Akt-dependent enhanced migratory capacity of Th17 cells from children with lupus nephritis.

Th17 cells infiltrate the kidneys of patients with lupus nephritis (LN) and are critical for the pathogenesis of this disease. In this study, we show that enhanced activity of Stat3 in CD4(+)CD45RA(-)Foxp3(-) and Foxp3(low) effector T cells from children with LN correlates with increased frequencies of IL-17-producing cells within these T cell populations. The levels of retinoic acid-related orphan receptor c and IL-17 mRNA are significantly higher in PBMCs from children with LN than in those from controls. Mammalian target of rapamycin inhibition by rapamycin reduces both Stat3 activation in effector T cells and the frequency of IL-17-producing T cells in lupus patients. Complement factor C5a slightly increases the expression of IL-17 and induces activation of Akt in anti-CD3-activated lupus effector T cells. Th17 cells from children with LN exhibit high Akt activity and enhanced migratory capacity. Inhibition of the Akt signaling pathway significantly decreases Th17 cell migration. These findings indicate that the Akt signaling pathway plays a significant role in the migratory activity of Th17 cells from children with LN and suggest that therapeutic modulation of the Akt activity may inhibit Th17 cell trafficking to sites of inflammation and thus suppress chronic inflammatory processes in children with LN.

Enhanced activity of Akt in Teff cells from children with lupus nephritis is associated with reduced induction of tumor necrosis factor receptor-associated factor 6 and increased OX40 expression.

OBJECTIVE: The breakdown of peripheral tolerance mechanisms is central to the pathogenesis of systemic lupus erythematosus (SLE). Although true Treg cells in patients with SLE exhibit intact suppressive activity, Teff cells are resistant to suppression. The underlying mechanisms are incompletely understood. This study was undertaken to examine the Akt signaling pathway and molecules that may alter its activity in T cells in lupus patients. METHODS: The Akt pathway and its regulators were analyzed in Teff and Treg cells from children with lupus nephritis and controls using flow cytometry and real-time quantitative polymerase chain reaction. T cell proliferation was assessed by analysis of 5,6-carboxyfluorescein succinimidyl ester dilution. RESULTS: CD4+CD45RA-FoxP3(low) and FoxP3- Teff cells from children with lupus nephritis expressed high levels of activated Akt, resulting in the down-regulation of the proapoptotic protein Bim and an enhanced proliferative response. The induction of tumor necrosis factor receptor-associated factor 6 (TRAF6) was impaired, and TRAF6 levels inversely correlated with Akt activity. Although the expression of OX40 was enhanced on Teff cells from children with lupus nephritis compared to controls, OX40 stimulation failed to significantly increase TRAF6 expression in cells from patients, in contrast to those from healthy controls, but resulted in further increased Akt activation that was reversed by blockade of OX40 signaling. Moreover, inhibition of Akt signaling markedly decreased the proliferation of Teff cells from lupus patients. CONCLUSION: Our findings indicate that hyperactivation of the Akt pathway in Teff cells from children with lupus nephritis is associated with reduced induction of TRAF6 and up-regulation of OX40, which may cause Teff cell resistance to Treg cell-mediated suppression.

Use of biomarkers in the management of children with lupus.

Childhood systemic lupus erythematosus (SLE) is known to have a worse prognosis than adult-onset disease, and monitoring and treatment of the disease are still a challenge. Thus, there is an urgent need for highly reliable, non-invasive biomarkers for early detection of relapses, to avoid long-term complications and to optimize the management of children with LN. Recent studies of pediatric patients have yielded novel specific biomarkers for SLE diagnosis which can be used for monitoring disease activity and response to treatment. The most promising biomarkers in juvenile-onset SLE include cell-bound complement activation products, some genomic profiles, and urinary proteins such as neutrophil gelatinase-associated lipocalin, monocyte chemoattractant protein-1, and alpha-1-acid glycoprotein. None of these might be suitable for use as a single SLE-biomarker. More likely a combination of novel biomarkers with traditionally used data, including autoantibodies and complement, might help to enhance sensitivity and specificity for early diagnosis, disease monitoring, and prediction of relapses.cp.

The transverse musculocutaneous gracilis flap for chest wall reconstruction in male patients with Poland's syndrome.

Poland's syndrome represents a congenital unilateral deformity of the breast, chest wall, and upper limb with extremely variable manifestations. In most cases, the problem is mainly cosmetic, and the reconstruction of the chest wall should use a method designed to be performed easily and to achieve minimal scarring and donor site morbidity. We describe using a transverse musculocutaneous gracilis (TMG) flap for chest wall and anterior maxillary fold reconstruction in three male patients. In two patients, only the pectoralis major muscle was missing. In the third case, the ipsilateral latissimus dorsi muscle was also absent. The indication for surgical treatment was purely cosmetic. In all patients, a free TMG flap was performed to reconstruct the anterior axillary fold and the soft tissue defect. There was no flap loss, and all three patients had a clearly improved appearance of the chest wall. In this article, we demonstrate our experience with the use of a TMG flap for chest wall reconstruction in male patients with Poland's syndrome.

Activity of childhood lupus nephritis is linked to altered T cell and cytokine homeostasis.

PURPOSE: Standard therapy for lupus nephritis is based on non-specific immunosuppression. We aimed to identify specific alterations in T cell and cytokine homeostasis and possible associations with disease activity in children with lupus nephritis (LN). METHODS: The phenotype of circulating T cells from children with LN and healthy controls (HC) was analyzed by flow cytometry. Intracellular expression of IL-17 and INF-γ was assessed after stimulation with anti-CD3 and anti-CD28. Serum concentrations of IP10, CCL2, TGF-ß, IL-17, and IL-23 were measured by ELISA. Disease activity was determined using the Systemic Lupus Erythematosus Disease Activity Index 2000 update (SLEDAI-2K). RESULTS: Children with active LN displayed increased frequencies of effector memory CD4(+)CD45RO(+)CCR7(-) and terminal differentiated CD4(+)CD45RA(+)CCR7(-) T cells and reduced naive CD4(+)CD45RA(+)CCR7(+) T cells compared to those with inactive LN or HC. Circulating CD4(+)CXCR3(+) and CD4(+)CCR2(+) T cells correlated inversely with the renal SLEDAI-2K, whereas IP10 and CCL2 showed a positive correlation. Reduced CD4(+)Foxp3(+) T cells and serum TFG-ß levels in active LN were associated with high serum IL-17 and IL-23 levels and correlated inversely with the renal SLEDAI-2K (r = -0.5855, p = 0.0013 and r = -0.6246, p = 0.0005, respectively), whereas IL-17 and IL-23 correlated positively (r = 0.5516, p = 0.0029 and r = 0.6116, p = 0.0007, respectively). Expansion of Th17 and Th1/Th17 cells in children with LN was significantly greater than in HC (p = 0.0304 and p = 0.0067, respectively). CONCLUSION: Children with active LN display high levels of pro-inflammatory cytokines associated with an increase in effector T cells and reduction of regulatory T cells. Therapeutic regulation of the aberrant cytokine profile might specifically interrupt pathogenic mechanisms.

Effect of vascular endothelial growth factor and its receptor KDR on the transendothelial migration and local trafficking of human T cells in vitro and in vivo.

In these studies, we find that the vascular endothelial growth factor (VEGF) receptor KDR is expressed on subsets of mitogen-activated CD4(+) and CD8(+) T cells in vitro. We also found that KDR colocalizes with CD3 on mitogen-activated T cells in vitro and on infiltrates within rejecting human allografts in vivo. To evaluate whether VEGF and KDR mediate lymphocyte migration across endothelial cells (ECs), we used an in vitro live-time transmigration model and observed that both anti-VEGF and anti-KDR antibodies inhibit the transmigration of both CD4(+) and CD8(+) T cells across tumor necrosis factor α (TNFα)-activated, but not unactivated ECs. In addition, we found that interactions among CD4(+) or CD8(+) T cells and TNFα-activated ECs result in the induction of KDR on each T cell subset, and that KDR-expressing lymphocytes preferentially transmigrate across TNFα-activated ECs. Finally, using a humanized severe combined immunodeficient mouse model of lymphocyte trafficking, we found that KDR-expressing lymphocytes migrate into human skin in vivo, and that migration is reduced in mice treated with a blocking anti-VEGF antibody. These observations demonstrate that induced expression of KDR on subsets of T cells, and locally expressed VEGF, facilitate EC-dependent lymphocyte chemotaxis, and thus, the localization of T cells at sites of inflammation.

Cutting edge: Vascular endothelial growth factor-mediated signaling in human CD45RO+ CD4+ T cells promotes Akt and ERK activation and costimulates IFN-gamma production.

In this study, we find that CD45RO+ memory populations of CD4+ T lymphocytes express the vascular endothelial growth factor (VEGF) receptors KDR and Flt-1 at both the mRNA and protein levels. Furthermore, by Western blot analysis, we find that VEGF increases the phosphorylation and activation of ERK and Akt within CD4+CD45RO+ T cells. These VEGF-mediated signaling responses were inhibited by a KDR-specific small interfering RNA in a VEGF receptor-expressing Jurkat T cell line and by SU5416, a pharmacological KDR inhibitor, in CD4+CD45RO+ T cells. We also find that VEGF augments mitogen-induced production of IFN-gamma in a dose-dependent manner (p < 0.001) and significantly (p < 0.05) increases directed chemotaxis of this T cell subset. Collectively, our results for the first time define a novel function for VEGF and KDR in CD45RO+ memory T cell responses that are likely of great pathophysiological importance in immunity.

Soluble VEGF receptor 1 promotes endothelial injury in children and adolescents with lupus nephritis.

BACKGROUND: Endothelial cell injury plays a key role in the pathogenesis of lupus nephritis (LN) and atherosclerosis. The aim of this study was to identify factors involved in the process of endothelial damage in children and adolescents with LN. METHODS: We evaluated the relationship between plasma vascular endothelial growth factor (VEGF), its soluble receptors sVEGFR-1 and sVEGFR-2 and markers of endothelial inflammation and injury (angiopoietin-2 and thrombomodulin, respectively) in 23 children and adolescents with LN (active LN, n = 14; inactive LN, n = 9; mean age 15 years) and 20 healthy controls (HC; mean age 12 years). RESULTS: VEGF, sVEGFR-1, angiopoietin-2 and thrombomodulin levels were significantly higher in children and adolescents with active LN than in patients in remission or HC. In active LN, however, VEGF was inversely related to sVEGFR-1 (r = -0.802, p < 0.001), angiopoietin-2 (r = -0.684, p = 0.007) and thrombomodulin (r = -0.697, p = 0.006). There was a significant positive correlation between sVEGFR-1 and thrombomodulin (r = 0.814, p < 0.0001), but sVEGFR-2 did not significantly differ between the patient groups and did not correlate with thrombomodulin (r = 0.046, p = 0.833). CONCLUSIONS: sVEGFR-1 may play an important role in promoting endothelial damage in children and adolescents with active LN and could possibly be used to monitor disease severity.

Markers of childhood lupus nephritis indicating disease activity.

Current treatment regimens for childhood lupus nephritis (LN) are associated with significant side-effects and toxicity in vulnerable phases of growth and development. The paucity of biomarkers particularly in childhood impedes the appropriate clinical management and the development of new therapeutics. We analyzed markers of immune system (BAFF, RANTES), complement (Bb, C1q, C3d-CIC, C5a) and endothelial cell activation (sVCAM-1) in children with LN (n=22, mean age 14.8±4.7 years), nephrotic syndrome (n=13) and age-matched healthy controls (n=20) to define parameters that correlate with LN activity. Complement fragments of the alternative (Bb, p=0.0004) classical (C3d-CIC, p<0.0001) and common pathway (C5a, p<0.0001) and the levels of BAFF (p<0.0001), RANTES (p=0.0002) and sVCAM-1 (p=0.0004) were significantly higher in active compared to inactive LN. Activation of complement was associated with the occurrence of anti-C1q antibodies and reduced complement C1q. Complement-activation fragments highly correlated with the markers for immune system and endothelial cell activation. The ensemble of these parameters may be of great value in identifying early flares or remissions of childhood LN, and moreover may prove useful in the assessment of new treatments and in determining the optimization of their use.

C-reactive protein and leukocytes do not reliably indicate severity of influenza a infection in childhood.

Influenza in children may mimic other infections leading to insufficient treatment. Determination of parameters that facilitate diagnosis and indicate severity would be useful to optimize treatment modalities. We prospectively screened 432 children for influenza infection. Forty-six children at the age of 4 weeks to 14 years (median, 18 months) with confirmed influenza A infection were analyzed. A clinical score of illness severity was calculated from the symptoms presented. To evaluate the dependency of the clinical score on C-reactive protein value, leukocyte count, age, and days of hospitalization, correlation and regression analyses were carried out. Neither the C-reactive protein values (median, 0.85 mg/dL; range, 0.2-18.6; r=0.14; p=0.35) nor the leukocyte counts (median, 7.95 G/L; range, 3.5-17.6; r=-0.14, p=0.34) correlated significantly with the clinical score of influenza severity. Thus, in daily clinical practice, C-reactive protein and leukocytes seem to be insufficient parameters to describe the clinical severity of influenza A infection in children.

Clinical Applications of the Transverse Musculocutaneous Gracilis Flap for Secondary Breast Reconstruction after Simple Mastectomy.

BACKGROUND: In secondary autologous breast reconstruction, the current standard is a flap derived from the lower abdomen or the back. If these donor sites are not available because of lack of tissue, prior operations, or simply the patient's desire to avoid these donor sites, the authors use the transverse musculocutaneous gracilis flap if feasible. METHODS: The authors retrospectively evaluated only patients where secondary autologous breast reconstruction was performed with a transverse musculocutaneous gracilis flap because of the prior mentioned reasons. Indications, limitations, advantages, and technique are discussed by sharing the authors' experience in 23 patients using 26 transverse musculocutaneous gracilis flaps. RESULTS: No flap loss could be observed in this series. In four patients, minimal lateral skin necrosis could easily be managed by débridement and primary wound closure. In 12 cases, subsequent lipofilling was performed for a better breast shape. On average, patient satisfaction was high. CONCLUSIONS: Secondary reconstruction after simple mastectomy using the transverse musculocutaneous gracilis flap requires a little more experience than after skin-sparing mastectomy but, especially combined with later lipofilling, can lead to an optimally shaped breast in selected patients with substantial skin laxity and fat distribution at the inner thigh. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

Molecular evaluation of renal biopsies: a search for predictive and prognostic markers in lupus nephritis.

The therapeutic management of patients with lupus nephritis (LN) remains a major challenge. The availability of biomarkers that accurately predict renal flares, response to immunosuppressive treatment and risk of progression to end-stage renal disease would allow the more effective use of currently available immunosuppression, with less toxicity. The molecular analysis of renal biopsy samples provides direct insights into pathologic processes in LN, and constitutes a valuable approach to discover biomarkers that may be used to improve the outcome of LN patients. Reich et al. recently described a method for simultaneously detecting multiple mRNA transcripts in archived formalin-fixed renal biopsy samples. The authors identify three transcripts (EGF, MMP7 and COL1A1) that relate to pathological indices of kidney injury and function.

Lower limb salvage in a 7-month-old infant using free tissue transfer.

Free flap reconstruction in infants is extremely rare. A seven-and-a-half-month-old male infant sustained an extensive soft tissue defect on his left knee caused by extravasation of an intraosseous arterenol infusion. A free latissimus dorsi flap was successfully performed for soft tissue reconstruction. Indications, advantages, and outcome of the procedure are discussed.

Rituximab in childhood systemic lupus erythematosus refractory to conventional immunosuppression: case report.

Rituximab, a chimeric monoclonal antibody specific for human CD20, has recently been used for the treatment of autoimmune diseases. A 14-year-old patient with severe systemic lupus erythematosus (SLE) and class IV glomerulonephritis presented with immunologic and clinical resistance to conventional immunosuppressive therapy for 10 months after diagnosis. To induce remission of active SLE, treatment with 6 monthly rituximab at 375 mg/m(2), oral mycophenolate and prednisone was initiated followed by maintenance rituximab every 3 months. The SLEDAI decreased significantly from 31 at diagnosis to 14 after nine applications of rituximab. Extrarenal symptoms of SLE improved significantly. However, after induction therapy with rituximab the patient presented a reversible intrinsic acute renal insufficiency for a period of 3 weeks. The discontinuation of the daily medication (oral prednisone and mycophenolate) by the patient herself may explain the progression of active SLE associated with the reversible acute renal failure. Under intensive immunosuppressive therapy improvement of active disease manifestations and stabilization of plasma creatinine concentrations to normal values was observed. However, proteinuria remained elevated and improved only after a protracted period (median protein-to-creatinine ratio 5.2 g/g, range 0.8-11.2 g/g). Hematuria and urinary cell casts persisted. In conclusion, the extrarenal symptoms of the patient responded particularly well to rituximab. However, despite complete B-cell elimination, renal remission of SLE was not achieved. Thus, it may be possible that humoral and cellular immune mechanisms have a fundamental involvement in the pathogenesis of SLE nephritis.

Glucocorticoids enhance interleukin-4 production to neo-antigen (hyaluronidase) in children immunocompromised with cytostatic drugs.

Immunoglobulin E (IgE)-mediated immediate-type allergic reactions to hyaluronidase have been observed in children with central nervous system (CNS) tumors. Glucocorticoids, used as therapy for brain edema, are discussed controversially as T helper 2 (Th2) stimulatory factors. In this study we investigated the role of glucocorticoids on a Th2 cytokine-promoting effect in children with CNS tumors. Peripheral blood mononuclear cells (PBMCs) from: 29 children suffering from malignant brain tumors, of whom 23 received short-term glucocorticoid treatment (for 3-4 days) during the course of chemotherapy; 18 children with nephrotic syndrome or renal transplantation receiving long-term glucocorticoid treatment; and 13 healthy children, were incubated with phytohemagglutinin (PHA) and/or anti-CD28 monoclonal antibody (mAb) and, in a second approach, with hyaluronidase. The concentrations of Th cell-mediated cytokines - interleukin (IL)-4, IL-10, and interferon-gamma (IFN-gamma) - were measured in supernatants. The IL-4 production of PBMCs incubated with PHA/anti-CD28 mAb from children with repeated co-administration of glucocorticoids, hyaluronidase, and cytostatic drugs (median: 249.9 pg/ml; range: 234.4-261.7) was significantly higher (p < 0.0001) than IL-4 production of PBMC from children of all the other groups (median: 86.18; range: 16.0-212.5). There was no significant difference in the levels of IL-10 and IFN-gamma within the groups. PBMCs stimulated only with hyaluronidase failed to produce detectable levels of cytokines. The results of this study indicate that repeated co-administration of glucocorticoids and hyaluronidase (a neo-antigen) enhance IL-4 production in vitro and thus may induce the production of specific IgE antibodies in children immunocompromised with cytostatic drugs. Hyaluronidase itself does not stimulate in vitro IL-4 synthesis in PBMCs of children receiving cytostatic drugs.

Dose-dependent and preterm- accentuated diaplacental transport of nutritive allergens in vitro.

OBJECTIVE: The mechanisms by which nutritive allergens are transported from mother to fetus and the ensuing immunological response are incompletely understood. We investigated the role of different allergen concentrations in influencing the diaplacental allergen transport in preterm and term placentas. METHOD: Twenty-seven human term placentas and 12 preterm placentas were dually perfused in vitro for up to 4 h by adding alternately two different nutritive allergens, beta-lactoglobulin (BLG) or ovalbumin (OVA), at four different allergen concentrations (0.02, 0.2, 2 and 20 mg/ml) to the maternal perfusate medium. Allergen concentrations in fetal venous outflow samples collected during perfusion were measured by using specific ELISAs. RESULTS: Perfusion of increasing allergen concentrations via the maternal circulation resulted in a concentration-dependent increase of fetal allergen uptake in all term and preterm placentas. A mean maternal-to-fetal ratio of 20,000/1 and 3,000/1 for BLG, and 40,000/1 and 5,000/1 for OVA was found in term and preterm placentas, respectively. Preterm placentas (27-36 weeks of gestation) were found to favor the diaplacental passage of nutritive allergens compared with placentas at term (>36 weeks of gestation). CONCLUSION: Maternal-to-fetal allergen transport occurs in a dose-dependent and molecular weight-dependent manner with clear accentuation in preterm placentas.