Thymus cell maturation: II. Differentiation of three “mature” subclasses in Vivo

Several thymus cell subclasses may be defined on the basis of their sedimentation velocity, their light-scattering properties (a measure of cell volume), or binding of a fluoresceinated anti-Thy 1.2 antiserum. Using a multiparameter fluorescence-activated cell sorter (FACS), cells with distinguishable light-scattering or fluorescence intensity (after staining with fluorescein anti-Thy 1.2) were separable for analysis of intrathymic maturation pathways. Outer thymic cortical large and medium lymphocytes were the only cells labeled within 1 hr after transcapsular diffusion of administered [³H]thymidine. These labeled cells were also entirely contained in the brightest fluorescence intensity (with fluorescein anti-Thy 1.2) subclass. Under conditions of [¹H] thymidine “chase” in vivo, label shifted proportionately and apparently in parallel to three “mature” subclasses: (1) small thymocytes with high surface concentrations of Thy 1.2, representing ~ 80% of all thymus cells; (2) slightly larger cells, with very low surface Thy 1.2, which are indistinguishable from cortisone-resistant thymocytes, and which make up less than 10% of all thymus cells; (3) dead or fragile cells.     

Dual immunofluorescence studies of cortisone-induced thymic involution: evidence for a major cortical component to cortisone-resistant thymocytes

Cortisone-resistant thymocytes (CRT) have been used as the experimental equivalent of medullary thymocytes for the past 15 yr. Studies with CRT have provided evidence that the medullary population is similar to mature T cells in phenotype and function and may therefore be the major source of thymus emigrants. However, we have recently demonstrated that CRT differ from medullary thymocytes in their expression of the homing receptor molecule recognized by the monoclonal antibody MEL-14. Thus, many CRT express high levels of the MEL-14-defined homing receptor, whereas medullary thymocytes are MEL-14- to MEL-14lo. In normal adult mice, only 1 to 3% of thymocytes are MEL-14hi; these cells are located exclusively in the cortex and many are phenotypically and functionally mature. In this study we have used dual immunofluorescence techniques to further characterize those thymocytes resistant to cortisone treatment. Aside from being of mature phenotype with respect to expression of peanut agglutinin binding sites and the cell surface molecules H-2K, Ly-1, Lyt-2, and L3T4, CRT can be divided into MEL-14lo and MEL-14hi subpopulations, suggesting that they may actually be derived from both the medullary and the MEL-14hi cortical thymocyte subsets.     

Molecular tools for inactivating a yeast enzyme in vivo.

As part of an effort to develop a new means of inducibly inactivating cellular proteins in vivo, three monoclonal antibodies which neutralize yeast alcohol dehydrogenase (ADH) activity were isolated and characterized with respect to criteria important for the inactivation strategy. The significance of these criteria is considered, and a general means of generating appropriate antibodies is suggested. All three antibodies described here were specific for ADH I; they did not recognize the closely related isozyme ADH II in a plate-binding assay and did not immunoprecipitate molecules other than ADH from a Saccharomyces cerevisiae extract. Neutralization occurred in a yeast extract and, for two antibodies, was blocked by high concentrations of the coenzyme NAD+. This finding suggests that the antibodies may block enzyme activity by stabilizing an inactive form of ADH lacking bound NAD+. These results provide a foundation for the use of these antibodies to inactivate ADH in vivo.     

Peyer''s patch-specific lymphocyte homing receptors consist of a VLA-4-like alpha chain associated with either of two integrin beta chains, one of which is novel.

Lymphocytes home to various lymphoid organs by adhering to and migrating through specialized high endothelial venules (HEV). The murine cell surface heterodimer LPAM-1 is involved in the homing of lymphocytes to mucosal sites (Peyer's patches). LPAM-1 has an alpha subunit (alpha 4m) analogous to the alpha chain of the human integrin molecule VLA-4. Here we show that the LPAM-1 beta subunit (beta p) is immunochemically and biochemically distinct from previously defined integrin beta subunits, suggesting that beta p represents a novel integrin beta subunit. Depending on the cellular source two alternative beta subunits, beta p and integrin beta 1, can be isolated in association with alpha 4m. Therefore, alpha 4m is the common subunit of the unique integrin LPAM-1 (alpha 4m beta p) and of the heterodimer LPAM-2 (alpha 4m beta 1), which is analogous to VLA-4. Antibody-blocking experiments suggest that, in addition to LPAM-1, LPAM-2 is also involved in the organ-specific adhesion of lymphocytes to Peyer's patch HEV.     

Role of the zeta chain in the expression of the T cell antigen receptor: genetic reconstitution studies.

The zeta (zeta) chain plays a central role in T cell antigen receptor assembly and signal transduction. From previous work in murine T cell hybridomas we have inferred that the zeta subunit is limiting in receptor assembly. Partial receptors made in excess of zeta are assembled in the endoplasmic reticulum, transported through the Golgi, but then rapidly and efficiently degraded in lysosomes. zeta would therefore seem to play a unique role in targeting receptors from the Golgi to the cell surface. To determine directly whether zeta limits receptor assembly we have reconstituted a zeta-deficient T cell line by transfection of the murine zeta cDNA. Transfection results in restoration of expression of surface T cell receptor. In addition, increasing zeta expression results in a commensurate increase in the survival of previously excess subunits. This is reflected in an increased surface expression of complete receptors. Finally, transfection of the zeta cDNA fails to produce detectable zeta-eta heterodimers. The implications of these findings with regard to receptor assembly, and the relationship between zeta and eta, are discussed.     

Predictors of multidrug- and extensively drug-resistant tuberculosis in a high HIV prevalence community

Background

Multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) have emerged in high-HIV-prevalence settings, which generally lack laboratory infrastructure for diagnosing TB drug resistance. Even where available, inherent delays with current drug-susceptibility testing (DST) methods result in clinical deterioration and ongoing transmission of MDR and XDR-TB. Identifying clinical predictors of drug resistance may aid in risk stratification for earlier treatment and infection control.

Methods

We performed a retrospective case-control study of patients with MDR (cases), XDR (cases) and drug-susceptible (controls) TB in a high-HIV-prevalence setting in South Africa to identify clinical and demographic risk factors for drug-resistant TB. Controls were selected in a 1∶1∶1 ratio and were not matched. We calculated odds ratios (OR) and performed multivariate logistic regression to identify independent predictors.

Results

We enrolled 116, 123 and 139 patients with drug-susceptible, MDR, and XDR-TB. More than 85% in all three patient groups were HIV-infected. In multivariate analysis, MDR and XDR-TB were each strongly associated with history of TB treatment failure (adjusted OR 51.7 [CI 6.6-403.7] and 51.5 [CI 6.4–414.0], respectively) and hospitalization more than 14 days (aOR 3.8 [CI 1.1–13.3] and 6.1 [CI 1.8–21.0], respectively). Prior default from TB treatment was not a risk factor for MDR or XDR-TB. HIV was a risk factor for XDR (aOR 8.2, CI 1.3–52.6), but not MDR-TB. Comparing XDR with MDR-TB patients, the only significant risk factor for XDR-TB was HIV infection (aOR 5.3, CI 1.0–27.6).

Discussion

In this high-HIV-prevalence and drug-resistant TB setting, a history of prolonged hospitalization and previous TB treatment failure were strong risk factors for both MDR and XDR-TB. Given high mortality observed among patients with HIV and drug-resistant TB co-infection, previously treated and hospitalized patients should be considered for empiric second-line TB therapy while awaiting confirmatory DST results in settings with a high-burden of MDR/XDR-TB.     

Dissecting the diverse functions of the metastasis suppressor CD82/KAI1

The recent identification of metastasis suppressor genes, the products of which inhibit metastasis but not primary tumor growth, distinguishes oncogenic transformation and tumor suppression from a hallmark of malignancy, the ability of cancer cells to invade sites distant from the primary tumor. The metastasis suppressor CD82/KAI1 is a member of the tetraspanin superfamily of glycoproteins. CD82 suppresses metastasis by multiple mechanisms including inhibition of cell motility and invasion, promotion of cell polarity as well as induction of senescence and apoptosis in response to extracellular stimuli. A common feature of these diverse effects is CD82 regulation of membrane organization as well as protein trafficking and interactions, which affects cellular signaling and intercellular communication.