Addictive drugs and their relationship with infectious diseases

The use of drugs of abuse, both recreationally and medicinally, may be related to serious public health concerns. There is a relationship between addictive drugs of abuse such as alcohol and nicotine in cigarette smoke, as well as illegal drugs such as opiates, cocaine and marijuana, and increased susceptibility to infections. The nature and mechanisms of immunomodulation induced by such drugs of abuse are described in this review. The effects of opiates and marijuana, using animal models as well as in vitro studies with immune cells from experimental animals and humans, have shown that immunomodulation induced by these drugs is mainly receptor-mediated, either directly by interaction with specific receptors on immune cells or indirectly by reaction with similar receptors on cells of the nervous system. Similar studies also show that cocaine and nicotine have marked immunomodulatory effects, which are mainly receptor-mediated. Both cocaine, an illegal drug, and nicotine, a widely used legal addictive component of cigarettes, are markedly immunomodulatory and increase susceptibility to infection. The nature and mechanism of immunomodulation induced by alcohol, the most widely used addictive substance of abuse, are similar but immunomodulatory effects, although not receptor-mediated. The many research studies on the effects of these drugs on immunity and increased susceptibility to infectious diseases, including AIDS, are providing a better understanding of the complex interactions between immunity, infections and substance abuse.     

WT p53, but not tumor-derived mutants, bind to Bcl2 via the DNA binding domain and induce mitochondrial permeabilization

The induction of apoptosis by p53 in response to cellular stress is its most conserved function and crucial for p53 tumor suppression. We recently reported that p53 directly induces oligomerization of the BH1,2,3 effector protein Bak, leading to outer mitochondrial membrane permeabilization (OMMP) with release of apoptotic activator proteins. One important mechanism by which p53 achieves OMMP is by forming an inhibitory complex with the anti-apoptotic BclXL protein. In contrast, the p53 complex with the Bcl2 homolog has not been interrogated. Here we have undertaken a detailed characterization of the p53-Bcl2 interaction using structural, biophysical, and mutational analyses. We have identified the p53 DNA binding domain as the binding interface for Bcl2 using solution NMR. The affinity of the p53-Bcl2 complex was determined by surface plasmon resonance analysis (BIAcore) to have a dominant component KD 535 +/- 24 nm. Moreover, in contrast to wild type p53, endogenous missense mutants of p53 are unable to form complexes with endogenous Bcl2 in human cancer cells. Functionally, these mutants are all completely or strongly compromised in mediating OMMP, as measured by cytochrome c release from isolated mitochondria. These data implicate p53-Bcl2 complexes in contributing to the direct mitochondrial p53 pathway of apoptosis and further support the notion that the DNA binding domain of p53 is a dual function domain, mediating both its transactivation function and its direct mitochondrial apoptotic function.     

CXCL12 limits inflammation by localizing mononuclear infiltrates to the perivascular space during experimental autoimmune encephalomyelitis

The inflammatory response in the CNS begins with the movement of leukocytes across the blood-brain barrier in a multistep process that requires cells to pass through a perivascular space before entering the parenchyma. The molecular mechanisms that orchestrate this movement are not known. The chemokine CXCL12 is highly expressed throughout the CNS by microendothelial cells under normal conditions, suggesting it might play a role maintaining the blood-brain barrier. We tested this hypothesis in the setting of experimental autoimmune encephalomyelitis (EAE) by using AMD3100, a specific antagonist of the CXCL12 receptor CXCR4. We demonstrate that the loss of CXCR4 activation enhances the migration of infiltrating leukocytes into the CNS parenchyma. CXCL12 is expressed at the basolateral surface of CNS endothelial cells in normal spinal cord and at the onset of EAE. This polarity is lost in vessels associated with an extensive parenchymal invasion of mononuclear cells during the peak of disease. Inhibition of CXCR4 activation during the induction of EAE leads to loss of the typical intense perivascular cuffs, which are replaced with widespread white matter infiltration of mononuclear cells, worsening the clinical severity of the disease and increasing inflammation. Taken together, these data suggest a novel anti-inflammatory role for CXCL12 during EAE in that it functions to localize CXCR4-expressing mononuclear cells to the perivascular space, thereby limiting the parenchymal infiltration of autoreactive effector cells.     

Adrenalectomy Blocks the Compensatory Increases in UT-A1 and AQP2 in Diabetic Rat Kidney

In normal rats we showed that glucocorticoids participate in the downregulation of UT-A1 protein abundance in the inner medullary tip and in lowering of basal and vasopressin-stimulated facilitated urea permeability in terminal IMCDs. To examine the relevance of this response to a rat model of human disease, we studied rats with uncontrolled diabetes mellitus (DM) induced by streptozotocin (STZ), since these rats have increased corticosterone production and urea excretion. We found that at 3 days of DM, UT-A1 protein abundance is downregulated in the inner medullary tip compared to pair-fed control rats, while DM for more than 7 days caused an increase in UT-A1. To test whether adrenal steroids could be a mechanism contributing to the latter increase, we studied adrenalectomized rats (ADX), ADX rats given STZ to induce diabetes (ADX + STZ), and ADX + STZ rats receiving exogenous aldosterone or dexamethasone. In contrast to control rats, UT-A1 protein abundance was not increased by prolonged DM in the ADX rats. Aquaporin 2 (AQP2) was not increased in the inner medullas of 10-day DM rats either. However, UT-A1 protein abundance was significantly reduced in the inner medullary tips from both diabetic aldosterone-treated (40 ± 2%) and dexamethasone-treated (43 ± 2%) ADX rats compared to diabetic ADX rats without steroid replacement. AQP2 was unaffected by steroid hormone treatments. Thus, both mineralocorticoids and glucocorticoids downregulate UT-A1 protein abundance in rats with uncontrolled diabetes mellitus for 10 days. These results suggest that: 1) the increase in UT-A1 observed in DM is dependent upon having adrenal steroids present; and 2) adrenal steroids are not sufficient to enable the compensatory rise in UT-A1 to a steroid-deficient diabetic animal.     

Characterization of native and reconstituted exosome complexes from the hyperthermophilic archaeon Sulfolobus solfataricus

The eukaryotic exosome is a protein complex with essential functions in processing and degradation of RNA. Exosome-like complexes were recently found in Archaea. Here we characterize the exosome of Sulfolobus solfataricus. Two exosome fractions can be discriminated by density gradient centrifugation. We show that the Cdc48 protein is associated with the exosome from the 30S-50S fraction but not with the exosome of the 11.3S fraction. While only some complexes contain Cdc48, the archaeal DnaG-like protein was found to be a core exosome subunit in addition to Rrp4, Rrp41, Rrp42 and Csl4. Assays with depleted extracts revealed that the exosome is responsible for major ribonucleolytic activity in S. solfataricus. Various complexes consisting of the Rrp41-Rrp42 hexameric ring and Rrp4, Csl4 and DnaG were reconstituted. Dependent on their composition, different complexes showed variations in RNase activity indicating functional interdependence of the subunits. The catalytic activity of these complexes and of the native exosome can be ascribed to the Rrp41-Rrp42 ring, which degrades RNA phosphorolytically. Rrp4 and Csl4 do not exhibit any hydrolytic RNase activity, either when assayed alone or in context of the complex, but influence the activity of the archaeal exosome.     

Pathogenesis of Epstein-Barr virus (EBV)-carrying lymphomas

The EBV carrier state is almost general in men. The virus induces B lymphocyte proliferation in vitro, but this is counteracted in vivo by the immune response. Therefore, EBV-induced malignancies occur only when the immune response is impaired, e.g. in transplant recipients. The versatility of the viral gene expression strategy secures the consistent maintainance of the virus in healthy individuals. The viral proteins required for transformation render the cell immunogenic. Expression of the transforming genes leads to rejection, but these genes are not required for the maintenance of the viral genome. EBV is an important contributor for malignant transformation, even when it does not directly induce cell proliferation. Several mechanisms have been unravelled in EBV-associated tumors whereby the virus may modify the cellular phenotype and may influence the interaction of tumor cells with their microenvironment. The virus carrier state can lead to the evasion of apoptosis and can intensify the response to growth promoting signals, too.     

Protein expression pattern in experimental pneumococcal meningitis

In this study, we investigated cytokine expression during experimental pneumococcal meningitis. Mice were intracisternally infected with Streptococcus pneumoniae and treated with ceftriaxone starting at 24 h after infection. At different time points before and after antibiotic therapy, the cytokine expression pattern was determined in mouse brains using protein arrays. Underlining the power of this method, the meningitis-relevant cytokines interleukin-1beta (IL-1beta), IL-6, KC, macrophage inflammatory protein-2 (MIP-2), and monocyte chemoattractant protein-1 (MCP-1/CCL2) were markedly elevated in infected animals. Newly identified proteins during the acute stage of the disease (until 30 h after infection) included lymphotactin (XCL-1), MIP-1gamma (CCL9) and MCP-5 (CCL12), cytokine responsive gene- 2 (CRG-2/CXCL10) and CXCL16, and insulin-like growth factor binding protein 3 (IGFBP3). During later stages, an induction of T-cell activation-3 (TCA-3/CCL1), platelet factor-4 (PF-4/CXCL4) and stromal derived factor-1alpha (SDF-1alpha/CXCL13), and IL-4 was observed. The validity of this method was supported by an additional ELISA analysis of the expression profile of CXCL16 and IGFBP3, which was identical to that observed by protein array. In conclusion, the use of protein array technology led to an extension of the current picture of protein expression in pneumococcal meningitis. Most important, new factors that might play a role in pneumococcal meningitis were identified.     

Monokine induced by interferon gamma and IFN-gamma response to a fusion protein of Mycobacterium tuberculosis ESAT-6 and CFP-10 in Brazilian tuberculosis patients

IFN-gamma responses to Mycobacterium tuberculosis antigens ESAT-6 and CFP-10 have been proposed as specific markers of M. tuberculosis infection. Monokine induced by gamma interferon (MIG/CXCL9) has been shown to be expressed by IFN-gamma stimulated mononuclear cells and to attract activated T-cells through the chemokine receptor CXCR3. Since MIG is induced early in the response to IFN-gamma, measuring MIG may provide an interesting marker to assess downstream IFN-gamma induced responses, in contrast to assays that mainly focus on quantifying production of IFN-gamma per se. We, therefore, investigated MIG and IFN-gamma responses to a fusion protein of ESAT-6 and CFP-10, and compared responses to the conserved mycobacterial antigen 85B (Ag85B) and purified protein derivative (PPD) of M. tuberculosis, in 29 BCG vaccine controls and 24 TB patients. IFN-gamma secreting cells were determined by ELISPOT, and MIG production was measured by ELISA and flow cytometry. Production of MIG in response to ESAT-6/CFP-10, Ag85B and PPD correlated overall with increased numbers of IFN-gamma secreting cells (r=0.55, P<0.0001). A significant increase was noted among patients compared to controls in the secretion of IFN-gamma and MIG following stimulation with ESAT-6/CFP-10 or PPD (P<0.05). Moreover, MIG intracellular expression was higher in TB patients compared to BCG vaccines (P<0.05) in response to ESAT-6/CFP-10 or PPD. We conclude that MIG production correlates significantly with enhanced T-cell IFN-gamma production induced by M. tuberculosis-specific antigens ESAT-6/CFP-10. These results point to MIG as a potential novel biomarker that may be helpful in assessing downstream responses induced by IFN-gamma in TB.