Dipetalonema vitae: survival of adult females and microfilarial release in both a chemically defined and serum-supplemented medium

Studies were conducted on survival and microfilarial release of afult Dipetalonema viteae in culture, using worms of various ages derived from jirds. In chemically defined NI medium (a 1:1 mixture of NCTC 135 and Iscove's modified Dulbecco's medium) under a gas phase of 5% CO2 in nitrogen (pO2 of medium approximately 40 mm Hg), the peak of microfilarial release of several thougsand microfilariae per female per 24 hr occurred at approximately day 10. Thereafter, microfilarial release declined and generally ended about 1 mo after the start of culture. The adult females moved actively for about 50 days or more and survived up to 82 days in NI medium alone. The females in NI medium supplemented with fetal bovine serum showed serpentine movement for approximately 2 mo. Some of the worms survived more than 83 days. The total number of microfilariae deposited in culture by D. viteae increased as adult females grew in size (volume) over time. Microfilarial deposition continued to increase after worms reached maximum size, deposition reaching a plateau between approximately 300 and 400 days of age. Thereafter, microfilarial deposition decreased as females continued to age. Addition of fetal bovine serum to the NI medium increased the number of microfilariae released and extended the period of release.     

Behavior of primary and serially transplanted estrogen-dependent renal carcinoma cells in monolayer and in collagen gel culture

Summary When estrogen is present in culture medium, enzyme-dissociated cells from estrogen-induced primary renal tumors in Syrian hamsters and from 1st to 4th serially transplanted carcinomas in monolayer culture contain progesterone receptor levels that are similar and comparable to those in tumors in vivo (i.e., 2 pmol/3×10⁶ cells). Despite the similarity of receptor levels in cultured cells isolated from primary and transplanted tumors, the ability of cells to be maintained in culture differs considerably from one tumor stage to another. When cultured as monolayers in plastic flasks, isolated cells from primary tumors exhibit a marked decline in cell number after 4 to 6 d in culture. On the other hand, monolayer-cultured cells from first and second transplantation tumors remain essentially constant in cell number over a 2 wk culture period and cells from third transplantation tumors undergo a two- to threefold increase in cell number during 2 wk in culture. When primary tumor cells are cultured in collagen gels, the decline in cell number over a 2 wk culture period is prevented and progesterone receptor levels remain elevated. Cells cultured from first transplantation tumors exhibit a delayed decline in cell number beginning after 2 wk in monolayer culture. The decline in cell number in monolayer culture, like that for cells from primary tumors, can be prevented by culturing cells from first transplantation tumors in collagen gels. Neither cells from primary nor first transplantation tumors exhibit significant increases in cell number in collagen gels. Increasing the serum concentration of growth medium to 30% does not stimulate growth of cells under these conditions. Cells isolated from fourth transplantation tumors undergo a fourfold increase in cell number over a 1 month culture period whether cells are cultured as monolayers or in collagen gels. This investigation was supported by Grant CA 22008 awarded by the National Cancer Institute, Department of Health, Education and Welfare, and by institutional research funds from Marquette University.     

Cryopreservation of infective larvae of Dipetalonema viteae.

infective larvae of Dipetalonema viteae produced infections in Mongolian jirds (Meriones unguiculatus) after storage of infected ticks (Ornithodoros tartakovskyi) in the presence of dimethyl sulfoxide (DMSO, 5%) for 7 or 595 days in liquid nitrogen (-196 C). Infectivity of these larvae was only partially impaired. Microfilaremias of test jirds were generally lower than those of control jirds given nonfrozen larvae; however, the majority of test jirds developed microfilarial counts suitable for use in infecting ticks. In contradistinction, larvae frozen free of the tick failed to retain infectivity. Apparently the tick, in conjunction with DMSO, protects the larvae during freezing and thawing.     

Transplanted Dipetalonema viteae in the jird: effect of worm burden on parturition rates and microfilaremia

Dipetalonema viteae was studied in the jird, Meriones unguiculatus, to determine the mechanism controlling the level of peripheral microfilaremia. Jirds killed 40 days after infection served as donors of female worms of known age and reproductive status. These worms were transplanted into uninfected jirds and the resultant microfilaremias were monitored. After approximately 100 days, the recipient jirds were killed and 58% of the transplanted worms were recovered alive but depleted of sperm and microfilariae, regardless of the total number implanted in a given host. A direct linear relationship between microfilaremia and the number of recovered adult worms was found. Based on the uniform absence of sperm and microfilariae in the recovered worms it was concluded that female worms, under the conditions of the present study, do not control the peripheral microfilaremia in multi-worm infections through a reduced parturition rate.     

Anticancer agents suppressive for adult parasites of filariasis in Mongolian jirds

Eight chemical structures not previously reported to possess antifilarial activity have been identified. A total of 79 compounds with anticancer properties were evaluated for possible macrofilaricidal activity against Brugia pahangi and Acanthocheilonema viteae transplanted into male Mongolian jirds (Meriones unguiculatus). All eight active compounds were suppressive for the onchocerciasis type (Acanthocheilonema viteae) of the disease. None was macrofilaricidal for the lymphatic form (Brugia pahangi). These new structures may represent a nucleus around which effective drugs can be synthesized.     

Glucagon production by cultured pancreatic islets: effects of different culture conditions and media

Various conditions for tissue culture of collagenase-isolated mouse pancreatic islets were studied for their effects on the glucagon production of the cultured specimens. Culture media containing heat-treated bovine calf serum degraded [125I]glucagon to a much less extent than those supplemented with untreated serum. Addition of aprotinin to the heat-treated serum gave a further reduction of the [125I]glucagon degradation in the culture medium. A similar supplementation of Medium 199, used for culture of isolated islets, resulted in the most extensive glucagon accumulation in the culture medium. Islets cultured free-floating or attached to the bottom of the culture dishes contained similar amounts of glucagon. However, the free-floating islets released less glucagon when tested in short-term experiments performed at the end of the 1 wk culture period. A comparison between different culture media showed that islets cultured in RPMI-1640 had the highest glucagon content and released most glucagon to the culture medium. Moreover, these islets responded most actively to an acute arginine challenge at the end of the culture period. The present data suggest that the optimal conditions for culture of isolated islets aimed at studies of glucagon production may be obtained by using a culture medium consisting of RPMI-1640 supplemented with both a proteinase inhibitor and heat-inactivated serum.     

周期型马来丝虫微丝蚴寿命的观察

将实验感染周期型马来丝虫的长爪沙鼠的微丝蚴蚴阳性腹腔稀释液,移注于正常沙鼠腹腔内,微丝蚴除能在腹腔内长期生存外,还可出现于外周血液中,其在外周血液内末次阳性检出时间最长可超过32周,在腹腔液内末次阳性检出时间最长为77周,故马来微丝蚴在沙鼠外周血液中的最长寿命不短于7.5月,而在腹腔液内的最长寿命可超过1.5年以上。     

Glucagon production by cultured pancreatic islets: Effects of different culture conditions and media

Summary Various conditions for tissue culture of collagenase-isolated mouse pancreatic islets were studied for their effects on the glucagon production of the cultured specimens. Culture media containing heat-treated bovine calf serum degraded [¹²⁵I]glucagon to a much less extent than those supplemented with untreated serum. Addition of aprotinin to the heattreated serum gave a further reduction of the [¹²⁵I]glucagon degradation in the culture medium. A similar supplementation of Medium 199, used for culture of isolated islets, resulted in the most extensive glucagon accumulation in the culture medium. Islets cultured free-floating or attached to the bottom of the culture dishes contained similar amounts of glucagon. However, the free-floating islets released less glucagon when tested in short-term experiments performed at the end of the 1 wk culture period. A comparison between different culture media showed that islets cultured in RPMI-1640 had the highest glucagon content and released most glucagon to the culture medium. Moreover, these islets responded most actively to an acute arginine challenge at the end of the culture period. The present data suggest that the optimal conditions for culture of isolated islets aimed at studies of glucagon production may be obtained by using a culture medium consisting of RPMI-1640 supplemented with both a proteinase inhibitor and heat-inactivated serum. This work was supported by grants from the Swedish Medical Research Council (12X-109), the Nordic Insulin Fund, and the Swedish Diabetes Association.     

In vitro cultivation of Dipetalonema viteae third-stage larvae: evaluation of culture media, serum, and other supplements

Third-stage larvae of Dipetalonema viteae obtained from the tick vector developed to the fourth stage in several cell-free culture systems. Survival and development of larvae in a number of commercially available cell culture media, supplemented with serum and other defined and undefined components, were compared. All cultures were gassed with 5% carbon dioxide in nitrogen. Best survival, growth and development were obtained in stationary cultures containing 1:1 (v/v) mixtures of NCTC 135, either RPMI 1640 or Iscove's Modified Dulbecco's Medium, and a supplement of 20% non-heat-inactivated fetal bovine serum. The importance of the medium composition and physical environment of the culture system, for the survival, growth and development of D. viteae was demonstrated.     

Biological activity of cryopreserved bovine spermatogonial stem cells during in vitro culture

Functional roles of spermatogonial stem cells in spermatogenesis are self-renewing proliferation and production of differentiated daughter progeny. The ability to recapitulate these actions in vitro is important for investigating their biology and inducing genetic modification that could potentially lead to an alternative means of generating transgenic animals. The objective of this study was to evaluate the survival and proliferation of frozen-thawed bovine spermatogonial stem cells in vitro and investigate the effects of exogenous glial cell line-derived neurotrophic factor (GDNF). In order to accomplish this objective we developed a bovine embryonic fibroblast feeder cell line, termed BEF, to serve as feeder cells in a coculture system with bovine germ cells. Bovine spermatogonial stem cell survival and proliferation in vitro were evaluated by xenogeneic transplantation into the seminiferous tubules of immunodeficient mice. Bovine germ cells cocultured for 1 wk resulted in significantly more round cell donor colonies in recipient mouse testes compared to donor cells transplanted just after thawing. Bovine germ cells cocultured for 2 wk had fewer colony-forming cells than the freshly thawed cell suspensions or cells cultured for 1 wk. Characterization of the feeder cell line revealed endogenous expression of Gdnf mRNA and protein. Addition of exogenous GDNF to the culture medium decreased the number of stem cells present at 1 wk of coculture, but enhanced stem cell maintenance at 2 wk compared to cultures without added GDNF. These data indicate that frozen-thawed bovine spermatogonial stem cells survive cryopreservation and can be maintained during coculture with a feeder cell line in which the maintenance is influenced by GDNF.     

Culture conditions for maintaining the survival and mitotic activity of rainbow trout transplantable type A spermatogonia

Germ-cell transplantation is a powerful tool for studying gametogenesis in many species. We previously showed that spermatogonia transplanted into the peritoneal cavity of trout hatchlings were able to colonize recipient gonads, and produced fully functional sperm and eggs in synchrony with the germ cells of the recipient. An in vitro-culture system enabling spermatogonia to expand, when combined with transplantation, would be valuable in both basic and applied biology. To this end, we optimized culture conditions for type A spermatogonia in the present study using immature rainbow trout at 8-10 month of age. Spermatogonial survival and mitotic activity were improved during culture in Leibovitz's L-15 medium (pH 7.8) supplemented with 10% fetal bovine serum at 10 degrees C compared with culture under standard conditions for salmonids (Hank's MEM (pH 7.3) supplemented with 25 mM HEPES and 5% FBS, and culture at 20 degrees C). Elimination of testicular somatic cells promoted spermatogonial mitotic activity. In addition, insulin, trout embryonic extract, and basic fibroblast growth factor promoted the mitosis of purified spermatogonia in an additive manner. Mitotic activity increased nearly sevenfold over 19 days of culture compared with growth factor-free conditions and was maintained for >1 month. Furthermore, the cultured spermatogonia could colonize and proliferate in recipient gonads following transplantation. This study represents the first step towards establishing a cell line that can be transplanted for use in surrogate broodstock technology and cell-mediated gene-transfer systems.     

Possible direct effect of diethylcarbamazine on the infective larvae of Brugia pahangi

The direct action of diethylcarbamazine (DEC) on the infective larvae of Brugia pahangi was studied. The larvae were cultured in RPMI 1640 supplemented with foetal bovine serum and antibiotics for 22 days. Most of the larvae remained alive for 8 days, but survival rate of larvae decreased rapidly from day 10 onwards. The larvae did not grow in the culture system. The addition of DEC did not affect the morbidity of the larvae and no difference was observed in the morphological characteristics between the larvae cultured in the presence or absence of DEC. The infective larvae were cultured in vitro for 5 days in the presence or absence of DEC, and inoculated into jirds. The animals were necropsied at intervals, and developing larvae and adult worms were recovered. When the larvae were cultured without DEC and then inoculated subcutaneously into jirds, 29.8% of the inoculum was recovered 3-15 days, and 25% 19-22 weeks, post-inoculation. However, when the larvae were exposed to DEC in vitro and inoculated into jirds, the rate of recovery was reduced to 25% 3-15 days post-inoculation and 2% after 19-22 weeks. When the control larvae cultured in vitro were inoculated intraperitoneally into jirds, 41.3% of inoculum was recovered 3-15 days, and 42.8% 19-22 weeks, post-inoculation. Again the corresponding value for larvae exposed to DEC in vitro was reduced to 19.8% 3-15 days, and 8% 19-22 weeks, post-inoculation. It was observed that the larvae exposed to DEC in vitro were retarded in their development in jirds.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of protoplasts,and culture and regeneration into plants in <Emphasis Type="Italic">Alstroemeria</Emphasis>

Summary An efficient system for the regeneration of plants from protoplasts was developed in Alstroemeria. Friable embryogenic callus (FEC) proved to be the best source for protoplast isolation and culture when compared with leaf tissue and compact embryogenic callus. Protoplast isolation was most efficient when FEC was cultured under vacuum for 5 min in an enzyme solution consisting of 4% cellulase, 0.5% Driselase and 0.2% Macerozyme, followed by culture for 12–16h in the dark at 24°C. Cell wall formation and colony formation were better in a liquid medium than on a semi-solid agarose medium. Micro-calluses were formed after 4 wk of culture. Ninety percent of the micro-calluses developed into FEC after 12wk of culture on proliferation medium. FEC cultures produced somatic embryos on a regeneration medium and half of these somatic embryos developed shoots. Protoplast-derived plants showed more somaclonal variation than vegetatively propagated control plants.     

The transmission of an avian filarioid Cardiofilaria nilesi to the jird, Meriones unguiculatus

This paper reports the experimental transmission of a bird parasite into jirds. Infective larvae of Cardiofilaria nilesi obtained from laboratory colonized Coquillettidia crassipes mosquitoes which had fed on an infected chicken were inoculated subcutaneously into jirds. The number of larvae per jird varied from 10 to 228. Microfilaraemia appeared 22 to 89 days after inoculation of the infective larvae. Experiments were carried out with 24 jirds through six generations extending over a period of 22 months and 17 produced patent infections. At present 8 infected jirds are being maintained in the laboratory; their patent periods ranging from 6 to 13 months. However, the longest patent period observed was about thirteen months. The percentage of adults recovered in autopsied jirds ranged from 0 to 40 with an average of 16. The chicken showed a microfilarial periodicity with the peak microfilarial density around 2200 hours. However, in jirds there was a change in sub-periodicity. This model in the jird may be very useful for the screening of filaricides and in immunological work.     

Concomitant immunity in a rodent model of filariasis: the infection of Meriones unguiculatus with Acanthocheilonema viteae

In an attempt to study the occurrence of concomitant immunity in filarial infections, jirds (Meriones unguiculatus) were experimentally infected with Acanthocheilonema viteae, and patent animals were superinfected with a defined dose of A. viteae stage 3 larvae (L3). Infected animals harbored significantly less worms deriving from the superinfection than the control group (P < 0.05, 56.2%, and 63.4% protection), as shown by analysis of female worms 6 wk after superinfection on the basis of their developmental status and their length. This protection was not due to contact with L3 antigens because a significant reduction of worm burdens deriving of a superinfection was also observed after subcutaneous implantation of a single female worm (P < 0.05, 40.2% and 64.9% protection). The induced protective responses target L3 and restrict their migration because an established infection resulted in a reduction of L3 recovery (95.6% and 94.3%, P < 0.001) from tissues of jirds at day 5 after superinfection. Other data show that L3 from a superinfection are trapped within eosinophil-rich granulomas, which is likely to create unfavorable conditions for the worms and to lead to later death. Taken together, established A. viteae-infections partially protect hosts against homologous superinfection by an immune-mediated mechanism and, thus, regulate the population density of the parasites within the host by concomitant immunity.     

Development in vitro of the metacercaria of Bucephaloides gracilescens (Trematoda: Bucephalidae)

The development of Bucephaloides gracilescens metacercariae was studied using a range of cultivation conditions. The most rapid development occurred at 18°C in a medium containing NCTC 135 supplemented with 25% chicken serum, 25% hen egg yolk and 25% hen egg albumen, with a gas phase of air. Under these conditions, shell-protein synthesis was triggered by day 3 in culture; secondary oocytes were apparent in the ovary by day 10; and egg production began by day 14. Survival of worms in media containing chicken serum was twice as long as that achieved with either whiting or angler fish serum. The ingestion of yolk (feeding) appeared to be a necessary prerequisite to development and egg production. The presence of yolk in the culture medium greatly increased the amount of ³H-thymidine incorporated by the reproductive system of freshly excysted metacercariae but had little effect on the uptake and incorporation of tyrosine. The eggs produced in vitro failed to embryonale and were abnormal in appearance, being non-operculate with irregularly thickened shells.     

Production of lipoprotein(a) by primary baboon hepatocytes

Primary baboon hepatocytes were cultured in a serum-free medium formulation that permitted the analysis of lipoprotein(a) (Lp(a] production by the cells. The hepatocytes were determined to synthesize Lp(a) on the basis of the following observations: (1) the culture medium reacted in an ELISA designed for detection of baboon Lp(a) in serum samples; (2) the Lp(a)-specific protein, apo(a), was detected in the culture medium by immunoblotting techniques; (3) the unique protein structure of Lp(a) was demonstrated (i.e., association of apo(a) with apoB via interchain disulfide bonds to form apoLp(a]; and (4) the Lp(a) proteins occurred in the medium at a density of about 1.05 g/ml when subjected to density gradient ultracentrifugation. De novo synthesis of Lp(a) by cultured hepatocytes was demonstrated by incorporation of [35S]cysteine. Lp(a) was produced by the hepatocytes throughout a 20 day culture period. Finally, apo(a) isoform patterns in the hepatocyte culture medium and the hepatocyte donors' serum were indistinguishable.     

Characteristics of strawberry plants propagated by in vitro bioreactor culture and ex vitro propagation method

Reproducible protocol for regeneration of complete plantlets from ‘Bounty’ strawberry (Fragaria ananassa Duch.), using a combination of gelled medium and bioreactor system, has been standardized. Sepals, leaf discs, and petiole halves produced multiple buds and shoots when cultured on semi solid‐gelled medium containing 4 μM thidiazuron (TDZ) for 4 wk followed by transferring in liquid medium containing 2 μM TDZ in a bioreactor system and cultured for another 4 wk. TDZ induced shoot proliferation at 0.1 μM in the bioreactor system but inhibited shoot elongation. TDZ‐induced shoots were elongated and rooted in vitro on gelled medium containing 2 μM zeatin. Such bioreactor‐derived tissue culture (BC) plantlets obtained from sepal explants were grown ex vitro and compared with those propagated by tissue culture on gelled medium (GC) and by conventional runner cuttings (RC), for growth, morphology, anthocyanin content, and antioxidant activity after three growth seasons. The BC and GC plants produced more crowns, runners, leaves, and berries than the RC plants although berry weight per plant did not differ significantly. BC and GC plants produced berries with more anthocyanin contents and antioxidant activities than those produced by the RC plants. However, intersimple sequence repeat (ISSR) marker assay produced a homogenous amplification profile in the tissue culture and donor control plants confirming the clonal fidelity of micropropagated plants. In vitro culture on TDZ and zeatin‐containing nutrient media apparently induced the juvenile branching characteristics that favored enhanced vegetative growth with more crown, runners, leaf, and berry production.     

Induction of protection against Brugia malayi infection in jirds by microfilarial antigens

The development of immunologic methods to reduce transmission of human lymphatic filariasis depends on measures that will enhance the host's ability to eliminate infective larvae, adult worms, or blood-borne microfilariae (mf). The present study was designed to assess the capacity of a crude extract of Brugia malayi mf to decrease the level of microfilaremia and adult worm burden in jirds inoculated with infective larvae, and to identify the filarial antigens that elicit antibody responses in these animals. Thirty weeks after subcutaneous inoculation with 75 infective larvae, 100% of control jirds were patent (i.e., had microfilaremia) compared with 60% of the group immunized with 10 micrograms of crude microfilarial extract (p less than 0.05). In addition, microfilaremia was lower in patent immunized animals compared with controls (p less than 0.05). The mean total number of adult female B. malayi per jird recovered at necropsy in control animals was 16.0 vs 7.0 in immunized jirds (p less than 0.05). Serum of immunized jirds contained anti-mf antibodies with an end titer of 1:8000, a value similar to that of animals with chronic B. malayi infection. Microfilarial antigens of Mr approximately 150,000, 75,000, 42,000, and 25,000 were identified in immunoblotting studies by reactivity with antibodies in sera of immunized jirds. Antibodies induced by immunization with microfilarial extract were not specific for this stage of the parasite life cycle, as jird anti-mf antibodies reacted with a Mr approximately 150,000 and several Mr 50,000 to 110,000 antigens derived from immature and mature adult parasites of both sexes. These data indicate that immunization of jirds with a water soluble microfilarial extract enhances the host's ability to eliminate adult worms and blood-borne mf. The filarial antigens that induce antibodies in immunized jirds have been identified.