Leucine-Rich Glioma-Inactivated 1 versus Contactin-Associated Protein-like 2 Antibody Neuropathic Pain: Clinical and Biological Comparisons.

Pain is a under-recognized association of leucine-rich glioma-inactivated 1 (LGI1) and contactin-associated protein-like 2 (CASPR2) antibodies. Of 147 patients with these autoantibodies, pain was experienced by 17 of 33 (52%) with CASPR2- versus 20 of 108 (19%) with LGI1 antibodies (p = 0.0005), and identified as neuropathic in 89% versus 58% of these, respectively. Typically, in both cohorts, normal nerve conduction studies and reduced intraepidermal nerve fiber densities were observed in the sampled patient subsets. In LGI1 antibody patients, pain responded to immunotherapy (p = 0.008), often rapidly, with greater residual patient-rated impairment observed in CASPR2 antibody patients (p = 0.019). Serum CASPR2 antibodies, but not LGI1 antibodies, bound in vitro to unmyelinated human sensory neurons and rodent dorsal root ganglia, suggesting pathophysiological differences that may underlie our clinical observations. ANN NEUROL 2021;90:683-690.

Evaluation of recombinant monoclonal antibody SVmab1 binding to Na V1.7 target sequences and block of human Na V1.7 currents.

Identification of small and large molecule pain therapeutics that target the genetically validated voltage-gated sodium channel Na V1.7 is a challenging endeavor under vigorous pursuit. The monoclonal antibody SVmab1 was recently published to bind the Na V1.7 DII voltage sensor domain and block human Na V1.7 sodium currents in heterologous cells. We produced purified SVmab1 protein based on publically available sequence information, and evaluated its activity in a battery of binding and functional assays. Herein, we report that our recombinant SVmAb1 does not bind peptide immunogen or purified Na V1.7 DII voltage sensor domain via ELISA, and does not bind Na V1.7 in live HEK293, U-2 OS, and CHO-K1 cells via FACS. Whole cell manual patch clamp electrophysiology protocols interrogating diverse Na V1.7 gating states in HEK293 cells, revealed that recombinant SVmab1 does not block Na V1.7 currents to an extent greater than observed with an isotype matched control antibody. Collectively, our results show that recombinant SVmab1 monoclonal antibody does not bind Na V1.7 target sequences or specifically inhibit Na V1.7 current.

Production of high-quality particulate methane monooxygenase in high yields from Methylococcus capsulatus (bath) with a hollow-fiber membrane bioreactor.

In order to obtain particulate methane monooxygenase (pMMO)-enriched membranes from Methylococcus capsulatus (Bath) with high activity and in high yields, we devised a method to process cell growth in a fermentor adapted with a hollow-fiber bioreactor that allows easy control and quantitative adjustment of the copper ion concentration in NMS medium over the time course of cell culture. This technical improvement in the method for culturing bacterial cells allowed us to study the effects of copper ion concentration in the growth medium on the copper content in the membranes, as well as the specific activity of the enzyme. The optimal copper concentration in the growth medium was found to be 30 to 35 micro M. Under these conditions, the pMMO is highly expressed, accounting for 80% of the total cytoplasmic membrane proteins and having a specific activity as high as 88.9 nmol of propylene oxide/min/mg of protein with NADH as the reductant. The copper stoichiometry is approximately 13 atoms per pMMO molecule. Analysis of other metal contents provided no evidence of zinc, and only traces of iron were present in the pMMO-enriched membranes. Further purification by membrane solubilization in dodecyl beta-D-maltoside followed by fractionation of the protein-detergent complexes according to molecular size by gel filtration chromatography resulted in a good yield of the pMMO-detergent complex and a high level of homogeneity. The pMMO-detergent complex isolated in this way had a molecular mass of 220 kDa and consisted of an alphabetagamma protein monomer encapsulated in a micelle consisting of ca. 240 detergent molecules. The enzyme is a copper protein containing 13.6 mol of copper/mol of pMMO and essentially no iron (ratio of copper to iron, 80:1). Both the detergent-solubilized membranes and the purified pMMO-detergent complex exhibited reasonable, if not excellent, specific activity. Finally, our ability to control the level of expression of the pMMO allowed us to clarify the sensitivity of the enzyme to NADH and duroquinol, the two common reductants used to assay the enzyme.