Development and migration of protective CD8+ T cells into the nervous system following ocular herpes simplex virus-1 infection

After infection of epithelial surfaces, HSV-1 elicits a multifaceted antiviral response that controls the virus and limits it to latency in sensory ganglia. That response encompasses the CD8(+) T cells, whose precise role(s) is still being defined; immune surveillance in the ganglia and control of viral spread to the brain were proposed as the key roles. We tracked the kinetics of the CD8(+) T cell response across lymphoid and extralymphoid tissues after ocular infection. HSV-1-specific CD8(+) T cells first appeared in the draining (submandibular) lymph node on day 5 and were detectable in both nondraining lymphoid and extralymphoid tissues starting on day 6. However, although lymphoid organs contained both resting (CD43(low)CFSE(high)) and virus-specific cells at different stages of proliferation and activation, extralymphoid sites (eye, trigeminal ganglion, and brain) contained only activated cells that underwent more than eight proliferations (CD43(high)CFSE(neg)) and promptly secreted IFN-gamma upon contact with viral Ags. Regardless of the state of activation, these cells appeared too late to prevent HSV-1 spread, which was seen in the eye (from day 1), trigeminal ganglia (from day 2), and brain (from day 3) well before the onset of a detectable CD8(+) T cell response. However, CD8(+) T cells were critical in reducing viral replication starting on day 6 and for its abrogation between days 8 and 10; CD8-deficient animals failed to control the virus, exhibited persisting high viral titers in the brain after day 6, and died of viral encephalitis between days 7 and 12. Thus, CD8(+) T cells do not control HSV-1 spread from primary to tertiary tissues, but, rather, attack the virus in infected organs and control its replication in situ.     

Cardiopulmonary effects of hemorrhagic shock in splenic autotransplanted pigs: a new surgical model

The spleen is an important organ for hemodynamic compensation during hemorrhagic shock. The aim of the study was to compare the hemodynamic and metabolic responses of sham-operated pigs with intact spleen, splenectomized pigs, and splenic autotransplanted pigs during hemorrhagic shock. Hemorrhagic shock was induced by 30% total blood volume bleed in sham-operated, splenectomized and splenic autotransplanted pigs (n = 20). Cardiopulmonary and metabolic variables were measured before, immediately after, and at 20, 60 and 100 minutes after hemorrhage. Upon hemorrhagic shock induction, body temperature, mean arterial pressure, mean pulmonary arterial pressure, cardiac output, cardiac index and oxygen delivery decreased, while lactate and shock index increased. Hemoglobin and hematocrit were significantly lower in the splenectomized and splenic autotransplant groups as compared with the control group at 60 and 100 minutes after hemorrhage (p < 0.05). Unlike intact spleen, splenic autotransplant could not improve hemodynamic parameters in hemorrhagic shock in pigs. In comparison to mice, rats or dogs, this species could be an interesting investigation model to test new surgical procedures during splenic related hemorrhagic shock, with potential applications in human medicine.     

Versatile Loops in Mycocypins Inhibit Three Protease Families

Mycocypins, clitocypins and macrocypins, are cysteine protease inhibitors isolated from the mushrooms Clitocybe nebularis and Macrolepiota procera. Lack of sequence homology to other families of protease inhibitors suggested that mycocypins inhibit their target cysteine protease by a unique mechanism and that a novel fold may be found. The crystal structures of the complex of clitocypin with the papain-like cysteine protease cathepsin V and of macrocypin and clitocypin alone have revealed yet another motif of binding to papain like-cysteine proteases, which in a yet unrevealed way occludes the catalytic residue. The binding is associated with a peptide-bond flip of glycine that occurs before or concurrently with the inhibitor docking. Mycocypins possess a β-trefoil fold, the hallmark of Kunitz-type inhibitors. It is a tree-like structure with two loops in the root region, a stem comprising a six-stranded β-barrel, and two layers of loops (6 + 3) in the crown region. The two loops that bind to cysteine cathepsins belong to the lower layer of the crown loops, whereas a single loop from the crown region can inhibit trypsin or asparaginyl endopeptidase, as demonstrated by site-directed mutagenesis. These loops present a versatile surface with the potential to bind to additional classes of proteases. When appropriately engineered, they could provide the basis for possible exploitation in crop protection.     

Intracellular proteolytic activity of cathepsin B is associated with capillary-like tube formation by endothelial cells in vitro

The lysosomal cysteine protease cathepsin B is implicated in degradation of extracellular matrix (ECM), a crucial step in a variety of physiological and pathological processes, including tumor dissemination and angiogenesis. In this study, we analyzed the contribution of extracellular and intracellular cathepsin B activity on the formation of capillary-like tubular structures by human umbilical vein endothelial cells (HUVECs) grown on Matrigel matrix, using general and specific cysteine protease inhibitors. We demonstrated, by confocal assay using quenched fluorescent protein substrate DQ-collagen IV, that endothelial cells degrade ECM both intracellularly and pericellularly. Intracellular cathepsin B activity detected by degradation of Z-Arg-Arg cresyl violet substrate was co-localized with the products of DQ-collagen IV degradation in the perinuclear region and in the capillary-like tubular structures. Treatment of cells with membrane-permeable CA-074 Me effectively abolished intracellular cathepsin B activity, and resulted in reduced tube length (32.3+/-9.4% at 10 microM), total tubule area (49.6+/-12.4% at 10 microM), and the number of branch points of tubules (47.5+/-7.7% at 10 microM) in a dose-dependent manner. In contrast, CA-074 (0.1-10 microM), a membrane-impermeable cathepsin B specific inhibitor, general cysteine protease inhibitors chicken cystatin (5 microM) and E-64 (10 microM), and the metalloprotease inhibitor Minocycline (10 microM) showed no significant inhibitory effect in our angiogenesis model. These results show that, besides multiple regulatory molecules, intracellular cathepsin B also contributes to the neovascularization process and should be considered as a potential therapeutic target.     

The influence of differential processing of procathepsin H on its aminopeptidase activity, secretion and subcellular localization in human cell lines

Cathepsin H is a unique member of the cysteine cathepsins that acts primarily as an aminopeptidase. Like other cysteine cathepsins, it is synthesized as an inactive precursor and activated by proteolytic removal of its propeptide. Here we demonstrate that, in human cells, the processing of the propeptide is an autocatalytic, multistep process proceeding from an inactive 41kDa pro-form, through a 30kDa intermediate form, to the 28kDa mature form. Tyr87P and Gly90P were identified as the two major endopeptidase cleavage sites, converting the 30kDa form into the mature 28kDa form. The level of processing differs significantly in different human cell lines. In monocyte-derived macrophages U937 and prostate cancer cells PC-3, the 28kDa form is predominant, whereas in osteoblasts HOS the processing from the 30kDa form to the 28kDa form is significantly lower. The aminopeptidase activity of the enzyme and its subcellular localization are independent of the product, however the 30kDa form was not secreted in HOS cells. The activity of the resulting cathepsin H in U937 cells was significantly lower than that in HOS cells, presumably due to the high levels of endogenous cysteine protease inhibitor cystatin F present specifically in this cell line. These results provide an insight into the dependence of human cathepsin H processing and regulation on cell type.     

CNTNAP2 and Language Processing in Healthy Individuals as Measured with ERPs

The genetic FOXP2-CNTNAP2 pathway has been shown to be involved in the language capacity. We investigated whether a common variant of CNTNAP2 (rs7794745) is relevant for syntactic and semantic processing in the general population by using a visual sentence processing paradigm while recording ERPs in 49 healthy adults. While both AA homozygotes and T-carriers showed a standard N400 effect to semantic anomalies, the response to subject-verb agreement violations differed across genotype groups. T-carriers displayed an anterior negativity preceding the P600 effect, whereas for the AA group only a P600 effect was observed. These results provide another piece of evidence that the neuronal architecture of the human faculty of language is shaped differently by effects that are genetically determined.     

Using the Simulated Patient Methodology to Assess Paracetamol-Related Counselling for Headache

Objectives

Firstly, to assess paracetamol-related counselling. Secondly, to evaluate the patient’s approach as a determinant of counselling and to test the acceptability of the simulated patient method in Slovenian pharmacies.

Methods

The simulated patient methodology was used in 17 community pharmacies. Three scenarios related to self-medication for headaches were developed and used in all participating pharmacies. Two scenarios were direct product requests: scenario 1: a patient with an uncomplicated short-term headache; scenario 2: a patient with a severe, long-duration headache who takes paracetamol for too long and concurrently drinks alcohol. Scenario 3 was a symptom-based request: a patient asking for medicine for a headache. Pharmacy visits were audio recorded and scored according to predetermined criteria arranged in two categories: counselling content and manner of counselling. The acceptability of the methodology used was evaluated by surveying the participating pharmacists.

Results

The symptom-based request was scored significantly better (a mean 2.17 out of a possible 4 points) than the direct product requests (means of 1.64 and 0.67 out of a possible 4 points for scenario 1 and 2, respectively). The most common information provided was dosage and adverse effects. Only the symptom-based request stimulated spontaneous counselling. No statistically significant differences in the duration of the consultation between the scenarios were found. There were also no significant differences in the quality of counselling between the Masters of Pharmacy and Pharmacy Technicians. The acceptability of the SP method was not as high as in other countries.

Conclusion

The assessment of paracetamol-related counselling demonstrates room for practice improvement.     

Cytomegalovirus Infection Impairs Immune Responses and Accentuates T-cell Pool Changes Observed in Mice with Aging

Prominent immune alterations associated with aging include the loss of naïve T-cell numbers, diversity and function. While genetic contributors and mechanistic details in the aging process have been addressed in multiple studies, the role of environmental agents in immune aging remains incompletely understood. From the standpoint of environmental infectious agents, latent cytomegalovirus (CMV) infection has been associated with an immune risk profile in the elderly humans, yet the cause-effect relationship of this association remains unclear. Here we present direct experimental evidence that mouse CMV (MCMV) infection results in select T-cell subset changes associated with immune aging, namely the increase of relative and absolute counts of CD8 T-cells in the blood, with a decreased representation of the naïve and the increased representation of the effector memory blood CD8 T-cells. Moreover, MCMV infection resulted in significantly weaker CD8 responses to superinfection with Influenza, Human Herpes Virus I or West-Nile-Virus, even 16 months following MCMV infection. These irreversible losses in T-cell function could not be observed in uninfected or in vaccinia virus-infected controls and were not due to the immune-evasive action of MCMV genes. Rather, the CD8 activation in draining lymph nodes upon viral challenge was decreased in MCMV infected mice and the immune response correlated directly to the frequency of the naïve and inversely to that of the effector cells in the blood CD8 pool. Therefore, latent MCMV infection resulted in pronounced changes of the T-cell compartment consistent with impaired naïve T-cell function.