Reduced Chance of Hearing Loss Associated with Therapeutic Drug Monitoring of Aminoglycosides in the Treatment of Multidrug-Resistant Tuberculosis.

Hearing loss and nephrotoxicity are associated with prolonged treatment duration and higher dosage of amikacin and kanamycin. In our tuberculosis center, we used therapeutic drug monitoring (TDM) targeting preset pharmacokinetic/pharmacodynamic (PK/PD) surrogate endpoints in an attempt to maintain efficacy while preventing (oto)toxicity. To evaluate this strategy, we retrospectively evaluated medical charts of tuberculosis (TB) patients treated with amikacin or kanamycin in the period from 2000 to 2012. Patients with culture-confirmed multiresistant or extensively drug-resistant tuberculosis (MDR/XDR-TB) receiving amikacin or kanamycin as part of their TB treatment for at least 3 days were eligible for inclusion in this retrospective study. Clinical data, including maximum concentration (Cmax), Cmin, and audiometry data, were extracted from the patients' medical charts. A total of 80 patients met the inclusion criteria. The mean weighted Cmax/MIC ratios obtained from 57 patients were 31.2 for amikacin and 12.3 for kanamycin. The extent of hearing loss was limited and correlated with the cumulative drug dose per kg of body weight during daily administration. At follow-up, 35 (67.3%) of all patients had successful outcome; there were no relapses. At a median dose of 6.5 mg/kg, a correlation was found between the dose per kg of body weight during daily dosing and the extent of hearing loss in dB at 8,000 Hz. These findings suggest that the efficacy at this lower dosage is maintained with limited toxicity. A randomized controlled trial should provide final proof of the safety and efficacy of TDM-guided use of aminoglycosides in MDR-TB treatment.

T helper cell recognition of muscle acetylcholine receptor in myasthenia gravis. Epitopes on the gamma and delta subunits.

We tested the response of CD4+ cells and/or total lymphocytes from the blood of 22 myasthenic patients and 10 healthy controls to overlapping synthetic peptides, 20 residues long, to screen the sequence of the gamma and delta subunits of human muscle acetylcholine receptor (AChR). The gamma subunit is part of the AChR expressed in embryonic muscle and is substituted in the AChRs of most adult muscles by an epsilon subunit. The delta subunit is present in both embryonic and adult AChRs. Adult extrinsic ocular muscles, which are preferentially and sometimes uniquely affected by myasthenic symptoms, and thymus, which has a still obscure but important role in the pathogenesis of myasthenia gravis, express the embryonic gamma subunit. Anti-AChR CD4+ responses were more easily detected after CD8+ depletion. All responders recognized epitopes on both the gamma and delta subunits and had severe symptoms. In four patients the CD4+ cell response was tested twice, when the symptoms were severe and during a period of remission. Consistently, the response was only detectable, or larger, when the patients were severely affected.

Epitopes on the beta subunit of human muscle acetylcholine receptor recognized by CD4+ cells of myasthenia gravis patients and healthy subjects.

We investigated the sequence regions of the human muscle acetylcholine receptor (AChR) beta subunit forming epitopes recognized by T helper cells in myasthenia gravis (MG), using overlapping synthetic peptides, 20 residues long, which screened the sequence of the AChR beta subunit. Since CD4+ lymphocytes from MG patients' blood did not respond to the peptides, we attempted propagation of beta subunit-specific T lines from six MG patients and seven healthy controls by cycles of stimulation of blood lymphocytes with the pooled peptides corresponding to the beta subunit sequence. CD4+ T lines were obtained from four patients and three controls. They secreted IL-2, not IL-4, suggesting that they comprised T helper type 1 cells. The T lines from MG patients could be propagated for several months. Three lines were tested with purified bovine muscle AChR and cross-reacted well with this antigen. All T lines were tested with the individual synthetic peptides present in the pool corresponding to the beta subunit sequence. Several beta subunit peptide sequences were recognized. Each line had an individual pattern of peptides recognition, but three sequence regions (peptides beta 181-200, beta 271-290, and the overlapping peptides beta 316-335 and beta 331-350) were recognized by most MG lines. The beta subunit-specific T lines from controls could be propagated for < 5 wk. Each line recognized several peptides, which frequently included the immunodominant regions listed above.

Myasthenia gravis. CD4+ T epitopes on the embryonic gamma subunit of human muscle acetylcholine receptor.

In myasthenia gravis (MG) an autoimmune response against muscle acetylcholine receptor (AChR) occurs. Embryonic muscle AChR contains a gamma subunit, substituted in adult muscle by a homologous epsilon subunit. Antibodies and CD4+ cells specific for embryonic AChR have been demonstrated in MG patients. We identified sequence segments of the human gamma subunit forming epitopes recognized by four embryonic AChR-specific CD4+ T cell lines, propagated from MG patients' blood by stimulation with synthetic peptides corresponding to the human gamma subunit sequence. Each line had an individual epitope repertoire, but two 20-residue sequence regions were recognized by three lines of different HLA haplotype. Most T epitope sequences were highly diverged between the gamma and the other AChR subunits, confirming the specificity of the T cells for embryonic AChR. These T cells may have been sensitized against AChR expressed by a tissue other than innervated skeletal muscle, possibly the thymus, which expresses an embryonic muscle AChR-like protein, containing a gamma subunit. Several sequence segments forming T epitopes are similar to regions of microbial and/or mammalian proteins unrelated to the AChR. These findings are consistent with the possibility that T cell cross-reactivity between unrelated proteins ("molecular mimicry"), proposed as a cause of autoimmune responses, is not a rare event.

Venous thrombotic events in patients admitted to a tuberculosis centre.

BACKGROUND: Considering the relationship between inflammation and thrombosis, patients with tuberculosis (TB) patients might be at high risk of venous thrombosis. AIM: To evaluate the risk of venous thromboembolism in patients admitted to the Beatrixoord Tuberculosis Centre (BTBC), a tertiary centre for TB. We specifically explored which cofactors elevate the risk of venous thrombosis (VTE), and whether the timing of venous thrombotic events would justify extended primary prophylaxis. DESIGN: retrospective cohort study. METHODS: We performed a retrospective chart review of all patients with TB discharged from BTBC between 2000 and 2010. We excluded patients who were already on therapeutic anticoagulation before their TB episode, below the age of 18 years and patients in which TB diagnosis was withdrawn. For evaluating the timing of venous thrombosis, we calculated the time between commencement of anti TB therapy and the VTE. RESULTS: Of 750 included in the final analysis, 18 (2.4%) suffered a venous thrombotic event. 3 of these events were not related to classic risk factors or hospitalization. Most (13/18) VTE's occurred in the time window of two weeks before starting TB medication.In the multivariate analysis, only Human Immunodeficiency Virus (HIV) infection was strongly associated with risk of VTE (adjusted Odds ratio 8.2 (95% confidence interval: 2.9-22.7)). CONCLUSIONS: This high risk in HIV co-infected TB patients suggests that standard thrombo-prophylaxis should be routinely considered in this group. However, our findings might not be generalizable due to referral bias. Further prospective studies in unselected HIV co-infected TB patients are needed to corroborate our findings.

Intravenous immunoglobulin for the treatment of acquired myasthenia gravis.

Acquired myasthenia gravis (MG) is an autoimmune disorder characterized by exertional fatigue and weakness that is made worse with activity and improved with rest, only to recur with the resumption of activity. The pathology results from an antibody-mediated attack to several different epitopes of the acetylcholine receptor (AChR) complex. The consensus of an expert panel is that intravenous immunoglobulin (IVIg) is effective in reversing myasthenic weakness. Although the mechanism of action is not known, it is likely that there is a downregulation of antibody production. IVIg appears to have a role as an acute treatment intervention in rapidly progressive weakness or as a chronic maintenance therapy when all other treatment modalities have failed. Its response is similar to but slower than the response of plasma exchange (PE), but it offers advantages when therapeutic apheresis is not available or when vascular access is problematic.

Passive transfer of human myasthenia gravis to rats: 1. Electrophysiology of the developing neuromuscular block.

Adult female Lewis rats were rendered immunologically tolerant to human gamma globulin, and were given a single intravenous injection of human myasthenic or normal control serum containing 7.5 to 12 mg of immunoglobulin G. The mean amplitude of miniature endplate potentials (MEPPs) in the forelimb flexor digitorum longus muscles from treated animals did not differ from control values during the first 24 hours after serum injection. Subsequently, MEPP amplitude was reduced in muscles from animals that had received myasthenic serum; maximum reduction was reached by 6 days after transfer. Mean MEPP amplitude at maximum reduction was 30 to 40% below the amplitude of controls and returned to control values 14 weeks after transfer. Similar reductions in endplate potential amplitudes were found in immunologically tolerant animals receiving myasthenic serum. No significant reduction of MEPP amplitude was seen in recipients that were not immunologically tolerant or that had received cobra venom factor to reduce complement activity. The delayed development of reduced MEPP amplitude indicated that the defect of neuromuscular transmission produced by myasthenic serum was not due entirely to a simple curare-like block of the acetylcholine receptor site by an IgG antibody.

Single-fiber electromyography in myasthenia gravis.

One-hundred-sixty single-fiber EMG studies of the extensor digitorum communis muscle were performed on 127 patients with myasthenia gravis; 131 demonstrated defective neuromuscular transmission. Jitter determinations in the biceps, deltoid or frontalis muscles increased the diagnostic yield significantly. Evoked-potential EMG studies were abnormal in less than 50 percent of patients in whom they were performed. The most sensitive criterion of abnormality was the percentage of fibers with increased jitter; the sensitivity of the test was enhanced, however, if the mean jitter of the tested muscle was also used as a criterion of abnormality. Since increased jitter may also be seen in primary muscle and nerve disease, these disorders must be excluded by other means before diagnosing myasthenia gravis on the basis of the single-fiber studies.

T cells in myasthenia gravis specific for embryonic acetylcholine receptor.

In myasthenia gravis (MG) there is an autoimmune response against muscle acetylcholine receptor (AChR). Embryonic and adult muscles express different AChRs; embryonic AChR contains a gamma subunit, instead of the homologous epsilon subunit that contributes to form adult AChR. We report propagation from the blood of MG patients of T helper (TH) cell lines specific for human embryonic AChR, by cycles of stimulation with a pool of synthetic peptides corresponding to the complete sequence of the gamma subunit (gamma pool). The TH lines strongly recognized AChR from embryonic mammalian muscle, and reacted less or not at all with adult muscle AChR. The existence of TH cells specific for embryonic AChR strongly suggests that the primary anti-AChR sensitization in MG occurs in a tissue other than the innervated skeletal muscle. This may be within the thymus, which expresses an AChR similar or identical to embryonic muscle AChR.

Race, sex, and puberty influence onset, severity, and outcome in juvenile myasthenia gravis.

We assessed the influence of race, sex, and puberty upon clinical features and outcome in 115 patients with autoimmune juvenile myasthenia gravis (JMG). These demographic variables influenced not only disease incidence but also disease severity, response to therapy, and outcome, despite comparable therapeutic strategies. Among white patients, those with prepubertal onset had low incidence and equal sex ratio; the incidence in females increased during and after puberty; males had lesser disease severity than females during and after puberty (p < 0.05); spontaneous remissions were most frequent (44%, p = 0.001) and persistence of active JMG for more than 10 years was least frequent (p = 0.05) in patients with prepubertal onset; remissions were more frequent after early than late thymectomy (p = 0.03); and final disease severity was less after early than late thymectomy. Black patients had similar incidence, disease severity, and sex ratio (F:M = 2:1) with pre-, peri-, or postpubertal disease onset; infrequent spontaneous or treatment-induced remissions; and the same final disease severity after early or late thymectomy. These observations imply that race and sex hormones modify the clinical features and outcome of JMG; spontaneous remissions are common in white patients with prepubertal disease onset; early thymectomy may be more beneficial than late thymectomy in white patients; and the role of thymectomy in the youngest patients is uncertain. We suggest that demographic factors should be considered when evaluating past and future therapeutic strategies for JMG.

CD4+ T-epitope repertoire on the human acetylcholine receptor alpha subunit in severe myasthenia gravis: a study with synthetic peptides.

The alpha subunit of the nicotinic acetylcholine receptor (AChR) seems crucial in the pathogenesis of the autoimmune paralysis myasthenia gravis (MG) because it contains both the epitopes that dominate the antibody response against the AChR and those recognized by CD4+ AChR-specific T helper (Th) cells. To define the repertoire of anti-AChR Th cells, we investigated the response of unselected blood CD4+ cells or total lymphocytes, or both, from 22 MG patients to 20-residue overlapping synthetic peptides, screening the complete sequence of human-muscle AChR alpha subunit. Several epitopes were identified. Only the most severely affected patients recognized alpha subunit epitopes, and they were mainly young women. Detection of in vitro AChR-specific CD4+ response was facilitated by removal of the CD8+ cells because in two patients a clear response to several alpha subunit peptide sequences could be detected when CD(8+)-depleted cells were used, while their total peripheral blood mononuclear cell population did not respond to any alpha subunit peptide. Although each patient had a unique pattern of peptide recognition, four immunodominant regions recognized by long-term AChR-specific CD4+ T-cell lines, or flanking peptide sequences, were recognized most frequently (residues 48-67, 101-137, 293-337, and 308-437).

Spinal cord infarction: etiology and outcome.

We reviewed 44 cases of ischemia and infarction of the spinal cord at two university hospitals. Three patients experienced transient ischemic attacks. Etiologies of completed strokes were diverse and included rupture and surgical repair of aortic aneurysms, aortic dissection, aortic rupture and thrombosis, global ischemia, anterior spinal artery embolism, repair and thrombosis of spinal arteriovenous malformations, hematomyelia, epidural hematoma, cervical osteophytosis, celiac plexus block, systemic lupus erythematosus, coagulopathy, and decompression sickness. Motor function improved in 12 patients, was substantial in only one, and occurred largely within the first 2 to 4 weeks. Favorable ambulatory outcome correlated with improving neurologic examinations and relatively preserved strength in hip abductors and knee extensors. More extensive deficits without initial improvement portended a more severe prognosis. Autonomic dysfunction, pain, paresthesia, and depression were common and impeded recovery in some patients. The mean level of deficit was at T-8 and in cases of global ischemia was at T-9, which leads us to dispute the classical view of a midthoracic watershed zone of ischemic vulnerability near T-4.

Myasthenia in SCID mice grafted with myasthenic patient lymphocytes: role of CD4+ and CD8+ cells.

OBJECTIVES: Acetylcholine receptor (AChR)-specific CD4+ cells are present in MG patients, and synthesis of the high-affinity immunoglobulin G (IgG) autoantibodies (autoAb) against the muscle AChR that causes MG symptoms requires intervention of CD4+ cells. The role of CD4+ cells in MG pathogenesis has been postulated but never proven. MG patients do not have anti-AChR cytotoxic phenomena, and it has been assumed that CD8+ cells do not have a pathogenic role in MG. However, CD8+ cells may facilitate rodent experimental MG, raising the possibility that CD8+ cells might be necessary also in MG. In this study we examined whether CD4+ and CD8+ cells play a role in the pathogenesis of MG and whether CD4+ cells specific for AChR epitope sequences recognized by most MG patients ("universal" epitopes) drive the synthesis of pathogenic antibodies. METHODS: First we characterized a chimeric human-mouse model of MG in severe combined immunodeficiency (SCID) mice engrafted with blood lymphocytes (BL) from MG patients. We used that model to determine whether CD4+ and CD8+ cells are necessary for transfer of MG symptoms. We engrafted SCID mice intraperitoneum with BL from 19 MG patients and 5 healthy controls. We engrafted some mice with either BL, BL depleted in CD4+ or CD8+ cells from the same patient, or CD4+ depleted BL reconstituted with CD4+ T cells from the same patient, specific for "universal" AChR epitopes or for two unrelated antigens, tetanus and diphtheria toxoids. We tested the mice for myasthenic symptoms for 7 to 18 weeks. RESULTS: Mice transplanted with BL, or CD8+ depleted BL, or CD4+-depleted BL reconstituted with anti-AChR CD4+ cells from MG patients frequently developed myasthenic weakness. The mice had human anti-AChR Ab in the serum and bound to muscle AChR. Mice transplanted with BL from controls, or CD4+-depleted BL from MG patients, or CD4+-depleted BL from an MG patient reconstituted with CD4+ cells specific for tetanus or diphtheria toxoids did not develop myasthenic weakness or anti-AChR Ab. CONCLUSIONS: CD4+ cells are necessary for MG pathogenesis; CD8+ cells may not be. CD4+ cells specific for "universal" AChR epitopes help the synthesis of pathogenic Ab.

CD4+ T cell repertoire on the epsilon subunit of muscle acetylcholine receptor in myasthenia gravis.

We have identified sequence regions of the human muscle acetylcholine receptor epsilon subunit recognized by CD4+ T cells from myasthenia gravis patients. We tested the proliferative response in vitro of blood CD4+ cells from 18 myasthenic patients and 5 controls, to individual overlapping synthetic peptides spanning the epsilon subunit sequence. All patients recognized a complex epitope repertoire. The peptides recognized by the CD4+ cells included sequence regions of the epsilon subunit that were diverged as compared to the homologous sequences of the other receptor subunits. Recognition of epitopes formed by sequence regions unique to the epsilon subunit suggests a direct role of this subunit in sensitizing the CD4+ cells. Several epsilon subunit peptides were recognized by many patients. Thus the epsilon subunit, like other acetylcholine receptor subunits, forms 'universal' CD4+ epitopes. The healthy subjects recognized some epsilon subunit peptides sporadically and at a low level.

T cell recognition of muscle acetylcholine receptor in ocular myasthenia gravis.

We examined the proliferative response of blood CD4(+) cells to muscle acetylcholine receptor (AChR) subunits and the epitope repertoire of the epsilon and gamma subunits, in ocular myasthenia gravis (oMG) patients and healthy subjects. oMG patients seldom recognized all subunits. The frequency and intensity of recognition was the same for all subunits, irrespective of the disease duration. The responses in oMG were lower than in generalized myasthenia gravis. Healthy subjects had frequent, low responses to one or more subunits. oMG patients recognized several epitopes on the gamma and epsilon subunits, that partially overlapped those recognized in gMG. The subunits and epitopes recognized by individual oMG patients changed over time. Thus, oMG patients have minimal and unstable sensitization of anti-AChR CD4(+) cells, in agreement with their low and inconsistent synthesis of anti-AChR antibody.

Intercostal muscle biopsy studies in myasthenia gravis: clinical correlations and the direct effects of drugs and myasthenic serum.

We have found a wide range of mean MEPP amplitude in intercostal muscle biopsies from 43 patients with MG, including several values in the normal range. There was no correlation between MEPP amplitude and the severity of clinical disease as assessed by manual muscle testing or by single-fiber EMG measurements of jitter in arm muscles. Through most of these patients were in a state of clinical remission or marked improvement after treatment with prednisone, we could not attribute the difference between our results and those of others to this factor alone. The application of morphine, meperidine and aminoglycoside antibiotics to intercostal muscle in vitro confirms effects previously demonstrated in rat muscle: (1) At equal therapeutic concentrations, meperidine has greater neuromuscular blocking effects than does morphine, but neither has significant effects at concentrations achieved in the serum clinically. (2) Tobramycin, netilmicin and neomycin have varying severity and sites of action, but their effects are the same in human myasthenic muscle as in normal rat muscle. Bath application of serum from myasthenic patients produces an acute, reversible worsening of neuromuscular blockade in myasthenic muscle. Electrophysiologic measurements in intercostal biopsies from patients with MG can provide information about the basic abnormality of neuromuscular transmission in this disease and can confirm the relevance of studies made in animal muscle.

T-helper epitopes on human nicotinic acetylcholine receptor in myasthenia gravis.

The synthesis of AChR antibodies requires intervention of AChR-specific Th cells. Because of the paucity of anti-AChR Th cells in the blood of myasthenia gravis (MG) patients, direct studies of these autoimmune cells in the blood are seldom possible. Propagation in vitro of anti-AChR T cells from MG patients by cycles of stimulation with AChR antigens selectively enriches and expands the autoimmune T-cell clones, allowing investigation of their function and epitope specificity. Torpedo electroplax AChR was initially used for propagation of anti-AChR T-cell lines. Those studies demonstrated the feasibility of in vitro propagation of AChR-specific T cells. These are bona fide CD4+ Th cells, which stimulate production in vitro of anti-AChR antibodies by B cells of myasthenic patients and recognize equally well denatured and native AChR, suggesting the usefulness of synthetic human AChR sequences as antigens for propagation of the autoimmune Th cells. We used pools of overlapping synthetic peptides, corresponding to the complete sequences of the human AChR alpha-, beta-, gamma-, and delta-subunits, to propagate AChR-specific Th cells from the blood of MG patients. The AChR sequence regions forming epitopes recognized by the autoimmune T cells were determined by challenging the lines with individual synthetic peptides, 20 residues long, screening the AChR subunit sequences. Although each line had an individual pattern of epitope recognition--as expected from their different HLA-DR haplotype--some peptides were recognized by most of all the CD4+ T-cell lines, irrespective of their DR haplotype. The existence of immunodominant regions of the AChR sequence was verified by investigating the response of unselected CD4+ cells from the blood of a relatively large number of MG patients to the individual peptides screening the human alpha-, gamma-, and delta-subunit sequences. Those studies confirmed that each patient has an individual pattern of peptide recognition. The studies also identified a large number of T epitopes of the human AChR and verified the existence of sequence regions immunodominant for T-helper sensitization, because a limited number of sequence regions, including all those immunodominant for the T-helper lines, were recognized by most patients. Anti-AChR CD4+ T lines could be propagated from some healthy controls only for a brief period of time. They recognized AChR sequences poorly, suggesting a low affinity of their T-cell receptors for the corresponding AChR epitopes.(ABSTRACT TRUNCATED AT 400 WORDS)

One hundred consecutive thymectomies for myasthenia gravis.

BACKGROUND: Between June 1997 and November 1993, 100 consecutive thymectomies for myasthenia gravis were performed at University of North Carolina Hospitals in Chapel Hill. METHODS: A consistent, planned protocol involving preoperative, intraoperative, and postoperative care was followed. All thymectomies were performed through a median sternotomy with removal of all visible thymus and perithymic fat in the anterior mediastinum. RESULTS: There was no perioperative mortality or longterm morbidity. Mean postoperative hospital stay was 6.3 days (range, 3 to 18 days). Ninety-six percent of the patients were extubated the day of the operation, and all patients were extubated within 24 hours. Mean postoperative intensive care unit stay was 1.2 days (range, 1 to 4 days). After a mean follow-up of 65 months (range, 1 to 199 months), 78% of all patients are improved by at least one modified Osserman classification when their current status is compared with their worst preoperative disease severity. In fact, 69% of patients with mild disease preoperatively (class I, II, or III maximal severity) are in pharmacologic remission (asymptomatic without regular medication), whereas 29% of patients with severe disease (class IV or V) are in remission (p = 0.0001). CONCLUSIONS: Our programmatic approach to thymectomy through a sternotomy has shown minimal morbidity and mortality. It is beneficial to myasthenics at both ends of the age and severity spectrum.

Measurement of laryngeal resistance in the evaluation of botulinum toxin injection for treatment of focal laryngeal dystonia.

In the past, there has been no consistent, objective method of following patients undergoing botulinum toxin injections for treatment of laryngeal dystonia. Herein, the application of translaryngeal resistance measurements to 15 dysphonic patients is described. Laryngeal resistance is calculated from analysis of translaryngeal pressure and airflow during the utterance /pi/, and found to fall predictably after successful toxin injection. In our series of patients, laryngeal resistance dropped by 69.1% after initial toxin injection. The changes in resistance over time correlate with subjective impressions of voice quality. Translaryngeal resistance measurements can be used objectively to follow patients longitudinally after injection and to collect objective data for analysis. No previously described measurements have met all these criteria. Laryngeal resistance measurement is an ideal method of documenting the results of botulinum toxin injection for the treatment of focal laryngeal dystonia.

The electrodiagnosis of myasthenia gravis and the Lambert-Eaton myasthenic syndrome.

Electrodiagnostic studies are valuable in confirming the diagnosis of a disorder of neuromuscular transmission. They are used to distinguish presynaptic and postsynaptic abnormalities. These studies provide an objective measure of the severity of the illness and may be useful in assessing the response to therapy. This article reviews the electrodiagnostic techniques that are commonly used today and highlights their specificity, sensitivity, and pitfalls.