Mutations in the N-terminal domains of nectin-1 and nectin-2 reveal differences in requirements for entry of various alphaherpesviruses and for nectin-nectin interactions

Nectin-1 and nectin-2 are related molecules that can function with different specificities as entry receptors for mammalian alphaherpesviruses through interaction with viral glycoprotein D (gD). The normal function of members of the nectin family is to mediate cell-cell adhesion through homotypic and heterotypic nectin-nectin interactions in cadherin-based adherens junctions. We examined mutations in three equivalent regions of the N-terminal V-like domains of nectin-1 and nectin-2 to test the effects on entry of various alphaherpesviruses, nectin-nectin interactions, and interactions of the mutant nectins with gD. Mutations in region I previously shown to severely impair herpes simplex virus (HSV) entry activity, but not pseudorabies virus (PRV) or bovine herpesvirus 1 (BHV-1) entry, did not reduce homotypic trans interactions for either nectin-1 or nectin-2 or binding of nectin-3 to nectin-1. Mutations in region II, patterned after a reported single-nucleotide polymorphism in nectin-2, enhanced intracellular accumulation of both nectin-1 and nectin-2 and had a deleterious effect on all of the activities under study. Mutations in region III previously shown to reduce homotypic trans interactions of nectin-2 impaired the entry of PRV and BHV-1 when introduced into either nectin-1 or nectin-2, but only the nectin-2 mutation reduced HSV entry activity. Binding of nectin-1 to nectin-3 was not affected. Effects of the nectin-1 and nectin-2 mutations on interactions with gD did not necessarily correlate with entry activity of the mutant receptors. We can conclude that structural requirements for HSV entry, PRV and BHV-1 entry, and homotypic and heterotypic trans interactions are all different despite the previously reported ability of HSV and HSV gD to inhibit trans interactions.     

Centrin: its secondary structure in the presence and absence of cations

Centrin is a low molecular mass (20 kDa) protein that belongs to the EF-hand superfamily of calcium-binding proteins. Local and overall changes were investigated for interactions between cations and Chlamydomonas centrin using Fourier transform infrared (FT-IR) and circular dichroic (CD) spectroscopies. FT-IR spectral features studied included the amide I' band and the side-chain absorbances for aspartate residues located almost exclusively at the calcium-binding sites in the spectral region of 1700-1500 cm(-1). The amide I' band is exquisitely sensitive to changes in protein secondary structure and is observed to shift from 1626.5 to 1642.7 cm(-1) in the presence and absence of calcium. These spectral bands are complex and were further studied using two-dimensional Fourier transform infrared (2D-FT-IR) correlation along with curve-fitting routines. Using these methods the secondary structure contributions were determined for holocentrin and apocentrin. The alpha-helical content in centrin was determined to be 60%-53% in the presence and absence of cations, respectively. Furthermore, the beta-strand content was determined to be 12%-36%, while the random coil component remained almost constant at 7%-13.5% in the presence and absence of cations, respectively. Changes in the side-chain band are mostly due to the monodentate coordination of aspartate to the cation. A shift of approximately 4 cm(-1) (for the COO- antisymmetric stretch in Asp) from 1565 to 1569 cm(-1) is observed for apocentrin and holocentrin, respectively. Thermal dependence revealed reversible conformational transition temperatures for apocentrin at 37 degrees C and holocentrin at 45 degrees C, suggesting greater stability for holocentrin.     

Cellular and cytokine immunoregulation in patients with chronic obstructive pulmonary disease and bronchial asthma

BACKGROUND: Different forms of chronic airway inflammation may involve diverse pathogenic elements. In general, deficient defence response is a feature of chronic obstructive pulmonary disease (COPD), whereas distorted immunoregulatory mechanisms lead to development of asthmatic symptoms. In addition to diverse effector mechanisms, the cellular and humoral elements participating in the development of immune response may appear to be different in COPD and bronchial asthma (BA) patients. AIMS: To evaluate the immunoregulatory properties of T cells and monocytes in cultures of peripheral blood mononuclear cells (PBMC) and to determine the chosen cytokine profiles in COPD and BA patients. METHODS: The microcultures of PBMC from COPD and BA patients were assessed for the T-cell response to mitogens, saturation of interleukin (IL)-2 receptors, T-cell suppressive activity and monokine influence on lymphocyte proliferation. Concomitantly, the cytokine (IL-1beta, interleukin-1 receptor antagonist, tumour necrosis factor-alpha, IL-4, IL-6, IL-8) concentrations were determined in the serum, the broncho-alveolar lavage fluid and in the culture supernatants. RESULTS: The T-lymphocyte reactions (response to phytohaemagglutinin, IL-2 receptor saturation, suppressive activity) were lower in BA patients than in COPD patients. Reversely, the immunogenic activity of monocytes (IL-1beta versus IL-1ra production) was higher in BA patients than in COPD patients. The highest values of cytokine concentrations were found in the culture supernatants. The concentrations of tumour necrosis factor-alpha, IL-4, IL-6 and IL-8 were significantly higher and the concentration of IL-1ra was lower in BA patients than in COPD patients. CONCLUSION: The assessments of cellular immunoregulatory properties and cytokine profiles in the cultures of blood mononuclear cells may prove helpful for diagnostic and therapeutic discrimination between BA and COPD patients.     

Small intestinal submucosa in abdominal wall repair after TRAM flap harvesting in a rat model

The strength of porcine small intestinal submucosa in abdominal wall repair after transverse rectus abdominis myocutaneous flap harvesting was examined in a rat model. Changes in the levels of selected molecular markers of inflammation after small intestinal submucosa implantation were also studied. Eighty-three rats were divided into three groups. In experimental group I, an abdominal wall defect created by removal of the rectus abdominis muscle was repaired with placement of a 1.5 x 5-cm2 patch of small intestinal submucosa. In experimental group II, the muscle defect was repaired with a combination of small intestinal submucosa patch placement and fascial closure. In the control group, the defect was repaired with direct fascial closure. At postoperative times of 3 days, 2 weeks, 1 month, and 2 months, the muscle tissues adjacent to the abdominal wall repair site were subjected to biopsies for assessment of inflammation markers. Full-thickness sections of the abdominal wall from the repair site in each animal were removed for tensile strength testing and histological examinations. The results demonstrated that interleukin-6 and interferon-gamma levels were increased in the two experimental, small intestinal submucosa-treated groups at 3 days and 2 weeks postoperatively. The results of mechanical testing demonstrated that the average tensile strength of the repaired abdominal wall in the repair model with combined small intestinal submucosa placement and fascial repair was significantly greater than the values for repairs with fascial closure or small intestinal submucosa placement alone. The use of small intestinal submucosa placement in combination with fascial repair can significantly improve the strength of the repaired abdominal wall after transverse rectus abdominis myocutaneous flap harvesting.     

Usefulness of testing histamine release from isolated human basophils in anti-allergic and anti-asthmatic drug assessment]

Histamine release from isolated human basophils test was used to evaluate an activity of: histamine receptors H1 and H2 blockers, agonists of beta-receptors, calcium channel blocking agents, hydrocortisone, and disodium cromoglycate (Intal). The study involved 84 patients hospitalized for the bronchial asthma. Basophils were isolated with Day's technique modified by Shov and Norn. Histamine was measured with Shov's spectrofluorimetric technique. It was found that histamine release from isolated human basophils may be used in both evaluation of the mechanism of action and efficiency of drugs used in allergic diseases therapy.     

Generation of Influenza Virus from Avian Cells Infected by Salmonella Carrying the Viral Genome

Domestic poultry serve as intermediates for transmission of influenza A virus from the wild aquatic bird reservoir to humans, resulting in influenza outbreaks in poultry and potential epidemics/pandemics among human beings. To combat emerging avian influenza virus, an inexpensive, heat-stable, and orally administered influenza vaccine would be useful to vaccinate large commercial poultry flocks and even migratory birds. Our hypothesized vaccine is a recombinant attenuated bacterial strain able to mediate production of attenuated influenza virus in vivo to induce protective immunity against influenza. Here we report the feasibility and technical limitations toward such an ideal vaccine based on our exploratory study. Five 8-unit plasmids carrying a chloramphenicol resistance gene or free of an antibiotic resistance marker were constructed. Influenza virus was successfully generated in avian cells transfected by each of the plasmids. The Salmonella carrier was engineered to allow stable maintenance and conditional release of the 8-unit plasmid into the avian cells for recovery of influenza virus. Influenza A virus up to 10⁷ 50% tissue culture infective doses (TCID₅₀)/ml were recovered from 11 out of 26 co-cultures of chicken embryonic fibroblasts (CEF) and Madin-Darby canine kidney (MDCK) cells upon infection by the recombinant Salmonella carrying the 8-unit plasmid. Our data prove that a bacterial carrier can mediate generation of influenza virus by delivering its DNA cargoes into permissive host cells. Although we have made progress in developing this Salmonella influenza virus vaccine delivery system, further improvements are necessary to achieve efficient virus production, especially in vivo.     

IL-33-Mediated Protection against Experimental Cerebral Malaria Is Linked to Induction of Type 2 Innate Lymphoid Cells,M2 Macrophages and Regulatory T Cells

Cerebral malaria (CM) is a complex parasitic disease caused by Plasmodium sp. Failure to establish an appropriate balance between pro- and anti-inflammatory immune responses is believed to contribute to the development of cerebral pathology. Using the blood-stage PbA (Plasmodium berghei ANKA) model of infection, we show here that administration of the pro-Th2 cytokine, IL-33, prevents the development of experimental cerebral malaria (ECM) in C57BL/6 mice and reduces the production of inflammatory mediators IFN-γ, IL-12 and TNF-α. IL-33 drives the expansion of type-2 innate lymphoid cells (ILC2) that produce Type-2 cytokines (IL-4, IL-5 and IL-13), leading to the polarization of the anti-inflammatory M2 macrophages, which in turn expand Foxp3 regulatory T cells (Tregs). PbA-infected mice adoptively transferred with ILC2 have elevated frequency of M2 and Tregs and are protected from ECM. Importantly, IL-33-treated mice deleted of Tregs (DEREG mice) are no longer able to resist ECM. Our data therefore provide evidence that IL-33 can prevent the development of ECM by orchestrating a protective immune response via ILC2, M2 macrophages and Tregs.