Assessment Of Cell-mediated Immunity Research Articles

B Cell and Antibody Responses in Bovine Tuberculosis.

The development of vaccines and effective diagnostic methods for bovine tuberculosis requires an understanding of the immune response against its causative agent, Mycobacterium bovis. Although this disease is primarily investigated and diagnosed through the assessment of cell-mediated immunity, the role of B cells and antibodies in bovine tuberculosis has been relatively undervalued and understudied. Current evidence indicates that circulating M. bovis-specific antibodies are not effective in controlling the disease. However, local humoral immune responses may contribute to either defence or pathology. Recent studies in animal models and cattle vaccine trials suggest a potential beneficial role of B cells in tuberculosis control. This review discusses the role of B cells and antibodies in bovine tuberculosis and explores antibody-based diagnostics for the disease, including traditional techniques, such as different ELISA, new platforms based on multiple antigens and point-of-care technologies. The high specificity and sensitivity values achieved by numerous antibody-based tests support their use as complementary tests for the diagnosis of bovine tuberculosis, especially for identifying infected animals that may be missed by the official tests.

Evaluation of influenza vaccine-induced cell-mediated immunity: Comparison between methods using peripheral blood mononuclear cells and whole blood.

Assessment of cell-mediated immunity (CMI) may be critical to evaluating the ability of individuals to protect themselves against influenza virus infection. However, it has been difficult to evaluate CMI because no simple means of measuring it is currently available. The aim of this study was to compare the performance of a CMI measurement method developed by us, which involves reacting whole blood with antigen, with the conventional method, which is based on isolating peripheral blood mononuclear cells (PBMCs). Correlations between these methods before and after vaccination of 26 healthy adults (aged 28-58 years; 12 men and 14 women) were assessed and changes in CMI after influenza vaccination in PBMCs cultured with antigen for 48 and 96 hr and whole blood cultured with antigen for 48 hr were studied. Results of CMI measurement using whole blood on Day 2 and PBMCs on Day 4 were found to be correlated. Spearman's correlation coefficients with four antigens (A [H1N1], A [H3N2], B [Yamagata lineage], and B [Victoria lineage]) before vaccination were 0.55, 0.61, 0.58, and 0.70, respectively and 0.40, 0.45, 0.62, and 0.52, respectively, after vaccination. CMI was detected sooner when whole blood was reacted with antigen than when PBMCs were reacted with antigen. The rate of positive reaction of influenza A (H1N1 and H3N2) in whole blood on Day 2 was higher than that in PBMCs on Day 2. Our method is simple and may be useful for vaccine development because it can measure CMI in a small amount of blood without separating off PBMCs.

Hepatitis B Virus Vaccine–Induced Cell-Mediated Immunity Correlates with Humoral Immune Response following Primary Vaccination during Infancy

Abstract Data on hepatitis B virus (HBV) vaccine–induced cell-mediated immunity (CMI) and humoral immune response during infancy is scarce. We assessed HBV vaccine–induced CMI among infants stratified as nonresponders (Ab against HBV surface Ag [anti-HBs] levels <10 IU/l), low-responders (anti-HBs levels 10–100 IU/l), and high-responders (anti-HBs levels ≥100 IU/l) following their primary vaccination against HBV. Moreover, we assessed the association between HBV vaccine–induced CMI and anti-HBs levels. Infants were immunized with HBV vaccine at ages 2, 4, and 6 mo. Hepatitis B surface Ag (HBsAg)-specific proliferation, intracellular cytokine production, and bulk cytokine secretion were assessed on PBMCs collected at 1 y and anti-HBs levels were measured at 1 and 2 y of age. Infants classified at 2 y of age as low-responders (n = 28) had lower median levels of secreted IFN-γ than high-responders (n = 29), 17.15 pg/ml versus 33.16 pg/ml, respectively, p = 0.009. Infants classified at 2 y of age as nonresponders (n = 15) had lower median levels of secreted TNF-α than high-responders (n = 29), 116.11 pg/ml versus 162.27 pg/ml, respectively, p = 0.032. There was a positive correlation between HBsAg-specific secreted IFN-γ levels at 1 y and anti-HBs levels at 1 and 2 y of age, rho = 0.269 and 0.302, respectively, (p = 0.019 and p = 0.01, respectively). There was a positive correlation between anti-HBs levels at age 1 y and the levels of secreted IL-10, rho = 0.297, p = 0.009. HBsAg-specific IFN-γ, IL-10, and TNF-α secretion correlated with HBV vaccine–induced humoral immune response. Assessment of CMI is a useful adjunct in demonstrating the persistence of HBV vaccine–induced memory immune response.

Improved Clearance during Treatment of HPV-Positive Head and Neck Cancer through mTOR Inhibition

Human papillomavirus (HPV)-related head and neck squamous cell carcinoma (HNSCC) incidence is increasing at a near epidemic rate. We investigated whether the mammalian (or mechanistic) target of rapamycin (mTOR) inhibitor, rapamycin, can be used as a concurrent agent to standard-of-care cisplatin/radiation therapy (CRT) to attenuate tumor lactate production, thus enhancing CRT-induced immune-mediated clearance of this antigenic tumor type. A C57Bl/ 6-derived mouse oropharyngeal epithelial cell line retrovirally transduced with HPV type 16 E6/E7 and human squamous cell carcinoma cell lines were evaluated for their response to rapamycin in vitro with proliferation assays, Western blots, and lactate assays. Clonogenic assays and a preclinical mouse model were used to assess rapamycin as a concurrent agent to CRT. The potential of rapamycin to enhance immune response through lactate attenuation was assessed using quantitative tumor lactate bioluminescence and assessment of cell-mediated immunity using E6/E7-vaccinated mouse splenocytes. Rapamycin alone inhibited mTOR signaling of all cancer cell lines tested in vitro and in vivo. Furthermore, rapamycin administered alone significantly prolonged survival in vivo but did not result in any long-term cures. Given concurrently, CRT/rapamycin significantly enhanced direct cell killing in clonogenic assays and prolonged survival in immunocompromised mice. However, in immunocompetent mice, concurrent CRT/rapamycin increased long-term cures by 21%. Preliminary findings suggest that improved survival involves increased cell killing and enhanced immune-mediated clearance in part due to decreased lactate production. The results may provide rationale for the clinical evaluation of mTOR inhibitors concurrent with standard-of-care CRT for treatment of HPV-positive HNSCC.

Ex VivoRestimulation of Human PBMC Expands aCD3+CD4-CD8-γδ+T Cell Population That Can Confound the Evaluation of CD4 and CD8 T Cell Responses to Vaccination

The measurement of vaccine-induced humoral and CD4+ and CD8+ cellular immune responses represents an important correlate of vaccine efficacy. Accurate and reliable assays evaluating such responses are therefore critical during the clinical development phase of vaccines. T cells play a pivotal role both in coordinating the adaptive and innate immune responses and as effectors. During the assessment of cell-mediated immunity (CMI) in subjects participating in a large-scale influenza vaccine trial, we identified the expansion of an IFN-γ-producing CD3+CD4−CD8− γδ + T cell population in the peripheral blood of 90/610 (15%) healthy subjects. The appearance of CD3+CD4−CD8− γδ + T cells in the blood of subjects was transient and found to be independent of the study cohort, vaccine group, subject gender and ethnicity, and ex vivo restimulation conditions. Although the function of this population and relevance to vaccination are unclear, their inclusion in the total vaccine-specific T-cell response has the potential to confound data interpretation. It is thus recommended that when evaluating the induction of IFN-γ-producing CD4+ and CD8+ immune responses following vaccination, the CD3+CD4−CD8− γδ + T cells are either excluded or separately enumerated from the overall frequency determination.

Drug-induced interstitial lung disease: mechanisms and best diagnostic approaches

Drug-induced interstitial lung disease (DILD) is not uncommon and has many clinical patterns, ranging from benign infiltrates to life-threatening acute respiratory distress syndrome. There are two mechanisms involved in DILD, which are probably interdependent: one is direct, dose-dependent toxicity and the other is immune-mediated. Cytotoxic lung injury may result from direct injury to pneumocytes or the alveolar capillary endothelium. Drugs can induce all types of immunological reactions described by Gell and Coombs; however, most reactions in immune-mediated DILD may be T cell-mediated.DILD can be difficult to diagnose; diagnosis is often possible by exclusion alone. Identifying the causative drug that induces an allergy or cytotoxicity is essential for preventing secondary reactions.One method to confirm the diagnosis of a drug-induced disease is re-exposure or re-test of the drug. However, clinicians are reluctant to place patients at further risk of illness, particularly in cases with severe drug-induced diseases. Assessment of cell-mediated immunity has recently increased, because verifying the presence or absence of drug-sensitized lymphocytes can aid in confirmation of drug-induced disease. Using peripheral blood samples from drug-allergic patients, the drug-induced lymphocyte stimulation test (DLST) and the leukocyte migration test (LMT) can detect the presence of drug-sensitized T cells. However, these tests do not have a definite role in the diagnosis of DILD. This study explores the potential of these new tests and other similar tests in the diagnosis of DILD and provides a review of the relevant literature on this topic.

Histopathology, immunology, and serology of oral yeast infections: Diagnosis of oral candidosis

For diagnostic purposes it is normally not important to obtain a biopsy specimen, since an oral smear from the lesion will yield blastospores and pseudohyphae in abundance. However, in lesions that respond poorly to antimycotic treatment a biopsy should be carried out to detect possible malignant changes in the epithelium. Assessment of cell-mediated immunity against Candida albicans and other antigens may be important in patients with severe chronic candidosis to assess the degree of immunocompetence and prognosis. Usually, patients with oral candidosis show only moderately elevated antibody titers in serum and saliva against C. albicans, and serologic tests are normally not a diagnostic tool for oral candidosis. However, such tests may be a prognostic instrument in patients with severe oral candidosis who respond poorly to antimycotic therapy.

Evaluation of the delayed skin reaction in chronically ill children

The evaluation of delayed cutaneous hypersensitivity by using a standardized test system offers an assessment of cell-mediated immunity. The number and diameter of positive responses induced by seven antigens result in a score. The study included 21 healthy children, 20 children with bacterial infections as well as 20 patients with chronic renal insufficiency. In addition, 16 children with cystic fibrosis were also studied. No differences in the skin reactions was found in children with bacterial infections, showing a score of 13 +/- 6 mm. But children with cystic fibrosis or with renal insufficiency exhibited a severe impaired cell-mediated immunity. To what extent the alteration of cell-mediated immunity affects the course of the various diseases, has to be investigated by further prospective studies.

Standardization of lymphocyte transformation to antigen.

In vitro lymphocyte transformation to non-specific stimulant or specific stimulant is frequently used in the assessment of cell-mediated immunity so that a better understanding of the immune response must be gained before use. Lymphocyte transformation to bovine serum albumin (BSA) or human serum albumin (HSA) immunogen was carefully studied. The standard conditions were: (1) 100 μg of BSA or HSA immunogen; (2) 120 hours of incubation time after addition of antigen; (3) 1/30 dilution of whole blood and 0.5X106 lymphocytes per culture; (4) 1 μCi of3H-thymidine per culture.

Application of the leucocyte adherence inhibition test to the assessment of cell mediated immunity in invasive amoebiasis

The leucocyte adherence inhibition (LAI) test was assessed as a means for measuring specific cell mediated immunity (CMI) in invasive amoebiasis. The LAI test and the amoebic gel diffusion (AGD) test were performed on 20 patients admitted to hospital with amoebic liver abscess (ALA) and 24 control subjects with no demonstrable clinical amoebiasis. The AGD results were acceptable for such a study in an endemic area as all the proven ALA subjects gave positive responses while all except one of the controls gave a negative reaction. A percentage adherence in the LAI test of more than 80% was considered to be negative and the mean percentage adherence of leucocytes for the control subjects was found to be 86·0 ± 4·9 while that for the experimental group was found to be 76·1 ± 15·4; the difference between these groups is statistically significant. It was noticed that the LAI test response was possibly linked with severity of disease in the ALA subjects. The LAI test has been found to be a relatively rapid test of specific CMI and although fastidious, it does not require the use of radio-isotopes.

Assessment of cell-mediated immunity against coxsackievirus B3-induced myocarditis in a primate model (Papio papio)

Delayed hypersensitivity and myocarditis were induced in five baboons (Papio papio) after inoculation with myocarditic murine coxsackievirus B3 (CVB3m). By means of the [3H]thymidine incorporation assay and the agarose droplet cell migration inhibition assay, specific cell-mediated immunity was detected in all five animals against viral antigens and/or KCl-extractable soluble antigens extracted from the heart of a CVB3m-inoculated baboon. KCl-extracted antigens from normal baboon heart tissues, as well as KCl-extracted antigens from spleen or liver tissues from a CVB3m-inoculated baboon, failed to stimulate baboon peripheral blood lymphocytes or inhibit the migration of baboon peritoneal exudate cells. The appearance of dissimilar antigen(s) in extracts of heart tissues from CVB3m-inoculated baboons which did not contain infectious CVB3m parallels the appearance of novel KCl-extractable antigen(s) induced by CVB3m in murine heart tissues (Paque et al., J. Immunol. 120:1672-1678). The animal model described represents the first systematic evaluation of cell-mediated immunity in CVB3m-induced myocarditis in primates and suggests that a similar phenomenon may occur in humans.

Simple methods for nutritional assessment in hemodialyzed patients

Uremic patients undergoing hemodialysis are often catabolic and malnourished. To treat malnutrition effectively, a preliminary nutritional assessment is needed. Available techniques should enable the clinician to readily detect the presence of malnutrition and to follow the response to nutritional therapy. In a group of chronic uremic patients undergoing maintenance hemodialysis, the authors evaluated the nutritional status with the following indices: 1) assessment of the somatic fat and protein compartments by means of anthropometric measurements (weight/height ratio, triceps and subscapular skinfold thickness, and arm muscle circumference); 2) assessment of the visceral protein compartment (serum total protein, albumin, transferrin, pseudocholinesterase, C3, and immunoglobulin content); 3) assessment of cell-mediated immunity by means of skin tests ("skin window," PPD and phytohemagglutinin) and blood lymphocyte content; and 4) assessment of the dietary intake of nutrients with dietary diaries. Anthropometric indices, serum protein content (except immunoglobulins), and the immune response was generally lower than in normal subjects, suggesting a mixed marasmus-like and kwashiorkor-like pattern of protein-calorie malnutrition. The protein intake was normal, whereas the energy intake tended to be low. Protein intake was significantly correlated with the predialysis serum urea nitrogen. Due to the difficulties in improving oral energy intake and the negative nitrogen balance reported during the days of dialysis therapy, patients were given intravenous supplements of essential or essential and nonessential amino acids for 2 months. The effects of this short-term supplementation were limited.

Assessment of cell-mediated immunity in the rat utilizing the agarose-droplet cell-migration-inhibition correlate of delayed-type hypersensitivity

The agarose-droplet cell-migration-inhibition assay was developed for measuring specific cell-mediated immunity in rats. Fischer #344 female rats were sensitized to mono(p-azobenzene-arsonate)-N-chloroacetyl-L-tyrosine (ARSNAT), keyhole-limpet-hemocyanin (KLH), or a BCG cell wall preparation. The optimal conditions for assay were determined by testing varying concentrations of antigens against normal and sensitized peritoneal exudate cells induced with 5% thioglycolate medium. Specific cell-mediated immunity to each of three different antigens was detected, which correlated with skin tests observed in vivo . Our adaptation of the agarose-droplet assay should provide a useful model for studying other aspects of cell-mediated and/or tumor immunity in the rat.

Announcement

Abstract An Advanced Institute in Methods of Immunological Diagnosis, June 14–25, 1976 under the auspices of the World Health Organization Collaborating Laboratory for the Serology of Autoimmune Diseases at the Department of Immunology and Microbiology, Wayne State University, is to be held at the Cranbrook Educational Community, Lone Pine Road, Bloomfield Hills, Michigan. The object of this two-week course is to present an up-to-date survey of immunologic methods that are applicable to the diagnosis of human immunological diseases. The course comprises practical laboratory sessions, demonstrations, lectures and discussions. It is designed primarily for pathologists, clinicians, and investigators interested in clinical applications of immunology. The following topics are to be included: Electroimmunodiffusion and newer immunoelectrophoretic methods; Passive hemagglutination; Immunofluorescence and immunoperoxidase; Measurement of complement and its components; Quantitation of lymphocytes; Lymphocyte stimulation; Lymphocyte mediated cytotoxicity; and Assessment of cell-mediated immunity.