HIF-1 mediated activation of antimicrobial peptide LL-37 in type 2 diabetic patients.

Infections are common in patients with diabetes, but increasing antibiotic resistance hampers successful bacterial clearance and calls for alternative treatment strategies. Hypoxia-inducible factor 1 (HIF-1) is known to influence the innate immune defense and could therefore serve as a possible target. However, the impact of high glucose on HIF-1 has received little attention and merits closer investigation. Here, we show that higher levels of proinflammatory cytokines and CAMP, encoding for the antimicrobial peptide cathelicidin, LL-37, correlate with HIF-1 in type 2 diabetic patients. Chemical activation of HIF-1 further enhanced LL-37, IL-1ß, and IL-8 in human uroepithelial cells exposed to high glucose. Moreover, HIF-1 activation of transurethrally infected diabetic mice resulted in lower bacterial load. Drugs activating HIF-1 could therefore in the future potentially have a therapeutic role in clearing bacteria in diabetic patients with infections where antibiotic treatment failed. KEY MESSAGES: • Mohanty et al. "HIF-1 mediated activation of antimicrobial peptide LL-37 in type 2 diabetic patients." • Our study highlights induction of the antimicrobial peptide, LL-37, and strengthening of the innate immunity through hypoxia-inducible factor 1 (HIF-1) in diabetes. • Our key observations are: 1. HIF-1 activation increased LL-37 expression in human urothelial cells treated with high glucose. In line with that, we demonstrated that patients with type 2 diabetes living at high altitude had increased levels of the LL-37. 2. HIF-1 activation increased IL-1ß and IL-8 in human uroepithelial cells treated with high glucose concentration. 3. Pharmacological activation of HIF-1 decreased bacterial load in the urinary bladder of mice with hereditary diabetes. • We conclude that enhancing HIF-1 may along with antibiotics in the future contribute to the treatment in selected patient groups where traditional therapy is not possible.

Glycemia-reducing effects of Bolivian nutraceutical plants / Efectos reductores de la glucemia de las plantas nutracéuticas bolivianas

INTRODUCTION: The prevalence of diabetes type 2 is increasing worldwide, thus the search of novel alternative ther¬apies is needed. According to their traditional use, we selected five Bolivian plants Chenopodium quinoa (CQ) Ama¬ranthus caudatus (AC), Chenopodium pallidicaule (CP), Lupinus mutabilis (LM) and Smallanthus sonchifolius (SS) that are traditionally used to control glycemia. METHODS: The effect of a single oral administration of Ethanolic (EtOH), hydro-ethanolic (EtOH70) and aqueous (Aq) extracts from all plant species were tested for their effect on blood glucose in non-fasted mice and during the oral glucose tolerance test (OGTT). The effect on insulin secretion was evaluated in mice pancreatic islets. RESULTS: EtOH70 extracts of all the plants showed glucose-reducing effect at the highest dose evaluated (2000 mg/ kg b.w.). EtOH70 extracts improved the glucose tolerance evaluated by the OGTT in mice fasted for 12 hours. The extracts have different effects on glucose homeostasis since just extracts of AC, LM and CQ but not CP and SS in¬creased insulin secretion as shown on mice pancreatic islets. The phytochemical qualitative characterization of EtOH70 extracts detected phenolic acids and flavonoids in AC, CP and CQ; alkaloids in LM and anthocyanidins in SS. None of EtOH70 extracts tested showed in vitro or in vivo acute toxicity at concentrations where they exhibit glucose lowering effects. CONCLUSIONS: We report here that extracts from AC, CQ, CP, LM and SS exhibit glucose lowering effect while just AC, CQ and LM stimulate directly the insulin secretion

INTRODUCCIÓN: La prevalencia de diabetes tipo 2 está aumentando en todo el mundo, por lo que se necesita la búsqueda de nuevas terapias alternativas. Según su uso tradicional, seleccionamos cinco plantas bolivianas Chenopodium quinoa (CQ) Amaranthus caudatus (AC), Chenopodium pallidicaule (CP), Lupinus mutabilis (LM) y Smallanthus sonchifolius (SS) que se usan tradicionalmente para controlar la glucemia. MÉTODOS: Se evaluó el efecto de la administración oral única de extractos etanólicos (EtOH), hidroetanólicos (EtOH70) y acuosos (Aq) de las plantas mencionadas para determinar su efecto sobre la glucosa en sangre en ratones en o sin ayunas y durante la prueba de tolerancia a la glucosa oral (PTGO). El efecto sobre la secreción de insulina se evaluó en islotes pancreáticos de ratones. RESULTADOS: Los extractos de EtOH70 de todas las plantas disminuyeron la glucemia a la dosis más alta evaluada (2000 mg / kg b.w.). Los extractos de EtOH70 mejoraron la tolerancia a la glucosa evaluada mediante la PTGO en ratones con ayuno de 12 horas. Los extractos tienen diferentes efectos sobre la homeostasis de la glucosa, ya que solo los extractos de AC, LM y CQ pero no CP y SS aumentaron la secreción de insulina como se muestra en los islotes pancreáticos de los ratones. La caracterización cualitativa fitoquímica de extractos de EtOH70 detectó ácidos fenólicos y flavonoides en AC, CP y CQ, alcaloides en LM y antocianidinas en SS. Ninguno de los extractos de EtOH70 probados mostró toxicidad aguda in vitro o in vivo a concentraciones en las que exhiben efectos reductores de glucosa. CONCLUSIÓN: Los extractos de AC, CQ, CP, LM y SS exhiben un efecto reductor de la glucosa, mientras que solo AC, CQ y LM estimulan directamente la secreción de insulina

Lupinus mutabilis Extract Exerts an Anti-Diabetic Effect by Improving Insulin Release in Type 2 Diabetic Goto-Kakizaki Rats.

Lupinus mutabilis (LM) is a legume part of Bolivian traditional diet that has a nutraceutical property reducing blood glucose levels. The prevalence of type 2 diabetes is increasing worldwide thus; the search for novel anti-diabetic drugs is needed. Based on its traditional use, we evaluated the anti-diabetic effect of LM in the spontaneously diabetic Goto-Kakizaki (GK) rat, a model of type 2 diabetes and in Wistar (W) rats as healthy control. LM seeds hydroethanolic extract, analyzed by gas chromatography-mass spectrometry and high-performance liquid chromatography-high resolution mass spectrometry, is a complex mixture of volatile and non-volatile components. A single oral administration of LM extract (2000 mg/kg b.w.) improved glucose tolerance during the oral glucose tolerance test (OGTT) (30⁻120 min) in GK and W rats (p < 0.0001). The long-term treatment with LM (1000 mg/kg b.w.), for 21 days, improved the area under the curve (AUC) of glucose during OGTT at day 20, in both GK (p < 0.01) and W rats (p < 0.01). The HbA1c (GK rats, p < 0.05 and W rats, p < 0.0001) and the non-fasting glucose (GK rats, p < 0.05) were also reduced. LM increased both serum insulin levels (2.4-fold in GK rats and 2.5-fold W rats), and the glucose-induced (16.7 mM glucose) insulin release in isolated islets from treated animals (6.7-fold in GK rats, and 6.6-fold in W rats). Moreover, LM (10 mg/mL) stimulated in vitro glucose induced (16.7 mM glucose) insulin release in batch incubated GK and W rat islets (p < 0.0001). In perifused GK rat islets, insulin release in 16.7 mM glucose was increased 95.3-fold compared to untreated islets (p < 0.0001), while no significant differences were found in perifused W rat islets. The LM mechanism of action, evaluated using inhibitory compounds of the insulin secretion pathway, showed that LM-dependent insulin secretion was reduced 42% by diazoxide (p < 0.001), 70% by nifedipine (p < 0.001), 86.7% by H89 (p < 0.0001), 70.8% by calphostine-C (p < 0.0001) and 93% by pertussis toxin (p < 0.0001). A similar effect was observed in W rats islets. Our findings provide evidence that LM has an anti-diabetic effect through stimulation of insulin release. The effect is-dependent on L-type calcium channel, protein kinase A and C systems, and G protein-coupled exocytosis and is partially mediated by K-ATP channels.

Amaranthus caudatus extract inhibits the invasion of E. coli into uroepithelial cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Amaranthus caudatus is traditionally used to treat infections. Based on its traditional usage, we investigated the effect of A. caudatus on the bladder epithelial cells in the protection of E. coli infection. MATERIALS AND METHODS: The direct antimicrobial effects of A. caudatus on uropathogenic bacteria were investigated using minimum inhibitory concentration (MIC) assay. Bladder epithelial cell lines T24 and 5637 and uropathogenic E. coli strain #12 were used to investigate the effect of A. caudatus. Bacterial adhesion and invasion into bladder cells treated with A. caudatus was analyzed. Expression of uroplakin-1a (UPK1A), ß1 integrin (ITGB1), caveolin-1 (CAV1) and the antimicrobial peptides human ß defensin-2 (DEFB4A) and LL-37 (CAMP) was evaluated using RT-PCR. RESULTS: No direct antibacterial effect on E. coli or any of the tested uropathogenic strains was observed by A. caudatus. However, we demonstrated reduced mRNA expression of uroplakin-1a and caveolin-1, but not ß1 integrin after treatment of uroepithelial cells, mirrored by the decreased adhesion and invasion of E. coli. A. caudatus treatment did not induce increased gene expression of the antimicrobial peptides, LL-37 and human ß-defensin-2. CONCLUSIONS: Our results showed that A. caudatus has a protective role on bladder epithelial cells against uropathogenic E. coli infection by decreasing the bacterial adhesion and invasion, thereby preventing infection.

Amaranthus caudatus Stimulates Insulin Secretion in Goto-Kakizaki Rats, a Model of Diabetes Mellitus Type 2.

Diabetes Mellitus Type 2 prevalence is increasing worldwide; thus efforts to develop novel therapeutic strategies are required. Amaranthus caudatus (AC) is a pseudo-cereal with reported anti-diabetic effects that is usually consumed in food preparations in Bolivia. This study evaluated the anti-diabetic nutraceutical property of an AC hydroethanolic extract that contains mainly sugars and traces of polyphenols and amino acids (as shown by nalysis with liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR)), in type 2 diabetic Goto-Kakizaki (GK) rats and healthy Wistar (W) rats. A single oral administration of AC extract (2000 mg/kg body weight) improved glucose tolerance during Oral Glucose Tolerance Tests (OGTT) in both GK rats and in W rats. Long-term treatment (21 days) with AC (1000 mg/kg b.w.) improved the glucose tolerance evaluated by the area under the curve (AUC) of glucose levels during the OGTT, in both GK and W rats. The HbA1c levels were reduced in both GK (19.83%) and W rats (10.7%). This effect was secondary to an increase in serum insulin levels in both GK and W rats and confirmed in pancreatic islets, isolated from treated animals, where the chronic AC exposure increased the insulin production 4.1-fold in GK and 3.7-fold in W rat islets. Furthermore, the effect of AC on in vitro glucose-dependent insulin secretion (16.7 mM glucose) was concentration-dependent up to 50 mg/mL, with 8.5-fold increase in GK and 5.7-fold in W rat islets, and the insulin secretion in perifused GK and W rat islets increased 31 and nine times, respectively. The mechanism of action of AC on insulin secretion was shown to involve calcium, PKA and PKC activation, and G-protein coupled-exocytosis since the AC effect was reduced 38% by nifedipine (L-type channel inhibitor), 77% by H89 (PKA inhibitor), 79% by Calphostine-C (PKC inhibitor) and 20% by pertussis toxin (G-protein suppressor).

Lupinus mutabilis Edible Beans Protect against Bacterial Infection in Uroepithelial Cells.

Lupinus mutabilis is a South American herb with edible beans, known to reduce serum glucose levels in diabetic patients. Furthermore, L. mutabilis contains phytochemicals known to decrease bacterial load. Based on the increased urinary tract infections experienced among patients with diabetes, we investigated the effect of L. mutabilis on bladder epithelial cells in the protection of E. coli infection during normal and high glucose concentrations. We did not observe any direct antibacterial effect by L. mutabilis extract. Instead we observed an influence on the host cells, with indirect impact on bacteria and their possibility of causing infection. L. mutabilis extract decreased adhesion to bladder epithelial cells of uropathogenic bacteria, including drug-resistant strains. Moreover, uroplakin1a, involved in adhesion, was downregulated while the antimicrobial peptide RNase 7 was upregulated in L. mutabilis treated cells irrespectively of glucose concentration. This supports an early effect fighting bacteria. Additionally, L. mutabilis prevented bacterial biofilm formation, which is used by bacteria to evade the immune system and antibiotics. In summary, L. mutabilis protects against bacterial infection in uroepithelial cells by preventing adhesion through alteration of the cell surface, increasing antimicrobial peptide expression, and reducing biofilm formation. Together, this promotes bacterial clearance, suggesting that L. mutabilis as extract or as a dietary item can contribute to the prevention of urinary tract infections, which is of importance in an era of increasing antibiotic resistance.

Extract of Clinopodium bolivianum protects against E. coli invasion of uroepithelial cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Clinopodium bolivianum is a South American plant with anti-inflammatory and anti-infective activities. The increasing antibiotic resistance urges for alternative therapy. Based on its use in traditional medicine, we investigated the effect of C. bolivianum on the ability to defend bladder epithelial cells from E. coli infection. MATERIALS AND METHODS: The extract was analyzed by LC-MS. Bladder epithelial cell lines T24 and 5637 and uropathogenic E. coli No. 12, its isogenic mutant WE16 csgBA bscA::Cm and CFT073 were used to investigate the effect of C. bolivianum on uroepithelial infection. Bacterial adherence and invasion to cells treated with C. bolivianum were analyzed. Expression of uroplakin 1a, ß1 integrin, caveolin-1, IL-8 and antimicrobial peptides in response to C. bolivianum treatment was assessed using RT-PCR. Protein expression was confirmed by Western blot analysis or ELISA. The antimicrobial effects of C. bolivianum on bacteria and fungus were investigated using minimum inhibitory concentration. Furthermore, the formation of biofilm was investigated with crystal violet assay. RESULTS: C. bolivianum extract consisted of more than 70 different types of phytochemicals including sugars and phenolic compounds. The extract decreased the uroplakin 1a expression and E. coli adhesion and invasion of uroepithelial cells while up-regulated caveolin-1. In uninfected C. bolivianum treated cells, IL-8 was lower than in non-treated cells. In infected cells, however, no difference was observed between treated and non-treated cells. Further, C. bolivianum treatment reduced uropathogenic E. coli (UPEC) biofilms but did not inhibit bacterial growth. CONCLUSIONS: Our results show that C. bolivianum has a protective role on bladder epithelial cells against UPEC infection by decreasing the bacterial adhesion, invasion and biofilm formation.

Molecular characterization of enterotoxigenic Escherichia coli isolates recovered from children with diarrhea during a 4-year period (2007 to 2010) in Bolivia.

Enterotoxigenic Escherichia coli (ETEC) is an important cause of childhood diarrhea. This study aimed to characterize ETEC strains isolated from Bolivian children aged <5 years according to enterotoxin profile, colonization factors (CFs), suggested virulence genes, and severity of disease. A total of 299 ETEC isolates recovered from children with diarrhea and 55 ETEC isolates from children without diarrhea (controls) were isolated over a period of 4 years. Strains expressing heat-labile toxin (LT) or heat-stable toxin (ST) alone were about equally common and twice as common as ETEC producing both toxins (20%). ETEC strains expressing human ST (STh) were more common in children aged <2 years, while ETEC strains expressing LT plus STh (LT/STh) were more frequent in 2- to 5-year-old children. Severity of disease was not related to the toxin profile of the strains. CF-positive isolates were more frequently identified in diarrheal samples than in control samples (P = 0.02). The most common CFs were CFA/I and CS14. CFA/I ETEC strains were more frequent in children aged <2 years than CS1+CS3 isolates and CS14 isolates, which were more prevalent in 2- to 5-year-old children. The presence of suggested ETEC virulence genes (clyA, eatA, tia, tibC, leoA, and east-1) was not associated with disease. However, east-1 was associated with LT/STh strains (P < 0.001), eatA with STh strains (P < 0.001), and tia with LT/STh strains (P < 0.001). A minor seasonal peak of ETEC infections was identified in May during the cold-dry season and coincided with the peak of rotavirus infections; this pattern is unusual for ETEC and may be important for vaccination strategies in Bolivia.

Comparación cliiico - laboratorial en pacientes con hipertensión inducida por el embarazo / Clinicl comparisson - laboratory in patients with pregnancy induced hypertension

Objetivos. Determinar si existe congruencia entre los diagnósticos clínico y laboratorial en pacientes que cursan con hipertensión inducida por el embarazo. Métodos. Se analizaron las historias clínicas perinatales del Sistema Informático Perinatal, correspondientes a mujeres que cursando un embarazo mayor de 20 semanas se internaron en el Hospital "La Paz". Se trata de un estudio de corte transversal hospitalario entre el 1 de junio de 2007 y el 31 de mayo de 2009 en el departamento de La Paz. Resultados. Se observó una asociación importante entre adolescencia y primiparidad vs. la ocurrencia de trastornos hipertensivos del embarazo (p<0,003). Un porcentaje diverso de usuarias presentan edema, cefalea y otros signos de vasoespasmo. Se omiten muchos exámenes de laboratorio al acceder una mujer con hipertensión al Hospital "La Paz". Conclusiones. La preeclampsia/eclampsia es un problema de salud pública que no siempre se aborda con seriedad. Las políticas de salud debieran desarrollar programas de educación continua y actualización para el personal de salud del país, a fin de promover la elaboración de protocolos y procedimientos actualizados.

Objectives. Determine if there is agreement between the clinical and laboratory diagnoses on patients with gestational hypertension. Methods. We evaluated the medical records of the Perinatal Data System, for pregnant women after 20 weeks gestation that were admitted to the Hospital "La Paz". It was a facility-based cross-sectional study between 1 June 2007 and 31 May 2009 in La Paz. Results. There was a significant association between adolescence and primiparity vs. the occurrence of hypertensive disorders of pregnancy (p <0.003). A different percentage of users have edema, headache and other signs of vasospasm. Providers use to omit many laboratory tests to access a woman with hypertension at Hospital La Paz". Conclusions. Preeclampsia/eclampsiais a public health problem not always addressed seriously. Health policies should develop continuing education programs and refresher courses for health personnel in the country, to develop protocols and procedures to date.

Seroprevalencia del Virus de la Hepatitis B y C mediante la reactividad a los marcadores serológicos / Seroprevalence of the Virus of hepatitis B and C through the reactivity to the sexological markers

En Bolivia estudios epidemiológicos sobre patologías producidas por virus hepatotróficos son escasos, debido a ello sus patrones de circulación son practicamente desconocidos. En el presente trabajo se reporta la seroprevalencia de infecciones por virus por virus de la hepatitis B y C.