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PubMed1991

Therapy of recurrent high-grade gliomas with surgery, autologous mitogen-activated IL-2-stimulated (MAK) killer lymphocytes, and rIL-2: II. Correlation of survival with MAK cell tumor necrosis factor production in vitro.

Seven patients with recurrent high-grade glioma were treated in a Phase I/II trial with surgical debulking, after which mitogen-activated IL-2-stimulated killer (MAK) lymphocytes and 10(5) units rIL-2 were implanted in the surgical defect. The therapy was well tolerated, and the mean survival of this group of patients was 29 weeks. Tumor necrosis factor (TNF) production by MAK lymphocytes stimulated with IL-2 in vitro was measured. A significant (r = .78, p = .04) correlation between survival of patients after therapy and the ability of the MAK lymphocytes to produce TNF in vitro was noted. A significant negative correlation (r = -.82, p = .02) was found when comparing TNF production and increasing tumor size measured on MRI. No correlation was found between TNF production in vitro and MAK lymphocytes lytic activity on K562 and U373 target cells. No correlation was found between survival and MAK cell lytic activity measured on K562 and U373 target cells. We conclude that TNF production in vitro and cytotoxic activity measured in vitro are measures of different antitumor activity in vivo and in vitro. TNF production during IL-2-stimulated proliferation may be an important in vitro assay in terms of predicting length of survival of recurrent high-grade gliomas.

Adult↗
PubMed1990

Comparison of in vitro and in vivo models of drug transcytosis through the blood-brain barrier.

Drug and solute transport through in vitro and in vivo models of the blood-brain barrier (BBB) were compared to provide a measure of how well the in vitro model predicted BBB permeability found in vivo. The in vitro model employed bovine brain capillary endothelial cells in either primary tissue culture or as a continuous line grown on Transwells and placed in side-by-side diffusion chambers. The in vivo model of BBB transport utilized an internal carotid artery perfusion/capillary depletion method in anesthetized rats. BBB permeability in vivo and in vitro was measured for 15 radiolabeled drugs and for L-[3H]dopa, D-[14C]glucose and [3H]albumin. [3H]- or [14C]sucrose was used in vivo as a blood volume reference. Lipid solubility of each drug was measured based on the 1-octanol/Ringer's partition coefficient. The morphology of the endothelial cell in primary tissue culture was spindle-shaped and the morphology of the endothelial cell in continuous culture was cuboid-shaped. The cuboidal morphology demonstrated a 2-fold greater resistance to solute transport and was used for the majority of the in vitro studies. Drug and solute permeability coefficients (Pe) ranged from 3.9 X 10(-3) to 2.5 X 10(-1) cm/min in vitro and from 1.0 X 10(-5) to 2.1 X 10(-2) cm/min in vivo. The In of the permeability.surface area product in vitro correlated with the In partition coefficient (r = 0.62, P less than .0125) and the In permeability.surface area product in vivo correlated with the In partition coefficient (r = 0.84, P less than .0005).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Comparison of in vitro cell binding characteristics of four monoclonal antibodies and their individual tumor localization properties in mice.

Although many antibodies are being used for imaging studies, it is not clear which in vitro properties of antibodies will best reflect their in vivo characteristics. The ability to correlate in vitro binding characteristics of monoclonal antibodies to tumor antigens with their in vivo localization characteristics, particularly with respect to tumor localization properties, is desirable for rapid selection of monoclonal antibodies with potential for clinical use. The in vitro binding characteristics of three monoclonal antibodies to the murine Ly-2.1 antigen and one to the Ly-3.1 antigen have been studied on cultured tumor cells bearing these antigens. The association and dissociation rate constants, apparent affinity, and immunoreactivity of each antibody in vitro were compared with their ability to localize the s.c. tumors from the same cell line growing in Ly-2.1-/Ly-3.1-mice. The antibody with the highest affinity and fastest association rate localized to tumor at the earliest time (16-20 h after injection) and had the highest percentage of the injected dose/g in the tumor (greater than 25%). The antibody with the lowest affinity showed significantly less localization to tumor cells, compared with the other three antibodies. The ranking of the antibodies by affinity agreed with the ranking in terms of their ability to localize to tumors, but the in vitro immunoreactivity of the antibodies, as measured by a cell binding assay, did not correlate with their tumor localization properties. Immunoscintigraphic studies did not precisely correlate with biodistribution data or in vitro binding characteristics, because tumors could be satisfactorily imaged with each antibody, although it was noted that the antibody with the highest affinity gave the best image. The results indicate that kinetic binding parameters and affinity of monoclonal antibodies may be useful in predicting their potential for tumor localization and that kinetics of association of antibodies in vitro may predict localization kinetics in vivo.

Animals↗
PubMed1987

Application of in vivo and in vitro pharmacokinetics for physiologically relevant drug exposure in a human tumor clonogenic cell assay.

The biological half-lives and decay rate constants under the conditions of a human brain tumor clonogenic cell assay were determined for six clinically used anticancer agents. The agents studied were: 1,3-bis(2-chloroethyl)-1-nitrosourea; 3-(2-chloroethyl-3-nitrosoureido-2-deoxy-D-glucopyranose; cis-diaminedichloroplatinum(II); 2,5-diaziridinyl-3,6-bis-(carboethoxyamino)-1,4-benzoquinone; 4-demethylepipodophylotoxin-D-thylidene glucoside; and 9-hydroxy-2-N-methylellipticine. In vitro decay of all six drugs was found to be according to first order kinetics. The half-lives of two drugs, namely, 1,3-bis(2-chloroethyl-1-nitrosourea and 3-(2-chloroethyl-3-nitrosoureido-2-deoxy-D-glucopyranose under the human tumor clonogenic cell assay (HTCA) conditions were found to be similar to their terminal in vivo half-lives in humans. For the other drugs, however, there was a very large difference between their in vitro and in vivo pharmacokinetics. In the case of 2,5-diaziridinyl-3,6-bis(carboethoxyamine)-1,4-benzoquinone, we observed about an 80-fold difference between its in vitro half-life of 40.76 h and its in vivo terminal half-life of 0.52 h. We describe the principles upon which these data can be used to design clinically more relevant in vitro drug exposure protocols in HTCAs. Since, generally, tumor cells are exposed to drugs in the HTCA either continuously or for a specified duration, e.g., 1 or 2 h, we computed the initial in vitro drug concentrations to which tumor cells should be exposed such that the resulting in vitro (c X t) after a 2-h or a continuous exposure will be within clinically achievable levels. The application of these in vivo and in vitro pharmacokinetic principles will provide for more physiological testing of patient tumor cell sensitivity to anticancer drugs in the HTCA, and is likely to result in lower rates of false positive responses in clinical trials using clonogenic cell assays.

Antineoplastic Agents↗
PubMed1986

In vivo and in vitro expression of an interleukin 2 receptor by murine B and T lymphocytes.

Interleukin 2 (IL 2), which is well established to be a T cell growth factor, has more recently been shown to stimulate B lymphocyte growth and differentiation in vitro. Responsiveness of B and T cells to IL 2 has been associated with expression of a cell membrane IL 2 receptor (IL 2R). To investigate the role of IL 2 in B cell growth and differentiation in vivo, a system was used in which the injection of mice with a goat antibody to mouse IgD (GaM delta) induces polyclonal T-independent B cell proliferation first, and later induces polyclonal T-dependent B cell proliferation and IgG secretion. IL 2R expression by splenic B and T lymphocytes from GaM delta injected mice was studied by a dual label immunofluorescence technique. Although GaM delta was found to be a strong inducer of B cell IL 2R expression in vitro, even in serum-free medium, and stimulated up to 50% of splenic T cells to express considerable quantities of IL 2R in vivo, it failed to induce more than minimal B cell IL 2R expression in vivo. Concanavalin A and bacterial lipid A also induced B cells to express IL 2R to a much greater extent in vitro than in vivo. Although these agents and GaM delta acted synergistically to stimulate B cell IL 2R expression both in vitro and in vivo, a single agent induced B cell IL 2R expression to a considerably greater extent in vitro than did all three agents acting together in vivo. In vitro GaM delta-induced B cell IL 2R expression was not suppressed by inclusion of IL 2 in the culture medium but was suppressed by the presence of 10% normal mouse serum or plasma. These observations suggest that polyclonal T-dependent B cell proliferation and antibody secretion may not require an interaction between B cells and IL 2; the in vivo environment may downregulate IL 2R expression by B cells: and in vivo B cell IL 2R expression and consequently, induction of B cell responsiveness to IL 2, may require stimuli beyond those sufficient to induce B cell IL 2R expression and IL 2 responsiveness in vitro.

Animals↗
PubMed1987

In vitro evidence that carbohydrate moieties derived from uromodulin, an 85,000 dalton immunosuppressive glycoprotein isolated from human pregnancy urine, are immunosuppressive in the absence of intact protein.

Our laboratory recently reported the purification of a unique immunosuppressive glycoprotein isolated from human pregnancy urine (7). This glycoprotein, which we term uromodulin, has a m.w. of 85,000 as assessed on SDS-PAGE and is 30% carbohydrate. Uromodulin blocks in vitro antigen-specific T cell proliferation to recall antigens such as tetanus toxoid at concentrations as low as 100 pM. This glycoprotein also blocks the in vitro generation of spontaneous monocyte-mediated cytotoxicity (7, 36). Recent evidence strongly suggests that the primary action of uromodulin is to act as a specific ligand and modulator of IL 1 (10, 33). We now report additional biochemical characterization of uromodulin, and based on three independent lines of evidence, find that its immunologic activity appears to result from its glycosylation. First, measures to alter the tertiary folding of the protein backbone of uromodulin, including succinylation or reduction and carboxymethylation, fail to significantly affect its in vitro bioactivity. Second, after extensive digestion of intact uromodulin with pronase, the majority of the in vitro bioactivity can be recovered in a single carbohydrate-rich fraction. Finally, digestion with N-glycanase (N-glycosidase F-, an enzyme specific for N-asparagine-linked oligosaccharides) and subsequent purification on thin layer chromatography yields a single complex oligosaccharide that appears to be responsible for the majority of the in vitro immunosuppression mediated by uromodulin. These data suggest that uromodulin displays N-linked carbohydrate sequences capable of down-regulating antigen-specific T cell responses in vitro. It has been suggested that endogenous lectins may play an important role as recognition molecules in mammalian, as well as more primitive immune systems (23, 24). Our in vitro biologic data strongly suggest that the carbohydrate portion of uromodulin is an excellent candidate to function as a potential lectin receptor.

Alkylation↗
PubMed1986

[In vivo experiments, in vitro assays in experimental oncology: establishment of LA 795 Vv-Vt].

In vivo experiment, in vitro assays (in vivo-in vitro system) is a kind of experimental technique which is different from both in vitro cell line and tumor line. The new model can be grown and studied either as an animal tumor or a cell culture. The specimen could be assayed for cell survival in vitro. This model can be used to study the response of malignant cells to the treatment in a precision and depth that is impossible in tumors grown in vivo. LA 795 Vv-Vt is the first in vivo-in vitro system developed in China. It was established with lung adenocarcinoma of T-739 mouse. The tumor cells could grow freely both in vivo and in vitro with a plating efficiency of 20%. Characteristics of LA 795 Vv-Vt tumor and cell in culture were described and compared. In order to estimate the radiation response of different doses, cell survival curves of tumors irradiated under oxic and hypoxic conditions were drawn and compared. The oxygen enhance ratio was 2.98. The experiments indicate that in vivo-in vitro system has a good dependent relation in dose and effect and is worth extensive application.

Adenocarcinoma↗
PubMed1986

[Results of in vitro chemosensitivity assays: human tumor clonogenic and scintillation assay].

The authors reviewed their experiences to date with chemosensitivity testing of 629 tumors by human tumor clonogenic assay (HTCA) and of 199 tumors by scintillation assay (SA). HTCA and SA were both performed using a double-layer-soft-agar system with continuous exposure of cells to one concentration of standard anticancer drugs. Overall, 60% of specimens in HTCA and 58% in SA produced significant growth in vitro. HTCA was 52% (13/25) reliable for predicting in vivo sensitivity, and 95% (36/38) reliable for in vivo resistance, whereas SA was 40% (8/20) reliable for in vivo sensitivity and 88% (21/24) for in vivo resistance. The current results indicate that there is no particular difference in the success rate and in the predictive accuracy for in vivo sensitivity and resistance between HTCA and SA. Therefore, it is doubtful whether only clonogenic tumor cells among whole tumor cell populations should be selected in assessing the chemosensitivities of human tumors. In vitro success rates were variable, depending on the tumor histology. In vitro growth of gastric cancer specimens was characteristically lower than that of colon cancer specimens (48% and 60% in HTCA, and 46% and 68% in SA, respectively). (p less than 0.005). Optimal in vitro-in vivo drug concentrations and culture conditions are still being defined. Correlation studies of in vitro-in vivo responses of gastrointestinal cancers suggested that in vitro concentrations of 5-fluorouracil and mitomycin C used in this study were considerably higher than their optimal doses. Tumor cell heterogeneity poses significant problems in the clinical use of chemosensitivity assays. The question arises as to whether a single biopsy specimen is representative of a patient's disease. In this last study, we sought evidence of tumor heterogeneity by comparing chemosensitivity responses between: 1) different portions of a single tumor, 2) a primary and a metastatic biopsy taken from a patient on the same day, and 3) different metastases from a patient taken on the same day. The results demonstrated the presence of considerable heterogeneity of response to chemotherapy among different tumors from the same patient, and even within the same tumor. The reported discrepancies of in vitro and in vivo sensitivity may be due to such therapeutic heterogeneity among tumors.

Antineoplastic Agents↗
PubMed1984

FcR epsilon+ lymphocytes and regulation of the IgE antibody system. III. Suppressive factor of allergy (SFA) is produced during the in vitro FcR epsilon expression cascade and displays corollary physiologic activity in vivo.

Exposure of lymphoid cells to IgE induces the expression of Fc receptors for IgE (FcR epsilon) and the production of soluble mediators, termed IgE-induced regulants (EIR). Conventional suppressive factor of allergy (SFA) and enhancing factor of allergy (EFA), derived from mouse ascites fluids, both inhibit IgE-induced FcR epsilon expression in vitro in cultures of unfractionated and T cell-enriched, but not B cell-enriched, lymphoid cells. This indicates that the inhibitory activities of both entities are T cell dependent, and distinguishes them from the inhibitory EIRI, which inhibits FcR epsilon induction in the absence of T cells. Moreover, SFA and EFA can be distinguished from one another by differences in the T cell subsets required for the inhibitory activity of each respective mediator on in vitro IgE-induced FcR epsilon expression. Thus, SFA requires the presence of Lyt-1+ T cells, whereas EFA requires the presence of Lyt-2+ T cells. Supernatant fluids from IgE-stimulated unfractionated lymphoid cell cultures suppress in vivo IgE synthesis in mice, indicating that SFA is produced along with the other species of EIR. To define conditions required for SFA production in vitro, EIR-rich supernatant fluids were tested for the presence of SFA by using Lyt-2+ cell-blocked indicator cells in the in vitro FcR epsilon induction assay system (this eliminates the inhibitory activity of EFA). SFA production in vitro by IgE-stimulated lymphoid cells was shown to result from cooperative interactions between B cells and Lyt-1+ T cells. In addition, as observed with the induction of FcR epsilon in general, induction of SFA requires the initial interaction of B cells with IgE, and the release of the B cell-selective EIRB. Once produced, EIRB can directly stimulate Lyt-1+ cells, but not Lyt-2+ cells, to produce SFA. The physiologic significance of the in vitro induction of SFA by the action of EIRB on Lyt-1+ cells was confirmed by the demonstration that EIRB, devoid of detectable SFA, selectively suppressed in vivo IgE synthesis after administration to intact mice. This indicates that EIRB can stimulate resident T cells of irradiated SJL mice to produce SFA. Finally, as shown previously with conventional ascites-derived SFA, the SFA produced in vitro after stimulation of lymphoid cells with IgE is devoid of IgE-binding properties, because its inhibitory effects on in vivo IgE antibody synthesis are not removed by passage over IgE affinity columns.

Animals↗
PubMed1983

Human myeloma in vitro colony growth: interrelationships between drug sensitivity, cell kinetics, and patient survival duration.

Ninety-seven patients with multiple myeloma evaluated serially had both a tritiated thymidine labeling index of bone marrow plasma cells (LI%) and in vitro myeloma stem cell culture performed. Thirty-three patients with myeloma colony growth had in vitro drug sensitivity testing carried out, 18 having in addition in vitro thymidine suicide determinations. The LI% and the likelihood of in vitro myeloma colony growth were highly correlated: the higher the LI%, the more likely was colony or cluster growth (p less than 0.001). The tritiated thymidine suicide of myeloma stem cells was usually very high. There was excellent correlation between in vitro and in vivo drug sensitivity. Both pretreatment drug resistance and selective sensitivity (e.g., interferon, bisantrene, methotrexate, vinblastine) at the time of relapse were accurately detected and correlated well with survival duration (p = 0.01 Wilcoxan). Although LI% and in vitro sensitivity were clearly independent variables, a high LI% (greater than 3%) plus in vitro resistance were associated with a subsequent survival duration of less than 6 mo. The studies allowed dissection of the complex interrelationship between cell kinetics and drug sensitivity.

Bone Marrow↗
PubMed1993

In vitro and in vivo effects of 5-aza-2'-deoxycytidine (Decitabine) on clonogenic cells from acute myeloid leukemia patients.

5-Aza-2'-deoxycytidine (Decitabine) is an analog of deoxycytidine now entering clinical trials in acute myeloid leukemia (AML) owing to a defined antileukemic activity mediated at least in part by DNA hypomethylation, altered gene expression, and induction of cell differentiation. In the present study, we examined the relationship between the in vitro sensitivity to Decitabine of blast progenitors and the clinical outcome, in nine AML patients treated in vivo with Decitabine within a phase II trial carried out at two different institutions. Leukemic blast progenitors in acute myeloid leukemia (AML) undergo terminal divisions giving rise to colonies in methylcellulose. The self-renewal capacity of blast progenitors is conversely reflected by a secondary methylcellulose assay after exponential growth of clonogenic cells in suspension cultures. Three out of four patients, in which clonogenic cells in methylcellulose were strongly suppressed by Decitabine and clonogenic growth of blasts cultured in suspension was only slightly affected, failed on Decitabine treatment in vivo. Two subjects, whose blast progenitors in suspension culture were significantly inhibited by Decitabine, obtained a positive hematological response (complete or partial remission, CR or PR) and an additional patient showing a similar in vitro pattern died in induction with an hypoplastic marrow without morphological evidence of persistant leukemia. Interestingly two patients displaying an unfavourable in vitro pattern (i.e. a minor suppression of self-renewal mitoses as evinced from suspension cultures) achieved a hematological response (CR and PR) upon in vivo therapy with Decitabine. The in vitro response to Decitabine of clonogenic progenitors from both these patients shifted to a favourable pattern (i.e. major suppression of self-renewal versus terminal mitoses) following manipulation of culture conditions by the addition or removal of exogenous growth factors. In addition, in a further patient refractory to treatment with Decitabine in vivo, similar alterations of the culture conditions were unable to modify the unfavourable pattern of response to the drug in vitro. Our results indicate that the sensitivity of blast progenitors in suspension cultures strongly correlates with the remission outcome of the patients. From our data, it also appears that alterations of culture microenvironment are able to modify the response of AML blasts to Decitabine, unveiling the 'hidden' sensitivity of leukemic progenitors to the drug in cases characterized by a discrepancy between in vivo and in vitro results, i.e. apparent in vitro resistance and favourable clinical outcome.

Acute Disease↗
PubMed1995

Clinical efficiency of in vitro and in vivo tests for allergic diseases.

BACKGROUND: Specific serum IgE determination is widely used in the diagnosis of IgE-mediated allergic diseases but the relative merits of in vitro measurement of IgE antibody in comparison to in vivo skin tests are still debated. OBJECTIVE: The aim of this study was to investigate the clinical efficiency of a "second generation" technique for in vitro analysis of IgE antibody (Pharmacia CAP System). METHODS: Eighty-six patients with suspected inhalant and/or food allergies and 20 asymptomatic subjects for a total of 655 tests were evaluated. Sera with divergent results between in vitro and in vivo techniques were further analyzed by using ImmunoCAP inhibition and immunoblotting. For the calculation of sensitivity and specificity of both in vitro and in vivo tests we considered as true value (reference value) either the concordant results or, in case of discordance, the datum confirmed by ImmunoCAP inhibition or immunoblot (ie, vitro positive, vivo negative, ImmunoCAP inhibition positive; true result: positive). RESULTS: The obtained results demonstrate that the in vitro results correlate well in terms of specificity and sensitivity to this new reference standard. In particular a higher specificity for Pharmacia CAP System in comparison to in vivo skin prick test for grass pollens and a better sensitivity for mites and cat allergens were found. CONCLUSIONS: Our results suggest that the in vitro "second generation" testing provides reliable results in all clinical situations.

Adolescent↗
PubMed1993

p53 mutations in mouse mammary epithelial cells: instability in culture and discordant selection of mutations in vitro versus in vivo.

The phenotypes of p53 mutations found in human and murine tumors often are analyzed using a variety of transformation assays in vitro, but data have not been available to correlate in vitro effects with in vivo activities. We have assessed the effects of p53 mutations using mouse mammary epithelial cell lines which can be analyzed both in vitro and in vivo. Parental mammary epithelial cell lines (FSK series) injected into cleared mammary fat pads of syngeneic mice frequently give rise to preneoplastic lesions (HAN) which can be reestablished in culture (TM lines) to permit analysis of genetic changes important in the development of preneoplasia. Characterization of the FSK3 cell line revealed a cell population mixed with respect to p53 genotypes. One subpopulation of mutant (Ser233-234) p53-expressing cells was selected in a preneoplastic mammary outgrowth in vivo (TM3), whereas another minor population of mutant (Pro135) p53-expressing cells was selected during culturing of FSK3 cells in vitro. When FSK3 cells were subdivided and passaged in parallel in vitro, p53 genotypes continued to evolve and diverge. These findings reveal that selective pressures exerted on mammary epithelial cell populations in vivo are different from pressures present in vitro. Selective forces may vary from one cell culture environment to another. The growth advantage conferred by a specific p53 mutation appears to differ in vivo versus in vitro. We propose that caution should be exercised when attempting to correlate the effects of p53 mutations assayed in cell culture systems with the in situ phenotypes of mutant p53 in cancer.

Amino Acid Sequence↗
PubMed1993

[Quality control of platelet concentrates. Functional assessment of stored platelets in vitro].

Platelet concentrates transfused for correction of thrombocytopenia or reduced platelet function do not consistently improve primary haemostasis in the recipient. Insufficient therapeutic effects may be caused by impaired donor platelet function and by unfavourable donation and storage conditions, as well as by immunological interactions with the recipient blood. The present study was designed to investigate whether the effect of platelet transfusion on recipient platelet function can be predicted by in vitro methods. METHODS. Blood samples were taken from 12 thrombocytopenic patients before (20 ml, P0) and after (10 ml, P(vivo)) transfusion of one unit of platelets previously stored for 24-120 h in acid citrate dextrose. An additional sample was taken from the platelet concentrate (TK) immediately before transfusion. P0 was divided into two specimens and TK platelets were added to one of them (P(vitro) in order to obtain a platelet count similar to that in P(vivo). Bleeding time (BT) and bleeding volume (BV) of the samples P0, P(vivo) and P(vitro) were measured using the method of Kratzer and Born (Fig. 2); mean values were calculated for each sample from six measurements. Aggregability of TK platelets was determined in addition by aggregometry. In contrast to previous studies, physiological Ca2+ concentrations were restored and secondary haemostasis was inhibited by low-molecular-weight heparin (Fragmin P, Pfrimmer Kabi GmbH und Co. KG, Erlangen) in the platelet-rich plasma used for aggregometry. RESULTS. Platelet counts increased in all patients after transfusion (P(vivo) vs P0, Table 1) and were nearly identical in P(vitro) and P(vivo) (r = 0.94, P < 0.001; Fig. 3). Parameters of primary haemostasis were significantly improved by addition of platelets to P0 in vitro (BT P < 0.05, BV P < 0.01) as well as by platelet transfusion (BT P < 0.05, BV P < 0.01). Direct comparison of P(vitro) and P(vivo) yielded a very close correlation of BT (r = 0.88, P < 0.001) and BV (r = 0.89, P < 0.01) in both samples. Although aggregometry revealed decreasing platelet function with increased storage time, aggregability was considerably higher compared to previous studies of platelet concentrates stored for 2-5 days. CONCLUSION. A new technique has been developed which allows reliable prediction of the effect of platelet concentrates on primary haemostasis of the recipient by in vitro measurement of bleeding time and bleeding volume prior to transfusion using the method of Kratzer and Born.

Blood Platelets↗
PubMed1996

Innervation of the chick cornea analyzed in vitro.

PURPOSE: During the early stages (embryonic day 3 [E3]) of avian corneal development, nerve fibers extend from the trigeminal ganglion to the corneal limbus. On E11, these nerve fibers enter the cornea and extend through the secondary stroma to begin innervation of the epithelium on E13. This process of innervation is concomitant with the cornea's dehydration and transition from opacity to transparency; thus, suggesting a link between innervation and the attainment of corneal function. This investigation attempts to ascertain whether the developing cornea can support its innervation in vitro and whether there is a possible developmental interrelationship between corneal innervation and dehydration, with the associated transition from opacity to transparency. METHODS: Isolated corneas from either E8 or E14 chicks were co-cultured with E8 dorsal root ganglia. After 4 days of culture, innervation was visualized by silver staining and immunohistochemistry. Changes in corneal composition and organization associated with this innervation in vitro were analyzed by measuring changes in specific hydration, thickness and compaction, and incorporation of [35S]sulfate into glycosaminoglycans during co-culture. RESULTS: The E8 and E14 corneas support extensive innervation in vitro. Developing nerve fibers extend through the secondary stroma to innervate the epithelium. In vitro innervation of E8, but not E14, corneas was associated with a decrease in corneal specific hydration, whereas control corneas (without dorsal root ganglia) failed to show any such changes. E8 corneas also showed a significant increase in compaction when innervated in vitro. Corneal innervation in vitro did not significantly change the overall incorporation of [35S]sulfate into glycosaminoglycans. Furthermore, incorporation of [35S]sulfate into corneal sulfated glycosaminoglycans (sGAG) is not influenced by either the number of nerve fibers innervating the cornea or nerve growth factor (NGF). In addition, the distribution of staining of the corneal glycosaminoglycans, chondroitin sulfate and keratan sulfate, and peanut agglutinin-binding epitopes, suggests that these molecules are not associated with inhibition of axonal development. CONCLUSIONS: The in vitro system described here is a useful model to understand the process of corneal development. Co-culture has shown that corneal innervation promotes the process of dehydration, which is dependent on the age of the cornea. However, other functionally related refinements necessary for transparency-notably proteoglycan synthesis-may not be linked to innervation or NGF production. The authors conclude that the development of transparency is dependent on corneal innervation, though not exclusively, and that other controlling factors also are required.

Animals↗
PubMedDate not supplied

In vitro sensitivity of chronic lymphocytic leukemia B-cells to fludarabine, 2-chlorodeoxyadenosine and chlorambucil: correlation with clinico-hematological and immunophenotypic features.

BACKGROUND: Chlorambucil (CLB), 2-chlorodeoxyadenosine (2-CDA) and fludarabine (FAMP) are among the most widely used drugs in chronic lymphocytic leukemia (CLL). Therefore we evaluated in vitro sensitivity to these drugs and cross-resistance of purine analogs. In addition, we correlated the in vitro data with the main clinico-hematological variables and surface markers. PATIENTS AND METHODS: Eighty CLL samples obtained from 63 untreated and 17 treated CLL patients were tested in vitro with the MTT assay. Lethal dose (LD)50 values were calculated to determine sensitivity to CLB, 2-CDA and FAMP. RESULTS: Samples were clustered either for a one-log increase of LD50 values or for LD50 threshold values of 3 microM for FAMP, 0.3 microM for 2-CDA and 7 microM for CLB, which correspond to the therapeutically achievable plasmatic levels of these drugs. A higher number of samples sensitive to 2-CDA were identified by the first approach; with the second method the relative order of sensitivity was FAMP > 2-CDA > CLB. Concerning 2-CDA and FAMP cross-resistance, out of 61 samples resistant to 2-CDA, 29.5% were sensitive to FAMP. Conversely, 13.9% out of 43 samples resistant to FAMP were sensitive to 2-CDA. No correlation was found between the main clinico-hematological features and the LD50 values of each drug either considering the whole series or only the untreated cases. In vitro drug sensitivity was also evaluated during the steady-state of the disease and at disease progression in six untreated cases. We observed a mean increase in the LD50 values of about 13, 38 and 22 times for CLB, FAMP and 2-CDA, respectively. Among the treated cases, the LD50 values of both purine analogs and CLB correlated with bone marrow histology. CLL cells expressing CD14, CD11c, CD11b, and FMC7 were more resistant in vitro to purine analogs but not to CLB. CONCLUSIONS: This study suggests that i) the purine analogs exert a greater cytotoxic effect on CLL cells; ii) 2-CDA and FAMP are not cross-resistant in vitro in a percentage of CLL samples, iii) a possible change in LD50 values may be related to modification of the disease status, and iv) the expression of certain surface markers, which are CLL-unrestricted, identifies samples with higher in vitro resistance to purine analogs.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1997

Cytokine regulation of mesothelial cell proliferation in vitro and in vivo.

Previous studies have demonstrated mitogenic effects of several mediators on mesothelial cells in vitro, but their effects in vivo have not been investigated. The aim of this study was to examine the effects of various cytokines on normal mesothelial cell proliferation in vitro and in vivo and correlate the findings in both assay systems. In vitro proliferation was assessed using a technique based on the uptake and subsequent release of methylene blue. Autoradiographic methods were applied in a murine model to assess mitogenic activity of these factors on mesothelium in vivo. In vitro data demonstrated a dose-dependent increase in human mesothelial cell proliferation by all mediators examined: at optimal concentrations, proliferation was enhanced between 26.53 +/- 3.77% standard deviation (SD), p < 0.001 for fibroblast growth factor-2 (FGF-2) and 114.58 +/- 6.97%, p < 0.001 for platelet-derived growth factor-AB (PDGF-AB) above control medium. In vivo, DNA synthesis in mesothelial cells was stimulated by FGF-2 (29.52 +/- 5.85% labeled cells, compared with 7.04 +/- 4.36% for control medium; p < 0.001), tumor necrosis factor-alpha (TNF-alpha; 13.14 +/- 4.55% compared with 7.23 +/- 2.85; p < 0.005) and PDGF-BB (11.53 +/- 4.74% compared with 4.67 +/- 3.48%; p < 0.005). Transforming growth factor-beta1 (TGF-beta1) and epidermal growth factor (EGF) had no effect on DNA synthesis in mesothelial cells in vivo. It is concluded that FGF2, TNF-alpha, and PDGF stimulate mesothelial cell proliferation in vitro and in vivo, whereas TGF-beta1 and EGF only had a mitogenic effect in vitro at the concentrations examined. The mitogenic potency of the different PDGF isoforms in vitro was consistent with PDGF-alpha and beta receptor expression.

Animals↗
PubMed1976

In vitro activation of mouse macrophages by rat lymphocyte mediators.

Macrophage-activating factors (MAF)3 were released by presensitized rat lymphocytes stimulated in vitro with the appropriate antigens. Different supernatants of presensitized rat lymphocytes specifically stimulated in vitro with several different mouse, dog, and rat tumor or normal cells were capable of rendering normal rat and mouse macrophages nonspecifically cytotoxic in vitro to their respective syngeneic tumor cells. The release of active mediators by rat lymphocytes sensitized in vivo was dependent upon immunologically specific recognition of an antigen in vitro. When rat lymphocytes were incubated in vitro with antigens unrelated to the in vivo sensitizing antigens, no release of MAF occurred. Once rat MAF was released, it activated both syngeneic (rat) and xenogeneic (mouse) macrophages to kill tumor cells in vitro. These activated marcophages destroyed all syngeneic tumor targets. Such cytotoxicity was obtained even when the cells used to elicit release of MAF were totally unrelated to the target tumor cells. The data thus demonstrated that MAF can cross strain and even species specificities and can activate macrophages to kill tumors in a nonspecific manner. The cytotoxicity mediated by in vitro activated mouse macrophages decreased with time once the macrophages were removed from MAF; and by 7 days postactivation, the macrophages were not cytotoxic. However, when incubated again with MAF, significant reactivation was observed. This suggested that activation of macrophages in vivo may be a continuous process of lymphocyte-macrophage interaction.

Animals↗