Biomedical subjects

J W Knight

Publications and source records attributed to J W Knight.

At least 37 records · Page 2Linked to original sources

PubMed1997

Plasmodium forresteri n. sp., from raptors in Florida and southern Georgia: its distinction from Plasmodium elongatum morphologically within and among host species and by vector susceptibility.

Plasmodium forresteri n. sp. naturally infects eastern screech-owls (Otus asio), great horned owls (Bubo virginianus), barred owls (Strix varia), bald eagles (Haliaeetus leucocephalus), red-shouldered hawks (Buteo lineatus), broad-winged hawks (Buteo platypterus), and red-tailed hawks (Buteo jamaicensis) in Florida and southern Georgia. Schizonts occur in mature or nearly mature erythrocytes, produce 2-6 merozoites arranged most commonly in fan or cruciform configuration, with mean dimensions among host species varying from 3.7 to 4.8 x 2.5 to 3.4 microns. Gametocytes are elongate, with mean dimensions among host species varying from 11.5 to 13.1 x 2.0 to 2.4 microns. One or both gametocyte margins are irregular and often crenulate. Gametocytes seldom fill the space between the erythrocyte nucleus and margin. Species characteristics were maintained in isodiagnostic Japanese quail (Coturnix japonica) and Pekin ducks (Anas platyrhynchos). In mosquito infection studies, only Culex restuans could support sporogony of P. forresteri, in contrast to Plasmodium elongatum of raptor origin that completed sporogony in both Cx. restuans and Culex nigripalpus.

Animals↗

PubMed1996

Rate limitations in posttranslational processing by the mammary gland of transgenic animals.

Our studies in transgenic animal bioreactors sought to determine the rate limitations in posttranslational processing of recombinant human protein C (rhPC) made in mammary gland of mice and pigs. Human protein C (hPC) is a complex plasma protein containing nine gamma-carboxylated glutamic acid (gla) residues that bind calcium at about 1 to 3 mM. Gamma carboxylation is a vitamin K-dependent posttranslational modification. The effect of rhPC synthesis rate on the extent of gamma-carboxylation of glutamic acid was studied. We have perturbed the biosynthesis of rhPC by using two different transgenes to direct mammary gland-specific expression. Promoter elements of the murine whey acid protein (mWAP) gene were used to drive the expression of hPC-cDNA and hPC-genomic transgenes. Transgenic mice with hPC-cDNA and hPC-genomic sequences gave expression levels of 11 +/- 4 micrograms rhPC/ml of milk and 895 +/- 21 micrograms rhPC/ml of milk, respectively. Transgenic pigs with hPC-cDNA and hPC-genomic sequences gave expression levels of 100 to 500 micrograms rhPC/ml of milk and 800 to 2000 micrograms rhPC/ml of milk, respectively. A monoclonal antibody (7D7B10-mAb) that binds an epitope in the gla domain of hPC in the absence of calcium was used to study the conformational behavior of immunopurified rhPC. Immunopurified rhPC from lower expressing mice and pigs gave a calcium-dependent binding inhibition by 7D7B10-mAb similar to that of hPC. Immunopurified rhPC from higher expressing mice and pigs gave a less calcium-dependent response. This study suggests that a rate limitation in gamma-carboxylation by the mammary gland occurs at expression levels about > 20 micrograms/ml in mice and > 500 micrograms/ml in pigs.

Amino Acid Sequence↗

PubMed1995

Comparison of migration and encapsulation of Brugia malayi microfilariae from the midgut to the hemocoel between Anopheles quadrimaculatus and Aedes aegypti.

Comparisons were made of migration and encapsulation of ingested sheathed microfilariae of Brugia malayi from the midgut into the hemocoel between Anopheles quadrimaculatus (refractory and susceptible strains to B. malayi) and Aedes aegypti (Black-eyed, Liverpool strain susceptible to B. malayi). Encapsulation and melanization of microfilarial sheaths and microfilariae occurred in both strains of An. quadrimaculatus and in Ae. aegypti. In both strains of An. quadrimaculatus, by 4 hr and by 24 hr after the ingestion of sheathed microfilariae of B. malayi in the infected bloodmeal, significantly more sheathed microfilariae penetrated the midgut and reached the hemocoel and thoracic muscles compared with those in Ae. aegypti. During the same time periods significantly more encapsulated and melanized microfilarial sheaths and a larger percentage of encapsulated and melanized microfilariae were observed in the hemocoel of both strains of An. quadrimaculatus than in Ae. aegypti. The results suggest that differences observed in the numbers of encapsulated and melanized microfilarial sheaths and percentages of melanized microfilariae between An. quadrimaculatus (both strains) and Ae. aegypti are due to different rates of penetration of the sheathed microfilariae from the midgut to the hemocoel.

Aedes↗

PubMed1995

Lectin binding to extracellularly melanized microfilariae of Brugia malayi from the hemocoel of Anopheles quadrimaculatus.

Binding patterns of fluorescein isothiocyanate (FITC)- and gold-conjugated lectins to extracellularly melanized sheathed and exsheathed microfilariae of subperiodic Brugia malayi, isolated from and in situ in the abdominal hemocoel of Anopheles quadrimaculatus 72-hr postinfection, were examined. Five FITC-conjugated lectins [Helix pomatia agglutinin (HPA), Arachis hypogaea (peanut agglutinin-PNA), Triticum vulgaris (wheat germ agglutinin-WGA), Lens culinaris (lentil-LCH), and Concanavalin A (Con A)] with specificities for different carbohydrate moieties were tested for binding to isolated melanized microfilariae and observed with transmitted light and fluorescence microscopy. All five FITC-lectins bound strongly to the acellular material accompanying the melanin deposits on the surface of isolated melanized microfilariae. Significant inhibition of FITC-lectin binding occurred when lectins were preincubated with their complementary carbohydrates before testing. H. pomatia agglutinin binding was totally inhibited by N-acetyl-D-glucosamine and N-acetyl-D-galactosamine. Other lectins were partially inhibited, such as PNA by galactose and lactose; WGA by N-acetylneuraminic acid; LCH by N-acetyl-D-glucosamine, mannose, glucose, and methyl alpha-D-mannopyranoside; and Con A by mannose and methyl alpha-D-mannopyranoside. Three gold-conjugated lectins (HPA, PNA, and Con A), examined by using transmission electron microscopy, bound to the outer surface of the acellular material associated with the melanin deposits on isolated melanized microfilarial sheaths and melanized microfilariae and to the remnants of lysed hemocytes found in the proximity of the melanized deposits. Con A in the presence of gold-labeled horseradish peroxidase, examined by using transmission electron microscopy, showed random binding within the melanized capsule formed around the microfilarial sheath in situ. These results indicate that the acellular material accompanying melanin deposits on melanized microfilarial sheaths and sheathed and exsheathed microfilariae contain several glycoconjugates with exposed carbohydrate moieties and are possibly glycoproteins. These glycoproteins could be the by-products of the activation of the prophenoloxidase by the microfilariae.

Animals↗

PubMed1995

Effect of uterine space and fetal sex on conceptus development and in vitro release of progesterone and estrone from regions of the porcine placenta throughout gestation.

Intact (n = 17) and unilaterally hysterectomized-ovariectomized (UHOX, n = 19) gilts were used to study conceptus development and the steroidogenic capability of three regions of the porcine placenta under normal and crowded intrauterine conditions. Gilts were hysterectomized on either Days 30, 50, 70, or 90 of gestation. Placentas were combined according to the sex of the fetus associated with the placental unit, except at Day 30, when all were combined. Placentas were cut into three regions: middle, inner, and polar. Placental tissues were incubated for 0, 1, 5, and 3 hr, and concentrations of progesterone (P4) and estrone (E1) in the incubation medium were determined. Fetal survival rate was greater (P < 0.01, 0.05, and 0.001 at days 50, 70, and 90, respectively) in intact than in UHOX gilts. Placental length and weight, fetal length and weight, and allantoic fluid volume were greater (P < 0.05, 0.001, 0.05, and 0.001, respectively) in intact than in UHOX gilts. P4 concentration was lower (P < 0.001, 0.002, and 0.001 at Days 50, 70, and 90, respectively) in the incubation medium of polar region placenta compared with the other regions. The release of P4 was greater (P < 0.005) from the placentas of intact versus UHOX gilts at Day 90 but was not different at Days 30, 50, or 70. The sex of the fetus did not affect placental P4 release. The region of the placenta affected E1 release at Day 30 (P < 0.01, greater in polar section) and Day 50 (P < 0.06, less in polar section). There was no effect of uterine status on E1 release.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Effect of intrauterine position of conceptus development, placental and endometrial release of progesterone and estrone in vitro, and concentration of steroid hormones in fetal fluids throughout gestation in swine.

Twenty intact and 25 unilaterally hysterectomized-ovariectomized gilts were used to study the effect of fetal intrauterine position on conceptus development, concentrations of endogenous progesterone (P4) and estrone (E1) in the placenta and endometrium, and steroid hormone concentrations in amniotic and allantoic fluids during gestation. Gilts were hysterectomized at 40, 60, 80, or 100 d of gestation. Placental and endometrial tissues were pooled individually by the intrauterine position of the associated fetus and incubated separately, and concentrations of P4 and E1 in the medium were determined. Uterine status (intact or unilaterally hysterectomized-ovariectomized) did not affect any of the variables measured. Intrauterine position affected fetal and placental weights (P < 0.02 and 0.01, respectively) only at Day 40 of gestation. The weights of female fetuses bordered in utero by two males and their associated placentas were lower than those from other intrauterine positions. Intrauterine position had no effect on placental and endometrial P4 release or on E1, P4, and androgen concentrations in fetal fluids at any stage of gestation. At Day 100 of gestation, placentas associated with fetuses bordered by those of the same sex released more E1 than did placentas associated with fetuses bordered by those of the same opposite sex (P < 0.01). This study indicates that intrauterine position in swine has a limited effect on conceptus development and on placental and endometrial steroidogenic activity.

Allantois↗

PubMed1995

Nature of endothelin binding in the porcine ovary.

We investigated the nature of endothelin (ET) binding in the porcine ovary. We demonstrated the presence of high affinity (Kd = 0.72; 95% confidence interval = 0.43-1.1 nM) binding sites for ET-1 in the porcine ovary. The binding capacity for this ET-1-specific binding site was 97 pmol/micrograms DNA (95% confidence interval = 90-107). Autoradiographic studies showed that putative ET receptors reside in the granulosa cell layer of the maturing Graafian follicle and in the vascular components of the corpora lutea. The relative abundance of ET receptors was greatest in granulosa cells of large antral follicles, whereas ET binding was absent in granulosa cells of preantral follicles and in luteal cells. ET binding by cultured granulosa cells was further characterized by RRA and shown to exhibit a rank order of binding affinities for different ET isopeptides. The observed rank order indicates that the ET receptors present on granulosa cells are of the ET(A) receptor subtype. The radioreceptor studies also indicated that granulosa cells collected from large antral follicles (9-10 mm in diameter) have a greater binding capacity for [125I]ET-1 in culture than granulosa cells collected from smaller follicles. When cultured granulosa cells were exposed to 100 nM ET-1 or 14 nM 12-O-tetradecanoylphorbol 13-acetate overnight, the percentage of specific [125I]ET-1 binding was reduced (12% and 50%, respectively), indicating a down-regulation of the ET receptor by these treatments. In summary, we have characterized the distribution, isopeptide specificity, relative abundance, and down-regulation of putative ovarian endothelin receptors of subtype ET(A) on swine granulosa cells. Such results in conjunction with other available literature strongly suggest that granulosa cells of maturing Graafian follicles are targeted by ET-1. An additional physiological role for ET-1 in the ovary is suggested by the presence of putative ET receptors in the vasculature of the corpus luteum.

Animals↗

PubMed1994

Expression of a functional human complement inhibitor in a transgenic pig as a model for the prevention of xenogeneic hyperacute organ rejection.

The serious shortage of human organs available for transplantation has engendered a heightened interest in the use of animal organs (xenografts) for transplantation. However, the major barrier to successful discordant xenogeneic organ transplantation is the phenomenon of hyperacute rejection. Hyperacute rejection results from the deposition of high-titer preformed antibodies that activate serum complement on the luminal surface of the vascular endothelium, leading to vessel occlusion and graft failure within minutes to hours. Although endogenous membrane-associated complement inhibitors normally protect endothelial cells from autologous complement, they are species restricted and thus confer limited resistance to activated xenogeneic complement. To address the pathogenesis of hyperacute rejection in xenotransplantation, transgenic mice and a transgenic pig were engineered to express the human terminal complement inhibitor hCD59. High-level cell surface expression of hCD59 was achieved in a variety of murine and porcine cell types, most importantly on both large vessel and capillary endothelium. hCD59-expressing porcine cells were significantly resistant to challenge with high-titer anti-porcine antibody and human complement. These experiments demonstrate a strategy for developing a pig-to-primate xenogeneic transplantation model to test whether the expression of a human complement inhibitor in transgenic pigs could render xenogeneic organs resistant to hyperacute rejection.

Animals↗

PubMed1994

The porcine mammary gland as a bioreactor for complex proteins.

The similar biological activity of rhPC and hPC indicates that porcine mammary gland can perform many of the processing reactions necessary for recombinant synthesis of complex human proteins and produce them at levels suitable for industrial bioreactor applications. The health of the transgenic pigs appeared unaffected by the expression of high levels of the heterologous protein. We suggest that one of the advantages of using the mammary gland as a bioreactor appears to be the high cell density relative to that of cell culture.

Animals↗

PubMed1994

Aspects of placental estrogen synthesis in the pig.

Conceptus estrogen synthesis in the pig begins with the d 11 blastocyst and continues throughout pregnancy. Estrogens have been implicated as regulators of numerous in utero events related to conceptus survival and development. Studies conducted in our laboratory indicate that progesterone (P4) production by the porcine placenta increases steadily throughout gestation. Estrone (E1) production is triphasic with peaks between d 14-18, around d 30, and a sustained increase from d 70 until parturition. Addition of pregnenolone (P5) augments in vitro P4 and E1 production by both the placenta and the endometrium. Both estrogen concentrations and the ratio between conjugated and free estrogens change drastically during gestation. Evidence from our laboratory suggests that enhanced and prolonged sulfatase activity by d 90 of gestation, coupled with an increase in sulfatase activity-not a change in aromatase activity-contributes to the rise in free estrogens as parturition approaches. We assessed the relative importance of ovarian versus placental production of P4 as a mediator of prenatal survival, conceptus development, and steroidogenesis by the placenta and endometrium. Among other findings, exogenous administration of the non-aromatizable progestagen medroxyprogesterone acetate (MPA) to ovariectomized (OVX) gilts between either d 20-30 or d 60-70 of gestation did not adversely affect concepts survival or development, in vivo estrogen measurements, or in vitro placental steroidogenesis. We also demonstrated that pregnancy may also be maintained during d 20-30 and d 60-70 in OVX gilts administered large quantities of P5 exogenously, presumably due to placental P4 production. Recent studies have examined variables of region of the placenta and intrauterine position on conceptus development, and placental and endometrial steroidogenesis under normal and crowded intrauterine conditions. Results indicated 1) a differential release of P4 and E1 by different regions of the placenta at certain days of gestation, 2) no compensatory increase in steroidogenic activity of the pig placenta when total placental mass was reduced, and 3) a less pronounced effect of intrauterine position on steroidogenic activity in the pig compared with other litter-bearing species.

Animals↗

PubMed1994

Effect of culture conditions, donor age, and injection site on in vitro development of DNA microinjected porcine zygotes.

A series of experiments evaluated development of porcine zygotes microinjected with DNA in three culture media and two incubation temperatures, from postpubertal and prepubertal donors, and between zygotes injected with DNA into the pronucleus and the cytoplasm. Zygotes recovered from 36 postpubertal gilts in Exp. 1 were injected and cultured in modified NCSU-23, modified NCSU-37, and CZB media at 37 degrees C or 39 degrees C for 7 d. In Exp. 2, zygotes were collected from postpubertal or prepubertal gilts, microinjected with DNA, and cultured in modified NCSU-23. In Exp. 3 superovulated prepubertal gilts had DNA injected into the cytoplasm or pronucleus of zygotes. Mean percentages developing to the expanded or hatched blastocyst stage in modified NCSU-23 (42.9) and modified NCSU-37 (40.1) did not differ, but development was greater than that for zygotes cultured in CZB (8.8; P < .05). Development was greater at 39 degrees C (P < .05) than at 37 degrees C (36.5 vs 24.6%). Microinjection of DNA decreased development (P < .05) from that of noninjected controls (18.1 vs 43.1%). Zygotes from postpubertal gilts had a higher percentage (68.0) of expanded and hatched blastocysts than zygotes from prepubertal donors (29.0; P < .05). No development difference was found between DNA injection into the pronucleus (23.1%) or cytoplasm (17.4%), but development was less than for control embryos (64.9%; P < .05). DNA microinjected porcine zygotes can be successfully cultured to the expanded blastocyst stage in modified NCSU-23 and modified NCSU-37 media at 39 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Ultrastructural comparison of extracellular and intracellular encapsulation of Brugia malayi in Anopheles quadrimaculatus.

Ultrastructural aspects of extracellular humoral encapsulation of microfilariae of Brugia malayi in the hemocoel of Anopheles quadrimaculatus were compared with those of intracellular encapsulation of first-stage larvae (L1) of the same parasite species, in the thoracic muscle cells of the same species of mosquito. The results showed that extracellular humoral encapsulation of microfilarial sheaths, and sheathed and exsheathed microfilariae, in the hemocoel of mosquitoes occurs around the parasite within the first 6 hr postingestion, apparently without initial participation of hemocytes. Hemocytes and their remnants were observed near the parasite during the first 6 hr postingestion. Within the next 24 hr, hemocytes attach to the initial humoral capsule. By contrast, intracellular encapsulation of L1S is initiated by the accumulation of a dense cytoplasmic layer derived from the infected thoracic muscle cell. Melanin deposits accumulate in this layer adjacent to the parasite cuticle, again without visible participation of hemocytes.

Animals↗

PubMed1993

Intracellular development of subperiodic Brugia malayi influenced by mosquito thoracic muscle cells.

The in vitro development of 1-day-old intracellularly lodged larvae of Brugia malayi cultured in infected excised thoraces of selected susceptible and refractory strains of Aedes aegypti and Anopheles quadrimaculatus was compared with larvae reared in vivo. In susceptible mosquitoes, both in vitro and in vivo, larvae developed normally and abnormally. In refractory mosquitoes this pattern of both normal and abnormal development was also observed, except that comparatively fewer larvae developed to the infective third-stage larvae (L3) in vitro than in vivo and that more first-stage larvae (L1) were intracellularly melanized in vivo than in vitro. These studies indicate that factors in the thoracic muscle cells of the mosquito greatly affect the development of B. malayi microfilariae to L3. Intracellular melanization of L1 in An. quadrimaculatus, previously demonstrated in vivo, rarely occurred in vitro. These studies therefore suggest that refractoriness and melanization of B. malayi larvae in the thoraces of An. quadrimaculatus are controlled by two different and separate mechanisms.

Aedes↗

PubMed1992

Evaluation of systems for collection of porcine zygotes for DNA microinjection and transfer.

Crossbred gilts and sows (n=116) were used for the collection of 1-cell zygotes for DNA microinjection and transfer. Retrospectively, estrus synchronization and superovulation schemes were evaluated to assess practicality for zygote collection. Four synchronization and superovulation procedures were used: 1) sows were observed for natural estrous behavior; 1000 IU human chorionic gonadotrophin (hCG) was administered at the onset of estrus (NAT); 2) cyclic gilts were synchronized with 17.6 mg altrenogest (ALT)/day for 15 to 19 days followed by superovulation with 1500 IU pregnant mares serum gonadotropin (PMSG) and 500 IU hCG (LALT); 3) gilts between 11 and 16 days of the estrous cycle received 17.6 mg ALT for 5 to 9 days and PMSG and hCG were used to induce superovulation (SALT); and 4) precocious ovulation was induced in prepubertal gilts with PMSG and hCG (PRE). A total of 505 DNA microinjected embryos transferred into 17 recipients produced 7 litters and 50 piglets, of which 8 were transgenic. The NAT sows had less (P < 0.05) ovarian activity than gilts synchronized and superovulated by all the other procedures. Synchronization treatments with PMSG did not differ (P > 0.05) in the number of corpora hemorrhagica or unovulated follicles, but SALT and PRE treaments had higher ovulation rates than LALT (24.7 +/- 2.9, 24.3 +/- 1.8 vs 11.6 +/- 2.7 ovulations; X +/- SEM). The SALT and PRE treatments yielded 12.3 +/- 2.6 and 17.7 +/- 1.7 zygotes. Successful transgenesis was accomplished with SALT and PRE procedures for estrus synchronization and superovulation.

Journal Article↗

PubMed1992

In vitro development of zygotes from prepubertal gilts after microinjection of DNA.

The effect of pronuclear microinjection of DNA and culture in excised mouse oviducts on the development of porcine zygotes was assessed in this study. Precocious ovulation was induced in prepubertal gilts with pregnant mare's serum gonadotrophin and hCG. Zygotes received either pronuclear microinjection of buffer alone, buffer containing a DNA construct, or no microinjection. Zygotes were cultured in vitro in either modified Krebs-Ringer bicarbonate medium (KRB) for 144 h or in mouse oviduct (MO) explant culture with KRB for 48, 72, 96, or 120 h. Pronuclear microinjection of DNA resulted in a lower (P less than .05) cleavage index (CI) than did buffer or no microinjection (CI 2.16 +/- .10 vs 2.80 +/- .13 and 2.93 +/- .10). The CI loss was greatest for DNA-injected zygotes at the two-cell stage of development. Coculture of zygotes in MO resulted in a higher CI (P less than .01) than did culture in KRB. Culture in MO for 72 h was the most beneficial system compared with MO for 48, 96, or 120 h (P less than .05; CI 3.25 +/- .12 vs 2.66 +/- .18, 2.79 +/- .14, and 2.40 +/- .14, respectively). Microinjection of DNA, not merely the mechanical procedure, was detrimental to early zygote development and may be the cause of low pregnancy rates.

Animals↗

PubMed1992

Characterization of the intracellular melanization response in Anopheles quadrimaculatus against subperiodic Brugia malayi larvae.

Intracellular melanization, a defense or an immune response in the thoracic muscle cells, was investigated in a refractory strain of Anopheles quadrimaculatus infected with larvae of Brugia malayi. In mosquitoes fed on B. malayi-infected jirds, intracellular melanization against first-stage larvae (L1) was better expressed when fewer than 40 microfilariae reached the thoracic muscle cells than when more than 40 microfilariae reached the thoracic muscle cells. This result suggests that when large numbers of microfilariae invade the thoracic muscle cells, the immune response of the mosquito may become overloaded. Intracellular melanization response against L1 in the thoracic muscle cells also showed a significant decrease in older females (14-16-day-old) as compared to the younger ones (4-9-day-old). A comparison is made between intracellular and extracellular responses of mosquitoes to filarial larvae. It is significant that in both cases high rate of infection can reduce both the number and percentage of larvae melanized.

Animals↗

PubMed1992

Comparative susceptibility of species A, B and C of Anopheles quadrimaculatus complex to infection with subperiodic Brugia malayi and Brugia pahangi (Nematoda: Filarioidea).

Susceptibilities of natural populations of sibling species A, B and C of the Anopheles quadrimaculatus complex and the colonized strain A to subperiodic Brugia malayi and Brugia pahangi were compared. All 3 sibling species showed varying degrees of susceptibility to both B. pahangi and B. malayi, and they were considerably more susceptible to B. pahangi than to B. malayi. The rate and intensity of infection to B. pahangi were highest for species A (66.2% and 7.4 L3/female, respectively) and lowest for species B (21.3% and 1.7 L3/female). For B. malayi these values were higher for species A (29.7% and 1.84 L3/female) than for species B (13.3% and 0.86 L3/female) and C (12.6% and 0.75 L3/female). The colonized strain A of An. quadrimaculatus was significantly more susceptible to both Brugia species than the natural populations of sibling species A, B and C.

Animals↗

PubMed1991

In vitro development of Brugia pahangi and Brugia malayi in cultured mosquito thoraces.

In vitro cultivation of Brugia pahangi and subperiodic Brugia malayi one-day old larvae to infective stage larvae (L3) within thoraces excised from Aedes aegypti (Black Eye, Liverpool) and Anopheles quadrimaculatus was attempted. The mosquito thoraces were excised under aseptic conditions, 24 h after a blood meal on either B. pahangi- or B. malayi-infected jirds. The excised thoraces were washed aseptically and inoculated into a diphasic media. A nutrient agar base was overlaid with either Grace's insect cell culture medium or Schneider's Drosophila medium or a mixture (1:1) of these two media. Each overlay medium contained a 1 x concentration of antibiotic/antimycotic mixture plus 20% fetal bovine serum. The excised thoraces provided the intracellular milieu for development of Brugia larvae. In Grace's or Schneider's insect tissue culture medium alone, the filaria larvae of both species developed only to the second larval stage after 12 days; whereas, in a mixture (1:1) of Grace's and Schneider's media, some one-day old larvae of both Brugia species developed to the infective larval (L3) stage after 12 days. However, large numbers of both species of larvae developed to the infective larval stage when, prior to providing an infective blood meal, the mosquitoes of both species were fed 1 x concentration of antibiotic/antimycotic mixture in a 10% sucrose solution containing 0.1% p-aminobenzoic acid for 6 days. These results showed for the first time that if one-day old Brugia larvae are confined intracellularly in excised thoraces, they can then develop in insect tissue culture media without adding a feeder layer of mosquito cells or conditioning the media with mosquito cell lines.

Aedes↗