CGM Metrics Identify Dysglycemic States in Participants From the TrialNet Pathway to Prevention Study.

OBJECTIVE: Continuous glucose monitoring (CGM) parameters may identify individuals at risk for progression to overt type 1 diabetes. We aimed to determine whether CGM metrics provide additional insights into progression to clinical stage 3 type 1 diabetes. RESEARCH DESIGN AND METHODS: One hundred five relatives of individuals in type 1 diabetes probands (median age 16.8 years; 89% non-Hispanic White; 43.8% female) from the TrialNet Pathway to Prevention study underwent 7-day CGM assessments and oral glucose tolerance tests (OGTTs) at 6-month intervals. The baseline data are reported here. Three groups were evaluated: individuals with 1) stage 2 type 1 diabetes (n = 42) with two or more diabetes-related autoantibodies and abnormal OGTT; 2) stage 1 type 1 diabetes (n = 53) with two or more diabetes-related autoantibodies and normal OGTT; and 3) negative test for all diabetes-related autoantibodies and normal OGTT (n = 10). RESULTS: Multiple CGM metrics were associated with progression to stage 3 type 1 diabetes. Specifically, spending ≥5% time with glucose levels ≥140 mg/dL (P = 0.01), ≥8% time with glucose levels ≥140 mg/dL (P = 0.02), ≥5% time with glucose levels ≥160 mg/dL (P = 0.0001), and ≥8% time with glucose levels ≥160 mg/dL (P = 0.02) were all associated with progression to stage 3 disease. Stage 2 participants and those who progressed to stage 3 also exhibited higher mean daytime glucose values; spent more time with glucose values over 120, 140, and 160 mg/dL; and had greater variability. CONCLUSIONS: CGM could aid in the identification of individuals, including those with a normal OGTT, who are likely to rapidly progress to stage 3 type 1 diabetes.

Affinity purification of serum-derived anti-IA-2 autoantibodies in type 1 diabetes using a novel MBP-IA-2 fusion protein.

Autoantibodies targeting epitopes contained within the intracellular domain (IC) of the protein phosphatase-like islet antigen 2 (IA-2) are a common marker of autoimmune type 1 diabetes (T1D), however the isolation of genuine, serum derived anti-IA-2 autoantibodies has proven challenging due to a lack of suitable bioassays. In the current study, an ELISA format was developed for affinity purification of human anti-IA-2ic autoantibodies utilizing a fusion protein (FP) incorporating maltose binding protein and the full-length IA-2IC domain. Using a T1D patient cohort validated for anti-IA-2ic autoantibodies by commercial ELISA, we demonstrate the MBP-IA-2ic FP ELISA detects serum anti-IA-2IC autoantibodies from 3 of 9 IA-2 positive patients. Further to this, a multi-plate MBP-IA-2ic FP ELISA protocol specifically affinity purifies IgG enriched for anti-IA-2ic autoantibodies. Interestingly, serum derived autoantibodies immobilised on the MBP-IA-2ic FP ELISA demonstrate increased Kappa light chain usage when compared to the respective total IgG derived from donor patients, suggesting a clonally restricted repertoire of anti-IA-2ic autoantigen specific B plasma cells is responsible for autoantibodies detect by the MBP-IA-2ic FP ELISA. This study is the first to demonstrate the generation of specific, genuine human derived anti-IA-2ic autoantibodies, thereby facilitating further investigation into the origin and functional significance of IA-2 autoantibodies in T1D.

Therapeutic Targeting of Macrophage Plasticity Remodels the Tumor-Immune Microenvironment.

SIGNIFICANCE: Comprehensive single-cell proteomics analyses of lung adenocarcinoma progression reveal the role of tumor-associated macrophages in resistance to PD-1 blockade therapy. See related commentary by Lee et al., p. 2515.

Incidence of an Insulin-Requiring Hyperglycemic Syndrome in SARS-CoV-2-Infected Young Individuals: Is It Type 1 Diabetes?

Pancreatic ACE2 receptor expression, together with increased prevalence of insulin-requiring hyperglycemia in patients with coronavirus disease 2019 (COVID-19), suggested that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pancreatic infection might trigger a ß-cell-selective inflammation precipitating autoimmune type 1 diabetes (T1D). We examined T1D incidence in patients with COVID-19 inside a large, global population using a "big data" approach. The incidence in 0-30-year-old patients with confirmed COVID-19 over an ∼15-month period from the beginning of the COVID-19 pandemic was compared with an age-matched population without COVID-19 inside the TriNetX COVID-19 Research Network (>80 million deidentified patient electronic medical records globally). The cohorts were used to generate outcomes of T1D postindex. In those up to 18 years of age, the incidence of insulin-requiring diabetes that could represent T1D in patients with already diagnosed, confirmed COVID-19 was statistically indistinguishable from the control population without COVID-19. In contrast, in those aged 19-30 years, the incidence was statistically greater. These data suggest that the incidence of T1D among patients with COVID-19 <30 years of age, at least up to this time since the beginning of the pandemic, is not greater when compared with an age-, sex-, and BMI-matched population without COVID-19. Nevertheless, we caution that patients with COVID-19 could be asymptomatic of a diabetic/prediabetic state and therefore would not be expected to come to medical attention, remaining undiagnosed. Hence, it is still possible that asymptomatic virus-infected individuals could acquire ß-cell autoimmunity, eventually progressing to dysglycemia and clinical T1D at higher rates.

Coding variants identified in patients with diabetes alter PICK1 BAR domain function in insulin granule biogenesis.

Bin/amphiphysin/Rvs (BAR) domains are positively charged crescent-shaped modules that mediate curvature of negatively charged lipid membranes during remodeling processes. The BAR domain proteins PICK1, ICA69, and the arfaptins have recently been demonstrated to coordinate the budding and formation of immature secretory granules (ISGs) at the trans-Golgi network. Here, we identify 4 coding variants in the PICK1 gene from a whole-exome screening of Danish patients with diabetes that each involve a change in positively charged residues in the PICK1 BAR domain. All 4 coding variants failed to rescue insulin content in INS-1E cells upon knock down of endogenous PICK1. Moreover, 2 variants showed dominant-negative properties. In vitro assays addressing BAR domain function suggested that the coding variants compromised BAR domain function but increased the capacity to cause fission of liposomes. Live confocal microscopy and super-resolution microscopy further revealed that PICK1 resides transiently on ISGs before egress via vesicular budding events. Interestingly, this egress of PICK1 was accelerated in the coding variants. We propose that PICK1 assists in or complements the removal of excess membrane and generic membrane trafficking proteins, and possibly also insulin, from ISGs during the maturation process; and that the coding variants may cause premature budding, possibly explaining their dominant-negative function.

Inhibition of Cyclin Dependent Kinase 4/6 Overcomes Primary Resistance to Programmed Cell Death 1 Blockade in Malignant Mesothelioma.

BACKGROUND: Despite the profound number of malignant pleural mesothelioma (MPM) patients now treated with programmed cell death 1 (PD-1) blockade, insight into the underpinnings of rational therapeutic strategies to treat resistance to checkpoint immunotherapy remains unrealized. Our objective was to develop a novel therapeutic approach to overcome primary resistance to PD-1 blockade in MPM. METHODS: We generated a transcriptome signature of resistance to PD-1 blockade in MPM patients treated with nivolumab (4 responders and 4 nonresponders). We used The Cancer Genome Atlas MPM cohort (n = 73) to determine what genomic alterations were associated with the resistance signature. We tested whether regulation of identified molecules could overcome resistance to PD-1 blockade in an immunocompetent mouse malignant mesothelioma model. RESULTS: Immunogenomic analysis by applying our anti-PD-1 resistance signature to The Cancer Genome Atlas cohort revealed that deletion of cyclin dependent kinase inhibitor 2A (CDKN2A) was highly associated with primary resistance to PD-1 blockade. Under the hypothesis that resistance to PD-1 blockade can be overcome by cyclin dependent kinase 4/6 (CDK4/6) inhibition, we tested whether CDK4/6 inhibitors could overcome resistance to PD-1 blockade in subcutaneous tumors derived from Cdkn2a-/- AB1 malignant mesothelioma cells, which were resistant to PD-1 blockade. The combination of daily oral administration of CDK4/6 inhibitors (abemaciclib or palbociclib) and intraperitoneal anti-PD-1 treatment markedly suppressed tumor growth compared with anti-PD-1 or CDK4/6 inhibitor alone. CONCLUSIONS: We identified a therapeutic target, CDK4/6, to overcome primary resistance to PD-1 blockade through comprehensive immunogenomic approaches. These data provide a rationale for undertaking clinical trials of CDK4/6 inhibitors in more than 40% of patients with MPM who demonstrate loss of CDKN2A.

Autoimmune Inflammation and Insulin Resistance: Hallmarks So Far and Yet So Close to Explain Diabetes Endotypes.

PURPOSE OF REVIEW: Diabetes mellitus can be categorized into two major variants, type 1 and type 2. A number of traits such as clinical phenotype, age at disease onset, genetic background, and underlying pathogenesis distinguish the two forms. RECENT FINDINGS: Recent evidence indicates that type 1 diabetes can be accompanied by insulin resistance and type 2 diabetes exhibits self-reactivity. These two previously unknown conditions can influence the progression and outcome of the disease. Unlike most conventional considerations, diabetes appears to consist of a spectrum of intermediate phenotypes that includes monogenic and polygenic loci linked to inflammatory processes including autoimmunity, beta cell impairment, and insulin resistance. Here we discuss why a shift of the classical bi-modal view of diabetes (autoimmune vs. non-autoimmune) is necessary in favor of a model of an immunological continuum of endotypes lying between the two extreme "insulin-resistant" and "autoimmune beta cell targeting," shaped by environmental and genetic factors which contribute to determine specific immune-conditioned outcomes.

Islet autoantibody types mark differential clinical characteristics at diagnosis of pediatric type 1 diabetes.

BACKGROUND: We aimed to study whether islet autoantibody type marks differential characteristics at the time of type 1 diabetes (T1D) diagnosis. METHODS: We studied 711 children with newly diagnosed autoimmune T1D. We compared demographic (sex, age, race/ethnicity), clinical (pubertal development, BMI percentile, diabetic ketoacidosis [DKA]) and laboratory (glucose, hemoglobin A1c [HbA1c], C-peptide, tissue transglutaminase antibodies [tTGA], thyroglobulin antibodies, and thyroid peroxidase antibodies [TPOA]) characteristics by presence/absence of autoantibodies to insulin (IAA), GAD65 (GADA), or IA-2/ICA512 (IA-2A). Islet autoantibody titers were evaluated among the children positive for the relevant autoantibody type. We used multivariable analysis to adjust for potential confounders. RESULTS: IAA+ was statistically associated with younger age (p < 0.0001) and lower HbA1c (p = 0.049) while Tanner stage, GADA status and number of positive islet autoantibodies were not significant in the multivariable model. GADA+ was associated with female sex (OR = 4.0, p = 0.002) and negatively with elevated tTGA titers (>50 U/mL) (OR = 0.21, p = 0.026) but not with age, IAA status, IA-2A status, islet autoantibody number, or thyroid autoimmunity. None of the associations with IA-2A positivity was statistically significant in the multivariable analysis. In multivariable models, IAA titer was significantly associated with younger age (p = 0.006), DKA (p = 0.017) and higher tTGA levels (p = 0.002); GADA titer with female sex (p = 0.028), racial minority (p = 0.046) and TPOA positivity (p = 0.021); and IA-2A titer with older age (p = 0.001) and not being African American (p = 0.024). CONCLUSIONS: Islet autoantibody type is associated with differential characteristics at diagnosis of pediatric T1D. Longitudinal and mechanistic studies are needed to evaluate T1D endotypes by autoantibody type.

Modulation of Leukocytes of the Innate Arm of the Immune System as a Potential Approach to Prevent the Onset and Progression of Type 1 Diabetes.

Type 1 diabetes (T1D) is characterized by insulin deficiency resulting from the selective destruction of pancreatic ß-cells by self-reactive T cells. Recent evidence demonstrates that innate immune responses substantially contribute to the pathogenesis of T1D, as they represent a first line of response to danger/damage signals. Here we discuss evidence on how, in a relapsing-remitting pattern, pancreas remodeling, diet, microbiota, gut permeability, and viral/bacterial infections induce the accumulation of leukocytes of the innate arm of the immune system throughout the pancreas. The subsequent acquisition and presentation of endocrine and exocrine antigens to the adaptive arm of the immune system results in a chronic progression of pancreatic damage. This process provides for the generation of self-reactive T-cell responses; however, the relative weight that genetic and environmental factors have on the etiopathogenesis of T1D is endotype imprinted and patient specific. With this Perspectives in Diabetes, our goal is to encourage the scientific community to rethink mechanisms underlying T1D pathogenesis and to consider therapeutic approaches that focus on these processes in intervention trials within new-onset disease as well as in efforts seeking the disorder's prevention in individuals at high risk.

Checking the Checkpoint Inhibitors: A Case of Autoimmune Diabetes After PD-1 Inhibition in a Patient with HIV.

Immune checkpoint inhibitor-associated diabetes mellitus (ICI-DM) is a known immune-related adverse event (irAE) following treatment with programmed cell death protein 1 (PD-1), with a reported 0.9% incidence. We hereby present the first case, to our knowledge, of ICI-DM following ICI use in a human immunodeficiency virus (HIV) patient. In this case, a 48-year-old man with HIV stable on highly active antiretroviral therapy (HAART) was diagnosed with Hodgkin lymphoma and initiated treatment with the PD-1 inhibitor nivolumab. His lymphoma achieved complete response after 5 months. However, at month 8, he reported sudden polydipsia and polyuria. Labs revealed a glucose level of 764 mg/dL and glycated hemoglobin A1c (HbA1c) of 7.1%. Low C-peptide and elevated glutamic acid decarboxylase 65 (GAD65) antibody levels confirmed autoimmune DM, and he was started on insulin. Major histocompatibility complex class II genetic analysis revealed homozygous HLA DRB1*03-DQA1*0501-DQB1*02 (DR3-DQ2), which is a known primary driver of genetic susceptibility to type 1 DM. Autoimmune DM has been reported as an ICI-associated irAE. However, patients with immunocompromising conditions such as HIV are usually excluded from ICI trials. Therefore, little is known about such irAEs in this population. In this case, risk of ICI-DM as an irAE was likely increased by several factors including family history, a high-risk genetic profile, islet-related immunologic abnormalities, active lymphoma, and HIV infection with a possible immune reconstitution event. Clinicians should maintain a high index of suspicion for development of irAEs associated with ICI, particularly as use of these therapies broadens. Thorough investigation for presence of higher-risk features should be conducted and may warrant inclusion of pre-therapy genetic and/or autoantibody screening.

Management of Latent Autoimmune Diabetes in Adults: A Consensus Statement From an International Expert Panel.

A substantial proportion of patients with adult-onset diabetes share features of both type 1 diabetes (T1D) and type 2 diabetes (T2D). These individuals, at diagnosis, clinically resemble T2D patients by not requiring insulin treatment, yet they have immunogenetic markers associated with T1D. Such a slowly evolving form of autoimmune diabetes, described as latent autoimmune diabetes of adults (LADA), accounts for 2-12% of all patients with adult-onset diabetes, though they show considerable variability according to their demographics and mode of ascertainment. While therapeutic strategies aim for metabolic control and preservation of residual insulin secretory capacity, endotype heterogeneity within LADA implies a personalized approach to treatment. Faced with a paucity of large-scale clinical trials in LADA, an expert panel reviewed data and delineated one therapeutic approach. Building on the 2020 American Diabetes Association (ADA)/European Association for the Study of Diabetes (EASD) consensus for T2D and heterogeneity within autoimmune diabetes, we propose "deviations" for LADA from those guidelines. Within LADA, C-peptide values, proxy for ß-cell function, drive therapeutic decisions. Three broad categories of random C-peptide levels were introduced by the panel: 1) C-peptide levels <0.3 nmol/L: a multiple-insulin regimen recommended as for T1D; 2) C-peptide values ≥0.3 and ≤0.7 nmol/L: defined by the panel as a "gray area" in which a modified ADA/EASD algorithm for T2D is recommended; consider insulin in combination with other therapies to modulate ß-cell failure and limit diabetic complications; 3) C-peptide values >0.7 nmol/L: suggests a modified ADA/EASD algorithm as for T2D but allowing for the potentially progressive nature of LADA by monitoring C-peptide to adjust treatment. The panel concluded by advising general screening for LADA in newly diagnosed non-insulin-requiring diabetes and, importantly, that large randomized clinical trials are warranted.

Commercially Available Insulin Products Demonstrate Stability Throughout the Cold Supply Chain Across the U.S.

OBJECTIVE: A recent publication questioned the integrity of insulin purchased from U.S. retail pharmacies. We sought to independently validate the method used, isotope dilution solid-phase extraction (SPE) liquid chromatography mass spectrometry (LC-MS), and expand analysis to two U.S. Pharmacopeia (USP) methods (high-performance LC with ultraviolet detection and LC-MS). RESEARCH DESIGN AND METHODS: Each method was used to evaluate nine insulin formulations, purchased at four pharmacies, within five geographic locations in the U.S. RESULTS: All human and analog insulins measured by the USP methods (n = 174) contained the expected quantity of active insulin (100 ± 5 units/mL). When using isotope dilution SPE-LC-MS, units-per-milliliter values were well below product labeling due to unequal recovery of the internal standard compared with target insulin. CONCLUSIONS: Insulin purchased from U.S. pharmacies is consistent with product labeling.

Time to dissect the autoimmune etiology of cancer antibody immunotherapy.

Immunotherapy has transformed the treatment landscape for a wide range of human cancers. Immune checkpoint inhibitors (ICIs), monoclonal antibodies that block the immune-regulatory "checkpoint" receptors CTLA-4, PD-1, or its ligand PD-L1, can produce durable responses in some patients. However, coupled with their success, these treatments commonly evoke a wide range of immune-related adverse events (irAEs) that can affect any organ system and can be treatment-limiting and life-threatening, such as diabetic ketoacidosis, which appears to be more frequent than initially described. The majority of irAEs from checkpoint blockade involve either barrier tissues (e.g., gastrointestinal mucosa or skin) or endocrine organs, although any organ system can be affected. Often, irAEs resemble spontaneous autoimmune diseases, such as inflammatory bowel disease, autoimmune thyroid disease, type 1 diabetes mellitus (T1D), and autoimmune pancreatitis. Yet whether similar molecular or pathologic mechanisms underlie these apparent autoimmune adverse events and classical autoimmune diseases is presently unknown. Interestingly, evidence links HLA alleles associated with high risk for autoimmune disease with ICI-induced T1D and colitis. Understanding the genetic risks and immunologic mechanisms driving ICI-mediated inflammatory toxicities may not only identify therapeutic targets useful for managing irAEs, but may also provide new insights into the pathoetiology and treatment of autoimmune diseases.

Autoantibodies Directed Toward a Novel IA-2 Variant Protein Enhance Prediction of Type 1 Diabetes.

We identified autoantibodies (AAb) reacting with a variant IA-2 molecule (IA-2var) that has three amino acid substitutions (Cys27, Gly608, and Pro671) within the full-length molecule. We examined IA-2var AAb in first-degree relatives of type 1 diabetes (T1D) probands from the TrialNet Pathway to Prevention Study. The presence of IA-2var-specific AAb in relatives was associated with accelerated progression to T1D in those positive for AAb to GAD65 and/or insulin but negative in the standard test for IA-2 AAb. Furthermore, relatives with single islet AAb (by traditional assays) and carrying both IA-2var AAb and the high-risk HLA-DRB1*04-DQB1*03:02 haplotype progress rapidly to onset of T1D. Molecular modeling of IA-2var predicts that the genomic variation that alters the three amino acids induces changes in the three-dimensional structure of the molecule, which may lead to epitope unmasking in the IA-2 extracellular domain. Our observations suggest that the presence of AAb to IA-2var would identify high-risk subjects who would benefit from participation in prevention trials who have one islet antibody by traditional testing and otherwise would be misclassified as "low risk" relatives.

Chimeric antigen receptor (CAR) T cells targeting a pathogenic MHC class II:peptide complex modulate the progression of autoimmune diabetes.

A primary initiating epitope in the NOD mouse model of Type 1 Diabetes (T1D) lies between residues 9 and 23 of the insulin B chain. The B:9-23 peptide can bind to the NOD MHC class II molecule (I-Ag7) in multiple registers, but only one, (register 3, R3), creates complexes able to stimulate the majority of pathogenic B:9-23-specific CD4+ T cells. Previously we generated a monoclonal antibody (mAb287) that targets this critical I-Ag7-B:9-23(R3) complex. When given weekly to pre-diabetic mice at either early or late stages of disease, mAb287 was able to delay or prevent T1D in the treated animals. Although the precise mechanism of action of mAb287 remains unclear, we hypothesized that it may involve deletion of antigen presenting cells (APCs) bearing the pathogenic IAg7-B:9-23(R3) complexes, and that this process might be rendered more efficient by re-directing cytotoxic T cells using a mAb287 chimeric antigen receptor (287-CAR). As anticipated, 287-CAR T cells secreted IFN-γ in response to stimulation by I-Ag7-B:9-23(R3) complexes expressed on artificial APCs, but not I-Ag7 loaded with other peptides, and killed the presenting cells in vitro. A single infusion of 287-CAR CD8+ T cells to young (5 week old) NOD mice significantly delayed the onset of overt hyperglycemia compared to untreated animals (p = 0.022). None of the 287-CAR CD8+ T cell treated mice developed diabetes before 18 weeks of age, while 29% of control-CAR T cell treated mice (p = 0.044) and 52% of the un-treated mice (p = 0.0001) had developed T1D by this time. However, the protection provided by 287-CAR CD8+ T cells declined with time, and no significant difference in overall incidence by 30 weeks between the 3 groups was observed. Mechanistic studies indicated that the adoptively transferred 287-CAR T cells selectively homed to pancreatic lymph nodes, and in some animals could persist for at least 1-2 weeks post-transfer, but were essentially undetectable 10-15 weeks later. Our study demonstrates that CAR T cells specific for a pathogenic MHC class II:peptide complex can be effective in vivo, but that a single infusion of the current iteration can only delay, but not prevent, the development of T1D. Future studies should therefore be directed towards optimizing strategies designed to improve the longevity of the transferred cells.

CD19+IgM+ cells demonstrate enhanced therapeutic efficacy in type 1 diabetes mellitus.

We describe a protective effect on autoimmune diabetes and reduced destructive insulitis in NOD.scid recipients following splenocyte injections from diabetic NOD donors and sorted CD19+ cells compared with NOD.scid recipients receiving splenocytes alone. This protective effect was age specific (only CD19+ cells from young NOD donors exerted this effect; P < 0.001). We found that the CD19+IgM+ cell is the primary subpopulation of B cells that delayed transfer of diabetes mediated by diabetogenic T cells from NOD mice (P = 0.002). Removal of IgM+ cells from the CD19+ pool did not result in protection. Notably, protection conferred by CD19+IgM+ cotransfers were not dependent on the presence of Tregs, as their depletion did not affect their ability to delay onset of diabetes. Blockade of IL-10 with neutralizing antibodies at the time of CD19+ cell cotransfers also abrogated the therapeutic effect, suggesting that IL-10 secretion was an important component of protection. These results were strengthened by ex vivo incubation of CD19+ cells with IL-5, resulting in enhanced proliferation and IL-10 production and equivalently delayed diabetes progression (P = 0.0005). The potential to expand CD19+IgM+ cells, especially in response to IL-5 stimulation or by pharmacologic agents, may be a new therapeutic option for type 1 diabetes.

Autoantibodies to the IA-2 Extracellular Domain Refine the Definition of "A+" Subtypes of Ketosis-Prone Diabetes.

OBJECTIVE: Autoantibodies directed against tyrosine phosphatase IA-2 antibody (IA-2 Ab) are diagnostic for autoimmune type 1 diabetes. Conventional assays target the intracellular domain of IA-2. Among patients with ketosis-prone diabetes (KPD), characterized by presentation with diabetic ketoacidosis (DKA), >60% of adults lack three classic islet autoantibodies-IA-2, GAD65, and ZnT8 Abs-associated with type 1 diabetes. We aimed to determine whether apparently autoantibody-negative ("A-") KPD patients possess occult IA-2 Ab directed against full-length IA-2 (IA-2FL) or its extracellular domain (IA-2EC). RESEARCH DESIGN AND METHODS: We developed an assay that targets IA-2FL and IA-2EC and used it to analyze 288 subjects with A- KPD. RESULTS: Ten A- KPD patients were positive for IA-2EC Ab (3.5%), and three were also positive for IA-2FL Ab (1.0%), similar to frequencies in type 1 and type 2 diabetes. CONCLUSIONS: Measurement of IA-2FL Ab and IA-2EC Ab improves the accuracy of the Aß classification of KPD patients.