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We have recently described an algorithm to design, among others, peptides with complementarity contour to autoimmune epitopes. Immunization with one such peptide resulted in a monoclonal antibody (mAb), termed CTCR8, that specifically recognized Vbeta15 containing TCR on acetylcholine receptor (AChR) alpha-chain residue 100-116-specific T cells. CTCR8 was found to label the cell surface of AChR100-116-specific T cell lines and clones, immunoprecipitate the TCR from such cells, and block their proliferative responses to AChRalpha100-116. In the present report, we have found that there is a marked reduction in IFN-gamma and no effect on IL-10 production in a CTCR8-treated AChRalpha100-116-specific T cell line. Interestingly, when AChR100-116-primed, primary T cells were stimulated with peptide and treated with CTCR8, there was once again inhibition of IFN-gamma but also marked stimulation of IL-10 production. The change in the Th1/Th2 cytokine profile was paralleled by a reduction in AChR-specific IgG2a and IgM with no effect on IgG1. Remarkably, the most profoundly inhibited Ab population was that which causes experimental autoimmune myasthenia gravis (EAMG) by reaction with the main immunogenic region (alpha61-76) of the AChR. Based on these results, CTCR8 was tested for prophylactic and therapeutic effects in EAMG. EAMG induced by immunization with purified native Torpedo AChR was both inhibited and reversed by CTCR8. These findings suggest a means to produce therapeutic mAb for the treatment of autoimmune diseases., (Copyright 2001 Academic Press.)

Release of allergic mediators from mast cells is enhanced by very late Ag (VLA)-5-mediated interaction of these cells with fibronectin. In this report, we show that VLA-5-mediated adhesion of bone marrow-derived mast cells to fibronectin can be induced by two different pathways: first, FcepsilonRI clustering, which depends on calmodulin activation and extracellular Ca(2+), and, second, by Mn(2+) stimulation, which is independent of calmodulin activation and antagonized by Ca(2+). Previous studies have shown the presence of several cation-binding domains in VLA-5 that are homologous to the calcium-binding EF-hands of calmodulin. To show a role for EF-hands of different proteins in VLA-5-mediated adhesion, we used calcium-like peptides (CALP), CALP1 and CALP2, designed to bind to EF-hands based on inverted hydropathy. CALP1 and, more potently, CALP2 inhibited FcepsilonRI-induced adhesion to fibronectin via different mechanisms. The target for the effects of CALP1 and 2 on FcepsilonRI-induced adhesion and degranulation was intracellular and likely involved calmodulin. Interestingly only CALP2 was able to inhibit Mn(2+)-induced calmodulin-independent adhesion by interfering with an extracellular target, which is probably VLA-5. We conclude that CALP1 and 2 can inhibit VLA-5-mediated adhesion of mast cells to fibronectin through binding to EF-hands of multiple proteins, and that these peptides can be used as lead compounds for the development of future therapy against allergy.

We have used the method of inverted hydropathy to develop peptides that interact with EF hands of calmodulin (CaM). Previously we have shown these peptides specifically interact with their desired target in a productive manner, in that they activated CaM in the absence of Ca(2+). Therefore, we sought to determine whether these peptides would enter cells, remain intact, and interact with CaM in the interior of the cell. Using several techniques we have demonstrated cellular uptake, stability, and an intracellular interaction with CaM with fluorescein-labeled and radiolabeled peptides in Jurkat T cells. The results suggest that these peptides may be useful in the study and the manipulation of Ca(2+)-mediated pathways in cells., (Copyright 2000 Academic Press.)

Calmodulin (CaM), as well as other Ca(2+) binding motifs (i.e., EF hands), have been demonstrated to be Ca(2+) sensors for several ion channel types, usually resulting in an inactivation in a negative feedback manner. This provides a novel target for the regulation of such channels. We have designed peptides that interact with EF hands of CaM in a specific and productive manner. Here we have examined whether these peptides block certain Ca(2+)-permeant channels and inhibit biological activity that is dependent on the influx of Ca(2+). We found that these peptides are able to enter the cell and directly, as well as indirectly (through CaM), block the activity of glutamate receptor channels in cultured neocortical neurons and a nonselective cation channel in Jurkat T cells that is activated by HIV-1 gp120. As a consequence, apoptosis mediated by an influx of Ca(2+) through these channels was also dose-dependently inhibited by these novel peptides. Thus, this new type of Ca(2+) channel blocker may have utility in controlling apoptosis due to HIV infection or neuronal loss due to ischemia.

Purpose: We have previously presented evidence that the neutrophil chemoattractant, N-acetyl-proline-glycine-proline (N-acetyl-PGP), triggers the initial polymorphonuclear leukocyte (PMN) invasion into the alkali-injured eye. In this study, sense-antisense methodology was used to develop novel complementary peptides to be potential inhibitors of N-acetyl-PGP., Methods: The polarization assay was used to measure the potential chemotactic response of PMNs to synthetic N-acetyl-PGP, the ultrafiltered tripeptide chemoattractants obtained from alkali-degraded rabbit corneas, or leukotriene B4 (LTB4). Inhibition was expressed as the peptide concentration producing 50% inhibition (ID50) of polarization. Five complementary peptides were tested as potential inhibitors of N-acetyl-PGP: arginine-threonine-arginine (RTR), RTR-glycine-glycine (RTRGG), RTR dimer, RTR tetramer, and alanine-serine-alanine (ASA) tetramer. In addition, the RTR tetramer and both monomeric peptides (RTR and RTRGG) were separately tested for inhibition of the ultrafiltered tripeptide chemoattractants or LTB4., Results: The complementary RTR tetrameric peptide was a powerful antagonist of N-acetyl-PGP-induced PMN polarization (ID50 of 200 nM). The RTR dimer was much less potent (ID50 of 105 microM). Both monomeric peptides, RTR and RTRGG, were only antagonistic at millimolar concentrations. The ASA tetramer showed no capacity to inhibit N-acetyl-PGP. The RTR tetramer also inhibited PMN activation by the ultrafiltered tripeptide chemoattractants (ID50 of 30 microM) but had no effect on LTB4., Conclusions: A complementary peptide (RTR) was designed which is an effective inhibitor of the neutrophil chemoattractant, N-acetyl-PGP. The potency of the RTR complementary peptide is dramatically enhanced by tetramerization. Inhibition of N-acetyl-PGP by complementary peptides offers great promise for control of the inflammatory response in the alkali-injured eye.

Myasthenia gravis (MG) and its animal model, experimental autoimmune (EA) MG, are caused by T cell-dependent autoantibodies that react with the nicotinic acetylcholine receptor (AChR) on muscle and interfere with neuromuscular transmission. Thus, selective inactivation of CD4(+) AChR-specific T helper cells should lower AChR Ab levels and ameliorate disease. In the Lewis rat model of EAMG, alpha chain residues 100-116 of the AChR represent the dominant T cell epitope, which is important in helping Ab responses to this autoantigen. In the present report, we have applied a new design technique that requires no knowledge of Ag receptor sequences on errant T cells in order to develop a synthetic peptide vaccine against T cells reactive with the aforementioned T cell epitope. Immunization with the peptide 1) induced polyclonal and monoclonal Ab, which inhibited AChR 100-116 stimulation of AChR-sensitized lymphocytes and recognized Vbeta15 containing T cell receptors on AChR 100-116-specific T cell lines and clones; 2) lowered AChR Ab levels; 3) reduced the loss of muscle AChR; and 4) lessened the incidence and severity of EAMG. These findings suggest a new strategy for the functional abrogation of epitope-specific T cells that could have potential application to human autoimmune diseases.

The cystic fibrosis gene encodes a chloride channel, CFTR (cystic fibrosis transmembrane conductance regulator), that regulates salt and water transport across epithelial tissues. Phosphorylation of the cytoplasmic regulatory (R) domain by protein kinase A activates CFTR by an unknown mechanism. The amino-terminal cytoplasmic tail of CFTR was found to control protein kinase A-dependent channel gating through a physical interaction with the R domain. This regulatory activity mapped to a cluster of acidic residues in the NH(2)-terminal tail; mutating these residues proportionately inhibited R domain binding and CFTR channel function. CFTR activity appears to be governed by an interdomain interaction involving the amino-terminal tail, which is a potential target for physiologic and pharmacologic modulators of this ion channel.

Purpose: The release of N-acetyl-proline-glycine-proline (PGP), a chemoattractant resulting from direct alkaline hydrolysis of corneal proteins, is believed to be the initial trigger for neutrophil invasion into the alkali-injured cornea. The purpose of this study is twofold: (1) to compare the activity of N-acetyl-PGP with the bioactivities of other similar synthetic peptides in an effort to uncover information about this chemoattractant molecule, and (2) to test these peptide analogs as potential antagonists of N-acetyl-PGP., Methods: The polarization assay was used to measure the potential chemotactic response of human neutrophils to peptides. Bioactivity was expressed as the peptide concentration required to produce 50% neutrophil polarization (EC50). Antagonist activity was expressed as the peptide concentration required to produce 50% inhibition (ID50) of polarization activated by N-acetyl-PGP., Results: Peptide bioactivities (EC50) were ranked as follows: APGPR (0.34 mM) > N-acetyl-PGP (0.5 mM) > N-(PGP)4-PGLG (3 mM) = t-Boc-PGP (3 mM) > N-acetyl-PG (3.4 mM) > N-methyl-PGP (15 mM) = PGP (15 mM) > peptides without detectable activity (t-Boc-PGP-OMe, N-acetyl-P, PG, PGG, GP, GG and gly-pro-hyp). Peptides with no detectable bioactivity were tested as potential antagonists of neutrophil polarization induced by N-acetyl-PGP. Gly-Pro-Hyp inhibited N-acetyl-PGP activation of polarization at 20 mM (ID50). No other synthetic peptide demonstrated a capacity for inhibition., Conclusions: The minimum requirement to elicit bioactivity was the presence of PGP alone or derivatives of PG in which the N-terminal proline is blocked. Using this approach, active and inactive mimetic peptides of N-acetyl-PGP were produced. The most active peptide, APGPR, was equal to or slightly greater than N-acetyl-PGP, suggesting that more potent analogs might be designed. Gly-pro-hyp was the only inactive peptide analog to inhibit the chemoattractant.

Papilledema from benign intracranial hypertension can cause severe loss of visual acuity and visual field, with an optic neuropathy. We report a study of 5 patients with benign intracranial hypertension, and severe visual loss (visual field loss and visual acuity reduced to 1/10 or less) not improved by medical therapy (acetazolamide). We performed unilateral orbital decompression of the optic nerve sheath. No major operative complication was noted. Follow-up ranged from 11 months to 23 months, with an average of sixteen months. Visual function improvement was noted within 3 months after operation in 4 patients (for 2 eyes on the surgical side improvement of 2/10 and 5/10, for 3 eyes on the opposite surgical side average improvement of 4.6/10). The other patient showed improvement, but no significant. Optic nerve sheath decompression in benign intracranial hypertension seems to be a safe procedure and a therapeutic option in the management of raised intracranial pressure complicated by optic neuropathy with severe visual loss.

A 25-year-old man sustained bilateral ocular trauma from an explosion, which resulted in ocular injuries from the blast, mineral projections, and heat. Before the accident, the patient had eight-incision radial keratotomy in the left eye followed 1 week later by photorefractive keratectomy in the right eye. After the accident, the left cornea had a full-thickness rupture of four incisions; the fellow cornea had a full-thickness laceration from a mineral projection. Five months after the accident, the left eye had an uncorrected visual acuity of 20/25; the right eye did not achieve an acuity of 20/200 until 20 months after the accident.

Pharmacological experiments have suggested that ocular ischemia, induced by high intraocular pressure in the rabbit, provokes an oxidative stress responsible for functional alteration of the retina. However, the nature of the oxidant chemical species and their mode of generation were not elucidated. The aim of the present studies was to characterize the oxygen-derived free radicals produced during and/or after the hyperpressure period. The technique used was based on electron spin resonance spin trapping analysis of the signals obtained in microdialysates of the retina perfused with the nitrone 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO). The oxidative stress was also evaluated under ischemia and reperfusion periods by measuring the level of ascorbate in the retina via electron spin resonance detection of the ascorbyl free radical-dimethyl sulfoxide (AFR-DMSO) complex. Electroretinograms were recorded to determine the functional consequences of high intraocular pressure and free radical generation. Our results show that superoxide dismutase-inhibitable DEPMPO/hydroxyl radical adducts were generated during the high intraocular pressure period and that the oxidative stress was not increased at reperfusion as assessed by spin trapping and AFR-DMSO measurements. Functional protection provided by free radical scavengers (superoxide dismutase+catalase, TEMPO nitroxide+catalase and dimethylthiourea) against high intraocular pressure-induced electroretinogram alteration confirmed these observations. In conclusion, these experiments demonstrate for the first time by direct measurement that oxygen-derived free radicals are produced by the retina during acute ischemia. This generation could be the explanation for electroretinogram alteration.

General anesthesia was obtained using a laryngeal mask in 53 patients with nasal fractures. The advantages of this procedure are: no need for curarization, easy removal, and awakening in the operating room. No complications were observed although orotracheal intubation was necessary in 3 patients.

Vital capacity and residual lung volume (in terms of 1/min or ml/m 2 of body surface area) of 357 subjects (205 males, 152 females) was evaluated in La Paz, Bolivia, situated at 3,750 m. The sample included: (1) 37 high altitude rural natives (all male), (2) 125 high altitude urban natives (69 male, 58 female), (3) 85 Bolivians of foreign ancestry acclimatized to high altitude since birth (40 male, 45 female), (4) 63 Bolivians of foreign ancestry acclimatized to high altitude during growth (30 male, 33 female), and (5) 47 non-Bolivians of either European or North American ancestry acclimatized to high altitude during adulthood (24 male, 23 female). Results indicate that (1) all samples studied, irrespective of origin or acclimatization status, have larger lung volumes than those predicted from sea level norms; (2) the high altitude rural natives have significantly greater lung volumes (vital capacity and residual lung volume) than the high altitude urban natives and all the non-native high altitude samples; (3) males acclimatized to high altitude since birth or during growth attain similar lung volumes as high altitude urban natives and higher residual lung volumes than subjects acclimatized to high altitude during adulthood but lower than the high altitude rural natives; (4) females acclimatized to high altitude since birth or during growth attain similar lung volumes as subjects acclimatized to high altitude during adulthood; (5) age at arrival to high altitude is inversely related to residual lung volume but not vital capacity; (6) among subjects acclimatized to high altitude during growth, approximately 20-25% of the variability in residual lung volume can be explained by developmental factors; (7) among high altitude rural and urban natives, it appears that approximately 20-25% of the variability in residual lung volume at high altitude can be explained by genetic traits associated with skin reflectance and genetic traits shared by siblings; and (8) vital capacity, but not the residual lung volume, is inversely related to occupational activity level. Together these data suggest that the attainment of vital capacity at high altitude is influenced more by environmental factors, such as occupational activity level, and body composition than developmental acclimatization. On the other hand, the attainment of an enlarged residual volume is related to both developmental acclimatization and genetic factors. Am. J. Hum. Biol. 9:191-203, 1997. © 1997 Wiley-Liss, Inc., (Copyright © 1997 Wiley-Liss, Inc.)

Dirofilaria repens is an endemic canine parasite in the Mediterranean area. Human infestation is rare. We observed a case in an HIV-positive patient who presented an ocular filaria which was successfully removed under local anesthesia. In man, filariae may be expulsed without a parasite cycle or complete a growth cycle, depending on the individual's immunological status. The disease has been known in southern France for several centuries, mainly in lagoons near the Mediterranean seaside. A review of the literature however shows that observation of clinical cases is rare, only 46 cases having been reported to date. Outcome is favorable.

The purpose of the study was to compare the retinal sensitivity of pigmented and albino rabbits to ischemia/reperfusion-induced electroretinogram (ERG) alterations and optic nerve morphological changes. High intraocular pressure (HIOP) was induced by applying a suction-cup on the eye and a depression with an ophthalmodynamometer. HIOP was maintained for lengths of time (30-75 min). Flash ERGs were recorded in dark-adapted animals for ischemia and 2 h reperfusion periods. Two weeks later, histological examination of the retinas and optic nerves was done. Albino rabbits submitted to 45 min HIOP failed to recover b-wave ERG amplitude after 2 h reperfusion, whereas pigmented animals presented a total ERG recovery even if ischemia was maintained as long as 75 min. Intravenous treatment of albino animals with Lazaroid U74389G led to significant ERG recovery at reperfusion. Histological studies show that pigmented rabbit optic nerves suffered less damage than the albino ones. These results emphasize the role of the pigmentary status of the animals in the retinal sensitivity to ischemia. Neuroprotection afforded by the antioxidant U74389G suggests that ocular pigments could also protect the retinal functional integrity through a free radical scavenging activity.

Amidating mouse pituitary cells (AtT-20) have been engineered to secrete human calcitonin (hCT) in the fully active amidated form, without the need of additional enzymatic or chemical modifications. The 141-residue human calcitonin precursor has first been cloned in the eucaryotic expression vector pRc/RSV, and the resulting plasmid pRc/RSV/hCT introduced in AtT-20 cells. After transfection, 122 independent clones resistant to G-418 were selected and screened for calcitonin production using a competitive ELISA specifically designed to detect the amidated form of calcitonin. One of these clones was amplified and showed expression of 17 ng/ml of hCT, with a 70% increase in productivity after cAMP treatment. Calcitonin was partially purified from culture medium by two sequential steps of reverse-phase chromatography and characterized in terms of immunoreactivity and molecular weight by TOF-MALDI mass spectroscopy, which confirmed the intended chemical nature and the presence of the C-terminal amidated residue.

Radial keratotomy reduces the strength of the cornea front of a trauma by blast. The authors present and analyze the case of a 25-year-old patient having been submitted to an ocular bilateral trauma by explosion. Twenty-two months earlier he had undergone radial keratotomy (RK) in the left eye followed 8 days later by photorefractive keratectomy (PKR) in the right eye for a -6,00 d. myopia RLE. Though the trauma by blast essentially reached the right eye, the corneal anatomical damages were more important in the left eye. This is according to us the first case report of trauma on eye subjected to PRK. We appreciate the clinical differences presented by each eye.

Cavernous sinus exploration, anterior middle fossa transpetrous approach, and saphenous vein graft bypass require proximal control of the horizontal segment of the petrous internal carotid artery. Exposing the petrous portion of the internal carotid artery is not without the potential for serious complications (cochlea, facial nerve, auditory tube, musculus tensor tympani). With guidance from the classicaly landmarks within Glasscock's triangle, the bony petrous carotid canal can be unroofed. The authors describe an alternative method for obtaining direct vascular control under the trigeminal ganglion, safety unroofing of the carotid canal and control of the posterior face of the carotid bend. The indications, advantages, and disadvantages of this approach are described in details, along with its use in seven patients.

Antibodies raised in rabbits against multimeric all-L peptides (MAP's) were first made monospecific by affinity chromatography on immobilized antigen columns and then tested for their ability to cross-react with topologically related variants of the parent antigen, where the chirality of each amino-acid residue (inverso derivatives), or the peptide sequence orientation (retro derivatives), was inverted, or where both modifications were simultaneously introduced (retro-inverso derivatives). Retro, inverso, and retro-inverso forms of the parent peptide were prepared, both in the linear as well as in the BSA-conjugated form, and found to cross-react to a significant extent with affinity purified polyclonal antibodies raised against the parent peptide. Peptide variants displayed similar dose-dependent inhibitory effects on the interaction between immobilized parent antigen and affinity purified antibodies. Analysis of molecular models of the peptide variants in the trans-configuration suggested that the topological equivalence of alternating side chains in the series of related peptides may be responsible for the observed cross-recognition, leading to the formation of similar recognition surfaces which could mimic the parent peptide antigenic structure.

This work is based on the microscopic study of 30 trochlear nerve trunks (15 heads). In 17 cases, the trunk arose from two nerve bundles, in 8 cases from one bundle, and for the other 5 nerves, three or four bundles. The mean total length of the trochlear nerve was 86 mm. The nerve may be separated into the 3 following parts: infratentorial, intracavernous, intraorbital. In all 30 cases studied, the first part of the nerve was infratentorial, thus leading us to suggest the term "infratentorial part" for this segment of the nerve. In 27 cases, contact was found with the superior cerebellar artery, in the infratentorial part. In the intracavernous part of ten nerves we found two rami tentorii and in eight cases fibers were exchanged with the ophthalmic nerve. In the orbit, 18 trochlear nerves crossed the posterior ethmoidal artery. 23 trochlear nerves ended on the medial face of the superior oblique muscle. The remaining 7 ended at the superior border of the muscle.