The potential role of artificial intelligence-assisted chest X-ray imaging in detecting early-stage lung cancer in the community-a proposed algorithm for lung cancer screening in Malaysia.

INTRODUCTION: The poor prognosis of lung cancer has been largely attributed to the fact that most patients present with advanced stage disease. Although low dose computed tomography (LDCT) is presently considered the optimal imaging modality for lung cancer screening, its use has been hampered by cost and accessibility. One possible approach to facilitate lung cancer screening is to implement a risk-stratification step with chest radiography, given its ease of access and affordability. Furthermore, implementation of artificial-intelligence (AI) in chest radiography is expected to improve the detection of indeterminate pulmonary nodules, which may represent early lung cancer. MATERIALS AND METHODS: This consensus statement was formulated by a panel of five experts of primary care and specialist doctors. A lung cancer screening algorithm was proposed for implementation locally. RESULTS: In an earlier pilot project collaboration, AI-assisted chest radiography had been incorporated into lung cancer screening in the community. Preliminary experience in the pilot project suggests that the system is easy to use, affordable and scalable. Drawing from experience with the pilot project, a standardised lung cancer screening algorithm using AI in Malaysia was proposed. Requirements for such a screening programme, expected outcomes and limitations of AI-assisted chest radiography were also discussed. CONCLUSION: The combined strategy of AI-assisted chest radiography and complementary LDCT imaging has great potential in detecting early-stage lung cancer in a timely manner, and irrespective of risk status. The proposed screening algorithm provides a guide for clinicians in Malaysia to participate in screening efforts.

Competitive immunoassay for recombinant hirudin using capillary electrophoresis with laser-induced fluorescence detection.

A competitive immunoassay based on capillary electrophoresis (CE) with laser-induced fluorescence (LIF) has been developed for the determination of recombinant hirudin (r-hirudin) in biological mixtures. Hirudin, a thrombin inhibitor, is a polypeptide of 65 amino acids. To check purity levels and perform pharmacokinetic studies of (r-hirudin), specific and reproducible analysis methods are demanded. The work involved the development of separation conditions allowing for routine analysis of plasma samples. In this study, r-hirudin was labeled with fluorescein isothiocyanate (FITC), and FITC-labeled r-hirudin was purified using high-performance liquid chromatography. The purified product was then mixed with the sample followed with the addition of anti-hirudin antibody. Free, antibody-bound, and tagged r-hirudin could be separated within 5 min by CE analysis using uncoated fused-silica capillary with high reproducibility. The developed method can be used to determine r-hirudin with good precision and a detection limit lower than 20 nM. This result demonstrates the feasibility of the CE-LIF immunoassay method for the determination of r-hirudin in plasma samples.

ClpA mediates directional translocation of substrate proteins into the ClpP protease.

The intracellular degradation of many proteins is mediated in an ATP-dependent manner by large assemblies comprising a chaperone ring complex associated coaxially with a proteolytic cylinder, e.g., ClpAP, ClpXP, and HslUV in prokaryotes, and the 26S proteasome in eukaryotes. Recent studies of the chaperone ClpA indicate that it mediates ATP-dependent unfolding of substrate proteins and directs their ATP-dependent translocation into the ClpP protease. Because the axial passageway into the proteolytic chamber is narrow, it seems likely that unfolded substrate proteins are threaded from the chaperone into the protease, suggesting that translocation could be directional. We have investigated directionality in the ClpA/ClpP-mediated reaction by using two substrate proteins bearing the COOH-terminal ssrA recognition element, each labeled near the NH(2) or COOH terminus with fluorescent probes. Time-dependent changes in both fluorescence anisotropy and fluorescence resonance energy transfer between donor fluorophores in the ClpP cavity and the substrate probes as acceptors were measured to monitor translocation of the substrates from ClpA into ClpP. We observed for both substrates that energy transfer occurs 2--4 s sooner with the COOH-terminally labeled molecules than with the NH(2)-terminally labeled ones, indicating that translocation is indeed directional, with the COOH terminus of the substrate protein entering ClpP first.

CpG motifs induce Langerhans cell migration in vivo.

Cytosine-guanosine (CpG) oligonucleotide (CpG-oligo) sequences are immunostimulatory motifs that are present in bacterial DNA and their presence in plasmids might contribute to the immune response generated by DNA vaccination. The cell targets of CpG motifs in vivo have not been characterized yet. In this report we assessed the in vivo effects of CpG motifs on Langerhans cells (LC) migration. We showed that intradermal injection of 10 microg of CpG-containing oligonucleotides in mouse ear induced the local depletion of LC within 2 h of exposure as shown by CD11c and Ia immunohistological staining. To demonstrate that LC depletion was due to LC migration, CpG oligonucleotides were injected into the explants ex vivo, and the CD11c(+) cells emigrating from the cultured isolated skin within medium were evaluated by immunostaining and FACS analysis. Our findings demonstrate that CpG motifs induce LC/dendritic cell (DC) migration out of the skin. To assess whether CpG motifs may act directly on LC/DC to induce their emigration we next analyzed the effects of CpG motifs in vitro on the expression of adhesion molecules involved in LC/DC migration. The results of these experiments show that alpha(6) integrins, E-Cadherin, ICAM-1, CD11b and CD11c were differentially regulated upon CpG-oligo treatment of immortalized DC. CpG treatment (10 microg/ml for 8 h) resulted in a 100% increase in ICAM-1 staining intensity, a 50% decrease in E-Cadherin staining and a 25% decrease in alpha(6) integrins staining, while no changes in the levels of CD11b and CD11c expression were recorded. Changes in adhesion molecule expression were mirrored by concomitant changes in the cell morphology that included cell depolarization, the appearance of filopods and loss of adherence. This study provides the first in vivo evidence that CpG motifs signal the migration of LC from the epidermis.

Chaperone rings in protein folding and degradation.

Chaperone rings play a vital role in the opposing ATP-mediated processes of folding and degradation of many cellular proteins, but the mechanisms by which they assist these life and death actions are only beginning to be understood. Ring structures present an advantage to both processes, providing for compartmentalization of the substrate protein inside a central cavity in which multivalent, potentially cooperative interactions can take place between the substrate and a high local concentration of binding sites, while access of other proteins to the cavity is restricted sterically. Such restriction prevents outside interference that could lead to nonproductive fates of the substrate protein while it is present in non-native form, such as aggregation. At the step of recognition, chaperone rings recognize different motifs in their substrates, exposed hydrophobicity in the case of protein-folding chaperonins, and specific "tag" sequences in at least some cases of the proteolytic chaperones. For both folding and proteolytic complexes, ATP directs conformational changes in the chaperone rings that govern release of the bound polypeptide. In the case of chaperonins, ATP enables a released protein to pursue the native state in a sequestered hydrophilic folding chamber, and, in the case of the proteases, the released polypeptide is translocated into a degradation chamber. These divergent fates are at least partly governed by very different cooperating components that associate with the chaperone rings: that is, cochaperonin rings on one hand and proteolytic ring assemblies on the other. Here we review the structures and mechanisms of the two types of chaperone ring system.

Global unfolding of a substrate protein by the Hsp100 chaperone ClpA.

The bacterial protein CIpA, a member of the Hsp100 chaperone family, forms hexameric rings that bind to the free ends of the double-ring serine protease ClpP. ClpA directs the ATP-dependent degradation of substrate proteins bearing specific sequences, much as the 19S ATPase 'cap' of eukaryotic proteasomes functions in the degradation of ubiquitinated proteins. In isolation, ClpA and its relative ClpX can mediate the disassembly of oligomeric proteins; another similar eukaryotic protein, Hsp104, can dissociate low-order aggregates. ClpA has been proposed to destabilize protein structure, allowing passage of proteolysis substrates through a central channel into the ClpP proteolytic cylinder. Here we test the action of ClpA on a stable monomeric protein, the green fluorescent protein GFP, onto which has been added an 11-amino-acid carboxy-terminal recognition peptide, which is responsible for recruiting truncated proteins to ClpAP for degradation. Fluorescence studies both with and without a 'trap' version of the chaperonin GroEL, which binds non-native forms of GFP, and hydrogen-exchange experiments directly demonstrate that ClpA can unfold stable, native proteins in the presence of ATP.

Rat interleukin-1 beta binding sites in rat hypothalamus and pituitary gland.

In this study, radiolabeled recombinant rat interleukin-1 beta (r125I-IL-1 beta) was used to localize and characterize IL-1 beta binding in rat hypothalamus and pituitary gland by quantitative autoradiography. The ability of this ligand to bind to type I IL-1 receptor was first tested on murine lymphoma cells (EL-4). In the rat-tissue sections, high densities of specific r125I-IL-1 beta binding sites were localized in the anterior as well as the posterior pituitary and in the choroid plexus. A fine labeling was observed in meninges and third ventricle walls while no binding was detected in the hypothalamic nuclei. Saturation experiments, in the anterior and posterior pituitary, revealed one specific binding site with an affinity constant (Kd) of 0.5 nM. Competition experiments were achieved using either rat IL-1 beta (rIL-1 beta) or human IL-1s (hIL-1 alpha, hIL-1 beta and IL-1 receptor antagonist: hIL-1a). Affinity constants (Ki) were drastically different according to the ligand used, while Ki values were found similar in anterior and posterior pituitary. Competition with rIL-1 beta revealed one binding affinity (Ki of 0.1 nM range). In contrast, competition with hIL-1 beta revealed two binding affinities: a high (Ki: 0.1 pM range) and a low one (Ki: 1 nM range). Competition with hIL-1ra was obtained for high concentrations only (Ki: 10-100 nM range), whereas human IL-1 alpha (hIL-1 alpha) was unable to compete at 1-100 nM.(ABSTRACT TRUNCATED AT 250 WORDS)

Suppressin: an endogenous negative regulator of immune cell activation.

We have recently identified a new suppressor molecule we named suppressin (SPN) that has all the characteristics of a global negative regulator of the immune system. SPN is a unique 63-kD monomeric polypeptide with a pI of 8.1 that is produced and secreted under basal conditions by murine splenocytes, human peripheral mononuclear cells, and hormone-secreting pituitary cells. The biological actions of SPN in vitro include the inhibition of mitogen-induced proliferation and immunoglobulin synthesis of lymphocytes and the suppression of interleukin-2-dependent CTLL-2 cell proliferation. In addition, SPN enhances natural killer cell activity by eliciting interferon-alpha and -beta synthesis and secretion. SPN effects are reversible, nontoxic, and require the continuous presence of exogenous SPN. T lymphocytes stimulated with concanavalin A or phytohemagglutinin are more sensitive to SPN (90% inhibition) than are lipopolysaccharide-stimulated B cells (60% inhibition). SPN arrests lymphocytes in the G0/G1 phase of the cell cycle after reduction of their RNA, protein and DNA synthesis, suggesting that SPN inhibits the processes required for G0 transition to G1. SPN is found intracellularly in all unstimulated lymphocyte subsets, monocytes, and in phytohemagglutinin-activated T lymphocytes immunopositive for the low affinity interleukin-2 receptor. These results suggest that SPN may be a major negative regulator of cell proliferation in the immune system. All SPN-producing cell types are also sensitive to SPN. Collectively, the results of these experiments provide the foundations for a model in which SPN regulates lymphocyte proliferation in an autocrine and/or paracrine manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of suppressin-producing cells in the rat pituitary.

Suppressin (SPN) is a novel polypeptide that is synthesized and secreted by normal rat pituitary cells and a rat pituitary tumor cell line, GH3. Specifically, SPN is a negative regulator of growth that inhibits lymphoid and neuroendocrine cell proliferation. The objective of the present study was to identify the cells in the normal rat pituitary that produce SPN. A double immunofluorescence technique, using antibodies to SPN in conjunction with antibodies to the six major adenohypophyseal hormones, was used to colocalize SPN and a specific hormone in a single dispersed pituitary cell. The results of these experiments showed that, on the average, 42% of the cells in the pituitary produce SPN. Suppressin production in the pituitary was restricted to the adenohypophysis. The SPN-producing population in the pituitary was composed of somatotrophs, lactotrophs, corticotrophs, thyrotrophs, and mammosomatotrophs, while gonadotrophs did not produce SPN. Additionally, a PRL reverse hemolytic plaque assay was used to examine SPN production in lactotrophs. The results of these experiments showed that SPN production and the amount of PRL secreted covaried. Specifically, SPN production was observed primarily in non-PRL-secreting lactotrophs or in lactotrophs secreting a high amount of PRL. The results of these experiments suggest a potential regulatory relationship between the synthesis and secretion of SPN and PRL. In summary, this report provides the first identification of SPN-producing cells in the pituitary and shows that SPN production occurs primarily in somatotrophs and lactotrophs.

Insulin-like growth factor 1 receptors in human breast tumour: localisation and quantification by histo-autoradiographic analysis.

To assess the precise role of IGF1 in benign and malignant breast diseases, we analysed the tissular localisation, characterised, and quantified specific insulin-like growth factor 1 (IGF1) binding sites in these heterogenous tissues, using histo-autoradiographic analysis (HAA). The 125I-IGF1 binding was performed on frozen tissue sections and analysed using 3H Ultrofilm autoradiography coupled to computerised image analysis. Competitive binding experiments using unlabelled IGF1, IGF2 and insulin showed that the tissues exhibited typical type I IGF binding sites. This specificity was confirmed by the use of alpha IR-3 monoclonal antibody, as inhibitor of 125I-IGF1 binding. IGF1 binding sites were detected in 18 human primary breast cancers, 12 benign breast tumours and two normal breast tissues. Using HAA we found that the human breast carcinomas studied exhibit a specific and high binding capacity for 125I-IGF1 exclusively localised on the proliferative epithelial component. The 125I-IGF1 binding activity of benign breast tumours or normal breast tissue was significantly lower than in cancerous tissues. There was a significant correlation between IGF1-R concentrations detected with HAA and those detected with a classical biochemical method. Moreover, HAA could be useful in further detailing whether a tumour is IGF1-R positive or negative HAA appears to be a useful method for the detection of growth factor receptors, specially in small biopsy specimens.

Specific binding sites for growth hormone in cultured mouse thymic epithelial cells.

Growth Hormones bound specifically to murine Thymic epithelial cells, which represent the major component of thymic micro-environment and can be modulated by pituitary hormones. The Kds found with human growth hormone and bovine growth hormone were 0.14 and 0.27 nM with a Bmax 0.56 and 0.35 fmol/10(6) cells respectively. Competition experiment analysis showed ED50 of 0.24 nM for hGH, 0.46 nM for rGH, 0.71 nM for bGH, 11.8 nM for hPRL and 11.2 nM for oPRL. No specific binding of [125I]-oPRL was observed under the same conditions. Both hPRL and bGH showed a negative regulatory effect on the number of the hGH binding sites when incubated with the culture for three days. The presence of GH receptors on Thymic epithelial cells provides biochemical evidence for the effect of GH on thymic function.

GnRH complementary peptide antibodies: outcome in GnRH receptor immunoanalysis.

The aim of this study was to obtain gonadoptropin-releasing hormone (GnRH) receptor antibodies of high affinity for receptor immunoanalysis. According to the complementary peptide theory, complementary nucleic acid segments encode for the hormone ligand and the receptor binding site, respectively. On this premise, we used as immunogen, GnRH complementary peptide [N-terminal]Ser-Arg-Ala-Gln-Ser-Ile-Gly-Pro-Val-Leu conjugated with a carrier protein. High antibody titers were obtained in rabbits, rats and mice. Our antisera recognized the hydrophobic middle part of the GnRH complementary peptide. A band of protein with a molecular weight similar to that of the GnRH receptor (60 kDa) was specifically detected by immunoblot of solubilized rat pituitary membranes with the highest titering rabbit antiserum. In bioassays on sheep pituitary cells in vitro, some antisera inhibit basal or GnRH-induced LH secretion. In order to elicit antibodies of high affinity, we used a selective receptor assay on rat brain and pituitary sections where the ligand was the labeled agonist Des-Gly10-D-Ala6 GnRH. None of the highest titering antisera prevented the binding of such a high affinity ligand. The complementary peptide approach thus appears not to be optimal for obtaining high affinity antibodies against the GnRH receptor binding site.

GnRH receptors in rat brain, pituitary and testis; modulation following surgical and gonadotropin-releasing hormone agonist-induced castration.

The regulation of rat gonadotropin-releasing hormone (GnRH) receptors in male rat pituitary, hippocampus and testis was studied, in vivo, under steady-state conditions during treatment with D-Trp6 GnRH (triptorelin, slow-release form, at 300 micrograms/kg/month). GnRH receptors were characterized on tissue sections by quantitative autoradiography using 125I-GnRHa as a tracer. Castrating doses of triptorelin strongly down-regulated pituitary GnRH receptors (50% of reduction after 8 h, 80% on days 1-30); in contrast, only a transient decrease (20% at 8 h) was observed in the hippocampus with a rapid return to control levels. Triptorelin induced a marked (2-fold) increase in GnRH receptors in testicular interstitial tissue during 5 days with a return to control value by day 20. Administration of a GnRH antagonist (BIM 21009, 1 mg/kg/24 h) induced a rapid reduction of pituitary and testicular receptors to undetectable levels at 24 h, while hippocampal receptors were strongly reduced only. This indicates that GnRH receptors with similar pharmacology are differently controlled in various tissues and that brain receptors are likely to be also regulated by GnRH agonists and antagonists.

[Isolation of Corynebacterium JK from the blood of neutropenic patients with septic diseases]. / Corynebacterium JK isolálása neutropéniás szeptikus betegek véréböl.

The authors report on the isolation of Corynebacterium JK from the blood of two neutropenic patients with hematological malignancy. In Hungary this paper is the first to discuss the microbiology of the organism and clinical manifestations of the group JK Coryneform infection. The clinical significance of this organism in the nosocomial infections of compromised patients and the association of the infection with the use of plastic devices has been emphasized. The sensitivity of the multiresistant coryneform bacterium to vancomycin may help to make the microbiological diagnosis and to select the drug for therapy.

Meningitis caused by Gordona aurantiaca (Rhodococcus aurantiacus).

In a case of hairy cell leukemia, Gordona aurantiaca (Rhodococcus aurantiacus) was isolated from cerebrospinal fluid as the pathogen responsible for lethal infection of the central nervous system. The pathogen had been isolated previously from one case of pulmonary infection process only.