Modulation of catecholamine metabolism in synaptosomes by a neuroregulatory factor from mammalian brain

Incubation of synaptosomes from rat brain with bovine brain extract caused inhibition of oxidative deamination of dopamine, decreased formation of 3,4-dihydroxyphenylacetic acid, increased formation of norepinephrine and its N-methyl derivatives and increased release of catecholamines. Omission of Ca2+ from the extrasynaptosomal medium completely blocked the brain extract induced release of [3H]catecholamines and decreased, by about 80%, the effect on changes in catecholamine metabolism. Chromatography of the brain extract on Sephadex-G25 columns resulted in an active compound eluting at the position expected for compounds with molecular weights in the region of 1500 to 2500 Da.     

Selective parathyroid auto‐transplantation during thyroidectomy

Aim: Permanent hypoparathyroidism is a debilitating morbidity following thyroidectomy and parathyroid auto‐transplantation has been shown to be effective in preventing permanent hypoparathyroidism. Controversy exists regarding the benefit of routine versus selective auto‐transplantation. We evaluate the outcome of selective parathyroid auto‐transplantation in our hospital. Methods: A retrospective study was conducted to assess the incidence of postoperative hypocalcaemia. Indication for parathyroid auto‐transplant was doubtful viability of parathyroid gland during thyroidectomy. From 1 July 2000 to 30 June 2005, all patients who underwent total, subtotal and completion thyroidectomy were included. Other outcome measures including recurrent laryngeal nerve injury and operative time were also analyzed. Results: A total of 170 bilateral or completion thyroidectomies were performed within this period. Total, subtotal, and completion total thyroidectomies were performed in 103 (60.6%), 62 (36.5%), and five (2.9%) patients, respectively. Median age was 45 years (range 19–82). One hundred and twenty‐four patients (73%) had benign thyroid disease, and 46 patients (27%) had thyroid carcinoma. Parathyroid auto‐transplant was performed in 35 patients (20.6%). Mean operation time was 204 min (range 95–510 min). There was no difference in the operation time between the patients with parathyroid auto‐transplant and those without auto‐transplant (217 vs 200 min, P = 0.229). Transient hypocalcaemia occurred in 31 patients (18.2%) whereas two patients had permanent hypocalcaemia (1.2%). Permanent recurrent laryngeal nerve injury occurred in one patient (0.6%). Conclusions: The adoption of selective parathyroid auto‐transplant during thyroidectomy achieves an extremely low incidence of permanent hypoparathyroidism without excessive transient hypoparathyroidism.     

Dosage forms of cyclosporine.

Cyclosporine can be given intravenously or orally, as a solution or soft gelatin capsule. Because of the drug's poor water solubility, ethoxylated plant fats and ethanol are added to the commercially available dosage forms.     

Age-dependent cyclosporine: pharmacokinetics in marrow transplant recipients

We evaluated the effect of age on cyclosporine pharmacokinetics in 69 nonobese patients aged 10 months to 56 years (median 22 years) undergoing allogeneic bone marrow transplantation for treatment of aplastic anemia or hematologic malignancy. Cyclosporine pharmacokinetics were studied during the first 2 posttransplant weeks after an intravenous dose of 2.6 to 3.5 mg/kg. Serum cyclosporine concentrations were measured by HPLC. Cyclosporine concentration-time data were fitted to a two-compartment model with a nonlinear regression program. There was a significant inverse linear correlation between age and both total systemic clearance (CL) (r = 0.42; P less than 0.001) and volume of distribution at steady-state (Vss) (r = 0.33; P less than 0.01). Mean (+/- SE) cyclosporine CL was 82 +/- 21, 45 +/- 5, 38 +/- 9, 44 +/- 8, and 20 +/- 3 ml/min/kg and mean cyclosporine Vss was 34 +/- 11, 28 +/- 10, 15 +/- 4, 14 +/- 5, and 4.7 +/- 0.7 L/kg in patients 0 to 10 (n = 12), 11 to 20 (n = 19), 21 to 30 (n = 12), 31 to 40 (n = 17), and greater than 40 (n = 9) years old, respectively. Patients 0 to 10 years old had a significantly higher cyclosporine CL than those 11 to 40 or greater than 40 years old and also had a significantly larger Vss than those greater than 40 yrs old (P less than 0.05). Age-related differences in CL or Vss were also observed when these parameters were normalized by body surface area.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vertical saccades in superior oblique palsy

Vertical saccadic velocities in 10 patients who had unilateral superior oblique muscle palsy and 14 normal subjects were measured with the magnetic scleral search coil. The authors sought to determine whether downward saccades in patients who had superior oblique palsy are slow. Peak velocities of 10 degrees and 20 degrees saccades performed in the superior and inferior fields of the orbit, and 10 degrees, 20 degrees, and 30 degrees saccades performed across the center of the orbit were recorded with the eye in center gaze, 30 degrees of adduction, and 30 degrees of abduction. Paired t-tests did not show statistically significant differences between upward and downward saccades in patients with superior oblique palsy; no effects of orbital field or position of horizontal gaze were found (P greater than 0.01). Comparison of similar saccades between normal subjects and patients with superior oblique palsy by two-sample t-tests did not show significant differences between the two groups (P greater than 0.01).     

Specific T Helper Cell Requirement for Optimal Induction of Cytotoxic T Lymphocytes against Major Histocompatibility Complex Class II Negative Tumors

This study shows that induction of tumor-specific CD4⁺ T cells by vaccination with a specific viral T helper epitope, contained within a synthetic peptide, results in protective immunity against major histocompatibility complex (MHC) class II negative, virus-induced tumor cells. Protection was also induced against sarcoma induction by acutely transforming retrovirus. In contrast, no protective immunity was induced by vaccination with an unrelated T helper epitope. By cytokine pattern analysis, the induced CD4⁺ T cells were of the T helper cell 1 type. The peptide-specific CD4⁺ T cells did not directly recognize the tumor cells, indicating involvement of cross-priming by tumor-associated antigen-presenting cells. The main effector cells responsible for tumor eradication were identified as CD8⁺ cytotoxic T cells that were found to recognize a recently described immunodominant viral gag-encoded cytotoxic T lymphocyte (CTL) epitope, which is unrelated to the viral env-encoded T helper peptide sequence. Simultaneous vaccination with the tumor-specific T helper and CTL epitopes resulted in strong synergistic protection. These results indicate the crucial role of T helper cells for optimal induction of protective immunity against MHC class II negative tumor cells. Protection is dependent on tumor-specific CTLs in this model system and requires cross-priming of tumor antigens by specialized antigen-presenting cells. Thus, tumor-specific T helper epitopes have to be included in the design of epitope-based vaccines.Adequate T helper cell activation is essential in the initiation of an immune reaction. The inability to control tumor outgrowth can be due to inadequate T helper responses underlying poor tumor-specific immunity. In the cellular immune response, specialized APCs process protein and present antigenic peptide fragments in MHC class II molecules to CD4⁺ T helper lymphocytes. These provide “help” to effector cells via the production of cytokines. Although tumor cells can directly present endogenously processed antigenic peptide in surface MHC class I molecules to CD8⁺ CTL precursors, initiation of tumor-specific CTL responses is likely to involve indirect presentation of tumor antigens by specialized APCs.Evidence for a role of T helper cell–mediated immunity comes from studies with genetically modified tumor cells. CD4⁺ cells can be directly activated by transfection of MHC class II α and β chain genes in mouse tumor cells (1–4). These cells become immunogenic, lose their tumorigenicity, and even induce protection against wild-type MHC class II negative tumors, indicating that direct MHC class II presentation of tumor expressed antigens can induce efficient anti–tumor responses.A central role of CD4⁺ T cells emerged from studies of immunity against FMR (Friend, Moloney, Rauscher)¹ murine leukemia virus (MuLV) type tumors by Greenberg (5). Transfer of purified polyclonal T cells from FBL (Friend MuLV-induced erythroleukemia cell line) vaccinated mice in naive animals can protect these mice against subsequent tumor challenge. Both purified CD4⁺ and CD8⁺ T cells transfer protection to FBL tumors (6). FBL cells do not express MHC class II molecules, but CD4⁺ T cells can protect mice even in the absence of CD8⁺ T cells. In this case, macrophages seem to play an important effector role. CD8⁺ T cells can only be effective if CD4⁺ T cells are present or if exogenous IL-2 is administered. Neither B cells nor NK cells seem to exert a significant role in the FBL sytem. These data suggest involvement of APCs, presenting tumor antigens, and a crucial regulatory role of Ths, which was strongly supported by experiments performed in Friend MuLV env-transgenic mice (7). These mice were rendered tolerant for env-specific Th responses and it was not possible to protect these mice against FBL tumors by vaccination.Immune responsiveness to MuLV is classically regulated by the genes of the H-2 (MHC) complex (8). In particular, the H-2^b haplotype confers resistance, and studies using H-2 recombinant and H-2 mutant mouse strains have mapped the protective effects to the class II I-A^b locus (9, 10). This MHC class II association indicates an important role of T helper cells influencing both CTL activity as well as class switching of antiviral antibodies from IgM to IgG. The H-2 I-A^b phenotype protects against early lymphomagenesis. The identification of two Friend MuLV env-derived epitopes, presented by MHC class II, I-A^b and I-E^b/d, respectively, indicated that tumor-directed T helper immunity is virus specific (11). The few lymphomas that arise in H-2^b mice have abrogated viral antigen or (more rarely) MHC class I expression (12), indicating that CTLs also play a crucial role. CTLs have been proven to recognize viral antigens, both gag and env proteins encode CTL epitopes (13). We have identified a K^b-presented, env-derived Moloney and Rauscher CTL epitope that is subdominant in C57BL/6 mice making use of the D^b mutant BM13 mouse strain (14). The D^b-presented gag-leader (gag-L) derived immunodominant CTL epitope for the FMR type of MuLV has been identified only recently (15).Vaccination with a synthetic peptide comprising a relevant T cell epitope is a powerful method to induce highly specific T cells. Protective vaccination using CTL peptide epitopes has been achieved in pathogenic viral models (16, 17) and tumor models (18–20). Peptide vaccination in IFA led to measurable specific CTL induction and protective immunity against virulent virus or tumor cells. Importantly, peptide vaccination can also be applied succesfully for therapy of established tumors by presenting the peptide in IFA, on RMA-S cells, or on activated dendritic cells (21, 22).We now report the induction of tumor-protective immunity by a single vaccination with a tumor-specific MuLV env-encoded T helper peptide. Strong protection can be achieved against highly aggressive tumor cells that lack MHC class II expression. This indicates the requirement of cross-priming of tumor antigens by local APCs. We show that CD8⁺ T cells, recognizing the gag-L–encoded CTL epitope, are crucial effector cells that are efficiently activated with help from peptide-primed tumor-specific CD4⁺ T cells. Vaccination with a mixture of the T helper peptide and the immunodominant CTL epitope resulted in synergistic, long-term tumor protection.     

Effects of amiloride on pH regulation in canine cardiac Purkinje fibers

Myocardial cells utilize membrane transport systems for proton extrusion as well as internal buffers to preserve pH homeostasis. Our laboratory had shown previously that amiloride (0.01-1.0 mM) causes a time- and dose-dependent increase in action potential duration, early after depolarizations and enhanced automaticity. Ion-selective microelectrode technique was used to evaluate whether the observed electrophysiologic effects of amiloride are linked to inhibition of Na/H exchange and subsequent inability of the myocardial cell to maintain steady-state intracellular pH (pHi), either under normal physiological conditions or in the presence of an imposed acid load. We analyzed different components of intracellular pH transients that occur in response to NH4Cl exposure and washout, which allowed us to quantitatively describe the effects of Na/H exchange inhibition in a multicellular preparation. Amiloride (0.01-1.0 mM) did not change the steady-state pHi, but did cause a dose-dependent increase in both the time for the pHi to reach a minimum value (time-to-peak) during washout of NH4Cl as well as in the absolute minimum value of pHi (peak acid). The effects of amiloride on pHi transients are rapidly reversible and antagonized by physiologic values of extracellular sodium activity. We conclude that Na/H exchange inhibition by amiloride does not cause intracellular acidosis under normal physiologic conditions, despite the dramatic changes in action potential characteristics. However, amiloride affected the time-to-peak and the peak acid value of the pHi transient during NH4Cl washout at concentrations that had no discernible effect on the overall time course of pHi recovery.     

Use of spiral computed tomography contrast angiography and ultrasonography to exclude the diagnosis of pulmonary embolism in the emergency department

Spiral computed tomography (CT) contrast angiography is a promising imaging modality for the diagnosis of pulmonary embolism but the negative predictive value of this test remains controversial. We performed a multi-center prospective cohort study to determine the safety of relying on a negative spiral CT contrast angiography scan to exclude pulmonary embolism. Patients presenting to the Emergency Departments of three tertiary care institutions with clinically suspected pulmonary embolism were potentially eligible for the study. Patients underwent a clinical evaluation to categorize pretest probability into low, moderate, and high categories, and had D-dimer testing performed. Patients at low pretest probability with normal D-dimer were considered to have pulmonary embolism excluded. The remaining patients underwent spiral CT contrast angiography scan of the pulmonary arterial circulation and bilateral venous ultrasound of the proximal leg veins. Patients who were confirmed to have pulmonary embolism or deep vein thrombosis were treated with anticoagulant therapy. Patients in whom the diagnosis of pulmonary embolism was excluded did not receive anticoagulant therapy and were followed for a 3-month period for the development of venous thromboembolic complications. Eight hundred fifty-eight (858) patients were enrolled in this study. Three-hundred sixty-nine (369) patients had low pretest probability and negative D-dimer results and no further diagnostic tests were performed. None of these patients subsequently developed venous thromboembolic complications (0%, 95% confidence interval [CI] 0% to 1.0%). The remaining 489 were referred for spiral CT contrast angiography scan and ultrasound. Sixty-seven patients were confirmed to have pulmonary embolism and an additional 15 patients with negative CT scans had proximal deep vein thrombosis (DVT) on ultrasound for a total prevalence of venous thromboembolism of 82/489 (16.8%). Two of 409 patients who had pulmonary embolism excluded in the initial evaluation phase developed proximal venous thromboembolism (0.5%; 95% CI 0% to 1.8%) in the 3-month follow-up period. These findings suggest that the combination of a negative spiral CT contrast angiography scan and normal venous ultrasound imaging safely excludes the diagnosis of pulmonary embolism in the Emergency Department setting.