In vivo hybridisation of cultured melanoma cells and isogenic normal mouse cells.

Somatic cell hybrids were derived by fusing tumourigenic and melanogenic melanoma (PAZG) cells with normal diploid male mouse cells in vivo. Their chromosomal composition was equivalent to the sum of both parental genomes and included a Y chromosome lacking in the melanoma parent. Our study showed that in PAZG X C57BL hybrids (MP), tumourigenicity was suppressed but pigmentation was expressed.     

Natural killer-type lysis of thymocytes from young mice by normal spleen cells in vitro

Normal spleen cells from 6- to 10-week-old mice, enriched for natural killer (NK) cells on a discontinuous polyvinylpyrrolidone-silica (Percoll) gradient, lyse thymocytes of young mice (less than or equal to 19 days old) in a short-term 51Cr release assay. The highest NK-type activity was found in band 3 (density less than or equal to 1.077 g/ml) of a four-step gradient. In some experiments band 2 (density less than or equal to 1.070 g/ml) also showed NK activity. Activity was not unequivocably detectable in cells before separation or in bands 1 and 4. These results also show that the thymocyte sensitivity is dependent on the age of the target cell. Sensitivity of very young thymocytes (less than or equal to 7 days old) was higher than that of thymus cells from 8- to 19-day-old donors. Moreover, it seemed that syngeneic target thymocytes were lysed more effectively than allogeneic. Thus, an NK-type cell population may have the ability to lyse immature thymic target cells at an early stage of their differentiation. This could be of importance as a physiological mechanism for controlling the T cell repertoire and its reactivity.     

In vitro and in vivo treatment of colon cancer by VIP antagonists

Vasoactive intestinal peptide (VIP) is secreted from many cancer lines and VIP binding was observed in many tumors. We have shown before that VIP antagonists are potent inhibitors of neoplastic growth of neuroblastoma, lung and breast cancer cells in vitro. Here, the cultured colon cancer cell line HCT-15 that exhibited VIP receptor expression was treated with the VIP hybrid antagonist neurotensin(6-11)VIP(7-28). The antineoplastic activity was assessed by thymidine incorporation. Neurotensin(6-11)VIP(7-28) efficiently inhibited cancer growth with a maximal effect at nanomolar concentrations. Once the inhibitory properties of the VIP antagonist on colon cancer cells were established, the in vivo curative effects were analyzed. Sprague-Dawley rats were injected with azoxymethane (AOM) (15 mg/kg/week) for 2 weeks, providing artificial induction of colon tumors. The rats were then allocated into four experimental groups: (1) receiving no treatment; (2) receiving treatment with saline; (3, 4) receiving treatment with 10 or 20 microg of neurotensin(6-11)VIP(7-28), respectively. After 10 weeks of daily injections, rats were sacrificed and tumors assessed for stage, volume, location, differentiation and lymphocytic infiltrate. Embedded mucosa was assessed for dysplastic crypts. Results showed that the antagonist treatment reduced the tumor volume, staging, lymphocyte infiltrate and the number of dysplastic crypts. Thus, neurotensin(6-11)VIP(7-28) could serve as an effective cancer treatment and a preventing agent.     

In vivo and in vivo/in vitro kinetics of cyclophosphamide-induced sister-chromatid exchanges in mouse bone marrow and spleen cells

In several acute and chronic exposures to various chemicals in vivo and in vitro, the average sister-chromatid exchange (SCE) frequencies in human, mouse, rat, and rabbit lymphocytes generally decrease with time following treatment. The rate of this decline varies, but little data have been published pertaining to the comparative kinetics of SCEs both in vivo and in vivo/in vitro (exposure of animals to the test compound and culturing of cells) simultaneously in the same tissues. In this study, a single dose of cyclophosphamide (40 mg/kg) was injected for varying periods (6-48 h) and its effects, as assessed by the induction of SCEs, were analyzed under both in vivo and in vivo/in vitro conditions in mouse bone marrow and spleen cells. In vivo, the cyclophosphamide-induced SCEs increased with increasing time up to 12 h, stayed at approximately the same level until 24 h, and then decreased with increase in post-exposure time. However, the SCE levels remained significantly higher than controls at 48 h post-exposure time in both bone marrow and spleen cells. Under in vivo/in vitro conditions, the SCEs in bone marrow decreased with increase in post-exposure time until reaching control values by 48 h post exposure. However, in spleen cells, the decrease in SCE level was gradual, and by 48 h post-exposure time, the cells still had approximately 6 times higher SCEs than the control values. These results suggest that there are pharmacokinetic differences for cyclophosphamide in mouse bone marrow and spleen. Also, there is a differential SCE response to cyclophosphamide under in vivo and in vivo/in vitro conditions.     

In vivo shedding of apical plasma membrane in the thyroid follicle cells of the mouse

Summary Clusters of luminal dense bodies, limited by a triple-layered membrane, were found in all follicle lumina in thyroid glands of mice. After thyroxine treatment the number of luminal dense bodies increased, especially in the periphery of the lumen, where the intraluminal bodies often displayed a striking resemblance to microvilli. In hyperplastic goiters, obtained by feeding mice with propylthiouracil, luminal dense bodies were replaced by intraluminal vesicles. During goiter involution the vesicles were gradually replaced by luminal dense bodies; the presence of intermediate forms suggests that vesicles and dense bodies are basically the same formations. Luminal dense bodies were observed in colloid droplets indicating their removal by endocytosis. As demonstrated by electron-microscopic cytochemistry, luminal dense bodies contain a membranebound peroxidase, and electron-microscopic autoradiography after administration of ¹²⁵I indicate that they possess an iodinating capacity.Our observations on mouse thyroid glands suggest that the luminal dense bodies, which appear as vesicles in hyperplastic glands, are formed by shedding of the apical plasma membrane of the follicle cell. The shedding process might be of importance for the turnover of plasma-membrane material.This study was supported by Grant No. 12X-537 from the Swedish Medical Research Council.     

Stimulation by normal and leukemic mouse sera of colony formation in vitro by mouse bone marrow cells

Using a modification of the agar gel method for bone marrow culture, serum from various strains of mice has been tested for colony stimulating activity. Ninety percent of sera from AKR mice with spontaneous or transplanted lymphoid leukemia and 40–50% of sera from normal or preleukemic AKR mice stimulated colony formation by C57B1 bone marrow cells. Sera from 6% of C3H and 30% of C57B1 mice stimulated similar colony formation. The incidence of sera with colony stimulating activity rose with increasing age. All colonies were initially mainly granulocytic in nature but later became pure populations of mononuclear cells. Bone marrow cells exhibited considerable variation in their responsiveness to stimulation by mouse serum. Increasing the serum dose increased the number and size of bone marrow cell colonies and with optimal serum doses, 1 in 1000 bone marrow cells formed a cell colony. Preincubation of cells with active serum did not stimulate colony formation by washed bone marrow cells. The active factor in serum was filterable, non-dialysable and heat and ether labile.     

In vitro and in vivo germ line potential of stem cells derived from newborn mouse skin

We previously reported that fetal porcine skin-derived stem cells were capable of differentiation into oocyte-like cells (OLCs). Here we report that newborn mice skin-derived stem cells are also capable of differentiating into early OLCs. Using stem cells from mice that are transgenic for Oct4 germline distal enhancer-GFP, germ cells resulting from their differentiation are expected to be GFP(+). After differentiation, some GFP(+) OLCs reached 40-45 μM and expressed oocyte markers. Flow cytometric analysis revealed that ～ 0.3% of the freshly isolated skin cells were GFP(+). The GFP-positive cells increased to ～ 7% after differentiation, suggesting that the GFP(+) cells could be of in vivo origin, but are more likely induced upon being cultured in vitro. To study the in vivo germ cell potential of skin-derived cells, they were aggregated with newborn ovarian cells, and transplanted under the kidney capsule of ovariectomized mice. GFP(+) oocytes were identified within a subpopulation of follicles in the resulting growth. Our finding that early oocytes can be differentiated from mice skin-derived cells in defined medium may offer a new in vitro model to study germ cell formation and oogenesis.     

In vitro transformation of mouse testis cells by oncogene transfection

Germ cell tumors (GCTs) are unique in that they exhibit diverse biological characteristics and pathological features. Although several in vivo GCT models are available, studies on GCTs are hampered because in vivo development of GCTs is time consuming and prevents a detailed molecular analysis of the transformation process. Here we developed a novel strategy to transform mouse testis cells in vitro. Lentivirus-mediated transfection of dominant negative Trp53, Myc, and activated Hras1 into a CD9-expressing testis cells caused tumorigenic conversion in vitro. Although these cells resembled embryonic stem (ES) cells, they were aneuploid and lacked Nanog expression, which is involved in the maintenance of the undifferentiated state in ES cells. Euploid ES-like cells were produced by transfecting the Yamanaka factors (Pou5f1, Myc, Klf4, and Sox2) into the same cell population. Although these cells expressed Nanog, they were distinct from ES cells in that they expressed CD44, a cancer stem cell antigen. Both treatments induced similar changes in the DNA methylation patterns in differentially methylated regions of imprinted genes. Moreover, despite the differences in their phenotype and karyotype, both cell types similarly produced mixed GCTs on transplantation, which were composed of teratomas, seminomas, and embryonal carcinomas. Thus, in vitro testis cell transformation facilitates an analysis of the GCT formation process, and our results also suggest the close similarity between GCT formation and reprogramming.     

In vitro inhibition of natural-killer-mediated lysis by chromatin fragments

A qualitative impairment of natural killer (NK) function and the presence of circulating DNA have been independently reported in clinical situations such as cancer and lupus. The existence of receptors for chromatin fragments at the leukocyte membrane raised the question of the relation between the presence of chromatin fragments in the extracellular medium and the impairment of NK function. The present study shows that plasmas from patients with metastatic cancer and with pathological DNA concentrations inhibited significantly the NK activity of normal lymphocytes as compared to cancer plasmas with DNA concentrations in the normal range. In vitro, it was demonstrated that chromatin fragments inhibited the NK-mediated cytotoxicity in a dose-dependent manner. Inhibitory concentrations of nucleosomes (2.5–10 g/ml) were lower than those of DNA and histones alone (100 g/ml). Inhibitory effects of nucleosomes, DNA and histones differed also according to the effector population used: nucleosomes were effective whatever the CD56⁺ cell enrichment of the effector population, while DNA inhibition needed T cells, and histone inhibition probably resulted from a subtoxic effect, prevented by the presence of adherent cells. Finally we found that nucleosomes could inhibit the NK function only when they were present in the extracellular medium. Taken together, these data suggest that the persistence of nucleosomal DNA at sites of cell death or in the blood might be responsible, at least partly, for the NK activity impairment observed in pathological circumstances characterized by a high rate of cell death phenomena such as cancer.     

In vivo growth stimulation of collagen gel embedded normal human and mouse primary mammary epithelial cells

A new system for studying growth of normal human mammary epithelial cells in an in vivo environment using athymic nude mice is described. Human mammary epithelial cells dissociated from reduction mammoplasty specimens were embedded within collagen gels and subsequently transplanted subcutaneously into nude mice. Histological sections of recovered collagen gels showed epithelial cells arranged as short tubules with some branching. Proliferation of mammary epithelial cells was quantitated in vivo by 3 days' continuous infusion with 5 bromo-2′-deoxy-uridine followed by immunostaining of sections from recovered gels. Ovarian steroids administered to the host animals, resulting in blood serum levels normally found in the human female, had little or no effect on the proliferation of human mammary epithelial cells. Collagen gel embedded mouse mammary epithelial cells, mouse mammary explants, and host mammary glands all responded similarly to ovarian steroids, suggesting that the unresponsiveness of the human mammary epithelial cells under these conditions was not due to dissociation per se. However, an increased dose of 17β-estradiol or a growth factor combination containing epidermal growth factor, cholera toxin, and cortisol significantly stimulated the proliferation of human outgrowths. The growth factor response was dependent on the location of the cells, with the greatest response seen in the part of the gel proximal to the osmotic pump delivering the growth factors and the effect gradually waning in area more distal to the pump. The effect was especially striking since the mitotic figures could be easily identified and the labeling index was as high as 75%. The host mouse mammary gland also responded to growth factors, resulting in ductal hyperplasia. The proliferative and morphogenetic effects of various agents on normal human mammary epithelial cells embedded in collagen gel can be studied in vivo in nude mice. © 1995 Wiley-Liss, Inc.     

In vivo and in vitro stimulatory effects of Cordyceps sinensis on testosterone production in mouse Leydig cells

The in vivo and in vitro effects of Cordyceps sinensis (CS) and its extracted fractions on the secretion of testosterone in mice were studied. CS, F2 (water soluble protein), and F3 (poorly water soluble polysaccharide and protein) significantly stimulated in vitro testosterone production in purified mouse Leydig cells. However, F1 (water soluble polysaccharide) had no effect (p>0.05). F2 and F3 stimulated in vitro testosterone production in dose- and time-dependent relationships with maximal responses at 3 mg/ml for 3 h (p<0.05). An in vivo study illustrated that testosterone levels in plasma were significantly increased by CS, F2, and F3, respectively (p<0.05). Because CS, F2, and F3 stimulated both in vitro and in vivo testosterone secretions in mice, it is possible that CS might contribute to an alternative medicine for the treatment of some reproductive problems caused by insufficient testosterone levels in human males.     

In vitro synthesis of primary specific anti-breast cancer antibodies by normal human peripheral blood mononuclear cells

In this study, we developed a unique in vitro model to mimic the endogenous tumor microenvironment to understand the effect of immunotherapy with activated T-cells (ATC) armed with anti-CD3 × anti-Her2 bispecific antibody (aATC) on antibody response by naive immune cells. This model contained a co-culture of naïve peripheral blood mononuclear cells (PBMC), breast cancer cells (SK-BR-3), ATC or aATC and CpG ODNs. Culture supernatants were tested at various time points for anti-SK-BR-3 antibodies by ELISA, Western blot and flow cytometry. PBMC cocultured with non-irradiated aATC or irradiated (*) aATC showed significant increases in anti-tumor antibody production at day 14 (P < 0.0001) in the presence of CpG-ODN compared to unstimulated PBMC cultures (n = 9). Antibody specificity was confirmed by ELISA, Western blot and flow cytometry. Co-cultures containing *aATC and CpG showed significantly enhanced levels of IgG₂ (P < 0.001) and cytokines that promote IgG₂ synthesis including IL-13 (P < 0.02), IFNγ (P < 0.01) and GM-CSF (P < 0.05) compared to unstimulated PBMC control (n = 3). We show that aATC targeting and lysis of tumor cells induces an anti-tumor antibody response in our in vitro model. This model provides a unique opportunity to evaluate the interactions of T-cells, B-cells, and antigen-presenting cells leading to specific anti-tumor antibody responses.     

Enhanced lysis of herpes simplex virus type 1-infected mouse cell lines by NC and NK effectors

Spontaneously cytotoxic murine lymphocytes lysed certain cell types infected by herpes simplex virus type 1 (HSV-1) better than uninfected cells. The levels of virus-directed lysis varied widely from target to target, and we found that differences in virus-directed lytic efficiency could be attributed both to the characteristics of HSV-1 replication in the different targets and to the subgroup of natural effector cells which mediated lysis. Although HSV-1 adsorbed to the surface of all the target cells, those in which the virus replicated more efficiently were lysed to a greater extent. As targets, we used cell lines that, when uninfected, were spontaneously lysed by NK cells (YAC-1) or by NC cells (WEHI-164). We also used a fibroblastoid cell line (M50) and a monocytic tumor line (PU51R), which were not spontaneously killed. Using complement-mediated elimination of Qa-5-positive or asialo-GM1-positive NK cells to distinguish NK from NC activity, we found that NK cells lysed HSV-1-infected YAC cells better than uninfected cells, and an NC-like activity selectively lysed HSV-1-infected WEHI cells. In addition, we showed that both NK and NC cytotoxicities contributed to the lysis against the HSV-1-infected fibroblastoid line, M50, but the infected PU51R cells were killed by only NK effectors. These findings were consistent with the results of experiments performed to define the role of interferon in induction of virus-augmented cytolysis. Increased lysis of YAC-HSV and PU51R-HSV was entirely due to interferon activation and was completely abolished by performing the 51Cr-release assay in the presence of anti-interferon serum. Because NC activity was not augmented by interferon, virus-enhanced NC lysis of M50-HSV and WEHI-HSV was not due to this nonspecific mechanism. Together, our data show that HSV-1 infection of NK/NC targets induces increased cytotoxicity, but the effector cell responsible for lysis is determined by the uninfected target, or by an interaction between the virus and target cell, rather than by a viral determinant alone.     

In vitro and in vivo characterization of neural stem cells

Neural stem cells are defined as clonogenic cells with self-renewal capacity and the ability to generate all neural lineages (multipotentiality). Cells with these characteristics have been isolated from the embryonic and adult central nervous system. Under specific conditions, these cells can differentiate into neurons, glia, and non-neural cell types, or proliferate in long-term cultures as cell clusters termed "neurospheres". These cultures represent a useful model for neurodevelopmental studies and a potential cell source for cell replacement therapy. Because no specific markers are available to unequivocally identify neural stem cells, their functional characteristics (self-renewal and multipotentiality) provide the main features for their identification. Here, we review the experimental and ultrastructural studies aimed at identifying the morphological characteristics and the antigenic markers of neural stem cells for their in vitro and in vivo identification.     

In vitro and in vivo interactions of homocysteine with human plasma transthyretin

Hyperhomocysteinemia is an independent risk factor for cardiovascular disease and an emerging risk factor for cognitive dysfunction and Alzheimer's disease. Greater than 70% of the homocysteine in plasma is disulfide-bonded to protein cysteine residues. The identity and functional consequences of protein homocysteinylation are just now emerging. The amyloidogenic protein transthyretin (prealbumin), as we now report, undergoes homocysteinylation at its single cysteine residue (Cys10) both in vitro and in vivo. Thus, when human plasma or highly purified transthyretin was incubated with 35S-L-homocysteine followed by SDS-PAGE and PhosphorImaging, two bands corresponding to transthyretin dimer and tetramer were observed. Treatment of the labeled samples with beta-mercaptoethanol prior to SDS-PAGE removed the disulfide-bound homocysteine. Transthyretin-Cys10-S-S-homocysteine was then identified in vivo in plasma from normal donors, patients with end-stage renal disease, and homocystinurics by immunoprecipitation and high performance liquid chromatography/electrospray mass spectrometry. The ratios of transthyretin-Cys10-S-S-homocysteine and transthyretin-Cys10-S-S-sulfonate to that of unmodified transthyretin increased with increasing homocysteine plasma concentrations, whereas the ratio of transthyretin-Cys10-S-S-cysteine to that of unmodified transthyretin decreased. The hyperhomocysteinemic burden is thus reflected in the plasma levels of transthyretin-Cys10-S-S-homocysteine, which in turn may contribute to the pathological consequences of amyloid disease.