Proteomic Analyses of the Effects of Drugs of Abuse on Monocyte-Derived Mature Dendritic Cells

Drug abuse has become a global health concern. Understanding how drug abuse modulates the immune system and how the immune system responds to pathogens associated with drug abuse, such hepatitis C virus (HCV) and human immunodeficiency virus (HIV-1), can be assessed by an integrated approach comparing proteomic analyses and quantitation of gene expression. Two-dimensional (2D) difference gel electrophoresis was used to determine the molecular mechanisms underlying the proteomic changes that alter normal biological processes when monocyte-derived mature dendritic cells were treated with cocaine or methamphetamine. Both drugs differentially regulated the expression of several functional classes of proteins including those that modulate apoptosis, protein folding, protein kinase activity, and metabolism and proteins that function as intracellular signal transduction molecules. Proteomic data were validated using a combination of quantitative, real-time PCR and Western blot analyses. These studies will help to identify the molecular mechanisms, including the expression of several functionally important classes of proteins that have emerged as potential mediators of pathogenesis. These proteins may predispose immunocompetent cells, including dendritic cells, to infection with viruses such as HCV and HIV-1, which are associated with drug abuse.     

Acute appendicitis in the developing world is a morbid disease

IntroductionAcute appendicitis in the developing world has a markedly different disease profile to that in the developed world.MethodsA retrospective study was undertaken over a four-year period at a university hospital in South Africa to review the disease spectrum and the clinical outcome of acute appendicitis.ResultsA total of 1,004 patients (54% male, median age: 18 years) with intraoperatively confirmed appendicitis were reviewed. Over half (56%) were from the urban district within the city of Pietermaritzburg and the remaining 44% were from the rural health district. The median duration of illness from onset to definitive care was 4 days. Sixty per cent of appendices were perforated and associated with intra-abdominal contamination. Forty per cent of patients required reoperation to control intra-abdominal sepsis. Ten per cent required admission to the intensive care unit. The median overall length of hospital stay was 5 days. The mortality rate was 1%.Rural patients had a longer median duration of illness (3 vs 5 days, p<0.001) as well as a more advanced disease profile associated with perforation and severe intra-abdominal sepsis (19% vs 71%, p<0.001). Female patients had a longer median duration of illness (3 vs 4 days, p<0.001), were more likely to present with severe intra-abdominal sepsis (31% vs 54%, p<0.001) and were more likely to require a laparotomy (50% vs 73%, p<0.001). The total cost of managing the entire cohort of 1,004 patients over the 4-year period was £2,060,972.ConclusionsAcute appendicitis in South Africa is a serious disease associated with significant morbidity. Late presentation is common. Female and rural patients have the worst clinical outcomes, with significant cost to the health system.     

Activated natural killer cells and interleukin-2 promote granulocytic and megakaryocytic reconstitution after syngeneic bone marrow transplantation in mice

Purified populations of natural killer (NK) cells were obtained from mice with severe combined immune deficiency (SCID). SCID spleen cells were cultured and activated with recombinant human interleukin-2 (rhIL- 2) in vitro. The activated NK cells were then transferred with syngeneic BALB/c bone marrow cells (BMC) and rhIL-2 into lethally irradiated syngeneic recipients to determine their effect on long-term hematopoietic reconstitution. On analysis, the transfer of rhIL-2- activated NK cells along with BMC resulted in significant increases in splenic and BM hematopoietic progenitor cells when compared with those for mice not receiving NK cells. Histologic and flow cytometric analysis showed a marked increase in granulocytic and megakaryocytic lineage cells present in the spleens of the mice receiving activated NK cells. Analysis of the peripheral blood indicated that the transfer of activated NK cells with BMC also significantly improved platelet and total white blood cell counts, with increases in segmented neutrophils. Erythroid recovery was not affected. Finally, lethally irradiated mice receiving activated NK cells and rhIL-2 along with limiting numbers of syngeneic BMC showed a marked increase in survival rate. These results show that the use of populations enriched for activated NK cells after syngeneic BM transplantation (BMT) has a profound enhancing effect on engraftment primarily affecting megakaryocytic and granulocytic cell reconstitution. Therefore, the transfer of activated NK cells and rhIL- 2 may be of clinical use to promote hematopoietic reconstitution after BMT.     

The metabolomics of asthma: novel diagnostic potential

Asthma is one of the most common chronic illnesses, especially in children. Reaching the diagnosis of asthma and its management are more difficult than for other chronic illnesses. For example, asthma is a heterogeneous syndrome with many clinical classifications based on patient symptoms, lung function, and response to therapy. The symptoms and objective measurements of lung function, often used to guide therapy, are largely based on the inflammation of the airways. Because measuring airway dysfunction and inflammation in a typical clinical setting is difficult, it is often not done. Metabolomics is the study of small molecules generated from cellular metabolic activity. It is possible that the metabolic profile of a patient with a chronic illness such as asthma is different from that of a healthy patient or from a patient with another respiratory illness. Furthermore, if this metabolome could be measured, it might also vary with disease severity. The pattern of metabolites becomes the diagnostic representing the disease. This article outlines the more recent work that has been done to develop the metabolomic profile of asthma.     

Serial head circumference and neurodevelopmental screening after surgical correction for single- and multiple-suture craniosynostosis

Objective : To evaluate serial head circumference (HC) measurements and neurodevelopmental (ND) screening before and after surgical revision for craniosynostosis. Design : Retrospective assessment. Setting : Tertiary institutional. Patients, Participants : All children treated with single-stage frontal-orbital advancement or total calvarial expansion for single-suture (SS) or multiple-suture (MS) craniosynostosis over a 7-year period. Main Outcome Measures : Changes in ND and HC were measured over postoperative visits after the primary surgery. More importantly, ND and HC changes were measured prerevision and postrevision. Results : Of 183 patients undergoing primary surgery, complete records and adequate follow-up were available for 112 patients. The overall revision rate was 21% (n = 23). Postrevision follow-up was adequate for 18 of the 23 revisions. After primary surgery, but prior to revision, children demonstrated a larger decline in HC (z-score, median = -1) along with higher ND findings (median = 2) from one postoperative visit to the next than those who did not go on revision (HC z-score median = 0, ND median = 0). After revision, patients demonstrated a significant improvement in ND screening findings (median ΔND findings = -2) compared with prerevision ND (p < .001). Head circumference also significantly increased by a z-score of +1 postrevision (p = .001). Conclusions : Patients chosen for revision surgery display not only a larger decline in HC but also more ND findings prior to revision. Furthermore, surgical revision has a significant association with both improved ND screening and HC.     

Computer-Aided Drug Discovery Approach Finds Calcium Sensitizer of Cardiac Troponin

In the fight against heart failure, therapeutics that have the ability to increase the contractile power of the heart are urgently needed. One possible route of action to improve heart contractile power is increasing the calcium sensitivity of the thin filament. From a pharmaceutical standpoint, calcium sensitizers have the distinct advantage of not altering cardiomyocyte calcium levels and thus have lower potential for side-effects. Small chemical molecules have been shown to bind to the interface between cTnC and the cTnI switch peptide and exhibit calcium-sensitizing properties, possibly by stabilizing cTnC in an open conformation. Building on existing structural data of a known calcium sensitizer bound to cardiac troponin, we combined computational structure-based virtual screening drug discovery methods and solution NMR titration assays to identify a novel calcium sensitizer 4-(4-(2,5-dimethylphenyl)-1-piperazinyl)-3-pyridinamine (NSC147866) which binds to cTnC and the cTnC-cTnI_147–163 complex. Its presence increases the affinity of switch peptide to cTnC by approximately a factor of two. This action is comparable to that of known levosimendan analogues.     

The protein product of the proto-oncogene c-cbl forms a complex with phosphatidylinositol 3-kinase p85 and CD19 in anti-IgM-stimulated human B-lymphoma cells

Multiple signal transduction cascades, consisting of multiple interacting proteins, are activated following stimulation through most cell surface receptors, including the immunoglobulin receptor of B lymphocytes. In this report, we investigated the multimolecular complexes formed following anti-Ig stimulation of a human B-lymphoma cell line, resulting in activation of phosphatidylinositol 3-kinase (PI3K). PI3K is a lipid kinase that consists of an 85-kD regulatory subunit, bound to a 110-kD catalytic subunit. CD19 is a 95-kD B-cell surface marker that contains a consensus binding motif for PI3Kp85 in the cytoplasmic domain and recruits PI3K activity in activated B cells. The protein product of the c-cbl protooncogene is a 120-kD protein that is expressed in early B-lineage cells and in myeloid cells and is phosphorylated on tyrosine following receptor-mediated signaling in T and B lymphocytes. We demonstrate here that phosphorylated c-cbl complexes with CD19 and with PI3Kp85 via its C-terminal SH2 domain, and that both c-cbl and CD19 are associated with active PI3K in anti-Ig- stimulated cells. Although we cannot differentiate between a three- component, c-cbl/CD19/p85 complex and individual two-component complexes, these studies suggest that c-cbl may function as a docking protein, possibly linking distinct signal transduction pathways.