Occult metastasis is no burden factor in oral squamous cell carcinoma patients when adhering to a standardized approach in neck dissection.

OBJECTIVES: Management of the neck in patients with oral squamous cell carcinoma (OSCC) is pivotal to oncologic control and survival. However, there is controversy regarding necessity of neck dissection (ND) in patients with clinically node-negative neck. We aimed to assess risk factors for occult metastasis and to explore whether the presence of occult lymph node metastases (LNMs) has an impact on recurrence and survival. MATERIAL AND METHODS: A retrospective cohort study was performed including patients with primary OSCC who underwent radical tumor resection and ND in a high-volume center adhering to the prevailing German guideline. The ND was performed according to a standardized approach. RESULTS: Four hundred twenty-one patients with primary surgically treated OSCC were included. The incidence of occult metastasis was 14.49%. A pathological T stage > 1 (multivariate analysis, odds ratio (OR) 3.958, p = 0.042) and the presence of extranodal extension in LNMs (multivariate analysis, OR 0.287, p = 0.020) were identified as independent risk factors for occult metastasis. When comparing patients with and without occult metastasis, there were no significant differences in terms of progression-free survival (log-rank, p = 0.297) and overall survival (log-rank, p = 0.320). There were no cases of ipsilateral neck recurrence. One patient developed contralateral neck metastasis; however, he initially presented with a unilateral pT1 pN0 tumor. CONCLUSIONS: Overall, our findings suggest that conducting a standardized approach in ND should be applied in terms of management of the neck in order to maintain survival rates and to prevent neck recurrence in OSCC patients. CLINICAL RELEVANCE: None of the risk factors for occult metastasis can be reliably assessed preoperatively. Although elective ND does not guarantee the complete prevention of neck recurrence, it increases the likelihood of either timely removal of micrometastases or strengthens the justification for adjuvant therapy. Consequently, this approach leads to improvements in clinical outcomes.

Histodenz Separation of Lysosomal Subpopulations for Analysis of Chaperone-mediated Autophagy.

Chaperone-mediated autophagy (CMA) is the most selective form of lysosomal proteolysis, in which proteins are individually selected for lysosomal degradation. CMA degradation targets bear a pentapeptide consensus motif that is recognized by the cytosolic chaperone HSPA8 (Hsc70), which participates in the trafficking of the target to the lysosomal surface. From there, it is translocated into the lysosomal lumen, independent of vesicle fusion, in a process dependent upon the lysosomal transmembrane protein LAMP2A. There are limited tools for studying CMA in whole cells and tissues, and many of the best techniques for studying CMA rely on the preparation of lysosome enriched fractions. Such experiments include (1) the in vitro evaluation of CMA substrate uptake activity, (2) the characterization of changes to lysosomal resident and CMA regulatory proteins, and (3) lysosomal targetomics, i.e., the use of quantitative proteomics to characterize lysosomal degradation targets. Previous studies using discontinuous metrizamide gradients have shown that a subpopulation of liver lysosomes is responsible for the majority of CMA activity ("CMA+ "). These CMA+ lysosomes are low density and have higher levels of MTORC2 relative to the "CMA- " lysosomes, which are high density and have higher levels of MTORC1. Because of safety concerns surrounding metrizamide, however, this compound is difficult to obtain, and it is impractically expensive. Here, we have provided protocols for isolation of lysosomal subpopulations for CMA-related analyses from mouse liver using Histodenz, a safe and affordable alternative to metrizamide. Supplementary protocols show how to perform CMA activity assays with appropriate statistical analysis, and how to analyze for lysosomal breakage/membrane integrity. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Isolation of lysosomal subpopulations from mouse liver using discontinuous Histodenz gradients Alternate Protocol: Isolation of lysosomes from cultured cells using discontinuous Histodenz gradients Support Protocol 1: Verifying enrichment of lysosomal markers in lysosome-enriched fractions Support Protocol 2: Measuring in vitro uptake of CMA substrates Support Protocol 3: Measuring lysosomal membrane integrity by hexosaminidase assay.

Quantitative subcellular study of apical pole membranes from chicken oxyntic cells in resting and HCl secretory state.

Vertebrate oxyntic cells, responsible for gastric HCl production, undergo a remarkable morphological reorganization in relation to their secretory cycle. In resting state, the luminal surface of the cells is smooth; a peculiar system of endocellular membranes, the tubular system, occupies the luminal cytoplasm. Actin filaments frame a cortical network between the tubular system and the luminal plasma membrane. With the onset of HCl secretion, the tubular system becomes incorporated into the luminal plasma membrane. Villous processes containing microfilaments fill the secretory surface. This morphological reorganization of membranes and cytoskeletal matrix could regulate HCl secretion by translocation of membranes containing the proton pump from the endocellular compartment to the secretory surface. In this paper, we describe the isolation of membranes that selectively belong to the tubular system or to the cytoplasmic processes of the secretory surface of chicken oxyntic cells. Chicken oxyntic cells are the main cellular component of the proventricular glands. A resting state was obtained after cimetidine treatment, whereas the HCl-secretory state was induced by histamine. We present a comparative analysis of resting and stimulated chicken gastric glands by quantitative subcellular fractionation. The HCl secretory state was related to specific modifications in membrane fractions derived from the secretory pole of oxyntic cells. Morphological and functional reorganization of oxyntic cells was closely correlated with changes in: the sedimentation pattern of the marker enzyme of the apical pole membrane (K-NPPase), the total activity of K-NPPase and nonmitochondrial Mg-ATPase, the valinomycin dependence of K-ATPase, and polypeptides that cosediment in purified membrane fractions. Changes in the distribution pattern of K-NPPase after fractionation of histamine-stimulated glands were consistent with the replacement of the small vesicles typical of resting glands by dense membrane profiles, analogous to the luminal processes of stimulated oxyntic cells. SDS-PAGE showed that, in purified membrane fractions of stimulated glands, the concentration of 28-, 43-, and 200-kD polypeptides increased while that of 95- and 250-kD polypeptides decreased. The present results define the tubular system of oxyntic cells as an organelle with properties different from those of endoplasmic reticulum, mitochondria, and plasma membrane. The biochemical and physico-chemical properties of this membraneous system changed when the organization of the membranes and the cytoskeletal matrix of the apical pole was modified by the onset of HCl secretion.

Isolation of rat liver lysosomes by isopycnic centrifugation in a metrizamide gradient.

A preparation, similar to the light mitochondrial fraction of rat liver (L fraction of de Duve et al, (1955, Biochem. J. 60: 604-617), was subfractionated by isopycnic centrifugation in a metrizamide gradient and the distribution of several marker enzymes was established. The granules were layered at the top or bottom of the gradient. In both cases, as ascertained by the enzyme distributions, the lysosomes are well separated from the peroxisomes. A good separation from mitochondria is obtained only when the L fraction if set down underneath the gradient. Taking into account the analytical centrifugation results, a procedure was devised to purify lysosomes from several grams of liver by centrifugation of an L fraction in a discontinuous metrizamide gradient. By this method, a fraction containing 10--12% of the whole liver lysosomes can be prepared. As inferred from the relative specific activity of marker enzymes, it can be estimated that lysosomes are purified between 66 and 80 times in this fraction. As ascertained by plasma membrane marker enzyme activity, the main contaminant could be the plasma membrane components. However, cytochemical tests for 5'AMPase and for acid phosphatase suggest that a large part of the plasma membrane marker enzyme activity present in the purified lysosome preparation could be associated with the lysosomal membrane. The procedure for the isolation of rat liver lysosomes described in this paper is compared with the already existing methods.

Innate immune function of eosinophils: from antiparasite to antitumor cells.

Eosinophils are multifunctional leukocytes classically described as being involved in helminth parasitic infections and allergic diseases. Previously restricted to an exclusive role in the release of cytotoxic mediators, they are now also considered to be immunoregulatory cells and potential effectors in innate immune responses. Eosinophils are mainly found in tissues, so specific procedures are needed for their isolation from venous blood and for functional assays. Murine models are very useful for the dissection of eosinophil physiology in vivo. But murine eosinophils significantly differ from human ones. A complete understanding of eosinophil biology therefore requires comparative study of eosinophils from different mammalian species. We summarize here the main experimental protocols used to study human, mouse, and rat eosinophil biology. We focus on technical improvements of existing methods that optimize purification and in vitro functional studies of eosinophils.

Removal of inhibitors against RNA-directed DNA polymerase activity in human milk.

Milk from a number of species (e.g., man, mouse, rat, dog, and cow) contains inhibitors of the RNA-directed DNA polymerase. When attempts are made to isolate virions from the milk, part of the inhibitors follow the virions in the purification. The amount of inhibitors varies in different milk samples. These inhibitors can probably account for the large discrepancies reported in studies of the presence of oncornaviruses in human milk. Phosphatases bound to subcellular particles or fragments seem to be the most important inhibitors in the milk interfering with the RNA-directed DNA polymerase assay. It is shown that the inhibitory enzymes can be completely removed by sedimentation of the milk through a Metrizamide gradient.

Studies on metrizamide-protein interactions.

1. The apparent density of catalase after isopycnic centrifugation in metrizamide gradients is dependent on the metrizamide concentration into which the enzyme is dissolved at the beginning of the centrifugation. 2. This different behaviour of the enzyme in metrizamide gradients is due to the formation of a metrizamide-protein complex which is more dense than the uncomplexed catalase. 3. A bimodal distribution of the catalase, with additional heavy bands, was only observed in metrizamide gradients in light water, where rather high metrizamide concentrations are needed even for a banding of the uncomplexed enzyme. 4. The half-life of the metrizamide-protein complex is less than 5 min. This was shown by spectroscopical measurements and band sedimentation analysis in an analytical ultracentrifuge.

Interactions between the macromolecule and the gradient-forming solute in isopycnic sedimentation.

Interactions between the macromolecules and the gradient-forming solute in isopycnic sedimentation equilibrium experiments affect the distribution of the macromolecules in a number of ways. A description is given of the effects that may be expected, and some recently reported results are interpreted in the light of these. Suggestions are made for the recognition of such interactions, and for their possible effects, both good and bad, on experimental results.

Metabolic and ultrastructural characterization of guinea pig alveolar type II cells isolated by centrifugal elutriation.

Direct biochemical studies of the whole lung have been quite misleading because of the heterogeneity of the lung cell types. One of the advantages of studying the isolated cells is to be able to correlate specific metabolic functions with intracellular molecular events and to differentiate factors that affect the type II cell function directly. In the present study we have isolated type II cells from guinea pig lung with elastase and purified them by centrifugal elutriation. These cells fluoresce with phosphine 3R as the dye is specifically taken up by the lamellar bodies. In the electron micrographs, the type II cells display punctate villi, which underwent fragmentation in those cases where metrizamide density gradient was used. Mitochondria are scattered throughout the cytoplasm, and smooth endoplasmic reticulum is sparse. Type II cells possess large irregularly shaped nuclei with peripheral areas of dense hemochromatin and at least one prominent nucleolus. Ovoid lamellar bodies are the most prominent cellular inclusions. These bodies are present throughout the cytoplasm and contain a substructure of whorling and concentric laminations. Biochemical studies indicate that type II cells prepared by centrifugal elutriation are metabolically well preserved as seen from incorporation of [14C]leucine into cellular proteins, [methyl-14C]choline into cellular disaturated phosphatidylcholine and CDP[methyl-14C]choline into mitochondrial and microsomal phosphatidylcholine. Superiority of centrifugal elutriation over the commonly employed combination of discontinuous metrizamide gradient and cell elutriation is evident from the present study.

Effect of salts and chromatin concentrations on the buoyant density of chromatin in metrizamide gradient.

Using isopycnic centrifugation in metrizamide gradient, effect of ions and chromatin concentration on the buoyant density of chromatin was quantitatively examined. An elevation followed by gradual decline and secondary increase of the density occurred in accordance with increase in MgCl2 or NaCl concentration. Maximum density was observed at a concentration of these salts known to result in the condensation of chromatin. Release of protein occurred during the phase of density decline. The second increase in density is mainly due to the density increment of DNA in the chromatin. The density was dependent upon the concentration of chromatin in a band formed in the metrizamide gradient, while the density of free DNA and protein was not so greatly affected by their concentration. The density of chromatin in the presence of 0.14 M NaCl was less affected by the chromatin concentration than that in the absence of salt. Calculation of results indicates that grade of hydration of chromatin at concentrations lower than 400 microgram/ml in 1 mM Tris-HCl (pH 8.0) is higher than that expected from its DNA and protein components.

Properties of ribosomes centrifuged in metrizamide gradients.

The banding of ribonucleoproteins in metrizamide has been characterised using yeast ribosomes as a model system. Metrizamide does not dissociate ribosomes but it can facilitate the loss of loosely bound proteins. The buoyant density of fixed ribosomes in metrizamide gradients increases dramatically in the presence of low concentrations of Mg2+, whilst high Mg2+ concentrations give rise to multiple bands of higher density. These phenomena can be explained in terms of the binding of Mg2+ to high and low affinity sites as proposed for Escherichia coli ribosomes.

Isolation and initial characterization of a lymphocyte cap structure.

A method for isolating the cap structure induced by polycationized ferritin on the surface of mouse T-lymphoma cells is described. The procedure, based on the 'density perturbation' approach designed by Wallach and co-workers (Wallach, D.F.H., Kranz, B., Ferber, E. and Fischer, H. (1972) FEBS Lett. 21, 29-33), involves a simple, one-step density gradient centrifugation using metrizamide as the gradient material. The isolated polycationized ferritin cap fraction is approx. 20-fold enriched in plasma membrane relative to the whole cell homogenate and is apparently free of all uncapped membrane. Our initial analysis of the protein composition of the isolated cap structure indicates that there are approx. 30 membrane-bound polypeptides specifically associated with the polycationized ferritin cap fraction. Interestingly, there are at least four phosphorylated membrane-bound polypeptides (mol.wt. approximately 130 000, 100 000, 30 000 and 20 000) which are preferrentially accumulated in the cap fraction. These findings provide further evidence for the selective redistribution of certain surface membrane proteins during lymphocyte capping.

The preparation and characterization of liposomes containing X-ray contrast agents.

Unilamellar phospholipid vesicles loaded with the water-soluble, ionic X-ray contrast agent diatrizoate (Hypaque, Renografin) were manufactured by reverse-phase evaporation for use as organ-enhancing agents in X-ray computed tomography. Encapsulation efficiency was determined as a function of various diatrizoate concentrations in vesicles of varying lipid composition. Loss of encapsulated diatrizoate over 24 h was examined in vesicles composed of several egg phosphatidylcholine/cholesterol ratios. Size estimates for loaded vesicles were obtained by negative-stain electron microscopy, Millipore filtration and light microscopy. Intravenous in vivo injection of loaded vesicles in the rat resulted in significant enhancement of both spleen and liver on subsequent scans. Vesicles were similarly prepared with the water-soluble, nonionic agent metrizamide (Amipaque). Encapsulation efficiency was determined, and in vivo behavior was observed.

A high molecular weight RNA fraction in chromatin.

Utilizing a new chromatin isolation and fractionation technique we have obtained a high molecular weight RNA fraction from L-929 cell chromatin. The synthesis of this RNA is not greatly inhibited by concentrations of 0.04 mug/ml actinomycin D in the medium. Its synthesis appears to be strongly inhibited by 2 mug/ml of alpha-amanitin. The RNA appears to be quickly degraded (or removed from the chromatin) and does not contain a poly(A) sequence at its 3'-OH terminal end. Our working hypothesis is that this RNA is "nascent" heterogenous nuclear RNA partially transcribed from regions of the chromatin.

Spinal computed tomography and computed tomographic metrizamide myelography in the early diagnosis of metastatic disease.

New lesions were shown by Tc99m bone scans to have developed in sixty patients with known metastatic cancer or high-risk primary cancer and normal neurologic examinations; they were further evaluated with plain radiographs, spinal computed tomography (CT), and CT myelography (CT-M) according to an algorithm. Three groups were identified based on plain radiographs: group 1 (normal radiograph), group 2 (compression fracture as indicated by radiograph), group 3 (evidence of metastasis as indicated by radiograph). In group 1 (n = 18), spinal CT revealed that 33% of the patients had benign disease and 67%, metastases; epidural compression was seen in 25% of the patients with metastasis as indicated by CT-M. In group 2 (n = 26), CT-M disclosed that 38% had a benign compression fracture and 62% had metastases and that 63% of the patients with metastases had an epidural compression. In group 3 (n = 16), spinal CT revealed that 15 patients had metastases (one patient had benign disease). Epidural cord compression was seen in 47% of the patients with metastatic disease. In all groups, the presence of cortical bone discontinuity around the neural canal (seen in 31 patients) was highly associated with epidural compression (seen in 20 patients). Our approach allowed the early and accurate diagnosis of spinal metastasis and epidural tumor as well as the diagnosis of benign disease and was useful in planning optimal local therapy.

Electron microscopic analysis supports a dual role for the mitochondrial telomere-binding protein of Candida parapsilosis.

Linear mitochondrial genomes exist in several yeast species which are closely related to yeast that harbor circular mitochondrial genomes. Several lines of evidence suggest that the conversion from one form to another occurred accidentally through a relatively simple mechanism. Previously, we (L.T. & J.N.) reported the identification of the first mitochondrial telomere-binding protein (mtTBP) that specifically binds a sequence derived from the extreme end of Candida parapsilosis linear mtDNA, and sequence analysis of the corresponding nuclear gene MTP1 revealed that mtTBP shares homology with several bacterial and mitochondrial single-stranded (ss) DNA-binding (SSB) proteins. In this study, the DNA-binding properties of mtTBP in vitro and in vivo were analyzed by electron microscopy (EM). When M13 ssDNA was used as a substrate, mtTBP exhibited similar DNA binding characteristics as human mitochondrial SSB: mtTBP formed protein globules along the DNA substrate, and the bound proteins were randomly distributed, indicating that the binding of mtTBP to M13 ssDNA is not highly cooperative. EM analysis demonstrated that mtTBP is able to recognize the 5' single-stranded telomeric overhangs in their natural context. Using isopycnic centrifugation of mitochondrial lysates of C. papsilosis we show that mtTBP is a structural part of mitochondrial nucleoids of C. parapsilosis and is predominantly bound to the mitochondrial telomeres. These data support a dual role of mtTBP in mitochondria of C. parapsilosis, serving both as a typical mitochondrial SSB and as a specific component of the mitochondrial telomeric chromatin.

Use of colloidal silica (Sepracell-MN) for enrichment of dendritic cells from human peripheral blood: comparison with other methods.

Three methods are described for enrichment of dendritic cells from human peripheral blood. In method 1, mononuclear cells were incubated in plastic tissue culture flasks for two h. Nonadherent cells were removed. Adherent cells were washed to remove floating cells and incubated for 14 h at 37 degrees C in 5% CO2. Carbonyl iron was added, and the flasks were incubated for another 2 h. Nonadherent cells were subjected to centrifugation over metrizamide gradient. Phagocytic cells containing ingested carbonyl iron, small lymphocytes, and free carbonyl iron particles passed through the metrizamide, while the interface cell population was enriched for dendritic cells. The purity and yield of enriched dendritic cells were 52.8% and 0.05%, respectively. In method 2, adherent mononuclear cells were cultured overnight, and the released cells (released adherent cells) were centrifuged over metrizamide to separate low-density cells. Monocytes from this low-density cell population were removed by panning over human gamma globulin-coated plastic Petri dishes. In this method the average purity and yield of DC were 63.8% and 0.1%, respectively. In method 3, released adherent cells were treated with anti-CD5 and anti-CD14 monoclonal antibodies plus baby rabbit complement for 15 min, washed, and centrifuged with colloidal silica (Sepracell-MN). Centrifugation with Sepracell-MN was repeated three times. Low-density cells were panned twice over human gamma globulin-coated plastic Petri dishes. Nonadherent cells were highly enriched for DC. Contamination of T cells, B cells, and NK cells was undetectable by flow cytofluorometry. Contamination of monocytes was less than 2%. This method provided greater than 85.0% purity and 0.4% yield. This method (method 3) combines adherence, complement-dependent lysis, centrifugation with colloidal silica, and panning and provides the best yield and purity; it is therefore recommended for optimal purification of DC.

Cytotoxic tumor targeting with scFv antibody-modified liposomes.

Specific targeting of liposome-formulated cytotoxic drugs or antigens to receptors expressed selectively on target cells represents an effective strategy for increasing the pharmacological efficacy of the delivered molecules. We have developed a feasible technique to selectively attach antibodies and fragments thereof, but also small-mol-wt ligands such as peptides, carbohydrates, or any molecules that recognize and bind target antigens or receptors to the surface of small unilamellar liposomes. Our concept is based on the site-specific functionalization of the ligands to be attached to the liposomes by thiol groups. These thiol groups can easily be introduced to antibodies or peptides by addition of cysteines, preferably at sites that do not interfere with the receptor binding domains. Optimally, the site-specific modification is introduced at the C-terminal end of the ligand, separated by an inert spacer sequence located between the thiols and the specific part of the ligand. The thiol-reactive molecules on the liposome surface are maleimides that are linked to phospholipids composing the liposome bilayer membrane. We illustrate the coupling method of a functionalized single-chain antibody fragment with binding specificity to ED-B fibronectin, an isoform of fibronectin exclusively expressed in tumor tissues, to long circulating small unilamellar poly(ethylene glycol) liposomes.

Isolation and culture of hepatic stellate cells.

Hepatic stellate cells (HSCs) are routinely prepared by collagenase/pronase digestion of liver using a perfusion system and subsequent fractionation of the heterogeneous cell suspension on continuous density gradients made out of Nycodenz, metrizamide, stractan, or percoll. Because of their lipid content, stellate cells are the least dense fraction of the nonparenchymal cells, and during centrifugation they float effectively away from other hepatic cells resulting in preparations containing almost 80% stellate cells. The degree of purity can be increased by further enrichment of cells by methods like centrifugal elutriation or Scatter-activated cell sorting. We present a detailed protocol from our laboratory to obtain a high number of pure, viable, freshly isolated hepatic stellate cells from rat liver. This two-step protocol (collagenase/pronase digestion and Nycodenz gradient) yields a preparation of approx 4-5 x 107 cells enriched in 74% HSC having a viability of at least 76% as estimated by Trypan blue exclusion test. Further purification by centrifugal elutriation results in virtually pure HSC preparations ( >98%).

Metrizamide cisternography in the investigation of the empty sella syndrome.

Pneumoencephalography, the usual method for demonstrating air within the sella turcica in empty sella syndrome (ESS), has been approached with reluctance by most physicians because of its technical difficulty and patient morbidity. For these reasons, neuroradiologists have investigated other contrast media in search of an ideal agent; metrizamide seems to be such an agent. Metrizamide is a nondissociable, water-soluble glucose amide containing three iodine molecules. This agent is miscible with CSF, and small recesses of the CSF-brain interface can be delineated with hypocycloidal tomography without performing cumbersome patient maneuvers to fill the cisterns. Furthermore, morbidity has been minimal, particularly with use of lower concentrations of metrizamide, allowed by the sensitivity of computerized tomographic (CT) scanning. Thus, at the present time, metrizamide cisternography (especially in conjunction with CT scanning) appears useful in evaluating an enlarged sella turcica, particularly when considering an entity such as ESS.