Lymphocyte Transformation Assay Research Articles

Immunomodulatory triterpenoids from the oleogum resin of Boswellia carterii Birdwood.

The immunomodulatory bioassay-guided fractionation of the oleogum resin of frankincense (Boswellia carterii Birdwood) resulted in the isolation and identification of 9 compounds; palmitic acid and eight triterpenoids belonging to lupane, ursane, oleanane, and tirucallane skeleta were isolated form the resin. These triterpenoids are lupeol, beta-boswellic acid, 11-keto-beta-boswellic acid, acetyl beta-boswellic acid, acetyl 11-keto-beta-boswellic acid, acetyl-alpha-boswellic acid, 3-oxo-tirucallic acid, and 3-hydroxy-tirucallic acid. The structures of the isolated compounds were deduced based on spectroscopic evidences. The lymphocyte transformation assay of the isolated compounds proved that the total extract retained more activity than that of any of the purified compounds.

Propionibacterium acnes and inflammation in acne; P. acnes has T-cell mitogenic activity.

Circumstantial evidence suggests that Propionibacterium acnes has a role in the inflammation of acne. This could be effected by antigenic or superantigenic or mitogenic reactions. The purpose of this investigation was to determine whether P. acnes had only antigenic activity or additional superantigenic and mitogenic activity. A lymphocyte transformation assay was used to detect responses to a mixture of eight P. acnes whole cell isolates, and their supernatant culture fluids. In order to determine the nature of T-cell reactions to P. acnes cells a mouse-antihuman major histocompatibility complex class II monoclonal antibody was used in the lymphocyte transformation assay to inhibit the antigenic stimulation of lymphocytes. An analysis of the T-cell receptor (TCR) variable region beta (BV) repertoire was undertaken using flow cytometry of the unstimulated and stimulated cells. Peripheral blood mononuclear cells (PBMNC) from adults with no history of acne responded strongly to stationary growth phase cells of P. acnes, less strongly to cells in the exponential growth phase. No response was detected to supernatant culture fluids. PBMNC from five cord blood samples (CBMNC) responded maximally after 3 and 7 days of incubation with stationary growth phase cells of P. acnes. The reaction of CBMNC to P. acnes cells was not suppressed completely by the blocking antibody. The analysis of the TCRBV repertoire indicated that P. acnes induced no deletion or over-representation of certain BV element-bearing T cells. The TCRBV analysis was repeated after preincubation with the blocking antibody. Deletion of T cells bearing certain BV components occurred and there was no over-representation of T cells carrying certain BV components. Two mechanisms of lymphocyte activation by P. acnes cells are proposed, antigen and mitogen driven. These results are consistent with the histological evidence of inflammation in acne lesions.

Posterior scleritis associated with Borrelia burgdorferi (Lyme disease) infection

Objective To report on the clinical findings in a patient with posterior scleritis associated with infection with Borrelia burgdorferi. Design Interventional case report. Participant A 39-year-old male ranger who experienced posterior scleritis after several tick bites with erythema migrans. Testing Extensive ophthalmic and systemic workup, including serologic testing and imaging techniques. Results Sonography and contrast-enhanced computed tomography showed a large scleral mass (16 × 12 × 13 mm) in a patient with painful proptosis in the left eye with episcleral vascular dilation, reduction in bulbar motility, and chorioretinal folds in the upper temporal quadrant. Treatment with high-dose corticosteroids resulted in rapid regression of clinical symptoms and of the scleral mass. Intensive workup revealed immunoglobulin M antibodies (enzyme-linked immunoassay, Western immunoblot) and a positive lymphocyte transformation assay against B. burgdorferi. No other cause for posterior scleritis could be identified. Conclusions Posterior scleritis should be added to the list of ocular manifestations associated with Lyme disease. Because corticosteroids alone resulted in rapid improvement of clinical symptoms, the scleritis might be mediated by autoimmunologic mechanisms.

Improving the in vitro antigen specific T cell proliferation assay: the use of interferon-alpha to elicit antigen specific stimulation and decrease bystander proliferation

The measurement of the proliferative response of primed T cells to an antigenic stimulus (lymphocyte transformation assay: LTT) is commonly used for determining T cell immune responsiveness. However, the ratio between the spontaneous and the antigen-triggered response (stimulation index) is frequently quite low (<3–5) making the interpretation difficult. We modified the assay by the addition of interferon-α and the use of fresh autologous serum instead of human AB pool serum. These measures significantly enhanced the stimulation index following stimulation with tetanus toxoid, Candida albicans and tick-borne encephalitis (TBE) viral antigen in studies of sensitized patients. There was no concomitant increase in false positive results. Kinetic studies showed a reduced nonspecific background proliferation of non-stimulated cultures particularly between days 4 and 6 of culture. Furthermore, the positive effect of interferon-α were confirmed in studies of patients with contact allergy to nickel and gold. We conclude that this modified form of proliferation assay significantly increases the signal to noise ratio which can be attained. This may be of particular value when looking at T cell responses in immunocompromised patients or in diagnostic attempts to detect very low frequencies of antigen-specific T cells.

Biological effects of ionizing radiation on human blood compounds ex vivo

Blood compounds are irradiated ex vivo to prevent transfusion-associated graft-versus-host-disease. Recently, ex vivo irradiation of re-transfused wound blood has been proposed to prevent metastatic spread in patients with malignant tumors, an issue requiring different dose concepts. To determine effects on blood cells we examined the impact of various doses of ionizing radiation. Full blood was irradiated with doses of 10-150 Gy. Potassium, LDH and hemoglobin levels were determined 2 h-96 h after irradiation. The lymphocyte proliferation after irradiation was measured by means of a lymphocyte-transformation assay. The impact of irradiation on mitogen-induced secretion of cytokines was determined by the ELISA technique, and P-selectin expression as an indicator of platelet activation was analyzed by flow-cytometry. Potassium levels increase with aging and irradiation dose. Mitogenic capacity is reduced by over 90% with moderate doses of 10-20 Gy, but a residual proliferation is still detectable up to 50 Gy. No enhancement of extracellular cytokine levels is detectable, but the cytokine release is reduced by radiation. Neither induction of platelet activation nor abrogation of activation has been detected. Doses of 30-50 Gy abrogate lymphocyte proliferation almost completely. In this range we did not observe severe adverse effects on blood transfusions. Hemolysis might be enhanced when the samples are stored for a longer period after irradiation.

Toward the development of diagnostic assays to discriminate between Mycobacterium bovis infection and bacille Calmette-Guérin vaccination in cattle.

A scientific review of the recent sharp increase in bovine tuberculosis in Great Britain has concluded that the development of a cattle vaccine holds the best prospect for long-term disease control. It is important to develop a diagnostic test that differentiates between vaccinated and Mycobacterium bovis-infected animals, to ensure that test-and-slaughter control strategies can continue alongside vaccination. The mycobacterial antigens ESAT-6, MPB64, and MPB83 are expressed at high levels in M. bovis but are expressed at low levels or not at all in bacille Calmette-Guérin (BCG) Pasteur. Promiscuous bovine T cell epitopes of these antigens were identified and formulated into a peptide cocktail. This cocktail and a cocktail composed of recombinant forms of the 3 antigens was able to distinguish cattle infected with virulent M. bovis from those vaccinated with BCG and from those sensitized to avian tuberculin in lymphocyte transformation and interferon-gamma assays.

Cellular immune responses in human neurocysticercosis.

Studies on the role of cell-mediated immune responses in human neurocysticercosis (NCC) are lacking. Various cell-mediated immune responses such as lymphocyte subpopulation, lymphocyte transformation to cysticercus antigens and cytokine profile were carried out in NCC patients. Lymphocyte transformation assays using larval antigens showed significantly higher (3)H-thymidine uptake. Immunophenotyping analysis showed an insignificant increase in B cells and a decrease in total T cells. However, there was a significant decrease (P < 0.05) in CD8+ T cells whereas there was no change in other cells like CD4+, HLA-DR+ and CD16+/CD56+. Cytokine profile revealed significantly higher (P < 0.01) production of Th1 cytokines (gamma-IFN and IL-2) using cysticercal antigens as stimulants for peripheral blood mononuclear cells, while there was no difference in IL-4 levels between NCC patients and healthy controls. The cytokine profile indicated the involvement of Th-1-like responses in NCC patients.

Assessment of the immunological surveillance value of humoral and lymphocyte assays in severe human cystic echinococcosis

Thirty cystic echinococcosis (CE) patients in Uruguay with severe bone or secondary disseminated echinococcosis were immunologically assessed using cellular (lymphocyte transformation assay, LTA) and humoral (specific antibody and subclass responses, circulating antigen and circulating immune-complexes) immunological assays during the course of chemotherapy (albendazole and/or praziquantel). CE patients were divided into 4 groups, according to clinical treatment and outcome: (I) surgery and chemotherapy, (II) chemotherapy with outcome unchanged, (III) chemotherapy with outcome improved, and (IV) chemotherapy considered cured. Increased circulating antigen was of prognostic value in some severe CE cases where levels remained high and/or increased. The lymphoproliferative response in vitro to Echinococcus granulosus antigen was statistically greater in all patient groups compared to normal individuals but at lower levels in improved or cured CE patients. Levels of non-specific LTA response were significantly lower than controls for all groups during albendazole treatment ( P < 0·001) but returned to normal levels in cured patients, a result consistent with parasite-induced suppression of cellular responses. This study suggests that, at least in severe osseous and secondary CE, immunosurveillance by specific antibodies, especially total specific immunoglobulin, was overall of more practical use than antigen-specific in-vitro lymphocyte transformation assays.

Common variable immunodeficiency in miniature dachshunds affected with Pneumonocystis carinii pneumonia.

Seven miniature dachshunds, all under the age of 1 year, were presented with polypnea, tachypnea, and exercise intolerance as a result of Pneumocystis carinii pneumonia, which was diagnosed on transtracheal aspirate cytology. In all of the dogs, historical and clinical signs were suggestive of immune incompetence. Immunological studies undertaken were leukogram parameters, serum immunoglobulin fraction quantification, lymphocyte transformation assay. CD3 and CD79a lymphocyte markers on lymphoid tissue, and anti-canine immunoglobulin G immunoperoxidase staining. The immunological studies showed hypogammaglobulinemia, deficiency of serum immunoglobulins A, G, and M, decreased lymphocyte transformation response to phytohemagglutinin and pokeweed mitogens and absence of B lymphocytes with presence of T lymphocytes in the lymphoid tissue stained with CD3 and CD79a lymphocyte markers. The preceding findings suggest that P. carinii pneumonia occurring in the miniature dachshund is a result of both a T- and B-cell deficiency. This presentation is not the classic primary severe combined immunodeficiency syndrome but rather combined variable immunodeficiency, which has been well documented in humans but never in the dog.

Epstein-barr virus transformation: involvement of latent membrane protein 1-mediated activation of NF-kappaB.

Epstein-Barr virus (EBV) transforms resting primary human B lymphocytes into indefinitely proliferating lymphoblastoid cell lines in vitro and is associated with several human malignancies in vivo. Recombinant EBV genetic analyses combined with in vitro B lymphocyte transformation assays demonstrate that latent infection membrane protein 1 (LMP1) is essential for EBV-mediated lymphocyte transformation. LMP1 has no intrinsic enzymatic activity but instead aggregates cellular proteins of the tumor necrosis factor receptor signaling pathway to activate transcription factor NF-kappaB. Mutants rendering LMP1 defective in these protein interactions are impaired in their abilities to activate NF-kappaB in reporter gene assays. Concordantly, EBV recombinants with LMP1 mutations that are compromised for NF-kappaB activation are impaired for growth transformation. Thus, EBV-mediated growth transformation is genetically and biochemically linked to LMP1-mediated activation of NF-kappaB.

A lymphocyte transformation assay for the detection of Mycobacterium bovis infection in the Eurasian Badger ( Meles meles)

The Eurasian badger ( Meles meles) is a significant wildlife reservoir of Mycobacterium bovis in Great Britain. Improved control strategies against the disease in badgers require the development of diagnostic tests and vaccines. Here, we report the development of a comparative lymphocyte transformation assay (LTA) using bovine and avian tuberculin as antigen to detect cell-mediated responses in M. bovis-infected badgers. In a pilot study, the performance of this assay was compared with the existing indirect ELISA assay for the detection of tuberculous badgers. The sensitivity of the Comparative LTA was 87.5% compared with 62.5% for the indirect ELISA whereas the ELISA test gave a greater specificity (100% compared with 84.6% for the comparative LTA). Preliminary evidence suggests that for the comparative LTA, the blood may be stored overnight prior to testing and that this procedure might improve the specificity of the assay without compromising the sensitivity.

The pathology of Psoroptes ovis infestation in cattle with a special emphasis on breed difference

In cattle, infestations with P. ovis are responsible for a severe dermatitis. The disease is very common in some breeds of beef cattle whereas dairy cattle such as Frisian Holstein are considered as resistant. In order to investigate the factors responsible for this marked breed susceptibility, the immune response to P. ovis under experimental and field conditions has been studied using serology, lymphocyte transformation assay, intradermal tests and passive cutaneous anaphylaxis in susceptible Belgian White and Blue (BWB) and resistant Frisian Holstein (FH) cattle. The results of published studies are reviewed in this paper; original data on specific hypersensitivity reactions to P. ovisare also presented. The use of a sandwich ELISA allowed the detection of specific antibodies as early as 7 days post-infestation and very high titres were observed. There was a clear positive correlation between the antibody titre and the extent of lesions. This antibody response was associated with a marked in vitro lymphocyte proliferation but there was no difference between susceptible and resistant cattle. The passive cutaneous anaphylaxis test revealed the presence of specific Ig E in actively infested animals irrespective of the breed. Specific Ig E were detected fairly late during the course of an experimental infestation with P. ovis.The intradermal injection of P. cuniculi antigenic extract induced an immediate reaction in all animals including the controls; in contrast, a delayed hypersensitivity reaction was observed in infected BWB animals only. It is concluded that immunological as well as non-immunological factors may be responsible for the breed related susceptibility or resistance to P. ovis.

Lymphocyte subsets in an experimental model of chronic pancreatitis in rats

The role of immunocompetent cells in chronic pancreatitis is poorly understood. Therefore, lymphocyte subsets were analyzed in pancreatic tissue, in peripheral blood, and in regional lymph nodes using an experimental chronic pancreatitis in rats. Methods: Pancreatitis was induced by intravenous application of dibutyltin dichloride (DBTC, 8mg/kg body weight). 5 rats per group were killed 1, 3, 5, 7, 14, 28, and 60 days after treatment. Pancreata were removed and examined immunohistochemicaUy with the APAAP technique. Peripheral blood and regional lymph nodes were obtained for isolation of lymphocytes followed by analysis of lymphocyte subsets using FACScan, Antibodies were directed to T lymphocytes, CD4+ helper T cells, CD8+ suppressor/cytotoxic T ceils, interleukin-2 receptor (CD25+), and B cells, Lymphocyte function was determined using the Concanavalin A (Con A) stimulated lymphocyte transformation assay with 3H-thymidine. Results: In DBTC-treated animals, we could demonstrate persistent infiltration of pancreatic tissue with lymphocytes. There was a predominance of CD4+ T cells compared to CD8+ T cells. However, CD8+ cells as well as CD25+ cells increased during the observation period. Number of B cells was low. During the observation period, no differences in the lymphocyte subsets in peripheral blood or in lymph nodes, were observed. Regarding lymphocyte function, we found significantly increased values of Con A induced lymphocyte transformation in regional lymph nodes in the course of pancreatitis. In peripheral blood, these changes could not be demonstrated. Conclusions: DBTC-pancreatitis was characterized by persistent infiltration of pancreatic tissue with CD4+ cells. CD8+ cells as well as activated lymphocytes increased. This indicates that suppressor/cytotoxic T cells play a role in the pathogenesis of experimental chronic pancreatitis. Regional lymph nodes were involved in the local inflammatory process. Supported by BMBF, FKZ 01/ZZ/9102

Natural infection of red deer with bovine tuberculosis

Six bovine tuberculosis-free red deer hinds were introduced in October 1993 to a 1.8 ha enclosure, within a larger field study site known to contain tuberculous possums, and kept there for 9 months. A Mycobacterium bovis-infected possum was found in the vicinity of the deer enclosure 3 weeks after the introduction. Subsequently, a further eleven infected possums were found in the area. The deer were monitored by repeated composite antibody detection ELISA and lymphocyte transformation assays for tuberculosis, interpreted in parallel, by skin testing and by routine culturing of samples collected from potential excretion sites. Lymphocyte transformation assay evidence of M. bovis infection in four hinds was first observed 4 months after introduction. One other hind became bovine tuberculin lymphocyte transformation assay positive in the 5th month. Positive or equivocal bovine reactivity remained evident at most test episodes. A comparative cervical skin test performed in July 1994, shortly before slaughter, was positive in these five hinds. Mycobacterium bovis was recovered off swabs from the oropharyngeal tonsils of two hinds during routine sampling. Detailed necropsy of the six deer revealed a single typical tuberculous lesion in only one, but culturing of various tissue specimens ascertained that the five blood test and comparative cervical skin test-positive animals were all infected. iMycobucterium bovis was cultured from the oropharyngeal tonsils of four and medial retropharyngeal lymph nodes of two of the deer with no typical gross lesions. Six additional tuberculosis-free hinds were introduced to the enclosure in April 1994 and kept there for 12 months. Four of these animals showed a positive lymphocyte transformation assay response to M. bovis after 9 weeks, but no significant reactivity thereafter. Concurrent observational studies suggest that five of the first six deer probably became infected through close inspection and investigation of the tuberculous possums, although the possibility of deer-to-deer transmission cannot be totally excluded. The likely deer-possum contact, and thus exposure to M. bovis, was related to the curiosity and social ranking of the hinds. The second group appear to have had transient exposure to M. bovis, possibly caused by direct contact with the infected hinds introduced earlier. This group never showed any curiosity toward, or interaction with, possums during the periods of observation.

Adenoid lymphocyte responses to outer membrane protein P6 of nontypable haemophilus influenzae in children with and without otitis media: Kodama H.; Faden H.; Harabuchi Y.; Kataura A.; Bernstein J.M.; Brodsky L.Acta Oto-Laryngol. Suppl. (USA) (1996) -/523 (153–154)

The cellular immune responses to nontypable Haemophilus influenzae in adenoids were determined in children by measuring lymphocyte blast transformation and antibody secretion in response to the P6 outer membrane protein. In the lymphocyte transformation assay, stimulation index of adenoid lymphocytes stimulated by P6 in otitis children (2.34 +/- 0.25) was significantly lower than that in non-otitis children (3.91 +/- 0.64, p <0.05). The number of IgM as well as IgA secreting cells after 8 days' culture with P6 were significantly smaller in otitis children (IgM: 7,534 +/- 2,843/10(6) lymphocyte; IgA: 1,573 +/- 620/10(6) lymphocyte) than those in non-otitis children (IgM: 12,994 +/- 2,533, p <0.05; IgA: 2,828 +/- 528, p <0.05). These data suggest that P6 protein is a target for the cellular immune response of the adenoid, and failure of adenoid lymphocytes to recognize it as a specific immunogen may be one of the causes of recurrent otitis media.

Cellular immunity and hypersensitivity as components of periodontal destruction.

Cellular immunity has been implicated in periodontal destruction for over 25 years. Studies in the 1970s used lymphocyte transformation and lymphokine assays to establish a role for cell-mediated mechanisms in periodontal disease. Immunohistological studies subsequently showed that the formation of gingivitis followed a similar pattern to the formation of a delayed type hypersensitivity reaction. Further functional studies suggested that a T cell/macrophage immunoregulatory imbalance may exist locally in the periodontitis lesion and that this imbalance may be antigen specific. RECENT EVIDENCE: More recently, T cell subsets have been dichotomised on the basis of their cytokine profiles. In general, ThI cells produce IL-2 and IFN-gamma while Th2 cells produce IL-4, IL-5 and IL-6. The major function of Th1 cells is to mediate delayed type hypersensitivity. In contrast the major function of Th2 cells is to provide B cell help. A model for periodontal disease has now been developed based on this functional dichotomy which provides a framework for the study of cytokine profiles in periodontal disease. Early studies in this context have demonstrated a higher proportion of IL-4 producing cells in periodontitis tissues suggesting a role for Th2 cells in the progressive lesion. Clonal studies have shown that the selection of a particular cytokine profile is not antigen dependent and that differences may be due to the host susceptibility although this remains to be determined. These emerging data clearly establish a role for cell-mediated mechanisms in the control of periodontal destruction and raise the possibility that in the future cytokine therapy for the treatment of periodontal disease in susceptible subjects may become a viable option.

Study on immune response of goats vaccinated with a live strain of Mycobacterium paratuberculosis

A study of the immune response of goats vaccinated with a live strain (316-F) of Mycobacterium paratuberculosis has been performed, taking into consideration both cellular and serological responses using a lymphocyte transformation assay (LTA) and a counterimmuno-electrophoresis test (CIET). The analyses made at three monthly intervals reveal an in vitro proliferative response of blood lymphocytes to specific antigen and lymphocytes T mitogen over a year of post-vaccination, but no humoral response was observed when CIET was used.

Identification and Characterization of an Orf Virus Homologue of the Vaccinia Virus Gene Encoding the Major Envelope Antigen p37K

DNA sequence analysis of a 1.55-kb region located 10 kb from the left end of the orf virus NZ-2 strain (OV NZ2) genome revealed an open reading frame, B2L, encoding a protein with a predicted molecular weight of 41.67 kDa. This protein (p42K) shows 42% amino acid sequence identity to the vaccinia virus (VAC) major envelope antigen p37K. In addition, p42K shows homology to a protein encoded by molluscum contagiosum virus (42.8% identity) and another encoded by fowlpox virus (38.3% identity). These proteins are themselves homologues of the VAC p37K. B2L is actively transcribed after the onset of DNA replication and S1 nuclease analysis mapped the 5′ end of the transcript to within the sequence TAAATG. A VAC recombinant capable of expressing the p42K gene was constructed and used as an antigen in radioimmune precipitations and lymphocyte transformation assays. These assays demonstrated that OV p42K is one of a limited number of OV proteins to which sheep mount a strong antibody response and which stimulate lymphocytes derived from draining lymph nodes following a natural infection with OV NZ2.

Cell‐mediated immune responses to Malassezia furfur serovars A, B and C in patients with pityriasis versicolor, seborrheic dermatitis and controls

It has been postulated that patients with Malassezia furfur-associated dermatoses have a deficient cell-mediated immune response to M. furfur. This study examined the cell-mediated immune responses to M. furfur serovars A, B and C of 10 patients with pityriasis versicolor and 10 age- and sex-matched controls; and 10 patients with seborrheic dermatitis and 10 age- and sex-matched controls. The responses to each serovar of M. furfur were assessed using the lymphocyte transformation assay and the leukocyte migration inhibition assay. The lymphocyte transformation responses of the patients with pityriasis versicolor to M. furfur serovars A, B and C (0/10, 6/10 and 5/10 respectively) were not significantly different from those of controls (0/10, 2/10 and 1/10). However, for patients with seborrheic dermatitis, significantly more patients' lymphocytes responded to serovars B and C (6/10 and 6/10 respectively) than those of controls (1/10 and 1/10). No patient or control responded to serovar A. In the leukocyte migration inhibition assay, the leukocytes from a greater proportion of patients with pityriasis versicolor (5/7) responded to serovar B than controls (2/10); and the leukocytes from a greater proportion of patients with seborrheic dermatitis (4/10) responded to serovar C than controls (0/9). Thus, this data did not indicate the presence of any cell-mediated immune deficiency to M. furfur in patients with pityriasis versicolor or seborrheic dermatitis, as measured by the lymphocyte transformation assay or the leukocyte migration inhibition assay. The greater responsiveness of T lymphocytes from patients may indicate that T lymphocytes might be involved in the pathogenesis of these diseases.

Virologic investigation of a case of suspected haemorrhagic fever

After travelling in subSaharan Africa, an area known for sporadic cases of Marburg virus infection, a young Swedish man presented with a classical picture of severe viral haemorrhagic fever complicated by disseminated intravascular coagulation and septicaemia. Serum samples examined by electron microscopy revealed particles of a size compatible with filovirions. Indirect fluorescent antibody tests indicated transient seroconversion to Marburg virus. In lymphocyte transformation assays of cells isolated from the patient 11 months after the onset of acute disease, Marburg viral antigen was able to stimulate lymphocyte proliferation 3.9-fold; however, exhaustive attempts to isolate virus from acute phase blood cultured in vitro or in vivo from guinea pigs and monkeys failed. Data suggest that this patient may have been infected with a filovirus. This case demonstrates the difficulties that may occur in laboratory diagnosis of viral haemorrhagic fevers.