Search

25 years after the declaration of a Global Emergency by the World Health Organization, tuberculosis (TB) remains a major enemy to the humankind. During this period, much progress has been done to better understand its natural history, revealing its huge complexity, which highlighted the need for implementing systems immunology approaches. Recent advances focused in understanding the role of macrophage subtypes and dendritic cells role, the importance of cytokine balance, and the antigenic repertoire. Identification of early irruption of polymorphonuclear neutrophils and extracellular growth of the bacilli seem to be the most disruptive factors to understand the evolution towards active TB. Their inclusion in future models will provide new tools for the better understanding of the tuberculosis.

Despite the existence of the prophylactic Bacille Calmette-Guérin (BCG) vaccine, infection by Mycobacterium tuberculosis (Mtb) remains a major public health issue causing up to 1.8 million annual deaths worldwide. Increasing prevalence of Mtb strains resistant to antibiotics represents an urgent threat for global health that has prompted a search for alternative treatment regimens not subject to development of resistance. Immunotherapy constitutes a promising approach to improving current antibiotic treatments through engagement of the host’s immune system. We designed a multi-antigenic and multiphasic vaccine, based on the Modified Vaccinia Ankara (MVA) virus, denoted MVATG18598, which expresses ten antigens classically described as representative of each of different phases of Mtb infection. In vitro analysis coupled with multiple-passage evaluation demonstrated that this vaccine is genetically stable, i.e. fit for manufacturing. Using different mouse strains, we show that MVATG18598 vaccination results in both Th1-associated T-cell responses and cytolytic activity, targeting all 10 vaccine-expressed Mtb antigens. In chronic post-exposure mouse models, MVATG18598 vaccination in combination with an antibiotic regimen decreases the bacterial burden in the lungs of infected mice, compared with chemotherapy alone, and is associated with long-lasting antigen-specific Th1-type T cell and antibody responses. In one model, co-treatment with MVATG18598 prevented relapse of the disease after treatment completion, an important clinical goal. Overall, results demonstrate the capacity of the therapeutic MVATG18598 vaccine to improve efficacy of chemotherapy against TB. These data support further development of this novel immunotherapeutic in the treatment of Mtb infections.

The aim of this paper was to arrive at an approximation of the value of non-professional (informal) care provided to disabled people living within a household in Spain.We used the Survey on Disabilities, Autonomy and Dependency carried out in 2008 to obtain information about disabled individuals and their informal caregivers. We computed the total number of informal caregiving hours provided by main caregivers in Spain in 2008. The monetary value of informal care time was obtained using three different approaches: the proxy good method, the opportunity cost method and the contingent valuation method.Total hours of informal care provided in 2008 were estimated at 4193 million and the monetary value ranged from EUR23,064 to EUR50,158 million depending on the method used. The value of informal care was estimated at figures equivalent to 1.73-4.90 % of the gross domestic product for that year.Informal care represents a very high social cost regardless of the estimation method considered. A holistic approach to care of dependent people should take into account the role and needs of informal caregivers, promote their social recognition and lead to policies that enhance efficient use of formal and informal resources.

This work sets out to analyze the motivations adult children may have to provide informal care, considering the monetary transfers they receive from their parents. Traditional motivations, such as altruism and exchange, are matched against more recent social bond theories. Our findings indicate that informal caregivers receive less frequent and less generous transfers than non-caregivers; that is, caregivers are more prone to suppress their self-interested motivations in order to prioritize the well being of another person. Additionally, long-term public care benefits increase both the probability of receiving a transfer and its amount, with this effect being more intense for both the poorest and richest households. Our findings suggest that if long-term care benefits are intended to increase the recipients' welfare and represent a higher fraction of total income for the poorest households, the effectiveness of these long-term care policies may be diluted.

This paper studies if a situation of formal care unmet needs is a strong motivation for the onset of caregiving behavior, and if becoming caregiving is a compelling argument for leaving current job (in the presence/absence of formal care unmet needs). We use data from the Eurobarometer 67.3 for 18 European countries and estimate a three simultaneous equations model taking into account the potential endogeneity of labor participation and formal care unmet needs and assuming non-zero correlation among the error terms of the three equations. Results show that individuals who anticipate that becoming caregiver can suppose an obstacle for continuing working feel more refractory and are more prone to avoid caregiving responsibilities. Knowing someone with an unmet needs problem increases the probability of becoming caregiver by +19.23 pp (with a maximum of +39.39 pp for difficult access unmet needs) and raises the probability of leaving employment by 5.77 pp. Having to possibility of receiving economic benefits for caregivers encourage more labor market exit as compared to payment of social security contributions during care leaves.

The development of a new tuberculosis vaccine is an urgent need due to the failure of the current vaccine, BCG, to protect against the respiratory form of the disease. MTBVAC is an attenuated Mycobacterium tuberculosis vaccine candidate genetically engineered to fulfil the Geneva consensus requirements to enter human clinical trials. We selected a M. tuberculosis clinical isolate to generate two independent deletions without antibiotic-resistance markers in the genes phoP, coding for a transcription factor key for the regulation of M. tuberculosis virulence, and fadD26, essential for the synthesis of the complex lipids phthiocerol dimycocerosates (DIM), one of the major mycobacterial virulence factors. The resultant strain MTBVAC exhibits safety and biodistribution profiles similar to BCG and confers superior protection in preclinical studies. These features have enabled MTBVAC to be the first live attenuated M. tuberculosis vaccine to enter clinical evaluation.

Este trabajo aborda la integracion del sistema Especial de Empleados del Hogar en el regimen Ge-neral de la seguridad social utilizando informacion sobre empleados del hogar que atienden a per-sonas dependientes. se han comparado los efectos del real Decreto 1620/2011 y real Decreto-ley29/2012. Esta segunda reforma supone una reduccion del ahorro en las cuotas por contingencias co-munes y profesionales de las que se beneficiaban empleados y empleadores con la primera reforma.las simulaciones de ingresos de la Tesoreria de la seguridad social durante 2013-2019, en los dife-rentes escenarios, muestran un incremento de los ingresos con el segundo texto legislativo.

Altres ajuts: Programa HIVACAT, programa CERCA (Generalitat de Catalunya), Red Temática Cooperativa de Investigación en SIDA (RD12/0017/0002)), Govern Basc (IT838-13), Spanish Supercomputing Network (grant numbers BCV-2015-2-0009 and BCV-2016-2-0005) The HIV-1 gp41 Membrane Proximal External Region (MPER) is recognized by broadly neutralizing antibodies and represents a promising vaccine target. However, MPER immunogenicity and antibody activity are influenced by membrane lipids. To evaluate lipid modulation of MPER immunogenicity, we generated a 1-Palmitoyl-2-oleoylphosphatidylcholine (POPC)-based proteoliposome collection containing combinations of phosphatidylserine (PS), GM3 ganglioside, holesterol (CHOL), sphingomyelin (SM) and the TLR4 agonist monophosphoryl lipid A (MPLA). A recombinant gp41-derived miniprotein (gp41-MinTT) exposing the MPER and a tetanus toxoid (TT) peptide that favors MHC-II presentation, was successfully incorporated into lipid mixtures (>85%). Immunization of mice with soluble gp41-MinTT exclusively induced responses against the TT peptide, while POPC proteoliposomes generated potent anti-gp41 IgG responses using lower protein doses. The combined addition of PS and GM3 or CHOL/SM to POPC liposomes greatly increased gp41 immunogenicity, which was further enhanced by the addition of MPLA. Responses generated by all proteoliposomes targeted the N-terminal moiety of MPER overlapping the 2F5 neutralizing epitope. Our data show that lipids impact both, the epitope targeted and the magnitude of the response to membrane-dependent antigens, helping to improve MPER-based lipid carriers. Moreover, the identification of immunodominant epitopes allows for the redesign of immunogens targeting MPER neutralizing determinants.

Background Nyaditum resae® (NR) is a galenic preparation of heat-killed Mycobacterium manresensis, a new species of the fortuitum complex, that is found in drinkable water, and that has demonstrated to protect against the development of active TB in a murine experimental model that develop human-like lesions. Methods Double-blind, randomized, placebo-controlled Clinical Trial (51 volunteers included). Two different doses of NR and a placebo were tested, the randomization was stratified by Latent Tuberculosis Infection (LTBI)-positive (n = 21) and LTBI-negative subjects (n = 30). Each subject received 14 drinkable daily doses for 2 weeks. Results All patients completed the study. The 46.3% of the overall reported adverse events (AE) were considered related to the investigational treatment. None of them were severe (94% were mild and 6% moderate). No statistical differences were found when comparing the median number of AE between the placebo group and both treatment groups. The most common AE reported were gastrointestinal events, most frequently mild abdominal pain and increase in stool frequency. Regarding the immunogenic response, both LTBI-negative and LTBI-positive volunteers treated with NR experienced a global increase on the Treg response, showed both in the population of CD25+CD39-, mainly effector Treg cells, or CD25+CD39+ memory PPD-specific Treg cells. Conclusion This clinical trial demonstrates an excellent tolerability profile of NR linked to a significant increase in the population of specific effector and memory Tregs in the groups treated with NR in both LTBI-positive and negative subjects. NR shows a promising profile to be used to reduce the risk of active TB.

Este trabajo aborda la integración del sistema Especial de Empleados del Hogar en el Régimen General de la Seguridad Social utilizando información sobre empleados del hogar que atienden a personas dependientes. Se han comparado los efectos del Real Decreto 1620/2011 y Real Decreto-Ley 29/2012. Esta segunda reforma supone una reducción del ahorro en las cuotas por contingencias comunes y profesionales de las que se beneficiaban empleados y empleadores con la primera reforma. Las simulaciones de ingresos de la Tesorería de la Seguridad Social durante 2013-2019, en los diferentes escenarios, muestran un incremento de los ingresos con el segundo texto legislativo.

The aim of this study was to evaluate the evolution and role of corded cell aggregation in Mycobacterium tuberculosis cultures according to growth time and conditions. Thus, in standard culture using aerated 7H9 Middlebrook broth supplemented with 0.05% Tween 80, a dramatic CFU decrease was observed at the end of the exponential phase. This phase was followed by a stable stationary phase that led to dissociation between the optical density (O.D.) and CFU values, together with the formation of opaque colonies in solid culture. Further analysis revealed that this was due to cording. Scanning electron microscopy showed that cording led to the formation of very stable coiled structures and corded cell aggregations which proved impossible to disrupt by any of the physical means tested. Modulation of cording with a high but non-toxic concentration of Tween 80 led to a slower growth rate, avoidance of a sudden drop-off to the stationary phase, the formation of weaker cording structures and the absence of opaque colonies, together with a lower survival at later time-points. An innovative automated image analysis technique has been devised to characterize the cording process. This analysis has led to important practical consequences for the elaboration of M. tuberculosis inocula and suggests the importance of biofilm formation in survival of the bacilli in the extracellular milieu.

ResumenObjetivoEl objetivo de este trabajo es analizar una parte del beneficio social que proporcionan los cuidados no profesionales (informales), planteando el escenario hipotético de los recursos que habría que movilizar si hubiera que sustituir su labor.Métodos y datosEmpleando información de la Encuesta sobre Discapacidades, Autonomía personal y situaciones de Dependencia 2008 (EDAD-08) se ha realizado un ejercicio de simulación sobre el coste que tendría que asumir la sociedad si reemplazara los cuidados informales a personas de 65 y más años de edad por servicios sociales profesionales. Para tal fin se estiman las horas de cuidados informales prestadas en España durante el año 2008 y se valoran monetariamente mediante el método de coste de sustitución.ResultadosLa traducción monetaria de las horas de cuidados informales prestados durante el año 2008 nos lleva a cifras que oscilan entre los 25.000 y los 40.000 millones de euros, dependiendo del precio sombra asignado a la hora de cuidado. Estas cifran serían equivalentes a entre un 2,3% y un 3,8% del producto interior bruto (PIB) del mismo año. Cuando nos trasladamos al ámbito regional, la valoración oscila de manera muy importante entre comunidades autónomas, llegando alguna a alcanzar cifras que equivalen a cerca de un 6% de su PIB.ConclusionesEl abordaje integral de los cuidados de las personas dependientes exige incluir el papel y la atención a las necesidades de las personas cuidadoras y avanzar en su reconocimiento social.AbstractObjectiveTo analyze one part of the social benefit derived from non-professional (informal) caregivers by analyzing the hypothetical amount of resources that would need to be invested if informal care were substituted by formal care.Methods and dataUsing data from the Survey of Disabilities, Personal Autonomy and Situations of Dependency (EDAD-2008), we estimated the cost to society if informal care were substituted by formal care of the population aged 65 years and older. For this purpose, first we computed the total amount of informal caregiving hours provided in Spain in 2008, and then we obtained its monetary worth by using the proxy good method.ResultsThe monetary worth of informal care provided in 2008 ranged from 25,000 and 40,000 million euros, depending on the shadow price used to value one hour of care. These figures represented between 2.3% and 3.8% of the GDP for the same year. In regional terms, the valuation of informal care across Spain's autonomous regions showed a significant degree of dispersion, and in some regions, amounted to 6% of their GDP.ConclusionsThe comprehensive approach to the care of the elderly should take the role and needs of informal caregivers into consideration. Caregivers should be given greater social recognition.

Based on the tuberculin skin test it is estimated that latent tuberculosis infection is present in one-third of the world's population. The new strategies in public health and research are aimed to reduce and eradicate this enormous reservoir. However, the absence of effective biomarkers for diagnosis and treatment of latent tuberculosis limits the development of new drugs and vaccines. Some components are present in both, the PPD (used in the tuberculin skin test) and the BCG vaccine. This increases the number of false positives in vaccinated individuals. Nowadays, there is not an immune diagnostic method that can differentiate latent tuberculosis and tuberculosis disease. New studies have addressed some strategies including specific antibodies, new cytokines and / or antigens as candidates for biomarkers. However, the high costs of these studies, the low number of participants and their different methodology make difficult a future meta-analysis and more conclusive results.

Background: The multifactorial mechanisms associated with radical reductions in HIV-1 reservoirs after allogeneic hematopoietic stem cell transplant (allo-HSCT), including a case of HIV cure, are not fully understood. Objective: To investigate the mechanism of HIV-1 eradication associated with allo-HSCT. Design: Nested case series within the IciStem observational cohort. Setting: Multicenter European study. Participants: 6 HIV-infected, antiretroviral-treated participants who survived more than 2 years after allo-HSCT with CCR5 wildtype donor cells. Measurements: HIV DNA analysis, HIV RNA analysis, and quantitative viral outgrowth assay were performed in blood, and HIV DNA was also measured in lymph nodes, ilea, bone marrow, and cerebrospinal fluid. A humanized mouse model was used for in vivo detection of the replication-competent blood cell reservoir. HIV-specific antibodies were measured in plasma. Results: Analysis of the viral reservoir showed that 5 of 6 participants had full donor chimera in T cells within the first year after transplant, undetectable proviral HIV DNA in blood and tissue, and undetectable replication-competent virus (

The cell cycle G1-restriction point is frequently deregulated in HR+ BC by alterations of cyclin D1 (CCND1), p16 (CDKN2A) or pRb (RB1). CDK4/6i (ribociclib, abemaciclib and palbociclib) have shown clinical activity in metastatic HR+ BC, both as single agents and in combination with endocrine therapy. Currently, no biomarkers of response to CDK4/6i have been identified beyond HR expression and little is known about mechanisms of acquired resistance. Twenty-one PDXs were established from HR+, HER2+ or HR+/HER2+ BC patient biopsies and their response to ribociclib was evaluated in vivo and ex vivo in matrigel cultures. Acquired-resistance was generated in vivo by isolating tumors that escaped therapy overtime. In order to identify response biomarkers, genetic and proteomic analysis of PDXs were performed and correlated with ribociclib antitumor activity. Candidates were validated in a cohort of 8 tumor samples from patients treated with abemaciclib monotherapy and in vitro. Combination with the PI3K-alpha inhibitor (PI3Ki) BYL719 was explored in vivo. In vivo, ribociclib exhibited antitumor activity in five out of 21 PDXs (24%), two of which acquired resistance after continuous dosage (75mg/kg, 6IW). Ex vivo matrigel cultures recapitulated the in vivo response with 75% sensitivity and 92% specificity (p=0.01), providing a novel approach for high throughput screening. Baseline levels of ER, PR and Ki67 protein or PIK3CA/ESR1 mutations did not discriminate between ribociclib-resistant/sensitive PDXs, whereas CCND1/D2-amplification/overexpression were only found in ribociclib-resistant models. Importantly, sensitive PDXs exhibited significant Ki67 reduction upon ribociclib treatment, higher baseline pRb- and lower p16-staining compared to ribociclib-resistant PDXs (p=0.004, 0.02 and 0.03, respectively). Three out of 8 acquired-resistant tumors (37.5%) exhibited pRb loss. In vitro, RB1 knockdown and cyclin D1/D2-overexpression resulted in higher BrdU incorporation and higher IC50 than control cells upon ribociclib treatment. p16 expression was significantly lower in samples of patients exhibiting clinical benefit with abemaciclib monotherapy (p=0.04). Remarkably, combination of ribociclib with a PI3Ki resulted in appreciable antitumor activity in 18 out of 20 PDXs (90%), including two models resistant to fulvestrant given in combination with ribociclib. In conclusion, HR+, HER2+ and HR+/HER2+ BC PDXs expressing both high Rb- and low p16-protein levels are sensitive to CDK4/6i whereas deregulation of the G1-restriction point due to low pRb or high cyclin D1/D2 protein levels is associated with resistance to ribociclib monotherapy. Addition of a PI3Ki markedly improves the antitumor response of ribociclib in most of PDXs, suggesting that the PI3K pathway may play a pivotal role in limiting the efficacy of CDK4/6 inhibition. Citation Format: Marta Palafox, María Teresa Herrera-Abreu, Meritxell Bellet, Mafalda Oliveira, Alejandra Bruna, Olga Rodriguez, Marta Guzmán, Judit Grueso, Cristina Vilaplana, Joaquín Arribas, Emmanuelle di Tomaso, Faye Su, Carlos Caldas, Nicholas C. Turner, Rodrigo Dienstmann, José Baselga, Maurizio Scaltriti, Javier Cortés, Cristina Saura, Violeta Serra. Biomarkers of response to CDK4/6 inhibitor (CDK4/6i) in hormone receptor (HR) positive and HER2-positive breast cancer (BC) patient-derived xenografts (PDX) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3596.

The use of tuberculin for the therapy of tuberculosis was attempted more than 100 years ago and abandoned because of its adverse reactions. In this historical review we point out that some of the intensive efforts to avoid the reactions were based on the best scientific rationale available at that time. Balancing the dosage and intervals of tuberculin delivery with clinical and laboratory monitoring of patients achieved a limited success, with implications, toward current research in the field. The role of economical and social aspects at that time is also a lesson to be learned toward current approaches to tuberculosis control.

The evolution of dependency in Spain will have a significant effect over long-term care demand. With the purpose of getting knowledge of the dimension of the dependent population, we apply the EDAD2008. Next, using the Short Term Demographic Projections drew up by INE, we develop different scenarios of dependency projections. We ascertain a growth of the institutionalization rate, a reduction of moderate dependency and an increase of high dependency, as well, as an acute process of dependency feminisation for the cohort older than 75 years. On the other hand, dependency projections point in favour of the morbidity compression model and exhibit a lesser dependency growth in comparison with previous estimations.

A Phase I interventional Clinical Trial was performed with a potential tuberculosis vaccine, based on detoxified cellular fragments of M. tuberculosis, named RUTI. The objective was to evaluate the safety profile and T-cell immune responses over a 6-month period following subcutaneous inoculation. The double-blind, randomized and placebo-controlled trial was conducted in healthy volunteers, all recruited at one site. RUTI, at each of the four tested doses, starting from 5microg and going up to 200microg, and placebo were inoculated to groups of 4 and 2 volunteers respectively, consecutively. RUTI appeared to be well tolerated as judged by local and systemic clinical evaluation, though vaccine dose dependent local adverse reactions were recorded. T-cell responses of blood lymphocytes to PPD and a number of antigen subunits were elevated, when compared with controls subjects. These results support the feasibility of future evaluation, to be targeted at subjects with latent tuberculosis infection (LTBI).

The identification of a herd of goats with tuberculosis let us test a new treatment regimen against latent tuberculosis infection (LTBI). Using large animal experimental models allows a better approach to understanding human tuberculosis according to immunopathological parameters. Based on an initial study showing a correlation between the ESAT-6-specific interferon (IFN)-gamma secretion and the severity of pulmonary lesions, this parameter was used in combination with an X-ray examination to screen the animals to be included in the efficacy and safety studies. All the animals proved to be infected with Mycobacterium caprae. The efficacy study was run in animals distributed in three experimental groups according to treatment: untreated (CT), treated with isoniazid (INH), and treated with INH + RUTI (a vaccine based on M. tuberculosis cell fragments) inoculated twice. RUTI temporarily increased the IFN-gamma production after stimulating the peripheral blood with ESAT-6, purified protein derivative and RUTI in vitro. The INH chemotherapy reduced both pulmonary and extra pulmonary affectation, but not disease in pulmonary lymph nodes. The addition of RUTI may have decreased extrapulmonary disease further but had no benefit to lung or lung lymph-nodes itself. Safety studies showed that inoculation of RUTI caused a temporary increase of rectal temperature (1-2 degrees C) and local swelling, both adverse effects being well tolerated. Neither systemic toxicity nor mortality was induced by the vaccination. The control of goats' infection by the therapeutic regimen consisting in INH chemotherapy + RUTI as well as its safety, represented a further step towards testing its effects in human LTBI in a future.

The chronic phase of Mycobacterium tuberculosis infection in mouse experimental models is characterized by the accumulation of foamy macrophages (FM)--which shape the outer ring of the granuloma - in the alveolar spaces, as detected in paraffin-embedded tissues stained with hematoxylin-eosin. In this study, the use of semi- and ultra-thin sections offers more detailed information about the origin of FM both in mouse and guinea-pig experimental models. Lipid bodies (LB) are present in macrophages from the beginning of infection and accumulate in the chronic phase. LB progress from an early (ELB) to a late (LLB) stage, defined according to their progressive capacity to generate cholesterol crystals, resembling atherosclerotic lesions. FM arise from massive accumulation of LLB. Electronic microscopy reveals intracellular lipophilic inclusions (ILIs) in those M. tuberculosis bacilli inside FM. It is our hypothesis that the accumulation of lipids in M. tuberculosis concomitant to the establishment of the non-replicating state prepares the bacilli for future reactivation and for facing future stressful environments.

RUTI is a therapeutic vaccine that is generated from detoxified and liposomedMycobacterium tuberculosiscell fragments that has demonstrated its efficacy in the control of bacillus reactivation after short-term chemotherapy. The aim of this study was to characterize the cellular immune response generated after the therapeutic administration of RUTI and to corroborate the lack of toxicity of the vaccine. Mouse and guinea pig experimental models were infected with a low-doseM. tuberculosisaerosol. RUTI-treated animals showed the lowest bacillary load in both experimental models. RUTI also decreased the percentage of pulmonary granulomatous infiltration in the mouse and guinea pig models. This was not the case afterMycobacterium bovisBCG treatment. Cellular immunity was studied through the characterization of the intracellular gamma interferon (IFN-γ)-producing cells after the splenocytes' stimulation withM. tuberculosis-specific structural and growth-related antigens. Our data show that the difference between the therapeutic administration of BCG and RUTI resides mainly in the stronger activation of IFN-γ+CD4+cells and CD8+cells against tuberculin purified protein derivative, ESAT-6, and Ag85B that RUTI generates. Both vaccines also triggered a specific immune response against theM. tuberculosisstructural antigens Ag16kDa and Ag38kDa and a marked mRNA expression of IFN-γ, tumor necrosis factor, interleukin-12, inducible nitric oxide synthase, and RANTES in the lung. The results show that RUTI's therapeutic effect is linked not only to the induction of a Th1 response but also to the stimulation of a quicker and stronger specific immunity against structural and growth-related antigens that reduces both the bacillary load and the pulmonary pathology.

RUTI is a vaccine consisting of Mycobacterium tuberculosis bacilli grown in stress conditions that is fragmented, detoxified and liposomed. RUTI was designed to shorten the treatment of latent tuberculosis infection (LTBI) with isoniazid from 9 months to just 1 month, by additional treatment with two inoculations of RUTI 4 weeks apart. During the validation process for monitoring the immunogenicity of administration of RUTI in a Phase I clinical trial, the question arose whether to introduce the tuberculin skin test (TST) in the screening of non-LTBI volunteers. This study was designed to evaluate the effect of TST on subsequent different T-cell interferon-gamma release assay (TIGRA) responses, using a spectrum of M. tuberculosis-related antigens (ESAT-6, CFP-10, 16 kDa, 19 kDa, MPT64, Ag 85B, 38 kDa, hsp65, PPD and BCG). The results showed an increase in post-TST response even in non-LTBI subjects for most antigens tested, as measured both by whole blood assay (WBA) and ELISPOT. Increased ELISPOT response decreased toward pre-TST levels within 1 month whereas the WBA response did not. Taking into account that there is no definitive correlation between TST and TIGRA tests to diagnose LTBI and the feasibility that TST might alter the immune monitoring included in clinical trials, these data suggest that TST determination should be carefully planned to avoid any interference with TIGRA.

Despite advances in medium formulations and pretreatment techniques, recovery of Legionella from water samples can still be quite low, difficult and time consuming. The aim of this study was to evaluate the utility of a Legionella urinary antigen enzyme immunoassay (Bartels ELISA, Trinity Biotech, Ireland) for the detection of Legionella in water samples. Reference ATCC Legionella strains were used to spike water samples to a final concentration of 10 4 –10 5 cfu/ml. The lower detection limit of the test for all Legionella pneumophila serogroups was assessed by serial dilutions of spiked water samples. Legionella antigen was detected in all filtered samples except for those spiked with L. bozemanii and L. longbeachae . The lower detection limit for soluble L. pneumophila serogroup 1 antigen was 780 cfu/ml. Bartels ELISA could be a useful method for antigen detection in water samples when a high recovery of L. pneumophila is suspected. The test could be used as a rapid screening method for the detection of Legionella in a large number of samples. However, the low sensitivity of the test requires to keep on performing conventional culture for isolation and for further studies on isolated bacteria.

The aim of this paper is to apply a rigorous econometric approach to analyze the benefits derived from participation in Third Age University Programs (3AUP). Two waves from the SHARE (Survey of Health, Ageing and Retirement in Europe) corresponding to 2007 and 2011 are used. This survey contains information concerning the degree of satisfaction with life and allows to define indicators for cognitive achievement in memory, vocabulary and fluency. A sample of individuals aged between 50 and 85 years old is selected. The percentage of participants in 3AUP has increased from 3.25 % to 5.47 %, which represents an average growth rate of 13.90 %. For the sample of all respondents we appreciate that: (i) the probability of retaining 10-14 words in the memory test increases by 0.34 points for participants in 3AUP, (ii) the probability of developing a list with at least twenty words increases by 0.45 points for participants in 3AUP, and it attains a maximum of 0.88 point for participants with high school education. El objetivo de este trabajo es aplicar un enfoque econométrico riguroso para analizar los beneficios derivados de la participación en programas universitarios para mayores (PUM) en España. Se utilizan dos olas de la encuesta SHARE (Survey of Health, Ageing and Retirement in Europe) para 2007 y 2011. Esta encuesta contiene información sobre el nivel de satisfacción general y permite construir indicadores para pruebas cognitivas de memoria. Se selecciona una muestra de personas de entre 50 y 85 años. El porcentaje de participantes ha aumentado del 3,25 % al 5,47 %. Por tanto, en el intervalo de cuatro años la tasa de participación ha crecido a una tasa media anual acumulada del 13,90 %. Para el conjunto de todos los individuos se constata que: i) la probabilidad de recordar entre 10 y 14 palabras en la prueba de memoria aumenta en 0,34 puntos si participa en PUM, y ii) la probabilidad de que la lista confeccionada en la prueba de fluidez contenga 20 o más nombres aumenta en 0,45 puntos, si participa en un PUM, con un máximo de 0,88 puntos de diferencia si tiene estudios secundarios.

Altres ajuts: Miguel Servet CP13/00174 The evolution of a tuberculosis (TB) infection toward active disease is driven by a combination of factors mostly related to the host response. The equilibrium between control of the bacillary load and the pathology generated is crucial as regards preventing the growth and proliferation of TB lesions. In addition, some experimental evidence suggests an important role of both local endogenous reinfection and the coalescence of neighboring lesions. Herein we propose a mathematical model that captures the essence of these factors by defining three hypotheses: (i) lesions grow logistically due to the inflammatory reaction; (ii) new lesions can appear as a result of extracellular bacilli or infected macrophages that escape from older lesions; and (iii) lesions can merge when they are close enough. This model was implemented in Matlab to simulate the dynamics of several lesions in a 3D space. It was also fitted to available microscopy data from infected C3HeB/FeJ mice, an animal model of active TB that reacts against Mycobacterium tuberculosis with an exaggerated inflammatory response. The results of the simulations show the dynamics observed experimentally, namely an initial increase in the number of lesions followed by fluctuations, and an exponential increase in the mean area of the lesions. In addition, further analysis of experimental and simulation results show a strong coincidence of the area distributions of lesions at day 21, thereby highlighting the consistency of the model. Three simulation series removing each one of the hypothesis corroborate their essential role in the dynamics observed. These results demonstrate that three local factors, namely an exaggerated inflammatory response, an endogenous reinfection, and a coalescence of lesions, are needed in order to progress toward active TB. The failure of one of these factors stops induction of the disease. This mathematical model may be used as a basis for developing strategies to stop the progression of infection toward disease in human lungs

This paper analyzes the reasons for the scarce development of the private long-term care insurance market in Spain, and its relationship with health insurance. We are also interested in the effects the crisis has had both on the evolution of the demand for long-term care insurance and on the existence of regional disparities. We estimate bivariate probit models with endogenous variables using Spanish data from the Survey on Health and Retirement in Europe. Our results confirm that individuals wishing to purchase long-term care insurance are, in a sense, forced to subscribe a health insurance policy. In spite of this restriction in the supply of long-term care insurance contracts, we find its demand has grown in recent years, which we attribute to the budget cuts affecting the implementation of Spain's System of Autonomy and Attention to Dependent People. Regional differences in its implementation, as well as the varying effects the crisis has had across Spanish regions, lead to the existence of a crowding-in effect in the demand for long-term care insurance in those regions where co-payment is based on income and wealth, those that have a lower percentage of public long-term care beneficiaries, or those with a smaller share of cash benefits over total public benefits. Copyright © 2016 John WileySons, Ltd.

In this paper we evaluate the effect of the Program School 2.0 on both Financial Education and Mathematics performance using data from PISA 2012. The Program Shool 2.0 was implemented in 2009 in some Spanish Autonomoous Communities. This program promoted the use of computers, both in school and at home, among elementary and high school students. We detect that a greater benefit is obtained when the contents of Financial Educaton are taught in conjunction with the contents of the subject of Mathematics, although the mean effect of Financial Education over Mathematics is more intense in the Communities that have not participated in the Program School 2.0. This result may be related to the fact that only a moderate use of computers for personal use increases Mathematics and Financial Education performance. Nevertheles, given the recent implementation of the Program School 2.0, we should expect some "learning effects" that should be confirmed with future data. DOI: http://dx.doi.org/10.4995/HEAd15.2015.238

Real-time RT-PCR was used to quantify the expression of genes possibly involved in Mycobacterium tuberculosis latency in in vitro and murine models. Exponential and stationary phase (EP and SP) bacilli were exposed to decreasing pH levels (from 6.5 to 4.5) in an unstirred culture, and mRNA levels for 16S rRNA, sigma factors sigA,B,E,F,G,H and M, Rv0834c, icl, nirA, narG, fpbB, acr, rpoA, recA and cysH were quantified. The expression of acr was the one that best correlated with the CFU decrease observed in SP bacilli. In the murine model, the expressions of icl, acr and sigF tended to decrease when bacillary counts increased and vice versa. Values from immunodepressed mice (e.g. alpha/beta T cells, TNF, IFN-gamma and iNOs knock out strains), with accelerated bacillary growth rate, confirmed this fact. Finally, the expression of acr was maintained in mice following long-term treatment with antibiotics. The quantification of acr expression could be useful for monitoring the presence of latent bacilli in some murine models of tuberculosis.

Intragranulomatous necrosis is a primary feature in the natural history of human tuberculosis (TB). Unfortunately, this phenomenon is not usually seen in the experimental TB murine model. Artificial induction of this necrosis in pulmonary granulomas (INPG) may be achieved through aerosol inoculation of lipopolysaccharide (LPS) 3 weeks after Mycobacterium tuberculosis infection. At week 9 post-infection, the centre of primary granulomas became larger, showing eosinophilic necrosis. Interestingly, INPG induction was related to mice strains C57BL/6 and 129/Sv, but not to BALB/c and DBA/2. Furthermore, the same pattern was obtained with the induction of infection using a clinical M. tuberculosis strain (UTE 0335R) that naturally induces INPG. In all the mice strains tested, the study of pulmonary mRNA expression revealed a tendency to increase or to maintain the expression of RANTES, interferon-gamma, tumour necrosis factor and iNOS, in both LPS- and UTE 0335R-induced INPG, thus suggesting that this response must be necessary but not sufficient for inducing INPG. Our work supports that INPG induction is a local phenomenon unrelated to the resistant (C57BL/6 and BALB/c) or susceptible (129/Sv and DBA/2) background of mice strains against M. tuberculosis infection.

Low dose aerosol infection of C57BL/6 mice with a clinical strain of Mycobacterium tuberculosis (UTE 0335 R) induced intragranulomatous necrosis in pulmonary granulomas (INPG) at week 9 postinfection. Infection of different knockout (KO) mouse strains with UTE 0335 R induced INPG in all strains and established two histopathological patterns. The first pattern was seen in SCID mice and in mice with deleted alpha/beta T receptor, TNF R1, IL-12, IFN-gamma, or iNOS genes, and showed a massive INPG with a high granulomatous infiltration of the lung, a large and homogeneous eosinophilic necrosis full of acid-fast bacilli, with marked karyorrhexis, coarse basophilic necrosis, and surrounded by patches delimited by partially conserved alveolar septum full of PMNs. The second pattern was seen in mice with deleted IL-1 R1, IL-6, IL-10, CD4, CD8 or gamma/delta T cell receptor genes, and showed more discrete lesions with predominant homogeneous eosinophilic necrosis with few bacilli and surrounded by a well-defined lymphocyte-based ring. Local expression of IFN-gamma, iNOS, TNF and RANTES showed no significant differences between these mouse strains generating a discrete INPG. Mouse strains showing a massive INPG showed higher, lower or equal expression values compared to the control strain. In conclusion, the severity of the INPG pattern correlated with pulmonary CFU counts, irrespective of the genetic absence or the infection-induced levels of cytokine mediators.

In this work we study older workers’ (50—64) labor force transitions after a health/disability shock. We find that the probability of keeping working decreases with both age and severity of the shock. Moreover, we find strong interactions between age and severity in the 50—64 age range and none in the 30–49 age range. Regarding demographics we find that being female and married reduce the probability of keeping work. On the contrary, being main breadwinner, education and skill levels increase it. Interestingly, the effect of some demographics changes its sign when we look at transitions from inactivity to work. This is the case of being married or having a working spouse. Undoubtedly, leisure complementarities should play a role in the latter case. Since the data we use contains a very detailed information on disabilities, we are able to evaluate the marginal effect of each type of disability either in the probability of keeping working or in returning back to work. Some of these results may have strong policy implications.

Background Tuberculosis (TB) is a disease of poverty that contributes significantly to ill-health in developing countries. Drug resistant TB is a major challenge to disease control. Early diagnosis and rapid determination of drug sensitivity is of paramount importance in eradication of TB. Although automated liquid culture based methods are available for rapid detection of drug resistance, the high cost of these tests prevent them from being used routinely in low resource settings. This study compares two phenotypic methods, the manual Mycobacteria Growth Indicator Tube (MGIT) and the Nitrate Reductase Assay (NRA) in liquid medium, with the agar proportion method (APM), the gold standard for susceptibility testing of Mycobacterium tuberculosis. Methodology Fourteen day old M. tuberculosis strains (n=373) grown on solid media were used for drug susceptibility testing by APM, NRA and the manual MGIT method. Rifampicin free and rifampicin incorporated (final concentration, 1 μg/ml) media were inoculated with the recommended concentrations of mycobacterial suspensions and incubated at 37°C in 5% CO2. In the APM, the proportion of colonies in the drug containing medium was determined. In the NRA, the colour change in the medium was compared with a standard colour series after day 6 and day 12 of incubation. Growth in the MGIT was detected using the manual MGIT reader from day 2 onwards. The 2 methods were compared with the gold standard, APM to determine sensitivity and specificity and agreement between the methods was calculated using kappa statistics. Results Thirty one (31) rifampicin resistant isolates were identified. When compared with the APM, the sensitivity of detection of rifampicin resistance was 85% for the NRA and 93% for the manual MGIT and the specificity was 99% and 100% respectively. Both assays, NRA (κ=0.86) and manual MGIT method (κ= 0.94) were in excellent agreement with the APM. The mean turnaround time for manual MGIT method and NRA were 08 days and 10 days respectively. Conclusion The NRA in liquid medium and manual MGIT are useful alternatives to APM for drug susceptibility testing of M. tuberculosis in low resource settings.

Having demonstrated previously that deletion of zinc metalloprotease zmp1 in Mycobacterium bovis BCG increased immunogenicity of BCG vaccines, we here investigated the protective efficacy of BCG zmp1 deletion mutants in a guinea pig model of tuberculosis infection. zmp1 deletion mutants of BCG provided enhanced protection by reducing the bacterial load of tubercle bacilli in the lungs of infected guinea pigs. The increased efficacy of BCG due to zmp1 deletion was demonstrated in both BCG Pasteur and BCG Denmark indicating that the improved protection by zmp1 deletion is independent from the BCG sub-strain. In addition, unmarked BCG Δzmp1 mutant strains showed a better safety profile in a CB-17 SCID mouse survival model than the parental BCG strains. Together, these results support the further development of BCG Δzmp1 for use in clinical trials.

The treatment of tuberculosis is based on combinations of drugs that directly target Mycobacterium tuberculosis. A new global initiative is now focusing on a complementary approach of developing adjunct host-directed therapies.\ud \ud Despite the availability of effective antibiotics for tuberculosis (TB) for the past half century, it remains an important global health problem; there are ~9 million active TB cases and ~1.5 million TB-induced deaths per year (see the World Health Organization (WHO) Global Tuberculosis Report in Further information). Health services around the world face major barriers to achieving optimal outcomes from current TB treatment regimens. These barriers include: the spread of multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB); complex and toxic treatment regimens for MDR-TB; HIV co-infection; pharmacokinetic interactions between TB drugs and antiretroviral drugs; relapse; permanent damage to lung and other tissues; long-term functional disability; immune reconstitution inflammatory syndrome (IRIS); and co-morbidity with non-communicable diseases such as diabetes and chronic obstructive airway diseases. Another fundamental problem is the long duration of TB drug treatment (6 months for drug-sensitive TB and at least 18 months for drug-resistant TB) to achieve a cure, owing to the presence of dormant Mycobacterium tuberculosis bacilli that are phenotypically resistant to current classes of anti-TB drugs, which can only target bacterial replication.\ud \ud There is therefore an urgent need for new TB treatments. However, the TB drug pipeline is thin1, 2. For the past 60 years, efforts to develop new treatments have focused on compounds and regimens that target M. tuberculosis directly. Recently, however, attention has focused on investigating a range of adjunct treatment interventions known as host-directed therapies (HDTs) that instead target the host response to infection. Here, we highlight the rationale for HDTs, the current portfolio of HDTs and their mechanisms of action, and a consortium-based approach to drive forward their evaluation in clinical trials.

Hydroxytyrosol is one of the main phenolic components of olive oil. It is present in the fruit and leaf of the olive (Olea europaea L.). During the past decades, it has been well documented that this phenolic compound has health benefits and a protective action has been found in preclinical studies against several diseases. Here, we review its bioavailability in human beings and several assays showing significant results related with cardiovascular diseases, cancer, and acquired immunodeficiency syndrome (AIDS). Mechanisms of action include potent anti-oxidant and anti-inflammatory effects, among others. The importance of hydroxytyrosol in protection of low-density lipoproteins and consequently its implication in the reduction of cardiovascular disease risk has been highlighted by the European Food Safety Authority, concluding that 5 mg of hydroxytyrosol and its derivatives should be consumed daily to reach this effect at physiological level. We discuss the potential uses of this compound in supplements, nutraceutic foods, or topical formulations in the disease risk reduction. Finally, we conclude that more studies are needed to sustain or reject many other health claims not yet fully documented and to validate these newly available hydroxytyrosol-based products, because it seems to be a good candidate to reduce the risk of diseases mentioned., This study was supported by the projects AGL2011-23690 (CICYT), and CSIC 201170E041 (Spanish Ministry of Economy and Competitiveness). Libia A. García-Flores was granted a pre-doctoral FPI, fellowship program from the Spanish government.

This work sets out to analyze the motivations adult children may have to provide informal care, considering the monetary transfers they receive from their parents. Traditional motivations, such as altruism and exchange, are matched against more recent social bond theories. Our findings indicate that informal caregivers receive less frequent and less generous transfers than non-caregivers; that is, caregivers are more prone to suppress their self-interested motivations in order to prioritize the well being of another person. Additionally, long-term public care benefits increase both the probability of receiving a transfer and its amount, with this effect being more intense for both the poorest and richest households. Our findings suggest that if long-term care benefits are intended to increase the recipients' welfare and represent a higher fraction of total income for the poorest households, the effectiveness of these long-term care policies may be diluted.

In a world in which the welfare state is under pressure, understanding the dynamic effects of money transfers from parents to adult children and their relationship with informal care can be relevant for policy purposes. We use the first two waves of the Survey of Health and Retirement in Europe (SHARE) to estimate a double-hurdle model for a parental decision to provide financial support for adult children and the amount involved, taking into account the potential endogeneity of informal caregiving. We find that informal caregivers receive less frequent transfers and less generous amounts than non-caregivers. This offers support for the idea of a form of sophisticated altruistic behavior, according to which caregiving costs are outweighed by the parent’s benefits. Regarding public policies, we find that while increased unemployment benefits would not generate any crowding-out effect in parental transfers, a reduction in long-term public care benefits has a negative multiplier effect on parental transfers.

Summary Tuberculosis was studied using an experimental model based on the C3HeB/FeJ mouse strain, which mimics the liquefaction of caseous necrosis occurring during active disease in immunocompetent adults. Mice were intravenously infected with 2 × 10 4 Colony Forming Units of Mycobacterium tuberculosis and their histopathology, immune response, bacillary load, and survival were evaluated. The effects of the administration of drugs with anti-inflammatory activity were examined, and the C3H/HeN mouse strain was also included for comparative purposes. Massive intra-alveolar neutrophilic infiltration led to rapid granuloma growth and coalescence of lesions into superlesions. A central necrotic area appeared showing progressive cellular destruction, the alveoli cell walls being initially conserved (caseous necrosis) but finally destroyed (liquefactive necrosis). Increasing levels of pro-inflammatory mediators were detected in lungs. C3HeB/FeJ treated with anti-inflammatory drugs and C3H/HeN animals presented lower levels of pro-inflammatory mediators such as TNF-α, IL-17, IL-6 and CXCL5, a lower bacillary load, better histopathology, and increased survival compared with untreated C3HeB/FeJ. The observation of massive neutrophilic infiltration suggests that inflammation may be a key factor in progression towards active tuberculosis. On the basis of our findings, we consider that the C3HeB/FeJ mouse model would be useful for evaluating new therapeutic strategies against human tuberculosis.

C3HeB/FeJ mice infected with Mycobacterium tuberculosis were used in an experimental animal model mimicking active tuberculosis in humans to evaluate the effect of antiinflammatory agents. No other treatment but ibuprofen was given, and it was administered when the animals' health started to deteriorate. Animals treated with ibuprofen had statistically significant decreases in the size and number of lung lesions, decreases in the bacillary load, and improvements in survival, compared with findings for untreated animals. Because antiinflammatory agents are already on the market, further clinical trials should be done to evaluate this effect in humans as soon as possible, to determine their suitability as coadjuvant tuberculosis treatment.

This paper analyses the effect of unmet formal care needs on informal caregiving hours in Spain using the two waves of the Informal Support Survey (1994, 2004). Testing for double sample selection from formal care receipt and the emergence of unmet needs provides evidence that the omission of either one of these two variables would causes underestimation of the number of informal caregiving hours. After controlling for these two factors the number of hours of care increases with both the degree of dependency and unmet needs. In the presence of unmet needs, the number of informal caregiving hours increases when some formal care is received. This result refutes the substitution model and supports complementarity or task specificity between both types of care. For the same combination of formal care and unmet needs, informal caregiving hours increased between 1994 and 2004. Finally, in the model for 2004, the selection term associated with the unmet needs equation is larger than that of the formal care equation, suggesting that using the number of formal care recipients as an indicator of the goodness of the long-term care system may be confounding, if we do not complete this information with other quality indicators.

A chemo-enzymatic strategy for the preparation of 2-aminomethyl derivatives of (2R,3R,4R)-2-(hydroxymethyl)pyrrolidine-3,4-diol (also called 1,4-dideoxy-1,4-imino-D-arabinitol, DAB) and its enantiomer LAB is presented. The synthesis is based on the enzymatic preparation of DAB and LAB followed by the chemical modification of their hydroxymethyl functionality to afford diverse 2-aminomethyl derivatives. This strategy leads to novel aromatic, aminoalcohol and 2-oxopiperazine DAB and LAB derivatives. The compounds were preliminarily explored as inhibitors of a panel of commercial glycosidases, rat intestinal disaccharidases and against Mycobacterium tuberculosis, the causative agent of tuberculosis. It was found that the inhibitory profile of the new products differed considerably from the parent DAB and LAB. Furthermore, some of them were active inhibiting the growth of M. tuberculosis.

Schizophrenia is a disease that causes strong societal rejection and requires a significant allocation of healthcare and social resources. The chronicity and characteristics of the disease require continued care, often provided by nonprofessionals close to the person diagnosed with schizophrenia.To analyze the value of informal care associated with the loss of personal autonomy (dependency) caused by schizophrenia in Spain; to study the association between the level of dependency and the number of hours of informal care provided; and to examine the association between the level of dependency/hours of informal care and the burden borne by caregivers.We used the Survey on Disabilities, Autonomy and Dependency to obtain information on the characteristics of disabled individuals with schizophrenia and the individuals who provide them with personal care. Assessment of informal care time was performed using the proxy good method. Statistical multivariate analyses using ordered probit models were conducted to study the impact of the degree of dependency on the hours of care provided and probit models to study the burden placed on caregivers in terms of health, professional, and leisure/social dimensions.The economic value of care varies between 523.06 and 866.7 euro per week censoring at 16 hours of care per day (667.22-1,105.66 when no time censored). A primary informal caregiver of a severe or greatly dependent individual with schizophrenia was between 20.5% and 23.8% more likely to provide a high level of informal care (over 70 hours per week), between 6.1% and 6.4% less likely to provide a medium level of informal care (between 31 and 70 hours per week), and between 14.4% and 17.6% less likely to provide low-level care (30 hours or less per week). Informal caregivers who provide care for severely/greatly dependent or moderately dependent people had a higher probability of suffering from health, professional, and family/socially related problems than caregivers who cared for non-dependent people.The results show a part of the high social cost of schizophrenia in Spain. At the same time, the study provides evidence that more severe levels of dependency are positively associated with more hours of informal care and higher caregiver burden.The results show that health or social care programs targeted at persons with autonomy limitations cannot overlook the importance of informal care, since doing so would lead to inefficiencies or inequities and reduce the well-being of citizens.Information on informal care should be incorporated in the design of health and social care policies geared toward improving equity and efficiency in the allocation of social resources. Longitudinal studies of patients diagnosed with schizophrenia would provide further information on the characteristics and impact of informal care.

Robert Koch was an eminence on Tuberculosis and a german hero worldwide since he discovered and described the ethiologic cause of the white plague. Soon afterwards, he devoted his work to study the effect of inoculating M.tuberculosis bacilli (Mtb) to guinea pigs, either healthy or already infected. From his experiments, as reported by himself, he learnt the infection was able to develop protection against reinfection in some extent and that using small doses of dead bacilli could help to heal the tuberculous lesions in the infected animals, while large doses could kill them. The 4th of July, 1890, during the International Medical Congress in Berlin, he announced he might have found a cure, and pointed out it should not be used in severe cases as it could do more harm than good. However, tuberculosis was one of the main causes of death at that moment, and the word of Koch’s finding its cure rapidly spread. Many physicians from all over the world traveled to Berlin to learn how to use the new remedy, called tuberculin for being obtained from Mtb bacilli, as Sir Conan Doyle did himself. Koch, probably scared of the great expectancy generated by his cure still not too well-known, decided to write a manuscript entitled “A further report on a remedy for tuberculosis“ (Koch 1890), which was published on midNovember of the same year. With this paper, he intended to clarify and to give a review of the subject, in order to avoid the public to get distorted knowledge on the remedy proposed by him. In the manuscript, he tried to explain why tuberculin worked: he believed the treatment was able to destroy the necrotic tissue of the tuberculous lesions, and this was the cause for the bacilli to die subsequently. But still more important, he cautioned about the fact of existing reactions following the inoculation of tuberculin, which severity depended on the patients and their previous illness status, and suggested it should be applied as early as possible to obtain a positive outcome (Koch 1890).