Autologous method for ex vivo expansion of human limbal epithelial progenitor cells based on plasma rich in growth factors technology.

PURPOSE: Develop an autologous culture method for ex vivo expansion of human limbal epithelial progenitor cells (LEPCs) using Plasma Rich in Growth Factors (PRGF) as a growth supplement and as a scaffold for the culture of LEPCs. METHODS: LEPCs were cultivated in different media supplemented with 10% fetal bovine serum (FBS) or 10% PRGF. The outgrowths, total number of cells, colony forming efficiency (CFE), morphology and immunocytochemistry against p63- α and cytokeratins 3 and 12 (CK3-CK12) were analyzed. PRGF was also used to elaborate a fibrin membrane. The effects of the scaffold on the preservation of stemness and the phenotypic characterization of LEPCs were investigated through analysis of CK3-CK12, ABCG-2 and p63. RESULTS: LEPCs cultivated with PRGF showed a significantly higher growth area than FBS cultures. Moreover, the number of cells were also higher in PRGF than FBS, while displaying a better morphology overall. CFE was found to be also higher in PRGF groups compared to FBS, and the p63-α expression also differed between groups. LEPCs cultivated on PRGF membranes appeared as a confluent monolayer of cells and still retained p63 and ABCG-2 expression, being negative for CK3-CK12. CONCLUSIONS: PRGF can be used in corneal tissue engineering, supplementing the culture media, even in a basal media without any other additives, as well as providing a scaffold for the culture.

PRGF exerts more potent proliferative and anti-inflammatory effects than autologous serum on a cell culture inflammatory model.

Ocular graft versus host disease (oGVHD) is part of a systemic inflammatory disease that usually affects ocular surface tissues manifesting as a dry eye syndrome. Current treatments provide unsatisfactory results. Blood-derived products, like plasma rich in growth factors (PRGF) emerge as a potential therapy for this disease. The purpose of this study was to evaluate the tissue regeneration and anti-inflammatory capability of PRGF, an autologous platelet enriched plasma eye-drop, compared to autologous serum (AS) obtained from oGVHD patients on ocular surface cells cultured in a pro-inflammatory environment. PRGF and AS were obtained from four GVHD patients. Cell proliferation and inflammation markers, intercellular adhesion molecule-1 (ICAM-1) and cyclooxygenase-2 (COX-2), were measured in corneal and conjunctival fibroblastic cells cultured under pro-inflammatory conditions and after treatment with PRGF or AS eye drops. Moreover, cell proliferation increased after treatment with PRGF and AS, though this enhancement in the case of keratocytes was significantly higher with PRGF. PRGF eye drops showed a significant reduction of both inflammatory markers with respect to the initial inflammatory situation and to the AS treatment. Our results concluded that PRGF exerts more potent regenerative and anti-inflammatory effects than autologous serum on ocular surface fibroblasts treated with pro-inflammatory IL-1ß and TNFα.

Platelet rich plasma in ocular surface. / Plasma rico en plaquetas en superficie ocular.

The use of platelet-rich preparations has experienced a significant increase in recent years due to its role in tissue-repair and regeneration. The aim of this study is to examine the available evidence regarding the application of plasma rich in growth factors, and its variations, on the ocular surface. A review is also presented on the effects of platelet-derived growth factors, the implications of the preparation methods, and the existing literature on the safety and efficacy of these therapies in ocular surface diseases. Despite the widespread use of platelet preparations there is no consensus on the most appropriate preparation method, and growth factors concentration vary with different systems. These preparations have been used in the treatment of ocular surface diseases, such as dry eye or persistent epithelial defects, among others, with good safety and efficacy profiles, but further studies are needed to compare to the currently available alternatives.

Effects of new biomimetic regenerating agents on corneal wound healing in an experimental model of post-surgical corneal ulcers.

OBJECTIVE: The purpose of this study is to assess the effectiveness of the topical application of cacicol regenerating agent (RGTA) in an experimental model of corneal ulcer after photorefractive keratectomy (PRK) in mice. METHODS: Mice were subjected to PRK surgery with a 2.0mm ablation zone on the central cornea and 45mm of depth on a VISX Star S2 excimer laser. Corneas were treated topically with cacicol drops 1hour and 48hours after injury. Control groups received balanced salt solution (BSS) in the same dosage. Clinical and histopathological events were evaluated at 1, 2, 3 and 7 days after surgery. Sections obtained through the central region of the corneas were used to analyze the histopathological events of injured and healed corneas. αSMA (myofibroblast transformation), E cadherin (assembly of epithelial cells) and neuronal class III ß-tubulin (innervation) were performed. RESULTS: Corneas treated topically with cacicol for 7 days showed a greater degree of transparency compared to controls. cacicol treated corneas showed improved epithelial cytoarchitecture. Analysis of αSMA profiles in the stroma showed that cacicol reduced or delayed the presence of myofibroblasts in the stroma compared to BSS (P<0.001). Finally, a putative neuroregenerative effect of cacicol was found in corneas subjected to an experimental PRK lesion. In some cases some interindividual variability could be observed due to the design of the experimental model. This is a limitation to consider, despite the statistical significance of the data. CONCLUSIONS: In a model of laser induced surgical lesions in the cornea, topical application of an RGTA (i.e. cacicol) could be involved in avoiding myofibroblast scarring formation and promoting nerve regeneration.

Plasma rich in growth factors (PRGF) eye drops stimulates scarless regeneration compared to autologous serum in the ocular surface stromal fibroblasts.

Autologous serum (AS) eye drops was the first blood-derived product used for the treatment of corneal pathologies but nowadays PRGF arises as a novel interesting alternative to this type of diseases. The purpose of this study was to evaluate and compare the biological outcomes of autologous serum eye drops or Plasma rich in growth factors (PRGF) eye drops on corneal stromal keratocytes (HK) and conjunctival fibroblasts (HConF). To address this, blood from healthy donors was collected and processed to obtain autologous serum (AS) eye drops and plasma rich in growth factors (PRGF) eye drops. Blood-derivates were aliquoted and stored at -80°C until use. PDGF-AB, VEGF, EGF, FGFb and TGF-ß1 were quantified. The potential of PRGF and AS in promoting wound healing was evaluated by means of proliferation and migration assays in HK and HConF. Fibroblast cells were induced to myofibroblast differentiation after treatment with 2.5ng/mL of TGF-ß1. The capability of PRGF and AS to prevent and inhibit TGF-ß1-induced differentiation was evaluated. Results showed significant higher levels of all growth factors analyzed in PRGF eye drops compared to AS. Moreover, PRGF eye drops enhanced significantly the biological outcomes of both HK and HConF, and reduced TGF-ß1-induced myofibroblast differentiation in contrast to autologous serum eye drops (AS). In summary, these results suggest that PRGF exerts enhanced biological outcomes than AS. PRGF may improve the treatment of ocular surface wound healing minimizing the scar formation compared to AS. Results obtained herein suggest that PRGF protects and reverses the myofibroblast phenotype while promotes cell proliferation and migration.

Treatment of ballism and pseudobulbar affect with sertraline.

BACKGROUND: The pathogenesis of ballism is uncertain and may involve more than one mechanism; treatment is not always efficacious. OBJECTIVE: To provide evidence of a nondopaminergic mechanism and the potential for a prompt and nearly complete response to a serotonergic agent. METHODS: Report of 2 separate trials of sertraline hydrochloride in a single patient. RESULTS: Complete remission of symptoms within 48 hours of each drug trial. CONCLUSION: Sertraline may offer an alternative with a better adverse effect profile than dopamine receptor blockers in the treatment of patients with ballism.

Extinction, working memory, and line bisection in spatial neglect.

The authors studied four patients with spatial neglect, using a task in which lines contain an off-centered bisection mark and a task in which the right and left segments of these bisected lines are presented independently and sequentially. In the prebisected line task, subjects reported the position of the bisection. In the segments task, subjects compared the length of the segments. Accuracy was greater with the sequential presentation of line segments, suggesting that an extinction-like phenomenon plays a role in line bisection bias.

Contribution of nitric oxide to exercise-induced changes in healthy volunteers: effects of acute exercise and long-term physical training.

Endothelium plays a central role in the regulation of regional blood flow through the release of certain vasoactive substances. We conducted this study to test whether an increase in the production of nitric oxide (NO) metabolites, atrial natriuretic peptide (ANP) and plasma and intraplatelet cyclic guanosine 3':5' monophosphate (cGMP) is involved in the adaptation to chronic exercise in physically trained people and in the vasodilatation induced by acute physical exercise. We studied one group of 10 trained athletes and another group of 10 untrained people. We measured plasma levels of nitrites, nitrates and cGMP and intraplatelet levels of cGMP, as an indicator of intracellular guanylate cyclase activity, and ANP before and after a maximal treadmill test. Resting cardiac rate (CR) and systolic blood pressure (SBP) were lower in the athlete group than in the control group (73.8 +/- 3.6 vs. 92 +/- 5.9; P < 0.02 and 110 +/- 2.58 vs. 118 +/- 3.27; P < 0.02 respectively). SBP did not show differences between groups after the exercise test. Diastolic blood pressure (DBP) at rest was lower in the athlete group (71 +/- 1.79 vs. 80.5 +/- 3.53; P < 0.03) and the decrease after maximal exercise was more pronounced in this group (64 +/- 2.67 vs. 74.5 +/- 3.2; P < 0.02). Basal plasma nitrites were 4.9 +/- 0.8 in the athlete group and 1.9 +/- 0.3 in the control group (P < 0.05). After exercise, test differences between groups remained (P < 0.05). Nitrates were significantly higher in the group of athletes and did not show exercise-related changes. Plasma levels of cGMP and ANP increased in both groups after the treadmill test, with no differences between groups. Among the athletes, cGMP increased from 1.11 +/- 0.1 to 2.6 +/- 0.4 (P < 0.001), whereas in the untrained group plasma cGMP rose from 1.14 +/- 0.09 to 1.86 +/- 0.2 (P < 0.01). There was a significant correlation between the increases in plasma cGMP and the atrial natriuretic peptide in both groups (r = 0.91, P < 0.0002, for athletes; and r= 0.68, P < 0.04, for control group). The intraplatelet concentration of cGMP did not show differences between groups and did not change after exercise. In conclusion, we have found increased basal levels of plasma nitrite and nitrate in trained subjects. Exercise does not produce differences in the increments of these metabolites. Therefore, we speculate the release of nitric oxide is not augmented by exercise in trained athletes.

Nitric oxide production during the hyperacute vascular rejection.

A complete understanding of the biological mechanisms that take part in the hyperacute vascular rejection is necessary to prevent its development. Nitric Oxide (NO) release by the graft could be one of those mechanisms as cytokine induction of NO synthase in reticuloendothelial cells produces NO with cytotoxic activity. We have performed 20 heterotopic pig-to-dog kidney xenotransplantations in order to determine if cytotoxic NO is released during the hyperacute rejection. Nitrites and cGMP levels were determined in plasma samples taken from the graft artery and vein until vessel thromboses. NO synthase activity was measured in pieces from renal artery and renal tissue. The results show that after discordant xenotransplantation, inducible NO synthase is activated in the transplanted organ, indicating that NO can mediate host tissue damage during the hyperacute rejection.

Effect of isosorbide-5-mononitrate on plasma and urine levels of cyclic GMP in relation to exercise in coronary patients compared with control subjects.

Nitric oxide (NO) and atrial natriuretic peptide (ANP) relax vascular smooth muscle increasing levels of cyclic guanosine 3':5' monophosphate (cGMP). Nitrovasodilators act as exogenous nitric oxide donors. The aim of this study was to ascertain the response of cGMP to exercise without medication and after the administration of 20 mg of isosorbide-5-mononitrate (IS-5-MN) in coronary patients (n = 8) and healthy control subjects (n = 9). A third group of 10 normal volunteers was studied to test plasma cGMP response to second exercise test without IS-5-MN administration. Plasma cGMP increased significantly in both patients (P < 0.02) and controls (P < 0.01) after the first ergometry. After IS-5-MN administration, plasma cGMP did not increase either before or after exercise. In normal volunteers without IS-5-MN plasma cGMP increased significantly after first (P < 0.004) and second (P < 0.0008) exercise test. In conclusion, plasma cGMP increases during exercise. Administration of IS-5-MN does not raise plasma cGMP and neither does performance of further exercise after its administration.

[The effect of the ingestion of nougat on blood glucose and insulin in healthy subjects]. / Efecto de la ingesta de turrón sobre la glucemia e insulinemia en voluntarios sanos.

The importance of the treatment of diabetes mellitus in the diet, is well known. Given that the glycemic response of the foods can not be predicted simply based on their chemical composition, prior to advising diabetics, one must have comparative data on the physiologic effects of the different foods. In this work we have studied the effect of nougat on glycemia and insulinemia, because this is a common food in our culture, and its knowledge may be of great use to diabetic patients. For the study we chose 7 healthy volunteers who were asked to ingest, on different days, a portion of whole wheat bread and one of Jijona nougat with an equivalent number of carbohydrates, and blood samples were taken before breakfast and at different times after initiating the ingestion, and in these samples we determined the plasma concentrations of glucose and insulin. We calculated the peak distances of glycemia and insulinemia, that is to say, the differences between the minimum and maximum values, and the areas under the curves. The difference between the glycemia peak distance between the trial done with nougat and that done with whole wheat bread, was statistically significant with a p < 0.02. The area under both curves did not show statistically significant differences. The same was true for the insulinemia figures. In conclusion, nougat, which traditionally is banned from the diet of diabetics, in this study does not show to produce a greater functional overload of the pancreatic islets than whole wheat bread does, and, although it has a high caloric value, it can probably be added to the diet of diabetic in small quantities, without producing great metabolic alterations.

[Acute eosinophilic pneumonia: presentation of a case and review of the literature]. / Neumonía eosinófila aguda: presentación de un caso y revisión de la literatura.

Pulmonary eosinophilias are characterized by the appearance of lung infiltrates by eosinophils and the presence, commonly, of peripheral blood eosinophilia. Among idiopathic pulmonary eosinophilias, with no evidence of any cause or underlying disease, chronic eosinophilic pneumonia is the most characteristic. Recently, there has been described a few cases of eosinophilic pneumonia with many similarities to chronic eosinophilic pneumonia but with a shorter clinical course, a very good response to corticoid therapy, and without tendency to relapse. This process has been named acute eosinophilic pneumonia. We present a case of this entity and review the literature, emphasizing on the potential severity of this disease, which often conduce to progressive respiratory failure, and its excellent prognosis after appropriate treatment.

Lesiones sincrónicas en el cáncer colorrectal avanzado / Synchronous lesions in the advanced colorectal cancer

Desde ene'82 a jun'91 fueron realizados 3701 fibrocolonoscopía, diagnosticándose cáncer colorrectal (CCR) en 554 pacientes (pac.). De ellos, se llegó a ciego en 283 pac (51,1// ) (Masc: 175 pac; Fem: 108 pac) ya sea en el momento del diagnóstico o dentro de los 6 primeros meses posteriores a la cirugía. La distribución de pólipos sincrónicos (p) en 87 pac (30,7//) = 61 pac. tuvieron 1p; 16 pac, 2p; y 10 pac, 3p. En total: 105 p. Adenomas: 59/105p. 11/105p representaron carcinoma temprano, hsitológicamente confirmado (representando 11/59 adenomas). La distribución de los mismos fue: 5/11 en el mismo segmento colorrectal; 6/11 en diferentes segmento. 1pac. tuvo 3 carcinomas tempranos. Cáncer colorrectal avanzado sincrónico fue presentado por 9 pac (Masc./Fem = 2/1). Todos ellos localizados en distinto segmento colorrectal que el tumor primario. En conclusión, la alta frecuencia de presentación de lesiones premalignas (adenomas) y malignas (cáncer temprano y/o avanzado), y el alto riesgo de presentación de las mismas en diferentes segmentos que el tumor primario, muestra a la colonoscopía como el método más eficiente en la detección ( y eventual extracción) de lesiones sincrónicas del cáncer colorrectal

Lesiones sincrónicas en el cáncer colorrectal avanzado / Synchronous lesions in the advanced colorectal cancer

Desde ene82 a jun91 fueron realizados 3701 fibrocolonoscopía, diagnosticándose cáncer colorrectal (CCR) en 554 pacientes (pac.). De ellos, se llegó a ciego en 283 pac (51,1// ) (Masc: 175 pac; Fem: 108 pac) ya sea en el momento del diagnóstico o dentro de los 6 primeros meses posteriores a la cirugía. La distribución de pólipos sincrónicos (p) en 87 pac (30,7//) = 61 pac. tuvieron 1p; 16 pac, 2p; y 10 pac, 3p. En total: 105 p. Adenomas: 59/105p. 11/105p representaron carcinoma temprano, hsitológicamente confirmado (representando 11/59 adenomas). La distribución de los mismos fue: 5/11 en el mismo segmento colorrectal; 6/11 en diferentes segmento. 1pac. tuvo 3 carcinomas tempranos. Cáncer colorrectal avanzado sincrónico fue presentado por 9 pac (Masc./Fem = 2/1). Todos ellos localizados en distinto segmento colorrectal que el tumor primario. En conclusión, la alta frecuencia de presentación de lesiones premalignas (adenomas) y malignas (cáncer temprano y/o avanzado), y el alto riesgo de presentación de las mismas en diferentes segmentos que el tumor primario, muestra a la colonoscopía como el método más eficiente en la detección ( y eventual extracción) de lesiones sincrónicas del cáncer colorrectal (AU)

[Synchronous lesions in advanced colorectal cancer]. / Lesiones sincrónicas en el cáncer colorrectal avanzado.

From jan '82 to jun '91 were done 3701 colonoscopic procedures. Of these, we arrived to cecum in 283 patients (pat) (51.1%) (male: 175 pat; female: 108 pat) in the first six months after the diagnostic of the colorectal cancer (CRC). The localization of the primary CRC was: rectum: 17.7%; left colon: 37.8% and right colon: 44.5%. 105 synchronical polyps (p) were seen in 87 pat (30.7%): 61 pat with 1p; 16 pat with 2 p and 10 pat with 3 p. Adenomas: 50/105 p (56.19%). Early cancer was histologically confirmed in 11/105 p (11/59 p adenomas). The distribution of early were 5/11 in the same colon segment ant 6/11 in the different colon segment. 1 pat has 3 early cancer. Synchronic advanced colorectal cancer (ACC) was seen in 9 pat (M/F = 2/1). All of these in different segments. In conclusion, the high frequency of the premalignant (adenomas) an malignant (early and advanced cancer) lesions, and the high risk of these in different segments of the large bowel, shows the colonoscopy is the most efficient method in the detection of the synchronical lesions of the colorectal cancer.

Lesiones sincrónicas en el cáncer colorrectal avanzado. / [Synchronous lesions in advanced colorectal cancer]

From jan 82 to jun 91 were done 3701 colonoscopic procedures. Of these, we arrived to cecum in 283 patients (pat) (51.1

) (male: 175 pat; female: 108 pat) in the first six months after the diagnostic of the colorectal cancer (CRC). The localization of the primary CRC was: rectum: 17.7

; left colon: 37.8

and right colon: 44.5

. 105 synchronical polyps (p) were seen in 87 pat (30.7

): 61 pat with 1p; 16 pat with 2 p and 10 pat with 3 p. Adenomas: 50/105 p (56.19

). Early cancer was histologically confirmed in 11/105 p (11/59 p adenomas). The distribution of early were 5/11 in the same colon segment ant 6/11 in the different colon segment. 1 pat has 3 early cancer. Synchronic advanced colorectal cancer (ACC) was seen in 9 pat (M/F = 2/1). All of these in different segments. In conclusion, the high frequency of the premalignant (adenomas) an malignant (early and advanced cancer) lesions, and the high risk of these in different segments of the large bowel, shows the colonoscopy is the most efficient method in the detection of the synchronical lesions of the colorectal cancer.