IgG Light Chain Research Articles

Novel HPV-E7 immune modulators to activate HPV-specific T cells to eliminate cervical cancer.

e18027 Background: Human papilloma virus (HPV) infection can lead to several types of cancers in both men and women. HPV+ tumor cells constitutively express the HPV-E7 antigen which can act as an oncogene to promote tumor growth and malignant transformation. Here, we report the application of novel Tavo Immune Modulator (TIM) biologics molecules which are consisted of a pMHC complex with an epitope peptide derived from HPV-E7 and co-stimulatory modulators of T cell activity. The HPV-E7 TIM molecules can specifically recognize and activate HPV-E7-specific T cells for the elimination of HPV affected cells. Methods: HPV-E7 TIM molecules were engineered as fusion molecules with HLA-A*02:01 MHC complexed with an HPV-E7 (11-20) epitope peptide at the N-termini, and various T cell costimulatory modulators at the C-termini of IgG heavy and light chains. TIM molecules were expressed in Expi293 cells and purified by Protein A affinity chromatography. Specific binding of TIM with HPV-E7 specific T cells was confirmed by immunostaining and flow cytometry. The activation and expansion of antigen specific CD8+ T cells were elucidated in T cell activation and recall assays. Results: HPV-E7 TIM molecules with various T cell co-stimulator molecules were engineered to specifically recognize HPV-specific T cells. Activation of T cells was antigen-specific and depended on the presence of an engineered T cell modulatory component on the TIM framework. The effects of various costimulatory molecules in different combinations on T cell activation were explored and an optimal combination was identified which facilitated high potency antigen-specific T cell activation. Such molecular combinations could facilitate T cell expansion and activation in T cell recall assays. Efficacy of HPV-E7 TIM molecules by inhibiting tumor growth in a syngeneic tumor model is ongoing. Conclusions: This study demonstrates that HPV-E7 TIM molecules selectively recognize and activate HPV-specific CD8+ T cells in the presence of a combination of two T cell costimulatory factors. Such novel biologics provide distinctive approaches in the treatment of HPV-related cancers and warrant further investigation. Additional in vitro and in vivo studies are ongoing to demonstrate the utility in eliminating HPV-infected tumor cells. Full data will be presented at the meeting.

Regulation of epidermal differentiation through KDF1-mediated deubiquitination of IKKα.

Progenitor cells at the basal layer of skin epidermis play an essential role in maintaining tissue homeostasis and enhancing wound repair in skin. The proliferation, differentiation, and cell death of epidermal progenitor cells have to be delicately regulated, as deregulation of this process can lead to many skin diseases, including skin cancers. However, the underlying molecular mechanisms involved in skin homeostasis remain poorly defined. In this study, with quantitative proteomics approach, we identified an important interaction between KDF1 (keratinocyte differentiation factor 1) and IKKα (IκB kinase α) in differentiating skin keratinocytes. Ablation of either KDF1 or IKKα in mice leads to similar but striking abnormalities in skin development, particularly in skin epidermal differentiation. With biochemical and mouse genetics approach, we further demonstrate that the interaction of IKKα and KDF1 is essential for epidermal differentiation. To probe deeper into the mechanisms, we find that KDF1 associates with a deubiquitinating protease USP7 (ubiquitin-specific peptidase 7), and KDF1 can regulate skin differentiation through deubiquitination and stabilization of IKKα. Taken together, our study unravels an important molecular mechanism underlying epidermal differentiation and skin tissue homeostasis.

Investigating the role of ERAD on antibody processing in glycoengineered Saccharomyces cerevisiae.

N-glycosylation plays an important role in the endoplasmic reticulum quality control (ERQC). N-glycan biosynthesis pathways have been engineered in yeasts and fungi to enable the production of therapeutic glycoproteins with human-compatible N-glycosylation, and some glycoengineering approaches alter the synthesis of the lipid-linked oligosaccharide (LLO). Because the effects of LLO engineering on ERQC are currently unknown, we characterized intracellular processing of IgG in glycoengineered Δalg3 Δalg11 Saccharomyces cerevisiae strain and analyzed how altered LLO structures affect endoplasmic reticulum-associated degradation (ERAD). Intracellular IgG light and heavy chain molecules expressed in Δalg3 Δalg11 strain are ERAD substrates and targeted to ERAD independently of Yos9p and Htm1p, whereas in the presence of ALG3 ERAD targeting is dependent on Yos9p but does not require Htm1p. Blocking of ERAD accumulated ER and post-Golgi forms of IgG and increased glycosylation of matα secretion signal but did not improve IgG secretion. Our results show ERAD targeting of a heterologous glycoprotein in yeast, and suggest that proteins in the ER can be targeted to ERAD via other mechanisms than the Htm1p-Yos9p-dependent route when the LLO biosynthesis is altered.

Bi-functional IgG-lysosomal enzyme fusion proteins for brain drug delivery

Most lysosomal storage disorders affect the central nervous system. However, lysosomal enzymes do not cross the blood-brain barrier (BBB), and intravenous enzyme infusion is not effective for the brain. Lysosomal enzymes can be re-engineered for BBB transport as IgG-enzyme fusion proteins, where the IgG domain is a monoclonal antibody (MAb) against an endogenous BBB receptor/transporter, and which acts as a molecular Trojan horse to deliver the enzyme to brain. However, the problem is retention of high enzyme activity following enzyme fusion to the IgG. The present investigation shows this is possible with a versatile approach that employs fusion of the enzyme to either the IgG heavy chain or light chain using a long flexible linker. The model IgG is a chimeric monoclonal antibody (MAb) against the human insulin receptor (HIR). The enzyme activity of the HIRMAb-enzyme fusion protein is preserved for hexosaminidase A, which is mutated in Tay Sachs disease, for protein palmitoylthioesterase-1, which is mutated in Batten disease type 1, acid sphingomyelinase, which is mutated in Niemann Pick disease type A, and beta galactosidase-1, which is mutated in GM1 gangliosidosis.

Serum amyloid P deposition is a sensitive and specific feature of membranous-like glomerulopathy with masked IgG kappa deposits

Membranous-like glomerulopathy with masked IgG kappa deposits (MGMID) is a recently described pattern of glomerulonephritis with a unique histopathology. The pattern is characterized by subepithelial and/or mesangial immune deposits that are “masked”, to immunoglobulin staining by routine immunofluorescence but strongly stain for IgG and kappa light chain after protease digestion. Patients with this pattern of glomerulonephritis are most commonly young females presenting with proteinuria and a vague history of autoimmune disease such as low titer antinuclear antibodies. Here we compared the mass spectrometry profile of laser capture microdissected glomeruli from nine MGMID renal biopsies with eight biopsies showing other patterns of membranous glomerulopathy. The protein most significantly increased in MGMID was serum amyloid P. Immunostaining showed serum amyloid P colocalized with IgG in the glomeruli of MGMID but not with PLA2R-associated membranous glomerulopathy. Serum amyloid P was positive in the glomeruli of all 32 MGMID biopsies but negative in biopsies of other types of membranous glomerulopathies such as those associated with PLA2R and THSD7A. There were four biopsies with glomerular serum amyloid P staining among the 173 biopsies that did not fulfill criteria for MGMID or amyloidosis. All four of these biopsies with positive serum amyloid P staining had a membranous pattern of glomerulopathy with IgG kappa deposits that only differed from MGMID by the lack of “masking”. Thus, positive staining within glomerular deposits for serum amyloid P identifies a unique form of glomerulonephritis likely sharing a common pathophysiologic mechanism of disease.

A case of acute monocytic leukemia complicated with multiple myeloma

A case of acute monocytic leukemia complicated with multiple myeloma

The role of routine pentavalent screening immunofixation with serum electrophoresis: A retrospective cost-benefit analysis

The reliable identification of serum paraproteins requires an immunofixation (IFE) using specific antisera and an electrophoretogram (EPG) on the same sample. Although serum IFE is more labour-intensive, requiring at least six sample spaces per sample, paraproteins may be missed if clinicians request a serum EPG only. There is presently no consensus amongst laboratories on the optimum testing algorithm to minimise missed paraproteins. Performing a single-lane screening IFE with pooled pentavalent antisera (IgG, IgA, IgM,kappa and lambda light chains) routinely may minimise the risk of missed paraproteins.

Oligoclonal IgG and free light chains – comparison between agarose and polyacrylamide isoelectric focusing

Oligoclonal IgG and free light chains – comparison between agarose and polyacrylamide isoelectric focusing

Long Term Outcomes of High Dose Therapy and Autologous Hematopoietic Cell Transplant for Lymphoplasmacytic Lymphoma-Associated Systemic Amyloidosis

Background Lymphoplasmacytic lymphoma (LPL) is associated with production of monoclonal paraproteins. Rarely, these paraproteins misfold, aggregate and form amyloid fibrils which deposit in organs and disrupt their structure and function. Several reports have described this association; however, transplant outcomes in patients with LPL and co-existing systemic amyloidosis are not well described. Methods We identified fifty patients with co-existing LPL and systemic amyloidosis diagnosed between 1997 and 2018 who were treated at the Memorial Sloan Kettering Cancer Center. Of these, eight patients underwent high dose therapy (HDT) followed by autologous hematopoietic cell transplant (AHCT) and are subjects of the current study. Results Among the eight patients, the median age was 60 years (range 55-70 years) with 37.5% females. Lymph nodes were the most commonly involved organ (N=6), followed by the heart (N=5), Kidneys (N=4), lungs (N=4) and peripheral nervous system (N=4). Median number of organs involved per patient was 4 (range 1-6). The monoclonal protein was IgM (N=5), IgG (N=2) and free lambda light chain (N=1); lambda light chain was involved in seven of eight patients. Median M spike was 0.43 gm/dL (range 0.2-1.3 gm/dL) and dFLC was 7.93 mg/dL (range 1.89-22.87 mg/dL). All patients had less than 10% plasma cell involvement of bone marrow. Induction regimens varied. Five patients received HDT/AHCT as part of initial therapy and three patients were transplanted at relapse (range 3-6 lines of prior therapy). CD34+ cells were mobilized using GCSF with Plerixafor (N=5), Rituximab, cyclophosphamide and GCSF (N=2) and cyclophosphamide alone (N=1). Median CD34+ cell yield was 8.74 × 106 cells/kg (range 4.5-12.2 × 106 cells/kg). High dose therapy was BCNU, etoposide, Ara-C and melphalan (BEAM) (N=2) and melphalan alone (N=6). Among patients conditioned with melphalan, 3 each received 140 mg/m2 and 200 mg/m2 based on risk adapted dosing. Median number of CD34+ cells infused was 5.325 × 106 cells/kg (range 3.2-5.9 × 106 cells/kg). Hematologic response following HDT/AHCT was very good partial response (VGPR) or better in five of the six patients with long term follow up. At median follow up of 64.5 months (range 3-116 months), all patients were alive at last contact. Only one patient relapsed more than 4 years post-transplant and has responded to salvage Daratumumab. Conclusion Amyloidosis can complicate lymphoplasmacytic lymphoma and pose a diagnostic and therapeutic challenge. Lymph node and lung involvement are common and may serve as distinguishing features. With over 5 years of follow up, all patients are alive with only a single patient who progressed. While numbers are small and patients were selected, these data indicate excellent long-term outcomes in patients with LPL-associated amyloidosis treated with risk adapted melphalan and suggest BEAM conditioning may not be essential.

Immunohistochemical expression of secretory proteins in pneumonic rat lungs

The present study evaluated the expression of secretory proteins involving surfactant proteins (SP-A, SP-B, proSP-C), Clara cell secretory protein (CCSP), and thyroid transcription factor-I (TTF-I), together with lambda light chain IgG (λ-IgG), proliferating cell nuclear antigen (PCNA), and lymphocytic phenotypes (T and B cells) in the pneumonic rat lungs. The most prominent gross lesion was severe pulmonary abscession. The lungs showed severe parenchymal destruction and necrosis encapsulated by fibrous tissue. Immunostaining for SPs displayed evident hyperplasia of type II pneumocytes. Immunoreaction to SP-A and SP-B occurred strongly on the luminal surface of type II cells, and to proSP-C in the perinuclear area. CCSP immunolabeling was seen in the cytoplasm of the lining epithelium of the distal airways. The nuclei of type II cells labeled positively for TTF-I. The λ-IgG positive reaction was found in the cytoplasm of plasmacytes and B cells. PCNA staining confirmed proliferation of type II cells and fibroblasts. Moderate numbers of CD3+ T cells, but few CD79αcy+ B cells, were found in the lungs. Statistical analysis found high significance (P < 0.001, except for λ-IgG) between groups. The results of the study revealed that type II cells proliferate highly to restore the damaged lungs and that CCSP and SPs may be used to identify tumors originating from Clara cells and type II pneumocytes of rats, respectively.

Serum markers in the differential diagnosis of Waldenstrom macroglobulinemia and other IgM monoclonal gammopathies.

IgM monoclonal gammopathy can be present in a broad spectrum of diseases. We evaluated the value of serum markers in the differential diagnosis of Waldenstrom macroglobulinemia (WM) and other types of IgM monoclonal gammopathies. We included patients who were first admitted to hospital and identified as having IgM monoclonal gammopathy by serum immunofixation electrophoresis (sIFE). We evaluated basic clinical features, sIFE, diagnosis, and serum markers. Furthermore, we applied the receiver operating characteristic (ROC) curve to analyze the differential diagnosis value of serum markers for WM. Finally, we used logistic regression and ROC curve to analyze the differential diagnosis value of multimarker combinations to identify WM. IgM monoclonal gammopathy was most frequently found in patients with Waldenstrom macroglobulinemia, followed by monoclonal gammopathy of undetermined significance (MGUS), B-cell non-Hodgkin Lymphoma (B-NHL), and multiple myeloma (MM). Serum markers showed significant differences among the four diseases. The diagnostic markers LDH, IgM, IgG, IgA, and serum light chain К had higher diagnostic efficiency. Among these markers, serum IgM provided the highest diagnostic efficiency. Additionally, the combined use of all five serum markers provided the most effective diagnosis. The five serum markers, LDH, IgM, IgG, IgA, and К, each yielded a specific efficacy in differential diagnosis of WM. The single marker with the highest diagnostic efficiency was the serum IgM level. However, a combination of multiple serum markers was better than the use of a single marker in diagnosing WM. The combined use of all five serum markers provided the most effective diagnosis, with an AUC of .952 and sensitivity and specificity of 87.8% and 86.9%, respectively.

Polyoxometalate-Coated Magnetic Nanospheres for Highly Selective Isolation of Immunoglobulin G.

Polyoxometalate [{a-PW11O39Zr(μ-OH)(H2O)}2]8- (POM1) is first prepared by sandwiching ZrIV among 2 mono-lacunary α-Keggin polyoxometalates, and then novel magnetic nanoparticles (NPs), Fe3O4@polyethyleneimine (PEI)@POM1, are fabricated by coating POM1 onto the surface of magnetic Fe3O4@PEI NPs under electrostatic interaction. The obtained Fe3O4@PEI@POM1 NPs are characterized by Fourier transform infrared, zeta potential, vibrating sample magnetometer, transmission electron microscopy, energy-dispersive X-ray spectroscopy, and X-ray diffraction. Ascribed to the hydrogen-bonding and electrostatic interactions, the NPs exhibit high adsorption selectivity toward IgG, and the adsorption capacity is high up to 304 mg g-1 under optimal adsorption conditions. By using 0.01% cetyl trimethylammonium bromide to strip the adsorbed protein species, an elution efficiency of 95% is achieved. The feasibility of Fe3O4@PEI@POM1 NPs in real-world sample assay has been demonstrated by the selective isolation of IgG heavy chain and light chain from human serum, as confirmed by the sodium dodecyl sulfate polyacrylamide gel electrophoresis assay.

Human and murine IL2 receptors differentially respond to the human-IL2 component of immunocytokines

ABSTRACTThe humanized immunocytokine, hu14.18-IL2 (ICp), leads to the immune cell-mediated destruction of GD2-expressing tumors in mouse models, resulting in potent antitumor effects with negligible IL2-related toxicity. In contrast, when ICp is used clinically, antitumor activity is accompanied by dose-limiting IL2-related toxicities. These species-specific differences in ICp toxicity may be linked to differential binding to mouse vs. human IL2 receptors (IL2Rs). We evaluated immunocytokines genetically engineered to preferentially bind either high-affinity αβγ-IL2Rs or intermediate-affinity βγ-IL2Rs. These ICs have the IL2 fused to the C-terminus of the IgG light chains rather than the heavy chains. We found that IC35, containing intact huIL2, maintained activation of human and mouse αβγ-IL2Rs but exhibited a 20-fold reduction in the ability to stimulate human βγ-IL2Rs, with no activation of mouse βγ-IL2Rs at the concentrations tested. The reduced ability of IC35 to stimulate human βγ-IL2Rs (associated with IL2-toxicities) makes it a potential candidate for clinical trials where higher clinical IC doses might enable better tumor targeting and increased antitumor effects with less toxicity. Contrastingly, ICSK (IC with an IL2 mutein that has enhanced binding to the IL2R β-chain) showed increased activation over ICp on mouse βγ-IL2Rs, with a dose-response curve similar to that seen with IC35 on human βγ-IL2Rs. Our data suggest that ICSK might be used in mouse models to simulate the anticipated effects of IC35 in clinical testing. Understanding the differences in species-dependent IL2R activation should facilitate the design of reagents and mouse models that better simulate the potential activity of IL2-based immunotherapy in patients.

Metaphylactic effect of minerals on the immune response, biochemical variables and antioxidant status of newborn calves

The aim of this study was to evaluate whether injectable zinc and copper affect host immune responses and antioxidant status of newborn calves. For this study, 19 newborn calves were divided into two groups. The control group consisted of 10 animals; and the treated group consisted of nine animals that received copper edetate (Cu-ed) and zinc edetate (Zn-ed) subcutaneously at the first day of life at doses of 0.3mg/kg and 1.0mg/kg respectively. Blood and faecal samples were collected for laboratory analyses (seric biochemistry, proteinogram, antioxidant enzymes and parasitological examination) on days 10, 20 and 30 after birth. On day 10, treated animals showed increased levels of total proteins, as well as increased globulin levels compared to the control group, a finding probably related to the increase in ceruloplasmin and IgG heavy chain. Thirty days after mineral metaphylactic administration, IgG light chain and acid glycoprotein levels significantly increased in treated animals (p<.05). There were no significant differences between groups regarding the biochemical analyses (triglycerides, cholesterol, glucose and urea). On the other hand, the superoxide dismutase and catalase activities increased on day 10 after treatment. In the control group, eight animals showed severe diarrhoea and one died 8days after birth. Two animals from this group showed mild diarrhoea. Only three treated animals had severe diarrhoea, and six showed signs of mild diarrhoea. All animals that showed severe diarrhoea (control=8; treated=3) had hyperthermia (over 39.5°C), and therefore, antibiotic therapy was administered (sulfadiazine and trimethoprim) for five consecutive days. In summary, Zn-ed and Cu-ed decreased the frequency and intensity of diarrhoea, modulated superoxide dismutase (SOD) and catalase (CAT) enzymes and also heightened the immune responsiveness of newborn calves, suggesting a new approach to improve cattle performance and minimize the occurrence of diarrhoea.

328 EBV and HHV1 Positive CNS Inflammatory Pseudotumor: A Case Report

Inflammatory pseudotumor (IPT) is a rare benign lesion of unknown cause, commonly involving the lung and orbit but found in virtually every body site. IPT was first described in 1954 based on imaging findings that mimicked those of malignant tumors. This rare entity, which consists of mixed inflammatory cells and myofibroblastic spindle cells, is often associated with Epstein-Barr virus (EBV) positivity. Here we are presenting a case of a 56-year-old Hispanic woman with a history of headaches, dizziness, and frequent falls. MRI of the head demonstrated a right temporal lesion with enhancement along the temporal convexity, extending into the insula. A lumbar puncture yielded atypical lymphocytes with increased nuclear to cytoplasmic ratio, irregular nuclear contours, and coarse chromatin. Cerebral spinal fluid flow cytometry revealed a mixed population of cells, including B-cells expressing CD19, CD20, kappa, and lambda. A subsequent open biopsy revealed areas of dense, mixed infiltrates consisting of lymphocytes, histiocytes, plasma cells and eosinophils. Immunohistochemical staining demonstrated positivity for IgG, IgG4, IgA, kappa and lambda light chains, CD3, CD10, CD15, CD20, CD79A, BCL-2, MUM-1, and CD138. A neu N stain disclosed focal, minimal neuronal loss while GFAP demonstrated background reactive astrocytes. The lesion showed positivity for EBV, which is consistent with current literature, but also for human herpesvirus 1 (HHV1) proven by polymerase chain reaction (PCR). This case is noteworthy for the rarity of presentation, diagnostic challenges and positivity for EBV and HHV1 by PCR.

Purification and characterization of monoclonal IgG antibodies recognizing Ebola virus glycoprotein.

PurposeThe goal of this study was to purify and characterize Ebola virus glycoprotein (GP)-specific IgG antibodies from hybridoma clones.Materials and MethodsFor hybridoma production, mice were injected by intramuscular-electroporation with GP DNA vaccines, and boosted with GP vaccines. The spleen cells were used for producing GP-specific hybridoma. Enzyme-linked immunosorbent assay, Western blot assay, flow cytometry, and virus-neutralizing assay were used to test the ability of monoclonal IgG antibodies to recognize GP and neutralize Ebola virus.ResultsTwelve hybridomas, the cell supernatants of which displayed GP-binding activity by enzyme-linked immunosorbent assay and the presence of both IgG heavy and light chains by Western blot assay, were chosen as a possible IgG producer. Among these, five clones (C36-1, D11-3, D12-1, D34-2, and E140-2) were identified to secrete monoclonal IgG antibodies. When the monoclonal IgG antibodies from the 5 clones were tested for their antigen specificity, they recognized GP in an antigen-specific and IgG dose-dependent manner. They remained reactive to GP at the lowest tested concentrations (1.953–7.8 ng/mL). In particular, IgG antibodies from clones D11-3, D12-1, and E140-2 recognized the native forms of GP expressed on the cell surface. These antibodies were identified as IgG1, IgG2a, or IgG2b kappa types and appeared to recognize the native forms of GP, but not the denatured forms of GP, as determined by Western blot assay. Despite their GP-binding activity, none of the IgG antibodies neutralized Ebola virus infection in vitro, suggesting that these antibodies are unable to neutralize Ebola virus infection.ConclusionThis study shows that the purified IgG antibodies from 5 clones (C36-1, D11-3, D12-1, D34-2, and E140-2) possess GP-binding activity but not Ebola virus-neutralizing activity.

A Case of Immunotactoid Glomerulopathy (ITG) Associated with Monoclonal B-Cell Lymphocytosis (MBL) with Chronic Lymphocytic Leukemia (CLL) Phenotype Treated with Obinutuzumab

Abstract Introduction: Although 44 to 90% of CLL cases have kidney infiltration on autopsy, renal involvement generally goes undetected and renal failure is unusual (Da'As N et al, Eur J Haematol 2001). There is no standard therapy for nephrotic syndrome (NS) in CLL. Treatment of CLL itself may result in regression of NS in most patients and treatment with alkylating agents, cyclosporine A, fludarabine and even splenectomy have been shown to be effective. Controversy exists regarding effectiveness of rituximab in CLL-associated nephrotic syndrome. (Yahata et al, Am J Nephrol 2000). Here we report the first case, to our knowledge, of MBL/CLL-associated NS successfully treated with obinutuzumab. Case Report: 71 years old female presented to ER with hypertensive urgency and anasarca. Past medical history was relevant for progressive hypertension (HTN) for 1 year prior. Creatinine (Cr) was 1.9mg/dL, BUN 45mg/dL, urine protein was >300mg/dl on urinalysis and subsequent 24hr urine protein was 9824mg/24hrs. Patient (pt) had renal ultrasound revealing morphologically normal kidneys without evidence of renal artery stenosis. Echocardiogram was normal. CBC revealed anemia (Hgb 9.2mg/dl) and mild thrombocytopenia (plt 121), WBC count was 7.6 x 10e3/mcl with relative lymphocytosis (47.46 %, ALC 3.594 x10e3 /mcl). On left kidney core biopsy 25 glomeruli were seen by light microscopy, 1 of which was globally sclerotic. The remaining glomeruli showed diffuse duplication of basement membranes with cellular interposition and mild to moderate intracapillary hypercellularity with endothelial cell swelling and/or leukocyte accumulation (Figure 1). Proximal tubules displayed patchy degenerative and regenerative changes including loss of apical brush border, nuclear enlargement and prominent nucleoli, and few interstitial foam cells (Figure 2). By immunofluorescence microscopy, glomeruli stained for IgG, C3, C1 and kappa light chain. There was no staining for lambda light chain. Stains for IgG heavy chain subtypes were positive for IgG1 only (Figure 3), consistent with monoclonal IgG1-kappa deposition. Electron microscopy revealed electron dense deposits in the mesangium, also in subendothelial and subepithelial locations in the capillary walls. On high-power magnification, these deposits revealed a microtubular substructure (i.e., with a central lumen) (Figure 4). These pathologic findings were consistent with ITG. Findings of ITG with IgG1 deposit as well as bicytopenia with relative lymphocytosis warranted further workup for gammopathy. SPEP revealed nonselective protein loss (total protein was 4.5g/dL, Albumin 2.9g/dL, total globulin 1.6g/dL, Albumin/Globulin ratio 1.8) with non-detectable M spike. UPEP was negative. IgG was 203mg/dL, IgA 45mg/dL, IgM 50mg/dL. Serum free kappa light chain was 35.79mg/L with normal Lambda light chain level of 20.08 mg/L with Kappa/Lambda ratio 1.78. Beta-2 microglobulin level was 6.4mg/L. Peripheral blood flow cytometry was ordered, which revealed 20% monoclonal B-cells (CD5+/CD23+) with CLL phenotype. Pt was started on prednisone 60mg daily for 7 days for ITG and decision was made to start CLL-targeted treatment with Obinutuzumab for 6 cycles, with the intent of improving the glomerulopathy, as pt did not meet IWCLL-2008 criteria for initiation of treatment for CLL. Obinutuzumab was given in the usual schedule of 100 mg on day 1, 900 mg on day 2 and 1000 mg on day 8 and 15 of first month, followed by 1000 mg every 28 days for 5 additional cycles. Repeat flow cytometry in 6 months after diagnosis showed complete response of monoclonal lymphocytosis (minimal residual disease negative). There was complete normalization of kidney function (Cr 0.73mg/dl and glomerular filtration rate 78ml/min) and no further evidence of proteinuria or edema. Hypertension has fully resolved, without additional need for anti-hypertensives. 30 months post treatment pt remains in remission for both MBL and nephrotic syndrome. Discussion: Treatment with obinutuzumab may lead to complete resolution and sustained remission of nephrotic syndrome associated with CLL. The more efficient B-cell depletion achieved with obinutuzumab may make this a better treatment choice for CLL associated NS than rituximab. Further evaluation with clinical trials is warranted. Download : Download high-res image (128KB) Download : Download full-size image Disclosures Skarbnik: Novartis: Speakers Bureau; Abbvie: Other: Ad board, Speakers Bureau; Genentech: Speakers Bureau; Gilead: Speakers Bureau; Seattle Genetics: Speakers Bureau.

Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find neurodegenerative diseases such as Alzheimer's and Parkinson's disease afflicting primarily the central nervous system, and systemic amyloidosis where serum amyloid A, transthyretin and IgG light chains deposit as amyloid in liver, carpal tunnel, spleen, kidney, heart, and other peripheral tissues. Amyloid has been known and studied for more than a century, often using amyloid specific dyes such as Congo red and Thioflavin T (ThT) or Thioflavin (ThS). In this paper, we present heptamer-formyl thiophene acetic acid (hFTAA) as an example of recently developed complements to these dyes called luminescent conjugated oligothiophenes (LCOs). hFTAA is easy to use and is compatible with co-staining in immunofluorescence or with other cellular markers. Extensive research has proven that hFTAA detects a wider range of disease associated protein aggregates than conventional amyloid dyes. In addition, hFTAA can also be applied for optical assignment of distinct aggregated morphotypes to allow studies of amyloid fibril polymorphism. While the imaging methodology applied is optional, we here demonstrate hyperspectral imaging (HIS), laser scanning confocal microscopy and fluorescence lifetime imaging (FLIM). These examples show some of the imaging techniques where LCOs can be used as tools to gain more detailed knowledge of the formation and structural properties of amyloids. An important limitation to the technique is, as for all conventional optical microscopy techniques, the requirement for microscopic size of aggregates to allow detection. Furthermore, the aggregate should comprise a repetitive β-sheet structure to allow for hFTAA binding. Excessive fixation and/or epitope exposure that modify the aggregate structure or conformation can render poor hFTAA binding and hence pose limitations to accurate imaging.

Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging.

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find neurodegenerative diseases such as Alzheimer's and Parkinson's disease afflicting primarily the central nervous system, and systemic amyloidosis where serum amyloid A, transthyretin and IgG light chains deposit as amyloid in liver, carpal tunnel, spleen, kidney, heart, and other peripheral tissues. Amyloid has been known and studied for more than a century, often using amyloid specific dyes such as Congo red and Thioflavin T (ThT) or Thioflavin (ThS). In this paper, we present heptamer-formyl thiophene acetic acid (hFTAA) as an example of recently developed complements to these dyes called luminescent conjugated oligothiophenes (LCOs). hFTAA is easy to use and is compatible with co-staining in immunofluorescence or with other cellular markers. Extensive research has proven that hFTAA detects a wider range of disease associated protein aggregates than conventional amyloid dyes. In addition, hFTAA can also be applied for optical assignment of distinct aggregated morphotypes to allow studies of amyloid fibril polymorphism. While the imaging methodology applied is optional, we here demonstrate hyperspectral imaging (HIS), laser scanning confocal microscopy and fluorescence lifetime imaging (FLIM). These examples show some of the imaging techniques where LCOs can be used as tools to gain more detailed knowledge of the formation and structural properties of amyloids. An important limitation to the technique is, as for all conventional optical microscopy techniques, the requirement for microscopic size of aggregates to allow detection. Furthermore, the aggregate should comprise a repetitive β-sheet structure to allow for hFTAA binding. Excessive fixation and/or epitope exposure that modify the aggregate structure or conformation can render poor hFTAA binding and hence pose limitations to accurate imaging.

Abolishment of proximal tubule albumin endocytosis does not affect plasma albumin during nephrotic syndrome in mice

The megalin/cubilin receptor complex is required for proximal tubular endocytosis and degradation of filtered albumin. An additional high-capacity retrieval pathway of intact albumin for the recovery of large amounts of filtered albumin has been proposed, possibly involving cooperation between megalin/cubilin and the neonatal Fc receptor. To clarify the potential role of such a pathway, we examined the effects of megalin/cubilin gene inactivation on tubular albumin uptake and plasma albumin levels in nephrotic, podocin knockout mice. Immunofluorescence microscopy of megalin/cubilin/podocin knockout mouse kidneys demonstrated abolishment of proximal tubule albumin uptake, in contrast to the excessive albumin accumulation observed in podocin knockout mice compared to controls. Correspondingly, urinary albumin excretion was increased 1.4 fold in megalin/cubilin/podocin compared to podocin knockout mice (albumin/creatinine: 226 vs. 157 mg/mg). However, no difference in plasma albumin levels was observed between megalin/cubilin/podocin and podocin knockout mice, as both were reduced to approximately 40% of controls. There were no differences in liver albumin synthesis by mRNA levels and protein abundance. Thus, megalin/cubilin knockout efficiently blocks proximal tubular albumin uptake in nephrotic mice but plasma albumin levels did not differ as a result of megalin/cubilin-deficiency, suggesting no significance of the megalin/cubilin-pathway for albumin homeostasis by retrieval of intact albumin.