Purified Protein Derivative Antigen Research Articles

Intralesional injection of tuberculin purified protein derivative (PPD) versus measles, mumps, and rubella (MMR) vaccine in treatment of molluscum contagiosum: a comparative study

Molluscum contagiosum (MC) is a skin and mucous membrane infection caused by the molluscum virus (MCV). To evaluate safety and efficacy of intralesional injection of tuberculin purified protein derivative (PPD) antigen injection versus MMR (mumps, measles, rubella) antigen for the treatment of molluscum contagiosum (MC). A total of thirty clinically confirmed patients of molluscum were recruited for this trial. Patients who were divided into three groups (A, B and C). Each group consisted of (30) patients. Group (A) subjects received intralesional MMR injections, group (B) subjects received intralesional PPD injection and group (C) received intralesional saline injection. The results of the present study revealed complete clearance of the injected lesions in 12 patients (80%), partial response in 3 patients (20%) of group (A). In group (B), complete clearance of the treated warts was observed in 11 patients (73.3%) and partial response in 4 (26.7%) of patients. In group (C), the majority of patients 8 (53.3%) demonstrated no response while 7 (46.7%) patients showed only partial clearance. We established a good safety and efficacy profile for tuberculin PPD and MMR antigens in treatment of molluscum contagiosum.

Intralesional immunotherapy for multiple recalcitrant plantar warts: Candida antigen is superior to intralesional purified protein derivative.

Treatment of recalcitrant plantar warts represent a highly challenging issue for both patients and physicians. Candida antigen and purified protein derivative (PPD) have shown promising efficacy in the treatment of warts, however no previous studies have compared both antigens for recalcitrant plantar warts. To assess the efficacy and safety of intralesional Candida antigen versus intralesional PPD in the management of recalcitrant plantar warts. The study included 120 adult patients with multiple recalcitrant plantar warts. They were randomly assigned to one of three groups; Candida antigen, PPD, or normal saline. Injections into the largest wart were repeated every 2 weeks until clearance or for a maximum of five sessions. Complete wart clearance was reported in 33 patients (82.5%) of the Candida antigen group, in 22 patients (55.6%) of the PPD group, and in one patient (5%) of the control saline group. A statistically significant difference was found between the studied groups in favor of Candida antigen. Adverse effects were mild and insignificant in the three groups. Intralesional antigen immunotherapy by Candida antigen or PPD is a promising, safe, and cost-effective therapeutic option for multiple recalcitrant plantar warts, with statistically significant superiority of Candida antigen.

Label-free quantitative lymphocyte activation profiling using microfluidic impedance cytometry

Circulating lymphocytes are integral components of our adaptive immunity with emerging clinical applications in immune status monitoring in infectious diseases and cell-mediated cancer immunotherapies. Herein we present a novel impedance-based microfluidic assay for label-free lymphocyte activation profiling based on native or antigen-specific T-lymphocyte biophysical responses. Single cell impedance profiling of T-lymphocytes first revealed distinct biophysical differences in cell size and membrane electrical impedance of healthy, activated (CD3/CD28) and dead lymphocyte populations. Impedance characterization of peripheral blood mononuclear cells (PBMCs) stimulated with mitogen phytohemagglutinin (PHA) or Tuberculin Purified Protein Derivative antigen (PPD) after 24 h also showed an increase in lymphocyte cell size (∼8 to 10 μm) which corresponded to activated lymphocytes (CD69+CD137+). We next developed a spiral inertial microfluidics cell sorter integrated with coplanar electrodes for direct impedance quantification of activated lymphocytes. By removing non-activated smaller lymphocytes (< 8 μm) and employing hydrodynamic-based single stream particle focusing, we demonstrated significant enrichment of activated lymphocytes (∼11.7-fold) to electrically detect low levels of lymphocyte activation (< 5%). Finally, the developed biochip is coupled with magnetic activated cell sorting (MACS) to quantify CD4+ T-lymphocytes response in PBMCs stimulated with PPD. A differential impedance cell count ratio (stimulated/unstimulated) was defined to distinguish activated T-lymphocytes, which showed better sensitivity as compared to immunophenotyping by flow cytometry. Taken together, the integrated impedance biosensor can be further developed as a rapid multiplexed screening assay to detect antigen-specific T-lymphocyte responses to characterize host immunity and diagnosis of infectious diseases (e.g tuberculosis, dengue and COVID-19).

Improving the diagnosis of bovine tuberculosis using gold nanoparticles conjugated with purified protein derivative: special regard to staphylococcal protein A and streptococcal protein G.

Different ancillary immunodiagnostic tests were traditionally-established for diagnosis of bovine tuberculosis (BTB) either cellular or humoral as tuberculin skin test (TST), gamma interferon (INF-γ), and indirect enzyme-linked immunosorbent assay (iELISA). These tests had been consumed more time and expensive, and needed sophisticated equipment. To dissolve these problems, serological diagnosis depending on humoral immunity is the aim of this work. Herein, slide-based agglutination test was chosen as a rapid and simple field test based on purified protein derivative (PPD) antigen in addition to some supplementation materials such as Staphylococcal protein A (SPA) and Streptococcal protein G (SPG) to improve detection of BTB antibody in serum samples. Gold nanoparticles (Au NPs) were synthesized by gamma ray, and after complete characterization, the synthesized Au NPs were spherical, small-sized, and stable without any impurities. Addition of such supplementation reagents for serodiagnosis of tuberculosis is of paramount important for the detection of serum antibodies against tuberculosis (TB) and it was considered an easily simple and possible way for improving TB diagnosis. In this work, 70 animals tested positive for TST as well as 20 animals tested negative for TST were used for the diagnosis of BTB depending on humoral immune response based on PPD slide agglutination test using reporter regents (SPA and/or SPG) either native or recombinant. The agglutination density was recorded and read in 4 degrees of positivity with scores ranging from negative (-) to very strong reaction (++++) occurred in different times of agglutination. Groups showed 100% positive reactivates employed in Exp. 1, 2, and 3 with differentiation of slide agglutination test density and was rated from moderate positivity (2+) to very strong (4+), with predominant positivity in density of (3+). Pink-colored intensity is associated with the strengthened reactions between PPD-conjugated Au NPs and serum antibody of each tested samples, which allows for visual rapid, simple, and effective attractive diagnosis of BTB. The specificity and sensitivity of the serological tests were characterized. TST offers the highest sensitivity (83.6%) among the other immunoassays, while the lowest specificity was recorded in TST (57.4%). SPA/SPG offers the best performance in term of combined sensitivity and specificity (performance index) of 175.4. Therefore, the development and uses of detection reagent (such as SPA and/or SPG) slide co-agglutination test (COAT), either native or recombinant (rSPA/SPG) for the detection of TB antibodies based on PPD antigen, as well as the uses of Au NPs rSPA/SPG as detection conjugate based on the same antigen, were also performed as a simple, rapid, sensitive, specific, eco-friendly, and low cost, which shows a great potential in field and lab diagnosis of BTB. So, high reduction in reagents that yields reactions similarly as traditional techniques was needed.

Prevalence and risk factors of swine tuberculosis in central Ethiopia

There is paucity of information in the epidemiology and pathology of tuberculosis in swine though the endemic occurrence of bovine tuberculosis was elucidated in Ethiopia. A cross-sectional study was employed to investigate the epidemiology of Mycobacterium tuberculosis complex and Mycobacterium avium complex in swine. The study was conducted from September 2016 to December 2017 using single intradermal comparative tuberculin test, gross pathology and histopathology. Tuberculin test was carried out in farmed swine of central Ethiopia while the necropsy at Addis Ababa Abattoirs Enterprise, Ethiopia. Of the total 329 heads of swine tuberculin tested by bovine purified protein derivative antigen, an animal level prevalence of 3% (95% CI: 2-6) and a herd level prevalence of 11% (95% CI: 1-49) were observed at a cut-off value of &gt;2mm. Multivariable logistic regression analysis revealed the protective exposure effect of body condition to tuberculin test positivity in swine (Adjusted OR: 0.06; 95% CI: 0.0-1.1; P: 0.055). Swine were predominantly in close proximity with dairy cattle which is an important risk factor for the transmission of bovine tuberculosis from cattle to swine. Necropsy study of tuberculosis-like lesions in 640 heads of swine revealed an overall lesion prevalence of 4.1 % (95 CI: 2.8-6.0). In conclusion, the present study demonstrated the occurrence of swine tuberculosis in low prevalence in farmed swine and swine slaughtered atAddis Ababa Abattoirs Enterprise in central Ethiopia. Further in-depth study covering larger sample size and wider areas is warranted so as to identify the prevalent species and their zoonotic importance.&#x0D; Keywords: Abattoir; Epidemiology; Ethiopia; Pathology; Swine; Tuberculosis

Intralesional immunotherapy with purified protein derivative antigen in the treatment of multiple cutaneous warts: an open label study in an urban teaching hospital

&lt;p&gt;&lt;strong&gt;Background:&lt;/strong&gt; Viral warts caused by human papilloma virus are a group of oncogenic viruses that result in verrucous growths on the skin and mucosae. Intralesional immunotherapy has been tried in recent years with success with the use of a variety of antigens to non-specifically stimulate the cell mediated immune response. Similarly, we undertook a study to evaluate the efficacy and safety of tuberculin purified protein derivative as an immunotherapeutic modality.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Methods:&lt;/strong&gt; A total of 25 patients were included in the study. Each patient was injected with 2.5 TU of tuberculin PPD (0.04 ml) intralesionally into most of the warts at 2 weekly intervals for a maximum of six sessions. It is an interventional study with follow-up upto 6 months. Descriptive analysis was done with use of SPSS version 23 tool. Complete resolution of warts was considered as the clinical end point. Response was graded as excellent response (&amp;gt;80% resolution), good response (50-80%), fair response (20-50%) and no response (&amp;lt;20%).&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Results:&lt;/strong&gt; Out of the 25 patients, 14 patients showed excellent response (56%), 4 patients showed good response (16%), 5 patients showed fair response (20%) and 2 patients did not show any response (8%). Over 22 patients (88%) showed features of pain, erythema and inflammation at the injection site, whereas 3 patients (12%) developed itching at the site. The ‘faces’ pain rating scale was used to measure pain tolerance. An overall response rate (fair/good/ excellent) was given by 23 out of the 25 patients (92%), which proved satisfactory.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Conclusions:&lt;/strong&gt; Intralesional immunotherapy with PPD effectively cures warts which are present locally and also at distant sites. It is safe, economic and efficacious.&lt;/p&gt;

Risk factors for increased immune reconstitution in response to Mycobacterium tuberculosis antigens in tuberculosis HIV-infected, antiretroviral-na\xefve patients

BackgroundLittle is known regarding the restoration of the specific immune response after combined antiretroviral therapy (cART) and anti-tuberculosis (TB) therapy introduction among TB-HIV patients. In this study, we examined the immune response of TB-HIV patients to Mycobacterium tuberculosis (Mtb) antigens to evaluate the response dynamics to different antigens over time. Moreover, we also evaluated the influence of two different doses of efavirenz and the factors associated with immune reconstitution.MethodsThis is a longitudinal study nested in a clinical trial, where cART was initiated during the baseline visit (D0), which occurred 30 ± 10 days after the introduction of anti-TB therapy. Follow-up visits were performed at 30, 60, 90 and 180 days after cART initiation. The production of IFN-γ upon in vitro stimulation with Mtb antigens purified protein derivative (PPD), ESAT-6 and 38 kDa/CFP-10 using ELISpot was examined at baseline and follow-up visits.ResultsSixty-one patients, all ART-naïve, were selected and included in the immune reconstitution analysis; seven (11.5%) developed Immune Reconstitution Inflammatory Syndrome (IRIS). The Mtb specific immune response was higher for the PPD antigen followed by 38 kDa/CFP-10 and increased in the first 60 days after cART initiation. In multivariate analysis, the variables independently associated with increased IFN-γ production in response to PPD antigen were CD4+ T cell counts <200 cells/mm3 at baseline, age, site of tuberculosis, 800 mg efavirenz dose and follow-up CD4+ T cell counts. Moreover, the factors associated with the production of IFN-γ in response to 38 kDa/CFP-10 were detectable HIV viral load (VL) and CD4+ T cell counts at follow-up visits of ≥200 cells/mm3.ConclusionsThese findings highlight the differences in immune response according to the specificity of the Mtb antigen, which contributes to a better understanding of TB-HIV immunopathogenesis. IFN-γ production elicited by PPD and 38 kDa/CFP-10 antigens have a greater magnitude compared to ESAT-6 and are associated with different factors. The low response to ESAT-6, even during immune restoration, suggests that this antigen is not adequate to assess the immune response of immunosuppressed TB-HIV patients.

Skin test results with common and melanoma antigens compared to clinical outcomes of melanoma patients treated with intralesional (IL) dinitrochorobenzene (DNCB) or IL bacillus Calmette-Guerin (BCG).

59 Background: In a previous study involving in vitro serial testing in patients receiving IL BCG or IL DNCB we determined that measurements of serial in vitro phytohemagglutinin stimulation levels correlated with the patients’ clinical courses. The current evaluation attempted to determine which in vivo skin test studies were useful in this setting. Methods: All patients had locally progressive melanoma satellosis, or in-transit metastases, but no evidence of distant metastasis past regional lymph nodes. Patients had been randomized to repeatedly receive either IL BCG (9 patients), or IL DNCB (9 patients) into cutaneous or subcutaneous metastases. A "BCG worse outcome group" consisted of 6 patients in whom distant metastasis was detected within 2 to 20 months, and who died within 9 to 24 months. A "BCG better outcome group" consisted of 3 patients who were alive and in whom no distant metastases were detected after 12 to 39 months. Similarly, the 9 patients randomized to be treated with intralesional DNCB were also subsequently divided into two groups: A "DNCB worse outcome group" consisted of 6 patients in whom distant metastasis was detected within 5 to 11 months, and who died within 6 to 12 months. A "DNCB better outcome group" consisted of 3 patients who were alive and in whom no distant metastases were detected after 8 to 20 months. Following informed consent, patients were serially skin tested, as possible, with Purified Protein Derivative antigen (PPD), Mumps antigen, Streptokinase/Streptodornase (SKSD), and inactivated melanoma extracts, using coded syringes. Results: In comparing the two "better outcome" groups with the two "worse outcome" groups, patients who prior to treatment had a positive reaction to melanoma extracts appeared to fare better than those who did not. All patient groups tended to react positively to SKSD, a general sign of immune capability. Conclusions: Among the skin tests compared, testing with melanoma extract appeared to correlate best with clinical courses in patients undergoing BCG or DNCB intralesional treatments for progressive melanoma satellosis or in-transit metastases.

Intralesional immunotherapy with tuberculin purified protein derivative (PPD) in recalcitrant wart: A randomized, placebo-controlled, double-blind clinical trial including an extra group of candidates for cryotherapy

Background: Due to paucity of randomized clinical trials, intralesional immunotherapy has not been yet accepted as a standard therapeutic method. Objective: To examine the efficacy and safety of intralesional immunotherapy with tuberculin purified protein derivative (PPD) for treating recalcitrant wart. Methods: In this randomized, placebo-controlled, double-blind clinical trial, a total of 69 patients with recalcitrant warts received either intralesional PPD antigen (n = 35) or intralesional saline (n = 34) for six times at 2-week intervals. A third group of candidates for cryotherapy (n = 33) was also included. The decrease in lesion size (good: complete response, intermediate: 50–99% improvement, poor: <50% improvement), adverse effects and recurrence within 6-month follow-up were documented. Results: At the final session, good, intermediate and poor responses were observed in 77.1%, 22.9% and 0% of the PPD patients; 0%, 14.7% and 85.3% of the placebo patients and 18.2%, 33.3% and 48.5% of the cryotherapy patients, respectively (PPD versus placebo: p < 0.001; PPD versus cryotherapy: p < 0.001). No significant complication was seen in the PPD group. The recurrence rate was 8.6%, 5.9% and 24.2% in the PPD, placebo and cryotherapy groups, respectively (p > 0.05). Conclusion: Intralesional immunotherapy with PPD antigen is highly effective and safe for treating recalcitrant warts. Clinical trial registration: IRCT201407089844N3 in the Iranian Registry of Clinical Trials (IRCT).

Comparison of Tuberculin Skin Test result and interferon gamma response to human PPD in BCG scar positive and negative children

BackgroundThe aim of this study is to compare Tuberculin Skin Test (TST) result and interferon gamma response to human PPD (purified protein derivative), in scar positive and scar negative BCG-vaccinated children. MethodsBetween August 2007 and May 2008 a total of 236 children aged 1–168months (mean 21months) admitted to Mofid Children’s Hospital, Tehran, Iran, were enrolled in a cross-sectional study. Each patient was examined for BCG vaccine scar and tested with TST and human PPD-based Interferon Gamma Release Assay (IGRA). ResultsTwo hundred and twenty one cases out of 236 (44% female, 1–168months, mean age 21months) were scar positive of whom 95% TST result was negative. Human PPD-based IGRA was positive in 110 (49.8%), negative in 85 (38.4 %) and indeterminate in 26 (11.8%) of scar positive patients.Fifteen children (40% female, 1–156months; mean age 42months) were scar negative. All the scar negative cases were TST negative. Human PPD-based IGRA was positive in 10 (66.7%), negative in 4 (26.7%) and indeterminate in 1 (6.7%) of scar negative patients. ConclusionsImmune responsiveness to human PPD antigens in scar positive and negative children may not correspond with results of the Tuberculin Skin Test.

Evaluation of IL-12 serum level in patients with recalcitrant multiple common warts, treated by intralesional tuberculin antigen

Background: No universal consensus about optimal modality for treating the recalcitrant multiple common warts (RMCW). Objective: To evaluate the immunological mechanisms and clinical therapeutic effect of using of intralesional purified protein derivative (PPD) in the treatment of RMCW. Methods: The study included 40 patients with RMCW. They were randomly assigned to 2 groups: first group (20 patients) received intralesional PPD antigen, and second group (20 patients) received intralesional saline as a control group. In both groups, injections were made into single lesions, or largest wart in case of multiple lesions, at weekly intervals, until complete clearance or for a maximum of six treatments. Blood serum was taken at pre-study and at week 6 to measure IL-12 level. Results: A significant difference was found between the therapeutic responses of RMCW to PPD antigen and saline control group (p < 0.001). In the PPD group, complete response was achieved in 75% after 5.8 ± 0.7 sessions' patients presenting with RMCW. There was a statistically significant increase in IL-12 of PPD group versus saline group. No recurrence was observed in the PPD group. Conclusion: Intralesional immunotherapy by PPD antigen is an effective and a safe treatment for RMCW in previously immunized patients.

Tuberculosis After Commencing Antiretroviral Therapy for HIV Infection Is Associated With Elevated CXCL9 and CXCL10 Responses to Mycobacterium tuberculosis Antigens

Commencing antiretroviral therapy (ART) in human immunodeficiency virus-infected patients with treated or unrecognized Mycobacterium tuberculosis disease may trigger tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) or ART-associated tuberculosis (ART-TB). We have shown that whole-blood interferon-gamma release assays may aid in the prediction and diagnosis of ART-TB. Here, we investigate interferon-gamma-inducible chemokines CXCL9 and CXCL10. CXCL9 and CXCL10 responses to region of difference 1 (RD1) antigens and purified protein derivative (PPD) were assayed in plasma from whole-blood cultures collected before and after 4, 12, and 24 weeks of ART from 15 TB-IRIS cases, 11 ART-TB cases, and matched controls. Relative to matched controls, ART-TB cases had elevated CXCL10 responses to RD1 antigens pre-ART (P = 0.02) and to PPD and RD1 antigens over 24 weeks of ART (P ≤ 0.02 and P ≤ 0.03). In contrast, TB-IRIS cases had higher CXCL10 responses to RD1 antigens before and after 4 weeks of ART only (P = 0.04 for both). CXCL9 responses to PPD and RD1 antigens were similar but less pronounced in ART-TB cases and did not differ between TB-IRIS cases and controls. CXCL10 responses to RD1 antigens performed as well as, or better than, IFN-γ responses in the prediction and diagnosis of ART-TB. Tuberculosis after commencing ART is associated with increased CXCL10 and, to a lesser extent, CXCL9 responses to M. tuberculosis antigens. Assessment of antigen-induced CXCL10 responses to RD1 antigens may assist in the prediction and diagnosis of ART-TB.

S43 Polyfunctional T cells reveal the spectrum of tuberculosis in HIV co-infection through the identification of immunological correlates of latent and active disease

BackgroundApproximately one-third of the world's population is infected with Mycobacterium tuberculosis (MTB), mostly as latent tuberculosis infection (LTBI). HIV co-infection confers the single greatest risk of progression to active tuberculosis...

Plasma biomarkers in the prediction and diagnosis of tuberculosis-associated immune reconstitution inflammatory syndrome

Tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) is the most common form of immune restoration disease (IRD) complicating early antiretroviral therapy (ART) in patients from resource-limited countries with HIV infection [1]. Increasing attention is being paid to the definition of plasma biomarkers that might assist in the prediction and diagnosis of TB-IRIS, as well as other forms of IRD [2,3]. The findings of the study by Haddow et al.[4] are, therefore, an important contribution to the literature on IRD but might have been more informative if considered in the context of data from other studies of plasma biomarkers. We described a prospective study of the immunopathogenesis of TB-IRIS and ART-associated TB (ART-TB; equivalent to ‘unmasking’ TB-IRIS [1] reported by Haddow et al.) undertaken using plasma from whole blood interferon (IFN)-gamma release assays (IGRAs) [5,6]. The advantage of using whole blood IGRAs in this context is that plasma from antigen-stimulated and antigen-unstimulated tubes can be analyzed simultaneously. However, this advantage must be balanced against the uncertainty of knowing whether biomarkers in plasma from unstimulated tubes reflect the in-vivo state or the production or consumption of biomarkers during the 24-h culture. The demonstration by Haddow et al.[4] that plasma CCL2 (monocyte chemotactic protein-1) levels are low in patients with ‘paradoxical’ TB-IRIS is supported by our finding of low CCL2 levels in unstimulated IGRA plasma from patients with TB-IRIS [6]. In addition, we demonstrated low levels of CCL2 before ART. In contrast to the findings of Haddow et al.[4], we found similar levels of interleukin (IL)-10 in unstimulated IGRA plasma from TB-IRIS cases and controls [6]. However, low production of IL-10 by antigen-stimulated peripheral blood mononuclear cells from patients with IRD associated with infection by nontuberculous mycobacteria has been demonstrated previously [7,8]. In the study by Seddiki et al.[8], low IL-10 production was associated with increased numbers of circulating regulatory T (Treg) cells, so the authors suggested that low IL-10 production might reflect dysfunctional Treg cells. As CCL2 and IL-10 are both produced by monocytes and macrophages, an alternative explanation is that CCL2 and IL-10 deficiency reflect monocyte/macrophage dysfunction. We agree with Haddow et al.[4] that there may be differences in the immunopathogenesis of TB-IRIS and ART-TB [5,6]. They reported that pre-ART plasma IFN-gamma levels were approximately 10-fold higher in patients who subsequently developed ‘unmasking’ TB IRIS compared with controls, but that there was no difference in pre-ART IFN-gamma levels when ‘paradoxical’ TB-IRIS cases were compared with controls [4]. We reported that pre-ART IFN-gamma responses to both region of difference 1 and purified protein derivative antigens of Mycobacterium tuberculosis in whole blood IGRAs, corrected for CD4+ T cell counts, were increased in patients who developed ART-TB and had strong performance characteristics in the prediction of ART-TB by receiver operator curve (ROC) analysis [5]. We also reported that levels of CXCL10 (IP-10) in unstimulated IGRA plasmas were higher in TB-IRIS patients than ART-TB patients during 24 weeks of ART and, furthermore, that pre-ART levels of CXCL10 in combination with CCL2 and IL-18 levels predicted the occurrence of TB-IRIS by ROC analysis. In contrast, Haddow et al.[4] did not demonstrate a difference in plasma levels of CXCL10 levels between ‘paradoxical’ TB-IRIS or ‘unmasking’ TB-IRIS cases and their controls [4], perhaps suggesting that in our study, these biomarkers were produced in cell culture. Unlike previous studies of various forms of IRD [2], Haddow et al.[4] did not demonstrate increased plasma levels of IL-6 in patients with ‘paradoxical’ TB-IRIS. A possible explanation for this comprises differences in the assay method. In most previous studies of IRD, plasma IL-6 levels have been assayed using enzyme-linked immunosorbent assays (ELISAs), whereas Haddow et al.[4] used a multiplex bead array assay (MBAA). Plasma IL-6 levels in HIV patients may differ significantly when assayed by ELISA or MBAA [9]. It is now clear that assays of selected biomarkers can assist in both the prediction and diagnosis of TB-IRIS and ART-TB. Large-scale studies are now needed to delineate the value of these assays as diagnostic tests. In doing this, consideration should be given to the use of both plasma and IGRA plasma from antigen-stimulated and antigen-unstimulated tubes and also to the method used to assay cytokines and chemokines. Acknowledgements Conflicts of interest There are no conflicts of interest.

CTLA-4 is expressed by human monocyte—derived dendritic cells and regulates their functions

Cytotoxic T lymphocyte antigen–4 (CTLA-4) is the major negative regulator of T-cell responses, although growing evidence supports its wider role as an immune attenuator that may also act in other cell lineages. Here, we have analyzed the expression of CTLA-4 in human monocytes and monocyte-derived dendritic cells (DCs), and the effect of its engagement on cytokine production and T-cell stimulatory activity by mature DCs. CTLA-4 was highly expressed on freshly isolated monocytes, then down-modulated upon differentiation toward immature DCs (iDCs) and it was markedly upregulated on mature DCs obtained with different stimulations (lipopolysaccharides [LPS], Poly:IC, cytokines). In line with the functional role of CTLA-4 in T cells, treatment of mDCs with an agonistic anti–CTLA-4 mAb significantly enhanced secretion of regulatory interleukin (IL)–10 but reduced secretion of IL-8/IL-12 pro-inflammatory cytokines, as well as autologous CD4 + T-cell proliferation in response to stimulation with recall antigen purified protein derivative (PPD) loaded-DCs. Neutralization of IL-10 with an anti–IL-10 antibody during the mDCs-CD4 + T-cell co-culture partially restored the ability of anti–CTLA-4–treated mDCs to stimulate T-cell proliferation in response to PPD. Taken together, our data provide the first evidence that CTLA-4 receptor is expressed by human monocyte–derived mDCs upon their full activation and that it exerts immune modulatory effects.

Modulation of Human Peripheral Blood Mononuclear Cell Proliferative Response by Diltiazem

The gradual aging of population has increased the number of elderly patients receiving kidney transplants. In elderly transplant recipients, careful immunosuppression has to be maintained to avoid both rejection and adverse effects. Clinical protocols after kidney transplantation include use of the calcium channel antagonist diltiazem to ameliorate the hypertensive effect and nephrotoxicity of the immunosuppressant agent ciclosporin (cyclosporine). Since immune response can be impaired by senescence, we evaluated the influence of diltiazem on lymphocyte proliferation both alone and in the presence of ciclosporin in younger versus older subjects. Peripheral blood mononuclear cells (PBMC) from younger healthy donors (aged 19-24 years) and older subjects (aged 59-65 years) were isolated and stimulated with mitogens, recombinant human interleukin-2 (IL-2), purified protein derivative (PPD) antigen from Mycobacteriumtuberculosis, and anti-CD3 monoclonal antibody (alphaCD3 moAb) in the presence or absence of 10(-4), 10(-5), 10(-6), 10(-7) mol/L concentrations of diltiazem. In some experiments, lymphocytes from younger and older subjects were used as responder cells in an allogeneic mixed lymphocyte reaction (MLR) in the presence of different concentrations of diltiazem and 10 ng/mL of ciclosporin. We found that PBMC from older subjects were more susceptible to immunosuppression induced by low concentrations of diltiazem when mitogens were used to stimulate cells. In particular, when pokeweed mitogen was used, diltiazem 10(-7) mol/L was associated with statistically significant immunosuppression in older subjects compared with younger subjects. This effect was not observed when IL-2, PPD antigen and alphaCD3 moAb were used as stimulators. Moreover, in the allogeneic MLR, we found no differences between younger and older subjects when the 10(-)(5), 10(-)(6) and 10(-7) mol/L concentrations of diltiazem were used alone or in the presence of ciclosporin. Only addition of the supratherapeutic 10(-4) mol/L concentration of diltiazem to ciclosporin was associated with statistically significant immunosuppression in older versus younger subjects. Our results show that PBMC from older subjects are no more susceptible than PBMC from younger subjects to therapeutic doses of diltiazem when T-cell receptors are directly or indirectly involved. On the contrary, when PBMC activation was not mediated by T-cell receptor involvement, as in the case of pokeweed mitogen, susceptibility to a therapeutic concentration of diltiazem in older subjects was enhanced. Moreover, co-administration of therapeutic doses of diltiazem and ciclosporin in an MLR showed no significant differences between younger and older subjects in an in vitro model of lymphocyte response to allogeneic transplantation. Since we found no variations in immunosuppression between older and younger subjects when therapeutic doses of diltiazem were added to ciclosporin, our data do not discourage the use of diltiazem in older kidney transplant recipients receiving ciclosporin therapy.

Failure in antigen responses by T cells from patients with common variable immunodeficiency (CVID).

Antigen-driven responses by T cells from patients with CVID and normal subjects have been assessed. Low-density cells enriched for antigen-presenting dendritic cells were cultured with T cells using a 20-microliters hanging drop system. T cells from all subgroups of CVID patients showed markedly reduced responses to the recall antigens purified protein derivative (PPD) or tetanus toxoid, whereas responses by cells from patients with X-linked agammaglobulinaemia, used as a disease control, were in the normal range. However, primary allo-stimulation of CVID T cells was normal. CVID cells from two patients failed to respond to stimulation with a neoantigen, an HIV env peptide, under conditions where normal T cells did respond. These data illustrate a profound defect in antigen-stimulated T cell proliferation in vitro in all groups of CVID patients, but do not distinguish whether the defect is in the presenting cell or in the T lymphocyte. In vivo, germinal centre B cells are thought to present antigen to primed T cells to obtain essential signals (e.g. CD40 ligand and IL-2) for B cell survival and progression to immunoglobulin secretion. A failure of antigen-specific T cell function in vivo in CVID would thus not provide the primed T cells needed for B cell rescue, and could be the primary defect leading to the low immunoglobulin production in this condition.

Up-regulation of alveolar macrophage platelet-derived growth factor-B (PDGF-B) mRNA by interferon-gamma fromMycobacterium tuberculosisantigen (PPD)-stimulated lymphocytes

Macrophage production of PDGF-B is believed to be important in the pathogenesis of diseases where chronic lung inflammation develops into fibrosis. Since tuberculosis is characterized by chronic inflammation and tissue fibrosis, we asked if lymphokines from lymphocytes stimulated by the Mycobacterium tuberculosis antigen PPD, contained factors capable of increasing human alveolar macrophage PDGF-B mRNA. Supernatants from both phytohaemagglutinin (PHA)- and purified protein derivative (PPD)-stimulated lymphocytes, when added to macrophages, induced an increase in the mRNA of PDGF-B, but not transforming growth factor-beta (TGF-beta). When lymphocytes from contacts of patients with tuberculosis, patients with tuberculosis, and normal subjects were compared following PPD stimulation, the lymphocytes from the contacts had the greatest proliferation response, the greatest production of interferon-gamma (IFN-gamma), and their lymphokines induced the greatest increase in PDGF-B mRNA in macrophages. Recombinant human IFN-gamma reproduced this ability of lymphokines to increase macrophage PDGF-B mRNA. Finally, the increase in macrophage PDGF-B mRNA following incubation with supernatants from PPD-stimulated lymphocytes was shown to be due to IFN-gamma, when the increase in macrophage PDGF-B mRNA was prevented by addition of anti-human IFN-gamma antibody to the lymphocyte supernatant. This study indicated that antigen-stimulated lymphocytes released IFN-gamma, which in turn resulted in an increase in PDGF-B mRNA in alveolar macrophages. Such a mechanism provides a link between the DTH response and the first stages of a fibrotic reaction, and may offer an explanation for the progression of chronic inflammation to fibrosis, as occurs in the lungs of patients with untreated pulmonary tuberculosis.

THE DYNAMICS OF ANTIGEN SPECIFIC PROLIFERATIVE RESPONSES OF LYMPHOCYTES AT EARLY STAGES OF BOVINE PARATUBERCULOSIS INFECTION

The present study was aimed to quantify the dynamics of early antigen specific proliferative responses of lymphocytes to (protein) antigens associated with experimentalMycobacterium avium subsp. paratuberculosis (Mp) infection cattle. The data were collected from20 experimentally infected calves, and 10 uninfected control animals, during the first 2 years oftheir lives. Several purified protein derivative antigens (Ppdp, Ppda, and Ppdb), tworecombinant Mp heatshock proteins (Hsp65 and Hsp70) and whole bacteria (sonicated Mpstrain 316F) were used to measure lymphocyte proliferation in a lymphocyte proliferationassay. Data were analyzed using a linear mixed effect (LME) model. The results showedsignificant group and timed effects for all antigens tested. At several time points, the responsesin the infected group were found significantly higher as compared to control group. The Ppdantigens induced similar lymphocyte proliferation patterns, as compared to whole bacteriaantigen and Hsp70. These results indicated that the antigen specific proliferative responses oflymphocytes differs for different antigens, probably related to differences in their availabilityduring different stages of infection. The application of LME model is a useful tool for analyzingthe quantitative longitudinal datasets. Keywords: dynamics, Mp, antigen, LME

Evidence for Antigen-Specific Immune Deviation in Patients With Acute Retinal Necrosis

Background: Because experimental acute retinal necrosis (ARN) induced by herpes simplex virus in mice develops only if mice fail to acquire virus-specific delayed hypersensitivity (DH), although they produce antiviral antibodies (ie, anterior chamber–associated immune deviation), we sought to determine whether a similar inverse correlation exists for patients with varicellazoster virus (VZV)-induced ARN. Design: Patients with acute, VZV-induced ARN and agematched control subjects were skin tested with VZV and purified protein derivative antigens to evaluate DH. Varicella-zoster virus–induced ARN was diagnosed using polymerase chain reaction and intraocular antibody quotient. Serum samples were collected and analyzed for anti-VZV and anti–herpes simplex virus antibody titers. Acute retinal necrosis activity was assessed clinically, and DH skin tests were repeated 3 months after onset when ocular recovery had taken place. Result: Whereas controls displayed intense DH when tested with VZV and purified protein derivative antigens, a subset of patients with ARN displayed absent VZV-specific DH (although their purified protein derivative responses were normal). Patients with the most severe ARN had the lowest DH responses to VZV antigens. Serum anti-VZV antibody titers were higher in patients with ARN than in controls, and antiviral titer correlated inversely with the intensity of anti-VZV DH responses. Varicella-zoster virus–specific DH responses were restored in patients who recovered from ARN. Conclusion: Varicella-zoster virus-ARN develops in a setting where DH reactivity to viral antigens is absent, implying that virus-specific DH might ameliorate the severity of ARN. Clinical Relevance: Linking virus-specific DH to vulnerability to ARN in individuals infected with VZV might reveal an underappreciated pathogenic mechanism.