An electron microscopic study of penetration by Trypanosoma rangeli into midgut cells of Rhodnius prolixus

The process of interaction of the Choachi strain of Trypanosoma rangeli with intestinal epithelial cells of Rhodnius prolixus was analyzed in experiments carried out in vitro and in vivo. For the in vitro experiments small fragments of the anterior region of the posterior midgut were incubated in the presence of the parasites, fixed, and processed for observation with the scanning electron microscope. Parasites attached to the surface of some epithelial cells, especially to the extracellular membrane layers (perimicrovillar membranes), were observed. For the in vivo experiments insects were infected with cultures of T. rangeli, sacrificed at different time intervals, and then processed for scanning and transmission electron microscopy. An intimate contact between the parasites and the membrane layers was observed. The parasites penetrated into cells that showed an electronlucent cytoplasm and a damaged surface, moved within the cytoplasm of the epithelial cell, reached the basal region, crossed the basal lamina, and entered the hemocoel.     

Localization of a Toxoplasma gondii rhoptry protein by immunoelectron microscopy during and after host cell penetration.

We immunolocalized a Toxoplasma gondii rhoptry protein (ROP1) before and after parasite host cell invasion of human fibroblasts and TG180 murine sarcoma cells by electron microscopy and immunogold labeling using either a monoclonal antibody (Tg49) or a monospecific rabbit antiserum (alpha 249). At all stages of parasite growth ROP1 was found within the body but rarely within the peduncle of rhoptries, even in those that appeared empty. Immediately after host cell invasion ROP1 was associated with the parasitophorous vacuole membrane. Within hours after invasion the amount of ROP1 immunodetectable on the parasitophorous vacuole membrane was markedly decreased. The localization of ROP1 suggests a role in the early establishment of infection in host cells, consistent with previous work that has indicated that monoclonal antibodies to ROP1 (including the one used in these studies) interfere with the phenomenon of penetration enhancement.     

Synthesis and antitumor activity of duocarmycin derivatives: A-ring pyrrole compounds bearing beta-(5',6',7'-trimethoxy-2'-indolyl)acryloyl group

A series of A-ring pyrrole derivatives of duocarmycin bearing beta-(5',6',7'-trimethoxy-2'-indolyl)acryloyl group were synthesized, and evaluated for in vitro anticellular activity against HeLa S3 cells and in vivo antitumor activity against murine sarcoma 180 in mice. New Seg-B analogues bearing beta-(5',6',7'-trimethoxy-2'-indolyl)acryloyl group containing double bond as spacer had lower peripheral blood toxicity than the derivatives bearing 5',6',7'-trimethoxyindole-2'-carboxyl group in Seg-B of the natural type. Moreover, most of them exhibited potent antitumor activity against in vivo murine tumor models.     

Ultrastructural damage to the malaria parasite in the sickled cell.

The process by which malaria parasites are killed in sickled erythrocytes was studied by electron microscopy. In vitro cultures of Plasmodium falciparum in sickle cell hemoglobin (HbS) homozygous (SS) and heterozygous (SA) red cells were deoxygenated for up to 6 h and fixed under anaerobic conditions. Parasites in SS cells appeared to be disrupted by intrusions of needle-like deoxyHbS aggregates; disintegration of cytoplasm and membranes followed. In SA red cells, the parasites were generally not disrupted. Instead, extensive vacuolization occurred, a sign of metabolic inhibition. The resistance of HbS gene carriers to malaria results partly from these causes of intracellular parasite death.     

Anthracycline resistance and consequences of the in situ-in vitro transfer

Adriamycin-resistant and normal cells of the sarcoma 180 of the mouse undergo qualitatively different deflections from the in situ state when prepared for an experiment. Resistant cells perform a fast reactive decline in the proliferative activity. They are capable of quiescence as defined by the time needed for the induction of the proliferation. Sensitive cells seem to be unable to quiesce and are only slowed down. These facts must be taken into account in interpretation of similar results. Differences in experiments need not necessarily imply differences in situ. Such in vitro appearing differences between sensitive and adriamycin-resistant cells of the murine sarcoma 180 include the retention of the mitochondria-specific stain rhodamine 123 and the uptake of anthracyclines, both being reduced in resistant cells. After labeling sensitive cells with thymidine in vivo and sorting them according to their rhodamine 123-derived fluorescence, the label was only found in the major, highly fluorescing fraction. A small low-fluorescing fraction remained unlabeled. We were able to demonstrate similar results with labeled anthracyclines applied to both the sensitive and the resistant cells in a short period between the removal of the cells from the ascites and the cell sorting. The adriamycin resistance seems to be joined with the ability of the cells to reduce their proliferative activity following changes to unfavorable conditions in vitro. Quiescent cells of the resistant line demonstrate the "anthracycline pump." Substances which are known to increase the sensitivity of anthracycline-resistant cells (TWEEN, verapamil) also shift the cells from low to high rhodamine 123-fluorescence.     

Anti-CD3 + IL-2-stimulated murine killer cells. In vitro generation and in vivo antitumor activity

The growth, phenotype, in vitro cytolytic characteristics, and in vivo antitumor activity of murine splenocytes stimulated with anti-murine CD3 mAb in combination with IL-2 as compared with IL-2 alone was investigated. When cultured for 12 days with anti-CD3 mAb + IL-2, murine splenocytes increased 100- to 4000-fold in number compared with only 6- to 20-fold for cultures stimulated with IL-2 alone. Anti-CD3 mAb + IL-2 activated cultures developed high lymphokine-activated killer activity against NK-resistant targets including the P815 mastocytoma cell line and fresh MCA 106 sarcoma. Peak cytotoxicity on a per cell basis developed by day 8 after anti-CD3 mAb + IL-2 activation. A large proportion of the total cytolytic activity of long term anti-CD3 mAb + IL-2-stimulated cultures was related to the presence of anti-CD3 in the assay, indicating enhancement of cytotoxicity by activated CD3+ T cells. Phenotypic analysis indicated that anti-CD3 mAb + IL-2-stimulated cultures contained heterogeneous populations of T cells with increased percentages of both CD4+ and CD8+ phenotypes compared with cultures stimulated with IL-2 alone. Anti-CD3 mAb + IL-2-stimulated cells were tested for their in vivo antitumor activity by using C57BL/6 mice bearing MCA 106 sarcoma pulmonary metastases. IL-2-activated murine killer cells were given in combination with in vivo IL-2 and indomethacin, the latter of which was shown to potentiate the antitumor effect of IL-2. When given on day 5 after tumor inoculation, cell doses as low as 5 x 10(6) anti-CD3 mAb + IL-2-stimulated cells per mouse significantly reduced the number of pulmonary metastases (p less than 0.005). Thus, activation with the combination of anti-CD3 mAb + IL-2 produces rapidly expanding cultures of cytolytic cells with demonstrated in vivo antitumor efficacy.     

Adaptation of chimeric retroviruses in vitro and in vivo: isolation of avian retroviral vectors with extended host range

We have designed and characterized two new replication-competent avian sarcoma/leukosis virus-based retroviral vectors with amphotropic and ecotropic host ranges. The amphotropic vector RCASBP-M2C(797-8), was obtained by passaging the chimeric retroviral vector RCASBP-M2C(4070A) (6) in chicken embryos. The ecotropic vector, RCASBP(Eco), was created by replacing the env-coding region in the retroviral vector RCASBP(A) with the env region from an ecotropic murine leukemia virus. It replicates efficiently in avian DFJ8 cells that express murine ecotropic receptor. For both vectors, permanent cell lines that produce viral stocks with titers of about 5 x 10(6) CFU/ml on mammalian cells can be easily established by passaging transfected avian cells. Some chimeric viruses, for example, RCASBP(Eco), replicate efficiently without modifications. For those chimeric viruses that do require modification, adaptation by passage in vitro or in vivo is a general strategy. This strategy has been used to prepare vectors with altered host range and could potentially be used to develop vectors that would be useful for targeted gene delivery.     

Effects of bone morphogenetic protein-7 stimulation on osteoblasts cultured on different biomaterials

The objective of the present study was to investigate the effects of an in vitro stimulation of human osteoblasts by recombinant human bone morphogenetic protein-7 (rhBMP-7) on the collagen types and the quantity of the collagen cross-links synthesized in a three-dimensional culture on various biomaterials for bone replacement. Trabecular bone chips were harvested from human iliac crests, and cell cultures were established at standard conditions. One hundred and fifty nanograms per milliliter of rhBMP-7 was added. For the second passage a cell scraper was used to bring the cells into suspension, and 100 microl osteoblasts (at a density of 3.3 x 10(5)) were transferred onto nine blocks of either Bio-Oss, Tutoplast, or PepGen p-15. Blocks incubated with cells that were not treated with rhBMP-7 served as controls. Cell colonization of the biomaterials was observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) after a period of 2, 4, and 6 weeks. Throughout the experiment medium, supernatants were collected and collagen was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Finally, the collagen cross-link residues hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) were quantified by HPLC. Within 4 weeks the cells became confluent on all of the studied biomaterials. All samples synthesized bone specific LP and collagen type I. However, in rhBMP-7-stimulated samples, the amount of HP and LP found was increased by 45% compared to non-stimulated samples. Cell proliferation and collagen synthesis was similar on the different biomaterials, but was consistently reduced in specimen not stimulated with rhBMP-7. In vitro stimulation of osteoblasts on Bio-Oss, Tutoplast, or PepGen p-15 with rhBMP-7 and subsequent transplantation of the constructs might lead to an enhanced osseointegration of the biomaterials in vivo.     

Synthesis and antitumor activity of duocarmycin derivatives: modification at the C-7 position of segment-A of A-ring pyrrole compounds

A series of the C7-substituted A-ring pyrrole derivatives of duocarmycin were synthesized, and evaluated for in vitro anticellular activity against HeLa S3 cells and in vivo antitumor activity against murine sarcoma 180 in mice. All of the C7-substituted A-ring pyrrole compounds decreased potency in vitro and in vivo. However, some showed strong antitumor activity with T/C values less than 0.3. Among them, the 7-formyl compound 5d showed remarkable potent in vivo antitumor activity and low peripheral blood toxicity, which were equal to 2c.     

Localization of a Toxoplasma gondii Rhoptry Protein by Immunoelectron Microscopy During and After Host Cell Penetration

ABSTRACT We immunolocalized a Toxoplasma gondii rhoptry protein (ROP1) before and after parasite host cell invasion of human fibroblasts and TG180 murine sarcoma cells by electron microscopy and immunogold labeling using either a monoclonal antibody (Tg49) or a monospecific rabbit antiserum (α249). At all stages of parasite growth ROP1 was found within the body but rarely within the peduncle of rhoptries, even in those that appeared empty. Immediately after host cell invasion ROP1 was associated with the parasitophorous vacuole membrane. Within hours after invasion the amount of ROP1 immunodetectable on the parasitophorous vacuole membrane was markedly decreased. The localization of ROP1 suggests a role in the early establishment of infection in host cells, consistent with previous work that has indicated that monoclonal antibodies to ROP1 (including the one used in these studies) interfere with the phenomenon of penetration enhancement.     

Development of Hammondia heydorni in cultured bovine and ovine cells

Sporozoites were excysted from oocysts of Hammondia heydorni obtained from a naturally-infected dog and inoculated into monolayer cultures of bovine pulmonary artery endothelial cells (CPA), Madin-Darby bovine kidney (MDBK) cells, bovine monocytes (M617), or ovine monocytes (WOMO). Sporozoites penetrated all four cell lines and underwent asexual reproduction by endodyogeny (as determined by electron microscopy) to form cyst-like structures at four to nine days after sporozoite inoculation (DAI). At 4-10 DAI, considerably more zoites were harvested from M617 cultures (80.1 x 10(6) zoites) than from CPA (17.4 x 10(6], MDBK (47.3 x 10(6], and WOMO (53.5 x 10(6]. Little or no parasite multiplication occurred at 10-16 DAI. Zoites harvested at 7 DAI and transferred to freshly prepared cultures did not penetrate cells nor develop further.     

Enumeration of 6-thioguanine-resistant peripheral blood lymphocytes in man as a potential test for somatic cell mutations arising in vivo.

An autoradiographic method to enumerate variant 6-thiogunanine-resistant (TGr) peripheral blood lymphocytes (PBLs) that occur in vivo in man is described. Variant cells are detected in PBL cultures stimulated to tritiated thymidine (3HTdr) incorporation in vitro with phytohemagglutinin (PHA) in the presence of TG. Cells with the naturally-occurring Lesch--Nyhan (LN) mutation served as prototype-variant cells. PBLs from a LN hemizygous male were found to be resistant to TG inhibition of PHA-stimulated 3HTdr in corporation in vitro while a LN heterozygous female was found to be a mosaic with 2/1000 PBLs resistant to 2 X 10(-4) M TG. Experiments with artificial mixtures of LN and normal PBLs showed that the LN cells were virtually all detectable even when present in low frequency (10(-5)). TGr PBLs were found in healthy non-LN individuals at median frequencies of 1.0 X 10(-4) and 1.1 X 10(-4) when determined at 2 X 10(-3) M TG and 2 X 10(-4) M TG respectively. Their frequencies were not age-related. TGr PBL-variant frequencies (Vf's) were determined in 47 cancer patients who were being treated with cytotoxic agents that are known to be mutagens. The median TGr PBL Vf determined at 2 X 10(-3) M TG in cancer patients was 2.2 X 10(-4) while, when determined at 2 X 10(-4) M TG, it was 8.5 X 10(-4). The distribution of Vf's for the treated cancer-patient group differed from that for the normal control group in that more than half of the treated cancer patients had TGr PBL Vf's greater than the highest seen for controls. Unlike those of the normal controls, the TGr PBL Vf's of treated cancer patients differed if determined at 2 X 10(-3) M TG and 2 X 10(-4) M TG, a behavior that suggested partial resistance and mimicked that seen with LN TGr PBLs. PBLs resistant to 2,6-diaminopurine (DAPr) were not found in two individuals, although the TGr PBL Vf was elevated in one. TGr PBL Vf's were greatly elevated under conditions of in vivo selection in patients receiving purine-analogue immunosuppressive therapy. The TGr PBL enumerative assay system is presented as one of potential value to detect somatic cell mutations occurring in vivo in man.     

First isolation of Neospora caninum from an aborted bovine fetus in Spain

Neospora caninum was isolated from the brain of a 6-mo-old aborted bovine fetus from Galicia, Spain. The fetal brain homogenate was inoculated intraperitoneally into cortisonized mice. The peritoneal exudate from the infected mice, along with mouse sarcoma cells (Tg180), was inoculated into a second group of mice, and parasites were harvested from the peritoneal exudate. The parasites were adapted to in vitro growth in Vero monolayers. The tachyzoites from the peritoneal exudate reacted positively with anti-N. caninum antibodies and not with anti-Toxoplasma gondii antibodies on indirect fluorescent antibody test. The tachyzoites were lethal to interferon gamma gene knock out (KO) mice and could be identified immunohistochemically in the tissues. The identity of the parasite was also confirmed by polymerase chain reaction amplification of N. caninum-specific fragments. The sequences of the amplified gene 5 fragments (GenBank AY494944) were found to be identical to that of an Austrian isolate of N. caninum but not to that of NC-1. This is the first isolation of viable N. caninum from Spain.     

Trypanosoma cruzi: alpha-2-macroglobulin regulates host cell apoptosis induced by the parasite infection in vitro

A2M is a broad spectrum proteinase inhibitor and cytokine carrier, besides presenting anti-apoptotic activity through the binding to its receptor, LRP. During Trypanosoma cruzi infection, apoptosis of host cells and intracellular parasites is commonly observed both in vivo and in vitro. Since plasma as well as tissue A2M levels are increased in both murine and human acute T. cruzi infection, we evaluated the possible role of A2M (its methylamine transformed Fast form-A2M-F) in regulating apoptotic events in peritoneal macrophages and cardiomyocytes during in vitro interaction with the parasite. Our data showed that DNA fragmentation (a hallmark of apoptosis) of both host cells and parasites was inhibited by A2M-F. Impaired apoptosis was also noted when A2M-F was added to the cultures maintained under serum deprivation. In addition, macrophages from C57/BL6 mice, known to display higher LRP levels as compared to those of C3H lineage, displayed higher reduction in the apoptotic levels during the A2M-F treatment.     

Demonstration of virus particles in Moloney murine sarcoma virus-induced periosteal bone in mice

Balb/c mice were inoculated intramuscularly with Moloney murine sarcoma virus in one of the hind legs. This led to the rapid development of a regressive sarcoma and also to the proliferation and osteogenic differentiation of cells in the adjacent periosteum. Examination of the tissues by transmission electron microscopy revealed the presence of type A and C virus particles within the sarcoma cells as well as within the cells of the newly formed bone. Extracellular type C virus particles were formed by budding from the cell surface and by release from disintegrating cells. No virus particles were found in the bone or the surrounding soft tissues of the contralateral, noninfected leg. These observations suggest that viral infection of periosteal cells are at least partly responsible for the osteogenic response associated with the virus-induced sarcoma. Production of growth factors by the sarcoma cells could also contribute to this process.     

Mesothelial cell apoptosis is confirmed in vivo by morphological change in cytokeratin distribution

Apoptosis of mesothelial cells has been demonstrated in vitro but not in vivo. To identify apoptotic pleural cells as mesothelial, we used cytokeratin as a marker and found a striking spheroid, aggregated appearance of cytokeratin in apparently apoptotic mesothelial cells. In in vitro studies, we found that the aggregated cytokeratin pattern correlated with apoptosis in primary mesothelial cells from mice, rabbits, and humans and was not seen with necrosis. In in vivo studies in mice, we then used this cytokeratin pattern to identify and quantitate apoptotic mesothelial cells. Apoptotic mesothelial cells were best harvested by pleural lavage, indicating that they were loosely adherent or nonadherent. Instillation of RPMI 1640 medium or wollastonite for 24 h induced apoptosis in 0.1 +/- 0. 1 (SE) and 1.0 +/- 0.7%, respectively, of all mesothelial cells recovered, whereas instillation of known apoptotic stimuli, crocidolite asbestos (25 microg) for 24 h or actinomycin D plus murine tumor necrosis factor-alpha for 12 h, induced apoptosis in 5. 1 +/- 0.5 and 22.4 +/- 4.5%, respectively (significantly greater than in control experiments, P < 0.05). By analysis of cytokeratin staining, mesothelial cell apoptosis has been confirmed in vivo.     

Biochemical analysis of retroviral structural proteins to identify and quantify retrovirus expressed by an NS0 murine myeloma cell line

A subclone of the NS0 murine myeloma cell line, frequently used to produce recombinant monoclonal antibodies, was found by a transmission electron microscopy method to express a surprisingly high titer of 10(11) retroviral particles per ml of culture supernatant. Infectivity assays showed a very low infectious titer with the restricted host range expected for a murine amphotropic retrovirus. A Western blot assay for the viral capsid protein was developed to confirm the high titer values and provide a means for monitoring batch consistency and virus removal during the purification process. Mass spectrometry of several of the viral Gag proteins demonstrated that the cell line appeared to produce at least two closely related retroviruses. N-terminal sequencing of three of the Gag proteins demonstrated that these retroviruses were members of the murine leukemia retroviral family. Western blot detection with an antibody for the capsid protein gave a linear standard curve over the range of 0.1-3 ng per lane. This allows the detection of viral titers as low as 6x10(7) virions per ml without the need to concentrate the sample. The Western blot method has higher throughput and less variability than transmission electron microscopy methods and has potential for monitoring viral titer and clearance during development of manufacturing processes.     

波形纤维蛋白与Nup 180的体外结合

本文作者采用大肠杆菌表达的波形纤维蛋白与大鼠肝细胞分离的核孔蛋白进行体外结合实验,以分析波形纤维与核孔的关系。实验结果显示,细菌表达的波形纤维蛋白在体外能组装成10 nm纤维,在体外反应体系中加入核孔蛋白后,室温反应30 min,SDS-聚丙烯酰胺凝胶电泳及免疫印迹法检测,结果表明180 kD核孔蛋白(Nup 180)与波形纤维蛋白有亲和反应。结合免疫胶体金标记与电镜负染色方法显示,核孔蛋白结合于体外装配的10 nm波形纤维上。本文结果提示在细胞内波形纤维可能通过与Nup 180的结合锚定于核孔复合体上。     

Highly efficient and economical baculovirus expression system for preparing human papillomavirus type16 virus-like particle

To improve the existing human papillomavirus type16 (HPV16) virus-like particle (VLP) preparation, a highly efficient, economical and timesaving system was established. Sf-9 cells were infected with recombinant baculovirus containing the target gene encoding HPV16L1 protein with 6xHis tag, and harvested 72 h postinfection (p.i.) at 27 degrees. The ProBond(TM) purification system was used for protein purification. The molecular weight of expressed HPV16L1 protein was 58 kD as revealed by SDS-PAGE, and confirmed by Western blot. The purity of denatured and native HPVL1 proteins that were prepared were 91.9% and 71.5%, respectively, which corresponded to a yield of 2.26 mg denatured protein and 1.84 mg native protein per 2x10(7) cells. The proteins were further analyzed by mouse erythrocyte hemagglutination assay and hemagglutination inhibition assay, and there effects on VLP formation were also visualized by transmission electron microscopy. Results showed that the native protein purified was biologically active as natural HPVL1 protein, inducing the murine erythrocyte agglutination and VLP formation. In addition, the purified recombinant HPV16L1 native protein with 6xHis tag could self-assemble into virions in vitro. Hopefully, the present expression and purification system is promising to be convenient, timesaving and economical for preparation of HPV16 VLP vaccine.     

Functional characterization of lymphoid cells generated in serum-deprived culture stimulated with stem cell factor and interleukin 7 from normal and autoimmune mice

We have investigated the phenotypic and functional characteristics of murine pre-B cells obtained in semisolid and liquid culture with stem cell factor (SCF) and interleukin 7 (IL-7). Both serum-supplemented and serum-deprived culture conditions were used. The source of bone marrow cells was either normal mice (CD1 and C3H) or the lupus strain of mice MRL/Ipr and its congenic strain MRL/+. SCF (100 ng/ml) and IL-7 (250 ng/ml) supported murine B cell proliferation in vitro from all the murine strains analyzed both in serum-supplemented and serum-deprived conditions. Maximal colony growth was observed in both cases when the factors were used in combination. The growth factors alone induced some colony growth in serum-supplemented cultures but were either ineffective or had modest activity in serum-deprived cultures. Cells harvested from the colonies or generated in liquid cultures and stimulated with SCF + IL-7 in the absence of serum had almost exclusively a pre-B cell phenotype (BP-1+, B220+, slg-, CD4-, CD8-, Mac-1, RB-6-). Both the maximal colony growth in semisolid culture and the maximal number of cells in liquid culture were observed at day 12–14. At this time, the pre-B cells failed to differentiate further and started to die. Pre-B cells generated in vitro were, however, capable of differentiating in vivo. SCID mice injected with 2 × 10⁶ pre-B cells had readily detectable serum levels of IgM (54 ± 26 m?g/ml) and IgG (60 ± 95 m?g/ml) at 4 weeks and 6 weeks posttransplantation, respectively. Mature B and T cells of the donor major histocompatibility complex type were detected in the SCID mice at sacrifice 14 weeks posttransplantation. These data indicate that purified (>80% BP-1+) populations of functional pre-B cells can be grown from murine bone marrow of normal mice as well as of lupus mice in serum-deprived cultures stimulated with SCF and IL-7. These cultures, therefore, provide a highly enriched source of pre-B cells but also contain T cell precursors that differentiate upon adoptive transfer into SCID mice. © 1995 Wiley-Liss, Inc.