Recombinant HIV-1 vaccine candidates based on replication-defective flavivirus vector.

Multiple approaches utilizing viral and DNA vectors have shown promise in the development of an effective vaccine against HIV. In this study, an alternative replication-defective flavivirus vector, RepliVax (RV), was evaluated for the delivery of HIV-1 immunogens. Recombinant RV-HIV viruses were engineered to stably express clade C virus Gag and Env (gp120TM) proteins and propagated in Vero helper cells. RV-based vectors enabled efficient expression and correct maturation of Gag and gp120TM proteins, were apathogenic in a sensitive suckling mouse neurovirulence test, and were similar in immunogenicity to recombinant poxvirus NYVAC-HIV vectors in homologous or heterologous prime-boost combinations in mice. In a pilot NHP study, immunogenicity of RV-HIV viruses used as a prime or boost for DNA or NYVAC candidates was compared to a DNA prime/NYVAC boost benchmark scheme when administered together with adjuvanted gp120 protein. Similar neutralizing antibody titers, binding IgG titers measured against a broad panel of Env and Gag antigens, and ADCC responses were observed in the groups throughout the course of the study, and T cell responses were elicited. The entire data demonstrate that RV vectors have the potential as novel HIV-1 vaccine components for use in combination with other promising candidates to develop new effective vaccination strategies.

Sensitization and symptoms associated with soybean exposure in processing plants in South Africa.

BACKGROUND: Following the results of a previous study that highlighted the potential for significant levels of dust exposure in South African soybean processing plants, a clinical investigation was undertaken to study the respiratory health of workers in this industry. METHODS: Workers from three soybean-processing plants were studied with a respiratory questionnaire and estimation of atopy and specific soybean IgE. RESULTS: A total of 144 of the 181 (79.6% participation rate) plant employees completed the questionnaire and 136 (75.1%) gave blood samples for analysis of specific IgE. There was a significant association between work-related chest tightness (OR 4.0 [95% CI 1.3-12.6]), work-related nasal symptoms (OR 4.3 [95% CI 1.3-14.6]) and cough or chest tightness after handling soybean (OR 3.6 [95% CI 1.1-11.6]) and soybean sensitization. There was a significant association between current exposure to dust during soybean off-loading and "flu-like" illness (OR 2.7 [95% CI 1.0-7.2]), and cough or chest tightness after such work (OR 7.4 [95% CI 2.4-23.6]). The strongest predictor of work related nasal symptoms was sensitization to soybean, the latter strongly predicted by the presence of atopy (OR 34.7 [95% CI 6.6-182.5]). CONCLUSIONS: Exposure and sensitization to soybean were associated with the presence of work related symptoms, including flu-like symptoms, cough, chest tightness, and nasal symptoms. The aetiology of these symptoms and more particularly the best intervention strategies require more detailed investigation.

CD8+ T cell responses following replication-defective adenovirus serotype 5 immunization are dependent on CD11c+ dendritic cells but show redundancy in their requirement of TLR and nucleotide-binding oligomerization domain-like receptor signaling.

Replication-defective adenovirus serotype 5 (rAd5) is the most potent recombinant vector for eliciting CD8 T cell responses in humans. In this study, the innate mechanisms that influence T cell responses following rAd5 immunization were assessed in mice. Using rAd5 expressing enhanced GFP (eGFP-rAd5), we show that rAd5 transfects CD11c(+) dendritic cells (DCs) in draining lymph nodes in vivo following s.c. or i.m. immunization. Among distinct DC subsets, eGFP expression was highest in CD11c(+)CD8(-)B220(-) with a lower frequency detected in CD11c(+)CD8(+)B220(-) and CD11c(+)B220(+) plasmacytoid DCs. CD11c(+) DCs but not CD11c(-) cells from mice immunized with rAd5 encoding the SIINFEKL peptide induced proliferation of naive OT-I CD8 T cells. Furthermore, CD11c(+)CD8(+)B220(-) was the most potent DC subset for eliciting naive OT-I CD8 T cell proliferation. Of note, mice with pre-existing immunity to rAd5 had a substantial decrease in eGFP expression in DCs, which was associated with approximately 2-fold decrease in Th1 and complete inhibition of CD8 responses. Thus, pre-existing rAd5 immunity has a greater influence on CD8 compared with CD4 T cell responses. In terms of how innate cytokines and signaling pathways influenced T cell immunity following rAd5 immunization, we show that the magnitude and quality of CD8 T cell responses are partially dependent on MyD88 but independent of IL-12, type I IFN, apoptosis-associated speck-like protein, nucleotide-binding oligomerization domain-like receptor protein 3, and IL-1. Taken together, these data demonstrate a critical role for CD11c(+) DCs for CD8 responses but striking redundancy for innate cytokines and signaling by TLR and nucleotide-binding oligomerization domain-like receptor pathways.

Identification of naturally processed CD8 T cell epitopes from prostein, a prostate tissue-specific vaccine candidate.

Prostein is a prostate tissue-specific protein that is uniquely and abundantly expressed in normal and cancerous prostate tissues. Due to this expression profile, we examined the immunogenicity of prostein as a potential vaccine candidate for prostate cancer. To determine the presence of CD8 T cells specific for naturally processed prostein-derived epitopes in healthy individuals, we developed and applied an in vitro stimulation protocol. Using this protocol, we identified CD8 T cells specific for prostein in the peripheral blood of a male and a female donor. Prostein-specific CD8 T cell clones specifically recognized prostein-expressing targets, including prostate tumor cell lines expressing the relevant HLA alleles. CD8 T cell clones isolated from the male donor were significantly less effective in recognizing target cells compared to cells isolated from the female donor and appeared to recognize subdominant epitopes. The identification of a prostein-specific CD8 T cell repertoire supports the development of prostein in vaccination strategies against prostate cancer. Furthermore, the naturally processed peptide epitopes identified provide tools for the development of peptide-based vaccination strategies against prostate cancer and for monitoring of prostein-specific responses in vaccinated patients.

Sensate radial forearm free flaps in tongue reconstruction.

BACKGROUND: Successful rehabilitation after ablative surgery requires not only the reconstruction of 3-dimensional form but also the restoration of physiologic function. OBJECTIVE: To assess sensory recovery of reinnervated radial forearm flaps used for tongue reconstruction. PATIENTS AND METHODS: Seventeen patients, who underwent reconstruction of glossectomy defects with reinnervated radial forearm free flaps, formed the study group. Recovery of sensation was measured by both subjective and detailed objective tests 8 months after surgery. Sensory function of the flap was compared with that of the normal residual tongue or the adjacent oral mucosa and the contralateral forearm donor site. RESULTS: All patients involved in this study had tongue defects of hemiglossectomy or greater and adjacent floor of the mouth. Sensory recovery was observed in all of the 17 patients within 8 months. Detailed sensory testing showed that median static 2-point discrimination, moving 2-point discrimination, and pressure sensitivity (1.2 cm, 0.8 cm, and 3.7 psi, respectively) were subjectively greater in the innervated forearm flaps than in the contralateral forearm donor site (2.3 cm, 1.7 cm, and 4.6 psi, respectively) (P= .064) and similar to those of the normal tongue (0.9 cm, 0.5 cm, and 3.6 psi). CONCLUSIONS: In all modalities examined, sensate free flaps proved superior in sensory fidelity to the native forearm donor site and closely approached that of the normal tongue. Microsurgical reinnervation of flaps should be considered in tongue reconstruction.

An atypical case of choroidal osteomas.

BACKGROUND: A choroidal osteoma is a benign choroidal tumor It usually occurs in young, healthy women and is typically unilateral and located in the juxtapapillary region. CASE REPORT: A case of bilateral choroidal osteomas occurring in a 32-year-old woman is reported. The visual symptoms coincided with her third pregnancy. The choroidal osteoma in her right eye was elevated with blurred margins and had signs of subretinal fluid. The lesion in her left eye was flat and dry. Both were located superior temporal to the macula. Fluorescein angiography and indocyanine green angiography showed evidence of subretinal fluid in her right eye. CONCLUSIONS: The atypical features of the osteoma in her right eye include its height, evidence of diffuse leakage of subretinal fluid with no definable neovascular membrane, and its possible growth associated with pregnancy. In addition, the location, indistinct borders, and bilateral presentation are not characteristic clinical features of choroidal osteomas.

Postpartum proptosis with ophthalmopathy.

BACKGROUND: Postpartum thyroid disease presents in two forms: postpartum thyroiditis (PPT) and postpartum Graves' disease (PPGD). CASE REPORT: A case of postpartum thyroid dysfunction with ophthalmopathy is presented. DISCUSSION: In women diagnosed with Graves' disease during the ages of 20 to 35 years, 66% have a postpartum onset; women with a previous history of Graves' disease frequently relapse in the postpartum period. Additionally, up to 25 to 30% of postpartum women can develop permanent hypothyroidism from postpartum thyroiditis. The signs and symptoms of PPT and PPGD may be indefinite after delivery and often go undiagnosed. Because complications can be significant and may become irreversible, proper diagnosis and timely treatment is important.

The effects of body weight and long ahemeral days on early production parameters and morphological characteristics of broiler breeder hens.

Shaver Starbro broiler breeder pullets were used to study the effect of day length (24 and 28 h) on egg production parameters and egg traits from 22 to 30 wk of age, and ovarian and morphological characteristics at 30 wk of age. Floor-housed pullets were raised in a light-tight facility from 1 d of age until housing in individually illuminated cages at time of photostimulation (22 wk). Cages were equipped with hardware to monitor egg laying time. The photoschedule during rearing was 24 h light: 0 h dark for the first 3 d followed by 8 h light: 16 h dark from 3 d to photostimulation. Body weight was monitored throughout the trial to maintain weights at targets set by Shaver Poultry Breeding Farms, Ltd. The experimental design was a 2 x 2 factorial with two day lengths [hemeral (24 h) and ahemeral (28 h)] and two BW groups [high (HBW) and low BW (LBW)], with the LBW weighing 327 g less than the HBW birds at 22 wk of age. The hemeral photoschedule was 14 h light: 10 h dark, and the ahemeral photoschedule was 14 h light: 14 h dark. Differences in BW remained throughout the trial. Egg numbers were not increased by the 28-h day (24 h = 25.4 eggs; 28 h = 23.1 eggs). The 24-h hens laid more double-yolked eggs (0.9% of total egg production) than did the 28-h birds (0.3%). Egg formation time was consistently longer for the 28-h hens (26.8 h at 25 wk of age and 25.8 h at 29 wk of age) compared with the 24-h hens (25.4 h at 25 wk of age and 24.8 h at 29 wk of age). Mean egg weight was higher for the 28-h birds (55.1 g) compared with the 24-h birds (53.0 g). Egg specific gravity was higher in eggs from the 28-h hens than eggs from the 24-h hens. Some of the increase in egg weight between the 28- and 24-h birds was due to shell weight. Absolute and proportional weights of the egg shell were 5.5 g and 10.0% of the total egg weight for the 28-h birds and 4.9 g and 9.3% for the 24-h birds, respectively. The 28-h hens had higher proportional breast muscle weight, smaller livers and oviducts, and lower ovary weight compared with the 24-h birds. The data indicated that, although egg size can be increased with the use of long ahemeral days early in lay, this result may be at the expense of egg numbers. Early in lay, follicular maturation rates are fast, and egg production may be limited to one ovulation per 28 h.

Clinical experience in end-to-side venous anastomoses with a microvascular anastomotic coupling device in head and neck reconstruction.

BACKGROUND: Microvascular anastomosis remains one of the most technically sensitive aspects of free-tissue transfer reconstructions. Despite the availability of various mechanical anastomotic coupling systems for human clinical use during the last 8 years, reported clinical series remain rare. OBJECTIVE: To describe a clinical experience in applying a mechanical microvascular anastomotic coupling device (MACD) to end-to-side anastomotic configurations in head and neck free-flap reconstruction. METHODS: The MACD is a readily available high-density polyethylene ring-stainless steel pin system that has been found to be highly effective in clinical studies of end-to-end arterial and venous anastomosis and in laboratory studies of end-to-side anastomosis of rabbit arteries. RESULTS: Thirty-seven end-to-side venous anastomoses were attempted, of which 33 (89%) were completed. Of these, 9 patients had critical anastomoses (only 1 venous anastomosis per patient). In patients undergoing parallel venous anastomoses, 6 had both anastomoses performed using the MACD; in the remaining 12 patients, 1 of the anastomoses was performed using the MACD. A variety of donor flaps and clinical contexts were encountered. Flap survival in the MACD series was 100%. Four anastomoses were converted to conventional suture technique intraoperatively. CONCLUSIONS: The MACD is well suited to end-to-side venous anastomosis when carefully and selectively used by experienced microvascular surgeons. The most common clinical situation requiring this configuration and technique was the lack of ipsilateral recipient veins for end-to-end anastomosis or a ligated internal jugular vein stump that required this approach for device application. Previous radiation therapy does not appear to be a contraindication to its use.

Identification of rat prostatic steroid-binding protein as a target antigen of experimental autoimmune prostatitis: implications for prostate cancer therapy.

The long term goal of this study is to develop autoimmune prostatitis as a therapy for prostate cancer. An immune attack capable of destroying normal prostate epithelial cells should also destroy malignant prostate tissue and provide therapeutic benefit in cancer patients. The current study was initiated to identify antigenic targets for experimental autoimmune prostatitis on the assumption that such proteins might also be suitable targets for immunotherapy of prostate cancer. Male Lewis rats were immunized with syngeneic prostate homogenates, and the immune sera were used to screen prostate proteins for immunoreactivity by Western blot analysis. The dominant protein recognized by the immune sera was purified by ion exchange chromatography and reverse phase HPLC. Microsequence analysis of two polypeptide components of this immunodominant protein demonstrated N-terminal sequences identical with two of the three component chains of rat prostatic steroid-binding protein (PSBP). T cell responses to PSBP were also detected in rats immunized with prostate homogenate. Immunizing male rats with purified PSBP induced vigorous Ab and T cell responses. Significant prostate inflammation was observed in some rats immunized with PSBP. Adoptive transfer of T cells immune to PSBP induced rapid and severe destructive autoimmune prostatitis. These results demonstrate that PSBP is a major target Ag of experimental autoimmune prostatitis in a rat model and may serve as a target Ag for vaccine and T cell therapy against prostate cancer.

Clinical experience with a microvascular anastomotic device in head and neck reconstruction.

BACKGROUND: Despite numerous refinements in microsurgical technique and instrumentation, the microvascular anastomosis remains one of the most technically sensitive aspects of free-tissue transfer reconstructions. MATERIALS AND METHODS: Concurrent with the development of microsurgical techniques, various anastomotic coupling systems have been introduced in an effort to facilitate the performance and reliability of microvascular anastomoses. The microvascular anastomotic coupling device (MACD) studied here is a high-density, polyethylene ring-stainless steel pin system that has been found to be highly effective in laboratory animal studies. Despite its availability for human clinical use over the last 5 years, reported clinical series remain rare. Our clinical experience with this MACD in 29 head and neck free-tissue transfers is reported herein. RESULTS: Thirty-five of 37 (95%) attempted anastomoses were completed with 100% flap survival with a variety of donor flaps, recipient vessels, and clinical contexts. Two anastomoses were converted to conventional suture technique intraoperatively, and one late postoperative venous thrombosis occurred after fistulization and vessel exposure. CONCLUSIONS: We conclude that the MACD studied here is best suited for the end-to-end anastomosis of soft, pliable, minimally discrepant vessels. Previous radiation therapy does not appear to be a contraindication to its use. Interpositional vein grafts may also be well suited to anastomosis with the device. When carefully and selectively employed by experienced microvascular surgeons, this MACD can be a safe, fast, and reliable adjunct in head and neck free-tissue transfer reconstructions, greatly facilitating the efficiency and ease of application of these techniques.

A subunit interaction in chloroplast ATP synthase determined by genetic complementation between chloroplast and bacterial ATP synthase genes.

F1F0-ATP synthases utilize protein conformational changes induced by a transmembrane proton gradient to synthesize ATP. The allosteric cooperativity of these multisubunit enzymes presumably requires numerous protein-protein interactions within the enzyme complex. To correlate known in vitro changes in subunit structure with in vivo allosteric interactions, we introduced the beta subunit of spinach chloroplast coupling factor 1 ATP into a bacterial F1 ATP synthase. A cloned atpB gene, encoding the complete chloroplast beta subunit, complemented a chromosomal deletion of the cognate uncD gene in Escherichia coli and was incorporated into a functional hybrid F1 ATP synthase. The cysteine residue at position 63 in chloroplast beta is known to be located at the interface between alpha and beta subunits and to be conformationally coupled, in vitro, to the nucleotide binding site > 40 A away. Enlarging the side chain of chloroplast coupling factor 1 beta residue 63 from Cys to Trp blocked ATP synthesis in vivo without significantly impairing ATPase activity or ADP binding in vitro. The in vivo coupling of nucleotide binding at catalytic sites to transmembrane proton movement may thus involve an interaction, via conformational changes, between the amino-terminal domains of the alpha and beta subunits.

Differential regulation of proto-oncogenes c-jun and c-fos in T lymphocytes activated through CD28.

The T cell surface molecule CD28 binds to ligands on accessory cells and APCs, playing an important costimulatory role in the response of T cells to Ags. Our knowledge of the intracellular signaling pathways coupled to this receptor is incomplete. In addition to activation of phospholipase C gamma 1, ligation of this receptor also seems to activate a calcium-independent, CD28-specific pathway. In this paper, we report that cross-linking of CD28 (but not CD2, CD5, LFA-1, or CD7) leads to an elevation of c-jun mRNA, with only minimal activation of c-fos expression. CD28-dependent induction of c-jun expression requires protein tyrosine kinase activity, but does not depend on activation of a phorbol ester-responsive protein kinase C or elevation of cytosolic calcium. Furthermore, CD28-dependent elevation of c-jun mRNA does not appear to be mediated at the level of mRNA stability. A mechanism is suggested whereby expression of c-jun and junB, in the absence of members of the fos family, can prevent inappropriate activation of T cells caused by ligation of CD28 in the absence of a specific antigenic stimulus.

Cell-associated haemolytic activity of Helicobacter pylori.

Helicobacter pylori cells cultured on solid medium were quantitatively tested for haemolytic activity against erythrocytes of man, sheep, the guinea pig and rabbit. Using 4-day and 8-day cultures of two standard strains (ATCC 43504, IMMi 676), human erythrocytes were not lysed by 10% bacterial suspensions. Rabbit erythrocytes were the most sensitive to 8-day cultures. Hot-cold incubation yielded the highest haemolysis titres. The extent of haemolysis strongly correlated with the number of bacterial cells. Supplementation of the test medium (PBS, pH 7.4) with L-cysteine, dithiothreitol, MgCl2, EDTA, cholesterol, lecithin or sphingomyelin did not influence the haemolysis titres. They were significantly reduced in the presence of pronase E, human serum, bovine serum albumin or CaCl2, and by heat treatment of the bacteria. Supplementation of the test medium with cardiolipin strongly increased the haemolysis titres. Comparing the cell-associated haemolytic activity of 18 strains, the titres ranged from < 2 to 64, with a median titre of 16. No correlation was found between the haemolytic activity and phospholipase C activity of the cell suspensions. It was concluded that the formation of lysophosphatides and non-enzymatic factors rather than a sulphydryl-activated cytolysin or phospholipase C are responsible for the cell-associated haemolytic activity. This property may be involved in the pathogenicity and virulence of Helicobacter pylori.

Bovine interleukin 2: regulatory mechanisms.

A cDNA clone of the bovine interleukin-2 (IL-2) gene has been isolated and demonstrated to produce a functional bovine IL-2 protein when transfected into either CV-1 or COS-1 monkey cells. Homology comparisons of both the nucleotide and predicted amino acid sequences of bovine IL-2 with those of the human and mouse show extensive regions of sequence conservation between the species. The amino acid sequence of the mature bovine IL-2 protein shares about 60-63% homology with those of the human and mouse, but the 3' untranslated regions of the human and mouse gene share as much, if not greater, sequence homology with the 3' untranslated regions of the human and mouse genes. In particular, a tandemly repeated sequence (TATT), n, found in the 3' untranslated tail of the bovine IL-2 clone is also found in the 3' untranslated region of a large group of cytokine genes and other inducible genes of the lymphoid and immune response systems. This sequence may serve a specific regulatory function in the immune system.

Molecular cloning of a functional bovine interleukin 2 cDNA.

A cDNA clone of the bovine interleukin 2 (IL-2) gene has been isolated and demonstrated to be functional in the production of secreted bovine IL-2 protein when transfected into monkey cells. The bovine IL-2 clone is 791 base pairs in length and contains an open reading frame of 474 base pairs coding for a bovine IL-2 precursor polypeptide of 158 amino acids with an estimated molecular weight of 17,884. The putative hydrophobic leader or signal sequence of the precursor protein is 23 amino acid residues long, suggesting that, after removal by processing, the mature secreted bovine IL-2 protein contains 135 amino acids and has a molecular weight of 15,464. Comparisons of both the nucleotide sequence and the predicted amino acid sequence of bovine IL-2 with those of the human and mouse IL-2 show extensive regions of sequence conservation between the species, interspersed with other regions of less similarity. The 3' untranslated region of the bovine IL-2 gene shares as much, if not greater, sequence homology with the 3' untranslated regions of the human and mouse genes as do the transcribed coding regions of these genes, suggesting an involvement of this region in regulation. In particular, a tandemly repeated sequence, (TATT)n, found in the 3' untranslated tail of the bovine IL-2 clone is also found in the 3' untranslated region of the other known interleukin and interferon genes, as well as in similar regions of many other inducible genes of the lymphoid and immune response systems, suggesting a cell or tissue-specific regulatory function for these evolutionarily conserved sequences.

Antibacterial hemolymph proteins of Manduca sexta.

Exclusion column fractionated immune hemolymph of the M. sexta larva contains five peaks of anti-E. coli activity with molecular weights of greater than 140 kD and approximately 91, 54, 14 and 4 kD, plus one peak of lysozyme activity with a molecular weight of 17 kD. Purification of the 54 kD peak showed that this peak consists of the previously described M18 proteins which have monomeric weights of approximately 20 kD and had antibacterial activity against certain gram negative bacteria. Approximately 80% of the total hemolymph antibacterial activity was detected in the 14 and 4 kD peaks. These proteins, which kill both gram negative and gram positive bacteria, appeared to be directly analogous to the cecropins of H. cecropia. The greater than 140 and 91 kD peaks constituted only a minor part of the total antibacterial activity.

Effect of sublethal Bacillus thuringiensis crystal endotoxin treatment on the larval midgut of a moth, Manduca: SEM study.

The effect of a single, sublethal dose of B. thuringiensis crystal endotoxin on the midgut of the moth Manduca sexta larvae was monitored during acute and recovery stages. Initially both goblet and columnar cells swelled. Many columnar cells produced membrane extrusions. In some cases the affected cells ruptured, extruding cellular debris into the midgut lumen. Following the acute stage, the midgut tissue recovered, the damaged cells being extruded into the midgut lumen apparently as newly regenerated cells rose to take their place. The insects appeared to recover completely and continue normal development.