The immunogenicity of dendritic cell-derived exosomes

Exosome production represents an alternate endocytic pathway for secretion. Multivesicular endosomes (MVE) fuse with the plasma membrane expelling internal vesicles or exosomes from cells. Exosome production has been recently described for immune cells including B cells, dendritic cells (DC), mast cells, macrophages and T cells. Exosomes derived from some DC populations stimulate T lymphocyte proliferation in vitro and have potent capacity to generate anti-tumour immune responses in vivo. These reported studies have involved in vitro grown mature DC expanded from precursors with cytokines. However, immature DC produce higher numbers of exosomes than mature DC and this is thought to be due to a reduction in endocytosis as DC mature, associated with reduced reformation of MVE and reduced exosome formation. This lab pioneered a method to generate immature DC in spleen long-term cultures (LTC). DC produced in cultures represent immature myeloid DC, highly endocytic but with weak capacity to stimulate T cells. LTC-DC produce exosomes and contain many MVE. This prompted a study of immunogenic potential with a view to the potential use of exosomes in vaccination and immunotherapy. DC produced in cultures represent immature myeloid DC, highly endocytic but with weak capacity to stimulate T cells. Exosomes were isolated by differential centrifugation from LTC-DC and shown by marker expression to arise by budding from the LAMP-1+ limiting endosomal membrane of MVE. These LTC-derived exosomes appear however to lack immunostimulatory markers like CD86, CD40, MHC-I and MHC-II. While LTC-DC can stimulate antigen-specific proliferation of CD4+ T cells, exosome preparations derived from antigen-pulsed DC were unable to stimulate purified na?ve T cells in vitro. They were however found to weakly activate allogeneic CD8+ T cells in vitro. Tumour antigen-pulsed LTC-DC or their exosomes could induce a protective response in mice against growth of a transplanted tumour but could not induce a response to clear an existing tumour. Exosomes derived from immature DC can modulate immune responses, but do not function in direct T cell activation in vitro. Modulation of immune responses by exosomes produced by immature DC may be dependent on the presence of other antigen presenting DC subsets in the animal. The possible function of immature DC and their exosomes in maintenance of tolerance and in the induction of immunity is discussed.     

复方861对肝星状细胞的增殖和凋亡的干预作用

目的 研究ＨＳＣ在体外和体内的增殖和凋亡及中药的干预作用。方法 体外研究对象为ＨＳＣ系。结果增殖采用ＭＴＴ比色法,细胞凋亡采用电镜观察,流式细胞仪和ＴＵＮＥＬ法检测。临床研究对象是量慢性乙型肝炎患者。结果 复方８６１显著抑制体外培养的ＨＳＣ增殖,随着药物剂量的增加和时间的延长,ＨＳＣ的凋亡明显增多,凋亡率增加,呈剂量和时间依赖,ＴＵＮＥＬ法检测,用５ｍｇ／ｍｌ复方８６１作用４８ｈ二,ＨＳＣ凋亡率为     

Growth and differentiation of B lymphocytes of patients with juvenile rheumatoid arthritis

We studied growth and differentiation of B lymphocytes of patients with juvenile rheumatoid arthritis (JRA) using B cell enriched populations. Mitogen stimulation led to similar proportionate increases in proliferation and immunoglobulin (Ig) secretion in cultures of patient and control lymphocytes. While there was no increase in proliferation, IgG secretion was increased in the absence of mitogen. Nonmitogen activated Ig synthesis could be reduced by replacing culture medium with fresh medium after 16-20 h in culture. It was partly reconstituted by addition of recombinant cytokines, interleukin (IL), IL-2, IL-4, or IL-6. Our results suggest there may be a population of B circulating B cells in patients with JRA and other rheumatic diseases which is sufficiently mature to differentiate and secrete Ig in response to cytokines alone.     

Effect of transforming growth factor-beta 1 on proliferation and death of rat prostatic cells

The ability of transforming growth factor B1 (TGF beta 1) to inhibit proliferation and activate death of rat ventral prostatic glandular cells was tested both in vivo and in vitro. In vivo administration of 50 ng TGF beta 1/day directly to the regressed ventral prostate of previously castrated male rats had no effect on the proliferative regrowth of the prostatic glandular cells induced by exogeneous androgen replacement. In addition, androgen-stimulated ventral prostatic cell proliferation in vitro in organ culture was not affected by exposure to 0.1-20 ng/ml TGF beta 1. In contrast in vivo administration of 50 ng TGF beta 1/day directly to the ventral prostate of intact noncastrated male rats resulted in the death of about 25% of the prostatic glandular cells within 7 days of treatment. Such TGF beta 1 treatment did not lower serum testosterone, nor did it affect the size or DNA content of the seminal vesicles, demonstrating the local nature of the response. Likewise, in androgen-maintained ventral prostate organ cultures in vitro, there was a dose-response relationship between glandular cell death and TGF beta 1 concentration in the medium. These results demonstrate that TGF beta 1 can induce the death of androgen-dependent prostatic glandular cells even when physiological levels of androgen are present. Previous studies have demonstrated that both the receptor and the mRNA for TGF beta 1 increase rapidly in the ventral prostate after castration. Taken with the present data, these results suggest that TGF beta 1 may be a physiological intermediate in the programmed cell death of rat prostatic glandular cells activated after androgen ablation.     

Detection and characterization of apoptotic peripheral blood lymphocytes in human immunodeficiency virus infection and cancer chemotherapy by a novel flow immunocytometric method

We have developed a quantitative and sensitive flow cytometric method for the detection of human apoptotic lymphocytes that, unlike previously described assays, allows their identification in mixed populations of peripheral blood leukocytes as well as their immunophenotyping. Apoptotic lymphocytes are identified on the basis of peculiar light scatter changes, reflecting their smaller size and their modified nucleus/cytoplasm organization, and of the decreased expression of surface CD45 molecules. Based on these criteria, apoptotic lymphocytes generated by exposure to ionizing radiation can be easily distinguished from viable cells and from necrotic lymphocytes generated by treatment with antibody and complement. Using this assay, we reappraised the phenomenon of the in vitro apoptosis of lymphocytes from patients with human immunodeficiency virus (HIV) infection. Lymphocytes from HIV patients, unlike those from normal HIV-negative subjects, undergo apoptosis upon simple in vitro culture. We found that the percentages of lymphocytes undergoing apoptosis were significantly higher in patients with low CD4 cell counts (< 400/microL) than in patients at earlier stages (> 400 CD4 cells/microL). However, phenotypic analysis disclosed that apoptotic lymphocytes generated in these cultures were mostly CD8+ T cells and CD19+ B cells. Thus, in contrast to what has been previously suggested, the phenomenon of in vitro lymphocyte apoptosis might not be pathogenetically related to the depletion of CD4+ T cells in acquired immunodeficiency syndrome. Nevertheless, it might represent an useful marker of disease progression. Our assay allows the analysis of unfractionated peripheral blood leukocytes and thus the identification of apoptotic lymphocytes circulating in vivo. Apoptotic lymphocytes could indeed be detected in the circulation of a patient with cancer shortly after high-dose cytotoxic chemotherapy. By contrast, no apoptotic lymphocytes could be detected in vivo in patients with early or advanced HIV infection.     

Growth factor-mediated proliferation in B cell non-Hodgkin's lymphomas

The non-Hodgkin's lymphomas (NHLs) are a heterogeneous group of human lymphoid tumors, primarily of B cell lineage, which appear to represent arrested stages in B lymphocyte differentiation. Control of cell proliferation is a fundamentally important but poorly understood area of study in these tumors. We have studied a representative group of B cell NHLs to assess their potential for growth factor-mediated proliferation in vitro. Our results show that purified monoclonal NHL B cells of the small cell (well-differentiated lymphocytic lymphoma, nodular poorly differentiated lymphocytic lymphoma, etc) type, that were positive for the human malignancy-associated nucleolar antigen could be stimulated by human B cell growth factor (BCGF) to proliferate in vitro. Other B cell activators such as insoluble anti-Ig and the mitogen protein A also could stimulate thymidine incorporation in the lymphoma cell populations. In vitro lymphoma cell growth could be maintained in the presence of the growth factor for up to five weeks. The large B cell type NHL, however, appeared to be refractory to in vitro stimulation by BCGF as well as other stimulators of normal B cells. These studies suggest that human B cell lymphoid tumors are not only phenotypically similar to their normal B lymphocyte counterparts, but are also sensitive in some cases, to the same types of immunoregulatory molecules that control normal lymphoid cell growth.     

Rickettsia rickettsii infection of cultured human endothelial cells induces tissue factor expression

Microvascular thrombi underlie many of the clinical manifestations of Rocky Mountain spotted fever (RMSF), a disease characterized by Rickettsia rickettsii infection of vascular endothelial cells. Studies were designed to determine whether R rickettsii-infection of cultured human umbilical vein endothelial cells results in tissue factor (TF) induction, a process that could directly activate coagulation in infected vessels. Whereas uninfected endothelial cell cultures showed essentially undetectable TF mRNA and activity, both TF mRNA and activity were present after R rickettsii infection. TF mRNA levels were transient, peaking at 4 hours after the initiation of infection, whereas the peak of TF activity occurred at 8 hours. Induction of the TF response requires the intracellular presence of R rickettsii organisms, because uninfected rickettsia were ineffective and the response was blocked by inhibiting rickettsial entry using cytochalasin B. TF induction was not mediated by endothelial cell release of soluble factor, because no response was induced using culture medium conditioned by R rickettsii-infected cells. Furthermore, preadsorption of suspensions of R rickettsii with polymyxin B to remove contaminating lipopolysaccharide did not eliminate the TF response. Induction of TF in vital endothelial cells during R rickettsii infection could be the trigger for vascular thrombus formation of RMSF.     

The relationship between in vitro cellular aging and in vivo human age.

Differences between early and late passage cell cultures on the organelle and macromolecular levels have been attributed to cellular "aging". However, concern has been expressed over whether changes in diploid cell populations after serial passage in vitro accurately reflect human cellular aging in vivo. Studies were therefore undertaken to determine if significant differences would be observed in the in vitro lifespans of skin fibroblast cultures from old and young normal, non-hospitalized volunteers and to examine if parameters that change with in vitro "aging" are altered as a function of age in vivo. Statistically signigificant (P less than 0.05) decreases were found in the rate of fibroblast migration, onset of cell culture senescence, in vitro lifespan, cell population replication rate, and cell number at confluency of fibroblast cultures derived from the old donor group when compared to parallel cultures from young donors. No significant differences were observed in modal cell volumes and cellular macromolecular contents. The differences observed in cell cultures from old and young donors were quantitatively and qualitatively distinct from those cellular alterations observed in early and late passage WI-38 cells (in vitro "aging"). Therefore, although early and late passage cultures of human diploid cells may provide an important cell system for examining loss of replicative potential, fibroblast cultures derived from old and young human donors may be a more appropriate model system for studying human cellular aging.     

Clonally related but phenotypically divergent human cancer cell lines derived from a single follicular thyroid cancer recurrence (TT2609).

Starting from different regional samples taken from a heterogeneous follicular thyroid cancer recurrence in a male patient, a series of cell cultures was initiated. Three stable cancer cell lines were successfully established (TT2609-A02, TT2609-B02, and TT2609-C02) and kept in continuous culture for more than 3 years. The lines are each characterized by a unique set of biological parameters such as morphology, ploidy state, cell proliferation rate, ultrastructure, thyroid marker expression, p53 expression, karyogram, agar clonogenic capacity and tumorigenicity as xenografts in nude mice. These characterization studies point to a marked heterogeneity at the level of the clinical tumor recurrence. Karyotype analysis of the cell lines showed a pattern of aberrations indicating that the lines are clonally related and that the A02 and C02 lines are subsequently derived from the more "original" tumor cell type B02 after a tetraploidization event. It is concluded that the obtained cell lines represent an in vitro/in vivo model for human follicular thyroid cancer. The availability of a series of cell lines for human follicular thyroid cancer, mimicking the biological heterogeneity observed in patient tumors, enables both detailed fundamental investigation of thyroid cancer cell biology and the experimental exploration of new treatment approaches.     

Enucleation of differentiated murine erythroleukemia cells in culture.

Friend murine leukemia cells induced to undergo erythrocytic differentiation by dimethyl sulfoxide give rise to progeny resembling ortho- or polychromatic normoblasts, which usually do not complete the maturation process to yield forms analogous to erythrocytes. Treatment of these differentiated cells with cytochalasin B can lead to a high proportion (i.e., 80-85%) of enucleated cells in vitro. Nuclear extrusion in cells induced to differentiate by dimethyl sulfoxide and subsequently treated with cytochalasin B began within 24-36 hr of exposure to the antibiotic, with the appearance of a pre-enucleated stage in which the cell nucleus became pycnotic and eccentrically located. Maximum enucleation occurred after 72-96 hr of exposure to cytochalasin B and was significantly enhanced when dimethyl sulfoxide was included in the culture medium during the period of treatment with cytochalasin B. Enucleation appeared to take place only in differentiated progeny, because nondifferentiated cells treated with cytochalasin B alone yielded a population of multinucleated cells. The findings indicate that highly tumorigenic nondifferentiated Friend erythroleukemia cells can be converted in high yield to mature enucleated forms that are unable to proliferate in vitro.     

Interleukin-4 inhibits apoptotic cell death and loss of the bcl-2 protein in B-chronic lymphocytic leukaemia cells in vitro

Summary. When monoclonal B cells from B-chronic lymphocytic leukaemia (B-CLL) patients are cultured in vitro , they die by apoptosis. Apoptotic cell death occurred in the B cells from 20/24 B-CLL patients after 26–30 h in in vitro culture, with 14.3–59.0% (mean 33.6%) of their DNA being fragmented in ∼180 base pair multimers. After 8–10 d culture, 90–100% of the B-CLL cells were dead. Cell death and DNA fragmentation were inhibited in the presence of 0.5–5 ng/ml human recombinant interleukin-4 (IL-4) and viable monoclonal B cells could be maintained in culture up to 3 weeks. At 5 ng/ml. IL-4 reduced DNA fragmentation after a 26–30 h culture to 2.2–33.3% (mean 14.9%). IL-4 inhibited apoptosis without stimulating cell proliferation. In four patients the cells were resistant to apoptosis in vitro and they could be maintained for up to 4 weeks in culture medium alone. DNA fragmentation in all patients was increased in the presence of the RNA synthesis inhibitor actinomycin-D. Western blot analysis of cell lysates showed expression of the bcl-2 protein in all 11 B-CLL patients studied. However, during culture, bcl-2 protein levels were preserved only in patients resistant to apoptosis and were reduced in those susceptible to apoptosis. Reduction of bcl-2 protein levels was inhibited in cells cultured in the presence of IL-4. These data offer an explanation for the difference between the long life in vivo and rapid death in vitro of B-CLL cells and indicate that IL-4 may participate in the extended survival of these non-dividing cells in vivo .     

Chemical transformation of Swiss mouse embryo fibroblasts in primary culture

Trypsinized secondary cultures of various cell systems have been frequently used by different investigators as models to study, in vitro chemical transformation. In the present study, non-trypsinized primary cultures of fibroblasts derived from fetuses of 14 days gestational age have been used to find out the timings of in vitro chemical transformation by 20-methylcholanthrene and has been designated as PMM-14 cells. These PMM-14 cells have been compared with trypsinized cultures of the same cell systems. The criteria for neoplastic transformation considered in the study involved the appearance of morphological changes, indefinite growth in tissue culture, acquisition of tumorigenic potential in vitro as evidenced by in vivo tests and in vivo latency period for palpable tumor formation. The time span of neoplastic transformation of non-trypsinized embryo fibroblasts in culture was remarkably reduced in comparison with trypsinized cultures of same cell systems. Such discrepancy seems to be due to repeated use of trypsin which may replace many of vital cell surface molecules causing a delay in the expression of carcinogen-induced malignancy. Similarly, repeated subculture before transformation may also have a role in the delayed expression of malignancy.     

Ovarian aromatase activity in the domestic fowl (Gallus domesticus)

The effect of excessive tri-iodothyronine (T3) in vivo was assessed using normal human lymphocytes. Cells from normal subjects were frozen in liquid nitrogen before and after oral administration of T3 for 1 week to permit a direct comparison under identical culture conditions. Within the group of individuals studied, some subjects did show changes in B or T cell function but hypertri-iodothyroninaemia produced no consistent effect for the whole group on circulating T cell subsets or T and B cell activation measured by short-term culture or stimulation of lymphocyte cultures with phytohaemagglutinin or pokeweed mitogen. Tri-iodothyronine supplementation of cultures in vitro did not affect pokeweed mitogen stimulation. These findings suggest that the immunological abnormalities in Graves' disease are not the result of increased circulating thyroid hormone levels and that remission following medical treatment is due to an immuno-suppressive effect of the drug rather than the restoration of euthyroidism.     

An analysis of the multilineage production of human hematopoietic progenitors in long-term bone marrow culture: evidence that reactive oxygen intermediates derived from mature phagocytic cells have a role in limiting progenitor cell self-renewal

To better understand the limited hematopoietic life span of human marrow "Dexter" cultures, we developed a miniaturized, two-stage culture system with which in vitro production of hematopoietic progenitors could be reproducibly detected and quantified. Light- density, gradient-separated human marrow cells were inoculated into Leighton slide tubes, and adherent ("stromal") cell layers were allowed to develop on the removable coverslips within these tubes during an initial 4 weeks of culture. Once stromal cell layers were established, cultures were irradiated (800 cGy) to eliminate all residual hematopoietic progenitors. The cultures were then recharged with autologous, cryopreserved marrow cells (enriched for BFU-E and CFU-GM) to reconstitute stem cell populations and to initiate in vitro hematopoiesis. Most progenitor cells added to irradiated cultures were no longer detectable by clonal assays within one to four days after recharge. Nonetheless, stable populations of adherent BFU-E and CFU-GM became established in these cultures within 24 to 48 hours, and when the total numbers of progenitors (adherent and nonadherent) were measured at weekly intervals thereafter, it was evident that both BFU-E and CFU-GM were generated in vitro. However, progenitor cell production declined as neutrophils and macrophages accumulated in the cultures. Moreover, with this accumulation of mature myeloid cells, increasing levels of O2- and H2O2 could be detected in the cultures, and it was found that the addition of oxidant scavengers (catalase and mannitol) to culture media enhanced the weekly expansions of progenitor cell numbers that could be measured. These findings support the conclusion that reactive O2 intermediates generated by mature myeloid cells have a role in limiting the duration and extent of hematopoietic progenitor cell self-renewal in long-term "Dexter" cultures of human marrow.     

Feasibility of cord blood stem cell manipulation with high-energy shock waves: an in vitro and in vivo study

OBJECTIVE: Cord blood CD34+ cells are more uncommitted than their adult counterparts as they can be more easily maintained and expanded in vitro and transduced with lentiviral vectors. The aim of this study was to evaluate whether pretreatment with high-energy shock waves (HESW) could further enhance the expansion of cord blood progenitors and the transduction efficiency with lentiviral vectors. METHODS: Human cord blood CD34+ cells underwent HESW treatment with a wide range of energy and number of shots (from 0.22 mJ/mm2 to 0.43 mJ/mm2 and from 200 to 1500 shots). Cells were then evaluated both for their in vitro expansion ability and in vivo engraftment in primary, secondary, and tertiary NOD/SCID mice. The transduction efficiency with a lentiviral vector (LV) was also evaluated in vitro and in vivo. RESULTS: Cell viability following HESW ranged from 75 to 92%. Pretreatment with HESW significantly improved early progenitor cell expansion after short-term suspension culture. Upon transplantation in primary NOD/SCID mice, the HESW treatment enhanced progenitor cell engraftment (total human CD45(+)CD34+ cells were 10% in controls and 14.5% following HESW, human CD45(+)CD34(+)CD38(-) cells were 0.87% in controls and 1.8% following HESW). HESW treatment enhanced the transduction of a GFP+ lentiviral vector (e.g., at day 42 of culture 6.5% GFP+ cells in LV-treated cell cultures compared to 11.4% of GFP+ cells in HESW-treated cell cultures). The percentage of human GFP+ cell engrafting NOD/SCID mice was similar (34% vs 26.4% in controls); however, the total number of human cells engrafted after HESW was higher (39.6% vs 15%). CONCLUSION: The pretreatment of CD34+ cells with HESW represents a new method to manipulate the CD34+ population without interfering with their ability to both expand and engraft and it might be considered as a tool for genetic approaches.     

Effects of cytochalasin D-eluting stents on intimal hyperplasia in a porcine coronary artery model

OBJECTIVE: To investigate whether cytochalasin D-eluting stents (CDES) suppress intimal hyperplasia in porcine coronary arteries and to compare the efficacy of paclitaxel and cytochalasin D as inhibitors of vascular smooth muscle cell (SMC) proliferation and platelet aggregation in vitro. METHODS: Rabbit platelet-rich plasma and SMC cultures derived from rabbit aortas were exposed to 10(-8)-10(-5) M cytochalasin D or paclitaxel. Stents directly coated with 2 microg cytochalasin D (low-dose CDES, n=12) and bare stents (n=12) were randomly deployed in the right and left coronary artery of 12 pigs. Six weeks later, neointima was studied using quantitative coronary angiography (QCA) and morphometry. To examine a ten-fold higher dose, polybutyl methacrylate/polyvinyl acetate-coated stents were loaded with 20 microg cytochalasin D. High-dose CDES (n=10) and polymer-only stents (n=11) were deployed in 11 pigs. RESULTS: After 7 days, cytochalasin D (IC(50) 9.9+/-0.4 10(-8) M) and paclitaxel (IC(50) 1.1+/-0.4 10(-8) M) inhibited SMC proliferation in vitro (n=4). In contrast, cytochalasin D (10(-6)-10(-5) M, n=5), but not paclitaxel, attenuated platelet shape change and aggregation induced by ADP. In vivo QCA showed less late lumen loss in low-dose CDES (0.08+/-0.07 vs. 0.32+/-0.08 mm, P=0.05), but morphometry demonstrated only a tendency toward a decreased intimal area. High-dose CDES inhibited both late lumen loss (0.31+/-0.08 vs. 0.91+/-0.06 mm, P<0.01) and intimal area (1.57+/-0.20 vs. 2.46+/-0.22 mm(2), P<0.01). Immunohistochemistry revealed that CDES suppressed peri-strut macrophage recruitment (CD68, P=0.04) and cell proliferation (Ki67, P=0.03) as compared to polymer-only stents without interfering with endothelial cell recovery or the density of alpha-SMC actin staining. Thromboses or edge effects were not observed in either study. CONCLUSIONS: CDES inhibited in-stent hyperplasia. The reduction (39%) with 20 mug CDES was equivalent to that reported for paclitaxel-eluting stents in pigs. Interference with platelet aggregation, SMC migration, SMC proliferation, and leukocyte recruitment could contribute to the benefit. The data indicate that targeting of actin microfilaments has a potential to suppress in-stent restenosis.     

Functional comparison of spleen dendritic cells and dendritic cells cultured in vitro from bone marrow precursors

We have compared dendritic cells (DC) isolated from mouse spleen, or generated in vitro from bone marrow (BM) precursors cultured in granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4), for the ability to process and present soluble antigen and stimulate major histocompatibility complex (MHC) Class II- restricted T cells. DC from spleen or BM cultures were equally able to stimulate the in vitro proliferation of allogeneic T cells or of antigen-specific T-cell receptor (TCR)-transgenic T cells. Both DC populations also induced comparable levels of IL-2 secretion by a T- cell hybridoma. Therefore, splenic and BM-derived DC express comparable levels of (Antigen + MHC Class II) ligands and/or costimulatory molecules and have comparable ability to stimulate T-cell responses. When presentation of a native protein antigen, rather than peptide, was evaluated, BM-derived DC were at least 50 times better than splenic DC at stimulating the proliferation of TCR-transgenic T cells. The antigen processing ability of the two populations was similar only when splenic DC were used immediately ex vivo. Therefore, unlike spleen DC, BM- derived DC maintain the capacity to process protein antigen for MHC Class II presentation during in vitro culture. Due to these characteristics, BM-derived DC may represent a useful tool in immunotherapy studies, as they combine high T-cell stimulatory properties with the capacity to process and present native antigen.     

Cryopreservation and culture of human primordial and primary ovarian follicles

Cryopreservation of ovarian cortical tissue containing high numbers of primordial and primary follicles would benefit young women who are going to undergo chemotherapy or radiotherapy, or anticipated premature ovarian failure. Human ovarian tissue has been successfully cryopreserved using dimethyl sulphoxide, propanediol and ethylene glycol as cryoprotectants. The viability after thawing has been shown morphologically, using viability tests, by transplanting the tissue to immunodeficient mice, and by culturing them in vitro. Maturation of oocytes in in vitro cultures from early follicles would be better than replantation for girls with malignancies which could be replanted with the tissue. For the time being we have managed to culture cryopreserved human primordial and primary follicles to secondary, and occasionally to early antral stages in organ culture within slices of cortical tissue in extracellular matrix. The culture conditions have to be improved to get systematically early antral follicles for a second step of maturation of cumulus-oocyte-complexes.     

Computer simulation of leukemia therapy: combined pharmacokinetics, intracellular enzyme kinetics, and cell kinetics of the treatment of L1210 leukemia by cytosine arabinoside.

An integrated mathematic computer-based model of the pharmacokinetics, intracellular enzyme kinetics, and cell kinetics of the treatment of L1210 leukemia by cytosine arabinoside (ara-C) is described. The compartment model of Bischoff and Dedrick is extended to the intracellular level by inclusion of equations describing the phosphorylation, dephosphorylation, and deamination of ara-C with enzymatic feedback control. The activities of kinase, deaminase, and phosphatase are explicitly included in the models and are estimated from relevant data. Cell proliferation is described by a continuous-flow mathematic model in which cellular maturation and cell-to-cell variability in maturation rates are key variables. Cell proliferation is related to intracellular biochemistry through mathematic expressions which relate cell lethality and progression delay to the time course of intracellular ara-CTP. In vitro and in vivo experiments performed in a number of laboratories are compared by simulation. The most sensitive parameters in dose-response and cell-survival simulations are deoxycytidine kinase activity, ara-CTP half-life, renal clearance of ara-C, and cell-kinetic parameters for proliferation and cell killing. Progression delay is vital to the realistic simulation of divided-dose schedules. By comparative simulation we have identified areas of uncertainty which can be classified by a few additional measurements. The applications of simulations combining pharmacokinetic, biochemical, and cell-kinetic data in vitro and in vivo are discussed, exploring consistency among different measurements, and relating experimental protocols to clinical treatment.     

Efficacy of topical treatment for herpes simplex virus infections: predictions from an index of drug characteristics in vitro

Topical antiviral treatments for recurrent infection with herpes simplex virus in immunocompetent patients have been generally ineffective. We investigated whether in vitro drug measures could predict in vivo efficacy. Twelve topical antiviral formulations were evaluated in vitro by measuring inhibition of viral plaque formation in cell culture (ID50) and drug penetration through excised guinea pig skin. In vivo efficacy for each treatment was determined in an experimental cutaneous infection with herpes simplex virus type 1 in guinea pigs and expressed as the percent reduction in lesion number, lesion area, and virus titer in the lesions. The in vitro findings were correlated with the results in the animal model. ID50 was a poor predictor of in vivo efficacy, whereas stronger correlations were found between the degree of skin penetration and in vivo activity. The best correlation was noted by using a summary expression of the in vitro results as follows: the ratio of drug penetration through skin at 37 C to ID50 (r = .95, .94, and .92 for lesion number, area, and virus titer, respectively, P less than .0005). Determination of this in vitro index should be included in the preclinical evaluation of new topical antiviral formulations.