Cutaneous lymphocyte antigen expression on human effector B cells depends on the site and on the nature of antigen encounter

In contrast to T cells, information on skin-homing B cells expressing the cutaneous lymphocyte antigen (CLA) is sparse. CLA expression on human B cells was investigated among circulating immunoglobulin-secreting cells (ISC) and among antigen-specific antibody-secreting cells (ASC) elicited by parenteral, oral or rectal primary immunization, or by parenteral or oral secondary immunization with Salmonella typhi Ty21a. CLA expression was examined by combining cell sorting with an enzyme-linked immunospot assay. Among all ISC, the proportion of CLA(+) cells was 13-21%. Parenteral immunization induced antigen-specific ASC of which 13% were CLA(+), while oral and rectal immunizations were followed by only 1% of CLA(+) ASC (p<0.001). Oral re-immunization was followed by an up-regulation of CLA (34-48%) regardless of the route of priming. Parenteral re-immunization elicited ASC of which 9-14% were CLA(+). In conclusion, the expression of CLA on human effector B cells depends on the site of antigen encounter: intestinal stimulation elicits cells with no CLA, while parenteral encounter elicits significant numbers of CLA(+) cells. Even though primary antigen encounter in the intestine failed to stimulate CLA expression, up-regulation of CLA was found upon intestinal antigen re-encounter. These findings may be of relevance in the pathogenesis of some cutaneous disorders.     

Immunity induced shortly after DNA vaccination of rainbow trout against rhabdoviruses protects against heterologous virus but not against bacterial pathogens.

It was recently reported that DNA vaccination of rainbow trout fingerlings against viral hemorrhagic septicaemia virus (VHSV) induced protection within 8 days after intramuscular injection of plasmid DNA. In order to analyse the specificity of this early immunity, fish were vaccinated with plasmid DNA encoding the VHSV or the infectious haematopoietic necrosis virus (IHNV) glycoprotein genes and later challenged with homologous or heterologous pathogens. Challenge experiments revealed that immunity established shortly after vaccination was cross-protective between the two viral pathogens whereas no increased survival was found upon challenge with bacterial pathogens. Within two months after vaccination, the cross-protection disappeared while the specific immunity to homologous virus remained high. The early immunity induced by the DNA vaccines thus appeared to involve short-lived non-specific anti-viral defence mechanisms.     

Alternative signaling pathways regulating type I interferon-induced apoptosis.

Type I interferons (IFNs) are pleiotropic cytokines that exert multiple effects on normal and tumor cells. These effects are supposedly mediated through the stimulation of several signal transduction pathways by type I IFNs. These include the well-studied canonical Jak-Stat pathway, largely responsible for the antiviral and growth-inhibitory activities of IFNs, as well as the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways, whose importance in IFN-induced biologic outcomes has not been precisely established. One of the effects of type I IFNs on tumor cells is the induction of programmed cell death, apoptosis, which has been studied extensively over the last decade and has been suggested to be an important effector mechanism for IFN's antitumor effects in the treatment of cancer. The aim of this review is to summarize and discuss the recent data in the field of type I IFN-induced apoptosis, with special emphasis on the molecular mechanisms of apoptosis and on the role of alternative noncanonical signaling pathways stimulated by type I IFNs in this process.     

Quantitation of cytomegalovirus (hCMV) DNA and β-actin DNA by duplex real-time fluorescence PCR in solid organ (liver) transplant recipients

Even now rare human cytomegalovirus (hCMV) reactivation is still a life-threatening complication after solid organ transplantation. Although PCR techniques are regarded as the most sensitive detection methods for hCMV, their accuracy and reproducibility are limited. This is a major disadvantage with quantitative PCR assays, which are thought to provide valuable information about hCMV latency or active viral replication in transplant patients. To enhance the diagnostic safety of quantitative hCMV PCR, we developed a duplex real-time fluorescence PCR that is capable of quantifying hCMV DNA and beta-actin DNA as internal control simultaneously within one reaction. By the use of 6-carboxyfluorescein and hexa-chloro-6-carboxyfluorescein as reporter fluorophores and 4-(4'-dimethylamino-phenylazo) benzoic acid as dark quencher dye, hCMV DNA and beta-actin DNA could be quantified in parallel in a wide linear range from 10(1) to 10(7) copies, each. To test the clinical applicability of this approach, we investigated hCMV DNA kinetics in peripheral leukocytes of three hCMV antigen-positive and four antigen-negative patients after liver transplantation, as assessed by intracellular hCMV pp65 alkaline phosphate-anti-alkaline phosphate (APAAP) complex. While all APAAP-negative individuals remained PCR negative, kinetics of HCMV DNA in leukocyte DNA samples of APAAP-positive patients correlated closely with hCMV antigen tests. Here, comparison of separate and simultaneous target quantitation revealed identical results. It is of interest that, while single hCMV antigen positivity is commonly not regarded as a reliable parameter of viral reactivation, in our study a viral load greater than 10(4) copies/2x10(5) beta-actin DNA copies clearly indicated a subsequent increase in APAAP-positive leukocytes. We conclude that with the presented method the reliability of hCMV quantitation via real-time PCR can be substantially increased and may be used to monitor hCMV kinetics in vivo.     

Opponent appetitive-aversive neural processes underlie predictive learning of pain relief

Termination of a painful or unpleasant event can be rewarding. However, whether the brain treats relief in a similar way as it treats natural reward is unclear, and the neural processes that underlie its representation as a motivational goal remain poorly understood. We used fMRI (functional magnetic resonance imaging) to investigate how humans learn to generate expectations of pain relief. Using a pavlovian conditioning procedure, we show that subjects experiencing prolonged experimentally induced pain can be conditioned to predict pain relief. This proceeds in a manner consistent with contemporary reward-learning theory (average reward/loss reinforcement learning), reflected by neural activity in the amygdala and midbrain. Furthermore, these reward-like learning signals are mirrored by opposite aversion-like signals in lateral orbitofrontal cortex and anterior cingulate cortex. This dual coding has parallels to 'opponent process' theories in psychology and promotes a formal account of prediction and expectation during pain.     

Sleep on the Night Shift: 24-Hour EEG Monitoring of Spontaneous Sleep/Wake Behavior

The present study sought to objectively describe the spontaneous sleep/wakefulness pattern of shift workers during a 24-hour period. Portable Medilog tape-recorders were used for ambulatory EEG monitoring of 25 male papermill workers (25-55 years) during days with night and afternoon work. The results showed that sleep after night work was two hours shorter than after afternoon work. The sleep reduction affected mainly Stage 2 and REM sleep while slow wave sleep was unchanged. In connection with night work 28% of the workers took a nap in the afternoon. These naps contained a large proportion of slow wave sleep and were, apparently, caused by the sleep deficit after the short main sleep period. The EEG recordings also revealed that 20% of the participants had sleep episodes during night work. These naps were as long as the afternoon naps, were experienced as "dozing offs" rather than naps, occurred at the time of the trough of the circadian wakefulness rhythm, and were concomitant with extreme subjective sleepiness and low rated work load. It was concluded that not only the sleep of shift workers was disturbed, but also the wakefulness--to the extent that sleepiness during night work sometimes reached a level where reasonable wakefulness could not be maintained. The latter observation is probably of special importance in work situations demanding a great responsibility for human lives or for great economic values.     

Transplantation of in vitro-generated<Emphasis Type="Italic"> Schistosoma mansoni</Emphasis> mother sporocysts into<Emphasis Type="Italic"> Biomphalaria glabrata</Emphasis>

Specific studies on schistosome gene functions require both access to the parasite stages, preferably the larvae, and to complete the life cycle. In the present study, we investigated whether short-term in vitro cultivation of sporocysts and surgical transplantation into snails could be combined to produce cercariae. Miracidia were maintained in vitro in the presence of Biomphalaria glabrata embryonic (Bge) cells or, alternatively, in Bge-cell-conditioned medium. The transformation of miracidia to mother sporocysts was observed in both cases. Two day-old sporocysts were transplanted into the cephalopedal sinus of recipient snails. Transplantation efficiencies varied between 16% and 43%, depending on the culture of the sporocysts in terms of the number of cercariae producing snails. Cercariae recovered from these snails were used to successfully infect hamsters, demonstrating that short term in vitro-generated sporocysts undergo normal cercariogenesis following transplantation. This combination of in vitro cultivation and transplantation may be useful for novel experimental approaches to investigate the genes involved in larval development or host-parasite molecular interactions.     

Somatic translocation and differential expression of Ig mu transgene copies implicate a role for the Igh locus in memory B cell development

Memory B cells of mice with Ig mu transgenes often carry transgene copies that have moved into the Igh locus via somatic translocation. This phenomenon has been attributed to a selection pressure for somatic hypermutations, which generally are observed at much higher frequencies in translocated copies than in ectopic copies. We tested this idea by immunizing Ig-mu transgenic mice in a manner designed to select B cells that required only one V(H) mutation for a switch in antigenic specificity and recruitment into the memory pool. Despite the minimal mutation requirement, hybridomas carrying somatic translocations to the Igh locus were obtained. Importantly, this occurred despite the fact that translocated and untranslocated mu-transgenes were mutated comparably. Evidently, a strong selection advantage was conferred upon B cells by the somatic translocations. Among the hybridomas, translocated mu-transgenes were active, while ectopic mu-transgenes were uniformly silent. The translocated copy that had conferred an affinity-based selection advantage was expressed at the highest level. Moreover, translocated copies were differentially expressed among hybridoma members, which belonged to a common post-mutational lineage. This suggests that adjustments in transgene expression levels had occurred during memory cell development. These results indicate that, apart from their potential influences on somatic hypermutagenesis and class switch recombination, elements in the Igh locus promote the selection of memory B cells in another way, possibly by regulating the level of Ig expression at various stages of antigen-driven differentiation.     

DNA immunization efficiently targets conserved functional domains of protease and ATPase/helicase of nonstructural 3 protein (NS3) of human hepatitis C virus

Nonstructural protein 3 (NS3) of human hepatitis C virus (HCV) is a conserved multi-functional protein essential for replication and translation of viral RNA and polyprotein processing. Early T-cell response against NS3 is capable of restricting viremia. We aimed at characterizing the immunogenicity in gene immunization of the conserved regions of NS3 critical for protein folding and activity. C57BL/6 mice were injected with NS3 gene of Russian HCV 1b isolate 274933RU. Immunization did not exert any overt histological changes and had no long-term effects on the immune status of NS3 gene-recipients. The immune response in NS3 gene-recipients was screened by antibody ELISA, T-cell proliferation test and immune assays for specific cytokine production. T-lymphocytes of NS3 gene-recipients proliferated in response to peptides representing conserved regions of protease and ATPase/helicase. Stimulated T-lymphocytes produced IL-2, and in response to protease-derived peptides, also IFN-gamma. Potent and long-lasting antibody response was raised against conserved NS3 regions including "Greek-key" motif of protease, motifs II, V and polynucleotide-binding domains of ATPase/helicase. Thus, gene immunization effectively targeted conserved regions critical for NS3 protease and helicase function. In type and specificity, immune response of NS3 gene-immunized mice mimicked immunity achieved in the acute self-limiting HCV infection of human and primates and in virus-exposed healthy individuals, indicating promiscuity of NS3 as immunogen.     

Robustness of the tuning of fly visual interneurons to rotatory optic flow

The sophisticated receptive field organization of motion-sensitive tangential cells in the visual system of the blowfly Calliphora vicina matches the structure of particular optic flow fields. Hypotheses on the tuning of particular tangential cells to rotatory self-motion are based on local motion measurements. So far, tangential cells have never been tested with global optic flow stimuli. Therefore we measured the responses of an identifiable neuron, the V1 tangential cell, to wide-field motion stimuli mimicking optic flow fields similar to those the fly encounters during particular self-motions. The stimuli were generated by a "planetarium-projector," casting a pattern of moving light dots on a large spherical projection screen. We determined the tuning curves of the V1-cell to optic flow fields as induced by the animal during 1) rotation about horizontally aligned body axes, 2) upward/downward translation, and 3) a combination of both components. We found that the V1-cell does not respond as specifically to self-rotations, as had been concluded from its receptive field organization. The neuron responds strongly to upward translation and its tuning to rotations is much coarser than expected. The discrepancies between the responses to global optic flow and the predictions based on the receptive field organization are likely due to nonlinear integration properties of tangential neurons. Response parameters like orientation, shape, and width of the tuning curve are largely unaffected by changes in rotation velocity or a superposition of rotational and translational optic flow.