DNA ploidy and cell cycle protein expression in oral squamous cell carcinomas with and without lymph node metastases.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is the most frequently occurring malignant tumour in the oral cavity. OSCC arises because of multiple genetic alterations. Cell cycle aberrations and aneuploidy are reportedly among the main characteristics of cancer cells and are associated with aggressive growth and poor prognosis. METHODS: The study sample included 47 non-metastasised and 39 metastasised primary OSCC, with matched positive cervical lymph nodes and 17 normal oral mucosa samples. Tissue microarrays (TMAs) were prepared with a minimum of three cores from each case. TMA sections were cut and immunostained with MCM2, Ki-67, geminin and cyclin D1 antibodies. DNA image analysis was performed on the whole tissue section before TMAs were created. RESULTS: The results revealed that there were no differences in cell cycle protein expression in different areas of the tumours or between the metastatic and non-metastatic carcinomas. None of the cell cycle proteins showed significant differences between the lymph node metastasis and the primary OSCC, except for Ki-67. Geminin/Ki-67 ratio showed significant difference between metastatic and non-metastatic tumours. Aneuploidy was detected in all (100%) cases of OSCC. Similarly, all lymph node samples (39 cases) were aneuploid. CONCLUSION: The results suggest that although there was dysregulation of cell cycle regulatory proteins, only Ki-67 and the MCM2/Ki-67 and geminin/Ki-67 ratios may have prognostic significance in oral cancer. DNA ploidy alone was not specific and may not be a good tool to evaluate prognosis or metastatic progression in oral cavity carcinomas.

Elevated sputum BPIFB1 levels in smokers with chronic obstructive pulmonary disease: a longitudinal study.

A previous study involving a proteomic screen of induced sputum from smokers and patients with chronic obstructive pulmonary disease (COPD) demonstrated elevated levels of bactericidal/permeability-increasing fold-containing protein B1 (BPIFB1). The aim of the present study was to further evaluate the association of sputum BPIFB1 levels with smoking and longitudinal changes in lung function in smokers with COPD. Sputum BPIFB1 was characterized by two-dimensional gel electrophoresis and mass spectrometry. The expression of BPIFB1 in COPD was investigated by immunoblotting and immunohistochemistry using sputum and lung tissue samples. BPIFB1 levels were also assessed in induced sputum from nonsmokers (n = 31), smokers (n = 169), and patients with COPD (n = 52) via an ELISA-based method. The longitudinal changes in lung function during the 4-year follow-up period were compared with the baseline sputum BPIFB1 levels. In lung tissue samples, BPIFB1 was localized to regions of goblet cell metaplasia. Secreted and glycosylated BPIFB1 was significantly elevated in the sputum of patients with COPD compared with that of smokers and nonsmokers. Sputum BPIFB1 levels correlated with pack-years and lung function as measured by forced expiratory volume in 1 s (FEV1) % predicted and FEV1/FVC (forced vital capacity) at baseline and after the 4-year follow-up in all participants. The changes in lung function over 4 years were significantly associated with BPIFB1 levels in current smokers with COPD. In conclusion, higher sputum concentrations of BPIFB1 were associated with changes of lung function over time, especially in current smokers with COPD. BPIFB1 may be involved in the pathogenesis of smoking-related lung diseases.

PLUNC protein expression in major salivary glands of HIV-infected patients.

OBJECTIVE: To analyse and compare the expression of Palate, Lung, and Nasal Epithelium Clone (PLUNC) proteins in salivary glands from patients with and without AIDS (control group) using autopsy material. METHODS: We analysed the expression of PLUNCs using immunohistochemistry in parotid (n = 45), submandibular (n = 47) and sublingual gland (n = 37) samples of AIDS patients [30 with normal histology, 21 with mycobacteriosis, 14 with cytomegalovirus (CMV) infection, 30 with chronic non-specific sialadenitis, and 30 HIV-negative controls. In situ hybridization (ISH) for SPLUNC 2 in the HIV-negative group was performed. RESULTS: SPLUNC 1 expression was detected in the mucous acini of submandibular and sublingual glands, and SPLUNC 2 were seen in the serous cells. LPLUNC 1 expression was only positive in the salivary ducts. There was a higher expression of SPLUNC 2 in AIDS patients with CMV infection and mycobacteriosis when compared with all other groups. The intensity of staining for SPLUNC 2 was greater around the lesions than the peripheral ones. ISH for SPLUNC 2 showed perinuclear positivity in the serous cells in all HIV-negative cases. CONCLUSIONS: SPLUNC 1 and LPLUNC 1 proteins were similarly expressed in the salivary glands of AIDS patients and non-HIV patients. CMV infection and mycobacteriosis increase SPLUNC 2 expression in serous cells in the salivary gland of AIDS patients.

Expression of PLUNC family members in benign and malignant salivary gland tumours.

OBJECTIVES: The aim of this study was to determine the expression of PLUNC proteins in benign and malignant salivary gland tumours and thus their potential use as diagnostic and / or prognostic tools. MATERIALS AND METHODS: A tissue microarray was assembled from 64 salivary gland tumours including adenoid cystic carcinoma, carcinoma ex-pleomorphic adenoma, mucoepidermoid carcinoma, polymorphous low-grade adenocarcinoma, pleomorphic adenoma, acinic cell carcinoma, myoepithelial carcinoma and papillary cystadenocarcinoma. Clinicopathological data were collected retrospectively and immunohistochemical analysis of three PLUNC proteins (SPLUNC1, SPLUNC2 and LPLUNC1) was performed. Immunoreactivity was assessed as positive or negative. RESULTS: PLUNC expression was only found in mucoepidermoid carcinomas and papillary cystadenocarcinoma; all other tumours studied were negative. Mucin plugs, mucous and intermediate cells of mucoepidermoid carcinomas were positive for LPLUNC1 and SPLUNC2, but areas composed of epidermoid and clear cells were negative for all PLUNCs. Papillary cystadenocarcinoma was positive for all PLUNCs. No correlation was found with tumour grade or outcome. CONCLUSIONS: Intense expression of two PLUNC proteins in mucous cells and mucin plugs of mucoepidermoid carcinoma and papillary cystadenocarcinoma indicate that they could be used as additional diagnostic tools in some equivocal cases, but further studies are needed to understand the biological processes involved in PLUNC expression.

An innate defense peptide BPIFA1/SPLUNC1 restricts influenza A virus infection.

The airway epithelium secretes proteins that function in innate defense against infection. Bactericidal/permeability-increasing fold-containing family member A1 (BPIFA1) is secreted into airways and has a protective role during bacterial infections, but it is not known whether it also has an antiviral role. To determine a role in host defense against influenza A virus (IAV) infection and to find the underlying defense mechanism, we developed transgenic mouse models that are deficient in BPIFA1 and used these, in combination with in vitro three-dimensional mouse tracheal epithelial cell (mTEC) cultures, to investigate its antiviral properties. We show that BPIFA1 has a significant role in mucosal defense against IAV infection. BPIFA1 secretion was highly modulated after IAV infection. Mice deficient in BPIFA1 lost more weight after infection, supported a higher viral load and virus reached the peripheral lung earlier, indicative of a defect in the control of infection. Further analysis using mTEC cultures showed that BPIFA1-deficient cells bound more virus particles, displayed increased nuclear import of IAV ribonucleoprotein complexes, and supported higher levels of viral replication. Our results identify a critical role of BPIFA1 in the initial phase of infection by inhibiting the binding and entry of IAV into airway epithelial cells.

An innate defense peptide BPIFA1/SPLUNC1 restricts influenza A virus infection.

This corrects the article DOI: 10.1038/mi.2017.45.

Regulatory circuits for plasmid survival.

Bacterial plasmids deploy a diverse range of regulatory mechanisms to control expression of the functions they need to survive in the host population. Understanding of the mechanisms by which autoregulatory circuits control plasmid survival functions, in particular plasmid replication, has been advanced by recent studies. At a molecular level, structural understanding of how certain antisense RNAs control replication and stability functions is almost complete. Control circuits linking plasmid transfer functions to the status of the bacterial population have been dissected, uncovering a complex and hierarchical organisation. Coordinate or global regulation of plasmid replication, transfer and stable maintenance functions is becoming apparent across a range of plasmid families.

Characterisation of the human plunc gene, a gene product with an upper airways and nasopharyngeal restricted expression pattern.

Here we report the cloning and characterization of the human homologue of plunc, a murine gene expressed specifically in the upper airways and nasopharyngeal regions. The human plunc cDNA codes for a leucine-rich protein of 256 amino acids which is 72% identical to the murine protein. RNA blot analysis suggests that expression of plunc is restricted to the trachea, upper airway, nasopharyngeal epithelium and salivary gland. The human plunc gene contains nine exons and is localised to chromosome 20q11.2. The unique expression pattern of the human plunc suggest that it may prove a useful model gene with which to study the regulatory mechanisms which direct expression of genes specifically to the upper airways.

Design and validation of anti-inflammatory peptides from human parotid secretory protein.

Parotid secretory protein (PSP) and palate-lung-nasal epithelium clone (PLUNC) are novel secretory proteins that are expressed in the oral cavity and upper airways. Both proteins are related to bactericidal/permeability increasing protein (BPI). Cationic peptides derived from BPI exhibit anti-inflammatory activity. To test if PSP (C20orf70 gene product) also contains anti-inflammatory peptides, we designed 3 cationic peptides based on the predicted structure of PSP and known active regions of BPI. Each peptide inhibited the lipopolysaccharide (LPS)-stimulated secretion of TNFalpha from RAW 264.7 macrophage cells. At 200 microg/mL, the peptide GK-7 exhibited inhibition similar to that achieved with 10 microg/mL of polymyxin B. PSP peptides directly inhibited the binding of LPS to LPS-binding protein. The cationic peptide Substance P had no inhibitory effect in these assays, confirming the specificity of the PSP peptides. These findings suggest that PSP peptides can serve as templates for the design of novel anti-inflammatory peptides.

Structure and sequence variation of the trypanosome spliced leader transcript.

We have assessed the potential of using the spliced leader (SL) or mini-exon gene as a marker for molecular phylogenetic analysis of genus Trypanosoma. A total of 27 trypanosome sequences were compared, 18 of these being newly reported. In contrast to genus Leishmania, we found the non-transcribed spacer region of the SL locus in trypanosomes to be far too variable for informative comparison of all but the most closely related species. At the other extreme, the short (39 nt) SL exon was usually completely conserved and hence uninformative. The SL RNA showed variation in both length (97-152 nt) and sequence among different trypanosome species, with most variation occurring in stem-loop II. Consequently, this region could not be aligned with confidence in multiple sequence alignment, severely reducing the number of phylogenetically informative nucleotide positions. In computer simulation, most of the SL RNAs readily folded into the 3 stem-loop secondary structure predicted previously, but again stem-loop II was highly variable. No obvious correlation could be seen between the length of this stem-loop and trypanosome biology. We conclude that the SL repeat is not an informative phylogenetic marker for long range evolutionary studies of genus Trypanosoma.

The taxonomic position and evolutionary relationships of Trypanosoma rangeli.

This paper presents a re-evaluation of the taxonomic position and evolutionary relationships of Trypanosoma (Herpetosoma) rangeli based on the phylogenetic analysis of ssrRNA sequences of 64 Trypanosoma species and comparison of mini-exon sequences. All five isolates of T. rangeli grouped together in a clade containing Trypanosoma (Schizotrypanum) cruzi and a range of closely related trypanosome species from bats [Trypanosoma (Schizotrypanum) dionisii, Trypanosoma (Schizotrypanum) vespertilionis] and other South American mammals [Trypanosoma (Herpetosoma) leeuwenhoeki, Trypanosoma (Megatrypanum) minasense, Trypanosoma (Megatrypanum) conorhini] and an as yet unidentified species of trypanosome from an Australian kangaroo. Significantly T. rangeli failed to group with (a) species of subgenus Herpetosoma, other than those which are probably synonyms of T. rangeli, or (b) species transmitted via the salivarian route, although either of these outcomes would have been more consistent with the current taxonomic and biological status of T. rangeli. We propose that use of the names Herpetosoma and Megatrypanum should be discontinued, since these subgenera are clearly polyphyletic and lack evolutionary and taxonomic relevance. We hypothesise that T. rangeli and T. cruzi represent a group of mammalian trypanosomes which completed their early evolution and diversification in South America.

Type II pneumocytes in mixed cell culture of human lung: a light and electron microscopic study.

Alveolar Type II epithelial cells dedifferentiate rapidly in vitro. Studies with animal tissue suggest that cell-cell and extracellular matrix-cell interactions are important in the retention of Type II cell morphology in vitro. Thus, in this study with human tissue, alveolar Type II cells, alveolar macrophages, and spindle cells were prepared from the same sample of lung (obtained following lobectomy for cancer, n = 3), cocultured on glass cover slips or tissue culture plastic, and studied by light microscopy with scanning (SEM) and transmission (TEM) electron microscopy for 8 days. The primary cell isolates contained approximately 45% Type II cells; the remainder were macrophages or unidentifiable cells. Clusters, made up of a single layer of cuboidal Type II cells around a central core of connective tissue (largely collagen and some elastic tissue), formed above a monolayer of spindle cells. The Type II cells were morphologically similar to those seen in vivo. The cells were still cuboidal at 8 days but had lost their lamellar bodies, which were released into the medium via the apical surface. The clusters increased in size with time (area, microns 2: day 1, 29(5-143) x 10(2); day 8, 63(10-311) x 10(2); mean(range); p less than 0.02) without changing in number per culture, suggesting Type II cell proliferation. This may have been due to factors produced by the other cells and adherence to the extracellular matrix (ECM); (free collagen fibers, present in the original preparation, spindle cells, and/or Type II cells could be responsible for presence of ECM). We propose this as a useful model for the study of human Type II epithelial cells in vitro.

High-throughput whole-genome sequencing to dissect the epidemiology of Acinetobacter baumannii isolates from a hospital outbreak.

Shared care of military and civilian patients has resulted in transmission of multidrug-resistant Acinetobacter baumannii (MDR-Aci) from military casualties to civilians. Current typing technologies have been useful in revealing relationships between isolates of A. baumannii but they are unable to resolve differences between closely related isolates from small-scale outbreaks, where chains of transmission are often unclear. In a recent hospital outbreak in Birmingham, six patients were colonised with MDR-Aci isolates indistinguishable using standard techniques. We used whole-genome sequencing to identify single nucleotide polymorphisms in these isolates, allowing us to discriminate between alternative epidemiological hypotheses in this setting.

Macrophages promote angiogenesis in human breast tumour spheroids in vivo.

An in vivo model has been established to study the role of macrophages in the initiation of angiogenesis by human breast tumour spheroids in vivo. The extent of the angiogenic response induced by T47D spheroids implanted into the dorsal skinfold chamber in nude mice was measured in vivo and compared to that induced by spheroids infiltrated with human macrophages prior to implantation. Our results indicate that the presence of macrophages in spheroids resulted in at least a three-fold upregulation in the release of vascular endothelial growth factor (VEGF) in vitro when compared with spheroids composed only of tumour cells. The angiogenic response measured around the spheroids, 3 days after in vivo implantation, was significantly greater in the spheroids infiltrated with macrophages. The number of vessels increased (macrophages vs no macrophages 34+/-1.9 vs 26+/-2.5, P<0.01), were shorter in length (macrophages vs no macrophages 116+/-4.92 vs 136+/-6.52, P<0.008) with an increased number of junctions (macrophages vs no macrophages 14+/-0.93 vs 11+/-1.25, P<0.025) all parameters indicative of new vessel formation. This is the first study to demonstrate a role for macrophages in the initiation of tumour angiogenesis in vivo.

Radiosensitivity and characterisation of a newly established cell line from an epithelioid sarcoma.

A new human tumour cell line (designated HX 165c) has been established from an epithelioid sarcoma presenting in a 28 year old male. The cells grew as an adherent monolayer with a doubling time of 38 h and had mainly epithelial morphology but with areas of mesenchymal-like cytoplasmic extensions. The mixed epithelial-mesenchymal phenotype was also apparent by intermediate filament analysis which showed reactivity to vimentin and keratin. The cells were tumorigenic in nude mice and aneuploid, possessing a mean chromosome number of 65. In vitro cloning determinations gave colony-forming efficiencies of 0.01% in an anchorage-independent soft agar assay and 34% in a monolayer anchorage-dependent assay. The cells were in the mid-range for radiosensitivity of human tumour cells (surviving fraction at 2 Gy of 0.39). In addition, experiments utilising continuous low dose rate irradiation at 3.2 cGy min-1, showed that the cells possessed only a small capacity to recover from radiation damage (dose reduction factor at 1% cell survival of 1.15 for 150 versus 3.2 cGy min-1). This cell line, being only the second we are aware of to be established from this rare soft tissue sarcoma, should be useful in helping to ascertain the histogenesis of epithelioid sarcoma.

Secretory leukoprotease inhibitor: partnering alpha 1-proteinase inhibitor to combat pulmonary inflammation.

Secretory leukoprotease inhibitor (SLPI) is a low molecular weight serine proteinase inhibitor, notably of neutrophil elastase (NE), which is synthesised and secreted by the pulmonary epithelium. SLPI plays an important role in limiting NE-induced pulmonary inflammation and, significantly, it also possesses anti-HIV activity. SLPI is a significant component of the anti-NE shield in the lung which has different reactivity from, and is therefore complementary to, the anti-NE action of alpha 1-proteinase inhibitor (alpha 1-PI). Inhaled recombinant SLPI (rSLPI) could prove beneficial in partnership with alpha 1-PI in the treatment of a number of inflammatory lung disorders including emphysema, chronic bronchitis, cystic fibrosis, and adult respiratory distress syndrome.

Ketosynthase domain probes identify two subclasses of fungal polyketide synthase genes.

Analysis of fungal polyketide synthase gene sequences suggested that these might be divided into two subclasses, designated WA-type and MSAS-type. Two pairs of degenerate PCR primers (LC1 and LC2c, LC3 and LC5c) were designed for the amplification of ketosynthase domain fragments from fungal PKS genes in each of these subclasses. Both primer pairs were shown to amplify one or more PCR products from the genomes of a range of ascomycetous Deuteromycetes and Southern blot analysis confirmed that the products obtained with each pair of primers emanated from distinct genomic loci. PCR products obtained from Penicillium patulum and Aspergillus parasiticus with the LC1/2c primer pair and from Phoma sp. C2932 with both primer pairs were cloned and sequenced; the deduced protein sequences were highly homologous to the ketosynthase domains of other fungal PKS genes. Genes from which LC1/2c fragments were amplified (WA-type) were shown by a phylogenetic analysis to be closely related to fungal PKS genes involved in pigment and aflatoxin biosynthetic pathways, whereas the gene from which the LC3/5c fragment was amplified (MSAS-type) was shown to be closely related to genes encoding 6-methylsalicylic acid synthase (MSAS). The phylogenetic tree strongly supported the division of fungal PKS genes into two subclasses. The LC-series primers may be useful molecular tools to facilitate the cloning of novel fungal polyketide synthase genes.

Cytokine-mediated induction of the human elafin gene in pulmonary epithelial cells is regulated by nuclear factor-kappaB.

Elafin, a low molecular-weight proteinase inhibitor, is a member of the recently described trappin gene family. These proteins are thought to play important roles in the regulation of inflammation and are expressed in multiple epithelia. Elafin is found within the lung, and its expression can be induced by inflammatory mediators. The molecular mechanisms that mediate its induction are not understood. In this study we investigated the transcriptional regulation of the elafin gene in pulmonary epithelial cell lines. Transfection of elafin promoter constructs into the elafin-expressing pulmonary epithelial cell line A549 identified a number of positive-acting elements. Cytokine-mediated inducibility of the elafin gene promoter was shown to occur through a nuclear factor (NF)-kappaB site present within the minimal promoter. This site was shown to bind to NF-kappaB proteins within nuclear extracts from cytokine stimulated cell lines as well as to in vitro-translated RelA. Cotransfection with both RelA and NF-kappaB-inducing kinase induced reporter gene activation via this site, and mutagenesis experiments confirmed that it was crucial for induction of elafin gene activity. These results clearly identify a role for elafin in the inflammatory response of the airway epithelium to pathogenic insult and show that this response is mediated by an NF-kappaB site within the proximal promoter.

The role of tumour-associated macrophages in tumour progression: implications for new anticancer therapies.

The role of macrophages in tumour growth and development is complex and multifaceted. Whilst there is limited evidence that tumour-associated macrophages (TAMs) can be directly tumouricidal and stimulate the anti-tumour activity of T cells, there is now contrasting evidence that tumour cells are able to block or evade the activity of TAMs at the tumour site. In some cases, tumour-derived molecules even redirect TAM activities to promote tumour survival and growth. Indeed, evidence has emerged for a symbiotic relationship between tumour cells and TAMs, in which tumour cells attract TAMs and sustain their survival, with TAMs then responding to micro-environmental factors in tumours such as hypoxia (low oxygen tension) by producing important mitogens as well as various growth factors and enzymes that stimulate tumour angiogenesis. This review presents evidence for the number and/or distribution of TAMs being linked to prognosis in different types of human malignancy. It also outlines the range of pro- and anti-tumour functions performed by TAMs, and the novel therapies recently devised using TAMs to stimulate host immune responses or deliver therapeutic gene constructs to solid tumours.

Characterization of the human surfactant protein D promoter: transcriptional regulation of SP-D gene expression by glucocorticoids.

We have previously described the characterization of genomic clones encoding the entire translated sequence of human pulmonary surfactant protein D (SP-D). We now describe the characterization of a genomic fragment (H5E7) that encodes the entirety of the first translated exon (Exon 2), Intron 1, a short transcribed untranslated sequence (Exon 1; 39 bp), and approximately 4 kb of sequence upstream from the transcription initiation site. The start site was identified by 5'-RACE-PCR cloning and primer extension. A putative TATA box (CATAAATA) was identified approximately 30 bp upstream of the start site. Complete sequencing of a HindIII/SacI fragment (HS-1674) encoding approximately 1.7 kb of sequence 5' to the TATA demonstrated multiple potential cis-regulatory elements including half-site glucocorticoid response elements (GRE), a canonical AP-1 consensus, several AP-1 like sequences, E-box sequences, NF-IL-6 and PEA3 motifs, and putative interferon response elements. H441 lung adenocarcinoma cells, which express low levels of SP-D mRNA, and liver HepG2 cells, were transiently co-transfected with chloramphenicol acetyl transferase (CAT) reporter constructs containing up to 3,000 base pairs of upstream sequence, and with constructs encoding beta-gal. H441 cells transfected with constructs containing at least 161 bp of upstream sequence gave normalized levels of CAT activity greater than or equal to that obtained for parallel positive control transfections using pTK-CAT. Treatment of the cells for 48 h with 50 nM dexamethasone (Dex) gave a 2- to 5-fold increase in CAT activity. Interestingly, a 5'-deletion construct containing 161 bp of upstream sequence (pFS161-CAT) conferred both cell type-restricted and dexamethasone-responsive expression. These studies emphasize the potential complexity of SP-D gene regulation, and further support the hypothesis that the effects of glucocorticoids on SP-D production in vivo are regulated at the level of transcription.