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.

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.

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.

Evaluation of donor-specific transfusion sources: unique failure of bone marrow cells to induce prolonged skin allograft survival with anti-CD154 monoclonal antibody.

BACKGROUND: Treatment with anti-CD154 monoclonal antibody (mAb) plus a donor-specific transfusion (DST) of spleen cells prolongs skin allograft survival in mice through a mechanism involving deletion of host alloreactive CD8(+) T cells. It is unknown if other lymphohematopoietic cell populations can be used as a DST. METHODS: Murine recipients of allogeneic skin grafts on day 0 were either untreated or given a DST on day -7 plus 4 doses of anti-CD154 mAb on days -7, -4, 0, and +4. Deletion of CD8(+) alloreactive cells was measured using "synchimeric" CBA recipients, which circulate trace populations of TCR transgenic alloreactive CD8(+) T cells. RESULTS: Transfusion of splenocytes, thymocytes, lymph node cells, or buffy coat cells led to prolonged skin allograft survival in recipients treated with anti-CD154 mAb. In contrast, bone marrow DST failed to delete host alloreactive CD8(+) T cells and was associated with brief skin allograft survival. Transfusions consisting of bone marrow-derived dendritic cells or a mixture of splenocytes and bone marrow cells were also ineffective. CONCLUSIONS: Donor-specific transfusions of splenocytes, thymocytes, lymph node cells, or buffy coat cells can prolong skin allograft survival in recipients treated with costimulation blockade. Bone marrow cells fail to serve this function, in part by failing to delete host alloreactive CD8(+) T cells, and they may actively interfere with the function of a spleen cell DST. The data suggest that transplantation tolerance induction protocols that incorporate bone marrow cells to serve as a DST may not be effective.

Clinical significance of phenotypic features of blasts in patients with myelodysplastic syndrome.

Knowledge of the blast phenotype in myelodysplastic syndrome (MDS) would be valuable, as in other malignancies, but remains sparse. This is mainly because MDS blasts are a minor population in clinical samples, making analysis difficult. Thus, for this blast phenotype study, we prepared blast-rich specimens (using a new density centrifugation reagent for harvesting blasts) from blood and marrow samples of 95 patients with various MDS subtypes and 21 patients with acute leukemia transformed from MDS (AL-MDS). Flow cytometry revealed that a high proportion of the enriched blast cells (EBCs) from almost all patients showed an immunophenotype of committed myeloid precursors (CD34(+)CD38(+)HLA-DR(+)CD13(+)CD33(+)), regardless of the disease subtype. The cytochemical reaction for myeloperoxidase was negative in 58% of the cases. Thus, the EBC phenotype is more immature in MDS than in de novo acute myeloid leukemia. MDS EBCs often coexpressed stem cell antigens and late-stage myeloid antigens asynchronously, but rarely expressed T- and B-lymphoid cell-specific antigens. Markers for myeloid cell maturation (CD10 and CD15) were more prevalent on EBCs from low-risk MDS (refractory anemia [RA] and RA with ringed sideroblasts), whereas markers for myeloid cell immaturity (CD7 and CD117) were more prevalent on EBCs from high-risk MDS (chronic myelomonocytic leukemia, RA with excess blasts [RAEB], and RAEB in transformation) and AL-MDS. A shift to a more immature phenotype of EBCs, accompanying disease progression, was also documented by sequential phenotyping of the same patients. Further, CD7 positivity of EBCs was an independent variable for a poor prognosis in MDS. These data represent new, valuable information regarding MDS.

A simple centrifugation method for harvesting myeloblasts.

Myeloblast-rich samples, required for investigation of myeloid malignancies, can be obtained only during the untreated stage of leukemia. Existing methods for myeloblast enrichment have various prerequisites that limit their application. In this new method, a mixture of peripheral blood (Mixed PB) from an acute myeloid leukemia (AML) patient and from a healthy control containing 5% myeloblasts was subjected to density gradient centrifugation using a 14.5% metrizamide solution. Both high purity (86.3% +/- 1.5%) and high recovery of viable myeloblasts were achieved. Close to 100-fold blast enrichment, even from Mixed PB containing only 0.15% myeloblasts, was achieved. Similarly, this method highly enriched myeloblasts from unprocessed samples, including marrow cells, from patients with AML, myelodysplastic syndromes, and chronic myeloid leukemia (purity: 2.7% +/- 2.0% before separation, 56.6% +/- 28.3% after separation) (n = 22). The enriched blasts were suitable for various analyses, eg, flow cytometry, immunocytochemistry, cytochemistry, fluorescence in situ hybridization, and gene analysis.

The uptake of calcium by isolated chromaffin granules of the adrenal medulla.

Bovine chromaffin secretory granules were purified by isopycnic Metrizamide gradient centrifugation and their Ca2+ sequestration pathways were characterized. The rate of Ca2+ sequestration at 37 degrees C was first order, with a maximal uptake of 26.9 +/- 0.46 (mean +/- S.D., n = 3) nmol Ca2+/mg protein and a first order rate constant (k) of 0.046 +/- 0.002 min-1. At 4 degrees C the rate of uptake was substantially attenuated, with only 2.47 +/- 0.2 (mean +/- S.D, n = 3) nmol Ca2+/mg protein sequestered in 60 min. Ca2+ sequestration was 93% inhibited by 180 mM NaCl [I50% of 78.7 +/- 9.3 mM NaCl (mean +/- S.D., n = 11)] but only slightly inhibited by KCl or MgCl2. Ca2+ sequestration was not stimulated by incubation with MgATP but was inhibited by 57% after incubation with 30 microM monensin. Ca2+ sequestration was dependent on extravesicular Ca2+ with half-maximal sequestration at pCa2+ 6.81 +/- 0.028 (mean +/- S.D., n = 3). Sequestered Ca2+ could be exchanged with external 45Ca2+, the exchange rate was first order (k of 0.042 +/- 0.004: mean +/- S.D., n = 3) and saturated at 27.7 +/- 1.1 nmol Ca2+/mg (mean +/- S.D., n = 3). The Ca2+/Ca2+ exchange system was totally inhibited by NaCl or KCl but only slightly by MgCl2. About 75% of sequestered 45Ca2+ could be released by incubation with NaCl, but only 8% was released by incubation with KCl. Half-maximal release of sequestered 45Ca2+ required 69.3 +/- 12.2 mM NaCl (mean +/- S.D., n = 3). The Na+-induced release of sequestered 45Ca2+ was rapid, t0.5 of 2.80 +/- 0.63 min (mean +/- S.D., n = 3) and inhibited at 4 degrees C. The concurrent incubation of chromaffin granules with 45Ca2+ and either annexin proteins V or VI resulted in attenuated uptake of 45Ca2+. These results suggest that Ca2+ uptake in adrenal chromaffin granules is regulated by Na+ and Ca2+ gradients and also possibly by annexins V and VI.

Large intrathoracic meningocele in a patient with neurofibromatosis: technical report.

The authors describe a case of large intrathoracic meningocele (16 cm) associated with neurofibromatosis. Computed tomography with metrizamide myelography proved valuable in locating the lesion and in reformation after surgery. The authors make some comments about surgical excision of large meningocele.

Interfacial catalysis by phosphoinositide 3'-hydroxykinase.

Phosphorylation of phosphoinositides by phosphoinositide 3'-hydroxykinase (PI3K) occurs at a lipid/water interface. We have determined that highly purified recombinant human P13K binds tightly to vesicle interfaces composed primarily of phosphatidylinositol (PI) or 1,2-dimyristoyl-sn-glycero-3-phosphomethanol (DMPM). The rate of desorption of PI3K from the vesicle interface is slow and does not significantly affect the observed product formation kinetics. Observations which demonstrate that PI3K is tightly bound to the vesicle lipid/water interface include the following: (1) product formation plateaus rapidly, even in the presence of active enzyme and excess substrate; (2) total product formation is proportional to the amount of PI3K; (3) initial product formation rates are unaffected by bulk lipid concentration but are dependent on the interfacial substrate concentration; and (4) PI3K partitions with lipid vesicles in sedimentation gradients. This enzymatic profile has been referred to as catalysis in the "scooting" mode (Berg et al., 1991). A kinetic analysis of PI3K catalysis in the scooting mode is presented. The interfacial Km,app for PI was determined to be approximately 6.0 mol % in PI/DMPM vesicles. The ratio of specificity constants (kcat/Km) for PI, phosphatidylinositol 4-monophosphate (PIP), and phosphatidylinositol 4,5-diphosphate (PIP2) utilization was determined to be near unity. These results provide a rigorous enzymological framework for the kinetic analysis of PI3K inhibitors.

Techniques of facial nerve block.

The efficacy of different techniques of facial nerve block for cataract surgery was investigated. Forty four patients underwent either modified O'Brien, Atkinson, van Lint, or lid blocks. Intentional muscle activity of the orbicularis oculi muscle was recorded and the area under the EMG curve calculated for quantitative comparison of muscle activity between the groups before and after injection of lignocaine with the vasoconstrictor naphazoline nitrate. In addition, the force of lid closure was measured and lid motility determined on a subjective score scale. Whereas the modified O'Brien and lid blocks nearly abolished the muscle activity recorded in the EMG (p < 0.003), the Atkinson and van Lint blocks did not significantly affect these variables. The O'Brien and lid blocks decreased the force of lid closure and lid movements far more effectively than the Atkinson and van Lint blocks (p < 0.0001). The topographic distribution of a mixture of metrizamide and lignocaine solutions was evaluated radiographically in eight additional patients, to assess potential causes for differences in the efficacy of the block techniques. The radiological results showed involvement of the region of the facial nerve trunk and its temporal and cervical divisions by the modified O'Brien block. The lid block, on the other hand, affected terminal branches of the facial nerve's temporal division. In this study, complete lid akinesia was achieved by both the modified O'Brien block and the lid block. However, because the modified O'Brien block involves the risk of neural injury to the facial nerve or its main divisions, the lid block is recommended as the most effective and safe method to achieve akinesia of the orbicularis oculi muscle.

Computed tomography of an intracranial lipoma confined in the suprasellar cistern.

A case of large intracranial lipoma involving the suprasellar cistern is presented in a 50-yr-old man with a history of occipital headaches. Evaluations by routine computed tomography (CT) combined with metrizamide CT cisternography were extremely useful in the diagnosis of this rare condition. Intracranial lipomas are reviewed in the literature and the role of CT evaluations is discussed.

Effect of thyroid hormones and other iodinated compounds on the transition of monocytes into veiled/dendritic cells: role of granulocyte-macrophage colony-stimulating factor, tumour-necrosis factor-alpha and interleukin-6.

Stimulation of human peripheral blood monocytes with the thyroid hormones tri-iodothyronine (T3) and thyroxine (T4) enhanced their ability to mature into cytologically and functionally characteristic veiled/dendritic cells. Veiled/dendritic cell transition induced by T3 and T4 was dependent on the production of granulocyte-macrophage colony-stimulating factor (GM-CSF), tumour necrosis factor-alpha (TNF alpha) and interleukin-6 (IL-6) in the culture, since the addition of antibodies specific for GM-CSF, TNF alpha and IL-6 to the culture system had blocking effects. The addition of antibodies to macrophage colony-stimulating factor and IL-1 had no effects. Contaminating T cells and B cells did not contribute to the transition of monocytes to veiled/dendritic cells, and it is therefore likely that the GM-CSF, TNF alpha and IL-6 produced in the culture system were derived from the monocytes themselves. Stimulation of the blood monocytes with an optimal concentration of metrizamide (14.5%), reverse T3 (rT3; 2 x 10(-10) M) or highly iodinated thyroglobulin (Tg; 2 x 10(-11) M) also resulted in an increased transition of monocytes to veiled/dendritic cells, but to a lesser extent in comparison with the thyroid hormones (T3, 31 +/- 6% and T4, 25 +/- 5% vs rT3, 22 +/- 8% and Tg with an iodination grade of 0.37%: 20 +/- 4% veiled/dendritic cells). Administration of anti-GM-CSF, anti-TNF alpha and anti-IL-6 to the culture system also had blocking effects on the transition from monocytes to veiled/dendritic cells induced by the iodinated compounds. The mechanisms by which such iodinated compounds act on the monocyte to veiled/dendritic cell transition can only be speculated on (interference H2O2-generating system?).

Intraneoplastic application of metrizamide-containing liposomes: kinetic studies with computed tomography.

Liposomes may serve as drug carriers not only for systemic chemotherapy but also for intraneoplastic drug therapy because they show a sustained drug release. In the present study, the in vivo kinetics of intraneoplastic deposits of large multilamellar vesicles containing metrizamide was followed up in a rat tumor model with computed tomography. The influence of four different lipid compositions on the retardation capacity of large multilamellar liposomes was investigated. By comparing the dynamic data of X-ray attenuation and volume of liposome deposits, a rank order for the in vivo stability of metrizamide containing multilamellar vesicles could be established: the least stable liposomes were made of pure dimyristoyl-phosphatidyl-choline, the most stable type was made of equimolar parts of stearoyl-palmitoyl-phosphatidyl-choline and cholesterol. Of intermediate stability were liposomes made of equimolar parts of dimyristoyl-phosphatidyl-choline and cholesterol, and those made of pure stearoyl-palmitoyl-phosphatidyl-choline. The addition of 50% cholesterol increased the membrane stability of both dimyristoyl-phosphatidyl-choline and stearoyl-palmitoyl-phosphatidyl-choline liposomes. No diffusion of large multilamellar liposomes away from the injection site was observed. The in vivo stability of the liposomes was considerably less than that observed in vitro, suggesting active degradation processes. It is concluded that large, multilamellar liposomes may be suitable carriers for intraneoplastic chemotherapy. The present model is easily adaptable to be transferred into clinical conditions, and may allow direct monitoring of intraneoplastic liposome-mediated chemotherapy in human brain tumors.

Lung tumor incidence after intrabronchial administration of the nonionic contrast agent metrizamide.

RATIONALE AND OBJECTIVES: Metrizamide has been used for examination of the gastrointestinal tract and tracheobronchial tree of infants. Contrast agents may enter the lungs during such examinations. The current study was undertaken to determine whether there would be any later pulmonary effects when metrizamide was administered to the lungs of weanling mice. METHODS: One hundred fifty mice (18-21 days old), divided into groups, received either 75 microL of metrizamide, using the manufacturer's diluent (190 mg iodine [I]/mL), or saline solution administered to the lungs by injection into the trachea. The mice were observed for the duration of their lives. Moribund animals were killed. At death, all animals underwent necropsy. The lungs were fixed in formalin, and histologic sections were examined for pathologic changes. RESULTS: The incidence of lung tumors was increased (P less than .05) in the lungs of mice receiving metrizamide compared with those receiving saline. Eighteen percent of the lung tumors in the metrizamide-treated mice were lymphomas, a histologic type not found in the saline-treated controls. CONCLUSIONS: A hypothesis proposing that metrizamide may be an initiator of carcinogenic transformation rather than a carcinogen was developed.

Correlaçäo entre tomografia computadorizada e diagnóstico cirúrgico dos tumores intrarraquianos / Correlation between computed tomography and surgical diagnosis of intraspinal tumors

Relatamos a contribuiçäo da tomografia computadorizada (TC) no diagnóstico e tratamento cirúrgico de 10 tumores intrarraquianos. A TC sem contraste, realizada em 2 pacientes, näo foi suficiente para demonstrar a patología. Salientamos a necessidade da utilizaçäo de contraste para o diagnóstico em todos os casos estudados. Em 9 pacientes o diagnóstico foi confirmado pela TC realizada após injeçäo intratecal de metrizamida (TC-metrizamida). No caso restante, o diagnóstico foi feito mediante administraçäo de contraste iodado (conray) por via endovenosa, que contrastou o tumor em toda sua extensäo

A species/strain comparison of hepatic natural lymphocytotoxic activities in rats and mice.

The natural killer (NK) and natural cytotoxic (NC) cell activities in livers from certain rat and mouse strains were compared. This included the two rodent strains used in animal carcinogenicity bioassays, i.e. Fischer 344 and B6C3F1 mice. Sprague-Dawley and Fischer 344 rats exhibited high hepatic NK activity, which was greater than the levels seen in all of the five mouse strains studied. However, the hepatic NC activity in rats was comparable to the activities observed in C57BL and BALB/c mice. An inverse relationship was observed between the two tumoricidal activities in all but one of the mouse strains examined; that is (at 8 weeks of age), NK activity: C3H greater than B6C3F1 greater than CBA greater than BALB/c; NC activity: BALB/c much greater than CBA greater than B6C3F1 greater than C3H. The C57BL mouse strain was the only strain to express both activities at comparatively high levels. Female mice exhibited a similar profile of cytotoxic activities. Rats also possessed high activities of a presently ill-defined tumoricidal activity, this being the spontaneous P815 mastocytoma killing by unstimulated effector cells, over an 18 h period. Both adherent and nonadherent effector cells from rat livers, but only the nonadherent cell population isolated from male mouse livers, exhibited this activity which may represent a distinct hepato-specific population of natural lymphocytotoxic effector cells. The tumoricidal activities in liver-derived cells were greater than those of effector cells isolated from the spleen. The differences in natural immunity reported in this study may be related to the varying background incidences of hepatic tumors, i.e. the mouse strains susceptible to high background incidences of liver tumors have relatively low natural immunity, whereas the two mouse strains resistant to hepatic tumors possess high levels of at least one hepatic NLC activity. Similarly, rats have relatively low hepatic tumor rates and high levels of hepatic natural immunity.

A neurite-promoting factor from muscle supports the survival of cultured chicken spinal motor neurons.

During embryonic development, spinal motor neurons require muscle-derived trophic factors for their survival and growth. We have recently isolated a protein from muscle that is not laminin but that still stimulates neurite outgrowth from embryonic neurons in culture. In the present study, we investigated whether this protein, which we refer to as muscle-derived neurite-promoting factor (NPF), could also promote the survival and growth of motor neurons in culture. Spinal motor neurons were isolated from 6-day-old chicken embryos by a metrizamide step-gradient centrifugation protocol. Most large cells (putative motor neurons) were found in the upper metrizamide fraction (0%-6.8% interface; fraction I). Motor neurons were identified by increased specific activity of choline acetyltransferase (CAT) and by their propensity to transport retrogradely either wheat germ agglutinin-horseradish peroxidase or the fluorescent dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine per chlorate (diI), when those substances were injected into the target field. Labeled motor neurons were 2.6-fold enriched in fraction I and the specific CAT activity was 4.4-fold increased in fraction I as compared to unfractionated cells. When motor neurons were grown on muscle-derived NPF, the protein supported the survival of at least 21% of the neurons for as long as 6 days in culture. The protein showed no significant effect on either CAT specific activity or on high-affinity choline uptake by neurons. There was a substantial increase from 21% to 38% of the survival of motor neurons when a combination of muscle-derived NPF and laminin was used as the substrate. Muscle-derived NPF also promoted the survival of sensory neurons and sympathetic neurons in culture. Our results demonstrate that a neurite-promoting protein derived from muscle promotes both the survival and the outgrowth of neurites from cultured spinal motor neurons as well as from sensory and sympathetic neurons.

Bilateral middle cranial fossa arachnoid cysts. A case report.

The temporal lobe agenesis syndrome is a rare congenital abnormality. This syndrome frequently has been described in association with arachnoid cysts or abnormal collections of cerebrospinal fluid. Arachnoid cysts develop most frequently in the middle cranial fossa and almost all these cysts are unilateral. Bilateral middle cranial fossa arachnoid cysts are extremely rare and only 9 cases have been reported in the literature. We present an adult case with bilateral arachnoid cysts and temporal lobe agenesis whose mental examination and neurologic assessment is normal. The cysts are demonstrated by CT and metrizamid CT cysternography.