Early-life dietary spray-dried plasma influences immunological and intestinal injury responses to later-life Salmonella typhimurium challenge.

Increasing evidence supports the concept that early-life environmental influences, including nutrition and stress, have an impact on long-term health outcomes and disease susceptibility. The objective of the present study was to determine whether dietary spray-dried plasma (SDP), fed during the first 2 weeks post-weaning (PW), influences subsequent immunological and intestinal injury responses to Salmonella typhimurium challenge. A total of thirty-two piglets (age 16-17 d) were weaned onto nursery diets containing 0, 2·5 % SDP (fed for 7 d PW) or 5 % SDP (fed for 14 d PW), and were then fed control diets (without SDP), for the remainder of the experiment. At 34 d PW (age 50 d), pigs were challenged with 3 × 109 colony-forming units of S. typhimurium. A control group (non-challenged) that was fed 0 % SDP in the nursery was included. At 2 d post-challenge, the distal ileum was harvested for the measurement of inflammatory, histological and intestinal physiological parameters. S. typhimurium challenge induced elevated ileal histological scores, myeloperoxidase (MPO), IL-8 and TNF, and increased intestinal permeability (indicated by reduced transepithelial voltage (potential difference) and elevated 4 kDa fluorescein isothiocyanate dextran (FD4) flux rates). Compared with S. typhimurium-challenged controls (0 % SDP), pigs fed the 5 % SDP-14 d diet exhibited reduced ileal histological scores, MPO levels, IL-8 levels and FD4 flux rates. Pigs fed the 5 % SDP-14 d nursery diet exhibited increased levels of plasma and ileal TNF-α in response to the challenge, compared with the other treatments. These results indicate that inclusion of SDP in PW diets can have an influence on subsequent immunological and intestinal injury responses induced by later-life S. typhimurium challenge.

Morphologic and molecular analysis of 39 spontaneous feline pulmonary carcinomas.

The present study was performed to determine the morphologic change and selected molecular features of spontaneous lung tumors in cats examined at the North Carolina State University Veterinary Teaching Hospital. Thirty-nine primary lung carcinomas represented 0.69% of all feline cases admitted to the hospital. Most lung tumors were observed in aged cats (P < .0001), and no sex predilection was found (P < .4241). Persian cats with pulmonary carcinoma were overrepresented in the data set, at least 4 times more frequently than other breeds. The histologic tumor types included adenocarcinoma (64.1%), bronchioloalveolar carcinoma (20.5%), and adenosquamous carcinoma (15.4%). Metastasis was observed in about 80% of 39 cases, with decreasing order of intrapulmonary metastasis, intrathoracic carcinomatosis, regional lymph nodes, and distant organs, including digits. The size of the largest tumor mass was significantly associated with metastatic potential (P < .001). Based on immunohistochemistry, more than 80% (20 of 24) of feline lung tumors were positively labeled with either surfactant protein A or thyroid transcription factor 1. Epidermal growth factor receptor mutant and p53 proteins were detected in approximately 20% (5 of 24) and 25% (6 of 24) of the feline lung tumor cases, respectively. Limited sequencing analysis of K-ras and p53 genes in 3 selected normal and neoplastic lung tissues did not reveal any alteration. Results indicate that primary lung carcinomas are rare but aggressive tumors in cats, thereby warranting further studies on molecular carcinogenesis.

Morphometry of A1 segment of the anterior cerebral artery and its clinical importance.

BACKGROUND AND AIM: Anterior cerebral artery, one of the terminal branches of the internal carotid artery is an important vessel taking part in the formation of circle of Willis. It supplies a large part of the medial surface of the cerebral hemisphere containing the areas of motor and somatosensory cortices of the lower limb. Aim of this study was the morphometry of A1 segment of the anterior cerebral artery. MATERIALS AND METHODS: 93 formalin fixed brain specimen of either sex and of Indian origin were studied. The mean length, mean external diameter and the anomalies present in A1 segment of the vessel were studied in detail and photographed. RESULTS: The mean length of A1 segment of the vessel was 14.49+/-0.28 mm and 14.22+/-0.22 mm on right and left side respectively. The mean external diameter of the vessel on right and left side was 2.12+/-0.07 mm and 2.32+/-0.06 mm respectively. Narrowing, aneurysm formation, buttonhole formation and median anterior cerebral artery were the anomalies seen with an occurrence of 15.05%, 5.37%, 3.22% and 12.9%, respectively. The above anomalies did not have any sex or side predilection. CONCLUSION: Knowledge of morphometry of the vessel will be of use to neurosurgeons while performing the shunt operation, in assessing the feasibility of such operations and in the choice of patients. From this study we infer that the morphometry of anterior cerebral artery varies in different population and that the neurosurgeons operating should have a thorough knowledge of the possible variations.

A rare variation of the profunda femoris vein in the popliteal fossa.

The profunda femoris artery is normally accompanied by a profunda femoris vein (deep femoral vein), which begins at the adductor magnus with various tributaries and drains into the femoral vein at the femoral triangle. Very rarely, the profunda femoris vein establishes communication with the popliteal vein. We present an anomalous profunda femoris vein in a 62-year-old male cadaver whose vein was located in the popliteal fossa as a direct communicating channel between the popliteal vein and the femoral vein.

Clinically-relevant variations of the carotid arterial system.

INTRODUCTION: Developmental anomalies in the origin and branching pattern of the external carotid artery are not common. The level of the bifurcation of the common carotid artery and also the variations in the origin/branching pattern of the external carotid artery are well known and documented. METHODS: The variations in the level of bifurcation of the common carotid artery and the branching pattern of the external carotid artery were studied on 95 cadavers (52 male and 43 female). The common, external and internal carotid arteries were dissected on both sides. The level of carotid bifurcation was determined by comparison with the cervical vertebrae. Branching patterns of the carotid arteries were examined. RESULTS: Apart from the textbook description of the arteries, we came across several interesting variations. The bifurcation level of the common carotid artery was determined to be 50 percent at the C3 level, 40 percent at the C4 level and ten percent at the C2 level on the right side, and 55 percent at the C3 level, 35 percent at the C4 level, one percent at the C5 level and nine percent at the C2 level on the left side. CONCLUSION: Anatomical knowledge of the origin, course, and branching pattern of the external carotid artery, as well as the level of bifurcation of the common carotid artery, will be useful to surgeons when ligating the vessels in the head and neck regions during surgery and to avoid unnecessary complications during carotid endarterectomy.

Induction of apoptosis by iridovirus virion protein extract.

Chilo iridescent virus (CIV; IIV-6) is the type member of the genus Iridovirus (family Iridoviridae, large icosahedral cytoplasmic DNA viruses). CIV induces death and deformity in the cotton boll weevil, Anthonomus grandis, replicates productively in larvae of the cotton boll weevil, and significantly reduces laboratory populations of the cotton aphid, Aphis gossypii. CIV virion protein extract (CVPE) shuts down host protein synthesis in several insect cell lines and induces mortality in neonate boll weevil larvae. We report here that CVPE induces apoptosis in spruce budworm and boll weevil cell lines, as detected by blebbing, DNA fragmentation, and TUNEL assay. Tissue culture toxicity dose assays (TCTD(50)) showed that spruce budworm cells were eight times more sensitive to CVPE than boll weevil cells. Pancaspase inhibitor suppressed apoptosis but had marginal effect on inhibition of host protein synthesis. Moreover, the CVPE dose for apoptosis was 1000-fold lower than the dose for shutdown of host synthesis. We also detected protein kinase activity in CVPE. Heating CVPE at 60 degrees C for 30 min destroyed all three activities. Our results suggest that one or more polypeptides in CIV induce apoptosis. This is the first study demonstrating apoptosis induction by a member of the genus Iridovirus and by virion extracts of a member of the family Iridoviridae.

Cadavers as teachers in medical education: knowledge is the ultimate gift of body donors.

In most of the medical colleges in India, unclaimed bodies from various mortuaries reach the dissection hall; and here, the body donors club has yet to gain the desired dimensions. In spite of all the adverse circumstances, the cadaver and the dissection both have survived the most rigorous test of pedagological fitness--the test of time. Today, many of the Western countries have long donor waiting lists where cadavers are acquired as anatomical gifts or through body donor programmes. Thailand's approach to body donors offers a role model for resolving the present situation. The spirit of volunteerism reflects the drastic shift in public perception and a global change in approach is needed in the present time.

Transcriptional mapping in Chilo iridescent virus infections.

Chilo iridescent virus (CIV) belongs to the family Iridoviridae, which are icosahedral cytoplasmic DNA viruses with large, linear, and circularly permuted genomes. Previous studies on infected-cell-specific polypeptides suggested temporal regulation of CIV gene expression. Recently, we demonstrated three temporal classes at the transcriptional level, in CIV infections of a spruce budworm cell line. We also demonstrated a transcriptional cascade with positive and negative control. In this paper, we assign all detectable viral transcripts into respective temporal classes and map them using restriction fragments from a genomic library. More than 90 percent of the genome is transcriptionally active with at least four major clusters of immediate-early transcription and at least three delayed-early clusters. Late transcripts were observed throughout the genome. There was at least one exclusive region in the genome for each of the three temporal classes. We correlated transcribed regions with ORFs on the CIV genome and showed that known ORFs in the exclusive regions are generally consistent with phase-specific requirements of large DNA viruses. Our data also suggest the presence of 5' or 3' coterminal transcripts. This is the first complete transcription map for a member of the genus Iridovirus.

Antibody and pre- plus post-transplant prednisone treatments support T cell-depleted stem cell engraftment without drug-induced morbidity.

Rigorous T cell depletion methods can now be used to reduce the risk of graft-versus-host disease (GVHD) associated with allogeneic, hematopoietic stem cell transplantation (HSCT). However, full T cell depletion is also associated with a significant risk of graft failure. Here we hypothesize that engraftment failures after T cell-depleted HSCT may be due, in part, to the absence of GVHD prophylaxis. To test this hypothesis, we used a haploidentical mouse model to systematically measure the effects of immunosuppressive drug treatments and anti-T cell antibodies on engraftment. Results showed that engraftment was supported in all animals when hosts were pre-treated with anti-T cell antibodies, but donor chimerism was significantly improved when hosts were also treated with prednisone. Interestingly, when hosts received only pre-HSCT prednisone treatments, engraftment was not improved; when hosts received only post-HSCT prednisone (initiated near the time of irradiation), the animals became extremely ill. Results therefore demonstrated the need for both pre- and post-HSCT prednisone treatments as a means to ensure engraftment without morbidity in all host animals.

Effect of extended immunosuppressive drug treatment on B cell vs T cell reconstitution in pediatric bone marrow transplant recipients.

Allogeneic bone marrow transplantation (BMT) is an effective therapy for a variety of malignancies and blood disorders, but rarely serves as a frontline treatment because of numerous, potential complications. Important and frequent complications relate to the profound immunosuppression that inevitably occurs during the first several months following treatment. To better elucidate and subsequently improve immune reconstitution, we examined T and B cell subsets among 43 pediatric BMT recipients in a retrospective study. We found that the relative numbers of T cells and B cells (T:B ratios) were discordant and highly variable among patients at day approximately 100 after BMT. Further investigation of BMT parameters identified a strong correlation between T:B ratios and immunosuppressive drug treatments, providing an explanation for variable lymphocyte reconstitution profiles. Results suggest that: (1) immunosuppressive therapy inhibits B cell expansion more strongly than T cell expansion following BMT; (2) WBC and absolute lymphocyte counts fail to reveal profound B cell immunodeficiencies in some BMT patients; and (3) routine analyses of T:B ratios serve to identify patients warranting close follow-up and extended supportive immunotherapy.

Structural features of HIV envelope defined by antibody escape mutant analysis.

Two neutralizing antibodies specific for the V3 sequence of HIV envelope were used to generate escape variants from the HTLV(IIIB) founder virus. The full gp120 sequence of each variant was then analyzed to identify mutations responsible for immune escape. As predicted, most escape variants harbored amino acid changes in the V3 crown sequence. However, one variant differed from its founder only within the conserved C2 region. These findings, when analyzed in conjunction with crystallographic data, suggest a new three-dimensional model for HIV envelope folding, in which the V3 loop extends across the CD4-binding face of gp120 to associate with discontinuous C2 residues. This envelope configuration may provide an effective immune defense mechanism for HIV, as the highly variable residues of the V3 loop may shield conserved amino acids pertinent to viral infection.

Transcription and temporal cascade in Chilo iridescent virus infected cells.

Chilo iridescent virus (CIV) is the type species for genus Iridovirus, and belongs to the family Iridoviridae. Members of this family are large, isometric, cytoplasmic DNA viruses. Our laboratory has established that CIV replicates productively in the cotton boll weevil, Anthonomus grandis. Given the economic importance of this host and the dearth of knowledge on this virus, we have initiated host-virus interaction and molecular studies on CIV. This report focuses on regulation of transcription in CIV infections. We carried out northern analyses on total cellular RNA from infections of IPRI-CF-124T cells, using a complete genomic library of CIV and several putative gene-specific probes. Our data show a temporal cascade based on analysis of 137 detectable transcripts comprising 38 immediate-early (IE), 34 delayed-early (DE), and 65 late (L) transcripts. Analysis with gene-specific probes supported the cascade pattern. Both helicase and RNA polymerase were immediate-early; major capsid protein was late. The CIV gene expression cascade appears to operate primarily at the transcriptional level. Temporal classes observed are consistent with earlier studies at the polypeptide level and with transcriptional patterns in frog virus 3, genus Ranavirus in the Iridoviridae. Our results provide an important basis for understanding mechanisms driving the CIV temporal cascade.

Analysis of expression of a cytosolic enzyme on the surface of Streptococcus pyogenes.

The normally cytosolic glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase, (GAPDH) has been reported to be expressed on the surface of Streptococcus pyogenes, group A, where it can act as a plasmin binding protein (Plr), and potentially a signaling molecule. In studies of wild-type and isogenic mutants, an association between surface expression of antigenic GAPDH/Plr and M and M-related fibrinogen-binding proteins was identified. Inactivation of the mga gene, whose product controls expression of M and M-related proteins also influenced expression of surface GAPDH/Plr. Revertants or pseudorevertants of mga mutants led to concomitant re-expression of surface GAPDH/Plr and M and M-related proteins. Using surface enhanced laser desorption ionization (SELDI) mass spectroscopy, a physical association between GAPDH/Plr and streptococcal fibrinogen-binding proteins was demonstrated. These studies support the hypothesis that surface M and M-related proteins are involved in anchoring GAPDH/Plr on the surface of group A streptococci.

Multi-envelope HIV vaccine safety and immunogenicity in small animals and chimpanzees.

A significant obstacle to HIV vaccine development lies in the remarkable diversity of envelope proteins, the major targets of neutralizing antibody. That envelope diversity must be targeted is demonstrated by results from nonhuman primate studies in which single-envelope vaccines have protected against homologous, but rarely against heterologous virus challenges. Similarly, in clinical trials, single-envelope vaccines have failed to prevent break-through infections when challenge viruses were inevitably mismatched with the vaccine. To protect humans from infection by any isolate of HIV, we have prepared vaccine cocktails combining multiple envelopes from distinct viral isolates. We have tested several vehicles for vaccine delivery in small animals and have shown that successive immunizations with envelope, presented first as a DNA recombinant, then as a vaccinia virus (VV) recombinant, and finally as purified protein elicited strong neutralizing antibody responses. We have also tested the VV recombinant vaccine in chimpanzees. Pairs of animals received either single- or multi-envelope VV recombinant vaccines administered by the subcutaneous route. Results showed that the multi-envelope vaccine was safe, immunogenic, and superior to the single-envelope vaccine in eliciting HIV-specific antibody measurable in a standard clinical, immune assay. The promise of this system has led to the initiation of clinical trials, with which the hypothesis that cocktail vaccines will prevent human HIV infections may ultimately be tested.

Interaction of group A streptococci with human plasmin(ogen) under physiological conditions.

A series of methods for analyzing the interaction of group A streptococci with the human plasminogen system are described. Examples of group A streptococcal isolates capable of assembling surface plasminogen activator activity when grown in human plasma are presented and the key requirements for this process are evaluated. The stabilities of cell-associated plasmin and plasminogen activator complexes are compared and a model for the interaction of group A streptococci with the plasminogen system in an infected host is presented.

Expression and purification of biologically active recombinant quail stem cell factor in E. coli.

Stem cell factor (SCF) is a multifunctional cytokine involved in hematopoiesis, melanogenesis and gametogenesis. Previous studies have demonstrated that avian SCF is a requirement for the proliferation and survival of various cell types in vivo and in vitro. In the current study, recombinant quail stem cell factor was produced in Escherichia coli using a prokaryotic expression system. SCF was expressed as a fusion protein with a histidine hexamer tag at the N-terminal end of the protein. Following expression, the protein was purified by affinity chromatography on the Ni-NTA column. The uninduced and induced protein lysates and the purified protein were separated by SDS-PAGE and transferred onto nitrocellulose membrane. Western blot analysis with the monoclonal antibody to the histidine tag identified SCF in the induced cell lysates and the purified sample. The recombinant SCF was approximately 22-23 kD in size. This protein was generated devoid of the signal peptide, the transmembrane domain, and the intracellular domain and, hence, resembles the soluble form of SCF. Biological activity was assayed using the in vitro survival of E12 chicken dorsal root ganglion-derived sensory neurons. The addition of recombinant quail SCF improved neuronal survival. Survival (20.6%) was the highest at the 50 ng/ml concentration of SCF. The availability of quail SCF will be a valuable tool to further resolve the function of stem cell factor in birds.

Analysis of the virus-specific and nonspecific B cell response to a persistent B-lymphotropic gammaherpesvirus.

Respiratory challenge of mice with murine gammaherpesvirus 68 (gammaHV68) results in acute replication in respiratory epithelial cells and persistent, latent infection of B cells and macrophages. gammaHV68 elicits virus-specific Ab, and also nonspecifically activates B cells to Ab production through a CD4+ T cell-dependent process. The current analysis characterizes virus-specific and nonspecific Ab production at the single cell level and investigates the requirements and nature of the nonspecific response. Virus-specific Ab-forming cell (AFC) numbers were dwarfed by the increase in total AFC in all sites examined, indicating substantial nonspecific Ab production. Clear increases and decreases in specific and total AFC numbers occurred in the lymph nodes draining the respiratory tract and the spleen, but AFC numbers in the bone marrow (BM) increased to a plateau and remained constant. The longevity of the BM response was reflected in a sustained increase in virus-specific and total serum Ab levels. Generally, the IgG2a and IgG2b isotypes predominated. Analysis of cytokine-deficient mice, CD40 ligand-deficient mice, and radiation BM chimeras lacking MHC class II expression specifically on B cells indicated that nonspecific Ab production is independent of IL-6 or IFN-gamma, and dependent on cognate CD4+ T cell help. Several observations were consistent with polyclonal B cell activation by gammaHV68, including the induction of durable serum levels of IgG reactive with mammalian dsDNA and murine type II collagen. Our findings indicate new directions for studies of this valuable model of gamma-herpesvirus pathogenesis.

Characterization of stage-specific embryonic antigen-1 (SSEA-1) expression during early development of the turkey embryo.

SSEA-1 is a carbohydrate epitope associated with cell adhesion, migration and differentiation. In the present study, SSEA-1 expression was characterized during turkey embryogenesis with an emphasis on its role in primordial germ cell development. During hypoblast formation, SSEA-1 positive cells were identified in the blastocoel and hypoblast and later in the germinal crescent. Based on location and morphology, these cells were identified, as PGCs. Germ cells circulating through embryonic blood vessels were also SSEA-1 positive. During the active phase of migration, PGCs in the dorsal mesentery and gonad could no longer be identified using the SSEA-1 antibody. The presence of PGCs at corresponding stages was verified using periodic acid Schiff stain. Pretreatment of PGCs with trypsin, alpha-galactosidase and neuraminidase did not restore immunoreactivity to SSEA-1. In general, expression was not limited to the germ cell lineage. SSEA-1 was also detected on the ectoderm, yolk sac endoderm, gut and mesonephric tubules. During neural tube closure, SSEA-1 was expressed by the neural epithelium of the fusing neural folds. Later SSEA-1 was detected in regions of the developing spinal cord. Enzyme pretreatment unmasked the epitope on some neural crest cells and cells in the sympathetic ganglion. The temporal and spatial distribution of SSEA-1 in the turkey embryo suggests a role in early germ cell and neural cell development. The absence of SSEA-1 on turkey gonadal germ cells was different from that observed for the chick. Therefore, while features of avian germ cell development appear to be conserved, expression of SSEA-1 can vary with the species.

Interaction of a group A Streptococcus within human plasma results in assembly of a surface plasminogen activator that contributes to occupancy of surface plasmin-binding structures.

Group A streptococcal isolate 187061 incubated in human plasma or serum reconstituted with fibrinogen but not plasminogen-depleted plasma or serum alone acquired a surface plasminogen activator activity. Assembly of the surface plasminogen activator was inhibited by the presence of neutralizing antibodies to streptokinase. Once assembled, the bacterial-associated plasminogen activator could generate plasmin when incubated in human plasminogen, plasmin or serum which could bind to bacterial surface plasmin-binding structures despite the presence of host physiological inhibitors. These studies provide evidence that the pathways by which group A isolates interact with human plasmin(ogen) are potentially linked and may provide a mechanism for bacteria to acquire host enzymatic activity efficiently in the infected host.