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OBJECTIVE: The coronavirus disease 2019 pandemic has affected healthcare systems around the globe and massively impacted patients with various non-infectious, life-threatening conditions. Stroke is a major neurological disease contributing to death and disability worldwide, and is still an ongoing issue during the pandemic. Here we investigate the impact of the coronavirus disease 2019 outbreak on stroke manifestations, treatment courses, the outcome of stroke patients, and the hospitalization rate in a referral center for stroke management in Tehran, Iran. METHODS: We extracted data regarding 31 stroke patients (10 patients with laboratory-confirmed coronavirus disease 2019) and compared the demographic and pathological characteristics of the patients with or without coronavirus disease 2019 infection. The association of demographic/pathological characteristics of stroke patients during the coronavirus disease 2019 pandemic and a corresponding period during the previous year (49 patients) and an earlier period during the same year as the pandemic (50 patients) was also evaluated. RESULTS: The absolute number of admissions decreased about 40% during the coronavirus disease 2019 pandemic. Except for the stroke severity (P = 0.002), there were no significant changes in the demographic and pathological characteristics of the stroke patients during the three studied periods. A significantly higher mean of age (75.60 ± 9.54 versus 60.86 ± 18.45; P = 0.007), a significant difference in the type of stroke (P = 0.046), and significantly higher stroke severity (P = 0.024) were observed in stroke patients with coronavirus disease 2019 compared with those of stroke patients without coronavirus disease 2019. Treatment approaches, duration of hospitalization, and mortality rates did not differ significantly. CONCLUSIONS: This report shows that the pandemic caused the number of acute stroke admissions to plummet compared to other periods. Although the pandemic did not affect the treatment plans and care of the patients, stroke cases with coronavirus disease 2019 had higher age, more large vessel ischemic stroke, and more severe stroke. Further studies are urgently needed to realize the probable interaction of the coronavirus disease 2019 pandemic and the neurologic disease.

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BACKGROUND: Low intensity ultrasound (US) has some well-known bio-effects which are of great importance to be considered. Objective: We conducted the present study to investigate the effects of low intensity continuous ultrasound on blood cells count in rat. METHODS: Rats were anesthetized and blood samples were collected before US exposure. Then, they were exposed to US with nominal intensity of 0.2 W/cm2 at frequency of 3 MHz for a period of 10 minutes and this protocol was repeated for 7 days. Twenty four hours after the last US exposure, secondary blood samples were collected and the changes in blood parameters were evaluated. RESULTS: Analysis revealed that platelets, hematocrit (HCT) and hemoglobin (HGB) were significantly different between experimental and sham groups but no difference between sham and control groups was observed. The results show that HCT and HGB of exposed rats were significantly reduced. Conclusion: This study shows that low intensity US may lead to side effects for hematological parameters such as reduction in the levels of HGB and HCT.

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Malignant breast tissue contains a rare population of multi-potent cells with the capacity to self-renew; these cells are known as cancer stem-like cells (CSCs) or tumor-initiating cells. Primitive mammary CSCs/progenitor cells can be propagated in culture as floating spherical colonies termed 'mammospheres'. We show here that the expression of the autophagy protein Beclin 1 is higher in mammospheres established from human breast cancers or breast cancer cell lines (MCF-7 and BT474) than in the parental adherent cells. As a result, autophagic flux is more robust in mammospheres. We observed that basal and starvation-induced autophagy flux is also higher in aldehyde dehydrogenase 1-positive (ALDH1(+)) population derived from mammospheres than in the bulk population. Beclin 1 is critical for CSC maintenance and tumor development in nude mice, whereas its expression limits the development of tumors not enriched with breast CSCs/progenitor cells. We found that decreased survival in autophagy-deficient cells (MCF-7 Atg7 knockdown cells) during detachment does not contribute to an ultimate deficiency in mammosphere formation. This study demonstrates that a prosurvival autophagic pathway is critical for CSC maintenance, and that Beclin 1 plays a dual role in tumor development.

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BACKGROUND: Prion protein (PrPc) has been previously reported to be associated with resistance to proapoptotic stimuli. We evaluated whether the expression of PrPc was associated with the resistance to adjuvant chemotherapy in patients with estrogen receptor (ER) -negative breast cancer. PATIENTS AND METHODS: The expression of PrPc by primary tumors was assessed by immunohistochemistry in a series of 756 patients included in two randomized trials that compared anthracycline-based chemotherapy to no chemotherapy. The PrPc expression was correlated with ER expression and the benefit of adjuvant chemotherapy was assessed according to PrPc expression in patients with ER-negative tumors. RESULTS: Immunostaining analysis showed that PrPc was mainly expressed by myoepithelial cells in normal breast tissue. Tissue microarray analysis from 756 breast tumors showed that PrPc was associated with ER-negative breast cancer subsets (P < 0.001). Adjuvant chemotherapy was not associated with a significant risk reduction for death in patients with ER-negative/PrPc-positive disease [adjusted hazard ratio (HR) for death = 0.98, 95% confidence interval (CI) 0.45-2.1, P = 0.95], while it decreased the risk for death (HR = 0.39, 95% CI 0.2-0.74, P = 0.004) in patients with ER-negative/PrPc-negative tumors. CONCLUSION: These data indicate that ER-negative/PrPc-negative phenotype is associated with a high sensitivity to adjuvant chemotherapy.

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In order to define genetic determinants of primary and metastatic melanoma cell susceptibility to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), we have applied oligonucleotide microarrays to TRAIL-sensitive primary T1 cells and TRAIL-resistant metastatic G1 cells treated or not with TRAIL. T1 and G1 cells are isogenic melanoma cell subclones. We examined 22 000 spots, 4.2% of which displayed differential expression in G1 and T1 cells. Cell susceptibility to TRAIL-mediated apoptosis was found to be correlated with gene expression signatures in this model. Some of the differentially expressed genes were identified as involved in ATP-binding and signaling pathways, based on previously published data. Further analysis provided evidences that c-kit was overexpressed in G1 cells while it was absent in T1 cells. The c-kit inhibitor, imatinib, did not restore TRAIL sensitivity, excluding a role for c-kit in TRAIL resistance in G1 cells. Surprisingly, imatinib inhibited cell proliferation and TRAIL-mediated apoptosis in melanoma cells. We investigated the possible involvement of several molecules, including c-ABL, platelet-derived growth factor receptor (PDGFR), cellular FADD-like interleukin-1 alpha-converting enzyme-like inhibitory protein (c-FLIP)(L/S), Fas-associated DD kinase, p53, p21(WAF1), proteins of B-cell leukemia/lymphoma 2 (Bcl-2) family and cytochrome c. Imatinib did not modulate the expression or activation of its own targets, such as c-ABL, PDGFRalpha and PDGFRbeta, but it did affect the expression of c-FLIP(L), BCL2-associated X protein (Bax) and Bcl-2. Moreover, c-FLIP(L) knockdown sensitized T1 cells to TRAIL-mediated apoptosis, with a sensitivity similar to that of cells previously treated with imatinib. More notably, we found that the resistance to TRAIL in G1 cells was correlated with constitutive c-FLIP(L) recruitment to the DISC and the inhibition of caspase 8, 3 and 9 processing. Moreover, c-FLIP(L) knockdown partly restored TRAIL sensitivity in G1 cells, indicating that the expression level of c-FLIP(L) and its interaction with TRAIL receptor2 play a crucial role in determining TRAIL resistance in metastatic melanoma cells. Our results also show that imatinib enhances TRAIL-induced cell death independently of BH3-interacting domain death agonist translocation, in a process involving the Bax:Bcl-X(L) ratio, Bax:Bcl-X(L)/Bcl-2 translocation, cytochrome c release and caspase activation. Our data indicate that imatinib sensitizes T1 cells by directly downregulating c-FLIP(L), with the use of an alternative pathway for antitumor activity, because PDGFRalpha is not activated in T1 cells and these cells do not express c-kit, c-ABL or PDGFRbeta. Caspase cascade activation and mitochondria also play a key role in the imatinib-mediated sensitization of melanoma cells to the proapoptotic action of TRAIL.

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Cytotoxic free radicals and release of several neurotransmitters such as bradykinin contribute to the pathogenesis of hypoxic-ischemic brain damage. We have studied the efficacy of noscapine, an opium alkaloid and a bradykinin antagonist, in reducing post-hypoxic-ischemic damage in developing brain of 7-d-old rat pups. Hypoxic-ischemic injury to the right cerebral hemisphere was produced by legation of the right common carotid artery followed by 3 h of hypoxia with 8% oxygen. Thirty to 45 min before hypoxia the rat pups received noscapine (dose = 0.5-2 mg/kg) or saline. Pups were scarified at 24 h post recovery for the assessment of cerebral damage by histological methods. Our results showed that noscapine was an effective agent in reducing the extent of brain injury after hypoxic-ischemic insult to neonatal rats. Therefore, it is concluded that noscapine may be a useful drug in the managements of patients after stroke.

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The inter-alpha-trypsin inhibitor (ITI) family is a group of plasma proteins built up from heavy (HC1, HC2, HC3) and light (bikunin) chains synthesized in the liver. In this study we determined the distribution of ITI constitutive chains in normal and cancerous lung tissues using polyclonal antibodies. In normal lung tissue, H2, H3, and bikunin chains were found in polymorphonuclear cells, whereas H1 and bikunin proteins were found in mast cells. Bikunin was further observed in bronchoepithelial mucous cells. In lung carcinoma, similar findings were obtained on infiltrating polymorphonuclear and mast cells surrounding the tumor islets. Highly differentiated cancerous cells displayed strong intracytoplasmic staining with H1 and bikunin antiserum in both adenocarcinoma and squamous cell carcinoma. Moreover, weak but frequent H2 expression was observed in adenocarcinoma cells, whereas no H3-related protein could be detected in cancer cells. Local lung ITI expression was confirmed by RT-PCR. Although the respective role of inflammatory and tumor cells in ITI chain synthesis cannot be presently clarified, these results show that heavy chains as well as bikunin are involved in malignant transformation of lung tissue.(J Histochem Cytochem 47:1625-1632, 1999)

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The inter-alpha-trypsin inhibitor (ITI) family is a group of structurally related plasma serine protease inhibitors. The ITI family members consist of combinations of mature heavy chains named HC1, HC2, HC3 linked to bikunin (a Kunitz-type protease inhibitor) by a covalent interchain protein-glycosaminoglycan-protein cross-link. The biosynthesis of the ITI family members takes place in the liver. In this report we examine the biosynthesis of these proteins using transient transfected COS-7 cells expressing one or more combinations of human ITI chains. The processing and secretion of alpha1-microglobulin and bikunin does not require the ITI heavy chains. A small proportion of the H3 chain seems to be processed into the HC3 form in the absence of the other ITI chains. In contrast, the processing of H2 into HC2 needs the presence of the L chain. The COS-7 cells are able to link the HC2 and HC3 heavy chains with bikunin by means of a chondroitin sulfate bridge, and thus to generate 260-kDa ITI-like proteins as well as pre-alpha-trypsin inhibitor (PalphaI). However, the maturation of the Hl chain into HC1 and the assembly of HC1 inside multichain proteins may take place according to a mechanism which differs from that of the H2 and H3 chains. These results indicate that the assembly of the constituent chains of the ITI-like proteins and PalphaI is not dependent on the liver machinery.

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The authors describe a model of spontaneous lung metastases in nude mice using green fluorescent protein (GFP) expression as a marker. The human lung cell line H460M was transfected with the humanised GFP-S65T cDNA and a stable fluorescent cell line termed H460M(GFP) was obtained. The latter kept in vitro biological features when compared to the parental H460M cell line, which suggests that GFP-expression does not influence H460M(GFP) cell line behaviour. In order to evaluate their metastatic potential and to determine the number of spontaneous metastases, H460M(GFP) cells were subcutaneously inoculated into nude mice. Animals were sacrificed at time intervals and tissues (lung, liver, spleen, node, and kidney) were analysed under fluorescence microscopy. These experiments demonstrated that 2 weeks after subcutaneous inoculation, 75% of animals exhibited fluorescent spontaneous lung micrometastases. From the third week, 100% of animals exhibited an increasing number of metastases (10-16) which were only localised in the lungs. At the end of the study, the number of lung metastases had dramatically increased (42-400 at 7 weeks). Although these metastases were mainly localised in lung, a few mice had an invasion of neighbouring lymph nodes. The H460M(GFP) cell line allowed to follow the seeding and development of spontaneous lung metastases and may be considered a simple and powerful tool to study each step of the metastasis to screen new anticancer drugs.

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To understand more about the human inter-alpha-trypsin inhibitor heavy chain H3 (ITIH3) expression and the relationship between this gene and the family of other ITI heavy chain genes, an analysis of the structure of the ITIH3 gene and its promoter region was performed. This gene is a single copy gene, 14 kilobase pair in length and consists of 22 exons. ITIH3 shares highly conserved exon size and intron-exon borders with other ITI heavy chain genes. We determined that the human ITIH1, ITIH3, and ITIH4 genes are closely linked within a 45-kilobase pair. They are arranged in the order of H1-H3-H4, with the ITIH4 gene transcribed in the opposite direction. A model for the evolution of the ITI heavy chain gene family is presented that involves multiple rounds of gene duplication plus inversion events. The minimum promoter region (-135 to +75) is identified in HepG2 cells. The transient transfection study in various cell lines indicates that the activity of the ITIH3 promoter is not liver-specific. DNase I footprinting, mobility shift assays, and cotransfection experiments reveal a functional CCAAT/enhancer-binding protein site (C/EBP, -1344 to -1305) which interacts with C/EBPalpha and C/EBPbeta factors. The latter factors control the transcription of the ITIH3 gene positively.

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Although widely used, the detection of DNA mutations by the single-strand conformation polymorphism (SSCP) method is often hampered by the need to examine a large set of electrophoretic conditions in order to select the one suited to the DNA sequence under study. We show here that the use of transverse chemical gradient gels allows for a quick and easy optimisation of SSCP analysis, as exemplified on two mutations in exon 2 of the alpha-1-antitrypsin gene.

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Extracellular matrix (ECM) is composed of several macromolecules associated in a complex network. This structure allows cells to adhere, migrate and interact. Hyaluronic acid (HA) is a glycosaminoglycan (GAG) and a major representative of ECM. HA-binding proteins such as CD44, aggrecan, and versican, have been implicated in structuring the ECM by stabilizing large macromolecular aggregates. They also play an important role in tumor metastasis and cell motility. Recently, further HA-binding proteins were identified: the inter-alpha-trypsin inhibitor(ITI)-related proteins. ITI is a glycoprotein composed of three polypeptides: two heavy chains (HC1 and HC2) and one light chain (bikunin). Bikunin confers the protease-inhibitor function. The heavy chains' function was unknown. Recent studies have shown that HC1 and HC2 are linked in vivo and in vitro to hyaluronic acid. This linkage greatly improves extracellular matrix stability. It also demonstrates that ITI-related proteins might be considered as HA-binding proteins (HABP). The ITI related proteins are composed of four polypeptides (HC1, HC2, HC3 and the bikunin) encoded by four genes H1, H2, H3 and L. Unlike the majority of plasma protein a non-disulfide covalent linkage exists between heavy chains and bikunin. This review presents the recent progress concerning the interactions between ITI and ECM showing that ITI-related proteins are HABP members. We will focus on the heavy chain linkage with HA, which represents the demonstration of covalent binding between proteins and HA.

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Previous loss of heterozygosity (LOH) studies of chromosome 3p loci have displayed a 60% deletion frequency in non-small-cell lung cancers (NSCLC), as opposed to small-cell lung cancers, in which the 3p deletion is consistently found. However, the high stromal-cell admixture found in NSCLC and the use of the Southern-blot method lead to under-evaluation of this frequency. In this study, we used a very precise microdissection technique followed by PCR amplification of 6 3p21-22 polymorphic genomic sequences to analyze LOH in 86 NSCLC and in normal adjacent tissue. We found the sensitivity of the microdissection-PCR-based LOH technique higher than the sensitivity of the Southern-blot technique: 87% of the squamous-cell carcinomas and 84% of the large-cell undifferentiated carcinomas showed a clear LOH for a 3p21-22 locus. All doubly informative cases but 4 showed concordant deletion at all 3p21-22 loci. The analysis of 3p microsatellite sequences displayed only 2 cases of genomic instability, one of them also displaying features of tumoral heterogeneity as regards the instability genotype. Four carcinomas in situ adjacent to these NSCLC showed the same allelic profile as the invasive tumors. The only prognostic factors in this study were the disease stage and histology. The 3p21-22 deletion was not related to the stage of the disease and did not appear to be a significant prognostic factor of survival. 3p21 loss appears, so far, to be the most frequent and the earliest genetic alteration described in NSCLC, but does not seem to carry significant prognostic information in invasive tumors.

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Partial cDNAs coding for each of the three human inter-alpha-trypsin inhibitor (ITI) heavy chains were expressed in a bacterial plasmid system and rabbits were immunised with the fusion peptides obtained. Despite the strong sequence homology of these chains, the antisera turned out to be highly specific in the analysis of corresponding mRNA translation products or partially digested serum ITI. Besides classical serum ITI members, their use in Western blotting made it possible to evidence an H3-related ITI form and a low-amount H1-related HC/bikunin component. The relative levels of ITI family members was further studied in baboon and foetal calf sera.

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In standard culture conditions, three human hepatoma cell lines, Hep3B, PLC/PRF/5 and HepG2, were characterised by a predominant transcription of only two (H2 and L) among the four genes involved in the synthesis of inter-alpha-trypsin inhibitor (ITI)-related proteins. Pulse-chase experiments followed by immuno-precipitation with specific anti-L and anti-H ITI antisera showed that the proteins synthesised displayed a restricted L and/or H2 antigenic reactivity. Furthermore, while Hep3B and PLC/PRF/5 lines only synthesised ITI precursors (mainly the L-form), HepG2 cells were able to secrete an ITI-like protein. Immunocytochemical analyses substantiated these results with uneven distribution of heavy and light-chain polypeptide reactivity among the cells. The use of hepatoma cell models for the study of protein synthesis and assembly must therefore be considered cautiously.

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The effects of interleukin 6 (IL-6), the major inducer of the acute-phase reaction, on the expression of inter-alpha-trypsin inhibitor (ITI) genes were examined using human HepG2 hepatoma cells. The three ITI heavy-chain genes H1, H2 and H3 were transcriptionally regulated by IL-6 in a dose- and time-dependent manner. The treatment of HepG2 cells with IL-6 resulted in an increase of H1 and H3 mRNA levels and a decrease of H2 and L mRNA levels. Actinomycin D blocked the action of IL-6, suggesting that IL-6 regulated the H1, H2, H3 gene expression. Moreover, the kinetics of the ITI mRNA degradation in untreated and IL-6-treated cells confirmed these data. The nuclear run-on assay supports the regulatory effect of IL-6 at the transcription level of the L and H2 genes. Primer extension experiments showed that the effect of IL-6 on L, H2 and H3 mRNA synthesis was not related to the transcription starting point. Although H1, H2, H3 and L gene products are supposedly present in similar amounts in the ITI and pre-alpha-trypsin inhibitor molecules, the present work shows that these genes are regulated in a different manner, at least under the influence of IL-6.

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The inter-alpha-trypsin inhibitor H1 (ITIH1) and inter-alpha-trypsin inhibitor H3 (ITIH3) genes have both previously been mapped to chromosomes 3 and 14 in the human and mouse, respectively. We now present evidence that these genes are physically linked. By using cDNA probes, a recombinant DNA phage has been isolated from a bacteriophage DNA library, which contains sequences flanking the 5' end of the ITIH3 gene and the 3' end of the ITIH1 gene. Restriction endonuclease mapping, PCR analysis and DNA sequence determination of the recombinant phage and comparison to genomic DNA revealed that the genes are in tandem, 2721 base pairs apart, with the ITIH1 gene to the 5' side of the ITIH3 gene. Their respective transcriptional units are thus on the same strand of DNA and most probably arose in evolution as the consequence of a duplication of a common ancestral gene.

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In human hepatoma HepG2 cells, the serum inter-alpha-trypsin inhibitor (ITI)-like protein is synthesized from two protein precursors, the heavy chain (H) H2 and the light chain (L). Both of them carry sulphate groups involved in the chondroitin sulphate glycosaminoglycan (GAG) linkage, as demonstrated by [35S]sulphate labelling, chondroitinase digestion and inhibition with beta-D-xyloside, an artificial GAG acceptor. While inhibition of N-glycosylation prevented neither the maturation nor the secretion of the ITI-related entities, brefeldin A induced the accumulation of H and L precursors in the cells, therefore blocking subsequent association and maturation of the precursors before their secretion. The enzyme system involved in the ester linkage between H and L chains is localized in the trans-Golgi network since no ITI-like protein could be obtained in the presence of monensin; instead free heavy-chain protein forms and bikunin were secreted in culture supernatants. The ITI-like protein synthesized by HepG2 cells is therefore composed of two heavy chains HC2 linked to two bikunin chains by chondroitin sulphate bridges, although the GAG linkage between HC2 chains is presumably different. Further, a different maturation route leading to restricted heavy-chain forms, Hm and Hd, could be shown.

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We investigated the ITI protein polymorphism in linkage analysis, using DraI and SstI as restriction fragment length polymorphism (RFLP) markers for the ITIH1 gene. Isoelectric focusing (IEF) classification from 76 individual plasma samples and RFLP analysis from the corresponding DNA preparations disclosed linkage disequilibrium between the phenotypic IEF patterns of the two common ITI alleles, ITI*1 and ITI*2, and the diallelic DNA polymorphisms of two ITIH1 RFLPs, represented by DraI 4.0 kb and DraI 2.4 + 1.6 kb, and by SstI 6.7 kb and SstI 6.0 + 0.7 kb, for the ITI 1 and ITI 2 IEF phenotypes, respectively, and by DraI 4.0/2.4 + 1.6 kb and SstI 6.7/6.0 + 0.7 kb for the heterozygous ITI 1-2 IEF phenotype. Linked segregation between either of the RFLPs and the polymorphic ITI plasma protein locus has been established in nine informative family pedigrees. The less frequent allele in Europeans, ITI*3, is not represented by a further allelic restriction fragment in either RFLP. The significant linkage disequilibrium observed in this genetic study indicates that the ITI locus, with the alleles ITI*1 and ITI*2, must be close to, or reside within, the ITIH1 gene. The diallelic ITI protein polymorphism therefore provides an informative phenotypic marker system for chromosome 3p211-212.

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The Hep G2 hepatoma cell line synthesizes the inter-alpha-trypsin inhibitor (ITI). This protease inhibitor and the other proteins of this family include four polypeptides chains: three heavy chains (HC1, HC2, HC3) and one light chain (bikunin). In the present study, we have demonstrated by immunofluorescence that ITI is detected mainly in perinuclear cytoplasmic zones comparable to those of albumin or alpha-1-antitrypsin. The presence of the mRNAs of the four polypeptide chains in all Hep G2 cells of a non-synchronized culture have been demonstrated by in situ hybridization. An evaluation of the transcription of the four ITI genes through an analysis of markings brings to the fore a clearly much higher rate of mRNAs from the light chain than from the heavy chains. The mRNAs corresponding to the HC2 chains are more heavily represented than are those corresponding to the HC1 and HC3 chains. In Hep G2 cells in culture, a quantification of mRNAs based on the in situ hybridization technique shows that their relative quantities, in decreasing order, are those of L, HC2, HC3 and HC1.

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