In vitro effects of sodium lauryl sulfate on DNA synthesis and fine structure of human thymocytes

Sodium lauryl sulfate, a model substance in testing skin irritability was examined with regard to its effects on DNA synthesis and fine structure of human thymocytes incubated in vitro. At 5 h, incorporation of 3H-thymidine into DNA was inhibited with a discernable effect at 3 X 10(-4) M in serum-free medium and at 6 X 10(-4) M in medium to containing 15% serum. Under the former conditions, the effect on DNA synthesis was accompanied by disintegration of the cells as observed electron-microscopically. In contrast, no clear effects on cell morphology were seen in the latter case, in spite of a strongly depressed replicative activity. After 48 h of culture in medium containing 15% serum, there was a marked decrease in cell number due to spontaneous cell death and, consequently, a low rate of DNA synthesis. Sodium lauryl sulfate suppressed the incorporation of 3H-thymidine into DNA at 1.2 X 10(-3) M. No clear alterations in cell morphology were observed at 3 X 10(-4) M, whereas signs of irreversible cell damage, including pyknotic nuclei, were seen at 6 X 10(-4) M and complete disruption of most cells occurred at 1.2 X 10(-3) M. The results show that sodium lauryl sulfate inhibits DNA synthesis in human thymocytes and that there is a partial discrepancy between this inhibition and the adverse effects on the structural organization of the cell. It is proposed that the irritant effect of sodium lauryl sulfate in vivo is due to the breaking up of lymphoid or other cells and a consequent release of material that gives rise to an inflammatory response.     

Morphology of the male genitalia and sperm fine structure in siluroid fish

Morphology of male genitalia and sperm fine structure of 4 siluroid fish, Callichthys thoracatus (Callichthyidae), Dysichthys coracoideus (Aspredinidae), Mystus armatus (Bagridae) and Ancistrus triradiatus (Loricariidae) were studied. In Dysichthys coracoideus and Ancistrus triradiatus the male genitalia consisted of paired testes and spermatic ducts but had no seminal vesicles. In Callichthys thoracatus the male genitalia had also paired seminal vesicles which were separated from the testis and emptied into the spermatic duct. Also in Mystus armatus seminal vesicles were found. They were not separated from the testes but the seminiferous tubules changed into seminal vesicle lobes which finally emptied into the spermatic ducts. All the investigated species had simply organized aquasperms typical for external fertilizing teleosts which were characterized by an ovoid or elliptoid head, by a small midpiece and by the absence of an acrosome. The spermatozoa of Dysichthys coracoideus and Mystus armatus were biflagellate, while they were monoflagellate in Ancistrus triradiatus and Callichthys thoracatus. The proximal centriole was reduced in Dysichthys coracoideus and Mystus armatus and the basal body attached to the nucleus. Single mitochondria were found in Dysichthys coracoideus, Ancistrus triradiatus, and Callichthys thoracatus. On the contrary, in Mystus armatus mitochondria were fused to a more complex chondriosome.     

In vitro assessment of antiborrelial activity of OspA vaccine sera

Prevention of Lyme disease by the recombinant OspA-based vaccine reportedly works by preventing transmission of spirochetes from ticks to humans. We report on an in vitro microculture assay, which can be used to provide an indicator of the need for booster doses of vaccine.     

In vitro study of cell survival following dynamic MLC intensity-modulated radiation therapy dose delivery

The possibility of reduced cell kill following intensity-modulated radiation therapy (IMRT) compared to conventional radiation therapy has been debated in the literature. This potential reduction in cell kill relates to prolonged treatment times typical of IMRT dose delivery and consequently increased repair of sublethal lesions. While there is some theoretical support to this reduction in cell kill published in the literature, direct experimental evidence specific to IMRT dose delivery patterns is lacking. In this study we present cell survival data for three cell lines: Chinese hamster V79 fibroblasts, human cervical carcinoma, SiHa and colon adenocarcinoma, WiDr. Cell survival was obtained for 2.1 Gy delivered as acute dose with parallel-opposed pair (POP), irradiation time 75 s, which served as a reference; regular seven-field IMRT, irradiation time 5 min; and IMRT with a break for multiple leaf collimator (MLC) re-initialization after three fields were delivered, irradiation time 10 min. An actual seven-field dynamic MLC IMRT plan for a head and neck patient was used. The IMRT plan was generated for a Varian EX or iX linear accelerator with 120 leaf Millenium MLC. Survival data were also collected for doses 1X, 2X, 3X, 4X, and 5x 2.1 Gy to establish parameters of the linear-quadratic equation describing survival following acute dose delivery. Cells were irradiated inside an acrylic cylindrical phantom specifically designed for this study. Doses from both IMRT and POP were validated using ion chamber measurements. A reproducible increase in cell survival was observed following IMRT dose delivery. This increase varied from small for V79, with a surviving fraction of 0.8326 following POP vs 0.8420 following uninterrupted IMRT, to very pronounced for SiHa, with a surviving fraction of 0.3903 following POP vs 0.5330 for uninterrupted IMRT. When compared to IMRT or IMRT with a break for MLC initialization, cell survival following acute dose delivery was significantly different, p < 0.05, in three out of six cases. In contrast, when cell survival following IMRT was compared to that following IMRT with a break for MLC initialization the difference was always statistically insignificant. When projected to a 30 fraction treatment, dose deficit to bring cell survival to the same value as in POP was calculated as 4.1, 24.9, and 31.1 Gy for V79, WiDr, and SiHa cell lines, respectively. The dose deficit did not relate to the alpha/beta ratio obtained in this study for the three cell lines. Clinical data do not show reduction in local control following IMRT. Possible reasons for this are discussed. The obtained data set can serve as a test data set for models designed to explore the effect of dose delivery prolongation/fractionation in IMRT on radiation therapy outcome.     

Photoreceptor fine structure in light- and dark-adaptation in the butterfly fish (Pantodon buchholzi)

The morphology of the retinal photoreceptors of the butterfly fish Pantodon buchholzi has been studied by electron microscopy in both light- and dark-adaptation. The photoreceptors in this species are readily divisible into rods and cones based on morphological criteria. No double or twin cones are present. The rod photoreceptors show marked retinomotor movements. In light-adaptation they are extremely elongate cells while in the dark-adapted state they are much shorter. Cones seem to respond but minimally to the circadian cycle. Rod outer segments are composed of membranous discs of uniform diameter displaying several incisures. The inner segment has a small distal ellipsoid and a thin myoid region which is lost in dark-adaptation. The nuclei of rods are condensed and always located vitread to the external limiting membrane. The rod synaptic spherule displays 2 or 3 invaginated sites. The single cones display a tapering outer segment. The wider inner segment contains a large electron-dense ellipsoid with small glycogen deposits located peripherally. The cone nuclei are large and vesicular and usually located sclerad to the external limiting membrane. The synaptic pedicle of cones is larger and more electron-lucent and contains more synaptic sites than do the rods. No mosaic pattern of arrangement of the photoreceptors is apparent. Except for the obvious lengthening or shortening of the rods, the morphology of the photoreceptors changes but little during the circadian cycle.     

In vitro effect of immune complex positive human sera on antibody dependent cellular cytotoxicity

The effect of immune complex positive and negative (control) sera on the antibody dependent cytotoxicity of normal human lymphocytes was studied in a xenogeneic assay in vitro. Significant inhibitions were obtained with the sera of immune complex positive patients as compared with age and sex matched controls. The possible significance of these findings in autoimmune diseases and malignancies is discussed.     

A comparative study on fine structure of inflammatory and neoplastic dysplasia of the esophagus

To clarify the fine structure of esophageal dysplasia, desmosomes and nuclei were evaluated numerically and morphologically using the electron microscope. Thirteen punch biopsies were taken from patients with esophageal dysplasia ascertained by iodine staining and showing up as unstained or faintly stained areas. We conducted a morphological study of two types of esophageal dysplasia, one with reactive immature epithelium (RIE) and the other classified as an intra-epithelial neoplastic lesion (INL). The ultrastructural differences observed between the RIE and INL were the degree of the cellular or nuclear atypia, the number of desmosomes or hemidesmosomes, and the amount of intercellular space. The nucleocytoplasmic ratio in INL was higher than in RIE. In the cells of RIE, the number of desmosomes was smaller than in normal epithelium, but larger than in INL. The hemidesmosomes were found predominantly between the RIE cells and basal laminae, but were very few or absent in INL and tumor cells. This study was presented at a symposium of the 23rd Annual Meeting of the Clinical Electron Microscopy Society of Japan, September 12, 1991.     

A fine structure study of passive transfer thyroiditis

The fine structural changes of passive transfer thyroiditis were observed in guinea-pigs injected intravenously with homologous anti-thyroglobulin serum. Eosinophils and mononuclear cells entered the interstitial area of the thyroid within 1 hour following the infusion of antisera. This cellular reaction reached its maximum intensity at 24 hours following antibody administration. Eosinophils were seen adjacent to and appeared to enter within the cytoplasm of the thyroid epithelial cells. Mononuclear cells ingest many of the eosinophils. Polymorpho-nuclear leucocytes were not present in significant numbers. Platelet aggregates were present in small blood vessels and endothelial cells showed increased pinocytotic activity during this period. This inflammatory reaction subsided rapidly and at 8 days the thyroid glands appeared histologically normal.

Passive transfer thyroiditis produced by homologous antithyroglobulin antiserum appears to be an unique experimental model in which eosinophils play a very prominent role.

     

In vitro studies of the impact of maribavir on CMV-specific cellular immune responses

BackgroundGanciclovir has demonstrated immunosuppressive effects in vitro which may lead to delayed cytomegalovirus (CMV)-specific immune reconstitution when the drug is given prophylactically. Maribavir is a new and more potent anti-CMV drug that is under evaluation for therapeutic use in transplant recipients.ObjectivesThe objective of this study was to evaluate the potential effect of maribavir on CMV-specific T cell function in comparison to ganciclovir.Study designIn ten immunocompetent CMV seropositive donors, maribavir and ganciclovir were compared over a broad range of concentrations (0.2–500 μM) regarding their effects on lymphoproliferation, CMV-specific CD4+ and CD8+ cytokine expression, T cell multifunctionality, degranulation and apoptosis.ResultsMaribavir inhibited lymphocyte proliferation at concentrations of 50 μM and above, however, cytokine expression, cellular degranulation and multifunctionality of CD4+ and CD8+ T cells in response to CMV lysate and pp65 peptide mix were not impaired except at the highest concentration of 500 μM. Ganciclovir inhibited lymphoproliferative responses starting at 10 μM. As with maribavir, other cellular responses following stimulation with CMV lysate and pp65 peptide mix were only impaired at the highest concentration of 500 μM of ganciclovir. Neither maribavir nor ganciclovir showed induction of lymphocyte apoptosis.ConclusionsMaribavir exhibits a low potential to suppress CMV-specific T cell function. This finding supports the use of higher doses in the prophylactic setting than originally proposed.     

Fine structure of the semicompact formation of the nucleus ambiguus of the rat

 We investigated the fine structure of the semicompact formation of the nucleus ambiguus (AmS), which was identified by retrogradely labeled pharyngeal (PH) motoneurons. When cholera toxin subunit B-conjugated horseradish peroxidase was injected into the lower pharyngeal muscle, many retrogradely labeled PH neurons were found throughout the AmS. Besides the PH neurons, two types of neurons were recognized in the AmS: unlabeled medium-sized neurons and unlabeled small neurons. The PH neuron was large (27.6 × 44.1 μm) and polygonal, and contained many Nissl bodies and well-developed cell organelles with a prominent spherical nucleus. The medium-sized neuron was dark and oval (19.3 × 33.2 μm), and contained many free ribosomes and much swollen rough endoplasmic reticulum with a distorted oval nucleus. The small neuron was spindle-shaped (12.3 × 20.2 μm), and had poorly developed cell organelles with an irregularly shaped nucleus. The average number of axosomatic terminals in a sectional plane was largest in the PH neurons (32.8), smaller in the medium-sized neurons (23.1), and smallest in the small neurons (6.3). The number of axo-somatic terminals containing round vesicles (Gray’s type I) was almost equal to that of terminals containing pleomorphic vesicles (Gray’s type II) in the PH neuron, and slightly smaller in the small and the medium-sized neurons. About 60% of the axodendritic terminals were Gray’s type I, and 40% were type II. These results indicate that there are two different types of interneurons besides the PH motoneurons in the AmS. Accepted: 24 June 1997     

In vitro andin vivo impact of a new glycosphingolipid on neutrophils

A new water-soluble, orally absorbable de-N-acetyl-lysoganglioside (WILD20), breakdown product of the monosialoganglioside GM₁, was found to influence some parameters of neutrophil response to inflammation stimuli. Superoxide anion production appears inhibited, along with neutrophil killing properties. A block of both pathways of arachidonic acid cascade and PAF was also found, as well as neutrophil ICAM-1-mediated adhesion to endothelial cells. Of particular interest was the significant reduction of neutrophils observed at the site of inflammation, whichever agonist was used. The effects on neutrophil physiology found in normal or in pathological conditions, are in favour of a WILD20-related inhibitory effect on neutrophil contribution to inflammation.     

In vitro and in vivo studies of the properties and effects of antimacrophage sera (AMS)

Antimacrophage sera (AMS) were prepared in rabbits by injection of mouse peritoneal cells with or without purification by in vitro culturing. These AMS showed high cytotoxic activity against macrophages in culture. The antimacrophage sera were not specific for macrophages; they cross-reacted and were cytotoxic to lymphocytes and granulocytes in the in vitro assay. Injection of AMS into mice did not affect antiShigella antibody production, nor did it prolong skin allograft survival. Furthermore, AMS-treated macrophages did not lose their ability to induce antiShigella antibody production in irradiated mice. On the other hand, AMS inhibited phagocytosis of Bacillus subtilis by macrophages and likewise increased the synthesis of DNA in cultured macrophages.     

In vivo effect of benzothiazole and amoscanate derivatives on the fine structure of adultBrugia spp. andLitomosoides carinii

Alterations in the fine structures of femaleBrugia spp. andLitomosoides carinii were investigated after in vivo treatment with curative doses of 4 compounds: CGP 20376 [2-tert-butyl-benzothiazole-5-methoxy-6-dithiocarbamic-S-(2-carboxyethyl)-ester], CGP 21833 (2-tert-butyl-benzothiazole-5-methyl-6-N-methylamino-piperazinylthiocarbonylamide), CGP 6140 [4-Nitro-4-(N-methyl-piperazinylthiocarbonylamido)-diphenylamine] and amoscanate (4-isothiocyanato-4-nitrodiphenylamine). All compounds caused early alterations in the somatic muscle cells. These alterations usually appeared within 24 h after treatment; they occurred later only after treatment ofL. carinii with amoscanate. InBrugia spp., swelling of the muscle cells occurred in which the glycogen deposits considerably increased in size. The electron density of the cytoplasm surrounding the myofilaments in the fibrillar protion of the muscle cells increased, and light zones appeared between the fibrils. The muscle cell mitochondria swelled, particularly their inner matrix, which became more electron-lucent, with some dense spots. InL. carinii the muscle cells were not increased in size, but their mitochondria were considerably swollen before disintegration; this was followed by disintegration of the myofilaments and vacuolization of the cytoplasm. Vacuolization before mitochondrial swelling was observed only after treatment with CGP 6140. Other tissues of this species were not altered before the 2nd day after treatment. InBrugia spp., electron-lucent areas appeared in the hypodermis either simultaneously with the alterations in the muscle cells or a few hours later. At 24 h after treatment with amoscanate, blebs were formed on the luminal side of the intestinal membrane. Although the pathological pattern is slightly different in the two species, the characteristic and very early alterations in the muscle mitochondria indicate that all compounds interfere with the energy metabolism of the filariae, which is particularly high in the very actively working muscle cells. The variations in sensitivity of hypodermal structures ofBrugia spp. to the various compounds may be due to compound differences.Abbreviations amu afibrillar portion of muscle cells - bc body cavity - bl blebs - cu cuticle - ep epithelium - fmu fibrillar portion of muscle cells - gd glycogen deposit - hy hypodermis - lz light zones between the myofibrils - mf myofilaments - mi mitochondria - mu muscle cells - mv microvilli - rmu ring muscle cells - ut uterus - va vacuoles