Unveiling renal pathology's potential: exploring a rare subtype of amyloid - apolipoprotein CII amyloidosis in the youngest patient: a case report and literature review.

In this clinical case report, we present a rare subtype of amyloidosis, apolipoprotein CII (apo CII), which was diagnosed through a renal biopsy and subsequently confirmed by identifying the p.K41T mutation via germline DNA sequencing. Upon reviewing the literature, five patients exhibiting identical mutation were identified via renal biopsy, while an additional patient was diagnosed through biopsies of the fat pad and bone marrow. Notably, our patient is the youngest recorded case. We pioneered the application of immunofluorescence and immunogold electron microscopy techniques for apo CII evaluation. Our report provides a detailed description of this case, supplemented by an extensive review encompassing apo CII, documented instances of apo CII amyloidosis with renal or systemic involvement, and potential underlying mechanisms.

Punctate IgG staining particles localize in the budding ballooning clusters of reactive foot processes in minimal change disease.

The etiology of minimal change disease (MCD) remains a mystery as the only characteristic findings are the diffuse effacement of foot processes seen on electron microscopy (EM). Punctate IgG staining found floating outside glomerular capillary loops in MCD cases was recently identified as autoimmune antibodies against nephrin of podocytes. We hypothesized that the punctate IgG staining is located on budding ballooning clusters (BBC) of reactive foot processes in Bowman's space found on EM. We identified seven patients with MCD cases showing IgG staining that were subsequently evaluated for BBC on EM. We concurrently examined 12 negative controls, either unremarkable cases or tubulointerstitial diseases, by EM. Immunogold labeling was performed to confirm the presence of IgG and determine localization. In seven MCD cases, there were positive punctate IgG staining particles outside of the glomerular basement membranes (GBM) along with concurrent punctate staining for C3, kappa, and lambda. By EM, all seven (100%) MCD cases revealed BBC that was characterized by ballooning foot processes ranging from 1 to 6 µm and was either budding or detached from the GBM in 3-7 clusters; no electron-dense materials were seen in BBC. BBC was also seen in only 1 of 12 (8%) negative controls. Immunogold labeling identified IgG particles within BBC of MCD by EM, but not in the negative control. Our data suggest that BBC are EM structures of reactive foot processes that are most likely correlated with punctate IgG staining seen in cases of MCD, supported by immunogold labeling for IgG.

Large-scale purification of functional AAV particles packaging the full genome using short-term ultracentrifugation with a zonal rotor.

Adeno-associated virus (AAV) vector-based gene therapy is potentially curative for various genetic diseases; however, the development of a scalable purification method for full-genome AAV vectors remains crucial to increase productivity and reduce cost of GMP production. In this study, we developed a large-scale short-term purification method for functional full-genome AAV particles by using 2-step cesium chloride (CsCl) density-gradient ultracentrifugation with a zonal rotor. The 2-step CsCl method with a zonal rotor improves separation between empty and full-genome AAV particles, reducing the ultracentrifugation time (4-5 h) and increasing the AAV volume for purification. The highly purified full-genome AAV particles were confirmed by analytical ultracentrifugation (AUC), droplet digital PCR (ddPCR) in the whole region of the AAV vector genome, transduction efficiency in target cells, and transmission electronic microscopy (TEM). The high-purity AAV9 particles were obtained using culture supernatant during vector preparation rather than cell lysate. CsCl could be simply removed by a hydroxyapatite column. Interestingly, ddPCR analysis revealed that "empty" AAV particles contain small fragments of the inverted terminal repeat (ITR), probably due to unexpected packaging of Rep-mediated ITR fragments. This large-scale functional AAV vector purification with ultracentrifugation would be effective for gene therapy.

Clinicopathologic predictors of renal outcomes in light chain cast nephropathy: a multicenter retrospective study.

Light chain cast nephropathy (LCCN) in multiple myeloma often leads to severe and poorly reversible acute kidney injury. Severe renal impairment influences the allocation of chemotherapy and its tolerability; it also affects patient survival. Whether renal biopsy findings add to the clinical assessment in predicting renal and patient outcomes in LCCN is uncertain. We retrospectively reviewed clinical presentation, chemotherapy regimens, hematologic response, and renal and patient outcomes in 178 patients with biopsy-proven LCCN from 10 centers in Europe and North America. A detailed pathology review, including assessment of the extent of cast formation, was performed to study correlations with initial presentation and outcomes. Patients presented with a mean estimated glomerular filtration rate (eGFR) of 13 ± 11 mL/min/1.73 m2, and 82% had stage 3 acute kidney injury. The mean number of casts was 3.2/mm2 in the cortex. Tubulointerstitial lesions were frequent: acute tubular injury (94%), tubulitis (82%), tubular rupture (62%), giant cell reaction (60%), and cortical and medullary inflammation (95% and 75%, respectively). Medullary inflammation, giant cell reaction, and the extent of cast formation correlated with eGFR value at LCCN diagnosis. During a median follow-up of 22 months, mean eGFR increased to 43 ± 30 mL/min/1.73 m2. Age, ß2-microglobulin, best hematologic response, number of cortical casts per square millimeter, and degree of interstitial fibrosis/tubular atrophy (IFTA) were independently associated with a higher eGFR during follow-up. This eGFR value correlated with overall survival, independently of the hematologic response. This study shows that extent of cast formation and IFTA in LCCN predicts the quality of renal response, which, in turn, is associated with overall survival.

Congenital granular cell epulis: 24 new cases with more differences than similarities to granular cell tumor.

Congenital granular cell epulis (CGCE) is a rare tumor of gingiva that is exclusive to newborns, has marked female predominance, and is rarely associated with other abnormalities. Although benign in behavior, CGCE can be lethal by obstruction of respiration and/or deglutition and can require a multidisciplinary team of specialist at birth for survival of an otherwise normal infant. Histologically, CGCE resembles granular cell tumor (GCT), but unlike GCT, which is Schwannian-derived, derivation of CGCE remains an enigma, largely because of its low prevalence. This study presents 24 new cases of CGCE, the largest series since the original description 150 years ago and permits detailed study of homogeneity of cases diagnosed as CGCE as well as detailed comparisons of CGCE with GCT by clinical, morphological, immunohistochemical, and ultrastructural studies. The data show homogeneity within the CGCE cases, more differences than similarities between CGCE and GCT, and no immunohistochemical staining for common placental proteins/hormones in CGCE. The findings support a primitive mesenchymal cell origin, and a progressive degenerative process in CGCE, rather than neoplasia. Prenatal detection of this lesion is important to facilitate adequate preparations for support of these infants during labor and delivery.

Renal amyloidosis: Pathogenesis.

For many years amyloidosis was considered an extremely rare, somewhat mysterious disease. However, in the last 2-3 decades its pathogenesis, particularly that of renal amyloidosis has been carefully dissected in the research laboratory using in-vitro and, to a lesser extent, in-vivo models. These have provided a molecular understanding of sequential events that take place in the renal mesangium leading to the formation of amyloid fibrils and eventual extrusion into the mesangial matrix, which itself becomes seriously damaged and, in due time, replaced by the fibrillary material. Amyloid, once considered to be an "inert" substance, has been proven to be involved in crucial biological processes that result in the destruction and eventual replacement of normal renal constituents. This review centers on mechanisms involved in the renal glomerular amyloidosis to understand its pathogenesis.

Electron microscopy in renal pathology: overall applications and guidelines for tissue, collection, preparation, and stains.

Electron microscopy is a mainstay in the analysis of renal biopsies, where it is typically employed in a correlative fashion along with light and immunofluorescence microscopy. Despite the development of a growing armamentarium of molecular and biochemical analytic methods as well as new immunostains with a widening panel of immunoreactants, electron microscopy remains crucial to the diagnosis of a number of disorders involving the renal glomerulus, vasculature, and tubulointerstitial compartment. The number of renal biopsies continues to grow and the indications for these biopsies continue expanding together with our understanding of disease processes. Proper collection of biopsies and careful analysis of data emanating from diagnostic modalities, clinical information, imaging, gross and microscopic tissue analysis, including a wide range of ancillary studies, represent the essential paradigm for generating detailed diagnoses with clinical significance. This communication offers a guide to the pre-analytic and analytic process for renal biopsy examination, discusses diagnostic keys and pitfalls for an important category of renal diseases (immune complex disorders), and provides an introduction to a useful adjunct diagnostic method (ultrastructural immunolabeling). Renal pathologists should render expert diagnoses that guide patient management, provide prognostic information and lead to targeted new therapeutic interventions that are currently available.

Renal amyloidosis with emphasis on the diagnostic role of electron microscopy.

Our understanding of renal diseases with structured deposits has improved in the last two decades with the development of new diagnostic techniques that also changed the role of ultrastructural pathology in diagnostic decision-making. This review article addresses the current role of electron microscopy in the evaluation of structured deposits and discusses the impact of new developments. The diagnosis in a subset of structured deposits, amyloidosis, relies on morphologic and tinctorial characteristics at the light microscopic level. Congo red staining of tissue with demonstrable birefringence upon polarization has been regarded as the mainstay during tissue evaluation; however, there are pitfalls that must be considered, and electron microscopy remains a crucial adjunct investigative tool. Ultrastructurally the amyloid fibrils are unique with their characteristic appearance. They are randomly arranged, rigid, criss-crossing, non-branching, 7-15 nm (0.07-0.15 um) in diameter and of variable length. The morphology of fibrils is very similar in the different types of amyloidosis. By scanning electron microscopy amyloid fibrils appear artfully displayed. Immunofluorescence and immunohistochemical stains can be used to characterize the type of amyloidosis while mass spectroscopy is extremely useful in cases where typing of the amyloid using the above-mentioned techniques is difficult or equivocal.

Monoclonal glomerulopathy with features of cryoglobulinemic glomerulopathy in murine multiple myeloma model.

In vivo and animal models of monoclonal light chain-associated renal diseases are limited. The Vk*MYC transgenic model with multiple myeloma in 50-70 weeks old mice with renal involvement has been reported before, but detailed renal pathologic changes have not been well documented. This study fully investigated pathologic changes in the kidneys of Vk*MYC transgenic model using light microscopy, immunofluorescence stains for kappa and lambda light chains, and electron microscopy. Compared to the kidneys of wild-type mice, the kidneys of transgenic mice showed either mesangial segmental expansion, some with associated hypercellularity, and/or thrombotic obstruction of glomerular capillaries. The glomeruli revealed stronger lambda staining than kappa light chain staining. Six out of 12 kidneys from transgenic mice showed abundant electron-dense deposits when examined ultrastructurally. The deposits were located in glomerular capillary lumina in three cases. Large luminal and subendothelial deposits were characterized by randomly disposed microtubular structures measuring up to 16 nm in diameter, with overall features most consistent with cryoglobulins. In summary, about 50% of kidneys from the Vk*MYC mice with myeloma had features most consistent with monoclonal cryoglobulinemic glomerulopathy.

A Substantial Structural Conversion of the Native Monomer Leads to in-Register Parallel Amyloid Fibril Formation in Light-Chain Amyloidosis.

Amyloid light-chain (AL) amyloidosis is a rare disease in which plasma-cell-produced monoclonal immunoglobulin light chains misfold and become deposited as fibrils in the extracellular matrix. λ6 subgroup light chains are particularly fibrillogenic, and around 25 % of amyloid-associated λ6 light chains exist as the allotypic G24R variant that renders the protein less stable. The molecular details of this process, as well as the structures of the fibrils, are unknown. We have used solid-state NMR to investigate different fibril polymorphs. The secondary structures derived from NMR predominantly show ß-strands, including in former turn or helical regions, and provide a molecular basis for previously identified fibrillogenic hotspots. We have determined, by using differentially 15 N:13 C-labeled samples, that the ß-strands are stacked in-register parallel in the fibrils. This supramolecular arrangement shows that the native globular folds rearrange substantially upon fibrillization, and rules out the previously hypothesized fibril formation from native monomers.

High-seas fish wars generate marine reserves.

The effective management of marine fisheries is an ongoing challenge at the intersection of biology, economics, and policy. One way in which fish stocks-and their habitats-can be protected is through the establishment of marine reserves, areas that are closed to fishing. Although the potential economic benefits of such reserves have been shown for single-owner fisheries, their implementation quickly becomes complicated when more than one noncooperating harvester is involved in fishery management, which is the case on the high seas. How do multiple self-interested actors distribute their fishing effort to maximize their individual economic gains in the presence of others? Here, we use a game theoretic model to compare the effort distributions of multiple noncooperating harvesters with the effort distributions in the benchmark sole owner and open access cases. In addition to comparing aggregate rent, stock size, and fishing effort, we focus on the occurrence, size, and location of marine reserves. We show that marine reserves are a component of many noncooperative Cournot-Nash equilibria. Furthermore, as the number of harvesters increases, (i) both total unfished area and the size of binding reserves (those that actually constrain behavior) may increase, although the latter eventually asymptotically decreases; (ii) total rents and stock size both decline; and (iii) aggregate effort used (i.e., employment) can either increase or decrease, perhaps nonmonotonically.

Kidney carcinoid tumor: Histological, immunohistochemical and ultrastructural features.

Primary carcinoid tumor of the kidney is uncommon. Only limited morphologic studies have been reported in the literature. In general, renal carcinoid tumor reveals typical morphological features as present in carcinoid tumors in the other organs, i.e. growing as trabeculae intermixed with nests of monotonous cuboidal cells that show "salt and pepper" nuclear appearance. As reported in other organs, different morphological expressions may also present in renal carcinoid tumors, such as papillary, pseudoglandular or solid growth patterns and clear/eosinophilic cytoplasmic changes. These morphological variations may mimic other benign and malignant primary neoplasms in the kidney. Therefore, in some cases, ancillary studies are necessary or even crucial to differentiate carcinoid tumors from other primary renal neoplasms. Herewith, we report two primary renal carcinoid tumors with long follow-up, one showed typical morphological features of carcinoid tumor, and the second one revealed some uncommon histological findings. To differentiate these carcinoid tumors from other primary renal neoplasms and confirm neuroendocrine differentiation, immunohistochemical studies are valuable. In case 2 immunohistochemical stain results were not definitive and electron microscopy was performed confirming the diagnosis of carcinoid tumor.

A case of HIV associated cryptococcal nephritis: Ultrastructural findings and literature review.

Acute renal failure in the setting of infection with human immunodeficiency virus can be due to various causes including pre-renal causes such as hypotension, sepsis, and nephrotoxic agents; thrombotic microangiopathy; or direct renal parenchymal infections by opportunistic organisms. We present a case of cryptococcal nephritis in a patient with systemic cryptococcosis and discuss the clinical findings as well as the histological, immunofluorescent, and transmission electron microscopy findings in the renal biopsy.

Metastatic TFE3-overexpressing renal clear cell carcinoma with dense granules: a histological, immunohistochemical, and ultrastructural study.

This is a case report of a 46-year-old white male who presented with dyspnea. Thoracic and abdominal examinations showed a heterogeneously enhancing mass in the right kidney, multiple pulmonary nodules, and left pleural thickening with large pleural effusion. Pleura biopsy revealed a malignant neoplasm composed of cells with predominantly clear cytoplasm. Considering the large mass in the right kidney, clear cell renal cell carcinoma (RCC) was the main differential diagnosis. The diagnosis in this case was not definitive by histology alone since clear cell RCC markers such as RCC and AE1/AE3 were negative, and CD10 was only focally positive. Transcription factor E3 (TFE3) immunohistochemistry was positive, while the XP11.2 translocation testing was negative. Electron microscopy demonstrated that the tumor cells had abundant cytoplasmic glycogen and lipid, focal long microvilli lining rare lumina, and adjacent interdigitating cell membranes joining the neoplastic cells, indicating a diagnosis of renal clear cell carcinoma. In addition, numerous crystalline-like dense granules were identified in the cytoplasm of the neoplastic cells, which are reminiscent of those typically seen in alveolar soft part sarcoma and rarely described in XP11.2 translocation RCC. Overall, this renal tumor likely represents a variant of XP11.2 translocation RCC, overexpressing TFE3 with dense granules.

Phenotypic plasticity of mesenchymal stem cells is crucial for mesangial repair in a model of immunoglobulin light chain-associated mesangial damage.

Mesangiopathies produced by glomerulopathic monoclonal immunoglobulin light chains (GLCs) acting on the glomerular mesangium produce two characteristic lesions: AL-amyloidosis (AL-Am) and light chain deposition disease (LCDD). In both cases, the pathology is centered in the mesangium, where initial and progressive damage occurs. In AL-Am the mesangial matrix is destroyed and replaced by amyloid fibrils and in LCDD, the mesangial matrix is increased and remodeled. The collagen IV rich matrix is replaced by tenascin. In both conditions, mesangial cells (MCs) become apoptotic as a direct effect of the GLCs. MCs were incubated in-vitro with GLCs and animal kidneys were perfused ex-vivo via the renal artery with GLCs, producing expected lesions, and then mesenchymal stem cells (MSCs) were added to both platforms. Each of the two platforms provided unique information that when put together created a comprehensive evaluation of the processes involved. A "cocktail" with growth and differentiating factors was used to study its effect on mesangial repair. MSCs displayed remarkable phenotypic plasticity during the repair process. The first role of the MSCs after migrating to the affected areas was to dispose of the amyloid fibrils (in AL-Am), the altered mesangial matrix (in LCDD) and apoptotic MCs/debris. To accomplish this task, MSCs transformed into facultative macrophages acquiring an abundance of lysosomes and endocytotic capabilities required to engage in phagocytic functions. Once the mesangial cleaning was completed, MSCs transformed into functional MCs restoring the mesangium to normal. "Cocktail" made the repair process more efficient.

Experimental evidence of masculinization by continuous illumination in a temperature sex determination teleost (Atherinopsidae) model: is oxidative stress involved?

The present study evaluates the influence of continuous light on phenotypic sex ratios in Chirostoma estor, a temperature sex determination animal model. Relative gene expression levels of 5 day old larvae were performed on two early gonad differentiation genes (sox9 and foxl2), two stress axis activation genes (gcr1 and crf) and four reactive oxygen species (ROS) antagonist effector genes (sod2, ucp2, gsr and cat). Two light treatments were applied from fertilization; control (12L:12D) simulated natural photoperiod and a continuous illumination photoperiod. By the end of the trial (12 weeks after hatching), differentiated and normal gonads were clearly identifiable in both treatments by histological observations. Regarding sex ratio, 73% of phenotypic males were found in continuous illumination compared with 40% in controls. Consistently, the sox9 gene (involved in early testis differentiation) showed an over expression in 64% of the individual larvae analysed compared with foxl2 (ovarian differentiation) suggesting a masculinization tendency in continuous illumination. On the other hand, only 36% of individuals showed the same tendency in the control treatment consistent with phenotypic sex ratios found under normal culture conditions. Relative gene expression results did not show significant difference in sod2, ucp2 and gcr1 levels, but cat, gsr and crf showed significantly higher expression levels in the continuous illumination treatment suggesting that both, the stress axis and ROS response mechanisms were activated at this time. This study suggests, a link between continuous light, oxidative stress and environmental sex determination in vertebrates. However, further research is necessary to describe this possible upstream mechanism that may drive some aspects of sexual plasticity in vertebrates.

Highly curved image sensors: a practical approach for improved optical performance.

The significant optical and size benefits of using a curved focal surface for imaging systems have been well studied yet never brought to market for lack of a high-quality, mass-producible, curved image sensor. In this work we demonstrate that commercial silicon CMOS image sensors can be thinned and formed into accurate, highly curved optical surfaces with undiminished functionality. Our key development is a pneumatic forming process that avoids rigid mechanical constraints and suppresses wrinkling instabilities. A combination of forming-mold design, pressure membrane elastic properties, and controlled friction forces enables us to gradually contact the die at the corners and smoothly press the sensor into a spherical shape. Allowing the die to slide into the concave target shape enables a threefold increase in the spherical curvature over prior approaches having mechanical constraints that resist deformation, and create a high-stress, stretch-dominated state. Our process creates a bridge between the high precision and low-cost but planar CMOS process, and ideal non-planar component shapes such as spherical imagers for improved optical systems. We demonstrate these curved sensors in prototype cameras with custom lenses, measuring exceptional resolution of 3220 line-widths per picture height at an aperture of f/1.2 and nearly 100% relative illumination across the field. Though we use a 1/2.3" format image sensor in this report, we also show this process is generally compatible with many state of the art imaging sensor formats. By example, we report photogrammetry test data for an APS-C sized silicon die formed to a 30° subtended spherical angle. These gains in sharpness and relative illumination enable a new generation of ultra-high performance, manufacturable, digital imaging systems for scientific, industrial, and artistic use.

Reduction of animal suffering in rabies vaccine potency testing by introduction of humane endpoints.

Potency controls of inactivated rabies vaccines for human use are confirmed by the National Institutes of Health challenge test in which lethal infection with severe neurological symptoms should be observed in approximately half of the mice inoculated with the rabies virus. Weight loss, decreased body temperature, and the presence of rabies-associated neurological signs have been proposed as humane endpoints. The potential for reduction of animal suffering by introducing humane endpoints in the potency test for inactivated rabies vaccine for human use was investigated. The clinical signs were scored and body weight was monitored. The average times to death following inoculation were 10.49 and 10.99 days post-inoculation (dpi) by the potency and challenge control tests, respectively, whereas the average times to showing Score-2 signs (paralysis, trembling, and coma) were 6.26 and 6.55 dpi, respectively. Body weight loss of more than 15% appeared at 5.82 and 6.42 dpi. The data provided here support the introduction of obvious neuronal signs combined with a body weight loss of ≥15% as a humane endpoint to reduce the time of animal suffering by approximately 4 days.

Contribution of human smooth muscle cells to amyloid angiopathy in AL (light-chain) amyloidosis.

OBJECTIVE: Amyloid light-chain (AL) amyloidosis is a disease process that often compromises the peripheral vascular system and leads to systemic end-organ dysfunction. Although amyloid formation in vessel walls is a multifaceted process, the assembly of the native light chains (LCs) into amyloid fibrils is central to its pathogenesis. Recent evidence suggests that endocytosis and endolysosomal processing of immunoglobin LCs by host cells is essential to the formation of amyloid fibrils that are deposited in at least some tissues. The aim of this study was to elucidate the role of vascular smooth muscle in amyloid angiopathy. METHODS: Human coronary artery smooth muscle cells (SMCs) were grown on coverslips, four chamber glass slides, and growth factor-reduced Matrigel matrix in the presence of 10 µg/ml of ALs (λ and κ isotypes), nonamyloidogenic LCs, and culture medium (negative control) for 48 and 72 hours. Thereafter, a detailed light microscopic, immunohistochemical, and ultrastructural evaluation was conducted to verify amyloid deposition and characterize the role of SMCs in the formation of amyloid deposits in the various experimental conditions. RESULTS: Amyloid deposits were detected extracellulary as early as 48 hours after exposure of vascular smooth muscle cells (VSMCs) to AL-LCs (amyloidogenic light chains) as confirmed by affinity to Congo red dye, thioflavin T fluorescence, and transmission electron microscopy. No amyloid was present in the cultures of SMCs treated with medium alone or nonamyloidogenic LCs. SMCs associated with amyloid deposits exhibited CD68, lysosome-associated membrane protein 1-1, and intracellular lambda light chain expression and only focal smooth muscle actin and muscle-specific actin positivity. Electron microscopy revealed these cells to have an expanded mature lysosomal compartment closely associated with deposits of newly formed amyloid fibrils. CONCLUSIONS: The interaction of amyloidogenic LCs with VSMCs is necessary for the formation of amyloid fibrils that are deposited in peripheral vessels. VSMCs participate in the formation of amyloid by the intracellular processing of AL-LCs, which is possible due to their transformation from a smooth muscle to a macrophage phenotype. The formation of amyloid fibrils occurs in the mature lysosomal compartment of transformed cells. The amyloid that is formed is then extruded into the extracellular matrix.