AAPS Perspective on the EURL Recommendation on the use of Non-Animal-Derived Antibodies.

In May 2020, the EU Reference Laboratory for alternatives to animal testing (EURL ECVAM) published a recommendation report entitled "Recommendation on nonanimal-derived antibodies". In this report, the EURL ECVAM specifically states: "Therefore, taking into consideration the ESAC Opinion on the scientific validity of replacements for animal-derived antibodies, EURL ECVAM recommends that animals should no longer be used for the development and production of antibodies for research, regulatory, diagnostic and therapeutic applications. The provisions of Directive 2010/63/EU should be respected, and EU countries should no longer authorise the development and production of antibodies through animal immunisation, where robust, legitimate scientific justification is lacking." (1). Here, we are providing the American Association of Pharmaceutical Scientists (AAPS) opinion on the EURL ECVAM recommendation report. In brief, there has been a clear and strong progress in reduction of animal use in the drug discovery and development process, including significant reduction of animal use in production of antibody reagents. Yet, it is proposed that more data need to be generated, shared and discussed within the scientific community before a decision to implement the change to non-animal derived antibodies is made.

High-efficiency 2 µm Tm:YAP laser with a compact mechanical Q-switch.

We describe a compact, highly efficient, diode-pumped, mechanically Q-switched Tm:YAP laser operating near 2 µm. The Q-switch, based on a torsion spring resonant mirror scanner, had negligible optical loss and required very low electrical drive power. At a 10 kHz pulse repetition frequency, the laser generated an average output power of 10.5 W at 1.94 µm, Q-switched pulse energy of 1.05 mJ, a pulse length of 31 ns, and a peak power of 34 kW. The Q-switched laser exhibited maximum optical and electrical efficiencies of 51% and 26%, respectively.

Narrow-band 1 W source at 257 nm using frequency quadrupled passively Q-switched Yb:YAG laser.

We describe generation of 1.1 W of 257 nm emission by frequency quadrupling the 1030 nm emission from a compact passively Q-switched Yb:YAG laser. The laser utilized a volume Bragg grating to achieve a 0.1 nm linewidth required for UV-Raman spectroscopic applications, generated 100 kW peak power, 250 µJ pulses and 3.6 W of average power at 1030 nm. Fourth harmonic generation (FHG) was carried out using a 10 mm lithium triborate (LBO) crystal to generate 515 nm second harmonic with 70% conversion efficiency, followed by a 7 mm beta-barium borate (BBO) crystal to generate 257 nm fourth harmonic with 45% efficiency, resulting in an overall nonlinear conversion efficiency of 31%. Far-field and near-field of the FHG emission were characterized.

Improved Monoblock laser brightness using external reflector.

The Monoblock laser has become the laser of choice in long-range, eye-safe laser range finders. It is eye-safe with emission at 1570 nm, high pulse energy, simple construction, and high efficiency when pumped by a laser-diode stack. Although the output beam divergence of a typical Monoblock with a 3 mm×3 mm cross section is relatively large (10-12 mrad), it can be reduced to <1 mrad using a telescope with large magnification. In this paper we present a simple and compact technique for achieving significant reduction in the Monoblock beam divergence using a partial reflector that is placed a short distance from the optical parametric oscillator (OPO). Using a 38 mm long Monoblock with a 10 mm long potassium titanyl phosphate OPO, we achieved a beam divergence of <4 mrad, corresponding to a >2.5× reduction from the unmodified laser. Performance using this technique with various feedback and etalon spacings is presented.

Quantitative objective markers for upper and lower motor neuron dysfunction in ALS.

OBJECTIVE: To investigate the value of objective biomarkers for upper (UMN) and lower (LMN) motor neuron involvement in ALS. METHODS: We prospectively studied 64 patients with ALS and its subsets using clinical measures, proton MR spectroscopic imaging ((1)H MRSI), diffusion tensor imaging, transcranial magnetic stimulation, and the motor unit number estimation (MUNE) at baseline and every 3 months for 15 months and compared them with control subjects. RESULTS: (1)H MRSI measures of the primary motor cortex N-acetyl-aspartate (NAA) concentration were markedly reduced in ALS (p = 0.009) and all UMN syndromes combined (ALS, familial ALS [fALS], and primary lateral sclerosis; p = 0.03) vs control values. Central motor conduction time to the tibialis anterior was prolonged in ALS (p < 0.0005) and combined UMN syndromes (p = 0.001). MUNE was lower in ALS (p < 0.0005) and all LMN syndromes combined (ALS, fALS, and progressive muscular atrophy; p = 0.001) vs controls. All objective markers correlated well with the ALS Functional Rating Scale-Revised, finger and foot tapping, and strength testing, suggesting these markers related to disease activity. Regarding changes over time, MUNE changed rapidly, whereas neuroimaging markers changed more slowly and did not significantly differ from baseline. CONCLUSIONS: (1)H MR spectroscopic imaging measures of the primary motor cortex N-acetyl-aspartate (NAA) concentration and ratio of NAA to creatine, central motor conduction time to the tibialis anterior, and motor unit number estimation significantly differed between ALS, its subsets, and control subjects, suggesting they have potential to provide insight into the pathobiology of these disorders.

End-pumped 1.5 microm monoblock laser for broad temperature operation.

We describe a next-generation monoblock laser capable of a greater than 10 mJ, 1.5 microm output at 10 pulses/s (pps) over broad ambient temperature extremes with no active temperature control. The transmitter design is based on a Nd:YAG laser with a Cr4+ passive Q switch and intracavity potassium titanyl phosphate optical parametric oscillator. To achieve the repetition rate and efficiency goals of this effort, but still have wide temperature capability, the Nd:YAG slab is end pumped with a 12-bar stack of 100 W (each) diode bars. Different techniques for focusing the pump radiation into the 4.25 mmx4.25 mm end of the slab are compared, including a lensed design, a reflective concentrator, and a lens duct. A wide temperature operation (-20 degrees C to 50 degrees C) for each end-pumped configuration is demonstrated.

Characteristics of patients with sensory neuropathy diagnosed with abnormal small nerve fibres on skin biopsy.

Clinical, laboratory and electrodiagnostic (EDX) characteristics of 62 patients with sensory neuropathy with abnormal skin biopsies were reviewed. Reduced epidermal nerve fibre density (ENFD) was seen in 71% and morphological changes with normal ENFD were seen in 29% of the patients. Patients with small fibre sensory neuropathy may have associated large fibre loss undetected by routine EDX. Identified associations included abnormal glucose metabolism, Lyme vaccination, monoclonal gammopathy, vitamin B12 deficiency, coeliac disease, and diseases of the connective tissue, inflammatory bowel and thyroid. Sensory neuropathy remained undetermined in 50% of the patients.

Randomized controlled phase II trial of glatiramer acetate in ALS.

The authors conducted a randomized controlled trial to test the safety and immunology of glatiramer acetate in ALS. Twenty treated patients were randomly assigned to daily or biweekly injections. Ten control patients were selected from another trial and followed up concurrently. Injection reactions were the only common adverse event (p = 0.01). Treated patients showed enhanced lymphocyte proliferation (p = 0.02). The safety profile and immune effects support conducting larger trials of dose selection and efficacy.

The natural history of primary lateral sclerosis.

OBJECTIVE: To define the syndrome of primary lateral sclerosis (PLS) and disorders that contain features of both ALS and PLS, to determine the time beyond which PLS is less likely to become ALS clinically, and to determine the outcome of people with PLS and those who develop lower motor neuron (LMN) signs. METHODS: The authors reviewed the records of all 39 patients initially diagnosed with PLS in 1984 to 2004. Diagnostic subgroups were defined based on clinical features. The authors used Kaplan-Meier methods to estimate the time to diagnosis, linear regression analyses to assess function, and a Cox proportional hazard model to assess survival in subgroups. RESULTS: Of the 39 patients, 29 had only upper motor neuron (UMN) signs on initial evaluation. Thirteen of the 29 were later classified as having UMN-dominant ALS (UMN-D) because they acquired evidence of denervation by EMG (3.17 years) or examination (3.67 years). Sixteen of the 29 patients, classified as clinically pure PLS, retained only UMN signs and a normal EMG (mean follow-up 8.7 years). Ten patients who met criteria for ALS at the initial visit were used as controls. The UMN-dominant ALS group had lower functional scores (p = 0.033) than the PLS group, and similar scores to those with ALS. Survival was longer in both the PLS group (p = 0.027) and the UMN-D group (p = 0.067) than the ALS group. CONCLUSIONS: Clinically pure PLS can be defined by isolated UMN signs 4 years after symptom onset, and is a syndrome of slow progression with high levels of function. Prior to the fourth year, the diagnosis of PLS cannot be made with certainty because many patients develop LMN signs. UMN-dominant ALS, defined by predominantly UMN disease with minor LMN signs, has disability similar to ALS, but slower progression.

Objective tests for upper motor neuron involvement in amyotrophic lateral sclerosis (ALS).

OBJECTIVE: To develop objective markers for upper motor neuron (UMN) involvement in ALS, the value of single-voxel MR spectroscopy (MRS) and transcranial magnetic stimulation (TMS) was studied. METHODS: Test results of 164 ALS patients who had MRS only (n = 91), TMS only (n = 13), or both (n = 60) were analyzed; also, 11 autopsy examinations were evaluated. RESULTS: Abnormal test results consistent with UMN involvement were found in 134 patients with clinical UMN signs: 86% on MRS, 77% on TMS, and 70% on MRS and TMS together. Among 30 patients with solely LMN signs (progressive muscular atrophy), UMN results were found in 63% on MRS, 63% on TMS, and 46% on both tests together. There was a significant association of the degree of abnormal N-acetyl aspartate/creatine ratios with UMN signs (p = 0.01). The sensitivity to detect UMN involvement was 0.86 for MRS (specificity 0.37) and 0.77 for TMS (specificity 0.38). At autopsy, all 11 patients had pathologic UMN abnormalities, including 4 with normal MRS and 1 with normal TMS in life. CONCLUSIONS: MRS is highly sensitive, somewhat more than TMS, and shows good correlation with clinical UMN signs. Combining MRS and TMS results in the same patient with further refinement may help in the early diagnosis of ALS.

Celiac neuropathy.

BACKGROUND: Celiac disease (CD) is a chronic inflammatory enteropathy resulting from sensitivity to ingested gluten. Neurologic complications are estimated to occur in 10% of affected patients, with ataxia and peripheral neuropathy being the most common problems. The incidence and clinical presentation of patients with CD-associated peripheral neuropathy have not previously been investigated. OBJECTIVE: To determine the incidence of CD in patients with neuropathy and to characterize the clinical presentation. METHODS: The records of 20 patients with neuropathy and biopsy-confirmed CD were reviewed. RESULTS: Six of the 20 patients had neuropathic symptoms alone without gastrointestinal involvement, and neuropathic symptoms preceded other CD symptoms in another 3 patients. All patients had burning, tingling, and numbness in their hands and feet, with distal sensory loss, and nine had diffuse paresthesias involving the face, trunk, or lumbosacral region. Only two had weakness. Results of electrophysiologic studies were normal or mildly abnormal in 18 (90%) of the patients. Sural nerve biopsies, obtained from three patients, revealed mild to severe axonopathy. Using the agglutination assay, 13 (65%) of the patients were positive for ganglioside antibodies. Excluding patients who were referred with the diagnosis of celiac neuropathy, CD was seen in approximately 2.5% of all neuropathy patients and in 8% of patients with neuropathy and normal electrophysiologic studies seen at our center. CONCLUSION: CD is commonly associated with sensory neuropathy and should be considered even in the absence of gastrointestinal symptoms.

Dynamic allocation of attention in aging and Alzheimer disease: uncoupling of the eye and mind.

CONTEXT: Visual attention can be distributed focally, in the direction of gaze, or globally, throughout the extrapersonal space. Aging, and especially Alzheimer disease (AD), may influence global attention, resulting in shifts of gaze to attend to the global workspace. OBJECTIVE: To determine if subjects who have AD and cognitively intact older subjects shift their gaze more often than young subjects while viewing a dynamic stimulus that emphasizes global attention. DESIGN: Experimental study of eye fixation patterns in response to a simulated driving scene with stationary and moving distractors. SETTING: Urban, medical school, National Institute on Aging-funded Alzheimer's Disease Center. PARTICIPANTS: Thirteen subjects with mild probable AD, 13 age-comparable cognitively intact older control subjects, and 11 young control subjects. MAIN OUTCOME MEASURE: Proportion of eye fixations within and outside of a central region of interest encompassing the "road" surface. RESULTS: Young controls made significantly more eye fixations (mean number of eye fixations, 47.5) than either of the other 2 groups (older controls mean, 33.2; patients with AD mean, 32.2). However, 76% of their fixations remained within the central region of interest. Older controls and subjects with AD made proportionately fewer fixations within this region (48% and 49%, respectively) than young controls and moved their eyes more often to the periphery but did not differ from one another. CONCLUSIONS: Young controls maintain central eye position regardless of peripheral distraction. Older controls move their eyes to the periphery, presumably to widen the window of attention. Subjects with mild AD did not experience an additional disadvantage beyond that associated with aging.

Electrophysiologic studies in critical illness associated weakness: myopathy or neuropathy--a reappraisal.

OBJECTIVE: Unexplained weakness in critically ill patients is recognized with increasing frequency. However, it is debated whether the condition is a peripheral neuropathy or a myopathy. Diagnostic difficulties can arise from multiple sources that are not generally a factor in other neuromuscular conditions. Conventional electrodiagnostic techniques may provide only non-specific data, clinical examination is often hampered, and muscle biopsy is not a practical screening tool. METHOD: To improve diagnostic yield, we studied 22 consecutive patients with critical illness associated weakness with additional electrodiagnostic techniques, including direct muscle stimulation, quantitative electromyography, and motor unit number estimation. RESULTS: The applied techniques supported an underlying myopathy in all the patients examined. The diagnosis was confirmed by muscle biopsy in 9 patients. Additional lesser features of neuropathy were concomitantly present in one patient who also underwent sural nerve biopsy. CONCLUSIONS: The study suggests that myopathy is much more common than polyneuropathy in critical illness. Suspicion of this entity should be high in this setting even without exposure to corticosteroids or non-depolarizing blocking agents.

Inclusion body myositis mimicking motor neuron disease.

OBJECTIVE: To describe the clinical and electrophysiologic features of patients with inclusion body myositis that was misinterpreted as motor neuron disease. PATIENTS AND METHODS: We retrospectively retrieved the medical records of 70 patients with a pathologic diagnosis of inclusion body myositis. From this group, we selected those who had been first diagnosed as having motor neuron disease or amyotrophic lateral sclerosis. We reviewed the clinical, electrophysiologic, laboratory, and morphologic studies. RESULTS: Nine (13%) of 70 patients with inclusion body myositis had been diagnosed as having motor neuron disease. Six of the 9 patients had asymmetric weakness; in 4 the distal arm muscles were affected. Eight patients had finger flexor weakness. Tendon reflexes were preserved in weak limbs in 6, hyperactive in 2, and absent in 1. Four patients had dysphagia. Fasciculation was seen in 2 patients. None had definite upper motor neuron signs or muscle cramps. Routine electromyographic studies showed fibrillation potentials and positive sharp waves in all 9. Fasciculation potentials were seen in 7 and long-duration polyphasic motor unit potentials were seen in 8. There was no evidence of a myogenic disorder in these 9 patients. Muscle biopsy was done because of slow progression or prominent weakness of the finger flexors and was diagnostic of inclusion body myositis. A quantitative electromyogram was myopathic in 4 of the 5 patients studied. CONCLUSIONS: Inclusion body myositis may mimic motor neuron disease. Muscle biopsy and quantitative electromyographic analysis are indicated in patients with atypical motor neuron disease, especially those with slow progression or early and disproportionate weakness of the finger flexors.

Recruitment of the mitochondrial-dependent apoptotic pathway in amyotrophic lateral sclerosis.

Molecular mechanisms of apoptosis may participate in motor neuron degeneration produced by mutant superoxide dismutase-1 (mSOD1), the only proven cause of amyotrophic lateral sclerosis (ALS). Consistent with this, here we show that the proapoptotic protein Bax translocates from the cytosol to the mitochondria, whereas cytochrome c translocates from the mitochondria to the cytosol in spinal cords of transgenic mSOD1 mice during the progression of the disease. Concomitantly, caspase-9 is activated in the spinal cord of transgenic mSOD1 mice. Only in end-stage transgenic mSOD1 mice is the downstream caspase-7 activated and the inhibitor of apoptosis, XIAP, cleaved. These results indicate a sequential recruitment of molecular elements of the mitochondrial-dependent apoptotic pathway in transgenic mSOD1 mice. We also provide immunohistochemical evidence that cytochrome c translocation occurs in the spinal cord of sporadic ALS patients. Collectively, these data suggest that the mitochondrial-dependent apoptotic pathway may contribute to the demise of motor neurons in ALS and that targeting key molecules of this cascade may prove to be neuroprotective.

Myopathy with tubulin-reactive crystalline inclusions.

A 61-year-old man with muscle aches and persistently elevated serum creatine kinase had aggregates of randomly oriented, rhomboidal or rectangular protein crystalline inclusions in the sarcoplasm of type II fibers. Immunochemical studies showed strong reactivity of the inclusions to tubulin antibodies, suggesting that these unique crystalline inclusions may be a consequence of altered synthesis, processing, or degradation of tubulin.

Repression of glucocorticoid receptor gene transcription by c-Jun.

The regulation of glucocorticoid receptor gene expression by members of the AP-1 family was examined in glucocorticoid-free NIH3T3 cells transfected with the human glucocorticoid receptor gene promoter driving expression of a CAT reporter gene. c-Jun inhibited the promoter activity by 80% and JunB by 30%, whereas c-Fos and JunD had no inhibitory effect. Electrophoretic mobility shift assays showed that c-Jun is unable to efficiently interact with the AP-1-like site present in the human glucocorticoid receptor promoter. Moreover, c-Jun was still able to repress promoter mutants in which the region containing the AP-1-like site was deleted. NIH3T3 cell clones overexpressing c-Jun exhibited lower glucocorticoid receptor mRNA levels, which suggests that the murine glucocorticoid receptor gene can also be regulated by AP-1. These results provide a new mechanism for cross-talk between the glucocorticoid receptor and the AP-1 family of transcription factors in the absence of glucocorticoid ligands.

Increased expression of the pro-inflammatory enzyme cyclooxygenase-2 in amyotrophic lateral sclerosis.

Mutations in the copper/zinc superoxide dismutase (mSOD1) gene are associated with a familial form of amyotrophic lateral sclerosis (ALS), and their expression in transgenic mice produces an ALS-like syndrome. Recent observations suggest a role for inflammatory-related events in the progression and propagation of the neurodegenerative process in ALS. Consistent with this view, the present study demonstrates that, during the course of the disease, the expression of cyclooxygenase type 2 (Cox-2), a key enzyme in the synthesis of prostanoids, which are potent mediators of inflammation, is dramatically increased. In both early symptomatic and end-stage transgenic mSOD1 mice, neurons and, to a lesser extent, glial cells in the anterior horn of the spinal cord exhibit robust Cox-2 immunoreactivity. Cox-2 mRNA and protein levels and catalytic activity are also significantly increased in the spinal cord of the transgenic mSOD1 mice. The time course of the spinal cord Cox-2 upregulation parallels that of motor neuronal loss in transgenic mSOD1 mice. We also show that Cox-2 activity is dramatically increased in postmortem spinal cord samples from sporadic ALS patients. We speculate that Cox-2 upregulation, through its pivotal role in inflammation, is instrumental in the ALS neurodegenerative process and that Cox-2 inhibition may be a valuable therapeutic avenue for the treatment of ALS.

Absence of echovirus sequences in brain and spinal cord of amyotrophic lateral sclerosis patients.

The role of enteroviruses in pathogenesis of amyotrophic lateral sclerosis (ALS) is controversial. A recent study, based on reverse transcription-polymerase chain reaction (RT-PCR) analysis of spinal cord, reported identification of a novel echovirus in 15 of 17 French subjects with ALS and only 1 of 29 subjects with other neurologic diseases. We established a real-time RT-PCR method based on this novel echovirus sequence and used this method and that previously employed for analysis of the French subjects to determine the prevalence of echoviral sequences in spinal cord and motor cortex of sporadic ALS subjects from the United States. No echoviral sequences were found in 20 spinal cord and 10 motor cortex samples from autopsy-confirmed cases of ALS or 13 spinal cord and 5 motor cortex samples from subjects with no motor neuron disease.

Primary central nervous system lymphoma in childhood presenting as progressive panhypopituitarism.

We report a 15-year-old boy who had isolated central diabetes insipidus initially diagnosed at age 11 years. A brain magnetic resonance imaging (MRI) was normal at the time. At age 12 years, growth hormone (GH) testing was performed because of a decline in linear growth rate and demonstrated GH deficiency. After a repeat normal brain MRI, GH therapy was begun. Three years later, hormonal testing revealed prepubertal gonadotropins and low testosterone levels, free thyroxine index, and morning cortisol levels. Repeat brain MRI demonstrated a 9-mm enhancing lesion in the region of the pituitary stalk. The pathologic diagnosis was that of a high-grade malignant B-cell lymphoma, suggestive of Burkitt Lymphoma. Growth hormone therapy has not been associated with an increased incidence of lymphoma. This report underscores the need for vigilance in follow-up brain imaging and hormonal evaluation in children with diabetes insipidus, especially those with evolving anterior hormone deficiencies.