T-lymphocyte-enriched murine peritoneal exudate cells. II. Genetic control of antigen-induced T-lymphocyte proliferation.

The recent introduction of a reliable, T-lymphocyte proliferation assay, which utilizes thioglycollate-induced, nylon wool column-passed, peritoneal exudate lymphocytes from immune mice (PETLES), allowed us to investigate the genetic control of murine immune responses at the T-lymphocyte level. Examination of the blast cells generated in this population 5 days after stimulation with antigen, revealed that 85% of the cells bore the Thy 1 antigen on their surface, whereas only 5% bore immunoglobulin. Thus, the assay can be considered to measure almost exclusively T-lymphocyte function. This assay was used to examine the T-lymphocyte proliferative responses to seven different antigens: poly(Glu60Ala30Tyr10), poly(Glu58Lys38Tyr4), poly-(Tyr,Glu)-poly-D,L-Ala--poly-Lys, poly-(Phe,Glu)-poly-D,L-Ala--poly-Lys, staphylococcal nuclease, lactate dehydrogenase H4, and the BALB/c IgA myeloma protein, TEPC-15. PETLES from a large number of different inbred mouse strains, including H-2 congenic resistant lines and H-2 recombinants, were studied. The strains could be classified as high responders, low responders, or nonresponders to a particular antigen as judged by the magnitude of the T-lymphocyte proliferative response. In every case but one this classification corresponded to the responder status given the strain based on its ability to mount an in vivo antibody response to the same antigen. For two of the antigens, poly-(Tyr,Glu)-poly-D,L-Ala--poly-Lys and TEPC-15, the immune response genes controlling the T-lymphocyte proliferative response were mapped to the K region or I-A subregion of the major histocompatibility complex, as had previously been shown for the control of the antibody responses to these antigens. This tight linkage of the two phenotypic responses very strongly suggests that the same immune response gene controls the expression of both the proliferative and antibody responses. Since there is essentially no contribution from B lymphocytes in the T-lymphocyte proliferation assay, it seems reasonable to conclude that none of the seven immune response genes studied are expressed solely in B lymphocytes.

B lymphocyte immune response gene phenotype is genetically determined.

We examined the effects of the developmental milieu on the capacity of B cells to undergo immune response gene-controlled, T cell-dependent polyclonal proliferation. Although I-Aq poly(Glu60 Ala30 Tyr10)n (GAT)-nonresponder T cells developing in a responder environment become phenotypic GAT-responders, I-Aq B cells remain unresponsive to GAT, even after maturation in a GAT-responder animal. Conversely, (B10.A x B10.Q)F1 ([GAT responder x GAT nonresponder]F1) T cells developing in a B10.Q GAT nonresponder host fail to respond to GAT, but F1 B cells from the same F1 leads to parent chimeras make excellent proliferative responses in the presence of GAT and responder T cells. Thus, by this assay, B cell immune response gene function is genetically determined and is not affected by the developmental milieu.

T lymphocyte-dependent B lymphocyte proliferative response to antigen. I Genetic restriction of the stimulation of B lymphocyte proliferation.

For the purpose of examining more closely the interaction between T and B lymphocytes, we have developed an in vitro T lymphocyte-dependent B lymphocyte proliferation assay. Proliferation of B lymphocytes in response to antigen was found to depend on the presence of primed T lymphocytes; the B lymphocytes could be derived from nonprimed animals. It appears that these B cells were nonspecifically recruited to proliferate. This nonspecific recruitment, however, was found to be Ir-gene restricted in that B lymphocytes from B10.S mice, which are genetic nonresponders to the polymer Glu60-Ala30-Tyr10 (GAT), could not be stimulated by GAT-primed (responder X nonresponder) F1 T cells. The apparent lack of antigen specificity in the face of Ir gene-restricted T-B interaction may have important implications in our understanding of the recognition unit(s) on T lymphocytes.

Antigen-specific T cell clones restricted to unique F1 major histocompatibility complex determinants. Inhibition of proliferation with monoclonal anti-Ia antibody.

The existence of T cells specific for soluble antigens in association with unique F(1) or recombinant major histocompatibility complex (MHC) gene products was first postulated from studies on the proliferative response of whole T cell populations to the antigen poly(Glu(55)Lys(36)Phe(9))(n) (GLphi). In this paper we use the newly developed technology of T lymphocyte cloning to establish unequivocally the existence of such cells specific for GLphi and to generalize their existence by showing that F(1)- specific cells can be isolated from T cell populations primed to poly(Glu(60)Ala(30)Tyr(10))(n) (GAT) where such clones represent only a minor subpopulation of cells. Gl.4b-primed B10.A(5R) and GAT-primed (B10.A x B10)F(1) lymph node T cells were cloned in soft agar, and the colonies that developed were picked and expanded in liquid culture. The GLphi-specific T cells were then recloned under conditions of high-plating efficiency to ensure that the final colonies originated from single cells. T cells from such rigorously cloned populations responded to stimulation with GILphi but only in the presence of nonimmune, irradiated spleen cells bearing (B10.A x B10)F(1) or the syngeneic B 10.A(5R) recombinant MHC haplotype. Spleen cells from either the B10 or B 10.A parental strains failed to support a proliferative response, even when added together. (B10 x B10.D2)F(1) and (B10 x B10.RIII)F(1) spleen cells also supported a proliferative response but (B10 x B10.Q)F(1) and (B10 X B10.S)F(1) spleen cells did not. These results suggested that the T cell clones were specific for GL[phi} in association with the beta(AE)(b)-alpha(E) (k,d,r,) Ia molecule and that recognition required both gene products to be expressed in the same antigen-presenting cells. Support for this interpretation was obtained from inhibition experiments using the monoclonal antibody Y-17 specific for a determinant on the beta(AE)(b)-alphaE Ia molecule. Y-17 completely inhibited the proliferative response of a GLphi-specific clone but had no effect on the response of either a PPD-specific or GAT-specific clone, both of which required the beta(A)-alpha(A) Ia molecule as their restriction element. No evidence could be found for the involvement of suppressor T cells in this inhibition. We therefore conclude that the phenomenon of F(1)-restricted recognition by proliferating T cells results from the presence of antigen- specific clones that must recognize unique F(1) or recombinant Ia molecules on the surface of antigen-presenting cells in addition to antigen in order to be stimulated.

Trypanosoma cruzi: the immunological induction of macrophage plasminogen activator requires thymus-derived lymphocytes.

In this article we describe methods in which unstimulated mouse peritoneal macrophages were induced to secrete high livels of plasminogen activator under in vitro conditions. The exposure of sensitized peritoneal or spleen cell populations from Trypanosoma cruzi-infected animals to either viable or heat-killed trypanosomes lead to the release of an inducing factor(s). Maximal levels of plasminogen activator secretion are achieved by the incubation of such factors (s) with unstimulated macrophages for 48 h. A significant increase in enzyme secretion was already observed after a 24 h incubation. The production of the inducing factor(s) by sensitized cells was immunologically specific and unrelated antigens did not stimulate the production of the factor(s) by sensitized peritoneal or spleen cell populations. The inducing factor(s) was produced by nylon-wool-fractionated spleen and peritoneal cells which had been depleted of marcrophages. Pretreatment of sensitized spleen cells with anti-theta serum and C abolished the production of the activating factor(s). The active supernatant fluids were able to induce secretion of macrophage plasminogen activator across H-2 barriers. Attempts to induce trypanocidal activity in unstimulated macrophages have not been successful.

Tuberculosis: infection control/exposure control issues for oral healthcare workers.

AIM: The aim is to present the essential elements of an infection control/exposure control plan for the oral healthcare setting with emphasis on tuberculosis (TB). METHODS AND MATERIALS: A comprehensive review of the literature was conducted with special emphasis on TB infection-control issues in the oral healthcare setting. RESULTS: Currently available knowledge related to TB infection-control issues is supported by data derived from well-conducted trials or extensive controlled observations. In the absence of supportive data the information is supported with the best-informed, most authoritative opinion available. CONCLUSION: Essential elements of an effective TB infection-control plan include a three-level hierarchy of administrative, environmental, and respiratory-protection controls. CLINICAL SIGNIFICANCE: Standard precautions provide the fabric for strategies to prevent or reduce the risk of exposure to bloodborne pathogens and other potentially infectious material. However, standard precautions are inadequate to prevent the spread of organisms through droplet nuclei 1-5 micron in diameter and additional measures are necessary to prevent the spread of Mycobacterium tuberculosis. Oral healthcare settings have been identified as outpatient settings in which patients with suspected or confirmed infectious TB disease are expected to be encountered. Therefore, oral healthcare settings must have a written TB infection-control program.

Direct toxic effects of immunopotentiators on monocytic, myelomonocytic, and histiocytic or macrophage tumor cells in culture.

Four murine monocyte, myelomonocyte, and histiocyte or macrophage tumor cell lines adapted to culture were growth inhibited by one or more of the following macrophage-activating substances: Mycobacterium bovis, Bacillus Calmette-Guérin strain, zymosan, lipopolysaccharide, and dextran sulfate, as well as tuberculin purified protein derivative, but not latex beads. Lipopolysaccharide was effective with one line at 4 ng/ml. All four lines actively phagocytosed zymosan and latex beads. In many cases the growth inhibition was apparently immediate but only cytostatic, and cell proliferation resumed upon removal of the drug. Bacillus Calmette-Guérin, live or boiled, was toxic to some of the tumor lines. Synthesis of lysozyme by all the cell lines in the monocyte series and production of granulocyte colony-stimulating factor by the myelomonocytic leukemia were not inhibited during several days of zero growth conditions in the presence of drugs. Since these agents had no direct effect on other hematopoietic tumor types (myeloma, T-lymphoma, mastocytoma) at the same or up to 10(4) higher concentrations, it is proposed that the sensitive tumors retain specific receptors for immunostimulants, either at the cell surface or within the cell in the case of phagocytosable particles. The binding of these agents to physiological receptors leads to stimulation and mitogenesis in normal macrophages and lymphocytes but leads to growth inhibition without affecting differenetiated functions in the corresponding tumor lines.

Safety and immunogenicity of a V3 loop synthetic peptide conjugated to purified protein derivative in HIV-seronegative volunteers.

OBJECTIVES: To develop a peptide-based model for a preventive vaccine for HIV-1 infection. DESIGN: Phase I trial in HIV-1-seronegative volunteers. PARTICIPANTS: Adult healthy subjects HIV-1-antibody-seronegative in an enzyme-linked immunosorbent assay, screened for tuberculin [purified protein derivative (PPD)] reactivity with 2 tuberculin units PPD-administered intradermally. INTERVENTIONS: Submicrogram doses of a PPD conjugate with a peptide of the primary neutralizing domain (PND) of HIV-1MN (PPD-MN-PND) were administered intradermally to tuberculin skin-test-positive and -negative volunteers. RESULTS: Antibodies to the MN-PND were measured after two immunizations in 10 out of 11 PPD skin-test-positive volunteers. After the fourth immunization high-affinity antibodies were detected, which persisted for over 1 year. High titers of MN-PND-specific immunoglobulin (Ig) G and IgA were detected in the serum and saliva of all volunteers tested. Serum antibodies were cross-reactive with PND peptide from some other HIV-1 strains but neutralized only the HIV-1MN prototype. Human leukocyte antigen (HLA)-B7-restricted MN-PND-specific cytotoxic T lymphocytes (CTL) were also detected. CONCLUSIONS: The PPD-MN-PND vaccine at submicrogram doses is safe and immunogenic in PPD skin-test-positive healthy adult volunteers. Long lasting humoral immune responses in the serum and saliva were possibly accompanied by HLA-B7-restricted CTL responses. This is a vaccine prototype that can be rapidly and inexpensively modified to include other peptide epitopes. It is especially suitable for use in a worldwide multibillion Bacillus Calmette-Guérin (BCG)-primed or tuberculosis-exposed population at risk for HIV-1 infection.

Detection of lytic antimycobacterial antibodies by immune lysis of liposomes sensitized to tuberculin.

Two procedures were used in order to incorporate purified protein derivative tuberculin (PPD) from M. tuberculosis, strain H37Rv, into calcein-containing liposomes: formation of multilamellar vesicles (MLV) in a PPD solution or exposure of preformed MLV to this solution. Immune lysis of these PPD-sensitized MLV was studied in the presence of a hyperimmune anti-M. tuberculosis sheep serum using a specific pathogen-free rabbit serum as a source of complement. A 50% release of encapsulated calcein was observed spectrofluorometrically after 30 min and remained unchanged up to 2 h. The release of calcein in the absence of complement or of anti-H37Rv serum or by liposomes which did not contain PPD never exceeded 1-2%. Liposomes formed in PPD solution were more sensitive to anti-H37Rv serum than preformed liposomes exposed to PPD. Trials with human sera from ten tuberculous patients revealed the presence of specific lytic immunoglobulins. In the presence of sera from skin test negative, non-tuberculous subjects, calcein release was significantly lower. This opens the way to a new method for the study of the humoral immunity in tuberculosis.

Development of a mucosal challenge test for leprosy using leprosin A.

There is little information about the mucosal immune response in leprosy. We have developed a nasal provocation test with leprosin A which will be used to investigate mucosal immunity to Mycobacterium leprae. Initial studies were performed with increasing doses of leprosin A (1.0 pg/ml-10 micrograms/ml) to determine the optimal safe dose of leprosin A. Anti-M. leprae IgA antibody and normal IgA concentrations were measured in the saliva of leprosy contacts and controls before and after instillation of leprosin A. Nasal leprosin A was well tolerated up to a concentration of 10 micrograms/ml without side effects. None of the six subjects who had not been exposed to leprosy had salivary IgA against whole M. leprae, whereas IgA was detected from 64 h to 140 h following instillation of leprosin A in all of the leprosy hospital workers and in 15 out of 18 healthy household contacts tested. There was no correlation between serum and salivary anti-M. leprae IgA levels before and after testing. Salivary IgA anti-lipoarabinomannan responses were seen in 12 out of 20 household contacts. Normal salivary IgA concentrations varied from 8 to 240 mg/l. The leprosin A nasal provocation test appears to be a safe method for the investigation of the role of mucosal immunity in the pathogenesis of leprosy.

Selective elimination of the delayed-type hypersensitivity response by extracorporeal thoracic duct filtration.

Calves were sensitized to tuberculin and histoplasmin. The delayed-type hypersensitivity skin response to these antigens was produced and the diameter of induration measured. Repeated skin tests prior to filtration demonstrated that the amount of induration produced by these skin tests was closely reproducible. Histoplasmin or tuberculin-coated columns were then introduced into a closed circuit extracorporeal thoracic duct circulation. A significant (P is less than 0.01) reduction or ablation of the delayed-type hypersensitivity response was obtained to the antigen used to coat the column. In contrast, no significant reduction occurred in the skin test response to the other antigen. Repeated skin tests to both antigens after the cessation of filtration showed a gradual rise toward prefiltration levels in the skin test to the filtered antigen. The results of these experiments indicate that a selective population of T lymphocytes can be removed from an in vivo system. The removal of these cells can selectively reduce a delayed-type hypersensitivity skin test response to a particular antigen. These results are consistent with the hypothesis that a type of in vivo selective immunosuppression can be produced by antigen-coated columns when they are placed in an extracorporeal thoracic duct lymph circulation system.

Suppression of experimental allergic encephalomyelitis by the encephalitogenic peptide, in solution or bound to liposomes.

We have investigated the ability of liposome-bound encephalitogenic peptide to suppress experimental allergic encephalomyelitis (EAE) in the guinea pig. EAE was induced by challenge with the encephalitogenic peptide, residues 113-122 of human myelin basic protein (MBP) in complete Freund's adjuvant. The peptide was acylated with stearic acid in order to anchor it to the lipid bilayer. The liposomal-bound peptide effectively suppressed clinical signs of EAE at relatively low doses, when given subcutaneously or intraperitoneally without incomplete Freund's adjuvant, several days after challenge. In vitro proliferation of lymphocytes from treated, protected animals in response to the peptide was greatly decreased but that to the purified protein derivative of tuberculin antigen was not, indicating an antigen-specific effect. However, histological signs of EAE were not reduced. The free peptide in solution was somewhat less effective when given intraperitoneally but was as or nearly as effective as liposome-bound peptide when given subcutaneously. Binding to liposomes may decrease the rate of clearance or degradation of the peptide when given intraperitoneally.

Liposomal encapsulation augments delayed hypersensitivity reactions to tuberculin proteins.

Encapsulation of purified tuberculoproteins in liposomes augmented their ability to elicit delayed hypersensitivity reactions in BCG-immune rats. The effect was most marked with a low-molecular-weight tuberculopeptide that was relatively poor at eliciting reactions when in free form. These findings indicate that, in addition to the antigenic nature of the material, the physical form of presentation of mycobacterial test antigens can influence their ability to elicit hypersensitivity reactions.

Isolation and partial characterisation of the Triton X-100 solubilised protein antigen from Mycobacterium tuberculosis.

This report describes extraction of a new native antigen fraction from Mycobacterium tuberculosis without massive degradation of proteins by Triton X-100. The Triton X-100 solubilised protein (TSP) antigen showed a characteristic antigen profile and reproducible extraction pattern. To characterise the nature of their composition, the TSP antigen was fractionated by Triton X-114 phase partitioning. The TSP antigen contained a variety of lipids and glycoconjugates as well as diverse proteins. Most proteins were partitioned into the aqueous phase during phase fractionation, whereas non-protein molecules and lipoproteins were recovered in the detergent phase. The lymphoproliferative responses to the TSP aqueous fraction in healthy tuberculin reactors were significantly higher than those to the purified protein derivative (PPD) and unfractionated TSP. In contrast, the antibody responses to TSP aqueous fraction in tuberculosis patients showed weak reactivity. This study suggests that the TSP aqueous fraction can be used as a T-cell antigen associated with protective immunity against tuberculosis.

Depressed responsiveness of peripheral blood mononuclear cells to heat-shock proteins in periodontitis patients.

The extensive homology between human and bacterial heat shock proteins (HSPs) may play a role in autoimmune reactions in periodontitis. Thus, we questioned whether peripheral blood mononuclear cell (PBMC) proliferative responses to HSPs are different between periodontitis patients and control subjects with gingivitis. The proliferative responses of PBMCs of patients (n = 10) and controls (n = 12) to recombinant mycobacterial HSP60 (MycHSP60) and HSP70 (MycHSP70), as well as recombinant human HSP60 (HumHSP60) and HSP70 (HumHSP70), were investigated. In addition, the proliferative responses to Candida albicans and purified protein derivatives of Mycobacterium (PPD) were included. Mean responses to HumHSP60, MycHSP60, and HumHSP70 were significantly lower for patients compared with controls. The responses to MycHSP70 showed a similar trend. However, when Candida and PPD were used as antigens, there was no difference in responses of the PBMCs between the periodontitis patients and controls. The level of IFN-gamma in the supernatants of the cells stimulated with HSPs was lower in the patients compared with controls. This concurs with the current hypothesis that periodontitis patients have a depressed Th1 response. Furthermore, we found that with an increasing estimated subgingival bacterial load, periodontitis patients mount a decreasing immune response to HSPs, while the controls showed a positive correlation between these two parameters. From these findings, we speculate that poor reactivity to HSPs may be a susceptibility factor for destructive periodontal disease and may need to be considered in the pathogenesis of this condition.

The stimulation of human peripheral blood lymphocytes by solubilized dental plaque: macrophage and T-cell dependence.

This study compared the ability of human peripheral blood lymphocyte subpopulations from individuals with moderate periodontitis to proliferate in response to stimulation with solubilized dental plaque, phytohemagglutinin (PHA), pokeweed mitogen (PWM) and purified protein derivative (PPD). Unseparated mononuclear lymphocytes and recombined purified T and B lymphocytes (4/1) responded significantly to all of the stimulants. DNA synthesis in response to solubilized dental plaque was maximal after 6 to 7 days of culture; the optimum dose was usually 10 to 20 micrograms/ml. T lymphocyte subpopulations purified by rosetting with sheep red blood cells and density gradient centrifugation responded well to PHA and PWM, but were unresponsive to solubilized dental plaque and PPD unless supplemented with 2% autologous macrophages, demonstrating that T cell responses to solubilized dental plaque and to PPD are macrophage-dependent. T cell-depleted enriched B lymphocyte subpopulations were poorly responsive to all of the tested stimulants; however, the responses of these cells to solubilized dental plaque and to the known T cell-dependent B cell mitogens PWM and PPD were increased significantly by the presence of 10% mitomycin C-treated T cells, demonstrating that B cell proliferation to solubilized dental plaque is T cell-dependent. Thus, cellular interactions between macrophages, T lymphocytes and B lymphocytes are required to obtain optimal proliferative lymphocyte responses to solubilized dental plaque. Since both T and B lymphocytes respond to dental plaque stimulants, they both have the potential to mediate periodontal inflammation and tissue destruction whenever dental plaque stimulants gain entrance into the periodontal tissues.

Mercury-reactive lymphocytes in peripheral blood are not a marker for dental amalgam associated disease.

OBJECTIVES: The popular press and publications associated with alternative medicine increasingly report that chronic ill health, particularly myalgic encephalitis like conditions, are associated with mercury amalgam fillings. There are no scientifically proven definitive tests to support these claims. One of the more scientific tests in vogue is to assess the level of blood-borne mercury-reactive lymphocytes and to conclude that patients with high levels have developed a hypersensitivity reaction to mercury. The objective of this study was to determine the diagnostic value of this test. METHODS: This study represents an open comparison of mercury-reactive lymphocyte levels in healthy control individuals with those in patients complaining of symptoms associated with adverse effects of dental metal amalgam fillings. The healthy control group consisted of 51 male and female individuals, aged between 12 and 82 years, with and without dental amalgam fillings. The patient group consisted of 70 male and female individuals, aged between 12 and 87 years, and with the exception of one patient, with three or more mercury amalgam fillings of more than 1 year's duration. In vitro lymphocyte responses to mercury, and to nickel, as an example of a metal commonly associated with hypersensitivity reactions, and to more conventional protein antigens were determined. RESULTS: In the blood of patients and controls, there were similar levels of specifically reactive lymphocytes to all of the in vitro stimulating agents, but there were significantly higher numbers of sub-normal and non-responders within the patient group. CONCLUSIONS: The incidence and quantity of mercury-reactive lymphocytes in the blood are not pathogenic markers of illness associated with dental metal amalgams, but may rather reflect exposure to mercury. The clinical relevance of the decreased in vitro lymphocyte responses in the patient group needs further investigation.

[Tuberculous infection in nursing students: prevalence and conversion during a 3-year follow-up]. / Infección tuberculosa en estudiantes de enfermería. Prevalencia y virajes durante 3 años de seguimiento.

BACKGROUND: The aim of this study was to ascertain the positive tuberculin prevalence among the nursing students at the beginning of their studies; to assess the annual tuberculin conversion rate during their studies; to obviate the possibility of false conversions, studying the booster effect. PATIENTS AND METHODS: Cohort study, prospective, 3 groups not parallels (n = 316), 32 months follow-up in all students. Mean age 21 years (SD = 4; range = 17-39). First phase: before beginning clinical practice, a tuberculin test was undertaken by Mantoux technique with PPD type RT-23 with Tween 80 of 2 TU; this was repeated after 7-10 days, to BCG vaccinated and PPD negative on the first one, to study the booster effect. Second phase: at 18 months we repeated the tuberculin test to PPD negatives including the vaccinates with booster effect but PPD negative. The end of the study was at 32 months, repeating the test to PPD negatives at the end of their nursing studies. RESULTS: Tuberculin prevalence of 12% (38/316); CI 95%: 8.4%-15.6%. There were no significant differences of prevalence between vaccinated and unvaccinated nurse students. The prevalence in women was 8.9% (24/267) and 28.6% (14/49) in men. The only variables with statistical significance for being tuberculin positive were, age (p = 0.002) and sex (p = 0.003). 3/42 vaccinated with BCG (7%) had initial PPD (+); 2/39 of the remaining (5%) showed to be tuberculin positive after the booster effect and 37 tuberculin negative and booster negative. In the conversion study there were 259 valid students at the end of 3 years; there were 14 (5.4%) converters in the second year and 9 (3.5%) in the third year. Tuberculin conversion annual rate was 3.8 per 100 people/year. CONCLUSIONS: The tuberculous infection prevalence in nursing students was 12% (38/316). The annual frequency of tuberculin conversion (3.8 per 100 people/year) was higher in our students than in the general population, reinforcing the suitability of making periodical tuberculin control tests in PPD negative student nurses with continuous hospital contact.