Multifunctional Nanofibrous Scaffolds with Angle-Ply Microstructure and Co-Delivery Capacity Promote Partial Repair and Total Replacement of Intervertebral Disc.

There is an urgent clinical need for the treatment of annulus fibrosus (AF) impairment caused by intervertebral disc (IVD) degeneration or surgical injury. Although repairing injured AF through tissue engineering is promising, the approach is limited by the complicated angle-ply microstructure, inflammatory microenvironment, poor self-repairing ability of AF cells and deficient matrix production. In this study, electrospinning technology is used to construct aligned core-shell nanofibrous scaffolds loaded with transforming growth factor-ß3 (TGFß3) and ibuprofen (IBU), respectively. The results confirm that the rapid IBU release improves the inflammatory microenvironment, while sustained TGFß3 release enhances nascent extracellular matrix (ECM) formation. Biomaterials for clinical applications must repair local AF defects during herniectomy and enable AF regeneration during disc replacement, so a box defect model and total IVD replacement model in rat tail are constructed. The dual-drug delivering electrospun scaffolds are assembled into angle-ply structure to form a highly biomimetic AF that is implanted into the box defect or used to replace the disc. In two animal models, it is found that biomimetic scaffolds with good anti-inflammatory ability enhance ECM formation and maintain the mechanical properties of IVD. Findings from this study demonstrate that the multifunctional nanofibrous scaffolds provide inspirations for IVD repair.

Effects of budlein A on human neutrophils and lymphocytes.

OBJECTIVE: In this study, we evaluated whether budlein A modulates the activation of innate and adaptive immune cells such as neutrophils and lymphocytes. MATERIAL AND METHODS: Our research group has investigated several plant species and several compounds have been isolated, identified, and their medical potential evaluated. Budlein A is a SL isolated from the species Aldama buddlejiformis and A. robusta (Asteraceae) and shows anti-inflammatory and anti-nociceptive activities. Advances in understanding how plant-derived substances modulate the activation of innate and adaptive immune cells have led to the development of new therapies for human diseases. RESULTS: Budlein A inhibited MPO activity, IL-6, CXCL8, IL-10, and IL-12 production and induces neutrophil apoptosis. In contrast, budlein A inhibited lymphocyte proliferation and IL-2, IL-10, TGF-ß, and IFN-γ production, but it did not lead to cell death. CONCLUSIONS: Collectively, our results indicate that budlein A shows distinct immunomodulatory effects on immune cells.

Effects of budlein A on human neutrophils and lymphocytes

ABSTRACT Sesquiterpene lactones (SLs) are the active constituents of a variety of medicinal plants used in traditional medicine for the treatment of inflammatory diseases and other ailments. Objective In this study, we evaluated whether budlein A modulates the activation of innate and adaptive immune cells such as neutrophils and lymphocytes. Material and Methods Our research group has investigated several plant species and several compounds have been isolated, identified, and their medical potential evaluated. Budlein A is a SL isolated from the species Aldama buddlejiformis and A. robusta (Asteraceae) and shows anti-inflammatory and anti-nociceptive activities. Advances in understanding how plant-derived substances modulate the activation of innate and adaptive immune cells have led to the development of new therapies for human diseases. Results Budlein A inhibited MPO activity, IL-6, CXCL8, IL-10, and IL-12 production and induces neutrophil apoptosis. In contrast, budlein A inhibited lymphocyte proliferation and IL-2, IL-10, TGF-β, and IFN-γ production, but it did not lead to cell death. Conclusions Collectively, our results indicate that budlein A shows distinct immunomodulatory effects on immune cells.

Cytokine Expression in Patients Hospitalized for Severe Odontogenic Infection in Brazil.

INTRODUCTION: Severe odontogenic infections remain an important public health concern and a significant economic burden to public health care facilities. Despite this, several aspects of the disease, such as its immune response profile, remain poorly understood. The aim of this study was to search for an association between mRNA levels of the cytokines interferon-γ, interleukin (IL)-1ß, tumor necrosis factor-α, IL-17A, IL-10, and transforming growth factor-ß and the chemokines IL-8, CCL2/MCP-1, and CCL5 and odontogenic infection. METHODS: The case group was composed of 12 patients hospitalized in consequence of severe odontogenic infection, and our control group included 12 individuals with healthy periapical tissues. Clinical samples were taken from the case (drainage site) and control (periapical interstitial fluid) groups with the aid of paper points. Total RNA was extracted, complementary DNA was synthesized, and mRNA levels were determined by quantitative polymerase chain reaction. Data analysis was performed by using SPSS, and the Wilcoxon signed rank test was used to determine statistical significance (P < .05). RESULTS: Data generated showed a significantly increased expression of proinflammatory cytokines (interferon-γ, IL-1ß, tumor necrosis factor-α, and IL-17A), IL-8, and CCL2/MCP-1 in odontogenic infection patients. The mRNA levels of IL-10, transforming growth factor-ß, and CCL5 were similar in both study groups. CONCLUSIONS: In general, individuals presenting with odontogenic infections exhibited extraordinary proinflammatory cytokine profiles paralleled with unaltered expression of regulatory mediators.

Effects of calcium and vitamin D3 on transforming growth factors in rectal mucosa of sporadic colorectal adenoma patients: a randomized controlled trial.

Transforming growth factor alpha (TGFα) and TGFß1 are growth-promoting and -inhibiting autocrine/paracrine growth factors, respectively, that may (1) affect risk for colorectal cancer and (2) be modifiable by anti-proliferative exposures. The effects of supplemental calcium and vitamin D3 on these two markers in the normal-appearing colorectal mucosa in humans are unknown. We conducted a pilot, randomized, double-blind, placebo-controlled, 2 × 2 factorial clinical trial (n = 92; 23/treatment group) of calcium 2 g and/or vitamin D3 800 IU/d versus placebo over 6 mo. TGFα and TGFß1 expression was measured in biopsies of normal-appearing rectal mucosa using automated immunohistochemistry and quantitative image analysis at baseline and 6-mo follow-up. In the calcium, vitamin D3 , and calcium plus vitamin D3 groups relative to the placebo group (1) the mean overall expression of TGFß1 increased by 14% (P= 0.25), 19% (P = 0.17), and 22% (P = 0.09); (2) the ratio of TGFα expression in the upper 40% (differentiation zone) to that in the lower 60 (proliferation zone) of the crypts decreased by 34% (P = 0.11), 31% (P = 0.22), and 26% (P = 0.33); and (3) the TGFα/TGFß1 ratio in the upper 40% of the crypts decreased by 28% (P = 0.09), 14% (P = 0.41), and 22% (P = 0.24), respectively. These preliminary results, although not statistically significant, suggest that supplemental calcium and vitamin D3 may increase TGFß1 expression and shift TGFα expression downward from the differentiation to the proliferation zone in the crypts in the normal-appearing colorectal mucosa of sporadic colorectal adenoma patients, and support further investigation in a larger clinical trial.

Implications of cytokine genes in allergic asthma

Asthma is a complex disease involving numerous mediator molecules and effector cells, in combination with a range of environmental determining factors. Cytokines play a key role in the physiopathological mechanisms of asthma; the study of the structure, regulation and variations of the genes that encode for these molecules is therefore crucial. Cytokines have extremely diverse roles, and exert effects both as activators and inhibitors of the innate and adaptive immune response. Certain modifications in the expression or structure of these molecules, resulting from the presence of polymorphisms, may give rise to deregulation of the mentioned effects, and therefore to a predisposition to develop concrete asthma phenotypes

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Chronic aspiration of gastric fluid induces the development of obliterative bronchiolitis in rat lung transplants.

Long-term survival of a pulmonary allograft is currently hampered by obliterative bronchiolitis (OB), a form of chronic rejection that is unique to lung transplantation. While tracheobronchial aspiration from gastroesophageal reflux disease (GERD) has clinically been associated with OB, no experimental model exists to investigate this problem. Using a WKY-to-F344 rat orthotopic left lung transplant model, the effects of chronic aspiration on pulmonary allograft were evaluated. Recipients received cyclosporine with or without 8 weekly aspirations of gastric fluid into the allograft. Six (66.7%) of 9 allografts with aspiration demonstrated bronchioles with surrounding monocytic infiltrates, fibrosis and loss of normal lumen anatomy, consistent with the development of OB. In contrast, none of the allografts without aspiration (n = 10) demonstrated these findings (p = 0.002). Of the grafts examined grossly, 83% of the allografts with chronic aspiration but only 20% without aspiration appeared consolidated (p = 0.013). Aspiration was associated with increased levels of IL-1 alpha, IL-1 beta, IL-6, IL-10, TNF-alpha and TGF-beta in BAL and of IL-1 alpha, IL-4 and GM-CSF in serum. This study provides experimental evidence linking chronic aspiration to the development of OB and suggests that strategies aimed at preventing aspiration-related injuries might improve outcomes in clinical lung transplantation.

Biología molecular del carcinoma de células claras renales: principios para un tratamiento selectivo / Molecular biology of the clear cell renal cell carcinoma: selective treatment

El cáncer de células renales (CCR) y más concretamente el subtipo más frecuente de células claras, se he mostrado resistente al tratamiento con quimioterapia y radioterapia cuando afecta a pacientes con metástasis a distancia. En los últimos años se han realizado grandes avances en el campo de la biología molecular que origina este tipo de tumores. Esto ha conducido a un mejor conocimiento del origen de la enfermedad y ha permitido el desarrollo de nuevos fármacos que actúan sobre los factores de crecimiento implicados en el desarrollo del tumor. En este artículo de revisión se resumen de manera concisa los hitos a nivel molecular que originan el desarrollo de los tumores renales de células claras

Renal cell carcinoma (RCC) and its most frequent subtype, the clear cell hystology type, has shown resistance to chemotherapy and radiotherapy treatment when disease was already spread in patients. Recently, a huge advance in the molecular biology of this tumor has been performed. This fact allowed a deeper and better knowledge of the disease and the development of new drugs that work against the growth factors involved in tumor origin. In this review article it is summarized the molecular milestones that are involved in the development of clear cell renal cell carcinomas

Balance entre citocinas pro y antiinflamatorias en estados sépticos / The balance between pro-inflammatory and anti-inflammatory citokines in septic states

Para que la defensa contra la infección se inicie de manera eficaz es necesaria la participación de citocinas con función fundamentalmente proinflamatoria (TNF-α, IL-1 ß, IL-12, IFN-γ, IL-6). La respuesta proinflamatoria inicial está controlada por moléculas antiinflamatorias (el antagonista del receptor de la IL-1 [IL-1 ra], el factor transformador del crecimiento beta [TGF-ß], las interleucinas 4, 6, 10, 11 y 13), y los receptores específicos para la IL-1, el TNF y la interleucina 18. En condiciones fisiológicas, todas estas moléculas sirven como inmunomoduladoras y, por lo tanto, limitan el efecto potencialmente dañino de la reacción inflamatoria. Sin embargo, en condiciones patológicas, la respuesta antiinflamatoria puede ser insuficiente para contrarrestar la actividad inflamatoria o, por el contrario, ser sobrecompensadora e inhibir el sistema inmune y dejar al huésped a merced de la infección. Desde el principio del cuadro de sepsis hasta el vigesimo octavo día y cuando ya han desaparecido la clínica de síndrome de respuesta inflamatoria sistémica (SIRS), el balance global entre las moléculas pro y antiinflamatorias estudiadas apunta hacia un incremento muy importante de estas últimas. El papel del TNF-α y de la IL-1 ß en la sepsis parece ser el de desencadenante, pero ambas carecen de valor patogénico posteriormente. El sTNFR-I, sTNFR-II e IL-1 ra, tienen un gran valor pronóstico y sus niveles se relacionan con el desarrollo de síndrome de disfunción multiorgánica (SDMO) y con cada fallo de órganos. Niveles elevados de IL-10 y de TGF-ß también se relacionan con la mortalidad, aunque de modo más tardío. Futuras intervenciones terapéuticas deberán tener en cuenta que la sepsis es un proceso dinámico

The participation of citokines with a predominant pro-inflammatory function (TNF-α, IL-1 ß, IL-12, INF-γ, IL-6) is necessary for the effective beginning of defense mechanisms against infection. Initial pro-inflammatory response is controlled by anti-inflammatoriy molecules (IL-1 receptor antagonist [IL-1 ra], transforming growth factor-beta [TGF-ß], interleukins 4, 6, 10, 11 and 13), and the specific receptors for IL-1, TNF, and interleukin 18. In physiological conditions, all these molecules serve as immunemodulators and as a result they limit the potentially harmful effect of the inflammatory reaction. However, in pathological conditions anti-inflammatory response can be insufficient in order to counteract the inflammatory activity or, on the contrary, can be excesive with immune system inhibition so that fails to help the host faced with the infection. From the start of sepsis symptomatology until the day 28, and when clinical disturbances of SIRS are already gone, the global balance among the pro- inflammatory and anti-inflammatory molecules studied aims at a very important increase of anti-inflammatory molecules. The role of TNF-α and IL-1 ß in sepsis seems to be that of triggering factors, but subsequently both lack of pathogenic role. sTNFR-I, sTNFR-II and IL-1 ar have great prognostic value and their levels are related to the development of MODS and to the failure of every organ system. Higher levels of IL-10 and TFR-ß are also related to mortality, although in late phases. Future therapeutic interventions should take into account the fact that sepsis is a dynamic process

The aggressive nature of the odontogenic keratocyst: is it a benign cystic neoplasm? Part 3. Immunocytochemistry of cytokeratin and other epithelial cell markers.

Numerous studies of keratin expression by the more common odontogenic cysts were done to determine whether patterns of cytokeratin staining could provide accurate diagnostic markers for the different varieties; to see whether comparative studies with oral mucosa and developing odontogenic epithelium could explain the pathogenesis of the cysts; and whether cytokeratin patterns could provide clues in elucidating the aggressive nature of the OKC. This review was a complex task with a range of at least 19 different cytokeratins being studied and also a broad range of antibodies in use for the same cytokeratin or group of cytokeratins. Moreover, there was not always standardisation of laboratory techniques in the selection and preparation of material. These difficulties were, in general, recognised by the different workers in the field, particularly when there was disagreement on results and caution was expressed about drawing conclusions from some positive findings. It would be fair to conclude that cytokeratin immunocytochemistry has not advanced to any meaningful extent, its use as a diagnostic marker for the OKC nor in eludidating its pathogenesis. With regard to OKC behaviour, it has been pointed out that there was strong reaction of OKC lining for keratin 16, a cytokeratin that has been associated with high proliferative activity. Yet other studies have also shown keratin 16 expression in dentigerous and radicular cysts. Differences in cytokeratin, EMA and CEA immunocytochemical reactivity between the parakeratinised and orthokeratinised varieties of cyst were demonstrated and the suggestion made that the orthokeratinised type has a considerably less aggressive behaviour, is a different entity and should bear a different name. Furthermore, Ki67 positive cells in the parakeratinised OKC linings were considerably more frequent than in the orthokeratinised linings.OKC, dentigerous and radicular cyst epithelium reacted positively for epithelial growth factor receptor (EGFr) but a trend indicating the most intense staining in the OKCs, followed by the dentigerous and then the radicular cyst linings led to the conclusion that the OKCs have an intrinsic growth potential not present in other odontogenic cysts.

Different preparation methods to obtain platelet components as a source of growth factors for local application.

BACKGROUND: Autologous platelet components were recently used as part of tissue-engineering strategies in oral and maxillofacial surgery. Various preparation methods were investigated to define standardized blood bank components and to collect data on the growth factor content of human platelets before and after storage. STUDY DESIGN AND METHODS: Apheresis platelets (AP), buffy coat-derived platelets (BCP), platelets prepared by tube method (TP), and highly concentrated samples prepared from AP and from BCP were evaluated for standard quality criteria of platelet components and for their concentration of transforming growth factor (TGF)-ss1, platelet-derived growth factor (PDGF)-AB, and PDGF-BB. AP were stored for 5 days. On Days 3 and 5, these components and freshly prepared, highly concentrated samples were evaluated for the same measures. RESULTS: Platelet concentration in TP was lower than that in the other groups (p<0.05). However, the concentrations of PDGF-AB, PDGF-BB, and TGF-ss1 were comparable in the three groups. TP showed higher spontaneous CD62 expression than did AP and BCP. The three preparation procedures resulted in significantly different WBC contamination, with the highest levels in TP. For the whole series of measurements, there was a strong correlation between growth factor levels and platelet concentration (p<0.05), which was due to the face that the growth factor content of concentrated platelet samples was tenfold that of AP, BCP, and TP. In TP, the WBC concentration was correlated with PDGF levels (p<0.05). After 5-day storage, the mean levels of PDGF-AB, PDGF-BB, and TGF-ss1 were 57.1, 43.0, and 72.0 percent of the initial values in AP. Overall, multiple regression analysis revealed the following factors influencing the measured growth factor concentrations: platelet concentration, baseline CD62 expression, lactate production, and WBC contamination. CONCLUSION: Various methods enable the preparation of platelet components and of highly concentrated components for local use according to standard blood banking criteria. The obtained components differ, particularly in their WBC content and in vitro platelet activation. These findings are relevant for planning and evaluating further studies of locally usable autologous platelet components.

Pseudocarcinomatous change in lymphomatoid papulosis and primary cutaneous CD30+ lymphoma: a clinicopathologic and immunohistochemical study of 6 patients.

We report 6 cases of pseudoepitheliomatous hyperplasia (PEH) mimicking squamous cell carcinoma in association with an atypical CD30+ dermal infiltrate. Three patients had lymphomatoid papulosis type A, and 3 patients had cutaneous CD30+ lymphoma. All 6 cases showed histologic evidence of PEH with keratinocyte atypia. In 4 cases there was significant atypia to prompt a diagnosis of squamous cell carcinoma. Three of these received treatment with wide local excision and 2 had been engrafted. Immunohistochemical staining for epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha) showed similar expression in lesional and perilesional skin. Epidermal growth factor receptor (EGFR) expression by the proliferating epithelium was similar to that of the suprabasal adjacent normal epidermis. There was no aberrant expression of EGF, TGF-alpha, and EGFR by atypical lymphocytes. These cases demonstrate that PEH associated with CD30+ lymphoproliferative disease may closely resemble squamous cell carcinoma, thereby leading to inappropriate diagnosis and treatment.

A chronic inflammatory infusion model of peritoneal dialysis in rats.

OBJECTIVES: Peritoneal membrane changes are related to daily exposure to non physiologic dialysate and recurrent acute inflammation. We modified a daily infusion and inflammation model and evaluated it for fibrotic and angiogenic features. The feasibility of adenovirus-mediated gene transfer in the model was also assessed. METHODS: Peritoneal catheters were implanted in rats. Over a period of 4 weeks, the animals received a daily infusion of Dianeal 4.25% (Baxter Healthcare Corporation, Deerfield, IL, U.S.A.) with an initial three doses of lipopolysaccharide (LPS) or physiologic saline. Peritoneal fluid was assayed for transforming growth factor beta (TGFbeta) and vascular endothelial growth factor (VEGF). Animals were humanely killed at week 5. Net ultrafiltration was then measured, and tissue samples were immunostained for factor VIII. Mesenteric tissue was assayed for hydroxyproline content. Adenovirus-mediated gene transfer of beta-galactosidase was assayed by intraperitoneal administration of the virus, 4 days before the end of the experiment. RESULTS: Animals treated with either Dianeal or physiologic saline showed peritoneal membrane thickening and increased vascularity. Fibrosis was demonstrated by increased hydroxyproline concentration. Ultrafiltration was impaired. We found increased concentrations of VEGF and TGFbeta in the peritoneal fluid of animals treated with LPS and daily infusion. Adenovirus-mediated gene transfer to the peritoneal membrane was demonstrated in the model. CONCLUSIONS: Exposure to LPS and daily Dianeal or physiologic saline leads to peritoneal fibrosis and neoangiogenesis. Vascularization and glucose transport correlate with ultrafiltration failure. The present animal model mimics changes seen in humans on peritoneal dialysis and may be valuable for evaluating short-term interventions to prevent membrane damage.

Reduction in left ventricular messenger RNA for transforming growth factor beta(1) attenuates left ventricular fibrosis and improves survival without lowering blood pressure in the hypertensive TGR(mRen2)27 Rat.

Angiotensin II recruits transforming growth factor beta(1) (TGFbeta(1)) and is related to left ventricular fibrosis. However, it is unclear whether chronic in vivo reduction in left ventricular TGFbeta(1) expression blunts fibrosis and improves outcome in angiotensin II-dependent hypertension. Four-week-old male hypertensive TGR(mRen2)27 (Ren2) rats received either normal food, low-dose losartan (0.5 mg. kg(-1). d(-1)), or tranilast (a nonspecific TGFbeta inhibitor; 400 mg. kg(-1). d(-1)) (n=10 for each group) for 12 weeks and were compared with Sprague-Dawley control rats. The effect of tranilast on survival was evaluated in 34 additional untreated homozygous Ren2 rats. Tranilast or low-dose losartan did not lower blood pressure. However, the increase in left ventricular weight (Ren2 versus SD 3.1+/-0.16 versus 2.1+/- 0.06 mg/g body wt; P<0.05) was significantly (P<0.05) blunted by both tranilast (2.7+/-0.05) and losartan (2.7+/-0.07). Both drugs prevented the increase in left ventricular TGFbeta(1) mRNA and fibronectin mRNA and blunted the increase in hydroxyproline content and the increase in perivascular fibrosis. The perivascular fibrosis score correlated significantly with the level of expression of TGFbeta(1) (r=0.62; P=0.019). In situ hybridization demonstrated increases in TGFbeta(1) mRNA, predominantly in perivascular and nonmyocyte areas. Both drugs did not prevent the decrease in systolic or diastolic dP/dt, but tranilast significantly improved the survival of untreated Ren2 rats (P=0.029). In conclusion, TGFbeta(1) mRNA expression is increased predominantly in nonmyocyte regions in the hypertrophied left ventricle in this angiotensin II-dependent model of hypertension. This increase is probably due to high angiotensin II levels rather than to hypertension. This is the first study to suggest that chronic inhibition of TGFbeta(1) expression attenuates left ventricular hypertrophy and fibrosis, even without lowering blood pressure.

Homologous acellular matrix graft for urethral reconstruction in the rabbit: histological and functional evaluation.

OBJECTIVE: To evaluate urethral replacement by a free homologous graft of acellular urethral matrix in a rabbit model. MATERIALS AND METHODS: In 30 male New Zealand rabbits, a 0.8 to 1.1 cm. segment of the urethra was resected, replaced with an acellular matrix graft of 1.0 to 1.5 cm. (mean 1.3 cm.), and placed on an 8F feeding tube. Additionally 4 animals underwent sham operation. At varying intervals before sacrifice (from 10 days to 8 months), the animals underwent urodynamic evaluation and retrograde urethrography (for which 4 untreated rabbits served as control). The grafted specimens were prepared for evaluation histologically and by reverse-transcription polymerase chain reaction (RT-PCR). RESULTS: In all animals, the acellular matrix graft remained in its original position. Histological examination showed complete epithelialization and progressive vessel infiltration. At 3 months, smooth muscle bundles were first observed infiltrating the matrix at the end-to-end anastomosis; after 6 months, the smooth muscle bundles had grown into one-third of the matrix. Urodynamics did not detect any difference between the control and matrix-grafted animals in bladder volume, leak-point pressure and residual volume. RT-PCR detected an increase in IGF mRNA in the graft between week 3 and month 6 and in HB-EGF mRNA after day 10 through month 3. TGF-alpha mRNA was not detected; TGF-beta mRNA was unchanged from normal urethral tissue. By 8 months, the host and implant could not be differentiated by urethrography. CONCLUSION: The acellular urethral matrix allows single-stage urethral reconstruction. All tissue components were seen in the grafted matrix after 3 months, with further improvement over time; however, the smooth muscle in the matrix was less than in normal rabbit urethra and was not well oriented. RT-PCR revealed the importance of time-dependent growth factor influences during regeneration.