The neuroretina is a novel mineralocorticoid target: aldosterone up-regulates ion and water channels in Müller glial cells.

Glucocorticoids reduce diabetic macular edema, but the mechanisms underlying glucocorticoid effects are imperfectly elucidated. Glucocorticoids may bind to glucocorticoid (GR) and mineralocorticoid (MR) receptors. We hypothesize that MR activation may influence retinal hydration. The effect of the MR agonist aldosterone (24 h) on ion/water channel expression (real-time PCR, Western blot, immunofluorescence) was investigated on cultured retinal Müller glial cells (RMGs, which contribute to fluid homeostasis in the retina), in Lewis rat retinal explants, and in retinas from aldosterone-injected eyes. We evidenced cell-specific expression of MR, GR, and 11-beta-hydroxysteroid dehydrogenase type II. Aldosterone significantly enhances expression of sodium and potassium channels ENaC-alpha (6.5-fold) and Kir4.1 (1.9-fold) through MR and GR occupancy, whereas aquaporin 4 (AQP4, 2.9-fold) up-regulation is MR-selective. Aldosterone intravitreous injection induces retinal swelling (24% increase compared to sham-injected eyes) and activation of RMGs. It promotes additional localization of Kir4.1 and AQP4 toward apical microvilli of RMGs. Our results highlight the mineralocorticoid-sensitivity of the neuroretina and show that aldosterone controls hydration of the healthy retina through regulation of ion/water channels expression in RMGs. These results provide a rationale for future investigations of abnormal MR signaling in the pathological retina.

Development of T-B cell collaboration in neonatal mice.

The neonatal immune response is impaired during the first weeks after birth. To obtain a better understanding of this immaturity, we investigated the development of T cell interactions with B cells in mice. For this purpose, we analyzed the immune response to three T-dependent antigens in vivo: (i) the polyclonal antibody response induced by vaccinia virus; (ii) the production of polyclonal and specific antibodies following immunization with hapten-carrier conjugates; (iii) the mouse mammary tumor virus superantigen (sAg) response involving an increase in sAg-reactive T cells and induction of polyclonal antibody production. After vaccinia virus injection into neonates, the polyclonal antibody response was similar to that observed in adult mice. The antibody response to hapten-carrier conjugates, however, was delayed and reduced. Injection with sAg-expressing B cells from neonatal or adult mice allowed us to determine whether B cells, T cells or both were implicated in the reduced immune response. In these sAg responses, neonatal T cells were stimulated by both neonatal and adult sAg-presenting B cells but only B cells from adult mice differentiated into IgM- and IgG-secreting plasma cells in the neonatal environment in vivo. Injecting neonatal B cells into adult mice did not induce antibody production. These results demonstrate that the environment of the neonatal lymph node is able to support a T and B cell response, and that immaturity of B cells plays a key role in the reduced immune response observed in the neonate.

Kallikrein genes are associated with lupus and glomerular basement membrane-specific antibody-induced nephritis in mice and humans.

Immune-mediated nephritis contributes to disease in systemic lupus erythematosus, Goodpasture syndrome (caused by antibodies specific for glomerular basement membrane [anti-GBM antibodies]), and spontaneous lupus nephritis. Inbred mouse strains differ in susceptibility to anti-GBM antibody-induced and spontaneous lupus nephritis. This study sought to clarify the genetic and molecular factors that maybe responsible for enhanced immune-mediated renal disease in these models. When the kidneys of 3 mouse strains sensitive to anti-GBM antibody-induced nephritis were compared with those of 2 control strains using microarray analysis, one-fifth of the underexpressed genes belonged to the kallikrein gene family,which encodes serine esterases. Mouse strains that upregulated renal and urinary kallikreins exhibited less evidence of disease. Antagonizing the kallikrein pathway augmented disease, while agonists dampened the severity of anti-GBM antibody-induced nephritis. In addition, nephritis-sensitive mouse strains had kallikrein haplotypes that were distinct from those of control strains, including several regulatory polymorphisms,some of which were associated with functional consequences. Indeed, increased susceptibility to anti-GBM antibody-induced nephritis and spontaneous lupus nephritis was achieved by breeding mice with a genetic interval harboring the kallikrein genes onto a disease-resistant background. Finally, both human SLE and spontaneous lupus nephritis were found to be associated with kallikrein genes, particularly KLK1 and the KLK3 promoter, when DNA SNPs from independent cohorts of SLE patients and controls were compared. Collectively, these studies suggest that kallikreins are protective disease-associated genes in anti-GBM antibody-induced nephritis and lupus.

An indirect immunofluorescence antibody test employing whole eggs as the antigen for the diagnosis of abdominal angiostrongyliasis

Abdominal angiostrongyliasis is a potentially fatal zoonotic disease with a broad geographical distribution throughout Central and South America. This study assessed the performance of Angiostrongylus costaricensis eggs as the antigen in an indirect immunofluorescence assay for the determination of parasite-specific IgG and IgG1 antibodies. For prevalence studies, an IgG antibody titre > 16 was identified as the diagnostic threshold with the best performance, providing 93.7% sensitivity and 84.6% specificity. Cross reactivity was evaluated with 65 additional samples from patients with other known parasitic infections. Cross reactivity was observed only in samples from individuals infected with Strongyloides stercoralis. For clinical diagnosis, we recommend the determination of IgG only as a screening test. IgG1 determination may be used to increase the specificity of the results for patients with a positive screening test.

Single-injection or continuous femoral nerve block for total knee arthroplasty?

BACKGROUND: The ideal local anesthetic regime for femoral nerve block that balances analgesia with mobility after total knee arthroplasty (TKA) remains undefined. QUESTIONS/PURPOSES: We compared two volumes and concentrations of a fixed dose of ropivacaine for continuous femoral nerve block after TKA to a single injection femoral nerve block with ropivacaine to determine (1) time to discharge readiness; (2) early pain scores and analgesic consumption; and (3) functional outcomes, including range of motion and WOMAC scores at the time of recovery. METHODS: Ninety-nine patients were allocated to one of three continuous femoral nerve block groups for this randomized, placebo-controlled, double-blind trial: a high concentration group (ropivacaine 0.2% infusion), a low concentration group (ropivacaine 0.1% infusion), or a placebo infusion group (saline 0.9% infusion). Infusions were discontinued on postoperative Day (POD) 2. The primary outcome was time to discharge readiness. Secondary outcomes included opioid consumption, pain, and functional outcomes. Ninety-three patients completed the study protocol; the study was halted early because of unanticipated changes to pain protocols at the host institution, by which time only 61% of the required number of patients had been enrolled. RESULTS: With the numbers available, the mean time to discharge readiness was not different between groups (high concentration group, 62 hours [95% confidence interval [CI], 51-72 hours]; low concentration group, 73 hours [95% CI, 63-83 hours]; placebo infusion group 65 hours [95% CI, 56-75 hours]; p = 0.27). Patients in the low concentration group consumed significantly less morphine during the period of infusion (POD 1, high concentration group, 56 mg [95% CI, 42-70 mg]; low concentration group, 35 mg [95% CI, 27-43 mg]; placebo infusion group, 48 mg [95% CI, 38-59 mg], p = 0.02; POD 2, high concentration group, 50 mg [95% CI, 41-60 mg]; low concentration group, 33 mg [95% CI, 24-42 mg]; placebo infusion group, 39 mg [95% CI, 30-48 mg], p = 0.04); however, there were no important differences in pain scores or opioid-related side effects with the numbers available. Likewise, there were no important differences in functional outcomes between groups. CONCLUSIONS: Based on this study, which was terminated prematurely before the desired sample size could be achieved, we were unable to demonstrate that varying the concentration and volume of a fixed-dose ropivacaine infusion for continuous femoral nerve block influences time to discharge readiness when compared with a conventional single-injection femoral nerve block after TKA. A low concentration of ropivacaine infusion can reduce postoperative opioid consumption but without any important differences in pain scores, side effects, or functional outcomes. These pilot data may be used to inform the statistical power of future randomized trials. LEVEL OF EVIDENCE: Level II, therapeutic study. See Guidelines for Authors for a complete description of levels of evidence.

A male with unilateral microphthalmia reveals a role for TMX3 in eye development.

Anophthalmia and microphthalmia are important birth defects, but their pathogenesis remains incompletely understood. We studied a patient with severe unilateral microphthalmia who had a 2.7 Mb deletion at chromosome 18q22.1 that was inherited from his mother. In-situ hybridization showed that one of the deleted genes, TMX3, was expressed in the retinal neuroepithelium and lens epithelium in the developing murine eye. We re-sequenced TMX3 in 162 patients with anophthalmia or microphthalmia, and found two missense substitutions in unrelated patients: c.116G>A, predicting p.Arg39Gln, in a male with unilateral microphthalmia and retinal coloboma, and c.322G>A, predicting p.Asp108Asn, in a female with unilateral microphthalmia and severe micrognathia. We used two antisense morpholinos targeted against the zebrafish TMX3 orthologue, zgc:110025, to examine the effects of reduced gene expression in eye development. We noted that the morphant larvae resulting from both morpholinos had significantly smaller eye sizes and reduced labeling with islet-1 antibody directed against retinal ganglion cells at 2 days post fertilization. Co-injection of human wild type TMX3 mRNA rescued the small eye phenotype obtained with both morpholinos, whereas co-injection of human TMX3(p.Arg39Gln) mutant mRNA, analogous to the mutation in the patient with microphthalmia and coloboma, did not rescue the small eye phenotype. Our results show that haploinsufficiency for TMX3 results in a small eye phenotype and represents a novel genetic cause of microphthalmia and coloboma. Future experiments to determine if other thioredoxins are important in eye morphogenesis and to clarify the mechanism of function of TMX3 in eye development are warranted.

In vivo expansion of T reg cells with IL-2-mAb complexes: induction of resistance to EAE and long-term acceptance of islet allografts without immunosuppression.

Via a transcription factor, Foxp3, immunoregulatory CD4(+)CD25(+) T cells (T reg cells) play an important role in suppressing the function of other T cells. Adoptively transferring high numbers of T reg cells can reduce the intensity of the immune response, thereby providing an attractive prospect for inducing tolerance. Extending our previous findings, we describe an in vivo approach for inducing rapid expansion of T reg cells by injecting mice with interleukin (IL)-2 mixed with a particular IL-2 monoclonal antibody (mAb). Injection of these IL-2-IL-2 mAb complexes for a short period of 3 d induces a marked (>10-fold) increase in T reg cell numbers in many organs, including the liver and gut as well as the spleen and lymph nodes, and a modest increase in the thymus. The expanded T reg cells survive for 1-2 wk and are highly activated and display superior suppressive function. Pretreating with the IL-2-IL-2 mAb complexes renders the mice resistant to induction of experimental autoimmune encephalomyelitis; combined with rapamycin, the complexes can also be used to treat ongoing disease. In addition, pretreating mice with the complexes induces tolerance to fully major histocompatibility complex-incompatible pancreatic islets in the absence of immunosuppression. Tolerance is robust and the majority of grafts are accepted indefinitely. The approach described for T reg cell expansion has clinical potential for treating autoimmune disease and promoting organ transplantation.

A pilot study of delayed versus immediate serial casting after botulinum toxin injection for partially reducible spastic equinus.

BACKGROUND: Serial casting is often prescribed after botulinum toxin injections to improve joint ranges of motion and to potentiate the decrease in hypertonia. The aim of this study was to compare delayed versus immediate serial casting as an adjunct to botulinum toxin therapy for partially reducible spastic equinus. METHODS: Twelve children who presented spastic equinus associated with mild gastrosoleus contracture took part. Five of them had a diagnosis of spastic diplegia, whereas 7 had a diagnosis of congenital hemiplegia. Children were randomized to immediate serial casting (same day) or delayed serial casting (4 weeks later) after botulinum toxin injection to their gastrosolei. Casts were replaced weekly for 3 weeks. RESULTS: Three children complained of pain that required recasting in the immediate casting group versus none in the delayed casting group (P = 0.08). At 3 months, there was a 27-degree improvement in the fast dorsiflexion angle (Tardieu R1) in the delayed casting group versus 17 degrees in the immediate casting group (P = 0.029). At 6 months, a 19-degree improvement persisted in the delayed group compared with 11 degrees in the immediate group (P = 0.010). CONCLUSIONS: There is a clear benefit in delaying serial casting after the injection of botulinum toxin in the recurrence of spasticity at the gastrosoleus that may also offer an advantage regarding the incidence of painful episodes associated with casting. Most importantly, reducing the recurrence of spasticity by delayed serial casting may offer the possibility of decreasing the frequency of botulinum toxin reinjections.

Prevalence of antibody to hantaviruses in humans and rodents in the Caribbean region of Colombia determined using Araraquara and Maciel virus antigens

We tested sera from 286 agricultural workers and 322 rodents in the department of Córdoba, northeastern Colombia, for antibodies against two hantaviruses. The sera were analysed by indirect ELISA using the lysate of Vero E6 cells infected with Maciel virus (MACV) or the N protein of Araraquara virus (ARAV) as antigens for the detection of antibodies against hantaviruses. Twenty-four human sera were IgG positive using one or both antigens. We detected anti-MACV IgG antibodies in 10 sera (3.5%) and anti-ARAV antibodies in 21 sera (7.34%). Of the 10 samples that were positive for MACV, seven (70%) were cross-reactive with ARAV; seven of the 21 ARAV-positive samples were cross-reactive with MACV. Using an ARAV IgM ELISA, two of the 24 human sera (8.4%) were positive. We captured 322 rodents, including 210 Cricetidae (181 Zygodontomys brevicauda, 28 Oligoryzomys fulvescens and 1 Oecomys trinitatis), six Heteromys anomalus (Heteromyidae), one Proechimys sp. (Echimyidae) and 105 Muridae (34 Rattus rattus and 71 Mus musculus). All rodent sera were negative for both antigens. The 8.4% detection rate of hantavirus antibodies in humans is much higher than previously found in serosurveys in North America, suggesting that rural agricultural workers in northeastern Colombia are frequently exposed to hantaviruses. Our results also indicate that tests conducted with South American hantavirus antigens could have predictive value and could represent a useful alternative for the diagnosis of hantavirus infection in Colombia.

Effect of treatment with cyclophosphamide in low doses upon the onset of delayed type hypersensitivity in mice chronically infected with Trypanosoma cruzi: involvement of heart interstitial dendritic cells

Acute infection with Trypanosoma cruzi results in intense myocarditis, which progresses to a chronic, asymptomatic indeterminate form. The evolution toward this chronic cardiac form occurs in approximately 30% of all cases of T. cruzi infection. Suppression of delayed type hypersensitivity (DTH) has been proposed as a potential explanation of the indeterminate form. We investigated the effect of cyclophosphamide (CYCL) treatment on the regulatory mechanism of DTH and the participation of heart interstitial dendritic cells (IDCs) in this process using BALB/c mice chronically infected with T. cruzi. One group was treated with CYCL (20 mg/kg body weight) for one month. A DTH skin test was performed by intradermal injection of T. cruzi antigen (3 mg/mL) in the hind-footpad and measured the skin thickness after 24 h, 48 h and 72 h. The skin test revealed increased thickness in antigen-injected footpads, which was more evident in the mice treated with CYCL than in those mice that did not receive treatment. The thickened regions were characterised by perivascular infiltrates and areas of necrosis. Intense lesions of the myocardium were present in three/16 cases and included large areas of necrosis. Morphometric evaluation of lymphocytes showed a predominance of TCD8 cells. Heart IDCs were immunolabelled with specific antibodies (CD11b and CD11c) and T. cruzi antigens were detected using a specific anti-T. cruzi antibody. Identification of T. cruzi antigens, sequestered in these cells using specific anti-T. cruzi antibodies was done, showing a significant increase in the number of these cells in treated mice. These results indicate that IDCs participate in the regulatory mechanisms of DTH response to T. cruzi infection.

Asymptomatic infection in individuals from the municipality of Barcelos (Brazilian Amazon) is not associated with the anti-Plasmodium falciparum glycosylphosphatidylinositol antibody response

Anti-glycosylphosphatidylinositol (GPI) antibodies (Abs) may reflect and mediate, at least partially, anti-disease immunity in malaria by neutralising the toxic effect of parasitic GPI. Thus, we assessed the anti-GPI Ab response in asymptomatic individuals living in an area of the Brazilian Amazon that has a high level of malaria transmission. For comparative purposes, we also investigated the Ab response to a crude extract prepared from Plasmodium falciparum, the merozoite surface protein (MSP)3 antigen of P. falciparum and the MSP 1 antigen of Plasmodium vivax (PvMSP1-19) in these individuals and in Angolan patients with acute malaria. Our data suggest that the Ab response against P. falciparum GPI is not associated with P. falciparum asymptomatic infection in individuals who have been chronically exposed to malaria in the Brazilian Amazon. However, this Ab response could be related to ongoing parasitaemia (as was previously shown) in the Angolan patients. In addition, our data show that PvMSP1-19may be a good marker antigen to reflect previous exposure to Plasmodium in areas that have a high transmission rate of P. vivax.

Antibody recognition of Plasmodium falciparum infected red blood cells by symptomatic and asymptomatic individuals in the Brazilian Amazon

In the Amazon Region, there is a virtual absence of severe malaria and few fatal cases of naturally occurring Plasmodium falciparum infections; this presents an intriguing and underexplored area of research. In addition to the rapid access of infected persons to effective treatment, one cause of this phenomenon might be the recognition of cytoadherent variant proteins on the infected red blood cell (IRBC) surface, including the var gene encoded P. falciparum erythrocyte membrane protein 1. In order to establish a link between cytoadherence, IRBC surface antibody recognition and the presence or absence of malaria symptoms, we phenotype-selected four Amazonian P. falciparum isolates and the laboratory strain 3D7 for their cytoadherence to CD36 and ICAM1 expressed on CHO cells. We then mapped the dominantly expressed var transcripts and tested whether antibodies from symptomatic or asymptomatic infections showed a differential recognition of the IRBC surface. As controls, the 3D7 lineages expressing severe disease-associated phenotypes were used. We showed that there was no profound difference between the frequency and intensity of antibody recognition of the IRBC-exposed P. falciparum proteins in symptomatic vs. asymptomatic infections. The 3D7 lineages, which expressed severe malaria-associated phenotypes, were strongly recognised by most, but not all plasmas, meaning that the recognition of these phenotypes is frequent in asymptomatic carriers, but is not necessarily a prerequisite to staying free of symptoms.

Anti-vascular endothelial growth factor acts on retinal microglia/macrophage activation in a rat model of ocular inflammation.

PURPOSE: To evaluate whether anti-vascular endothelial growth factor (VEGF) neutralizing antibodies injected in the vitreous of rat eyes influence retinal microglia and macrophage activation. To dissociate the effect of anti-VEGF on microglia and macrophages subsequent to its antiangiogenic effect, we chose a model of acute intraocular inflammation. METHODS: Lewis rats were challenged with systemic lipopolysaccharide (LPS) injection and concomitantly received 5 µl of rat anti-VEGF-neutralizing antibody (1.5 mg/ml) in the vitreous. Rat immunoglobulin G (IgG) isotype was used as the control. The effect of anti-VEGF was evaluated at 24 and 48 h clinically (uveitis scores), biologically (cytokine multiplex analysis in ocular media), and histologically (inflammatory cell counts on eye sections). Microglia and macrophages were immunodetected with ionized calcium-binding adaptor molecule 1 (IBA1) staining and counted based on their differential shapes (round amoeboid or ramified dendritiform) on sections and flatmounted retinas using confocal imaging and automatic quantification. Activation of microglia was also evaluated with inducible nitric oxide synthase (iNOS) and IBA1 coimmunostaining. Coimmunolocalization of VEGF receptor 1 and 2 (VEGF-R1 and R2) with IBA1 was performed on eye sections with or without anti-VEGF treatment. RESULTS: Neutralizing rat anti-VEGF antibodies significantly decreased ocular VEGF levels but did not decrease the endotoxin-induced uveitis (EIU) clinical score or the number of infiltrating cells and cytokines in ocular media (interleukin [IL]-1β, IL-6, tumor necrosis factor [TNF]-α, and monocyte chemoattractant protein [MCP]-1). Eyes treated with anti-VEGF showed a significantly decreased number of activated microglia and macrophages in the retina and the choroid and decreased iNOS-positive microglia. IBA1-positive cells expressed VEGF-R1 and R2 in the inflamed retina. CONCLUSIONS: Microglia and macrophages expressed VEGF receptors, and intravitreous anti-VEGF influenced the microglia and macrophage activation state. Taking into account that anti-VEGF drugs are repeatedly injected in the vitreous of patients with retinal diseases, part of their effects could result from unsuspected modulation of the microglia activation state. This should be further studied in other ocular pathogenic conditions and human pathology.

Antitumour effects of single or combined monoclonal antibodies directed against membrane antigens expressed by human B cells leukaemia.

Background: The increasing availability of different monoclonal antibodies (mAbs) opens the way to more specific biologic therapy of cancer patients. However, despite the significant success of therapy in breast and ovarian carcinomas with anti-HER2 mAbs as well as in non-Hodkin B cell lymphomas with anti-CD20 mAbs, certain B cell malignancies such as B chronic lymphocytic leukaemia (B-CLL) respond poorly to anti-CD20 mAb, due to the low surface expression of this molecule. Thus, new mAbs adapted to each types of tumour will help to develop personalised mAb treatment. To this aim, we analyse the biological and therapeutic properties of three mAbs directed against the CD5, CD71 or HLA-DR molecules highly expressed on B-CLL cells. Results: The three mAbs, after purification and radiolabelling demonstrated high and specific binding capacity to various human leukaemia target cells. Further in vitro analysis showed that mAb anti-CD5 induced neither growth inhibition nor apoptosis, mAb anti-CD71 induced proliferation inhibition with no early sign of cell death and mAb anti-HLA-DR induced specific cell aggregation, but without evidence of apoptosis. All three mAbs induced various degrees of ADCC by NK cells, as well as phagocytosis by macrophages. Only the anti-HLA-DR mAb induced complement mediated lysis. Coincubation of different pairs of mAbs did not significantly modify the in vitro results. In contrast with these discrete and heterogeneous in vitro effects, in vivo the three mAbs demonstrated marked anti-tumour efficacy and prolongation of mice survival in two models of SCID mice, grafted either intraperitoneally or intravenously with the CD5 transfected JOK1-5.3 cells. This cell line was derived from a human hairy cell leukaemia, a type of malignancy known to have very similar biological properties as the B-CLL, whose cells constitutively express CD5. Interestingly, the combined injection of anti-CD5 with anti-HLA-DR or with anti-CD71 led to longer mouse survival, as compared to single mAb injection, up to complete inhibition of tumour growth in 100% mice treated with both anti-HLA-DR and anti-CD5. Conclusions: Altogether these data suggest that the combined use of two mAbs, such as anti-HLA-DR and anti-CD5, may significantly enhance their therapeutic potential.