Subcutaneous immunization with a novel immunogenic candidate (urease) confers protection against Brucella abortus and Brucella melitensis infections

Brucellosis is a world prevalent endemic illness that is transmitted from domestic animals to humans. Brucella spp. exploits urease for survival in the harsh conditions of stomach during the gastrointestinal infection. In this study, we examined the immune response and the protection elicited by using recombinant Brucella urease (rUrease) vaccination in BALB/c mice. The urease gene was cloned in pET28a and the resulting recombinant protein was employed as subunit vaccine. Recombinant protein was administered subcutaneously and intraperitoneally. Dosage reduction was observed with subcutaneous (SC) vaccination when compared with intraperitoneal (IP) vaccination. rUrease induced mixed Th1–Th2 immune responses with high titers of specific IgG1 and IgG2a. In lymphocyte proliferation assay, splenocytes from IP and SC‐vaccinated mice displayed a strong recall proliferative response with high amounts of IL‐4, IL‐12 and IFN‐γ production. Vaccinated mice were challenged with virulent Brucella melitensis, B. abortus and B. suis. The SC vaccination route exhibited a higher degree of protection than IP vaccination (p value ≤ 0.05). Altogether, our results indicated that rUrease could be a useful antigen candidate for the development of subunit vaccines against brucellosis.     

Kombucha tea ameliorates experimental autoimmune encephalomyelitis in mouse model of multiple sclerosis

Experimental autoimmune encephalomyelitis (EAE) is a mouse model for multiple sclerosis (MS), in which an inflammatory demyelination and axonal damage occurs. Kombucha tea is a fermented beverage made from kombucha mushroom, brewed tea, and sugar. In recent years kombucha tea has attracted interest due to its pharmacological properties like antioxidant effects. The aim of the present research was to test the therapeutic effect of kombucha tea in EAE. We induced EAE model in 18 female C57BL/6 mice by inoculation of myelin oligodendrocyte glycoprotein-35-55 (MOG_35-55) in complete Freund’s adjuvant emulsion. Then, in order to ameliorate EAE symptoms, we used kombucha tea. During the course of study clinical evaluation was assessed, and on the day 21 post-immunization, for evaluation of nitric oxide (NO), total antioxidants capacity and tumor necrosis factor-alpha (TNF-α), blood samples were taken from the heart of mice. The mice were sacrificed and brains and cerebellums of mice were removed for histological analysis. Our findings demonstrated that kombucha tea had beneficial effects on EAE by lower incidence, attenuation in the severity, and also a delay in the onset of disease. Histological analysis showed that inflammatory criteria including the number of infiltrated immune cells and plaques as well as demyelination in kombucha tea dosed mice were significantly lower than the control group. Also, in comparison with control mice, the serum levels of NO and TNF-α in kombucha tea-treated mice were significantly decreased. Kombucha tea with its potential therapeutic effects and immunomodulatory properties might be proposed, after additional necessary tests and trials, for treatment of MS.     

Expression of human immunodeficiency virus type 1gag gene using genetically engineered herpes simplex virus type 1 recombinants

Infectious herpes simplex virus type 1 (HSV-1) recombinants were constructed by inserting the cDNA sequence of the human immunodeficiency virus type 1 (HIV-1)gag gene (from nucleotide position 675 [SacI] to 3859 [Asp 718] of the cDNA sequences of HIV-1 strain BH-10) within the DNA sequences of theBamHI DNA fragment B of the genome of an apathogenic HSV-1 strain HFEM. This HSV-1 strain possesses a 4.1-kbp deletion within theBamHI DNA fragment B between 0.762 and 0.789 map units of the viral genome, which allows the insertion of at least 4 kbp of foreign genetic material into this particular region. The DNA sequences of the immediate early promoter (IE4) of HSV-1 that were inserted upstream from thegag gene were used as a promoter. The screening of 205 virus stocks derived from individual plaques revealed that 46 recombinant viruses harbor HIV-1gag-specific DNA sequences. However, it was found that only six of the recombinant viruses are able to express thegag gene product of HIV-1. This indicates that the ratio of the positive recombination events is about 2.9%.     

Identification of a Thymidylate Synthase Gene within the Genome of Chilo Iridescent Virus

The thymidylate synthase (TS, EC 2.1.1.45) is essential for the de novo synthesis of dTMP in pro- and eucaryotic organisms. Consequently it plays a major role in the replication of the DNA genome of a cell or a DNA virus. The gene encoding the TS of Chilo iridescent virus (CIV) was identified by nucleotide sequence analysis of the viral genome and was mapped within the EcoRI CIV DNA fragments G and R. Computer assisted analysis of the DNA nucleotide sequence between the genome coordinates 0.482 and 0.489 revealed an open reading frame (ORF) of 885 nucleotides. This ORF was found to encode a polypeptide of 295 amino acid residues (33.9 kDa) that showed significant homologies to known TS of different species including mammals, plants, fungi, protozoa, bacteria, and DNA viruses. The highest amino acid homologies were found between the CIV-TS and the TS of herpesvirus ateles (54.0%), Saccharomyces cerevisiae (51.8%), herpesvirus saimiri (51.0%), rhesus monkey rhadinovirus (50.7%), mouse (50.5%), rat (50.2%), varicella-zoster virus (50.2%), equine herpesvirus 2 (50.0%), and the human TS (48.4%). The CIV-TS contains six amino acid domains that are highly conserved in the TS of other species. Within these domains the major amino acid residues are present for which a functional role has been reported. The CIV-TS was found to be more closely related to the TS of eucaryotes than to the TS of procaryotes indicating the phylogenetic origin of the CIV-TS gene. The identification of a TS gene in the genome of CIV is the first report of a viral TS that is not encoded by a herpesvirus or a bacteriophage.     

Glial cell and fibroblast cytotoxicity study on 4026-cyclotene photosensitive benzocyclobutene (BCB) polymer films

Photosensitive benzocyclobutene (photo-BCB) is a class of polymers with the trade name Cyclotene. The photoimagable property of Cyclotene makes it suitable for the manufacture of microelectronic devices. The motivation behind this study is that we see an exciting application of photo-BCB as substrates in implantable microelectronic biomedical devices due to several desirable properties distinctive from other polymer materials. To our knowledge, however, photo-BCB has never been tested for biomedical implant applications, as evidenced by the lack reported data on its biocompatibility. This study takes the first step towards assessing photo-BCB biocompatibility by evaluating the cytotoxicity and cell adhesion behavior of Cyclotene 4026 coatings exposed to monolayers of glial and fibroblast cells in vitro. It can be concluded from these studies that photo-BCB films deposited on silicon wafers using microfabrication processes did not adversely affect 3T3 fibroblast and T98-G glial cell function in vitro. We also successfully rendered photo-BCB films non-adhesive (no significant fibroblast or glial cell adhesion) with surface immobilized dextran using methods developed for other biomaterials and applications. Future work will further develop prototype photo-BCB microelectrode devices for chronic neural implant applications.     

Early responses of skeletal muscle in recovery from hypothyroidism

Postnatal growth of skeletal muscle (m. gastrocnemius) was compared in rats under euthyroid, hypothyroid and hypothyroid-rehabilitated conditions. In normal (euthyroid) animals, gastrocnemius muscle grows significantly in terms of weight (150 x) from birth to the young adult and, in terms of total contractile myofibril protein (15 x) and myosin ATPase activity (10 x) between days 25 and 90. Rats made hypothyroid (with 0.1% w/v propylthiouracil, PTU) from birth show reduced growth. At 25 days (weaning), compared with euthyroid, muscle weight is only 25% of normal, and a similar reduction is found in total DNA, RNA, protein, myofibril protein, and myosin ATPase activity. These deficits, already significant by day 10, are more marked by day 50 due to the near arrest of growth. Hypothyroid rats allowed to recover by PTU withdrawal after day 25 (rehabilitated) undergo marked compensatory muscle growth. By day 90, muscle weight and protein content increase 50 x, DNA 7 x and RNA 17 x. Over this period, total myofibrillar protein and myosin ATPase increase 20-40 x, but are still below those of 90-day controls, suggesting that the severe growth retardation had not yet been fully compensated. Early thyroid deficiency drastically reduces the normal age-related growth of skeletal muscle and severely retards the development of contractile elements, affecting muscle hypertrophy (protein content) more than cell proliferation (DNA content). Rehabilitation compensates to a major degree for this growth retardation. These results underline the key role of thyroid hormones in regulating development and maturation of skeletal muscle throughout the preweaning and postweaning phases of growth.     

Molecular characterization and determination of the coding capacity of the genome of equine herpesvirus type 2 between the genome coordinates 0.235 and 0.258 (theEcoRI DNA fragment N; 4.2 kbp)

The complete DNA nucleotide sequence of theEcoRI DNA fragment N (0.235 to 0.258 viral map units) of equine herpes virus type 2 (EHV-2) strain T400/3 was determined. This DNA fragment comprises 4237 bp with a base composition of 55.23% G+C and 44.77% A+T. Nineteen open reading frames (ORFs) of 50-287 amino acid (aa) residues were detected. ORF number 10 is located between the nucleotide position 2220 and 2756 coding for a protein of 179 amino acid residues. This protein shows significant homology to the cytokine synthesis inhibitory factor (CSIF; interleukin 10) of human (76.4%) and mouse (68.5%), and to the Epstein-Barr virus (EBV) protein BCRF1 (70.6%). The existence of an interleukin 10 (IL-10) analogous gene within the genome of the EHV-2 was confirmed by screening the genome of nine EHV-2 strains using specific oligonucleotide primers corresponding to the 5 and 3 region of this particular gene by polymerase chain reaction. In all experiments an 870 bp DNA product was amplified. The specifity of the amplified DNA fragments obtained from individual EHV-2 strains was confirmed by DNA-DNA hybridization experiments. The DNA sequence analysis of the amplified DNA products of the EHV-2 strain LK was carried out. This analysis revealed the identity of the corresponding IL-10 gene (540 bp) of this strain to the IL-10 gene of EHV-2 strain T400/3. The presented data indicate that the EHV-2 genome harbors a viral interleukin 10-like gene. This is further evidence that the IL-10 gene can be present in the genomes of members of the Herpesviridae family.     

Contribution of CD4+ and CD8+ T lymphocyte subsets to the cytokine secretion patterns induced in mice during sensitization to contact and respiratory chemical allergens.

Chemical allergens of different types, those that cause in humans allergic contact dermatitis or occupational asthma induce in mice divergent immune responses characteristic, respectively, of T-helper 1 (Th1)- and Th2-type cell activation. Such responses are associated with the development of different cytokine secretion patterns by draining lymph node cells (LNC), such that contact allergens stimulate vigorous interferon-gamma (IFN-gamma) production, but little secretion of the Th2 cytokines interleukin-4 and interleukin-10 (IL-4 and IL-10), whereas the converse pattern is provoked by respiratory allergens. Using selective depletion with antibody and complement we have here examined the relative contribution of CD4+ and CD8+ T lymphocytes to the cytokine secretion patterns of draining LNC isolated from mice sensitized to chemical allergens. Mice received repeated topical applications of respiratory allergens, trimellitic anhydride (TMA) or diphenylmethane diisocyanate (MDI), or of contact allergens 2,4-dinitrochlorobenzene (DNCB) or formaldehyde. Thirteen days following the initiation of exposure the production by draining LNC of IL-10, IFN-gamma and mitogen (concanavalin A)-inducible IL-4 was measured by enzyme-linked immunosorbent assay (ELISA) after various periods of culture. It was found that the high levels of IL-4 and IL-10 secretion stimulated by TMA or MDI, and the lower levels of these cytokines induced by DNCB or formaldehyde, were in all cases dependent upon the presence of CD4- cells. In contrast, the comparatively high concentrations of IFN-gamma observed following exposure to contact allergens were found to be derived from CD4+ cells, and in the case of DNCB from CD8+ cells also. The low levels of IFN-gamma induced by treatment with TMA or MDI were associated largely or wholly with CD8+ cells. These data indicate that the type 2 cytokine responses induced to different extents by both contact and respiratory chemical allergens are almost exclusively a function of CD4+ cells, but that IFN-gamma is produced by either CD4+ cells in the case of contact allergens or largely by CD8+ cells in the case of chemical respiratory allergens.     

DNA polymerase gene locus of <Emphasis Type="Italic">Cercopithecine herpesvirus 1</Emphasis> is a suitable target for specific and rapid identification of viral infection by PCR technology

The family Herpesviridae comprises at least 100 herpesviruses. Numerous human and animal pathogenic herpesviruses have been identified so far, including Cercopithecine herpesvirus 1 (CeHV-1). This virus is a member of the subfamily Alphaherpesvirinae and is the most hazardous herpesvirus to man. CeHV-1 is also known as B-virus or monkey B virus and as Herpesvirus simiae. In order to gain more genetic information, the viral DNA polymerase (DPOL) gene was identified using polymerase chain reaction (PCR) and DNA nucleotide sequence analysis. The deduced amino acid sequence contains the motifs and signatures that are typical for the B-family of DPOLs. The DPOL gene of CeHV-1 was found to be a suitable target for the specific and rapid identification of the Cercopithecine herpesvirus 1 infection by PCR technology. Comparative analysis of the DNA sequences of the DPOL gene loci of CeHV-1, Human herpesvirus 1 and 2 (HHV-1 and HHV-2), and other herpesviruses was carried out for determination of unique genomic regions of the individual DPOL genes. A primer set of 12 primers was used for screening the DNA of CeHV-1, HHV-1, and HHV-2 by detailed PCR. It was found that six out of twelve primer combinations are able to detect specifically the CeHV-1 genome without cross reactivity with the genome of HHV-1 and/or HHV-2. The specificity of the individual amplified DNA fragments was confirmed by DNA nucleotide sequence analysis. The results of these studies indicate that the six primer combinations of the specific CeHV-1 DPOL primer set is the method of choice for a rapid, precise and specific identification of a CeHV-1 infection by PCR. Due to the fact that this specific CeHV-1 DPOL primer set does not amplify any DNAs of HHV-1 or HHV-2 genome this technology is stressing and can be successfully used unlimited and more credible in all laboratories with PCR technical facility routinely for detection of a CeHV-1 infection in vivo or in vitro.The GenBank Accession No. of the sequence of DNA polymerase gene of Cercopithecine herpesvirus 1 (CeHV-1) reported in this study is AY568415, DPOL protein ID AAT67222; nuclear phosphoprotein ID AAT67223     

Differential expression of FIZZ1 and Ym1 in alternatively versus classically activated macrophages

Alternatively activated macrophages (aaMphi) display molecular and biological characteristics that differ from those of classically activated macrophages (caMphi). Recently, we described an experimental model of murine trypanosomosis in which the early stage of infection of mice with a Trypanosoma brucei brucei variant is characterized by the development of caMphi, whereas in the late and chronic stages of infection, aaMphi develop. In the present study, we used suppression subtractive hybridization (SSH) to identify genes that are expressed differentially in aaMphi versus caMphi elicited during infection with this T. b. brucei variant. We show that FIZZ1 and Ym1 are induced strongly in in vivo- and in vitro-elicited aaMphi as compared with caMphi. Furthermore, we demonstrate that the in vivo induction of FIZZ1 and Ym1 in macrophages depends on IL-4 and that in vitro, IFN-gamma antagonizes the effect of IL-4 on the expression of FIZZ1 and Ym1. Collectively, these results open perspectives for new insights into the functional properties of aaMphi and establish FIZZ1 and Ym1 as markers for aaMphi.