Breadth of T Cell Responses After Immunization with Adenovirus Vectors Encoding Ancestral Antigens or Polyvalent Papillomavirus Antigens.

Oncogenic human papillomaviruses (HPVs) are in most cases eliminated by intervention of T cells. As many other pathogens, these oncogenic HPVs belong to an ancient and diverse virus family. Therefore, we found it relevant to investigate the potential and limitations of inducing a broad response-either by inducing cross-reactive T cells or by administering a polyvalent vaccine. To test these strategies, we designed three ancestral and two circulating sequences based on the two domains of the E1 and E2 proteins of papillomaviruses (PVs) that exhibit the highest degree of conservation in comparison with the other PV proteins. The PV sequences were fused to a T cell adjuvant, the murine invariant chain and encoded in a recombinant adenoviral vector which was administered to naïve outbred mice. By measuring T cell responses induced by these different vaccines and towards peptide pools representing three circulating strains and a putative ancestor of oncogenic HPVs, we showed that the ancestral vaccine antigen has to be approximately 90% identical to the circulating PVs before a marked drop of ~90% mean CD8+ T cell responses ensues. Interestingly, the combination of two or three type-specific PV vaccines did not induce a significant decrease in the CD8+ T cell response to the individual-targeted PV types. Polyvalent HPV vaccine based on the E1 and E2 proteins seem to be capable of triggering responses towards more than one type of PV while the cross-reactivity of ancestral vaccine seems insufficient in consideration of the sequence diversity between HPV types.

IFNγ and perforin cooperate to control infection and prevent fatal pathology during persistent gammaherpesvirus infection in mice.

Infection with murine gammaherpesvirus 68 has become an accepted model for studying the virus/host interactions with regard to gammaherpesvirus infections. Previous studies using gene-deficient mice have revealed that neither IFNγ nor perforin is essential in controlling the outcome of infection or the virus load during chronic infection in C57BL/6 mice. However, pronounced multiorgan fibrosis and splenic atrophy are observed in mice lacking IFNγ or the IFNγ receptor. To study the interplay between perforin and IFNγ in controlling the virus-induced pathology and the viral load during chronic gammaherpesvirus infection, we infected IFNγ/perforin double-deficient C57BL/6 mice and followed the course of infection. While absence of perforin prevented the splenic atrophy in IFNγ-deficient mice, fibrosis did not disappear. Moreover, double-deficient mice developed extreme splenomegaly, were unable to control the viral load and displayed chronic immune activation. Thus, IFNγ and perforin act in concert to minimize pathology and control the viral load in mice chronically infected with MHV68. Furthermore, while certain aspect of the virus-induced pathology in IFNγ-deficient mice may be alleviated in double-deficient mice, other aspects are exaggerated, and the normal architecture of the spleen is completely destroyed. We believe that these findings add to the understanding of the virus/host interaction during chronic gammaherpes virus infection.

Glucose metabolism is altered after loss of L cells and α-cells but not influenced by loss of K cells.

The enteroendocrine K and L cells are responsible for secretion of glucose-dependent insulinotropic polypeptide (GIP) and glucagon like-peptide 1 (GLP-1), whereas pancreatic α-cells are responsible for secretion of glucagon. In rodents and humans, dysregulation of the secretion of GIP, GLP-1, and glucagon is associated with impaired regulation of metabolism. This study evaluates the consequences of acute removal of Gip- or Gcg-expressing cells on glucose metabolism. Generation of the two diphtheria toxin receptor cellular knockout mice, TgN(GIP.DTR) and TgN(GCG.DTR), allowed us to study effects of acute ablation of K and L cells and α-cells. Diphtheria toxin administration reduced the expression of Gip and content of GIP in the proximal jejunum in TgN(GIP.DTR) and expression of Gcg and content of proglucagon-derived peptides in both proximal jejunum and terminal ileum as well as content of glucagon in pancreas in TgN(GCG.DTR) compared with wild-type mice. GIP response to oral glucose was attenuated following K cell loss, but oral and intraperitoneal glucose tolerances were unaffected. Intraperitoneal glucose tolerance was impaired following combined L cell and α-cell loss and normal following α-cell loss. Oral glucose tolerance was improved following L cell and α-cell loss and supernormal following α-cell loss. We present two mouse models that allow studies of the effects of K cell or L cell and α-cell loss as well as isolated α-cell loss. Our findings show that intraperitoneal glucose tolerance is dependent on an intact L cell mass and underscore the diabetogenic effects of α-cell signaling. Furthermore, the results suggest that K cells are less involved in acute regulation of mouse glucose metabolism than L cells and α-cells.

Pregabalin and dexamethasone for postoperative pain control: a randomized controlled study in hip arthroplasty.

BACKGROUND: Optimal pain treatment with minimal side-effects is essential for early mobility and recovery in patients undergoing total hip arthroplasty. We investigated the analgesic effect of pregabalin and dexamethasone in this surgical procedure. METHODS: One hundred and twenty patients were randomly allocated to either Group A (placebo), Group B (pregabalin 300 mg), or Group C (pregabalin 300 mg+dexamethasone 8 mg). The medication and acetaminophen 1 g were given before operation. Spinal anaesthesia was performed. Postoperative pain treatment was with acetaminophen 1 g three times daily and patient-controlled i.v morphine, 2.5 mg bolus. Nausea was treated with ondansetron. Morphine consumption, pain intensity at rest and during mobilization, nausea and vomiting, sedation, dizziness, and consumption of ondansetron were recorded 2, 4, and 24 h after operation. P<0.05 was considered statistically significant. RESULTS: Twenty-four hour morphine consumption was significantly reduced in Groups B [mean (SD) 24 (14) mg] and C [25 (19) mg] compared with Group A [47 (28) mg]. Vomiting was reduced in Group C compared with Group B (P=0.03). Sedation was significantly increased in Group B compared with the other groups. CONCLUSIONS: Pregabalin resulted in a 50% reduction in 24 h postoperative morphine requirements. This was not associated with a reduced incidence of nausea or vomiting. Pregabalin resulted in increased levels of sedation. Combining pregabalin and dexamethasone provided no additional effects on pain or opioid requirements.

EBI2 in splenic and local immune responses and in autoimmunity.

The seven transmembrane G protein-coupled receptor EBV-induced gene 2 (EBI2), also known as GPR183, is expressed in particular in immune cells. Activated by its endogenous ligands, which are a group of oxysterols, it functions as a chemo-attractant receptor, mediating cell migration. In coordination with other receptors, EBI2 plays important roles in controlling the migration of immune cells during the course of a T-dependent Ab response in the spleen. In recent years, it has become clear that EBI2 also has other roles to play in the immune system. Thus, EBI2 seems to be involved in innate immune responses, such as those mediated by TLR signaling, and it has been implicated in regional immune responses, including immune responses in the CNS. In this review, we describe the functions of EBI2 in B cells, T cells, and dendritic cells during the course of a T-dependent Ab response in the spleen. Furthermore, we review the existing evidence supporting a role for EBI2 in local immune responses and in autoimmune diseases, with a special focus on immune responses in the CNS. Finally, we discuss which type of role EBI2 may play in autoimmune diseases, and we give our opinion about the paths of future research in EBI2.

Tumorigenesis induced by the HHV8-encoded chemokine receptor requires ligand modulation of high constitutive activity.

ORF74 (or KSHV-vGPCR) is a highly constitutively active G protein-coupled receptor encoded by HHV8 that is regulated both positively and negatively by endogenous chemokines. When expressed in transgenic mice, this chemokine receptor induces an angioproliferative disease closely resembling Kaposi sarcoma (KS). Here we demonstrate that several lines of mice carrying mutated receptors deficient in either constitutive activity or chemokine regulation fail to develop KS-like disease. In addition, animals expressing a receptor that preserves chemokine binding and constitutive activity but that does not respond to agonist stimulation have a much lower incidence of angiogenic lesions and tumors. These results indicate that induction of the KS-like disease in transgenic mice by ORF74 requires not only high constitutive signaling activity but also modulation of this activity by endogenous chemokines.

Proteins involved in the production and perception of oligosaccharides in relation to plant and animal development.

Chitin oligosaccharides and their derivatives are involved in developmental and defence-related signalling pathways. Major advances include the structural identification of lectins involved in development that bind chitin oligosaccharides and the links between chitin oligosaccharide and hyaluronan synthesis. Also, recent advances in the understanding of the biological role of oligosaccharides are summarised in a model for multistep glycan recognition.

cDNA cloning and tissue distribution of five human EPH-like receptor protein-tyrosine kinases.

We have isolated cDNA clones from a human fetal brain library that encode five members of the EPH sub-family of receptor protein tyrosine kinases (PTKs). Comparison of the DNA sequences of these receptors to the Genbank database reveals that two of our clones correspond to the previously identified HEK and ERK receptors, two are apparently human homologues of the mouse receptors Sek and Bsk and one is novel. With these additions, the number of known human EPH sub-family members is nine and the total in all vertebrate species is 13 making it the largest known sub-family of PTKs. Analysis of the expression pattern of EPH sub-family mRNAs reveals that some are expressed in a wide variety of adult tissues while others are quite restricted. Consistent with the amplification of these sequences from a fetal brain cDNA library, all five members which we have isolated are expressed in the brain. We have named these receptors HEK4, HEK5, HEK7, HEK8 and HEK11, following the nomenclature of Wicks et al. (1992) and the numbering convention set forth by Sajjadi et al. (1991). Analysis of these new EPH sub-family members will increase our understanding of the biology of this receptor family and their isolation will provide reagents for the identification of ligands for this large family of orphan receptors.

Hereditary resistance to 1,25-dihydroxyvitamin D: defective function of receptors for 1,25-dihydroxyvitamin D in cells cultured from bone.

UNLABELLED: The syndrome of rickets, alopecia, hypocalcemia, and high circulating levels of 1,25-dihydroxyvitamin D (1,25-(OH)2D) apparently is caused by resistance of target tissues to 1,25-(OH)2D. To evaluate this, we cultured cells from explants of long bone of one patient with this syndrome and from a control without any preexisting disorder of mineral metabolism. The cultured cells showed morphological features of fibroblasts but contained alkaline phosphatase activity without detectable acid phosphatase activity, indicating an osteoblastic origin for some or all of the cultured cells. Receptors for 1,25-(OH)2D were assessed by three methods: high affinity uptake of hormone in nuclei of dispersed cells, high affinity binding in hypertonic extracts (herein termed cytosol) from cells, and sedimentation velocity of bound [3H]1,25-(OH)2D3 in extracts of cell nuclei. With cells cultured from bone of the normal control, receptors for 1,25-(OH)2D exhibited properties indistinguishable from those found with cultured skin fibroblasts. With cells cultured from bone of the patient with resistance to 1,25-(OH)2D, high affinity uptake of 1,25-(OH)2D into nuclei was unmeasurable, but high affinity binding of hormone with cytosol was normal; these abnormal findings also were indistinguishable from abnormal findings obtained with fibroblasts cultured from skin of that patient. IN CONCLUSION: 1) Cells cultured from explants of human bone showed morphological features of fibroblasts but retained a marker enzyme characteristic of osteoblasts. Significant admixture of osteoblast-like cells with fibroblasts was possible. 2) Cells cultured from bone of a patient with familial resistance to 1,25-(OH)2D exhibit a defect in vitamin D metabolism, indistinguishable from the defect observed with cells cultured from skin of the same patient.

Determination of follicle regulatory protein levels in urine during the normal menstrual cycle using an enzyme-linked immunosorbant assay.

A protein from follicular fluid [referred to as follicle regulatory protein (FRP)] which inhibits aromatase activity in granulosa cells was recently isolated and partially characterized. The purified FRP was used to produce a monoclonal antibody which was used to develop an enzyme-linked immunosorbant assay suitable for quantitation of FRP in urine. Twelve normal premenopausal women underwent daily collection of blood and first morning urine samples, beginning on the 1st day of menses, as well as daily ultrasonographic evaluation of follicular diameter, beginning on the 10th day of the menstrual cycle, until the onset of the next menses. Serum estradiol, progesterone, LH, and FSH levels were determined by RIA. Urinary FRP levels increased in the midfollicular phase, reached their zenith in the midluteal phase [mean, 0.38 +/- 0.03 (+/- SE) immunoreactive units; 1 immunoreactive unit = approximately 1 ng FRP/mL.mg creatinine], and then declined to reach their nadir (not detectable) during the early follicular phase. Immunohistochemical evaluation of ovarian tissue demonstrated that anti-FRP localized to mural granulosa cells in viable follicles, to all follicular epithelial cells in atretic follicles, and to the large cells of the corpus luteum. These findings indicate that immunoreactive FRP levels in urine change during the menstrual cycle and suggest a relationship among FRP, follicular maturation, and corpus luteum formation.

A viral antigen-bearing cell line derived from culture of Paget's bone cells.

This study documents the characteristics of a viral antigen-bearing cell line derived from co-culture of the bone from a 71-year-old woman with Paget's disease of bone and HEp-2 cells. The cell line has survived in continuous culture for 3 1/2 years and 185 subcultures. The cells are epithelioid in appearance, produce alkaline and acid phosphatase, increase alkaline phosphatase activity in response to 1,25-(OH)2-D3 and contain receptors for 1,25-(OH)2-D3. Immunofluorescent studies utilizing antisera to respiratory syncytial virus and measles virus reveal antigens of both viruses in the cells. These cells do not produce bone in culture and the adenylate cyclase activity found in their plasma membrane does not increase significantly in response to parathyroid hormone or calcitonin. The cells are not contact inhibited and form spherical colonies in agarose. They are aneuploid and have a modal number of 62-74 as well as HeLa markers. When injected into athymic mice, osteosarcomas are produced. These tumors continue to bear viral antigens. The availability of this cell line should aid in further studies of the viral antigens associated with Paget's disease of bone.

Speed and flexibility on word fluency tasks after focal brain lesions.

It was predicted that frontal lobe damaged patients are slower on word fluency tasks, especially on the generation of words beginning with a particular letter, and less flexible ("stuck-inset") on category alternation than patients with posterior lesions, whereas the latter commit a higher number of repetitions ("recurrent perseverations") than the former. Twenty-nine anterior and 31 posterior brain damaged patients were requested to say as quickly as possible (1) 20 animal names, (2) 10 words beginning with the letter S, and (3) alternately animals and S-words, 10 from each category without repeating the words already used in these tasks. The results failed to confirm the predictions.

Deficient programming in spatial learning after frontal lobe damage.

Patients with anterior or posterior brain damage and control subjects performed a spatial sequence learning task in which the score obtained depended on the subject's ability to set sub-goals appropriate for his learning capacity. The anterior group obtained lower scores and more frequently set inadequate sub-goals than the posterior group. No anterior vs posterior difference was found on a similar learning task in which another sequence was learnt by predetermined sub-goals. The result supported the hypothesis that frontal lobe lesions disturb programming or goal-based search for action structure on spatial learning.

Presence of brain-derived neurotrophic factor in brain and human and rat but not mouse serum detected by a sensitive and specific immunoassay.

Brain-derived neurotrophic factor (BDNF) is one of several endogenous proteins that play key roles in neuronal development and homeostasis. We describe here the characterization and use of a sensitive and specific enzyme-linked immunoassay (EIA) for BDNF protein. Recombinant BDNF was detected at concentrations as low as 10 pg/ml, whereas the EIA did not detect NT-3, NT-4/5, or NGF at concentrations as high as 100 ng/ml. Because BDNF protein sequences are identical among humans, mice, and rats, we utilized the BDNF EIA to detect BDNF in the circulation or brain regions of these species. High concentrations of BDNF were detected in human and rat serum, and up to 50-fold lower BDNF levels were present in citrated human or rat plasma. The BDNF signal (66-141 pg/ml) in 20% human plasma was completely blocked by pre-exposure of plasma to a monoclonal antibody (Mab) specific for BDNF but not by exposure to 5-fold greater concentrations of an irrelevant Mab of the same isotype (IgG1). There was a significant and positive correlation (r = +0.86) between plasma levels of BDNF and serotonin, an indoleamine that is specifically released from activated platelets. These results are consistent with the view that the BDNF detected in human and rat plasma is derived from platelet degranulation, and that circulating levels of BDNF are negligible. In contrast to human or rat serum, mouse serum contained no detectable BDNF. However, BDNF protein was readily detectable at 108-256 ng/g of tissue in hippocampus, frontal cortex, and neostriatum of mice and rats. Thus, the failure to detect BDNF in murine serum was not due to an assay defect but highlights a significant species difference in the tissue-specific expression of BDNF that may be of biological importance. The presence of BDNF protein in blood and brain regions at quantities which greatly exceed those described for NGF confirm the abundant distribution of this broadly-acting neurotrophic factor.

Mental programming after frontal lobe lesions: results on digit symbol performance with self-selected goals.

The aim of this study was to demonstrate that the inability to set adequate sub-goals in a cognitive task is a sensitive indicator of programming deficit after frontal lobe lesion. Sixty-one patients with focal cerebral lesions and 25 control subjects were studied with a modified Digit Symbol task, in which the score depended on the adequacy of the sub-goals set by the subject. This score was compared to that on the standard condition, in which the subject was requested to work as quickly as possible without self-selected goals. The results confirmed the prediction that patients with anterior lesions set less adequate sub-goals than patients with posterior lesions and, unlike the latter patients, have a more pronounced deficit on the performance with self-selected goals than on the standard condition. In particular patients with left frontal lobe lesions underestimated their capabilities in relation to task requirements.

Speed-accuracy optimization in acquisition of figures after focal cerebral lesion.

Seventy-seven brain damaged patients and 14 control subjects performed a visual memory test in which the subjects had to reproduce geometric figures from memory after studying the design for 10 seconds (standard condition) and after an observation period subjectively necessary and sufficient for accurate reproduction (self-timed condition). The brain damaged patients were less efficient than the control subjects on the self-timed acquisition even when the covariance on the standard condition was taken into account. The prediction that frontal lobe damaged patients are inferior to posterior brain damaged patients on the speed-accuracy optimization of acquisition was true only in the right hemisphere damaged patients on the simple trials, suggesting that right frontal lobe damage disturbs voluntary attention. Visuospatial errors were typical for right hemisphere damaged patients, perservations for left frontal lobe damaged patients.

Cognitive deficits related to computed tomographic findings after surgery for a ruptured intracranial aneurysm.

A consecutive series of 118 patients operated on for ruptured intracranial arterial aneurysms was studied. Ninety-six of them could be adequately examined with a battery of psychological tests and computed tomographic scans 1 year after a subarachnoid hemorrhage. Seventeen orthopedic control patients with no history of brain damage were also tested. The pattern of cognitive deficits was strongly related to the findings on the computed tomographic scans. Patients with left lateral infarctions had deficits on performances requiring verbal efficiency, including memory and classification tasks, whereas patients who had right lateral infarctions were poor on a visuoconstructional task (the copying of Rey's Figure). These deficits were pronounced when lateral infarction was associated with diffuse brain damage. Patients with frontal medial infarctions had low scores on memory tests; the inefficiency in verbal fluency, categorical reasoning, and memory was related to diffuse brain damage. The patients who had no infarctions did not differ significantly from the control group. Cognitive impairments after left lateral and frontal medial infarctions, as well as diffuse brain damage, correlated with the Glasgow Outcome Scale.

Social outcome related to cognitive performance and computed tomographic findings after surgery for a ruptured intracranial aneurysm.

A series of 83 patients was examined with a battery of cognitive tests, a clinical interview, and computed tomography 1 year after surgery for a ruptured intracranial aneurysm. Disability on the Glasgow Outcome Scale (33%), failure to return to work (25%), impaired social relations (25%), and subjective or clinical mental impairment (56%) were found to be related to each other and to poor performance on cognitive tests, especially to verbal impairments in patients with left lateral infarctions and to memory deficits and cognitive inflexibility in patients with frontal medial infarctions. Furthermore, cognitive deficits and poor outcome were associated with diffuse brain damage. Depression and anxiety were unrelated to test performances, but were frequently reported by patients with right lateral infarctions.

In vivo incorporation of radioactive 36Cl, a method for monitoring chloro compounds in biological material.

A new method for fast and easy monitoring of the presence, isolation, and separation of natural chloro compounds in plants is described. The method relies on the in vivo incorporation of radioactive 36Cl and new enhancement methods in autoradiographic technology. The method allows the time of exposure to be limited to 4 days and is thus suitable for routine purposes.

Comparison of nedocromil sodium at two dosage frequencies with placebo in the management of chronic asthma.

Nedocromil sodium (4 mg b.d. or q.i.d.) was added to the therapy of 76 chronic asthmatic patients in a four-centre, double-blind cross-over, placebo-controlled trial. Patients had troublesome symptoms uncontrolled by high doses of inhaled corticosteroids (mean 1450 micrograms). In 54 patients who completed the study, nedocromil sodium was significantly more efficacious than placebo (P < 0.01) in relieving morning chest-tightness and cough, in reducing total diary card score and nocturnal bronchodilator usage, and in increasing morning and evening peak flow. Asthma severity at clinic visits decreased significantly (P = 0.001) following treatment with nedocromil sodium, which was globally rated more effective than placebo (P < 0.01). Treatment differences favored q.i.d. over b.d. dosage but without statistical significance. There were no serious adverse effects. Although the pulmonary function changes were small, these findings suggest that the addition of nedocromil sodium may benefit asthmatic patients who are inadequately controlled by high doses of inhaled corticosteroids.