Human Oral Mucosa as a Potentially Effective Source of Neural Crest Stem Cells for Clinical Practice.

We report in this study on the isolation and expansion of neural crest stem cells (NCSCs) from the epithelium of oral mucosa (OM) using reagents that are GMP-certified and FDA-approved for clinical use. Characterization analysis showed that the levels of keratins K2, K6C, K4, K13, K31, and K15-specific to OM epithelial cells-were significantly lower in the experimental NCSCs. While SOX10 was decreased with no statistically significant difference, the earliest neural crest specifier genes SNAI1/2, Ap2a, Ap2c, SOX9, SOX30, Pax3, and Twist1 showed a trend in increased expression in NCSCs. In addition, proteins of Oct4, Nestin and Noth1 were found to be greatly expressed, confirming NCSC multipotency. In conclusion, our study showed that the epithelium of OM contains NCSCs that can be isolated and expanded with clinical-grade reagents to supply the demand for multipotent cells required for clinical applications in regenerative medicine. Supported by Emmaus Medical Inc.

Thrombospondin-1 induction and VEGF reduction by proteasome inhibition.

The present study focuses on investigating the expression of thrombospondin-1 (TSP-1), a natural inhibitor of neovascularization. Immunofluorescent staining was used to detect the expression of TSP-1 in rabbit corneal tissue with vascularization induced by limbectomy. TSP-1 was detected in healthy and Cultured Autologous Oral Mucosal Epithelial Cell Sheet (CAOMECS) grafted rabbit corneas. TSP-1 was not detected in diseased corneas. Rabbit and human primary oral mucosal and corneal epithelial cells were cultured and treated with proteasome inhibitor (PI) in vitro. Changes in the expression of TSP-1, HIF-1 alpha and 2 alpha, VEGF-A, and VEGF receptor were analyzed by Western blotting. Neovascularization developed in rabbits' corneas as early as 1 month after limbectomy and was stable for at least 3 months. HIF-1 alpha and VEGF-A expression was reduced in CAOMECS grafted corneas, as compared to sham corneas. While TSP-1 expression was decreased in injured corneas, it was expressed in CAOMECS grafted corneas, but still less expressed compared to healthy corneas. PI treatment, of human oral mucosal and corneal epithelial cells increased TSP-1 expression and reduced VEGF-A expression. The results showed that TSP-1 expression was lost in injured corneal surface and that CAOMECS grafting restored TSP-1 expression to certain extent. Proteasome inhibition treatment increased TSP-1 and decreased VEGF-A expression in human oral mucosal and corneal epithelial cells. The result suggests that corneal neovascularization could be managed with the inhibition of the proteasome after CAOMECS grafting and increase corneal transparency.

Posology and Serum-/Xeno-Free Engineered Adipose Stromal Cells Cell Sheets.

Well-characterized adipose stem cells and chemically defined culture media are important factors that control the production of the cell sheet, used in translational medicine. In this study, we have developed and engineered multilayer adipose stem cell cell sheets (ASCCSs) using chemically defined/serum-free culture media: undifferentiated or differentiated into osteoblasts and chondrocytes. In addition, using the cell sheet transmittance, we estimated the number of cells per cell sheet. Undifferentiated ASCCSs were engineered in 10 days, using serum-free/xeno-free culture media. They were CD29+, CD73+, CD90+, CD105+, HLA-A+, and HLA-DR-. ASCCSs differentiated into chondrocytes and osteoblasts were also engineered using chemically defined and animal-free culture media, in only 14 days. The addition of an ROCK inhibitor improved the chondrocyte cell sheet engineering. The decrease in the cell sheet transmittance rate was higher for the osteoblast cell sheets due to the intracellular Ca2+ accumulation. The estimation of cell number per cell sheet was carried out with the transmittance, which will provide important information for cell sheet posology. In conclusion, three types of ASCCSs were engineered using serum-free, xeno-free culture media, expressing their specific markers. Their transmittance measurement allowed estimating the number of cells per cell sheet, with a non-invasive methodology.

Aldehyde Dehydrogenases Expression in Corneal Epithelial Cells with Limbal Stem Cell Deficiency.

PURPOSE: The purpose of the present study is to investigate the expression of aldehyde dehydrogenases (ALDHs) in rabbit corneas with limbal stem cell deficiency (LSCD) and corneas treated with cultured autologous oral mucosa epithelial cell sheet CAOMECS designed to reconstruct the ocular surface with LSCD. METHODS: New Zealand white rabbit autologous oral mucosal epithelial cells were isolated from a buccal biopsy and cultured to be grafted back onto corneas of rabbit model of LSCD. Immunofluorescent staining and Western blot analysis were used to compare the expression of ALDH1A1 and ALDH1A3 in healthy, LSCD-diseased, CAOMECS treated corneas. Human oral mucosal and corneal epithelial cells (OMECS and CECs) were cultured and treated with retinoic acid (RA) to further investigate the expression of ALDHs. RESULTS: In healthy corneas, ALDH1A1 and ALDH1A3 were markedly expressed in basal cells of corneal epithelium. In LSCD diseased corneas, ALDH1A1 and ALDH1A3 were markedly expressed in the conjunctivalized apical epithelial cells, the goblet cells, and the stroma. CAOMECS grafted corneas showed a decreased expression of ALDHs as compared to LSCD diseased corneas. Western blot analysis confirmed the up regulation of ALDH1A1 and ALDH1A3 expression in LSCD-diseased corneal epithelial cells. CAOMECS expressed low levels of ALDH1A1 and ALDH1A3, as compared to diseased CECs (D-CEC). When ALDH1A3 was up regulated by retinoic acid treatment in OMECS, Pax-6 expression was down regulated, suggesting a decrease in regenerative capacity when ALDH enzymes are up regulated. CONCLUSIONS: These findings report for the first time the up regulation of ALDH1A1 and ALDH1A3 in rabbit corneas with LSCD and document that CAOMECS grafting used to reconstruct corneal epithelium may reduce the expression levels of ALDH enzymes.

Measurement of the Adipose Stem Cells Cell Sheets Transmittance.

In the field of cell therapy, the interest in cell sheet technology is increasing. To determine the cell sheet harvesting time requires experience and practice, and different factors could change the harvesting time (variability among donors and culture media, between cell culture dishes, initial cell seeding density). We have developed a device that can measure the transmittance of the multilayer cell sheets, using a light emitting diode and a light detector, to estimate the harvesting time. The transmittance of the adipose stromal cells cell sheets (ASCCS) was measured every other day as soon as the cells were confluent, up to 12 days. The ASCCS, from three different initial seeding densities, were harvested at 8, 10, and 12 days after seeding. Real-time PCR and immunostaining confirmed the expression of specific cell markers (CD29, CD73, CD90, CD105, HLA-A, HLA-DR), but less than the isolated adipose stromal cells. The number of cells per cell sheets, the average thickness per cell sheet, and the corresponding transmittance showed no correlation. Decrease of the transmittance seems to be correlated with the cell sheet maturation. For the first time, we are reporting the success development of a device to estimate ASCCS harvesting time based on their transmittance.

cG-CAOMECS-clinical-grade cultured autologous oral mucosal epithelial cell sheet.

The present study reports the feasibility and successful production of rabbit cG-CAOMECS, designed to reconstruct corneal epithelium of patients with bilateral limbal stem cell deficiency. To produce a safe, chemically defined and FDA compliant cG-CAOMECS, oral mucosal epithelial cells were isolated from a biopsy of rabbit buccal tissue and seeded on a cGMP-certified cell culture surface coated with GMP-grade extracellular matrix. A newly designed clinical-grade medium (KaFa™ medium) was utilized to carry out cell expansion. Detachment and harvesting of the produced cell sheet was accomplished using collagenase treatment. Live cell imaging and morphological analysis techniques were used to examine cell growth. Cells attached onto the surface and self-assembled into colony-forming units (CFUs). Microscopic examination showed that CFUs formed during the first 5 days, and basal monolayer cell sheet formed in less than 10 days. Cells expanded to form a multilayered epithelial cell sheet that was harvested after 17-19 days in culture. Immunostaining and Western blot analyses showed that deltaNp63 was expressed in the basal cells and K3/K12 was expressed in the apical cells, indicating the presence of corneal epithelial-like cells in the produced cell sheet. Adhesion molecules, E-cadherin, beta-catenin, and Cnx43 were also expressed and exhibited the epithelial integrity of the cell sheet. The expression of integrin-beta1 and beta4 confirmed that the collagenase treatment used for detaching and harvesting the cell sheet did not have adverse effects. Our results showed that the utilization of clinical-grade and FDA-approved reagents successfully supported the production of cG-CAMECS.

Clinical Trials of Limbal Stem Cell Deficiency Treated with Oral Mucosal Epithelial Cells.

The corneal surface is an essential organ necessary for vision, and its clarity must be maintained. The corneal epithelium is renewed by limbal stem cells, located in the limbus and in palisades of Vogt. Palisades of Vogt maintain the clearness of the corneal epithelium by blocking the growth of conjunctival epithelium and the invasion of blood vessels over the cornea. The limbal region can be damaged by chemical burns, physical damage (e.g., by contact lenses), congenital disease, chronic inflammation, or limbal surgeries. The degree of limbus damage is associated with the degree of limbal stem cells deficiency (partial or total). For a long time, the only treatment to restore vision was grafting part of the healthy cornea from the other eye of the patient or by transplanting a cornea from cadavers. The regenerative medicine and stem cell therapies have been applied to restore normal vision using different methodologies. The source of stem cells varies from embryonic stem cells, mesenchymal stem cells, to induced pluripotent stem cells. This review focuses on the use of oral mucosa epithelial stem cells and their use in engineering cell sheets to treat limbal stem cell deficient patients.

Vitrification and storage of oral mucosa epithelial cell sheets.

Shipping time and shipping delays might affect the quality of the stem cells based engineered "organs." In our laboratory, we have developed a limbal stem cell deficient (LSCD) rabbit model. To reverse the LSCD, we cultured oral mucosal epithelial cells for 2-3 weeks and engineered cultured autologous oral mucosa epithelial cell sheets (CAOMECS), which were grafted on the LSCD cornea. The purpose of this study was to vitrify CAOMECS and to store it until the CAOMECS can be grafted onto patients. CAOMECS were vitrified in LN2 for up to 204 days. We tested two different methods of vitrification with different solutions; however, CAOMECS were only viable when they were not stored in a vitrification solution; results were only reported from this CAOMECS. On the basis of hematoxylin and eosin staining, we showed that the CAOMECS morphology was well preserved after long-term storage in LN2 . Most of the preservation solutions maintained the CAOMECS phenotype (Ki67, proliferating cell nuclear antigen (PCNA), Beta-Catenin, ZO-1, E-Cadherin, CK3, CK4, CK13). The exception was the solution composed with ethylene glycol and Dimethyl sulfoxide (DMSO): this resulted in loss of DeltaN-p63 expression. DeltaN-p63 is an important marker for cell proliferation. The expression of proteins involved in cell-cell connection and the differentiation markers were maintained. Apoptosis was not detected in the thawed CAOMECS. We demonstrated that CAOMECS can be stored long-term in LN2 without affecting their morphology and phenotype.

Engineering, differentiation and harvesting of human adipose-derived stem cell multilayer cell sheets.

Aim: The study goals were to engineer and harvest scaffold-free undifferentiated/differentiated multilayer human adipose-derived stem cell (hADSC) cell sheets, in absence of treatment. Materials & methods: The hADSC are seeded in 35 mm culture dishes. At confluence or when multilayer cell sheets are formed, hADSC are treated with predefined differentiation culture media (adipocyte, chondrocyte and osteoblast). Results: Undifferentiated hADSC and differentiated adipocyte, osteoblast and chondrocyte hADSC multilayer cell sheets (hADSCmCS) have been harvested. Hematoxylin & eosin showed the formation of multilayer cell sheets. Undifferentiated hADSC multilayer cell sheets preserve their stem cell markers. Differentiated adipocyte, osteoblast and chondrocyte hADSCmCS expressed specific markers. Conclusion: This simple protocol opens possibilities to engineer scaffold-free hADSCm cell sheet to transplant them on damaged organs.

A Phase 3 Trial of l-Glutamine in Sickle Cell Disease.

BACKGROUND: Oxidative stress contributes to the complex pathophysiology of sickle cell disease. Oral therapy with pharmaceutical-grade l-glutamine (USAN, glutamine) has been shown to increase the proportion of the reduced form of nicotinamide adenine dinucleotides in sickle cell erythrocytes, which probably reduces oxidative stress and could result in fewer episodes of sickle cell-related pain. METHODS: In a multicenter, randomized, placebo-controlled, double-blind, phase 3 trial, we tested the efficacy of pharmaceutical-grade l-glutamine (0.3 g per kilogram of body weight per dose) administered twice daily by mouth, as compared with placebo, in reducing the incidence of pain crises among patients with sickle cell anemia or sickle ß0-thalassemia and a history of two or more pain crises during the previous year. Patients who were receiving hydroxyurea at a dose that had been stable for at least 3 months before screening continued that therapy through the 48-week treatment period. RESULTS: A total of 230 patients (age range, 5 to 58 years; 53.9% female) were randomly assigned, in a 2:1 ratio, to receive l-glutamine (152 patients) or placebo (78 patients). The patients in the l-glutamine group had significantly fewer pain crises than those in the placebo group (P=0.005), with a median of 3.0 in the l-glutamine group and 4.0 in the placebo group. Fewer hospitalizations occurred in the l-glutamine group than in the placebo group (P=0.005), with a median of 2.0 in the l-glutamine group and 3.0 in the placebo group. Two thirds of the patients in both trial groups received concomitant hydroxyurea. Low-grade nausea, noncardiac chest pain, fatigue, and musculoskeletal pain occurred more frequently in the l-glutamine group than in the placebo group. CONCLUSIONS: Among children and adults with sickle cell anemia, the median number of pain crises over 48 weeks was lower among those who received oral therapy with l-glutamine, administered alone or with hydroxyurea, than among those who received placebo, with or without hydroxyurea. (Funded by Emmaus Medical; ClinicalTrials.gov number, NCT01179217 .).

Corneal keratin aggresome (CKAGG) formation and clearance by proteasome activation.

PURPOSE: To understand the mechanism of corneal keratin expression and clearance in corneal epithelium with Limbal Stem Cell Deficiency (LSCD). The hypothesis is that LSCD-induced proteasome dysfunction is a contributing factor to keratin aggregation, causing corneal keratin aggresome (CKAGG) formation. METHOD: LSCD was surgically induced in rabbit corneas. LSCD corneal epithelial cells (D-CEC) were collected to investigate keratin K4 and K13 expression and CKAGG formation. Oral mucosal epithelial cells (OMECS) were isolated and cultured to study K4 and K13 expression. Cultured cells were treated with proteasome inhibitor to induce CKAGG formation. RESULTS: K4 and K13 were strongly expressed in D-CEC, with additional higher molecular weight bands of K4 and K13, suggesting CKAGG formation. Double staining of K4/K13 and ubiquitin showed co-localization of these keratins with ubiquitin in D-CEC. Proteasome inhibition also showed K4/K13 modification and accumulation in cultured OMECS, similar to D-CEC. Proteasome activation was then performed in cultured OMEC. There was no accumulation of keratins, and levels of unmodified keratins were found significantly reduced. CONCLUSION: Results showed an abnormal expression of K4 and K13 after LSCD-induced proteasome dysfunction, which coalesce to form CKAGG in Corneal Epithelial Cells (CEC). We propose that CKAGG formation may be one of the causative factors of morphological alterations in the injured corneal epithelium, and that CKAGG could potentially be cleared by enhancing proteasome activity.

Feeder Cells Free Rabbit Oral Mucosa Epithelial Cell Sheet Engineering.

The optimal cell culture method of autologous oral mucosal epithelial cell sheet is not well established for a safe transplantation on to the patients' ocular surface. Animal serum and 3T3 mouse feeder cells are currently being used to stimulate the growth of the epithelial cells. However, the use of animal compounds can have potential side effects for the patient after transplantation of the engineered cell sheet. In the present study, we focused on engineering a rabbit oral mucosal epithelial cell sheet without 3T3 mouse feeder cells using a mix of Dulbecco's Modified Eagle Medium/Bronchial Epithelial Cell Growth Medium culture media (DMEM/BEGM). Autologous oral mucosal epithelial cell sheets, engineered with DMEM/BEGM feeder cell free culture media, were compared to those cultured in presence of serum and feeder cells. Using a DMEM/BEGM mix culture media, feeder cell free culture condition, autologous oral mucosal epithelial cells reached confluence and formed a multilayered sheet. The phenotype of engineered cell sheets cultured with DMEM/BEGM were characterized and compared to those cultured with serum and feeder. Hematoxylin and eosin staining showed the formation of a similar stratified multilayer cell sheets, in both culture conditions. The expression of deltaN-p63, ABCG2, PCNA, E-cadherin, Beta-catenin, CK3, CK4, CK13, Muc5AC, was similar in both culture conditions. We demonstrated that rabbit autologous oral mucosal epithelial cell sheet can be engineered, in feeder cell free conditions. The use of the DMEM/BEGM culture media to engineer culture autologous oral mucosa epithelial cell sheet will help to identify key factors involved in the growth and differentiation of oral mucosal epithelial cells.

Proteasomes in corneal epithelial cells and cultured autologous oral mucosal epithelial cell sheet (CAOMECS) graft used for the ocular surface regeneration.

PURPOSE: This study focuses on characterizing proteasomes in corneal epithelial cells (CEC) and in cultured autologous oral mucosal epithelial cell sheets (CAOMECS) used to regenerate the ocular surface. METHODS: Limbal stem cell deficiency (LSCD) was surgically induced in rabbit corneas. CAOMECS was engineered and grafted onto corneas with LSCD to regenerate the ocular surface. RESULTS: LSCD caused an increase in inflammatory cells in the ocular surface, an increase in the formation of immunoproteasomes (IPR), and a decrease in the formation of constitutive proteasome (CPR). Specifically, LSCD-diseased CEC (D-CEC) showed a decrease in the CPR chymotrypsin-like, trypsin-like and caspase-like activities, while healthy CEC (H-CEC) and CAOMECS showed higher activities. Quantitative analysis of IPR inducible subunit (B5i, B2i, and B1i) were performed and compared to CPR subunit (B5, B2, and B1) levels. Results showed that ratios B5i/B5, B2i/B2 and B1i/B1 were higher in D-CEC, indicating that D-CEC had approximately a two-fold increase in the amount of IPR compared to CAOMECS and H-CEC. Histological analysis demonstrated that CAOMECS-grafted corneas had a re-epithelialized surface, positive staining for CPR subunits, and weak staining for IPR subunits. In addition, digital quantitative measurement of fluorescent intensity showed that the CPR B5 subunit was significantly more expressed in CAOMECS-grafted corneas compared to non-grafted corneas with LSCD. CONCLUSION: CAOMECS grafting successfully replaced the D-CEC with oral mucosal epithelial cells with higher levels of CPR. The increase in constitutive proteasome expression is possibly responsible for the recovery and improvement in CAOMECS-grafted corneas.

The Role of E-Cadherin in Maintaining the Barrier Function of Corneal Epithelium after Treatment with Cultured Autologous Oral Mucosa Epithelial Cell Sheet Grafts for Limbal Stem Deficiency.

The role of E-cadherin in epithelial barrier function of cultured autologous oral mucosa epithelial cell sheet (CAOMECS) grafts was examined. CAOMECS were cultured on a temperature-responsive surface and grafted onto rabbit corneas with Limbal Stem Cell Deficiency (LSCD). E-cadherin levels were significantly higher in CAOMECS compared to normal and LSCD epithelium. Beta-catenin colocalized with E-cadherin in CAOMECS cell membranes while phosphorylated beta-catenin was significantly increased. ZO-1, occludin, and Cnx43 were also strongly expressed in CAOMECS. E-cadherin and beta-catenin localization at the cell membrane was reduced in LSCD corneas, while CAOMECS-grafted corneas showed a restoration of E-cadherin and beta-catenin expression. LSCD corneas did not show continuous staining for ZO-1 or for Cnx43, while CAOMECS-grafted corneas showed a positive expression of ZO-1 and Cnx43. Cascade Blue® hydrazide did not pass through CAOMECS. Because E-cadherin interactions are calcium-dependent, EGTA was used to chelate calcium and disrupt cell adhesion. EGTA-treated CAOMECS completely detached from cell culture surface, and E-cadherin levels were significantly decreased. In conclusion, E cadherin high expression contributed to CAOMECS tight and gap junction protein recruitment at the cell membrane, thus promoting cellular adhesion and a functional barrier to protect the ocular surface.

Carrier-free cultured autologous oral mucosa epithelial cell sheet (CAOMECS) for corneal epithelium reconstruction: a histological study.

This study investigates the therapeutic effects of carrier-free cultured autologous oral mucosa epithelial cell sheet (CAOMECS) transplantation for experimentally induced severe rabbit limbal stem cell deficiency (LSCD). Buccal biopsies were performed and CAOMECS were cultured and transplanted onto diseased corneas. Six-month follow-up examinations indicated that three out of four corneas with CAOMECS grafts showed a decrease in superficial vascularization, while almost all the sham corneas did not show a similar decrease. H&E staining of corneas showed that CAOMECS transplantation reduced blood vessel invasion of central cornea, reduced lymphocyte infiltration and fibrotic tissue formation. DeltaNp63 stained markedly in the grafted cornea and to a lesser extent in the sham corneas. PCNA and Ki-67 staining were much greater in the sham corneas than in the grafted and normal corneas. K3 and K13 staining demonstrated that CAOMECS transplanted corneas had much more K3- and less K13- positive cells compared to the sham corneas. Muc5AC was decreased in the central region of grafted corneas. Very little alpha-smooth muscle actin (aSMA) staining was detected in grafted corneas, while there was a greater amount of aSMA staining in sham corneas. Staining for anti-angiogenic factor TIMP -3 was also increased, and pro-angiogenic factor MMP-3 was decreased in grafted corneas compared to sham corneas. Our results indicate that CAOMECS grafts resulted in improved epithelialization of the corneal surface and decreased vascularization and fibrosis of the diseased corneas.

Characterization of the anaemia associated with Graves' disease.

BACKGROUND: Graves' disease (GD) is associated with hyperthyroidism. Thyrotoxicosis adversely affects multiple organ systems including haematopoiesis. Anaemia occurring specifically in GD has not been systematically studied previously. OBJECTIVE: To define the prevalence and characteristics of the anaemia associated with GD. DESIGN: Eighty-seven newly diagnosed patients with GD were recruited. Haematological indices, thyroid function and inflammatory parameters were examined at presentation and following successful treatment of hyperthyroidism. SETTING: Tertiary care academic referral centre. RESULTS: Thirty-three per cent of subjects presented with anaemia. The prevalence of anaemia not attributable to other causes (GD anaemia) was 22%. GD anaemia affected 41.6% (10/24) of men compared to 17.5% of women (11/63). Mean erythropoietin (EPO) levels (15.5 +/- 5.3 mIU/ml) were within normal reference limits but significantly higher (P = 0.004) than those of the non-anaemic controls. Hgb correlated inversely with EPO (P = 0.05) and CRP (P = 0.04) levels, a relationship that persisted after multivariate adjustment for TT3 or TT4. With antithyroid therapy for 16 +/- 6.3 weeks, Hgb levels normalized in 8 out of 9 subjects with GD anaemia (10.7 +/- 0.8 to 13.5 +/- 1.3 g/dl, P = 0.0001). After normalization of Hgb, mean MCV and TIBC were significantly increased, and median ferritin and mean EPO were significantly decreased. CONCLUSIONS: GD anaemia is common, resembles the anaemia of chronic disease, and is associated with markers of inflammation. It corrects promptly with return to the euthyroid state following treatment.

Aged garlic extract may be safe for patients on warfarin therapy.

Garlic has been known to have antiplatelet properties. Because of the lack of major clinical data regarding the safety of concomitant use of garlic supplements and anticoagulants, we decided to evaluate the safety of using garlic extract along with oral anticoagulation therapy. During this project we tested aged garlic extract (AGE), a commercial garlic preparation, with warfarin (Coumadin). Sixty-six (66) patients were screened for a double-blind, randomized, placebo-controlled pilot study. Fifty-two (52) patients were randomized for the project. Forty-eight patients (30 men and 18 women, with a mean age of 56+/-10 years) completed the study. Eighteen patients (14 before randomization, 4 after randomization) were dropped from the study. The study medication (AGE or placebo) was administered at a dose of 5 mL twice a day for 12 wk. Potential bleeding and thromboembolic episodes were monitored. There was no evidence of increased hemorrhage in either the placebo or the AGE group. Adverse events included headache, fatigue, colds, and dizziness. However, no significant difference was found in the incidence of these minor adverse events between the groups. Thus, the adverse events are unlikely to be attributable to AGE. The results suggest that AGE is relatively safe and poses no serious hemorrhagic risk for closely monitored patients on warfarin oral anticoagulation therapy. Although the risk-benefit ratio of AGE use needs to be considered carefully when warfarin therapy is necessary, its positive effects may be beneficial to people with a high-risk background or who are taking cardiovascular medications.

Aged garlic extract is a potential therapy for sickle-cell anemia.

Sickle-cell anemia is one of the most prevalent hereditary disorders with prominent morbidity and mortality. Oxidative phenomena play a significant role in the disorder's pathophysiology. A forumlation of garlic (Allium sativum), AGE, has been reported to exert an antioxidant effect in vitro. We evaluated the antioxidant effect of AGE on sickle red blood cells (RBCs). Five patients (two men and three women, mean age 40+/-15 years, range 24-58 years) with sickle-cell anemia participated in the study. AGE was administered at a dose of 5 mL daily. Whole blood samples were obtained at baseline and at 4 wk, primarily for Heinz body analysis. In all patients, the number of Heinz bodies decreased over the 4-wk period (58.9+/-20.0% at baseline to 29.8+/-15.3% at follow-up; P=0.03). These data suggest that AGE has a significant antioxidant activity on sickle RBCs. AGE may be further evaluated as a potential therapeutic agent to ameliorate complications of sickle-cell anemia.

L-glutamine therapy reduces endothelial adhesion of sickle red blood cells to human umbilical vein endothelial cells.

BACKGROUND: We have previously demonstrated that therapy with orally administered L-glutamine improves nicotinamide adenosine dinucleotide (NAD) redox potential of sickle red blood cells (RBC). On further analysis of L-glutamine therapy for sickle cell anemia patients, the effect of L-glutamine on adhesion of sickle RBC to human umbilical vein endothelial cells (HUVEC) was examined. METHODS: The first part of the experiment was conducted with the blood samples of the 5 adult sickle cell anemia patients who had been on L-glutamine therapy for at least 4 weeks on a dosage of 30 grams per day compared to those of patient control group. In the second part of the experiment 6 patients with sickle cell anemia were studied longitudinally. Five of these patients were treated with oral L-glutamine 30 grams daily and one was observed without treatment as the control. t-test and paired t-test were used for determination of statistical significance in cross-sectional and longitudinal studies respectively. RESULTS: In the first study, the mean adhesion to endothelial cells with the autologous plasma incubated cells were 0.97 +/- 0.45 for the treated group and 1.91 +/- 0.53 for the nontreated group (p < 0.02). Similarly with lipopolysaccharide (LPS) incubated cells the mean adhesion to endothelial cells were 1.39 +/- 0.33 for the treated group and 2.80 +/- 0.47 for the untreated group (p < 0.001). With the longitudinal experiment, mean decrease in the adhesion to endothelial cells was 1.13 +/- 0.21 (p < 0.001) for the 5 treated patients whereas the control patient had slight increase in the adhesion to endothelial cells. CONCLUSION: In these studies, oral L-glutamine administration consistently resulted in improvement of sickle RBC adhesion to HUVEC. These data suggest positive physiological effects of L-glutamine in sickle cell disease.