The role of oxygen availability in the embryonation of Heterakis gallinarum eggs.

The importance of oxygen availability in the embryonation of the infective egg stages of the gastrointestinal nematode parasite Heterakis gallinarum was studied in the laboratory. Unembryonated H. gallinarum eggs were kept under either aerobic conditions by gassing with oxygen, or anaerobic conditions by gassing with the inert gas nitrogen, under a range of constant temperatures. Oxygenated eggs embryonated at a rate influenced by temperature. Conversely, eggs treated with nitrogen showed no embryonation although when these eggs were transferred from nitrogen to oxygen gas after 60 days of treatment, embryonation occurred. This demonstrated that oxygen is an essential requirement for H. gallinarum egg development, although undeveloped eggs remain viable, even after 60 days in low oxygen conditions. The effects of climate on the biology of free-living stages studied under constant laboratory conditions cannot be applied directly to the field where climatic factors exhibit daily cycles. The effect of fluctuating temperature on development was investigated by including an additional temperature group in which H. gallinarum eggs were kept under daily temperature cycles between 12 and 22°C. Cycles caused eggs to develop significantly earlier than those in the constant mean cycle temperature, 17°C, but significantly slower than those in constant 22°C suggesting that daily temperature cycles had an accelerating effect on H. gallinarum egg embryonation but did not accelerate to the higher temperature. These results suggest that daily fluctuations in temperature influence development of the free-living stages and so development cannot be accurately predicted on the basis of constant temperature culture.     

Burn Injury Reduces Neutrophil Directional Migration Speed in Microfluidic Devices

Thermal injury triggers a fulminant inflammatory cascade that heralds shock, end-organ failure, and ultimately sepsis and death. Emerging evidence points to a critical role for the innate immune system, and several studies had documented concurrent impairment in neutrophil chemotaxis with these post-burn inflammatory changes. While a few studies suggest that a link between neutrophil motility and patient mortality might exist, so far, cumbersome assays have prohibited exploration of the prognostic and diagnostic significance of chemotaxis after burn injury. To address this need, we developed a microfluidic device that is simple to operate and allows for precise and robust measurements of chemotaxis speed and persistence characteristics at single-cell resolution. Using this assay, we established a reference set of migration speed values for neutrophils from healthy subjects. Comparisons with samples from burn patients revealed impaired directional migration speed starting as early as 24 hours after burn injury, reaching a minimum at 72–120 hours, correlated to the size of the burn injury and potentially serving as an early indicator for concurrent infections. Further characterization of neutrophil chemotaxis using this new assay may have important diagnostic implications not only for burn patients but also for patients afflicted by other diseases that compromise neutrophil functions.     

Antibody-targeted photolysis: in vitro immunological, photophysical, and cytotoxic properties of monoclonal antibody-dextran-Sn(IV) chlorin e6 immunoconjugates.

A set of anti-melanoma immunoconjugates were prepared which contained chlorin e6: antibody molar ratios of 18.9:1, 11.2:1, 6.8:1, and 1.7:1. All immunoconjugates retained antigen binding activity regardless of the chromophore:antibody substitution ratio that was attained. In contrast, the ground-state absorption spectra of the immunoconjugates showed features which appeared to be dependent on the chromophore:antibody molar ratio. In addition, the quantum yield of singlet oxygen generated by the conjugated chromophores was observed to be significantly less than that observed with the unbound dye. Time-resolved absorbance spectroscopy of the chromophore excited triplet state indicated that the loss of singlet oxygen quantum yield resulted from diminished chromophore triplet yield. Analysis of data obtained from in vitro photolysis of target melanoma cells, in combination with that obtained from the immunochemical and photochemical studies, indicates that the observed immunoconjugate phototoxicity can be reasonably quantitatively represented by (1) the ability of the immunoconjugate to bind SK-MEL-2 cell surface antigen, (2) the amount of chromophore localized at the target cells by immunoconjugate binding, (3) the delivered dose of light at 634 nm, and (4) the singlet oxygen quantum yield of the antibody-bound photosensitizer. Though these data argue strongly for photolysis by the cumulative dosage of singlet oxygen at the cell membrane, nonetheless, the concurrent photoinduced release of other cytotoxic agents should not be ruled out.     

Developmental mutants isolated from wild-caught Xenopus laevis by gynogenesis and inbreeding

Xenopus laevis obtained from indigenous African populations are a rich source of mutants affecting development. Gynogenesis and inbreeding were used to isolate mutants affecting development from wild-caught Xenopus laevis females. Fourteen mutants were recovered from eight females tested. One mutant was recovered from each of two females. This load of 1.875 developmental mutants per female is similar to that found in the axolotl (Ambystoma mexicanum), a urodele amphibian, and is only slightly less than the load of mutants with major developmental effects found in Drosophila and man. These results suggest that the anuran amphibian Xenopus laevis, an ancestrally tetraploid species, has undergone extensive diploidization of developmentally important loci and that gynogenesis and inbreeding of wild-caught animals can provide adequate mutants at diploid loci for developmental genetic studies.     

Effects of in vivo ultrasound hyperthermia on natural killer cell cytotoxicity in the hamster

The effects of in vivo ultrasound irradiation of the spleen on immunological functions were assessed with an in vitro natural killer (NK) cell cytotoxic assay. Anesthetized hamsters were exposed to 1 MHz ultrasound at intensity levels currently being used clinically for therapeutic diathermy and hyperthermia (1-5 W/cm2, for 500 sec with constant beam scanning). Hyperthermic levels in the spleen ranged from 38-43 degrees C. Significant depression of natural killer (NK) cell activity was seen 4 h after spleen irradiation as compared to sham irradiated and normal animals. A return towards normal levels was observed in experimental groups at 24 h after exposure. Sham and normal animals were not significantly different in NK activity, indicating no significant stress-related immunosuppressive effects due to handling. Differential leukocyte counts taken for each exposure condition showed significant lymphopenia at 4, 8, and 16 h after exposure, near normal levels at 24 h, and complete recovery by 48 h. The number of circulating mononuclear cells at 4 h showed a dose-related suppression as the exposure intensities were increased.     

Long-term in vitro function of adult hepatocytes in a collagen sandwich configuration.

In an effort to reconstruct the cellular polarity normally found in the liver, adult rat hepatocytes were sandwiched between two layers of hydrated rat tail tendon collagen matrix. Functionally, sandwiched hepatocytes maintained the secretion of albumin, transferrin, fibrinogen, bile acids, and urea for at least 6 weeks, whereas cells cultured on a single layer of collagen gel ceased such secretion in 1-2 weeks. After 1 week of culture on a single layer of collagen gel, hepatocytes could still recover these lost functions when a second layer of collagen gel was applied. The exact nature of the substrate for constructing the sandwich system appeared to be unimportant as long as it allowed cellular attachment. Hepatocytes cultured in the sandwich system appeared to maintain a distribution of actin filaments similar to the in vivo state, whereas cells cultured on a single layer of collagen gel showed abnormal formation of stress fibers. These studies suggest that simple manipulations of the configuration of extracellular elements can dramatically alter the behavior of cultured hepatocytes.     

Transmission of feline immunodeficiency virus in domestic cats via artificial insemination.

The objective of this study was to determine whether semen from male domestic cats infected with feline immunodeficiency virus (FIV) can transmit virus to females. Twelve inseminations were performed by an intrauterine laparoscopic technique with fresh or cryopreserved electroejaculates from asymptomatic males chronically infected with the NCSU1 strain of FIV. Of six inseminations performed with fresh semen, three resulted in infection of queens, as indicated by seroconversion, expression of FIV gag provirus in peripheral blood leukocytes, and reduced peripheral CD4+/CD8+ T-lymphocyte ratios. None of the six inseminates with thawed cryopreserved semen resulted in infection. Two infected queens and one uninfected queen became pregnant. Virus was not evident in the seven offspring. We conclude that FIV can be transmitted horizontally by artificial insemination with fresh semen.     

Engineering organ perfusion protocols: NMR analysis of hepatocyte isolation from perfused rat liver

The development and use of an extracorporeal liver support device depends upon the isolation of a large number of viable, functioning hepatocytes from whole or partial livers. Current practice, however, produces nonoptimal yields, given that a large percentage of hepatocytes initially present are not successfully isolated. The normal hepatocyte isolation protocol consists of sequential perfusion with calcium chelating and collagenase buffers, and then separation of viable hepatocytes from non-viable and nonparenchymal cells, usually on the basis of cell density. In order to improve understanding regarding the metabolic and perfusion state of the liver during this perfusion protocol, ATP, pH, and tissue perfusion were evaluated using nuclear magnetic resonance (NMR). Perfusion with calcium chelating buffer was found to have minimal effect on the metabolic and perfusion parameters, whereas subsequent perfusion with collagenase buffer produced large declines in ATP, pH, and homogeneity of perfusion within 3 min. Perfusion with calcium-chelating buffer alone, or perfusion with calcium chelating buffer followed by a short period of ischemia to mimic the perfusion disruption of collagenase, did not produce the same decline in metabolic parameters. This NMR data suggested that enhancing the early perfusion and penetration of collagenase or prolonging the nontoxic calcium-chelation step may improve the yield and/or functionality of isolated cells. Therefore, several altered perfusion protocols were evaluated in terms of yield of viable parenchymal hepatocytes and hepatocyte albumin production. Although increasing the perfusion flow rate and initial perfusion with inactive (cold) collagenase did not produce significant improvements when compared with the control protocol (control cell yield 226 +/- 42 x 10(6) viable hepatocytes for 10- to 14-week-old female Lewis rat), prolonging and enhancing the calcium-chelating perfusion step or increasing the collagenase concentration did yield a significantly great number of viable parenchymal hepatocytes (393 +/- 44 and 328 +/- 39 x 10(6) viable hepatocytes, respectively) with no change in albumin production per seeded viable cell. (c) 1994 John Wiley & Sons, Inc.