Generation of superoxide anion by alveolar macrophages in sarcoidosis: evidence for the activation of the oxygen metabolism in patients with high-intensity alveolitis.

We studied superoxide anion (O2) generation by alveolar macrophages (AM) isolated from bronchoalveolar lavages (BAL) of patients with sarcoidosis, and assayed immediately after the isolation or after maintenance in culture for 2 days. In assays of cells freshly isolated from BAL, AM of patients with active sarcoidosis with a high-intensity lymphocytic alveolitis produced more O2- in response to phorbol myristate acetate than AM of patients with inactive sarcoidosis. Also, after 2 days of cultivation sarcoid AM were heterogeneous in their capability to metabolize oxygen, although both AM of active and inactive sarcoid patients produced higher amounts of O2- than AM of healthy subjects. In vitro treatment with recombinant interferon-gamma (rIFN-gamma) caused an enhancement of the capability of AM of inactive sarcoid patients to produce O2- in response to PMA. AM of patients with active sarcoidosis did not respond to rIFN-gamma when they already produced O2- vigorously. However, they became sensitive to the activating effect of rIFN-gamma after the down-modulation of their capability to produce O2-, that occurred upon prolonged cultivation. Monocytes isolated from blood of sarcoid patients and assayed immediately or after different times of cultivation did not produce more O2- than control monocytes and monocyte-derived macrophages, thus indicating that the activation of AM in sarcoidosis is likely a local phenomenon. These studies strengthen the notion that T lymphocyte-macrophage interaction is a critical event in the pathogenesis of sarcoidosis and establish that the enhanced capability to metabolize oxygen to highly reactive intermediates by AM is one of the consequence of this interaction.     

Uncovering the multifaceted roles played by neutrophils in allogeneic hematopoietic stem cell transplantation

Allogeneic hematopoietic stem cell transplantation (alloHSCT) is a life-saving procedure used for the treatment of selected hematological malignancies, inborn errors of metabolism, and bone marrow failures. The role of neutrophils in alloHSCT has been traditionally evaluated only in the context of their ability to act as a first line of defense against infection. However, recent evidence has highlighted neutrophils as key effectors of innate and adaptive immune responses through a wide array of newly discovered functions. Accordingly, neutrophils are emerging as highly versatile cells that are able to acquire different, often opposite, functional capacities depending on the microenvironment and their differentiation status. Herein, we review the current knowledge on the multiple functions that neutrophils exhibit through the different stages of alloHSCT, from the hematopoietic stem cell (HSC) mobilization in the donor to the immunological reconstitution that occurs in the recipient following HSC infusion. We also discuss the influence exerted on neutrophils by the immunosuppressive drugs delivered in the course of alloHSCT as part of graft-versus-host disease (GVHD) prophylaxis. Finally, the potential involvement of neutrophils in alloHSCT-related complications, such as transplant-associated thrombotic microangiopathy (TA-TMA), acute and chronic GVHD, and cytomegalovirus (CMV) reactivation, is also discussed. Based on the data reviewed herein, the role played by neutrophils in alloHSCT is far greater than a simple antimicrobial role. However, much remains to be investigated in terms of the potential functions that neutrophils might exert during a highly complex procedure such as alloHSCT.     

On the co-purification of 6-sulfo LacNAc dendritic cells (slanDC) with NK cells enriched from human blood

The ability of NK cells to directly recognize pathogens and be activated via Toll-like receptors (TLR) is increasingly recognized. Nevertheless, controversial results on the NK cell ability to be directly activated by lipopolysaccharide (LPS), the ligand of TLR4, have been recently reported. To start elucidating the reasons explaining the contrasting observations of the literature, we focused on the potential role of currently used NK cell purification procedures to condition putative NK cell responsiveness to LPS. To do so, human NK cells were isolated by negative selection, using three different commercial kits, to be comparatively evaluated for the production of IFNγ in response to ultra-pure LPS and/or IL-2. Despite the lack of surface TLR4, we found that two out of the three NK cell-enriched populations released IFNγ (and one of the two, IL-12p70 as well) in response to the LPS plus IL-2 combination, whereas the last one did not. However, the two LPS plus IL-2-responsive NK cell populations were found variably contaminated with 6-sulfo LacNAc⁺ dendritic cells (slanDC), demonstrated responsible for triggering, via the production of IL-12p70 in response to LPS, the release of IFNγ by IL-2-stimulated NK cells. Accordingly, slanDC depletion completely abrogated the capacity to produce both IL-12p70 and IFNγ in response to LPS plus IL-2 by slanDC-containing NK cells. Taken together, our data uncover that two commercially available kits, specifically designed to isolate NK cells by negative selection, also co-purify variable amounts of slanDC. The latter cells may dramatically affect the outcome of experiments carried on to evaluate NK cell responsiveness to TLR agonists such as LPS.     

Interference of antibiotic therapy on blood cultures time-to-positivity: analysis of a 5-year experience in an oncological hospital

This study performed a retrospective analysis on the relationship between blood culture time-to-positivity (TP) and type of isolated microorganism, antibiotic administration, and immunological status of the patients. We analyzed the data related to 1,218 positive blood cultures. When compared to Gram positive bacteraemia, the percentage of Gram negative growth was higher and the mean TP significantly shorter (p < 0.0001). In patients receiving antibiotics, median and mean TPs of blood culture were different for Gram positive bacteraemia (log-rank p = 0.0022, Wilcoxon p < 0.0001) but not for Gram negative (log-rank p = 0.4011, Wilcoxon p = 0.1585). No statistically significant effect on TP was found for sampling site, interaction between sampling site and antibiotic administration, and immunological status of the patient. In conclusion, TP is independent of antibiotic therapy in cases of Gram negative bacteraemia, while for Gram positive bacteraemia a prolongation of TP occurs.     

Acute lower limb thrombosis caused by a congenital fibrous ring of the superficial femoral artery

Segmentary isolated stenosis or obstructions of the superficial femoral artery in young people are rarely reported. In patients, most of them women, affected by chronic symptomatology of the lower limbs, the aetiology has been referred to fibromuscular dysplasia with unusual localization. We report a case of acute lower limb thrombosis in a young woman caused by a congenital fibrous ring of the superficial femoral artery and the treatment we performed in this situation including the complication that happened after the percutaneous transluminal angioplasty that we carried out in order to reduce the stenosis of the femoral superficial artery. Histological examination of the lesion demonstrated the nature of the fibrous ring caused by an embryological anomaly, followed by a secondary thrombosis in a woman not using oral contraceptives and without any alteration of the coagulation chain. Fibromuscular dysplasia of the femoral artery is commonly caused by previous thigh injuries, thromboembolic events with recanalization of the artery or arteritis, but in some cases appears to be the consequence of primitive intimal dysplasia. When a fibromuscular dysplasia is suspected, all authors agree on the necessity for a screening of the two preferential localizations of the disease: common carotid artery and renal artery, in the case reported the result was negative.     

On the detection of neutrophil-derived vascular endothelial growth factor (VEGF)

In recent years, several investigators have addressed the question of whether mature polymorphonuclear neutrophils (PMN) are able to secrete cytokines. Their studies have brought forward new and exciting discoveries, by establishing that the release of inflammatory cytokines constitutes a novel and important aspect of the neutrophil biology, thereby emphasizing that PMN should no longer be regarded as cells that only release preformed mediators. Although it is still premature to assess the true biological significance of cytokine production by neutrophils, this new aspect of neutrophil biology opens novel perspectives as to the potential role of these cells in the inflammatory and immune responses. In this context, a correct methodological analysis and a detailed molecular investigation of the mechanisms regulating cytokine production by neutrophils in vitro is a critical and fundamental step to better understand how the release of cytokines by PMN may influence pathophysiological processes in vivo. We now describe and discuss the approach that we typically used throughout most of the last decade to characterize cytokine production by human neutrophils, as illustrated herein for a protein that is expressed and released by PMN, namely, vascular endothelial growth factor (VEGF).     

CCL20/macrophage inflammatory protein-3alpha production in LPS-stimulated neutrophils is enhanced by the chemoattractant formyl-methionyl-leucyl-phenylalanine and IFN-gamma through independent mechanisms

We have recently demonstrated that polymorphonuclear neutrophils (PMN), when cultured with LPS or TNF-alpha, have the capacity to release CCL20, a chemokine primarily chemotactic for immature dendritic cells and specific lymphocyte subsets. Here, we report that the chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP), as well as the immunoregulatory cytokine IFN-gamma, can significantly up-modulate the production of neutrophil-derived CCL20 through entirely unrelated mechanisms. We found that fMLP dramatically up-regulates CCL20 mRNA expression and synthesis in neutrophils stimulated with LPS for 2-3 h, and that its effect takes place through CCL20 mRNA stabilization. In contrast, IFN-gamma potentiates CCL20 gene expression and production only after 21 h of LPS treatment, its effect being mediated by endogenous TNF-alpha in an autocrine fashion, as revealed using neutralizing anti-TNF-alpha antibodies added to IFN-gamma plus LPS-treated PMN. Finally, we demonstrate that activation of p38 mitogen-activated protein kinase (MAPK) plays an important role in mediating the production of CCL20 induced by LPS (with or without IFN-gamma), whereas activation of p42/44 extracellular signal-regulated kinases (ERK) is involved in the enhancing effect of fMLP. Taken together, these findings identify novel biological actions exerted by fMLP and IFN-gamma, potentially involved in the orchestration of inflammatory and immune responses within epithelial and mucosal tissue.