Guidelines for the genetic diagnosis of hereditary recurrent fevers.

Hereditary recurrent fevers (HRFs) are a group of monogenic autoinflammatory diseases characterised by recurrent bouts of fever and serosal inflammation that are caused by pathogenic variants in genes important for the regulation of innate immunity. Discovery of the molecular defects responsible for these diseases has initiated genetic diagnostics in many countries around the world, including the Middle East, Europe, USA, Japan and Australia. However, diverse testing methods and reporting practices are employed and there is a clear need for consensus guidelines for HRF genetic testing. Draft guidelines were prepared based on current practice deduced from previous HRF external quality assurance schemes and data from the literature. The draft document was disseminated through the European Molecular Genetics Quality Network for broader consultation and amendment. A workshop was held in Bruges (Belgium) on 18 and 19 September 2011 to ratify the draft and obtain a final consensus document. An agreed set of best practice guidelines was proposed for genetic diagnostic testing of HRFs, for reporting the genetic results and for defining their clinical significance.

Prognostic value of genomic alterations in minimal residual cancer cells purified from the blood of breast cancer patients.

The prognostic value of disseminated tumour cells derived from 353 breast cancer patients was evaluated. Disseminated tumour cells were purified from blood using a newly established method and nucleic acids were subsequently isolated. We investigated genomic imbalances (GI) such as mutation, amplification and loss of heterozygosity of 13 tumour suppressor genes and 2 proto-oncogenes using DNA from isolated minimal residual cancer cells. Significant correlations were found between genomic alterations of the DCC - and c-erbB-2 genes in disseminated breast cancer cells and actuarial relapse-free survival. Furthermore, increasing numbers of genomic imbalances measured in disseminated tumour cells were significantly associated with worse prognosis of recurrent disease. Logistic regression and Cox multivariate analysis led to the identification of genomic imbalances as an independent prognostic factor. Determination of disseminated tumour cells by genotyping of oncogenes and tumour suppressor genes seems not only to be a useful adjunct in follow up of carcinoma patients but provides also valuable additional individualized prognostic and predictive information in breast cancer patients beyond the TNM system.

Activation induces rapid and profound alterations in the trafficking of T cells.

Activation and differentiation of lymphocytes have profound effects on their trafficking. Whereas naive T cells recirculate through lymphoid organs, activated cells localize predominantly in other compartments. Here, we report that changes in migratory properties of T cells occur immediately upon activation via the TCR. One hour stimulation is enough to target T cells into lung and liver following i.v. injection. The high localization within lung and liver and the lack of recirculation through lymphoid tissues are key features of activated lymphocytes. the source, in vitro as well as in vivo activated lymphocytes show this behavior, which is not caused by increased cell size. Accumulation in the lung requires protein synthesis and is partly mediated by LFA-1, in contrast to the acquisition of liver "homing" properties. Intravital microscopy reveals firm adhesion of activated cells within periportal sinusoids of the liver. Selective homing to other organs, such as skin or mucosa, was not observed, regardless of the cell's origin. These data indicate that activation quickly switches the trafficking program of lymphocytes from recirculation to sequestration; it is tempting to speculate that especially the induced trapping in the liver has a distinct role in limiting systemic T cell responses.

Detection of mammaglobin expressing cells in blood of breast cancer patients.

Expression of human mammaglobin (hMAM) was published to be exclusively expressed in mammary tissue, in solid tumors, axillary lymph nodes and disseminated cancer cells in blood of breast cancer patients. A quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) test was applied to investigate hMAM expression in blood of breast cancer patients. Mammaglobin mRNA expression was found not only in breast cancer cell lines but also in cell lines of other cancer origin. In our patient cohort hMAM expression in 11/98 (11%) samples of breast cancer and 3/12 (25%) ovarian cancer patients could be detected. hMAM mRNA expression as a candidate marker for the detection of disseminated cancer cells in blood of breast cancer patients showed low sensitivity and reduced tissue specificity. A prognostic significance of hMAM expression could not be demonstrated.

Independent prognostication and therapy monitoring of breast cancer patients by DNA/RNA typing of minimal residual cancer cells.

Clinical relevance, purification techniques and molecular characterization of minimal residual cancer cells (MRCC) is a controversial topic in the literature. An analytical concept including a novel isolation procedure and a panel of tests for DNA and RNA typing of MRCCs is described and clinically evaluated in this paper. The purification procedure exploiting the physical characteristics of MRCCs shows superior performance leading to > 50% pure and viable tumor cells. Proof of the presence and purity of MRCCs in an isolated sample is given by multiparametric DNA typing (amplifications, mutations, losses of heterozygosity). On the basis of the proven presence of MRCCs tumor-relevant mRNAs can be adequately analyzed by normalized quantitative real-time RT-PCR. The molecular characterization of MRCCs isolated from blood of breast cancer patients could have a strong clinical impact on prognostication, drug targeting and therapy monitoring.

Analysis and sorting of T cells according to cytokine expression.

Functional distinct populations of T helper cells can be defined according to the expression of cytokines. A remarkable diversity of cytokine expression has been demonstrated in single T cells even from clonal populations and up to now no stable surface markers have been described which on the single-cell level directly correlate with the secretion of a certain cytokine. Since cytokines are the major parameters of T cell effector function we have developed strategies which now allow to separate cells according to the specific cytokines they secrete. The "affinity matrix technology"--secreted molecules are relocated to the cell surface by an artificially created antibody matrix--allows to isolate cells according to a distinct secreted product. In addition to this universal, but laborious technology, we could demonstrate by high-sensitivity immunofluorescence that IFN-gamma and IL-10 but not IL-2 and IL-4 are specifically expressed in low copy number on the surface of cells secreting these cytokines. Surface IFN-gamma and IL-10 are the first unambiguous surface markers for pro-inflammatory IFN-gamma-secreting Th 0/1 cells and IL-10-producing anti-inflammatory Th2/3 cells. We have used purified cytokine-secreting T cells to study in individual T cells the sequential production of IL-2, IFN-gamma, and IL-10, the stability of IFN-gamma expression and the selective homing of IFN-gamma-producing cells into inflamed tissues.

P- and E-selectin mediate recruitment of T-helper-1 but not T-helper-2 cells into inflammed tissues.

When activated, T helper cells differentiate into one of two subsets, Th1 and Th2, characterized by distinct profiles of cytokine production. Th1 cells activate pro-inflammatory effector mechanisms involved in protection and autoimmunity, whereas Th2 cells induce humoral and allergic responses and downregulate local inflammation. Apart from differences in the repertoire of cytokines, no phenotypic attributes are established that distinguish the two subsets. Here we show that Th1 cells, but not Th2 cells, are able to bind to P-selectin and E-selectin. Moreover, only Th1 cells can efficiently enter inflamed sites in Th1-dominated models, such as sensitized skin or arthritic joints, but not in a Th2-dominated allergic response. Immigration of Th1 cells into inflamed skin can be blocked by antibodies against P- and E-selectin. These results provide evidence for adhesion mechanisms to distinguish between the two T helper subsets and mediate their differential trafficking. They indicate that selective recruitment is an additional level of regulation for both effector function profile and character of a local immune response.

Transforming growth factor-beta 1-induced expression of the mucosa-related integrin alpha E on lymphocytes is not associated with mucosa-specific homing.

The integrin alpha E (HML-1, alpha IEL, alpha M290) is largely expressed on lymphocytes in epithelial sites, especially the gut mucosa. We investigated whether alpha E has any role in homing or delineates a phenotype with distinct migratory behavior. Lymph node T cells were stimulated for 5 days with anti-CD3 in the presence or absence of transforming growth factor (TGF)-beta 1 to generate alpha E+ or alpha E- cells, respectively. The two populations were then tested for their homing properties in mice. Both alpha E+ (TGF-beta-treated) and alpha E- (control) cells of either CD4+ or CD8+ subset had a low capacity to enter the gut and showed the same homing behavior with respect to a variety of other organs. The same was true for alpha E+ and alpha E- cells that had been briefly stimulated with anti-CD3 (24 h) and then allowed to return to a resting state before injection, though in this case both populations showed a greater capacity to recirculate through lymphoid tissue than was seen with fully activated cells. The results indicate that alpha E beta 7 does not act as a homing receptor, and that the expression of the site-specific marker alpha E does not correlate with a distinct homing behavior.

Characterization of idiotype-specific I-Ed-restricted T suppressor lymphocytes which confine immunoglobulin class expression to IgM in the anti-alpha (1- > 3) Dextran B 1355 S response of BALB/c mice.

The humoral immune response of conventionally raised BALB/c mice to the so-called "thymus independent" antigen alpha (1- > 3) Dextran B 1355 S (Dex) is predominantly of the IgM class. The response is further characterized by Igha allotype linkage and the dominance of the public idiotypes (Id) J558 and MOPC 104. In germfree raised BALB/c and in BALB/c nu/nu mice immunized with the same antigen an additional IgG response of the public Id is observed. Analysis of the regulation of the class expression reveals existence of specific Ts cells in euthymic mice which must have been activated pre- or perinatally by exposure to environmental bacterial antigens. They permit or enforce differentiation of Dex-specific B cells into B gamma memory cells without allowing further development into IgG producing plasma cells. An analogue of these splenic Ts cells has now been cloned and identified as an I-Ed restricted Id-specific T cell with exactly the properties ascribed above to the splenic Ts cells. This paper describes phenotypical and functional properties of the Ts cell clone 178-4. It evaluates this clone's role in controlling efficient anti-bacterial IgM-mediated immunity under conditions where a class switch to IgG antibody production is actively suppressed; possibly as a measure to avoid hazardous autoimmune reactions on the basis of crossreaction and antigenic mimicry between polysaccharide antigens.

Lymphokine profile and activation pattern of two unrelated antigen- or idiotype-specific T suppressor cell clones.

Two T suppressor (Ts) clones of different specificity have been analyzed for their lymphokine spectrum. BVI/5 is an I-Ek-restricted bovine serum albumin (BSA)-specific Ts cell clone from a CBA/J mouse tolerized by low doses of BSA. It affects directly or indirectly the function of BSA-specific T helper (Th) cells. The Ts cell clone 178-4 from a BALB/c mouse is I-Ed restricted and recognizes the public J558 Id on B cells. It prevents alpha(1----3)dextran B 1355S (Dex)-specific IgG antibody production and drives Dex-specific J558 idiotype-bearing B cells into an anergic B IgG memory cell state. Both Ts cell clones thus cause specific suppression, yet in different experimental systems using different effector mechanisms. Upon stimulation with concanavalin A or fixed CD3-specific monoclonal antibody, both clones produce high levels of interferon (IFN)-gamma and tumor necrosis factor (TNF) but in contrast to Th1 cells no interleukin (IL)-2. Both clones produce low levels of IL-3 and IL-6 but no IL-4, IL-5 and IL-9. Furthermore, unlike Th2 cells, both clones do not respond to IL-1. The mechanism of the idiotype-specific induction of anergy in Dex-specific B IgG memory cells by 178-4 Ts cells is not yet understood. BVI/5 Ts cells suppress in vitro the BSA-specific proliferation of the BSA-specific Th cell clone 83/1, as well as the response of BSA-primed CBA/LN cells. Whereas the suppressive effect on 83/1 cells is due to IFN-gamma alone the suppression of BSA-specific lymph node cells can be simulated neither by IFN-gamma nor the combination of IFN-gamma and TNF. Thus these mediators cannot account for the antigen-specific suppression by BVI/5 Ts cells in polyclonal in vitro responses from lymph node cells and probably not for the induction of in vivo unresponsiveness.

Cremophor EL as an adjuvant affecting immunoglobulin class switch in the immune response to the thymus-independent antigen alpha(1- greater than 3) dextran B 1355 S.

The humoral immune response to the so-called thymus independent antigen dextran B 1355 S in conventionally raised BALB/c mice consists solely of IgM antibodies. Expression of IgG anti-Dex antibodies in these mice is prevented by pre- or perinatally activated idiotype-specific T-suppressor lymphocytes. IgG B-memory cells nevertheless develop during the course of immunization, but are arrested in an anergic state. In the presence of Cremophor EL the induction of this anergic state is inhibited and the immune response shifts fully to an IgG anti-Dex response.

Regulation of the anti-alpha(1----3) dextran IgG antibody response of BALB/c mice by idiotype-specific T suppressor lymphocytes.

The immune response of BALB/c mice against the so-called thymus-independent bacterial Ag alpha(1----3) dextran (Dex) is restricted to the expression of few major idiotypes (Id). It is furthermore under the control of T lymphocytes which regulate the isotype expression in such a way that they prevent anti-Dex IgG antibody production upon immunization. At the same time these T cells are part of a regulatory system for Dex-specific B cell memory formation. The underlying Ts cell activity has previously been analyzed by using euthymic and athymic congenic animals. Now we have isolated CD4-positive Id-specific T cell lines and clones which by several criteria are representatives of the above Ts cells. They inhibit in vitro proliferation and antibody secretion of Dex-specific hybridoma B cells. They prevent Id-restricted in vivo IgG anti-Dex antibody formation in T cell-reconstituted BALB/c nu/nu mice. At the same time they enforce, again Id-specific, accumulation of Dex-specific B memory cells. As has been shown previously under the influence of splenic Ts cells, these B memory cells are arrested in the original host but can be expanded and activated for anti-Dex IgG antibody formation upon adoptive transfer into X-irradiated allotype congenic nonresponder BALB.Ighb mice. The data show that the regulatory influence of T cells on the anti-Dex response is Id specific. It can now be studied by means of cloned Ts cells.