Global and local characterization explains the different mechanisms of failure of the human ribs.

Knowledge of the mechanics and mechanistic reasons inducing rib fracture is fundamental for forensic investigations and for the design of implants and cardiopulmonary resuscitation devices. A mechanical rationale to explain the different rib mechanisms of failure is still a challenge. The aim of this work was to experimentally characterize human ribs to test the hypothesis that a correlation exists between the ribs properties and the mechanism of failure. 89 ribs were tested in antero-posterior compression. The full-field strain distribution was measured through Digital Image Correlation. The fracture load ranged 7-132 N. Two main different mechanisms of failure were observed: brittle and buckling. The strain analysis showed that the direction of principal strains was either aligned with the ribs, or oblique, around 45°, with a rather uniform direction in the most strained area. The maximum principal strains were in the range between 1000 and 30000 microstrain and the minimum principal strain between -30000 and -800 microstrain. The ribs undergoing brittle fracture had significantly thicker cortical bone than those undergoing buckling. Also, larger tensile strains were observed in the specimens with brittle fracture than in the buckling ones. These findings support the focus of cortical thickness modelling which could help in sharpening computational models for the aforesaid purposes.

Effects Induced by Osteophytes on the Strain Distribution in the Vertebral Body Under Different Loading Configurations.

The mechanical consequences of osteophytes are not completely clear. We aimed to understand whether and how the presence of an osteophyte perturbs strain distribution in the neighboring bone. The scope of this study was to evaluate the mechanical behavior induced by the osteophytes using full-field surface strain analysis in different loading configurations. Eight thoracolumbar segments, containing a vertebra with an osteophyte and an adjacent vertebra without an osteophyte (control), were harvested from six human spines. The position and size of the osteophytes were evaluated using clinical computed tomography imaging. The spine segments were biomechanically tested in the elastic regime in different loading configurations while the strains over the frontal and lateral surface of vertebral bodies were measured using digital image correlation. The strain fields in the vertebrae with and without osteophytes were compared. The correlation between osteophyte size and strain alteration was explored. The strain fields measured in the vertebrae with osteophytes were different from the control ones. In pure compression, we observed a mild trend between the size of the osteophyte and the strain distribution (R 2 = 0.32, p = 0.15). A slightly stronger trend was found for bending (R 2 = 0.44, p = 0.075). This study suggests that the osteophytes visibly perturb the strain field in the nearby vertebral area. However, the effect on the surrounding bone is not consistent. Indeed, in some cases the osteophyte shielded the neighboring bone, and in other cases, the osteophyte increased the strains.

Type, size, and position of metastatic lesions explain the deformation of the vertebrae under complex loading conditions.

BACKGROUND: Bone metastases may lead to spine instability and increase the risk of fracture. Scoring systems are available to assess critical metastases, but they lack specificity, and provide uncertain indications over a wide range, where most cases fall. The aim of this work was to use a novel biomechanical approach to evaluate the effect of lesion type, size, and location on the deformation of the metastatic vertebra. METHOD: Vertebrae with metastases were identified from 16 human spines from a donation programme. The size and position of the metastases, and the Spine Instability Neoplastic Score (SINS) were evaluated from clinical Quantitative Computed Tomography images. Thirty-five spine segments consisting of metastatic vertebrae and adjacent healthy controls were biomechanically tested in four different loading conditions. The strain distribution over the entire vertebral bodies was measured with Digital Image Correlation. Correlations between the features of the metastasis (type, size, position and SINS) and the deformation of the metastatic vertebrae were statistically explored. RESULTS: The metastatic type (lytic, blastic, mixed) characterizes the vertebral behaviour (Kruskal-Wallis, p = 0.04). In fact, the lytic metastases showed more critical deformation compared to the control vertebrae (average: 2-fold increase, with peaks of 14-fold increase). By contrast, the vertebrae with mixed or blastic metastases did not show a clear trend, with deformations similar or lower than the controls. Once the position of the lytic lesion with respect to the loading direction was taken into account, the size of the lesion was significantly correlated with the perturbation to the strain distribution (r2 = 0.72, p < 0.001). Conversely, the SINS poorly correlated with the mechanical evidence, and only in case of lytic lesions (r2 = 0.25, p < 0.0001). CONCLUSION: These results highlight the relevance of the size and location of the lytic lesion, which are marginally considered in the current clinical scoring systems, in driving the spinal biomechanical instability. The strong correlation with the biomechanical evidence indicates that these parameters are representative of the mechanical competence of the vertebra. The improved explanatory power compared to the SINS suggests including them in future guidelines for the clinical practice.

Reconstruction of proximal humeral fractures without screws using a reinforced bone substitute.

Multi-fragment fractures are still a challenge: current clinical practice relies on plates and screws. Treatment of fractures of the proximal humerus has the intra-operative risk of articular damage when inserting multiple screws. Distal-varus collapse of the head is a frequent complication in osteoporotic patients. The aim of this biomechanical study was to investigate if an Innovative-cement-technique (the screws are replaced by injection of cement) provides the same or better stability of the reconstructed head compared to the Standard-technique (locking screws). A four-fragment fracture was simulated in twelve pairs of humeri, with removal of part of the cancellous bone to simulate osteoporotic "eggshell" defect. One humerus of each pair was repaired either with a Standard-technique (locking plate, 2 cortical and 6 locking screws), or with the Innovative-cement-technique (injection of a partially-resorbable reinforced bone substitute consisting of PMMA additivated with 26% beta-TCP). Cement injection was performed both in the lab and under fluoroscopic monitoring. The reconstructed specimens were tested to failure with a cyclic force of increasing amplitude. The Innovative-cement-technique withstood a force 3.57 times larger than the contralateral Standard reconstructions before failure started. The maximum force before final collapse for the Innovative-cement-technique was 3.56 times larger than the contralateral Standard-technique. These differences were statistically significant. The Innovative-cement-technique, based on the reinforced bone substitute, demonstrated better biomechanical properties compared to the Standard-technique. These findings, along with the advantage of avoiding the possible complications associated with the locking screws, may help safer and more effective treatment in case of osteoporotic multi-fragment humeral fractures.

Dual tropism of HIV-1 envelopes derived from renal tubular epithelial cells of patients with HIV-associated nephropathy.

The phenotype of HIV-1 gp120 envelope derived from renal epithelium and peripheral blood mononuclear cells (PBMC) of patients with HIV-associated nephropathy was investigated in vitro. Chimeric viruses were derived from kidney or blood and used to infect primary CD4+T cells, cell lines expressing single co-receptors and a renal epithelial cell line HPT-1. HIV-1 variants derived from renal epithelium were dual tropic whereas simultaneously derived viruses from PBMC were R5-tropic. Utilization of alternative co-receptors CCR3, BONZO and BOB, also differed.

Lentiviral gene delivery to CNS by spinal intrathecal administration to neonatal mice.

BACKGROUND: Direct injection of lentivectors into the central nervous system (CNS) mostly results in localized parenchymal transgene expression. Intrathecal gene delivery into the spinal canal may produce a wider dissemination of the transgene and allow diffusion of secreted transgenic proteins throughout the cerebrospinal fluid (CSF). Herein, we analyze the distribution and expression of LacZ and SEAP transgenes following the intrathecal delivery of lentivectors into the spinal canal. METHODS: Four weeks after intrathecal injection into the spinal canal of newborn mice, the expression of the LacZ gene was assessed by histochemical staining and by in situ polymer chain reaction (PCR). Following the spinal infusion of a lentivector carrying the SEAP gene, levels of enzymatically active SEAP were measured in the CSF, blood serum, and in brain extracts. RESULTS: Intrathecal spinal canal delivery of lentivectors to newborn mice resulted in patchy, widely scattered areas of beta-gal expression mostly in the meninges. The transduction of the meningeal cells was confirmed by in situ PCR. Following the spinal infusion of a lentivector carrying the SEAP gene, sustained presence of the reporter protein was detected in the CSF, as well as in blood serum, and brain extracts. CONCLUSIONS: These findings indicate that intrathecal injections of lentivectors can provide significant levels of transgene expression in the meninges. Unlike intracerebral injections of lentivectors, intrathecal gene delivery through the spinal canal appears to produce a wider diffusion of the transgene. This approach is less invasive and may be useful to address those neurological diseases that benefit from the ectopic expression of soluble factors impermeable to the blood-brain barrier.

Genetic analysis of HIV by in situ PCR-directed laser capture microscopy of infected cells.

Behind the exponential expansion of the acquired immunodeficiency syndrome (AIDS) epidemic, there is a continuous and progressive molecular evolution of human immunodeficiency virus (HIV)-1. In this regard, the molecular analysis of viral strains infecting several anatomic compartments in humans has become critical to understanding AIDS-related pathologies and to improving emerging therapeutic protocols. Laser capture microdissection provides outstanding results in the genetic analysis of HIV-1 variants detectable in AIDS patients. The ability of the instrument to microdissect infected cells from a heterogeneous tissue compartment allows the investigator to obtain critical information regarding the genetic nature of a specific viral strain. To perform laser capture microdissection with better accuracy, a priori detection techniques may provide useful information about HIV distribution in the tissue specimen. An in situ polymerase chain reaction (PCR) assay on a serial slide results in a detailed map of the viral infection specific for the case under analysis. The knowledge of HIV distribution in the tissue section is critical for improving the dissection of infected cells by laser capture microscopy. This chapter describes laser capture microdissection and in situ PCR and its role in the analysis of the genetic nature of HIV-1 variants and quasispecies.

Homophilic interaction of NTBA, a member of the CD2 molecular family: induction of cytotoxicity and cytokine release in human NK cells.

NK-T-B antigen (NTBA) is a CD2 family member that functions as a coreceptor in human NK cell activation. Several receptor/ligand interactions occur between different members of this molecular family. In this study, in order to identify the natural ligand of NTBA, we produced a chimeric protein formed by the NTBA extracellular region fused with the Fc portion of human IgG1 (termed NTBA-Fc*). NTBA-Fc* specifically binds to NTBA cell transfectants but not to cells transfected with other CD2 family members including CD2, CD48, CD84, CD150, CD229, and CD244. Moreover, NTBA-Fc* also binds to NTBA(+) but not to NTBA(-) T cell lines. Enzyme-linked immunosorbent assays, plasmon resonance analysis, as well as NTBA-Fc*-mediated down-regulation of NTBA surface expression further confirmed the occurrence of NTBA/NTBA homophilic interaction. Functionally, in NK cells, NTBA-Fc* promoted a strong production of IFN-gamma and TNF-alpha. Moreover, NTBA-transfected targets displayed increased susceptibility to NK-mediated killing as compared to untransfected cells and this effect was specifically inhibited by anti-NTBA mAb. Altogether our data indicate that NTBA is characterized by self recognition.

Podocan, a novel small leucine-rich repeat protein expressed in the sclerotic glomerular lesion of experimental HIV-associated nephropathy.

Growing evidence suggests that human immunodeficiency virus (HIV)-1 infection of podocytes plays a central role in the glomerular disease of HIV-associated nephropathy (HIVAN). As an approach to identify host genes involved in the pathogenesis of the sclerotic glomerular lesion in HIVAN, representational difference analysis of cDNA was used to identify differentially expressed genes in HIV-1 transgenic and nontransgenic podocytes. We isolated a novel member of the small leucine-rich repeat (SLR) protein family, podocan, that is expressed at high levels in the HIV-1 transgenic podocytes. In normal embryonic kidney, a 3.2-kb podocan transcript was detected at low levels, and expression increased dramatically within 24 h following birth. Expression of a 2.3-kb transcript became evident after birth and gradually increased to 50% of the total podocan RNA in the mature kidney. Phylogenetically, podocan represents a new class in the SLR protein gene family, an expanding protein family sharing homology with the small leucine-rich repeat proteoglycans. The 3.2-kb transcript encodes a predicted 611-amino acid secretory protein with 20 leucine-rich repeats, a unique N-terminal cysteine-rich cluster pattern and a highly acidic C-terminal domain. In situ hybridization of normal kidney revealed podocan mRNA expression in podocytes and likely vascular endothelial cells within the glomerulus. The immunohistochemical staining pattern of podocan protein in normal kidney glomeruli was consistent with that of the glomerular basement membrane, and staining was markedly increased in sclerotic glomerular lesions in the transgenic HIVAN model. Thus, podocan defines a new class within the SLR protein family and is a previously unrecognized component of the sclerotic glomerular lesion that develops in the course of experimental HIVAN.

Replication and compartmentalization of HIV-1 in kidney epithelium of patients with HIV-associated nephropathy.

HIV-associated nephropathy is a clinicopathologic entity that includes proteinuria, focal segmental glomerulosclerosis often of the collapsing variant, and microcystic tubulointerstitial disease. Increasing evidence supports a role for HIV-1 infection of renal epithelium in the pathogenesis of HIV-associated nephropathy. Using in situ hybridization, we previously demonstrated HIV-1 gag and nef mRNA in renal epithelial cells of patients with HIV-associated nephropathy. Here, to investigate whether renal epithelial cells were productively infected by HIV-1, we examined renal tissue for the presence of HIV-1 DNA and mRNA by in situ hybridization and PCR, and we molecularly characterized the HIV-1 quasispecies in the renal compartment. Infected renal epithelial cells were removed by laser-capture microdissection from biopsies of two patients, DNA was extracted, and HIV-1 V3-loop or gp120-envelope sequences were amplified from individually dissected cells by nested PCR. Phylogenetic analysis of kidney-derived sequences as well as corresponding sequences from peripheral blood mononuclear cells of the same patients revealed evidence of tissue-specific viral evolution. In phylogenetic trees constructed from V3 and gp120 sequences, kidney-derived sequences formed tissue-specific subclusters within the radiation of blood mononuclear cell-derived viral sequences from both patients. These data, along with the detection of HIV-1-specific proviral DNA and mRNA in tubular epithelium cells, argue strongly for localized replication of HIV-1 in the kidney and the existence of a renal viral reservoir.

Lentiviral gene transduction of kidney.

Gene transfer into kidney holds great potential as a novel therapeutic approach. We have studied the transduction of kidney in vivo after delivery of lentiviral vectors by various routes of administration. A lentiviral vector expressing the bacterial lacZ gene from the cytomegalovirus early promoter was used. The lentiviral vector was delivered into the kidneys of BALB/c mice by retrograde infusion into the ureter, by injection into the renal vein or artery, or by direct injection into the renal parenchyma. Expression of the reporter gene was achieved independently of the route of administration, although it appeared more efficient after parenchymal or ureteral administration. After parenchymal or ureteral infusion, expression of the transgene was localized to the outer medulla and corticomedullary junction. In the case of parenchymal injection, expression of the reporter gene extended to the cortex. Detection of the transgene in the renal proximal tubules was confirmed by in situ polymerase chain reaction after parenchymal or ureteral infusion. On delivery of the lentiviral vector through the renal artery or vein, expression of the reporter gene was markedly lower than was observed with parenchymal or ureteral infusion and was limited to the inner medullary collecting ducts. No apparent histological abnormality was observed after virus administration and transgene expression was stable for at least 3 months. These results provide the first evidence that lentiviral vectors can stably transduce renal cells in vivo and may be effective vehicles for gene delivery to the kidney.

In vivo gene transfer to kidney by lentiviral vector.

BACKGROUND: The growing understanding of the molecular basis of renal diseases makes the development of gene therapy for kidney disorders a potential treatment alternative. Work aimed at determining the feasibility and the efficiency of gene transfer to the kidney using different viral and nonviral transduction systems is a necessary component to understanding the full potential. Lentiviral vectors have been shown to transduce stably different tissues and cell types that are refractory to other gene transfer approaches. To date, the potential of lentiviral vectors to transfer genes in kidney has not been investigated. The scope of this work was to analyze the efficiencies of in vivo transduction of kidney by a lentiviral vector. METHODS: A pseudotyped lentiviral vector carrying the gene for the enhanced green fluorescent protein (EGFP) was delivered into one kidney of experimental mice by retrograde infusion through the ureter. The presence of the virus and the expression of the reporter protein were monitored over time. RESULTS: Both viral DNA and EGFP expression were measurable in the kidney infused with the lentiviral vector but not in the contralateral kidney. Protein expression was detected by immunostaining, as EGFP fluorescence was masked by the high background fluorescence of the kidney. Expression of EGFP persisted for the entire two-month duration of the experiments. CONCLUSIONS: Lentiviral vectors can effectively deliver exogenous genes to the kidney in vivo, resulting in persistent expression of the introduced gene.