Ultrasensitive and Multiplexed Protein Imaging with Clickable and Cleavable Fluorophores.

Single-cell spatial proteomic analysis holds great promise to advance our understanding of the composition, organization, interaction, and function of the various cell types in complex biological systems. However, the current multiplexed protein imaging technologies suffer from low detection sensitivity, limited multiplexing capacity, or are technically demanding. To tackle these issues, here, we report the development of a highly sensitive and multiplexed in situ protein profiling method using off-the-shelf antibodies. In this approach, the protein targets are stained with horseradish peroxidase (HRP) conjugated antibodies and cleavable fluorophores via click chemistry. Through repeated cycles of target staining, fluorescence imaging, and fluorophore cleavage, many proteins can be profiled in single cells in situ. Applying this approach, we successfully quantified 28 different proteins in human formalin-fixed paraffin-embedded (FFPE) tonsil tissue, which represents the highest multiplexing capacity among the tyramide signal amplification (TSA) methods. Based on their unique protein expression patterns and their microenvironment, ∼820,000 cells in the tissue are classified into distinct cell clusters. We also explored the cell-cell interactions between these varied cell clusters and observed that different subregions of the tissue are composed of cells from specific clusters.

Delineation of the healthy rabbit tonsil by immunohistochemistry - A short communication.

Situated in the oral cavity, the rabbit palatine tonsils are part of the mucosal immune system and help to defend the body against foreign pathogens. Expressed as two oval protrusions in the wall of the oropharynx, the rabbit palatine tonsils are characterized by excretory ducts and trabeculae. We here compare paraffin embedded and cryosections of the healthy rabbit tonsils. This analysis centers on evaluating the differential outcomes resulting from the application of these fixation methodologies in conjunction with immunohistochemical assays targeting collagen I, collagen III, fibronectin, α-smooth muscle actin (α-SMA), and ki67. Subsequent recommendations are provided based on our findings. Furthermore, we demonstrate the advantage of an antigen retrieval step in immunohistochemical labeling of paraffin sections. Basic classical histological stainings as HE, GT and elastin were also performed. Comparison of different stainings and labelings was furthermore performed in serial sections, showing that adjacent to the excretory ducts, the tonsillar tissue was particularly composed of collagen I and fibronectin, while the vessel walls were predominantly α-SMA positive. Moreover, PAR-2 immunohistochemical staining was performed, where a small fraction of the cells found in the tonsillar connective tissue were PAR-2 positive (probably a subpopulation of mast cells), as well as the lumen of some excretory ducts and trabeculae. Collagen III on the other hand was only weakly expressed in the tonsils. Proliferating ki67 positive cells were rare. This endeavor serves to furnish the scientific community with reference imagery pertinent to researchers opting for the rabbit palatine tonsil model. The diversity of staining techniques employed herein establishes a foundational repository of images, primed for comparative analysis against pathological conditions. Furthermore, these images hold the potential to illustrate inter-species variations. For instance, they can be juxtaposed against murine or rodent tonsils, or even offer insights into the human context.

Distribution of periodontopathic bacterial species between saliva and tonsils.

Periodontopathic bacteria cause an inflammatory disease localized in the periodontal tissue and are associated with various conditions in other body parts. The distribution of periodontopathic bacterial species in the tonsils is unknown, even though the tonsils are located close to the oral cavity, and inflammation of the tonsils causes various systemic diseases. We detected the major periodontopathic bacterial species residing in saliva and tonsil specimens from 25 subjects undergoing tonsillectomy. Nine of the ten major periodontopathic bacterial species were detected by polymerase chain reaction of tonsil specimens, among which Campylobacter rectus was the most common (80.0%), followed by Porphyromonas gingivalis (36.0%). The other seven types of periodontopathic bacterial species were distributed with 0% to 25.0% abundance in the tonsil specimens. C. rectus had a high detection rate in tonsil specimens (> 75.0%), regardless of whether it was detected in the corresponding saliva specimens. However, the detection rate for P. gingivalis in tonsil specimens was significantly higher in subjects with P. gingivalis-positive saliva (77.8%) than in those with P. gingivalis-negative saliva (6.3%; P < 0.001). Furthermore, 75.0% of P. gingivalis in tonsil specimens did not have the known fimA gene that encodes the 41-kDa filamentous appendage protein FimA, which is expressed on the cell surface of the bacteria. Our results suggest that certain periodontopathic bacterial species are detected in the tonsils either independently of or depending on their distribution in the oral cavity and may be involved in tonsil-related diseases.

Ultrasensitive and Multiplexed Protein Imaging with Cleavable Fluorescent Tyramide and Antibody Stripping.

Multiplexed single-cell analysis of proteins in their native cellular contexts holds great promise to reveal the composition, interaction and function of the distinct cell types in complex biological systems. However, the existing multiplexed protein imaging technologies are limited by their detection sensitivity or technical demands. To address these issues, here, we develop an ultrasensitive and multiplexed in situ protein profiling approach by reiterative staining with off-the-shelf antibodies and cleavable fluorescent tyramide (CFT). In each cycle of this approach, the protein targets are recognized by antibodies labeled with horseradish peroxidase, which catalyze the covalent deposition of CFT on or close to the protein targets. After imaging, the fluorophores are chemically cleaved, and the antibodies are stripped. Through continuous cycles of staining, imaging, fluorophore cleavage and antibody stripping, a large number of proteins can be quantified in individual cells in situ. Applying this method, we analyzed 20 different proteins in each of ~67,000 cells in a human formalin-fixed paraffin-embedded (FFPE) tonsil tissue. Based on their unique protein expression profiles and microenvironment, these individual cells are partitioned into different cell clusters. We also explored the cell-cell interactions in the tissue by examining which specific cell clusters are selectively associating or avoiding each other.

Highly Multiplexed Immunohistochemical MALDI-MS Imaging of Biomarkers in Tissues.

Immunohistochemistry (IHC) combined with fluorescence microscopy provides an important and widely used tool for researchers and pathologists to image multiple biomarkers in tissue specimens. However, multiplex IHC using standard fluorescence microscopy is generally limited to 3-5 different biomarkers, with hyperspectral or multispectral methods limited to 8. We report the development of a new technology based on novel photocleavable mass-tags (PC-MTs) for facile antibody labeling, which enables highly multiplexed IHC based on MALDI mass spectrometric imaging (MALDI-IHC). This approach significantly exceeds the multiplexity of both fluorescence- and previous cleavable mass-tag-based methods. Up to 12-plex MALDI-IHC was demonstrated on mouse brain, human tonsil, and breast cancer tissues specimens, reflecting the known molecular composition, anatomy, and pathology of the targeted biomarkers. Novel dual-labeled fluorescent PC-MT antibodies and label-free small-molecule mass spectrometric imaging greatly extend the capability of this new approach. MALDI-IHC shows promise for use in the fields of tissue pathology, tissue diagnostics, therapeutics, and precision medicine.

Transcriptome Analysis of the Cecal Tonsil of Jingxing Yellow Chickens Revealed the Mechanism of Differential Resistance to Salmonella.

Salmonella is one of the most common food-borne pathogens. It can be transmitted between chickens, as well as to people by contaminated poultry products. In our study, we distinguished chickens with different resistances mainly based on bacterial loads. We compared the cecal tonsil transcriptomes between the susceptible and resistant chickens after Salmonella infection, aiming to identify the crucial genes participating in the antibacterial activity in the cecal tonsil. A total of 3214 differentially expressed genes (DEGs), including 2092 upregulated and 1122 downregulated genes, were identified between the two groups (fold change ≥ 2.0, padj < 0.05). Many DEGs were mainly involved in the regulation of two biological processes: crosstalk between the cecal tonsil epithelium and pathogenic bacteria, such as focal adhesion, extracellular-matrix-receptor interaction, and regulation of the actin cytoskeleton and host immune response including the cytokine-receptor interaction. In particular, the challenged resistant birds exhibited strong activation of the intestinal immune network for IgA production, which perhaps contributed to the resistance to Salmonella infection. These findings give insight into the mRNA profile of the cecal tonsil between the two groups after initial Salmonella stimulation, which may extend the known complexity of molecular mechanisms in chicken immune response to Salmonella.

Tissue-Specific Gene Expression during Productive Human Papillomavirus 16 Infection of Cervical, Foreskin, and Tonsil Epithelium.

Epidemiological data confirm a much higher incidence of high-risk human papillomavirus 16 (HPV16)-mediated carcinogenesis of the cervical epithelium than for other target sites. In order to elucidate tissue-specific responses to virus infection, we compared gene expression changes induced by productive HPV16 infection of cervical, foreskin, and tonsil organotypic rafts. These rafts closely mimic persistent HPV16 infection, long before carcinogenesis sets in. The total number of gene expression changes varied considerably across the tissue types, with only 32 genes being regulated in common. Among them, we confirmed the Kelch-like family protein KLHL35 and the laminin-5 complex to be upregulated and downregulated, respectively, in all the three tissues. HPV16 infection induces upregulation of genes involved in cell cycle control, cell division, mitosis, DNA replication, and DNA damage repair in all the three tissues, indicative of a hyperproliferative environment. In the cervical and tonsil epithelium, we observe significant downregulation of genes involved in epidermis development, keratinocyte differentiation, and extracellular matrix organization. On the other hand, in HPV16-positive foreskin (HPV16 foreskin) tissue, several genes involved in interferon-mediated innate immunity, cytokine signaling, and cellular defenses were downregulated. Furthermore, pathway analysis and experimental validations identified important cellular pathways like STAT1 and transforming growth factor ß (TGF-ß) to be differentially regulated among the three tissue types. The differential modulation of important cellular pathways like TGF-ß1 and STAT1 can explain the sensitivity of tissues to HPV cancer progression.IMPORTANCE Although the high-risk human papillomavirus 16 infects anogenital and oropharyngeal sites, the cervical epithelium has a unique vulnerability to progression of cancer. Host responses during persistent infection and preneoplastic stages can shape the outcome of cancer progression in a tissue-dependent manner. Our study for the first time reports differential regulation of critical cellular functions and signaling pathways during productive HPV16 infection of cervical, foreskin, and tonsil tissues. While the virus induces hyperproliferation in infected cells, it downregulates epithelial differentiation, epidermal development, and innate immune responses, according to the tissue type. Modulation of these biological functions can determine virus fitness and pathogenesis and illuminate key cellular mechanisms that the virus employs to establish persistence and finally initiate disease progression.

Marginal Effects of Systemic CCR5 Blockade with Maraviroc on Oral Simian Immunodeficiency Virus Transmission to Infant Macaques.

Current approaches do not eliminate all human immunodeficiency virus type 1 (HIV-1) maternal-to-infant transmissions (MTIT); new prevention paradigms might help avert new infections. We administered maraviroc (MVC) to rhesus macaques (RMs) to block CCR5-mediated entry, followed by repeated oral exposure of a CCR5-dependent clone of simian immunodeficiency virus (SIV) mac251 (SIVmac766). MVC significantly blocked the CCR5 coreceptor in peripheral blood mononuclear cells and tissue cells. All control animals and 60% of MVC-treated infant RMs became infected by the 6th challenge, with no significant difference between the number of exposures (P = 0.15). At the time of viral exposures, MVC plasma and tissue (including tonsil) concentrations were within the range seen in humans receiving MVC as a therapeutic. Both treated and control RMs were infected with only a single transmitted/founder variant, consistent with the dose of virus typical of HIV-1 infection. The uninfected RMs expressed the lowest levels of CCR5 on the CD4+ T cells. Ramp-up viremia was significantly delayed (P = 0.05) in the MVC-treated RMs, yet peak and postpeak viral loads were similar in treated and control RMs. In conclusion, in spite of apparent effective CCR5 blockade in infant RMs, MVC had a marginal impact on acquisition and only a minimal impact on the postinfection delay of viremia following oral SIV infection. Newly developed, more effective CCR5 blockers may have a more dramatic impact on oral SIV transmission than MVC.IMPORTANCE We have previously suggested that the very low levels of simian immunodeficiency virus (SIV) maternal-to-infant transmissions (MTIT) in African nonhuman primates that are natural hosts of SIVs are due to a low availability of target cells (CCR5+ CD4+ T cells) in the oral mucosa of the infants, rather than maternal and milk factors. To confirm this new MTIT paradigm, we performed a proof-of-concept study in which we therapeutically blocked CCR5 with maraviroc (MVC) and orally exposed MVC-treated and naive infant rhesus macaques to SIV. MVC had only a marginal effect on oral SIV transmission. However, the observation that the infant RMs that remained uninfected at the completion of the study, after 6 repeated viral challenges, had the lowest CCR5 expression on the CD4+ T cells prior to the MVC treatment appears to confirm our hypothesis, also suggesting that the partial effect of MVC is due to a limited efficacy of the drug. New, more effective CCR5 inhibitors may have a better effect in preventing SIV and HIV transmission.

EVALUATION OF A TEST AND CULL STRATEGY FOR REDUCING PREVALENCE OF CHRONIC WASTING DISEASE IN MULE DEER ( ODOCOILEUS HEMIONUS).

We evaluated a test and cull strategy for lowering chronic wasting disease (CWD) prevalence in a naturally-infected, free-ranging mule deer ( Odocoileus hemionus) herd wintering in the town of Estes Park, Colorado, US and in nearby Rocky Mountain National Park. We tested 48-68% of the estimated number of adult (≥1 yr old) deer annually for 5 yr via tonsil biopsy immunohistochemistry (IHC), collecting 1,251 samples from >700 individuals and removing IHC-positive deer. Among males, CWD prevalence during the last 3 yr of selective culling was lower (one-sided Fisher's exact test P=0.014) than in the period prior. In contrast, CWD prevalence among females before culling and after culling were equivalent ( P=0.777). Relatively higher annual testing of males (mean 77%) compared to females (mean 51%) might have contributed to differences seen in responses to management. A more intensive and sustained effort or modified spatial approach might have reduced prevalence more consistently in both sexes. Limitations of this technique in wider management application include cost and labor as well as property access and animal tolerance to repeated capture. However, elements of this approach could potentially be used to augment harvest-based disease management.

A Fluorescent Probe to Unravel Functional Features of Cannabinoid Receptor CB1 in Human Blood and Tonsil Immune System Cells.

The human endogenous cannabinoid system (ECS) regulates key physiological processes and alterations in its signaling pathways, and endocannabinoid levels are associated with diseases such as neurological and neuropsychiatric conditions, cancer, pain and inflammation, obesity, and metabolic and different immune related disorders. Immune system cells express the G-protein coupled cannabinoid receptor 1 (CB1), but its functional role has not been fully understood, likely due to the lack of appropriate tools. The availability of novel tools to investigate the role of CB1 in immune regulation might contribute to identify CB1 as a potential novel therapeutic target or biomarker for many diseases. Herein, we report the development and validation of the first fluorescent small molecule probe to directly visualize and quantify CB1 in blood and tonsil immune cells by flow cytometry and confocal microscopy. We coupled the cannabinoid agonist HU210 to the fluorescent tag Alexa Fluor 488, generating a fluorescent probe with high affinity for CB1 and selectivity over CB2. We validate HU210-Alexa488 for the rapid, simultaneous, and reproducible identification of CB1 in human monocytes, T cells, and B cells by multiplexed flow cytometry. This probe is also suitable for the direct visualization of CB1 in tonsil tissues, allowing the in vivo identification of tonsil CB1-expressing T and B cells. This study provides the first fluorescent chemical tool to investigate CB1 expression and function in human blood and tonsil immune cells, which might well pave the way to unravel essential features of CB1 in different immune and ECS-related diseases.

Fully automated 5-plex fluorescent immunohistochemistry with tyramide signal amplification and same species antibodies.

The ability to simultaneously visualize the presence, abundance, location and functional state of many targets in cells and tissues has been described as a true next-generation approach in immunohistochemistry (IHC). A typical requirement for multiplex IHC (mIHC) is the use of different animal species for each primary (1°Ab) and secondary (2°Ab) antibody pair. Although 1°Abs from different species have been used with differently labeled species-specific 2°Abs, quite often the appropriate combination of antibodies is not available. More recently, sequential detection of multiple antigens using 1°Abs from the same species used a microwaving treatment between successive antigen detection cycles to elute previously bound 1°Ab/2°Ab complex and therefore to prevent the cross-reactivity of anti-species 2°Abs used in subsequent detection cycles. We present here a fully automated 1°Ab/2°Ab complex heat deactivation (HD) method on Ventana's BenchMark ULTRA slide stainer. This method is applied to detection using fluorophore-conjugated tyramide deposited on the tissue and takes advantage of the strong covalent bonding of the detection substrate to the tissue, preventing its elution in the HD process. The HD process was characterized for (1) effectiveness in preventing Ab cross-reactivity, (2) impact on the epitopes and (3) impact on the fluorophores. An automated 5-plex fluorescent IHC assay was further developed using the HD method and rabbit 1°Abs for CD3, CD8, CD20, CD68 and FoxP3 immune biomarkers in human tissue specimens. The fluorophores were carefully chosen and the narrow-band filters were designed to allow visualization of the staining under fluorescent microscope with minimal bleed through. The automated 5-plex fluorescent IHC assay achieved staining results comparable to the respective single-plex chromogenic IHC assays. This technology enables automated mIHC using unmodified 1°Abs from same species and the corresponding anti-species 2°Ab on a clinically established automated platform to ensure staining quality, reliability and reproducibility.

Tonsillar solitary fibrous tumour: A interesting rare case.

A solitary fibrous tumour (SFT) is a rare mesenchymal tumour that frequently originates in the mesothelium-covered surfaces, such as the pleura and peritoneum. It may develop in various body parts, including the head and neck. These tumours may arise in several different patterns, which results in difficulties in diagnosing them. This case is a solitary tumour developing from the palatine tonsil in a 17-year-old male patient; it is the second case in the literature. The tumour has the histopathological characteristics of a patternless pattern, a slight pleomorphism, and a composition of hypercellular and hypocellular sites. Immunohistochemically, the tumour cells showed a strong positive staining with CD34, Bcl-2, and vimentin. No recurrence developed in the patient's approximately 18-month-long follow-up period.

Advanced oxidation protein product levels as a marker of oxidative stress in paediatric patients with chronic tonsillitis.

We aimed to determine whether advanced oxidation protein product (AOPP) levels can serve as a marker of oxidative stress in paediatric patients with chronic tonsillitis. Thirty children with chronic tonsillitis and 30 healthy children (control group) were recruited from the Otorhinolaryngology (ORL) and Paediatric Surgery departments, respectively, of Dumlupinar University Hospital. In the patient group, blood samples were collected before tonsillectomy, and tonsil tissue was sampled during the operation. Blood samples were also obtained from the control subjects. AOPP levels in the serum and tonsil tissue were measured by the spectrophotometric method. Serum AOPP levels were significantly higher in the patient group (13.1 ± 3.3 ng/ml) than in the control group (11.6 ± 2.3 ng/ml; P < 0.05). In addition, the mean AOPP level (41.9 ± 13.5 ng/mg protein) in the tonsil tissue in the patient group was significantly higher than the mean serum AOPP levels in the control and patient groups (P < 0.05). AOPP levels are elevated in the tonsil tissue and serum of patients with chronic tonsillitis compared to the serum AOPP levels in healthy controls. AOPPs may represent a novel class of pro-inflammatory molecules that are involved in oxidative stress in chronic tonsillitis. AOPPs may be used as a marker of oxidative stress in paediatric patients with chronic tonsillitis.

Selenium-binding protein 1 in head and neck cancer is low-expression and associates with the prognosis of nasopharyngeal carcinoma.

BACKGROUND: Selenium-binding protein 1 (SELENBP1) expression is reduced markedly in many types of cancers and low SELENBP1 expression levels are associated with poor patient prognosis. METHODS: SELENBP1 gene expression in head and neck squamous cell carcinoma (HNSCC) was analyzed with GEO dataset and characteristics of SELENBP1 expression in paraffin embedded tissue were summarized. Expression of SELENBP1 in nasopharyngeal carcinoma (NPC), laryngeal cancer, oral cancer, tonsil cancer, hypopharyngeal cancer and normal tissues were detected using immunohistochemistry, at last, 99 NPC patients were followed up more than 5 years and were analyzed the prognostic significance of SELENBP1. RESULTS: Analysis of GEO dataset concluded that SELENBP1 gene expression in HNSCC was lower than that in normal tissue (P < 0.01), but there was no significant difference of SELENBP1 gene expression in different T-stage and N-stage (P > 0.05). Analysis of pathological section concluded that SELENBP1 in the majority of HNSCC is low expression and in cancer nests is lower expression than surrounding normal tissue, even associated with the malignant degree of tumor. Further study indicated the low SELENBP1 expression group of patients with NPC accompanied by poor overall survival and has significantly different comparing with the high expression group. CONCLUSION: SELENBP1 expression was down-regulated in HNSCC, but has no associated with T-stage and N-stage of tumor. Low expression of SELENBP1 in patients with NPC has poor over survival, so SELENBP1 could be a novel biomarker for predicting prognosis.

Homogenization of tissues via picosecond-infrared laser (PIRL) ablation: Giving a closer view on the in-vivo composition of protein species as compared to mechanical homogenization.

Posttranslational modifications and proteolytic processing regulate almost all physiological processes. Dysregulation can potentially result in pathologic protein species causing diseases. Thus, tissue species proteomes of diseased individuals provide diagnostic information. Since the composition of tissue proteomes can rapidly change during tissue homogenization by the action of enzymes released from their compartments, disease specific protein species patterns can vanish. Recently, we described a novel, ultrafast and soft method for cold vaporization of tissue via desorption by impulsive vibrational excitation (DIVE) using a picosecond-infrared-laser (PIRL). Given that DIVE extraction may provide improved access to the original composition of protein species in tissues, we compared the proteome composition of tissue protein homogenates after DIVE homogenization with conventional homogenizations. A higher number of intact protein species was observed in DIVE homogenates. Due to the ultrafast transfer of proteins from tissues via gas phase into frozen condensates of the aerosols, intact protein species were exposed to a lesser extent to enzymatic degradation reactions compared with conventional protein extraction. In addition, total yield of the number of proteins is higher in DIVE homogenates, because they are very homogenous and contain almost no insoluble particles, allowing direct analysis with subsequent analytical methods without the necessity of centrifugation. BIOLOGICAL SIGNIFICANCE: Enzymatic protein modifications during tissue homogenization are responsible for changes of the in-vivo protein species composition. Cold vaporization of tissues by PIRL-DIVE is comparable with taking a snapshot at the time of the laser irradiation of the dynamic changes that occur continuously under in-vivo conditions. At that time point all biomolecules are transferred into an aerosol, which is immediately frozen.

Characterization of B-Cells in tonsils of patients diagnosed with pediatric autoimmune neuropsychiatric disorder associated streptococcus.

OBJECTIVE: To determine if Pediatric Autoimmune Neuropsychiatric Disorder Associated with Streptococcus (PANDAS) patients demonstrate a significantly different number of B-Cells or markers of activity when compared to recurrent Group A Streptococcus or Obstructive Sleep Apnea patients. STUDY DESIGN: Prospective Cohort Study. STUDY SETTING: Academic University Hospital. METHODS: Tonsil tissue was collected from twenty-two patients in the operating room and organized into three groups. Ten clinically diagnosed PANDAS, six Group A Streptococcus and six Obstructive Sleep Apnea patients were included in this study. Each tissue sample was extracted with MSD Tris Lysis Buffer and protein lysates were analyzed for CD 19, B-Cell Activating Factor and B-Cell Activating Receptor by western blot methods. RESULTS: Based on ANOVA analysis, there was no significant difference in the expression of B-Cell Activating Factor, B-Cell Activating Receptor or CD 19 when comparing the three study groups by western blot analysis methods. CONCLUSIONS: In this prospective cohort study, it appears that PANDAS patients do not demonstrate increased number of B-Cells, expression of B-Cell Activating Factor or B-Cell Activating Receptor when compared to Group A Streptococcus or Obstructive Sleep Apnea cohorts. As a result, further evaluation of the cell-mediated immune system is warranted to provide further insight into the pathophysiology of PANDAS. In addition, we must investigate if PANDAS patients only demonstrate increased B-Cell number or activity when undergoing an acute Tic/OCD exacerbation.

Postmortem diagnosis of cytomegalovirus and accompanying other infection agents by real-time PCR in cases of sudden unexpected death in infancy (SUDI).

As an opportunistic pathogen with high mortality rates, Cytomegalovirus (CMV) may lead to fatal disseminated CMV infection of the premature and newborn; thus necessitating the demonstration of CMV-DNA with clinical history and/or histopathological findings of CMV infection and defining other bacterial and viral infection agents with real-time polymerase chain reaction (RT-PCR) in udden unexpected death in infancy (SUDI) cases as we aimed in this study. 314 (144 female, 170 male) SUDI cases were prospectively investigated from January 2013 to January 2015 in Istanbul Forensic Medicine Institution. The study includes 87 tissue samples of 39 cases for post-mortem histopathological examination of interstitial pneumonia, myocarditis, meningitis, encephalitis, hepatitis, colitis or tubulointerstitial nephritis and/or accompanying chronic sialadenitis. CMV-DNA was found positive in 35 (40.2%) salivary gland, 19 (21.8%) lung, 1 (1.1%) tonsil, and 1 (1.1%) brain tissues. CMV sialadenitis and/or CMV pneumonia associated with other viral and/or bacterial agents were detected in 23 (60%) of 39 infant cases. The demonstration of CMV-DNA would significantly clarify the cause of death and collection of epidemiological data in SUDI cases with clinical history and histopathological findings of CMV infection accompanying chronic CMV sialadenitis. Furthermore, CMV suppresses the immune system, and may predispose to other bacterial and/or viral infections in these cases. Post-mortem molecular investigations are useful in explaining cause of death in SUDI with a suspicion of infection in forensic autopsies.

Combining multiset resolution and segmentation for hyperspectral image analysis of biological tissues.

Hyperspectral images can provide useful biochemical information about tissue samples. Often, Fourier transform infrared (FTIR) images have been used to distinguish different tissue elements and changes caused by pathological causes. The spectral variation between tissue types and pathological states is very small and multivariate analysis methods are required to describe adequately these subtle changes. In this work, a strategy combining multivariate curve resolution-alternating least squares (MCR-ALS), a resolution (unmixing) method, which recovers distribution maps and pure spectra of image constituents, and K-means clustering, a segmentation method, which identifies groups of similar pixels in an image, is used to provide efficient information on tissue samples. First, multiset MCR-ALS analysis is performed on the set of images related to a particular pathology status to provide basic spectral signatures and distribution maps of the biological contributions needed to describe the tissues. Later on, multiset segmentation analysis is applied to the obtained MCR scores (concentration profiles), used as compressed initial information for segmentation purposes. The multiset idea is transferred to perform image segmentation of different tissue samples. Doing so, a difference can be made between clusters associated with relevant biological parts common to all images, linked to general trends of the type of samples analyzed, and sample-specific clusters, that reflect the natural biological sample-to-sample variability. The last step consists of performing separate multiset MCR-ALS analyses on the pixels of each of the relevant segmentation clusters for the pathology studied to obtain a finer description of the related tissue parts. The potential of the strategy combining multiset resolution on complete images, multiset segmentation and multiset local resolution analysis will be shown on a study focused on FTIR images of tissue sections recorded on inflamed and non-inflamed palatine tonsils.

Fibronectin in the palatine tonsil as a susceptibility marker in Egyptian rheumatic families: histological and immunohistochemical studies.

Rheumatic fever (RF) and rheumatic heart disease (RHD) are the multisystem autoimmune sequel of group A streptococci (GAS) infection of the upper respiratory passages, mainly tonsillopharyngitis. The major receptor on the surface of human palatine tonsil for GAS is fibronectin (FN; adhesin receptor). Early detection of RF susceptibility is considered as an important aim of this study. Therefore, the present study aimed to use FN immunoreactivity (FN-ir) as a marker for early detection of rheumatic susceptible children with palatine tonsil crypts surface epithelium. A total of 30 palatine tonsillar specimens were obtained from children aged from 3 to 15 years. Histological studies showed moderate vascular changes and more than four apparent epithelial disruptions in the crypt epithelium. FN-ir showed a significant increase in FN in the basal layers of surface epithelium, subepithelial connective tissue and interfollicular areas. Tonsils of children in rheumatic families showed significant increase in FN in subepithelial connective tissue areas with more than one apparent crypt epithelial disruption compared to normal children. We can conclude that FN plays a central role in the RF and differentially distributed in the functional compartments of the palatine tonsil in children with RHD, so FN-ir can be used as a marker for rheumatic susceptibility.

Elution of High-affinity (>10-9 KD) Antibodies from Tissue Sections: Clues to the Molecular Mechanism and Use in Sequential Immunostaining.

Inconsistent results obtained with published methods for the elution of antibodies from tissue sections prompted the assessment of both old and new methods in combination with monoclonal rabbit antibodies of known, increased affinity (above 1×10(-9) KD). We tested an acidic (pH 2) glycine buffer, a 6 M urea hot buffer and a 2-Mercaptoethanol, SDS buffer (2-ME/SDS). Some antibodies were not removed by the glycine pH 2 or 6 M urea hot buffers, indicating that antibodies survive much harsher conditions than previously believed. We found that the elution is dependent upon the antibody affinity and is reduced by species-specific crosslinking via a dimeric or Fab fragments of a secondary antibody. The high affinity bond of exogenous streptavidin with the endogenous biotin can be removed by 6 M urea but not by the other buffers. 2-ME/SDS buffer is superior to glycine pH 2 and 6 M urea hot elution buffers for all antibodies because of its irreversible effect on the structure of the antibodies. It also has a mild retrieving effect on some antigens present on routinely treated sections and no detrimental effect on the immunoreactivity of the tissue. Therefore, 2-ME/SDS buffer is the method of choice to perform multiple rounds of immunostaining on a single routine section.