Suppression of Hela cell growth and increase in nuclear size by chemical carcinogens: a possible screening method.

HeLa cell monolayers were "pulse" treated with either carcinogenic or "non-carcinogenic" chemicals. Pre-carcinogens were added with a liver homogenate to provide an appropriate metabolizing system. All proximate carcinogens and a proportion of pre-carcinogens were able to inhibit cell division, and in all cases examined, this was accompanied by nuclear enlargement. Although several "non-carcinogenic" chemicals also arrested cell division, nuclear enlargment was not produced. The possibility that growth inhibition and nuclear enlargement in cells treated briefly with a chemical could provide a rapid indication of carcinogenic activity is discussed.     

Two types of chloride cells in the gill epithelium of a freshwater-adapted euryhaline fish: Lebistes reticulatus; their modifications during adaptation to saltwater

Two types of chloride cells were identified in the gill epithelium of freshwater-adapted guppies. One type, referred to as an "alpha-chloride cell," was a pale, elongated cell located at the base of the secondary lamella in close contact with the arterioarterial pillar capillaries. In its cytoplasm, membranous tubules in continuity with its basolateral plasma membrane formed an extended tridimensional network. The vesiculotubular system (Pisam: Anat. Rec. 200:401-414, 1981) consisted of a few tubules and vesicles located next to the apical plasma membrane. A second type, referred to as a "beta-chloride cell," was a darker, ovoid cell located in the interlamellar region of the primary epithelium facing the central venous sinus. Membranous tubules in continuity with the basolateral plasma membrane were unevenly distributed in the cytoplasm. A prominent vesiculotubular system composed of numerous vesicles and tubules was found between the Golgi apparatus and the apical surface. During seawater adaptation, the alpha-chloride cells increased in size and progressively transformed into characteristic "seawater alpha-chloride cells" with a well-developed, regular, tight tubular network and numerous vesicles and tubules of the vesiculotubular system accumulated below the apical pit. The beta-chloride cells underwent a progressive degeneration and disappeared. Thus, in freshwater-adapted guppies, there are two types of chloride cells, alpha and beta, respectively, related to the arterial and the venous vessels, whereas in seawater-adapted fishes, a single type of cell, the alpha-chloride cell, was related to both the arterial and venous channels.     

Bladder cancer diagnosis by computer image analysis of cells in the sediment of voided urine using a video scanning system

A video-based computerized semiautomated image analysis system was applied to the diagnostic evaluation of 119 sediments of voided urine: 103 from patients with a broad variety of neoplastic and nonneoplastic disorders of the lower urinary tract and 16 normal controls. Each specimen was presented to the machine as a single cytocentrifuge preparation, preserved in 2% Carbowax in 50% ethanol and stained-by the Papanicolaou method. Five hundred sequential "objects" were scanned within an area of 9 sq mm on each slide. "Objects" of no diagnostic value, such as dirt, debris, inflammatory cells, cell clusters, poorly preserved cells, etc., were eliminated from the final diagnostic analysis by a computer-based hierarchic triage system. The final specimen classifier was based on the cell images identified by the computer as well-preserved normal (NEG), atypical (ATY I), suspicious (ATY II) and malignant (POS) cells. For specimen classification by computer, the four categories of "abnormal," "inadequate," "acellular" and "negative" were defined. For high-grade tumors, the performance of the specimen classifier was generally comparable to the visual diagnosis. The specimen classifier unexpectedly identified twice as many low-grade papillary urothelial tumors as abnormal than did the visual analysis. Several false "alarms" were recorded by computer in patients with benign prostatic hypertrophy and prostatic carcinoma, some of whom had atypical urothelium. One of the 16 negative controls was misdiagnosed by the computer as abnormal. The possibility that the video system recognizes nuclear abnormalities not perceived by the human eye is being investigated further. The details of the computer analysis are reported, and the value of the system is discussed. The system appears to be promising as a future laboratory instrument, although it requires further extensive testing.     

Preselection of alarms in a hybrid system for screening of cervical cancer.

In this report, a preselection of alarms in a system for automated screening of cervical cancer based on depositing the cell sample linearly as a "cell trace" on a tape and analyzing it at different decision levels with increasing complexity, and preliminary results on analyzing cervical material with this system are discussed. The "cell trace" is analyzed with the slit-scan technique. Six parameters are computed: 1) cellular diameter; 2) nuclear diameter; 3) nuclear fluorescence (acriflavin-Feulgen) as nuclear DNA; 4) cellular fluorescence; 5) nuclear to cytoplasm ratio (N/C ratio); and 6) nuclear density. At present, only nuclear fluorescence is used to define a decision boundary between normal and potentially atypical cells. Under this criteria the slit-scan analysis leaves 5% of the events in a sample that must be rechecked at a second decision level in normal cell samples. A further reduction is expected when several slit-scan parameters are used at the first decision step. All events declared suspicious will be investigated in more detail by a two dimensional image analyzing system where the fluorescence image is generated by a laser scanning system. Results obtained in preliminary experiments are discussed in this paper.     

Hybridoma cells in a protein-free medium within a composite gel perfusion bioreactor

A composite gel system has been developed combining the chemical and physical properties of calcium alginate and agarose gels. The results of growing composite gel immobilized hybridoma SPO1 cells in a protein-free medium within a fluidized-bed perfusion bioreactor are presented in this paper. During the continuous operation of this system, the total cell density reached 3.9×10⁷ cells per ml of beads (viability 79.6%). The specific productivity of monoclonal antibody of the immobilized hybridoma cells reached more than 1.5 g per 10⁶ viable cells per hour, compared with 0.5 for non-immobilized viable cells grown in a one liter agitated bioreactor with the same medium. Significant increases in cell metabolic activities, including substrate utilization and byproduct formation, were also observed. Leaching of materials from the beads was evident and the major fraction of released materials was alginate.     

Multi-walled carbon nanotube-induced inflammatory response and oxidative stress in a dynamic cell growth environment

Background

Rapid increase in multi-walled carbon nanotube (MWCNT) production for their industrial and biomedical applications has led to concerns over the effects of MWCNTs on human health and the environment. Both animal and in vitro studies have provided important findings about MWCNT-induced effects on the lung cells or tissues. In vitro studies have provided a considerable amount of fundamental information on MWCNT-induced effects on the specific lung cells. However, the cell culture systems used in those studies were limited by the absence of dynamic nature of lung tissues. We hypothesized that MWCNT-induced cellular responses such as proliferation, inflammation, and oxidative stress under dynamic cell growth environment may differ from those under static cell growth environment.

Results

In this study, we used a dynamic cell growth condition to mimic mechanically dynamic environment of the lung and characterized interleukin 8 (IL-8), reactive oxygen species (ROS), glutathione (GSH), and cell proliferation for three days following exposure of MWCNTs at different concentrations (5, 10, and 20 μg/ml) to A549 cell monolayer under both static and dynamic cell growth conditions. Our results demonstrated the distinct differences in the levels of inflammatory response and oxidative stress between static and dynamic cell growth conditions.

Conclusions

In conclusion, the dynamic cell growth system used in this study provided important changes in cellular responses that were not found in the static cell growth system and were similar to animal studies. The dynamic cell growth system can be considered as a viable alternative to in vivo test system in combination with existing in vitro static cell growth systems to evaluate the effect of MWCNTs on cellular responses in the respiratory system.

     

ENDOXY - Development of a Biomimetic Oxygenator-Test-Device

Objective

This study focusses on the development of a biomimetic oxygenator test device. Due to limited biocompatibility, current oxygenators do not allow mid- to long-term therapy. Tissue engineering uses autologous cell sources to overcome the immunogenic barriers of biomaterials. Surface coating with endothelial cells might improve hemocompatibility and thus prevent immunogenic reactions of the body. In this study this concept is applied to endothelialise a gas-permeable membrane to develop a biomimetic oxygenator test-device (ENDOXY).

Methods

ENDOXY—a multifunctional test-system was developed to endothelialise a gas-permeable membrane suitable for cell culture and to test the cell retention under shear stress and to measure gas transfer through it.

Results

Successful endothelialisation of the membrane was achieved and cells showed characteristic endothelial morphologies. They stained positive for endothelial markers. The number of cells aligned with shear stress and cell retention after blood perfusing experiments was high. Gas transfer is observed via uncoated and endothelialised membranes.

Conclusion

The study showed promising results with regard to system design, endothelialisation, and cell retention under shear stress conditions. It strongly encourages further research into the system by testing different membrane materials to design a biomimetic membrane surface and pave way for a fully hemocompatible oxygenator.     

High-level expression of human BSF-2/IL-6 cDNA in Escherichia coli using a new type of expression-preparation system

BSF-2 (B cell stimulatory factor-2/IL-6) is a member of the lymphokine family and responsible for B cell differentiation. Expression plasmids of human BSF-2 cDNA were constructed using a trp promotor/operator and a trpA terminator. In an extract of Escherichia coli HB101 holding "direct" expression plasmid pBSF-2D, activity of BSF-2 was detected, but overproduction was not observed. A "fused" expression system was therefore developed to prepare the recombinant protein. In this system, cDNA was expressed as a fused protein with human IL-2 N-terminal peptide. In the case of the fused BSF-2 expression plasmid, pBSF-2F, inclusion bodies were observed and overproduction of the protein occurred. As this fused protein had a Phe-Arg-Ala sequence at the junction of hIL-2 and BSF-2, it was possible to process mature BSF-2 from the fused BSF-2 by treatment with kallikrein and aminopeptidase P. From 1 liter of E. coli culture, 45 mg of mature BSF-2 was purified; it had a relative biological activity equal to that of natural BSF-2 purified from T cells.     

Size-Based Isolation of Circulating Tumor Cells in Lung Cancer Patients Using a Microcavity Array System

Background

Epithelial cell adhesion molecule (EpCAM)-based enumeration of circulating tumor cells (CTC) has prognostic value in patients with solid tumors, such as advanced breast, colon, and prostate cancer. However, poor sensitivity has been reported for non-small cell lung cancer (NSCLC). To address this problem, we developed a microcavity array (MCA) system integrated with a miniaturized device for CTC isolation without relying on EpCAM expression. Here, we report the results of a clinical study on CTCs of advanced lung cancer patients in which we compared the MCA system with the CellSearch system, which employs the conventional EpCAM-based method.

Methods

Paired peripheral blood samples were collected from 43 metastatic lung cancer patients to enumerate CTCs using the CellSearch system according to the manufacturer’s protocol and the MCA system by immunolabeling and cytomorphological analysis. The presence of CTCs was assessed blindly and independently by both systems.

Results

CTCs were detected in 17 of 22 NSCLC patients using the MCA system versus 7 of 22 patients using the CellSearch system. On the other hand, CTCs were detected in 20 of 21 small cell lung cancer (SCLC) patients using the MCA system versus 12 of 21 patients using the CellSearch system. Significantly more CTCs in NSCLC patients were detected by the MCA system (median 13, range 0–291 cells/7.5 mL) than by the CellSearch system (median 0, range 0–37 cells/7.5 ml) demonstrating statistical superiority (p = 0.0015). Statistical significance was not reached in SCLC though the trend favoring the MCA system over the CellSearch system was observed (p = 0.2888). The MCA system also isolated CTC clusters from patients who had been identified as CTC negative using the CellSearch system.

Conclusions

The MCA system has a potential to isolate significantly more CTCs and CTC clusters in advanced lung cancer patients compared to the CellSearch system.     

Continuous production of tissue plasminogen activator from recombinant CHO cells in a depth filter perfusion system

Summary A depth filter perfusion system (DFPS), equipped with a 40-m polypropylene depth filter for cell immobilization, was used for the continuous production of tissue plasminogen activator (t-PA) from recombinant Chinese hamster ovary cells. Final cell density in the DFPS with oxygen control was 1.8×10⁷ cells/mL of the total working volume and maximum t-PA productivity was 2.63 mg/L/day. Dissolved oxygen concentration in the filter matrix was successfully controlled by air sparging and stable operation was possible for more than 20 days.     

Profile control scheme in a Bakers' yeast fed-batch culture

This article develops and discusses a practical and useful computer control scheme so that the biomass concentration or the specific growth rate will as accurately as possible follow a desired profile specified in advance. Many computer simulations certified the validity of the proposed control scheme. The control scheme proposed, called "programmed-controller/feedback-compensator (PF) system," consists of a programmed controller that will follow the desired profile unless there is noise or disturbance and a feedback compensator that will compensate the noise and correct error in the model parameters. As the feedback compensator, the model reference adaptive control (MRAC) algorithm was also proposed. The PF system with MRAC, named PF-MRAC, could be used sufficiently for the profile control of the specific growth rate. For the profile control of the cell concentration, "predictive control algorithm" should be added to the PF system, and the consequent control scheme was named as the PFP system. Many numerical examples showed that the PFP system with MRAC, named PFP-MRAC, proposed here worked sufficiently well.     

Measurement of the adhesive strength of a gingival cell line to agar

A new technique is described for measuring the adhesive strength of a gingival cell line to an agar substratum by the modification of the original "blister" test for adhesives. A cell monolayer was developed on a Petri dish with a hole in the center of the growing surface, overlayed with agar, and the system pressurized to debond the cells from the agar surface. Pressure changes were measured by a capacitance pressure transducer the output of which was measured by a strip-chart recorder. The modulus (E) of the agar overlay was determined and used in the calculation of the adhesive-bond strength (gammaalpha). The gammaalpha yield for the gingival cell line (cell-agar debond) was 48.8 ergs per cm2, and for the control (no cells) (agar-polystyrene debond) was 30.0 ergs per cm2.     

The vacuolar-tubular continuum in living trichomes of chickpea (Cicer arietinum) provides a rapid means of solute delivery from base to tip

Summary A vacuolar continuum exists from base to tip in the secretory trichomes of chickpea (Cicer arietinum). This continuum is seen in living trichomes which have been labeled with Lucifer yellow CH and examined with confocal microscopy. It encompasses the large vacuole of the lower stalk cell, the vacuoles and tubules of the central stalk cell, the thin tubules of the upper stalk cell, and the tubules and vacuoles of the secretory head cells. The vacuolar-tubular system is structurally distinct within each cell, forming a gradient of large vacuoles in the lower stalk cell, thick tubules in the central stalk cell, and thin anastamozing tubules in the upper stalk cell. This membrane system appears to be continuous between trichome cells, as thin tubules emanate from plasmodesmata between stalk cells and between the upper stalk and lower head cell. In the upper stalk cell, the thin tubules of this continuum are streaming up and down the long axis of the cell at 0.67 m/s. The larger vacuolar-tubular system in the central and lower stalk cells is also slowly moving, with apparent peristalsis occurring in the central cell. The vacuolar-tubular system of the secretory head cells is completely labeled with Lucifer yellow when the dye has only partly diffused up the long walls of the trichome, indicating that the streaming tubular system delivers solute through the stalk cells to the secretory head cells faster than diffusion through the trichome walls. In the lower head cells, tubules emanate from the plasmodesmata connecting to the upper stalk cell, and these tubules are continuous with the head cell vacuoles. In addition, another layer of thin tubules forms along the edges of the secretory head cells, at the site of exocytotic secretion. We propose that the continuous vacuolar-tubular system in these trichomes functions to rapidly deliver solute from the base of the trichome to the secretory head cells. This system provides a pathway for the transport of secretory material.Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

Use of a cyclodextrin for increased protective activity of volatile allyl sulfides against benzo[a]pyrene-induced toxicity in human cell model

The lower inhibitory effect of allyl sulfides on benzo[a]pyrene (B[a]P)-induced toxicity in a cell culture model, rather than in an animal model, was related to their volatile natures. An improved assay system for these volatile chemicals is now suggested. When hydroxypropyl--cyclodextrin was used as an inclusion vehicle of sulfur chemicals, cell viabilities of B[a]P-treated cells were significantly increased by 30–100% and 30–80% at 100–1000 M diallyl disulfide (DADS) and 10–100 M diallyl trisulfide (DATS) treatments, respectively.     

Construction of a cultivation system of a yeast single cell in a cell chip microchamber

A novel single cell screening system was constructed using a yeast cell chip in combination with the yeast cell surface engineering [NanoBiotechnology 2005, 1, 105-111]. Enzymes or functional proteins displayed on a yeast cell surface can be used as a protein cluster. To achieve high-throughput screening of protein libraries on the cell surface, a catalytic reaction by a single cell-surface-engineered yeast cell was successfully carried out in the microchamber on the yeast cell chip. After screening, to replicate a target cell for use in measuring of activity, DNA sequencing, and preservation, a novel single cell cultivation system in the yeast cell chip was constructed. To avoid damage of the rapid dry up of medium in the microchamber array, the yeast cell chip was modified with a protection sheet, so that the modified chip was like a micro-culture tank constructed on the yeast cell chip microchamber. As a result, single yeast cell cultivation in the yeast cell chip microchamber was observed, and the modified yeast cell chip was evaluated to be good for a single cell selection. The improvement showed that the single cell screening system coupled with the single cell cultivation using the modified yeast cell chip may be superior to that by a cell sorter for the isolation of a target cell and its practical use.     

Organization of multiple nucleoids and DNA molecules in mitochondria of a human cell.

Mitochondrial nucleoids (mt-nucleoids) of the A2780 line of cultured human cells were stained with DAPI and observed using an epifluorescence microscope. The mt-nucleoids appeared to be organized compactly in mitochondria. Numbers of mt-nucleoids per mitochondrion ranged from 1 to more than 10, and 70% were "multinucleated" mitochondria. Intensities of fluorescence of mt-nucleoids in each mitochondrion were measured by a video-intensified microscope system (VIM system) and copy numbers of mitochondrial DNA (mtDNA) in each mitochondria were determined. The copy numbers of mtDNA per mitochondrion ranged from 1 to 15, and the average was 4.6. Because the cells had 107 mitochondria on average, the copy number of mtDNA per cell was estimated to be about 500.     

Bowenoid papulosis. Classification as a low-grade in situ carcinoma of the epidermis on the basis of histomorphologic and DNA ploidy studies

The nature of bowenoid papulosis was investigated by a comparative investigation of 12 biopsy specimens of this lesion, 19 biopsy specimens of Bowen's disease, 14 biopsy specimens of squamous cell carcinoma and 10 biopsy specimens of seborrheic keratosis. In addition to conventional histomorphologic and cytomorphologic studies, nuclear DNA measurements on single cells isolated from tissue blocks were performed using a TV image analysis system combined with an automatic microscope. Two parameters, the "5c exceeding rate" (5cER) and the "2c deviation index" (2cDI), were computed from the single-cell DNA values to arrive at a "DNA diagnosis" and a "DNA malignancy grade" (DNA-MG). All specimens of bowenoid papulosis and Bowen's disease were morphologically diagnosed as in situ carcinomas of the epidermis; a DNA diagnosis of malignant was rendered in all of these specimens due to the detection of aneuploid nuclei (5cER greater than or equal to 1). DNA diagnoses of malignant were also rendered on all specimens of squamous cell carcinoma (100% sensitivity) while DNA diagnoses of benign were rendered in all specimens of seborrheic keratosis (100% specificity). The mean DNA-MG for bowenoid papulosis (0.69) was significantly lower than that for Bowen's disease (1.04) and squamous cell carcinoma (1.15). The mean morphologic (Broder's) grade of malignancy was also lower for bowenoid papulosis than for Bowen's disease and squamous cell carcinoma. HPV 16 DNA was detected in 10 of 12 specimens of bowenoid papulosis. Thus, the results of DNA image cytometry and morphologic investigation suggest that bowenoid papulosis is a low-grade carcinoma in situ of the epidermis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monitoring of recombinant protein production using bioluminescence in a semiautomated fermentation process

On-line optimization of fermentation processes can be greatly aided by the availability of information on the physiological state of the cell. The goal of our "BioLux" research project was to design a recombinant cell capable of intracellular monitoring of product synthesis and to use it as part of an automated fermentation system. A recombinant plasmid was constructed containing an inducible promoter that controls the gene coding for a model protein and the genes necessary for bioluminescence. The cells were cultured in microfermenters equipped with an on-line turbidity sensor and a specially designed on-line light sensor capable of continuous measurement of bioluminescence. Initial studies were done under simple culture conditions, and a linear correlation between luminescence and protein production was obtained. Such specially designed recombinant bioluminescent cells can potentially be applied for model-based inference of intracellular product formation, as well as for optimization and control of recombinant fermentation processes.     

Anti-CCR7 therapy exerts a potent anti-tumor activity in a xenograft model of human mantle cell lymphoma

Background

The chemokine receptor CCR7 mediates lymphoid dissemination of many cancers, including lymphomas and epithelial carcinomas, thus representing an attractive therapeutic target. Previous results have highlighted the potential of the anti-CCR7 monoclonal antibodies to inhibit migration in transwell assays. The present study aimed to evaluate the in vivo therapeutic efficacy of an anti-CCR7 antibody in a xenografted human mantle cell lymphoma model.

Methods

NOD/SCID mice were either subcutaneously or intravenously inoculated with Granta-519 cells, a human cell line derived from a leukemic mantle cell lymphoma. The anti-CCR7 mAb treatment (3 × 200 μg) was started on day 2 or 7 to target lymphoma cells in either a peri-implantation or a post-implantation stage, respectively.

Results

The anti-CCR7 therapy significantly delayed the tumor appearance and also reduced the volumes of tumors in the subcutaneous model. Moreover, an increased number of apoptotic tumor cells was detected in mice treated with the anti-CCR7 mAb compared to the untreated animals. In addition, significantly reduced number of Granta-519 cells migrated from subcutaneous tumors to distant lymphoid organs, such as bone marrow and spleen in the anti-CCR7 treated mice. In the intravenous models, the anti-CCR7 mAb drastically increased survival of the mice. Accordingly, dissemination and infiltration of tumor cells in lymphoid and non-lymphoid organs, including lungs and central nervous system, was almost abrogated.

Conclusions

The anti-CCR7 mAb exerts a potent anti-tumor activity and might represent an interesting therapeutic alternative to conventional therapies.