Golgi staining of neurons: Oxidation-state dependent spread of chemical reaction identifies a testable property of the connectome

Camillo Golgi observed reticular nature of the nervous system by his staining method. Ramon Cajal modified this protocol to obtain staining restricted to individual neurons, in support of the cell theory. Close examination shows that Golgi used an oxidizing agent to pre-treat the brain tissue before the staining reaction and Cajal used an additional oxidizing agent for the same step. It shows that oxidation state of the tissue has a crucial role in determining the spread of Golgi chemical reaction between neurons. The correct structure-function mechanism of brain functions may reveal the nature of the route through which the staining reaction spreads between neurons under decreasing oxidation states. Present work examines the chemical reaction behind the staining, explores the role of oxidizing agents in limiting the stain to individual neurons, and discusses a probable property of the connectome that can provide a gateway for an oxidation state-dependent spread of staining reaction.

P02.06 Semi-automated validation and quantification of CTLA-4 in 90 different Tumor entities using multiple antibodies and artificial intelligence

BackgroundCTLA-4 is an inhibitory immune checkpoint receptor and a negative regulator of anti-tumor T-cell function. This study aimed at a comparative analysis of CTLA-4+ cells between different tumor entities.Materials and MethodsTo quantify CTLA-4+ cells, 4,582 tumor samples from 90 different tumor entities as well as 608 samples of 76 different normal tissue types were analyzed by immunohistochemistry in a tissue microarray format. Two different antibody clones (MSVA-152R and CAL49) were validated and quantified using a deep learning framework for automated exclusion of unspecific immunostaining.ResultsComparing both CTLA-4 antibodies revealed a clone dependent unspecific staining pattern in adrenal cortical adenoma (63%) for MSVA-152R and in pheochromocytoma (67%) as well as hepatocellular carcinoma (36%) for CAL49. After automated exclusion of non-specific staining reaction (3.6%), a strong correlation was observed for the densities of CTLA-4+ lymphocytes obtained by both antibodies (r=0.87; p<0.0001). The mean density of CTLA-4+ cells was 674±1482 cells/mm2 and ranged from 71±175 cells/mm2 in leiomyoma to 5916±3826 cells/mm2 in Hodgkin’s lymphoma. Within epithelial tumors, the density of CTLA-4+ lymphocytes were higher in squamous cell (421±467 cells/mm2) and urothelial carcinomas (419±347 cells/mm2) than in adenocarcinomas (269±375 cells/mm2) and renal cell neoplasms (256±269 cells/mm2). A high CTLA-4+ cell density was linked to low pT category (p<0.0001), absent lymph node metastases (p=0.0354), and PD-L1 expression in tumor cells or inflammatory cells (p<0.0001 each). A high CTLA-4/CD3-ratio was linked to absent lymph node metastases (p=0.0295) and to PD-L1 positivity on immune cells (p<0.0026).ConclusionsMarked differences exist in the number of CTLA-4+ lymphocytes between tumors. Analyzing two independent antibodies by a deep learning framework can facilitate automated quantification of immunohistochemically analyzed target proteins such as CTLA-4.Disclosure InformationD. Dum: None. T.L.C. Henke: None. T. Mandelkow: None. E. Bady: None. R. Simon: None. G. Sauter: None. S. Steuerer: None. W. Wilczak: None. E. Burandt: None. J. Raedler: None. M. Lennartz: None. N.C. Blessin: None.

Semi-automated validation and quantification of CTLA-4 in 90 different Tumor entities using multiple antibodies and artificial intelligence

Introduction/Objective Introduction: CTLA-4 is an inhibitory immune checkpoint receptor and a negative regulator of anti-tumor T-cell function. This study aimed at a comparative analysis of CTLA-4+ entities. cells between different tumor Methods/Case Report Methods: To quantify CTLA-4+ cells, 4,582 tumor samples from 90 different tumor entities as well as 608 samples of 76 different normal tissue types were analyzed by immunohistochemistry in a tissue microarray format. Two different antibody clones (MSVA-152R and CAL49) were validated and quantified using a deep learning framework for automated exclusion of unspecific immunostaining. Results (if a Case Study enter NA) Results: Comparing both CTLA-4 antibodies revealed a clone dependent unspecific staining pattern in adrenal cortical adenoma (63%) for MSVA-152R and in pheochromocytoma (67%) as well as hepatocellular carcinoma (36%) for CAL49. After automated exclusion of non-specific staining reaction (3.6%), a strong correlation was observed for the densities of CTLA-4+ lymphocytes obtained by both antibodies (r=0.87; p&lt;0.0001). The mean density of CTLA-4+cells was 674±1482 cells/ mm2 and ranged from 71±175 cells/mm2 in leiomyoma to 5916±3826 cells/mm2 in Hodgkin’s lymphoma. Within epithelial tumors, the density of CTLA-4+ lymphocytes were higher in squamous cell (421±467 cells/ mm2) and urothelial carcinomas (419±347 cells/ mm2) than in adenocarcinomas (269±375 cells/ mm2) and renal cell neoplasms (256±269 cells/ mm2). A high CTLA-4+ cell density was linked to low pT category (p&lt;0.0001), absent lymph node metastases (p=0.0354), and PD-L1 expression in tumor cells or inflammatory cells (p&lt;0.0001 each). A high CTLA-4/CD3-ratio was linked to absent lymph node metastases (p=0.0295) and to PD-L1 positivity on immune cells (p&lt;0.0026). Conclusion Marked differences exist in the number of CTLA-4+ lymphocytes between tumors. Analyzing two independent antibodies by a deep learning framework can facilitate automated quantification of immunohistochemically analyzed target proteins such as CTLA-4.

The study the formation of silver nanoparticles by bacteria of the genus Bacillus

Nanotechnology studies materials with a size of 1-100 nm. Various microorganisms (bacteria, fungi. actinomycetes and others) are involved in the synthesis of nanoparticles in a biological way. Nanoscale materials are important as new antimicrobials due to their unusual physical and chemical properties - the activity of intracellular and extracellular enzymes plays a key role in the synthesis of nanoparticles by microorganisms. Was studied the ability of Bacillus sp strains to formation of silver nanoparticles. During the study, silver nitrate solution was added to biomass and culture medium of Bacillus sp.l, sp.2, sp.3. It was found that the culture medium of the Bacillus sp. l strain capableof producing silver nanoparticles, but the biomass does not have the property of forming silver nanoparticles. Its ability to form silver nanoparticles was detected by staining reaction medium in a dark color. However, the ivestigation the ability to formation silver nanoparticles by strains Bacillus sp2 and Bacillus sp3 showed that they cannot biosynthesis the silver nanoparticles. Was investigated the morpho-cultural features of the bacterial strain Bacillus sp.l. Was shown that Bacillus sp.l strain belonged to Bacillus cereus specie. Key words: bacteria, silver nanoparticles, Bacillus sp, culture fluid, biomass

Expression of Immune Checkpoint Regulators, Cytotoxic T Lymphocyte Antigen 4 (CTLA-4) and CD137 in Cervical Carcinomas

Introduction: Immune checkpoint inhibitors in cancer therapy has a significant role in oncology. One of these immune checkpoint mediators is cytotoxic T-lymphocyte associated protein 4 (CTLA-4). Inhibition of the CTLA-4 pathway has already led to the FDA approval of Ipilimumab (anti-CTLA-4), a targeted therapy for melanoma and other malignancies. CD137 is an inducible, costimulatory receptor of the tissue necrosis factor receptor superfamily expressed on activated immune cells. Clinical trials had also been set for anti-CD137 in several malignancies. We investigated the expression of CTLA-4 and CD137 antibodies in benign and malignant uterine cervical tissues. Method: We assessed CTLA-4 and CD137 expression on a tissue microarray (TMA) comprising of 100 normal, non-neoplastic, and neoplastic cervical tissues. When detected as strong granular cytoplasmic reaction in the epithelial cells, CTLA-4 expression was scored as positive. For CD137, the results were recorded based on the presence or absence of staining reaction on the cell membranes of the lymphoplasmacytic infiltrate. Result: Overall, CTLA-4 was positive in 30% (30/100) of the cervical malignancies. Subcategorically, 20% of invasive endocervical adenocarcinomas, 62.5% of adenosquamous carcinomas, and 31% of squamous cell carcinomas were positive for CTLA-4 with a tendency toward lower grades SCCs. CD137 was positive in lymphoplasmacytic infiltrates of all endocervical adenocarcinomas, 90.5% of squamous cell carcinoma, and 87.5% cores of adenosquamous carcinomas. Conclusion: This study has found a significant expression of CTLA-4 in cervical cancer cells and CD137 positivity of lymphoplasmacytic infiltrates with potentials for future targeted immunotherapy.

Myelin basic protein and neurofilament H in postmortem cerebrospinal fluid as surrogate markers of fatal traumatic brain injury

The aim of this study was to investigate if the biomarkers myelin basic protein (MBP) and neurofilament-H (NF-H) yielded informative value in forensic diagnostics when examining cadaveric cerebrospinal fluid (CSF) biochemically via an enzyme-linked immunosorbent assay (ELISA) and comparing the corresponding brain tissue in fatal traumatic brain injury (TBI) autopsy cases by immunocytochemistry versus immunohistochemistry. In 21 trauma and 19 control cases, CSF was collected semi-sterile after suboccipital puncture and brain specimens after preparation. The CSF MBP (p = 0.006) and NF-H (p = 0.0002) levels after TBI were significantly higher than those in cardiovascular controls. Immunohistochemical staining against MBP and against NF-H was performed on cortical and subcortical samples from also biochemically investigated cases (5 TBI cases/5 controls). Compared to the controls, the TBI cases showed a visually reduced staining reaction against MBP or repeatedly ruptured neurofilaments against NF-H. Immunocytochemical tests showed MBP-positive phagocytizing macrophages in CSF with a survival time of > 24 h. In addition, numerous TMEM119-positive microglia could be detected with different degrees of staining intensity in the CSF of trauma cases. As a result, we were able to document that elevated levels of MBP and NF-H in the CSF should be considered as useful neuroinjury biomarkers of traumatic brain injury.

Assessment of Bacteria and Parasite Contamination of Dried Sliced Beef (Kilishi) Sold within Birnin Kebbi Metropolis, Kebbi State, Northern Nigeria

Kilishi is a version of jerky that originated in Hausa land Nigeria. It is made from deboned cow, sheep or goat meat. The dried sliced beef (kilishi) is often left open in a basin thereby exposing it to flies, dust and other effects of the environment by so doing the product can be contaminated. This study was carried out to investigate the bacteria and parasite contaminants of dried slice beef (kilishi) sold in different locations within Birnin Kebbi metropolis. The samples were analyzed using pour plate method. The bacterial species were characterized and identified on the basis of their colonial morphology; gram’s staining reaction and biochemical characteristics. The protozoans and helminthes cyst/eggs morphology were identified using microscopy techniques. The total bacteria plate count for each sample ranges from the highest (8x109) to the lowest (3.5x103) CFU/g from Birnin Kebbi Kalgo, Jega and Aliero samples respectively. The organisms isolated include; Staphylococcus aureus (35.2%), Escherichia coli showed 30(21.1%) percentage of occurrence, Bacillus species occurred 17(12%) Klebsiella spp 16(11.3%), Pseudomonas spp 13(9.2%), Shigella spp 10(7.04%) and the least was P. vulgaris 6(4.2%). The kilishi meat product was also contaminated with some Protozoans and helminthes contaminants which include; Acaris lumbricoides (14.2%), Entamoeba histolytica (35%), Girdia lambila (42.8%), and Taenia spp. (7.1%). It was concluded that the high bacteria count and frequency of isolates from the kilishi samples tested is an indication of high contamination of the meat by potential pathogens due to poor handling and sanitary conditions which may pose a potential source of food borne diseases.

Aerobic Bacteriology of Burn Wound Infections in Burn Patients and Their Antibiotic Susceptibility Pattern in a Tertiary Care Hospital in Kashmir Valley

Aims: The burn wound represents a susceptible site for opportunistic colonization by organisms of endogenous and exogenous origin. Burn wound infections are an important cause of mortality, morbidity and prolonged hospitalization in burn patients and the causative agent is generally a multidrug resistant organism. The pattern of microbial flora infecting burn wound varies according to geographical pattern as well as with duration of hospital stay. The main aim of the study was to determine the bacteriological profile and antimicrobial susceptibility patterns of burn wound isolates. Study Design: It was a prospective cross-sectional hospital-based study. Place and Duration of Study: The present study was conducted in the Department of Microbiology, Government Medical College, Srinagar, from December 2019 to November 2020. Methodology: Swabs were taken from burn wound of 351 patients and cultured aerobically. Samples were processed for identification and sensitivity. Bacteria isolated were identified using their morphological characteristics, Gram staining reaction and biochemical tests. The antimicrobial susceptibility testing was done using Kirby-Bauer disc diffusion method. Results: A total of 351 samples were obtained for the study out of which, the most common isolate was Klebsiella pneumoniae – 154 (38.3%), followed by Pseudomonas aeruginosa - 133 (33.08%), Proteus sp – 42( 10.44%) , Acinetobacter sp- 26 (6.46%), Staphylococcus aureus - 26 (6.46%), Escherichia coli - 17 (4.22%), Enterococcus - 2 (0.49%), and Providencia sp- 2 (0.49%). Colistin was the most effective drug against Gram negative organisms while as linezolid was most effective against Gram positives. Conclusion: The finding of the study will be helpful for identifying the common bacteria causing burn wound infection and also to take proper precautions to prevent the emergence of antibiotic-resistant bacteria.

Histochemical demonstration of protein concentration in intestinal tissue of coccidiosis infected broiler chicks treated with Mercurius corrosivus

The present investigation includes the study of protein concentration in intestinal tissue of coccidiosis infected broiler chicks orally inoculated by the infection of Eimeria tenella sporulated oocysts and treated with Mercurius corrosivus by using the methods of histochemical techniques. The treatment of homoeopathic medicine Mercurius corrosivus was administered to the coccidiosis infected group of broiler chicks.For the study of protein concentration in intestinal tissue of the coccidiosis infected and non-infected broiler chicks, the histochemical technique was used. Histochemical study indicates that the presence of different concentration of protein content found in the intestinal tissue of the broiler chicks.Intestinal tissue of all the chicks of different groups have presence of protein content in variable concentrations. It is also observed that the presence of protein concentration was variable in different region and shows different traces like minimum, medium and maximum amount of protein content found in different region of the intestinal tissue.The amount of protein concentration was observed high in group B(INC) in comparison to the other group of chicks observed during the experimental period. The concluded results of the study by using histochemical techniques shows that the protein concentration in the intestinal tissue is demonstrated by the variable staining reaction. The concentration of protein content is maximum in the parasitic stages showing the utilization of the protein from the host tissue. Whereas the infected and treated group with homoeopathic medicine Mercurius corrosivus also showed variable staining reaction according to the presence of protein in the intestinal tissue.

Application of histochemical stains for rapid qualitative analysis of the lignin content in multiple wood species

Rapid qualitative analysis was used to determine the influence of the lignin content of wood cell walls on the compression and bending properties of multiple wood species. The lignin type and cell wall content of Cunninghamia lanceolate, Fagus longipetiolata, Betula alnoides, Fraxinus mandshurica, and Tectona grandis was analyzed via histochemical staining, which included: the Mäule staining reaction, the Weisner staining reaction, and a fluorescence reaction. The results showed that the more red the Mäule staining reaction was, the greater the Syringyl lignin (S-type lignin) content was, and the more yellowish-brown the Mäule staining reaction was, the greater the Guaiacyl lignin (G-type lignin) content was. In addition, the more reddish-purple the Wiesner staining reaction was, the greater the lignin content was. The greater the brightness value of the fluorescence reaction was, the greater the lignin content was. Due to the negative correlation between the lignin content of the wood cell wall and the bending and compression properties of the wood, the application of histochemical stains for the analysis of wood lignin content could provide a reference and experimental basis for bending and compression treatments of various woods.