Biallelic mutations in DYNC2LI1 are a rare cause of Ellis-van Creveld syndrome.

Ellis-van Creveld syndrome (EvC) is a chondral and ectodermal dysplasia caused by biallelic mutations in the EVC, EVC2 and WDR35 genes. A proportion of cases with clinical diagnosis of EvC, however, do not carry mutations in these genes. To identify the genetic cause of EvC in a cohort of mutation-negative patients, exome sequencing was undertaken in a family with 3 affected members, and mutation scanning of a panel of clinically and functionally relevant genes was performed in 24 additional subjects with features fitting/overlapping EvC. Compound heterozygosity for the c.2T>C (p.Met1?) and c.662C>T (p.Thr221Ile) variants in DYNC2LI1, which encodes a component of the intraflagellar transport-related dynein-2 complex previously found mutated in other short-rib thoracic dysplasias, was identified in the 3 affected members of the first family. Targeted resequencing detected compound heterozygosity for the same missense variant and a truncating change (p.Val141*) in 2 siblings with EvC from a second family, while a newborn with a more severe phenotype carried 2 DYNC2LI1 truncating variants. Our findings indicate that DYNC2LI1 mutations are associated with a wider clinical spectrum than previously appreciated, including EvC, with the severity of the phenotype likely depending on the extent of defective DYNC2LI1 function.

mTOR mutations in Smith-Kingsmore syndrome: Four additional patients and a review.

Smith-Kingsmore syndrome (SKS) OMIM #616638, also known as MINDS syndrome (ORPHA 457485), is a rare autosomal dominant disorder reported so far in 23 patients. SKS is characterized by intellectual disability, macrocephaly/hemi/megalencephaly, and seizures. It is also associated with a pattern of facial dysmorphology and other non-neurological features. Germline or mosaic mutations of the mTOR gene have been detected in all patients. The mTOR gene is a key regulator of cell growth, cell proliferation, protein synthesis and synaptic plasticity, and the mTOR pathway (PI3K-AKT-mTOR) is highly regulated and critical for cell survival and apoptosis. Mutations in different genes in this pathway result in known rare diseases implicated in hemi/megalencephaly with epilepsy, as the tuberous sclerosis complex caused by mutations in TSC1 and TSC2, or the PIK3CA-related overgrowth spectrum (PROS). We here present 4 new cases of SKS, review all clinical and molecular aspects of this disorder, as well as some characteristics of the patients with only brain mTOR somatic mutations.

Role of T cell recruitment and chemokine-regulated intra-graft T cell motility patterns in corneal allograft rejection.

Keratoplasty is the primary treatment to cure blindness due to corneal opacification. However, immune-mediated rejection remains the leading cause of keratoplasty failure. Here, we utilize an in vivo imaging approach to monitor, track, and characterize in real-time the recruitment of GFP-labeled allo-specific activated (Bonzo) T cells during corneal allograft rejection. We show that the recruitment of effector T cells to the site of transplantation determined the fate of corneal allografts, and that local intra-graft production of CCL5 and CXCL9/10 regulated motility patterns of effector T cells in situ, and correlated with allograft rejection. We also show that different motility patterns associate with distinct in vivo phenotypes (round, elongated, and ruffled) of graft-infiltrating effector T cells with varying proportions during progression of rejection. The ruffled phenotype was characteristic of activated effectors T cells and predominated during ongoing rejection, which associated with significantly increased T cell dynamics within the allografts. Importantly, CCR5/CXCR3 blockade decreased the motility, size, and number of infiltrating T cells and significantly prolonged allograft survival. Our findings indicate that chemokines produced locally within corneal allografts play an important role in the in situ activation and dynamic behavior of infiltrating effector T cells, and may guide targeted interventions to promote graft survival.

The eye: A window to the soul of the immune system.

The eye is considered as an immune privileged site, and with good reason. It has evolved a variety of molecular and cellular mechanisms that limit immune responses to preserve vision. For example, the cornea is mainly protected from autoimmunity by the lack of blood and lymphatic vessels, whereas the retina-blood barrier is maintained in an immunosuppressive state by the retinal pigment epithelium. However, there are several scenarios in which immune privilege is altered and the eye becomes susceptible to immune attack. In this review, we highlight the role of the immune system in two clinical conditions that affect the anterior and posterior segments of the eye: corneal transplantation and age-related macular degeneration. Interestingly, crosstalk between the innate and adaptive immune systems is critical in both acute and chronic inflammatory responses in the eye, with T cells playing a central role in combination with neutrophils and macrophages. In addition, we emphasize the advantage of using the eye as a model for in vivo longitudinal imaging of the immune system in action. Through this technique, it has been possible to identify functionally distinct intra-graft motility patterns of responding T cells, as well as the importance of chemokine signaling in situ for T cell activation. The detailed study of ocular autoimmunity could provide novel therapeutic strategies for blinding diseases while also providing more general information on acute versus chronic inflammation.

Mutations in a new gene in Ellis-van Creveld syndrome and Weyers acrodental dysostosis.

Ellis-van Creveld syndrome (EvC, MIM 225500) is an autosomal recessive skeletal dysplasia characterized by short limbs, short ribs, postaxial polydactyly and dysplastic nails and teeth. Congenital cardiac defects, most commonly a defect of primary atrial septation producing a common atrium, occur in 60% of affected individuals. The disease was mapped to chromosome 4p16 in nine Amish subpedigrees and single pedigrees from Mexico, Ecuador and Brazil. Weyers acrodental dysostosis (MIM 193530), an autosomal dominant disorder with a similar but milder phenotype, has been mapped in a single pedigree to an area including the EvC critical region. We have identified a new gene (EVC), encoding a 992-amino-acid protein, that is mutated in individuals with EvC. We identified a splice-donor change in an Amish pedigree and six truncating mutations and a single amino acid deletion in seven pedigrees. The heterozygous carriers of these mutations did not manifest features of EvC. We found two heterozygous missense mutations associated with a phenotype, one in a man with Weyers acrodental dysostosis and another in a father and his daughter, who both have the heart defect characteristic of EvC and polydactyly, but not short stature. We suggest that EvC and Weyers acrodental dysostosis are allelic conditions.

The anterior chamber of the eye as a clinical transplantation site for the treatment of diabetes: a study in a baboon model of diabetes.

AIMS/HYPOTHESIS: The aim of this study was to provide evidence that the anterior chamber of the eye serves as a novel clinical islet implantation site. METHODS: In a preclinical model, allogeneic pancreatic islets were transplanted into the anterior chamber of the eye of a baboon model for diabetes, and metabolic and ophthalmological outcomes were assessed. RESULTS: Islets readily engrafted on the iris and there was a decrease in exogenous insulin requirements due to insulin secretion from the intraocular grafts. No major adverse effects on eye structure and function could be observed during the transplantation period. CONCLUSIONS/INTERPRETATION: Our study demonstrates the long-term survival and function of allogeneic islets after transplantation into the anterior chamber of the eye. The safety and simplicity of this procedure provides support for further studies aimed at translating this technology into the clinic.

Role of interleukin 12 and costimulators in T cell anergy in vivo.

The induction of T cell anergy in vivo is thought to result from antigen recognition in the absence of co-stimulation and inflammation, and is associated with a block in T cell proliferation and Th1 differentiation. Here we have examined the role of interleukin (IL)-12, a potent inducer of Th1 responses, in regulating this process. T cell tolerance was induced by the administration of protein antigen without adjuvant in normal mice, and in recipients of adoptively transferred T cells from T cell receptor transgenic mice. The administration of IL-12 at the time of tolerance induction stimulates Th1 differentiation, but does not promote antigen-specific T cell proliferation. Conversely, inhibiting CTLA-4 engagement during anergy induction reverses the block in T cell proliferation, but does not promote full Th1 differentiation. T cells exposed to tolerogenic antigen in the presence of both IL-12 and anti-CTLA-4 antibody are not anergized, and behave identically to T cells which have encountered immunogenic antigen. These results suggest that two processes contribute to the induction of anergy in vivo; CTLA-4 engagement, which leads to a block in the ability of T cells to proliferate to antigen, and the absence of a prototypic inflammatory cytokine, IL-12, which prevents the differentiation of T cells into Th1 effector cells. The combination of IL-12 and anti-CTLA-4 antibody is sufficient to convert a normally tolerogenic stimulus to an immunogenic one.

Cytokine transcriptional events during helper T cell subset differentiation.

The molecular basis for changes in cytokine expression during T helper (Th) cell subset differentiation is not well understood. We have characterized transcriptional events related to cytokine gene expression in populations of naive T cell receptor-transgenic T cells as they are driven in vitro toward Th1 or Th2 phenotypes by interleukin (IL)-12 or IL-4 treatment, respectively. Quantitative reverse transcriptase-polymerase chain reaction analysis of cytokine transcripts indicates that interferon (IFN) gamma, IL-4, and IL-2 mRNA are expressed with distinct kinetics after naive T cells are stimulated with antigen and either IL-4 or IL-12. IFN-gamma mRNA appears as early as 6 h in IL-12-treated cultures, IL-4 appears only after 48 h in IL-4-treated cultures, and IL-2 is equivalently expressed in both types of cultures. Analyses were performed to determine if there were any differences in activation of IL-2 or IL-4 transcription factors that accompanied Th1 versus Th2 differentiation. These studies demonstrated that signal transducer and activator of transcription 6 (STAT6) binds to a sequence in the IL-4 promoter and that this STAT6-binding site can support IL-4-dependent transcription of a linked heterologous promoter. Prolonged activation of STAT6 is characteristic of populations undergoing Th2 differentiation. Furthermore, STAT6 is activated in an autocrine manner when differentiated Th2 populations are stimulated by antigen receptor ligation. Th1 populations derived from IL-12 plus antigen treatment of naive T cells remain responsive to IL-4 as indicated by induction of STAT6 and IL-4 mRNA. These data indicate that Th1 and Th2 differentiation represents the combination of different, apparently independently regulated transcriptional events. Furthermore, among transcription factors that bind to the IL-4 or IL-2 promoters, STAT6 is the one whose activation distinguishes Th2 versus Th1 development.

Dry eye is matched by increased intrasubject variability in tear osmolarity as confirmed by machine learning approach. / El ojo seco está relacionado con un aumento intrasujeto de la variabilidad de osmolaridad lagrimal confirmado por tecnología de aprendizaje de máquinas.

OBJECTIVE: Because of high variability, tear film osmolarity measures have been questioned in dry eye assessment. Understanding the origin of such variability would aid data interpretation. This study aims to evaluate osmolarity variability in a clinical setting. MATERIAL AND METHODS: Twenty dry eyes and 20 control patients were evaluated. Three consecutive osmolarity measurements per eye at 5min intervals were obtained. Variability was represented by the difference between both extreme readings per eye. Machine learning techniques were used to quantify discrimination capacity of tear osmolarity for dry eye. RESULTS: Mean osmolarities in the control and dry eye groups were 295.1±7.3mOsm/L and 300.6±11.2mOsm/L, respectively (P=.004). Osmolarity variabilities were 7.5±3.6mOsm/L and 16.7±11.9mOsm/L, for the control and dry eye groups, respectively (P<.001). Based on osmolarity, a logistic classifier showed an 85% classification accuracy. CONCLUSIONS: In the clinical setting, both mean osmolarity and osmolarity variability in the dry eye group were significantly higher than in the control group. Machine learning techniques showed good classification accuracy. It is concluded that higher variability of tear osmolarity is a dry eye feature.

VEGF increases retinal vascular ICAM-1 expression in vivo.

PURPOSE: Intraocular injections of vascular endothelial growth factor (VEGF), a peptide implicated in the pathogenesis of diabetic retinopathy, can induce retinal ischemia. Diabetic retinal ischemia may be caused, in part, by the adhesion of leukocytes to the retinal vasculature. In this study, the ability of VEGF to increase the expression of intercellular adhesion molecule-1 (ICAM-1) and other adhesion molecules in capillary endothelium and the retinal vasculature was examined. METHODS: The expression of ICAM-1, vascular cell adhesion molecule-1 (VCAM-1), E-selectin, and P-selectin on human brain capillary endothelial cell monolayers exposed to VEGF was quantitated by immunoassay. The effect of VEGF on retinal vascular ICAM-1 expression was determined in ICAM-1 immunofluorescence studies of retinal flat-mounts and in RNase protection assays. RESULTS: VEGF increased capillary endothelial cell ICAM-1 levels in a dose- and time-dependent manner (6-24 hours, plateau after 6 hours; EC50, 25 ng/ml). VEGF failed to alter E-selectin, P-selectin, or VCAM-1 levels under the conditions tested. Intravitreal injections of pathophysiologically relevant concentrations of VEGF increased ICAM-1 protein and mRNA levels in the retinal vasculature. CONCLUSIONS: VEGF increases retinal vascular ICAM-1 expression. VEGF-induced increases in ICAM-1 may promote retinal leukostasis in diabetic eyes.

Mechanisms of peripheral T cell tolerance.

Peripheral tolerance to self proteins is induced because these antigens are presented to T lymphocytes under conditions that do not allow effective immune responses to develop, or because the responses of the specific T cells are tightly regulated. The two principal mechanisms of peripheral tolerance are activation-induced cell death (AICD) and anergy. In CD4+ T lymphocytes, AICD is induced by repeated stimulation, with high levels of interleukin (IL)-2 production. Under these conditions, the T cells co-express Fas (CD95) and Fas ligand (FasL), and engagement of Fas triggers apoptotic death of the T cells. Mice with defects in Fas, FasL, IL-2R alpha or beta chain exhibit defects in AICD and develop autoimmune disease. The induction of T cell anergy is dependent on the recognition of B7 co-stimulators by the inhibitory T cell counter-receptor, CTLA-4. Failure of anergy is the likely basis for the fatal autoimmune disease of CTLA-4 knockout mice. The single-gene defects that result in autoimmunity are all defects in lymphocyte regulation, indicating that tolerance is often maintained by the control of lymphocyte responses to self antigen. The existence of distinct pathways of T cell tolerance suggest that different types of antigens induce tolerance by distinct mechanisms.

HPLC analysis of 5H-benzo[a]carbazole with antifungal activity.

A sensitive and simple high-performance liquid chromatographic (HPLC) method for the assay of 6,11-dihydro-2-methoxy-5H-benzo[a]carbazole (1) and 6,11-dihydro-2-methoxy-11-[2-(1-piperidinyl)]ethyl-5H-benzo[a]carbazole (2) was developed. The procedure is based on the use of the reversed-phase high-performance liquid chromatographic (RP-HPLC) method with UV detector. Each analysis required no longer than 11 min. A linear relationship between the concentration of both the drugs and the UV absorbance at 254 nm was obtained. This linearity was maintained over the concentration ranged from 5 to 80 microg/ml. The detection limits were found to be 1.6 and 0.7 ng for compounds 1 and 2. The quantitation limits were found to be 5.3 and 2.5 ng for compounds 1 and 2, respectively. For recovery studies, several determinations were carried out. Recovery values ranged from 98 to 102.1% for compound 1 and from 98.4 to 101.6% for compound 2. Method precision was also evaluated and RSD% found was less than 2%. This method was applied without any interference from degradation products.

In-vivo impaired T helper 1 cell development in submandibular lymph nodes due to IL-12 deficiency following antigen injection into the anterior chamber of the eye.

PURPOSE: To determine the functional properties of antigen-specific T cells that accumulate in lymph nodes following injection of antigen into the anterior chamber (AC). METHODS: Ovalbumin (OVA)-specific, CD4(+) transgenic T cell receptor (Tcr) T cells (KJ-126(+)) from DO11.10 mice were adoptively transferred into unirradiated syngeneic BALB/c mice who then received an injection of OVA in the AC or subcutaneously (SC). Three days later, KJ126(+) T cells in draining and non-draining lymph nodes were analyzed for clonal expansion, activation, and cell-cycle status by flow cytometry. In addition, the cells' functional phenotype was assessed in vitro by proliferation assays and cytokine production by ELISA. RESULTS: Tcr T cells localized exclusively to draining lymph nodes after injection of OVA into the AC (neck) and SC (neck, axilla, brachial). In both cases, the KJ126(+) T cells displayed evidence of in-vivo activation and proliferation. T cells from AC-injected donors when stimulated in vitro with OVA produced small amounts of IL-2, but no IFN-gamma, IL-4, IL-5, IL-10, or TGFbeta. By contrast, T cells from SC-injected animals displayed a Th1-like phenotype (IL-2, IFN-gamma). The draining lymph nodes of mice that received systemic or intraocular administration of exogenous IL-12 at the time of the AC injection of OVA also contained KJ126(+) T cells that secreted IFN-gamma and IL-2. CONCLUSION: Antigen attracts antigen-specific T cells to lymph nodes that drain the injection site. Whereas responding T cells in nodes draining SC-injection sites differentiated into Th1-like cells, T cells in nodes draining AC injection failed to differentiated beyond the capacity to secrete IL-2 in response to antigen. Since the failure was reversed by exogenous IL-12, we propose that orderly differentiation of antigen-specific T cells down the Th1 pathway is aborted by the eye, because of a deficit in IL-12 production.

Uveitis with neurological manifestations.

Neurological symptoms are not rare in patients with various uveitic syndromes, and they can be used as a guide to diagnosis. Some of the findings are nonspecific, but others can be typical of certain pathological conditions (table). After combining the information obtained in the medical history with the physical examination, target-oriented laboratory and diagnostic tests may then be obtained to confirm a diagnosis. Neurological manifestations can be of great value throughout this process and clearly should be carefully evaluated in every patient with uveitis.

Computed tomography in pulmonary tuberous sclerosis.

Pulmonary disease is a rare manifestation of tuberous sclerosis. Respiratory symptoms often progress to severe pulmonary insufficiency and death may ensue within a few years of diagnosis. We describe a case where a computerized tomographic scan aided early diagnosis in a patient with spontaneous pneumothorax and subtle, nonspecific changes on a plain x-ray film.

[Being a nurse in a transcultural context]. / O ser enfermeiro num contexto transcultural.

This is a descriptive and exploratory study developed according to a qualitative approach, aiming at discovering how male nurses are seen by the family and other professionals regarding their profession. Also, it has observed their technical vision about this problem. This research was done with male nurses from the Master Degree Course in Public Health Nursing at the Federal University of Paraíba. The population of this study was composed of six male nurses from which four agreed in taking part in this research. The data were collected through a questionnaire with open questions, based on the Leininger Transcultural Theory. The results showed that a great shock and cultural impositions among the male nurses, who have suffered a great variety of misconceptions and stigmatized assumptions from different social actors.

Evc regulates a symmetrical response to Shh signaling in molar development.

Tooth morphogenesis involves patterning through the activity of epithelial signaling centers that, among other molecules, secrete Sonic hedgehog (Shh). While it is known that Shh responding cells need intact primary cilia for signal transduction, the roles of individual cilia components for tooth morphogenesis are poorly understood. The clinical features of individuals with Ellis-van Creveld syndrome include various dental anomalies, and we show here that absence of the cilial protein Evc in mice causes various hypo- and hyperplasia defects during molar development. During first molar development, the response to Shh signaling is progressively lost in Evc-deficient embryos and, unexpectedly, the response consistently disappears in a buccal to lingual direction. The important role of Evc for establishing the buccal-lingual axis of the developing first molar is also supported by a displaced activity of the Wnt pathway in Evc mutants. The observed growth abnormalities eventually manifest in first molar microdontia, disruption of molar segmentation and symmetry, root fusions, and delayed differentiation. Analysis of our data indicates that both spatially and temporally disrupted activities of the Shh pathway are the primary cause for the variable dental anomalies seen in patients with Ellis-van Creveld syndrome or Weyers acrodental dysostosis.

[New options in the treatment of painful shoulder syndrome]. / Nuevas alternativas en el tratamiento del síndrome de hombro doloroso.

Shoulder pain is a common complaint in clinical practice in Primary Care and affects 20% of the general population. The usual form of treatment is based on NSAIDs, rest, rehabilitation and, as an alternative, a local injection into the joint. There are also radiofrequency techniques on the suprascapular nerve in the cases of refractory pain to these therapies. Radiofrequency can be used in two ways: Conventional Radiofrequency, using high temperatures to the target tissue with the aim of producing a thermal neurolysis and Pulsed Radiofrequency where the temperatures are lower and produces a temporary non-destructive blockage; the latter being the most common technique in the management of shoulder pain. Although the analgesic mechanism of action of Radiofrequency is unknown, recent studies have shown that it is safe, effective and Lasting. Radiofrequency of the suprascapular nerve is a valid, effective and with few complications in the treatment of shoulder pain refractory to other therapies.

Bilateral episcleritis as a manifestation of cutaneous leukocytoclastic vasculitis.

PURPOSE: The authors describe how bilateral episcleritis can be a sign of active systemic disease and can respond to treatment in a patient with cutaneous leukocytoclastic vasculitis. DESIGN: Case report. METHODS: Comprehensive ophthalmic and physical examination and color photography were used to monitor inflammation and its response to systemic immunosuppression. RESULTS: Systemic cyclophosphamide caused regression of systemic symptoms, cutaneous lesions, and episcleritis. After an 8-month follow-up, the patient has not had a systemic or ocular recurrence. CONCLUSION: Episcleritis may be a manifestation of cutaneous leukocytoclastic vasculitis. Careful examination of ocular inflammation is important in monitoring systemic disease and treatment.