A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood.

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide's concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for (125)I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p <0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p = 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p = 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

A Cross-Species Analysis in Pancreatic Neuroendocrine Tumors Reveals Molecular Subtypes with Distinctive Clinical, Metastatic, Developmental, and Metabolic Characteristics.

UNLABELLED: Seeking to assess the representative and instructive value of an engineered mouse model of pancreatic neuroendocrine tumors (PanNET) for its cognate human cancer, we profiled and compared mRNA and miRNA transcriptomes of tumors from both. Mouse PanNET tumors could be classified into two distinctive subtypes, well-differentiated islet/insulinoma tumors (IT) and poorly differentiated tumors associated with liver metastases, dubbed metastasis-like primary (MLP). Human PanNETs were independently classified into these same two subtypes, along with a third, specific gene mutation-enriched subtype. The MLP subtypes in human and mouse were similar to liver metastases in terms of miRNA and mRNA transcriptome profiles and signature genes. The human/mouse MLP subtypes also similarly expressed genes known to regulate early pancreas development, whereas the IT subtypes expressed genes characteristic of mature islet cells, suggesting different tumorigenesis pathways. In addition, these subtypes exhibit distinct metabolic profiles marked by differential pyruvate metabolism, substantiating the significance of their separate identities. SIGNIFICANCE: This study involves a comprehensive cross-species integrated analysis of multi-omics profiles and histology to stratify PanNETs into subtypes with distinctive characteristics. We provide support for the RIP1-TAG2 mouse model as representative of its cognate human cancer with prospects to better understand PanNET heterogeneity and consider future applications of personalized cancer therapy.

Axon guidance factor SLIT2 inhibits neural invasion and metastasis in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) metastasizes by neural, vascular, and local invasion routes, which limit patient survival. In nerves and vessels, SLIT2 and its ROBO receptors constitute repellent guidance cues that also direct epithelial branching. Thus, the SLIT2-ROBO system may represent a key pinch point to regulate PDAC spread. In this study, we examined the hypothesis that escaping from repellent SLIT2-ROBO signaling is essential to enable PDAC cells to appropriate their local stromal infrastructure for dissemination. Through immunohistochemical analysis, we detected SLIT2 receptors ROBO1 and ROBO4 on epithelia, nerves, and vessels in healthy pancreas and PDAC specimens, respectively. SLIT2 mRNA expression was reduced in PDAC compared with nontransformed pancreatic tissues and cell lines, suggesting a reduction in SLIT2-ROBO pathway activity in PDAC. In support of this interpretation, restoring the SLIT2 expression in SLIT2-deficient PDAC cells inhibited their bidirectional chemoattraction with neural cells, and more specifically, impaired unidirectional PDAC cell navigation along outgrowing neurites in models of neural invasion. Restoring autocrine/paracrine SLIT2 signaling was also sufficient to inhibit the directed motility of PDAC cells, but not their random movement. Conversely, RNA interference-mediated silencing of ROBO1 stimulated the motility of SLIT2-competent PDAC cells. Furthermore, culture supernatants from SLIT2-competent PDAC cells impaired migration of endothelial cells (human umbilical vein endothelial cells), whereas an N-terminal SLIT2 cleavage fragment stimulated such migration. In vivo investigations of pancreatic tumors with restored SLIT2 expression demonstrated reduced invasion, metastasis, and vascularization, with opposing effects produced by ROBO1 silencing in tumor cells or sequestration of endogenous SLIT2. Analysis of clinical specimens of PDAC showed that those with low SLIT2 mRNA expression exhibited a higher incidence and a higher fraction of tumor-infiltrated lymph nodes. Taken together, our findings argue that disrupting SLIT2-ROBO signaling in PDAC may enhance metastasis and predispose PDAC cells to neural invasion.

Locking reconstruction double plating of distal humeral fractures: how many screws in the distal ulnar column segment in A3 fracture provide superior stability? A comparative biomechanical in vitro study.

OBJECTIVES: Two 90-degree configurations of locking reconstruction plates with different numbers of screws in the distal ulnar column segment of distal extra-articular humeral fractures with metaphyseal comminution (A3) were biomechanically investigated. METHODS: Eight pairs of fresh-frozen human humeri were used. For paired comparison, the humeri were divided into 2 randomized groups. In both groups, double-plate osteosyntheses with locking reconstruction plates were performed in 90-degree configurations. In group 1, the posteriorly placed radial column plate exceeded the capitellum and the ulnar column plate extended into the ulnar sulcus. The ulnar plate was molded around the medial epicondyle and fixed with 3 short angular stable screws distally. In group 2, the posteriorly placed radial column plate was applied analogous to group 1. The locking reconstruction plate placed on the ulnar column was used reaching to the ulnar epicondyle, fixed with 1 long, angular, stable screw in the distal fragment. Stiffness testing for axial load and bending in static and cyclic tests were performed. In static test mode, a load was applied with a frequency of 0.1 Hz. For cyclic loading conditions, a load was applied at 1 Hz for 5000 cycles. RESULTS: All tested specimens adequately resisted simulated physiologic loading conditions with no failure. Comparable stiffness values for axial load (P = 0.161) and significant lower stiffness values for bending (P = 0.017) in group 2 under static bending conditions were found. Considering cyclic loading conditions, no significant alterations in stiffness in each group under axial load occurred. In bending conditions, stiffness values for group 2 were significantly lower than that for group 1 (P = 0.036). CONCLUSIONS: Under static and cyclic bending conditions, stiffness in group 2 was significantly lower than that in group 1. Nevertheless, both implant configurations showed no failure of the constructs. Based on these data, when applying locked plates in the clinical setting, more than 1 locked screw applied into the distal ulnar column of the articular segment is recommended.

Structural plasticity of an acid-activated chaperone allows promiscuous substrate binding.

HdeA has been shown to prevent acid-induced aggregation of proteins. With a mass of only 9.7 kDa, HdeA is one of the smallest chaperones known. Unlike other molecular chaperones, which are typically complex, multimeric ATP-dependent machines, HdeA is known to undergo an acid-induced dimer to monomer transition and functions at low pH as a disordered monomer without the need for energy factors. Thus, HdeA must possess features that allow it to bind substrates and regulate substrate affinity in a small and energy-independent package. To understand better how HdeA accomplishes this, we studied the conformational changes that accompany a shift to low pH and substrate binding. We find that the acid-induced partial unfolding and monomerization that lead to HdeA activation occur very rapidly (k >3.5 s(-1)). Activation exposes the hydrophobic dimer interface, which we found to be critical for substrate binding. We show by intramolecular FRET that the partially unfolded character of active HdeA allows the chaperone to adopt different conformations as required for the recognition and high-affinity binding of different substrate proteins. These efficient adaptations help to explain how a very small protein is rapidly activated and can bind a broad range of substrate proteins in a purely pH-regulated manner.

Three-dimensional distribution of trabecular bone density and cortical thickness in the distal humerus.

BACKGROUND: One major barrier to osteosynthesis in distal humeral fractures is poor bone quality. This study was an attempt to measure the bone quality in the distal humerus. METHODS: We measured the distribution of total bone mineral density (BMD), trabecular BMD (tBMD), and cortical thickness (CTh) in the distal humerus using peripheral quantitive computed tomography. Four slices in the infracondylar, supracondylar, and distal disphyseal regions of 25 human cadaver humeri were investigated. RESULTS: Total BMD decreased continuously from the distal diaphysis to the trochlea. Within the infracondylar region, the capitellum was the region of lowest tBMD and CTh (P < .001). Measurements in anterior regions were higher than in most others (P < .001). The tBMD of the medial column in the infracondylar and supracondylar regions was 31% and 36% higher vs the lateral column (P < .001). The medial column had an average 22% higher CTh in the supracondylar and 38% higher CTh in distal diaphyseal regions vs the lateral sides (P < .001). CONCLUSIONS: Distal humeral bone properties vary widely, providing stronger bone stock on the medial side. This may improve understanding of implant failure and techniques in surgical treatment.

[Angle-fixed plate fixation or double-plate osteosynthesis in fractures of the proximal humerus: a biomechanical study]. / Osteosynthese am proximalen Humerus mittels winkelstabiler Platte oder Doppelplatte: eine vergleichende biomechanische Untersuchung.

Internal fixation of fractures of the proximal humerus needs a high stability of fixation to avoid secondary loss of fixation. This is especially important in osteoporotic bone. In an experimental study, the biomechanical properties of the angle-fixed Philos plate (internal fixator) and a double-plate osteosynthesis using two one-third tubular plates were assessed. The fracture model was an unstable three-part fracture (AO type B2). Eight pairs of human cadaveric humeri were submitted to axial load and torque. In the first part of the study, it was assessed to which degree the original stiffness of the humeri could be restored after the osteotomy by the osteosynthesis procedure. Subsequently, subsidence during 200 cycles of axial loading and torque was analysed. During axial loading, the Philos plate was significantly stiffer and showed less irreversible deformation. Two double-plate fixations, but none of the Philos plate osteosynthesis, failed. During torsion, there were no significant differences between the two implants. From the biomechanical point of view, the angle-fixed Philos plate represents the implant of choice for the surgical fixation of highly unstable three-part fractures of the proximal humerus, as the internal fixator system is characterised by superior biomechanical properties.

Mechanical comparison in cadaver specimens of three different 90-degree double-plate osteosyntheses for simulated C2-type distal humerus fractures with varying bone densities.

OBJECTIVES: To investigate the bone-implant-anchorage of 90-degree double-plate osteosynthesis in simulated complete intra-articular distal humerus fractures using conventional reconstruction plates (CRP), locking compression plates (LCP), and distal humerus plates (DHP), depending on the bone mineral density (BMD) of the cadaver specimens. METHODS: Groups (CRP, LCP, DHP, n=8; LCP, DHP, n=13) in distal humerus cadaver bones were created based on BMD. The fracture model was an unstable intraarticular distal humerus fracture with a transverse osteotomy gap representing metaphyseal comminution (AO type 13-C2.3). Flexion and extension stiffness as well as cycles until failure due to screw pullout under cyclic loading were evaluated. Estimates of BMD values, below which failure was likely to occur, were determined. RESULTS: Stiffness values were not significantly different between groups (extension: P=0.881, flexion: P=0.547). Under cyclic loading, consistent screw pullout failure occurred at BMD values below about 400 mg/cm for CRP and below about 300 mg/cm for LCP constructs. Comparing BMD-matched groups of 8 and 13 specimens respectively, the failure rate was significantly lower for the DHP (0/8) than for the CRP (5/8; P=0.026) and tended to be lower for the DHP (0/13) as compared to the LCP (4/13; P=0.096). CONCLUSION: Bone-implant anchorage was different between locking and nonlocking plate constructs and depended on BMD. While in good bone quality implant choice was not critical, both locking plates provided superior resistance against screw loosening as compared to the CRP at low BMD values (<420 mg/cm). Based on our laboratory results, we conclude that locking plates such as the LCP and DHP are constructs designed to keep anatomical reduction in the presence of comminution and poor bone quality in a low intra-articular fracture of the distal humerus.

Assessment of bone quality in the proximal humerus by measurement of the contralateral site: a cadaveric analyze.

INTRODUCTION: The presence of osteoporosis decreases the success of osteosynthesis, especially in the proximal humerus. Estimation of the bone mineral density (BMD) at the fracture site could aid in the decision making for surgical treatment and potential implant choice with regard to the individual bone properties. BMD measurement at a fracture site is prone to inaccuracies and alternative measurement sites need to be identified. In the case of a proximal humerus fracture, promising alternative measurement sites are at the same contralateral or at a different ipsilateral location. The aim of this study was to determine if the BMD for the humeral head can be predicted by BMD measurements from the ipsilateral distal humerus or the contralateral proximal humerus. MATERIAL AND METHODS: Cancellous BMD values were obtained from 88 paired human cadaver humeri (age 75.8+/-13.5 years) at the humeral head and at the distal metaphyseal area by pQCT. Correlations between BMD values of the ipsi- and contralateral sites were computed. RESULTS: Correlations between proximal and distal BMD values within one bone were moderate for both left (R2=0.37) and right humeri (R2=0.40). BMD comparison between left and right humeri revealed high correlations for both the distal (R2=0.90) and the proximal humerus (R2=0.74) (all P<0.01). Elderly specimen (>or=70 years) showed better intersite correlations between all regions than younger specimen (<70 years). CONCLUSION: High correlations between contralateral BMD values may be the result of similar biomechanical loading conditions. Although a relationship between proximal and distal bone quality of the same bone was found, the moderate coefficient suggests that ipsilateral measurements do not provide a good prediction of humeral head BMD. Bone quality at the humeral head is best predicted by BMD measurements at the contralateral location rather than the ipsilateral distal site.

Distal humerus fractures in elderly patients: results after open reduction and internal fixation.

PROBLEM: Fractures of the distal humerus are difficult to treat. In elderly patients, diminished bone mineral quality and increased trauma-associated joint destruction may make stable joint reconstruction even more problematic. Furthermore, comorbidities and poor tolerance of joint immobilization might be additional factors which influence elbow function negatively. Until now, disagreement has existed on how to treat these fractures in elderly patients. Recommendations range from conservative treatment to primary total elbow replacement. So far, reports in the literature on whether or not open reduction and internal fixation in these patients is justified are very rare. AIM OF THE STUDY: To analyze fracture patterns, surgical approach, complications, and functional results after open reduction and internal fixation in patients of age 60 years and older. PATIENTS AND METHODS: Retrospective clinical study of two university level 1 trauma centers, including 45 patients (median age 73 years; range, 61-92 years) with surgically treated distal humerus fractures. Fracture patterns were recorded according to their AO classification. All patients were treated by open reduction and internal fixation. A clinical and radiological follow-up was obtained after a minimum of 24 months following surgery (median 87 months; range, 24-121 months). Functional results were evaluated according to the Mayo Elbow Score. RESULTS: Fractures with complete joint involvement were seen most often. Taking the fracture type into consideration, functional results deteriorated with degree of joint involvement. Postoperative complication rate was high, predominantly seen as screw loosening and/or implant failure at the lateral column. Neverthless, functional results were preponderating good or excellent. Factors negatively influencing outcome were joint immobilization longer than 14 days and severe joint involvement. DISCUSSION: In elderly patients, distal humerus fractures, which are often considered "osteoporotic fractures," still remain one of the most demanding challenges in trauma surgery. The present study demonstrates that despite diminished bone quality and a high complication rate, open reduction and internal fixation in elderly patients is justified. CONCLUSION: Open reduction and internal fixation of distal humerus fractures in elderly patients should be the main goal, since good elbow function can be achieved in the majority of patients. Elbow immobilization longer than 14 days should be avoided. Stable implant anchorage at the lateral column remains problematic, reflecting a general potential for further implant improvements.

Spontaneous defect remodeling in a distal humerus fracture with extensive osseous loss: a case report of a complex elbow fracture.

Sideswipe injuries to the elbow cause complex fracture patterns and remain difficult management problems. The authors report a case of spontaneous bone remodeling of an extensive metaphyseal defect zone.

A biomechanical evaluation of methods of distal humerus fracture fixation using locking compression plates versus conventional reconstruction plates.

OBJECTIVES: To examine the biomechanical behavior of 2 techniques of double-plate osteosynthesis for fractures of the adult distal humerus using conventional reconstruction plates and locking compression plates. DESIGN: Basic science study. SETTING: Experimental in vitro study. PATIENTS/PARTICIPANTS: Forty fresh-frozen human distal humeri specimens. INTERVENTION: Four matched groups with 10 humeri each, median age 74 years (46-95), were created using similar bone mineral density values. Two standard configurations of double-plate osteosynthesis (dorsal or 90 degrees configuration) with either conventional reconstruction plates or locking compression plates were studied for biomechanical properties of the constructs. A fracture model with a 5-mm supracondylar osteotomy gap simulating metaphyseal comminution (AO type 13-A3.3) was used. MAIN OUTCOME MEASUREMENT: Stiffness testing of the constructs in anterior/posterior bending, torsion, and axial compression loading. Evaluation of alterations of the bone-implant interface and failure patterns under cyclic loading and strength testing. RESULTS: The study demonstrates that primary stiffness in anterior/posterior bending and torsional loading is significantly increased by using locking compression plates in a 90 degrees configuration (P < 0.05) as compared with dorsally applied plates. The differences between the different plate types are insignificant if applied in the same configuration. It is demonstrated that none of the tested implants failed under cyclic loading within the number of cycles expected for 3 months of use. The bone-implant interface is less likely to fail during strength testing with locking compression plates. CONCLUSION: The biomechanical behavior of the osteosynthesis depends more on plate configuration than plate type. Advantages of locking compression plates are only significant if compared with dorsal plate application techniques. Nevertheless, locking compression plates are helpful supplementary tools for achieving primary stable fracture fixation. This might be of considerable clinical relevance in patients with diminished bone mineral quality or in the presence of metaphyseal comminution.

Where should implants be anchored in the humeral head?

To determine histomorphometric and bone strength distribution of the proximal humerus, analyses were done on 24 freshly harvested human cadaveric humeri. Median ages of 46 and 69 years were recorded respectively for the male group (n = 11; minimum, 34 years; maximum, 76 years) and the female group (n = 13; minimum, 46 years; maximum, 90 years). The humeral head was sliced into four equal horizontal levels (Levels 1-4). Five regions of interest were defined in each cutting plane: anterior, posterior, lateral, medial, and central. Histomorphometric analyses evaluated structural parameters (tissue volume to bone volume ratio, trabecular thickness), connectivity (number of nodes, node to node length), and trabecular orientation (mean bone length). The peak values of histomorphometric parameters and bone strength were identified for the cranial section and decreased caudally. The medial and dorsal aspects of the proximal humeral head were found to be the areas of highest bone strength. The trabecular network formed a pattern that connected the center of the gleaned cavity. The structural and connectivity parameters, bone strength, and trabecular orientation showed region- and level-related characteristics. Knowledge of distribution, microstructure, and quality of bone in the humeral head allows the remaining bone stock to be used effectively, even in elderly patients, with a minimally invasive approach and maximum mechanical stability.

The LCP-concept in the operative treatment of distal humerus fractures--biological, biomechanical and surgical aspects.

Distal humerus fractures in adults are treated by open reduction and internal fixation, which produces good results in the majority of patients. However, in elderly patients or in cases with metaphyseal comminution, stable fracture fixation still remains problematic. Anatomical joint reconstruction and primary stable osteosynthesis are often particularly difficult to achieve in this group of patients since poor bone mineral quality is frequently encountered. Consequently, longer immobilization is necessary, which is known to negatively influence functional outcome. Over the past few years, double-plate osteosynthesis techniques using different configurations have become the treatment of choice. Nevertheless, complications due to inappropriate primary stability and/or implant failure have been described. Investigations on whether the recently introduced Locking Compression Plates (LCPs) could enhance primary stability are rare. On the basis of clinical and biomechanical experiences, the authors consider LCPs a helpful tool for increasing primary stability in osteosynthesis of distal humerus fractures. LCPs might be of substantial advantage in patients with diminished bone mineral quality or in the presence of metaphyseal comminution.

Anterosuperior labrum lesions of the shoulder joint: pathogenesis, arthroscopic treatment, and results.

The anterosuperior labrum lesion of the shoulder without biceps anchor involvement is a rare injury, and it is unclear whether the refixation has advantages over resection. We describe the pathophysiology and treatment, and compare the functional outcome of these procedures of refixation and resection in 21 patients (median follow-up 6 months, range 5-16). The labrum was refixated in 11 cases. Clinical evaluation used the Constant score. Synovialitis in the area of the labral tear was detected in all cases. In refixation patients there was a significant postoperative improvement in Constant score (91.5 vs. 70). Débridement showed a greater increase in the Constant score range (92 vs. 48). An anterosuperior labrum lesion without involvement of the biceps anchor is a separate entity. The pathology is the consequence of degenerative labral lesions without loss of stability in the glenohumeral joint. In view of this background and the results of this study refixation of loose labral tears in anterosuperior labral lesions without loss of stability should not considered.