is not enough evidence or reproducibility to make this marker the gold standard of islet graft rejection diagnosis. This is a noninvasive alternative to biopsies. Toti et  al. obtained microparticles from platelet-free plasma and measured content as well as functional capacity by phosphatidylserine content.29

Autoimmune recurrence There is a risk, when transplanting islets into a T1D recipient, of autoimmune recurrence. The donor beta cells express specific antigens that are recognized and targeted by the T and B cells of the recipient.30 These T and B cells are antigen experienced and respond to lower antigen concentrations; they do not require costimulation, and they possess memory surface markers.30 The classic biomarkers of T1D are autoantibodies against (pro)insulin, GAD65, IA-2, and ZnT8 zinc transporter.4,30,31 When two or more of these autoantibodies are present in the patient, this asymptomatic disease stage is a strong indicator of future hyperglycemia.4,32,33

In a recent study, Burke et al. found that 6%–8% of islet allotransplant recipients developed T1D recurrence.34 He also found that 25% of patients experienced autoantibody conversion: recipients who were autoantibody negative pre-transplant became autoantibody positive posttransplant.34 Autoantibody detection is a strong indicator of islet transplant outcome: it is highly reliable and it is consistent between different patient cohorts.4 The autoantibodies are present for years after diabetes onset, and therefore, there is no time limit for measurement.4 Yet, T-cell responses can be detected by different methods, and a standardized method has not yet been developed.30 In addition, autoimmune-positive measurements lack the ability to reveal the pathological rate of progression or tissue of origin.35

Proteins C-peptide Connecting peptide (C-peptide) is a short polypeptide connecting chain within the proinsulin molecule. It is released in equimolar amounts to insulin and, accordingly, C-peptide is recognized as a measurement of islet durability.3 Patients with chronic pancreatitis exhibit a lower stimulated C-peptide and disposition index, a measure of islet dysfunction.3 Lundberg et  al. reported that stimulating C-peptide to more than 4 ng/mL preoperation positively correlates with the number of recovered islet equivalents per kilogram of patient body weight.3 In contrast, C-peptide is not a reliable source for the assessment of islet damage because viable islets secrete it in response to glucose or serum. These components are present in media during islet isolation, and Saravanan et  al. found femtomolar levels of C-peptide abundant in the transport medium, recombination medium, and cap and bagging samples during the islet isolation procedure.36 Decreased C-peptide expression may indicate graft decline, but provides no information on the percentage of islet graft function or rejection (Fig. 2).

GAD65 GAD65 is a protein released by damaged islets and has a relatively short half-life of about 180  min.23,37 Unpublished data in dogs found high levels of GAD65 only 1 h after transplant, which correlated with poor long-term metabolic outcome.24 In  vivo administration of streptozotocin to induce diabetes in mice was followed by peak expression of GAD65 after 5–10 h.38 Recent data have shown GAD65 expressed at much greater concentrations due to human beta-cell death as compared to dogs.38 Thus, GAD65 is a measure of islet damage and can assess early beta-cell loss in T1D.38 GAD65 detection

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44.  Predicting the function of islets after transplantation

is not a reliable detection method for multiple reasons: (1) up to 40% of T1D patients have circulating anti-GAD65 antibodies that interfere with GAD65 measurement; (2) a low molar concentration of GAD65 is released by damaged islets20,24,39; and (3) there are conflicting results about the sensitivity of the assay (Fig. 2).24

damaged beta cells.39 Currently, detection is measured by liquid chromatography and mass spectrometry, but immunoprecipitation assays are limited by their sensitivity (Fig. 2).20,39

Doublecortin

Protein phosphatase 1, regulatory (inhibitor) subunit 1A (PPP1R1A) is a beta-cell enzyme originally identified as an inducible regulator of muscle activity by muscle glycogen content.40 This marker is beta-cell specific and has a high molar content in beta cells, which is released into the plasma after damage to beta cells. Solimena et al. also showed decreased insulin release in a type 2 diabetes setting due to PPP1R1A inhibition.41 PPP1R1A is measured within the nanomolar range by immunoprecipitation assays.39 There is a high molar concentration in beta cells but a half-life of 15 min once released from the beta cells, making it difficult to measure in vivo (Fig. 2). PPP1R1A measurement is relatively new and is not yet consistent and reproducible.

Doublecortin is a microtubule-associated protein that was first studied in a neuroscience context due to its role in neurogenic processes and as a neuronal migration marker.23,39 Doublecortin is released by injured beta cells when discharged from intracellular to extracellular space. Thus, doublecortin has strong beta-cell selectivity and is present at high molar concentrations within the cytoplasm of all beta cells.39 Affinity capture is possible 60 min after infusion and is present in plasma for about 3 h.39 Doublecortin is expressed at levels five times greater than GAD65, but there is still a lack of immune assays that are sensitive enough to detect its release from

PPP1R1A

FIG. 2  Comparison of transplant biomarkers based on indication of damage or inflammation in the islet graft, indication of islet functional change, and sensitivity of the assay. The orange circles indicate biomarkers best for function or damage and inflammation.

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Nucleic acids

UCH-L1 UCH-L1 is a protein with expression restricted to neural and pancreatic beta cells. It serves a role in the ubiquitin-proteasome system of insulin production.24 ­ This intracellular protein is released from the INS-1 cell line and human islets in response to injury. There is double the amount of UCH-L1 mRNA expression in beta cells as there is in brain tissue, and UCH-L1 is detected at three- to sixfold greater amounts than GAD65 in rat and human beta cells (Fig. 2).24 Brackeva et al. showed these results in a hypoxia-induced necrosis model. In mouse studies, UCH-L1 expression in plasma peaks 2–4 h after a dose of streptozotocin, a diabetes-inducing drug.20,24 This protein has a half-life of 20 min in rats.24 The amount of UCHL1 in human beta cells is about 10 femtomol/1000 cells. A potential problem addressed by Brackeva et al. is inconsistent detection in vivo, likely due to rapid clearance by the liver and spleen.24 Rapid clearance leaves no time for UCH-L1 to accumulate, particularly when beta cells are first releasing low-grade amounts due to initial injury.24

plant outcomes (Fig. 2).44 CXCL10 is present in the blood serum within hours of infusion at levels >100 pmol/ mL. This expression attracts aggressive immune cells and further destroys pancreatic beta cells.45 A blockade of CXCL10 by neutralizing antibodies prolongs the survival of heart allografts in fully major histocompatibility complex-mismatched mice and has the potential to increase the survival of islet transplants.45

CCL2 Kanak et al. identified the significantly increased expression of CCL2 chemokine expression after islet isolation.46 This expression induces migration and infiltration of macrophages that result in islet destruction.46 Thus, CCL2 is a predictive inflammatory biomarker of pancreas transplantation outcome. After a simultaneous kidney-pancreas transplant, Monti et al. found that high levels of CCL2 in the donor directly correlate with the amount released in the graft after revascularization and are negatively associated with long-term graft survival (Fig. 2).30 Furthermore, inhibition of CCL2 in an islet graft recipient was necessary to ease additional chemokine production.46

HMGB1 High-mobility group box  1 (HMGB1) is a DNAbinding, nonhistone protein. Its functions include maintenance of chromosomal structure, regulation of transcription, and general support of nucleosomal stability.42,43 HMGB1 is released by dying islet cells and is thus a biomarker of islet damage that correlates to poor islet transplant outcomes in animals and human autotransplantation.30 The limitations of this assay include lack of tissue specificity and current disagreement regarding its correlation with islet damage (Fig. 2).

CXCL10 CXCL10 is the chemokine released in highest quantities by islets immediately following islet perfusion in the transplant procedure, as identified by serum samples from 34 patients undergoing islet transplantion.44 Inflammatory mediators such as physical and chemical stress by isolation and infusion procedures stimulate the release of CXCL10 through an NFAT-MAPK signaling pathway.44 In particular, interferon-γ release at high concentrations during islet infusion contributes to the stimulation of CXCL10 and the loss of up to 50% of the islet graft immediately following transplant. The induction of additional chemokines, including IL-1β, IL-10, MCP-1, MIP-1β, and TNFα, has been identified by Luminex multiplex protein screening of patient blood. The induction of CXCL10 is tissue specific to insulin-producing pancreatic beta cells, and increased expression was recently identified as an indicator of poor long-term islet trans-

Nucleic acids Circulating cell-free DNA Circulating cell-free DNA was first proposed by Lo in 1998 as a biomarker of acute graft damage in multiple organ types.47 In a clinical islet transplant setting study, Gadi et al. found high levels of donor-derived cell-free DNA immediately after infusion, which was a precursor for insulin dependency soon after infusion.48 Cell-free DNA is not tissue specific and expression could be due to damage in cells other than islets.20

Ratio of unmethylated to methylated insulin DNA This ratio is a marker of beta-cell death and new-­onset T1D.35,49 Unmethylated and methylated insulin DNA are detectable in patient serum using droplet digital PCR within 2 h of cell injury or death and after transplant.3,49 Unmethylated and methylated DNA are differentiated by bisulfite treatment of circulatory DNA.49 About 50% of patients have high methylated and unmethylated insulin DNA 90 days posttransplant, which is associated with a higher hyperglycemia risk (Fig.  3).49,50 It was later recognized that unmethylated DNA is specific to beta-cell death, while methylated DNA can arise from any non-beta islet cell or exocrine tissue.49 Further study is necessary to determine the impact of isolation, instant blood-mediated inflammatory reaction, and

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44.  Predicting the function of islets after transplantation

the islet transplant procedure.36 It is released at multiple stages, but is significantly increased after 10–20 min of pancreas digestion. Patients with strong miRNA-375 release also required exogenous insulin after 6 months due to poor posttransplant islet graft function and higher HbA1c levels.36 In addition, miRNA-375 is free of confounders of islet stress on insulin secretion.20 Before Micro RNAs TPIAT, miRNA-375 showed significant correlation with Micro RNAs (miRNAs) are short noncoding RNAs C-peptide levels.52 approximately 18–24 nucleotides in length that particimiRNA-200c is also a significant miRNA enriched in pate in gene expression, cell-to-cell communication, dif- beta cells and is used to anticipate pre- and posttransferentiation, immune activation, mRNA regulation, and plant graft function in TPIAT patients.46,52 The volume protein translation.23 Advantages of miRNA biomarkers of circulating miRNA-200c is significantly positively corinclude tissue and pathology specificity. They are sta- related with 1-year posttransplant insulin requirement, ble for at least 2 months within microvesicles or bound C-peptide levels, and SUITO index.50,52 The maximum to argonaute2 complex.20,23 miRNAs also make good release is measured after 10–20 min of enzymatic digesbiomarker candidates because they are easily obtained tion. It is also significant in the bagging stage; expression from serum and amplified by PCR for quantification.20 is alleviated and then upregulated again at purification miRNA quantification is limited by the tedious sample or recombination.36 processing as well as expensive quantitative PCR maVallabhajosyula et  al. have shown that chine required for analysis.23 Multiple miRNAs show miRNA-3613-5p is highly upregulated in islet exosomes increased expression in chronic pancreatitis patients, in- after islet transplantation. It binds targets that mediate cluding miRNA-200a, −200c, −320, and −375 (Fig. 3).50 insulin receptor isoforms.53 Exosomal miRNAs are a relmiRNA-375 regulates glucose-mediated insulin se- atively new and popular method of biomarker detection. cretion in islets by targeting the myotrophin protein-­ These are stable and contain tissue-specific proteomic producing gene.36,51 Kanak et al. identified miRNA-375 and RNA signature profiles that reflect the tissue condias a biomarker to anticipate pre- and posttransplant tional state.53 Therefore, in a transplant setting, exosomal graft function in total pancreatectomy islet autotrans- miRNAs allow accurate monitoring of the condition of plant (TPIAT) patients. Saravanan et al. documented the the transplanted organ using its own exosomes.53 Plasma expression of miRNA-375 at different time points during exosomes are analyzed on NanoSight nanoparticle dec­ otransplanted acinar tissue on the expression of unmethylated and methylated insulin DNA. This ratio does not currently predict the insulin secretory potential of beta cells after revascularization. This biomarker is also limited by a short half-life.49

Pancreatic b-cell Pancreatic a-cell

Pancreatic b-cell Pancreatic a-cell

Legend Circular RNA Unmethylated DNA Methylated DNA miRNA lncRNA

Pre transplant

Post transplant

FIG. 3  Islet nucleic acids are potential biomarkers due to a significant change in expression after the stress of transplantation. Depletion of circular RNAs circHIPK3 and ciRS-7/CDR1 results in impaired beta cell function. High levels of methylated insulin DNA in alpha cells and unmethylated insulin DNA in beta cells is associated with a higher hyperglycemia risk. miRNA-375 and miRNA-200c anticipate graft function in TPIAT. Βlinc2 and βlinc3 lncRNAs contribute to beta-cell dysfunction after transplant.

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Current noninvasive imaging techniques for pancreatic islet transplantation

tector fluorescence mode for human leukocyte antigen (HLA)-positive exosomes and confirmed by Western blot HLA signal.53 Vallabhajosyula et  al. compared the kinetics of an HLA exosome signal with blood glucose monitoring in the rejection of a xenoislet model.53 They found that fasting blood glucose remained stable during the first 6 days after infusion, but the HLA exosomes decreased significantly by day 1. This is indicative of the acute rejection process and is an advantage in detecting islet graft stress by early minimal levels of T-cell infiltration before the graft is completely destroyed. It was reported that exosomal miRNA is more accurate and time sensitive for detecting acute rejection than other noninvasive biomarkers.53 A high noise-to-signal ratio remains a challenge when extracting whole plasma exosomes. Thus, there is a need to quantitate and characterize tissue-­ specific exosome profiles from bodily fluids.53

Long noncoding RNAs Long noncoding RNAs (lncRNAs) are present in diverse gene-regulatory mechanisms, and dysregulation of these RNAs has been identified in many diseases. lncRNAs exceed 200 nucleotides in length and recruit chromatin-modifying enzymes to regulate gene transcription. lncRNAs are a potential islet transplant outcome biomarker because they contribute to beta-cell development, glucose homeostasis, and beta-cell depletion at the early stages of T1D.54 In a recent study, Motterle et  al. identified, by RNA sequencing, βlinc2 and βlinc3, which contribute to beta-cell dysfunction and type 2 diabetes development.54 These lncRNAs are expressed at levels capable of strong detection and are highly abundant in beta cells (Fig. 3). There are limited isoforms, and they are conserved in humans.54

Circular RNAs Circular RNAs are a class of noncoding RNAs recently discovered to constitute a significant portion of the mammalian transcriptome.55 Circular RNAs are abundant in islets and are conserved between mouse, rat, and human.55 Stoll et  al. identified two circular RNAs important in determining islet transplantation outcomes: circHIPK3 and ciRS-7/CDR1.55 Depletion of these circRNAs in the islet results in impaired beta-cell function, proliferation, and survival (Fig. 3).55 Stoll et al. depleted circHIPK3 in islets, which reduced glucose-stimulated insulin secretion and insulin content.55 Genes specific to insulin secretion and the PI3K-Akt signaling pathway (proliferation and stress response) were significantly reduced in circHIPK-depleted islets of mouse, rat, and human.55 Similarly, the overexpression of ciRS-7/CDR1 increased glucose-stimulated insulin secretion potential

557

and insulin content.55 Under diabetic conditions, these circular RNAs and their control of beta-cell function is depleted.55 To date, no clinical trials have tested the detection of circular RNAs after transplant or their correlation to graft success.

Current noninvasive imaging techniques for pancreatic islet transplantation An ideal noninvasive imaging methodology is one that (1) locates and measures the islet graft in the liver and (2) measures the viability level of islets to estimate their functionality overtime. This information is critical in assessing the effectiveness of pancreatic islet transplantation. Additionally, ideal imaging techniques would allow researchers to gather information over longer time periods depending on graft survival. This section reports on different imaging methods in practice and in the research phase; Table 2 summarizes the information on imaging methods.

Bioluminescence imaging Bioluminescence imaging is a technique for imaging islets using light-generating enzymes. Bioluminescence cells are modified to express the enzyme luciferase (injected just before image acquisition) in transgenic mouse strains. Luciferase-labeled islets emit visible light detected by bioluminescence imaging.56 While this technique is very sensitive, it is dependent on light attenuation through tissue before reaching the detector and the sensitivity of the charge-coupled device that captures light emission.57 Extracellular glucose influences these measurements as well.58 This is a useful research tool in a mouse model but is not suitable for clinical application.59 As few as 100 bioluminescent cells should be detected in subcutaneous sites when implanting, and 106 are required to generate signal through 2 cm of tissue.57 Islets, as the light source, currently need to be transplanted within a few centimeters beneath the surface because increased depth weakens the signal readout.57 Future improvements include luciferase gene introduction via gene transfer methods that are nonimmunogenic, stable, and long lasting.57 For best prediction of graft outcome, bioluminescence imaging should be used in combination with blood glucose measurement and immunohistochemistry.57

Fluorescence imaging Fluorescence imaging is an optical technique in human islet transplantation into mice. It uses near infrared fluorescent dye combined with dextran-coated superparamagnetic nanoparticles.59 The intravenous injection

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44.  Predicting the function of islets after transplantation

TABLE 2  Noninvasive imaging techniques in islet transplantation Techniques

Pros

Cons

Optical imaging

• No ionizing radiation • High sensitivity

• Not clinically applicable • Low spatial resolution

Ultrasonography

• No ionizing radiation • No adverse effects • Low cost

• Cannot visualize single islets • Operator dependency

SPECT

• High resolution • Unlimited depth penetration

• Ionizing radiation, short imaging time • No anatomical information • Low spatial resolution

PET

• High sensitivity

• Islet and recipient irradiation • Low spatial resolution

[18F] FDG

• Imaging just after islet infusion

• Pretransplant labeling • Short half-life

Noninvasive reporter probe imaging • Imaging of islet functionality

• Pretransplant islet transfection • Time-limited technique • Gut uptake

GLP-1 receptor PET imaging

• Imaging of endocrine pancreas • Islet specificity and sensibility in intraportal • Potentially applicable to intraportal liver transplant transplantation unclear

MRI

• No radiation • Good spatial resolution

• Low sensitivity

SPIO-labeled islets

• Feasibility in humans • Quantification • Correlated to the islet mass

• Pretransplantation labeling • Long-term labeling persistence in intraportal transplantation unclear • Correlation with islet function less demonstrated

Gadolinium-labeled islets

• Feasibility in the intraportal model

• Pretransplant labeling • Nonpersistence of the labeling agent after 60 days

Encapsulated islets

• Better sensitivity • No or less immunosuppressive drugs

• No correlation with islet function

Manganese enhanced

• No pretransplant labeling • Correlated to the islet functional mass

• Use for native pancreas imaging • Not accurate for intraportal transplantation

Islet vascularization imaging

• Potentially a good surrogate marker for early graft function

• Requires high-field MRI • Difficult to quantify in the intraportal model

[18F] FDG, 18F-fluorodeoxyglucose; BLI, bioluminescence imaging; GLP-1, glucagon-like peptide-1; MRI, magnetic resonance imaging; PET, positron emission tomography; SPECT, single-photon emission computed tomography; SPIO, superparamagnetic iron oxide.

is restricted to beta cells and excludes exocrine pancreatic cells. These optical imaging modalities cannot visualize deep tissues and are currently restricted to preclinical studies.

islets for 15 min after injection compared to the other organs’ echogenicity.60 Challenges include visualized enhancement of individual islets and depth of penetration. This technique requires further clinical research.

Ultrasonography

Positron emission tomography

Ultrasonography is the most widely used technique. It is readily available, relatively easy to use, and does not involve dangerous ionizing radiation exposure. Recent enhancements allow differentiation between islet cells and infiltrating immune cells by this technique. Recent studies show that enhanced ultrasonography combined with fluorescent acoustic liposome nano or microbubbles could increase the echogenicity of transplanted

Positron emission tomography (PET) is a highly sensitive, noninvasive imaging methodology in biomedical research. It uses gamma rays associated with positron annihilation events to localize the specific site of islet engraftment in the liver. In addition, this technique can quantify viable islets and functionality overtime. Many imaging agents have been developed to improve the sensitivity and specificity of PET in evaluating the effi-

B.  Islet allo-transplantation



References

ciency of islet graft function. The most current updates include carbon-11, flourine-18, copper-64, and gallium68-­labeled radioligands. Earlier studies in islet transplantation used the classic tracer 18F-fluorodeoxyglucose ([18F]FDG), a glucose analogue that can be taken up by beta cells.56 However, because liver uptake is very high, this tracer cannot discriminate islets in the liver if injected intravenously before imaging. Also, due to the short half-life of 18F (110 min), this technique can be used to study islet survival and engraftment only immediately after islet infusion. Recently, [11C]5-hdroxytryptophan has shown a positive correlation to hepatic uptake and function of intraportally transplanted islets, suggesting a promising tool for monitoring visible pancreatic islets.59 To be considered the gold standard of transplant outcome measurement, PET traces must be nontoxic and have high beta-cell specificity.

Single-photon emission computed tomography Single-photon emission computed tomography (SPECT) is a nuclear medical imaging modality using gamma rays. It can evaluate islet function based on the tracer enhancement as a marker of radioactivity, but the spatial resolution is not good. In the recent studies, the SPECT quantification of 111In-Extendin-3 uptake was positively correlated with the insulin-positive areas of islet transplants in the muscle of mice, suggesting potential for in vivo monitoring of ­beta-cell mass in islet grafts.61 Further studies are needed for a nontoxic tracer for SPECT.

Magnetic resonance imaging Magnetic resonance imaging (MRI) is of growing interest in the field of islet transplantation due to its high spatial resolution and penetration depth. To overcome its inability to distinguish transplanted islets from surrounding liver tissue, addition of contrast agents such as superparamagnetic iron oxide nanoparticles is required. These particles are established as MRI contrast agents, and MRI signal by iron oxide labeling is able to image a single cell. Recently, clinical-grade iron nanoparticles like ferucarbotran have been used due to their low toxicity and signal stability.59 Currently, multiwalled carbon nanotubes (MWCNTs) are being studied as labeling compounds for islet transplantation.62 Because of the carbon backbone that protects iron from degradation, MWCNTs may represent a useful tool for human islet MRI labeling and posttransplant monitoring.

Summary Accurate measurement of islet graft function remains a work in progress. In patients who have achieved

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insulin-­independent status following either autologous or allogeneic islet transplants, the function of islet grafts can be measured using methods normally employed for endocrine assessment of the pancreas. However, in patients with partial graft function who use exogenous insulin to maintain normoglycemia, graft assessment using protein markers alone is not adequate. Researchers have identified biomarkers of favorable molarity and specificity as well as their stable peak expression points during or after transplant. Recent progress using nucleic acid markers for beta-cell function has tremendous promise for sensitive and accurate assessment of islet graft function that highly correlates with clinical outcomes.

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