Studies on Sperm Antigenicity 6. In vivo and in Vitro Cellular Reactivity in Guinea Pigs Sensitized with Fractions of Guinea Pig Spermatozoa

Normal guinea pig spermatozoa cells were homogenized by a French pressure cell. Three soluble and three insoluble fractions were obtained by ultracentrifugation and (emulsified in CFA) were used for guinea pig sensitization. The following were observed: 1) all fractions were immunogenic except one; 2) in vivo and in vitro delayed hypersensitivity was elicited in animals immunized with these fractions; 3) two distinctive histopathologic lesions were observed in the testes of sensitized animals: lesions of orchitis type developed in animals injected with some fractions. Other fractions induced lesions of aspermatogenic type. These results correlated well with delayed hypersensitivity results obtained by in vivo and in vitro tests. Although some other spermatozoal fractions did not cause severe changes in the testes, the lack of sperm accumulation in the epididymis was obvious.     

Studies on Sperm Antigenicity 6. In vivo and in Vitro Cellular Reactivity in Guinea Pigs Sensitized with Fractions of Guinea Pig Spermatozoa

Normal guinea pig spermatozoa cells were homogenized by a French pressure cell. Three soluble and three insoluble fractions were obtained by ultracentrifugation and (emulsified in CFA) were used for guinea pig sensitization. The following were observed: 1) all fractions were immunogenic except one; 2) in vivo and in vitro delayed hypersensitivity was elicited in animals immunized with these fractions; 3) two distinctive histopathologic lesions were observed in the testes of sensitized animals: lesions of orchitis type developed in animals injected with some fractions. Other fractions induced lesions of aspermatogenic type. These results correlated well with delayed hypersensitivity results obtained by in vivo and in vitro tests. Although some other spermatozoal fractions did not cause severe changes in the testes, the lack of sperm accumulation in the epididymis was obvious.     

In Vitro Effect of Oxidized Dextrans on Peritoneal Cells

We studied the dependence of in vitro dextran biocompatibility on the method of oxidation of 35-kDa dextran. The biocompatibility of dextran oxidized with potassium permanganate was higher compared to that obtained by radiochemical oxidation. It was related to the formation of peroxide compounds during radiochemical oxidation. Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Suppl. 1, pp. 120–122, 2008     

Antiviral Activity of Bordetella Pertussis Vaccine-Elicited Peritoneal Exudate Cells

Peritoneal exudate cells collected from mice 7 days after treatment with Bordetella pertussis vaccine exhibited significant in vitro antiviral activity against vesicular stomatitis virus (VSV). Vaccine-induced peritoneal exudate cells exhibited both intrinsic and extrinsic antiviral activity in culture with target VSV-infected L cells. Virus replication was poor in the vaccine-induced exudate cells. Coculture of vaccine-induced exudate cells and VSV-infected L cell targets decreased virus yield. The activity appeared specific for infected cells and at least a portion of the antiviral activity was directed against the initial infection cycle. Nonadherent vaccine-induced exudate cells showed an increase in antiviral activity over total vaccine-induced exudate cells.     

In Vitro Proliferation of T Lymphocytes from Listeria-Infected Rodents: Assay Conditions for Rat Peritoneal Exudate Cells and Characterization of an Inhibitor

Conditions favorable to [³H]thymidine incorporation into antigen-stimulated T lymphocytes from Listeria-infected rats have been established. In cultures of peritoneal exudate (T) lymphocytes purified twice with nylon-wool vigorous antigen-specific proliferation was observed within 2 days. Cultures of lymphocytes from nodes draining a subcutaneous Listeria-infection site differed in that back-ground proliferation was higher than for peritoneal exudate lymphocytes, and [³H]thymidine incorporation was maximal at day 3. A critical factor for the rate of proliferation was the lymphocyte-to-macrophage ratio; optimal cultures of peritoneal exudate lymphocytes contained 2 to 5% macrophages. Macrophages exceeding a proportion of 10% strongly, if not completely, inhibited [³H]thymidine incorporation into antigen-stimulated lymphocytes. Inhibition was associated with mononuclear cells, adherent to plastic or nylon-wool, of the stimulated or unstimulated peritoneal cavity. It was neither attributable to release of cold thymidine from macrophages nor to rapid degradation of particulate antigen by macrophages. The degree of inhibition reflected the metabolic activity of macrophages; on a cell-for-cell basis, heat-killed and glutaraldehyde-fixed macrophages were less inhibitory, and stimulated macrophages were more inhibitory than macrophages from the unstimulated peritoneal cavity.     

In Vitro and In Vivo Effects of Endotoxin on Mouse Peritoneal Cells

The in vitro effect of endotoxin (LPS) on unfractionated mouse peritoneal cells and cells fractionated into glass adherent and nonadherent populations was studied. LPS caused blast transformation and deoxyribonucleic acid synthesis in the nonadherent, nonphagocytic cells. There was no evidence of a mitogenic effect on macrophages. Instead, a cytotoxic effect was noted. When incubated with unfractionated peritoneal cells, LPS was still cytotoxic for macrophages, but the mitogenic effect for nonadherent cells was decreased or ablated. The intraperitoneal administration of LPS to mice resulted in an acute inflammatory response with a transient depletion of mononuclear cells. There was no stimulation of division of macrophages. Data were obtained which indicated that local cell division is an important factor in the normal turnover of peritoneal macrophages.     

Comparative Study of the In Vitro Effect of Nanoliposomes with Oxidized Dextrans on Peritoneal Cells

We studied the in vitro effect of hybrid molecular-nanosomal biocompatible compositions on cultured peritoneal cells. The compositions consisted of oxidized dextrans with a mean molecular weight of 35 and 60 kDa, which were obtained by chemical and radiochemical oxidation of dextran. Hybrid nanoliposomal compositions of chemically oxidized dextran (permanganate method) had greater biocompatibility and tropic activity to macrophages compared to nanoliposomes of radiochemically oxidized dextran. Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Suppl. 1, pp. 123–126, 2008     

Interleukin-4 Induces In Vitro Migration in Naive B Cells Through Direct Mechanisms

Interleukin-4 (IL-4) mediated locomotor responses by marine B cells in vitro were examined in this paper. The IL-4 induced migration was found to act directly on purified, splenic B cells, without involvement of secondary mediators. It appeared that only a subpopulation of B cells was able to respond in migration assays. Flowcytometric analysis showed that the migrating cells had the characteristics of naive B cells: I-A¹⁰, J 11d^hi, IgD^hi. They also displayed high expression of the adhesion molecule L-selectin and made predominantly IgM antibodies. This is contrary to what has previously been observed regarding motile responses to chemotactic factors by T cells, which mostly affect memory or activated T cells. However, this is in accordance with other studies indicating that IL-4 is a cytokine that exerts its effect mainly on resting B cells.     

Imaging Bioluminescent Exogenous Stem Cells in the Intact Guinea Pig Cochlea

Stem-cell-based therapy may be used to replace damaged or lost neurons in the cochlear nerve of patients suffering from severe-to-profound sensorineural hearing loss. In order to achieve functional recovery in future clinical trials, knowledge about survival of grafted cells and their differentiation into functional neurons is a prerequisite. This calls for non-invasive in vivo visualization of cells and long-term monitoring of their survival and fate after cochlear transplantation. We have investigated if molecular optical imaging enables visualization of exogenous cells in the intact cochlea of guinea pig cadaver heads. Transduced (stem) cells, stably co-expressing fluorescent (copGFP) and bioluminescent (Luc2) reporter molecules, were injected into the internal auditory meatus or directly into the cochlea through the round window. After injection of the cells into the internal auditory meatus, a bright bioluminescent signal was observed in the cavum conchae of the auricle, indicating that light generated by Luc2 is passing through the tympanic membrane and the external auditory meatus. Similar results were obtained after injection of the cells through the round window membrane, either directly into the scala tympani or in Rosenthal's canal within the modiolus of the basal cochlear turn. Imaging of the auditory bulla demonstrated that the bioluminescent signal passes through the tympanic membrane and crevices in the bony wall of the bulla. After opening the auditory bulla, the bioluminescent signal was emanating from the round window. This is the first study demonstrating that bioluminescence imaging enables visualization of luciferase-expressing cells injected into the intact guinea pig cochlea. Anat Rec, 303:427–440, 2020. © 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.     

Migration in Vitro by Blood and Exudate Neutrophils Assessed Serially During an Inflammatory Response

Migration in vitro by blood and inflammatory neutrophils has been compared serially during an inflammatory response. Using an experimental pig model, neutrophils are isolated from the peripheral blood and from the pleural space at hourly intervals after an intrapleural challenge with zymosan activated pig serum (ZAS). Following acepromazine sedation and halothane anesthesia, blood neutrophil migration was transiently reduced. By 1 hour random and directed migration of blood neutrophils returned to normal. Directed and random migration of exudate neutrophils was markedly decreased to both a stimulus-specific (ZAS) and an unrelated (LTB₄) chemoattractant. After 3 hours, migration by exudate neutrophils was similar to migration by blood neutrophils examined in parallel. These findings emphasize the importance of performing serial evaluations of cell function during an inflammatory response.     

Demonstration of a Complement-Dependent Migration Inhibitory Activity in Normal Guinea Pig Serum

A cell migration inhibitory effect was evidentiated in normal guinea pig serum as compared with heat inactivated serum, Granuloeytes when used as target showed a greater sensitivity to this effect than lymphomonocytes or macrophages. The migration inhibitory activity of GPS is abrogated or decreased by using complement destroying agents such as: heating at 56°C for as little as 5 min, absorption on immune complexes in presence of calcium, on zymosan, on Sephadex G-50 or by adding EDTA or heparin to culture medium. The GPS dialysation fractions while exhibiting neither complement haemolytic effect nor migration inhibitory activity when tested alone, restored these functions by recombination. Absorption of GPS on homologous blood cells abrogated the migration inhibitory effect but retained the complement haemolytic function. When GPS absorbed on homologous blood cells was mixed 1:5 with heat-inactivated serum (5min at 56°G), the migration inhibitory activity was regained, suggesting that the complement factors from the first sample were necessary for manifestation for the migration inhibitory activity from the heat-inactivated serum.     

Altered Expression of Matrix Metalloproteinases Within Mineralizing Bone Cells In Vitro in the Presence of Fluoride

Fluoride is known to alter mineralization within bone, although the mechanism for its action is unclear. An important stage in the formation of mineralized tissues is the remodeling of the osteoid, facilitating mineral deposition. Using a bone mineralizing culture system derived from rat femur washes, this study investigated the influence of fluoride on MMP expression at a developmental stage relating to the onset of mineralization. Bone cells cultured in the absence of fluoride synthesized an active form of a 45-kD MMP, which was immunoreactive with an antibody to human MMP-1 (although full characterization of this MMP was not achieved), trace levels of an MMP immunoreactive with anti-MMP-3, and a 66-kD proteolytic species. Incubation in 10 &#109 7 and 10 &#109 5 M fluoride resulted in a decrease in expression of the 45-kD MMP, sharp increases in the expression of MMP-3, and the appearance of a band at 110 kD, which showed immunoreactivity for MMP-9. The influence of fluoride on MMP expression is likely to influence the composition of the remodeling matrix and subsequent mineralization and offers a potential mechanism by which fluoride alters mineralization.     

Studies On Chemiluminescence and Protein Kinase-C Activity of Cisplatin Treated Mouse Peritoneal Exudate Cells

A single i.p. injection of cisplal-in (10 mg/kg body weight) into mice results in a significant increase in chemiluminescence and ATP contents of the peritoneal exudate cells (PEC) than that of PEC from untreated mice. It is also observed that in vitro treatment of macrophages with cisplatin, rIFN-γ and LPS show increased activity of the protein kinase-C.(PK-C). the activation of PK-C could result in stimulation of NADPH-oxidase resulting in increased levels of chemi luminescence. Increased contents of ATP in PEC after cisplatin treatment also suggests that this activation is energy dependent.     

In Vitro Effect of an Acute Nonspecific Inflammatory Exudate on Tritiated-Thymidine Incorporation by Unstimulated and Pha-Stimulated Spleen Cells

The effect of an acute nonspecific inflammatory exudate with mitogenic activity for macrophages in culture has been tested on the spontaneous and PHA-induced DNA synthesis by spleen cells in vitro.

Stimulatory effect of this exudate was observed on the spontaneous DNA synthesis which was detectable over a range of 1:4 to 1:4096 concentrations. After optimal PHA stimulation, an inhibition of mitogen-induced DNA synthesis was observed when the cells were exposed to the highest concentrations (up to 1:128) of the exudate. Thereafter, the phenomenon could be reversed and the stimulation was maximal at the concentration of 1:2048. When a suboptimal dose of PHA was used, the stimulatory effect was more pronounced and detected from 1:8 up to 1:4096 concentrations.     

Increased Tissue Conductance and Ion Transport in Guinea Pig Ileum After Exposure to Staphylococcus aureus Delta-Toxin In Vitro

Prior studies had shown that Staphylococcus aureus delta-toxin was able to inhibit water absorption in guinea pig ileum and to elevate the cyclic AMP content of this tissue, but was unable to elicit certain cyclic AMP-mediated changes in Y-1 adrenal or Chinese hamster ovary cells. Because water movement passively follows the net movement of electrolytes in the gut, this study investigated the effect of delta-toxin on ion transport in guinea pig ileum maintained in vitro. The transmural potential difference (PD) of guinea pig ileum was measured and nullified with an automatic voltage clamp. The short circuit flowing under these conditions (I_sc) was measured, and the conductance was calculated (I_sc/PD). Unidirectional ²²Na⁺ and ³⁶Cl⁻ fluxes were measured. In a glucose-free Ringer solution, delta-toxin caused an immediate spike in PD and I_sc, and the extent and duration of the spike generally increased with increasing toxin concentration. The conductance of ileum was increased by delta-toxin, and this effect on conductance could be blocked by lecithin, a known inhibitor of delta-toxin. Tissue in the presence of glucose did not exhibit a spike in PD or I_sc when exposed to delta-toxin. In a glucose-free medium, delta-toxin caused a 1.5- to 2.5-fold increase in both the unidirectional absorption and secretion of Na⁺ and Cl⁻, whereas the net secretion of Na⁺ increased above basal levels. The observation that delta-toxin causes a prompt increase in intestinal ion flux lends credence to the concept that the elevation in cellular cyclic AMP, which occurs later, is a secondary response to the toxin. The rapid increase in ion flux may reflect the ability of delta-toxin to augment intercellular movement of ions across the mucosa rather than the stimulation of transcellular processes.     

Inhibition of Pseudomonas aeruginosa by Hyperbaric Oxygen: Interaction with Mouse Peritoneal Exudate Cells

High-pressure oxygen (HPO) therapy for Pseudomonas aeruginosa infections of burn wounds has not been as effective as in vitro studies predicted. Mitigation of HPO toxicity for P. aeruginosa by nutrients present at the burn site could explain the lack of in vivo success. Alternatively, HPO-induced depression of host defense mechanisms could negate beneficial effects arising from HPOs known toxicity for P. aeruginosa. Accordingly, mouse peritoneal exudate cells (PEC), preincubated for 24 h in 1 atm of air-CO(2), were used to study the in vitro effects of HPO or air-CO(2) on phagocytosis of P. aeruginosa or sheep erythrocytes (SRBC). Subsequent 2-h exposures of PEC to increasing numbers of bacteria, in an air-CO(2) atmosphere, decreased the percentage of bacteria cleared as well as PEC viability. Similar exposures of PEC to bacteria in an HPO atmosphere prevented the loss of PEC viability and increased bacterial clearance. In control experiments, increasing the number of SRBC relative to PEC decreased the percentage of SRBC cleared without decreasing PEC viability, as determined under air-CO(2); short (2 h) exposure to HPO did not affect SRBC clearance. Microscopic examination of PEC indicated that a 24-h preincubation in HPO decreased the percentage of PEC which could ingest SRBC during subsequent experimental exposures (2 h) to air-CO(2) or HPO. These data suggest that short periods of exposure to HPO promote the ability of PEC to clear pseudomonads by adversely affecting the bacteria. This in turn prevents a pseudomonad-induced depression of PEC viability and function. In contrast, prolonged HPO exposure may be detrimental to phagocytic activity.     

豚鼠肠系膜淋巴管平滑肌细胞的培养

目的 :培养豚鼠肠系膜淋巴管平滑肌细胞。方法 :肠鼠肠系膜淋巴管用胰蛋白酶消化二次 ,第一次消化 15min ,除去肠系膜、淋巴管外膜等组织 ,第二次消化中膜的平滑肌 ,3 0～ 45min后 ,吸出未消化的组织 ,获得平滑肌细胞。常规培养、传代。结果 :培养细胞经光镜、电镜和荧光显微镜可观察到平滑肌的典型的形态和结构。结论 :所培养细胞为平滑肌细胞 ,此法可应用于管径较小的淋巴管和血管平滑肌细胞的培养。     

Corynebacterium parvum-Induced Peritoneal Exudate Cells with Rapid Cytolytic Activity against Tumour Cells are Non-Phagocytic Cells with Characteristics of Natural Killer Cells

Peritoneal exudate cells taken from mice 3 days after intraperitoneal treatment with Corynebacterium parvum (Cp) have been shown to kill specifically certain tumour targets in vitro. We have analysed in detail such Cp-induced cytotoxic cells as to their cellular characteristics, considering the fact that size and charge characteristics of cellular subgroups are useful markers in describing their representative characteristics. We could thus show that the cytolytic cell could not be classified as a macrophage. They behaved, in every manner analysed, exactly as the previously defined natural killer cells found in the lymphoid organs of normal mice.     

Adherence and Migration of Guinea Pig Granulocytes After Enzyme Treatment of the Cell Surface

Glycogen-induced polymorphonuclear granulocytes (PMN) from the peritoneal cavity ofguinea pigs were examined (1) for their adherence to nylon fibers in the absence and presence of the adherence-enhancing chemotactic peptide formyl-methionyl-leucyl-phenylalanine (f-MLP), (2) for their random migration through the filter of a Boyden chamber and (3) for their chemotactic migration towards f-MLP. The cells were analyzed before and after treatment with the enzymes neuraminidase, papain and trypsin. PMN adhesiveness was increased by neuraminidase digestion but reduced by treatment with the proteolytic enzymes. Neuraminidase and trypsin had no effect on cell migration, while papain reduced random migration without affecting f-MLP-induced chemotaxis. The data suggest that the type of adherence measured by the nylon fiber method differs from the temporary attachment of cells migrating through a chemotaxis filter towards an attracting substance.