Protein Derivative Research Articles

Characterization of early immune responses elicited by live and inactivated vaccines against Johne's disease in goats.

Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is the causative agent of Johne's disease, a chronic debilitating condition affecting ruminants causing significant economic losses to the dairy industry. Available inactivated vaccines are not effective in controlling the disease and vaccinated animals can continue to infect newly born calves. Recently, we have shown that a live-attenuated vaccine candidate (pgsN) is protective in goats and calves following challenge with virulent strains of M. paratuberculosis. To decipher the dynamics of the immune responses elicited by both live-attenuated and inactivated vaccines, we analyzed key immunological parameters of goats immunized through different routes when a marker-less pgsN vaccine was used. Within a few weeks, the inactivated vaccine triggered the formation of granulomas both at the site of inoculation and in regional lymph nodes, that increased in size over time and persisted until the end of the experiment. In contrast, granulomas induced by the pgsN vaccine were small and subsided during the study. Interestingly, in this vaccine group, histology demonstrated an initial abundance of intra-histiocytic mycobacterial bacilli at the site of inoculation, with recruitment of very minimal T lymphocytes to poorly organized granulomas. Over time, granulomas became more organized, with recruitment of greater numbers of T and B lymphocytes, which coincided with a lack of mycobacteria. For the inactivated vaccine group, mycobacterial bacilli were identified extracellularly within the center of caseating granulomas, with relatively equal proportions of B- and T-lymphocytes maintained across both early and late times. Despite the differences in granuloma-specific lymphocyte recruitment, markers for cell-mediated immunity (e.g., IFN-γ release) were robust in both injected pgsN and inactivated vaccine groups. In contrast, the intranasal live-attenuated vaccine did not elicit any reaction at site of inoculation, nor cell-mediated immune responses. Finally, 80% of animals in the inactivated vaccine group significantly reacted to purified protein derivatives from M. bovis, while reactivity was detected in only 20% of animals receiving pgsN vaccine, suggesting a higher level of cross reactivity for bovine tuberculosis when inactivated vaccine is used. Overall, these results depict the cellular recruitment strategies driving immune responses elicited by both live-attenuated and inactivated vaccines that target Johne's disease.

Predictive performance of interferon-gamma release assays and the tuberculin skin test for incident tuberculosis: an individual participant data meta-analysis

Evidence on the comparative performance of purified protein derivative tuberculin skin tests (TST) and interferon-gamma release assays (IGRA) for predicting incident active tuberculosis (TB) remains conflicting. We conducted an individual participant data meta-analysis to directly compare the predictive performance for incident TB disease between TST and IGRA to inform policy. We searched Medline and Embase from 1 January 2002 to 4 September 2020, and studies that were included in previous systematic reviews. We included prospective longitudinal studies in which participants received both TST and IGRA and estimated performance as hazard ratios (HR) for the development of all diagnoses of TB in participants with dichotomised positive test results compared to negative results, using different thresholds of positivity for TST. Secondary analyses included an evaluation of the impact of background TB incidence. We also estimated the sensitivity and specificity for predicting TB. We explored heterogeneity through pre-defined sub-group analyses (e.g. country-level TB incidence). Publication bias was assessed using funnel plots and Egger's test. This review is registered with PROSPERO, CRD42020205667. We obtained data from 13 studies out of 40 that were considered eligible (N=32,034 participants: 36% from countries with TB incidence rate ≥100 per 100,000 population). All reported data on TST and QuantiFERON Gold in-Tube (QFT-GIT). The point estimate for the TST was highest with higher cut-offs for positivity and particularly when stratified by bacillus Calmette-Guérin vaccine (BCG) status (15mm if BCG vaccinated and 5mm if not [TST5/15 mm]) at 2.88 (95% CI 1.69-4.90). The pooled HR for QFT-GIT was higher than for TST at 4.15 (95% CI 1.97-8.75). The difference was large in countries with TB incidence rate <100 per 100,000 population (HR 10.38, 95% CI 4.17-25.87 for QFT-GIT VS. HR 5.36, 95% CI 3.82-7.51 for TST5/15 mm) but much of this difference was driven by a single study (HR 5.13, 95% CI 3.58-7.35 for TST5/15 mm VS. 7.18, 95% CI 4.48-11.51 for QFT-GIT, when excluding the study, in which all 19TB cases had positive QFT-GIT results). The comparative performance was similar in the higher burden countries (HR 1.61, 95% CI 1.23-2.10 for QFT-GIT VS. HR 1.72, 95% CI 0.98-3.01 for TST5/15 mm). The predictive performance of both tests was higher in countries with TB incidence rate <100 per 100,000 population. In the lower TB incidence countries, the specificity of TST (76% for TST5/15 mm) and QFT-GIT (74%) for predicting active TB approached the minimum World Health Organization target (≥75%), but the sensitivity was below the target of ≥75% (63% for TST5/15 mm and 65% for QFT-GIT). The absolute differences in positive and negative predictive values between TST15 mm and QFT-GIT were small (positive predictive values 2.74% VS. 2.46%; negative predictive values 99.42% VS. 99.52% in low-incidence countries). Egger's test did not show evidence of publication bias (0.74 for TST15 mm and p=0.68 for QFT-GIT). IGRA appears to have higher predictive performance than the TST in low TB incidence countries, but the difference was driven by a single study. Any advantage in clinical performance may be small, given the numerically similar positive and negative predictive values. Both IGRA and TST had lower performance in countries with high TB incidence. Test choice should be contextual and made considering operational and likely clinical impact of test results. YH, IA, and MXR were supported by the National Institute for Health and Care Research (NIHR), United Kingdom (RP-PG-0217-20009). MQ was supported by the Medical Research Council [MC_UU_00004/07].

Evaluation of the efficacy and safety of the intralesional purified protein derivative of tuberculin versus intralesional vitamin D3 for the management of recalcitrant warts

Introduction: Warts are caused by the human papillomavirus (HPV), which infects the epidermal cell layers leading to their hyperproliferation. The concept of immunotherapy has recently come to light. It acts by mounting delayed cell-mediated and humoral immunity against the HPV in the host. Objective: This study aimed to evaluate the efficacy and safety of the intralesional PPD versus intralesional vitamin D3 for the management of recalcitrant warts. Materials and Methods: This prospective randomized trial was conducted on patients between the ages of 12 and 65 years with two or more recalcitrant extragenital warts. The subjects were randomly divided into two groups, namely, group A (PPD) and group B (vitamin D3). In group A, a purified protein derivative (PPD) of tuberculin was employed without any prior pre-sensitization testing. Each patient in group I received 0.1–0.2 mL of antigen at the base of the largest wart. In group B, the patients received 0.1–0.2 mL in a similar manner. Results: In group A, 39 (78%) patients had complete clearance while, in group B, 38 (76%) had complete clearance in injected warts (p = 0.8). As far as distant warts were concerned, in group A, 35 (70%) had a complete response while, in group B, 29 (48%) had a complete response. There was a statistically significant difference between the two groups with a greater degree of clearance in distant warts with the tuberculin PPD (p = 0.04). Conclusion: Both the tuberculin PPD and vitamin D3 are reliable and safe management options for recalcitrant, treatment-resistant, and extensive warts.

Comparative characteristics of oxidative modification intensity of proteins in infant milk formulas

Cow's milk is mainly used in the production of infant milk formulas. However, the protein composition of cow's milk differs significantly from the proteome of breast milk. In addition, various technological factors significantly affect the properties and structure of proteins, susceptibility to oxidative processes. This article uses a method of complex evaluation of the products of oxidative modification of proteins to characterize the total level of carbonyl compounds with the analysis of the ratio of aldehyde-dinitrophenylhydrazones (ADNPH) and ketone-dinitrophenylhydrazones (CDNPH), which increases the possibility of determining the severity of protein damage. The purpose of the study was to compare the level of indicators of oxidative modification of proteins in adapted milk infant formulas. Material and methods. The research objects were 4 dry adapted milk infant formulas, as well as ultra-pasteurized cow's milk. The intensity of oxidative modification of milk proteins was determined spectrophotometrically by the reaction of carbonyl compounds with dinitrophenylhydrazine (DNFG). Results. With spontaneous oxidation in infant formula, the total area of carbonyl derivatives of proteins (Sомб) was increased compared to the indicators of ultra-pasteurized cow's milk. The greatest change occurred in relation to ADNFG indicators (SАДНФГ), the level of which increased in 3 formulas by 48.6-59.4%. The content of ketone derivatives (SКДНФГ) did not differ significantly in the studied mixtures from milk indicators. The level of carbonyl derivatives of proteins in milk infant formulas was even more elevated during the induction of oxidation by the addition of iron ions and hydrogen peroxide to the incubation medium. The content of both total and various fractions of carbonyl compounds exceeded the corresponding indicators of cow's milk by 2.0-2.6 times. Conclusion. The results of the study indicate a greater susceptibility to oxidative damage of proteins in milk infant formulas, compared with cow's milk.

Contact Investigations With a Single Tuberculin Skin Test on Infants Exposed to Tuberculosis in a Postpartum Care Center During the Neonatal Period.

Tuberculosis (TB) exposure in congregate settings related to neonates is a serious medical and social issue. TB exposure happens during the neonatal period, but contact investigations for exposed infants are usually conducted after the neonatal period. Generally, recommendations for screening and managing close contact are different for neonates and children. Thus, there are challenges in contact investigations. We aimed to report contact investigations with a single tuberculin skin test (TST) on infants exposed to infectious TB in a postpartum care center. The index case was a healthcare worker with active pulmonary TB: sputum acid-fast bacilli smear negative, culture positive, and no cavitary lesion. All exposed infants underwent medical examinations and chest X-ray. After TB disease was ruled out, contacts received window period prophylaxis with isoniazid (INH) until three months after the last exposure. TST was performed only once after completing the prophylaxis. A total of 288 infants were selected as high-priority contacts. At the initial contact investigation, the age of infants ranged from 8 to 114 days. None of these exposed infants had TB disease. The prevalence of latent TB infection (LTBI) was 25.3% (73/288; 95% confidence interval [CI], 20.7-30.7). There were no serious adverse events related to the window period prophylaxis or LTBI treatment with INH. During the 1-year follow-up period, no infants progressed to overt TB disease. The size of TST induration in infants vaccinated with percutaneous Bacillus Calmette-Guérin (BCG) vaccine was significantly larger than that of infants vaccinated with intradermal BCG vaccine (median, 8 mm vs. 5 mm; P = 0.002). In multiple logistic regression analysis, independent factors associated with TST positivity (≥ 10 mm induration) were male (adjusted odds ratio [aOR], 2.98; 95% CI, 1.6-5.64), percutaneous BCG vaccination (aOR, 3.30; 95% CI, 1.75-6.48), TST reading between 60 and 72 hours after injecting purified protein derivative (aOR, 2.87; 95% CI, 1.53-5.49), and INH prophylaxis more than four weeks (aOR, 0.49; 95% CI, 0.25-0.94). A single TST at three months after the last TB exposure with INH prophylaxis could be used as a main protocol in contact investigations for infants exposed to infectious TB during the neonatal period in congregate settings in Korea.

Effect of Demineralization on Gelatin Production from Grasshopper (Valanga nigricornis) as an Alternative of Halal Gelatin

Gelatin is a protein derivative product resulting from the collagen breakdown in a material. The protein content in dried grasshoppers, which are halal insects, reaches 40%. Hence, they have the potential to be produced as a substitute for non-halal gelatin. This study aims to determine the effect of various acid and base solvents and soaking time in the demineralization process on the characteristics of the gelatin. This study used a completely randomized design. The treatment of solvent types in the demineralization process used 6% HCl, 1% citric acid, 3% acetic acid, pineapple extract, and NaOH; while soaking times for treatment were 24 hours, 36 hours, and 48 hours. Each soaking time and solvent type in the demineralization process gave a significant effect on the yield value (P &lt; 0.05). Demineralization treatment using 6% HCl for 48 hours showed gelatin with the best yield than other treatments, i.e., gelatin with a moisture content of 9.93%, ash content of 1.11%, and pH 5. The characteristics of gelatin in this study are under SNI 06-3735 of 1995.

A Study of Comparison and Evaluation of Various Intralesional Therapies in Cutaneous Warts.

The study compares the efficacy of four immunotherapeutic agents, measles mumps and rubella (MMR), purified protein derivative (PPD), Candida extract, and vitamin D3, in the treatment of multiple cutaneous warts. To observe the clinical responses and safety of different intralesional immunotherapeutic agents and compare their efficacy. Hundred patients with multiple (>5) cutaneous warts were enrolled in the study and randomized into four groups: Group A: MMR, Group B: PPD, Group C: Candida extract, and Group D: Vitamin D. Target wart was selected, and the intralesional injections were given at three weekly intervals for a maximum of three doses. Response was observed in target and distant warts three months after the last injection. Intralesional vitamin D3 had the highest efficacy, while MMR had the lowest efficacy in clearance of target wart. Intralesional Candida extract had the highest efficacy, while vitamin D3 had the lowest efficacy in clearance of distant warts. Intralesional Candida extract was the most effective treatment for both local and distant warts. Side effects were minimal and transitory in nature. Intralesional immunotherapy is a safe, affordable, and efficacious treatment for warts.

Application of the mineral-binding protein fetuin-A for the detection of calcified lesions.

Rationale: Calcium plays an essential role in the biology of vertebrates. Calcium content in body fluids is maintained within a narrow physiologic range by feedback control. Phosphate is equally important for metabolism and is likewise controlled, albeit over a wider range. This results in a nearly supersaturated state of calcium phosphate in body liquids driving mineral precipitation in soft tissues, which is actively prevented by calcification inhibitors. The hepatic plasma protein fetuin-A is a circulating mineralization inhibitor regulating calcium phosphate crystal growth and calcified matrix metabolism. Ectopic mineralization is associated with many pathological conditions aggravating their outcome. Current diagnostic methods lack sensitivity towards microcalcifications representing the initial stages of the process. Given the irreversibility of established calcifications, novel diagnostic tools capable of detecting nascent calcium phosphate deposits are highly desirable. Methods: We designed fluorescent fusion proteins consisting of fetuin-A coupled to a green or red fluorescent protein derivate, mEmerald or mRuby3, respectively. The proteins were expressed in mammalian cell lines. Sequence optimization resolved folding issues and increased sensitivity of mineral binding. Chimeric proteins were tested for their ability to detect calcifications in cell cultures and tissue sections retrieved from calcification-prone mice. Results: We employed novel genetically labeled fetuin-A-based fluorescent proteins for the detection of ectopic calcifications. We show that fetuin-A-based imaging agents are non-toxic and suitable for live imaging of microcalcifications beyond the detection limit of conventional staining techniques. The ability of fetuin-A to preferentially bind nascent calcium phosphate crystals allowed the resolution of histopathological detail of early kidney damage that went previously undetected. Endogenous expression of fetuin-A fluorescent fusion proteins allowed extended live imaging of calcifying cells with unprecedented sensitivity and specificity. Conclusion: Ectopic microcalcifications represent a major clinical concern lacking effective diagnostic and treatment options. In this paper, we describe novel highly sensitive fetuin-A-based fluorescent probes for imaging microcalcifications. We show that fusion proteins consisting of a fetuin-A mineral binding moiety and a fluorescent protein are superior to the routine methods for detecting calcifications. They also surpass in continuous live cell imaging the chemically fluorescence labeled fetuin-A, which we established previously.

Purified Protein Derivative Response in Type 1 Diabetics

Aim: The aims of our study were to identify the role of suppressor T1 helper cells in the immunopathogenesis of type 1 diabetes mellitus (T1D) and to investigate the purified protein derivative (PPD) response, which is a delayed type of skin sensitivity response that develops secondarily to the effect of the T1 helper cell response. Method: Twenty-four newly diagnosed patients with T1D were included in the study (12 girls, 12 boys), and 30 (18/12) age/sex matched healthy children were included as controls. All patients underwent the PPD skin test (TST) in the first diagnostic 24–48 h (study group-1) and again 1 month after diagnosis (study group-2). The PPD response was compared with the response of healthy children. The lower limit of PPD positivity was accepted as ≥ 10 mm. Results: The mean PPD induration value taken in the study group-2 was statistically greater than both the study group-1 and the control group PPD (p=0.014, p=0.001). Regarding the increase in duration, the PPD positivity rate was statistically higher after the first month than in the control group (p= 0.005) and the initial PPD positivity rate (p=0.023). Conclusion: Interesting results emerged for the PPD response of the study group-2, because the PPD positivity rate increased over time.

Treatment of warts with intralesional immunotherapy: factors predicting clinical response and role of mannose-binding lectin-gene polymorphism

Background Warts are skin disease caused by human papilloma virus infection and characterized by high rate of recurrence and resistance. The use of purified protein derivatives (PPD) for treatment of warts achieved good results, however, some patients do not respond to it. Deficiency of mannose-binding lectin (MBL) may be a risk for repeated viral infection.Objective To investigate the factors that may affect and predict the clinical response of warts to the treatment with PPD.Patients and methods This study was conducted on 50 patients with warts and 50 apparently healthy-control volunteers. Blood samples from all participants were investigated for the polymorphism in MBL2-gene exon-1 codon 54 by PCR before treatment. All patients were injected intralesionally in the biggest wart every 2 weeks for five sessions.Results About 56% of the patients achieved complete response, 8% attained partial response, and 36% did not show any response. We did not detect recurrence or progression during the follow-up period. There was significantly higher clinical response in patients with less than five warts and in those with warts of less than 1-year duration. There was significantly lower clinical response in patients with warts over the dorsum of hand, while higher clinical response was observed in the warts over the sole. Although higher number (48%) of patients compared with controls showed polymorphism of MBL2 gene, it has no relation of the effect of PPD injection.Conclusion Intralesional immunotherapy with PPD is effective and safe for treatment of warts. Duration, number, and site of warts might have an impact on the clinical response to PPD immunotherapy, while MBL2-gene exon-1 codon-54 polymorphism has no effect.

Mass Casualty Incident Involving Rapid Acting Insulin

We report on an unusual prehospital incident involving the inadvertent administration of short-acting insulin among a group of high school students. Sixteen students iatrogenically received 10 units of insulin lispro intradermally instead of tuberculin purified protein derivative (PPD), resulting in several students experiencing symptomatic hypoglycemia. A mass casualty incident was declared and the local poison center consulted. An incident command system, with the support of on-scene EMS physicians, was established to track, treat, and transport the involved patients.

Development of an enzyme-linked immunosorbent assay for the diagnosis of feline tuberculosis

Cases of feline tuberculosis (TB) can be challenging to diagnose. Currently, this is achieved through a combination of mycobacterial culture, polymerase chain reaction (PCR), or interferon-gamma release assay (IGRA); however, these each have limitations. There is limited data regarding the use of humoral immunodiagnostics for TB in cats. Therefore, we sought to develop an enzyme-linked immunosorbent assay (ELISA) to further facilitate the diagnosis of feline TB. A comparative PPD (purified protein derivative) antibody ELISA was optimised for use on serum and plasma, and was tested against samples from 14 cats with culture-confirmed TB and 24 uninfected controls. Selection of an appropriate positive cut-off value based on receiver-operator characteristic curve analysis gave test sensitivity of 64.3 % and specificity of 100 %. When tested on further samples from cats with strongly suspected mycobacteriosis, 32.9 % (23/70) were antibody positive. Notably, positive results were recorded in cats that failed to respond to the IGRA, and in one PCR and IGRA negative cat. No positive responses were identified in cats with non-tuberculous mycobacterial infections, or with non-mycobacterial diseases (n = 12). Therefore, antibody-based diagnostics may be useful adjunctive tests for cases of TB missed by the IGRA, helping protect both feline and, in turn, human health.

Analyzing the postulated inhibitory effect of Manumycin A on farnesyltransferase.

Manumycin A is postulated to be a specific inhibitor against the farnesyltransferase (FTase) since this effect has been shown in 1993 for yeast FTase. Since then, plenty of studies investigated Manumycin A in human cells as well as in model organisms like Caenorhabditis elegans. Some studies pointed to additional targets and pathways involved in Manumycin A effects like apoptosis. Therefore, these studies created doubt whether the main mechanism of action of Manumycin A is FTase inhibition. For some of these alternative targets half maximal inhibitory concentrations (IC50) of Manumycin A are available, but not for human and C. elegans FTase. So, we aimed to 1) characterize missing C. elegans FTase kinetics, 2) elucidate the IC50 and Ki values of Manumycin A on purified human and C. elegans FTase 3) investigate Manumycin A dependent expression of FTase and apoptosis genes in C. elegans. C. elegans FTase has its temperature optimum at 40°C with KM of 1.3µM (farnesylpyrophosphate) and 1.7µM (protein derivate). Whilst other targets are inhibitable by Manumycin A at the nanomolar level, we found that Manumycin A inhibits cell-free FTase in micromolar concentrations (Ki human 4.15μM; Ki C. elegans 3.16μM). Furthermore, our gene expression results correlate with other studies indicating that thioredoxin reductase 1 is the main target of Manumycin A. According to our results, the ability of Manumycin A to inhibit the FTase at the micromolar level is rather neglectable for its cellular effects, so we postulate that the classification as a specific FTase inhibitor is no longer valid.

Site-Directed Spin Labeling EPR Spectroscopy for Determination of Albumin Structural Damage and Hypoalbuminemia in Critical COVID-19.

The main factors in the COVID-19 pathology, which can initiate extensive structural changes at the cellular and molecular levels, are the generation of free radicals in abnormal amounts, and oxidative stress. Under "oxidative shock" conditions, the proteins undergo various modifications that affect their function and activity, and as a result distribute malfunctioning protein derivatives in the body. Human serum albumin is a small globular protein characterized by a high overall binding capacity for neutral lipophilic and acidic dosage forms. The albumin concentration is crucial for the maintenance of plasma oncotic pressure, the transport of nutrients, amino acids, and drugs, the effectiveness of drug therapy, and the prevention of drug toxicity. Hypoalbuminemia and structural defects molecule in the protein suggest a risk of changed metabolism and increased plasma concentration of unbound drugs. Therefore, the albumin structural and functional changes accompanied by low protein levels can be a serious prerequisite for ineffective therapy, frequent complications, and high mortality in patients with SARS-CoV-2 infection. The current opinion aims the research community the application of Site-Directed Spin Labeling Electron Paramagnetic Resonance spectroscopy (SDSL-EPR) and 3-Maleimido-PROXYL radical in determining abnormalities of the albumin dynamics and protein concentrations in COVID-19 critical patients.

Fibrosing mediastinitis in a child with Mendelian susceptibility to mycobacterial disease possibly due to Bacillus Calmette-Guérin

BackgroundMendelian susceptibility to mycobacterial disease (MSMD) is an uncommon disorder with increased susceptibility to less virulent mycobacteria including bacillus Calmette-Guérin (BCG). Fibrosing mediastinitis (FM) is also a rare condition defined by excessive fibrotic reactions in the mediastinum. So far, some infectious organisms and autoimmune diseases have been introduced as possible etiologies of FM. However, no study has ever discussed the possible association of BCG infection and FM.Case presentationIn this study, we report a 3-year-old female presenting with persistent fever, weakness, and bloody diarrhea in addition to mediastinal lymphadenopathy, hepatosplenomegaly, and pleural and pericardial effusion. Further examinations established a diagnosis of MSMD based on her clinical condition, immunologic data, positive tests for mycobacterial species, positive family history, and genetic study (IL12RB1 gene, c.G1193C, p.W398S). A year and a half later, she was referred with submandibular lymphadenitis and underwent immunologic work-up which revealed high inflammatory indices, a slight reduction in numbers of CD3 + and CD4 + cells as well as elevated CD16/56 + cell count and hyperimmunoglobulinemia. Purified protein derivative (PPD), QuantiFERON, and gastric washing test were all negative. Her chest computed tomography (CT) scan revealed suspicious para-aortic soft tissue and her echocardiography was indicative of strictures in superior vena cava and pulmonary veins. She further underwent chest CT angiography which confirmed FM development. Meanwhile, she has been treated with anti-mycobacterial agents and subcutaneous IFN-γ.ConclusionIn summary, we described a novel case of MSMD in a child presenting with granulomatous FM possibly following BCG infection. This is the first report introducing aberrant BCG infection as the underlying cause of FM. This result could assist physicians in identifying early-onset FM in suspicious cases with MSMD. However, more studies are required to support this matter.

Effects of Paratuberculosis Vaccination at Different Ages in a Dairy Goat Herd: A 2-Year Follow-Up.

Vaccination could be considered as an effective method for paratuberculosis control, although controversial, with a need for investigation in some aspects. The objective of this study was to evaluate the effect of vaccination, depending on the age of the animals, on their immune response, the reduction of paratuberculosis cases, mortality and culled animals in a commercial dairy herd. Goats from three different ages were immunized with the inactivated Gudair® vaccine. Peripheral antibody and IFN-γ output were evaluated for 21 months post-vaccination (mpv) and intradermal skin tests (IDSTs) for tuberculosis, with avian- and bovine-purified protein derivatives (PPD), were carried out at 6 and at 18 mpv to evaluate the humoral and cellular immune peripheral responses, respectively. The number of dead or culled animals, regardless of the reason, was also monitored and the causes of death determined by pathological examination. A significant increase in the production of IFN-γ was observed in all the vaccinated groups when the blood samples were stimulated with avian PPD, from 3 mpv to 18 mpv, and with bovine PPD, between 3 and 21 mpv. Moreover, serum antibody levels increased between 3 and 21 mpv in all vaccinated groups. The highest levels were found in animals vaccinated at 5 months, and the lowest in adult individuals. No positive reactants to tuberculosis were found by intradermal skin test. No animal losses associated with clinical paratuberculosis were detected in any of the groups. The number of total culled animals was significantly lower in the vaccinated than in the unvaccinated groups, especially on 1.5-month-old vaccinated kids. These results suggest that vaccination of paratuberculosis, especially in young animals, could induce heterologous protection.

Dynamic analysis of tuberculosis specific mononuclear cells in peripheral blood of HIV patients with tuberculosis

Objective: To evaluate the response of peripheral blood mononuclear cells (PBMCs) in patients with human immunodeficiency virus (HIV) combined with active tuberculosis (TB) to TB-specific antigen stimulation. Methods: From January to December, 2018, individuals infected with both HIV and TB (HIV/TB group) were taken as the study subjects. Individuals infected with HIV alone (HIV group), individuals infected with TB alone (TB group) and healthy people (Health control group, HC group) were taken as the control groups. PBMCs were isolated and stimulated with purified protein derivative of bacillus calmette-guerin (BCG-PPD). The expression of surface molecules in T cells (CD3+, CD4+, CD8+) and monocytes (CD14+) and the percentages of Interferon (IFN)-γ and tumor necrosis factor (TNF)-α were detected by cell surface molecular staining, direct intracellular cytokine staining and flow cytometry (CD3- lymphocytes were mainly B lymphocytes and NK cells). Analysis of non-parametric data was used to compare the data between the two groups, and paired t-test was used to compare the data before and after PPD stimulation in each group. Results: Before PPD stimulation, the percentage of IFN-γ+ CD8+ cells in the peripheral blood of HIV/TB group(mean 0.52%) was significantly lower than that in TB group(mean 0.94%, P=0.010). The TNF-α+cell percentages in CD3+, CD4+, CD8+, or CD14+ cells in the HIV/TB group(mean 19.2%) were significantly lower than those in the HIV group(mean 31.9%, P=0.002). The percentage of TNF-α secreted by monocytes in the HIV group was significantly lower than that in the HC group. The percentages of IFN-γ+ CD8+ and IFN-γ+ CD3- cells in the peripheral blood of the TB group (mean 0.94%) were significantly higher than thoset in the HC group(mean 0.51%, P=0.020), while the percentages of TNF-α+ cells in each subsets of PBMCs were significantly lower than those in the HC group. After PPD stimulation, the percentage of IFN-γ+ CD8+ cells in the HIV/TB group was significantly lower than that in the TB group(P=0.008), and the change was more marked than that before stimulation. The percentage of IFN-γ+ CD8+ cells in the HIV group(mean 0.20%) was lower than that in the HC group (mean 0.52%, P=0.044). The percentage of IFN-γ+ CD3- in the TB group was significantly higher than in the HC group. There were no significant differences in TNF-α+ cell percentages in the 3 groups compared with the control group after PPD stimulation. The percentages of IFN-γ+ CD4+ cells in the HC and the TB groups were significantly increased after PPD stimulation in each group (P=0.002, P=0.001, respectively). However, there were no significant differences of IFN-γ+ CD4+ cell percentages in the HIV/TB group and the HIV group. The percentages of TNF-α production by monocytes were significantly increased after PPD stimulation in all groups. Conclusions: Chronic Mycobacterium tuberculosis (MTB) infection reduced the ability of PBMCs to produce TNF-α. For patients with TB infection, the production of TNF-α was reduced when combined with HIV infection. The capacity of CD8+ and CD3- lymphocytes to produce IFN-γ was increased in TB patients, while the capacity of CD8+ T cells to produce IFN-γ was decreased with co-infection of HIV. Infection of HIV weakened the immune response to MTB infection, which made the clinical diagnosis and treatment of TB more difficult.

Effect of a recent parenteral dexamethasone and ketoprofen administration on the immunological diagnosis of tuberculosis in goats.

Caprine tuberculosis (TB) is a zoonosis caused by members of the Mycobacterium tuberculosis complex (MTBC). Caprine TB eradication programmes are based mainly on intradermal tuberculin tests and slaughterhouse surveillance. Different factors may affect the performance of the TB diagnostic tests used in caprine herds and, therefore, their ability to detect infected animals. The present study evaluates the effect of the fraudulent administration of two anti-inflammatory substances, dexamethasone and ketoprofen, on the performance of the TB diagnostic techniques used in goats, as well as the suitability of high performance liquid chromatography (HPLC) for their detection in hair samples. The animals (n = 90) were distributed in three groups: (1) a group treated with dexamethasone (n = 30); a second group treated with ketoprofen (n = 30); and a third non-treated control group (n = 30). Both dexamethasone and ketoprofen groups were subjected to intramuscular inoculation with the substances 48 h after the administration of bovine and avian purified protein derivatives (PPDs), that is, 24 h before the tests were interpreted. All the animals were subjected to the single and comparative intradermal tuberculin (SIT and CIT, respectively) tests, interferon-gamma release assay (IGRA) and P22 ELISA. The number of SIT test reactors was significantly lower in the dexamethasone (p = 0.001) and ketoprofen (p < 0.001) groups 72 h after the bovine PPD inoculation compared with the control group. A significantly higher number of positive reactors to IGRA was detected within the dexamethasone group (p = 0.016) 72 h after PPD administration compared to the control group. Dexamethasone and ketoprofen detection in either hair or serum samples was challenging when using HPLC since these substances were not detected in animals whose skin fold thickness (SFT) was reduced, what could be an issue if they are used for fraudulent purposes. In conclusion, the parenteral administration of dexamethasone or ketoprofen 48 h after the PPDs administration can significantly reduce the increase in SFT (mm) and subsequently the number of positive reactors to SIT test.

Subsequent AS01-adjuvanted vaccinations induce similar transcriptional responses in populations with different disease statuses.

Transcriptional responses to adjuvanted vaccines can vary substantially among populations. Interindividual diversity in levels of pathogen exposure, and thus of cell-mediated immunological memory at baseline, may be an important determinant of population differences in vaccine responses. Adjuvant System AS01 is used in licensed or candidate vaccines for several diseases and populations, yet the impact of pre-existing immunity on its adjuvanticity remains to be elucidated. In this exploratory post-hoc analysis of clinical trial samples (clinicalTrials.gov: NCT01424501), we compared gene expression patterns elicited by two immunizations with the candidate tuberculosis (TB) vaccine M72/AS01, between three groups of individuals with different levels of memory responses to TB antigens before vaccination. Analyzed were one group of TB-disease-treated individuals, and two groups of TB-disease-naïve individuals who were (based on purified protein derivative [PPD] skin-test results) stratified into PPD-positive and PPD-negative groups. Although TB-disease-treated individuals displayed slightly stronger transcriptional responses after each vaccine dose, functional gene signatures were overall not distinctly different between groups. Considering the similarities with the signatures found previously for other AS01-adjuvanted vaccines, many features of the response appeared to be adjuvant-driven. Across groups, cell proliferation-related signals at 7 days post-dose 1 were associated with increased anti-M72 antibody response magnitudes. These early signals were stronger in the TB-disease-treated group as compared to both TB-disease-naïve groups. Interindividual homogeneity in gene expression levels was also higher for TB-disease-treated individuals post-dose 1, but increased in all groups post-dose 2 to attain similar levels between the three groups. Altogether, strong cell-mediated memory responses at baseline accelerated and amplified transcriptional responses to a single dose of this AS01-adjuvanted vaccine, resulting in more homogenous gene expression levels among the highly-primed individuals as compared to the disease-naïve individuals. However, after a second vaccination, response heterogeneity decreased and was similar across groups, irrespective of the degree of immune memory acquired at baseline. This information can support the design and analysis of future clinical trials evaluating AS01-adjuvanted vaccines.

Mixed pulmonary infection with four isolates of nontuberculous mycobacteria: a case report of mycobacterium bacteremicum infection

BackgroundA mixed pulmonary infection of Mycobacterium bacteremicum and three different isolates of nontuberculous mycobacteria (NTM) is an unusual clinical manifestation and have not yet been indicated. In this case report, we reported four isolates of NTM using phenotypic and genotypic test of pulmonary sample in Tehran, Iran.Case presentationWe report a case of severe pulmonary disease in a 19-year-old male patient with productive cough, shortness of breath, and low-grade fever for several weeks. The C-reactive protein (CRP) level (80.2 mg/L) and erythrocyte sedimentation rate (ESR) (95 mm/h) were high. The computed tomographic scan indicated bronchiectasis, nodular opacities, consolidation, and cavitary lesions on both sides. The result of purified protein derivative (PPD) test was equal to 15 mm. The sequences of hsp65, rpoB, and 16S rDNA genes indicated more than 99% homology to four isolates of nontuberculous mycobacteria (NTM), including Mycobacterium fortuitum, M. chelonae, M. mucogenicum, and M. bacteremicum. We found that all four strains were susceptible to amikacin, cefoxitin, ciprofloxacin, clarithromycin, imipenem, and linezolid. The patient was treated with ciprofloxacin, clarithromycin, and amikacin, along with Montelukast, for five months.ConclusionWe report a case of severe pulmonary infection by four isolates of NTM. After treatment, the patient reported complete resolution of the signs and a weight gain of 5 kg; also, the CRP and ESR were normal. Nine months after the infection diagnosis, a new CT scan revealed further improvements.