Circulating Unmethylated Insulin DNA As a Biomarker of Human Beta Cell Death: A Multi-laboratory Assay Comparison.

CONTEXT: There is an unmet need for biomarkers of pancreatic beta-cell death to improve early diagnosis of type 1 diabetes, enroll subjects into clinical trials, and assess treatment response. To address this need, several groups developed assays measuring insulin deoxyribonucleic acid (DNA) with unmethylated CpG sites in cell-free DNA. Unmethylated insulin DNA should be derived predominantly from beta-cells and indicate ongoing beta-cell death. OBJECTIVE: To assess the performance of three unmethylated insulin DNA assays. DESIGN AND PARTICIPANTS: Plasma or serum samples from 13 subjects undergoing total pancreatectomy and islet autotransplantation were coded and provided to investigators to measure unmethylated insulin DNA. Samples included a negative control taken post-pancreatectomy but pretransplant, and a positive control taken immediately following islet infusion. We assessed technical reproducibility, linearity, and persistence of detection of unmethylated insulin DNA for each assay. RESULTS: All assays discriminated between the negative sample and samples taken directly from the islet transplant bag; 2 of 3 discriminated negative samples from those taken immediately after islet infusion. When high levels of unmethylated insulin DNA were present, technical reproducibility was generally good for all assays. CONCLUSIONS: The measurement of beta cell cell-free DNA, including insulin, is a promising approach, warranting further testing and development in those with or at-risk for type 1 diabetes, as well as in other settings where understanding the frequency or kinetics of beta cell death could be useful.

Islet Harvest in Carbon Monoxide-Saturated Medium for Chronic Pancreatitis Patients Undergoing Islet Autotransplantation.

Stresses encountered during human islet isolation lead to unavoidable ß-cell death after transplantation. This reduces the chance of insulin independence in chronic pancreatitis patients undergoing total pancreatectomy and islet autotransplantation. We tested whether harvesting islets in carbon monoxide-saturated solutions is safe and can enhance islet survival and insulin independence after total pancreatectomy and islet autotransplantation. Chronic pancreatitis patients who consented to the study were randomized into carbon monoxide (islets harvested in a carbon monoxide-saturated medium) or control (islets harvested in a normal medium) groups. Islet yield, viability, oxygen consumption rate, ß-cell death (measured by unmethylated insulin DNA), and serum cytokine levels were measured during the peri-transplantation period. Adverse events, metabolic phenotypes, and islet function were measured prior and at 6 months post-transplantation. No adverse events directly related to the infusion of carbon monoxide islets were observed. Carbon monoxide islets showed significantly higher viability before transplantation. Subjects receiving carbon monoxide islets had less ß-cell death, decreased CCL23, and increased CXCL12 levels at 1 or 3 days post transplantation compared with controls. Three in 10 (30%) of the carbon monoxide subjects and none of the control subjects were insulin independent. This pilot trial showed for the first time that harvesting human islets in carbon monoxide-saturated solutions is safe for total pancreatectomy and islet autotransplantation patients.

ß cell responses to inflammation.

BACKGROUND: The extended and clinically silent progression of Type 1 diabetes (T1D) creates a challenge for clinical interventions and for understanding the mechanisms that underlie its pathogenesis. Over the course of the development of Type 1 diabetes, studies in animal models and of human tissues have identified adaptive changes in ß cells that may affect their immunogenicity and susceptibility to killing. Loss of ß cells has traditionally been identified by impairment in function but environmental factors may affect these measurements. SCOPE OF REVIEW: In this review we will highlight features of ß cell responses to cell death, particularly in the setting of inflammation, and focus on methods of detecting ß cell death in vivo. MAJOR CONCLUSIONS: We developed an assay to measure ß cell death in vivo by detecting cell free DNA with epigenetic modifications of the INS gene that are found in ß cells. This assay has robust technical performance and identifies killing in individuals at very high risk for disease, but its ability to identify ß cell killing in at-risk relatives is limited by the short half-life of the cell free DNA and the need for repeated sampling over an extended course. We present results from the Diabetes Prevention Trial-1 using this assay. In addition, recent studies have identified cellular adaptations in some ß cells that may avoid killing but impair metabolic function. Cells with these characteristics may aggravate the autoimmune response but also may represent a potentially recoverable source of functional ß cells.

Unmethylated Insulin as an Adjunctive Marker of Beta Cell Death and Progression to Type 1 Diabetes in Participants at Risk for Diabetes.

Islet autoantibody (iAb)-positive individuals have a high risk of progression to type 1 diabetes (T1D), although the rate of progression is highly variable and factors involved in the rate of progression are largely unknown. The ratio of unmethylated/methylated insulin DNA levels (unmethylated INS ratio) has been shown to be higher in participants at high risk of T1D compared to healthy controls. We aimed to evaluate whether an unmethylated INS ratio may be a useful biomarker of beta cell death and rate of progression to T1D. In TrialNet participants who were followed in the Pathway to Prevention Study and progressed to diabetes (n = 57, median age of onset 15.3 years), we measured unmethylated INS ratio and autoantibodies by electrochemiluminescence (ECL) assays (ECL-IAA, ECL-GADA, and ECL-IA2) and radioimmunoassays (RIA) (mIAA, GADA, IA2A, and ZnT8A) longitudinally for 24 months prior to diagnosis. Linear models were used to test the association between unmethylated INS ratio and the age at T1D diagnosis and unmethylated INS ratio and iAb over time. Close to diabetes onset, the unmethylated INS ratio was associated with mIAA (p = 0.003), ECL-IAA (p = 0.002), and IA2A (p = 0.01) levels, but not with GADA, ECL-GADA, ECL-IA2, or ZnT8A levels. No significant associations were found at baseline (24 months prior to T1D diagnosis). Only mIAA levels were significantly associated with an unmethylated INS ratio over time, with a 0.24 change in the ratio for each 0.1 change in mIAA z-score (p = 0.02). Adjusting for a baseline unmethylated INS ratio, an increased rate of change in unmethylated INS ratio from baseline to diabetes onset was associated with a five-year decrease in age at T1D diagnosis (p = 0.04).

Genome-wide diversity and gene expression profiling of Babesia microti isolates identify polymorphic genes that mediate host-pathogen interactions.

Babesia microti, a tick-transmitted, intraerythrocytic protozoan parasite circulating mainly among small mammals, is the primary cause of human babesiosis. While most cases are transmitted by Ixodes ticks, the disease may also be transmitted through blood transfusion and perinatally. A comprehensive analysis of genome composition, genetic diversity, and gene expression profiling of seven B. microti isolates revealed that genetic variation in isolates from the Northeast United States is almost exclusively associated with genes encoding the surface proteome and secretome of the parasite. Furthermore, we found that polymorphism is restricted to a small number of genes, which are highly expressed during infection. In order to identify pathogen-encoded factors involved in host-parasite interactions, we screened a proteome array comprised of 174 B. microti proteins, including several predicted members of the parasite secretome. Using this immuno-proteomic approach we identified several novel antigens that trigger strong host immune responses during the onset of infection. The genomic and immunological data presented herein provide the first insights into the determinants of B. microti interaction with its mammalian hosts and their relevance for understanding the selective pressures acting on parasite evolution.

Methylation of insulin DNA in response to proinflammatory cytokines during the progression of autoimmune diabetes in NOD mice.

AIMS/HYPOTHESIS: Type 1 diabetes is caused by the immunological destruction of pancreatic beta cells. Preclinical and clinical data indicate that there are changes in beta cell function at different stages of the disease, but the fate of beta cells has not been closely studied. We studied how immune factors affect the function and epigenetics of beta cells during disease progression and identified possible triggers of these changes. METHODS: We studied FACS sorted beta cells and infiltrating lymphocytes from NOD mouse and human islets. Gene expression was measured by quantitative real-time RT-PCR (qRT-PCR) and methylation of the insulin genes was investigated by high-throughput and Sanger sequencing. To understand the role of DNA methyltransferases, Dnmt3a was knocked down with small interfering RNA (siRNA). The effects of cytokines on methylation and expression of the insulin gene were studied in humans and mice. RESULTS: During disease progression in NOD mice, there was an inverse relationship between the proportion of infiltrating lymphocytes and the beta cell mass. In beta cells, methylation marks in the Ins1 and Ins2 genes changed over time. Insulin gene expression appears to be most closely regulated by the methylation of Ins1 exon 2 and Ins2 exon 1. Cytokine transcription increased with age in NOD mice, and these cytokines could induce methylation marks in the insulin DNA by inducing methyltransferases. Similar changes were induced by cytokines in human beta cells in vitro. CONCLUSIONS/INTERPRETATION: Epigenetic modification of DNA by methylation in response to immunological stressors may be a mechanism that affects insulin gene expression during the progression of type 1 diabetes.

Efficient Detection of Pathogenic Leptospires Using 16S Ribosomal RNA.

Pathogenic Leptospira species cause a prevalent yet neglected zoonotic disease with mild to life-threatening complications in a variety of susceptible animals and humans. Diagnosis of leptospirosis, which primarily relies on antiquated serotyping methods, is particularly challenging due to presentation of non-specific symptoms shared by other febrile illnesses, often leading to misdiagnosis. Initiation of antimicrobial therapy during early infection to prevent more serious complications of disseminated infection is often not performed because of a lack of efficient diagnostic tests. Here we report that specific regions of leptospiral 16S ribosomal RNA molecules constitute a novel and efficient diagnostic target for PCR-based detection of pathogenic Leptospira serovars. Our diagnostic test using spiked human blood was at least 100-fold more sensitive than corresponding leptospiral DNA-based quantitative PCR assays, targeting the same 16S nucleotide sequence in the RNA and DNA molecules. The sensitivity and specificity of our RNA assay against laboratory-confirmed human leptospirosis clinical samples were 64% and 100%, respectively, which was superior then an established parallel DNA detection assay. Remarkably, we discovered that 16S transcripts remain appreciably stable ex vivo, including untreated and stored human blood samples, further highlighting their use for clinical detection of L. interrogans. Together, these studies underscore a novel utility of RNA targets, specifically 16S rRNA, for development of PCR-based modalities for diagnosis of human leptospirosis, and also may serve as paradigm for detection of additional bacterial pathogens for which early diagnosis is warranted.

ß cell death and dysfunction during type 1 diabetes development in at-risk individuals.

UNLABELLED: Role of the funding source: Funding from the NIH was used for support of the participating clinical centers and the coordinating center. The funding source did not participate in the collection or the analysis of the data. BACKGROUND: The ß cell killing that characterizes type 1 diabetes (T1D) is thought to begin years before patients present clinically with metabolic decompensation; however, this primary pathologic process of the disease has not been measured. METHODS: Here, we measured ß cell death with an assay that detects ß cell-derived unmethylated insulin (INS) DNA. Using this assay, we performed an observational study of 50 participants from 2 cohorts at risk for developing T1D from the TrialNet Pathway to Prevention study and of 4 subjects who received islet autotransplants. RESULTS: In at-risk subjects, those who progressed to T1D had average levels of unmethylated INS DNA that were elevated modestly compared with those of healthy control subjects. In at-risk individuals that progressed to T1D, the observed increases in unmethylated INS DNA were associated with decreases in insulin secretion, indicating that the changes in unmethylated INS DNA are indicative of ß cell killing. Subjects at high risk for T1D had levels of unmethylated INS DNA that were higher than those of healthy controls and higher than the levels of unmethylated INS DNA in the at-risk progressor and at-risk nonprogressor groups followed for 4 years. Evaluation of insulin secretory kinetics also distinguished high-risk subjects who progressed to overt disease from those who did not. CONCLUSION: We conclude that a blood test that measures unmethylated INS DNA serves as a marker of active ß cell killing as the result of T1D-associated autoimmunity. Together, the data support the concept that ß cell killing occurs sporadically during the years prior to diagnosis of T1D and is more intense in the peridiagnosis period. TRIAL REGISTRATION: Clinicaltrials.gov NCT00097292. FUNDING: Funding was from the NIH, the Juvenile Diabetes Research Foundation, and the American Diabetes Association.

Analysis of ß-cell death in type 1 diabetes by droplet digital PCR.

Type 1 diabetes (T1D) and other forms of diabetes are due to the killing of ß-cells. However, the loss of ß-cells has only been assessed by functional studies with a liquid meal or glucose that can be affected by environmental factors. As an indirect measure of ß-cell death, we developed an assay using a novel droplet digital PCR that detects INS DNA derived from ß-cells. The release of INS DNA with epigenetic modifications (unmethylated CpG) identifies the ß-cellular source of the DNA. The assay can detect unmethylated DNA between a range of approximately 600 copies/µL and 0.7 copies/µL, with a regression coefficient for the log transformed copy number of 0.99. The assay was specific for unmethylated INS DNA in mixtures with methylated INS DNA. We analyzed the levels of unmethylated INS DNA in patients with recent onset T1D and normoglycemia subjects at high risk for disease and found increased levels of unmethylated INS DNA compared with nondiabetic control subjects (P < .0001). More than one-third of T1D patients and one-half of at-risk subjects had levels that were more than 2 SD than the mean of nondiabetic control subjects. We conclude that droplet digital PCR is a useful method to detect ß-cell death and is more specific and feasible than other methods, such as nested real-time PCR. This new method may be a valuable tool for analyzing pathogenic mechanisms and the effects of treatments in all forms of diabetes.

Borrelia miyamotoi sensu lato seroreactivity and seroprevalence in the northeastern United States.

Borrelia miyamotoi sensu lato, a relapsing fever Borrelia sp., is transmitted by the same ticks that transmit B. burgdorferi (the Lyme disease pathogen) and occurs in all Lyme disease-endemic areas of the United States. To determine the seroprevalence of IgG against B. miyamotoi sensu lato in the northeastern United States and assess whether serum from B. miyamotoi sensu lato-infected persons is reactive to B. burgdorferi antigens, we tested archived serum samples from area residents during 1991-2012. Of 639 samples from healthy persons, 25 were positive for B. miyamotoi sensu lato and 60 for B. burgdorferi. Samples from ≈10% of B. miyamotoi sensu lato-seropositive persons without a recent history of Lyme disease were seropositive for B. burgdorferi. Our results suggest that human B. miyamotoi sensu lato infection may be common in southern New England and that B. burgdorferi antibody testing is not an effective surrogate for detecting B. miyamotoi sensu lato infection.

Monitoring human babesiosis emergence through vector surveillance New England, USA.

Human babesiosis is an emerging tick-borne disease caused by the intraerythrocytic protozoan Babesia microti. Its geographic distribution is more limited than that of Lyme disease, despite sharing the same tick vector and reservoir hosts. The geographic range of babesiosis is expanding, but knowledge of its range is incomplete and relies exclusively on reports of human cases. We evaluated the utility of tick-based surveillance for monitoring disease expansion by comparing the ratios of the 2 infections in humans and ticks in areas with varying B. microti endemicity. We found a close association between human disease and tick infection ratios in long-established babesiosis-endemic areas but a lower than expected incidence of human babesiosis on the basis of tick infection rates in new disease-endemic areas. This finding suggests that babesiosis at emerging sites is underreported. Vector-based surveillance can provide an early warning system for the emergence of human babesiosis.

Quantitative PCR for detection of Babesia microti in Ixodes scapularis ticks and in human blood.

Babesia microti, the primary cause of human babesiosis in the United States, is transmitted by Ixodes scapularis ticks; transmission may also occur through blood transfusion and transplacentally. Most infected people experience a viral-like illness that resolves without complication, but those who are immunocompromised may develop a serious and prolonged illness that is sometimes fatal. The geographic expansion and increasing incidence of human babesiosis in the northeastern and midwestern United States highlight the need for high-throughput sensitive and specific assays to detect parasites in both ticks and humans with the goals of improving epidemiological surveillance, diagnosis of acute infections, and screening of the blood supply. Accordingly, we developed a B. microti-specific quantitative PCR (qPCR) assay (named BabMq18) designed to detect B. microti DNA in tick and human blood samples using a primer and probe combination that targets the 18S rRNA gene of B. microti. This qPCR assay was compared with two nonquantitative B. microti PCR assays by testing tick samples and was found to exhibit higher sensitivity for detection of B. microti DNA. The BabMq18 assay has a detection threshold of 10 copies per reaction and does not amplify DNA in I. scapularis ticks infected with Babesia odocoilei, Borrelia burgdorferi, Borrelia miyamotoi, or Anaplasma phagocytophilum. This highly sensitive and specific qPCR assay can be used for detection of B. microti DNA in both tick and human samples. Finally, we report the prevalence of B. microti infection in field-collected I. scapularis nymphs from three locations in southern New England that present disparate incidences of human babesiosis.

Plasmodium falciparum phosphoethanolamine methyltransferase is essential for malaria transmission.

Efficient transmission of Plasmodium species between humans and Anopheles mosquitoes is a major contributor to the global burden of malaria. Gametocytogenesis, the process by which parasites switch from asexual replication within human erythrocytes to produce male and female gametocytes, is a critical step in malaria transmission and Plasmodium genetic diversity. Nothing is known about the pathways that regulate gametocytogenesis and only few of the current drugs that inhibit asexual replication are also capable of inhibiting gametocyte development and blocking malaria transmission. Here we provide genetic and pharmacological evidence indicating that the pathway for synthesis of phosphatidylcholine in Plasmodium falciparum membranes from host serine is essential for parasite gametocytogenesis and malaria transmission. Parasites lacking the phosphoethanolamine N-methyltransferase enzyme, which catalyzes the limiting step in this pathway, are severely altered in gametocyte development, are incapable of producing mature-stage gametocytes, and are not transmitted to mosquitoes. Chemical screening identified 11 inhibitors of phosphoethanolamine N-methyltransferase that block parasite intraerythrocytic asexual replication and gametocyte differentiation in the low micromolar range. Kinetic studies in vitro as well as functional complementation assays and lipid metabolic analyses in vivo on the most promising inhibitor NSC-158011 further demonstrated the specificity of inhibition. These studies set the stage for further optimization of NSC-158011 for development of a class of dual activity antimalarials to block both intraerythrocytic asexual replication and gametocytogenesis.

Neurological manifestations of human babesiosis.

Babesiosis is a worldwide emerging infectious disease caused by intraerythrocytic protozoa that are transmitted by Ixodid ticks, or less commonly through blood transfusion or transplacentally. Although headache and lethargy are common symptoms, babesiosis is uncommonly associated with specific neurological dysfunction in humans. Decreased level of consciousness or coma are rare complications that are associated with severe and often fatal disease but the pathogenesis is unclear.

Identification and functional analysis of the primary pantothenate transporter, PfPAT, of the human malaria parasite Plasmodium falciparum.

The human malaria parasite Plasmodium falciparum is absolutely dependent on the acquisition of host pantothenate for its development within human erythrocytes. Although the biochemical properties of this transport have been characterized, the molecular identity of the parasite-encoded pantothenate transporter remains unknown. Here we report the identification and functional characterization of the first protozoan pantothenate transporter, PfPAT, from P. falciparum. We show using cell biological, biochemical, and genetic analyses that this transporter is localized to the parasite plasma membrane and plays an essential role in parasite intraerythrocytic development. We have targeted PfPAT to the yeast plasma membrane and showed that the transporter complements the growth defect of the yeast fen2Δ pantothenate transporter-deficient mutant and mediates the entry of the fungicide drug, fenpropimorph. Our studies in P. falciparum revealed that fenpropimorph inhibits the intraerythrocytic development of both chloroquine- and pyrimethamine-resistant P. falciparum strains with potency equal or better than that of currently available pantothenate analogs. The essential function of PfPAT and its ability to deliver both pantothenate and fenpropimorph makes it an attractive target for the development and delivery of new classes of antimalarial drugs.

Development of a real-time polymerase chain reaction assay for sensitive detection and quantitation of Babesia microti infection.

BACKGROUND: Babesia microti, the most frequently implicated pathogen in transfusion-transmitted babesiosis, is widely endemic in the Northeast and upper Midwestern United States. High seroprevalence in endemic areas limits antibody-based donor screening. A high-performance molecular test is needed to identify donors in the preseroconversion window phase as well as to discriminate past serologic exposure with parasite clearance from continued parasitemia. STUDY DESIGN AND METHODS: Frozen Babesia-spiked whole blood was microcentrifuged, and the supernatant transferred and microcentrifuged again to concentrate the parasite. The DNA was extracted and amplified using real-time polymerase chain reaction (PCR) using Babesia-specific primers. The assay was employed in three series of experiments: 1) a validation and optimization spiking experiment, 2) a blinded serial dilution probit analysis to determine the limit of detection, and 3) evaluation of two blinded panels of clinical samples from possible babesiosis cases. RESULTS: At a decreasing inoculum of 445, 44.5, and 4.45 copies/mL, the assay had positive rates of 100, 97.5, and 81%, respectively. The blinded probit analysis demonstrated a detection rate of 95 and 50% at 12.92 and 1.52 parasites/2 mL of whole blood, respectively. Evaluation of clinical samples showed 13 of 21 samples to be positive, with a range of 85 to 4.8 million parasites/mL. There were no positives detected among 48 healthy donors CONCLUSION: We have developed a highly sensitive and specific, quantitative real-time PCR-based assay for detection of B. microti that could have a useful role in blood screening. It can also be employed broadly to understand Babesia epidemiology, disease pathogenesis, and host immunology.

Sequencing of the smallest Apicomplexan genome from the human pathogen Babesia microti.

We have sequenced the genome of the emerging human pathogen Babesia microti and compared it with that of other protozoa. B. microti has the smallest nuclear genome among all Apicomplexan parasites sequenced to date with three chromosomes encoding ∼3500 polypeptides, several of which are species specific. Genome-wide phylogenetic analyses indicate that B. microti is significantly distant from all species of Babesidae and Theileridae and defines a new clade in the phylum Apicomplexa. Furthermore, unlike all other Apicomplexa, its mitochondrial genome is circular. Genome-scale reconstruction of functional networks revealed that B. microti has the minimal metabolic requirement for intraerythrocytic protozoan parasitism. B. microti multigene families differ from those of other protozoa in both the copy number and organization. Two lateral transfer events with significant metabolic implications occurred during the evolution of this parasite. The genomic sequencing of B. microti identified several targets suitable for the development of diagnostic assays and novel therapies for human babesiosis.

Population Genetics of Aedes albopictus (Diptera: Culicidae) Invading Populations, Using Mitochondrial nicotinamide Adenine Dinucleotide Dehydrogenase Subunit 5 Sequences.

Aedes albopictus (Skuse) (Diptera: Culicidae), the Asian tiger mosquito indigenous to Asia, now an invasive species worldwide, is an important vector for several arboviruses. Genetic analysis using the mitochondrial nicotinamide adenine dinucleotide dehydrogenase subunit 5 (ND5) gene was carried out in populations from Cameroon (n = 50), Hawaii (n = 38), Italy (n = 20), the continental United States, Brazil, and its native range. Data for Brazil, the continental United States, and the native range was obtained from Birungi and Munstermann (2002). Direct sequencing was used to identity unique haplotypes. The limited phylogeographic partitioning of haplotypes with low levels of sequence divergence in both Cameroon and Hawaii was consistent with the population structure of Ae. albopictus in the United States and Brazil. Four new haplotypes were identified from the samples from Cameroon and Hawaii, adding to previously described haplotypes. Hawaii shared a haplotype with Cameroon that was unique to these two regions. Hawaii also had higher overall haplotype diversity than seen in previous continental United States, Brazil, or native range populations. Hawaiian, Cameroon, and Italian populations did not share haplotypes with Brazil, which validates the earlier mitochondrial DNA studies indicating a separate introduction of this species into Brazil.