Immunogold Labeling of Human Leukocytes for Scanning Electron Microscopy and Light Microscopy: Quantitative Aspects of the Methodology

When cell surface antigens are labeled with the colloidal gold marker, backscattered electron images (BEI) reveal all the gold particles and, therefore, permit total counts. Secondary electron images (SEI) show only a small percentage of the gold particles and are inadequate for quantitative evaluation. For determination of the cellular labelinq index, a time-consuming method implies the screening of 100 cells by scanninq electron microscopy, at a magnification of approximately 12,000 to 15,000x, with continuous SE/BE shifts. A much more efficient method is to transfer the SEM sample or its equivalent under the light microscope and to count the total number of gold labeled cells in the epi-polarization mode. The total cell count can be evaluated under UV light, taking advantage of the autofluorescence of the glutaraldehyde fixed cells

Immuno-Scanning Electron Microscopy of Normal and Leukemic Leukocytes Labeled with Colloidal Gold

The immunogold method, utilizing 40 nm colloidal gold particles which can be selectively visualized with the scanning electron microscope (SEM) in the backscattered electron imaging mode was used for the study of blood cells incubated with various monoclonal antibodies. Numerous antileukocyte monoclonal antibodies still recognize lightly glutaraldehyde prefixed antigens and can be used to identify various blood cell types and even to recognize their different maturation stages. Clearcut differences in surface morphology exist among peripheral blood normal leukocytes and even among the principal lymphocyte subclasses. Marked heterogeneity in surface morphology is, on the other hand, evident when studying precursors or leukemic cells. Immature cells show, nevertheless, relatively smooth surfaces while some distinct surface features appear on cells already committed toward a specific differentiation lineage. Hairy cells can also be precisely identified, especially when in small number in heterogeneous populations, combining their typical surface morphology with their positivity for B1 and Leu M5 monoclonal antibodies

Immuno-Cytochemistry with Backscattered Electrons

Some cytochemical reaction products are visible inside the cytoplasm of cells observed with the scanning electron microscope (SEM) using the backscattered electron imaging (BEI) mode. Methods can be utilized whenever they result in the deposition of heavy metal, like silver, lead or osmium at the sites of the enzymatic reaction. More recently the BEI mode of the SEM has been demonstrated to improve the detection of immunogold labeled cell surface antigens. Colloidal gold particles, 40 to 15 nm in diameter can be efficiently used for immuno-specific labeling. Moreover, cytochemical reactions can be applied to previously immunogold labeled cells, therefore combining the results of enzyme cytochemistry and of surface labeling at the level of each individual cell. The choice of fixative, incubation media, dehydration and drying methods should be guided by considerations on the sample characteristics for optimal electron scattering. Cytochemical as well as immuno-labeling reactions are not used per se but in combination with the study of cell surface morphology which needs, therefore, to be sufficiently well preserved. Coating should provide good conductivity and secondary electron emission, while emitting a minimal number of backscattered electrons. The application of these methods considerably enhances our capacity to characterize with the SEM the surface morphology of precisely identified subpopulations of many cell types

A bovine lymphosarcoma cell line infected with theileria annulata exhibits an irreversible reconfiguration of host cell gene expression

Theileria annulata, an intracellular parasite of bovine lymphoid cells, induces substantial phenotypic alterations to its host cell including continuous proliferation, cytoskeletal changes and resistance to apoptosis. While parasite induced modulation of host cell signal transduction pathways and NFκB activation are established, there remains considerable speculation on the complexities of the parasite directed control mechanisms that govern these radical changes to the host cell. Our objectives in this study were to provide a comprehensive analysis of the global changes to host cell gene expression with emphasis on those that result from direct intervention by the parasite. By using comparative microarray analysis of an uninfected bovine cell line and its Theileria infected counterpart, in conjunction with use of the specific parasitacidal agent, buparvaquone, we have identified a large number of host cell gene expression changes that result from parasite infection. Our results indicate that the viable parasite can irreversibly modify the transformed phenotype of a bovine cell line. Fifty percent of genes with altered expression failed to show a reversible response to parasite death, a possible contributing factor to initiation of host cell apoptosis. The genes that did show an early predicted response to loss of parasite viability highlighted a sub-group of genes that are likely to be under direct control by parasite infection. Network and pathway analysis demonstrated that this sub-group is significantly enriched for genes involved in regulation of chromatin modification and gene expression. The results provide evidence that the Theileria parasite has the regulatory capacity to generate widespread change to host cell gene expression in a complex and largely irreversible manner

Cell Surface Changes of Hemopoietic Cells During Normal and Leukemic Differentiation: An Immuno-Scanning Electron Microscopy Study

Hemopoietic cells display a wide range of cell surface antigens which are either lineage specific or acquired during differentiation. Monoclonal antibodies can be used, in conjunction with colloidal gold markers, to identify under the scanning electron microscopy (SEM) at the single cell level, specific lineage or maturation stages in the hemopoietic bone marrow. Normal bone marrow cells, either gradient separated or purified by immuno-magnetic methods and leukemic cell samples, which can be considered as frozen stages of hemopoietic differentiation, have been studied with this method. Typical cell surface morphologies, which characterize immature progenitor cells and cells committed or differentiated towards the lymphoid, myeloid, erythroid and megakaryocytic lineage have been identified. Correlations between cell surface features and some hemopoietic cells functions have been attempted on the basis of these findings

Scanning Electron Microscope Cytochemistry of Blood Cells

The backscattered electron imaging (BEI) mode of scanning electron microscopy (SEM) has been applied to study various histo-cytochemical reactions in biological specimens since the early seventies. Due to numerous, recent technical improvements the BEI mode of SEM now belongs to the routine of many SEM laboratories. For cytochemistry, BEI has been mainly used to: visualize intracellular structures and organelles; recognize the different cell types in heterogeneous populations or tissues; study the correlations between enzymatic activities and cell surface features. We have evaluated the most relevant results obtained in the study of blood cells and the possible future applications of these techniques

Indoor Radon Surveying and Mitigation in the Case-Study of Celleno Town (Central Italy) Located in a Medium Geogenic Radon Potential Area

Indoor radon surveying and remediation were implemented in a single-family home affected by high levels of indoor radon in the Celleno municipality (central Italy) with the aim of identifying the contribution of radon sources, evaluating the factors affecting radon entry into the building, and reducing radon risk. Average radon levels were relatively low at the ground floor (286 ± 202 Bq m−3) and first floor (167 ± 84 Bq m−3) in autumn when the temperature was still warm and the windows were open, but increased up to 2776 ± 1768 Bq m−3 and 970 ± 202 Bq m−3 in the first half of December, when the heating system was on and the windows were closed. The inner walls of the pilot room at the ground floor, semi buried on one side, were then treated with a waterproof product (a silane terminated polymer) and the average radon was halved (1475 ± 1092 Bq m−3) in the following month, which was still characterised by winter conditions. Radon entry in the room was identified and sealed with the same product, and a radon accumulation space behind a NE-SW oriented wall was naturally ventilated, reducing radon below the reference level in April with northerly winds conditions

The impact of COVID-19 pandemic on pornography habits: a global analysis of Google Trends

As the COVID-19 spread globally, social distancing, self-isolation/quarantine, and national lockdowns have become crucial to control the pandemic. However, these measures may also lead to increases in social isolation, loneliness, and stress, which can alter the consumption of pornography habits. The aim of the study was thus to explore the interest pattern in pornography and coronavirus-themed pornography during the COVID-19 outbreak. Google Trends\uae was employed to determine the most popular porn websites (Porn, XNXX, PornHub, xVideos, and xHamster), and coronavirus-themed pornography worldwide and in six nations with different COVID-19 outbreak and self-isolation recommendations. We analyzed every search trend on Google\uae from January 9, 2020 to May 25, 2020 using \u201cjoint point regression analysis\u201d. Comparisons of week relative search volume (WRSV) and temporal patterns were analyzed to assess the change of interest in search terms during nations lockdowns. Paired t-test was used to compare WRSV values among the porn websites during the national lockdowns and the equivalent timespan of the weeks in the previous 4 years. The research trend of almost every keyword increased with significant inflection points for those nations with a straight \u201cstay at home orders\u201d (China, Italy, Spain, and France). \u201cPornHub\u201d and \u201cPorn\u201d showed the highest increase of interest worldwide with an average weekend percentage change (AWPC) of 4.9 and 3.8, respectively. The mean WRSV for keywords in USA and Sweden did not show a similar increase as the other nations. The WRSV percentage change with the historical data had a peak during the straight nations\u2019 lockdowns (p < 0.01). All the nations had a significant increase in WRSV coronavirus-themed pornography for each keyword (p < 0.01) with an AWPC, ranging worldwide between 18.5 and 61.8 (p < 0.01), after the beginning of self-quarantine. As strengths this study uses a big data technology to collect worldwide trend of interest, however, data are anonymous and do not allow analysis of subpopulation groups. In conclusion, we demonstrated an increased interest in pornography and coronavirus-themed pornography after the outbreak of COVID-19 in nations with a straight \u201cstay at home orders\u201d

Reduced intensity conditioning allogeneic transplant for advanced chronic lymphocytic leukemia

We report the preliminary results of 12 patients with advanced stage chronic lymphocytic leukemia (CLL) transplanted following reduced intensity conditioning (RIC. With a median of 22 months of follow-up, 9 patients are alive and 3 have died of progressive disease, graft-versus-host disease (GVHD) or toxic hepatitis. Acute grade I-III GVHD occurred in 33% of patients and chronic GVHD in 50%. Eight of the 12 patients achieved a complete remission (CR) and 2 patients a partial remission (PR). Donor lymphocyte infusion was effective in 6 patients. Event-free survival, progression-free survival and non-relapse mortality at 3 years were 68%, 42% and 16%, respectively. Our results show successful immunomodulation and reduction in tumor burden in high risk CL