Ana Gonçalves

The involvement of the chemokine receptor CXCR2 in neutrophil recruitment in LPS-induced inflammation and in Mycobacterium avium infection.

Knockout mice for CXC receptor 2 (CXCR2) chemokine receptor were used to study the recruitment of neutrophils during acute and chronic inflammatory responses. When treated with lipopolysaccharide (LPS), either intraperitoneally or intratracheally, these animals had a significant reduction in the neutrophil recruitment in the first 24–48 h as compared with control mice. During 15 days of intraperitoneal infection by Mycobacterium avium, the knockout mice showed significantly reduced numbers of neutrophils in the peritoneal cavity as compared with the control mice. In contrast, the recruitment of neutrophils to the lungs during an aerogenic M. avium infection was not affected by the CXCR2 mutation throughout the 60 days of the study. Finally, we could not find any impact of the mutation on the mycobacterial growth of the infected animals. These findings indicate that CXCR2 may be essentially involved in acute inflammatory responses where an early and rapid recruitment of neutrophils is observed.

CD40 is required for the optimal induction of protective immunity to Mycobacterium avium.

C57Bl/6 mice and mice deficient in the CD40 molecule were infected with three strains of Mycobacterium avium. Two of the M. avium strains proliferated more extensively in CD40‐deficient (CD40−/−) mice than in control mice. The increased susceptibility to infection of CD40−/− mice was associated with the generation of poorer interleukin‐12 (IL‐12) p40 and interferon‐γ (IFN‐γ) responses as compared to the controls, suggesting a role for CD40 in the development of protective immunity. In contrast, direct triggering of CD40 on infected macrophages failed to induce any anti‐mycobacterial activity in infected macrophages.

New variants, challenges and pitfalls in DMD genotyping: implications in diagnosis, prognosis and therapy

Molecular characterization of patients with Duchenne or Becker muscular dystrophies is essential for establishing a differential diagnosis, allowing appropriate clinical follow-up, patient management and genetic counseling. In light of the recent mutation-based therapeutic approaches, DMD gene analysis has gained further relevance. Owing to the size and complexity of the DMD gene and the diversity of mutation types, molecular analysis is not always a straightforward task requiring the combination of several methodologies. Our national genetic diagnostic service genetically characterized 308 dystrophinopathy patients (284 unrelated families), leading to the identification of 175 distinct mutations, including 39 unpublished variants. These studies revealed several potential diagnostic pitfalls (because of technical limitations or related with DMD’s genetic heterogeneity) that may be overlooked even considering the international disease-specific diagnostic guidelines. Comprehensive analysis involved expression studies at the mRNA level, the identification of splicing changes and ultimately providing evidence for apparent exceptions to the reading-frame rule. Besides increasing the mutation detection rate, this detailed molecular characterization is indispensable for the identification of suitable candidates for the new mutation-centered therapies. As patient registries are internationally recognized as essential for clinical trial recruitment, this led us to develop the Portuguese Duchenne and Becker Muscular Dystrophy registry in collaboration with the Translational Research in Europe—Assessment and Treatment of Neuromuscular Diseases network.

Effects of recombinant granulocyte-colony stimulating factor administration during Mycobacterium avium infection in mice

Granulocyte colony‐stimulating factor (G‐CSF) administration in vivo has been shown to improve the defence mechanisms against infection by different microbes. Here we evaluated a possible protective role of this molecule in a mouse model of mycobacterial infection. The administration of recombinant G‐CSF promoted an extensive blood neutrophilia but failed to improve the course of Mycobacterium avium infection in C57Bl/6 or beige mice. G‐CSF administration also failed to improve the efficacy of a triple chemotherapeutic regimen (clarithromycin + ethambutol + rifabutin). G‐CSF treatment did not protect interleukin‐10 gene disrupted mice infected with M. avium. Spleen cells from infected mice treated with G‐CSF had a decreased priming for antigen‐specific production of interferon gamma compared to control infected mice. Our data do not substantiate previous reports on the protective activity of G‐CSF in antimycobacterial immunity using mouse models.

Modulation of neutrophil influx with cell adhesion molecule specific antibodies during nonspecific and immune mediated inflammatory reactions.

Neutrophils are essential for the host defence against infection. However, neutrophils may also mediate damage namely during immune mediated pathologies. We therefore tested whether targeting of different cell adhesion molecules with specific monoclonal antibodies might reduce immune mediated neutrophil recruitment but spare the nonspecific accumulation of neutrophils that is essential for the resistance against acute infections. Neutrophil recruitment was induced by either intraperitoneal injection of casein as a nonspecific phlogistic agent or by i.p. injection of antigen in Mycobacterium bovis Bacille Caimette‐Guérin (BCG) immune mice. Similar degrees of inhibition of neutrophil accumulation were observed in both models of inflammation with antibodies directed at CD11a, ICAM‐1 and CD11b with the latter showing the most marked effects. Individual targeting of selectins was without effect in immune mediated responses whereas targeting of L or E selectin inhibited nonspecific recruitment of neutrophils. This was apparently not owing to a dosage effect nor to a kinetic difference. The inhibitory effect of anti‐CD11b antibodies was most likely as a result of activation of circulating neutrophils rather than the blocking of receptor–ligand interactions. We were therefore unable to selectively abrogate immune mediated neutrophil recruitment with the use of the antibodies selected in this study.

Long term performance evaluation of small-diameter vascular grafts based on polyvinyl alcohol hydrogel and dextran and MSCs-based therapies using the ovine pre-clinical animal model.

The functional and structural performance of a 5cm synthetic small diameter vascular graft (SDVG) produced by the copolymerization of polyvinyl alcohol hydrogel with low molecular weight dextran (PVA/Dx graft) associated to mesenchymal stem cells (MSCs)-based therapies and anticoagulant treatment with heparin, clopidogrel and warfarin was tested using the ovine model during the healing period of 24 weeks. The results were compared to the ones obtained with standard expanded polyetetrafluoroethylene grafts (ePTFE graft). Blood flow, vessel and graft diameter measurements, graft appearance and patency rate (PR), thrombus, stenosis and collateral vessel formation were evaluated by B-mode ultrasound, audio and color flow Doppler. Graft and regenerated vessels morphologic evaluation was performed by scanning electronic microscopy (SEM), histopathological and immunohistochemical analysis. All PVA/Dx grafts could maintain a similar or higher PR and systolic/diastolic laminar blood flow velocities were similar to ePTFE grafts. CD14 (macrophages) and α-actin (smooth muscle) staining presented similar results in PVA/Dx/MSCs and ePTFE graft groups. Fibrosis layer was lower and endothelial cells were only detected at graft-artery transitions where it was added the MSCs. In conclusion, PVA/Dx graft can be an excellent scaffold candidate for vascular reconstruction, including clinic mechanically challenging applications, such as SDVGs, especially when associated to MSCs-based therapies to promote higher endothelialization and lower fibrosis of the vascular prosthesis, but also higher PR values.

LAMA2 gene mutation update: Toward a more comprehensive picture of the laminin-α2 variome and its related phenotypes

Congenital muscular dystrophy type 1A (MDC1A) is one of the main subtypes of early‐onset muscle disease, caused by disease‐associated variants in the laminin‐α2 (LAMA2) gene. MDC1A usually presents as a severe neonatal hypotonia and failure to thrive. Muscle weakness compromises normal motor development, leading to the inability to sit unsupported or to walk independently. The phenotype associated with LAMA2 defects has been expanded to include milder and atypical cases, being now collectively known as LAMA2‐related muscular dystrophies (LAMA2‐MD). Through an international multicenter collaborative effort, 61 new LAMA2 disease‐associated variants were identified in 86 patients, representing the largest number of patients and new disease‐causing variants in a single report. The collaborative variant collection was supported by the LOVD‐powered LAMA2 gene variant database (https://www.LOVD.nl/LAMA2), updated as part of this work. As of December 2017, the database contains 486 unique LAMA2 variants (309 disease‐associated), obtained from direct submissions and literature reports. Database content was systematically reviewed and further insights concerning LAMA2‐MD are presented. We focus on the impact of missense changes, especially the c.2461A > C (p.Thr821Pro) variant and its association with late‐onset LAMA2‐MD. Finally, we report diagnostically challenging cases, highlighting the relevance of modern genetic analysis in the characterization of clinically heterogeneous muscle diseases.