Delphi survey of intercontinental experts to identify areas of consensus on the use of indocyanine green angiography for tissue perfusion assessment during plastic and reconstructive surgery.

BACKGROUND: In recent years, indocyanine green angiography (ICG-A) has been used increasingly to assist tissue perfusion assessments during plastic and reconstructive surgery procedures, but no guidelines exist regarding its use. We sought to identify areas of consensus and non-consensus among international experts on the use of ICG-A for tissue-perfusion assessments during plastic and reconstructive surgery. METHODS: A two-round, online Delphi survey was conducted of 22 international experts from four continents asking them to vote on 79 statements divided into five modules: module 1 = patient preparation and contraindications (n = 11 statements); module 2 = ICG administration and camera settings (n = 17); module 3 = other factors impacting perfusion assessments (n = 10); module 4 = specific indications, including trauma debridement (n = 9), mastectomy skin flaps (n = 6), and free flap reconstruction (n = 8); and module 5 = general advantages and disadvantages, training, insurance coverage issues, and future directions (n = 18). Consensus was defined as ≥70% inter-voter agreement. RESULTS: Consensus was reached on 73/79 statements, including the overall value, advantages, and limitations of ICG-A in numerous surgical settings; also, on the dose (0.05 mg/kg) and timing of ICG administration (∼20-60 seconds preassessment) and best camera angle (61-90o) and target-to-tissue distance (20-30 cm). However, consensus also was reached that camera angle and distance can vary, depending on the make of camera, and that further research is necessary to technically optimize this imaging tool. The experts also agreed that ambient light, patient body temperature, and vasopressor use impact perfusion assessments. CONCLUSION: ICG-A aids perfusion assessments during plastic and reconstructive surgery and should no longer be considered experimental. It has become an important surgical tool.

Lessons learned in allergy and immunology training: a survey analysis.

BACKGROUND: There is currently little Canadian data to assess how well traditional time-based residency training programs have prepared residents for careers in Clinical Immunology and Allergy (CIA). This study aims to identify the perceived preparedness of residents in various areas of practice upon the completion of a Canadian CIA residency training program. METHODS: In the summer of 2020, an electronic survey was sent to 2018 and 2019 graduates of Canadian CIA training programs by the Canadian Society of Allergy and Clinical Immunology (CSACI). RESULTS: Former residents felt well prepared in most Medical Expert areas. Residents felt less prepared for the intrinsic roles of Leader, Communicator, Collaborator, Health Advocate, Scholar, and Professional. The majority of the intrinsic competencies were learned through mentorship and on the job after finishing training. CONCLUSIONS: Upon completion of training, Canadian CIA residents felt well prepared for many competencies, particularly in Medical Expert areas. Training programs may wish to focus on various intrinsic competencies in order to better prepare residents for transition to practice. Academic half-day was not identified as a primary learning centre for intrinsic competencies, suggesting that new teaching strategies may be required.

A Cadaveric Anatomical and Histological Study of Recipient Intercostal Nerve Selection for Sensory Reinnervation in Autologous Breast Reconstruction.

BACKGROUND: Autologous breast reconstruction (ABR) has grown in popularity due to improved aesthetic and long-term patient reported outcomes, but data regarding sensory reinnervation of autologous flaps remain limited. Traditionally, the lateral cutaneous branch of the fourth intercostal nerve has been used for flap neurotization, but the use of the anterior cutaneous branch of the intercostal nerves (ACB) offer a more optimal location to the microsurgical field when using internal mammary vessels for the microanastomosis. This study aimed to evaluate the optimum ACB recipient site level for sensory nerve coaptation in ABR. METHODS: Twelve hemi-chests were dissected from six fresh cadaveric females. Costal cartilages were removed and the anterior cutaneous intercostal nerve (ACB) and the lateral (subcutaneous) division of the anterior cutaneous branch (LACB) of the intercostal nerve were exposed. Anatomical measurements were recorded, and nerve samples were evaluated histologically with carbonic anhydrase staining to differentiate sensory fascicles. Assessment of fascicular diameter, axonal counts, and fascicular area were compared. RESULTS: A total of 75 nerve specimens were assessed. The ACB was identified at all levels (100%) and the subcutaneous LACB was noted consistently in the second to fourth rib space (96% cadavers), with a median length of 43, 37.5, and 37 mm, respectively. Across all rib spaces, the fascicular and axonal counts were comparable between the LACB and ACB. Nerves in the second intercostal space had a significantly larger mean fascicular area mean (112,816 ± 157,120 µm2) compared with that in the fourth (mean 26,474 ± 38,626 µm2), p = 0.03. Axonal count of sensory fascicles was the highest in the second intercostal nerves (p < 0.05). CONCLUSION: This study provides anatomical and histological basis to determine the optimum recipient site choice for sensory coaptation in microsurgical breast reconstruction. This would aid in operative decision-making regarding the ideal recipient anterior cutaneous intercostal nerve branches for recipient site coaptation in ABR.

Toll-like receptor-mediated eosinophil-basophil differentiation: autocrine signalling by granulocyte-macrophage colony-stimulating factor in cord blood haematopoietic progenitors.

Eosinophils are multi-functional leucocytes that play a role in inflammatory processes including allergy and infection. Although bone marrow (BM) inflammatory cells are the main source of eosinophil-basophil (Eo/B) differentiation-inducing cytokines, a recent role has been demonstrated for cytokine induction through Toll-like receptor (TLR)-mediated signalling in BM progenitors. Having previously demonstrated that cord blood (CB) progenitors induce Eo/B colony-forming units (CFU) after lipopolysaccharide (LPS) stimulation, we sought to investigate the intracellular mechanisms by which LPS induces Eo/B differentiation. Freshly isolated CD34-enriched human CB cells were stimulated with LPS (and/or pharmacological inhibitors) and assessed for alterations in haematopoietic cytokine receptor expression and signalling pathways by flow cytometry, Eo/B CFU in methylcellulose cultures, and cytokine secretion using Luminex assays. The LPS stimulation resulted in a significant increase in granulocyte-macrophage colony-stimulating factor (GM-CSF)-responsive, as opposed to interleukin-5-responsive, Eo/B CFU, which also correlated with significant increases in CD34(+) cell GM-CSFRα expression. Functionally, CB CD34(+) cells secrete abundant amounts of GM-CSF following LPS stimulation, via a p38 mitogen-activated protein kinase (MAPK)-dependent mechanism; this secretion was responsible for Eo/B CFU formation ex vivo, as shown by antibody blockade. We show for the first time that LPS stimulation of CB progenitor cells results in autocrine activation of p38 MAPK-dependent GM-CSF secretion facilitating Eo/B differentiation ex vivo. This work provides evidence that early life exposure to products of bacterial agents can modulate Eo/B differentiation, representing a novel mechanism by which progenitor cells can respond to microbial stimuli and so affect immune and inflammatory responses.

Cord blood hemopoietic progenitor profiles predict acute respiratory symptoms in infancy.

Atopy is characterized by eosinophilic inflammation associated with recruitment of eosinophil/basophil (Eo/B) progenitors. We have previously shown that Eo/B progenitor phenotypes are altered in cord blood (CB) in infants at high risk of atopy/asthma, and respond to maternal dietary intervention during pregnancy. As respiratory tract viral infections have been shown to induce wheeze in infancy, we investigated the relationship between CB progenitor function and phenotype and acute respiratory illness (ARI), specifically wheeze and fever. CB from 39 high-risk infants was studied by flow cytometry for CD34(+) progenitor phenotype and by ex vivo Eo/B-colony forming unit (CFU) responses to cytokine stimulation in relation to ARI in the first year of life. A consistent relationship was observed between increased numbers of granulocyte/macrophage (GM)-colony-stimulating factor (CSF)- and IL-3-responsive Eo/B-CFU in CB and the frequency/characteristics of ARI during infancy. Comparable associations were found between ARI and CB IL-3R(+) and GM-CSFR(+)CD34(+) cell numbers. Conversely, a reciprocal decrease in the proportion of CB IL-5R(+) cells was found in relation to the clinical outcomes. The elevation of IL-3/GM-CSF-responsive Eo/B progenitors in high-risk infants in relation to ARI outcomes suggests a mechanism for the increased severity of inflammatory responses in these subjects following viral infection.

In vitro effects of budesonide on eosinophil-basophil lineage commitment.

UNLABELLED: IL-5 is the primary cytokine that stimulates the production and survival of eosinophils and basophils from progenitor cells. The inhaled glucocorticoid, budesonide, has been shown to exert a therapeutic effect via suppression of eosinophil/basophil progenitors in vivo. Since various steroids have exhibited the ability to enhance eosinophil/basophil progenitor differentiation, we examined the effects of budesonide in vitro. Bone marrow and cord blood samples were obtained and cultured in the presence of IL-5 alone or IL-5 plus budesonide. Eosinophil/basophil colony-forming units were enumerated from cultured nonadherent mononuclear cells and from purified CD34⁺ cells. CD34⁺ cells with and without budesonide were also examined for up-regulation of ERK1/2, MAPK and GATA-1 using real time-PCR. RESULTS: i) up-regulation of eosinophil/basophil colony-forming units is due to the direct effects of budesonide on IL-5-stimulated progenitors; ii) GATA-1 is likely involved in the early amplification of eosinophil/basophil progenitor commitment leading to increased differentiation. A potential transcriptional pathway has been identified which may mediate the effects of budesonide on eosinophil/basophil lineage commitment.

The effect of desloratadine on eosinophil/basophil progenitors and other inflammatory markers in seasonal allergic rhinitis: a placebo-controlled randomized study.

BACKGROUND: Eosinophil/basophil (Eo/B) progenitors fluctuate in the peripheral circulation during seasonal allergen exposure in atopic subjects. Several drugs have been shown to modulate Eo/B progenitor levels in the peripheral blood but, to date, the possible effect of antihistamines on Eo/B progenitors has not been explored. Our objective was to evaluate whether the antihistamine desloratadine (DL) can modulate peripheral blood Eo/B progenitors or other markers of allergic inflammation. METHODS: We performed a randomized double-blind placebo-controlled study on the effects of DL on peripheral blood Eo/B progenitors in subjects with symptomatic, seasonal allergic rhinitis during a ragweed pollen season. Forty-five subjects were randomized to treatment for 4 weeks with DL 20 mg daily or placebo. RESULTS: The expected fall in the number of Eo/B progenitors from baseline to 2 weeks of treatment was seen in the placebo group [median drop of 1.0 colony-forming unit (CFU)/10(6) cells], and was greater than in the DL group (median drop of 0.0 CFU/10(6) cells) (p = 0.013). The change in histamine concentration per colony from baseline to 2 weeks of treatment was lower in the DL group (median decrease of 6.1 pg/colony) compared to placebo (median increase of 1.8 pg/colony) (p = 0.01). An increase in the nasal lavage eotaxin concentration from baseline to 4 weeks of treatment was statistically significant in the placebo group but not in the DL group. Eo/B CFU were not affected by varying in vitro concentrations of DL. CONCLUSION: These results suggest that DL can modulate aspects of allergic inflammation in vivo through mechanisms other than simple blockade of H1 histamine receptors.

Haemopoietic mechanisms in murine allergic upper and lower airway inflammation.

Eosinophil recruitment to the airways, including involvement of haemopoietic eosinophil-basophil progenitors (Eo/B-CFU), is primarily regulated by interleukin-5 (IL-5) and eotaxin. In this study, we investigated the haemopoietic mechanisms in upper and lower airway eosinophilic inflammation. Ovalbumin (OVA) sensitized and challenged BALB/c mice were used to establish isolated upper (UAC), isolated lower (LAC), or combined upper and lower airway (ULAC) inflammation. Airway, blood and bone marrow responses were evaluated in each model. Numbers of airway eosinophils and CD4(+) cells were increased significantly in the nasal mucosa in UAC and ULAC mice, and in the lung tissue in LAC and ULAC groups. Levels of IL-5 and eotaxin were increased significantly in the nasal lavage fluid (NL) in UAC and ULAC mice, and in the bronchoalveolar lavage fluid (BAL) in LAC and ULAC groups. The proportion of IL-5-responsive bone marrow Eo/B-CFU was significantly higher than the control in all treatment groups, but peaked much earlier in the ULAC group. Kinetic studies revealed that IL-5 and eotaxin in NL, BAL and serum peaked between 2 and 12 hr after OVA challenge in ULAC mice, and at 24 hr in UAC mice, related to the timing of maximal progenitor responses. These data support the concept that the systemic mechanisms linking rhinitis to asthma depend on the location and extent of airway allergen exposure.

Fish oil supplementation in pregnancy modifies neonatal progenitors at birth in infants at risk of atopy.

Dietary n-3 polyunsaturated fatty acids (PUFA) may represent a mode of allergy prevention. Cord blood (CB) CD34+ hemopoietic progenitors are altered in infants at risk of atopy. We therefore studied the effects of dietary n-3 PUFA supplementation during pregnancy on numbers and function of progenitors in neonates at high risk of atopy. In a double-blind study, atopic, pregnant women were randomized to receive fish oil capsules or placebo from 20 wk gestation until delivery. At birth, CB CD34+ cells were isolated and analyzed by flow cytometry for expression of cytokine (IL-5Ralpha, IL-3Ralpha, granulocyte/macrophage colony-stimulating factor Ralpha) or chemokine (CXCR4 and CCR3) receptors. CB cells were also cultured in methylcellulose assays for eosinophil/basophil colony-forming cells. At age 1 y, infants were clinically assessed for atopic symptoms and skin tests. Percentages of CB CD34+ cell numbers were higher after n-3 PUFA than placebo. Co-expression of cytokine or chemokine receptors on CD34 cells was not altered by n-3 PUFA supplementation. However, there were significantly more IL-5-responsive CB eosinophil/basophil colony forming units (Eo/B-CFU) in the fish oil, compared with the control, group. Overall, there was a positive association between CD34+ cells and IL-5-responsive Eo/B-CFU in CB and 1 y clinical outcomes, including atopic dermatitis and wheeze. Dietary n-3 PUFA supplementation during pregnancy in atopic mothers alters infant cord blood hemopoietic progenitor phenotype. This may have an impact on development of atopic disease.

Effects of a cysteinyl leukotriene receptor antagonist on eosinophil recruitment in experimental allergic rhinitis.

The cysteinyl leukotrienes (cysLTs) are potent lipid mediators in allergic disease, acting through a receptor (cysLT1-R) which can be targeted in rhinitis and asthma. We investigated the effects of cysLT1-R antagonism in experimental allergic rhinitis, focusing on bone marrow eosinophil progenitor responses. BALB/c mice were sensitized, then given daily intranasal ovalbumin for 2 weeks, with montelukast sodium (5 mg/kg or 2.5 mg/kg) or placebo by gavage. Bone marrow eosinophil/basophil colonies were enumerated, and colony cells were morphologically assessed as indices of eosinophil differentiation and maturation. Montelukast treatment resulted in a significant decrease of eosinophils in the nasal mucosa, and in either bone marrow interleukin (IL)-5-, but not IL-3-, or granulocyte-macrophage colony-stimulating factor-responsive eosinophil/basophil colony-forming units, and IL-5-stimulated eosinophil maturation. These results indicate that cysLT1-R antagonism in vivo limits both IL-5-responsive eosinophilopoiesis, acting at several stages of eosinophil differentiation and maturation. The anti-allergic effects of cysLT1-R antagonists are consistent with the concept that cysLTs and IL-5 act together in the recruitment of eosinophils and eosinophil progenitors from the marrow during upper airway allergic inflammation.

Hippocampal damage in mouse and human forms of systemic autoimmune disease.

Systemic lupus erythematosus (SLE) is frequently accompanied by neuropsychiatric (NP) and cognitive deficits of unknown etiology. By using autoimmune MRL-lpr mice as an animal model of NP-SLE, we examine the relationship between autoimmunity, hippocampal damage, and behavioral dysfunction. Fluoro Jade B (FJB) staining and anti-ubiquitin (anti-Ub) immunocytochemistry were used to assess neuronal damage in young (asymptomatic) and aged (diseased) mice, while spontaneous alternation behavior (SAB) was used to estimate the severity of hippocampal dysfunction. The causal relationship between autoimmunity and neuropathology was tested by prolonged administration of the immunosuppressive drug cyclophosphamide (CY). In comparison to congenic MRL +/+ controls, SAB acquisition rates and performance in the "reversal" trial were impaired in diseased MRL-lpr mice, suggesting limited use of the spatial learning strategy. FJB-positive neurons and anti-Ub particles were frequent in the CA3 region. Conversely, CY treatment attenuated the SAB deficit and overall FJB staining. Similarly to mouse brain, the hippocampus from a patient who died from NP-SLE showed reduced neuronal density in the CA3 region and dentate gyrus, as well as increased FJB positivity in these regions. Gliosis and neuronal loss were observed in the gray matter, and T lymphocytes and stromal calcifications were common in the choroid plexus. Taken together, these results suggest that systemic autoimmunity induces significant hippocampal damage, which may underlie affective and cognitive deficits in NP-SLE.

Anti-allergic therapies: effects on eosinophil progenitors.

Marked eosinophilic infiltration is the typical inflammatory response associated with allergic inflammation. Previous research involving animal and human models has established a role for the eosinophil/basophil hematopoietic progenitor in a systemic process of allergic inflammation. In this article, we will review the evidence implicating eosinophil/basophil progenitors in this systemic response and will discuss the rationale for targeting this cell in the treatment of allergic disease. In this context, we discuss corticosteroid treatment of allergic diseases, such as asthma and its effects on hematopoietic mechanisms, the effects of therapies that inhibit the actions of cysteinyl leukotrienes, the effects of in vivo blockade of the eosinophil-active cytokine interleukin-5, and the effects of antihistamines on hematopoiesis. It is suggested that several anti-allergic therapies exert their beneficial effects on allergic inflammation by influencing eosinophil production systemically. Therefore, targeting the systemic hematopoietic response may provide additional, more beneficial, therapeutic effects.