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Summary Geographically dispersed patients, inconsistent treatment tracking, and limited infrastructure slow research for many orphan diseases. We assess the feasibility of a patient-powered study design to overcome these challenges for Castleman disease, a rare hematologic disorder. Here, we report initial results from the ACCELERATE natural history registry. ACCELERATE includes a traditional physician-reported arm and a patient-powered arm, which enables patients to directly contribute medical data and biospecimens. This study design enables successful enrollment, with the 5-year minimum enrollment goal being met in 2 years. A median of 683 clinical, laboratory, and imaging data elements are captured per patient in the patient-powered arm compared with 37 in the physician-reported arm. These data reveal subgrouping characteristics, identify off-label treatments, support treatment guidelines, and are used in 17 clinical and translational studies. This feasibility study demonstrates that the direct-to-patient design is effective for collecting natural history data and biospecimens, tracking therapies, and providing critical research infrastructure., Graphical Abstract, Highlights Partnership with the patient community supports recruitment and results dissemination A patient-powered design enables high enrollment from a rare disease population Extensive clinical data reveal >40 off-label treatments used in Castleman disease De-identified linkage with a biobank supports translational research discoveries, Pierson et al. describe the feasibility of a patient-powered natural history registry for studying Castleman disease. They pair a traditional registry with a patient-powered approach, in which patients self-enroll and data collection is centralized. Clinical insights support treatment guidelines, and de-identified linkage to a biobank enables translational discoveries.

A sensitive and specific double antibody radio- immunoassay for the major apolipoprotein (apoB) of rhesus (Macaca muIatta) serum very low density lipoprotein (VLDL) and low density lipoprotein (LDL) is described. The anti- serum was raised to LDL (d 1.030-1.040 g/ml) and the LDL, (d 1.020-1.050 g/ml) was labeled with '=I by the chloramine-T or iodine monochloride method. The assay, which was sensitive to 0.02-0.5 pg of LDL2, had an inter- assay coefficient of variation of 4.5%. This assay was suc- cessfully used to measure apoB in the whole serum and low density lipoproteins of control monkeys maintained on a standard Purina monkey chow (PMC) diet and of three groups of monkeys fed atherogenic diets: an "average American diet," a 25% peanut oil and 2% cholesterol-sup- plemented PMC diet, and a 25% coconut oil and 2% choles- terol-supplemented PMC diet. The control monkeys (n = 13) had a serum cholesterol of 146 f 28 mg/dl and an apoB of 50 f 18 mg/dl. In the monkeys maintained on the atherogenic diets the serum apoB was elevated: 103 +- 28 mg/dl (American), 102 f 35 mg/dl (peanut oil), and 312 f 88 mg/dl (coconut oil). The values for serum total choles- terol were 333 f 65 mg/dl (American), 606 f 212 mg/dl (peanut oil), and 864 f 233 mg/dl (coconut oil) and were elevated relative to controls (P < 0.001). For each of the diets, total serum cholesterol correlated with serum apoB (E' < 0.001). The slopes of the regression lines of serum apoB vs. cholesterol for the monkeys on the PMC, Ameri- can, and coconut oil diets were similar (m = 0.531, 0.401, and 0.359, respectively), but differed from that of monkeys on the peanut oil diet (m = 0.12 1). The immunoreactivities of rhesus and human LDL were compared using specific antisera raised against these antigens. In homologous assay systems, monkey and human LDL exhibited unique im- munological determinants. The same results were obtained with the delipidated preparations of the two LDLs using antisera raised against either monkey or human apoB. Crossover studies using a heterologous tracer with each anti- serum resulted in the selection of a specific population of antibodies directed against antigenic sites shared by these two LDL species.

The development of a radioimmunoassay for apolipoprotein A-II (apo A-II) is described. Initial studies revealed a lack of immunological identity between purified apo A-II used as the standard and serum or HDL. Extensive testing of different buffers, standards, antisera, tracers, utilization of a detergent, and heating of sera failed to resolve the problem. Gel filtration of iodinated and non-iodinated apo A-II on Sephadex G-100 columns showed that apo A-II, in dilute solution, elutes in a higher molecular zone than expected with a broad, assymetrical profile. The use of a subfraction of the tracer in the assay resulted in parallelism in the serum and standard dilution curves. The apo A-II assay was sensitive, specific, and reproducible. Apo A-II added to sera was fully recovered and delipidation did not affect the immunoreactivity of either serum or HDL. Apo A-II contributed approximately 20% to the protein mass of HDL. Comparison of these results with those obtained by radial immunodiffusion, and with previously reported data, indicates that the reactivity of apo A-II in its native and delipidated forms may be markedly influenced by different immunologic methodologies and their specific reagents. Caution should thus be shown at present in assigning absolute concentrations to apo A-II in serum or HDL.

A sensitive and reproducible method has been developed for separation of the major serum lipoproteins from 1 ml or less of human serum by isopycnic density gradient ultracentrifugation. The serum, applied to a step gradient (total volume 12.8 ml), was spun for 48 hr at 38,000 rpm at 10°C and, in each of the fractions, apoli- poproteins B and A-I were quantified by the respective radioimmunoassays. The markers for lipid distribution used were (4-I4C )cholesterol and (U-'4C)lecithin, each in- cubated with an aliquot of serum at 20°C for 75 min prior to ultracentrifugation. In control sera, three main frac- tions, very low density (VLDL), low density (LDL), and high density (HDL) lipoproteins were clearly separated from a bottom fraction. Their flotational, electrophoretic, and chemical properties were in good agreement with those reported for the corresponding lipoproteins separated by conventional ultracentrifugation. Both apo B and apo A-I were fully recovered. Essentially all of the apo B was found in VLDL (9.3 2 3.5%) and LDL (87 & 4.6%); of the apo A-I, 81.0 rt 5.7% was in HDL and the remainder (17.0 +- 5.8%) was in the bottom fraction. The peak activities of (14C)cholesterol coincided with the peak of apo B in both LDL and VLDL, and with the peak of apo A-I in HDL. The results with the radiolabeled cholesterol were in good agreement with those obtained by chemical analyses. Carbon 14-labeled lecithin, although fully recovered, was not an accurate marker of phospholipid distribution be- cause, under our experimental conditions, a significant amount of the lecithin was converted into its lyso deriva- tive. The mechanism of the conversion was not established; it appeared to be unrelated to the activities of either lecithin-cholesterol acyl transferase or a Caz+-dependent phospholipase. Besides its validity in the study of control sera, our method also proved successful in the separation of the serum lipoproteins of the few patients with dyslipo- proteinemia (abetalipoproteinemia and familial hyper- cholesterolemia) who were examined. However, the ap- plicability of the method to all dyslipoproteinemias was not assessed. Taken together, the results indicate that the single-spin method could be useful in clinical studies as a complement to other established techniques.

Isolated livers from rhesus monkeys (Macaca mulatta) were perfused in order to asses the nature of newly synthesized hepatic lipoprotein. Perfusate containing [3H]leucine was recirculated for 1.5 hr, followed by an additional 2.5-hr perfusion with fresh perfusate. Equilibrium density gradient ultracentrifugation clearly separated VLDL from LDL. The apoprotein composition of VLDL secreted by the liver was similar to that of serum VLDL. The perfusate LDL contained some poorly radiolabeled, apoB-rich material, which appeared to be contaminating serum LDL. There was also some material of an LDL-like density, which was rich in radiolabeled apoE. Rate zonal density gradient ultracentrifugation fractionated HDL. All perfusate HDL fractions had a decreased cholesteryl ester/unesterified cholesterol ratio, compared to serum HDL. Serum HDL distributed in one symmetric peak near the middle of the gradient, with coincident peaks of apoA-I and apoA-II. The least dense fractions of the perfusate gradient were rich in radiolabeled apoE. The middle of the perfusate gradient contained particles rich in radiolabeled apoA-I and apoA-II. The peak of apoA-I was offset from the apoA-II peak towards the denser end of the gradient. The dense end of the HDL gradient contained lipoprotein-free apoA-I, apoE, and small amounts of apoA-II, probably resulting from the relative instability of nascent lipoprotein compared to serum lipoprotein. Perfusate HDL apoA-I isoforms were more basic than serum apoA-I isoforms. Preliminary experiments, using noncentrifugal methods, suggest that some hepatic apoA-I is secreted in a lipoprotein-free form. In conclusion, the isolated rhesus monkey liver produces VLDL similar to serum VLDL, but produces LDL and HDL which differ in several important aspects from serum LDL and HDL.

Publisher Summary This chapter discusses the clinical significance of circulating pro-insulin and c-peptide. Till 1967, it was believed that the two chains of insulin were synthesized separately and then joined by means of two disulfide bonds at a post-ribosomal site. However, studies by Steiner and his colleagues using slices of a pancreatic islet cell tumor demonstrated the presence of a large molecular weight, single chain precursor molecule, which was named pro-insulin. This protein consists of the insulin A and B chains linked by an additional polypeptide segment of approximately 30 to 35 amino acids, depending on the species. The enzymes involved in the transformation of pro-insulin to insulin are probably localized in the granules or their membranes, and the conversion process takes place as the granules mature and move toward the plasma membrane of the cell. These pro-teolytic enzymes cleave pro-insulin at specific sites where two pairs of basic amino acids join the connecting peptide (C-peptide) to the insulin chains. The major products of this reaction are insulin and the C-peptide.

Publisher Summary The discovery of proinsulin, the biosynthetic precursor of insulin, has stimulated a search for the existence of precursors of other polypeptides. Growing evidence suggests that such forms exist not only for many peptide hormones including, along with insulin, parathyroid hormone, glucagon, and gastrin, but also for structural proteins including collagen, and virus capsule proteins. It is difficult to provide specific methods for the identification and analysis of all polypeptide precursors because of the divergent origin and chemical properties of the possible forms. The approach in each ease must consist of an imaginative synthesis of currently available methods for detecting and separating related proteins. A critical assessment of the precursor–product relationship of any pair of polypeptides requires both biochemical and chemical approaches. The methods used for the determination of this relationship and for the final characterization of the precursor form are largely those traditionally used in the detection and isolation of a variety of proteins and peptides. The final proof for the existence of a precursor polypeptide must rely on several carefully met criteria, each of which may only be suggestive in the absence of the others.

Proinsulin is a single polypeptide chain precursor of insulin having a molecular weight of about 9000 and containing the B and A chains of insulin linked through a connecting peptide segment of about 30 residues. Proinsulin is produced on the ribosomes of the β-cells, is transformed to insulin within the islet cells, is stored mainly as insulin in the granules, and is secreted mainly as such from the islets. The transformation of proinsulin to insulin most likely occurs in the Golgi apparatus or in the initial phases of granule maturation. The role of proinsulin appears to be to facilitate the formation of the disulfide bonds during biosynthesis. However, under some circumstances small amounts of proinsulin appear to be secreted from the islets and small amounts have been found to circulate in the plasma and to be excreted in the urine. The possible relationship of proinsulin to disorders of carbohydrate metabolism in man such as diabetes represents an interesting area for further study.