Clinical and experimental studies of intraperitoneal lipolysis and the development of clinically relevant pancreatic fistula after pancreatic surgery.

BACKGROUND: Visceral obesity is one of the risk factors for clinically relevant pancreatic fistula after pancreatic resection. The objective of this study was to evaluate the impact of intraperitoneal lipolysis on postoperative pancreatic fistula. METHODS: The degree of intraperitoneal lipolysis was investigated by measuring the free fatty acid concentration in drain discharge in patients after pancreatic resection. An experimental pancreatic fistula model was prepared by pancreatic transection, and the impact of intraperitoneal lipolysis was evaluated by intraperitoneal administration of triolein (triglyceride) with, or without orlistat (lipase inhibitor). RESULTS: Thirty-three patients were included in the analysis. The free fatty acid concentration in drain discharge on postoperative day 1 was significantly associated with the development of a clinically relevant pancreatic fistula (P = 0·004). A higher free fatty acid concentration in drain discharge was associated with more visceral adipose tissue (P = 0·009). In the experimental model that included 98 rats, intraperitoneal lipolysis caused an increased amount of pancreatic juice leakage and multiple organ dysfunction. Intraperitoneal administration of a lipase inhibitor reduced lipolysis and prevented deterioration of the fistula. CONCLUSION: Intraperitoneal lipolysis significantly exacerbates pancreatic fistula after pancreatic resection. Inhibition of lipolysis by intraperitoneal administration of a lipase inhibitor could be a promising therapy to reduce clinically relevant postoperative pancreatic fistula. Surgical relevance Clinically, there are two types of pancreatic fistula after pancreatic resections: harmless biochemical leak and harmful clinically relevant pancreatic fistula. Visceral obesity is one of the known risk factors for clinically relevant pancreatic fistula; however, the underlying mechanisms remained to be elucidated. Patients with clinically relevant pancreatic fistula had a higher free fatty acid concentration in the drain discharge, suggesting a relationship between intraperitoneal lipolysis and pancreatic fistula. The experimental model of pancreatic fistula demonstrated that intraperitoneal lipolysis caused deterioration in pancreatic fistula, suggesting that intraperitoneal lipolysis is one of the mechanisms that drives biochemical leakage to clinically relevant pancreatic fistula. Intraperitoneal administration of a lipase inhibitor prevented lipolysis as well as pancreatic fistula deterioration in the experimental model, suggesting a future clinical application for lipase inhibitors in prevention of clinically relevant pancreatic fistula.

Long-term outcomes of clinical transplantation of pancreatic islets with uncontrolled donors after cardiac death: a multicenter experience in Japan.

BACKGROUND: Pancreatic islet transplantation has emerged as an effective treatment for type 1 diabetes mellitus, but its use is limited due to an insufficient supply of cadaveric pancreata. In Japan, uncontrolled donors after cardiac death (DCD) are not deemed to be suitable for whole-organ pancreatic transplantation, and can provide a source of pancreas for islet transplantation. However, the long-term outcomes and utility of uncontrolled DCD in the clinical setting remain controversial. Here, we summarize the long-term outcomes of islet transplantation employing uncontrolled DCD as reported to the Japan Islet Transplantation Registry. METHODS: Sixty-four isolations and 34 transplantations of pancreatic islets were conducted in 18 subjects with type 1 diabetes mellitus under the cover of immunosuppression with basiliximab, sirolimus, and tacrolimus. All donors were uncontrolled DCD at the time of harvesting. The mean follow-up time was 76 months. RESULTS: Of the 18 recipients, 8, 4, and 6 recipients received 1, 2, and 3 islet infusions, respectively. Overall graft survivals (defined as a C-peptide level ≥0.3 ng/mL) were 72.2%, 44.4%, and 22.2% at 1, 2, and 5 years, respectively, whereas the corresponding graft survivals after multiple infusions were 90.0%, 70.0%, and 30.0%, respectively. Three of these recipients achieved insulin independence in 14, 79, and 215 days. HbA1c levels and the requirement of exogenous insulin were improved before loss of graft function. All recipients became free of severe hypoglycemia unawareness, however, at least 5 of 14 patients who had graft failure experienced recurrence of severe hypoglycemia after the loss of graft function. CONCLUSIONS: Islet transplantation from DCD can relieve glucose instability and problems with hypoglycemia when the graft is functioning. However, islets from uncontrolled DCD may be associated with reduced long-term graft survival. Further improvements in the clinical outcome by modification of islet isolation/transplantation protocols are necessary to establish islet transplantation using DCD.

Severely fibrotic pancreases from young patients with chronic pancreatitis: evidence for a ductal origin of islet neogenesis.

While it is known that islet cell mass increases considerably after birth, general uncertainty surrounds the source of new beta cells in humans. Chronic pancreatitis (CP) presents a natural injury model for studying postnatal beta-cell regeneration in the human pancreas. In this report, we present histological evidence from human CP pancreases to support the theory that islet neogenesis can occur from ductal precursor cells after birth. Three young patients (ages 16, 12, and 28 years) underwent total pancreatectomy for the management of CP followed by islet isolation and autologous transplantation to prevent or minimize postsurgical diabetes. In all cases, the pancreases had extensive fibrosis, a rock-like consistency, and calcifications in the ducts. During islet isolations, we observed the unusual release of islets with many ductal fragments. In histopathological evaluation of these pancreases, solid cords of cells sometimes formed islet like structures intraductally or extending from ductal structures. Immunofluorescence staining for chromogranin, insulin, proinsulin, PDX1, glucagon, and cytokeratins confirmed these structures to be composed of chromogranin-positive endocrine cells which included both ß-cells and α-cells. Labeling for Ki67 to demonstrate mitotic activity showed frequent labeling of duct epithelial cells and of some periductal cells. Using insulin and wide-spectrum cytokeratin double immunofluorescent labeling, we found insulin-positive cells to be present within the ductal lumens, among the cytokeratin-positive ductal epithelium, and extending from the ductal epithelium into surrounding connective tissues, providing evidence for a ductal origin of islet neogenesis.

Actions of the Japanese Pancreas and Islet Transplantation Association regarding transplanted human islets isolated using Liberase HI.

PURPOSE: The potential for introducing transmissible spongiform encephalopathy (TSE) into islet cells was indicated by recognizing that Liberase HI is isolated from Clostridium histolyticum grown in media containing brain-heart infusion broth. A national team within the Japanese Pancreas and Islet Transplantation Association implemented an islet transplantation program in Japan using Liberase HI. The program comprised 65 islet isolations from non-heart-beating donors and 34 transplants into 18 patients. Herein, we have summarized how the Association followed these recipients over the long term. PROCEDURES: We established an ad hoc committee to follow recipients transplanted with islets isolated using Liberase HI after becoming informed of the associated dangers of using this enzyme. We also stopped islet transplantations using Liberase. The committee addressed the major concerns of the risk of the collagenase being contaminated with TSE and of the recipient follow-up. All recipients were examined by diffusion MRI and EEG and then scheduled for evaluation and follow-up by specialists in Creutzfeldt-Jakob disease (CJD). Bioassays of bovine spongiform encephalopathy prions in the enzyme proceeded using knock-in mice expressing bovine prion protein. These assays could detect contaminating prions at a dilution of 1 × 10(4). After inactivating its collagenase activity, Liberase HI was injected into the abdominal cavities of knock-in mice. Four months later, prion infectivity in Liberase HI was evaluated by immunohistochemical staining and Western blotting of spleen homogenates using anti-prion protein antibodies. MAIN FINDINGS: Western blotting and immunohistochemical staining did not detect prions in Liberase HI. Diffusion MRI and EEG evaluations performed by CJD specialists confirmed that none of the transplanted recipients had CJD. CONCLUSIONS: Three years of follow-up revealed that none of the Japanese recipients of islet transplants developed CJD. Prion bioassays showed that the Liberase HI used to isolate islets for transplantation was free of infectious TSE prions.

Pancreas oxygen persufflation increases ATP levels as shown by nuclear magnetic resonance.

BACKGROUND: Islet transplantation is a promising treatment for type 1 diabetes. Due to a shortage of suitable human pancreata, high cost, and the large dose of islets presently required for long-term diabetes reversal; it is important to maximize viable islet yield. Traditional methods of pancreas preservation have been identified as suboptimal due to insufficient oxygenation. Enhanced oxygen delivery is a key area of improvement. In this paper, we explored improved oxygen delivery by persufflation (PSF), ie, vascular gas perfusion. METHODS: Human pancreata were obtained from brain-dead donors. Porcine pancreata were procured by en bloc viscerectomy from heparinized donation after cardiac death donors and were either preserved by either two-layer method (TLM) or PSF. Following procurement, organs were transported to a 1.5-T magnetic resonance (MR) system for (31)P nuclear magnetic resonance spectroscopy to investigate their bioenergetic status by measuring the ratio of adenosine triphosphate to inorganic phosphate (ATP:P(i)) and for assessing PSF homogeneity by MRI. RESULTS: Human and porcine pancreata can be effectively preserved by PSF. MRI showed that pancreatic tissue was homogeneously filled with gas. TLM can effectively raise ATP:P(i) levels in rat pancreata but not in larger porcine pancreata. ATP:P(i) levels were almost undetectable in porcine organs preserved with TLM. When human or porcine organs were preserved by PSF, ATP:P(i) was elevated to levels similar to those observed in rat pancreata. CONCLUSION: The methods developed for human and porcine pancreas PSF homogeneously deliver oxygen throughout the organ. This elevates ATP levels during preservation and may improve islet isolation outcomes while enabling the use of marginal donors, thus expanding the usable donor pool.

Persufflation improves pancreas preservation when compared with the two-layer method.

Islet transplantation is emerging as a promising treatment for patients with type 1 diabetes. It is important to maximize viable islet yield for each organ due to scarcity of suitable human donor pancreata, high cost, and the large dose of islets required for insulin independence. However, organ transport for 8 hours using the two-layer method (TLM) frequently results in low islet yields. Since efficient oxygenation of the core of larger organs (eg, pig, human) in TLM has recently come under question, we investigated oxygen persufflation as an alternative way to supply the pancreas with oxygen during preservation. Porcine pancreata were procured from donors after cardiac death and preserved by either TLM or persufflation for 24 hours and subsequently fixed. Biopsies collected from several regions of the pancreas were sectioned, stained with hematoxylin and eosin, and evaluated by a histologist. Persufflated tissues exhibited distended capillaries and significantly less autolysis/cell death relative to regions not exposed to persufflation or to tissues preserved with TLM. The histology presented here suggests that after 24 hours of preservation, persufflation dramatically improves tissue health when compared with TLM. These results indicate the potential for persufflation to improve viable islet yields and extend the duration of preservation, allowing more donor organs to be utilized.

Rapid quantitative assessment of the pig pancreas biopsy predicts islet yield.

BACKGROUND: The cost of islet procurement from donor pigs is increased by the use of organs that produce low yields. We developed an assessment system using dithizone-stained pig pancreas biopsies to enable the preselection of donor organs. METHODS: Pig pancreas biopsy slices were soaked in dithizone solution. The islets were evaluated before islet isolation by converting the islet counts (IC) to islet equivalents (IE), and then determining the IE/cm(2), IE/IC, % islets >150 microm, and % islets >200 microm. These parameters were evaluated in 3 different areas of the pancreas (duodenal, splenic, and connecting lobe; n = 42 each). Stepwise multivariate linear regression analysis was performed to assess for correlations with islet yield and decide which area of the pancreas had the most predictive value. To identify other predictors, including donor and islet isolation variables, we performed binary logistic regression analysis with significant variables from the univariate analysis (n = 67). For this analysis, the pigs were categorized into high (n = 23) and low (n = 44) yield groups. RESULTS: Stepwise multivariate linear regression analysis revealed that IE/cm(2) of the splenic lobe significantly predicted islet yield. Binary logistic regression analysis indicated that the IE/mm(2) of the splenic lobe was the only parameter that significantly correlated with successful pig islet isolations (P = .01; odds ratio 3.605). Variables associated with donor and islet isolation, such as age, gender, ischemic time, or enzyme lot, were not significantly correlated with islet yield. CONCLUSION: Our study suggests that the islet distribution of splenic lobe biopsies can be a reliable predictor of islet yield from pig pancreata.

Improved method of porcine pancreas procurement with arterial flush and ductal injection enhances islet isolation outcome.

BACKGROUND: Several pancreas procurement procedures have been used for porcine islet isolation; however, their impact on outcomes has not been extensively studied. We evaluated an advanced procurement technique for porcine islet isolation designed to reduce warm ischemia, to remove blood content, and enhance cooling of the pancreas by implementing a vascular flush and ductal preservation. METHOD: Pancreata procured from adult Landrace pigs were divided into 3 different surgical protocols: Pancreatectomy utilizing only surface cooling (group 1; n = 24); surface cooling and ductal injection with cold preservation solution before pancreatectomy (group 2; n = 12); or surface cooling, ductal injection, and an approach by selectively flushing through the celiac trunk and the superior mesenteric artery (group 3; n = 14). We assessed the islet isolation results and quality using in vitro and in vivo assays. RESULTS: Significantly higher overall yield and islet yield per gram pancreas were obtained from group 3 pigs compared with the other groups. Measurements of islet viability after 7 days of culture, as assessed by oxygen consumption rate per DNA, showed that group 3 islets displayed the highest values. Sustained normoglycemia was observed in diabetic nude mice transplanted with 2000 islet equivalents from all 3 groups. DISCUSSION: This study demonstrated that an advanced pancreas procurement technique including ductal preservation and selective arterial flush with cold preservation solution provided significant improvements in porcine islet isolation outcomes.

Human islet isolation for autologous transplantation: comparison of yield and function using SERVA/Nordmark versus Roche enzymes.

Islet autotransplantation (IAT) is used to preserve as much insulin-secretory capacity as possible in patients undergoing total pancreatectomy for painful chronic pancreatitis. The enzyme used to dissociate the pancreas is a critical determinant of islet yield, which is correlated with posttransplant function. Here, we present our experience with IAT procedures to compare islet product data using the new enzyme SERVA/Nordmark (SN group; n = 46) with the standard enzyme Liberase-HI (LH group; n = 40). Total islet yields (mean +/- standard deviation; 216,417 +/- 79,278 islet equivalent [IEQ] in the LH group; 227,958 +/- 58,544 IEQ in the SN group; p = 0.67) were similar. However, the percentage of embedded islets is higher in the SN group compared to the LH group. Significant differences were found in pancreas digestion time, dilution time, and digested pancreas weight between the two groups. Multivariate linear regression analysis showed the two groups differed in portal venous pressure changes. The incidence of graft function and insulin independence was not different between the two groups. The SN and LH enzymes are associated with similar outcomes for IAT. Further optimization of the collagenase/neutral protease ratio is necessary to reduce the number of embedded islets obtained when using the SN enzyme.

Bone marrow traffic to regenerating islets induced by streptozotocin injection and partial pancreatectomy in mice.

The role of bone marrow (BM)-derived cells in the process of pancreatic islet regeneration remains unclear. The purpose of this study was to determine the role of BM cells in the repair process or regeneration of pancreatic islets in mice using chimeric green fluorescent protein (GFP) expressing BM cells. BM-infused chimeric mice were made diabetic by streptozotocin (STZ) injection or 60% partial pancreatectomy. GFP-positive cells within the islets and pancreas were studied immunohistologically. STZ treatment induced a 10-fold increase in PCNA-positive cells within the islets on day 7 posttreatment. GFP-positive cells increased in number within the islets as well as in the pancreatic parenchyma immediately after STZ injection. The partial pancreatectomy induced 2- to 3-fold increases on day 7 to 28 posttreatment. GFP-positive cells increased in number in pancreatic parenchyma but not within the islets. BM traffic to the pancreas significantly increased in the 2 models inducing islet regeneration. In both models, GFP-positive cells were not positive for antibodies against insulin, glucagon, or somatostatin, but were positive for markers of macrophages or fibroblasts, suggesting their involvement in the initiation of islet regeneration.

Evaluation of energy state of islet independent of size using a newly developed ATP bioluminescence assay.

ATP content or energy charge (EC) of islets may be a good parameter to assess viability. In this study, we examined adenine nucleotides and EC of freshly isolated or 24-hour cultured rat islets of various diameters using a novel bioluminescent enzymatic cycling assay system. For freshly isolated islets, ATP content and islet diameter showed a high correlation (r = 0.842, P < .001), but a significant correlation was not observed for cultured islets (r = 0.284) when all islets were included for the analysis. When only the cultured islets with a diameter <350 microm were included for analysis, a significant correlation was observed (r = 0.719). EC of freshly isolated islets fluctuated widely irrespective of diameter, in contrast with results of 24-hour cultured islets, which showed a stable, high EC, regardless of diameter. These data suggest that the ATP content of islets correlates with the islet size and that EC of islets widely fluctuates following isolation, indicating a significant role of monitoring ATP and EC of islets before transplantation.

Treatment of external iliac artery dissection with endovascular stent placement in a patient with simultaneous pancreas and kidney transplantation.

Dissection of the external iliac artery is a rare but serious complication after transplantation, leading to graft loss. Stent angioplasty has been accepted for iliac artery dissection, but the safety and benefits of this procedure in transplantation are unclear. We report a case of right external iliac artery dissection with simultaneous kidney and pancreas transplantation, which was noticed during the operation. A self-expandable metallic stent was inserted from the right femoral artery to the dissected area and arterial blood flow was immediately restored. The postoperative clinical course was uneventful and soon after transplantation both the kidney and pancreas graft functioned well. Together with a previously reported six cases of stent replacement with transplantation and our case, no complication related to this procedure was reported, and in six cases the arterial flow was restored and graft function was improved. Endovascular stent replacement may be a safe and effective procedure for iliac artery dissection in transplantation.

Effects of combined emergency percutaneous cardiopulmonary support and reperfusion treatment in patients with refractory ventricular fibrillation complicating acute myocardial infarction.

OBJECT: We conducted a prospective study to determine whether or not combined emergency percutaneous cardiopulmonary support (PCPS) and coronary reperfusion treatment are useful for acute myocardial infarction (MI) patients with unsynchronized electric shock-resistive ventricular fibrillation (VF). PATIENTS AND METHODS: Thirty-two acute MI patients who lapsed into the refractory VF were entered into the study. Group 1 consisted of 19 patients with VF outside the hospital, and Group 2 consisted of 13 patients with VF immediately after arrival at the hospital. The primary endpoint was successful reperfusion, return of spontaneous circulation and good recovery without neurologic disability. RESULTS: The infarct-related arteries showed a significant difference between Groups 1 and 2. However, the two groups had similar rates of successful reperfusion (84.6% vs 94.7%, respectively) and return of spontaneous circulation (89.5% vs 84.6%, respectively). The rates of good recovery were similarly low in both groups (5.3% vs 15.4%, respectively). CONCLUSION: Combined emergency PCPS and reperfusion treatment produced high return of spontaneous circulation, however the neurologic outcome was low.

Radiation immobilization of poly(methyloctylsiloxane) on silica for use in HPLC: a uniform layer model.

Poly(methyloctylsiloxane) (PMOS) was sorbed into the pores of HPLC silica by a solvent evaporation procedure, then irradiated with gamma rays from a cobalt-60 source to absorbed doses in the range from 0 to 200 kGy (1Gy = 1J kg-1). Non-irradiated and irradiated samples were characterized by solvent extraction, specific surface area determination, infrared spectroscopy and reversed-phase column performance. Solvent extraction data reveal that about 40% of the PMOS is not extractable prior to irradiation and this increases to about 75% with radiation doses of 50 kGy or higher. Column performance was improved by the radiation treatment, reaching a maximum efficiency in the dose range of 80-140 kGy while the peak symmetry changed from As = 1.7 to 1.1. The improvement is attributed to the increased mass of polymer immobilized by the radiation treatment and to a more uniform distribution of the immobilized polymer in the silica pore system. A multi-layer stationary-phase model is presented in which the first layer consists of an adsorbed monolayer of PMOS and the second layer is immobilized by gamma radiation.

A capillary-array electrophoresis system using side-entry on-column laser irradiation combined with glass rod lenses.

We have developed a simple and high-throughput capillary-array electrophoresis system that uses side-entry on-column laser irradiation. The number of capillaries in an array is generally limited by laser-power attenuation along the array due to reflection and divergence. We overcame these problems by placing the capillaries in water and adding glass rod lenses between the capillaries. As a result, up to 45 capillaries could be simultaneously irradiated with a single laser beam and the fluorescence from all the capillaries could be detected with high sensitivity. We demonstrated the high throughput of 12 kbp/h with a 45 capillary array using this system.

[The statistics of inpatients at Department of Neuropsychiatry in Sapporo Medical University Hospital (October, 1983 - March, 1996)].

The role of university hospitals should be re-examined considering the recent situation in psychiatrical health care. In this report, we investigated inpatients of neuropsychiatry in Sapporo Medical University Hospital from various aspects. Statistics were gathered on 1) age and sex, 2) address, 3) admission form based on the mental health law, 4) the number of admissions and discharges per year, 5) hospitalization term, 6) diagnostic group, 7) diagnosis, 8) rates of re-admission and re-admission within three months, 9) age and sex of schizophrenia patients and 10) age and sex of patients with affective disorders from October 31, 1983 to March 31, 1996. In our hospital, the rates of the inpatients with dementia and personality disorders are higher than those in other university hospitals. The reason for the high rate of personality disorders is elusive; however, most of the dementia patients enter our hospital mainly because we concentrate on a special research project about dementia. These data indicate that a specific function is required in university hospitals. In recent years, however, the surroundings of people with mental disorders have become more complicated, and the services for them have become diversified. However, it is very difficult for university hospitals to provide them with all such services, as the hospitals fulfill just one specialized function among the necessary services.

A capillary array gel electrophoresis system using multiple laser focusing for DNA sequencing.

A very simple and highly sensitive capillary array gel electrophoresis system is constructed to analyze DNA fragments. On-column detection of DNA migration in a large number of gel-filled capillaries is carried out using side-entry laser irradiation and with a CCD camera, although it has been considered impossible because the irradiation laser is scattered strongly at the surfaces of the first few capillaries. By optimizing optical conditions, the laser beam can be focused repeatedly to irradiate all the capillaries held on a plate by working each capillary as a cylindrical convex lens. DNA sequencing samples migrating in 24 capillaries can simultaneously be analyzed with the system.

Microscopic analysis of the elastic properties of nebulin in skeletal myofibrils.

The elastic properties of nebulin were studied by measuring the elasticity of single skeletal myofibrils, from which the portion of the thin filament located at the I band had been selectively removed by treatment with plasma gelsolin under rigor conditions. In this myofibril model, a portion of each nebulin molecule at the I band was expected to be free of actin filaments and exposed. The length of the exposed portion of the nebulin molecule was controlled by performing the gelsolin treatment at various sarcomere lengths. The relation between the passive tension and extension of the exposed portion of the nebulin showed a convex curve starting from a slack length, apparently in a fashion similar to that of wool. The slack sarcomere length shifted depending on the length of the exposed portion of the nebulin, however, the relation being represented by a single master curve. The elastic modulus of nebulin was estimated to be two to three orders of magnitude smaller than that of an actin filament. Based on these results, we conclude that nebulin attaches to an actin filament in a side-by-side fashion and that it does not significantly contribute to the elastic modulus of thin filaments. The relation between the passive tension and extension of connectin (titin) was obtained for a myofibril from which thin filaments had been completely removed with gelsolin under contracting conditions; this showed a concave curve, consistent with the previous results obtained in single fibers.

Structural and functional reconstitution of thin filaments in skeletal muscle.

Thin filaments were reconstituted by incorporating exogenous actin, tropomyosin and troponin into glycerinated skeletal muscle fibres or myofibrils. Firstly, thin filaments except short fragments at the Z line were selectively removed by treatment with plasma gelsolin, an actin severing protein. As a result, the fibres (or fibrils) lost the ability to generate active tension. Next, actin filaments were reconstituted by adding purified G-actin which polymerizes onto the actin fragments which remained at the Z line. Rhodamine phalloidin staining of myofibrils showed that exogenous actin was incorporated into the position where the intrinsic thin filaments located. Thin section electron micrographs of fibres showed that reconstituted actin filaments ran from the Z line to the inside of the A band, with some reaching the H zone. The number density of reconstituted actin filaments in the A band was about 20% of that found in intact fibres. The actin filament-reconstituted fibres (or fibrils) generated active tension in a Ca(2+)-insensitive manner and the tension was reversibly suppressed by 2,3-butanedione 2-monoxime. The recovered active tension was about 20% of tension developed by intact fibres. These results indicate that reconstituted actin filaments bear active tension similar to that borne by intact thin filaments. Thin filament-reconstituted fibres, which were prepared by adding purified tropomyosin-troponin complexes into actin filament-reconstituted fibres, showed Ca(2+)-sensitive tension generation. The maximum tension generated was not affected by the presence of tropomyosin and troponin. SDS-PAGE analysis showed that more than 25% of actin and 20% of tropomyosin and troponin was incorporated into the reconstituted fibres. These results indicate that the structure and function of thin filaments are substantially reconstituted by self-assembly of actin, tropomyosin and troponin. The reconstituted fibres and fibrils will be useful for studying the molecular mechanism of muscle contraction and its regulation.