A novel methodology for NETs visualization under light microscopy.

Neutrophils are the most abundant leukocytes in the bloodstream and are very important for the resolution of infection. One of the strategies used by neutrophils to eliminate a microorganism is the formation of extracellular traps. Different methods for neutrophil extracellular traps (NETs) visualization have been described along the years, usually requiring the use of a fluorescent, confocal or scanning electron microscope. This research aimed to visualize NETs using light microscopy as another way to study NETs prior to using the more expensive techniques, making NETs research more cost effective. We evaluated neutrophil purity, viability and function by analyzing the formation of NETs comparing DAPI with safranin. When evaluating NETs formation, neutrophils that were not stimulated did not form NETs and when neutrophils were exposed to PMA or S. aureus NETs were formed and visualized with safranin under light microscopy and DAPI under fluorescence microscopy. Our method demonstrates another way to visualize NETs that can be added to the standard methods of visualization of NETs, increasing the opportunities to generate knowledge in the topic in any lab around the world.

Fatal Strongyloides stercoralis hyperinfection syndrome in an alcoholic diabetic patient from México / Síndrome de hiperinfección mortal por Strongyloides stercoralis en un paciente diabético y alcohólico de México

Strongyloides stercoralis hyperinfection syndrome is a medical emergency that requires a high level of suspicion. Immunocompromised patients are at high risk of hyperinfection syndrome; however, malnutrition, alcoholism, and diabetes mellitus also need to be considered as predisposing factors. The diagnosis and treatment of Strongyloides hyperinfection are challenging and patients often have severe complications. Consequently, mortality is overwhelmingly high, with proportions above 60%. Herein, we report a case of Strongyloides hyperinfection in a 40-year-old alcoholic diabetic patient living in México. Unfortunately, the late diagnosis resulted in his death despite the treatment and supportive measures. Increased awareness is needed to prevent the dire consequences of strongyloidiasis.

El síndrome de hiperinfección por Strongyloides stercoralis es una emergencia médica que requiere una aguda sospecha clínica. Los pacientes inmunocomprometidos tienen alto riesgo de sufrir el síndrome de hiperinfección; sin embargo, la desnutrición, el alcoholismo y la diabetes mellitus también deben considerarse factores predisponentes. El diagnóstico y el tratamiento de la hiperinfección por S. stercoralis constituyen un desafío y los pacientes a menudo tienen complicaciones graves. Como consecuencia, la mortalidad es abrumadoramente alta, con proporciones superiores al 60 %. Se presenta un caso de hiperinfección por S. stercoralis en un paciente diabético y alcohólico de 40 años que vivía en México. Infortunadamente, el diagnóstico tardío causó su muerte a pesar del tratamiento y las medidas de soporte. Se necesita un mayor conocimiento para prevenir las terribles consecuencias de la estrongiloidiasis.

Fatal Strongyloides stercoralis hyperinfection syndrome in an alcoholic diabetic patient from México / Síndrome de hiperinfección mortal por Strongyloides stercoralis en un paciente diabético y alcohólico de México

Strongyloides stercoralis hyperinfection syndrome is a medical emergency that requires a high level of suspicion. Immunocompromised patients are at high risk of hyperinfection syndrome; however, malnutrition, alcoholism, and diabetes mellitus also need to be considered as predisposing factors. The diagnosis and treatment of Strongyloides hyperinfection are challenging and patients often have severe complications. Consequently, mortality is overwhelmingly high, with proportions above 60%. Herein, we report a case of Strongyloides hyperinfection in a 40-year-old alcoholic diabetic patient living in México. Unfortunately, the late diagnosis resulted in his death despite the treatment and supportive measures. Increased awareness is needed to prevent the dire consequences of strongyloidiasis.

El síndrome de hiperinfección por Strongyloides stercoralis es una emergencia médica que requiere una aguda sospecha clínica. Los pacientes inmunocomprometidos tienen alto riesgo de sufrir el síndrome de hiperinfección; sin embargo, la desnutrición, el alcoholismo y la diabetes mellitus también deben considerarse factores predisponentes. El diagnóstico y el tratamiento de la hiperinfección por S. stercoralis constituyen un desafío y los pacientes a menudo tienen complicaciones graves. Como consecuencia, la mortalidad es abrumadoramente alta, con proporciones superiores al 60 %. Se presenta un caso de hiperinfección por S. stercoralis en un paciente diabético y alcohólico de 40 años que vivía en México. Infortunadamente, el diagnóstico tardío causó su muerte a pesar del tratamiento y las medidas de soporte. Se necesita un mayor conocimiento para prevenir las terribles consecuencias de la estrongiloidiasis.

Inflammatory and Anti-inflammatory Responses Co-exist Inside Lung Granuloma of Fatal Cases of Coccidioidomycosis: A Pilot Report.

Coccidioidomycosis is a fungal disease caused by Coccidioides immitis or Coccidioides posadasii. These fungi are endemic in the southern USA and northern Mexico. Immunocompromised patients are susceptible to develop severe forms of this fungal infection. Cytokines play an important role in controlling the fungal infection, but little is known about the predominant immunological environment in human lung tissue from fatal cases. Our aim was to analyze the pro-inflammatory and anti-inflammatory cytokines and monocyte/macrophages markers (CD14 and CD206) in the granulomas of six fatal cases of coccidioidomycosis. Cytokines and surface markers were higher in coccidioidomycosis cases when compared to control (P < 0.05). CD14 positive cells were increased inside the coccidioidal granuloma when compared to the outside (P < 0.05). No differences were found in the number of CD206+ cells inside the granuloma when compared to the outer population (P > 0.05). Interestingly, an analysis of stain intensity signals showed an increased signaling of CD14, CD206, IL-10 and TNFα inside the granuloma when compared to the outside (P < 0.05). iNOS and IL-12 gene expression were not detected in coccidioidomycosis cases, while IL-10, IL-6 and TGFß gene expression were detected, but the differences when compared to healthy lungs were not significant (P > 0.05). TNFα gene expression was lower in coccidioidomycosis cases when compared to healthy lung (P = 0.05). In conclusion, pro- and anti-inflammatory responses co-exist inside of the granulomas of fatal cases of coccidioidomycosis and the absent of iNOS and IL-12 gene expression may be related with patient's outcome.

Interferon γ and interleukin 10 responses in immunocompetent and immunosuppressed New Zealand White rabbits naturally infected with Encephalitozoon cuniculi.

Levels of interferon (IFN)-γ and interleukin (IL)-10 were measured in the serum of immunocompetent and immunosuppressed New Zealand White rabbits naturally infected with Encephalitozoon cuniculi. IFN-γ levels were elevated in infected rabbits, and a synergic effect was observed in animals treated with the immunosuppressive agent dexamethasone (Dex). The role of IL-10 in infected rabbits remains unclear, as IL-10 levels were similar to those of negative controls. Dex appeared to exhibit a proinflammatory effect, as IFN-γ levels were elevated in infected immunosuppressed rabbits. Similarly, Dex exhibited a synergic effect in infected immunosuppressed rabbits, as evidenced by the elevation in IFN-γ production. These data indicate that the immune response to this glucocorticoid should be considered in the design of future animal model studies of immunosuppression.

Possible association of rare polymorphism in the ABCB1 gene with rifampin and ethambutol drug-resistant tuberculosis.

Human P-glycoprotein (P-gp) is a membrane transporter encoded by ABCB1 (also known as MDR1) that plays a critical role in pharmacokinetics of many unrelated drugs. Rifampin (RMP) and ethambutol (ETB), two anti-tubercular agents, are substrates of P-gp. Single nucleotide polymorphisms (SNPs) in ABCB1 have been associated with resistance to several drugs; however, their association with RMP and ETB resistance in tuberculosis patients has not yet been studied. Genotype/allele frequencies in C1236T, G2677T/A and C3435T SNPs of ABCB1 were obtained from 99 tuberculosis patients susceptible or resistant to RMP and ETB (NoRER or RER). 2677G>A allele prevalence was found to be significantly higher in the RER group compared to NoRER (5 resistant vs 2 non-resistant patients, P < 0.01; OR, 11.0; 95% CI, 2.00-56.00). No differences were found in genotype/allele frequencies in C1236T and C3435T SNPs of ABCB1 and resistance to RMP and ETB in tuberculosis patients (P > 0.05). The present study suggests the 2677G>A allele of ABCB1 could be associated with simultaneous resistance to RMP and ETB in pulmonary tuberculosis patients. Further studies with larger sample sizes are needed to confirm this association and explore its nature.

Enhancement of Ad-CRT/E7-mediated antitumor effect by preimmunization with L. lactis expressing HPV-16 E7.

Although current polyvalent vaccines can prevent development of cervical cancer, they cannot be used to treat patients who already have the disease. Adenovirus expressing calreticulin-E7 (Ad-CRT-E7) has shown promising results in the cervical cancer murine model. We also demonstrated that immunization with Lactococcus lactis encoding HPV-16 E7 (Ll-E7) anchored to its surface induces significant HPV-16 E7-specific immune response. Here, we assessed the combination of both approaches in the treatment of a cervical cancer animal model. Intranasal preimmunization of Ll-E7, followed by a single Ad-CRT/E7 application, induced ∼80% of tumor suppression in comparison with controls. Mice treated with a combination of Ll-E7 and Ad-CRT/E7 resulted in a 70% survival rate 300 days post-treatment, whereas 100% of the mice in the control groups died by 50 days. Significant CD8+ cytotoxic T-lymphocytes infiltration was detected in the tumors of mice treated with Ll-E7+Ad-CRT/E7. Tumors with regression showed a greater number of positive cells for in situ TUNEL staining than controls. Our results suggest that preimmunization with Ll-E7 enhances the Ad-CRT/E7-mediated antitumor effect. This treatment provides an enormous advantage over repeated applications of Ad-CRT/E7 by maintaining the effectiveness of the three-dose application of Ad-CRT/E7, but avoiding the high systemic toxicities associated with such repeat treatments.

Expression of CDllc in blood monocytes as biomarker for favorable response to antituberculosis treatment.

CD11c is involved in Mycobacterium tuberculosis phagocytosis by human macrophages. CD11c has not been evaluated during antituberculosis (anti-TB) treatment. CD11c expression was evaluated on monocytes from peripheral blood leukocytes of pulmonary TB (PTB) patients and healthy controls by flow cytometry, whereas CD11c/Arg47Trp polymorphism was analyzed using polymerase chain reaction-restriction fragment length polymorphism. PTB patients had increased levels of CD11c on blood monocytes as compared to healthy controls. CD11c levels decreased in response to anti-TB treatment. CD11c/Arg47Trp polymorphism is not associated with PTB.

CD14 C(-159)T polymorphism is a risk factor for development of pulmonary tuberculosis.

BACKGROUND: Neither the expression of CD14 and Toll-like receptor 4 (TLR4) on monocytes' surface nor the mutations CD14 -159TT and TLR4 Asp299Gly have yet been evaluated as risk factors for development of pulmonary tuberculosis (TB) in the Mexican population. METHODS: Level of membrane CD14 (mCD14) and membrane TLR4 (mTLR4) were determined by flow cytometry, in 104 patients with pulmonary TB (before and after treatment), 67 household contacts, and 114 healthy control subjects. Genotype/allele frequencies in CD14 -159 and TLR4 Asp299Gly were obtained by polymerase chain reaction-restriction-fragment length polymorphism. Levels of soluble CD14 (sCD14) in sera were quantified by ELISA. RESULTS: Higher levels of mCD14/sCD14 and mTLR4 were observed in the patients and the household contacts than in the control subjects (P<.05) and decreased in the patients after the infection was resolved. The frequency of the CD14 -159TT genotype was higher in the patients than in the control subjects (35.6% vs. 12.3%, respectively). Patients who were homozygous for allele T of the CD14 promoter gene had a significantly higher risk for development of pulmonary TB, with an odds ratio of 2.267 (95% confidence interval, 1.5%-3.3%). Levels of sCD14 or mCD14 were not associated with the CD14 -159TT genotype (P>.05). CONCLUSIONS: No association between TLR4 Asp299Gly and pulmonary TB was found. CD14 -159TT is a risk factor for development of pulmonary TB, whereas mCD14/sCD14 and mTLR4 are possible biomarkers for the prognosis for TB disease. CLINICAL TRIAL PROTOCOL ID: SA1168-05.

Mycobacterium tuberculosis upregulates coreceptors CCR5 and CXCR4 while HIV modulates CD14 favoring concurrent infection.

Tuberculosis is the most frequent coinfection in humans infected with HIV-1, but little is known about mechanisms that favors coinfection. The aim of this work is to understand tuberculosis and HIV infections. We determined the pattern of expression of CD11c, CD14, CD40, CCR5, and CXCR4 and quantified IL-1beta, IL-6, IL-8, TNF-alpha, and RANTES in tuberculosis patients and HIV patients. Monocytes from healthy PPD+ volunteers (HP(+)V) stimulated with intracellular proteins (IP), lipids, and polysaccharides (PLS) from Mycobacterium tuberculosis down regulate CD11c expression (p < 0.05). On the contrary, CD14 expression was elevated in tuberculosis patients (p < 0.05) and HIV-infected patients (p > 0.05). CD14 expression was elevated on monocytes from HP(+)V stimulated with PLS and lipids (p < 0.05). CD40 low expression was found in tuberculosis patients and on monocytes from HP(+)V stimulated with lipids, but it was elevated in HIV-infected patients (p < 0.05). CXCR4 and CCR5 expression was high in pulmonary tuberculosis patients and low in HIV-infected patients (p < 0.05). Finally, CCR5+ monocytes from HP(+)V after stimulation with PLS and CXCR4+ lymphocytes were elevated after stimulation with IP (p < 0.05). In general, high levels of IL-1beta, IL-6, and TNF-alpha were found in all groups, but low levels of RANTES were found in pulmonary tuberculosis patients. In conclusion, the pulmonary tuberculosis patients have a microenvironment that facilitates the HIV infection through three possible mechanisms: (1) increasing the coreceptor for HIV entrance, (2) increasing proinflammatory cytokines, and (3) down-regulating RANTES. At the same time, HIV patients have a microenvironment that facilitates entry of M. tuberculosis into macrophages through CD14.