The advancement in molecular techniques has been attributed to the quality and significance of cancer research. Pancreatic cancer (PC) is one of the rare cancers with aggressive behavior and a high mortality rate. The asymptomatic nature of the disease until its advanced stage has resulted in late diagnosis as well as poor prognosis. The heterogeneous character of PC has complicated cancer development and progression studies. The analysis of bulk tissues of the disease was insufficient to understand the disease, hence, the introduction of the single-cell separating technique aided researchers to decipher more about the specific cell population of tumors. This review gives an overview of the Laser Capture Microdissection (LCM) technique, one of the single-cell separation methods used in PC research.

• MeSH
• Carcinoma, Pancreatic Ductal * pathology   MeSH
• Laser Capture Microdissection MeSH
• Humans MeSH
• Pancreatic Neoplasms * pathology   MeSH
• Pancreas pathology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

Molecular assessment of renal allografts has already been suggested in antibody-mediated rejection (ABMR), but little is known about the gene transcript patterns in particular renal compartments. We used laser capture microdissection coupled with quantitative RT-PCR to distinguish the transcript patterns in the glomeruli and tubulointerstitium of kidney allografts in sensitized retransplant recipients at high risk of ABMR. The expressions of 13 genes were quantified in biopsies with acute active ABMR, chronic active ABMR, acute tubular necrosis (ATN), and normal findings. The transcripts were either compartment specific (TGFB1 in the glomeruli and HAVCR1 and IGHG1 in the tubulointerstitium), ABMR specific (GNLY), or follow-up specific (CXCL10 and CX3CR1). The transcriptional profiles of early acute ABMR shared similarities with ATN. The transcripts of CXCL10 and TGFB1 increased in the glomeruli in both acute ABMR and chronic active ABMR. Chronic active ABMR was associated with the upregulation of most genes (SH2D1B, CX3CR1, IGHG1, MS4A1, C5, CD46, and TGFB1) in the tubulointerstitium. In this study, we show distinct gene expression patterns in specific renal compartments reflecting cellular infiltration observed by conventional histology. In comparison with active ABMR, chronic active ABMR is associated with increased transcripts of tubulointerstitial origin.

• MeSH
• Biopsy MeSH
• Chronic Disease MeSH
• Adult MeSH
• Kidney Glomerulus immunology   metabolism   pathology   MeSH
• HLA Antigens immunology   MeSH
• Isoantibodies immunology   MeSH
• Real-Time Polymerase Chain Reaction MeSH
• Laser Capture Microdissection MeSH
• Kidney immunology   metabolism   pathology   MeSH
• Middle Aged MeSH
• Humans MeSH
• Reverse Transcriptase Polymerase Chain Reaction MeSH
• Graft Rejection genetics   immunology   metabolism   pathology   MeSH
• Aged MeSH
• Gene Expression Profiling MeSH
• Case-Control Studies MeSH
• Transcriptome * MeSH
• Kidney Transplantation adverse effects   MeSH
• Treatment Outcome MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH

• Keywords
• Hematologie,
• MeSH
• Cytological Techniques MeSH
• Hematologic Tests MeSH
• Hematopoiesis MeSH
• Histological Techniques MeSH
• Bone Marrow MeSH
• Blood MeSH

• NML Fields
• hematologie a transfuzní lékařství

The platform for precise proteomic profiling of targeted cell populations from heterogeneous tissue sections is developed. We demonstrate a seamless and systematic integration of LCM with an automated cap-IA for the handling of a very small-sized dissected tissues section from the kidney, liver and pancreatic Langerhans islet of rats. Our analysis reveals that the lowest LCM section area ≥ 0.125 mm2 with 10 µm thickness can be optimized for the detection of proteins through LCM-cap-IA integration. We detect signals ranging from a highly-abundant protein, β-actin, to a low-abundance protein, LC-3AB, using 0.125 mm2 LCM section from rat kidney, but, so far, a relatively large section is required for good quality of results. This integration is applicable for a highly-sensitive and accurate assessment of microdissected tissue sections to decipher hidden proteomic information of pure targeted cells. To validate this integration, PCK2 protein expression is studied within Langerhans islets of normal and diabetic rats. Our results show significant overexpression of PCK2 in Langerhans islets of rats with long-term diabetes.

• Publication type
• Journal Article MeSH

• MeSH
• Cytological Techniques MeSH
• Hematologic Tests MeSH
• Hematopoiesis MeSH
• Histological Techniques MeSH
• Bone Marrow MeSH
• Blood MeSH

• Conspectus
• Patologie. Klinická medicína
• Učební osnovy. Vyučovací předměty. Učebnice
• NML Fields
• hematologie a transfuzní lékařství
• NML Publication type
• učebnice vysokých škol

Members of neuropeptide B/W signaling system have been predominantly detected and mapped within the CNS. In the rat, this system includes neuropeptide B (NPB), neuropeptide W (NPW) and their specific receptor NPBWR1. This signaling system has a wide spectrum of functions including a role in modulation of inflammatory pain and neuroendocrine functions. Expression of NPB, NPW and NPBWR1 in separate heart compartments, dorsal root ganglia (DRG) and stellate ganglia was proven by RT-qPCR, Western blot (WB) and immunofluorescence. Presence of mRNA for all tested genes was detected within all heart compartments and ganglia. The presence of proteins preproNPB, preproNPW and NPBWR1 was confirmed in all the chambers of heart by WB. Expression of preproNPW and preproNPB was proven in cardiac ganglionic cells obtained by laser capture microdissection. In immunofluorescence analysis, NPB immunoreactivity was detected in nerve fibers, some nerve cell bodies and smooth muscle within heart and both ganglia. NPW immunoreactivity was present in the nerve cell bodies and nerve fibers of heart ganglia. Weak nonhomogenous staining of cardiomyocytes was present within heart ventricles. NPBWR1 immunoreactivity was detected on cardiomyocytes and some nerve fibers. We confirmed the presence of NPB/W signaling system in heart, DRG and stellate ganglia by proteomic and genomic analyses.

• MeSH
• Gene Expression MeSH
• Fluorescent Antibody Technique MeSH
• Stellate Ganglion metabolism   MeSH
• Myocardium metabolism   MeSH
• Neuropeptides genetics   immunology   metabolism   MeSH
• Rats, Zucker MeSH
• Receptors, Neuropeptide genetics   immunology   metabolism   MeSH
• Receptors, G-Protein-Coupled genetics   immunology   metabolism   MeSH
• Reproducibility of Results MeSH
• Signal Transduction MeSH
• Ganglia, Spinal metabolism   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Eudiplozoon nipponicum (Goto, 1891) is a hematophagous monogenean ectoparasite which inhabits the gills of the common carp (Cyprinus carpio). Heavy infestation can lead to anemia and in conjunction with secondary bacterial infections cause poor health and eventual death of the host. This study is based on an innovative approach to protein localization which has never been used in parasitology before. Using laser capture microdissection, we dissected particular areas of the parasite body without contaminating the samples by surrounding tissue and in combination with analysis by mass spectrometry obtained tissue-specific proteomes of tegument, intestine, and parenchyma of our model organism, E. nipponicum. We successfully verified the presence of certain functional proteins (e.g. cathepsin L) in tissues where their presence was expected (intestine) and confirmed that there were no traces of these proteins in other tissues (tegument and parenchyma). Additionally, we identified a total of 2,059 proteins, including 72 peptidases and 33 peptidase inhibitors. As expected, the greatest variety was found in the intestine and the lowest variety in the parenchyma. Our results are significant on two levels. Firstly, we demonstrated that one can localize all proteins in one analysis and without using laboratory animals (antibodies for immunolocalization of single proteins). Secondly, this study offers the first complex proteomic data on not only the E. nipponicum but within the whole class of Monogenea, which was from this point of view until recently neglected.

• MeSH
• Carps parasitology   MeSH
• Cathepsins analysis   metabolism   MeSH
• Laser Capture Microdissection MeSH
• Parenchymal Tissue metabolism   MeSH
• Platyhelminths metabolism   MeSH
• Peptide Hydrolases analysis   metabolism   MeSH
• Proteome analysis   MeSH
• Proteomics methods   MeSH
• Intestinal Mucosa metabolism   MeSH
• Tandem Mass Spectrometry MeSH
• Chromatography, High Pressure Liquid MeSH
• Gills parasitology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The dentin-enamel junction (DEJ) is the border where two different mineralized structures - enamel and dentin - meet. The protein-rich DEJ, together with the inner enamel region of mature teeth, is known to exhibit higher fracture toughness and crack growth resistance than bulk phase enamel. However, an explanation for this behavior has been hampered by the lack of compositional information for the DEJ and the adjacent enamel organic matrix (EOM). We studied proteomes of the DEJ and EOM of healthy human molars and compared them with dentin and enamel proteomes from the same teeth. These tissues were cut out of tooth sections by laser capture microdissection, proteins were extracted and cleaved by trypsin, then processed by liquid chromatography coupled to tandem mass spectrometry to analyze the proteome profiles of these tissues. This study identified 46 proteins in DEJ and EOM. The proteins identified have a variety of functions, including calcium ion-binding, formation of extracellular matrix, formation of cytoskeleton, cytoskeletal protein binding, cell adhesion, and transport. Collagens were identified as the most dominant proteins. Tissue-specific proteins, such as ameloblastin and amelogenin, were also detected. Our findings reveal new insight into proteomics of DEJ and EOM, highly mineralized tissues that are obviously difficult to analyze.

• MeSH
• Chromatography, Liquid MeSH
• Dentin * MeSH
• Humans MeSH
• Microdissection MeSH
• Molar * MeSH
• Proteins metabolism   MeSH
• Proteome analysis   MeSH
• Proteomics methods   MeSH
• Tandem Mass Spectrometry MeSH
• Dental Enamel * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

AL amyloidosis (light chain; previously also called primary amyloidosis) is a systemic disease characterized by an amyloid deposition process affecting many organs, and which still has unsatisfactory survival of patients. The monoclonal light chains kappa (κ) or lambda (λ) or their fragments form the fibrils that deposit and accumulate in different tissues. Renal involvement is very frequent in AL amyloidosis and can lead to the development of nephrotic syndrome followed by renal failure in some cases. AL amyloidosis ultimately leads to destruction of tissues and progressive disease. With recent advances in the treatment, the importance of an early diagnosis of amyloidosis and correct assessment of its type is high. Histologic confirmation is based on Congo red detection of amyloid deposits in tissues but AL amyloidosis must also be distinguished from other systemic forms of amyloidoses with renal involvement, such as AA amyloidosis, amyloidosis with heavy chain deposition, fibrinogen Aα or ALECT2 (leukocyte chemotactic factor 2) deposition. Immunofluorescence (IF) plays a key role here. IF on formalin-fixed paraffin-embedded tissue after protease digestion, immunohistochemistry or laser microdissection with mass spectrometry should complete the diagnosis in unclear cases. Standard treatment with melphalan and prednisolone or with cyclophosphamide and dexamethasone has been replaced with newer drugs used for the treatment of multiple myeloma-bortezomib, carfilzomib and ixazomib or thalidomide, lenalidomide and pomalidomide. High-dose melphalan supported by autologous stem cell transplantation remains the therapeutic option for patients with low-risk status. These new treatment options prolong survival from months to years and improve the prognosis in a majority of patients.

• MeSH
• Combined Modality Therapy MeSH
• Humans MeSH
• Immunoglobulin Light-chain Amyloidosis diagnosis   therapy   MeSH
• Prognosis MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

We have determined patient's amyloid subtype through immunohistochemical and proteomic analyses of formalin-fixed, paraffin-embedded (FFPE) tissue samples from two affected organs per patient. Amyloid typing, via immunohistochemistry (IHC) and laser microdissection followed by the combination of liquid chromatography with mass spectrometry (LMD-LC-MS), was performed using tissue samples of the human heart, liver, kidney, tongue, and small intestine from 11 patients, and the results were compared with clinical data. LMD-LC-MS correctly typed AL amyloidosis in all 22 FFPE tissue samples despite tissue origin. In contrast, IHC was successful only in the analysis of eight FFPE tissue samples with differences between the examined organs. In the majority of LMD-LC-MS typed samples, the level of IHC staining intensity for transthyretin and serum amyloid A was the same as that for Ig κ and Ig λ antibodies, suggesting low Ig κ or Ig λ antibodies reactivity and the additional antibody clones were essential for correct typing. Both methods used in the study were found to be suitable for amyloid typing, although LMD-LC-MS yielded more promising results than IHC.

• MeSH
• Amyloid genetics   isolation & purification   metabolism   MeSH
• Amyloidosis genetics   metabolism   pathology   MeSH
• Chromatography, Liquid MeSH
• Formaldehyde MeSH
• Mass Spectrometry MeSH
• Liver metabolism   pathology   MeSH
• Tongue metabolism   pathology   MeSH
• Kidney metabolism   pathology   MeSH
• Middle Aged MeSH
• Humans MeSH
• Myocardium metabolism   pathology   MeSH
• Proteomics * MeSH
• Antibodies immunology   MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Intestine, Small metabolism   pathology   MeSH
• Tissue Distribution genetics   MeSH
• Paraffin Embedding MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH