The association of dietary animal and plant protein with putative risk markers of colorectal cancer in overweight pre-diabetic individuals during a weight-reducing programme: a PREVIEW sub-study.

PURPOSE: Diets with increased protein content are popular strategies for body weight regulation, but the effect of such diets for the colonic luminal environment is unclear. We aimed to investigate the associations between putative colorectal cancer-related markers and total protein intake, plant and animal proteins, and protein from red and processed meat in pre-diabetic adults (> 25 years). METHODS: Analyses were based on clinical and dietary assessments at baseline and after 1 year of intervention. Protein intake was assessed from 4-day dietary records. Putative colorectal cancer-related markers identified from 24-h faecal samples collected over three consecutive days were: concentration of short-chain fatty acids, phenols, ammonia, and pH. RESULTS: In total, 79 participants were included in the analyses. We found a positive association between change in total protein intake (slope: 74.72 ± 28.84 µmol per g faeces/E%, p = 0.01), including animal protein intake (slope: 87.63 ± 32.04 µmol per g faeces/E%, p = 0.009), and change in faecal ammonia concentration. For change in ammonia, there was a dose-response trend from the most negative (lowest tertile) to the most positive (highest tertile) association (p = 0.01): in the high tertile, a change in intake of red meat was positively associated with an increase in ammonia excretion (slope: 2.0 ± 0.5 µmol per g faeces/g/day, p < 0.001), whereas no such association was found in the low and medium tertile groups. CONCLUSION: Increases in total and animal protein intakes were associated with higher excretion of ammonia in faeces after 1 year in overweight pre-diabetic adults undertaking a weight-loss intervention. An increase in total or relative protein intake, or in the ratio of animal to plant protein, was not associated with an increase in faeces of any of the other putative colorectal cancer risk markers. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01777893.

Persistence, adherence, and medication-taking behavior in women with postmenopausal osteoporosis receiving denosumab in routine practice in Germany, Austria, Greece, and Belgium: 12-month results from a European non-interventional study.

UNLABELLED: Persistence with and adherence to osteoporosis therapy are critical for fracture reduction. This non-interventional study is evaluating medication-taking behavior of women with postmenopausal osteoporosis (PMO) receiving denosumab in Germany, Austria, Greece, and Belgium. Patients were representative of the PMO population and highly persistent with and adherent to denosumab at 12 months. INTRODUCTION: Persistence with and adherence to osteoporosis therapy are important for optimal treatment efficacy, namely fracture reduction. This ongoing, non-interventional study will evaluate medication-taking behavior of women with postmenopausal osteoporosis (PMO) receiving denosumab in routine practice in four European countries. METHODS: The study enrolled women who had been prescribed subcutaneous denosumab (60 mg every 6 months) in accordance with prescribing information and local guidelines. Persistence was defined as receiving the subsequent injection within 6 months + 8 weeks of the previous injection. Adherence was defined as receiving two consecutive injections within 6 months ± 4 weeks of each other. Medication coverage ratio (MCR) was calculated using the time a patient was covered with denosumab, as assessed from prescription records. Treatment was assigned prior to and independently of enrollment; outcomes are recorded during routine practice. RESULTS: These planned 12-month interim analyses included data from 1500 patients from 141 sites. Mean age was 66.4-72.4 years, mean baseline total hip T-scores ranged from -2.0 to -2.1 and femoral neck T-scores from -2.2 to -2.6, and 30.7-62.1% of patients had prior osteoporotic fracture. Persistence was 87.0-95.3%, adherence 82.7-89.3%, and MCR 91.3-95.4%. In a univariate analysis, increased age, decreased mobility, and increased distance to the clinic were associated with significantly decreased persistence; parental history of hip fracture was associated with significantly increased persistence. CONCLUSIONS: These data extend the real-world evidence regarding persistence with and adherence to denosumab, both of which are critical for favorable clinical outcomes, including fracture risk reduction.

CD24(hi)CD27(+) B cells from patients with allergic asthma have impaired regulatory activity in response to lipopolysaccharide.

BACKGROUND: Regulatory B cells have been identified that strongly reduce allergic and auto-immune inflammation in experimental models by producing IL-10. Recently, several human regulatory B-cell subsets with an impaired function in auto-immunity have been described, but there is no information on regulatory B cells in allergic asthma. OBJECTIVE: In this study, the frequency and function of IL-10 producing B-cell subsets in allergic asthma were investigated. METHODS: Isolated peripheral blood B cells from 13 patients with allergic asthma and matched healthy controls were analyzed for the expression of different regulatory B-cell markers. Next, the B cells were activated by lipopolysaccharide (LPS), CpG or through the B-cell receptor, followed by co-culture with endogenous memory CD4(+) T cells and house dust mite allergen DerP1. RESULTS: Lower number of IL-10 producing B cells were found in patients in response to LPS, however, this was not the case when B cells were activated through the B-cell receptor or by CpG. Further dissection showed that only the CD24(hi)CD27(+) B-cell subset was reduced in number and IL-10 production to LPS. In response to DerP1, CD4(+) T cells from patients co-cultured with LPS-primed total B cells produced less IL-10 compared to similar cultures from controls. These results are in line with the finding that sorted CD24(hi)CD27(+) B cells are responsible for the induction of IL-10(+) CD4(+) T cells. CONCLUSIONS: Taken together, these data indicate that CD24(hi)CD27(+) B cells from allergic asthma patients produce less IL-10 in response to LPS leading to a weaker IL-10 induction in T cells in response to DerP1, which may play a role in allergic asthma.

A common atopy-associated variant in the Th2 cytokine locus control region impacts transcriptional regulation and alters SMAD3 and SP1 binding.

BACKGROUND: Type 2 immune responses directed by Th2 cells and characterized by the signature cytokines IL4, IL5, and IL13 play major pathogenic roles in atopic diseases. Single nucleotide polymorphisms in the human Th2 cytokine locus in particular in a locus control region within the DNA repair gene RAD50, containing several RAD50 DNase1-hypersensitive sites (RHS), have been robustly associated with atopic traits in genome-wide association studies (GWAS). Functional variants in IL13 have been intensely studied, whereas no causative variants for the IL13-independent RAD50 signal have been identified yet. This study aimed to characterize the functional impact of the atopy-associated polymorphism rs2240032 located in the human RHS7 on cis-regulatory activity and differential binding of transcription factors. METHODS: Differential transcription factor binding was analyzed by electrophoretic mobility shift assays (EMSAs) with Jurkat T-cell nuclear extracts. Identification of differentially binding factors was performed using mass spectrometry (LC-MS/MS). Reporter vector constructs carrying either the major or minor allele of rs2240032 were tested for regulating transcriptional activity in Jurkat and HeLa cells. RESULTS: The variant rs2240032 impacts transcriptional activity and allele-specific binding of SMAD3, SP1, and additional putative protein complex partners. We further demonstrate that rs2240032 is located in an RHS7 subunit which itself encompasses repressor activity and might be important for the fine-tuning of transcription regulation within this region. CONCLUSION: The human RHS7 critically contributes to the regulation of gene transcription, and the common atopy-associated polymorphism rs2240032 impacts transcriptional activity and transcription factor binding.

Identification of biomarkers for apoptosis in cancer cell lines using metabolomics: tools for individualized medicine.

BACKGROUND: Metabolomics is a versatile unbiased method to search for biomarkers of human disease. In particular, one approach in cancer therapy is to promote apoptosis in tumour cells; this could be improved with specific biomarkers of apoptosis for monitoring treatment. We recently observed specific metabolic patterns in apoptotic cell lines; however, in that study, apoptosis was only induced with one pro-apoptotic agent, staurosporine. OBJECTIVE: The aim of this study was to find novel biomarkers of apoptosis by verifying our previous findings using two further pro-apoptotic agents, 5-fluorouracil and etoposide, that are commonly used in anticancer treatment. METHODS: Metabolic parameters were assessed in HepG2 and HEK293 cells using the newborn screening assay adapted for cell culture approaches, quantifying the levels of amino acids and acylcarnitines with mass spectrometry. RESULTS: We were able to identify apoptosis-specific changes in the metabolite profile. Moreover, the amino acids alanine and glutamate were both significantly up-regulated in apoptotic HepG2 and HEK293 cells irrespective of the apoptosis inducer. CONCLUSION: Our observations clearly indicate the potential of metabolomics in detecting metabolic biomarkers applicable in theranostics and for monitoring drug efficacy.

Discovery of phosphatidylcholines and sphingomyelins as biomarkers for ovarian endometriosis.

BACKGROUND: Current non-invasive diagnostic methods for endometriosis lack sensitivity and specificity. In search for new diagnostic biomarkers for ovarian endometriosis, we used a hypothesis-generating targeted metabolomics approach. METHODS: In a case-control study, we collected plasma of study participants and analysed their metabolic profiles. We selected a group of 40 patients with ovarian endometriosis who underwent laparoscopic surgery and a control group of 52 healthy women who underwent sterilization at the University Clinical Centre Ljubljana, Slovenia. Over 140 targeted analytes included glycerophospholipids, sphingolipids and acylcarnitines. The analytes were quantified by electrospray ionization tandem mass spectrometry. For assessing the strength of association between the metabolite or metabolite ratios and the disease, we used crude and adjusted odds ratios. A stepwise logistic regression procedure was used for selecting the best combination of biomarkers. RESULTS: Eight lipid metabolites were identified as endometriosis-associated biomarkers due to elevated levels in patients compared with controls. A model containing hydroxysphingomyelin SMOH C16:1 and the ratio between phosphatidylcholine PCaa C36:2 to ether-phospholipid PCae C34:2, adjusted for the effect of age and the BMI, resulted in a sensitivity of 90.0%, a specificity of 84.3% and a ratio of the positive likelihood ratio to the negative likelihood ratio of 48.3. CONCLUSIONS: Our results suggest that endometriosis is associated with elevated levels of sphingomyelins and phosphatidylcholines, which might contribute to the suppression of apoptosis and affect lipid-associated signalling pathways. Our findings suggest novel potential routes for therapy by specifically blocking highly up-regulated isoforms of phosphpolipase A2 and lysophosphatidylcholine acyltransferase 4.

Correlated phases of bosons in the flat lowest band of the dice lattice.

We study correlated phases occurring in the flat lowest band of the dice-lattice model at flux density one-half. We discuss how to realize this model, also referred to as the T(3) lattice, in cold atomic gases. We construct the projection of the model to the lowest dice band, which yields a Hubbard Hamiltonian with interaction-assisted hopping processes. We solve this model for bosons in two limits. In the limit of large density, we use Gross-Pitaevskii mean-field theory to reveal time-reversal symmetry breaking vortex lattice phases. At low density, we use exact diagonalization to identify three stable phases at fractional filling factors ν of the lowest band, including a classical crystal at ν = 1/3, a supersolid state at ν = 1/2, and a Mott insulator at ν = 1.

Fractional quantum Hall effect of lattice bosons near commensurate flux.

We study interacting bosons on a lattice in a magnetic field. When the number of flux quanta per plaquette is close to a rational fraction, the low-energy physics is mapped to a multispecies continuum model: bosons in the lowest Landau level where each boson is given an internal degree of freedom, or pseudospin. We find that the interaction potential between the bosons involves terms that do not conserve pseudospin, corresponding to umklapp processes, which in some cases can also be seen as BCS-type pairing terms. We argue that in experimentally realistic regimes for bosonic atoms in optical lattices with synthetic magnetic fields, these terms are crucial for determining the nature of allowed ground states. In particular, we show numerically that certain paired wave functions related to the Moore-Read Pfaffian state are stabilized by these terms, whereas certain other wave functions can be destabilized when umklapp processes become strong.

An association between chronic obstructive pulmonary disease and abdominal aortic aneurysm beyond smoking: results from a case-control study.

OBJECTIVES: It is currently unclear whether the parallels between abdominal aortic aneurysms (AAAs) and chronic obstructive pulmonary disease (COPD) are explained by common risk factors alone, such as cigarette smoking, or by a predetermined cause. Given the persistent controversy with regard to the association between AAA and COPD, we studied this association in depth. METHODS: We conducted a case-control study comparing patients with a small AAA (maximum infrarenal diameter 35-50 mm, n = 221) with controls diagnosed with peripheral artery disease (PAD, n = 87). The controls were matched to the cases for lifetime cigarette smoking. Pulmonary function was measured by spirometry, and all subjects completed a questionnaire on medical history and smoking habits (current, former and never smokers). RESULTS: Aneurysm patients were similar to controls with respect to gender (p = 0.71), lifetime cigarette smoking (39 vs. 34 pack years, p = 0.23) and history of cardiovascular disease (45% vs. 55%, p = 0.12). Aneurysm patients had more airway obstruction (forced expiratory volume in 1 s/forced vital capacity (FEV1/FVC) (0.69 ± 0.12 vs. 0.78 ± 0.11, p < 0.001)), which was most pronounced in never smokers (0.73 ± 0.07 vs. 0.86 ± 0.07, p < 0.001). COPD was more prevalent in aneurysm patients (44%; 98/221) than in controls (20%; 17/87) (adjusted odds ratio (OR) 3.0; 95% confidence interval (95%CI) 1.6-5.5, p < 0.001). In particular, a major proportion of AAA patients was newly diagnosed with COPD; only 40 of 98 patients (41%) with COPD (mild, moderate or severe/very severe) were known before with obstructive pulmonary defects and received treatment. CONCLUSIONS: This study confirms an association between AAA and COPD and shows that this association is independent from smoking. Findings also demonstrate that COPD is under-diagnosed in AAA patients.

Prevalence of osteoporosis and fractures among women prescribed osteoporosis medication in five European countries: the POSSIBLE EU study.

UNLABELLED: European observational 1-year study assessed osteoporosis and fracture patterns in 3,402 postmenopausal women prescribed osteoporosis medication. Almost 40% of patients had a previous fracture, while 25% had neither fracture nor dual energy X-ray absorptiometry (DXA) diagnosis and were prescribed medication, probably due to other risk factors. INTRODUCTION: This analysis assessed osteoporosis and fracture prevalence in postmenopausal women prescribed osteoporosis treatment in the Prospective Observational Study Investigating Bone Loss Experience in Europe(POSSIBLE EU). METHODS: Women in this observational, multicenter 1-year study were categorized by fracture history and location at baseline. Baseline characteristics were analyzed according to no DXA and DXA diagnosis (osteoporosis or osteopenia). Fractures occurring during the 1-year follow-up period were recorded. RESULTS: Of the 3,402 women enrolled, 39% had a previous fracture, of whom 30% had ≥ 2 fractures. One thousand seven hundred and eighty-four (52%) patients had a DXA diagnosis (osteoporosis 68%, osteopenia 31%, and unknown 1%). Among the osteoporosis patients, 37% had a previous fracture (hip 2.9%, vertebral 8.8%, and non-hip, non-vertebral 25%) and 35% had fractures associated with major trauma. Of the 3,402 women, 1,476 (43%) had no DXA diagnosis; of these, 57% had no fracture (25% of all women). Risk factors varied across patients with and without DXA diagnosis. During the 1-year follow-up period, the fracture incidence in patients with or without a previous fracture at baseline was 4.7% and 1.6%, respectively. CONCLUSION: Almost 40% of patients prescribed osteoporosis medication had a previous fracture, highlighting a population with advanced disease. In contrast, 25% of patients had neither a previous fracture nor DXA diagnosis and were prescribed treatment, probably due to other risk factors. There is a need for continued improvement of disease management in European women.

Machine learning approach to muon spectroscopy analysis.

In recent years, artificial intelligence techniques have proved to be very successful when applied to problems in physical sciences. Here we apply an unsupervised machine learning (ML) algorithm called principal component analysis (PCA) as a tool to analyse the data from muon spectroscopy experiments. Specifically, we apply the ML technique to detect phase transitions in various materials. The measured quantity in muon spectroscopy is an asymmetry function, which may hold information about the distribution of the intrinsic magnetic field in combination with the dynamics of the sample. Sharp changes of shape of asymmetry functions-measured at different temperatures-might indicate a phase transition. Existing methods of processing the muon spectroscopy data are based on regression analysis, but choosing the right fitting function requires knowledge about the underlying physics of the probed material. Conversely, PCA focuses on small differences in the asymmetry curves and works without any prior assumptions about the studied samples. We discovered that the PCA method works well in detecting phase transitions in muon spectroscopy experiments and can serve as an alternative to current analysis, especially if the physics of the studied material are not entirely known. Additionally, we found out that our ML technique seems to work best with large numbers of measurements, regardless of whether the algorithm takes data only for a single material or whether the analysis is performed simultaneously for many materials with different physical properties.

Regulation of cellular antibody synthesis. Cellular 7S production and longevity of 7S antigen-sensitive cells in the absence of antibody feedback.

Transfer of spleen cells from mice immunized against sheep red blood cells (SRBC) into irradiated (600 R) nonimmune, syngeneic mice in the presence of antigen resulted in excessive cellular 7S production 7 days later. The number of 7S plaque-forming cells usually exceeded 10(6) per spleen and the mean proportion varied between 1 and 70%. In occasional animals all spleen cells were producing antibodies to SRBC. Serum antibody synthesis was also excessively increased, the titers in agglutination after 2-ME treatment and in hemolysis varying between 2(15) and 2(25). The generation time of the 7S PFC was found to be 9.6 hr in the secondary hosts. It seemed possible that the excessive production of 7S PFC and antibodies in the irradiated nonimmune recipients was caused by the absence of feedback inhibition of the immune response by antibody, a mechanism which would normally function to restrict antibody synthesis. This conclusion was strengthened by the demonstration that transfer of antigen-stimulated immune cells into actively or passively immunized irradiated recipients resulted in a marked suppression of cellular 7S synthesis. Serial transfers of antigen-stimulated immune cell populations in irradiated hosts resulted in an equally high number of 7S PFC during the first four transfer generations. However, after the fifth to seventh transfer generation the number of 7S PFC rapidly declined and disappeared within one to three passages. Serum antibodies and 7S PFC declined in parallel during the last transfer generations. Further passages of antigen-stimulated spleen cells lacking 7S PFC did not lead to reappearance of PFC. Thus, antigen-sensitive cells have a limited lifespan and/or multiplication capacity. From the hypothesis that the 7S PFC developed by division from antigen-sensitive precursors it was calculated that 38-40 divisions occurred, Thus, one antigen-sensitive precursor has the potential to give rise to 10(12) 7S PFC.

Effect of B-cell mitogens on lymphocyte subpopulations possesing C'3 and Fc receptors.

By the use of a rosette method allowing the detection at the cellular level of lymphocytes simultaneously binding Fc- and C'3-sensitized red cells it was found that about 70% of the rosette-forming cells from spleens of nude and normal mice possessed receptors for both Fc and C'3, whereas 30% only had Fc receptors. Very few, if any, lymphocytes possessed only C'3 receptors. The B-cell mitogens, purified-protein derivative of tuberculin (PPD), lipopolysaccharide from Escherichia coli (LPS), and pneumococcal polysaccharide type SIII (SIII), induced marked changes of these receptor-bearing lymphocytes. PPD caused a rapid loss of cells capable of binding C'3 and a concomitant increase of only Fc-binding cells, which was detected after only 24 h. LPS and SIII induced analogous changes, but they were not detected until 48 h and were not complete until after 72 h. It is suggested that immature lymphocytes possess both Fc and C'3 receptors and lose the latter receptor upon differentiation induced by B-cell mitogens PPD. and LPS would affect different populations of B cells, PPD-activating cells that have already reached a higher differentiation stage, whereas LPS and SIII would activate more immature B cells.

Immunological unresponsiveness to native dextran B512 in young animals of dextran high responder strains is due to lack of Ig receptors expression. Evidence for a nonrandom expression of V-genes.

Young mice of dextran high responder strains were found to be complete nonresponders to the alpha-1-6 epitope of dextran during 30-40 days after birth. They also failed to respond to thymus-dependent dextran-protein conjugates. Cells from young and adult mice were activated equally well to polyclonal antibody synthesis by the polyclonal B-cell-activating property of dextran. There was no age difference in the immune response to haptens conjugated to dextran, indicating that dextran can function as an efficient carrier also in young mice. Unresponsiveness could not be attributed to suppressor T cells or to a suppressive environment in young animals, as shown by transfer experiments, in which living or irradiated cells from young and adult mice were admixed in various ways and transferred to irradiated recipients of different ages. Cells from young mice did not affect response of adult cells (and the reverse), nor did the age of the irradiated recipient influence the response. When lymphocytes from young and adult mice were polyclonally activated in vitro by lipopolysaccharide, only cells from young mice failed to synthesize antibodies against the alpha-1-6 epitope of dextran, although they produced antibodies of all other specificities tested for. It was concluded that young animals fail to express immunoglobulins directed against the alpha-1-6 epitope during the first 30-40 days after birth. Since the mice possess the VH gene coding for antibodies against this particular epitope, it was concluded that the timing of V gene expression is regulated during development, possibly at the V-C gene translocation level.

Role of C'3 and Fc receptors in B-lymphocyte activation.

Attempts were made to identify the non-Ig lymphocyte receptor responsible for B-cell induction by antigen and polyclonal B-cell activators (PBA). As a first step, the role of C'3 and Fc receptors was analyzed. It was shown that complement could be fixed onto B cells to such an extent that the lymphocytes could not bind complement-coated red cells, but this did not result in induction of polyclonal antibody synthesis, nor did it inhibit the lymphocytes response to PBA. However, the C'3 receptros possessed a passive focussing role in the induction of polyclonal antibody responses. Thus, PBA that had fixed complement activated polyclonal responses at lower concentrations than the same substances that had not fixed complement. Most likely the dual binding of PBA molecules to B cells by the PBA and the C'3 receptors caused more PBA molecules to be bound to each cell. However, the focussing function of the C'3 receptors was several orders of magnitude smaller than that of the Ig receptors. Analogous studies were carried out with Fc receptors. Binding of different types of antigen-antibody complexes did not cause activation of polyclonal or specific antibody synthesis, nor did it significantly interfere with induction of antibody synthesis by PBA substances.

Mechanism of unresponsiveness to the alpha 1-6 epitope of dextran B512 in a C57BL substrain.

C57BL/10ScCr mice are low responders to the alpha 1-6 epitope of dextran B512, although other C57BL mice are high responders. Both thymus-independent and thymus-dependent forms of dextran failed to induce an immune response in C57BL/10ScCr mice, but dextran functioned as a good carrier for antihapten responses in this strain. Dextran is a potent polyclonal B cell activator for cells from C57BL/10ScCr mice, although such cells are not activated by LPS. The C57BL/10ScCr mice possess the Igh-V gene coding for antibodies against dextran and the antidextran antibodies induced in (A X C57BL/10ScCr)F1 hybrids share an idiotype with antidextran antibodies produced in C57BL/10 mice. Bone marrow cells from C57BL/10ScCr mice do not respond to dextran when transferred into lethally irradiated C57BL/10 mice and C57BL/10 cells transferred into C57BL/10ScCr mice give a strong antidextran response. Thus, B cells having both the Igh-V gene coding for antibodies against dextran and activation receptors for dextran cannot be activated into antibody synthesis against any form of this immunogen. This determinant specific immunodeficiency suggests the existence of as yet unknown regulatory influences on Igh-V gene expression or B cell activation.

T cell growth factor abrogates concanavalin A-induced suppressor cell function.

Concanavalin A (Con-A)-induced suppressor T cells were found to respond to T cell growth factor (TCGF) by proliferation. TCGF abrogated the suppressor activity exerted by these cells on phytohemagglutinin (PHA)- and alloantigen- induced lymphocyte proliferation and on pokeweed mitogen (PWM)-driven immunoglobulin secretion. The Con-A-activated suppressor T cells absorbed the TCGF activity, preincubation of these active suppressor cells with TCGF abolished their suppressor activity and addition of increasing numbers of Con-A-activated T cells reverted the abrogator,/ effect of TCGF. Altogether, these findings suggest that Con-A-induced suppressor T cells exert their function by decreasing the available levels of TCGF. Cyclosporin-A (CYA), which is known to inhibit the expression of receptors for TCGF on T cells, also inhibited the suppressor activity as determined in both indicator systems, namely PHA- or alloantigen-induced DNA synthesis and PWM-induced immunoglobulin synthesis. CYA made Con-A-treated T cells unresponsive to TCGF and unable to absorb the growth factor, supporting the notion that CYA inhibits the expression of TCGF receptors on T cells, a mechanism by which this drug seems to abrogate Con-A-induced suppressor T cell function.

Generation of cytotoxic lymphocytes in mixed lymphocyte reactions. I. Specificity of the effector cells.

Generation of cytotoxic effector cells by a unidirectional mixed lymphocyte reaction (MLR) in the mouse H-2 system was studied using labeled YAC (H-2(a)) leukemia cells as targets. The responding effector cell displayed a specific cytotoxic effect against target cells of the same H-2 genotype as the stimulating cell population. Killing of syngeneic H-2 cells was not observed, even when the labeled target cells were "innocent bystanders" in cultures where specific target cells were reintroduced. Similar results were found with spleen cells taken from mice sensitized in vivo 7 days earlier. The effector cell was not an adherent cell and was not activated by supernatants from MLR. The supernatants were not cytotoxic by themselves. When concanavalin A or phytohemagglutinin was added to the cytotoxic test system, target and effector cells were agglutinated. Under these conditions, killing of H-2(a) target cells was observed in mixed cultures where H-2(a) lymphocytes were also the effector cells. These findings indicate that specifically activated, probably thymus-derived lymphocytes, can kill nonspecifically once they have been activated and providing there is close contact between effector and target cells. Thus, specificity of T cell killing appears to be restricted to recognition and subsequent binding to the targets, the actual effector phase being nonspecific.

Immunological unresponsiveness to thymus-independent antigens: two fundamentally different genetic mechanisms of B-cell unresponsiveness to dextran.

The immune response of mice to the alpha-l-6 epitope of dextran (Dx) B512 was found to be under genetic control. The congenic mouse strains A, A.CA, A.SW, A.TH, and A.TL exhibited a specific defect in their response to alpha-l-6. Also strain CBA/N was unresponsive to alpha-1-6, but the mechanism of unresponsiveness was found to be different. Unresponsiveness to alpha-l-6 in congenic A strains was not due to suppressor cells. Although these strains failed to respond to the alpha-l-6 epitope, they responded strongly to the hapten Fluorescein isothiocyanate (FITC) conjugated to Dx, indicating that the Dx can function as an efficient carrier in these strains. Dx was a potent polyclonal B-cell activator in congenic A strains as well as in high responder strains. Polyclonally-activating concentrations of lipopolysaccharide (LPS) failed to induce the synthesis of anti-alpha- l-6 antibodies in congenic A strains, although antibodies of all other specificities studied were produced. However, in high responder strains, LPS induced the synthesis of anti-alpha-l-6 antibodies. It was concluded that congenic A strains do not express V genes coding for antibodies against alpha-l-6. In contrast, strain CBA/N failed to respond to both the alpha-l-6 and FITC epitope on Dx, whereas they could respond to FITC conjugated to horse erythrocytes. Dx induced a very small, if any, polyclonal antibody response in B cells from CBA/N mice or male CBA/N x DBA hybrids, whereas Dx was a very potent polyclonal B-cell activator in female hybrids. It is concluded that CBA/N mice are nonresponders to Dx or haptenated Dx, because the cell population that can respond to the polyclonal B-cell activating properties of Dx is severely depleted.

Cytotoxic T-cell activation by polyribonucleotides: DNA synthesis is not required.

We have shown that cytotoxic T cells can be polyclonally activated by a short exposure to complexes of polyadenylic:polyuridylic acid (poly A:U). Activation is optimal at a dose of 100 microgram/ml poly A:U and occurs during a 2 h incubation period in the absence of antigen. Killing of allogeneic, but not syngeneic, target cells can be observed after 12 h in culture and peaks after 21-24 h in the absence of any nonspecific binding ligand. The observed cytotoxicity is mediated by T lymphocytes and dose not require accessory macrophages or DNA synthesis for the activation or expression of effector functions. These results suggest that few requirements exist for the activation of cytotoxic T cells.