A Retrospective Study on the Start and End of Continuous Hemodialysis using a Polymethylmethacrylate Hemofilter for Severe Acute Pancreatitis.

Objective We previously reported the successful outcomes in severe acute pancreatitis (SAP) after continuous hemodialysis using a polymethylmethacrylate hemofilter (PMMA-CHD). The present study makes informative suggestions regarding the initiation and termination of PMMA-CHD. Methods We retrospectively studied 63 patients with SAP admitted to the intensive-care unit between January 1, 2011, and December 31, 2022, including 30 who received PMMA-CHD therapy for renal dysfunction. Statistical significance was evaluated using a multiple logistic regression analysis for severity scores, prognostic factor scores in the Japanese severity criteria, the Kidney Disease: Improving Global Outcomes (KDIGO) stage, and the lung injury score (LIS). Results At the onset of blood purification therapy using PMMA-CHD, a significant increase in the KDIGO stage was shown, with a cutoff value of 2.0. The prognostic factor score and LIS at the start of blood purification therapy were significantly high, with a cutoff value of 3.0. Analyses of severity scores, the KDIGO stage, and the LIS before the start of PMMA-CHD were also increased significantly, with cutoff values of +2.0, +1.0, and +3.0, respectively. Furthermore, on analyses of improvements in values after starting PMMA-CHD, the value of KDIGO staging significantly decreased, and the cutoff value was -2.0. The prognostic factor score was also significantly decreased, with a cutoff value of -2.0. Conclusion Prognostic factor scores of the Japanese severity criteria and LIS, as well as the KDIGO stage, are valuable indicators for determining the start and end of PMMA-CHD therapy.

Clinical efficacy of blood purification using a polymethylmethacrylate hemofilter for the treatment of severe acute pancreatitis.

BACKGROUND: Severe acute pancreatitis (SAP) is a systemic inflammatory disease, and it can often complicate into acute kidney injury (AKI) and acute lung injury/acute respiratory distress syndrome (ALI/ARDS). This study aimed to evaluate the clinical effectiveness of blood purification using a polymethylmethacrylate (PMMA) hemofilter. METHODS: We retrospectively examined 54 patients, who were diagnosed with SAP according to the Japanese criteria from January 2011 to December 2019. RESULTS: Of a total of 54 SAP patients, 26 patients progressively developed AKI and required continuous hemodialysis with a PMMA membrane hemofilter (PMMA-CHD). Acute Physiology and Chronic Health Evaluation (APACHE) II score and Sequential Organ Failure Assessment (SOFA) score were significantly higher in patients requiring PMMA-CHD than in patients not requiring hemodialysis. The lung injury scores were also significantly higher in patients requiring PMMA-CHD. Of the 26 patients, 16 patients developed ALI/ARDS and required mechanical ventilation. A total of seven patients developed severe ALI/ARDS and received additional intermittent hemodiafiltration using a PMMA hemofilter (PMMA-HDF). Although the length of intensive care unit stay was significantly longer in patients with severe ALI/ARDS, blood purification therapy was discontinued in all the patients. The survival rates at the time of discharge were 92.3% and 92.9% in patients with and without PMMA-CHD, respectively. These real mortality ratios were obviously lower than the estimated mortality ratios predicted by APACHE II scores. CONCLUSIONS: These finding suggest that the blood purification using a PMMA hemofilter would be effective for the treatment of AKI and ALI/ARDS in SAP patients.

Synaptogenesis and amino acid release from long term embryonic rat spinal cord neuronal culture using tissue culture inserts.

In the present study, using tissue culture inserts (TCI) coupled with a primary spinal cord neuronal culture, we characterize a new perfusion system, which permits continuous perfusate collection from cultured neurons. Primary spinal cord neurons were isolated from the lumbar portion of E14 spinal cords of Sprague-Dawley rats, plated on TCI and fed with DMEM/B27/10% FBS. At 1-4 weeks after isolation the development of synapses and neurotransmitter phenotype in cultured neurons was verified using immunofluorescence. A time-dependent development of synapses (Syn) was seen with a dense Syn-positive network identified at 3-4 weeks after plating. A sub-population of plated neurons (35-40%) showed GABA immunoreactivity and expressed NMDAR1 receptor. To measure neurotransmitter release, a chamber accommodating TCI was constructed permitting perfusion of the insert across the membrane. To evoke amino acid release from cultured neurons, NMDA (10 mmol/l) was added into the perfusion buffer. Stimulation with NMDA evoked a significant GABA (4050 +/- 950%) and glutamate release (130 +/- 42%) during first 10 min after exposure. In control non-stimulated cells no significant changes were measured. These data show that by using TCI it is possible to maintain embryonic spinal cord neurons for an extended period and that this system may represent a simple tool to identify neurotransmitter and/or peptides associated with a specific population of cultured brain and/or spinal cord neurons.

An antibody that binds to primary specific pocket-associated structure in the active site of bovine thrombin.

We attempted to produce a monoclonal antibody (MAb) against the active site of native thrombin. Bovine thrombin was treated with diisopropyl fluorophosphate, and prepared diisopropylphosphoryl-thrombin was used for the immunization to BALB/c mice. Spleen cells of immunized mice were hybridized with mouse myeloma cells P3U1, and a hybridoma clone CC2, which produced a MAb against bovine thrombin was established. The MAb produced by hybridoma clone CC2 (MAb(CC2)), consisting of IgG(1) and kappa light chain, was purified using protein A affinity chromatography. Purified MAb(CC2) prolonged the fibrin forming time of bovine thrombin and inhibited the release of fibrinopeptide A from rabbit fibrinogen. In addition, it was found that argatroban partially, but competitively, interfere the binding between MAb(CC2) and bovine thrombin. It was then considered that MAb(CC2) would bind to the molecular structure associating primary specific pocket in the active site of bovine thrombin.

Bound thrombin from crushed clots is composed of alpha-thrombin and the N-terminal regions of alpha- and gamma-chains of fibrinogen.

We aimed at clarifying the structural characteristics of the bound thrombin that is liberated by mechanical breakdown of fibrin clots. Fibrin clots were prepared with bovine thrombin and rabbit fibrinogen, and were crushed mechanically with a glass rod. The supernatant of the crushed clots was subjected to immunoaffinity chromatography to isolate the bound thrombin. Western blotting analysis revealed that the bound thrombin could be reacted with both antithrombin and antifibrinogen under unreduced conditions. SDS-PAGE under reduced conditions revealed that there were three bands, two of which were found to be the N-terminal fragments of the alpha- and gamma-chains of fibrinogen. The bound thrombin could be dissociated into three distinct fibrin fragments and bovine alpha-thrombin when denatured by 8 M urea. Thus, the bound thrombin liberated from crushed clots is a stable complex between bovine alpha-thrombin and fibrin fragments of the N-terminal regions of rabbit alpha- and gamma-chains.