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PubMed2000

Relationship between permeability status of the blood-brain barrier and in vitro permeability coefficient of a drug.

OBJECTIVE: The aim was to test the hypothesis that the assessment of basal and drug-induced changes in permeability of the blood-brain barrier (BBB) during in vitro drug transport assays is essential for an accurate estimation of the permeability coefficient of a drug. METHODS: An in vitro BBB model was used, comprising of brain capillary endothelial cells (BCEC) and astrocytes co-cultured on semi-permeable filter inserts. Experiments were performed under control and challenged experimental circumstances, induced to simulate drug effects. The apparent BBB permeability coefficient for two markers for paracellular drug transport, sodium fluorescein (P(app,FLU), M(w) 376 Da) and FITC-labeled dextran (P(app,FD4), M(w) 4 kDa), was determined. Transendothelial electrical resistance (TEER) was used to quantify basal and (simulated) drug-induced changes in permeability of the in vitro BBB. The relationship between P(app) and TEER was determined. Drug effects were simulated by exposure to physiologically active endogenous and exogenous substances (i.e., histamine, deferroxamine mesylate, adrenaline, noradrenaline, bradykinin, vinblastine, sodium nitroprusside and lipopolysaccharide). RESULTS: P(app,FLU) and P(app,FD4) in control experiments varied from 1.6 up to 17.6 (10(-6)cm/s) and 0.3 up to 7. 3 (10(-6)cm/s), respectively; while for individual filters P(app, FLU) was 4 times higher than P(app,FD4) (R(2)=0.97). As long as TEER remained above 131.Omega cm(2) for FLU or 122.Omega cm(2) for FD4 during the transport assay, P(app) remained independent from the basal permeability of the in vitro BBB. Below these TEER values, P(app) increased exponentially. This nonlinear relationship between basal BBB permeability and P(app) was described by a one-phase exponential decay model. From this model the BBB permeability status independent permeability coefficients for FLU and FD4 (P(FLU) and P(FD4)) were estimated to be 2.2+/-0.1 and 0.48+/-0.03 (10(-6)cm/s), respectively. In the experimentally challenged experiments, a reliable indication for P(FLU) and P(FD4) could be estimated only after the (simulated) drug-induced change in BBB permeability was taken into account. CONCLUSIONS: The assessment of basal BBB permeability status during drug transport assays was essential for an accurate estimation of the in vitro permeability coefficient of a drug. To accurately extrapolate the in vitro permeability coefficient of a drug to the in vivo situation, it is essential that drug-induced changes in the in vitro BBB permeability during the drug transport assay are determined.

Animals↗
PubMed1985

In vitro formation of ookinetes and functional maturity of Plasmodium berghei gametocytes.

In vitro formation of Plasmodium berghei ookinetes was studied. Gametocytes produced in vitro were obtained from heart and tail blood of Swiss mice and from blood removed from mosquitoes directly after feeding on these mice. In vitro produced gametocytes were obtained from short-term cultures of the erythrocytic stages of P. berghei. Reproducible ookinete production was obtained in medium RPMI 1640, pH 7.8-8.0, using in vivo and in vitro produced gametocytes. The morphology of developmental stages of ookinetes and degenerate forms at the light microscope level is described. More ookinetes were produced in medium RPMI 1640 compared to MEM and ookinete yield--defined as the ratio between the number of in vitro produced ookinetes/10(5) erythrocytes and the number of exflagellations/10(5) erythrocytes in the infected blood--increased with lower erythrocyte densities in the cultures within the range of dilutions tested. A linear relationship existed between gametocytaemia and the number of ookinetes produced. The methods for in vitro ookinete formation and for estimating ookinete yields enabled us to study aspects of functional maturity of gametocytes independent of mosquitoes. The numbers of exflagellating gametocytes and in vitro ookinete yields in tail blood corresponded with those in heart blood and blood ingested by mosquitoes, suggesting a random distribution of functionally mature gametocytes within the vertebrate host.

Animals↗
PubMed1979

Nuclear transcription in vitro. Sensitivity to inhibition by ribosyldichlorobenzimidazole and rifamycin AF/013.

L cell nuclear preparations were shown to transcribe RNA for periods up to 1 h at 37 degrees C. Nearly 70% of the transcription products were sensitive to inhibition by 1 microgram/mL of alpha-amanitin, indicating that they were transcribed by RNA polymerase II. Analysis of polyphosphorylated termini of in vitro synthesized RNA showed the presence of a phosphatase activity which prevents quantitative recovery of these termini. The finding of in vitro labeled polyphosphorylated termini in RNA greater than 12 S after short periods of incubation shows initiation in vitro for this size class. The labeling of these polyphosphorylated termini is decreased in the presence of rifamycin AF/013. The use of two apparent inhibitors of initiation, rifamycin AF/013 and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), has allowed detection of in vitro initiated transcripts of heterogeneous nuclear RNA. Both of these inhibitors act primarily at later times of incubation, in contrast to alpha-amanitin which acts on elongation and inhibits in vitro RNA synthesis immediately. The selective pattern of DRB inhibition on hnRNA is retained in vitro and some accumulation of large-size molecules is observed. It can be estimated that about 30% of the greater than 12S hnRNA sequences transcribed in vitro are sensitive to DRB and 48% of greater than 12S RNA are sensitive to rifamycin AF/013 inhibition.

Cell Nucleus↗
PubMed2002

Lack of in vitro-in vivo correlation of a novel investigational anticancer agent, SH 30.

In solid tumors, the reasons for the lack of in vitro and in vivo correlation of drug activities are multifold and includes permeability to the tumor cells, interstitial hypertension and metabolic degradation. So, it is important to study the permeability and metabolic disposition of new compounds early in discovery and development of anticancer drugs. An experimental anti-cancer drug, SH 30 demonstrated highly selective and potent cytotoxic activity against a number of multi-drug resistant tumor cell lines in vitro. However, it was inactive in a murine tumor model. This study was conducted to identify the barriers that result in lack of correlation between in vitro and in vivo cytotoxic activity of novel anticancer agents. Two important barriers: physical (permeability) and metabolic (enzymatic inactivation) to poor delivery of SH 30 to solid tumors were investigated in this study. Tumors were sliced to separate the vascular and avascular sections. The concentrations of the drug at various regions of the tumor after single and multiple doses were investigated to determine the permeability barrier. The permeability barrier was also probed using two in vitro model systems, namely, matrigel films representing extracellular matrix and caco-2 multilayer cell cultures that simulate solid tumors. The drug and its metabolite concentrations were determined in the plasma and tumors to determine the metabolic barrier to the drug cytotoxic action. The metabolic barrier was further probed using in vitro mouse hepatocytes and liver microsome preparations. Our examination revealed the metabolic barrier to be the major contributor to the ineffectiveness of SH 30 in vivo. Examination of concentration of the drug across various regions of the tumor corroborated by data from in vitro permeation studies suggested that, for SH 30, permeability barrier did not exist. After single injection, the concentrations of SH 30 and its metabolites in plasma and tumor were comparable to another investigational drug with similar features (XK 469). Contrary to day 1, after 8 consecutive days of administration, SH 30 concentrations were significantly lower, while the metabolites concentrations were higher, suggesting extensive metabolism due to induction of enzyme(s). The in vitro hepatocytes and liver microsome results also showed SH 30 biotransformation to the same metabolites. Neither drug penetration, nor drug distribution into regions of the tumors distal to vasculature were impeded. The inactivity of SH 30 in vivo is primarily due to induction of extensive metabolism to inactive metabolites. This metabolism prevents adequate drug levels being achieved in the tumor.

Administration, Oral↗
PubMed1999

Relationship of the human cumulus-free oocyte maturational profile with in vitro outcome parameters after intracytoplasmic sperm injection.

PURPOSE: We investigated whether the human oocyte maturational profile at the removal of cumulus/corona cells affects the fertilization rate and subsequent embryo quality after intracytoplasmic sperm injection. METHODS: A total of 1011 oocytes from 150 cycles was included in this retrospective analysis. Cumulus-free oocytes that were in prophase or metaphase I of meiosis at the removal of cumulus/corona cells were incubated in vitro until they reached metaphase II (in vitro-matured oocytes) and were then immediately injected with a single spermatozoa. Oocytes that were in metaphase II at the removal of cumulus/corona cells (MII oocytes) received sperm injection after 3-4 hr of preinjection incubation. RESULTS: The fertilization rate of the MII oocytes was significantly higher than that of in vitro-matured oocytes (81 vs 62%; P < 0.001). The cleavage rates were similar in the two groups (MII oocytes, 94%; in vitro-matured oocytes, 91%). However, MII oocytes had significantly higher percentages of good-quality embryos (grade 1-3 embryos, 87 vs 58%, P < 0.001) and embryos with high cumulative embryo scores (score 10-32 embryos, 62 vs 33%, P < 0.001). The mean cumulative embryo score of MII oocytes after fertilization was also higher than that of in vitro-matured oocytes (12.1 +/- 3.8 vs 8.8 +/- 3.4; P = 0.014). CONCLUSIONS: MII oocytes that extruded the first polar body at the removal of cumulus/corona cells had better fertilization rates and embryo morphology than in vitro-matured oocytes that extruded the first polar body following the removal of cumulus/corona cells and in vitro culture.

Embryo, Mammalian↗
PubMed1975

Fresh explant culture of human tumours in vitro and the assessment of sensitivity to cytotoxic chemotherapy.

Ninety-seven fresh explants of human tumours have been cultured in vitro in an attempt to predict their sensitivity to subsequent cytotoxic chemotherapy. Only 3/26 solid ovarian tumours were cultured successfully although 12 of the 23 which failed to grow proved later to have benign histology. Of 10 solid tumours from other sites, only 2/4 renal tumours and one melanoma were successfully grown and tested in vitro. A higher success rate was achieved in culturing carcinoma cells of ovarian (10/22) and breast (10/22) origin from ascitic and pleural fluids. Using increase in cell number after 7 days' growth in vitro as the biological end-point, concentrations of cytotoxic drugs which are achievable in patients produced significant effects on some tumour explants. Detailed studies of serial subcultures in vitro from an ovarian tumour showed that large changes in chemosensitivity occur within about 2 passages, in vitro, so that sensitivity testing can only be carried out using fresh explants or their first subcultures if any possible correlation between in vitro cytotoxicity and in situ response is to be studied. Clinical effectiveness and in vitro chemosensitivity are compared for a limited number of patients with ovarian and breast carcinomata for whom follow-up information was available; no useful correlation was found.

Antineoplastic Agents↗
PubMed1978

In vitro induction of tumour-specific immunity V. Detection of common antigenic determinatnts of murine fibrosarcomas.

Two 3-methylcholanthrene and a spontaneous BALB/c fibrosarcoma were examined for tumour-associated antigens (TAA) by in vivo and in vitro induction of tumour-immune responses. When BALB/c mice were immunized to these fibrosarcomas by surgical tumour removal, cross-reacting tumour-associated transplantation antigens (TATA) were detected on all 3 tumours. Cytotoxic effector cells (CL) were then induced in vitro by co-culture of BALB/c spleen cells with the spontaneous, or one of the carcinogen-induced fibrosarcomas. These CL were shown to be cytotoxic T cells (Tc) and to be directed against cross-reacting TAA on all 3 tumours, by two in vitro 51Cr-release assay systems, direct 51Cr-release cytotoxicity and cellular competitive inhibition of 51Cr release. Further studies demonstrated that the fibrosarcoma TAA involved in in vitro induction of Tc were not present on normal adult or foetal tissues. A secondary cytotoxic response was also detected in vitro when spleen cells from mice immunized to a carcinogen-induced fibrosarcoma were tested. The patterns of cross-reactivity detected by the in vivo and primary in vitro tumour-immune responses suggested that the TAA detected in vivo (TATA) were not identical to the TAA detected in vitro.

Animals↗
PubMed1983

Studies on the in vivo production of a lymphokine activity, interleukin 3 (IL-3) elaborated by lymphocytes and a myeloid leukaemic line in vitro and the fate of IL-3 dependent cell lines.

Interleukin 3 (IL-3) is produced constitutively by WEHI-3b leukaemic cells and stimulated lymphoid cell populations in vitro. We have investigated the in vivo production of IL-3 in mice rendered leukaemic with WEHI-3b cells and mice stimulated by acute graft versus host disease (GVHD). In leukaemic mice, IL-3 was not found in serum or sonicates of 18-day spleens or bone marrow, although cells from the leukaemic organs were fully competent to elaborate IL-3 in vitro. Further, elaboration of IL-3 by WEHI cells in vitro was not affected by co-culture with normal haemopoietic cells. However, intracellular IL-3 was detected in leukaemic nodules isolated from the liver. Inhibitors specific for IL-3 were not found, although liver-cell conditioned medium and leukaemic nodule sonicates contained potent non-specific inhibitors of cell growth. At 21 days, intracellular IL-3 was also present in spleens and correlated with the presence of non-specific inhibitors. In GVHD, no evidence for IL-3 elaboration in vivo was found, nor did lymphoid populations affected by GVHD spontaneously elaborate it in vitro; however, their competence to produce it was unaffected, as IL-3 was elaborated on subsequent mitogen stimulation in vitro. We also investigated the recovery and circulation of in vitro 111Indium-labelled IL-3 dependent cells after injection in vivo and the half-life of semi-purified IL-3. Dependent cells were not recovered after injection into irradiated recipients, although the cells recirculated for at least 24 hours. Inability to recover dependent cells was explicable on general cytotoxicity which masked potential recovery. The serum half-life of injected partially purified material with IL-3 activity was short (less than 30 min). We conclude that the elaboration of IL-3 by leukaemic WEHI-3b is not an in vitro artifact and these results are discussed in relationship to other growth factors and the leukaemic state, and the origin of IL-3 dependent lines.

Animals↗
PubMed1998

In vitro invasion of small-cell lung cancer cell lines correlates with expression of epidermal growth factor receptor.

Formation of metastasis is a multistep process involving attachment to the basement membrane, local proteolysis and migration into surrounding tissues, lymph or bloodstream. In the present study, we have analysed the correlation between in vitro invasion and presence of the epidermal growth factor receptor (EGFR) in a panel of 21 small-cell lung cancer (SCLC) cell lines. We have previously reported that ten of these cell lines expressed EGFR protein detected by radioreceptor and affinity labelling assays. In 11 small-cell lung cancer (SCLC) cell lines, EGFR mRNA was detected by Northern blot analysis. In vitro invasion in a Boyden chamber assay was found in all EGFR-positive cell lines, whereas no invasion was detected in the EGFR-negative cell lines. Quantification of the in vitro invasion in 12 selected SCLC cell lines demonstrated that, in the EGFR-positive cell lines, between 5% and 16% of the cells added to the upper chamber were able to traverse the Matrigel membrane. Expression of several matrix metalloproteases (MMP), of tissue inhibitor of MMP (TIMP) and of cathepsin B was evaluated by immunoprecipitation, Western blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR). However, in vitro invasive SCLC cell lines could not be distinguished from non-invasive cell lines based on the expression pattern of these molecules. In six SCLC cell lines, in vitro invasion was also determined in the presence of the EGFR-neutralizing monoclonal antibody mAb528. The addition of this antibody resulted in a significant reduction of the in vitro invasion in three selected EGFR-positive cell lines. Our results show that only EGFR-positive SCLC cell lines had the in vitro invasive phenotype, and it is therefore suggested that the EGFR might play an important role for the invasion potential of SCLC cell lines.

Antibodies, Monoclonal↗
PubMed1992

Effect of sodium stibogluconate and pentamidine on in vitro multiplication of Leishmania donovani in peritoneal macrophages from infected and drug-treated BALB/c mice.

The in vitro antileishmanial activity of sodium stibogluconate (SSG) and pentamidine in peritoneal macrophages from three different groups of animals (i.e. normal, Leishmania donovani-infected and drug-treated BALB/c mice) is reported. Peritoneal macrophages were extracted from all these animals and infected in vitro with L. donovani promastigotes. After 24 h, the infected macrophages (with amastigotes) were exposed to various concentrations of SSG (10-90 micrograms/mL) and pentamidine (0.1-5.0 micrograms/mL). The in vitro infection could be cured readily with 80 micrograms/mL of SSG and 4 micrograms/mL of pentamidine in macrophages from normal animals. But even higher dosages of these drugs added in vitro could not reduce the amastigote loads in macrophages from infected animals. In contrast, incubation in vitro of infected macrophages with very low dosages of these drugs (40 micrograms/mL of SSG and 1.0 mu/mL of pentamidine) could eliminate the parasites present within macrophages obtained from drug-treated animals. This was probably because the macrophages from drug-treated animals tackled the parasites themselves by their microbicidal mechanisms and the in vitro infection was tackled by the drug in vitro. This implies that a well-developed specific immunity in leishmaniasis helps in the antileishmanial activity of these drugs.

Animals↗
PubMed2002

Beta-lactamase protein fragment complementation assays as in vivo and in vitro sensors of protein protein interactions.

We have previously described a strategy for detecting protein protein interactions based on protein interaction assisted folding of rationally designed fragments of enzymes. We call this strategy the protein fragment complementation assay (PCA). Here we describe PCAs based on the enzyme TEM-1 beta-lactamase (EC: 3.5.2.6), which include simple colorimetric in vitro assays using the cephalosporin nitrocefin and assays in intact cells using the fluorescent substrate CCF2/AM (ref. 6). Constitutive protein protein interactions of the GCN4 leucine zippers and of apoptotic proteins Bcl2 and Bad, and the homodimerization of Smad3, were tested in an in vitro assay using cell lysates. With the same in vitro assay, we also demonstrate interactions of protein kinase PKB with substrate Bad. The in vitro assay is facile and amenable to high-throughput modes of screening with signal-to-background ratios in the range of 10:1 to 250:1, which is superior to other PCAs developed to date. Furthermore, we show that the in vitro assay can be used for quantitative analysis of a small molecule induced protein interaction, the rapamycin-induced interaction of FKBP and yeast FRB (the FKBP-rapamycin binding domain of TOR (target of rapamycin)). The assay reproduces the known dissociation constant and number of sites for this interaction. The combination of in vitro colorimetric and in vivo fluorescence assays of beta-lactamase in mammalian cells suggests a wide variety of sensitive and high-throughput large-scale applications, including in vitro protein array analysis of protein protein or enzyme protein interactions and in vivo applications such as clonal selection for cells expressing interacting protein partners.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2005

A novel in vitro system for gamete fusion in maize.

Various systems by using electric pulse, calcium, or polyethylene glycol have been developed in the past decade for the in vitro fusion of plant gametes. These in vitro systems provide a new way to study the fertilization mechanisms of plants. In this study, we developed a bovine serum albumin (BSA)-mediated fusion system for the in vitro fusion of maize gametes. The in vitro fusion of the isolated single egg cell and sperm cell of maize was observed microscopically in the BSA solution and the fertilized egg cell showed normal cell wall regeneration and nuclear division. The effects of the BSA concentration, pH value and calcium level on the efficiency of the maize gamete fusion were also assessed. BSA concentration and pH value did significantly affect the efficiency of the gamete fusion. Calcium was not necessary for the gamete fusion when BSA was present. The optimal solution for the gamete fusion contained 0.1% BSA, pH 6.0. The fusion frequency was as high as 96.7% in that optimal solution. This new in vitro fertilization system offers an alternative tool for the in vitro study of fertilization mechanisms with much simpler manipulating procedure than PEG system, and it will be especially useful for the in vitro study of the calcium dynamics during plant fertilization.

Botany↗
PubMed2003

In vitro follicular growth affects oocyte imprinting establishment in mice.

In vitro folliculogenesis of cryopreserved ovarian tissue could be an effective method for insuring fertility for patients who receive gonadotoxic treatment. Although several culture systems have been described for growing female gametes in vitro, the production of competent oocytes for further development remains a considerable challenge. The purpose of our study was to determine whether maternal primary imprinting progresses normally during mouse oocyte growth in vitro. We analysed the DNA methylation status of differentially methylated regions of the imprinted genes H19, Mest/Peg1 and Igf2R using fully grown germinal vesicle-stage oocytes (fg oocytes) produced by in vitro folliculogenesis from early preantral follicles. When compared to fg oocytes removal from control females, we observed after in vitro development, a loss of methylation at the Igf2R locus in six out of seven independent experiments and Mest/Peg1 locus (one out of seven), and a gain of methylation at the H19 locus (one out of seven). These results provide insight into the dysregulation of the process of primary imprinting during oocyte growth in vitro and highlight the need for effective new biomarkers to identify complete nuclear reprogramming competence after in vitro folliculogenesis.

Animals↗
PubMed2001

Quantitative assessment of primary skin irritants in vitro in a cytotoxicity model: comparison with in vivo human irritation tests.

BACKGROUND: While great efforts have been made in recent years to develop in vitro methods for assessing skin irritation potential, there are relatively few data that correlate in vitro data with in vivo data. OBJECTIVES: To expand our previously reported investigations on in vitro vs. in vivo correlation of a series of homologous N-alkyl sulphates of different alkyl chain length to include primary skin irritants of different chemical classes. METHODS: Anionic surfactants (three different sodium alkyl sulphonates and sodium lauryl sulphate), cationic surfactants (three alkyl trimethyl ammonium bromides), non-ionic surfactants (polyoxyethylene-20-cetyl ether and Tween 20), benzoic acid, dimethyl sulphoxide and phenol were chosen as model irritants. A spontaneously immortalized human keratinocyte line, HaCaT, was used as an in vitro model to predict the cutaneous irritation. The end-point used to assess toxicity was uptake of the vital dye neutral red (NR) 24 h after dosing. The cytotoxicity data from these assays were compared with the irritant responses (as evaluated by measurement of erythema and transepidermal water loss) obtained after 24-h application of the same compounds (100 microL of 20 mmol L(-1) aqueous solution) to the volar forearm of human volunteers. RESULTS: All tested irritants had cytotoxic effects as demonstrated by a decreased NR uptake, which showed a clear dose-response relationship. Concentrations resulting in 50% inhibition of NR uptake (IC(50)) ranged from 8 micromol L(-1) (hexadecyl trimethyl ammonium bromide) to 328 mmol L(-1) (dimethyl sulphoxide). We found a good overall correlation between in vitro cytotoxicity (NR uptake IC(50) values) and in vivo irritation potential in humans. Only the high molecular weight compounds Tween 20 and polyoxyethylene-20-cethyl ether were problematic, as their irritation potential was overestimated by the in vitro assay. This non-conformity of these high molecular weight (> 1000) compounds was expected, and can be largely attributed to the epidermal permeability barrier. The epidermal barrier, which greatly limits the percutaneous penetration of xenobiotics in vivo, does not exist in cell culture models. CONCLUSIONS: The in vitro cytotoxicity model is a useful screening tool, but data should be interpreted critically and require confirmation by appropriate in vivo studies.

Cell Culture Techniques↗
PubMed1996

In vitro immunization of naive human B cells yields high affinity immunoglobulin G antibodies as illustrated by phage display.

In vitro antibody responses to a synthetic immunogen, consisting of both a B cell [V3 loop of gp120 from human immunodeficiency virus type 1 (HIV-1)] and a T-helper epitope (15 amino acids of tetanus toxoid) was studied. The in vitro activation was performed by primary and secondary in vitro immunizations, using lymphocytes obtained from uninfected, seronegative donors. Analysis of the in vitro immune response demonstrated an antigen-specific isotype switch, which was dependent on the presence of antigen-specific T-helper cells, CD40 ligation and antigen. Antibody libraries were constructed from cells derived directly from the naive donors, or from primary or secondary in vitro immunized B cells. Five libraries were displayed on filamentous phage and selected for anti-V3-specific Fab fragments, using a selection approach that linked recognition and phage replication. A panel of 19 recombinant antigen-specific Fab. representing different phases of the humoral in vitro immune response were sequenced, expressed and analysed using a biosensor. Recombinant Fab fragments derived from cultures on day 12 exhibited an increase in affinity of close to two orders of magnitude compared to those obtained from cells primary immunized for 7 days. This study provides the first evidence that an antigen-specific in vitro immune response can yield high-affinity immunoglobulinG antibodies.

Amino Acid Sequence↗
PubMed1999

The diagnostic role of the in vitro drug-induced interferon-gamma release test in Stevens-Johnson syndrome.

BACKGROUND: Drug-related T-cell activity in cutaneous drug reactions may be assessed by in vitro cytokine release tests. The diagnostic role of in vitro drug-induced interferon-gamma (IFN-gamma) release was evaluated in a patient with Stevens-Johnson syndrome. CASE REPORT: Stevens-Johnson syndrome was diagnosed in a 58-year-old man, treated with colchicine (1 mg daily for 39 days) and allopurinol (300 mg daily for 13 days). Based on a clinical-epidemiologic score, allopurinol was more likely to be the causative agent. In vitro drug-induced IFN-gamma release test was conducted on this patient and on two controls, using an enzyme-linked immunoabsorbent assay (ELISA) technique. Increased IFN-gamma release was observed following an in vitro challenge of the patient's lymphocytes with allopurinol, but not following in vitro challenge with colchicine. An in vitro challenge with allopurinol in two control patients, treated with allopurinol without adverse drug reactions, did not induce a significant increase in IFN-gamma release. CONCLUSIONS: The role of allopurinol as the drug responsible for the induction of Stevens-Johnson syndrome in our patient was confirmed by in vitro allopurinol-induced IFN-gamma release, which may indicate a drug-specific immune response.

Allopurinol↗
PubMed1998

Naïve, hapten-specific human T lymphocytes are primed in vitro with derivatized blood mononuclear cells.

Our goal is to develop an in vitro system for priming naïve, hapten-specific human T cells similar to the effector cells that mediate contact hypersensitivity in vivo. Using strategies based on results of experiments conducted with murine cells, we stimulated peripheral blood T cells from normal human beings in vitro with peripheral blood mononuclear cells that had been pretreated with granulocyte-macrophage colony stimulating factor and then derivatized with dinitrochlorobenzene. T cells activated in this manner in vitro were compared functionally with immune T cells from individuals sensitized to dinitrochlorobenzene. We found that dinitrochlorobenzene-derivatized fresh peripheral blood mononuclear cells induced significant proliferation among immune T cells. Large amounts of interferon-gamma, but no interleukin-4, were detected in supernatant of these cultures. More interestingly, a significant number of T cells were activated when nave, hapten-specific T cells were stimulated with dinitrochlorobenzene-modified, cultured autologous peripheral blood mononuclear cells. Again, copious amounts of interferon-gamma were detected in the culture supernatants. Furthermore, this subset of T cells proliferated and secreted interferon-gamma, but not interleukin-4, when restimulated in vitro with dinitrochlorobenzene-derivatized fresh peripheral blood mononuclear cells. In these regards, in vitro activated T cells functionally resembled T cells that were harvested from the blood of individuals previously sensitized with dinitrochlorobenzene. This in vitro system for activation of naive, hapten-specific T cells offers opportunities on the one hand to study the earliest T cell and antigen-presenting cell events in the induction of human contact hypersensitivity, and on the other hand to simulate in vitro therapies with molecules designed to abort or prevent contact hypersensitivity when it causes disease.

Animals↗
PubMed2000

Cyclical changes in rat vibrissa follicles maintained In vitro.

In mammals hair growth is cyclical; however, the factors that regulate the hair growth cycle are still poorly understood. The recent development of methods for culturing hair follicles in vitro has proved an important tool to investigate many aspects of the regulation of hair follicle growth. At present, however, these models are based on the culture of anagen hair follicles and have only partially been used to address the cyclical nature of hair growth. In this study we have made use of the fact that in rodents the hair growth cycle is synchronized, well characterized, and relatively short. We have isolated vibrissa follicles from 12 d old rats and confirmed by histology that these follicles are in the anagen stage of their first hair growth cycle. We have then maintained these follicles in vitro, on Gelfoam supports, for up to 23 d (35 d of age) and compared their histology with in vivo follicles from equivalent age littermates. We observed that 12 d old follicles maintained in vitro for up to 23 d show changes in morphology that suggest that cultured rat vibrissa follicles retain cyclical activity in vitro. Cyclical changes in hair follicle morphology were only seen in follicles maintained on gelfoam supports and moreover, hair follicle size appears to be a key feature in determining the ability of the follicle to cycle in vitro. All follicles that showed cyclical changes in vitro, however, appeared to remain blocked in pro-anagen. These data suggest that the vibrissa follicle is a in vitro good model system with which to investigate hair cycle control. J Invest Dermatol 115:1152-1155 2000

Animals↗