Expanded allogeneic adipose-derived stem cells (eASCs) for the treatment of complex perianal fistula in Crohn's disease: results from a multicenter phase I/IIa clinical trial.

PURPOSE: The management of perianal fistula in patients with Crohn's disease is an extremely challenging medical problem as many fistulas do not respond to available treatments. The objectives were to assess the safety and efficacy of a suspension of expanded adipose-derived allogeneic mesenchymal stem cells (eASCs) for the treatment of complex perianal fistula in Crohn's disease METHODS: An open-label, single-arm clinical trial was conducted at six Spanish hospitals. Twenty-four patients were administered intralesionally with 20 million eASCs in one draining fistula tract. A subsequent administration of 40 million eASCs was performed if fistula closure was incomplete at week 12. Subjects were followed until week 24 after the initial administration. RESULTS: Treatment-related adverse events did not indicate any clinical safety concerns after 6 months follow-up. The full analysis of efficacy data at week 24 showed 69.2 % of the patients with a reduction in the number of draining fistulas, 56.3 % of the patients achieved complete closure of the treated fistula achieved, and 30 % of the cases presenting complete closure of all existing fistula tracts. Of note, closure was strictly defined as: absence of suppuration through the external orifice and complete re-epithelization, plus absence of collections measured by magnetic resonance image scan (MRI). Furthermore, MRI Score of Severity showed statistically significant differences at week 12 with a marked reduction at week 24. CONCLUSIONS: Locally injected eASCs appear to be a simple, safe, and beneficial therapy for perianal fistula in Crohn's disease patients. Additional studies are needed to further confirm the efficacy of the eASCs.

Overproduction of Native and Click-able Colanic Acid Slime from Engineered Escherichia coli.

The fundamental biology and application of bacterial exopolysaccharides is gaining increasing attention. However, current synthetic biology efforts to produce the major component of Escherichia sp. slime, colanic acid, and functional derivatives thereof have been limited. Herein, we report the overproduction of colanic acid (up to 1.32 g/L) from d-glucose in an engineered strain of Escherichia coli JM109. Furthermore, we report that chemically synthesized l-fucose analogues containing an azide motif can be metabolically incorporated into the slime layer via a heterologous fucose salvage pathway from Bacteroides sp. and used in a click reaction to attach an organic cargo to the cell surface. This molecular-engineered biopolymer has potential as a new tool for use in chemical, biological, and materials research.

Angiotensinase and vasopressinase activities in hypothalamus, plasma, and kidney after inhibition of angiotensin-converting enzyme: basis for a new working hypothesis.

Reducing angiotensin II (Ang II) production via angiotensin-converting enzyme (ACE) inhibitors is a key approach for the treatment of hypertension. However, these inhibitors may also affect other enzymes, such as angiotensinases and vasopressinase, responsible for the metabolism of other peptides also involved in blood pressure control, such as Ang 2-10, Ang III, Ang IV, and vasopressin. We analyzed the activity of these enzymes in the hypothalamus, plasma, and kidney of normotensive adult male rats after inhibition of ACE with captopril. Aspartyl- (AspAP), glutamyl- (GluAP), alanyl- (AlaAP) and cystinyl-aminopeptidase (CysAP) activities were measured fluorimetrically using arylamides as substrates. Systolic blood pressure (SBP), water intake, and urine flow were also measured. Captopril reduced SBP and increased urine flow. In the hypothalamus, GluAP and AspAP increased, without significant changes in either AlaAP or CysAP. In contrast with effects in plasma, GluAP was unaffected, AspAP decreased, while AlaAP and CysAP increased. In the kidney, enzymatic activities did not change in the cortex, but decreased in the medulla. These data suggest that after ACE inhibition, the metabolism of Ang I in hypothalamus may lead mainly to Ang 2-10 formation. In plasma, the results suggest an increased formation of Ang IV together with increased vasopressinase activity. In the kidney, there is a reduction of vasopressinase activity in the medulla, suggesting a functional reduction of vasopressin in this location. The present data suggest the existence of alternative pathways in addition to ACE inhibition that might be involved in reducing BP after captopril treatment.

Distribution of HLA alleles and haplotypes in the Maldivian population.

The study of human leukocyte antigen (HLA), allele and haplotype frequencies within populations provides an important source of information for anthropological investigation, organ and hematopoietic stem-cell transplantation purposes as well as disease association studies. As of today, there are no data available in the literature on the HLA structure of the Maldivian population. Altogether 106 families were studied. We used the parents of each family (212 unrelated individuals) to analyze the frequencies of HLA class I and class II allele groups and haplotypes.

Airborne study of grass allergen (Lol p 1) in different-sized particles.

BACKGROUND: The Poaceae family is considered one of the main causes of pollen allergy in industrialized countries. The aim of this study is to establish the dynamics of the Poaceae allergens and determine their distribution in the different-sized particles in the atmosphere. METHODS: The air of Granada (southern Spain) was sampled during the pollination period of Poaceae using a cascade impactor and a Hirst-type volumetric collector simultaneously. The sampled airborne allergens were analyzed by indirect ELISA and field emission scanning electron microscopy. Airborne pollen was evaluated with the Spanish Aerobiological Network methodology. RESULTS: Poaceae pollen and allergenic activity have parallel dynamics during the period of maximum pollination, which is reflected in the positive correlations between the 2 variables. In addition, the highest Lol p 1 concentrations were recorded in particle sizes lower than 3.3 mum (stage 4-F). The Spearman correlation test showed that airborne allergens are not dependent on meteorological factors, such as humidity, wind direction or sunshine, however, Lol p 1 allergen correlated positively with Poaceae pollen. CONCLUSIONS: The results of the present study confirm that the Lol p 1 allergen is detected more frequently with pollutants than with coarse particles with similar dynamics and a positive correlation between airborne pollen and aeroallergens. Moreover, Lol p 1 is released in stable weather conditions without large changes in humidity or temperature.

Hypothalamic and plasmatic angiotensin metabolism in L-NAME treated rats.

In order to study the interaction between the renin-angiotensin system (RAS) and nitric oxide (NO), we analyzed the activity of aspartyl- (AspAP), glutamyl- (GluAP), alanyl- (AlaAP), and cystinylaminopeptidase (CysAP) enzymes involved in the RAS cascade, in the hypothalamus, and plasma of normotensive adult male rats after the inhibition of NO production with the NO synthase inhibitor L-NAME (L-N (G)-nitroarginine methyl ester). L-NAME treatment produced a significant increase of systolic blood pressure (SBP). In plasma, while GluAP activity decreased significantly, suggesting a lower Ang III formation, the other aminopeptidases did not change after L-NAME treatment. In hypothalamus, the activities of AspAP and CysAP were not affected after L-NAME treatment. In contrast, GluAP and AlaAP increased significantly. These results suggested mainly a higher formation of Ang III, but also higher levels of Ang IV in the hypothalamus of L-NAME treated rats. Both peptides have hypertensive properties at central level. On the contrary, Ang III may counteract the hypertensive action of Ang II at the periphery. Therefore, the increased SBP in L-NAME treated rats may be due in part to the increased activity of GluAP and AlaAP in hypothalamus and to a decreased activity of GluAP in plasma.

Identification of a new HLA DRB1 allele (HLA-DRB1*1167) in a potential hematopoietic stem cell donor from Iraqi Kurdistan.

High-resolution polymerase chain reaction sequence-specific primer typing and sequence-based typing of the human leukocyte antigen (HLA) gene DRB1 in a potential hematopoietic stem cell donor of Kurdish ethnicity revealed a new allelic variant of HLA-DRB1*11. The sequence was named DRB1*1167, and comparison with previously described DRB1 alleles demonstrated a mixed pattern shared with some DRB1*08 alleles.

Fluorescent labeling of proteins and its application to SDS-PAGE and western blotting.

This chapter describes very simple fluorescent methods developed in our laboratory allowing the rapid monitoring of total protein patterns on both sodium dodecyl sulfate (SDS) polyacrylamide gels and western blots. The noncovalent dye Nile red (9-diethylamino-5H-benzo[alpha]phenoxazine-5-one) is used for the sensitive staining of proteins in SDS gels. This method is compatible with the electroblotting of protein bands and with the staining of the resulting blot with the covalent dye MDPF (2-methoxy-2,4-diphenyl-3(2H)-furanone). These staining procedures are applied sequentially; there is no need to run a duplicate unstained gel for protein blotting. Furthermore, since only the adduct formed by the reaction of MDPF with proteins is fluorescent, there is no need to destain the membrane after protein labeling. In addition, MDPF staining is compatible with further immunodetection of specific bands with polyclonal antibodies. Finally, using the adequate conditions described later, MDPF staining does not preclude the N-terminal sequence analysis of proteins in selected bands.

The Evaluation of Angiogenesis Markers in Hepatocellular Carcinoma and Precursor Lesions in Liver Explants From a Single Institution.

Hepatocellular carcinoma (HCC) is a global health problem associated with chronic liver disease. Precursor lesions are described, and the correct diagnosis of liver nodules is paramount when considering liver transplantation. We evaluated the immunohistochemical expression of vascular endothelial growth factor (VEGF) and angiopoietin-2 in HCC and precursors lesion in a single institution series of whole liver explants between 2013 and 2015, evaluating morphologic and clinical variables. The study comprised 67 patients (32.8% female) and 107 nodules. The mean age of the patients was 52.7 years (29 to 70 y). There were no significant epidemiologic differences among malignant lesions, dysplastic nodules, and regenerative nodules. Angiopoietin-2 expression was significantly more expressed in carcinoma when compared with regenerative lesions (P<0.0001). A statistically significant relationship was noted between the expression of VEGF in hepatocytes and Ang-2 expression in the small vasculature (P=0.006). VEGF expression also correlated significantly with the number of nonpaired arteries (P=0.03), although it was not useful in separating benign from malignant cases. We identified a sensitivity of 54% and a specificity of 96% using angiopoietin-2, and a sensitivity of 68.7% and a specificity of 31.2% when using VEGF for the diagnosis of HCC. There was no significant correlation between the immunohistochemical parameters and the clinical staging, the number of gross lesions, and the histologic grade in cases of HCC. Angiopoietin-2 may be a candidate biomarker in assessing liver nodules in transplant patients, and may assist in the diagnosis of difficult lesions and in small biopsies pretransplant.

Aerobiological and allergenic analysis of cupressaceae pollen in Granada (Southern Spain).

Cupressaceae pollen has been cited in recent years as one of the major airborne allergens of the Mediterranean region, prompting us to conduct an exhaustive analysis on the aerobiological behaviour of this pollen in the Iberian Peninsula and the repercussion that it has had on the atopic population. The aerobiological study, performed from 1996 to 2003 in the city of Granada (S. Spain), used a volumetric Hirst collector. The results indicate that this pollen is present in the air most of the year, registering a high incidence during the winter months. This type of pollen behaved irregularly in the air, fluctuating yearly, seasonally, and within the same day. Temperature and humidity were the parameters that most directly influence the variability of this allergen, while rainfall prior to flowering increased pollen production. The predictive models used estimated a high percentage of the levels reached over the short term by this pollen in the atmosphere of Granada. The clinical study performed with atopic patients showed that some 30% of the population with pollinosis are sensitive to Cupressaceae pollen, affecting people of both genders equally. On the other hand, the most sensitive age group was 21-40 years of age, while children and the elderly registered almost negligible values. Most of the sensitive subjects resided within the city or in the metropolitan area, where environmental pollution reached high levels, while the pathology was found to be less frequent in rural zones. The most frequent symptoms were upper-respiratory ailments and an asthmatic profile.

Angiotensinase activity is asymmetrically distributed in the amygdala, hippocampus and prefrontal cortex of the rat.

There are important asymmetries in brain functions such as emotional processing and stress response in humans and animals. Knowledge of the bilateral distribution of brain neurotransmitters is important to appropriately understand its functions. Some peptides such as those included in the renin-angiotensin system (RAS) and cholecystokinin (CCK) are related to modulation of behavior and stress. However, although angiotensin AT1 and CCK type 2 receptors were found in adult rat brain, there are no studies of their bilateral distribution in stress-related areas. The function of angiotensin peptides is depending on the action of several aminopeptidases (AP) called angiotensinases, some of them being also involved in the metabolism of CCK. We have studied the bilateral distribution of soluble (SOL) and membrane-bound (MEM) alanyl- (AlaAP), cystinyl- (CysAP), glutamyl- (GluAP) and aspartyl- (AspAP) AP activities in stress-related areas such as amygdala, hippocampus and medial prefrontal cortex of adult male rats in resting conditions. These enzymes are involved in the metabolism of angiotensins (AlaAP, CysAP, GluAP, AspAP) and CCK (GluAP, AspAP). In the amygdala, all the activities studied showed a right predominance with a significant difference ranging from 30% for SOL CysAP to 125% for SOL GluAP. In the hippocampus, there was a left predominance for SOL AlaAP, SOL and MEM CysAP and MEM AspAP activities (100, 80, 300 and 100% higher, respectively). In contrast, GluAP predominated remarkably in the right hippocampus (eight-fold for SOL and three-fold for MEM). In the prefrontal cortex, SOL and MEM CysAP and SOL AspAP predominated in the left hemisphere (40, 100 and 40% higher, respectively). These results demonstrated a heterogeneous bilateral pattern of angiotensinase activities in motivation and stress-related areas. This may reflect an uneven asymmetrical distribution of their endogenous substrates depending on the brain location and consequently, it would be also a reflect of the asymmetries in the functions they are involved in.

Fluorescent Labeling of Proteins and Its Application to SDS-PAGE and Western Blotting.

This chapter describes very simple fluorescent methods developed in our laboratory allowing the rapid monitoring of total protein patterns on both sodium dodecyl sulfate (SDS) polyacrylamide gels and western blots. The noncovalent dye Nile red (9-diethylamino-5H-benzo[α]phenoxazine-5-one) is used for the sensitive staining of proteins in SDS gels. This method is compatible with the electroblotting of protein bands and with the staining of the resulting blot with the covalent dye MDPF (2-methoxy-2,4-diphenyl-3(2H)-furanone). These staining procedures are applied sequentially; there is no need to run a duplicate unstained gel for protein blotting. Furthermore, since only the adduct formed by the reaction of MDPF with proteins is fluorescent, there is no need to destain the membrane after protein labeling. In addition, MDPF staining is compatible with further immunodetection of specific bands with polyclonal antibodies. Finally, using the adequate conditions described below, MDPF staining does not preclude the N-terminal sequence analysis of proteins in selected bands.

Absence of plasma melatonin circadian rhythm during the first 72 hours of life in human infants.

To assess whether the circadian rhythm of melatonin (MT) described in umbilical cord blood of term babies is due to an active pineal in the newborn, we analyzed 119 normal neonates during the first 72 h of life. Plasma MT was measured by RIA in different neonates at different hours of the day. Statistical analysis consisted of comparison of the means of MT values grouped in two time periods of 12 h each [day period, 0900-2100 h (77 neonates); night period, 2100-0900 h (42 neonates)] and cosinor analyses to determine the existence of a circadian rhythm of MT. Mean MT levels did not vary greatly during the first 72 h of life, and no differences were found between day and night periods. These results suggest that the pineal gland in the neonate actively secretes MT, but not in a rhythmic manner, implying that the circadian rhythm of MT described previously in cord blood is a reflection of the maternal rhythm.

Mediation by neurotensin-receptors of effects of neurotensin on self-stimulation of the medial prefrontal cortex.

1 Intracortical microinjections of neurotensin (NT) selectively decreased intracranial self-stimulation (ICSS) of the medial prefrontal cortex in the rat. 2 To elucidate whether this effect is mediated by NT receptors or by the formation of NT-dopamine complexes, we investigated the effects on ICSS of intracortical microinjections of neurotensin (1-11), an NT fragment that forms extracellular complexes with dopamine but does not bind to NT receptors. 3 We also studied the effects of the peripheral administration of SR 48692, a selective antagonist of NT receptors, on the inhibition of ICSS produced by the intracortical administration of NT. 4 Unilateral microinjections of neurotensin (1-11) at doses of 10, 20 and 40 nmol into the medial prefrontal cortex did not change the basal ICSS rate of this area. 5 The intraperitoneal administration of SR 48692 at doses of 0.08 and 0.16 mg kg-1 30 min before microinjection of 10 nmol of NT into the medial prefrontal cortex, antagonized the inhibition of ICSS produced by the neuropeptide. 6 These results demonstrate that the inhibitory effect of NT on ICSS is mediated by NT receptors.

Peptidase inhibitors potentiate the effects of neurotensin and neuromedin N on self-stimulation of the medial prefrontal cortex.

The purpose of this study was to examine the possible role of endogenous peptidases in the inhibition of intracranial self-stimulation (ICSS) produced by injections of neurotensin (NT) and neuromedin N (NN) into the medial prefrontal cortex (MPC) of the rat. We studied the effects on ICSS of the MPC of the administration of thiorphan and bestatin, two specific inhibitors of the peptidases that inactivate NT and NN respectively. Microinjections into MPC of thiorphan (10 micrograms) and bestatin (25 micrograms) potentiated in inhibition of ICSS produced by the intracortical administration of NT (10 nmol) and NN (20 nmol) respectively. This potentiation affected both the amplitude and the duration of the inhibition of ICSS produced by the neuropeptides. Our data indicate that endogenous peptidases are involved in the inactivation of NT and NN in the prefrontal cortex.

Properties of rat brain dipeptidyl aminopeptidases in the presence of detergents.

Rat brain dipeptidyl aminopeptidases I to IV were assayed in the soluble and membrane-bound fractions of rat brain, and the effects of the detergents Triton X-100 and sodium deoxycholate on their activities were studied. Dipeptidyl aminopeptidases I and II were significantly inhibited in the presence of sodium deoxycholate, but were not affected by the presence of Triton X-100. However, dipeptidyl aminopeptidase III was not influenced by either detergent, whereas the activity of dipeptidyl aminopeptidase IV was stimulated in the presence of Triton X-100, but remained unaffected by deoxycholate. These effects were partially or totally reversed after detergents were removed from the medium with adsorbent polymeric beads. Although detergents may have different effects on each DAP activity, the behavior of each enzyme activity in the presence of these substances was similar regardless of their subcellular location. These findings suggest that, as with other aminopeptidases, each of these proteins corresponds to the same molecular species in two different cell compartments.

Sex differences and in vitro effects of steroids on serum aminopeptidase activities.

We studied the possible existence of physiological sex differences in serum aminopeptidase activities in mice, by evaluating the effect of gonadectomy and the in vitro response to the presence in the medium of cholesterol or steroid hormones. Alanyl- and glutamyl-aminopeptidase activities were measured in sera from male, female, orchiectomized and ovariectomized mice, incubated with substrate solutions, and compared with the same groups of serum incubated with substrate solutions including cholesterol, 17-beta-estradiol, testosterone, progesterone or hydrocortisone. Our results demonstrated highly significant sex differences, and an influence of cholesterol and steroid hormones on aminopeptidase activity. Depending on the nature of the aminopeptidase, these enzymes responded in different ways to the presence of these substances and also responded differently to gonadectomy. For alanyl-aminopeptidase activity, but not for glutamyl-aminopeptidase activity, there was a clear difference in response between males and females to incubation of the serum with steroid hormones.

Angiotensinase activity in mice fed an olive oil-supplemented diet.

We evaluated the influence of a diet supplemented with olive oil (20% by weight) (OO) on the activity of glutamyl aminopeptidase (GluAP) and aspartyl aminopeptidase (AspAP), which are involved in angiotensin metabolism. Serum concentrations of total cholesterol and fatty acids were also measured. Animals fed on the OO diet gained significantly more weight than did controls from the second week until the end of the feeding period. Serum total cholesterol concentration was significantly higher in the OO group than in control mice. Total monounsaturated fatty acids increased in OO-fed animals, but total saturated fatty acids decreased. No differences between the two groups were observed for total polyunsaturated fatty acids. Serum from animals fed on the OO diet contained significantly lower proportions of myristic, pentadecanoic, palmitic, palmitoleic, vaccenic, alpha-linolenic, gamma-linolenic, and 11,14-eicosadienoic acids than did serum from control animals. In contrast, the OO group had higher levels of oleic, stearic, and gondoic acids. GluAP activity decreased significantly in the serum of OO-fed animals. In these animals soluble AspAP activity was significantly higher in the testis, and significantly lower in the lung and adrenal, in comparison to controls. Membrane-bound AspAP activity was higher in the testis and atrium, and lower in lung, in the OO group. Soluble GluAP activity was significantly lower in the testis of OO-fed animals. Membrane-bound GluAP activity did not differ between the two groups in any of the tissues analyzed. Serum AspAP and GluAP activities correlated negatively with palmitoleic and vaccenic acid respectively in the OO group. However, no significant correlations were found in the control group. These results may reflect functional changes in the renin-angiotensin system in the serum, adrenal, testis, lung and atrium after feeding with a diet enriched in olive oil.

Renal aminopeptidase activities in animal models of hypertension.

Aminopeptidase activity (AP) has been implicated in the metabolism of renal and circulating vasoactive peptides. This activity is involved in the pathogenia of hypertension, essentially in spontaneously hypertensive rats. However, no other animal models, which develop hypertension by other different ways, have been used to study the possible role of aminopeptidase activity. To investigate the role of this activity in the pathogenesis of hypertension, angiotensinase A activity (glutamyl-AP and aspartyl-AP), aminopeptidase M activity (alanyl-AP), aminopeptidase B activity (arginyl-AP), pyroglutamyl-AP, and cystinyl-AP were measured in the serum and kidney of two experimental animal models of renovascular hypertension: Goldblatt two-kidney one clip (G2K-1C) and low renal mass rats (LRM). No differences were found in serum levels of AP in LRM or G2K-1C in comparison with their respective controls. In LRM rats there was a significant decrease in membrane-bound angiotensinase A (glutamyl-AP), arginyl-AP and alanyl-AP activities. In G2K-1C rats there was a significant decrease in soluble and membrane-bound angiotensinase A activity (aspartyl-AP). Our results suggest that AP activities play a role in the regulation of renal vasoactive peptides, and respond differently depending on the cause of hypertension.

Effects of dehydration on renal aminopeptidase activities in adult male and female rats.

Aminopeptidases (APs) are important regulators of peptides directly involved in water homeostasis such as angiotensins (Ang) and vasopressin (AVP). Sex differences in water balance and differences in the effects of gonadal steroids on osmotic stimulation of vasopressin secretion have been reported. Since sex steroids may be involved, the gonadotropin response to osmotic stimuli may be different between males and females. The purpose of this study was to determine the behavior of angiotensinases, vasopressin-degrading activity and gonadotropin-releasing hormone (GnRH)-degrading activity in the cortex and medulla of the kidney of dehydrated male and female rats. In the renal cortex, our results demonstrated an increase in Ang III-degrading activity in dehydrated males but not in females. This response may lead to an increased formation of Ang IV. This occurs with an increase in AspAP activity (which metabolizes Ang I to des-Asp(1)-Ang I), with no changes in Ang II-degrading activity and also with increased levels of AVP-degrading activity in dehydrated animals. These results may suggest an increased cortical blood flow due to enhanced formation of Ang IV together with reduced availability of the vasoconstrictor agents Ang II and AVP in the renal cortex of dehydrated males. The results obtained in the renal medulla suggest the inhibition of the metabolism of Ang I to des-Asp(1)-Ang I, together with a reduced metabolism of Ang II and AVP in dehydrated males but not in females. These results suggest a prolonged action of Ang II and AVP, which could stimulate sodium and water reabsorption in the medulla of dehydrated males. Changes in APs after dehydration occur preferentially in males, which may explain in part the reported sex differences in water homeostasis. The present results suggest a physiologically relevant role for AP activities in water homeostasis.