Erythropoiesis equivalence, pharmacokinetics and immune response following repeat hematide administration in cynomolgus monkeys.

Hematide is a synthetic PEGylated peptidic erythropoiesis stimulating agent (ESA) that is presently being developed for the correction of anemia in patients with chronic renal failure. Unlike currently marketed ESAs, Hematide does not possess any sequence homology to erythropoietin (EPO) and has not elicited moribund immune responses in animal safety studies thereby allowing the generation of a robust safety package. Animals administered marketed ESAs develop anti-EPO antibodies that null the effect of the administered ESA and neutralize endogenous EPO, resulting in severe anemia that precludes the interpretation of chronic safety studies. The primary objective of this study is to determine whether Hematide-specific antibodies are generated when male monkeys are exposed to high Hematide doses (10 mg/kg, intravenous [IV] and subcutaneous [SC]) administered at frequent dosing intervals (every two weeks) for a total of 9 doses; secondary objectives are to evaluate whether developed antibodies impact pharmacokinetics (PK) and pharmacology. In this study, no Hematide-specific antibodies were detected. Hematide exhibits a prolonged plasma half-life and slow clearance by either IV or SC administration. Hematide induced significant erythropoiesis with reticulocytosis and subsequent increases in red blood cells, hematocrit and hemoglobin (Hgb) levels. No erythropoietic differences were noted between the IV and the SC dosed groups with mean +/- SD Hgb levels of 20.9 +/- 2.5 and 20.3 +/- 2.1 g/dL, respectively, occurring on Day 48, corresponding to Hgb increases of 6.5 and 6.7 g/dL, respectively, over pre-dose levels. In conclusion, Hematide is a potent erythropoiesis stimulating agent that exhibits plasma persistence in monkeys. Similar erythropoietic responses were produced following IV and SC administration. The absence of antibody development suggests that Hematide, at the doses and regimen described, has a low immunogenic potential in cynomolgus monkeys.

Sex differences in baseline neuropsychological function and concussion symptoms of collegiate athletes.

OBJECTIVE: To investigate sex differences in baseline neuropsychological function and concussion symptoms between male and female collegiate athletes. METHODS: A post-test only design was used to examine baseline neuropsychological test scores and concussion symptoms. A total of 1209 NCAA Division I collegiate athletes from five northeastern universities in the USA completed a baseline ImPACT test. ImPACT, a computerised neuropsychological test battery, was administered during an athlete's pre-season. RESULTS: Female athletes performed significantly better than male athletes on baseline verbal memory scores (p = 0.001), while male athletes performed significantly better than female athletes on baseline visual memory scores (p = 0.001). Female athletes endorsed a significant number of mild baseline symptoms as compared to male athletes. CONCLUSIONS: Male and female athletes exhibit differences on baseline neuropsychological test performance and concussion symptoms.

Outcome of treatment of ruptured abdominal aortic aneurysms depending on the type of hospital.

OBJECTIVE: To compare the outcome of patients operated on acutely for ruptured abdominal aortic aneurysms (AAA) or otherwise symptomatic aortic aneurysms in a university hospital and in two county hospitals by the same group of vascular surgeons. DESIGN: Retrospective study. SETTING: 1 university and 2 county hospitals, Sweden. SUBJECTS: 108 patients operated on urgently for AAA, 81 at the university hospital, and 27 at the county hospitals between January 1992 and December 1998. INTERVENTION: Repair of the AAA. MAIN OUTCOME MEASURES: Morbidity and mortality. RESULTS: 21 of the 81 patients having urgent repair of an AAA at the university hospital (26%) had been transferred from the county hospitals. Thirteen patients were not operated on, 7 because of their poor general condition and great age (median 84 years), 3 who refused operation, and 3 in whom the diagnosis was incorrect. During the same time period a further 27 haemodynamically unstable patients were operated on by the same vascular surgeons at the county hospitals. The on-table mortality for patients with ruptured AAA and shock was 5/43 (12%) at the university hospital and 4/27 (15%) at the county hospitals. The corresponding in-hospital rates were 11/43 (26%) and 11/27 (41%). Mortality was significantly higher if the operation was delayed by more than 45 minutes. The incidence of postoperative complications was the same in both hospitals. CONCLUSION: If a patient with a ruptured AAA and shock is admitted to the county hospital and operated on by a specialist vascular surgeon the outcome is fully acceptable. The difference seems to be related to the postoperative period. To what extent the delay caused by the surgeon's journey to the county hospital has any influence on the outcome is not possible to evaluate.

The adolescent sexual world and AIDS prevention: a democratic approach to programme design in Zimbabwe.

The main aim of this study was to redress the under-representation of adolescent attitudes in AIDS prevention programme design and to discuss the implications of a democratic sexuality education approach within a health promotion context. The study surveyed the attitudes of adolescents (average age 16 years) on sex-related topics such as culture, marriage, sexual behaviour and sex education, identified similarities and differences in attitudes according to gender and socio-economic environments, and examined sources of students' knowledge of selected sex-related topics. Questionnaires were completed by 3429 secondary school students from different backgrounds. Findings showed significant differences in attitude related to gender and socio-economic settings and also in sources of information. The study drew on a democratic sexuality education approach. Using this approach, adolescents review ideological perspectives and decide which are most appropriate for them as guides in making decisions about their own lives. This type of education is fully consistent with the principles of democratic living and gives guidance to teenagers who are trying to decide how to live a healthy life. Study results suggest that programmers would be well advised to collect information from the adolescents' psycho-social-economic environment and link it with good governance and civil society strategies in developing AIDS prevention programmes that involve the adolescent in making lifestyle decisions. To achieve a more supportive environment for AIDS prevention, this approach can link adolescent attitudes and health promotion action to advocate for public policy reform, gender equality, multi-dimensional partnerships and social marketing.

Structural requirements for the stabilization of metarhodopsin II by the C terminus of the alpha subunit of transducin.

The retinal receptor rhodopsin undergoes a conformational change upon light excitation to form metarhodopsin II (Meta II), which allows interaction and activation of its cognate G protein, transducin (G(t)). A C-terminal 11-amino acid peptide from transducin, G(talpha)-(340-350), has been shown to both bind and stabilize the Meta II conformation, mimicking heterotrimeric G(t). Using a combinatorial library we identified analogs of G(talpha)-(340-350) that bound light-activated rhodopsin with high affinity (Martin, E. L., Rens-Domiano, S., Schatz, P. J., and Hamm, H. E. (1996) J. Biol. Chem. 271, 361-366). We have made peptides with key substitutions either on the background of the native G(talpha)-(340-350) sequence or on the high affinity sequences and used the stabilization of Meta II as a tool to determine which amino acids are critical in G protein-rhodopsin interaction. Removal of the positive charge at the N termini by acylation or delocalization of the charge by K to R substitution enhances the affinity of the G(talpha)-(340-350) peptides for Meta II, whereas a decrease was observed following C-terminal amidation. Cys-347, a residue conserved in pertussis toxin-sensitive G proteins, was shown to interact with a hydrophobic site in Meta II. These studies provide further insight into the mechanism of interaction between the G(talpha) C terminus and light-activated rhodopsin.

Seatbelts contribute to location of lesion in moderate to severe closed head trauma.

The relationship between seatbelt use and injury severity, brain lesion location, and functional outcome was investigated in 163 individuals who sustained traumatic brain injuries in motor vehicle collisions. Of this group, 31 were using a seatbelt at the time of the accident and 132 were not. Restrained motor vehicle occupants were significantly more likely to sustain damage to subcortical brain structures than unrestrained occupants. Conversely, unrestrained occupants sustained a greater frequency of posterior brain lesions. In addition, demographic and behavioral variables were significantly related to increased likelihood of seatbelt use. Analyses revealed no significant differences between groups for injury severity variables and functional outcome measures. Seatbelts alter the body's response to forces applied in motor vehicle collisions, creating disparities in lesion sites between restrained and unrestrained motor vehicle occupants. The relationship between seatbelt use and injury severity and functional outcome is discussed.

Underutilization of neuropsychology in traumatic brain injury rehabilitation: is managed care to blame?

We evaluated factors determining which individuals received neuropsychological evaluations (NPEs) following traumatic brain injury (TBI). Comprehensive records from a State-wide/sponsored Head Injury Program were followed from 1985--1995 to monitor effects of managed care on provision (or absence) of formal NPEs and ultimately on rehabilitation outcome. Only 26% of 273 individuals received NPEs (within their first three years post-injury). In the years prior to and after large changes in managed care, there were no differences in the provision of formal NPEs. Discriminant analysis identified functional status at discharge from primary rehabilitation and total number of rehabilitation facilities as the two variables that most distinguished those who had received NPEs with 69% classification accuracy. Between group analyses revealed that individuals were more likely to receive NPEs if they were young, involved in liability claims, attended multiple rehabilitation facilities, or had higher functional status at discharge from primary rehabilitation, regardless of the nature or severity of their TBI. Individuals receiving formal NPEs ultimately achieved higher levels of functional independence, suggesting a potential selection bias. Individuals were no more likely to receive NPEs according to insurance status (private versus government assisted) or as a function of the decade of their injury (1980's versus 1990's). It appears that health-care reform has had no deleterious effect on neuropsychologists' ability to provide consultative services for this population, and following TBI, only a discrete sample of individuals receive and benefit from NPEs.

Selection of estrogen receptor beta- and thyroid hormone receptor beta-specific coactivator-mimetic peptides using recombinant peptide libraries.

Steroid and thyroid hormone receptors are members of the superfamily of nuclear receptors (NR) that participate in developmental and homeostatic mechanisms by changes in the transcription of specific genes. These activities are governed by the receptors' cognate ligands and through interaction with the components of the transcriptional machinery. A number of coactivator molecules of the steroid receptor coactivator (SRC)/nuclear receptor coactivator (NCoA) family interact with activation functions within NRs through a conserved region containing helical domains of a core LXXLL sequence and, thereby, participate in transcriptional regulation. Using a mammalian-two-hybrid assay, we show that the thyroid hormone receptor beta (TRbeta) and estrogen receptor beta (ERbeta) have different LXXLL motif preferences for interactions with SRC-1. Using large random and focused (centered on the LXXLL motif) recombinant peptide diversity libraries, we have obtained novel peptide sequences that interact specifically with ERbeta or with TRbeta in a ligand-dependent manner. Random sequence libraries yielded LXXLL-containing peptides, and sequence analysis of selected clones revealed that the preferred residues within and around the LXXLL motif vary significantly between these two receptors. We compared the receptor binding of library-selected peptides to that of peptides derived from natural coactivators. The affinities of selected peptides for the ligand binding domains of ERbeta and TRbeta were similar to the best natural LXXLL motifs tested, but showed a higher degree of receptor selectivity. These selected peptides also display receptor-selective dominant inhibitory activities when introduced into mammalian cells. Finally, by directed mutations in specific residues, we were able to alter the receptor binding preference of these peptides.

A potent dimeric peptide antagonist of interleukin-5 that binds two interleukin-5 receptor alpha chains.

Two series of peptides that specifically bind to the extracellular domain of the alpha chain of the human interleukin-5 receptor (IL-5Ralpha), but share no primary sequence homology to IL-5, were identified from libraries of random recombinant peptides. Affinity maturation procedures generated a 19-aa peptide that binds to the IL-5 receptor alpha/beta heterodimer complex with an affinity equal to that of IL-5 and is a potent and specific antagonist of IL-5 activity in a human eosinophil adhesion assay. The active form of the peptide is a disulfide-crosslinked dimer that forms spontaneously in solution. Gel filtration analysis, receptor-binding studies, and analytical ultracentrifugation reveal that the dimeric peptide binds simultaneously to two receptor alpha chains in solution. Furthermore, the dimer peptide, but not IL-5, can activate a chimeric receptor consisting of the IL-5Ralpha extracellular domain fused to the intracellular domain of the epidermal growth factor receptor, thus demonstrating that the peptide also promotes receptor dimerization in a cellular context. The functional antagonism produced by the bivalent interaction of the dimeric peptide with two IL-5R alpha chains represents a distinctive mechanism for the antagonism of cytokines that use heteromeric receptors.

Response of inflammatory markers to balloon angioplasty in peripheral arterial occlusive disease.

OBJECTIVES: To study the inflammatory response to balloon angioplasty (PTA). DESIGN: Prospective study. MATERIALS: Blood samples were drawn for cytokine analysis from 10 patients undergoing PTA before, after 60 min and 6 h after the balloon inflation. Adhesion molecules were analysed in 14 patients undergoing PTA and in seven patients undergoing angiography only. Arterial samples were taken in eight patients, before PTA, immediately after and 15 min later. Venous samples were taken in six patients and in the group undergoing angiography only. The sampling was before, 60, 90 and 120 min after the procedure. As controls served 15 patients with no signs of peripheral arterial disease. METHODS: Cytokines (IL-6, TNF-alpha) were analysed using ELISA. Adhesion molecule expression on WBC was measured by flow cytometry. RESULTS: A significant increase of IL-6 in the sample taken 6 h after the last balloon inflation was seen in five patients. TNF-alpha was raised only in one patient. The group of patients with peripheral arterial occlusive disease (PAOD) expressed pre-interventionally a higher level of adhesion molecules on WBC compared to the controls. The expression of adhesion molecules (CD11b/CD18) was significantly decreased after PTA. CONCLUSION: Only a very limited cytokine response is caused by PTA reflecting the small surgical trauma. PTA results in a downregulation of detectable CD11b/CD18 expression on WBC in the circulation, which may reflect removal of activated cells through adhesion and extravasation.

Using the Lac repressor system to identify interacting proteins.

The present protocol describes a series of procedures to identify peptides interacting with PDZ domains. It is conceivable that the procedures can be applied to other purified protein modules or intact proteins without substantial modifications. With the deduced consensus combined with sequence information, it is possible to identify proteins present in the database with compatible sequences. If the expression of target protein and potential interacting candidate overlap temporally and spatially, biochemical and molecular experiments can be designed to study their physical and functional interactions.

A minimal peptide substrate in biotin holoenzyme synthetase-catalyzed biotinylation.

The Escherichia coli biotin holoenzyme synthetase, BirA, catalyzes transfer of biotin to the epsilon amino group of a specific lysine residue of the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase. Sequences of naturally biotinylated substrates are highly conserved across evolutionary boundaries, and cross-species biotinylation has been demonstrated in several systems. To define the minimal substrate requirements in BirA-catalyzed biotinylation, we have measured the kinetics of modification of a 23-residue peptide previously identified by combinatorial methods. Although the sequence of the peptide bears little resemblance to the biotinylated sequence in BCCP, it is enzymatically biotinylated in vivo. Rates of biotin transfer to the 23-residue peptide are similar to those determined for BCCP. To further elucidate the sequence requirements for biotinylation, transient kinetic measurements were performed on a series of amino- and carboxy-terminal truncations of the 23-mer. The results, determined by stopped-flow fluorescence, allowed identification of a 14-residue peptide as the minimum required sequence. Additional support was obtained using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometric analysis of peptides that had been incubated with an excess of biotinyl-5'-adenylate intermediate and catalytic amounts of BirA. Results of these measurements indicate that while kinetically inactive truncations showed no significant shift in molecular mass to the values expected for biotinylated species, kinetically active truncations exhibited 100% biotinylation. The specificity constant (k(cat)/Km) governing BirA-catalyzed biotinylation of the 14-mer minimal substrate is similar to that determined for the natural substrate, BCCP. We conclude that the 14-mer peptide efficiently mimics the biotin acceptor function of the much larger protein domain normally recognized by BirA.

Peptide binding consensus of the NHE-RF-PDZ1 domain matches the C-terminal sequence of cystic fibrosis transmembrane conductance regulator (CFTR).

The Na+-H+ exchanger regulatory factor (NHE-RF) is a cytoplasmic phosphoprotein that was first found to be involved in protein kinase A mediated regulation of ion transport. NHE-RF contains two distinct protein interaction PDZ domains: NHE-RF-PDZ1 and NHE-RF-PDZ2. However, their binding partners are currently unknown. Because PDZ domains usually bind to specific short linear C-terminal sequences, we have carried out affinity selection of random peptides for specific sequences that interact with the NHE-RF PDZ domains and found that NHE-RF-PDZ1 is capable of binding to the CFTR C-terminus. The specific and tight association suggests a potential regulatory role of NHE-RF in cystic fibrosis transmembrane conductance regulator (CFTR) function.

Peptide agonists of the thrombopoietin receptor.

We have screened a variety of L-amino acid peptide libraries against the extracellular domain of the human thrombopoietin (HuTPO) receptor, c-Mpl. A large number of peptide ligands were recovered and categorized into two families. Peptides from each family compete with the binding of HuTPO and with the binding of peptides from the other familiy. Representative peptides were synthesized and found to activate the full-length HuTPO receptor expressed in Ba/F3 cells to promote proliferation. These peptide families show no apparent homology to the primary sequence of TPO. We have focused our optimization efforts on one of the peptides, a linear 14-mer (IEGPTLRQWLAARA) with an IC50 of 2 nM in a competition binding assay and an EC50 of 400 nM in the proliferation assay. In order to enhance the potency of the compound, we constructed dimeric peptides by linking the carboxy-termini of the 14-mers to a lysine branch. These molecules exhibited slightly higher affinity (0.5 nM) and greatly increased potency (0.1 nM). The EC50 of the dimeric peptide was equivalent to that of the 332 aa form of baculovirus-expressed recombinant HuTPO. As previously shown for the erythropoietin-mimetic peptides, the TPO-mimetic peptides probably activate the TPO receptor by binding and inducing receptor dimerization. This supposition is supported by the observation that covalent dimerization of the peptide enhances its potency by 4,000-fold over that of the monomer. The peptide dimer is also active in stimulating in vitro proliferation of progenitors and maturation of megakaryocytes from human bone marrow, and in promoting an increase in platelet count when administered to normal mice.

Peptide agonist of the thrombopoietin receptor as potent as the natural cytokine.

Two families of small peptides that bind to the human thrombopoietin receptor and compete with the binding of the natural ligand thrombopoietin (TPO) were identified from recombinant peptide libraries. The sequences of these peptides were not found in the primary sequence of TPO. Screening libraries of variants of one of these families under affinity-selective conditions yielded a 14-amino acid peptide (Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala) with high affinity (dissociation constant approximately 2 nanomolar) that stimulates the proliferation of a TPO-responsive Ba/F3 cell line with a median effective concentration (EC50) of 400 nanomolar. Dimerization of this peptide by a carboxyl-terminal linkage to a lysine branch produced a compound with an EC50 of 100 picomolar, which was equipotent to the 332-amino acid natural cytokine in cell-based assays. The peptide dimer also stimulated the in vitro proliferation and maturation of megakaryocytes from human bone marrow cells and promoted an increase in platelet count when administered to normal mice.

PDZ domain of neuronal nitric oxide synthase recognizes novel C-terminal peptide sequences.

PDZ domains are multifunctional protein-interaction motifs that often bind to the C-terminus of protein targets. Nitric oxide (NO), an endogenous signaling molecule, plays critical roles in nervous, immune, and cardiovascular function. Although there are numerous physiological functions for neuron-derived NO, produced primarily by the neuronal NO synthase (nNOS), excess nNOS activity mediates brain injury in cerebral ischemia and in animal models of Parkinson's disease. Subcellular localization of nNOS activity must therefore be tightly regulated. To determine ligands for the PDZ domain of nNOS, we screened 13 billion distinct peptides and found that the nNOS-PDZ domain binds tightly to peptides ending Asp-X-Val. This differs from the only known (Thr/Ser)-X-Val consensus that interacts with PDZ domains from PSD-95. Preference for Asp at the -2 peptide position is mediated by Tyr-77 of nNOS. A Y77D78 to H77E78 substitution changes the binding specificity from Asp-X-Val to Thr-X-Val. Guided by the Asp-X-Val consensus, candidate nNOS interacting proteins have been identified including glutamate and melatonin receptors. Our results demonstrate that PDZ domains have distinct peptide binding specificity.

Inhibition of PDC-E2 human combinatorial autoantibodies by peptide mimotopes.

Immunohistochemical studies have shown that a unique immunoreactive molecule is present near the apical region of human biliary epithelial (BE) cells in patients with primary biliary cirrhosis (PBC). This can be visualized by confocal microscopy in PBC livers using a number of unique monoclonal antibodies to the E2 component of pyruvate dehydrogenase complex (PDC-E2), the autoantigen most commonly recognized by antimitochondrial antibodies (AMA). One such antibody, the murine mAb C355.1 was used to identify peptide mimotopes of PDC-E2 by screening a random dodecapeptide phage library ON 159.2 to identify the possible biochemical nature of this apical staining molecule. Out of 36 independent clones, 29 showed a common sequence and seven other sequences were singly represented. Three common amino acid motifs (SYP, TYVS and VRH) were found among these eight sequences. Similar to C355.1, the human combinatorial antibodies derived from a patient with PBC, SP1 and SP4, recognize the inner lipoyl domain of PDC-E2. However, when these antibodies are used to stain PBC BE cells, SP4 stains the apical region of PBC BE cells with high intensity whereas SP1 produces only cytoplasmic staining. Competitive inhibition of immunohistochemical staining using PDC-E2 specific human combinatorial antibodies SP1 and SP4 was performed using five of the above dodecapeptides. Interestingly, the peptides selected with C355.1 differentially inhibited the binding of SP1 and SP4 to PBC BE cells. Finally, rabbit sera raised against one such peptide (WMSYPDRTLRTS) stained BE cells from patients with PBC with a higher intensity than controls. Comparable data was obtained with immunoelectronmicroscopy. These data suggest that a molecular mimic of PDC-E2 is present at the external aspect of PBC BE cells.

Random phage mimotopes recognized by monoclonal antibodies against the pyruvate dehydrogenase complex-E2 (PDC-E2).

Dihydrolipoamide acetyltransferase, the E2 component of the pyruvate dehydrogenase complex (PDC-E2), is the autoantigen most commonly recognized by autoantibodies in primary biliary cirrhosis (PBC). We identified a peptide mimotope(s) of PDC-E2 by screening a phage-epitope library expressing random dodecapeptides in the pIII coat protein of fd phage using C355.1, a murine monoclonal antibody (mAb) that recognizes a conformation-dependent epitope in the inner lipoyl domain of PDC-E2 and uniquely stains the apical region of bile duct epithelium (BDE) only in patients with PBC. Eight different sequences were identified in 36 phage clones. WMSYPDRTLRTS was present in 29 clones; WESYPFRVGTSL, APKTYVSVSGMV, LTYVSLQGRQGH, LDYVPLKHRHRH, AALWGVKVRHVS, KVLNRIMAGVRH and GNVALVSSRVNA were singly represented. Three common amino acid motifs (W-SYP, TYVS, and VRH) were shared among all peptide sequences. Competitive inhibition of the immunohistochemical staining of PBC BDE was performed by incubating the peptides WMSYPDRTLRTS, WESYPDRTLRTS, APKTYVSVSGMV, and AALWGVKVRHVS with either C355.1 or a second PDC-E2-specific mAb, C150.1. Both mAbs were originally generated to PDC-E2 but map to distinct regions of PDC-E2. Two of the peptides, although selected by reaction with C355.1, strongly inhibited the staining of BDE by C150.1, whereas the peptide APKTYVSVSGMV consistently inhibited the staining of C355.1 on biliary duct epithelium more strongly than the typical mitochondrial staining of hepatocytes. Rabbit sera raised against the peptide WMSYPDRTLRTS stained BDE of livers and isolated bile duct epithelial cells of PBC patients more intensively than controls. The rabbit sera stained all size ducts in normals, but only small/medium-sized ductules in PBC livers. These studies provide evidence that the antigen present in BDE is a molecular mimic of PDC-E2, and not PDC-E2 itself.