Guidelines on good clinical laboratory practice: bridging operations between research and clinical research laboratories.

A set of Good Clinical Laboratory Practice (GCLP) standards that embraces both the research and clinical aspects of GLP were developed utilizing a variety of collected regulatory and guidance material. We describe eleven core elements that constitute the GCLP standards with the objective of filling a gap for laboratory guidance, based on IND sponsor requirements, for conducting laboratory testing using specimens from human clinical trials. These GCLP standards provide guidance on implementing GLP requirements that are critical for laboratory operations, such as performance of protocol-mandated safety assays, peripheral blood mononuclear cell processing and immunological or endpoint assays from biological interventions on IND-registered clinical trials. The expectation is that compliance with the GCLP standards, monitored annually by external audits, will allow research and development laboratories to maintain data integrity and to provide immunogenicity, safety, and product efficacy data that is repeatable, reliable, auditable and that can be easily reconstructed in a research setting.

Quantitative collection and proteolytic activity of preacetabular gland enzyme (s) of cercariae of schistosoma mansoni.

Protease-containing preacetabular gland secretion can be collected from cercariae of Schistosoma mansoni by stimulation with penetration-inducing skin surface lipid, but the method lacks quantitative control because of variability of the lipid. Two commercially available free fatty acid fractions of skin lipid active in stimulating cercariae to penetrate skin, linolenic and linoleic acids, were substituted for skin surface lipid in a technique which provides an improved method of secretion collection. The pattern of protease activity of secreted enzyme (s) was followed throughout patency of infection of a group of snails with the same date of exposure. Day-to-day variability was a characteristic feature of all parameters studied. Major trends were elevated enzyme activity and cercarial emergence during midpatency.

Effect of a cryopreserved live vaccine on resistance in mice with a pre-existing Schistosoma mansoni infection.

Experiments were conducted to determine the effect of anti-schistosomal vaccine exposure on the level of pre-existing resistance in mice with a bisexual Schistosoma mansoni infection. C57BL/6 mice with light S. mansoni infections of 8 weeks duration were injected with 10 Krad-irradiated, cryopreserved and thawed schistosomules. Four weeks later the mice were exposed to normal cercariae, and adult worms collected by perfusion 6 weeks post-challenge. In 4 experiments, the baseline levels of resistance in infected mice ranged from 19% to 50% reduction in challenge worm burden (mean of 36%). Although vaccine administration slightly raised the overall level of resistance in infected mice (mean of 49%), in only 1 experiment was the increase over that in infected mice statistically significant. The levels of resistance attributed to the patent infection and vaccine exposure were not additive. Vaccine exposure had no effect on recovery of the adult worms of the primary infection.

Proteolytic, antigenic and immunogenic properties of Schistosoma mansoni cercarial secretion material.

Schistosoma mansoni cercariae secrete preacetabular gland material in association with skin penetration. The material is heterogeneous and contains proteolytic enzyme(s) which comprise a small proportion of the total protein. We have immunized mice with cercarial secretion material in several protocols designed to induce a variety of antibody responses. The cercarial secretion material is sufficiently immunogenic to induce precipitating and reaginic antibody production. The antibodies obtained were not of the necessary quantity and/or quality to afford protection from a challenge infection.

Schistosoma mansoni: radiation dose and morphologic integrity of schistosomules as factors for an effective cryopreserved live vaccine.

The effectiveness of a cryopreserved, irradiated schistosomule vaccine against an homologous Schistosoma mansoni cercarial challenge was tested in C57B1/6 mice. Highly significant levels of protection developed consistently when mice were immunized with the vaccine irradiated at 10-20 Krad, i.e., doses below that considered optimal for irradiated cercariae (50 Krad). Cryopreserved schistosomules irradiated at 10 or 20 Krad induced greater levels of protection than did schistosomules irradiated at 2, 5, 30, or 50 Krad. Protective immunity developed as early as 3 weeks post-immunization. When immunizing inocula were injected at various times post-thaw, or when schistosomule subpopulations of normal-appearing, damaged or dead organisms were injected, those populations which had appeared to sustain the least degree of damage were those most capable of stimulating protective immunity. These findings highlight the hazards of extrapolating conditions considered standard for an irradiated cercarial vaccine to one in which cryopreservation, for storage of the schistosomules, is an added stress.

Cryopreservation of schistosomules of Schistosoma mansoni in quantity.

Large quantities of Schistosoma mansoni schistosomules were cryopreserved in 35% ethanediol, and attempts were made to improve their morphology and infectivity after thawing. Several variables affected the appearance of the thawed organisms. These included: 1) the particular in vitro method used to transform cercariae to schistosomules; 2) addition of serotonin to the thawing medium; 3) changing the thawing temperature; and 4) culturing schistosomules for extended post-thaw times before assessing their condition. Varying either the postemergence age of the cercariae before transformation or the concentration of organisms in the freezing suspension had no effect on their survival. Although damage to many of the thawed schistosomules became apparent only upon extended in vitro cultivation, the addition of fetal calf serum to the thawing medium usually retarded this deterioration. When injected into mice, approximately 5% of cryopreserved schistosomules matured to adult worms, representing 71% of the value for maturation of unfrozen schistosomules. These studies define conditions of importance for the successful preservation of very large quantities of schistosomules (up to 500,000 in 1 ml volume) in liquid nitrogen.

Pathology of a live attenuated anti-schistosome vaccine in mice.

Tissue responses of mice to intramuscular injection of 50 kR 60Co-attenuated schistosomula of Schistosoma mansoni were studied. Controls included injection of unattenuated schistosomula, medium alone, antigen-coated beads, and alum-adsorbed tetanus/diphtheria toxoids. Primary reactions to tissue-confined deposits of injected schistosomula, whether attenuated or not, were relatively intense and prolonged. Parasite attrition proceeded steadily, with most destroyed by the 7th day; however, a few intact organisms persisted up to 4 weeks. Cryopreservation did not alter the course of parasite attrition nor host reaction. Irradiated larvae were not found in lymph nodes, lungs, or liver. Neutrophils dominated the early reactions and were gradually replaced by mononuclear phagocytes, lymphoid cells, and eosinophils. Fibroblast proliferation and muscle regeneration began by day 3; reaction size and intensity peaked by day 7. From weeks 1-4, inflammatory infiltrates and regenerative proliferation underwent gradual involution, and injection sites were healed with no scarring by the end of 4-5 weeks. Mice primed by infection or by prior injection showed an accelerated course of inflammation, enhanced tissue eosinophilia, and more rapid healing. An unwanted, but prominent, feature of schistosomular vaccine reactions in mice was tracking of the inflammatory infiltrate along connective tissue septal and nerve sheaths, the latter raising the question of the pain potential of the vaccine. To conclude, in mice, attenuated schistosomular vaccines cause relatively marked local inflammatory responses but no systemic lesions at all, and their injection sites heal without permanent damage.

Resistance of mice to secondary infection with Schistosoma mansoni. I. Comparison of bisexual and unisexual initial infections.

Mice receiving a unisexual primary infection with either sex of Schistosoma mansoni did not develop detectable resistance to reinfection. In contrast, mice receiving a bisexual primary infection developed a high degree of resistance. The number of adult worms developing from the challenge infection was reduced, relative to controls, by 72--100% at challenge times of 6 weeks or greater.

Large-scale laboratory maintenance of Schistosoma mansoni, with observations on three schistosome/snail host combinations.

This review discusses the large-scale laboratory maintenance of Schistosoma mansoni. Emphasized are features which increase efficiency in such facilities, and problems most frequently encountered. Profiles are given of the long-term, high-level production of 3 strains of S. mansoni. Two of the strains, NMRI and PR-1, were of Puerto Rican origin and the other, LE, was from Brazil. Three to 8 million cercariae of each strain were usually obtained per week. The most obvious differences between the 3 strains were cercarial output per snail and snail mortality rates. Maintenance problems encountered were usually related to water quality, temperature, genetics of the parasite or snail host, predators or contaminants, feeding, or crowding of snails. Examination of the production data from these 3 life cycles led to identification of features that could be of benefit for increasing the productivity and efficiency of other S. mansoni life cycles used in research activities.

Schistosoma mansoni: localization of calcium-detecting reagents in electron-lucent areas of specific preacetabular gland granules.

In an attempt to establish the exact location of calcium within the preacetabular glands of cercariae of Schistosoma mansoni, these larvae were exposed to reagents (potassium oxalate, potassium pyroantimonate, chloranilic acid, and silver nitrate) useful in the detection of calcium, and were subsequently observed with the aid of light and electron microscopes. Cercariae incubated in potassium oxalate and viewed in polarized light showed birefringence only in the preacetabular gland funduses. At the ultrastructural level, the preacetabular glands of potassium oxalate-treated cercariae had no electron-dense precipitate, but instead had translucent, irregularly shaped inclusions, similar to spaces left by volatilized calcium oxalate as described by others. Pyroantimonate treatment, on the other hand, localized the reaction in the electron-lucent areas of the light-spotted granules. The von Kossa silver nitrate procedure destroyed the secretory granules; therefore, an electron-dense precipitate was distributed throughout the gland. However, pretreatment with chloranilic acid before fixation preserved the granules, and subsequent exposure to the von Kossa silver nitrate gave a reaction identical to that obtained with the pyroantimonate alone. When viewed in polarized light, chloranilic acid-incubated cercariae showed birefringence in the fundus and duct areas.

Schistosoma mansoni: stimulus and transformation of cercariae into schistosomules.

Schistosoma mansoni schistosomules prepared from cercariae by seven in vitro techniques had not all reached the same state of development at the end of the incubation period as scored by seven parameters: water tolerance; Cercarienhüllen Reaktion; presence of the glycocalyx; condition of the surface membrane; nuclear state; granule migration; and cryopreservability. At the end of the specific incubation period for each technique, the level of development was judged with respect to schistosomules which had developed in situ for 1 hr after penetration of the ear skin of mice. In descending order of their correspondence to in vivo schistosomules, those derived in vitro (by the procedures listed) ranked as follows: first, penetration of dried rat skin; second, centrifuging and vortexing, or incubation in serum-supplemented medium; and third, syringe passage, omnimixing, centrifuging, and incubating, or incubating alone. The only treatment common to all techniques was incubation in 37 C culture medium for 2 hr or more. This is suggested as the stimulus for the cercaria-to-schistosomule transformation.