Novel approach to gene expression profiling in Sézary syndrome.

BACKGROUND: Microarray hybridization studies in Sézary syndrome (SS) have compared T lymphocytes from patients with cutaneous T-cell lymphoma with those of normal controls; a major limitation of this design is that significant inherent genetic variability of lymphocyte populations between individuals may produce differences in gene expression unrelated to disease state. OBJECTIVE: The objective of this study was to minimize the heterogeneity of information derived from whole-genome expression analysis and to identify specific genetic differences between highly purified malignant and nonmalignant (control) T cells from the same patient with SS. METHODS: Peripheral blood mononuclear cells were obtained from a patient with SS, stained with anti-T-cell receptor Vb (TCR-Vb) antibodies, and sorted by multiparameter flow cytometry. Malignant cells expressed the dominant TCR-Vb; control T cells lacked the dominant TCR-Vb but were otherwise phenotypically identical (CD3+CD4+CD45RO+). These cell populations were compared using the Illumina Inc. Sentrix Human-6 expression BeadChip system. RESULTS: Transcriptome analysis using the J5 test, which was selected for data analysis based on an efficiency analysis of competing statistical methods, showed differential expression of 44 genes between the malignant and nonmalignant cell subsets. Promyelocytic leukaemia zinc finger protein (ZBTB16) was the most profoundly upregulated gene in the malignant cell population, while interferon regulatory factor 3 (IRF3) and interferon-induced protein 35 (IFI35), which are important elements of the cellular response to viral infection, were significantly downregulated. CONCLUSIONS: The results of this study suggest the feasibility of this novel comparative approach to genomic profiling in SS. Using this method, we identified several differentially expressed genes and pathways not previously described in SS. While these findings require validation in larger studies, they may be important in SS pathogenesis.

Failure to detect immunocytochemically reactive endogenous lectin on the cell surface of Dictyostelium discoideum.

The endogenous lectins of Dictyostelium discoideum, called discoidins I and II, have been implicated in cell cohesion during the associative phase of this organism. In an effort to repeat and extend the studies of these putative cell-surface proteins, we attempted a variety of immunocytochemical techniques. Antibodies to a mixture of the purified discoidins were raised in rabbit. Both living and fixed cells were examined by indirect immunoferritin labeling using whole antiserum and by direct immunolabeling using purified specific IgG adsorbed to colloidal gold. Cells, at the appropriate stage, of strains A3, NC-4, and WS-582 were tested. In no instance were cell surface antigens detected despite meticulous efforts to duplicate the published techniques and to extend and refine them. Specific localization was found only in the cytosol and on the cytoplasmic face of certain endomembrane vesicles, and much less so on outer nuclear and mitochondrial membranes, in inadvertently disrupted cells. In no case was specific label found on either side of the plasma membrane or on food vacuoles. Exogenously supplied discoidins, bound to cells, were successfully localized by our technique. We conclude that the discoidins are not present on the cell surface, or are there in undetectable quantities, during the associative phase. We suggest that previous demonstrations of these proteins at the cell surface were artifacts resulting from the way in which the cells were handled, which caused the binding of externalized discoidins, possibly those released from lysed cells. We believe that the current notion that the discoidins play a direct role in cell cohesion by virtue of their carbohydrate-binding capacity should be reexamined. We suggest that the true role of the discoidins is solely intracellular.

Incorporation of protein into spore coats is not cell autonomous in Dictyostelium.

At maturity, the spores of Dictyostelium are suspended in a viscous fluid droplet, with each spore being surrounded by its own spore coat. Certain glycoproteins characteristic of the spore coat are also dissolved in this fluid matrix after the spore coat is formed. To determine whether any proteins of the coat reside in this fluid phase earlier during the process of spore coat assembly, pairs of strains which differed in a spore coat protein carbohydrate marker were mixed and allowed to form spore coats in each other's presence. We reasoned that proteins belonging to an early, soluble, extracellular pool would be incorporated into the spore coats of both strains. To detect trans-incorporation, spores were labeled with a fluorescent antibody against the carbohydrate marker and each spore's fluorescence was analyzed by flow cytometry. Several proteins of both the outer and inner protein layers of the coat appeared to be faithfully and reciprocally trans-incorporated and hence judged to belong to a soluble, assembly-phase pool. Western blot analysis of sorted spores, and EM localization, confirmed this conclusion. In contrast, one outer-layer protein was not trans-incorporated, and was concluded to be insoluble at the time of secretion. Three classes of spore coat proteins can be described: (a) Insoluble from the time of secretion; (b) present in the early, soluble pool but not the late pool after spore coat formation; and (c) present in the soluble pool throughout spore coat assembly. These classes may, respectively: (a) Nucleate spore coat assembly; (b) comprise a scaffold defining the dimensions of the nascent spore coat; and (c) complete the assembly process by intercalation into the scaffold.

[Neuromonitoring and neuroprotection in cardiac anaesthesia. Nationwide survey conducted by the Cardiac Anaesthesia Working Group of the German Society of Anaesthesiology and Intensive Care Medicine]. / Neuromonitoring und Neuroprotektion in der Kardioanästhesie: Bundesweite Umfrage des Arbeitskreises Kardioanästhesie der Deutschen Gesellschaft für Anästhesiologie und Intensivmedizin e.V.

OBJECTIVE: The primary objective of this nationwide survey carried out in department of cardiac anesthesia in Germany was to identify current practice with regard to neuromonitoring und neuroprotection. METHODOLOGY: The data are based on a questionnaire sent out to all departments of cardiac anesthesia in Germany between October 2007 und January 2008. The anonymized questionnaire contained 26 questions about the practice of preoperative evaluation of cerebral vessels, intra-operative use of neuromonitoring, the nature und application of cerebral protective measures, perfusion management during cardiopulmonary bypass, postoperative evaluation of neurological status, and training in the field of cerebral monitoring. RESULTS: Of the 80 mailed questionnaires 55% were returned and 90% of department evaluated cerebral vessels preoperatively with duplex ultrasound. The methods used for intra-operative neuromonitoring are electroencephalography (EEG, 60%) for type A dissections (38.1%), for elective surgery on the thoracic and thoraco-abdominal aorta (34.1% and 31.6%, respectively) and in carotid surgery (43.2%) near infrared spectroscopy (40%), evoked potentials (30%) and transcranial Doppler sonography (17.5%), with some centers using combined methods. In most departments the central nervous system is not subjected to monitoring during bypass surgery, heart valve surgery, or minimally invasive surgery. Cerebral protective measures used comprise patient cooling on cardio-pulmonary bypass (CPB 100%), extracorporeal cooling of the head (65%) and the administration of corticosteroids (58%), barbiturates (50%) and antiepileptic drugs (10%). Neuroprotective anesthesia consists of administering inhalation anesthetics (32.5%; sevoflurane 76.5%) and intravenous anesthesia (20%; propofol and barbiturates each accounting for 46.2%). Of the departments 72.5% cool patients as a standard procedure for surgery involving cardiovascular arrest and 37.5% during all surgery using CPB. In 84.6% of department CPB flow equals calculated cardiac output (CO) under normothermia, while the desired mean arterial pressure (MAP) varies between 60 and 70 mmHg (43.9%) and between 50 and 60 mmHg (41.5%), respectively. At body temperatures less than 18 degrees C CPB flow is reduced below the calculated CO (70%) while 27% of departments use normothermic flow rates. The preferred MAP under hypothermia is between 50 and 60 mmHg (59%). The results of intra-operative neuromonitoring are documented on the anesthesia record (77%). In 42.5% of the departments postoperative neurological function is estimated by the anesthesiologist. Continuing education sessions pertaining to neuromonitoring are organized on a regular basis in 32.5% of the departments and in 37.5% individual physicians are responsible for their own neuromonitoring education. CONCLUSION: The present survey data indicate that neuromonitoring and neuroprotective therapy during CPB is not standardized in cardiac anesthesiology departments in Germany. The systemic use of available methods to implement multimodal neuromonitoring would be desirable.

[Anesthesia in patients with glucose-6-phosphate dehydrogenase deficiency: case report and perioperative anesthesiologic management]. / Anästhesie bei Patienten mit Glukose-6-Phosphat-Dehydrogenase-Mangel: Fallbericht und perioperatives anästhesiologisches Management.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, a frequent congenital human enzyme defect, is the most frequent cause of hemolytic anemia triggered by drugs or infectious diseases. Drugs which induce acute hemolysis in patients with G6PD deficiency are often used in anesthesia and perioperative pain therapy. Considering the fact that patients from geographic regions with a high prevalence of the disease are often treated in European hospitals, special attention should be paid to this problem. We report a case of a 30-year-old female patient with favism and review the disease and anesthesia-related implications.

Fluctuations and ultrastructural localization of oncoproteins and cell cycle regulatory proteins during growth and apoptosis of synchronized AGF cells.

AGF cells were synchronized by blocking the cell cycle at the G1/S boundary with high concentrations of thymidine (thymidine block) for 11 h. Prolongation of the thymidine block from 11 h to 20 h resulted in apoptosis. Early changes in cellular and nuclear morphology were monitored by confocal microscopy, transmission electron microscopy, and scanning electron microscopy. The fluctuations in the levels of the proliferation cell nuclear antigen (PCNA), cyclin A, CDC-2, c-myc, and p53 proteins were monitored in synchronized cultures and in cells undergoing apoptosis by immunofluorescence staining, flow cytometry, and Western immunoblotting. When assayed by immunofluorescence staining and flow cytometry, the levels of cyclin A and PCNA increased about 2-fold during the S phase, and the level of CDC-2 was fairly constant during S and slightly decreased during late S/G2. The level of c-myc also increased about 2-fold during the S phase, whereas the level of p53 increased only slightly during S. Most importantly, however, the level of staining for c-myc, p53, cyclin A, CDC-2, and PCNA increased 50%-150% during apoptosis compared to the levels observed in cells at G1/S. In contrast, the levels of actin and vimentin, although increased during S, were decreased during apoptosis compared to the levels observed at G1/S. Western blot analysis of the steady state levels of PCNA, cyclin A, and CDC-2 revealed an increase in the levels of all three proteins during S, with higher levels of these proteins observed in apoptotic cells compared to the levels observed in cells at G1/S. Similarly, the levels of p53 and c-myc proteins increased during S and were also high in apoptotic cells. Interestingly, high levels of these two proteins were observed also in cells arrested at G1/S. AGF cells undergoing apoptosis were immunostained for c-myc, p53, PCNA, cyclin A, and CDC-2 and were viewed by confocal microscopy. Apoptotic cells exhibited increased staining for c-myc and p53 in the blebbing nuclei. Furthermore, we observed for the first time that CDC-2, cyclin A, and PCNA proteins were associated mostly with the plasma membrane and the cytoplasm of log phase cells. However, in cells undergoing apoptosis, these proteins were found exclusively in the nuclei of apoptotic cells. These results suggest a possible active role for c-myc, p53, and the cell cycle regulatory proteins in the process of nuclear blebbing and apoptosis.

Ultrastructural localization and fluctuation in the level of the proliferating cell nuclear antigen and myc oncoproteins in synchronized neuroblastoma cells.

A method for rapid synchronization of neuroblastoma cells was developed using the thymidine block to arrest cells in the G1-S boundary. Following release from the thymidine block, cells traversed to G2-M in 7-8 h with 85% cell synchrony. Determination of the steady-state level of proliferating cell nuclear antigen (PCNA) mRNA and protein by Northern and Western blots revealed an accumulation of the PCNA messenger RNA transcripts and PCNA protein at G1-S and a rapid decrease when cells entered S phase. The level of both the messenger RNA transcripts and protein increased as the cells moved to late-S and G2-M. Similarly, the steady-state level of c-myc and N-myc messenger RNA transcripts and proteins increased during the G1-S block, decreased when the cells entered S, and increased as the cells moved through S phase to G2-M. However, immunofluorescence staining for PCNA and myc protein indicated a low level of staining for all three proteins at G1-S and a significant increase in staining intensity during S phase. Similarly, immunoelectron microscopy revealed low levels of N-myc and c-myc staining during G1-S and increased staining during mid-S and late S phase of the cell cycle. These results suggest differential cell cycle-dependent accessibility of myc protein and PCNA to staining in the intact cells compared to the whole cell extract. Furthermore, using immunofluorescence staining, confocal microscopy, and immunoelectron microscopy, we demonstrate for the first time that myc proteins are associated with the chromosomes during mitosis.

Identification and characterization of a testis-specific isoform of a chaperonin in a moth, Heliothis virescens.

Two relatively abundant proteins having subunit molecular weights of 60,000 and 63,000 (p60 and p63, respectively) have been purified as a 16 to 18S complex from sperm mitochondria of a moth. Heliothis virescens. Although the function of these proteins had heretofore not been established, interest in the p63 polypeptide stemmed from its sperm-specific expression and its striking occurrence as a net charge variant among several insect species surveyed, using two-dimensional gel electrophoresis. Genomic and cDNA clones corresponding to the p63 protein have now been isolated and their sequencing has revealed extensive amino acid sequence identity with both the Escherichia coli GroEL protein and its eukaryotic homologues, the chaperonins. Immunoblot studies with a Tetrahymena chaperonin antiserum demonstrated that the p60 protein, which is expressed in all cell types, is structurally related to p63 and is itself a chaperonin subunit. While the chaperonin complex from Heliothis sperm shares certain properties with GroEL, including the ability to hydrolyze ATP and organization of its subunits into a seven-member ring, electron microscopic analysis revealed that its higher-order structure differed from GroEL (and other lower eukaryotic chaperonins) in that the native particle comprises one such ring rather than a doublet. It is not yet known whether the two chaperonin isoforms coexpressed in moth sperm assemble separately or give rise to hybrid particles. In either case, the existence of multiple chaperonin subunits in sperm leaves open the possibility that some aspect of mitochondrial biogenesis that is dependent upon the activity of these proteins is qualitatively or quantitatively different in this cell type.

Hearts and mouths: perceptions of oral hygiene by at-risk heart surgery patients.

OBJECTIVE: To assess and use the attitudes of patients who are placed at risk after valvular heart surgery due to the connection between poor oral hygiene, valvular heart disease/surgery and the risk of developing infective endocarditis. DESIGN: A qualitative (focus group) design based study carried out on subjects three months post heart surgery. METHOD: There were five focus groups of five participants each convened by an experienced moderator. RESULTS: These portrayed an apparent pressing desire by most patients to talk about their experiences. However, patients did not accept the link between their oral health and their general health. Oral hygiene practices were not necessarily oral health related. CONCLUSIONS: The importance of the study in understanding the reasons for a patient's behaviour is evident when there is a clear need to modify the behaviour patterns of the patients effectively. Clinical trials can now be developed based on these results.

Toxic effects of Solanum malacoxylon on sheep bone.

Solanum malacoxylon (Sm), a calcinogenic plant that contains 1,25-(OH)2D3 glycoside, was administered orally to sheep. Fifty milligrams of air-dried leaves three times a week caused an increased volume density of cancellous bone within lumbar vertebrae and an increased trabecular thickness. There was little remodeling activity at the end of a 180-day treatment period, and few trabecular bone surfaces had tetracycline double labels at this time. Bone biopsies taken at the end of a 1-month treatment demonstrated increased extent of bone-forming surfaces and osteoid volume. Sm caused a mineralization defect that was transitory but resulted in unmineralized lines and foci in osteones. These remaining foci of unmineralized bone were associated with the deposition of acid mucopolysaccharide, and acid mucopolysaccharide accumulation could be identified on all bone envelopes in 30-day biopsy specimens. A similar hyperostosis in the metaphyses of rats was produced by parenteral administration of 1,25-(OH)2D3 for 10 days.

Toxic effects of solanum malacoxylon on sheep bone.

Solanum malacoxylon (Sm), a calcinogenic plant that contains 1,25-(OH)2D3 glycoside, was administered orally to sheep. Fifty milligrams of air-dried leaves three times a week caused an increased volume density of cancellous bone within lumbar vertebrae and an increased trabecular thickness. There was little remodeling activity at the end of a 180-day treatment period, and few trabecular bone surfaces had tetracycline double labels at this time. Bone biopsies taken at the end of a 1-month treatment demonstrated increased extent of bone-forming surfaces and osteoid volume. Sm caused a mineralization defect that was transitory but resulted in unmineralized lines and foci in osteones. These remaining foci of unmineralized bone were associated with the deposition of acid mucopolysaccharide, and acid mucopolysaccharide accumulation could be identified on all bone envelopes in 30-day biopsy specimens. A similar hyperostosis in the metaphyses of rats was produced by parenteral administration of 1,25-(OH)2D3 for 10 days.

Development of a two color immunofluorescence stain and immunolocalization method for N-myc and c-myc oncoproteins with a newly generated mouse IgM anti N-myc antibody.

A new mouse monoclonal antibody specific for N-myc oncoprotein was generated and used in combination with an anti-c-myc antibody to develop two color immunofluorescence staining and ultrastructural immunolocalization of N-myc and c-myc in well established (SK-N-SH; CHP 126) and in newly established neuroblastoma (NB) cell lines. Analysis and quantitation of c-myc and N-myc in dually stained cells was done by flow cytometry. Immunolocalization was done by staining with immunogold secondary antibodies and transmission electron microscopy. The results obtained from analysis of 13 newly established NB cell lines revealed, great heterogeneity in the expression of N-myc oncoprotein with 10/13 cell lines over expressing the protein. C-myc oncoprotein was also expressed in all cell lines, however, the level of expression was 4-10-fold lower than the N-myc oncoprotein. Localization studies of c-myc and N-myc oncoproteins on the level of light microscopy and electron microscopy revealed exclusive nuclear localization of c-myc whereas N-myc was localized to the nucleus and to the cytoplasm.

Differential effect of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) on alpha B-crystallin and hsp70 gene expression in murine cell lines.

We studied the effect of isoquinolinesulfonamide derivatives (H-7, H-8, and HA1004) on the expression of two heat shock genes (alpha beta-crystallin and hsp70) in NIH 3T3 and Swiss 3T3 cells after heat shock at 45 degrees for 10 min. Western blots and northern blots showed that H-7 effectively suppressed the accumulation of HSP70 and alpha B-crystallin mRNA as well as the synthesis of their proteins. The degree of suppression was dependent upon the concentration of the drug. Moreover, the expression of the hsp genes was differentially suppressed by H-7. The expression of the alpha B-crystallin gene was more effectively inhibited than that of the hsp70 gene by H-7. Nuclear run-on assay demonstrates that this difference was due to the differential effect of H-7 on the elongation of transcription of different hsp genes.

Effect of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) on HSP70 and HSP28 gene expression and thermotolerance development in human colon carcinoma cells.

The effect of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), a potent protein kinase C (PKC) inhibitor, on the development of thermotolerance and expression of heat shock genes (HSP70 and HSP28) was investigated in human colon carcinoma HT-29 cells. After acute heating at 45 degrees for 15 min, cells became resistant to a challenge heat shock. The development of thermotolerance was suppressed by adding H-7 after heat shock. Northern blots show that the levels of HSP70 and HSP28 mRNA increased rapidly and reached maximal values within 6 hr. H-7 suppressed the accumulation of HSP70 and HSP28 mRNA as well as their protein synthesis, and the level of suppression was concentration dependent. However, little effect was observed if the drug was added 1 hr before and during heat shock. These results suggest that PKC is involved in the regulation of heat shock gene expression after acute heat shock.

Fluctuations and nuclear translocation of p53, C-myc, cdc-2 and pcna proteins during growth and commitment to differentiation of agf cells.

Transcription regulatory proteins such as c-myc and p53 play an important role in the regulation of cell growth, differentiation and apoptosis. Similarly, cell cycle regulatory proteins such as CDC-2, cyclin A and the proliferation cell nuclear antigen (PCNA), play an important role in regulation of cell growth. Yet, there are contradictory reports as for the exact mechanism by which these proteins affect antagonistic processes like growth, differentiation and apoptosis. In the present study, we report, for the first time a detailed analysis of the steady-state level and the nuclear/cytoplasmic distribution of oncoproteins and cell cycle regulatory proteins, in the early phase of PMA induced differentiation of AGF cells. Particular emphasis was put on the first 6 h of commitment to differentiation as well as on the committed/differentiated cells (24 h post induction). Using Western blots of protein extracted from the cytosolic, membranal and nuclear fraction we document an early intranuclear influx of p53 with concomitant extranuclear efflux of c-myc as early as 1-6 h post induction. Similarly, intranuclear sequestration of CDC-2 occurs throughout the commitment phase. General down regulation of p53 and partial down regulation of CDC-2, PCNA and c-myc occur in committed/differentiated cells (24 h post induction). The results obtained by Western blots were further supported by immunolocalization using confocal microscopy.

Regulation of cell-growth and apoptosis in synchronized agf cells - involvement of oncogenes and cell-cycle regulatory proteins.

Cell synchrony was induced in AGF cells by blocking of the cell cycle at GI-S boundary with high concentrations (2 mM) of thymidine for 11 h. Prolonged arrest of cells in GI-S (15 h-20 h) induced progressive and time dependent apoptosis. Early morphological changes in cellular and nuclear morphology (blebbing) were monitored by Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM) and by staining of nuclei with Hoechst and propidium iodide and stained cells viewed by fluorescence and confocal microscopy. The fluctuations in the levels of the proliferation cell nuclear antigen (PCNA), cyclin A, CDC-2, c-myc and p53 proteins were monitored in synchronized cultures and in cells undergoing apoptosis by immunofluorescence staining and flow cytometry. As expected, the levels of cyclin A and PCNA increased during the S phase and the level of CDC-2 decreased during late S/G2. Similarly, the level of c-myc increased during the S phase, whereas the level of p53 did not change much during S phase. Most importantly, however, the level of staining for c-myc, p53, cyclin A, CDC-2 and PCNA increased markedly during apoptosis. In contrast, the level of actin vimentin and tubulin, although increased during S phase, were markedly decreased during apoptosis. AGF cells stained for c-myc during apoptosis, and viewed by confocal microscopy, revealed increased staining for c-myc in the blebbing nuclei. These results, taken together, suggest a possible active role for c-myc in the process of nuclear blebbing and apoptosis.

Differential expression and subcellular-localization of protein-kinase-C, alpha, gamma, delta, xi and zeta isoforms in agf T-cells - modification during pma-induced differentiation.

The steady-state level and translocation of the protein kinase C (PKC) isozymes during the early stages of phorbol 12-myristate 13-acetate (PMA)-induced differentiation, was followed in AGF cells by Western blot analysis of various cell fractions, immunofluorescence staining and by confocal microscopy. By Western blot analysis, uninduced AGF cells express four PKC isoforms, PKC-alpha, PKC-gamma, PKC-epsilon and PKC-zeta with no expression of PKC-beta or PKC-delta. PKC-alpha was exclusively localized to the cytosol, whereas PKC-epsilon was localized predominantly in the cytosol. PKC-gamma and PKC-zeta were found in the cytosolic, as well as in the nuclear and the membrane fractions. Following stimulation with PMA from 15 min to 24 h, cytosolic PKC-alpha did not translocate to the membrane or nuclear fractions. PKC-gamma expression in the membrane and nuclear fractions was decreased following 1 h of PMA stimulation. The expression of PKC-zeta in the membrane and nuclear fractions was transiently increased (2-3 fold) between 3-6 h after PMA stimulation. The expression of PKC epsilon and delta was also affected by PMA treatment. While PKC epsilon, in the membrane fraction, was down-regulated by PMA treatment (3 h), the expression of PKC delta was induced by PMA. Confocal microscopy of the translocation of PKC isoforms during PMA-induced differentiation, confirmed the results obtained by Western blot analysis. Our results indicate that both Ca2+-dependent (PKC-alpha and PKC-gamma) and Ca2+-independent (PKC-epsilon, delta and PKC-zeta) isozymes are expressed in AGF cells and their pattern of expression differ in response to short and prolonged stimulation with phorbol ester. The demonstrated heterogeneity of PKC isozymes in AGF cells, suggests that each PKC isoform may provide a unique contribution to signal transduction pathways and growth control.

Interactions Between Antibody-Coated Iron Micro-Particles and Leukemic Cells as seen by Electron Microscopy.

An electron microscopy study was performed to visualize the interactions between monoclonal antibodies (MoAbs) coated to ferromagnetic microparticles and leukemic cells. The properties of this specific reagent used for the efficient purging of residual leukemic cells from autografts before autologuous bone marrow transplantation (ABMT) have been previously reported. Incubation of a CD10+ NALM6- cell suspension with CD10 or CD8 MoAb conjugated particles was performed following a time-course schedule ranging from 0 to 90 min. at 4°C and 37°C. Transmission electron microscopy demonstrated that, although the CD10 MoAb-particles complex linked to the cell surface only at 4°C, they readily penetrated into cells at 37°C. In contrast, after incubation with CD8 MoAb-particles, no iron particles were seen at the cell surface, or within the cells no matter what the temperature and duration of the incubation were. This observation offers the unique opportunity to deliver a pharmaceutical agent crosslinked to the same magnetic carrier inside a cell, using an interesting site-specific drug delivery concept.

The genotoxicity and cytotoxicity of dermally-administered cadmium: effects of dermal cadmium administration.

Cadmium, unlike zinc, selenium and copper, has no known biological importance, and therefore, it is classified as a carcinogen in humans, as well as in animals. The effect(s) of levels of dermally-administered cadmium on cadmium genotoxicity and cytotoxicity was investigated in Harlan Sprague-Dawley rats for 14, 21, 28, 35 and 42 days at concentrations of 14 and 28 mg/kg/day. Exposure of rats to cadmium via dermal application caused lesions on the skin (hyperkeratosis, acanthosis and scabbing, alopecia and erythema) and tumors in the scrotum. Anatomical changes, such as distention of the stomach, atrophy of kidney and liver and loss of body weight were also observed in these rats. The toxic effects of cadmium on cell ultrastructure were nuclear membrane damage, chromatin condensation, regression of mitochondrial cristae and ultimately cell death. Analyses of the brain, kidney and liver cells of rats exposed to cadmium, clearly showed DNA damage. Of the three organs examined, DNA from kidney cells sustained the most damage followed by DNA in liver cells. There is a positive correlation between Cd dose(s) and duration of exposure and the extent of DNA damage.

Enhanced uptake of liposomes by bovine macrophages after opsonization with antibodies to Brucella abortus.

Phosphatidylcholine:cholesterol liposomes containing 4(5) carboxyfluorescein were modified by incorporation of Brucella abortus lipopolysaccharide. These vesicles were opsonized with bovine anti-B abortus or anti-B abortus plus complement. The fluorescent marker entrapped in antibody-opsonized liposomes was taken up by cultured bovine macrophages more effectively than was the same compound which had been loaded into nonopsonized liposomes. The addition of bovine complement to antibody-opsonized liposomes did not increase the extent of 4(5) carboxyfluorescein uptake over that caused by antibodies alone. The system, described in this report, could be used as a model for evaluation of the efficacy of selectively directing liposomes containing antibiotics to macrophages infected with intracellular B abortus, since macrophages possess Fc receptors capable of binding antibody-opsonized liposomes.