Development of a method to analyze single cell activity by using dielectrophoretic levitation.

In cell fusion and genetic recombination, although the activity of single cells is extremely important, there is no method to analyze single cell activity. Development of a quick analyzing method for single cell activity is desired in various fields. Dielectrophoresis (DEP) refers to the force exerted on the induced dipole moment of an uncharged dielectric and/or conductive particle by a nonuniform electric field. By applying DEP, we obtained experimentally a relationship between the cell activity and the dielectric property, Re[K(omega)], and examined how to evaluate the single cell activity by measuring Re[K(omega)] of a single cell. A cone and plate electrode geometry was adapted in order to achieve the feedback-controlled DEP levitation. The single cell is exposed to a nonuniform field induced by the cone and plate electrode, and a more polarizable cell is moved to the direction of the cone electrode by the DEP force. The cell settles in the position where the DEP force and gravity are balanced by controlling applied voltage. This settled position, measured on the center axis of the cone electrode, depended on the dielectric constant of the cell. From these results, the relationship between the specific growth rates in cell growth phase and the dielectric properties Re[K(omega)] was obtained. Furthermore, the effect on the cell activity of various stresses, such as concentration of carbon dioxide, temperature, etc., was examined.

In vitro characterization of rat bone marrow-derived dendritic cells and their precursors.

Although the rat is commonly used for basic immunology and transplantation research, phenotypic and functional characterization of rat dendritic cells (DCs) lags behind similar studies in the human and mouse. Therefore, these features were examined using DCs propagated from cultures of rat bone marrow maintained in a medium supplemented with granulocyte-monocyte colony-stimulating factor. Analysis of cytospin preparations of cultured cells showd that DCs arise from OX7+ myelomonocytic precursors. Typical mature rat DCs were morphologically similar to their mouse and human counterparts and expressed major histocompatibility complex (MHC) class II (common part determinant of Ia), OX62 (integrin molecule), OX7 (CD90), ICAM-1 (CD54), and CTLA4 counterreceptor, but were negative for OX8 (CD8), OX19 (CD5), W3/25 (CD4), and ED2, a rat macrophage marker. Functional analysis of OX62+ sorted DCs showed that they could effectively present the soluble antigen ovalbumin to naive T cells in vitro. A combination of anti-MHC class II monoclonal antibody and CTLA4-immunoglobulin inhibited allostimulatory ability more effectively than either reagent alone. Implications for studying the role of DCs in immune responses in the rat are discussed.

Adjuvant chemoimmunotherapy for hepatocellular carcinoma patients. Adriamycin, interleukin-2, and lymphokine-activated killer cells versus adriamycin alone.

To determine improved postresection survival in patients with hepatocellular carcinoma, two postoperative protocols were compared: adoptive chemoimmunotherapy versus chemotherapy. Following resection, 24 patients were allocated at random to receive (1) arterial infusion of Adriamycin, recombinant interleukin-2 and lymphokine-activated killer cells or (2) arterial infusion of Adriamycin alone. The spleen was removed at operation and used to prepare lymphokine-activated killer cells. Each group had 12 patients. They were followed until signs of recurrence appeared. The overall survival rates of the patients were 91.7%, 82.9%, and 72.5% at 1, 2, and 3 years, respectively, and slightly higher than would be expected with surgery alone. No statistically significant difference was found between the two groups either in the survival rate (generalized Wilcoxon test, P = .936) or in the cumulative disease free rate (P = .182). However, when patients who had had hepatic resection with negative margin (> or = 1 cm) were separated, the 2-year cumulative disease-free rate in the adoptive chemoimmunotherapy was higher (83.3%, n = 6) than that in chemotherapy (37.5%, n = 8). Toxicity to adoptive chemoimmunotherapy was moderate; no severe side effects were observed. Totally no statistical difference between the two groups was found. Although only one of six patients in adoptive chemoimmunotherapy experienced recurrence after hepatic resection with negative margin, it was not feasible to determine the role of interleukin-2 and lymphokine-activated killer cells. We conclude that the adoptive chemoimmunotherapy in this study is not an ideal adjuvant protocol after hepatic resection.

A new protocol for the propagation of dendritic cells from rat bone marrow using recombinant GM-CSF, and their quantification using the mAb OX-62.

Bone marrow (BM)-derived dendritic cells (DC) are the most potent known antigen (Ag) presenting cell in vivo and in vitro. Detailed analysis of their properties and mechanisms of action requires an ability to produce large numbers of DC. Although DC have been isolated from several rat tissues, including BM, the yield is uniformly low. We describe a simple method for the propagation of large numbers of DC from rat BM and document cell yield with the rat DC marker, OX-62. After depletion of plastic-adherent and Fc+ cells by panning on dishes coated with normal serum, residual BM cells were cultured in gelatin coated flasks using murine rGM-CSF supplemented medium. Prior to analysis, non-adherent cells were re-depleted of contaminating Fc+ cells. Propagation of DC was monitored by double staining for FACS analysis (major histocompatibility complex (MHC) class II+/OX-62+, OX-19-). Functional assay, morphological analysis and evaluation of homing patterns of cultured cells revealed typical DC characteristics. MHC class II and OX-62 antigen expression increased with time in culture and correlated with allostimulatory ability. DC yield increased until day 7, when 3.3 x 10(6) DC were obtained from an initial 3 x 10(8) unfractionated BM cells. Significant numbers of DC can be generated from rat BM using these simple methods. This should permit analysis and manipulation of rat DC functions in vivo and in vitro.

[Changes of arterial CO2 (PaCO2) and urine output by carbon dioxide insufflation of the peritoneal cavity during laparoscopic cholecystectomy].

We studied on the effects of carbon dioxide insufflation during laparoscopic cholecystectomy on the arterial blood gas analysis and urine output. Intra-abdominal pressure was increased up to either 10cmH2O or 15cmH2O, and we compared the PaCO2 values before and during insufflation. Both increase of PaCO2 and decrease in pH were larger in intra-abdominal pressure of 15cmH2O than 10cmH2O. In the intra-abdominal pressure 15cmH2O group, the increase of PaCO2 by CO2 peritoneal insufflation was significantly larger in operative time of more than 60 minutes group than in less than 60 minutes group, but no significant changes were observed in 10cmH2O group. In the group of obesity index of more than 120, elevation levels of PaCO2 by CO2 insufflation were significant, but in the group of less than 120 no significant elevation were observed. The tendency that the urine output during operation was decreased as increase of intra-abdominal pressure or operative time, but the obesity had no definite effects on urine output. Insufflation of the abdomen with CO2 caused large changes in PaCO2 or pH in the patients with a previous history of major cardiopulmonary disorder. It was shown that the low insufflation pressure and short operative time is good for the maintenance of normal physiological state.

[Study on the complete parathyroidectomy and parathyroid autoimplantation for secondary parathyroidism in chronic renal failure].

We studied on the operative indication and therapeutic effect of complete parathyroidectomy and autoimplantation for chronic hemodialysis. Twenty three surgical resections were performed on 21 patients who had been received long-term hemodialysis. Total resected glands from these patients were 83 and the mean total parathyroid's weight was 3.2 gram. The detection rate of hyperparathyroidism was 50.0% by scintigram, and 72.9% by computed tomography (CT). The mean diameter of resected glands proved to be 5 to 6mm larger than that estimated by CT. There was no statistical correlation between the durations of hemodialysis and the total parathyroid's weights. Our study revealed that the mean weight of 5 cases without 1,25(OH)2D3-plus therapy was 5.8 gram, which was significantly heavier than that with this therapy. Histopathologically, the larger glands often showed nodular growth, but the smaller glands tended to show diffuse hyperplasia. There was a positive correlation (r = 0.93, p < 0.01) between the weights of the resected glands and serum CPTH values only in those cases with the glands weighing equal or less than 4 gram.

[Host immunological status modification by various chemotherapeutic drugs].

There are many unsolved problems about the effects on a host's cellular immunologic status by various chemotherapeutics used routinely. Thus, we performed an experimental study mainly on these effects using mouse effector cells. We administered the usual therapeutic dosage of various chemotherapeutic agents to 8-week female C57BL/6 mice, and 3 days after these administrations all mice were sacrificed and splenectomised. The number of splenocytes in whole spleen, LAK precursor cell activity, NK cell activity, and IL-2 productivity were determined, and these results were compared with those for normal mice. Although CY, 5-FU, MMC and LIB exhibited immunologic depressive effects, BLM and PEP showed augmentative effects, and ADR and CDDP no effects. It seemed important that the appropriate drug selection be made on the basis of the above data in order to be the best therapeutic regimen for the individual patient's malignancy.

Augmented accumulation of transferred lymphokine-activated killer (LAK) cells at murine tumor sites through production of LAK-attractant facilitated by chemotherapy.

We observed that effects of adoptive immunotherapy with lymphokine-activated killer (LAK) cells on BMT-11, a fibrosarcoma in C57BL/6 mice were improved by combination with cyclophosphamide (CY)-chemotherapy corresponding to enhanced accumulation at tumor sites of LAK cells. On the other hand, cytotoxic T lymphocytes (CTLs) which were able to accumulate at tumor sites more densely than LAK cells produced significant therapeutic effects by themselves. We have also found observed that LAK-attractant activity was detected in conditioned medium (CM) of CY-treated tumor tissue but not in the CM of untreated tumor tissue. These findings reveal that CY-chemotherapy facilitates LAK-attractant-production and enhances the accumulation in tumor tissue of LAK cells and that therapeutic effects of adoptive transfer of LAK cells are augmented by cancer chemotherapy through the enhanced accumulation of LAK cells.

[Augmentation of LAK cell-accumulation in tumor tissue and its therapeutic effect after chemotherapy].

We investigated the therapeutic effects of a combination of chemotherapy with adoptive immunotherapy on BMT-11 fibrosarcoma in C57BL/6 mice. Although no significant therapeutic effects were brought about by CY alone or LAK.rIL-2 alone, CY plus LAK/rIL-2 brought about significant therapeutic effects such as a reduced proportion of dead mice (63%) and prolongation of the mean survival times of dead mice (64.4 days). We transferred radioactive 111In-labeled effector cells into untreated and CY-treated tumor bearing mice. LAK cell-accumulation (% dose/g) at the tumor site was only 2.7% in untreated mice and 19.7 in CY-treated mice. On the other hand, CTL.rIL-2 therapy exhibited significant therapeutic effects by itself. The accumulation of CTL was 9.1% in untreated tumors and only slightly enhanced by CY-chemotherapy. These results suggest that the therapeutic effects of adoptive immunotherapy depend on the accumulation of transferred effector cells at the tumor site and are augmented by the enhanced accumulation of effector cells after chemotherapy.

[Studies on lymphokine-activated killer (LAK) cell: accumulation in tumor tissue and the therapeutic effects of adoptive immunotherapy].

We studied the therapeutic effects of adoptive immunotherapy with lymphokine-activated killer (LAK) cells combined with chemotherapy on BMT-11 fibrosarcoma in C57BL/6 mice. Compared with the untreated group, no significant therapeutic effect was brought about by CY therapy alone or LAK.rIL-2 alone and all mice belonging to these three groups died with a mean survival time (MST) of 45.3, 51.8 and 45.9 days respectively. CY plus LAK.rIL-2 brought about complete cures in 3 out of 8 mice (37.5%) and a significant prolongation of MST of mice which died (64.4 days) and the accumulation of LAK cells (% Dose/g) at tumor sites was enhanced more than 7-fold by combination with CY. On the other hand, the therapeutic effects of cytotoxic T lymphocytes (CTLs) was sufficiently high even in the CTL.rIL-2 alone and were only slightly enhanced by combination with CY compared with LAK cells. Also, we detected LAK-attractant activity in the conditioned medium (CM) of CY-treated tumor tissues but not in that of untreated tumor tissues, and peak activity was reached 5 days after CY-treatment. This attractant activity was located in two major 10,000-50,000 M. W. fractions of CM. We then observed that LAK-attractant was produced in CM of host reactive cell enriched fractions from CY-treated tumor tissues, but not in that of tumor cell enriched fractions. The above findings imply that the effects of adoptive immunotherapy depend upon the accumulation of transferred effector cells at tumor sites, and we believe that the production of LAK-attractant by tumor tissue, facilitated by chemotherapy, is one of the mechanisms responsible for enhanced LAK-cell-accumulation at tumor sites. We performed a preliminary clinical trial with adriamycin, autologous spleen-LAK cells and rIL-2 on 30 hepatocellular carcinoma patients after radical resection on the basis of the experimental results above. There were no significant therapeutic effects after adoptive immunotherapy during the postoperative course but tendency for temporary inhibition of recurrence. Thus it is shown that this method has probable value as effective adjuvant postoperative therapy.

A study on conformationally restricted sangivamycins and their inhibitory abilities of protein kinases.

Conformational restrictions of sangivamycin, a rather selective inhibitor of PKC, could be achieved by the use of the steric effect and the gauche effect of the substituents on the ribofuranose moiety. The conformational deviations obtained by these methods were found to nicely correlate with the inhibitory ability of PKC.

Continuous flow determination of chloride in the non-linear response region with a tubular chloride ion-selective electrode.

A microcomputer-aided continuous-flow system was constructed for the determination of chloride in the non-linear electrode-response region from 10 to 0.1 mg/l. Interpolation by spline function was used to calculate the concentration from the measured potential. As few as four points were enough to obtain a practical calibration curve, plotted as Evs. log c. The interferences of bromide and iodide (at weight ratios <0.1 to chloride) could be removed by adding colloidal silver chloride continuously in the flow stream. Analysis of rain and snow containing 0.07-8.8 mg/l. chloride showed fairly good agreement with that by ion-chromatography.