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Attenuation of Eimeria mivati (= mitis) by selection for precocious development.

By selection for early development of oocysts during serial passage through chickens the pre-patent period of the Houghton (H) strain of Eimeria mivati (= mitis) was reduced by over 20 h. The precocious parasite is less pathogenic than the H strain and has a reduced reproductive potential. Chicks inoculated with the precocious parasite were protected against challenge with the H strain. The pathogenicity, immunogenicity and reproduction of precocious E. mivati and an attenuated egg-adapted line of E. mivati were compared. Although the precocious parasite produced more oocysts than the egg-adapted parasite in chickens, neither parasite induced loss of weight and both were immunogenic.

Animals↗

A preliminary study of the nature of infection and immunity in chickens given an attenuated line of Eimeria acervulina.

The Houghton (H) strain of Eimeria acervulina was attenuated by serial passage through chickens of the first oocysts produced during infection. This selection pressure resulted in a reduction in the pre-patent period of the parasite, shown to be due to the selection of a line predominantly with only 3 instead of 4 generations of schizonts. The precocious line had a reproductive potential much lower than that of the parent strain and it was significantly less pathogenic. Chickens given oocysts of the precocious line were almost completely immune to challenge with the Houghton strain.

Animals↗

Studies on the growth, chemotherapy and enzyme variation of Eimeria acervulina var. diminuta and E. acervulina var. mivati.

Eimeria acervulina var. diminuta was serially passaged 12 times in chicken embryos, but growth in cultured chick kidney cells was limited to 2 generations of schizonts. After 7 embryo passages the sensitivities of E. acervulina var. diminuta and an embryo-adapted strain of E. acervulina var. mivati to the anticoccidial drugs amprolium, methyl benzyquate, robenidine and sulphaquinoxaline were compared. Both parasites were sensitive to all the anticoccidials but E. acervulina var. diminuta was more sensitive to sulphaquinoxaline and amprolium. The chicken-maintained strain of E. acervulina var. diminuta was extremely sensitive to clopidol, sulphaquinoxaline and decoquinate. Electrophoretic analyses of several enzymes from E. acervulina var. diminuta revealed enzyme profiles with similarities and differences to the embryo-adapted strain of E. acervulina var. mivati.

Animals↗

Theileria parva: influence of vector, parasite and host relationships on the epidemiology of theileriosis in southern Africa.

The protozoan parasite Theileria parva, transmitted by the ixodid tick Rhipicephalus appendiculatus, is the cause of East Coast fever (ECF) and the related syndromes of Corridor disease and January disease in cattle of eastern, central and southern Africa. It is likely that buffalo (syncerus caffer) are the natural host of T. parva. In eastern and southern Africa, there exists both buffalo-adapted and cattle-adapted T. parva. Disease caused by buffalo-adapted parasites is called Corridor disease, and that caused by cattle-adapted parasites is termed East Coast fever. In eastern Africa, it has been shown experimentally that buffalo-adapted T. parva can, after serial passage in cattle, become adapted to cattle, in which it can then be maintained and cause ECF. This adaptation has been termed transformation. The transformation of buffalo-adapted T. parva to a cattle-adapted parasite has not been reported in southern Africa, and ECF, eradicated from South Africa, Swaziland and southern Mozambique by 1960, has not reappeared in the subcontinent. This paper discusses the possible reasons for this, and hypothesizes that vector population dynamics and the susceptibility of the vector population to infection with T. parva are among the most important factors which influence the expression of ECF as a disease entity, and the likelihood of transformation occurring. It also considers the possibility that disappearance of ECF from southern Africa resulted from the extinction, as a result of vigorous control measures and unfavourable climatic conditions, of non-diapausing populations of R. appendiculatus that may have been introduced from eastern Africa with cattle imported in 1901.

Africa, Southern↗

Human infectivity trait in Trypanosoma brucei: stability, heritability and relationship to sra expression.

Some Trypanosoma brucei lines infect humans whereas others do not because the parasites are lysed by human serum. We have developed a robust, quantitative in vitro assay based on differential uptake of fluorescent dyes by live and dead trypanosomes to quantify the extent and kinetics of killing by human serum. This method has been used to discriminate between 3 classes of human serum resistance; sensitive, resistant and intermediate. TREU 927/4, the parasite used for the T. brucei genome project, is intermediate. The phenotype is expressed in both bloodstream and metacyclic forms, is stably expressed during chronic infections and on cyclical transmission through tsetse flies. Trypanosomes of intermediate phenotype are distinguished from sensitive populations of cells by the slower rate of lysis and by the potential to become fully resistant to killing by human serum as a result of selection or long-term serial passaging in mice, and to pass on full resistance phenotype to its progeny in a genetic cross. The sra gene has been shown previously to determine human serum resistance in T. brucei but screening for the presence and expression of this gene indicated that it is not responsible for the human serum resistance phenotype in the trypanosome lines that we have examined, indicating that an alternative mechanism for HSR exists in these stocks. Examination of the inheritance of the phenotype in F1 hybrids for both bloodstream and metacyclic stages from 2 genetic crosses demonstrated that the phenotype is co-inherited in both life-cycle stages in a manner consistent with being a Mendelian trait, determined by only one or a few genes.

Animals↗

Analysis of the host-specific haemagglutination of influenza A(H1N1) viruses isolated in the 1995/6 season.

Two phenotypes of human influenza A(H1N1) virus are currently circulating in Japan. One (group 1) agglutinates both chicken and goose red blood cells (CRBC and GRBC), the other (group 2) agglutinates GRBC but not CRBC. In the 1995/6 season, group 2 viruses accounted for 70% of the H1N1 viruses isolated in MDCK cells. The 1995/6 viruses were located on two branches of the genetic tree. One branch contained both group 1 and group 2 viruses and the other branch contained only group 2 viruses. Group 2 viruses had aspartic acid at residue 225 in the haemagglutinin (HA) protein, the key amino acid residue for group 2 phenotype. The HA protein of group 1 viruses had a change from aspartic acid to asparagine at residue 225 and the expressed HA protein of these viruses adsorbed CRBC. Serial passage of group 2 viruses in MDCK cells or embryonated chicken eggs caused these viruses to gain the ability to agglutinate CRBC. MDCK-adapted viruses had the same amino acid sequences of HA polypeptide as the original ones, but egg-adapted viruses had changed amino acid sequences. The expressed HA protein from one egg-adapted virus that originally belonged to group 2 adsorbed CRBC.

Amino Acid Sequence↗

An additional copy of the homologous region (hr1) sequence in the Autographa californica multinucleocapsid polyhedrosis virus genome promotes hyperexpression of foreign genes.

The Autographa californica multinucleocapsid nuclear polyhedrosis virus genome contains nine homologous region (hr1, hr1a, hr2, hr2a, hr3, hr4a, hr4b, hr4c, and hr5) sequences that are thought to be involved in viral replication and activation of transcription. Our results show that the 750 bp hr1 sequence is capable of functioning as an enhancer of transcription of foreign genes from the homologous late polyhderin gene promoter and the heterologous Drosophila heat shock protein (hsp70) promoter in insect cells. Introduction of an additional copy of the complete hr1 element downstream to the polyhedrin locus in the viral genome, while not affecting the stability of the recombinant virus for at least 30 serial passages, led to hyperexpression of reporter genes. The enhancement in the expression levels of foreign genes varied from 40 to 90-fold depending on the promoter used.

Animals↗

Differences in immunoreactivity of estrogen receptor (ER) in tamoxifen-sensitive and -resistant breast carcinomas: preclinical and first clinical investigations.

Inherited or acquired tamoxifen resistance is a major constraint in the endocrinological treatment of breast carcinomas. We developed an enzyme-immunoassay that discriminates between tamoxifen-sensitive and -resistant tumors. The procedure was established and standardized using two xenografted breast carcinomas--3366 (highly sensitive to tamoxifen) and 3366/TAM (acquired tamoxifen resistance). The latter model was developed by treatment of 3366 tumor-bearing nude mice during serial passaging over 3 years with tamoxifen. Both lines were estrogen receptor (ER) positive (101 or 82 fmol/mg protein), and revealed no differences in the nucleotide sequences of the hormone binding domain of the ER protein. However, while in the sensitive tumors an upregulation of ER levels was registered after estradiol treatment of tumor bearing nude mice, the ER expression in the resistant line remained unchanged. The tamoxifen sensitive and -resistant breast carcinoma 3366 differed, additionally, in their immunoreactivity of ER to mAB H222. While an incubation with estradiol or tamoxifen of immobilized ER prepared from cytosols of the sensitive tumors 3366 led to a significant increase in immunoreactivity, samples of resistant tumors failed in the exposition of additional immunologically reactive epitopes. These results were the basis for the development of an assay for determination of the tamoxifen response in patients. Our retrospective results with 38 breast tumors from a tumor bank indicated that patients with an increase of immunoreactivity of ER more rarely had a recurrence while under going tamoxifen therapy compared with patients expecting no increase. However, the data indicate interesting changes occurring with the ER of tam-resistant tumors that are to be explained by further mutational or protein-chemical analysis.

Animals↗

A defective RNA associated with bamboo mosaic virus and the possible common mechanisms for RNA recombination in potexviruses.

A naturally occurring 1.1 kb RNA was isolated from purified virions of bamboo mosaic potexvirus isolate S (BaMV-S). This RNA is a defective RNA (D RNA) derived from a single internal deletion of the BaMV genome. A cDNA clone representing the complete nucleotide sequence of the BaMV-S D RNA was generated and its nucleotide sequence was determined. The BaMV D cDNA is 1015 nts in length [excluding the poly(A) tail] and consists of two regions corresponding to 867 nts of the 5' terminus and 148 nts of the 3' terminus of the BaMV genomic RNA. BaMV D cDNA contains a single open reading frame (ORF) encoding a putative 29.7 kDa protein comprised of a fusion of the first 258 amino acids of BaMV ORF 1 and the last 2 amino acids of coat protein. The coding capacity of D RNA was verified by in vitro translation of native BaMV-S D RNA and of 1.1 kb RNA transcribed in vitro from the full-length D cDNA. BaMV D RNA can be reproducibly generated by serial passages of BaMV-S in Nicotiana benthamiana and is the first D RNA in the potexvirus group shown to be generated de novo. Alignments of sequences surrounding the 5' and 3' junction borders of reported potexvirus D RNAs reveal a 65.2-84.6% sequence identity, suggesting that common mechanisms for viral RNA recombination are involved in the generation of potexvirus D RNAs.

Amino Acid Sequence↗

Characterization of bipotent mammary epithelial progenitor cells in normal adult human breast tissue.

The purpose of the present study was to characterize primitive epithelial progenitor populations present in adult normal human mammary tissue using a combination of flow cytometry and in vitro colony assay procedures. Three types of human breast epithelial cell (HBEC) progenitors were identified: luminal-restricted, myoepithelial-restricted and bipotent progenitors. The first type expressed epithelial cell adhesion molecule (EpCAM), alpha6 integrin and MUC1 and generated colonies composed exclusively of cells positive for the luminal-associated markers keratin 8/18, keratin 19, EpCAM and MUC1. Bipotent progenitors produced colonies containing a central core of cells expressing luminal markers surrounded by keratin 14+ myoepithelial-like cells. Single cell cultures confirmed the bipotentiality of these progenitors. Their high expression of alpha6 integrin and low expression of MUC1 suggests a basal position of these cells in the mammary epithelium in vivo. Serial passage in vitro of an enriched population of bipotent progenitors demonstrated that only myoepithelial-restricted progenitors could be readily generated under the culture conditions used. These results support a hierarchical branching model of HBEC progenitor differentiation from a primitive uncommitted cell to luminal- and myoepithelial-restricted progenitors.

Antigens, Neoplasm↗

Purification and partial amino acid sequence of proteins from human epidermal keratinocyte conditioned medium.

Keratinocytes are the main cell type of the epidermis. They secrete a variety of proteins and peptides that have diverse roles in epidermal physiology. In this report, we present purification and partial amino acid sequence of LEKTI, a serine proteinase inhibitor, and DAN (NO3) zinc-finger protein, a tumor suppressor protein of neuroblastoma, from human keratinocyte conditioned medium. Epidermal keratinocytes were isolated from human foreskin and serially passaged in a defined medium (MSBM). At confluence of the fourth passage, MSBM medium was replaced with protein-free Dulbecco's modified Eagle medium/F12 (DMEM:F12) 3:1 base medium and collected every 24 h for 4 days. Medium was pooled and concentrated using a stirred cell concentrator. Concentrated medium was diluted 1:1 in 50 mM sodium phosphate, pH 8 buffer, and loaded onto a preparative heparin affinity column. Proteins/peptides were purified from heparin column passthrough by the combination of preparative and analytical FPLC-based gel filtration chromatography and reversed-phase HPLC. Samples electroblotted onto a PVDF support were sequenced by Edman degradation in a gas-phase sequencing system.

Amino Acid Sequence↗

Stimulation of the growth of human tumor by low-power laser irradiation.

We studied the effect of low-power laser on the growth of human gastric adenocarcinoma transplanted to athymic mice. Irradiation shortened the latency of tumor growth in recipients from 4-6 months to 21-24 days. After 17 serial passages on athymic mice, the size of tumor node in irradiated recipients on day 33 after transplantation was 161.1 mm(3) (vs. 10.2 mm(3) in nonirradiated mice). These findings suggest that low-power laser irradiation can stimulate the growth of metastases in patients with a history of malignancy.

Adenocarcinoma↗

The Madin Darby canine kidney (MDCK) epithelial cell monolayer as a model cellular transport barrier.

Two strains of Madin Darby canine kidney (MDCK) cells were grown on a polycarbonate membrane with 3-micron pores without any extracellular matrix treatment. The membrane, 2.45 cm in diameter, which is part of a commercially obtained presterilized culture insert, provides two chambers when placed in a regular six-well culture plate. This device was found to be convenient for investigating transport of a few selected fluid-phase markers across the MDCK cell monolayer. Both the strain from the American Type Culture Collection (ATCC) and the so-called highly resistant strain I, at a serial passage between 65 and 70, showed a seeding concentration-dependent lag phase followed by a growth phase with a 21-hr doubling time. When seeded at 5 x 10(4) cells/cm2, cell confluence was achieved in 5 days in a modified Eagle's minimum essential medium (MEM) containing 10% fetal bovine serum under a 5% CO2 atmosphere. Similarly, transepithelial electrical resistance (TEER) also reached a plateau value in 5 days. Both light and electron microscopic examinations revealed well-defined junctional structures. Transport of the fluid-phase markers, sucrose, lucifer yellow CH (LY), inulin, and dextran across the MDCK cell monolayers was studied primarily at 37 degrees C following the apical-to-basolateral as well as the basolateral-to-apical direction. Large variations in the steady-state transport rate were observed for a given marker between the cell layer preparations. Thus, the present study proposes an "internal standard" procedure for meaningful comparisons of the transport rate. When normalized to the rate of sucrose, the rate ratio was 1.00:0.80:0.67:0.15 for sucrose:LY:inulin:dextran.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

A novel spontaneous metastasis model of human osteosarcoma developed using orthotopic transplantation of intact tumor tissue into tibia of nude mice.

Evaluation of potential new treatment strategies requires adequate experimental tumor models which resemble the clinical situation as closely as possible. The purpose of the present study was to establish a new human osteosarcoma spontaneous metastasis model using orthotopic transplantation of histologically intact tumor tissue into the tibia of nude mice. Intact tumor pieces, obtained from the 32nd serial passage of subcutaneously growing human osteosarcoma xenografts, were implanted into the proximal tibia in 31 nude mice. Animals were sacrificed and autopsied 2, 4, 6, and 8 weeks after transplantation and examined macroscopically and microscopically for local tumor growth and metastases. All mice developed local intratibial bone tumors that were radiographically and histologically similar to primary human osteosarcoma. Lung metastases were observed in all mice, local and distant lymph node metastases in 15 (48%), and liver metastases in 6 (19%) mice. The microscopic appearance of the metastases was similar to that observed in the donor patient's tumor, corresponding subcutaneous xenografts and orthotopically transplanted intratibial tumors. This spontaneous metastasis model of human osteosarcoma in nude mice may resemble a clinical situation and could thus be useful for studies on local tumor growth, metastasis formation and therapy.

Animals↗

Experimental reproduction of the Jorge Lobo's disease in BAlb/c mice inoculated with Lacazia loboi obtained from a previously infected mouse.

Long-term maintenance of Lacazia loboi in the laboratory has not been reported. We report here the use BALB/c mice to maintain the Lacazia loboi for extended period of time. Eight to ten week-old mice were inoculated intradermally in both hind footpads with a fungal suspension from a macerated footpad obtained from an original mouse previously infected with the fungi and sacrificed 8 months after inoculation. The inoculated animals were sacrificed at different time intervals, footpads were excised, the right one was submitted to histopathological examination and the left one was macerated in sterile saline for fungal count and viability index determination. The inoculated animals presented the histopathological picture identical to the mice previously inoculated with material from human lesion. Granulomatous infiltrates with predominance of macrophages and giant cells were observed. The granulomas evolved progressively as observed in the different times of sacrifice. After 7 months of inoculation, macroscopic lesions were observed, and the number of fungi obtained from macerated footpads was higher than the number of inoculated fungi. The pattern of lesion development was similar to what was observed in animals infected with a fungal suspension obtained from a human lesion. Considering the histopathological findings, the clinical manifestations, and the finding of a higher number of fungi obtained than the inoculated into footpads of each mice, we believe the BALB/c mice strain is as an excellent way to maintain L. loboi in laboratory. Moreover, even after serial passages of the fungi, the granulomatous lesions are reproduced consistently in laboratory conditions.

Animals↗

Comparative levels of DNA breaks and sensitivity to oxidative stress in aged and senescent human fibroblasts: a distinctive pattern for centenarians.

Basal and H(2)O(2)-induced DNA breaks as well as DNA repair activity and efficacy of the antioxygenic system were determined in human dermal fibroblasts explanted from either (i) young donors and passaged serially to reach replicative senescence or (ii) young, old and centenarian donors and shortly propagated in culture. These fibroblasts have been employed as an in vitro and ex vivo model, respectively, to evaluate comparatively DNA integrity during senescence (increasing population doubling levels) and aging (increasing donor age). Constitutive levels of DNA total strand breaks, as determined by the alkaline extraction procedure, changed moderately among the different cell lines, which exhibited, however, significant differences in the amount of either single or double strand breaks. The former decreased along with both aging and senescence; the latter augmented during senescence while being virtually steady during aging. Moreover, fibroblasts from centenarians showed to be less sensitive to H(2)O(2)-induced DNA damage than other ex vivo fibroblasts. This feature could not account for either increased DNA repair activity or higher efficacy of the antioxygenic system and pointed, instead, to an intrinsic nuclear stability which might be typical of centenarian fibroblasts and potentially functional to longevity.

Adolescent↗

Expansion and neural differentiation of embryonic stem cells in adherent and suspension cultures.

The embryonic stem cell line, S25, is a genetically modified line that allows lineage selection of neural cells (M. Li, L. Lovell-Badge, A. Smith (1998) Current Biology 8: 971-974). Here, the growth parameters of this cell line were analysed. Serial passaging in adherent conditions enabled these cells to grow rapidly (average specific growth rates of 0.035 h-1) and generate high viable cell densities (above 90%). The aggregation of the S25 cells into embryoid bodies (EBs) was also studied, indicating limited cell growth (maximum cell densities of 2.7 x 10(5) cells ml-1) and a high variability of aggregate size (70-400 microns after 8 d). Enzymatic dissociation of EBs with 1% (v/v) trypsin gave highest cell viability (91%) and density (1.4 x 10(4) cells ml-1) and the cells thus obtained are able to differentiate into neurons.

Animals↗

Measurements of hydrogen peroxide induced premature senescence: senescence-associated beta-galactosidase and DNA synthesis index in human diploid fibroblasts with down-regulated p53 or Rb.

Human diploid fibroblasts (HDFs) inevitably undergo replicative senescence in culture after serial passages. Recent work indicates that early passage HDFs undergo irreversible growth arrest and develop features of senescence after being treated with oxidants and other agents. Senescence is usually measured by a decrease in DNA synthesis index and an increase in the activity of senescence-associated beta-galactosidase (SA beta-gal). We compared these two measurements here and found that IMR-90 HDFs lost the ability to synthesize DNA immediately but did not activate SA beta-gal until 4 days after the treatment with 75 microM or 0.75 pmol/cell H2O2. Expression of human papillomaviral E6 or/and E7 genes results in reduction of p53 or/and Rb protein levels and increases in ED50 for DNA synthesis inhibition or SA beta-gal expression. A small fraction of wild type and E7 expressing cells could not synthesize DNA and did not express SA beta-gal one week following the treatment with H2O2 at doses lower than 150 microM or 1.5 pmol/cell. The dose response curve of SA beta-gal activation overlapped with that of DNA synthesis inhibition in E6 and E6E7 expressing cells. The results indicate that the expression of SA beta-gal correlates with inability of DNA synthesis in the majority of wild type, E6, E7 or E6E7 expressing cells one week after H2O2 treatment.

Cell Line↗