Specific Cell-mediated Responses Research Articles

Identification of asymptomatic Leishmania infection in patients undergoing kidney transplant using multiple tests

ObjectivesIn immunocompromised patients, asymptomatic Leishmania infection can reactivate, and evolve to severe disease. To date, no test is considered the gold standard for the identification of asymptomatic Leishmania infection. A combination of methods was employed to screen for Leishmania infection in patients undergoing kidney transplant (KT). MethodsWe employed polymerase chain reaction for the detection of parasitic DNA in peripheral blood, Western blot to identify serum immunoglobulin G and whole blood assay to detect cytokines/chemokines after stimulation of whole blood with parasitic antigen. ResultsOne-hundred twenty patients residing in Italy were included in the study at the time of KT. Each patient that tested positive to at least one test was considered as Leishmania positive. Fifty out of 120 patients (42%) tested positive for one or more tests. The detection of specific cell-mediated response (32/111, 29%) was the most common marker of Leishmania infection, followed by a positive serology (24/120, 20%). Four patients (3%) harbored parasitic DNA in the blood. ConclusionOur findings underline the high prevalence of asymptomatic Leishmania infection in patients undergoing KT in Italy, who are potentially at-risk for parasite reactivation and can benefit from an increased vigilance. Understanding the clinical relevance of these findings deserves further studies.

Test combination to detect latent Leishmania infection: A prevalence study in a newly endemic area for L. infantum, northeastern Italy.

BackgroundMost people infected with Leishmania remain asymptomatic, which is a common element that may promote the resurgence of clinically evident leishmaniasis in individuals with impaired cell-mediated immune responses. Unfortunately, there is no universally accepted assay to identify asymptomatic infection. This cross-sectional study focuses on the employment of three methods targeting different features of the parasitic infection to be used in combination for the screening of latent leishmaniasis in a newly endemic area of northeastern Italy.Methodology/principal findingsThe selected methods included highly sensitive Real-Time PCR for detection of parasitic kinetoplast (k)DNA in peripheral blood, Western Blot (WB) for detection of specific IgG, and Whole Blood stimulation Assay (WBA) to evaluate the anti-leishmanial T-cell response by quantifying the production of IL-2 after stimulation of patients’ blood with Leishmania specific antigens. Among 145 individuals living in a municipality of the Bologna province, northeastern Italy, recruited and screened for Leishmania infection, 23 subjects tested positive (15.9%) to one or more tests. Positive serology was the most common marker of latent leishmaniasis (15/145, 10%), followed by the detection of specific cell-mediated response (12/145, 8%), while only few individuals (6/145, 4%) harbored parasitic DNA in the blood.Conclusions/significanceCombining different tests substantially increased the yield of positivity in detecting latent Leishmania infection. The test combination that we employed in this study appears to be effective to accurately identify latent leishmaniasis in an endemic area.

Abstract 14442: Tuberculosis-Specific Cell-Mediated Immune Responses Are Associated With Coronary Artery Atherosclerosis Among Persons With and Without HIV in Uganda

Introduction: Latent tuberculosis infection (LTBI) has been associated with increased odds of acute myocardial infarction. Here we assessed the relationship between subclinical coronary atherosclerosis, LTBI status, and Mycobacterium tuberculosis ( Mtb )-specific cell-mediated responses. Hypothesis: LTBI and the magnitude of Mtb -specific cell-mediated responses are associated with increased rates of subclinical coronary artery disease (CAD). Methods: Cross-sectional study within a cohort of HIV-infected and -uninfected participants enrolled at the Joint Clinical Research Centre in Kampala, Uganda. Participants were ≥45 years with at least one risk factor for cardiovascular disease. Persons living with HIV (PLWH) were on stable antiretroviral therapy with HIV viral load ≤1,000 copies/mL within the 6 months prior to study entry. Participants underwent QuantiFERON®-TB Gold (QFT) testing to define LTBI status. Interferon-γ (IFN-γ) cell-mediated responses to Mtb antigens contained in the QFT TB tube were extracted. Participants underwent coronary computed tomography angiography (CCTA) utilizing a 128-slice Siemen’s scanner. Obstructive CAD was defined as having at least one coronary segment with plaque causing ≥ 50% luminal stenosis (CAD-RADS ≥3). Results: Of 138 participants, 66 (48%) had LTBI (17 PLWH and 49 HIV-uninfected), and 72 (52%) were non-LTBI (38 PLWH and 34 HIV-uninfected). Median age was 57 years; 91 (66%) were female; 120 (87%) had hypertension, 45 (33%) had diabetes mellitus, 24 (17%) had dyslipidemia, and 7 (5%) used tobacco. Median CD4 count was 569 cells/uL in PLWH. Prevalence of obstructive CAD was 9% in LTBI vs. 3% in non-LTBI; p =0.152. Mtb -specific responses were higher in persons with obstructive CAD compared to those without it (IFN-γ IU/mL median [IQR]; 5 [0.4 - 9.6] vs. 0.3 [0.1 - 2.9]; p =0.04). Mtb -specific responses remained associated with obstructive CAD after adjusting for age, sex, HIV status, cholesterol, hypertension, tobacco use, and diabetes mellitus (adjusted OR, 1.33; 95% CI; 1.04 - 1.7). Conclusions: Higher Mtb -specific cell-mediated responses were associated with obstructive CAD. Our findings suggest that Mtb contributes to coronary plaque burden independently of HIV and cardiovascular risk factors.

Dynamics and Differences in Systemic and Local Immune Responses After Vaccination With Inactivated and Live Commercial Vaccines and Subsequent Subclinical Infection With PRRS Virus.

The goals of our study were to compare the immune response to different killed and modified live vaccines against PRRS virus and to monitor the antibody production and the cell mediated immunity both at the systemic and local level. In the experiment, we immunized four groups of piglets with two commercial inactivated (A1—Progressis, A2—Suivac) and two modified live vaccines (B3—Amervac, B4—Porcilis). Twenty-one days after the final vaccination, all piglets, including the control non-immunized group (C5), were i.n., infected with the Lelystad strain of PRRS virus. The serum antibody response (IgM and IgG) was the strongest in group A1 followed by two MLV (B3 and B4) groups. Locally, we demonstrated the highest level of IgG antibodies in bronchoalveolar lavages (BALF), and saliva in group A1, whereas low IgA antibody responses in BALF and feces were detected in all groups. We have found virus neutralization antibody at DPV 21 (days post vaccination) and higher levels in all groups including the control at DPI 21 (days post infection). Positive antigen specific cell-mediated response in lymphocyte transformation test (LTT) was observed in groups B3 and B4 at DPV 7 and in group B4 at DPV 21 and in all intervals after infection. The IFN-γ producing lymphocytes after antigen stimulation were found in CD4−CD8+ and CD4+CD8+ subsets of all immunized groups 7 days after infection. After infection, there were obvious differences in virus excretion. The virus was detected in all groups of piglets in serum, saliva, and occasionally in feces at DPI 3. Significantly lower virus load was found in groups A1 and B3 at DPI 21. Negative samples appeared at DPI 21 in B3 group in saliva. It can be concluded that antibodies after immunization and infection, and the virus after infection can be detected in all the compartments monitored. Immunization with inactivated vaccine A1—Progressis induces high levels of antibodies produced both systemically and locally. Immunization with MLV-vaccines (Amervac and Porcilis) produces sufficient antibody levels and also cell-mediated immunity. After infection virus secretion gradually decreases in group B3, indicating tendency to induce sterile immunity.

Early immune responses and profiling of cell-mediated immunity-associated gene expression in response to rHVT-IBD vaccination

Infectious bursal disease (IBD) remains a major threat to the poultry industry. Recombinant herpesvirus of turkey (rHVT)-IBD vaccines have been successfully used to induce a protective immune response against IBD. However, the capacity for rHVT-IBD vaccines to induce early protection without detectable antibodies, and the underlying mechanisms mediating specific cell-mediated responses in the early stages following vaccination, have been poorly investigated. Therefore, in this study, specific pathogen-free (SPF) chickens were vaccinated with rHVT-IBD and T-cell subsets were analyzed. Both splenic and circulating CD8+ cell populations increased at 7 days postvaccination (dpv). Next, the expression of adaptive immunity-related genes was analyzed in the spleen and lung of rHVT-IBD-vaccinated chickens. Upregulation of CD8 expression was observed at 7 dpv. Interestingly, a parallel increase in the transcription of granzymes A and K was also detected from 7 dpv. To our knowledge, the latter result is the first to be reported, and it suggests that cytotoxic activity of CD8+ T lymphocytes is activated. In contrast, expression of the innate genes examined remained largely unchanged following vaccination. To further investigate the IBD virus (IBDV)-specific responses triggered by rHVT-IBD vaccination, vaccinated chickens were inoculated with an attenuated IBDV strain with the aim of restimulating induced immune responses in vivo. The expression profiles of various genes associated with adaptive immune responses were subsequently analyzed in lung, spleen, and bursa of Fabricius samples. Significant upregulation of CD4, CD8, perforin, and IFNγ expression were observed in the bursa samples 7 days postinoculation (dpi). In the lung, transcript levels of CD8, granzymes and perforin were also significantly higher in the rHVT-IBD-vaccinated chickens at 7 dpi, thereby suggesting that specific cellular immune responses were activated. Overall, these results support the hypothesis that stimulation of specific CD8+ cell-mediated immunity contributes to the response against IBDV in rHVT-IBD-vaccinated chickens.

Biodegradable nanoparticle delivery of inactivated swine influenza virus vaccine provides heterologous cell-mediated immune response in pigs

Abstract Swine influenza virus (SwIV) causes considerable economic loss to pig industry, and some SwIV are zoonotic. This study was conducted to evaluate the cross-protective efficacy of PLGA (poly lactic-co-glycolic acid) nanoparticles (NPs) encapsulated SwIV vaccine in pigs. Killed SwIV H1N2 (δ lineage) antigens (KAg) were encapsulated in PLGA NPs of 200–300 nm (PLGA-KAg NPs), and influenza antibody-free pigs were prime-boost vaccinated intranasally as mist and challenged using a heterologous, virulent and zoonotic SwIV H1N1 (γ lineage). PLGA-KAg NPs induced maturation of pig macrophages and dendritic cells in vitro. In vaccinated pigs, PLGA-KAg NPs induced antigen specific lymphocyte proliferation and enhanced the frequency of T-helper/memory cells and cytotoxic T cells in peripheral blood mononuclear cells (PBMCs). In virus challenged pigs, the PLGA-KAg NPs vaccine rescued virus induced clinical fever, reduced the gross lung pathology, reduced the virus load in the lung sections with complete clearance of the virus from the lungs of most of the pigs; but the nasal virus shedding was not reduced. Immunologically, at post-challenge day 6 in a recall response in PBMCs of PLGA KAg NPs vaccinated pigs, a significant increase in IFN-γ secreting T cells against both vaccine and challenge viruses were detected. However, humoral immune response in those pigs was not augmented. In conclusion, intranasal delivery of PLGA NPs based SwIV induced cross-protective response through specific cell-mediated response. Future studies are aimed at boosting the mucosal antibody response.

Cell phenotypic change due to Cryptosporidium parvum infection in immunocompetent mice

Cryptosporidium parvum is an intracellular parasite causing enteritis which can become life-threatening in immunocompromised host. Immunoregulatory T cells play a central role in the regulatory network of the host. Here, we proposed to characterize the populations of immune cells during infection and reinfection with C. parvum. Four-week-old BALB/C mice were inoculated with oocysts of C. parvum at days 0 and 22. Fecal and blood samples, spleens, and small intestines were collected for analysis. Peripheral blood and spleen cell populations were characterized by flow cytometry. After infection (days 0 to 21), mice presented higher values of neutrophils, eosinophils, NK cells and CD4(+)CD25(high) T cells in peripheral blood. After reinfection, this upward trend continued in the following days for all four populations in infected mice. At day 35, infected mice presented similar values to the control group, except for CD4(+)CD25(high) T cells, which remained higher in infected mice. A possible correlation between alterations in blood and spleen cell populations was also studied, but no consistent association could be established. Small intestine sections were screened for intracellular stages of the parasite but no evidence of pathology was observed. Here, we report information which may be important for the understanding of the specific cell-mediated response in immunocompetent mice to C. parvum infection. Although some questions remain unanswered and complementary studies are needed, our results are expected to contribute to a better understanding of innate and Treg cells role in the clearance process of this parasite.

Early induction of humoral and cellular immune responses during experimental Mycobacterium avium subsp. paratuberculosis infection of calves.

Johne's disease (paratuberculosis) of cattle is widespread and causes significant economic losses for producers due to decreased production and poor health of affected animals. The chronic nature of the disease and the lack of a reproducible model of infection hinder research efforts. In the present study, instillation of Mycobacterium avium subsp. paratuberculosis into the tonsillar crypts of neonatal calves resulted in peripheral colonization as detected by antemortem culture of feces and postmortem (320 days postchallenge) culture of intestinal tissues. Antigen-specific blastogenic, gamma interferon (IFN-gamma), and nitric oxide responses by blood mononuclear cells from infected calves exceeded prechallenge responses beginning 194 days postchallenge. Upon in vitro stimulation with paratuberculosis antigens, CD4(+) cells from infected calves proliferated, produced IFN-gamma, and increased expression of CD26 and CD45RO (indicative of an activated memory phenotype). Utilizing a lipoarabinomannan-based enzyme-linked immunosorbent assay, specific serum immunoglobulin was detected as early as 134 days postchallenge and generally increased after this time point. Two antigens of approximately 50 and approximately 60 kDa were particularly immunodominant early in infection, as shown by immunoblot with serum collected within 2 weeks postchallenge. Findings indicate that the intratonsillar inoculation route will prove useful as an experimental model for paratuberculosis infection. Additionally, this study confirms that mycobacteria-specific antibody is detectable early in the course of experimental Johne's disease, even preceding the development of specific cell-mediated responses.

Cell-mediated immune response to high-passage Borrelia spirochetes in C57bl/6 mice is strictly dependent on antigen specificity

Inbred C57bl/6 mice were challenged with high-passage Borrelia afzelii, Borrelia garinii and Borrelia burgdorferi sensu stricto and tested for antigen specific T-cell response in vitro. Sonicated preparations of washed spirochetes were potent cell activators, capable of stimulating polyclonal proliferation after 72h of culture while increasing the incubation time up to 120h provoked specific cell-mediated response. Isolated murine spleocytes previously sensitized to B. burgdorferi sensu lato but not those from control mice could be induced for antigen-specific proliferation in vitro, as revealed by [ 3H]thymidine incorporation assay, Moreover, in mice presensitized to B. burgdorferi sensu lato, detectable cell-mediated response could be induced only with antigen preparations derived from a corresponding strain but not with those obtained from other Borrelia genospecies. The current study emphasises that the B. burgdorferi antigen-specific response may also be expected in different genospecies infections in men.

The cell-mediated immune response to Borrelia afzelii, garinii and burgdorferi in C57BL/6 mice is dependent on antigen specificity.

Inbred C57BL/6 mice were challenged with Borrelia afzelii, Borrelia garinii and Borrelia burgdorferi sensu stricto and tested for antigen-specific T-cell response in vitro. The sonicated preparations of in vitro grown spirochetes were capable of stimulating polyclonal proliferation and specific cell-mediated response, depending on duration of the cell culture. Murine splenocytes previously sensitized to B. burgdorferi sensu lato (s.l. ), but not those from control mice, could be induced for antigen-specific proliferation in vitro. Moreover, detectable cell-mediated response could be induced only with antigen preparations derived from a corresponding strain but not with those obtained from other Borrelia genospecies as revealed by the [(3)H]thymidine incorporation assay. The current study considers that the strict B. burgdorferi s.l. antigen-specific response may also be expected in infections in humans and contributes to the explanation of the frequently poor antibody- and cell-mediated immune response observed in patients diagnosed with Lyme disease.

The cell-mediated immune response to Borrelia afzelii, garinii and burgdorferi in C57BL/6 mice is dependent on antigen specificity

Inbred C57BL/6 mice were challenged with Borrelia afzelii, Borrelia garinii and Borrelia burgdorferi sensu stricto and tested for antigen-specific T-cell response in vitro. The sonicated preparations of in vitro grown spirochetes were capable of stimulating polyclonal proliferation and specific cell-mediated response, depending on duration of the cell culture. Murine splenocytes previously sensitized to B. burgdorferi sensu lato (s.l.), but not those from control mice, could be induced for antigen-specific proliferation in vitro. Moreover, detectable cell-mediated response could be induced only with antigen preparations derived from a corresponding strain but not with those obtained from other Borrelia genospecies as revealed by the [ 3H]thymidine incorporation assay. The current study considers that the strict B. burgdorferi s.l. antigen-specific response may also be expected in infections in humans and contributes to the explanation of the frequently poor antibody- and cell-mediated immune response observed in patients diagnosed with Lyme disease.

Assessing humoral and cell-mediated immune response in Hawaiian green turtles, Chelonia mydas

Seven immature green turtles, Chelonia mydas, captured from Kaneohe Bay on the island of Oahu were used to evaluate methods for assessing their immune response. Two turtles each were immunized intramuscularly with egg white lysozyme (EWL) in Freund’s complete adjuvant, Gerbu, or ISA-70; a seventh turtle was immunized with saline only and served as a control. Humoral immune response was measured with an indirect enzyme linked immunosorbent assay (ELISA). Cell-mediated immune response was measured using in vitro cell proliferation assays (CPA) using whole blood or peripheral blood mononuclear cells (PBM) cultured with concanavalin A (ConA), phytohaemagglutinin (PHA), or soluble egg EWL antigen. All turtles, except for one immunized with Gerbu and the control, produced a detectable humoral immune response by 6 weeks which persisted for at least 14 weeks after a single immunization. All turtles produced an anamnestic humoral immune response after secondary immunization. Antigen specific cell-mediated immune response in PBM was seen in all turtles either after primary or secondary immunization, but it was not as consistent as humoral immune response; antigen specific cell-mediated immune response in whole blood was rarely seen. Mononuclear cells had significantly higher stimulation indices than whole blood regardless of adjuvant, however, results with whole blood had lower variability. Both Gerbu and ISA-70 appeared to potentiate the cell-mediated immune response when PBM or whole blood were cultured with PHA. This is the first time cell proliferation assays have been compared between whole blood and PBM for reptiles. This is also the first demonstration of antigen specific cell-mediated response in reptiles. Cell proliferation assays allowed us to evaluate the cell-mediated immune response of green turtles. However, CPA may be less reliable than ELISA for detecting antigen specific immune response. Either of the three adjuvants appears suitable to safely elicit a detectable immune response in green turtles.

Specific cell-mediated responses to bacterial antigens and clinical correlations in reactive arthritis, Reiter's syndrome and ankylosing spondylitis.

In 2 cases of ReA seen during the acute phase and shown serologically to be due to Y. enterocolitica 0:3, the LT test showed a marked response using as antigen a freeze dried preparation of the causative organism. The test result correlated with the activity of the disease when repeated during a flare in the 1st case, and during remission in both. Patients with ReA/RS in general showed a significantly higher response to the yersinia and klebsiella antigens tested when compared to AS, suggesting an overall difference in cell-mediated immunity to these enteric bacteria. AS cases reacted significantly less than controls to K. pneumoniae under suboptimal conditions. K. pneumoniae was shown to enhance the LT response to yersinia, possibly through an adjuvant effect. This was found with AS, ReA and in controls, though whether it is of relevance in the etiopathogenesis of AS or ReA/RS remains far from clear. Acute non-traumatic synovitis of the knee, occurring de novo, or in association with psoriasis, inflammatory bowel disease, or as part of RS, may be accompanied by evidence of heightened reactivity to streptococci both by blood and synovial fluid mononuclear cells. In 1 case with serological evidence of streptococcal infection and erythema nodosum, these changes were found to parallel disease activity. ReA can, it appears, follow recent streptococcal infection, and be associated with B27.

Macrophage procoagulant activity as a measure of cell-mediated immunity to P2 protein of peripheral nerves in the Guillain-Barré syndrome

Cell-mediated and humoral immunity to purified nerve proteins has been assessed in GBS patients and compared with that of patients with other neurological diseases and healthy controls. A strong and specific cell-mediated response to the neuritogenic basic protein P2 occurred in 13/16 GBS patients tested. Extremely low levels of P2 (0.01 micrograms/culture) induced monocyte/macrophage procoagulant activity (MPCA) on peripheral blood mononuclear cells (PBM) from GBS patients (P = 0.007) whereas higher concentrations (2 micrograms/culture) of myelin basic protein (MBP) and sciatic nerve myelin (SNM) were required to stimulate similar levels of activity. These concentrations of nerve antigens failed to induce significant MPCA on peripheral blood mononuclear cells of patients with other neuropathies or healthy controls. Lipopolysaccharide, a non-specific stimulant of macrophage procoagulant activity, induced similar procoagulant levels on PBM from each group. The MPCA assay was a sensitive, quantitative and specific indicator of cell-mediated immunity to the neuritogenic peptide, P2 in GBS patients. Serum antibodies to P2, P0 and SNM were detected by a sensitive solid-phase radioimmunoassay. Naturally occurring antibodies to peripheral nerve antigens were observed in sera of healthy subjects and these levels were not significantly different from patients with GBS or those with other neuropathies. Our results indicate that the autoimmune cell-mediated response to the neuritogenic peptide P2 plays a major role in the pathogenesis of GBS.

Impaired cell-mediated immunity in Plasmodium falciparum-infected patients with high-parasitemia and cerebral malaria

Several cell-mediated functions were studied in vivo and in vitro in 63 Thai patients with acute falciparum malaria, including 21 cases with cerebral manifestations and 10 cases with initial parasitemia over 10%. Initial delayed cutaneous reactions to phytohemagglutinin and soluble protein antigens were negative in most cerebral malaria cases. In other patients, skin reactions were impaired or abolished as a direct function of parasitemia. No major alteration in the numbers of blood T and B lymphocytes was found. In lymphocyte cultures, proliferative responses to lectins were generally found within normal ranges; in contrast, proliferative responses to candidin were suppressed in parallel with delayed cutaneous responses to the same antigen. From these data, it can be concluded that the alteration of specific cell-mediated responses are predominantly detectable in acute cases with major parasite invasion, i.e., high parasitemia and/or cerebral manifestations. A direct role of Plasmodium falciparum was further suggested by the rapid restoration of cell-mediated functions observed in several cases under successful antimalarial therapy. These results do not support any evidence in favor of a preexisting cellular immune deficiency in relation with the occurrence of cerebral or high-parasitemia acute malaria in these patients.

Cell-mediated immunity to varicella-zoster virus infection in subjects with lymphoma or leukemia

Normal subjects and patients with lymphoma or leukemia were tested for the levels of lymphocytes, E-rosette--forming T-cells, serum and vesicle fluid interferon, and specific in vitro proliferative response to varicella-zoster antigen after clinical varicella or herpes zoster illness. The effect of polyinosinic acid/polycytidilic acid on the immune response was also evaluated. The development of VZ specific cell-mediated response in normal subjects was characterized by intense proliferative activity eight to ten days after the onset of illness, with significant decline 70 to 80 days later. The responses in subjects with lymphoma or leukemia were much lower. Few subjects with chickenpox or zoster with lymphoma or leukemia died during the infection. Death was associated with significant depletion of E-rosette--forming T-cells, and grossly deficient specific cellular response to VZ antigen. Treatment with Poly IC frequently induced elevations in serum as well as vesicle fluid interferon levels, and increased the proliferative activity of lymphocytes against VZ antigen.

Circulatory and cellular immune responses to squamous cell carcinoma of the uterine cervix

The sera from 176 patients with cervical cancer were examined by indirect immunofluorescence against cervical cancer cells (imprints of cervical cancer tissue). The mean serum titer in these patients was significantly higher than the titer in two control groups of identical size. These tumor-related antibodies were also higher in titer in sera from a small sample of nine husbands of cancer patients than in 18 control group patients. A cell-mediated immune response was studied with the use of leukocyte-migration inhibition from 39 blood specimens, including 23 patients with invasive epithelial carcinoma, five with intraepithelial carcinoma of the uterine cervix, six with other gynecologic diseases, and five withou gynecologic disease. Migration inhibition was demonstrated for squamous cell carcinoma of the uterine cervix, suggesting a common specific cell-mediated response.