José Antonio Enciso-Moreno

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.

Molecular analysis of Mycobacterium isolates from extrapulmonary specimens obtained from patients in Mexico

BackgroundLittle information is available on the molecular epidemiology in Mexico of Mycobacterium species infecting extrapulmonary sites in humans. This study used molecular methods to determine the Mycobacterium species present in tissues and body fluids in specimens obtained from patients in Mexico with extrapulmonary disease.MethodsBacterial or tissue specimens from patients with clinical or histological diagnosis of extrapulmonary tuberculosis were studied. DNA extracts from 30 bacterial cultures grown in Löwenstein Jensen medium and 42 paraffin-embedded tissues were prepared. Bacteria were cultured from urine, cerebrospinal fluid, pericardial fluid, gastric aspirate, or synovial fluid samples. Tissues samples were from lymph nodes, skin, brain, vagina, and peritoneum. The DNA extracts were analyzed by PCR and by line probe assay (INNO-LiPA MYCOBACTERIA v2. Innogenetics NV, Gent, Belgium) in order to identify the Mycobacterium species present. DNA samples positive for M. tuberculosis complex were further analyzed by PCR and line probe assay (INNO-LiPA Rif.TB, Innogenetics NV, Gent, Belgium) to detect mutations in the rpo B gene associated with rifampicin resistance.ResultsOf the 72 DNA extracts, 26 (36.1%) and 23 (31.9%) tested positive for Mycobacterium species by PCR or line probe assay, respectively. In tissues, M. tuberculosis complex and M. genus were found in lymph nodes, and M. genus was found in brain and vagina specimens. In body fluids, M. tuberculosis complex was found in synovial fluid. M. gordonae, M. smegmatis, M. kansasii, M. genus, M. fortuitum/M. peregrinum complex and M. tuberculosis complex were found in urine. M. chelonae/M. abscessus was found in pericardial fluid and M. kansasii was found in gastric aspirate. Two of M. tuberculosis complex isolates were also PCR and LiPA positive for the rpo B gene. These two isolates were from lymph nodes and were sensitive to rifampicin.Conclusion1) We describe the Mycobacterium species diversity in specimens derived from extrapulmonary sites in symptomatic patients in Mexico; 2) Nontuberculous mycobacteria were found in a considerable number of patients; 3) Genotypic rifampicin resistance in M. tuberculosis complex infections in lymph nodes was not found.

1,25-Dihydroxyvitamin D3 Induces LL-37 and HBD-2 Production in Keratinocytes from Diabetic Foot Ulcers Promoting Wound Healing: An In Vitro Model

Diabetic foot ulcers (DFU) are one of the most common diabetes-related cause of hospitalization and often lead to severe infections and poor healing. It has been recently reported that patients with DFU have lower levels of antimicrobial peptides (AMPs) at the lesion area, which contributes with the impairment of wound healing. The aim of this study was to determine whether 1,25-dihydroxyvitamin D3 (1,25 (OH)2 D3) and L-isoleucine induced HBD-2 and LL-37 in primary cultures from DFU. We developed primary cell cultures from skin biopsies from 15 patients with DFU and 15 from healthy donors. Cultures were treated with 1,25 (OH)2D3 or L-isoleucine for 18 h. Keratinocytes phenotype was identified by western blot and flow cytometry. Real time qPCR for DEFB4, CAMP and VDR gene expression was performed as well as an ELISA to measure HBD-2 and LL-37 in supernatant. Antimicrobial activity, in vitro, wound healing and proliferation assays were performed with conditioned supernatant. The results show that primary culture from DFU treated with 1,25(OH)2D3, increased DEFB4 and CAMP gene expression and increased the production of HBD-2 and LL-37 in the culture supernatant. These supernatants had antimicrobial activity over E. coli and induced remarkable keratinocyte migration. In conclusion the 1,25(OH)2D3 restored the production of AMPs in primary cell from DFU which were capable to improve the in vitro wound healing assays, suggesting their potential therapeutic use on the treatment of DFU.

LL-37 Immunomodulatory Activity during Mycobacterium tuberculosis Infection in Macrophages

ABSTRACT Tuberculosis is one of the most important infectious diseases worldwide. The susceptibility to this disease depends to a great extent on the innate immune response against mycobacteria. Host defense peptides (HDP) are one of the first barriers to counteract infection. Cathelicidin (LL-37) is an HDP that has many immunomodulatory effects besides its weak antimicrobial activity. Despite advances in the study of the innate immune response in tuberculosis, the immunological role of LL-37 during M. tuberculosis infection has not been clarified. Monocyte-derived macrophages were infected with M. tuberculosis strain H37Rv and then treated with 1, 5, or 15 μg/ml of exogenous LL-37 for 4, 8, and 24 h. Exogenous LL-37 decreased tumor necrosis factor alpha (TNF-α) and interleukin-17 (IL-17) while inducing anti-inflammatory IL-10 and transforming growth factor β (TGF-β) production. Interestingly, the decreased production of anti-inflammatory cytokines did not reduce antimycobacterial activity. These results are consistent with the concept that LL-37 can modulate the expression of cytokines during mycobacterial infection and this activity was independent of the P2X7 receptor. Thus, LL-37 modulates the response of macrophages during infection, controlling the expression of proinflammatory and anti-inflammatory cytokines.

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.

Genetic diversity of the Mycobacterium tuberculosis Complex in San Luis Potosí, México

BackgroundAlthough epidemiologic and socioeconomic criteria and biomedical risk factors indicate high-priority for tuberculosis (TB) control in Mexico, molecular epidemiology studies of the disease in the country are scarce.MethodsComplete sociodemographic and clinical data were obtained from 248 of the 432 pulmonary TB (PTB) cases confirmed from 2006 to 2010 on the population under epidemiological surveillance in the state of San Luis Potosí, México. From most PTB cases with complete data Mycobacterium tuberculosis complex (MTC) isolates were recovered and their spoligotypes, lineages and families, geographic distribution and drug resistance determined.ResultsPulmonary tuberculosis incidence ranged from 2.4 to 33.4 (cases per 100,000 inhabitants) in the six state sanitary jurisdictions that were grouped in regions of low (jurisdictions I-II-III), intermediate (jurisdictions IV-V) and high incidence (jurisdiction VI) with 6.2, 17.3 and 33.4 rates, respectively. Most patients were poor, 50-years-median-age males and housewives. Among the 237 MTC spoligotyped isolates, 232 corresponded to M. tuberculosis (104 spoligotypes in 24 clusters) and five to M. bovis. The predominant Euro-American lineage was distributed all over the state, the East-Asian lineage (Beijing family) in the capital city, the Indo-Oceanic (Manila family) in eastern localities, and M. bovis in rural localities.ConclusionsIn San Luis Potosí TB affects mainly poor male adults and is caused by M. tuberculosis and to a minor extent by M. bovis. There is great genotypic diversity among M. tuberculosis strains, the Euro-American lineage being much more prevalent than the Indo-Oceanic and East-Asian lineages. The frequency of resistant strains is relatively low and not associated to any particular lineage.

Expression and vitamin D-mediated regulation of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in healthy skin and in diabetic foot ulcers

Diabetic foot ulcers (DFUs) are chronic wounds with high matrix metalloproteinase (MMP) activity, and are a frequent complication on diabetics. This work studied the expression of selected MMP and tissue inhibitor of metalloproteinases (TIMP) gene family members in DFU and normal skin biopsies, and in vitamin D-treated keratinocytes cultured from those biopsies. We report for the first time the expression of some of these genes in healthy skin. Our results suggest that vitamin D may modulate the expression of some MMP gene family members in keratinocytes. Gene expression in DFU and in non-diabetic healthy skin (control) biopsies was evaluated by RT-qPCR for MMP-1, MMP-3, MMP-8, MMP-9, MMP-10, MMP-19, TIMP-1 and TIMP-2, and also by immunohistochemistry for MMP-1 and MMP-9. Primary keratinocytes cultured from DFU and healthy skin biopsies were used for gene expression analyses of selected MMPs and TIMPs by RT-qPCR, both in the presence and absence of calcitriol. The expression of MMP-1, MMP-8, MMP-9, MMP-10, and TIMP-2 in healthy skin is reported here for the first time. DFUs showed increased MMP-1, MMP-9 and TIMP-1 expression, compared to healthy skin. Calcitriol down-regulated MMP-1 and MMP-10 expression in DFU-derived keratinocytes but not in those derived from healthy skin. Our data demonstrate the expression of certain MMPs that had not been previously described in healthy skin, and further support previous reports of MMP and TIMP up-regulation in DFUs. Our results point to calcitriol as a potential modulator for the expression of certain MMP members in DFUs.

Type I Interferon Gene Response Is Increased in Early and Established Rheumatoid Arthritis and Correlates with Autoantibody Production

Background Rheumatoid arthritis (RA) is an inflammatory debilitating disease that affects the joints in the early and productive phases of an individual’s life. Several cytokines have been linked to the disease pathogenesis and are known to contribute to the inflammatory state characteristic of RA. The participation of type I interferon (IFN) in the pathogenesis of the disease has been already described as well as the identity of the genes that are regulated by this molecule, which are collectively known as the type I IFN signature. These genes have several functions associated with apoptosis, transcriptional regulation, protein degradation, Th2 cell induction, B cell proliferation, etc. This article evaluated the expression of several genes of the IFN signature in different stages of disease and their correlation with the levels of anticitrullinated protein antibodies (ACPA) anticarbamylated protein (Anti-CarP) antibodies. Methods Samples from individuals with early and established RA, high-risk individuals (ACPA+ and ACPA−), and healthy controls were recruited at “Unidad de Artritis y Rheumatismo” (Rheumatism and Arthritis Unit) in Guadalajara Jalisco Mexico. Determinations of ACPA were made with Eurodiagnostica ACPA plus kit. Anti-CarP determinations were made according to previously described protocols. RNA was isolated, and purity and integrity were determined according to RNA integrity number >6. Gene expression analysis was made by RT-qPCR using specific primers for mRNAs of the type I IFN signature. Relative gene expression was calculated according to Livak and Schmitgen. Results Significant differences in gene expression were identified when comparing the different groups for MXA and MXB (P < 0.05), also when comparing established RA and ACPA− in both IFIT 1 and G15. An increased expression of ISG15 was identified (P < 0.05), and a clear tendency toward increase was identified for HERC5. EPSTRI1, IFI6, and IFI35 were found to be elevated in the chronic/established RA and early RA (P < 0.05). Significant correlations were identified for the IFN signature genes with the levels of ACPA and anti-CarP (P < 0.05). Conclusion Our data confirm previous observations in the role of IFN signature and the pathogenesis of RA. Also, we provide evidence of an association between several genes of the IFN signature (that regulate Th2 cells and B cell proliferation) with the levels of anti-CarP antibodies and ACPA.

A population-based study of first and second-line drug-resistant tuberculosis in a high-burden area of the Mexico/United States border

The resistance of 139 Mycobacterium tuberculosis (MTB) isolates from the city of Monterrey, Northeast Mexico, to first and second-line anti-TB drugs was analysed. A total of 73 isolates were susceptible and 66 were resistant to anti-TB drugs. Monoresistance to streptomycin, isoniazid (INH) and ethambutol was observed in 29 cases. Resistance to INH was found in 52 cases and in 29 cases INH resistance was combined with resistance to two or three drugs. A total of 24 isolates were multidrug-resistant (MDR) resistant to at least INH and rifampicin and 11 MDR cases were resistant to five drugs. The proportion of MDR-TB among new TB cases in our target population was 0.72% (1/139 cases). The proportion of MDR-TB among previously treated cases was 25.18% (35/139 cases). The 13 polyresistant and 24 MDR isolates were assayed against the following seven second-line drugs: amikacin (AMK), kanamycin (KAN), capreomycin (CAP), clofazimine (CLF), ethionamide (ETH), ofloxacin (OFL) and cycloserine (CLS). Resistance to CLF, OFL or CLS was not observed. Resistance was detected to ETH (10.80%) and to AMK (2.70%), KAN (2.70%) and CAP (2.70%). One isolate of MDR with primary resistance was also resistant to three second-line drugs. Monterrey has a high prevalence of MDR-TB among previously treated cases and extensively drug-resistant-MTB strains may soon appear.