Experimental and mathematical modelling of magnetically labelled mesenchymal stromal cell delivery.

A key challenge for stem cell therapies is the delivery of therapeutic cells to the repair site. Magnetic targeting has been proposed as a platform for defining clinical sites of delivery more effectively. In this paper, we use a combined in vitro experimental and mathematical modelling approach to explore the magnetic targeting of mesenchymal stromal cells (MSCs) labelled with magnetic nanoparticles using an external magnet. This study aims to (i) demonstrate the potential of magnetic tagging for MSC delivery, (ii) examine the effect of red blood cells (RBCs) on MSC capture efficacy and (iii) highlight how mathematical models can provide both insight into mechanics of therapy and predictions about cell targeting in vivo. In vitro MSCs are cultured with magnetic nanoparticles and circulated with RBCs over an external magnet. Cell capture efficacy is measured for varying magnetic field strengths and RBC percentages. We use a 2D continuum mathematical model to represent the flow of magnetically tagged MSCs with RBCs. Numerical simulations demonstrate qualitative agreement with experimental results showing better capture with stronger magnetic fields and lower levels of RBCs. We additionally exploit the mathematical model to make hypotheses about the role of extravasation and identify future in vitro experiments to quantify this effect.

Anxiety, depression and treatment adherence among HIV-infected migrants.

Diagnosing symptoms of psychological distress can be challenging in migrants living with HIV (MLWH) living in Western Europe. We evaluated the Hospital Anxiety and Depression Scale (HADS) as a screening tool for psychological distress. Additionally, the association between psychological distress and adherence to combination Antiretroviral Therapy (cART) was determined. Socio-demographic and clinical characteristics, psychosocial variables, and self-reported adherence to cART data were collected. 306/352 participants completed the HADS. A HADS+ (≥15, at risk for psychological distress) was found in 106/306. The Composite International Diagnostic Interview (CIDI) was completed by 60/106. The HADS was repeated in 58 participants as the time between the first HADS and the CIDI was more than three months. In 21/37 participants with a HADS+ (57%) within three months before the CIDI a diagnosis of depression or anxiety disorder based on the CIDI was found. Participants with a HADS+ were more likely to be non-adherent (71.3% vs. 43.6%). In a large group of MLWH in the Netherlands, 35% were at risk for symptoms of psychological distress. The HADS seems to be a suitable screening tool for MLWH.

Reducing resource utilization during non-operative treatment of pediatric proximal humerus fractures.

INTRODUCTION: The majority of proximal humeral fractures in the skeletally immature are treated non-operatively. Operative indications vary but are largely based on degree of displacement. Non-union is rare. Non-operatively treated fractures are typically monitored radiographically to assess healing. HYPOTHESIS: We hypothesize that the decision to treat fractures operatively is made at the time of first imaging and that follow-up X-rays do not lead to a change in management. MATERIAL AND METHODS: We retrospectively reviewed the records of 239 patients treated for proximal humerus fractures over a 5-year period. There were 225 who were treated non-operatively. Records were reviewed for the number of clinic visits and radiographs, as well as any change to operative management based on follow-up X-rays. RESULTS: The primary outcome of the study was the proportion of proximal humerus fractures, initially treated non-operatively, for which displacement or angulation on follow-up radiographs led to a change to operative treatment. Secondary outcomes were the number of follow-up radiographs obtained after the initial diagnosis and initiation of non-operative treatment. Of the 225 patients that were initially managed non-operatively, only 1 patient required subsequent surgical management. This patient underwent a proximal humerus epiphysiodesis 365 days from injury after development of a partial growth arrest. The mean number of fracture clinic visits for patients managed non-operatively was 2.67 (±1.24). The mean number of radiology department visits and radiographs obtained was 3.57 (±1.44) and 8.36 (±3.89) respectively. No clinical or radiographic non-unions were identified in these patients. No patients suffered a refracture during the review period. DISCUSSION: This study shows that of the 239 uncomplicated pediatric proximal humerus fractures treated at our hospital over a 5-year period, only 1 had a change in treatment plan, from non-operative to operative, based on follow-up radiographs. These data suggest that non-operative treatment of proximal humerus fractures seldom results in displacement that warrants operative intervention. Moreover, they suggest that there is little utility to the routine use of postoperative radiographs in follow-up of these patients. STUDY DESIGN: Retrospective case series. LEVEL OF EVIDENCE: IV.

Cryoballoon ablation of paroxysmal atrial fibrillation within the dilated coronary sinus in a case of persistent left superior vena cava.

Trigger sources of paroxysmal atrial fibrillation (PAF) are not limited to a pulmonary vein origin and may be achievable by cardiac vascular structures like the coronary sinus (CS), the vena cava superior and in some rare cases by a persistent left superior vena cava (LSVC). Cryoballoon ablation has been shown to be effective in pulmonary vein isolation. We report an unusual case of using this technique in the dilated CS in case of a persistent LSVC. A 64 year old patient presented PAF recurrences after cryo pulmonary vein isolation 4 months before. A maintaining pulmonary vein isolation could be demonstrated by transseptal mapping. Further bi-atrial mapping localized repetitive atrial trigger activity in a dilated CS proceeding to a LSVC. A cryoballoon was deployed in the CS target area and during cryoablation the triggered activity suspended. Ablation side effects were excluded by coronary angiography. During a follow up time of 8 months the patient has remained free of PAF recurrences. The current report underlines the importance of a patient-tailored ablation approach. Cryothermic balloon technology may be more applicable in delicate cardiac structures by developing new anatomically adapted balloon shapes and sizes.

Tyrosine sulfation of glycoprotein I(b)alpha. Role of electrostatic interactions in von Willebrand factor binding.

Glycoprotein I(b)alpha (GP I(b)alpha), the ligand binding subunit of the platelet glycoprotein Ib-IX-V complex, is sulfated on three tyrosine residues (Tyr-276, Tyr-278, and Tyr-279). This posttranslational modification is known to be critical for von Willebrand factor (vWF) binding; yet it remains unclear whether it provides a specific structure or merely contributes negative charges. To investigate this issue, we constructed cell lines expressing GP I(b)alpha polypeptides with the three tyrosine residues converted to either Glu or Phe and studied the ability of these mutants to bind vWF in the presence of modulators or shear stress. The mutants were expressed normally on the cell surface as GP Ib-IX complexes, with the conformation of the ligand-binding domain preserved, as judged by the binding of conformation-sensitive monoclonal antibodies. In contrast to their normal expression, both mutants were functionally abnormal. Cells expressing the Phe mutant failed to bind vWF in the presence of either ristocetin or botrocetin. These cells adhered to and rolled on immobilized vWF only when their surface receptor density was increased to twice the level that supported adhesion of cells expressing the wild-type receptor and even then only 20% as many rolled and rolled significantly faster than wild-type cells. Cells expressing the Glu mutant, on the other hand, were normal with respect to ristocetin-induced vWF binding and adhesion to immobilized vWF but were markedly defective in botrocetin-induced vWF binding. These results indicate that GP I(b)alpha tyrosine sulfation influences the interaction of this polypeptide with vWF primarily by contributing negative charges under physiological conditions and when the interaction is induced by ristocetin but contributes a specific structure to the botrocetin-induced interaction.

Novel gain-of-function mutations of platelet glycoprotein IBalpha by valine mutagenesis in the Cys209-Cys248 disulfide loop. Functional analysis under statis and dynamic conditions.

Platelet-type von Willebrand disease is a bleeding disorder resulting from gain-of-function mutations of glycoprotein (GP) Ibalpha that increase its affinity for von Willebrand factor (vWf). The two known naturally occurring mutations, G233V and M239V, both enrich the valine content of an already valine-rich region within the Cys(209)-Cys(248) disulfide loop. We tested the effect of converting other non-valine residues in this region to valine. Of 10 mutants expressed in CHO cells as components of GP Ib-IX complexes, four displayed a gain-of-function phenotype (G233V, D235V, K237V, and M239V) based on (125)I-vWf binding and adhesion to immobilized vWf. The remainder displayed loss-of-function phenotypes. The gain-of-function mutants bound vWf spontaneously and had a heightened response to low concentrations of ristocetin or botrocetin, whereas the loss-of-function mutants bound vWf more poorly than wild-type GP Ibalpha. No distinct gain- or loss-of-function conformations were identified with conformation-sensitive antibodies. Compared with cells expressing wild-type GP Ibalpha, cells expressing the gain-of-function mutants rolled significantly more slowly over immobilized vWf under flow than wild-type cells and were able to adhere to vWf coated at lower densities. In aggregate, these data indicate that the region of GP Ibalpha bounded by Asn(226) and Ala(244) regulates the affinity for vWf.

Necessity of conserved asparagine residues in the leucine-rich repeats of platelet glycoprotein Ib alpha for the proper conformation and function of the ligand-binding region.

The polypeptides of the platelet von Willebrand factor (vWf) receptor, the GP Ib-IX-V complex, each contain tandem repeats of a sequence that assigns them to the leucine-rich repeat protein family. Here, we studied the role of conserved Asn residues in the leucine-rich repeats of GP Ib alpha, the ligand-binding subunit of the complex. We replaced the Asn residue in the sixth position of the first or sixth leucine-rich repeat (of seven) either with a bulky, charged Lys residue or with a Ser residue (sometimes found in the same position of other leucine-rich repeats) and studied the effect of the mutations on complex expression, modulator-dependent vWf binding, and interactions with immobilized vWf under fluid shear stress. As predicted, the Lys substitutions yielded more severe phenotypes, producing proteins that either were rapidly degraded within the cell (mutant N158K) or failed to bind vWf in the presence of ristocetin or roll on immobilized vWf under fluid shear stress (mutant N41K). The binding of function-blocking GP Ib alpha antibodies to the N41K mutant was either significantly reduced (AK2 and SZ2) or abolished (AN51 and CLB-MB45). Ser mutations were tolerated much better, although both mutants demonstrated subtle defects in vWf binding. These results suggest a vital role for the conserved asparagine residues in the leucine-rich repeats of GP Ib alpha for the structure and functions of this polypeptide. The finding that mutations in the first leucine-rich repeat had a much more profound effect on vWf binding indicates that the more N-terminal repeats may be directly involved in this interaction.

Mutation in the leucine-rich repeat of platelet glycoprotein Ib alpha results in defects in its interaction with immobilized von Willebrand factor under flow.

OBJECTIVE: To characterize effects of the GP Ib alpha mutation (A156V) on its interaction with von Willebrand factor (vWf) under high fluid shear stress. METHODS: The residue A156 of GP Ib alpha was converted to a valine and the mutant expressed in CHO cells expressing wild-type GP Ib beta and GPIX. The transfected cells were tested for their interaction with a panel of GP Ib alpha antibodies and for rolling on immobilized vWf under high shear. RESULTS: The mutation led to surface expression of a GP Ib alpha polypeptide that adopted a different conformation at its N-terminus because binding of the GP Ib alpha antibody AN51, which has a binding epitope in the N-terminal 35 residues, was eliminated, whereas binding of the others (AK2, MB45, and SZ2, all of which bind to regions C-terminal to the AN51 epitope) was normal. Mutant-expressing cells could adhere and roll on immobilized vWf under high fluid shear stress and rolled significantly faster than wild-type cells. CONCLUSION: These studies demonstrate that the mutation A156V results in a conformation change at the N-terminus of GP Ib alpha, which leads to an increase in the dissociation rate of the bond between the GP Ib alpha mutant and vWf.

[A mutation of platelet glycoprotein I balpha results in defects in its interaction with immobilized vWF].

OBJECTIVE: To study the importance of glycoprotein (GP) I balpha mutation (A156V) in interaction between mutant expressing cell and immobilized vWF under fluid shear stress. METHODS: Mutant GP I balpha cDNA was cloned into the EcoR I site of the mammalian expression vector pDX, mutant cDNA was then transfected into CHO betaIX cells. Human vWF was purified from blood cryoprecipitate by glycine and NaCl precipitation and subsequent separation on sepharose 4B column. The purified vWF was immobilized onto a coverslip. Cell rolling was induced in a parallel-plate flow chamber and observed by phase-contrast video microscope. RESULTS: CHO cells expressing GP I b-IX-V complex could adhere to and roll on immobilized vWF. The A156V mutant cells retained the ability to adhere and roll on vWF matrix, but the rolling speed was significantly faster than that of wild type cells, indicating that the off-rate of the ligand-receptor bond between the mutant and vWF was impaired. Binding of monoclonal antibody AN51 to mutant GP I balpha decreased significantly, indicating that the A156V mutation altered the conformation of the N-terminal ligand-binding region of GP I balpha. CONCLUSION: The mutant GP I balpha has a faster off-rate for its interaction with immobilized vWF. The mutant polypeptide adopts an altered conformation in N-terminal ligand-binding region of GP I balpha. The parallel-plate flow chamber is an extremely useful system in evaluating interaction between GP I balpha and vWF.

The glycoprotein Ib-IX-V complex is a platelet counterreceptor for P-selectin.

We have identified platelet glycoprotein (GP) Ibalpha as a counterreceptor for P-selectin. GP Ibalpha is a component of the GP Ib-IX-V complex, which mediates platelet adhesion to subendothelium at sites of injury. Cells expressing P-selectin adhered to immobilized GP Ibalpha, and GP Ibalpha-expressing cells adhered to and rolled on P-selectin and on histamine-stimulated endothelium in a P-selectin-dependent manner. In like manner, platelets rolled on activated endothelium, a phenomenon inhibited by antibodies to both P-selectin and GP Ibalpha. Unlike the P-selectin interaction with its leukocyte ligand, PSGL-1 (P-selectin glycoprotein ligand 1), the interaction with GP Ibalpha required neither calcium nor carbohydrate core-2 branching or alpha(1,3)-fucosylation. The interaction was inhibited by sulfated proteoglycans and by antibodies against GP Ibalpha, including one directed at a tyrosine-sulfated region of the polypeptide. Thus, the GP Ib-IX-V complex mediates platelet attachment to both subendothelium and activated endothelium.

Bidirectional effect of interleukin-10 on early murine B-cell development: stimulation of flt3-ligand plus interleukin-7-dependent generation of CD19(-) ProB cells from uncommitted bone marrow progenitor cells and growth inhibition of CD19(+) ProB cells.

B-cell commitment and early development from multipotent hematopoietic progenitor cells has until recently been considered to be dependent on direct interaction with stromal cells. We recently showed that the flt3 ligand (FL) has a unique ability to interact with interleukin-7 (IL-7) to directly and selectively promote B-cell development from murine bone marrow progenitor cells with a combined myeloid and lymphoid potential. Here we report that whereas IL-10 alone has no ability to stimulate growth of primitive (Lin-Sca-1(+)c-kit+) bone marrow progenitor cells, it potently enhances FL + IL-7-induced proliferation (sevenfold). This enhanced proliferation results from recruitment of progenitors unresponsive to FL + IL-7 alone, as well as from increased growth of individual clones, resulting in a 7,000-fold cellular expansion over 12 days. Single cell cultures and delayed addition studies suggested that the stimulatory effect of IL-10 was directly mediated on the progenitor cells. The cells generated in response to FL + IL-7 + IL-10 appeared to be almost exclusively proB cells, as shown by their expression of B220, CD24, CD43, and lack of expression of c mu, myeloid, erythroid, and T-cell surface antigens. Although IL-10 also enhanced kit ligand (KL) + IL-7-induced proliferation of Lin-Sca-1(+)c-kit+ progenitor cells, the resulting cells were predominantly myeloid progeny. Accordingly, FL + IL-7 + IL-10 was 100-fold more efficient in stimulating production of proB cells than KL + IL-7 + IL-10. In contrast to its ability to stimulate the earliest phase of proB cell formation and proliferation, IL-10 inhibited growth of proB cells generated in response to FL + IL-7. Analysis of CD19 expression on cells generated in FL + IL-7 + IL-10 showed that almost all cells generated under these conditions lacked expression of CD19, in contrast to cells generated in the absence of IL-10, which were predominantly CD19(+). Replating of sorted CD19(+) and CD19(-) proB cells in FL + IL-7 or FL + IL-7 + IL-10 showed that IL-10 efficiently blocked growth of CD19(+), but not CD19(-) cells. Both CD19(-) and CD19(+) cells expressed lambda5 and VpreB , shown to be specific for B-cell progenitors. In addition, sorted CD19(-) cells generated CD19(+) cells in response to FL + IL-7. Thus, IL-10 has a dual regulatory effect on early B-cell development from primitive murine bone marrow progenitor cells in that it enhances FL + IL-7-induced proB-cell formation and growth before acquisition of CD19 expression, whereas growth of CD19(+) proB cells is inhibited.

[Immune response against modified low-density lipoproteins in patients with non-insulin-dependent diabetes mellitus]. / Respuesta inmune contra lipoproteínas de baja densidad modificadas en pacientes con diabetes mellitus no-insulino dependiente.

BACKGROUND: Diabetes mellitus is a risk factor for atherosclerosis. Low density lipoproteins are considered a key factor in the formation of atheroma and the immune system has an important contribution to this process. AIM: To quantify the immune response against modified low density lipoproteins in patients with non insulin dependent diabetes mellitus. MATERIAL AND METHODS: LDLs obtained from blood of healthy subjects, were glycated or altered with malondialdehyde and used as antigens. Serum autoantibodies against these LDLs were measured by ELISA in 22 patients with non insulin dependent diabetes mellitus aged 46 to 67 years old and 13 healthy controls aged 41 to 65 years old. Basal and LDL stimulated tumor necrosis factor a production in vitro, by peripheral leukocytes of diabetics and controls was also measured. RESULTS: The ratio of glycated LDL/native LDL antibodies was higher in diabetics than in controls (9.37 +/- 2.72 and 0.41 +/- 0.11 respectively p < 0.05) and the ratio of MDA modified LDL/native LDL antibodies was not significantly different (8.64 +/- 3.83 and 2.14 +/- 1.26 respectively, NS). Tumor necrosis or production by leukocytes was higher in diabetics than in controls in basal conditions (53.3 +/- 15.3 and 26.9 +/- 14.7 arbitrary units (a.u.) respectively), when stimulated with native LDL (46.5 +/- 5 and 24.3 +/- 9.4 a.u. respectively), when stimulated with malondialdehyde modified LDL (50 +/- 16.2 and 24.4 +/- 7.7 a.u. respectively) or when stimulated with glycated LDL (38.3 +/- 8.8 and 14.4 +/- 7.5 a.u. respectively). CONCLUSIONS: Diabetic patients have an enhanced immune response against low density lipoproteins, factor that could contribute to the accelerated atherogenesis of this disease.

Galactothermin, a reversibly heat-precipitable protein of human milk at neutral pH.

1. A protein, aggregating at body temperature and solubilizing when cooled, was isolated from fresh human milk at neutral pH and studied for some of its physical, chemical and immunological properties. The name ;galactothermin' is proposed for this protein. 2. Isolation and purification of galactothermin involved casein removal from skim milk at pH4.64 followed by centrifugal fractionation of residual protein-containing solutions repeatedly heated and cooled between 40 degrees C and 0 degrees C at pH7.3. 3. The molecular weight by ultracentrifugal analysis and the minimum molecular weight by sum of amino acid residues were 11400 and 14000 respectively. The sedimentation coefficient s(25,w) was 1.05S and the diffusion coefficient was 7.15x10(-7). Reversible aggregation is favoured by increase in protein concentration, ionic strength, temperature, time and approach to the isoionic point of 7.27 from either acidic or alkaline conditions. 4. Among the amino acid residues, proline predominates and non-polar species account for two-thirds of the total. Cysteine and cystine are absent. Analysis of galactothermin showed it to be essentially free of hexose, sialic acid, calcium and phosphate. 5. Galactothermin is antigenic in the rabbit as evidenced by the passive cutaneous anaphylaxis test. Single precipitin lines are produced in immunodiffusion tests. 6. By electrophoresis in polyacrylamide gel at pH4.0 only one sharp band is produced.