京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20251号 / 医博第4210号 / 新制||医||1020(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 妹尾 浩, 教授 湊谷 謙司, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20282号 / 医博第4241号 / 新制||医||1021(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 三森 経世, 教授 鈴木 茂彦, 教授 生田 宏一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(社会健康医学) / 甲第21456号 / 社医博第90号 / 新制||社医||10(附属図書館) / 京都大学大学院医学研究科社会健康医学系専攻 / (主査)教授 滝田 順子, 教授 小杉 眞司, 教授 三森 経世 / 学位規則第4条第1項該当 / Doctor of Public Health / Kyoto University / DFAM

Light Chain Deposition Disease (LCDD) is a type of monoclonal immunoglobulin deposition disease characterized by the non-amyloid deposition of monoclonal light chains in the tubular basement membranes and Bowman’s capsule. It was first described about 3 decades ago, but due to varied clinical presentations, many differential diagnoses and low incidence, it is both underrecognized and underreported. We present a case of 85-year-old female with past medical history significant for CKD and HTN, who presented with accelerated HTN, normocytic anemia and worsening renal function. Laboratory data showed Hgb <9.5 gm/dL, MCV 93 fL, Total protein 5.9, Albumin 3.2, Calcium 8.9, Serum Creatinine 2.37, BUN 45, Urine with hematuria (50–99 erythrocytes per high-power field) and nephrotic range proteinuria. Renal biopsy showed evidence of Membranoproliferative glomerulonephritis, with immunofluorescence features indicative of a monoclonal immunoglobulin deposition disease. Bone marrow biopsy showed mildly increased plasma cells (5-7%) confirmed to be clonal (lambda light chain) by flow cytometry, negative for Congo-Red stain. Although no underlying hematological abnormality like Multiple Myeloma or Amyloidosis was observed in this case, the renal pathological findings is consistent with proliferative glomerulonephritis with monoclonal IgG lambda deposits. There is no standard of care for the management of LCDD based on rarity of this condition. Many treatment modalities including chemotherapy and stem cell transplant have been tried. A combination of high dose melphalan or Cyclophosphamide with dexamethasone is preferred for Non-IgM type monoclonal protein kidney deposition, like in this case. Bortezomib and Thalidomide-based chemotherapy have been promising in recent research. For IgM type monoclonal protein deposition, Rituximab alone or in combination with cyclophosphamide and dexamethasone are used. This patient was not a good candidate for corticosteroid and chemotherapy or stem cell transplant due to old age (>77 years) and poor functional status, therefore, was started on hemodialysis. Following dialysis, improvement in renal function and general clinical condition was evident. The prognostic factors include age, degree of renal insufficiency at presentation affecting the renal prognosis, underlying hematologic disorder and extrarenal LC deposition. In this case, despite hemodialysis, long term survival and prognosis remain poor due to her inability to tolerate chemotherapy.

No description available.

Crystallizing RNA has been an imperative facet and a challenging task in the world of RNA research. Assistive methods such as Chaperone Assisted RNA Crystallography (CARC), employing monoclonal antibody fragments (Fabs) as crystallization chaperones have enabled us to obtain RNA crystal structures by increasing the crystal contacts and providing initial phasing information. Using this technology the crystal structure of [delta]C209 P4-P6 RNA (an independent folding domain of the self-splicing Tetrahymena group I intron) complexed to Fab2 (high affinity binding Fab) has been resolved to 1.95 Å (1). Although the complexed class I ligase ribozyme has also been crystallized using CARC (2), in practice, it has been found that the crystallization of, large RNA-Fab complex remains a confrontation. The possible reason for this difficulty is that Fabs have not been optimized for crystallization when complexed with RNA. Here we have used the Surface Entropy Reduction technique (SER) for the optimization process. Candidate residues for mutations were identified based on combining results from visual inspection of [delta]C209 P4-P6/Fab2 crystal structure complex using pyMOL software and a web-based SER software. The protruding lysine and glutamate residues were mutated to a set of alanine (Super Mutant Alanine SMA) and serine (Super Mutant Serine SMS) mutant clones. Filter binding assay studies confirmed that the mutant clones bind to [delta]C209 P4-P6 with similar binding affinities as that of the parent Fab2. Large scale expression of the mutants, parent clone and [delta]C209 P4-P6 RNA were optimised. Crystal trays for [delta]C209 P4-P6 complexed with Fab2, Fab2SMA and Fab2SMS were set-up side-by-side using Hampton crystal screen kits and ~600 conditions including temperature as a variable condition were screened. Crystal screening shows significantly higher crystal-forming ratios for the mutant complexes. As the chosen SER residues are far away from the CDR regions of the Fab, the same set of mutations can be potentially applied to other Fabs binding to a variety of ribozymes and riboswitches to improve the crystallizability of the Fab-RNA complex.

During the first days after parturition, mammalian milk (colostrum) is specifically formulated to nourish newborns. Immunoglobulins are a particularly important component for newborn New World camelids, as their immune system is almost totally dependent on the intestinal transfer of colostral immunoglobulins to acquire passive immunity. In this study, colostrum samples were collected from 20 alpaca mares in the first four days after parturition and analyzed for their immunoglobulin concentration. Sampling started on the day of parturition. The associations of immunoglobulins with other components were determined. The immunoglobulin G (IgG) concentrations decreased significantly within the first four days after parturition. The correlation coefficients between IgG content and the content of various minerals were significant but variable. The correlation between IgG content and fat and lactose content was negative but between IgG content and protein content was highly positive. This strong association could be used for a brief estimation of the IgG content of the colostrum based on the measured protein concentration. The results of the present study can be used for the development of colostrum replacers where motherless rearing is required. Abstract Colostrum provides the newborn with nutrients and immunoglobulins. Immunoglobulins and their intestinal transfer play a major role in the immune system of neonates since they are born agammaglobulinemic. In this study immunoglobulin G (IgG) content was determined in alpaca colostrum and the correlations of the IgG concentration by fat, protein, lactose and minerals were calculated. Colostrum samples were collected daily from 20 multiparous alpaca mares during the first four days after parturition. The IgG concentrations were determined by radial immunodiffusion using a Camelid IgG Test Kit. The IgG concentration decreased significantly from 26,319 mg/dL on day 1 to 3848.8 mg/dL on day 4. There were significant correlations between IgG concentration and the other components of the colostrum. While the correlations between IgG and fat (r = −0.69, p ≤ 0.001) and lactose (r = −0.64, p ≤ 0.001) were negative, the correlations with protein (r = 0.91, p ≤ 0.001), magnesium (r = 0.86, p ≤ 0.001) and cobalt (r = 0.87, p ≤ 0.001) were strongly positive. Due to the strong association, the colostrum protein concentration could be used for a brief estimation of the IgG content.

Immunoglobulin (Ig) proteins, also known as antibodies, are important molecules with great variability in their amino acid sequences. Aberrantly overproduced monoclonal Ig light chain (LC) proteins may aggregate into a β-sheet featured structure, and deposit in the extracellular space; this pathologic process, called primary amyloidosis or Ig LC amyloidosis (AL) causes problems to multiple organs during the course of the disease. Post-translational modifications (PTMs), which remain to be explored, are likely an important factor affecting the formation of AL fibrils. In addition, therapeutic monoclonal antibodies (mAbs) are widely employed because of their high specificity and low side effects. Using plants as the expression platform is commercially attractive although this approach has been hampered by low protein expression yield and undesired glycosylation patterns. The investigations detailed in this dissertation focus on the analyses of Ig proteins derived from several human and plant sources. A method combining 2D SDS-PAGE separation and mass spectrometry (MS) analysis was used for de novo sequencing of Ig in a fat biopsy for which the corresponding DNA was unavailable, and for characterizing the LC proteins found in autopsied kidney, serum and urine samples from patients with AL amyloidosis whose LC-DNA was sequenced. The PTMs of each LC were extensively characterized with different enzymes and various tandem MS techniques including collision-induced dissociation (CID), higher-energy collisional dissociation (HCD) and electron transfer dissociation (ECD). PTMs observed include truncations, mono-/di-chlorination of the tyrosine residues and a nitrile group formed from the primary amine on lysine side chains. All these may play critical roles in the fibrillogenesis and/or the disease pathogenesis. Experimental evidence supports the hypothesis that the proteolytic processing of amyloidogenic LCs occurs after deposition in the organ. Characterization of a plant-derived HSV8 mAb was accomplished using high-performance liquid chromatography separation and gel display followed by various MS methods. Three N-terminal and one C-terminal truncations were found. The N-glycan moiety attached to the heavy chain was also analyzed. The MS method established helps to elucidate important structural information on therapeutic mAbs in complex mixtures, potentially contributing to optimization of plant systems that may produce more stable mAbs.

Light chain (AL) amyloidosis is a disease that occurs due to the presence of a small plasma-cell clone, which produces amyloidogenic light chains. These chains can misfold and aggregate, leading to the deposition of amyloid fibrils in tissues. If left untreated or if treatment is ineffective, this can result in irreversible organ dysfunction and eventual death. Current therapeutic treatments generally target and remove the clonal plasma cell population responsible for secreting full-length light chains which is not always effective or safe, however, a different approach to halt pathological LC misfolding would be to inhibit the amyloidogenesis cascade at its starting point. Small molecules have been identified that have the ability to bind to highly conserved residues in the interface between heavy and light chains which can be used to potentially impede the process of amyloid fibril deposition before the native FL LC can misfold or undergo proteolysis to form amyloid fibrils. To test whether small-molecule kinetic stabilizers are effective in stabilizing light chains, we measured the ability of the small molecule to bind to LCs, and the ability of light chains to aggregate and unfold in the absence and presence of small-molecule. Our findings suggest that the binding of stabilizers to the interface between variable domains of the LC dimer can increase equilibrium stability and decrease the rate of aggregation, thereby delaying the onset of amyloid formation.

No description available.