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Preeclampsia is associated with increased levels of the circulating antiangiogenic factor sFlt-1 and with an excessive shedding of placenta-derived multinucleated syncytial aggregates into the maternal circulation. However, it remains unclear whether these aggregates are transcriptionally active in the maternal organs and can, therefore, contribute to the systemic manifestations of preeclampsia. In this study, we measured placental soluble fms-like tyrosine kinase-1 (sFlt-1) mRNA levels in preeclamptic- and control placentas and performed RNA in situ hybridization to localize the main placental expression site of sFlt-1 mRNA. Because the maternal lung is the first capillary bed that circulating syncytial aggregates traverse, we studied the presence and persistence of placental material in lungs of preeclamptic and control subjects. To confirm the placental origin of these aggregates in maternal lungs, immunohistochemistry for the placenta-specific marker hCG (human chorionic ghonadotropin) and Y chromosome in situ hybridization were performed. Using human placental tissue, we found that syncytial knots are the principal site of expression of the antiangiogenic factor sFlt-1. In addition, autopsy material obtained from women with preeclampsia (n=9) showed significantly more placenta-derived syncytial aggregates in the lungs than in control subjects (n=26). Importantly, these aggregates still contained the antiangiogenic factor sFlt-1 after their entrapment in the maternal lungs. The current study confirms the important role of syncytial knots in placental sFlt-1 mRNA production. In addition, it shows a significant association between preeclampsia and larger quantities of sFlt-1 containing syncytial aggregates in maternal lungs, suggesting that the transfer of syncytial aggregates to the maternal compartment may contribute to the systemic endothelial dysfunction that characterizes preeclampsia.

In situ transmission electron microscopy was performed on the electromigration in platinum (Pt) nanowires (14 nm thick, 200 nm wide, and 300 nm long) with and without feedback control. Using the feedback control mode, symmetric electrodes are obtained and the gap usually forms at the center of the Pt nanowire. Without feedback control, asymmetric electrodes are formed, and the gap can occur at any position along the wire. The three-dimensional gap geometries of the electrodes in the Pt nanowire were determined using high-angle annular dark-field scanning transmission electron microscopy; the thickness of the nanowire is reduced from 14 nm to only a few atoms at the edge with a gap of about 5-10 nm.

We report on a photodetector in which colloidal quantum dots directly bridge nanometer-spaced electrodes. Unlike in conventional quantum-dot thin film photodetectors, charge mobility no longer plays a role in our quantum-dot junctions as charge extraction requires only two individual tunnel events. We find an efficient photoconductive gain mechanism with external quantum efficiencies of 38 electrons-per-photon in combination with response times faster than 300 ns. This compact device-architecture may open up new routes for improved photodetector performance in which efficiency and bandwidth do not go at the cost of one another.

Since the Pap test was introduced in the 1940s, there has been an approximately 70% reduction in the incidence of squamous cell cervical cancers in many developed countries by the application of organized and opportunistic screening programs. The efficacy of the Pap test, however, is hampered by high interobserver variability and high false-negative and false-positive rates. The use of biomarkers has demonstrated the ability to overcome these issues, leading to improved positive predictive value of cervical screening results. In addition, the introduction of HPV primary screening programs will necessitate the use of a follow-up test with high specificity to triage the high number of HPV-positive tests. This paper will focus on protein biomarkers currently available for use in cervical cancer screening, which appear to improve the detection of women at greatest risk for developing cervical cancer, including Ki-67, p16(INK4a), BD ProEx C, and Cytoactiv HPV L1.

We report on a method to fabricate and measure gateable molecular junctions that are stable at room temperature. The devices are made by depositing molecules inside a few-layer graphene nanogap, formed by feedback controlled electroburning. The gaps have separations on the order of 1-2 nm as estimated from a Simmons model for tunneling. The molecular junctions display gateable I-V-characteristics at room temperature.

We present the formation of single-molecule devices based on nanometre-spaced platinum electrodes. The electrodes are fabricated using a self-breaking electromigration method which yields nanogaps with long-term stability at room temperature [Prins et al., APL, 2009, 94, 123108.]. The stability at room temperature allows for detailed comparison of the device electrical properties before and after deposition of the molecules. In this way, conductance as a result of direct tunneling between the electrodes can be distinguished from conductance through the molecule. After molecule deposition, some devices display transport in the strong coupling regime while others are in the weak-coupling Coulomb blockade regime. Gated transport is observed in the latter case.

Tumor involvement of resection margins is found in a large proportion of patients who undergo breast-conserving surgery. Near-infrared (NIR) fluorescence imaging is an experimental technique to visualize cancer cells during surgery. To determine the accuracy of real-time NIR fluorescence imaging in obtaining tumor-free resection margins, a protease-activatable NIR fluorescence probe and an intraoperative camera system were used in the EMR86 orthotopic syngeneic breast cancer rat model. Influence of concentration, timing and number of tumor cells were tested in the MCR86 rat breast cancer cell line. These variables were significantly associated with NIR fluorescence probe activation. Dosing and tumor size were also significantly associated with fluorescence intensity in the EMR86 rat model, whereas time of imaging was not. Real-time NIR fluorescence guidance of tumor resection resulted in a complete resection of 17 out of 17 tumors with minimal excision of normal healthy tissue (mean minimum and a mean maximum tumor-free margin of 0.2 ± 0.2 mm and 1.3 ± 0.6 mm, respectively). Moreover, the technique enabled identification of remnant tumor tissue in the surgical cavity. Histological analysis revealed that the NIR fluorescence signal was highest at the invasive tumor border and in the stromal compartment of the tumor. In conclusion, NIR fluorescence detection of breast tumor margins was successful in a rat model. This study suggests that clinical introduction of intraoperative NIR fluorescence imaging has the potential to increase the number of complete tumor resections in breast cancer patients undergoing breast-conserving surgery.

We report an in situ transmission electron microscopy (TEM) imaging of grain growth in a Pt nanobridge induced by a high electric current density. The change in morphology at the nanoscale was recorded in real time together with the electrical characterization of the Pt nanobridge. We find a drop in the differential resistance as the voltage across the bridge is increased; TEM inspection shows that this coincides with thermally induced grain growth, indicating that a reduction of grain boundary scattering is the cause of the resistance decrease.

Receptor endocytosis is critical for cell signaling. IGF1R mediates an autocrine loop that is de-regulated in Ewing Sarcoma (ES) cells. Here we study the impact of IGF1R internalization, mediated by clathrin and caveolin-1 (CAV1), in ES signaling. We used clathrin and CAV1-siRNA to interfere in clathrin- and caveolin-dependent endocytosis. Chlorpromazine (CPMZ) and methyl-beta-cyclo-dextrin (MCD) were also used in order to inhibit clathrin- and caveolin-dependent endocytosis, respectively. We analyzed IGF1R internalization and co-localization with clathrin and CAV1 upon ligand binding, as well as the status of the IGF1R pathway, cellular proliferation, and the apoptosis of interfered and inhibited ES cells. We performed a high-throughput tyrosine kinase phosphorylation assay to analyze the effects of combining the IGF1R tyrosine kinase inhibitor AEW541 (AEW) with CPMZ or MCD on the intracellular phospho-proteome. We observed that IGF1R is internalized upon ligand binding in ES cells and that this process is dependent on clathrin or CAV1. The blockage of receptor internalization inhibited AKT and MAPK phosphorylation, reducing the proliferative rate of ES cells and increasing the levels of apoptosis. Combination of AEW with CPMZ or MCD largely enhanced these effects. CAV1 and clathrin endocytosis controls IGF1R internalization and signaling and has a profound impact on ES IGF1R-promoted survival signaling. We propose the combination of tyrosine-kinase inhibitors with endocytosis inhibitors as a new therapeutic approach to achieve a stronger degree of receptor inhibition in this, or other neoplasms dependent on IGF1R signaling.

We have investigated charge transport in ZnTPPdT-Pyr (TPPdT: 5,15-di(p-thiolphenyl)-10,20-di(p-tolyl)porphyrin) molecular junctions using the lithographic mechanically controllable break-junction (MCBJ) technique at room temperature and cryogenic temperature (6 K). We combined low-bias statistical measurements with spectroscopy of the molecular levels in the form of I(V) characteristics. This combination allows us to characterize the transport in a molecular junction in detail. This complex molecule can form different junction configurations, having an observable effect on the trace histograms and the current-voltage (I(V)) measurements. Both methods show that multiple, stable single-molecule junction configurations can be obtained by modulating the interelectrode distance. In addition we demonstrate that different ZnTPPdT-Pyr junction configurations can lead to completely different spectroscopic features with the same conductance values. We show that statistical low-bias conductance measurements should be interpreted with care, and that the combination with I(V) spectroscopy represents an essential tool for a more detailed characterization of the charge transport in a single molecule.

Many types of molecular motors have been proposed and synthesized in recent years, displaying different kinds of motion, and fueled by different driving forces such as light, heat, or chemical reactions. We propose a new type of molecular motor based on electric field actuation and electric current detection of the rotational motion of a molecular dipole embedded in a three-terminal single-molecule device. The key aspect of this all-electronic design is the conjugated backbone of the molecule, which simultaneously provides the potential landscape of the rotor orientation and a real-time measure of that orientation through the modulation of the conductivity. Using quantum chemistry calculations, we show that this approach provides full control over the speed and continuity of motion, thereby combining electrical and mechanical control at the molecular level over a wide range of temperatures. Moreover, chemistry can be used to change all key parameters of the device, enabling a variety of new experiments on molecular motors.

During pregnancy, maternal lymphocytes at the fetal-maternal interface play a key role in the immune acceptance of the allogeneic fetus. Recently, CD4(+)CD25(bright) regulatory T cells have been shown to be concentrated in decidual tissue, where they are able to suppress fetus-specific and nonspecific immune responses. Decidual CD8(+) T cells are the main candidates to recognize and respond to fetal HLA-C at the fetal-maternal interface, but data on the characteristics of these cells are limited. In this study we examined the decidual and peripheral CD8(+) T cell pool for CD45RA, CCR7, CD28, and CD27 expression, using nine-color flow cytometry. Our data demonstrate that decidual CD8(+) T cells mainly consist of differentiated CD45RA(-)CCR7(-) effector-memory (EM) cells, whereas unprimed CD45RA(+)CCR7(+) naive cells are almost absent. Compared with peripheral blood EM CD8(+) T cells, the decidual EM CD8(+) T cells display a significantly reduced expression of perforin and granzyme B, which was confirmed by immunohistochemistry of decidual tissue sections. Interestingly, quantitative PCR analysis demonstrates an increased perforin and granzyme B mRNA content in decidual EM CD8(+) T cells in comparison with peripheral blood EM CD8(+) T cells. The presence of high levels of perforin and granzyme B mRNA in decidual EM T cells suggests that decidual CD8(+) T cells pursue alternative means of EM cell differentiation that may include a blockade of perforin and granzyme B mRNA translation into functional perforin and granzyme B proteins. Regulation of decidual CD8(+) T cell differentiation may play a crucial role in maternal immune tolerance to the allogeneic fetus.

Primary cilia are specialized cell surface projections found on most cell types. Involved in several signaling pathways, primary cilia have been reported to modulate cell and tissue organization. Although they have been implicated in regulating cartilage and bone growth, little is known about the organization of primary cilia in the growth plate cartilage and osteochondroma. Osteochondromas are bone tumors formed along the growth plate, and they are caused by mutations in EXT1 or EXT2 genes. In this study, we show the organization of primary cilia within and between the zones of the growth plate and osteochondroma. Using confocal and electron microscopy, we found that in both tissues, primary cilia have a similar formation but a distinct organization. The shortest ciliary length is associated with the proliferative state of the cells, as confirmed by Ki-67 immunostaining. Primary cilia organization in the growth plate showed that non-polarized chondrocytes (resting zone) are becoming polarized (proliferating and hypertrophic zones), orienting the primary cilia parallel to the longitudinal axis of the bone. The alignment of primary cilia forms one virtual axis that crosses the center of the columns of chondrocytes reflecting the polarity axis of the growth plate. We also show that primary cilia in osteochondromas are found randomly located on the cell surface. Strikingly, the growth plate-like polarity was retained in sub-populations of osteochondroma cells that were organized into small columns. Based on this, we propose the existence of a mixture ('mosaic') of normal lining (EXT(+/-) or EXT(wt/wt)) and EXT(-/-) cells in the cartilaginous cap of osteochondromas.

Purpose: To investigate the effect of intraepithelial tumor-infiltrating lymphocytes (ieTIL) and their ligands expressed by cervical tumor cells on the outcome of cervical cancer patients., Experimental Design: The prognostic value of ieTILs was investigated in 115 cases of cervical cancer. T-cell subsets, CD57(+) cells, and regulatory T cells (Treg) were enumerated. The associations of these different ieTIL subtypes with human leukocyte antigen (HLA) class I and MHC class I chain-related molecule A (MICA) expression were determined in relation to clinical variables and patient survival., Results: Survival analysis showed that a high number of intraepithelial Treg (FoxP3(+)), a low CD8(+)/regulatory T-cell ratio, and a weak HLA-A expression were all associated with worse survival (P=0.034, 0.025, and 0.033, respectively, log-rank test). Further stratification of patient groups based on HLA-A-MICA expression and HLA-A-MICA-CD8(+)/Treg ratio revealed an even poorer survival (P=0.005). In a multivariate Cox analysis, low CD8(+)/Treg ratio (P=0.047), weak HLA-A-MICA expression (P=0.003), and weak HLA-A-MICA expression combined with low CD8(+)/Treg ratio (P=0.002) were all found to be independent unfavorable prognostic predictors in cervical carcinoma (hazard ratios, 2.7, 4.0, and 4.9, respectively)., Conclusion: Weak HLA-A-MICA expression combined with low CD8(+)/Treg ratio reveals a patient group with the poorest survival in cervical cancer. As a single variable, low CD8(+)/Treg ratio was a significant independent unfavorable prognostic factor.

The influence of the counteranion on the structure and the spin-transition properties of original 1D bis(tetrazole) Fe(II) systems, namely [Fe(btzx)(3)]X(2) (X=PF(6) (-) (1), CF(3)SO(3) (-) (2) and ClO(4) (-) (3); btzx=m-xylylenebis(tetrazole)) is studied. The X-ray crystal structures of compounds 1 and 2 are described in detail. These structures present a solvent molecule encapsulated within pockets formed by btzx ligands along the 1D coordination chains. Compound 2 is shown to be the first structurally characterised alternating HS-LS 1D spin-transition system (HS=high spin, LS=low spin). The magnetic susceptibility measurements of all three compounds are compared. The degree of completion and the transition temperature are both drastically influenced by the counterion used, while surprisingly, the cooperative nature of the transition is not affected by the choice of counterion. Compounds 1 and 2 are further studied by Mössbauer spectroscopy and their distinct LIESST properties are compared.

Reflection contrast microscopy (RCM) is a light microscopic method to image cells at high definition and enhanced sensitivity compared to conventional bright-field microscopy. RCM images have very high contrast, which makes them easily applicable for digital image analysis. Because ultrathin sections are mostly used in this method, RCM also functions by bridging light with electron microscopy: the combination of ultrastructural with histochemical studies. RCM can also replace electron microscopy for rapid and simple screening of large quantities of samples for immunocytochemical staining. Special attention is paid to small biological objects, which have to be processed for RCM. If you encounter the limits of brightfield microscopy, in resolution, sensitivity or handling of the specimen, RCM will be a feasible option. Reflection contrast microscopy methods use only slightly adjusted electron microscopy methods for specimen preparation. Therefore, many familiar techniques for ultrathin specimen preparation can be applied. It is essential that only refractive index differences exist in those areas that are of interest and that the further specimen is as optically homogenic as possible, with a refractive index as close to that of glass as possible. Therefore, plastic embedding is recommended.

Hereditary head and neck paragangliomas are tumours associated with the autonomic nervous system. Recently, mutations in genes coding for subunits of mitochondrial complex II, succinate-ubiquinone-oxidoreductase (SDHB, SDHC, and SDHD), have been identified in the majority of hereditary tumours and a number of isolated cases. In addition, a fourth locus, PGL2, has been mapped to chromosome 11q13 in an isolated family. In order to characterize phenotypic effects of these mutations, the present study investigated the immunohistochemical expression of the catalytic subunits of complex II (flavoprotein and iron protein), SDH enzyme activity, and mitochondrial morphology in a series of 22 head and neck paragangliomas. These included 11 SDHD-, one SDHB-, two PGL2-linked tumours, and eight sporadic tumours. In the majority of the tumours (approximately 90%), the enzyme-histochemical SDH reaction was negative and immunohistochemistry of catalytic subunits of complex II showed reduced expression of iron protein and enhanced expression of flavoprotein. Ultrastructural examination revealed elevated numbers of tightly packed mitochondria with abnormal morphology in SDHD-linked and sporadic tumours. Immuno-electron microscopy showed localization of the flavoprotein on the remnants of the mitochondrial inner membranes, whereas virtually no signal for the iron protein was detected. These results indicate that the function of mitochondrial complex II is compromised in the majority of head and neck paragangliomas., (Copyright 2003 John Wiley & Sons, Ltd.)

Polycystin-2 is a predicted integral membrane protein with non-selective cation channel activity. The protein is encoded by the PKD2 gene, which is mutated in approximately 15% of patients with autosomal dominant polycystic kidney disease (ADPKD). Polycystin-2 can interact with the transmembrane protein polycystin-1, the product of the PKD1 gene. However, endoplasmic reticulum (ER) localization was reported for (heterologously expressed) polycystin-2 in cultured cells and baso-lateral localization has been reported in renal tissues. Using two polyclonal antisera raised against polycystin-2 we demonstrated distinct expression of the endogenous protein in the Golgi apparatus and the plasma membrane of MDCK cells. In contrast, most of the heterologously expressed polycystin-2 (PC2-EGFP) remained in the ER, substantially overlapping with the staining pattern of protein-disulfide isomerase (PDI), a marker for the ER. Only in a small subset of these cells weak plasma membrane signals were observed. Membrane staining was also suggested by immunoelectron microscopy and was confirmed by subcellular fractionation on sucrose density gradients. The plasma membrane staining disappeared following extraction with a buffer containing Triton X-100, whereas signals for polycystin-1 and E-cadherin remained visible, suggesting that polycystin-2 is neither tightly bound to the Triton X-100 insoluble cytoskeleton, nor to these proteins. We conclude that endogenous polycystin-2 is transported via the Golgi apparatus to the plasma membrane and has a broader membrane localization than polycystin-1. These data suggest that polycystin-2 can move freely in certain regions of the membrane where it probably functions as a channel, activated by, or in complex with, polycystin-1.

The authors recently showed variable subcellular immunoreactivity of the Bcl-2 and Bax proteins after fixation of cell monolayers with acetone, methanol, or paraformaldehyde (PF) followed by methanol (PF/methanol). Here, the authors demonstrate by reflection contrast microscopy and transmission electron microscopy that acetone or methanol fixation result in complete loss of integrity of intracellular structures in contrast with PF or glutaraldehyde fixation. Scanning electron microscopy revealed poor preservation of plasma membrane integrity after fixation in acetone or methanol. Fixation with PF before methanol reduced damage to intracellular and plasma membranes. In addition, Western blot analysis demonstrated loss of Bcl-2 and Bax protein during acetone or methanol fixation, whereas PF fixation before methanol permeabilization markedly reduced this loss. For studies on the intracellular localization of soluble or unknown types of antigen, the authors discourage the use of acetone and methanol as single fixatives.

Polycystin-1 is a novel protein predicted to be a large membrane-spanning glycoprotein with an extracellular N-terminus and an intracellular C-terminus, harboring several structural motifs. To study the subcellular localization, antibodies raised against various domains of polycystin-1 and against specific adhesion complex proteins were used for two-color immunofluorescence staining. In Madine Darby canine kidney (MDCK) cells, polycystin-1 was detected in the cytoplasm as well as co-localizing with desmosomes, but not with tight or adherens junctions. Using confocal laser scanning and immunoelectron microscopy we confirmed the desmosomal localization. By performing a calcium switch experiment, we demonstrated the sequential reassembly of tight junctions, subsequently adherens junctions and finally desmosomes. Polycystin-1 only stained the membrane after incorporation of desmoplakin into the desmosomes, suggesting that membrane-bound polycystin-1 may be important for cellular signaling or cell adhesion, but not for the assembly of adhesion complexes.

To clarify where and how beta-amyloid begins to deposit in senile plaques, we examined the ultrastructural localization of amyloid beta protein (Abeta) in diffuse plaques of brains with hereditary cerebral hemorrhage with amyloidosis-Dutch type. Alzheimer disease (AD), and from nondemented aged subjects. Serial ultrathin sections of osmium-plastic blocks were immunogold-labeled for Abetax-42 (Abeta42), and sections on grids were observed under the electron microscope (EM) after observing the exact localization of the diffuse plaques in sections on glass slides by the reflection contrast microscope. Abeta42 deposition, which was decollated with gold particles, appeared in 3 forms in all subjects under the EM: 1) Scattered small bundles of amyloid fibrils between cell processes, frequently seen in the densely stained area of diffuse plaques. 2) Scattered small foci of nonfibrillar materials between cell processes as a relatively minor form. 3) Abeta42 on a part of the cell surface plasma membrane of normal appearing cell processes, a major form in weakly immunostained areas. The last form was not associated with degenerative neurites or reactive glia. Abeta42 deposition on the cell surface plasma membrane appears to be an initial event in diffuse plaques, and then it develops into amorphous/fibrillar amyloid between cell processes.

The C-terminal profile and ultrastructure of small and presumably early capillary amyloid beta protein (Abeta) deposits were investigated in four patients with hereditary cerebral hemorrhage with amyloidosis, Dutch type. The C terminus of the 40 (Abeta40) or the 42 (Abeta42) amino acid form of Abeta was gold labeled in serial, ultrathin sections on glass slides for reflection contrast microscopy and on grids for electron microscopy. In all studied subjects, reflection contrast microscopy revealed capillaries with focal Abeta42 immunolabeling in the absence of Abeta40 labeling. In the adjacent electron microscopic section, Abeta42 labeling was confined to the capillary basement membrane. The majority of Abeta42(+)40(-) deposits showed no amyloid fibrils. Abeta42(+)40(-) deposits were sometimes observed in an unremarkable basement membrane but usually showed increased electron density and reticular structures. A small subset of Abeta42(+)40(-) deposits with basement membrane changes showed few amyloid fibrils. Abeta42(+)40(+) capillary deposits always showed definite fibrils and were larger than Abeta42(+)40(-) capillary deposits. The present findings suggest that in capillaries the accumulation and subsequent polymerization of Abeta42, possibly in conjunction with basement membrane changes, precedes the definite fibril formation with Abeta40.

Mesangial cell (MC) injury is a characteristic feature in the early phase of Thy.1 nephritis. The present study investigates the contribution of complement to MC apoptosis in this experimental model of kidney disease in rats. Thy.1 nephritis was induced by injection of mouse anti-Thy.1 monoclonal antibody (ER4G). To assess the contribution of the terminal sequence of complement on apoptosis, the studies were performed in complement-sufficient PVG/c (PVG/c+) rats and in rats deficient in complement C6 (PVG/c-). Apoptosis was monitored by assessment of the number of condensed nuclei in kidney sections stained with periodic acid-Schiff (PAS) and by the terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) method and expressed as number of apoptotic cells per 50 glomerular cross sections. In the PAS method, 1 h after intravenous injection of ER4G, PVG/c+ rats exhibited 160.9 +/- 49.5 apoptotic cells, whereas PVG/c- rats had only 3.2 +/- 1.4 apoptotic cells. Control rats exhibited 0.9 +/- 0.6 apoptotic cells. These findings were confirmed with the TUNEL method. In PVG/c- rats, a maximum number of 8.8 +/- 3.1 TUNEL-positive (TUNEL+) cells was found at 6 h followed by a decline thereafter. In PVG/c+ rats, apoptosis was associated with deposition of C6 and C5b-9. Restoration of the complement system of PVG/c- rats with purified human C6 resulted in an increase of apoptosis at 1 h after injection of ER4G from minimal numbers to 239.9 +/- 52.4 TUNEL+ cells. These studies appear to indicate for the first time that the terminal sequence of complement is involved in induction of apoptosis.

The clinical course of patients with polycythaemia vera (PV) and essential thrombocythaemia (ET) is frequently complicated by arterial thrombotic events. The pathogenesis is not clearly understood but attributed to abnormalities in platelet function. An increase in platelet thromboxane formation has been described in the majority of asymptomatic patients with thrombocythaemia, probably reflecting spontaneous platelet activation in vivo. In the present study we prospectively investigated whether an increase in platelet thromboxane formation actually precedes arterial microvascular thrombosis. In addition, we studied the effect of selective inhibition of platelet thromboxane formation on clinical outcome by reinstitution of low-dose aspirin (50 mg/ d). Six ET patients and one PV patient participated in this study. Within 10 d after withdrawal of aspirin, three patients developed arterial microvascular thrombosis of extremities (erythromelalgia), which was preceded by a 3-30-fold increase in urinary thromboxane excretion as compared with patients who remained asymptomatic. The increased urinary thromboxane excretion and clinical signs could be inhibited by a platelet-specific aspirin regimen of 50 mg/d without affecting vascular cyclooxygenase, indicating that platelets were the main source of the increased thromboxane generation. These data suggest that in symptomatic patients an enhanced formation of thromboxane by platelets, reflecting platelet activation in vivo, precedes the development of arterial microvascular thrombosis. These data provide a rationale for using low-dose aspirin as an antithrombotic agent in thrombocythaemia.

The epithelial cell adhesion molecule Ep-CAM is capable of mediating Ca2+-independent homotypic cell-cell adhesion when introduced into cells lacking their own means of cell-cell interactions. We used (confocal) immunofluorescent and (immuno-) electron microscopy to investigate the structural organization of Ep-CAM-mediated adhesions and their relation to other types of intercellular adhesions. Ep-CAM-transfected cell lines, cells of epithelial origin, and epithelial tissues were analyzed. In transfected L cells Ep-CAM brings the opposing intercellular membranes into a close proximity (approximately 10-14 nm) at sporadic contacts; however, no structures resembling junctional complexes were observed. In L cells cotransfected with Ep-CAM and E-cadherin, both molecules localize at the sites of cell-cell contact, forming independent adhesion sites with no Ep-CAM detectable within the structurally distinguishable cadherin-mediated adherens junctions. In well-differentiated carcinoma cell lines Ep-CAM colocalized with E-cadherin practically along the whole lateral domain; however, no colocalization was observed between Ep-CAM and the components of the tight junction complex (occludin and ZO-1), desmosomes (desmoplakins I/II), or cell-substrate adhesions (beta1 integrins). This was confirmed by analysis of polarized epithelium of normal colon where Ep-CAM was present at the lateral membrane including the adherens junction areas, but was fully excluded from the apical cell membrane (microvilli), tight junctions, and desmosomes. We conclude that (1) Ep-CAM does not form junctional complexes in L cells, (2) in epithelial cells, cell surface Ep-CAM is present at the lateral cell membrane, but is excluded from tight junctions and desmosomes, and (3) in epithelial cells, Ep-CAM is present within adhesions mediated by the classic cadherins (especially E-cadherin) with both types of molecules remaining as independent clusters. The colocalization with cadherins might be important for the modulating effect of Ep-CAM on cadherin-mediated adhesions., (Copyright 1999 Academic Press.)

The contribution of noncadherin-type, Ca2+-independent cell-cell adhesion molecules to the organization of epithelial tissues is, as yet, unclear. A homophilic, epithelial Ca2+-independent adhesion molecule (Ep-CAM) is expressed in most epithelia, benign or malignant proliferative lesions, or during embryogenesis. Here we demonstrate that ectopic Ep-CAM, when expressed in cells interconnected by classic cadherins (E- or N-cadherin), induces segregation of the transfectants from the parental cell type in coaggregation assays and in cultured mixed aggregates, respectively. In the latter assay, Ep-CAM-positive transfectants behave like cells with a decreased strength of cell-cell adhesion as compared to the parental cells. Using transfectants with an inducible Ep-CAM-cDNA construct, we demonstrate that increasing expression of Ep-CAM in cadherin-positive cells leads to the gradual abrogation of adherens junctions. Overexpression of Ep-CAM has no influence on the total amount of cellular cadherin, but affects the interaction of cadherins with the cytoskeleton since a substantial decrease in the detergent-insoluble fraction of cadherin molecules was observed. Similarly, the detergent-insoluble fractions of alpha- and beta-catenins decreased in cells overexpressing Ep-CAM. While the total beta-catenin content remains unchanged, a reduction in total cellular alpha-catenin is observed as Ep-CAM expression increases. As the cadherin-mediated cell-cell adhesions diminish, Ep-CAM-mediated intercellular connections become predominant. An adhesion-defective mutant of Ep-CAM lacking the cytoplasmic domain has no effect on the cadherin-mediated cell-cell adhesions. The ability of Ep-CAM to modulate the cadherin-mediated cell-cell interactions, as demonstrated in the present study, suggests a role for this molecule in development of the proliferative, and probably malignant, phenotype of epithelial cells, since an increase of Ep-CAM expression was observed in vivo in association with hyperplastic and malignant proliferation of epithelial cells.

Patients with essential thrombocythaemia (ET) exhibit a decrease of large von Willebrand factor (VWF) multimers in plasma, which is inversely related to the platelet count. In the present study we investigated whether the decrease of large VWF multimers in plasma with increasing platelet counts is the consequence of increased turnover of large VWF multimers in vivo. To that end we measured the half-life times of endogenously released VWF:Ag and VWF:CBA (collagen binding activity) after intravenous administration of desmopressin (DDAVP) to nine ET patients and nine control subjects (N). In addition, the half-life times of VWF:Ag and VWF:CBA were also measured in four ET patients after cytoreduction of the increased platelet count to normal or nearly normal values. Estimated half-life times of VWF:Ag did not differ between ET patients and normals (11.0+/-4.0 h v 12.4+/-2.5 h, P>0.05). Estimated half-life times of VWF:CBA were significantly lower in ET patients as compared with normal individuals (6.1+/-2.0 h v 8.4+/-2.5 h, P<0.05). After cytoreduction of the increased platelet count to (nearly) normal values in all four ET patients the half-life time of VWF:CBA significantly (P=0.014) increased from 5.2+/-1.2 h to 8.7+/-2.0 h. Our data suggest that platelets may play a role in the homeostasis of circulating von Willebrand factor, which may compromise normal haemostasis at fairly increased platelet counts.

Erythromelalgia, a characteristic aspirin-responsive microvascular thrombotic complication in essential thrombocythemia (ET), may develop despite oral anticoagulant treatment or treatment with heparin, suggesting that the generation of thrombin is not a prerequisite for its development. To study this, a cross-sectional comparison of the plasma levels of thrombomodulin (TM), platelet factor 4 (PF4), beta-thromboglobulin (beta-TG), prothrombin fragment 1 + 2 (F1 + 2) and total degradation products of fibrin(ogen) (TDP) was made between 5 ET patients suffering from erythromelalgia, 16 asymptomatic ET patients and 20 control subjects, and after treatment with aspirin, respectively. Furthermore, 2 ET patients with a history of erythromelalgia were studied at regular time intervals after discontinuation of aspirin until erythromelalgia recurred. As compared with asymptomatic ET patients and control subjects erythromelalgia was characterized by significantly higher beta-TG and TM levels but no significant differences were detected in either F1 + 2 or TDP levels. Treatment of erythromelalgia with aspirin resulted in disappearance of erythromelalgic signs and symptoms, which was paralleled by a significant decrease of beta-TG and TM levels. Histopathologic and immunohistochemical analysis of biopsies derived from erythromelalgic skin areas of 2 ET patients showed that erythromelalgic thrombi stained positively for von Willebrand factor opposed to only a weak fibrin staining. Our data suggest that erythromelalgia is caused by the intravascular activation and aggregation of platelets with subsequent sludging or occlusion of the acral arterial microvasculature. The generation of thrombin appears not to be essential for the formation of these platelet thrombi, thereby giving a plausible explanation for the inefficacy of coumadin derivatives and heparin in the prevention and treatment of erythromelalgia in essential thrombocythemia.

The polycystic kidney disease-1 gene, which is mutated in the majority of patients with autosomal dominant polycystic kidney disease, has been identified. The protein encoded by this gene, polycystin, has no homology with any gene known thus far. To gain more insight into the function of polycystin, we raised antibodies against synthetic peptides and a fusion protein corresponding to the sequence of two different fragments of polycystin. Two of the antibodies were capable of immunoprecipitating an in vitro transcription and translation product corresponding to a fragment of polycystin. In the cyst-lining epithelium of polycystic kidney disease-1 patients, a strong staining was observed. In normal adult and embryonic kidney tissues, expression was seen in the epithelium of all tubular structures and in the glomerular parietal and visceral epithelium (podocytes), although the podocytes were mainly recognized on cryosections and not on paraffin sections. A double-labeled immunofluorescence with one of the polycystin antibodies and the monoclonal antibody 8G8 ascertained that within the glomerular tuft podocytes were recognized.

Previous studies have revealed quantitative alterations in laminin-1 expression at the mRNA and protein levels during the development of glomerulonephritis and glomerulosclerosis in chronic graft-versus-host disease in mice, a model for lupus nephritis. We have now studied the qualitative alterations in laminin expression with two monoclonal antibodies that recognize epitopes on either the E8 or the P1 fragment of laminin-1. Both of these fragments are involved in cell-matrix and matrix-matrix interactions. In normal glomeruli these laminin epitopes are present only in the mesangial matrix; during embryogenesis, however, they are also present in the glomerular basement membrane. The distribution of laminin epitopes was first studied by using immunofluorescence in kidneys of mice with graft-versus-host disease at different points in time after disease induction. Reflection contrast and immunoelectron microscopy were performed after in vivo injection of the horseradish peroxidase-coupled monoclonal antibodies. In glomeruli of mice 8 weeks after disease induction, both injected antibodies bound specifically in electron-dense immune deposits in the mesangium and subepithelially along the glomerular basement membrane as well as in the expanded mesangial matrix. At 11 and 12 weeks after disease induction, when focal and segmental glomerulosclerosis had developed, the antibodies additionally bound in the matrix subendothelially along the glomerular basement membrane and at the periphery of end-stage sclerotic lesions. To study changes in the distribution of laminin epitopes over time, mice were injected with either monoclonal antibody before induction of graft-versus-host disease. The antibodies were detected 8 and 12 weeks later in the mesangial matrix of mice with lupus nephritis. Once segmental glomerulosclerosis had developed, the antibodies were additionally detected within the thickened glomerular capillary wall. The specific binding of anti-laminin monoclonal antibodies in electron-dense immune deposits further substantiates the hypothesis that anti-laminin autoantibodies participate in glomerular immune complex formation in this model, as suggested by earlier studies. Furthermore, our results show that the distribution of glomerular laminin epitopes in the matrix is altered during the development of glomerular disease. These changes in the structure of the glomerular basement membrane may contribute to the abnormal cell-matrix and matrix-matrix interactions during the development of glomerular disease in this model for lupus nephritis.

Proximal tubular cells (PTC) were isolated from porcine kidney by collagenase treatment, subsequently purified on a discontinuous density gradient and finally cultured. Porcine PTC (PPTC) in primary culture expressed keratin, characteristics of epithelia and brush border specific glycoproteins (FX1A). In addition, vimentin was present. All cells were negative for the endothelial marker pal-E. Less than 0.1% expressed the Tamm-Horsfall protein, characteristic of the distal tubule, while less than 0.3% of all cells in culture expressed desmin, characteristic of connective tissue (i.e. fibroblasts) and mesangial cells. Ultrastructural analysis revealed microvilli, tight junctions and abundant mitochondrial and lysosomes, all characteristics of proximal tubular cells. Freshly isolated PPTC were validated as in vitro model to detect nephrotoxicity by studying the effect of mercuric chloride, cis-platin, p-aminophenol and the halogenated alkenes 1,2 dichlorovinyl-l-cysteine, S-(1,1-difluoro-2,2-dichloroethyl)-L-cysteine (DCDFE-cys) and the glutathione conjugate of DCDFE on viability and mitochondrial membrane potential. The cells responded, time- and dose-dependently, to the nephrotoxic compounds with a decrease in mitochondrial membrane potential and loss of viability. The sensitivity of the porcine cells in detecting toxic effects corresponded favorably with in vitro systems derived from other animals.

Allogeneic islet transplantation in Type I diabetic patients is considerably hampered by the variable outcome of islet isolation and purification. After collagenase digestion of the pancreas, islet isolation is traditionally performed under hypothermic conditions in physiological solutions such as Hanks and RPMI. The University of Wisconsin solution (UWS) has been shown superior for hypothermic preservation of the pancreas. We, therefore, compared the UWS and RPMI for canine islet isolation and subsequent purification in either a conventional hyperosmotic density gradient of dextran in Hanks, or a novel normosmotic density gradient of Percoll in UWS. The isolation solution did not affect islet yield before purification (51% of the native islet mass). Loss of amylase (30%) and swelling of the acinar cells were observed in RPMI. In contrast, no loss of amylase and slight shrinkage of the acinar cells were observed in the UWS. Cell swelling affected the density separation and viability of the cells. Dextran density separation resulted in a 15% purity and 41% recovery of the islets isolated in RPMI, as compared to a 93% purity and 52% recovery of islets isolated in UWS. Percoll density separation improved the purity (99%) and recovery (74%) of islets isolated in UWS. Islets isolated in UWS demonstrated a superior basal and glucose stimulated insulin release during perifusion. Electron microscopy demonstrated a well-preserved islet ultrastructure after isolation in both solutions--except for slightly swollen mitochondria after isolation in RPMI. Autotransplantation of islets in pancreatectomised dogs was successful both after isolation in UWS and RPMI. We conclude that prevention of cell swelling during isolation and purification in the UWS resulted in an improved yield of viable and consistent virtually pure islets. Prevention of cell swelling during islet isolation should facilitate the analysis and control of other factors affecting outcome in man.

The bcl-2 oncogene is expressed in lymphoid and myeloid cells as well as in neurons and several types of epithelial cells and inhibits programmed cell death (apoptosis). Deregulation by the t(14;18) translocation in lymphoid malignancies induces inappropriate cell survival and serves as one of the steps toward a fully malignant behavior. Using pre- and postembedding immunoelectron microscopy in normal and neoplastic lymphocytes, we demonstrate bcl-2 immunoreactivity to the mitochondrial outer circumference and the nuclear envelope and to a lesser degree to the cell membrane. Mitochondrial staining was patchy, reminiscent of mitochondrial contact zones. Additionally, there was a suggestion of association with nuclear pores. In these regions, transmembrane transport is mediated. This may suggest that bcl-2 exerts its function in this process.

The outcome of islet isolation is considered uncertain because of the large variability of islet and insulin yield, but comparison of the isolated and native islet population has not been attempted. We therefore addressed the efficacy of collagenase digestion, and density gradient purification of islets from the splenic dog pancreas (n = 31) by morphometry of the islet volume and size distribution, and by extraction of insulin and amylase, in samples from the pancreas, the digest, and gradient fractions. In contrast to a approximately 90% recovery of pancreatic insulin and amylase after digestion, islet yield amounted to 50% of the islet content of the pancreas. After density separation, islets were mainly found in the purified fractions, while half of the recovered insulin was located in the acinar fraction of the gradients-indicating a substantial proportion of islets entrapped in acinar fragments. The islet and insulin content of the pancreas correlated well with islet and insulin yield after digestion (r = 0.7, p < .0001). The insulin content of digest suspensions did neither correlate with islet nor insulin recovery in the purified fraction of the gradients (r = 0.4) as opposed to the islet content of digest suspensions, which correlated with both (r = 0.7, p < .0001). After density separation near 100% purity was obtained, and no loss of insulin from isolated islets was demonstrated by extraction and microscopy. Size distributions of native and isolated islets demonstrated no fragmentation. We conclude that the variability of isolation outcome may be attributed to a large extent to the variability of the native endocrine pancreas. Isolation efficacy was best documented by morphometry, because insulin extraction did not discriminate between free and entrapped islets. However, assessment by both morphometry and extraction allowed the quantitation of entrapped islets, and demonstrated preservation of beta-cell granulation. Similar studies should facilitate the analysis of other factors affecting islet isolation in man.

In the present study the contribution of rat liver endothelial cells (EC) and Kupffer cells (KC) in the clearance of human (hu) C1q in rats was investigated. In untreated rats and rats depleted from KC the clearance kinetics and the tissue distribution of hu C1q were measured. In untreated rats, the clearance of hu C1q occurred in a monophasic manner with a half-life of 66 +/- 26.7 min. The clearance of hu C1q in KC-depleted rats was delayed significantly (p < 0.001) and occurred with a half-life of 217 +/- 78.8 min. Fifteen min after injection, 11 +/- 3.5% of hu C1q was found in the liver of untreated rats and 8 +/- 1.4% was found in the liver of KC-depleted rats. The percentage non-trichloroacetic acid precipitable activity in the circulation, as a measure for degradation of C1q, reached a level of 11.6 +/- 5.6% at 240 min in untreated rats compared with 4.6 +/- 5.8% in KC-depleted rats. Double immunofluorescence staining 5 min after administration of C1q in untreated rats, revealed that C1q was associated with KC and EC in the liver. Fifteen minutes after i.v. injection of hu C1q, there was an uptake of C1q in the hepatocytes. In KC-depleted rats, 5 min after administration of hu C1q, C1q was bound to the EC. Fifteen minutes after injection, C1q was also found in the hepatocytes. Electron microscopical studies revealed that C1q binds to EC, and that it is internalized in the hepatocytes and KC. The clearance of hu C1q in untreated rats was inhibited by preadministration of high concentrations of bovine C1q. These data show that rats depleted from KC are able to bind, internalize and degrade C1q, and that EC may play a role in the handling of C1q and C1q bound to immune complexes.

Reflection contrast microscopy (RCM) of ultrathin sections was recently introduced as a sensitive technique for visualization with enhanced definition in immunogold histochemistry. Experience of using RCM as a major tool in immunocytochemical research in different fields is summarized, e.g. oncology, nephrology and embryology. The sensitive visualization of immunocytochemical labels, gold particles or peroxidase-diaminobenzidine deposits in or on ultrathin sections, by RCM instead of electron microscopy is demonstrated. RCM of ultrathin sections is an adequate light microscopical alternative for immunoelectron microscopy, since an overview of both label and tissue is obtained with a high image definition and high contrast of label. In the studies presented, RCM is shown to provide a better gradation in staining intensity and staining pattern than other light microscopical methods. Moreover, a precise localization of multiple labels is obtained with this method. Besides the applications shown, ultrathin section visualization by RCM is very useful for correlative light- and electron microscopical studies of fine structures. Commercially available fluorescence microscopes can be adapted for proper RCM functioning; an adaptation scheme and list of microscopes tested is provided.

Background: In murine chronic graft-versus-host disease, an experimental model for lupus nephritis, autoantibodies against renal tubular epithelium can be found. Part of these antibodies are directed against a constituent of renal tubular epithelium, the enzyme Dipeptidyl peptidase IV (DPP IV). DPP IV is present on the cell membrane of glomerular epithelial and endothelial cells, and plays an important role in cell-extracellular matrix interactions. In mice and rats, administration of heterologous anti-DPP IV antibodies can induce proteinuria and podocyte effacement., Experimental Design: In this study, the glomerular DPP IV enzyme activity was investigated in the course of graft-versus-host disease in (C57BL/10 x DBA/2) F1 hybrids both by light- and electron microscopy using a DPP IV-specific substratum., Results: Light microscopical examination revealed an overall reduction of DPP IV activity and an altered distribution pattern in glomeruli as early as 4 weeks after induction of graft-versus-host disease. Immunofluorescence studies using anti-DPP IV antibodies showed actual redistribution, excluding antibody-mediated enzyme inactivation. Enzyme electron microscopy revealed an irregular deposition of reaction product characterized by a patchy, "moth eaten" appearance of the endothelial and epithelial membranes. This process occurred simultaneously with the development of albuminuria and preceded the effacement of epithelial foot processes. In control mice, DPP IV showed a continuous distribution along endothelial and podocyte membranes., Conclusions: In view of these findings we postulate that impairment of the function of DPP IV as a non integrin adhesion molecule may be one of the causative factors underlying the structural and functional lesions observed in this model for lupus nephritis.

The development of glomerulosclerosis was studied in murine chronic graft-versus-host disease (GvHD), which is a model for human systemic lupus erythematosus. The authors investigated the distribution patterns of six components of the extracellular matrix (ECM), i.e., laminin, fibronectin, collagen types I, III, IV, and VI during the course of the disease. All of these ECM components except collagen type I were found in the glomeruli of normal mice, where all of them were intrinsic constituents of the mesangium. Laminin, fibronectin, and collagen type IV were also found in the glomerular capillary walls. Starting 6 weeks after the induction of GvHD and continuing at week 8, the onset of an expansion of the mesangial matrix was observed. At the same time, the amounts of laminin, fibronectin, and collagen types IV and VI increased. Ten weeks after the onset of the disease, glomerulosclerosis developed. Traces of the interstitial collagen type I were found in sclerotic glomeruli. The levels of four ECM components, i.e., collagens III, IV, VI, and laminin were markedly decreased in the sclerotic glomeruli as compared with week 8. In contrast, the amount of fibronectin in the sclerotic glomeruli increased dramatically. Immunoelectron microscopic examination showed fibronectin in the sclerotic lesions, in contrast to laminin, collagen type I, and collagen type IV. It is concluded that the sclerotic lesions in murine chronic GvHD contain fibronectin. The small amounts of the ECM components laminin, as well as collagens III, IV, and VI in the sclerotic glomeruli in GvHD, might represent remnants of mesangial material and collapsed capillary walls. These components are probably replaced by increased production and/or accumulation of collagen type I and fibronectin.

The present study describes the development of membranous glomerulopathy (MGP) with high proteinuria in DZB rats exposed to mercuric chloride (HgCl2). IgG1 and IgG2a antibodies, eluted from glomeruli with subepithelial immune deposits, bind to the interface of the GBM and epithelial cells. High reactivity to GBM was demonstrated by ELISA and Western blotting, which could be absorbed for 30% by laminin or laminin-associated extracellular matrix components. No reactivity was found with type IV collagen, fibronectin, heparan sulfate proteoglycans, or tubular brush border antigens. Absorption to GBM removed the reactivity to renal antigens. Passively transferred eluted antibodies bind in a predominantly linear pattern along the GBM, causing focal ultrastructural transformations of the podocytes. These results suggest that this type of HgCl2-induced MGP, associated with epithelial cell injury and proteinuria, is caused by autoantibodies to basement membrane components which are located at the epithelial cell-basement membrane interface and may be involved in cell-matrix binding.

We investigated the clearance kinetics and tissue distribution of different sized IgA in normal and macrophage-depleted rats. Rats were injected iv with liposomes containing dichloromethylene diphosphonate (DMDP). DMDP treatment resulted in complete depletion of liver macrophages 24-48 h after administration. Normal and macrophage depleted rats were injected intravenously with monomeric, dimeric, polymeric or aggregated polymeric IgA (AIgA) and assessed for blood clearance and tissue distribution. In normal rats, clearance of IgA was size dependent, i.e. a faster clearance with increasing size. No differences in clearance kinetics were observed of the different sized IgA between normal and DMDP-treated rats. TCA non-precipitable radioactivity, a measure for degradation of IgA, was found in the circulation of normal and DMDP-treated rats after AIgA administration. The liver was the main organ responsible for the clearance of IgA in normal and DMDP-treated rats. Immunofluorescence studies on liver biopsies indicated that AIgA was associated with Kupffer cells in normal rats. Electron microscopical studies revealed that the AIgA was internalized and located in vesicles in Kupffer cells. In DMDP-treated rats the AIgA was associated with endothelial cells and electron microscopy studies showed that this AIgA was taken up by endothelial cells. These data show that rat liver endothelial cells are able to bind, internalize and degrade AIgA in situations where Kupffer cells are absent, and that these cells may play an important role in the handling of AIgA and IgA-immune complexes.

One simple post-embedding method for combined light- and electron microscopy is presented. Different types of antigens in normal rat and mouse kidneys as well as in tissues from cases of experimental induced nephritis were stained after Lowicryl K4M embedding by an immunogold (silver) method. The (silver-enhanced) gold particles were visualized by light microscopy, e.g. bright-field (BFM)- and reflection contrast (RCM) microscopy, as well as by electron microscopy. The potentials of RCM visualization in this field were investigated, resulting in the successful detection of colloidal gold (15 nm) particles, or silver enhanced gold particles, on ultrathin sections. Furthermore, an increased detection sensitivity of RCM compared with BFM together with an increase in the sensitivity of the immunostaining by RCM visualization was found. The different ways to use RCM, alone or in combination with bright-field- or phase contrast microscopy for visualization of plastic sections varying in thickness, type of plastic and staining, are discussed.

Alterations in glomerular function and structure were studied in protein-overload nephrosis in the rat induced by intraperitoneal administration of bovine serum albumin (BSA). Fractional clearance (C/GFR) studies using inulin and tracer proteins of different molecular size and charge revealed in proteinuric rats 1) unchanged glomerular filtration rate and renal plasma flow; 2) a 34-fold increase in C/GFR of rat serum albumin, reaching values similar to BSA; 3) a 2-fold increase in C/GFR for anionic horse radish peroxidase (HRP), but normal values for neutral and cationic HRP, and 4) an 11- and 3-fold increase for heterologous IgG and IgM, respectively. Glomerular epithelial cells showed degenerative changes, but the distribution of anionic sites in the glomerular basement membrane was found to be unaltered, as determined by polyethyleneimine binding studies. In summary, an elevation of serum albumin concentration resulted in an increased transcapillary albumin transport. This was found to lead to degenerative changes of glomerular epithelial cells with development of large pore defects, which were completely reversible.