Clinical implementation of artificial-intelligence-assisted detection of breast cancer metastases in sentinel lymph nodes: the CONFIDENT-B single-center, non-randomized clinical trial.

Pathologists' assessment of sentinel lymph nodes (SNs) for breast cancer (BC) metastases is a treatment-guiding yet labor-intensive and costly task because of the performance of immunohistochemistry (IHC) in morphologically negative cases. This non-randomized, single-center clinical trial (International Standard Randomized Controlled Trial Number:14323711) assessed the efficacy of an artificial intelligence (AI)-assisted workflow for detecting BC metastases in SNs while maintaining diagnostic safety standards. From September 2022 to May 2023, 190 SN specimens were consecutively enrolled and allocated biweekly to the intervention arm (n = 100) or control arm (n = 90). In both arms, digital whole-slide images of hematoxylin-eosin sections of SN specimens were assessed by an expert pathologist, who was assisted by the 'Metastasis Detection' app (Visiopharm) in the intervention arm. Our primary endpoint showed a significantly reduced adjusted relative risk of IHC use (0.680, 95% confidence interval: 0.347-0.878) for AI-assisted pathologists, with subsequent cost savings of ~3,000 €. Secondary endpoints showed significant time reductions and up to 30% improved sensitivity for AI-assisted pathologists. This trial demonstrates the safety and potential for cost and time savings of AI assistance.

Interleukin-2-dependent mechanisms are involved in the development of glomerulosclerosis after partial renal ablation in rats.

BACKGROUND: Glomerulosclerosis is a common feature of many end-stage renal diseases. The contribution of cellular immune mechanisms has been implicated in the development of glomerulosclerosis. We investigated whether the inhibition of lymphocyte activation influences this process in an established rat model of renal hyperfiltration. METHODS: After removal of two-thirds of their respective kidney mass, rats were treated with either tacrolimus (0.08 mg/kg/day) or vehicle until the end of the study (n = 10/group). The rats were pair-fed and proteinuria was assessed regularly. Twenty weeks after nephrectomy, creatinine clearance and systemic blood pressure were determined, and kidneys were harvested for morphological, immunohistological and PCR analysis. RESULTS: In control animals, renal function started to decline from week 12, as indicated by an elevated proteinuria. Interleukin (IL)-2 and IL-2 receptor synthesis was upregulated in control animals and inhibited by tacrolimus treatment. Transforming growth factor-beta (TGF-beta(1)), platelet-derived growth factor-AA (PDGF-AA) and macrophage chemoattractant protein-1 (MCP-1) mRNA levels were upregulated in control animals, but were significantly lower in immunosuppressed hosts. Additionally, tacrolimus treatment resulted in a significant reduction of proteinuria. Morphological analysis supported these functional results; glomerular sclerosis, tubular atrophy and intimal proliferation were more pronounced in controls than in the tacrolimus group. These morphological parameters were accompanied by reduced infiltration of CD5+ (rat T-cell marker) T cells, ED1+ (rat macrophage marker) macrophages, and less intense staining for laminin and fibronectin. CONCLUSION: A continuous treatment with tacrolimus - an inhibitor of lymphocyte proliferation - reduced the pace of glomerulosclerosis in the remnant kidney.

Predicting the effects of actions: interactions of perception and action.

Many theories in cognitive psychology assume that perception and action systems are clearly separated from the cognitive system. Other theories suggest that important cognitive functions reside in the interactions between these systems. One consequence of the latter claim is that the action system may contribute to predicting the future consequences of currently perceived actions. In particular such predictions might be more accurate when one observes one's own actions than when one observes another person's actions, because in the former case the system that plans the action is the same system that contributes to predicting the action's effects. In the present study participants (N = 104) watched video clips displaying either themselves or somebody' else throwing a dart at a target board and predicted the dart's landing position. The predictions were more accurate when participants watched themselves acting. This result provides evidence for the claim that perceptual input can be linked with the action system to predict future outcomes of actions.

The extent of traumatic damage determines a graded depression of the endotoxin responsiveness of peripheral blood mononuclear cells from patients with blunt injuries.

OBJECTIVE: To study whether the endotoxin responsiveness of peripheral blood mononuclear cells correlates with the severity of injury in trauma patients. DESIGN: Prospective, observational study. SETTING: University trauma center. PATIENTS: Fifty-nine patients with blunt trauma (Injury Severity Score [ISS] 4 to 57 points). INTERVENTIONS: Standard emergency department care, surgical care, and postoperative intensive care unit treatment. MEASUREMENTS AND MAIN RESULTS: Whole blood and serum were obtained 94+/-89 (SD) mins post trauma (day 0) and during a 14-day period postinjury. Endotoxin-induced tumor necrosis factor-alpha (TNF-alpha) synthesis of peripheral blood mononuclear cells ex vivo was tested using a whole blood assay. Serum samples were assayed for TNF-alpha concentrations. A reduced capacity of whole blood to produce TNF-alpha ex vivo with endotoxin treatment was found to be closely correlated with the ISS. The capacity to produce TNF-alpha on endotoxin stimulation of whole blood from patients with an ISS > or =16 points was depressed immediately after trauma and did not reach normal values during the observation period. In patients with an ISS >22 points, maximum depression of the capacity of whole blood to produce TNF-alpha occurs within 100 mins post injury. In contrast, in patients with an ISS <22 points, maximal depression of whole blood TNF-alpha production occurs with a delay of 24 to 48 hrs after trauma. Based on pre- and postoperative values, primary surgical intervention caused a decrease of the endotoxin-stimulated TNF-alpha production of whole blood in the latter patient subgroup, as well as in the entire patient population (ISS 4 to 57) when secondary surgical treatment was necessary 5 to 13 days after trauma. CONCLUSIONS: The extent of traumatic tissue damage leads to a graded depression of immunocyte function and appears to be amplified by surgical treatment. The endotoxin responsiveness of peripheral blood mononuclear cells displays a functional marker of the anatomically defined severity of injury and gives insights into the regulation of immunocyte function after severe blunt trauma.

Relation of ex vivo stimulated blood cytokine synthesis to post-traumatic sepsis.

The cytokine production in endotoxin stimulated blood of patients immediately after polytrauma with high risk for developing sepsis or multi organ failure was analysed. Forty patients sustaining traumatic injury with >/=317 pts according to the Injury Severity Score (ISS), 10 of whom developed severe sepsis (ACCP/SCCM conference 1992) were included in the study. Levels of interleukin 8 (IL-8), IL-6 and tumour necrosis factor (TNF) were measured by ELISA in endotoxin-stimulated whole blood and IL-10 and IL-6 in serum. The allotype for the bi-allelic Nco I restriction length polymorphism in the TNF locus was determined for each patient.Two to four hours after polytrauma endotoxin-stimulated synthesis of TNF and IL-6 was found to be reduced in whole blood from patients compared to healthy donors, whereas no such differences were found for IL-8 synthesis. At this time, however, the patients who developed sepsis at a later stage (day 4-6) showed significantly (P<0.05) enhanced IL-8 synthesis in endotoxin stimulated whole blood in comparison to healthy donors. The IL-6 and TNF production of their blood was significantly enhanced compared to patients with uncomplicated recovery. Ninety per cent of the patients developing sepsis were of the TNFB2/TNFB2 allotype, whereas this was the case for only 30% of the non-septic group. Assessment of endotoxin-stimulated cytokine synthesis may provide a prognostic indicator for patients at high risk for developing a sepsis syndrome.

Analysis of intragraft cytokine expression during early reperfusion after liver transplantation using semi-quantitative RT-PCR.

In the present study the intergraft mRNA formation immediately before and after transplantation of human livers was investigated by semi-quantitative polymerase chain reaction. The analysis was carried out with mRNA isolated from biopsies routinely taken perioperatively and included the determination of the expression of tumour necrosis factor alpha (TNF-alpha), interleukin (IL-6), IL-8, IL-10, transforming growth factor beta (TGF-beta) and beta-actin. It was found that biopsies obtained 30-60 min after reperfusion of the liver graft contained significantly higher levels of mRNA for TNF-alpha, IL-6 and IL-8 than biopsies collected subsequently to cold preservation. No such differences were obtained for TGF-beta and IL-10 mRNA. Considerable interindivdual differences were observed concerning the degree of inducibility, in particular for IL-6 mRNA. Retrospective comparison with the clinical course of the individual patients revealed a close and statistically significant correlation between low IL-6 expression and the occurrence of acute rejection episodes within 30 postoperative days, while high IL-6 mRNA levels coincided with the absence of rejection signs. High values for TNF-alpha mRNA were associated with ensuing acute rejection episodes.

Expression of HSP 70 as a potential prognostic marker for acute rejection in human liver transplantation.

In experimental models, the synthesis of heat shock protein 70 (HSP 70) has been recognized as an intracellular response to ischemia and reperfusion, insults inherent to transplantation. In this study, the HSP response in early stages of human liver transplantation was investigated. HSP 70 mRNA expression was detected by means of reverse transcriptase (RT)-PCR in liver biopsies (n = 28) and in cells obtained from the organ perfusate (n = 14) following cold preservation. The expression of HSP 70 differed substantially between individuals. Retrospective analysis revealed a close correlation of the amount of HSP 70 mRNA in perfusate cells and biopsies with the onset of organ dysfunction due to early graft rejection. Patients with early graft rejection had a significantly lower amount of HSP 70 mRNA than patients without rejection. These results suggest a protective role of HSP 70 expression. Low levels of HSP 70 may, therefore, represent a prognostic marker for early graft rejection.

[Modification of immune response after polytrauma and cardiopulmonary bypass by hematopoietic growth factors]. / Beeinflussung der Immunantwort nach Polytrauma und cardiopulmonalem Bypass durch hämatopoetische Wachstumsfaktoren.

Cardiac surgery and polytrauma result in an impaired immune response as it can be demonstrated by a reduced endotoxin-stimulated TNF alpha production of whole blood cultures ex vivo. The immune-stimulating hematopoetic growth factor GM-CSF is in vitro capable to antagonize the suppressed immune function after trauma and cardiac surgery and, therefore, GM-CSF represents a potential therapeutic for immune suppressed states.

[Endotoxin tolerance and hemorrhagic shock in rats]. / Endotoxintoleranz und hämorrhagischer Schock bei Ratten.

The aim of the presents study was to investigate the protective capacity of endotoxin tolerance in a hemorrhagic shock model in rats. A pretreatment with low dose endotoxin induces a state of tolerance, which is characterized by decreased TNF alpha production in vivo and in vitro upon subsequent high dose endotoxin challenge. This endotoxin tolerance improves survival after hemorrhagic shock from 22.8% in untreated controls to 68.8 in tolerant rats. The protection was accompanied with the appearance of n TNF alpha inhibitory activity in the serum of endotoxin tolerant animals, which might be responsible for the improved survival after hemorrhagic shock.

Regulation of whole blood tumor necrosis factor production upon endotoxin stimulation after severe blunt trauma.

BACKGROUND: Trauma has been recognized to be accompanied by alterations of leukocyte functions such as cytokine release. The regulatory principles involved in these changes are still poorly defined. To further characterize leukocyte function after multiple trauma, endotoxin-stimulated tumor necrosis factor (TNF) production of trauma patients' whole blood and a possible regulatory mechanism were studied. METHODS: Endotoxin responsiveness in trauma patients (n = 18, Injury Severity Score = 24 +/- 7) was assayed ex vivo using a whole blood model. TNF release and TNFalpha mRNA levels were determined during a 14-day period. Furthermore, the influence of patients' sera on whole blood TNF production was evaluated. MAIN RESULTS: The capacity of trauma patients' whole blood to produce TNF was reduced for 2 to 6 days after trauma and was equally evident for both TNF release and TNFalpha mRNA levels. The reduction of TNF coincides with the appearance of an inhibitory activity for TNF production in trauma patients' sera. No correlation was found between the inhibitory activity and soluble TNF receptors, endotoxin-neutralizing molecules, inhibitory cytokines (interleukin 10 and transforming growth factor beta), or prostaglandins. CONCLUSIONS: Major trauma leads to the appearance of a circulating inhibitory activity for TNF synthesis that may potentially contribute to an anti-inflammatory response in patients with multiple trauma. The elucidation of its structural and functional properties may contribute to the understanding of the pathogenesis of severely injured patients.

Organization of the 5'-End of the porcine gamma-glutamyl transpeptidase gene and identification of three different mRNAs in the kidney.

Three different mRNAs coding for the porcine gamma-glutamyl transpeptidase (GGT) in the kidney were identified by 5'-RACE-PCR. These differ in their 5'-noncoding region. Genomic Southern blot analysis has demonstrated the existence of a single GGT gene in the porcine genome. Thus, the existence of multiple mRNAs can only be explained by the use of different promoters or alternative splicing. Four GGT-specific genomic clones containing the complete 5'-end of the gene were isolated and characterized, revealing six exons common to all three mRNAs. Four of these exons were located in the coding region comprising the codons for amino acids 1 to 138. Two exons and an intervening sequence were identified upstream from these six common exons representing the unique 5'-ends of the three mRNAs. The coding exons show a significant sequence homology to mouse, rat, and human GGT cDNA, whereas exons 1 and 3 display no homology.

Multiple signal transduction pathways regulate clusterin (gp 80) gene expression in MDCK cells.

Clusterin (gp 80, apolipoprotein J, TRPM-2) is a widely expressed multifunctional glycoprotein. Its demonstrated and proposed functions include the transport of lipids and membrane fragments, the inhibition of the cytolytic action of the terminal complement complex and the modulation of cell-cell interactions. The expression of the gene is enhanced during tissue injury and remodelling and by hormone-withdrawal-induced apoptosis of prostate and mammary cells. We show here that, in the kidney-derived epithelial cell line MDCK, clusterin mRNA is repressed by glucocorticoids and by progesterone. Treatment with epidermal growth factor also represses clusterin gene expression in MDCK cells. Incubation with 12-O-tetradecanoyl-phorbol-13-acetate, which activates protein kinase C (PKC), induces clusterin mRNA, while chelerythrine, an inhibitor of PKC, represses clusterin gene expression, suggesting that the clusterin gene responds to signalling pathways involving PKC. These results open up the possibility of studying the complex regulation of the clusterin gene by multiple signal transduction pathways within a single cell type, and most importantly, of characterizing interactions between the individual signal transduction cascades.

Clusterin gene expression in apoptotic MDCK cells is dependent on the apoptosis-inducing stimulus.

Clusterin (Apolipoprotein J, complement lysis inhibitor) is a widely expressed multifunctional glycoprotein. The expression of clusterin mRNA has been reported to be elevated in a broad spectrum of apoptotic or degenerative tissues. More recently, it was shown that within these tissues clusterin is expressed in the surviving rather than in the dying cells, and that clusterin gene expression is actually down-regulated in the apoptotic cells. We have studied the expression of the clusterin gene in apoptotic MDCK cells. Cell death was initiated by three different stimuli: application of the steroid hormone antagonist RU 486, activation of protein kinase C by the application of the phorbol ester TPA, and--since clusterin is involved in lipid and cholesterol transport--perturbation of cell membranes by cholesterol. We show that clusterin gene expression is repressed in cells undergoing apoptosis in response to the application of RU 486 and TPA, but is unchanged in cells in which apoptosis has been triggered by cholesterol treatment.

Expression of apolipoprotein A-I in porcine brain endothelium in vitro.

Apolipoprotein (apo) A-I is the major protein component of high-density lipoproteins (HDLs), which are responsible for reverse cholesterol transport from peripheral tissues to the liver. A low level of plasma HDL is correlated with susceptibility to atherosclerosis and coronary heart disease. Mammalian apo A-I synthesis has been attributed mainly to liver and intestine. Recently, apo A-I expression has been shown in porcine brain capillaries, suggesting an independent lipid metabolism within the brain. In this study, protein synthesis and secretion were investigated in primary cultures of porcine brain microvascular endothelial cells and compared with those in large vessel endothelium. Active protein synthesis in vitro was demonstrated by metabolic labeling. Cerebral endothelial cells were shown to secrete apo A-I into the culture supernatant, whereas aortic endothelial cells were negative for apo A-I expression. Further studies of transcriptional regulation showed that cerebral endothelium was responsive to apo A-I-inducing agents, such as cholesterol, insulin, and retinoic acid, as previously shown in human hepatoma HepG2 cells. Thus, cultures of porcine cerebral endothelial cells may represent a suitable model for physiological studies of apo A-I-regulation with regard to brain lipid metabolism and blood-brain barrier function. To investigate the interspecies conservation of regulatory elements, 178 bp of the 5' flanking region of the porcine apo A-I gene was cloned using PCR techniques. Alignments of the cDNA, of the deduced apo A-I protein sequence, and of the 5' promoter region with the corresponding genomic sequences of different species show a high degree of similarity between the porcine and the primate apo A-I genes, thus indicating a similar function and possibly common regulatory mechanisms in those species. In contrast, the rodent and avian apolipoprotein A-I promoter sequences differed significantly.

Pericytes of the brain microvasculature express gamma-glutamyl transpeptidase.

The expression of gamma-glutamyl transpeptidase (GGT) is a specific property of the brain capillary endothelium that constitutes the blood-brain barrier. We report here the detection of GGT, not only in endothelial cells, but also in pericytes, demonstrating that a brain capillary-specific pericyte population exists. We raised antibodies to GGT using a porcine brain microvessel GGT-protein-A (staphylococcal protein A) fusion protein as antigen which was expressed in Escherichia coli. The immunohistochemical analysis of the subcapillary distribution of GGT in porcine brain cortex and cerebellum sections by both light and electron microscopy revealed the expression of GGT in the capillary-adjacent pericytes in addition to the GGT-positive endothelial layer. We confirmed these data for cultured porcine brain microvascular endothelial cells and pericytes. GGT immunofluorescence could be detected in both cell types in culture. Endothelial cells exhibited a weak staining, whereas pericytes were strongly positive for GGT. Due to the high phagocytotic activity of pericytes and their location on the abluminal surface of the microvessels, we propose a possible protective or detoxifying function of GGT in cerebrovascular pericytes.

Application of pressure scanning to the tuning of a high resolution dye laser.

Advantages of pressure scanning over other methods of tuning the wavelength of dye lasers are discussed. The construction of a pressure scanned high resolution dye laser is described. The laser has a scan range of more than 40 cm(-1) in its intermediate resolution mode, an improvement on the order of a hundredfold over previous methods of tuning. Preliminary tests have indicated a scan range of 4 cm(-1) in the high resolution mode. As a demonstration of its applicability and versatility, the laser was used to xesolve the ground state splitting of the chromium isotopes in ruby.