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At least 955 records · Page 53Linked to original sources
PubMed1999

A new approach to modelling the relationship between in vitro and in vivo drug dissolution/absorption.

A major goal of the pharmaceutical scientist is finding a relationship between an in vitro characteristic of an oral dosage form and its in vivo performance. One such relationship between drug dissolution (or absorption) in vivo and that in vitro is known as an 'in vitro-in vivo correlation' (IVIVC) whose importance stems from the fact that it may be used to minimize the number of human studies required during product development, assist in setting meaningful in vitro dissolution specifications and justify biowaivers for scale-up and post approval changes. A number of ways of describing an IVIVC have been reported with 'level A' being the most informative and therefore most desirable. In the majority of cases reported to date, both the model and the statistical methods employed for level A IVIVC are very simplistic. The model assumes that the rate and extent of dissolution in vivo are the same as those in vitro. The statistical methods ignore the repeated measures nature of the data and use a response variable as an independent variable without accounting for measurement error. This paper describes some new models which include the simple model as a special case. The modelling approach is based on considering the time at which a drug molecule enters solution (in vitro or in vivo) to be a random variable. The in vitro and in vivo distributions are then related to one another using a proportional odds, proportional hazards or proportional reversed hazards model. The models can be extended by adding a linear time component which describes a time varying relationship. Following the addition of random effects to these structural models in order to account for the repeated measures nature of the data collected, the models may be described as generalized linear mixed effects models. The models were fitted to some data sets using a maximum likelihood based method and the results indicate that these models have potential for describing an in vitro-in vivo relationship which cannot be described using the currently available models.

Absorption↗
PubMed2000

Analysis of in vivo regulatory properties of T cells activated in vitro by TGFbeta2-treated antigen presenting cells.

PURPOSE: To determine whether naive T cells activated in vitro by antigen-pulsed, transforming growth factor(beta) (TGFbeta)-treated antigen presenting cells (APCs) acquire the capacity to suppress the induction and expression of delayed hypersensitivity in vivo. METHODS: Naive ovalbumin (OVA)specific T cells from DO 11.10 Tcr transgenic mice were stimulated in vitro with OVA-pulsed TGF(beta2)-treated APCs. The cultured cells were harvested and assayed for in vitro production of mature TGFbeta. Similar cells were coinjected with primed OVA-specific BALB/c T cells plus OVA-pulsed APCs into ear pinnae of normal BALB/c mice (assay for delayed hypersensitivity expression) or coinjected with OVA-pulsed APCs into footpads of naive DO11.10 mice whose draining lymph node cells were harvested 4 days later and assayed in vitro for capacity to secrete interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) when stimulated with OVA (assay for induction of delayed hypersensitivity). RESULTS: DO11.10 T cells activated in vitro by OVA-pulsed TGFbeta2-treated APCs secreted large amounts of mature TGFbeta and suppressed the expression of delayed hypersensitivity in a local adoptive transfer assay. Suppression was reversed in the presence of neutralizing anti-TGFbeta antibodies. In addition, in vitro generated regulatory T cells influenced naive T cells in DO11.10 mice that were responding to an initial immunization with OVA to secrete IL-4, rather than IFN-gamma. This influence was independent of TGFbeta. CONCLUSIONS: OVA-pulsed APCs, pretreated in vitro with TGF(beta)2, activate DO11.10 T cells in a manner that endows the responding cells with the capacity to suppress the induction and then the expression of delayed hypersensitivity in vivo. In certain ways, these properties of in vitro-activated DO11.10 T cells resemble the properties of afferent and efferent regulatory T cells typically found in the spleens of animals with anterior chamber-associated immune deviation.

Animals↗
PubMed2001

In vitro substrate identification studies for p-glycoprotein-mediated transport: species difference and predictability of in vivo results.

Two different cellular assay models were assessed as in vitro systems for P-glycoprotein (P-gp) substrate identification: cellular accumulation studies with KB-V1, a human MDR1 P-gp-overexpressing multidrug-resistant human epidermoid carcinoma cell line; and transcellular transport studies with L-MDR1 (or L-mdr1a), a human MDR1 (or mouse mdr1a)-transfected porcine renal epithelial cell line. The in vitro-in vivo correlation for P-gp-mediated transport activity was also examined by comparing in vitro data obtained from L-mdr1a cell studies and in vivo data from mdr1a (-/-)/(+/+) CF-1 mice studies for several compounds. The results are summarized as follows: 1) two in vitro assay systems routinely identified the substrate for human MDR1 P-gp-mediated transport with similar quantitative results; 2) in vitro studies with L-MDR1 and L-mdr1a cells demonstrated that the P-gp substrate susceptibility is different between human and mouse for certain compounds (species difference); and 3) in vivo brain concentration ratios of mdr1a (-/-) to (+/+) CF-1 mice, either at a certain time point or up to 60 min, correlated well with the in vitro transcellular transport ratios from L-mdr1a cells (r(2) = 0.968 and 0.926, respectively). This indicates that, at least in mice, the in vitro data are valid predictors of the in vivo contribution of P-gp: the contribution of P-gp to the distribution of the compound to the brain up to 60 min post i.v. administration. These results provide a rationale for predicting in vivo relevance of P-gp in human from in vitro data using human P-gp-expressing cells.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2003

Growth and maturation of oocytes in vitro.

The development of technologies to grow and mature oocytes from the most abundant primordial follicles holds many attractions for clinical practice, animal production technology and research. However, despite much research attention, it has proved difficult to grow follicles from early stages to maturity in vitro, as relatively little is known about the biology of oogenesis. It is clear that throughout oocyte development in vivo, follicle cell support is fundamental to provide the germ cell with nutrients and growth regulators to ensure progression through the protracted growth phase. Conversely, the oocyte actively promotes growth and differentiation of the follicular cells. Both of these characteristics must be mimicked in vitro. Replication of the normal follicular growth span from the primordial to Graafian follicle stages and the changes in the trophic requirements of the cells, cellular interactions, morphogenesis and the sheer increase in bulk as the antrum forms present major challenges for follicle culture technology. These observations could explain why methods that have proved successful for the culture of isolated rodent follicles are unable to support the growth of larger human and ruminant follicles in vitro and are incompatible with the requirements for primordial follicle growth activation. At present, the best option available for the complete growth and maturation of oocytes in vitro is to develop an extended multistage culture strategy which will provide a complex support system that closely resembles the ovary in vivo. In an attempt to achieve this goal primordial follicle growth is first initiated and maintained to the preantral stages through the culture of thin slices of ovarian cortex. The isolation and continued culture of these preantral follicles will support antral cavity formation and the induction of differentiated function in the somatic cell compartment. Finally, after exposure to an appropriate steroid milieu in vitro it should be possible to induce nuclear and cytoplasmic maturation in the fully grown oocytes. The prospects of succeeding at each stage, and of finally producing a fertile gamete, are likely to be increased by preserving cellular interactions and the phenotype of follicle cells as these provide the physiological environment in which oocytes develop. Although the technology for the in vitro maturation (IVM) of fully grown oocytes has been exploited successfully in ruminants, in human assisted reproduction IVM is still experimental as the efficiency of IVM is low and only a small number of pregnancies and live births have been reported. Thus, although complete in vitro growth and maturation may be achieved eventually, immediate goals must include the optimization of methods for isolating and culturing oocytes at both ends of the size spectrum and the full evaluation of the normality of the oocytes grown for extended periods in vitro.

Animals↗
PubMed2004

[Digestive and absorptive characteristic of starches determined in vitro and in vivo].

OBJECTIVE: To compare the different digestive and absorptive characteristic of digestible starch (DS) and resistant starch (RS), using both in vitro and in vitro methods. METHODS: Firstly, DS and RS content in four starchy samples were determined in vitro according to Englyst's method. Secondly, DS and RS digestion and absorption were evaluated by determination of four-hour blood glucose level, after direct injection of DS and RS solution into rats' duodenum. And in another animal test, the effect of RS on apparatus absorption rate of carbohydrate and nitrogen and secretion of gastric acid and pepsin was observed. RESULTS: In vitro, it was showed that general cornstarch was rich of DS, while 76% of potato starch was RS. In vitro, it was tested that after injection of DS into duodenum, most of DS could be absorbed in 120 min with high peak value of plasma glucose. While, RS needed more time to complete its absorption, and its apparatus absorption was up to 96%. It was also showed that RS could increase fecal nitrogen excretion, decrease nitrogen apparatus absorption rate and change gastric acid and pepsin excretion. CONCLUSION: By both in vitro and in vitro methods, RS is identified to be absorbed slowly but completely. The results partly support Englyst's method, which regards 120min as the time point to separate DS with RS in vitro.

Animals↗
PubMed2004

[Study on relationship of biodegradable properties of PGLA film in vivo and in vitro].

This experimental study on the biodegradable properties of poly(glycolide-co-lactide)[PGLA] film in vivo and in vitro was aimed to investigate the relationship between in vivo degradation process and in vitro degradation process. First, PGLA film was cut to 1 cm x 1 cm in size. It was put into artificial saliva and PBS solution respectively in vitro, and was implanted into subcutaneous tissue in Wistar rat in vivo. Then the mass loss rate was calculated every week not only for sample in vitro but also in vivo. The molecular weight was measured every two weeks. The results showed that the degradable speed of PGLA was faster in artificial saliva than in PBS solution. The change of molecular weight was earlier than that of mass loss rate. The degradable period in vitro was about 9-10 weeks. The degradable period in vivo was about 8 weeks. The rate of in vivo degradation was 1.33 times faster than that of in vitro. In conclusion, the degradation of PGLA in vitro was mainly a chemical degradation process achieved by hydrolysis of ester bond. The degradation of PGLA in vivo would be affected by stress and by biological factors, thus the degradation process was apparently faster in vivo, but both accorded with the degradation kinetics model of aliphatic polyester. There was some relationship of biodegradation between in vitro and in vivo.

Absorbable Implants↗
PubMed1985

[Chemotherapy testing for human ovarian cancer using in vitro colony assay].

The in vitro evaluation of anticancer drug efficacy was performed using the human tumor clonogenic assay developed by Hamburger and Salmon, and correlation between the in vitro and clinical efficacy was analyzed retrospectively. The in vitro colony assay method used in this study was a minor modification of the above method. Thirty-two out of forty-eight samples from patients with ovarian cancer formed more than five colonies per plate on in vitro colony assay. The median plating efficiency was 0.06% (range 0.02-1.3%) and the median colony count per plate was 279 (range: 8-4,000). With regard to colony formation of ovarian cancer according to the source of the specimen, the colony-forming rate of solid tumor was high (72%) as compared with 63% for ascites and 43% for pleural effusion. In vitro chemosensitivity was defined as more than a 50% decrease in colony formation and the rates for standard drugs on ovarian cancer were as follows: adriamycin (0.04 micrograms/ml) 29%, bleomycin (0.1 micrograms/ml) 24% cisplatin (0.2 micrograms/ml) 31%, 5-FU (1.0 micrograms/ml) 22%, hexamethylmelamine (1.0 micrograms/ml) 19%, L-PAM (0.4 micrograms/ml) 44%, mitomycin C (0.1 micrograms/ml) 38% and THP-adriamycin (0.5 micrograms/ml) 36%. A group of patients who had not been exposed to any anticancer drug showed higher sensitivity in vitro as compared with a group of patients who had received prior chemotherapy (35% vs 22%, p less than 0.05). Correlation between in vitro drug sensitivity and clinical responses in patients treated with the same drugs were analysed retrospectively. In all twenty cases, two were true positive cases (29%), while in ten cases, the results were true negative (77%), The overall predictive accuracy was 60%. In conclusion, ovarian cancer cells can form colonies well when the soft agar method is used and this assay method is suitable for the evaluation of various anticancer drugs in vitro.

Altretamine↗
PubMed1987

Defective lymphopoiesis in the bone marrow of motheaten (me/me) and viable motheaten (mev/mev) mutant mice. II. Description of a microenvironmental defect for the generation of terminal deoxynucleotidyltransferase-positive bone marrow cells in vitro.

We have presented evidence in a previous paper that the development of prothymocytes, pre-B cells, and TdT+ lymphoid precursor cells in the bone marrow of motheaten (me/me) and viable motheaten (mev/mev) mice is defective. In the present study, we have used a selective culture system that supports the generation of rat- and mouse-origin TdT+ bone marrow lymphoid cells in vitro to further investigate the early stages of lymphopoiesis in me/me and mev/mev mice. The results demonstrate that bone marrow stromal cell feeder layers derived from me/me and mev/mev mice do not support the growth of rat TdT+ cells in vitro, whereas stromal cell feeder layers from heterozygous (+/-) littermates and wild type (+/+) control mice do. Moreover, composite feeder layers formed by mixing as few as one part me/me and mev/mev bone marrow cells with 7 to 9 parts +/- littermate bone marrow cells also fail to effectively support the generation of TdT+ cells in vitro. In contrast to me/me and mev/mev mice, other mutant mouse models of autoimmune (NZB, NZB/W), immunodeficient (nu/nu), and hemopoietic (W/Wv, Sl/Sld) disorders form feeder layers that support normal to elevated levels of TdT+ cell growth in vitro. Thus, to date, only the me/me and mev/mev mutant mice have been found to lack the appropriate microenvironment for the generation of TdT+ bone marrow cells. Histologic analysis of the stromal cell feeder layers that are formed in our culture system shows that multilayered cellular patches, which normally are the most active sites of TdT+ cell development in vitro, are absent in feeder layers of me/me and mev/mev cells. Moreover, feeder layers from mev/mev mice contain a population of MAC 1+, basophilic, nonvacuolated, macrophage-like cells; whereas feeder layers from control mice contain MAC 1+, eosinophilic, vacuolated macrophage-like cells. Stromal cell feeder layers formed by mixtures of me/me or mev/mev and control mouse bone marrow cells contain numerous multilayered cellular patches and vacuolated mononuclear cells, but also contain large numbers of basophilic mononuclear cells. These composite feeder layers have a disproportionately reduced capacity to support the generation of TdT+ cells in vitro. Although the stromal microenvironment of me/me and mev/mev bone marrow does not support the growth of TdT+ cells in vivo or in vitro, the bone marrow from these mutant mice contains detectable numbers of pre-TdT+ cells. Thus, when cultured on normal mouse feeder layers, mutant mouse bone marrow rapidly generates TdT+ cells in vitro, albeit at significantly reduced levels as compared to +/- littermate controls.(ABSTRACT TRUNCATED AT 400 WORDS)

Acid Phosphatase↗
PubMed1987

Degenerative changes in the reproductive organs of female schistosomes during maintenance in vitro.

Degenerative alteration of the reproductive organs of female schistosomes in correlation with the change in egg-laying rate of schistosome pairs in vitro was studied by electron microscopy. The production of normal eggs by adult S. japonicum pairs decreased after 4 days in vitro followed by an increase of abnormal egg laying up to day 8. In S. mansoni, the yield of both normal and abnormal eggs decreased gradually from the start of maintenance in vitro in spite of a much higher pairing rate than in S. japonicum. The vitelline gland of 14-day in vitro-maintained S. japonicum stained with Fast red B, while that of S. mansoni did not. The ovary of both species exhibited regressive features after 14 days of maintenance in vitro. Ultrastructural examination showed that the vitelline cells and oocytes of S. japonicum and S. mansoni had already lost their structural integrity after 2 days in vitro and continued to exhibit signs of structural degeneration throughout the 14-day in vitro maintenance period. The regressive changes in reproductive potential of female S. mansoni maintained in vitro for 4 days could be reversed by surgically implanting the parasites into mouse mesenteric veins.

Animals↗
PubMed1985

Development of an in vitro microtest for determining the susceptibility of Plasmodium falciparum to sulfadoxine-pyrimethamine: laboratory investigations and field studies in Port-au-Prince, Haiti.

An in vitro microtest for assessing the susceptibility of Plasmodium falciparum to sulfadoxine-pyrimethamine (S-P) was developed following WHO guidelines. Paraaminobenzoic acid and folic acid were depleted in the culture medium used, the test wells were predosed with sulfadoxine and pyrimethamine at a constant ratio of 80:1, and the parasites were incubated for 48 hours. Optimum parasite multiplication was obtained with a 2% erythrocyte suspension in medium supplemented with 12% serum. During in vitro studies with laboratory-adapted isolates, response patterns were obtained which distinguished 3 isolates with documented in vivo sensitivity to S-P from 2 isolates with documented in vivo resistance to S-P. In addition, among the three S-P-sensitive isolates, one isolate that was pyrimethamine-resistant in vitro had a higher S-P inhibitory endpoint than 2 isolates that were pyrimethamine-sensitive in vitro. The S-P microtest was further evaluated in combined in vivo and in vitro studies in Port-au-Prince, Haiti. Twenty-six patients infected with P. falciparum were treated with standard doses of S-P, resulting in prompt clearance of parasitaemia, with no recurrence in the 24 patients who completed a 28-day follow-up period. Parallel in vitro tests with pyrimethamine alone showed 3 pyrimethamine-resistant isolates out of 22 successful tests on the patients' blood samples. In 23 successful S-P tests, the known in vivo S-P-sensitive parasites were inhibited at S-P concentrations that were generally lower for in vitro pyrimethamine-sensitive isolates than for in vitro pyrimethamine-resistant ones.

Animals↗
PubMed1985

Comparison of in vivo and in vitro cholinergic responses of normal and denervated canine hearts.

Ten dogs were subjected to total extrinsic cardiac denervation. The negative inotropic responses of their hearts were compared with those of nine normal controls with respect to acetylcholine (ACh; 0.1-1.0 micrograms) and nicotine (0.5-100 micrograms) administered intra-coronary. The dogs were on cardiopulmonary bypass and atrial and ventricular contractility were measured by means of a four-chamber isovolumic technique. At the end of this phase of the experiment, atrial strips were removed for in vitro evaluation of negative inotropic responses to Ach (1 X 10(-8) to 1 X 10(-6) M) and nicotine (1 X 10(-6) to 1 X 10(-4) M). Both in vivo and in vitro methods demonstrated a leftward shift of the dose-response curve for nicotine in denervated hearts, indicative of denervation hypersensitivity. Hypersensitivity to ACh was seen in atria in vitro but not in vivo. An additional 13 dogs were used only for in vitro studies. There was no difference in response between these dogs and those which had been used for both in vivo and in vitro studies. The results support the in vivo observation that the intrinsic cardiac neurons become supersensitive to nicotinic activation after extrinsic denervation. In addition, in vitro testing revealed atrial hypersensitivity to ACh which was not detected by in vivo methods. Inasmuch as the in vivo and the in vitro testing were done on the same animals, a method for determining equivalence for in vivo doses and in vitro concentrations is presented.

Acetylcholine↗
PubMed1982

[Chemosensitivity of adriamycin, mitomycin C, and bleomycin with in vitro colony assay].

In vitro colony assay was used to examine the sensitivities of adriamycin (ADM), mitomycin C (MMC), and bleomycin (BLM), and the effects of a prior chemotherapy on in vitro chemosensitivities of the drugs. One hundred forty-seven tumor specimens from 106 patients with various malignancies were placed in culture. Seventy tumors (56.0%) from 125 evaluable specimens which had histologically or cytologically confirmed to be malignant, and were plated at 5 x 10(5) cells/plate formed more than 30 colonies/plate enough for sensitivity assays. The results of in vitro sensitivities of the drugs were nearly similar to those reported in the clinical trials. The in vitro antitumor activity of BLM was noted in 3 (21.4%) of 14 patients with ovarian cancer, therefore the results suggest that the assay is useful to select effective drugs against tumors in which phase II study has never been performed. In vitro sensitivity of BLM was markedly reduced by a prior chemotherapy containing the same drug (p less than 0.05), and prior therapeutic ADM exposure also reduced in vitro activity of the drug, whereas there was no significant influence on in vitro activity of MMC. These results indicate the usefulness of the in vitro colony assay for predicting clinical antitumor activities of ADM, MMC, and BLM.

Bleomycin↗
PubMed1984

In vitro human lymphocyte responses to Cryptococcus neoformans. Evidence for primary and secondary responses in normals and infected subjects.

Cryptococcus neoformans causes meningitis and disseminated infection in healthy individuals, but more commonly in hosts with defective immune responses. To study the role of immune lymphocytes in protection and recovery from this infection, in vitro lymphocyte proliferative responses to whole killed organisms were characterized in normal controls and in patients with cryptococcosis. Several differences were found between these two groups. All normal individuals were found to have significant proliferative responses to cryptococci in vitro; however, patients recovering from infection had accelerated and augmented responses. Patients' T cells, but not control T cells, were found to produce interleukin 2 (IL 2) during initial stimulation in vitro. After in vitro priming, mononuclear cells from normal controls rechallenged with cryptococci in vitro demonstrated accelerated kinetics and IL 2 production similar to that of cells from recovering patients. Proliferative responses in normals required T cells and antigen-presenting cells, and resulted in an increase in the proportion of T8+ and Tac+ cells in the responding population by the ninth day of primary stimulation. An increase in Tac+ cells, but not T8+ cells, was found by the fourth day of secondary stimulation. These results demonstrate in vivo acquisition of T cell responsiveness to the organism in this small group of recovering patients, and in vitro acquisition of T cell recognition of this antigen by normal T cells during primary stimulation. The data suggest that these responses represent in vitro priming to cryptococci and indicate a role for the T8+ lymphocyte subpopulation, both unusual findings for previously described in vitro responses to soluble antigens.

Antigens, Fungal↗
PubMed1983

Clinical correlations of leukemic clonogenic cell chemosensitivity assessed by in vitro continuous exposure to drugs.

This study was performed to assess the value of prolonged, as opposed to short-pulse, in vitro exposure of leukemic cells to chemotherapeutic drugs in leukemic clonogenic assay for prediction of clinical response. In 21 patients with acute nonlymphocytic leukemia treated with intensive combination chemotherapy based on an anthracycline and 1-beta-D-arabinofuranosylcytosine infusion, chemotherapy sensitivity of leukemic clonogenic cells was assessed in comparison with that of normal myeloid clonogenic cells by the in vitro continuous exposure to drugs throughout the entire culture period. Analysis of these in vitro data in terms of prediction of achieving clinical complete remission was carried out in comparison with data on 22 cases in which in vitro sensitivity was assessed by the pulse 1-hr exposure. The in vitro sensitivity index, expressed as a log odds ratio, was positive (greater than 0) in 8 of 11 patients achieving complete remission and negative (less than 0) in 7 of 10 patients failing to achieve complete remission, with an overall correlation of 71%. This is at least as good as the pulse exposure method, which has a correlation of 68%. If sensitivity indexes of marginal magnitudes (--1.0 approximately +1.0) are excluded, the correlation increases to 92% (12 of 13 patients). The correlation appears to improve especially for 1-beta-D-arabinofuranosylcytosine by the continuous exposure method (71%) as compared with the pulse method (57%). This study establishes the feasibility of an in vitro chemotherapy sensitivity testing of leukemic clonogenic cells by continuous in vitro drug exposure and suggests that the continuous exposure method may be better than the pulse method for antimetabolites such as 1-beta-D-arabinofuranosylcytosine. The data also suggest that simulation of the in vivo drug schedule may be important in this in vitro test.

Adult↗
PubMed1984

Production of human-human hybridomas secreting antibody to sheep erythrocytes after in vitro immunization.

This report describes the formation of human hybridomas after in vitro immunization of tonsillar lymphocytes and fusion of the stimulated lymphocytes with a human lymphoblastoid cell line, WI-L279HF2. Lymphocytes were stimulated in vitro with sheep erythrocytes (SRBC) and polyclonal activators (PCA). By varying the duration of in vitro immunization before fusion and varying the PCA concentrations, the optimal conditions for cell fusion, hybrid cell growth, nonspecific immunoglobulin (Ig) secretion, and secretion of antibody to SRBC were determined. Lymphocytes cultured allogeneically at 5 X 10(6) cells per well in 24-well plates with SRBC and PCA (PWM, 1/10,000 dilution or LPS, 5 micrograms/ml) fused with the greatest efficiency. PWM-stimulated cultures fused on day 6 of in vitro stimulation yielded the greatest number of wells containing Ig-secreting hybridomas, whereas LPS-stimulated cultures produced more wells with hybridomas secreting Ig when fused on day 7 of stimulation. In vitro stimulation conducted in tissue culture flasks produced fewer wells with hybrid cell growth and Ig secretion. A fusion ratio of 1:1 (lymphocyte:WI-L2729HF2) yielded the most hybrid formation. Ig levels in wells with hybrid cell growth varied greatly, from less than 10 ng/ml up to 30 micrograms/ml. Ig of IgG, IgA, and IgM isotypes were produced. The addition of lymphocyte conditioned medium to in vitro immunization cultures does not improve the yield of Ig-secreting hybridomas. Hybrid cells secreting antibody to SRBC were cloned and analyzed for HLA specificities and chromosome numbers. Additionally hybridoma cells were formed after in vitro stimulation of peripheral blood lymphocytes (PBL) of normal individuals by fusion of the stimulated lymphocytes with WI-L2-729HF2. Hybridoma formation with PBL was less successful, but hybridoma cells secreting antibody to SRBC after in vitro stimulation were isolated.

Animals↗
PubMed1982

Biological characterization of cells derived from a human breast carcinoma. I. Some characteristics of cells cultivated in vitro prior to and after transplantation into nude mice.

Single cells from a patient (B. E., 65 years) have been isolated by collagenase treatment and cultivated in vitro. The human tumour of the mammary gland showed predominantly simple undifferentiated and also tubular structures. The 5th in vitro passage of these cells was characterized by DNA-distribution pattern, cell doubling time, chromosome number, ultrastructure, hormone and drug sensitivity. Cells of the 5th in vitro passage were i.p. transplanted into nude mice. The characteristics of in vitro cultivated cells (5th in vitro passage) were compared with both ascitic cells (1703/A) and cells of solid tumour material (1703/S) grown in nude mice for several passages (0, 10, 21). DNA-distribution patterns, chromosome numbers and cell doubling times are in good correlation. The number of polyploid cells is increased in ascitic cells. These malignant cells are best able to proliferate in vitro after transplantation into nude mice. Ultrastructure examination of the 21st passage has shown similarity between cultured cells, ascitic cells and cells of solid tumour material grown in nude mice. Virus particles could be observed in cells of solid only. Estrogen binding could be observed in the original tumour material only. All other cell or tissue preparations contained no receptors. Drug sensitivity was changed in the case of Vinblastin, Daunoblastin and Sarkolysin treatment of cells of solid tumour material more than in ascitic cells grown in nude mice. The environment dependence of cells and biological differences between in vivo-in vitro tumours and human neoplasms has to be taken into account when using cells as in vivo or in vitro model systems in experimental and clinical cancer research.

Aged↗
PubMed1977

Passive transfer of systemic tumor immunity with cells generated in vitro by a secondary immune response to a syngeneic rat gross virus-induced lymphoma.

Spleen cells taken from W/Fu rats 4 to 6 weeks after immunization with the syngeneic Gross virus-induced lymphoma, (C58NT)D cells, at a time when they lack detectable activity in a short-term 51Cr release assay, were previously shown to retain the capacity to generate cytotoxic activity upon reexposure to mitomycin C-treated lymphoma (C58NT)D cells in vitro. In the studies presented here, we evaluated whether in vitro sensitization of immune lymphoid cells before systemic transfer to a nonimmune recipient allows for more effective transfer of tumor immunity. The results show that the passive transfer of immune spleen cells after in vitro cocultivation with mitomycin-treated (C58NT)D cells allows for inhibition of growth of a subcutaneous inoculum of lymphoma cells. In contrast, spleen cells obtained 4 to 6 weeks after primary sensitization or after secondary in vivo sensitization did not effectively confer anti-tumor immunity. As few as 5 x 107 in vitro sensitized cells permitted complete inhibition of 106 (C58NT)D cells and also allowed for inhibition of the growth of 107 (C58NT)D-F cells, which was lethal to control animals. Immune cells sensitized with syngeneic thymocytes or normal spleen cells sensitized with (C58NT)D cells in vitro did not confer in vivo anti-tumor immunity. After systemic transfer of in vitro sensitized cells, delayed hypersensitivity occurred at the site of tumor inoculation and tumor growth was suppressed. Specificity of the passive immunity was shown by the failure to inhibit growth of a polyoma virus-induced sarcoma in rats which inhibited growth of the Gross virus-induced lymphoma cells. In vitro sensitized cells were more effective in the transfer of anti-tumor protection after 5 days, as compared to 2 days, of cocultivation with tumor. Results show that in vitro sensitized cells can effectively transfer systemic tumor immunity.

AKR murine leukemia virus↗
PubMed1993

Protein folding and assembly in vitro parallel intracellular folding and assembly. Catalysis of folding and assembly of the human chorionic gonadotropin alpha beta dimer by protein disulfide isomerase.

Although purified proteins will refold and assemble in vitro, it is not known if cellular factors change the mechanisms of these processes. Based on the gel migration of folding intermediates, the kinetic relationships between these intermediates, and on the order of formation of six disulfide bonds, we have found that the in vitro folding pathway of the human chorionic gonadotropin beta subunit (hCG-beta) is indistinguishable from the intracellular folding pathway. The same rate-limiting event was found in both folding environments; however, the t1/2 for this step in a cell is 4 min, whereas in vitro the t1/2 was > or = 80 min. Protein disulfide isomerase (PDI) increased the in vitro rate of this event (t1/2 = 25 min) without changing the order of disulfide bond formation. PDI also catalyzed the in vitro rate of assembly of hCG subunits. In intact cells, assembly of the alpha beta heterodimer occurs before all of the intramolecular disulfide bonds of beta are formed. In vitro, assembly was increased after reduction of two of the carboxyl-terminal disulfide bonds of hCG-beta by PDI. These results strongly suggest that both in intact cells and in vitro, partially unfolded hCG-beta is more assembly-competent than is fully folded hCG-beta. The comparison of in vitro and intracellular hCG-beta folding and hCG subunit assembly which is shown in this report indicates that the assisted folding and assembly pathway that occurs in cells, where proteins such as PDI play a role, differs only in rate but not in the order of disulfide bond formation or in the precursor-product relationships among the folding intermediates.

Animals↗