Video-Assisted Thoracoscopy for Vertebral Body Tethering of Juvenile and Adolescent Idiopathic Scoliosis: Tips and Tricks of Surgical Multidisciplinary Management.

VATS (video assisted thoracoscopic surgery) is routinely and successfully performed in minor and major complex thoracic procedures. This technique has been recently introduced for the treatment of severe forms of idiopathic scoliosis (IS) with the aim to repair the deformity, reduce morbidity and to prevent its progression in patients with skeletal immaturity. This study aims to present VATS in anterior vertebral body tethering (AVBT) approach to support the pediatric orthopedic surgeons during vertebral body fixation. Surgical and anesthesiologic tips and tricks are reported to assure a safe procedure. The study includes preadolescents with IS and a grade of scoliosis >40° that had a high probability of deterioration due to remaining growth (December 2018 to April 2021). Skeletal immaturity of enrolled patients was assessed by Sanders classification and Risser sign. Patients had a Risser score between 0 and 1 and a Sanders score >2 and <5. AVBT technique using VATS was performed by a senior pediatric surgeon assisting the pediatric orthopedic surgeon. Twenty-three patients have been submitted to VATS AVBT in the period of study (age range 9-14 years). The patients had a classified deformity Lenke 1A or B convex right and all types of curves were treated. In all patients, the vertebrae submitted to tethering surgery ranged from D5 to D12; mean curve correction was 43%. Three postoperative complications occurred: one late postoperative bleeding requiring a chest tube positioning on 12th postoperative day; one screw dislodged and needed to be removed; one child showed worsening of the scoliosis and needed a posterior arthrodesis. Initial results of VATS AVBT in growing patients with spinal deformities are encouraging. An appropriate selection of patients and a pediatric dedicated multidisciplinary surgical approach decrease intraoperative complications, time of operation and postoperative sequelae and guarantee an optimal outcome.

Stat 6-dependent induction of myeloid derived suppressor cells after physical injury regulates nitric oxide response to endotoxin.

OBJECTIVE: To delineate the role of T-helper 2 (Th2) cytokines in the induction of trauma induced myeloid suppressor cells (TIMSC) and the regulation of nitric oxide production. BACKGROUND: Trauma induces myeloid cells that express CD11b+/Gr1+ and arginase 1 and exhibit an immune suppressing activity. This article explores the mechanisms that induce TIMSC and the effects on nitric oxide production in response to endotoxin. METHODS: TIMSC were studied in response to Th2 cytokines and a subsequent challenge to endotoxin. The role of Th2 cytokines was studied in STAT6-/- mice. Accumulation of TIMSC in spleens was studied using flow cytometry and immunhistochemistry. Plasma was recovered to measure accumulation of nitric oxide metabolites. RESULTS: TIMSC accumulated in the spleen of injured mice and were particularly sensitive to IL-4 and IL-13 with large inductions of arginase activity. Significant blunting in both the accumulation of TIMSC in the spleen and induction of arginase 1 was observed in STAT6-/- mice after physical injury. Accumulation of nitric oxide metabolites to endotoxin was observed in STAT6-/- mice. CONCLUSION: This study shows that induction of CD11b+/Gr1+ cells after physical injury play an essential role in the regulation of nitric oxide production after a septic challenge. The accumulation and induction of arginase 1 in TIMSC is Th2 cytokine dependent. To our knowledge, the role of TIMSC in the regulation of nitric oxide is a novel finding. This observation adds to the possibility that TIMSC could play an important role in immunosuppression observed after physical injury.

Nature of myeloid cells expressing arginase 1 in peripheral blood after trauma.

BACKGROUND: Myeloid cells that express arginase 1 are upregulated by different stimuli, including trauma, and are capable of depleting arginine from the surrounding environment. Through arginine depletion, myeloid cells are capable of regulating T-cell function. We have previously reported increased arginase 1 expression in the peripheral blood mononuclear cells (PBMCs) after injury. The nature of the cells expressing arginase in humans after trauma is unknown and is the focus of this article. METHODS: PBMCs were isolated using a Ficoll-Hypaque gradient. Arginase activity was measured by conversion of arginine to ornithine, and arginase 1 protein expression was measured by Western blot. The percent CD16 granulocytes and phenotypical analysis of the cells present in PBMCs were determined by flow cytometry. Magnetic microbeads were used for isolation and exclusion of specific cell subpopulations. RESULTS: Trauma patients exhibited a dramatic increase in arginase activity (p < 0.05) and an increased percentage of CD16 granulocytes in the PBMC layer (p < 0.05) compared with control volunteers. Increased arginase activity in the PBMC layer was due to the contamination of this layer by granulocytes, as their exclusion decreased arginase activity back to baseline (p < 0.05). Granulocytes isolated from the PBMC layer expressed increased CD11b (p < 0.05) and CD66b (p < 0.05), markers of granulocyte activation. Furthermore, these granulocytes were significantly more swollen and degranulated compared with noncontaminating granulocytes. CONCLUSION: In humans, increased arginase 1 expression after trauma observed in the PBMC layer seems to be exclusively the result of an increased number of activated granulocytes.

Effect of citrulline and glutamine on nitric oxide production in RAW 264.7 cells in an arginine-depleted environment.

BACKGROUND: Nitric oxide (NO) is a highly reactive free radical essential for antimicrobial and tumor immunity as well as endothelial function. Arginine is a limiting factor in NO synthesis. Citrulline can be converted to arginine and might restore NO production when arginine availability is limited, while glutamine may competitively inhibit citrulline availability. We aimed to assess how these amino acids interact to generate NO using an in vitro model. METHODS: RAW 264.7 cells were exposed to various amino acid concentrations before and after lipopolysaccharide (LPS) stimulation, and NO production was assessed. RESULTS: NO production directly correlated up to 200 microM with arginine available after LPS stimulation (R(2) = 0.99). Provided the same arginine concentrations following LPS stimulation, low arginine precultured cells produced significantly less NO than high arginine precultured cells (P < .01). Citrulline added to low arginine preculture significantly increased NO production compared to cells in low arginine alone (P < .01). When glutamine was withdrawn before and after LPS stimulation, cells precultured in low arginine and citrulline produced NO equivalent to that of high arginine precultured cells. Additional citrulline provided after LPS stimulation additionally improved NO production beyond that observed in cells precultured in high arginine (P < .01), and NO production became less dependent on arginine availability (R(2) = 0.78). CONCLUSION: Arginine availability is a limiting factor for NO production. Citrulline is a potential substitute to restore NO production when arginine availability is limited. Glutamine appears to be an important modulator that interferes with citrulline-mediated NO production.

High mobility group box I (HMGB1) release from tumor cells after treatment: implications for development of targeted chemoimmunotherapy.

We have recently demonstrated that cytolysis of human melanoma cells by immune effectors (both NK and T cells) is associated with release of the nuclear chromatin protein, high mobility group box I (HMGB1). Extracellular HMGB1 mediates a number of important functions including endothelial cell activation, stromagenesis, recruitment and activation of innate immune cells, and also dendritic cell maturation that, in the setting of cancer, lead to a chronic inflammatory response. This reparative inflammatory response promotes tumor cell survival, expansion, and metastases. Release of HMGB1 after chemotherapy-induced cytotoxicity has not been well characterized. We measured the release of HMGB1 after chemotherapy or immune cytolysis and demonstrated that this did not correlate with conventional markers of apoptosis and necrosis in several human colorectal, pancreatic, and melanoma tumor cell lines. Rather, we observed that tumor cells incubated with the platinating agent oxaliplatin, retained HMGB1 within the nucleus for significantly longer periods than other agents used at comparable cytotoxic concentrations or even with potent cytolytic cells. Thus, release of HMGB1 from dying tumor cells treated with chemotherapy or cells with lymphokine activated killer cell activity is not dependent solely on the mode of cell death. Sequestration of the damage associated molecular pattern molecule, HMGB1, may play a role in the clinical efficacy of platinating agents and suggests this as a superior agent for coupling with immunotherapeutic strategies, possibly enhancing their effectiveness.

Arginine and immunity.

For many years, dietary arginine supplementation, often combined with other substances, has been used as a mechanism to boost the immune system. Considerable controversy, however, exists as to the benefits and indications of dietary arginine due in part to a poor understanding of the role played by this amino acid in maintaining immune function. Emerging knowledge promises to clear this controversy and allow for arginine's safe use. In myeloid cells, arginine is mainly metabolized either by inducible nitric oxide (NO) synthases (iNOS) or by arginase 1, enzymes that are stimulated by T helper 1 or 2 cytokines, respectively. Thus, activation of iNOS or arginase (or both) reflects the type of inflammatory response in a specific disease process. Myeloid suppressor cells (MSC) expressing arginase have been described in trauma (in both mice and humans), intra-abdominal sepsis, certain infections, and prominently, cancer. Myeloid cells expressing arginase have been shown to accumulate in patients with cancer. Arginase 1 expression is also detected in mononuclear cells after trauma or surgery. MSC efficiently deplete arginine and generate ornithine. Through arginine depletion, MSC may control NO production and regulate other arginine-dependent biological processes. Low circulating arginine has been documented in trauma and cancer, suggesting that MSC may exert a systemic effect and cause a state of arginine deficiency. Simultaneously, T lymphocytes depend on arginine for proliferation, zeta-chain peptide and T-cell receptor complex expression, and the development of memory. T-cells cocultured with MSC exhibit the molecular and functional effects associated with arginine deficiency. Not surprisingly, T-cell abnormalities, including decreased proliferation and loss of the zeta-chain, are observed in cancer and after trauma.

High mobility group B1 protein suppresses the human plasmacytoid dendritic cell response to TLR9 agonists.

Plasmacytoid dendritic cells (PDC) are innate immune effector cells that are recruited to sites of chronic inflammation, where they modify the quality and nature of the adaptive immune response. PDCs modulate adaptive immunity in response to signals delivered within the local inflammatory milieu by pathogen- or damage-associated molecular pattern, molecules, and activated immune cells (including NK, T, and myeloid dendritic cells). High mobility group B1 (HMGB1) is a recently identified damage-associated molecular pattern that is released during necrotic cell death and also secreted from activated macrophages, NK cells, and mature myeloid dendritic cells. We have investigated the effect of HMGB1 on the function of PDCs. In this study, we demonstrate that HMGB1 suppresses PDC cytokine secretion and maturation in response to TLR9 agonists including the hypomethylated oligodeoxynucleotide CpG- and DNA-containing viruses. HMGB1-inhibited secretion of several proinflammatory cytokines including IFN-alpha, IL-6, TNF-alpha, inducible protein-10, and IL-12. In addition, HMGB1 prevented the CpG induced up-regulation of costimulatory molecules on the surface of PDC and potently suppressed their ability to drive generation of IFN-gamma-secreting T cells. Our observations suggest that HMGB1 may play a critical role in regulating the immune response during chronic inflammation and tissue damage through modulation of PDC function.

The enhanced tumor selectivity of an oncolytic vaccinia lacking the host range and antiapoptosis genes SPI-1 and SPI-2.

The ability of cancer cells to evade apoptosis may permit survival of a recombinant vaccinia lacking antiapoptotic genes in cancer cells compared with normal cells. We have explored the deletion of two vaccinia virus host range/antiapoptosis genes, SPI-1 and SPI-2, for their effects on the viral replication and their ability to induce cell death in infected normal and transformed cells in vitro. Indeed, in three paired normal and transformed cell types, the SPI-1 and SPI-2 gene-deleted virus (vSP) preferentially replicates in transformed cells or p53-null cells when compared with their normal counterparts. This selectivity may be derived from the fact that vSP-infected normal cells died faster than infected cancer cells. A fraction of infected cells died with evidence of necrosis as shown by both flow cytometry and detection of high-mobility group B1 protein released from necrotic cells into the culture supernatant. When administered to animals, vSP retains full ability to replicate in tumor tissues, whereas replication in normal tissues is greatly diminished. In a model of viral pathogenesis, mice treated with vSP survived substantially longer when compared with mice treated with the wild-type virus. The mutant virus vSP displayed significant antitumoral effects in an MC38 s.c. tumor model in both nude (P < 0.001) and immunocompetent mice (P < 0.05). We conclude that this recombinant vaccinia vSP shows promise for oncolytic virus therapy. Given its enhanced tumor selectivity, improved safety profile, and substantial oncolytic effects following systemic delivery in murine models, it should also serve as a useful vector for tumor-directed gene therapy.

Dendritic cells and natural killer cells interact via multiple TNF family molecules.

Dendritic cells (DC) and natural killer (NK) cells are essential components of the innate immune system, which rapidly sense and eliminate invading pathogens and transformed cells, mediate inflammation, and initiate adaptive immune responses. During the early immune events, DC and NK cells interact and regulate each other. The cellular "cross talk" and its molecular mediators are believed to be critical to the quality and magnitude of innate and adaptive immune responses. The goal of the present manuscript is to identify and initially assess major molecular mediators of DC-NK cell interaction. We have previously shown that DC and NK cells constitutively express several tumor necrosis factor family ligands (TNFfLs) and corresponding TNF family receptors (TNFfRs). Therefore, DC and NK cells might be able to interact via cognate interplays of TNFfLs and TNFfRs. Here, we provide initial experimental evidence supporting this possibility. We found that combined but not individual ligation of several TNFfRs induced substantial increases in secretion of interleukin-12 and interferon-gamma by DC and NK cells, respectively. In contrast, specific, individual disruptions of the engagements of the corresponding TNfL-TNFfR pairs greatly impaired DC and NK cell abilities to reciprocally mediate the increases in cytokine secretion. These findings indicate that multiple TNFfLs mediate DC-NK cell interaction.

R-MC46 monoclonal antibody stimulates adhesion and phagocytosis by rat macrophages.

BACKGROUND: In our previous experiments it was shown that R-MC46 monoclonal antibody (mAb), produced at our Institute, stimulated homotypic aggregation of rat granulocytes and production ofproinflammatory cytokines. The aim of this study was to examine antigen expression and function, recognized by R-MC46 mAb on macrophages. METHODS: The expression of R-MC46 antigen on thymic and peritoneal macrophages was investigated using immunocytochemistry and flow cytometry methods. Its biochemical characterization was performed by Western blot. The ability of R-MC46 mAb to modulate adhesion and phagocytosis by macrophages was studied by using co-culture experiments with autologous thymocytes. RESULTS: R-MC46 mAb stained thymic macrophages more strongly than peritoneal macrophages. After in vivo treatment of peritoneal macrophages with Pristane, a significant up-regulation of the R-MC46 antigen expression was observed Western blot analysis showed that the mAb recognized a low molecular weight antigen of about 5.5 kDa. R-MC46 mAb significantly enhanced binding and phagocytosis of thymocytes by both thymic and peritoneal macrophages. These processes were completely blocked by WT.3 (anti-CD18) mAb. The stimulation of binding thymocyte to macrophages was higher with the use of thymic macrophages,while the phagocytosis of these cells was higher in the presence of peritoneal macrophages. CONCLUSION: R-MC46 mAb recognized a new molecule expressed by rat macrophages. The antigen is most probably involved in 82 integrin-mediated adhesion and phagocytosis, as well as proinflammatory functions of macrophages.

Differentiation of human dendritic cells from monocytes in vitro using granulocyte-macrophage colony stimulating factor and low concentration of interleukin-4.

Several laboratories have developed culture systems that allow the generation of large numbers of human dendritic cells (DC) from monocytes using granulocyte-macrophage colony stimulating factor (GM-CSF), and interleukin-4 (IL-4). In this work we provided evidence that GM-CSF (100 ng/ml) in combination with a low concentration of IL-4 (5 ng/ml) was efficient in the generation of immature, non-adherent, monocyte-derived DC as the same concentration of GM-CSF, and ten times higher concentration of IL-4 (50 ng/ml). This conclusion was based on the similar phenotype profile of DC, such as the expression of CD1a, CD80, CD86, and HLA-DR, down-regulation of CD14, and the absence of CD83, as well as on their similar allostimulatory activity for T cells. A higher number of cells remained adherent in cultures with lower concentrations of IL-4 than in cultures with higher concentrations of the cytokine. However, most of these adherent cells down-regulated CD14 and stimulated the proliferation of alloreactive T cells. In contrast, adherent cells cultivated with GM-CSF alone were predominantly macrophages, as judged by the expression of CD14 and the inefficiency to stimulate alloreactive T cells. DC generated in the presence of lower concentrations of IL-4 had higher proapoptotic potential for the Jurkat cell line than DC differentiated with higher concentrations of IL-4, suggesting their stronger cytotoxic, anti-tumor effect.

Different roles of a rat cortical thymic epithelial cell line in vitro on thymocytes and thymocyte hybridoma cells: phagocytosis, induction of apoptosis, nursing and growth promoting activities.

In this work, the interaction between a rat cortical thymic epithelial cell (TEC) line (R-TNC.1) with nursing activity and thymocytes as well as BWRT 8 thymocyte hybridoma (TH) cells has been studied. The R-TNC.1 cell line significantly bound thymocytes and TH. Binding was stronger during the first 30 min of cell incubation and was followed by a progressive deadhesion. Among adherent thymocytes the proportion of apoptotic cells increased with culture time which was a consequence of higher capacity of the line for binding of apoptotic than viable cells and induction of apoptosis in a subset of adherent thymocytes. Emperiopolesis activity of this thymic nurse cell (TNC) line was manifested by engulfment of thymocytes as well as TH cells. A subset of viable intra-TNC thymocytes has been triggered to die by apoptosis, whereas other internalized thymocytes have been stimulated to proliferate, as measured by an increase in the percentage of cells in mitosis and higher incorporation of bromodeoxyuridine (BrdU), in comparison to thymocytes cultivated alone. A significant stimulation of proliferation of engulfed TH cells was also observed. The R-TNC.1 cell line efficiently phagocytosed both apoptotic thymocytes and TH, and the process is followed by intra-TNC destruction of ingested cells. Cumulatively, these results suggest different role of the R-TNC.1 clone: phagocytosis of apoptotic cells; induction of apoptotic cell death in a subset of both bound and internalized thymocytes and stimulation of proliferation of a subset of intra-TNC thymocytes or TH cells.