A Heterotopic Mouse Model for Studying Laryngeal Transplantation.

Laryngeal heterotopic transplantation, although a technically challenging procedure, offers more scientific analysis and cost benefits compared to other animal models. Although first described by Shipchandler et al. in 2009, this technique is not widely used, possibly due to the difficulties in learning the microsurgical technique and time required to master it. This paper describes the surgical steps in detail, as well as potential pitfalls to avoid, in order to encourage effective use of this technique. In this model, the bilateral carotid arteries of the donor larynx are anastomosed to the recipient carotid artery and external jugular vein, allowing for blood flow through the graft. Blood flow can be confirmed intraoperatively by the visualization of blood filling in the graft bilateral carotid arteries, reddening of the thyroid glands of the graft, and bleeding from micro vessels in the graft. The crucial elements for success include delicate preservation of the graft vessels, making the correct size arteriotomy and venotomy, and using the appropriate number of sutures on the arterial-arterial and arterial-venous anastomoses to secure vessels without leakage and prevent occlusion. Anyone can become proficient in this model with sufficient training and perform the procedure in approximately 3 h. If performed successfully, this model allows for immunologic studies to be performed with ease and at low cost.

Trilayered Hydrogel Scaffold for Vocal Fold Tissue Engineering.

The lamina propria within the vocal fold (VF) is a complex multilayered tissue that increases in stiffness from the superficial to deep layer, where this characteristic is crucial for VF sound production. Tissue-engineered scaffolds designed for VF repair must mimic the biophysical nature of the native vocal fold and promote cell viability, cell spreading, and vibration with air flow. In this study, we present a unique trilayered, partially degradable hydrogel scaffold that mimics the multilayered structure of the VF lamina propria. Using thiol-norbornene photochemistry, trilayered hydrogel scaffolds were fabricated via layer-by-layer stacking with increasing polymer concentration from the top to middle to deep layer. Mechanical analysis confirmed that hydrogel modulus increased with increasing polymer concentration. Partially degradable hydrogels promoted high cell viability and cell spreading in three dimensions as assessed via live/dead and cytoskeleton staining, respectively. Importantly, partially degradable hydrogels maintained some degree of the three dimensional polymer network following protease exposure, while still enabling encapsulated cells to remodel their local environment via protease secretion. Finally, the trilayered hydrogel scaffold successfully vibrated and produced sound in proof-of-concept air flow studies. This work represents a critical first step toward the design of a multilayered, hydrogel scaffold for vocal fold tissue engineering.

Human amniotic epithelial cell transplantation improves scar remodeling in a rabbit model of acute vocal fold injury: a pilot study.

OBJECTIVE: To gain insight into the molecular mechanisms underlying the early stages of vocal fold extracellular matrix (ECM) remodeling after a mid-membranous injury resulting from the use of human amniotic epithelial cells (hAEC), as a novel regenerative medicine cell-based therapy. METHODS: Vocal folds of six female, New Zealand White rabbits were bilaterally injured. Three rabbits had immediate bilateral direct injection of 1 × 106 hAEC in 100 µl of saline solution (hAEC) and three with 100 µl of saline solution (controls, CTR). Rabbits were euthanized 6 weeks after injury. Proteomic analyses (in-gel trypsin protein digestion, LC-MS/MS, protein identification using Proteome Discoverer and the Uniprot Oryctolagus cuniculus (Rabbit) proteome) and histological analyses were performed. RESULTS: hAEC treatment significantly increased the expression of ECM proteins, elastin microfibril interface-located protein 1 (EMILIN-1) and myocilin that are primarily involved in elastogenesis of blood vessels and granulation tissue. A reactome pathway analysis showed increased activity of the anchoring fibril formation by collagen I and laminin, providing mechanical stability and activation of cell signaling pathways regulating cell function. hAEC increased the abundance of keratin 1 indicating accelerated induction of the differentiation programming of the basal epithelial cells and, thereby, improved barrier function. Lastly, upregulation of Rab GDP dissociation inhibitor indicates that hAEC activate the vesicle endocytic and exocytic pathways, supporting the exosome-mediated activation of cell-matrix and cell-to-cell interactions. CONCLUSIONS: This pilot study suggests that injection of hAEC into an injured rabbit vocal fold favorably alters ECM composition creating a microenvironment that accelerates differentiation of regenerated epithelium and promotes stabilization of new blood vessels indicative of accelerated and improved repair.

A Tissue Engineered Construct for Laryngeal Regeneration: A Proof-of-Concept Device Design Study.

OBJECTIVES/HYPOTHESIS: Develop a patient-specific tissue engineered construct for laryngeal reconstruction following a partial laryngectomy. STUDY DESIGN: Bench and animal research. METHODS: A construct made from a porous polyethylene scaffold shaped in a canine-specific configuration and seeded with autologous canine adipose-derived stem cells in fibrin glue was implanted in a canine following a partial laryngectomy. After 1 year, the construct was first evaluated in vivo with high-speed imaging and acoustic-aerodynamic measures. It was then explanted and evaluated histologically. RESULTS: The canine study at 1 year revealed the construct provided voicing (barking) with acoustic and aerodynamic measures within normal ranges. The canine was able to eat and breathe normally without long-term support. The construct was integrated with epithelialization of all areas except the medial portion of the vocal fold structure. No anti-infective agents were needed after the standard perioperative medications were completed. CONCLUSION: This study provided a successful first step toward developing a patient-specific composite construct for patients undergoing partial laryngectomies. LEVEL OF EVIDENCE: NA Laryngoscope, 132:S1-S11, 2022.

High calories but not fat content of lard-based diet contribute to impaired mitochondrial oxidative phosphorylation in C57BL/6J mice heart.

PURPOSE: High calorie intake leads to obesity, a global socio-economic and health problem, reaching epidemic proportion in children and adolescents. Saturated and monounsaturated fatty acids from animal (lard) fat are major components of the western-pattern diet and its regular consumption leads to obesity, a risk factor for cardiovascular disease. However, no clear evidence exists whether consumption of diet rich in saturated (SFAs) and monounsaturated (MUFAs) fatty acids has detrimental effects on cardiac structure and energetics primarily due to excessive calories. We, therefore, sought to determine the impact of high calories versus fat content in diet on cardiac structure and mitochondrial energetics. METHODS: Six-week-old C57BL/6J mice were fed with high calorie, high lard fat-based diet (60% fat, HFD), high-calorie and low lard fat-based diet (10% fat, LFD), and lower-calorie and fat diet (standard chow, 12% fat, SCD) for 10 weeks. RESULTS: The HFD- and LFD-fed mice had higher body weight, ventricular mass and thickness of posterior and septal wall with increased cardiomyocytes diameter compared to the SCD-fed mice. These changes were associated with a reduction in the mitochondrial oxidative phosphorylation (OXPHOS) complexes I and III activity compared to the SCD-fed mice without significant differences between the HFD- and LFD-fed animals. The HFD-fed animals had higher level of malondialdehyde (MDA) than LFD and SCD-fed mice. CONCLUSIONS: We assume that changes in cardiac morphology and selective reduction of the OXPHOS complexes activity observed in the HFD- and LFD-fed mice might be related to excessive calories with additional effect of fat content on oxidative stress.

Computed Tomography Data to Generate a Reproducible, Anatomically Accurate Hemilaryngeal Model.

OBJECTIVE: The study aims to demonstrate the reproducibility and feasibility of creating a hemilaryngeal model with a medialized vocal fold (VF) using 3-dimensional (3D) modeling techniques in both healthy larynges and those affected by cancer. STUDY DESIGN: Three-dimensional modeling of human larynges. SETTING: Tertiary academic referral center and regenerative medicine laboratory. SUBJECTS AND METHODS: Computed tomography (CT) scans from 10 healthy control and 10 patients with laryngeal cancer were segmented and imported into 3D modeling software. The larynx was cut sagittally to create a hemilaryngeal model and the vocal fold medialized. Measurements were taken from the CT and 3D model data and compared. RESULTS: All control modeling data closely matched the CT data and were not statistically different from each other. There was a significant correlation between subglottic anteroposterior diameter and VF length (r2 = 0.78, P = .0008), and it may be a valuable tool to infer true VF dimension in cases where disruption has occurred. The modeling data from patients with cancer did not show statistical difference to the control data, showing that accurate modeling can also be achieved in patients with laryngeal cancer. CONCLUSION: CT scan-based 3D modeling of the larynx and VF is possible and reproducible. The results closely match those previously reported in the literature and can also be replicated in cases with laryngeal cancer. This study paves the way for future de novo fabricated laryngeal scaffolds that can be synthesized using 3D printers and tailored to meet surgical demands.

Chemotherapy can induce weight normalization of morbidly obese mice despite undiminished ingestion of high fat diet.

Morbidly obese patients who accomplish substantial weight loss often display a long-term decline in their resting metabolism, causing even relatively restrained caloric intake to trigger a relapse to the obese state. Paradoxically, we observed that morbidly obese mice receiving chemotherapy for cancer experienced spontaneous weight reduction despite unabated ingestion of their high fat diet (HFD). This response to chemotherapy could also be achieved in morbidly obese mice without cancer. Optimally dosed methotrexate (MTX) or cyclophosphamide (CY) enabled the mice to completely and safely normalize their body weight despite continued consumption of obesogenic quantities of HFD. Weight reduction was not attributable to decreased HFD intake, enhanced energy expenditure or malabsorption. MTX or CY dosing significantly depleted both adipose tissue and preadipocyte progenitors. Remarkably, however, despite continued high fat feeding, a compensatory increase in hepatocyte lipid storage was not observed, but rather the opposite. Gene microarray liver analyses demonstrated that HFD mice receiving MTX or CY experienced significantly inhibited lipogenesis and lipid storage, whereas Enho (energy homeostasis) gene expression was significantly upregulated. Further metabolic studies employing a human hepatocellular line revealed that MTX treatment preserved robust oxidative phosphorylation, but also promoted mitochondrial uncoupling with a surge in proton leak. This is the first report that certain optimally dosed chemotherapeutic agents can induce weight loss in morbidly obese mice without reduced dietary intake, apparently by depleting stores of adipocytes and their progenitors, curtailment of lipogenesis, and inconspicuous disposal of incoming dietary lipid via a steady state partial uncoupling of mitochondrial oxidative phosphorylation.

Duloxetine prophylaxis for episodic migraine in persons without depression: a prospective study.

OBJECTIVE: The objective of this pilot study is to evaluate the effects of daily duloxetine, 60-120 mg, on the frequency, duration, and severity of migraine attacks and the level of disability in episodic migraineurs. BACKGROUND: There is a need for more proven effective migraine preventive medications. Two antidepressants, both of which block serotonin and norepinephrine reuptake, have been shown to be effective in the preventive treatment of migraine. Neither has earned a level A recommendation in the 2012 guidelines of the American Academy of Neurology. Duloxetine also blocks serotonin and norepinephrine reuptake. METHODS: This was a prospective, 5-visit study on duloxetine treatment of episodic migraine headache with 4-10 migraine days, and less than 15 headache days per month. Patients were titrated to a goal dose of 120 mg. They were excluded if they had depression. RESULTS: There were 22 completers plus 5 subjects who took at least 1 dose of drug. The mean duloxetine dose was 110 mg. In a modified intent-to-treat analysis, subjects went from 9.2 ± 2.7 headache days per month at baseline to 4.5 ± 3.4 headache days per month (P < .001). There were no significant differences in the average headache duration, average headache severity, maximum headache attack severity, and level of functioning. Fifty-two percent of subjects had a 50% or greater improvement in headache days. CONCLUSIONS: Migraine prophylactic treatment with high-dose duloxetine may be effective in a nondepressed individual. The reported treatment response is in line with other commonly used migraine preventives.

CTL recognition of a novel HLA-A*0201-binding peptide derived from glioblastoma multiforme tumor cells.

Genetic instability of tumor cells can result in translation of proteins that are out of frame, resulting in expression of neopeptides. These neopeptides are not self-proteins and therefore should be immunogenic. By eluting peptides from human glioblastoma multiforme (GBM) tumor cell surfaces and subjecting them to tandem mass spectrometry, we identified a novel peptide (KLWGLTPKVTPS) corresponding to a frameshift in the 3' beta-hydroxysteroid dehydrogenase type 7 (HSD3B7) gene. HLA-binding algorithms predicted that a 9-amino acid sequence embedded in this peptide would bind to HLA-A*0201. We confirmed this prediction using an HLA-A*0201 refolding assay followed by live cell relative affinity assays, but also showed that the 12-mer binds to HLA-A*0201. Based on the 9-mer sequence, optimized peptide ligands (OPL) were designed and tested for their affinities to HLA-A*0201 and their abilities to elicit anti-peptide and CTL capable of killing GBM in vitro. Wild-type peptides as well as OPL induced anti-peptide CTL as measured by IFN-γ ELISPOTS. These CTL also killed GBM tumor cells in chromium-51 release assays. This study reports a new CTL target in GBM and further substantiates the concept that rational design and testing of multiple peptides for the same T-cell epitope elicits a broader response among different individuals than single peptide immunization.

Immunity, cancer and aging: lessons from mouse models.

The deterioration of immune function with advancing age is associated with an increased incidence of cancer. Most of the studies to evaluate the effect of immunotherapy on cancer have been conducted in the young without considering the effect of age-associated changes in immune function. Studies from my laboratory and others groups indicate that immunotherapeutic interventions could be effective in young animals, but that the same therapies are not as effective in old animals. The present review summarizes some defects found in the old immune system affecting the activation of antitumor immune responses, the strategies used to activate a more robust antitumor immune response in the old and the description of a preclinical tumor model indicating possible strategies for optimization of immunotherapeutic interventions in the old.

Proteomic identification of an MHC-binding peptidome from pancreas and breast cancer cell lines.

Peptides bound to cell surface MHC class I molecules allow the immune system to recognize intracellular pathogens and tumor-derived peptides. Our goal was to learn what the immune system "sees" on the surfaces of tumor cells by acid-eluting peptides from HLA molecules for extended time periods. We determined how long peptides would continue to elute over time from a pancreatic tumor cell line, Panc-1, and a breast cancer cell line, MCF-7, at pH 3.0 in citrate buffer while monitoring viability. Both cell lines demonstrated greater than 90% viability after 25min at pH 3.0. Panc-1 remained >90% intact after 45min at pH 3.0. Acid eluted peptide sequences were identified using LC-MS/MS and searching the NCBI refseq database. The total number of peptides eluted peaked between 40 and 45min for Panc-1, but continued to increase over time from MCF-7. A total of 131 peptides were identified from Panc-1 while 101 peptides were identified from MCF-7 elutions. Two classes of peptides were eluted: (1) 8-10 amino acid peptides fitting the HLA-binding motifs of each cell line, and (2) peptides longer than 10 amino acids containing HLA-binding motifs of each cell line. W6/32 antibody affinity purification of intact MHC molecules after papain cleavage of MHC class I from tumor cell surfaces also indicated that peptides longer than 10 amino acids bind to class I proteins. A peptide-MHC-refolding assay further substantiated the binding of longer peptides to HLA-A*0201. Our findings provide sequences and gene names of peptides presented by MHC class I molecules from common pancreas and breast cancer cell lines. We utilized a novel refolding assay to demonstrate that peptides longer than the canonical 8-10 amino acids commonly bind in MHC class I cell surface molecules.

Correlation of increase in phosphene threshold with reduction of migraine frequency: observation of levetiracetam-treated subjects.

OBJECTIVES: To correlate the reduction in migraine frequency with change in phosphene threshold of transcranial magnetic stimulation during levetiracetam treatment. BACKGROUND: Several case series have suggested levetiracetam efficacy may be effective in the management of migraine. Phosphene threshold is reduced in patients with migraine with aura, migraine without aura, and menstrual migraine. Preventive treatment may raise phosphene threshold while reducing headache frequency. METHODS: Subjects experiencing 4-10 migraine attacks per month and not currently receiving preventive treatment for the indication of migraine were recruited into an open-label trial using levetiracetam, and asked to record headache symptoms, severity, duration, and acute medication use in a daily diary. Following a 28-day qualifying baseline period, subjects were titrated over 6 weeks to either a total daily dose of 3000 mg or their maximum tolerated dose (minimum tolerated daily dose of 1000 mg required). Transcranial magnetic stimulation was performed at day 28 and days 26, 28, 84, and 154. The visual cortex of each subject was stimulated 2 times at 20% power. Power was increased by 10% increments until at least one of the 2 stimulations produced a positive phosphene response. Once a positive response was achieved, a random order of 5 stimulation intensities surrounding the initial positive threshold was generated and given 3 times per session. Stimulation intensities were -10%, -5%, 0%, +5%, and +10% in relation to the positive threshold achieved. To eliminate a learning curve distortion, only observations at days 28, 84, and 154 were used for analysis. The mean phosphene threshold was defined as the average of the lowest positive threshold of the 3 stimulation sequences per visit. Ordinary least squares regression was used to evaluate the association between the change in mean daily headache rate from visit 3 to visit 7 and the change in mean transcranial magnetic stimulation threshold during the same period. RESULTS: Sixty-one subjects were enrolled. Twenty-one subjects were discontinued (because of poor study compliance or attack frequency) during the baseline phase prior to study drug initiation, and an additional subject whose data were not analyzed because of suspect quality. During the first 6 weeks on study drug (titration phase), 8 subjects dropped out (20.5%). Full analysis of the remaining 31 subjects, who reached a maintenance dose after 6 weeks on study medication, was performed. Subjects were largely white, female, and had a mean age of 41 +/- 13 years. Increasing age (beta = 1.27, P = .09), nonwhite race (beta = 6.90, P = .03), and diagnosis of tension-type headache (beta = 6.12, P = .095) were found to be associated with a higher mean transcranial magnetic stimulation threshold. Conversely, increasing body mass index was found to be associated with a lower mean transcranial magnetic stimulation threshold (beta = -1.19, P = .005). The number of migraine attacks decreased from 4.24 during the baseline interval to 2.53 during the interval preceding visit 7 (P = .001). There was a small but significant increase in transcranial magnetic stimulation threshold from visit 3 to visit 5 (P = .03) and visit 3 to visit 7 (P = .03 omnibus test). However,the difference between visit 5 and visit 7 was not statistically significant (P = .88). The mean transcranial magnetic stimulation threshold did not change from visit 5 to visit 7. CONCLUSION: Phosphene threshold increased during treatment with levetiracetam. At the 10% significance level, headache frequency and phosphene threshold were negatively correlated.

Variation in cytotoxic T-lymphocyte responses to peptides derived from tyrosinase-related protein-2.

In this study, we developed three optimized peptide ligands (OPL) that demonstrate increased affinities for HLA-A*0201 compared with wild-type tyrosinase-related protein-2 (TRP-2) peptide. The OPL contain amino acids from TRP-2((180-188)) and preferred primary and auxiliary HLA-A*0201 anchor residues. Cytotoxic T lymphocyte (CTL) lines were generated against wild-type TRP-2 peptide and OPL by multiple rounds of peptide stimulation of peripheral blood mononuclear cells from HLA-A2*0201(+) healthy individuals. CTL reactivity profiles to three different OPL were donor-dependent. Among donors, at least one OPL was particularly stimulatory and elicited high levels of CTL that cross-reacted with wild-type TRP-2 peptide. Cytotoxicity assays using CTL raised on wild-type TRP-2 peptide or OPL demonstrated lysis of HLA-A2-positive glioblastoma cells. Molecular models of TRP-2 and OPL peptides docked with HLA-A*0201 demonstrated that substitution of F for S at position 1 (P1) oriented the peptides favoring a pi-pi aromatic interaction with W 167 of HLA-A*0201. This in turn positions P5 and P8 aromatic rings to face solvent that may promote binding to the T-cell receptor, leading to a robust T-cell activation. The results of this study further substantiate the concept that rational design and testing of multiple peptides for the same T-cell epitope should elicit a broader response among different individuals than single peptide immunization. Our results may partially explain why some patients have better clinical responses to peptide-based immunotherapy, whereas others respond poorly.

Dihydroergotamine for early and late treatment of migraine with cutaneous allodynia: an open-label pilot trial.

OBJECTIVE: To explore whether dihydroergotamine (D.H.E. 45) is equally effective and safe for migraine with allodynia, when administered either early or late in an attack. BACKGROUND: Central sensitization may account for the extracranial tenderness and cutaneous allodynia that can occur with migraine. Once allodynia is established, triptans are less effective. Dihydroergotamine is often effective for patients whose refractory headaches have failed prior triptan therapy. METHODS: In this single-center, open-label pilot trial, patients with episodic migraine associated with cutaneous allodynia were treated on 2 occasions with dihydroergotamine 1.0 mg intramuscularly. One attack was treated within 2 hours (early) and a second attack at 4 hours (late) after the onset of throbbing pain. Headache pain and any associated symptoms, subjective cutaneous allodynia, and mechanical (brush) allodynia were assessed. All data were analyzed using the Fisher's exact test. RESULTS: Thirteen patients met the entry criteria; however, data from only 9 patients, those who completed treatment for 2 migraine attacks, were used to evaluate the efficacy and safety of dihydroergotamine. Whether they took dihydroergotamine early or late in the attack, most patients (>55%) had headache relief within 2 hours, and at least 44% of patients achieved headache-free status by 8 hours postdose. Subjective cutaneous allodynia started to decline after 30 minutes postdose in the early treated group and after 120 minutes postdose in the late-treated group. Brush allodynia began to decline after 15 minutes postdose in the early treated group and after 90 minutes postdose in the late-treated group. Six of 9 patients (67%) reported at least 1 adverse event. CONCLUSIONS: The results of this pilot trial provide proof of concept for the headache-relief benefit of dihydroergotamine in patients with migraine headache and allodynia. A large, placebo-controlled trial of dihydroergotamine in allodynic patients is warranted.

Patients' preference for migraine preventive therapy.

OBJECTIVE: Preventive treatment is an important part of migraine therapy. When prescribing medication, physicians should understand patients' treatment preferences and select drugs that most closely meet their patients' needs. Understanding the factors that influence patients' preference increases physicians' ability to select appropriate migraine therapy. However, unlike acute migraine treatment, patients' preferences for migraine preventive treatment have never been studied. METHODS: We enrolled 250 patients who attended the Jefferson Headache Center and Sao Paulo Headache Center and had a primary headache diagnosis. Patients' age, gender, body mass index (BMI), headache diagnosis, headache frequency, duration, and intensity, headache disability (by MIDAS), and current preventive treatments were ascertained. Patients were asked to rate 7 aspects of headache prevention (efficacy, speed of onset, out-of-pocket expenses, adverse events, formulation of therapy, type of treatment, and frequency of dosing) in order of importance (1-7). Each patient also evaluated 12 different clinical scenarios, each one containing a simulation of 2 hypothetical headache preventive treatments, wherein patients could choose Product A, Product B, or neither. Patients were informed of each product's efficacy data (50%, 75%, or 100% headache elimination), adverse event profile (weight gain, concentration difficulty, and/or fatigue), and dosing frequency (once every 3 months, once per day, or twice per day). RESULTS: Most patients were Caucasian. Mean BMI was 26.55 +/- 5.34, range (17-45). Mean history of headache was 20.93 years. Fifty patients (40%) had 45 or more headache days in the past 3 months. Mean headache intensity score (0-10 scale) was 5.7 +/- 1.8. Patients were on various preventive treatments, including beta-blockers (48 [41%]), calcium-channel blockers (19 [16%]), antidepressants (52 [44%]), antiepileptics (46 [39%]), neurotoxins (16 [14%]), vitamins/herbal therapies (28 [24%]), and nonmedicinal therapy (38 [32%]). Of the 7 aspects of migraine prevention that patients were asked to rate, 72% rated effectiveness the most important aspect. Twelve percent rated speed of onset most important, 6% rated absence of adverse events most important, 3% rated formulation of therapy most important, 3% rated out-of-pocket expenses most important, and 2% rated type of treatment (prescription/vitamin) most important. None rated frequency of dosing as the most important factor. In the area of preventive treatment scenarios, patients were more likely to choose treatments with higher efficacy rates, fewer adverse events and less frequent dosing schedule. Patients indicated that they preferred the treatment options with higher efficacy rates even if side effects were present and a more frequent dosing schedule was necessary. CONCLUSION: Patients' preference regarding migraine prevention is very important in headache management. Patients rated efficacy the most important aspect in preventive therapy and preferred treatment options with higher efficacy rates. Future studies are needed for a better understanding of patients' preference for migraine prevention.

Evaluation of an electronic diary as a diagnostic tool to study headache and premenstrual symptoms in migraineurs.

OBJECTIVE: To evaluate an electronic diary as a tool to evaluate the occurrence and relationship of headaches and premenstrual syndrome (PMS) symptoms throughout the menstrual cycle in women with migraine. BACKGROUND: Menstrually related headache and PMS significantly impact the quality of life of many women. The time relationship of these 2 menstrually related problems is not well understood and not well described. METHODS: Twenty women with migraine experiencing regular menstrual cycles were enrolled in a prospective study designed to date- and time-stamp data, both self- and computer-prompted, headache and PMS symptoms, for 3 consecutive months. A previously validated PMS score was calculated by grading 23 PMS criteria on a scale of 0 to 3 (0 = no symptoms, 3 = severe symptoms). RESULTS: The total number of data entries recorded was 2009, composed of 56 menstrual cycles in 20 migraineurs. Five hundred forty-four entries reported a current, prodromal, or previous headache. The mean daily occurrence of headache increased beginning on cycle day -5, peaked on days +1 to +5, and returned to baseline by day +7. Mean daily PMS scores ranged from 2.4 to 12. Mean daily PMS scores peaked on days -6 to +2 and returned to baseline by day +8. CONCLUSIONS: An electronic diary may have potential as a diagnostic tool in studying headaches and PMS symptoms throughout the menstrual cycle. The occurrence of headache and PMS symptoms in migraineurs follows similar time courses.

Consecutive transcranial magnetic stimulation: phosphene thresholds in migraineurs and controls.

OBJECTIVE: To characterize the temporal course of transcranial magnetic stimulation-induced phosphene thresholds in subjects with migraine and in controls. METHODS: Eleven subjects with migraine with aura, 10 subjects with migraine without aura, 9 subjects with menstrual migraine, and 15 controls (no history of migraine and without migraine during the study) were studied. Subjects were not on preventive medication. Transcranial magnetic stimulation was performed, and a phosphene threshold was measured 3 times a week over 3 weeks in a manner timed to incorporate the menstrual period in females. A headache calendar was kept during the study. RESULTS: Mean transcranial magnetic stimulation thresholds were lower for each migraine group compared with controls (P <.001) for each comparison. There was a trend for lower thresholds among subjects with migraine with aura compared with subjects with migraine without aura (P <.10), but not subjects with menstrual migraine. There was consistent lowering of thresholds from the first to the last stimulation in all migraine groups and in the controls. Maximum and minimum thresholds did not predict headache occurrence, nor did the occurrence of headache predict an ensuing maximum or minimum phosphene threshold. CONCLUSIONS: Transcranial magnetic stimulation thresholds are lower in subjects with migraine compared with controls. The reported phosphene threshold is lowered with repeated measurement. Neither high nor low phosphene thresholds predict a subsequent headache, nor do migraines predict a subsequent high or low threshold.

Her-2/ neu altered peptide ligand-induced CTL responses: implications for peptides with increased HLA affinity and T-cell-receptor interaction.

In this study, we developed two Her-2/ neu-derived E75 altered peptide ligands (APLs) that demonstrate increased affinities for the HLA-A*0201 allele compared with wild-type E75 peptide. The APLs contain amino acids from E75(369-377), an immunodominant Her-2/ neu-derived peptide, and preferred primary and auxiliary HLA-A*0201 molecule anchor residues previously identified from combinatorial peptide library screening with the recombinant molecule. CTL lines were generated against wild-type E75 peptide (KIFGSLAFL) and APLs by multiple rounds of peptide stimulation of peripheral blood mononuclear cells (PBMCs) from HLA-A2+ antigen normal individuals. CTL lines raised on wild-type E75 peptide cross-reacted with APLs and similarly, CTL lines raised on APLs cross-reacted with wild-type E75 peptide, as measured by IFN-gamma ELISpot and target cell lysis assays. One of five individuals demonstrated specificity for APL 2 (FLFGSLAFL), whereas APL 5 (FLFESLAFL)-specific responses were observed from all five individuals tested. Molecular models of the E75, APL 2, and APL 5/HLA-A2 complexes indicated that the substitution of glycine with glutamic acid at position four of APL 5 resulted in the presentation of a large, negatively charged side chain that interacts with the outer edge of the HLA-A2 antigen alpha helix and is freely available to interact with cognate T-cell receptors. The results of this study further substantiate the concept that rational design of T-cell epitopes may lead to stronger peptide immunogens than natural, wild-type peptides.