Rapid decline in 51Cr-ethylenediaminetetraacetic acid-measured renal function during the first weeks following liver transplantation.

BACKGROUND: Renal dysfunction is a serious late complication after liver transplantation (LTX), but there are no studies addressing the early changes associated with this complication. METHODS: We prospectively studied glomerular filtration rate (GFR) before and at 1, 3 and 12 weeks after LTX using 51Cr-labelled ethylenediaminetetraacetic acid clearance in 37 adult consecutive patients who underwent non-acute first LTX. RESULTS: The mean (±SD) age was 49.5 ± 9.5 years, and the male:female sex ratio was 21:16. Diagnoses were autoimmune liver diseases (17), alcoholic cirrhosis (10) and other diseases (10). Immunosuppressive treatment consisted predominantly of triple-drug therapy. A total of 27 of the 37 patients were eligible for GFR analysis at all times. The mean (±SD) GFR was 86 ± 26 mL/min/1.73 m2 before LTX, and 77 ± 30 mL/min/1.73 m2 at 1 week, 64 ± 27 mL/min/1.73 m2 at 3 weeks and 64 ± 23 mL/min/1.73 m2 at 12 weeks after LTX, comparable to a reduction in mean GFR compared with baseline values of 10% (P = 0.1907), 25% (P = 0.0010) and 26% (P = 0.0007). Age and number of blood transfusions during surgery were identified as risk factors for this decline as well as gender, but not pre-transplant diagnosis, model of end-stage liver disease score, cold ischaemia time or post-transplant area under the curve tacrolimus during Days 0-14. CONCLUSIONS: Using measured rather than estimated GFR, our results show that severe renal impairment occurs during the first week after LTX. These results emphasize the need for more studies addressing renoprotective treatment strategies.

Improvement of in vitro potency assays by a resting step for clinical-grade chimeric antigen receptor engineered T cells.

BACKGROUND: Chimeric antigen receptor engineered T (CAR-T) cell therapy is a promising approach currently revolutionizing the field of cancer immunotherapy. However, data concerning clinical-grade CAR-T cell stability and functionality after months of cryopreservation have not been released by companies so far. To investigate the effect of cryopreservation on CAR-T cells and to further optimize the potency assays, we performed this study. METHODS: A third generation of CD19 CAR-T cells was manufactured according to Good Manufacturing Practice (GMP) requirements, which is applied to patients in an ongoing clinical phase 1 study. Quality control tests for sterility, endotoxin and mycoplasma were performed for each batch. Stability in terms of viability, recovery, transduction efficiency and functional capacity was determined using microscopy, multiparametric flow cytometry as well as chromium-51 release tests. RESULTS: Up to 90days of cryopreservation had no influence on viability, recovery and transduction efficiency of CAR-T cells. However, higher cell concentration for cryopreservation could alter the cell viability and recovery but not the transduction efficiency. Moreover, directly after thawing, both the quantity and quality of the functionality of CAR-T cells were transiently hampered by the negative effects of cryopreservation. Notably, the impaired functionality could be fully restored and even strengthened after an overnight resting process. DISCUSSION: Cryopreservation is a challenge for the functional activity of CAR-T cells. However, CAR-T cells regain their potency by overnight incubation at 37°C, which mimics the clinical application setting. Therefore, an overnight resting step should be included in in vitro potency assays.

Further studies to evaluate methods of leucoreduction to prevent alloimmune platelet refractoriness and induce tolerance in a dog platelet transfusion model.

OBJECTIVES: Three leucoreduction filters were evaluated - when used alone or combined with centrifuge leucoreduction (C-LR) - to prevent alloimmune platelet refractoriness in a dog platelet transfusion model. MATERIALS AND METHODS: Donor platelet-rich plasma (PRP) or buffy coat (BC) platelets were either filter leucoreduced (F-LR) or F-LR/C-LR, (51) Cr radiolabelled and transfused. Weekly transfusions were given for up to 8 weeks or until platelet refractoriness. Recipients who accepted treated transfusions were then given non-leucoreduced (non-LR) platelets to determine whether donor-specific tolerance had been induced. RESULTS: Acceptance of F-LR PRP transfusions ranged from 29% to 66%. F-LR/C-LR transfusions prepared from PRP were accepted by 92%, from BC by 63% and from pooled PRP by 75% of recipients (p=NS); overall acceptance rate of F-LR/C-LR transfusions was 83%. Tolerance to subsequent non-LR transfusions occurred in 45% of the F-LR-/C-LR-accepting recipients unrelated to DR-B compatibility between donors and recipients (P = 0·18). CONCLUSION: In a dog platelet transfusion model, acceptance of donor platelets required combining F-LR with C-LR as apparently each process removes different immunizing WBCs.

Roles of crustacean hyperglycaemic hormone in ionic and metabolic homeostasis in the Christmas Island blue crab, Discoplax celeste.

There is a growing body of evidence implicating the involvement of crustacean hyperglycaemic hormone (CHH) in ionic homeostasis in decapod crustaceans. However, little is known regarding hormonally influenced osmoregulatory processes in terrestrial decapods. As many terrestrial decapods experience opposing seasonal demands upon ionoregulatory physiologies, we reasoned that these would make interesting models in which to study the effect of CHH upon these phenomena. In particular, those (tropical) species that also undergo seasonal migrations might be especially informative, as we know relatively little regarding the nature of CHHs in terrestrial decapods, and hormonally mediated responses to seasonal changes in metabolic demands might also be superimposed or otherwise integrated with those associated with ionic homeostasis. Using Discoplax celeste as a model crab that experiences seasonal extremes in water availability, and exhibits diurnal and migratory activity patterns, we identified two CHHs in the sinus gland. We biochemically characterised (cDNA cloning) one CHH and functionally characterised (in terms of dose-dependent hyperglycaemic responses and glucose-dependent negative feedback loops) both CHHs. Whole-animal in situ branchial chamber (22)NaCl perfusion experiments showed that injection of both CHHs increased gill Na(+) uptake in a seasonally dependent manner, and (51)Cr-EDTA clearance experiments demonstrated that CHH increased urine production by the antennal gland. Seasonal and salinity-dependent differences in haemolymph CHH titre further implicated CHH in osmoregulatory processes. Intriguingly, CHH appeared to have no effect on gill Na(+)/K(+)-ATPase or V-ATPase activity, suggesting unknown mechanisms of this hormone's action on Na(+) transport across gill epithelia.

Endogenous cannabinoid system regulates intestinal barrier function in vivo through cannabinoid type 1 receptor activation.

The deleterious effects of stress on the gastrointestinal tract seem to be mainly mediated by the induction of intestinal barrier dysfunction and subsequent subtle mucosal inflammation. Cannabinoid 1 receptor (CB1R) is expressed in the mammalian gut under physiological circumstances. The aim of this investigation is to study the possible role of CB1R in the maintenance of mucosal homeostasis after stress exposure. CB1R knockout mice (CB1R(-/-)) and their wild-type (WT) counterparts were exposed to immobilization and acoustic (IA) stress for 2 h per day during 4 consecutive days. Colonic protein expression of the inducible forms of the nitric oxide synthase and cyclooxygenase (NOS2 and COX2), IgA production, permeability to (51)Cr-EDTA, and bacterial translocation to mesenteric lymph nodes were evaluated. Stress exposure induced greater expression of proinflammatory enzymes NOS2 and COX2 in colonic mucosa of CB1R(-/-) mice when compared with WT animals. These changes were related with a greater degree of colonic barrier dysfunction in CB1R(-/-) animals determined by 1) a significantly lower IgA secretion, 2) higher paracellular permeability to (51)Cr-EDTA, and 3) higher bacterial translocation, both under basal conditions and after IA stress exposure. Pharmacological antagonism with rimonabant reproduced stress-induced increase of proinflammatory enzymes in the colon described in CB1R(-/-) mice. In conclusion, CB1R exerts a protective role in the colon in vivo through the regulation of intestinal secretion of IgA and paracellular permeability. Pharmacological modulation of cannabinoid system within the gastrointestinal tract might be therapeutically useful in conditions on which intestinal inflammation and barrier dysfunction takes place after exposure to stress.

In vivo muscle electroporation threshold determination: realistic numerical models and in vivo experiments.

In vivo electroporation is used as an effective technique for delivery of therapeutic agents such as chemotherapeutic drugs or DNA into target tissue cells for different biomedical purposes. In order to successfully electroporate a target tissue, it is essential to know the local electric field distribution produced by an application of electroporation voltage pulses. In this study three-dimensional finite element models were built in order to analyze local electric field distribution and corresponding tissue conductivity changes in rat muscle electroporated either transcutaneously or directly (i.e., two-plate electrodes were placed either on the skin or directly on the skeletal muscle after removing the skin). Numerical calculations of electroporation thresholds and conductivity changes in skin and muscle were validated with in vivo measurements. Our model of muscle with skin also confirms the in vivo findings of previous studies that electroporation "breaks" the skin barrier when the applied voltage is above 50 V.

Naegleria fowleri lysate induces strong cytopathic effects and pro-inflammatory cytokine release in rat microglial cells.

Naegleria fowleri, a ubiquitous free-living ameba, causes fatal primary amebic meningoencephalitis in humans. N. fowleri trophozoites are known to induce cytopathic changes upon contact with microglial cells, including necrotic and apoptotic cell death and pro-inflammatory cytokine release. In this study, we treated rat microglial cells with amebic lysate to probe contact-independent mechanisms for cytotoxicity, determining through a combination of light microscopy and scanning and transmission electron microscopy whether N. fowleri lysate could effect on both necrosis and apoptosis on microglia in a time- as well as dose-dependent fashion. A (51)Cr release assay demonstrated pronounced lysate induction of cytotoxicity (71.5%) toward microglial cells by 24 hr after its addition to cultures. In an assay of pro-inflammatory cytokine release, microglial cells treated with N. fowleri lysate produced TNF-α, IL-6, and IL-1ß, though generation of the former 2 cytokines was reduced with time, and that of the last increased throughout the experimental period. In summary, N. fowleri lysate exerted strong cytopathic effects on microglial cells, and elicited pro-inflammatory cytokine release as a primary immune response.

Antibody-dependent antitumor cytotoxicity by human monocytes cultured with recombinant macrophage colony-stimulating factor. Induction of efficient antibody-mediated antitumor cytotoxicity not detected by isotope release assays.

Macrophage colony-stimulating factor (M-CSF) is known to stimulate proliferation of monocyte/macrophage progenitors and enhance in vitro antitumor cytotoxicity by murine macrophages. In this paper we have shown that recombinant human M-CSF causes human peripheral blood monocytes to differentiate in culture into metabolically active macrophage-like cells. These cells mediate very efficient antibody-dependent cellular cytotoxicity (ADCC) against human melanoma and neuroblastoma cell lines in the presence of two murine IgG3 mAbs (3F8 and R24). They also mediate antibody-independent cytotoxicity (or cytostasis) to a lesser extent. Human serum had an inconsistent effect on ADCC, but often induced similar high levels of ADCC. Cytotoxicity was measured using a novel ELISA to detect surviving tumor cells after ADCC. Two conventional isotope-release assays (51Cr and [3H]TdR) underestimated or entirely failed to detect ADCC by M-CSF-activated monocytes. Optimal activation occurred with 100-300 U/ml of M-CSF, and required 9-11 d for completion. Most of the M-CSF cultured monocytes expressed the low-affinity Fc receptor (CD16). ADCC by cells of the monocyte/macrophage lineage using murine IgG3 mAbs may have significance for the immunotherapy of human malignancies.

No difference in Gag and Env immune-response profiles between vaccinated and non-vaccinated rhesus macaques that control immunodeficiency virus replication.

Recent advances in human immunodeficiency virus (HIV) vaccine design have resulted in induction of strong CD4 T-cell proliferative and polyfunctional cytokine responses, which are also characteristic for long-term non-progressing (LTNP) HIV-infected individuals. However, limited information is available on the persistence of these responses after infection. Results from studies in non-human primates indicate that vaccine-induced immune responses are partially maintained upon viral infection and differ from the responses seen in non-vaccinated animals that typically progress to disease. However, it is unclear how these partially preserved responses compare to immune responses that are acquired naturally by LTNP animals. In this study, immune-response profiles were compared between vaccinated animals that, upon SHIV89.6 challenge, became infected but were able to control virus replication, and a group of animals having spontaneous control of this viral infection. Both groups were found to develop very similar immune responses with regard to induction of CD4 and CD8 T-cell polyfunctional cytokine responses, proliferative capacity and cytotoxic capacity, as measured by a standard 51Cr release assay and more direct ex vivo and in vivo CTL assays. Hence, vaccinated animals that become infected, but control infection, appear to establish immune responses that are similar to those elicited by long-term non-progressors.

Red cell volume measurement - using indium as a replacement for chromium.

The withdrawal of Cr-chromate has meant that the technique commonly used for direct measurement of red cell volume has had to be replaced. Most centres moved to a Tc erythrocyte label, however, Tc is known to dissociate over time. We have investigated an alternative technique using an In-chloride and tropolone solution and tested this both in vitro and in vivo. Initial in-vitro and in-vivo work, which included a check of the stability of the radio-labelled product at one hour, demonstrated this label to be stable over this time period. To date, 20 patients have undergone this technique and results show that this technique is a viable alternative to Cr-chromate particularly for patients with splenomegaly who require late sampling. This procedure is now in routine use in our institution.

Evidence for passing down of postnasal drip into respiratory organs.

UNLABELLED: Postnasal drip is believed to be one of the main sources of serious respiratory diseases, such as sinobronchial syndrome. However, there is little direct evidence showing that postnasal drip flows into the trachea and results in the development of inflammatory responses in the lower airway. In the present study, whether postnasal drip entered the respiratory organs and whether material in the trachea moved toward the lungs and the digestive organs were examined by using an experimental model with mice. MATERIALS AND METHODS: In the first set of experiments, 1.0 microL of 51Cr-labeled pseudo-postnasal drip in a normal saline or a glycerin solution was instilled into the nasal cavity of male ICR mice anesthetized with sodium barbital by intraperitoneal injection. In the second set of experiments, the destination of 51Cr-labeled red blood cells (RBCs) after intratracheal instillation was examined in the anesthetized mice. The lungs, the stomach and the intestines were removed from mice killed under anesthesia at various intervals after instillation, and measured for radioactivity. RESULTS: When glycerin solution containing 51Cr (but not normal saline) was instilled in mice, the presence of much higher levels of 51Cr was observed in the lungs. Although the presence of high levels of 51Cr-labeled RBCs was observed in the lungs one hour after instillation radioactivity in the lungs gradually decreased as time went by. On the other hand, radioactivity in the digestive organs gradually increased and peaked three hours after instillation with 51Cr-labeled RBC. CONCLUSION: These results suggest that thicker viscous postnasal drip can flow into the respiratory organs when the host is asleep. In addition, postnasal drip which flows into the trachea can move gradually to the oral side by mucociliary transportation of the tracheal mucosa and thus be swallowed.

Evaluating Antibody-Dependent Cell-Mediated Cytotoxicity by Chromium Release Assay.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is a mechanism in which immune cell activation is induced by the cross-linking of CD16 with the Fc region of antibodies that at the same time bind specifically to cell surface antigens. ADCC stimulates the secretion of perforin, granzymes, and cytokines leading to lysis of the malignant cells. Natural killer (NK) cells express the CD16 receptor and can therefore be activated by ADCC to kill tumor cells. To study the cytotoxicity of NK cells against cancer cells, an ADCC-based assay is described: the chromium release assay. In this method, the antibody trastuzumab, which binds specifically to HER2-positive malignant cells, is used to trigger ADCC.

Uptake studies of environmentally hazardous (51)Cr in Mung beans.

Attempt has been made to study the accumulation behaviour of a common plant, Mung bean (Vigna radiata) towards Cr(III) and Cr(VI) to have an insight on the migration and bio-magnification of Cr. For this purpose healthy germinated Mung bean seeds were sown in the sand in the presence of Hoagland's nutrient solution containing measured amount of K(2)(51)Cr(2)O(7) and (51)Cr(NO(3))(3).9H(2)O. Growth rate was also studied in the presence and absence of phosphate salts in the medium. It has been found that the transfer of chromium from soil to plant is significantly low (maximum 5% for both Cr(III) and Cr(VI)). Maximum accumulation of Cr occurs in the root with respect to the total chromium accumulation by the plant. Other parts of the Mung bean plant, e.g. cotyledons, shoot and leaves, show negligible accumulation. Therefore, the chance of direct intake of Cr through food as well as through the grazing animals to human body is less.

Cytotoxic activity of rheumatoid and normal lymphocytes against allogeneic and autologous synovial cells in vitro.

The possibility that lymphocytes from patients with rheumatoid arthritis (RA) might be sensitized to RA synovial cell antigens was investigated with a 51Cr release cytotoxicity assay. Peripheral blood lymphocytes from rheumatoid and normal donors were tested for cytotoxic activity against their own synovial cells and against allogeneic rheumatoid and nonrhemuatoid synovial cells. In the allogeneic studies, the degree of cytotoxicity was significantly influenced by the age in culture (passage number) of the synovial target cells (P less than 0.001). When the passage number of the target cells was considered in the analysis, rheumatoid lymphocytes were found to have greater cytotoxic activity than normal lymphocytes against young cultures (low passage number) of both RA and non-RA synovial cells (P = 0.0042). Differences in susceptibility to lysis between RA and non-RA synovial cells were more susceptible to both RA and normal lymphocyte-induced lysis than were non-RA synovial cells (P = 0.0048). No evidence of cytotoxicity was detected when lymphocytes from nine RA patients and two osteoarthritis patients were reacted against their own synovial cells. Although the data demonstrated an increased cytotoxic activity of peripheral blood lymphocytes from some RA patients against allogeneic synovial cells, the fact that this reactivity was seen against both non-RA and RA synovial cells and was not demonstrated against autologous synovial cells argues against the presence of an immunospecific response of RA lymphocytes to RA synovial cell antigens.

Enhancement of platelet function by superoxide anion.

During the aerobic conversion of xanthine to uric acid by xanthine oxidase, superoxide anion and hydrogen peroxide are produced along with the hydroxyl radical. Our studies demonstrate that washed human platelets incubated with xanthine and xanthine oxidase aggregated and released [14C]serotonin. Aggregation and release were dependent on the duration of exposure to xanthine oxidase as well as the concentration of enzyme. Both reactions were inhibited by the superoxide scavenger enzyme superoxide dismutase but not by catalase, or the free radical scavenger mannitol, suggesting that they were induced by superoxide anion. Superoxide-dependent release was inhibited by prior incubation of platelets with 1 mM EDTA, 1 micronM prostaglandin E1, or 1 mM dibutyryl cyclic AMP, but was unaffected by 1 mM acetylsalicylic acid or 1 micronM indomethacin. After prolonged incubation with xanthine and xanthine oxidase there was also efflux of up to 15% of intraplatelet 51Cr, a cytosol marker. This leakage was prevented by the addition of catalase to the media but not by superoxide dismutase. Incubation with xanthine and xanthine oxidase did not produce malonyldialdehyde, the three-carbon fatty acid fragment produced during prostaglandin endoperoxide synthesis and lipid peroxidation. Prior exposure of platelets to low fluxes of superoxide anion lowered the threshold for release by subsequent addition of thrombin, suggesting a synergistic effect. We conclude that superoxide-dependent aggregation and release may be a physiologically important method to modulate hemostatic reactions particularly in areas of inflammation or vessel injury which could have high local concentrations of superoxide anion.

IL-1alpha, IL-1beta, and IFN-gamma mark beta cells for Fas-dependent destruction by diabetogenic CD4(+) T lymphocytes.

Cytokines such as IL-1alpha, IL-1beta, and IFN-gamma have long been implicated in the pathogenesis of autoimmune diabetes, but the mechanisms through which they promote diabetogenesis remain unclear. Here we show that CD4(+) T lymphocytes propagated from transgenic nonobese diabetic (NOD) mice expressing the highly diabetogenic, beta cell-specific 4.1-T-cell receptor (4.1-TCR) can kill IL-1alpha-, IL-1beta-, and IFN-gamma-treated beta cells from NOD mice. Untreated NOD beta cells and cytokine-treated beta cells from Fas-deficient NOD.lpr mice are not targeted by these T cells. Killing of islet cells in vitro was associated with cytokine-induced upregulation of Fas on islet cells and was independent of MHC class II expression. Abrogation of Fas expression in 4.1-TCR-transgenic NOD mice afforded nearly complete protection from diabetes and did not interfere with the development of the transgenic CD4(+) T cells or with their ability to cause insulitis. In contrast, abrogation of perforin expression did not affect beta cell-specific cytotoxicity or the diabetogenic potential of these T cells. These data demonstrate a novel mechanism of action of IL-1alpha, IL-1beta, and IFN-gamma in autoimmune diabetes, whereby these cytokines mark beta cells for Fas-dependent lysis by autoreactive CD4(+) T cells.

Inhibition of prostaglandin synthesis after metabolism of menadione by cultured porcine endothelial cells.

We have examined the effects of menadione on porcine aortic endothelial cell prostaglandin synthesis. Addition of 1-20 microM menadione caused a dose- and time-dependent inhibition of stimulated prostaglandin synthesis with an IC50 of 5 microM at 15 min. Concentrations greater than 100 microM menadione were necessary to increase 51Cr release from prelabeled cells. Recovery of enzyme inactivated by menadione required a 6-h incubation in 1% serum. In a microsomal preparation, menadione was shown to have no direct effect on conversion of arachidonic acid to prostaglandins. In intact cells menadione caused only a 40% inhibition of the conversion of PGH2 to prostacyclin. Enzymes involved in the incorporation and the release of arachidonic acid were not affected by menadione (20 microM, 15 min). Menadione undergoes oxidation/reduction reactions in intact cells leading to partial reduction of oxygen-forming, reactive oxygen species. In our cells menadione was found to increase KCN-resistant oxygen consumption. Further, an increased accumulation of H2O2 was observed with a time course consistent with menadione-induced inhibition of prostaglandin synthesis. We conclude that menadione at sublethal doses caused inhibition of prostaglandin synthesis. The mechanism involves inactivation of PGH2 synthase by a reactive species resulting from metabolism of menadione by endothelial cells.

A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: comparisons to a 4 h 51Cr-release assay.

Natural killer (NK) cell-or T cell-mediated cytotoxicity traditionally is measured in 4-16 h (51)Cr-release assays (CRA). A new four-color flow cytometry-based cytotoxicity assay (FCC) was developed to simultaneously measure NK cell cytotoxicity and NK cell phenotype (CD3(-)CD16(+)CD56(+)). Target cells, K562 or Daudi, were labeled with Cell Tracker Orange (CTO) prior to the addition of effector cells. Following co-incubation, 7 amino-actinomycin D (7-AAD) was added to measure death of target cells. The phenotype of effectors, viability of targets, the formation of tumor-effector cell conjugates and absolute numbers of all cells were measured based on light scatter (FSC/SSC), double discrimination of the fluorescence peak integral and height, and fluorescence intensity. Kinetic studies (0.5 and 1 to 4 h) at different effector to target (E:T) cell ratios (50, 25, 12, and 6) confirmed that the 3 h incubation was optimal. The FCC assay is more sensitive than the CRA, has a coefficient of variation (CV) 8-13% and reliably measures NK cell-or lymphokine-activated killer (LAK) cell-mediated killing of target cells in normal controls and subjects with cancer. The FCC assay can be used to study a range of phenotypic attributes, in addition to lytic activity of various subsets of effector cells, without radioactive tracers and thus, it is relatively inexpensive. The FCC assay has a potential for providing information about molecular interactions underlying target cell lysis and thus becoming a major tool for studies of disease pathogenesis as well as development of novel immune therapies.

Evaluation of tetrazolium-based semiautomatic colorimetric assay for measurement of human antitumor cytotoxicity.

A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based colorimetric assay was developed and compared with 51Cr release from different adherent tumor cell targets (human squamous cell carcinoma lines of the head and neck established in our laboratory, melanoma, and colorectal carcinoma) using 5-7-day human lymphokine-activated killer cells and monocyte-depleted peripheral blood lymphocytes as effectors. With adherent tumor cell targets, MTT colorimetry was more sensitive than the 51Cr release assay in measuring the antitumor activity of effectors: median, 4385 (range, 988-8144) versus median, 1061 (range, 582-7294) lytic units (the number of effector cells required to lyse 20% of 5 x 10(3) targets)/10(7) effectors (P less than 0.01). Background effects (without effector cells) were comparable in 4-h assays (9% versus 10%) between MTT colorimetry and 51Cr release. In 24-h assays, MTT colorimetry showed higher antitumor activity (70-100% versus 40-60% lysis at 1:1 effector:target cell ratio) but lower background effects (6% versus 38%) than 51Cr release assay. Thus, MTT colorimetry was more sensitive, did not use radiolabeled targets, required fewer effector cells, and was easier, less expensive, and better adaptable to serial monitoring of effector cell function in cancer patients. This colorimetric assay is especially well suited to adherent tumor cell targets. The use of adherent tumor cell monolayers, as opposed to trypsinized single cell suspensions, provides an opportunity to measure interactions of effector cells with enzymatically unaltered solid tumor targets. Because of the greater sensitivity of the colorimetric assay, the transformation of MTT data into lytic units, as commonly used for 51Cr release assays, required an adjustment to avoid the extrapolation based on the exponential fit equation.

Pathogenesis of malignant ascites formation: initiating events that lead to fluid accumulation.

Initiating events leading to the accumulation of malignant ascites in the peritoneal cavity were investigated in two syngeneic transplantable murine ascites-producing tumors, MOT mouse ovarian tumor and the TA3/St mammary carcinoma. The transport of two tracers, 125I-labeled human serum albumin (125I-HSA) and 51Cr-labeled red blood cells (51Cr-RBC), into and out of the peritoneal cavity was studied at early times after i.p. tumor cell injection, prior to abundant fluid accumulation, and at intervals of 5 to 360 min after i.v. or i.p. tracer injection. Tracer influx and efflux rates were estimated from the mass of tracer passing into or out of the peritoneal cavity following a bolus injection of tracer into either the blood or the peritoneal cavity. Efflux of 125I-HSA from the peritoneal cavity was markedly reduced (3- to 5-fold) within 1 day of i.p. injection of either type of tumor cell. Significantly reduced efflux preceded any increase in tumor cell number and by itself did not induce peritoneal fluid accumulation. 125I-HSA tracer influx from plasma to peritoneal fluid did not increase detectably until 5 to 7 days after tumor cell injection, when the tumor cell number had increased by 10- to 100-fold. Only at relatively late stages of ascites tumor growth, when the flow rate into the peritoneal cavity had increased relative to the flow rate out of the peritoneum, was there net peritoneal fluid accumulation. Thus, increased influx, in addition to impaired efflux, were required for malignant ascites accumulation. Following i.p. injection, the efflux rates of 125I-HSA always exceeded those of 51Cr-RBC, even in ascites tumor-bearing animals. Furthermore, 125I-HSA tracer disappeared from the peritoneal cavity more rapidly than it appeared in the plasma, suggesting that 125I-HSA moves more rapidly through the channels by which 51Cr-RBC egress from the peritoneum (primarily diaphragmatic lymphatics) and/or has access to additional pathways not open to 51Cr-RBC. Finally, flow rates into and out of the blood and peritoneum were used to obtain kinetic parameters that characterized tracer transport: k1, the rate constant for tracer transport from the blood to the peritoneum; k2, the rate constant for tracer transport from the peritoneal cavity to the blood; and k6, the rate constant for tracer transport from the peritoneal cavity to surrounding interstitial tissue.(ABSTRACT TRUNCATED AT 400 WORDS)