In vitro hemolysis of autologous erythrocytes caused by immune murine spleen cells and spherules of the fungus Coccidioides immitis.

This communication describes an in vitro system wherein mouse erythrocytes are lysed in the presence of spherules of the fungus Coccidioides immitis and spleen cells from syngeneic mice immunized with a variety of antigens. The antigens include: tobacco mosaic virus in complete Freund's adjuvant (CFA), CFA alone, separate components of CFA, sheep erythrocytes, and allogeneic tumor. Spleen cells from mice sublethally infected with C. immitis are also capable of participating in this response. The lytic phenomenon, which does not require complement, is dependent upon the number of spleen cells per culture, the number of spherules per culture, the time of culture incubation, the amount of antigen injected into the animal and the time after immunization at which spleen cells are recovered. Live spherules or spherules killed with heat, with dimethylsulfoxide, or with formalin were effective participants, together with immune spleen cells, in the lytic reaction.     

Cellular immunity to Coccidioides immitis: In vitro lymphocyte response to spherules,arthrospores, and endospores

In vitro stimulation of incorporation of tritiated thymidine by human peripheral lymphocytes in response to two soluble antigens and three different intact but nonviable fungal forms of Coccidioides immitis was studied. Lymphocytes were obtained from three groups of subjects: healthy skin test positive, healthy skin test negative, and disseminated disease. Dose-response relationships to the intact forms (endospores, arthrospores, and spherules) were determined. Responses of lymphocytes from healthy skin test-positive subjects and subjects with disseminated disease were similar. Ranking of antigens by “potency” gave the following results: endospores = spherulin > mycelial filtrate > arthrospores = spherules. Endospores were the most potent of the intact forms in 10 of 11 subjects. The clear superiority of endospores over spherules is not due to differences in the total particle surface area available for presentation to the leukocytes. All antigens tested except spherules could discriminate between skin test-positive and skin test-negative subjects in this in vitro system. A T-cell-enriched, B-cell- and mono-cyte-depleted cell population demonstrated an active response to spherulin and to endospores. The variance of these finding with animal studies demonstrating spherules to be immunogenically superior when compared to endospores is discussed. This may have importance in future studies in humans of vaccines to C. immitis.     

A study of adipocyte precursor cells derived from brown adipose tissue: the expression of specific cell surface antigens during their differentiation in culture

Using cell specific anti-adipocyte sera and an immuno-precipitation procedure, the nature of the cell surface antigens characterizing adipocytes from rat brown adipose tissue was investigated. Initially the ability of anti-sera, raised against adipose plasma membrane preparations of white or brown adipose tissue, to distinguish between membrane preparations derived from either tissue was confirmed. Analysis of the plasma membranes derived from brown adipose and similar preparations labelled with 125I revealed the presence of specific externally disposed mature brown adipocyte-specific antigens. The specifically immunoprecipated antigens had molecular weights of 70,000, 56,000 and 23,000. None of these antigens were cross immunoprecipated by antisera to mature white adipocyte membranes. The presence of the brown adipose specific antigens on the surface of differentiating adipocyte precursor cells derived from rat brown adipose tissue was demonstrated using a labelled-secon antibody cellular immunoassay. The expression of the immunoreactivity associated with these antigens was shown to be an early event in the differentiation programme of the cells in vitro. The functional identity and possible roles of these antigens in the control of brown adipocyte differentiation now becomes accessible to further experimental investigation.     

Temperature-dependent protein folding in vivo--lower growth temperature increases yield of two genetic variants of Xenopus laevis Cu,Zn superoxide dismutase in Escherichia coli.

Two genetic variants of Xenopus laevis Cu,Zn superoxide dismutase, XSODA and XSODB, have been expressed in Escherichia coli by recombinant DNA techniques. Production of both proteins was obtained, although with different yields, XSODB being more abundant than XSODA in all the conditions tested. Lowering the temperature of growth was found to be a specific factor, decisive in obtaining quantitatively abundant, active Xenopus enzymes. Impaired folding of these proteins in the E.coli cytoplasm was found to parallel their in vitro properties.     

Production of a hyperimmune antitoxic donor plasma against Pseudomonas aeruginosa

Research work has been carried out with the aim of obtaining hyperimmune antitoxic plasma against Pseudomonas aeruginosa. Hyperimmune plasma active against P. aeruginosa toxin has been obtained from donors immunized with P. aeruginosa toxoid. The preparation is highly specific and active, which is revealed by in vivo and in vitro tests. The clinical study of the preparation indicates its high efficacy in the treatment of diseases caused by different P. aeruginosa serotypes.     

Interaction of Staphylococcal α-Toxin with Artificial and Natural Membranes

Comparison of hemolytic activity and chromate-releasing activity of partially purified preparations of staphylococcal alpha-toxin indicated the presence of a lytic factor other than alpha-toxin. This lytic release factor (RF) was isolated from the preparations and was shown to be active against both lipid spherules and erythrocytes. Heat-purified alpha-toxin (HP alpha-toxin) disrupted spherules, with the formation of fragments which always showed the presence of ring structures similar in dimensions (ca. 90 A) to pure alpha 12S-toxin. The interaction of HP alpha-toxin with spherules was accompanied by loss of hemolytic activity and adsorption of toxic protein. The alpha 12S-toxin, although only weakly hemolytic, was shown to be lytic for spherules. An alpha 12S-free toxin rapidly disrupted spherules, with formation of fragments with attached rings similar in dimensions to the alpha 12S molecule. Lipid monolayer experiments showed that HP alpha-toxin could penetrate lipid monolayers by virtue of a hydrophobic interaction. Effects of HP alpha-toxin on rabbit and human erythrocyte ghosts were similar to its effects on spherules, in that rings appeared on membrane fragments. Toxin-lysed rabbit erythrocytes showed similar rings on the resulting membrane fragments. However, rings were not seen on toxin-treated rabbit erythrocytes in the prelytic lag phase; this result and the fact that human erythrocytes are largely insensitive to alpha-toxin were interpreted as evidence against a lytic mechanism involving ring formation as the primary event. Rings were interpreted as toxin polymers similar to alpha 12S molecules, formed from specifically orientated active toxin molecules at the surface of lipid structures. Possible mechanisms for toxin lysis of spherules and erythrocytes are discussed.     

Conversion of Coccidioides immitis in tissue culture

Summary Utilization of a pre-treatment resulted in the establishment of a simple technique to enhance spherulation ofC. immitis in tissue culture.Establishment of the fact that culture spherules are produced from mycelial elements was also recognized and should be included in the life cycle ofC. immitis when cultured in vitro.     

Polyp regeneration from isolated tentacles of Aurelia scyphistomae: a role for gating mechanisms and cell division

The record of magnetic spherules in sediments from Crummock Water (the English Lake District) is presented and a method for obtaining magnetic extracts described. It is shown that magnetic spherule concentrations in the Crummock Water sediments increases by nearly two orders of magnitude following the Industrial Revolution, and this can be attributed to increases in fossil fuel combustion particularly after about 1900 A.D. Associated with the increase in spherule numbers are changes in their size distributions, which may reflect the changing sources for the spherules.It is demonstrated that magnetic spherules are distributed regionally and hence form widespread cryptic marker horizons in lacustrine sediments. Location of this horizon in a lake sediment profile provides an approximate dating method and this horizon may be located using susceptibility measurements corroborated by microscopic spherule counts.     

Surface antigen structure of Neisseria meningitidis. I. The isolation of a serotypic antigenic complex of N. meningitidis strain B16B6 by direct detergent treatment and its immunochemical characteristics

The method for obtaining a serotyping antigenic complex from N. meningitidis B16B6 by their direct treatment with the mixture of detergents (0.5% sodium desoxycholate and 0,5% cholic acid in the proportion 1 : 1) in 0.5 M KCl solution is proposed. Such treatment has been found to increase the yield of the preparation in terms of protein more than 4 times in comparison with earlier methods for obtaining serotyping antigens. The immunochemical study of the preparation has demonstrated its serological specificity and high immunological activity, not inferior to that of serotyping antigenic preparations from group B meningococci, obtained by the heretofore known methods.     

Outer root sheath cells of human hair follicle are able to regenerate a fully differentiated epidermis in vitro

During wound healing, interfollicular epidermis can be regenerated from the outer root sheath of hair follicles, showing that the cells of this structure can shift toward an interfollicular epidermal phenotype. Similarly, it has been shown that a multilayered epithelium originating from outer sheath cells can be obtained in vitro by culturing hair follicles. However, in the culture systems developed so far, the phenotypical shift was incomplete since the cells retained some of their original characteristics and did not acquire several key markers of terminally differentiated epidermis. In this paper, we describe a new tissue culture method for obtaining a multilayered epithelium from outer sheath cells. This is performed by implanting human hair follicles vertically into dermal equivalents and then raising the culture at the air-liquid interface. The morphological, immunological, and biochemical features of the in vitro reconstructed tissue are very similar to those observed in normal interfollicular epidermis, including those specific for terminally differentiated keratinocytes. Thus, under appropriate in vitro conditions, outer root sheath cells are able to express an interfollicular epidermal phenotype as occurs in vivo during wound healing.     

Morphological Diversity of the Rod Spherule: A Study of Serially Reconstructed Electron Micrographs

Purpose

Rod spherules are the site of the first synaptic contact in the retina’s rod pathway, linking rods to horizontal and bipolar cells. Rod spherules have been described and characterized through electron micrograph (EM) and other studies, but their morphological diversity related to retinal circuitry and their intracellular structures have not been quantified. Most rod spherules are connected to their soma by an axon, but spherules of rods on the surface of the Mus musculus outer plexiform layer often lack an axon and have a spherule structure that is morphologically distinct from rod spherules connected to their soma by an axon. Retraction of the rod axon and spherule is often observed in disease processes and aging, and the retracted rod spherule superficially resembles rod spherules lacking an axon. We hypothesized that retracted spherules take on an axonless spherule morphology, which may be easier to maintain in a diseased state. To test our hypothesis, we quantified the spatial organization and subcellular structures of rod spherules with and without axons. We then compared them to the retracted spherules in a disease model, mice that overexpress Dscam (Down syndrome cell adhesion molecule), to gain a better understanding of the rod synapse in health and disease.

Methods

We reconstructed serial EM images of wild type and Dscam^GoF (gain of function) rod spherules at a resolution of 7 nm in the X-Y axis and 60 nm in the Z axis. Rod spherules with and without axons, and retracted spherules in the Dscam^GoF retina, were reconstructed. The rod spherule intracellular organelles, the invaginating dendrites of rod bipolar cells and horizontal cell axon tips were also reconstructed for statistical analysis.

Results

Stereotypical rod (R1) spherules occupy the outer two-thirds of the outer plexiform layer (OPL), where they present as spherical terminals with large mitochondria. This spherule group is highly uniform and composed more than 90% of the rod spherule population. Rod spherules lacking an axon (R2) were also described and characterized. This rod spherule group consists of a specific spatial organization that is strictly located at the apical OPL-facing layer of the Outer Nuclear Layer (ONL). The R2 spherule displays a large bowl-shaped synaptic terminal that hugs the rod soma. Retracted spherules in the Dscam^GoF retina were also reconstructed to test if they are structurally similar to R2 spherules. The misplaced rod spherules in Dscam^GoF have a gross morphology that is similar to R2 spherules but have significant disruption in internal synapse organization.

Conclusion

We described a morphological diversity within Mus musculus rod spherules. This diversity is correlated with rod location in the ONL and contributes to the intracellular differences within spherules. Analysis of the Dscam^GoF retina indicated that their R2 spherules are not significantly different than wild type R2 spherules, but that their retracted rod spherules have abnormal synaptic organization.     

Immunological mechanisms of the radioprotective effects of ceruloplasmin

The aim of the work was to examine immunotropic features of celluroplasmine++ (Cp) and estimate its capacity as radioprotector. The pulse radiolysis method was used to set the mechanism of elementary reactions, which were responsible for antioxidant activity, and to demonstrate a particular role of reversible oxycomplexis, Cp...O2. Within in vitro test-systems the effects of Cp were examined on the model of interaction between lymphocytes from Shelter staff which constantly contacts with ionizing irradiation, and autologous erythrocytes in rosset-forming phenomenon (223 Shelter employees in comparison with 253 donors of Kyiv blood transfucion station). Expression of this Index in the presence of antigens with known poly-specificity was determined (antigens were taken from cortical and pyramid sections of kidney, liver, lungs, myocard, pancreatic gland, grey matter and aorta). Simultaneously the presence of analogous autoantibodies was determined (ELISA). It has been shown that Cp can reprogram the level of expression of immunovaluable receptors towards to norm. It has been also defined that Cp presence in blood irradiated in vitro (1-15 rem) promotes the masking of active centers of autoantibodies of different tissue specificity.     

The active E-rosette test: a sensitive in vitro correlate for human delayed-type hypersensitivity.

The "active" rosette test was adapted as an in vitro assay and correlated with human delayed cutaneous hypersensitivity (DCH) to two microbial antigens. Peripheral lymphocytes were purified from donors known to be responders or nonresponders to PPD-tuberculin or tularemia on the basis of prior DCH reactions. Skin test antigen, incubated with lymphocytes from antigen-sensitive donors, produced a significant increase (+2 S.D.) in the ability of the lymphocytes to form active rosette-forming cells (A-RFC) when compared to lymphocytes cultured without antigen. Skin test antigen incubated with lymphocytes from nonsensitive donors produced no increase in their A-RFC. The optimal dose of each antigen was approximately 100 ng/ml. The percentage of A-RFC rose to maximum levels between 3 and 4 hr after the addition of antigen to the lymphocytes incubated at 37 degrees C. The assay appears to be specific for the antigen to which the individual demonstrates DCH. This assay may provide a new in vitro method for investigating mechanisms of cell-mediated immunity and a rapid diagnostic test for sensitization to microbial antigens.     

Scanning electron microscopy of freeze-fractured sclerotia of Physarum polycephalum

The ultrastructure and architecture of freeze-fractured sclerotia of Physarum polycephalum was studied by a scanning electron microscope (SEM). The sclerotia are built of many spherules grouped together in a common outer coat. Each spherule has hard walls which separate it from its neighbors. The spherules are rounded in 2-day-old sclerotia, and have a lobe-like structure 3 weeks later. A new simple technique for obtaining freeze-fractured biological material is described and compared with freeze-fracture in a freeze-etching apparatus. The ultrastructural details of the fractured sclerotia are described.     

The role of non-type-specific protein antigens of the cell wall in Streptococcus group A in developing delayed hypersensitivity

The role of the type-nonspecific (TNS) cell-wall antigens of group A streptococci has been determined. The study has been made on guinea pigs sensitized with whole microbial cells or HCl extracts containing TNS antigens. To determine delayed hypersensitivity, the in vitro cytotoxic test on adhering lymph-node cells in the autologous system has been used. The study has shown that sensitization with group A streptococci of different types or with TNS antigens induces the development of delayed hypersensitivity to TNS antigens (or antigen), common for different types of group A streptococci, but specific for this group. HCl extracts containing TNS antigens can be recommended as the preparation for testing delayed hypersensitivity to antigens, specific for group A streptococci.     

Radioimmunochemical methods for the quantitative autoradiographic determination of antigens in brain and other tissues

1. We have used horseradish peroxidase-conjugated protein A- and 125I-protein A to develop immunohistochemical and radioimmunohistochemical methods for the localization of antigens in brain and other tissues of the rat. 2. We visualized methionine-enkephalin fibers in the rat brain by incubating tissue sections with a specific polyclonal antibody and peroxidase-conjugated protein A. The method is simple, fast, and less expensive and more sensitive than classical immunohistochemical techniques and the principle could be used to visualize many other tissue antigens. 3. Incubation of tissue samples with specific polyclonal antibodies and 125I-protein A, followed by autoradiography, allows the permanent recording of the radioimmunohistochemical localization of brain methionine-enkephalin, tyrosine hydroxylase, and angiotensin-converting enzyme and of pituitary vasopressin and could be applied to the localization of many other tissue antigens. 4. A new quantitative radioimmunohistochemical technique for methionine-enkephalin allows the determination of the endogenous peptide content in discrete brain nuclei from 16-microns-thick sections. The method is based on the quantitative determination of the amount of 125I-protein A bound to specific tissue areas after incubation with a specific polyclonal antibody, followed by autoradiography and computerized microdensitometry. To quantify the endogenous peptide content, the values obtained are interpolated into a methionine-enkephalin internal standard curve. This standard curve was constructed by measuring endogenous concentrations of methionine-enkephalin by radioimmunoassay in specific brain regions and correlating these values with quantitative autoradiographic determinations in homologous areas of adjacent sections. Similar methods can be developed for other tissue antigens. 5. These new methods allow for the localization and quantification of tissue antigens in very discrete areas of the brain and other tissues and have a wide application in neurobiology and pathology.     

Strategies in cancer vaccines development

The recent definition of tumour-specific immunity in cancer patients and the identification of tumour-associated antigens have generated renewed enthusiasm for the application of immune-based therapies for the treatment of malignancies. Recent developments in cancer vaccines have also been based on an improved understanding of the cellular interactions required to induce a specific anti-tumour immune response. Consequently, a number of cancer vaccines have entered clinical trials. Targeting broad-spectrum tumour-associated antigens has emerged as a strategy to lower the risk of tumour escape due to the loss of specific nominal antigen. Amongst the most challenging of tumour-associated antigens to which to target in active specific immunotherapy applications are carbohydrate antigens. As carbohydrates are intrinsically T-cell-independent antigens, more novel approaches are perhaps needed to drive specific-T-cell-dependent immune responses to carbohydrate antigens. In this context peptide mimetics of core structures of tumour-associated carbohydrate antigens might be developed to augment immune responses to these broad-spectrum antigens.     

Cytotoxic properties of the splenocytes of C3HA mice following treatment with exogenous RNA

The influence of fractions of exogenous RNA, isolated from spleens of C3HA mice and of rats, both intact (control--cRNA) and immunized with homogenate of normal syngenic, allogenic and xenogenic tissues (immune--immRNA), on the cytotoxic properties of splenocytes of C3HA intact mice was compared in the in vitro cytotoxic experiments. The splenocytes treated with different RNA fractions were used as effector-cells. In vitro cultivated MGXXIIa cells of strain specific C3HA mice hepatoma, and K562 cells of human erythroleukemia, both labeled with 3H-uridine, served as target cells. Thus, it is only the cytoplasmic fraction of immRNA isolated from the spleens of rats immunized with tissue antigens of C3HA mice that induced a more pronounced stimulation of cytotoxic activity of splenocytes.     

Down regulation of ITGA4 and ITGA5 genes after formation of 3D spherules by human Wharton’s jelly stem cells (hWJSCs)

Human Wharton’s jelly mesenchymal stem cells (hWJSCs) are multipotent stem cells that could be aggregated into 3D spherules. ITGA4 and ITGA5 genes encode α4 and α5 subunits of integrins, respectively. In this study, we analyzed expression levels of ITGA4 and ITGA5 gene mRNAs in undifferentiated and 3D spherules forming hWJSCs in order to determine their expression pattern for possible future treatment of cancer cells in a co-culture fashion. For the purpose of obtaining hWJSCs, umbilical cords were collected from patients with caesarian section at full term delivery. The cells were then characterized according to cell surface markers using flow cytometry. Furthermore pluripotency of the obtained cells was verified. Subsequently the cells were aggregated in 3D spherules using hanging drop cultures. Expression levels of ITGA4 and ITGA5 gene mRNAs were determined by RT-PCR and Real time PCR, both in the initial undifferentiated cells and those aggregated in the spherules. The obtained hWJSCs demonstrated pluripotency, differentiating to adipogenic and osteogenic cells. They also expressed mesenchymal stem cell surface markers. Following the aggregation of these cells and formation of 3D spherules, mRNA expression levels of both genes were significantly reduced (P?<?0.05) compared with the initial undifferentiated state. The results of this study demonstrated that aggregation of hWJSCs into spherules alters their expression of ITGA4 and ITGA5. The implications of such an alteration would require further research.