Dual functionality of diacylglycerols in water-in-oil emulsion gel systems.

Distearin (DS) can be used as an emulsifier, due to its surface activity derived from the amphiphilic nature of the molecule, moreover, it can also crystallize and form a 3D crystal network that can induce oil gelation. The current research aimed to examine the ability to combine both emulsifying and oil gelation properties to structure and stabilize water-in-oil emulsion gel system. Different water contents and DS concentrations produce emulsion gels with different textural attributes while incorporating up to 30% of water in a 15% wt. DS-based oleogel resulted in stable white gels. Microscopy imaging confirmed the formation of a water-in-oleogel type emulsion gel characterized by DS crystallization in the continuous phase and at the interface through Pickering mechanism. A positive relation was observed between the G' and hardness values and water content, suggesting gel strengthening resulted from interactions between the DS crystals at the interface and the continuous phase, as suggested by the active filler theory. Thermal analysis revealed two broad melting events at the temperature range of 42.2-44.9 °C and 55.9-58.6 °C for emulsion gels with 10-30% water content, suggesting initial melting of ß' polymorph and transition to ß during melting, which was confirmed by XRD. The results showed that homogenization significantly improved the oil retention of the gels due to increased crystal surface area, while water addition slightly reduced it. Compared with traditional emulsions or oleogels, this water-in-oil gel system demonstrated prolonged stability and enhanced mechanical properties due to the dual functionality of DS at the water/oil interface and bulk.

Skin Microbiome Modulates the Effect of Ultraviolet Radiation on Cellular Response and Immune Function.

The skin is colonized by a diverse microbiome intricately involved in various molecular and cellular processes within the skin and beyond. UV radiation is known to induce profound changes in the skin and modulate the immune response. However, the role of the microbiome in UV-induced immune suppression has been overlooked. By employing the standard model of contact hypersensitivity (using germ-free mice) we found diminished UV-induced systemic immune suppression in the presence of microbiome. Upon UV exposure, we found enhanced epidermal hyperplasia and neutrophilic infiltration in the presence and enhanced numbers of mast cells and monocyte or macrophages in the absence of microbiome. Transcriptome analysis revealed a predominant expression of cytokine genes related to pro-inflammatory milieu in the presence versus immunosuppressive milieu (with increased interleukin-10) in the absence of microbiome. Collectively, microbiome abrogates the immunosuppressive response to UV by modulating gene expression and cellular microenvironment of the skin.

Cbl-b deficiency provides protection against UVB-induced skin damage by modulating inflammatory gene signature.

Exposure of skin to ultraviolet (UV) radiation induces DNA damage, inflammation, and immune suppression that ultimately lead to skin cancer. However, some of the pathways that regulate these events are poorly understood. We exposed mice to UVB to study its early effects in the absence of Cbl-b, a known suppressor of antitumor immune response in the skin. Cbl-b-/- mice were protected from UV-induced cell damage as shown by the lower number of cyclobutane pyrimidine dimers and sunburn cells in exposed skin compared to wild-type mice. Microarray data revealed that deficiency of Cbl-b resulted in differential expression of genes involved in apoptosis evasion, tumor suppression and cell survival in UV-exposed skin. After UVB, Cbl-b-/- mice upregulated gene expression pattern associated with regulation of epidermal cell proliferation linked to Wnt signaling mediators and enzymes that relate to cell removal and tissue remodeling like MMP12. Additionally, the skin of Cbl-b-/- mice was protected from chronic inflammatory responses and epidermal hyperplasia in a 4-weeks UVB treatment protocol. Overall, our results suggest a novel role for Cbl-b in regulating inflammation and physiologic clearance of damaged cells in response to UVB by modulating inflammatory gene signature.

8-Methoxypsoralen Plus Ultraviolet A Reduces the Psoriatic Response to Imiquimod in a Murine Model.

The effects of 8-Methoxypsoralen plus ultraviolet A (PUVA) or ultraviolet B (UVB) alone on imiquimod-induced psoriasis were examined in a mouse model. Mouse skin was treated with repetitive sub-phototoxic doses of PUVA or UVB before or during the induction of toll-like receptor 7/8 activation and psoriasis through the application of imiquimod. PUVA, to a greater degree than UVB, suppressed the established imiquimod-induced psoriatic phenotype, but pretreatment with PUVA prior to administration of imiquimod also reduced the susceptibility of murine skin to respond to imiquimod to a greater degree than did pretreatment with UVB. PUVA downregulated baseline levels of miRNA27a and 29a, as well as interferon-γ, interleukin-17 and -9, cytokines, which drive psoriatic inflammation. Microarray analysis showed enrichment of senescence pathway genes linked to upregulation of p16/p21 proteins after PUVA pretreatment. However, the anti-psoriatic effect of PUVA was lost when there was an interval of 7 days between final exposure to PUVA and the start of administration of imiquimod. This indicated that (UVB and) PUVA diminished imiquimod-induced established psoriatic inflammation, but also primed the skin in favour of a reduced responsiveness to toll-like receptor activation.

Patients with sporadic and familial amyotrophic lateral sclerosis found value in genetic testing.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is increasingly recognized as a genetic disease. There is no consensus, however, as to the role of genetic testing in the care of the ALS patient. METHODS: We conducted a survey to study patient access, attitudes, and experience with ALS genetic testing among patients enrolled in a US ALS registry. RESULTS: Among 449 survey respondents, 156 (34.7%) were offered testing and 105 of 156 (67.3%) completed testing. The majority of respondents with familial ALS (fALS) (31/45, 68.9%) were offered testing, while a minority of respondents with sporadic ALS (sALS) (111/404, 27.5%) were offered testing (p = .00001). Comparison of mean test experience scores between groups revealed that respondents with fALS were no more likely to report a favorable experience with genetic testing than those with sALS (p = .51). Respondents who saw a genetic counselor did not have significantly different test experience scores, compared to those who did not (p = .14). In addition, no differences in test experience scores were observed between those who received positive or negative genetic test results (p = .98). CONCLUSION: These data indicate that patients with ALS found value in clinical genetic testing.

MUG-Mel2, a novel highly pigmented and well characterized NRAS mutated human melanoma cell line.

NRAS mutation in melanoma has been associated with aggressive tumor biology and poor prognosis. Although targeted therapy has been tested for NRAS mutated melanoma, response rates still appear much weaker, than in BRAF mutated melanoma. While plenty of cell lines exist, however, only few melanogenic cell lines retain their in vivo characteristics. In this work we present an intensively pigmented and well-characterized cell line derived from a highly aggressive NRAS mutated cutaneous melanoma, named MUG-Mel2. We present the clinical course, unique morphology, angiogenic properties, growth characteristics using in vivo experiments and 3D cell culture, and results of the exome gene sequencing of an intensively pigmented melanogenic cell line MUG-Mel2, derived from a cutaneous metastasis of an aggressive NRAS p. Q61R mutated melanoma. Amongst several genetic alterations, mutations in GRIN2A, CREBP, PIK3C2G, ATM, and ATR were present. These mutations, known to reinforce DNA repair problems in melanoma, might serve as potential treatment targets. The aggressive and fast growing behavior in animal models and the obtained phenotype in 3D culture reveal a perfect model for research in the field of NRAS mutated melanoma.

NorUrsodeoxycholic acid ameliorates cholemic nephropathy in bile duct ligated mice.

BACKGROUND & AIMS: Severe cholestasis may cause cholemic nephropathy that can be modeled in common bile duct ligated (CBDL) mice. We aimed to explore the therapeutic efficacy and mechanisms of norursodeoxycholic acid (norUDCA) in cholemic nephropathy. METHODS: In 8-week CBDL mice fed with norUDCA (prior or post CBDL) or chow we evaluated serum urea levels, urine cytology and urinary neutrophil gelatinase associated lipocalin (uNGAL), kidney and liver tissue quantification of fibrosis by hydroxyproline content and gene chip expression looking at key genes of inflammation and fibrosis. Moreover, we comprehensively analysed bile acid profiles in liver, kidney, serum and urine samples. RESULTS: NorUDCA-fed CBDL mice had significantly lower serum urea and uNGAL levels and less severe cholemic nephropathy as demonstrated by normal urine cytology, significantly reduced tubulointerstitial nephritis, and renal fibrosis as compared to controls. NorUDCA underwent extensive metabolism to produce even more hydrophilic compounds that were significantly enriched in kidneys. CONCLUSION: NorUDCA ameliorates cholemic nephropathy due to the formation of highly hydrophilic metabolites enriched in kidney. Consequently, norUDCA may represent a medical treatment for cholemic nephropathy. LAY SUMMARY: The term cholemic nephropathy describes renal dysfunction together with characteristic morphological alterations of the kidney in obstructive cholestasis that can be mimicked by ligation of the common bile duct in mice. Feeding the hydrophilic bile acid norUDCA to bile duct ligated mice leads to a significant amelioration of the renal phenotype due to the formation of highly hydrophilic metabolites enriched in the kidney and may therefore represent a medical treatment for cholemic nephropathy.

Zinc intake and status in Austria in the light of different reference values.

Zinc has been identified as a critical micronutrient also in high-income countries. There is still some uncertainty about the evaluation of zinc sufficiency due to divergent daily intake reference values. We wanted to exemplify this issue using data from the Austrian Study on Nutritional Status 2012. Plasma zinc concentrations were measured in a nationally representative sample of 872 persons aged 6-80 years (55.5 % female). Dietary zinc intake was estimated from two 24h dietary recalls. Additionally, parameters of the antioxidative status (plasma malondialdehyde (MDA), total antioxidative capacity) and activities of alkaline phosphatase (AP), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px)) were determined. Zinc status was marginal in schoolchildren (40 % of boys and 22 % of girls) and in elderly (28 % of men and 33 % of women). Dietary zinc intake was also unsatisfactory in these groups with 38 % of boys and 32 % of girls and 64.5 % of older men below the nationally recommended intake levels. However, the adequacy of zinc intake varied with different reference values. Adults were more likely to meet the D-A-CH reference values and those from the European Food Safety Authority than the recommendations of the International Zinc Nutrition Consultative Group (IZiNCG) and the Institute of Medicine, whereas children met the IZiNCG values best. Zinc status correlated weakly with AP activity (r = -0.298, p < 0.001) and some antioxidant status markers (CAT, MDA, GSH-PX, SOD), especially in the elderly (MDA: r = -0.527, p < 0.001, and SOD: r = -0.466, p = 0.002). Our results suggest a suboptimal zinc supply in Austria particularly among schoolchildren and older adults.

Patients with Amyotrophic Lateral Sclerosis Have High Interest in and Limited Access to Genetic Testing.

Although genetic testing for amyotrophic lateral sclerosis (ALS) is widely available, it is unknown what proportion of patients with ALS have access to genetic counseling and testing, and patient attitudes towards ALS genetic testing have not been studied. We conducted a national survey of ALS patients enrolled in the Agency for Toxic Substances and Disease Registry, which consisted of multiple choice questions and two 12 item Likert scale series assessing respondents' experience with and attitude toward genetic testing. The survey had an 8 % response rate, with 449 completed responses. Genetic testing was offered to 33.4 % and completed by 67.1 % of those offered. A minority of respondents (12.5 %) saw a genetic counselor, and were much more likely to be offered genetic testing (p = 0.0001). Respondents with a family history of ALS (8.4 %) were more likely to be offered testing (p = 0.0001) and complete testing (p = 0.05). Respondents with a family history of ALS were more likely to report a favorable attitude towards genetic testing (p = 0.0003), as were respondents who saw a genetic counselor (p = 0.02). The majority of respondents (82.7 %) felt that genetic testing should be offered to all patients with ALS. Our results indicate that ALS patients may have limited access to genetic testing, but perceive benefit from this service. Development of practice guidelines for genetic testing in ALS, to include the routine offer of genetic counseling, may result in broader and more consistent access to these services.

Chondrocyte cultures from human proximal interphalangeal finger joints.

Osteoarthritis (OA) of the hand is a common disease resulting in pain and impaired function. The pathogenesis of hand OA (HOA) is elusive and models to study it have not been described. Chondrocyte culture has been essential to understand cartilage degeneration, which is a hallmark of OA. We investigated the feasibility of human chondrocyte culture derived from proximal interphalangeal (PIP) finger joints. Hyaline cartilage of the PIP and knee joints was obtained from human cadavers. Chondrocytes harvested up to 236 h after death of the donors were viable and expressed chondrocyte-specific genes. Gene expression comparing chondrocytes from PIP and knee joints using Affymetrix GeneChip arrays resulted in a unique PIP-specific gene expression pattern. Genes involved in developmental processes including the WNT pathway were differentially expressed between the joints. These findings suggest that our knowledge on chondrocyte biology derived mainly from knee and hip joints may not apply to chondrocytes of the PIP joints and some of the distinctive features of HOA may be caused by the specific properties of PIP chondrocytes. Chondrocyte culture of PIP cartilage is a novel tool to study cartilage degeneration in HOA. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1569-1575, 2016.

Effect of Laminin-A4 inhibition on cluster formation of human osteoarthritic chondrocytes.

Formation of chondrocyte clusters is not only a morphological sign of osteoarthritis but it is also observed in cell culture. Active locomotion of chondrocytes is controlled by integrins in vitro. Integrins bind to Laminin-A4 (LAMA4), a protein that is highly expressed in vivo in clusters of hypertrophic chondrocytes. We tested if LAMA4 is relevant for cluster formation. Human chondrocytes were cultured in a 2D matrigel model and treated with different concentrations of a monoclonal inhibitory anti-LAMA4-antibody. Migration and cluster formation was analysed using live cell imaging technique. Full genome gene expression analysis was performed to assess the effect of LAMA4 inhibition. The data set were screened for genes relevant to cell motility. F-actin staining was performed to document cytoskeletal changes. Anti-LAMA4 treatment significantly reduced the rate of cluster formation in human chondrocytes. Cells changed their surface morphology and exhibited fewer protrusions. Expression of genes associated with cellular motility and migration was affected by anti-LAMA4 treatment. LAMA4-integrin signalling affects chondrocyte morphology and gene expression in vitro, thereby contributing to cluster formation in human osteoarthritic chondrocytes.

Low spinophilin expression enhances aggressive biological behavior of breast cancer.

Spinophilin, a putative tumor suppressor gene, has been shown to be involved in the pathogenesis of certain types of cancer, but its role has never been systematically explored in breast cancer. In this study, we determined for the first time the expression pattern of spinophilin in human breast cancer molecular subtypes (n = 489) and correlated it with survival (n = 921). We stably reduced spinophilin expression in breast cancer cells and measured effects on cellular growth, apoptosis, anchorage-independent growth, migration, invasion and self-renewal capacity in vitro and metastases formation in vivo. Microarray profiling was used to determine the most abundantly expressed genes in spinophilin-silenced breast cancer cells. Spinophilin expression was significantly lower in basal-like breast cancer (p<0.001) and an independent poor prognostic factor in breast cancer patients (hazard ratio = 1.93, 95% confidence interval: 1.24 -3.03; p = 0.004) A reduction of spinophilin levels increased cellular growth in breast cancer cells (p<0.05), without influencing activation of apoptosis. Anchorage-independent growth, migration and self-renewal capacity in vitro and metastatic potential in vivo were also significantly increased in spinophilin-silenced cells (p<0.05). Finally, we identified several differentially expressed genes in spinophilin-silenced cells. According to our data, low levels of spinophilin are associated with aggressive behavior of breast cancer.

Synergistic effects of NOD1 or NOD2 and TLR4 activation on mouse sickness behavior in relation to immune and brain activity markers.

Toll-like receptors (TLRs) and nuclear-binding domain (NOD)-like receptors (NLRs) are sensors of bacterial cell wall components to trigger an immune response. The TLR4 agonist lipopolysaccharide (LPS) is a strong immune activator leading to sickness and depressed mood. NOD agonists are less active but can prime immune cells to augment LPS-induced cytokine production. Since the impact of NOD and TLR co-activation in vivo has been little studied, the effects of the NOD1 agonist FK565 and the NOD2 agonist muramyl dipeptide (MDP), alone and in combination with LPS, on immune activation, brain function and sickness behavior were investigated in male C57BL/6N mice. Intraperitoneal injection of FK565 (0.001 or 0.003mg/kg) or MDP (1 or 3mg/kg) 4h before LPS (0.1 or 0.83mg/kg) significantly aggravated and prolonged the LPS-evoked sickness behavior as deduced from a decrease in locomotion, exploration, food intake and temperature. When given alone, FK565 and MDP had only minor effects. The exacerbation of sickness behavior induced by FK565 or MDP in combination with LPS was paralleled by enhanced plasma protein and cerebral mRNA levels of proinflammatory cytokines (IFN-γ, IL-1ß, IL-6, TNF-α) as well as enhanced plasma levels of kynurenine. Immunohistochemical visualization of c-Fos in the brain revealed that NOD2 synergism with TLR4 resulted in increased activation of cerebral nuclei relevant to sickness. These data show that NOD1 or NOD2 synergizes with TLR4 in exacerbating the immune, sickness and brain responses to peripheral immune stimulation. Our findings demonstrate that the known interactions of NLRs and TLRs at the immune cell level extend to interactions affecting brain function and behavior.

Extended ultrastructural characterization of chordoma cells: the link to new therapeutic options.

Chordomas are rare bone tumors, developed from the notochord and largely resistant to chemotherapy. A special feature of this tumor is the heterogeneity of its cells. By combining high pressure freezing (HPF) with electron tomography we were able to illustrate the connections within the cells, the cell-cell interface, and the mitochondria-associated endoplasmic reticulum membrane complex that appears to play a special role among the characteristics of chordoma. These lipid raft-like regions are responsible for lipid syntheses and for calcium signaling. Compared to other tumor cells, chordoma cells show a close connection of rough endoplasmic reticulum and mitochondria, which may influence the sphingolipid metabolism and calcium release. We quantified levels of ceramide and glycosylceramide species by the methyl tert-butyl ether extraction method and we assessed the intracellular calcium concentration with the ratiometric fluorescent dye Fura-2AM. Measurements of the changes in the intracellular calcium concentration revealed an increase in calcium due to the application of acetylcholine. With regard to lipid synthesis, glucosylceramide levels in the chordoma cell line were significantly higher than those in normal healthy cells. The accumulation of glycosylceramide in drug resistant cancer cells has been confirmed in many types of cancer and may also account for drug resistance in chordoma. This study aimed to provide a deep morphological description of chordoma cells, it demonstrated that HPF analysis is useful in elucidating detailed structural information. Furthermore we demonstrate how an accumulation of glycosylceramide in chordoma provides links to drug resistance and opens up the field for new research options.

Monocytic cell differentiation from band-stage neutrophils under inflammatory conditions via MKK6 activation.

During inflammation, neutrophils are rapidly mobilized from the bone marrow storage pool into peripheral blood (PB) to enter lesional sites, where most rapidly undergo apoptosis. Monocytes constitute a second wave of inflammatory immigrates, giving rise to long-lived macrophages and dendritic cell subsets. According to descriptive immunophenotypic and cell culture studies, neutrophils may directly "transdifferentiate" into monocytes/macrophages. We provide mechanistic data in human and murine models supporting the existence of this cellular pathway. First, the inflammatory signal-induced MKK6-p38MAPK cascade activates a monocyte differentiation program in human granulocyte colony-stimulating factor-dependent neutrophils. Second, adoptively transferred neutrophils isolated from G-CSF-pretreated mice rapidly acquired monocyte characteristics in response to inflammatory signals in vivo. Consistently, inflammatory signals led to the recruitment of osteoclast progenitor cell potential from ex vivo-isolated G-CSF-mobilized human blood neutrophils. Monocytic cell differentiation potential was retained in left-shifted band-stage neutrophils but lost in neutrophils from steady-state PB. MKK6-p38MAPK signaling in HL60 model cells led to diminishment of the transcription factor C/EBPα, which enabled the induction of a monocytic cell differentiation program. Gene profiling confirmed lineage conversion from band-stage neutrophils to monocytic cells. Therefore, inflammatory signals relayed by the MKK6-p38MAPK cascade induce monocytic cell differentiation from band-stage neutrophils.

Use of whole genome expression analysis in the toxicity screening of nanoparticles.

The use of nanoparticles (NPs) offers exciting new options in technical and medical applications provided they do not cause adverse cellular effects. Cellular effects of NPs depend on particle parameters and exposure conditions. In this study, whole genome expression arrays were employed to identify the influence of particle size, cytotoxicity, protein coating, and surface functionalization of polystyrene particles as model particles and for short carbon nanotubes (CNTs) as particles with potential interest in medical treatment. Another aim of the study was to find out whether screening by microarray would identify other or additional targets than commonly used cell-based assays for NP action. Whole genome expression analysis and assays for cell viability, interleukin secretion, oxidative stress, and apoptosis were employed. Similar to conventional assays, microarray data identified inflammation, oxidative stress, and apoptosis as affected by NP treatment. Application of lower particle doses and presence of protein decreased the total number of regulated genes but did not markedly influence the top regulated genes. Cellular effects of CNTs were small; only carboxyl-functionalized single-walled CNTs caused appreciable regulation of genes. It can be concluded that regulated functions correlated well with results in cell-based assays. Presence of protein mitigated cytotoxicity but did not cause a different pattern of regulated processes.

Hypomorphic PCNA mutation underlies a human DNA repair disorder.

Numerous human disorders, including Cockayne syndrome, UV-sensitive syndrome, xeroderma pigmentosum, and trichothiodystrophy, result from the mutation of genes encoding molecules important for nucleotide excision repair. Here, we describe a syndrome in which the cardinal clinical features include short stature, hearing loss, premature aging, telangiectasia, neurodegeneration, and photosensitivity, resulting from a homozygous missense (p.Ser228Ile) sequence alteration of the proliferating cell nuclear antigen (PCNA). PCNA is a highly conserved sliding clamp protein essential for DNA replication and repair. Due to this fundamental role, mutations in PCNA that profoundly impair protein function would be incompatible with life. Interestingly, while the p.Ser228Ile alteration appeared to have no effect on protein levels or DNA replication, patient cells exhibited marked abnormalities in response to UV irradiation, displaying substantial reductions in both UV survival and RNA synthesis recovery. The p.Ser228Ile change also profoundly altered PCNA's interaction with Flap endonuclease 1 and DNA Ligase 1, DNA metabolism enzymes. Together, our findings detail a mutation of PCNA in humans associated with a neurodegenerative phenotype, displaying clinical and molecular features common to other DNA repair disorders, which we showed to be attributable to a hypomorphic amino acid alteration.

Resolving tumor heterogeneity: genes involved in chordoma cell development identified by low-template analysis of morphologically distinct cells.

The classical sacrococcygeal chordoma tumor presents with a typical morphology of lobulated myxoid tumor tissue with cords, strands and nests of tumor cells. The population of cells consists of small non-vacuolated cells, intermediate cells with a wide range of vacuolization and large heavily vacuolated (physaliferous) cells. To date analysis was only performed on bulk tumor mass because of its rare incidence, lack of suited model systems and technical limitations thereby neglecting its heterogeneous composition. We intended to clarify whether the observed cell types are derived from genetically distinct clones or represent different phenotypes. Furthermore, we aimed at elucidating the differences between small non-vacuolated and large physaliferous cells on the genomic and transcriptomic level. Phenotype-specific analyses of small non-vacuolated and large physaliferous cells in two independent chordoma cell lines yielded four candidate genes involved in chordoma cell development. UCHL3, coding for an ubiquitin hydrolase, was found to be over-expressed in the large physaliferous cell phenotype of MUG-Chor1 (18.7-fold) and U-CH1 (3.7-fold) cells. The mannosyltransferase ALG11 (695-fold) and the phosphatase subunit PPP2CB (18.6-fold) were found to be up-regulated in large physaliferous MUG-Chor1 cells showing a similar trend in U-CH1 cells. TMEM144, an orphan 10-transmembrane family receptor, yielded contradictory data as cDNA microarray analysis showed up- but RT-qPCR data down-regulation in large physaliferous MUG-Chor1 cells. Isolation of few but morphologically identical cells allowed us to overcome the limitations of bulk analysis in chordoma research. We identified the different chordoma cell phenotypes to be part of a developmental process and discovered new genes linked to chordoma cell development representing potential targets for further research in chordoma tumor biology.

Mutations in KPTN cause macrocephaly, neurodevelopmental delay, and seizures.

The proper development of neuronal circuits during neuromorphogenesis and neuronal-network formation is critically dependent on a coordinated and intricate series of molecular and cellular cues and responses. Although the cortical actin cytoskeleton is known to play a key role in neuromorphogenesis, relatively little is known about the specific molecules important for this process. Using linkage analysis and whole-exome sequencing on samples from families from the Amish community of Ohio, we have demonstrated that mutations in KPTN, encoding kaptin, cause a syndrome typified by macrocephaly, neurodevelopmental delay, and seizures. Our immunofluorescence analyses in primary neuronal cell cultures showed that endogenous and GFP-tagged kaptin associates with dynamic actin cytoskeletal structures and that this association is lost upon introduction of the identified mutations. Taken together, our studies have identified kaptin alterations responsible for macrocephaly and neurodevelopmental delay and define kaptin as a molecule crucial for normal human neuromorphogenesis.

Identification of bone morphogenetic protein 7 (BMP7) as an instructive factor for human epidermal Langerhans cell differentiation.

Human Langerhans cell (LC) precursors populate the epidermis early during prenatal development and thereafter undergo massive proliferation. The prototypic antiproliferative cytokine TGF-ß1 is required for LC differentiation from human CD34(+) hematopoietic progenitor cells and blood monocytes in vitro. Similarly, TGF-ß1 deficiency results in LC loss in vivo. However, immunohistology studies revealed that human LC niches in early prenatal epidermis and adult basal (germinal) keratinocyte layers lack detectable TGF-ß1. Here we demonstrated that these LC niches express high levels of bone morphogenetic protein 7 (BMP7) and that Bmp7-deficient mice exhibit substantially diminished LC numbers, with the remaining cells appearing less dendritic. BMP7 induces LC differentiation and proliferation by activating the BMP type-I receptor ALK3 in the absence of canonical TGF-ß1-ALK5 signaling. Conversely, TGF-ß1-induced in vitro LC differentiation is mediated via ALK3; however, co-induction of ALK5 diminished TGF-ß1-driven LC generation. Therefore, selective ALK3 signaling by BMP7 promotes high LC yields. Within epidermis, BMP7 shows an inverse expression pattern relative to TGF-ß1, the latter induced in suprabasal layers and up-regulated in outer layers. We observed that TGF-ß1 inhibits microbial activation of BMP7-generated LCs. Therefore, TGF-ß1 in suprabasal/outer epidermal layers might inhibit LC activation, resulting in LC network maintenance.