Pathways followed by membrane recovered from the surface of plasma cells and myeloma cells.

Evidence for recovery of surface membrane and its fusion with Golgi cisternae has been obtained previously in several glandular cells. This study was conducted to determine whether or not membrane is similarly retrieved from the surfaces of plasma cells from lymph nodes (of rats immunized with horseradish peroxidase [HRP]) and mouse myeloma cells (RPC 5.4 and X63 Ag 8 cell lines). Electron-dense tracers (cationic and anionic ferritin, HRP) were used to trace the pathways followed by surface membrane recovered by endocytosis, and immunocytochemistry was used to identify the secretory compartments. When plasma cells or myeloma cells were incubated with cationized ferritin (CF), it bound to the cell surfaces and was taken up in endocytic vesicles, for the most part bound to the vesicle membrane. After 30-60 min, it was found increasingly within lysosomes and in several secretory compartments- notably in multiple stacked Golgi cisternae and secretory vacuoles. By immunocytochemistry the secretory product (immunoglobulins) and CF could be demonstrated in the same Golgi components. When myeloma cells were incubated with native (anionic) ferritin or in HRP, these tracers were taken up in much smaller amounts, primarily within the contents of endocytic vesicles. With continued incubation, they appeared only in lysosomes. When cells were doubly incubated, first in CF and then in HRP, both tracers were taken up (often within the same endocytic vesicle), but they maintained their same destinations as when incubated in a single tracer alone: the content marker, HRP, was localized exclusively within the lysosomal system, whereas the membrane marker, CF, was found within elements along the secretory pathway as well as within lysosomes. The findings demonstrate the existence of considerable membrane traffic between the cell membrane and the Golgi cisternae and lysosomes in both normal plasma cells and myeloma cells. Because myeloma cells behave like the glandular cells studied previously with regard to pathways of retrieved surface membrane, they represent a suitable and promising system for further studies of mechanisms and pathways of membrane retrieval and recycling in secretory cells.

Lithium-induced downregulation of aquaporin-2 water channel expression in rat kidney medulla.

Lithium, a widely used treatment for bipolar affective disorders, often causes nephrogenic diabetes insipidus. The effect of chronic lithium therapy on the expression of the vasopressin-regulated water channel Aquaporin-2 (AQP2) in rat kidney was examined. Membranes were prepared from inner medulla of one kidney from each rat, while the contralateral one was fixed for immunofluorescence and immunoelectronmicroscopy. Immunoblotting revealed that lithium treatment reduced AQP2 expression dramatically, to 31 +/- 8% after 10 d and to 4 +/- 1% after 25 d, coincident with development of severe polyuria. Immunofluorescence and immunogold quantitation confirmed the lithium-induced decrease in AQP2 expression (from 11.2 +/- 1.0 to 1.1 +/- 0.2 particles/microns 2). The downregulation was only partly reversed by return to lithium-free diet for 1 wk (40 +/- 8% of control). Furthermore, immunoblotting and immunogold quantitation revealed that 2 d of thirsting or 7 d of dDAVP treatment, in the continued presence of lithium, increased AQP2 expression by six- and threefold, respectively, coincident with increased urinary osmolality. Thirsting increased AQP2 immunolabeling mainly of vesicles, whereas dDAVP caused accumulation of AQP2 predominantly in the subapical region and plasma membrane. Thus, lithium causes marked downregulation of AQP2 expression, only partially reversed by cessation of therapy, thirsting or dDAVP treatment, consistent with clinical observations of slow recovery from lithium-induced urinary concentrating defects.

Cellular localization of PNA binding in colorectal adenomas: comparison with differentiation, nuclear:cell height ratio and effect of desialylation.

The lectin Arachis Hypogaea (Peanut Agglutinin, PNA) was used to study the cellular localization of the Thomsen-Friedenreich (T) disaccharide Gal-beta (1-3)-GalNAc alpha 1-R in 22 formalin-fixed paraplast-embedded colorectal adenomas of varying cellular dysplasia. An indirect immunoperoxidase method was used prior to and after neuraminidase treatment. Detailed information on the cellular localization of PNA binding was obtained. In addition, morphometric measurements of the nuclear: cell height ratios were performed on staining-filtered micrographs of crypts from all adenomas. We found 1) a statistically significant increase in the nuclear:cell height ratio with increasing grade of dedifferentiation (p less than 0.003), 2) a statistically significant smaller nuclear:cell height ratio in crypts that were PNA-positive in the Golgi region when these were compared to crypts that were PNA-positive on luminal cell membranes, 3) a decreasing number of crypts expressing PNA binding sites in the Golgi region with increasing dedifferentiation, leading to complete absence of PNA binding sites in Grade IV adenomas, 4) neuraminidase pretreatment increased the number of crypts expressing PNA binding sites in cytoplasm and on luminal membranes, whereas no changes were detected in crypts expressing PNA binding sites in the Golgi region. Our results confirm the general concept of accumulation of precursors of carbohydrate antigens in dedifferentiated cells. On the basis of the results presented, we conclude that the nuclear:cell height ratio shows a good correlation with the cellular localization of PNA binding, cellular differentiation and classic pathologic grading.

The number and size of glomeruli in long-term lithium-induced nephropathy in rats.

Chronic renal failure was induced in 10 Wistar rats using a lithium-containing (40 mmol/kg) diet from time of birth until an age of 55-65 weeks. Nine Wistar rats served as controls. The plasma lithium, the plasma urea, and the inulin clearance were measured, and one kidney was fixed by vascular perfusion with glutaraldehyde. The number of glomeruli was estimated stereologically by the fractionator method. The total number of glomeruli per kidney was 23.9 x 10(3) +/- 3.65 x 10(3) (+/- SD) in controls and 22.0 x 10(3) +/- 1.48 x 10(3) in the lithium-treated group, showing no statistically significant difference. The mean glomerular volume was also estimated using stereological methods. The number-weighted mean volume was reduced by 42% in the lithium-treated group, whereas the volume-weighted mean volume was unchanged. This can be attributed to the occurrence of many small glomeruli and a few very large glomeruli in the lithium-treated group. The many small glomeruli have in a previous study been shown to be atubular. The present study showed that the glomerular population is quite resistant to the deleterious effect of lithium; thus glomerular atrophy was seen, but no loss of glomeruli occurred.

GLUT4 expression in human muscle fibres is not correlated with intracellular triglyceride (TG) content. Is TG a maker or a marker of insulin resistance?

We have recently reported a progressive decline in the expression of glucose transporter isoform 4 (GLUT4) from control subjects through obese non-diabetics to obese type 2 diabetic subjects, indicating that the reduced GLUT4 in slow twitch fibres could be secondary to obesity. In this study we investigate the association of GLUT4 expression with the intracellular triglyceride (TG) content in the same muscle fibres and with plasma lipid parameters. We used histochemistry and stereology to study the relationship between TG content and GLUT4 expression in muscle fibres from obese, obese type 2 diabetic subjects, and young lean controls. TG density was significantly higher in slow compared to fast fibres in all studied subjects (p<0.05). We found an increased TG density in slow twitch fibres of obese diabetic subjects compared to obese (p<0.05) and lean controls (p<0.008). Intracellular TG densities in slow and fast fibres did not correlate with the corresponding GLUT4 density in the same fibres in our study groups (p>0.05). Plasma TG and FFA did not correlate with GLUT4 expression in slow or fast fibres (p>0.05). In conclusion, TG content was increased in diabetic slow fibres with a reduced GLUT4 expression. The GLUT4 expression was not associated with an increased intracellular triglyceride content or with increased plasma FFA levels. Thus, intracellular TG content and circulating FFA may not influence glucose transport directly through GLUT4 expression.

Cholesteatoma: a morphologic study of stratum corneum lipids.

Two different lipid-retaining fixation techniques permitted electron microscopic visualization of both intra- and extracellular lipids in cholesteatoma epithelium obtained from 25 patients. An increased number of intracellular lipid-containing Odland bodies was demonstrated, with a maximum in the basal layer of the stratum granulosum, while the superior layer contained substantially fewer organelles than are found in normal skin. At the stratum granulosum/stratum corneum interface lipids secreted from Odland bodies were found in sac-like invaginations along the cell membrane but premature exocytosis was also frequently observed. In the intercellular spaces of the stratum corneum, multiple long sheets of lamellar structures interrupted by slits or pores enclosed the keratinized corneocytes. The intercellular spaces seemed narrow and an extracellular barrier was not found until well above the stratum granulosum/stratum corneum interface. A similar distribution of Odland bodies and structure of intercellular lipids can occur in several dermatoses, where the formation and maintenance of the cutaneous permeability barrier are defective.

Culture of rabbit middle ear epithelial cells. A method for primary culture and subculture with identification, characterization and growth specification.

During the last decade middle ear epithelium has been cultured from various species. Until now, subcultivation has been achieved only with the use of a feeder-cell layer or conditioned medium. These factors are possible confounders in the in vitro model. On the other hand, subcultivation is necessary for exact quantitative studies. We present a reproducible culture method allowing subcultivation without feeder-cells or conditioned medium. The main features in our method are a low-serum, hormone-supplemented medium, an incubation temperature of 34 degrees C, fixation of explants, gentle trypsinization and replating with high cell density. Cells were identified by immunohistochemistry through a battery of monclonal antibodies. The percentage of epithelial cells in the subculture was 99.2%. To our knowledge, this is the first report describing subcultivation of middle ear epithelial cells exclusively in a completely controlled environment. These are optimal circumstances for future investigation and quantification of various factors influencing proliferation and differentiation of middle ear epithelium.

SMARCC1 expression is upregulated in prostate cancer and positively correlated with tumour recurrence and dedifferentiation.

BACKGROUND: The identification of new prognostic markers in prostate cancer (PC) is essential to improve patient treatment and management. Data suggest that SMARCC1 protein, a part of the intranuclear SWI/SNF complex which enhances the transactivation of the androgen receptor, is upregulated in PC and therefore a possible candidate marker for PC progression. MATERIALS: Expression of SMARCC1 immunostaining was analysed on a tissue microarray containing specimens from 327 patients with prostate cancer and clinical follow-up information. Furthermore, 30 specimens from patients with benign prostate hyperplasia were included as controls as well as 30 specimens of benign prostate tissue from PC patients. Also, 18 specimens from lymph node metastases were analysed. RESULTS: All benign specimens showed no or minimal staining for SMARCC1. In contrast, 20% of the specimens from patients with non-metastatic and non-recurrent disease showed moderate to marked staining. In 31% of the patients with recurrent disease and in 31% of the patients with metastatic disease we found moderate to strong SMARCC1 immunostaining. In total, 23% of lymph node metastases expressed SMARCC1. SMARCC1 expression was also positively correlated to Gleason score (p<0.05), clinical T stage (p<0.01) and time to recurrence (p<0.001). In a logistic regression analysis, patients with a marked SMARCC1 immunostaining had a significantly elevated odds ratio (OR) of 16 for recurrent cancer and an OR of 4.5 for metastatic disease. Conclusions. Our present results demonstrate an increased expression of SMARCC1 protein in prostate cancer and reveal a positive correlation with tumour dedifferentiation, progression, metastasis and time to recurrence.

Effect of intraluminal pressure on the ultrastructure and protein transport in the proximal tubule.

Proteins filtered in the kidney are generally considered to be catabolized by the renal lysosomes, but it has also been suggested that some protein may pass the tubule wall intact via the intercellular spaces. To examine the latter possibility, electron-dense tracers were in one part of the study injected retrograde into the kidney of living flounders and in another part by micropuncture directly into the tubule lumen of isolated flounder kidney tubules incubated in vitro. In both cases disruptions in the tubule wall were demonstrated and this was shown to be associated with the passage of tracer into the lateral intercellular spaces. From the in vitro studies it was further shown that only when the intraluminal pressure is increased do tracers pass into the intercellular spaces and they do so via the disruptions in the tubule wall. It is concluded that no passage of tracer occurs at intraluminal pressures in the physiological range.

Reversible peritubular binding of a cationic protein (lysozyme) to flounder kidney tubules.

Proteins filtered in the renal glomeruli are reabsorbed by the proximal tubule and catabolized in the lysosomes. On the basis of studies on isolated flounder tubules it has been suggested that, in addition to this catabolism, a transtubular transport of intact protein (lysozyme) also occurs. The present study demonstrates that significant amounts of lysozyme are reversibly bound to the peritubular side of isolated tubules. Electron microscopic autoradiography demonstrates that the protein is located in the basement membrane and intercellular spaces. After in vivo injection, 125I-lysozyme is mainly located in endocytic vacuoles of the first proximal segment, but also over the basal part of the cells. Since a significant peritubular binding of lysozyme is demonstrated in vitro, it is suggested that a similar binding of tracer protein originating from the peritubular capillaries might occur in vivo and that subsequent release of this protein in vitro might simulate transtubular transport. It is therefore concluded that release of tracer protein from isolated kidney tubules does not conclusively demonstrate transtubular transport of intact protein in experimental systems in which peritubular binding of protein can be demonstrated.

Ultrastructure and segmentation of microdissected kidney tubules in the marine flounder, Pleuronectes platessa.

A new method combining electron microscopy with microdissection was used to study the segmental variation along the tubule of a marine flounder. Two different nephron types were present. One type had long tubules with the glomeruli located close to the kidney surface. The other type had shorter and more coiled tubules with the glomeruli located close to the terminal end of the same nephron. Five different segments were present: (1) neck segment, (2) first proximal segment, (3) second proximal segment, (4) third proximal segment, and (5) collecting tubule. The third proximal segment was not present in all tubules. An extensive system of infoldings from the plasma membrane was present in all segments, except the neck segment and the collecting tubule. Tight junctions impermeable to lanthanum were present in all segments. The collecting duct cells also had extensive infoldings from the plasma membrane and tight junctions impermeable to lanthanum were also present here.

Lithium-induced uremia in rats - a new model of chronic renal failure.

Three groups of new-born rats were studied: Group Li/Li treated with Li for 16 weeks, group Li/C treated for 8 weeks followed by 8 weeks without Li, and Group C/C 16 weeks old controls. Both Li-treated groups showed severe reduction of renal function, particularly group Li/Li, where the mean GFR was reduced by 80%. Plasma urea, creatinine, and osmolality were increased, blood hemoglobin and hematocrit were reduced, whereas plasma Na, K, and standard bicarbonate were unchanged. Na clearance was maintained and fractional Na excretion thus increased. Fractional Li excretion was also increased, indicating inhibition of proximal tubular salt and water reabsorption. Renal concentrating ability was markedly reduced. When Li was withdrawn, plasma urea levels remained unchanged or continued to rise, and the concentrating defect persisted. The results demonstrate that Li administration to new-born rats causes irreversible chronic renal failure which may progress even in the absence of Li. This model of chronic renal failure has several characteristics in common with chronic renal failure in humans.

Stereological estimation of nuclear volume in benign and malignant melanocytic lesions of the skin. Inter- and intraobserver variability of malignancy grading.

The volume-weighted, mean nuclear volume (nuclear vv) may be estimated without any assumptions regarding nuclear shape using modern stereological techniques. As a part of an investigation concerning the prospects of nuclear vv for classification and malignancy grading of cutaneous melanocytic tumors, the observer variability of estimates of nuclear vv is studied. Routinely processed, paraffin embedded tissue specimens from 22 malignant melanomas and nine benign melanocytic cutaneous lesions are retrospectively investigated. The sampling scheme for estimation of nuclear vv is easy to use and robust, with more than 85% of the associated observed variance explained by differences among different tumors. Inter- and intraobserver reproducibilities are high, showing correlation coefficients of .86 and .96, respectively, with slopes of the regression lines close to unity. It is concluded that estimates of the three-dimensional nuclear vv in melanocytic cutaneous tumors are objective, unbiased, and highly reproducible.

Lithium-induced uraemia in rats: survival and renal function and morphology after one year.

Chronic renal failure was induced in Wistar rats by administration of a LiCl-containing (40 mmol/kg) diet from birth until an age of 55-65 weeks. The 55 weeks mortality was 51% in Li-uraemic rats versus 6% in control rats. In surviving rats the mean plasma Li levels were 0.6-0.7 mmol/l after 16 weeks and 1.0-1.1 mmol/l after 48 weeks. The mean plasma urea level was 14 mmol/l after 16 weeks and 22 mmol/l after 48 weeks of treatment compared with 8 mmol/l in the controls rats. In 55 weeks old Li-uraemic rats inulin clearance was reduced by 62% and Li clearance by 39%. Thus, fractional Li excretion was increased (from 20 to 34%) in rats with chronic Li-uraemia. Li-uraemic rats also had polyuria and failed to concentrate their urine in response to exogenous vasopressin. Systolic and mean arterial blood pressures were not significantly changed in rats with Li-uraemia. Morphological examinations of the kidneys showed large cortical cysts formed by dilated distal tubules and collecting ducts and widespread interstitial fibrosis. Proximal tubular mass was reduced by 50% and glomerular volume was also significantly reduced. The results indicate that in rats with Li-induced uraemia renal function and morphology deteriorate during Li-exposure up to an age of one year, associated with increased mortality.

Ultrastructural quantitation of atubular and hypertrophic glomeruli in rats with lithium-induced chronic nephropathy.

The very heterogeneous population of glomeruli in rats with lithium-induced chronic nephropathy which includes small glomeruli without connection to a proximal tubule (atubular glomeruli) and large hypertropic glomeruli with connection to a normal proximal tubule, was studied at the ultrastructural level, using stereological methods. After 8 weeks of lithium treatment followed by 8 weeks without lithium the hypertrophic glomeruli showed no changes in their relative ultrastructural composition, including normal mesangium, basement membrane-like material and peripheral basement membrane. The absolute quantities of each component were, however, increased due to the increased volume of the glomeruli. The atubular glomeruli had increased volume fractions of mesangium, peripheral basement membrane, basement membrane-like material and epithelium, whereas the absolute quantities were decreased due to the decreased volume. The thickness of the basement membrane was within normal limits in the group of hypertrophic glomeruli but increased by 31% above controls in the group of atubular glomeruli. Both groups of glomeruli in lithium-treated animals showed normal mean foot process width, but with a slightly abnormal distribution. The atubular glomeruli showed a disproportionate large decrease in peripheral filtration surface and capillary length, compared with the reduction in glomerular volume, whereas the hypertrophic glomeruli showed changes in proportion with the increased volume.

Early renal changes in lithium-treated normal and vasopressin-deficient rats.

The effects of lithium (Li) on the kidney were studied in normal Long Evans (LE) rats and in rats of the Brattleboro strain (DI rats) which have a congenital (hypothalamic) diabetes insipidus. The rats received a moderate daily oral dose of Li for 4 weeks. The renal cortex of untreated DI rats showed no changes compared with LE rats, but in their medullary collecting ducts (CD) the cells appeared more voluminous, and by light microscope morphometry (outer medulla) their volume fraction was increased. Furthermore, there was an increase in the enzyme histochemical activities of succinate and alpha-glycerophosphate dehydrogenases, most pronounced in the inner medullary zone. Li-treated LE rats and DI rats both showed the same degree of cortical and medullary changes. In the cortex, the light microscope and enzyme histochemical changes were confined to the connecting tubules (CNT) and CD. In the outer and inner medullary zones the increase in CD volume fraction and enzyme activity was much more pronounced than in untreated DI rats. Morphometry of the inner stripe of the outer medullary zone showed the same decrease in the volume fraction of the distal straight tubules (DST) in DI rats with and without Li treatment, as well as in Li-treated LE rats compared with normal LE rats. This decrease may be due to the lack of vasopressin-mediated cyclic AMP generation in the DST of these three groups of animals. It is concluded that the changes induced by Li in the DST are likely to be due to impairment of the vasopressin response known to be present in this segment. However, in the CNT and in the cortical and medullary CD there are changes which are effects of Li not related to the cellular actions of vasopressin.

Lithium-induced structural changes in the cortical distal nephron localized by computer-assisted three-dimensional reconstruction.

Lithium treatment is known to cause tubule dilation in distal nephron segments both in rat and in man. However, due to the heterogeneous cell composition of the distal nephron and the cellular changes following lithium treatment, it has been difficult to identify the structurally changed segments. In this study we have therefore applied computer-assisted reconstruction of cortical distal nephron segments. Tubule dilation was demonstrated in connecting and initial collecting tubules and in the first part of cortical collecting ducts (CCD) whereas it was absent from distal straight and distal convoluted tubules. Principal cells (P cells) in the CCD showed swelling of the cytoplasm, accumulation of actin-like microfilaments, and abnormal arrangements of basolateral membranes. Connecting tubule cells (CNT cells) showed similar but less pronounced changes. Intercalated cells (I cells) showed an accumulation of vesicles in the apical cytoplasm and a reduced luminal surface area. Lesions in P and CNT cells may, at least in part, explain the diabetes insipidus and sodium loss found during lithium treatment. Proton secretion in I cells is probably mediated by an ATPase present in the luminal membrane. The reduction in area of this membrane may explain why lithium-treated animals have a lowered ability to excrete an acid load.

Severe functional and structural changes caused by lithium in the developing rat kidney.

Lithium (Li) was administered to rats during maternal pregnancy and/or 8 weeks post-natally, to study the effects on renal function and structure in the developing kidney. Plasma Li was 0.5-1.0 mmol/l 3 and 8 weeks post-natally. Functionally, post-natal Li leads to growth retardation, polyuria with lowering of renal concentration ability, and uremia associated with as much as 80% lowering of the normal glomerular filtration rate (GFR). Pre-natal Li alone did not affect the concentrating ability but caused a 20% increase in GFR when evaluated 8 weeks post-natally. Post-natal Li caused very severe structural changes, consisting of up to 3 mm cortical cysts (= dilated distal convoluted tubules), extensive interstitial fibrosis with cell infiltration, and atrophy of the cortical collecting ducts. Morphometric measurements showed a significant reduction in the volume of the proximal tubular cells. Pre-natal Li caused only slight structural changes, and animals treated both pre- and post-natally were less affected than animals treated post-natally only. The structural changes caused by post-natal Li were unrelated to changes in the concentrating ability but showed a significant correlation with the lowering of the GFR. It is concluded that the post-natally developing rat kidney is particularly sensitive to the nephrotoxic effects of Li, which in low concentrations causes impairment of renal function, leading to uremia. Pre-natal Li exposure by maternal lithium treatment had little effect on renal function and structure when evaluated post-natally.

Lithium-induced morphological changes in the rat kidney at different levels of urine flow.

Lithium treated rats become polyuric and at the same time develop pronounced dilatations of distal tubular segments and characteristics enzyme histochemical changes. In the present study we have compared lithium-polyuric Wistar rats with lithium treated rats in which the polyuria was prevented either by administration of a vasopressin analogue or by water restriction. The kidneys were studied using enzyme histochemistry and light microscope morphometry. The characteristic lithium induced changes were present in all groups irrespective of the presence or absence of polyuria. It is concluded therefore, that the morphological and enzyme histochemical changes are induced by the lithium ion per se and not by the accompanying polyuria.

Human papilloma virus (HPV) and carcinomas of the head and neck.

Increasing evidence for a causal link between human papilloma virus and carcinomas of the cervix has emerged in recent research. This group of species-specific, epitheliotropic viruses has also been associated with tumours of the head and neck, but the individual reports deal only with relatively small sample numbers. In the present review these reports are considered in relation to the methods employed, and it is concluded that HPV is associated with more than 50% of oral and nasal carcinomas, as well as with carcinomas of the larynx and oesophagus. The clinical relevance and strategies for future work are outlined.