Deep Characterization of the Human Antibody Response to Natural Infection Using Longitudinal Immune Repertoire Sequencing.

Human antibody response studies are largely restricted to periods of high immune activity (e.g. vaccination). To comprehensively understand the healthy B cell immune repertoire and how this changes over time and through natural infection, we conducted immune repertoire RNA sequencing on flow cytometry-sorted B cell subsets to profile a single individual's antibodies over 11 months through two periods of natural viral infection. We found that 1) a baseline of healthy variable (V) gene usage in antibodies exists and is stable over time, but antibodies in memory cells consistently have a different usage profile relative to earlier B cell stages; 2) a single complementarity-determining region 3 (CDR3) is potentially generated from more than one VJ gene combination; and 3) IgG and IgA antibody transcripts are found at low levels in early human B cell development, suggesting that class switching may occur earlier than previously realized. These findings provide insight into immune repertoire stability, response to natural infections, and human B cell development.

AutoGate: automating analysis of flow cytometry data.

Nowadays, one can hardly imagine biology and medicine without flow cytometry to measure CD4 T cell counts in HIV, follow bone marrow transplant patients, characterize leukemias, etc. Similarly, without flow cytometry, there would be a bleak future for stem cell deployment, HIV drug development and full characterization of the cells and cell interactions in the immune system. But while flow instruments have improved markedly, the development of automated tools for processing and analyzing flow data has lagged sorely behind. To address this deficit, we have developed automated flow analysis software technology, provisionally named AutoComp and AutoGate. AutoComp acquires sample and reagent labels from users or flow data files, and uses this information to complete the flow data compensation task. AutoGate replaces the manual subsetting capabilities provided by current analysis packages with newly defined statistical algorithms that automatically and accurately detect, display and delineate subsets in well-labeled and well-recognized formats (histograms, contour and dot plots). Users guide analyses by successively specifying axes (flow parameters) for data subset displays and selecting statistically defined subsets to be used for the next analysis round. Ultimately, this process generates analysis "trees" that can be applied to automatically guide analyses for similar samples. The first AutoComp/AutoGate version is currently in the hands of a small group of users at Stanford, Emory and NIH. When this "early adopter" phase is complete, the authors expect to distribute the software free of charge to .edu, .org and .gov users.

Blood basophils from cystic fibrosis patients with allergic bronchopulmonary aspergillosis are primed and hyper-responsive to stimulation by aspergillus allergens.

INTRODUCTION: Fifteen to sixty percent of cystic fibrosis patients harbor Aspergillus fumigatus (Af) in their airways (CF-AC) and some will develop allergic bronchopulmonary aspergillosis (CF-ABPA). Since basophils play a key role in allergy, we hypothesized that they would display alterations in CF-ABPA patients compared to CF-AC or patients without Af colonization (CF). METHODS: Using flow cytometry, we measured CD203c, CD63 and CD123 levels on basophils from CF-ABPA (N=11), CF-AC (N=14), and CF (N=12) patients before and after ex vivo stimulation with Af allergens. RESULTS: Baseline CD203c was increased in basophils from CF-ABPA compared to CF-AC and CF patients. Af extract and recombinant Aspf1 stimulated basophils from CF-ABPA patients to markedly upregulate CD203c, along with modest upregulation of CD63 and a CD123 downward trend. Plasma TARC/CCL17 at baseline and post-stimulation cell supernatant histamine levels were similar in the three groups. CONCLUSIONS: In CF-ABPA, blood basophils are primed and hyperresponsive to Af allergen stimulation.

Syrbactin class proteasome inhibitor-induced apoptosis and autophagy occurs in association with p53 accumulation and Akt/PKB activation in neuroblastoma.

Syrbactins belong to a new class of proteasome inhibitors which include syringolins and glidobactins. These small molecules are structurally distinct from other, well-established proteasome inhibitors, and bind the eukaryotic 20S proteasome by a novel mechanism. In this study, we examined the effects of syringolin A (SylA) and glidobactin A (GlbA) as well as two synthetic SylA-analogs (SylA-PEG and SylA-LIP) in human neuroblastoma (SK-N-SH), human multiple myeloma (MM1.S, MM1.RL, and U266), and human ovarian cancer (SKOV-3) cells. While all four syrbactins inhibited cell proliferation in a dose-dependent manner, GlbA was most potent in both dexamethasone-sensitive MM1.S cells (IC(50): 0.004microM) and dexamethasone-resistant MM1.RL cells (IC(50): 0.005microM). Syrbactins also inhibited the chymotrypsin-like proteasome activity in a dose-dependent fashion, and GlbA was most effective in SK-N-SH cells (IC(50): 0.015microM). The GlbA-promoted inhibition of proteasomal activity in SK-N-SH cells resulted in the accumulation of ubiquitinated proteins and tumor suppressor protein p53 and led to apoptotic cell death in a time-dependent manner. GlbA treatment also promoted the activation of Akt/PKB via phosphorylation at residue Ser(473) and induced autophagy as judged by the presence of the lipidated form of microtubule-associated protein 1 light chain 3 (LC3) and autophagosomes. Collectively, our data suggest that syrbactins belong to a new and effective proteasome inhibitor class which promotes cell death. Proteasome inhibition is a promising strategy for targeted anticancer therapy and syrbactins are a new class of inhibitors which provide a structural platform for the development of novel, proteasome inhibitor-based drug therapeutics.

Drugs from slugs--past, present and future perspectives of omega-conotoxin research.

Peptides from the venom of carnivorous cone shells have provided six decades of intense research, which has led to the discovery and development of novel analgesic peptide therapeutics. Our understanding of this unique natural marine resource is however somewhat limited. Given the past pharmacological record, future investigations into the toxinology of these highly venomous tropical marine snails will undoubtedly yield other highly selective ion channel inhibitors and modulators. With over a thousand conotoxin-derived sequences identified to date, those identified as ion channel inhibitors represent only a small fraction of the total. Here we discuss our present understanding of conotoxins, focusing on the omega-conotoxin peptide family, and illustrate how such a seemingly simple snail has yielded a highly effective clinical drug.

Sudden death due to nontraumatic diaphragmatic hernia in an adult.

Sudden death in an adult due to an undiagnosed congenital diaphragmatic hernia (CDH) is a very rare occurrence. The majority of adults who experience discomfort related to their condition have experienced some kind of trauma prior to symptom manifestation; however, there can be the cases that are exceptions to a medical trend. In the following, we present a case of an adult female who died from an undiagnosed congenital diaphragmatic hernia without any prior traumatic event or significant medical history. In these situations, early diagnosis and rapid surgical intervention are necessary for successful treatment of affected individuals.

Cracking the code: a decode strategy for the international business machines punch cards of Korean war soldiers.

During the Korean War, International Business Machines (IBM) punch cards were created for every individual involved in military combat. Each card contained all pertinent personal information about the individual and was utilized to keep track of all soldiers involved. However, at present, all of the information known about these punch cards reveals only their format and their significance; there is little to no information on how these cards were created or how to interpret the information contained without the aid of the computer system used during the war. Today, it is believed there is no one available to explain this computerized system, nor do the original computers exist. This decode strategy is the result of an attempt to decipher the information on these cards through the use of all available medical and dental records for each individual examined. By cross-referencing the relevant personal information with the known format of the cards, a basic guess-and-check method was utilized. After examining hundreds of IBM punch cards, however, it has become clear that the punch card method of recording information was not infallible. In some cases, there are gaps of information on cards where there are data recorded on personal records; in others, information is punched incorrectly onto the cards, perhaps as the result of a transcription error. Taken all together, it is clear that the information contained on each individual's card should be taken solely as another form of personal documentation.