"Tau Protein Targeting Via Radioiodinated Azure A For Brain Theranostics: Radiolabeling, Molecular Docking, in vitro And in vivo Biological Evaluation".

Azure-A is one of the phenothiazines (PTZs) derivatives which for decades have been used as antipsychotic drugs due to good lipophilic characteristics which enable them to pass through the blood brain barrier (BBB), besides the important property of enabeling investigation of the pathological forms of aggregated tau protein found in the neurons of the central nervous system. Radioiodination of Azure-A was carried out via an electrophilic substitution reaction using chloramine-T as oxidizing agent. The influence of various reaction parameters and conditions on radioiodination efficiency was investigated, and a high radiochemical yield of 92.07 ± 0.9 % was obtained. An in vitro cytotoxicity study of iodinated Azure-A on three cell lines (HCT-116, human colon carcinoma cell line; Hep-G2, liver carcinoma cell line and HFB-4, normal human melanocytes) was carried out, and the data revealed that ioiodinated Azure A has no to very low toxic effect. The in vivo biodistribution study of 131 I-Azure A showed a high brain uptake of 6.15 ± 0.09 % injected dose/g tissue organ at 30 minutes post-injection, and its retention in brain remained high up to 2 hours, whereas the clearance from the body appeared to proceed via the renal system. The experimental data were confirmed by the molecular docking studies to predict the effect of radioiodination on the binding affinity of the parent molecule (Azure A) to tau paired helical filaments (PHFs). Both ligands showed better binding to S2 and S3 pockets of (PHFs). Consequently, radioiodinated Azure A seems to be a good candidate as an imaging agent for taupathies such as Alzheimer's disease, chronic traumatic encephalopathy, and corticobasal degeneration. Furthermore, it could be a very potent theranostics agent for brain tumors.

Comprehensive high-resolution genomic profiling and cytogenetics of human chondrocyte cultures by GTG-banding, locus-specific FISH, SKY and SNP array.

In the development of cell-based medicinal products, it is crucial to guarantee that the application of such an advanced therapy medicinal product (ATMP) is safe for the patients. The consensus of the European regulatory authorities is: "In conclusion, on the basis of the state of art, conventional karyotyping can be considered a valuable and useful technique to analyse chromosomal stability during preclinical studies". 408 chondrocyte samples (84 monolayers and 324 spheroids) from six patients were analysed using trypsin-Giemsa staining, spectral karyotyping and fluorescence in situ hybridisation, to evaluate the genetic stability of chondrocyte samples from non-clinical studies. Single nucleotide polymorphism (SNP) array analysis was performed on chondrocyte spheroids from five of the six donors. Applying this combination of techniques, the genetic analyses performed revealed no significant genetic instability until passage 3 in monolayer cells and interphase cells from spheroid cultures at different time points. Clonal occurrence of polyploid metaphases and endoreduplications were identified associated with prolonged cultivation time. Also, gonosomal losses were observed in chondrocyte spheroids, with increasing passage and duration of the differentiation phase. Interestingly, in one of the donors, chromosomal aberrations that are also described in extraskeletal myxoid chondrosarcoma were identified. The SNP array analysis exhibited chromosomal aberrations in two donors and copy neutral losses of heterozygosity regions in four donors. This study showed the necessity of combined genetic analyses at defined cultivation time points in quality studies within the field of cell therapy.

Exploring the interaction of Azure dyes with t-RNA by hybrid spectroscopic and computational approaches and its applications toward human lung cancer cell line.

In the present study, in depth characterization of binding aspects of Azure A (AZA) and Azure B (AZB) with transfer Ribonucleic acid (t-RNA) from Escherichia coli (E.coli) is investigated using spectroscopic techniques. The absorbance and fluorescence properties of these dyes have been remarkably changed upon binding with t-RNA. Significant changes in the absorption maxima of the dyes evidence the t-RNA induced metachromasy and the binding clearly revealed the high affinity of AZA and AZB to t-RNA. Strong emission polarization of the bound dyes and strong energy transfer from the guanine base pairs of t-RNA suggested intercalative binding interaction. The stoichiometry of AZA and AZB with t-RNA complexes are determined by the Benesi-Hildebrand plot from emission data. The negative values of free energy change indicated the involvement of hydrophobic forces and noncovalent interactions in the complexation of both the dyes with t-RNA. The 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay in A-549 human lung cancer cell lines reveals that binding of t-RNA reduces the toxicity of AZA and AZB. The utility of the present work explores the potential binding applicability of these dyes to t-RNA for their development as effective therapeutic agents and its target at molecular level for the treatment of diseases like cancer.

Azure C Targets and Modulates Toxic Tau Oligomers.

Alzheimer's disease (AD) is the most common age-related neurodegenerative disorder affecting millions of people worldwide. Therefore, finding effective interventions and therapies is extremely important. AD is one of over 20 different disorders known as tauopathies, characterized by the pathological aggregation and accumulation of tau, a microtubule-associated protein. Tau aggregates are heterogeneous and can be divided into two major groups: large metastable fibrils, including neurofibrillary tangles, and oligomers. The smaller, soluble and dynamic tau oligomers have been shown to be more toxic with more proficient seeding properties for the propagation of tau pathology as compared to the fibrillar Paired Helical Filaments (PHFs). Therefore, developing small molecules that target and interact with toxic tau oligomers can be beneficial to modulate their aggregation pathways and toxicity, preventing progression of the pathology. In this study, we show that Azure C (AC) is capable of modulating tau oligomer aggregation pathways at micromolar concentrations and rescues tau oligomers-induced toxicity in cell culture. We used both biochemical and biophysical in vitro techniques to characterize preformed tau oligomers in the presence and absence of AC. Interestingly, AC prevents toxicity not by disassembling the oligomers but rather by converting them into clusters of aggregates with nontoxic conformation.

Thinprep plus Papanicolaou stain method is more sensitive than cytospin-coupled Wright Giems stain method in cerebrospinal fluid cytology for diagnosis of leptomeningeal metastasis from solid tumors.

BACKGROUND: The present study was designed to determine whether the Thinprep plus Papanicolaou stain (Thinprep) method is more sensitive than the Cytospin-coupled Wright-Giemsa (WG) stain (Cytospin) method in diagnosis of leptomeningeal metastasis (LM) from malignant solid tumors in cerebrospinal fluid (CSF). We also explored if the Thinprep method could be used in the differential diagnosis of the type of primary tumor cells based on the morphology of tumor cells in CSF samples. METHODS: The morphological features of tumor cells in fresh CSF samples were analyzed using both methods. The tumor cell detection rates were compared between the two methods. RESULTS: Using the Thinprep method, we found that each type of tumor cells in the CSF samples had specific identifiable morphological features linked to their primary cancer origins, such as adenocarcinomas originated from the lungs, breast, and stomach, and lung squamous cell carcinomas, small cell lung cancer, large-cell neuroendocrine lung cancer, hepatocellular carcinoma, and malignant melanoma. In a retrospective study with 88 LM patients, cancer cells were detected in 80 out of the 88 CSF samples. In the comparative study with 45 LM patients, the initial detection rate of the Thinprep method was significantly higher than that of the Cytospin method (73.3% vs. 57.8%, P<0.01). The cell morphology was better preserved and subcellular structures were clearer using the Thinprep method, compared to the Cytospin method. CONCLUSIONS: The Thinprep method is more sensitive and suitable for LM diagnosis in CSF in patients with malignant solid tumors than the Cytospin method. The Thinprep method may facilitate primary tumor detection and help design early treatment regimens for LM patients with tumors of unknown primary origin.

Peripheral blood smears, bone marrow aspiration, trephine and clot biopsies: methods and protocols.

Maximum diagnostic information is obtained when peripheral blood smears, bone marrow aspiration smears, trephine biopsy imprints, trephine and clot biopsy sections are simultaneously examined. Peripheral blood smears reflect end organ function and provide clues to underlying hematolymphoid pathology that may prompt additional studies including bone marrow examination. Bone marrow aspiration alone has diagnostic utility in the evaluation of a limited number of primary hematological conditions including: megaloblastic anemias, hyporegenerative anemias, certain hemolytic anemias, normochromic normocytic anemias, neutropenias, thrombocytopenias, immunoglobulin disorders, storage diseases, and leukemias (Bain, J Clin Pathol 54:657-663, 2001). Bone marrow trephine biopsy is indicated in those situations where marrow aspiration is unsuccessful; in evaluation of cytopenias, myelofibrosis, suspicion of lymphoma, metastatic tumor, granulomatous disease, evaluation of myeloproliferative neoplasms, and for the examination of trabecular bone in metabolic diseases (Bain, J Clin Pathol 54:737-742, 2001). Many of the indications for marrow aspiration overlap with those for trephine biopsy. Because it is not possible to predict which patients will have diagnostic aspiration biopsies and which will have diagnostic trephine biopsies, both procedures are routinely performed together (Brynes et al., Am J Clin Pathol 70:753-759, 1978; Cotelingam, Adv Anat Pathol 10:8-26, 2003; Lee et al., Int J Lab Hematol 30:349-364, 2008; Peterson et al., Arch Pathol Lab Med 126:1050-1056, 2002).

Cytogenetics in myelodysplastic syndromes.

Cytogenetic information in patients with myelodysplastic syndrome (MDS) is important in predicting prognosis and therapeutic direction. In MDS, the detection of numerical type abnormalities, either whole chromosome or partial chromosomal segments, is important. In general, conventional banding chromosome analysis is useful in detecting chromosome changes in MDS and is able to predict prognosis. More recently, uniparental disomy at various loci has been found in some MDS patients and target genes located within the deleted chromosome regions; these deletions are either cytogenetically detectable resulting in partial monosomy, or cryptic. Further therapeutic approaches for MDS patients may require more precise cytogenetic information in the near future.

Sex chromosome rearrangements in Polyphaga beetles.

The presence of a parachute sex chromosome bivalent (Xyp) at metaphase I of male meiosis is a well-known characteristic of Coleoptera, present in almost all families of this order and assumed to represent their ancestral sex chromosome formula. Sex chromosomes appear to be manifold more frequently involved in inter-chromosomal rearrangements than the average of the nine autosomal pairs usually forming their karyotype. This leads to various formulae such as neo-sex, multiple sex and perhaps unique sex chromosomes. These rearrangements alter the intimate association between sex chromosomes and nucleolar proteins, which are usual components of the Xyp. Different situations, selected in a series of 125 mitotic and meiotic cytogenetic studies of Polyphaga beetle species, are reported and discussed, with the aim to improve our knowledge on the mechanisms of sex chromosome rearrangements, the relationships with nucleoli and the consequences on dosage compensation and chromosome segregation.

Immunoperoxidase in the interpretation of discordant histologic and urease findings for Helicobacter pylori.

Azure A and methylene blue ("Diff-Quik," DQ) and tissue urease (U) tests are popular methods to diagnose Helicobacter pylori. These tests usually correlate well but sometimes produce discordant results. This study evaluates the DQ and U tests by comparing them with the immunoperoxidase reference method to resolve discordant results. DQ and U tests were performed on gastric biopsies. Results were tabulated as DQ(+)/U(+), DQ(+)/U(-), DQ(-)/U(+), and DQ(-)/U(-). Cases that were DQ(+)/U(+) were recorded as positive and not tested with immunoperoxidase. Cases that had discordant DQ/U results were tested by immunoperoxidase to resolve the discordance. Cases which were negative for both DQ/U were evaluated by immunoperoxidase to confirm the validity of DQ(1-)/U(-). The groups were compared with concordant results (DQ(1-)/U(-) group) and immunoperoxidase versus discordant DQ/U results and immunoperoxidase. There were 56 gastric biopsy specimens. Among all cases, 6 were DQ(+)/U(+). Of the remaining 50 cases, 38 were concordant DQ(-)/U(-), whereas 12 showed discordant DQ/U results. All 38 concordant DQ(-)/U(-) specimens were confirmed negative, 11 discordant DQ/U cases were confirmed negative, and 1 DQ(+)/U(-) specimen was confirmed positive by immunoperoxidase. Comparison of concordant versus discordant results was not statistically significant (P=0.10). Among all discordant DQ and U, 11/12 (92%) were confirmed negative by immunoperoxidase. Thus, both concordant negative results and discordant results can be considered negative. Such interpretation of discordant results might prevent unnecessary additional procedures or treatment.

Classification of haematopoietic cells and haemocytes in Chinese prawn Fenneropenaeus chinensis.

It is commonly believed that crustacean haemocytes originate from a specialised haematopoietic tissue (HPT), whereas the differentiation relationship between HPT cells and circulating haemocytes is still not clearly understood. The HPT cells and haemocytes of Fenneropenaeus chinensis were characterised using morphological and histochemical methods. Three types of HPT cells were identified under the transmission electron microscope (TEM). Type 1 cells had high N/C ratios, developed dispersed chromatins and no cytoplasmic granules. Type 2 cells had smaller size, developed condensed chromatins and cytoplasmic granules, which were homogeneous or striated in type 2a cells, and homogeneous in type 2b cells. We deduce that type 1 cells may give rise to type 2 cells in terms of the presence of possible intermediates between type 1 and type 2 cells. The circulating haemocytes were divided into three populations, i.e. hyaline haemocytes (HH), small granular haemocytes (SHG) and large granular haemocytes (LGH), based on Wright-Giemsa staining and TEM observation. Comparing the HPT cells with the circulating haemocytes, type 2a cells of HPT may represent the HH due to similar granule types, cell size and N/C ratios, and type 2b cells may be the young and immature LGH. By Wright-Giemsa and acid alpha-naphthyl acetate esterase staining, the intermediates between the HH and SGH were observed, which indicates that the SGH may be derived from the HH in the circulatory system. Therefore, it is suggested that the F. chinensis haemocytes could be divided into two haemocyte lineages, i.e. the HH-SGH and LGH lineage.

Mott cell (Russell bodies) Barrett's oesophagitis.

The first case of Barrett's oesophagus with chronic inflammation having predominantly (> 50%) Mott cells, i.e. plasma cells with stored immunoglobulins, known as Russell bodies, is reported. Biopsies from two oesophagoscopies revealed similar changes, suggesting that the predominance of Mott cells is not a fortuitous event but a more long-lasting microscopic process. Periodic acid-Schiff (PAS) stain ruled out Candida albicans and immunostains, plasma cell neoplasia. Mott cells were not present in biopsies from the gastric mucosa or the urinary bladder, suggesting that this phenomenon was not widespread but localized to the Barrett's mucosa. The retention of immunoglobulins (Russell bodies) suggests that the mechanism of protein transport in those plasma cells is incompetent, and that the proteins are neither degraded nor secreted, but remain stored in dilated cisternae. Increased awareness of the existence of this subgroup of Barrett's oesophagitis may result in similar cases being reported in the future.

Detection of Leishmania infantum kinetoplast DNA in laryngeal tissue from an immunocompetent patient.

Mucosal leishmaniasis of the upper respiratory tract is usually associated with the visceral form or is found in immunosuppressed individuals. This report presents a case of isolated mucosal leishmaniasis in an immunocompetent patient, whose diagnosis mainly rested on histology and positive polymerase chain reaction result for Leishmania donovani in the laryngeal tissue. A 59-year-old man, who never lived outside Italy, showed a subglottic mucosal polypoid-like lesion. The typical morphological picture and positive polymerase chain reaction result for L donovani by DNA extracted from laryngeal biopsy specimens allowed the diagnosis of mucosal leishmaniasis. Specific amphotericin B therapy was started, resulting in clinical and endoscopic improvement. Increased knowledge about the histological and molecular tissue analysis of Leishmania enhances the diagnostic testing for mucosal leishmaniasis, as primary mucosal leishmaniasis may occur in both immunosuppresed and immunocompetent patients who travel to or reside in areas endemic for Leishmania.

Identification of a high frequency of chromosomal rearrangements in the centromeric regions of prostate cancer cell lines by sequential giemsa banding and spectral karyotyping.

BACKGROUND: Currently, prostate cancer (CaP) cytogenetics is not well defined, largely because of technical difficulties in obtaining primary tumor metaphases. METHODS AND RESULTS: We examined three CaP cell lines (LNCaP, DU145, PC-3) using sequential Giemsa banding and spectral karyotyping (SKY) to search for a common structural aberration or translocation breakpoint. No consistent rearrangement common to all three cell lines was detected. A clustering of centromeric translocation breakpoints was detected in chromosomes 4, 5, 6, 8, 11, 12, 14, and 15 in DU145 and PC-3. Both these lines were found to have karyotypes with a greater level of complexity than LNCaP. CONCLUSION: The large number of structural aberrations present in DU145 and PC-3 implicate an underlying chromosomal instability and subsequent accumulation of cytogenetic alterations that confer a selective growth advantage. The high frequency of centromeric rearrangements in these lines indicates a potential role for mitotic irregularities associated with the centromere in CaP tumorigenesis.

Novel technique for the direct flow cytofluorometric analysis of human basophils in unseparated blood and bone marrow, and the characterization of phenotype and peroxidase of human basophils.

BACKGROUND: No technique has been reported to analyze directly the antigen expression on basophil leukocytes when using a flow cytometer; therefore, the exact phenotype of human basophils and the character of the peroxidase in basophils are not well understood. METHODS: Human blood basophils were purified by using an antibody against high-affinity Fc epsilon receptor (hFcepsilonR) and a MACS magnetic cell sorting system and then cytochemically stained. The phenotype and peroxidase of the human basophils were flow cytofluorometrically analyzed directly in unseparated blood and bone marrow samples as hFcepsilonR+/MBP+ (major basic protein)/Hist+ (histamine) light-density cells distributed in the high sidescatter area of lymphocytes on light scattergrams. RESULTS: The peroxidase granules of human basophils were stained by an anti-eosinophil peroxidase (EPO) antibody. The human blood basophils had common granulocyte markers plus CD25, i.e., they were CD11a/ CD11b/CD11c/CD25/CD38/CD13/CD33/hFcepsi lonR/MBP/Hist/ EPO positive, CD71 dim positive, CD14/CD15 partially positive, and CD2/CD3/CD7/CD122/CD16/CD56/CD57/ CD10/CD19/CD20/CD22/HLA-DR/MPO (myeloperoxidase)/CD23 negative. Further examination was done to analyze the expression of colony-stimulating factor receptors on three lineages of granulocytes, i.e., basophils, eosinophils, and neutrophils. The neutrophils were CD114 (G-CSFR)/CD116 (GM-CSFR)/CD124 [interleukin (IL)-4R]/CD126 (IL-6R) positive and CD123 (IL-3R)/CD125 (IL-5R) negative. In contrast, the eosinophils and basophils were CD116/CD123/CD125/CD126 positive and CD114/CD124 negative. CONCLUSIONS: This novel technique for directly characterizing human basophil leukocytes with flow cytometry may be a convenient way to screen the expression of surface antigens and the cytoplasmic expression of CD antigens and other proteins in human blood basophils and to analyze alterations of the character of basophils by cytokines and other biological substances in vivo and in vitro.

Abnormalities of chromosomes 8, 11, 14, and X in T-prolymphocytic leukemia studied by fluorescence in situ hybridization.

Twenty-one patients with T-prolymphocytic leukemia (T-PLL) were studied by FISH to characterize abnormalities of chromosomes 8, 11, 14, and X. A higher percentage of abnormalities of these chromosomes was detected by FISH than by cytogenetics. Seventy-one percent had inv(14) (q11q32)/t(14;14)(q11;q32). Four patients had abnormalities involving Xq28 (MTCP-1 locus) resulting from t(X;14)(q28;q11) or t(X;7)(q28;q35). These abnormalities have also been described in persistent expanding pre-malignant T-cell clones in patients with ataxia telangiectasia (AT). We have previously reported that in T-PLL and AT developing T-cell leukemia, the above abnormalities occur with additional abnormalities, mainly trisomy for 8q resulting predominantly from an i(8)(q10) and an increased expression of MYC. In this series, 81% of cases had chromosome 8 abnormalities including i(8)(q10)[43%]/t(8;8)(p12;q11)[14%], + 8[14%], and 8p + [14%]. The use of probes for MYC (8q24) and chromosome 8 centromere on metaphase chromosomes revealed that cases with i(8)(q10) were dicentric and t(8;8) monocentric. These abnormalities are not only associated with increase in dosage of 8q and the MYC gene, but also involved 8p. 8p is known to have several suppressor genes associated with solid tumors. Our findings suggest that the possible loss of a tumor suppressor gene plus the increased dosage of the q arm and/or the high expression of TCL-1/MTCP-1, which results from inv(14)/t(14;14), allows the malignant phenotype to emerge.

Chromosomal instability in in vivo radiation exposed subjects.

PURPOSE: To investigate whether delayed chromosomal instability arises in human peripheral T lymphocytes exposed in vivo to gamma-irradiation. MATERIALS AND METHODS: Long-term cultures were established from lymphocytes obtained from subjects involved in the radiological accident in Estonia in 1994. Two individuals exposed to a high dose, one individual with low exposure and one apparently unexposed person were studied. Two Estonian controls not involved with the accident were also analysed. Cells were grown for 6-42 days and chromosomal aberrations were assessed from G-banded metaphases. In addition, FISH chromosome painting analysis was performed on short-term cultures established from whole blood. RESULTS: No obvious sign of chromosomal instability was observed in the in vivo follow-up of the frequencies of chromosomal aberrations in lymphocytes of radiation accident victims performed by the FISH technique (48 h cultures established at different time intervals after the exposure). However, when the lymphocytes were cultured long term in vitro, chromosomal instability was observed. There was no dose-response, and the appearance of chromosomally unstable cells in long-term cultures was also observed in a subject exposed to a dose of less than 0.1 Gy. Moreover, in contrast with previous findings, chromosomal instability was also observed in cells from non-exposed control individuals. The chromosomal changes observed in the controls were less complex than the aberrations in the cultures derived from individuals exposed to high doses. CONCLUSIONS: Chromosomal instability was observed in long-term cultures of donors with in vivo exposure to gamma-radiation. No dose-response was apparent. However, in contrast with previous findings, signs of chromosomal instability were observed also in long-term cultures from non-exposed controls. Further studies are needed to assess possible inter-individual differences in the induction of chromosomal instability.

Laboratory correlates and prognostic significance of granular acute lymphoblastic leukemia in children. A Pediatric Oncology Group study.

As part of a comprehensive prospective clinicopathologic study by the Pediatric Oncology Group (POG), 2,092 children with acute lymphoblastic leukemia (ALL) were evaluated by uniform morphologic, cytochemical, and immunologic methods to assess the frequency and implications of granular lymphoblasts. All cases were Sudan black or myeloperoxidase negative and met French-American-British (FAB) morphologic criteria for ALL. Granular ALL, characterized by the presence of more than 5% marrow blasts with at least three clearly defined azurophilic cytoplasmic granules, was identified in 56 of the 1,252 fully studied cases (4.5%). The frequency of granular features did not differ among early pre-B (4.3%), pre-B (3.6%), and T (5.8%) ALL; no cases were identified among the 12 patients with B ALL. Within the early pre-B/pre-B group, granular ALL was equally distributed between good- and poor-risk clinical groups but was more frequent among FAB L2 than FAB L1 cases (12% vs. 2%; P less than or equal to 0.001). Patients were treated with standard POG protocols for early pre-B/pre-B and T ALL. Complete remission (CR) rates were significantly lower for those with granular lymphoblasts, regardless of risk group, immunophenotype, or FAB type. Analysis of event-free survival (EFS) showed a significantly poorer outcome for granular early pre-B/pre-B cases with FAB L2 morphologic characteristics (P less than 0.001) and for those classified as poor risk (P = 0.015). These findings suggest a relationship between granules and L2 morphologic characteristics in childhood ALL and indicate that the presence of granular lymphoblasts conveys a worse prognosis for certain subgroups of children with ALL.