Expression of nestin, transcription factor SOX3 and homeobox protein DLX-4 in the facial tissue of children with cleft lip and palate / Expresión de nestina, factor de transcripción SOX3 y proteína homeobox DLX-4 en el tejido facial de niños con fisura labiopalatina

The failure of fusion of nasal and maxillary processes results in cleft lip and palate (CLP), which is one of the most common birth defects. The morphopathogenesis of this pathology is multifactorial and still largely unclear. The aim of this study was to evaluate the presence of nestin, transcriptor factor SOX3 (Sox3) and homeobox protein DLX-4 (Dlx-4) in complete unilateral (CU) and complete bilateral (CB) CLP affected facial tissue. Oral mucosa tissue samples were obtained from 17 CUCLP and 13 CBCLP patients during surgical cleft correction and 6 unaffected control subjects. Obtained tissue sections were stained with hematoxylin and eosin and by immunohistochemistry for nestin, Sox3 and Dlx-4. The intensity of staining was graded semiquantitatively. Nestin-positive structures were detected in all CUCLP and CBCLP patients' tissue samples, varying from moderate number of nestin-positive structures to numerous. Sox3 immunoreactivity was more prominent in epithelial cells in both patient groups with frequently patchy distribution. Mainly moderate number of Dlx-4-positive cells was observed in most of tissue samples. Statistically significant moderate positive correlation was found between nestin and Sox3 factors in CUCLP patient group (Spearman's rank correlation coefficient = .517, P = .034). Increase of nestinpositive structures suggests its role in the regulation of the remodeling of tissue in both CUCLP and CBCLP affected tissue. Dominance of Sox3 positivity in cleft affected epithelium indicates its possible role in (compensatory) formation of defective oral epithelium of CUCLP and CBCLP patients. The reduced expression of Dlx-4 implicates its limited regulatory role on the craniofacial development in CUCLP and CBCLP affected tissue.

La falla en la fusión de los procesos nasal y maxilar son causante de la fisura labiopalatina (FLP), que es uno de los defectos congénitos más comunes. La morfopatogenia de esta patología es multifactorial y aún poco clara. El objetivo de este estudio fue evaluar la presencia de nestina, el factor transcriptor SOX3 (Sox3) y la proteína homeobox DLX-4 (Dlx-4) en todo el tejido facial afectado por FLP bilateral unilateral (FU) y bilateral completa (FB). Se obtuvieron muestras de tejido de mucosa oral de 17 pacientes FUFLP y 13 FBFLP durante la corrección quirúrgica de la fisura y de 6 sujetos de control no afectados. Las secciones de tejido obtenidas se tiñeron con hematoxilina y eosina y mediante inmunohistoquímica para nestina, Sox3 y Dlx-4. La intensidad de la tinción fue graduada semicuantitativamente. Se detectaron estructuras positivas para nestina en todas las muestras de tejido de pacientes FUFLP y FBFLP, variando desde un número moderado a numerosas estructuras positivas para nestina. La inmunorreactividad de Sox3 fue más prominente en las células epiteliales en ambos grupos de pacientes con distribución frecuentemente irregular. Se observó un número principalmente moderado de células Dlx-4-positivas en la mayoría de las muestras de tejido. Se encontró una correlación positiva moderada estadísticamente significativa entre los factores de nestina y Sox3 en el grupo de pacientes FUFLP (coeficiente de correlación de rangos de Spearman = 0,517, P = 0,034). El aumento de estructuras positivas para nestina sugiere su papel en la regulación de la remodelación de tejido, tanto en tejido afectado por FUFLP como FBFLP. La dominancia de la positividad de Sox3 en el epitelio afectado de la fisura, indica su posible papel en la formación (compensatoria) del epitelio oral defectuoso de pacientes FUFLP y FBFLP. La expresión reducida de Dlx-4 implica su función reguladora limitada en el desarrollo craneofacial en tejido afectado por FUFLP y FBFLP.

Local antimicrobial, protease and cytokine defense systems in psoriatic skin.

Background: Psoriasis vulgaris is an infl ammatory skin condition characterized by dramatic biochemical and immunological changes. Aims: The aim of the study was to evaluate antimicrobial response, tissue degeneration reactions and distribution of infl ammatory cytokines in untreated psoriatic skin as well as the correlations between these factors and infl uence on the course of the disease. Methods: We evaluated skin samples obtained from routine punch biopsies in 40 patients with psoriasis vulgaris. All tissue specimens were examined by hematoxylin and eosin staining and immunohistochemistry for human beta defensin 2 (HBD-2), matrix metalloproteinase 2 (MMP-2), tumor necrosis factor-alpha (TNF-alpha), interleukin 6 (IL-6) and IL-8. The staining intensity was semi-quantitatively graded. Results: Numerous keratinocytes, fibroblasts and macrophages expressed HBD-2 while the number of MMP-2-positive macrophages, fi broblasts and epitheliocytes varied. TNF-alpha-positive cells varied from a few to numerous in each microscopic fi eld. IL-6-positive cells varied from a few to abundant and IL-8-positive cells from numerous to abundant in each fi eld. Limitations: This study had a rather small patient number. Conclusions: Psoriatic skin shows a strong correlative increase in skin antimicrobial proteins and enzymes mediating tissue degeneration suggesting that the skin maintains compensatory mechanisms during persistent remodeling. While individual notable decrease in antimicrobial proteins was observed in some tissue samples, generally the increased human beta defensin associated with psoriasis is likely to be due to an altered immune status. TNF-alpha, IL-6 and IL-8 are common cytokines expressed in psoriatic skin plaques to maintain the infl ammatory cycle. HBD-2, MMP-2 and TNF-alpha positively correlate with the severity of psoriasis. Meanwhile, the expression of IL-8 signifi cantly decreases with clinically more severe psoriasis, perhaps making these factors candidate prognostic factors for psoriatic inflammation.

IRF6, RYK, and PAX9 expression in facial tissue of children with cleft palate / Expresión de IRF6, RYK y PAX9 en el tejido facial de niños con paladar fisurado

Cleft lip and palate (CLP) is a congenital anomaly characterized by the inappropriate fusion of the upper lip, alveolus, and secondary palate. This study investigated whether expression of interferon regulatory fac tor 6 (IRF6), receptor-like tyrosine kinase (RYK), and paired-box 9 (PAX9), which are essential for the normal development and morphogenesis of craniofacial structures, is dysregulated in children with CLP. Oral mucosa tissue samples were obtained from patients with complete bilateral (CB) CLP (n= 19) during corrective plastic surgery and unaffected control subjects (n= 7). IRF6, RYK, and PAX9 expression was assessed by immunohistochemistry, and data were analyzed with the Mann-Whitney test. In patients, IRF6 immunoreactivity in the connective tissue was moderate to high, but the overall number of IRF6-positive oral epithelial cells was lower than that in controls (z= -3.41; P= 0.01). RYK expression was observed only sporadically in the oral epithelium of 4 patients, in contrast to the control group (z= -3.75; P< 0.001). PAX9-positive epithelial cells were present in low to moderate numbers in patients with CBCLP, while an abundance of these cells was observed in the basal layer of the oral epithelium in controls (z= -3.60; P<0.001). IRF6 is the main connective tissue regulatory factor in CBCLP, and its low level of expression in the oral epithelium suggests a reduced potential for epitheliocyte differentiation, while low PAX9 and RYK expression may explain the decreased cell migration and cleft remodeling in CBCLP.

La fisura labial y palatina (FLP) son anomalías congénitas caracterizadas por la fusión inadecuada del labio superior, alvéolo y paladar secundario. En este estudio se investigó si en niños con FLP hay una desregulación de la expresión del factor regulador de interferón 6 (IRF6), del receptor de la tirosina quinasa (RYK), y del factor de transcripción PAX9, que son esenciales para el desarrollo normal y la morfogénesis de las estructuras craneofaciales. Se obtuvieron muestras de la mucosa oral de pacientes con FLP completa bilateral (CB) (n= 19), tomadas durante la realización de cirugía plástica correctiva, y de sujetos de control no afectados (n= 7). Se evaluó la expresión de IRF6, RYK y PAX9 por inmunohistoquímica, y los datos se analizaron con la prueba de Mann-Whitney. En los pacientes, la inmunoreactividad de IRF6 en el tejido conectivo fue de moderada a alta, pero el número total de células epiteliales orales positivas para IRF6 fue menor que en los controles (z= -3,41; P= 0,01). La expresión de RYK se observó sólo esporádicamente en el epitelio oral de 4 pacientes, en contraste con el grupo control (z= -3,75; P<0.001). Células epiteliales positivas para PAX9 estaban presentes en números bajos a moderados en pacientes con FLP completa bilateral, mientras que se observó una abundante cantidad de estas células en la capa basal del epitelio oral en los controles (z= -3,60; P<0,001). IRF6 es el principal factor regulador del tejido conectivo con FLP completa bilateral, y su bajo nivel de expresión en el epitelio oral sugiere un potencial reducido para la diferenciación del epitelio, mientras que la expresión baja de PAX9 y RYK pueden explicar la disminución de la migración celular y la remodelación de la fisura con FLP completa bilateral.

Basic Fibroblast Growth Factor (bFGF), Fibroblast Growth Factor Receptor 1 (FGFR1), Transforming Growth Factor Beta (TGF-β) and Chromogranin A (CgA) Appearance in Congenital Intra-abdominal Adhesions in Children under One Year of Age.

Aim: Of this work was to determine dispersion of TGF-b, fibrosis modulating factor (bFGF and FGR1) and granule marker chromogranin A in case of intra-abdominal adhesion which could be essential factors in disease pathogenesis. Thus than could be used as possible biomarker. Study Design: Observational study. Place of Study: Institute of Anatomy and Anthropology and Department of Children Surgery, Rīga Stradiņš University. Materials and Methods: The specimens used for research were obtained from 50 patients aged 1 to 292 days. They underwent abdominal surgery due to obstructive gut malrotation and several additional pathologies. Tissues were processed for bFGF, FGFR1, TGF-β and CgA by means of biotin-streptavidin immunohistochemistry. Results: In adhesion tissue bFGF positive connective tissue cells varied from a few to an abundant amount, but in 15 specimens no positive structure was observed. Few connective tissue fibers and moderate to numerous fibroblasts and macrophages contained FGFR1. A moderate number of TGF-β positive connective tissue fibers were observed. Some specimens also contained positive fibroblasts, macrophages and endotheliocytes. Few connective tissue cells contained CgA. A moderate correlation was observed between bFGF and FGFR1 (Spearman’s rank correlation coefficient = .500, P < .001) as well as between bFGF and CgA (Spearman’s rank correlation coefficient = .311, P = .03). Conclusions: The connection between the less distinct bFGF and more prominent FGFR1 proves the compensatory stimulation of receptors as a response on the lack of the same factor in case of adhesion disease. Persisting appearance of TGF-β positive structures in congenital adhesions indicates the continuing growth/regeneration potential of loose connective tissue. Positive CgA structures indicate the involvement of the neuroendocrine system in case of adhesion disease.

Cytokines and Defensins in Tissue Biopsies Obtained by Bronchoscopy from Patients with Post-Intubation Tracheal Stenosis.

Aims: The objective of our study was to perform the routine analysis of bronchoscopically obtained tracheal samples to determine the appearance and relative distribution of cytokines and antimicrobial proteins in patients with post-intubation tracheal stenosis (PITS). Study Design: Retrospective. Place and Duration of Study: Rīga Stradiņš University, Institute of Anatomy and Anthropology, Pauls Stradiņš Clinical University Hospital, between May 2014 and May 2015. Methodology: Five patients with PITS were involved in this study. Tissue samples were obtained by bronchoscopy from the upper part of trachea, then proceeded for routine histological staining with hematoxylin and eosin. Interleukine-1 (IL-1), interleukine-10 (IL-10) and tumor necrosis factor alpha (TNFα), as well as beta defensin-2 (β def-2) were detected by use of immunohistochemistry (IMH) method. The number of immunoreactive (positive) structures was graded semi-quantitatively. Results: Squamous metaplasia, inflammatory cell infiltration and formation of granulation tissue were observed in all cases. Significant expression of IL-10 and β def-2 was seen as various number of immunoreactive structures in tracheal tissue. Only few scattered IL-1 and TNFα positive macrophages were found in part of cases. Conclusions: The leading role in pathogenesis of post-intubation tracheal stenosis is assumed to be the chronic inflammation, fibrous scarring, as well as the remodeling of tracheal wall due to the ischemia. Compensatory expression of antimicrobial peptide β def-2 and anti-inflammatory cytokine IL-10 indicates the intense local tissue defense reactions. TNFα and IL-1 are not among the most significant factors in pathogenesis of PITS.

The Distribution of Matrix Metalloproteinase-2, Tissue Inhibitor of Metalloproteinase-2 and Tissue Inhibitor of Metalloproteinase-4 in Psoriatic Skin.

Aims: To evaluate the appearance and distribution of matrix metalloproteinase-2 (MMP-2), tissue inhibitor of metalloproteinase-2 (TIMP-2) and tissue inhibitor of metalloproteinase-4 (TIMP-4) in lesional skin biopsies of psoriasis patients. Study Design: Observational study. Place and Duration of Study: Institute of Anatomy and Anthropology and Department of Infectology and Dermatology, Rīga Stradiņš University, between September 2013 and June 2014. Methodology: We included 40 patients (31 men, 9 women; age range 18-70 years) with Psoriasis vulgaris, with present characteristic psoriatic eruptions in typical localization sites and no treatment received. Skin samples were obtained using routine punch biopsy method. 10 clinically healthy skin samples obtained during nevus excision procedure were used as control material. All tissue specimens were stained with hematoxylin and eosin and by immunohistochemistry for MMP- 2, TIMP-2 and TIMP-4. The intensity of staining was graded semiquantitatively. Spearman’s rank correlation coefficient was calculated. Results: In psoriasis patients numerous MMP-2-containing keratinocytes were found in epidermis, MMP-2 positive dermal fibroblasts and inflammatory cells varied from few to abundant. Few epidermal cells and moderate to numerous dermal cells contained TIMP-2. Moderate to numerous epidermal and dermal cells contained TIMP-4. Statistically significant strong positive correlation was found between MMP-2 in epidermis and dermis (Spearman’s rank correlation coefficient = .878, P = .000). Statistically significant moderate positive correlation was found between TIMP-2 and TIMP-4 in dermis (Spearman’s rank correlation coefficient = .639, P = .000) and between TIMP-2 in epidermis and dermis (Spearman’s rank correlation coefficient = .564, P = .000). Conclusion: TIMP-4 seems to be most important inhibitor of psoriatic skin degeneration, richly raised by MMP-2. Its moderate correlation with TIMP-2 proves involvement of other tissue inhibitors in the degeneration inhibition and gives evidence about possible patterning between the tissue inhibitors of metalloproteinases.