Erythropoietin improves skin wound healing and activates the TGF-β signaling pathway

We could recently report that erythropoietin (EPO) accelerates skin wound healing in mice. Now, we provide insight into the molecular mechanisms of this non-hematopoietic property of EPO analyzing the transforming growth factor (TGF)-β signaling pathway. EPO receptor was found expressed in both non-wounded and wounded skin tissue as well as in fibroblasts and keratinocytes. In saline-treated control animals, wounds exhibited a significant upregulation of TGF-β1 and of α-smooth muscle actin (α-SMA) compared with non-wounded skin. EPO treatment accelerated wound epithelialization and induced mRNA expression of TGF-β1 and α-SMA. In addition, EPO significantly enhanced phosphorylation of Smad2 and Smad3 in fibroblasts and also elevated phosphorylation of Smad3 in wound tissue. Blockade of TGF-β using a neutralizing anti-TGF-β antibody attenuated EPO-induced acceleration of wound epithelialization in vivo and markedly reversed EPO effects on mRNA expression of TGF-β1 and α-SMA. In conclusion, EPO caused activation of the Smad-dependent TGF-β signaling pathway, enhanced differentiation of myofibroblasts, and accelerated skin wound closure.     

Antibody-binding epitope differences in the nucleoprotein of avian and mammalian influenza A viruses

Abstract Influenza virus nucleoprotein (NP) binds to the viral genome RNA and forms the internal ribonucleoprotein complex of the virus particle. Avian and human influenza virus NP have characteristic differences at several amino acid positions. It is not known whether any of these differences can be recognized by antibodies. In the present study five monoclonal antibodies (MAbs) were produced against NP of A/Duck/Novosibirsk/56/05 (H5N1) influenza virus. Two MAbs discerned human and avian influenza strains on ELISA testing. The NP expressed in a prokaryotic system was used for the analysis of site-specific mutants carrying amino acid substitutions in the relevant positions. Amino acid residues in positions 100 and 101 were shown to be recognized by the MAbs. The residue in position 100 is host-specific, and its recognition by the MAb 2E6 may be useful for the differentiation of human and avian viruses. The data are discussed in view of the effects of amino acid substitutions in influenza virus NP affecting both host range and antibody-binding specificity.     

Accurate determination of brain metabolite concentrations using ERETIC as external reference

Magnetic Resonance Spectroscopy (MRS) can provide in vivo metabolite concentrations in standard concentration units if a reliable reference signal is available. For ¹H MRS in the human brain, typically the signal from the tissue water is used as the (internal) reference signal. However, a concentration determination based on the tissue water signal most often requires a reliable estimate of the water concentration present in the investigated tissue. Especially in clinically interesting cases, this estimation might be difficult. To avoid assumptions about the water in the investigated tissue, the Electric REference To access In vivo Concentrations (ERETIC) method has been proposed. In this approach, the metabolite signal is compared with a reference signal acquired in a phantom and potential coil‐loading differences are corrected using a synthetic reference signal. The aim of this study, conducted with a transceiver quadrature head coil, was to increase the accuracy of the ERETIC method by correcting the influence of spatial B₁ inhomogeneities and to simplify the quantification with ERETIC by incorporating an automatic phase correction for the ERETIC signal. Transmit field ( ) differences are minimized with a volume‐selective power optimization, whereas reception sensitivity changes are corrected using contrast‐minimized images of the brain and by adapting the voxel location in the phantom measurement closely to the position measured in vivo. By applying the proposed B₁ correction scheme, the mean metabolite concentrations determined with ERETIC in 21 healthy subjects at three different positions agree with concentrations derived with the tissue water signal as reference. In addition, brain water concentrations determined with ERETIC were in agreement with estimations derived using tissue segmentation and literature values for relative water densities. Based on the results, the ERETIC method presented here is a valid tool to derive in vivo metabolite concentration, with potential advantages compared with internal water referencing in diseased tissue.     

Comprehensive Assessment of Current Management Strategies for Patients With Diabetes and Chronic Limb-Threatening Ischemia

Chronic limb-threatening ischemia (CLTI) is the most severe form of peripheral artery disease. It is estimated that 60% of all nontraumatic lower-extremity amputations performed annually in the United States are in patients with diabetes and CLTI. The consequences of this condition are extraordinary, with substantial patient morbidity and mortality and high socioeconomic costs. Strategies that optimize the success of arterial revascularization in this unique patient population can have a substantial public health impact and improve patient outcomes. This article provides an up-to-date comprehensive assessment of management strategies for patients afflicted by both diabetes and CLTI.

More than 30 million Americans have diabetes and are presumably at higher risk of developing peripheral arterial disease (PAD) (1,2). Advanced PAD can manifest as chronic limb-threatening ischemia (CLTI), which is defined as limb pain at rest and/or the presence of ischemic ulceration or gangrene (3,4). CLTI affects ∼2 million Americans >40 years of age and is associated with higher risk of limb loss due to above-ankle (major) amputations (5–10). It is estimated that 60% of all nontraumatic lower-extremity amputations performed annually in the United States are in patients with diabetes and CLTI (11,12). These procedures are associated with substantial morbidity, considerable mortality, and high socioeconomic costs.On a per-patient basis, the cost of treating CLTI in patients with diabetes is higher than the treatment of both coronary artery disease (CAD) and cerebrovascular disease (13–15). These increased costs are likely the result of higher rates of hospital admissions, procedures, and complications. CLTI and its significant financial burdens are anticipated to increase as the preva-lence of diabetes continues to increase globally from 450 million living with diabetes in 2017 to an estimated 700 million by 2045 (16).Accordingly, strategies that optimize successful revascularization in patients with diabetes and CLTI can have a substantial public health impact and improve patient outcomes. Here, we review the medical and modern surgical management strategies for patients with diabetes and CLTI. We specifically reviewed studies with cohorts that were at least 50% patients with diabetes or CLTI, had subanalyses relevant for patients with diabetes or CLTI, and reported standard clinical outcomes relevant to patients with severe PAD.     

Deficits in motor abilities and developmental fractionation of imitation performance in high-functioning autism spectrum disorders

The co-occurrence of motor and imitation disabilities often characterises the spectrum of deficits seen in patients with autism spectrum disorders (ASD). Whether these seemingly separate deficits are inter-related and whether, in particular, motor deficits contribute to the expression of imitation deficits is the topic of the present study and was investigated by comparing these deficits’ cross-sectional developmental trajectories. To that end, different components of motor performance assessed in the Zurich Neuromotor Assessment and imitation abilities for facial movements and non-meaningful gestures were tested in 70 subjects (aged 6–29 years), including 36 patients with high-functioning ASD and 34 age-matched typically developed (TD) participants. The results show robust deficits in probands with ASD in timed motor performance and in the quality of movement, which are all independent of age, with one exception. Only diadochokinesis improves moderately with increasing age in ASD probands. Imitation of facial movements and of non-meaningful hand, finger, hand finger gestures not related to social context or tool use is also impaired in ASD subjects, but in contrast to motor performance this deficit overall improves with age. A general imitation factor, extracted from the highly inter-correlated imitation tests, is differentially correlated with components of neuromotor performance in ASD and TD participants. By developmentally fractionating developmentally stable motor deficits from developmentally dynamic imitation deficits, we infer that imitation deficits are primarily cognitive in nature.     

SENSE imaging with a quadrature half-volume transverse electromagnetic (TEM) coil at 4T

PURPOSE: To demonstrate the feasibility of high-field SENSE imaging of large objects, such as the human head, using a semicircular (half-volume) coil for both transmission and multi-channel reception. MATERIALS AND METHODS: As a proof of concept, we present experimental data obtained using a seven-element half-volume (180 degrees of arc) transmit/receive quadrature transverse electromagnetic (TEM) coil. SENSE images of the human brain were acquired with a reduction factor of R=2, using two degenerate linear modes of the same coil as independent receive channels at 4T. Since the need for additional hardware (i.e., a separate set of receive coils) is eliminated, the design can be substantially simplified. RESULTS: The experimental data demonstrate that linear modes of the half-volume TEM coil have essentially no noise correlation, and their sensitivity profiles satisfy the requirement for small g-factors. Also, this type of coil provides efficient transmission with a relatively large uniform region and a reception profile that is more uniform than that of the surface coils. CONCLUSION: We demonstrate the feasibility of SENSE imaging using a half-volume coil. Half-volume coils allow reduced total power deposition compared to full-volume coils, and may replace the latter in body imaging applications in which the target region of interest (ROI) is smaller than the entire torso.     

Release of pig leukocytes and reduced human NK cell recruitment during ex vivo perfusion of HLA‐E/human CD46 double‐transgenic pig limbs with human blood

Background

In pig‐to‐human xenotransplantation, interactions between human natural killer (NK) cells and porcine endothelial cells (pEC) are characterized by recruitment and cytotoxicity. Protection from xenogeneic NK cytotoxicity can be achieved in vitro by the expression of the non‐classical human leukocyte antigen‐E (HLA‐E) on pEC. Thus, the aim of this study was to analyze NK cell responses to vascularized xenografts using an ex vivo perfusion system of pig limbs with human blood.

Methods

Six pig forelimbs per group, respectively, stemming from either wild‐type (wt) or HLA‐E/hCD46 double‐transgenic (tg) animals, were perfused ex vivo with heparinized human blood for 12 hours. Blood samples were collected at defined time intervals, cell numbers counted, and peripheral blood mononuclear cells analyzed for phenotype by flow cytometry. Muscle biopsies were analyzed for NK cell infiltration. In vitro NK cytotoxicity assays were performed using pEC derived from wt and tg animals as target cells.

Results

Ex vivo, a strong reduction in circulating human CD45 leukocytes was observed after 60 minutes of xenoperfusion in both wt and tg limb groups. NK cell numbers dropped significantly. Within the first 10 minutes, the decrease in NK cells was more significant in the wt limb perfusions as compared to tg limbs. Immunohistology of biopsies taken after 12 hours showed less NK cell tissue infiltration in the tg limbs. In vitro, NK cytotoxicity against hCD46 single tg pEC and wt pEC was similar, while lysis of double tg HLA‐E/hCD46 pEC was significantly reduced. Finally, circulating cells of pig origin were observed during the ex vivo xenoperfusions. These cells expressed phenotypes mainly of monocytes, B and T lymphocytes, NK cells, as well as some activated endothelial cells.

Conclusions

Ex vivo perfusion of pig forelimbs using whole human blood represents a powerful tool to study humoral and early cell‐mediated rejection mechanisms of vascularized pig‐to‐human xenotransplantation, although there are several limitations of the model. Here, we show that (i) transgenic expression of HLA‐E/hCD46 in pig limbs provides partial protection from human NK cell‐mediated xeno responses and (ii) the emergence of a pig cell population during xenoperfusions with implications for the immunogenicity of xenografts.