Julio E. Castañeda-Delgado

Ability of innate defence regulator peptides IDR-1002, IDR-HH2 and IDR-1018 to protect against Mycobacterium tuberculosis infections in animal models.

Tuberculosis is an ongoing threat to global health, especially with the emergence of multi drug-resistant (MDR) and extremely drug-resistant strains that are motivating the search for new treatment strategies. One potential strategy is immunotherapy using Innate Defence Regulator (IDR) peptides that selectively modulate innate immunity, enhancing chemokine induction and cell recruitment while suppressing potentially harmful inflammatory responses. IDR peptides possess only modest antimicrobial activity but have profound immunomodulatory functions that appear to be influential in resolving animal model infections. The IDR peptides HH2, 1018 and 1002 were tested for their activity against two M. tuberculosis strains, one drug-sensitive and the other MDR in both in vitro and in vivo models. All peptides showed no cytotoxic activity and only modest direct antimicrobial activity versus M. tuberculosis (MIC of 15–30 µg/ml). Nevertheless peptides HH2 and 1018 reduced bacillary loads in animal models with both the virulent drug susceptible H37Rv strain and an MDR isolate and, especially 1018 led to a considerable reduction in lung inflammation as revealed by decreased pneumonia. These results indicate that IDR peptides have potential as a novel immunotherapy against TB.

Activity of LL-37, CRAMP and antimicrobial peptide-derived compounds E2, E6 and CP26 against Mycobacterium tuberculosis

Tuberculosis (TB) is a major worldwide health problem in part due to the lack of development of new treatments and the emergence of new strains such as multidrug-resistant (MDR) and extensively drug-resistant strains that are threatening and impairing the control of this disease. In this study, the efficacy of natural and synthetic cationic antimicrobial (host defence) peptides that have been shown often to possess broad-spectrum antimicrobial activity was tested. The natural antimicrobial peptides human LL-37 and mouse CRAMP as well as synthetic peptides E2, E6 and CP26 were tested for their activity against Mycobacterium tuberculosis both in in vitro and in vivo models. The peptides had moderate antimicrobial activities, with minimum inhibitory concentrations ranging from 2 μg/mL to 10 μg/mL. In a virulent model of M. tuberculosis lung infection, intratracheal therapeutic application of these peptides three times a week at doses of ca. 1mg/kg led to significant 3-10-fold reductions in lung bacilli after 28-30 days of treatment. The treatments worked both against the drug-sensitive H37Rv strain and a MDR strain. These results indicate that antimicrobial peptides might constitute a novel therapy against TB.

Differential expression of antimicrobial peptides in active and latent tuberculosis and its relationship with diabetes mellitus.

Tuberculosis (TB) is one of the most important infectious diseases, causing 1.8 million deaths annually worldwide. This problem has increased because of the association with human immmunodeficiency virus and diabetes mellitus type 2, mainly in developing countries. In the past few years it has been highlighted the significance of antimicrobial peptides in the immunopathogenesis of TB ex vivo and in experimental models studies. In this study we analyzed the expression of CAMP, DEFA1, DEFB4, and DEFB103A in patients with latent TB and progressive TB with and without comorbidity with diabetes mellitus type 2. Antimicrobial peptide gene expression increased during progressive TB, which could be used as a biomarker for reactivation. By contrast, patients with diabetes mellitus type 2 have lower antimicrobial peptides gene expression, suggesting that the lack of its proper production in these patients contribute to enhance the risk for TB reactivation.

Prime-boost BCG vaccination with DNA vaccines based in β-defensin-2 and mycobacterial antigens ESAT6 or Ag85B improve protection in a tuberculosis experimental model.

The World Health Organization (WHO) has estimated that there are about 8 million new cases annually of active Tuberculosis (TB). Despite its irregular effectiveness (0-89%), the Bacillus Calmette-Guérin) BCG is the only vaccine available worldwide for prevention of TB; thus, the design is important of novel and more efficient vaccination strategies. Considering that β-defensin-2 is an antimicrobial peptide that induces dendritic cell maturation through the TLR-4 receptor and that both ESAT-6 and Ag85B are immunodominant mycobacterial antigens and efficient activators of the protective immune response, we constructed two DNA vaccines by the fusion of the gene encoding β-defensin-2 and antigens ESAT6 (pDE) and 85B (pDA). After confirming efficient local antigen expression that induced high and stable Interferon gamma (IFN-γ) production in intramuscular (i.m.) vaccinated Balb/c mice, groups of mice were vaccinated with DNA vaccines in a prime-boost regimen with BCG and with BCG alone, and 2 months later were challenged with the mild virulence reference strain H37Rv and the highly virulent clinical isolate LAM 5186. The level of protection was evaluated by survival, lung bacilli burdens, and extension of tissue damage (pneumonia). Vaccination with both DNA vaccines showed similar protection to that of BCG. After the challenge with the highly virulent Mycobacterium tuberculosis strain, animals that were prime-boosted with BCG and then boosted with both DNA vaccines showed significant higher survival and less tissue damage than mice vaccinated only with BCG. These results suggest that improvement of BCG vaccination, such as the prime-boost DNA vaccine, represents a more efficient vaccination scheme against TB.

Induction of β‐defensins by l‐isoleucine as novel immunotherapy in experimental murine tuberculosis

Tuberculosis is a worldwide health problem, and multidrug‐resistant (MDR) and extensively multidrug‐resistant (XMDR) strains are rapidly emerging and threatening the control of this disease. These problems motivate the search for new treatment strategies. One potential strategy is immunotherapy using cationic anti‐microbial peptides. The capacity of l‐isoleucine to induce beta‐defensin expression and its potential therapeutic efficiency were studied in a mouse model of progressive pulmonary tuberculosis. BALB/c mice were infected with Mycobacterium tuberculosis strain H37Rv or with a MDR clinical isolate by the intratracheal route. After 60 days of infection, when disease was in its progressive phase, mice were treated with 250 µg of intratracheal l‐isoleucine every 48 h. Bacillary loads were determined by colony‐forming units, protein and cytokine gene expression were determined by immunohistochemistry and reverse transcription–quantitative polymerase chain reaction (RT–qPCR), respectively, and tissue damage was quantified by automated morphometry. Administration of l‐isoleucine induced a significant increase of beta‐defensins 3 and 4 which was associated with decreased bacillary loads and tissue damage. This was seen in animals infected with the antibiotic‐sensitive strain H37Rv and with the MDR clinical isolate. Thus, induction of beta‐defensins might be a potential therapy that can aid in the control of this significant infectious disease.

Expression of antimicrobial peptides in diabetic foot ulcer

BACKGROUND Foot ulcers are one of the main diabetes complications due to its high frequency and difficulty of complete healing. There are several factors that participate in diabetic ulcers development and limited information exists about the role of antimicrobial peptides (AMP) in its pathogenesis. OBJECTIVE The aim of this study was to analyze the expression pattern of the main AMPs: Human Neutrophil Peptide (HNP)-1, Human β-defensin (HBD)-1, HBD-2, HBD-3, HBD-4 and cathelicidin LL-37 in biopsies from diabetic foot ulcers (DFU). METHODS 20 biopsies from DFU grade 3 according to Wagners classification and 20 biopsies from healthy donors were obtained. Real time PCR, immunohistochemistry and primary cell cultures were performed. RESULTS β-Defensins were overexpressed in DFU, whereas LL-37 has low or none expression in comparison with healthy skin. When primary cell culture from these biopsies were performed and infected with Staphylococcus aureus, epidermal cell from diabetic ulcers showed lower LL-37 expression compared with cell cultures from healthy donors skin. CONCLUSION These results suggest that though most AMPs are expressed in DFU, this production is not appropriate to promote wound healing and contain secondary infections.

Bacterial expression and antibiotic activities of recombinant variants of human β-defensins on pathogenic bacteria and M. tuberculosis

Five variants of human β-defensins (HBDs) were expressed in Escherichia coli using two vector systems (pET28a(+) and pQE30) with inducible expression by IPTG. The last vector has not been previously reported as an expression system for HBDs. The recombinant peptides were different in their lengths and overall charge. The HBDs were expressed as soluble or insoluble proteins depending on the expression system used, and the final protein yields ranged from 0.5 to 1.6 mg of peptide/g of wet weight cells, with purities higher than 90%. The recombinant HBDs demonstrated a direct correlation between antimicrobial activity and the number of basic charged residues; that is, their antimicrobial activity was as follows: HBD3-M-HBD2 > HBD3 = HBD3-M = HB2-KLK > HBD2 when assayed against E. coli, Staphylococcus aureus and Pseudomonas aeruginosa. Interestingly, HBD2 had the best antimicrobial activity against the Mycobacterium tuberculosis strain H37Rv (1.5 μM) and the heterologous tandem peptide, HBD3-M-HBD2, had the best minimal inhibitory concentration (MIC) value (2.7 μM) against a multidrug resistance strain (MDR) of M. tuberculosis, demonstrating the feasibility of the use of HBDs against pathogenic M. tuberculosis reported to be resistant to commercial antibiotics.

Kinetics and cellular sources of cathelicidin during the course of experimental latent tuberculous infection and progressive pulmonary tuberculosis

In spite of advances in immunology on mycobacterial infection, there are few studies on the role of anti‐microbial peptides in tuberculosis. The cathelin‐related anti‐microbial peptide (CRAMP) is the only cathelicidin isolated from mice. In this work we investigated the cellular sources and the production kinetics of this molecule during experimental tuberculosis, using two well‐characterized models of latent or chronic infection and progressive disease. The lung of non‐infected control mice expressed CRAMP at very low levels. In both models of experimental tuberculosis the main cells immunolabelled for CRAMP were bronchial epithelial cells, macrophages and pneumocytes types II and I. After intratracheal infection with a high bacilli dose (H37Rv strain) in Balb/c mice to produce progressive disease, a high CRAMP gene expression was induced showing three peaks: very early after 1 day of infection, at day 21 when the peak of protective immunity in this model is raised, and at day 28 when the progressive phase starts and the immunoelectronmicroscopy study showed intense immunolabelling in the cell wall and cytoplasm of intracellular bacilli, as well as in cytoplasmic vacuoles. Interestingly, at day 60 post‐infection, when advanced progressive disease is well established, characterized by high bacillary loads and extensive tissue damage, CRAMP gene expression decreased but strong CRAMP immunostaining was detected in vacuolated macrophages filled with bacilli. Thus, cathelecidin is highly produced during experimental pulmonary tuberculosis from diverse cellular sources and could have significant participation in its pathogenesis.

Vitamin D supplementation promotes macrophages' anti-mycobacterial activity in type 2 diabetes mellitus patients with low vitamin D receptor expression.

The increasing number of people with type 2 diabetes (DM2) is alarming and if it is taken into account that the relative odds of developing tuberculosis in diabetic patients ranges from 2.44 to 8.33 compared with non-diabetic patients, thus in developing countries where these two diseases are encountering face to face, there is a need for prophylaxis strategies. The role of vitamin D has been widely implicated in growth control of Mycobacterium tuberculosis (Mtb) during primary infection mainly through the induction of certain antimicrobial peptides (AMPs). In this study we evaluated the vitamin D serum levels, CYP27B1-hydroxylase enzyme, vitamin D receptor (VDR) and AMPs gene expression in Healthy donors, DM2 and TB patients. Results showed that DM2 group has lower VDR and AMPs expression levels. When Monocytes Derived Macrophages (MDM) from DM2 patients with low VDR expression were supplemented with vitamin D, MDMs eliminate efficiently M. tuberculosis. This preliminary study suggests the use of vitamin D as prophylaxis for tuberculosis in high DM2 endemic countries.

Metformin Induces Cell Cycle Arrest, Reduced Proliferation, Wound Healing Impairment In Vivo and Is Associated to Clinical Outcomes in Diabetic Foot Ulcer Patients.

Background Several epidemiological studies in diabetic patients have demonstrated a protective effect of metformin to the development of several types of cancer. The underlying mechanisms of such phenomenon is related to the effect of metformin on cell proliferation among which, mTOR, AMPK and other targets have been identified. However, little is known about the role that metformin treatment have on other cell types such as keratinocytes and whether exposure to metformin of these cells might have serious repercussions in wound healing delay and in the development of complications in diabetic patients with foot ulcers or in their exacerbation. Material and Methods HaCaT Cells were exposed to various concentrations of metformin and cell viability was evaluated by a Resazurin assay; Proliferation was also evaluated with a colony formation assay and with CFSE dilution assay by flow cytometry. Cell cycle was also evaluated by flow cytometry by PI staining. An animal model of wound healing was used to evaluate the effect of metformin in wound closure. Also, an analysis of patients receiving metformin treatment was performed to determine the effect of metformin treatment on the outcome and wound area. Statistical analysis was performed on SPSS v. 18 and GraphPad software v.5. Results Metformin treatment significantly reduces cell proliferation; colony formation and alterations of the cell cycle are observed also in the metformin treated cells, particularly in the S phase. There is a significant increase in the area of the wound of the metformin treated animals at different time points (P<0.05). There is also a significant increase in the size and wound area of the patients with diabetic foot ulcers at the time of hospitalization. A protective effect of metformin was observed for amputation, probably associated with the anti inflammatory effects reported of metformin. Conclusions Metformin treatment reduces cell proliferation and reduces wound healing in an animal model and affects clinical outcomes in diabetic foot ulcer patients. Chronic use of this drug should be further investigated to provide evidence of their security in association with DFU.